First detection of Leishmania major in dogs living in an endemic area of zoonotic cutaneous leishmaniasis in Tunisia.

BACKGROUND: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL. METHODS: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR. RESULTS: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL. CONCLUSIONS: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation.

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana.

Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i) in North Africa caused by Leishmania (L.) major, L. tropica, and L. infantum, which caused in most cases localized autoresolutives forms, and ii) in French Guiana resulting from L. guyanensis and L. braziliensis, two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL.

Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Enriched PUFA environment of Leishmania infantum promastigotes promotes the accumulation of lipid mediators and favors parasite infectivity towards J774 murine macrophages.

Leishmania parasites are the causative agents of visceral or cutaneous leishmaniasis in humans and of canine leishmaniosis. The macrophage is the predilected host cell of Leishmania in which the promastigote stage is transformed into amastigote. We previously showed changes in the fatty acid composition (FA) of lipids in two strains of Leishmania donovani upon differentiation of promastigote to amastigote, including increased proportions of arachidonic acid (AA) and to a less extent of docosahexaenoic acid (DHA). Here, we carried out supplementation with AA or DHA on two Leishmania infantum strains, a visceral (MON-1) and a cutaneous (MON-24), to evaluate the role of these FA in parasite/macrophage interactions. The proportions of AA or DHA in total lipids were significantly increased in promastigotes cultured in AA- or DHA-supplemented media compared to controls. The content of FA-derived oxygenated metabolites was enhanced in supplemented strains, generating especially epoxyeicosatrienoic acids (11,12- and 14,15-EET) and hydroxyeicosatetraenoic acids (5- and 8- HETE) from AA, and hydroxydocosahexaenoic acids (14- and 17-HDoHE) from DHA. For both MON-1 and MON-24, AA-supplemented promastigotes showed higher infectivity towards J774 macrophages as evidenced by higher intracellular amastigote numbers. Higher infectivity was observed after DHA supplementation for MON-24 but not MON-1 strain. ROS production by macrophages increased upon parasite infection, but only minor change was observed between control and supplemented parasites. We propose that under high AA or DHA environment that is associated with AA or DHA enrichment of promastigote lipids, FA derivatives can accumulate in the parasite, thereby modulating parasite infectivity towards host macrophages.

ITS1 and cpb genetic polymorphisms in Algerian and Tunisian Leishmania infantum isolates from humans and dogs.

Leishmania (L.) infantum strains, isolated from varying hosts and clinical manifestations (cutaneous, visceral and canine leishmaniasis), were investigated in order to understand the genetic polymorphisms within this species in Algeria and Tunisia. Two DNA-based typing methods were tested in order to evaluate their effectiveness against Multilocus enzyme electrophoresis (MLEE), widely considered as the reference method for Leishmania parasite typing. On the other hand, MLEE is cumbersome, high-cost, time consuming and frequently does not detect intra-species genetic polymorphisms. In this work, we used two molecular target regions to discriminate L. infantum strains, Internal transcribed spacer 1 (ITS1) and the cysteine proteinase B (cpb). The ITS1 region offers good resolution for Leishmania discrimination but does not spotlight intra-species polymorphisms. In contrast, cpbE and cpbF PCR-Sequencing demonstrated a certain variability within CL and VL Algerian and Tunisian L. infantum isolates. Following phylogenetic analyses of 44 L. infantum isolates, two main groups were identified, a group with 39 bp deletion in the cpb sequence, composed of cutaneous, visceral and canine isolates from both countries with no significant clinical or geographic distribution; these samples were typed as MON-1, MON-24, and MON-80 zymodemes. A second group which presents a clear clusterization of Tunisian cutaneous strains belonging to the L. infantum MON-24. This group, with no deletion in the mature domain of the cpb gene sequence, should be further explored with a higher number of samples.

Mediterranean visceral leishmaniasis : update on biological diagnosis.

Visceral leishmaniasis (VL) is a severe life threatening parasitosis requiring early management of cases. It is an emerging disease in the Mediterranean region with a spread of endemic areas and an increase in case incidence. The patient profile has also evolved with more affected adults, presenting generally non-specific symptoms. Hence the interest of a systematic biological confirmation. The microscopic detection of Leishmania amastigotes in bone marrow aspirates (BMA) smears is the gold standard diagnostic technique. However, it requires invasive sampling. Serological tests searching for specific antibodies remain highly contributory, but their interpretation must always take into account the epidemiological context and the patient's clinical and biological features. Currently, the Western-Blot represents the most specific serological technique for diagnostic confirmation. VL diagnosis has greatly improved by the introduction of both rapid diagnostic tests (RDTs) and molecular biological techniques. RDTs using recombinant rk39 antigen are easy to perform and deliver results in less than 30 minutes. Real-time PCR (Polymerase Chain Reaction) is currently retained as the best technique for VL diagnosis. It is efficient on simple blood samples, allowing to avoid invasive BMA needed for microscopy. In addition, real time PCR estimates parasite load which is helpful for the post-treatment follow-up. In any case, the choice of techniques to be used should be strategic and adapted to the local epidemiology as well as to the means available.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

[Knowledge of malaria and preventive attitudes of Tunisian travellers toward malaria endemic areas]. / Connaissances du paludisme et attitudes préventives des voyageurs tunisiens vers les zones d'endémie palustre.

Introduction: travellers to endemic areas must know malaria, its risk factors and prophylactic measures. This can help to avoid severe cases of malaria and to prevent transmission in countries that are malaria-free. The purpose of this study is to assess Tunisian travellers´ knowledge about malaria, its transmission and prevention and their adherence to prophylactic measures. Methods: we conducted a survey based on two anonymous questionnaires (pre- and post-trip) among adults travelling to endemic countries. The 1st questionnaire was followed by a medical interview focusing on level of risk and recommended prophylactic measures. Results: two hundred and eighty-nine travellers were recruited. They mainly moved within sub-Saharan Africa (99%) for professional reasons (84,4%). The average age of subjects was 42.3 years and sex ratio (male/female) was 3.1. Prior to departure, only 53.3% of subjects were aware of the risk of malaria, and only 28% gave correct answers about modes of transmission. Recommendations for chemoprophylaxis were only known by 62.3% of subjects and only 43.6% intended to use chemoprophylaxis (p < 0.01). Better adherence to protective measures, including chemoprophylaxis, was reported after the trip, with attitudes qualified as good or excellent by 64.2% on return against 23.7% before the interview (<0.001). Conclusion: Tunisian travellers knowledge of malaria is insufficient. Strengthening information through specialized consultations (whose usefulness has been demonstrated) is required.

rs401502 and rs11575934 Polymorphisms of the IL-12 Receptor Beta 1 Gene are Protective Against Colorectal Carcinogenesis.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia. It ranks among the main cancers in terms of incidence and cancer-related cause of death. Its pathogenesis is currently considered to be multifactorial involving genetic and environmental factors. Recent studies have suggested that the gene encoding the ß1 subunit of the IL-12 receptor, an important pro-inflammatory cytokine of the anti-tumor response, could be involved in the susceptibility to inherited CRC. Hence, it would be interesting to study the role of single nucleotide polymorphisms (SNPs) within the IL-12RB1 gene (rs401502 and rs11575934) in CRC susceptibility. Aim: Our purpose was to assess whether genetic variants IL-12RB1 +1196G/C (rs401502) and IL-12RB1 +705A/G (rs11575934) within the IL-12RB1 gene are associated with the sporadic CRC risk. Methods: A total of 110 Tunisian patients with sporadic CRC and 141 healthy control subjects were included in this study. Genotyping was performed by high-resolution melting (HRM) analysis. All results were confirmed by direct DNA sequencing or PCR-RFLP methods. Later, the allele frequencies and genotype distribution were established and compared between the control group and CRC patients. Results: The obtained results showed that the two target SNPs were in Hardy-Weinberg equilibrium (HWE) in both patients and controls. Minor allele frequencies of rs401502 SNP were 16.4% in CRC cases and 23.8% in controls. Mutant allele of rs11575934 SNP was present with 21.4% in CRC patients and 29.8% in control group. An association study showed a significant association of two target polymorphisms with CRC, according to the dominant genetic model with OR = 0.577, 95% CI = [0.343 to 0.972], p = 0.038 and OR = 0.547, 95% CI = [0.328 to 0.911], p = 0.02, respectively. Conclusion: In this study, we found, for the first time, a potential protective effect of two SNPs in the IL-12RB1 gene, namely rs401502 and rs11575934, in sporadic colorectal cancer in Tunisians.

Diversity, Abundance and Leishmania infantum Infection Rate of Phlebotomine Sandflies in an Area with Low Incidence of Visceral Leishmaniasis in Northern Tunisia.

We report the study of sandfly Leishmania infection in an area of low incidence of visceral leishmaniasis in Tunisia. Sandflies were collected monthly using CDC light-traps set in houses and animal shelters during May-November 2016 and 2017. All males were identified at the species level. A sample of 878 females including all gravid specimens was subjected to kDNA qPCR for Leishmania detection and parasite load estimation. Leishmania species were determined by ITS1 PCR sequencing, and species identification of infected sandflies was performed by DNA barcoding. Phlebotomus perfiliewi and P. perniciosus were the dominant species during the two-year period. However, comparison of their relative abundances showed that P. perniciosus was more abundant during peaks of 2017 with longer activity duration. Real-time kDNA PCR did not detect Leishmania infection in 2016, although it identified four positive specimens (0.7%) in 2017. All four infected specimens were identified as P. perniciosus. ITS1 PCR sequencing allowed L. infantum identification in one kDNA qPCR-positive specimen. This was a P. perniciosus gravid female with a high parasite load caught during the long-lasting peak of 2017. This work highlights the usefulness of multi-seasonal studies of sandfly dynamics and kDNA qPCR in screening Leishmania infection and determining L. infantum vectors in hypo-endemic foci of human leishmaniasis.

Amplicon-based next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection of single-celled parasites in human faecal samples.

Comprehensive detection and differentiation of intestinal protists mostly rely on DNA-based methods. Here, we evaluated next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection and differentiation of intestinal eukaryotic protists in the stool of healthy Tunisian individuals. Thirty-six faecal DNA samples previously evaluated by microscopy and ameboid species-specific PCRs were tested. The hypervariable regions V3-V4 and V3-V5 of the 18S rRNA gene were amplified using three universal eukaryotic primer sets and sequenced using Illumina®MiSeq sequencing. In addition, real-time PCR assays were used to detect Dientamoeba fragilis, Giardia duodenalis, and Cryptosporidium spp. The metabarcoding assay detected Blastocystis (subtypes 1, 2, and 3) and archamoebid species and subtypes (Entamoeba dispar, Entamoeba hartmanni, Entamoeba coli RL1 and RL2, Endolimax nana, Iodamoeba bütschlii RL1) in 27 (75%) and 22 (61%) of the 36 stool samples, respectively. Meanwhile, the assay had limited sensitivity for flagellates as evidenced by the fact that no Giardia-specific reads were found in any of the five Giardia-positive samples included, and Dientamoeba-specific reads were observed only in 3/13 D. fragilis-positive samples. None of the samples were positive for Cryptosporidium by any of the methods. In conclusion, a large variety of intestinal eukaryotic protists were detected and differentiated at species and subtype level; however, limited sensitivity for common flagellates was observed.

Rapid high-resolution melting method to identify human leukocyte antigen-G (HLA-G) 3' untranslated region polymorphism +3142C/G (rs1063320).

BACKGROUND: HLA-G is a non-classical class I gene of the human Major Histocompatibility encoding molecules with immune-modulatory properties. Expression of HLA-G is being largely studied in pathological conditions, such as tumors, viral infections, inflammation, and autoimmune diseases, grafted tissues, among others. HLA-G +3142C/G (rs1063320: dbSNP database) polymorphism is located in 3' UTR of HAL-G and plays a key role in determining the magnitude of gene and protein expression. The detection of HLA-G +3142C/G polymorphism in the most published report is done through polymerase chain reaction followed by enzymatic digestion. Therefore, it is so interesting to develop a rapid and sensitive assay to genotype HLA-G +3142C/G polymorphism. High-resolution melt analysis (HRM) is a technology that is based on the analysis of the melting profile of PCR products through gradual temperature increase. The aim of this work is to apply high-resolution melt method for genotyping the HLA-G +3142C/G polymorphism. METHODS: DNA from 118 individuals was extracted from whole blood with QIAamp® DNA blood mini kit (Qiagen, Germany). Primer couple was designed using Primer 3 online tools so as to have only one SNP in the target sequence for high HRM efficiency. Positive Controls were identified using DNA sequencing and used as reference when assigning genotypes for trial samples. RESULTS: We were able to recognize the three genotypes with similar accuracy than DNA sequencing using high resolution melting method. Hardy-Weinberg equilibrium test shows that our population is in equilibrium for the studied SNP. Genotypes frequencies of +3142C/G polymorphism in Tunisian general population are 0.475 for heterozygote G/C, 0.186 for homozygote G/G and 0.339 for homozygote C/C. CONCLUSION: HRM is a cost-effective method suitable for SNP genotyping.

Cutaneous Leishmaniasis in Algeria; Highlight on the Focus of M'Sila.

Algeria ranks second after Afghanistan for the incidence of cutaneous leishmaniasis (CL) worldwide. Here, we report a 34-years retrospective analysis of CL in Algeria and focused on the most affected region, the M'Sila province. All 66 cutaneous isolates corresponded to Leishmania (L.) major. Our study of the sandfly and rodent fauna further highlighted the high density of Phlebotomus papatasi and additional phlebotomine species of medical importance, not previously identified in M'Sila. Wild rodents belonging to nine species were trapped in M'Sila, and Psammomys obesus and Meriones shawi were found infected by L. major. In addition, Leishmania infantum was isolated from two visceral leishmaniasis cases, one dog and its proven vectors (P. perniciosus, P. longicuspis, and P. perfiliewi) inventoried during the survey. The high incidence of CL in the M'Sila province is likely a consequence of the increase in minimum temperatures recorded that constitutes suitable conditions for establishing a high endemicity and leads to an explosive rise in leishmaniases cases in this region. A thorough investigation of the underlying risk factors is urgently needed to detect new cases earlier. All these would improve the preparedness to fight the disease.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. MATERIALS AND METHODS: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. RESULTS: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). CONCLUSION: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.

Nuclear and mitochondrial genome sequencing of North-African Leishmania infantum isolates from cured and relapsed visceral leishmaniasis patients reveals variations correlating with geography and phenotype.

Although several studies have investigated genetic diversity of Leishmania infantum in North Africa, genome-wide analyses are lacking. Here, we conducted comparative analyses of nuclear and mitochondrial genomes of seven L. infantum isolates from Tunisia with the aim to gain insight into factors that drive genomic and phenotypic adaptation. Isolates were from cured (n=4) and recurrent (n=3) visceral leishmaniasis (VL) cases, originating from northern (n=2) and central (n=5) Tunisia, where respectively stable and emerging VL foci are observed. All isolates from relapsed patients were from Kairouan governorate (Centre); one showing resistance to the anti-leishmanial drug Meglumine antimoniate. Nuclear genome diversity of the isolates was analysed by comparison to the L. infantum JPCM5 reference genome. Kinetoplast maxi and minicircle sequences (1 and 59, respectively) were extracted from unmapped reads and identified by blast analysis against public data sets. The genome variation analysis grouped together isolates from the same geographical origins. Strains from the North were very different from the reference showing more than 34 587 specific single nucleotide variants, with one isolate representing a full genetic hybrid as judged by variant frequency. Composition of minicircle classes within isolates corroborated this geographical population structure. Read depth analysis revealed several significant gene copy number variations correlating with either geographical origin (amastin and Hsp33 genes) or relapse (CLN3 gene). However, no specific gene copy number variation was found in the drug-resistant isolate. In contrast, resistance was associated with a specific minicircle pattern suggesting Leishmania mitochondrial DNA as a potential novel source for biomarker discovery.

Assessment of Incubation Period of Cutaneous Leishmaniasis due to Leishmania major in Tunisia.

The period between the infective sandfly bites and appearance of cutaneous leishmaniasis (CL) lesions is still hypothetical and little studied. This work aimed at assessing the incubation time of zoonotic CL (ZCL) due to Leishmania major using a standardized methodology. The retrospective analysis used the epidemiological, clinical, and biological information available in the database recording all the CL cases diagnosed at the Parasitology Department of the Pasteur Institute of Tunis during 2015-2019. It allowed for the selection of 92 privileged observations 1) of confirmed CL cases with presentation suggestive of ZCL form 2) living in northern regions free of ZCL 3) with a single infective trip of less than a week to ZCL foci during transmission season and 4) with accurate dates of travel and onset of lesions. Incubation length computed in this population ranged from 1 to 21 weeks, with a median of 5 weeks (interquartile range: 3-8.5 weeks).

Evaluation of six commercial kits for the serological diagnosis of Mediterranean visceral leishmaniasis.

BACKGROUND: Zoonotic visceral leishmaniasis (VL) is endemic in the Mediterranean basin. However, large-scale comparative analyses of the commercial kits for the serological diagnosis of this neglected disease are lacking. This study compared the performances of four enzyme-linked immunosorbent assays (ELISA) and two immunochromatographic tests (ICT) as screening tests for the serodiagnosis of human VL in the Mediterranean region. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples from 319 patients living in France, Tunisia or Morocco were tested using two ICT (IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four ELISA reagents (NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania). The population with proven VL (n = 181) included 65 immunocompromised patients. Significantly higher percentages of false-negative results were obtained with all assays in immunocompromised patients, compared with the immunocompetent population. In the whole population, sensitivity and specificity ranged from 80.7% to 93.9% and from 95.7% to 100%, respectively. The maximum accuracy was observed with the Bordier and Vircell ELISA kits (96.2%), and the lowest accuracy with Ridascreen reagent (88.7%). New thresholds of positivity are proposed for the Bordier, Vircell and NovaLisa ELISA kits to achieve 95% sensitivity with the highest possible specificity. Western blot (WB), used as a confirmation method, showed 100% sensitivity and identified 10.1% of asymptomatic carriers among the control population from the South of France. CONCLUSIONS/SIGNIFICANCE: This is the first study that compared commercially available kits for VL serodiagnosis in the endemic region of the Mediterranean basin. It provides specific information about the tests' performance to help clinicians and biologists to select the right assay for VL screening.

Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic Leishmania proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects.

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 multi-epitope 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-II-restricted 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.