An outbreak of leptospirosis among reserve military recruits, Hulu Perdik, Malaysia.

Here, we investigated an outbreak of leptospirosis among reserve military recruits that occurred following a survival exercise in the Hulu Perdik forest within the Hulu Langat district, Kuala Lumpur, Malaysia. Blood samples from the 12 patients that presented symptoms for febrile illness on clinical examination were subjected to laboratory investigation, comprising Lepto IgM rapid test, IgM ELISA, and microscopic agglutination test (MAT). All these patients were interviewed for possible risk factors for leptospirosis. Rodent trapping and environmental sampling for possible isolation of leptospires in the outbreak site was performed. The isolated leptospires were genetically characterized and investigated for the potential epidemiological link with human leptospirosis. Among the 12 patients, two (2/12; 16.6%) were confirmed positive for leptospirosis by microscopic agglutination test (MAT with titers 400-800; serovar autumnalis and hardjobovis). Two Leptospira species from rodents (L. interrogans and L. borgpetersenii) and two from the environment (L. kmetyi and L. wolffii) were identified. The possible epidemiological link between human serovars and animal Leptospira species indicates rodents as the potential reservoir while the environment (soil and water) serves as a transmission route. This investigation highlights the robust presence of pathogenic leptospires on Malaysian environment and rodents which may present the risk of infection, especially among high-risk individuals. Hence, occupational risk individuals are cautioned to observe appropriate preventive measures including prophylaxis and seek immediate medical attention for any illness following similar activities.

A versatile isothermal amplification assay for the detection of leptospires from various sample types.

Background: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene. Results: Several reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 104 copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease. Conclusions: In short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study.

In vivo and in silico Virulence Analysis of Leptospira Species Isolated From Environments and Rodents in Leptospirosis Outbreak Areas in Malaysia.

The zoonotic disease leptospirosis is caused by pathogenic species of the genus Leptospira. With the advancement of studies in leptospirosis, several new species are being reported. It has always been a query, whether Leptospira species, serovars, and strains isolated from different geographical locations contribute to the difference in the disease presentations and severity. In an epidemiological surveillance study performed in Malaysia, we isolated seven novel intermediate and saprophytic species (Leptospira semungkisensis, Leptospira fletcheri, Leptospira langatensis, Leptospira selangorensis, Leptospira jelokensis, Leptospira perdikensis, Leptospira congkakensis) from environments and three pathogenic species from rodents (Leptospira borgpetersenii strain HP364, Leptospira weilii strain SC295, Leptospira interrogans strain HP358) trapped in human leptospirosis outbreak premises. To evaluate the pathogenic potential of these isolates, we performed an in vivo and in silico virulence analysis. Environmental isolates and strain HP364 did not induce any clinical manifestations in hamsters. Strain SC295 caused inactivity and weight loss with histopathological changes in kidneys, however, all hamsters survived until the end of the experiment. Strain HP358 showed a high virulent phenotype as all infected hamsters died or were moribund within 7 days postinfection. Lungs, liver, and kidneys showed pathological changes with hemorrhage as the main presentation. In silico analysis elucidated the genome size of strain HP358 to be larger than strains HP364 and SC295 and containing virulence genes reported in Leptospira species and a high number of specific putative virulence factors. In conclusion, L. interrogans strain HP358 was highly pathogenic with fatal outcome. The constituent of Leptospira genomes may determine the level of disease severity and that needs further investigations.

Genomic data of Leptospira interrogans HP358 isolated from rodent captured from the human leptospirosis suspected areas of Selangor state, Malaysia.

The data provided in this article is the genomic sequence of new Leptospira isolate, Leptospira interrogans strain HP358 (L. interrogans HP358) isolated from rodent, Sundamys muelleri (S. muelleri), captured from the human leptospirosis suspected area, in forest environment, Hulu Perdik, Selangor. The kidney of the rodent was cultured, and the genomic DNA of pure Leptospira isolate was extracted and sequenced. The de novo assembly of genome generated 118 contigs with N50 of 133,176bp. The genome size of the L. interrogans HP358 was determined with a length of 4,808,724 and 35.01% G+C content with 229 subsystems, 5236 coding sequences and 39 RNAs. The whole genome shotgun project has been deposited in NCBI GenBank under the accession number JAFCYY000000000.1.

Gamification, a Successful Method to Foster Leptospirosis Knowledge among University Students: A Pilot Study.

Leptospirosis is a zoonotic disease that has been reported in Malaysia and has been associated with a recent trend of recreational activities among the youth. Thus, efforts such as educational interventions among high-risk populations, especially the youth, are key to increasing public awareness regarding leptospirosis. This paper presents the findings of a pilot study wherein an educational intervention using a gamification intervention method was used to determine changes in leptospirosis knowledge among youth. On this note, students from a public university in Seremban district, Malaysia, were recruited and were asked to complete questionnaires before and after gamification activities. Baseline and immediate post-intervention data on leptospirosis knowledge were obtained. The total knowledge score was calculated, and differences in the mean pre- and post-intervention knowledge score were determined. Of the total 185 questionnaires that were completed at baseline and immediately post-intervention, only 168 that belonged to respondents who had heard of leptospirosis were analysed in this paper. A significant increase in leptospirosis knowledge was observed for the students following health education by gamification (p < 0.01). The results demonstrate the effectiveness of an educational intervention using gamification in improving leptospirosis knowledge among youth and suggest that gamification could become an efficient tool to prevent the disease within university-age demographics.

Microhabitat Factors Influenced the Prevalence of Pathogenic Leptospira spp. in Small Mammal Host.

Leptospirosis, a widespread zoonotic disease, is a public health problem, especially in major urban centres, and is mainly reported to be associated with rats. In Malaysia, focus has been primarily given to the Leptospira prevalence in rodents per se, but there is lack of information on the microhabitat structure of the outbreak areas. We aimed to determine the diversity of small mammal species, microhabitat types, and their prevalence of pathogenic Leptospira spp. in the outbreak areas, which were categorized as urban, semi-urban, and recreational forests. Sampling involved deploying 100 to 300 live traps at each study site. Kidney samples were extracted from selected individuals, for screening of pathogenic Leptospira spp. by PCR. Out of 537 individuals from 15 small mammal species captured, 4 species were recorded from urban, 13 from semi-urban, and 11 from recreational forest sites. From 389 individuals screened, 58 were tested positive for pathogenic Leptospira. Recreational forests recorded the highest prevalence with 19.4% (n = 93), followed by urban, 16.6% (n = 163) and semi-urban sites with 9.8% (n = 133). Seven rodent species were tested positive for pathogenic Leptospira from all areas. R. norvegicus was found to harbour the highest prevalence (66.7%) in urban, R. rattus (53.8%) in semi-urban, whereby M. whiteheadi (44.4%) in recreational forest sites. Microhabitat analysis revealed that rubbish quantity contributed especially strongly to a high prevalence of Leptospira. This study contributes to understanding of the host and microhabitat preferences of Leptospira, which is important in controlling the spread of this disease in human's landscapes.

Molecular characterization of pathogenic Leptospira sp. in small mammals captured from the human leptospirosis suspected areas of Selangor state, Malaysia.

Leptospirosis is caused by the spirochetal bacterium Leptospira of which rodents are considered the most important reservoir. This study aims to determine and characterize virulent Leptospira species among rodents and small mammals found in human settlements and recreational spots within the Hulu Langat and Gombak districts of Selangor, Malaysia; regions that frequently report probable human leptospirosis cases. Molecular analysis revealed an overall Leptospira detection rate of 14.3% among the 266 small mammals captured, and the human settlements were found to have the highest number of isolates (15.1%), followed by recreational sites (14.5%). The molecular characterization conducted based on the lipL32, secY genes and MLST revealed that the strains belonged to four different species, including; Leptospira interrogans (29; 76.3%; ST50, ST238, ST243), L. kirschneri (5; 13.15%; ST110), L. borgpetersenii (3; 8%; ST143) and L. weilii (1; 2.63%; ST242). The study revealed genotypes of circulating strains among small mammals in Malaysia, which include Leptospira locus ST110 L. kirschneri, ST 50 L. interrogans, ST143 L. borgpetersenii and ST242 L. weilii. Among the small mammals studied, 17/105 (16.2%) Rattus norvegicus, 7/59 (11.9%) of Rattus rattus, 5/24 (20.8%) of Maxomys whiteheadi, 4/18 (22.2%) of Sundamys muelleri, 2/22 (9%), Tupaia gliss, 2/16 (12.5%) Rattus tiomanicus and 1/4 (25%) of Suncus murinus carried pathogenic leptospires. The data from the present study may imply that, in addition to rodents, other small mammals also serve as maintenance hosts for Leptospira. Hence, much remains unknown about Leptospira maintenance hosts, and there is need for further investigation to ascertain the prevailing serovars of pathogenic Leptospira in Malaysia. This will assist in the development of efficient diagnostic assays with improved microscopic agglutination test (MAT) panels, and in the implementation of suitable prevention and control measures.