A Study on the Presence of Pathogenic Leptospira spp. in Environmental Water Samples Obtained from Selected Recreational Areas in Terengganu, Malaysia
Salwani Ismail*, Noor Zarina Abd Wahab, Norhidayah Badya, Nor Iza A. Rahman, Chew Chieng Yeo, Ahmad Zubaidi A. Latif, Mainul Haque
Faculty of Medicine and Health Sciences, Universiti Sultan Zainal Abidin, 20400 Kuala Terengganu, Malaysia
*Corresponding Author E-mail: salwani@unisza.edu.my
ABSTRACT:
Leptospirosis is a zoonosis of worldwide distribution. It is endemic in Malaysia and is caused by infection with pathogenic Leptospira species. Leptospires can survive in water for many months hence water is an important vehicle in their transmission. Recreational activities such as swimming, fishing, canoeing and rafting place a person at risk for the infection. The objective of this study was to detect the presence of pathogenic Leptospira spp. in environmental water samples obtained from selected recreational areas in Terengganu. A total of 40 water samples (2000 ml) were collected from two recreational areas in north Terengganu namely Lata Tembakah and Lata Belatan Waterfalls. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) media, incubated at 30°C for one month and examined under the dark-field microscope. Positive growth for the pathogenic leptospires was then confirmed by Polymerase Chain Reaction (PCR) assay using G1/G2 primers. Pathogenic leptospires were detected in both recreational areas, 5% (1 out of 20) samples and 10% (2 out of 20) samples from Lata Tembakah Waterfall and Lata Belatan Waterfall respectively and were confirmed positive for the presence of pathogenic leptospires. As a conclusion, pathogenic leptospires were detected at two recreational areas in Terengganu and this would pose a risk of infection to human and lead to leptospirosis outbreak. Therefore, based on the result of this study, precautionary steps should be taken by responsible authorities to prevent human infection or leptospirosis outbreak.
KEYWORDS: Leptospirosis, Recreational, Water, Pathogenic-Leptospires, Malaysia
INTRODUCTION:
Leptospirosis is a re-emerging zoonotic disease of global importance as because it ‘affects humans and animals in all continents, in both rural and urban areas’ [1-3]. It is caused by infection with pathogenic Leptospira species and is characterized by a broad spectrum of clinical manifestations, varying from unapparent infection to fulminant, fatal disease[1,4]. Rodents and domestic mammals, such as cattle, pigs and dogs, serve as major reservoir hosts [1]. Infected animals may excrete leptospires intermittently or regularly for months or years, or for their lifetime [1]. Human infection results from direct or indirect exposure to the urine of carrier animals [1].
Since leptospires are excreted in the urine and can survive in water for many months, water is an important vehicle in their transmission [5]. The first case of leptospirosis from India was reported in 1929 from Andaman and Nicobar Islands [6]. Leptospires gain entry into the human blood stream via cuts, skin abrasions or mucous membranes [1].
Recreational activities such as swimming, fishing, canoeing and rafting place a person at risk for the infection[7-8]. Malaysia is an endemic area for Leptospirosis [9]. The first case of leptospirosis in Malaysia was reported in 1925 [9]. In June, 2010 there was an outbreak of leptospirosis involving visitors at Lubuk Yu Recreation Centre in Maran, Pahang and laboratory tests conducted on the water samples confirmed the presence of leptospires[9-11]. In Bangkok, Thailand, there were 59 cases from January 1994 to December 2000 are documented [12]. Poor hygienic condition at the recreational area was identified as a contribution factor for the occurrence of the outbreak [12-13]. Poor hygienic condition will attract the attention of rodents to come to the area and pollute the water and soil which in turn pose a risk of infection to human [13].
Table 1. Identification of Leptospira isolates by microscopic observation and PCR assay from Lata Tembakah Waterfall
|
Sample Types |
First sampling |
Second sampling |
|||
|
Detection method |
|||||
|
Microscopic observation |
PCR |
Microscopic observation |
PCR |
||
|
LT1 |
LT1A |
-- |
-- |
-- |
-- |
|
LT1B |
-- |
---- |
-- |
-- |
|
|
LT1C (spin) |
-- |
-- |
-- |
-- |
|
|
LT2 |
LT2A |
-- |
-- |
-- |
-- |
|
LT2B |
-- |
-- |
-- |
-- |
|
|
LT2C (spin) |
-- |
-- |
-- |
-- |
|
|
LT3 |
LT3A |
-- |
-- |
-- |
-- |
|
LT3B |
-- |
-- |
-- |
-- |
|
|
LT3C (spin) |
-- |
-- |
-- |
-- |
|
|
LT4 |
LT4A |
-- |
-- |
-- |
-- |
|
LT4B |
-- |
-- |
-- |
-- |
|
|
LT4C (spin) |
-- |
-- |
+ve |
-ve |
|
|
LT5 |
LT5A |
-- |
-- |
-- |
-- |
|
LT5B |
-- |
-- |
-- |
-- |
|
|
LT5C (spin) |
-- |
-- |
+ve |
+ve |
|
|
LT6 |
LT6A |
-- |
-- |
-- |
-- |
|
LT6B |
-- |
-- |
-- |
-- |
|
|
LT6C (spin) |
-- |
-- |
-- |
-- |
|
|
LT7 |
LT7A |
-- |
-- |
-- |
-- |
|
LT7B |
-- |
-- |
-- |
-- |
|
|
LT7C (spin) |
-- |
-- |
-- |
-- |
|
|
LT8 |
LT8A |
-- |
-- |
-- |
-- |
|
LT8B |
-- |
-- |
-- |
-- |
|
|
LT8C (spin) |
+ve |
-ve |
-- |
-- |
|
|
LT9 |
LT9A |
-- |
-- |
-- |
-- |
|
LT9B |
-- |
-- |
-- |
-- |
|
|
LT9C (spin) |
+ve |
-ve |
-- |
-- |
|
|
LT10 |
LT10A |
-- |
-- |
-- |
-- |
|
LT10B |
-- |
-- |
-- |
-- |
|
|
LT10C (spin) |
+ve |
-ve |
+ve |
-ve |
|
* A and B are un-centrifuged water samples, Csas centrifuged water sample, LT: Lata Tembakah Waterfall
Terengganu is famous with its recreational areas namely Sekayu Waterfall, Lata Tembakah Waterfall, Lata Belatan Waterfall and Kenyir Lake. There was no published data so far about the presence of pathogenic Leptospira spp. at these recreational areas of Terengganu, Malaysia. This present study was to detect the presence of pathogenic Leptospira spp. in water samples obtained from selected recreational areas in Terengganu. The data of this study thus may help in controlling and preventing the disease by alerting the responsible authorities to take further actions and instilling public awareness.
MATERIALS AND METHODS:
Samples Collection
The environmental water samples were obtained from two recreational areas in north region of Terengganu, Malaysia known as Lata Tembakah and Lata Belatan Waterfalls. Sample collection at each recreational area was done on 2 occasions with 1 month apart dated 8th February 2012 and 21st March 2012. At each recreational area, 10 sampling sites were selected and at each sampling site 50 ml of water was collected. Thus a total of 1000 ml water was collected at each recreational area on 2 sampling occasions. The sampling was done by dipping a 100 ml sterile water container directly into the stagnant water. The samples were then transported to the laboratory and processed within 24 hours of sample collection. pH and temperature of the water samples were also recorded.
Samples Filtration, Inoculation and Incubation:
5 ml water sample from each sampling site were syringed out and 2.5 ml was filtered through a sterile syringe filter with 0.20 µm pore size membrane filter into duplicated of 5 ml Ellinghausen McCullough Johnson Harris (EMJH) media [14]. The rest of the water sample was centrifuged at 14,000 rpm for 30 min, the pellet then was inoculated into 5 ml EMJH media. All the culture media were then incubated aerobically at 30°C in shaking incubator for a maximum of 30 days.
Isolate Detection:
The presence of Leptospira spp. in the culture media was detected by a dark-field microscope. Leptospires have a hook-like end, very thin and motile. If the leptospires were not detectable after 30 days of incubation, the sample was considered to be negative. Presence of at least four leptospires in a microscopic field was considered as a positive result [15]. The positive samples were separated and were later used for identification of pathogenic species by PCR method.
Identification of Pathogenic Leptospira Spp. by PCR:
DNA extraction was performed on all leptospires isolates by using the WizardTM Genomic DNA purification kit (PROMEGA). The G1/G2 primers [16] were used in this PCR assay. Amplification of the DNA was conducted in a total volume of 25 μl. The reaction mixture consisted of 1X
PCR buffer, 3.0 mM of MgCl2, 250 μM of dNTP’s premixed, 1 μM of each primer and 0.5 unit of Taq DNA Polymerase. There were 3 steps that consisted of denaturation at 94ºC for 1.5 minutes, annealing at 55ºC for 1 minute and extension at 72ºC for 2 minutes. The steps were repeated for 30 cycles with addition of 5 minutes before cycles and 10 minute after the last cycles for final extension of the primers. The PCR product together with loading buffer were subjected to electrophoresis on a 1.0% agarose gel in 1X TBE buffer at 65 V for 2 hours. The gel was pre-stained with ethidium bromide and photographed under Alpha Innotech Gel Doc.
Table 2. Identification of Leptospira isolates by microscopic observation and PCR assay from Lata Belatan Waterfall
|
Sample Types |
First sampling |
Second sampling |
|||
|
Detection methods |
|||||
|
Microscopic observation |
PCR |
Microscopic observation |
PCR |
||
|
LB1 |
LB1A |
-- |
-- |
-- |
-- |
|
LB1B |
-- |
-- |
-- |
-- |
|
|
LB1C (spin) |
-- |
-- |
-- |
-- |
|
|
LB2 |
LB2A |
-- |
-- |
-- |
-- |
|
LB2B |
-- |
-- |
-- |
-- |
|
|
LB2C (spin) |
-- |
-- |
-- |
-- |
|
|
LB3 |
LB3A |
-- |
-- |
-- |
-- |
|
LB3B |
-- |
-- |
-- |
-- |
|
|
LB3C (spin) |
-- |
-- |
-- |
-- |
|
|
LB4 |
LB4A |
-- |
-- |
-- |
-- |
|
LB4B |
-- |
-- |
-- |
-- |
|
|
LB4C (spin) |
-- |
-- |
+ve |
+ve |
|
|
LB5 |
LB5A |
-- |
-- |
-- |
-- |
|
LB5B |
-- |
-- |
-- |
-- |
|
|
LB5C (spin) |
-- |
-- |
-- |
-- |
|
|
LB6 |
LB6A |
-- |
-- |
-- |
-- |
|
LB6B |
-- |
-- |
-- |
-- |
|
|
LB6C (spin) |
-- |
-- |
+ve |
-ve |
|
|
LB7 |
LB7A |
-- |
-- |
-- |
-- |
|
LB7B |
-- |
-- |
-- |
-- |
|
|
LB7C (spin) |
-- |
-- |
+ve |
+ve |
|
|
LB8 |
LB8A |
-- |
-- |
-- |
-- |
|
LB8B |
-- |
-- |
-- |
-- |
|
|
LB8C (spin) |
+ve |
-ve |
-- |
-- |
|
|
LB9 |
LB9A |
-- |
-- |
-- |
-- |
|
LB9B |
-- |
-- |
-- |
-- |
|
|
LB9C (spin) |
-- |
-- |
-- |
-- |
|
|
LB10 |
LB10A |
-- |
-- |
-- |
-- |
|
LB10B |
-- |
-- |
-- |
-- |
|
|
LB10C (spin) |
-- |
-- |
-- |
-- |
|
*A and B samples denoted as un-centrifuged water sample (duplicate), C sample denoted as centrifuged water sample, LB: Lata Belatan Waterfall
RESULTS:
The overall results for detection of pathogenic leptospiral isolates from Lata Tembakah and Lata Belatan Waterfall were summarized in Table 1 and Table 2 respectively. Observation of positive growth of leptospires under the dark field microscope is a preliminary step to identify leptospires. Leptospires have a hook-like end, very thin and motile. Observation through dark field microscope revealed 30% (6 out of 20) and 20% (4 out of 20) samples from Lata Tembakah and Lata Belatan respectively were positive for the growth of leptospires.
This study showed that pathogenic Leptospires were present in both Lata Tembakah and Lata Belatan Waterfalls. Lata Belatan Waterfall however revealed a higher detection rate compared to Lata Tembakah with 10% and 5% respectively while observing in dark field microscope. Subsequently, Polymerase Chain Reaction (PCR) assay was done for confirmation of pathogenic leptospires by using G1/G2 primers. The result showed that pathogenic leptospires were detected in both recreational areas with 5% (1 out of 20) samples and 10% (2 out of 20) samples from Lata Tembakah and Lata Belatan respectively were confirmed positive for the presence of pathogenic leptospires through formation of 285 base pairs product in 1% agarose gel (Figure 1).
Figure 1. Detection of pathogenic leptospires by PCR assay using G1/G2 primer Mw: molecular weight (100bp), - Ctrl: negative control, + Ctrl: positive control. LB: Lata Belatan Waterfall, LT: Lata Tembakah Waterfall
The isolation rate of Leptospires from both areas was quite low with 30% for Lata Tembakah and 20% for Lata Belatan Waterfall. All of the culture media with positive growth for leptospires came from centrifuged samples. It showed that centrifugation of the water samples before inoculating into the culture media will concentrate the leptospires thus increased the isolation rate. Other than that the isolation rate is also based on few other factors like the pH of the culture media, temperature, presence of bacterial contamination and the duration for which the cultures are kept.
This study also showed that in Lata Tembakah Waterfall, out of 6 positive isolate only one (17%) isolate was confirmed positive for pathogenic leptospires whereas 50% (2 out of 4) for Lata Belatan Waterfall. This indicated that the rest of the isolates were saprophytic leptospires. Lata Tembakah Waterfall, even though 6 samples were positive for the growth of leptospires but only one sample was confirmed positive for the presence of pathogenic leptospires by PCR, the rest of the isolates were non-pathogenic. Meanwhile for the Lata Belatan Waterfall, out of 4 samples that were positive for the growth of leptospires, 2 samples were positive for the pathogenic leptospires and another 2 were non-pathogenic.
DISCUSSION AND CONCLUSIONS:
Appropriate conditions such as pH of water, temperature, characteristics of the water and availability of wild animals which act as reservoirs would sustain the presence of leptospires in the environment [17]. Poor hygienic condition at the recreational area was identified as a contribution factor for the occurrence of the outbreak. Poor hygienic condition will attract the attention of rodents to come to the area and pollute the water and soil which in turn pose a risk of infection to human [13]. Isolation of pathogenic leptospires from environment in bacterial media is difficult due too rapid growth of free living saprophytic leptospires in culture which hinder the pathogenic leptospires from being isolated [18]. Furthermore saprophytic leptospires are more abundant in environment as compared to pathogenic leptospires because pathogenic species can thrive well under certain environmental conditions only [19-22]. Pathogenic Leptospira was also isolated long before in 1966 from ‘surface water’ of recreational site as like current study [23-24]. There has been recently similar reports isolation of pathogenic Leptospira in number countries [25-27].
Pathogenic leptospires were detected at two recreational areas in Terengganu and this would pose a risk of infection to human and lead to a leptospirosis outbreak. Therefore, based on the result of this study, precautionary steps should be taken by responsible authorities to prevent human infection of leptospirosis. Well-designed prospective studies are advocated to prevent communicable diseases from such beautiful natural resorts areas of Terengganu and Malaysia with attention to safeguard health of local and foreign tourists.
ACKNOWLEDGEMENTS:
The authors would like to thank the Director of Institute of Medical Research (IMR) Malaysia and staffs for supporting this project. This project was financed by UniSZA Research Grant Scheme No. UniSZA/11/GU (006). Authors do not possess any conflict of interest.
REFERENCES:
1. Victoriano AFB, Smythe LD, Glorianin-Barzaga N, Cavinta LL, Kasai TK, Limpakarnjanarat K, Ong BL, Gyanendra G, Julie H, Caroline AC, Yasutake Y, Yoshida S, Alder B. Leptospirosis in the Asia Pacific region. BMC Infectious Diseases. 9(147); 2009: Available from: URL:http://www.biomedcentral.com/ content/pdf/1471-2334-9-147.pdf
2. Biswas A, Kumar R, Chaterjee A, Pan A. A case of leptospirosis in a child with unusual clinical manifestation. Journal of College of Medical Sciences-Nepal. 8 (2); 2012: 37-41. Available from: URL: http://www.nepjol.info/index.php/JCMSN/article/view File/6836/5544
3. Vein J, Perrin A, Berny PJ, Benoit E, Leblond A, Kodjo A. Adaptation of a real-time PCR method for the detection and quantification of pathogenic leptospires in environmental water. Can J Microbiol. 58(7); 2012: 828-835. doi: 10.1139/w2012-060. Epub 2012 Jun 14. Available from URL: https://www. researchgate.net/publication/225373504_Adaptation_of_a_real-time_PCR_method_for_the_detection_and_quantification_of_pathogenic_leptospires_in_environmental_water
4. Forouhar E, Mitsani D. Sporadic urban leptospirosis. Journal of Community Hospital Internal Medicine Perspectives. 1 (1); 2011: 7042. DOI: 10.3402/jchimp.v1i1.7042. Available from: URL: http://www.jchimp.net/index.php/jchimp/article/download/ 7042/pdf_139
5. Peter S, Rudy H. Leptospirosis. Harrison’s Principles of Internal Medicine 17th Edition, 2008 164: 1199-1202.
6. Faines S. Guidelines for control of leptospirosis. Geneva, World Health Organisation offset Publication 1982; 67. Available from: URL: http://whqlibdoc.who.int/offset/WHO_OFFSET_67_(p1-p98).pdf
7. Unchalee T, Chanchai T, Duangporn P, Charnchudhi C. Duplex PCR-Hybridization Based Detection of Pathogenic Leptospira In Environmental Water Samples Obtained From Endemic Areas In Northeast Region Of Thailand. Southeast Asian J Trop Med Public Health. 37(4); 2006: 729-741.
8. Narita M, Fujitani S, Haake DA, Paterson DL. Leptospirosis after recreational exposure to water in the Yaeyama islands, Japan. Am J Trop Med Hyg. 73(4); 2005: 652-6. Available from: URL: http://www.ajtmh.org/content/73/4/652.full.pdf+html
9. El-Jalii IM, Bahaman AR. A review of human leptospirosis in Malaysia. Trop Biomed. 21; 2004:113-119. Available from: URL: http://www.msptm.org/files/Leptospirosis_Review_ Humans_Isam_2.pdf
10. How SH, Ng KH, Jamalludin AR, Shah A, Rathor Y. Melioidosis in Pahang. Med J Malaysia. 2005; 60 (5): 606-13. Available from: URL: http://www.e-mjm.org/2005/v60n5/Melioidosis_ Pahang.pdf
11. Sapian M, Khairi MT, How SH, Rajalingam R, Sahhir K, Norazah A, Khebir V, Jamalludin AR. Outbreak of Melioidosis and Leptospirosis Co-infection Following a Rescue Operation. Med J Malaysia. 67 (3); 2012: 293-297. Available from: URL: http://www.e-mjm.org/2012/v67n3/Melioidosis-and-Leptospirosis.pdf
12. Ariyapruchya B, Sungkanuparph S, Dumrongkitchaiporn S. Clinical presentation and medical complication in 59 cases of laboratory-confirmed leptospirosis in Bangkok. Southeast Asian J Trop Med Public Health. 34(1); 2003: 159-64.
13. Weese JS, Peregrine AS, Armstrong J. Occupational health and safety in small animal veterinary practice: Part I — Nonparasitic zoonotic diseases. Can Vet J. 43(8); 2002; 631–636. PMCID: PMC339409. Available from: URL: http://www.ncbi.nlm. nih.gov/pmc/articles/PMC339409/pdf/20020800s00035p631.pdf
14. Johnson RC, Harris VG. Differentiation of Pathogenic and Saprophytic Leptospires I. Growth at Low Temperatures. J Bacteriol. 94(1); 1967: 27–31. Available from: URL: http://jb.asm.org/content/94/1/27.full.pdf
15. Issazadeh K, Amirmozaffari N, Mehrabian S, Oryan S. Assessment of Distribution Leptospira spp. in Surface Waters of Guilan Province. American-Eurasian Journal of Scientific Research. 3(2); 2008: 147-152. Available from: URL: http://www.idosi.org/aejsr/3(2)08/6.pdf
16. Gravekamp C, Van de Kemp H, Franzen M, Carrington D, Schoone GJ, Van Eys GJ, Everard CO, Hartskeerl RA, Terpstra WJ. Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol. 139(8); 1993: 1691-1700. Available from: URL: http://mic.sgmjournals.org/ content/139/8/1691.long
17. Ridzlan FR, Bahaman AR, Khairani-Bejo S, Mutalib AR. Detection of pathogenic Leptospira from selected environment in Kelantan dan Terengganu, Malaysia. Trop Biomed. 27(3); 2010: 632-8. Available from: URL: http://www.msptm.org/files/632_-_638_Ridzlan_FR.pdf
18. Wilson R, Fujioka R. Development of a method to selectively isolate pathogenic Leptospira from environmental samples. Water Science and Technology. 5-6; 1995: 275–282.
19. Picardeau M, Bulach DM, Bouchier C, Zuerner RL, Zidane N, Wilson PJ, Creno S, Kuczek ES, Bommezzadri S, Davis JC, McGrath A, Johnson MJ, Boursaux-Eude C, Seemann T, ´ Rouy Z, Coppel RL, , Rood JI, Lajus AL, Davies JK, Me ´digue C, Adler B. Genome Sequence of the Saprophyte Leptospira biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis. PLoS ONE. 3 (2); 2008: e1607. Available from: URL: http://www.plosone.org/article/fetch Object.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0001607&representation=PDF
20. Muñoz-Zanzi C, Mason MR, Encina C, Astroza A, Romero A. Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile. Int J Environ Res Public Health. 11(7); 2014: 6666–6680. doi: 10.3390/ijerph110706666. PMCID: PMC4113836. Available from: URL: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC4113836/pdf/ijerph-11-06666.pdf
21. Yasouri SR, R. Moghadam RG, Ghane M. Identification of Pathogenic and Saprophytic Leptospira spp from the Rice Fields of Tonekabon Township Using PCR Technique. Advanced Studies in Biology. 5 (10); 2013: 437-445. Available from: URL: http://m-hikari.com/asb/asb2013/asb9-12-2013/ghane ASB9-12-2013.pdf
22. Trueba G, Zapata S, Madrid K, Cullen P, Haake D. Cell aggregation: a mechanism of pathogenic Leptospirato survive in fresh water. Int Microbiol. 7; 2004: 35-40. Available from: URL: http://www.im.microbios.org/25march04/06%20Trueba.pdf
23. Diesch SL, McCulloch WF. Isolation of pathogenic leptospires from waters used for recreation. Public Health Rep. 81(4); 1966: 299–304. PMCID: PMC1919800. Available from: URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919800/pdf/pubhealthreporig00040-0005.pdf
24. Braun JL, McCulloch WF. Use of 8-azaguanine to differentiate leptospires isolated from Iowa surface waters. Appl Microbiol. 16(1); 1968: 174–175. PMCID: PMC547355. Available from: URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC547355/ pdf/applmicro00237-0196.pdf
25. Ganoza CA, Matthias MA, Collins-Richards D, Brouwer KC, Cunningham CB, Segura ER, Gilman RH, Gotuzzo E, Vinetz JM. Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira. PLoS Medicine. 3 (8); 2006: 1329-1340. Available from: URL: http://web.b.ebscohost.com/ehost/pdfviewer/ pdfviewer?sid=898c0c49-b110-4524-a60f-77d51bc6f395%40sessionmgr114&vid=0&hid=119
26. Schneider MC, Jancloes M, Buss DF, Aldighieri S, Bertherat E, Najera P, Galan DI, Durski K, Espinal MA. Leptospirosis: A silent epidemic disease. Int J Environ Res Public Health. 10; 2013:7229–7234. doi:10.3390/ijerph101207229 Available from: URL: http://www.mdpi.com/1660-4601/10/12/7229#tabs-5
27. Abela-Ridder B, Sikkema R, Hartskeerl RA. Estimating the burden of human leptospirosis. Int J Antimicrob Agents. 36; 2010: S5–S7. doi: 10.1016/j.ijantimicag.2010.06.012. Available from: URL: http://www.sciencedirect.com/science/article/pii/ S0924857910002530
Received on 03.09.2014 Modified on 20.09.2014
Accepted on 24.09.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 7(10): Oct. 2014 Page 1153-1157