serodiagnosis of lyme borreliosis

Clinical Microbiology Newsletter

Vol. 15, No. 11

Serodiagnosis of Lyme Borreliosis Paul S. Duffey, Ph.D. Jesse Salugsugan, B.S. Microbial Diseases Laboratory California Department of Health Services Berkeley, CA 94704

Lyme borreliosis (Lyme disease) is a tick-borne, multisystem, infectious disease caused by Borrelia burgdorferi (1). It is distributed worldwide and currently is regarded as the most prevalent vector-borne disease in the United States, where it is endemic in 41 of 50 states (2). The reported incidence car- rently ranges from 0.9 per 100,000 to 42 per 100,000 population, although rates above 100 per 100,000 have been recorded (3). However, nine states ac- count for over 97% of the reported cases (1, 2). Paradoxically, Lyme disease is regarded as overdiagnosed, yet underreported (3, 4). This, in part, may be due to incorrect use and interpretation of laboratory tests.

Diagnosis Diagnosis depends on identifying

characteristic clinical signs, together with demonstration of a compatible history of exposure to presumptively infected Ixodes spp. vectors (1, 5). Like other spirochetal diseases, Lyme borre- liois exhibits early, intermediate, and late stages (5). Vigorous antibiotic therapy is advised, since >_80% of early disease progress to late forms of disease (5, 6).

Early Lyme disease may be unrecog- nized or asymptomatic, so that diagnosis may not occur until the intermediate or late stages (1, 4). Also, the causative agent, B. burgdorferi, may be found in

patients who do not exhibit concurrent inflammatory signs compatible with Lyme disease (7). Intermediate and late stages, especially, may mimic other diseases that are not relieved by appropri- ate antibiotic therapy (6). Under surveillance case reporting guidelines issued by the U.S. Public Health Serv- ice, Centers for Disease Control (CDC) (8), laboratory confirmation is required in the absence of known or probable exposure to infected ticks. Also, without obviously compatible clinical signs, laboratory tests may be essential (5).

Labo ra to ry Tests Nonspecific laboratory methods are

useful in diagnosing Lyme disease (4, 9, 10). Specific methods acceptable under CDC guidelines include identification of B. burgdorferi in or its isolation from patients and demonslration of a serological reaction with B. burgdorferi antigens (8).

Spirochetes in tissue may be visual- ized with histochemical stains (10); or spirochetes resembling B. burgdorferi may be identified through immunohisto- chemical methods using antibodies reactive with B. burgdorferi and related antigens (10); or an antigen-capture assay may be used to find antigens resembling those ofB. burgdorferi in urine or other specimens (11). B. burgdorferi also may be isolated by culture (12). These methods have low sensitivity (not greater than 15%) (13). Only isolation ofB. burgdorferi is def'mitive, but requires up to 6 wk to complete (12). Polymerase chain reaction (PCR) assays that identify nucleic acids unique

June 1,1993

to B. burgdorferi (14) may be sensitive and specific but require further evaluation.

A serological response to spirochetal antigens may be demonstrated in several ways (10). Barbour and associates (15) reported on an immunofluorescence assay (IFA) in 1983 using B. burgdorferi as antigen and Russell et al. (16) reported on an enzyme-linked immunosorbent assay (ELISA) in 1984. Commercial assay kits soon appeared. By 1990, more than 16 manufacturers marketed over 20 different test kits (17). Although other procedures exist, the most common tests are ELISAs and IFAs.

ELISA The ELISAs are generally per-

formed, using a predetermined serum dilution, in microtest plates or on plas- tic "dipstick"-type devices, and are read

In This Issue

Serodiagnosis of Lyme Borreliosis . . . . . . . . . . . . . . . . . . . . 81 An overview of the advantages and limi- tations on the available tests for the serodiagnosis of L yme disease

Pulmonary Infection due to Myco- bacterium malmoense in an Im- munocompromised Patient . . . . . . 86 A case report

Letters to the Editor . . . . . . . . . . . . 87

CMNEE/15(11)81-88,1993 Elsevier 0196-4399/93/$0.00 + 06.00

visually, by comparison with a color standard, or photometrically, with values related to the absorbance of an internal, positive control (16, 17). For the instrumented methods, absorbance above a threshold level is considered positive, and the ratio of test to control is calculated with a specified algorithm and used to express the result. One vari- ety of ELISA, the Western blot (WB) assay, employs electrophoretically sepa- rated antigens electroblotted to a mem- brane support, on which the patient's serum or CSF is reacted (18). Interpreta- tion is based on the number, molecular size, and staining intensity of individual antigen bands found. The WB is generally read visually, but can be read using a scanning deusitometer. Its advantage is that one can determine which of several different antigens is recognized by antibodies in the patient's serum (19). ELISA antigens are proprietary whole cell sonicates of B. burgdorferi, of unre- ported composition, which may have been altered by either enriching or re- moving some antigen fractions. Recom- binant antigens also are under study (20-22).

IFA The IFAs use dried, whole B. burg-

dorferi cells as antigens. The test is read using a fluorescence microscope by reference of the test fluorescence to a standard (15). Either a series of dilu- tions or a single dilution of the patient's serum, representing the threshold dilution, is tested. The endpoint of a posi- five, semiquantitative test is the highest dilution of specimen that is estimated to reproduce the reactivity of the positive control. A fluorometric IFA test, first reported by Hechemy et al. (23, 24), uses a sonieated antigen adsorbed to a plas- tic "dipstick" device. A predetermined serum dilution is used, and the test is read in a proprietary fluorometer. Fluorescence above a threshold value is positive.

Since sera from most uninfected indi- viduals react in this test (25), a reactivity threshold must be set to reduce positives due to nonspecific reactions. Where this threshold is set determines the sensitivity and specificity of the test (19). Unfortunately, each manufacturer may specify a different dilution of serum, and use different antigens and dilu- ents, all of which affect reactivity. Moreover, each evaluates test reactivity using unique reference specimens and methods for setting the threshold. Thus, the tests are not equivalent, although their reactivities overlap. This being so, one cannot expect that assays of a given serum using any two tests will necessar- ily yield agreement. Although it is expected that marginally reactive specimens would most often provide discrepant results, sera found strongly positive in one brand of test may be negative in another (26). Several re- ports have indicated that both intra- and interlaboratory agreement using commercial test kits and in-house assays are poor. In a cooperative interlaboratory evaluation of seven different commercial test kits using a large panel of sera (17, 27), no two kits tested agreed com- pletely. Yet some laboratories using different assays also showed good agreement (27), suggesting a need for improved quality control.

Test Reproducibi l i ty Good test reproducibility requires a

strict quality control regimen. Pho- tometrically read, microplate-based ELISAs are preferred, as they are not interpreted subjectively (28). Experience gained in our laboratory confirms that applying consistent methods is impor- tant in reproducing results. An auto- mated microplate washer and micro#ate agitator should be used. Regular validation of proper equipment function within predetermined toler- ances is essential. Achieving reprodnc- ible incubation temperatures requires

that cold reagents be allowed to warm to a consistent temperature before use, and that a tolerance be established for the permissible range of ambient room temperature in which the assay is per- formed. Reagents used many times should, when possible, be distributed into small, tightly capped vials containing sufficient reagent for a single day's use (i.e., control sera, conjugate, sorbent). The pH and clarity of all reagents should also be within acceptable tolerance limits at the time of use (if the tol- erances are not specified in the manufacturer's data sheet, the laboratory must establish them). Evaporation from microplates occurs unevenly, generally being greater near the edges of the plate, which may affect test results. Accordingly, all wells, whether used or not, should be filled uniformly with buffer and the plates sealed during incubation. Also, the plates should be visually inspected before and after each step. Wells noticeably different in volume should be marked and results not reported.

Although the commercial test kits contain internal standards, use of an external control serum panel also is advisable. The external panel should include at least a known, strongly positive sera, a minimally reactive serum with mean reactivity approximately 1 to 2 standard deviations above the positive threshold. and a negative control reactive between 1 and 2 standard deviations below the threshold. These control sera, which should be validated by a trusted reference laboratory, are also used to vali- date new kits or reagents by testing them in parallel with the current reagents. It is advisable to include sera from previous test runs that are reactive at various levels to serve as reproducibility controls. Specimens and controls should be tested at least in duplicate, distributed randomly on the plate (i.e,, duplicates not in adjacent wells). To be reported, the test run must reproduce

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82 0196-4399/93/$0.00 + 06.00 © 1993 Elsevier Science Publishing Co., Inc. Clinical .Microbiology Newsletter 15:11,1993

TABLE 1. Reproducibility of an IgG-specific Lyme ELISA: Optical densities (O.D.)" Standards Mean S.D. Range +1 S.D. -1 S.D.

P1-T b'c 0.43 0.06 0.34 to 0.55 0.49 0.37 P1-Pa 0.41 0.04 0.35 to 0.49 0.46 0.37 P2-T 0.42 0.06 0.33 to 0.57 0.48 0.37 P2-P 0.41 0.04 0.35 to 0.48 0.45 0.37 M1-T 0.11 0.00 0.10 to 0.12 0.12 0.11 M1-P 0.11 0.00 0.10 to0.11 0.11 0.11 M2-T 0.11 0.01 0.09 to 0.12 0.12 0.10 M2-P 0.11 0.00 0.09 to 0.11 0.11 0.10 Neg-T 0.00 0.00 - - 0.00 0.00 Neg-P 0.00 0.00 - - 0.00 0.00 R3250 e 0.01 0.00 0.01 to 0.02 0.01 0.01 R3077 0.03 0.00 0.03 to 0.03 0.03 0.03 R4176 0.11 0.01 0.10 to 0.12 0.12 0.10 R2794 0.11 0.00 0.09 to 0.12 0.11 0.10 R2868 0.17 0.01 0.09 to 0.18 0081 0.16

a Values have the blank subtracted, and are rounded to the nearest 0.0l.

b p 1, P2 are positive controls; M 1, M2 are minimally reactive controls; Neg is the negative control serum.

c -T indicates results for 37 duplicate experiments, which include control values that exceed the range of+l S.D.

d _p indicates results for 31/37 duplicate experiments, excluding six experiments for which the control values exceeded +1 S.D.

• R prefix samples are specimens assayed in 14-27 of the assays.

the manufacturer's controls within tolerance, reproduce the known positive within specified limits (e.g., +1 S.D.), and reproduce the expected minimal reactive and high non-reactive control re- suits (+1 S.D.). Participation in a proficiency evaluation program serves as an external validation of test accu- racy. A well-controlled assay should closely reproduce specimen results on repeated testing (Table 1). Large deviations indicate that the test run should be repeated after determining the cause. Graphs of controls should be used to detect drift in reactivity that may be caused by reagent evaporation (for ex- ample, see Fig. 1). Individual specimen results are reported if both members of a pair yield the same result, i.e., both are negative or positive and statistically equivalent. Otherwise, they must be retested.

Test Interpretation Interpretation of serologic tests re-

quires knowledge of their sensitivity, specificity, and reproducibility. How- ever, these estimates are also subject to error. Control procedures establish the reproducibility limits of the test as per- formed in a particular laboratory. Data

provided by manufacturers have been obtained in good faith on random pan-

els of sera by reference to existing IFAs, ELISAs, or WB assays. The reference tests, in turn, were most likely standardized by reference to other existing tests. Test sensitivity and specificity as determined by reference to a confined clinical diagnosis of Lyme borreliosis (28, 29) were 53 to 80% and 37 to 84%, respectively, depending on the stage of the disease. Thus, tests with sensitivities and specificities claimed to be 80 to 97% are, using the combined probabilities (30), 45 to 65% and 30 to 67%, respectively, at most (27). These values suggest (30) that between 8 of 1,000 and 20 of 1,000 positive tests obtained at random in a population with a 1% disease preva- lence (greater than that found anywhere in the United States) would, statistically, be Lyme disease. Put bluntly, the currently available tests are useless for screening large populations, and should be used only when highly suggestive clinical signs ace present. Even then, the test does not stand alone, and should be interpreted only after ancillary testing has been done and the re- suits of which support or refute the serological result.

Figure 1. Pos#ive control serum reactivity. Duplicate positive control optical density (O.D.) values (vertical) were plotted against run date (horizontal)for 37 runs. New vials o f control serum were opened on 3-27, 6-5, 8-20, and 12-18. The vertical drift of the plotted points between those dates is consistent with evaporation o f the control serum, indicating the need to use fresh via!s of control sera. This behavior also suggested the need to aliquot control sera in smaller portions in order to avoid this problem. Points that feU above or below the dotted lines were considered to be outside the control limits for the test, and results from those runs were not reported.

Lyme Elisa Positive (IgG) Control (January 8, 1991 - December 27, 1991)

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Clinical Microbiology Newsletter 15:11,1993 @ 1993 Elsevier Science Publishing Co., Inc. 0196-4399/93/$0.00 + 06.00 83

Competitive Inhibition of Lyme IgG Ab. W i t h B. b u r g d o r f e r i ( B 3 1 A n t i g e n )

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Figure 2. Effect of soluble B. burgdorferi antigen-antibody aggregates on ELISA reactivity. A positive serum with an ELISA optical density ( 0 29. ) ratio of ca. 1.00 was diluted 1:500 in diluent containing graded amounts OfB. burgdofferi B31 sonicate, and allowed to react for 2 h at room temperature to generate soluble antigen-antibody aggregates. A 1:500 dilution in buffer with no B31 sonicate was also prepared as a control. These samples were tested in quadruplicate by the standard L yme disease ELISA. The percent inhibition was calculated as 10011 - (0 2). test/ O 29. control)] and plotted (vertical axis) against the amount Of B31 sonicate (horizontal axis). As the amount of antigen available to aggregate with antibody increased, the ELISA react&ity decreased. Sera that contain a high proportion of soluble B. burgdorferi antigen relative to the amount of antibody available, so that most of the antibody is contained in antigen-antibody aggregates, may be expected to react poorly or not at all in serological assays.

Currently available tests fast detect antibodies in serum about 2 to 4 wk fol- lowing the primary infection in fewer than half the patients (31), but antibiotic therapy may delay or abrogate this early response (31). The response exceexls 80% in later stages of disease (31). Pres- ence of an erythema migrans rash corre- lates negatively with detectable antibodies while the rash is present (27). The sera of some patients with early stage disease and the CSF of oth- ers with CNS involvement contain antigen-antibody aggregates rather than free antibodies (32, 33). Also, about 20% of confirmed intermediate and late stage Lyme disease patients fail to de- velop detectable serum antibodies, although patients with CNS involvement may, nonetheless, have oligoclonal anti- B. burgorferi antibodies in the CSF (34, 35). Unfortunately, commercial test kits available in the United States are not presently approved for use with CSF.

The earliest serum antibodies detected are typically IgM-class, and react al- most exclusively with the 41 kD endo- flagellar antigen of B. burgdorferi (3 I). An IgM-specific test that uses a purified 41 kD antigen alone improves the likeli- hood of detecting a response during early disease (19). As the disease progresses past 6 to 8 wk, IgG-, then IgA- class antibodies appear as IgM-class antibodies decline (31). However, IgM- class antibodies also may be found in intermediate- and late-stage disease (31). As the disease progresses, the number of different antigens recognized also increases (31, 36), but the recognized mix differs among patients (36, 37). The ap- propriate test for suspected intermediate-to-late-stage disease is IgG-specific or polyvalent and uses flagella or a com- plex B. burgdorferi antigen mixture (19). Negative results must be considered in light of the probable disease stage, and with knowledge of the kind

i i

of test used (i.e., IgM-specific/flageUar, etc.). Also, in early disease with compatible clinical signs, an evaluation of complement levels and (whole, or C3) total IgM levels and an assay for aggre- gated immunoglobulins should be done (5, 31, 38). Positives in these assays are suggestive of Lyme disease.

Cross React ions Antibodies to other members of the

genera Treponema and Borrelia may cross-react in Lyme disease tests. Known cross-reactors include Tre- ponema pallidum, the causative agent for syphilis (39), Borrelia recurrentis, and B. hermsii and B. parkeri, the causative agents of louse-borne and tick-borne relapsing fever (39, 40), the latter endemic in the western United States; and also Treponema vincentii, B. buccale, and T. denticola, found in the mouth, where they may be associ- ated with ulcerative or inflammatory disease (31). Cross-reactions with T. pallidum are most common (39, 40) (ca. 22% on the average). Cross-reactions also occur with human transplanta- tion antigens (HLA) (41, 42). Elimination of cross-reactive syphilitic sera from syphilis patients, alone, increases the test specificity to ,297% (39). Thus, all Lyme test-positive specimens should be retested by the VDRL or RPR tests before reporting (39, 40), since B. burgdorferi infection does not cause significant increases in non-treponemal antibodies (43). Positive VDRL or RPR tests may indicate either present or past syphilis, or a biological false-positive reaction (44). Positive VDRL or RPR tests should be confirmed using the MHA-TP, which does not react with Lyme antibodies (39). A positive MHA-TP indicates current or prior syphilis (notwithstanding, the patient may also have Lyme disease), while a negative test argues against syphilis. In this case, a WB showing no reactivity against the 41 kD antigen would argue against Lyme disease as well (31), while a strongly stained 41 kD band, together with at least three other bands in the 16to 21, 34 to 38, and 60 to 100 kD ranges, is consistent with a specific response to B. bur gdor- feri (18, 19). The possibility of current or past relapsing fever must be consid-

84 0196-4399/93/$0.00+ 06.00 © 1993 Elsevier Sclence Publishing Co.. Inc. Climca! Microbiology Newsletter 15:11A993

ered in persons who may have been ex- posed to B. recurrentis (39), and persons with a recent history of necrotizing ulcerative gingivitis or peri- odontitis should also be confh'med by WBA (31, 39, 40). Similarly, multiply transfused patients, multiparous female patients, or patients with autoimmune diseases may have cross-reactive anti- HLA antibodies. Positive ELISA re- suits in these patients should be conf'mned by WB, if possible, and ap- propriate tests to rule out autoimmune diseases should be done. Patients who are HIV positive, or who have been re- cently immunized against certain vi- ruses, may produce polyclonal antibodies reactive with B. burgdorferi antigens (45, 46).

Many sera contain antibodies reactive with B. burgdorferi antigens (31, 36). These may be due to shared epitopes present on related or unrelated bacteria (31). However, recombinant antigens that contain only the B. burgdorferi-specif ic epitopes, when used in ELISA assays, do not exhibit nonspecific- or cross-reactivity (20-22). Test kits using such antigens promise specificity approaching 100% with improved sensitivity. Nonetheless, careful quality control, validation of test results with ancillary methods, and consideration of the patient's history remain essential in effectively using serological assays for Lyme disease diagnosis.

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22. Simpson, W.J. et al. 1991. Antibodies to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals. J. Clin. Microbiol. 29: 236-243.

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in immune complexes in seronegative Lyme disease. Lancet 335:412-315.

33. Coyle, P. K. et al. 1990. Cerebrospinal fluid immune complexes in patiaats ex- posed to Borrelia burgdorferi: detection of Borrelia-specific and -nonspecific complexes. Ann. Neurol. 28:739-744.

34. Hansen, K., N. Cruz, and H. Link. 1990. Oligoclonal Borrelia burgdorferi- specific IgG antibodies in eerebrospinal fluid in Lyme neuroborreliosis. L In- fect. Dis. 161:1194-1202.

35. Steere, A. C. et al. 1990. Evaluation of the intzathecal antibody response to Bor- relia burgdorferi as a diagnostic test for Lyme neuroborreliosis. L lnfecL Dis. 161:1203-1209.

36. Ma, B. et al. 1992. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies. L Clin. Microbiol. 30:370-376.

37. Nadal, D., Ch. Tavema, and W. H. Hitzig. 1989. Immtmoblot analysis of antibody binding to polypeptides of Borrelia burg- doffed in children with different clinical manifestations of Lyme disease. Pediaa'. Rcs. 26: 377-382.

38. Hardin J. A. ct al. 1989. Circulating immune c~nplcxes in Lyrnc arthritis: detection by the 1251-Cl-binding, Clq solid phase, and RAJI cell assays. J. Clin. In- vest. 63:468---477.

39. Wilkinson, H. et al. 1985. Caveats on using nonstandardized serologic tests for Lyme disease. J. Clin. Microbiol. 21:291.

40. Magnarelli L. et al. 1990. Cross-reactivity of nonspccific treponemal antibody in serologic testing for Lyme disease. J. Clin. Microbiol. 28:1276-1279.

41. Abcrer, E. et al. 1989. Molecular mim- icry and Lymc borreliosis: A shared an- tigenic determinant between Borrelia burgdorferi and human tissue. Ann.

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42. Stccrc, A. C., E. Dwycr, and R. Win- chester. 1990, Association of chronic Lyme arthritis with HLA-DR4 and HLA-DR2 alleles. N. Engl. L Med. 323:219-223~

43. Mackworth-Young, E.N. et. al. 1988. Anficardiolipin antibodies in Lyme disease. Arthritis Rheum. 31:1052-1056.

44. Catterall, R.D. 1972. Presidential ad- dress to the H.&S.V.D. Systemic disease and the biological false positive reaction. Br. L Vener. Dis. 48:1-12.

45. Raolt, D. et al. 1989. Cross-reaction with Borrelia burgdorferi antigen of sera from patients with human immu- nodcficiency virus infection, syphilis and leptospirosis. J. Clin. Microbiol. 27:21522155

46. Feder, H.M. Jr., eL al. 1991. False positive serologic tests for Lyme disease after variceUa infection. N. EngL J. Med. 325:1886-1887.

Case Report i

Pulmonary Infection due to Mycobacterium malmoense in an Immunocompromised Patient

Christophe Burucoa, M.D. Anne Bourgoin, M.D. Monique Casters, M.D. Laboratory of Microbiology Regional Hospital Centre Poitiers, France

Pierre Dot6, M.D. Didier Recart, M.D. Department of Pneumology Regional Hospital Centre Poitiers, France

V6ronique Vincent, Ph.D. Tuberculosis and Mycobacteria Unit Pasteur Institute Paris, France

Mycobacterium malmoense was first described in 1977 by Schr6der and Juh- lin (1) as a new pathogenic, nonphoto- chromogenic Mycobacterium sp. They reported four cases of pulmonary dis-

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ease caused by this species in Maim& Sweden. Since 1977, M. malmoense has been isolated with increasing fre- quency from clinical specimens, especially in northwestern Europe (2). To our knowledge, only one patient infected with M. malmoense has been documented in France (3), We report here another French case of M. malmoense pulmonary infection.

Case Report A 40-yr-old man was admitted to the

hospital with a 3-wk history of weight loss (7 kg). Four years before, he had undergone a bilateral pleurectomy for a spontaneous pneumothorax, and the bi- opsy of pleura had been negative on smear for acid-fast bacilli. At admission, he presented with productive cough, fever, and chest pain on the right side. A chest radiograph showed multiple cavities in the two upper lobes. He had a nonreactive tuberculin skin test and was negative for HIV antibody. Bronchial secretions and two sputum smears contained few acid-fast bacilli (1+ according to the International Un-

© 1993 E l sev ie r Sc ience Publ i sh ing Co. , Inc.

J

ion Against Tuberculosis standard). De- spite treatment ini'dated with isoniazid, rifampin, and ethambutol, the patient did not improve and continued to lose weight. A tetraparesisand pyramidal syndrome appeared and the patient died 1 mo after admission. The necropsy showed metastatic cerebral epidermoid carcinoma of unknown origin.

Microbiology Middlebro~ 7H12 broth was used

for culturing the clinical specimens and mycobacterial growth was detected in a BACTEC 460 instrument (Becton Dick- inson, Sparks, MD). In addition, LOwen- stein-Jeusen medium was inoculated. Three positive cullmcs were detected using the BACTF_~ system after 14, 27 and 56 days of incubation at 37°C, whereas no growth was seen on solid medium, No other pathogen was isolated. The positive prinmry cultures were referred to the mycobacteriology laboratory of the Pasteur Institute in Paris for complete identification. Thin- layer chromotography of the surface lipids and the cellular fatty acid compo-

Clinical Mic rob io logy News l e t t e r 15:1 ! , | 993

serodiagnosis of lyme borreliosis

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