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A hospital-based study on seroprevalence of leptospirosis among febrile cases in northeastern Malaysia

Open AccessPublished:January 28, 2013DOI:https://doi.org/10.1016/j.ijid.2012.12.012

      Summary

      Objective

      To determine the seroprevalence of leptospirosis among febrile inpatient cases in northeastern Malaysia.

      Methods

      A hospital-based cross-sectional study was conducted among 999 febrile cases admitted to 10 hospitals in northeastern Malaysia. A survey using a proforma sheet was used to obtain sociodemographic and occupational information. Serum samples were screened for leptospirosis by IgM enzyme-linked immunosorbent assay test (IgM ELISA) and confirmed by microscopic agglutination test (MAT).

      Results

      There was an equivalent distribution of males and females in the 999 respondents enrolled in the study. The majority were Malay (94.7%) and their mean age was 39.4 (standard deviation 17.6) years. The overall seroprevalence of leptospirosis was 8.4% (95% confidence interval (CI) 6.8–10.3) (n = 84). The high-risk occupational group was found to have a higher seroprevalence, which was 56% (95% CI 45.3–66.1) (n = 47). The predominant serogroup was Sejroe (82.1%, 95% CI 72.6–88.8) (n = 69).

      Conclusions

      This study revealed a possible high seroprevalence of leptospirosis among febrile cases, indicating the need to review the importance of adding leptospirosis to the case investigation of febrile illness, especially among high-risk occupational groups in Malaysia, as well as in other endemic countries.

      Keywords

      1. Introduction

      Leptospirosis is reported as a frequent cause of undifferentiated febrile illness in developing countries. Multicenter investigations in Indian hospitals have indicated that leptospirosis accounts for 12.7% of acute febrile illnesses.
      • Sehgal S.
      • Sugunan A.
      • Vijayachari P.
      Leptospirosis: disease burden estimation and surveillance networking in India. South East Asian.
      Leptospirosis is the cause of a significant proportion of cases of non-hepatitis A and E jaundice, non-malarial febrile illnesses, and non-dengue hemorrhagic fever in Southeast Asia.
      • Laras K.
      • Van C.B.
      • Bounlu K.
      • Tien N.T.
      • Olson J.G.
      • Thongchanh S.
      • et al.
      The importance of leptospirosis in Southeast Asia.
      Leptospirosis was formerly considered to be a primarily occupational disease, and it has been associated with activities such as mining, sewer maintenance, livestock farming and butchering, veterinary medicine, and military maneuvers.
      • Bharti A.R.
      • Nally J.E.
      • Ricaldi J.N.
      • Matthias M.A.
      • Diaz M.M.
      • Lovett M.A.
      • et al.
      Leptospirosis: a zoonotic disease of global importance.
      A survey for leptospiral antibodies was conducted throughout West Malaysia from 1961 through 1971 on 18 occupational groups. A total of 4646 serum samples were tested, of which 592 (12.7%) were positive using the sensitized erythrocyte lysis test. High antibody rates (23.2% to 32.6%) were found among oil palm estate and rubber estate workers and hospital staff. Moderately high rates (13.0% to 17.9%) were observed in laborers, the army, tin miners (of open cast, dredge, and gravel pump mines), farmers, and paddy planters. Moderate rates (11.6% to 12.2%) were found among shop owners, policemen, and veterinary staff, and low rates (1.5% to 9.4%) were detected in school teachers, housewives, office workers, and tin miners of underground or lode mines.
      • Tan D.S.
      Occupational distribution of leptospiral (SEL) antibodies in West Malaysia.
      Although leptospirosis cases have been reported in Malaysia since the 1920s, the actual disease burden in the country remains underestimated. This is partly because it was not on the list of notifiable diseases under the Prevention and Control of Communicable Diseases Act 1988 until recently. In addition, notified cases only cover clinical cases, however a seroprevalence study can better estimate disease burden by detecting both clinical and subclinical phases. Thirteen serogroups have reportedly been identified in Malaysia, namely Australis, Autumnalis, Bataviae, Canicola, Celledoni, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Pomona, Pyrogenes, Sejroe, and Tarassovi.
      • Bahaman A.R.
      • Ibrahim A.L.
      A review of leptospirosis in Malaysia.
      The objective of this study was to determine the seroprevalence of leptospirosis among febrile cases in northeastern Malaysia.

      2. Methods

      A hospital-based cross-sectional study was conducted in 10 hospitals in northeastern Malaysia. The proportionate sampling method based on the proportion of the number of beds in each study hospital was used, and a total of 1200 respondents were estimated.
      Cases were febrile adults aged 18 years and older, admitted to the medical wards with an oral temperature above 37 °C. HIV patients, patients on immunosuppressive drugs, and patients with autoimmune diseases were excluded from this study.
      Informed written consent was obtained from all subjects based on the approved study protocol, by the Research Ethics Committee (Human), Universiti Sains Malaysia (Reference No. USMKK/PPP/JEPeM (219.3.(03))) and the Medical Review and Ethics Committee, National Institute of Health, Ministry of Health Malaysia (NMRR-10-318-5389). A patient proforma sheet was used to collect sociodemographic and occupation data. For this study, occupation types with high and moderate antibody rates were grouped into a high-risk occupation group and those with low antibody rates were grouped into a low-risk occupation group, as stated in Tan.
      • Tan D.S.
      Occupational distribution of leptospiral (SEL) antibodies in West Malaysia.
      Single venous samples of 5–8 ml were collected from the study subjects. The samples were centrifuged and stored at −20 °C. The PanBio Leptospira IgM ELISA test (PanBio, Queensland, Australia) was used for the qualitative detection of IgM antibodies to Leptospira. All serology samples were analyzed in the Microbiology Laboratory of Universiti Sains Malaysia. One operator-trained laboratory technician performed the ELISA test to avoid bias. A score of less than 9 units indicates a negative result or no detectable IgM antibodies, a score of 9–11 units indicates an indeterminate result, and a score of more than 11 units indicates a positive result, i.e. the presence of Leptospira-specific IgM antibodies.
      The microscopic agglutination test (MAT) was performed among positive and indeterminate samples of the ELISA test. The frozen sera were sent to the Institute of Medical Research (IMR), Malaysia, and all were tested for the presence of anti-leptospiral antibodies using the MAT following standard methods.
      World Health Organization
      Leptospirosis: laboratory manual.
      A panel battery of 18 live reference serovars, representing 18 serogroups, were used as antigen. Leptospira reference cultures were obtained from the World Health Organization (WHO) Collaborating Centre, Queensland, Australia, with three additional local pathogenic serovars (unidentified). The serovars included in the antigen panel were Leptospira biflexa serovar Patoc, Leptospira interrogans serovar Australis, L. interrogans serovar Autumnalis, Leptospira borgpetersenii serovar Ballum, L. interrogans serovar Bataviae, L. interrogans serovar Canicola, Leptospira weilii serovar Celledoni, L. interrogans serovar Hebdomadis, Leptospira kirschneri serovar Cynopteri, L. interrogans serovar Grippotyphosa, L. interrogans serovar Icterohaemorrhagiae, L. borgpetersenii serovar Javanica, L. interrogans serovar Pomona, L. interrogans serovar Pyrogenes, L. borgpetersenii serovar Tarassovi, L. interrogans serovar Hardjo, L. borgpetersenii serovar Sejroe, and L. interrogans serovar Djasiman.
      Live Leptospira cell suspensions representing the 18 serovars were added to serially diluted serum specimens in microtiter plates and incubated at 30 °C for 2 h. Each serum was tested at three dilutions, 1:25, 1:50, and 1:100, and when agglutination was observed at the 1:100 dilution, it was titrated to determine the highest agglutination titer. Agglutination was examined using dark-field microscopy at a magnification of ×100. Positive agglutination was considered when the approximate numbers of free leptospires was <50% of leptospires in the control wells. The titer result was taken as the last dilution that showed <50% of free leptospires compared to control wells. A positive MAT was defined as a MAT titer of ≥1:400 in a single, acute specimen. This titer was specified by the IMR as the national reference laboratory, and it was based on the background exposure and seroprevalence of leptospirosis in the country. This cut-off MAT titer has also been applied in several studies on leptospirosis.
      • Suttinont C.
      • Losuwanaluk K.
      • Niwatayakul K.
      • Hoontrakul S.
      • Intaranongpai W.
      • Silpasakorn S.
      • et al.
      Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study.
      • Dassanayake D.L.
      • Wimalaratna H.
      • Agampodi S.B.
      • Liyanapathirana V.C.
      • Piyarathnaa T.A.
      • Goonapienuwala B.L.
      Evaluation of surveillance case definition in the diagnosis of leptospirosis, using the microscopic agglutination test: a validation study.
      • Cumberland P.
      • Everard C.O.
      • Wheeler J.G.
      • Levett P.N.
      Persistence of anti-leptospiral IgM, IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados 1979–1989.
      • Pradutkanchana J.
      • Pradutkanchana S.
      • Kemapanmanus M.
      • Wuthipum N.
      • Silpapojakul K.
      The etiology of acute pyrexia of unknown origin in children after a flood.
      • Bharadwaj R.
      • Bal A.M.
      • Joshi S.A.
      • Kagal A.
      • Pol S.S.
      • Garad G.
      • et al.
      An urban outbreak of leptospirosis in Mumbai, India.
      • Thammakumpee K.
      • Silpapojakul K.
      • Borrirak B.
      Leptospirosis and its pulmonary complications.
      • Sanders E.J.
      • Rigau-Perez J.G.
      • Smits H.L.
      • Deseda C.C.
      • Vorndam V.A.
      • Aye T.
      • et al.
      Increase of leptospirosis in dengue-negative patients after a hurricane in Puerto Rico in 1996 [correction of 1966].
      In this study, the sensitivity, specificity, positive predictive value, and negative predictive value between the MAT and ELISA could not be calculated due to the fact that only indeterminate and positive ELISA results (n = 111) were subjected to the MAT.
      The statistical analysis was performed using PASW Statistics 18 (formerly SPSS Statistics). All continuous variables are described using the mean and standard deviation (SD), whereas for categorical variables, frequencies and percentages are presented. The seroprevalence of leptospirosis was computed using positive MAT over total samples and is presented as a percentage with 95% confidence interval (CI).

      3. Results

      A total of 999 subjects were recruited to participate in this study. The majority of study subjects were Malay, and their mean age was 39.4 (SD 17.6) years. Thirty-six percent of the subjects were grouped into the high-risk occupational group (Table 1).
      Table 1Sociodemographic characteristics of respondents (n = 999)
      Variable
      Age, years
       Mean (SD)30.4 (17.6)
       Range18–94
      Sex, n (%)
       Female456 (45.6)
       Male543 (54.4)
      Race, n (%)
       Non-Malay53 (5.3)
       Malay946 (94.7)
      Occupational classification, n (%)
       Low-risk occupational group
      Low-risk: indoor worker, teacher, student, housewife/unemployed, retired, production operator, and others.
      638 (63.9)
       High-risk occupational group
      High-risk: agriculture worker, military, medical personnel, and outdoor worker.
      361 (36.1)
      SD, standard deviation.
      a Low-risk: indoor worker, teacher, student, housewife/unemployed, retired, production operator, and others.
      b High-risk: agriculture worker, military, medical personnel, and outdoor worker.
      Eighty-eight samples tested positive by IgM ELISA and 23 samples were indeterminate. Eighty-four respondents were confirmed positive for leptospirosis and the overall seroprevalence was 8.4% (95% CI 6.8–10.3) (Table 2). The seroprevalence of leptospirosis in the high-risk occupational group was 56% (95% CI 45.3–66.1) (n = 47) and in the low-risk group was 44% (95% CI 33.8–54.6) (n = 37).
      Table 2ELISA vs. MAT results
      ELISAMATTotal
      Negative frequency (%)Positive frequency (%)
      Indeterminate7 (30.4)16 (69.6)23 (100.0)
      Positive20 (22.7)68 (77.3)88 (100.0)
      Total27 (24.3)84 (75.7)111 (100.0)
      ELISA, enzyme-linked immunosorbent assay; MAT, microscopic agglutination test.
      Table 3 shows the level and the frequency of the MAT titer from a total of 111 IgM ELISA (indeterminate and positive) results. The cut-off point for a positive MAT was a titer of ≥1:400, giving a total number of 84 positive MAT cases. The MAT titer range was from 1:400 to 1:1600.
      Table 3MAT titer among ELISA indeterminate and positive cases (n = 111)
      MAT titerFrequency%
      1:5010.9
      1:10054.5
      1:2002118.9
      1:4003127.9
      1:8003027.0
      1:16002320.7
      Total111100
      ELISA, enzyme-linked immunosorbent assay; MAT, microscopic agglutination test.
      The highest frequency was 1:400, which was 28.0% of the total MAT.
      The highest frequency of leptospirosis cases was found among agricultural workers, followed by housewives or unemployed and outdoor workers. None of the leptospirosis cases were noted among the retired. Figure 1 shows the frequency of leptospirosis cases based on type of occupation.
      Figure thumbnail gr1
      Figure 1Occupation types of the leptospirosis cases confirmed by MAT (n = 84).
      Table 4 shows the distribution of serogroups that were determined by the positive MAT among the 84 confirmed leptospirosis cases. Among these, eight serogroups were identified and the most common serogroup was Sejroe, which contributed 82.1% (95% CI 72.6–88.8) (n = 69) of the leptospirosis cases in the northeastern state of Malaysia.
      Table 4Identified serogroups for leptospirosis cases (n = 84)
      SerogroupFrequency
      Sejroe69
      Pyrogenes5
      Icterohaemorrhagiae3
      Celledoni2
      Javanica2
      Australis1
      Bataviae1
      Autumnalis1
      Total84

      4. Discussion

      This study of febrile inpatient cases from 10 hospitals in northeastern Malaysia has an adequate coverage for publication, and a hospital-based study is the best method to capture febrile cases. Seroprevalence data from a retrospective study of human leptospirosis in Malaysia based on the MAT showed a 13% overall prevalence of infection for the period 1983 to 1998; this has been repeatedly cited in recent reviews and studies.
      • Bahaman A.R.
      • Ibrahim A.L.
      A review of leptospirosis in Malaysia.
      • El Jalii I.M.
      • Bahaman A.R.
      A review of human leptospirosis in Malaysia.
      • Lim J.K.
      • Murugaiyah V.A.
      • Ramli A.
      • Rahman H.A.
      • Mohamed N.
      • Shamsudin N.
      • et al.
      A case study: leptospirosis in Malaysia.
      • Lim V.K.
      Leptospirosis: a re-emerging infection.
      In this study, leptospirosis was confirmed in 84 febrile subjects (8.4%), which is in line with other tropical areas. A similar prevalence of leptospirosis among febrile patients has been reported previously in urban Bangladesh,
      • Kendall E.A.
      • LaRocque R.C.
      • Bui D.M.
      • Galloway R.
      • Ari M.D.
      • Goswami D.
      • et al.
      Leptospirosis as a cause of fever in urban Bangladesh.
      which is also a tropical climate area. In that study, the seroprevalence of leptospirosis was calculated based on definite and probable diagnoses in which a definite diagnosis was defined as patients with either a single MAT titer ≥1:800 for any serovar at either phase, or a serovar-specific four-fold increase from acute phase to convalescent phase with a minimum convalescent titer of 1:400. Meanwhile a MAT titer ≥1:100 for any serovar in either an acute-phase or convalescent-phase serum sample was categorized as probable leptospirosis. In our study, the prevalence of leptospirosis was based on a single MAT titer of ≥1:400, as we used only a single serum sample, and the subjects had an acute febrile illness as well as the presence of background antibody as part of the population of an endemic area.
      However, the seroprevalence in this study was lower than findings in a rural Thai population,
      • Suttinont C.
      • Losuwanaluk K.
      • Niwatayakul K.
      • Hoontrakul S.
      • Intaranongpai W.
      • Silpasakorn S.
      • et al.
      Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study.
      which showed seroprevalences of 36.9% and 17.5% among adults on the Thai–Myanmar border.
      • Ellis R.D.
      • Fukuda M.M.
      • McDaniel P.
      • Welch K.
      • Nisalak A.
      • Murray C.K.
      • et al.
      Causes of fever in adults on the Thai–Myanmar border.
      Suttinont et al. used more stringent clinical criteria, with at least two serum specimens comprised of acute and convalescent samples.
      • Suttinont C.
      • Losuwanaluk K.
      • Niwatayakul K.
      • Hoontrakul S.
      • Intaranongpai W.
      • Silpasakorn S.
      • et al.
      Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study.
      Ellis et al. recruited both inpatients and outpatients with a temperature of 38 °C over the previous 48 h and obtained paired samples.
      • Ellis R.D.
      • Fukuda M.M.
      • McDaniel P.
      • Welch K.
      • Nisalak A.
      • Murray C.K.
      • et al.
      Causes of fever in adults on the Thai–Myanmar border.
      In contrast, a previous study by Shaheen and Shah reported that the prevalence of leptospirosis among cases with pyrexia of unknown origin (PUO) in the Kashmir Valley was 2.8%.
      • Shaheen R.
      • Shah A.
      Prevalence of leptospirosis among the PUO cases in a tertiary care hospital of Kashmir Valley.
      This lower finding could be due to the smaller sample size and different diagnostic criteria. In addition, climatic factors might also have contributed to the finding.
      We found that agriculture and outdoor workers contributed about 50% of the leptospirosis cases. This finding is in keeping with some previous studies.
      • Cumberland P.
      • Everard C.O.
      • Wheeler J.G.
      • Levett P.N.
      Persistence of anti-leptospiral IgM, IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados 1979–1989.
      • Sethi S.
      • Sharma N.
      • Kakkar N.
      • Taneja J.
      • Chatterjee S.S.
      • Banga S.S.
      • et al.
      Increasing trends of leptospirosis in northern India: a clinico-epidemiological study.

      Russ AK, Jali IE, Bahaman AR, Tuen AA, Ismail G. Seroepidemiological study of leptospirosis among the indigenous communities living in periphery of Croker Range Park Sabah, Malaysia. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC); January–March 2003.

      However, a study by Myint et al. in Thailand showed that more than 80% of confirmed leptospirosis cases were farmers and agricultural workers, which is higher than in our study.
      • Myint K.S.
      • Gibbons R.V.
      • Murray C.K.
      • Rungsimanphaiboon K.
      • Supornpun W.
      • Sithiprasasna R.
      • et al.
      Leptospirosis in Kamphaeng Phet, Thailand.
      They conducted passive surveillance during the rainy season and paired serum samples were taken from the suspected cases. Besides, more than 75% of the subjects worked in the agricultural sector and nearly 60% of the land of the province was used for agriculture. In the current study, employment in the agricultural sector was 50%.

      Malaysia, Kelantan Darul Naim. Information Malaysia 2002 yearbook. Kuala Lumpur: Berita Publishing; 2002, p. 841–55.

      Unexpectedly, 20% of the cases were in the group of housewives and unemployed. This could be the result of various factors such as walking barefoot, the presence of rats in the house, previous occupation, type of water supply, and waste disposal, which were not explored in the current study.
      In this study, leptospirosis cases reacted to a broad range of Leptospira serogroups. All of them had positive MAT results for serogroups previously identified as common in Malaysia, but overall, the most reactive serogroup in our study population was Sejroe, which accounted about 82.1% of the leptospirosis cases; this is higher than in other studies done in Vietnam,
      • Van C.T.
      • Thuy N.T.
      • San N.H.
      • Hien T.T.
      • Baranton G.
      • Perolat P.
      Human leptospirosis in the Mekong Delta, Viet Nam.
      rural Thailand,
      • Suttinont C.
      • Losuwanaluk K.
      • Niwatayakul K.
      • Hoontrakul S.
      • Intaranongpai W.
      • Silpasakorn S.
      • et al.
      Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study.
      Reunion Island,
      • Paganin F.
      • Bourdin A.
      • Dalban C.
      • Courtin J.P.
      • Poubeau P.
      • Borgherini G.
      • et al.
      Leptospirosis in Reunion Island (Indian Ocean): analysis of factors associated with severity in 147 confirmed cases.
      and Bulgaria.

      Taseva E, Christova I, Gladnishka T. Epidemiological, clinical and serological features of human leptospirosis in Bulgaria in 2005. 17th European Congress of Clinical Microbiology and Infectious Disease, Munich, Germany, March 31–April 4, 2007.

      Our finding of the most prevalent serogroup was different from the serogroups that have been reported elsewhere in Southeast Asia,
      • Laras K.
      • Van C.B.
      • Bounlu K.
      • Tien N.T.
      • Olson J.G.
      • Thongchanh S.
      • et al.
      The importance of leptospirosis in Southeast Asia.
      the Western Pacific,
      • Berlioz-Arthaud A.
      • Kiedrzynski T.
      • Singh N.
      • Yvon J.F.
      • Roualen G.
      • Coudert C.
      • et al.
      Multicentre survey of incidence and public health impact of leptospirosis in the Western Pacific.
      and Israel.
      • Bishara J.
      • Amitay E.
      • Barnea A.
      • Yitzhaki S.
      • Pitlik S.
      Epidemiological and clinical features of leptospirosis in Israel.
      In India, studies done among acute undifferentiated febrile illness patients and during leptospirosis outbreaks revealed the most common infecting serogroup to be Icterohaemorrhagiae.
      • Bharadwaj R.
      • Bal A.M.
      • Joshi S.A.
      • Kagal A.
      • Pol S.S.
      • Garad G.
      • et al.
      An urban outbreak of leptospirosis in Mumbai, India.
      • Bal A.M.
      • Bharadwaj R.S.
      • Joshi S.A.
      • Kagal A.S.
      • Arjunwadkar V.P.
      Common infecting leptospiral serovar in and around Pune, Mahastra.
      However, these two studies used a lower MAT cut-off titer for diagnosis and fewer panel antigens compared to our study.
      Our study had a few limitations. Firstly, our seroprevalence estimation was limited by the use of a single acute phase serum sample, which might have led to an underestimation in febrile cases. This was mainly due to the difficulty in tracing cases post-hospitalization in the large geographical area covered. At least two serum specimens should be collected from each patient (one ‘acute’ sample, on admission, and a ‘convalescent’ sample, 2–4 weeks post-discharge). Secondly, the cut-off value of the MAT usually depends on the baseline in the community in a particular geographical area
      • Tan D.S.
      Occupational distribution of leptospiral (SEL) antibodies in West Malaysia.
      and varies from laboratory to laboratory.
      • Sanders E.J.
      • Rigau-Perez J.G.
      • Smits H.L.
      • Deseda C.C.
      • Vorndam V.A.
      • Aye T.
      • et al.
      Increase of leptospirosis in dengue-negative patients after a hurricane in Puerto Rico in 1996 [correction of 1966].
      Currently, there is no consensus regarding the standard cut-off titer in view of the local scenario. The cut-off titer of ≥1:400 was set by the IMR based on background exposure and seroprevalence of leptospirosis in the country.
      In conclusion, this study revealed a possible high seroprevalence of leptospirosis among febrile cases, indicating the need to review the importance of adding leptospirosis to the case investigation of febrile illness, especially among those in the high-risk occupational group in Malaysia, as well as in other endemic countries.

      Acknowledgements

      The authors gratefully acknowledge the help and cooperation of Dr Fairuz binti Amran (IMR) and all the directors and staff of the participating hospitals. We also thank all the subjects who made this study a success.
      Funding: The study was funded by the Research University Grant Universiti Sains Malaysia (1001/PPSP/812057) and was ethically approved by Research and Ethic Committee of Universiti Sains Malaysia.
      Conflict of interest: No conflict of interest to declare.

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