Cutaneous Leishmaniasis in Algeria; Highlight on the Focus of M’Sila

Algeria ranks second after Afghanistan for the incidence of cutaneous leishmaniasis (CL) worldwide. Here, we report a 34-years retrospective analysis of CL in Algeria and focused on the most affected region, the M’Sila province. All 66 cutaneous isolates corresponded to Leishmania (L.) major. Our study of the sandfly and rodent fauna further highlighted the high density of Phlebotomus papatasi and additional phlebotomine species of medical importance, not previously identified in M’Sila. Wild rodents belonging to nine species were trapped in M’Sila, and Psammomys obesus and Meriones shawi were found infected by L. major. In addition, Leishmania infantum was isolated from two visceral leishmaniasis cases, one dog and its proven vectors (P. perniciosus, P. longicuspis, and P. perfiliewi) inventoried during the survey. The high incidence of CL in the M’Sila province is likely a consequence of the increase in minimum temperatures recorded that constitutes suitable conditions for establishing a high endemicity and leads to an explosive rise in leishmaniases cases in this region. A thorough investigation of the underlying risk factors is urgently needed to detect new cases earlier. All these would improve the preparedness to fight the disease.


Introduction
Algeria ranks second after Afghanistan in cutaneous leishmaniasis (CL) incidence [1]. Initially confined in the high plateaus' steppe regions in the semi-arid and arid bioclimatic zones [2,3], CL has undergone a worrying geographic extension toward the north of Algeria [4]. Such an expansion also occurs in the Maghreb and the Middle East [1,[5][6][7].
The province of M'Sila is located in Central-Eastern Algeria and is most affected by the disease, with nearly 90,000 cumulative cases recorded since 1982 [8]. Cutaneous leishmaniasis is first described in 1860 in Algeria [9] and 1924 in the Hodna basin [10]. Starting from a longitudinal survey on the incidence of CL cases from 1982 to 2020 in the M'Sila focus, we gathered data on the diversity of the phlebotomine sandfly and rodent fauna and ecological factors in this region. We put them in perspective with the overall CL epidemiological situation in Algeria. These analyses are helpful to improve future public health strategies aimed at controlling ZCL.

Study Area and Study Design
The province of M'Sila locates between 35 • 18 and 35 • 32 North and 4 • 15 and 5 • 06 East, 240 km south-east of Algiers, in the country's highlands. Rainfall varies from 69 mm to 200 mm/year, and the average annual temperatures between 13 • C and 23 • C, with extremes of −5 • C in winter to 46 • C in summer [20]. The province encompasses 15 Daïras (districts), and the most populated one is M'Sila, a city followed by Boussaâda, called "the gateway to the Sahara" (Figure 2).
In the endemic foci of M'Sila, we collected data on ZCL incidence from 1982 to 2020 and other associated information, including clinical aspects of lesions, the dynamic and diversity of the phlebotomine, and the diversity of the wild rodent's population. In parallel, data on CL incidence in Algeria were computed for the 48 provinces of the country from 1982 to 2017 [8]. A phlebotomine fauna survey was carried out from 2003 to 2004 and in 2018 in various sites: stables, sheepfolds, gardens, rodent burrows, palm groves, crevices, and dwellings (Table 1).    In five stations (Nouara, Ouled Madhi, Maadid, Ain El Hadjel, Boussaâda), the trapping effort was carried out twice a month over the transmission season (March to October). The other trapping sites were sampled one time along east-west and northsouth transects. Sandflies were collected using adhesive paper (20 cm × 20 cm papers soaked in castor oil) and CDC light traps (John W., Hock Co., Gainesville, FL, USA) as described by Rioux et al. [21]. Specimens were collected and stored in glass vials containing 70% ethanol for morphological identification, using dichotomous keys of Abonnenc [22], Croset et al. [23], and Dedet et al. [24]. Seasonal fluctuation of the sandfly population was ascertained in the station of Ain El Hadjel (Table 1).

Rodent Capture
Trapping of rodents was performed during 1996,2003,2004,2007,2010,2012, and 2018, based on indications of their presence (burrows, fragments of Chenopodiaceae plants, droppings, and traces of urine). Sherman traps were placed along a transect of the salt-lake (sebkha) and down the hills' piedmont (Table 2).
Rodents were anesthetized with carbon monoxide (CO); morphological identification was performed according to identification keys described by Bernard [25]. Auricle, muzzle, legs, and tail exposed to insect bites were carefully examined. A biopsy of lesions was taken and smeared on a glass slide and stained with Giemsa. A portion of the sample was inoculated into an NNN medium supplemented with few Penicillin antibiotic drops (1U/mL) solution under sterile conditions [26,27].
The spleen was collected and homogenized in microtubes containing PBS with singleuse pellet pestles (Polylabo, France). Aliquots of the suspension were stored at −20 • C until DNA extraction. According to the manufacturer protocol, the total DNA was purified using the QIAamp DNA mini kit (QIAGEN). Parasite DNA was detected by PCR amplification of the ribosomal internal transcribed spacer 1 (ITS1), using LITSR and L5.8S primers [28].

Patient: Skin Wound Sampling Collection
Ninety-six patients with evocative CL lesions were recruited during a routine diagnosis of the disease. Sixty patients were from Boussaâda and 36 from M'Sila city. Skin lesions were scarified using a curved sterile scalpel blade (Swann-Morton-Sinner, France), as described in the WHO guidelines for diagnosing human CL [29]. Exudates from dermal lesions were spotted onto microscope slides, inoculated into a culture medium, and processed for DNA extraction. Blood samples from two children with symptoms of visceral leishmaniasis and 18 members of their family and a dog living in the same house were sampled in an EDTA coated flask (Vacutest Kima, Arzergrand, Italy).

Parasitological Diagnosis
Direct microscopic examination of smears was performed for all suspected CL patients. Thus, smears were stained with Giemsa and examined at 100× magnification. Exudates were seeded in an NNN medium supplemented with few drops of antibiotic (Penicillin 1 U/mL) in sterile saline solution [26,27] and then sub-cultured every week for four weeks. The buffy coat extracted from the blood of VL cases, their family members, and the dog samples were inoculated into both NNN and rabbit's coagulated serum medium [30].  [28]. DNAs of reference strains and water were used as positive and negative controls. The amplification product was analyzed on a 2% agarose gel and visualized under ultraviolet light. The presence of the 300 and 350 base pair bands compatible with those of the reference strains was analyzed. Then, positive samples were incubated at 37 • C for 24 h with the endonuclease enzyme HaeIII (Hybaid GmbH, Heidelberg, Germany) to identify the parasite at the species level. After electrophoresis on 2% agarose gel and visualization under ultraviolet light, the lengths of the bands obtained were compared with those of the reference strains [28].

Molecular Diagnostic and Typing
Real-time qPCR amplification of the kDNA was performed were carried out in a volume of 25 µL containing 1 µL of DNA in 24 µL of a master mix (Taq Man Universal Master mix II with UNG (Applied)) having 100 nM of each primer (5 -CTTTTCTGGTCCTCCGGGT AGG-3') and (5'-CCACCCGGCCCTATTTTACACCAA-3 ), 50 nM the probe (FAM 5 -TTTTCGCAGAACGCCCCTACCCGC-3 ). The DNA was amplified with a Taq Man Applied Biosystems 1 apparatus (Applied Biosystems, Foster City, CA, USA). The amplification conditions were 95 • C for 2 min, followed by 40 cycles of 95 • C for 15 s, 60 • C for 1 min, and 60 • C for 1 min. Reactions were validated with positive and negative control samples. The DNA of the samples was quantified against a serial dilution of Leishmania DNA extracted from a known number of parasites (0.5 to 0.005 parasites per reaction tube). Real-Time PCR was considered positive when the test sample's cycle threshold was below that of the last point of the range, in a valid reaction [31].

Sequencing and Phylogeny of Chosen Isolates
ITS1 amplicons were purified using ExoSAP (ThermoScientific, EU). Leishmania species were identified by DNA sequencing using an ABI Prism1 Big Dye™ Terminator, Cycle Sequencing Ready Reaction Kit, and AB1 3130 sequencing system (ABI, PE Applied Biosystems), with the same primers used for PCR. DNA sequences from both strands were aligned and edited using the Staden software package [32]. The MEGA version 7 software [33] was used to conduct multiple sequence alignments (ClustalW option) and construct a phylogenic tree. Homology searches were performed using BLAST (Basic Local Alignment Search Tool) using the NCBI (National Center for Biotechnology Information) server [34]. The generated sequences were aligned using SeaView4 [35] to study the targeted genes' genetic variability. Phylogenetic analysis was carried out using maximum likelihood analysis with the online phylogeny website (www.phylogeny.fr, accessed on

Multilocus Enzymatic Electrophoresis (MLEE) Typing
Eighty-seven isolates were analyzed with MLEE (66 from humans and 21 from rodents, including 23 isolates collected during the survey performed in 2018/2019. The remaining 43 human isolates were taken from the cryobank of the Institut Pasteur of Algeria. The isoenzyme characterization of isolates was carried out using 15 enzyme systems, according to the protocol previously described by Rioux et al. [37].

Data Management and Statistical Analysis
Data management and analysis were performed using Excel 365 (Microsoft office, USA); Graph-prism (8.02, GraphPad Software Inc., San Diego, CA, USA); XL STAT (v2020, Addinosoft) and Stata 11 (v16.1, StataCorp, TX, USA). Maps that relate the spatial distribution of CL cases and the cumulative incidence within the 48 provinces and the cumulative number of patients in the 15 districts of M'Sila were constructed using Photoshop (v2015, Adobe). A one-factor ANOVA analysis used to test CL per province's incidences per year was performed using the Graph-prism (v8.02, GraphPad Software Inc). and Stata 11 software. Dunnett's post-test was used when the ANOVA p-value was less than 0.05. A principal component analysis (PCA) was performed using XL STAT.

Ethical Considerations
The study protocol, informed consent forms, and sampling/experimental procedure were reviewed and approved by the Ethical Committee of Institut Pasteur of Algeria (the study began after obtaining the informed consent from patients under Helsinki's declaration (1964)). All animal experiments carried out in vivo comply with the guidelines of the Veterinary Inspection department of Algiers (veterinary health declaration established by the chief veterinary inspector, N • 2505 / IVWA / 2019 in April 2020 and approved by the Research Ethics Committee of the Institut Pasteur of Algeria, and also approved by the Algerian Association for the Sciences of Animal Experimentation (AAAES).

Dynamics of Cutaneous Leishmaniasis in the Province of M'Sila Compared to the National Epidemiological Status of the Disease
From 1982 to 2017 (34-year period), more than a quarter of a million cases (252,659) of CL were registered in the country ( Figure 4A). During this period, five provinces (Bechar, El Oued, Batna, Biskra, and M'Sila) recorded more than 70% of the total cases ( Figure 4B).
Since 1982, the dynamics of CL reveals an endemic-epidemicity status of the disease, with several peaks observed, the most critical being in 2005 with more than 25,511 cases recorded in the country ( Figure 5B). Statistical analysis disclosed a highly significant difference (ANOVA 1-way test, p < 0.0001) in the annual distribution of recorded cases over the 34 years. As compared to 2005, the incidence appears to be significantly different to   The district of M'Sila is significantly more affected in terms of the number of cumulative cases than the rest of the districts (1-way ANOVA test, p < 0.0001). A higher CL case incidence was reported in M'Sila than in the former foci of Biskra (Dunnett's post, p < 0.05) ( Figure 6A,B).

Leishmania Detection and Identification in Patients and Reservoirs
The results of the diagnostic methods performed on skin samples of humans suspected of CL are summarized in Table 3. Among the suspected CL patients, 45.83% were recorded positive based on direct examination and 53.68% based on culture. A higher positivity rate was recorded for molecular methods, reaching 73.40% using qPCR. In total, 71 patients were diagnosed as CL positive (Table 3). Molecular identification (PCR-RFLP) disclosed that all 42 CL samples and 21 gerbil samples tested were positive for L. major. Samples collected from humans with visceral symptoms (two cases) and from a dog (one case) belong to L. infantum (see Figure 8). Isolates characterized by MLEE (66 from humans and 21 from rodents) were identified as L. major zymodeme MON-25. A minor variation in 3 enzymatic complexes for G6PD, PGD, and ME is recorded in 10 isolates, suggesting the possible existence of L. major variants in this region. No discrepancy was observed between PCR-RFLP and MLEE results. A fragment amplified from the ITS1 gene was sequenced for seven isolates (1 VL, 1 canine leishmaniasis, and 5 CL). Reference strains used were L. infantum MHOM/FR/78/LEM75, L. killicki (syn L. tropica) MHOM/TN/80/LEM163, and L. major MHOM/MA/81/LEM265 (7). L. infantum was identified in visceral leishmaniasis and canine leishmaniasis cases, whereas L. major was identified in CL cases. The identification of L. major in lesions of CL and the presence of L. infantum in dog and human suspected cases of VL were confirmed by the phylogenetic analysis of the ITS1 sequences ( Figure 8B).      Within the M'Sila province, P. papatasi is highly prevalent, particularly in the districts with high CL incidence, P. longicuspis being abundant in districts where few cases of VL are recorded. The sandfly dynamics recorded in the province of M'Sila highlight a relation between sandfly density and the average temperature recorded. The sandfly population density reaches its first peak in June and a second in September (see Figure 9). In Boussaâda, 12 species were identified; P. papatasi is highly abundant in this station (74.83%), followed by P. longicuspis (18.65%). Two species were detected for the first time in this region, P. chabaudi (4.29%) and S. lewisi (0.06%). In the district of M'Sila, the specimens collected belonged to 12 species. The Phlebotomus genus represents 86.78% of the total sample, and P. longicuspis (53.25%) was the most prevalent. Notably, P. papatasi represented 33.84% of the specimens captured, and S. clydei was identified for the first time in this region. In Chellal, specimens belonging to 10 sandfly species were recorded. Sergentomyia were prevalent and represented 71.38%, including S. fallax (46.53%), S. antennata (38.79%), S. schwetzi (8.78%), and S. minuta (5.77%). Phlebotomus papatasi represented 27.12% of sandflies captured in rodent burrows. In this district, P. perniciosus (3%), P. longicuspis (1.31%), P. alexandri and P. chabaudi (0.65%) were also recorded. In Magra, 6 species were identified, with P. papatasi (53.85%), P. perniciosus (19.23%), and P. longicuspis (6.73%) being the most frequently trapped. At Ain El Hadjel, 10 species were identified. Phlebotomus papatasi is the most abundant (91%), followed by P. perniciosus (6%). Four specimens of P. perfiliewi were collected for the first time in this arid region. A total of 7 species were identified at Ouled Derradj. Phlebotomus perniciosus is the most frequently identified (66%), followed by P. sergenti (11.96%), P. papatasi, and P. longicuspis (both < 10%). In the districts of Sidi Aissa, Ain El Melh, and Hammam Delaa, a low number of sandflies were collected, with a large majority represented by P. papatasi (78%). The factorial plane displays that P. papatasi is the main contributor of the F1 inertia, and its abundance correlates with arid steppe, plains, and foothills, and less frequently with mountains (9.3%). Steppes offer optimal ecological conditions for this species [7,24,38,39]. Phlebotomus perniciosus and P. longicuspis are the main contributors to the axis 2 inertia. A third group encompasses other sandfly species that, for the majority, are not of medical importance (Figure 10, lower panel).

Survey of the Reservoir Hosts of Cutaneous Leishmaniasis
More than 445 wild rodents belonging to 5 families, 7 genera, and 9 species were collected during the study (Table 2). Gerbils are abundant in the plains and the Chott ( Table 2). Ctenodactylus gundi (Ctenodactylidae) is frequent in the Rocky Mountains of Boussaâda and Ain Farez (Daïra of Ain el melh), located 30 km south-east of Boussaâda city. Psammomys obesus and Meriones shawi often overlap in terms of territories. Psammomys obesus was trapped frequently in the steppe where Chenopodiaceae develop, the Chellal region, and the road shoulders along the Sebkha (around the Chott). This rodent species was also trapped at the piedmont of Jebel Choukchot's mountains near Sidi Aissa and in peripheral areas of M'Sila. Like M. shawi, it settled on the slopes at the edge of cereal fields. It is also abundant in orchards and agricultural areas. It was captured near the salt-lake (Sebkha) of M'Sila, in the countryside and its suburbs. Molecular diagnosis by PCR-RFLP revealed the infection by Leishmania in only two species, P. obesus, and M. shawi. Among the 186 P. obesus and the 37 M. shawi collected during the survey, 29.57% and 27% were positive for Leishmania DNA, respectively ( Table 6). The 21 isolates of Leishmania from rodents (P. obesus = 17, M. shawi = 4) were identified as L. major MON-25 (see above).

Epidemiology of Leishmaniases in Algeria and M'Sila
In Algeria, the first case of human CL was described in 1860 by Hamel in the former focus of Biskra [9] and 1924 in M'Sila [10]. The disease evolved slowly until the early 1980s when outbreaks occurred suddenly in M'Sila, which is very close to Biskra. Since then, the disease has become a real public health problem, and its incidence has increased significantly in the recent decades [4,40]. After 34 years (1982-2017), more than a quarter of a million cases were recorded in all the country (252,659 cases), which equals an incidence of 1372 cases/100,000 inhabitants. Five provinces are highly endemic for CL, with more than 10,000 cases recorded between 1982 and 2017. Three are located in the west of the country and are adjacent to Biskra (Batna, M'Sila, and El Oued). M'Sila and its seven neighboring provinces account for 74.5% of the total CL cases reported in Algeria during the 34 years of our study. M'Sila and Biskra alone account for 37.22% of the recorded cases during these three decades. A Dunnett's statistical analysis discloses no significant differences in terms of incidence between these two provinces. The temporal dynamics of the CL case number follow that of M'Sila, showing the endemic-epidemic profile of the disease with a high proportion of cases in the direct neighborhood of patients (56.1%).

Leishmania Species and Clinical Presentation in M'Sila
Parasite identifications carried out during our prospective and retrospective surveys (87 by MLEE, 42 by PCR-RFLP) confirmed L. major as the etiological agent of CL in the M'Sila region and are in accordance with previous studies [3,12,41]. The most common zymodem in Algeria (MON-25) [12,41] was the sole identified. However, variability was recorded for three enzymatic systems, G6PD, PGM, and ME, in some isolates from Boussaâda, Sidi Aissa, and M'Sila. Leishmania major MON-269 as a causative agent of CL was previously reported in Algeria [41]. This zymodem differs from MON-25 by a single enzyme system (PGD 122/94) and could correspond to a recent mutation or a posttranslational modification [42]. The combination of four diagnostic methods (microscopy, NNN culture, ITS1-PCR, and RT-PCR) allowed diagnosis confirmation of a maximum number of cases among the suspected CL patients, resulting in a more accurate clinical description of the disease. As reported [31,43], RT-PCR was the most sensitive (100%) technique. On the other hand, the high sensitivity of cultures (72.9%) is related to the adaptation of L. major to the NNN medium [27]. The clinical appearance is characterized by a predominance of childhood infection and multiple lesions (61.4%), preferentially localized to the limbs (70.4%) [44]. Children of less than or equal to 15 years (76.1% of cases) considered as "naive" are likely to be the most susceptible to L. major infection [45,46].
Otherwise, M'Sila does not fall into the group of provinces where VL is endemic; therefore, the identification of L. infantum in dogs and humans requires further studies to delineate the endemic occurrence of the transmission cycle.

Sandfly Diversity and Dynamics in Algeria and M'Sila
Out of the 24 species reported in Algeria [24,47,48], 16 were identified during this study, mostly in sympatry in the various districts investigated. The comparable richness in sandfly fauna was reported in the north/north-eastern and middle eastern regions of Algeria [49][50][51]. Our study enriched the sandfly entomofauna of M'Sila [52] [4] indicates that sandfly richness might not have changed over time in this area. Likewise, P. papatasi being the proven vector of L. major, zoonotic CL incidence, is high in this area [15,53].
Interestingly, some identified sandfly species with high densities in some districts, such as P. perniciosus and P. longicuspis, are suspected or proven vectors for visceral leishmaniasis. The sandfly seasonal dynamics revealed a bell-shaped density curve with a broad peak encompassing the July-September period and between early October to late May. The season for leishmaniasis transmission risk took place from early June through the end of September when maximal density is achieved. This kind of bell-shaped density curve was described for sandflies in the Mediterranean area [54]. During this period, the second and third generations of sandflies are more abundant, increasing the infection risk [53]. The ZCL seasonal dynamic is under minimal temperature control and presents a 2-month lag between the reported infection date and the presumed date of CL lesion onset [55,56].

Ecology, Diversity, and Medical Importance of Sandflies in M'Sila
The 5 stations analyzed with PCA revealed a predominance of P. papatasi in stations above 300 m of elevation, plains, and piedmonts and coincided with the ZCL foci of M'Sila. In these areas, steppes constitute the optimal ecological niche for P. papatasi. Phlebotomus papatasi can also be present at a higher altitude, up to 1000 m, like in Maadid, where it accounted for 9.30% of the total specimen trapped. In lowland rural areas, P. papatasi reach up to 91% (Ain El Hadjel, Sidi Aissa, and Boussaâda). The low sanitation level would favor the presence of manure and organic matter that, along with high humidity, are adequate for sandfly breeding [57]. Phlebotomus perniciosus and P. longicuspis are abundant mainly in the piedmont area of Nouara (M'Sila) and the mountain of Maadid (Ouled derradj), respectively. Phlebotomus longicuspis is abundant in humid peri-urban regions, like in Nouara, or Boussaâda palm grove, representing 51.82% and 18.65% of the total trapped sandflies, respectively. It is a suspected L. infantum vector in the arid zones of Morocco and Tunisia [58,59]. Leishmania infantum has been detected in P. longicuspis in Algeria [60]. During our survey in 2018, two autochthonous VL cases and one canine case were detected in Maarif (a few kilometers from Boussaâda city). However, the potential vectorial role of P. longiscupis for L. infantum requires to be proved more thoroughly in M'Sila. Only seven specimens of P. perfiliewi, a proven vector of CL caused by L. infantum in Algeria [61,62] and Tunisia [63], and of VL in Algeria [64], were captured during our survey. Common in the humid and subhumid areas, this sandfly was collected in the highlands' arid region, where conditions might not be optimal for this species [24]. This species is abundant in the semi-arid bioclimatic stages, like in Constantine [50,65]. Specimen of the paraphlebotomus genus were less frequently trapped. Phlebotomus sergenti represented 0.68% of the total specimen, but it reached up to 12% in the Maadid station in Ouled Derradj. In Algeria, this species is mainly encountered in the south of the country in the Saharan Atlas' foothills, the pre-Saharan steppes, and the Tell regions [24]. It was found in crevices of walls, rodent burrows, and rocky areas [24]. It is the proven vector of L. tropica [66] in the Middle East and probably of L. killicki (syn: L. tropica) in the Maghreb countries [67]. Leishmania killicki has been identified in many CL cases in Ghardaia in the center of Algeria [68]. Phlebotomus chabaudi was observed in the Boussaâda palm grove, and the Chellal region (Ouled Madhi). Since its description in Tunisia [23], the presence of this species is documented in Ghardaïa in Algeria [69] and Morocco [70]. The presence of P. chabaudi in the biotopes of Ctenodactylus gundi (rocky biotopes) would make them a potential vector of L. tropica. Phlebotomus (Par). Alexandri, collected in the district of Ain El Hadjel, is considered as a ZCL vector in the former USSR and Iran [71,72]. Also, in this district a single specimen of Phlebotomus (Lar) langeroni was collected. It is considered as a vector of L. infantum, responsible for sporadic CL in Northern Tunisia [73] and of VL in Egypt [74,75]. It is particularly abundant in rocky and dry biotopes where Ctenodactylus gundi and reptiles roost [24].

Rodents and Reservoirs in the M'Sila Province
In Algeria, the endemicity of the ZCL is linked to a regular epidemiological cycle maintained by P. obesus, whose populations are geographically restricted. In contrast, epidemic outbreaks correlate with the proliferation of M. shawi and food abundance during rainy years. The surveys have made possible an update of mammalian fauna inventory and a more accurate spatial and micro-zonal distribution of rodents playing a role in ZCL transmission. Several ecological niches were identified, and nine rodent species were inventoried. This number does not reflect the rodent fauna richness in this region since the inventory performed in 1991 by Kowalski and Rzebik-Kowalska [76] reported 19 rodent species in the Hodna basin. During our survey, only P. obesus and M. shawi were found infected by L. major MON-25 ( Table 2). The presence of L. major in M. shawi trapped in Sidi Aissa (1996), and Ain El Khadra (2000) ( Table 2) represent the first report of such events in the M'Sila province. The molecular diagnosis detected L. major in 29.57% of P. obesus and 27% of M. shawi trapped. This confirms the primary reservoir's role of these two species in the province of M'Sila.
Interestingly, in a previous study, a Leishmania isolates from Meriones spp. was categorized as non-susceptible (S−) toward trivalent and pentavalent antimony [6,77]. Further experiments are required to address the risk of chemotherapeutic failure in link with the transmission of such variants.

Conclusions
Zoonotic CL caused by L. major is highly prevalent in the M'Sila region for more than 30 years, with an average of around 3000 cases per year. This focus is characterized by an arid bioclimate, a high density of P. papatasi sandflies, and a high Leishmania infection rate of Psammomys obesus and Meriones shawi rodents. The clinical profile of the disease is similar to that described in other North African countries, CL being most frequently encountered mainly in childhood and presenting as multiple lesions preferentially localized on the limbs. Identifying ZCL risk factors in M'Sila could allow more efficient control of the disease.