Updates on Geographical Dispersion of Leishmania Parasites Causing Cutaneous Affections in Algeria

Leishmaniases are neglected tropical diseases of public health concern in Algeria. To update the geographical distribution of Leishmania spp. causing cutaneous affection, we examined a set of Giemsa-stained smears prepared from skin lesions of the patients suspected to have cutaneous leishmaniasis (CL) in various geographical areas in Algeria. The identification of Leishmania parasites was performed using microscopy, conventional PCR, and PCR–RFLP (PCR-Restriction Fragment Length Polymorphism) targeting ITS1-rDNA. Among 32 smears provided from 27 suspected patients with cutaneous lesions, no trace of parasites was observed in the smear of three patients using microscopy and molecular approaches. Furthermore, four patients presented at least two lesions. PCR–RFLP confirmed the presence of Leishmania in 29 smears prepared from 24 patients. Two biopsies, negative after microscopic examination, were found positive by PCR. Of these 29 PCR positive smears (24 patients), 20 were identified using RFLP–PCR as L. major, two as L. tropica, and two as L. infantum. We found L. major infected patients from Ain skhouna, Biskra, El M’hir, Ghardaïa, M’Sila, and Saida, in agreement with previously reported cases. Furthermore, we highlighted for the first time, the identification of L. major in the patients from Bourkika, Bou Kremissa, Bou Saada Clef, Hajout, Maghnia, Médéa, Menaceur, Messad, Mostaghanem, Nador, Oran, and Sidi Okba. A phylogenetic reconstruction performed with sequences collected from the PCR products confirmed these identifications. Our data provide additional information on the geographical extension of CL caused by L. tropica and L. infantum in Algeria.


Introduction
Leishmaniases are vector-borne diseases caused by obligate protozoan parasites from the genus Leishmania (Trypanosomatida: Trypanosomatidae), and transmitted by the bite of infected female phlebotomine sandflies (Diptera: Psychodidae), whose hosts/reservoirs are animals such as canids, rodents, marsupials, hyraxes, or humans [1]. Epidemiological cycles of leishmaniases fall into two broad categories: the zoonotic forms of leishmaniases (ZL), where the primary reservoirs are wild or domestic mammals, and anthroponotic forms (AL) for which humans are the primary reservoirs. Two clinical presentations are distinguished: visceral (VL) and cutaneous (CL). Leishmaniases are endemic in large areas of the tropics, subtropics, and the Mediterranean basin. In 2018, 92 and 83 countries or territories were considered endemic or previously reported for CL and VL, respectively [2]. spacer 1 (ITS1) was performed using LITSR (forward: 5 -CTGGATCATTTTCCGATG-3 ) and L5.8S (reverse: 5 -TGATACCACTTATCGCACTT-3 ) primers [27]. Negative (absence of target DNA) and positive (presence of DNA from reference Leishmania strains) controls were used for each PCR batch. Amplicons were analyzed after electrophoresis in a 1.5% agarose gel containing ethidium bromide. Endonuclease digestion was performed following a previously published protocol [27]. Briefly, 10 µL of the PCR product was incubated at 37 • C in a final volume of 30 µL, containing 2 µL of BsuRI (HaeIII) (Fermentas, Vilnius, Lithuania), 2 µL of 10× buffer, and 16 µL of distilled water. After 4 h, digested fragments were run on a 3% agarose gel containing ethidium bromide. A DNA ladder of 50 bp (Fermentas) was used to identify diagnostic DNA fragments.

Sequencing and Typing of Leishmania Isolates
Leishmania DNA was subjected to conventional PCR targeting ITS1 (partial sequence), 5.8S (complete sequence), and ITS2 (partial sequence), using forward (ITS1F: 5 -GCAGCTGGATCATTTTCC-3 ) and reverse (ITS2R4: 5 -ATATGCAGAAGAGAGGAGG C-3 ) primers with an expected length of 430 bp [28,29]. Double-distilled water and purified DNA from L. major, L. tropica, and L. infantum were used as negative and positive controls for each PCR batch. Amplicon quality was analyzed after electrophoresis in a 1.5% agarose gel with ethidium bromide. PCR products were purified using an Invisorb Fragment CleanUp kit (Stratec Molecular, Berlin, Germany) and sequenced using the same primers for PCR amplification. The sequences were compared to homologous sequences collected in the GenBank database and aligned with the Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST). All sequences were identified as L. major, L. tropica, or L. infantum, based on ≥99% identity with GenBank sequences. The phylogenetic analysis was carried out using MEGA v.6 software. A phylogenetic tree of Leishmania species (identified in this study) and GenBank sequences was constructed using neighbor-joining (NJ) with bootstrap values of 1000 replicates.

Results
A total of 32 Giemsa stained smears were prepared from active skin lesions of suspected 27 CL patients referred to the Hadjout, Biskra, and Saida health centers in Algeria ( Figure 1). Biopsies were taken from all lesions (one to three lesions) from patients of ages ranging from 3 to 82. After microscopic examination, 27 smears from the 32 lesions processed were positive for Leishmania sp. (including four patients with at least two lesions). Five patients were negative for Leishmania infection after a microscopic examination. See Table 1 for epidemiological and clinical information of all patients.
All biopsies were subjected to molecular characterization by PCR-RFLP. A schematic representation of the PCR-RFLP restriction profile is given in Figure 2, along with the restriction profiles generated for selected samples. The twenty-seven smears (24 patients), which were positive after microscopic examination, were also positive for PCR (Table 1). Two lesions, considered as negative after microscopic examination, were positive with PCR. Most lesions caused by L. major were located on feet (9/20 cases), whereas lesions due to L. tropica were on the head (forehead and face) ( Table 1).
The identification of Leishmania at the species level was further confirmed by direct sequencing of each isolate's PCR product. All the sequences were deposited in GenBank under the accession numbers of XN348129 to XN348154. This analysis pinpoints that Leishmania sequences from Algerian patients clustered into three well-differentiated and supported clades of L. major, L. tropica, and L. infantum ( Figure 3). They gathered with Leishmania sequences of various Mediterranean origins collected from GenBank. The two L. infantum sequences clustered with L. infantum isolated from humans or dogs in different Mediterranean countries, with a bootstrap value of 65% ( Figure 3). All biopsies were subjected to molecular characterization by PCR-RFLP. A schematic representation of the PCR-RFLP restriction profile is given in Figure 2, along with the restriction profiles generated for selected samples. The twenty-seven smears (24 patients), which were positive after microscopic examination, were also positive for PCR (Table 1). Two lesions, considered as negative after microscopic examination, were positive with PCR. Most lesions caused by L. major were located on feet (9/20 cases), whereas lesions due to L. tropica were on the head (forehead and face) ( Table 1). The identification of Leishmania at the species level was further confirmed by direct sequencing of each isolate's PCR product. All the sequences were deposited in GenBank under the accession numbers of XN348129 to XN348154. This analysis pinpoints that Leishmania sequences from Algerian patients clustered into three well-differentiated and supported clades of L. major, L. tropica, and L. infantum ( Figure 3). They gathered with Leishmania sequences of various Mediterranean origins collected from GenBank. The two L. infantum sequences clustered with L. infantum isolated from humans or dogs in different Mediterranean countries, with a bootstrap value of 65% ( Figure 3).

Discussion
The first reported cases of cutaneous and visceral leishmaniases in Algeria d to 1860 by Hamel, and 1911 by Lemaire [30]. Besides, Edmond and Etienne Serg

Discussion
The first reported cases of cutaneous and visceral leishmaniases in Algeria date back to 1860 by Hamel, and 1911 by Lemaire [30]. Besides, Edmond and Etienne Sergent and their collaborators were the first, in 1921, to prove sandflies' vector role. They incriminated the phlebotomus papatasi as transmitting the "Clou de Biskra" agent [31,32]. For a long time, L. major and L. infantum foci were geographically separated in Algeria by the Tell Atlas Mountains, representing a natural barrier. The leishmaniasis epidemiological features seem to be in continuous evolution, resulting in more reports [33].
ZCL due to L. major is the oldest leishmaniasis, with Biskra in the east and Abadla in the west as the formerly known foci in Algeria [34]. It is prevalent over the entire North-Saharan fringe, corresponding to the arid and semi-arid areas with a progression towards the North. Three CL outbreaks occurred between 2004 and 2006, with 14,822, 25,511, and 14,714 cases, respectively. Besides Biskra and Ababla, Msila experienced an epidemic in 1982, with 8000 recorded cases [35]. In recent years, several new foci of CL due to L. major, namely those of El M'hir, Batna, and Bordj Bou Arreridj have emerged on the Northern part of the chain of the Tell Atlas [12,33]. In the present study, in agreement with previously reported cases, we found L. major infected patients coming from Ain skhouna, Biskra, El M'hir, Ghardaïa, M'Sila, and Saida [12,13,[36][37][38]. Furthermore, we highlight for the first time, the identification of L. major in the patients from Bourkika, Bou Kremissa, Bou Saada, Chlef, Hajout, Maghnia, Médéa, Menaceur, Messad, Mostaghanem, Nador, Oran, and Sidi Okba (Figure 1, Table 1). Due to limited information on the medical records of some patients, together with the multiple trips of some of them to ZCL endemic regions, mostly due to seasonal works or vacations, it is quite difficult to justify the precise location of some patients when infected by Leishmania parasites (Table 1). Nevertheless, these results confirm the extension of L. major in northern Algeria [12]. Studied patients had an age range between 3 to 82 years old, with most lesions located on the feet (45%) ( Table 1). Men exhibited the most cutaneous lesions caused by L. major (13 out of 20 cases, 65%). Based on the phylogenic tree, we recorded some slight intraspecific heterogeneity for L. major (AVC03, AVC05, and AVC06, originating from Biskra, Bou Kremissa, and Bou Saada). Such a genetic diversity has also been reported in other L. major endemic regions; Iran [39], Tunisia [40], and Morocco [41]. On the other hand, ZCL has been the subject of multiple studies, mostly isoenzymatic investigations. The characterization of parasites circulating in Algeria using isoenzymatic analysis started in 1981 [42]. Isoenzymatic characterization of L. major, the causative agent of zoonotic cutaneous leishmaniasis, evidenced the zymodeme MON-25 in patients, sandfly vectors (P. papatasi), and animal reservoirs (Psammomys and Meriones) [10,[43][44][45]. Some years later, a new and less prevalent zymodeme, the MON-269, was identified. It differs from MON-25 by the PGD (phosphogluconate dehydrogenase) enzymatic system [45] (Table 2).
ACL due to L. tropica has been reported in the southern part of the country, particularly in the Oasis of Ghardaia [13]. The MON-301 and MON-306 zymodemes of Leishmania tropica are restricted to Constantine [15], Ghardaï [10], and Tipaza [16]. They present some intriguing characteristics, like their inherent lower susceptibility towards antimonialcontaining drugs [8,46], or the physiopathological alteration recorded in murine infection models [47]. In the present study, we identified two L. tropica cases from Ghardaia and Constantine, which grouped in the same clade with other L. tropica sequences from other Mediterranean countries.
Since the discovery of VL's first case in 1911, the Kabylie has been known for many years as an active focus of the visceral form in particular. Located in the north of the country, it presents a very large geodiversity, with very contrasting portions, both from a bioclimatic, geomorphological, and vegetation point of view, thus offering very diverse biotopes for the different species of sand flies and animal reservoirs. For many years, the highest number of VL cases registered in Algeria occurred in the region of Tizi ouzou (Kabylie). In the recent years, an extension of VL from the old foci in Kabylie (Tizi-Ouzou, Bejaı a) to the center (Blida, Chlef, Medea, Tipaza) and the north-eastern part of northern Algeria, with scattered cases occurring in the West (Oran, Tlemcen) [7] have been recorded. The MON-1 and MON-24 zymodemes of L. infantum were responsible for zoonotic visceral leishmaniasis and sporadic cutaneous leishmaniasis [48]. They were the most frequently characterized zymodemes in patients, sand flies vectors, and animal reservoirs [9,10,21,22,49]. Although the isoenzymatic characterization allows Leishmania species identification, its complexity and prohibitive costs restrict its use in clinical settings [50]. See Table 2 for a synthetic overview of Leishmania zymodemes characterized in Algeria. Although most VL and sporadic CL cases due to L. infantum are primarily reported in humid regions in northern Algeria, L. infantum infection cases are sporadically reported in arid areas [10]. In Algeria, L. infantum is associated with diverse clinical and eco-epidemiological situations that raised genetic diversity concerns. The occurrence of three L. infantum populations was recorded in Algeria, with two clades encompassing the isolates belonging to the zymodeme MON-1, and a third one, with mainly zymodeme MON-24 isolated from cutaneous leishmaniasis cases [51]. Occasionally recombination events and a generation of hybrid genotypes between MON-1 and MON-24/80 in Algeria have been suspected [52]. In the present study, we identified two SCL cases caused by L. infantum in the patients originating from Tizi Ouzu and Cherchell. Due to the restriction in SCL case numbers processed in the present study, our L. infantum sequences clustered tightly with other Mediterranean strains, with no significant heterogeneity ( Figure 3).
Parasitological methods (direct examination and in vitro culture) have several limitations regarding their positivity and sensitivity rate. This poor performance of parasitological methods is related to low parasitic load or irregular distribution of amastigotes in lesions [53]. The use of DNA amplification by PCR has allowed Leishmania parasites to be identified, and clarified the taxa's distribution [4,33,54]. In analyzing the phylogenic tree generated with specimens isolated from Algerian patients, we recorded a high level of genetic homogeneity in the isolates of L. major, L. tropica, and L. infantum, which cluster with their counterparts identified in various Mediterranean basin areas (Figure 3). This confirms the identification performed using PCR-RFLP and agrees with Leishmania genotyping carried out by Gherbi et al. [55], El Baidouri et al. [56], and Schonian et al. [57] using multilocus microsatellite typing (MLMT) on North African specimens. Table 2. Leishmania zymodemes reported in human, sand fly, and animal reservoirs in Algeria.

Zymodemes Reference Human
Vector Reservoir