Title: Revisiting the Lyme Disease Serodiagnostic Algorithm: The Momentum Gathers

Transcription

1 JCM Accepted Manuscript Posted Online 13 June 2018 J. Clin. Microbiol. doi: /jcm Copyright 2018 American Society for Microbiology. All Rights Reserved. 1 Title: Revisiting the Lyme Disease Serodiagnostic Algorithm: The Momentum Gathers Author: Adriana R. Marques, MD Affiliation: Chief, Lyme Disease Studies Unit, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Address correspondence to: amarques@niaid.nih.gov Acknowledgement: This research was supported by the Intramural Research Program of the NIH, NIAID. Disclaimer: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 1

2 18 Abstract: Lyme disease is a tickborne illness caused by Borreliella (Borrelia) burgdorferi and it is the most common vector-borne disease in the United States, with an estimated incidence of 300,000 cases per year. The currently recommended approach for laboratory support of the diagnosis of Lyme disease is the standard 2-tiered algorithm (STT), comprised of an enzyme-linked immunoassay (EIA) or immunofluorescence assay (IFA), followed by a Western blot (WB). The STT algorithm has low sensitivity in early infection, and there are drawbacks associated with the WB use in practice. Modified 2- tiered algorithms (MTT) have been shown to improve the sensitivity of the testing in early disease while maintaining high specificity. In this issue of the Journal of Clinical Microbiology, A. Pegalajar-Jurado et al (J Clin Microbiol :e report the results of their evaluation of the LIAISON VlsE CLIA, the Captia B. burgdorferi IgG/IgM EIA, and the C6 B. burgdorferi (Lyme) EIA as MTT algorithms, compared with STT algorithm using the same tests as the first-tier, and the ViraStripe IgM and IgG WBs as the second-tier test. The results showed that all MTTs had higher sensitivities when compared with STTs and were highly specific. These results showed that MTT approaches are a valid alternative to the currently recommended STT algorithm for serodiagnosis of Lyme disease, opening the door for the development of rapid diagnostics and point-of-care testing that can provide diagnostic information during the initial patient visit. 2

3 Commentary: Lyme disease, or Lyme borreliosis, is caused by the spirochete Borreliella (Borrelia) burgdorferi and is transmitted by the bite of infected ticks of the Ixodes ricinus complex. Lyme disease is the most common vector-borne illness in the United States (US) and Europe. It is estimated that over 300,000 new cases of Lyme disease occur per year in the US, with the majority of cases occurring in the mid-atlantic, Northeast, and upper Midwest regions. B. burgdorferi causes a complex, multi systemic infection in humans. For clinical purposes, Lyme disease is divided into early localized, early disseminated and late stages. The disease typically begins with erythema migrans (EM), an expanding skin lesion that usually develops between 7 to 14 days at the site of the tick bite. If untreated, spirochetes may disseminate from the site, and patients may present with additional skin lesions (multiple EM), neurologic, cardiac and/or rheumatologic manifestations (1). The vast majority of laboratory tests used to support the diagnosis of Lyme disease are based on the detection of the antibody responses against B. burgdorferi in serum and this is the only type of diagnostic testing approved by the US Food and Drug Administration (FDA). Ten years ago, an editorial asked if it was time for a change in laboratory testing for Lyme disease (2). It accompanied a study comparing the two-tier algorithm for the serodiagnosis of Lyme disease with a peptide-based enzyme-linked immunoassay (EIA) test (3). Since then, there have been other studies that investigated this question, with the study by Pegalajar-Jurado et al (4) being the most recent example. In order to fully appreciate how this study fits in the historical context and on the discussion regarding the 3

4 need to change the currently recommended two-tier algorithm for serodiagnosis of Lyme disease, one needs to go back to the early years after the identification of the disease. Lyme disease was first recognized in the US in 1977, with the discovery of the pathogen, B. burgdorferi, in Because direct tests for B. burgdorferi are challenging and have low sensitivity in most presentations (5), the vast majority of laboratory tests used to support the diagnosis of Lyme disease are based on the detection of the antibody responses against B. burgdorferi in serum. The initial (first-generation) tests used antigen consisting of B. burgdorferi whole cell sonicates (WCS) to detect immunoglobulin M (IgM) and/or immunoglobulin G (IgG) responses. Early studies showed very poor agreement between tests and laboratories (6), and demonstrated the potential of employing Western blots (WB) to supplement a primary EIA or indirect immunofluorescence assay (IFA) test (7, 8), as well as the need for established standardized interpretation criteria for the tests. In 1994, the Second National Conference on Serologic Diagnosis of Lyme Disease was held in Dearborn, Michigan to address the problems of precision and accuracy in serodiagnosis of Lyme disease in the US. The results led to the current standard 2-tiered testing (STT) algorithm (Figure 1), which was recommended by the Centers for Disease Control and Prevention (CDC) in 1995 (9). The first step of the algorithm uses a sensitive EIA (or rarely, an IFA). If the initial test result is borderline or positive, the sample is retested using separate IgM and IgG WB as the second step. The WB is interpreted using standardized criteria, requiring at least two of three signature bands for a positive IgM WB, and 5 of 10 signature bands for a positive IgG WB. The IgM WB results are used only for disease of less or equal to 30 days of duration. This two-tier testing algorithm has been the basis for the national 4

5 standardization of Lyme disease serologic testing methods in the US and represented a major improvement in the field. Since its introduction, many studies have evaluated the STT algorithm in practice (3, 10-14). These studies have shown that, if used as recommended, the approach works relatively well, particularly for later manifestations of the infection. However, the current approach has low sensitivity during early infection because the sensitivity of the IgM WB component is half of the first-tier component (3, 11, 12, 15, 16). Due to the subjective interpretation of immunoblot bands, there is still inter and intra-laboratory variability, and false positive IgM WBs are common in commercial laboratories (17). Other issues include the high cost, substantial training requirement, and the labor-intensive process to perform the test, leading to an increased turnaround time for the WB. Finally, a major problem is the confusion by health care providers and patients regarding how to interpret the results from the tests. Laboratories will report the presence of individual IgG and IgM scored bands, plus the result (positive or negative) for the IgM and IgG WB, independent of the duration of the disease, leading to much confusion, misinterpretation and uncertainty(18). The need to improve the testing algorithm has been recognized for a long time, including making testing simpler and independent from disease duration, and increasing sensitivity in early disease while still maintaining a very high specificity. High specificity is an essential feature for any stand-alone test or test algorithms for serodiagnosis of Lyme disease. It is estimated that about 3.4 million Lyme serologic tests are done in the US every year (19). Therefore, it is likely that most testing is done in situations with a low pretest probability of Lyme disease, which increases the likelihood of false positive 5

6 tests (20, 21). If one estimates a scenario where at least 3 million tests are being performed in cases with a low pretest predictive value, a 1% decrease in specificity can lead to an increase of almost 30,000 false-positive tests. After the standardization afforded by the implementation of the two-tier testing, the second major advance in the serodiagnosis of B. burgdorferi infection was the discovery of the antibody response against VlsE, the variable surface antigen of B. burgdorferi, and to the C6 peptide, which is derived from the invariable region 6 of VlsE (14). This response is immunodominant and highly conserved among B. burgdorferi strains. Because the plasmid containing VlsE is rapidly lost during in vitro passage, and poorly expressed in culture, it was not a significant part of tests using WCS. These second-generation tests using recombinant VlsE or synthetic C6 peptide have comparable sensitivity to WCS-based EIAs, but with a much higher specificity, most markedly in patients with illnesses other than Lyme disease. When compared with the STT algorithm, the sensitivity of these assays was significantly higher in patients with earlier manifestations of the infection, while specificity of the assays was slightly lower (3, 10-12, 14, 16). The use of an alternative (or modified) two-tiered (MTT) strategy (Figure 1) was suggested in 2004 (22) in a letter commenting on a study comparing VlsE1, C6 and pepc10 (a 10-aa segment of outer surface protein C of B. burgdorferi) to the STT approach using a WCS EIA as the initial test (10). A re-analysis of the dataset using the proposed approach (WCS EIA first, followed by the two peptide EIA assays if positive or equivocal) showed that the strategy performed quite well (23). Later, a follow up study using a WCS EIA as the initial tests and a multiplex assay with VlsE1-IgG and pepc10-6

7 IgM as the second-tier test showed again the advantage when comparing with the WB (13). None of these tests were approved by the FDA. An approach using two commercially available EIAs was proposed in Europe, with a study published in 2005 showing that if the results of an IgG EIA assay with 4 recombinant antigens and the C6 peptide EIA were concordant, the immunoblot could be omitted (24). Initial studies had added a VlsE band to the WB, and an alternative approach was proposed using the VlsE band alone as the second-tier test for patients with early or early disseminated disease, while requiring 5 of 11 bands for late disease(12). While this approach increased sensitivity compared to the two-tier and removed the problems of IgM testing, it still required different interpretation criteria based on disease stage, and the use of the WB. Based on the favorable results of the initial studies of the 2-EIA approach, this strategy was evaluated in the US using two FDA approved first-tier tests, a polyvalent WCS EIA followed by a C6 EIA, for those with a positive or equivocal first-tier result (16). The results showed that this 2-EIAs algorithm had similar sensitivity to the C6 EIA, while preserving the specificity of the STT testing. This strategy was shown to be cost effective in a reanalysis of the data from a large study of the C6 EIA (25). Follow up studies continue to show the MTT strategy works well (26-29), confirming that MTT algorithms provided similar or greater sensitivity compared with STTs, particularly for early disease, while retaining similar specificity. Noteworthy, in a recent study of patients with EM (26), an MTT using the Liaison B. burgdorferi chemiluminescent immunoassay (CLIA), that detects IgM and IgG antibodies against VlsE from B. burgdorferi B31 and B. garinii strain PBi, followed by the C6 EIA, had the highest sensitivity of the MTTs in 7

8 the acute phase(27). This bring us to the current study (4), where the authors compared three MTTs with 3 STTs using the same assays as the initial tier test, followed by WB. The samples used in the study were from the CDC Lyme Serum repository, and they have been described and analyzed in other studies (28, 29). The tests chosen included the LIAISON VlsE CLIA and the Captia B. burgdorferi IgG/IgM EIA, that detects antibodies against whole cell lysate from B. burgdorferi B31. These were analyzed with results from testing using the C6 B. burgdorferi (Lyme) EIA and the ViraStripe IgM and IgG WBs (read using densitometry) that were performed on the same samples under previous studies (28). Again, it showed that all MTTs had higher sensitivities when compared with STTs, due to the increase in sensitivity in early stages of the infection. There were no significant differences between the sensitivity of the three MTTs in this study, with high agreement between the results. For patients with EM, VlsE/C6, WCS/C6 and WCS/VlsE MTTTs sensitivity were 50%, 55% and 58% for acute samples, and 76%, 79% and 76% for convalescent samples. STT sensitivity for VlsE/ ViraStripe, C6/ViraStripe and WCS/ViraStripe were 43%, 43% and 50% for acute samples, and 61%, 61% and 63% for convalescent samples. There were no differences for patients with later manifestations of Lyme disease. The three MTTs also had high specificity, similar to the SSTs. There was improved specificity when comparing the MTT VlsE/C6 with the STT WCS/Virastripe in controls with other diseases. Pegalajar-Jurado et al. study results are similar to previous studies (16, 26-29), and again prove that MTT algorithms that included VlsE or C6, have improved sensitivity in early Lyme disease presenting with erythema migrans while maintaining 8

9 high specificity. The need for the algorithm to be highly specific is the reason that two EIA assays are necessary. While the WCS EIA and C6 EIA are not completely independent tests (30), the use of two assays increases the specificity when compared with either test alone. As shown here and in previous studies (4, 16, 26-29), MTT algorithms will be positive in only about 50% of acute samples from patients with EM, comparing with 40% for the STT algorithm (Table 1). Therefore, patients with EM should receive treatment based on the clinical diagnosis (1). While a positive serological test is not necessary for the diagnosis of Lyme disease in patients with EM, this group is an important benchmark for tests development, and changes that reduce the window period between time of infection and an accurate test result are welcome improvements. The sensitivity of antibody-based tests increases with the duration of the infection, and MTTs and SSTs have similar sensitivity in patients with later manifestations of Lyme disease. There are many advantages to the MTT approach when compared to the STT algorithms, and they are discussed in detail on a report from a conference on diagnostic tests for Lyme disease which was held in 2016 (31). Overall, the MTT approach is more sensitive than the STT for early disease, has similar sensitivity for later infection, and maintains similar specificity. Additionally, the MTT approach is much simpler than the STT algorithm in all aspects. The MTT algorithm gives an overall single result (positive or negative) for seropositivity in Lyme disease, facilitating the interpretation of the test. The tests are less expensive, less labor intensive, can be performed using automated instruments, and have objective, quantitative results. The MTT approach can also be used in patients who acquired the infection in Europe, when such cases are evaluated in the 9

10 US. Alternatively, the WB has the advantage of providing information regarding the expansion of the immune response, and the WB will still have a place in evaluating difficult cases. But for the vast majority of tests performed, this information is not necessary and only increases the confusion regarding interpretation of the results. Certain MTT algorithms may be positive in cases of Borrelia miyamotoi infection, as well as other relapsing fever Borrelia infections (32). Moreover, the MTTs algorithms opens the way for a sensitive and specific pointof-care diagnostic for Lyme disease. These tests would be particularly useful in evaluating patients with stage 2 manifestations of Lyme disease like facial palsy or carditis, as well as children with arthritis. At this point, if these patients do not have other manifestations of Lyme disease (erythema migrans) or a very suggestive history, the diagnosis may depend on serological tests results. This can cause delay in appropriate therapy and lead to unnecessary treatment, including surgical procedures in pediatric patients with Lyme arthritis (33), and permanent pacemakers in Lyme carditis. In summary, MTT approaches are a valid substitute for the STT algorithm for the majority of tests performed for the serodiagnosis of Lyme disease and they are a welcome addition to the currently recommended algorithm for serodiagnosis of Lyme disease. The MTT approach has the potential for rapid diagnostics, that substantially improve upon the time to result, and point-of-care diagnostic testing that could be used in many clinical settings. These improvements can lead to better management of the individual patient. Hopefully, further advances in diagnostic technologies will continue to refine and expand first-line tests, possibly with the development of combination technologies to detect both antibodies and antigens, which could shorten the detection window period and the time to 10

11 results, provide information about disease stage, and possibly serve as biomarkers for active infection. 11

12 References 1. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, Krause PJ, Bakken JS, Strle F, Stanek G, Bockenstedt L, Fish D, Dumler JS, Nadelman RB The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis 43: Weinstein A Editorial commentary: laboratory testing for Lyme disease: time for a change? Clin Infect Dis 47: Steere AC, McHugh G, Damle N, Sikand VK Prospective study of serologic tests for lyme disease. Clin Infect Dis 47: Pegalajar-Jurado A, Schriefer ME, Welch RJ, Couturier MR, MacKenzie T, Clark RJ, Ashton LV, Delorey MJ, Molins CR Evaluation of Modified Two- Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well- Characterized Serum Samples. J Clin Microbiol doi: /jcm Marques AR Laboratory diagnosis of Lyme disease: advances and challenges. Infect Dis Clin North Am 29: Bakken LL, Case KL, Callister SM, Bourdeau NJ, Schell RF Performance of 45 laboratories participating in a proficiency testing program for Lyme disease serology. Jama 268: Dressler F, Whalen JA, Reinhardt BN, Steere AC Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 167:

13 Craven RB, Quan TJ, Bailey RE, Dattwyler R, Ryan RW, Sigal LH, Steere AC, Sullivan B, Johnson BJ, Dennis DT, Gubler DJ Improved serodiagnostic testing for Lyme disease: results of a multicenter serologic evaluation. Emerg Infect Dis 2: Centers for Disease Control and Prevention Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep 44: Bacon RM, Biggerstaff BJ, Schriefer ME, Gilmore RD, Jr., Philipp MT, Steere AC, Wormser GP, Marques AR, Johnson BJ Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates. J Infect Dis 187: Wormser GP, Schriefer M, Aguero-Rosenfeld ME, Levin A, Steere AC, Nadelman RB, Nowakowski J, Marques A, Johnson BJ, Dumler JS Singletier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease. Diagn Microbiol Infect Dis 75: Branda JA, Aguero-Rosenfeld ME, Ferraro MJ, Johnson BJ, Wormser GP, Steere AC tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test. Clin Infect Dis 50: Porwancher RB, Hagerty CG, Fan J, Landsberg L, Johnson BJ, Kopnitsky M, Steere AC, Kulas K, Wong SJ Multiplex immunoassay for Lyme disease 13

14 using VlsE1-IgG and pepc10-igm antibodies: improving test performance through bioinformatics. Clin Vaccine Immunol 18: Liang FT, Steere AC, Marques AR, Johnson BJ, Miller JN, Philipp MT Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlse. J Clin Microbiol 37: Wormser GP, Nowakowski J, Nadelman RB, Visintainer P, Levin A, Aguero- Rosenfeld ME Impact of clinical variables on Borrelia burgdorferi-specific antibody seropositivity in acute-phase sera from patients in North America with culture-confirmed early Lyme disease. Clin Vaccine Immunol 15: Branda JA, Linskey K, Kim YA, Steere AC, Ferraro MJ Two-tiered antibody testing for Lyme disease with use of 2 enzyme immunoassays, a wholecell sonicate enzyme immunoassay followed by a VlsE C6 peptide enzyme immunoassay. Clin Infect Dis 53: Seriburi V, Ndukwe N, Chang Z, Cox ME, Wormser GP High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice. Clin Microbiol Infect 18: Conant JL, Powers J, Sharp G, Mead PS, Nelson CA Lyme Disease Testing in a High-Incidence State: Clinician Knowledge and Patterns. Am J Clin Pathol 149: Hinckley AF, Connally NP, Meek JI, Johnson BJ, Kemperman MM, Feldman KA, White JL, Mead PS Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis 59:

15 Lantos PM, Branda JA, Boggan JC, Chudgar SM, Wilson EA, Ruffin F, Fowler V, Auwaerter PG, Nigrovic LE Poor Positive Predictive Value of Lyme Disease Serologic Testing in an Area of Low Disease Incidence. Clin Infect Dis 61: Botman E, Ang CW, Joosten JHK, Slottje P, van der Wouden JC, Maarsingh OR Diagnostic behaviour of general practitioners when suspecting Lyme disease: a database study from BMC Fam Pract 19: Porwancher RB Cost-effectiveness of peptide-antigen immunoassays for Lyme disease. J Infect Dis 189:1962; author reply Johnson BJB, Biggerstaff BJ, Bacon RM, Schriefer ME Reply. The Journal of Infectious Diseases 189: Jansson C, Carlsson SA, Granlund H, Wahlberg P, Nyman D Analysis of Borrelia burgdorferi IgG antibodies with a combination of IgG ELISA and VlsE C6 peptide ELISA. Clin Microbiol Infect 11: Wormser GP, Levin A, Soman S, Adenikinju O, Longo MV, Branda JA Comparative cost-effectiveness of two-tiered testing strategies for serodiagnosis of lyme disease with noncutaneous manifestations. J Clin Microbiol 51: Lipsett SC, Branda JA, McAdam AJ, Vernacchio L, Gordon CD, Gordon CR, Nigrovic LE Evaluation of the C6 Lyme Enzyme Immunoassay for the Diagnosis of Lyme Disease in Children and Adolescents. Clin Infect Dis 63:

16 Branda JA, Strle K, Nigrovic LE, Lantos PM, Lepore TJ, Damle NS, Ferraro MJ, Steere AC Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease. Clin Infect Dis 64: Molins CR, Delorey MJ, Sexton C, Schriefer ME Lyme Borreliosis Serology: Performance of Several Commonly Used Laboratory Diagnostic Tests and a Large Resource Panel of Well-Characterized Patient Samples. J Clin Microbiol 54: Molins CR, Delorey MJ, Replogle A, Sexton C, Schriefer ME Evaluation of biomerieux's Dissociated Vidas Lyme IgM II and IgG II as a First-Tier Diagnostic Assay for Lyme Disease. J Clin Microbiol 55: Wormser GP, Molins CR, Levin A, Lipsett SC, Nigrovic LE, Schriefer ME, Branda JA Evaluation of a sequential enzyme immunoassay testing algorithm for Lyme disease demonstrates lack of test independence but high diagnostic specificity. Diagn Microbiol Infect Dis doi: /j.diagmicrobio Branda JA, Body BA, Boyle J, Branson BM, Dattwyler RJ, Fikrig E, Gerald NJ, Gomes-Solecki M, Kintrup M, Ledizet M, Levin AE, Lewinski M, Liotta LA, Marques A, Mead PS, Mongodin EF, Pillai S, Rao P, Robinson WH, Roth KM, Schriefer ME, Slezak T, Snyder J, Steere AC, Witkowski J, Wong SJ, Schutzer SE Advances in Serodiagnostic Testing for Lyme Disease Are at Hand. Clin Infect Dis 66: Sudhindra P, Wang G, Schriefer ME, McKenna D, Zhuge J, Krause PJ, Marques AR, Wormser GP Insights into Borrelia miyamotoi infection from an 16

17 untreated case demonstrating relapsing fever, monocytosis and a positive C6 Lyme serology. Diagn Microbiol Infect Dis 86: Dart AH, Michelson KA, Aronson PL, Garro AC, Lee TJ, Glerum KM, Nigrovic PA, Kocher MS, Bachur RG, Nigrovic LE Hip Synovial Fluid Cell Counts in Children From a Lyme Disease Endemic Area. Pediatrics 141(5). DOI: /peds Downloaded from on November 7, 2018 by guest 17

18 Figure 1: Standard 2-tiered algorithm (STT) and modified 2-tiered algorithms (MTT) for serodiagnosis of Lyme disease. Figure 1 legend: *For patients with signs or symptoms consistent with Lyme disease for less than or equal to 30 days, the provider may treat the patient and follow up with a convalescent serum. Patients with erythema migrans should receive treatment on the basis of the clinical diagnosis. 18

19 Table 1 Sensitivity of modified 2-tiered algorithms (MTT) versus standard 2-tiered algorithm (STT) in acute samples from patients with erythema migrans. Study Test Algorithm Acute EM Test Algorithm Acute EM MTT Sensitivity STT Sensitivity Branda, Linskey et al. 2011(16) WCS Vidas f/b C6 EIA 52.6% WCS Vidas f/b WB 42.1% Branda, Strle et al. 2017(27) WCS W EIA f/b C6 EIA 38.2% C6 EIA f/b WB 36.4% WCS W EIA f/b VlsE CLIA 36.4% VlsE CLIA f/b WB 34.5% VlsE CLIA f/b C6 EIA 54.5% WCS W f/b WB 25.4% Molins, Delorey et al. WCS Vidas f/b C6 EIA 50% WCS Vidas f/b WB 47.5% 2016(28) a 19

20 Molins, Delorey et al. 2017(29) a Pegalajar-Jurado, Schriefer et al. 2018(4) a WCS Vidas f/b C6 EIA 50% LYM/G Vidas f/b WB 42.5% LYM/LYG f/b C6 EIA 55% WCS Vidas f/b WB 47.5% WCS Captia f/b C6 EIA 55% WCS Captia f/b WB 50% WCS Captia f/b VlsE CLIA 57.5% VlsE CLIA f/b WB 42.5% VlsE CLIA f/b C6 EIA 50% C6 EIA f/b WB b 42.5% Downloaded from Wormser, Levin et al. 2013(11), Wormser, Schriefer et al. 2013(25) WCS EIA f/b C6 EIA c 58.4% C6 EIA f/b WB 37.6% WCS f/b WB 38.3% 20 on November 7, 2018 by guest

21 WCS: whole cell sonicate. EIA: enzyme-linked immunoassay. WB: Western blot. f/b: followed by a These studies evaluated the same serum samples, derived from the CDC Lyme Serum repository. b This analysis was also presented in Molins, Delorey et al (28) and Molins, Delorey et al. 2017(29). c A. Levin and G.P. Wormser, unpublished data. Standard 2-tiered algorithm (STT): Samples that test positive or equivocal in the initial EIA test are tested by IgG and IgM WB, interpreted using standardized criteria. The IgM WB results is only used for disease of 30 days or less of duration. Modified 2-tiered algorithms (MTT): Samples that test positive or equivocal in the initial first-tier EIA are sequentially tested by a different first-tier EIA. The result is positive if both tests are positive or equivocal. WCS Vidas: detects IgM and IgG antibodies against a WCS preparation from B. burgdorferi strain B31 (biomérieux). WCS W: Wampole B. burgdorferi IgG/IgM enzyme-linked immunosorbent assay II (Alere). C6 EIA: detects both IgM and IgG antibodies to the C6 peptide, derived from the invariable region 6 of VlsE from B. burgdorferi B31 (Immunetics). VlsE CLIA: Liaison B. burgdorferi chemiluminescent immunoassay (CLIA) detects IgM and IgG antibodies against VlsE from B. burgdorferi B31 and B. garinii strain PBi (DiaSorin). WCS Captia: B. burgdorferi IgG/IgM EIA detects antibodies against whole cell lysate from B. burgdorferi B31 (Trinity Biotech). 21

22 LYM/G Vidas: Lyme IgM II (LYM) and IgG II (LYG) EIAs, separately detects IgM antibodies (LYM assay) to recombinant antigens DbpA and OspC, and IgG antibodies (LYG assay) to recombinant antigens VlsE, DbpA, and OspC of B. burgdorferi (biomérieux). 22

23 IgG and IgM Western blot Positive or Equivocal Standard 2-tiered algorithm (STT) First-tier immunoassay Negative No further testing* Positive or Equivocal Modified 2-tiered algorithm (MTT) Different first-tier immunoassay First-tier immunoassay Negative No further testing* Downloaded from Interpretation uses standardized criteria: at least 2 of 3 bands for a positive IgM Western blot, and 5 of 10 bands for a positive IgG Western blot Illness duration less or equal to 30 days Use both IgM and IgG Western blot results Illness duration more than 30 days Use only IgG Western blot results Positive or Equivocal Positive overall result Negative Negative overall result* on November 7, 2018 by guest