Research Article: Genetic Diversity and Population Structure of Leishmania infantum from Southeastern France: Evaluation Using Multi-Locus Microsatellite Typing

Date Published: January 25, 2016

Publisher: Public Library of Science

Author(s): Christelle Pomares, Pierre Marty, Anne Laure Bañuls, Emmanuel Lemichez, Francine Pratlong, Benoît Faucher, Fakhri Jeddi, Sandy Moore, Grégory Michel, Srikanth Aluru, Renaud Piarroux, Mallorie Hide, Gabriele Schönian.

Abstract: In the south of France, Leishmania infantum is responsible for numerous cases of canine leishmaniasis (CanL), sporadic cases of human visceral leishmaniasis (VL) and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL, respectively). Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). Within these endemic areas, the two cities of Nice (AM) and Marseille (P), which are located 150 km apart, and their surroundings, concentrate the greatest number of French autochthonous leishmaniasis cases. In this study, 270 L. infantum isolates from an extended time period (1978–2011) from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT). MLMT revealed a total of 121 different genotypes with 91 unique genotypes and 30 repeated genotypes. Substantial genetic diversity was found with a strong genetic differentiation between the Leishmania populations from AM and P. However, exchanges were observed between these two endemic areas in which it seems that strains spread from AM to P. The genetic differentiations in these areas suggest strong epidemiological structuring. A model-based analysis using STRUCTURE revealed two main populations: population A (consisting of samples primarily from the P and AM endemic areas with MON-1 and non-MON-1 strains) and population B consisting of only MON-1 strains essentially from the AM endemic area. For four patients, we observed several isolates from different biological samples which provided insight into disease relapse and re-infection. These findings shed light on the transmission dynamics of parasites in humans. However, further data are required to confirm this hypothesis based on a limited sample set. This study represents the most extensive population analysis of L. infantum strains using MLMT conducted in France.

Partial Text: Leishmaniases are a group of diseases caused by obligatory intracellular protozoan parasites of the genus Leishmania. Among the species of Leishmania, Leishmania infantum is mostly responsible for canine leishmaniasis (CanL), although it also causes sporadic cases of human visceral leishmaniasis (VL), and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL) throughout the Mediterranean basin [1]. Transmission to humans is caused by the bite of infected phlebotomine sandflies, and dogs are considered to be the principal domestic reservoir. In France, the parasite is currently only endemic in the south of France, along the Mediterranean coast, where several foci have been clearly identified: Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO) [2]. In the Provence-Alpes-Côtes d’Azur (PACA) region, which comprises the AM and P endemic areas, transmission has been reported for 100 years [3,4]. The two cities of Nice and Marseille, which are located 150 km apart, and their surroundings concentrate the greatest number of French autochthonous leishmaniasis cases [2,5]. Although the same species of L. infantum (primarily zymodeme MON-1), the same predominant vector (Phlebotomus perniciosus) and the same and unique reservoir (dog) are found in both regions, the transmission environment of VL is heterogeneous in these two foci [5]. Disease transmission in Nice and the surrounding area is associated with scattered habitation and mixed forest in the foothills [5]. In contrast, around Marseille, VL transmission is associated with an urban environment [5]. Regarding the main vector; Phlebotomus pernicious; the population is quite homogeneous and belongs mainly to the same haplogroup (for 88% pern01) in Provence, France [6]. The isolates of L. infantum from AM and P endemic foci have been characterized using Multi-Locus Enzyme Electrophoresis (MLEE), which is the current reference method. However, MLEE based analyses are limited at the intrinsic level of polymorphisms. Thus, differentiating between isolates in PACA region is impossible using the MLEE method [7]. Epidemiological studies on L. infantum require the use of highly discriminative techniques that can differentiate between MON-1 strains. Multi-Locus Microsatellite Typing (MLMT) has been shown to be a powerful tool for population genetics and epidemiological studies of Leishmania spp. [8]. This tool has been already applied to genotype L. infantum isolates from healthy blood donors, sandflies, dogs and human patients in Southern France [9]. Genetic differentiations were evidenced between asymptomatic carrier strains and non-asymptomatic carrier strains and especially between asymptomatic carrier and HIV+ populations [9]. However, due to the weak sample size, these results must be confirmed on a larger sample set [9].

Leishmaniasis due to L. infantum is endemic in Southern France. In this study, we used MLMT, a molecular tool useful for population genetic studies, to analyze an extensive set of isolates from four endemic areas in Southern France (AM, P, CE and CO). To the best of our knowledge, this study is the first to investigate a large number of L. infantum isolates from different endemic areas in Southern France. We also focused on the AM and P endemic areas over an extended period of time. A greater number of samples came from AM than P (AM = 178 versus P = 75) because AM is the most active foci in France with the greatest number of leishmaniasis cases per year [2]. The study period was also longer for the AM area than for the P endemic area (AM: 1978–2011; P: 1993–2009). This aspect may generate a sampling bias, although no significant genetic differentiation was found when comparing isolates from AM and P during the same time period. Although MON-1 is the most prevalent zymodeme, other zymodemes also circulate in the south of France [7]. Microsatellite characterization of L. infantum isolates revealed a total of 121 different genotypes. Overall, 91 unique genotypes and 30 repeated genotypes were found. A greater number of repeated genotypes were observed in AM compared with P for the same period, thereby suggesting variations in the transmission cycle between the two areas such as outbreak, vector diversity or density, or host density.



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