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Previous in vitro works focusing on virulence determinants of the spirochete Leptospira implicated metalloproteinases as putative contributing factors to the pathogenicity of these bacteria. Those proteins have the capacity to degrade extracellular matrix components (ECM) and proteins of host's innate immunity, notably effectors of the complement system. In this study, we gained further knowledge on the role of leptolysin, one of the leptospiral-secreted metalloproteinases, previously described as having a broad substrate specificity. We demonstrated that a proportion of human patients with mild leptospirosis evaluated in the current study produced antibodies that recognize leptolysin, thus indicating that the protease is expressed during host infection. Using recombinant protein and a knockout mutant strain, Manilae leptolysin - , we determined that leptolysin contributes to Leptospira interrogans serum resistance in vitro, likely by proteolysis of complement molecules of the alternative, the classical, the lectin, and the terminal pathways. Furthermore, in a hamster model of infection, the mutant strain retained virulence; however, infected animals had lower bacterial loads in their kidneys. Further studies are necessary to better understand the role and potential redundancy of metalloproteinases on the pathogenicity of this important neglected disease., (© 2024 John Wiley & Sons Ltd.)

Background: Leptospirosis is a zoonosis caused by pathogenic species of bacteria belonging to the genus Leptospira. Most studies infer the epidemiological patterns of a single serogroup or aggregate all serogroups to estimate overall seropositivity, thus not exploring the risks of exposure to distinct serogroups. The present study aims to delineate the demographic, socioeconomic and environmental factors associated with seropositivity of Leptospira serogroup Icterohaemorraghiae and serogroup Cynopteri in an urban high transmission setting for leptospirosis in Brazil., Methods/principal Findings: We performed a cross-sectional serological study in five informal urban communities in the city of Salvador, Brazil. During the years 2018, 2020 2021, we recruited 2.808 residents and collected blood samples for serological analysis using microagglutination assays. We used a fixed-effect multinomial logistic regression model to identify risk factors associated with seropositivity for each serogroup. Seropositivity to Cynopteri increased with each year of age (OR 1.03; 95% CI 1.01-1.06) and was higher in those living in houses with unplastered walls (exposed brick) (OR 1.68; 95% CI 1.09-2.59) and where cats were present near the household (OR 2.00; 95% CI 1.03-3.88). Seropositivity to Icterohaemorrhagiae also increased with each year of age (OR 1.02; 95% CI 1.01-1.03) and was higher in males (OR 1.51; 95% CI 1.09-2.10), in those with work-related exposures (OR 1.71; 95% CI 1.10-2.66) or who had contact with sewage (OR 1.42; 95% CI 1.00-2.03). Spatial analysis showed differences in distribution of seropositivity to serogroups Icterohaemorrhagiae and Cynopteri within the five districts where study communities were situated., Conclusions/significance: Our data suggest distinct epidemiological patterns associated with the Icterohaemorrhagiae and Cynopteri serogroups in the urban environment at high risk for leptospirosis and with differences in spatial niches. We emphasize the need for studies that accurately identify the different pathogenic serogroups that circulate and infect residents of low-income areas., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: The objectives of this study were to determine the seroprevalence and risk factors of leptospirosis among slaughterhouse workers in Burkina Faso., Methods: We performed a cross-sectional survey of slaughterhouse workers from Ouagadougou and Bobo Dioulasso between March and April 2021. Blood was collected by venipuncture and serum samples were tested using enzyme-linked immunosorbent assay and microscopic agglutination test. Questionnaires were used to collect information from these workers on sociodemographic characteristics, work activities, knowledge of zoonosis, and risky behaviors., Results: Of the 172 subjects investigated, 28 (16.28%) were found seropositive for leptospirosis using enzyme-linked immunosorbent assay or microscopic agglutination test. The main Leptospira infecting serogroup were Mini, Autumnalis, Canicola, Copenhageni, L. mayottensis (ND), Icterohaemorrhagiae, Pyrogenes/Tarassovi (cross reaction), Panama, and Ballum. Risk factors according to multivariate analysis, included residence ( P  = 0.02), working at the bleeding station ( P  = 0.03), contact with feces and urine ( P  = 0.04), and the practice of agriculture outside the slaughterhouse ( P  = 0.05)., Conclusion: These findings indicate that a significant proportion of slaughterhouse workers are being exposed to pathogenic Leptospira . Public-health interventions against leptospirosis will need to target this occupational group. Proper personal protective equipment and information about the disease should be disseminated among slaughterhouses., Competing Interests: The authors have no competing interests to declare., (© 2023 The Authors.)

Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Over the last century, outbreaks and pandemics have occurred with disturbing regularity, necessitating advance preparation and large-scale, coordinated response. Here, we developed a machine learning predictive model of disease severity and length of hospitalization for COVID-19, which can be utilized as a platform for future unknown viral outbreaks. We combined untargeted metabolomics on plasma data obtained from COVID-19 patients (n = 111) during hospitalization and healthy controls (n = 342), clinical and comorbidity data (n = 508) to build this patient triage platform, which consists of three parts: (i) the clinical decision tree, which amongst other biomarkers showed that patients with increased eosinophils have worse disease prognosis and can serve as a new potential biomarker with high accuracy (AUC = 0.974), (ii) the estimation of patient hospitalization length with ± 5 days error (R 2  = 0.9765) and (iii) the prediction of the disease severity and the need of patient transfer to the intensive care unit. We report a significant decrease in serotonin levels in patients who needed positive airway pressure oxygen and/or were intubated. Furthermore, 5-hydroxy tryptophan, allantoin, and glucuronic acid metabolites were increased in COVID-19 patients and collectively they can serve as biomarkers to predict disease progression. The ability to quickly identify which patients will develop life-threatening illness would allow the efficient allocation of medical resources and implementation of the most effective medical interventions. We would advocate that the same approach could be utilized in future viral outbreaks to help hospitals triage patients more effectively and improve patient outcomes while optimizing healthcare resources., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leptospira was investigated in kidneys ( n = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate Leptospira , followed by the LipL32 qPCR to detect Leptospira DNA. The SecY gene region was amplified, sequenced, and analyzed for LipL32 qPCR-positive samples or Leptospira isolates. The overall frequency of isolation of Leptospira spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively ( p > 0.05). However, with LipL32 qPCR, the overall frequency of Leptospira DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively ( p = 0.03). Based on 22 SecY sequences, the phylogenetic tree identified the L. interrogans cluster with serovar Icterohaemorrhagiae and the L. borgpetersenii cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of Leptospira spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which L. borgpetersenii serovar Hardjo bovis is not part. Our data show that pathogenic L. interrogans and L. borgpetersenii are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.

The objective of this study was to analyze the eco-epidemiological aspects of Leptospira seroprevalence and seroincidence and its associated factors in two municipalities of northwest Colombia. A prospective study was performed in rural areas of Urabá, Antioquia, Colombia. The study enrolled 597 people between November 2015 and January 2016, of which 274 were followed up 1 year later. Serologic testing was performed by a microscopic agglutination. The outcomes were seroprevalent and seroincident cases, and the primary exposure was an outdoor occupation. A binary and mixed-effect multinomial logistic regression model was used to estimate factors associated with seroprevalent or seroincident cases of Leptospira infection. The overall Leptospira seroprevalence was 27.81% (95% confidence interval: 23.62-32.49), and the overall cumulative seroincidence for Leptospira was 14.60% (95% confidence interval: 10.33-20.23). Multivariable analysis showed that factors associated with L. interrogans serogroups seropositivity were outdoor occupation, male gender, older age, the presence of dirt floor in the household, and the presence of piglets and opossums. It also showed that factors associated with other Leptospira species serogroups were the presence of pit latrines and of turkeys. In addition, the multivariable model of seroincident cases of L. interrogans serogroups evidenced outdoor occupations, the presence of rats, and corn cultivation as risk factors. Likewise, the multivariable model for seroincident cases of other Leptospira species showed that the presence of hunting canines and cassava cultivation were risk factors. We found specific factors associated with the transmission of Leptospira serogroups contribute to the understanding of the epidemiology of Leptospira infection in rural areas of Urabá, Colombia.

Background: Zoonotic spillover from animal reservoirs is responsible for a significant global public health burden, but the processes that promote spillover events are poorly understood in complex urban settings. Endemic transmission of Leptospira , the agent of leptospirosis, in marginalised urban communities occurs through human exposure to an environment contaminated by bacteria shed in the urine of the rat reservoir. However, it is unclear to what extent transmission is driven by variation in the distribution of rats or by the dispersal of bacteria in rainwater runoff and overflow from open sewer systems., Methods: We conducted an eco-epidemiological study in a high-risk community in Salvador, Brazil, by prospectively following a cohort of 1401 residents to ascertain serological evidence for leptospiral infections. A concurrent rat ecology study was used to collect information on the fine-scale spatial distribution of 'rattiness', our proxy for rat abundance and exposure of interest. We developed and applied a novel geostatistical framework for joint spatial modelling of multiple indices of disease reservoir abundance and human infection risk., Results: The estimated infection rate was 51.4 (95%CI 40.4, 64.2) infections per 1000 follow-up events. Infection risk increased with age until 30 years of age and was associated with male gender. Rattiness was positively associated with infection risk for residents across the entire study area, but this effect was stronger in higher elevation areas (OR 3.27 95% CI 1.68, 19.07) than in lower elevation areas (OR 1.14 95% CI 1.05, 1.53)., Conclusions: These findings suggest that, while frequent flooding events may disperse bacteria in regions of low elevation, environmental risk in higher elevation areas is more localised and directly driven by the distribution of local rat populations. The modelling framework developed may have broad applications in delineating complex animal-environment-human interactions during zoonotic spillover and identifying opportunities for public health intervention., Funding: This work was supported by the Oswaldo Cruz Foundation and Secretariat of Health Surveillance, Brazilian Ministry of Health, the National Institutes of Health of the United States (grant numbers F31 AI114245, R01 AI052473, U01 AI088752, R01 TW009504 and R25 TW009338); the Wellcome Trust (102330/Z/13/Z), and by the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB/JCB0020/2016). MTE was supported by a Medical Research UK doctorate studentship. FBS participated in this study under a FAPESB doctorate scholarship., Competing Interests: ME, FS, TC, NN, Dd, JC, GS, HK, EW, KH, JH, JC, MR, MB, PD, EG, FC No competing interests declared, AK has received funding from Serimmune and Zoetis for work related to leptospirosis. AIK also received payment and honoraria from Reckit Global Health Institute for participating in a non-profit panel. AIK received travel support from World Health Organisation and Brazilian Ministry of Health. AIK is listed as co-inventor on an issued patent (US 7,718,183 B2) and pending patent (US 61/951,732) related to leptospirosis vaccines. AIK is also on the following boards: Board of Directors, American Society of Tropical Medicine and Hygiene; Executive Board Member (2009-present), International Leptospirosis Society; Member, Inaugural Expert Panel, Reckitt Global Hygiene Institute; Steering Committee Member, Global Leptospirosis Environmental Action Network (GLEAN), WHO. The author has no other competing interests to declare, (© 2022, Eyre et al.)

Background: The structural environment of urban slums, including physical, demographic, and socioeconomic attributes, renders inhabitants more vulnerable to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Yet, little is known about the specific determinants that contribute to high transmission within these communities. We therefore aimed to investigate SARS-CoV-2 seroprevalence in an urban slum in Brazil., Methods and Findings: We performed a cross-sectional serosurvey of an established cohort of 2,041 urban slum residents from the city of Salvador, Brazil between November 2020 and February 2021, following the first Coronavirus Disease 2019 (COVID-19) pandemic wave in the country and during the onset of the second wave. The median age in this population was 29 years (interquartile range [IQR] 16 to 44); most participants reported their ethnicity as Black (51.5%) or Brown (41.7%), and 58.5% were female. The median size of participating households was 3 (IQR 2 to 4), with a median daily per capita income of 2.32 (IQR 0.33-5.15) US Dollars. The main outcome measure was presence of IgG against the SARS-CoV-2 spike protein. We implemented multilevel models with random intercepts for each household to estimate seroprevalence and associated risk factors, adjusting for the sensitivity and specificity of the assay, and the age and gender distribution of our study population. We identified high seroprevalence (47.9%, 95% confidence interval [CI] 44.2% to 52.1%), particularly among female residents (50.3% [95% CI 46.3% to 54.8%] versus 44.6% [95% CI 40.1% to 49.4%] among male residents, p < 0.01) and among children (54.4% [95% CI 49.6% to 59.3%] versus 45.4% [95% CI 41.5% to 49.7%] among adults, p < 0.01). Adults residing in households with children were more likely to be seropositive (48.6% [95% CI 44.8% to 52.3%] versus 40.7% [95% CI 37.2% to 44.3%], p < 0.01). Women who were unemployed and living below the poverty threshold (daily per capita household income <$1.25) were more likely to be seropositive compared to men with the same employment and income status (53.9% [95% CI 47.0% to 60.6%] versus 32.9% [95% CI 23.2% to 44.3%], p < 0.01). Participation in the study was voluntary, which may limit the generalizability of our findings., Conclusions: Prior to the peak of the second wave of the COVID-19 pandemic, cumulative incidence as assessed by serology approached 50% in a Brazilian urban slum population. In contrast to observations from industrialized countries, SARS-CoV-2 incidence was highest among children, as well as women living in extreme poverty. These findings emphasize the need for targeted interventions that provide safe environments for children and mitigate the structural risks posed by crowding and poverty for the most vulnerable residents of urban slum communities., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.I.K serves as an expert panel member for Reckitt Global Hygiene Institute, scientific advisory board member for Revelar Biotherapeutics and a consultant for Tata Medical and Diagnostics and Regeneron Pharmaceuticals, and has received grants from Merck, Regeneron Pharmaceuticals and Tata Medical and Diagnostics for research related to COVID-19, all of which are outside the scope of the submitted work. D.A.T.C. has received a grant from Merck for research unrelated to COVID-19, outside of the scope of this work. M.O.F has past stock ownership in GlaxoSmithKline. Other authors declare no conflict of interest.

Introduction: Leptospirosis is a zoonosis with a worldwide spread that leads to clinical manifestations ranging from asymptomatic infection to a life-threatening disease. The immune response is predominantly humoral mediated limited to the infecting serovar. Individuals living in an area endemic for leptospirosis are often exposed to an environment contaminated with leptospires and there is a paucity of information on naturally acquired immunity. In the present study, we evaluated the kinetics of agglutinating antibodies in individuals from an endemic area for leptospirosis in Salvador, Brazil comparing two different intersample collection times., Methods: Between 2017-2018, we carried out a biannual prospective cohort with 2,086 individuals living in an endemic area for leptospirosis in Salvador, Brazil. To compare agglutinating antibody kinetics using microscopic agglutination test (MAT) with different collection times, a subcohort of 72 individuals with quarterly follow-up was carried out in parallel., Results: The results revealed that using a shorter time for intersample collection led to the detection of a higher number of infections and reinfection events. Furthermore, we observed a higher rate of titer decay indicating partial and short protection. However, there was no indication of major changes in risk factors for the disease., Conclusions: We evaluated antibody kinetics among residents of an endemic area for leptospirosis comparing two sample collection times. The constant exposure to the contaminated environment increases the risk for leptospirosis infection with reinfection events being more common than expected. This indicates that the burden of leptospirosis might be underestimated by serological surveys, and further studies are necessary to better characterize the humoral response after infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cruz, Nery, Sacramento, Victoriano, Montenegro, Santana, Costa, Ko, Reis and Wunder.)

Background: Leptospirosis is an important public health problem affecting vulnerable urban slum populations in developing country settings. However, the complex interaction of meteorological factors driving the temporal trends of leptospirosis remain incompletely understood., Methods and Findings: From March 1996-March 2010, we investigated the association between the weekly incidence of leptospirosis and meteorological anomalies in the city of Salvador, Brazil by using a dynamic generalized linear model that accounted for time lags, overall trend, and seasonal variation. Our model showed an increase of leptospirosis cases associated with higher than expected rainfall, lower than expected temperature and higher than expected humidity. There was a lag of one-to-two weeks between weekly values for significant meteorological variables and leptospirosis incidence. Independent of the season, a weekly cumulative rainfall anomaly of 20 mm increased the risk of leptospirosis by 12% compared to a week following the expected seasonal pattern. Finally, over the 14-year study period, the annual incidence of leptospirosis decreased significantly by a factor of 2.7 (8.3 versus 3.0 per 100,000 people), independently of variations in climate., Conclusions: Strategies to control leptospirosis should focus on avoiding contact with contaminated sources of Leptospira as well as on increasing awareness in the population and health professionals within the short time window after low-level or extreme high-level rainfall events. Increased leptospirosis incidence was restricted to one-to-two weeks after those events suggesting that infectious Leptospira survival may be limited to short time intervals., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2 remdesivir resistance mutations have been generated in vitro but have not been reported in patients receiving treatment with the antiviral agent. We present a case of an immunocompromised patient with acquired B-cell deficiency who developed an indolent, protracted course of SARS-CoV-2 infection. Remdesivir therapy alleviated symptoms and produced a transient virologic response, but her course was complicated by recrudescence of high-grade viral shedding. Whole genome sequencing identified a mutation, E802D, in the nsp12 RNA-dependent RNA polymerase, which was not present in pre-treatment specimens. In vitro experiments demonstrated that the mutation conferred a ~6-fold increase in remdesivir IC 50 but resulted in a fitness cost in the absence of remdesivir. Sustained clinical and virologic response was achieved after treatment with casirivimab-imdevimab. Although the fitness cost observed in vitro may limit the risk posed by E802D, this case illustrates the importance of monitoring for remdesivir resistance and the potential benefit of combinatorial therapies in immunocompromised patients with SARS-CoV-2 infection., (© 2022. The Author(s).)

In this study, we genotyped samples from environmental reservoirs (surface water and soil), colonized rat specimens, and cases of human severe leptospirosis from an endemic urban slum in Brazil, to determine the molecular epidemiology of pathogenic Leptospira and identify pathways of leptospirosis infection. We identified a well-established population of Leptospira interrogans serovar Copenhageni common to human leptospirosis cases, and animal and environmental reservoirs. This finding provides genetic evidence for a potential environmental spillover pathway for rat-borne leptospirosis through the environment in this urban community and highlights the importance of environmental and social interventions to reduce spillover infections., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Leptospirosis is an environmentally transmitted zoonotic disease caused by pathogenic Leptospira spp. that affects poor communities worldwide. In urban slums, leptospirosis is associated with deficient sanitary infrastructure. Yet, the role of sewerage in the reduction of the environmental contamination with pathogenic Leptospira has not been explored. Here, we conducted a survey of the pathogen in soils surrounding open and closed sewer sections in six urban slums in Brazil. We found that soils surrounding conventionally closed sewers (governmental interventions) were 3 times less likely to contain pathogenic Leptospira (inverse OR 3.44, 95% CI = 1.66-8.33; p < 0.001) and contained a 6 times lower load of the pathogen (0.82 log 10 units difference, p < 0.01) when compared to their open counterparts. However, no differences were observed in community-closed sewers (poor-quality closings performed by the slum dwellers). Human fecal markers (BacHum) were positively associated with pathogenic Leptospira even in closed sewers, and rat presence was not predictive of the presence of the pathogen in soils, suggesting that site-specific rodent control may not be sufficient to reduce the environmental contamination with Leptospira . Overall, our results indicate that sewerage expansion to urban slums may help reduce the environmental contamination with the pathogen and therefore reduce the risk of human leptospirosis.

Background: Leptospirosis is a zoonotic disease transmitted through the urine of wild and domestic animals, and is responsible for over 50,000 deaths each year. In East Africa, prevalence varies greatly, from as low as 7% in Kenya to 37% in Somalia. Transmission epidemiology also varies around the world, with research in Nicaragua showing that rodents are the most clinically important, while studies in Egypt and Chile suggest that dogs may play a more important role. There are no published studies of leptospirosis in Rwanda., Methods & Findings: We performed a cross-sectional survey of asymptomatic adults recruited from five occupational categories. Serum samples were tested using ELISA and Microscopic Agglutination Test (MAT). We found that 40.1% (151/377) of asymptomatic adults had been exposed to Leptospira spp. Almost 36.3% of positive subjects reported contact with rats (137/377) which represent 90.7% among positive leptospira serology compared with 48.2% of negative subjects (182/377) which represent 80.5% among negative leptospira serology (OR 2.37, CI 1.25-4.49) and 1.7 fold on prevalence ratio and 2.37 of odd ratio. Furthermore, being a crop farmer was significantly associated with leptospirosis (OR 2.06, CI 1.29-3.28). We identified 6 asymptomatic subjects (1.6%) who met criteria for acute infection., Conclusions: This study demonstrates a high prevalence of leptospiral antibodies infection among asymptomatic adults in rural Rwanda, particularly relative to neighboring countries. Although positive subjects were more likely to report rat contact, we found no independent association between rats and leptospirosis infection. Nonetheless, exposure was high among crop farmers, which is supportive of the hypothesis that rats together with domestic livestock might contribute to the transmission. Further studies are needed to understand infecting Leptospira servers and elucidate the transmission epidemiology in Rwanda and identify means of host transmitters., Competing Interests: The authors have declared that no competing interests exist.

As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19., (© 2021. The Author(s).)

Objective: To describe the neurological and neurodevelopmental outcomes of children with Congenital Zika Syndrome (CZS) associated microcephaly beyond 2 years of age., Method: We followed children with CZS-associated microcephaly in an outpatient clinic in Salvador, Brazil. Neurological and neurodevelopmental assessments were performed using the Hammersmith Infant Neurological Examination (HINE) and Bayley Scales of Infant and Toddler Neurodevelopment (Bayley-III) respectively., Results: Of the 42 children included, 19 were male (45.2%); median (interquartile range) age at neurological evaluation was 28 (25-32) months, and 36 (85.7%) had severe microcephaly. HINE and Bayley-III results were completed for 35/42 (83.3%) and 33/42 (78.5%) children respectively. Bayley-III identified a severe developmental delay in 32/33 (97.0%) children while 1/33 (3.0%) had only a mild delay. In the multivariable analysis, we found that Bayley-III and HINE scores were correlated. Better HINE scores were associated with higher Bayley-III cognitive raw scores (β = 0.29; CI 95% = 0.02-0.57) and motor raw scores (β = 0.43; CI 95% = 0.04-0.82) after adjusting for head circumference, prematurity, and age at neurodevelopmental evaluation. Furthermore, we found that greater head circumference at follow up was associated with higher cognitive (β = 1.27; CI 95% = 0.01-2.53) and motor raw scores (β = 2.03; CI 95% = 0.25-3.81)., Conclusion: Children with CZS-associated microcephaly demonstrate severe neurodevelopmental delays and slower growth rates than their peers over time. Still, they have remarkably heterogeneous neurodevelopmental profiles according to neurological exam scores which correlate with their long-term outcomes. We found that HINE scores effectively captured the heterogeneity of neurological capabilities among these children and could be predictive of cognitive and motor development progress., Competing Interests: The authors have declared that no competing interests exist.

To understand the disease burden of sexually transmitted Zika virus (ZIKV), we prospectively followed a cohort of 359 adult and adolescent residents of an urban community in Salvador, Brazil, through the 2015 ZIKV epidemic. Later, in 2017, we used a retrospective survey to associate sexual behavior during the epidemic with ZIKV infection as defined by immunoglobulin G3 NS1 enzyme-linked immunosorbent assay. We found that males who engaged in casual sexual encounters during the epidemic were more likely (adjusted odds ratio, 6.2 [95% confidence interval, 1.2-64.1]) to be ZIKV positive, suggesting that specific groups may be at increased risk of sexually transmitted infections., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

This study aims to describe the sociodemographic determinants associated with exposure to Zika Virus (ZIKV) in pregnant women during the 2015-2016 epidemic in Salvador, Brazil., Methods: We recruited women who gave birth between October 2015 and January 2016 to a cross-sectional study at a referral maternity hospital in Salvador, Brazil. We collected information on their demographic, socioeconomic, and clinical characteristics, and evaluated their ZIKV exposure using a plaque reduction neutralization test. Logistic regression was then used to assess the relationship between these social determinants and ZIKV exposure status., Results: We included 469 pregnant women, of whom 61% had a positive ZIKV result. Multivariate analysis found that lower education (adjusted Prevalence Rate [aPR] 1.21; 95%CI 1.04-1.35) and food insecurity (aPR 1.17; 95%CI 1.01-1.30) were positively associated with ZIKV exposure. Additionally, age was negatively associated with the infection risk (aPR 0.99; 95%CI 0.97-0.998)., Conclusion: Eve after controlling for age, differences in key social determinants, as education and food security, were associated with the risk of ZIKV infection among pregnant women in Brazil. Our findings elucidate risk factors that can be targeted by future interventions to reduce the impact of ZIKV infection in this vulnerable population., Competing Interests: The authors declare that there is no conflict of interest.

Land use change can elevate disease risk by creating conditions beneficial to species that carry zoonotic pathogens. Observations of concordant global trends in increased pathogen prevalence or disease incidence and landscape change have generated concerns that urbanization could increase transmission risk of some pathogens. Yet host-pathogen relationships underlying transmission risk have not been well characterized within cities, even where contact between humans and species capable of transmitting pathogens of concern occurs. We addressed this deficit by testing the hypothesis that areas in cities experiencing greater population loss and infrastructure decline (i.e., counter-urbanization) can support a greater diversity of host species and a larger and more diverse pool of pathogens. We did so by characterizing pathogenic Leptospira infection relative to rodent host richness and abundance across a mosaic of abandonment in post-Katrina New Orleans (Louisiana, USA). We found that Leptospira infection loads were highest in areas that harboured increased rodent species richness (which ranged from one to four rodent species detected). Areas with greater host co-occurrence also harboured a greater abundance of hosts, including the host species most likely to carry high infection loads, indicating that Leptospira infection can be amplified by increases in overall and relative host abundance. Evidence of shared infection among rodent host species indicates that cross-species transmission of Leptospira probably increases infection at sites with greater host richness. Additionally, evidence that rodent co-occurrence and abundance and Leptospira infection load parallel abandonment suggests that counter-urbanization can elevate zoonotic disease risk within cities, particularly in underserved communities that are burdened with disproportionate concentrations of derelict properties., (© 2020 John Wiley & Sons Ltd.)

Background: The prevalence of developmental alterations associated with in-utero Zika virus (ZIKV) exposure in children is not well understood. Furthermore, estimation of the Population Attributable Fraction (PAF) of developmental alterations attributed to ZIKV has not been performed due to lack of population-based cohorts with data on symptomatic and asymptomatic ZIKV exposures and an appropriate control group. The aim of this study was to characterize neurodevelopmental outcomes of children at 11 to 32 months of age with intrauterine ZIKV exposure and estimate the PAF of alterations secondary to ZIKV exposure., Methodology/principal Findings: We performed a cohort of biannual community-based prospective serosurveys in a slum community in Salvador, Brazil. We recruited women participating in our cohort, with a documented pregnancy from January 2015 to December 2016 and children born to those mothers. Children were classified as ZIKV exposed in utero (born from women with ZIKV seroconversion during pregnancy) or unexposed (born from women without ZIKV seroconversion or that seroconverted before/after pregnancy) by using an IgG monoclonal antibody blockade-of-binding (BoB). We interviewed mothers and performed anthropometric, audiometric, ophthalmological, neurologic, and neurodevelopmental evaluations of their children at 11 to 32 months of age. Among the 655 women participating in the cohort, 66 (10%) were pregnant during the study period. 46 (70%) of them completed follow-up, of whom ZIKV seroconversion occurred before, during, and after pregnancy in 25 (54%), 13 (28%), and 1 (2%), respectively. The rest of women, 7 (21.2%), did not present ZIKV seroconversion. At 11 to 32 months of life, the 13 ZIKV-exposed children had increased risk of mild cognitive delay (RR 5.1; 95%CI 1.1-24.4) compared with the 33 children unexposed, with a PAF of 53.5%. Exposed children also had increased risk of altered auditory behavior (RR 6.0; 95%CI 1.3-26.9), with a PAF of 59.5%., Conclusions: A significant proportion of children exposed in utero to ZIKV developed mild cognitive delay and auditory behavioral abnormalities even in the absence of gross birth defects such as microcephaly and other neurodevelopmental domains. Furthermore, our findings suggest that over half of these abnormalities could be attributed to intrauterine ZIKV exposure., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA - mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA - mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity., Competing Interests: EW, HA, CH, KO, LL, CR, VB, JN, DA, MR, PD, PF, AK No competing interests declared

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leptospirosis is an important economical disease of livestock globally, especially in Asia, the Caribbean, and the African continent. Its presence has been reported in a wide range of livestock. However, information on leptospirosis in South Africa is scanty. We conducted a cross-sectional study in 11 randomly selected abattoirs to determine the seroprevalence and risk factors for leptospirosis in slaughtered cattle in Gauteng province, South Africa. During abattoir visits to selected abattoirs, blood samples were collected from 199 cattle and demographic data obtained on the slaughtered animals. The microscopic agglutination test (MAT) was performed on all sera using a 26-serotype panel using cutoff titer ≥ 1:100. Animal- and abattoir-level risk factors were investigated for their association with seropositivity for leptospirosis. The seroprevalence of leptospirosis in the cattle sampled was 27.6% (55/199). The predominant serogroups detected in seropositive cattle were Sejroe (sv. Hardjo) (38.2%) and Mini sv. Szwajizak) (14.5%) but low to Canicola (sv. Canicola) (1.8%) and Pomona (sv. Pomona) (1.8%). The differences were statistically significant (P < 0.05). Of the five variables investigated, only one (abattoirs) had statistically significantly (P < 0.001) differences in the seroprevalence of leptospirosis among abattoirs. The study documented for the first time in South Africa, the occurrence of serogroups Sejroe (Hardjo bovis strain lely 607), Tarassovi, Hebdomadis, and Medanensis in slaughtered cattle. It was concluded that six of the nine serovars (representing seven serogroups) of Leptospira spp. circulating in cattle population in South Africa are not vaccine serogroups. The clinical, diagnostic, and public health importance of the findings cannot be ignored.

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira . Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro , and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response., (Copyright © 2020 Holzapfel, Bonhomme, Cagliero, Vernel-Pauillac, Fanton d’Andon, Bortolussi, Fiette, Goarant, Wunder, Picardeau, Ko, Werling, Matsui, Boneca and Werts.)

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum., Competing Interests: KG, FT, EW, MB, FS, AM, ZS, JL, MP, AK, AB, CS No competing interests declared, (© 2020, Gibson et al.)

Four spirochetes (F1 T , B21, Yale T and AMB6-RJ) were isolated from environmental sources: F1 T and B21 from soils of an urban slum community in Salvador (Brazil), Yale T from river water in New Haven, Connecticut (USA) and AMB6-RJ from a pond in a horse farm in Rio de Janeiro (Brazil). Isolates were helix-shaped, aerobic, highly motile and non-virulent in a hamster model of infection. Draft genomes of the strains were obtained and analysed to determine the relatedness to other species of the genus Leptospira . The analysis of 498 core genes showed that strains F1 T /B21 and Yale T /AMB6-RJ formed two distinct phylogenetic clades within the 'Pathogens' group (group I). The average nucleotide identity (ANI) values of strains F1 T /B21 and Yale T /AMB6-RJ to other previously described Leptospira species were below <84 % and <82 %, respectively, which confirmed that these isolates should be classified as representatives of two novel species. Therefore, we propose Leptospirayasudae sp. nov. and Leptospirastimsonii sp. nov. as new species in the genus Leptospira . The type strains are F1 T (=ATCC-TSD-163=KIT0259=CLEP00287) and Yale T (=ATCC-TDS-162=KIT0258=CLEP00288), respectively.

The incidence of hospitalized leptospirosis patients was positively associated with increased precipitation in Salvador, Brazil. However, Leptospira infection risk among a cohort of city residents was inversely associated with rainfall. These findings indicate that, although heavy rainfall may increase severe illness, Leptospira exposures can occur year-round.

An essential property associated with leptospiral virulence is the pathogen's ability to translocate across host cells, enabling Leptospira to evade the host immune response, disseminate, and establish infection. Cell monolayer translocation assay allows for the quantification of Leptospira strain's competence to cross cell barriers while measuring the integrity of the polarized eukaryotic cell monolayer during this process.

Zika virus (ZIKV) infection during pregnancy causes congenital abnormalities, including microcephaly. However, rates vary widely, and the contributing risk factors remain unclear. We examined the serum antibody response to ZIKV and other flaviviruses in Brazilian women giving birth during the 2015-2016 outbreak. Infected pregnancies with intermediate or higher ZIKV antibody enhancement titers were at increased risk to give birth to microcephalic infants compared with those with lower titers (P < 0.0001). Similarly, analysis of ZIKV-infected pregnant macaques revealed that fetal brain damage was more frequent in mothers with higher enhancement titers. Thus, features of the maternal antibodies are associated with and may contribute to the genesis of ZIKV-associated microcephaly., (© 2019 Robbiani et al.)

Leptospirosis is a widespread and potentially life-threatening zoonotic disease caused by spirochaetes of the genus Leptospira. Humans become infected primarily via contact with environmental reservoirs contaminated by the urine of shedding mammalian hosts. Populations in high transmission settings, such as urban slums and subsistence farming communities, are exposed to low doses of Leptospira on a daily basis. Under these conditions, numerous factors determine whether infection occurs, including the route of exposure and inoculum dose. Skin wounds and abrasions are risk factors for leptospirosis, but it is not known whether broken skin is necessary for spillover, or if low-dose exposures to intact skin and mucous membranes can also cause infection. To establish a quantitative relationship between dose, route and probability of infection, we performed challenge experiments in hamsters and rats, developed mechanistic dose-response models representing the spatial dynamics of within-host infection and persistence, and fitted models to experimental data. Results show intact skin is a strong barrier against infection, and that broken skin is the predominant route by which low-dose environmental exposures cause infection. These results identify skin integrity as a bottleneck to spillover of Leptospira and underscore the importance of barrier interventions in the prevention of leptospirosis. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Background: The burden of leptospirosis in Puerto Rico remains unclear due to underreporting., Methods: A cross-sectional survey and rodent trapping was performed in a community within San Juan, Puerto Rico to determine the seroprevalence and risk factors for Leptospira infection. The microscopic agglutination test was used to detect anti-Leptospira antibodies as a marker of previous infection. We evaluated Leptospira carriage by quantitative polymerase chain reaction among rodents trapped at the community site., Results: Of 202 study participants, 55 (27.2%) had Leptospira agglutinating antibodies. Among the 55 seropositive individuals, antibodies were directed most frequently against serogroups Icterohaemorrhagiae (22.0%) and Autumnalis (10.6%). Of 18 captured rodents, 11 (61.1%) carried pathogenic Leptospira (Leptospira borgpetersenii, 7 and Leptospira interrogans, 2). Four participants showed their highest titer against an isolate obtained from a rodent (serogroup Ballum). Increasing household distance to the canal that runs through the community was associated with decreased risk of infection (odds ratio = 0.934 per 10-meter increase; 95% confidence interval, .952-.992)., Conclusions: There are high levels of Leptospira exposure in an urban setting in Puerto Rico, for which rodents may be an important reservoir for transmission. Our findings indicate that prevention should focus on mitigating risk posed by infrastructure deficiencies such as the canal., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Leptospirosis is the leading cause of zoonotic morbidity and mortality globally, yet little is known about the immune mechanisms that may contribute to pathogenesis and severe disease. Although neutrophils are a key component of early immune responses to infection, they have been associated with tissue damage and inflammation in some febrile infections. To assess whether neutrophils contribute to the pathogenesis observed in severe leptospirosis, we quantitated levels of neutrophil activation markers in patients with varying disease severities. Hospitalized leptospirosis patients had significantly higher levels of toll-like receptors 2 and 4 (TLR2 and TLR4, respectively) on peripheral neutrophils than healthy controls, with the highest levels detected in patients with organ dysfunction. We observed no significant differences in other neutrophil baseline activation markers (CD62L and CD11b) or activation capacity (CD62L and CD11b levels following stimulation), regardless of disease severity. Our results provide preliminary evidence supporting the hypothesis that higher initial bacterial loads or inadequate or delayed neutrophil responses, rather than TLR-driven inflammation, may drive severe disease outcomes.

Leptospira dzianensis and Leptospira putramalaysiae were recently described as novel species and published almost concurrently with Leptospira yasudae and Leptospira stimsonii . Genome comparisons based on average nucleotide identity of the type strain genomes indicate that L. dzianensis and L. putramalaysiae are conspecific with L. yasudae and L. stimsonii , respectively. Based on the rules of priority, L. dzianensis should be reclassified as a later heterotypic synonym of L. yasudae , and L. putramalaysiae should be reclassified as a later heterotypic synonym of L. stimsonii .

The clinical outcomes associated with Zika virus (ZIKV) in the Americas have been well documented, but other aspects of the pandemic, such as attack rates and risk factors, are poorly understood. We prospectively followed a cohort of 1453 urban residents in Salvador, Brazil, and, using an assay that measured immunoglobulin G3 (IgG3) responses against ZIKV NS1 antigen, we estimated that 73% of individuals were infected during the 2015 outbreak. Attack rates were spatially heterogeneous, varying by a factor of 3 within a community spanning 0.17 square kilometers. Preexisting high antibody titers to dengue virus were associated with reduced risk of ZIKV infection and symptoms. The landscape of ZIKV immunity that now exists may affect the risk for future transmission., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Leptospirosis is a worldwide zoonosis, responsible for more than 1 million cases and 60,000 deaths every year. Among the 13 pathogenic species of the genus Leptospira , serovars belonging to L. interrogans serogroup Icterohaemorrhagiae are considered to be the most virulent strains, and responsible for majority of the reported severe cases. Serovars Copenhageni and Icterohaemorrhagiae are major representatives of this serogroup and despite their public health relevance, little is known regarding the genetic differences between these two serovars. In this study, we analyzed the genome sequences of 67 isolates belonging to L. interrogans serovars Copenhageni and Icterohaemorrhagiae to investigate the influence of spatial and temporal variations on DNA sequence diversity. Out of the 1072 SNPs identified, 276 were in non-coding regions and 796 in coding regions. Indel analyses identified 258 indels, out of which 191 were found in coding regions and 67 in non-coding regions. Our phylogenetic analyses based on SNP dataset revealed that both serovars are closely related but showed distinct spatial clustering. However, likelihood ratio test of the indel data statistically confirmed the presence of a frameshift mutation within a homopolymeric tract of lic12008 gene (related to LPS biosynthesis) in all the L. interrogans serovar Icterohaemorrhagiae strains but not in the Copenhageni strains. Therefore, this internal indel identified can genetically distinguish L. interrogans serovar Copenhageni from serovar Icterohaemorrhagiae with high discriminatory power. To our knowledge, this is the first study to identify global sequence variations (SNPs and Indels) in L. interrogans serovars Copenhageni and Icterohaemorrhagiae.

Leptospira interrogans is the etiological agent of leptospirosis, a globally distributed zoonotic disease. Human infection usually occurs through skin exposure with water and soil contaminated with the urine of chronically infected animals. In this study, we aimed to quantitatively characterize the survival of Leptospira interrogans serovar Copenhageni in environmental matrices. We constructed laboratory microcosms to simulate natural conditions and determined the persistence of DNA markers in soil, mud, spring water and sewage using a quantitative PCR (qPCR) and a propidium monoazide (PMA)-qPCR assay. We found that L. interrogans does not survive at high concentrations in the tested matrices. No net growth was detected in any of the experimental conditions and in all cases the concentration of the DNA markers targeted decreased from the beginning of the experiment following an exponential decay with a decreasing decay rate over time. After 12 and 21 days of incubation the spiked concentration of 10 6 L. interrogans cells/ml or g decreased to approximately 100 cells/ml or g in soil and spring water microcosms, respectively. Furthermore, culturable L. interrogans persisted at concentrations under the limit of detection by PMA-qPCR or qPCR for at least 16 days in soil and 28 days in spring water. Altogether, our findings suggest that the environment is not a multiplication reservoir but a temporary carrier of L. interrogans Copenhageni, although the observed prolonged persistence at low concentrations may still enable the transmission of the disease. IMPORTANCE Leptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira that primarily affects impoverished populations worldwide. Although leptospirosis is transmitted by contact with water and soil, little is known about the ability of the pathogen to survive in the environment. In this study, we quantitatively characterized the survival of L. interrogans in environmental microcosms and found that although it cannot multiply in water, soil or sewage, it survives for extended time periods (days to weeks depending on the matrix). The survival parameters obtained here may help to better understand the distribution of pathogenic Leptospira in the environment and improve the predictions of human infection risks in areas where such infections are endemic., (Copyright © 2018 American Society for Microbiology.)

The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete Leptospira . By screening a library of random transposon mutants in the spirochete Leptospira biflexa , we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene fcpB for flagellar-coiling protein B. fcpB is conserved in all members of the Leptospira genus, but not present in other organisms including other spirochetes. Complementation of the fcpB - mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are Leptospira -specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.

Background: Leptospirosis is an important zoonotic disease that causes considerable morbidity and mortality globally, primarily in residents of urban slums. While contact with contaminated water plays a critical role in the transmission of leptospirosis, little is known about the distribution and abundance of pathogenic Leptospira spp. in soil and the potential contribution of this source to human infection., Methods/principal Findings: We collected soil samples (n = 70) from three sites within an urban slum community endemic for leptospirosis in Salvador, Brazil. Using qPCR of Leptospira genes lipl32 and 16S rRNA, we quantified the pathogenic Leptospira load in each soil sample. lipl32 qPCR detected pathogenic Leptospira in 22 (31%) of 70 samples, though the median concentration among positive samples was low (median = 6 GEq/g; range: 4-4.31×102 GEq/g). We also observed heterogeneity in the distribution of pathogenic Leptospira at the fine spatial scale. However, when using 16S rRNA qPCR, we detected a higher proportion of Leptospira-positive samples (86%) and higher bacterial concentrations (median: 4.16×102 GEq/g; range: 4-2.58×104 GEq/g). Sequencing of the qPCR amplicons and qPCR analysis with all type Leptospira species revealed that the 16S rRNA qPCR detected not only pathogenic Leptospira but also intermediate species, although both methods excluded saprophytic Leptospira. No significant associations were identified between the presence of pathogenic Leptospira DNA and environmental characteristics (vegetation, rat activity, distance to an open sewer or a house, or soil clay content), though samples with higher soil moisture content showed higher prevalences., Conclusion/significance: This is the first study to successfully quantify the burden of pathogenic Leptospira in soil from an endemic region. Our results support the hypothesis that soil may be an under-recognized environmental reservoir contributing to transmission of pathogenic Leptospira in urban slums. Consequently, the role of soil should be considered when planning interventions aimed to reduce the burden of leptospirosis in these communities.

Leptospirosis has emerged as an important urban health problem as slum settlements have expanded worldwide. Yet the dynamics of the environmentally transmitted Leptospira pathogen has not been well characterized in these settings. We used a stratified dense sampling scheme to study the dynamics of Leptospira abundance in surface waters from a Brazilian urban slum community. We collected surface water samples during the dry, intermediate and rainy seasons within a seven-month period and quantified pathogenic Leptospira by quantitative PCR (qPCR). We used logistic and linear mixed models to identify factors that explained variation for the presence and concentration of Leptospira DNA. Among 335 sewage and 250 standing water samples, Leptospira DNA were detected in 36% and 34%, respectively. Among the 236 samples with positive results geometric mean Leptospira concentrations were 152 GEq/mL. The probability of finding Leptospira DNA was higher in sewage samples collected during the rainy season when increased leptospirosis incidence occurred, than during the dry season (47.2% vs 12.5%, respectively, p = 0.0002). There was a marked spatial and temporal heterogeneity in Leptospira DNA distribution, for which type of water, elevation, and time of day that samples were collected, in addition to season, were significant predictors. Together, these findings indicate that Leptospira are ubiquitous in the slum environment and that the water-related risk to which inhabitants are exposed is low. Seasonal increases in Leptospira presence may explain the timing of leptospirosis outbreaks. Effective prevention will need to consider the spatial and temporal dynamics of pathogenic Leptospira in surface waters to reduce the burden of the disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira . In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator ) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one ( lvrB ) or both genes ( lvrA/B ) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.