Multilocus Microsatellite Typing reveals intra-focal genetic diversity among strains of Leishmania tropica in Chichaoua Province, Morocco

AbstractIn Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is a major public health threat. Strains of this species have been shown to display considerable serological, biochemical, molecular biological and genetic heterogeneity; and Multilocus Enzyme Electrophoresis (MLEE), has shown that in many countries including Morocco heterogenic variants of L. tropica can co-exist in single geographical foci. Here, the microsatellite profiles discerned by MLMT of nine Moroccan strains of L. tropica isolated in 2000 from human cases of CL from Chichaoua Province were compared to those of nine Moroccan strains of L. tropica isolated between 1988 and 1990 from human cases of CL from Marrakech Province, and also to those of 147 strains of L. tropica isolated at different times from different worldwide geographical locations within the range of distribution of the species. Several programs, each employing a different algorithm, were used for population genetic analysis. The strains from each of the two Moroccan foci separated into two phylogenetic clusters independent of their geographical origin. Genetic diversity and heterogeneity existed in both foci, which are geographically close to each other. This intra-focal distribution of genetic variants of L. tropica is not considered owing to in situ mutation. Rather, it is proposed to be explained by the importation of pre-existing variants of L. tropica into Morocco

Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

Leishmania infantum causes Visceral and cutaneous leishmaniasis in northern Morocco. It predominantly affects children under 5 years with incidence of 150 cases/year. Genetic variability and population structure have been investigated for 33 strains isolated from infected dogs and humans in Morocco. A multilocus microsatellite typing (MLMT) approach was used in which a MLMtype based on size variation in 14 independent microsatellite markers was compiled for each strain. MLMT profiles of 10 Tunisian, 10 Algerian and 21 European strains which belonged to zymodeme MON-1 and non-MON-1 according to multilocus enzyme electrophoresis (MLEE) were included for comparison. A Bayesian model-based approach and phylogenetic analysis inferred two L.infantum sub-populations; Sub-population A consists of 13 Moroccan strains grouped with all European strains of MON-1 type; and subpopulation B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses subpopulations were significantly different from each other and from the Tunisian, Algerian and European non MON-1 strains which constructed one separate population. The presence of these two sub-populations co-existing in Moroccan endemics suggests multiple introduction of L. infantum from/to Morocco; (1) Introduction from/to the neighboring North African countries, (2) Introduction from/to the Europe. These scenarios are supported by the presence of sub-population B and sub-population A respectively. Gene flow was noticed between sub-populations A and B. Five strains showed mixed A/B genotypes indicating possible recombination between the two populations. MLMT has proven to be a powerful tool for eco-epidemiological and population genetic investigations of Leishmania.The fieldwork and data collection was granted by Pastur Institute in Morocco for Dr. Meryem Lemrani. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

New Clinicoepidemiologic Profile of Cutaneous Leishmaniasis, Morocco

During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. We describe distribution of Leishmania major and L. tropica in Morocco and a new focus of cutaneous leishmaniasis due to L. infantum. We recommend using molecular techniques to diagnose suspected leishmaniasis cases

Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

Background Many cases of cutaneous leishmaniasis (CL) have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. Methods Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA) sequence. Excreted factor (EF) serotyping and multilocus enzyme electrophoresis (MLEE) were also applied. Results This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT) done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains. Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria. Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains came. Conclusions The Palestinian strains that were assigned to different genetic groups differed in their MLEE profiles and their EF types. A new zymodeme, zymodeme MON-307 was discovered that seems to be unique to the northern part of the Palestinian West Bank. What seemed to be a straight forward classical situation of L. tropica causing anthroponotic CL in the Jenin District might be a more complex situation, owing to the presence of two separate sub-types of L. tropica that, possibly, indicates two separate transmission cycles involving two separate types of phlebotomine sand fly vector

Intra and peridomiciliary comparison of density, sex ratio and gonotrophic stage of Phlebotomus sergenti in an active anthroponotic cutaneous leishmaniasis focus in Morocco

This study was funded by the University of Granada (Centro de Ini-ciativas de Cooperacion al Desarrollo, CICODE, 2013) . We thanks the Delegation du Ministe`re de la Sante, province de Settat, Morocco, and the colleagues from the El Borouj health center. Funding for open access charge: Universidad de Granada/CBVA.Cutaneous leishmaniasis due to Leishmania tropica represents a major public health problem due to its ability to spread into non-endemic areas by means of its vectors, and the associated dramatic psychosocial impact. The objective of this work was to compare the intra and extradomiciliary density, sex ratio and gonotrophic stage of sand flies from a recent active focus in Morocco. This field study is based on the need to optimize the effectiveness of control programs. Two different capture methods, CDC light traps and sticky traps, were used at two different times of the year, corresponding with the peaks of sand fly abundance. 7,815 sand flies were captured and classified into 13 species belonging to genera Sergentomyia (50.8%) and Phlebotomus (49.2%). Phlebotomus sergenti was the most abundant and frequent species of the genus Phlebotomus both inside (49.3%) and outside houses (52.1%) and it showed the highest density in extradomiciliary captures in June. The proportion of bloodfed females was similar indoors and outdoors (21.5% and 26.3%, respectively). Females in the three gonotrophic stages were found in 26% houses and this was significantly associated with some factors related to housing conditions. Therefore, P. sergenti seems well adapted to both indoors and outdoors biotopes where these females coexist with males. These findings suggest that the adoption of additional measures could benefit the strategy of the Moroccan health authorities, currently consisting of indoor insecticide spraying, given that transmission may also occur outdoors.University of Granada (Centro de Ini-ciativas de Cooperacion al Desarrollo, CICODE, 2013

First Molecular Epidemiological Study of Cutaneous Leishmaniasis in Libya

Cutaneous leishmaniasis (CL) is caused by protozoan parasites of the genus Leishmania. The disease is characterized by the formation of chronic skin lesions followed by permanent scars and deformation of the infected area. It is distributed in many tropical and subtropical countries with more than 2 million cases every year. During the past few years CL has emerged as a major public health problem in Libya. So far, diagnosis was based on clinical symptoms and microscopic observation of parasites. Disease outbreaks were not investigated and the causative leishmanial species of CL were not identified so far. Our study indicates the presence of two coexisting species: Leishmania major and Leishmania tropica. These results are crucial in order to provide accurate treatment, precise prognosis and appropriate public health control measures. The recent armed conflict in Libya that ended with the Gadhafi regime collapse on October 2011 has affected all aspects of the life in the country. In this study we discussed multiple risk factors that could be associated with this conflict and present major challenges that should be considered by local and national health authorities for evaluating the CL burden and highlighting priority actions for disease control

Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Protozoal parasites of the genus Leishmania are transmitted by sand fly bites to humans and animals. Three major forms of disease are caused by these parasites: cutaneous leishmaniasis, responsible for disfiguring skin wounds; mucocutaneous leishmaniasis, causing non-healing ulceration around the mouth and nose; and the potentially fatal visceral leishmaniasis, involving internal organs such as the spleen and liver. More than 2 million new human infections are caused annually by leishmaniasis globally, it is endemic in more than 88 countries and prevalent also as an imported disease in non-endemic regions due to travel and tourism. Most species of Leishmania that infect humans are zoonotic and transmitted from animal reservoir hosts. As various leishmanial parasites cause disease with similar symptoms, but require different therapeutic regimens and have dissimilar prognoses, reliable, sensitive and rapid diagnostic assays are needed. This study focuses on the five main species that cause leishmaniasis in the Old World. It presents a new assay for rapid detection, species identification and quantification of leishmanial parasites in clinical samples, reservoir hosts and sand flies. This technique could be especially valuable in regions where several leishmanial species exist, in non-endemic regions where infected patients require a rapid diagnosis, and for epidemiological host and vector studies leading to prevention programs

Insecticide susceptibility status of Phlebotomus (Paraphlebotomus) sergenti and Phlebotomus (Phlebotomus) papatasi in endemic foci of cutaneous leishmaniasis in Morocco

<p>Abstract</p> <p>Background</p> <p>In Morocco, cutaneous leishmaniasis is transmitted by <it>Phlebotomus sergenti </it>and <it>Ph. papatasi</it>. Vector control is mainly based on environmental management but indoor residual spraying with synthetic pyrethroids is applied in many foci of <it>Leishmania tropica</it>. However, the levels and distribution of sandfly susceptibility to insecticides currently used has not been studied yet. Hence, this study was undertaken to establish the susceptibility status of <it>Ph. sergenti </it>and <it>Ph. papatasi </it>to lambdacyhalothrin, DDT and malathion.</p> <p>Methods</p> <p>The insecticide susceptibility status of <it>Ph. sergenti </it>and <it>Ph. papatasi </it>was assessed during 2011, following the standard WHO technique based on discriminating dosage. A series of twenty-five susceptibility tests were carried out on wild populations of <it>Ph. sergenti </it>and <it>Ph. papatasi </it>collected by CDC light traps from seven villages in six different provinces. Knockdown rates (KDT) were noted at 5 min intervals during the exposure to DDT and to lambdacyhalothrin. After one hour of exposure, sandflies were transferred to the observation tubes for 24 hours. After this period, mortality rate was calculated. Data were analyzed by Probit analysis program to determine the knockdown time 50% and 90% (KDT50 and KDT90) values.</p> <p>Results</p> <p>Study results showed that <it>Ph.sergenti </it>and <it>Ph. papatasi </it>were susceptible to all insecticides tested. Comparison of KDT values showed a clear difference between the insecticide knockdown effect in studied villages. This effect was lower in areas subject to high selective public health insecticide pressure in the framework of malaria or leishmaniasis control.</p> <p>Conclusion</p> <p><it>Phlebotomus sergenti </it>and <it>Ph. papatasi </it>are susceptible to the insecticides tested in the seven studied villages but they showed a low knockdown effect in Azilal, Chichaoua and Settat. Therefore, a study of insecticide susceptibility of these vectors in other foci of leishmaniasis is recommended and the level of their susceptibility should be regularly monitored.</p

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

Old world cutaneous leishmaniasis is a vector-borne disease occurring in rural areas of developing countries. The main reservoirs are the rodents Psammomys obesus and Meriones shawi. Zoonotic Leishmania transmission cycle is maintained in the burrows of rodents where the sand fly Phlebotomus papatasi finds the ideal environment and source of blood meals. In the present study we showed seasonality of the incidence of disease during the same cycle with an inter-epidemic period ranging from 4 to 7 years. We evaluated the impact of climate variables (rainfall, humidity and temperature) on the incidence of zoonotic cutaneous leishmaniais in central Tunisia. We confirmed that the risk of disease is mainly influenced by the humidity related to the months of July to September during the same season and mean rainfall lagged by 12 to 14 months