Evolutionary insights into the emergence of virulent Leptospira spirochetes.

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the mammalian complement system and the adaptations to avoid activation of the innate immune cells by the highly-virulent Leptospira species (also called P1+ species), unlike other species belonging to the phylogenetically related P1- and P2 groups, as well as saprophytes. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.

Inter-species Transcriptomic Analysis Reveals a Constitutive Adaptation Against Oxidative Stress for the Highly Virulent Leptospira Species.

Transcriptomic analyses across large scales of evolutionary distance have great potential to shed light on regulatory evolution but are complicated by difficulties in establishing orthology and limited availability of accessible software. We introduce here a method and a graphical user interface wrapper, called Annotator-RNAtor, for performing interspecies transcriptomic analysis and studying intragenus evolution. The pipeline uses third-party software to infer homologous genes in various species and highlight differences in the expression of the core-genes. To illustrate the methodology and demonstrate its usefulness, we focus on the emergence of the highly virulent Leptospira subclade known as P1+, which includes the causative agents of leptospirosis. Here, we expand on the genomic study through the comparison of transcriptomes between species from P1+ and their related P1- counterparts (low-virulent pathogens). In doing so, we shed light on differentially expressed pathways and focused on describing a specific example of adaptation based on a differential expression of PerRA-controlled genes. We showed that P1+ species exhibit higher expression of the katE gene, a well-known virulence determinant in pathogenic Leptospira species correlated with greater tolerance to peroxide. Switching PerRA alleles between P1+ and P1- species demonstrated that the lower repression of katE and greater tolerance to peroxide in P1+ species was solely controlled by PerRA and partly caused by a PerRA amino-acid permutation. Overall, these results demonstrate the strategic fit of the methodology and its ability to decipher adaptive transcriptomic changes, not observable by comparative genome analysis, that may have been implicated in the emergence of these pathogens.

Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.

Identification of circulating miRNA biomarkers in leptospirosis for early detection: A promising diagnostic approach.

Leptospirosis is a zoonotic disease of global significance, contributing to morbidity and mortality worldwide. It is endemic to tropical regions, with outbreaks during monsoons. The disease manifestations are similar to that of other febrile illness such as dengue, malaria hence often misdiagnosed and underreported. The zoonoses if undetected, progresses to cause severe life-threatening complications also known as Weil's disease. Routine diagnostic tests are based on the detection of antibodies in patient serum and are not accurate during the initial phase of the infection. Therefore, it is necessary to detect novel biomarkers that can be used in early detection of leptospirosis. Circulating miRNAs are known to be promising biomarkers for various diseases including cancer, tuberculosis, influenza; hence in this study the potential of miRNAs as biomarkers for leptospirosis was evaluated. A total of 30 leptospirosis cases were screened for the differential expression of 10 miRNA by RT-qPCR assay. The differential expression was calculated by relative quantification using healthy individuals as controls. Among the 10 miRNA,3 miRNA, miR-28-5p, miR-302c-3p and miR-302a-3p were reported to exhibit a significant trend of upregulation. Further their role in immune pathways and biological processes was investigated by KEGG analysis and Gene Ontology. The 3 miRNAs were observed to target various immune response pathways, thus confirming their role in host immune response. Based on the results obtained in this study, miR-28-5p, miR-302c-3p and miR-302a-3p can be considered as potential biomarkers for the detection of leptospirosis.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Isolation and characterization of Leptospira licerasiae in Austrian swine - a first-time case report in Europe.

BACKGROUND: Leptospiraceae comprise a diverse family of spirochetal bacteria, of which many are involved in infectious diseases of animals and humans. Local leptospiral diversity in domestic animals is often poorly understood. Here we describe the incidental detection of Leptospira (L.) licerasiae in an Austrian pig. CASE PRESENTATION: During an experiment to characterize the pathogenesis of L. interrogans serovar Icterohaemorrhagiae in pigs, cultivation of a urine sample from a non-challenged contact pig resulted in growth of a spirochetal bacterium that tested negative for pathogenic Leptospira (LipL32 gene). PCR, Sanger sequencing and standard serotyping further confirmed that the recovered isolate was clearly different from the challenge strain L. interrogans serovar Icterohaemorrhagiae used in the animal experiment. Whole genome sequencing revealed that the isolate belongs to the species L. licerasiae, a tropical member of the Leptospiraceae, with no prior record of detection in Europe. CONCLUSIONS: This is the first report describing the occurrence of L. licerasiae in Europe. Since L. licerasiae is considered to have intermediate pathogenicity, it will be important to follow the geographical distribution of this species and its pathogenic and zoonotic potential in more detail.

Detection of Leptospira kirschneri in a short-beaked common dolphin (Delphinus delphis delphis) stranded off the coast of southern California, USA.

BACKGROUND: Pathogenic Leptospira species are globally important zoonotic pathogens capable of infecting a wide range of host species. In marine mammals, reports of Leptospira have predominantly been in pinnipeds, with isolated reports of infections in cetaceans. CASE PRESENTATION: On 28 June 2021, a 150.5 cm long female, short-beaked common dolphin (Delphinus delphis delphis) stranded alive on the coast of southern California and subsequently died. Gross necropsy revealed multifocal cortical pallor within the reniculi of the kidney, and lymphoplasmacytic tubulointerstitial nephritis was observed histologically. Immunohistochemistry confirmed Leptospira infection, and PCR followed by lfb1 gene amplicon sequencing suggested that the infecting organism was L.kirschneri. Leptospira DNA capture and enrichment allowed for whole-genome sequencing to be conducted. Phylogenetic analyses confirmed the causative agent was a previously undescribed, divergent lineage of L.kirschneri. CONCLUSIONS: We report the first detection of pathogenic Leptospira in a short-beaked common dolphin, and the first detection in any cetacean in the northeastern Pacific Ocean. Renal lesions were consistent with leptospirosis in other host species, including marine mammals, and were the most significant lesions detected overall, suggesting leptospirosis as the likely cause of death. We identified the cause of the infection as L.kirschneri, a species detected only once before in a marine mammal - a northern elephant seal (Mirounga angustirostris) of the northeastern Pacific. These findings raise questions about the mechanism of transmission, given the obligate marine lifestyle of cetaceans (in contrast to pinnipeds, which spend time on land) and the commonly accepted view that Leptospira are quickly killed by salt water. They also raise important questions regarding the source of infection, and whether it arose from transmission among marine mammals or from terrestrial-to-marine spillover. Moving forward, surveillance and sampling must be expanded to better understand the extent to which Leptospira infections occur in the marine ecosystem and possible epidemiological linkages between and among marine and terrestrial host species. Generating Leptospira genomes from different host species will yield crucial information about possible transmission links, and our study highlights the power of new techniques such as DNA enrichment to illuminate the complex ecology of this important zoonotic pathogen.

Molecular and serological prevalence of Leptospira spp. among slaughtered cattle and associated risk factors in the Bahr El Ghazal region of South Sudan.

INTRODUCTION: Leptospirosis is a neglected emerging and zoonotic disease reported worldwide. This study sought to determine the molecular and serological prevalence of Leptospira spp. and the associated risk factors in slaughtered cattle from the Bahr El Ghazal region of South Sudan. MATERIALS AND METHODS: Between January 16th and February 25th, 2023, blood and urine samples were collected from 402 cattle at the Lokoloko Municipal Slaughterhouse in Western Bahr El-Ghazal State. Serum samples were tested using the microscopic agglutination test (MAT), with a panel of 12 serovars (sv) from 12 serogroups (sg) and 4 species (spp) of Leptospira spp. These serovars had been previously identified in Sudan and the East African region. Simultaneously, 400 corresponding urine samples were screened using qualitative real-time polymerase chain reaction (PCR) to detect the shedding of Leptospira spp. in urine. To identify the associated risk factors, the age, sex, breed and body condition score of each sampled cattle was noted at the time of sampling and subsequently analysed using logistic regression models. RESULTS: Among the 402 serum samples screened, a substantial 81.8% (329/402, 95% CI 77.9-85.3) displayed seropositivity for Leptospira spp. with a MAT titre ≥ 100. The prevalence of urine shedding determined by PCR was 6% (23/400, 95% CI 3.8-8.4), while probable recent leptospirosis with a MAT ≥ 1:800 was observed in 33.1% (133/402, 95% CI 28.6-37.8) of the cattle. Multiple reactions were detected in 34.8% (140/402, 95% CI 30.6-39.5) serum samples. The seropositivity was against L. borgpetersenii sg. Tarassovi (78.6%; 316/402, 95% CI 74.4-82.3), followed by L. borgpetersenii sg. Ballum at 20.4% (82/402, 95% CI, 16.7-24.4%), L. kirschneri sg. Autumnalis At 8.7% (35/402, 95% CI 5.7-11.7), L. interrogans sg. of Pomona at 7.0% (28/402, 95% CI 4.5-9.5), and L. interrogans sg. Hebdomadis was 5.0% (20/402, 95% CI 2.8-7.2). Several risk factors are associated with seropositivity. Older animals (≥ 2 years) had 2.0 times greater odds (95% CI 1.14-3.5) of being seropositive than younger animals (< 2 years), P-value = 0.016. Female animals demonstrated 2.1 times greater odds (95% CI 1.2-3.6) of seropositivity than males did (P-value = 0.008). Additionally, Felata/Mbororo cattle exhibited 2.4 times greater odds (95% CI 1.3-4.5) of being seropositive than did local Nilotic cattle (P-value = 0.005). The agreement between the MAT and PCR results was poor, as indicated by a kappa statistic value of 0.001 and a P-value of 0.913. But there was a moderate agreement between MAT high titres ≥ 800 and PCR positivity with a kappa statistic value = 0.501 and a P-value < 0.001. CONCLUSION: In addition to the high seroprevalence, Leptospira spp. were found in the urine of slaughtered cattle, suggesting that leptospirosis is endemic to the study area. This finding underscores the significance of cattle as potential sources of infection for slaughterhouse workers, the general public, and other animal species. To address this issue effectively in the Bahr El Ghazal Region and South Sudan, a comprehensive strategy involving a multidisciplinary approach is essential to minimize disease among animals, hence reducing potential zoonotic risks to humans.

Spatiotemporal assessment of pathogenic Leptospira in subtropical coastal watersheds.

The World Health Organization classifies leptospirosis as a significant public health concern, predominantly affecting impoverished and unsanitary regions. By using the Pensacola Bay System as a case study, this study examines the underappreciated susceptibility of developed subtropical coastal ecosystems such as the Pensacola Bay System to neglected zoonotic pathogens such as Leptospira. We analyzed 132 water samples collected over 12 months from 44 distinct locations with high levels of Escherichia coli (>410 most probable number/100 mL). Fecal indicator bacteria (FIB) concentrations were assessed using IDEXX Colilert-18 and Enterolert-18, and an analysis of water physiochemical characteristics and rainfall intensity was conducted. The LipL32 gene was used as a quantitative polymerase chain reaction (qPCR) indicator to identify the distribution of Leptospira interrogans. The results revealed 12 instances of the presence of L. interrogans at sites with high FIB over various land cover and aquatic ecosystem types. Independent of specific rainfall events, a seasonal relationship between precipitation and elevated rates of fecal bacteria and leptospirosis was found. These findings highlight qPCR's utility in identifying pathogens in aquatic environments and the widespread conditions where it can be found in natural and developed areas.

Leptospira infection and carrier survey on rats from wet market areas in Kuala Lumpur, Malaysia.

BACKGROUND OBJECTIVES: Leptospirosis is an important zoonotic infection that has caused significant mortality and morbidity worldwide. This disease is endemic in Malaysia and as a developing tropical country, leptospirosis is concerning as it threatens Malaysian public health and the country's economic sectors. However, there is limited information on leptospirosis in Malaysia, especially regarding leptospiral seroepidemiology among carriers in Malaysia. Therefore, more epidemiological information on the source of the disease and reservoir are needed for better disease control and source intervention. The objectives of this study are to gather information on Leptospira infection and the carrier status of rats captured from selected wet markets of Kuala Lumpur metropolitan city in Malaysia. METHODS: Live rat trappings were performed in four major wet markets in Kuala Lumpur, namely, Pudu, Chow Kit, Datuk Keramat, and Petaling Street. Animal samplings were performed for 12 months in 2017, where blood and kidney samples were collected and tested for anti-leptospiral antibodies via Microscopic Agglutination Test (MAT) and pathogenic Leptospira screening via Polymerase Chain Reaction (PCR) amplification offlaB gene. RESULTS: MAT showed that 34.7% (n = 50/144) of the captured rats were positive for anti-leptospiral antibody of which the most prominent serovar was Malaya followed by a local strain, IMR LEP 175. In parallel, 50 rats were also positive for pathogenic Leptospira DNA. INTERPRETATION CONCLUSION: This study showed that there are persistent Leptospira infections among rats in Kuala Lumpur wet markets and these rats are important reservoir hosts for the bacteria.

Genetic structure and diversity of the rfb locus of pathogenic species of the genus Leptospira.

Leptospirosis is caused by pathogenic strains of the genus Leptospira and is considered the most widespread zoonotic bacterial disease. The genus is characterized by the large number of serology variants, which challenges developing effective serotyping methods and vaccines with a broad spectrum. Because knowledge on the genetic basis of the serological diversity among leptospires is still limited, we aimed to explore the genetic structure and patterns of the rfb locus, which is involved in the biosynthesis of lipopolysaccharides, the major surface antigen that defines the serovar in leptospires. Here, we used genomic data of 722 pathogenic samples and compared the gene composition of their rfb locus by hierarchical clustering. Clustering analysis showed that the rfb locus gene composition is species-independent and strongly associated with the serological classification. The samples were grouped into four well-defined classes, which cluster together samples either belonging to the same serogroup or from different serogroups but sharing serological affinity. Our findings can assist in the development of new strategies based on molecular methods, which can lead to better tools for serological identification in this zoonosis.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples.

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

Fatal leptospirosis in endangered Barbary macaques (Macaca sylvanus) kept in captivity: Assessing the role of sympatric rodents.

Between December 2020 and January 2021, an outbreak of acute mortality in endangered Barbary macaques (Macaca sylvanus) kept in captivity was detected in a zoo in Spain. The main findings observed in the two fatally affected animals at post-mortem evaluation were jaundice, renal tubular necrosis and interstitial nephritis. Leptospira spp. infection was confirmed by real time PCR (qPCR) in different tissues in both individuals. Analyses of secY gene from a positive individual showed 100% homology with a previously published sequence corresponding to Leptospira interrogans serovar Copenhageni. Free-living sympatric brown rats (Rattus norvegicus) from the affected zoo were also analyzed, and showed a prevalence and seroprevalence of Leptospira spp. of 18.2% (4/22; 95% CI: 2.1-34.3) and 41.9% (26/62; 95% CI: 29.7-54.2), respectively. We detected seropositive sera to five different serovars of Leptospira spp. (Copenhageni, Grippotyphosa, Pomona, Canicola and Hardjo) in the rodent population, with L. Copenhageni being the predominant one. This study describes for first time an outbreak of fatal leptospirosis in captive non-human primates in Europe. Our results show that Barbary macaques, an endangered species, are highly susceptible to Leptospira spp. infection, with sympatric wild rodents being the most likely reservoir animals involved in transmission in this outbreak. Our results suggest that rodent control could be an effective measure for minimizing exposure to Leptospira spp. in zoological collections. Given the potential implications for conservation, animal and public health, non-human primates and rodents should be included in surveillance programs for Leptospira spp. in zoos.

Investigation of anti-Leptospira spp. antibodies and leptospiruria in cats attended to a veterinary teaching hospital in southern Brazil.

Leptospirosis is a bacterial zoonosis that affects both humans and animals worldwide. Currently, it is known that cats may be susceptible to infection. This study aims to investigate the presence of anti-Leptospira spp. antibodies and leptospiruria in cats, using Microscopic Agglutination Test (MAT) and Real-time Polymerase Chain Reaction (PCR) techniques, respectively. A total of 76 cats, undergoing comprehensive anamnesis, general physical examination, and complementary exams were included in the investigation. Among the 76 cats tested, 9.2% (7/76) exhibited the presence of anti-Leptospira spp. antibodies, while Leptospira spp. DNA was detected in at 1.3% (1/76) of the evaluated urine samples. No significant associations were observed between the serological and molecular diagnostic results and the assessed variables, including clinical data and laboratory results of cats testing positive. This study provides insight into the occurrence of Leptospira spp. infection and leptospiruria in cats treated at a veterinary teaching hospital in southern Brazil.

Detection of pathogenic Leptospira spp. in unconventional pets.

Leptospirosis is a disease caused by Leptospira spp. responsible for considerable impacts on the public and animal health. In the past two decades, non-domesticated species of pets (unconventional pets) have become popular. However, the role of these unconventional pets on maintaining diseases still unclear. Therefore, the objective of this study was to survey the presence of Leptospira spp. DNA in unconventional pets. Samples of kidney tissues from 29 animals belonging to the Mammalia class (including Orders Carnivora, Lagomorpha and Rodentia) were analyzed for the presence of the gene lipL32. As a result, DNA of pathogenic Leptospira spp. from specie L. interrogans was detected in four (13,80%) of the analyzed samples: three from Oryctolagus cuniculus and one from Mesocricetus auratus. This study highlights the importance of epidemiological surveillance of leptospirosis, as it identified in species of unconventional pets, that may possibly act as reservoirs of Leptospira spp.

Use of Advanced Diagnostics for Timely Identification of Travel-associated Leptospira santarosai Infection in Four Adolescents Through Plasma Microbial Cell-free DNA Sequencing With the Karius Test.

BACKGROUND: Leptospirosis is an important zoonotic infection worldwide. Diagnosis of leptospirosis is challenging given its nonspecific clinical symptoms that overlap with other acute febrile illnesses and limitations with conventional diagnostic testing. Alternative advanced diagnostics, such as microbial cell-free DNA (mcfDNA), are increasingly being used to aid in the diagnosis of infections and can be applied to pathogens with public health importance such as Leptospira , a nationally notifiable disease. METHODS: The Karius Test uses plasma mcfDNA sequencing to detect and quantify DNA-based pathogens. This test offered through the Karius lab detected 4 cases of Leptospira santarosai during a 5-month period across the United States in 2021 and were clinically reviewed. RESULTS: In our case series, 4 adolescents with recent travel to Central America (Costa Rica, n = 3 and Belize, n = 1) from April to August 2021 were diagnosed with leptospirosis. While a large workup was performed in all cases, mcfDNA testing was the first test to detect L. santarosai as the microbiological diagnosis in all cases. CONCLUSIONS: Results of the Karius Test enabled rapid, noninvasive diagnosis of leptospirosis allowing for targeted therapy. Use of mcfDNA can be utilized for diagnosis of pathogens where conventional testing is challenging or limited. This in turn can enable quick diagnosis for targeted treatment and potentially aid in supporting case definitions of reportable diseases of public health concern.

Detection of pathogenic Leptospira with rapid extraction followed by recombinase polymerase amplification (RPA) and quantitative polymerase chain reaction (qPCR) assay-A comprehensive study from Sri Lanka.

Leptospirosis is the most widespread zoonosis in the world. The disease is more prevalent in tropical regions where the majority of developing countries are located. Leptospirosis is considered a protean manifestation zoonosis with severity of the disease ranging from a mild febrile illness to a severe and life-threatening illness. Clinical symptoms of leptospirosis overlap with other tropical febrile illnesses. Early, rapid, and definitive diagnosis is important for effective patient management. Since Polymerase Chain Reaction (PCR)-based assays are not readily available in most clinical settings, there is a need for an affordable, simple, and rapid diagnostic test. Quantitative PCR (qPCR) and Recombinase Polymerase Amplification (RPA) were implemented at the Faculty of Medicine, University of Kelaniya, and a prospective study to evaluate RPA for diagnosis of acute phase of leptospirosis was conducted. Results indicate that RPA and qPCR were positive in 81% (98/121) of the total positive and acute clinical samples. Of the 81 positive MAT confirmed patients 60 (74%) and 53 (65%) were positive with qPCR and RPA respectively. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity-70% and specificity-87%) of RPA compared to MAT as the reference gold standard. Results further suggest that there is no significant difference between the two assays, qPCR and RPA-SwiftX (P = 0.40). Laboratory procedures for the extraction and detection by qPCR in the laboratory have been optimized to obtain results within 6 hours. However, the RPA-SwiftX method under field conditions took 35 minutes. The RPA-SwiftX method could replace the qPCR which shows similar sensitivity and specificity. Therefore, RPA established under the current study presents a powerful tool for the early and rapid diagnosis of leptospirosis at point-of-care.

Prevalence, genetic diversity and eco-epidemiology of pathogenic Leptospira species in small mammal communities in urban parks Lyon city, France.

Rodents are recognized as the main reservoirs of Leptospira spp. Rats, in particular, serve as hosts for the widely predominant Leptospira interrogans serovar Icterohaemorrhagiae, found worldwide. Several studies have shown the importance of other reservoirs, such as mice or hedgehogs, which harbor other leptospires' serovars. Nevertheless, our knowledge of circulating Leptospira spp. in reservoirs other than rats remains limited. In this context, we proposed an eco-health approach to assess the health hazard associated with leptospires in urban green spaces, where contacts between human/small mammals and domestic animals are likely. We studied the prevalence, the diversity of circulating strains, and epidemiology of pathogenic Leptospira species in small terrestrial mammal communities (rodents and shrews), between 2020-2022, in two parks in Lyon metropolis, France. Our study showed a significant carriage of Leptospira spp. in small terrestrial mammals in these parks and unveiled a global prevalence rate of 11.4%. Significant variations of prevalence were observed among the small mammal species (from 0 to 26.1%), with Rattus norvegicus exhibiting the highest infection levels (26.1%). We also observed strong spatio-temporal variations in Leptospira spp. circulation in its reservoirs. Prevalence seems to be higher in the peri-urban park and in autumn in 2021 and 2022. This is potentially due to differences in landscape, abiotic conditions and small mammal communities' composition. Our study suggests an important public health relevance of rats and in a lesser extent of other rodents (Apodemus spp., Clethrionomys glareolus and Mus musculus) as reservoirs of L. interrogans, with rodent species carrying specific serogroups/serovars. We also emphasize the potential hazard associated between the shrew Crocidura russula and L. kirschneri. Altogether, these results improve our knowledge about the prevalence of leptospirosis in an urban environment, which is an essential prerequisite for the implementation of prevention of associated risks.

Study on the relation of the characteristics of the capture sites with the Leptospira spp. occurrence in bats and rodents from Yucatan, Mexico.

This study aims to describe the natural Leptospira occurrence in small mammals from Yucatan, Mexico, and to explore the relation between the characteristics of the capture sites and the Leptospira occurrence. Bats and rodents were captured in five sites of Yucatan state, and from them, a kidney fragment was collected that was used in the genomic DNA extraction. Leptospira DNA was identified by PCR targeting the 16S-rRNA and LipL32 genes. Additionally, a bioinformatic analysis was carried out to know the Leptospira species and was corroborated with a phylogenetic tree. The assemblage of small mammals was compound of 82 (51.2 %) bats and 78 (48.8 %) rodents. A global frequency (bats plus rodents) of Leptospira occurrence of 21.2 % (34/160) was observed; in bats, it was 21.9 % (18/82), and in rodents, 20.5 % (16/78). The phylogenetic trees based on LipL32 gene showed that the recovered sequences most closely resemble the species L. borgpetersenii and L. noguchii. The ordination of the capture sites with tropical deciduous forests as original vegetation is more related to the abundance of Leptospira-infected rodents. The ordination of the capture sites with tropical sub-deciduous forests as original vegetation is more related to the diversity of Leptospira-infected bat species. The canonical ordering of the capture sites is by the original vegetation type and the diversity and abundance of Leptospira-infected bat and rodent species.