A comparative genomics approach reveals a local genetic signature of Leishmania tropica in Morocco.

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is an important health problem. Despite the high incidence of CL in the country, the genomic heterogeneity of these parasites is still incompletely understood. In this study, we sequenced the genomes of 14 Moroccan isolates of L. tropica collected from confirmed cases of CL to investigate their genomic heterogeneity. Comparative genomics analyses were conducted by applying the recently established Genome Instability Pipeline (GIP), which allowed us to conduct phylogenomic and principal components analyses (PCA), and to assess genomic variations at the levels of the karyotype, gene copy number, single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) variants. Read-depth analyses revealed a mostly disomic karyotype, with the exception of the stable tetrasomy of chromosome 31. In contrast, we identified important gene copy number variations across all isolates, which affect known virulence genes and thus were probably selected in the field. SNP-based cluster analysis of the 14 isolates revealed a core group of 12 strains that formed a tight cluster and shared 45.1 % (87 751) of SNPs, as well as two strains (M3015, Ltr_16) that clustered separately from each other and the core group, suggesting the circulation of genetically highly diverse strains in Morocco. Phylogenetic analysis, which compared our 14 L. tropica isolates against 40 published genomes of L. tropica from a diverse array of locations, confirmed the genetic difference of our Moroccan isolates from all other isolates examined. In conclusion, our results indicate potential regional variations in SNP profiles that may differentiate Moroccan L. tropica from other L. tropica strains circulating in endemic countries in the Middle East. Our report paves the way for future research with a larger number of strains that will allow correlation of diverse phenotypes (resistance to treatments, virulence) and origins (geography, host species, year of isolation) to defined genomic signals such as gene copy number variations or SNP profiles that may represent interesting biomarker candidates.

Population Genetic Structure of Phlebotomus sergenti (Diptera: Psychodidae) Collected in Four Regions of Morocco Based on the Analysis of Cyt b and EF-1α Genes.

Phlebotomus (Ph.) sergenti is the main vector of Leishmania (L.) tropica (Trypanosomatida: Trypanosomatidae), the causative agent of anthroponotic cutaneous leishmaniasis in Morocco. This species has an extended geographical distribution, wider than that of the parasite. The main objective of our study was to analyze the genetic diversity of Ph. sergenti collected in four foci in Morocco: Taza, Foum Jemâa, El Hanchane, and Ouarzazate. We studied a set of diversity and population structure indices by sequencing two markers; nuclear EF-1α and mitochondrial Cyt b from 175 individual sand flies. Our results showed a considerable degree of intraspecific polymorphism with a high number of haplotypes identified in both genes. Many polymorphic sites detected in the Cyt b sequences (SCyt b = 45) indicate that it is the most polymorphic marker showing a distinct distribution of haplotypes according to their geographical origin, whereas the EF-1α marker showed no geographical isolation. Analysis by Tajima's D and Fu's Fs tests revealed a possible recent expansion of the populations, especially with the EF-1α marker, showing significant values in Taza and Ouarzazate sequences. The present study revealed significant genetic diversity within Ph. sergenti populations in Morocco. The results warrant further research using a combination of more than two markers including mitochondrial and non-mitochondrial markers, which may provide more information to clarify the genetic status of Ph. sergenti.

Genetic Diversity and Population Structure of Leishmania infantum in Morocco as Revealed by Multilocus Sequence Typing (MLST) Approach.

Leishmania infantum is endemic in Morocco, and it causes both visceral (VL) and cutaneous leishmaniasis (CL). In this study, the multilocus sequence typing (MLST) approach was used to investigate the phylogeny and population structure of Leishmania infantum strains isolated from CL and VL patients and the canine reservoir in different leishmaniasis endemic foci in Morocco. For this purpose, eight loci (pgm, alat, me, fh, g6pd, pgd, gpi and cytb) were amplified in 40 samples, out of which 31 were successfully sequenced. The genetic diversity analysis detected a high degree of intraspecific genetic variability among the studied strains. The phylogenetic and the haplotype analyses showed that most of the strains from the same geographical areas clustered together. The recombination among Leishmania infantum strains was revealed through a splits tree analysis and the number of recombination events. Moreover, the assessment of the gene flow between Leishmania infantum and Leishmania tropica through phylogenetic analysis and haplotype diversity in two endemic foci where the two species were sympatric showed no genetic exchange between the two species.

Environmental, Climatic, and Parasite Molecular Factors Impacting the Incidence of Cutaneous Leishmaniasis Due to Leishmania tropica in Three Moroccan Foci.

Cutaneous leishmaniasis (CL) occurring due to Leishmania tropica is a public health problem in Morocco. The distribution and incidence of this form of leishmaniasis have increased in an unusual way in the last decade, and the control measures put in place are struggling to slow down the epidemic. This study was designed to assess the impact of climatic and environmental factors on CL in L. tropica foci. The data collected included CL incidence and climatic and environmental factors across three Moroccan foci (Foum Jemaa, Imintanout, and Ouazzane) from 2000 to 2019. Statistical analyses were performed using the linear regression model. An association was found between the occurrence of CL in Imintanout and temperature and humidity (r2 = 0.6076, df = (1.18), p-value = 3.09 × 10-5; r2 = 0.6306, df = (1.18), p-value = 1.77 × 10-5). As a second objective of our study, we investigated the population structure of L.tropica in these three foci, using the nuclear marker internal transcribed spacer 1 (ITS1). Our results showed a low-to-medium level of geographic differentiation among the L.tropica populations using pairwise differentiation. Molecular diversity indices showed a high genetic diversity in Foum Jemaa and Imintanout; indeed, 29 polymorphic sites were identified, leading to the definition of 13 haplotypes. Tajima's D and Fu's F test statistics in all populations were not statistically significant, and consistent with a population at drift-mutation equilibrium. Further analysis, including additional DNA markers and a larger sample size, could provide a more complete perspective of L. tropica's population structure in these three regions. In addition, further research is needed to better understand the impact of climatic conditions on the transmission cycle of Leishmania, allowing both for the development of effective control measures, and for the development of a predictive model for this parasitosis.

Entomological study in an anthroponotic cutaneous leishmaniasis focus in Morocco: Fauna survey, Leishmania infection screening, molecular characterization and MALDI-TOF MS protein profiling of relevant Phlebotomus species.

In Morocco, leishmaniases are a major public health problem due to their genetic diversity and geographical distribution. Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted typically by bite of phlebotomine sand flies. This study identifies sand fly fauna in Ibaraghen village, province of Azilal, which is a focus of CL, by combination of morphological and molecular methods (sequencing of COI gene, MALDI-TOF MS protein profiling). Nested-kDNA PCR was used to detect and identify Leishmania species within potential vector species. 432 CDC light traps were placed at different heights above ground level at four capture sites during a whole year. Traps at 1.5 m above the ground yielded capture of sand flies almost double compared to above ground level (29.33%), while the collection reached 55.09% when the traps were placed 2.5 m above ground. A total of 2,830 sand flies were collected, 2,213 unfed specimens were morphologically identified, 990 males (44.73%) and 1,223 females (55.26%) of 13 species; ten Phlebotomus species and three Sergentomyia species. Six species were analysed by MALDI-TOF MS protein profiling (4 Phlebotomus and 2 Sergentomiya species), and their identification was confirmed by COI sequencing. 1,375 unfed females were screened for the presence of Leishmania by nested-kDNA PCR in pools, 11/30 pools of P. sergenti showing a single band of 750 bp corresponding to L. tropica. Our results confirm the role of P. sergenti as a proven vector in Azilal focus of cutaneous leishmaniasis; however, the relative abundance of other species known as vectors of Leishmania species emphasizes the risk of introduction of L. infantum and L. major in this province. For the first time in Morocco, a combined approach to identify sand flies by both morphology and molecular methods based on DNA barcoding and MALDI-TOF MS protein profiling was applied.

Multilocus sequence analysis provides new insight into population structure and genetic diversity of Leishmania tropica in Morocco.

Cutaneous leishmaniasis (CL) is one of the most neglected tropical diseases, caused by different Leishmania species. Despite its high incidence in Morocco, CL due to Leishmania tropica is poorly understood in terms of its epidemiological status and population structure. In this study, we used multilocus sequence typing (MLST) in order to explore the genetic heterogeneity of L. tropica strains. Samples (N = 48) were collected from CL patients in two localities in Morocco (Foum Jamaa in the Azilal province and Imintanoute in Chichaoua province). PCR-sequencing of 18 strains was carried out for six housekeeping genes (cytb, me, fh, g6pd, pgd and gpi), Genetic diversity indices showed a high population genetic differentiation between and among populations. There was no shared haplotypes between the two localities studied. Our results reveal a considerable degree of differentiation through the relatively high FST value (> 0.4) and remarkable intraspecific polymorphism (S = 29). Imintanoute strains have more polymorphisms (S = 22) than the Foum Jamaa strains despite their small sample size. These results provide crucial background information of epidemiology in Imintanoute which raises questions about animal involvement in L. tropica transmission cycle.

The role of sampling by cotton swab in the molecular diagnosis of cutaneous leishmaniasis.

An appreciated sampling technique is essential for achieving optimum results from diagnostic tests of cutaneous leishmaniasis (CL); conventional sampling methods, such as skin biopsy and dermal scrapping, are painful for the patients and require qualified staff and hospital facilities, while swabbing is patient-friendly more comfortable than invasive traditional techniques and can be carried out under field conditions. The aim of this study is to evaluate a non-invasive sampling method (swab) in the cutaneous leishmaniasis diagnosis, compared to microscopic examination. The recruitment of 205 patients was done during 3 years in six regions known as endemic CL foci in the south-east, the centre, the south-west and the north of Morocco. The results showed that molecular detection of the nuclear marker ITS1 on swab materials is to be less sensitive than microscopic examination, and the difference was statistically significant (p-value = .036). Thereafter, 55 patients were randomly selected to compare the results of two molecular techniques (ITS1-PCR and nested KDNA-PCR), performed both on swab and on stained smears used for microscopic examination; ITS1-PCR results from stained smears reached 87% positivity against 65,2% for cotton swab; and it was statistically significant (p-value = .019); on the other hand for the KDNA marker, results from cotton swab reached 93% of positivity against 91% for stained smears; and statistically the difference was not significant (p-value = .5113). One can presume that rubbing over the lesion with cotton swab implies a low parasitic load collection, but the choice of the amplification target greatly influences the results obtained from cotton swab; on top of that swab remains useful in the cases of patients with multiples lesions or the latter are located in sensitive places difficult to reach with an invasive method such as the eyelids, the lips or other mucous areas.

Population structure of leishmania major in southeastern morocco.

Cutaneous leishmaniasis (CL) is an infectious disease caused by various Leishmania species. It is among the most neglected tropical diseases and has been considered a major health threat over the past decades in the country. Its zoonotic form caused by Leishmania (L) major is the most prevalent in Morocco. This study investigated the population structure of L. major in southeastern Morocco. Samples (n = 67) were collected from patients with CL in five different endemic areas located in three provinces (Ouarzazate, Tinghir, and Zagora). These samples were then sequenced using two nuclear markers: internal transcribed spacer 1 (ITS1) and a fragment of the virulence factor GP63. Next, the sequences were edited and analyzed. Molecular diversity indices showed a high population genetic diversity but an overall low haplotype diversity. Our results suggest small population differentiation, indicating a low geographic structure. Tajima's D and Fu's Fs tests both suggested recent population expansion based on the significant deviations from neutrality in both tests for all populations except Tinghir, which may be due to a small sample size. Based on our findings, the region is experiencing rapid population expansion caused by recent CL outbreaks, and one of them has been recently studied. In addition, analysis of molecular variance and FST suggested gene flow between Zagora and both Ouarzazate and Tinghir. Nonetheless, no gene flow was observed between Tinghir and Ouarzazate. To the best of our knowledge, this is the first analysis of the population structure of L. major in Morocco. The results of this study provide crucial background information for epidemiological studies by showing the presence of gene flow between populations and clonal expansion in cases of an outbreak. This will drive authorities to reconsider the implemented control strategies.

Epidemiological features of a recent zoonotic cutaneous leishmaniasis outbreak in Zagora province, southern Morocco.

BACKGROUND: Leishmania major is an endemic vector-borne disease in Morocco that causes zoonotic cutaneous leishmaniasis (ZCL), especially in arid pre-Saharan regions where its unique vector and reservoir are Phlebotomus papatasi and Meriones shawi, respectively, and may cause epidemics. In late 2017, the Zagora province, an endemic focus for ZCL in southern Morocco, had CL outbreak. The main objective of our investigation was to analyze the epidemiological features of this latest ZCL outbreak. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed epidemiological features of this latest ZCL outbreak. The Regional Delegation of Health, Zagora, recorded 4,402 CL patients between October 2017 and end of March 2018. Our findings showed that 24 municipalities were affected and majority (55.1%) of infected cases belonged to the Tinzouline rural municipality. Majority of patients were females (57.2%). While all age group patients were affected, those aged <10 years were the most affected (42.1%). During this outbreak over 5 days in December 2017, we conducted a survey in Tinzouline and recruited and sampled 114 CL patients to confirm CL diagnosis by parasitological (direct examination and culture) and molecular (ITS1-PCR) methods and identify the etiological agent of infection using ITS1-PCR-RFLP and sequencing. We completed a detailed questionnaire including clinical and epidemiological data for each patient and found 72.8% of patients presenting multiple lesions (≥2), with an average number of lesions of 5.16 ± 0.5. Lesions were more prevalent in the upper limbs, with the most common type being the ulcerocrusted lesion (60.5%). We detected no associations between lesion type and patients' sex or age. CONCLUSIONS/SIGNIFICANCE: Among 114 clinically diagnosed CL patients, we confirmed 90.35% and identified L. major as the species responsible for this outbreak. Self-medication using various products caused superinfection and inflammation of lesions and complicated the diagnosis and treatment. Thus, ZCL remains a major public health problem in the Zagora province, and commitment of all stakeholders is urgently required to implement a sustainable regional control program.

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.