Multilocus Sequence Typing of Leptospira from ...

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Multilocus Sequence Typing of Leptospira from Clinical Isolates in Sri Lanka Karunanayake L.1*, Vimal Raj R.2, Anwesh M.2, Muruganandam N.2, Perera KCR.1, Fernanando N.3, Niloofa R.3, 3 4 5 2 Handunnetti S. , Premawansa S. , Rajapaksha S. , Vijayachari P. 1 National Reference Laboratory for Leptospirosis, Medical Research Institute, Colombo, Sri Lanka 2 Regional Medical Research Centre (RMRC), Andaman and Nicobar Islands, India 3 Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 4 Faculty of Science, University of Colombo, Sri Lanka 5 Faculty of Medicine, University of Colombo, Sri Lanka

Background: Leptospirosis is an endemic disease with a high case fatality and wide spectrum of clinical manifestations and complications in Sri Lanka. It is observed that the circulating serovars/serogroups has changed over time; with the presence of Icterohaemmorrhagiae in 1960’s, Pyrogenes in 2012 and Tarrassovi in 2015 by Microscopic Agglutination Test. In the present study, we compared clinical isolates of Leptospira spp. serogroups Icterohaemmorrhagiae, Javanica, Autumnalis, Louisiana and Sejroe recovered during 1964-1967 and clinical isolates from recent years to assess the genetic diversity of isolates from Sri Lanka. Method: A total of 14 isolates were recovered between 2012 and 2014 from leptospirosis patients and five isolates

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Conclusion: Although we observed a change in the circulating serovars / serogroups over time, genetic similarity of the strains belonging to diverse serovars is seen within the country. This data will be useful for epidemiological surveillance and control of leptospirosis in Sri Lanka.

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isolates belonged to the serogroup Pyrogenes whereas one isolate belongs to serogroup Autumnalis. Phylogenetic analysis using MLST showed that all isolates belonged to genomospecies Leptospira interrogans. Four isolates were closely related to serovar weerasinghe of the Autumnalis serogroup which was isolated from a patient in 1965 from Sri Lanka, and one isolate was closely related to serogroup Australis reported in 1934 from Australia.

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Result: The serogroup classification revealed that four

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were characterized. Group-specific rabbit antisera were used for the serotyping whereas molecular characterisation was performed using Multilocus Sequence Typing (MLST) as described by Ahmed N. et.al 2006.

MLST was completed using six genes viz., adk (Adenylate kinase), icdA (Isocitrate dehydrogenase), LipL41 (Outer membrane Lipoprotein LipL 41), rrs2 (16s Ribosomal RNA), Secy (Translocase pre protein SecY) and LipL32 (Outer membrane Lipoprotein LipL 32). Evolutionary analyses were conducted in MEGA6

Karunanayake L, Karunanayake SAAP, Perera KCR, Senarath U, Gunarathna HDNT, Peter D. Demographic, Clinical and Bacteriological characteristics of Human Leptospirosis in Sri Lanka: A retrospective analysis.J Bacteriol Mycol. 2016: 3(2) AhmedN, Devi SM, A’ ValverdeM de Los, Vijayachari P, Machang'uRS, Ellis WA, HartskeerlRA.Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira speciesAnnal. Clin.Microbio. Microbial 2006, 28 (5), doi: 10.1186/1476-0711-5-28

Acknowledgment Staff of National Reference Laboratory for Leptospirosis, Medical Research Institute, Sri Lanka and Regional Medical Research Centre (RMRC), Andaman and Nicobar Islands, India is gratefully acknowledged.

Funding and Ethics Approval Obtained under project No.24/2012, Ethics Review Committee, Medical Research Institute, Ministry of Health, Colombo, Sri Lanka.