ABSTBACT Knowledge of the host-feeding pattern of blood-sucking insects helps to understand the epidemiology of a vector-born disease. A set of primers was used to selectively amplify segment of vertebrates' prepronociceptin gene from abdomens of engorged sand flies. Vertebrate DNA was successfully amplified in 65% of blood-fed phlebotomines assayed. Direct sequencing and comparison of resultant sequences with sequences in GenBank, using Basic Local Alignment Search Tool, led to the specific identification of the host in 100% of the cases. In total, 249 blood-fed females belonging to five different sand flies species were captured thanks to Centers for Disease Control and Prevention light traps and sticky papers in different areas of Tunisia between 2007 and 2009. Bloodmeal origin was determined for 146 blood-fed midges: Phlebotomus sergenti Parrot sampled fed only on Ovis aries and Equus caballus, while bloodmeal origin of P. perniciosus Newstead, P. longicuspis Nitzulescu, and P. papatasi (Scopoli) was diversified. We found that midges were fed mainly on Homos sapiens (n = 37; 22.69%), Bos taunts (n = 11; 6.74%), Mus musculus (n = 2; 1.22%), Capra hircus (n = 4; 2.45%), Camelus dromedarius (n = 3; 1.84%), Ovis aries (n = 98; 60.12%), Equus caballus (n = 3; 1.84%), Felis catus (n = 1; 0.6%), Oryctolagus cuniculus (n = 3; 1.84%), and Rattus norvegicus (n = 1; 0.6%). In this study, interestingly, we found for the first time that Mus musculus DNA was found in one female of S. minuta (Rondani) specie and question about its possible vectorial role is opened.

Human cutaneous leishmaniasis, although designated as one of the most neglected tropical diseases, remains underestimated due to its misdiagnosis. The diagnosis is mainly based on the microscopic detection of amastigote forms, isolation of the parasite, or the detection of Leishmania DNA, in addition to its differential clinical characterization; these tools are not always available in routine daily practice, and they are expensive and time-consuming. Here, we present a simple-to-use, noninvasive approach for human cutaneous leishmaniasis diagnosis, which is based on the analysis of volatile organic compounds in exhaled breath with an array of specifically designed chemical gas sensors. The study was realized on a group of n = 28 volunteers diagnosed with human cutaneous leishmaniasis and a group of n = 32 healthy controls, recruited in various sites from Tunisia, an endemic country of the disease. The classification success rate of human cutaneous leishmaniasis patients achieved by our sensors test was 98.2% accuracy, 96.4% sensitivity, and 100% specificity. Remarkably, one of the sensors, based on CuNPs functionalized with 2mercaptobenzoxazole, yielded 100% accuracy, 100% sensitivity, and 100% specificity for human cutaneous leishmaniasis discrimination. While AuNPs have been the most extensively used in metal nanoparticle−ligand sensing films for breath sensing, our results demonstrate that chemical sensors based on ligand-capped CuNPs also hold great potential for breath volatile organic compounds detection. Additionally, the chemical analysis of the breath samples with gas chromatography coupled to mass spectrometry identified nine putative breath biomarkers for human cutaneous leishmaniasis.

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and-II (HLA-II)-restricted multiepitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-IIrestricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and-II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and-II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis.