PROS AND CONS OF LYME DISEASE TESTS: AN AGREEMENT ANALYSIS Waczulíková I.1, Mišenko P.1, Matlahová K.1,2, and Schwarzová K.3 1Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, 2Department of simulation and virtual medical education, and 3Institute of Microbiology, Faculty of Medicine, all Comenius University in Bratislava, Slovakia. (e-mail: waczulikova@fmph.uniba.sk) Introduction Lyme disease (LD) is diagnosed based on patient’s medical history, objective clinical symptoms, a history of tick exposure. Laboratory testings in support of the clinical diagnosis of LD are mainly based on assays of antibodies against Borrelia burgdorferi (B.b.) ˗ enzyme-linked immunosorbent assay (ELISA), Western blotting (WB). The mainstay is a two-step protocol, in which a reactive first-tier ELISA is supplemented by separate IgM and IgG immunoblots. As a result, patients with negative ELISA results, but affected with LD, might be missed. Aim of study To assess and discuss the degree of agreement between ELISA and WB performed in a paired design on patients presenting symptoms potentially associated with LD. More specifically, to identify groups of patients whose ELISA and WB results disagree, and to discuss possible scenarios. Methodology 638 samples of patients with suspected LD were tested using ELISA with the recombinant variable surface antigen (VlsE), and WB, also with the VlsE (a reference test). Dichotomised version of ELISA titre values based on the epidemiological cut-off was used to represent patients’ results. Agreement analysis was used to evaluate the degree of agreement and disagreement between ELISA and WB results summarised in 2×2 contingency tables. All tests were conducted at a significance level of 5% using StatsDirect® 2.8.0 software. Results Demographic characteristics: gender male 238 (37.3%) with median age of 52 years (min-max: 16-87 ); female 400 (62.7%), median age of 53 years (min-max: 7-92 ). Not significant. The overall observed agreement between ELISA and WB was 69% for IgM and 57% for IgG (P<<0.001). The agreement was also related to age with decreasing agreement for IgM and increasing agreement for IgG as it was expected in older individuals (Table 1). A kappa below 0.2 indicates poor agreement and a kappa above 0.8 indicates very good agreement beyond chance. From a practical point of view, a descriptive classification of patients into respective WB categories according both ELISA test results is more informative than statistical testing (Table 2). Information on how many patients would be missed when a patient is tested with ELISA on both, IgG and IgM, and with WB as a confirmatory test only, is summarised under Table 2. Table 1: Relationship of categorised age and degree of Cohen’s kappa agreement between ELISA and WB. Age category up to 50 51-74 75 and above mean kappa for IgM 0.322±0.059 0.304±0.062 0.276±0.061 mean kappa for IgG 0.160±0.073 0.263±0.069 0.329±0.150 Discussion Based on our results, there is a concern that sticking on the two-tiered testing procedure might not be an optimal strategy, since it could lead to a high rate of missed cases with acute or persistent/recurrent LD. Summarising Pros/Cons for ELISA and WB: ELISA tests provide an estimate of the magnitude of the IgG/IgM humoral antibody response to all of the antigens that are expressed under the culture conditions. ELISA results are objective and quantitative - they can be correlated with antibody titres. ELISA method is simple, easy to perform and relatively cheap. The limitation is that ELISA test detects only free antibody, thus a negative test might actually indicate a more serious infection. ELISA has low agreement/high disagreement with WB, which implies low ELISA’s specificity (and potentially not adequate sensitivity). On the other hand, WB is sufficiently specific and sensitive. However, it is “manual” and so-called over-reading is a frequent concern. It is also more expensive in comparison to ELISA. Table 2: Cross-classification table for ELISA IgG with nested IgM categories classified according the results from WB IgG with nested IgM categories. Each value in the table represents a matched pair of ELISA(IgG;IgM) × WB(IgG;IgM) result WB IgG WB IgM Negative Total Positive Grand Total ELISA IgG ELISA IgM 120 8 128 123 27 150 278 37 11 48 49 47 96 144 157 19 176 172 74 246 422 15 2 17 71 88 105 9 3 12 50 99 111 24 5 29 67 187 216 181 205 292 141 433 638   Both, ELISA IgG and ELISA IgM negative / WB IgG or WB IgM positive (158 out of 278; 57%) ELISA IgG negative, ELISA IgM positive / WB IgG positive (49 out of 144; 34%) ELISA IgG positive, ELISA IgM negative / WB IgM positive (2 out of 105; 2%) Conclusion Our results support the necessity of WB confirmation for both positive and negative ELISA. Antibody assays for LD will improve when recombinant antigens become available to the unique antigens of B.b. making ELISA assays highly sensitive (>95%) and more specific (>90%). For the time being, it seems more reasonable to test IgM and IgG in any new patient at any stage of disease by ELISA and WB to provide adequate support for clinical evaluation.