Methods Comparison for Diagnosis of Lyme Disease

Transcription

1 MICROBIOLOGY Philip M.Tierno, Jr, PhD Jessie Cadet-Legros, MS Methods Comparison for Diagnosis of Lyme Disease Unfortunately, the hallmark rash may be atypiabstract Despite the tendency to rely considerably on cal or absent, and the disease may be indistinlaboratory test results for the differential diagnosis of Lyme guishable from a nonspecific viral illness. Initially disease, considerable problems exist with such testing. In following a tick bite, B burgdorferi multiply in the skin and are found at the margins of the EM lesion this article we compare the effectiveness of five main categories of laboratory tests used for the detection of Lyme or secondary annular lesions. Simultaneously, B burgdorferi is disseminated through the blood to disease. Our goal is to enable the reader to better undertissues of the nervous, cardiac, musculoskeletal, stand the limitations of different test methods and to realize and urinary tract systems potentially resulting in that until better tests become available, diagnosis of Lyme more serious complications.5"8 For unknown readisease still must be made clinically. Clinical data, however,sons, B burgdorferi has an affinity for these sites. can be used to increase the predictive power of any labora- Because Lyme disease may mimic other diseases, clinicians rely heavily on laboratory test results for tory test as well as to help judge its validity. differential diagnosis.3 From the Department of Microbiology, New York University Medical Center-Tisch Hospital, New York. Reprint requests to Dr Tierno, Department of Microbiology, TH374, New York University Medical Center-Tisch Hospital, 560 First Ave, New York, NY According to results of a recent survey, Lyme disease (Lyme borreliosis) was misdiagnosed in almost 77% of all cases.1 Diagnosis of Lyme disease is difficult for clinicians and laboratorians alike. The disease's clinical presentation is indistinct and variable, and no definitive laboratory test exists. Lyme disease first was described in Europe in the early 1920s and in the United States in "3 The term Lyme disease was coined in '2 In 1982, Burgdorfer and Barbour isolated a previously unrecognized spirochete, now called Borrelia burgdorferi, from Ixodes dammini ticks. 4 This spirochete was recovered from patients with Lyme disease in the United States and Europe.4 Today, Lyme disease is the tickborne illness reported most often in the United States and Europe.5 Lyme disease manifests in many ways; thus, it is confused easily with other diseases.1'5"7 The earliest and most common clinical manifestations are the result of a localized infection. A characteristic rash called erythema migrans (EM) occurs in 60% to 80% of patients. The rash often is accompanied by mild, nonspecific, flulike symptoms such as malaise, headache, arthralgias, and myalgias. on 18 February 2018LABORATORY MEDICINE VOLUME 27, NUMBER The best clinical marker for the disease is the initial rash.9 The Centers for Disease Control and Prevention (CDC) clinical case definition for Lyme disease is fulfilled if either EM or at least one late manifestation of disease, such as musculoskeletal, nervous, or cardiovascular system involvement, and laboratory confirmation of infection, are present. Serology more appropriately confirms cases retrospectively, rather than prospectively, because available tests are neither sensitive nor specific. During the first few weeks after infection with B burgdorferi, Lyme serology may be nonreactive in about half of all cases.10'11 About one quarter of cases may be positive by serology, which can be confirmed by Western blot. The remaining quarter of cases may be weakly reactive by serology but negative by Western blot. This latter group of test results causes confusion. Many individuals have indigenous microflora that share common epitopes with B burgdorferi, which may cause a false-positive result. For example, initial immunoglobulin M (IgM) and IgG responses can occur to these common epitopes, particularly those that cross-react with the 41-kd flagellin antigen. This antigen has an amino acid sequence similar to other "normal flora" bacterial flagellins as well as to human tissue. The laboratory can

2 s u p p o r t only the clinical diagnosis of Lyme disease owing to variability in the sensitivity and specificity of these tests. Despite these problems, the most m e a n i n g f u l d i a g n o s t i c results are obtained when clinical findings are integrated with laboratory results. Two main types of laboratory procedures can be performed (Table 1): 1. Direct demonstration of 5 burgdorferi organism or its antigens 2. Serologic detection of anti-b burgdorferi antibody by immunoassay procedures. Direct Demonstration As with other infectious diseases, definitive diagnosis of Lyme disease can be made by recovering and culturing B burgdorferi from the patient. Culture of the causative agent remains the gold standard test for the diagnosis of Lyme disease. In the case of Lyme disease, however, culture of the specimen as well as direct visualization of the spirochete is difficult and therefore not practical. Borrelia burgdorferi also has been reported to be cultured from peripheral blood, cerebrospinal fluid (CSF), and s y n o v i u m, b u t the c u l t u r e p r o c e s s is difficult a n d yields i n c o n s i s t e n t results. 10 Borrelia burgdorferi can be isolated in a cell-free medium, although culture requires several weeks for growth to occur. While infected ticks can be triturated (rubbed to a powder) and cultured with some success, the yield from human specimens is too low to be useful diagnostically. The medium of choice for culture is Kelly's modification of Barbour's medium for spirochetes. 2 A sensitive polymerase chain reaction (PCR) technique has been developed for the detection of B burgdorferi, but it is of limited use owing to the lack of organisms found in humans. No matter how sensitive a PCR technique is, it is useless if no o r g a n i s m is p r e s e n t in t h e test s a m p l e. Nevertheless, a recent study demonstrated the potential importance of PCR. In this study, little or no B burgdorferi antibodies were found in patients who tested positive for this disease t h r o u g h culture or PCR tests w h o had been afflicted with late-stage Lyme disease for years. 12 Polymerase chain reaction or culture therefore might prove advantageous in such a patient population. In the near future, it may be possible to use culturing as an early detection method when combined with PCR. Polymerase chain reaction may prove to be the most sensitive method for detection of spirochetal DNA. Current research t e c h n i q u e s based on TABLE 1. LABORATORY DETECTION PCR have n o t b e e n OF LYME BORRELIOSIS a p p r o v e d by the Direct Methods Food and Drug Culture (Kelly's modification of Administration and Barbour's medium) c a n n o t be used for Histologic stain (eg, Warthin-Starry) diagnostic purposes in Capture antigen test t h e r o u t i n e clinical Polymerase chain reaction laboratory. Immunoassay In serial dilution Indirect immunofluorescence experiments with Enzyme-linked immunosorbent assay k n o w n n u m b e r s of Photometrically read 13 organisms, Rys has Fluorometrically read shown that the Capture antibody test genomic and plasmid Immunoblot (eg, Western blot) PCR targets provide comparable sensitivity. Synovial fluids provided the most consistently positive results by amplifyinga B burgdorferi plasmid target (OspA). For reasons unknown, genomic targets often are negative on these samples. The sensitivity of PCR synovial fluids from p a t i e n t s with Lyme a r t h r i t i s approaches 80%. Nocton recently has shown that PCR may replace culture in testing the synovial fluid in Lyme arthritis. 1 4 In addition, PCR results may guide the clinician in making therapeutic decisions for patients with persistent Lyme arthritis despite multiple courses of antibiotic therapy. 14 Positive results also may be confirmed by using multiple independent DNA target sequences, 13 including flagellin,15 DNA, 16 and OspA, 17 but the choice of target sequence will determine assay sensitivity and specificity and may not yield equivalent results for all specimen types. 13 Urine, blood, and tissue samples from patients may contain spirochete antigens. An antigen capture test uses antibodies with broad reactivities to multiple epitopes of B burgdorferi to capture spirochetal antigens on a solid surface. A specific antispirochete antibody, usually to the outer surface protein A (OspA) or outer surface protein B (OspB) of the spirochete, then is added to detect B burgdorferi antigens. A specific antigen test for urinary B burgdorferi also has been described, but this test has yet to prove its effectiveness.5 Histologic stains such as Warthin-Starry may be used to stain organisms for visualization with light microscopy. In many cases, however, too few organisms can be isolated to be stained. In addition, some host tissue components may stain similarly to loosely coiled Borrelia. on 18 February 2018 VOLUME 27, NUMBER 8 LABORATORY MEDICINE in C 0 \ 0 e 3 E o o u t4> I

3 Immunoassays By default, the diagnostic method of choice for the detection of B burgdorferi is immunoassay. Four categories of immunoassays exist: 1. Indirect immunofluorescence assays (IFA) 2. Enzyme-linked immunosorbent assays (ELISAs), either photometrically or fluorometrically read 3. Capture antibody assays 4. Immunoblot/Western blot techniques regarding serology, a small percentage of the general population does not produce antibodies as a result of infection. If Lyme disease still is suspected, B- and T-cell function studies should be performed. Indirect Immunofluorescence Assay The first clinical diagnostic method used to detect antibodies to B burgdorferi was IFA. As with most fluorescent microscopic techniques, the effectiveness of IFA can only be Borrelia burgdorferi is antigenically complex with ensured if individuals trained in immunofluoresstrains that vary considerably; this causes the lim- cence perform the procedure. Otherwise, itations of serologic procedures. 18 ' 19 Humoral considerable subjectivity can be introduced. In antibody response to B burgdorferi has been char- experienced hands, however, IFA is an acceptable, acterized as complex and variable for different accurate, and highly sensitive technique for individuals.5 Another confounding factor relates detecting antibodies to B burgdorferi. If used to the IgM response. Although the IgM response properly, IFA techniques are about 10 times more peaks on the first month after symptoms, IgM sensitive than photometric ELISA methods but 3 may remain high throughout the course of illness times less sensitive than fluorometric ELISA 22,23 While some patients with rheumatin some patients, while in others it may decline. methods. The earliest immunologic response to B burgdor- ic disease have been reported to exhibit falseferi is directed against the 22- to 25-kd OspC anti- positive reactions, an experienced fluorescence gens and the 41-kd flagellin antigen. As men- microscopist may find these results to be caused tioned previously, a cross-antigenicity exists by an abnormal fluorescence pattern (ie, beaded). between the flagellin antigens of B burgdorferi Thus, the results can be excluded as true and common indigenous gram-negative positives. bacilli as well as other spirochetes. This comunfortunately, other problems related to the pounds the difficulty in interpreting serologic use of IFA exist that are not controlled as easily as results. Antibodies to OspA (31 kd) and OspB (34 the above. The most serious problems are related kd) are highly specific and may be abundant. to variation among manufacturers' preparation Unfortunately, response to these protein antigens of kits. Most IFA kits use whole B burgdorferi varies and usually occurs late in the course fixed to a slide using one of several methods that of disease.10 can affect interpretation of results. Patient serum The 45- and 39-kd proteins are major B is added. Binding of human antibodies to antigen burgdorferi antigens and are fairly specific, if anti- is detected by the addition of fluorescein-conjubody to them appears at all. European strains are gated antihuman Ig. Indirect immunofluorescharacterized by 30-, 31-, and 94-kd proteins. cence assays are most useful in a relatively small Response to these proteins varies greatly as well. immunofluorescence laboratory with a low samheat shock proteins 60-to 72-kd do not appear to ple volume and skilled technical personnel. These be specific and are of limited use. About 30 dif- limitations make IFA impractical for use on a ferent antigens have been identified, but antibod- large scale. ies have only been detected against 18 of them.20 In addition to the variability in individual Enzyme-Linked Immunosorbent Assay response to B burgdorferi proteins, some appar- The subjectivity of IFA is eliminated through use ently healthy individuals have low levels of of an ELISA technique. Enzyme-linked indigenous cross-reacting antibodies. This adds immunosorbent assays are read colorimetrically to the error in interpreting results. or fluorometrically and can be automated. Some individuals appear to have a long delay The ELISA technique is useful for laboratories in developing antibodies to Lyme disease anti- with high volumes. The technique is used most gens.21 It therefore is important to draw repeated often for the diagnosis of Lyme disease and is samples for 1 to 3 months and for as long as 6 useful for detection of IgG and IgM. For this promonths after initial negative results have been cedure, microtiter wells are coated with different reported.21 In spite of the efforts one may make B burgdorferi antigens depending on the manufacturer. A great deal of strain variation and antigenic complexity intrinsic to B burgdorferi exists, 544 on 18 February 2018LABORATORY MEDICINE VOLUME 27, NUMBER 8

4 however. Even the same strain may or may not express specific antigens at different times. This fact, combined with the variation that exists with humoral host response (a type of immune response) and different manufacturing procedures, explains why results vary among laboratories. Lastly, some manufacturers "spike" their kits by adding specific antigen to the milieu to increase sensitivity, specificity, or both. This process introduces more variation. Nevertheless, once an antigenic cocktail is chosen, it is used to coat the microtiter plates. Patient serum then can be added to the wells and incubated. Antibody binding is detected using an enzyme-conjugated antihuman Ig. Substrate then is added to develop color for positive reactions. The amount of color is read by photometry. An alternative to photometrically read ELISA is a fluorometrically read ELISA, in which fluorescent substrates are used instead of color substrates. This ELISA is 100 times more sensitive than is a photometrically read ELISA.24 Capture Antibody Assay A variation of the ELISA method is the antibody capture enzyme immunoassay. 23 This test primarily is used for the diagnosis of neuroborreliosis (central nervous system or meningeal infection). Demonstration of intrathecal antibody production using conventional ELISA requires the dilution of serum IgG to a concentration that matches that of CSF, because IgG can cross the blood-brain barrier. Immunoglobulin M and IgA do not cross that barrier. They therefore offer a technical advantage over indirect systems, because they do not require adjustment of serum and CSF concentrations. Results of the capture procedure include a CSF/serum-specific antibody ratio determination for each antibody isotype (IgG, IgM, IgA) and are reported as the ratio of specific antibody to B burgdorferi in each sample. A CSF serum index greater than 1 is consistent with a clinical diagnosis of neuroborreliosis, that is, the CSF result must be higher in titer than the serum. The method involves the following five steps: 1. The patient's CSF or serum is added to wells that have been coated previously with goat antihuman Ig (G, M, or A). 2. The patient's antibodies then bind with the capture antibody. 3. Borrelia burgdorferi antigens are added. 4. Rabbit antibodies to B burgdorferi antigens are added. 5. Horseradish peroxidase-labeled goat IgG directed against rabbit IgG is added. TABLE 2. RECOMMENDATIONS FOR INTERPRETING POSITIVITY OF WESTERN BLOTS FOR IgG AND IgM* Test is considered significant for: IgG if IgM if any 5 of 10 bands* any 2 of 3 bands* listed below are positive listed below are positive (flagellin) 1.22 (OspC) (OspC) (flagellin) (OspA) * Centers for Disease Control and Prevention, Atlanta; IgG indicates immunoglobulin G; IgM, immunoglobulin M. t Kilodaltons. A color reaction occurs when the patient has antibodies to B burgdorferi. Intrathecal antibody synthesis is indicated when the proportion of antibodies to B burgdorferi is greater in the CSF than in serum. This is significant because the intrathecal production of anti-5 burgdorferi antibodies in the CSF appears to be the best available indicator of central nervous system or meningeal infection (neuroborreliosis). The antibody capture assay combined with Western blot is one of the best ways to separate true- and false-positive serologies, especially in late disseminated Lyme disease.26 Western Blot (Immunoblot) In the Western blot procedure, antigens of B burgdorferi are separated by electrophoresis in acrylamide gels, then transferred to a nitrocellulose or nylon membrane. Patient serum then is incubated with the membrane. Binding of antib burgdorferi antibodies found in the patient serum is detected using an enzyme-conjugated anti-human Ig. Substrate then is added, and any resultant bands are analyzed and interpreted. Western blots are demanding technically, and their interpretation is subjective and qualitative, requiring a trained analyst. Immunoblot kits for Lyme disease have become widespread, somewhat reducing the technical difficulties. Lack of standardization among kits can lead to significant variability in results, however. Interpretation of immunoblot results also is challenging owing to the debate regarding what determines positivity the number of bands, the on 18 February 2018 VOLUME 27, NUMBER 8 LABORATORY MEDICINE 545

5 type of band, or their intensity. Some researchers27 have reported that as many as 45% of healthy individuals have IgG antibodies to three or less polypetides (either 41-, 25-, 31-, 34-, or 66-kd proteins). It therefore is important to follow the CDC's recommendation27 of considering IgG blots positive only if five of the following 10 blots are present: 18, 22 (OspC), 28, 30 (OspA), 39, 41 (flagellin), 45, 58, 66, and 93 kd; and of performing an IgM immunoblot and considering it positive only if two of the following three bands are present: 22 (OspC), 39, and 41 (flagellin) kd (Table 2). Because immunoblot is less sensitive than other immunoassays, it may not detect low levels of IgM or IgG antibody. Despite these problems, the Western blot still is considered the gold standard serologic test in the laboratory confirmation of a clinical diagnosis of Lyme disease, especially when used in conjunction with one of the other immunoassays. 2. Barbour AG. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984;57: Lennhoff T. Spirochetes in etiology of obscure diseases. Acta Derm Venereol. 1948;28: Burgdorfer W, Barbour AG, Hayes SF. Lyme disease a tick-borne spirochetosis. Science. 1982;216: Golightly MG. Lab considerations in the diagnosis and management of Lyme borreliosis. Am J Clin Pathol. 1993;99: Golightly MG, Dattwyler R. Lyme disease vs rheumatic disease. Immunopathology. 1986;10: Stechenberg W. Lyme disease: the latest great imitator. Pediatr Infect Dis. 1988;7: Dattwyler RJ. Lyme borreliosis an overview of the clinical manifestations. Lab Med. 1990;21: Centers for Disease Control and Prevention. Lyme disease. MMWR. 1990;39: Rosenfeld MEA, Nowakowski J, McKenna DF, Carbonoro CA, Wormser GR Serodiagnosis of Lyme disease. ] Clin Microbiol. 1993;31: Rosenfeld MEA, Nowakowski J, Bittker S, Cooper D, Nadelman RB, Worman GP.; Clin Microbiol. 1996;34: Oski J, Uksila J, Marjamaki M. 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Many other tion of B burgdorferi associated DNA sequences in synovial fluids of patients with juvenile RA. Arthritis Rheum. diseases and infections, such as infectious 1992;35: mononucleosis (Epstein-Barr), syphilis, rheuma17. Pershing DH, Telford SA. Detection of Borrelia burgdortoid arthritis, acquired immunodeficiency feri infection with PCR. / Clin Microbiol. 1990;28: Zingy BC, Brown RN, Lane RS, LeFebure RB. Genetic syndrome (polyclonal Ig increases), systemic scle- diversity among B burgdorferi isolates from wood rats and rosis, Sjogren's syndrome, chronic nephritis, and kangaroo rats in Californians. / Clin Microbiol. systemic lupus erythematosus, as well as normal 1993;31: Schwan TG, Schrumpf ME, Karstens RH. Distribution indigenous microflora, can result in a high inci- and molecular analyses of Lyme disease spirochetes isolated dence of cross-reactivity with anti-b burgdorferi from ticks throughout California. / Clin Microbiol. antibodies. This can lead to false-positive results 1993;31: Dressier F, Whalen JA, Reinhardt BN, Steere AC. with IFA, ELISA, and antibody capture tests, Comparison ofwestern blot and ELISA for diagnosis of necessitating confirmation by Western blot analy- Lyme disease. ] Infect Dis. 1993;167:392^ Tierno PM. Lyme borreliosis: another mystery solved. sis. Lack of standardization and lack of uniformilab Med. 1990;21: ty among tests also pose problems. 22. Hemmilia IL. Fluorometric immunoassays. Clin Chem. Until better tests become available, diagnosis 1985;31: Hechemy KE, Harris HL, Benach JL. Fluoro immunoasof Lyme disease must be made clinically, based on say23.studies with solubilized antigens from B burgdorferi. } Clin a comprehensive patient history and physical Microbiol. 1989;27: Hemmilia I. Fluoroimmunoassays and immunofluoresexamination. For these reasons, clinical data must be used to increase the predictive power of any cence assays. Clin Chem. 1985;31: Berardi VD, Weeks Ke, Steere AL. Serodiagnosis of early laboratory test and to help judge its validity. It is Lyme disease: analysis of IgM and IgG responses using a caphoped that with further research, laboratory tests ture-enzyme immunoassay. J Infect Dis. 1988;158: Grodzicki RL, Steere LL. Comparison of immunoblotwill become front-line diagnostic tools and not ting and indirect ELISA using different antigen preparations just retrospective support for a diagnosis based for diagnosing early Lyme disease. / Infect Dis. largely on clinical judgment. 1988;157: ASTPHLD. Proceedings from the Second National Conference on Serologic Diagnosis of Lyme Disease. References Association for State and Territorial Public Health Laboratory 1. Rosenthal E. Flaws in diagnosis of Lyme disease. New York Directors; 1995; Washington, DC. Times. June 15, 1993; pp Al, cl 1.1. on 18 February 2018 LABORATORY MEDICINE VOLUME 27, NUMBER 8