article: 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. 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. 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.
  Leishmaniasis comprises a group of diseases that are caused by various intracellular protozoa species of the genus Leishmania and transmitted by sand flies (Diptera, Phlebotominae). Leishmaniasis is responsible for considerable rates of morbidity and mortality globally, and it affects Mediterranean and other endemic countries, putting a population of 350 million people at risk of infection. The overall prevalence of leishmaniasis is estimated as 12 million cases worldwide, and the global yearly incidence of all clinical forms of the disease is 1. 3 million [1] In the Mediterranean basin, two clinico-epidemiological forms of leishmaniasis are endemic: visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) [2]. VL is a zoonotic disease caused by Leishmania infantum infection. The disease tends to be relatively chronic, and children are especially affected [3]. Dogs represent the principal reservoir host in the entire Mediterranean basin [4,5]. CL is often described as a group of diseases because of the varied spectrum of clinical manifestations, which range from small cutaneous nodules to gross mucosal tissue destruction. CL can be caused by several Leishmania spp. Despite its increasing worldwide incidence, it is rarely fatal, and thus, CL has become a neglected disease. In Morocco, CL constitutes a major health threat consisting of three nosogeographic entities: anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica and transmitted by Phlebotomus (Paraphlebotomus) sergenti [6,7]; zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major, which has been known to exist in the vast area of the arid pre-Saharan region for over a century [8]. Phlebotomus papatasi and Meriones shawi are the vector and reservoir, respectively, of L. major [9]; and sporadic CL, which is caused by L. infantum MON-24 and is present mainly in Northern Morocco, in which ZVL caused by L. infantum MON-1 is endemic [10–12]. The phlebotomine sand fly vectors of L. infantum are considered to be Phlebotomus perniciosus, Phlebotomus longicuspis and Phlebotomus ariasi [13]. Whereas the last two species have been already reported to be infected by L. infantum [4,14], the role of P. perniciosus as a vector of L. infantum has never been confirmed in Morocco. Recently, the epidemiological profile of leishmaniasis in Morocco has changed. Specifically, the predominance of VL in Northern Morocco and CL in Southern Morocco is no longer accurate, as the geographic distribution areas of the three medically important Leishmania species overlap. Indeed, L. tropica has been found in established L. major foci [15]. In addition, cases of VL have been reported in arid endemic areas of ZCL [16], and the dermotropic variant of L. infantum has spread to areas considered foci of CL due to the presence of L. tropica [12,17]. This is the case in Taza province, considered the first focus of CL caused by L. tropica in Northern Morocco. Indeed, in the mid-1990s, this region experienced a rapid expansion of CL due to L. tropica MON-102 [18]. Recently reported data identified L. infantum as the causative agent of CL in 41% of infected patients in this province [17]. Despite the existence of both CL and VL and co-existence of L. tropica and L. infantum in this region [17,18], data on the vectors of Leishmania transmission is not available. Therefore, this study sought to detect and identify the Leishmania parasite responsible for the recent cases of CL and the putative vector species in Taza city, a mixed focus of CL and VL in Northern Morocco.
  This study was conducted in peri-urban areas of Taza, situated in North-eastern Morocco in the corridor between the Rif and Middle Atlas mountains (Fig 1). Taza is situation at 550-m elevation. The climate is seasonal, shifting from cool in winter to hot in summer. The temperature varies between 3. 2 and 44. 5°C. Although annual rainfall in the city has ranged between 84 and 120 mm over the last 8 years, the amount can reach 593 mm per year [19]. Phlebotomine sand flies were caught monthly on 3 consecutive nights between July and October 2015 in three peri-urban sites (Fig 1), using 3 CDC light traps per site placed inside houses and domestic animal shelters. The traps were set before sunset and collected at approximately 6 am the next day. The collected sand flies were then placed in 1. 5-ml Eppendorf tubes, transferred in dry ice to our laboratory and kept frozen in −80°C for further processing and identification. Sand fly specimens were washed in sterile distilled water, and the head and genitalia of each sand fly were removed and mounted on microscope slides using the solution of Marc-André (40 g of chloral hydrate, 30 ml glacial acetic acid and 30 ml of distilled water) for morphological identification using the Moroccan morphologic key [20,21]. The remainder of the sand fly body was stored in sterile Eppendorf microtubes for molecular use. To distinguish P. perniciosus and P. longicuspis, we used criteria based on the morphological features of their genitalia and the number of coxite hairs [22,23]. For the female subgenus Larroussius, they were separated by examining the dilatation of the distal part of spermathecal ducts [24]. In the case of males, the identification was based on the morphological features of the copulatory valve and by the number of coxite hair. P. perniciosus is characterized by two forms: (i) copulatory valves with a bifid apex; (ii) copulatory valves with a curved apex. The number of coxite hair is not higher than 18. P. longicuspis has a copulatory valve ending with a single and long point with a mild curve at the edge, and has 21 or more coxite hairs [23,25]. Under the National Program of Fight against Leishmaniasis conducted in Taza by regional health authorities, we participated in active screening for 4 days in April 2015. The screening involved schools, nurseries and houses in neighbourhoods in which cases of CL were commonly reported. We also collected clinical samples from patients at health centres. CL was suspected in 12 patients. Before sampling, we completed a questionnaire regarding information about each patient (code, age, gender, address, history of travel) and the lesions (number, location, onset of the disease, clinical characteristics). The lesions were cleaned by Alcohol 70% and the tissue samples were collected by dermal scraping from suspected CL patients from the edge of lesions, after which slide smears were prepared, fixed with absolute methanol and stained with Giemsa for microscopic examination. The whole slides were analysed using a ×100 immersion objective. Total DNA was extracted from slide smears and monospecific pools of sand flies using the phenol chloroform method. Pools of unengorged females of P. longicuspis, P. perniciosus and P. sergenti were homogenised in 200 μl of phosphate-buffered saline (PBS 1×) using a disposable pestle. After centrifugation at 6000 × g for 2 min, the supernatants were used for DNA extraction. DNA quantification was determined using a NanoDrop (Thermo Scientific). The ITS1 was amplified using primers LITSR and L5. 8S following the protocol of Schonian et al. [26]. A negative control (without DNA) was used for each PCR run. To identify Leishmania species, the positive PCR products of 350 bp in size were subjected to enzymatic restriction by HaeIII for 2 h at 37°C. RFLPs were analysed by electrophoresis on 3% agarose gel containing ethidium bromide. A 100-bp DNA size marker was used (HyperLadder 100bp Plus). The restriction profiles were compared to reference strains for L. infantum (MHOM/TN/80/IPT1), L. major (MHOM/SU/73/5ASKH) and L. tropica (MHOM/SU/74/K27). The detection and identification of Leishmania parasites were performed via nested PCR amplification of kDNA as previously described [27]. Two PCR stages were performed in two separate tubes. The first-stage used the forward primer CSB2XF and reverse primer CSB1XR and the second stage used the nested forward primer 13Z with the nested reverse primer LiR. These primers allowed the amplification of the variable region of kDNA of Leishmania species, giving a specific molecular weight for each species as follows: 560 bp for L. major, 680 bp for L. infantum and 750 bp for L. tropica [27,28]. Cross-contamination was monitored using negative controls for sample DNAs and solutions of all PCR reagents used. The amplification products of nested PCR were separated and confirmed by electrophoresis in an ethidium bromide-stained 2% agarose gel. The obtained 350-bp ITS1-PCR products were purified using exonuclease I/shrimp alkaline phosphatase (GE Healthcare, US). The sequencing reaction was performed in both directions using BigDye Terminator version 3. 1 (Applied Biosystems, Foster City, CA, USA). The resulting forward and reverse sequences were aligned. A total of nine partial ITS1 DNA sequences, in addition to the studied sequences, were selected from GenBank, including five sequences from L. infantum (AJ634361, GQ367486, KX664452, AJ634339 and AJ634355), four sequences from L. tropica (KP202104, KU194923, KX599337 and KP202102) and one sequence from L. major (AJ000310). Phylogenetic analysis was performed with MEGA version 7 software using the neighbour-joining and Kimura 2-parameter models. The tree topology was supported by 1000 bootstrap replicates. Written informed consent was obtained from all the adults who participated in the study. Consent for inclusion of young children, was obtained from parents or guardians. The study and the protocols were approved by the Ethics Committee for Biomedical Research (CERB) of the Faculty of Medicine and Pharmacy, Rabat, Morocco.
  A total of 3,583 sand flies (2,485 females and 1,098 males, Table 1) were collected in three peri-urban neighbourhoods of Taza (Fig 1). Our results revealed a monthly evolution that varied among the species. Specifically, the evolution was monophasic for some species and biphasic for others. P. longicuspis, the most abundant sand fly species, had one peak of activity in August, whereas P. sergenti, the second most prevalent species, exhibited two peaks of activity in July and September (Table 1). Morphological identification revealed the presence of 13 species, including 10 belonging to the genus Phlebotomus and 3 in the genus Sergentomyia (Table 1). The most prevalent species was P. longicuspis, with a relative abundance of 64. 08% of the total collected sand flies. P. sergenti and P. perniciosus were the next most abundant species, comprising 20. 78 and 8. 52%, respectively of the specimens. These three species constituted 93. 11% of the total number of collected sand flies. The remaining 3. 55% consisted of P. papatasi, P. bergeroti, P. ariasi, P. dreyfussi, P. kazeruni, P. langeroni, P. alexandri, P. perfiliewi, S. minuta and S. antennata (Table 1). The sex ratio was 0. 43, indicating that more females were collected by CDC light trap in this area. Unengorged female sand flies of the most prevalent species collected in this study, namely P. longicuspis, P. sergenti and P. perniciosus, were organised in a total of 54 pools, each containing up to 30 sand flies (39 P. longicuspis pools, 4 P. perniciosus pools and 11 P. sergenti pools, Table 2). The pools were screened for Leishmania infection using kDNA nested PCR. Leishmania DNA was amplified from 23 P. longicuspis and 4 P. perniciosus pools, giving a single band of 680 bp corresponding to L. infantum, as well as seven P. sergenti pools, giving an expected single band of 750 bp corresponding to L. tropica. Hence, the overall minimum L. infantum infection rates based on the positive PCR of P. longicuspis and P. perniciosus were 2. 51 and 7. 27%, respectively. The L. tropica infection rate within P. sergenti was evaluated as 3. 24% (Table 2). Among the 12 patients suspected to have CL based on clinical criteria, seven were found to be positive on microscopic examination. ITS1-PCR was more sensitive, as it identified nine CL-positive patients. Enzymatic digestion by HaeIII revealed two profiles. The first consisted of two bands (185 and 57/53 bp), as observed for L. tropica (MHOM/SU/74/K27), and the second profile (184,72 and 55 bp) was the most abundant band pattern; identical to L. infantum MHOM/FR/78/LEM75 and detected in eight patients. As for L. tropica it was observed in only one patient (Table 3). To confirm the PCR-RFLP identification results, the ITS1-PCR products were sequenced and subjected to NCBI-BLAST analysis for homology (http: //blast. ncbi. nlm. nih. gov). The ITS1 sequences exhibited a length range of 300–350 bp. The identity of all isolates from patients with CL was in agreement with the ITS1-PCR-RFLP results, indicating that the sequences KY973658, KY973656 and KY973661 were similar (97%) to the Spanish L. infantum sequence deposited in GenBank under accession number AJ634355, whereas KY973656, KY973657 and KY973662, were similar (98%) to the Moroccan L. infantum sequence deposited in GenBank under accession number KX664452. Conversely, the sequence KY974310 was similar to the Moroccan L. tropica sequence deposited in GenBank under accession number KP202104 (99%). The phylogenetic analysis based on ITS1 sequences generated in this work and other sequences from NCBI revealed that all L. infantum isolates are clustered together, whereas L. tropica and L. major each appear in different clusters. The obtained tree also revealed a level of polymorphism among the L. infantum Moroccan isolates (four distinct genotypes were identified among eight isolates, Fig 2).
  Based on our entomological and epidemiological findings, we believe that environmental changes may have shifted the sand fly distribution, making the subgenus Larroussius most prevalent. This has led to the establishment of a stable transmission cycle of L. infantum and subsequently to the emergence of CL due to L. infantum, which is invading the area of L. tropica. In fact, L. infantum is becoming a problem in Morocco because of its rapid spread throughout the country. Indeed, this Leishmania species was reported in several foci previously only considered L. tropica CL foci. L. infantum was also identified in L. major CL foci. Thus, more investigations and control measures are needed in Taza city in light of our findings to counter the spread of both L. infantum and L. tropica.
summary: Two types of leishmaniasis are endemic in Morocco: visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). VL is caused by Leishmania infantum, a species that is responsible for sporadic cutaneous leishmaniasis, in addition to L. major and L. tropica, which has the largest geographic distribution in Morocco. Taza province is considered as an area of mixed focus wherein VL and CL coexist. However, since the mid-1990s, this region is particularly focused upon for CL due to the presence of L. tropica. Recently, an L. infantum dermotropic variant has also been detected in CL patients. This situation calls for an in depth epidemiological investigation to identify with certainty the vectors of both L. infantum and L. tropica in this area. Our results highlight the dominance of L. infantum in cutaneous leishmaniasis patients, this species seems to invade L. tropica' s field. Moreover, two phlebotomines species have been incriminated in the transmission of L. infantum, whereas the L. tropica’s vector has been confirmed. Thus, in light of our findings, the health authorities should implement control measures to counter the spread of both L. infantum and L. tropica in Taza province.
keywords: invertebrates
  morocco
  medicine and health sciences
  tropical diseases
  geographical locations
  sand flies
  parasitic diseases
  parasitic protozoans
  animals
  diptera
  protozoans
  leishmania
  neglected tropical diseases
  molecular biology techniques
  insect vectors
  africa
  research and analysis methods
  infectious diseases
  zoonoses
  artificial gene amplification and extension
  epidemiology
  phlebotomus
  protozoan infections
  molecular biology
  disease vectors
  insects
  arthropoda
  people and places
  leishmania infantum
  eukaryota
  polymerase chain reaction
  leishmaniasis
  biology and life sciences
  species interactions
  organisms
title: Molecular identification of Leishmania infection in the most relevant sand fly species and in patient skin samples from a cutaneous leishmaniasis focus, in Morocco