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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 3  |  Page : 171-176

Leptospirosis: Seroprevalence, risk factors, and diagnostic view in a tertiary care center in North India


1 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
2 Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, AMU, Aligarh, Uttar Pradesh, India

Date of Web Publication20-Jul-2018

Correspondence Address:
Dr. Shyam Kishor Kumar
HN-95, Al-Hamd Apartment, Masjid 64 Khambha, Meerdard Road, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_25_18

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  Abstract 


INTRODUCTION: Leptospirosis is a zoonotic disease with clinical manifestations, ranging from subclinical infection to fulminant disease.
OBJECTIVE: To study the seroprevalence, various epidemiological risk factors, and diagnostic aspects of leptospirosis.
MATERIALS AND METHODS: One hundred and seventy-one clinically suspected individuals were enrolled in the study at Jawaharlal Nehru Medical College and Hospital, AMU, Aligarh, during January 2013–May 2014. A detailed history of patients was taken on a predesigned pro forma. Blood sample was collected from the patients, and immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and microscopic agglutination test (MAT) were done for diagnosis. The patients' details were also scored according to the modified Faine's criteria.
RESULTS: Of 171 patients, 24 (14.1%) cases were diagnosed positive by IgM ELISA. Contact with dirty surroundings, fieldworkers, history of animal contact, and use of public bathing facilities/ponds were associated with 20 (83.3%), 20 (83.3%) 19 (79.2%), and 18 (75%) cases, respectively. Fifteen (62.5%) cases were diagnosed during July–September (P = 0.004). Fever, myalgia, and abdominal pain were the common symptoms. Calf tenderness was seen in 22 cases (P < 0.0001). Of 24 seroreactive cases, 20 (83.3%) cases were positive by PCR. MAT was performed for only 15 serologically reactive patients. A titer of 1:40 was observed in only two cases. Faine's score (Part A + Part B + Part C) was calculated for all ELISA-reactive patients. Only one case had score between 20 and 25 and the rest had >25.
CONCLUSIONS: Dirty surroundings, fieldworkers, animal contact, and heavy rainfall are the major epidemiological risk factors for leptospirosis. Modified Faine's criteria may play a useful role in diagnosis for a patient with low clinical score.

Keywords: Calf tenderness, enzyme-linked immunosorbent assay, leptospirosis, rainfall


How to cite this article:
Ahmad N, Shukla I, Kumar SK, Rizvi M. Leptospirosis: Seroprevalence, risk factors, and diagnostic view in a tertiary care center in North India. Int J Health Allied Sci 2018;7:171-6

How to cite this URL:
Ahmad N, Shukla I, Kumar SK, Rizvi M. Leptospirosis: Seroprevalence, risk factors, and diagnostic view in a tertiary care center in North India. Int J Health Allied Sci [serial online] 2018 [cited 2022 Jul 2];7:171-6. Available from: https://www.ijhas.in/text.asp?2018/7/3/171/237262




  Introduction Top


Leptospirosis was first described as a disease in 1886 by Adolf Weil, and the pathogen Leptospira was described independently in the second half of the twentieth century by Inada and Ido in Japan and Uhlenhuth and Fromme in Germany.[1] Leptospirosis is a zoonotic disease and caused by spirochete, Leptospira. It is a worldwide public health problem, but more common in tropical and subtropical countries.[2] In India, it was first identified in 1931.[3] Several wild and domestic animals serve as reservoirs such as rodents, dogs, cattle, pigs, and horses. Rodents are considered the major reservoir of infections. Humans are accidental hosts which are infected by contact with urine of infected animals either directly or indirectly. Persons residing in proximity to animals; environmental factors such as high temperature, rainfall, and flood; and socioeconomic status such as low income, outdoor occupations, and improper health education are the risk factors for infection.[4],[5] Clinical manifestations of leptospirosis range from asymptomatic infection to severe fatal complications such as renal failure, meningitis, pulmonary hemorrhage, and multiorgan failure.[6] Early appropriate treatment can reduce the severity of disease. However, delayed diagnosis may be possible due to several other mimicking illnesses which can lead to high case fatality rate culminating up to 20%.[2]

Leptospira may be identified in laboratory mainly by three methods – by detection of leptospiral DNA by polymerase chain reaction (PCR), by culture of organism, and by serological tests.[7] Although PCR-based diagnostic methods can identify the disease in early period with high sensitivity and specificity, this facility is not available in common laboratories due to high cost. Culture of leptospires requires specialized media like Ellinghausen-McCullough-Johnson-Harris, and it may take a long time up to 13 weeks.[7]

Serology-based methods may be serogroup specific and genus specific. Microscopic agglutination test (MAT) is a serogroup-specific test which is also the gold standard method. However, it requires live leptospires and also complex in performance, so this test is done only in reference laboratory.[8] Immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is alternative to MAT which is a genus-specific test. It allows the detection of antibody in the 1st week of illness with sensitivity ranging from 68% to 100% and specificity up to 97%.[9] Hence, it may be a good alternative diagnostic method.

This study was carried out to study the seroprevalence of leptospirosis, its association with various risk factors, and various diagnostic methods along with significance of Faine's criteria in poor-setting areas.


  Materials and Methods Top


A prospective study was carried out on 171 individuals suspected with leptospiral infection at medical college and tertiary care hospital in India located at Aligarh from January 2013 to May 2014. All patients who fulfilled inclusion and exclusion criteria were included in the study [Table 1]. Twenty controls were also enrolled in this study. This study was approved by the Institutional Ethical Committee. Epidemiological factors, clinical history, and laboratory parameters were recorded from each patient after taking consent. On the basis of patient's details, the modified Faine's criteria were scored and assessed [Table 2].[10] Socioeconomic status was evaluated using Kuppuswamy index.[11]
Table 1: Inclusion and exclusion criteria for patients

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Table 2: Modified Faine's criteria

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Five to eight milliliters of blood was withdrawn in plain vial from each patient aseptically. It was allowed to clot and centrifuged at 2000–3000 rpm for 10 min to separate serum and then stored in sterile vials at −20°C until further used.

The collected serum sample was used for three procedures:

Immunoglobulin M enzyme-linked immunosorbent assay

Detection of IgM antibody against Leptospira was performed using PanBio IgM ELISA (PanBio Diagnostics, Australia). The test procedures and interpretation of the results were done as per the manufacturer's instructions. The values <9 PanBio ELISA units were considered negative, 9–11 equivocal, and >11 positive. Equivocal samples were repeated after 1 week and the test was repeated.

Polymerase chain reaction

PCR was performed on all ELISA-reactive patients only. DNA was extracted from the blood samples using QIAamp DNA Blood Mini Kit (Qiagen Gmbh, Germany) as per the manufacturer's instructions. Amplification of DNA was performed in 50 μl. Two sets of oligonucleotide primers used were G1 (5' CTG AAT CGC TGT ATA AAA GT) & G2 (5' GGA AAA CAA ATG GTC GGA AG) and B64-I (5' CTG AAT TCT CAT CTC AAC TC) & B64-II (5' GCA GAA ATC AGA TGG ACG AT).

The reaction mixture (50 μl) consisted of 5 μl of X10 assay buffer (10 mM Tris HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, and 0.01% gelatin), 200 μM each dNTPs, 20 pM of each primer, 0.5 U of Taq DNA polymerase, and template DNA (10–40 μl). 20 μl of mineral oil was added on the top of the reaction mixture, if required, to prevent evaporation. Amplification was carried out in a thermal cycler for 30 cycles. Each cycle consisted of denaturation of DNA at 94°C for 1 min, annealing of the primer at 55°C for 1–2 min, and extension for 2 min at 72°C. After the cycles completed, the final extension of the amplified product was done at 72°C for 7 min. The amplified PCR products were analyzed in 1.5% agarose gel with 0.1 μg/ml ethidium bromide in TAE buffer pH 8.0. At the end of the electrophoresis, the gel was visualized under Bio-Rad Gel Doc EZ imager and photographed. Amplification of 285 base pair DNA fragment indicated the presence of leptospiral DNA in the specimen [Figure 1]a and [Figure 1]b.
Figure 1: (a and b) Amplified product of polymerase chain reaction

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Microscopic agglutination test

MAT was performed for 15 ELISA-reactive samples at Regional Medical Research Centre, ICMR, Port Blair. MAT was carried out using 10 live leptospiral reference strains as antigens. The serogroup strains used for identification were australis, autumnalis, lai like, canicola, grippotyphosa, icterohaemorrhagiae, hebdo, hardjo, pomona, and pyrogenes.

Statistical analysis

Fisher's exact test (2 × 2 contingency table) was used to compare the data. P < 0.05 was taken as indicative of statistical significance, and a P < 0.01 was considered highly significant. The statistical calculations were done using GraphPad software 2014 (GraphPad Inc., La Jolla, California)


  Results Top


One hundred and seventy-one suspected cases of leptospirosis were enrolled in this study, of which 24 (14.1%) cases were reactive for leptospirosis by PanBio IgM ELISA. All 20 controls enrolled in this study were serologically negative. On analyzing epidemiological data of serologically confirmed cases, it was observed that 16 (66.7%) cases were male and 8 (33.3%) cases were female. The mean age of all cases was 45 years. Maximum cases ranged between 11 and 60 years [Table 3]. Seventeen (70.8%) cases were in low socioeconomic state (P = 0.04). Twenty (83.3%) cases were fieldworkers and having contact with dirty surroundings. History of animal contact, use of public bathing facilities/ponds, and barefooted were associated with 19 (79.2%), 18 (75%), and 16 (66.7%) cases, respectively [Table 4]. Fifteen (62.5%, P = 0.004) cases were diagnosed during July–September.
Table 3: Age distribution and socioeconomic status patients

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Table 4: Associated risk factors with cases of leptospirosis

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Acute febrile illness (18/24) was the most common associated clinical presentation followed by acute renal failure (4/24) and acute icteric illness (2/24).

All 24 patients had fever, mostly with chills and rigor. Calf tenderness was the most common associated symptom (22/24, P < 0.0001). Other symptoms and signs such as myalgia (83.3%), headache (62.5%), abdominal pain (58.3%), vomiting (41.7%), breathlessness (33.3%), hepatomegaly (37.5%), and subconjunctival effusion (25%) were also noted in this study [Table 5].
Table 5: Clinical presentations, symptoms and signs and laboratory parameters

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Leukocytosis and anemia are the most common hematological abnormalities [Table 5]. Hyperbilirubinemia and deranged hepatic enzymes were found in 4 (16.7%) and 2 (8.3%) cases, respectively.

Of 24 ELISA-confirmed cases, 20 (83.3%) cases were positive by PCR [Table 6]. MAT was performed for only 15 serologically reactive patients. A titer of 1:40 was observed in only two cases. The serovar Hebdo was present in both of the cases while the serovar pyrogenes was in one case. Faine's score (Part A + Part B + Part C) was calculated for all ELISA-reactive patients. Only one case had score between 20 and 25 and the rest had >25.
Table 6: Polymerase chain reaction results of 24 enzyme-linked immunosorbent assay reactive patients

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  Discussion Top


The minimum and maximum age of all included cases was 0.8 and 80 years. However, the serologically confirmed cases were ranged between 11 and 70 years. The mean age of all confirmed cases was 45 years. Most of the studies mention that the active age groups (<60 years) are more affected than the older age groups (>60 years).[3],[12],[13],[14] In our study, many patients were working still over the age of 60 years, so they were also infected as like the younger age groups. Male patients were more than female patients (16/8) in our study which is in accordance with other studies.[3],[12],[13] It may be due to the fact that men are more involved in animal handling and outdoor activities. Almost 70% patients in our study belonged to low socioeconomic status (P = 0.04). There was no patient in high socioeconomic group [Table 3]. Low socioeconomic group of people generally shows poor personal hygiene, so they are more prone to disease development. Similar findings are found in other studies.[5],[15]

All those factors which result in poor personal hygiene or environmental sanitation contribute to leptospiral infection. In this study, dirty environment and working in field (P = 0.012), animal contact (P = 0.001), and using public bathing facilities or ponds and barefooted (P < 0.0001) were the major contributing factors [Table 4]. The results of our study correlate well with other studies.[3],[15],[16] In our study, cattle was major source for the transmission rather than the rodents as reported by Loganathan et al.[16] Almost every study found cluster of cases during rainy seasons.[3],[12],[13],[16] Our study also found most cases during July–September which correspond to rainy seasons [Graph 1]. Rainfall can form water pools in which Leptospira survives and infects human or animals whoever come in contact with these water sources.[17] Fever was seen in 100% cases as like reported in other studies.[13],[18] Other associated symptoms and signs such as myalgia, headache, abdominal pain, vomiting, breathlessness, hepatomegaly, subconjunctival effusion, splenomegaly, altered sensorium, and oliguria were also found as other studies reported.[13],[19] An important point to be noted that calf tenderness was associated with leptospirosis very significantly (91.7%, P < 0.0001). De Francesco Daher et al. also found calf tenderness in leptospirosis patients to be significant.[20]



Leukocytosis and anemia were found in 62.5% and 45.8% cases, respectively. Other parameters such as thrombocytopenia, increased prothrombin time, hyperbilirubinemia, d-dimer, and deranged aspartate transaminase and alanine transaminase were also present which are also noted by other studies.[3],[13]

We identified 14% (24/171) cases using PanBio ELISA. The seroprevalence in our study is similar to other studies.[2],[18],[19] We also performed PCR for all ELISA-reactive samples. Although PCR is more sensitive as compared to ELISA for diagnosis of leptospirosis, our study found lesser PCR positivity [Graph 2]. The possible explanation for less PCR positivity (20/24) is that the study was conducted in a tertiary care center and patients might have already taken antibiotics from elsewhere. This finding is similar to Chaudhry et al., who found lesser number of PCR-positive samples as compared to ELISA.[12] MAT was done for 15 samples at Regional Medical Research Centre, Port Blair. Only two cases showed positive result – hebdo serovar associated in both cases while pyrogenes in one case. A wide variation in ELISA and MAT was seen in our study. This discrepancy may be due to prolonged transportation time which may lead to contamination of samples as well as deterioration of antibodies in samples. Another reason for low positivity may be due to sample collection in early stage of disease. The IgM ELISA is a genus-specific test, and it detects IgM antibody early in the disease. MAT is a serogroup/serovar specific test, and the detectable antibodies appear at the end of 1st week. Although MAT is a reference test, it has low sensitivity.[14]



As far as concerned diagnosis of leptospirosis by modified Faine's criteria, 23 (95.9%) patients scored >25 which is considered positive. Thus, it may be a valuable tool for a physician to diagnose leptospirosis with low clinical score as suggested by other studies too.[3],[6]


  Conclusions Top


Epidemiological risk factors such as low socioeconomic status, poor personal hygiene, occupational and behavioral habits, animal contact, and rainfall are strongly associated with leptospirosis. Hence, a physician should always exclude leptospirosis in endemic cases with these risk factors. Modified Faine's criteria may play a useful role in diagnosis for a patient with low clinical score. IgM ELISA may be a preferred diagnostic technique due to good sensitivity, easy availability, and cost-effective test. A general measure such as to improve lifestyle, to maintain clean environment, and to break the chain of disease transmission which are done for any communicable disease will also helpful to control leptospirosis.

Limitations

We did not collect second blood sample to observe increase in titer in paired sera. Another point is that the interpretation of MAT result requires a baseline titer value in a particular geographical area and we had no any reference value regarding this scenario.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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From the centers for disease control and prevention. Outbreak of leptospirosis among white-water rafters – Costa Rica, 1996. JAMA 1997;278:808-9.  Back to cited text no. 1
    
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Sethi S, Sharma N, Kakkar N, Taneja J, Chatterjee SS, Banga SS, et al. Increasing trends of leptospirosis in Northern India: A clinico-epidemiological study. PLoS Negl Trop Dis 2010;4:e579.  Back to cited text no. 3
    
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Faraji H, Mirzaei H, Afshar D, Nouri P, Roshanjo K, Mohamadi Bardebari A, et al. The epidemiology of Leptospira infection in Mazandaran Province, Northern Iran, during 2012 – 2013. Iran Red Crescent Med J 2016;18:e23194.  Back to cited text no. 4
    
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Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol 2015;387:65-97.  Back to cited text no. 7
    
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Brandão AP, Camargo ED, da Silva ED, Silva MV, Abrão RV. Macroscopic agglutination test for rapid diagnosis of human leptospirosis. J Clin Microbiol 1998;36:3138-42.  Back to cited text no. 8
    
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Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, et al. Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis. J Clin Microbiol 2003;41:803-9.  Back to cited text no. 9
    
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Philip RR, Indu PS, Anish TS, Shrinivasa BM, Joy TM, Sujina CM. Occupational and environmental risk factors of Leptospirosis – A case control study in a tertiary care setting in Kerala, India. Health Sci 2013;2:1-12.  Back to cited text no. 15
    
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Loganathan N, Ramalingam S, Shivakumar S. Epidemiological profile of human leptospirosis in an urban South Indian City. Nat J Res Com Med 2012;1:123-77.  Back to cited text no. 16
    
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Desvars A, Jégo S, Chiroleu F, Bourhy P, Cardinale E, Michault A, et al. Seasonality of human leptospirosis in reunion island (Indian Ocean) and its association with meteorological data. PLoS One 2011;6:e20377.  Back to cited text no. 17
    
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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