A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e., Bratislava, Sejroe and Pyrogenes. The ability of the test to diagnose acute leptospiral infection was assessed by testing 343 single serum samples from 96 laboratory-confirmed leptospirosis case patients with positive result in the standard microscopic agglutination test (MAT), 55 serum samples from patients with various diseases other than leptospirosis, and 192 serum samples from healthy individuals. Using the results of the MAT as a gold standard, the sensitivity and specificity of the test strip IgM dot-ELISA assay were 98.96 and 93.93 per cent, respectively. The assay offered relatively high negative predictive values (99.57%) thus making the assay ideally suited for rapid screening. The stability of the test strip was assessed with a panel of five positive and five negative control sera after storage at 4 degrees C and -20 degrees C at different times. The results showed a good performance of the test strip at both storage temperatures for up to one year. In conclusion, the test strip IgM dot-ELISA assay was sufficiently sensitive for use as a screening test for serodiagnosis of acute leptospirosis. The assay was simple, inexpensive, and easy to perform for both a single test format and a large number of specimens. However, further studies are still needed to improve the stability of the test strip and assay reagents at ambient temperature, and to make the assay more rapidly and more user friendly.

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L. interrogans were used as antigens in the MAT assay. The result of MAT at serum titers > or = 1:400 showed three pathogenic serovars of leptospira, Bratislava (71.88%), Sejroe (63.54%) and Pyrogenes (36.46%), were among the most commonly reacted serovars and they were selected for preparation of pool sonicated antigen for both IgM ELISA tests. The microplate IgM-ELISA, performed with sera at 1:80 dilution using the cutoff OD of 0.60, demonstrated sensitivity, specificity and efficiency of 87.50, 97.57, and 94.75%, respectively. The same values for IgM dot-ELISA performed with sera at 1:160 dilution were 98.96, 93.93, and 95.33%, respectively. Both ELISA methods showed results with statistically significant differences from MAT (p < 0.05). The agreement rate of IgM dot-ELISA compared with microplate IgM-ELISA was 0.85 by Kappa analysis. Both assays offered relatively high negative predictive values (95.26-99.57%), thus making the assays ideally suited for rapid screening. Future applications of the IgM dot-ELISA as a test kit would be suitable for use at the peripheral level as a rapid screening test for human leptospirosis.