Marie-Estelle Soupé-Gilbert

Though not Reservoirs, Dogs might Transmit Leptospira in New Caledonia

Leptospira has been a major public health concern in New Caledonia for decades. However, few multidisciplinary studies addressing the zoonotic pattern of this disease were conducted so far. Here, pig, deer and dog samples were collected. Analyses were performed using molecular detection and genotyping. Serological analyses were also performed for dogs. Our results suggest that deer are a reservoir of L. borgpetersenii Hardjobovis and pigs a reservoir of L. interrogans Pomona. Interestingly, 4.4% of dogs were renal carriers of Leptospira. In dog populations, MAT results confirmed the circulation of the same Leptospira serogroups involved in human cases. Even if not reservoirs, dogs might be of significance in human contamination by making an epidemiological link between wild or feral reservoirs and humans. Dogs could bring pathogens back home, shedding Leptospira via their urine and in turn increasing the risk of human contamination. We propose to consider dog as a vector, particularly in rural areas where seroprevalence is significantly higher than urban areas. Our results highlight the importance of animal health in improving leptospirosis prevention in a One Health approach.

Sensitivity and Specificity of a New Vertical Flow Rapid Diagnostic Test for the Serodiagnosis of Human Leptospirosis.

Background: Leptospirosis is a growing public health concern in many tropical and subtropical countries. However, its diagnosis is difficult because of non-specific symptoms and concurrent other endemic febrile diseases. In many regions, the laboratory diagnosis is not available due to a lack of preparedness and simple diagnostic assay or difficult access to reference laboratories. Yet, an early antibiotic treatment is decisive to the outcome. The need for Rapid Diagnostic Tests (RDTs) for bedside diagnosis of leptospirosis has been recognized. We developed a vertical flow immunochromatography strip RDT detecting anti-Leptospira human IgM and evaluated it in patients from New Caledonia, France, and French West Indies. Methodology/Principal Findings: Whole killed Leptospira fainei cells were used as antigen for the test line and purified human IgM as the control line. The mobile phase was made of gold particles conjugated with goat anti-human IgM. Standards for Reporting of Diagnostic Accuracy criteria were used to assess the performance of this RDT. The Microscopic Agglutination Test (MAT) was used as the gold standard with a cut-off titer of ≥400. The sensitivity was 89.8% and the specificity 93.7%. Positive and negative Likelihood Ratios of 14.18 and 0.108 respectively, and a Diagnostic Odds Ratio of 130.737 confirmed its usefulness. This RDT had satisfactory reproducibility, repeatability, thermal tolerance and shelf-life. The comparison with MAT evidenced the earliness of the RDT to detect seroconversion. When compared with other RDT, the Vertical Flow RDT developed displayed good diagnostic performances. Conclusions/Significance This RDT might be used as a point of care diagnostic tool in limited resources countries. An evaluation in field conditions and in other epidemiological contexts should be considered to assess its validity over a wider range of serogroups or when facing different endemic pathogens. It might prove useful in endemic contexts or outbreak situations.

Severity markers in severe leptospirosis: a cohort study

We aimed to evaluate parameters for their value as severity markers in hospitalized leptospirosis patients. We recruited 47 informed adult consenting patients and assessed a number of clinical, hematological, biochemical, and biological variables. Patients were sorted according to severity based on fatality or the requirement of mechanical ventilation or dialysis; the parameters studied were compared between groups on inclusion and the next day. Beside septic shock presentation or a high severity score (Simplified Acute Physiology Score; SAPS II), increased lactate, total bilirubin, lipase, and AST/ALT ratio or a decreased cytokines IL-10/TNF-α ratio were all significantly associated with severity. The gene expression of the IL-1 receptor antagonist IL-1ra, IL-1α, and the long pentraxin PTX-3 were also transcribed at higher levels in most severe cases. Patients could rapidly improve or deteriorate, highlighting the need for a new assessment the next day. Our results add to the limited body of knowledge about severity markers in leptospirosis. They also suggest that patients should be reassessed the next day before being possibly discharged from the hospital. Further studies are needed in order to confirm relevant and reliable prognostic parameters in leptospirosis that would be helpful for the purpose of triage.

Cytokine and Chemokine Expression in Kidneys during Chronic Leptospirosis in Reservoir and Susceptible Animal Models.

Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. Humans can be infected after exposure to contaminated urine of reservoir animals, usually rodents, regarded as typical asymptomatic carriers of leptospires. In contrast, accidental hosts may present an acute form of leptospirosis with a range of clinical symptoms including the development of Acute Kidney Injury (AKI). Chronic Kidney Disease (CKD) is considered as a possible AKI-residual sequela but little is known about the renal pathophysiology consequent to leptospirosis infection. Herein, we studied the renal morphological alterations in relation with the regulation of inflammatory cytokines and chemokines, comparing two experimental models of chronic leptospirosis, the golden Syrian hamster that survived the infection, becoming carrier of virulent leptospires, and the OF1 mouse, a usual reservoir of the bacteria. Animals were monitored until 28 days after injection with a virulent L. borgpetersenii serogroup Ballum to assess chronic infection. Hamsters developed morphological alterations in the kidneys with tubulointerstitial nephritis and fibrosis. Grading of lesions revealed higher scores in hamsters compared to the slight alterations observed in the mouse kidneys, irrespective of the bacterial load. Interestingly, pro-fibrotic TGF-β was downregulated in mouse kidneys. Moreover, cytokines IL-1β and IL-10, and chemokines MIP-1α/CCL3 and IP-10/CXCL-10 were significantly upregulated in hamster kidneys compared to mice. These results suggest a possible maintenance of inflammatory processes in the hamster kidneys with the infiltration of inflammatory cells in response to bacterial carriage, resulting in alterations of renal tissues. In contrast, lower expression levels in mouse kidneys indicated a better regulation of the inflammatory response and possible resolution processes likely related to resistance mechanisms.

Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils

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.

Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence

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.

High level of IL-10 expression in the blood of animal models possibly relates to resistance against leptospirosis

HighlightsHigher IL‐10/TNF‐&agr; and IL‐10/IL‐1&bgr; expression ratios in mouse compared to hamster.Differential expression independently of the Leptospira strain.Neutralisation of IL‐10 led to increased susceptibility in Leptospira‐infected mouse.IL‐10 has a role in regulation of IL‐1&bgr; during leptospirosis.IL‐10 neutralization did not interact with TNF‐&agr; expression level. Abstract Leptospirosis is a severe zoonosis which immunopathogenesis is poorly understood. We evaluated correlation between acute form of the disease and the ratio of the anti‐inflammatory cytokine IL‐10 to the pro‐inflammatory TNF‐&agr; and IL‐1&bgr; expression during the early phase of infection comparing resistant mice and susceptible hamsters infected with two different species of virulent Leptospira. The IL‐10/TNF‐&agr; and IL‐10/IL‐1&bgr; expression ratios were higher in mouse compared to hamster independently of the Leptospira strain, suggesting a preponderant role of the host response and notably these cytokines in the clinical expression and survival to leptospirosis. Using an IL‐10 neutralization strategy in Leptospira‐infected mouse model, we also showed evidence of a possible role of this cytokine on host susceptibility, bacterial clearance and on regulation of cytokine gene expression.

Experimental Hamster Infection with a Strain of Leptospira borgpetersenii Ballum Isolated from a Reservoir Mouse in New Caledonia.

Leptospirosis is a neglected zoonosis caused by pathogenic Leptospira. In this study, we characterized the virulence of isolate B3-13S obtained from a wild mouse (Mus musculus) captured in New Caledonia, subsequently identified as a bacterium belonging to the L. borgpetersenii serogroup Ballum. Hamsters were infected with an intraperitoneal injection of 2 × 10(8) bacteria, resulting in severe histopathological organ damages consistent with tissue lesions previously observed with other strains. Hamsters were also injected with 1 × 10(8) or 5 × 10(7) bacteria and animals that recovered showed renal carriage of leptospires in concentrations similar to the bacterial load quantified in mouse kidneys, with urinary shedding of bacteria up to 4 weeks postinfection. The serogroup Ballum is increasingly reported in human leptospirosis, and these results highlight the use of the B3-13S isolate for the development of models resulting in either severe acute or chronic forms of the infection, allowing for better characterization of its pathogenesis.

An exotic case of leptospirosis imported into an endemic area

a Institut Pasteur in New Caledonia, Leptospirosis Research and Expertise Unit, BP 61, 98845 Noumea cedex, New Caledonia b Institut Pasteur in New Caledonia, Medical Biology Laboratory, Noumea, New Caledonia c Polyvalent Medicine Department, Centre Hospitalier Territorial, Noumea, New Caledonia d Intensive Care Unit, Centre Hospitalier Territorial, Noumea, New Caledonia e Internal Medicine Department, Centre Hospitalier Territorial, Noumea, New Caledonia

Biodiversity of Environmental Leptospira: Improving Identification and Revisiting the Diagnosis

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.