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Leptospirosis in a midland rural area of Kerala State.

Background & objectives: Leptospirosis has been repeatedly reported from the State of Kerala since 1989 and is one of the commonest diseases among the 14 notifiable diseases in the district level communicable disease surveillance system. As there are no field studies on human leptospirosis in Kerala, we undertook this seroepidemiological study of ieptospirosis in the two hamlets of Ernakulam district in Kerala to identify locally prevalent serogroups, and to understand local epidemiological features of the disease.

Methods: Blood samples were collected for five consecutive years from persons willing to subject themselves for the study from the selected areas (Kunnackal and Kunnakkurudy hamlets). Animal studies were conducted among samples from rodents captured from the area by the human volunteers themselves. Antibody titres were determined by microscopic agglutination test (MAT). The investigators inspected all the houses and surrounding land of the study subjects in this period.

Results: Of the 385 persons enrolled, 29.6 per cent showed evidence of past leptospiral infection. Many locally prevalent serogroups and changing patterns of prevalence in humans were identified in the five consecutive years of the study. The major serogroups were autumnalis, louisiana, australis, and grippotyphosa. Rodents showed 11 per cent seropositivity. Bandicoota indica showed the highest seropositivity (44.4%).

Interpretation & conclusions: Our findings showed that the physical environment of the study area was favourable for prolonged survival of leptospires. Farming practices exposed the human volunteers to soil and surface water and leptospirosis is endemically present here. Majority of seropositive individuals in the community had only subclinical infection. Rodents were abundant and contributed to enzootic and endemic prevalence of leptospirosis.

Key words Bandicoota indica--Kerala--leptospirosis--MAT--seroepidemiology

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Leptospirosis is the most frequently reported notifiable infectious disease in Kerala (1). The District Medical officer had reported 20 confirmed leptospiral deaths in Ernakulam district alone in 2006 (monthly reports of the District Medical Officer, Ernakulam, Kerala). There must be many more unreported and unconfirmed cases. Leptospiral infection may be subclinical or overtly clinical depending on the level of exposure and general immunity of people.

Leptospira interrogans which causes human leptospirosis, consists of over 24 serogroups made up of a large and expanding number of serovars. Serogroups and their member serovars causing leptospirosis differ from region to region. These are identified by specific laboratory tests like isolation of organism by culture, specific antigen detection by gene amplification by polymerase chain reaction (PCR) or antibody detection by microscopic agglutination test (MAT). MAT uses whole leptospire as antigen and detects both lg M and lg G antibodies. IgM antibodies may disappear after six months and Ig G antibodies persist for two to 10 years after infection (4). MAT has a high degree of specificity, identifies serogroups and is accepted worldwide; but it is expensive, time consuming and labour intensive.

Knowledge of high level of exposure and prevalence of leptospirosis in an area helps the primary physician in deciding to start early antibiotic treatment on clinical assessment. Antibiotic treatment should be started as soon as possible on clinical suspicion of leptospirosis because organ damage sets in by the second half of first week itself and late antibiotic treatment does not influence the outcome (5) (some strains like lai may cause earlier mortality in the first three days (6)). Presently available culture and antibody based tests become positive only after the end of first week of infection and are unhelpful in deciding to start antibiotics early.

Developing vaccines for domestic animals and humans to bring down the number of cases in endemic areas is important. China, Japan and Cuba have reported the effectiveness of regionally made vaccines (7). Vaccines should be specific for the group of strains particular to a region, hence the identification of local serogroup(s) is important (6,8). Documenting high level of seroprevalence among rodents is also important to impress upon the general public the need for rodent control and protective barriers for farmers as important methods of control of leptospirosis.

Kolenchery is a village in Ernakulam district situated in midland Kerala between the Western Ghats and the coastal strip of Arabian Sea. Several cases of leptospirosis have been diagnosed among the local people here since 1989 (2,3). In MOSC hospital in Kolenchery we diagnosed 155 cases of human leptospirosis in 1993 and 888 cases in 1994 (as per hospital records), many of them were from Kunnackal (KL) and Kunnakkurudy (KR) hamlets at the periphery of Kolenchery. KL and KR are regularly visited by the Community health project volunteers of our hospital. These hamlets are typical of midland rural Kerala and the major occupations of all families are agriculture and animal rearing. These factors favour endemicity for leptospirosis. Hence these two hamlets were chosen for the present study.

We carried out this study to estimate seroprevalence of leptospirosis among humans and rodents in the community living in the two hamlets (KL and KR) in Ernakulam district in Kerala, and to identify locally prevalent leptospiral serogroups.

Material & Methods

Human study: The purpose of this study was conveyed to the members of the community in the two selected hamlets through the community health project volunteers of our hospital after getting approval from the local ethics committee. Apparently healthy male and female volunteers without any clinical illness above the age of 15 yr assembled on a prefixed day at the local community health centre. After taking verbal informed consent, blood samples (5ml each) were collected from all who volunteered. Each of them was registered into the study and a unique number provided taking into consideration address and house number. Samples were collected once a year for 5 consecutive years from 1993 to 1997. A total of 1325 samples were collected from 385 persons in 5 years. Largest number of samples was collected in the 1st year (376) and the least (99) in 1997. The blood collection was done in every February. Serum was separated, labelled, frozen and stored for MAT. The procedures and MAT were performed in the microbiology department of MOSC Hospital, Kolenchery.

MAT was performed on the samples using a stock of local reference strains (3) developed from samples of our earlier patients with the help of the Royal Tropical Institute, Amsterdam, as antigen. It was assumed that the same serogroups that caused clinical disease would be causing subclinical infection in the community. The following serogroups were tested: grippotyphosa (G), australis (As), autunmalis (At), louisiana (L), pomona (P), sarmin (Sa), panama (Pa), sejroe (Se), icterohaemorrhagiae (I), patoc (Pt), pyrogenes (Py), ballum (Ba), bataviae (Bt), canicola (C), djasiman (D), javanica (J). Of these, the last 2 were tested only on rodent samples since their cultures became available only later in the study.

MAT was performed by standard methods (9). All chemicals used for the preparation of media and test procedures were purchased from Hi-Media Laboratories, Mumbai. Bovine albumin fraction V was purchased from Merck India (manufacturers- E Merck, Germany).

Since this study was not conducted during an outbreak but in normal situation, exposure to pathogen may not be very recent. So a MAT titre of 50 or more was decided as the cut-off point for this study. A titre of 1:25 was ignored to avoid ambiguity.

When multiple serogroups were tested positive in the same sample, only the highest titre was taken as significant. This has eliminated representation of serogroups with lower titres. If more than one serogroup showed the same highest titre in a serum sample, the sample was considered positive for all the serogroups.

Studies in rodents: Participants in the study were requested to trap rodents from their farmland and peridomestic areas and bring the rodents live to the investigators. The rodents were anaesthetized and blood was collected for antibody detection and kidney and urine samples for culture. MAT (as in human samples) was used to detect antibodies in blood. Urine was examined under dark field microscopy for leptospires. Kidneys pulverized aseptically as well as urine were inoculated into separate numbered bottles containing Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and incubated at room temperature. The cultures were examined by dark-field microscopy at weekly intervals for a period of two months and identified by MAT using monoclonal antibodies/ antisera. In rodents also, antibody levels of 50 and above were taken as indicator of past contact with leptospires.

Follow up study: Several field visits were conducted to the habitat of individuals found seropositive on completing MAT on all the sera. The primary aim was to identify factors that exposed them to a leptospire contaminated environment including farming activities, irrigation practices and animal rearing. Subjects were questioned for past history suggestive of leptospirosis in them or their family members.

Results

The number of persons contacted and samples collected in this study during the last (fifth) year of the study was low due to migration of people and timing constraints of the investigators. A total of 385 persons (11% of the population) belonging to 110 families took part in the study (Table I). One hundred fourteen persons participating in the study (29.6% of the sample group) had at least one sample tested positive during the period of study. Seropositivity was almost the same in males and females.

The percentage positivity in each year and the variety and numbers of serogroups identified each year are given in Table II. Only those serogroups that showed highest titre or multiple equal titres were reported in a sample. Different serovars belonging to the same serogroup have been counted as a single serogroup.

Human sera sample showed positivity to 13 out of 14 serogroups tested. Of the positive 229 samples from 114 persons, autumnalis was most commonly identified serogroup followed by louisiana, and australis grippotyphosa. Ballum and canicola were the least common with a single positive in each. Autumnalis was the commonest serogroup from 1993 to 1995, grippotyphosa was the largest group in 1996 and 1997. 17 samples showed multiple equal reactions (MER) as follows; grippotyphosa-lousiana and sarmin-1, grippotyphosa-autamnalis and panama-1, grippotyphosa and autumnalis-3, icterohaemorrhagiae and canicola-2, icterhaemorrhagiae and autumnalis-1, autumnalis and louisiana-3, auumnalis and patoc-1, australis and panama-3, australis and louisiana-1, louisiana and sarmin-1. No particular combination of serogroups was discernible.

Seroconversion rate was calculated only in the 74 persons who gave blood samples in all 5 years to get as accurate a picture as possible (Table III). Largest conversion rate was seen in 1994. No negative seroconversion was found in this year.

Seropositive individuals brought 54 rodents caught by them from in and around their houses and crop land. Of these 54, 39 belonged to species Rattus rattus the common peridomestic Indian rat, but only 2 of them showed seropositivity. In contrast, Bandicoota indica were small in number but showed a higher seropositivity (4 out of 9=44.4%). Suncus murinus and Neokia indica did not show seropositivity. Urine aspirated from the bladder of three bandicoots showed leptospires, but urine cultures were contaminated on incubation making serogroups unidentifiable. Urine sample from one Rattus rattus grew serogroup javanica (Table IV).

Field visit to households with seropositive persons confirmed that all their families were engaged in farming and kept animals (cows, goats, dogs). All family members participated in farming activities regardless of other occupations. Farms were small and at subsistence level. The land had a network of natural streams, ponds and irrigation canals, and were interlinked in some areas. Fields were irrigated from all these water bodies during dry seasons and cultivated throughout the year with food crops like tapioca, pineapple and rice. Cattle were grazed in the surrounding fields in daytime and housed and handfed in sheds built close to human dwellings at night.

Discussion

In the present study 29.4 per cent of persons studied showed antileptospiral antibodies against varying serogroups during a period of five years. Among these, 21.92 per cent gave history suggestive of past leptospiral disease. Some of the factors which might have contributed to this high rate of exposure are discussed.

Kerala is in the wet tropical geographical zone with annual rainfall of 3107 mm and ambient temperature ranges from 20 to 32[degrees]C throughout the year except April and May when temperature may rise to 36[degrees]C. This climate is conducive to a group II epidemiology pattern where many hosts act as carriers and multiple serogroups are present in a limited geographical area and cases occur throughout the year (10). Industrialized countries in temperate climates in contrast have a group 1 epidemiology pattern where exposure is occupational or recreational and the animal reservoir and infecting serogroups are limited in number except during natural calamities like floods due to a breakdown of infrastructure (11,12). Kerala gets heavy rains and intermittent floods from April to October. Intermittent flooding of low lying areas leads to repeated flushing out of the forests and farmlands and the rodent burrows there into all water sources including ponds, streams, rivers and canals where leptospires can survive for months. This contamination of surface water can cause monsoon outbreaks as well as sporadic cases throughout the year. Epidemics of leptospirosis during monsoon months have been reported from Kerala (13,14).

KL and KR are low lying regions in Ernakulam district and have many natural streams, ponds and irrigation canals which confluence during monsoon flooding and are fed by the same irrigation project namely the Periyar Valley Irrigation Project during dry months. Moist soil is suitable for prolonged survival of leptospires. Uninterrupted cultivation and continued availability of food crops like rice and pineapple provide rodents with food and cover enabling them to thrive.

All serogroups tested for except pomona were identified in the study at least in one sample. Autumnalis, louisiana, grippotyphosa and australis were the commonest serogroups generally. In our previous study, australis and autumnalis were the serogroups found by MAT in majority of patients with severe infections; icterohaemorrhagiae was less commonly found. Grippotyphosa was the most frequently identified serogroup among patients with milder disease. We have observed a relative decrease in the number of fulminant leptospirosis in our hospital over the last decade (unpublished observation) indicating the population developing partial immunity to leptospirosis or virulent strains like autumnalis, louisiana and australis being replaced by milder strains like grippotyphosa (15).

Three samples showed positivity only to patoc antigen. Patoc is a non-pathogenic leptospire belonging to the biflexa group that cannot invade human tissue but shares common antigens with several pathogenic leptospires. Since positivity only to patoc antigen indicates exposure to pathogenic leptospires belonging to serogroups not represented in the antigen groups tested, it is certain that unidentified serogroups exist in the study area.

The highest sero-conversion rate was observed in 1994 in our study. The maximum number of clinical cases was also recorded in our hospital during 1994. Negative seroconversion in the group was noticed only from 1995 indicating that antibodies persist for a minimum of 2 years.

Immunity to leptospirosis is serovar--specific and conferred by specific antibodies consisting of IgM and IgG. IgG antibodies persist in the blood for many years and provide specific protection. Clinical and subclinical leptospirosis result in the same type of antibody response (8). A polyvalent vaccine made of antigens from locally prevalent serogroups causing severe disease may protect both animals and humans.

All the serogroups identified in humans must necessarily be carried by animal hosts. But the serogroups identified in rodents were more limited in number compared to humans. This may be because of the small number of rodents studied, single sampling and their short lifespan compared to humans. Autumnalis, which is the predominant serogroup, identified among humans formed one third of the serogroups identified among rodents. Domestic animals grazing in the fields and housed close to human habitats may be possible carriers and their role in the cycle of transmission needs to be studied.

The culture method used for isolation of leptospires from urine and kidneys of animals was cumbersome, time consuming and prone to contamination in our experience. Better and sensitive methods like polymerase chain reaction (16) that can eliminate false positive results need to be developed for future studies.

In conclusion, this study detected antileptospiral antibodies in 29.4 per cent persons and 11 per cent of rodents in the two areas surveyed. Majority of the positive humans did not have a history of clinical leptospirosis. A wide variety of serogroups have been demonstrated in humans. Serogroups other than detected in this study may be present in the area. Environmental hygiene and self-protection measures should be the first priorities in prevention of leptospirosis. A comprehensive study on a large sample to identify and characterize local strains is needed.

Acknowledgment

Authors acknowledge Dr T. Jacob John Vellore, for his valuable suggestions in writing this article, Dr Punnen Kurien Manarcaudu, for identifying the rodents, Smt. Anjaly Vijayan G, Smt Jeeva for data processing, Shri C.R. Anandan, Programme Coordinator, Community Health Department for organizing the field work, and the community health volunteers and people of Kunnakurudy and Kunnackal villages for help and co-operation. Authors thank the MOSCMM hospital management for all the material support and the hospital ethics committee for their kind permission to conduct this study.

Received April 13, 2007

References

(1.) John TJ, Rajappan K, Arjunan KK. Communicable diseases monitored by Disease Surveillance in Kottayam district, Kerala state, India. Indian J Med Res 2004; 120: 86-93.

(2.) Kuriakose M, Eapen CK, Punnoose E, Koshy G. Leptospirosis-clinical spectrum and correlation with seven simple laboratory tests for early diagnosis in the Third World. Trans R Soc Trop Med Hyg 1990; 84: 419-21.

(3.) Kuriakose M, Eapen CK, Paul R. Leptospirosis in Kolenchery, India: epidemiology, prevalent serogroups and serovars and a new serovar. Eur J Epidemiol 1997; 13: 691-7.

(4.) Everard COR, Bennett S. Persistence of leptospiral agglutinins in Trinidadian survey subjects. Eur J Epidemiol 1990; 1: 40-4.

(5.) Speelman P. Leptospirosis. In: Kasper DL, Fauci AS, Braunwald E, Lougo DL, Houser SL Jameson JL, editors. Harrison's principles of internal medicine 16th ed. New York: Mcgraw-Hill Medical Publishing Division; 2005. p. 988-91.

(6.) Faine SB, Adler B, Bolin C, Perolat P. Appendix 2. Species and serovar list. Leptospira and leptospirosis 2nd ed. Melbourne, Australia: Medisci; 1999. p. 193-9.

(7.) Faine S, Adler B, Bolin C, Perolat P. Appendix 2. Species and serovar List in Leptospira and Leptospirosis 2nd ed. Melbourne, Australia: Medisci; 1999. p. 159-62.

(8.) Prevention and intervention in human leptospirosis, guidance for diagnosis, surveillance and control International Leptospirosis Society-Geneva: World Health Organization; 2003. p. 23-24.

(9.) Guidelines for the control of leptospirosis, S. Faine WHO Offset publication No. 67. Geneva: World Health Organization; 1982. p. 115-6.

(10.) Faine S, Adler B, Bolin C, Perolat P. Appendix 2 Species and serovar list. Leptospira and leptospirosis 2nd ed. Melbourne, Australia Medisci; 1999. p.138-9.

(11.) Kariv R, Klempfner R, Barnea A, Sidi Y, Schwartz E. The changing epidemiology of leptospirosis in Israel, CDC vol 7 No6 Nov-Dec 2001:4. Available at: http://www.cdc.gov/ncidod/ eid/vol7no6/kariv.html, accessed on November 4, 2005.

(12.) Vinetz JM, Glass GE, Flexner CE, Mueller P, Kaslow DC. Sporadic urban leptospirosis. Ann Intern Med 1996; 125: 794-8.

(13.) Pappachen MJ, Sheela M, Aravindan KP. Relation of rainfall pattern and epidemic leptospirosis in the Indian state of Kerala. J Epidemiol Community Health 2004; 58: 1054.

(14.) Pappachan MJ, Mathew S, Aravindan KP, Khader A, Bhargavan PV, Kareem MMA, et al. Risk factors for mortality in patients with leptospirosis during an epidemic in northern Kerala. Nat Med J India 2004; 17: 240-2.

(15.) Kosovin IL, Chernukha YP. Serological classification of leptospirae of Grippotyphosa serogroup: A new serological ratnapura. J Microbiol (Moscow) 1970; 7: 102-5.

(16.) Bal AE, Gravekamp C, Hartskeerl RA, De, Meza-Brewster, Korver H, Terpstra WJ. Detection of leptospires in urine by PCR for early diagnosis of leptospirosis. J Clin Microbiol 1994; 32: 1894-8.

Reprint requests: Dr Mariamma Kuriakose, Associate Professor, Department of General Medicine Malankare Orthodox Syrian Church Medical College, Kolenchery, 682 311, India

e-mail: lisammal@yahoo.co.in

Mariamma Kuriakose *, Regi Paul *, M.R. Joseph **, Sheela Sugathan ([dagger]) & T.N. Sudha **

Departments of * Internal Medicine, **Community Medicine, ([dagger]) Microbiology, Malankara Orthodox Syrian Church Medical College, Ernakulam, India
Table I. The demographic details of the population studied
in the study area

Total number of
population in the
study area (ages Male Female Total
15-60 yr) 1840 1660 3500 (100)

Sample size 197 (10.7) 188 (11.3) 385 (11)
Number of persons with
 at least one sample
 positive 58 (29.4) 56 (29.8) 114 (29.6)
Number of persons with
 past history
 suggestive of
 clinical disease 15/114 10/114 25/114
 of leptospirosis (13.15) (8) (21.9)

Values in parentheses are percentages

Table II. Pattern of frequency of serogroups detected
each year (1993-1997)

 Total
 no. of Total
 No. of samples Samples positive
 of tested with for one
Year samples positive MER serogroup

1993 376 38 2 36
1994 330 68 5 63
1995 300 75 7 68
1996 220 29 2 27
1997 99 19 1 19
Total 1325 229 17 212

 Number of samples positive for
 representative serovars of different
 serogroups in samples positive for
 one serogroup only

Year At L I Py Sa Pa As

1993 15 9 2 2 1 1 1
1994 19 24 4 3 2 1 5
1995 26 19 4 3 2 1 7
1996 3 1 0 0 0 0 5
1997 1 0 1 0 0 0 2
Total 64 53 11 8 5 3 20

 Number of samples positive for
 representative serovars of different
 serogroups in samples positive for
 one serogroup only

Year G Pt Se Ba Bt Ca

1993 1 0 0 0 0 0
1994 8 0 1 1 1 0
1995 10 1 1 0 1 1
1996 10 6 2 0 0 0
1997 10 4 0 0 0 0
Total 39 11 4 1 2 1

At, Autumnalis; L, Louisiana; I, Icterohaemorrhagiae, Py, Pyrogenes;
Sa, Sarmin; Pa, Panama; As, Australis; G, Grippotyphosa; Pt, Patoc,
Se, Sejroe; Ba, Ballum, Bt, Bataviae, C, Canicola; MER, multiple
equal reaction

Table III. Seroconversion among persons followed up for
5 years (N=74)

 No. of
 samples New
Year seropositive positives

1993 9 9
1994 22 13
1995 19 3
1996 17 7
1997 16 3

 Positive
 seroconversion * Negative
Year (%) seroconversion **

1993 NA NA
1994 17.5 NIL
1995 4 1 (1.3%)
1996 9.45 5 (6.7%)
1997 4 2 (2.7%)

* Proportion of persons who were seronegative and turned
seropositive each year; **Proportion of persons seropositive and
turned seronegative each year

Table IV Rodents showing evidence of leptospiral infection

 Number
 Number showing
Rodent species studied seropositivity Serogroups

Bandicoota 9 4 Autumnalis--2
 indica Djasiman--1
 Pomona--1
Rattus rattus 39 2 Javanica--2
Suncus murinus 5 Nil --
Neokia indica 1 Nil --
Total 54 6 6

 Presence of leptospires
 in urine

 Direct Culture
 % darkfield
Rodent species positivity microscopy

Bandicoota 44.4 2 nil
 indica

Rattus rattus 5.1 1 1 (javanica)
Suncus murinus -- nil nil
Neokia indica -- nil nil
Total 11.1 3 1
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Author:Kuriakose, Mariamma; Paul, Regi; Joseph, M.R.; Sugathan, Sheela; Sudha, T.N.
Publication:Indian Journal of Medical Research
Geographic Code:9INDI
Date:Sep 1, 2008
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