Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.

Abstract. Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.

Leptospirosis is an emerging disease, especially in countries with a tropical climate such as Malaysia. A dramatic increase in the number of cases has been reported over the last decade; however, information on the epidemiological trends of this disease is lacking. The objective of this study is to provide an epidemiological description of human leptospirosis cases over a 9-year period (2004-2012) and disease relationship with meteorological, geographical, and demographical information. A retrospective study was undertaken to describe the patterns of human leptospirosis cases and their association with intrinsic (sex, age, and ethnicity) and extrinsic (location, rainfall, and temperature) factors. Data was grouped according to age, sex, ethnicity, seasonality and geographical distribution, and analyzed using statistical tools to understand the influence of all the different factors on disease incidence. A total of 12,325 cases of leptospirosis were reported between 2004 and 2012 with an upward trend in disease incidence, with the highest in 2012. Three hundred thirty-eight deaths were reported with an overall case fatality rate of 2.74%, with higher incidence in males (9696; 78.7%) compared with female patients (2629; 21.3%), and overall male to female ratio of 3.69:1. Patients aged cohorts between 30-39 years old (16.22 per 100,000 population) had the highest disease incidence while the lowest incidence occurred between <1 to 9 years old (3.44 per 100,000 population). The average incidence was highest amongst Malays (10.97 per 100,000 population), followed by Indians (7.95 per 100,000 population). Stratification according to geographical distribution showed that the state of Malacca had the highest average disease incidence (11.12 per 100,000 population) followed by Pahang (10.08 per 100,000 population). The states of Terengganu, Kelantan, and Perak recorded similar rates of incidence (≈8.00 per 100,000 population), while Johor with the least number of reported cases (1.80 per 100,000 population). Positive relationships were recorded between the number of reported cases with the number of raining days per month and monthly average temperature (p-value<0.05). However, no significant association was noted between rainfall volume and number of reported Leptospirosis cases. This collaborative efforts between medical, academic and governmental institutions has enabled the construction of this comprehensive database that is essential to understand the disease trends in Malaysia and add insights into the prevention and control of this disease.

BackgroundLeptospirosis is an emerging infectious disease of global significance, and is endemic in tropical countries, including Malaysia. Over the last decade, a dramatic increase of human cases was reported; however, information on the primary vector, the rat, and the Leptospira serovars circulating among the rat population is limited. Therefore, the present study was undertaken to isolate Leptospira and characterise the serovars circulating in the urban rat populations from selected main cities in Peninsular Malaysia.MethodsRat trappings were carried out between October 2011 to February 2014 in five urban cities which were chosen as study sites to represent different geographical locations in Peninsular Malaysia. Microscopic agglutination test (MAT) and PCR were carried out to identify the Leptospiral serogroup and determine the pathogenic status of the isolates, respectively while pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR were used to characterize the isolates.ResultsThree rat species were identified from the three hundred and fifty seven rats captured with Rattus rattus, being the dominant rat species (285, 80 %) followed by Rattus norgevicus (53, 15 %) and Rattus exulans (19, 5 %). Only 39 samples (11.0 %) were positive by culture and further confirmed as pathogenic Leptospira by PCR. Significant associations were shown between host infection with locality, season, host-age and species. Based on MAT, two serogroups were identified in the population namely; L. borgpetersenii serogroup Javanica (n = 16) and L. interrogans serogroup Bataviae (n = 23). Pulsed-field gel electrophoresis (PFGE) distinguished the two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (41 %), and L. interrogans serovar Bataviae (59 %). RAPD-PCR yielded 14 distinct patterns and was found to be more discriminative than PFGE.ConclusionsThis study confirms two Leptospira serovars circulating among the urban rats population in Peninsular Malaysia namely; L. borgpetersenii serovar Javanica and L. interrogans serovars Bataviae. Despite the low number of isolates obtained from the rat population, this study suggests that rodent control programs and disease surveillance may help to reduce the possible risk of disease transmission.

The history and epidemiology of human leptospirosis in Malaysia from 1925 to 2012 are described. Previous studies have demonstrated that leptospirosis is an endemic disease in Malaysia occurring in both urban and rural locations. The number of cases has risen dramatically since the Ministry of Health Malaysia highlighted leptospirosis as a notifiable disease in 2010, with reported cases increasing from 248 cases in 2004 to 3604 in 2012. The incidence of infection among the population suggests that occupation, sex, age, ethnic background, water recreational activities, and sporting events are risk factors. A robust surveillance system is now in place to monitor temporal and spatial changes in the incidence and prevalence of infection and to identify risk areas and disease behavior. Despite extensive studies over the past decade, there is a still a need to describe local serovars in host carriers and the human population, with the view to develop an effective vaccine against leptospirosis.

Rodents have historically been associated with zoonotic pandemics that claimed the lives of large human populations. Appropriate pathogen surveillance initiatives could contribute to early detection of zoonotic infections to prevent future outbreaks. Bordetella species are bacteria known to cause mild to severe respiratory disease in mammals and, some have been described to infect, colonize and spread in rodents. There is a lack of information on the population diversity of bordetellae among Malaysian wild rodents. Here, bordetellae recovered from lung tissues of wild rats were genotypically characterized using 16S rDNA sequencing, MLST and nrdA typing. A novel B. bronchiseptica ST82, closely related to other human-derived isolates, was discovered in three wild rats (n=3) from Terengganu (5.3333° N, 103.1500° E). B. pseudohinzii, a recently identified laboratory mice inhabitant, was also recovered from one rat (n=1). Both bordetellae displayed identical antimicrobial resistance profiles, indicating the close phylogenetic association between them. Genotyping using the 765-bp nrdA locus was shown to be compatible with the MLST-based phylogeny, with the added advantage of being able to genotype non-classical bordetellae. The recovery of B. pseudohinzii from wild rat implied that this bordetellae has a wider host range than previously thought. The findings from this study suggest that bordetellae surveillance among wild rats in Malaysia has to be continued and expanded to other states to ensure early identification of species capable of causing public health disorder.

Forty-seven Salmonella Typhimurium (33 zoonotic, 14 clinical) strains were tested for antimicrobial resistance using the standard disk diffusion method. The presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) and plasmid profiling were carried out to determine the genomic diversity of Salmonella Typhimurium. Approximately 57.4% of the S. Typhimurium strains were multidrug resistant (MDR) and showed high resistance rates to tetracycline (70.2%), sulfonamides (57.4%), streptomycin (53.1%), ampicillin (29.7%), nalidixic acid (27.6%), kanamycin (23.4%), chloramphenicol (21.2%), and trimethoprim (19.1%). Resistance towards cephalosporins was noted for cephalothin (27.6%), cephradine (21.2%), amoxicillin clavulanic acid (17.0%), and cephalexin (17.0%). Resistance genes, bla TEM , strA, aadA, sul1, sul2, tetA, tetB, and tetC, were detected among the drug-resistant strains. Thirtythree strains (70.2%) carried class 1 integrons, which were grouped in 9 different profiles. DNA sequencing identified sat, aadA, pse-1, and dfrA genes in variable regions on class 1 integrons. Thirty-five strains (74.4%) were subtyped to 22 different plasmid profiles, each with 1-6 plasmids (2.0 to 95 kb). PFGE subtyped the 47 strains into 39 profiles. In conclusion, high rates of multidrug resistance were found among the Malaysian Salmonella Typhimurium strains. The emergence of multidrug-resistant Salmonella Typhimurium to cephalosporin antibiotics was also observed. The strains were very diverse and no persistent clone was observed. The emergence of MDR Salmonella Typhimurium is a worldwide problem, and this report provides information for the better understanding of the prevalence and epidemiology of MDR S. Typhimurium in Malaysia.

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