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Leptospirosis is a bacterial zoonosis that occurs in tropical and subtropical regions worldwide. Chiroptera are known to be a formidable reservoir of zoonotic pathogens, including leptospires. The epidemiology of leptospirosis in bats in the Pacific Islands is poorly known, both in terms of prevalence and in terms of the bacterial strains involved. A strong host specificity between leptospiral strains and their mammalian reservoir is recognized. This phenomenon has notably been studied recently in bat communities, providing strong evidence of co-evolution. In New Caledonia, a biodiversity hotspot where leptospirosis is endemic and enzootic, Chiroptera are the only indigenous terrestrial mammals. In this study, we aimed to investigate leptospires associated with three flying fox species in New Caledonia. Kidneys and urine samples of Pteropus spp. from captures and seizures were analysed. Among 254 flying foxes analysed, 24 harboured pathogenic leptospires corresponding to an observed prevalence of 9.45% with 15.8% on the Main Island and 4.3% on Loyalty Islands. The analysis of the rrs gene, lfb1, and MLST sequences evidenced four distinct clusters of undescribed strains, likely corresponding to undescribed species. All four strains belong to the Group I of pathogenic Leptospira spp., which includes Leptospira interrogans, Leptospira noguchii, and Leptospira kirschneri. We detected pathogenic leptospires in all three Pteropus spp. studied (including two endemic species) with no evidence of host specificity in two co-roosting species. For a better understanding of Leptospira-host co-evolution, notably to genetically characterize and evaluate the virulence of these original bat-associated leptospires, it is essential to improve isolation techniques. Flying foxes are traditionally hunted and eaten in New Caledonia, a massive cause of bat-human interactions. Our results should encourage vigilance during these contacts to limit the spillover risk of these pathogens to humans.

International audience; Mostly studied as a zoonosis, leptospirosis is also an environment-borne infection and most human cases originate from soil or water contaminations. Yet, only few studies have been interested in the survival of pathogenic Leptospira in freshwater. In this study, water microcosms were designed to evaluate the survival and virulence of Leptospira spp. for 2 years. Four commercial bottled drinking waters and a non-ionized water, all previously filter-sterilized, were studied. Either one of two Leptospira interrogans strains, one Leptospira borgpetersenii strain, or a saprophytic Leptospira biflexa was inoculated in these waters under nutrient-deprived conditions. Molecular, microscopic and cultural approaches were used to study Leptospira survival. Direct virulence of the pathogens was assessed using animal challenge without re-culturing. Our results confirmed the capacity of pathogenic Leptospira to survive for more than a year in water. In addition, we showed the ability of L. interrogans in nutrient-deprived conditions to directly cause systemic infection in susceptible animals. To our knowledge, this is the first report of direct infection of a susceptible host with Leptospira following a long starvation and survival period in nutrient-deprived water. Our results also suggest that Leptospira turned into a physiological "survival" state in harsh freshwater conditions. These data are of prime importance considering that freshwater is a major source of Leptospira infections. Environmental survival and virulence of pathogenic Leptospira spp. are becoming a crucial challenge to determine the environmental risk and adopt relevant prevention and control strategies.

Background Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. Methods/principal findings The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. Conclusions/significance Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC., Author summary Dengue represents a risk for almost half of the world’s population, especially throughout the tropics. In New Caledonia, dengue outbreaks have become more frequent in the past decade along with the recent circulation of chikungunya and Zika viruses. The opportunity to use the biocontrol method involving the release of Wolbachia-infected Ae. aegypti mosquitoes has been investigated as an alternative solution to the traditional control methods, like elimination of larval habitats and pyrethroid insecticide application to kill adults, which are becoming insufficient. A local strain of Ae. aegypti carrying Wolbachia (NC-wMel) has been generated and tested to evaluate its pathogen blocking capacity for the four dengue virus serotypes as well as chikungunya and Zika viruses. The fitness of NC-wMel strain has also been assessed to estimate its ability to compete with the wild-type strain in the field. Noumea city, where a third of the population of New Caledonia resides, has been chosen as the first site to implement the method in New Caledonia. As Ae. aegypti is the only proven vector in New Caledonia, we expect a significant impact on dengue outbreaks occurring in Noumea as soon as a high frequency of NC-wMel is established in the population.

International audience; Dengue virus (DENV) evolutionary dynamics are characterized by frequent DENV genotype/lineage replacements, potentially associated with changes in disease severity and human immunity. New Caledonia (NC) and Cambodia, two contrasted epidemiological settings, respectively experienced a DENV-1 genotype IV to I replacement in 2012 and a DENV-1 genotype I lineage 3 to 4 replacement in 2005-2007, both followed by a massive dengue outbreak. However, their underlying evolutionary drivers have not been elucidated. Here, we tested the hypothesis that these genotype/lineage switches reflected a higher transmission fitness of the replacing DENV genotype/lineage in the mosquito vector using in vivo competition experiments. For this purpose, field-derived Aedes aegypti from NC and Cambodia were orally challenged with epidemiologically relevant pairs of four DENV-1 genotype I and IV strains from NC or four DENV-1 genotype I lineage 3 and 4 strains from Cambodia, respectively. The relative transmission fitness of each DENV-1 genotype/lineage was measured by quantitative RT-PCR for infection, dissemination, and transmission rates. Results showed a clear transmission fitness advantage of the replacing DENV-1 genotype I from NC within the vector. A similar but more subtle pattern was observed for the DENV-1 lineage 4 replacement in Cambodia. Our results support the hypothesis that vector-driven selection contributed to the DENV-1 genotype/lineage replacements in these two contrasted epidemiological settings, and reinforce the idea that natural selection taking place within the mosquito vector plays an important role in DENV short-term evolutionary dynamics.

International audience; Dengue virus (DENV) serotype-2 was detected in the South Pacific region in 2014 for the first time in 15 years. In 2016-2020, DENV-2 re-emerged in French Polynesia, Vanuatu, Wallis and Futuna, and New Caledonia, co-circulating with and later replacing DENV-1. In this context, epidemiological and molecular evolution data are paramount to decipher the diffusion route of this DENV-2 in the South Pacific region. In the current work, the E gene from 23 DENV-2 serum samples collected in Vanuatu, Fiji, Wallis and Futuna, and New Caledonia was sequenced. Both maximum likelihood and Bayesian phylogenetic analyses were performed. While all DENV-2 strains sequenced belong to the Cosmopolitan genotype, phylogenetic analysis suggests at least three different DENV-2 introductions in the South Pacific between 2017 and 2020. Strains retrieved in these Pacific Islands Countries and Territories (PICTs) in 2017-2020 are phylogenetically related, with strong phylogenetic links between strains retrieved from French PICTs. These phylogenetic data substantiate epidemiological data of the DENV-2 diffusion pattern between these countries.

Leptospires, the etiological agents of leptospirosis, are fastidious slow-growing organisms. Here we describe the isolation and routine maintenance of leptospires from clinical (blood, urine, or tissue) and environmental (water or soil) samples. Using combinations of filtration, agar plating, and selective agents, leptospires can be isolated in pure cultures even from complex contaminated sources in standard EMJH culture medium.

Medical microbiology has used phenotypical and metabolic criteria to identify bacterial pathogens for decades. However, no such criteria have been applied to identify leptospires at the species level. In the recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has emerged as new tool for the identification of bacterial species in the medical microbiology laboratory. This technology has rapidly gained more and more popularity. Actually, this technique is sensitive and economic, saving both labor and bench costs, but also rapid, significantly reducing turnaround time from isolation to identification. MALDI-ToF MS provides an unprecedented tool for the rapid identification of Leptospira at the species level.

International audience; Medical microbiology has used phenotypical and metabolic criteria to identify bacterial pathogens for decades. However, no such criteria have been applied to identify leptospires at the species level. In the recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has emerged as new tool for the identification of bacterial species in the medical microbiology laboratory. This technology has rapidly gained more and more popularity. Actually, this technique is sensitive and economic, saving both labor and bench costs, but also rapid, significantly reducing turnaround time from isolation to identification. MALDI-ToF MS provides an unprecedented tool for the rapid identification of Leptospira at the species level.

Zika virus (ZIKV) has caused severe epidemics in South America beginning in 2015, following its spread through the Pacific. We comparatively assessed the vector competence of ten populations of Aedes aegypti and Ae. albopictus from Brazil and two of Ae. aegypti and one of Culex quinquefasciatus from New Caledonia to transmit three ZIKV isolates belonging to African, Asian and American lineages. Recently colonized mosquitoes from eight distinct sites from both countries were orally challenged with the same viral load (107 TCID50/mL) and examined after 7, 14 and 21 days. Cx. quinquefasciatus was refractory to infection with all virus strains. In contrast, although competence varied with geographical origin, Brazilian and New Caledonian Ae. aegypti could transmit the three ZIKV lineages, with a strong advantage for the African lineage (the only one reaching saliva one-week after challenge). Brazilian Ae. albopictus populations were less competent than Ae. aegypti populations. Ae. albopictus generally exhibited almost no transmission for Asian and American lineages, but was efficient in transmitting the African ZIKV. Viral surveillance and mosquito control measures must be strengthened to avoid the spread of new ZIKV lineages and minimize the transmission of viruses currently circulating.

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm’s cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium’s ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.

International audience; With the increasing use of real time PCR techniques, Leptospira isolation has mostly been abandoned for the diagnosis of human leptospirosis. However, there is a great value of collecting Leptospira isolates to better understand the epidemiology of this complex zoonosis and to provide the researchers with different isolates. In this study, we have successfully isolated different Leptospira strains from BacT/Alert aerobic blood culture bottles and suggest that this privileged biological material offers an opportunity to isolate leptospires.

International audience; BACKGROUND: In spite of a local favorable environment, leptospirosis has never been described in Central African Republic so far mainly because of the weakness of diagnostic tests and differential diagnostic strategy for febrile jaundice cases negative for yellow fever virus. Here we bring a complementary insight to conclusions of Gadia CLB et al. regarding the presence of leptospirosis in Central African Republic in YFV-negative febrile icteric patients.METHODS: Our study included 497 individuals presenting with fever and jaundice but negative for yellow fever infection, retrospectively selected from the national surveillance biobank for yellow fever in Institut Pasteur de Bangui, Central African Republic. A combination of serological (ELISA, agglutination) and molecular biology techniques (quantitative real-time polymerase chain reaction) was used to identify Leptospira or the patient's immune response to the bacteria. Statistical analyses were done using the non parametric Mann-Withney U test with a 5% statistical threshold.RESULTS: ELISA test results showed 46 positive serum samples while 445 were negative and 6 remains equivocal. In addition, the reference microscopic agglutination test for leptospirosis diagnostic confirmed that 7 out of 32 samples tested were positive. Unfortunately, all 497 serum samples tested for leptospirosis were negative using the molecular techniques.CONCLUSIONS: Unlike Gadia et al., we confirmed that leptospirosis is circulating in Central African Republic and therefore may be responsible for some of the unexplained cases of febrile jaundice in the country. Thus, leptospirosis needs to be investigated to improve identification of aetiological pathogens. Our study also suggests a need to improve sample transportation and storage conditions.

Background Estimates of leptospirosis morbidity identified Oceania as the region with highest burden. Besides Australia and New Zealand, Oceania is home of Pacific Island Countries and Territories, most of which are developing countries facing a number of challenges. Their archipelago geography notably affects health infrastructure and access to healthcare. Although human leptospirosis was formerly identified in Vanuatu, there is a lack of knowledge of this disease in the country. We aimed to identify leptospirosis in outpatients visiting the hospital. Methodology/Principal findings We conducted a clinical study to investigate leptospirosis as a cause of non-malarial acute febrile illness in Vanuatu. A total 161 outpatients visiting the outpatient clinics at Port Vila Central Hospital for internal medicine were recruited over 20 month. We showed that leptospirosis significantly affects humans in Vanuatu: 12 cases were confirmed by real-time PCR on acute blood samples (n = 5) or by high serology titers evidencing a recent infection (MAT titer ≥800 or ELISA≥18 Units, n = 7). A high rate of positive serology was also evidenced, by MAT (100, Author summary Leptospirosis is thought to impose its highest burden to tropical island populations, especially in the Pacific region of Oceania. Yet, very few information has ever been reported from some of the Pacific Island Countries and Territories, including Vanuatu. In this study, we aimed at evidencing leptospirosis in a convenience sample of outpatients visiting Port Vila Central Hospital, the major hospital of this archipelago country in the South Pacific. Using reference laboratory techniques, our study confirms a significant number of acute leptospirosis cases, as well as a high level of previous exposure in outpatients visiting this hospital. Risk factor analysis point to livestock and lifestyle as important factors exposing to leptospirosis in this developing country. Furthermore, there is evidence that the disease probably has a higher incidence in remote islands outside of the capital of Efate in this very wide archipelago. Taken together, our results point to a need of increased awareness, notably in peripheral health centers and of better access to laboratory confirmation for surveillance.

International audience; Leptospirosis is an important environmental disease and a major threat to human health causing at least 1 million clinical infections annually. There has recently been a growing interest in understanding the environmental lifestyle of Leptospira. However, Leptospira isolation from complex environmental samples is difficult and time-consuming and few tools are available to identify Leptospira isolates at the species level. Here, we propose a polyphasic isolation and identification scheme, which might prove useful to recover and identify environmental isolates and select those to be submitted to whole-genome sequencing. Using this approach, we recently described 12 novel Leptospira species for which we propose names. We also show that MALDI-ToF MS allows rapid and reliable identification and provide an extensive database of Leptospira MALDI-ToF mass spectra, which will be valuable to researchers in the leptospirosis community for species identification. Lastly, we also re-evaluate some of the current techniques for the molecular diagnosis of leptospirosis taking into account the extensive and recently revealed biodiversity of Leptospira in the environment. In conclusion, we describe our method for isolating Leptospira from the environment, confirm the usefulness of mass spectrometry for species identification and propose names for 12 novel species. This also offers the opportunity to refine current molecular diagnostic tools.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral ‘pathogens’ cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.

International audience; Zika virus (ZIKV) infection has been associated with neurologic disorders including Guillain-Barré syndrome (GBS). In New Caledonia during the ZIKV outbreak (2014-2015), case-control and retrospective studies have been performed to assess the link between ZIKV and GBS. Among the 15 cases included, 33% had evidence of a recent ZIKV infection compared to only 3.3% in the 30 controls involved. All patients were Melanesian, had facial diplegia and similar neurophysiological pattern consistent with acute inflammatory demyelinating polyneuropathy, and recovered well. Furthermore, during the peak of ZIKV transmission, we observed a number of GBS cases higher than the calculated upper limit, emphasizing the fact that ZIKV is now a major trigger of GBS.

Background Dengue virus (DENV) is the arbovirus with the highest incidence in New Caledonia and in the South Pacific region. In 2012–2014, a major DENV-1 outbreak occurred in New Caledonia. The only known vector of DENV in New Caledonia is Aedes aegypti but no study has yet evaluated the competence of New Caledonia Ae. aegypti populations to transmit DENV. This study compared the ability of field-collected Ae. aegypti from different locations in New Caledonia to transmit the DENV-1 responsible for the 2012–2014 outbreak. This study also aimed to compare the New Caledonia results with the vector competence of Ae. aegypti from French Polynesia as these two French countries have close links, including arbovirus circulation. Methods Three wild Ae. aegypti populations were collected in New Caledonia and one in French Polynesia. Female mosquitoes were orally exposed to DENV-1 (106 FFU/ml). Mosquito bodies (thorax and abdomen), heads and saliva were analyzed to measure infection, dissemination, transmission rates and transmission efficiency, at 7, 14 and 21 days post-infection (dpi), respectively. Results DENV-1 infection rates were heterogeneous, but dissemination rates were high and homogenous among the three Ae. aegypti populations from New Caledonia. Despite this high DENV-1 dissemination rate, the transmission rate, and therefore the transmission efficiency, observed were low. Aedes aegypti population from New Caledonia was less susceptible to infection and had lower ability to transmit DENV-1 than Ae. aegypti populations from French Polynesia. Conclusion This study suggests that even if susceptible to infection, the New Caledonian Ae. aegypti populations were moderately competent vectors for DENV-1 strain from the 2012–2014 outbreak. These results strongly suggest that other factors might have contributed to the spread of this DENV-1 strain in New Caledonia and in the Pacific region. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2319-x) contains supplementary material, which is available to authorized users.

Background Leptospirosis is an important re-emerging infectious disease that affects humans worldwide. Infection occurs from indirect environment-mediated exposure to pathogenic leptospires through contaminated watered environments. The ability of pathogenic leptospires to persist in the aqueous environment is a key factor in transmission to new hosts. Hence, an effort was made to detect pathogenic leptospires in complex environmental samples, to genotype positive samples and to assess leptospiral viability over time. Methodology/Principal findings We focused our study on human leptospirosis cases infected with the New Caledonian Leptospira interrogans serovar Pyrogenes. Epidemiologically related to freshwater contaminations, this strain is responsible for ca. 25% of human cases in New Caledonia. We screened soil and water samples retrieved from suspected environmental infection sites for the pathogen-specific leptospiral gene lipL-32. Soil samples from all suspected infection sites tested showed detectable levels of pathogenic leptospiral DNA. More importantly, we demonstrated by viability qPCR that those pathogenic leptospires were viable and persisted in infection sites for several weeks after the index contamination event. Further, molecular phylogenetic analyses of the leptospiral lfb-1 gene successfully linked the identity of environmental Leptospira to the corresponding human-infecting strain. Conclusions/Significance Altogether, this study illustrates the potential of quantitative viability-PCR assay for the rapid detection of viable leptospires in environmental samples, which might open avenues to strategies aimed at assessing environmental risk., Author summary Leptospirosis is an emerging zoonotic disease caused by infection with pathogenic strains of Leptospira. Most human infections arise from environmental exposure to contaminated freshwater environments or watered soils where pathogenic Leptospira are considered as able to survive for prolonged periods. Therefore, a good understanding of Leptospira survival strategy in the environment is a key step to identifying crucial factors amenable to interventions and public health actions to lower leptospirosis burden. In this study, we investigated the environmental presence and survival of pathogenic leptospires in areas where recent human leptospirosis cases had been reported. Although detection of Leptospira from complex environmental samples is difficult, we successfully detected the presence of pathogenic Leptospira in soils of suspected infection sites. In addition, we showed that these pathogenic leptospires were alive and present in soils several weeks after the infecting event. Typing of leptospiral DNA retrieved from the environment revealed identities between environmental pathogenic Leptospira and the causative strains involved in human leptospirosis index cases. Interestingly, we also identified yet unreported genotypes. Altogether, our work illustrates the potential of quantitative molecular assays for the rapid detection and typing of viable leptospires in environmental samples, which could prove useful to assess the risk of environmental exposure.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species., Author summary Leptospirosis which is an emerging zoonotic disease worldwide, is transmitted to humans through contact with soils or surface waters contaminated with the urine of reservoir animals. Species of Leptospira, which include infectious and non-infectious strains, are ubiquitous in the environment. In this study we have sequenced the genomes of strains of Leptospira isolated from several environmental sources worldwide. Comparison of these genomes with other members of the Leptospira genus revealed the existence of 30 novel Leptospira species. A comparative genomic analysis of species of the genus allowed us to identify genes or genome features that are specific of infectious species, providing insights into virulence evolution in these atypical bacteria but also allow refinement of the Leptospira classification.