Investigation of natural infection of Phlebotomine (Diptera: Psychodidae) by Leishmania in Tunisian endemic regions.

Leishmaniases are caused by protozoan parasites of the genus Leishmania transmitted by females blood-feeding phlebotomine insects (Diptera: Psychodidae). In Tunisia, cutaneous and visceral leishmaniases are of public health concern. In Tunisia, 17 species of phlebotomine sand flies are described. Here we investigate natural infection in Tunisian mixed foci regions of leishmaniases. We trap female sandflies during the Leishmania transmission season in the country's central-eastern and northern parts. We investigate Leishmania infection using PCR-RFLP targeting the ITS1 ribosomal DNA, followed by enzymatic digestion with HaeIII; then, we identify sand flies using molecular methodologies. We confirm the presence of Phlebotomus papatasi and Phlebotomus perniciosus infected by L. major and L. infantum parasites in Tunisia.

Development and Assessment of Leishmania major and Leishmania tropica Specific Loop-Mediated Isothermal Amplification Assays for the Diagnosis of Cutaneous Leishmaniasis in Tunisia.

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica, respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.

First detection of Leishmania DNA in Psammomys obesus and Psammomys vexillaris: Their potential involvement in the epidemiology of leishmaniasis in Tunisia.

Leishmaniasis, a public health problem in Tunisia, are diseases caused by different Leishmania species. Cutaneous leishmaniasis is present from the North to the South under different forms, due to Leishmania (L.) major, L. infantum or L. tropica. Whereas, Psammomys (P.) obesus is the confirmed reservoir host of L. major, those of L. tropica and dermotropic L. infantum wait to be identified. Importantly, P. vexillaris species have been recently highlighted; however, no studies have been carried out to explore its potential role in leishmaniasis epidemiology. Seventy two rodents were collected from Central and South-West of Tunisia between 2007 and 2010. Using several methods, 43 animals were identified as P. obesus and 29 as P. vexillaris. Leishmania kinetoplast DNA was detected in liver samples by real-time PCR in 18 P. obesus and in 8 P. vexillaris. Then, the direct sequencing of the amplified internal transcribed spacer 1, allowed the identification of L. infantum DNA in five P. obesus and in three P. vexillaris, as well as L. tropica DNA in three other P. vexillaris. Whereas, PCR fluorescent fragment length analysis of the 7 spliced leaders, allowed identifying L. major among infected P. obesus and P. vexillaris, and interestingly co-infection (L. major/L. infantum) among two P. obesus. We report here for the first time, the infection of P. obesus, from Central Tunisia, by L. infantum. Suggesting that P. obesus the known reservoir host of L. major, may also serve as reservoir host for L. infantum and thus play a role in the spread of sporadic cutaneous or visceral leishmaniasis in this region. Of equal importance, this work establish for the first time, the natural infection of P. vexillaris by different Leishmania species, suggesting its potential epidemiological role as reservoir host.

First detection of Leishmania major DNA in Sergentomyia (Sintonius) clydei (Sinton, 1928, Psychodidae: Phlebotominae), from an outbreak area of cutaneous leishmaniasis in Tunisia.

In recent years there has been growing interest in Sergentomyia species. Their role in the spread of mammalian leishmaniasis appears repeatedly in the literature and the possibility of its implication in Leishmania transmission to humans remains controversial. Sergentomyia (Sintonius) clydei is one of several cryptic species sharing therefore common morphologic criteria with others species of the subgenera Sintonius. Little is known about this specie in Tunisia. We sampled and identified different specimens including four specimens of S. clydei collected from Sidi Bouzid and Kairouan areas (center of Tunisia) using morphological tools. Male Sergentomyia clydei and Sergentomyia christophersi are known to share several morphological characters and can be mistaken for. Consequently we took advantage of 5 male S. christophersi available in our collection (Tataouin, South of Tunisia). In our study morphological tools were completed by molecular study of cytochrome b gene to identify S. clydei. For the detection of Leishmania spp. that might infect our specimens, Leishmania DNA was analyzed by amplification of kinetoplast minicircle DNA using real-time PCR and nested-PCR. Obtained result was confirmed by restriction analysis of the amplified ribosomal internal transcribed spacer 1 (ITS1). We provide in our study, the first molecular identification of S. clydei, in Tunisia. Our Neighbor Joining tree based on mitochondrial cytochrome b gene shows two different clusters. The first includes the Tunisians S. clydei and other specimens from Africa, Middle East and the Arabic peninsula, and the second cluster containing the specimens from Seychelle. Unexpectedly, we also demonstrate the infection of one anthropophilic female S. clydei by Leishmania major DNA. This finding shows that more attention should be paid when identifying parasites by molecular tools within sandfly vector.