Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp. circulating in Sri Lanka.
ABSTRACT: BACKGROUND:Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. METHODOLOGY/PRINCIPAL FINDINGS:We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. CONCLUSIONS/SIGNIFICANCE:This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.
Project description:Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospira strains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka. Leptospira interrogans, Leptospira borgpetersenii and Leptospira kirschneri strains were identified to be circulating in western and southern provinces. L. interrogans was the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure.
Project description:BACKGROUND:Leptospirosis is one of the most important neglected tropical bacterial diseases worldwide. However, there is limited information on the genetic diversity and host selectivity of pathogenic Leptospira in wild small mammal populations. METHODOLOGY/PRINCIPAL FINDINGS:Jiangxi Province, located in southern China, is a region highly endemic for leptospirosis. In this study, among a total of 3,531 trapped rodents dominated by Apodemus agrarius (59.7%), 330 Leptospira strains were successfully isolated from six different sites in Jiangxi between 2002 and 2015. Adding 71 local strains from humans, various kinds of livestock and wild animals in Jiangxi, a total of 401 epidemic strains were characterized using 16S rRNA gene senquencing, multilocus sequence typing (MLST) and the microscopic agglutination test (MAT). Among them, the most prevalent serogroup was Icterohaemorrhagiae (61.10%), followed by Javanica (19.20%) and Australis (9.73%); the remaining five serogroups, Canicola, Autumnalis, Grippotyphosa, Hebdomadis and Pomona, accounted for 9.97%. Species identification revealed that 325 were L. interrogans and 76 were L. borgpetersenii. Moreover, L. interrogans was the only pathogenic species in Fuliang and Shanggao and was predominant in Shangrao (95.0%); L. borgpetersenii was the most common in the remaining three sites. Twenty-one sequence types (STs) were identified. Similarly, ST1 and serogroup Icterohaemorrhagiae were most prevalent in Shangrao (86.0% and 86.4%) and Fuliang (90.4% and 90.4%), ST143 and serogroup Javanica in Shangyou (88.5% and 90.4%) and Longnan (73.1% and 73.1%), and ST105 and serogroup Australis in Shanggao (46.3% and 56.1%). Serogroup Icterohaemorhagiae primarily linked to A. agrarius (86.9%), serogroup Canicola to dogs (83.3%). There were significant differences in the distribution of leptospiral species/serogroups/STs prevalence across host species/collected locations among the 394 animal-associated strains (Fisher's exact test, p<0.001). CONCLUSIONS/SIGNIFICANCE:Our study demonstrated high genetic diversity of pathogenic Leptospira strains from wild small animals in Jiangxi from 2002 to 2015. A. agrarius was the most abundantly trapped animal reservoir, and serogroup Icterohaemorrhagiae and ST1 were the most dominant in Jiangxi. Significant geographic variation and host diversity in the distribution of dominant species, STs and serogroups were observed. Moreover, rat-to-human transmission might play a crucial role in the circulation of Leptospirosis in Jiangxi. Details of the serological and molecular characteristics circulating in this region will be essential in implementing prevention and intervention measures to reduce the risk of disease transmission in China. However, phylogenetic analysis of more Leptospira isolates should explore the impact of ecological change on leptospirosis transmission dynamics and investigate how such new knowledge might better impact environmental monitoring for disease control and prevention at a public health level.
Project description:Leptospirosis is an important cause of acute undifferentiated fever and complex multisystem febrile diseases in the tropics and subtropics. Understanding the evolution of Leptospira especially as related to the clinical pathogenesis of leptospirosis is facilitated by systematic comparative genomic analysis of human-infecting isolates. Here, we announce the complete genome sequences of three Leptospira strains that were isolated from blood of humans with undifferentiated fever in Sri Lanka.
Project description:INTRODUCTION:Sri Lanka has one of the highest incidences of leptospirosis worldwide. We hypothesised that different geographical locations and patient context will have a distinct molecular epidemiology of leptospirosis, based on microgeographical characteristics related to regiona-specific Leptospira predominance. Our objective is to characterise the clinical, epidemiological and molecular aspects of leptospirosis in Sri Lanka to understand disease progression, risk factors and obtain isolates of Leptospira. METHODS AND ANALYSIS:We designed a multicentre prospective study in Sri Lanka to recruit undifferentiated febrile patients and conduct follow-ups during hospital stays. Patients will be recruited from outpatient departments and medical wards. This study will be conducted at two main sites (Anuradhapura and Peradeniya) and several additional sites (Awissawella, Ratnapura and Polonnaruwa). Blood and urine will be collected from patients on the day of admission to the ward or presentation to the outpatient department. Bedside inoculation of 2-4 drops of venous blood will be performed with Ellinghausen-McCullough-Johnson-Harris (EMJH) semisolid media supplemented with antibiotics. Regionally optimised microscopic agglutination test, culture and qPCR-evidence will be performed to confirm the presence of Leptospira in blood which in turn will confirm the presence of disease. Whole genome sequencing will be carried out for all isolates recovered from patients. Multilocus sequence typing (MLST) will be used for the genotyping of new isolates. Sri Lankan isolates will be identified using three published MLST schemes for Leptospira. ETHICS AND DISSEMINATION:Ethical clearance for the study was obtained from Ethics Review Committees (ERC), Medicine and Allied Sciences (FMAS), Rajarata University of Sri Lanka (RUSL) and University of Peradeniya. All genomic data generated through this project will be available at GenBank. Anonymised data will be deposited at the ERC, FMAS, RUSL.
Project description:Molecular data are required to improve our understanding of the epidemiology of leptospirosis in Africa and to identify sources of human infection. We applied molecular methods to identify the infecting Leptospira species and genotypes among patients hospitalized with fever in Tanzania and compared these with Leptospira genotypes detected among animals in Tanzania to infer potential sources of human infection. We performed lipL32 real-time PCR to detect the presence of pathogenic Leptospira in acute-phase plasma, serum, and urine samples obtained from study participants with serologically confirmed leptospirosis and participants who had died with febrile illness. Leptospira blood culture was also performed. In positive specimens, we performed species-specific PCR and compared participant Leptospira secY sequences with Leptospira reference sequences and sequences previously obtained from animals in Tanzania. We detected Leptospira DNA in four (3.6%) of 111 participant blood samples. We detected Leptospira borgpetersenii (one participant, 25.0%), Leptospira interrogans (one participant, 25.0%), and Leptospira kirschneri (one participant, 25.0%) (one [25%] undetermined). Phylogenetic comparison of secY sequence from the L. borgpetersenii and L. kirschneri genotypes detected from participants was closely related to but distinct from genotypes detected among local livestock species. Our results indicate that a diverse range of Leptospira species is causing human infection. Although our analysis suggests a close relationship between Leptospira genotypes found in people and livestock, continued efforts are needed to obtain more Leptospira genetic material from human leptospirosis cases to help prioritize Leptospira species and genotypes for control.
Project description:BACKGROUND:Leptospirosis is one of the most significant zoonoses across the world not only because of its impact on human and animal health but also because of the economic and social impact on agrarian communities. Leptospirosis is endemic in Sri Lanka where paddy farming activities, the use of draught animals in agriculture, and peridomestic animals in urban and rural areas play important roles in maintaining the infection cycle of pathogenic Leptospira, especially concerning animals as a potential reservoir. In this study, an environmental DNA (eDNA) metabarcoding methodology was applied in two different agro-ecological regions of Sri Lanka to understand the eco-epidemiology of leptospirosis. METHODOLOGY/PRINCIPAL FINDINGS:Irrigation water samples were collected in Kandy District (wet zone mid-country region 2) and Girandurukotte, Badulla District (intermediate zone low-country region 2); and analysed for the presence of pathogenic Leptospira, associated microbiome and the potential reservoir animals. Briefly, we generated PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. The analysis of eDNA showed different environmental microbiomes in both regions and a higher diversity of Leptospira species circulating in Kandy than in Girandurukotte. Moreover, the number of sequence reads of pathogenic Leptospira species associated with clinical cases such as L. interrogans was higher in Kandy than in Girandurukotte. Kandy also showed more animal species associated with pathogenic bacterial species than Girandurukotte. Finally, several pathogenic bacterial species including Arcobacter cryaerophilus, responsible for abortion in animals, was shown to be associated with pathogenic Leptospira. CONCLUSIONS/SIGNIFICANCE:Leptospirosis has been considered to be endemic in wet regions, consistently, leptospiral sequences were detected strongly in Kandy. The great Leptospira species diversity in Kandy observed in this study shows that the etiological agents of leptospirosis in Sri Lanka might be underestimated. Furthermore, our eDNA metabarcoding can be used to discriminate bacterial and animal species diversity in different regions and to explore environmental microbiomes to identify other associated bacterial pathogens in the environment.
Project description: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.
Project description:Leptospirosis is known to be an important cause of weather disaster-related infectious disease epidemics. In 2011, an outbreak of leptospirosis occurred in the relatively dry district of Anuradhapura, Sri Lanka where diagnosis was resisted by local practitioners because leptospirosis was not known in the area and the clinical presentation was considered atypical. To identify the causative Leptospira associated with this outbreak, we carried out a cross-sectional study. Consecutive clinically suspected cases in this district were studied during a two-and-a-half-month period. Of 96 clinically suspected cases, 32 (33.3%) were confirmed by qPCR, of which the etiological cause in 26 cases was identified using 16S rDNA sequencing to the species level. Median bacterial load was 4.1 × 10(2)/mL (inter-quartile range 3.1-6.1 × 10(2)/mL). In contrast to a 2008 Sri Lankan leptospirosis outbreak in the districts of Kegalle, Kandy, and Matale, in which a predominance of Leptospira interrogans serovars Lai and Geyaweera was found, most cases in the 2011 outbreak were caused by Leptospira kirschneri. Seven (21.9%) confirmed cases had acute renal failure; five (15.6%) had myocarditis; severe thrombocytopenia (<20,000/uL) was seen in five (15.6%) cases. This outbreak of leptospirosis in the relatively dry zone of Sri Lanka due primarily to L. kirschneri was characterized by markedly different clinical presentations and low leptospiremia. These observations and data demonstrate the public health relevance of molecular diagnostics in such settings, possibly related to the microgeographic variations of different Leptospira species, but of particular value to public health intervention in what appears to have been a regionally neglected tropical disease.
Project description:Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.
Project description:BACKGROUND:Although leptospirosis is a zoonosis of major concern on tropical islands, the molecular epidemiology of the disease aiming at linking human cases to specific animal reservoirs has been rarely explored within these peculiar ecosystems. METHODOLOGY/PRINCIPAL FINDINGS:Five species of wild small mammals (n = 995) as well as domestic animals (n = 101) were screened for Leptospira infection on Reunion Island; positive samples were subsequently genotyped and compared to Leptospira from clinical cases diagnosed in 2012-2013 (n = 66), using MLST analysis. We identified two pathogenic species in human cases, namely Leptospira interrogans and Leptospira borgpetersenii. Leptospira interrogans was by far dominant both in clinical samples (96.6%) and in infected animal samples (95.8%), with Rattus spp and dogs being its exclusive carriers. The genetic diversity within L. interrogans was apparently limited to two sequence types (STs): ST02, identified among most clinical samples and in all rats with complete MLST, and ST34, identified in six humans, but not in rats. Noteworthy, L. interrogans detected in two stray dogs partially matched with ST02 and ST34. Leptospira borgpetersenii was identified in two clinical samples only (3.4%), as well as in cows and mice; four haplotypes were identified, of which two seemingly identical in clinical and animal samples. Leptospira borgpetersenii haplotypes detected in human cases were clearly distinct from the lineage detected so far in the endemic bat species Mormopterus francoismoutoui, thus excluding a role for this volant mammal in the local human epidemiology of the disease. CONCLUSIONS/SIGNIFICANCE:Our data confirm rats as a major reservoir of Leptospira on Reunion Island, but also pinpoint a possible role of dogs, cows and mice in the local epidemiology of human leptospirosis. This study shows that a comprehensive molecular characterization of pathogenic Leptospira in both clinical and animal samples helps to gaining insight into leptospirosis epidemiology within a specific environmental setting.