CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

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.

Leptospira spp. and Rickettsia spp. as pathogens with zoonotic potential causing acute undifferentiated febrile illness in a central-eastern region of Peru.

OBJETIVE: this study was to determine the relationship between acute febrile illness and bacterial pathogens with zoonotic potential that cause emerging and re-emerging diseases in a central-eastern region of Peru. RESULTS: Out of the 279 samples analyzed, 23 (8.2%) tested positive for infection by Rickettsia spp., while a total of 15 (5.4%) tested positive for Leptospira spp. Women had a higher frequency of infection by Rickettsia spp., with 13 cases (53.3%), while men had a higher frequency of infection by Leptospira spp., with 10 cases (66.7%). The most frequently reported general symptom was headache, with 100.0% (n = 23) of patients with Rickettsia (+) and 86.7% (n = 13) of patients with Leptospira (+) experiencing it. Arthralgia was the second most frequent symptom, reported by 95.6% (n = 22) and 60% (n = 9) of patients with Rickettsia (+) and Leptospira (+), respectively. Myalgia was reported by 91.3% (n = 21) and 66.7% (n = 10) of patients with Rickettsia (+) and Leptospira (+), respectively. Retroocular pain, low back pain, and skin rash were also present, but less frequently. Among the positives, no manifestation of bleeding was recorded, although only one positive case for Leptospira spp. presented a decrease in the number of platelets.

Leptospirosis: predictores de mala evolución clínica en pacientes hospitalizados, 25 años de experiencia / Leptospirosis: predictors of poor outcomes in hospitalized patients, 25 years of experience

Introducción: La leptospirosis es una zoonosis que cons-tituye un problema emergente de salud pública. La insufi-ciencia renal, plaquetopenia y compromiso respiratorio se describen como predictores de mortalidad.Objetivos: Describir características clínicas, radiológicas y de laboratorio de individuos hospitalizados por leptos-pirosis y evaluar los predictores de mala evolución clínica (MEC).Materiales y métodos: Estudio de cohorte de inclusión ambispectiva de pacientes con leptospirosis internados en un hospital de la ciudad de Santa Fe entre 1997 y 2022. Se definió MEC como la admisión a Unidad de Cuidados Intensivos (UCI), requerimiento de asistencia respiratoria mecánica (ARM) y/o muerte. Se utilizaron las pruebas de Chi2, test T de Student o la U de Mann-Whitney, según co-rrespondiera. Se construyó una regresión logística binaria con las variables con p<0,05.Resultados: 101 pacientes, 87,1% (n=88) hombres, media-na de edad de 29 (RIC 20-44) años. La fiebre fue el síntoma más frecuente [83,2% (n=84)], seguido del compromiso di-gestivo [62,4% (n=63)]. Las alteraciones de laboratorio más frecuentes fueron: eritrosedimentación elevada [91,9% (n=79)] y leucocitosis [61% (n=61)]. Se observó MEC en el 25,7% (n=26). El 25,7% (n=26) fue admitido en UCI, el 13,9% (n=14) requirió ARM y el 5% (n=5) falleció. La presencia de plaquetopenia (OR=13,3, IC95% 2-80), las alteraciones en la radiografía de tórax (OR=33,5, IC95% 5-225) y la ausencia de cefalea (OR=6,8, IC95% 1-32) fueron predictores inde-pendientes de MEC.Conclusiones: En concordancia con la bibliografía, la afec-tación pulmonar y plaquetopenia son factores de riesgo para la mala evolución clínica. En nuestra serie, la cefalea constituyó un síntoma protector

Introduction: Leptospirosis is an emerging zoonotic di-sease that poses a public health problem. Renal failu-re, thrombocytopenia, and respiratory involvement have been described as predictors of mortality.Objectives: To describe the clinical, radiological, and la-boratory characteristics of hospitalized individuals with leptospirosis and evaluate predictors of poor clinical outcomes (PCO).Materials and methods: A prospective cohort study was conducted including patients with leptospirosis admit-ted to a hospital in the city of Santa Fe between 1997 and 2022. PCO was defined as admission to the Intensive Care Unit (ICU), requirement for mechanical respiratory assistance (MRA), and/or death. The chi-square test, Student>s t-test, or Mann-Whitney U test were used as appropriate. A binary logistic regression was performed with variables having p<0.05.Results: Out of the 101 patients included, 87.1% (n=88) were male, with a median age of 29 (IQR 20-44) years. Fever was the most common symptom [83.2% (n=84)], followed by digestive involvement [62.4% (n=63)]. The most frequent laboratory abnormalities were elevated erythrocyte sedimentation rate [91.9% (n=79)] and leuko-cytosis [61% (n=61)]. PCO was observed in 25.7% (n=26) of patients, with 25.7% (n=26) admitted to the ICU, 13.9% (n=14) requiring MRA, and 5% (n=5) resulting in death. The presence of thrombocytopenia (OR=13.3, 95% CI 2-80), abnormalities in chest X-rays (OR=33.5, 95% CI 5-225), and absence of headache (OR=6.8, 95% CI 1-32) were predictors of PCO. Conclusions: Consistent with the literature, pulmonary involvement and thrombocytopenia are independent risk factors for poor clinical outcomes. In our series, the pre-sence of headache was a protective symptom

Blocking pathogenic Leptospira invasion with aptamer molecules targeting outer membrane LipL32 protein.

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).

Molecular detection of leptosipira in synanthropic and wild rodents from Villavicencio municipality, Colombia / Detección molecular de leptospiras en roedores del municipio de Villavicencio, Colombia

Introduction: Rodents are potential transmitters of Leptospira spp. In the municipality of Villavicencio, Colombia, leptospirosis is a disease that, although notifiable, is still underreported. In this region, rodent species that can host pathogenic leptospira remain unknown. Objective: To detect the presence of Leptospira spp. through molecular analysis in rodents (Rodentia) from peri-urban and rural areas belonging to the municipality of Villavicencio in Colombia. Methods: Peri-urban and rural areas of the townships belonging to Villavicencio municipality were selected for sampling. These areas presented similar ecological conditions: they were near water bodies and peridomiciliary areas, and some of them included fields of agricultural crops. Rodents´ kidneys were removed and frozen in liquid nitrogen. DNA was extracted using a commercial kit and subsequently amplified through conventional polymerase chain reaction. Results: The rodent species collected were: Rattus rattus, Mus musculus, Zygodontomys brevicauda, Oligoryzomys sp, Hylaeamys (formerly Oryzomys) and Proechimys cf. oconnelli. Leptospira DNA was amplified in six rodents and the purified amplicons were sent to Macrogen Inc. (Seoul, Korea) for sequencing. The alignment analysis of the sequenced products demonstrated 98.64 percent of coverage and identity with Leptospira interrogans. Conclusions: This is the first study carried out on wild and synanthropic rodents in the municipality of Villavicencio. The incidence of leptospirosis raises the alarm due to the important role of these small mammals in the transmission of this zoonosis, which is considered the second cause, after dengue, of undifferentiated febrile illness in Villavicencio(AU)

Introducción: Los roedores son potenciales transmisores de Leptospira spp. En el municipio de Villavicencio, Colombia, la leptospirosis es una enfermedad que, aunque debe notificarse obligatoriamente, sigue subreportada. En esta región, algunas especies de roedores pueden ser reservorios de leptospiras patógenas, situación que se desconoce. Objetivo: Detectar la presencia de Leptospira spp. a través del análisis molecular en roedores (Rodentia) de áreas periurbanas y rurales del municipio de Villavicencio, Colombia. Métodos: Para el trampeo se seleccionaron áreas periurbanas y rurales de las veredas pertenecientes al municipio de Villavicencio. Las áreas escogidas presentaban condiciones ecológicas similares: cerca de cuerpos de agua y áreas peridomiciliarias; algunas de ellas localizadas en campos de cultivos de la agricultura. Se extirparon los riñones de los roedores y se conservaron en nitrógeno líquido. Se extrajo el ADN usando un estuche comercial y posteriormente se amplificó mediante reacción en cadena de la polimerasa convencional. Resultados: Las especies de roedores colectadas fueron: Rattus rattus, Mus musculus, Zygodontomys brevicauda, Oligoryzomys sp., Hylaeamys (ahora Oryzomys) y Proechimys oconnelli. El ADN de leptospira se amplificó en seis roedores y los amplicones purificados se enviaron a Macrogen Inc. (Seoul, Korea) para secuenciación. El análisis de alineamiento de los productos secuenciados demostró un 98,64 por ciento de cobertura e identidad con Leptospira interrogans. Conclusiones: Este es el primer estudio llevado a cabo en roedores silvestres y sinantrópicos en el municipio de Villavicencio. La incidencia de la leptospirosis genera una alarma con respecto a la importancia del papel de esos pequeños mamíferos en la transmisión de esta zoonosis, la cual es la segunda causa de los síndromes febriles indiferenciados en Villavicencio, después del dengue(AU)

Motility of the Zoonotic Spirochete Leptospira: Insight into Association with Pathogenicity.

If a bacterium has motility, it will use the ability to survive and thrive. For many pathogenic species, their motilities are a crucial virulence factor. The form of motility varies among the species. Some use flagella for swimming in liquid, and others use the cell-surface machinery to move over solid surfaces. Spirochetes are distinguished from other bacterial species by their helical or flat wave morphology and periplasmic flagella (PFs). It is believed that the rotation of PFs beneath the outer membrane causes transformation or rolling of the cell body, propelling the spirochetes. Interestingly, some spirochetal species exhibit motility both in liquid and over surfaces, but it is not fully unveiled how the spirochete pathogenicity involves such amphibious motility. This review focuses on the causative agent of zoonosis leptospirosis and discusses the significance of their motility in liquid and on surfaces, called crawling, as a virulence factor.

Galleria mellonella infection model to evaluate pathogenic and nonpathogenic Leptospira strains.

The Galleria mellonella larvae infection model is emerging as a valuable tool for studying various characteristics of infectious agents and host-pathogen interaction. This system has been widely recognized as a high throughput, ethical, and cost-effective invertebrate infection model to study the virulence and pathogenesis of various bacterial pathogens. In this study, we compared the effect of Leptospira infection in G. mellonella larvae infected with Leptospira interrogans serovar Copenhageni (pathogenic) or Leptospira biflexa serovar Patoc (saprophytic) strains. We observed significant pathologic changes such as decreased activity, complete melanization, and lower survival rate in the G. mellonella larvae infected with a pathogenic strain L. interrogans serovar Copenhageni compared to those infected with a nonpathogenic strain L. biflexa serovar Patoc. Our study demonstrates the feasibility and the potential of using G. mellonella larvae as an alternative model to study virulence mechanisms and pathogenesis of Leptospira strains. Once optimized, the G. mellonella infection model can be a potential substitute for hamsters to explore various host and pathogen-related mechanistic events in Leptospira infection.

Laboratory diagnostic, epidemiological, and clinical characteristics of human leptospirosis in Okinawa Prefecture, Japan, 2003-2020.

BACKGROUND: Leptospirosis is considered an endemic disease among agricultural workers in Okinawa Prefecture, which is the southernmost part of Japan and has a subtropical climate, but data on the current status and trend of this disease are scarce. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective study of clinically suspected leptospirosis patients whose sample and information were sent to the Okinawa Prefectural Institute of Health and Environment from November 2003 to December 2020. Laboratory diagnosis was established using culture, nested polymerase chain reaction (PCR), and/or microscopic agglutination test (MAT) with blood, cerebrospinal fluid, and/or urine samples. Statistical analyses were performed to compare the epidemiological information, clinical features, and sensitivities of diagnostic methods among laboratory-confirmed cases. Serogroups and the species of Leptospira isolates were determined by MAT using 13 antisera and flaB sequencing. A total of 531 clinically suspected patients were recruited, among whom 246 (46.3%) were laboratory confirmed to have leptospirosis. Among the confirmed cases, patients aged 20-29 years (22.4%) and male patients (85.7%) were the most common. The most common estimated sources of infection were recreation (44.5%) and labor (27.8%) in rivers. Approximately half of the isolates were of the L. interrogans serogroup Hebdomadis. The main clinical symptoms were fever (97.1%), myalgia (56.3%), and conjunctival hyperemia (52.2%). Headache occurred significantly more often in patients with Hebdomadis serogroup infections than those with other serogroup infections. The sensitivities of culture and PCR exceeded 65% during the first 6 days, while the sensitivity of MAT surpassed that of culture and PCR in the second week after onset. PCR using blood samples was a preferable method for the early diagnosis of leptospirosis. CONCLUSIONS/SIGNIFICANCE: The results of this study will support clinicians in the diagnosis and treatment of undifferentiated febrile patients in Okinawa Prefecture as well as patients returning from Okinawa Prefecture.

The oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

Crosstalk between E-Cadherin/ß-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis.

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor ß-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/ß-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay.

Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.

Pathogenesis of Pulmonary Hemorrhagic Syndrome in Human Leptospirosis.

Based on a previous study and by incorporating new knowledge, the goal of our study was to understand more fully the pathogenesis of hemorrhagic pneumonia of severe human leptospirosis, highlighting the onset of capillary lesions by Leptospira itself and/or its antigenic/toxic products acting on the endothelium and binding to cadherins. Both events lead to loss of endothelial integrity, alter permeability, cause rupture, and open intercellular junctions, contributing to the hemorrhagic phenomena associated with severe leptospirosis.

Next-Generation Sequencing Analysis of Pathogenic Leptospira: A Way Forward for Understanding Infectious Disease Dynamics in Low/Middle-Income, Disease-Endemic Settings.

In the current genomic era, knowledge of diversity of Leptospira, the spirochetal agents of leptospirosis, is changing rapidly. Next-generation sequencing has decreased in price and increased in scale, with the potential to democratize large-scale analysis of pathogens in resource-limited, low/middle-income (LMIC) regions. Consequently, the molecular classification of Leptospira, a pathogen disproportionately affecting LMIC countries, has changed dramatically over the last decade. Leptospira classification and molecular understandings of pathogen diversity have rapidly evolved, now most precisely based on core genome analysis supplemented by new insights provided by culture-independent methods directly using body fluids such as blood and urine. In places where leptospirosis disease burden is highest, genomic technologies have not been available, and serology-based methods remain the mainstay of leptospiral classification. Understanding the epidemiology, pathogenesis, and ultimately new approaches to treating and preventing leptospirosis requires detailed knowledge of regionally circulating Leptospira in highly endemic settings. Next-generation sequencing-based, culture-independent typing overcomes the limitation of culture isolation of Leptospira from clinical samples, with promise of providing public health-actionable information applicable to leptospirosis-endemic LMIC settings.

Epidemiology of human leptospirosis in urban and rural areas of Brazil, 2000-2015.

BACKGROUND: Leptospirosis is one of the most widespread zoonosis in the world and Brazil has the highest number of cases in Latin America. Transmission occurs mainly through exposure to water and soil contaminated by the urine of infected animals. The goals of this study are to describe the geographic distribution, demographic characteristics and exposure factors of urban and rural cases of leptospirosis, and identify spatial clusters in urban and rural areas of Brazil. METHODS/RESULTS: A retrospective epidemiological study was carried out using 16 years (2000-2015) of surveillance data from the Brazilian Ministry of Health. Cases were described by age, sex and race, and exposure factors were characterized in urban and rural areas. A spatial autocorrelation analysis was conducted using local Moran's I to identify urban and rural clusters of disease. On average 3,810 leptospirosis cases were reported annually with higher numbers in urban areas. National urban and rural incidence rates were the same (1.9 cases/100,000 population), however, regional differences were observed. Urban incidence rates were higher in the North and Northeast regions, while rural incidence rates were higher in the Southeast and South. The main exposure factor reported in urban and rural areas was exposure to places with signs of rodents, followed by flood in urban areas and agriculture and animal farming in rural areas. Clusters of leptospirosis were identified in densely populated urban areas of the North, Southeast and South regions, while rural clusters were concentrated in of the Southern region with large agriculture and animal farming practices. CONCLUSIONS: This study highlights that leptospirosis is an important public health problem in both urban and rural areas of Brazil. The results provide decision-makers with detailed information about where disease incidence is high and can be used in the development of prevention and control strategies for priority areas and risk groups.

Molecular Detection of Pathogens in Negative Blood Cultures in the Lao People's Democratic Republic.

Bloodstream infections cause substantial morbidity and mortality. However, despite clinical suspicion of such infections, blood cultures are often negative. We investigated blood cultures that were negative after 5 days of incubation for the presence of bacterial pathogens using specific (Rickettsia spp. and Leptospira spp.) and a broad-range 16S rRNA PCR. From 190 samples, 53 (27.9%) were positive for bacterial DNA. There was also a high background incidence of dengue (90/112 patient serum positive, 80.4%). Twelve samples (6.3%) were positive for Rickettsia spp., including two Rickettsia typhi. The 16S rRNA PCR gave 41 positives; Escherichia coli and Klebsiella pneumoniae were identified in 11 and eight samples, respectively, and one Leptospira species was detected. Molecular investigation of negative blood cultures can identify potential pathogens that will otherwise be missed by routine culture. Patient management would have been influenced in all 53 patients for whom a bacterial organism was identified, and 2.3-6.1% of patients would likely have had an altered final outcome. These findings warrant further study, particularly to determine the cost-benefit for routine use, ways of implementation, and timing of PCR for organisms such as Rickettsia and Leptospira, which are important pathogens in rural Asia.

Knowledge, Attitude, and Practices regarding Leptospirosis among Visitors to a Recreational Forest in Malaysia.

Leptospirosis is a zoonotic disease and a worldwide public health problem that affects mainly high-risk groups. Characterizing knowledge, attitude, and practice (KAP) among high-risk groups is important to develop appropriate prevention programs. Here, we performed a cross-sectional study among 300 visitors of a recreational forest in Malaysia to examine leptospirosis KAP and demographics. These variables were integrated to create knowledge and practice scores for each respondent. All respondents had heard about leptospirosis, and 87% of them correctly identified it as a disease. The majority of respondents had high knowledge (63%), positive attitude, and good practice (68%) toward prevention of the disease. However, there were gaps in knowledge, with 78% of the respondents indicating eating without washing hands as the major cause of leptospirosis transmission. Our final model identified that higher knowledge score was associated with higher practice score. Our results indicate that it is important to increase knowledge, especially on transmission routes of leptospirosis, among visitors in recreational areas. Moreover, more attention needs to be paid to promote good practice habits among visitors, targeting those at higher risk of being infected by leptospirosis to prevent potential outbreaks in the recreational areas.

High exposure to pathogenic leptospires by the population residing in dairy farms in Hidalgo, Mexico.

Leptospirosis is a neglected zoonotic disease of unknown magnitude that has been overlooked and underreported, influenced by complex interactions established among humans, animals, and the environment; certain occupations, such as working with livestock, have an increased risk of exposure. We conducted a cross trans-sectional study in 374 serum samples obtained from workers and residents of dairy farms in the Tizayuca Basin, Hidalgo, Mexico, to determine the prevalence of anti-Leptospira antibody and the risk factors associated to this type of environment. The determination of anti-Leptospira antibodies was obtained by microscopic agglutination test. Seropositivity was defined from titles > 1:100. Seropositivity of anti-Leptospira antibodies among the population was 46.8% (176/374) (95% Cl 41.9-52.1). Thirty-nine percent (146/74) of the analyzed serum reacted to the Hardjo serovar (Sejröe serogroup). Eighty-eight percent (8/9) slaughterhouse workers tested were seropositive. Those who belonged to an ethnic group had OR 1.78 (IC 1.02-3.11, P = 0.041). Seropositivity was associated with having a secondary school level or lower, with OR 1.79 (IC 0.97-3.29, P = 0.058). Exposure to Leptospira in a dairy production farm is a risk factor for humans. Our findings can contribute to strengthening the intervention of the Public Health System to prevent this zoonosis that prevails in dairy farm environments.

Deciphering the Role of Leptospira Surface Protein LigA in Modulating the Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate (activate or evade) the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in modulating the host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.