Reviews: July 2005, Volume 18, Issue 3

Clinical Microbiology Reviews Reviews: July 2005, Volume 18, Issue 3 Alexander Mathis, Rainer Weber, and Peter Deplazes Zoonotic Potential of the Mic...
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Clinical Microbiology Reviews

Reviews: July 2005, Volume 18, Issue 3 Alexander Mathis, Rainer Weber, and Peter Deplazes Zoonotic Potential of the Microsporidia Clin. Microbiol. Rev. 2005 18: 423-445. Eduardo Villamor and Wafaie W. Fawzi Effects of Vitamin A Supplementation on Immune Responses and Correlation with Clinical Outcomes Clin. Microbiol. Rev. 2005 18: 446-464. Firdausi Qadri, Ann-Mari Svennerholm, A. S. G. Faruque, and R. Bradley Sack Enterotoxigenic Escherichia coli in Developing Countries: Epidemiology, Microbiology, Clinical Features, Treatment, and Prevention Clin. Microbiol. Rev. 2005 18: 465-483. Maria E. Aguero-Rosenfeld, Guiqing Wang, Ira Schwartz, and Gary P. Wormser Diagnosis of Lyme Borreliosis Clin. Microbiol. Rev. 2005 18: 484-509. Thomas S. Murray, M. Elizabeth Groth, Carol Weitzman, and Michael Cappello Epidemiology and Management of Infectious Diseases in International Adoptees Clin. Microbiol. Rev. 2005 18: 510-520. Bénédicte Fournier and Dana J. Philpott Recognition of Staphylococcus aureus by the Innate Immune System Clin. Microbiol. Rev. 2005 18: 521-540. Peter J. M. Openshaw and John S. Tregoning Immune Responses and Disease Enhancement during Respiratory Syncytial Virus Infection Clin. Microbiol. Rev. 2005 18: 541-555. Brad Spellberg, John Edwards, Jr., and Ashraf Ibrahim Novel Perspectives on Mucormycosis: Pathophysiology, Presentation, and Management Clin. Microbiol. Rev. 2005 18: 556-569. William E. Collins and Geoffrey M. Jeffery Plasmodium ovale: Parasite and Disease Clin. Microbiol. Rev. 2005 18: 570-581.

Author's Correction: Gabriel A. Schmunis and Jose R. Cruz Safety of the Blood Supply in Latin America Clin. Microbiol. Rev. 2005 18: 582.

CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 423–445 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.423–445.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 18, No. 3

Zoonotic Potential of the Microsporidia Alexander Mathis,1* Rainer Weber,2 and Peter Deplazes1 Institute of Parasitology, Vetsuisse Faculty, University of Zurich,1 and Division of Infectious Diseases and Hospital Epidemiology, University Hospital,2 Zurich, Switzerland INTRODUCTION .......................................................................................................................................................423 The Organisms ........................................................................................................................................................424 Diagnostic Techniques ...........................................................................................................................................424 Phylogeny .................................................................................................................................................................425 Natural History .......................................................................................................................................................426 ENTEROCYTOZOON BIENEUSI ..............................................................................................................................426 Infections in Humans.............................................................................................................................................426 Infections in Animals .............................................................................................................................................428 Animal Models ........................................................................................................................................................428 Molecular Epidemiology ........................................................................................................................................428 Sources of Human Infections and Transmission ...............................................................................................429 ENCEPHALITOZOON ................................................................................................................................................430 ENCEPHALITOZOON CUNICULI............................................................................................................................431 Infections in Humans.............................................................................................................................................431 Infections in Animals .............................................................................................................................................432 Rabbits .................................................................................................................................................................432 Rodents.................................................................................................................................................................432 Carnivores............................................................................................................................................................433 Monkeys ...............................................................................................................................................................433 Molecular Epidemiology ........................................................................................................................................433 Sources of Human Infections and Transmission ...............................................................................................434 ENCEPHALITOZOON HELLEM ..............................................................................................................................434 Infections in Humans.............................................................................................................................................434 Infections in Animals .............................................................................................................................................434 Molecular Epidemiology ........................................................................................................................................435 Sources of Human Infections and Transmission ...............................................................................................435 ENCEPHALITOZOON INTESTINALIS....................................................................................................................435 Infections in Humans.............................................................................................................................................435 Infections in Animals .............................................................................................................................................436 Molecular Epidemiology ........................................................................................................................................436 Sources of Human Infections and Transmission ...............................................................................................436 OTHER MICROSPORIDIA......................................................................................................................................436 Vittaforma spp. .........................................................................................................................................................436 Pleistophora spp. ......................................................................................................................................................437 Trachipleistophora spp.............................................................................................................................................437 Brachiola spp. ..........................................................................................................................................................437 Microsporidium spp. ................................................................................................................................................438 Microsporidium ceylonensis .................................................................................................................................438 Microsporidium africanum ..................................................................................................................................438 CONCLUDING REMARKS ......................................................................................................................................438 ACKNOWLEDGMENT..............................................................................................................................................438 REFERENCES ............................................................................................................................................................438 agents of economically important diseases in insects (silk worms and honey bees) (14), fish (179, 256), and mammals (rabbits, fur-bearing animals, and laboratory rodents) (37), and they emerged as important opportunistic pathogens when AIDS became pandemic (320). Thus, the question of whether animal reservoirs are the sources of human infections is reasonable. The focus of this review is to discuss the most recent perceptions on the zoonotic potential of the various microsporidia with proven vertebrate hosts. Furthermore, we provide a brief

INTRODUCTION Microsporidia are an exceptionally diverse group of organisms, comprising more than 1,200 species which parasitize a wide variety of invertebrate and vertebrate hosts. These organisms have long been known to be causative * Corresponding author. Mailing address: Institute of Parasitology, Winterthurerstr 266a, CH-8057 Zurich, Switzerland. Phone: 41 (0)44 635 85 36. Fax: 41 (0)44 635 89 07. E-mail: [email protected] .unizh.ch. 423

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FIG. 1. Electron micrograph of the developmental stages of the microsporidian species Encephalitozoon cuniculi in a host cell-derived vacuole in in vitro-cultivated human fibroblast cells. K, nucleus of host cell; M, meront; P, sporont, which divides into two sporoblasts; B, sporoblast (2 ␮m in length), with cross sections of the polar tube; S, mature spore.

update on other microsporidia which have no known vertebrate host or an invertebrate host and cause rare infections in humans.

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ery organ in the body and a broad spectrum of cell types (217) (Table 1). The most common microsporidial infections in humans are due to E. bieneusi and Encephalitozoon intestinalis. Both species have been found worldwide, mainly in HIV-infected patients with chronic diarrhea but also in immunocompetent persons with acute, self-limited diarrhea. Encephalitozoon cuniculi and Encephalitozoon hellem have been diagnosed, with very few exceptions, in immunocompromised patients and as causing local (e.g., ocular) or disseminated infections. Other rare microsporidial species infecting immunodeficient patients include Vittaforma corneae (formerly Nosema corneum), Pleistophora ronneafiei, Trachipleistophora spp., and Brachiola spp. Furthermore, V. corneae, Trachipleistophora hominis, Brachiola algerae, Nosema ocularum, Microsporidium ceylonensis, and Microsporidium africanum have been described in single cases as agents of ocular infections in immunocompetent persons. Due to the administration of antiretroviral combination therapy, which restores immunity in HIV-infected persons, the number of clinically manifest microsporidial infections has markedly decreased in affluent countries (322). However, it is estimated that two-thirds of all HIV-infected persons live in sub-Saharan Africa, where antiretroviral therapy is not widely available due to the costs, and consequently, opportunistic complications continue to cause severe morbidity and mortality. Indeed, a recent study showed that 13% of HIV-positive diarrheic adults in Mali were positive for E. bieneusi, which thus was the most prevalent intestinal parasite in this African country (10). Furthermore, microsporidial infections are increasingly being diagnosed in affluent countries in immunosuppressed patients who undergo organ transplantation (39, 115, 119, 130, 132, 156, 167, 197, 200, 228, 229, 244, 260, 283), as well as causing ocular infections in nonimmunocompromised persons (47, 171, 206, 259, 274, 284).

The Organisms

Diagnostic Techniques

Microsporidia are unicellular, obligate intracellular eukaryotes. Their life cycle includes a proliferative merogonic stage, followed by a sporogonic stage resulting in characteristically small (1 to 4 ␮m), environmentally resistant, infective spores (Fig. 1) (95). The spores contain a long, coiled tubular extrusion apparatus (“polar tube”), which distinguishes microsporidia from all other organisms and has a crucial role in host cell invasion: Upon extrusion from the spore, it injects the sporoplasm along with its nucleus into the cytoplasm of a new host cell after piercing the plasmalemma of the host cell or the membrane of the phagosomes containing the endocytosed spores (58, 106). Before the onset of the AIDS pandemic, only eight cases of human microsporidial infections had been reported (reviewed in reference 320). In most cases, species identification of the causative agents was not conclusive. In 1985, as early as 2 years after the identification of human immunodeficiency virus (HIV) as the causative agent of AIDS, the microsporidial species Enterocytozoon bieneusi was discovered in HIV-infected patients with chronic diarrhea (76). Subsequently, several new genera and species of microsporidia were found to be important opportunistic pathogens in humans, infecting virtually ev-

Considerable progress has been made in improving the repertoire of techniques for detection of microsporidia. Previously, diagnosis was based on laborious electron-microscopic examinations because of the small size of the organisms. The introduction of staining techniques allowed routine, light-microscopic diagnosis of microsporidial spores, at least to the genus level (319). Diagnosis to the species level is achieved by using antibodies (polyclonal or monoclonal) and by molecular methods based on the PCR (reviewed in references 116, 322, and 327). This latter sensitive and specific method has, in addition, the intrinsic advantage that upon further analysis of the PCR products with various methods (restriction fragment length polymorphism, SSCP, or sequencing), identification at the subspecies level (strains or genotypes) can be achieved (86, 154). Serologic tests have been useful in detecting antibodies of E. cuniculi in several species of animals, but the value of detecting antibodies against Encephalitozoon spp. in humans has been controversial because of possible nonspecificity of the tests when spore walls are used as the antigen (322). By employing recombinant antigens of the polar tube of E. cuniculi, a high specificity was recently demonstrated, and the development of

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TABLE 1. Microsporidial species infecting humans Discovery

No. of confirmed reported patients

Species Host

Yr

Reference(s)

Immunocompromiseda

Animal host(s)

Immunocompetentb

Site(s) of infection (reference[s])

Intestine, biliary tract, respiratory tract (322) Eye, respiratory tract, urinary tract, systemic infection (322) Intestine, biliary tract, respiratory tract, bone, skin, systemic infection (322)

Vertebrates

Systemic infection, eye, respiratory tract, urinary tract, liver, peritoneum, brain (322) Eye, urinary tract (75, 101, 231)

Mammals Unknown

Intestine (278) Muscle (169)

Unknown Unknown

Muscle, eye (100, 231)

Unknown

Systemic infection, eye (151, 335)

Unknown

Eye, muscle (61, 307)

Mosquito

Enterocytozoon bieneusi

Human

1985

76

⬎1,000

⬍20

Encephalitozoon hellem

Human

1991

78

⬍50

3

Encephalitozoon intestinalis (originally named Septata intestinalis) Encephalitozoon cuniculi

Human

1993

30, 139

⬍200

2

Rabbit

1923

170

⬍20

—c

Vittaforma corneae (originally named Nosema corneum) Vittaforma-liked Pleistophora ronneafiei (originally named Pleistophora sp.) Trachipleistophora hominis Trachipleistophora anthropophthera Brachiola algerae (originally named Nosema algerae) Brachiola connori (originally named Nosema connori) Brachiola vesicularum Nosema ocularum Microsporidium ceylonensis Microsporidium africanum

Human

1990

65, 258

1

3

Human Human

2003 1985

278 32, 169

22 1e

Human

1996

145

1

Human

1998

305

3

3 — 1 —

f

1

Birds Mammals

Mosquito

1970

304

1

Human

1974

33, 273

1g



Systemic infection (273)

Unknown

Human Human Human

1998 1991 1973

33 31 11, 12, 36

1

— 1

Muscle (33) Eye (31) Eye (12)

Unknown Unknown Unknown

Human

1981

223

1 (unknown)

Eye (223)

Unknown

— 1 (unknown)

a

HIV-seropositive persons, AIDS patients, and organ transplant recipients. Immunocompetent, otherwise healthy. Two patients with unknown immunostatus, presumably cellular immunodeficiency (see Table 4). d PCR/sequencing results only, needs confirmation. e Cellular immunodeficiency (HIV antibody negative). f Patient was taking immunosuppressive agents for rheumatoid arthritis for decades. g Thymic aplasia. b c

appropriate serodiagnostic tools seems feasible (298). No tests are available for the serodiagnosis of E. bieneusi. Many but not all microsporidia infecting humans can be continuously cultivated in vitro in various cell lines (184, 306). This facilitates investigations of their basic biology (102, 127) and also allows for easy assessment of drugs (80) or disinfection schemes (118, 147, 150, 173), which has become an issue since human-pathogenic microsporidia have been discovered in surface waters (57, 89, 90, 103, 150, 255, 272). Phylogeny Investigations on the basic biology have unearthed highly exceptional characteristics of the microsporidia. Although they are true eukaryotes (i.e., they possess a typical eukaryotic nucleus, endomembrane system, and cytoskeleton), they also display molecular and cytological characteristics reminiscent of prokaryotes, including features of the translational apparatus, genome size (which is in the range of that of bacteria), and lack

of recognizable mitochondria, peroxisomes, and typical Golgi membranes (reviewed in reference 189). Because of their prokaryotic features, microsporidia were initially classified within the Archezoa, together with other amitochondriate organisms (Giardia, trichomonads, and Entamoeba) which were thought to have evolved prior to the acquisition of mitochondria by endosymbiosis and consequently to represent the most primitive eukaryotes (42). However, the genome sequence of E. cuniculi revealed that it contains genes related to some mitochondrial functions, implying that microsporidia have retained a mitochondrion-derived organelle (152). Indeed, tiny organelles with double membranes could be demonstrated by using antibodies against a mitochondrial protein (Hsp70) in the human microsporidial parasite T. hominis (330). Furthermore, sophisticated phylogenetic analyses revealed that the microsporidia evolved from within the fungi, being most closely related to the zygomycetes (43, 155). Microsporidia share additional features with fungi, e.g., the presence of chitin (although chitin is also found in other phyla, e.g., mol-

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mans (Fig. 2) illustrates their polyphyletic nature. Of particular interest is the fact that the closest relatives of three species infecting humans and belonging to the genera Trachipleistophora and Vittaforma are microsporidia that infect insects, and it is tempting to speculate that the insects might serve as reservoirs for these microsporidia. Indeed, another microsporidian of arthropod origin, Brachiola (formerly Nosema) algerae, was demonstrated to be a causative agent in human infections (61). The two new species of the genus Encephalitozoon, E. intestinalis and E. hellem, are very closely related to the widespread parasite of mammals E. cuniculi. The most prevalent species, E. bieneusi, is most closely related to a fish pathogen. For all four major microsporidial species infecting humans (E. bieneusi and the three Encephalitozoon spp.), animal hosts are known (Table 1; Fig. 2) implying a zoonotic nature of these parasites. Molecular studies have identified phenotypic and/or genetic variability within these species, indicating that they are not uniform. Whereas the significance of this variability remains largely unknown for E. hellem and E. intestinalis, strain variation in E. bieneusi and E. cuniculi provided new insights into the biology, origin and distribution of these parasites and has allowed the question of their zoonotic potential to be addressed. ENTEROCYTOZOON BIENEUSI

FIG. 2. Dendrogram generated from the small subunit ribosomal RNA (ssrRNA) gene of microsporidian species identified in humans (underlined) and selected other species (Kimura’s distance, unweighted pair group method of analysis). Known animal hosts are indicated in brackets; the brewer’s yeast Saccharomyces cerevisiae serves as an outgroup. No corresponding gene sequences are known for the human-infecting microsporidian species Pleistophora ronneafiei, Trachipleistophora anthropophthera, Brachiola (formerly Nosema) connori, B. vesicularum, Nosema ocularum, Microsporidium ceylonensis, and M. africanum (Table 1).

lusks) and trehalose, similarities between the cell cycles, and the organization of certain genes (35, 211). Therefore, microsporidia are nowadays considered to be highly derived fungi that underwent substantial genetic and functional losses resulting in one of the smallest eukaryotic genomes described to date. The placement of microsporidia among the fungi might have implications for the discovery of new therapeutic strategies. Although microsporidiosis in general can be successfully treated with albendazole and fumagillin, therapy for the most prevalent species, E. bieneusi, is difficult (129, 204). Indeed, a few studies have demonstrated that inhibitors of chitin synthase enzymes are effective against microsporidia (19, 270). Natural History A fundamental question that arose with the discovery of new microsporidial species in humans was that of their natural origin. The phylogram generated with small subunit ribosomal RNA (ssrRNA) gene sequences of microsporidia infecting hu-

There are two genera in the family Enterocytozoonidae: (i) Nucleospora, with the two characterized species N. salmonis, an intranuclear microsporidian of salmonid fish (87), and N. secunda, a parasite of a warm-water African fish (180), and (ii) Enterocytozoon, with E. bieneusi, infecting the cytoplasm of enterocytes and other epithelial cells in humans and mammals (76). E. bieneusi, the most common microsporidial species known to cause human disease, was first described as an HIV-associated opportunistic intestinal pathogen in 1985 and was morphologically characterized using electron microscopy (76). In 1996, morphologically identical spores were detected in feces of pigs (74), and subsequently they also were detected in fecal samples and intestinal tissue of other mammals. Infections in Humans Several hundred HIV-infected patients with chronic diarrhea attributed to this organism have been reported from all over the world. The prevalence of E. bieneusi infections among HIV-infected patients reached up to 50% as documented by selected studies in Table 2. Human infections are well documented in all inhabited continents. In most studies, prevalences were significantly higher in patients with chronic diarrhea (92, 99, 128, 271, 324, 332). The prevalences presented in Table 2, however, do not allow for comparative analyses because there were considerable differences with regard to the selection of the patient groups, the patients’ characteristics (age, sex, sociodemographic data, degree of immunodeficiency, and clinical features), and the specimens investigated (biopsies or stool samples). Furthermore, the improvements of diagnostic methods achieved over the last 15 years have to be considered.

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TABLE 2. Selected studies on prevalence rates of E. bieneusi in HIV-infected persons Geographic area

Africa Cameroon (Yaunde) Niger (Niame) Zimbabwe (Harare) Zimbabwe Zambia (Lusaka) Mali (Bamako) Tanzania Zimbabwe (Harare) Mali (Bamako) Americas United States (Washington, D.C.) United States (Texas)

Specimen; diagnostic methoda

9b 7 13 46 23 32

1997 1993 1995 1997 1995 1997

86; chronic diarrhea 88; diarrhea longer than 1 week 61; diarrhea

3 18 (LM), 51 (PCR) 13.1

1999 (44) 1999 (133) 2002 (10)

Intestinal biopsies; EM, TEM Duodenal biopsies; TEM

67; chronic unexplained diarrhea 55; chronic diarrhea 51; without chronic diarrhea 65; diarrhea 65; without diarrhea 34; AIDS patients 194; diarrhea 68; diarrhea

30 33 25 11 2 44b 39b 37

1990 (218) 1993 (227)

United States (New York)

Intestinal biopsies; TEM

United States (New York)

Duodenal biopsies; PCR, confirmation by TEM

United States (California)

Stool; LM

Brazil (Fortaleza)

Stool; LM

Brazil (Rio de Janeiro) Brazil (Rio de Janeiro)

Stool; LM Stools, duodenal or ileal biopsies Stool; LM

Australia (Victoria) Thailand Thailand (Bangkok) Thailand India Europe The Netherlands The Netherlands France France (Paris) France (Nice) Italy (Apulia) Italy Germany (Cologne) Germany (Hamburg) England (London) England (northwest) Spain (Madrid) Sweden (Stockholm)

Yr (reference[s])

66; chronic diarrhea 60; 41 with diarrhea 129; hospitalized with diarrhea 74; 45 with diarrhea 69; chronic diarrhea 88; 80% with chronic diarrhea

Stool; LM

Australasia Australia (New South Wales)

Prevalence (%)

Stool; LM Stool; LM Stool; LM Formalin-fixed stool; PCR Stool; LM Stool; LM, partially confirmed by TEM Stool; LM, TEM Stool; LM, PCR Stool; LM, IFAT, PCR

United States (Atlanta, Ga.)

Peru (Lima)

No. of patients examined; patients’ characteristics

43; without diarrhoea 8,439; diarrhea, yr 1993, 1994, 1995, 1996. 79; with diarrhea 82; without diarrhea 13; chronic unexplained diarrhea 40; chronic diarrhea 2672; diarrhea

2.3 8.8, 9.7, 6.6, 2.9

(241) (27) (300) (41) (91) (185)

1993 (128) 1994 (160, 161) 1996 (62) 1998 (53)

6b 1b 46b 27.5

1996 (25) 2000 (24)

3.9

2003 (275)

1994 (332)

Duodenal biopsies; LM confirmed by EM Stool; LM, TEM Stool; LM, TEM Stool; LM; TEM Stool; LM, TEM Stool; LM

109; chronic diarrhea 71; without diarrhea 139; diarrhea 66; chronic diarrhea 288; diarrhea 95; children with diarrhea 120; diarrhea

29 1.4 3.5 33.3 11 25.3 2.5

1993 (99)

Duodenal biopsies; LM partially confirmed by TEM Stool; LM Duodenal biopsies; LM Stool, intestinal biopsies; LM Stool; LM Stool; LM Intestinal biopsies; EM, TEM Intestinal biopsies; PCR, Southern hybridization Stool; LM

55; unexplained diarrhea 38: without diarrhea

27b 3b

1991 (92)

143; diarrhea 66; chronic diarrhea 18; chronic unexplained diarrhea 46; chronic diarrhea 56; diarrhea 72; chronic diarrhea 46; diarrhea

7.7 2 50 24 2b 4.2 22

1993 1993 1993 1995 1995 1996 1996

50; 47; 59; 63;

32 4 14b 14

1998 (271)

2

1997 (69)

Intestinal biopsies; LM, EM Stool, intestinal biopsies; LM confirmed by TEM Stool; LM, confirmation by PCR Duodenal biopsies; LM

Switzerland

Stool; LM partially confirmed by TEM and PCR

Portugal

Stool; LM, PCR

diarrhea, hospitalized patients without diarrhea diarrhea diarrhea

48 children; chronic diarrhea 72; abdominal symptoms of unknown cause 164; chronic diarrhea (1992–1994) 156; chronic diarrhea (1994–1996) 949; without diarrhea 215; diarrhea

1993 1998 2001 2002 2002

(239) (313) (317) (312) (199)

(301) (56) (203) (16) (187) (309) (110)

1991 (221) 1995 (166)

b

3

1998 (281)

10.7 5.3 0.4 42.8c

1999 (324) 2001 (98)

a

LM, light microscopy; TEM, transmission electron microscopy. Intestinal microsporidia, species not stated. c A total of 92 samples were positive for microsporidia; 20 of 69 isolates that were further characterized by PCR were E. bieneusi, and 49 were E. intestinalis. b

A few studies from affluent countries indicate that the prevalence of E. bieneusi in HIV-infected patients is decreasing (53, 324). Recent studies have shown that administration of antiretroviral combination therapy can result in remission of HIV-

associated intestinal microsporidiosis (40, 54, 120, 198, 214). A decrease of 50% in E. bieneusi cases in Switzerland was also interpreted as being associated with antiretroviral therapy (324).

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Although predominantly described among adults suffering from immunodeficiency due to HIV infection, E. bieneusi infections were also reported from HIV-negative patients who were immunocompromised due to an underlying disease or due to therapeutic immunosuppression when undergoing organ transplantation (119, 130, 197, 228, 229, 260, 318). Furthermore, a few E. bieneusi infections in HIV-negative, immunocompetent, and otherwise healthy persons that were associated with self-limited diarrhea were reported, mostly in the context of traveler’s diarrhea in Europe (9, 71, 114, 181, 182, 209, 242, 268, 282, 314) but also in single cases in Africa (44, 131). Hence, E. bieneusi was detected by PCR in stool samples from 7 of 148 travelers with diarrhea returning to Germany (209). A recent study performed in Spain revealed that 8 of 47 (17%) geriatric persons with diarrhea were infected with E. bieneusi (182), and it was speculated that agerelated diminishment of the immunological capacities might predispose elderly persons to microsporidial infections. On the other hand, no data so far indicate that children might be more susceptible to E. bieneusi infections (300). Over the last decade, evidence has accumulated that E. bieneusi might also persist as an asymptomatic infection in immunocompetent humans. E. bieneusi was recovered in 8 of 990 stool samples from African children who were not considered HIV positive, suggesting enteric carriage among immunocompetent persons in tropical countries (27). Asymptomatic infections in children were reported in another study from Africa (44) and in a study from Asia, where not only healthy orphans (5.9%) but also child-care workers (1.9%) were infected (213). In all these studies, light microscopy, partly combined with transmission electron microscopy for confirmation of positive cases, was the diagnostic method, which might not be sensitive enough to detect subclinical infections under all circumstances. Therefore, more sensitive diagnostic tools such as PCR are required to elucidate the question of whether this parasite is a common organism of the human intestinal flora, causing severe disease only under immunodeficiency, or whether zoonotic transmission is of considerable importance (see below). Infections in Animals Eleven years after its discovery as a human pathogen, E. bieneusi was for the first time detected in animals, namely, in pigs (74), and a prevalence of 35% was determined by PCR in a subsequent investigation (26) of 109 randomly selected pigs from 22 farms located in different parts of Switzerland. A significantly (P ⱕ 0.05) higher occurrence of E. bieneusi was found in weaned piglets. The feces of three infected pigs, which did not show any clinical signs, were examined weekly by PCR, revealing excretion of E. bieneusi spores in 67% of the samples. Hence, E. bieneusi seems to be a common parasite in asymptomatic pigs. The low pathogenicity of E. bieneusi for pigs was further substantiated by the lack of intestinal lesions in immunosuppressed and immunocompetent gnotobiotic piglets experimentally infected with E. bieneusi of human or macaque origin (159). This study, however, demonstrated that immunosuppression of piglets did lead to an increased excretion of spores. Subsequent studies have confirmed the occurrence of E.

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bieneusi in pigs with high prevalence (32%) (29) and also in calves (9.5 to 11.5%) (237, 243, 277). The parasite has also been detected in cats (72, 191), dogs (182, 191), a goat (182), a llama (72), a variety of species of wild fur-bearing mammals (beavers, foxes, muskrats, otters, and raccoons) (72, 276), hedgehogs (A. Mathis, unpublished data), and, recently, nonmammalian hosts (chickens and pigeons) (233; M. Haro et al., unpublished data [GenBank accession number AY668953]). Natural infections with E. bieneusi were documented in captive monkeys, namely, rhesus macaques. Prevalences were 16.7% (n ⫽ 131) in normal, asymptomatic animals, in which the infection persisted for 262 days, and 33.8% (n ⫽ 53) in animals which were experimentally infected with the simian immunodeficiency virus (186). A screening of 42 wild monkeys from Central Africa (Cameroon) by microscopy and PCR did not yield a single E. bieneusi-positive result (225). Animal Models For various reasons including mass production of the parasite for basic research, development of diagnostic tools, drug screening, and studies on disease pathogenesis, an animal model of enterocytozoonosis is desirable. E. bieneusi of human origin has been established in immunocompromised rhesus monkeys (126, 293), immunosuppressed gnotobiotic piglets (159), Sprague-Dawley rats (2), and New Zealand rabbits (3). In all animals, only chronic asymptomatic infections were observed, similar to the infections in naturally infected, immunocompetent pigs (26). Many attempts to establish E. bieneusi in immunocompetent and immunodeficient mice were unsuccessful (83). Hence, all hitherto-described experimental animal models appear not to be adequate to mimic the pathological intestinal situation in HIV-infected patients. Molecular Epidemiology Analyses of the single internal transcribed spacer of the rRNA genes (ITS) have revealed that there is considerable genetic variation within E. bieneusi isolates of human and animal origins, and more than 50 genotypes have so far been described based on subtle differences within this 243-bp sequence. An overview of human-infecting genotypes is provided in Table 3. In contrast to the situation with microsporidia of the genus Encephalitozoon (see below), no other genetic markers are available. However, classification of isolates of E. cuniculi and E. hellem based on ITS sequences has largely been confirmed by data for other genetic loci. Nevertheless, additional independent markers for E. bieneusi are highly desirable in order to clarify the genetic structure of the parasite’s populations. Five different ITS genotypes of E. bieneusi infecting humans have been confirmed in independent studies and another 12 were discovered in single studies, with one of these studies accounting for eight of these novel genotypes (275). Limited information is available on the geographic distribution of human-derived genotypes of E. bieneusi. Except for the abovementioned eight genotypes identified in a study in South America (275), all other genotypes have been found in Europe, where most of the studies aiming at genotyping this parasite have been conducted (26, 72, 174, 235, 240). In additions,

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TABLE 3. Enterocytozoon bieneusi genotypes in humans: number of described cases and animal hosts No. of reported human cases (reference) E. bieneusi genotype designation

Genotypes identified in independent studies A B, I C, II

D IV

Genotypes identified in single studies Q (identical to C, II but with 2 nt insertion) III V Peru3 Peru4 Peru5 Peru6 Peru7 Peru8 Peru10 Peru11

HIV-infected or otherwise immunocompromised patients

3 (235), 3 (26), 1 (193), 32 (275) 3 (235), 8 (26), 66 (174), 2 (193), 11 (240) 2 (235), 2 (26), 1 (transplant recipient) (260), 9 (174), 7 (transplant recipient) (174), 1 (lymphoma) (174), 1 (72) 1 (236), 9 (275), 1 (240) 9 (174), 1 (transplant recipient), (174), 1 (myeloma) (174), 18 (275), 1 (240)

Immunocompetent patients

Animal host, genotype designation (reference[s])

1 (174)

1 (174), 10 (children with diarrhea, immunostatus not determined; estimated to be 18–29% among these children) (292)

Macaque (45, 126); pig, EBITS9 (29); wildlife, WL8 (29, 45, 276) Cat, K (72); cattle, BEB5 (72, 277)

1 (72) 3 1 1 1

(174) (174) (275) (275)

3 1 8 4 3 6

(275) (275) (275) (275) (275) (275)

genotypes A, B, D, and IV have been identified in HIV-infected patients from South America and the United States (193, 275). In single studies from Asia and Africa, genotype A was found in 20 asymptomatic children in Thailand (I. Subrungruang et al., unpublished data [GenBank accession numbers AY357185 to AY357404]) and genotype IV in children with diarrhea in Uganda (292). Of all 17 human-infecting E. bieneusi genotypes identified so far, four seem to have a zoonotic potential, as they have also been discovered in vertebrate hosts (Table 3). For the three genotypes A, B, and C, which account for the overwhelming number of isolates from humans, no animal host is known, and one might speculate that it is simply a matter of time until such hosts will be identified. On the other hand, several lines of evidence suggest that there is a certain degree of host specificity, at least for some of the E. bieneusi genotypes. First, a dendrogram based on ITS sequences of human-infecting E. bieneusi genotypes (confirmed in independent studies) and selected genotypes with animal hosts reveals a clustering of genotypes according to host species, although this grouping is not absolute (Fig. 3). Interestingly, the eight novel E. bieneusi genotypes recently identified in a single study of HIV patients (275) (Table 3) cluster within the branch containing all other

Pig, EbpC (26); pig E (237); wildlife, WL13 (276) Wildlife, WL11 (276)

human-derived genotypes (for a detailed dendrogram, see reference 275). Further evidence for some degree of host specificity originates from experimental animal models using immunodeficient or immunosuppressed animals (see above). With E. bieneusi of human origin, only chronic asymptomatic infections which do not appropriately mimic the pathological intestinal situation in HIV-infected patients were observed in rhesus monkeys, rats, piglets, and rabbits (see above), and many attempts to establish human-derived E. bieneusi in immunodeficient mice were unsuccessful (83). A minor role of animals as sources of human infections is also substantiated by epidemiological data (see below). Taken together, the picture of E. bieneusi with respect to its zoonotic potential is reminiscent of that of another intestinal parasite, Giardia lamblia, which comprises zoonotic as well as nonzoonotic genotypes (205).

Sources of Human Infections and Transmission Extensive intestinal and rare respiratory tract involvement as described for patients with disseminated E. bieneusi infections suggest that different modes of transmission are possible, in-

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FIG. 3. Dendrogram of ITS sequences of human-infecting E. bieneusi genotypes (confirmed in independent studies) and selected genotypes with animal hosts. All sequences are deposited in GenBank with the same designations (Kimura’s distance, unweighted pair group method of analysis).

cluding the fecal-oral or oral-oral route, inhalation of aerosols, or ingestion of food contaminated with fecal material. In addition, direct human-to-human transmission is substantiated by studies that implicate male homosexuality or having an HIVinfected cohabitant as risk factors for acquiring intestinal microsporidiosis (148, 316). Person-to-person transmission was also suggested in a study which revealed that 9 of 13 infected orphans, who were HIV negative, were confined to two houses, whereas HIV-positive children inhabiting another house were not infected (213). The detection of E. bieneusi in immunocompetent human carriers indicates that this parasite, or at least some of its genotypes, could naturally persist in the human population. Infections in organ transplant recipients or otherwise immunocompromised HIV-negative patients as well as in immunocompetent individuals were probably unrelated to direct contact with infected patients with AIDS. No seasonal variation was obvious in the prevalence of human intestinal microsporidiosis in Brazil, as had been found for the intestinal parasite Cryptosporidium parvum (332). De-

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spite differences in climate and sociodemographic factors, similar results were obtained in a study in southern California investigating 8,439 HIV-infected patients over a period of 4 years (53). In both studies it was suggested that contaminated drinking water was not likely to be the major source of microsporidial infections. However, other investigators have suggested that water contact may be an independent risk factor for enterocytozoonosis. In a prospective case-control study including 30 HIV-infected patients with intestinal microsporidiosis (28 with E. bieneusi infection and 2 with E. intestinalis infection) and 56 HIV-infected controls (148), “swimming in a pool” during the preceding 12 months was identified as one of three risk factors for intestinal microsporidiosis (besides male homosexuality and a CD4 lymphocyte count of ⱕ100/mm3). Other factors, such as consumption of different sorts of beverages or undercooked food, exposure to animals (cats, dogs, birds, bees, or fish), or recreational activities (swimming in freshwater or in the sea, trips abroad in the past 36 months, or visits to the countryside) were found not to be related to infection risk (148). Another study done in the United States included 12 HIV-infected patients with intestinal microsporidiosis and 54 CD4-matched controls. Risk factors for the acquisition of microsporidia were different kinds of water contacts (recreational swimming in rivers and lakes, drinking unfiltered tap water, or use of humidifiers) and close contact with another HIV-infected person (316). Lastly, a study investigating an urban cohort of HIV-infected patients revealed occupational contact with water or use of a hot tub or spa as risk factors for acquiring intestinal microsporidiosis (64), whereas contact with companion animals was not related to infection risk. Infection risk associated with traveling was suggested for E. bieneusi infections of HIV-infected and noninfected travelers in one study (55) but not in another one (64). A comparative study on diarrheic HIV-infected patients from the Paris area (France), including 26 patients with intestinal microsporidial infection (species not determined) and 28 patients with cryptosporidiosis, revealed that trips to tropical countries were strongly associated only with microsporidial infections (55). It is not known whether particular factors are associated with microsporidial transmission in tropical countries where HIVnegative adult and children were found to be infected (27). Detection of E. bieneusi and confirmation to the species level was achieved by PCR and subsequent sequence analysis of part of the ssrRNA gene in surface water but not in groundwater samples (89, 272) and recently, by species-specific PCR, also in zebra mussels from a river (123). Contamination of surface water may be from discharged domestic wastewater or from animal sources. As no genotyping was performed in these studies, the potential infection risk for humans from such sources needs further clarification. ENCEPHALITOZOON Three species of the genus Encephalitozoon have been identified as human pathogens: (i) E. cuniculi, which has a wide host range among mammals (37) and a worldwide distribution in domestic rabbits and is found in distinct geographic areas in carnivores and monkeys; (ii) E. hellem, which was distinguished from E. cuniculi in 1991 (78) and which has been reported in a few cases in avian hosts in the United States and Indonesia;

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TABLE 4. Single cases of human infections due to E. cuniculi confirmed by molecular analyses Immune status and country

HIV-infected patients United Kingdom

Clinical manifestations

Abdominal pain, anorexia, nausea, vomiting, fever, cough, renal failure Fever, cough, emesis, insomnia, sinus congestion, severe dry eyes, blurred vision Keratoconjunctivitis, sinusitis, rhinitis

United States Germany Switzerland

Headache, visual impairment, cognitive impairment, nausea, vomiting Dizziness, fever, nausea, abdominal pain, diarrhea

United States Switzerland Mexico Switzerland Switzerland Switzerland Switzerland Italy France

None Pneumonitis, otitis media None Conjunctivitis, sinusitis, seizure disorder Renal insufficiency, leucocyturia, erythrocyturia None Chronic sinusitis, bilateral keratoconjunctivitis Visual impairment

Chile

Cough, fever

Spain Italy

Fever, asthenia, abdominal pain, diarrhea Fever, myalgia, poor general condition

HIV-negative patients, immunocompromised (undergoing organ transplantation) Canada Mexico United States HIV-negative patients, otherwise immunocompromisedc Switzerland

Site(s) of infection and/or specimen

E. cuniculi strain

Kidney, urine

III

Urine, sputum

III a

Yr

Reference(s)

1994

1, 141, 143

1995

63, 66, 85

1995

111

I

1997

321

III

1997

196

I III I I I

1997 1997 1997 1997 1997

192 73, 192 73, 192 192 192

Urine, respiratory specimen Nasal epithelium Cerebrospinal fluid, urine, sputum, stool, duodenal biopsy Bronchoalveolar lavage, transbronchial biopsy Stool, urine, sputum Kidney, liver, lymph nodes, spleen, adrenal medulla, brain, ovary

I I ND

1997 1998 2000

192 238 105

ND (IIIb)

2001

328

III III

2001 2002

68 288

Urine, sputum stool, nasal discharge, duodenal biopsy Cerebrospinal fluid, stool, sputum, urine Adrenal glands, kidneys, brain, heart, trachea, urinary bladder, spleen, lymph nodes Urine Urine, respiratory specimen, stool Urine Urine Urine

ND

Fever, keratoconjunctivitis, allograft tenderness, Cough, fever, diarrhea, thoracic pain, extreme weakness Respiratory distress

Urine, stool, sputum, conjunctival scrapings Liver, kidney

III

2002

200

III

2003

115

Lung biopsy

III

2004

283

Iris tumor

Tumor biopsy

I

2005

158

a

ND, not determined. E. cuniculi strain III deduced from clinical and epidemiological findings (see the text). c Idiopathic CD4⫹ T-lymphocytopenia. b

and (iii) E. intestinalis (originally designated Septata intestinalis), which first was described in 1993 (30) and which was diagnosed in feces of farm animals in Mexico and in gorillas in Africa. All three species have spores that are morphologically indistinguishable. Intraspecies genetic variation has so far been described for E. cuniculi and E. hellem. In E. cuniculi, three strains (I, II, and III) are recognized, which, according to the host of the originally characterized isolates, are also designated “rabbit strain,” “mouse strain,” and “dog strain.” The fourth known species from this genus, E. lacertae (37, 162), was identified in reptiles only and is most closely related to E. cuniculi. ENCEPHALITOZOON CUNICULI Infections in Humans The first Encephalitozoon infection reported in humans, in 1959 (195), as well as a few subsequent cases were diagnosed based on spore morphology only. Therefore, these cases cannot unambiguously be attributed to E. cuniculi, as species differentiation was not possible at that time. Recent findings of E. cuniculi infections, as determined by immunological and/or

molecular methods, in several patients (HIV infected, undergoing organ transplantation, or with idiopathic CD4⫹ T-lymphocytopenia) from Europe and from the United States prove the infectivity of E. cuniculi for immunodeficient humans (Table 4). In seroepidemiological studies with enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent-antibody test (IFAT) using spore antigens of E. cuniculi or parasite cell suspensions, prevalences of up to 42% have been reported for patients with a history of tropical diseases or a stay in tropical countries and for patients with renal diseases, psychiatric disorders, or neurological disorders (15, 142, 146, 261). Despite Western blot analysis, which potentially increases the specificity of the ELISA (146), it is uncertain whether detection of antibodies to E. cuniculi reflects true infections or antigen exposure without establishment of the parasite, cross-reactivity, or reactions due to polyclonal B-cell stimulation, particularly in patients with tropical diseases. More recent studies (50, 121, 135, 165, 224, 302) suffered from the same methodological limitations. However, all these serological studies suggest that human exposure to microsporidia may be common but without clinical significance. In a very recent serodiagnostic study of an

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FIG. 4. Rabbit with torticollis (head tilt) due to cerebral infection with E. cuniculi.

immunocompetent laboratory worker who was accidentally infected with E. cuniculi when drops containing spores were spilled in his eyes, cross-reactivity of his serum with spore walls of E. hellem and E. intestinalis was demonstrated. However, only little cross-reactivity was observed when recombinant polar tube proteins of these parasites were used as antigens (298). Infections in Animals Beyond the numerous reports of E. cuniculi infections in rabbits, carnivores, and monkeys (see below), the parasite was identified as the causative agent of placentitis and abortion in a horse (220). Serological investigations (IFAT) revealed antibodies against E. cuniculi in goats and cattle (51, 136), but, as outlined above, the test specificity was not ascertained. Rabbits. Encephalitozoonosis in laboratory and pet rabbits is of clinical significance worldwide. E. cuniculi usually causes chronic infections which can persist asymptomatically for years. Severe neurological disease due to granulomatous encephalitis can occur unrelated to the age and sex of the animals (210) (Fig. 4). Until microsporidian-negative rabbit colonies were established, encephalitozoonosis was an important confounding variable in rabbit-based biomedical research on a

variety of diseases (reviewed in reference 315). In the past, high prevalences of encephalitozoonosis were recorded for laboratory rabbit colonies (37, 315), but these infections can be controlled by serological screening and high hygienic standards. On the other hand, the disease is still highly endemic in the pet rabbit population. In seroepidemiological surveys in Switzerland and the United Kingdom, specific antibodies against E. cuniculi spores were detected in 7.5% (n ⫽ 292) and 23% (n ⫽ 26) of healthy rabbits and in 85% (n ⫽ 72) and 71% (n ⫽ 65) of rabbits (mainly kept as pets) with neurological symptoms or with direct contact with symptomatic animals (74, 137, 210), respectively. In rabbits, horizontal transmission by ingestion of spores is regarded to occur most frequently, but intrauterine infection has also been documented (13, 37). After experimental oral infections of rabbits, regular spore excretion in the urine was observed between days 38 and 63 postinfection and later intermittently at very low density (59). Spore excretion was reported in 9 of 11 symptomatic rabbits (60), indicating that such animals may play an important epidemiological role. Based upon serological evidence, it was suggested that wild rabbits (Oryctolagus cuniculus) represent the natural host of E. cuniculi (331). Other studies revealed seroprevalences of 3.9% among 204 wild European rabbits in France (46) and 25% in the wild rabbit population in Western Australia (285). To our knowledge, E. cuniculi infections have so far not been reported for other free ranging lagomorphs. Rodents. E. cuniculi has been diagnosed in the past in numerous cases as a common parasite of laboratory rodents such as mice, rats, hamsters, and guinea pigs (reviewed in references 37 and 315), but nowadays, with high hygienic standards for the maintenance of laboratory rodents being applied, infections with microsporidia should no longer be a significant problem in these animals. On the other hand, rodent models have gained more attention for immunological research in the field of microsporidiosis (reviewed in reference 77). Information about the possible significance of microsporidiosis in rodents kept as pet animals and about the epidemiology of microsporidiosis in wild rodents is scarce. Until 1986, only three reports of E. cuniculi infections in wild rats from Japan and the United Kingdom had been published (37). Recently, we isolated E. cuniculi strain II (“mouse strain”) from one of 30 wild rats (Rattus norvegicus) caught in the city of Zurich, Switzerland (212) (Table 5). In wild mice, specific antibodies against E. cuniculi spores

TABLE 5. Hosts and geographical distribution of Encephalitozoon cuniculi strains a

E. cuniculi strain

I (“rabbit strain”)

II (“mouse strain”) III (“dog strain”) a

Host

Geographic area (no. of isolates)

Reference(s)

Rabbit

Switzerland (21), United States (3), Germany (1), Australia (1), Italy (1), Japan (1)

Human Mouse Wild rat Blue fox Dog Prosimian

Switzerland (6), Italy (1), United States (1) Czech Republic (1), United Kingdom (1), United States (1) Switzerland (1) Norway (8), Finland (1) United States (10), South Africa (1)

86, 113, 153, 192, 210; P. Deplazes and A. Mathis, unpublished data. 73, 238, 321, 334 86, 334 212 8, 190 86, 141, 264

As determined by the number of 5⬘-GTTT-3⬘ repeats in the ITS (86).

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were found in Iceland in 4% and 9% of Apodemus sylvaticus and Mus musculus animals, respectively (140). The authors suggested that mice are a potential “reservoir” of E. cuniculi for arctic foxes and feral minks. Indeed, recent molecular characterization of E. cuniculi isolates from Norway and Finland (8, 190) revealed that all foxes originating from four different farms were infected with strain II (“mouse strain”) of E. cuniculi (see “Molecular Epidemiology” below). Carnivores. The clinical manifestation of canine encephalitozoonosis is an encephalitis-nephritis syndrome which had previously been confused with canine distemper (23). Encephalitozoonosis in domestic dogs, which is caused by strain III (“dog strain”) of E. cuniculi, has been described from Tanzania, South Africa, and the United States (23, 37, 252, 264). In domestic cats, only three cases of E. cuniculi infections have been reported (37, 178). In captive carnivores, disseminated infections similar to those found in dogs occurred in fox cubs (215). This disease, which is still a major problem and causes heavy losses of blue foxes in northern European countries (5), is caused by strain II (“mouse strain”) of E. cuniculi (190). Outbreaks of encephalitozoonosis in carnivores in zoos were previously reviewed (37). Few data are available about the disease in wild carnivores. Encephalitozoon-like organisms were detected by light microscopy in brain tissues from a wild hand-reared red fox (Vulpes vulpes) with neurological symptoms from the United Kingdom (331) and from captive wild dog (Lycaon pictus) pups which died of a fatal disease resembling canine distemper 13 days after vaccination with a live attenuated strain of canine distemper virus (303). Serological investigations with ELISA revealed no seropositive animals in 86 wild red foxes from Switzerland (210). In Iceland, seroprevalences were 12% among 372 wild arctic foxes (Alopex lagopus) and 8% in feral mink (Mustela vison) (140). Histopathological findings for a seropositive fox pup with a neurological disorder which died 2 days after capture were consistent with encephalitozoonosis described for farmed foxes. The authors suggested that encephalitozoonosis contributed to the decline of the arctic fox population size by depressing fetal and pup survival and that mice may represent a potential reservoir for microsporidia (140). Indeed, in Greenland, where rodents are absent from the diet of these arctic foxes, none of 230 foxes investigated had antibodies to E. cuniculi (7). In all mentioned reports of encephalitozoonosis in wild carnivores (140, 303, 331), Encephalitozoon-like organisms were detected by light microscopy only. Therefore, species and strain determinations were not conclusive. In dogs and foxes, encephalitozoonosis is being perpetuated in the population by horizontal and vertical transmission (23, 201). Dogs and foxes infected by ingestion of spores showed moderate clinical symptoms, and the chronically infected animals represented the main source of infections for the offspring. In fur farms, food contaminated with spores from infected rodents or rabbits was assumed to be a possible source of infection for foxes and minks (140, 215). Monkeys. Disseminated natural infections resulting in high morbidity and severe encephalitis caused by Encephalitozoonlike organism have been reported for stillborn and young squirrel monkeys (Saimiri sciureus) in the United States (28, 337). Although parasite identification was based on electron micros-

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copy only, which does not allow one to distinguish E. cuniculi from E. hellem, the neuropathological symptoms strongly suggested that E. cuniculi was the species involved. Only recently, strain III (“dog strain”) of E. cuniculi was identified in tamarin colonies (Saguinus imperator, Oedipomidas oedipus, and Leontopithecus rosalia rosalia) in two zoos in Europe, causing severe disseminated infection with high mortality in infants (134, 234, 329). In experimental infections of vervet monkeys with dog-derived E. cuniculi isolates, transmission from infected infants to their nursing dams as well as transplacental transmission was established (297). In naturally infected monkeys, transplacental transmission was shown by the presence of multifocal granulomatous encephalitis with invasion of the brain by Encephalitozoon in stillborn monkeys or monkeys only a few days old, as well as by the reported placentitis in an animal and the isolation of Encephalitozoon-like parasites from placental tissue of a baboon (reviewed in reference 37). The hypothesis that animals with silent infections can perpetuate the disease in a colony is supported by a serological survey of a squirrel monkey colony over 3 years. More than half of these 250 animals tested positive at least once, and asymptomatic young animals were also seropositive (251). Export of such seropositive asymptomatic animals represents a high risk for the introduction of the parasite in other colonies, as was recently observed in an Emperor tamarin colony in Europe (134). So far, no sources of infection have been elucidated for E. cuniculi infections in monkeys, and it is not known whether this microsporidial species is prevalent in free-living monkeys. In contrast to natural infections causing neonatal death or lethal infections in young monkeys, experimental infections of immunocompetent monkeys with dog- or rabbit-derived isolates resulted in asymptomatic infections (84, 254, 297). Therefore, many factors, including host species, E. cuniculi strain, age and immune status of the host, and mode of transmission (e.g., intrauterine or oral), may influence the outcome of infections in monkeys. Molecular Epidemiology In E. cuniculi, the existence of three strains (I, II, and III; also named “rabbit strain,” “mouse strain,” and “dog strain”) was established using immunological and molecular methods (86). The repeated element 5⬘-GTTT-3⬘ in the ITS is a reliable and widely used genetic marker: strain I contains three such repeats, strain II contains two repeats, and strain III contains four repeats. A recent multilocus analysis yielded additional markers for the three strains, namely, the genes coding for the polar tube protein and spore wall protein 1 (333). This strain differentiation helped to elucidate the complex epidemiological situation of E. cuniculi infections in different hosts and in different parts of the world. Table 5 shows the host species and the geographical origins of those E. cuniculi isolates which were determined to the strain level and for which comprehensive information about the host animal was available. These data confirm earlier observations which suggested from circumstantial evidence that different strains of E. cuniculi with different host preferences might exist in nature (reviewed in reference 37). Strain II (“mouse strain”) was identified in rodents (mouse and rat) and

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in farmed blue foxes, supporting the hypothesis that mice are a reservoir of E. cuniculi for blue foxes (140). All E. cuniculi isolates from rabbits characterized so far belong to strain I (“rabbit strain”), and all isolates from dogs belong to strain III (“dog strain”). Dogs indeed seem to be susceptible to strain III only. In Switzerland, where strain I (“rabbit strain”) is highly prevalent in the rabbit population (210) and strain II (“mouse strain”) was detected in a rat (212), no positive antibody reactions were detected by ELISA in 104 healthy dogs and in 108 dogs with neurological disorders (210). E. cuniculi strain II is known to have a remarkable pathogenicity for the blue fox and mink in Norway (6, 37), but encephalitozoonosis of dogs has not been reported from Norway, and a recent serological survey with 1,104 canine serum samples revealed no indications for an infection (6). The absence of canine encephalitozoonosis in Europe indicates that strains I and II of E. cuniculi do not play a significant role as pathogens in the dog population. Host preference of the strains is further substantiated by the observations that strain II (“mouse strain”) was recently identified from a wild rat in an area in Switzerland (212) where strain I (“rabbit strain”) of E. cuniculi is highly endemic in pet rabbits and that, vice versa, a considerable number of rabbits from all over Switzerland all were infected with strain I but never with strain II. It is unlikely that strains I and II simply are epidemiologically separated, as E. cuniculi-positive rabbits were identified from stables where rodents were freely roaming. On the other hand, a strict host specificity of the strains was not demonstrated under experimental conditions (reviewed in reference 192). E. cuniculi strains II (“mouse strain”) and III (“dog strain”), for example, were also infective to rabbits that were given spores from an in vitro culture by oral administration (192; P. Deplazes, unpublished data). The strains of E. cuniculi seem to differ in their geographical distributions. Hence, strain II has so far been identified in Europe only, strain I infects rabbits from at least three continents (America, Australia, and Europe), and strain III was identified in dogs in America and South Africa. Strain III was recently identified for the first time in Europe in tamarin colonies from zoos (134, 234, 329). However, it was suggested in one of these cases that this infection had been imported with ancestor animals originating from the United States and has been perpetuated in the population by transplacental infections (134). A potential for spreading of this imported strain to the autochthonous carnivore population in the future has to be considered. Immunocompromised humans were found to be infected with strain I (“rabbit strain”) in Europe and with strain III (“dog strain”) in the Americas (Table 5). In Europe, too, several HIV-infected patients were diagnosed to be infected with E. cuniculi strain III (Table 4), but no travel histories for these patients were provided. Sources of Human Infections and Transmission It is unlikely that E. cuniculi is a natural pathogen in humans, and its zoonotic origin is evident. Indeed did two of the six patients from Switzerland, all of whom were infected with E. cuniculi strain I (“rabbit strain”), recall exposure to rabbits

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in the past (192, 321). Two patients from the United States who were infected with strain III (“dog strain”) did own a pet dog, but microsporidial infection in the animals was not proven (85, 283). Seroconversion in one of three children exposed to puppies with overt encephalitozoonosis has been reported (79). In another case of an E. cuniculi infection in an HIVinfected person where no material was available for genotyping, strain III (“dog strain”) was suspected to be the causative agent based on geographical (Chile) and clinical reasons and on the fact that the patient had a high risk of exposure as she was working as a pet dog groomer (328). Patient-to-patient transmission was considered to be unlikely in the only study to date investigating several patients, because these patients neither knew each other nor had obvious personal contacts such as hospitalization at the same time (73, 192). As spores of E. cuniculi are highly resistant in the environment and can survive several months under humid conditions (172, 311) direct contact with infected animals or humans is not required, and waterborne infections seem feasible. However, none of the two E. cuniculi strains found in humans have so far been detected in surface waters, in contrast to the case for strain II (“mouse strain”), which in a single study was identified by PCR in one of a total 50 samples from Switzerland (A. Mathis, unpublished data). ENCEPHALITOZOON HELLEM Infections in Humans E. hellem has so far been diagnosed in around 50 HIVinfected persons in a relatively few countries: Most cases were reported from the United States (52, 78, 81, 248–250, 308). E. hellem was diagnosed in European patients from Italy (247), Switzerland (73, 109, 323), Germany (109), and the United Kingdom (144) and in one case in Africa (Tanzania) (75). It is not clear whether epidemiological factors are responsible for a restricted distribution of this microsporidian or whether the relatively difficult identification of this species by immunological or molecular methods hampers its detection. E. hellem causes disseminated and ocular infections in HIV-positive patients, but asymptomatic infections of the respiratory tract have also been described (245). To our knowledge, E. hellem has so far been identified on two occasions in nonimmunosuppressed and HIV-seronegative patients, namely, from bronchoalveolar lavage of a patient with a simultaneous Mycobacterium tuberculosis coinfection (246) and in fecal samples from two diarrheic travelers returning from Singapore, who were coinfected with E. bieneusi (209) as diagnosed by PCR and confirmed by sequencing. Infections in Animals Before the description of E. hellem in 1991 (78), Encephalitozoon-like microsporidia were described on several occasions from psittacine birds (parrots). With the exception of a peachfaced lovebird from Australia, all cases were from birds kept in aviaries in the United States (37, 216, 230). Molecular analyses recently allowed identification of E. hellem in psittacine birds in the United States as the etiological agent of lethal infections in budgerigar chicks (Melopsittacus undulatus) in an aviary

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(20), in tissues from two eclectus parrots (Eclectus roratus) (226), and in droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) (267). The first detection of E. hellem in a wild psittacine bird was demonstrated by molecular analyses of spores from the intestinal contents of a yellowstreaked lory (Chalopsitta scintillata) captured on Aru Island (Indonesia) (279). However, microsporidial infections have been reported from nonpsittacine birds in the last few years. Horned puffin (Fratercula corniculata) chicks, which were caught in Alaska and which suffered a high mortality rate in quarantine at a zoo, were found to be infected with nonspecified microsporidian-like organisms (287). An intestinal microsporidiosis was diagnosed postmortem in a clinically normal ostrich (Struthio camelus) in Texas (125), and the causative agent was identified as E. hellem (263). Several species of wild, migratory hummingbirds from California, which suffered from enteritis, were infected with E. hellem (262). Finally, the parasite was diagnosed in a Gouldian finch from a flock with high nestling mortalities in Australia (38). A single epidemiological survey reported a prevalence of 25% of 198 clinically normal lovebirds from three flocks (265). Experimental inoculations have resulted in disseminated infections in athymic nude mice (266). In an attempt to assess whether avian species with close contacts with humans can serve as hosts of E. hellem, young chickens and turkey poults (3 to 5 days of age) were inoculated by gastric gavage with 107 spores (97). All birds remained asymptomatic, and no microsporidia were detected by microscopy in fecal smears and in histological specimens of the intestine. However, PCR, which was shown to be considerably more sensitive for detection of microsporidia in feces than microscopy (96), was positive with fecal material from chicks only. Taken together, these results suggest that several avian species might serve as asymptomatic carriers of E. hellem. The means of transmission of E. hellem infections in birds remain unexplored. As spores were found in kidney and intestine, spore excretion probably occurs with feces (20, 37, 216, 279). Black and colleagues (20) observed severe outbreaks of encephalitozoonosis with high mortality in young chicks, whereas adult birds in the same aviary appeared unaffected. In several reports, avian microsporidiosis leading to high morbidity and mortality was observed shortly after birds were brought into a new environment (37, 279, 287). These observations suggest that latent infections as observed with E. cuniculi in other animals also occur in adult birds and that the stress of translocation may reduce the degree of resistance to this infection. Immunosuppressive viral infections such as that with the psittacine beak and feather disease virus were found in two unrelated cases of lethal microsporidiosis in eclectus parrots (226), and a significant correlation was identified between shedding of E. hellem spores and psittacine beak and feather disease virus infection of lovebirds (265). Molecular Epidemiology Comparable to the situation in E. cuniculi, different genotypes as assessed by the sequence of the ITS were identified in E. hellem (194). Genotype 1, which represents the overwhelming number of E. hellem isolates characterized so far, was determined for isolates from several patients from the United

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States (153, 310, 333) and from Europe (109, 333) and for one E. hellem isolate each from a parrot caught in the wild in Indonesia (279) and from a psittacine bird from the United States (267). Genotypes 2 and 3 (194) and 2b (333), which differ from each other only by short insertions, consist of human-derived E. hellem isolates from Switzerland (three isolates) and Tanzania (one isolate) (194, 333). Differences among the E. hellem genotypes were also detected by Western blot analysis, but there was no absolute match between genotype and antigen profile. Indeed, karyotyping (17, 70) and multilocus analyses targeting coding (polar tube protein gene and ssrRNA gene) and noncoding (intergenic spacer) loci identified a least six different human-derived genotypes (138, 222, 333). From all these analyses, it appears that E. hellem isolates from the United States and from Europe are different populations. No isolates from birds have as yet been subjected to this more detailed genetic analysis; therefore, the biological and epidemiological significance of these findings remains to be elucidated. Sources of Human Infections and Transmission To our knowledge no epidemiological studies to demonstrate risk factors for E. hellem infections have been conducted, and the parasite has never been identified in surface waters. However, it is worth noting that some patients with ocular microsporidiosis owned or were exposed to pet birds (112, 219, 336). The identification of E. hellem as etiological cause of severe renal and intestinal infections in parrots and the excretion of spores in feces indicate that birds are a potential, but yet unproven, source of zoonotic infections. Once introduced into a susceptible human population, transmission of spores from person to person might be of importance. In HIV-infected patients with disseminated E. hellem infections, spores were isolated from sputum, nasal secretions, sinus aspirate, and urine (219, 323). The occurrence of upper and lower respiratory tract infections suggests that this species can be transmitted via the aerosol route (248, 323). Furthermore, oral or ocular autoinoculation, perhaps by contaminated fingers, may occur (249). For drug addicts, a hematogenous mode of transmission with contaminated syringes has been suggested but was not proven (117). ENCEPHALITOZOON INTESTINALIS Infections in Humans E. intestinalis is the second most prevalent microsporidial species infecting humans. Infections in HIV-positive patients have been reported from the Americas (62, 208, 257), from Europe (21, 98, 107, 175, 177, 202, 271, 280, 299, 325), from Australia (88, 168, 208), and from Africa (10, 122, 157, 167). Most reports are descriptions of single case; in a few studies larger groups were examined, and prevalences for E. intestinalis were 7.3% for 68 AIDS patients with diarrhea from the United States (62), 2% for 97 consecutive HIV-infected patients in Germany (271), 3% for 75 patients with chronic diarrhea from Zambia (157), and 0.9% for 320 patients with chronic diarrhea in Switzerland (324). A study investigating 216 AIDS patients with gastrointestinal complaints in Portugal

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revealed 92 patients being positive for microsporidia by microscopy (trichrome stain, uvitex, and calcofluor; prevalence, 42.8%). Of the positive specimens, 69 were further investigated by species-specific PCR, resulting in the diagnosis of E. intestinalis in 49 of them and of E. bieneusi in 20 (98). When 63 stool samples from HIV-infected patients with diarrhea from England were investigated by the calcofluor method, no E. intestinalis infections were found, whereas in 9 samples (14.3%) E. bieneusi was detected (166). Similarly, no E. intestinalis infections were detected in 61 HIV-positive persons from Mali (Africa) by using a monoclonal antibody, but 8 (13.1%) were positive for E. bieneusi (10). Two studies have proposed that double infections with E. bieneusi and E. intestinalis might occur. Five such double infections were identified in 10 cases of E. intestinalis infections in AIDS patients as proven by PCR on biopsy material (109). van Gool and colleagues (299), when attempting to cultivate E. bieneusi from four stool samples from biopsy-proven patients, surprisingly ended up with cultures of E. intestinalis in all four cases. They suggested that this microsporidian might be more common than suspected; however, laboratory contamination of the cultures cannot be excluded. E. intestinalis had been identified in two HIV-negative travelers with diarrhea (232). In a cross-sectional survey of two rural villages in Mexico, a monoclonal antibody (3B6) which recognizes E. intestinalis, E. hellem, E. cuniculi, and the other microsporidial species Nosema and Vairimorpha spp., but neither Enterocytozoon bieneusi nor yeast from stool sample (93), was used to investigate 255 persons. Twenty (7.8%) of them were positive, with 21% of 70 households having at least one member who was positive (94). Indications for high prevalences in immunocompetent persons were also found in another study (302). Using serologic techniques with E. intestinalis as antigens, high seroprevalences among 300 Dutch blood donors (8%) and 276 pregnant French women (5%) were found. However, the tests used were not strictly specific for E. intestinalis but presumably were genus specific. Infections in Animals Experimentally infected wild-type mice were shown to excrete spores intermittently and in low numbers (4), a situation that is reminiscent of the one with E. bieneusi infections in pigs (26). In a survey using the monoclonal antibody 3B6 (which is not E. intestinalis specific; see above) in IFAT, fecal samples from domestic animals from a rural area in central Mexico were investigated, resulting in 19 (11%) of 172 mammalian and 16 of 99 avian samples being positive. Transmission electron microscopy on specimens from mammals revealed microsporidian-like structures. PCR using a contamination-prone protocol (with a cloned E. intestinalis target sequence as positive control) was done several months later on total DNA extracted from the formalin-fixed specimens, which generally is considered not to be suitable for sensitive detection due to timedependent, formalin-induced degradation of DNA (149, 291). Six of the eight investigated fecal samples from goat, pig, cow, dog, and donkey were positive for E. intestinalis but negative when tested with primers specific for E. cuniculi and E. hellem (22). Such a high prevalence in animals has not been confirmed in

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other studies. In a recent study, by employing microscopy and PCR on nonfixed material, low prevalences (3%) of E. intestinalis were identified in free-ranging gorillas and in people who share gorilla habitats in Uganda (122). Hence, a zoonotic potential was suggested, but further studies are needed to investigate whether E. intestinalis indeed is a common pathogen of a wide variety of (domestic) animals and whether animal-borne E. intestinalis is epidemiologically related to the human disease. Molecular Epidemiology In contrast to the situation in other human-infecting Encephalitozoon species, for which distinct strains with differences in their biology and epidemiology were identified, E. intestinalis seems to be a very homogenous species. Analysis of five human-derived isolates revealed only minor differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot profiles (67). Whereas isolates from both E. cuniculi and E. hellem show considerable intraspecies variability in their karyotypes (17, 18, 70, 269), isolates of E. intestinalis displayed identical karyotypes when analyzed by pulsed-field gel electrophoresis (17). No variation of the ITS sequences was detected in 16 isolates of E. intestinalis (82, 176). Sources of Human Infections and Transmission Modes of transmission of E. intestinalis spores are uncertain. Considering the various sites of infections (intestinal, respiratory, and ocular) (108) and spore release into the environment via stool, transmission via the fecal-oral route, by inhalation, or by contaminated fingers seems plausible. Waterborne infection, at least focally, can be suspected, as the parasite was identified by sequence analysis of PCR amplicons from surface water and groundwater (89, 286), from 6 of 12 investigated samples of source water used for consumption (90), and recently, by species-specific PCR, also in zebra mussels from a river (123). Case-control studies indeed demonstrated that contact with water might be a risk factor for acquiring intestinal microsporidiosis (E. bieneusi and E. intestinalis), but either microsporidial species were not specified or E. bieneusi was the predominant one (see “E. bieneusi: Sources of Human Infections and Transmission” above). OTHER MICROSPORIDIA Several microsporidial species have been identified in rare cases of patients with ocular infections, myopathies, intestinal infections, or disseminated infections (Table 1). We briefly discuss the features of those microsporidia for which ultrastructural and/or molecular information is available. With the exception of B. algerae, which infects mosquitoes, no host other than humans is known for these species. Vittaforma spp. Vittaforma corneae (258), which originally was designated Nosema corneum (253), was the first human-derived microsporidial isolate which could be established in an in vitro culture. Up to now, four cases of human infections are on record, three

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of them related to eye infections in immunocompetent patients from the United States and Europe (101, 231, 253) and one presenting as an urinary tract infection in a patient with AIDS living in Africa, who had a concurrent infection with E. hellem (75). Phylogenetic analysis based on rRNA gene sequences (Fig. 2) revealed arthropod-infecting species as being most closely related to V. corneae and the human-infecting E. bieneusi as a close taxonomic relative. Although no common natural hosts of Vittaforma have yet been identified, these microsporidia seem to be ubiquitous. Investigations of surface waters in the United States and in Europe by PCR and sequencing revealed gene sequences with highest scores of identity (91 to 98%) with V. corneae in, e.g., seven of eight samples analyzed (89, 104). The Vittaforma-like organisms (96% identity with V. cornea) identified in feces from 25 diarrheic patients (22 HIV positive and 3 HIV negative) in Portugal (278) might therefore simply reflect intestinal passages of microsporidial spores. Pleistophora spp. Three cases of myositis in immunodeficient patients were identified as being caused by Pleistophora spp. (49, 124, 169). The correct taxonomic position of the organisms in two of the cases (49, 124) was not possible but it was assumed (48) that they belong to the subsequently described genus Trachipleistophora (see below). Ultrastructural analysis of the third isolate (169) allowed its classification as the new species Pleistophora ronneafiei (32). Pleistophora spp. have nearly exclusively lower (poikilothermic) vertebrate hosts, in particular fish and reptiles, but no other Pleistophora species has spores with the dimensions of P. ronneafiei, and hence a possible animal reservoir remains obscure. Trachipleistophora spp. Closer examination of a morphologically Pleistophora-like microsporidian isolated from an patient with AIDS with infection of skeletal muscle and also the nasal sinus cavities and conjunctiva (100) led to the establishment of the new genus and species Trachipleistophora hominis (145). Apart from this first case from Australia, T. hominis was identified in an immunocompetent African immigrant in the United Kingdom as causal agent of a stromal keratitis (231). Phylogenetic analysis of T. hominis based on rRNA gene sequences revealed that its closest relationship is with species of the genus Vavraia, which are pathogenic to insects (48) (Fig. 2). T. hominis indeed could be readily propagated in mosquito larvae, and the spores produced were again infective for athymic mice. Furthermore, spores were transferred from infected adult mosquitoes to a feeding substrate (326). Insects being a potential source of T. hominis was substantiated by experimental infections of severe combined immunodeficient (SCID) mice. Whereas oral inoculation was ineffective for establishing an infection, injection of the parasites, mimicking insect stings, led to disseminated infections, including infection of urinary bladder, liver, and spleen but not the brain (163). Severe ocular and milder disseminated infections were also observed upon eye contamination with spores, providing a direct way of infection independent of insect bites.

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A second species of this genus, Trachipleistophora anthropophthera, was differentiated based on ultrastructural features and clinical syndromes (305). In contrast to T. hominis infections in humans and in SCID mice, T. anthropophthera also infected the brain, as was reported for the first two infections in AIDS patients from the United States (335). This microsporidial species was identified in a single additional human case in an HIV-infected person from Thailand suffering from chronic keratitis, and the isolate was successfully cultivated in mouse fibroblast cells (151). No genetic information allowing phylogenetic analysis is available for T. anthropophthera. Based on the common characteristic feature of bisporous sprorophorous vesicles, it was suggested that this species might be related to the genus Telomyxa, which infects insects (mayflies) (289). Brachiola spp. Brachiola algerae (183) (formerly Nosema algerae) is has long been known as a pathogen of different orders of insects, e.g., mosquitoes (304), and was a candidate agent for biological control of these insects (296). Within this context, it was evaluated in the 1970s whether B. algerae could pose a biohazard for mammalians when injected by a mosquito during a blood meal. Later, during the AIDS pandemic and before the first case of a human infection with this parasite was reported (307), the potential of B. algerae to infect immunocompromised vertebrates was assessed. Hence, this microsporidial species was shown to proliferate in mammalian cell lines, initially only at temperatures of below 37°C (294) but later also after adaptation to 37°C (207, 289). Intravenous injection of spores failed to induce infections in immunocompetent and immunocompromised mice, and subcutaneous injections led to limited development close to the infection site in extremities only (290, 295). Furthermore, ocular, but not oral, intraperitoneal, intramuscular, or subcutaneous, inoculation of SCID mice with spores of B. algerae led to severe infection of the liver as the only affected internal organ (164). It was concluded that an initial infection of the eye allowed the parasite to adapt to the conditions within the mammalian host and to spread to internal organs, presumably via macrophages. In the first case of a human B. algerae infection, the parasite was isolated from the cornea of an immunocompetent patient (307), and the infection was resolved after appropriate treatment. In the second case (61), a deep-tissue infection in muscles in a patient with diabetes and rheumatoid arthritis was documented. The patient, who was HIV negative and who eventually died from a massive cerebrovascular infarcation, was taking immunosuppressive agents for rheumatoid arthritis. Diagnosis from muscle biopsies was confirmed by light and electron microscopy, by IFAT using rabbit antiserum against B. algerae, and by PCR and sequencing. Rather perplexing is the finding that the parasite isolate proliferated in RK13 (rabbit kidney) cells in vitro at 30°C but not at 37°C, a fact that was not further discussed by the authors. Two further species of the genus Brachiola have been described based on ultrastructural features from single human infections: B. connori (33) (originally named Nosema connori [273]), causing disseminated infection in an immunologically compromised child (188, 273), and B. vesicularum, causing

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myositis in an AIDS patient (33). The validity of the latter species name was doubted, and it was suggested that B. vesicularum, for which no genetic information was provided, may be identical to B. algerae based on the ultrastructure of B. algerae when growing in human muscle fibroblasts at 38°C (289). However, examination of muscle biopsies from the above-mentioned B. algerae case (61) confirmed the existence of ultrastructural differences, implying that B. algerae and B. vesicularum indeed are different species (34).

Microsporidium spp. Microsporidium ceylonensis. In the original case report (12) of this ocular infection of a Tamil boy, spores were the only parasite stages identified, and it was suggested that either E. cuniculi, which had been confirmed as a human pathogen at that time (195), or Nosema helminthorum was the causative agent. The latter species, a parasite of tapeworms of goats, was suggested because the boy had suffered an injury of the affected eye when he had been gored by a goat. In a subsequent ultrastructural study published in 1998 (36), a generic classification of this isolate could not be achieved and it was named Microsporidium ceylonensis. The ultrastructural details identified should facilitate the recognition of this parasite in future cases. Microsporidium africanum. Microsporidial spores were identified in histiocystes of a corneal ulcer of an African woman (223), with a tentative identification as a Nosema sp. The isolate was later transferred to the collective group Microsporidium upon reexamination of the electron micrographs and was designated Microsporidium africanum (37). There are no clues whatsoever on other hosts of this species.

CONCLUDING REMARKS Microsporidia are identified as parasitic organisms of almost every animal group, including numerous invertebrates and vertebrates. In humans, fewer than 10 human infections were reported until microsporidia had emerged as important opportunistic pathogens when AIDS became pandemic. Meanwhile, their significance has dramatically decreased in areas where antiretroviral combination therapy is available, but the prevalence of opportunistic infections in HIV-infected patients remains still high or is increasing in resource-poor countries. In affluent countries, the focus has shifted to microsporidial infections in otherwise immunocompromised patients, particularly organ transplant recipients, and in immunocompetent persons with cornea infection. Human microsporidial infections might be underdiagnosed due to the difficulties of detection. The sources of most microsporidia infecting humans and modes of transmission are still uncertain. Because microsporidial spores are released into the environment via stool, urine, and respiratory secretions, possible sources of infection may be persons or animals infected with microsporidia. Furthermore, because some microsporidia infecting humans are morphologically identical to organisms known in animal reservoirs, the question arose as to whether these intracellular parasites are of zoonotic origin. Molecular techniques have been applied to

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address this question, but at present, conclusive results are available for only a few microsporidial species. Using genetic analyses, the zoonotic potential has clearly been documented for several Encephalitozoon species including E. cuniculi and E. intestinalis, which infects mammals and humans, as well as E. hellem, which infects birds and humans. Epidemiological evidence, however, is lacking because the number of reported human infections is still low. Most animal and human isolates of E. bieneusi, the most prevalent human microsporidian, causing mainly severe and chronic HIV-associated diarrhea, are genetically distinct. Nevertheless, identical E. bieneusi genotypes were found in mammals and in a few human patients, suggesting that zoonotic transmission is possible. The molecular results strongly support the hypothesis that most human-infecting E. bieneusi genotypes are “human adapted” and not zoonotic. Because other microsporidial species are only very rarely found even in severely immunocompromised humans, it appears likely that humans do not represent a major parasite reservoir. Although the natural hosts have not been found yet for several of these rare human pathogens, an arthropod or fish host reservoir might be considered. Ingestion of microsporidial spores is the most probable mode of acquisition, and it is assumed that infection is most likely transmitted either directly from human to human via the fecal-oral route or indirectly via water or food. Although some epidemiological studies indicated the possibility of human-tohuman or waterborne infections, convincing epidemiological results are still lacking. Transmission by the aerosol route has also been considered, because spores have been found in respiratory specimens of patients with Encephalitozoon spp. or E. bieneusi infection. Patients who have severe cellular immunodeficiency appear to be at highest risk of developing microsporidial disease, but little is known about immunity to microsporidial infection. It is not understood whether microsporidial infection in these patients is primarily a reactivation of latent infection acquired before the state of suppressed immunity or whether microsporidial disease is caused by recently acquired infection. Based on the scarce current epidemiological knowledge concerning transmission of microsporidia, no recommendations to prevent infections can be suggested. ACKNOWLEDGMENT Work on microsporidia in the authors’ groups was supported by grants from the Swiss National Science Foundation (3200-049751.96/1 and 3237399.93). REFERENCES 1. Aarons, E. J., D. Woodrow, W. S. Hollister, E. U. Canning, N. Francis, and B. G. Gazzard. 1994. Reversible renal failure caused by a microsporidian infection. AIDS 8:1119–1121. 2. Accoceberry, I., J. Carriere, M. Thellier, S. Biligui, M. Danis, and A. Datry. 1997. Rat model for the human intestinal microsporidian Enterocytozoon bieneusi. J. Eukaryot. Microbiol. 44:83S. 3. Accoceberry, I., P. Greiner, M. Thellier, A. Achbarou, S. Biligui, M. Danis, and A. Datry. 1997. Rabbit model for human intestinal microsporidia. J. Eukaryot. Microbiol. 44:82S. 4. Achbarou, A., C. Ombrouck, T. Gneragbe, F. Charlotte, L. Renia, I. Desportes-Livage, and D. Mazier. 1996. Experimental model for human intestinal microsporidiosis in interferon gamma receptor knockout mice infected by Encephalitozoon intestinalis. Parasite Immunol. 18:387–392. 5. Akerstedt, J. 2002. An indirect ELISA for detection of Encephalitozoon cuniculi infection in farmed blue foxes (Alopex lagopus). Acta Vet. Scand. 43:211–220.

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ceedings of the 34th Annual Meeting of the Infectious Diseases Society of America, New Orleans, La., p. 67. Infectious Diseases Society of America, New Orleans, La. Waywa, D., S. Kongkriengdaj, S. Chaidatch, S. Tiengrim, B. Kowadisaiburana, S. Chaikachonpat, S. Suwanagool, A. Chaiprasert, A. Curry, W. Bailey, Y. Suputtamongkol, and N. J. Beeching. 2001. Protozoan enteric infection in AIDS related diarrhea in Thailand. Southeast Asian J. Trop. Med. Public Health 2:151–155. Weber, R., and R. T. Bryan. 1994. Microsporidial infections in immunodeficient and immunocompetent patients. Clin. Infect. Dis. 19:517–521. Weber, R., R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara. 1992. Improved light-microscopical detection of Microsporidia spores in stool and duodenal aspirates. N. Engl. J. Med. 326:161–166. Weber, R., R. T. Bryan, D. A. Schwartz, and R. L. Owen. 1994. Human microsporidial infections. Clin. Microbiol. Rev. 7:426–461. Weber, R., P. Deplazes, M. Flepp, A. Mathis, R. Baumann, B. Sauer, H. Kuster, and R. Luthy. 1997. Cerebral microsporidiosis due to Encephalitozoon cuniculi in a patient with human immunodeficiency virus infection. N. Engl. J. Med. 336:474–478. Weber, R., P. Deplazes, and D. Schwartz. 2000. Diagnosis and clinical aspects of human microsporidiosis. Contrib. Microbiol. 6:166–192. Weber, R., H. Kuster, G. S. Visvesvara, R. T. Bryan, D. A. Schwartz, and R. Lu ¨thy. 1993. Disseminated microsporidiosis due to Encephalitozoon hellem: pulmonary colonization, microhematuria, and mild conjunctivitis in a patient with AIDS. Clin. Infect. Dis. 17:415–419. Weber, R., B. Ledergerber, R. Zbinden, M. Altwegg, G. E. Pfyffer, M. A. Spycher, J. Briner, L. Kaiser, M. Opravil, C. Meyenberger, M. Flepp, et al. 1999. Enteric infections and diarrhea in human immunodeficiency virusinfected persons: prospective community-based cohort study. Arch. Intern. Med. 159:1473–1480. Weber, R., B. Sauer, M. A. Spycher, P. Deplazes, R. Keller, R. Ammann, J. Briner, and R. Lu ¨thy. 1994. Detection of Septata intestinalis in stool specimens and coprodiagnostic monitoring of successful treatment with albendazole. Clin. Infect. Dis. 19:342–345. Weidner, E., E. U. Canning, C. R. Rutledge, and C. L. Meek. 1999. Mosquito (Diptera: Culicidae) host compatibility and vector competency for the human myositic parasite Trachipleistophora hominis (phylum microspora). J. Med. Entomol. 36:522–525. Weiss, L. M. 2000. Molecular phylogeny and diagnostic approaches to microsporidia. Contrib. Microbiol. 6:209–235. Weitzel, T., M. Wolff, J. Dabanch, I. Levy, C. Schmetz, G. S. Visvesvara, and I. Sobottka. 2001. Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. Infection 29:237–239. Wenker, C. J., J.-M. Hatt, D. Ziegler, A. Mathis, I. Tanner, and P. Deplazes. 2002. Microsporidiosis (Encephalitozoon spp.) of new world primates—an emerging disease? A seroepidemiological, pathological and therapeutical survey in the Zurich zoo. In Proceedings of the 4th Scientific Meeting of the European Association of Zoo and Wildlife Vets, Heidelberg, Germany, p. 503–508. European Association of Zoo and Wildlife Vets, Heidelberg, Germany. Williams, B. A., R. P. Hirt, J. M. Lucocq, and T. M. Embley. 2002. A mitochondrial remnant in the microsporidian Trachipleistophora hominis. Nature 418:865–869. Wilson, J. M. 1979. Encephalitozoon cuniculi in wild European rabbits and a fox. Res. Vet. Sci. 26:114. Wuhib, T., T. M. Silva, R. D. Newman, L. S. Garcia, M. L. Pereira, C. S. Chaves, S. P. Wahlquist, R. T. Bryan, R. L. Guerrant, Q. Sousa Ade, et al. 1994. Cryptosporidial and microsporidial infections in human immunodeficiency virus-infected patients in northeastern Brazil. J. Infect. Dis. 170: 494–497. Xiao, L., L. Li, H. Moura, I. Sulaiman, A. A. Lal, S. Gatti, M. Scaglia, E. S. Didier, and G. S. Visvesvara. 2001. Genotyping Encephalitozoon hellem isolates by analysis of the polar tube protein gene. J. Clin. Microbiol. 39:2191–2196. Xiao, L., L. Li, G. S. Visvesvara, H. Moura, E. S. Didier, and A. A. Lal. 2001. Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences. J. Clin. Microbiol. 39:2248–2253. Yachnis, A. T., J. Berg, A. Martinez-Salazar, B. S. Bender, L. Diaz, A. M. Rojiani, T. A. Eskin, and J. M. Orenstein. 1996. Disseminated microsporidiosis especially infecting the brain, heart, and kidneys. Report of a newly recognized pansporoblastic species in two symptomatic AIDS patients. Am. J. Clin. Pathol. 106:535–543. Yee, R. W., F. O. Tio, J. A. Martinez, K. S. Held, J. A. Shadduck, and E. S. Didier. 1991. Resolution of microsporidial epithelial keratopathy in a patient with AIDS. Ophthalmology 98:196–201. Zeman, D. H., and G. B. Baskin. 1985. Encephalitozoonosis in squirrel monkeys (Saimiri sciureus). Vet. Pathol. 22:24–31.

CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 446–464 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.446–464.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 18, No. 3

Effects of Vitamin A Supplementation on Immune Responses and Correlation with Clinical Outcomes Eduardo Villamor1* and Wafaie W. Fawzi1,2 Departments of Nutrition1 and Epidemiology,2 Harvard School of Public Health, Boston, Massachusetts INTRODUCTION .......................................................................................................................................................446 VITAMIN A SUPPLEMENTATION AND INNATE IMMUNE RESPONSES..................................................447 Barrier Function and Mucosal Immunity ...........................................................................................................447 Acute-Phase Response and Complement System ...............................................................................................447 Monocytes/Macrophages ........................................................................................................................................447 Natural Killer Cells and Neutrophils ..................................................................................................................449 VITAMIN A SUPPLEMENTATION AND ADAPTIVE IMMUNE RESPONSES .............................................453 T and B Lymphocytes.............................................................................................................................................453 T-cell counts ........................................................................................................................................................453 T-cell function .....................................................................................................................................................453 B cells ...................................................................................................................................................................457 T-Cell-Dependent Humoral Responses................................................................................................................457 Tetanus and diphtheria......................................................................................................................................457 Measles.................................................................................................................................................................458 Polio ......................................................................................................................................................................458 Influenza...............................................................................................................................................................459 T-Cell-Independent Humoral Responses.............................................................................................................459 VITAMIN A SUPPLEMENTATION AND CLINICAL OUTCOMES .................................................................459 CONCLUSIONS .........................................................................................................................................................460 ACKNOWLEDGMENT..............................................................................................................................................461 REFERENCES ............................................................................................................................................................461 before the structure of vitamin A was deduced in 1931 (70). These included the discoveries that fat in butter improved the outcome of infections in malnourished animals (98) and that vitamin A-deficient rats appeared to be more susceptible to infection (55, 80). In 1932, Joseph B. Ellison discovered that a vitamin A extract reduced the measles case fatality rate in children, and up to 1940, at least 30 therapeutic trials were conducted on the effect of vitamin A on infection-related outcomes (123). After the discovery of antibiotics, research on the mechanisms through which vitamin A improved the immune function was revitalized in the 1960s by the landmark review by Scrimshaw et al. of the interactions between nutrition and infection (119) and later in the 1980s by the finding of protective effects of supplementation on overall child mortality in Indonesia (142), which were confirmed in subsequent large randomized clinical trials (9, 150). More recently, the discovery of the nuclear receptors for the vitamin A active metabolites all-trans- and 9-cis-retinoic acids (retinoic acid receptor and retinoic X receptor) (53, 87, 101), which regulate gene transcription, provided fundamental evidence for the understanding of the mechanisms through which retinoids affect immunity. The field of vitamin A immunology has greatly benefited from animal and in vitro experiments. These studies have provided a vast body of knowledge on the cellular and molecular mechanisms by which vitamin A and its metabolites influence the immune function at various levels. Excellent reviews of the available literature on these mechanisms have been recently published (121, 122, 145); however, there are fewer critical

INTRODUCTION The term vitamin A designates a group of retinoid compounds with the biologic activity of all-trans-retinol. Retinoids usually consist of four isoprenoid units with five conjugated carbon-carbon double bonds (141). Preformed vitamin A can be obtained mostly from dietary animal sources (liver, fish liver oils, eggs, and dairy products) as retinyl palmitate, whereas carotenoids that can be converted into retinol are obtained from vegetable foodstuffs (dark-green leafy vegetables and deep-orange fruits). Vitamin A plays an essential role in a large number of physiological functions that encompass vision, growth, reproduction, hematopoiesis, and immunity (143). Despite major advances in the knowledge of vitamin A biology, its deficiency is still a serious public health problem that affects an estimated 127 million preschool children and 7.2 million pregnant women worldwide (153). In children, vitamin A deficiency results in increased risks of mortality and morbidity from measles and diarrheal infections (150), blindness (156), and anemia (125), and among women it is likely to be associated with high mortality related to pregnancy (24, 154). Many of these effects can be linked to the immunological functions of vitamin A. Vitamin A is one of the most widely studied nutrients in relation to immune function. The first observations that suggested a link between vitamin A and immunity were made even

* Corresponding author. Mailing address: Department of Nutrition, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Phone: (617) 432-1238. Fax: (617) 432-2435. E-mail: evillamo @hsph.harvard.edu. 446

VOL. 18, 2005

reviews on the impact of vitamin A supplementation as a preventive or therapeutic intervention on indicators of immunity as measured in population studies. In this paper we present a review of the randomized, controlled clinical trials of vitamin A that have been conducted in humans and which included direct measurements of innate or adaptive aspects of the immune function as study end points. We also provide a correlation of the results from these trials with the effect of supplementation on clinical end points. We consider only the trials in which preformed vitamin A or related retinoids have been tested; trials with pro-vitamin A carotenoids alone are not included in this paper. Results from the human studies reviewed here are interpreted in light of available knowledge from basic research; however, this paper is not aimed at comprehensively reviewing animal or in vitro experimentation. VITAMIN A SUPPLEMENTATION AND INNATE IMMUNE RESPONSES Barrier Function and Mucosal Immunity Vitamin A is fundamental in maintaining the integrity of epithelia. Vitamin A deficiency is associated with pathological alterations in ocular (60, 143), respiratory (91, 160), gastrointestinal (114, 152), and genitourinary (93) epithelial tissues. A number of clinical trials examined the effect of vitamin A supplementation in humans on indicators of mucosal immunity, which included measurements of gut integrity and secretion of immune factors in the genital tract, saliva, and breast milk (Table 1). The lactulose/mannitol (L/M) urinary excretion test was used in some trials as an indicator of gut permeability. One dose of vitamin A administered to Indian children who were hospitalized with diarrhea resulted in a more rapid recovery of intestinal integrity within 30 days after discharge (147); among nonhospitalized children receiving weekly doses, however, there was no effect after 2 months. Vitamin A and ␤-carotene supplementation to human immunodeficiency virus (HIV)-infected pregnant women in South Africa was associated with lower L/M ratios in their infants at 14 weeks, but only in those who became HIV infected (49). In a nonrandomized, non-placebo-controlled study of children with a history of persistent diarrhea or underweight, supplementation with a single 200,000-IU vitamin A dose (100,000 if ⬍12 mo) was related to a significant decrease in the L/M ratio after 2 weeks compared to the baseline level (22). These children also received 20 mg zinc daily, and therefore it is not possible to ascertain whether the apparent treatment effect could be attributable to vitamin A alone. In the same study, serum retinol was negatively correlated to the urinary L/M ratio, whereas no correlation was found with zinc, which could suggest that if there was a treatment effect, it may have been due to vitamin A. In a small (n ⫽ 20), non-placebo-controlled trial of acitretin (a retinoid) in children with severe gastrointestinal mucosal lesions associated with chemotherapy, no effect on the L/M ratio was found after 4 to 5 weeks (75). Two clinical trials reported the effect of vitamin A supplementation during pregnancy (128) or the early postpartum period (48) on the concentration of mucosal anti-infective proteins in breast milk. No effects were found on secretory immunoglobulin A (IgA), lactoferrin, lysozyme, or interleukin-8 (IL-

VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE

447

8). Vitamin A supplementation had no effect on the concentration of immune factors in cervicovaginal fluids of HIV-infected pregnant women, including lactoferrin, lysozyme, and secretory leukocyte proteinase inhibitor, an innate protein produced by mucosal epithelial and acinar cells (102). There was also no effect on the concentration of IL-1␤ in genital fluids of HIV-infected women in another trial (40). Fecal IL-8 following infections with enterotoxigenic Escherichia coli was reduced by vitamin A supplementation according to preliminary results from a trial with Mexican children (82). One noncontrolled trial among children showed an apparent decrease in the concentration of secretory IgA in saliva after 4 weeks of supplementation with 100,000 IU vitamin A with respect to baseline values, but comparisons with children who did not receive vitamin A were not made (16). In summary, the available evidence indicates a beneficial effect of vitamin A supplementation on intestinal integrity among children suffering from severe infections or who are undernourished. A few supplementation studies have not shown a consistent effect on the concentration of mucosal anti-infective or inflammatory markers in milk, saliva, or genital fluids. Acute-Phase Response and Complement System The impact of vitamin A on circulating effectors of innate immunity, including acute-phase response proteins and the complement system, was studied in trials from Ghana, Indonesia, and South Africa (Table 1). In the Ghana study of preschool children, large doses of vitamin A every 4 months for 1 year resulted in significantly increased serum amyloid A and C-reactive protein among children with symptoms of gastrointestinal infections, including severe diarrhea and vomiting (47). By contrast, no significant effect was found on C-reactive protein concentrations after 5 weeks of a single oral dose in the Indonesia study (120). Plasma C3 complement was not affected by four doses of vitamin A administered within a 42-day period to South African children (28). Monocytes/Macrophages Experiments in vitro and animal studies suggest that retinoids are important regulators of monocytic differentiation and function. When added to monocytic, myelomonocytic or dendritic cell line cultures, retinoic acid promotes cellular differentiation (19, 52, 69, 92) and influences the secretion of key cytokines produced by macrophages, including tumor necrosis factor (TNF-␣), IL-1␤, IL-6, and IL-12. All-trans-retinoic acid skewed the differentiation of human peripheral blood monocytes to IL-12-secreting dendritic cells in one in vitro study (92), whereas in another it inhibited lipopolysaccharide-induced IL-12 production by mouse macrophages (96). All-transretinoic acid was shown to decrease secretion of TNF-␣ in murine peripheral blood mononuclear cells (73) and myelomonocytic (95) and macrophage (88, 94) cell lines. On the other hand, retinoids appear to enhance the secretion of IL-1␤ (59, 89) and IL-6 (2) by macrophages and monocytes. In rats (61) and in experiments in vitro (35), vitamin A increased the phagocytic capacity of macrophages. A few supplementation studies with humans included indi-

Lactulose/mannitol ratio:

238 infants born to 728 HIV⫹ women

212 postpartum women

60 HIV⫹ pregnant women

Vitamin A (5,000 IU) ⫹ ␤-carotene (30 mg) daily during pregnancy ⫹ vitamin A (200,000 IU) at delivery to mothers vs placebo

Vitamin A (10,000 IU) daily during pregnancy vs placebo

Vitamin A (200,000 IU) single dose vs 7.6 mg ␤-carotene daily vs placebo started at 1–3 wk postpartum for 9 mo

Vitamin A (5,000 IU) ⫹ ␤-carotene (30 mg) daily during pregnancy ⫹ vitamin A (200,000 IU) at delivery vs placebo

Vitamin A (45,000 IU, or 20,000 IU if ⬍12 mo) vs placebo every 2 mo for 1 yr

South Africa (49, 115)

Malawi (128)

Bangladesh (48)

South Africa (102)

Mexico (82)

188 infants, 6–15 mo

Breast milk lactoferrin at 6 wk postpartum

334 HIV⫹ pregnant women

IL-8 IL-10 TNF-␣ IFN-␥ MCP-1

No effect No effect No effect

0.79 3.04 124 26.2

␤-Carotene

No effect

Placebo

Placebo

0.86 3.40 109 33.6

Placebo

Placebo

No significant effect No effect Higher during summer in vitamin A group Lower after enterotoxigenic E. coli Lower after enterotoxigenic E. coli No effect Higher during summer in vitamin A group No effect No significant effect

Vitamin A

Vitamin A

0.74 3.06 118 29.6

Vitamin A

Vitamin A

Higher Higher

Lower Lower No significant effect

0.09 0.50

Placebo

0.11 0.17

Vitamin A

No difference at any time point

Vitamin A Placebo Significantly different in both vitamin A-treated groups compared to control group

Result for the indicated measure of effectb

⬎0.05

⬍0.05

⬍0.05 ⬍0.05

⬍0.05

⬎0.05

⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05

0.009 0.014

⬎0.05 ⬍0.05

⬍0.05

P value

VILLAMOR AND FAWZI

IL-4 IL-5 IL-6

Cytokines in stool

Lactoferrin after 6 wk Lysozyme after 6 wk SLPI after 6 wk

Immune factors in CVL

Secretory IgA (g/liter) Lactoferrin (g/liter) Lysozyme (mg/liter) IL-8 (ng/liter)

Breast milk immune factors at 3 mo from first dose

Urinary neopterin excretion in infants at 0, 1 wk, 6 wk, 6 mo

242 infants

Among HIV⫺ infants at 14 weeks Among HIV⫹ infants at 14 weeks Lactulose/creatinine ratio at: 1 wk 14 wk

Lactulose/mannitol ratio at 4 and 8 wk

80 infants

Vitamin A (16,700 IU) weekly for 8 wk vs placebo

Lactulose/mannitol ratio at 10 and 30 days after discharge

End pointa

94 infants hospitalized, with diarrhea or respiratory infection

Population

TABLE 1. Randomized, controlled trials of vitamin A in relation to innate immune responses

Intervention groups

Vitamin A (200,000 IU) on admission vs 200,000 IU at discharge, vs placebo, single dose

India (147)

Study site (reference[s])

448 CLIN. MICROBIOL. REV.

b

a

SLPI, secretory leukocyte proteinase inhibitor; MCP, macrophage/monocyte chemoattractant protein; CVL, cervicovaginal lavage. All values presented are means unless noted otherwise. Verbal descriptions of results correspond to those in the indicated references when numerical data were not presented.

⬎0.05 ⬎0.05 1.11 1.41 1.25 1.48

Vitamin A (200,000 IU, or 100,000 IU if ⬍12 mo) vs placebo at days 0, 2, 8, and 42 South Africa (28)

8 42

Placebo Vitamin A

No significant differences No significant differences ␣1-Glycoprotein C-reactive protein

Vitamin A Change from baseline at 5 wk

Vitamin A (200,000 IU) vs placebo, single dose, 2 wk before antitetanus immunization Indonesia (120)

236 children age 3–6 yr, 50% xerophtalmic

Plasma complement C3 (g/liter) at days

⬎0.05 ⬎0.05 1.15 1.18

Serum amyloid A Children with vomiting Children with diarrhea C-reactive protein Children with vomiting Children with diarrhea ␣1-Glycoprotein Children with vomiting Children with diarrhea

Vitamin A Ratio of acute-phase proteins between vitamin A and placebo groups 329 children age 6–59 mo Vitamin A (200,000 IU, or 100,000 IU if ⬍ 12 mo) vs placebo every 4 mo for 1 yr Ghana (47)

60 infants age 4–24 mo hospitalized with measles

⬍0.05 ⬎0.05 2.91 2.11

Placebo

Placebo

⬎0.05 ⬍0.05

VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE

2.15 4.20

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449

rect measures of monocyte and macrophage function, mostly related to cytokine secretion. In a non-placebo-controlled, nonrandomized study of six patients with common variable immunodeficiency who had low serum retinol concentrations, supplementation with vitamin A at 6,500 IU/day for 6 months resulted in decreased concentrations of TNF-␣ compared to their baseline levels (3). Preliminary results from a trial with Mexican infants showed that the concentration of IL-6 in stool was lower in those who received vitamin A, but this effect appeared to be limited to the period following an infection with enterotoxigenic Escherichia coli (82). Vitamin A and ␤-carotene supplementation to HIV-infected pregnant women had no effect on the concentration of IL-1␤ in cervicovaginal secretions (40) or on urinary neopterin excretion, an indicator of macrophage activation (115), in studies from Tanzania and South Africa, respectively. The trials described above suggest that supplementation with preformed vitamin A might down-regulate the secretion of specific proinflammatory cytokines (e.g., TNF-␣ and IL-6) by macrophages, but seemingly only in response to infections by particular pathogens. Additional, more robust data from human trials would be needed to support this potential mechanism.

Natural Killer Cells and Neutrophils NK cells are important in the first line of defense against tumors and viral infections. The number of circulating NK cells is reduced during experimental vitamin A deficiency in animals (161). In humans, one cross-sectional study found that children with low serum retinol concentrations had a greater proportion of NK cells than those with higher retinol concentrations (68); however, vitamin A-deficient children were also more likely to be infected with HIV, which is likely to have confounded the association observed. In a clinical trial from South Africa among HIV-infected children, vitamin A supplementation was related to increased number of cells with the CD56 receptor, mostly expressed by NK cells (66). The development of neutrophils in the bone marrow is controlled by retinoic acid receptor-modulated genes (90), and retinoic acid in cultures accelerates neutrophil maturation (113). Treatment with retinoic acid (162) or vitamin A (62) was shown to restore the number of neutrophils and the superoxide-generating capacity in rats and calves, respectively. There are limited data on the relationship between vitamin A and neutrophil function in humans. In a cross-sectional study of non-HIV-infected pregnant women, low serum retinol concentrations were not predictive of the neutrophil count (54), a very unspecific indicator. A study among preschool children reported no significant differences in neutrophil hydrogen peroxide (H2O2) or superoxide (O2⫺) production according to serum retinol concentrations (16). In the same study, the oral administration of 100,000 IU vitamin A was related to a significant increase in H2O2 production over baseline values after 4 weeks, but no comparisons with children not receiving supplementation were made. The apparent benefit of vitamin A supplementation on NK cells among immunosuppressed children deserves confirmation in future investigations.

T-cell subset change at 18 mo % CD4 % CD8 CD4/CD8 ratio

Absolute lymphocyte count at 4 wk

276 infants

185 infants

Vitamin A (100,000 IU) at age 6 mo vs placebo

Vitamin A (100,000 IU) at age 6 and 9 mo vs placebo

T-helper cell responses to HIV in cord blood (infants)

Woman’s T-cell subset counts (/␮l) CD4 at 6 wk postpartum CD4 at 30 wk postpartum Overall effect CD8 at 6 wk postpartum CD8 at 30 wk postpartum Overall effect CD3 at 6 wk postpartum CD3 at 30 wk postpartum Overall effect

33 infants born to 728 HIV⫹ women 1,078 HIV⫹ pregnant women

Vitamin A (5,000 IU) ⫹ ␤-carotene (30 mg) daily during pregnancy ⫹ vitamin A (200,000 IU) at delivery to mothers vs placebo

Vitamin A (5,000 IU) ⫹ ␤-carotene (30 mg) daily during pregnancy and breast feeding to mothers vs placebo

South Africa (76)

Tanzania (40, 44–46)

CD4 cell count at 4 wk CD56 cell count at 4 wk CD29 cell count at 4 wk

75 HIV⫹ infants

Vitamin A (200,000 IU) on 2 consecutive days vs placebo

South Africa (66)

T-cell subset change between 6 and 9 mo of age % CD4 % CD8 CD4/CD8 ratio

% CD8⫹ CD45RO⫹

% CD8⫹ CD45RA⫹

% CD4⫹ CD45RO⫹

% CD4⫹ CD45RA⫹

CD4/CD8 ratio at 5 wk from supplementation % CD4 naive T cells at 5 wk Change in T-cell subsets after 5 wk CD4/CD8 ratio

Guinea-Bissau (13)

55 children age 3–6 yr. 30 xerophthalmic

Vitamin A (200,000 IU) vs placebo, single dose, 2 wk before antitetanus immunization

Lymphocyte count (109/liter) Difference at day 42 Change from day 0 to day 42 IL-2 (IU/ml) at day: 8 42

End point

Indonesia (132)

60 infants age 4–24 mo hospitalized with measles

Population

Vitamin A (200,000 IU, or 100,000 IU if ⬍12 mo) vs placebo on days 0, 2, 8, and 42

Intervention groups

South Africa (28)

Study site (reference[s])

Placebo 6.45 1.96 1.72 1.70

Vitamin A Placebo 1.32 0.97 Higher in vitamin A group

Vitamin A 8.10 3.40 1.76 1.74

Result for the indicated measure of effectb

Placebo 562 509 1,094 909 1,741 1,494

1,061 943 1,698 1,515

0.13 0.23 0.29 0.32 0.26 0.13 0.20 0.68 0.25

0.05 0.03 0.05 0.07

0.07 0.12 0.06

0.31 0.21 0.26

⬍0.0001 ⬍0.01

0.05 0.06 ⬎0.05 ⬎0.05

P value

VILLAMOR AND FAWZI

Vitamin A 558 496

Vitamin A Placebo Slightly more frequent in vitamin A arm but not significant

Placebo

No vitamin A ⫺2.72 0.49 ⫺0.20

Vitamin A 2.50 ⫺1.93 0.28

Increase Increase Increase Increase

0.45 1.61 ⫺0.07

⫺0.11 1.21 ⫺0.15

Vitamin A

No vitamin A

Vitamin A

Significant increase, No significant change P ⬍ 0.007 Significant increase, No significant change P ⬍ 0.003 No change in any group, no significant difference in change No change in any group, no significant difference in change Significant decrease No significant change

TABLE 2. Randomized, controlled trials of vitamin A in relation to T-cell functiona 450 CLIN. MICROBIOL. REV.

Vitamin A (200,000 IU) single dose vs placebo

Vitamin A (300,000 IU) single dose vs placebo

Vitamin A (10,000 IU) daily for 6 wk vs placebo

Vitamin A (⬇1,500 IU) daily for 3 mo vs. placebo, in both groups, half also received 25 mg zinc zulfate

United States (129)

United States (65)

Kenya (5)

Italy (50)

Vitamin A (5,000 IU) ⫹ ␤-carotene (30 mg) daily for ⬇3–5 yr

118 men and women age ⬎65

400 HIV⫹ women

40 HIV⫹ women

120 HIV⫹ injection drug users

1,078 HIV⫹ women

393 HIV⫹ pregnant women 664 infants born to 1,078 HIV⫹ women

Total leucocytes (/␮l) Baseline Change from baseline Lymphocytes (/␮l) Baseline Change from baseline CD3⫹ (/␮l) Baseline Change from baseline CD4⫹ (/␮l) Baseline Change from baseline CD4⫹ DR⫹ (/␮l) Baseline Change from baseline CD8⫹ (/␮l) Baseline Change from baseline CD8⫹ DR⫹ (␮l) Baseline Change from baseline CD4/CD8 ratio Baseline Change from baseline Cytotoxic T lymphocytes Baseline Change from baseline

T-cell subsets at 6 wk (/␮l) CD4 CD8

% CD4 at 8 wk % CD8 that are CD38⫹ Lymphocyte proliferation in response to: PHA Candida

CD4 cells (/␮l) at 4 wk

T-cell subsets (/␮l) at ⬎4 yr after enrollment CD4 CD8 CD3

Infant’s CD4 cell count during first 2 yr

IL-1␤ in CVL (␮g/ml)

1,942 62 1,123 49 711 ⫺23 70 18 629 30 116 ⫺0.7 1.16 ⫺0.05 84 51

1,977 ⫺73 1,260 ⫺109 712 ⫺125 76 6 665 ⫺70 139 ⫺0.8 1.17 ⫺0.10 70 23

0.19

0.55

0.64

0.12

0.12

0.01

0.01

0.05

0.30

0.04 0.08

⬎0.05 ⬎0.05

0.56 ⬎0.05

0.17

0.30 0.07 0.05

0.41

Continued on following page

No Vitamin A 6,206 361

Vitamin A 6,149 138

Placebo

Placebo

449 968 1,497

Placebo 225 581

No effect

219c

Vitamin A 272 719

Vitamin A

Vitamin A

434 920 1,425

218c

VOL. 18, 2005 VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE 451

Vitamin A (50,000 IU) vs placebo at each time of DPT/ OPV vaccination (0, 4, and 8 wk)

Bangladesh (106)

c

b

120 infants age 6–17 wk

200 children age 5 mo–17 yr hospitalized with measles

91 children 1–6 yr

Population

End point

Cutaneous DTH response (% children) after 1 mo after last dose Tetanus Diphtheria Tuberculin (PPD) Streptococcus Candida Trichophyton Proteus Total (sum induration) Total among children with baseline retinol ⱖ0.7 ␮mol/liter Avg induration (mm) among children with baseline retinol ⱖ0.7 ␮mol/liter % Anergic infants

Cutaneous DTH response at 2 wk % Children with reactions Sum of indurations ⱖ2 mm Mean induration per reaction % of children unresponsive to specific antigens Tetanus Diphtheria Tuberculin (PPD) Streptococcus Candida Trichophyton Proteus IL-4, % change from baseline to 2 wk

% of positive cutaneous DTH responses (ⱖ5 mm) to a protein derivative and Candida after 4 wk

NK cells (/␮l) Baseline Change from baseline

TABLE 2—Continued

252 45

Control 31.9

Placebo 61.3 8.4 3.9 81.0 82.5 72.1 90.1 85.5 92.8 86.8 35.0

Placebo 32.8 22.4 46.6 6.9 8.6 3.4 1.7 67.2 57.9 4.2 32.8

225 ⫺4

Vitamin A 29.5

Vitamin A 44.4 6.9 4.1 82.8 81.8 86.6 100.0 97.1 97.1 97.1 9.0

Vitamin A 30.6 30.6 59.7 4.8 3.2 1.6 4.8 72.6 93.3 7.0 27.4

Result for the indicated measure of effectb

PHA: phytohemagglutinin; CVL: cervicovaginal lavage; DPT: diphtheria, pertussis, and tetanus vaccine; OPV: oral polio vaccine; PPD: purified protein derivative; All values presented are means unless noted otherwise. Verbal descriptions of results correspond to those in the indicated references when numerical data were not presented. Median.

Vitamin A (200,000 IU) single dose vs placebo

Zambia (116)

a

Vitamin A (200,000 IU intramuscularly) vs no intervention at the time of first of 3 antitetanus immunizations

Intervention groups

Bangladesh (20)

Study site (reference[s])

⬎0.05

0.01

⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 0.008

⬎0.05 ⬎0.05 ⬍0.05 ⬎0.05 ⬍0.05 ⬎0.05 ⬍0.05 0.18

0.04 0.20 0.50

⬎0.05

0.11

P value

452 VILLAMOR AND FAWZI CLIN. MICROBIOL. REV.

VOL. 18, 2005

VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE

VITAMIN A SUPPLEMENTATION AND ADAPTIVE IMMUNE RESPONSES T and B Lymphocytes T-cell immunocompetence can be affected by vitamin A deficiency at various levels, including lymphopoiesis, distribution, expression of surface molecules, and cytokine production. The end points examined in human clinical trials could be grouped into T-lymphocyte counts and function. T-cell counts. A potential effect of vitamin A on human lymphopoiesis has been suggested in pediatric supplementation trials (Table 2). Among infants from South Africa, vitamin A supplementation significantly increased the total lymphocyte count after 42 days (28), whereas in Indonesia, supplementation was related to a higher proportion of CD4 naive T cells (CD4⫹ CD45RA⫹) after 5 weeks, compared to controls (132). In a trial conducted in Guinea-Bissau, vitamin A supplementation at age 6 months was not associated with significant changes in T-cell subpopulations; however, supplementation at both 6 and 9 months resulted in a borderline significant increase in the proportion of CD4 T cells at age 18 months (13). Some trials examined the effect of vitamin A on T-cell counts among HIV-infected children. In South Africa, supplementation in HIV-positive infants was related to a significant increase in total lymphocyte counts as well as specific T-cell subpopulations, including CD4, after 4 weeks (66). By contrast, supplementation in HIV-infected women during pregnancy had no effect on the babies’ T lymphocytes in Tanzania (46) or South Africa (76). The comparison between the direct supplementation trial (66) and the maternal supplementation studies (46, 76) is limited not only by the way of administration but also because supplements in the latter trials included ␤-carotene. The potential effect of vitamin A on lymphocyte counts in adults has been studied among HIV-infected individuals, who are at high risk of developing profound nutritional deficiencies (51, 71, 135). Single-dose supplementation with vitamin A in nonpregnant HIV-positive women (65) and injection drug users (129) did not have a significant effect on CD4 cell counts; however, daily supplementation with vitamin A during 6 weeks in Kenyan HIV-infected women resulted in a modest, marginally significant greater mean CD4 cell count (5). Daily vitamin A and ␤-carotene supplementation of Tanzanian HIV-infected women during pregnancy had no effect on their CD4 cell counts (44). In extended analyses of the same study, daily supplementation for ⬎3 years resulted in a statistically significant small negative effect on CD3 and a borderline significant effect on CD8 (45). It is not possible to distinguish whether these effects were due to the preformed vitamin A or the pro-vitamin A carotenoid. One study among elderly men and women reported an apparent adverse effect of daily vitamin A supplementation on total lymphocyte counts at the expense of CD4 (50). This effect seemed to be ameliorated by the concomitant administration of zinc. In summary, vitamin A supplementation to children has the potential to increase T-cell counts, particularly of the CD4 subpopulation. Studies have included children who are at high risk of vitamin A deficiency or who are infected with HIV.

453

There is little evidence to support an effect of preformed vitamin A supplementation to adults on lymphopoiesis. T-cell function. Some evidence is available from human studies regarding the role of vitamin A in lymphocyte immunocompetence. The ex vivo production of gamma interferon (IFN-␥), a Th1 proinflammatory cytokine, was found to be depressed in vitamin A-deficient children from Indonesia (159). In the South African trial among presumably non-HIVinfected infants (28), no vitamin effect was observed on the serum concentration of IL-2, another Th1 cytokine produced mainly by CD4 lymphocytes that plays a key role in proliferation and activation of T, B, and natural killer cells. IL-2 production is transient and peaks after 8 to 12 h after lymphocyte activation (1), and therefore it is unlikely that single measures of serum concentrations accurately reflect a potential treatment effect. In another study in Zambia among children hospitalized with measles, no effect on the change from baseline in IL-4 concentration after 2 weeks of a single vitamin A dose was noted (116), and a study in Mexico found no differences by treatment arm in IL-4 from stool samples (82). IL-4 is produced mostly by CD4 cells of the Th2 subset. A number of studies indicate a role for vitamin A in the regulation of IL-10 secretion. IL-10 produced by Th2-helper T cells inhibits the synthesis of proinflammatory Th1-type cytokines, including IFN-␥ and IL-2, in both T and NK cells. This mechanism is important in limiting inflammatory responses to some pathogens. Venezuelan children with subclinical vitamin A deficiency had significantly lower circulating concentrations of IL-10 than nondeficient controls in a cross-sectional study (81). In the study of patients with common variable immunodeficiency (3), vitamin A supplementation increased IL-10 concentrations in patients with low serum vitamin A levels. In contrast, preliminary results from a trial of pregnant and lactating women in Ghana suggest that vitamin A supplementation increased the ratios of proinflammatory IFN-␥ and TNF-␣ to IL-10 in the postpartum period (31). The authors speculate that, by reducing IL-10, vitamin A supplementation postpartum could reverse the anti-inflammatory Th2 bias induced by pregnancy and therefore diminish the risk of perinatal infections. This hypothesis would be consistent with studies showing reductions in morbidity from infections and infant mortality in the offspring of mothers supplemented with vitamin A; although some studies suggest a decrease in the incidence of febrile episodes (118), an effect on infant mortality is not apparent (86). The trial among HIV-infected women from the United States (65) examined the effect of vitamin A supplementation on ex vivo lymphocyte proliferation in response to phytohemagglutinin and Candida mitogens and on lymphocyte activation markers (CD8⫹ CD38⫹) at 2, 4, and 8 weeks after a single dose; no effect on any of these parameters was observed. Three studies among children assessed the effect of vitamin A supplementation on the cutaneous delayed-type hypersensitivity (DTH) response, an indicator of T-cell-dependent macrophage activation. There were no differences by treatment arm in the percentage of children with a DTH response to a protein derivative or Candida in a non-placebo-controlled trial of intramuscular vitamin A from Bangladesh (20); however, in a different trial among younger infants in the same country,

Vitamin A (200,000 IU intramuscularly) vs no intervention at the time of first of 3 anti tetanus immunizations

Vitamin A (200,000 IU) vs placebo, single dose, 2 wk before antitetanus immunizations

Vitamin A (30,000 IU) for 3 days together with DPT at 2, 3, and 4 mo of age (half also received 150 mg vitamin E one dose at each time

Vitamin A (50,000 IU) vs placebo at each time of DPT/OPV vaccination (0, 4, and 8 wk)

Vitamin A (200,000 IU, or 100,000 IU if ⬍12 mo) vs placebo at days 0, 2, 8, and 42

Vitamin A (200,000 IU) single dose vs placebo

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (age 6 mo), single dose

Indonesia (131)

Turkey (79)

Bangladesh (108)

South Africa (28)

Zambia (116)

Indonesia (133)

Intervention groups

Bangladesh (20)

Study site (reference[s])

336 infants age 6 mo

200 children age 5 mo–17 yr hospitalized with measles

60 infants age 4–24 mo hospitalized with measles

56 infants

89 infants

236 children age 3–6 yr, 50% xerophtalmic

95 children 1–6 yr

Population

Placebo 11.0 5.7 119

Placebo 222.8 654.8

Placebo 4.30

Placebo 79.3% 100.0% 77.2%

Vitamin A 22.9 4.1 112

Vitamin A 384.2 869.5

Vitamin A 4.12

Vitamin A 66.3% 100.0% 61.8%

GMT IgG anti-measles hemagglutinin protein at 2 wk from vitamin A dose

Seroconversion to measlesd after 6 mo from immunization among infants with titer of: ⱖ8 ⬍8 Protective titers (⬎120) after 6 mo in infants with baseline titers ⱖ8 GMT to measles vaccine after: 1 mo 6 mo

IgG anti-measles nucleocapsid protein (mIU/ml) at day: 8 42

GM IgG (mg/liter) after 1 mo from last dose Antidiphteria Antipertussis Antitetanus

0.05 0.09

⬍0.04 ⬍0.03

0.25

0.01 ⬎0.05

0.029 ⬎0.05 ⬎0.05

⬎0.05 ⬎0.05

⬎0.05 ⬎0.05

⬍0.009 ⬍0.07

⬎0.05

P value

VILLAMOR AND FAWZI

25% lower in vitamin A group 25% lower in vitamin A group

100% 92%

100% 92%

Anti-tetanus GMT IgG (mIU/ml) at age: 5 mo 16–18 mo Protective antitetanus titers (ⱖ100 mIU/ ml) at age: 5 mo 16–18 mo

Placebo only 880.6 314.4

Placebo

Vitamin A

Vitamin A only 1,126.6 314.7

0.011

0.016

24 891

Control

Vitamin A

Result for the indicated measure of effectb

62 1,903

Tetanus-naive childrenc Tetanus-exposed childrenc

Anti-tetanus GM IgG (␮g/liter) change after 3 wk from immunization in:

Anti-tetanus GMT IgG (IU/ml) at 12 wk from first immunization (8 from second immunization)

End point

TABLE 3. Randomized, controlled trials of vitamin A in relation to antibody responsesa

454 CLIN. MICROBIOL. REV.

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (9 mo), single dose

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (age 9 mo)e single dose

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (9 mo), single dose

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (9–12 mo), single dose

Indonesia (124)

India (7)

India (23)

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (age 9 mo only), single dose

Vitamin A (100,000 IU) vs placebo at 2 times of measles immunization (ages 6 and 9 mo)

India (17)

Guinea-Bissau (10, 11, 14)

395 infants

618 infants

394 infants age 9 mo

100 infants

152 infants followed up

312 infants age 9 mo

79 infants followed up

150 infants age 6 mo

1,048

1,366

Protective titers (⬎120) after 6 mo GMT to measles vaccine after: 1 month 6 months

Seroconversion to measlesi 1 mo postimmunization

Seroconversion to measlesd 12 wk postimmunization among infants with titers ⱕ2,500 GMT 12 wk postimmunization GMT change from baseline to 12 wk postimmunization GMT 12 wk postimmunization among children with low weight

Baseline titer ⬍8 Baseline titer 8–120 Baseline titer ⬎120 Protective titer (⬎120) after 6 mo Baseline titer ⬍8 Baseline titer 8–120 Baseline titer ⬎120 GMT to measles vaccine after: 1 mo 6 mo

Seroconversion to measlesd after 1 mo from immunization

Among infants with titers ⱖ8 Among infants with titers ⬍8 Overall

Seroconversion to measlesg after 4 wk

2439 91%

3704 100%

GMT at age 18 mo Protective titers (ⱖ125 mlU/liter) at age 6–8 yr GMT at age 6–8 yr

178.2 95.3 160.3

Placebo 99%

211.8 116.4 251.2

Vitamin A 99%

0.57 0.31

0.14

1.00

⬍0.05

⬎0.05 ⬎0.05

0.50f

0.18 0.03

⬎0.05 ⬎0.05 ⬎0.05

⬎0.05 ⬎0.05 ⬎0.05

⬍0.01h ⬎0.05 ⬍0.001h

0.09

⬍0.01 0.0095

0.15f

0.51

⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 0.36

VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE Continued on following page

160.7 212.8

Placebo 87.6%

Vitamin A 89.5%

169.6 187.4

2,298 1,900

1,772 1,164

85%

98.7% 100.0% 100.0%

99.4% 100.0% 100.0%

78%

99.0% 100.0% 9.1%

Placebo

63.0% 75.0% 64.0%

Placebo

98.5% 100.0% 9.5%

Vitamin A

83.7% 100.0% 84.0%

Vitamin A

93%

783

670

20% 79% 65% 98% 1,768 100%

20% 88% 74% 98% 1,431 95%

97%

Placebo

Seroconversion to measlese at age 18 mo

Vitamin A

At age 9 mo Baseline titer ⱖ32 mlU Baseline titer ⬍32 mlU Overall At age 18 mo GMT at age 18 mo Protective titers (ⱖ125 mIU/liter) at age 6–8 yr GMT at age 6–8 yr

Seroconversion to measlese

VOL. 18, 2005 455

Vitamin A (100,000 IU) vs placebo at the time of measles immunization (9–12 mo), single dose

Vitamin A (50,000 IU) vs placebo at each time of DPT/OPV vaccination (0, 4, and 8 wk)

Vitamin A (200,000 IU) vs placebo to mothers within 24 h of delivery; all newborns received OPV dose within 72 h of birth

Vitamin A (50,000 IU) vs vitamin A (25,000 IU) vs placebo at each time of OPV vaccination (6, 10, and 14 wk)

Vitamin A (200,000 IL) to mothers between 18 and 28 days postpartum plus 25,000 IU to infants at the time of DPT/OPVI (6, 10, 14 wks) vs placebo to mothers and children

Bangladesh (107)

India (15)

Indonesia (130)

India (6)

Intervention groups

India (23)

Study site (reference[s])

399 motherinfant pairs

467 infants

100 motherinfant pairs

57 infants

395 infants

Population

Protective titer (ⱖ4) against poliovirus after 12 wk from last dose Type 1 Type 2 Type 3 GMT against poliovirus Type 1 Type 2 Type 3

Seroconversion to OPVk at age 9 mo Type 1 Type 2 Type 3 Protective titer (ⱖ8) against poliovirus Type 1 Type 2 Type 3 Loge antibody titers against poliovirus at age 9 mo Type 1 Type 2 Type 3

Seroconversion to OPVd at 6 wk Type 1 Type 2 Type 3 GMT against poliovirus at 6 wk Type 1 Type 2 Type 3

Seroconversion to OPVj after 1 mo from last dose Type 1 Type 2 Type 3

Protective titers (⬎120) after 6 mo GMT to measles vaccine after: 1 month 6 months

Seroconversion to measlesi 1 mo postimmunization

End point

TABLE 3—Continued

71.2% 93.2% 80.5% 17.6 289.1 60.5

27.2 287.1 66.5

5.26 5.95 5.36

82.0% 92.3% 84.5%

5.63 6.02 5.44

5.32 5.96 5.21

94.5% 99.1% 95.6%

Placebo

94.5% 98.4% 98.3%

93.1% 98.3% 96.5%

98.9% 100.0% 99.1%

Placebo

Vitamin A

Vitamin A, 25,000 IU 99.1% 100.0% 100.0%

23.4 21.6 9.7

18.4 17.9 9.6

Vitamin A, 50,000 IU 98.0% 100.0% 100.0%

Placebo 77.5% 79.6% 65.3%

83% 91% 87%

79% 82% 82%

Vitamin A 67.3% 83.6% 63.3%

Placebo

160.7 212.8

169.6 187.4

Vitamin A

85%

Placebo 99%

78%

Vitamin A 99%

Result for the indicated measure of effectb

VILLAMOR AND FAWZI ⬍0.05 ⬎0.05 ⬎0.05

0.01f 0.73f 0.29f

⬎0.05 ⬎0.05 ⬎0.05

0.88 0.84 0.45

0.77 1.00 0.99

⬎0.05 ⬎0.05 ⬎0.05

⬎0.05 ⬎0.05 ⬎0.05

0.76 0.34 0.64

0.57 0.31

0.14

1.00

P value

456 CLIN. MICROBIOL. REV.

GMT, geometric mean titers (ln 1/liter); GM, geometric mean; DPT, diphtheria, pertussis, and tetanus vaccine; OPV, oral polio vaccine. All values presented are means unless noted otherwise. Verbal descriptions of results correspond to those in the indicated references when numerical data were not presented. Tetanus-naive subjects were children without an immunization history and low baseline anti-tetanus IgG titers (⬍10 ␮g/liter). Tetanus-exposed children had a history of tetanus immunization and high baseline titers. d Seroconversion defined as a rise in antibody titer of ⱖ4-fold over the calculated expected titer. e A positive antibody titer (⬎ 32 mlU) and no record of measles infection. f From ␹2 test calculated with data presented in the reference. g Twofold rise in titers from baseline h P value from ␹2 test recalculated with data presented in the reference ⫽ 0.02. i Titers of ⱖ8 in children with no measurable antibodies at baseline, or a rise of ⱖ4-fold over the calculated expected titer at 4 weeks postimmunization in children with preexisting antibodies. j Antibody titer of ⱖ16 in a previously negative infant or a ⱖ4-fold rise over baseline level. k Positive antibody titer (ⱖ2) minus the calculated expected titer of passively acquired maternal antibody, assuming a half-life of 4 weeks for IgG. c

b

50 70 66 66

VITAMIN A SUPPLEMENTS AND IMMUNE RESPONSE

a

18.9 36.4 19.8 28.8

0.23 0.71

Placebo United States (57)

Vitamin A (200,000 IU) vs placebo on days 0 and 1; all received inactivated influenza vaccine and hemagglutinin antigens on day 14

59 HIV⫹ children 2–17 yr

Increase in influenza antibodies, GMT (U/liter) day 28 H1N1 H3N2 % with titers ⱖ1:32 after vaccination H1N1 H3N2

Vitamin A

0.68 0.71

VOL. 18, 2005

457

monthly oral doses of vitamin A resulted in a significantly greater DTH response, but the effect was limited to the subset of children who had serum retinol concentrations of ⬎0.70 ␮mol/liter at baseline (106). In the Zambia study among children who had been hospitalized with measles, vitamin A supplements appeared to diminish the proportion of children with DTH responses and seemed to increase the proportion of children who were unresponsive to three antigens (tuberculin [purified protein derivative], Candida, and Proteus) (116). Children in this study had low mean retinol concentrations at baseline. In light of the results from several in vitro experiments and animal studies, it has been proposed that vitamin A deficiency induces a shift in the immune response towards Th1-cell-mediated activity whereas vitamin A supplementation would tend to boost Th2-type responses, as recently reviewed by Stephensen (145). Results from trials that examined the effect of vitamin A on clinical outcomes from infections that elicit either a Th1 or a Th2 response suggest that the immunological mechanisms through which vitamin A exerts an effect are pathogen specific and may involve aspects other than the Th1/Th2 balance. Future studies are warranted to assess the role of vitamin A supplementation in humans on differential Th1/Th2 responses according to the baseline vitamin A status of the population and the specific pathogens causing infection. In summary, there is no conclusive evidence to date for a direct effect of vitamin A supplementation on cytokine production or lymphocyte activation. One of the reasons why the results vary widely across studies is that the potential effect of vitamin A on T-cell function may depend on the specific immune response that each particular pathogen elicits. Also, vitamin A could have transient effects on intermediary markers of T-cell-dependent immunity that may be missed by few and relatively random assessments in population studies. B cells. There is very little evidence from randomized clinical trials regarding the potential effect of vitamin A supplementation on the proliferation or activation of B lymphocytes. An indirect measure of the potential effects of vitamin A supplementation on B-cell function is the production of antibodies; however, this effect would most likely represent an influence of vitamin A on antigen-presenting cells, as suggested in experiments (33, 63) and human studies reviewed here. T-Cell-Dependent Humoral Responses The synthesis of antibodies to T-cell-dependent antigens is typically depressed during vitamin A deficiency, as shown in several animal models (58, 100, 140, 158). The evidence for an association between vitamin A status and T-cell-dependent antibody response is reviewed below. Tetanus and diphtheria. In two small observational studies, no relationship was found between low retinol concentrations (⬍0.7 ␮mol/liter) at the time of immunization with diphtheria and tetanus toxoids and the antibody response after 2 (78) or 4 (16) weeks, respectively. In a vitamin A trial from Indonesia, among children who received a placebo the anti-tetanus toxoid response of xerophthalmic children was the same as that of those who were nonxerophthalmic (131); in that study, both xerophthalmic and nonxerophthalmic children had low serum

458

VILLAMOR AND FAWZI

retinol concentrations. Low retinol concentrations have been widely used as an indicator of vitamin A deficiency; however, they may also be the result of the acute-phase response during generalized inflammatory states. The effect of vitamin A on the antibody response to tetanus or diphtheria has been examined in five clinical trials (16, 20, 79, 108, 131), four of which were randomized (20, 79, 108, 131) (Table 3) and three of which (79, 108, 131) used a placebo group as control. In the first trial, conducted among Bangladeshi children 1 to 6 years of age, the intramuscular administration of 200,000 IU vitamin A at the time of the first tetanus immunization was not associated with the vaccine response after 4 or 12 weeks (a second dose of the toxoid had been administered 4 weeks after the first) (20). The intervention was not placebo controlled, and the IgG detection assay seemed to have low sensitivity. In an apparently nonrandomized trial, the oral administration of 100,000 versus 200,000 IU vitamin A to 50 Indian children 1 to 6 years of age at the time of diphtheria and tetanus immunization did not result in significantly different antibody responses to either toxoid after 4 weeks (16). Although no formal comparisons with the response of children who did not receive any vitamin A were made, it seems from the data presented that there was not a significant vitamin effect. In the Indonesian study (131), the oral administration of 200,000 IU vitamin A to tetanus-naive children 3 to 6 years of age resulted in significantly higher titers of anti-tetanus toxoid after immunization compared to those in the placebo group, independent of whether the children were xerophthalmic at baseline or not. It was concluded that, since both xerophthalmic and nonxerophthalmic children had low retinol concentrations at baseline and the vitamin was administered 2 weeks before the toxoid, the correction of vitamin A deficiency may be related to improved antibody responses. Vitamin A had no significant effects on the response to anti-tetanus toxoid in two subsequent clinical trials conducted among younger children (⬍2 months) in Bangladesh (108) and Turkey (79). The Turkish trial was a two-by-two factorial design with four arms (vitamin A alone, vitamin E alone, vitamins A and E, or placebo), with limited statistical power to make comparisons between separate arms. There appeared to be a higher antibody response in the two arms that received vitamin A (alone or with vitamin E) than in those without vitamin A (placebo or vitamin E only), but a statistical comparison between the arms regrouped in this manner was not presented. Contradictory results between the Indonesian trial and others have been attributed to differences in the underlying prevalence of vitamin A deficiency; however, this would not explain the contrast with the Bangladesh studies (16, 108), in which the concentrations of serum retinol at baseline were similarly low. One alternative explanation is that in the Turkish (79) and the latest Bangladeshi (108) trials subjects were young infants in whom the antibody response to tetanus may have been affected by passive immunity, whereas in Indonesia, children in whom the vitamin effect was observed were older and naive to tetanus. The mean preimmunization concentration of tetanus-specific IgG in the latter study was between 6.0 and 15.1 ␮g/liter, much lower than that in Bangladesh (between 6.1 and 7.5 mg/liter). It is also possible that the administration of vitamin A some time before the immunization, such as in the Indonesian study, allows for a partial correction of deficiency that may be necessary for an

CLIN. MICROBIOL. REV.

enhanced response to be observed. In the most recent trial from Bangladesh (108), vitamin A had a positive, significant effect on the antibody response to diphtheria toxoid antigen; this effect has not been examined in other randomized trials. Measles. The effect of vitamin A on the antibody response to measles infection has been studied in a number of clinical trials. Studies in South Africa (28) and Zambia (116) examined the antibody response of children who had been hospitalized with severe measles. In the South African study, the IgG titers to measles virus at day 8 were significantly higher among children who received vitamin A than among those who received a placebo, while no difference was observed at day 42. In the Zambian study, vitamin A had no effect on measles virus antibodies at day 14. The higher dose used in South Africa (twice that in the Zambian trial before the assessment of antibodies) could explain the differences in results. Other trials assessed the effect of vitamin A supplementation at the time of immunizations on the antibody response. An apparent adverse effect of vitamin A was suggested by a study in Indonesia in which infants who received the supplement at age 6 months together with the Schwarz measles vaccine had a lower seroconversion rate at age 12 months (133). This effect was limited to infants with high antibody titers at baseline, and it was suggested that vitamin A-related enhancement of the immune function together with circulating maternal antibodies could neutralize the vaccine before it induced protective immunity. However, a similar study in Guinea-Bissau did not show a significant effect of vitamin A on measles titers after 3 months (14); it was not possible to compare the effect by levels of maternal antibody concentrations at age 6 months with those in the Indonesian study due to the use of different cutoff points to define baseline titers. It was suggested that the differential effect observed could have been attributed to a higher prevalence of vitamin A deficiency in Indonesia than in Guinea-Bissau (14). In the Guinea-Bissau study, an additional dose of vitamin A administered during a second immunization round at 9 months did not affect seroconversion at age 18 months (10) or the proportion of children with protective titers at age 6 to 8 years (11). Several other studies have evaluated the impact of vitamin A supplementation simultaneous with measles vaccination at age 9 months only. In a study from Guinea Bissau, the proportion of children who seroconverted to measles virus at age 18 months was not significantly different by treatment arm; however, the geometric mean titer was significantly higher for children given vitamin A supplementation than for those who received a placebo (10) and was particularly higher among boys. In the same study, the proportion of children with protective titers at age 6 to 8 years was significantly higher in those who had received vitamin A together with their measles vaccine at age 9 months (11). In a trial from India among children who received the Edmonston Zagreb strain of measles vaccine at age 9 months, vitamin A resulted in higher seroconversion rates after 1 month (17); however, in two other studies in India (6, 23) and in a trial from Indonesia (124), no effects were observed. Polio. Vitamin A supplementation has also been studied in relation to poliovirus vaccine seroconversion in Bangladesh, India, and Indonesia. Trials in which vitamin A supplements were administered to infants at each time of oral polio vaccine

VOL. 18, 2005

vaccination in Bangladesh (107) and Indonesia (130) or to Indian mothers in the early postpartum period (15) showed no effect on titers to any of the three poliovirus types. In the Bangladeshi and Indian trials, seroconversion rates were measured within 2 months of the exposure to vitamin A, whereas in Indonesia the outcomes were assessed about 6 months thereafter. In the latter study, late assessment of seroconversion could have decreased statistical power to detect an effect of treatment, since the rates were close to 100% overall. In a recent trial in India in which the vitamin was administered to both mothers and children, the proportion of children with protective titers against type 1 poliovirus was significantly higher in the experimental group than in the placebo group (6). Influenza. One study among HIV-infected children found no effect of supplementation on the antibody response to inactivated influenza vaccine (57); further studies with the larger population of non-HIV-infected children are necessary. In summary, vitamin A may have the potential to increase the antibody response to tetanus toxoid when administered some time before immunization, particularly in children with vitamin A deficiency who have not been exposed to tetanus. Whether a similar effect exists in response to the diphtheria toxoid needs further examination. Vitamin A administered at 9 months of age does not decrease the antibody response to measles virus, but when administered at 6 months together with a dose of measles vaccine that is not to be repeated at 9 months, supplementation could potentially decrease the antibody response. More studies on the effect of vitamin A on the antibody responses to other vaccines are needed. T-Cell-Independent Humoral Responses Some animal studies have suggested a role of vitamin A in the antibody response to T-cell-independent antigens such as pneumococcal polysaccharide (100), but a causal link cannot be established with certainty since interventions other than vitamin A repletion may trigger normal antibody responses in animals (99). It would be relevant to examine whether vitamin A supplementation in humans could enhance the response to this and other T-cell-independent antigens from encapsulated bacteria, including meningococcus and Haemophilus. VITAMIN A SUPPLEMENTATION AND CLINICAL OUTCOMES A major motivation to study the effects of vitamin A on the immune function is the search for mechanistic explanations of the impact of supplementation on mortality and morbidity among children and pregnant women that has been documented in clinical trials (25, 138, 150, 154). Although this review is focused primarily on the effect of vitamin A on immunological parameters, we consider it relevant to briefly correlate the effects on immunity with those on clinical outcomes reported to date. Vitamin A supplementation after 6 months of age is associated with a reduction in all-cause child mortality of about 23 to 30% (9, 38). Supplementation at birth appeared to decrease mortality in two trials from Indonesia and India (64, 111) but not in a trial from Zimbabwe (86). Supplementation at between 1 and 5 months does not seem to have a beneficial effect

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(32, 109, 155, 157). The specific immunological mechanisms behind these contrasting effects are not fully understood, and it is likely that they depend on the underlying nutritional and immunological status and the nature of infectious agents causing disease in children. Recently, Benn and colleagues hypothesized that the reason why vitamin A decreases mortality when given to newborns and children at 6 months but not in the 1- to 5-month age bracket is that vitamin A amplifies the nonspecific immune modulation induced by live vaccines (BCG and measles), which are routinely administered at birth and 6 months (12). Additional data to confirm this hypothesis are needed. The effects of vitamin A supplementation on child morbidity include a reduction in the severity of measles that could be correlated with the enhanced T-cell-dependent antibody production that was observed in the South African study (27, 28). A decrease in the severity of measles morbidity could explain an overall average reduction in measles-specific mortality of about 60% (38) as shown in trials from England (37), South Africa (27, 67), and Tanzania (8). The benefits of vitamin A on measles-related outcomes may go beyond the correction of underlying deficiencies and could actually represent adjuvant therapeutic effects (117). Vitamin A also appears to decrease the severity of some diarrheal episodes in childhood and their incidence when administered in combination with zinc (110). This outcome could be the consequence of the improvement in gut integrity during severe diarrheal episodes that was documented in South Africa (49) and India (147). Preliminary results from a trial in Mexico indicate that bimonthly vitamin A supplementation reduces the incidence of Giardia lamblia infections and diarrheal episodes associated with Ascaris lumbricoides (83); the mechanisms are still unknown but could be related to enhanced Th2 immune reactions, which are important in the line of defense against some parasitic infections. A few studies on the effect of vitamin A supplementation on outcomes related to malaria infection have been conducted. A trial in Ghanaian children found no effect on death from malaria, fever episodes, malaria parasitemia, or probable malaria illness (18); however, statistical power in this study may have been limited. In Papua New Guinea, a trial was conducted to specifically examine the effect of vitamin A supplementation on malaria outcomes. In this trial, supplementation with 200,000 IU at 3-month intervals resulted in a significant 30% reduction in clinical Plasmodium falciparum episodes, particularly in children age 12 to 36 months (138). Vitamin A also decreased parasite density and spleen enlargement. In a study of preschool children in Tanzania, vitamin A supplementation every 4 months resulted in a decreased risk of death from malaria (42) and improved weight gain among children who had malaria at baseline (151) but was not associated with the incidence of malaria parasitemia during a 4- to 8-month follow-up period (149). A trial in Mozambique among children admitted to hospital with severe malaria found a non-statistically significant benefit of a single vitamin A dose on hospital death. There were no significant effects on duration of hospital stay, time to resolution of fever, clearance of parasitemia, or development of neurological sequelae (148). A marginally significant reduction of active placental malaria infection at delivery was reported in relation to vitamin A supplementation during pregnancy in a study from Ghana (31). It has been

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proposed that the beneficial effects of vitamin A supplementation on malaria could be due to increased phagocytosis of nonopsonized erythrocytes mediated through up-regulation of CD36 cytoadherence receptors and decreased secretion of TNF-␣ through down-regulation of the peroxisome proliferator activated receptor ␥-retinoic X receptor (137). The effect of vitamin A on respiratory infections has been examined in several randomized clinical trials, with varying results. Most hospital-based studies on vitamin A supplementation and the severity of pneumonia have not shown significant overall effects (36, 41, 74, 97, 139), as recently reviewed by Brown and Roberts (21). In fact, some of the hospital-based studies suggest an apparent increase in indicators of severity associated with vitamin A supplementation (41, 85, 105, 146). A number of community-based trials have also found an apparent increase in respiratory symptoms in relation to vitamin A supplementation (34, 39, 103, 104, 112, 136, 144), particularly among children who are not undernourished. It is not clear whether this apparent increase in respiratory symptoms may be due to a proinflammatory immune response associated with the supplements. The effect of vitamin A supplementation on tuberculosis outcomes was studied in a clinical trial among hospitalized children who received 200,000 IU vitamin A during two consecutive days or a placebo (56). No effects were found on radiological or other outcomes after 3 months. In a small study of adults in Indonesia, 5,000 IU vitamin A together with 15 mg zinc daily for 6 months resulted in faster sputum conversion and resolution of the X-ray lesion area (72), but it is not possible to attribute the effect to vitamin A alone. Potential effects of vitamin A on tuberculosis could be correlated with the results of trials on DTH responses, but stronger evidence for both clinical and immunological outcomes is lacking. Vitamin A deficiency is common among HIV-infected persons and appeared to be a predictor of mortality in observational studies (126, 127). A number of clinical trials have examined the effect of vitamin A supplementation on health and survival outcomes in the course of HIV infection. Among HIVinfected children, vitamin A decreases mortality (42, 134) and morbidity from diarrheal disease (26), improves growth (151), and reduces viral load (57); however, among HIV-infected adults, no beneficial effects on mortality, disease progression (45), or viral load (5, 29, 65, 129) have been observed, and only a modest effect on CD4 cell counts has been noted, as reviewed above (5). The effects on child mortality, morbidity from diarrheal disease, and growth could be related in part to the vitamin’s action in maintaining the integrity of the intestinal mucosa (49, 147). Positive effects through the enhancement of cellular immunity or antibody production are possible but are not yet consistently supported by results from randomized clinical trials in humans. When administered daily during pregnancy and lactation, combined vitamin A and ␤-carotene could increase the risk of mother-to-child transmission (MTCT) of HIV as shown in one study in Tanzania (43). In a smaller study from South Africa, daily administration of vitamin A and ␤-carotene during pregnancy and at delivery was not significantly related to early MTCT, although the 95% confidence interval included the possibility of a harmful effect (30). In a third trial of daily supplementation during pregnancy in Malawi (77), vitamin A

CLIN. MICROBIOL. REV.

alone had no effect on MTCT. One potential mechanism to explain the adverse effect on MTCT is that vitamin A increases viral shedding in genital secretions, as shown in the Tanzanian trial in which supplementation resulted in greater MTCT (40); in a study of nonpregnant women, there was no effect on genital shedding (5). In one recent study, vitamin A had no effect on genital shedding of herpes simplex virus (4). An alternative mechanism is that retinol may increase the expression of CCR5 receptors in monocytes/macrophages, which would increase the susceptibility of cells to M-type HIV infection (84). The suggestion that daily vitamin A supplementation to HIV-infected mothers increases MTCT (43) and the recent finding that vitamin A reduces the benefits of multivitamins (B, C, and E) on HIV-related outcomes (45) cast some doubts on the safety of providing vitamin A/␤-carotene to HIV-infected adults. CONCLUSIONS Vitamin A may have the potential to increase the antibody response to tetanus toxoid when administered some time before immunization; this effect seems to be limited to children with vitamin A deficiency who have not been exposed to tetanus. A similar positive effect in response to the diphtheria toxoid might exist and needs to be confirmed. When administered with the measles vaccine at age 9 months, vitamin A supplementation does not decrease the antibody response and may actually increase it among boys or in children with low weight. However, when administered at 6 months together with a dose of measles vaccine that is not to be repeated at 9 months, vitamin A could potentially decrease the antibody response. Vitamin A supplementation to the child or the mother alone does not appear to affect the antibody response to oral polio vaccine; confirmation of a potential beneficial effect when administered to both the mother and the child is needed. The effect of vitamin A on the immune response to other vaccines, including BCG, polysaccharides from encapsulated bacteria, and hepatitis virus, has not been studied in randomized clinical trials. In addition to the impact of vitamin A supplementation on antibody production against selected antigens, known effects on cellular immunity are apparent on T-cell lymphopoiesis or lymphocyte differentiation among vitamin A-deficient or HIVinfected children. Effects on cytokine production have not been well documented in clinical trials, and the evidence on lymphocyte activation as measured by the DTH response is mixed. There is limited research on the effects of vitamin A supplementation to adults and the elderly on their immune function; currently available data provide no consistent evidence for beneficial effects. Additional studies with these age groups are needed. Periodic vitamin A supplementation to children ⱖ6 months is a useful public health strategy to improve child survival and to decrease the risk of nutritional blindness and of morbidity of infectious origin from measles, severe diarrhea, HIV, and possibly malaria and intestinal helminthiases. The beneficial effects of vitamin A supplementation among children with severe measles could be mediated by a short-term increase in antibody production, possibly as a result of increased lymphocyte

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proliferation. The effect on severe diarrhea is likely due to the role of vitamin A in restoring and maintaining gut mucosal integrity. The apparent benefit for survival and growth among HIV-infected children could also be related to the latter, through decreased nutrient losses and improved nutritional status. Other immunological pathways through which vitamin A could exert an effect against severe diarrhea are likely to depend on the causative microorganisms and warrant investigation in future studies. Specific mechanisms for the potential effect of vitamin A on malaria and other parasitic infections are yet to be examined in randomized clinical trials. Similarly, explanatory mechanisms for the apparent harmful impact of vitamin A on respiratory infection among nonundernourished children, MTCT of HIV, and T-cell counts in the elderly deserve further assessment. Data are needed from human trials about the role of vitamin A supplementation in modulating the Th1/Th2 response as a potential explanatory mechanism for the vitamin’s observed effects on clinical outcomes. ACKNOWLEDGMENT This work was supported by the National Institute of Child Health and Human Development (grant 1R01HD045134-01A1). REFERENCES 1. Abbas, A. K., and A. H. Lichtman. 2003. Cellular and molecular immunology. Saunders, Philadelphia, Pa. 2. Arena, A., A. B. Capozza, D. Delfino, and D. Iannello. 1997. Production of TNF alpha and interleukin 6 by differentiated U937 cells infected with Leishmania major. New Microbiol. 20:233–240. 3. Aukrust, P., F. Muller, T. Ueland, A. M. Svardal, R. K. Berge, and S. S. Froland. 2000. Decreased vitamin A levels in common variable immunodeficiency: vitamin A supplementation in vivo enhances immunoglobulin production and downregulates inflammatory responses. Eur. J. Clin. Investig. 30:252–259. 4. Baeten, J. M., R. S. McClelland, L. Corey, J. Overbaugh, L. Lavreys, B. A. Richardson, A. Wald, K. Mandaliya, J. J. Bwayo, and J. K. Kreiss. 2004. Vitamin A supplementation and genital shedding of herpes simplex virus among HIV-1-infected women: a randomized clinical trial. J. Infect. Dis. 189:1466–1471. 5. Baeten, J. M., R. S. McClelland, J. Overbaugh, B. A. Richardson, S. Emery, L. Lavreys, K. Mandaliya, D. D. Bankson, J. O. Ndinya-Achola, J. J. Bwayo, and J. K. Kreiss. 2002. Vitamin A supplementation and human immunodeficiency virus type 1 shedding in women: results of a randomized clinical trial. J. Infect. Dis. 185:1187–1191. 6. Bahl, R., N. Bhandari, S. Kant, K. Molbak, E. Ostergaard, and M. K. Bhan. 2002. Effect of vitamin A administered at Expanded Program on Immunization contacts on antibody response to oral polio vaccine. Eur. J. Clin. Nutr. 56:321–325. 7. Bahl, R., R. Kumar, N. Bhandari, S. Kant, R. Srivastava, and M. K. Bhan. 1999. Vitamin A administered with measles vaccine to nine-month-old infants does not reduce vaccine immunogenicity. J. Nutr. 129:1569–1573. 8. Barclay, A., A. Foster, and A. Sommer. 1987. Vitamin A supplements and mortality related to measles: a randomised clinical trial. Br. Med. J. 294: 294–296. 9. Beaton, G. H., R. Martorell, and K. J. Aronson. 1993. Effectiveness of vitamin A supplementation in the control of young child morbidity and mortality in developing countries. ACC/SCN State-of-the-Art Series policy discussion paper no. 13. World Health Organization, Geneva, Switzerland. 10. Benn, C. S., P. Aaby, C. Bale, J. Olsen, K. F. Michaelsen, E. George, and H. Whittle. 1997. Randomised trial of effect of vitamin A supplementation on antibody response to measles vaccine in Guinea-Bissau, West Africa. Lancet 350:101–105. 11. Benn, C. S., A. Balde, E. George, M. Kidd, H. Whittle, I. M. Lisse, and P. Aaby. 2002. Effect of vitamin A supplementation on measles-specific antibody levels in Guinea-Bissau. Lancet 359:1313–1314. 12. Benn, C. S., C. Bale, H. Sommerfelt, H. Friis, and P. Aaby. 2003. Hypothesis: vitamin A supplementation and childhood mortality: amplification of the non-specific effects of vaccines? Int. J. Epidemiol. 32:822–828. 13. Benn, C. S., I. M. Lisse, C. Bale, K. F. Michaelsen, J. Olsen, K. Hedegaard, and P. Aaby. 2000. No strong long-term effect of vitamin A supplementation in infancy on CD4 and CD8 T-cell subsets. A community study from Guinea-Bissau, West Africa. Ann. Trop. Paediatr. 20:259–264. 14. Benn, C. S., H. Whittle, P. Aaby, C. Bale, K. F. Michaelsen, and J. Olsen. 1995. Vitamin A and measles vaccination. Lancet 346:503–504.

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man, and J. O. Alvarez. 1998. Adverse effects of high-dose vitamin A supplements in children hospitalized with pneumonia. Pediatrics 101:E3. Thurnham, D. I., C. A. Northrop-Clewes, F. S. McCullough, B. S. Das, and P. G. Lunn. 2000. Innate immunity, gut integrity, and vitamin A in Gambian and Indian infants. J. Infect. Dis. 182:S23–S28. Varandas, L., M. Julien, A. Gomes, P. Rodrigues, W. Van Lerberghe, F. Malveiro, P. Aguiar, P. Kolsteren, P. Van Der Stuyft, K. Hilderbrand, D. Labadarios, and P. Ferrinho. 2001. A randomised, double-blind, placebocontrolled clinical trial of vitamin A in severe malaria in hospitalised Mozambican children. Ann. Trop. Paediatr. 21:211–222. Villamor, E., M. R. Fataki, R. L. Mbise, and W. W. Fawzi. 2003. Malaria parasitaemia in relation to HIV status and vitamin A supplementation among pre-school children. Trop. Med. Int. Health 8:1051–1061. Villamor, E., and W. Fawzi. 2000. Vitamin A supplementation: implications for morbidity and mortality in children. J. Infect. Dis. 182:S122–S133. Villamor, E., R. Mbise, D. Spiegelman, E. Hertzmark, M. Fataki, K. Peterson, G. Ndossi, and W. Fawzi. 2002. Vitamin A supplements ameliorate the adverse effect of HIV-1, malaria, and diarrheal infections on child growth. Pediatrics 109:E6. Warden, R. A., M. J. Strazzari, P. R. Dunkley, and E. V. O’Loughlin. 1996. Vitamin A-deficient rats have only mild changes in jejunal structure and function. J. Nutr. 126:1817–1826. West, K. P., Jr. 2002. Extent of vitamin A deficiency among preschool children and women of reproductive age. J. Nutr. 132:2857S–2866S. West, K. P., Jr., J. Katz, S. K. Khatry, S. C. LeClerq, E. K. Pradhan, S. R. Shrestha, P. B. Connor, S. M. Dali, P. Christian, R. P. Pokhrel, A. Sommer, et al. 1999. Double blind, cluster randomised trial of low dose supplementation with vitamin A or beta carotene on mortality related to pregnancy in Nepal. Br. Med. J. 318:570–575. West, K. P., Jr., J. Katz, S. R. Shrestha, S. C. LeClerq, S. K. Khatry, E. K. Pradhan, R. Adhikari, L. S. Wu, R. P. Pokhrel, and A. Sommer. 1995. Mortality of infants ⬍ 6 mo of age supplemented with vitamin A: a randomized, double-masked trial in Nepal. Am. J. Clin. Nutr. 62:143–148. Whitcher, J. P., M. Srinivasan, and M. P. Upadhyay. 2001. Corneal blindness: a global perspective. Bull. W. H. O. 79:214–221. W.H.O./CHD Immunization-Linked Vitamin A Supplementation Study Group. 1998. Randomized trial to assess benefits and safety of vitamin A supplementation linked to immunization in early infancy. Lancet 352:1257– 1263. Wiedermann, U., L. A. Hanson, H. Kahu, and U. I. Dahlgren. 1993. Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats. Immunology 80:581–586. Wieringa, F. T., M. A. Dijkhuizen, C. E. West, J. van der Ven-Jongekrijg, and J. W. van der Meer. 2004. Reduced production of immunoregulatory cytokines in vitamin A- and zinc-deficient Indonesian infants. Eur. J. Clin. Nutr. 58:1498–1504. Wong, Y. C., and R. C. Buck. 1971. An electron microscopic study of metaplasia of the rat tracheal epithelium in vitamin A deficiency. Lab. Investig. 24:55–66. Zhao, Z., D. M. Murasko, and A. C. Ross. 1994. The role of vitamin A in natural killer cell cytotoxicity, number and activation in the rat. Nat. Immun. 13:29–41. Zhao, Z., and A. C. Ross. 1995. Retinoic acid repletion restores the number of leukocytes and their subsets and stimulates natural cytotoxicity in vitamin A-deficient rats. J. Nutr. 125:2064–2073.

CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 465–483 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.465–483.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 18, No. 3

Enterotoxigenic Escherichia coli in Developing Countries: Epidemiology, Microbiology, Clinical Features, Treatment, and Prevention Firdausi Qadri,1 Ann-Mari Svennerholm,2 A. S. G. Faruque,1 and R. Bradley Sack3* International Centre for Diarrhoeal Disease Research, Bangladesh, and Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh1; Department of Medical Microbiology and Immunology, Go ¨teborg University, 40530 Go ¨teborg, Sweden2; and Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland3 INTRODUCTION .......................................................................................................................................................465 HISTORICAL ASPECTS ...........................................................................................................................................466 From Discovery to Present Understanding of Public Health Importance......................................................466 BIOLOGY ....................................................................................................................................................................466 LT and ST Enterotoxins ........................................................................................................................................466 Colonization Factors ..............................................................................................................................................467 ETEC Serotypes ......................................................................................................................................................468 ETEC Strains in Animals ......................................................................................................................................469 EPIDEMIOLOGY .......................................................................................................................................................469 Age-Related Infections in Children and Adults..................................................................................................469 Relation to Presence of LT, ST, and Colonization Factors ..............................................................................470 Single Versus Mixed Infections ............................................................................................................................471 Seasonality of ETEC...............................................................................................................................................471 Comparison of ETEC Diarrhea and Cholera in Children and Adults ...........................................................471 Presence of ETEC in Food and Water in the Environment .............................................................................472 ETEC Infections and Malnutrition ......................................................................................................................473 Infections in International Travelers ...................................................................................................................473 CLINICAL FEATURES .............................................................................................................................................474 Disease Severity.......................................................................................................................................................474 Mortality from ETEC Diarrhea............................................................................................................................474 DIAGNOSIS ................................................................................................................................................................474 Laboratory Assays...................................................................................................................................................474 TREATMENT AND MANAGEMENT .....................................................................................................................475 Rehydration .............................................................................................................................................................475 Antimicrobials .........................................................................................................................................................475 Multidrug Resistance Patterns .............................................................................................................................476 Nutritional and Micronutrient Therapy ..............................................................................................................477 PREVENTION.............................................................................................................................................................477 Vaccine Development..............................................................................................................................................477 Purified CFs and Enterotoxoids ...........................................................................................................................477 Inactivated Whole-Cell Vaccines...........................................................................................................................478 Live Oral ETEC Vaccines......................................................................................................................................478 CONCLUSIONS .........................................................................................................................................................478 ACKNOWLEDGMENTS ...........................................................................................................................................479 REFERENCES ............................................................................................................................................................479 for the estimated 1.5 million deaths per year are enterotoxigenic Escherichia coli (ETEC), rotavirus, Vibrio cholerae, and Shigella spp. (88, 96); all are known to be endemic in essentially all developing countries. Whereas V. cholerae, Shigella spp., and rotavirus can be readily detected by standard assays, ETEC is more difficult to recognize and therefore is often not appreciated as being a major cause of either infantile diarrhea or of cholera-like disease in all age groups. Since ETEC is a major cause of traveler’s diarrhea in persons who travel to these areas, the organism is regularly imported to the developed world (18, 58, 75).

INTRODUCTION Acute infectious diarrhea is the second most common cause of death in children living in developing countries, surpassed only by acute respiratory diseases accounting for approximately 20% of all childhood deaths (96). The major etiologic agents that account * Corresponding author. Mailing address: Department of International Health, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Room W5035, Baltimore, MD 21205. Phone: (410) 955-2719. Fax: (410) 502-6733. E-mail: [email protected] 465

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TABLE 1. Enterotoxin profiles and presence of colonization factors in enterotoxigenic E. coli strains isolated from symptomatic children in different regions of the worlda Parameter

Bangladesh

Mexico

Prevalence of ETEC (% of subjects) Toxin profile (% of subjects) ST LT/ST LT Prevalence of CF (%) No. of subjects tested

18

33

50 25 25 56b 435a, 662b

19 57 41 21 75c

Peru

7.4 31 13 56 NT 377d

Egypt

Argentina

India

Nicaragua

20

18.3

12–24

38

32 27 41 76 1,220h

32 49 19 61 235i

53 34 13 24 242e, 211f

40 7 53 55 145g

a Data are from Bangladesh (24[a], 132[b]), Mexico (38[c]), Peru (19[d]), Egypt (1[e], 139[f]), Argentina (207[g]), India (180[h]), and Nicaragua (126[i]). NT, not tested.

Among the six recognized diarrheagenic categories of Escherichia coli (118), ETEC is the most common, particularly in the developing world (214). Specific virulence factors such as enterotoxins and colonization factors differentiate ETEC from other categories of diarrheagenic E. coli. ETEC belongs to a heterogeneous family of lactose-fermenting E. coli, belonging to a wide variety of O antigenic types, which produce enterotoxins, which may be heat labile and/or heat stable, and colonization factors which allow the organisms to readily colonize the small intestine and thus cause diarrhea (118, 155, 211). This review summarizes data on the recognition and importance of ETEC diarrhea in developing countries, emphasizing on its prevalence, toxin types, colonization factors, and morbidity in different population groups at risk. We have reviewed information on ETEC since its discovery almost 50 years ago (43) and used clinical and laboratory data from hospital and community-based studies around the world, in both urban and rural settings, to present a comprehensive picture of ETECmediated diarrheal disease with regard to epidemiology, diagnosis, treatment, and prevention through the use of vaccines. We hope that this review may increase the knowledge and awareness of the importance of ETEC infections, particularly in the developing world. HISTORICAL ASPECTS From Discovery to Present Understanding of Public Health Importance E. coli was first suspected as being a cause of children’s diarrhea in the 1940s, when nursery epidemics of severe diarrhea were found to be associated with particular serotypes of E. coli (27). These specific serotypes, designated enteropathogenic E. coli, were epidemiologically incriminated as the cause of the outbreaks. Studies of rabbit ileal loops with these strains in 1961 (199) showed only a poor correlation of fluid accumulation with the incriminated E. coli serotypes and were not definitive. Later, however, in volunteer experiments it was confirmed that ingestion of large numbers of these organisms resulted in diarrhea, and the ingested strains were recovered in the feces (98). Extensive work has been done with this group of enteropathogenic E. coli and has recently been reviewed (118). The history of enterotoxigenic E. coli begins in 1956 in Calcutta (43). De and his colleagues injected live strains of E. coli, isolated from children and adults with a cholera-like illness, into isolated ileal loops of rabbits and found that large

amounts of fluid accumulated in the loops, similar to that seen with Vibrio cholerae. However, they did not test the filtrates of these cultures to determine whether they produced an enterotoxin. These findings were not followed up until 1968, when Sack reported studies, also in Calcutta, of adults and children with a cholera-like illness, who had almost pure growth of E. coli in both stool and the small intestine (154). These E. coli isolates were found to produce a strong cholera-like secretory response in rabbit ileal loops, both as live cultures and as culture filtrates (74). The patients were also found to have antitoxin responses to the heat-labile enterotoxin produced by these organisms (163). At about the same time, similar studies were being done with animals that also demonstrated strains of E. coli to be responsible for diarrheal disease in several animal species: pigs, calves, and rabbits (80, 177, 178). Studies of these animal enterotoxigenic E. coli paralleled and sometimes preceded those done with human strains; these organisms were also found to produce enterotoxins and specific colonization factors. These findings from Calcutta were soon confirmed by oral challenge of human volunteers (49, 100) and by corroboration of studies in Dhaka, Bangladesh (61, 113, 117, 149). ETEC were shown in these studies to be most frequently found in children; such findings have been subsequently corroborated in multiple studies in developing countries (23, 24). Thus, in most studies in the developing world, ETEC have been shown to be the most common bacterial enteric pathogen, accounting for approximately 20% of cases, as shown in Table 1, which summarizes findings from some of the more detailed studies done in several different countries. BIOLOGY LT and ST Enterotoxins Following the initial discovery of ETEC in humans, there was an intensive effort to further characterize its mechanisms of pathogenesis and means of laboratory identification. Over a period of several years, its major virulence mechanisms were identified. ETEC produce one or both of two enterotoxins, heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), that have been fully characterized, cloned, and sequenced, and their genetic control in transmissible plasmids was identified (70, 138, 179). ETEC also produces one more of many defined colonization factors (pili/fimbrial or nonfimbrial), also under plasmid control (65, 66). The full sequence of laboratory events leading to the better understanding of virulence factors

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TABLE 2. Examples of case-control studies of symptomatic versus asymptomatic children infected with ETEC of different toxin types Study area

Yr

Method useda

Sao Paulo, Brazil

1982

A

Ethiopia

1982

A

New Caledonia

1993

B

Arizona

1995

A

Bangladesh

1999

B

Argentinab

Egyptb

a b

1999

1999

C

C

No. of subjects (% positive) P

Toxin type

N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC N LT only LT/ST ST only All ETEC

cases

cases

cases

cases

cases

isolates

isolates

Patients

Controls

245 3.3% 4.9% 4.9% 13% 86 11% 11% 4% 23% 488 1% 0.4% 3.4% 5.36% 1042 3.1% 1.3% 3.2% 7.5% 814 4.6% 3.9% 8.4% 16.8% 371 9.7% 1.3% 7.3% 18.5% 628 10.7% 2.6% 14.5% 30.1%

96 11.4% 0 0 11% 60 3% 7% 0 10% 88 0 0 0 0 1059 1.1% 0.2% 0.7% 3.2% 814 5% 1.6% 2.2% 8.8% 595 9.8% 1.2% 1.7% 13.3% 1027 8.5% 3.9% 6.9% 19.4%

⬍.01 0.0175 0.0175 ns ⬍0.05 ns ns ⬍0.05

Reference

143

184

13 ns ns ns 0.031 ⬍0.05 ⬍0.05 ⬍0.001 ⬍0.001

167

7 0.64 0.004 0.0001 0.0001 207 ns ns ⬍0.001 ⬍0.05 1 ns ns ⬍0.01 ⬍0.01

The assays used for detection of enterotoxins on ETEC were adrenal cell and infant mouse (A), DNA probes (B), and GM-1 ELISA (C). Based on number of specimens analyzed; the rest are based on number of subjects tested.

in ETEC has been reviewed in a number of publications (66, 157, 116, 118, 158, 211) and will not be elucidated further in this article. Only a brief summary of the actions of these two enterotoxins will be given. LT was found to be very similar physiologically, structurally, and antigenically to cholera toxin and to have a similar mode of action. The molecular mass (84 kDa) and the subunit structure of the two toxins were essentially identical, with an active (A) subunit surrounded by five identical binding (B) subunits (70, 83). Following colonization of the small intestine by ETEC and release of the LT, the LTB subunits bind irreversibly to GM1 ganglioside, and the A subunit activates adenylate cyclase, which results in increases in cyclic AMP, which stimulates chloride secretion in the crypt cells and inhibits neutral sodium chloride in the villus tips. When these actions exceed the absorptive capacity of the bowel, purging of watery diarrhea results (70). ST is a nonantigenic low-molecular-weight peptide, consisting of 18 to 19 amino acids. There are two variants, STp and STh, named from their initial discovery from pigs and humans, respectively, and which have identical mechanisms of action. Released in the small intestine, ST binds reversibly to guanylate cyclase, resulting in increased levels of cyclic GMP (138).

ST has also been implicated in the control of cell proliferation via elevation of intracellular calcium levels (174). As with LT, chloride secretion by the crypt cells is then increased and inhibition of neutral sodium chloride absorption occurs, leading to outpouring of diarrheal stool. The relative proportions of LT, ST, and LT/ST toxin-producing ETEC seems to vary from one geographic area to another in patients with ETEC diarrhea or asymptomatic carriers (Tables 1, 2, and 3). The rate of isolation from asymptomatic children has varied between 0% and 20% in numerous studies carried out with children in different settings but has in most instances been lower than the rates in children with diarrhea (7, 8, 19, 38, 79, 88, 143). On average ETEC is seen at least two to three times more frequently in symptomatic than asymptomatic children (Table 2). Colonization Factors More than 22 colonization factors (CFs) have been recognized among human ETEC and many more are about to be characterized (66). The CFs are mainly fimbrial or fibrillar proteins, although some CFs are not fimbrial in structure (60, 61, 66). Notable among these is CS6, an antigen increasingly

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TABLE 3. Toxin profile of ETEC strains isolated from children with diarrhea selected during different years in various developing countries

Country

Bangladesh

Thailand Mexico Nicaragua Argentina Egypt Guinea Bissaua a

Yr

1980 1992 1995 1999 2000 2004 1983 1989 1997 1988 1990 1997 1991 1999 1993 1999 2003 2002

% of strains producing:

No. of strains

88 48 54 137 662 727 35 112 107 31 241 310 109 68 57 107 933 1018

Reference LT

ST

LT and ST

4 58 15 27 27 20 23 39 49 19 34 24 21 53 21 31 26 56

34 38 57 50 48 66 69 36 27 32 39 34 65 40 35 47 58 18

62 4 28 23 25 14 9 25 24 49 27 42 14 7 44 22 16 26

113 11 8 7 132 215 52 55 120 37 103 126 16 207 212 128 139 183

ETEC from children with diarrhea or asymptomatic carriers.

being isolated in recent studies. The CFs allows the organisms to colonize the small bowel, thus allowing expression of either or both LT and ST in close proximity to the intestinal epithelium. Studies with humans as well as experimental animals have shown that CF-positive bacteria but not their isogenic CF-negative mutants colonize and induce diarrhea (60, 66, 100, 195). A nomenclature for the CFs designating them as coli surface antigen (CS) was introduced in the mid-1990s (66). A list showing the old and new classifications of the CFs can be seen in Table 4. All except CFA/I have the CS designation in the present designation. Some of the better-characterized CFs can be subdivided into different families, i.e., the colonization factor I-like group (including CFA/I, CS1, CS2, CS4, CS14, and CS17) (66) and the coli surface 5-like group (with CS5, CS7, CS18, and CS20) (204) and those that are unique (CS3, CS6, and CS10 to CS12). Within each of these families there are cross-reactive epitopes that have been considered as candidates for vaccine development (147). Of the wide range of CFs, the most commonly present on diarrheagenic strains include CFA/1, CS1, CS2, CS 3, CS4, CS5, CS6, CS7, CS14, CS17, and CS21 (66). These have been found on ETEC strains worldwide in various frequencies (Table 5). However, CFs have not been detected on all ETEC, and on roughly 30 to 50% of strains worldwide no known CFs could be detected. This could be due to the absence of CFs, to loss of CF properties on subculture of strains, or to lack of specific tools for their detection. ETEC Serotypes Besides determination of the toxins and CFs, serotyping, i.e., determination of O serogroups associated with the cell wall lipopolysaccharides and H serogroups of the flagella, has been

TABLE 4. Past and present designations for colonization factors of ETECa Nomenclature Old

New

Type of antigen

CFA/1 CS1 CS2 CS3 CS4 CS5 CS6 CS7 CFA/III 2230 PCF0148 PCF0159 PCF09 PCF0166 8786 CS17 PCF020 CS19 CS20 Longus CS22

CFA/I CS1 CS2 CS3 CS4 CS5 CS6 CS7 CS8 CS10 CS11 CS12 CS13 CS14 CS15 CS17 CS18 CS19 CS20 CS21 CS22

F F F f F H nF H F nF f F f F nF F F F F F f

a Abbreviations: F, fimbrial; f, fibrillae; nF, nonfimbrial; H, helical. All except CFA/I have the CS designation (66).

used to identify and characterize ETEC (124). In early studies in, e.g., Bangladesh, it was suggested that typing of the most prevalent serotypes might be used to identify ETEC (112). However, as shown in numerous studies in different countries, clinical ETEC isolates may belong to a large number of serotypes. Furthermore, ETEC serotype profiles may change over time (186). Based on an extensive database analysis of ETEC from a number of different countries all over the world, Wolf reported that in the ETEC antigen the largest variety was the O antigen (211). Thus, 78 different O groups were detected in the 954 ETEC isolates included in the study (hereafter called the ETEC database). In addition, there were several rough strains that lacked side chains, thus being nontypeable with regard to O antigen, or strains that had unknown serogroups. The most common O groups in this retrospective study were O6, O78, O8, O128, and O153; these accounted for over half of the ETEC strains. In a more recent study in Egypt, a large variation of O groups was also recorded, with 47 O groups being represented among the 100 ETEC strains isolated; however, an entirely different O group pattern was recorded than in the database, the most common O groups in the Egyptian study being O159 and O43 (128). Considerably fewer H serogroups than O serogroups are associated with ETEC. Thus, a total of 34 H groups were identified among the 730 ETEC strains included in the ETEC database (211). Five H types accounted for over half of those strains and they were widespread. Similarly, five different H types accounted for almost half of 100 ETEC strains isolated prospectively from Egyptian children, although different H types predominated from those reported for the ETEC database (128).

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TABLE 5. Colonization factor profiles of ETEC isolated from children with diarrhea in different countries % of strains expressing indicated CF Country

n

% CFsa

CFA/II

Bangladesh Egypt India Guatemala Chile Guinea Bissaub a b

109 68 662 10,000 100 933 111 96 93 795

52 55 56 59 23 76 51 47 58

Reference CS7

CS1⫹3

Argentina

CFA/IV

CFA/I

23 10 13 27 3 42 14 10 12 6

CS2⫹3

CS3

CS4⫹6

CS5⫹6

CS17

CS14

Other

7 4 7 3

1 4 6

CS6

11 1 1 0 15 3 0 2 0 0 7 9 5 5 1 6 6 5 11 8 3 6 13 10 4 1 0 2 8 CS1⫹3⬎CS2⫹3⬎CS3 CS4⫹CS6⫽CS5⫹CS6⬍CS6 5 0 15 11 9 1 1 1 8 12 27 24 1 3 2 3 11

3 6 6 2 4 1

NT 9 2 3 17 4 NT

7 3 3

4

16 207 132 215 128 139 180 201 99 183

Indicates % of ETEC with any CF. ETEC from children with diarrhea or asymptomatic carriers.

There are clearly preferred combinations of serotypes, CFs, and toxin profiles in ETEC. For instance, certain H groups are strongly associated with an O serogroup, such as O8:H9, O78: H12, and O25:H42, and some O and H serogroups are associated with one or more CFs (112, 214). In a study of ETEC isolated from children in Argentina, it was shown that most CFs were expressed by strains exhibiting a limited array of serotypes, while the ETEC strains that lacked detectable CFs belonged to many different serotypes (207). However, the significance of these different combinations regarding enhanced virulence (211) or for vaccine development is still unclear. Serotyping appears to give an indication of the variety of strains that are present in a particular ETEC type in a certain geographical area. A close genetic relationship has been found within ETEC strains belonging to a certain serotype, which is different from that noted in other serotypes, and the pattern of genetic relatedness did not change over a period of 15 years (125). The loss of CFs and toxin phenotypes did not affect the genetic relatedness of these strains and their clonal relationship, which suggests that serotype analysis can be coupled to genetic typing for studying the clustering of strains for epidemiological and pathogenetic studies of ETEC. Altogether, the great variation in O and H serogroups in ETEC makes serotyping less suitable for identification of these bacteria and makes O and H antigens less attractive as putative candidate antigens in an ETEC vaccine. ETEC Strains in Animals ETEC is also a major cause of severe diarrheal disease in suckling and weanling animals (66, 115). Animal ETEC strains are known to produce enterotoxins similar to those of human strains and to possess species-specific CFs. The LT from animal strains, designated LT1, is similar to the LT produced by human ETEC; however, another variety designated LTII is only found in animals and is not associated with clinical disease. Animal strains produce two major types of ST, designated STa (STI) and STb (STII). STa, which is a small molecule of ca. 2.0 kDa, was the first of the enterotoxins to be identified in animals (177). As in humans, both STh (STIb) and STp (STIa) may be produced by animal strains. Animal strains (rarely human strains) can also produce STb, a slightly larger

ST (ca. 5 kDa), which does not activate intracellular nucleotide levels and whose mechanism of action is poorly understood (115). Like human ETEC, animal strains also have distinct binding proteins (adhesins and fimbriae), which allow the organisms to attach and colonize the small intestinal mucosa. Indeed, ETEC CFs display a remarkable species specificity, and colonization factors are clearly different from those of human and animal ETEC. Their genetic control may be in plasmids or in the chromosome. The most common of these have been designated K88, K99, and 987P, but there are at least another eight or more, which have other designations. The animal colonization factors are now being identified by F numbers, such as F4, F5, and F6 instead of K88, K99, and 987P (65). Because of the specificity of these adhesins, animal ETEC strains normally do not infect humans. This is in contrast to other diarrheagenic E. coli, such as those that produce Shiga-like toxins, e.g., O157: H7, which are found in animals, mainly cows, and produce severe disease in humans (92) EPIDEMIOLOGY Age-Related Infections in Children and Adults Studies over the last few years have documented that ETEC is usually a frequent cause of diarrhea in infants younger than 2 years of age (132, 139, 183). In Egypt it was found to be the most common cause of diarrhea in the study infants, accounting for about 70% of the first episodes (139). The incidence was higher in males than females. In a detailed investigation in children 0 to 5 years in Bangladesh, 90% of cases of ETEC diarrhea reporting to the hospital were children aged from 3 months to 2 years (132). In an ongoing birth cohort study in Bangladesh, it was found to be the most common cause of diarrhea in children 0 to 2 years of age, accounting for 18% of all diarrheas. The susceptibility of infants and young children has also been observed in other settings which have poor public health and hygiene conditions. The characteristics of the toxin types and CFs present on ETEC strains isolated from young children vary among countries where ETEC is endemic (139, 183, 215). A comparison is shown for Bangladesh and other developing

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TABLE 6. Toxin profiles of ETEC isolated from patients of different age groups in a hospital in Bangladesha No. (%) of subjects aged:

Toxin type

0–23 mo

24–59 mo

5–14 yr

ⱖ15 yr

Total

ST LT LT/ST Total

182 (55) 73 (22) 75 (23) 330

31 (51) 15 (25) 15 (25) 61

21 (44) 8 (17) 19 (40) 48

72 (40) 55 (31) 52 (29) 179

306 (50) 151 (24) 161 (26) 618

a Data are from the surveillance for ETEC from 1996 to 2000 from patients with diarrhea enrolled in the 2% systemic routine surveillance system at the ICDDR in Bangladesh (185). The numbers of patients and percent positive for the respective toxins are shown. Data are from patients infected with ETEC as a single pathogen.

countries (Tables 1 to 5). Studies to better understand the natural infection pattern of ETEC are being conducted with cohorts of infants to discern the infection and reinfection pattern as well as the age group most at risk for infection. In studies of infants in West Africa, Egypt, and Bangladesh, the rate of ETEC infections in community-based studies increased from about 3 to 6 months of age, similar to the surveillance data of hospitalized patients in a diarrheal hospital in Bangladesh (139, 183, 215). The age at which a primary ETEC infection can be documented depends to some extent on the phenotype of ETEC that is infecting the child. In a study in Guinea Bissau, it was reported that in the youngest age group, 3 months, ETEC strains producing STh and LT were most common, whereas at 6 to 7 months ETEC strains producing STp, STpLT, and SThLT predominated (183). The incidence of ETEC infections in developing countries decreases after 5 years of age with a decrease of infections between the ages of 5 to 15 years (Table 6). The incidence increases again in those over 15 years of age and about 25% of ETEC illness is seen in adults (113, 132). Limited epidemiological information is available for adults, and those available are mostly from India and Bangladesh. It was in these settings that ETEC was first described extensively and was shown to be a cause of adult diarrhea resembling cholera in the severity of

infection (113, 149, 154). It thus became obvious that adults with severe dehydrating cholera-like illness attributable to ETEC infections are not uncommon (132, 214). In hospitalized patients, adults often present with more severe forms of ETEC diarrhea than children and infants (Table 7). Interestingly, further analyses have shown that the elderly are also susceptible to ETEC infections requiring hospitalization (62). ETEC was found to be the second most frequently isolated (13%) bacterial pathogen after V. cholerae O1 (20%). In this age group (⬎65 years), patients also presented with more severe dehydration than children. The reason why ETEC infections decrease after infancy and increase at adulthood may be due to both environmental and immunological factors. Data obtained from studies in animals indicated changes with age in the presence of intestinal cell receptors for K99 fimbrial antigen produced by ETEC infecting animals (148). Another factor could be the immunogenetics and diversity between individuals, which may prevent or predispose to ETEC infections (33, 69) or increased immune responses due to repeated infections in early childhood, which may decrease due to fewer infections during adolescence (Table 6).

Relation to Presence of LT, ST, and Colonization Factors Indeed, ETEC expressing LT only have been considered less important as pathogens, especially since they are more frequently isolated (than the other two toxin types) from healthy persons than from patients (33). This could be related to the low prevalence of CFs on the LT-producing ETEC strains (66, 110). Thus, in many epidemiological studies, the CFs have been detected on less than 10% of LT-producing ETEC strains, compared to over 60% of the ST- and LT/ST-expressing ETEC. However, it cannot be excluded that LT-producing ETEC strains may be highly pathogenic, given that they may have been isolated from sick patients with severe dehydrating diarrhea (Table 8). A comparison of the toxin pattern of the infecting strains in

TABLE 7. Clinical characteristics of adults and children hospitalized with ETEC, V. cholerae O1, and rotavirus diarrhea in Dhaka, Bangladesh, from 1996 to 2002a % of adults Variable

Vomiting Fever Severe dehydration Moderate dehydration None/mild dehydration Intravenous management Oral rehydration Outpatient stay Inpatient stay Watery stool Nonwatery stool Stool with blood

ETEC (N ⫽ 478)

81 7 36*‡ 41 23 54* 46* 14 86 95 5 2.3

% of children

V. cholerae O1 (N ⫽ 1,417)

ETECa (N ⫽ 1107)

V. cholerae O1 (N ⫽ 865)

Rotavirus (N ⫽ 3,406)

92 2 64* 29 7 84* 16* 4 96 99 1 1

80 19 3†‡ 24 73 10† 90† 31 69 97 3 17

89 15 23† 40 37 43† 57† 13 87 98 2 1

87 22 2† 19 79 5† 95 24 76 97 3 1

a Data are from 15,558 patients seen at the diarrhea hospital in Bangladesh based on the routine surveillance for enteric pathogens (185). The age of the children ranged from 0.1 to 36 months (median, 0.83 years) and that of the adults from 15 to 80 years (median, 30 years), *, P ⬍ 0.001 in comparisons (*) between adults with ETEC or V. cholerae O1 diarrhea; (†) between children with ETEC or rotavirus diarrhea in comparison to V. cholerae O1 diarrhea; (‡) between adults and children with ETEC diarrhea. For rotavirus diarrhea only, data from children are given.

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TABLE 8. Association of severity of disease with toxin type and presence of known colonization factors on ETEC in children up to 3 years of age in rural Bangladesh during a 2-year surveillancea No. (%) with symptoms that were:

CF and toxin type

Mild

Moderate

Severe

Total

CF positive CF negative ST LT/ST LT

142 (56) 140 (64) 131 (56) 69 (52) 82 (79)

90 (36) 56 (26) 78 (33) 54 (41) 14 (13)

20 (8) 22 (10) 24 (11) 10 (7) 8 (8)

252 218 233 133 104

a CF types were studied with a panel of 13 different monoclonal antibodies against the most prevalent CFs (66, 132). The degree of dehydration of the child from whom the ETEC strain was isolated is indicated. The strains were isolated from children in the hospital at Matlab in Bangladesh. Data are from patients infected with ETEC as a single pathogen. A significant difference (P ⬍ 0.05) was only seen between ETEC strains positive for ST (P ⬍ 0.001) or LT/ST (P ⬍ 0.001) and LT. The latter were isolated at significantly lower frequencies from children with moderate dehydration. Chi square for trends was used for statistical analysis.

patients hospitalized with diarrhea in the different age groups shows that the toxin phenotype did not change with age (132) (Table 6). In longitudinal studies with infants, both LT and ST phenotypes of ETEC were found to be associated with diarrhea (1, 37). This has been shown to be the case also in hospital- (7, 8) as well as community-based studies (1, 37). However, in hospital-based studies (132), ETEC producing both LT and ST or ST alone were found to cause relatively more severe disease than that caused by LT-producing ETEC strains (Table 8). Although over 22 CFs have been detected on ETEC (Table 4), only six to eight are more frequently isolated from diarrheal stools (Table 5). Of these, CFA/I and CS1 to CS6 are the predominant types (66). These CFs are mostly present on ETEC producing ST or both LT and ST. It is believed that immunity to strains that express the nonimmunogenic ST is derived from the anti-CF response to the protein adhesins. Thus, in the development of vaccines, these CFs as well as LT are being included to give a broad-spectrum protection (189, 192). The relationship between the presence of colonization factors and the disease-producing capability in ETEC diarrhea has been analyzed in many different epidemiological settings. In community-based studies the risk of diarrhea increased when a CF was present on the infecting strain (1). In Bangladesh the presence or absence of CFs on ETEC could not be associated with the severity of diarrhea in hospitalized patients (Table 8) (132). Studies in Mexico suggest that there is a reduced risk of diarrhea in infants if there was reinfection with ETEC producing the same compared with different CFs (38). In volunteer challenge studies, protection was observed to ETEC with the same CF as that present on the vaccine strain (97). Some CFs are seen more often in infants than in adults, suggesting that natural immunity to infection may develop. Thus, studies in Bangladesh have shown that almost all ETEC expressing CS7 and CS17 were isolated from children less than 3 years of age (132). LT-producing ETEC strains expressing CS7 were also most pathogenic in a birth cohort in West Africa whereas CF-negative strains were not, suggesting that the presence of a CF, even in the absence of ST, enhances the virulence of ETEC (183).

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Single Versus Mixed Infections Coinfection with ETEC and other enteric pathogens is common, which may lead to problems in determining whether the symptoms are caused by the actual ETEC infection and understanding the actual pathogenesis of the infection (134, 182). Mixed infections are frequent and may be seen in up to 40% of cases (7, 17, 127, 132, 139). The presence of enteric pathogens in asymptomatic persons is also known to be high in areas of poor sanitation. The incidence of mixed infections seems to increase with age in studies in Bangladesh and fewer copathogens were seen in infants than in older children and adults with ETEC diarrhea (134). In cases of mixed infections in children, rotavirus is the most common, followed by other bacterial enteropathogens, e.g., V. cholerae, Campylobacter jejuni, Shigella spp., Salmonella spp., and Cryptosporidium (7, 67, 166). In traveler’s diarrhea, enteroaggregative E. coli and Campylobacter spp. have been common pathogens together with ETEC (2, 127). Seasonality of ETEC Several studies have reported that ETEC diarrhea and asymptomatic infections are most frequent during warm periods of the year (1, 8, 79, 103, 132, 139, 167, 183), suggesting that travelers to these regions are also more at risk to develop ETEC infections during the warm seasons. In Bangladesh, ETEC follows a very characteristic biannual seasonality with two separate peaks, one at the beginning of the hot season, that is, the spring, and another peak in the autumn months, just after the monsoons, but it remains endemic all year (8, 94, 132, 146) (Fig. 1). Such a seasonality may be initiated by climate and spread by environmental factors. As the atmospheric temperature increases when spring sets in after the cooler winter months, there is increased growth of bacteria in the environment and this continues in the summer months. Furthermore, with the advent of rains in the monsoon season, there is enhanced contamination of surface water with fecal material and the surface water can thus become heavily contaminated (146). A seasonality for the different toxin phenotypes has also been suggested, with ST-producing ETEC strains being more common in the summer (1, 139) whereas LT-producing ETEC strains are present all year round and do not show any seasonality (Fig. 2). Comparison of ETEC Diarrhea and Cholera in Children and Adults In Bangladesh cholera and ETEC diarrhea are still endemic (134, 165). Both diseases share a biannual periodicity, peaking once in the spring and again in the autumn (Fig. 1) and remaining endemic all year (63). In the spring ETEC infections appear to be more prevalent than V. cholerae O1 infections (Fig. 1). In a recent 4-year study, carried out for the surveillance for cholera in rural Bangladesh (165), it was found that ETEC and not V. cholerae was often the cause of the diarrhea in some of these field areas (215). It is also not surprising to have concomitant outbreaks of both ETEC and V. cholerae during peak seasons and during outbreaks (31, 63). Active screening for ETEC needs to be carried out in outbreaks and

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FIG. 1. Estimated numbers of enterotoxigenic E. coli and V. cholerae O1 isolated from diarrheal stools of children under 5 years of age from surveillance carried out from 1996 to 2002 at the International Centre for Diarrheal Disease Research hospital in Dhaka, Bangladesh. The figure indicates monthly isolation of ETEC (Œ) and Vibrio cholerae O1 (■).

epidemics for both epidemiological and public health purposes. A large proportion of patients with ETEC infection have short stays in the hospital and only about 5% of patients need to be hospitalized for longer periods of time. The length of stay at the hospital is similar for patients infected with any of the three toxin phenotypes of ETEC. Few patients go on to chronic illness (⬎14 days). Patients with ETEC diarrhea and cholera have similar clinical characteristics and differ mainly in the rates of severe dehydration (Table 7). Presence of ETEC in Food and Water in the Environment Diarrhea due to ETEC, like other diarrheal illnesses, may be the result of ingestion of contaminated food and water (26, 40, 87, 99, 121, 122). In any situation where drinking water and sanitation are inadequate, ETEC is usually a major cause of

FIG. 2. Monthly incidence of ETEC diarrhea in Abu Homas, Egypt, showing that ST-producing ETEC was more common in warmer months, while LT-producing ETEC was present at similar levels throughout the year. Reproduced from reference 139 with permission.

diarrheal disease. Surface waters in developing countries have been found to harbor these organisms (14, 121) and transmission can occur while bathing and/or using water for food preparation. These forms of transmission are common in area where it is endemic both in the local populations and in international travelers to these areas (see the later section on traveler’s diarrhea). Transmission of ETEC by processed food products outside of the developing world is less commonly seen but well documented. In 1977, Sack et al. found that of 240 isolates of E. coli from food of animal origin in the United States, 8% were found to contain ETEC which produced either or both LT and ST (164). None of these food products were associated with diarrheal outbreaks. In studies carried out in the 1970s in Sweden, however, outbreaks of diarrhea due to food-borne ETEC were reported (41). Similar findings were reported from Brazil in 1980 (144); 1.5% of 1,200 E. coli strains from processed hamburger or sausage were found to be ETEC. ETEC transmission on cruise ships has now been reported on several occasions (40). These findings suggested that since ETEC is not uncommonly found in meat and cheese products, these organisms have the potential for producing diarrheal outbreaks in different parts of the world. Contaminated weaning food is also a likely cause of ETEC diarrhea in infants (139, 146). Contaminated food and water sources both contribute to seasonal outbreaks which affect tourists. Thus, ETEC is a cause of traveler’s diarrhea more often in the warm than in the cool season. In a study in Bolivia it has been shown that ETEC could be isolated from a sewagecontaminated river (121). Furthermore, contaminated food and water were found to be the source of ETEC infections in Peru (23). Surface water sources in Bangladesh, in both rural and urban areas, are highly contaminated with ETEC. Thus, recently in a study in Bangladesh, ETEC strains were obtained from clinical samples as well from ponds, rivers, and lakes around the clinical field site. In this study it was found that 32% of water samples obtained from the surface water sources were contaminated with ETEC and that the toxin and CF pheno-

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types of strains isolated from the clinical and environmental samples were comparable (14). Furthermore, pulsed-field gel electrophoretic analysis of the ETEC strains showed that those present in the environment were similar to the clinical isolates, supporting that, as seen for V. cholerae, surface waters may be a major source for the survival and spread of ETEC. Studies in communities where personal hygiene, education, and general living conditions are poor have shown that infection can spread within family groups. In one study on ETEC infections in Bangladesh, the bacteria were spread to 11% of contacts in a 10-day study period (25); transmission was dependent on socioeconomic status and living conditions. Contaminated food and water and the mothers themselves, who are food handlers, seem to be the reservoirs for such infections (54). It is not surprising therefore that the possession of a sanitary latrine significantly decreased the risk of ETEC diarrhea in children in Egypt (1). On the White Mountain Apache reservation in Whiteriver, Arizona, where ETEC was found to be an important cause of diarrhea in children, these organisms were also found in river water, sites of large gatherings of Apaches on festive occasions (162). Although ETEC has been detected as a cause of diarrhea in Apache children in Arizona (162), where water and sanitation were suboptimal, subsequent studies in the developed world where water supplies and sanitation are optimal show very low frequencies of ETEC in children with diarrhea (156). ETEC Infections and Malnutrition As for other diarrheal diseases, preexisting malnutrition can lead to more severe enteric infections, including those caused by ETEC, possibly due to the immunocompromised nature of the host that also predisposes these individuals to a greater bacterial load on the mucosal surfaces of the gut than the well-nourished child (28). In a study in India, diarrheal illness including that caused by ETEC was found to be more severe in children with malnutrition (107). Micronutrient deficiency such as vitamin A and zinc is quite common in developing countries and generally increases the morbidity due to diarrheal illnesses (137, 140), although the effect on the morbidity of ETEC diarrhea has not yet specifically been studied. It has been estimated that in Bangladesh over 40% of children younger than 5 years of age may have zinc deficiency (131, 168). Supplementation with zinc increases the adaptive immune responses to cholera vaccination in children and adults (9, 91, 131) and in children with shigellosis (140). The effect of micronutrient deficiency on the morbidity and protective immune responses in ETEC diarrhea has not been specifically studied but is an area that needs attention. However, repeated diarrheal episodes including those induced by ETEC may be an important cause in predisposing the child to malnutrition (22, 106). Other factors, such as breast feeding, may have the capacity to prevent ETEC diarrhea. Factors in milk such as specific secretory immunoglobulin A antibodies and receptor analogues (85) as well as innate and anti-inflammatory factors may all contribute to decrease the infection. Hyperimmune bovine colostrum containing high titers of ETEC CF antibodies has been shown to provide temporary protection against ETEC challenge (64) but is not suitable for public health application

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(198). Breast feeding reduces overall diarrhea and mortality (71, 205). A reduction in diarrheal episodes has been seen in infants who had been breastfed for the first 3 days of life, irrespective of other dietary practices, emphasizing the positive effects of colostrum (34). Studies in Bangladesh have shown that breast milk antibodies against cholera toxin and lipopolysaccharide do not protect children from colonization with V. cholerae but do protect against disease in those that are colonized (72). Protection from cholera in breastfed infants of mothers immunized with killed cholera vaccine could not be correlated to antibacterial and antitoxic antibodies in breast milk, suggesting that the reduced transmission of pathogens from the mother to the infant had a protective effect (35). Since secretory immunoglobulin A antibodies to CFs and enterotoxin are present in breast milk samples from mothers in developing countries (39, 84, 187), it would be natural to assume that breastfed infants should be protected from ETEC diarrhea. However, epidemiological studies show that partial breast feeding does not result in a reduced risk of ETEC diarrhea. However, in data obtained in various studies, it appears that exclusive breast feeding practices have a positive effect of decreasing the severity and/or incidence of ETEC infections (34, 102, 136). This effect is short term and does not last long after infancy, and an overall protection is not seen in the crucial first 2 to 3 years of life (1, 34, 139). The limited capacity of breast milk to protect against ETEC diarrhea in developing countries can also be attributed to other social and behavioral factors. These include the introduction of contaminated water and weaning food in the child’s diet, leading to increased symptomatic as well as asymptomatic ETEC infections. In Mexico, the incidence of diarrhea increased even in the first 3 months of age if a barley drink was given to the infant (102). Since mixed feeding is started quite early in life in a majority of infants in developing countries, sometimes as soon as after birth, contaminated water may also be the cause of a multitude of infections (146). The importance of personal hygiene rather than breastfeeding appeared to be more protective against ETEC diarrhea in Egypt (1). Infections in International Travelers ETEC remains endemic all year round but is highest during the warm season, reflecting the seasonal difference of ETEC and other bacterial enteropathogens in the country visited (109, 175), suggesting that travelers are more vulnerable to the diarrheal illnesses at these times. In travelers, the phenotypes of ETEC strains vary from country to country, e.g., LT-only ETEC was more commonly isolated from visitors to Jamaica, 58% (90), and LT/ST ETEC was most often seen in visitors to India, 45% (90), and ST-only ETEC in visitors to Kenya, 51% (175). Thus, strains that are circulating in a particular country, infecting primarily children, and contaminating the water and food sources (as well as the hands of the food handlers) may determine the type of ETEC infecting the travelers. Travelers to such countries do not know the cause of their diarrheal illness since it cannot be identified on site, outside of research studies. The data available suggest that from 20 to 40% of traveler’s diarrhea cases (18, 90, 159, 175) may be caused by ETEC, and the children resident in those countries have rates

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of 20% of hospitalized diarrheal episodes caused by ETEC. Thus, ETEC seems to be the most frequent cause of traveler’s diarrhea in North Americans and Europeans visiting developing countries (58, 145, 150, 175, 213).

CLIN. MICROBIOL. REV.

It should be mentioned that the adult form of ETEC-related disease (of considerable severity) seems to be identified more in the Indian subcontinent. There are few (if any) reports of ETEC in adults, other than in traveler’s diarrhea. This may be due to the lack of diagnosis in adults, again because of the lack of easily available laboratory techniques.

CLINICAL FEATURES Disease Severity

Mortality from ETEC Diarrhea

The diarrheal disease caused by ETEC that was first recognized consisted of a cholera-like illness in both adults and children in Calcutta (161). Since then, many studies around the world have shown that ETEC-induced diarrhea may range from very mild to very severe. There are, however, short-term, asymptomatic carriers of the organisms (20). The diarrhea produced by ETEC is of the secretory type: the disease begins with a sudden onset of watery stool (without blood or inflammatory cells) and often vomiting, which lead to dehydration from the loss of fluids and electrolytes (sodium, potassium, chloride, and bicarbonate) in the stool (25, 157). The loss of fluids progressively results in a dry mouth, rapid pulse, lethargy, decreased skin turgor, decreased blood pressure, muscle cramps, and eventually shock in the most severe forms. The degree of dehydration is categorized from mild to severe, and this clinical distinction is important in the provision of adequate therapy. The patients are afebrile. Usually the diarrhea lasts only 3 to 4 days and is self-limited; if hydration is maintained, the patients survive, and without any sequelae. With adequate treatment, the mortality should be very low (⬍1%). The pathophysiology of the illness caused by ETEC is essentially the same as that caused by Vibrio cholerae (94) and the clinical picture is identical, especially in adults (Table 7). Studies with human volunteers have shown that the infecting dose is high for both diseases. For ETEC, the dose is around 106 to 1010 CFU, with lower doses being less pathogenic (100). The need for a large infectious dose, the proliferation of the bacteria in the small bowel through colonization factors and the production of enterotoxins, and the watery, secretory type of diarrhea which produces clinical dehydration are comparable in both diseases. Both organisms produce an immunologic protective response, reflecting the observation that the attack rates are higher in children and decrease with age (24, 134). In Bangladesh, the majority of cases of acute watery diarrhea, especially in children, are caused by three pathogens, rotavirus, V. cholerae, and ETEC (7, 8, 24). Hospital-based studies during the early 1980s have demonstrated that the purging rate is higher in cholera compared to the other two illnesses (114). A comparison of the clinical features of the disease in adults with ETEC and V. cholerae infections seeking care at the hospital in Bangladesh shows that ETEC disease differs significantly from V. cholerae infections in the severity of dehydration (Table 7), although both infections can result in severe dehydration. In comparison to children, adults with ETEC diarrhea seem to have more dehydrating illness, requiring longer hospitalization and more intravenous fluid management. This may be because of more delay in reaching a treatment facility. In children, rotavirus and ETEC diarrhea share similar clinical characteristics but differ from cholera in being less severe (Table 7).

Mortality data due to ETEC infections are difficult to estimate. Similar to cholera, if patients with severe ETEC disease reach an adequate treatment center, mortality should be very low, ⬍1%. Although untreated cholera patients may have a high mortality (⬃50%), untreated ETEC patients would be expected to have a lower mortality rate based on the lesser severity of illness overall. In a World Health Organization report it has been suggested that there are 380,000 deaths annually in children less than 5 years of age that are caused by ETEC (214). However, there are no well-documented mortality figures for ETEC-induced diarrhea, because the microbiologic diagnosis cannot be done easily in many settings, and therefore only rates for cholera, which is cultured easily, can be accurately determined. ETEC-related deaths at present would be counted as diarrheal deaths in many countries. It is presumed, however, that there is significant mortality in patients not receiving treatment. DIAGNOSIS Laboratory Assays Since ETEC must be recognized by the enterotoxins it produces, diagnosis must depend upon identifying either LT and/or ST. Because the assays necessary were very cumbersome, it was thought that some other marker could be a proxy in identification. Initially the serogroups of ETEC were identified and found to be relatively few, and therefore it was thought that perhaps serotyping could be used to differentiate ETEC from other E. coli, including the enteropathogenic strains whose characteristic serotypes were known (124). Serotyping was found to be of limited use in Bangladesh (113, 152, 186) and when it became clear that a very large number of E. coli serotypes could be enterotoxigenic, this was abandoned. Direct identification of the enterotoxins of ETEC has evolved over the past 35 years. Physiologic assays, the rabbit ileal loop model for LT (43), and the infant mouse assay (44) for ST were initially used as the gold standards before other simpler assays could be identified. Because LT was strongly immunogenic whereas ST was not, diagnostic assays developed along different lines. In 1974 the direct action of LT on two tissue culture cell lines, Y1 adrenal cells (46) and Chinese hamster ovarian cells (78), was found to be provide physiological responses that could be detected by morphological changes in tissue culture. These changes were specific for LT and could be neutralized by antitoxin. The two tissue culture assays were widely used for LT recognition until the development of the enzyme-linked immunosorbent assay technology in 1977 (217). Other assays such as staphylococcal coagglutination (32), passive latex agglutination (173), immunoprecipitation in agar, and the Biken test (86) were found to be specific but were not

VOL. 18, 2005

used widely for diagnostic purposes. Enzyme-linked immunosorbent assays became a widely used method for detecting LT, particularly using microtiter GM1 ganglioside methods (190, 196). Subsequently, combined GM1 enzyme-linked immunosorbent assays for ST and LT were developed (196, 197) and have been used in different epidemiological studies (1, 16, 126, 128, 132). ST testing in infant mice continued to be used widely and could be enhanced by the use of culture pools, thereby minimizing the numbers of infant mice. In 1981 Gianella developed a radioimmunoassay for ST which compared favorably with the infant mouse assay (68). In 1980, methods using molecular diagnostic techniques began. Moseley et al. (104) showed that the genes controlling the enterotoxins could be detected using 32P-labeled DNA probes derived from plasmids for both LT and ST. This method was shown to be specific and sensitive and could detect as few as 1 to 100 CFU per gram of material (53, 82). Variations of this technology, including both polynucleotide and oligonucleotide probes with both radioactive and nonradioactive labeling, have been found to be useful in detecting ETEC both in clinical and environmental samples and is widely used (7, 53, 82). In 1993, PCR was first used in ETEC diagnosis (123). It was found to be useful for diagnosis directly on fecal material as well as of isolated colonies (172). It was also adapted to a multiplex form so that the diagnosis of LT- and ST-producing organisms as well as other diarrheagenic E. coli can be made simultaneously (181, 200, 208, 209). During recent years DNA probes, with either radioactive or nonradioactive detections or GM1 enzyme-linked immunosorbent assays using monoclonal antibodies against ST or LT have been the most widely used methods for detection of ETEC toxins (7, 133, 183, 188). For detection of ETEC colonization factors a number of different methods have been used during the years. Initially the capacity of E. coli CFs to agglutinate certain species of erythrocytes in a mannose-resistant manner was used for demonstration of CFA/I and CS1, CS2, and CS3 (60). This nonprecise method was soon replaced by more specific slide agglutination and immunodiffusion tests initially using polyclonal sera and subsequently monoclonal antibodies against different CFs (5, 76, 110). Other methods that were used included nonspecific salting-out tests (16) and binding to tissue culture cell lines (42). These assays have now been replaced by different molecular methods, e.g., DNA probes and PCR methods against most of the known CFs or dot blot assays using several different anti-CF monoclonal antibodies (133, 139, 180, 182). The method of choice varies from one laboratory to another and is dependent on the capability of the investigator and the level of development of the laboratory where the work is being carried out. The phenotypic methods can be set up relatively easily in different laboratories and are useful for prospective studies; most reagents are not available commercially but may be obtained from different laboratories. One point to bear in mind is that the virulence antigens are encoded by plasmid genes and can be easily lost or become silenced due to the loss of regulatory genes (182). The more recently developed DNA probe methods have the capacity to detect the structural genes for toxins and CFs and thus have the advantage of detecting ETEC from samples which have been stored for long periods

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of time and where phenotypic changes may have taken place. These procedures are more difficult to adapt to field sites in developing countries, where laboratory facilities may be inadequate for molecular microbiological methods. Furthermore, in some instances ETEC CFs can only be detected by molecular but not phenotypic methods, since they are not exposed on the bacterial surfaces due to mutation of genes required for surface expression (119). Unfortunately, in spite of all these available techniques, there are still no simple, readily available methods that can be used to identify these organisms in minimally equipped laboratories. For that reason many laboratories conducting studies on diarrhea in developing countries do not include ETEC in their routine diagnostic capabilities, and special research or referral laboratories are necessary to identify these bacteria. TREATMENT AND MANAGEMENT The treatment of diarrheal disease due to ETEC is the same as that for cholera or any other acute secretory diarrheal disease. The correction and maintenance of hydration is always most important. Antimicrobials are useful only when the diagnosis or suspicion of ETEC-related diarrhea or cholera is made. Provision of adequate nutrition is critical in children in the developing world, where all diarrheal diseases are frequent. The guidelines for therapy of all diarrheas have been widely disseminated by the World Health Organization (216). Rehydration Rapid rehydration using intravenous fluids (such as Ringer’s lactate) is required initially for all patients with severe dehydration. After restoration of blood pressure and major signs of dehydration, patients can be put on oral rehydration solutions for the remainder of therapy. For all other patients with lesser degrees of dehydration, therapy with oral rehydration solutions alone can be used until the diarrhea ceases. Details of management of acute gastroenteritis in children have recently been summarized by King and colleagues (6, 95). Antimicrobials The use of antimicrobials in the treatment of ETEC diarrhea is problematic, since an etiologic diagnosis cannot be made rapidly. This differs from the treatment of cholera, an epidemic disease, where clinical findings and rapid laboratory tests can readily lead to correct diagnosis. In cholera treatment, antimicrobials are an integral part of therapy because they lead to a marked decrease in stool output and shortening of the disease (30, 95). Because childhood diarrheas, however, are caused not only by ETEC but also by other bacterial and viral agents, and the clinical presentations are not sufficient to differentiate them, it has been difficult to study the effect of antimicrobials in children with ETEC disease and antimicrobials are not used routinely in treatment of childhood diarrhea. One study in Bangladeshi adults in which tetracycline was used to treat ETEC diarrhea (determined retrospectively) showed only a minimal effect on the severity or duration of diarrhea (113). Antimicrobials, however, are of definite benefit in the treat-

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TABLE 9. Changing pattern of antimicrobial sensitivity of ETEC from its identification to the presenta Yr and subjectsa

Sensitivity

References

1968–1980, T/EP 1980–1990, T/EP 1990–present, T/EP

Sensitive to all antimicrobials (Mexico, India, U.S.A., Kenya, Morocco) Single antibiotic resistance appears (Mexico, Bangladesh) Multiple antimicrobial resistance appears (Tet, Amp, SXT, Doxy, Nal, Ery, S) (Somalia, Middle East, Bangladesh) Resistance to Cip and fluoroquinolones and multiple resistance (India, Japan)

49, 150, 154, 160 47, 48, 104, 186, 213 31, 89, 176

2001–present, T/EP

31, 108

a

T, travelers, including army and service personnel stationed in or visiting ETEC-endemic countries; EP, endemic population. Tet, tetracycline; Amp, ampicillin; SXT, trimethoprim-sulfamethoxazole; nal, nalidixic acid; Ery, erythromycin; S, streptomycin; Doxy, doxycyline; Cip, ciprofloxacin.

ment of diarrhea of travelers, a diarrheal syndrome in which the clinical symptom is well recognized and ETEC is known to be the most frequent pathogen (90). It should be noted, however, that antimicrobials used for traveler’s diarrhea will treat not only ETEC but also most of the other known causes (enteroaggregative E. coli, Shigella, and Campylobacter) of the diarrhea. The antimicrobial treatment of traveler’s diarrhea has changed over the years because of increasing antimicrobial resistance (58). When ETEC were first recognized, the bacteria were usually highly sensitive to all antimicrobials, including tetracyclines and trimethoprim-sulfamethoxazole (159). However, with time, antibiotic resistance emerged, necessitating the use of newer antimicrobials for treatment of traveler’s diarrhea. Antimicrobials that have been used in effective treatment include doxycycline, trimethoprim-sulfamethoxazole, erythromycin, norfloxacin, ciprofloxacin, ofloxacin, azithromycin, and rifamycin. A summary of these studies over the years is given in several references (58, 159). The general history of the evolving antibiotic resistance patterns in ETEC is given in Table 9. At present, recommendations for treating ETEC can only be stated for surety in the treatment of traveler’s diarrhea, where ETEC are known to be the most frequent cause (51). For adults we recommend a short course of ciprofloxacin, 500 mg every 12 h for 1 day, which usually stops the illness within 24 h. The new nonabsorbable antimicrobial rifaximin (50) has only recently become available and is effective for treatment of traveler’s diarrhea in adults, using 200 mg two times a day for 3 days. For children, we empirically recommend azithromycin, 10 mg/kg/day for 2 days, although there have been no studies to confirm this. In areas where ETEC is endemic, antimicrobial treatment is usually not given because the diagnosis cannot be easily made microbiologically and there are no controlled studies to provide recommendations. Multidrug Resistance Patterns Which antibiotic can be used has changed since the late 1970s, when doxycycline and trimethoprim-sulfamethoxazole were the drugs of choice. Due to increasing microbial resistance of ETEC, newer drugs have been used. A fluoroquinolone such as ciprofloxacin, levofloxacin, or ofloxacin is currently the drug of choice, since no significant resistance to these drugs has yet developed (58, 59). A newer nonabsorbed drug, rifaxamin, has also been shown to be as effective as a fluoroquinolone and has only recently been approved for use in the United States (50). Multidrug resistance is increasing in ETEC due to the widespread use of chemotherapeutic agents

in countries where diarrhea is endemic. Antimicrobial sensitivities, however, have only been studied extensively in international travelers and during common source outbreaks of disease or specific epidemiologic studies in areas where diarrhea is endemic. The primary reason for this is the difficulty of recognizing the organisms. Although no sensitivities were reported in ETEC strains first isolated in Calcutta in 1968, when ETEC strains were used in volunteer studies (49), they were sensitive to ampicillin, which was used for treatment. ETEC strains described for the first time in Apache children in Arizona in 1971 (162) showed a completely uniform sensitivity pattern. Because of the high sensitivity of ETEC to doxycycline, and because it has a long half-life and high levels in stool, this drug was first chosen to study antibiotic prophylaxis among travelers to developing countries (75, 111, 151). The first studies of doxycycline prophylaxis were done in Peace Corps volunteers in Kenya (150) and Morocco (160), who showed high degrees of protection (⬃85%). In the Kenyan study (150) all ETEC strains were sensitive to tetracycline, and only a few were resistant to streptomycin and sulfonamide in the Moroccan study (160). An interesting finding in these two traveler’s diarrhea studies (150, 160) and the study in Apaches (162) was that nontoxigenic E. coli strains showed more antimicrobial resistance than ETEC. This pattern was also seen in a study of large numbers of ETEC isolated before 1978 (45), suggesting that there may be some protective effect of harboring enterotoxin plasmids; it was also shown that ST-producing strains were more likely to be resistant to antimicrobials than either LT or LT/ST strains. In 1973, Gyles (81) found that a single conjugative plasmid carried genes for both antibiotic resistance and enterotoxin production, the result of recombination of an R factor with an enterotoxin-carrying plasmid. A few years later, Echeverria (56) found that antibiotic resistance and the ability to produce enterotoxin were frequently transferred together and suggested that the widespread use of antibiotics could result in an increase of enterotoxigenic strains. Plasmids coding for both antibiotic resistance and ST could be transferred in vitro to E. coli K-12 (56) and in vivo in suckling mice, suggesting that antibiotic selective pressure could result in a wider distribution of ETEC (105). This hypothesis, however, has never been conclusively verified. A marked increase in resistance in ETEC began to be reported in 1980, when it was found that during a cruise ship outbreak the epidemic strain O25:NM was resistant to tetracycline and sulfathiazole (104) and in a hospital outbreak, all

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isolates of the epidemic strain were also resistant to tetracycline (89). During a study of traveler’s diarrhea in Mexico, in 1989 to 1990, 49% of 74 ETEC strains were resistant to doxycycline, 9% to trimethoprim-sulfamethoxazole, 35% to ampicillin, but none to norfloxacin or aztreonam (47) and in studies of outbreaks of ETEC diarrhea aboard three different cruise ships during 1997 to 1998, tetracycline resistance as high as 84% (27/32) was reported while 30% were resistant to more than three antimicrobials (40). This was a marked change from previous outbreaks before 1990 when no ETEC were resistant to more than three antimicrobials. More recently, studies from Bangladesh and India have also shown multiple antimicrobial resistance of ETEC isolates. A comparison of the resistance pattern in strains isolated recently with those obtained 30 years back highlights the increase of resistance to commonly used drugs (48). Studies of ETEC strains isolated between 1999 and 2001 show intermediate to complete resistance to multiple drugs and combined resistance to four to six drugs (including erythromycin, ampicillin, cotrimoxazole, tetracycline, streptomycin, and doxycycline); however, not a single strain was found to be resistant to ciprofloxacin. In studies in India, multidrug resistance including resistance to nalidixic acid and to fluoroquinolones is increasing (31). In Bangladesh, ETEC strains are still sensitive to drugs which are generally used for the treatment of invasive diarrhea, but there needs to be more awareness of changing drug sensitivity patterns of ETEC when erythromycin is used for treatment of acute watery diarrhea in children. Nutritional and Micronutrient Therapy Recently it has been found that the addition of zinc to the therapy of diarrhea in children with diarrhea leads to shorter duration of illness and a decrease in mortality from diarrhea (15, 21). These studies have been done in areas of the world where chronic zinc deficiency in children is known to occur. Nutritional therapy for all childhood diarrheas, including those due to ETEC, is an integral part of diarrhea treatment. Episodes of diarrhea due to any cause, including ETEC, result in decreased nutritional status and thus inhibit growth in children (106). Attention to providing food, particularly breast milk, early in the course of therapy is essential. Additional food during and following the diarrheal episode will help in catch-up growth (3). PREVENTION Vaccine Development Prevention of ETEC infection is clearly related to water and sanitation, including food preparation and distribution. In the developing world, such major improvements will be a long time coming (57). It is estimated that it would take US$200 billion to make the improvements necessary to prevent fecally spread diseases in South America alone (135). Other methods on a microscale are presently being done: building safe-water tube wells, chlorination/filtration/heating of drinking water, and building and improving latrines. These attempts to block transmission are certainly effective if implemented but cannot solve

477

the problem quickly. Therefore, there is much interest in the development of vaccines for prevention of ETEC disease. Based on the great impact of ETEC infections on morbidity and mortality, and probably also on nutritional status (106), particularly of children in areas where they are endemic, an effective ETEC vaccine is highly desirable. Such a vaccine is feasible since epidemiologic evidence and results from experimental challenge studies with human volunteers have demonstrated that specific immunity against homologous strains follows ETEC infection. Furthermore, multiple infections with antigenically diverse ETEC strains seem to lead to broadspectrum protection against ETEC diarrhea (38). Experimental studies with animals and indirect evidence from clinical trials (191) suggest that protective immunity against ETEC is mediated by secretory immunoglobulin A antibodies directed against the CFs, other surface antigens, and LT; ST, which is a small peptide, does not elicit neutralizing antibodies following natural infection. To provide broad-spectrum protection, an ETEC vaccine should probably contain fimbrial antigens representative of the most prevalent ETEC pathogens. The great diversity of ETEC serotypes, with regard to both O and H antigens, makes such antigens less attractive as vaccine components. Since CFA/I and CS1 to CS6 are the most common human ETEC fimbriae, they are key candidate immunogens in an ETEC vaccine. Other fimbrial CFs may also be considered, based on their relative importance in certain geographic areas (see Table 5). Since a majority of ETEC strains that produce both LT and ST or ST only produce CFs, it has been postulated that a multivalent ETEC vaccine containing CFA/I and CS1 to CS6 may provide protection against approximately 50 to 80% of ETEC strains in most geographic areas (189). If an LT toxoid such as the nontoxic B subunit LTB or a mutant LT is included, a multivalent toxoid-CF vaccine might provide relatively broad protection against 80 to 90% of ETEC strains worldwide. Inclusion of, e.g., CS7, CS12, CS14, and CS17 might expand the potential spectrum of coverage to up to 90% of all ETEC strains (189). A number of different strategies have been taken to deliver fimbrial and toxin antigens of ETEC to the human immune system to elicit protective immune responses and functional immunological memory. Purified CFs and Enterotoxoids Various purified CFs have been tested as oral immunogens but have been considered less suitable since they are expensive to prepare and sensitive to proteolytic degradation (101). To protect the fimbriae from degradation in the stomach, purified CFs have been incorporated into biodegradable microspheres. However, no significant protection was induced by any formulation of purified CFs against subsequent challenge with ETEC expressing the homologous CFs, either when immunizing with high doses of a combination of CS1 and CS3 or recombinantly produced CS6 (93, 101). Since LTB as well as the immunologically cross-reactive cholera toxin B subunit are strongly immunogenic, lack toxicity, are stable in the gastrointestinal milieu, and are capable of binding to the intestinal epithelium, they are suitable candidate antigens to provide anti-LT immunity. The cholera toxin B subunit has also afforded significant protection against ETEC producing LT or LT/ST both in coun-

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tries where ETEC is endemic and in travelers (36, 127), but it is possible that an LT toxoid might be slightly more effective than cholera toxin B subunit. An alternative administration route that has been considered is to give an ETEC vaccine by the transcutaneous route. Such administration of E. coli CS6 together with LT has induced immune responses against CS6 in about half of the volunteers and anti-LT responses in all of them (77). Work is in progress to evaluate E. coli LT as a candidate vaccine after transcutaneous immunization (73). Inactivated Whole-Cell Vaccines Another approach that has been extensively attempted is to immunize orally with killed ETEC bacteria that express the most important CFs on the bacterial surface together with an appropriate LT toxoid, i.e., cholera toxin B subunit or LTB (189). A vaccine that consists of a combination of recombinantly produced cholera toxin B subunit and formalin-inactivated ETEC bacteria expressing CFA/1 and CS1 to CS5 as well as some of the most prevalent O antigens of ETEC has been extensively studied in clinical trials in travelers as well as in children in areas where ETEC is endemic. This recombinant cholera toxin B (rCTB)-CF ETEC vaccine has been shown to be safe and gave rise to significant immunoglobulin A immune responses in the intestine and increased levels of circulating antibody-producing cells in a majority of adult Swedish volunteers (4). The vaccine has also been well tolerated and given rise to mucosal immune responses against the different CFs of the vaccine in 70% to 100% of volunteers of different age groups from 18 months to 45 years in Egypt and Bangladesh (130, 134, 169–171). However, due to an increased frequency of vomiting in the youngest children (6 to 18 months), a reduced dose of the vaccine, i.e., a quarter dose that can be given safely and with retained immunogenicity to Bangladeshi infants, has been identified (129). In an initial pilot study, the rCTB-CF ETEC vaccine was shown to confer 82% protective efficacy (P ⬍ 0.05) against ETEC disease in European travelers going to 20 different countries in Africa, Asia, and Latin America (213). However, the number of cases fulfilling the inclusion criteria was low. In a large placebo-controlled trial in nearly 700 American travelers going to Mexico and Guatemala, the rCTB-CF ETEC vaccine was shown to be effective (protective efficacy, 77%; P ⫽ 0.039) against nonmild ETEC diarrheal illness, i.e., disease that interfered with the travelers⬘ daily activities (153, 193). However, in a recent pediatric study in rural Egypt, the vaccine did not confer significant protection in the 6- to 18-month-old children tested (194). Live Oral ETEC Vaccines The potential of live ETEC vaccines has been suggested based on previous findings in human volunteers that a live vaccine strain expressing different CSs afforded highly significant protection against challenge with wild-type ETEC expressing the corresponding CS factors (97). For example, different live multivalent Shigella/ETEC hybrid vaccines have been constructed in which important fimbrial CFs are expressed along with mutated LT (10). Such vaccine candidates

CLIN. MICROBIOL. REV.

have expressed CS2 and CS3 fimbriae or CFA/I, CS2, CS3, and CS4 as well as a detoxified version of human LT (12). These candidate vaccine strains are presently being evaluated for safety and immunogenicity in different animal models, including macaques. The ultimate goal is to produce five different Shigella strains that can express the most important CFs and an LT toxoid simultaneously in the gut. Another approach has been to utilize attenuated ETEC strains as vectors of key protective antigens, e.g., CS1 and CS3 (203). Evaluation of such mutated strains, PTL002 and PTL003, in human volunteers has shown that they are safe and immunogenic when given in a single dose. The only vaccine that has been evaluated for protective efficacy in a field trial in young children in areas where ETEC is endemic so far is the rCTB-CF ETEC vaccine. Since this vaccine did not induce significant protection in this important target group, intense efforts should be made to improve the immunogenicity of this or modified ETEC vaccine candidates. As yet, no alternative ETEC vaccine is within reach to be licensed within the next 3 to 5 years. CONCLUSIONS Based on the multitude of information presented in this review, we make the following conclusions. ETEC is an underrecognized but extremely important cause of diarrhea in the developing world where there is inadequate clean water and poor sanitation. It is the most frequent bacterial cause of diarrhea in infants, children, and adults living in developing countries and the most common cause of diarrhea in international travelers visiting these areas. ETEC diarrhea is most frequently seen in children, suggesting that a protective immune response occurs with age. The pathogenesis of ETEC-induced diarrhea, including the production of enterotoxins and colonization in the small intestine, is similar to that of cholera. ETEC diarrhea could well be misdiagnosed as cholera because the diseases have common clinical syndromes and seasonalities. Treatment of ETEC diarrhea by rehydration is similar to that for cholera, but antibiotics are used routinely for ETEC only in the specific circumstances of traveler’s diarrhea. The frequency and thus importance of ETEC in our understanding of diarrheal agents on a worldwide scale is partly hampered by the difficulty in recognizing the organisms; no

TABLE 10. Major improvements needed Develop simple, rapid diagnostic methods for ETEC Set up formal reference center for testing to help standardize procedures and make reagents available to regions where ETEC is endemic Active screening for ETEC in diarrhea epidemics and outbreaks in developing countries to better understand its role as a major cause of “non-vibrio cholera” Create awareness of the problems of ETEC diarrhea worldwide, especially in child health programs Direct efforts and funds directed to the development of a broadbased multivalent ETEC vaccine Evaluate markers of protective immunity Design strategies to develop effective ETEC vaccines for use in developing countries

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simple diagnostic tests are presently available, and identification depends on specific reagents and technological expertise. Like V. cholerae, ETEC is transmitted by the fecal-oral route from contaminated food and water. Since ETEC is a multivalent pathogen, it leads to repeated infections that may adversely affect the nutritional status of children. Protective strategies for infections are not simple, since they include improvements in hygiene and development of effective ETEC vaccines. Although antimicrobials are not used routinely in treatment of children with ETEC diarrhea, the emerging problem of multiple antimicrobial resistance will definitely affect the drugs used for traveler’s diarrhea. Serious efforts need to be made to improve the awareness of the importance of ETEC, particularly on the health of infants and children living in the developing world (Table 10).

14. 15.

16.

17. 18. 19. 20.

ACKNOWLEDGMENTS

21.

This work was supported by the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B), Centre for Health and Population Research. We acknowledge with gratitude the commitment of the National Institute of Allergy and Infectious Disease (NIH grant AI39129) and the Swedish Agency for Research and Economic Cooperation (Sida-SAREC, grant no. 2001-3970) to the Centre’s research efforts.

22. 23.

24.

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CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 484–509 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.484–509.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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Diagnosis of Lyme Borreliosis Maria E. Aguero-Rosenfeld,1,4* Guiqing Wang,2 Ira Schwartz,2 and Gary P. Wormser3,4 Departments of Pathology,1 Microbiology and Immunology,2 and Medicine,3 Division of Infectious Diseases, New York Medical College, and Westchester Medical Center,4 Valhalla, New York INTRODUCTION .......................................................................................................................................................484 CHARACTERISTICS OF B. BURGDORFERI........................................................................................................485 B. burgdorferi Genospecies .....................................................................................................................................485 LYME BORRELIOSIS: DISEASE SPECTRUM....................................................................................................486 LABORATORY DIAGNOSIS....................................................................................................................................486 Direct Detection of B. burgdorferi .........................................................................................................................486 Culture of B. burgdorferi sensu lato .................................................................................................................487 (i) Culture techniques ....................................................................................................................................487 (ii) Culture of clinical specimens .................................................................................................................487 (iii) Sensitivity of culture...............................................................................................................................489 (iv) Practical considerations .........................................................................................................................489 Molecular methods of detection of B. burgdorferi sensu lato .......................................................................490 (i) PCR analysis of clinical specimens ........................................................................................................490 (ii) Real-time quantitative PCR....................................................................................................................492 (iii) PCR sensitivity and specificity..............................................................................................................492 (iv) Applications and limitations of molecular methods...........................................................................492 Immunologic Diagnosis of B. burgdorferi Sensu Lato Infection .......................................................................492 B. burgdorferi sensu lato antigens of importance in immunodiagnosis.......................................................493 Antibody detection methods ..............................................................................................................................493 (i) IFA...............................................................................................................................................................494 (ii) Enzyme immunoassays ............................................................................................................................494 (iii) Western IB ...............................................................................................................................................495 (iv) Two-tier testing ........................................................................................................................................496 Newer EIA antibody tests ..................................................................................................................................497 (i) Enzyme immunoassays using recombinant antigens ...........................................................................497 (ii) Peptide-based immunoassays .................................................................................................................498 (iii) Use of a combination of recombinant or peptide antigens in immunoassays ...............................498 Other antibody detection methods ...................................................................................................................499 (i) Functional antibodies: borreliacidal antibody assays ..........................................................................499 (ii) Detection of antibodies bound to circulating immune complexes.....................................................499 Detection of antibodies in cerebrospinal fluid................................................................................................499 Cellular immune response in LB: T-lymphocyte and mononuclear cell proliferation assays .................500 TEST INTERPRETATION........................................................................................................................................500 ACKNOWLEDGMENTS ...........................................................................................................................................501 REFERENCES ............................................................................................................................................................501 for LB in 1982, and the Council of State and Territorial Epidemiologists adopted a resolution making LB a nationally notifiable disease in 1990. LB is the most common vector-borne disease in North America and represents a major public health challenge for the medical community. Since 1982, more than 200,000 LB cases in the United States have been reported to the CDC, with about 17,000 cases reported yearly between 1998 and 2001 (54). In 2002 the number of cases of LB in the United States increased to 23,763, with a national incidence of 8.2 cases per 100,000 population. Approximately 95% of the cases occurred in 12 states located in the northeastern, midAtlantic, and north central regions (54); these states were Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin. LB is widely distributed in European countries and also occurs in far eastern Russia and in some Asian countries (128, 288, 351).

INTRODUCTION Lyme borreliosis (LB), or Lyme disease, which is transmitted by ticks of the Ixodes ricinus complex, was described as a new entity in the United States in the late 1970s (318, 319, 324, 325). Many of its individual manifestations had been documented many decades earlier in Europe (355). The etiologic agent, Borrelia burgdorferi, was recovered first in 1982 from the vector tick Ixodes dammini (now I. scapularis) (41) and subsequently from skin biopsy, cerebrospinal fluid (CSF), or blood specimens of patients with LB in the United States (22, 323) and Europe (3, 14, 268). In the United States, the Centers for Disease Control and Prevention (CDC) initiated surveillance

* Corresponding author. Mailing address: Clinical Laboratories, Room 1J-04, Westchester Medical Center, Valhalla, NY 10595. Phone: (914) 493-7389. Fax: (914) 493-5742. E-mail: m_aguero [email protected] 484

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During the past 20 years, notable advances have been made in understanding the etiologic agent, B. burgdorferi, and the illness that it causes. Many excellent reviews have been published detailing progress in expanding the knowledge base on the microbiology of B. burgdorferi (20, 29, 50, 283, 307, 351, 353) and on the ecology and epidemiology (12, 40, 247, 297, 299), pathogenesis (127, 225, 321, 335, 357), clinical aspects (219, 253, 313–316, 372), and laboratory diagnosis (18, 39, 291, 360, 362, 365) of LB. It has been estimated that more than 2.7 million serum samples are tested each year for the presence of B. burgdorferi-specific antibodies in the United States alone (339). To meet the demand for laboratory-based diagnosis, various new tests for direct detection of the etiologic agent, or for detection of specific antibodies by using whole-cell lysates, recombinant antigens, or peptide antigens in enzyme immunoassays (EIA), have been introduced into the clinical laboratory. This review attempts to provide a comprehensive assessment of the development and application of currently available tests for the laboratory diagnosis of LB. Future directions for improvement of established tests and for development of new approaches are also discussed. CHARACTERISTICS OF B. BURGDORFERI B. burgdorferi is a helically shaped bacterium with multiple endoflagella. The cells, configured with 3 to 10 loose coils, are 10 to 30 ␮m in length and 0.2 to 0.5 ␮m in width (20). This spirochete possesses several morphological, structural, ecologic, and genomic features that are distinctive among prokaryotes. Cultured B. burgdorferi organisms are motile and swim in freshly prepared slides. Live organisms can be visualized by dark-field or phase-contrast microscopy. They can also be recognized by light microscopy after staining with silver stains or by fluorescent microscopic methods. The ultrastructure of B. burgdorferi is comprised of an outer slime surface layer (Slayer), a trilaminar outer membrane surrounding the periplasmic space that usually contains 7 to 11 periplasmic flagella and an innermost compartment, the protoplasmic cylinder (120). Detailed information on the cell structure and biology of B. burgdorferi is found in published reviews (20, 353). B. burgdorferi is the first spirochete whose complete genome was sequenced (98). The genome size of the type strain B. burgdorferi sensu stricto B31 is 1,521,419 bp. This genome consists of a linear chromosome of 910,725 bp, with a G⫹C content of 28.6%, and 21 plasmids (9 circular and 12 linear) which have a combined size of 610,694 bp (52, 98). Comparative analysis of the genome of the recently sequenced Borrelia garinii strain PBi with that of B. burgdorferi B31 reveals that most of the chromosome is conserved (92.7% identity with regard to both DNA and amino acids) in the two species. The chromosome and two linear plasmids (lp54 and cp26), which carry approximately 860 genes, seem to belong to the basic genome inventory of the Lyme Borrelia species (105). Not all strains of B. burgdorferi have the complete complement of plasmids, and thus the cumulative genome size may vary among different B. burgdorferi isolates. Genome analysis has revealed that B. burgdorferi possesses certain genetic structures that are uncommon among prokaryotes (98). These include (i) a linear chromosome and mul-

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tiple linear and circular plasmids in a single bacterium; (ii) a unique organization of the rRNA gene cluster, consisting of a single 16S rRNA gene (rrs) and tandemly repeated 23S (rrl) and 5S (rrf) rRNA genes; (iii) over 150 lipoprotein-encoding genes, which account for 4.9% of the chromosomal genes and 14.5% of the plasmid genes, significantly higher than that of any other bacterial genome sequenced to date; (iv) a substantial fraction of plasmid DNA that appears to be in a state of evolutionary decay; (v) evidence for numerous, potentially recent DNA rearrangements among the plasmid genes; and (vi) a lack of recognized genes that encode enzymes required for synthesis of amino acids, fatty acids, enzyme cofactors, and nucleotides. B. burgdorferi also lacks genes coding for tricarboxylic acid cycle enzymes or for compounds involved in electron transport, findings which, taken together with the preceding, indicate the parasitic nature of this microorganism (52, 98, 353). B. burgdorferi Genospecies Eleven Borrelia species within the B. burgdorferi sensu lato complex have been described worldwide (16, 49, 99, 149, 168, 194, 264, 350). Of these, three species (B. burgdorferi sensu stricto, Borrelia andersonii, and Borrelia bissettii) have been identified in North America, five species (B. burgdorferi sensu stricto, B. garinii, Borrelia afzelii, Borrelia valaisiana, and Borrelia lusitaniae) have been recognized in Europe, and seven species (B. garinii, B. afzelii, B. valaisiana, Borrelia japonica, Borrelia tanukii, Borrelia turdi, and Borrelia sinica) have been identified in Asian countries (e.g., China, Japan, or Korea). Identification and differentiation of B. burgdorferi sensu lato species can be achieved by using several molecular approaches, which are reviewed elsewhere (351). At least three B. burgdorferi sensu lato species, i.e., B. burgdorferi sensu stricto, B. garinii, and B. afzelii, are pathogenic to humans in Europe (342, 351). In contrast, B. burgdorferi sensu stricto is the sole species known to cause human infection in the United States (195). However, several subtypes of B. burgdorferi sensu stricto have been identified (175, 176), and an association between specific subtypes of B. burgdorferi sensu stricto and hematogenous dissemination and invasion in patients (304, 370) and experimentally infected animals (348, 349) has been reported. Of the seven B. burgdorferi sensu lato species identified in Asia, only B. garinii and B. afzelii have been confirmed definitively to be pathogenic in humans. Although studies with both patients and laboratory animals have indicated the potential for B. bissettii, B. valaisiana, or B. lusitaniae to cause clinical disease (65, 81, 106, 257, 334), the pathogenicity of these Borrelia species in humans is not well established. The genetic relatedness of B. burgdorferi sensu lato isolates has been compared at the species, subspecies, or single gene level by various molecular approaches. Earlier analyses of B. burgdorferi sensu lato isolates representing different species suggested that these species had highly conserved chromosomal gene orders (51, 232) with linear plasmid profiles similar to that described for the B. burgdorferi sensu stricto type strain B31 (243). Recent studies, however, have documented genetic heterogeneity among B. burgdorferi sensu lato isolates in the United States (173, 195) and in Europe (263, 288, 352). A large

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number of DNA sequences of B. burgdorferi sensu lato genes are now available in the GenBank database from the National Center for Biotechnology Information (http://www.ncbi.nlm .nih.gov/entrez/). Examples include fla, vlsE, bmpA, and dbpA and genes encoding B. burgdorferi sensu lato 16S rRNA and outer surface proteins A and C. It is well known that B. burgdorferi sensu lato expresses different surface proteins in adaptation to various microenvironments (79, 95, 299). For example, the spirochete expresses OspA but not OspC when residing in the midguts of unfed ticks. However, during a blood meal by the tick, some spirochetes stop expressing OspA and instead express OspC (230, 298). Certain B. burgdorferi sensu lato genes either are expressed only in a mammalian host or have significantly upregulated expression in that environment; such gene products include VlsE (71, 230), DbpA (71, 129), BBK32 (96), Erp (202), and Mlp (376) proteins. Recently, whole-genome microarrays were employed to analyze gene expression of B. burgdorferi sensu stricto grown under conditions analogous to those found in unfed ticks, fed ticks, and mammalian hosts (32, 220, 231, 278). Gene expression analysis of B. burgdorferi B31 grown at 23°C and 35°C, to simulate temperatures found in tick vectors and mammalian hosts, respectively, demonstrated that a total of 215 open reading frames were differentially expressed at the two temperatures. Strikingly, 136 (63%) of the differentially expressed genes were plasmid carried, which highlights the potential importance of plasmid-carried genes in the adjustment of B. burgdorferi sensu lato to diverse environmental conditions (231). Genetic diversity and differential expression of B. burgdorferi sensu lato genes in patients have important implications for development of molecular assays and serologic tests in the laboratory diagnosis of LB. As discussed in the following sections, the choice of PCR primers targeting different segments of the B. burgdorferi sensu lato genome, as well as the selection of particular antigens for serologic assays, may affect the sensitivities and specificities of these diagnostic assays (252, 291). LYME BORRELIOSIS: DISEASE SPECTRUM Infection with B. burgdorferi sensu lato can result in dermatological, neurological, cardiac, and musculoskeletal disorders. The basic clinical spectra of the disease are similar worldwide, although differences in clinical manifestations between LB occurring in Europe and North America are well documented (219, 316, 351). Such differences are attributed to differences in B. burgdorferi sensu lato species causing LB on the two continents. Furthermore, differences in clinical presentations exist between regions of Europe, presumably due to differences in the rates of occurrence of infection caused by distinct B. burgdorferi sensu lato species (128, 288, 342). Patients with B. burgdorferi sensu lato infection may experience one or more clinical syndromes of early or late LB. Usually, early infection consists of localized erythema migrans (EM), which may be followed within days or weeks by clinical evidence of disseminated infection that may affect the skin, nervous system, heart, or joints and subsequently, within months, by late infection (219, 314, 316, 318, 373). Arthritis appears to be more frequent in North American patients (326, 332), whereas lymphocytoma, acrodermatitis chronica atrophi-

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cans (ACA), and encephalomyelitis have been seen primarily in Europe (313). EM is the characteristic sign of early infection with B. burgdorferi sensu lato and the clinical hallmark of LB. In recent series it is recognized in at least 80% of patients with objective clinical evidence of B. burgdorferi sensu lato infection who meet the CDC surveillance definition of LB (327). The rash begins at the site of the tick bite as a red macule or papule, rapidly enlarges, and sometimes develops central clearing. The clinical diagnosis of early LB with EM relies on recognition of the characteristic appearance of a skin lesion of at least 5 cm in diameter. At this stage, patients may either be asymptomatic or, more commonly in the United States, experience flu-like symptoms, such as headache, myalgia, arthralgias, or fever (309, 332). The presence of constitutional signs and symptoms in a patient with EM has been considered evidence of dissemination by some investigators, but this is not evidence based (192). Instead, we will refer to this clinical presentation as symptomatic EM later in this review. Hematogenous dissemination of B. burgdorferi sensu lato to the nervous system, joints, heart, or other skin areas, and occasionally to other organs, may give rise to a wide spectrum of clinical manifestations of what is called early LB. Usually, patients with objective evidence of dissemination experience one or more of the following syndromes: multiple EM lesions, atrioventricular conduction defects, myopericarditis, arthritis, facial palsy, meningitis, and meningoradiculoneuritis (Bannwarth’s syndrome) (238, 333, 343). Late LB may develop among some untreated patients months to a few years after tick-transmitted infection. The major manifestations of late LB include arthritis, late neuroborreliosis (peripheral neuropathy or encephalomyelitis), and ACA. Lyme arthritis begins as intermittent attacks of mono- or pauciarticular arthritis, especially of large joints. In up to 10% of patients, arthritis may persist for months or a few years despite treatment with antimicrobials. Treatment-resistant arthritis is more frequently seen in patients with the certain HLA DRB alleles (112, 145). It has been suggested that autoimmunity plays a role in this clinical entity (320, 338). While Lyme arthritis is the most common late manifestation of LB in North America, ACA appears to be the most common manifestation of late LB in Europe. As mentioned earlier, these differences are likely due to the different species causing LB in the two continents (219, 316, 351). More details on the clinical spectra of LB are found in recent reviews (219, 313, 314, 316, 372). LABORATORY DIAGNOSIS Direct Detection of B. burgdorferi A variety of laboratory techniques have been developed for direct detection of B. burgdorferi sensu lato These assays provide evidence for the presence of intact spirochetes or spirochete components such as DNA or protein in tick vectors, reservoir hosts, or patients. Four different approaches have been used in the clinical laboratory: microscope-based assays, detection of B. burgdorferi-specific proteins or nucleic acids, and culture. Of these, culture of B. burgdorferi sensu lato undoubtedly offers the best

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TABLE 1. Selected features of various modifications of Kelly medium Name

Description (reference[s])

Stoenner modification......................................Addition of Yeastolate and CMRL 1066, without glutamine and without sodium bicarbonate (329) Barbour modification (BSK II) ......................Use of CMRL 1066 without glutamine; Yeastolate; neopeptone as the peptone preparation; and HEPES as a buffer (17, 19) Preac-Mursic modification (MKPa) ...............Removal of Yeastolate; different proportions of certain ingredients (267) BSK-H................................................................Deletion of gelatin; different proportions of certain ingredients (262) a

Also referred to as the Pettenkofer modification (256).

confirmation of active infection and has been increasingly used as a diagnostic modality by many researchers on both sides of the Atlantic. The availability of cultured organisms has also allowed investigation of the structural, molecular, antigenic, and pathogenetic properties of the different B. burgdorferi sensu lato species. Direct microscopic detection of B. burgdorferi sensu lato has limited clinical utility in laboratory confirmation of LB due to the sparseness of organisms in clinical samples (24, 27, 75–77, 125, 139, 153, 156, 190, 212, 221, 310). Antigen detection assays (aside from PCR) also suffer from the same limitations as microscopic detection. Although antigen capture tests have been used to detect B. burgdorferi sensu lato antigens in CSF of patients with neuroborreliosis (67, 69) and in urine samples from patients with suspected LB (83, 131, 155), their reliability is poor or at best questionable (155). Our review of direct methods of detection of B. burgdorferi sensu lato in clinical samples will focus on culture and molecular methods. Culture of B. burgdorferi sensu lato. (i) Culture techniques. The liquid media currently used to grow B. burgdorferi sensu lato were derived from the original Kelly medium (152) through various modifications made over time (17, 19, 267, 328, 329). Current versions of this medium (Barbour-Stoenner-Kelly II medium [BSK II] [17], BSK-H [262], and Kelly medium Preac-Mursic [MKP] [267]) are better able to support growth of B. burgdorferi sensu lato in terms of recovery from low inocula, shorter generation times of the spirochete, and maximal concentration of spirochetes in culture (⬃108 to 109/ ml) (Table 1). Key ingredients of BSK II include CMRL-1066, which is a standard medium used for growing various types of mammalian cells; bovine serum albumin fraction V, which serves as a rich source of protein and to stabilize the pH; N-acetylglucosamine, a precursor for bacterial cell wall biosynthesis; rabbit serum; citrate; pyruvate; and many others. The growth-promoting capability of BSK II and related media depends upon the careful selection of certain key components that may be highly variable in composition depending on their source (262), particularly the specific preparations of bovine serum albumin (43) and rabbit serum (262). For example, a minority of preparations of rabbit serum contain antispirochetal antibodies, and inclusion of such preparations in the medium will reduce or entirely abrogate growth of B. burgdorferi sensu lato (262). Consequently, as a necessary quality control measure, each new batch of liquid medium must be tested for its ability to support adequately the growth of laboratory-adapted strains of B. burgdorferi sensu lato. Once prepared, BSK-H medium can be preserved for future use at ⫺20°C for at least 8 months (262). Cultures in liquid medium are usually incubated at 30° to 34°C under microaerophilic conditions. Incubation at temper-

atures of 39°C or higher may reduce or prevent growth (17). Cultures are incubated for up to 12 weeks, which is much longer than is necessary to grow most other human bacterial pathogens, due in part to the spirochete’s prolonged generation time (7 to 20 h or longer) during log-phase growth (17, 266, 353). Detection of growth is accomplished by periodic examination of an aliquot of culture supernatant for the presence of spirochetes by dark-field microscopy or by fluorescence microscopy after staining with the fluorochrome dye acridine orange or a specific fluorescence-labeled antibody (277, 367). Visualized spirochete-like structures should be confirmed as B. burgdorferi sensu lato by demonstration of reactivity with specific monoclonal antibodies or by detection of specific DNA sequences by using PCR methodology (215, 312, 353, 367). Lack of experience in the microscopic detection of B. burgdorferi sensu lato can lead to false-positive readings, as other structures such as cellular debris may appear thread-like and thus be mistaken for B. burgdorferi sensu lato (110). B. burgdorferi sensu lato can also be grown on solid media with agarose to solidify the liquid media discussed above and incubated under microaerophilic or anaerobic conditions (17, 158, 266). An advantage of using solid media is that individual colonies can be identified as a means to select out particular clonal strains of B. burgdorferi sensu lato. Laboratory-propagated strains of B. burgdorferi sensu lato can be successfully cocultivated with tick cell lines (157, 213, 229) and with certain mammalian cell lines (121, 311, 341). Cocultivation techniques may prove to be useful for primary isolation of B. burgdorferi sensu lato from clinical specimens as well (311, 341). (ii) Culture of clinical specimens. B. burgdorferi sensu lato can be recovered from various tissues and body fluids of patients with LB, including biopsy (14, 25, 31, 140, 165, 178, 200, 204, 209, 214, 221, 227, 237, 256, 267, 303, 327, 333, 342, 369) and lavage (369) specimens of EM skin lesions, biopsy specimens of ACA skin lesions (14, 258, 267, 342), biopsy specimens of borrelial lymphocytoma skin lesions (188), cerebrospinal fluid specimens (60, 147, 237, 267, 342), and blood specimens (13, 22, 189, 216, 221, 322, 367, 368, 374). Anecdotally, recovery of B. burgdorferi sensu lato from other tissue or fluid specimens (189), such as synovial fluid (289), cardiac tissue (312), and iris (265), has also been reported. Aside from the late cutaneous manifestation ACA, from which B. burgdorferi sensu lato has been recovered more than 10 years after onset of the skin lesion (14), the vast majority of successful isolations have been from untreated patients with early disease (EM or early neuroborreliosis) (147, 219, 303, 351, 360). (a) Erythema migrans. Recovery of B. burgdorferi sensu lato from 2- to 4-mm skin biopsy samples of an EM skin lesion can

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TABLE 2. Cultivation of Lyme borreliae from clinical samples Representative culture yield (%) (reference[s]) Site United States

Europe

Skin Erythema migrans

⬎50a (25, 200, 204, 214, 227, 303, 327, 369)

Acrodermatitis chronica atrophicans Borrelial lymphocytoma Cerebrospinal fluid Synovial fluid Blood

NDh ND ND Anecdotal ⬎40e (367, 368)

ⱖ40b (14, 31, 140, 165, 209, 237, 256, 333, 342, 380) ⱖ22c (14, 258, 342) 24 (188) 10d (60, 147, 237, 342) Anecdotal 1.2f to 9g (13, 189)

a

Highest reported yield, 86% (25). Highest reported yield, 88% (342). Highest reported yield, 60% (342). d Highest reported yield, 17% (342). e Plasma (high volume, ⱖ9 ml) of untreated adult United States patients with erythema migrans. f Plasma (low volume, ⱕ3 ml) of European adults with erythema migrans. g Plasma (low volume, ⬍1 ml) of untreated European children with erythema migrans. h ND, no data available. b c

typically be achieved for at least 40% of untreated patients (Table 2). The highest reported success rates were 86% in a study of 21 U.S. patients for whom 4-mm skin biopsy specimens were cultured (25) and 88% in a study from The Netherlands of 57 patients who underwent a 4-mm skin biopsy (342). B. burgdorferi sensu lato can be recovered from both primary (site of tick bite) and secondary (presumed to arise by hematogenous dissemination) EM lesions (204, 213). Despite the clinical tradition of biopsying the advancing border of an EM lesion, a recent systematic study of this question conducted in Europe found comparable yields from biopsy specimens taken from the EM center (140). According to one report, positive cultures can also be obtained from clinically normal-appearing skin 4 mm beyond the EM border (25). B. burgdorferi sensu stricto can also be grown from culture of saline injected into and then withdrawn from an EM lesion. In one study of a group of U.S. patients with EM, cultures were established for both a 2-mm skin biopsy sample and a lavage sample in which nonbacteriostatic sterile saline was injected into the same EM lesion and then recovered using a novel two-needle technique (369). A version of this technique had previously been found to be successful in recovering B. burgdorferi sensu lato from the skin of infected animals (259). In the clinical study, excluding contaminated samples, B. burgdorferi sensu stricto was recovered from 20 (74%) of the 27 skin biopsy specimens, compared with 12 (40%) of the 30 lavage samples (P ⬍ 0.05) (369). Although less sensitive, cutaneous lavage has the advantage of being less invasive. Recovery of B. burgdorferi sensu stricto from solitary EM lesions is significantly more likely in U.S. patients with skin lesions of shorter duration (214), suggesting that the patient’s immune response is usually effective in clearing the spirochete from that skin site over a relatively short time interval. Consistent with this premise are the results of a recent study in which the number of spirochetes in 2-mm skin biopsy samples of the advancing border of an EM skin lesion was determined using a quantitative PCR (qPCR) technique (177). This study demonstrated that significantly fewer spirochetes were present in older or larger EM lesions (in the United States the size of an EM lesion correlates directly with its duration) (23, 214,

215). The same phenomenon probably explains a much earlier clinical observation that in the absence of antibiotic therapy, EM lesions will nevertheless resolve in a median time period of approximately 4 weeks (317). Clearance of spirochetes from the skin in the absence of antibiotic treatment has also been documented in a rhesus macaque model of B. burgdorferi sensu stricto infection (254). Borrelia burgdorferi sensu stricto usually cannot be recovered on culture of EM lesions of patients already receiving appropriate antibiotic treatment (372) and rarely, if ever, cannot be recovered from the prior site of a resolved EM lesion in U.S. patients who have completed a course of appropriate antimicrobial therapy (26, 214). Borrelia burgdorferi sensu stricto has been recovered, however, from cultures of EM lesions of patients treated with the narrow-spectrum cephalosporin cephalexin, an antibiotic which is inactive in vitro against B. burgdorferi sensu lato and ineffective clinically (4, 226). (b) Whole blood, serum, and plasma. The rate of recovery of B. burgdorferi sensu stricto from blood or blood components of untreated patients with EM had generally been 5% or less (22, 108, 323, 345), and until recently this source of culture material was largely abandoned. In past studies on the sensitivity of blood cultures for recovery of B. burgdorferi sensu stricto, only a very small volume of less than 1 ml of blood or blood components was cultured (22, 108, 216, 217, 322, 323, 345). For other bacterial infections, however, the volume of blood cultured is an important determinant of yield (201, 354, 356). The reason for this is that with more conventional pathogens the number of cultivable bacteria per milliliter of whole blood is less than 1 in 50% of bacteremic patients and less than 0.1 in nearly 20% (82, 151). Therefore, in view of the recommendation to culture quantities of blood as large as 20 to 30 ml for other bacterial infections, the rationale for culturing much smaller volumes for LB patients was open to challenge. In a series of recent experiments with adult patients with EM from the United States, it was demonstrated that recovery of B. burgdorferi sensu stricto was better from serum than from an identical volume of whole blood (374) and that the yield from plasma was significantly greater than that from serum (367). Yield directly correlated with the volume of material cultured.

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Recovery of B. burgdorferi sensu stricto from high-volume cultures of 9 ml of plasma inoculated into a modified BSK II preparation devoid of antimicrobials and gelatin, with a 20:1 ratio of medium to plasma, was consistently above 40% (367, 368). Unfortunately, high-volume plasma cultures are not appropriate for young children, since obtaining such a large quantity of blood would be unacceptable. Reported rates of recovery from blood of patients with EM in Europe have been ⬍10% (13, 189). This may be due to a low frequency of hematogenous dissemination of B. afzelii, the principal cause of EM in Europe (351), or to culturing an inadequate volume of blood. A study that addressed further increasing the volume of plasma from 9 ml to 18 ml for adult U.S. patients with EM found only a small increment of approximately 10% in culture yield (368), suggesting that if substantially greater yields from blood cultures are still possible, it will be by further modifying the culture medium rather than by increasing the volume of plasma cultured. In that study it was estimated that the average number of cultivable B. burgdorferi sensu stricto cells per milliliter of whole blood was approximately 0.1, which, if correct, would explain why blood cultures had such a consistently low yield in former studies in which the volume of blood or blood components cultured was extremely small (368). A recent study of patients with EM showed a greater recovery of B. burgdorferi sensu stricto from blood of symptomatic patients; nevertheless, the majority of symptomatic patients had negative blood cultures (371), raising doubts about the reliability of the assumption that the presence of symptoms indicates dissemination. Although less extensively studied, culture of blood samples is rarely positive in patients with any objective clinical manifestation of LB other than EM (191, 216; J. Nowakowski, unpublished observations). Blood cultures are also negative in patients with persistent subjective symptoms following completion of an appropriate course of antibiotic treatment (154, 191). (iii) Sensitivity of culture. Although a number of as low as one spirochete can be recovered on culture using laboratoryadapted and continuously propagated strains of B. burgdorferi sensu stricto (17, 254, 262, 331), the sensitivity of culture for clinical specimens is undefined. Compared to PCR for detection of spirochetes in cutaneous specimens, culture has proven to be slightly more sensitive in some studies but not in others (31, 165, 209, 227, 256, 303, 327, 380). Such disparate results are likely to be attributable to differences in the various studies in the PCR protocols employed, including type of PCR and/or primer and target selection and/or method of tissue preservation, as well as differences in culture techniques, including size of the skin biopsy sample cultured and/or choice of culture medium. In a recent U.S. study of skin biopsy samples of 47 untreated adult patients with EM lesions, culture grew B. burgdorferi sensu stricto in 51%, compared with an 81% detection rate using a qPCR method (P ⫽ 0.004 by Fisher’s exact test, two tailed) and a 64% detection rate using a nested PCR (P ⫽ 0.3) (227). Although all of the different B. burgdorferi sensu lato species and subspecies recognized to cause human infection can be cultivated, it is not clear whether the sensitivity of culture is identical for each. In one study, subtyping of B. burgdorferi sensu stricto was performed directly on 51 skin biopsy samples

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of patients with EM by using PCR-restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and also on the strains of B. burgdorferi sensu stricto that grew from culture of the same biopsy samples (176). No significant difference in the rate of recovery of any single subtype was observed. However, due to the relatively small number of isolates of B. burgdorferi sensu stricto in that study, further investigation of this question is needed. (iv) Practical considerations. Culture has been principally used as a diagnostic modality in research studies and has enabled a better understanding of the clinical, laboratory, and pathogenetic aspects of LB through identification of a group of culture-authenticated patients (7, 215, 309, 332, 333). For example, the recognition that substantive differences exist in the clinical manifestations of patients with EM caused by B. burgdorferi sensu stricto compared with those with EM caused by B. afzelii was based on analysis of culture-confirmed cases (332). In addition, culturing of ticks, EM skin lesions, and blood samples was instrumental in demonstrating that different subtypes of B. burgdorferi sensu stricto vary in their potential to spread to extracutaneous sites (304, 370). Patients with cultureconfirmed LB have also been a valuable source of specimens to assess the accuracy of other diagnostic tests (7, 203). Such well-defined patient populations have also played an important role in studying therapeutic regimens (375) and vaccines (327). Culture, however, has not been used in routine clinical practice as a diagnostic test for several reasons. One reason has been the lack of consistent availability of high-quality (i.e., borrelial growth-promoting) lots of liquid medium for growing B. burgdorferi sensu lato. Until fairly recently, this medium was not sold commercially, and although BSK-H is now available commercially, it has periodically been in short supply or of variable quality. Furthermore, by most conventional bacteriologic standards, borrelial cultures are more labor-intensive, more expensive, and much slower, requiring up to 12 weeks of incubation before being considered negative. The rapidity of identifying a positive culture, however, is directly dependent on the frequency with which the culture supernatant is examined microscopically, since macroscopic changes in the appearance of the culture medium tend to occur later, if at all. In our research studies, in which B. burgdorferi sensu stricto cultures are usually first examined only at 2 weeks, 70% of positive cultures of skin biopsy samples of EM lesions and approximately 85% of positive cultures of plasma from EM patients turned positive at 2 weeks, and approximately 95% of each type of sample turned positive by 4 weeks (G. P. Wormser, unpublished observations). The time to detection of a positive specimen can be reduced to a clinically relevant time frame if cultures are examined on a daily basis (203). For example, the Marshfield Laboratories identified 74 (82%) of 90 positive cultures within the first 7 days of incubation and 32 (35%) within the first 3 days (277). In the experience of that laboratory, the longest time to detection of a positive culture was 16 days. Culture positivity might be detected even more quickly if aliquots of culture supernatant were examined by PCR, rather than microscopy, to detect B. burgdorferi sensu lato DNA (301), and in the future it is conceivable that such an approach might be adaptable to automation.

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Another limiting factor is that culture is useful only for untreated patients. Culture positivity is rapidly aborted by even a few doses of appropriate antibiotic treatment (214, 303). Perhaps the most fundamental limitation is that culture is far too insensitive in patients with extracutaneous manifestations of LB, which is unfortunately the group of patients who pose the greatest diagnostic confusion (219, 314). Culture has been most successful for patients with early LB who have EM, but visual recognition of the characteristic appearance of the skin lesion is usually sufficient for an accurate diagnosis, and no laboratory testing is indicated (218, 219). Culture would be helpful only in a minority of cases in which the rash may be atypical or the patient was not known to have had tick exposure in an area where LB is endemic. Molecular methods of detection of B. burgdorferi sensu lato. For laboratory diagnosis of LB, the utilization of molecular techniques has focused mainly on PCR-based methods. The first PCR assay for specific detection of a chromosomally encoded B. burgdorferi sensu lato gene was reported in 1989 (282). Various other PCR protocols were subsequently developed for detection of B. burgdorferi sensu lato DNA in clinical specimens and were reviewed by Schmidt in 1997 (291). (i) PCR analysis of clinical specimens. Given that the number of spirochetes in infected tissues or body fluids of patients is very low, appropriate procedures for sample collection and transport and preparation of DNA from clinical samples are critical for yielding reliable and consistent PCR results. A variety of clinical specimens from patients with suspected Lyme disease have been analyzed by PCR assays (291). Of these, skin biopsy samples taken from patients with EM or ACA have been the most frequently tested specimens (346). Depending on the clinical manifestations of the patients, appropriate body fluid samples (e.g., blood, CSF, or synovial fluid) can be collected and analyzed by PCR. The sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi sensu lato DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes will have migrated from the skin biopsy to the culture medium. In this case, DNA should be prepared from both the skin biopsy and the medium and analyzed by PCR separately. Comparative analysis using a quantitative PCR assay of skin biopsy specimens placed in BSK overnight to 2 days (177) has demonstrated a higher copy number of B. burgdorferi sensu stricto DNA in samples extracted from the medium than in those extracted from the skin sample. This may be attributed to migration of the spirochetes out of the skin sample and/or to the presence of PCR inhibitors in skin (G. Wang, unpublished data). Alternatively, spirochetes that have migrated into the medium can be collected by centrifugation and subjected to DNA extraction together with the biopsy tissue. Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection, or they should be kept frozen. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that with DNA from paraffin-embedded, formalin-fixed tissues (163). As host DNA can interfere with PCR detection of B. burgdorferi sensu lato in clinical and tick samples (62, 302), an optimized DNA extraction procedure is essential to yield reli-

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able PCR results for certain clinical samples (28). Also, PCR inhibitors may be present in various biological samples (blood, urine, synovial fluid, and CSF) obtained from patients (31); this can usually be assessed by spiking negative samples with a known number of spirochetes or particular amounts of spirochetal DNA during DNA extraction or PCR amplification. In most cases, inhibition of PCR may be minimized by dilution of the extracted DNA (62). PCR results can be qualitative (conventional PCR and nested PCR) or quantitative (competitive PCR and real-time PCR). Each of these PCR methods has its advantages and disadvantages. For laboratory diagnosis of B. burgdorferi sensu lato infection, a qualitative PCR is usually sufficient. Nevertheless, several real-time PCR instruments such as the Sequence Detection System (Applied Biosystems, Inc.) and LightCycler (Roche Diagnostics, Inc.) are now commercially available and offer options for automation in a clinical laboratory setting (90). The efficiency of a PCR assay is determined by several factors. Among these, the selections of an appropriate gene target and primer set for PCR amplification are the most important in development of any new PCR protocols. In general, a PCR primer set yielding an amplicon of 100 to 300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can reduce the effects of DNA fragmentation during sample processing. Although PCR assays targeting numerous B. burgdorferi sensu lato genes have been employed in research laboratories, only a few of these genes have been utilized by clinical laboratories as targets for PCR analysis of B. burgdorferi sensu lato DNA in clinical specimens. These include chromosomally carried genes such as rRNA genes, flaB, recA, and p66 and the plasmid-carried gene ospA (291). (a) PCR analysis of skin biopsy samples from patients with cutaneous manifestations. B. burgdorferi sensu lato DNA was first detected by PCR in skin biopsies from three of four patients with EM and four of five patients with ACA in The Netherlands in 1991 (199). In 1992, Schwartz et al. reported on the detection of B. burgdorferi-specific rRNA genes in skin biopsies from EM patients in the United States (303). PCR targets that have been employed for detection of B. burgdorferi sensu lato DNA in skin biopsy specimens include p66 (31, 210, 211, 344, 358, 359), the 16S rRNA gene (296, 303), fla (177, 234, 256), the 23S rRNA gene (31), the 5S rRNA-23S rRNA gene spacer (279), recA (177), and ospA (209, 279, 327). The sensitivity of PCR for detection of B. burgdorferi sensu lato DNA in EM lesions is usually high, ranging from 36% to 88% (Table 3). A prospective study in the United States showed that 85 of 132 (64%) skin biopsies taken from EM patients during a phase III vaccine trial were positive by PCR (327). B. burgdorferi sensu lato-specific DNA has been detected in 54 to 100% of skin biopsy samples from patients with ACA in Europe (Table 3). The sensitivity of PCR for detection of B. burgdorferi sensu lato in skin biopsy samples from ACA patients appears to be dependent on the target sequences selected. Rijpkema et al. reported that 15 of 24 (63%) skin biopsies from patients with ACA in The Netherlands were positive by a nested PCR targeting ospA; only 10 of these

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TABLE 3. Sensitivities and specificities of PCR assays for detection of B. burgdorferi DNA in different clinical specimens from patients with LBa No. of studies included

Median % sensitivityb (range)

Reported % specificity range

16 4 12 8

69 (36–88) 64 (59–67) 73 (36–88) 76 (54–100)

98–100 98–100 100 100

6 3 3

14 (0–100) 18 (0–59) 10 (4–100)

100 NAc

CSF United States Europe

16 6 10

38 (12–100) 73 (25–93) 23 (12–100)

93–100 93–100 98–100

Synovial fluid United States Europe

8 4 4

78 (42–100) 83 (76–100) 66 (42–85)

100 100 100

Clinical specimen and region

Skin biopsy EM United States Europe ACA, Europe Blood, plasma, serum United States Europe

References

177, 301, 303, 327 31, 165, 209–211, 234, 256, 272, 279, 344, 358, 359 31, 199, 209, 210, 256, 279, 344, 359 108, 172, 345 78, 233, 234 134, 150, 172, 180, 223, 239 11, 57, 74, 88, 130, 162, 233, 236, 270, 377 30, 172, 224, 252 87, 133, 270, 293

Only studies published in MEDLINE-indexed periodicals during the years 1991 to 2003 and those examined by PCR assay for ⱖ5 cases are included. Median sensitivity of PCR assays based on included studies. For studies tested with multiple PCR primer sets, the highest sensitivity reported was selected for analysis. c NA, not available. a b

samples (42%) were positive if a fragment of the 5S-23S rRNA gene intergenic spacer was targeted (279). Results also depended on which genes were used as targets in PCR for skin biopsies from ACA patients in a small study reported from Germany. Four of five patients with ACA were detected by PCR targeting the p66 gene, versus two of five when the 23S rRNA gene was amplified (31). (b) PCR analysis of blood from patients with LB. B. burgdorferi sensu lato DNA has been detected by PCR in blood samples from patients with EM (108, 234) and early disseminated disease such as neuroborreliosis and carditis (78, 172). In a prospective study of U.S. patients with EM, B. burgdorferi sensu stricto DNA was detected by PCR in 14 of 76 (18.4%) plasma samples (108). In general, the sensitivity of PCR for detection of B. burgdorferi sensu lato DNA in blood, plasma, or serum samples from patients with Lyme disease is low (Table 3). The low yield could be a reflection of lack of spirochetemia or transient spirochetemia (22), a low level of spirochetes in blood (108), and/or the presence of PCR inhibitors in host blood (2, 62, 345). In one study, PCR-documented spirochetemia in patients with EM was correlated with symptomatic illness and with the presence of multiple EM lesions (108); multivariate analysis indicated that a high number of systemic symptoms was the strongest independent predictor of PCR positivity (108). None of 78 patients with post-Lyme disease syndromes (musculoskeletal pains, neurocognitive symptoms, dysesthesia, fatigue, malaise, headache, or sleep disturbance) had detectable DNA in blood specimens, despite a positive Western immunoblot (IB) for immunoglobulin G (IgG) antibodies against B. burgdorferi sensu stricto in 39 of these patients (154). (c) PCR analysis of CSF specimens from patients with neuroborreliosis. B. burgdorferi sensu lato DNA has been detected by PCR in CSF specimens from patients with a variety of neurological symptoms in the United States and in Europe

(Table 3). Lack of a gold standard method to support the diagnosis of neuroborreliosis makes it difficult to assess the performance of PCR with CSF. The sensitivity of PCR for detection of B. burgdorferi sensu lato DNA in CSF specimens may be dependent on the clinical presentation, CSF white cell counts, disease duration, and whether antibiotic treatment was given. In a study of 60 U.S. patients with neuroborreliosis (16 with early and 44 with late neuroborreliosis), the sensitivity of PCR in CSF was 38% in early and 25% in late neuroborreliosis, and an inverse correlation was found between duration of antimicrobial treatment and PCR results (223). In this study, four different PCR primer or probe sets were used, three targeting OspA genes and one targeting OspB genes, and concordance between the different assays was poor. In a Swedish study, B. burgdorferi sensu lato DNA was detectable only in LB patients with CSF pleocytosis (7/36; 19.4%). None of 29 patients with clinical signs of LB (EM, cranial neuritis, or radiculoneuritis) without CSF pleocytosis was positive by PCR analysis of CSF specimens (236). Another study found that 7 of 14 (50%) neuroborreliosis patients from Denmark with disease duration of less than 2 weeks yielded a positive PCR result, compared with only 2 of 16 (13%) patients in whom the illness duration was greater than 2 weeks (P ⫽ 0.045) (165). (d) PCR analysis of synovial fluid from patients with Lyme arthritis. PCR analysis of synovial fluid is a much more sensitive approach than culture for detection of B. burgdorferi sensu lato in affected joints of patients with Lyme arthritis (30, 87, 133, 172, 224, 252, 270). In a U.S. study of 88 patients, B. burgdorferi sensu stricto DNA was detected in synovial fluid of 75 (85%) patients with Lyme arthritis (224). The PCR positivity rate was lower in patients who had received antibiotic therapy than in untreated patients. Of 73 patients who were untreated or treated with only short courses of oral antibiotics, 70 (96%) had a positive PCR in synovial fluid samples. In contrast, B. burgdorferi sensu

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stricto DNA was demonstrated in only 7 of 19 (37%) patients who received either parenteral antibiotics or oral antibiotics for more than 1 month (224). Four PCR primer sets were used in this study, three amplifying DNA sequences encoding OspA and one targeting a portion of the gene encoding 16S rRNA. Among the 75 patients with positive PCR results, the most sensitive primer-probe set detected B. burgdorferi sensu stricto DNA in 89%, versus 56% for the least sensitive set. Only 48 of the 75 (64%) patients with a positive PCR result were positive with all three OspA primers (224). Therefore, the yield of PCR to detect B. burgdorferi sensu lato DNA in synovial samples will depend on the primer-probe set (s) used and the duration of antimicrobial therapy. It is noteworthy that B. burgdorferi DNA has been detected in synovial membrane samples of patients whose synovial fluid specimens were PCR negative after antibiotic treatment (269). The observation of higher sensitivity of PCR targeting B. burgdorferi sensu stricto plasmid-encoded OspA than of that targeting the 16S rRNA chromosomally encoded genes in synovial fluid specimens (224, 252) has been referred to as “target imbalance.” It has been speculated that B. burgdorferi sensu stricto present in the synovium may selectively shed OspA DNA segments into the synovial fluid. (e) PCR analysis of urine samples from patients with early Lyme borreliosis. B. burgdorferi sensu lato has been frequently recovered from culture of urinary bladder specimens of experimentally infected laboratory animals (21, 348), suggesting that this spirochete could be excreted into the urine. However, although there have been reports of detection of B. burgdorferi DNA by PCR in urine specimens from patients with EM (28, 164, 290, 292), with neuroborreliosis (130, 146, 164, 172, 187, 270), or with Lyme arthritis (109, 146, 270), the sensitivity was highly variable. In addition, nonspecific amplification in urine PCR using different targets has been documented (31, 146, 172, 290). Thus, it is not appropriate to use urine PCR for the laboratory diagnosis of LB. (ii) Real-time quantitative PCR. A real-time PCR assay was first used for quantitation of B. burgdorferi DNA in tissues from experimentally infected laboratory mice (208, 242). Subsequently, it has been employed to analyze the number of spirochetes in field mice (33, 349), dogs (330), field-collected or laboratory-infected tick vectors (103, 260, 347), and clinical specimens of patients with LB (177, 296; reviewed in reference 346). Also, real-time PCR assays have been utilized to genotype the pathogenic B. burgdorferi sensu lato species in both ticks and EM patients in Europe (207, 261, 276). In addition, real-time multiplex PCR assay has been applied for simultaneous detection of B. burgdorferi sensu stricto and Anaplasma phagocytophilum infections in ticks (66). Recently, the number of spirochetes in clinical specimens of patients with LB was determined by real-time qPCR assays (177, 296). In one study, B. burgdorferi sensu stricto-specific recA DNA was detected by a LightCycler qPCR assay in 40 (80%) skin biopsy samples from 50 untreated adult U.S. patients with EM (177). The number of spirochetes in a 2-mm biopsy ranged from 10 to 11,000, with a mean number of 2,462 spirochetes. Significantly higher numbers of spirochetes were detected in culture-positive than in culture-negative skin specimens (3,940 versus. 1,642 spirochetes; P ⬍ 0.01). In another study, B. burgdorferi sensu lato fla was detected using a Taq-

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Man probe in 5 of 28 (17.9%) synovial fluid specimens and 1 of 5 (20%) synovial membrane biopsies obtained from 31 patients with arthropathies in Switzerland (296). The numbers of spirochetes varied from 20 to 41,000/ml of synovial fluid. Of 56 CSF samples from 54 patients with a clinical suspicion of neuroborreliosis, only one (1.8%) was positive by real-time PCR (296). It is not clear whether these CSF specimens were simultaneously analyzed by conventional PCR or any other molecular assays. (iii) PCR sensitivity and specificity. Table 3 summarizes the sensitivities and specificities of PCR assays for the detection of B. burgdorferi DNA in different clinical samples as published in MEDLINE-indexed periodicals during the years 1991 to 2003. Of the 24 studies in which B. burgdorferi sensu lato DNA in skin biopsies was examined, the sensitivities of the PCR assays varied from 36 to 88% for patients with EM and from 54 to 100% for patients with ACA. The median sensitivities of the reported PCR assays for detection of B. burgdorferi DNA in skin biopsies from patients with EM and ACA were 69% and 76%, respectively. The sensitivity of PCR assays for detection of B. burgdorferi sensu lato in whole blood (plasma or serum) and CSF specimens is low (Table 3). By contrast, higher PCR sensitivities were reported in both U.S. and European studies with synovial fluid samples from patients with Lyme arthritis. A published meta-analysis also demonstrated that PCR is a very sensitive approach when it is employed to detect B. burgdorferi sensu lato DNA in skin biopsy and synovial fluid specimens from patients with LB, whereas the diagnostic value of PCR assays for detection of B. burgdorferi sensu lato DNA in blood (plasma or serum) and CSF specimens is low (85). (iv) Applications and limitations of molecular methods. PCR-based molecular techniques have been employed for (i) confirmation of the clinical diagnosis of suspected LB, (ii) molecular species identification and/or typing of the infecting spirochetes in clinical specimens or on cultured isolates, and (iii) detection of coinfection of B. burgdorferi sensu lato and other tick-borne pathogens. However, PCR assays have not been widely accepted for laboratory diagnosis of LB because of low sensitivity in CSF and blood. PCR as a diagnostic tool may be hampered by potential false-positive results due to accidental contamination of samples with a small quantity of target DNA. False-positive PCR results have been reported for LB (206). Although PCR is highly sensitive for detection of B. burgdorferi sensu lato DNA in skin biopsy samples from patients with EM (85), such testing is rarely necessary, as a clinical diagnosis can be easily made if the characteristic skin lesion is present. For patients with LB involving systems other than skin, PCR sensitivity is in general low, with the exception of patients with Lyme arthritis. Immunologic Diagnosis of B. burgdorferi Sensu Lato Infection The complexity of the antigenic composition of B. burgdorferi sensu lato has posed challenges for the serodiagnosis of LB. As described above, a sizable number of antigens are differentially expressed in the vector and the host (103, 300), and some are exclusively expressed in vivo in the infected mammalian host (72, 94). Furthermore, antigenic differences

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exist among the B. burgdorferi sensu lato species causing LB (117, 135, 281, 336). Due to limitations in direct detection of B. burgdorferi sensu lato in clinical specimens, antibody detection methods have been the main laboratory modality used to support a clinical diagnosis of LB. Although a cellular immune response is also elicited, most methods used in the laboratory confirmation of LB involve detection of serum antibodies. It is, however, important to emphasize that relatively few studies have evaluated serologic tests in culture-confirmed populations. Therefore, conclusions derived from published studies on the serology of patients with clinically defined Lyme borreliosis, discussed later in this review, have inherent limitations. B. burgdorferi sensu lato antigens of importance in immunodiagnosis. It is important to understand the antigenic composition of B. burgdorferi sensu lato, as it pertains to immunodiagnosis. Numerous early studies recognized the importance of the flagellar protein flagellin (41 kDa), or FlaB, as an immunodominant antigen (63, 70). Strong IgG and IgM responses to this protein are developed within a few days after infection with B. burgdorferi sensu lato (8, 84, 111). Thus, some immunoassays consist of purified flagella alone (114, 115, 138, 148), whereas in others, flagellin is added to enrich the antigenic mixture (174). Unfortunately, although highly immunogenic, this antigen is highly cross-reactive with antigens in other bacteria, particularly when denatured, as in immunoblots (35, 91, 179). Certain flagellin epitopes are also cross-reactive with antigens found in mammalian tissues such as neural tissues, synovium, and myocardial muscle (1, 179). The internal portion of the flagellin molecule, containing the variable, genus-specific immunodominant domain, is less cross-reactive with antigens of other bacteria than the whole protein (101, 179, 274). The flagellar outer sheath protein FlaA, with a molecular size of 37 kDa, is another immunodominant antigen, especially in early disease (7, 84, 104, 246). One of the most immunodominant antigens early after infection with B. burgdorferi sensu lato is the plasmid-encoded OspC protein (molecular size of about 21 to 25 kDa) (8, 84, 89, 241, 363), which begins to be expressed during tick feeding while the spirochete is still in the tick midgut. Highly passaged in vitro-cultured B. burgdorferi sensu lato does not express OspC, which explains why the importance of this antigen was unrecognized in early studies that used high-passage B. burgdorferi sensu lato as the source of antigen (63, 70). OspC is heterogeneous, and amino acid differences exist among the sequences of OspC proteins from the different B. burgdorferi sensu lato species (135, 197, 336). Furthermore, intraspecies differences also exist; for example, at least 13 OspC serotypes have been identified in B. garinii by using a panel of monoclonal antibodies (361). Sixty-nine ospC groups have been described among B. burgdorferi sensu lato species isolated from different sources in Europe and United States when assayed using the technique of single-strand conformational polymorphism (159). Of interest is that invasive B. burgdorferi sensu lato strains appear to belong to just 24 of the 69 ospC groups (159). It is postulated that OspC is an important virulence factor for both infectivity and invasiveness of B. burgdorferi sensu lato species (86, 193, 304). Antibodies elicited against OspC may be borreliacidal and thus may play a role in the functional antibody assays described below. The search for

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immunogenic, conserved epitopes within the OspC protein has led to the development of a synthetic peptide that contains the conserved C-terminal 10 amino acids of OspC (pepC10) (198). The chromosomally encoded 39-kDa protein BmpA (306) is also immunogenic. The gene encoding this protein is located in a bmp cluster that also includes genes for BmpB, BmpC, and BmpD; all have molecular sizes similar to that of BmpA (271). However, it is unclear if BmpB through -D have any utility as antigens in serologic assays. Genetic and antigenic differences have been found among BmpA sequences of different B. burgdorferi sensu lato species, which may limit the use of this antigen in serologic testing (281). Decorin binding protein A (DbpA) (also referred to as Osp17), which has a molecular size of approximately 17 kDa, is immunogenic (124, 136). DbpA is associated with binding of B. burgdorferi sensu lato to the host collagen-associated proteoglycan decorin. Similar to the case for other B. burgdorferi sensu lato proteins, considerable differences in amino acid sequences exist among DbpA proteins of different B. burgdorferi sensu lato species. Neither OspA (31 kDa) nor OspB (34 kDa) is significantly expressed by B. burgdorferi sensu lato during early stages of infection. OspA is down-regulated in the tick midgut during tick feeding (298, 299). Presumably OspA and OspB are eventually expressed in mammals, since antibodies to these antigens can be detected during late infection (10, 84). Antibodies to OspA or OspB may be borreliacidal (46, 132, 286, 287). OspA antibodies were readily generated after administration of the recombinant OspA vaccine, which was commercially available in the United States for use in humans until March 2002 (327). Recently, the Vmp-like sequence expressed (VlsE) protein, a surface-exposed lipoprotein encoded by the linear plasmid lp28-1 of B. burgdorferi B31, has been found to be highly immunogenic (378). Antigenic variation in B. burgdorferi sensu lato occurs when recombination between silent vls cassettes and vlsE takes place. VlsE in B. burgdorferi sensu lato has a predicted molecular mass of approximately 34 to 35 kDa and contains variable and invariable domains. Studies on the antigenicity of this protein demonstrated that one invariable region (IR6), which is found within the variable portion of VlsE, is highly immunogenic (161, 170). This sequence is conserved among B. burgdorferi sensu lato species, making it an appealing candidate as a broadly reactive immunodominant antigen. Several other antigens expressed in vivo in the mammalian host appear promising in the search for highly specific immunodominant antigens. Among these are the P35/BBK32 and P37 proteins (10, 94, 96). BBK32 is a fibronectin binding protein first described as P35 in a form lacking some of its Nterminal amino acids; it has an apparent molecular mass of about 47 kDa (96). The in vivo-expressed P35/BBK32 and P37 proteins are not equivalent to other B. burgdorferi sensu lato proteins of diagnostic significance of similar size, such as VlsE, FlaA, or the P35 scored by Engstrom et al. in their immunoblot evaluation (89). Expression of P37 or P35/BBK32 by B. burgdorferi sensu lato in clinical materials, as well as in ticks during a blood meal, has supported their presence in the early stages of infection (80, 96). Antibody detection methods. Several methods have been used for detection of antibodies to B. burgdorferi sensu lato. Early modalities included indirect immunofluorescent-anti-

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TABLE 4. Reactivities obtained using different methods to detect antibodies to B. burgdorferi in Lyme borreliosis in patients from the United States % Reactivity (reference[s]) in patients with: Test EM, acute phase

Whole-cell ELISA IgM IBb IgG IBb Two-tier testing

33–49 (7, 8, 89, 337) 43–44 (7, 89) 0–13 (7), 43.6d 29–40 (7, 15, 89, 227, 337)

EM, convalescent phasea

76–86 75–84 15–21 29–78

(7, 8, 89, 337) (7, 89) (7), 80d (7, 15, 89, 227, 337)

Neurological involvement

Arthritis

79 (IgG ELISA) (84) 80c (84) 64–72 (84) 87 (15)

100 (IgG ELISA) (84) 16c (84) 96–100 (84) 97 (15)

a

Sera obtained after antimicrobial treatment. IgM and IgG IB criteria are those of Engstrom et al. (89) and Dressler et al. (84), respectively, except as indicated. c IgM IB criteria of Dressler et al. (84). d IgG IB criteria of Engstrom et al. (89). b

body assays (IFA) and a variation of this assay using antigens attached to a membrane (FIAX) (251). These assays have for the most part been replaced by EIA, including enzyme-linked immunosorbent assay (ELISA) and enzyme-linked fluorescent assay (ELFA), that are more amenable to automation (366). Additional immunoassays in use are Western IBs and immunochromatographic and dot blot assays. Methods less frequently used are assays that detect borreliacidal (functional) antibodies, antibodies bound to immune complexes (IC), and hemagglutinating antibodies (46, 48, 248, 287). At least 70 different commercial immunoassays to detect B. burgdorferi sensu stricto antibodies have been approved for use in the United States by the Food and drug Administration (FDA) (6, 55). Several of these assays are sold under a label different from that under which they were originally marketed, and others are no longer available. Most of these assays use the B31 type strain of B. burgdorferi sensu stricto as the source of antigen. Other strains of B. burgdorferi sensu stricto have been used in assays developed by academic centers in the United States, and various B. burgdorferi sensu lato species have been employed in in-house or commercial assays available in Europe. (i) IFA. IFA uses cultured organisms fixed onto glass slides. Serum specimens are diluted in preparations that may include an absorbent such as material derived from Reiter treponema or egg yolk sac to remove nonspecific antibodies. After addition of fluorescein isothiocyanate-labeled anti-human IgG or IgM, the presence of antibodies is detected by fluorescence microscopy. Specimens testing reactive at screening dilutions are serially diluted, and titers of 128 or 256 for IgM or IgG, respectively, are usually considered positive (186, 284). Limitations of this assay include the need for fluorescence microscopy and for well-trained personnel and the subjectivity in reading and interpreting fluorescence microscopy. These issues were addressed in the modification of this assay that was available about a decade ago (FIAX; Whittaker), which used antigens applied to membranes, with the degree of fluorescence read by an automated system. (ii) Enzyme immunoassays. ELISA is the most frequently used format to test for antibodies to B. burgdorferi sensu lato. Most commonly, antigen mixtures comprised of whole-cell sonicates of B. burgdorferi sensu lato are used as the source of antigen for the detection of IgG, IgM, or IgA antibodies individually or in combination (most frequently IgG-IgM combinations). Purified antigens, such as flagellar components, or

recombinant antigens, such as P39, have been added to the antigen mixture in some kits (6). Recently, an ELISA using only a single synthetic peptide derived from the VlsE sequence (IR6 or C6 peptide) as the source of antigen has become commercially available. Using sonicated whole-cell preparations of low-passage B. burgdorferi sensu lato, the sensitivity of ELISA is in general less than 50% in acute-phase sera of patients with EM of a duration of less than 1 week. Sensitivity increases rapidly over time after the first week in untreated patients with EM. Sensitivity is also high in patients with EM who are symptomatic or who have multiple EMs. Sensitivity is very high in patients with objective evidence of extracutaneous involvement (e.g., carditis or neuroborreliosis) (84). ELISA is almost invariably positive in sera of patients with late disease such as arthritis (Table 4) (84). One of the limitations of ELISA for detection of B. burgdorferi sensu lato antibodies is lack of standardization. Variations exist between assays in terms of antigenic composition and in the detection of specific immunoglobulin classes, particularly in the detection of IgM antibodies (7, 8, 34, 167, 337). Such variations may occur among different commercial kits as well as between lots of the same kit. Unlike serological assays for detection of antibodies to human immunodeficiency virus, B. burgdorferi sensu stricto antibody assays cleared by the FDA have not been standardized against a panel of well-characterized sera. The regulatory process for clearance of B. burgdorferi sensu stricto serological assays requires only that the manufacturer provide information demonstrating that the new test is substantially equivalent to a test already approved by the FDA (34). Whole-cell antigen preparations lack specificity because of the presence of cross-reacting antigens of B. burgdorferi sensu lato. These include common bacterial antigens such as heat shock proteins, flagellar antigens, and others (35, 64, 89, 91). Specificity is also affected by the choice of absorbent material used to dilute the serum specimens. Sera of individuals who received OspA vaccination may react in ELISA using wholecell sonicates (9). Although the commercial availability of the vaccine has been discontinued, some previously vaccinated individuals may still have antibodies reacting with OspA. Specificity is in general better with substitution of selected recombinant or peptide antigens for whole-cell sonicates. ELISA has advantages over other immunoassays, including ease of testing, objective generation of numerical values that

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correlate in relative terms with the quantity of antibodies present, and the capability of automation. Due to lack of specificity of ELISA and other first-tier assays, such as ELFA or IFA, using whole-cell antigen preparations, a positive test is not indicative of seropositivity. A recent analysis of results obtained with an automated ELFA found that the specificity was 91% for 559 samples from a control population (137). Serum specimens testing positive or equivocal with a sensitive ELISA, ELFA, or IFA should be retested with IB; this approach is termed two-tier testing (53). First-tier assays should detect both IgG and IgM antibodies to B. burgdorferi sensu stricto, and separate IgG and IgM IBs should be done as the second tier. (iii) Western IB. The use of antigens separated by molecular size in IB assays has contributed to the determination of which antigens of B. burgdorferi sensu lato are immunodominant at different stages of LB. Academic centers in the United States have evaluated in-house IB assays using B. burgdorferi sensu stricto strains other than B31 (84, 89). The few commercial IB assays that are currently available in the United States use B. burgdorferi sensu stricto strains; two manufacturers use B31 (6). Various B. burgdorferi sensu lato species and strains isolated from different geographic locations have been studied in Europe (117, 118, 280). Based on recognized inter- and intraspecies differences among the immunodominant antigens of B. burgdorferi sensu lato, it is not surprising that the source of antigens in IB affects the detection of antibodies in European patients with LB (280). Whether B. burgdorferi sensu lato antigens elicit IgM versus IgG antibodies depends on the duration of infection and the manifestation of LB. Detection of reactivity is also affected by the quality of the antigen used in the immunoassays (including the type and source of antigen). In early LB, IgM antibodies are directed to OspC and the flagellar antigens, FlaB (41 kDa) and FlaA (37 kDa) (7) (Fig. 1, left). Variable rates of IgM response to BmpA (39 kDa) have been observed in sera of patients with early LB, which appears to be related in part to the source of antigen used and/or the duration of disease prior to testing for antibodies (7, 84, 89, 181). The highest rates for IgM reactivity to the 39-kDa protein were reported by Engstrom et al. (89), using B. burgdorferi sensu stricto strain 297 (84%), and by Ma et al. (181), using B31 (50%). In contrast, Dressler et al. (84), using B. burgdorferi sensu stricto strain G39/40, reported IgM reactivity to the 39-kDa protein in only 4 and 8% of acute- and convalescent-phase sera of patients with EM, respectively. The infrequency of reactivity to BmpA antigen reported by Dressler et al. could be attributed to the low expression of this antigen by the B. burgdorferi sensu stricto G39/40 strain used in their study. We have observed IgM reactivity to this antigen in only 3% of acute-phase sera of patients with EM of a duration of less than 1 week and in 35% of acute-phase sera of untreated patients with EM of a duration of more than 7 days, using a commercial IB kit prepared with the B. burgdorferi B31 strain; convalescent-phase sera from the same groups showed IgM reactivity to the 39-kDa protein in 37% and 31%, respectively (7). The number of antigens recognized in IgM IB and the intensity of the immune response as determined by band intensity are greater in sera of symptomatic patients with EM, those with multiple EMs, or those with EM of a duration of more

DIAGNOSIS OF LYME BORRELIOSIS

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FIG. 1. (Left) Selected IgM immunoblot reactivities. Lane 1, serum band locator control showing several bands, including the significant 41-kDa protein, 39-kDa protein, and OspC (arrows). Lane 2, serum sample from a patient with early LB with EM. Lane 3, serum sample from a patient with early disseminated LB with multiple EM lesions. Note the larger number of bands observed in serum of the patient with early disseminated LB. (Right) Selected IgG immunoblot reactivities. Lane 1, serum band locator control showing several immunoreactive bands, including those considered significant in the IgG blot criteria (arrows). Lane 2, serum sample from a patient with early disseminated LB with neurological involvement. Lane 3, serum sample from a patient with Lyme arthritis. Lane 4, serum sample from an individual who received three doses of OspA vaccine. In lane 4, note the strong reactivity with OspA (31 kDa) and other antigens below OspC.

than 2 weeks at presentation, compared to asymptomatic patients with a solitary EM of a duration of less than 2 weeks (7) (Fig. 1, left). An expanded immunologic response is also found in patients with early neuroborreliosis (84). In early LB, IgG antibodies are directed to OspC and flagellin (41 kDa). IgG reactivity to BmpA (39 kDa) was reported by Engstrom et al. to occur in 85% of acute-phase sera of patients with EM (89). In our experience, antibody to this antigen is detected in 35% of acute-phase sera of untreated patients with EM of a duration of more than 7 days and in 33% and 64% of convalescent-phase sera of treated patients with EM of durations of less than 7 days and more than 7 days, respectively (7). Other antigens that elicit IgG immunoreactivity detectable by IB prepared with B. burgdorferi B31 are the 93 (also referred as P83/100)-, 66-kDa, 45-, 35-, 30-, and 18-kDa antigens. IgG antibodies reacting with a large number of antigens are typically seen in sera of patients with neuroborreliosis or late LB (84, 280) (Fig. 1, right). In an attempt to standardize serologic diagnosis of LB, cri-

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teria for IB interpretation have been established in the United States (53). These guidelines were derived from the systematic evaluation of IB in LB by two academic centers. The IgM criteria adopted were those of Engstrom et al. (89) based on a study of patients with EM using B burgdorferi sensu stricto strain 297. According to these criteria, a positive IgM blot is defined by the presence of two of three particular immunoreactive bands (OspC, 41 or 39 kDa). The IgG criteria are based on a study by Dressler et al. (84), who used B. burgdorferi sensu stricto isolate G39/40 as the source of antigen for evaluation of sera from patients with various manifestations of LB. These criteria require the presence of at least 5 of 10 particular bands (93, 66, 58, 45, 41, 39, 30, 28, 21 [OspC], or 18 kDa). The guidelines for IB interpretation further state that IgM or IgG criteria can be used during the first 4 weeks of illness, but only IgG criteria can be used after 4 weeks after onset of disease. Guidelines for IB interpretation in Europe have recently been published, but consensus on criteria has not been reached (118). Criteria applicable to each species causing LB may be needed in Europe (280). IB studies using members of each of the three different B. burgdorferi sensu lato species causing LB in Europe as a source of antigen to test sera from German patients with various manifestations of LB indicated that the overall highest sensitivity was achieved with B. afzelii pKo (118). Levels of immunoreactivity are also a function of the specific manifestation of LB in Europe. For example, OspC of B. garinii strains has better immunoreactivity than OspC of other B. burgdorferi sensu lato species in detecting IgM antibodies in patients with neuroborreliosis. This is most likely explained by the fact that B. garinii is the species that most frequently causes neuroborreliosis in Europe (197). In general, IB and ELISA have similar sensitivities except in the detection of acute-phase antibodies in early LB (7, 8). In a study of well-characterized sera from 46 culture-confirmed U.S. patients with EM, IgM IB was positive in 43% of acutephase sera, compared with 33% by whole-cell ELISA (7). IB and ELISA have similar sensitivities when sera of patients with extracutaneous manifestations or late stages are tested (Table 4). The specificity of IB is greater than that of ELISA, as interpretation of IB relies on the presence of specific immunoreactive bands; nevertheless, it is important to emphasize that the specificity of IB is not 100%. Sera of individuals who received the recombinant OspA vaccination may show several bands on IB, depending on the source of antigen used in IB. Most frequently there is IgG reactivity to the 31-kDa antigen (OspA) and to other fragments of this antigen migrating below OspC (Fig. 1, right). These latter bands might be confused with the 18-kDa antigen included in the IgG criteria for IB interpretation (9, 205). Major limitations of IB include the visual scoring and subjective interpretation of band intensity that may lead to falsepositive readings, the cost, and the variability of antibody responses in patients with the same clinical manifestation of LB. False-positive readings are particularly seen in IgM IB due to the presence of low-level reactivities to the 41-kDa and OspC antigens in sera from individuals presenting with other infectious and noninfectious illnesses (84, 89). False-positive IgM IB results have been reported in 6% of patients with illnesses such as rheumatoid arthritis, infectious mononucleosis, and systemic lupus erythematosus (89). Furthermore, IgM reactiv-

CLIN. MICROBIOL. REV. TABLE 5. Comparison of the sensitivities of various diagnostic methods for 47 adult patients with erythema migrans evaluated in 2000 at the LB Diagnostic Center in Valhalla, N.Y.a Diagnostic method

Sensitivity (%)

Culture Skin biopsy of EM........................................................................ 51 Plasma ............................................................................................ 45 PCR on skin biopsy of EM Nested ............................................................................................ 64 Quantitative................................................................................... 81 Two-tier serologic testing Acute phase................................................................................... 40 Convalescent phase ...................................................................... 66 a

Modified from reference 227 with permission of the publisher.

ity may persist for prolonged periods of time after treatment of early Lyme borreliosis (7). In addition, there is lack of standardization of the antigen source and preparations used in IB. Important methodological considerations include the use of appropriate positive control sera. Alternatively, the use of monoclonal antibodies raised to immunodominant antigens may assist in band location and interpretation. In the absence of an objective means of determining band intensities (densitometry), the use of intensity cutoff materials (weak control sera) is recommended. (iv) Two-tier testing. The use of two-tier testing for serodiagnosis of LB was an attempt to improve test accuracy in the United States. It has increased the specificity of B. burgdorferi sensu stricto antibody testing while slightly decreasing the sensitivity, particularly when testing sera of patients with EM (7, 167, 337). Relatively few studies using currently available commercial tests have evaluated the performance of the recommended two-tier testing on well-characterized sera from patients with extracutaneous manifestations of LB. Comparison of sensitivities and specificities between studies is difficult due to the use of different antigen preparations and test methods and inclusion of sera from LB patients with undefined disease duration and treatment history. The most comprehensive study on the two-tier approach evaluated sera of 280 patients with various manifestations of LB (15). A sensitivity of 38% was observed for sera of patients with EM during the acute phase, which increased to 67% during convalescence after antimicrobial treatment (15). The sensitivity increased to 87% in sera of patients with early neuroborreliosis and to 97% in those with Lyme arthritis (Table 4). In this study specificity was set at 99%. Similar results were obtained for 47 patients with clinically defined EM, for whom the sensitivities of the two-tier test were 40.4% in acute-phase sera and 66% during the convalescent phase after treatment (Table 5) (227). The restriction of use of the IgM IB criteria to the first 4 weeks of early disease is directed mainly at increasing the specificity of IB, since IgM may persist for prolonged periods of time after treatment and after resolution of symptoms (7). Furthermore, a positive IgM IB using current guidelines for IB interpretation may be found in asymptomatic individuals living in areas of endemicity or in patients with non-Lyme disease illnesses (308). This time-restricted use of the IgM IB may,

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DIAGNOSIS OF LYME BORRELIOSIS

497

TABLE 6. Selected recombinant and peptide antigens used for detection of IgM, IgG, or polyvalent antibodies to B. burgdorferi Range % positive in the indicated LB disease stage Recombinant antigen or peptide

Location

EM IgM

OspC

IgG

Neurological Polyva

IgM

IgG

Late Polyv

IgM

IgG

References Polyv

United States Europe

41–73 35–44

35–43 5–35

47–53

6–33

7–53

3–60

100, 102, 183, 184, 241 144, 196, 244, 275

United States Europe

40–53 36

5

53 45

8

9 0

0

15 196

United States Europe

43–53

35–52 49

34–60

VlsE

United States

19–40

73

100

Invariable region 6 (IR-6, C6 peptide)

United States

pepC10 P41 internal fragment

Acute Convalescent Europe FlaA (37 kDa)

44–63

63

45–74 70–90 87

183, 184 119, 144, 273 100

39

60–95 64

97

87

15, 161, 185

94–100 83–98

15, 171, 255 122, 169

79

104 246

United States Europe

45–68 27

15

DbpA

Europe

9

17

100

93–98

122, 123

BBK32

Europe

13

74–100

100

96–100

122, 123, 160

P66

United States

80

24

75–100

228

a

58

50

74

57

37

35–63

Polyv, polyvalent enzyme immunoassay.

however, reduce the sensitivity of two-tier testing for confirmation of seroconversion in treated patients evaluated slightly beyond the 4-week time point in early convalescence, before IgG antibodies have fully developed (337). Newer EIA antibody tests. Because of the above-described limitations of current ELISA and IB testing, there is interest in developing simplified but accurate new approaches for serodiagnosis. The principal focus has been on the use of purified, recombinant, or synthetic peptides as the source of antigens in immunoassays. Unfortunately, so far no single antigen has demonstrated sufficient sensitivity and specificity to warrant replacing two-tier testing. As mentioned earlier in this report, antigenic variability among B. burgdorferi sensu lato species and the temporal appearance of antibodies to different antigens at various stages of LB make the choice of a single antigen a difficult task. (i) Enzyme immunoassays using recombinant antigens. Several immunoassays using recombinant antigens have been developed and evaluated for the serodiagnosis of LB. Recombinant antigens have included those containing the internal portion of the flagellin (P41-G or 41-i), as well as FlaA, BBK32, P39, P35, and outer surface proteins A, B, C, E, F, VlsE, and DbpA (92, 94, 97, 100, 102, 119, 122–124, 144, 160, 182–184, 235, 241, 244, 275, 294). In serodiagnosis, recombinant antigens have been used alone or in combination. Particularly in Europe they have been prepared from different B. burgdorferi sensu lato species and used in both ELISA and IB formats in an attempt to increase sensitivity. The performance of selected immunoassays using recombinant antigens is shown in Table 6. In general, preparations containing recombinant OspC have performed better with sera of patients with early LB and when

IgM antibodies are tested (100, 196). Padula et al., using a recombinant OspC derived from a B. burgdorferi sensu stricto strain, found that it detected IgM antibodies in 62% of U.S. patients with EM (241). However, some European studies have shown lower (30 to 44%) IgM reactivity to recombinant OspC in sera of patients with EM than was found in U.S. studies (196, 244). Studies using the recombinant internal region of the flagellin protein have demonstrated it to be less sensitive than purified whole flagellin preparations when testing sera from patients with any stage of LB (119). Moreover, although it shows less cross-reactivity than the whole protein when testing sera from non-LB patients, the internal region of this protein still suffers from this limitation (183, 184, 273). Assays using recombinant VlsE have shown that it has sensitivity comparable to that of recombinant OspC during early disease and superior sensitivity in sera of patients with neuroborreliosis or late manifestations of LB (15, 161, 185). Recombinant VlsE binds IgM antibodies more frequently than the VlsE invariable region 6 (IR6, C6 peptide) in sera of patients with EM (but IgG anti-C6 is already present in patients with EM) or early neuroborreliosis (161, 171, 185). Recombinant DbpA preparations obtained from different B. burgdorferi sensu lato species have been evaluated in Europe to detect antibodies in patients with LB (61, 122, 124). These recombinants detect mostly IgG antibodies and have the greatest sensitivities (93 to 100%) in sera from patients with neuroborreliosis and late stages of LB. They performed poorly with sera from European patients with EM (124). European studies have shown that recombinant BBK32 shows high sensitivity (74 to 100%) in the detection of anti-

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bodies at any stage of LB, including EM (122, 123, 160). Similar to DbpA, however, this antigen mostly binds IgG antibodies. Epitopes not included in these recombinant antigens may perhaps bind IgM antibodies, since the BBK32 antigen is expressed in vivo during the early stages of infection. Use of recombinant P66 in the serodiagnosis of LB in the United States has shown promising results. The 66-kDa protein is a candidate ligand for ␤3-chain integrins with apparent surface localization that is recognized by sera of patients with LB; IgG reactivity to the 66-kDa protein is one of the significant bands included in the IgG IB criteria (84). The central portion of this protein is involved in integrin recognition, while the C terminus contains a surface-exposed immunodominant loop. Studies using cloned segments of this protein as an antigen source in immunoassays show that different portions of P66 protein appear to be preferentially recognized by sera from patients with different manifestations of LB (228). Sera from patients with late disease manifestations most frequently had IgG antibodies to the C terminus. Specificities for IgM and IgG antibodies to full-length P66 were 94% and 91%, respectively (228). (ii) Peptide-based immunoassays. The generation of peptides containing selected immunoreactive epitopes of immunodominant protein antigens has led to the evaluation of pepC10 and C6 (IR6). The advantage of these two peptides is that they are highly conserved among different B. burgdorferi sensu lato species and should be less cross-reactive than the full-length antigens. (a) pepC10 peptide. pepC10 peptide has been evaluated both in Europe (196) and in the United States (15). This peptide preferentially binds IgM antibodies. The reported sensitivity in patients with EM was 40%, increasing to 53% in patients with early neuroborreliosis (15). This peptide does not detect antibodies in patients with late LB (Table 6). Reported specificities of pepC10 are 92 to 99% (15, 196). The presumed advantage of pepC10 over recombinant OspC is that pepC10 is less heterogeneous than recombinant OspC, suggesting that it should be more broadly reactive in samples of patients with LB from different geographic locations, particularly in Europe. (b)C6 peptide (IR6). The discovery of IR6, a highly immunogenic and highly conserved region of VlsE, brought great optimism to the prospect of finding a single-tier assay for serodiagnosis of LB. Although a C6 peptide assay is now commercially available, most studies published to date have used in-house assays rather than the commercially available test. Published studies have shown that despite not eliciting an IgM response, C6 peptide assays have high sensitivity in all stages of LB. The sensitivity of the C6 assay surpasses that of VlsE in patients with EM (IgG positivity in up to 90% versus 63%) (171, 185). Recombinant VlsE, however, may detect antibodies from patients with neuroborreliosis more frequently than C6 (15, 161). Using a kinetic ELISA, Bacon et al. found that IgG antibodies to recombinant VlsE were detected in 15 of 15 patients with acute neuroborreliosis, compared with 9 of 15 (60%) for IgG antibodies to C6 and 13 of 15 (87%) for two-tier testing (15). Whether additional epitopes present in VlsE but not contained in C6 are needed to detect antibodies in acute neuroborreliosis is currently unknown. Further investigation into the performance of C6 in sera of patients with neuroborreliosis is warranted.

CLIN. MICROBIOL. REV.

Overall similar sensitivities were found by Bacon et al. for an in-house synthetic peptide C6 assay and two-tier testing with 280 serum samples from patients with various manifestations of LB (66% versus 68%, respectively) (15). Assays using C6 peptide have also been evaluated in Europe on sera obtained from 23 culture-confirmed EM patients (10 from Austria and 13 from Italy) and on 41 sera from patients with late manifestations of LB (21 from Austrian patients with ACA and 20 from Italian patients with late neuroborreliosis). C6 seroreactivity was observed in 20 of 23 (87%) sera from patients with EM, in 20 of 21 (95%) sera of Austrian patients with ACA, and in 14 of 20 (70%) sera of Italian patients with late neuroborreliosis (169). Fifty-one of 52 (98%) German children with Lyme arthritis tested positive for C6 antibodies in another study (122) (Table 6). These findings further support the notion that C6-based enzyme immunoassays are broadly reactive in sera of patients with LB from different geographic localities. It has been claimed that the C6 ELISA can be used to assess the outcome of therapy for LB. Philipp et al. reported that 80% of a subset of patients treated for early localized or disseminated LB had a ⱖ4-fold decrease in their reciprocal geometric mean titers to C6 at 6 months or thereafter (255). Other studies have not confirmed these findings. Peltomaa et al. found that 33% and 86% of patients with early and late LB, respectively, had a ⬍4-fold decline in C6 titers (250). Those authors also reported that in another group of patients, 50% of those with early LB and 83% of those with late LB still had detectable C6 reactivity 8 to 15 years after treatment. Likely explanations for the discrepancies in the findings of Philipp and Peltomaa include differences in serum dilutions used to calculate the decline in antibodies, whether patients had sufficient titers at baseline to permit detection of a fourfold decline at least 6 months later, and/or the patient populations studied (250, 255). (iii) Use of a combination of recombinant or peptide antigens in immunoassays. Since no single antigen appears to have the desirable sensitivity to be used alone in the serodiagnosis of LB, some authors have evaluated their use in combination. Rauer et al. (275) investigated the combination of recombinant OspC and P41-i in sera of patients with early LB. Detection of IgM antibodies by the hybrid ELISA was 46%, similar to the sensitivity obtained with whole-cell ELISA (45%) using the B. afzelii pKo strain (275). Other combinations that have been evaluated include P41-i and a 59-kDa fragment of P83 (273) and P41-i, recombinant OspC, and recombinant P83 (144). The latter three-antigen combination showed a sensitivity of greater than 90% in sera of patients with neuroborreliosis. An immunochromatographic assay that includes recombinant, chimeric, truncated forms of OspA, OspB, OspC, P93, and flagellin as the antigen is currently commercially available in the United States to test for B. burgdorferi sensu stricto antibodies in serum or whole blood (107). This assay is being promoted as a method for first-step testing that can be performed in the health care provider’s office. A specificity of 85% was obtained with sera of syphilitic patients with this ELISA (107). Since it includes OspA, this assay would be expected to show reactivity with sera of individuals who have received OspA vaccination. The sensitivity of an ELISA with the same antigen mixture was 44%, compared with 39% for a commer-

VOL. 18, 2005

cial whole-cell ELISA, in a study of sera from 41 patients with culture-confirmed EM; sensitivities of 62% and 54% for the chimeric versus the whole-cell ELISA were observed with sera of patients with early disseminated LB (patients with multiple EMs or EM plus objective signs of neurologic or cardiac involvement), respectively. A sensitivity similar to that of wholecell ELISA was seen in patients with late LB. Other commercially available immunoassays that contain mixtures of purified or recombinant antigens have been formatted as dot blot assays on strips. One of these assays uses whole borrelial lysates plus recombinant high-molecularweight antigens, purified flagellin, recombinant flagellin, and recombinant OspC. Another uses whole borrelial lysates plus recombinant P39 and purified flagellin (6). Limited information is available on the performance of these assays. Bacon et al. (15) evaluated recombinant VlsE for detection of IgG and/or IgM antibodies, the peptide pepC10 for detection of IgM antibodies, and the peptide C6 for the detection of IgG antibodies, in comparison to the two-tier approach, for testing sera from 280 patients with LB. They analyzed the results of individual assays and of their potential combinations. The overall best sensitivity was seen with the combination of C6 IgG and pepC10 IgM compared with two-tier testing (78% versus 68%, respectively). The greatest difference was observed with sera of patients with EM; 63% of samples were positive for C6 IgG-pepC10 IgM during the acute phase, compared with 38% by two-tier testing. By convalescence after treatment, 80% and 67% were positive with the combined peptides and two-tier testing, respectively. Both VlsE IgGpepC10 IgM and VlsE IgG-VlsE IgM had higher sensitivity (100%) in sera of patients with early neuroborreliosis compared with C6 IgG-pepC10 IgM (73%) or two-tier testing (82%). All test combinations had high sensitivities in late LB. The specificity of these assays was set at ⱖ98%. Whether the use of immunoassays using recombinant or synthetic peptide antigens will become the standard of practice for diagnosis of LB is currently unknown. More studies are needed to determine the applicability of such antigens to the serodiagnosis of patients with various manifestations of LB in both the United States and Europe. A desirable feature is that they are potentially amenable to automation, which would avoid the subjectivity of immunoblot interpretation. The cost will depend on patent and ownership issues. Other antibody detection methods. (i) Functional antibodies: borreliacidal antibody assays. Antibodies to certain antigens of B. burgdorferi sensu lato, in particular to certain outer surface proteins, may have bactericidal activity (46, 132, 249, 286, 287). Borreliacidal antibodies have been used in the immunodiagnosis of early and late LB by a few centers (5, 47, 48). In these assays live B. burgdorferi sensu stricto is incubated with patient serum plus an exogenous source of complement for 16 to 72 h (47). Growth inhibition of B. burgdorferi sensu stricto can be determined by visual inspection of the percentage of nonmotile spirochetes, color changes by use of a pH indicator, or flow cytometry after staining with acridine orange. The advantage of these assays is their high specificity in untreated patients compared with matrix-based nonfunctional assays. Invariably these tests are nonreactive in healthy control populations or in sera of non-Lyme disease patients with rheumatological diseases (47, 48). Modifications of the assay with

DIAGNOSIS OF LYME BORRELIOSIS

499

reported higher sensitivities employ B. burgdorferi sensu stricto strain 50772, which lacks OspA and OspB (44, 45). Major disadvantages include the need for cultured live B. burgdorferi sensu stricto, interference from antimicrobials that might be present in patient sera, and the relatively cumbersome nature of the assays. (ii) Detection of antibodies bound to circulating immune complexes. It has been proposed that seronegativity in early LB is mainly due to the formation of specific antigen-antibody complexes that impede the detection of free antibodies by conventional methods. Some studies have suggested that IC are found not only in serum but also in other body fluids such as cerebrospinal and synovial fluids of patients with LB (68, 116, 295, 379). Detection of antibodies bound to immune complexes involves the treatment of serum with polyethylene glycol to precipitate the IC, followed by dissociation through alkalinization of the complexes to release antibodies that can then be detected by ELISA or IB. A recent modification of this assay, the enzyme-linked IgM capture IC biotinylated antigen assay, was compared to IgG and IgM ELISA and IB with sera collected from clinically defined patients with LB and a control population. The enzyme-linked IgM capture IC biotinylated antigen assay was found to be more sensitive and specific than the aforementioned tests and furthermore detected antibodies more consistently in those patients with clinical evidence of active disease (62 of 64 patients; 97%) than in those with past infection (4 of 28; 14%). In that study, standard IgM ELISA and IgM IB were reactive in 67% and 58%, respectively, of patients with active disease and in 43% and 39%, respectively, of patients with past infection (36). Potential utilities of this type of assay include detection of antibodies in seronegative patients during early disease and ascertainment of whether persistent seropositivity is due to ongoing infection, since IC are speculated to be present only in active infection (36). Although these assays appear to be helpful in certain clinical scenarios, only a few investigators have used them. Detection of antibodies in cerebrospinal fluid. There are currently no FDA-approved tests to measure intrathecal production of antibodies in CSF. Methods that have been used to detect antibodies in CSF include capture immunoassays, CSF/ serum indices determined by ELISA, and Western immunoblots (69, 113, 364). Determination of intrathecal production of antibodies can be accomplished by measuring the CSF/ serum index of B. burgdorferi sensu lato antibodies. CSF and serum samples diluted to match the total IgG concentration in CSF are run in parallel in an IgG ELISA. Positive intrathecal production is indicated by CSF/serum optical density ratios of ⬎1.3 (364). Intrathecal production can also be determined by testing CSF and serum at matching concentrations of IgG and IgM in IgG and IgM IB, respectively. Greater numbers and intensities of bands in CSF compared with the corresponding serum would be consistent with intrathecal antibody production, but this has not been well substantiated (356). Specific intrathecal production of IgG, IgA, and/or IgM B. burgdorferi sensu lato antibodies has been described by several investigators (56, 58, 59, 141, 340). Recombinant antigens have also been studied in Europe for evaluation of intrathecal antibody production (142, 143, 245). The use of recombinant internal

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fragment of flagellin, although of lower sensitivity than wholecell preparations, has shown greater specificity in the detection of B. burgdorferi sensu lato antibodies in CSF (142, 143). Detection of intrathecal antibodies by using three variants of the recombinant proteins DbpA, BBK32, and OspC, each variant originating from a different species of Lyme Borrelia, plus synthetic peptide IR6 as the source of antigens was superior to a commercial flagellum-based ELISA in a Finnish study of 89 patients with neuroborreliosis (245). In this study, the highest sensitivities for each antigen for the detection of IgG antibodies in CSF were 88% for DbpA, 80% for IR6, 76% for BBK32, 75% for OspC, and 52% for flagella. Intrathecal B. burgdorferi sensu lato antibody detection in patients with neuroborreliosis in the United States seems to be less frequent than that in such patients from Europe. A factor contributing to this discrepancy may be the difference in B. burgdorferi sensu lato species causing neuroborreliosis in Europe and the United States. Several European studies dealing with typing of B. burgdorferi sensu lato strains cultured from CSF samples or detected by PCR have demonstrated that the most frequent species is B. garinii (42, 166, 285). Only four of 36 (11%) CSF isolates from German patients and 1 of 40 (2.5%) CSF isolates from Slovenian patients were B. burgdorferi sensu stricto (42, 285). Cellular immune response in LB: T-lymphocyte and mononuclear cell proliferation assays. The development of a cellular immune response in Lyme borreliosis has been observed in assays using peripheral blood mononuclear cells or cells present in affected tissues of patients with LB (37, 38, 73, 240, 305). However, the initial enthusiasm for use of an in vitro lymphoproliferation assay for diagnosis of LB has been tempered. T-lymphocyte proliferation assays have seldom been used as diagnostic tests due to their cumbersome nature and concerns about specificity and standardization (126).

TEST INTERPRETATION Of the nonculture direct methods of detection, PCR is the most promising in providing assistance in the diagnosis of LB. A positive PCR result in a synovial fluid specimen of a patient with exposure to an area where LB is endemic and who also has a positive B. burgdorferi sensu lato ELISA and IgG IB is strongly supportive of a diagnosis of Lyme arthritis. Untreated patients with Lyme arthritis, as well as patients during the first days to weeks of therapy, may exhibit a positive PCR result in synovial fluid. Positive PCR results for B. burgdorferi sensu lato nucleic acids obtained in samples from patients with possible extracutaneous manifestations of LB in the absence of serological evidence of B. burgdorferi sensu lato infection, however, should be interpreted with caution. In those situations, PCRs are often false positive. One of the limitations of nucleic acid amplification methods is the generation of false-positive results due to contamination. Adherence to rigorous quality control steps is of utmost importance. Interpretation of serology in LB requires an understanding of the use and limitations of the currently available tests for B. burgdorferi sensu lato antibodies. (i) These tests detect antibodies reacting with B. burgdorferi

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sensu lato and can support the clinical suspicion of LB, but in and of themselves they do not diagnose LB. (ii) Antibodies may not be detectable at the time the patient presents with signs and symptoms of early LB with EM, and their presence correlates directly with the duration of disease prior to seeking medical attention, as well as with the presence of symptoms or objective signs of dissemination (for example, multiple EMs or cranial nerve palsies). For patients with early LB who have EM, the diagnosis is established on clinical grounds if the skin lesion is characteristic. In situations where the skin lesion is atypical or absent, tests to detect B. burgdorferi sensu lato antibodies may be necessary, as follows. (a) When antibodies are not detectable or not diagnostic in the acute-phase serum specimen, a convalescent-phase specimen should be collected 2 to 4 weeks later and tested for B. burgdorferi sensu lato antibodies (7, 89). (b) In patients treated with antimicrobials, testing of the convalescent-phase sample within 1 month of onset of symptoms will maximize sensitivity, since the criteria for test positivity at this time point include IgM IB seroconversion, whereas after 1 month IgG seroconversion is required for seropositivity. (iii) Testing for B. burgdorferi sensu lato antibodies should include the two-tier approach as currently recommended (53). In those specimens testing positive or equivocal by first-tier assays, second-tier IB is used to improve specificity. IB should not be used with sera testing negative by the first tier, as this would reduce specificity compared to the two-tier testing strategy (369a). Patients who received the OspA vaccine may be seropositive due to the presence of OspA in whole-cell B. burgdorferi sensu lato preparations or in other antigen preparations that include recombinant OspA in the mixture. Vaccine reactivity can often be distinguished from antibodies elicited by natural infection with B. burgdorferi sensu stricto by IB (9) (Fig. 1, right). (iv) B. burgdorferi sensu lato antibodies, both IgG and IgM, may persist for many years after successful treatment of LB (93). Thus, persistent seropositivity is not, per se, an indication of treatment failure. Persistence of antibodies to B. burgdorferi sensu lato in sera of individuals residing in areas where LB is endemic and who have been treated for LB, or who have resolved an asymptomatic infection, may limit the utility of future serologic testing as a diagnostic tool when such persons present with a new clinical event. In these circumstances, a change in reactivity between acute- and convalescent-phase specimens may be of assistance, although this has never been systematically evaluated. An increase in antibody concentration as determined by optical density in EIA, or by IFA titers, in first-tier assays or by an increase in intensity or appearance of new immunoreactive bands by IB might suggest a new or recent B. burgdorferi sensu lato infection. Physicians caring for these patients should store an aliquot of an acute-phase serum specimen to be submitted along with the convalescent-phase sample for testing in parallel for B. burgdorferi sensu lato antibodies. (v) Patients with late manifestations of LB usually have a high concentration of antibodies by first-step tests and have numerous immunoreactive bands in IgG blots, often far surpassing the number of bands required in the IgG IB interpre-

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tation criteria (84). A lack of seropositivity in patients suspected of having late LB practically excludes this diagnosis. (vi) Laboratories performing B. burgdorferi sensu lato serology should use assays of proven performance as determined by the summary of surveys run by the College of American Pathologists or local or state proficiency programs. (vii) Laboratorians should make an attempt to avoid scoring weak bands leading to false-positive readings in IB, particularly IgM IB. This can be avoided by strict adherence to comparison of band intensity to those of cutoff intensity control materials. (viii) The use of laboratory tests or interpretation strategies that have not been appropriately validated is of great concern and is strongly discouraged (53). Currently there are commercial laboratories offering B. burgdorferi sensu lato urine antigen tests, immunofluorescent staining for cell wall-deficient forms of B. burgdorferi sensu lato, and lymphocyte transformation tests. In addition, some laboratories are performing PCR for B. burgdorferi sensu lato DNA on inappropriate samples such as blood and urine or are interpreting IB by using criteria that have not been validated (53). (ix) The use of B. burgdorferi sensu lato antibody testing should be restricted to those patients with a 0.2 to 0.8 pretest probability of having LB as recommended by the American College of Physicians (339). The use of these tests in unselected populations with a low pretest probability of the disease is more likely to yield false-positive than true-positive results. Testing is not recommended for patients presenting with classic EM, since treatment without testing is more costeffective and B. burgdorferi sensu lato antibody assays have low sensitivity at this stage (222). Since the specificity of B. burgdorferi sensu lato antibody testing is not 100% and since approximately 2.7 million tests are estimated to be done yearly in the United States, for every 1% reduction in test specificity there will be approximately 27,000 false-positive results per year, dwarfing the true positive incidence of about 20,000 cases/year. ACKNOWLEDGMENTS We thank Charles Pavia, Susan Bittker, Denise Cooper, Carol Carbonaro, Janet Roberge, and Lois Zentmaier for technical assistance and Lisa Giarratano for secretarial help. REFERENCES 1. Aberer, E., C. Brunner, G. Suchanek, H. Klade, A. G. Barbour, G. Stanek, and H. Lassmann. 1989. Molecular mimicry and Lyme borreliosis: a shared antigenic determinant between Borrelia burgdorferi and human tissue. Ann. Neurol. 26:732–737. 2. Abu Al-Soud, W., and P. Radstrom. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485– 493. 3. Ackermann, R., J. Kabatzki, H. P. Boisten, A. C. Steere, R. L. Grodzicki, S. Hartung, and U. Runne. 1984. Spirochete etiology of erythema chronicum migrans disease. Dtsch. Med. Wochenschr. 109:92–97. 4. Agger, W. A., S. M. Callister, and D. A. Jobe. 1992. In vitro susceptibilities of Borrelia burgdorferi to five oral cephalosporins and ceftriaxone. Antimicrob. Agents Chemother. 36:1788–1790. 5. Agger, W. A., and K. L. Case. 1997. Clinical comparison of borreliacidalantibody test with indirect immunofluorescence and enzyme-linked immunosorbent assays for diagnosis of Lyme disease. Mayo Clin. Proc. 72:510– 514. 6. Aguero-Rosenfeld, M. E. 2004. Detection of Borrelia burgdorferi antibodies, p. 11.6.1–11.6.10. In H. D. Isenberg (ed.), Clinical microbiology procedures handbook, 2nd ed. ASM Press, Washington, D.C. 7. Aguero-Rosenfeld, M. E., J. Nowakowski, S. Bittker, D. Cooper, R. B. Nadelman, and G. P. Wormser. 1996. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J. Clin. Microbiol. 34:1–9.

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Caimano, J. D. Radolf, and M. V. Norgard. 1999. Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family. Infect. Immun. 67:6008–6018. Zbinden, R., D. Goldenberger, G. M. Lucchini, and M. Altwegg. 1994. Comparison of two methods for detecting intrathecal synthesis of Borrelia burgdorferi-specific antibodies and PCR for diagnosis of Lyme neuroborreliosis. J. Clin. Microbiol. 32:1795–1798. Zhang, J. R., J. M. Hardham, A. G. Barbour, and S. J. Norris. 1997. Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89:275–285. Zhong, W., P. Oschmann, and H. J. Wellensiek. 1997. Detection and preliminary characterization of circulating immune complexes in patients with Lyme disease. Med. Microbiol. Immunol. (Berlin) 186:153–158. Zore, A., E. Ruzic-Sabljic, V. Maraspin, J. Cimperman, S. Lotric-Furlan, A. Pikelj, T. Jurca, M. Logar, and F. Strle. 2002. Sensitivity of culture and polymerase chain reaction for the etiologic diagnosis of erythema migrans. Wien. Klin. Wochenschrift. 114:606–609.

CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 510–520 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.510–520.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 18, No. 3

Epidemiology and Management of Infectious Diseases in International Adoptees Thomas S. Murray, M. Elizabeth Groth, Carol Weitzman, and Michael Cappello* Yale International Adoption Clinic, Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue, New Haven, Connecticut 06520 INTRODUCTION .......................................................................................................................................................510 Trends in International Adoption ........................................................................................................................510 EPIDEMIOLOGY OF INFECTIOUS DISEASES IN INTERNATIONAL ADOPTEES ..................................510 Viral Infections........................................................................................................................................................510 HIV........................................................................................................................................................................510 Hepatitis B virus .................................................................................................................................................511 Hepatitis C virus.................................................................................................................................................512 Measles virus.......................................................................................................................................................512 CMV......................................................................................................................................................................512 SARS virus and avian influenza virus .............................................................................................................512 Bacterial Infections.................................................................................................................................................513 Mycobacterium tuberculosis .................................................................................................................................513 Syphilis .................................................................................................................................................................513 Helicobacter pylori ................................................................................................................................................513 Bacterial gastroenteritis.....................................................................................................................................513 Parasitic Infections.................................................................................................................................................514 Intestinal parasites .............................................................................................................................................514 Ectoparasites .......................................................................................................................................................514 (i) Scabies ........................................................................................................................................................514 (ii) Lice (pediculosis) .....................................................................................................................................514 INFECTIOUS DISEASES IN INTERNATIONAL ADOPTEES: THE YALE INTERNATIONAL ADOPTION CLINIC EXPERIENCE...............................................................................................................514 PREADOPTION MEDICAL RECORD REVIEW..................................................................................................515 POSTADOPTION MEDICAL SCREENING ..........................................................................................................515 RECOMMENDATIONS FOR IMMUNIZATION OF INTERNATIONAL ADOPTEES .................................517 ACKNOWLEDGMENTS ...........................................................................................................................................518 REFERENCES ............................................................................................................................................................518 records. However, in 1991 Hostetter et al. published a seminal prospective study documenting the high prevalence of infectious diseases and other serious medical problems in international adoptees, many of which were not identified by routine physical exams (36). Multiple studies have subsequently confirmed that international adoptees are at risk for viral, bacterial, and parasitic infections. This review focuses on the epidemiology of those infectious diseases most prevalent in international adoptees, as well as offer guidelines for the medical management of infections commonly encountered in this unique population of children.

INTRODUCTION Trends in International Adoption International adoption has become an increasingly common means of creating and growing families in the United States. As evidence of this phenomenon, the number of international adoptees in the United States has increased dramatically over the past decade. In 1995 10,641 orphan visas were issued by the U.S. State department. This number increased to 17,718 in 2000 and to 21,616 in 2003. Since 2000, China has been the leading source of international adoptees in the United States, followed by Russia, Guatemala, South Korea, and Kazakhstan (78) (Table 1). Given the distinct epidemiology of infectious diseases in these primarily developing countries, international adoptees represent a group of patients with unique health care needs. The majority of studies examining the health of international adoptees have relied on retrospective reviews of medical

EPIDEMIOLOGY OF INFECTIOUS DISEASES IN INTERNATIONAL ADOPTEES A summary of infectious diseases seen in international adoptees is given in Table 2. Viral Infections

* Corresponding author. Mailing address: Yale Program in International Child Health, 464 Congress Avenue, New Haven, CT 06520. Phone: (203) 737-4320. Fax: (203) 737-5972. E-mail: michael.cappello @yale.edu.

HIV. The human immunodeficiency virus (HIV) is a singlestranded RNA virus that is transmitted via intimate exposure to blood or body fluids. Potential modes of infection of inter510

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TABLE 1. Numbers of international adoptees in the United States No. of orphan visas issued 2003 (% of total)

Country of origin

China ................................................................................ Russia ............................................................................... Guatemala........................................................................ South Korea..................................................................... Kazakhstan....................................................................... Ukraine ............................................................................ India.................................................................................. Vietnam............................................................................ Colombia.......................................................................... Haiti..................................................................................

6,859 (31.7) 5,209 (24.1) 2,328 (10.7) 1,790 (8.2) 825 (3.8) 702 (3.2) 472 (2.1) 382 (1.7) 272 (1.2) 250 (1.1)

Total ................................................................................. 21,616

national adoptees include both mother-to-child (perinatal) transmission and transmission through contact with contaminated needles or sexual abuse within institutional settings. The risk of perinatal HIV transmission is approximately 25%, although the incidence can be dramatically reduced through the use of peripartum antiretroviral therapy (2). Although many children infected perinatally with HIV are asymptomatic, others exhibit a variety of clinical signs and symptoms, including growth delay, hepatosplenomegaly, and recurrent bacterial infections. Advanced HIV infection is also associated with a number of well described opportunistic infections, including recurrent oral candidiasis (thrush) and Pneumocystis carinii pneumonia, conditions that are also associated with malnutrition, a common finding in international adoptees. In Eastern Europe and Russia there were an estimated 280,000 new cases of HIV in 2003, mostly associated with injection drug use (23). Although the overall prevalence of HIV infection in China is low, estimates suggest that up to 1 million people may currently be infected (62). This number may rise to more than 10 million by 2010. Importantly, little is known about regional variation in seroprevalence, thus making the overall risk among Chinese adoptees difficult to estimate. HIV is also a major public health problem in other Asian countries, as recent estimates suggest that there are more than 100,000 active infections in Vietnam and Cambodia and more than 300,000 in India (65). In Vietnam, the seroprevalence of HIV among pregnant women may be as high as 0.28%, representing a small but measurable risk to international adoptees

TABLE 2. Infectious diseases in international adoptees Infection

Viral Varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, HIV Bacterial Mycobacterium tuberculosis (active or latent), syphilis, gastroenteritis, urinary tract infection Parasitic Intestinal protozoa (Giardia lamblia, Blastocystis hominis, Dientamoeba fragilis), ectoparasites (scabies, head lice), intestinal nematodes (Strongyloides stercoralis, Trichuris trichiura, Ascaris lumbricoides, hookworm), visceral larva migrans (Toxocara spp.)

511

(26). The HIV seroprevalence in South Korea is estimated to be less than 0.1% (84). Data on the prevalence of HIV in Guatemala is limited, but one U.S. government report estimated that in 2002 there were 203 children under the age of 14 years infected with HIV (77). The above data suggest that there might be a significant risk of HIV infection in international adoptees, especially those from Eastern Europe. One study published in 1993 reported an HIV seroprevalence of 20% in a Romanian orphanage, although the route of infection in these children was most likely related to exposure through contaminated needles (32). More recent studies have failed to identify a confirmed case of HIV infection in an international adoptee (56, 59). Although Saimen et al. identified 2 out of 490 adoptees (0.4%) with antibodies to HIV, both tested negative for infection by HIV PCR, suggesting that the initial test detected maternal antibody (66). The likely explanation for the very low rates of HIV infection among recent international adoptees is that preadoption testing is now common, especially in China and Eastern Europe. Thus, although concern about HIV infection remains a source of great anxiety for adoptive families, there is little evidence to date that this disease represents a major health risk for international adoptees. Hepatitis B virus. Hepatitis B is a double-stranded DNA virus of the Hepadnaviridae family. Most hepatitis B virus infections in underdeveloped countries are acquired during childbirth. The risk of transmission from a hepatitis B surface antigen (HBsAg)-positive mother to her newborn is approximately 10%, although this number rises to 85% in women who are HBeAg positive (70). While the incubation period for hepatitis B virus in adults ranges from 4 weeks to 4 months, those who acquire the infection perinatally may remain asymptomatic for decades (48, 51). Symptoms are often insidious in onset and may include jaundice, vomiting, and anorexia. Serum hepatic transaminase levels and bilirubin may be elevated, and anemia is also common. Up to 90% of children who acquire hepatitis B virus perinatally will progress to chronic infection, with an increased risk of cirrhosis and hepatocellular carcinoma. The lifetime risk of hepatocellular carcinoma is 50% for men infected at birth and 20% for women (71). Studies in Taiwan clearly indicate that hepatitis B vaccination dramatically decreases the incidence of hepatocellular carcinoma as well as other long-term sequelae of hepatitis B virus infection (12). According to estimates from the Centers for Disease Control and Prevention, 2 to 7% of the population in China, South America, and the southern part of Eastern Europe are HBsAg positive, consistent with chronic infection. In other countries of Eastern Europe, including Moldova, Bulgaria, Georgia, Armenia, and Azerbaijan, the prevalence of HBsAg is perhaps greater than 8% (11). Therefore, internationally adopted children should be considered at potentially significant risk of infection with hepatitis B virus. A variety of retrospective studies have evaluated international adoptees for serological evidence of hepatitis B virus infection. The percentage of infants with evidence of active infection (seropositive for HBsAg) ranged from 2.0% to 5.9% (36, 46, 56, 59, 66)., while serological evidence for previous infection ranged from 22% to as high as 53% (39, 40). Although these data suggest a significant risk of hepatitis B virus infection in international adoptees, it is also

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likely that an increased availability of preadoption medical screening has identified many infected children in orphanages. Hepatitis C virus. Hepatitis C virus is an RNA virus of the Flaviviridae family. Infection is acquired most commonly via exposure to blood and/or other body fluids, often through transfusions or the use of contaminated needles. The risk of perinatal transmission is estimated at 2 to 8% (21), although the rate in mothers coinfected with HIV may be as high as 15% (27). Up to 85% of infected individuals will eventually develop chronic hepatitis, as evidenced by elevated serum levels of hepatic transaminases (42). These patients are at significant risk of progressing to cirrhosis and/or developing hepatocellular carcinoma. The Global Burden of Hepatitis C Working Group of the World Health Organization estimates that the prevalence of hepatitis C in Russia, China, and Eastern Europe is 2 to 2.9% (28). In South America, the prevalence is estimated at 1 to 1.9%. When these prevalence rates are compared to those of hepatitis B, the prediction would be that the risk of hepatitis C is significantly lower. Limited data from published studies appear to confirm that hypothesis, with reported seroprevalence rates of well under 1% in international adoptees, despite the fact that few children are likely to be screened for hepatitis C virus infection in foreign orphanages (56, 66). Measles virus. Measles virus, a single-stranded RNA virus spread by contact with respiratory droplets, is common in many of the countries from which children are adopted into the United States. Following an incubation period of 8 to 12 days, initial symptoms include fever, cough, conjunctivitis, coryza, and Koplik’s spots, which are whitish plaques on the buccal mucosa. Patients are infectious for several days before the onset of the rash. The diagnosis is often made based on the constellation of clinical symptoms described above, although serologic studies can be helpful to confirm the diagnosis. Therapy for measles is primarily supportive (61). Although the World Health Organization estimates that 84% of children worldwide are presently vaccinated, millions of cases of measles occur annually (83). In 2001 there was an outbreak of measles in the United States that was traced to a group of adoptees from China (10). Epidemiologic investigations ultimately identified 10 adoptees from a single orphanage, as well as four additional cases resulting from exposure to one of the infected adoptees. In April 2004, adoptions from Hunan Province in China were temporarily suspended due to a separate outbreak among a group of adoptees (10). Overall, 9 of 12 children in a group of adoptees traveling to the United States developed clinical signs and symptoms of measles. All cases were ultimately traced to a single orphanage, which was in the midst of an outbreak involving many additional cases. This outbreak emphasizes that international adoptees, in particular those from Chinese orphanages, are at increased risk due to the failure of current immunization practices. CMV. Cytomegalovirus (CMV) is a double-stranded DNA virus transmitted by contact with infected bodily fluids, including saliva or urine. CMV infection can also be acquired in utero or during the neonatal period. Based on data from large, population-based seroepidemiologic studies, most individuals living in developing and industrialized nations acquire infection by young adulthood. Both primary infection and reactivation during pregnancy are associated with some risk of con-

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genital infection of the newborn (25). Congenitally infected newborns are frequently asymptomatic but may manifest a combination intracranial calcifications, retinitis, hepatitis, deafness, and thrombocytopenia. Importantly, the absence of clinical findings at birth does not necessarily predict a benign course, as sequelae may develop later in childhood. Ganciclovir has been evaluated in the treatment of congenitally acquired CMV, and small studies suggest that treatment initiated early in the postnatal period may reduce the incidence of deafness (54). The clinical significance of CMV infection in international adoptees is most likely low. While Hostetter et al. cultured CMV from the urine of 111/245 international adoptees (45%), only three children demonstrated clinical evidence of congenitally acquired infection (36). Of these, two children had hepatitis and one had deafness with intracranial calcifications. One published report described two Chinese infants with CMV pneumonia and immunodeficiency, and the authors hypothesized that malnutrition increased their susceptibility to infection (8). The current prevalence of infection in international adoptees is unknown, as most adoption specialists do not routinely screen for CMV. Since CMV isolated from adoptees in most cases represents postnatally acquired infection, viral shedding in the absence of physical findings suggestive of congenital CMV is of limited clinical significance. SARS virus and avian influenza virus. International adoptees, as well as prospective adoptive family members, are at potential risk for acquiring two newly emerging respiratory viral infections (44). From November 2002 to March 2003, approximately 300 cases of pneumonia of unknown etiology were reported in Guangdong Province, China (15). In the ensuing months, the illness, which became known as severe acute respiratory syndrome (SARS), rapidly spread to nearly 30 countries, with over 8,000 infections and a case fatality rate of approximately 10%. The etiologic agent of SARS is a novel coronavirus with genetic similarity to a strain identified in palm civets, which are commonly sold in live-animal markets in China (45, 64). Person-to-person transmission of SARS has occurred on commercial airplanes and within hospital settings, although the factors that contribute to the risk of secondary transmission have not been completely defined (60, 81). Because of the established risk of person-to-person transmission of the SARS virus, the potential exists for international adoptees to acquire (and hence transmit) the infection within the orphanage setting. Prospective parents or family members are also at risk for acquiring the infection in China, as are fellow travelers on commercial airplanes who are exposed to an infected adoptee or adult. The clinical signs and symptoms of SARS in children are often milder than those in infected adults, perhaps lowering the index of suspicion for younger individuals with respiratory illness (34, 49). Out of concern for the spread of SARS, adoptions in China were temporarily suspended in 2003. Fortunately, through aggressive public health measures, the incidence of SARS has been dramatically reduced over the past year, and adoptions are now proceeding regularly. In 2003, China and other Asian countries reported a number of cases of influenza caused by the H5N1 avian strain of influenza A virus (43, 44). These infections occurred primarily in individuals who had contact with infected poultry, although

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evidence also suggested some degree of person-to-person transmission (43). In children, infection with avian influenza is associated with high mortality (31). Because of concern about the potential for the virus to undergo a genetic reassortment, thus altering its pathogenicity, there remains some risk for this strain to evolve into one that is easily transmitted among humans (9, 43). Of note, the currently available influenza vaccines do not confer protection against the avian H5N1 strain, although the antiviral drug oseltamivir may be useful for therapy in some settings (30). Should this strain of influenza A spread throughout China, there would be risk of international adoptees acquiring the infection and transmitting it to prospective family members and travelers (44).

Bacterial Infections Mycobacterium tuberculosis. Mycobacterium tuberculosis is an acid-fast bacillus (AFB) transmitted via inhalation of airborne particles. More than one-third of the world’s population is infected with M. tuberculosis. Most children with M. tuberculosis have latent (asymptomatic) infection and are diagnosed on the basis of a positive intradermal (Mantoux) skin test using a purified protein derivative (PPD) of the M. tuberculosis bacterium (69). Symptoms of primary pulmonary tuberculosis in infants and children include cough, fever, and occasionally growth delay. A variety of radiographic findings may be present on chest X ray, including perihilar lymphadenopathy, lobar consolidation, atelectasis, or diffuse pneumonitis. Children are also at significant risk for extrapulmonary manifestations of tuberculosis, including lymphadenitis (scrofula), nephritis, meningitis, osteomyelitis, and disseminated (miliary) disease (74). The estimated incidences of all new cases (per 100,000 people) of M. tuberculosis infection in the most common countries of origin of international adoptees are as follows: China, 112.7; Russia, 126.4; Guatemala, 77.2; South Korea, 90.6; and Kazakhstan, 145.6 (82). These data suggest that exposure to M. tuberculosis is common in international adoptees and represents a potentially significant health risk to both the adoptees and their immediate families. This risk is illustrated in a published report documenting transmission of M. tuberculosis from an adopted child to family members in the United States (17). Numerous studies have examined rates of latent M. tuberculosis infection in international adoptees. The percentage of children with a positive PPD skin test has ranged from as low as 1.0% to as high as 19% (1, 36, 40, 46, 56, 66). Of note, the majority of children with evidence of M. tuberculosis infection were adopted from Russia and China. Studies from the early 1990s reported a relatively high incidence of active tuberculosis in international adoptees with positive skin tests. For example, a study by Nicholson et al. that found 3/9 children with a positive Mantoux test had evidence of active disease (59), while a second study identified 4 of 10 children with positive skin tests and evidence of pulmonary tuberculosis (36). More recent reports, however, suggest that most international adoptees with positive PPD skin tests are unlikely to have active tuberculosis (1, 36, 40, 46, 56, 66). Nonetheless, global trends in transmission of M. tuberculosis suggest that international adoptees will remain at increased risk for infection.

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Syphilis. Syphilis is caused by infection with the bacterial spirochete Treponema pallidum. In children, including international adoptees, the likely routes of T. pallidum transmission include perinatal acquisition and sexual abuse. Newborns frequently do not manifest the hallmark signs of congenital infection, which include anemia, thrombocytopenia, hepatosplenomegaly, and rash. If untreated, however, children with congenital syphilis may develop significant growth and cognitive delays, deafness, keratitis, and dental findings (Hutchinson teeth) (18). A recent increase in the incidence of syphilis in the former Soviet Union has been well documented (63). In China, the prevalence of syphilis, including congenital syphilis, may be increasing, although published data are incomplete and may not accurately represent what is likely to be substantial regional variation. Of note, one report suggests that the incidence of syphilis in China increased from 0.18/100,000 in 1993 to 4.31/100,000 in 1998, suggesting a potential increase in the risk of congenital infection in adoptees (13). By contrast, the prevalence of syphilis in South Korea is only 0.2% (14), while data from Guatemala are unavailable. Despite recent increases in the global incidence of syphilis, particularly in Eastern Europe, studies of international adoptees have reported a prevalence of less than or equal to 1.7% (36, 40, 46, 56, 59). As is the case with HIV infection, the relatively low prevalence suggests that many children are likely screened for syphilis before being referred for adoption. In support of this, many orphanage records, particularly for children adopted from Russia and Eastern Europe, contain references to syphilis treatment, in either the birth mother or child. Helicobacter pylori. H. pylori is a gram-negative bacillus that colonizes the gastric mucosa. Infection is often acquired during childhood and is frequently transmitted in institutional settings. Although the infection is more commonly diagnosed in adults with peptic ulcer disease, data suggest that children are also at risk for acquiring H. pylori. Although infection in children is usually asymptomatic, chronic infection has been associated with an increased risk of gastroesophageal reflux, growth delay, and gastric cancer. There are a variety of ways to diagnose H. pylori infection, including the [13C]urea breath test, a fecal antigen test, serologic testing, and endoscopy with gastric biopsy (29, 80). Treatment of H. pylori infection in otherwise asymptomatic children is not recommended, although combination antibiotic regimens are effective at eradicating the organism (29). Miller et al. have published the only study examining the seroprevalence of H. pylori antibodies in international adoptees (57). They reported that 31% of children had evidence of H. pylori infection. Risk factors included residence in an orphanage and older age at adoption. Of note, children with H. pylori antibodies were twice as likely to have intestinal parasites on fecal examination, although infection was not associated with anemia, gastrointestinal symptoms, or growth delay (57). Thus, the clinical significance of H. pylori antibodies in international adoptees remains to be determined. Bacterial gastroenteritis. Several studies have identified enteric bacterial pathogens by culturing stool samples from international adoptees. Published rates of bacterial gastroenteritis in international adoptees range from 2.5% to 7.0%. The two most common bacterial pathogens isolated on routine

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fecal culture include Salmonella spp. and Campylobacter jejuni (36, 56, 59, 66). The above data suggest that internationally adopted children are at modest risk for colonization with pathogens known to cause bacterial gastroenteritis. However, in the absence of specific gastrointestinal symptoms, the value of routine stool culture for bacteria has not been established.

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cases of head lice in their series of international adoptees, although the prevalence was not reported (36). Jenista and Chapman reported 13/124 (10.5%) children with head lice and/or scabies (39).

Parasitic Infections

INFECTIOUS DISEASES IN INTERNATIONAL ADOPTEES: THE YALE INTERNATIONAL ADOPTION CLINIC EXPERIENCE

Intestinal parasites. Gastrointestinal parasites, particularly protozoa, are frequently transmitted via contaminated drinking water but also may be spread from person to person through fecal-oral contact. As numerous outbreaks in institutionalized settings have been reported, it is not surprising that intestinal protozoa are frequently identified on routine screening of international adoptees. Examination of a fresh fecal specimen by microscopy is an effective means of diagnosing intestinal parasites, although the sensitivity increases markedly if multiple samples are evaluated. In the case of the protozoan parasite Giardia lamblia, a commercially available fecal antigen test has high sensitivity for detecting mild to moderate infections. Infection with intestinal parasites is common in international adoptees, with a prevalence of 14% to 33%. The most common intestinal parasite identified in adoptees is Giardia lamblia, which is found most often in children from Eastern Europe (40, 66). In addition to having giardiasis, international adoptees may also be infected with other parasites, including Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercoralis, Blastocystis hominis, and Dientamoeba fragilis (36, 39, 56, 59). Ectoparasites. (i) Scabies. Scabies is a skin infection caused by the mite Sarcoptes scabiei. Outbreaks have been reported in a variety of institutional settings, including orphanages. Early infection may be asymptomatic, after which the host becomes highly sensitized to the infestation. In contrast to primary infection, reinfection is associated with significant symptoms, including diffuse pruritus and the development of a diffuse pustular rash. Intertriginous areas, as well as the wrist, ankles, and web spaces between the fingers and toes are most commonly involved. Scrapings of the pustular lesions may reveal adult scabies when examined under light microscopy. Lange and Warnock-Eckhart identified 4/360 (1.1%) Korean adoptees with scabies (46). Not surprisingly, all four infected children were living in an orphanage, rather than in foster homes as is typical for Korean adoptees. Nicholson et al. diagnosed scabies in 7/99 (7%) adoptees evaluated in a hospital-based clinic in Australia (59). Otherwise, data on the prevalence of scabies in international adoptees are limited. (ii) Lice (pediculosis). Lice are bloodfeeding arthropods that infect the hair and scalp (Pediculus capitis), body (Pediculus corporis), or pubic area (Phthirus pubis). Risk factors for pediculosis include living in an institutionalized setting, such as an orphanage. Infection with Pediculus capitis may be asymptomatic, although pruritus of the scalp is a common feature. The diagnosis is made by identifying any of the major life cycle stages of the arthropod, including adults, nymphs, or eggs. The exact prevalence of head lice in international adoptees is unknown. However, Nicholson et al. reported that 4/99 (4%) of a group of international adoptees evaluated in their clinic were diagnosed with head lice (59). Hostetter et al. also identified

The Yale International Adoption Clinic (YIAC), located in New Haven, Conn., is a hospital-based specialty clinic that provides preadoption counseling and medical/developmental evaluations of international adoptees. To date, the YIAC has evaluated more than 800 children adopted into families living primarily in Connecticut, New York, Vermont, Massachusetts, and Rhode Island. In order to compare the published literature with our recent experience, we reviewed the infectious disease diagnoses for the last 105 consecutive patients who underwent medical screening at the YIAC. All children underwent a routine physical examination and standard medical screening (see “Postadoption Medical Screening” below). Roughly one-third of these children were born in Russia, while another one-third were from China. The remaining one-third were adopted from 10 other countries. All 105 children (72 females and 33 males) were evaluated between 8 September 2003 and 14 March 2005, and the average age was 27 months (range, 4 months to 11 years). Table 3 lists various markers of infectious diseases identified in these children. The data suggest that the YIAC experience is similar to that gleaned from published reports from clinics specializing in the evaluation of international adoptees. In particular, the data demonstrate that the risk of serious infections, including HIV, hepatitis B virus, and hepatitis C virus, in recent adoptees is low. Other infections, however, remain common in adoptees, including varicella-zoster virus (VZV) infection (25%), giardiasis (20%), and scabies (10%). In addition, the YIAC data also reveal a high prevalence of hepatitis B virus surface antibody seropositivity (70%) in international adoptees, with little evidence of prior infection. This finding most likely reflects a high rate of hepatitis B virus immunization and confirms that most international adoptees are at low risk of infection. In support of this, only one child out of the 100 tested (1%) was found to have chronic hepatitis B virus infection. The high seroprevalence rate (25%) for VZV is worth noting, as little is known about this infection in international adoptees. Further analysis revealed that those children who were seropositive tended to be older (mean age, 57 ⫾ 35 months) than those who were seronegative (mean age, 22 ⫾ 20 months), although the difference was not found to be statistically significant. These data confirm that, as expected, children acquire VZV over time in their country of origin and that routine screening for infection may be warranted. Other common infections in this cohort of recent adoptees included giardiasis and scabies, which were identified in 20% and 9.5% of adoptees, respectively. Taken together, these data confirm that adoptees, most of whom have lived in orphanages, are at risk for infections well known to be transmitted efficiently within institutional settings. Three out of 98 (3.1%) children evaluated in the YIAC were

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TABLE 3. Markers of selected infectious diseases (by country of origin) in children evaluated at the Yale International Adoption Clinic from September 2003 to March 2005a No. positive/no. tested (% positive) Country (n)

Russia (33) China (32) Guatemala (10) Kazakhstan (9) Ukraine (5) Poland (3) Ethiopia (3) Korea (3) Georgia (2) Belarus (2) India (2) Romania (1) Total (105)

Giardia antigen

HBsAb

VZV IgG

VDRL/FTA

Scabies

7/26 (27) 3/17 (17.6) 0/8 0/5 3/4 (75) 0/3 0/2 0/1 1/2 (50) 0/2

26/32 (81) 20/30 (66) 7/9 (77) 7/9 (77) 2/5 (40) 1/3 (33) 1/3 (33)b 3/3 (100) 0/2 1/2 (50) 2/2 (100)

3/12 (25) 1/18 (5.5) 0/6 4/8 (50) 0/3 3/3 (100) 2/3 (66) 0/1

1/30 (3.3) 0/29 1/10 (10) 0/9 0/5 0/3 0/3 0/3 1/2 (50) 0/2 0/2

5/33 (15) 4/32 (12.5) 0/10 1/9 (11) 0/5 0/3 0/3 0/3 0/2 0/2 0/2 0/1

14/70 (20)

70/100 (70)

3/98 (3.1)c

10/105 (9.5)

1/1 (100)

14/55 (25)

a

All patients were seronegative for antibodies to HIV and hepatitis C virus. Additional infections diagnosed included visceral larva migrans in one child from Guatemala and head lice in one child from Ethiopia. b One child was found to have circulating HBsAg and HBcAb in the absence of HBsAb, consistent with chronic hepatitis B virus infection. c One child was FTA positive and VDRL negative in serum, with a preadoption record of treatment for congenital syphilis. The other two children were positive for both VDRL and FTA.

diagnosed with congenital syphilis (one from Russia, one from Guatemala, and one from Georgia). Although this rate of infection is somewhat higher than those reported in other studies, the small sample size make it difficult to draw conclusions as to the significance of this finding. Of note, two of the three children diagnosed with congenital syphilis had no mention of this infection in the preadoption record, raising concerns about the reliability of preadoption medical information (see below).

(2%) and prior (14%) hepatitis B infection, tuberculosis (5%), and intestinal pathogens (51%). Additionally, this study found significant discordance between the postadoptive physical exam and preadoptive medical record for noninfectious medical conditions as well. These data suggest that the preadoption medical record is neither sensitive nor specific for accurately assessing the risk of infectious diseases in international adoptees. Therefore, parents should be counseled on the inherent uncertainty of medical record review as a means of evaluating the health of prospective adoptees.

PREADOPTION MEDICAL RECORD REVIEW Anecdotal reports suggest that preadoption medical records are often misleading and inaccurate, although some experts maintain that records from certain countries (e.g., South Korea) may be more reliable than those from others (e.g., China and Russia) (38). Over the past decade, it has become increasingly common for prospective parents to ask specialists in international adoption, as well as primary care pediatricians, to review video tapes and/or medical records in order to help inform their decision whether or not to adopt a particular child. However, foreign medical records are often difficult to interpret because of incomplete information and inaccurate translation, as well as the presence of multiple, often confusing diagnoses. While several articles refer to these difficulties, few studies have rigorously examined the accuracy of foreign medical records by comparing them to laboratory data and physical exam findings after adoption. Albers et al. reviewed preadoptive medical records for 43 children adopted from Eastern European orphanages (1). Pneumonia or bronchitis was listed as a prior medical diagnosis in 18/43 children (42%). Otitis media was listed in 7/43 records (16%), adenoidal hypertrophy in 2/43 (4.6%), diarrhea in 2/43 (4.6%), dysbacteriosis in 2/43 (4.6%), measles in 2/43 (4.6%), and pertussis in 2/43 (4.6%). Laboratory studies carried out postadoption also identified multiple infectious diseases not documented in the medical record. These included chronic

POSTADOPTION MEDICAL SCREENING In 1991, Hostetter et al. reported that 81% of the medical diagnoses for international adoptees were detected through the use of defined screening tests and not by routine history or physical examination (36). The authors concluded from that study that all children who are adopted from foreign countries should undergo thorough testing in order to identify occult conditions, most of which were infectious diseases. Original recommendations based on these findings included screening for infection with hepatitis B virus, HIV, tuberculosis, syphilis, CMV, and intestinal parasites. Additional medical tests included a complete blood count and urinalysis (36). These recommendations have been modified somewhat over the past decade (Table 4), although the principles upon which these original recommendations were based have not changed. In the YIAC, adoptees are tested for antibodies to HIV types 1 and 2 by serum enzyme-linked immunosorbent assay (ELISA), with a confirmatory Western blot if the ELISA is positive. Although serologic testing for antibodies to HIV is useful for screening, a positive antibody test (ELISA or Western blot) in children less than 1 year of age may reflect acquisition of maternal antibody, and thus this test lacks specificity in young adoptees. Conversely, a negative HIV antibody test does not rule out recent infection, particularly if there is a possibility that a child was exposed through the use of a con-

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TABLE 4. Medical screening tests for international adoptees Test

Routinely recommended Hepatitis B virus surface antigen, surface antibody, and core antibody Hepatitis C virus antibody HIV ELISA and RNA PCR if ELISA positive Varicella-zoster virus antibody Fecal examination for parasite ova and Giardia antigen Urinalysis VDRL (RPR) and FTA PPD skin test for M. tuberculosis Complete blood count with differential serum lead level Thyroid-stimulating hormone level Hearing Vision Developmental evaluation Occasionally recommended Stool culture for bacterial pathogens Urine culture Liver function tests Serum electrolytes Hemoglobin electrophoresis Serum G6P-Da level Generally not recommended H. pylori antibody CMV antibody and/or urine culture for CMV a

G6P-D, glucose 6-phosphate dehydrogenase deficiency.

taminated needle just prior to adoption. Therefore, for recent adoptees, we also test for HIV by using an RNA PCR assay. If PCR testing is not available, we recommend repeating the ELISA at 4 to 6 months postadoption to definitively rule out infection with HIV. We routinely test adoptees for hepatitis B virus infection by using standard serologic assays. Acute hepatitis B infection is characterized by the presence of HBsAg, HBeAg, and hepatitis B virus core antibody (HBcAb). Resolution of acute infection is associated with a rise in hepatitis B virus surface antibody (HBsAb) and the disappearance of HBsAg from the bloodstream. Chronic infection is characterized by the persistence of HBsAg, as well as HBcAb. The presence of HBcAb generally distinguishes those with past infection from those who have been previously immunized, as both groups will have measurable HBsAb (48, 51). In recent adoptees who do not have HBsAb on the initial screen, we recommend repeating the hepatitis B panel in 2 to 3 months in order to rule out infection immediately prior to adoption. For those children who are chronically infected with hepatitis B virus, interferon alfa-2b may be effective therapy (72). Testing for serum antibodies to hepatitis C virus is the recommended method of screening international adoptees. However, because maternal antibodies to hepatitis C virus may persist for at least 1 year after birth, a positive antibody test does not necessarily indicate ongoing infection in an infant. Therefore, in children with a positive antibody test for hepatitis C virus, we confirm the infection by using an RNA PCR assay. Dunn et al. studied the appropriate timing of diagnostic tests for hepatitis C virus in neonates and determined that serologic testing for perinatally acquired infection was not conclusive

until a child reaches 18 months of age (20). However, a negative PCR test in an infant older than 4 weeks of age, even in the presence of antibodies to hepatitis C virus, suggests that the child is not infected. For children evaluated less than 2 months following adoption, repeating the hepatitis C virus antibody test will identify those who might have been exposed just prior to adoption. Children who are infected may benefit from therapy with alpha interferon, either alone or in combination with ribavirin (19, 21, 42, 52, 75). Given the fact that children with congenital syphilis are often asymptomatic, serologic testing should be performed for all international adoptees. The Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests are highly sensitive, although false-positive results may occur in individuals with rheumatologic or inflammatory conditions. All positive nontreponemal tests (VDRL or RPR) must be confirmed using the fluorescent treponemal antibody (FTA) absorption test, which is more specific (6). Infected children should undergo lumbar puncture for VDRL testing of the cerebrospinal fluid in order to rule out neurosyphilis, as well as a complete blood cell count, liver function tests, long-bone radiographs, and vision and hearing screening (18). For therapy of children with untreated congenital syphilis, we recommend 10 days of intravenous penicillin G (100,000 to 150,000 U/kg/day). The response to therapy should be monitored by following VDRL test levels every 3 to 6 months for up to 24 months, with the goal of documenting a fourfold drop in titer or reversion to negative. Those children with a positive VDRL test in the cerebrospinal fluid require a repeat lumbar puncture in 6 months to document a reduction in titer (6). Importantly, the FTA absorption test will remain positive, even after therapy. Because of the difficulty in validating orphanage records, we recommend therapy for all seropositive adoptees, even if there is written or verbal documentation of treatment prior to adoption. In the absence of gastrointestinal symptoms, a single fecal sample should be evaluated for parasite ova and Giardia antigen. It is recommended that international adoptees receive treatment for Giardia infection because of the potential for transmission to close contacts (35). Metronidazole, tinidazole, and nitazoxanide are all effective (24, 85). Of note, Saimen et al. reported that 85/87 (97.7%) adoptees with giardiasis were cured after one course of therapy, while the remaining 2 responded to a second course (66). In our experience, some children may continue to experience gastrointestinal symptoms, particularly loose stools, for weeks to months following treatment. Treatment is also recommended for international adoptees infected with intestinal nematodes. Albendazole and mebendazole, two benzimidazole anthelminthics, are effective against Ascaris, Enterobius, hookworm, and Trichuris (58). Pyrantel pamoate is also approved for the treatment of intestinal nematodes, although it is not active against Trichuris. Nitazoxanide is also effective against a broad array of intestinal nematodes and may be a reasonable alternative (24). For those adoptees with evidence of gastroenteritis, we also obtain a stool culture for pathogenic bacteria and Cryptosporidium. Although many international adoptees have records and/or a cutaneous scar (most commonly in the left deltoid) consistent with prior Mycobacterium bovis BCG vaccination, we routinely recommend a PPD skin (Mantoux) test as part of their post-

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adoption medical screening. Interpreting the significance of a PPD skin test result requires an initial assessment of an individual’s pretest probability of being infected. Therefore, because of the high rates of tuberculosis in countries from which children are adopted into the United States, most experts recommend greater than or equal to 10 mm of induration as the positive threshold for international adoptees. The risk of a “false-positive” PPD skin test associated with prior BCG immunization is considered low if the skin test is placed at least 1 year following the vaccine (50), although recent data suggest that prior BCG immunization is associated with significant PPD reactions years later (76). False-negative results may be due a number of factors, including malnutrition, concurrent inflammatory/rheumatologic disease, underlying immune deficiency, active M. tuberculosis, inactive antigen, or poor technique when placing the PPD test. We recommend a chest radiograph for all children with a positive PPD skin test result. A child with a positive PPD skin test but no evidence of pulmonary disease should receive 9 months of therapy with isoniazid at a dose of 10 mg/kg/day. If the chest radiograph suggests active disease, then sputum or gastric aspirates should be obtained for AFB stain and culture (69, 86). All household contacts and close family members of any child with active pulmonary (or miliary) tuberculosis should also undergo PPD skin testing. A child with pulmonary or extrapulmonary disease should be treated with a regimen of at least three antituberculous medications for 2 months while the results of AFB cultures and sensitivity data are pending. The ultimate drug regimen and duration of therapy should be based on the sensitivity profile of the specific isolate (5). In order to detect those international adoptees whose initial PPD test might be negative due to malnutrition, a second PPD skin test at 4 to 6 months postadoption is recommended. Importantly, any child with a negative PPD skin test result who demonstrates clinical signs or symptoms of tuberculosis should undergo thorough evaluation, including gastric lavage, as active disease has been reported in this setting (47). In light of the high prevalence of scabies in international adoptees, many children are treated empirically in the appropriate clinical setting. The treatment of choice is topical 5% permethrin cream (79). A second application in 1 week is recommended for complete cure. Family members and close contacts should be treated if they develop symptoms or signs of infection. Oral ivermectin, administered in one or two doses, is also highly effective in the treatment of scabies in adults (53), although its efficacy in children is unknown. Treatment options for head lice (pediculosis) include topical 1% permethrin, 1% lindane, or 0.5% malathion, which are applied to the scalp or skin and rinsed off after 10 min (16). Although reported cure rates approach 95% with these therapies, resistance to topical agents may represent an emerging challenge (33). Two doses of oral ivermectin, administered 10 days apart, may be an alternative (41). In addition to the above screening for infectious diseases, we also obtain a complete blood count, routine urinalysis, serum lead level, and serum thyroid-stimulating hormone level to evaluate for noninfectious medical conditions. Additional screening tests recommended by adoption specialists include hemoglobin electrophoresis, serum liver transaminase levels, serum electrolytes, blood urea nitrogen and creatinine, and

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bacterial cultures of the stool and urine (7, 59). As outlined above, depending on the results of the initial medical screening, some tests may need to be repeated (7, 73). RECOMMENDATIONS FOR IMMUNIZATION OF INTERNATIONAL ADOPTEES The recent cases of imported measles traced to a group of Chinese adoptees underscore the notion that these children are at risk of both acquiring and transmitting vaccine-preventable infections. Strategies aimed at assessing the likelihood of an international adoptee having been appropriately immunized are problematic for a variety of reasons. First, many of the preadoption medical records are incomplete, and they often do not contain information about vaccinations. Albers et al. reviewed the medical records of 32 international adoptees and found that many lacked documentation of complete ageappropriate vaccination (1). For example, 27/32 records contained documentation of vaccination against measles, 17/32 for poliomyelitis, 5/32 for mumps, 25/32 for BCG, and 5/32 for diphtheria-pertussis-tetanus. No child was documented to be vaccinated against rubella, hepatitis B, or Haemophilus influenzae. Shulte et al. examined 504 children evaluated at an adoption clinic in 1997 and 1998. Only 178/504 (35%) children had written documentation of immunization prior to arrival in the United States (68). Of those with immunizations recorded and determined to be valid, 112/167 (67%) were found to be current for at least one vaccine series. When immunization records of children older than 6 months were examined, only 14/150 (9%) were current for all recommended vaccines (68). Whether these data suggest incomplete documentation or limited immunization practices in resource-poor countries is unknown, but they clearly reinforce concerns about the failure of institutional care settings to meet even the most basic health needs of these children. Data from studies of international adoptees with seemingly appropriate records suggest that children with documentation of vaccination may not have serologic evidence of protective immunity. Miller et al. reported data from a study of 70 children in whom serum antibodies against the standard vaccinepreventable infections were measured (55). Of these, 61% had levels of antibody that were consistent with immunity to tetanus, 88% to diphtheria, 50% to pertussis, 65% to polio, and 90% to measles. These children were adopted from a number of different countries, and the authors found no association between serologic evidence of vaccination and country of origin. Similarly, Schulpen et al. reported that only 60% of Chinese adoptees were positive for antibodies against diphtheria and tetanus (67). Of note, in both of these studies a substantial number of children had antibody levels that were considered to be in the marginal or borderline category. Saimen et al. found that a third of children reported to have received a complete hepatitis B virus immunization series were HBsAb negative upon testing in the United States (66). There are a number of potential explanations for the apparent discordance between documentation of vaccination and serum antibody levels in some international adoptees. One obvious reason is that the records are intentionally falsified in order to give the impression that a child has received adequate care. A second potential explanation is that orphanages may

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MURRAY ET AL. TABLE 5. Strategies for management of vaccine-preventable infections in international adoptees Strategy

1. Accept all appropriate preadoption records of immunization 2. Measure serum antibodies against vaccine-preventable pathogens for which reliable testing is available; if present, accept all corresponding preadoption records of immunization 3. Measure serum antibodies against vaccine-preventable pathogens for which reliable testing is available; if positive, consider the child to have received one immunization in the series 4. Accept no preadoption records of immunization; immunize all adoptees with age-appropriate vaccinations

not have the resources to ensure proper storage of vaccines, particularly those that require refrigeration. It is also possible that children may receive outdated or diluted vaccine, perhaps due to the limited resources available to a particular orphanage. Lastly, certain effects of institutionalization, attributable perhaps to malnutrition, may impair host immunity, thus leading to a less robust response to immunization. In support of this, it is interesting that Hostetter and Johnson reported that adoptees who had resided in orphanages were less likely to have serologic evidence of adequate vaccination than those children from foster homes (37). Regardless of the underlying causes, it is clear that many international adoptees whose vaccination records are accepted as accurate may be at risk for acquiring vaccine-preventable infections. Because of the above uncertainties, recommendations vary as to the appropriate management of international adoptees with regard to routine childhood immunizations. For children with questionable preadoption records, there seems to be little choice but to recommend that they receive all age-appropriate vaccinations according to current recommendations (3). For children with preadoption medical records that appear to document appropriate immunization, there are a variety of strategies to guide future decisions about vaccination (Table 5). One option is to accept the records as valid and complete the immunization schedule based on what is documented. A second strategy would be to test adoptees for antibodies to hepatitis B virus, diphtheria, tetanus, poliovirus (all three serotypes), measles virus, mumps virus, and rubella virus and then accept as valid those immunizations for which a child has appropriate levels of antibody. However, results of antibody testing must be interpreted with caution, in light of the possibility that a child might have received unrecorded immunizations immediately prior to adoption that could lead to a transiently elevated titer or antibody level. A third strategy is to consider a child to have received one immunization for each of the pathogens against which he or she has measurable antibody levels. This strategy would potentially eliminate one immunization in each of the multidose series, while still ensuring that adoptees are fully protected against vaccine-preventable illnesses. However, because it requires extensive testing with the potential for only a limited reduction in the number of vaccinations, this strategy is likely to be the least cost-effective. A fourth strategy is to ignore all preadoption records, and fully immunize each child based on age-appropriate recommenda-

tions (4). As there appears to be little evidence that immunization of international adoptees is associated with a substantial risk of adverse events, we have generally recommended that adopted children under 1 year of age receive the full series of most vaccinations. This recommendation is guided primarily by our concern about the potential risk of adoptees reaching adulthood without appropriate levels of immunity to vaccinepreventable infections. The above recommendations apply to those vaccines that are routinely administered in orphanages and foster home settings, namely, hepatitis B virus, diphtheria-pertussis-tetanus, poliovirus, and measles virus. However, few adoptees have records documenting immunization against VZV, Haemophilus influenzae type b, and Streptococcus pneumoniae. Vaccination against H. influenzae and S. pneumoniae should be administered to international adoptees according to age-specific recommendations (4). We recommend testing for antibodies to VZV in children older than 1 year of age, since their presence in these children suggests prior infection and hence immunity. It has been suggested that this practice may be cost-effective for children who are older than 5 years (22), and data from the Yale Clinic confirm high rates of infection in adoptees (Table 3). For those children without measurable antibodies, we recommend vaccination with the live attenuated vaccine. ACKNOWLEDGMENTS We express our appreciation for the advice and support of Margaret Hostetter, founder of the YIAC. We also thank the staff, patients, and families from YIAC for their continued support. This study was approved by the Yale University Human Investigations Committee. REFERENCES 1. Albers, L. H., D. E. Johnson, M. K. Hostetter, S. Iverson, and L. C. Miller. 1997. Health of children adopted from the former Soviet Union and Eastern Europe. Comparison with preadoptive medical records. JAMA 278:922–924. 2. American Academy of Pediatrics. 2003. HIV, p. 360–382. In L. Pickering (ed.), Red book: 2003 report of the Committee on Infectious Diseases, 26th ed. American Academy of Pediatrics, Elk Grove Village, Ill. 3. American Academy of Pediatrics. 2003. Immunization in special clinical circumstance, p. 92–93. In L. Pickering (ed.), Red book: 2003 report of the Committee on Infectious Diseases, 26th ed. American Academy of Pediatrics, Elk Grove Village, Ill. 4. American Academy of Pediatrics. 2003. Medical evaluation of internationally adopted children for infectious diseases, p. 173–180. In L. Pickering (ed.), Red book: report of the Committee on Infectious Diseases, 26th ed. American Academy of Pediatrics, Elk Grove Village, Ill. 5. American Academy of Pediatrics. 2003. Mycobacterium tuberculosis, p. 642– 660. In L. Pickering (ed.), Red book: 2003 report of the Committee on Infectious Diseases, 26th ed. American Academy of Pediatrics, Elk Grove Village, Ill. 6. American Academy of Pediatrics. 2003. Syphilis, p. 595–607. In L. Pickering (ed.), Red book: 2003 report of the Committee on Infectious Diseases, 26th ed. American Academy of Pediatrics, Elk Grove Village, Ill. 7. Aronson, J. 2000. Medical evaluation and infectious considerations on arrival. Pediatr. Ann. 29:218–223. 8. Boyce, T. G., and P. F. Wright. 1999. Cytomegalovirus pneumonia in two infants recently adopted from China. Clin. Infect. Dis. 28:1328–1330. 9. Capua, I., and D. J. Alexander. 2004. Human health implications of avian influenza viruses and paramyxoviruses. Eur. J. Clin. Microbiol. Infect. Dis. 23:1–6. 10. Centers for Disease Control and Prevention. 2004. Multistate investigation of measles among adoptees from China—April 2004. Morb. Mortal. Wkly. Rep. 53:309–310. 11. Centers for Disease Control and Prevention. 2004. www.cdc.gov/travel /diseases/maps/hbv_map.htm. 12. Chan, C. Y., S. D. Lee, and K. J. Lo. 2004. Legend of hepatitis B vaccination: the Taiwan experience. J. Gastroenterol. Hepatol. 19:121–126. 13. Chen, X. S., X. D. Gong, G. J. Liang, and G. C. Zhang. 2000. Epidemiologic trends of sexually transmitted diseases in China. Sex. Transm. Infect. 27: 138–142.

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D. Erdman, T. C. Peret, C. Burns, T. G. Ksiazek, P. E. Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor, K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, A. D. Osterhaus, C. Drosten, M. A. Pallansch, L. J. Anderson, and W. J. Bellini. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399. Ruxrungtham, K., T. Brown, and P. Phanuphak. 2004. HIV/AIDS in Asia. Lancet 364:69–82. Saiman, L., J. Aronson, J. Zhou, C. Gomez-Duarte, P. S. Gabriel, M. Alonso, S. Maloney, and J. Schulte. 2001. Prevalence of infectious diseases among internationally adopted children. Pediatrics 108:608–612. Schulpen, T. W., A. H. van Seventer, H. C. Rumke, and A. M. van Loon. 2001. Immunisation status of children adopted from China. Lancet 358:2131–2132. Schulte, J. M., S. Maloney, J. Aronson, P. San Gabriel, J. Zhou, and L. Saiman. 2002. 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CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 521–540 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.521–540.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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Recognition of Staphylococcus aureus by the Innate Immune System Be´ne´dicte Fournier1* and Dana J. Philpott2 Laboratoire des Listeria1 and Groupe d’Immunite´ Inne´e et Signalisation,2 Institut Pasteur, Paris, France INTRODUCTION .......................................................................................................................................................521 INFLAMMATORY RESPONSE TO S. AUREUS...................................................................................................522 TOLL-LIKE RECEPTOR 2.......................................................................................................................................523 Staphylococcal Structures Recognized by TLR2 ................................................................................................524 Teichoic acids ......................................................................................................................................................524 (i) Structure.....................................................................................................................................................524 (ii) Role in the inflammatory response........................................................................................................525 (iii) Interaction with TLR2............................................................................................................................525 Alanylation of teichoic acids .............................................................................................................................525 (i) Structure.....................................................................................................................................................525 (ii) Role in the inflammatory response........................................................................................................525 (iii) Interaction with TLR2............................................................................................................................526 Peptidoglycan.......................................................................................................................................................526 (i) Structure.....................................................................................................................................................526 (ii) Role in the inflammatory response........................................................................................................526 (iii) Interaction with TLR2............................................................................................................................528 Phenol-soluble modulin......................................................................................................................................528 (i) Structure.....................................................................................................................................................528 (ii) Role in the inflammatory response........................................................................................................528 (iii) Interaction with TLR2............................................................................................................................528 TLR2 Cooperation with Other Receptors ...........................................................................................................529 TLR1/TLR6 ..........................................................................................................................................................529 CD14 .....................................................................................................................................................................529 CD36 .....................................................................................................................................................................529 Asialo-GM1 ..........................................................................................................................................................529 Structure of TLR2...................................................................................................................................................530 TLR2 Signaling .......................................................................................................................................................530 NOD PROTEINS ........................................................................................................................................................532 Staphylococcal Structure Recognized by Nod Proteins .....................................................................................533 Structure of Nod2 ...................................................................................................................................................533 Nod Signaling ..........................................................................................................................................................533 RIP2 pathway ......................................................................................................................................................533 Interaction with the TLR pathway ...................................................................................................................533 TNFR1 ..........................................................................................................................................................................534 PEPTIDOGLYCAN RECOGNITION PROTEINS.................................................................................................534 OTHER STAPHYLOCOCCAL COMPONENTS INVOLVED IN THE INFLAMMATORY RESPONSE.....534 Hemolysins...............................................................................................................................................................535 Formylated Peptides ...............................................................................................................................................535 Staphylococcal DNA ...............................................................................................................................................535 CONCLUDING REMARKS ......................................................................................................................................535 ACKNOWLEDGMENTS ...........................................................................................................................................536 REFERENCES ............................................................................................................................................................536 The primary site of infection is often a breach in the skin that may lead to minor skin and wound infections, but S. aureus can also infect any tissue of the body, causing life-threatening diseases such as osteomyelitis, endocarditis, pneumonia, and septicemia. The pathogenicity of S. aureus is due to the repertoire of toxins, exoenzymes, adhesins, and immune-modulating proteins that it produces. With the exception of diseases caused by specific toxins, such as enterotoxins and exfoliative or toxic shock syndrome toxins, no single virulence factor has been shown to be sufficient to provoke a staphylococcal infection. Such infection is rather promoted by the coordinated action of

INTRODUCTION Staphylococcus aureus is a major pathogen in both community-acquired and nosocomial infections. S. aureus often colonizes hosts asymptomatically and lives as a commensal of the human nose. The anterior nares are the major reservoir of S. aureus: 20% of persons are persistently colonized and 60% are intermittent carriers, whereas 20% never carry S. aureus (86).

* Corresponding author. Mailing address: Laboratoire des Listeria, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 40 61 31 12. Fax: (33) 1 40 61 35 67. E-mail: [email protected] 521

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various virulence factors, which are cell wall associated and secreted bacterial proteins. Indeed, both localized infections, such as soft-tissue abscesses, and life-threatening systemic diseases, such as sepsis, result from the ability of this pathogen to attach to cells or tissues; escape the host immune system, i.e., factors that decrease phagocytosis and factors that interact with antistaphylococcal antibodies; and elaborate proteases, exotoxins, and enzymes, factors that specifically cause cell and tissue damage allowing dissemination of S. aureus (154). The expression of these virulence factor genes is controlled by several regulatory systems such as Agr, SarA, and Arl (26, 42, 129, 148). The accessory gene regulator (Agr) is one of the best-characterized global regulatory systems involved in the regulation of virulence factor genes. Indeed, Agr, which is a quorum-sensing regulatory system, regulates virulence by increasing the expression of exoprotein genes and decreasing the expression of cell wall-associated protein genes (129, 148). Staphylococcal sepsis differs from enterobacterial sepsis in that S. aureus infection often begins from infected loci at the body surface such as skin or soft tissue infections rather than enteric or genitourinary sources (159). Furthermore, staphylococcal infection induces an influx of neutrophils. Indeed, S. aureus is a pyogenic pathogen capable of tissue invasion and evasion of phagocytosis by neutrophils. Tissue invasion and killing of phagocytes are involved in the inflammatory response that leads to septic shock (178). Even though mortality rates in systemic nosocomial infections associated with S. aureus are higher than those due to enterobacteria, they are also lower than those due to aerobic gram-negative bacilli such as Pseudomonas aeruginosa (5, 151). S. aureus, followed by Enterococcus spp. in the United States and Streptococcus pneumoniae in Europe, is the organism most frequently isolated during invasive nosocomial infections (17, 41). The increasing frequency of gram-positive sepsis is probably due to the ability of S. aureus to colonize intravascular catheters or surgically implanted materials and to the spread of antibiotic-resistant S. aureus, such as methicillin-resistant S. aureus (143). The outer cell wall of staphylococci is composed of exposed peptidoglycan, lipoteichoic acids, and a range of other toxic secreted products, which are probably implicated in staphylococcal septic shock (178). However, no symptom of shock is observed when the serum concentration of tumor necrosis factor alpha (TNF-␣), one of the proinflammatory cytokines responsible for septic shock in gram-negative bacteria, is increased. This suggests that, unlike gram-negative-mediated shock, induction of TNF-␣ is not sufficient to cause symptoms of shock in staphylococcal infection (60). Thus, the mechanisms that lead to staphylococcal septic shock are probably multifactorial (178). Pathogens invading the host are controlled by innate and adaptive immune responses. Recognition of microorganisms is the first step of host defense. The adaptive immune system recognizes pathogens by antigen receptors that are expressed at the surface of B and T lymphocytes. These receptors are characterized by specificity and memory. However, gene rearrangement followed by T- and B-cell activation usually takes several days to be fully active and to eradicate pathogens (183). Therefore, more rapid defense mechanisms have been developed by the host through the innate immune system. This system is capable of recognizing pathogens and provides a first

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line of defense to the host. Indeed, the innate immune response initiates a sequence of events that results in the production and secretion of a wide range of inflammatory cytokines and chemokines, the activation of macrophages/ monocytes, and the initiation of adaptive immunity. The production of proinflammatory factors is due to several cell types, including white blood cells (neutrophils and macrophages), epithelial, and endothelial cells as well as platelets. White blood cells can phagocytose, kill, and degrade the pathogen. Compounds or molecules resulting from this degradation are then presented to T cells to activate adaptive immunity (3, 169). The role of the innate immune system is to recognize a large number of different pathogens and to discriminate them from self and also those bacteria composing the normal flora with a limited number of receptors. Furthermore, pathogens have the ability to mutate, altering phenotypic expression of virulence determinants and recognition by host receptors. Thus, the host has developed a variety of innate immune receptors that have the ability to detect different microbial motifs that are usually conserved among species (3, 77). Interestingly, the structures recognized by the innate immune system are usually essential for the invading organisms and are not present in the host cells (113). INFLAMMATORY RESPONSE TO S. AUREUS The complex mechanisms of the host response upon invasion by pathogens include the production and release of proinflammatory and immunomodulating cytokines. Cytokines are inducible proteins that are mainly produced on stimulation of white blood cells and other cells by pathogens (197). Thus, synthesis of cytokines is necessary for the host defense against infections. Indeed, the absence of cytokines in deficient mice has been shown to be deleterious to the host (22). However, an inflammatory response with excessive production of proinflammatory cytokines induces side effects and can even lead to multiple organ dysfunction syndrome and death (22, 197). Cells of the monocytic lineage are essential for innate immunity and also play a critical role in the pathophysiology of bacterial sepsis. Indeed, monocytes/macrophages are the main source of inflammatory cytokines responsible for septic shock (see Fig. 3). Among the different cytokines implicated in inflammation and septic shock, TNF-␣, interleukin-1␤ (IL-1␤), and IL-6 are proinflammatory cytokines. IL-10 is an antiinflammatory cytokine that inhibits proinflammatory cytokines such as IL-1␤, IL-6, IL-8, and TNF-␣. IL-8 is a strong chemoattractant for neutrophils, while MCP-1 (macrophage/ monocyte chemoattractant protein-1) and MIP-1 (macrophage-inflammatory protein-1) are chemoattractive mainly for monocytes: they elicit recruitment of phagocytes to the infectious site (109) (Table 1). Gram-positive infections such as those with S. aureus are capable of producing systemic cytokine responses. However, the peak cytokine response in gram-positive infections occurs 50 to 75 h after the challenge, whereas it occurs 1 to 5 h after in gram-negative infections (143). TNF-␣, IL-1␤, and IL-6 are produced from peripheral blood mononuclear cells and tissue macrophages. IL-12 is also increased in sepsis. Synthesis of the chemokine IL-8 is partly triggered by TNF-␣. Gamma interferon (IFN-␥) is also produced in response to IL-12 and IL-8

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TABLE 1. The six cytokine familiesa Family

Cytokines

Interleukins

Biological activities

IL-1␣ (intracellular) and IL-1␤ (extracellular)

Induces growth factors and inflammatory mediators Activates T cells

IL-6

Stimulates terminal differentiation of B cells Enhances immunoglobulin secretion by B cells Stimulates hepatocytes to produce acute-phase proteins

IL-10

Inhibits the production of cytokines Inhibits major histocompatibility complex class II molecule expression on monocytes Enhances B-cell proliferation and immunoglobulin synthesis

IL-12

Stimulates cytokine synthesis (IFN-␥)

Cytotoxic cytokines

TNF-␣ and TNF-␤

Pleiotropic and multifunctional stimulators of cellular responses Stimulates cytokines and cell adhesion molecules Regulates the proliferation/differentiation of lymphocytes

Colony-stimulating factors

Granulocyte colony-stimulating factor

Stimulates proliferation and differentiation of functionally active mature neutrophils

Interferons

IFN-␥

Induces antiviral activity in a wide variety of cells Involved in macrophage activation

Growth factors

Platelet-derived growth factor

Mitogenic Stimulates chemotaxis

Chemokines

IL-8 (or CXCL8) MIP-1␣ (or CCL3), MIP-1␤ (or CCL4), MCP-1 (or CCL2)

Chemotactic for neutrophils but not lymphocytes and monocytes Chemotactic for monocytes rather than neutrophils

a

Data are from references 22, 63, and 109.

(72, 106, 133, 178, 197). However, they are generally produced in lower amounts in gram-positive infections compared to gram-negative infections (43, 178). Although gram-positive and gram-negative bacteria have relatively different structural and pathogenic profiles, they induce a similar pattern of shock in the host (178). As the systemic inflammatory response is involved in sepsis, it was suggested that treatment modulating or inhibiting inflammatory mediators would improve survival of patients with staphylococcal sepsis. In contrast to some animal models of gram-negative sepsis, pretreatment of animals with corticosteroids does not modify mortality when animals are challenged with S. aureus. Similar results were observed when patients with staphylococcal sepsis were treated with anti-inflammatory agents (143). Furthermore, it appears that anticytokine therapies have a detrimental effect in gram-positive sepsis (2, 143). Thus, these results suggest that gram-positive infections are more difficult to cure than those with gram-negative bacteria (178). TOLL-LIKE RECEPTOR 2 The first members of the Toll family were identified in Drosophila melanogaster. Drosophila melanogaster is very resistant to microbial infections because it can synthesize antimicrobial peptides (7). Since the production of these peptides is regulated by a Toll receptor, adult flies that are mutated in Toll are susceptible to infection by fungi and bacteria (99). Thus, Toll is an essential receptor in the innate immune recognition in Drosophila melanogaster.

By a homology search of databases, 11 homologues of Toll designated Toll-like receptors (TLRs) have been found in humans (4, 114, 224). The role of TLRs in mammals is also to participate in innate immune recognition as pattern recognition receptors that detect common bacterial motifs. Several lines of evidence support the view that TLR2 has a broad role as a pattern recognition receptor for a variety of microbes and microbial structures. These include lipoproteins from pathogens such as mycobacteria, spirochetes, and mycoplasmas, lipoarabinomannan from mycobacteria, Trypanosoma cruzi glycosylphosphatidylinositol anchor, and zymosan from fungi (4). Furthermore, TLR2 has been reported to be involved in the recognition of staphylococcal peptidoglycan and lipoteichoic acid (LTA) (see section on staphylococcal structures recognized by TLR2) (37, 61, 101, 171, 185, 194, 195, 222). The involvement of TLR2 has been implicated in the host response to several staphylococcal infection models. For the most part, lack of TLR2 appears to increase the susceptibility of the host to staphylococcal infections. For example, TLR2deficient mice are highly susceptible to S. aureus when inoculated intravenously (67, 184). After subcutaneous injections, staphylococci can grow intradermally in TLR2⫺/⫺ mice, whereas they are cleared in wild-type mice (67). In addition, since nasal carriage is a major risk factor for staphylococcal infection, this parameter was investigated in TLR2-deficient mice (202). In a model of intranasal infection, TLR2-deficient mice showed 10-fold higher nasal carriage of S. aureus compared to that in wild-type mice (54). However, TLR2 deficiency has also been shown to be protective in certain infection

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FIG. 1. Structures of teichoic acid (A) and lipoteichoic acid (B) of S. aureus (data from references 39 and 163). NAG, N-acetylglucosamine.

models. One of the main components involved in the pathophysiology of sepsis is myocardial dysfunction, and it was found that in TLR2-deficient mice, the heart is protected against cardiac dysfunction caused by S. aureus. Furthermore, in this model, TNF-␣ and IL-1␤ production is significantly attenuated in TLR2deficient mice (87). Thus, from these many investigations, it appears that TLR2 plays an important role as an innate immune receptor in the response to staphylococcal infections. However, depending on the infectious model, deficiency of TLR2 can be either protective or detrimental to the host organism. TLR2 is expressed by different cells involved in the inflammatory response such as monocytes/macrophages, neutrophils, dendritic cells, astrocytes, and mast cells (38, 112, 164, 199). Interestingly, TLR2 and TLR6 are poorly expressed by human intestinal epithelial cells, which are broadly unresponsive to TLR2-dependent ligands such as staphylococcal phenol-soluble modulin or LTA (117).

Staphylococcal Structures Recognized by TLR2 Gram-positive bacterial cell walls are composed of multiple peptidoglycan layers, wall teichoic acids linked to the pepti-

doglycan and lipoteichoic acid (LTA) linked to the cytoplasmic membrane. In contrast, the cell envelope of a typical gramnegative bacterium is composed of a thin layer of peptidoglycan, an outer membrane, and lipopolysaccharides (LPS) and phospholipids. S. aureus does not contain lipopolysaccharide (endotoxin), which is the main cell wall component of gramnegative bacteria responsible for septic shock. However, S. aureus can cause septic shock and multiple organ failure (31). Indeed, in a canine model, infection by S. aureus provokes the same symptoms of septic shock as does Escherichia coli (134). The pathogenicity of S. aureus is postulated to depend on the expression of a wide range of cell wall-associated and secreted bacterial proteins. We will not describe in this review the role of superantigen toxins in the inflammatory response because these superantigens are encoded by accessory genetic elements not always present in the S. aureus genome. Although cell wall-associated and secreted proteins are keys for staphylococcal virulence, their role in innate immunity remains largely unknown. On the other hand, several cell wall components such as peptidoglycan and lipoteichoic acids have been well studied. Teichoic acids. (i) Structure. Both wall teichoic acids and lipoteichoic acids are highly charged polymers. They concen-

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trate cations at the cell wall surface and are associated with proteins to form complexes. They are involved in various biological activities: cation balance in the cell surface, cell division, and regulation of peptidoglycan autolysis (163). S. aureus wall teichoic acid is a water-soluble polymer composed of 40 ribitol phosphate units that are substituted with D-alanine ester and N-acetylglucosaminyl residues (Fig. 1A). The chain is attached to peptidoglycan by a phosphodiester bond through a linkage unit that is composed of three glycerophosphate residues linked to two amino sugars (Fig. 1A). LTA is the major macroamphiphile molecule of gram-positive bacteria. The physiochemical properties of LTA are similar to those of LPS in gram-negative bacteria. Staphylococcal LTA consists of about 25 poly(1–3)-glycerol phosphate linked to a diacylglycerolipid anchor (Fig. 1B). The hydrophilic polyglycerol phosphate chain is long enough to penetrate the peptidoglycan, and the lipid moiety attaches the polymer to the surface of the cytoplasmic membrane. The glycolipid structure resembles the bacterial membrane composition and usually diverges among gram-positive bacteria in a genus-specific manner (40). (ii) Role in the inflammatory response. Although crucial for bacterial life, purified wall teichoic acids of S. aureus are not very inflammatory (108). However, a number of studies suggest that the bacterial LTA of S. aureus may contribute to sepsis (83). LTA from S. aureus has been shown to provoke secretion of cytokines and chemoattractants (TNF-␣, IL-1␤, IL-10, IL12, IL-8, leukotriene B4, complement factor 5a, MCP-1, MIP-1␣ and granulocyte colony-stimulating factor) from monocytes or macrophages (14, 27, 30, 81, 179, 200). Complement factor 5a and leukotriene B4 are chemoattractants active on polymorphonuclear cells (PMNs) and monocytes. Thus, LTA induces an inflammatory response. However, very large amounts of LTA are necessary to induce responses of cells in vitro. Indeed, LTA, in the 1 to 10 ␮g/ml range is required to trigger cellular responses while LPS in the ng/ml range is sufficient to elicit responses (94). It must be taken into consideration that the active concentrations of LTA (1 ␮g or 105 to 107 CFU) as well as of LPS (20 ng or 107 CFU) are comparable when they are transposed to bacterial cell equivalents (170, 200), suggesting that LTA and LPS preparations may have similar potency. Comparison of the activity of LPS versus LTA showed that staphylococcal LTA is able to promote the same strong induction of chemoattractants (IL-8, MIP-1␣, MCP-1, complement factor 5a, and leukotriene B4), granulocyte colony-stimulating factor, and anti-inflammatory cytokines (IL-10) as LPS, whereas it is a weaker inducer of TNF-␣, IL-1␤, and IL-6 (200, 201). LTA also induces less IL-12 than LPS and subsequently IFN-␥ (64, 91). The cytokine pattern produced by LTA is similar to that induced by the whole bacterium (200, 210). Furthermore, when LTA is inoculated intranasally in mice, a strong neutrophil and macrophage infiltration is observed in the lung, suggesting that LTA elicits granulocyte recruitment by producing chemoattractants (200). It is likely, then, that staphylococcal LTA participates in the formation of pus by recruiting neutrophils (200). Thus, staphylococcal LTA is a strong inducer of chemoattractant and granulocyte colonystimulating factor release, suggesting that it is not just a weak

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LPS-like molecule but indeed displays activities distinct from LPS. (iii) Interaction with TLR2. LTA causes cytokine induction in mononuclear phagocytes. It was thus tempting to imagine that TLRs transduce the signal necessary for activation of immune cells by LTA. However, the role of TLRs in LTAinduced cell stimulation as well as the stimulation of cytokine production obtained in different cell systems has been controversial (14, 81, 94, 132, 171, 185). In most of these studies, commercial staphylococcal LTA preparations were used. It has been demonstrated that these preparations have a high degree of compositional heterogeneity and also contain significant amounts of endotoxin (the origin of this endotoxin is unknown) (45, 126). Furthermore, purification of LTA from S. aureus by standard methods using phenol extraction results in hydrolysis of D-alanine substituents. These substituents have been shown to be crucial for biological activity of LTA (125) (see section on alanylation of teichoic acids). Thus, taken together, the heterogeneity of the preparations, contamination by endotoxin, and inappropriate methods of purification that result in partial degradation of LTA might explain contradictory results in the literature concerning the role of TLR in the transduction of the signal and the production of cytokines after staphylococcal LTA stimulation (126). A novel purification method using butanol extraction produces staphylococcal LTA without LPS contamination. This LTA exhibits the same efficiency, pattern of cytokine induction (TNF-␣, IL-1␤, IL-6, and IL-10), and recognition by TLR as a chemically synthesized LTA (45, 98, 125, 127). By using TLR2deficient mice or monoclonal antibodies to TLR2, production of cytokines, e.g., IL-6 and TNF-␣, in response to LTA stimulation requires TLR2 (38, 59, 121, 185), suggesting that TLR2 is a key receptor in response to LTA of S. aureus. Although LTA exists as a heterogeneous family of related molecules, it appears that no relationship between LTA structure and efficiency is observed. However, several LTA compounds, such as D-Ala constituents, the glycosyl substituents, and the lipid anchor, modify LTA activity (64, 127, 128). Furthermore, LTAs from S. aureus and Bacillus subtilis exhibited similar TLR2 induction (144), whereas pneumococcal LTA is less active than staphylococcal LTA in stimulating TLR2 (59, 192). Taken together, LTA appears to constitute a broad immunostimulatory factor of gram-positive bacteria with possibly differing potencies depending on the constituents of the molecule (47, 64). Alanylation of teichoic acids. (i) Structure. The D-alanyl ester of teichoic acids results from a D-alanine substitution on the sugar (Fig. 1). The products of an operon of the S. aureus chromosome, dltABCD (for D-alanyl-LTA), catalyze the introduction of D-Ala into both wall teichoic acids and LTA. DAlanylation of teichoic acids modulates the properties of the envelope. Indeed, D-alanine-esterified teichoic acids protect S. aureus against cationic antimicrobial peptides produced by host (150). Furthermore, the degree of D-alanylation is affected by growth environment (90, 135). In a culture at low salt concentrations, the degree of alanylation is 60% for wall teichoic acids and 80% for staphylococcal LTA (39). (ii) Role in the inflammatory response. Alanylation of teichoic acids increases the release of TNF-␣ and MIP-2 (93): in mice, MIP-2 and keratinocyte chemoattractant may serve as a

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neutrophil chemotactic factors in the recruitment of PMNs to sites of infection and inflammation because IL-8 is not present in mice (97). D-Alanine has been shown to be an important substituent for LTA activity since hydrolysis of alanine substituents of active LTA dramatically decreases cytokine induction (125). Furthermore, alanylation of teichoic acids increases the virulence of S. aureus in mice (28). The inflammatory response due to D-alanylation is correlated with the number and survival of bacteria (93). Indeed, dlt-deficient S. aureus are cleared more rapidly than the wild-type strain after administration of a similar inoculum. The lack of alanylation has three considerable consequences: increased susceptibility to defensin-like antimicrobial peptides (150), reduced adherence to host cells (1), and reduced inflammatory activity of LTA. Thus, alanylation induces multiple changes affecting the outcome of in vivo experiments. (iii) Interaction with TLR2. The minimum infective doses for wild-type S. aureus and dlt-deficient bacteria in TLR2⫺/⫺ mice are 10-fold and 500-fold lower, respectively, than those observed in wild-type mice (93), suggesting that TLR2 is involved in murine host defense against alanylated staphylococcal teichoic acids. Since both wall teichoic acids and LTA are D-alanylated, this result confirms that TLR2 stimulates cytokine production in response to alanylated LTA (185, 201). However, the role of alanylated wall teichoic acids in TLR2 stimulation remains unknown. Even in TLR2⫺/⫺ mice, dlt-deficient bacteria have a higher minimum infective dose than the wild-type strain (93). Furthermore, release of MIP-2 and TNF-␣ occurs in TLR2-deficient mice. These results suggest that additional host defense mechanisms not related to TLR2 are involved. Thus, a receptor distinct from TLR2 or another mechanism also seems to be involved in the inflammatory response of mice to S. aureus alanylated teichoic acids (93). Peptidoglycan. (i) Structure. Peptidoglycan is a large polymer and the most conserved component of the gram-positive envelope. It provides shape-determining properties for bacteria. This polymer is constituted of glycan strands of two alternating sugar derivatives, N-acetylglucosamine and N-acetylmuramic acid, which form a dissacharide subunit. The carboxyl group of N-acetylmuramic acid is linked to a peptide subunit (or stem peptide) consisting of four or five alternating L- and D-amino acids (Fig. 2). This structure is highly cross-linked by peptide bridges (95). Whereas the structure of the glycan chains is highly conserved, the composition of the stem peptide varies among bacterial species. Indeed, most gram-negative bacteria and gram-positive bacilli possess m-diaminopimelic acid in position 3 of the stem peptide, which is usually directly cross-linked. In contrast, gram-positive cocci such as S. aureus have L-lysine in position 3 of the stem peptide that is crosslinked via an “interpeptide bridge,” the composition of which is different among bacteria (34, 167) (Fig. 2). Staphylococcal peptidoglycan belongs to this second type and has a pentaglycine interpeptide bridge (Fig. 2). Peptidoglycan hydrolases are capable of hydrolyzing bonds in the cell wall peptidoglycan. They are classified into the following four classes according to their hydrolytic bond specificities: N-acetylmuramidases, N-acetylglucosaminidases, Nacetylmuramyl-L-alanine amidases, and endopeptidases (174). The first class, muramidase, catalyzes the hydrolysis of the

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glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (Fig. 2). The enzymes that cleave the bond between N-acetylglucosamine and the adjacent N-acetylmuramic acid in dissacharide subunits have been defined as glucosaminidases (Fig. 2). Another class of peptidoglycan hydrolases, amidases, hydrolyze the bond between the lactoyl group of Nacetylmuramic acid and L-alanine. Finally, endopeptidases cleave peptide cross-links (Fig. 2). Peptidoglycan hydrolases are present in all bacteria, and some of them, called autolysins, digest their own protective cell wall peptidoglycan. Since peptidoglycan is essential for cellular integrity, the action of autolysins usually results in bacterial autolysis. Lysostaphin, which is an endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, cleaves staphylococcal peptidoglycans in general (189) (Fig. 2). When lysing staphylococci, the enzymatic reaction of lysostaphin is the specific hydrolysis of the Gly-Gly bond in the pentaglycine bridge of the staphylococcal peptidoglycan. Host enzymes are also implicated in peptidoglycan cleavage. Lysozyme is a muramidase present in various tissues (mucous membranes, respiratory and intestinal tracts) and fluids (serum, saliva, and tears). It is important for host defense because it can kill bacteria. Interestingly, S. aureus is resistant to lysozyme because N-acetylmuramic acid of staphylococcal peptidoglycan is O-acetylated at position C6-OH (Fig. 2) by an O-acetyltansferase that is an integral membrane protein (13). The resistance of S. aureus to lysozyme may contribute to its ability to colonize the skin and mucosal tissues such as the anterior nares. Peptidoglycan recognition protein L (PGRPL), also known as serum amidase, displays an N-acetylmuramoyl-L-alanine amidase activity towards peptidoglycan. PGRP-L is thought to function as a peptidoglycan-scavenging molecule that likely reduces peptidoglycan-induced inflammation (209) (see section on PGRPs below). (ii) Role in the inflammatory response. Staphylococcal peptidoglycan has been shown to stimulate the production of proinflammatory cytokines and chemokines (TNF-␣, IL-1␤, IL-6, and IL-8) in monocytes and macrophages (66, 110, 190, 205). It has also been observed that primary astrocytes produce numerous cytokines such as IL-1␤, TNF-␣, MIP-1␤, and MCP-1 in response to staphylococcal peptidoglycan (38) (Fig. 3). Larger amounts of peptidoglycan, in the 10 to 100 ␮g/ml range, are necessary to stimulate cellular responses compared to LPS, which induces responses in the ng/ml range (94). Since 1 ⫻ 106 to 6 ⫻ 106 CFU correspond to 1 ␮g of staphylococcal peptidoglycan (192), whereas 107 CFU correspond to 20 ng of LPS, this suggests that whole peptidoglycan is about 100-fold less active than LPS. Studies of S. aureus cell walls indicate that only part of peptidoglycan is active. Indeed, insoluble and soluble peptidoglycan chains of high molecular weight are not very inflammatory. Hydrolyzing these chains to sugar and peptide monomers completely abolishes inflammation. In staphylococcal peptidoglycan, three cross-linked stem peptides appear to be the minimal structural constraint to be inflammatory (128). Furthermore, treatment of S. aureus by lysostaphin, which cleaves the pentaglycine bridge, moderately attenuates release of cytokines, whereas digestion with cellosyl, a muramidase hydrolyzing glycosidic bonds, nearly abrogates the induction of cytokine (131, 223). This suggests that the glycan

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FIG. 2. Structure of peptidoglycan of S. aureus and several other bacteria. The peptidoglycan of gram-negative bacteria and gram-positive bacilli is indicated on the right. Digestion by different enzymes (glucosaminidase, muramidase, amidase, and endopeptidase) is shown by dotted arrows. MDP structure is indicated in the dotted square. NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; m-DAP, m-diaminopimelic acid.

strand is crucial for cytokine production, whereas stem peptide structure does not seem to be critical for the inflammatory activities of peptidoglycan. Although peptidoglycan by itself promotes a weak induction of cytokines, it shows synergistic effects with LTA or LPS (221). Indeed, intravenous administration of staphylococcal peptidoglycan or LTA alone cannot cause shock, whereas coadministration of peptidoglycan with LTA in rats induces the production of TNF-␣ and IFN-␥ in a synergistic way and provokes septic shock and multiple organ failure (31, 83, 188).

Furthermore, although intranasally inoculated LTA does not synergize with peptidoglycan to induce the production of TNF-␣ and the chemoattractants MIP-2 and keratinocyte chemoattractant, these staphylococcal components act in synergy to elicit recruitment of PMNs into mouse lung (97). Peptidoglycan of S. aureus also synergizes with low doses of LPS to cause multiple organ failure (188). Furthermore, coinjection of peptidoglycan with LPS induces synergistic production of TNF-␣ and IL-6 in the blood. Surprisingly, the release of IL-10 was not modified by the coadministration of pepti-

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FIG. 3. Action of staphylococcal components to promote immune responses from immune cells (data from reference 63). FPR, formylated peptide receptor.

doglycan with LPS (188, 207). Thus, peptidoglycan seems to synergize with LPS to induce the release of proinflammatory cytokines but not the anti-inflammatory cytokines. (iii) Interaction with TLR2. The role of TLR2 as a peptidoglycan receptor has been investigated extensively, and until recently, it was broadly accepted that TLR2 is a receptor for staphylococcal peptidoglycan (76, 164, 171, 185). Indeed, staphylococcal peptidoglycan binds strongly to a soluble form of recombinant TLR2 composed of its putative extracellular domain, suggesting that the extracellular TLR2 domain directly interacts with peptidoglycan (76). However, there are now contradictory results about the role of TLRs in peptidoglycan-induced cell stimulation and cytokine production. Most studies have been mainly carried out with commercial S. aureus peptidoglycan preparations, and a recent publication showed that highly purified peptidoglycan did not elicit TLR2-dependent activation and IL-6 and TNF-␣ production from mouse peritoneal macrophages, whereas lipoproteins and LTA did. It was hypothesized that the stimulation of TLR2 by peptidoglycan could be attributed to other inflammatory cell wall components contaminating the commercial peptidoglycan preparations (192). Indeed, treatment of peptidoglycan with hydrofluoric acid abolished TLR2 stimulation by gram-positive cell walls. As hydrofluoric acid hydrolyzes LTA, it was suggested that the peptidoglycan contaminant could be LTA (192). Thus, it seems that staphylococcal peptidoglycan is probably not recognized by TLR2 but rather

by Nod2, another innate immune receptor (see section on Nod proteins). Phenol-soluble modulin. (i) Structure. Although infections with Staphylococcus epidermidis are less severe than those with S. aureus, S. epidermidis can cause infections ranging from localized infections to sepsis. This bacterium releases phenolsoluble modulin (PSM) in the extracellular fluid (115). PSM is composed of at least three components, PSM␣, PSM␤, and PSM␥, which are very small proteins of 22 to 25 amino acids. PSM␥ is identical to S. epidermidis ␦-hemolysin, which is also present in S. aureus. ␦-Hemolysin can be formylated (see section on formylated peptides). PSM is released into the culture medium during overnight culture and is also detected in the supernatant fluid after vigorous vortexing of washed bacteria, suggesting that PSM is also partially bound to the cell surface (85). (ii) Role in the inflammatory response. The ␦-hemolysin of S. aureus may be involved in staphylococcal virulence. Indeed, it has been shown to lyse erythrocytes, probably by crossing the lipid membrane and inducing the release of their contents (152). It also binds to neutrophils and monocytes, inducing the production of TNF-␣ (168). S. epidermidis PSM also induces cytokine production (TNF-␣, IL-1␤, and IL-6) and activates NF-␬B in macrophages. Furthermore, PSM is a chemotactic agent for both neutrophils and monocytes (102). (iii) Interaction with TLR2. PSM has been shown to use TLR2 to modulate the immune response (58). Indeed, human

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dermal endothelial cells express only very little TLR2 and do not respond to several TLR2 ligands such as PSM. On the other hand, endothelial cells that are transfected with TLR2 are capable of responding to TLR2 ligands, including PSM (19). Although further studies are required to demonstrate conclusively the role of TLR2 in PSM recognition, it appears that this staphylococcal virulence factor is involved in the inflammatory response through its activation of TLR2. TLR2 Cooperation with Other Receptors TLR2 has been shown to detect various specific components of pathogens. However, it is not clear how a receptor like TLR2 has the capacity to recognize such a wide spectrum of stimuli. Antibodies to CD14, TLR1, or TLR2 (but not those to TLR4) significantly reduces TNF-␣ production by mononuclear cells in response to staphylococcal LTA, suggesting that these receptors are involved in LTA recognition (59). TLR1/TLR6. Ligand recognition is more complicated with TLR2 because it has been shown to cooperate with TLR1 and/or TLR6 to increase its range of pathogen-associated molecular patterns. Ozinski et al. (145) first suggested that TLR2 mainly recognizes its ligands by forming functional heterodimers with either TLR1 or TLR6 (84, 214). TLR2 appears to require a partner to stimulate cytokine production. Indeed, dimerization of the cytoplasmic domain of TLR2 does not elicit TNF-␣ induction (145). TLR1 and TLR6 have been shown to mediate the discriminatory recognition of triacyl and diacyl lipopeptides by TLR2. TLR6 is necessary for TLR2 to detect MALP2 (macrophage-activating lipopeptide 2 from Mycoplasma pneumoniae), which is only diacylated (186). In contrast, TLR1 is necessary for the response to triacyl lipopeptides and mycobacterial lipoproteins (165, 187). Since staphylococci also produce a set of lipoproteins, such as the ABC transporters, it will be interesting to examine the possibility that these lipoproteins are indeed detected by TLR2 and whether their form, either diacylated or triacylated, dictates the contribution of either TLR1 or TLR6 to their recognition. TLR6 has been shown to cooperate with TLR2 in the recognition of S. aureus (137, 145). Indeed, in the presence of a dominant negative form of TLR6, cytokine production induced by S. aureus is abolished (145). However, macrophages from TLR6-deficient mice stimulated by staphylococcal peptidoglycan still produce TNF-␣, suggesting that TLR6 is not absolutely necessary for peptidoglycan recognition (4, 186). In TLR6⫺/⫺ fibroblasts, the staphylococcal LTA response is significantly attenuated compared to wild-type cells, suggesting that TLR6 is required for LTA recognition by TLR2 (130). However, it has also been shown that TLR2 synergizes with TLR1 to sense LTA, consistent with the fact that this product is diacylated (192). The TLR2-mediated activation of NF-␬B by PSM is increased by TLR6 but attenuated by TLR1 (58). Although definitive experiments in TLR1-deficient mice are still lacking, these results confirm a functional interaction between these receptors and interaction of staphylococcal components. CD14. CD14 is a member of the family of glycosylphosphatidylinositol-anchored membrane proteins lacking an intracellular signaling domain. CD14 is a key coreceptor expressed on the surface of macrophages and PMNs and is necessary for the

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full induction of an inflammatory response by LPS-stimulated TLR4 (217). Several studies have demonstrated that different components of S. aureus such as peptidoglycan and LTA also interact with the CD14 molecule (27, 36, 56, 59, 78, 94, 155, 170, 171, 212). Thus, it has been suggested that CD14 is a functional receptor for staphylococcal peptidoglycan and LTA. However, as it has recently been shown that LTA contamination could be involved in the inflammatory activity due to commercial preparations of peptidoglycan (192), the question is whether or not it is indeed peptidoglycan that binds to CD14. An anti-CD14 antibody that abolishes cytokine release induced by LTA does not reduce the cytokine production caused by LPS, whereas two other antibodies have similar inhibitory effects. This suggests that the CD14 sites that recognize LTA and LPS are distinct with perhaps an overlap (56, 64). Furthermore, cytokine induction by LTA is inhibited by soluble CD14, suggesting that LTA trigger monocyte activation via CD14 (64). In addition, expression of CD14 in fibroblasts synergistically increases NF-␬B activation mediated by TLR2 in response to S. aureus (222). However, the same mortality and symptoms of shock are observed in both wild-type and CD14deficient mice challenged with various doses of S. aureus. This result suggests that CD14 does not have a significant role in septic shock caused by S. aureus (60). Thus, other CD14-independent mechanisms might be involved in TLR2 activation (60, 195). CD36. CD36, a single polypeptide membrane glycoprotein, is a member of a family of scavenger receptors. Recently, it has been shown that CD36-mutant mice are hypersusceptible in two models of staphylococcal infections (cutaneous and systemic) (67). Furthermore, cytokine production is abolished in CD36-deficient macrophages after stimulation by LTA and the R-enantiomer of MALP2 but is normal when stimulated by other TLR2 ligands such as S-MALP2, triacylated lipopeptide, and zymosan (67). As both LTA and MALP2 are diacylated, this suggests that CD36 is involved in the recognition of diacylglycerides. Interestingly, in TLR2-deficient cells, expression of CD36 or TLR6 alone does not induce NF-␬B activation by LTA, suggesting that these receptors need a partner to transduce the LTA signal to cellular responses. Indeed, coexpression of either CD36 or TLR6 with TLR2 significantly increases the TLR2 response to LTA. When TLR2, TLR6, and CD36 are expressed together, the highest NF-␬B activation is observed. Thus, it seems that CD36 may play a role analogous to that of CD14 by concentrating the diacylglyceride signal for transduction through TLR2 (67). Asialo-GM1. S. aureus is known to cause pulmonary infections. Once staphylococci are in the lung, they multiply and sometimes invade the epithelium of the bronchioli. The production and release of cytokines and chemokines, including IL-8, due to the presence of S. aureus elicit infiltration of PMNs and macrophages, leading to tissue damage and subsequent pneumonia (138). In airway epithelium, asialylated glycolipids such as asialoGM1 (asialoganglioside gangliotetraosylceramide) (153) are present on the epithelial surface and can act as receptors. Indeed, S. aureus binds to the GalNac␤1-4Gal moiety exposed at the cell surface and initiates IL-8 production through NF-␬B activation (158). A recent study found small amounts of TLR2 on the apical surfaces of airway epithelial cells. After stimula-

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FIG. 4. Domain structure of TLR2 (A) and Nod2 (B). Numbers correspond to amino acid residues. A. TLR2 structure (data from references 44 and 118). ECD, extracellular domain; TMD, transmembrane domain; ICD, intracellular domain; TIR, Toll/IL-1 receptor domain. The regions homologous to LRRs are indicated by square boxes, and LRR-like motifs are indicated by boxes with an asterisk. B. Nod2 structure (data from reference 141). CARD, caspase-activating and recruitment domain; NBS, nucleotide binding site and LRR domain.

tion with S. aureus, TLR2 and asialo-GM1 are mobilized into lipid raft structures on the surface of the epithelial cells. The lipid raft microdomains are necessary for IL-8 production (176). Thus, asialo-GM1, similar perhaps to CD36, serves as a coreceptor that amplifies the signaling function of TLR2 (176). Interestingly, the S. aureus strain lacking Agr, a staphylococcal virulence regulator (see the Introduction), shows attenuated IL-8 production through asialo-GM1, suggesting that the staphylococcal ligand recognized by asialo-GM1 is probably a surface molecule regulated by Agr such as these adhesins that interact with extracellular matrices (158). Structure of TLR2 Human TLRs are type I transmembrane proteins with an extracellular domain, a transmembrane domain, and an intracellular domain (Fig. 4A). The N-terminal domain of TLR2, which is located in the extracellular compartment, is composed of leucine-rich repeat (LRR) motifs. The LRR consensus sequence consists of a motif of 24 to 29 residues with a highly conserved region (12). The N-terminal domain of TLR2 possesses 10 canonical LRR sequences and 8 to 10 LRR-like motifs that are poorly defined (Fig. 4A) (118). Proteins that contain LRRs, such as the extracellular domain of TLR2, are involved in interaction with a great variety of ligands (160). The leucine residues at positions 107, 112, and 115 in an LRR motif (44) as well as the extracellular region between Ser40 and Ile64 are crucial for the detection of peptidoglycan by TLR2 (122). In another study, Meng et al. found that the seven LRR motifs located at the N terminus (Fig. 4A) were not implicated in TLR2 activation by bacterial polypeptides, whereas the integrity of the extracellular domain was necessary to induce a full response to S. aureus peptidoglycan (118). Thus, potential binding domains of bac-

terial polypeptides most probably differ from those of peptidoglycan. This result suggests that TLR2 probably possesses multiple binding domains for its various ligands, explaining its promiscuous nature in terms of the molecular patterns recognized (118). Although, in light of the recent data arguing that peptidoglycan is not a TLR2 ligand (192), these findings may have to be revisited in future studies. The cytoplasmic portion of Toll-like receptors shows a high similarity to that of the IL-1 receptor family and is now called the Toll/IL-1 receptor (TIR) domain (182). Upon extracellular stimulation, the TIR domain associates with an adaptor protein, which actuates a succession of signaling proteins, and this cascade of activation mediates signal transduction. Several polymorphisms located in the TIR domain of TLR2 have been detected. The presence of the Arg677Trp or Arg753Gln mutation in the TIR domain reduces NF-␬B activation and cytokine production in response to TLR2 ligands (16, 105, 201). Furthermore, the ArG753Gln polymorphism has been shown to be present in 2 out of 91 septic patients, and these patients developed staphylococcal infections (105). However, a study of 420 patients exhibiting severe S. aureus infection showed that the Arg753Gln polymorphism in the TIR domain of the TLR2 gene is not associated with susceptibility to severe disease caused by S. aureus (124). These contradictory results might be explained by a previous observation indicating that the presence of only one wild-type allele of TLR2 is sufficient in vitro to induce normal cytokine production in response to staphylococcal LTA (124, 201). TLR2 Signaling TLR and Nod (see section on Nod proteins) trigger the activation of the transcription factor NF-␬B (nuclear factor ␬B), which controls the expression of genes encoding numer-

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FIG. 5. Different signaling pathways of TLR2, Nod, and TNFR1 in response to ligand, resulting in activation of NF-␬B, the nuclear transcriptional factor responsible for regulation of the immune response genes (data from references 3, 113, and 194). RIP1, receptor-interacting protein 1; FADD, Fas-associated death domain protein; TRAF2, TNF receptor-associated factor 2; PI3K, phosphatidylinositol 3-kinase composed of two subunits (p85 and p110).

ous cytokines, chemokines, and costimulatory molecules necessary for the activation of the defense response. NF-␬B is a transcription factor regulated by nuclear-cytoplasmic shuttling. Indeed, NF-␬B dimerizes, interacts with DNA of the target genes located in the nucleus, and modifies their expression. However, NF-␬B contains a nuclear localization sequence that is masked when inhibitors, the I␬Bs (inhibitors of ␬B), bind to NF-␬B dimers. Thus, I␬Bs are responsible for NF-␬B retention in the cytoplasm (80). I␬B phosphorylated by I␬B kinases (IKKs) is degraded by the proteasome, and NF-␬B is then free to move to the nucleus, where it can directly regulate gene expression (Fig. 5) (113). Thus, stimulation of the pattern recognition receptor promotes the activation of IKKs and the release of NF-␬B. The molecular cascades from the receptor to

the release of NF-␬B have been extensively examined to identify signaling molecules implicated in the TLR response. The nature of the intracellular events following the stimulation of individual TLR is dependent on the adaptor molecules that interact with the different TLRs (Fig. 5). Although intracellular signaling induced by TLR ligands can utilize a shared group of molecules, distinct cellular responses can be generated (157). While some of the intracellular responses induced downstream of TLR2 and TLR4 are similar, for example, the activation of the NF-␬B pathway, distinct differences in the cellular signaling pathways activated by these two receptors are observed (21). One of the most striking differences in cell signaling downstream of TLR2 and TLR4 is the activation of the interferon response factor pathway by TLR4

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stimulation. This pathway requires the adaptors TRIF (TIR domain-containing adaptor inducing IFN-␤) and TRAM (TRIF-related adaptor molecule) that are not present in TLR2 pathway (147, 181). Indeed, TLR2 represents a more simplified signaling pathway mediated mainly by the adaptors MyD88 (myeloid differentiation protein) and TIRAP/Mal (TIR-associated proteins, also termed MAL for MyD88-associated ligand), which regulate the NF-␬B pathway and an additional adaptor, Tollip (Toll-interacting protein), which appears to play a regulatory role in the pathway. From these adaptors, a cascade of events involving different mediators such as IRAK (IL-1R-associated kinase), TRAF6 (TNF receptor-associated factor 6), TAK1 (Transforming growth factor-activated kinase), and IKKs leads to the activation of NF-␬B (Fig. 5) (208). Since there are many excellent recent reviews describing the activation of the NF-␬B pathway, the following will focus on a description of the upstream events involving the specific adaptors of the TLR2 pathways (Fig. 5). MyD88 also possesses a C-terminal TIR domain that interacts with the TIR domain of the TLR receptor (Fig. 5). Activation of a TLR by the ligand promotes its dimerization and the TIR domains of TLR and MyD88 bind. MyD88-deficient mice are more susceptible to systemic S. aureus infection than wild-type mice (184). Furthermore, cytokine production is attenuated in MyD88-deficient macrophages after S. aureus stimulation (183, 184). In contrast to systemic and in vitro infection, however, MyD88-deficient mice intranasally inoculated with S. aureus do not show an increase susceptibility to pulmonary infections and cytokine production as well as the neutrophil recruitment is not impaired, suggesting that lung tissues do not need MyD88 to respond to staphylococcal infections (173). Thus, the importance of MyD88 in the TLR2 signaling pathway appears to be tissue specific. This suggests that the pulmonary innate immune response to S. aureus implicates TLR2 signaling pathways distinct from MyD88 or recognition of staphylococcal ligands by a receptor independent of TLR2 (173). TIRAP/Mal, similar to MyD88, contains a C-terminal TIR domain that directly interacts with the TIR domain of TLR2 (183). Indeed, cells from TIRAP/Mal knockout mice are unresponsive to TLR2 ligands. TIRAP/Mal, therefore, seems to be essential for signaling pathways activated by TLR2 (69, 141, 219). An additional adaptor molecule, Tollip, has also been proposed to interact with TIR domains. Tollip was first identified as a molecule that associates with the cytoplasmic domain of IL-1R (20) and was shown to bind directly with TLR2 (225). In contrast to the other adaptor molecules, Tollip appears to suppress the TLR2 signaling pathway. Indeed, cells that overexpress Tollip cannot induce NF-␬B activation in response to TLR2 ligands (20). Tollip associates and interferes with IRAK, one of the key molecules involved in the TLR2 signaling pathway (Fig. 5) (20, 225). In addition to the MyD88-Tollip-TIRAP/Mal pathway of NF-␬B activation, it has been observed that activation of Rac1, which belongs to the Rho family of GTPases, is also implicated in the signaling pathway of TLR2 (Fig. 5). Indeed, stimulation by S. aureus leads to association of Rac1 with the TLR2 cytosolic domain and the activation of the phosphatidylinositol

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3-kinase pathway. This event induces the activation of protein kinases such as Akt (serine/threonine kinase) mediating NF-␬B transactivation (Fig. 5) (8). NOD PROTEINS As stated above, TLR2 is an essential receptor for the recognition of staphylococcal components. However, it is interesting that astrocytes or macrophages from TLR2-deficient mice stimulated by S. aureus still produce proinflammatory mediators (38, 100, 184), suggesting that alternative receptors are also implicated in S. aureus recognition. The TLRs are involved in recognition of pathogens in the extracellular compartment, whereas the NBS-LRR (for nucleotide-binding site and leucine-rich repeat) family of proteins is involved in intracellular sensing of microorganisms and their products (9, 23). In this family, nucleotide-binding oligomerization domain proteins Nod1 and Nod2 play a role in the innate immune response by regulating the cytokine induction initiated by bacterial ligands through pathways that are possibly independent of TLR signaling. The 302insC frameshift mutation in the nod2 gene is associated with the inflammatory bowel disease Crohn’s disease (71, 139). This disease is associated with a severe inflammatory reaction at the level of the intestinal mucosa. Contrary to the expected gain-of-function mutation that would be consistent with the auto-inflammatory nature of this disease, the frameshift mutation in Nod2 results in a loss of bacterial sensing and decreased activation of inflammatory pathways (49, 75, 139). Much research is now focused on trying to find the mechanisms of Nod2 function that reconcile the apparently paradoxical role of this pattern recognition receptor in the pathogenesis of disease as reviewed by Kelsall (82). Although much work is still required to fully understand Nod2 function in mediating disease, two recent papers have attempted to shed light on this issue (89, 107). One study used Nod2-deficient mice that have a “knock-in” of a mutation in Nod2 corresponding to the human Nod2 frameshift mutation associated with Crohn’s disease (107). Surprisingly, these mice have high background inflammatory signaling and amplified responses to muramyl dipeptide (MDP), the specific ligand of Nod2 (see section on staphylococcal structure recognized by Nod proteins). Although these observations fit very well conceptually with the phenotype of Crohn’s disease, i.e., increased levels of inflammation, they are diametrically opposed to findings in Crohn’s disease patients. Cells from patients with the Nod2 frameshift mutation have normal basal levels of inflammatory signaling and are refractory to MDP stimulation (75). Thus, any firm conclusions regarding this study await confirmatory findings. The other recent paper on mechanisms of Nod2 function demonstrated that Nod2-deficient mice have lower expression of alpha-defensins, or cryptins, at the level of the intestinal mucosa, and consequently, these mice have an increased susceptibility to oral infection with Listeria monocytogenes, a gram-positive pathogen of humans that crosses the intestinal barrier to enter the host (89). Alpha-defensins are a class of antimicrobial peptides secreted by specialized cells of the intestinal epithelium called Paneth cells. Decreased expression of alpha-defensins has also been observed in Crohn’s disease

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patients (211). Accordingly, Nod2 mutations may render the intestinal mucosa more accessible to bacterial translocation with a resulting overamplified inflammatory response in the afflicted patient. Staphylococcal Structure Recognized by Nod Proteins Nod1, which is ubiquitously expressed, is involved mainly in the recognition of gram-negative bacteria. Indeed, the Nod1 ligand is a bacterial peptidoglycan fragment containing an Nacetylglucosamine-N-acetylmuramic acid tripeptide motif with diaminopimelic acid (Fig. 2) (24, 48). Nod2, which is mainly expressed in monocytes/macrophages, detects peptidoglycan from gram-negative (E. coli and Shigella flexneri) and grampositive bacteria (B. subtilis and S. aureus) (49, 140). This protein recognizes the minimal peptidoglycan motif common to both classes of bacteria, muramyl dipeptide, corresponding to N-acetylmuramic acid-L-alanyl-D-isoglutamine (Fig. 2) (49). MDP is also the smallest unit of peptidoglycan capable of providing biological activities such as immunogenicity. MDP alone induces minimal TNF-␣ production, and the production of TNF-␣ induced by MDP is independent of CD14 or TLR2 (198, 215, 221), suggesting that another receptor is involved in the recognition of MDP. Furthermore, MDP binding sites were determined to be located within the intracellular compartment (180). Thus, it seems that Nod2 is the intracellular sensor for MDP, and it is therefore likely that Nod2 participates in the intracellular innate immune response to bacterial pathogens (50). Since Nod1 and Nod2 are cytoplasmic proteins, the question has been raised whether or not bacteria such as S. aureus could be sensed by this class of pattern recognition receptors. Indeed, highly purified peptidoglycan does not induce cytokine production in epithelial cells expressing Nod2, suggesting that peptidoglycan must penetrate the cells to stimulate Nod proteins (57, 192). Although classically thought of as an extracellular bacterium, several observations indicate that S. aureus may also be an intracellular pathogen. S. aureus has been shown to be internalized by different mammalian cells (pulmonary epithelial cells, enterocytes, fibroblasts, endothelial cells, osteoblasts, and neutrophils) (6, 10, 11, 18, 55, 65, 70, 79, 119) in a manner dependent on the expression of the virulence regulators Agr and SarA (213). Intracellular staphylococci are sometimes present within vacuoles, but the majority appear to be free and replicating within the cytoplasm, having escaped from the endosome (70, 119). Agr is necessary for endosomal escape, probably by regulating the expression of membraneactive toxins (156, 172). Interestingly, neutrophils of patients with Crohn’s disease show increased intracellular survival of S. aureus (29, 216). Thus, it is imaginable that peptidoglycan fragments from cytoplasm-dwelling bacteria could be available for recognition by Nod2 and initiate Nod-dependent cellular responses. For the most part, however, S. aureus is classically considered an extracellular pathogen because it is found within the extracellular space during the course of a bacterial infection. Once within the host, the action of host and/or bacterial enzymes somewhat modifies the structure and composition of peptidoglycan and thereby can participate in Nod-dependent detection. On the host side, lytic enzymes such as lysozyme and

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amidases are known to degrade peptidoglycan polymers. The peptidoglycan of S. aureus, however, is resistant to lysozyme, and amidase digestion liberates fragments that are not detected by Nod2 (51). On the bacterial side, autolysins may contribute to staphylococcal lysis. Thus, MDP that is released when bacteria lyse can then be internalized into phagocytic cells and activate Nod2. Indeed, it has been shown that MDP is also able to be taken up by peptide transporters such as human PepT1 (196). It is also possible that phagocytic cells contain intracellular hydrolases capable of digesting bacterial peptidoglycan from phagocytosed bacteria and then releasing muropeptides active towards Nod2 into the cytosol. Although the findings discussed here suggest that Nod2 should be capable of detecting S. aureus, it is not yet clear how this intracellular pattern recognition receptor contributes to innate immunity following infection by this organism. Studies of S. aureus infection of Nod2-deficient mice have yet to be described. Structure of Nod2 The Nod family of cytoplasmic proteins presents a tripartite domain structure with an amino-terminal domain caspaseactivating and recruitment domain (CARD), which is a protein-protein interaction domain, a central nuclear-binding site (NBS) domain, and a carboxy-terminal LRR domain (Fig. 4B). The CARD domain provides homophilic interactions with other molecules carrying these motifs, and its integrity is necessary for the activation of NF-␬B (140). In contrast to Nod1, which has one CARD domain, Nod2 has two of these domains. The NBS domain, which mediates oligomerization of Nod proteins, includes consensus nucleotide-binding motifs: the P-loop, which is also found in ATP/GTPases, and the Mg2⫹binding site (Fig. 4B) (23, 73). Similar to TLR, the LRR domain of Nod2 is involved in ligand sensing. Indeed, in the absence of the LRR domain, Nod2 is unresponsive to the bacterial ligand (23). Nod Signaling RIP2 pathway. Expression of Nod2 protein in mammalian cells is sufficient to promote NF-␬B activation (140). The LRR domain of Nod2 recognizes bacterial products. RIP2 (for receptor interacting protein 2, also known as RICK or CARDIAK), a CARD-containing protein kinase, is a common downstream signaling molecule (111). There is evidence that Nod interacts directly with RIP2 through homophilic CARDCARD interactions (Fig. 5) (140). Furthermore, in RIP2-deficient embryonic fibroblasts, NF-␬B activity is not induced by Nod ligands but is restored after addition of a RIP2 expression vector (88). A central region located between the CARD and the kinase domain of RIP2 associates with IKK␥ (74). IKK is the I␬B kinase complex composed of two catalytic subunits, IKK␣ and IKK␤, and a third regulator subunit, IKK␥ (80). Interaction of RIP2 with this kinase complex appears to be sufficient for its activation, leading to the subsequent activation of NF-␬B (Fig. 5) (140). Interaction with the TLR pathway. Another pathway involved in Nod signaling has recently been described. TAK1 is required for Nod2-induced NF-␬B activation. Indeed, activa-

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tion of NF-␬B by Nod2 is inhibited by a dominant negative form of TAK1 (25). Furthermore, the Nod2 LRR domain appears to interact directly with TAK1 and to inhibit TAK1induced NF-␬B activation. The wild-type LRR of Nod2 is more efficient to suppress TAK1-induced NF-␬B activation than the LRR with a 302insC mutation (Fig. 5) (25). For the moment, however, the role of TAK1 in Nod2 sensing of bacterial ligands has not been confirmed in in vivo studies. Interestingly, RIP2-deficient cells show reduced cytokine production when TLR2 is stimulated by its ligand, suggesting that RIP2 is necessary for optimal signaling through TLR2 (Fig. 5) (88). Taken together, these results suggest that the Nod2 and TLR2 pathways may interact with each other and explain one aspect of Nod-TLR cross talk. TNFR1 TNF-␣ receptor 1 (TNFR1) is a receptor for TNF-␣ that is widely expressed on the airway epithelium. An exciting recent study showed that protein A interacts directly with TNFR1. Protein A, which is a major surface protein of S. aureus strains, is covalently anchored to the peptidoglycan and belongs to the cell wall-associated virulence factors of S. aureus. Purified protein A elicits release of IL-1␤, IL-4, IL-6, IL-8, and IFN-␥ and weak release of TNF-␣ from monocytes and fibroblasts (149, 193). This virulence factor has also been shown to contribute to staphylococcal sepsis. Indeed, intravenous administration of protein A-deficient S. aureus to mice causes lower mortality than the wild-type strain (146). It has been shown that TNFR1 recognizes S. aureus and its cell wall-associated protein A. Indeed, protein A binds to TNFR1 and reproduces the effects of TNF-␣, the ligand of TNFR1. Activation of the TNFR1 pathway by protein A induces mobilization and shedding of TNFR1 into the extracellular compartment. It also induces IL-8 production and PMN recruitment through NF-␬B activation (Fig. 5) (53). In the presence of a dominant-negative form of TLR2 and TLR4, protein A still induces NF-␬B activation in airway epithelial cells, suggesting that TLR2 or TLR4 agonists do not contaminate protein A preparations (53). Challenge of TNFR1-deficient mice resulted in reduced pneumonia and mortality in a mouse pneumonia model of infection with wild-type S. aureus. Similarly, the absence of staphylococcal protein A also reduces pneumonia and mortality in the same model with wild-type mice (53). Moreover, protein A expression is induced in S. aureus isolated from patients with pulmonary infections (52). Thus, these results suggest that protein A may be involved in the pathophysiology of pneumonia caused by S. aureus by activating TNFR1 and inducing a strong inflammatory response characteristic of PMN infiltration that is deleterious to the host. Although molecular studies on the interaction of protein A with TNFR1 are still lacking, this study opens up the possibility of novel therapeutic strategies against staphylococcal disease. PEPTIDOGLYCAN RECOGNITION PROTEINS Peptidoglycan is an obvious target of the innate immune system since it is a structural cell wall molecule that is conserved among bacterial species and is not found in the host.

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Furthermore, it is essential for the survival of bacteria such as S. aureus (34). Peptidoglycan recognition proteins (PGRPs) were isolated first in Drosophila melanogaster because they are able to interact with peptidoglycan with high affinity and are implicated in resistance to gram-positive bacteria (120). The family of peptidoglycan recognition proteins is conserved from insects to mammals. Peptidoglycan is detected in different compartments depending on the type of the mammalian PGRPs, which can be membrane bound, stored in vesicles, or secreted into the extracellular space. Four PGRPs are present in humans, PGRP-S, PGRP-L, PGRP-I␣, and PGRPI␤, each containing at least three conserved peptidoglycan-binding domains (34, 50, 104). PGRP-S, the best-studied mammalian PGRP, is found in the neutrophil tertiary granules, attenuates the growth of grampositive bacteria, and induces their intracellular killing (35, 103). PGRP-S-deficient mice that are intraperitoneally inoculated are more susceptible to infections with nonpathogen gram-positive bacteria such as B. subtilis, but they show no enhanced susceptibility to virulent gram-positive bacteria such as S. aureus (35). Thus, PGRP-S is probably another antibacterial protein present in PMNs that contributes to innate immune activity against low-virulence gram-positive bacteria. Surprisingly, however, it does not seem to act on S. aureus. PGRP-L is the only PGRP that has the full complement of conserved amino acids necessary for enzyme activity; PGRP-L has N-acetylmuramoyl-L-alanine amidase activity (46, 209). The minimum peptidoglycan structure cleaved by PGRP-L is N-acetylmuramic acid tripeptide (209). PGRP-L resembles the serum amidase in its molecular mass and specificity of the substrate. Although PGRP-L is predicted to be a transmembrane protein, it has been found in the serum (104, 218). The serum amidase is present in the extracellular compartment but is also tissue bound. This suggests that PGRP-L is likely to be the serum/tissue amidase (34, 209). Predigestion with lysozyme is required for mouse PGRP-L to effectively hydrolyze staphylococcal peptidoglycan (46). Amidases inactivate the proinflammatory activities of peptidoglycan by degrading its structure (68, 116). Thus, the combined action of lysozyme and PGRP-L might inactivate staphylococcal peptidoglycan and reduce peptidoglycan-induced inflammation. However, a recent study shows that no differences in mortality are observed between wild-type and PGRPL-deficient mice challenged with S. aureus. Furthermore, S. aureus stimulation induces similar IL-6 and TNF-␣ production in PGRP-L-deficient and wild-type peritoneal macrophages (218). Therefore, these results suggest that PGRP-L probably plays a minor role in the innate immune response to S. aureus. OTHER STAPHYLOCOCCAL COMPONENTS INVOLVED IN THE INFLAMMATORY RESPONSE From the discussion presented above, it can be concluded that peptidoglycan and LTA are certainly important S. aureus components involved in the inflammatory response (Fig. 3). However, peptidoglycan from S. aureus causes cytokine production similar to that from B. subtilis and Curtobacterium flaccumfaciens, which are not pathogens. This suggests that the inflammatory property of peptidoglycan does not correlate

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with the pathogenicity of the bacteria (128, 131). Furthermore, several studies found a specificity between the bacterial species and the biological activity of LTA (31, 59, 83), whereas others do not observe any differences (45, 64, 144, 170). Thus, it is possible that peptidoglycan and LTA mainly act to enhance the response promoted by other staphylococcal products (131). Indeed, several other cytokine-stimulating cell components different from peptidoglycan and LTA have been described in staphylococcal species.

Hemolysins Much less is known about the impact of virulence factors on the inflammatory response to S. aureus. We previously discussed the important role of cell wall-associated protein A in the inflammatory response. However, the overall in vivo effect of other virulence factors on the innate immune system is not clearly understood. S. aureus produces several secreted virulence factors, among which are hemolysins such as ␦-hemolysin (see the section on PSM). Alpha-toxin is another hemolysin secreted into the extracellular supernatant during the postexponential phase of growth. This toxin associates to form pores in the cell membrane, inducing lysis of several types of mammalian cells such as erythrocytes and monocytes. It is an important pathogenicity factor of S. aureus. At sublytic concentrations, S. aureus alphatoxin can induce the production of IL-1␤, IL-6, and IL-8, and a weak release of TNF-␣ (15, 33, 142). After intraperitoneal inoculation, alpha-toxin also elicits neutrophil recruitment into the mouse peritoneal cavity (142). Furthermore, it has been shown that IL-8 production induced by alpha-toxin is dependent on NF-␬B (33). ␤-Hemolysin is also secreted during the postexponential phase and is an Mg2⫹-dependent sphingomyelinase C. It degrades sphingomyelin present in the phospholipid layer of the cell membrane, lysing sheep erythrocytes and human monocytes, but it has no action against human granulocytes, fibroblasts, lymphocytes, or erythrocytes. In contrast to alpha-toxin, which stimulates inflammatory cytokine and chemokine production at sublytic doses by activating NF-␬B, it seems that ␤-hemolysin provides an inflammatory response by lysing cells containing mediators. Indeed, cytolysis of monocytes induced by ␤-hemolysin releases IL-1␤ (206). To date, how these hemolysins induce an inflammatory response and which innate immune receptor they activate is not known (Fig. 3). However, as it is difficult to purify hemolysins without any contamination of inflammatory cell wall components such as lipopeptides or LTA, it is possible that these contaminants themselves rather than hemolysins cause cytokine induction (96). Interestingly, PSM production has been shown to be upregulated by Agr (203). Agr also upregulates production of hemolysins such as alpha-toxin and ␤-hemolysin in S. aureus (129, 148). The culture supernatant from an agr mutant strain of S. epidermidis which does not contain either PSM or hemolysins does not induce TNF-␣ production, whereas the supernatant of the wild-type strain promotes significant cytokine production (203). These results suggest that the components that are present in staphylococcal culture supernatant and mediate cytokine production are regulated by Agr.

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Formylated Peptides Bacteria produce formylmethionyl peptides that are chemoattractants for neutrophils and macrophages (166). It has been shown that S. aureus releases formylated peptides into the culture supernatant (161). One of these peptides was purified and its amino acid composition was determined to be fMet-Ile-Leu-Phe. This formylated peptide can inhibit the ability of a labeled formylmethionyl chemoattractant to bind human monocytes, suggesting that this peptide may bind to the formylated peptide receptor (Fig. 3) (162). S. aureus produces ␦-hemolysin in both formylated and deformylated forms. Interestingly, only the formylated form, which represents 90% of the ␦-hemolysin present in the culture supernatant, is able to elicit migration of PMNs (175). Thus, formylated peptides are important inflammatory mediators, although the exact nature of their innate immune receptor is not formally established. Staphylococcal DNA Bacterial DNA has inflammatory properties (191). Indeed, it is able to induce the production of IL-6, IL-12, IFN-␥, and TNF-␣ (92, 136). Staphylococcal DNA causes arthritis (32). Furthermore, injection of staphylococcal DNA in a mouse model of cutaneous inflammation causes a strong inflammatory response (32, 123). These results suggest that staphylococcal DNA may be an important inflammatory molecule of S. aureus. Staphylococcal DNA has been shown to induce the production of TNF-␣, IL-12, and IFN-␥, although its inflammatory effect is lower than that of E. coli (136, 177, 220). The inflammatory property of bacterial DNA is abolished by treatment with DNase but not RNase, confirming the deoxynucleotide nature of the stimulus. The inflammatory activity of bacterial DNA results from cytosine-phosphate-guanosine (CpG) motifs that are unmethylated at cytosine residues. In contrast, eukaryotic genomic DNA, which is not inflammatory, is mostly methylated and has less CpG than the bacterial genome (92, 220). TLR9 is a key receptor for bacterial DNA. Indeed, it has been shown that activation of B cells in TLR9-deficient mice is abolished when stimulated by CpG (Fig. 3) (62). TLR9 requires an adaptor protein such as MyD88 and promotes mitogen-activated protein kinase and NF-␬B activation. Interestingly, TLR9 is present in the intracellular endosomal compartment, whereas TLR2 is anchored to the cell surface (Fig. 3) (92, 204). Although DNA is an important inflammatory component of S. aureus, the role of TLR9 in staphylococcal infections is still not clear and awaits further study in S. aureus-challenged TLR9-deficient mice. CONCLUDING REMARKS It is clear that a large number of staphylococcal molecules (and probably many more than those that have been discovered so far) interact with the innate immune system to induce cytokine production and the inflammatory response, suggesting a complex interaction between Staphylococcus and eukaryotic cells. Although much work has been conducted with peptidoglycan and LTA on the level of responses to TLR2 and Nod2, much less is known about the impact of virulence factors

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such as hemolysins and adhesins on the inflammatory response to S. aureus. Indeed, the overall in vivo effect of these proteins on the innate immune system needs to be studied further. Furthermore, TLR2, Nod2, and TNFR1 probably have a key role in the inflammatory response to S. aureus since these receptors are necessary for full responses to S. aureus or its inflammatory components. However, other less-studied receptors such as TLR9 and formylated peptide receptor may also contribute to the activation of NF-␬B and production of cytokines in response to staphylococcal infection.

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21.

22.

23. 24.

ACKNOWLEDGMENTS We thank Ivo G. Boneca, Minou Adib-Conquy, and Andre´ Klier for critical reading of the manuscript.

25.

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Vol. 18, No. 3

Immune Responses and Disease Enhancement during Respiratory Syncytial Virus Infection Peter J. M. Openshaw* and John S. Tregoning Department of Respiratory Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London W2 1PG, United Kingdom INTRODUCTION .......................................................................................................................................................541 THE BURDEN OF RSV DISEASE ..........................................................................................................................541 Epidemiology and Clinical Presentation .............................................................................................................541 Acute and Delayed Pathology of RSV Infection .................................................................................................542 HOST FACTORS AFFECTING PATHOGENESIS ...............................................................................................543 Host Genetics ..........................................................................................................................................................543 Disease in Immunosuppressed Individuals.........................................................................................................543 Effects of Age ...........................................................................................................................................................543 VIRAL INTERACTIONS AFFECTING DISEASE PATHOGENESIS: THE INNATE IMMUNE RESPONSE AND VIRAL EFFECTS ON THE HOST.........................................................................................................544 AUGMENTATION OF DISEASE BY VACCINATION.........................................................................................546 Disease Enhancement by Vaccination with FI-RSV...........................................................................................546 Immune Enhancement in Dengue and Measles Virus Infection......................................................................546 FI-RSV IN ANIMAL MODELS ................................................................................................................................546 Mice ..........................................................................................................................................................................546 Cotton Rats..............................................................................................................................................................547 Calves .......................................................................................................................................................................547 Primates ...................................................................................................................................................................547 Immune Priming with Individual RSV Antigens................................................................................................548 DISSECTING IMMUNOPATHOGENESIS............................................................................................................548 Antigen-Presenting Cells........................................................................................................................................548 Role of CD4 Helper T Cells ..................................................................................................................................549 Role of CD8 Cytotoxic T Cells ..............................................................................................................................549 Unconventional T Cells..........................................................................................................................................550 Effects of Virus-Specific Antibody.........................................................................................................................550 SUMMARY OF IMMUNE MECHANISMS OF RSV DISEASE .........................................................................550 Vaccine Development and Future Therapies for RSV Disease ........................................................................551 CONCLUSION............................................................................................................................................................551 REFERENCES ............................................................................................................................................................551 and how this can cause disease. We will discuss the burden of disease caused by RSV infection, factors which affect disease severity, and what is known of the mechanisms of viral bronchiolitis. It is useful to consider inflammation in RSV disease in three distinct scenarios: (i) the response to first infections in previously nonexposed hosts, (ii) the pathogenesis of enhanced disease in RSV-infected recipients of formalin-inactivated RSV (FI-RSV) vaccines, and (iii) specific animal models of disease augmentation. By comparing and contrasting the immunopathogeneses of primary bronchiolitis and enhanced disease, we attempt to identify common mechanisms that are shared or distinct in these conditions.

INTRODUCTION It is increasingly appreciated that symptoms and signs of many viral diseases are caused less by viral cytopathic effects than by the host’s response to infection. The peak of viral infection often precedes the period of maximal illness, which coincides with cellular infiltration of infected tissues and the release of inflammatory mediators. In this review, we discuss the role of overexuberant immune responses in disease caused by respiratory syncytial virus (RSV). RSV is the most important cause of viral respiratory tract infection in infants. Previous reviews have described the clinical impact of RSV disease (63), its pathogenesis (102), and the molecular biology of paramyxoviruses (30) and have compared RSV to other paramyxoviruses (40). The aim of this review is to provide an up-to-date summary of the host-RSV interaction

THE BURDEN OF RSV DISEASE Epidemiology and Clinical Presentation

* Corresponding author. Mailing address: Department of Respiratory Medicine, National Heart and Lung and Wright Fleming Institutes, Faculty of Medicine, Imperial College London, Paddington, London W2 1PG, United Kingdom. Phone: (44) 20 7594 3854. Fax: (44) 20 7262 8913. E-mail: [email protected]

RSV is a negative-strand, nonsegmented RNA pneumovirus of the family Paramyxoviridae. It is the single most important cause of acute respiratory tract viral infections in infants (62). In 2002, the World Health Organization estimated that 18.3 541

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million people died of infectious diseases; of these, 3.96 million died of respiratory infections, over 95% of which were lower respiratory tract infections (LRTI) (188). Viral LRTI are particularly serious during infancy, during which time the lungs are adapting to extrauterine life. Viral bronchiolitis is the most common single cause of infantile hospitalization in the developed world, and about 70% of bronchiolitis hospitalizations are associated with RSV infection (70). RSV has been estimated to cause 91,000 hospital admissions per year in the United States, with associated costs of $300,000,000 per year. In Europe, RSV accounts for 42 to 45% of hospital admissions with LRTI in children younger than 2 years of age, with inpatient populations tending to be younger and experiencing greater disease severity (153). The potential burden of RSV reinfection of adults on the health care system has been underappreciated; RSV also causes significant disease in healthy adults (especially those with contact with children) and generally passes undiagnosed (63). RSV causes high morbidity and mortality in patients with underlying cardiopulmonary illnesses (178), the elderly (44), and the immunosuppressed, particularly bone marrow transplant patients (68, 135). Primary infantile RSV infection typically presents as a winter upper respiratory tract infection, which is followed by mild lower respiratory tract symptoms in about 40% of cases. Otitis media is common. The mechanism of progression may involve aspiration of virus-containing upper respiratory tract secretions (by, for example, inhalation of postnasal drip) or shortrange intercellular spread via the extracellular fluid or sol phase of the surface mucus (136). Lower respiratory tract signs include tachnypea, hyperinflation, recession, crackles, and expiratory wheezing (leading to a clinical diagnosis of bronchiolitis). RSV is extremely common in children; for example, in the Houston Family Study, the infection rate was 68.8/100 in children less than 12 months of age and 82.6/100 during the second year of life; virtually all children had been infected at least once by 24 months of age, and about half had experienced two infections (53). RSV infections usually pass in less than a week and tend to be more severe in children aged 8 to 30 weeks. About 1 to 2% of all infants require hospitalization for bronchiolitis; among these, mechanical ventilation is needed in 2 to 5% (92). In affluent countries, mortality from RSV infection has been estimated as 0.005% to 0.02%. However, very few previously healthy children suffer life-threatening infections, and deaths are practically confined to those who are immunocompromised or who have preexisting cardiorespiratory disease (70, 138). Risk factors for severe RSV disease include premature birth, male sex, concurrent heart or lung disease, the presence of multiple siblings in the household (especially those sharing a bedroom), day care attendance, having parents who smoke, lower family income, and lack of breast feeding (152). In recent years there have been some excellent studies of RSV in developing countries such as Indonesia (39), South Africa (97), and the Gambia (184). In these regions RSV infections are usually seasonal and are not necessarily most frequent at the coldest time of year (164). Risk factors differ from those in developed countries and include, for example, the presence or absence of a flushing toilet and exposure to cooking fires; however, high sibling number remains an important risk factor in all settings (183).

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Acute and Delayed Pathology of RSV Infection RSV bronchiolitis is pathologically similar to bronchiolitis caused by other respiratory viruses such as influenza virus, parainfluenza virus type 3, and adenovirus. RSV has a direct cytopathic effect on cells in the lung epithelium, leading to loss of specialized functions such as cillial motility and sometimes to epithelial destruction (4). In addition, a peribronchiolar mononuclear cell infiltrate forms and is accompanied by submucosal edema and mucus secretion. This inflammation leads to bronchiolar obstruction with patchy atelectasis and areas of compensatory emphysema (47). Syncytium formation is not often seen in vivo and varies considerably from one RSV strain to another in vitro (146). In explanted human epithelial cultures, RSV infects the apical surface of ciliated columnar cells, is shed exclusively from the luminal surface, and spreads to neighboring cells by cillial motion (191). It is important to recognize that only a small minority of RSV-infected children develop severe disease and that the disease in ventilated children (i.e., those from whom it is possible to obtain samples from the lung) may be very different from that in hospitalized nonventilated children; we know virtually nothing about the pathogenesis of disease in the great majority of children, who develop mild respiratory symptoms and are neither seen by doctors nor sent to hospital. Since only a few RSV-infected children get very ill, the causative factor or factors do not need to be universal or even common. Severe RSV infection in the first 6 months of life is often followed by recurrent childhood wheezing, an association which is lost by 11 to 13 years (100, 150, 163). In a study by Sigurs (148), it was shown that children with severe RSV bronchiolitis in infancy had a significantly higher rate of asthma than age- and sex-matched controls (11% versus 0% at age 1, 23% versus 1% at age 3, and 23% versus 2% at age 7.5). In this same cohort studied at 13 years of age, asthma had been diagnosed in 37% of the RSV bronchiolitics and 5.4% of the control group. Allergic rhinoconjuntivitis was present in 39% and 15%, respectively, and skin prick tests were also more often positive in ex-bronchiolitics (50% versus 28%; P ⫽ 0.022) (149). There are also variable reports of an association with atopic disease (124), with some studies reporting a positive association (112, 116), which was not found by others (154). Accepting that RSV bronchiolitis and recurrent childhood wheeze are associated, the fundamental question remains: is the association causal, or does bronchiolitis act as a marker for an increased risk of allergy and wheezing illness due to genetic predisposition or impaired respiratory reserve (11, 118)? Direct interventional studies demonstrating a causal relationship have not been published. However, administration of antiRSV immune globulin to children at high risk of RSV disease seems to improve asthma scores and reduce atopy (185). It is possible that an anti-RSV neutralizing monoclonal antibody (e.g., palivizumab) also has long-term beneficial effects, but studies are still to be published. The mechanisms that could account for delayed effects of RSV infection are not clear but could include immune “imprinting” (see “Effects of Age” below) and viral persistence. A sustained increase in interleukin-2 (IL-2) receptor levels is seen after RSV infection, suggesting that inflammation may continue after the acute symptoms and signs have resolved

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(155), but direct histological confirmation of persistent inflammation has not been possible. RSV has been shown to be persistent in vitro in a macrophage-like cell line (57, 58, 173) and in cattle (172), mice (143), and guinea pigs (23). If persistence occurs in humans, it could explain the apparent delayed effects and serve as a reservoir for future RSV outbreaks in infants. Although severe RSV disease in infancy may cause recurrent wheezing, this is not the case with most viral infections. Uncomplicated common colds (without wheeze), type I herpetic stomatitis, chicken pox, and exanthema subitum seem to protect against wheeze in children up to 7 years of age. The risk of asthma diagnosis by this age is reduced by about 50% in children with two or more reported common colds by the age of 1 year (79). Viral infections typically induce T-helper type 1 responses, characterized by high levels of gamma interferon (IFN-␥) production; by contrast, asthma and atopy are typically characterized by T-helper type 2 cells producing IL-4 and IL-5 (Th2 cells). In contrast, analysis of nasal lavage and peripheral blood samples from RSV-infected children shows elevated IL-4/IFN-␥ ratios in infants during the first week of acute bronchiolitis compared with infants with upper respiratory tract signs alone. These data are consistent with excessive type 2 and/or deficient type 1 immune responses in RSV bronchiolitis (93). Viral infection could act to permit inhaled antigen to penetrate the mucosal barrier of the respiratory tract and meet relevant antigen-presenting cells and specific T cells, thereby leading to systemic sensitization. Prior allergic sensitization potentiates the physiologic and structural changes induced by acute RSV bronchiolitis, suggesting that an allergic diathesis may increase the severity of RSV infections in children (137). Animal models favor a role for RSV bronchiolitis in triggering asthma and in promoting a Th2 bias in immune responses to other antigens (117). Using palivizumab to prevent infection reduces airway obstruction and airway hyperreactivity to methacholine challenge in mice (104). Whatever the role of RSV in the inception of asthma, it (and other viruses) can certainly lead to asthma exacerbations in older children and adults (63). Rhinoviruses are commonly found during acute exacerbations of chronic obstructive pulmonary disease, but there is intriguing preliminary data suggesting that RSV may also be present in some patients during remission (144). HOST FACTORS AFFECTING PATHOGENESIS Host Genetics Even in a single outbreak of RSV disease, the severity is highly variable. Viral strain variations seem to play only a minor role in causing this variation, implying that host factors are important in determining disease severity, even when taken in the context of age-specific effects, current or recent infection, atmospheric pollution, and concurrent allergen exposure. For example, bronchiolitis risk is linked to polymorphisms in the wide-spectrum chemokine receptor CCR5 (74) and the IL-8 locus (54, 75). Further studies on IL-8 gene haplotype variants suggest that variation in susceptibility to RSV-induced bronchiolitis occurs via an increase in IL-8 transcription, pos-

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sibly mediated by functional polymorphisms (59). Genetic associations have also been found with the IL-4 gene (28, 73); with promoter variants of IL-9, IL-10, and tumor necrosis factor (TNF) alpha (72); and with the protein surfactant D (91). An association has also been found between soluble CD14 and wheezing following RSV bronchiolitis (156). Tolllike receptor 4 (TLR4) and CD14 are part of a receptor complex involved in the innate immune response to RSV, and TLR4 mutations have been associated with severe disease (165). In animal models, the host genotype has a large effect on the severity of disease (77, 77, 162). Disease in Immunosuppressed Individuals RSV infections tend to be prolonged in patients with defects in immunity (102), and bone marrow transplant recipients are at particularly high risk of severe RSV disease. In one study 8.2% of adult hematological inpatients were diagnosed with RSV infection, of whom 50% developed LRTI. Two of the 16 patients (12.5%) died of respiratory failure due to RSV pneumonia, despite intensive care unit admission and supportive ventilation (1). Immunosuppression caused by human immunodeficiency virus also affects RSV pathogenesis, and patients with AIDS have an increased duration of viral shedding (26). It is an apparent paradox that RSV causes severe problems in immunodeficient individuals, given that the disease is in large part due to excessive immune responses. Clearly, if viral replication is unchecked, RSV causes progressive cytopathic damage to the lung, leading to viral pneumonia and respiratory failure, as seen in immunodeficient (athymic nu/nu or irradiated) mice (25). On the other hand, partial immune reconstitution (e.g., during engraftment of bone marrow transplantation) is associated with an exuberant immunopathogenic response, representing an unbalanced reaction that is poorly antiviral. Effects of Age RSV has its greatest effects at the extremes of age. First infections in neonates may be severe, particularly in premature infants (139); reinfections are generally milder in older children and in adults but again can have serious consequence in the elderly (44, 113, 168). In neonates, the onset of air breathing is associated with a relatively high dead space, inelastic lungs, and flexible ribs arrayed horizontally (123); in older persons, the lungs normally decline in elasticity and trap air, which limits expiration (35). The immune system has distinctive features which may account for increased disease susceptibility in the young and the old (10, 105). The effects are most evident in high-risk premature infants, particularly those born before 28 weeks of gestation, before the transfer of maternal antibody occurs (36). Immune immaturity in neonates and immune senescence in the elderly may be associated with imbalanced RSV-specific immune responses that favor disease enhancement. In the mouse model, subjecting animals to primary infection at up to 1 week of age leads to increased disease severity during adult reinfection with RSV. Mice that were infected with RSV neonatally are sicker and have greater cell recruitment to the lung, increased IL-4 production, and a mixture of lung eosin-

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FIG. 1. RSV binding and triggering of cellular responses. RSV is bound by surface glycosaminoglycans, and the F protein binds to TLR4, while G glycoprotein (virus associated or secreted) binds fractalkine receptor CX3CR1. The interaction with TLR4 leads to upregulation of NF-␬B via MyD88. RSV upregulates NF-␬B via I␬B and STAT1 and -3 via reactive oxygen species (ROS), and RSV RNA activates protein kinase R. Viral NS proteins inhibit the interferon response factor (IRF3) pathway.

ophilia and neutrophilia during adult RSV challenge (34). Therefore, in the mouse model at least, the timing of neonatal infection establishes and determines the subsequent “imprinted” pattern of T-cell responses and, consequently, the nature and severity of disease during of reinfection in adulthood. The practical implication of these studies is that delaying RSV infection beyond early infancy could have long-term benefits. VIRAL INTERACTIONS AFFECTING DISEASE PATHOGENESIS: THE INNATE IMMUNE RESPONSE AND VIRAL EFFECTS ON THE HOST RSV surface proteins bind glycosaminoglycans (e.g., heparin or chondroitin sulfate), removal of which reduces the infectibility of HEp2 cells in vitro (65). RSV can also interact with annexin II and L-selectin (99). The RSV glycoprotein G has been shown to have structural homologies with the CX3C chemokine fractalkine. G binds the human CX3CR1 receptor and mediates chemotaxis of cells that respond to CX3CL (169). It is possible that this interaction facilitates binding to CX3CR1-bearing cells, including mast cells and neuronal cells. The fusion (F) protein binds TLR4 (89), upregulating its surface expression and sensitizing airway epithelial cells to endo-

toxin (109). The frequency of TLR4⫹ monocytes is increased in the peripheral blood of some infants with RSV bronchiolitis (46), but the role of TLR4 in vivo is unclear (41). Events during the first minutes and hours after viral entry are of key importance, not only in determining the balance between viral multiplication and elimination but also in setting the pattern that will be followed by acquired immune responses. Early viral proteins therefore frequently interfere with innate immune mechanisms (Fig. 1). Once within cells, RSV upregulates the STAT pathway via reactive oxygen species (95). Nitric oxide production is associated with the upregulation of IL-8 (polymorphisms in the promoter of which have been shown to be associated with bronchiolitis severity [54]), leading to pulmonary neutrophilia (159). RSV infection also upregulates proapoptotic factors in the cell (88) and activates the nuclear factor ␬B (NF-␬B) pathway (19), which stimulates the transcription of genes directly involved in the antiviral response via I␬B kinase (60). NF-␬B is an upstream mediator of many of the innate responses, especially alpha/beta interferon and chemokine production, which leads to the recruitment and activation of cells and the production of further inflammatory mediators. RSV’s nonstructural proteins, NS1 and NS2, cause species-specific resistance to alpha/beta interferons (141, 157) via interferon

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FIG. 2. Cells involved in the immune response to RSV. Cellular infection triggers the release of early inflammatory mediators, e.g., TNF and IFN-␣/␤. NK cells and PMN are recruited in the first 3 days of infection, at which time DC carry viral antigen to local lymph nodes and present it to CD4⫹ T cells. Once primed, these cells migrate back to the infected epithelium, release further mediators, and recruit additional inflammatory cells, including mononuclear cells (including CD8⫹ T cells and B cells) and granulocytes (e.g., neutrophils [PMN] and eosinophils [Eo]). Ab, antibody.

regulatory factor 3 (IRF3) (21, 22). Similar effects have been demonstrated with other paramyxoviruses, simian virus 5, and Sendai virus (38). Chemokines are crucial in directing the recruitment of different cell subsets and make attractive targets for intervention. Double-stranded RNA selectively induces the secretion of chemokines such as CCL5 and IL-8, a factor that promotes neutrophils (49). Chemokines are produced in abundance during RSV infection in humans (90, 115, 145), and RSV infection of BALB/c mice induces expression of CXC, CC, and C chemokines in the lung (61, 106). Cytokine depletion, receptor blockade, or genetic deletion of chemokines or their receptors generally reduces disease severity and pathology during RSV infection. For example, antibody depletion of CCL5 (167) or CCL11 (101) reduces eosinophilia and disease severity in immune-augmented RSV disease, and MIP1␣ knockout mice have less severe disease during primary RSV infection (61). CCL5 (RANTES) seems of particular interest. It is produced in response to stimuli such as IFN-␥, IL-1␣, IL-1␤, and TNF by many cells, including fibroblasts, smooth muscle cells, and epithelial cells; in later stages of infection it is made by infiltrating cells, including ␥␦ T cells. It selectively recruits monocytes and memory T cells and eosinophils and (at high concentrations) activates T cells. Treatment of HEp-2 cells with recombinant human CCL5 inhibits infection with RSV in vitro, an effect not seen with other chemokines. This action

may result from blocking RSV fusion with host cells (42). CCL5 increases after RSV infection of mice and correlates with the severity of disease. Anti-CCL5 antibody administration decreases airway hyperreactivity and increases IL-12 production. Moreover, CCL5 production appears to be regulated by IL-13 which is also important in RSV-induced airway hyperreactivity (167). CCL5 may also be important in humans, since genetic studies show that polymorphisms of CCR5 affect disease severity (74). Moreover, CCL5 levels in nasal secretions during acute RSV bronchiolitis, although not correlated with disease severity, may be predictive of the later development of recurrent wheeze (29). Cytokine production has also been extensively studied in RSV bronchiolitis. For example, IL-9 is a cytokine associated with Th2 responses and with asthma (114). IL-9 mRNA and protein production is elevated in the lungs of infants with RSV bronchiolitis. Intriguingly, polymorphonuclear cells (PMN) seem to be a major source of IL-9 in this situation (103). RSV infection thus triggers innate immune responses that influence the developing acquired immune response (71). NK cells are an abundant source of IFN-␥, which has potent effects on developing ␣␤ T cells and thus on the immunopathology of RSV infection (81). IL-12 from antigen-presenting cells has potent effects on NK cells, enhancing IFN-␥ production (78). This cascade of innate and acquired events during RSV infection is outlined in Fig. 2.

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AUGMENTATION OF DISEASE BY VACCINATION Disease Enhancement by Vaccination with FI-RSV The best-studied model of RSV disease enhancement by the immune system is the formalin-inactivated vaccine FI-RSV. Following the success of other chemically inactivated viral vaccines (e.g., polio vaccine), studies using FI-RSV were conducted in 1966 and 1967. Vaccines were administered to infants and children aged 2 months to 9 years in two or three intramuscular doses separated by 1 to 3 months (27). After subsequent RSV exposure, the rate of virus infection in children receiving FI-RSV was no less (and was perhaps even greater) than that in a control group immunized with a control parainfluenza vaccine. Most remarkably, 80% of RSV vaccinees needed hospitalization, whereas only 5% of RSV-infected children given the control parainfluenza vaccine required admission. Illnesses among RSV-infected FI-RSV-vaccinated children included pneumonia, bronchiolitis, rhinitis, or bronchitis; two of the vaccinated children died (87). Importantly, these trials were conducted in the absence of prior animal testing. Postmortem examinations showed bronchopneumonia with emphysema and pneumothorax. Microscopically, there was an intense inflammatory infiltrate, including mono- and polymorphonuclear cells and eosinophilia. These changes suggested an immunopathological cause of enhanced disease. Analysis of sera from children immunized with FI-RSV shows that antibodies to the F and G proteins were generated but were poorly neutralizing (110). The severity of illness was remarkably dependent on the age of the vaccinees, with the younger children suffering more severe symptoms. The reasons for this are not clear, but animal models suggest that there is an age-dependent factor as a major determinant of the pathogenic immune response (34, 125).

TABLE 1. Advantages and disadvantages of animal models of RSV disease Characteristic of model

Ideal animal model: Determines which antigens to use in vaccines Allows optimization of route, dose, frequency, etc. Predicts effects of key genetic and environmental variables Anticipates vaccine failures and adverse effects Animal models can: Allow study of complex biological systems Allow controlled interventional experiments Test genetic influences Illustrate principles Generate hypotheses Animal models cannot: Give quantitative information about human responses Determine exactly which protective or pathogenic mechanisms operate in humans Accurately determine the effects of genetic variations Usually anticipate adverse effects in humans

inal antigenic sin” in the T-cell responses may suppress or delay viral elimination, leading to higher viral loads and increased immunopathology (108). FI-RSV IN ANIMAL MODELS Given the tragic results of the FI-RSV trials in human infants, it is essential to determine the nature of pathogenesis in animal models. Animal models have been used extensively to elucidate possible mechanisms linking RSV disease with subsequent wheezing (Table 1). These include the cotton rat, calf, monkey, mouse, and guinea pig models. Each has advantages and drawbacks.

Immune Enhancement in Dengue and Measles Virus Infection

Mice

Like FI-RSV, formalin-inactivated measles virus (FI-MV) can cause severe and generalized disease during subsequent natural infection (45). This ‘atypical’ measles is seen after a delay of about 7 years, whereas enhanced RSV disease occurs within 2 years of FI-RSV administration (128). Therefore, a window of protection may be followed by a phase of disease enhancement (111), followed by a final period during which immune memory is still measurable but neither protection nor enhancement is seen. The pathogenesis of dengue hemorrhagic fever is imperfectly understood, but epidemiological data suggest that it occurs when a dengue virus-immune person becomes infected with a second viral serotype (140). In that prior infection leads to a more severe disease on reinfection, this resembles RSV disease following FI-RSV immunization. Antibody-dependent enhancement has been suggested, whereby preexisting nonneutralizing antibodies may opsonize dengue virus and enhance its uptake and replication in macrophages. Higher viral loads have been demonstrated in preimmune primates (66). However, T-cell activation may also contribute to disease in that virus-specific CD8 T cells disappear from the peripheral blood during acute infection. It has been suggested that “orig-

Factors favoring the mouse model include the easy availability of immunological reagents (exceeding that for any other species); the many inbred and congenic strains, gene knockouts, and transgenics; and a complete genome sequence. The mouse and human genomes are very similar; each has about 30,000 genes, of which only 1% are species specific. Equivalent mouse genes have been found for all genes known to cause human disease, and 99% of mouse genes have a human homologue. Although the human and mouse immune systems have diverged during the 75 million years since separation, the same immunological niche is sometimes occupied by nonhomologous proteins (e.g., KIR and Ly49) because of convergent functional development (182). The role of T cells in augmented lung pathology has been highlighted in the mouse model of FI-RSV. Connors et al. showed that CD4⫹ T cells are crucial to the immunopathogenesis of FI-RSV disease and that RSV-specific antibodies (in the absence of CD4⫹ and CD8⫹ T cells) are not sufficient to cause disease enhancement (31). Further studies revealed a marked increase in the expression of Th2-type cytokines (IL-5, IL-13, and IL-10) and reduced expression of IL-12 in FI-RSV-immunized mice, indicating a Th2 bias in the increased inflammation

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(181). The same authors found a positive correlation between the signal for IL-5 mRNA and the eosinophil infiltration. Moreover, skewing to a T-helper 1 (Th1) pattern of cytokine production by priming with live RSV prevented subsequent enhanced disease (180).

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FI-bRSV therefore causes enhanced disease in bRSVinfected cattle in a pattern highly reminiscent of the disease seen in infants, mice, cotton rats, and monkeys.

Primates Cotton Rats FI-RSV-vaccinated cotton rats show disease augmentation during intranasal challenge with RSV. The lungs of immunized animals are infiltrated with a mixture of cells, including neutrophils, macrophages, and lymphocytes. Serum contained only low levels of neutralizing antibodies (134). It has been suggested that epitopes against which neutralizing antibody is directed could have been modified by formalin treatment, leaving nonprotective epitopes undamaged and therefore inducing high titers of antibodies binding in enzyme-linked immunosorbent assay that form immune complexes. However, FI-RSV does reduce RSV titers in the lung by 90% while increasing peribronchiolitis and alveolitis (132), demonstrating a dissociation between effects on viral load and immunopathology. While the cotton rat model demonstrates clear disease enhancement and immunopathology, it has hitherto been difficult to dissect the precise mechanisms because of the paucity of immune reagents. A good range of reagents are now being developed, enhancing the utility of the cotton rat model (20). Calves Bovine RSV (bRSV) is a natural infection of cattle that is of considerable economic importance. Primary bRSV infection can cause severe lower respiratory tract disease in calves, although asymptomatic infections also occur. The effects of bRSV are similar in some ways to those of human RSV in humans, but bRSV tends to cause an acute interstitial pneumonia with alveolitis, emphysema, and bronchiolitis, especially in calves and yearlings (174). Immunization with FI-bRSV generally results in strong immunoglobulin G antibody responses against F and G, without an adequate neutralizing antibody response (50). In one study, 6-month-old calves were vaccinated with FI-bRSV, live bRSV, or control material. One month after the second vaccination, vaccinees were infected with a field isolate of bRSV. The FI-bRSV recipients developed pyrexia and dyspnea more rapidly than controls but showed inconsistent changes in pulmonary pathology (186). Increased disease after vaccination was also shown during a bRSV outbreak in 60 calves less than 8 months old that were housed in barns. During this outbreak, FI-bRSV-vaccinated calves showed more severe disease than unvaccinated animals, with 30% of the immunized calves dying of respiratory distress. In the calves that died, an eosinophilic infiltrate was present in the lungs (142). Another study examined the role of antibody subtypes and suggested that FI-bRSV enhances Th2 immune responses (86). Antonis et al. also showed that immunization with FI-bRSV mainly primes a Th2like inflammatory response, which is associated with an eosinophilic influx into the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies (8).

Studies with nonhuman primates generally show patterns of FI-RSV-induced disease augmentation similar to those seen in other species. In rhesus macaques, FI-RSV vaccination leads to RSV-specific T cells, predominantly producing the Th2 cytokines IL-13 and IL-5. Intratracheal challenge with RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia, and two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation (37). This result is compatible with those of other studies showing decreased lung viral titer following RSV challenge in association with enhanced pathology (85). Bonnet monkeys also develop enhanced disease after FIRSV vaccination, but with increased viral replication in perivascular sites of the lung (130). The site of RSV replication may be mononuclear cells which have taken up RSV because of infection-enhancing antibodies. Enhancement of infection was not observed in animals with primary and tertiary infections or in those immunized with FI-Vero cell culture. Serum antibody from animals immunized with FI-RSV increased RSV infection of U937 cells, and the enhancement index correlated positively with the pathological scores of the FI-RSVvaccinated monkeys (131). This finding suggests that antibody may play a role in FI-RSV enhancement of disease. It has been suggested that waning levels of maternal antibody might cause spontaneous disease augmentation in children undergoing natural primary RSV infection between 2 and 6 months of age (43). However, maternal antibody is clearly protective in mice (166) and cotton rats (133). In many hundreds of studies of passive antibody transfer in animals, disease augmentation has not been observed. In a prospective study of 68 infants with RSV infection and 575 controls, neutralizing antibody titers in cord blood correlated with protection against disease, not with infection. Moreover, the level of antibody at birth directly correlated with the age at the time of infection and severity of disease (52). More conclusively, passive immunization with palivizumab induces solid protection in most infants and does not cause enhanced disease. It is possible that the anti-RSV antibody induced by FI-RSV blocks epitopes on the RSV surface proteins that bind receptors (for example, TLR4) on the target cells which normally elicit a downstream immune response. If this binding is blocked, the initial protection afforded by interferons or other early innate immune systems may also be blocked, leading to uncontrolled virus replication. The FI-RSV models are useful because they provide information pertinent to the design of future vaccines, especially as to what strategies may be unsuccessful; they give a guide towards how RSV interacts with the body; and they have led to the development of animal models of RSV disease, which can be used to better study it.

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FIG. 3. Immunopathology to RSV surface proteins. In the BALB/c mouse, different RSV proteins expressed by recombinant vaccinia viruses after infection by scarification of the skin cause markedly different effects on subsequent RSV challenge intranasally. The F protein primes CD4 and CD8 T cells and leads to intense inflammation characterized by efflux of PMN. The G attachment glycoprotein primes for CD4 T cells and no CD8 response and is associated with a relatively weak NK cell response; this leads to eosinophilia. The transcription antiterminator (M2) protein primes only CD8 T cells and induces almost no CD4 T-cell response and virtually no antibody. This is often the most illness inducing of the sensitizing protocols, but the effect is not as durable and that induced by G or F. Ab, antibody.

Immune Priming with Individual RSV Antigens Sensitization of BALB/c mice by dermal scarification with recombinant vaccinia viruses (rVV) expressing individual RSV proteins makes it possible to dissect the contribution of different RSV antigens and T-cell subsets to protection and pathology. The three most studied RSV proteins are the major surface glycoprotein (G), the fusion protein (F), and the transcription antiterminator (formerly called the second matrix protein, M2). These vectors induce contrasting outcomes (Fig. 3). While rVV-G primes Th2 cells and leads to secondary RSV disease characterized by lung eosinophilia (5), rVV-F primes cytotoxic T lymphocytes (CTL) and Th1 responses, resulting in secondary RSV disease with PMN efflux (122, 160), and rVV-M2 primes for a secondary RSV disease characterized by a strong CTL response (120). This situation is reminiscent of that seen in lymphatic filarial infection, where some antigens tend to prime Th1 cells and others prime Th2 cells (190). Using deletion mutations of regions of the G protein, a site within G which seems to be of key importance to induction of Th2 cells has been identified. Deletion mutants of this region of G no longer prime an eosinophilic response to infection but do prime the immune system to clear the virus more effectively (158). Sensitizing mice with recombinant vaccinia virus expressing the secreted soluble form of the G protein leads to a greater eosinophilic influx into the lungs following RSV challenge than in mice sensitized with vaccinia virus expressing only the membrane-anchored form (15, 82). Engineered recombinant RSV expressing only the membrane-bound form of G is immunogenic but grows poorly in vivo and is unable to generate the eosinophilic response seen with nonrecombinant virus (98). Antibody depletion of cells bearing T1/ST2 (a marker of the Th2 cells [96]) reduces eosinophilia in RSVinfected rVV-G-primed mice (179).

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rVV-G does not prime for eosinophilia in all strains of mice. In tests of 15 different inbred mouse strains, eosinophilia developed in all H-2d mice but not in H-2k mice. Among H-2b mice, 129 and BALB/B mice developed eosinophilia, whereas C57BL/6 and C57BL/10 mice did not. Testing of F1 crosses between sensitive and resistant strains showed that eosinophilia developed in all H-2d ⫻ H-2k mice but not in H-2d ⫻ H-2b mice, so that inheritance of the eosinophilic trait could be either recessive or dominant depending on the strain combination (77). Treatment of otherwise eosinophilia-resistant mice with anti-CD8 antibody (or use of mice genetically deficient in functional CD8⫹ cells) allows the development of eosinophilic inflammation in previously resistant strains (76). These results support the concept that RSV disease arises because of the balance between different subsets of T cells. If an engineered, secreted form of the F protein is used for vaccination, IL-4 and IL-5 production is increased but pulmonary eosinophilia following RSV challenge is not seen (16). The transcription antiterminator protein (M2) contains an immunodominant Kd-restricted peptide epitope (121) which can be used for mucosal vaccination in conjunction with the adjuvant LTK63 (151). M2 primes strong CTL responses and severe disease enhancement on RSV challenge. Crucially, this type of priming induces virtually no RSV-specific antibody, and transfer of CTL to naive mice results in accelerated viral clearance but greater disease (24). Immunization with vaccinia virus expressing individual RSV proteins gives valuable information about the possible pathways of RSV disease pathogenesis but does not reproduce the pathology seen after FI-RSV vaccination. Lung eosinophilia is seen in RSV-challenged mice primed with FI-RSV or with rVV-G, but G protein is not necessary for formalin inactivation-enhanced disease (129). In another study, formalin-inactivated mutant RSV strains with truncated or deleted G or deleted SH induced lower protective antibody levels, but immunopathological effects were still seen, with increased illness and eosinophilia (83). This suggests that immunity to G is important for protective immunity but is not necessary for FI-RSV-enhanced disease. DISSECTING IMMUNOPATHOGENESIS Many different cell types are involved in the responses to RSV. Some of these responses are clearly protective but can also cause increased damage. The interactions between these cell types occur through cognate interaction and by cytokines and chemokines (Fig. 4). Some of these mediators are produced early during virus infection, but others predominate in the later phases. They may therefore influence both primary and delayed diseases. Antigen-Presenting Cells Dendritic cells (DC) are able to initiate potent responses in naive T cells. Influenza A leads to an increase in DC numbers in the lung (189), which may in turn lead to excessive lymphocyte infiltration and immune augmentation. DC are also associated with atopic patients (107) and are seen in allergy models (161). In mice, there is a sustained increase in DC numbers following RSV infection (18). RSV has also been shown to

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FIG. 4. Interactions of T cells and cytokines. Normal mild or asymptomatic infections are cleared with a predominantly Th1 response, with IFN-␥ being generated from NK cells and CD4 and CD8 T cells (left). This inhibits the Th2 cytokine pathways, which are generally inactive. In disease enhanced by prior immunity or in some otherwise predisposed individuals, there is a rapid and strong Th2-type system (right). This Th2-type response may be caused by reduced levels of key cytokines (e.g., IL-12) at an early stage of infection or by vaccination (e.g., with FI-RSV) leading to preferential production of cells that make IL-4 and IL-5. IL-4 suppresses Th1 cytokines and upregulates Th2 cytokines, most importantly IL-5 and IL-13, which leads to eosinophilia and airway narrowing. However, the Th1 response can also be pathogenic if it exceeds that necessary for simple viral clearance, and it may be associated not with eosinophilia but with PMN efflux (see also Fig. 3). IgE, immunoglobulin E.

decrease the production of IFN-␥ (14) and to increase the production of prostaglandin E2, IL-10, and IL-11 in cord blood-derived DC, suggesting that RSV might drive the immune system towards a Th2-type environment by effects on DC (13). Role of CD4 Helper T Cells It seems some T cells enhance disease, while others control it. In mice, primary signs of disease are reduced by CD4 and/or CD8 T-cell depletion (56). Depletion of CD4⫹ T cells (32) or transfer of CD8⫹ T cells modulates the eosinophilia seen in rVV-G-primed RSV-infected mice (6), while eosinophilia can be made to appear in strains that normally do not develop it if CD8 T cells are depleted or impaired in function (76). This suggests that Th2 cells promote RSV-induced eosinophilia and that CD8 cells generally inhibit it (Fig. 3). As described above, RSV G-induced pathology is caused mainly by the overactive Th2 CD4⫹ T cells (5). It has been shown that these CD4 T cells are oligoclonal, with approximately half of the cells expressing V␤14, and that Th2-like pulmonary injury can be abolished by elimination of this CD4⫹ V␤14⫹ subpopulation (177). This intriguing finding suggests that this novel subset of CD4⫹ T cells is crucial to the development of pa-

thology and that G may have a “superantigen” effect. Investigation of this phenomenon has not been followed through in studies on bronchiolitic infants. Another subset of Th2 CD4 T cells, T1/ST2, has been shown to play a role in RSV-driven eosinophilia (179). As described above, the effects of RSV are mediated through the cytokines and chemokines that it induces, and these mediators will necessarily affect the outcome of an infection. The immune background of the neonatal lung is different from that of the adult lung (3, 119), with a general bias towards Th2 responses. For example, CD4⫹ T cells show hypermethylation in the promoter region of the IFN-␥ gene, affecting transcription efficiency (187), and IL-12 gene transcription is reduced in neonatal human monocyte-derived dendritic cells (55). It may be that these factors in part account for enhanced disease severity in RSV-infected infants. Role of CD8 Cytotoxic T Cells Although murine FI-RSV and rVV-G vaccination models of disease augmentation have focused on the pathogenesis of lung eosinophilia and have emphasized the role of Th2 cells, RSV immunopathology is certainly not solely Th2 related. During primary infection RSV, like most viral infections, tends

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to induce inflammation dominated by Th1 cytokine production even in BALB/c mice (which are prone to Th2 responses). Cell transfer studies show that CD8 T cells can cause viral clearance but also can result in remarkable disease enhancement (24). If rVV-M2 is used to prime BALB/c mice, RSV infection results in extreme sickness due to a massive pulmonary influx of CD8⫹ CTL and pathology reminiscent of acute respiratory distress syndrome. In a few cases, RSV bronchiolitis has also been associated with acute respiratory distress syndrome in humans (67), but the normal pathology of infantile bronchiolitis is very similar to that seen in mice with highly activated CD8 T-cell responses. CD8 T cells are a major source of IFN-␥. In the mouse model, IFN-␥ is produced following primary infection but not sufficiently to control RSV-induced allergy to OVA (12). The role of IFN-␥ in RSV disease is unclear; in the mouse model it has been shown to be necessary for protection (126), and airway obstruction is decreased in IFN-␥ knockout mice (175). There is a considerable evidence that IFN-␥ is produced following RSV infection (see, for example, reference 176) but possibly in an inverse relationship to bronchiolitis severity (48). Clinical data point either towards at least a balanced Th1/Th2 cytokine production (170) or towards a deficiency of Th1 cytokines characterized by infants with RSV producing less IFN-␥ (84). Compared to the case for other severe respiratory viral infections leading to LRTI, IFN-␥ production by peripheral blood mononuclear cells appears to be decreased in RSV disease (2).

Unconventional T Cells It seems that numerically small subsets of T cells may play a major role in regulating immune enhancement in RSV disease. NK cells are transiently present in the early stages of RSV infection of mice and are a major source of IFN-␥ at day 4, when cells of the acquired immune system are differentiating. Whereas CD8⫹ T cells can cause enhanced weight loss, IL-12activated NK cells inhibit lung eosinophilia without causing enhanced illness. However, depletion of both NK and CD8 T cells allows RSV to spread to mediastinal lymph nodes, showing that either subset alone can have antiviral effects (77). Cells of the innate immune system can therefore direct the pattern of subsequent specific immunity. ␥␦ T cells are defined by the use of the ␥␦ T-cell receptor instead of the more common ␣␤ type associated with classical T lymphocytes. While they are relatively scarce, ␥␦ T cells appear early in thymic ontogeny and are associated with mucosal surfaces (69). In virus-infected infants, ␥␦ T cells produce more IL-4 and less IFN-␥ during RSV infection than during reovirus infection (9). It seems possible that these small subsets of cells can be crucial to directing the form of pathology. CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack V␣14⫹ natural killer T cells. CD8 T-cell recruitment is reduced in CD1d⫺/⫺ mice, which show alterations in illness, viral clearance, and IFN-␥ production. Activation of NK T cells in normal mice by ␣GalCer results in reduced illness and delayed viral clearance (81). Thus, early IFN-␥ production and efficient induction of

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CD8 T-cell responses during primary RSV infection require CD1d-dependent events. Effects of Virus-Specific Antibody As described above, it was initially thought that FI-RSV pathology was antibody mediated. This view went out of favor but gained support from studies in vitro and in primates that suggested that antibody may increase viral replication (51, 130, 131). In addition to possible infection-enhancing antibody, antibody may enhance disease by forming immune complexes and activating complement. This has been shown to be important for both FI-RSV (129) and FI-MV (127). This effect appears to preferentially affect lung function, but it possibly also affects Th2 differentiation (7). SUMMARY OF IMMUNE MECHANISMS OF RSV DISEASE Studies of immune-augmented secondary disease are sometimes interpreted as being informative about the origins of disease in primary bronchiolitis, but the relationship between the pathogeneses of these conditions is disputed. It is important to resist overinterpreting studies of immune sensitization when attempting to explain the pathogenesis of primary bronchiolitis. However, there are common themes linking the two conditions. The incubation period of primary RSV is about 5 days; children hospitalized with bronchiolitis have usually already been ill for 3 to 6 days. Virus replicates to higher levels and remains detectable for longer during primary infection than after prior sensitization. It is therefore possible that “acquired” T- and B-cell responses are developing by this time and contribute to disease. This situation is exacerbated by prior sensitization or in those otherwise predisposed to particular specific immune responses. Important common and distinct factors in different forms of RSV disease are illustrated in Fig. 5. In primary infection of nonvaccinated individuals, the disease is characterized by a relatively high viral load and the delayed appearance of disease, the timing of which coincides with the development of specific acquired T-cell immunity (Fig. 5A). In previously sensitized individuals (or perhaps those in some way predisposed to enhanced disease), viral replication is more limited and viral elimination occurs more rapidly (Fig. 5B). However, the heightened immune response leads to even more disease than is typical during first infections. Age and host genetics certainly affect the balance of immune responses during primary infection, making first RSV infections sometimes resemble secondary disease. In the neonatal period, Th1 responses are generally poor or short-lived and IL-12 production weak. One possibility is that both Th1 and Th2 responses are formed during the initial infection; however, there is specific IL-4-dependent apoptosis of Th1 cells in the neonatal environment (94). This may explain why at this age, there is a natural tendency towards strong Th2 responses. An autocrine Th2 feedback loop driven by IL-4 might therefore tend to cause a cascade of events causing immune damage or future skewing of response to disease.

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CONCLUSION

FIG. 5. Overview of the sequence of immune events in viral clearance and disease. In primary infection of nonvaccinated individuals (A), virus peaks on about day 4, associated with recruitment of NK cells, which make IFN-␥. Virus is eliminated between days 5 and 8, during which time activated CD4 and CD8 T cells are recruited and produce local cytokines. The peak of disease coincides with this phase. Anti-RSV serum antibody appears relatively late. In previously vaccinated or sensitized individuals (B), the virus titer is typically 100- to 1,000-fold less than in primary infection and peaks earlier (e.g., day 2). However, the rapid and potent cellular response enhances disease severity, which is usually much greater than in primary infection. Notably, high levels of preexisting specific antibody can prevent infection completely and do not cause disease enhancement.

Vaccine Development and Future Therapies for RSV Disease Despite continuous efforts to develop safe and effective vaccines, spanning 40 years, none have been successful. The basic difficulties to be overcome include the fact that natural infection gives only transient and partial immunity to reinfection of the upper respiratory tract (64). Many established vaccines are ineffective in the first 6 months of life (147), and it is in this period that most children suffer from RSV bronchiolitis. Vaccines would be useful in the elderly, but many established vaccines are poorly immunogenic in older people. Nonetheless, the potential benefits of an effective vaccine are undoubted. Vaccines under development include cold-passaged live viruses (33), purified proteins (171), and conventional inactivated and DNA vaccines (17). Given that disease appears to be in some part caused by immune overactivity, it is logical to test specific short-lived immune inhibitors. Steroids appear to be of little or no value, possibly because they lack specificity and reduce the severity of the immune response while potentially increasing viral replication. Anti-TNF, anti-IL-9, and anti-immunoglobulin E therapy all merit consideration, perhaps in combination with antiviral treatments. Such experimental treatments would need to be carefully justified in patients with the most severe forms of bronchiolitis. Effective prophylaxis is available in the form of a biosynthetic humanized monoclonal anti-F antibody, palivizumab. This is administered as a monthly intramuscular injection and is highly effective in preventing infection. However, its cost prohibits its use in resource-poor settings.

Severe RSV disease appears to be associated with a misdirected immune response, characterized by enhanced release of mediators and infiltration of a range of monocytes and polymorphonuclear cells. Animal models are essential to understanding disease enhancement and to the development of safe and effective vaccines, but none is ideal in all aspects (Table 1). While it is clear that primary and immune-augmented RSV diseases are not identical, these models shed light on which components of the immune response warrant further study and give rise to important general conclusions about immunopathogenesis: (i) single clinical syndromes (bronchiolitis, asthma, etc.) can result from diverse pathogenic pathways, (ii) the time and place of sampling are critical in acute transient diseases, (iii) samples from remote locations may be misleading, and (iv) the most numerous cells may not be the most influential. The effects of formalin-inactivated RSV are remarkably similar in all species studied, including cattle, rodents, and primates. Such vaccines enhance disease by multiple pathways, leading to overactive acquired immune responses. In animal models, this disease is relatively hard to block by specific immunomodulation. By contrast, the immunopathogenesis of augmented disease in inbred mice vaccinated with single RSV proteins is highly specific and relatively easy to prevent by treatments that take out single components of the immune response (80, 83). It seems clear that the immunopathogenesis of RSV disease during primary infection varies considerably from one individual to another and is affected by the postnatal age. Severe RSV bronchiolitis occurs only in a small minority of children and is usually transient and self-limiting. However, studies of disease augmentation by prior sensitization are capable of reproducing many features of the severe primary disease seen in susceptible individuals and may therefore give indications of what type of immunomodulation should be attempted. A great deal has been learned in recent years, and it is to be hoped that clinical application of this knowledge will soon benefit the large numbers of children who suffer from RSV infections each year. REFERENCES 1. Abdallah, A., K. E. Rowland, S. K. Schepetiuk, L. B. To, and P. Bardy. 2003. An outbreak of respiratory syncytial virus infection in a bone marrow transplant unit: effect on engraftment and outcome of pneumonia without specific antiviral treatment. Bone Marrow Transplant. 32:195–203. 2. Aberle, J. H., S. W. Aberle, M. N. Dworzak, C. W. Mandl, W. Rebhandl, G. Vollnhofer, M. Kundi, and K. T. Popow. 1999. Reduced interferon-gamma expression in peripheral blood mononuclear cells of infants with severe respiratory syncytial virus disease. Am. J. Respir. Crit. Care Med. 160:1263– 1268. 3. Adkins, B., C. LeClerc, and S. Marshall-Clarke. 2004. Neonatal adaptive immunity comes of age. Nat. Rev. Immunol. 4:553–564. 4. Aherne, W., T. Bird, S. D. M. Court, P. S. Gardner, and J. McQuillin. 1970. Pathological changes in virus infections of the lower respiratory tract in children. J. Clin. Pathol. 23:7–18. 5. Alwan, W. H., W. J. Kozlowska, and P. J. M. Openshaw. 1994. Distinct types of lung disease caused by functional subsets of antiviral T cells. J. Exp. Med. 179:81–89. 6. Alwan, W. H., F. M. Record, and P. J. M. Openshaw. 1992. CD4⫹ T cells clear virus but augment disease in mice infected with respiratory syncytial virus: comparison with the effects of CD8⫹ cells. Clin. Exp. Immunol. 88:527–536. 7. Anderson, C. F., and D. M. Mosser. 2002. Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J. Immunol. 168:3697–3701. 8. Antonis, A. F., R. S. Schrijver, F. Daus, P. J. Steverink, N. Stockhofe, E. J. Hensen, J. P. Langedijk, and R. G. van der Most. 2003. Vaccine-induced

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CLINICAL MICROBIOLOGY REVIEWS, July 2005, p. 556–569 0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.3.556–569.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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Novel Perspectives on Mucormycosis: Pathophysiology, Presentation, and Management Brad Spellberg,1,2* John Edwards, Jr.,1,2 and Ashraf Ibrahim1,2 Department of Medicine, Los Angeles Biomedical Institute at Harbor-UCLA Medical Center, Torrance, California,1 and David Geffen School of Medicine at UCLA, Los Angeles, California2 INTRODUCTION .......................................................................................................................................................556 PATHOGENESIS........................................................................................................................................................556 Host Defenses ..........................................................................................................................................................556 Role of Iron in Pathogenesis.................................................................................................................................557 Fungal-Endothelial Interactions ...........................................................................................................................558 CLINICAL PRESENTATION ...................................................................................................................................558 General Principles ..................................................................................................................................................558 Epidemiology and Disease Manifestations..........................................................................................................558 Rhinocerebral mucormycosis ............................................................................................................................559 Pulmonary mucormycosis ..................................................................................................................................560 Cutaneous mucormycosis...................................................................................................................................560 Gastrointestinal mucormycosis.........................................................................................................................560 Disseminated mucormycosis..............................................................................................................................561 Miscellaneous forms...........................................................................................................................................561 TREATMENT..............................................................................................................................................................561 General Principles ..................................................................................................................................................561 Role of Surgery........................................................................................................................................................562 Antifungal Therapy.................................................................................................................................................562 Polyenes................................................................................................................................................................563 Azoles....................................................................................................................................................................564 Echinocandins .....................................................................................................................................................564 Novel Iron Chelators ..............................................................................................................................................565 Other Adjunctive Therapies ..................................................................................................................................565 PROGNOSIS ...............................................................................................................................................................565 CONCLUSIONS .........................................................................................................................................................565 REFERENCES ............................................................................................................................................................566 clude Rhizopus microsporus var. rhizopodiformis, Absidia corymbifera, Apophysomyces elegans, Mucor species, and Rhizomucor pusillus (61, 81, 129). Increasing cases of mucormycosis have been also reported due to infection with Cunninghamella spp. (in the Cunninghamellaceae family) (24, 78, 82, 161). To date, rare case reports have demonstrated the ability of species belonging to the remaining four families to cause mucormycosis (12, 68, 72, 89, 129).

INTRODUCTION The zygomycoses are infections caused by fungi of the class Zygomycetes, comprised of the orders Mucorales and Entomophthorales. The Entomophthorales are rare causes of subcutaneous and mucocutaneous infections known as entomophthoromycosis, which largely afflict immunocompetent hosts in developing countries. In contrast, fungi of the order Mucorales are causes of mucormycosis, a life-threatening fungal infection almost uniformly affecting immunocompromised hosts in either developing or industrialized countries. Fungi belonging to the order Mucorales are distributed into six families, all of which can cause cutaneous and deep infections (129). Species belonging to the family Mucoraceae are isolated more frequently from patients with mucormycosis than any other family. Among the Mucoraceae, Rhizopus oryzae (Rhizopus arrhizus) is by far the most common cause of infection (129). Other less frequently isolated species of the Mucoraceae family that cause a similar spectrum of infections in-

PATHOGENESIS Host Defenses Both mononuclear and polymorphonuclear phagocytes of normal hosts kill Mucorales by the generation of oxidative metabolites and the cationic peptides defensins (33, 166, 169) (Fig. 1). Clinical evidence demonstrates that these phagocytes are the major host defense mechanism against mucormycosis. For example, neutropenic patients are at increased risk of developing mucormycosis. Furthermore, patients with dysfunctional phagocytes are also at higher risk for developing mucormycosis. Hyperglycemia and acidosis are known to impair the ability of phagocytes to move toward and kill the organisms by both oxidative and nonoxidative mechanisms (23). Additionally, corticosteroid treatment affects the ability of mouse bron-

* Corresponding author. Mailing address: Division of Infect. Dis., Harbor-UCLA Medical Center, 1124 West Carson St. RB2, Torrance, CA 90502. Phone: (310) 222-5381. Fax: (310) 782-2016. E-mail: [email protected] 556

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FIG. 1. Pathogenetic mechanisms of and host defense mechanisms against mucormycosis. To cause disease, the agents of mucormycosis must scavenge from the host sufficient iron for growth, must evade host phagocytic defense mechanisms, and must access vasculature to disseminate. A) In a normal host, primary defense mechanisms against mucormycosis include sequestration of iron in serum by specialized iron-binding proteins (1), phagocytes including circulating neutrophils (2a) and tissue macrophages (2b), and endothelial cells (3), which regulate vascular tone and permeability. Acting in concert, these mechanisms prevent establishment of infection in tissue and subsequent endovascular invasion. B) In susceptible hosts, normal defense mechanisms break down. For example, in diabetic ketoacidosis (DKA), the acidic pH of the serum causes dissociation of free iron from sequestering proteins (1). This release of free iron allows rapid fungal growth. Defects in phagocytic defense mechanisms (2), for example, deficiency in cell number (neutropenia) or functional defects caused by corticosteroids or the hyperglycemia and acidosis of diabetic ketoacidosis, allow proliferation of the fungus. Finally, adherence to and damage of endothelial cells by the fungus (3) allows fungal angioinvasion and vessel thrombosis and subsequent tissue necrosis and dissemination of the fungal infection.

choalveolar macrophages to prevent germination of the spores in vitro or after in vivo infection induced by intranasal inoculation (169). The exact mechanisms by which ketoacidosis, diabetes, or steroids impair the function of these phagocytes remain unknown. Role of Iron in Pathogenesis A recently identified important clinical feature is the increased susceptibility to mucormycosis of patients with elevated available serum iron. It has been known for two decades that patients treated with the iron chelator deferoxamine have a markedly increased incidence of invasive mucormycosis (16). However, it is now clear that iron chelation is not the mechanism by which deferoxamine enables mucormycosis infections. While deferoxamine is an iron chelator from the perspective of the human host, Rhizopus spp. actually utilize deferoxamine as a siderophore to supply previously unavailable iron to the fungus (15, 30). Rhizopus spp. can accumulate 8- and 40-foldgreater amounts of iron supplied by deferoxamine than can Aspergillus fumigatus and Candida albicans, respectively, and this increased iron uptake by Rhizopus spp. is linearly corre-

lated with its growth in serum (15). Additionally, data from animal models emphasize the exceptional requirement of iron for Rhizopus pathogenicity since administration of deferoxamine or free iron worsens survival of animals infected with Rhizopus spp. but not Candida albicans (1, 16, 30, 158). Finally, animal models have demonstrated that other iron chelators, which are not used as siderophores by the fungus, do not similarly exacerbate mucormycosis infection (16). Patients with diabetic ketoacidosis are at high risk of developing rhinocerebral mucormycosis (61, 81, 129). Multiple lines of evidence support the conclusion that patients in systemic acidosis have elevated levels of available serum iron, likely due to release of iron from binding proteins in the presence of acidosis (9). For example, sera collected from patients with diabetic ketoacidosis supported growth of Rhizopus oryzae in the presence of acidic pH (7.3 to 6.88) but not in the presence of alkaline pH (7.78 to 8.38). Acidic sera that supported the growth of R. oryzae were found to contain increased available serum iron (69 ␮g/dl versus 13 ␮g/dl for sera which did not support the growth of R. oryzae). Finally, simulated acidotic conditions decreased the iron-binding capacity of sera collected from normal volunteers, suggesting that acidosis

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temporarily disrupts the capacity of transferrin to bind iron (9). Therefore, the increased susceptibility to mucormycosis of patients with diabetic ketoacidosis is likely due at least in part to an elevation in available serum iron during diabetic ketoacidosis. Fungal-Endothelial Interactions A hallmark of mucormycosis infections is the virtually uniform presence of extensive angioinvasion with resultant vessel thrombosis and tissue necrosis. This angioinvasion is associated with the ability of the organism to hematogenously disseminate from the original site of infection to other target organs. Hence, damage of and penetration through endothelial cells lining blood vessels is likely a critical step in the organism’s pathogenetic strategy. R. oryzae spores but not germlings (i.e., pregerminated spores) have the ability to adhere to subendothelial matrix proteins including laminin and type IV collagen in vitro (17). Similarly, we have recently found that R. oryzae spores adhere to subendothelial matrix proteins significantly better than do R. oryzae hyphae, however spores and hyphae adhere equivalently to human umbilical vein endothelial cells (64). The disparity of spore and germ tube adherence to subendothelial matrix proteins but equivalent adherence to endothelial cells indicates that R. oryzae adhesins to endothelial cells are likely distinct from the adhesins used to bind to subendothelial matrix proteins. We also found that germlings of R. oryzae damage endothelial cells in vitro. This damage is independent of serum factors and requires phagocytosis of R. oryzae by endothelial cells (64). Surprisingly, R. oryzae viability was not required for endothelial cell damage, but phagocytosis was required for dead R. oryzae to cause damage (64). In a subsequent pilot study, intravenous administration of four doses of heat-killed R. oryzae blastospores resulted in a 40% mortality in diabetic mice (unpublished observations). The precise mechanisms by which dead R. oryzae mediates tissue injury remain unclear. Nevertheless, the clinical implication is that simply killing R. oryzae once it has already established a presence in tissue may not prevent subsequent tissue injury, perhaps in part explaining the lack of efficacy of cidal antifungal agents during clinical disease. CLINICAL PRESENTATION General Principles As mentioned earlier, the clinical hallmark of mucormycosis is vascular invasion resulting in thrombosis and tissue infarction/necrosis. Mucormycosis virtually always occurs in patients with defects in host defense and/or with increased available serum iron, although rare cases have been reported in apparently normal hosts (83, 85, 119). In most cases, the infection is relentlessly progressive and results in death unless treatment with a combination of surgical debridement and antifungal therapy is initiated promptly. Epidemiology and Disease Manifestations Mucormuycosis is less common than other opportunistic fungal infections, such as those caused by Candida and Aspergillus spp. One population-based study estimated the incidence of mucormycosis to be 1.7 cases per million people per

CLIN. MICROBIOL. REV. TABLE 1. Relationship between predisposing condition and site of infection Predisposing condition

Diabetic ketoacidosis Neutropenia Corticosteroids Deferoxamine Malnutrition Trauma, catheter/injection site, skin maceration

Predominant site of infection

Rhinocerebral Pulmonary and disseminated Pulmonary, disseminated, or rhinocerebral Disseminated Gastrointestinal Cutaneous/subcutaneous

year, which translates to approximately 500 cases per year in the United States (126). In autopsy series, the prevalence of mucormycosis has ranged from 1 to 5 cases per 10,000 autopsies, making the infection 10- to 50-fold less common than invasive Candida or Aspergillus infections (56, 154, 178). Finally, in patients at higher risk, such as those undergoing allogeneic bone marrow transplantation, the prevalence of mucormycosis has been described to be as high as 2 to 3% (90, 96). Based on clinical presentation and the involvement of a particular anatomic site, mucormycosis can be divided into at least six clinical categories: (i) rhinocerebral, (ii) pulmonary, (iii) cutaneous, (iv) gastrointestinal, (v) disseminated, and (iv) miscellaneous. Of note, these categories of invasive mucormycosis tend to occur in patients with specific defects in host defense (Table 1). For example, diabetics in ketoacidosis typically develop the rhinocerebral form of the disease, and much more rarely develop pulmonary or disseminated disease (75, 99, 107, 118). The mechanism for ketoacidosis preferentially causing susceptibility to the rhinocerebral form of the disease remains unclear. As mentioned earlier, patients in ketoacidosis, or indeed any systemic acidosis, have increased available iron in serum due to dissociation of iron from sequestering proteins in acidic conditions (9). However, the predominant presentation of mucormycosis in the setting of deferoxamine therapy is disseminated disease (37, 46, 123, 151, 160), indicating that increased available iron cannot, by itself, explain the preferential occurrence of rhinocerebral disease in ketoacidosis. Furthermore, while it is known that hyperglycemia and acidosis negatively impact neutrophil chemotaxis and phagocytic activity (23), these observations cannot explain the preferential occurrence of rhinocerebral disease in diabetic ketoacidosis because neutropenic patients more commonly develop pulmonary mucormycosis than rhinocerebral disease (98, 150). Much more readily understood are the risk factors for invasive skin and soft tissue infections caused by the agents of mucormycosis. These infections occur in patients with disrupted cutaneous barriers, as a result of either traumatic implantation of soil, maceration of skin by a moist surface (5, 119), or even via direct access through intravenous catheters or subcutaneous injections (4, 73, 125). These cases illustrate an alarming trend in the epidemiology of mucormycosis. Mucormycosis, formerly virtually always community acquired and often in the setting of diabetic ketoacidosis, is rapidly becoming a nosocomial infection in patients with malignancy or undergoing organ or hematopoietic cell transplantation (108). Given the increasing incidence of diabetes and cancer in the

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increasingly obese and elderly United States population, it is not surprising that a recent review found a marked increase in reported cases of mucormycosis over the last two decades (45). As mentioned, there has also been a shift from community onset to nosocomial onset of disease. Nosocomial mucormycosis has been associated with iatrogenic immunosuppression (79, 108, 130) and a variety of procedures or devices used in hospitals, including antifungal prophylaxis (69, 130), bandages or medication patches (43, 119), intravenous catheters (4, 11, 74, 87), and even tongue depressors (50, 54, 92) (see below). At transplant centers there has also been an alarming rise in the incidence of mucormycosis (69, 140). For example, at the Fred Hutchinson Cancer Center, Marr et al. have described a doubling in the number of cases from 1985 to 1989 to 1995 to 1999 (94). Similarly, Kontoyianis et al. have described a greater than doubling in the incidence of mucormycosis in transplant patients over a similar time span (79). In patients undergoing hematological stem cell transplantation, mucormycosis develops at least as commonly in nonneutropenic periods as in neutropenic periods. For example, two major transplant centers have recently reported that more than half the cases of mucormycosis occurred more than 90 days after transplantation (90, 94). Major risk factors for mucormycosis in the transplant setting include underlying myelodysplastic syndrome (possibly due to iron overload from repeated blood transfusions) and graftversus-host disease treated with steroids (90, 94, 116, 140). Administration of antithymocyte globulin may also pose a risk for mucormycosis (140). Although less than half of these patients are neutropenic at the time of disease onset, prolonged neutropenia is a risk factor for mucormycosis in this setting (130), as are diabetes mellitus and steroid use (130). The role of antifungal prophylaxis in predisposing patients to developing mucormycosis is increasingly being described, as discussed further below. Prophylaxis with either itraconazole (130) or voriconazole (65, 66, 69, 96, 163) has been implicated in predisposing to mucormycosis. Rhinocerebral mucormycosis. Rhinocerebral mucormycosis continues to be the most common form of the disease, accounting for between one-third and one-half of all cases of mucormycosis (122). About 70% of rhinocerebral cases (occasionally referred to as craniofacial) are found in diabetic patients in ketoacidosis (99). More rarely, rhinocerebral mucormycosis has also occurred in patients who received a solid organ transplant or those with prolonged neutropenia (2, 45, 118, 119, 122, 179). Recently, rhinocerebral disease has been an increasing problem in patients undergoing hematopoietic stem cell transplantation (94, 102). These cases have largely been associated with steroid use for graft-versus-host disease. The initial symptoms of rhinocerebral mucormycosis are consistent with either sinusitis or periorbital cellulitis (32, 149) and include eye or facial pain and facial numbness, followed by the onset of conjunctival suffusion, blurry vision, and soft tissue swelling (75, 118, 153). Fever is variable and may be absent in up to half of cases (149); white blood cell counts are typically elevated, as long as the patient has functioning bone marrow (153). If untreated, infection usually spreads from the ethmoid sinus to the orbit, resulting in loss of extraocular muscle function and proptosis. Marked chemosis may also be seen. The infection may rapidly extend into the neighboring tissues. On-

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set of signs and symptoms in the contralateral eye, with resulting bilateral proptosis, chemosis, vision loss, and ophthalmoplegia, is an ominous sign that suggests the development of cavernous sinus thrombosis. Upon visual inspection, infected tissue may appear normal during the earliest stages of spread of the fungus. Infected tissue then progresses through an erythematous phase, with or without edema, before onset of a violaceous appearance, and finally the development of a black, necrotic eschar as the blood vessels become thrombosed and tissue infarction occurs (57, 119). Infection can sometimes extend from the sinuses into the mouth and produce painful, necrotic ulcerations of the hard palate (119). Cranial nerve findings represent extensive infection and signal a grave prognosis. Progressive vision loss and ultimately blindness may result either from involvement of the optic nerve or from arteriolar invasion resulting in infarction (58, 109, 143, 153) or from cavernous sinus thrombosis. Cranial nerves five and seven may also be affected, resulting in ipsilateral loss of facial sensation and ptosis and pupillary dilation (32, 118, 153). Infection can also spread posteriorly from either the orbit or sinuses to the central nervous system. A bloody nasal discharge may be the first sign that infection has invaded through the terbinates and into the brain. When there is extensive central nervous system involvement, the angioinvasive nature of the fungus may result in cavernous sinus thrombosis and internal carotid artery encasement and thrombosis with extensive resulting cerebral infarctions (7, 88, 104, 153). Occasionally cerebral vascular invasion may lead to hematogenous dissemination of the infection (84, 122), with or without development of mycotic aneurysms (135). A high index of suspicion is required to make the diagnosis of rhinocerebral mucormycosis, as evidenced by the fact that autopsy series have found up to half of cases are diagnosed postmortem (78, 101, 154). Imaging techniques may be suggestive of mucormycosis but are rarely diagnostic. Indeed, the initial imaging study is frequently negative or has only subtle findings. The most common finding on computerized tomography (CT) scanning of the head or sinuses is subtle sinus mucosal thickening or thickening of the extraocular muscles. It is also common to detect no abnormalities in the bones of the sinuses despite clinical evidence of progressive disease (149). However, when present, the finding of bony erosion of the sinuses is strongly suggestive of the diagnosis in the appropriate clinical context (e.g., patient in diabetic ketoacidosis with proptosis). It should be emphasized that it is very uncommon to visualize an organized retroorbital mass. Although evidence of infection of the soft tissues of the orbit may sometimes be seen by CT scan, magnetic resonance imaging is more sensitive (40). Still, as with CT scans, patients with early rhinocerebral mucormycosis may have a normal magnetic resonance imaging, and surgical exploration with biopsy of the areas of suspected infection should always be performed in high-risk patients. It is critically important to emphasize that if mucormycosis is suspected, initial empirical therapy with a polyene antifungal should begin while the diagnosis is being confirmed, rather than waiting while a protracted series of diagnostic tests are completed. Given the limitations of imaging studies, diagnosing mucormycosis almost always requires histopathologic evidence of fungal invasion of the tissues. Culturing organisms from a

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potentially infected site is rarely sufficient to establish the diagnosis of mucormycosis because the causative agent is ubiquitous, may colonize normal persons, and is a relatively frequent laboratory contaminant. Additionally, the organism may be killed during tissue grinding (168), which is routinely used to process tissue specimens for culture. Thus, a sterile culture does not rule out the infection (149). Furthermore, waiting for the results of the fungal culture may delay the institution of appropriate therapy. There are no reliable serologic, PCR-based, or skin tests for mucormycosis. Therefore, the diagnosis should be made by biopsy of infected tissues. The biopsy should demonstrate the characteristic wide, ribbon-like, aseptate hyphal elements that branch at right angles. The organisms are often surrounded by extensive necrotic debris. Other fungi, including Aspergillus, Fusarium, or Scedosporium spp, may look similar to the Mucorales on biopsy. However, these molds have septae, are usually thinner, and branch at acute angles. The genus and species of the infecting organism may be determined by culture of the infected tissue. However, the organism is rarely isolated from cultures of blood, cerebrospinal fluid, sputum, urine, feces or swabs of infected areas. Pulmonary mucormycosis. Mucormycosis of the lung occurs most commonly in leukemic patients who are receiving chemotherapy or in patients undergoing hematopoietic stem cell transplants. Indeed, the pulmonary form of the disease is the most common form found in neutropenic and stem cell-transplant patients (94, 102). These patients typically have severe neutropenia and are frequently receiving broad-spectrum antibiotics for unremitting fever (150), while in patients undergoing allogeneic stem cell transplantation, the disease often occurs postengraftment and is strongly associated with graftversus-host disease. Patients with diabetic ketoacidosis can also develop pulmonary mucormycosis, although infections in these patients are less common and less fulminant and follow a more subacute course than is typically seen in patients with neutropenia (132, 150). Pulmonary mucormycosis may develop as a result of inhalation or by hematogenous or lymphatic spread. Symptoms of pulmonary mucormycosis include dyspnea, cough, and chest pain (150). In a recent series of 32 cases of pulmonary mucormycosis, fever was present in the majority of patients (150). Angioinvasion results in necrosis of tissue parenchyma, which may ultimately lead to cavitation and/or hemoptysis, which may be fatal if a major blood vessel is involved (49, 171). Radiographically, a variety of findings may be present, including, in descending order of frequency: lobar consolidation, isolated masses, nodular disease, and cavitation (70, 98, 150). Wedge-shaped infarcts of the lung may also be seen, particularly following thrombosis of the pulmonary vessels due to fungal angioinvasion (93). High-resolution chest CT scan is the best method of determining the extent of pulmonary mucormycosis and may demonstrate evidence of infection before it is seen on the chest x-ray. One suggestive finding is expansion of a mass or consolidation across tissue planes, in particular towards the great vessels in the mediastinum (127). Unfortunately, sputum culture is highly unreliable. In two case series, sputum and bronchiolar alveolar lavage cultures were negative

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in 18 of 19 cases of biopsy-proven pulmonary mucormycosis (127, 150). Therefore, biopsy with histopathological assessment remains the best modality to diagnose pulmonary mucormycosis. If pulmonary infection is not treated, hematogenous dissemination to the contralateral lung and other organs frequently occurs. Patients with untreated pulmonary mucormycosis usually die from disseminated disease before respiratory failure occurs. The notable exception is the rare patient with massive hemoptysis (103, 113). The overall mortality of pulmonary mucormycosis is approximately 50 to 70% but is ⬎95% if the pulmonary mucormycosis is part of a disseminated process (45, 150). Cutaneous mucormycosis. As mentioned, patients who are at high risk of developing cutaneous mucormycosis are those with disruption of the normal protective cutaneous barrier. The agents of mucormycosis are typically incapable of penetrating intact skin. However, burns, traumatic disruption of skin, and persistent maceration of skin enable the organisms to penetrate into deeper tissues. A typical case results from traumatic implantation of soil, for example, as a result of a motor vehicle accident or penetrating injury with plant material (e.g., a thorn) (4, 119). In diabetic and immunocompromised patients, cutaneous lesions may also arise at insulin injection or catheter insertion sites (73, 125). Contaminated surgical dressings have also been implicated as a source of cutaneous mucormycosis (43, 100). Cutaneous mucormycosis has also occurred in the context of contaminated tape used to secure an endotracheal tube in a ventilated patient (5). Cutaneous disease can be very invasive locally and penetrate from the cutaneous and subcutaneous tissues into the adjacent fat, muscle, fascia, and even bone. Secondary vascular invasion may also lead to hematogenously disseminated infection of the deep organs. Cutaneous and subcutaneous disease may lead to necrotizing fasciitis, which has a mortality approaching 80% (18, 80, 115, 124). However, isolated cutaneous mucormycosis (i.e., not disseminated disease) has a favorable prognosis and a low mortality if aggressive surgical debridement is done promptly (4). Gastrointestinal mucormycosis. Mucormycosis of the gastrointestinal tract is rare. It mainly occurs in patients who are extremely malnourished (especially infants or children) and is thought to arise from ingestion of the fungi. In particular, gastrointestinal mucormycosis has been seen in premature neonates, often in association with widespread disseminated disease (6, 27, 71, 76, 128, 137). Necrotizing enterocolitis has been described largely in premature neonates (35, 71, 106, 128, 139, 157, 174, 177) and more rarely in neutropenic adults (146, 152). Rare cases of gastrointestinal mucormycosis have been described in association with other immune-compromising conditions, including AIDS (19), systemic lupus erythematosus (55), and organ transplantation (77, 95, 97, 138). The stomach, colon, and ileum are the most commonly involved sites. Cases of hepatic mucormycosis have also been associated with ingestion of herbal medications (111). Because this infection is acute and rapidly fatal, it is often diagnosed postmortem. The symptoms are varied and depend on the site affected. Nonspecific abdominal pain and distention associated with nausea and vomiting are the most common symptoms. Fever and hematochezia may also occur. The patient is often

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thought to have an intra-abdominal abscess. The diagnosis may be made by biopsy of the suspected area during surgery or endoscopy. Recently, a iatrogenic outbreak of gastric mucormycosis occurred due to contamination of the wooden applicators used to mix drugs that were poured down the patients’ nasogastric feeding tubes (92). These patients presented with massive gastric bleeds. The diagnosis was made by culture of gastric aspirates and culture of the box of wooden tongue depressors. This experience further underscores the alarming trend of increasing iatrogenic/nosocomial onset for mucormycosis. Disseminated mucormycosis. Hematogenously disseminated mucormycosis may originate from any primary site of infection. Pulmonary mucormycosis in severely neutropenic patients has the highest incidence of dissemination. Less commonly, dissemination can arise from the gastrointestinal tract, the sinuses, or cutaneous lesions, the last occurring particularly in burn patients. The most common site of dissemination is the brain, but metastatic lesions may also be found in the spleen, heart, skin, and other organs. Cerebral infection following dissemination is distinct from rhinocerebral mucormycosis and results in abscess formation and infarction. Patients present with sudden onset of focal neurological deficits or coma. The mortality associated with dissemination to the brain approaches 100% (144). Even without central nervous system involvement, disseminated mucormycosis has a mortality of ⬎90% (45). In patients undergoing hematopoietic stem cell transplantation, the 1-year mortality is ⬎95% due to a combination of underlying disease, graft-versus-host disease, and the infection (57, 94). Recent case series have described frequent dissemination in the context of voriconazole prophylaxis of transplant patients. In five recent case series, a total of 18 cases of disseminated mucormycosis have occurred in patients post-allogeneic hematopoietic stem cell transplantation who were receiving voriconazole either prophylactically or therapeutically for other infections (65, 66, 96, 140, 163). An additional patient who had leukemia and was treated with combination voriconazole plus caspofungin for proven Aspergillus pneumonia, who subsequently developed disseminated mucormycosis, has been described (13). Finally, three patients who developed mucormycosis while receiving voriconazole for empirical neutropenic fever (not in the transplant setting) or for prophylaxis postrenal transplantation have been described (112, 163). It has been pointed out that the increase in frequency of mucormycosis in transplant patients preceded the availability of voriconazole and that therefore the precise role of voriconazole in predisposing patients to mucormycosis is unclear (69). For example, increasingly intensive immunosuppressive regimens and broader availability of allogeneic transplantation (e.g., to patients of increasing age) may be playing a role. Furthermore, the increasing use of peripheral stem cell transplants (in lieu of bone marrow-derived cells), nonmyeloablative conditioning regimens, and unrelated donor and/or HLA-mismatched transplants have increased the incidence of graftversus-host disease. As mentioned, graft-versus-host disease and its treatment with corticosteroids are strongly linked with the risk of mucormycosis in the transplant setting (90, 94, 116, 140). Indeed, most of the reported cases of mucormycosis

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occurring during voriconazole therapy have occurred in patients receiving corticosteroids for graft-versus-host disease (66, 96, 140). Nevertheless, the uniformity of the reports of mucormycosis in patients receiving voriconazole infections implicates a link between the drug and the disease. Voriconazole has broad activity against Aspergillus, Candida, and Scedosporium spp. and the dematiaceous fungi, but has no clinically relevant activity against the agents of mucormycosis (34, 117, 120, 147). Therefore, it is possible that the predisposition to mucormycosis is due to selective inhibition of other fungi, which allows the agents of mucormycosis to colonize the patient. As well, it is also possible that voriconazole is preventing early-onset deadly infections caused by other species of fungi (i.e., Candida and Aspergillus), thereby allowing highly immunocompromised patients, who in the past would have died earlier posttransplant, to live long enough to become infected with the agents of mucormycosis. It should be reiterated that a similar phenomenon has been described with itraconazole prophylaxis, the use of which is also an independent risk factor for development of mucormycosis in this setting (130). Finally, there have been two case reports of breakthrough mucormycosis in patients receiving either caspofungin (133) or caspofungin plus voriconazole (13) for other infections. The diagnosis of disseminated disease is difficult because patients are usually severely ill from multiple diseases and virtually always have negative blood cultures. If there is evidence of infarction in multiple organs, the diagnosis of mucormycosis should be considered. However, aspergillosis is commonly associated with an identical clinical picture. When disseminated mucormycosis is suspected, a careful search should be made for cutaneous lesions that can be biopsied for diagnostic purposes. Miscellaneous forms. Agents of the Mucorales may cause infection in virtually any body site. Brain involvement in the absence of sinus infection, endocarditis, and pyelonephritis occur occasionally, mainly in intravenous drug abusers (156, 162, 164, 176). Other reports have described mucormycosis in bones (91, 121), mediastinum (25, 86), trachea (8, 175), kidneys (172), and peritoneum associated with dialysis (4, 136). Other unusual forms of infection include superior vena cava syndrome (51) and external otitis (110). Although mucormycosis is not commonly seen in AIDS patients, there have been a number of case reports of this infection in this patient population (41, 105, 134). TREATMENT General Principles Four factors are critical for eradicating mucormycosis: rapidity of diagnosis, reversal of the underlying predisposing factors (if possible), appropriate surgical debridement of infected tissue, and appropriate antifungal therapy. Early diagnosis is important because small, focal lesions can often be surgically excised before they progress to involve critical structures or disseminate (107). Unfortunately, there are no serologic or PCR-based tests to allow rapid diagnosis. As mentioned, autopsy series have reported that up to half the cases of mucormycosis are diagnosed postmortem (78, 101, 154), un-

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derscoring the critical need to maintain a high index of clinical suspicion and to aggressively pursue diagnostic biopsy. Correcting or controlling predisposing problems is also essential for improving the treatment outcome. In diabetic ketoacidotic patients, hyperglycemia and acidemia should be corrected. Discontinuation of deferoxamine or immunosuppressive therapy, particularly steroids, should be strongly considered when the diagnosis of mucormycosis is made. Given the rapidly progressive nature of rhinocerebral mucormycosis and the marked increase in mortality when the fungus penetrates the cranium, any diabetic patient with a headache and visual changes is a candidate for prompt evaluation with imaging studies and nasal endoscopy to rule out mucormycosis. Furthermore, to reiterate a concept that is frequently poorly grasped by clinicians inexperienced with mucormycosis, the initial imaging study is frequently negative or has subtle findings. Radiographic findings lag behind clinical progression in this disease, and a negative imaging study does not provide a rationale to delay more aggressive diagnostic maneuvers (e.g., endoscopy with biopsy) if clinical suspicion is high. The appearance of tissue at endoscopy may also lag behind invasion, as the mucosa can appear pink and viable during the initial phase of fungal invasion. Therefore, if the suspicion for disease is high, blind biopsies of sinus mucosa and/or thickened extraocular muscles are warranted to make the diagnosis. Finally, time is of the essence in the management of mucormycosis. Because patients with rhinocerebral disease may initially present with normal mental status and appear clinically stable, the urgency for establishing the diagnosis is frequently underappreciated. The key concept is that initial spread of the fungus to the brain may be relatively asymptomatic. Once the fungus has penetrated the cranium or entered the major intracranial vasculature, mortality increases substantially. Additionally, starting the patient on an antifungal is not definitive therapy, since surgery may be a key addition to the treatment strategy. The sensitivity of the organisms varies considerably, so that a patient on amphotericin B alone may be receiving completely ineffective therapy during the diagnostic period. Minutes and hours count, and if the clinical suspicion is high, the workup should proceed on an emergent basis even if the patient currently appears clinically stable. Indeed, delayed diagnosis has been associated with a dramatically worse outcome (75). One strategy to expedite the workup is to rely upon frozen sections to guide further diagnostic and therapeutic decisions rather than waiting for fixed and stained histopathology from a biopsy. Use of frozen sections in this setting has been shown to shorten the time to diagnosis and has been associated with improved outcomes in two recent case series (48, 53). Role of Surgery Mucormycosis is frequently rapidly progressive, and antifungal therapy alone is often inadequate to control the infection. The numerous agents of mucormycosis have a broad range of susceptibilities to antifungal agents; some strains may be highly resistant to amphotericin B. Furthermore, the hallmark angioinvasion, thrombosis, and tissue necrosis of this disease result in poor penetration of anti-infective agents to the site of in-

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fection. Therefore, even if the causative organism is susceptible to the treating antifungal agent in vitro, the antifungal may be ineffective in vivo. Finally, surgery is necessary due to the massive amount of tissue necrosis occurring during mucormycosis, which may not be prevented by killing the organism (63). Surgical debridement of infected and necrotic tissue should be performed on an urgent basis. In rhinocerebral mucormycosis, early surgical excision of the infected sinuses and appropriate debridement of the retroorbital space can often prevent the infection from extending into the eye, thereby obviating the need for enucleation and resulting in extremely high cure rates (⬎85%) (107). Repeated surgical exploration of the sinuses and orbit may be necessary to ensure that all necrotic tissue has been debrided and the infection has not progressed. Published case series continue to support the need for surgical debridement to optimize outcomes. For example, in a case series totaling 49 patients with rhinocerebral mucormycosis, the mortality was 70% in cases treated with antifungal agents alone versus 14% in cases treated with antifungal agents plus surgery (75, 118). Similarly, in a combined series of rhinocerebral, cutaneous, and pulmonary mucormycosis, 11 of 17 (65%) patients treated with surgery plus antifungal agents survived the infection, compared to zero of seven (0%) patients treated with antifungal agents alone (119). Clearly there is the potential for selection bias in these case series, as patients who do not undergo surgery may have fundamental differences in severity of illness or comorbidities. Nevertheless, the observational clinical data support the concept that surgical debridement is necessary to optimize cure rates. In patients with pulmonary mucormycosis, surgical treatment plus antifungal therapy also greatly improves outcome compared to the use of antifungal therapy alone (10, 79, 116, 127, 150). In one series, the mortality of patients treated with antifungal agents alone was 68%, versus 11% in patients treated with antifungal agents plus surgery (150). Finally, localized (nondisseminated) cutaneous mucormycosis treated with aggressive surgical debridement and adjunctive antifungal therapy has a mortality of ⬍10% (4, 73). A similar experience has been described with isolated renal mucormycosis (172). However, because surgical debridement of necrotic tissue is frequently highly disfiguring, if the patient survives the acute phase of the disease, major reconstructive surgery may be necessary.

Antifungal Therapy A major obstacle for clinicians to choose among the current available antifungal agents in treating mucormycosis is the lack of available clinical trials (Table 2). Prospective interventional study of mucormycosis has been impractical for several reasons. First, although the disease is unusually deadly, it occurs at a lower frequency relative to other opportunistic infections. By extrapolation from studies of Aspergillus infection (52), which is more common, dozens to possibly even hundreds of trial sites and multiple years would be required to accrue sufficient patients to adequately power a standard phase III superiority study. Such a study would undoubtedly cost tens of millions of dollars, and mucormycosis cases represent an insufficient po-

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TABLE 2. Antifungal strategies for mucormycosis Therapy

Antifungal

Pros

Cons

Amphotericin B deoxycholate (AmB)

50 years experience Cidal Less toxic than AmB Improved CNS penetration (47) High-dose LAmB (15 mg/kg/day) superior to AmB (1 mg/kg/day) in murine model (59) Superior to AmB in retrospective clinical study (45) Less toxic than AmB

Toxicity Resistance seen in individual isolates Most expensive polyene

Itraconazole

Superior toxicity profile Successful case reports (36, 125)

Posaconazole

More effective than itraconazole in animal models (28, 148) Successful case reports (65, 155) Possible combination with polyene therapy, but no data available Very low toxicity

Poor activity in animal models despite in vitro susceptibility (28, 29, 159) Breakthrough mucormycosis described during prophylactic itraconazole (130) Not yet FDA approved

Established therapies Liposomal amphotericin B (LAmB)

Amphotericin B lipid complex (ABLC)

Inferior CNS penetration vs. LAmB in one rabbit study (47) Not superior to placebo or AmB in murine model even at high doses (up to 30 mg/kg/day) (62, 141) No comparative clinical data published

Investigational/adjunctive therapies

Caspofungin

Iron chelation Hyperbaric oxygen Cytokine therapy

Synergistic with ABLC in murine model (141) FDA approved (not for mucormycosis) Theoretical benefit in combination with antifungals Nontoxic Successful case reports (22, 42) In vitro activity (44) Successful case reports (3)

tential market to spur any pharmaceutical company to sponsor such a study. An additional barrier to clinical trials of mucormycosis is the abysmal rate of success of monotherapy. Because of this low success rate, it might be considered unethical to randomize patients in a clinical trial to any “less intensive” regimen (i.e., standard-dose versus high-dose monotherapy, monotherapy versus combination therapy, etc.). For these reasons, prospective interventional trials have not been performed. Lacking any significant clinical trial data, physicians have been forced to rely upon anecdotal case reports, limited retrospective reviews, and unpublished observations in determining the first-line therapy for mucormycosis. Such reports are intrinsically subject to publication and observer bias and allow no comparison of the relative efficacies of various treatment strategies. For these reasons, animal models of mucormycosis are essential to provide well-controlled comparative analyses of antifungal therapies.

Static in vitro Activity inferior to AmB in murine models (28, 148) Virtually no clinical data for mucormycosis Minimal activity as monotherapy in murine model (60) No data available No effective agents are FDA approved Not widely available No controlled studies Limited data Expensive Toxicity profile unclear

Several murine models have been developed to study mucormycosis in vivo, including intravenous (59), intranasal (169), and intrasinus (167) diabetic mice models. Additionally, neutropenic (148), corticosteroid- (169), and deferoxamine-treated mouse models (1) and a deferoxamine-treated guinea pig model (15, 16) have been reported. More rarely, immunocompetent mice have been studied (28). A variety of species have been utilized in these models, including R. oryzae, R. microsporus, and Mucor and Absidia spp. There is no clear advantage to any one of these models in evaluating the efficacy of different antifungal regimens, and none of the models completely accurately recapitulates the normal route of infection (inhalation) of the majority of mucormycosis infections. Nevertheless, given the lack of controlled clinical trials for mucormycosis, these models are essential to evaluating the relative merits of different antifungal strategies. Polyenes. There have been no prospective randomized trials to define the optimal antifungal therapy for mucormycosis. Until recently, only members of the polyene class, including

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amphotericin B deoxycholate and its lipid derivatives, had been demonstrated to have activity against the agents of mucormycosis. Furthermore, the various species that cause mucormycosis have a broad range of susceptibilities to amphotericin. Therefore, the recommended dose of amphotericin B deoxycholate has been 1 to 1.5 mg/kg/day (61, 81, 145), which results in a very high toxicity rate. Unfortunately, given the underdeveloped state of the molecular biology of the Mucorales, virtually nothing is known about the mechanisms of drug resistance in these organisms. The molecular basis of drug resistance in these organisms is an area that is highly meritorious of future research. Fortunately, a series of new therapies that have the potential to impact the outcomes of mucormycosis have or may soon become available. The lipid formulations of amphotericin are significantly less nephrotoxic than amphotericin B deoxycholate and can be safely administered at higher doses for a longer period of time. However, the use of increased dosing for lipid-based amphotericin also increases costs enormously. For example, in contrast to US$5 per day for a 1-mg/kg daily dose of amphotericin B deoxycholate, 5 to 15 mg/kg of lipid-based amphotericins can cost between US$500 and US$3,000 per day (142). Nevertheless, several case reports and case series of patients with mucormycosis have documented successful outcomes with either liposomal amphotericin B or amphotericin B lipid complex (21, 38, 170, 173). In our murine model of disseminated R. oryzae infection in mice in diabetic ketoacidosis, high-dose liposomal amphotericin B (15 mg/kg/day) was considerably more effective than amphotericin B deoxycholate (1 mg/kg/day), nearly doubling the survival rate (59). Further in support of the first-line role of liposomal amphotericin are the results of a recent retrospective review of 120 cases of mucormycosis in patients with hematological malignancies (45). Treatment with liposomal amphotericin was associated with a 67% survival rate, compared to 39% survival when patients were treated with amphotericin B deoxycholate (P ⫽ 0.02, ␹2). Given the retrospective nature of this study, there is clear potential for several types of bias to affect the outcome. Nevertheless, based on the combination of these retrospective clinical data, the historically poor success rates with amphotericin B deoxycholate, and the available animal data showing superiority of liposomal amphotericin B over amphotericin B deoxycholate, there is a developing consensus that high doses of lipid formulation amphotericin are the preferred initial antifungal therapy for patients with mucormycosis. Recent data are useful for guiding the choice of liposomal amphotericin B versus amphotericin B lipid complex. A study in rabbits (47) demonstrated that liposomal amphotericin B penetrated brain parenchyma at levels more than fivefold above those of amphotericin B lipid complex. In fact, the brain levels of amphotericin B lipid complex were lower than the levels of amphotericin B deoxycholate, despite the fact that amphotericin B lipid complex was administered at a fivefoldhigher dose. Furthermore, in contrast to liposomal amphotericin B, amphotericin B lipid complex (5, 20, or 30 mg/kg/day) did not improve survival compared to placebo or amphotericin B deoxycholate in our murine model of disseminated R. oryzae infection (62, 141). Finally, in contrast to the recent review of

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the effect of liposomal amphotericin B in clinical mucormycosis, no comparable data set has been published reviewing the effect of amphotericin B lipid complex in this setting. Until direct comparisons of the efficacy of liposomal amphotericin B versus amphotericin B lipid complex are published, definitive conclusions regarding their relative efficacies for mucormycosis cannot be made. For now, the pharmacokinetic data, animal model data, and retrospective clinical data all support the first-line use of high-dose liposomal amphotericin B for mucormycosis, particularly for cases of central nervous system disease, with amphotericin B lipid complex serving as a reasonable second-line agent. Therefore, a rational approach to the treatment of life-threatening mucormycosis infections is emergent surgical consultation followed by immediate initiation of liposomal amphotericin B at 10 to 15 mg/kg/day. Azoles. Itraconazole is the only marketed azole drug that has in vitro activity against Mucorales (147). There are case reports of successful therapy with itraconazole alone (36, 125). However, as mentioned above, itraconazole prophylaxis has been described as a risk factor for breakthrough mucormycosis (130). Furthermore, animal studies revealed that itraconazole was completely ineffective against Rhizopus and Mucor spp. even though the isolates were susceptible in vitro (28, 29, 159). In contrast, itraconazole did have activity in vivo against a hypersusceptible strain of Absidia (MIC, 0.03 ␮g/ml). Therefore, itraconazole should not be considered a first-line agent against mucormycosis, but its use may be considered as adjunctive therapy in selected situations where highly susceptible fungi have been cultured. Voriconazole, a recently approved second-generation broad-spectrum triazole, is not active against the Mucorales in vitro (147). Conversely, posaconazole and ravuconazole, investigational triazoles, have promising in vitro activity against the agents of mucormycosis (120, 147). In experimental animal models of disseminated mucormycosis, posaconazole is more efficacious than itraconazole but less efficacious than amphotericin B deoxycholate (28, 148). There are increasing reports of salvage posaconazole therapy for refractory mucormycosis. Successful outcomes have been seen in patients with rhinocerebral mucormycosis in conjunction with amphotericin (65), and in a heart/kidney transplant patient who failed on amphotericin therapy (155). Further data are needed to determine whether posaconazole, alone or in combination with amphotericin, may be useful for the treatment of mucormycosis. Echinocandins. Caspofungin, the first member of the novel echinocandin class of antifungal drugs to be marketed in the United States, has minimal activity against the agents of mucormycosis when tested in vitro by standard techniques (31, 39). However, the accuracy of current in vitro testing of caspofungin activity against molds remains unclear. It is now known that R. oryzae expresses the target enzyme for caspofungin (60), and in the murine model of disseminated mucormycosis, caspofungin did have limited activity against R. oryzae (60). Furthermore, we found that the combination of caspofungin (1 mg/kg/day) plus amphotericin B lipid complex (5 mg/kg/day) was synergistic (141). While either therapy alone mediated no survival benefit, the combination significantly improved survival (50% survival for the combination versus 0% for placebo, caspofungin alone, or amphotericin B lipid complex alone). Clinical experience with caspofungin in the setting of mu-

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cormycosis is extremely limited. In a report of a patient with necrotizing pancreatitis and abdominal mucormycosis, the addition of caspofungin to liposomal amphotericin did not improve the outcome, but the patient had already clinically progressed on liposomal amphotericin prior to initiation of caspofungin (165). As mentioned, there have also been reports of breakthrough mucormycosis in patients receiving either caspofungin alone (133) or caspofungin plus voriconazole (13). Conversely, the related experimental echinocandin micafungin has been added on a salvage basis to a patient failing antifungal therapy for craniofacial mucormycosis (67). The patient began responding to therapy shortly after the addition of micafungin and was ultimately cured. These data suggest that echinocandins may have a role as a second agent, especially in combination with a polyene, in serious cases of mucormycosis. More study of the utility of echinocandins in this setting is needed. Novel Iron Chelators The central role of iron metabolism in the pathogenesis of mucormycosis suggests the possibility of utilizing effective iron chelators as adjunctive antifungal therapy. In fact, two experimental iron chelators have been studied in vitro against R. oryzae (16). In contrast to deferoxamine, the other iron chelators did not allow the organism to take up iron and did not support its growth in vitro in the presence of iron. Furthermore, while deferoxamine significantly worsened disseminated R. oryzae infection in guinea pigs, one of the other chelators had no impact on the in vivo infection and one of them more than doubled the mean survival time (16). The latter agent is approved for use in India and Europe and is available on a compassionate-use basis for iron overload in the United States and Canada. The potential for this iron chelator to serve as adjunctive therapy in combination with other antifungal agents is under active investigation. Other Adjunctive Therapies Case reports have suggested that hyperbaric oxygen may be a beneficial adjunct to the standard surgical and medical antifungal therapy of mucormycosis, particularly for patients with rhinocerebral disease (22, 42). It is hypothesized that hyperbaric oxygen might be useful for treating mucormycosis in conjunction with standard therapy because higher oxygen pressure improves the ability of neutrophils to kill the organism (26). Additionally, high oxygen pressure inhibits the germination of fungal spores and growth of mycelia in vitro (131). Whether hyperbaric oxygen actually improves the outcome of patients with mucormycosis remains to be established through appropriately controlled prospective clinical trials. The role of adjunctive cytokine therapy for mucormycosis has been understudied. Cytokines that activated phagocytic activity, such as gamma interferon and granulocyte-macrophage colony-stimulating factor, increase the ability of phagocytes to kill agents of mucormycosis in vitro (44). A recent case report suggested a favorable outcome in a leukemic child with rhinocerebral mucormycosis following the addition of gamma interferon and granulocyte-macrophage colony-stimulating factor to the regimen (3). Further studies of cytokines that activate host phagocyte function are warranted for this disease.

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PROGNOSIS Previously, cases of rhinocerebral mucormycosis were almost consistently fatal (84). Although the mortality rate of rhinocerebral disease remains high, the infection can be cured when diagnosed early and treated with aggressive surgery and antifungal agents (discussed further below) (20, 114, 119). Recent series have described a mortality of approximately 40% in diabetics with rhinocerebral mucormycosis (107, 118) and a similar survival rate for rhinocerebral disease in patients with hematological malignancies (45). Of note, the prognosis is much better if the disease has not penetrated beyond the sinus prior to surgical debridement; in local sinonasal disease, the mortality has been reported to be ⬍10% (107). The nature of the underlying disease and the reversibility of the immune dysfunction are also important determinants of survival. One study showed that 75% of patients with rhinocerebral disease who had no underlying immune compromise survived, while 60% of those with diabetes and only 20% of patients with other immunocompromised states were cured (14). The overall survival rate of patients with mucormycosis is approximately 50%, although survival rates of up to 85% have been reported more recently. Much of the variability in outcome is due to the various forms of the disease. Rhinocerebral mucormycosis has a higher survival rate than does pulmonary or disseminated mucormycosis because the rhinocerebral disease can frequently be diagnosed earlier and the most common underlying cause, diabetic ketoacidosis, can be treated readily (114). In contrast, pulmonary mucormycosis has a high mortality (⬇65% at 1 year) (94) because it is difficult to diagnose and it frequently occurs in neutropenic patients. For example, in one large study, only 44% with pulmonary mucormycosis were diagnosed premortem, and the overall survival rate was only 20% (150). In a separate study in which 93% of the infections were diagnosed premortem, the survival rate was 73% (114). Mortality in patients with disseminated disease approaches 100%, in large part because surgical removal of infected tissues is not feasible and in part because these patients tend to be the most highly immunocompromised (e.g., allogeneic stem cell transplantation). CONCLUSIONS Mucormycosis is an increasingly common infection in immunocompromised patients. The central role of iron in the organism’s pathogenesis has only recently been appreciated. The interaction between the Mucorales and endothelial cells is also beginning to be understood. Both of these pathogenetic features of disease may be amenable to novel therapeutic intervention in the future. Currently, novel regimens for the treatment of mucormycosis include combination lipid-based amphotericin plus either an echinocandin or itraconazole or both. As well, compassionate-use posaconazole is currently available, and its potential for combination therapy with a polyene, caspofungin, or both is meritorious for study. In the future, novel iron chelator therapy may be useful as an adjunct to standard antifungal therapy. Finally, prompt diagnosis, reversal of predisposing conditions, and aggressive surgical debridement remain cornerstones of therapy for this deadly disease.

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