CLINICAL AND DIAGNOSIS CONSIDERATIONS OF LYME DISEASE Doris Ionescu1,2, Ani Ioana Cotar2*, Daniela Bãdescu2, Silvia Dumitriu1

2National

1University

of Medicine and Pharmacy “Carol Davila” Bucharest, Romania Institute for Research and Development in Microbiology and Immunology Cantacuzino, Romania

1. ETIOLOGIC AGENTS AND EPIDEMIOLOGY OF LYME DISEASE

Numerous improvements have been made along the years in the prevention, diagnosis and treatment of Lyme borreliosis (LB). Nonetheless, LB remains the most common human infection transmitted by ticks in temperate regions of the northern hemisphere [1,2]. Lyme borreliosis is a multisystemic infection caused by the pathogenic genospecies of the Borrelia burgdorferi sensu lato (s.l.) complex, including B. burgdorferi sensu stricto (s.s.), Borrelia garinii, Borrelia afzelii, Borrelia bavariensis, and Borrelia spielmanii. All pathogenic genospecies can cause erythema migrans (EM), which is the classical target rash that is characteristic of Lyme disease but distinct genospecies possess diferent organotropism, and may preferentially cause distinct clinical manifestations of LB. B. burgdorferi s.s. is most commonly associated with arthritis, B. garinii with neuroborreliosis, and B. afzelii with cutaneous manifestations of LB (EM, lymphocytoma borreliosis, and acrodermatitis chronica atrophicans) [3]. B. burgdorferi s.s. and B. afzelii may also be associated with neurological manifestations, but at a lower rate than that of B. garinii [4-6]. Approximately 60% of patients with EM, treated with antibiotics, have long or short episodes of arthritis caused by B. burgdorferi s.s in North America [7]. By contrast, only 3-15% of patients with LB suffer from arthritis in Europe [8], where B. garinii and B. afzelii are the most commonly found genospecies. The average Lyme borreliosis annual incidence is 37.77, the maximum being registered in Slovenia, and the minimum value in Ireland (0.6) [9]. In some geographical regions it has had an increase in the rates of disease transmission and in the incidence implicitly [10], so that LB should continue to be regarded as an emerging disease. In Romania, aco-

ording to data reported by the National Center for Surveillance and Control of Communicable Diseases (CNSCBT) of the National Institute of Public Health (INSP), both institutions under the coordination of the Ministry of Health, the incidence of LB in 2011 was 2/100,000. B. burgdorferi sensu lato (s.l.) genospecies circulating in Romania are: B. burgdorferi s.s., B. afzelii and B. garinii [11]. 2. VECTORS AND HOSTS OF LYME BORRELIOSIS

B. burgdorferi s.l., the etiological agent of Lyme borreliosis, is transmitted to man through the bite of the ticks of the genus Ixodes. Maintaining B. burgdorferi sl in a complex enzootic tick-vertebrate cycle requires its successful persistence in multiple stages of development of arthropod, and coordinated dissemination through tick tissues to the new host [11, 12]. Given the absence of transovarial transmission, the pathogen must be acquired in one of the stages of development of ticks when these feed with the the infected vertebrate host blood [12-14]. Ixodes genus ticks of the family Ixodidae (hard bodied ticks) are vectors and reservoir hosts for all genospecies of B. burgdorferi s.l complex. I. ricinus, the tick of European origin is vector for B. burgdorferi s.s., B. afzelii and B. garinii genospecies. In Romania I. ricinus has an area of expanded spread all over the country, mainly in the deciduous forests and edge of forest. Ixodes tick life cycle includes four stages of development: eggs, larva, nymph and adult. Only larvae, nymphs and adults must feed with blood only once on different vertebrate hosts (birds, mammals). The ticks that acquire Borrelia in the course of blood feeding can maintain their infectivity and transmit borrelia to a subsequent host. The larvae and nymphs feed on birds and small rodents [15], whereas large mammals (sheep in Europa) are preferred hosts of I. ricinus adult stage.

*Corresponding author: Ani Ioana Cotar, NIRDMI Cantacuzino, 103 Spl. Independentei, Bucharest, Romania, e-mail: [email protected]; tel: 0040726305705

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3. THE CLINICAL STAGES OF LYME DISEASE

Clinical manifestations of Lyme disease generally have a progressive evolution, being divided into 3 stages: early localized, early disseminated, and late disseminated. The frequency of clinical manifestations is very variable, being dependent on the age patient, the pathogen species involved and other factors. All stages of Lyme disease have the potential to be healed through antibiotic treatment. The infection progresses to disseminated disease in approximately 50% of untreated patients. The three stages of the disease are not always clearly separated so that various signs and symptoms may overlap. Once B. burgdorferi is injected into the host, patients may eliminate the infection without developing any clinical sign, it happens in asymptomatic, but seropositive patients. A few days or weeks after the bite of an infected tick, the patient develops flu-like symptoms (pain in the muscles and joints), low fever, and/or fatigue [16].

Stage I, early localised Lyme borreliosis includes two clinical entities, erythema migrans (EM) and borrelial lymphocytoma. EM, the characteristic rash spreading from the site of a tick bite, typically starts as a red macula/papule, which extends over a period of several days or weeks to form a red spot or bluish-red, with central clarification when it looks like a ring or without central clarification [17]. EM, the earliest manifestation of Lyme disease, which generally occurs from 3 to 32 days after tick exposure, is the pathognomonic clinical sign, the most important of Lyme disease present in ~ 70% of patients with LB [18]. Borrelial lymphocytoma (lymphadenosis benigna cutis) is rare in Europe (< 1% of cases), and very rare in North America. It is a painless bluishred nodule or plaque, usually found on the ear lobe, ear helix, nipple or scrotum and occurs more frequently in children (especially on the ear) than in adults [17]. Stage II, LB early disseminated is characterized by hematogenous or direct dissemination of Borrelia to the other organs and tissues, with development of general systemic signs and symptoms, especially on the nervous system and joints. Also after hematogenous spread of Borrelia to other areas of the skin, multiple EM lesions can occur. Clinical pathological characteristics of this stage of the disease occur several weeks to months after the initial tick bite, being represented by neurologic abnormalities (acute lymphocytic meningitis, meningoradi134

culitis, cranial neuropathy, radiculonevritis), joint (mono- or oligoarthritis flashing at large joints: knee, ankle, shoulder, elbow, or other joints) and heart (block atriventricular). Ophthalmic manifestations rarely occur at this stage and include follicular conjunctivitis, keratitis and rarely uveitis. In stage III, late LB the pathological clinical manifestations appear after months/years from initial infection. Late manifestations are due to spreading of spirochetes under the skin, or through the bloodstream to other organs (brain, heart, joints), so late LB manifests as encephalomyelitis, peripheral neuropathy, carditis, acrodermatitis chronica atrophicans (ACA) or chronic arthritis with a history of the symptoms for more than 6-12 months [19].

Other manifestations of Lyme borreliosis The recognition of LB as a potential cause of ocular diseases is important for implementing appropriate treatment, although it is difficult to obtain reliable diagnosis. Ophthalmological changes are apparently rare and usually take the form of conjunctivitis during the early manifestations of BL. Uveitis (previous intermediate posterior panuveitis), papillitis, keratitis and episcleritis may occur occasionally [20].

Post-Lyme disease syndrome Some patients report ongoing, recurrent or persistent symptoms after appropriate treatment of a proven manifestation of LB. This problem, described as post-Lyme syndrome (PLS) is characterized by the persistence of a complex of symptoms for more than 6 months after treatment [21]. The symptoms are nonspecific and include reduced performance, increased fatigue, irritability, emotional lability, and disturbances in sleep, concentration, and memory. Thorough clinical and laboratory assessment of such patients is required to exclude the possibility of treatment failure or the presence of a new condition unrelated to previous LB. Various double-blind, placebo-controlled studies have so far failed to support the idea that persistence of borrelial infection is the cause of such symptoms and have failed to show any sustained benefit from prolonged treatment with antibiotics [22, 23].

Co-infections in Lyme disease Ixodes ricinus, the vector of B. burgdorferi s.l., may also transmit other zoonotic organsims (table 1). Some of them may co-infect with B. burgdorferi s.l. and thus affect LB diagnosis, treatment and epidemiology. Patients with co-infection may present

Clinical and diagnosis considerations of Lyme disease



Table 1. Zoonotic organisms potentially co-transmitted with Borrelia burgdorferi s.l. by Ixodes ricinus. (EUCALB-European Concerted Action on Lyme Borreliosis)

Organism Babesia divergens Babesia microti Coxiella burnetii Anaplasma spp Francisella tularensis Rickettsia helvetica TBEV

with a confusing mixture of clinical manifestations. There are reports of tick borne encephalitis (TBE) cases accompanied by thrombocytopenia, leukopenia and elevated transaminases, which are classic features of erlichial and babesial infections. Sometimes manifestations caused by different pathogens may overlap sufficiently for one of the infections to be overlooked, resulting in failure to provide appropriate treatment, as in the case of TBE and early neuroborreliosis [24]. Some of the zoonotic pathogens transmitted by I. ricinus, such as ehrlichias and babesias, are known to be potentially immunosuppressive and they may affect the severity and duration of infection of co-transmitted pathogens. Dual infection with Babesia microti and B. burgdorferi s.s. may result in more severe disease than that caused by infection with only one of the two agents. BL with TBE combination could cause a dilemma and hence delay in starting treatment with antibiotics. In Europe, coinfection with Anaplasma phagocytophilum, the agent of human granulocyte erlichiosis (HGE), affects the choice of antibiotics for the treatment of Lyme borreliosis. Thus, the use of doxycycline is an advantage compared to betalactam antibiotics, which are ineffective in the treatment of HGE. B. burgdorferi coinfection with Anaplasma phagocytophilum occurs in ~ 29% of patients with borreliosis in Austria. In conclusion, to give appropriate therapy it is important to assess the possible co-infections in patients bitten by ticks with LB suspected when clinical manifestations are consistent with co-infections transmitted by I. ricinus. 4. LYME BORRELIOSIS DIAGNOSIS

The diagnosis of LB is primarily a clinical one, since clinical criteria (symptoms and clinical signs) are decisive factors for diagnosis and for assessing diagnosis based on microbiology laboratory tests. However, the diagnosis of BL is very complex due to the diversity of clinical manifestations, very few

Boala babesiosis babesiosis Q fever erlichiosis tularaemia perimyocarditis, sarcoidosis tick-borne encephalitis

are exclusive to infection with B. burgdorferi. The diagnosis should be based on clinical criteria, exposure history and laboratory test results. In general, microbiological laboratory data is a major criterion for the clinical diagnosis of BL. History is very important for the diagnosis of the disease (exposure to ticks, living/outdoor operations in an endemic area, the existence of recent tick bite). B. burgdorferi infection may be subclinical, or have several clinical pictures, depending on the length of time after infection and the organs/systems affected. Some patients experience subsequent events without having passed through or observed symptoms of early stage. The appropriate criteria of clinical case definitions underlying standardized diagnosis are described by Stanek in 2011 and presented in detail by EUCALB (European Concerted Action on Lyme borreliosis) (Table 2). Case definitions are essential for reliable epidemiological studies and are of great value in clinical management. 4.1. THE CLINICAL DIAGNOSIS OF LYME BORRELIOSIS

The moment when signs and symptoms appear is important for diagnosis. The detailed description of the rash is very useful if the lesion is not present during the examination of the patient. Also, the history of the extent of the lesion, which may exceed 5 cm in diameter, is clinically useful. If not treated in most patients it progresses from the early to the late stage. The diagnosis of EM is clinic. Erythema migrans, the characteristic rash spreading from the site of a tick bite, typically starts about two to thirty days after the tick bite and is the direct result of the spirochaete migrating through the skin. Lesions which start earlier or during the first 24 hours could be due to a local reaction to the tick bite or an acute bacterial infection such as streptococcal or staphylococcal infection. In adults, EM generally develops at the site of the tick bite, only a small percentage 135

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Table 2. Summary of clinical case definitions for Lyme borreliosis (Stanek, 2011) Clinical case definition Expanding red or bluish-red patch Erythema (•5 cm in diameter)a, with or migrans (EM) without central clearing. Advancing edge typically distinct, often intensely coloured, not markedly elevated. Borrelial Painless bluish-red nodule or lymphocytoma plaque, usually on ear lobe, ear (rare) helix, nipple or scrotum; more frequent in children (especially on ear) than in adults. Acrodermatitis Long-standing red or bluish-red chronica lesions, usually on the extensor atrophicans surfaces of extremities. Initial (ACA) doughy swelling. Lesions eventually become atrophic. Possible skin induration and fibroid nodules over bony prominences. Lyme In adults mainly meningoneuroborreliosis radiculitis, meningitis; rarely encephalitis, myelitis; very rarely cerebral vasculitis. In children mainly meningitis and facial palsy. Term

Laboratory evidence: essential None

Laboratory/clinical evidence: supporting Detection of B. burgdorferi s.l. by culture and/or PCR from skin biopsy.

Seroconversion or positive serologyb Histology in unclear cases

Histology. Detection of B. burgdorferi s.l. by culture and/or PCR from skin biopsy. EM recent sau concomitent Histology. Detection of B. burgdorferi s.l. by culture and/or PCR from skin biopsy.

High level of specific serum IgG antibodiesb

Pleocytosis and demonstration of intrathecal specific antibody synthesis

Lyme arthritis

Recurrent attacks or persisting objective joint swelling in one or a few large joints. Alternative explanations must be excluded.

Specific serum IgG antibodies, usually in high concentrationsb

Lyme carditis (rare)

Acute onset of atrio-ventricular (I–III) conduction disturbances, rhythm disturbances, sometimes myocarditis or pancarditis. Alternative explanations must be excluded Conjunctivitis, uveitis, papillitis, episcleritis, keratitis.

Specific serum antibodiesb

Ocular manifestations (rare)

Specific serum antibodiesb

Detection of B. burgdorferi s.l. by culture and/or PCR from CSF. Intrathecal synthesis of total IgM, and/or IgG and/or IgA. Specific serum antibodies. Recent or concomitant EM. Synovial fluid analysis. Detection of B. burgdorferi s.l. by PCR and/or culture from synovial fluid and/or tissue. Detection of B. burgdorferi s.l. by culture and/or PCR from endomyocardial biopsy. Recent or concomitant erythema migrans and/or neurologic disorders Recent or concomitant Lyme borreliosis manifestations. Detection of B. burgdorferi s.l. by culture and/or PCR from ocular fluid.

If 55%) [44]. EFNS guidelines “the guideline on the diagnosis and management of European Lyme neuroborreliosis” recommends the following criteria for the diagnosis of early and late LNB [32] (Table 3) The clinical diagnosis of Lyme carditis Cardiac manifestations of acute LB are rare. Most commonly they can be seen together with EM or shortly after the occurrence of EM or in combi-

Clinical and diagnosis considerations of Lyme disease

nation with neurological symptoms or arthritis [19]. The frequency of Lyme carditis is higher in North America, where between 4-10% of LB patients have cardiac abnormalities unlike the situation encountered in Europe, where only 0.5-4% of patients with untreated LB have similar abnormalities [45], which can be explained by the fact that the etiologic agent responsible for Lyme carditis is B. burgdorferi s.s. Lyme carditis manifested as conduction defects (atrioventricular block, complete heart block, block the beam), tachyarrhythmias (atrial due to pericarditis, rarely ventricular) miopericarditis and average myocardial dysfunction. The predominant cardiac manifestation is partial heart block determined by preventing the electric signal, which controls the contraction of the upper and lower chambers of the heart. Heart block is usually medium with full recovery in six weeks of onset. The most common symptoms are light-headedness, fainting, shortness of breath, palpitations, and/or chest pain. Occasionally, heart block can be complete and permanent, requiring the introduction of a pacemaker. The course of the disease is usually favorable. In patients treated with antibiotics and even in the untreated, the symptoms of the heart and ECG abnormalities usually disappear within 3-6 weeks. Antibodies to B. burgdorferi s.l. should be present in the serum, but positive serology alone is not confirmatory for Lyme carditis, appropriate clinical signs should be considered [46, 47]. Clinical diagnosis of late manifestations in stage III Lyme borreliosis This stage appears after several years (after 6 months) after initial infection and may involve articulations (Lyme arthritis), skin (ACA), and rarely nervous system (chronic neurological symptoms).

Clinical diagnosis of Lyme arthritis Lyme arthritis is one of the manifestations of persistent Lyme disease, being rarely seen in Europe, but common in North America. It typically acts as a mono- or intermittent or chronic oligoarthritis large joints, rarely with the erosion of cartilage or bone. Sometimes it occurs in patients who have previously had intermittent arthralgia or oligoarthritis in the early stages of the disease and who have progressed to chronic arthritis. Knee joint is the most commonly affected. In the USA ~ 60% of patients treated in the early stage of LB may develop intermittent episodes of pain and joint swelling months or years after the onset of infection [17]. Some patients with Lyme arthritis do not show a decrease until disappearance

of symptoms even after adequate long-term therapy. This form of the disease was named by Steere and his colleagues, treatment-resistant Lyme arthritis [48]. Subsequently, it was found that there is a genetic predisposition to this form of disease, which was more frequently detected in individuals with DR2 or HLA-DR4 phenotypes, which suggested the involvement of immunogenetic factors in the pathogenesis. It was also observed that antibodies synthesized against Borrelia OspA protein may cross-react with the adhesion molecule on human lymphocytes, T-lymphocyte function-associated antigen-1 (hLFA1aL332-340). This molecular mimism between OspA-1 epitopes and hLFA can lead to autoimmunity in affected synovial tissues and may explain the persistence of symptoms after Borrelia eradication from the joints.

Clinical diagnosis of ACA –Pick-Herheimer disease ACA is almost exclusively seen in adults, predominantly women, though ACA-like lesions in children have been reported occasionally [49]. It is a long-lasting, usually progressive manifestation of LB, characterised by red or bluish-red lesions, usually on the extensor surfaces of the extremities. Initially there is a bluish-red discolouration, often with doughy swelling. Later on skin atrophy becomes more and more prominent. The skin becomes thin and wrinkled, colored bluish-red. Veins are prominent and there is no healing skin lesion. A fibrous induration can develop, which often takes the form of ulnar or tibial bands or nodules region when located above the bony prominences (patella, elbow). Some patients may develop sclerotic lesions, peripheral nerves and joints being frequently affected. Fibroid nodules may develop over bony prominences and sclerodermic changes may develop in atrophic skin areas [17]. Pathohistological findings frequently show a dermal infiltration of lymphocytes and plasma cells, and sometimes subcutaneous with or without atrophy. Involvement of peripheral nerves is not uncommon, locally at the site of the skin lesion, usually as large-fibre axonal polyneuropathy with predominantly mild sensory symptoms [50, 51]. ACA does not heal spontaneously. The specific diagnosis of ACA can be further supported by the isolation of borrelia from affected skin before treatment with antibiotics. B. afzelii genospecies is predominantly involved in the ACA but is not exclusively the etiological agent of this disease. This late manifestation of BL is rare in the USA, being described in particular in Scandinavia, where it is the most preva139

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lent form of Lyme disease. In Austria, Germany and Switzerland ACA prevalence is lower compared to that of arthritis.

Clinical diagnosis of chronic neuroborreliosis Chronic LNB is characterized by neurological symptoms that occur several months or years after borrelia inoculation. This form of late LB includes typical abnormalities of the CNS and PNS. CNS abnormalities are represented by encephalopathy, which is prevalent in North America, and encephalomyelitis, which is more common in Europe. Medium or moderate encephalopathy may persist for years if untreated and is characterized by the following symptoms: memory and concentration disturbances, insomnia, somnolence. Also average mental disturbances, such as depression, irritability, paranoia may be present. Chronic encephalomyelitis is one of late neurological sequelae of untreated infection, which may have clinical features similar to multiple sclerosis (MS), but no magnetic resonance imaging findings characteristic of MS. Some patients, usually elderly, have a gradual evolution radiculopathy pain that develops over many months after infection [52]. The use of antimicrobial agents leads to a decrease in the disappearance of pain and prevents the prolonged pain in patients with radiculopathy. In encephalomyelitis neurological signs may be present at various levels, which reflects brain, spinal cord and peripheral and cranial nerves damage. Insidious onset and progressive worsening over months to years allow a distinction between this form and the clinical manifestations observed in early LNB. In most cases, the clinical manifestations of late LNB does not disappear spontaneously. After treatment with antibiotics, the improvement of health is variable. 4.2. LABORATORY DIAGNOSIS OF LYME DISEASE

LB diagnosis should be based primarily on the clinical presentation and an assessment of tick-exposure risk. In most cases laboratory support is essential because of the nonspecific nature of many clinical manifestations [19]. At present, for the diagnosis of LB both direct and especially indirect methods (serology) are used.

4.2.1. Direct methods for detection of B. burgdorferi infections In laboratory diagnosis of LB two direct methods are used: culture and methods based on amplification of nucleic acids. 140

Culture of spirochaetes from patient material is still the gold standard for specificity in the laboratory diagnosis of LB, but is not used as routine diagnostic test, due to the low number of viable spirochaetes usually present in patient biopsies [53]. Also the fastidious nature of many B. burgdorferi strains makes the culture result to be negative, but not always rules out the active infection. The culture requires special media, which are difficult to standardize, and the duration of the culture incubation may be for 2-4 weeks, so the results are not provided in a clinically useful period of time. The highest rates of borrelia isolation were recorded for cultures from skin biopsies of patients with EM (up to 80%), and from patients with ACA (up to 60%). The lowest rates of isolation (17%) were recorded for samples of cerebrospinal fluid (CSF). Borrelia has been isolated from the blood of patients with early LB and in the synovial fluid of patients with Lyme arthritis. However, blood and synovial fluid samples are not a reliable source for Borrelia isolation by culture. In the European Lyme disease spirochaetemia is transient and spirochaetes are relatively difficult to harvest from tissues. For these reasons and also because successful culture demands expertise and specialist culture media that are often unavailable in diagnostic laboratories, culture is not used as first-line support for clinical diagnosis, but may be useful for confirmation and for uncertain cases. Also, culture is useful for research, as it provides proven pathogenic isolates. Borrelia grown from the patient sample can be observed at dark-field microscopy. Also borrelia present in the colored histological sections (skin) [54] can be seen in this type of microscope, but the sensitivity and specificity of this method of detection is low [55]. Focus floating microscopy was recently described as a sensitive method for detection of B. burgdorferi in skin biopsies [56]. Nevertheless, there is currently insufficient evidence to recommend any of these methods based on microscopy for routine diagnosis of LB. Molecular biology methods are based on nucleic acids amplification testing using PCR technology, and permit detection and identification of a large spectrum of fastidious pathogens. At present, there are PCR tests which have genospecies specificity, and that can detect a small number of B. burgdorferi s.l copies directly from clinical specimens [57-59]. Unlike culture, PCR detects Borrelia DNA not only from viable organisms, thus DNA detection, although suggestive, is unable to determine if infection is active or not. Detection of borrelial DNA by poly-

Clinical and diagnosis considerations of Lyme disease

merase chain reaction (PCR) is diagnostically useful in certain well-defined circumstances. PCR allows the detection of Borrelia DNA in over 90% of synovial fluid or synovial tissue from patients with ACA, untreated [60]. In patients with EM, the detection rate of Borrelia DNA is 70–80% from skin byopsies [59, 61], whereas in patients with acute LNB, Borrelia DNA was detected in 15-30% of CSF samples tested [57, 62]. For late LNB, PCR sensitivity is very low. The analytical specificity of PCR (the ability to identify exclusively Borrelia DNA) can be 98-100% when precautionary measures are taken to avoid contamination and amplification products are identified by appropriate methods such as sequencing [62-64]. PCR from CSF samples has a low sensitivity, but can be useful in very early LNB when antibody index (AI) is negative, or in patients with immunodeficiencies. PCR is not recommended for testing urine or blood samples [64]. PCRs are now available that detect and identify individual genospecies of B. burgdorferi sensu lato. These methods either use species-specific primers for PCR, or analyse the PCR product by DNA sequencing, hybridisation to species-specific probes, or restriction endonuclease polymorphism. Mixed infections of 2 or 3 different species have been detected in European neuroborreliosis patients and patients with an EM or ACA. Despite the occurrence of mixed infections, the association of B. afzelii with ACA remains unchallenged. In neuroborreliosis, about 60-70% of the strains belong to B. garinii species , and so far the majority of isolates from European Lyme arthritis cases have proved to be B. burgdorferi s.s., though several studies have reported genospecies heterogeneity in Lyme arthritis cases. Despite the increased use of PCR for DNA detection of B. burgdorferi s.l. in laboratory diagnosis, this method has significant limitations. Also, there is still no general consensus on the most appropriate choice for amplifying genomic targets, nor whether positive results are clinically significant in some manifestations of the disease [19]. Despite these disadvantages, molecular detection methods can provide useful support in the diagnosis of difficult cases.

4.2.2. Indirect methods for laboratory diagnosis Serological testing for detection of antibodies to B. burgdorferi remains the mainstay of diagnostic testing method, being usually the first and often the only method which supports the clinical diagnosis, because: it is relatively easy to obtain samples, the availability of laboratory facilities necessary for car-

rying it out, as well as of serological diagnostic kits. However, the limitations of antibody tests must be taken into account. On the one hand the antibody response in early LB may be weak or absent, especially in EM and early LNB [58, 65]. Furthermore, seroconversion in such patients may be absent because early antibiotic treatment can ablate antibody production [66, 67]. On the other hand, a positive specific antibody response may persist for months or even years after successful treatment of the infection, so follow-up of antibody titres in patients following therapy is not a reliable approach for monitoring success of treatment [49, 68]. Some European countries recommend diagnosis by EIA (enzyme immuno-assay) alone, whth a specificity of at least 95%, but preferably of 98%. In other European countries, including Romania, and also the USA, a two-step approach using two different tests is recommended. In the first step, an initial screening test is used (usually ELISA), which must have a specificity of more than 90%. In the second step, a western blot (immunoblot, IB) is used for reactive and equivocal samples to the first test. The specificity of IB test must be at least 97%. Appropriate use of immunoblots greatly increases specificity. Even so, IgM immunoblots remain problematic, as false-positive reactions can still occur in the presence of other acute infections and in autoimmune conditions. Use of IgM tests should be reserved for patients who have acute presentations and with a high probability of Lyme borreliosis [19]. ELISA (enzyme linked immunosorbent assay) is currently the most widely used serological diagnosis BL. The main advantages of ELISA are the relative ease of large-scale testing, coupled with avoidance necessity of subjective interpretation, as in the case of indirect immunofluorescence tests, which were the first used serological tests for LB serological diagnosis. The first generation of ELISA tests was based on sonicated antigens, and the second and third generation are characterized by an improved specificity, because they use semi-purified antigens, one or several purified antigens and synthetic peptides or recombinant antigens. The usefulness of an ELISA test depends on the the choice of cut-off levels, which should be confirmed by studies in target populations. Usually ELISA has a high sensitivity, and is used as a preliminary screening test, followed by an immunoblot test with high specificity for confirmation. ELISA tests can give false-positive reactions in the presence of other infections (syphilis, glandular fever, autoimmune and other inflammatory conditions). It is important that labora141

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tories are provided with appropriate clinical details, including onset date of illness and date of most recent tick exposure in order to avoid unnecessary and possibly detrimental IgM testing. Immunoblot is important for the characterization of immune response against specific proteins of B. burgdorferi s.l. Standardization of criteria for immunoblot interpretation in Europe was the subject of the study conducted by EUCALB [69]. This multicenter study which involved six European laboratories that have used immunoblotting protocols, to identify 8 bands, which were considered discriminatory for all laboratories, with variations in terms of their significance. Several rules were established which provide a common framework for interpreting immunoblot, and that can be adapted to the characteristics of LB in different geographical areas. The complete standardization of immunoblot protocols in Europe is not a realistic target today.

The importance of serological diagnosis in different stages of LB Serology is indicated in all cases of clinically suspected Lyme disease, excepting EM. However, the less specific the symptoms, the weaker the a priori probability of Lyme borreliosis and the lower the predictive value of microbiological methods (Table 4) [64, 65]. Specific IgG and/or IgM antibodies are present in only 40-60% of untreated cases, particularly in patients with signs of haematogenous spread, e.g.: multiple EM and broad general symptoms. A significant increase of the specific IgG and/or IgM between the pairs of serum samples, first of which was taken at the first visit to the doctor, and the second 3-6 weeks later, provides the highest specificity of diagnosis. Early treatment for EM can lead to lack of detectable specific antibody response. Therefore, for EM diagnosis the serology is not essential. For diagnosis of Borrelial lymphocytoma the following are essential: (1) the presence of a high titer IgM antibodies or (2) a substantial change in titers of specific IgG and/or IgM between the paired samples of serum, the first taken at the first visit to doctor, and the second 3-6 weeks later. The clinical relevance of a positive serological result should be interpreted in the context of clinical features presented by the patient. For example, in patients with high levels of exposure to ticks (forestry workers) a positive IgG result may reflect exposure in the past that was not related to current clinical problem. Seropositivity may persist indefinitely in some patients and therefore does not indicate continuation of disease or the need for re-treatment. 142

Laboratory diagnosis for early LNB is necessary only for clinical diagnosis confirmation. The diagnosis for neuroborreliosis should not be performed based only on serological tests. For laboratory diagnosis of early LNB the demonstration of antibody intrathecal synthesis is needed; serum and CSF sampling should be performed on the same day and they should be tested together. To prove intrathecal production of B. burgdorferi specific antibodies calculations that consider blood/CSF-barrier dysfunctions (AI) based on quantitative ELISA are used, which determine the level of specific antibodies from CSF and serum, and the total antibodies concentrations (from CSF and serum). A positive AI index along with typical inflamation signs in CSF confirms a clinical diagnosis of LNB [61]. The serological tests for early LNB diagnosis can have some limitations: (1) in some early LNB cases, the antibodies are detectable in CSF, but absent in serum, because specific response in intrathecally antibodies in CSF can be positive before seroconversion occurs in peripheral blood (especially in children), and in CSF inflammation signs are present [70], (2) Igm result can be false-positive due to oligoclonal stimulation, (3) IgG and IgM antibodies can persist for years after successful therapy [71]. Also, a positive AI index can persist for years after Borrelia infection remission from CNS, whereas other signs of inflammation tend to disappear in a few months (up to 12) [41, 72]. IgM antibodies intrathecally synthesized show a high sensitivity in early NBL of short duration especially in children [73]. However, a false positive IgM reactivity was observed in Eppstein-Barr virus meningitis [70]. The presence of specific antibodies for B. burgdorferi in the CSF with proofs of intrathecal synthesis is standard traditional diagnostic, but has limitations such as low sensitivity in the very early phase of the disease [74] and their persistence for years after infection eradication. [73]. Therefore, antibody testing should be performed only for patients who have symptoms suggestive of LNB (see case definition in Table 3). The diagnostic sensitivity of AI is about 80% for the LNB of short duration (80% positive at presentation) or paired sera for seroconversion or rise in antibody titre. Intrathecal specific antibodies and specific CSF/serum antibody index. Single serum IgG test. Intrathecal specific antibodies and specific CSF/serum antibody index Single serum test (>90% positive at presentation) or paired sera for seroconversion or rise in antibody titre Single serum test (>90% positive at presentation) or paired sera for seroconversion or rise in antibody titre Single serum IgG test

Occasionally in CSF on expert advice

immune complexes, lymphocyte transformation test, cyst formation, lymphocyte markers [32]. For the diagnosis of chronic LNB of CNS it is essential to demonstrate intrathecal antibody production by determination of the specific titers of antibodies in serum and CSF samples collected on the



PCR

Occasionally in synovial fluid on expert advice

Not indicated

Of tissue

Of tissue

High specific IgG antibody levels occur in serum. Granulocytic cell response in synovial fluid. PCR of synovial fluid may be useful in antibiotic-refractory arthritis. Lymphocytic CSF pleiocytosis is characteristic. Seroconversion within 2-3 weeks of initial seronegativity. PCR of CSF positive in only 10-30% of acute neuroborreliosis. Lymphocytic CSF pleiocytosis is usual. Raised CSF total protein and oligoclonal bands. PCR of CSF is rarely positive PCR Seroconversion occurs within 2-3 weeks of initial seronegativity PCR of tissue positive in about 80% of untreated cases. Histological analysis useful but not for diagnosis Very high characteristic IgG antibody levels. PCR of tissue positive in >90% of untreated cases. Histological analysis useful but not for diagnosis

same day. A positive IgM without IgG antibodies argues against the diagnosis of chronic NBL. In late LNB, the diagnostic sensitivity of an ELISA screening test for IgG (IgM is not useful in the diagnosis of late form) is > 90-100% [75-79]. 143

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