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Indian Journal of Medical Microbiology, Vol. 24, No. 4, October-December, 2006, pp. 349-352 Supplement Seroprevalence of leptospirosis in high risk groups in Calicut, North Kerala, India Swapna RN, Tuteja U, Nair L, Sudarsana J Department of Microbiology, Calicut Medical College, Kerala Code Number: mb06111 Leptospirosis is an anthropozoonotic infection of universal distribution caused by the pathogenic Leptospira spp.[1] It is indeed a successful culmination of the leptospira, the reservoir hosts and the environment. Leptospirosis is primarily an occupational disease, but a contaminated environment makes any person vulnerable to infection.[2] Calicut is an endemic area for leptospirosis. Clinical cases of leptospirosis have been recorded from the late 1980s in this geographical area and have steadily increased over the past decade. The disease typically occurs as an epidemic lasting a few weeks during the monsoon season.[3] Close interaction of humans, animals, soil and water in this region makes the spread of leptospirosis to humans easy. Certain groups of humans in a population are more likely to be exposed to the leptospiral infection as a result of either occupational or recreational activities. Since there are a large number of potential sources of infection and many different opportunities for transmission, risk groups may differ from one area to another. The major risk groups identified include agricultural workers, veterinarians, fishermen, workers in animal farms and poultry, butchers, conservancy staff, laboratory staff, construction workers, miners, soldiers, children, housewives and also those who participate in leisure and recreational activities.[4] Materials and Methods A cross-sectional study was carried out in the Department of Microbiology, Medical College, Calicut, during the year 2005 to assess the seroprevalence of leptospirosis among various high-risk groups in Calicut. Five hundred and eighty people belonging to different occupational groups participated in the study. Fifty people who belonged to low risk groups (students and blood donors) were also taken as healthy controls. Occupation and sex-wise distribution of the study population is given in [Table - 1]. The respondents were identified at random comprising people between the age group of 10 to 60 years. From each participant, 5 ml blood was collected by venipuncture in a sterile tube and serum was separated. One per cent merthiolate was added as preservative. The serum samples were subsequently stored in eppendorf vials at -20°C until use. All the blood samples were tested for anti-leptospiral IgM and IgG antibodies separately by enzyme-linked immunosorbent assay (ELISA). IgM antibodies were detected using a commercially available PanBio Leptospira IgM ELISA kit and IgG antibodies were detected using IVD Leptospira Microwell ELISA kit. Microscopic agglutination test (MAT) was performed on 200 sera samples. The study group consisted of agricultural workers (35), fishermen and fisher folk (43), hospital sanitary workers (37), sewage workers (35), laboratory staff (15), veterinarians (12), construction workers (10) and healthy controls (13). MAT titer of 1:100 was taken as positive. Sixteen serovars were used which included Australis, Autumnalis, Ballum, Bataviae, Cynopteri, Canicola, Djasiman, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Pomona, Sarmin, Shermani, Tarassovi and Patoc. The leptospiral strains were obtained from Defence Research and Development Establishment (DRDE), Gwalior. Results and Discussion The epidemiology of leptospirosis is dynamic. New risk groups may be formed as a result of changes in agricultural or social practices or in the reservoir animal population in the area. Rapid urbanization and man-made eco-environmental disturbances observed in Calicut in the past two decades like creation of bunds, construction of houses in continuous fashion, construction of roads with inadequate drainage system and discontinuation of water transport system may have contributed to water logging condition vis-a-vis increase in the number of cases of leptospirosis in this region. Intermittent rains and repeated flooding of rodent nests lead to the contamination of surface water and paddy fields. Leptospirosis occurs mainly by cutaneous exposure of the legs while walking in stagnant water or moist soil. This implies that leptospires multiplied in those areas where water remained stagnant for a period of two to three days after the rains. Most people wore rubber chappals or walked bare foot during the rainy season, which offered little protection against possible infection.[5] Overall prevalence of leptospirosis based on the detection of anti-leptospiral antibodies in the study population was calculated taking into consideration both IgM and IgG antibodies and it was found to be 37% as shown in [Table - 2]. The seroprevalence rate among the high-risk groups alone was 38.1% and among the healthy controls was 24% [Table - 3]. Similar seroprevalence studies have also been reported from different parts of the world including India.[6],[7],[8],[9],[10],[11],[12] The highest prevalence in the study was found in hospital sanitary workers (56.2%) and fishermen and fisher folk (52.8%) followed by construction workers (40%), agricultural workers (30%), sewage workers (28.2%), veterinarians (13.3%) and laboratory staff (3.3%). Healthy controls had a prevalence of 24%, which was comparatively high suggesting leptospirosis is predominantly an environmental disease, with an added component in the form of occupational activity. Pappachan et al (2002), also has reported that there is no occupational/recreational predilection for leptospirosis in Calicut.[3] Seropositivity rates were almost uniformly distributed among all age groups. Sex-wise prevalence was higher in females (46.3%) when compared to males (30.4%) [Table - 3]. This might be due to the sex bias in the study as majority of the participants among hospital sanitary workers (73%) and fisherfolk (63%) were women and most of the seropositives in these groups (60.7% and 60.8% respectively) were females. Females were exposed to the same risk as males in these occupational groups. Taking MAT titre of 1:100 as positive, 164 samples were MAT positive out of which 75 samples were positive for multiple serovars. Distribution of leptospiral serovars in the various occupational groups is given in [Table - 4][Table - 5]. The most common serovar identified in the high risk groups (26.5%) was Pomona, followed by Shermani (19.5%), Canicola (16%), Bataviae (13.5%), Autumnalis (11%), Djasiman (10.5%), Tarassovi (10%), Icterohaemorrhagiae (7%), Australis (6.5%), Hebdomadis (4.5%), Hardjo (3%), Ballum (2.5%), Cynopteri (2.5%), Sarmin (2.5%) and Patoc (2.5%). None of the samples were positive for the serovar Grippotyphosa. In an earlier study done here in 2003, Pomona was the predominant serovar (26.9%) with Hardjo, Hebdomadis, Tarassovi and Icterohaemorrhagiae being the other serovars.[13] In the present study, seroprevalence was determined by screening all serum samples for both IgM and IgG antibodies. Unlike in other human infections, the antibody response in leptospirosis does not seem to follow the classical IgM and IgG pattern: the unusually long persistence of anti-leptospiral IgM agglutinins and the different abilities of the individual patients to produce IgG agglutinins are well-documented.[14],[15] As evident from the various studies, leptospiral IgM and IgG antibodies are both useful for seroprevalence studies of leptospirosis in a particular geographical area.[16],[17],[18] The IgG antibody titres more or less paralleled the MAT antibody. Hence IgG antibody could contribute more to the MAT than IgM antibodies though both ELISA and MAT are considered to detect different types of antibodies. ELISA is genus specific, while MAT is serovar specific.[19] Analysis of the results using IgM ELISA, IgG ELISA and MAT either singly or in combination [Table - 6] showed that the sensitivity of IgM and IgG ELISA for seroprevalence of leptospirosis increased when used together (76.2%) than individually (25.6% and 60.9% respectively); however, the specificity decreased to 30.5%. Positive predictive value (PPV) became 83.3% and negative predictive value became 22% when both ELISA tests were used in combination. Tests with high sensitivity are better for prevalence studies as they will pick up most of the seropositive cases. a) The sensitivity and specificity of IgM ELISA as compared to MAT were 25.6% and 83.3% respectively with a positive and negative predictive values of 87.5% and 18% respectively b) The sensitivity and specificity of IgG ELISA as compared to MAT were 60.9% and 38% respectively with a positive and negative predictive values of 81.9% and 17.9% respectively c) The sensitivity and specificity of IgM and IgG ELISA as compared to MAT were 76.2% and 30.5% respectively with a positive and negative predictive values of 83.3%.and 22% respectively In this study, ELISA proved to be a moderately sensitive test for serodiagnosis of leptospirosis. Due to its reasonably good sensitivity and positive predictive value, it can be considered as a good screening test for leptospirosis. Significant false negativity and false positivity associated with ELISA methods leading to low specificity and negative predictive value as compared to MAT renders it unsuitable for use in studying seroprevalence in the community. Genus-specific ELISA tests suffice to diagnose all forms of leptospirosis but they are not infallible and are not likely to replace the MAT. Thus MAT still remains the reference standard for serodiagnosis of leptospirosis for the foreseen future.[2] The reasons for low specificity and false negativity associated with ELISA is related to multiple factors. This includes the individual differences in eliciting IgG and IgM antibody response and sometimes low titres making it undetectable by ELISA. Moreover, considering the significant number of participants with false negative ELISA results, the actual seroprevalence of leptospirosis is likely to be much higher. This reflects the alarming prevalence of leptospirosis in the society and calls for early necessary steps to prevent further outbreaks. The best measures that can be adopted to limit the effects of leptospirosis on humans and animals they depend on, is by identifying of sources and containing them. The prevalent serovars can likewise suggest animal reservoirs. Education can raise awareness and prevent infection in humans and animals. As the present study shows a prevalence rate of 24% even among healthy controls, it indicates that the sources of leptospirosis are so numerous and widespread, that it is impossible to prove whether or how the person contracted the disease. Money spent on trying to pinpoint the sources of infection in the tropics would be better spent persuading the public to improve general domestic hygiene, to wear simple protective clothing and to achieve some degree of rodent control. Acknowledgements We acknowledge Dr. H. V. Batra, Dr. Urmil Tudeja and Dr. Jyothi Shukla from Microbiology division DRDE, Gwalior for providing the leptospiral strains and technical assistance in performing MAT. References
Copyright 2006 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb06111t5.jpg] [mb06111t6.jpg] [mb06111t1.jpg] [mb06111t4.jpg] [mb06111t3.jpg] [mb06111t2.jpg] |
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