Evaluation of two commercially available rapid diagnostic tests for Lyme borreliosis

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1 DOI /s ARTICLE Evaluation of two commercially available rapid diagnostic tests for Lyme borreliosis P. W. Smit & S. Kurkela & M. Kuusi & O. Vapalahti Received: 26 June 2014 /Accepted: 17 July 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The diagnosis of Lyme disease is very complicated and a single diagnostic method cannot exclude infection. We assessed the performance of two commercially available Borrelia burgdorferi rapid diagnostic tests (RDT) in comparison to multiple laboratory-based diagnostic assays using specimens with a gradually increasing probability of Borrelia infection. Based on 200 specimens, the analytical sensitivities for IgG and IgM were 18 and 23 % for the Lyme RDT and 24 and 32 % for the Borreliose Complete RDT. The sensitivity for detecting diagnosed Lyme borreliosis cases was low (26 % Lyme RDT and 32 % with the Borreliose Complete RDT respectively), whereas the specificity was good (85 % Lyme RDT and 88 % Borreliose Complete). Based on this evaluation, the performance of RDTs in detecting Lyme borreliosis appeared to be below that of laboratory-based diagnostics. Introduction Borrelia burgdorferi sensu lato includes 18 closely related species, but only three cause Lyme disease (Lyme borreliosis): P. W. Smit European Program for Public Health Microbiology Training, European Centre for Disease Prevention and Control, Stockholm, Sweden P. W. Smit (*): M. Kuusi Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, Helsinki, Finland pieterwsmit@gmail.com S. Kurkela: O. Vapalahti Faculty of Medicine, University of Helsinki, Helsinki, Finland S. Kurkela: O. Vapalahti Helsinki University Central Hospital Laboratory, Helsinki, Finland O. Vapalahti Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland B. burgdorferi sensu stricto, B. afzelii and B. garinii [1, 2]. The number of reported Lyme borreliosis cases is steadily increasing in Finland. In 2013, a total of 1,707 cases were recorded in Finland, with the highest incidence in Åland (1,621/100,000) [3]. Diagnosis of Lyme disease is complex and early infections are particularly challenging for physicians as laboratory-based tests cannot be used [4]. For late phase infections, diagnosis often involves a two- or three-tier laboratory test setup combined with assessment of clinical symptoms by a physician [2, 5, 6]. Even though the diagnosis is complex, rapid diagnostic tests (RDTs) are commercially available at pharmacies in Europe, for use by non-clinicians. RDTs require only a drop of blood, can be performed with minimal training, and are suitable for use in a non-laboratory setting. There are no regulations governing the performance of RDTs and published evaluations are not available. Studies evaluating the performance of RDTs for other spirochete bacteria, such as Treponema pallidum, have shown reasonable sensitivity and specificity [7]. Because of the high incidence in Finland and wide concern regarding ticks, the general public may frequently use RDTs for self-diagnosis. This could affect healthcare-seeking behaviour in the case of tick bites. There is a clear need to evaluate the performance of Borrelia burgdorferi RDTs compared with standard laboratory-based diagnostic assays. To reflect the multistep decision-making process of the clinical and laboratory-based diagnoses of Borrelia, the performance of RDTs was assessed using specimens with a gradually increasing probability of Borrelia infection. Materials and methods Study setting and specimens Lyme disease is mandatorily recorded by physicians and microbiological laboratories at the National Infectious Disease

2 Registry in Finland. Lyme borreliosis laboratory diagnostics are centralised to a limited number of laboratories in Finland. The laboratory involved in this project, the Department of Virology and Immunology, Helsinki University Central Hospital Laboratory HUSLAB, receives serum specimens from suspected Lyme borreliosis cases from general practitioners and hospitals throughout Finland. Data on symptoms were occasionally provided by the treating physician when sending the specimens to the laboratory for analysis. Therefore, data on Lyme disease-related symptoms were included in the study where possible. No information on duration of disease at the time of sampling could be obtained. Specimens and reference tests The sampling frame comprised 13,510 serum specimens that were tested with six serological laboratory tests for routine borrelia diagnostics. Two enzyme linked immunosorbent assays (ELISA), the Borrelia afzelii and VlsE IgG (Sekisui Virotech, Rüsselsheim, Germany) and the Borrelia afzelii IgM ELISA kit (Sekisui Virotech), acted as primary tests. As secondary tests, the chemiluminescence immunoassays (CLIA) Liaison Borrelia IgG and the Liaison Borrelia IgM (DiaSorin) were used. In the event of discordance between these two tests or when requested by the treating physician, the Borrelia Europe Plus TpN17 Line IgG Line Immunoblot (Sekisui Virotech) and the Borrelia Europe LINE IgM Line Immunoblot (Sekisui Virotech) were performed. A positive reaction for the IgG immunoblot required distinct reactions against at least two of the antigens included in the assay (OspC, VlsE-mix, p39, DbpA-mix, p58 or p83). A positive reaction for the IgM immunoblot required distinct reactions against at least two of the antigens included in the assay (OspC, VlsE-mix, p39, or DbpA-Mix) or a distinct reaction against OspC alone. In order to assess the analytical sensitivity of the RDTs, antibody concentrations were estimated by categorising the combined CLIA and ELISA test result values into low/ intermediate concentration, , and high concentration, 180 [8]. The linear measurement ranges were 9 40 VE/ml for the Sekisui Virotech test, and AU/ml for the DiaSorin test. Cross reaction with syphilis may occur and we therefore selected syphilis-positive specimens, diagnosed by a reactive CLIA (LIASON, treponema screen, DiaSorin), and TPPA (Serodia, Fujirebio, Japan) with a non-reactive Borrelia IgM and IgG ELISA. The laboratory participates four times per year in EQA programs (Labquality) for both Lyme borreliosis and syphilis. Specimens were collected and routinely tested for Lyme borreliosis between April 2013 and March 2014 and stored at 20 C until used in this study in April The sera had therefore undergone one freeze thaw cycle before this study. The specimens were listed in the laboratory database according to date of specimen. To select specimens with a gradually increasing probability of Borrelia infection, a random starting point within the sampling frame was chosen and specimens were systematically selected until each possible combination of negative, low, or high IgM antibody concentration and negative, low, or high IgG antibody concentration was found (see Table 2). For specimens confirmed with immunoblot, however, a convenience sampling was conducted because of the limited number of specimens available within the sampling frame. According to the Finnish National Guidelines, serodiagnoses of Lyme borreliosis cases are based on high IgG values with reactive ELISA/CLIA test or positive immunoblot [IgM or IgG]). Rapid diagnostic tests The Lyme IgM (VEDA.LAB, Alecon, France; lot: 13042), Lyme IgG (VEDA.LAB, Alecon, France; lot: ), and the Keul-o-test Borreliose Complete IgM and IgG test (Borreliose Complete; BioGenTechnologies, Steinfurt, Germany); lot: ) were performed. The Lyme IgM and Lyme IgG RDTs are sold as separate tests, while the Borreliose Complete RDT combines IgG and IgM detection in one plastic cassette. According to the manufacturer s instructions, the test can be used with 50 μl of whole blood or 25 μl serum. The latter sample type was chosen for this evaluation and a timer was used to ensure that the test was read after 10 to 15 min. All tests were performed according to the manufacturer s instructions. Ethics Ethics approval was obtained from the Hospital District of Helsinki and Uusimaa committee. Data were analysed using Stata12 (StataCorp, College Station, TX, USA). Results Two Borrelia IgM and two Borrelia IgG RDTs were evaluated with a retrospective panel of 200 specimens. To assess the performance of these tests, specimens were divided into a spectrum of gradually increasing probability of infection (Table 1). The overall analytical sensitivity for IgG was 18 % for the Lyme RDTand 24 % for the Borreliose Complete RDT. For IgM, the analytical sensitivity was higher, 23 % for the Lyme RDT and 32 % for the Borreliose Complete RDT (Table 1). Based on the analysis of the 82 negative specimens, the analytical specificity of the RDTs ranged from %, none cross reacted with treponemal antibodies (Table 1).

3 Table 1 Performance of the Lyme and Borreliose Complete IgM and IgM-specific tests. Results (N=200) are given in comparison with reference tests, IgG positivity and with cases diagnosed with Lyme borreliosis Lyme RDTs Borreliose Complete RDT IgG IgM IgG IgM Number % Number % Number % Number % Reference test N=200 Negative (4 syphilis positive) Low antibody levels a High antibody levels a ELISA, CLIA and immunoblot positive b ELISA, CLIA, immunoblot positive b and clinical symptoms IgG N=200 IgG Neg IgGPos Diagnosed cases N=200 Lyme borreliosis-positive cases Lyme borreliosis-negative cases ELISA enzyme-linked immunosorbent assay, CLIA chemiluminescence immunoassays a Antibody levels based on ELISA and CLIA results without immunoblot b Immunoblot positive with high and/or low antibody levels (ELISA and CLIA results) Based on the 91 specimens that would be diagnosed and reported as Lyme borreliosis-positive cases (high IgG with ELISA and CLIA, or positive immunoblot [IgM or IgG]), 24 (26, 95 % confidence interval [CI]: %) were positive according to Lyme RDTs (IgG and/or IgM) and 29 (32, 95 %CI: %) were positive according to Borreliose Complete RDT (IgG and/or IgM). Specificity was 88 % (95 %CI: 81 % 94) for Lyme RDTs and 85 % (95 %CI: %) for Borreliose RDT. Of the 118 positive IgM specimens, 23 (19 %) were positive according to both RDTs (Fig. 1). For IgG, 19 out of 118 (16 %) were positive according to both RDTs. The performance of the RDTs was further assessed by dividing the 200 specimens into 18 categories with a gradually increasing probability of Borrelia infection using CLIA, ELISA, Immunoblot test results and clinical symptoms as a combined measure (Table 2). Out of the 28 specimens that were IgG-positive (,, or ) and IgM-negative, only 1 sample (3.6 %) was detected by RDTs. laboratory-based tests in various clinical settings and have become the backbone of national screening policies for sexually transmitted diseases in multiple countries [10]. Although RDTs are a clear success for some diseases, they may not achieve appropriate sensitivities for certain pathogens [11], and as shown in this study, not for the Borrelia burgdorferi sensu stricto either. However, it is essential to know the analytical performance of the tests in order to interpret the possible clinical diagnostic significance of the result. Discussion To the authors knowledge, this is the first study to evaluate the performance of Borrelia RDTs. Currently, RDTs are available for many infectious diseases caused by pathogens like HIVand EBVand have a major public health impact throughout the world [9]. Over the last few years, RDTs have replaced Fig. 1 Venn diagram showing distribution of positive specimens among the three assays. Numbers represent positive specimens detected by three assays (number is given in three overlapping circles), or by two assays (2 overlapping circles) or by one assay (given as number below the assay name). IgG values are represented in black; IgM values are given in purple

4 Table 2 Performance of the Borreliose rapid diagnostic test (RDT) and Lyme RDT. Distribution of reactive specimens (n=146) into 14 categories with increasing probability of Lyme borreliosis infection, using low or high ELISA and CLIA values, immunblot results and clinical symptoms together a Fourteen combinations of IgM and IgG values, aligned with an increasing probability of infection b Category IgG Clinical IgM n (13) (12) Percentage detected by Lyme RDTs Percentage detected by Borreliose Complete RDT negative, low IgM /IgG, high IgM/IgG, immunoblot positive (12) (13) (8) (6) (8) (6) (7) Clinical (21) a IgG was valued over low or high IgM results. A positive immunoblot, regardless of ELISA or CLIA value, was believed to have a high probability of Lyme borreliosis b Left low probability, right high probability Even though the performance of Borreliose Complete RDT was slightly better than that of the Lyme RDTs, the sensitivity in detecting Borrelia IgG or IgM was lower compared with laboratory-based tests. The sensitivity was particularly low for specimens that were IgG-positive and IgM-negative. Even for specimens with high antibody levels, less than 50 % of the positive specimens were detected by RDTs. Additionally, the sensitivity of the RDTs was not associated with our estimated increased probability of infection. It should be noted that serum specimens were used instead of whole blood specimens, limiting translation of the results to a non-laboratory setting. Based on other immunochromatographic test evaluations, performance usually seems to be better with plasma or serum than with whole blood [12], making it more likely that the test results obtained in this study overestimate performance when used with whole blood in a non-laboratory setting. Regardless of the performance of these RDTs, time after infection is essential for valid antibody testing and although speculating, it is very likely that RDTs are used too soon after a tick-bite by the general public to obtain valid results. The analytical specificity of these RDTs was nearly 100 % and did not cross react with treponemal antibodies. Although negative specimens obtained from suspected Lyme disease cases were used in this study, we did not use an extensive, well-characterised panel to assess the specificity in great depth owing to financial restrictions. Because of poor performance the usefulness of these RDTs in a clinical setting is questionable. However, these tests are being purchased by the general public at the local pharmacy for diagnosis at home. Owing to the complexity of Lyme disease diagnosis and the high incidence in Finland, many cases are missed when using RDTs. It is therefore likely that the number of patients seeking appropriate healthcare would be reduced or at least severely delayed because of the availability of these RDTs at the pharmacies. Untreated or very late detection of patients would increase healthcare demand in the long term as infection could lead to arthritis, late dermatological manifestations, myocarditis, meningitis, or neurological damage, such as facial nerve palsy, focal central nervous system damage or neuropathy [1]. Based on this evaluation, the performance of RDTs in detecting Lyme borreliosis appeared to be below that of laboratory-based diagnostics. Acknowledgements We would like to thank Aftab Jasir, Androulla Efstratiou, Triin Pärn and Jaana Vuopio for critically proofreading the manuscript. Conflict of interest interest. References The authors declare that they have no conflict of 1. Stanek G, Wormser GP, Gray J, Strle F (2012) Lyme borreliosis. Lancet 379: Aguero-Rosenfeld M, Wang Q, Schwartz I, Wormser GP (2005) Diagnosis of Lyme borreliosis. Clin Microbiol Rev 18: Jaakola S, Lyytikäinen O, Rimhanen-Finne R et al (2013) Infectious diseases in Finland National Institute of Health and Welfare, Helsinki 4. Busson L, Reynders M, Van den Wijngaert S et al (2012) Evaluation of commercial screening tests and blot assays for the diagnosis of Lyme borreliosis. Diagn Microbiol Infect Dis 73: Lee SH, Vigliotti JS, Vigliotti VS, Jones W, Shearer DM (2014) Detection of borreliae in archived sera from patients with clinically suspect Lyme disease. Int J Mol Sci 15: Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T (2011) Large differences between test strategies for the detection of anti-borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots. Eur J Clin Microbiol Infect Dis 30: Smit P, Mabey D, Changalucha J et al (2013) The trade-off between accuracy and accessibility of syphilis screening assays. PLoS One 8: e Mygland A, Ljøstad U, Fingerle V, Rupprecht T, Schmutzhard E, Steiner I (2010) EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis. Eur J Neurol 17(8 16):e1 e4

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