Risk factors associated with leptospirosis during an outbreak in Middle Andaman, India.

Background & objectives: Leptospirosis outbreaks occur frequently in North and South Andaman Islands but not in Middle Andaman. In 2002, an outbreak appeared in Middle Andaman for the first time. Although a study on risk factors was conducted in North Andaman, it used seropositivity to define leptospirosis. Since seropositivity might not indicate current leptospiral infection and as no study on risk factors was conducted in Middle Andaman, we carried out this study to identify the risk factors during the outbreak. Methods: A suspected outbreak of leptospirosis occurred in Rangat of Middle Andaman during October - November 2002. Suspected cases were screened for leptospirosis using microscopic agglutination test (MAT). Fifty two patients confirmed to have leptospirosis based on rising titres in MAT on paired sera, and 104 age, sex and neighbourhood seronegative matched controls, were included in the study. A conditional multiple regression by backward elimination process was carried out with acute leptospirosis as the dependent factor and various environmental, occupational and behavioural factors as independent factors. A stratified analysis was also carried out. Results: The presence of cattle in the house, drinking stream water, contact with garbage, walking barefoot and standing in water while working were identified as significant factors associated with leptospirosis. Stratified analysis showed a dose response relationship between number of cattle in the house and the risk of leptospiral infection suugesting that cattle could be a source of infection. Interpretation & conclusions: Identification of the potential risk factors would help understand the transmission dynamics of the disease and formulate public health interventions.

Transmission dynamics of diurnally subperiodic lymphatic filariasis transmitted by Ochlerotatus (Finlaya) niveus in the Andaman & Nicobar Islands.

BACKGROUND & OBJECTIVE: In India diurnally subperiodic filariasis is prevalent only in the Nicobar district of Andaman and Nicobar Islands, with significant health problem. Sample surveys indicated that this form of filariasis is restricted to a small region of Nancowry group of islands with Ochlerotatus niveus as the vector. We therefore carried out a comprehensive study to assess the transmission dynamics of LF in Teressa island in Nicobar district in view of its control and evaluation of interventions. METHODS: Entomological studies were carried out for a period of twelve months covering all the seasons in the year, by means of man landing catches in Teressa Island, an endemic island for this form of filariasis. Parameters viz., the annual biting rate (ABR), annual infective biting rate (AIBR), annual transmission index (ATI), risk of infection index (RII) and annual transmission potential (ATP), which reflect the dynamics and intensity of transmission of filariasis, were estimated. Host efficiency of Oc. niveus was also assessed. RESULTS: The number of vectors biting a person in a year was estimated to be 21851, of which 107 were harbouring infective parasite. Risk of infection intensity was 0.02332. Every person in this study area was at the risk of receiving an estimated number of 22 infective stage larvae per year. The host efficiency index of Oc. niveus indicated that over 40 per cent of the microfilariae ingested were able to develop into infective stages. The ATP was 169 with evidence of year round transmission. The pattern of monthly transmission potential suggested that the intensity of transmission was high during summer months. INTERPRETATION & CONCLUSION: Perennial transmission of subperiodic Wuchereria bancrofti in the typical forest ecosystem was evident in Teressa Island with transmission parameters suggesting that summer is a high risk season for transmission. Personal protection measure is the method of choice to protect from the risk of infection, because of day biting, exophilic and exophagic behaviour of the vector and larvae are not amenable to larvicidal measures. An alternative method to control the transmission would be to use selective or periodic mass chemotherapy to reduce the parasite load in this community.

Phenotypic & genotypic conservation of ompL1 & lipL41 among leptospiral isolates of Andaman Islands.

BACKGROUND & OBJECTIVES: The leptospiral antigens that are conserved among the diverse pathogenic leptospires have potential importance in the development of new serodiagnostic and immunoprotective strategies. The present study was therefore carried out to find out the phenotypic conservation of the leptospiral proteins OmpL1 and LipL41, and the genetic conservation of ompL1 and lipL41 genes among the leptospiral isolates of Andaman Islands and among the reference strains. METHODS: In one dimensional SDS-PAGE the leptospiral samples prepared from strains of various leptospiral serovars were run and transferred on to nitrocellulose paper and probed with pooled convalescent phase human sera to find out the phenotypic conservation of the protein fragments at 31 and 41 kDa. Further, the proteins were indirectly confirmed as OmpL1 and LipL41 by using specific rabbit hyperimmune sera. Specific primers were utilized to amplify the fragments to study the genetic conservation of ompL1 and lipL41. Further, these two fragments were sequenced and BLAST analysis was done with the whole genome of Leptospira interrogans serovar Lai for comparison. RESULTS: Analysis of individual immunoblots using patient sera showed that the OmpL1 and LipL41 were conserved among all the isolates used in the study. Further, these two proteins were probed with specific rabbit hyperimmune sera of OmpL1 and LipL41 for confirming the fragments and it was found to be conserved among all the isolates. The PCR based amplification further showed that the genes ompL1 and lipL41 were conserved among the leptospiral isolates studied. Sequencing followed by BLAST analysis of these showed 97 per cent similarity with the whole genome sequence and low score values in comparison with other bacterial species. INTERPRETATION & CONCLUSION: The antigenic and genetic conservation of the two proteins, OmpL1 and LipL41, indicated that these could be potential candidates for development of diagnostic test systems for leptospirosis.

Comparison of immunoreactive proteins of commonly circulating serogroups of Leptospira in Andaman Islands, India.

BACKGROUND & OBJECTIVE: Early diagnosis is the key to the treatment of leptospirosis. For development of rapid diagnostic kits, a thorough knowledge about the nature of the proteins expressed by the pathogen during infection is necessary. The present study was undertaken to understand the nature of immunoreactive proteins from commonly circulating serogroups of Leptospira in the endemic Andaman and Nicobar Islands, India. METHODS: Proteins were extracted from six strains of Leptospira representing five different serogroups following four different preparation methods, viz., whole cell lysis by sonication, detergent solubilization, outer and inner membrane isolations, and were subsequently characterized on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots were made from the sonicated proteins using hyperimmune rabbit antisera, homologous and heterologous patient sera separately. RESULTS: The 67, 65, 45, 43, 35, 32 and 18 kDa major proteins in the whole cell lysate were common among all the five serogroups of Leptospira. The 67, 41, 35, 32, 28 and 22 kDa were the major outer membrane proteins, while 94, 32, 25 and 18 kDa protein were in inner membrane. Immunoblots with hyperimmune rabbit antisera detected 67, 65, 60, 45, 43, 41 and 32 kDa common proteins from the whole cell lysates of all strains while homologous and heterologous patient sera detected 32 kDa as the major immunoreactive protein in all pathogenic serogroups. This protein reacted against specific LipL32 antisera indicating that this protein was LipL32. INTERPRETATION & CONCLUSION: The circulating serogroups of Leptospira have common nature of expression of proteins during human infection. Among several immunoreactive proteins, three (67, 45 and 32 kDa) were recognized as major antigens by both rabbit hyperimmune sera and patients sera while the 32 kDa protein was recognized as the major immunoreactive protein by homologous and heterologous patient sera. These conserved immunoreactive proteins could be utilized in developing indigenous diagnostic tests for leptospirosis.

Leptospiral proteins expressed during acute & convalescent phases of human leptospirosis.

BACKGROUND & OBJECTIVES: The available serological techniques for the diagnosis of leptospirosis have less sensitivity during the early stage of the disease. Understanding of leptospiral proteins expressed during acute and convalescent phases of leptospirosis, would be help the develop of new serodiagnostic strategies. Therefore, the present study was carried out to identify (i) an antigen that is conserved among the various pathogenic leptospira; (ii) best protein antigen to which immune response can be identified in the acute phase; and (iii) best protein antigen which is present in convalescent sera which can be used for seroepidemiological studies. METHODS: Quantitative immunoblot analysis was performed using acute and convalescent phase human sera along with sera from normal healthy individuals and from patients with typhoid, malaria and hepatitis as the controls. All the samples were analyzed for the leptospiral protein recognition by using IgM and IgG immunoblots. Leptospiral cell fractionation was performed using triton X-114 and lysozyme and further the conservation of leptospiral proteins was also performed. RESULTS: In confirmed cases of leptospirosis, the IgG recognition in acute phase sera was 30.2, 39.5, 27.9, 55.8 and 27.9 per cent for the leptospiral proteins p32, p41/42, p58, p62 and p82 respectively. The IgG has considerably increased to 65.1, 55.8, 46.5, 67.4 and 48.8 per cent against the same proteins during convalescent phase. The IgM recognition was 32.6 , 32.6, 30.2 and 37.2 per cent for acute phase sera and 32.6, 37.2, 44.2 and 41.9 per cent for convalescent phase sera for the leptospiral proteins p14, p25, p32 and p41/42, respectively. Leptospiral proteins like p62 and p82 were recognized among all the control groups with 3.3-15.3 per cent for IgG recognition. INTERPRETATION & CONCLUSION: Leptospiral protein p32 was found to be highly sensitive and specific and could be useful for the development of newer techniques for diagnosis and seroepidemiological studies. Combination of p32 and p41/42 for IgG and p14, p25, p32, p41/42 for IgM would increase the sensitivity of these techniques further.

Molecular tools in leptospirosis diagnosis and characterization of isolates.

The incidence of leptospirosis in human beings has been increasing in recent years. Early diagnosis and treatment can prevent complications and reduce mortality. The conventional laboratory methods for diagnosis rely on the demonstration of leptospires in clinical specimens, recovering the organisms in culture or the demonstration of antibodies to leptospires. Demonstration techniques have low sensitivity and specificity. Leptospires grow slowly and the positivity rate in culture is very low. Although microscopic agglutination test has been the cornerstone of serological diagnosis, the procedure is complex. New tests, like ELISA, dipstick test, lateral flow, etc, are relatively simple and rapid, but sensitivity is low during the early stages of the disease. The cross agglutination absorption test (CAAT) and typing with monoclonal antibodies (MCA) are the techniques used for serological characterization. These techniques are complicated and might not help in the case of certain serogroups. An alternate method for early diagnosis and characterization focuses on DNA-based techniques. Polymerase chain reaction (PCR), in situ hybridization etc are some of the methods used for early diagnosis, whereas restriction endonuclease analysis (REA), random amplified polymorphic DNA (RAPD) fingerprinting, arbitrarily primed PCR (AP-PCR), pulsed field gel electrophoresis (PFGE), ribotyping and DNA sequencing are useful for characterization. PCR is the most popular and quickest method for diagnosis. It can detect even if only a small number of organisms are present in a clinical sample. Fingerprinting tools such as RAPD, REA, RFLP, PFGE etc translate the complex genetic code into easily recognizable patterns, which facilitates characterization of the isolates up to sub-serovar level.

Leptospirosis disease burden estimation and surveillance networking in India.

Although leptospirosis is known to have occurred in India since the early years of the 20th century, no accurate data on disease burden exist. During the past two decades, leptospirosis cases have been reported with increasing frequency from different parts of the country. Several large outbreaks have occurred. In the year 2000, the Indian Council of Medical Research set up a Task Force on Leptospirosis. The Task Force conducted a multicentric study on disease burden due to leptospirosis. As part of the study, 3,682 patients with acute febrile illness, from 13 different centers in India, were investigated for the presence of current leptospiral infection using the Lepto-dipstick test. Of these patients, 469 (12.7%) were found to have leptospiral infection. The positivity rate ranged from 3.27% in the central zone to 28.16% in the southern zone. Fever, body aches and chills were the common symptoms observed. Urinary abnormalities, such as oliguria, yellow discoloration of urine and hematuria were found in 20%-40% of patients. Distribution of serogroups was studied based on microscopic agglutination test (MAT) titers. The southern zone had all the eleven serogroups in the panel, the eastern zone had nine, the northern zone eight, and the central and western zones had five circulating serogroups each. Among various risk factors studied, rat infestation of houses had the strongest association with leptospiral infection. Many other factors related to the environment, personal and occupational habits, etc, also had significant associations. The study had a few drawbacks. The Task Force has decided to continue the study with modified protocols to generate more accurate and detailed information about disease burden.

Outbreak of leptospirosis after the cyclone in Orissa.

BACKGROUND: Two weeks after the cyclone during October-November 1999, several persons in Orissa suffered from a febrile illness with haemorrhagic manifestations. Serum samples from a few such patients tested positive for anti-leptospiral IgM antibodies. We conducted a study in four villages that were flooded after the cyclone to examine the possibility of leptospirosis being the cause of the outbreak. METHODS: One hundred forty-two persons living in four flooded villages in the Jaipur district of Orissa were interviewed for their disease history and possible risk factors after the cyclone. Blood samples were collected and tested for anti-leptospiral antibodies using the microscopic agglutination test, IgM ELISA and lepto-dipstick. Follow up samples were collected from those who had inconclusive results on the first test and the microscopic agglutination test was repeated on these samples. RESULTS: Eighty-four of the 142 study subjects had suffered a febrile illness and 40 of them had positive results in one or more IgM-based tests and 28 had a positive microscopic agglutination test result as well. Thus, 19.2% of the study subjects (28/142) had serological evidence of symptomatic leptospiral infection after the cyclone. Also, 8.5% of the subjects had low levels of antibodies indicative of the level of background seroprevalence. CONCLUSIONS: The results indicate that there was an outbreak of leptospirosis in the flooded villages and the attack rate was high. A carrier state might have existed in the animal population and the cyclone and floods changed the environment drastically making it conducive for transmission of infection. Large numbers of persons were continuously exposed to flood waters and this resulted in the outbreak.

Survivability of Shigella dysenteriae type 1 & S. flexneri 2a in laboratory conditions simulating aquatic environment.

BACKGROUND & OBJECTIVES: Information about the duration of survival of Shigellae in water is speculative. The present study was undertaken to assess the duration of survival of Shigella dysenteriae type 1 and S. flexneri type 2a in the laboratory conditions simulating the aquatic environment, their invasive property and the association of different physico-chemical parameters in the survival process. METHODS: Five natural water sources were selected in a diarrhoea prone rural area. Collection of water, determination of physico-chemical parameters and bacteriology were carried out following standard procedures. Filter-sterilised water samples were inoculated with S. dysenteriae type 1 and S. flexneri 2a and survival was monitored by estimating total viable count at regular intervals. Bivariate correlations between the duration of survival and physico-chemical parameters were estimated. Multiple linear regression models were fitted for the duration of survival of the bacteria. RESULTS: All water sources were contaminated with faecal coliforms including Escherichia coli, S. dysenteriae type 1 survived for a mean duration of 3.33 days and S. flexneri 2a for mean 11.167 days in field water samples in laboratory condition. Duration of survival had positive correlation with the initial bacterial counts. In the multiple regression model the strongest predictor of the duration of survival of both S. dysenteriae type 1 and S. flexneri 2a was the concentration of bacteria. Other possible predictors for S. flexneri 2a were Mg and water temperature. INTERPRETATION & CONCLUSION: S. dysenteriae type 1 with epidemic potential survives for shorter duration than S. flexneri 2a. Although some of the physico-chemical parameters had positive relation with duration of survival, the variation of these in natural water samples studied has not caused much variation in the survival in case of S. dysenteriae type 1. In case of S. flexneri 2a, the observed variation in Mg concentration can cause up to 25 days difference in the duration of survival and thus could be a factor determining the endemicity of S. flexneri 2a infection.

Evaluation of microscopic agglutination test as a diagnostic tool during acute stage of leptospirosis in high & low endemic areas.

BACKGROUND & OBJECTIVES: Making a diagnosis on the results of a single microscopic agglutination test (MAT) is difficult because of the uncertainties about the cut-off titre. The present study was conducted to determine the significant titre for a single MAT in areas of high and low endemicity for leptospirosis. METHODS: A total of 1944 serum samples were collected from healthy individuals and confirmed patients residing in areas of high and low endemicity. All the sera were screened by MAT using 10 live leptospiral strains as antigens. From the distribution of titres among healthy individuals and in patients, the sensitivity and specificity at different cut-off titres were calculated. Likelihood ratio positive (LR+), likelihood ratio negative (LR-), and LR+/LR- were calculated. Receiver operating characteristics (ROC) curves were plotted for the early and late stages of the disease in both the areas. RESULTS: The ROC plot was totally below the no benefit line during the first week of illness in high endemic area. During the second to fourth weeks it showed better characteristics and the best cut-off titre was 1:200, where the sensitivity was 93.4 per cent and specificity 74.7 per cent LR+ LR- ratio was 41.82 indicating reasonable separation between the positive and negative test results. In the other states the ROC plot was above the no benefit line even during the first week, the best cut-off being 1:50 where the sensitivity was 56.7 per cent and specificity was 90.6 per cent. During the second to fourth weeks the test showed the best characteristics in the low endemicity regions with an ROC curve having the ideal shape. Best cut-off was at 1:100 where the sensitivity was 96.6 per cent and specificity 94.8 per cent LR+ LR- ratio was 523.25 indicating a wide separation between the positive and negative test results. INTERPRETATION & CONCLUSION: MAT does not have any diagnostic value during the first week, particularly in high endemic areas. The best cut-off to be used will be 1:50 in low endemicity areas during the first week, 1:100 during the second to fourth week and 1:200 in high endemicity regions during the second to fourth weeks.

Evaluation of darkground microscopy as a rapid diagnostic procedure in leptospirosis.

BACKGROUND & OBJECTIVES: Although darkground microscopy (DGM) is the method employed for examining the growth of leptospires in culture, it is not useful for diagnosis when used directly on body fluids. However, it continues to be used as a method for diagnosing leptospirosis in many centres in India. The present study was undertaken to evaluate the diagnostic accuracy of DGM by comparing it with established tests like culture, microscopic agglutination test (MAT), IgM ELISA and Lepto-Dipstick. METHODS: A total of 170 patients clinically suspected to have leptospirosis were included in the study. The gold standard for diagnosis was positive blood culture, or seroconversion, or a four-fold rise in titre in MAT. DGM was done on plasma after centrifugation at 1000 g and 3000 g and on serum. Indices of accuracy including sensitivity, specificity, predictive values and kappa value of agreement with the gold standard diagnostic criteria were calculated. RESULTS: DGM had a sensitivity of 40.2 per cent, specificity of 61.5 per cent, a positive predictive value of 55.2 per cent and a negative predictive value of 46.6 per cent. It had agreement with the gold standard in only 50 per cent cases the kappa value being 0.017141. INTERPRETATION & CONCLUSION: DGM has low indices of accuracy. The results obtained in patients who met the gold standard criteria for diagnosis and those who did not were identical indicating that the test results are not determined by the presence of leptospiral infection in the patient. DGM, therefore, is not recommended as a sole diagnostic procedure for early diagnosis of leptospirosis.

Lai-like leptospira from the Andaman Islands.

BACKGROUND & OBJECTIVES: Leptospirosis has been an important public health problem in the Andaman Islands since 1988. As information about the exact etiological agent is not available, the present study was undertaken to isolate and identify Leptospira from human patients. METHODS: An isolate coded AF61 was recovered from the blood of a patient clinically suspected to have leptospirosis, with fever, headache and body aches as the main symptoms. The isolation was done using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium following standard procedure. The isolate was identified using microscopic agglutination test (MAT) with 'groupsera', cross agglutination absorption test (CAAT) and monoclonal antibodies. RESULTS: Agglutination tests with rabbit antisera revealed that the isolate belonged to the serogroup icterohaemorrhagiae. The CAAT results showed that it was closely related to the serovar lai. Analysis of AF61 with monoclonal antibodies confirms our observation with CAAT that it is closely related to the reference strain Lai serovar lai. INTERPRETATION & CONCLUSIONS: Serovar lai, has been associated with pulmonary haemorrhage in China and Korea. However, the strain AF61 was not isolated from a patient with pulmonary symptoms. Further studies are needed to understand the possible relationship between serovars and clinical patterns and the distribution of serovar lai and lai-like strains in Asia.

Prevalence of hepatitis B infection among the primitive tribes of Andaman & Nicobar Islands.

BACKGROUND & OBJECTIVES: Andaman and Nicobar Islands, is the home of six primitive tribes. No information is available on the prevalence of hepatitis B virus (HBV) infection among them. Hence a study was undertaken with the objective of assessing the sero-prevalence of HBV infection among the four accessible tribes of these islands. METHODS: A total of 1266 serum samples were collected from four tribes i.e., Nicobarese, Shompens, Onges and Great Andamanese and tested for the presence of HBsAg and anti-HBs. Information about different risk factors associated with HBV infection was also collected from the Nicobarese tribe. RESULTS: The overall seropositivity rate of HBsAg among the Nicobarese was 23.3 per cent (95% C.I. 21.0-25.9). Hepatitis B was also found to be an important health problem among the Shompens and Onges with HBsAg positivity of 37.8 and 31.0 per cent respectively. The age-wise distribution of these serological markers among Nicobarese tribe indicate that the infection is very common in all the age groups. The sero-prevalence was also found to be very high among the children. CONCLUSIONS & INTERPRETATION: The findings of the present study indicate that hepatitis B infection is hyper endemic among the primitive tribes of these islands. Though none of the risk factors studied in the Nicobarese was found to be significantly associated with HBV infection, the fact that almost 20 per cent of the women in the reproductive age group were positive for HBsAg indicates the possibility of vertical transmission among the Nicobarese. Further studies are required to find out other modes of transmission.

Aedes aegypti (L) in Port Blair, Andaman and Nicobar islands-distribution and larval ecology.

A comprehensive survey was carried out in the Port Blair town in Andaman and Nicobar islands, in order to study the distribution and relative prevalence of Aedes aegypti during the monsoon (July'1997-October'1997) season using larval survey and adult collection methods. Ae. aegypti was found in all 21 localities surveyed. Spatial variations in distribution was evident which was closely related to population density. The nature of the larval habitats was observed to be similar in all the localities. For both outdoor and in-door containers, breeding preference ratio was highest for mud/brick/cement containers, followed by metal and plastic containers. These findings are correlated with water storage habits of the residents in the localities surveyed.

Clinico-epidemiological study of hospitalized cases of severe leptospirosis.

In an attempt to understand the clinical spectrum and pathological and biochemical abnormalities and their prognostic importance in leptospirosis, a prospective study was carried out in Port Blair during September 1996 to August 1997. Out of 80 patients suspected to have leptospirosis, 58 were proved to have current leptospiral infection using serological tests and among these, 14 died giving a case fatality rate of 24.1 per cent. The incidence of the disease showed two separate peaks roughly coinciding with the paddy sowing and harvesting season and the majority of the patients had history of exposure to wet and water logged environment prior to the attack of the disease. The disease presented as two separate clinical syndromes--the hepato-renal form and the pulmonary form though some degree of overlap was present. Hepatic and renal complications occurred in 30 patients each with 26 of them having both. These generally occurred late in the course of the disease and the mortality rate was relatively low. In contrast pulmonary complication occurred quite early and the case fatality rate in those patients was very high (6.7% vs 42.9%). The other complications encountered in the current series of cases were refractory hypotension probably due to myocarditis in 40 per cent and neck stiffness and altered sensorium due to central nervous involvement in 12.1 per cent of the patients. The chances of the patients developing complications were considerably low if treated early and very few of them developed any complications after 2 days of hospitalized treatment. The early occurrence of pulmonary complications indicates a pathogenesis totally different from that responsible for the other complications.