Leptospirosis in Takeo Province, Kingdom of Cambodia, 2003.

BACKGROUND: In Cambodia, epidemiology and disease burden of leptospirosis were not addressed as they do not have an existing surveillance system and have limitations on their laboratory diagnosis. OBJECTIVE: Define the existence of leptospirosis and determine the antibodies to serovars of leptospires in Cambodia. MATERIAL AND METHOD: One hundred and twenty-one suspected cases of leptospirosis were enrolled in this cross-sectional study, between September 8 and November 30, 2003 from Takeo Provincial Hospital in Doun Keo District, Cambodia. RESULTS: Common clinical manifestations were fever (96%), headache (92%), and myalgia (87%). Common risk behaviors were throwing garbage on the ground (84%), pulling out sprouts (77%), fertilizing (49%), and plowing (47%). Microscopic agglutination test result confirmed four cases and polymerase chain reaction test result confirmed seven cases. Two cases each showed antibodies to serovars Javanica and Australis. An estimated annual incidence of leptospirosis in Takeo province was 7.65 per 100,000 populations. Further studies to define epidemiology and burden of disease are needed. CONCLUSION: Increasing awareness and knowledge on leptospirosis among people are necessary to decrease the impact of leptospirosis in Cambodia.

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.

An outbreak of leptospirosis, Thailand--the importance of the laboratory.

The reported incidence of leptospirosis increased 30-fold in Thailand between 1995 and 2000. Despite many hypotheses to explain the increase, the true etiology remains unknown. We conducted a review of the national surveillance system for leptospirosis, examining the reporting practices, system attributes, and utilization of laboratory confirmation in two northeastern provinces. Using standard guidelines for evaluation of public health surveillance systems, we assessed the timeliness, completeness, and accuracy of data; the sensitivity and specificity of case ascertainment; and the overall usefulness of the Thai leptospirosis surveillance system. Physicians were interviewed to assess compliance and understanding of the case definition. Capacity for confirmation of leptospirosis by a Thai latex agglutination test was assessed. Completeness for variables critical for linking epidemiologic and laboratory data for leptospirosis was 69%. Twenty-eight percent of 208 provincial surveillance reports were considered timely. Interviewed physicians indicated that the national case definition was difficult to understand and apply, and that laboratory confirmation was infrequently used. Compared to a standardized microscopic agglutination test (MAT) panel, the Thai test was specific, but relatively insensitive. We found that a lack of a standardized case definition for leptospirosis, the infrequent use of confirmatory laboratory testing, and the inability to link clinical, epidemiologic, and laboratory data hindered system utility. This surveillance system for leptospirosis highlights difficulties with surveillance of febrile illnesses in general, and the importance of laboratory confirmation for infections that are difficult to diagnose clinically.

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.