Meryem Lemrani

Detection and molecular typing of Leishmania tropica from Phlebotomus sergenti and lesions of cutaneous leishmaniasis in an emerging focus of Morocco.

BackgroundCutaneous leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. In Morocco, anthroponotic cutaneous leishmaniasis due to Leishmania tropica is considered as a public health problem, but its epidemiology has not been fully elucidated. The main objective of this study was to detect Leishmania infection in the vector, Phlebotomus sergenti and in human skin samples, in the El Hanchane locality, an emerging focus of cutaneous leishmaniasis in central Morocco.MethodsA total of 643 sand flies were collected using CDC miniature light traps and identified morphologically. Leishmania species were characterized by ITS1 PCR-RFLP and ITS1-5.8S rRNA gene nested-PCR of samples from 123 females of Phlebotomus sergenti and 7 cutaneous leishmaniasis patients.ResultsThe sand flies collected consisted of 9 species, 7 of which belonged to the genus Phlebotomus and two to the genus Sergentomyia. Phlebotomus sergenti was the most predominant (76.67%).By ITS1 PCR-RFLP Leishmania tropica was found in three Phlebotomus sergenti females and four patients (4/7). Using nested PCR Leishmania tropica was identified in the same three Phlebotomus sergenti females and all the 7 patients. The sequencing of the nested PCR products recognized 7 haplotypes, of which 6 have never been described.ConclusionsThis is the first molecular detection and identification of Leishmania tropica in human skin samples and Phlebotomus sergenti in support of its vector status in El Hanchane. The finding of seven Leishmania tropica haplotypes underscores heterogeneity of this species at a high level in Morocco.

First detection of Toscana virus RNA from sand flies in the genus Phlebotomus (Diptera: Phlebotomidae) naturally infected in Morocco.

ABSTRACT In total, 656 sand flies were collected in June 2008 from Louata, a locality of Sefrou province, Morocco. Testing was conducted for the presence of phlebovirus by nested reverse transcriptase polymerase chain reaction and sequencing. We detected Toscana virus in four pools of male Phlebotomus perniciosus. This virus belongs to the genotype B previously recognized in France and Spain. This is the first time that Toscana virus has been detected in 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 sub-population B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses sub-populations 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.

Toscana virus isolated from sandflies, Morocco

To investigate the transmission of phleboviruses, a total of 7,057 sandflies were collected in well-known foci of cutaneous leishmaniasis and were identified to species level according to morphological characters.Collected sandflies were tested by Nested PCR for the presence of Phleboviruses and subsequently by viral isolation on Vero cells. The corresponding products were sequenced. Toscana virus was isolated, for the first time, from 5 pools of sandflies.Hence, Toscana virus should be considered a potential risk that threatens public health and clinicians should be aware of the role of Toscana virus in cases of meningitis and encephalitis in Morocco.

Phlebotomus sergenti in a Cutaneous Leishmaniasis Focus in Azilal Province (High Atlas, Morocco): Molecular Detection and Genotyping of Leishmania tropica, and Feeding Behavior

Background Phlebotomus (Paraphlebotomus) sergenti is at least one of the confirmed vectors for the transmission of cutaneous leishmaniasis caused by Leishmania tropica and distributed widely in Morocco. This form of leishmaniasis is considered largely as anthroponotic, although dogs were found infected with Leishmania tropica, suggestive of zoonosis in some rural areas. Methodology and Findings This survey aimed at (i) studying the presence of Leishmania in field caught Phlebotomus sergenti, (ii) investigating genetic diversity within Leishmania tropica and (iii) identifying the host-blood feeding preferences of Phlebotomus sergenti. A total of 4,407 sand flies were collected in three rural areas of Azilal province, using CDC miniature light traps. Samples collected were found to consist of 13 species: Phlebotomus spp. and 3 Sergentomyia spp. The most abundant species was Phlebotomus sergenti, accounting for 45.75 % of the total. 965 female Phlebotomus sergenti were screened for the presence of Leishmania by ITS1-PCR-RFLP, giving a positive rate of 5.7% (55/965), all being identified as Leishmania tropica. Nucleotide heterogeneity of PCR-amplified ITS1-5.8S rRNA gene-ITS2 was noted. Analyses of 31 sequences obtained segregated them into 16 haplotypes, of which 7 contain superimposed peaks at certain nucleotide positions, suggestive of heterozygosity. Phlebotomus sergenti collected were found to feed on a large variety of vertebrate hosts, as determined by Cytochrome b sequencing of the DNA from the blood meals of 64 engorged females. Conclusion Our findings supported the notion that Phlebotomus sergenti is the primary vector of Leishmania tropica in this focus, and that the latter is genetically very heterogeneous. Furthermore, our results might be suggestive of a certain level of heterozygosity in Leishmania tropica population. This finding, as well as the feeding of the vectors on different animals are of interest for further investigation.

Epidemiologic study and molecular detection of Leishmania and sand fly species responsible of cutaneous leishmaniasis in Foum Jamâa (Azilal, Atlas of Morocco).

The region of Foum Jamâa (province of Azilal) has become endemic for cutaneous leishmaniasis (CL) since 2006. The objective of this study was to investigate molecular identification of the etiological agent of CL in this region; we also carried out an entomological survey of Phlebotomine sand flies (Diptera: Psychodidae) in this focus to study the sand fly fauna, species composition, and the monthly prevalence of sand flies during 1 year. In the period between 2009 and 2010, skin scrapings spotted on glass slides were collected from 119 patients, aged from 9 months to 70 years old, who came from 43 localities distributed in 3 sectors in Foum Jamâa (FJ). The ITS1 PCR-RFLP was used to identify the Leishmania parasite responsible for the recent cases of CL in FJ. Our results revealed that the disease is caused by L. tropica. No significant association was observed between gender and the rate of CL in presenting patients, while the highest rate of positive lesions was found in the age group of 9 years old or under (86.67%). In this study, we found also that L. tropica infection mostly caused single lesions (67.90%) that were located in the face (96.30%). Morphological identification was performed on a total of 1152 sand flies (23% females and 77% males) collected by sticky paper traps. 57% of the total collected flies were identified as Phlebotomus (Paraphlebotomus) sergenti (Parrot).

A variant in the promoter of MBL2 is associated with protection against visceral leishmaniasis in Morocco

Progressive visceral leishmaniasis (VL) is fatal if not treated; yet, most infections with the causative agents are asymptomatic. We hypothesized that genetic factors contribute to this variable response to infection. The mannose-binding lectin 2 gene (MBL2) is a candidate that merits examination in the context of VL because it enhances infection with intracellular pathogens. Four functional MBL2 polymorphisms at codons 52, 54, 57 and in the promoter at the -221 position (X/Y) are known to be associated with the outcome of several diseases. The aim of the present study was to investigate whether these functional variants were associated with VL in Moroccan children. Here, we genotyped polymorphisms by sequencing and PCR-RFLP in 112 individuals with VL, 97 asymptomatic subjects and 42 healthy individuals who had no evidence of present or past infection. Regression analysis showed no significant association between polymorphisms in exon 1 genotypes and outcome of infection with Leishmania infantum. However, the genotype XY in -221 conferred a protective role against VL in our study population with a significant difference (OR=0.291; CI [0.158-0.538]; p=0.0006). Subjects with YY genotypes in -221 had a higher risk to developing VL. We concluded that MBL2 polymorphism at the -221 promoter region plays a protective role in L. infantum infection.

Intraspecific genetic variability in a population of Moroccan Leishmania infantum revealed by PCR-RFLP of kDNA minicircles

In Morocco, Leishmania infantum is the main etiologic agent of human and canine visceral leishmaniasis (VL). This species has been proven to be an opportunistic agent in HIV+ patients and is also responsible of sporadic cutaneous leishmaniasis (CL).This work aims to evaluate the genetic variability of Moroccan L. infantum strains based on PCR-RFLP analysis of the kinetoplastid DNA (kDNA) minicircles. A total of 75 DNA samples extracted from positive Giemsa-stained smears (n=32) and from L. infantum cultures (n=43) was studied. The samples have been taken from VL patients infected (n=7) or not (n=56) by HIV, patients with CL (n=2) and finally from infected dogs (n=10). An hypervariable region of kDNA was amplified using the primers MC1 and MC2; the PCR products were digested separately by a panel of nine restriction enzymes. The presence or absence of restriction fragments was scored in a binary matrix and the SplitsTree4 software was used for the construction of a Neighbor-Net network. Moroccan L. infantum population showed an important level of variability with the identification of 6 genotypes. For each genotype a PCR product was sequenced, confirming the presence of all the expected restriction sites. The predominant profile was the genotype B. A new genotype, named Q was detected for the first time, whereas the four other genotypes (G, K, N and O) were reported sporadically in the Mediterranean basin. The Neighbor-Net network segregates our L. infantum population into 3 clusters: Cluster I includes genotype B, cluster II grouping the genotypes O, Q and G and finally the cluster III contains the genotype N. The kDNA-PCR-RFLP assay is suitable for use directly on biological samples; it reveals an important degree of genetic variability among L. infantum strains even those belonging to the same zymodeme what is of great epidemiological interest.

The TLR2 and TLR4 gene polymorphisms in Moroccan visceral leishmaniasis patients.

Visceral leishmaniasis (VL) is endemic in the Mediterranean basin and leads to the most severe form of Leishmania infection, lethal if left untreated. However, most infections are sub-clinical or asymptomatic, reflecting the influence of host genetic background on disease outcome. This study aimed to investigate possible association of TLR4 Asp299Gly, TLR4 Thr399Ile and TLR2 Arg753Gln polymorphisms with VL in Moroccan children. We enrolled 119 children with VL caused by Leishmania infantum as well as 138 unrelated children, 95 asymptomatic subjects and 43 healthy individuals who had no evidence of present or past infection. Polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). Results showed significant differences in genotype Thr399Ile and recessive model frequencies between VL and delayed-type hypersensitivity (DTH+) groups (p=0.018, OR=0.414CI 0.195-0.880; p=0.029, OR=0.448CI 0.214-0.938], respectively) by having the amino-acid threonine polymorphism as a reference in the VL group. Concerning the Asp299Gly there were a significant associations when comparing VL vs DTH+ (Asp299Gly genotype p=0.002, OR=0.326CI 0.158-0.671, allele frequencies p=0.033, OR=0.396CI 0.164-0.959, recessive model p=0.002, OR=0.343CI 0.172-0.681) and DTH+ vs DTH- groups (Asp299Gly genotype p=2.160E-4, OR=3.065CI 1.672-5.618, Gly299Gly genotype p=0.047, OR=0.368CI 0.299-0.452, allele frequencies p=1.406E-7, OR=29.571CI 3.907-223.8, recessive model p=4.370E-14, OR=36.965CI 8.629-158.3), by having the aspartic acid polymorphism as a reference these results suggest that the allele A (savage) confer protection against the clinical manifestations but not against the infection. Furthermore, there was a significant association regarding the Arg753Gln genotype (p=0.002, OR=0.326CI 0.158-0.671), allele frequencies (p=0.033, OR=0.396CI 0.164-0.959) and when applying a recessive model (p=0.002, OR=0.343CI 0.172-0.681) in the VL vs DTH+ groups. The same results was observed when comparing DTH+ vs DTH- groups (p=4.136E-6, OR=0.211CI 0.104-0.428), allele frequencies (p=0.008, OR=0.327CI 0.137-0.779) and recessive model (p=1.748E-5, OR=0.244CI 0.124-0.480). The results provide evidence that allele C in Thr399Ile and allele G in Arg753Gln polymorphisms may lead to protection against the clinical disease. Our data provide insights into the possible role of TLR2 and TLR4 variations in VL susceptibility.

Molecular identification of Leishmania infection in the most relevant sand fly species and in patient skin samples from a cutaneous leishmaniasis focus, in Morocco

Background Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted by several species of sand flies. CL is among the most neglected tropical diseases, and it has represented a major health threat over the past 20 years in Morocco. The main objectives of this study were to identify relevant sand fly species and detect Leishmania infection in the most prevalent species and patient skin samples in Taza, a focus of CL in North-eastern Morocco. Methodology and finding A total of 3672 sand flies were collected by CDC miniature light traps. Morphological identification permitted the identification of 13 species, namely 10 Phlebotomus species and 3 Sergentomyia species. P. longicuspis was the most abundant species, comprising 64.08% of the total collected sand flies, followed by P. sergenti (20.1%) and P. perniciosus (8.45%). Using nested-kDNA PCR, seven pools of P. sergenti were positive to Leishmania tropica DNA, whereas 23 pools of P. longicuspis and 4 pools of P. perniciosus tested positive for Leishmania infantum DNA. The rates of P. longicuspis and P. perniciosus Leishmania infection were 2.51% (23/915) and 7.27% (4/55), respectively, whereas the infection prevalence of P. sergenti was 3.24%. We also extracted DNA from lesion smears of 12 patients suspected of CL, among them nine patients were positive with enzymatic digestion of ITS1 by HaeIII revealing two profiles. The most abundant profile, present in eight patients, was identical to L. infantum, whereas L. tropica was found in one patient. The results of RFLP were confirmed by sequencing of the ITS1 DNA region. Conclusion This is the first molecular detection of L. tropica and L. infantum in P. sergenti and P. longicuspis, respectively, in this CL focus. Infection of P. perniciosus by L. infantum was identified for the first time in Morocco. This study also underlined the predominance of L. infantum and its vector in this region, in which L. tropica has been considered the causative agent of CL for more than 20 years.