Chamidri Naotunna， Suneth Buddhika Agampodi， Thilini Chanchala Agampodi

Department of Community Medicine， Faculty of Medicine and Allied Sciences， Rajarata University of Sri Lanka， Saliyapura 50008， Sri Lanka

Etiological agents causing leptospirosis in Sri Lanka: A review

Chamidri Naotunna， Suneth Buddhika Agampodi*， Thilini Chanchala Agampodi

Department of Community Medicine， Faculty of Medicine and Allied Sciences， Rajarata University of Sri Lanka， Saliyapura 50008， Sri Lanka

Accepted 15 March 2016

Available online 20 April 2016

Leptospira

Leptospirosis

Sri Lanka

Serovar

Strain

Species

Objective: To systematically review the etiological agent causing human leptospirosis in Sri Lanka. Methods: Published articles on leptospirosis and Leptospira in Sri Lanka were all reviewed to determine serovar， strain and species level indentifi cation of Leptospira. After screening process，74 full text articles/reports were reviewed and among of them， 12 published papers describing isolation of Leptospira from Sri Lankan patients/animals， 5 molecular epidemiology papers on newer typing methods citing Sri Lanka isolates， with a descriptions of the isolates and 6 published papers reporting PCR based species level identification were identified. Results: Published literature showed that more than 40 strains classifi ed under at least 20 serovars and 10 serogroups have been isolated from Sri Lanka. These isolates belong to four species， namely， Leptospira interrogans， Leptospira kirschneri， Leptospira borgpetersenii， and Leptospira santarosai. In addition，recent studies on direct patient samples without culture and isolation showed Leptospira from Leptospira weilli is also circulating in Sri Lanka. Multi locus sequence typing showed 13 genotypes of Leptospira from Sri Lankan isolates. Conclusions: This review shows the diversity of Leptospira in Sri Lanka， but culture isolation data has not been published in Sri Lanka during last 30 years.

1. Introduction

Leptospirosis is one of the most widely spread zoonotic disease in the world with an estimated 1.03 million annual cases and 58 999 deaths［1］. Control and prevention of leptospirosis are often limited due to knowledge gaps on disease agent， hosts and environment enabling the disease transmission in local settings. The disease is caused by spirochetes belonging to genus Leptospira， which has more than 230 serovars classifi ed in to 31 serogroups based on the serology. Based on the DNA hybridization methods， 21 species has been identifi ed to date［2-12］. Because of the diversity of Leptospira，management and control of leptospirosis is a challenge especially for countries in tropical setting where the facilities for isolation and typing are limited.

Sri Lanka is having one of the highest incidence of leptospirosis and considered as a leptospirosis high endemic country［13］. During the fi ve year period from 2004 to 2008， the total number of cases reported to the Epidemiology Unit （National surveillance Centre）was around 4 000 cases［14］. In 2008， Sri Lanka experienced the worst recent outbreak of leptospirosis with more than 7 000 reported cases［15］. Cumulative annual incidence of leptospirosis during 2008-2014 period in Sri Lanka was more than 25 per 100 000 and during the last 6 years， an average of more than 5 000 cases were reported annually to the Epidemiology unit［16-20］. Despite all control measures taken by the national control programme， leptospirosis continues to aff ect lives of people in Sri Lanka. This could be partly attributed to the knowledge gap in leptospirosis transmission. There were considerable numbers of research carried out since 2008，however investigations on disease causing agents are limited.

The present standard diagnostic method for leptospirosis is microscopic agglutination test （MAT）［21］. Several extensive studies showed that MAT could not be considered as the gold standard due to its very low sensitivity［22］. We experienced same in Sri Lanka in 2008［15］ and 2011［23］. Though imperfect， MAT is still the standard test for leptospirosis diagnosis due to lack of other diagnostic facilities. Standard MAT panel should be based on the local knowledge on circulating serovars or it should be a broad MAT panel to cover all pathogenic serogroups. In high burden countries in South East Asia， including Sri Lanka， these standard MAT diagnostic facilities are still not available. As an example， till 2014 Sri Lankan diagnostic laboratory used genes specifi c Patoc serovar for MAT and still the MAT panel used in Sri Lankan reference center include only 11 serovars. This lack of diagnostic facilities are attributed due to lack of knowledge on circulating serovars or lack of resources to maintain labour intensive MAT diagnosis facilities. However， this may be partly due to lack of knowledge synthesis within the country. The purpose of this review is to identify the locally prevalent serovars of Leptospira in Sri Lanka based on published literature to fi ll this knowledge gap.

2. Materials and methods

We carried out a comprehensive search of literature to identify studies related to leptospirosis and Leptospira in Sri Lanka. Two main search platforms were used for internet based search: Pubmed and Google Scholar. The search strategies were as follows: Pubmed search string （56） （（（（“Leptospirosis”（Mesh） OR ‘Weil Disease’（Mesh） OR （‘Leptospira”（Mesh） OR ‘Leptospira interrogans（L. interrogans） serovar Pomona’（Mesh） OR ‘L. interrogans serovar Icterohaemorrhagiae’（Mesh） OR ‘L. interrogans serovar Hebdomadis’（Mesh） OR ‘L. interrogans serovar Canicola’（Mesh）OR ‘L. interrogans serovar Autumnalis’（Mesh） OR ‘L. interrogans serovar Australis’ （Mesh） OR ‘L. interrogans’（Mesh） ））） OR（（（‘Leptospirosis’ OR ‘Leptospira’ OR ‘Weils disease’ OR ‘Weil’s syndrome’ ））） AND （（‘Sri Lanka’ OR ‘Ceylon’））； Google Scholar search （2 320） （“Leptospirosis” OR “Leptospira”） AND （“Sri Lanka”O(jiān)R “Ceylon”）.

In PubMed search， we used a specifi c search strategy. However， in Google Scholar， we manually screened all titles appeared after the initial search to include grey literature and articles that are not listed in PubMed.

We also carried out manual search， specially to retrieve data from non-indexed local publications. For this purpose we used three bibliographic references: a Bibliography of medical publications related to Sri Lanka 1811-1976［24］ and its supplement Bibliography on health in Sri Lanka， 1977-1980［25］ by Peiris and Uragoda and Bibliography of Medical Literature 1980-2005 compiled by Post Graduate Institute of Medicine （PGIM） Library， Colombo. In addition， we hand searched titles of theses and dissertations， and casebooks submitted to PGIM， all issues of Ceylon Medical Journals prior to 2008 and archived issues of Sri Lanka Journal of Medical Sciences and Kandy Medical Journal in four libraries （Sri Lanka Medical Association Library， PGIM library and Colombo and Peradeniya Medical School libraries）. Further， we searched technical reports published by Medical Research Institute during 1960-1980 period and Quarterly Epidemiological Bulletins of Epidemiology Unit 1980-2014. Reference lists of relevant articles were also searched to identify any missing article. In addition， we searched for publications which included global collection of Leptospira isolates for typing to get information on Sri Lankan isolates.

The search was done in several steps. First we selected all Sri Lankan publications and publications on Sri Lankan patients or animals related to leptospirosis and Leptospira. This was done by three investigators. A title and abstract search was done to eliminate review articles or articles without primary data. Once the articles with original data were identifi ed， we searched the full text for any article reporting culture isolation or inoculation of patient samples to animal models to isolate Leptospira. We did not exclude any article based on quality of culture isolation details， rather we cross checked the reported isolations specially strain information with online databases on Leptospira published by Pasture institute and data published in speices classifi cation studies using large collection of global isolates to validate our fi nding. We did not limit our search to languages and online translations were used to translate the title and abstracts of selected articles.

3. Results

The fl ow diagram shows in Figure 1.

Figure 1. Flow diagram.

Through full text review of selected literature， we identifi ed 12 published papers［26-37］ describing isolation of Leptospira from Sri Lankan patients/animals， 5 molecular epidemiology papers on newer typing methods citing Sri Lanka isolates， with a descriptions of the isolates［38-42］ and 8 published papers［15， 23， 43-47］ reporting PCR based species level identifi cation.

First confirmed case of leptospirosis in Sri Lanka was reported by Rajasuriya et al［26］ and the investigators have done guinea pig inoculation and demonstration of Leptosires， but isolation was not done. First report on isolation of Leptospira from Sri Lankan patient was by Nityananda in 1962［27］ and L. interrogans serovar Icterroheamorragia was identified. Most of the early studies demonstrated Leptospires after guinea pig inoculation， but only few studies isolated and reported the serovars identifi ed.

Early publications on isolation were based on serological classifi cations only. More than 20 serovars have been documented as isolated from Sri Lanka from four speicies； L. interrogans， Leptospira kirschneri （L. kirschneri）， Leptospira borgpetersenii （L. borgpetersenii），and Leptospira santarosai （L. santarosai） （Table 1）. However， the classifi cation was not clear for some of the serovars and some of the recently isolated strains are listed as undetermined［40］. Of the strains isolated from Sri Lanka， eight strains are listed as the reference strains for the serovars.

In some of the published studies， only the serogroup was mentioned after the culture isolation but not the exact serovars. Combination of published serovars and serogroups showed that serovars belonging to at least 10 serogroups are circulating in Sri Lanka. The circulating serogroups of Leptospira include Icterohaemorragiae， Autumnalis，Sejroe， Grippotyphosa， Javanica， Louisiana， Canicola， Hebdomadis，Pomona and Pyrogenes.

Table 1Summary of Leptospira serovars isolated from Sri Lanka*.

Isolation of serovars was mainly from leptospirosis patients. However， several studies looked at the presence of Leptospira in mammals and rodents. The largest study on mammalian and rodent hosts were carried out in 1971 which included cattle， dog， swine，shrew， mongoose， hare， cat， bandicoots， sewer rats， rice fi eld rats，Asian house shrew， Ceylon flat country house rat， Ceylon hill country house rat and rock squirrels［32］. Cattle， swine， mongoose and hare kidney tissue and samples did not revealed leptospires after culture. All other animals showed leptospires belonged to several serogrups.

Recent studies using molecular techniques without culture or isolation also confi rmed the wide variation of species circulating in Sri Lanka （Table 2）.

Table 2Leptospira species identifi cation based on molecular techniques without culture and isolation.

These recent studies also show regional variations of circulating serovars， which is common among human and animal species.

Multi Locus Sequence based genotyping of Sri Lankan isolates was reported using three diff erent typing schemes［38， 40-42］. The combined typing scheme published in 2013 using seven MLST loci using 23 Sri Lankan isolates （Table 3） shows that at least 13 genotypes are causing human leptospirosis in this country［41］. Two of these genotypes （ST 1 & ST 44） were demonstrated in direct clinical samples from confi rmed cases of leptospirosis［48］.

Table 3Multi Locus Sequence based genotyping of Sri Lankan isolates using 7 loci published by Boonslip et al 2013.

4. Discussion

Contrary to the popular belief among Sri Lankan researchers and practitioners， a large number of Leptospira serovars， stains and serotypes have been already identified in Sri Lanka， majority in 1960’s and 70’s. These include more than 40 strains classifi ed in to more than 20 serovars from 10 serogroups representing fi ve species. However， no published data available on culture and isolation of Leptospira in the recent past， where the leptospirosis has become hyper endemic in this country.

This review clearly shows the diversity of Leptospira in Sri Lanka，a small country with 20 million population in 74 000 square kilometers. This diversity is only for the disease causing agent，however the disease transmission is more complicated with large number of reservoir animals and a range of different ecological systems that facilitate disease transmission in animal-human interface. As shown in a recent publication［23］， these complexities may lead to a completely diff erent clinical presentation by diff erent serovars through different reservoir hosts. This complicates the disease control activities even within a context of small country，where national level strategies may not be applicable in diff erent geographical regions in the country.

Though we reported large number of serovars in this review， it is important to note that this is not providing data on prevalent serovars or serotypes circulating in Sri Lanka at present. With the climate change， change of ecological systems and diff erent animal human interaction may completely change the circulating serovars and strain types which need to investigate using culture and isolation. Though the new robust methods of Leptospira classifi cation based on MLST［41］ is more scientific and provide more information on genetic relatedness of Leptospira， for MAT we need serovar-based identification with culture and isolation. This is a challenge in settings where the presentation is late due to initial treatment at out patient settings and also heavy use of antibiotics before coming to hospitals.

As we observed in Sri Lanka， knowledge synthesis of already available and published data will provide a strong platform for future research and baseline data for diagnosis and control activities of this globally important disease.

Conflict of interest statements

We declare that we have no confl ict of interest.

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ent heading

10.1016/j.apjtm.2016.03.009

15 January 2016

Suneth Buddhika Agampodi， Department of Community Medicine， Faculty of Medicine and Allied Sciences， Rajarata University of Sri Lanka，Saliyapura 50008， Sri Lanka.

Tel: +94777880096

Fax: +94252226252

E-mail: sunethagampodi@yahoo.com

in revised form 20 February 2016

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