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Single Toxin Detection Is Inadequate to Diagnose Clostridium difficile Diarrhea in Pediatric Patients HOWARD A. KADER,* DAVID A. PICCOLI,* ABBAS F. JAWAD,‡ KARIN L. MCGOWAN,§ and ERIC S. MALLER* Divisions of *Gastroenterology and Nutrition, ‡Biostatistics and Epidemiology, and §Infectious Disease, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

Background & Aims: Clostridium difficile is an important cause of symptomatic diarrhea in pediatric patients. The bacterium produces two toxins, although many laboratories assay for only one. We questioned this diagnostic approach when patients had positive results for C. difficile at our institution, but initially had tested negative at outside laboratories. Methods: We retrospectively analyzed relative frequencies of C. difficile toxin A alone, toxin B alone, and toxins A and B from pediatric patients with diarrhea. Results were stratified according to toxin detection and patient age. Results: Of 1061 specimens, 276 (26.8%) were positive for C. difficile toxin(s). Fifty-one (18.5%) were positive for toxin A alone, 133 (48.2%) for toxin B alone, and 92 (33.3%) for both toxins. Assaying for toxin B identified C. difficile infection more frequently than did assaying for toxin A (P F 0.0001). The frequency of toxin B detection was significantly higher for older children but not for infants. Conclusions: Testing for C. difficile toxin A or toxin B alone will result in more frequent misdiagnosis than testing for both toxins. This practice may lead to inappropriate further invasive investigations in children, although this finding may not be applicable to adults.

lostridium difficile is a common and important pathogen causing symptomatic diarrhea in pediatric patients.1 The bacterium produces two toxins that are involved in the pathogenesis of this diarrhea. Toxin A is believed to be the primary pathogenic toxin involved in clinical symptoms.2,3 To detect toxin A, an enzymelinked immunosorbant assay (ELISA) specific for the toxin is commonly used. ELISA provides inexpensive, sensitive (71%–99%), and specific (91%–100%) noninvasive means of identifying toxin A in stool specimens.4–9 However, toxin A ELISAs were validated in clinical laboratories by comparison of toxin A detection with the accepted toxin B tissue culture assay. This comparison assumes that toxin A and toxin B are produced equally in every C. difficile infection.10–12 Toxin B detection through cytotoxicity testing has a reported sensitivity of 67%–

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99% and a specificity of 95%–99% but is more expensive and labor intensive and requires 48 hours to complete.8,13 Toxin B detection by ELISA, although commercially available, still has poor sensitivity and specificity. Many hospital laboratories do not perform assays for both toxins on submitted stool specimens. The primary pathogenic C. difficile toxin remains controversial. The American Academy of Pediatrics (AAP) 1997 Red Book: Report of the Committee on Infectious Diseases addresses this issue in its recommendation to test for C. difficile toxin B alone or for both toxin A and toxin B. Our institution routinely tests all stool specimens for both C. difficile toxins. We sought to determine the frequency of C. difficile toxin A alone, toxin B alone, and both toxins A and B in the stool specimens of our symptomatic pediatric patients.

Materials and Methods We retrospectively analyzed results of all stool specimens submitted to The Children’s Hospital of Philadelphia’s laboratory for detection of C. difficile toxins A and B over a 20-month period from January 1, 1996, through August 31, 1997. Stool samples that were solid, not frozen if collected outside the hospital, or not placed on ice if collected in the hospital were not accepted by our laboratory. C. difficile toxin A was detected by Premier EIA (Meridian Diagnostics, Cincinnati, OH) and C. difficile toxin B by the Biowhittaker (Walkersville, MD) tissue culture assay. The Premier EIA has a 93% sensitivity, 100% specificity, 100% positive predictive value, and 99% negative predictive value.4 The Biowhittaker tissue culture assay has a 99% sensitivity and 99% specificity.13 The same toxin assays were used throughout the study period. Multiple stool specimens with either negative or positive results from the same patient were considered separately if they were collected more than 3 weeks apart, to allow for treatment response. We identified the overall percentage of C. difficile toxin–positive stool samples and stratified the results based on Abbreviations used in this paper: AAP, American Academy of Pediatrics; ELISA, enzyme-linked immunosorbent assay; NK-1, neurokinin 1. r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

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the toxins isolated into 3 categories: toxin A only, toxin B only, or toxins A and B. We then subgrouped the specimens into the following patient age categories—less than 6 months, 6 months to less than 1 year, 1 year to less than 16 years, and 16 years and older—to examine the effect of age on the results. The results, analyzed as proportions, were compared using the McNemar’s paired x2 test for statistical difference. The microbiology laboratories of 9 community hospitals and 4 pediatric tertiary care centers in the region were contacted by telephone to determine which C. difficile toxin(s) they routinely assayed.

Results During this 20-month period, 1668 stool specimens were submitted for detection of C. difficile toxin(s) as a possible etiology for symptomatic diarrhea in pediatric patients. There were 1061 samples that met criteria for analysis collected from 1031 patients. C. difficile toxin(s) was identified in 276 (26.8%) stool samples from 244 patients. Only 2 of 30 patients (7%) for whom multiple samples were submitted had a change from one toxin expression pattern to another. Positive specimens were obtained from 187 inpatients and 89 outpatients. Of these 276 positive stool samples, 51 (18.5%) were positive for toxin A alone, 133 (48.2%) were positive for toxin B alone, and 92 (33.3%) were positive for both toxins A and B (Table 1). The difference in frequencies of specimens that were positive only for toxin A (n 5 51) and those that were positive only for toxin B (n 5 133) was statistically significant (P , 0.0001). Results of identical analyses of these results grouped by inpatient and outpatient samples were similar and of comparable significance. Table 1 presents the frequency and percentage of C. difficile toxin results grouped by age. In patients younger than 6 months, 18.4% of samples (28 of 152) were positive for C. difficile toxin; 8 (28.6%) of these patients were positive for toxin A, 5 (17.8%) were positive for toxin B, and 15 (53.6%) were positive for both toxins A and B. From patients aged 6 months to less than 1 year, 29.6% of samples (32 of 108) were positive for C. difficile toxin; 9 (28.1%) of these patients were positive for toxin A, 17 (53.1%) were positive for toxin B, and 6 (18.8%) Table 1. Frequency of Positive C. difficile Toxin Results

Age group ,6 mo (n 5 28) 6 mo to ,1 yr (n 5 32) 1 yr to ,16 yr (n 5 182) $16 yr (n 5 34) All ages (n 5 276)

Positive toxin A only

Positive toxin B only

Positive toxins A and B

8 (28.6%) 9 (28.1%)

5 (17.8%) 17 (53.1%)

15 (53.6%) 6 (18.8%)

29 (15.9%) 5 (14.7%) 51 (18.5%)

92 (50.5%) 19 (55.9%) 133 (48.2%)

61 (33.5%) 10 (29.4%) 92 (33.3%)

Table 2. Inpatient Frequency of Positive C. difficile Toxin Results

Age group ,6 mo (n 5 18) 6 mo to ,1 yr (n 5 25) 1 yr to ,16 yr (n 5 126) $16 yr (n 5 18) All ages (n 5 187)

Positive toxin A only

Positive toxin B only

Positive toxins A and B

6 (33.3%) 9 (36.0%)

3 (16.7%) 12 (48%)

9 (50.0%) 4 (16%)

20 (15.9%) 2 (11.1%) 37 (19.8%)

63 (50.0%) 8 (44.4%) 86 (46.0%)

43 (34.1%) 8 (44.4%) 64 (34.2%)

were positive for both toxins A and B. In patients aged 1– 16 years, 26% of samples (182 of 687) were positive for C. difficile toxin; 29 (16%) of these patients were positive for toxin A, 92 (51%) were positive for toxin B, and 61 (34%) were positive for both toxins A and B. From patients older than 16 years, 30% of samples (34 of 114) were positive for C. difficile toxin; 5 (15%) of these patients were positive for toxin A, 19 (56%) were positive for toxin B, and 10 (29%) were positive for both toxins A and B. In each age group except for infants, toxin B was detected significantly more commonly than toxin A: less than 6 months, P 5 0.58; 6 months to less than 1 year, P 5 0.17; 1 year to 16 years, P , 0.0001; and more than 16 years, P 5 0.008. Tables 2 and 3 present these data arranged by inpatient and outpatient samples. The results of the telephone survey of microbiology laboratories in community hospitals and tertiary pediatric hospitals in the region were as follows: 7 of the 9 community hospitals contacted routinely perform C. difficile toxin A assay and do not routinely test for toxin B. Two community hospitals do not test for C. difficile toxin but refer specimens to local tertiary pediatric hospitals (which test for only toxin B). Three of the 4 pediatric tertiary hospitals routinely assay for toxin B alone. One other pediatric tertiary hospital (The Children’s Hospital of Philadelphia) routinely tests for both toxins.

Discussion C. difficile is an important pathogen that causes pediatric diarrhea.1,14–18 Illness associated with this organism varies from a mild, self-limiting, nonbloody diarrhea to severe abdominal cramps, tenderness, distention, fever, leukocytosis, hemorrhagic colitis, protein-losing enteropathy, toxic megacolon, colonic or cecal perforation, peritonitis, sepsis, shock, and death.1,15 A toxin-receptor mechanism, first described in animal studies,19,20 is believed to account for human C. difficile. The receptor, neurokinin 1 (NK-1), is required for C. difficile toxin A to induce human enteritis.19 The prevalence of carrier status for C. difficile in

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healthy, asymptomatic outpatients is reported to be up to 70% in healthy infants, 3% in children, and 2% in adults.21 Colonization with C. difficile is significantly less in infants treated with antibiotics but not in children more than 1 year of age treated with antibiotics.22 Breast-fed children similarly are less colonized by C. difficile than bottle-fed children at 6 weeks of age (21% vs. 47%) and at 6 months of age (19% vs. 39%).23 In a Nigerian study, children fed formula alone had a 50% frequency of cytotoxin positivity, those fed both breast milk and formula had a 19% frequency, and those fed only breast milk had a 17.5% frequency.24 Cytotoxin detection has also been reported to be more frequent in infants with diarrhea than in children more than 1 year of age with diarrhea.22 Either the immunoglobulin A secreted in breast milk or possibly 3 other nonimmunoglobulin components is able to inhibit C. difficile toxin A binding and may explain how breast-feeding protects against disease while reducing toxin detection. However, this breast milk factor does not prevent the cytotoxic activity of toxin A.25 Although the adherence properties of the C. difficile toxins are believed to be important virulence factors in adult diarrhea, they may not be in pediatric diarrhea.26 In our study, the prevalence of toxin A detection in infants only is not different from the prevalence of toxin B detection (Table 1). For all other symptomatic age groups, C. difficile toxin B is more likely to be detected. This may be related to the markedly increased carrier rate or to differences in infectivity or pathogenicity in this age group. Determination of cytotoxin production in C. difficile colonization shows that in infants younger than 6 months, the frequency of strains producing cytotoxin was low, whereas after age 6 months, approximately 50% of the colonizing strains isolated produced toxins.23 Another study found many different serotypes of C. difficile colonizing the infant intestine, with 62.5% of C. difficile cultures found not to secrete either toxin A or B, 37.5% to secrete both toxins, and only 3% to secrete toxin A alone in these asymptomatic patients.27 Cell culture studies show that the enzymatic activity of toxins A and B was the main determinant of cytotoxic potency, and the difference in receptor binding contributed to a lesser degree.28 These results suggest a mechanism that explains the clinical observations that infants can have disease caused by C. difficile. A recent study of nosocomial diarrhea showed that C. difficile accounted for 3% of all inpatient pediatric diarrhea infections.29 However, C. difficile was the most common pathogen identified in that study, occurring in 17.5% (7 of 40 inpatients) of patients in whom a pathogen was identified.29 Other studies have reported a

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14.8% incidence of toxin B in C. difficile–related diarrhea and incidence as high as 51%–61% for non–antibioticrelated and antibiotic-related diarrhea, respectively, in a study from Thailand.24,30 Our data show that 26.8% of specimens tested for C. difficile from symptomatic pediatric patients with diarrhea in our institution are related to C. difficile. Of the samples positive for C. difficile toxin A, B, or both toxins, 187 of 276 (67.8%) were inpatient and 89 of 276 (32.3%) were outpatient samples (Tables 2 and 3). When samples were grouped as inpatient or outpatient collections and stratified into the same age groups for the entire study population, the same results and significances were observed. The diagnosis of C. difficile is now made by detection of toxin A and/or toxin B produced by this organism.3,31 Cultures were once thought to be most sensitive, whereas detection of toxin B by tissue culture assay was considered most specific.32,33 Toxin A is an enterotoxin that causes mucosal inflammation, mucous discharge, increased vascular permeability, fluid secretion, epithelial necrosis, abdominal cramping, and gut motility abnormalities.34 Toxin A has been reported to be the primary mediator of illness.35 Toxin B is a cytotoxin that causes mucosal damage with 10-fold more potency than toxin A.36 It is associated with diarrhea, hemorrhagic colitis, and cytopathic effects. The relative importance of these toxins in pediatric diarrhea has not been fully established.36,37 In a study of acute diarrhea in 618 children, Cerquitti et al.38 identified C. difficile toxin B in 4.2% of all specimens. C. difficile toxin A was not evaluated. It has been suggested that C. difficile typically produces either both toxins concurrently or neither toxin.35 Several studies have demonstrated by C. difficile culture that certain strains produce varying amounts of toxin A or B or none at all.39 We did not perform C. difficile culture and strain identification on our samples. However, our data indicate that an assay for toxin A alone will identify only 51.8% of C. difficile infections. This discrepancy is greater than that which might be explained by the expected sensitivity and specificity of the toxin A assay, especially if both toxins are elaborated in clinical disease and since toxin A assay detects both active and inactive Table 3. Outpatient Frequency of Positive C. difficile Toxin Results

Age group

Positive toxin A only

Positive toxin B only

Positive toxins A and B

,6 mo (n 5 10) 6 mo to ,1 yr (n 5 7) 1 yr to ,16 yr (n 5 56) $16 yr (n 5 16) All ages (n 5 89)

2 (20.0%) 0 9 (16.1%) 3 (18.7%) 14 (15.7%)

2 (20.0%) 5 (71.4%) 29 (51.8%) 11 (68.8%) 47 (52.8%)

6 (60.0%) 2 (28.6%) 18 (32.1%) 2 (12.5%) 28 (31.5%)

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toxin A. The tissue culture assay for toxin B alone will identify 81.5% of C. difficile infections. This difference in detection between toxins A and B is significant (P , 0.0001). Laboratories that test for both C. difficile toxins identify significantly more infections than those performing either assay alone. The total incidence of C. difficile infection in our patient population is unknown but must equal or exceed the 26.8% identified in this study. To ensure an optimal identification rate, an adequate specimen must be obtained and properly handled. Although the organism itself is hardy and resistant to desiccation, the toxins are less hardy and can deteriorate at room temperature. The stool must be liquid, kept refrigerated between 2° and 8°C, or frozen and transported on ice to the laboratory.13,40 This may pose significant problems for laboratories or physicians’ offices that refer stool specimens to other institutions or commercial laboratories for toxin identification. Most community hospitals in this study routinely identify only toxin A. This can result in an unacceptably high false-negative rate. Even the identification of toxin B alone results in significant underdiagnosis of the disease. However, a large percentage of outpatients in this study were previously evaluated by primary care physicians in the community. If assay systems in the community had sufficient sensitivity, tertiary referral of these patients might have been unnecessary. Failure to identify C. difficile in the early stages of infection may result in substantial worsening of the disease because physicians may treat the diarrhea with further antibiotics or antisecretory agents. Furthermore, the subsequent evaluation of patients with bacterial culture and C. difficile toxin– negative chronic diarrhea frequently includes radiologic and endoscopic studies. Failure to diagnose C. difficile infection in these patients may result in substantially increased and unnecessary costs. These additional studies also result in increased patient risk and discomfort. Previously, C. difficile detection by culture, despite the absence of cytotoxin detection in certain diarrheal syndromes, was considered reason to treat.41 The role of culture is still controversial; one study showed that endoscopic pseudomembranous colitis was associated with 51% culture and toxin B assay–positive stool, whereas 11% of patients had positive culture results but negative toxin B results.33 Both groups responded similarly to metronidazole or vancomycin therapy.33 Toxin A was not evaluated in this study and could have accounted for the negative detection; however, if toxin A had been evaluated and was found to be negative, it would have provided further support for culturing for C. difficile. C. difficile cultures may still be clinically relevant in the diagnosis of cases in which diarrhea stools are negative for

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toxin A and toxin B. C. difficile culture is still important in laboratory research and epidemiology for identification of the various strains and their virulence patterns.42 Clinically, however, if a patient is symptomatic and C. difficile is detected, physicians probably will prescribe medical therapy of metronidazole or vancomycin, regardless of the relative virulence of the strain. This retrospective study has a number of limitations. A large number of the patients studied were inpatients when the stool specimens were obtained. Nosocomial infections could bias the frequency of positive results. The bias may be lessened by the large percentage of outpatient samples included in the analysis. Despite the criteria of 3 weeks between specimens for exclusion of samples that were from the same patient, it is still possible that a selection bias occurred because recurrent infections or incompletely treated infections could be included in these data. The actual number of patients for which this occurred is limited compared with the total patient population, so we believe any bias would be minimal. To determine the overall frequency of toxin identification in symptomatic patients in the community or to sequentially compare large numbers of patients is beyond the scope of this study. Patients who were taking antibiotics effective against C. difficile when stool specimens were assayed could theoretically change the pattern of toxin A and B elaboration specifically. Although there are no data in the literature to support this concern, it could be a minor, confounding factor. Nevertheless, these data illustrate significant difficulties in the current laboratory diagnosis and clinical management of C. difficile diarrhea infections. The implications of this study are that neither toxin A nor toxin B assay alone identifies all pediatric patients with C. difficile infection evaluated at a tertiary care center. These results may not apply to adult disease. Our data support the AAP 1997 Red Book recommendation of testing for C. difficile toxin B at a minimum. We would encourage those testing for toxin A alone to consider testing for toxin B, especially if results for toxin A are negative. A laboratory decision to identify only toxin A may be inappropriate. Results should be accompanied by the warning that a significant percentage of C. difficile infections will be undetected by a single toxin assay. Given such a warning, physicians might routinely order assays for both toxin A and toxin B or order screening for the second toxin if results for the first are negative to address the problem of underdiagnosis. Further studies are needed to determine what laboratory testing approach might be most efficient and cost effective in the diagnosis of C. difficile infection. We ideally support the detection of both toxins for best diagnosis of this infection in a

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noninvasive manner. Because some health maintenance organizations may only approve toxin A detection, physicians who order C. difficile toxin assays should understand the limitations of each assay available in their region.

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17. Medline A, Shin DH, Medline NM. Pseudomembranous colitis associated with antibiotics. Hum Pathol 1976;7:693–703. 18. Gryboski JD, Pellerano R, Young N, Edberg S. Positive role of Clostridium difficile infection and diarrhea in infants and children. Am J Gastroenterol 1991;86:685–689. 19. Castagliuolo I, Riegler M, Pasha A, Nikulasson S, Lu B, Gerard C, Gerard NP, Pothoulakis C. Neurokinin-1 (NK-1) receptor is required in Clostridium difficile–induced enteritis. J Clin Invest 1998;101:1547–1550. 20. Eglow R, Pothoulakis C, Itzkowitz S, Israel EJ, O’Keane CJ, Gong D, Gao N, Xu YL, Walker WA, LaMont JT. Diminished Clostridium difficile toxin A sensitivity in newborn rabbit ileum is associated with decreased toxin A receptor. J Clin Invest 1992;90:822–829. 21. Mitty RD, LaMont T. Pseudomembranous colitits. In: Walker WA, Durie PR, Hamilton JR, Walker-Smith JA, Watkins JB, eds. Pediatric gastrointestinal disease: pathophysiology, diagnosis, management. Volume 1. 2nd ed. St. Louis, MO: Mosby, 1996:726–739. 22. Camorlinga M, Munoz O, Guiscafre H, Torres J. Colonization by Clostridium difficile in hospitalized children: risk factor and typification of the isolated strains. Arch Invest Med 1991;22:19– 26. 23. Tullus K, Aronsson B, Marcus S, Mollby R. Intestinal colonization with Clostridium difficile in infants up to 18 months of age. Eur J Clin Microbiol Infect Dis 1989;8:390–393. 24. Emeruwa AC, Oguike JU. Incidence of cytotoxin producing isolates of Clostridium difficile in faeces of neonates and children in Nigeria. Microbiologica 1990;13:323–328. 25. Rolfe RD, Song W. Immunoglobulin and non-immunoglobulin components of human milk inhibit Clostridium difficile toxin A–receptor binding. J Med Microbiol 1995;42:10–19. 26. Gonzalez-Valencia G. Munoz O, Torres JF. Toxigenicity and adherence in Clostridium difficile strains isolated from patients with and without diarrhoea. Arch Invest Med 1991;22:189–196. 27. Collignon A, Ticchi L, Depitre C, Gaudelus J. Delmee M. Corthier G. Heterogenicity of Clostridium difficile isolates from infants. Eur J Pediatr 1993;152:319–322. 28. Chaves-Olarte E, Weidmann M, Eichel-Streiber C, Thelestam M. Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells. J Clin Invest 1997;100:1734–1741. 29. Meropol SB, Luberti AA, DeJong AR. Yield from stool testing of pediatric inpatients. Arch Pediatr Adolesc Med 1997;151:142– 145. 30. Wongwanich S, Ramsiri S, Vanasin B, Khowsaphit P, Tantipatayangkul P, Phan-urai R. Clostridium difficile associated disease in Thailand. SE Asian J Trop Med Public Health 1990;21:367–372. 31. Bond F, Payne G, Borriello SP, Humphreys H. Usefulness of culture in the diagnosis of Clostridium difficile infection. Eur J Clin Microbiol Infect Dis 1995;14:223–226. 32. Staneck JL, Weckbach LS, Allen SD, Siders JA, Gilligan PH, Coppitt G, Kraft JA, Willis DH. Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination. J Clin Microbiol 1996;34:2718–2721. 33. Gerding DN, Brazier JS. Optimal methods for identifying Clostridium difficile infections. Clin Infect Dis 1993;16(suppl 4):S439– S442. 34. Triadafilopoulos G, Pothoulakis C, O’Brien MJ, LaMont JT. Differential effects of Clostridium difficile toxins A and B on rabbit ileum. Gastroenterology 1987;93:273–279. 35. Fekety R. Antibiotic-associated colitis. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases. 4th ed. New York: Churchill Livingstone, 1995:978–987. 36. Riegler M, Feil W, Hamilton G, Bischof G, Cosentini E, Teleky B, Wenzl E, Pothoulakis C, LaMont JT, Schiessel R. Clostridium difficile toxin B is more potent than toxin A in damaging human

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41. Lashner BA, Todorczuk J, Sahm DF, Hanauer SB. Clostridium difficile culture-positive toxin-negative diarrhea. Am J Gastroenterol 1986;81:940–943. 42. Settle CD, Wilcox MH. Review article: antibiotic-induced Clostridium difficile infection. Aliment Pharmacol Ther 1996;10:835– 841.

Received May 18, 1998. Accepted August 24, 1998. Address requests for reprints to: Howard A. Kader, M.D., Division of Gastroenterology and Nutrition, Children’s Hospital of Philadelphia, 34th Street & Civic Center Boulevard, Philadelphia, Pennsylvania 19104. e-mail: [email protected]; fax: (215) 590-3606. The authors thank Dale Cranmer, Coordinator of the Clinical Laboratories, Laboratory Information Systems, and the clinical microbiology laboratory at The Children’s Hospital of Philadelphia for helping them retrieve the necessary data for this project.