Monitoring of Leptospira species diversity in freshwater bathing area and in rats in Paris, France.

Leptospirosis is a neglected zoonotic disease with a worldwide distribution caused by bacterial pathogenic Leptospira. Rodents are considered as the main reservoir of Leptospira and transmission usually occurs through exposure to urine-contaminated environment. However, interactions between environment, rodent reservoir and human leptospirosis remain poorly studied. Here, we evaluated the concentration of Leptospira in surface water and captured rats in the city of Paris (France) from 2018 to 2020 using an integrity qPCR (Quantitative Polymerase Chain Reaction). All environmental samples (n = 1031) were positive for saprophytic Leptospira but pathogenic Leptospira P1 group were only found in 40% (n = 363; 2018) to 0% (n = 264; 2020) of samples. In the same time, analysis of 200 brown rat corpses trapped in the city, showed about 15% of positivity for Leptospira but the different method used for rats conservation (based on presence or absence of conservative agent) showed important variations in the Leptospira prevalence. Metagenomic analysis, based on rrs gene sequencing, was also carried out to evaluate the distribution of Leptospira in samples. Results could indicate that some species of Leptospira are found in surface waters as well as rats, but further study is needed to accurately describe the nature of the link between these two reservoirs. Quantification of Leptospira and pathogenic species description circulating inside animal reservoir living in the vicinity of freshwater in urban areas, will be helpful to understand the eco-epidemiology of leptospirosis and to establish prevention and intervention strategies, especially in the context of organization of recreative activity events in these urban areas.

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

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