Southeast Asian J Trop Med Public Health

SEROPREVALENCE OF LEPTOSPIROSIS AMONG TOWN SERVICE WORKERS IN ,

Mohamad Azfar Zainuddin1, Nabilah Ismail2, Siti Asma’ Hassan2, Mohd Nazri Shafei1, Mohamed Rusli Abdullah1, Zahiruddin Wan Mohamad1, Nor Azwany Yaacob1, A Mohd Nikman3, Aziah Daud1 and Maizurah O4

1Department of Community Medicine, 2Department of Medical Microbiology and Parasitology, 3Department of Anaesthesiology, Universiti Sains Malaysia, , Malaysia, 4Informatics for Community Health, Universiti Sains Malaysia, Pulau Pinang

Abstract. Leptospirosis is presumed to be the most widespread re-emerging zoonotic disease of global distribution affecting humans in tropical, subtropical and temperate zones. Humans can be exposed to leptospirosis as a result of variety of occupations, such as town service worker, paddy planter, army and health care worker due to the presence of environmental determinants for human leptospirosis. This study determined seroprevalence of leptospirosis among 321 town service workers in Kelantan, a northeastern state of Malaysia, involving a cross sectional study in four districts, namely, , Gua Musang, , and Tanah Merah in Kelantan. Sociodemographic data were collected and sera samples screened for the presence of anti-leptospiral antibodies using IgM enzyme-linked immunosorbent assay (IgM ELISA) and confirmed by microscopic agglutination test (MAT) using a panel of 20 live leptospires obtained from Institute of Medical Research (IMR) , Malaysia. Serovars used were australis, autumnalis, bataviae, canicola, celledoni, copenhageni, djasiman, grippotyposa, hardjobovis, hardjoprajitno, icterohaemorrha- giae, javanica, lai, melaka, pomona, pyrogenes, patoc, , tarassov, and tereng- ganu. A MAT titer of ≥100 was used as cut-off for positive samples. All participants were of Malay race, with mean age (± SD) of 40 (± 10) years and mean duration of employment of 12 (± 10) years. Ninety-eight samples were positive or indetermi- nate by IgM ELISA, and MAT revealed an overall seroprevalence of leptospirosis of 25.5% (95% CI : 20.6-30.5). Garbage collectors had the highest seroprevalence (35%; 95% CI : 8.1-14.9), followed by town cleaners (30%; 95% CI : 43.6-54.8), lorry drivers/ mechanics (20%; 95% CI : 17.8-27.4), and landscapers (14%; 95% CI : 13.1-21.8). The most common serovar was against a strain of Leptospira spp isolated from Sarawak (60%) and none positive for serovars bataviae, djasiman, hardjoprajitno, melaka, pyrogenes, pomona and . The present study highlights the high risk of leptospiral infection among town service workers in Kelantan, Malaysia. Keywords: high risk occupation, leptospirosis, seroprevalence, town service worker

Correspondence: Aziah Binti Daud, Depart- INTRODUCTION ment of Community Medicine, School of Medi- cal Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Leptospirosis is considered to be the E-mail: [email protected]; aziahdaud68@gmail. most widespread re-emerging zoonotic com disease of global distribution affecting hu- Tel: +609 767 6633/6622; Fax: +609 767 6654 mans in tropical, subtropical and temper-

1222 Vol 48 No. 6 November 2017 Seroprevalence of Leptospirosis among Town Service Workers ate zones. The most significant foci of the Thus, this study was conducted to disease are The Caribbean, Latin America, determine the seroprevalence of lepto- the Indian subcontinent, Southeast Asia, spirosis among town service workers in Oceania, and Eastern Europe (Pappas et al, Kelantan, Malaysia. 2008). Seychelles was found to have the highest prevalence of leptospirosis in the MATERIALS AND METHODS world with an annual rate of 432.1 per million population while USA reported Study population the lowest annual rate of 0.1 per million A cross sectional study was carried population (Pappas et al, 2008). The exact out in four districts (Kota Bharu, Gua number of human cases worldwide is not Musang, Kuala Krai, Tanah Merah) in known due to a lack of comprehensive Kelantan, a northeastern state of Malaysia, surveillance (Hartskeerl et al, 2011). involving 321 town service workers: gar- Incubation period of leptospirosis bage collector, town cleaner, landscaper infection is usually 5-14 days, with a range and lorry driver/mechanic. Garbage col- of 2-30 days (WHO, 2012). In humans, lector collects garbage from residential, it can cause a wide range of symptoms, commercial and industrial areas and ranging from mild (influenza- like illness dumps garbage from containers onto symptoms) to severe (Weils’ syndrome), lorry (truck) for disposal at landfills; town while some infected persons may have cleaner duties are sweeping, collecting no symptoms at all. If the infection is and removing of litter, detritus and leaves not treated, progression of the disease from public spaces; landscaper performs will lead to complications, such as renal a range of duties, such as transporting failure, meningitis, liver damage, respira- and planting new vegetation, mulching, tory distress and widespread hemorrhage fertilizing, watering, as well as cutting (Dircio Montes Sergio et al, 2012). and trimming grass; and lorry driver, in Humans may be exposed to leptospi- addition to routine work, sometimes as- rosis through occupational, recreational sists garbage collectors to perform their or environmental factors (Levett, 2001). duties, while lorry mechanic repairs and Occupation such as town service worker, undertakes maintenance of the lorries paddy planter, army and health care used for garbage collection. Sample size, worker impose higher risk of leptospiral calculated based on 24.7% seropositiv- infection due to presence of environmen- ity for leptospirosis among town service tal determinants for human leptospirosis workers (Sulong et al, 2012) at 95% confi- (Hadad et al, 2006; Tiwari, 2008; Sulong dence interval (CI) and a drop-out of 20%, et al, 2012). Town service workers are more was estimated to be 346. Participants were likely to be exposed to the leptospiral in- interviewed for socio-demographic, job fection as a result from their occupational category and duration of employment. activities in every step of solid waste Data were analyzed using SPSS Version management process. Based on data from 20.0 (IBM, Armonk, NY). an in-depth, long term and country-wide Prior written consent was obtained studies in Denmark, disease and injury from participants and ethical clearance risks for solid waste management workers was approved by Research and Ethic are six times greater than those of control Committee (Human), School of Medical population (Cointreau, 2006). Sciences, Health Campus, Universiti Sains

Vol 48 No. 6 November 2017 1223 Southeast Asian J Trop Med Public Health

Table 1 test (MAT) following standard protocols Sociodemographic characteristics of (WHO, 2007). MAT was performed with a town service workers, Kelantan, panel of 20 live leptospires obtained from Malaysia. Institute of Medical Research (IMR), Kuala Lumpur, Malaysia. Serovars used were Characteristic Number australis, autumnalis, bataviae, canicola, (%) celledoni, copenhageni, djasiman, grip- (N = 321) potyposa, hardjobovis, hardjoprajitno, Age (year), mean (SD) 41 (10) icterohaemorrhagiae, javanica, lai, mela- Gender ka, pomona, pyrogenes, patoc, sarawak, Male 309 (96) tarassov, and terengganu. MAT titer of Female 12 (4) ≥100 was taken as cut-off for a positive Marital status sample. Seroprevalence of leptospirosis Married 268 (83) was described with 95% CI. Single 44 (14) Widower 9 (3) Number of children, mean (SD) 4 (2) RESULTS Monthly income (RM), mean (SD) 1,198 (419) Three hundred and twenty-one town Education level service workers were enrolled in the No formal education 4 (1) Primary school 64 (20) study. All participants were of Malay race Lower secondary school 88 (28) with a mean ± SD age of 41 ± 10 years, Upper secondary school 151 (47) with mean duration of employment of 12 Form 6/Diploma 14 (4) ± 10 years. Majority of them were from Town council Kota Bharu municipal council and work Kota Bharu Municipal Council 183 (57) as town cleaner. Their education status Council 48 (15) mainly at the level of upper and lower Council 49 (15) secondary school (Table 1). Council 41 (13) Ninety-eight serum samples from 321 Job category Town cleaner 157 (49) subjects (30.5%) were positive or inde- Lorry driver/mechanic 71 (22) terminate in the IgM-ELISA. Subsequent Landscaper 56 (17) MAT revealed an overall seroprevalence Garbage collector 37 (12) of leptospirosis of 25.5% (82/321) (95% CI: 20.6-30.5). Garbage collectors had the highest seroprevalence (35.1%; 95% CI: 8.1-14.9), followed by town cleaners (30%; Malaysia [Reference no: USMKK/PPP/ 95% CI: 43.6-54.8), lorry drivers/mechan- JEPeM 261.3(7)]. ics (20%; 95% CI: 17.8-27.4), and landscap- Sample analysis ers (14%; 95% CI: 13.1-21.8) (Table 2). Five ml of venous blood were ob- The most common serovar among the tained from each participant and stored 82 seropositive subjects as determined by at 4-8ºC until used. Serum samples were positive MAT with titer ≥100 was sarawak screened for the presence of anti-leptospir- (60%) (Table 3). None of the samples were al antibodies using IgM enzyme-linked positive for serovars bataviae, djasiman, immunosorbent assay (IgM ELISA) and hardjoprajitno, melaka, pyrogenes, po- confirmed by microscopic agglutination mona and terengganu.

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Table 2 Seroprevalence of leptospirosis using MAT among town service workers according to job categories.

Job category Number MAT ≥100

Number (%) 95% CI

Town cleaner 157 47 (30) 43.6-54.8 Lorry driver/mechanic 71 14 (20) 17.8-27.4 Landscaper 56 8 (14) 13.1-21.8 Garbage collector 37 13 (35) 8.1-14.9 Total 321 82 (25.5) 20.6-30.5

Table 3 seroprevalence survey, the cut-off value Serovar determined by positive MAT of MAT depends on endemicity of the (titer ≥100) among town service workers study area and varies from one reference (N = 82). laboratory to another (Plank and Dean, 2000; WHO, 2007). Currently, there is no Serovar tested Number consensus with regards to standard cut-off positive titer for leptospirosis seroprevalence stud- (%)a ies in Malaysia; however, the cut-off titer sarawak 49 (60) value of ≥100 is generally accepted (Plank grippotyphosa 20 (24) and Dean, 2000) and is frequently used in patoc 19 (23) other seroprevalence studies all around copenhageni 9 (11) the world including Malaysia (Gonçalves lai 8 (10) et al, 2006; Swapna et al, 2006; Majd et al, celledoni 4 (5) 2012; Sulong et al, 2012). javanica 4 (5) australis 3 (4) This seroprevalence study for lepto- canicola 3 (4) spirosis among town service workers in hardjobovis 2 (2) Kelantan, Malaysia revealed the highest icterohaemorrhagiae 2 (2) seroprevalence in the occupational groups autumnalis 1 (1) studied compared to previous similar tarrasovi 1 (1) surveys which reported seroprevalence of 16.2% to 25.5% (Tan, 1973; Sulong et al, aTotal >100 as workers can be positive for more than one serovar. 2012). MAT seroprevalence in our study was comparable to that of Sulong et al (2012). The highest prevalence was noted DISCUSSION among garbage collectors. In comparison to previous study (Sulong et al, 2012), MAT is the ‘gold standard’ test for the present study reported higher serop- the diagnosis of leptospirosis. It is also revalence in all job categories except for used for seroprevalence survey and is landscapers. The higher seroprevalence generally performed by reference labo- among garbage collectors was probably ratories (Chirathaworn et al, 2014). In a related to their daily occupational activi-

Vol 48 No. 6 November 2017 1225 Southeast Asian J Trop Med Public Health ties that prolonged exposure and contact serovar Sarawak is found in monkey, rat, with garbage, soil, water or environment bat, squirrel and mongoose (Thayaparan that might be contaminated with lepto- et al, 2013). Rats are well-known carriers spires from urine of infected animal hosts. of leptospirosis, which they spread among Seroprevalence for leptospirosis other animals and humans; however, their among town service workers in this study carrier status of the other infected animals was higher than among army personnel are not known. in Malaysia, 16.2% by Yaacob et al (2013, Previously, in a study among town unpublished data) and 17.2% in an earlier service workers of Kota Bharu Municipal study by Tan and Lopes (1972). Based on Council in 2008 the common serovars sensitized-erythrocyte-lysis (SEL) test, identified are patoc (39.7%), followed by 24.2% of blood samples from healthy bataviae (12.3%) and javanica (11.0%), and paddy planters in Kelantan showed lep- no sample was positive for serovar grip- tospiral antibodies in the wet season but potyphosa, but serovar sarawak was not only 7.2% during the dry season (Tan, tested (Sulong et al, 2012). Serovar patoc 1970). Oil palm estate workers recorded was used as a saprophytic strain that the highest seroprevalence of 32.6%, fol- cross-reacts with human antibodies gener- lowed by hospital staff (25.5%) and rub- ated by a number of pathogenic serovars, ber estate workers (23.2%) (Tan, 1973). including those of rare, unknown or new Leptospirosis seroprevalence ranged strains in the area concerned (WHO, 2003). from 11.6% to 16.4% in veterinary staff, This may suggest the circulation of other policemen, shop owners, anti-malarial pathogenic serogroups that were not in- laborers, farmers and tin miners, whereas cluded in the MAT antigens of 20 serovars in occupational low risk group (school used in the present study. These findings teachers, office workers and housewives), emphasize the need to validate or revise prevalence ranged from 1.5% to 9.4% (Tan, the MAT criteria for the specific epidemio- 1979). logical situation in which serosurveys are It was difficult to relate the findings or will be performed. of the predominant serovars among town A study involving army person- service workers in this study to local nel, one of the occupation risk group, animal reservoirs and environment as in four base camps (, there is limited study on human, animal Desa Pahlawan, Tanah Merah and Jeli) of and environmental samples (water and Kelantan reported the common serovars wet soils) leptospirosis in Malaysia and detected are pathogenic serovar tereng- in Kelantan in particular (Thayaparan ganu (38.3%) and non-pathogenic se- et al, 2013). L. interrogans serovar sarawak rovar patoc (35.2%), but MAT for serovar (also known as lepto 175), the most pre- tarrasovi, djasiman and grippotyphosa dominant serovar found in our study, is a were not performed (Yaacob et al, 2013, poorly known strain endemic to Malaysia unpublished data). Serovar terengganu, and its pathogenic potential is still un- together with serovars melaka, sarawak known. Ongoing research to obtain more and bejo, are local strains in Malaysia, information on serovar Sarawak espe- but very limited published studies are cially in human leptospirosis infection is available (Slack et al, 2009; Thayaparan currently being undertaken. In wildlife in et al, 2013). A hospital-based study involv- Sarawak state, Malaysia positive titer for ing 999 febrile cases from 10 hospitals

1226 Vol 48 No. 6 November 2017 Seroprevalence of Leptospirosis among Town Service Workers in Kelantan reported the most common that represent those present in the com- serogroup is Sejroe, which accounts about munity (local serovars), it will lead to an 82% of the leptospirosis cases (Rafizah incomplete panel resulting in false nega- et al, 2013). Seroprevalence of leptospiro- tive results (Plank and Dean, 2000; Musso sis in wild rats by MAT revealed 17.3% are and La Scola, 2013). positive for serovars icterohaemorrhagiae Leptospirosis is preventable if appro- (12.3%), canicola (2.5%), ballum (1.2%), priate measures are taken, especially for and pyrogenes (1.2%) (Mohamed Hassan modifiable risk factors. It is essential for et al, 2010). The findings from this animal people whose occupation involve expo- study may suggest that serovars ictero- sure to leptospirosis to have knowledge haemorhagiae and canicola infecting our of the disease, especially on prevention subjects might have originated from rats, of infection. Education and awareness but this requires further studies. campaign specifically directed to the high Our study has a number of limita- risk groups and the community should tions. Firstly, the town service workers be conducted. Workers in high risk oc- who participated in the study were pre- cupation must wear appropriate personal dominantly male and all of them were protective apparels, such as boots, gloves, of Malay race, which make our study goggles, overall, face mask and eye unable to examine the effect of gender protection based on their jobs and risks and race on the leptospirosis seroposi- involved (Rao et al, 2003). Eradication of tivity. Secondly, we used IgM ELISA as leptospires from the environment is obvi- a preliminary screening step to identify ously impractical, but measures to reduce seropositive samples before proceeding direct contact with infected animals and to MAT to identify the specific serovar. indirect contact with urine contaminated The limited specificity and high sensitiv- soil and water remain the most effective ity of IgM ELISA suggest that it would strategy (Sharma and Yadav, 2008). be Tore valuable as a ‘rule-out test’, with In conclusion, the present study high- confirmation of positivity using the more lights the high risk of leptospiral infection specific MAT at a reference laboratory in town service workers in Kelantan, and (Canal et al, 2013; Musso and La Scola, identifies the predominant serovar as 2013). A number of participants might Sarawak, a poorly known strain endemic have had leptospiral infection in recent to Malaysia. This warrants further local past and their IgM levels might have study in human leptospirosis, particu- become undetectable, a situation which larly in other occupation high risk groups MAT could have excluded resulting in and in the community in general, and of misclassification at the preliminary test animal leptospirosis to better understand stage. Thirdly, even though MAT is the local serovars and leptospirosis in Ma- reference method for the diagnosis and laysia. Such information could help in seroprevalence studies of leptospirosis, it developing better prevention and control has potential drawbacks similar to those measures of the disease locally and in of IgM ELISA, namely, misclassification other countries as well. of persons as seronegative and seroposi- tive resulting in a potential bias. Fourthly, ACKNOWLEDGEMENTS if certain Leptospira serovars were not included in the panel of live leptospires The authors would like to express

Vol 48 No. 6 November 2017 1227 Southeast Asian J Trop Med Public Health their deepest gratitude and thanks to all Emergence, control and re-emerging lep- participants who provided their invalu- tospirosis: dynamics of infection in the able responses. The study was funded by changing world. Clin Microbiol Infect 2011; the Universiti Sains Malaysia Research 17: 494-501. Grant No. 1001/PPSP/812131. Levett PN. Leptospirosis. Clin Microbiol Rev 2001; 14: 296-326. CONFLICTS OF INTEREST Majd NS, Darian EK, Khaki P, Bidhendi SM, Yahaghi E, Mirnejad R. Epidemiological The authors declare no conflicts of patterns of Leptospira spp among slaugh- interest. terhouse workers in Zanjan–Iran. Asian Pacific J Trop Dis2012; S550-2. REFERENCES Mohamed-Hassan SN, Bahaman AR, Mutalib AR, Khairani-Bejo S. Serological preva- Canal E, Pollett S, Heitzinger K, et al. Detection lence of leptospiral infection in wild rats of human leptospirosis as a cause of acute at the National Service Training Centres fever by capture ELISA using a leptospira in Kelantan and Terengganu. Trop Biomed interrogans serovar Copenhageni (M20) de- 2010; 27: 30-2. rived antigen. BMC Infect Dis 2013; 13: 438. Musso D, La Scola B. Laboratory diagnosis of Cointreau S. Occupational and environmental leptospirosis: a challenge. J Microbiol Im- health issues of solid waste management: munol Infect 2013; 46: 245-52. special emphasis on middle and lower- Pappas G, Papadimitriou P, Siozopoulou V, income countries. Washington, DC: Urban Christou L, Akritidis, N. The globaliza- Sector Sector Board, The World Bank, 2006. tion of leptospirosis: worldwide incidence [Cited 2017 Jun 15]. Available from: http:// trends. Int J Infect Dis 2008; 12: 351-7. www.worldbank.org/urban/ Plank R, Dean D. Overview of the epidemio- Chirathaworn C, Inwattana R, Poovorawan Y, logy, microbiology, and pathogenesis of Suwancharoen D. Interpretation of micro- Leptospira spp. in humans. Microbes Infect scopic agglutination test for leptospirosis 2000; 2: 1265-76. diagnosis and seroprevalence. Asian Pac J Trop Biomed 2014; 4(Suppl 1): S162-4. Rafizah AAN, Aziah BD, Azwany YN,et al. Risk factors of leptospirosis among febrile hos- Dircio Montes Sergio A, Gonzalez Figueroa E, pital admissions in northeastern Malaysia. Guzman Maria Saadia V, et al. Leptospi- Prev Med (Baltimore) 2013; 26: 17.11-17.13 rosis prevalence in patients with initial diagnosis of dengue. J Trop Med 2012; 2012: Rao RS, Gupta N, Bhalla P, Agarwal S. Lepto- ID519701. spirosis in India and the rest of the world. Gonçalves DD, Teles PS, Dos Reis CR, et al. Braz J Infect Dis 2003; 7: 178-93. Seroepidemiology and occupational and Sharma M, Yadav A. Leptospirosis: epidemi- environmental variables for leptospirosis, ology, diagnosis, and control. J Infect Dis brucellosis and toxoplasmosis in slaugh- Antimicrob Agents 2008; 25: 93-103. terhouse workers in the Paraná State, Slack AT, Khairani-Bejo S, Symonds ML, et al. Brazil. Rev Inst Med Trop Sao Paulo 2006; 48: Leptospira kmetyi sp. isolated from an en- 135-40. vironmental source in Malaysia. Int J Syst Hadad E, Pirogovsky A, Bartal C, et al. An out- Evol Microbiol 2009; 59(Pt 4): 705-8. break of leptospirosis among Israeli troops Sulong MR, Shafei MN, Yaacob NA, et al. Se- near the Jordan River. Am J Trop Med Hyg roprevalence of leptospirosis among town 2006; 74: 127-31. service workers in northeastern State of Hartskeerl RA, Collares-Pereira M, Ellis WA. Malaysia. Int J Collab Res Intern Med Public

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