Cyrille Goarant

Leptospira: The Dawn of the Molecular Genetics Era for an Emerging Zoonotic Pathogen

Leptospirosis is a zoonotic disease that has emerged as an important cause of morbidity and mortality among impoverished populations. One hundred years after the discovery of the causative spirochaetal agent, little is understood about Leptospira spp. pathogenesis, which in turn has hampered the development of new intervention strategies to address this neglected disease. However, the recent availability of complete genome sequences for Leptospira spp. and the discovery of genetic tools for their transformation have led to important insights into the biology of these pathogens and their pathogenesis. We discuss the life cycle of the bacterium, the recent advances in our understanding and the implications for the future prevention of leptospirosis.

Detection of Zika Virus in Urine

We describe the kinetics of Zika virus (ZIKV) detection in serum and urine samples of 6 patients. Urine samples were positive for ZIKV >10 days after onset of disease, which was a notably longer period than for serum samples. This finding supports the conclusion that urine samples are useful for diagnosis of ZIKV infections.

Experimental infection models for shrimp vibriosis studies: a review

Vibrio species have become a major source of concern for shrimp culture because of their close association with low survival rates in hatcheries or growout ponds. New shrimp pathogens belonging to the Vibrio genus have been described although their virulence is not yet fully understood. Indeed, they may act as opportunistic agents in secondary infections or be true pathogens. This review presents the usefulness of infection models with vibriosis pathogens for pathogenicity experiments, testing of curative or prophylactic treatments and the study of host-factors influencing bacterial virulence. Furthermore, some guidelines for experimental trials are given to evaluate the in vivo virulence of Vibrio isolates.

Rapid Leptospira identification by direct sequencing of the diagnostic PCR products in New Caledonia

BackgroundMost of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment.ResultsBoth the lfb1 and secY diagnostic PCR products displayed a sequence polymorphism that could prove useful in presumptively identifying the infecting leptospire. Using both this polymorphism and MLST results with New Caledonian isolates and clinical samples, we confirmed the epidemiological relevance of the sequence-based identification of Leptospira strains. Additionally, we identified one cluster of L. interrogans that contained no reference strain and one cluster of L. borgpetersenii found only in the introduced Rusa deer Cervus timorensis russa that is its probable reservoir.ConclusionsThe sequence polymorphism of diagnostic PCR products proved useful in presumptively identifying the infecting Leptospira strains. This could contribute to a better understanding of leptospirosis epidemiology by providing epidemiological information that cannot be directly attained from the use of PCR as an early diagnostic test for leptospirosis.

Risk factors and predictors of severe leptospirosis in new caledonia.

Background Leptospirosis is a major public health concern in New Caledonia (NC) and in other tropical countries. Severe manifestations of the disease are estimated to occur in 5–15% of all human infections worldwide and factors associated with these forms are poorly understood. Our objectives were to identify risk factors and predictors of severe forms of leptospirosis in adults. Methods and Findings We conducted a retrospective case-control study of inpatients with laboratory-confirmed leptospirosis who were admitted to two public hospitals in NC in 2008–2011. Cases were patients with fatal or severe leptospirosis, as determined by clinical criteria. This approach was meant to be pragmatic and to reflect the routine medical management of patients. Controls were defined as patients hospitalized for milder leptospirosis. Risk and prognostic factors were identified by multivariate logistic regression. Among the 176 patients enrolled in the study, 71 had criteria of severity including 10 deaths (Case Fatality Rate = 14.1%). Three risk factors were independently associated with severe leptospirosis: current cigarette smoking (OR = 2.94 [CI 1.45–5.96]); delays >2 days between the onset of symptoms and the initiation of antibiotherapy (OR = 2.78 [CI 1.31–5.91]); and Leptospira interrogans serogroup Icterohaemorrhagiae as the infecting strain (OR = 2.79 [CI 1.26–6.18]). The following post-admission laboratory results correlated with poor prognoses: platelet count ≤50,000/µL (OR = 6.36 [CI 1.79–22.62]), serum creatinine >200 mM (OR = 5.86 [CI 1.61–21.27]), serum lactate >2.5 mM (OR = 5.14 [CI 1.57–16.87]), serum amylase >250 UI/L (OR = 4.66 [CI 1.39–15.69]) and leptospiremia >1000 leptospires/mL (OR = 4.31 [CI 1.17–15.92]). Conclusions To assess the risk of developing severe leptospirosis, our study illustrates the benefit for clinicians to have: i) the identification of the infective strain, ii) a critical threshold of qPCR-determined leptospiremia and iii) early laboratory results. In New Caledonia, preventative measures should focus on early presumptive antibacterial therapy and on rodent (reservoir of Icterohaemorrhagiae serogroup) control.

Rodent Abundance Dynamics and Leptospirosis Carriage in an Area of Hyper-Endemicity in New Caledonia

Background Widespread but particularly incident in the tropics, leptospirosis is transmitted to humans directly or indirectly by virtually any Mammal species. However, rodents are recognized as the most important reservoir. In endemic regions, seasonal outbreaks are observed during hot rainy periods. In such regions, hot spots can be evidenced, where leptospirosis is “hyper-endemic”, its incidence reaching 500 annual cases per 100,000. A better knowledge of how rodent populations and their Leptospira prevalence respond to seasonal and meteorological fluctuations might help implement relevant control measures. Methodology/Principal Findings In two tribes in New Caledonia with hyper-endemic leptospirosis, rodent abundance and Leptospira prevalence was studied twice a year, in hot and cool seasons for two consecutive years. Highly contrasted meteorological situations, particularly rainfall intensities, were noted between the two hot seasons studied. Our results show that during a hot and rainy period, both the rodent populations and their Leptospira carriage were higher. This pattern was more salient in commensal rodents than in the sylvatic rats. Conclusions/Significance The dynamics of rodents and their Leptospira carriage changed during the survey, probably under the influence of meteorology. Rodents were both more numerous and more frequently carrying (therefore disseminating) leptospires during a hot rainy period, also corresponding to a flooding period with higher risks of human exposure to waters and watered soils. The outbreaks of leptospirosis in hyper-endemic areas could arise from meteorological conditions leading to both an increased risk of exposure of humans and an increased volume of the rodent reservoir. Rodent control measures would therefore be most effective during cool and dry seasons, when rodent populations and leptospirosis incidence are low.

Outbreak of leptospirosis in New Caledonia: diagnosis issues and burden of disease

A leptospirosis epidemic affected New Caledonia during the first semester of 2008. A total of 135 cases were diagnosed with a relatively low fatality rate of 3.7%. Heavy rainfalls, related to La Niña, favoured this epidemic. The PCR, routinely used, confirmed 54% of the cases, and the microagglutination test 56%. Epidemiological and economical data on this epidemic are presented and discussed.

A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection

Understanding of antimicrobial defence mechanisms of penaeid shrimp should help in the design of efficient strategies for the management and disease control in aquaculture. In this study, we have specifically analysed the expression in circulating hemocytes of antimicrobial peptides (AMPs) encoding genes, such as PEN2 and PEN3, ALF, crustin, lysozyme and a putative cysteine-rich peptide. We evidenced a relationship between the level of expression of some AMPs and the successful response of the shrimp, Litopenaeus stylirostris, to circumvent a pathogenic Vibrio penaeicida infection. Additionally, significant differences in some AMP transcript amounts are evidenced between control, non-selected shrimp line and the third generation breeding of shrimp selected for their survival to natural V. penaeicida infections. On the basis of these results, it will now be of great interest to determine if these AMPs are directly involved in the resistance of shrimp to infection or if they only reflect other acquired defence mechanisms which can confer a resistance.

Gene expression profiles of immune mediators and histopathological findings in animal models of leptospirosis: comparison between susceptible hamsters and resistant mice.

ABSTRACT Leptospirosis is a widespread zoonosis characterized by multiple organ failure and variable host susceptibility toward pathogenic Leptospira strains. In this study, we put the role of inflammatory mediators in parallel with bacterial burdens and organ lesions by comparing a susceptible animal model, the hamster, and a resistant one, the Oncins France 1 (OF1) mouse, both infected with virulent Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun. Histological observations evidenced edema, congestion, hemorrhage, and inflammatory infiltration in the organs of hamsters, in contrast to limited changes in mice. Using reverse transcription-quantitative PCR techniques, we showed that the relative Leptospira burden progressively increased in hamster tissues, while a rapid clearance was observed in mouse tissues. The early regulation of the proinflammatory mediators interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and cyclo-oxygenase-2 and the chemokines gamma interferon-inducible protein 10 kDa/CXCL10 and macrophage inflammatory protein-1α/CCL3 in mouse tissues contrasted with their delayed and massive overexpression in hamster tissues. Conversely, the induction of the anti-inflammatory cytokine IL-10 was faster in the resistant than in the susceptible animal model. The role of these cytokines in the pathophysiology of leptospirosis and the implications of their differential regulation in the development of this disease are discussed.

Differential cytokine gene expression according to outcome in a hamster model of leptospirosis.

Background Parameters predicting the evolution of leptospirosis would be useful for clinicians, as well as to better understand severe leptospirosis, but are scarce and rarely validated. Because severe leptospirosis includes septic shock, similarities with predictors evidenced for sepsis and septic shock were studied in a hamster model. Methodology/Principal Findings Using an LD50 model of leptospirosis in hamsters, we first determined that 3 days post-infection was a time-point that allowed studying the regulation of immune gene expression and represented the onset of the clinical signs of the disease. In the absence of tools to assess serum concentrations of immune effectors in hamsters, we determined mRNA levels of various immune genes, especially cytokines, together with leptospiraemia at this particular time-point. We found differential expression of both pro- and anti-inflammatory mediators, with significantly higher expression levels of tumor necrosis factor α, interleukin 1α, cyclo-oxygenase 2 and interleukin 10 genes in nonsurvivors compared to survivors. Higher leptospiraemia was also observed in nonsurvivors. Lastly, we demonstrated the relevance of these results by comparing their respective expression levels using a LD100 model or an isogenic high-passage nonvirulent variant. Conclusions/Significance Up-regulated gene expression of both pro- and anti-inflammatory immune effectors in hamsters with fatal outcome in an LD50 model of leptospirosis, together with a higher Leptospira burden, suggest that these gene expression levels could be predictors of adverse outcome in leptospirosis.