Jihene Bettaieb

Topical Paromomycin with or without Gentamicin for Cutaneous Leishmaniasis

BACKGROUND There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P<0.001 for each treatment group vs. the vehicle-control group). Cure of the index lesion was accompanied by cure of all other lesions except in five patients, one in each of the paromomycin groups and three in the vehicle-control group. Mild-to-moderate application-site reactions were more frequent in the paromomycin groups than in the vehicle-control group. CONCLUSIONS This trial provides evidence of the efficacy of paromomycin-gentamicin and paromomycin alone for ulcerative L. major disease. (Funded by the Department of the Army; ClinicalTrials.gov number, NCT00606580.).

Temporal Dynamics and Impact of Climate Factors on the Incidence of Zoonotic Cutaneous Leishmaniasis in Central Tunisia

Background Old world Zoonotic Cutaneous Leishmaniasis (ZCL) is a vector-borne human disease caused by Leishmania major, a unicellular eukaryotic parasite transmitted by pool blood-feeding sand flies mainly to wild rodents, such as Psammomys obesus. The human beings who share the rodent and sand fly habitats can be subverted as both sand fly blood resource. ZCL is endemic in the Middle East, Central Asia, Subsaharan and North Africa. Like other vector-borne diseases, the incidence of ZCL displayed by humans varies with environmental and climate factors. However, so far no study has addressed the temporal dynamics or the impact of climate factors on the ZCL risk. Principal Findings Seasonality during the same epidemiologic year and interval between ZCL epidemics ranging from 4 to 7 years were demonstrated. Models showed that ZCL incidence is raising i) by 1.8% (95% confidence intervals CI:0.0–3.6%) when there is 1 mm increase in the rainfall lagged by 12 to 14 months ii) by 5.0% (95% CI: 0.8–9.4%) when there is a 1% increase in humidity from July to September in the same epidemiologic year. Conclusion/Significance Higher rainfall is expected to result in increased density of chenopods, a halophytic plant that constitutes the exclusive food of Psammomys obesus. Consequently, following a high density of Psammomys obesus, the pool of Leishmania major transmissible from the rodents to blood-feeding female sand flies could lead to a higher probability of transmission to humans over the next season. These findings provide the evidence that ZCL is highly influenced by climate factors that could affect both Psammomys obesus and the sand fly population densities.

Risk Factors and Characteristics of Low Pathogenic Avian Influenza Virus Isolated from Commercial Poultry in Tunisia

Objective Estimate the seroprevalence of influenza A virus in various commercial poultry farms and evaluate specific risk factors as well as analyze their genetic nature using molecular assays. Materials and Methods This report summarizes the findings of a national survey realized from October 2010 to May 2011 on 800 flocks in 20 governorates. Serum samples were screened for the presence of specific influenza virus antibodies using cELISA test. Additionally, swab samples were tested by real time and conventional RT-PCR and compared with results obtained by others assays. Phylogenetic and genetic analyses of the glycoproteins were established for some strains. Results Out of the 800 chicken and turkey flocks tested by cELISA, 223 showed positive anti-NP antibodies (28.7%, 95% CI: 25.6–32.1). Significantly higher seroprevalence was found among the coastal areas compared to inland and during the autumn and winter. Broiler flocks showed significantly lower seroprevalence than layers and broiler breeders. The influenza virus infection prevalence increased after the laying phase among layer flocks. In addition, AIV seropositivity was significantly associated with low biosecurity measures. The Ag EIA and rRT-PCR tests revealed significantly higher numbers of AI positive samples as compared to cell cultures or egg inoculation. All new strains were subtyped as H9N2 by real time and conventional RT-PCR. Drift mutations, addition or deletion of glycosylation sites were likely to have occurred in the HA and NA glycoproteins of Tunisian strains resulting in multiple new amino acid substitutions. This fact may reflect different evolutionary pressures affecting these glycoproteins. The role of these newly detected substitutions should be tested. Conclusion Our findings highlight the potential risk of AIV to avian health. Strict enforcement of biosecurity measures and possible vaccination of all poultry flocks with continuous monitoring of poultry stations may ensure reduction of AIV prevalence and avoid emergence of more pathogenic strains.

Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the Vector of Leishmania major in Tunisia.

Background During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it. Methodology/Principal Findings Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva. Conclusions/Significance Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease.

Heterogeneity of hepatitis B transmission in Tunisia: Risk factors for infection and chronic carriage before the introduction of a universal vaccine program☆

UNLABELLED A population-based sero-epidemiological study enrolled 9486 volunteers in two governorates, Béja in the north and Tataouine in the south of Tunisia, in order to assess the magnitude of HBV transmission heterogeneity between the north and the south and within the same governorate, as well as the risk factors associated with infection and chronic carriage. RESULTS The overall prevalence of anti-HBc, HBsAg and chronic carriage was 28.5, 5.3 and 2.9%, respectively. Significant differences were observed between the two governorates according to anti-HBc (32.1% in Béja and 27.8% in Tataouine; p=0.005) and HBsAg prevalence (4.2% in Béja and 5.6% in Tataouine; p=0.001). Significant differences were noticed between districts revealing important heterogeneity in HBV transmission within the same governorate (HBsAg ranged from 12 to <2% within the same governorate). At the individual level, the presence of a family member infected with HBV, scarification practices, needle practices in the Primary Care Center and gender (male) significantly increased the risk of anti-Hbc, HBsAg positivity and chronic carriage of infection while existence of sanitation in the house was found to be protective. The basic reproductive number and the force of infection confirmed the heterogeneity of transmission. Horizontal transmission within the family explains hyperendemic clusters in Tunisia.

First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia

BackgroundMany sand fly species are implicated in the transmission cycle of Leishmania parasites around the world. Incriminating new sand flies species, as vectors of Leishmania is crucial to understanding the parasite–vector transmission cycle in different areas in Tunisia and surrounding countries.FindingsSeventy-four unfed females belonging to the genera Sergentomyia and Phlebotomus were collected in South Tunisia between June and November 2014, using sticky papers. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis of the internal transcribed spacer 1 (ITS1) was used for Leishmania parasites detection and identification. Leishmania (L.) major (Yakimoff & Shokkor, 1914) was identified within two Sergentomyia (S.) minuta (Rondani, 1843) and one Phlebotomus papatasi (Scopoli, 1786).ConclusionThis is the first report of L. major identified from S. minuta in Tunisia. This novel finding enhances the understanding of the transmission cycle of L. major parasites of cutaneous leishmaniasis in an endemic area in South Tunisia.

Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.

Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

Genotype Profile of Leishmania major Strains Isolated from Tunisian Rodent Reservoir Hosts Revealed by Multilocus Microsatellite Typing

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major parasites represents a major health problem with a large spectrum of clinical manifestations. Psammomys (P.) obesus and Meriones (M.) shawi represent the most important host reservoirs of these parasites in Tunisia. We already reported that infection prevalence is different between these two rodent species. We aimed in this work to evaluate the importance of genetic diversity in L. major parasites isolated from different proven and suspected reservoirs for ZCL. Using the multilocus microsatellites typing (MLMT), we analyzed the genetic diversity among strains isolated from (i) P. obesus (n = 31), (ii) M. shawi (n = 8) and (iii) Mustela nivalis (n = 1), captured in Sidi Bouzid, an endemic region for ZCL located in the Center of Tunisia. Studied strains present a new homogeneous genotype profile so far as all tested markers and showed no polymorphism regardless of the parasite host-reservoir origin. This lack of genetic diversity among these L. major isolates is the first genetic information on strains isolated from Leishmania reservoirs hosts in Tunisia. This result indicates that rodent hosts are unlikely to exert a selective pressure on parasites and stresses on the similarity of geographic and ecological features in this study area. Overall, these results increase our knowledge among rodent reservoir hosts and L. major parasites interaction.

Insight into the global evolution of Rodentia associated Morbilli-related paramyxoviruses

One portion of the family Paramyxoviridae is a group of Unclassified Morbilli-Related Viruses (UMRV) recently recognized in wild small mammals. At a global level, the evolutionary history of these viruses is not properly understood and the relationships between UMRV and their hosts still remain largely unstudied. The present study revealed, for the first time, that Rodentia associated UMRV emerged from a common ancestor in southern Africa more than 4000 years ago. Sequenced UMRV originating from different regions in the world, clustered into four well-supported viral lineages, which suggest that strain diversification occurred during host dispersal and associated exchanges, with purifying selection pressure as the principal evolutionary force. In addition, multi-introductions on different continents and islands of Rodentia associated UMRV and spillover between rodent species, most probably Rattus rattus, were detected and indicate that these animals are implicated in the vectoring and in the worldwide emergence of this virus group. The natural history and the evolution dynamics of these zoonotic viruses, originating from and hosted by wild animals, are most likely shaped by commensalism related to human activities.