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

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 muel- leri (S. muelleri) , captured from the human leptospirosis sus- pected 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 de- posited in NCBI GenBank under the accession number JAF- CYY0 0 0 0 0 0 0 0 0.1

Diagnostic accuracy of rapid diagnostic tests for the early detection of leptospirosis

Background: Leptospirosis is often misdiagnosed with several other tropical febrile illnesses in Malaysia due to similarities in clinical manifestations. Although treatment regimens could be started based on clinical judgments, early diagnosis has become paramount as a guide to chemotherapeutic interventions. Confirmed laboratory diagnosis through MAT or PCR is time consuming and usually available only in reference laboratories and not practical in healthcare settings. Rapid and easy to perform diagnostic tests are widely used in these settings as the point of care diagnosis. The present study was undertaken to compare the diagnostic performance of two IgM based immunodiagnostic assay kits for acute leptospirosis. Methods: A total of 50 serum samples were collected from patients clinically suspected for acute leptospirosis on admission in the Hospital Serdang, from June 2016 to June 2017. All the samples were subjected to MAT, lipL32 PCR and the two rapid tests (Leptocheck-WB and ImmuneMed Leptospira IgM Duo Rapid test). Results: Out of the 50 clinically suspected patients sampled, 19 were confirmed positive for leptospirosis. Six (12%) were confirmed by MAT and 13 (26%) by PCR. Similarly, of the 50 clinically suspected cases, 17 (34%) showed positivity for Leptocheck-WB and 7 (14%) for ImmuneMed Leptospira IgM Duo Rapid test. The overall sensitivity and specificity was 47.37% and 80.65% for Leptocheck-WB, and 21.05% and 90.32% for ImmuneMed Leptospira IgM Duo Rapid test. In another set of previously confirmed MAT positive samples (1:400-1:3600) obtained from a reference laboratory, Leptocheck-WB showed higher sensitivity (90.72%) than ImmuneMed Leptospira IgM Duo Rapid test (40.21%), and comparable specificity for ImmuneMed Leptospira IgM Duo Rapid test (88.89%) and Leptocheck-WB (82.86%). Conclusion: The sensitivity was higher for Leptocheck-WB and had a comparable specificity with ImmuneMed Leptospira IgM Duo Rapid test. Therefore, based on the present study, Leptocheck-WB is found to be a more sensitive rapid immunodiagnostic test for acute leptospirosis screening in hospital settings

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

Isolation and characterization of pathogenic leptospires from rodents and small mammals captured in human leptospirosis suspected areas in Selangor, Malaysia

Leptospirosis, previously known to be a neglected zoonotic disease in the tropical region is now re-emerging as a threat to public health in both urban and rural settings. It is known that rodents are the major carrier of pathogenic Leptospira sp. In Malaysia, the endemicity of leptospirosis in human is concentrated in areas where rats were highly populated such as in residential and recreational areas with improper trash management and poor sanitation. The statistics in Malaysia has shown an increasing trend of suspected leptospirosis cases and reported deaths since it has been gazetted as a notifiable disease in 2010. This makes an urgent call to study the carrier status of pathogenic leptospires infecting the human through the study of Leptospira sp. in rodents and small mammals in Malaysia. The objective of this study is to identify and characterize the predominant pathogenic Leptospira sp. circulating in the leptospirosis suspected areas in the Selangor state of Malaysia. The study was carried out from January 2016 to April 2017 in six suspected areas comprising of urban, semi-urban and recreational forest areas. The study sites were identified by the Selangor State Health Department as outbreak or hotspot areas. Rodents trapping was performed in all six study sites. The trapped rodents were dissected and kidneys harvested. The rodent kidneys were subjected to Leptospira isolation by culture and dark-field microscopy. The identification and pathogenic strain of the isolated leptospires were determined by PCR approach. The characterization included secY and lipL32 PCR and multi-locus sequence typing (MLST). A total of 14 small mammals species were identified from the 266 captured small mammals with Rattus norvegicus (66%, n=100) being the dominant rat species in the urban area while Maxomys whiteheadi (30%, n=23) dominated the recreational forest area. Among the 266 rodents captured, 217 kidney samples were cultured, while for the remaining 49 samples, DNA was directly extracted from the kidney. From 217 samples cultured 55 (25.3%) were positive for spirochetes examined under the dark-field microscope (DFM). Of the 55 culture and 49 DNA samples, 38/266 (14.3%) were identified to be positive for pathogenic Leptospira confirmed by secY and lipL32 PCR. Phylogenetic analysis by secY PCR showed unique clusters for each species and all isolates clustered according to the respective species. Leptospira interrogans dominated all studied sites followed by L. kirschneri (n=5), L. borgpetersenii (n=2) and L. weilii (n=1). However, no significant association was shown between the infection rate in small mammals with three different sites category (urban, semi-urban and recreational forest) (chi-square, x2 0.5296; p= 0.767). From MLST analysis, three clones of Leptospira sp. were found to dominate the study sites; L. interrogans serovar Bataviae ST50, L. kirschneri serovar Grippotyphosa ST110 and L. borgpetersenii serovar Javanica ST143. While with the help of curator of MLST database, a new ST number (ST238, ST242 and ST243) representing new L. interrogans and L. weilii species was curated for samples isolated mostly from the recreational forest area sites. From the present study, R. norvegicus was identified as the common pathogenic Leptospira host dominating the urban area followed by R. rattus. However although M. whiteheadi dominated the recreational forest area, S. muelleri was the major carrier of the pathogenic Leptospira. In conclusion two species of rats (R. norvegicus and S. muelleri) were identified to play a vital role in environmental contamination in all study sites. This study also confirms for the first time, carriers of Leptospira locus ST110 L. kirschneri, ST242 L. weilii, ST238 L. interrogans and ST243 L. interrogans among small mammals in Malaysia. Thus further research and attention on the serovar status, of all isolates should be carried out as these species may potentially become highly pathogenic serovars contributing to increased risk of severe leptospirosis in Malaysia. In addition, these strains should be included into MAT panels as to improve the diagnosis in the future

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

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

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