Molecular characterisation and disease severity of leptospirosis in Sri Lanka.
ABSTRACT: 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, commonly known as rat-urine disease, is a global but endemic zoonotic disease in the tropics. Despite the historical report of leptospirosis in Malaysia, the information on human-infecting species is limited. Determining the circulating species is important to understand its epidemiology, thereby to strategize appropriate control measures through public health interventions, diagnostics, therapeutics and vaccine development. METHODOLOGY/PRINCIPLE FINDINGS:We investigated the human-infecting Leptospira species in blood and serum samples collected from clinically suspected leptospirosis patients admitted to three tertiary care hospitals in Malaysia. From a total of 165 patients, 92 (56%) were confirmed cases of leptospirosis through Microscopic Agglutination Test (MAT) (n = 43; 47%), Polymerase Chain Reaction (PCR) (n = 63; 68%) or both MAT and PCR (n = 14; 15%). The infecting Leptospira spp., determined by partial 16S rDNA (rrs) gene sequencing revealed two pathogenic species namely Leptospira interrogans (n = 44, 70%) and Leptospira kirschneri (n = 17, 27%) and one intermediate species Leptospira wolffii (n = 2, 3%). Multilocus sequence typing (MLST) identified an isolate of L. interrogans as a novel sequence type (ST 265), suggesting that this human-infecting strain has a unique genetic profile different from similar species isolated from rodents so far. CONCLUSIONS/SIGNIFICANCE:Leptospira interrogans and Leptospira kirschneri were identified as the dominant Leptospira species causing human leptospirosis in Central Malaysia. The existence of novel clinically important ST 265 (infecting human), that is different from rodent L. interrogans strains cautions reservoir(s) of these Leptospira lineages are yet to be identified.
Project description: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 one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
Project description:Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.
Project description:Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.
Project description:Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.We used bioinformatics to evaluate pre-existing loci with the potential to increase the discrimination of outbreak strains. Previously deposited sequence data were evaluated by phylogenetic analyses using either single or concatenated sequences. We identified and evaluated the applicability of the ligB, secY, rpoB and lipL41 loci, individually and in combination, to discriminate between 38 pathogenic Leptospira strains and to cluster them according to the species they belonged to. Pairwise identity among the loci ranged from 82.0-92.0%, while interspecies identity was 97.7-98.5%. Using the ligB-secY-rpoB-lipL41 superlocus it was possible to discriminate 34/38 strains, which belong to six pathogenic Leptospira species. In addition, the sequences were concatenated with the superloci from 16 sequence types from a previous MLST scheme employed to study the association of a leptospiral clone with an outbreak of human leptospirosis in Thailand. Their use enhanced the discriminative power of the existing scheme. The lipL41 and rpoB loci raised the resolution from 81.0-100%, but the enhanced scheme still remains limited to the L. interrogans and L. kirschneri species.As the first aim of our study, the ligB-secY-rpoB-lipL41 superlocus demonstrated a satisfactory level of discrimination among the strains evaluated. Second, the inclusion of the rpoB and lipL41 loci to a MLST scheme provided high resolution for discrimination of strains within L. interrogans and L. kirschneri and might be useful in future epidemiological studies.
Project description:Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the genus Leptospira. We sought to determine if rodents in U.S. Virgin Islands (USVI) are carriers of Leptospira. In total, 140 rodents were sampled, including 112 Mus musculus and 28 Rattus rattus. A positive carrier status was identified for 64/140 (45.7%); 49 (35.0%) were positive by dark-field microscopy, 60 (42.9%) by culture, 63 (45.0%) by fluorescent antibody testing, and 61 (43.6%) by real-time polymerase chain reaction (rtPCR). Molecular typing indicated that 48 isolates were L. borgpetersenii and 3 were L. kirschneri; the remaining nine comprised mixed species. In the single culture-negative sample that was rtPCR positive, genotyping directly from the kidney identified L. interrogans. Serotyping of L. borgpetersenii isolates identified serogroup Ballum and L. kirschneri isolates as serogroup Icterohaemorrhagiae. These results demonstrate that rodents are significant Leptospira carriers and adds to understanding the ecoepidemiology of leptospirosis in USVI.
Project description:A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown.A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection.Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.
Project description:Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the Leptospira genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, Leptospira infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of Leptospira species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of Leptospira spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as L. interrogans serogroup Pomona serovar Kennewicki (20 strains), L. interrogans serogroup Canicola serovar Canicola (1 strain), L. borgpetersenii serogroup Sejroe serovar Hardjo (10 strains) and L. noguchii (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic Leptospira in their urine, uncovering a threat for public health that is being largely neglected. The two L. interrogans serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local Leptospira strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic Leptospira strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.
Project description:<h4>Background</h4>Reports of chronic hepatitis in dogs caused by Leptospira spp. are confined to small case series. Fluorescence in situ hybridization (FISH) allows the identification of spirochetes in liver samples. Consequently, this technique may help elucidate the role of Leptospira spp. in cases of chronic hepatitis.<h4>Objectives</h4>To describe cases of hepatic leptospirosis in dogs diagnosed by FISH and subsequent polymerase chain reaction (PCR) speciation, with the absence of clinically relevant renal involvement.<h4>Animals</h4>Ten client-owned dogs.<h4>Methods</h4>Retrospective case series from the University of Cambridge presented between 2013 and 2016 or cases consulted by telephone advice during this time period. Cases were selected based on histopathologically confirmed granulomatous hepatitis and leptospiral organisms identified by FISH and PCR speciation (Leptospira interrogans/kirschneri).<h4>Results</h4>All cases had increased liver enzyme activities, and FISH in combination with PCR speciation-confirmed infection with L. interrogans/kirschneri. Four dogs underwent repeat liver biopsy, FISH and PCR speciation 4-15?months after initial presentation and doxycycline treatment with 1 dog undergoing repeat sampling at necropsy. Three dogs that underwent repeat biopsy remained positive for L. interrogans/kirschneri infection. Six dogs were alive at the time of manuscript preparation and 4 dogs were euthanized as a result of progressive liver disease.<h4>Conclusions and clinical importance</h4>The presence of hepatic leptospiral organisms may be associated with chronic granulomatous hepatitis without clinical evidence of renal involvement. Further studies are necessary to elucidate the etiological role of these organisms in the disease.