Genetic diversity of Leishmania tropica in Morocco: does the dominance of one haplotype signify its fitness in both predominantly anthropophilic Phlebotomus sergenti and human beings?

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania tropica is endemic to locations where the predominantly anthropophilic blood-feeding Phlebotomus sergenti and humans co-perpetuate. The objective of this study was to explore whether the range of epidemiological features of CLcould be linked to the range of L. tropica genetic heterogeneity and to further explore the relationships between the genetic diversity of L. tropica in both P. sergenti and humans. L. tropica DNAwas extracted from dermal scarping smears of 125 CLpatients. Genetic polymorphisms were analyzed by sequencing the internal transcribed spacer (ITS) 1 and 5.8S rDNAgene. Nucleotide diversity (π), haplotype diversity (Hd) and Tajima's D test for neutrality, as well as overall and pairwise FSTvalues, were calculated using Arlequin ver 3.5 software. Out of the 125 amplified DNAsequences, 93 were completely sequenced and 13 L. tropica haplotypes were identified, which confirmed the significant genetic heterogeneity of L. tropica in Morocco. The most common haplotype included 74 out of 93 sequences; this haplotype is not only widely represented but was also detected in P. sergenti, which is known to be the most abundant species in the studied foci. Considering the negative value calculated using Tajima's D index, we briefly discussed the hypothesis that the L. tropica common haplotype propagation could be a sign of its fitness in P. sergenti and human hosts. Furthermore, analysis of molecular variance (AMOVA) shows significant correlations between intraspecific variants of L. tropica and patients' geographic origins. The long-term goals of the present pilot study are to further explore the relationships between the genetic diversity of L. tropica in human and P. sergenti populations.

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.

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.

Molecular detection and identification of Leishmania infection in naturally infected sand flies in a focus of cutaneous leishmaniasis in northern Morocco.

BACKGROUND: Cutaneous leishmaniasis is an infectious disease caused by various species of the flagellate protozoan Leishmania. During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. The main objective of this study was to study the occurrence of Leishmania infection in vectors and to identify sand fly blood meal sources in an endemic locality of cutaneous leishmaniasis within Sefrou province, where the vectors of leishmaniasis were still unknown. METHODS: 2650 sand flies were collected using CDC miniature light traps and identified morphologically. The identified sand flies were tested for Leishmania infection by nested PCR. The source of blood meal of 10 freshly engorged females: 6 Phlebotomus longicuspis and 4 Phlebotomus sergenti, was determined using the Cyt b sequence. RESULTS: The collected sand flies consisted of 10 species, seven of which belonged to the genus Phlebotomus and three to the genus Sergentomyia. The most abundant species was P. longicuspis, accounting for 72% of the total sand flies collected. In females of three P. longicuspis and four P. sergenti, Leishmania infantum and Leishmania tropica DNA was detected, respectively.The source of blood meal of engorged females showed that all sand flies tested fed on humans. CONCLUSIONS: We report for the first time the natural infection of P. longicuspis with L. infantum in Morocco. The high frequency of this species in this region, in addition to its anthropophilic character make P. longicuspis the putative vector of L. infantum in this cutaneous leishmaniasis focus where L. tropica is confirmed as the causative agent of the disease and P. sergenti as its vector. The presence of L. infantum, and its presumed vector in this area, makes this a site of high risk of visceral leishmaniasis, mostly because of the proximity of a focus of human and canine visceral leishmaniasis.

Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco.

BACKGROUND & OBJECTIVES: An entomological study using CDC miniature light-traps was performed in El Hanchane locality, where cutaneous leishmaniasis (CL) was emerging during the summer of 2011. The aim of this study is to identify the vectors of Leishmania and of phleboviruses. METHODS: In the field, a total of 643 sandfly specimens were collected, identified by morphological keys and categorized by sex and species. A total of nine distinct species were morphologically identified where seven belonged to the Phlebotomus genus and two species to the Sergentomyia genus. Phlebotomus sergenti was the most abundant species (76%). Phleboviruses were detected by nested RT-PCR using 30 pooled sandflies while P. sergenti females were tested individually for infections of Leishmania species. RESULTS: By using ITS1-PCR-RFLP approach, Leishmania tropica DNA was detected in 10 females, caught in this emerging focus, and provide additional evidence in favour of the role of P. sergenti as vector of L. tropica in Morocco. Real-time PCR screening for phlebovirus RNA, using an assay targeting the polymerase gene, showed positive result in one pool of male P. sergenti. INTERPRETATION & CONCLUSION: In this study, P. sergenti were infected by L. tropica and Toscana virus. To our knowledge, actually this is the first time that Toscana virus has been detected in P. sergenti.

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

BACKGROUND: Cutaneous 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: This 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.

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).