Rihab Yazidi

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.

First Report of Natural Infection of Least Weasel (Mustela nivalis Linnaeus, 1776) with Leishmania major in Tunisia

Zoonotic cutaneous leishmaniasis, caused by Leishmania major (L. major), is endemic in Tunisia. Several rodents have been identified as reservoir hosts of parasites. This study reports, for the first time, the natural infection with L. major zymodeme MON-25 in a specimen of least weasel: Mustela nivalis Linnaeus, 1776 (M. nivalis) collected in Sidi Bouzid. This finding justifies further research on larger samples of this animal to verify its role as a potential reservoir host for cutaneous leishmaniasis in 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.

Adaptive automatic segmentation of Leishmaniasis parasite in Indirect Immunofluorescence images.

This paper describes the first steps for the automation of the serum titration process. In fact, this process requires an Indirect Immunofluorescence (IIF) diagnosis automation. We deal with the initial phase that represents the fluorescence images segmentation. Our approach consists of three principle stages: (1) a color based segmentation which aims at extracting the fluorescent foreground based on k-means clustering, (2) the segmentation of the fluorescent clustered image, and (3) a region-based feature segmentation, intended to remove the fluorescent noisy regions and to locate fluorescent parasites. We evaluated the proposed method on 40 IIF images. Experimental results show that such a method provides reliable and robust automatic segmentation of fluorescent Promastigote parasite.

Hybrid segmentation of fluorescent leschmania-infected images using a watersched and combined region merging based method

Automatic parasite segmentation in fluorescent images is of high importance as it serves as an easier and faster tool for detecting and counting parasites in each focus. In this paper we present a hybrid segmentation for the Promastigote form of Leishmania parasites in Indirect Immunofluorescence (IIF) images, combining edge and region-based techniques through the morphological algorithm of watershed. The proposed approach deals first with a pre-processing step to correct illumination non-uniformities in the fluorescence Leishmania-infected images before performing the initial segmentation by means of the watershed algorithm. A merging step using joint region homogeneity and edge integrity criteria is then applied to improve the segmentation results. Segmentation tests of 1438 parasites from 40 collected IIF images illustrate the efficiency of our approach.Automatic parasite segmentation in fluorescent images is of high importance as it serves as an easier and faster tool for detecting and counting parasites in each focus. In this paper we present a hybrid segmentation for the Promastigote form of Leishmania parasites in Indirect Immunofluorescence (IIF) images, combining edge and region-based techniques through the morphological algorithm of watershed. The proposed approach deals first with a pre-processing step to correct illumination non-uniformities in the fluorescence Leishmania-infected images before performing the initial segmentation by means of the watershed algorithm. A merging step using joint region homogeneity and edge integrity criteria is then applied to improve the segmentation results. Segmentation tests of 1438 parasites from 40 collected IIF images illustrate the efficiency of our approach.

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.

First Report on Natural Infection of Phlebotomus sergenti with Leishmania tropica in a Classical Focus of Leishmania major in Tunisia

In Tunisia, chronic cutaneous leishmaniasis due to Leishmania tropica is an important health problem. Its spreading has not been fully elucidated. Information on sandfly vectors, as well as their associated Leishmania species, is of paramount importance since vector dispersion is one of the major factors responsible for pathogen dissemination. Ninety-seven unfed females belonging to the genera Sergentomyia and Phlebotomus were collected between June and August 2015 using sticky paper traps. Polymerase chain reaction-restriction fragment length polymorphism analysis of the internal transcribed spacer 1and sequencing were used for Leishmania detection and identification. In total, 650 sandflies were captured and identified (380 males and 270 females). Ninety-seven unfed females were tested for the presence of Leishmania parasite DNA. Six Phlebotomus sergenti were found positive for L. tropica. This novel finding enhances the understanding of the cycle extension of L. tropica outside its original focus of Tataouine.

Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia

Incriminating new rodent species, as reservoir hosts of Leishmania parasites is crucial for understanding the transmission cycle of cutaneous leishmaniasis in Tunisia. Ctenodactylus (C.) gundi was previously described as extremely abundant in all Tunisian Leishmania (L.) tropica foci in south Tunisia besides its presence in L. major endemic area. The aim of this study was to detect Leishmania species parasites among C. gundi in two endemic regions in Tunisia: Sidi Bouzid and Tataouine. Total DNA was isolated from the spleens and the livers of 92C. gundi. Leishmaniasis clinical manifestations were detected among 11 rodents (12%). Leishmania parasites were detected in 30 (32.6%) rodents using direct exam method. Leishmania DNA was detected in 40 (43.5%) C. gundi by combining results among spleens and livers using ITS1-PCR. Positive samples were confirmed to be L. major except for only one specimen which was L. tropica. These results demonstrated, for the first time, the high natural infection rate of C. gundi with L. major parasites in Tunisia. Hence, C. gundi should be considered as potential reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.

No Evidence of Interspecific Genetic Exchange by Multi-Locus MicrosatelliteTyping Between Leishmania Killicki and Leishmania Major in a Mixed Focus ofCutaneous Leishmaniasis in Southeast Tunisia

Sixty-four Leishmania samples were isolated from patients in several villages in the Tataouine governorate, southeast Tunisia. This region is known to be a mixed focus of human cutaneous Leishmaniasis caused by Leishmania (L.) killicki (synonymous L. tropica) and L. major. To identify the Leishmania species in this governorate, a nested polymerase chain reaction based on the variable region of the kinetoplast minicircle was performed on each isolate. Multi-locus microsatellite typing using markers selected for their ability to amplify the two species was used to explore patterns of interspecific genetic exchange. Thirteen L. major and 51 L. killicki isolates were identified. The analysis of microsatellite data showed very low genetic diversity in each species with this set of microsatellites but a high differentiation between the two species. Nine L. major and five L. killicki strains revealed heterozygous genotypes with no shared allele between the two species. These heterozygotes probably resulted from genetic mutation events and not from interspecific genetic exchange. Specific and different epidemiological cycles at the sympatric level might explain the absence of genetic exchange between the two Leishmania species in the Tataouine governorate.