The Associations of Leishmania major and Leishmania tropica Aspects by Focusing Their Morphological and Molecular Features on Clinical Appearances in Khuzestan Province, Iran

Cutaneous leishmaniasis has various phenotypic aspects consisting of polymorphic amastigotes with different genetic ranges. Samples were collected from suspected patients of Khuzestan province. Prepared smears were stained, scaled, and measured using ocular micrometer. The Cyt b, ITS-rDNA, and microsatellite genes of Leishmania were amplified and Leishmania species were identified by molecular analyses. Of 150 examined suspected patients, 102 were identified to Leishmania species (90 L. major, nine L. tropica, and three unidentified). The amastigotes of 90 L. major had regular and different irregular shapes within three clinical lesions with no and/or low genetic diversity. Three haplotypes of Cyt b of L. major were found but no variation was observed using ITS-rDNA gene. Interesting findings were that all nine L. tropica had regular amastigote shapes with more genetic variations, also a patient which had coinfection of L. major, L. tropica, and Crithidia. At least two L. major and L. tropica were identified in suspected patients of the regions. Different irregular amastigotes' shapes of L. major can be explained by various reservoir hosts and vectors. In contrast, more molecular variations in L. tropica could be justified by genetic characters. Unidentified Leishmania could be mixed pathogens or nonpathogens with mammals' Leishmania or Crithidia.


Introduction
Cutaneous leishmaniasis (CL) is caused by parasites belonging to the genus Leishmania parasitic and referred to neglected tropical diseases [1,2]. CL is distributed in more than half of the Iranian provinces (20 out of 32) with different prevalence (1.8%-37.9%), also, the number of reported CL cases/year and estimated annual CL incidence are 26,630 and 69,000 to 113,300, respectively [3,4].
Different clinical aspects are caused by CL and are observed in several parts of the world wide but many influencing factors of CL have not absolutely understood [8,9]. The spectrum of genetic diversity in the genus Leishmania is described as one of the most controversial issues [10][11][12][13][14][15][16]. The genetic heterogeneity/homogeneity of protozoan parasite may bring diverse phenotypic ranges including clinical manifestations or even diversity of morphometric characters in Trypanosomatidae [12,[17][18][19].
Currently, Leishmania species are unambiguously circulating in Khuzestan province, where it is a neglected focus of leishmaniasis in southwestern Iran, coborder with Iraq. The different Leishmania parasites' aspects have not been precisely elucidated yet such as ultrataxonomy (strain/haplotype), phenotypic features, and their phylogenetic inferences in Khuzestan province [20][21][22][23]. The complexities of these Leishmania criteria are associated with T u r k m e n i s t a n A f g h a n i s t a n T u r k e y N E S W K h u z e s t a n Iraq Iraq P e r s ia n G u lf P e r s i a n G u l f the intricacy of leishmaniasis epizoology, migration of people to Iraq and vice versa, recent reactivation of old foci, lack of using accurate diagnostic markers, developing geographical distribution with high temperature (50 ∘ C) and humidity (95%), long distance, remote distance from the equipped laboratory, and unsafe sampling in Iraq border [21].
Cyt is the central redox catalytic subunit of quinol which belongs to the mitochondrial genome and it is valuable in understanding phylogenetic study among individuals [33][34][35].
The ITS-rDNA marker is utilized to infer evolutionary relationships in genus Leishmania because of having conserved regions and low intracellular polymorphism [30,36].
Microsatellites are short tandemly repeated DNA sequence motifs which are distributed plentifully in Leishmania species. Microsatellites have a high specificity in identification of Leishmania infections owing to their allelic characters [37].
Molecular identification of Leishmania species has been conducted by targeting different DNA markers in Iran [29,[38][39][40]. But in ignored ZCL foci of Khuzestan province, few extensive studies were done on epidemiological and molecular aspects on morphological features of leishmaniases [20][21][22][23]. In this research, epidemiological, molecular, and morphological methods were combined with more details on morphological characters of Leishmania amastigote shapes, clinical appearances of lesions, and the type of ulcers using nuclear (ITS-rDNA) and mitochondrial (Cyt b) genes. The finding of prevalence of CL, molecular variations, and different morphological shapes of Leishmania parasite can height our knowledge about behavioral aspects of L. major and L. tropica in southwestern Iran.

Sampling and Identifying of Leishmania Parasites.
In southwest of Iran, within the ZCL foci, three geographical locations, in west (Dashte azadegan, Sosangerd and Hoveyze), central (Ahvaz), and east (Ramhormoz and Ramshir) along with their neighboring villages were sampled (31 ∘ 7 N to 32 ∘ 5 N and 47 ∘ 41 E to 48 ∘ 30 E) ( Figure 1). Some surveyed locations are situated in the border of Iran and Iraq within about 10,406 km 2 with 12-210 meters above sea level (A.S.L.). The environmental and climate conditions of the studied regions are different from other CL foci in Iran with temperature range: 20-60 ∘ C, humidity: 50-85%, and monthly average rainfall: 17-25 mm ( Figure 1, Table 1).

BioMed Research International
The lesions of suspected patients to CL were sampled in 2012-2013. The smears were obtained and prepared in two methods of passive and active for Leishmania parasites. The personal information was recorded for each suspected patient (sex, age, lesion duration, type and number of lesion, patients' travelling to endemic regions of ZCL, and drug consumption) ( Table 1) [7].
The samples were smeared on two microscopic slides, dried, and stained by Giemsa and Leishmania infections were identified under light microscope with high magnification (×1000). The positive slides of each patient were graded to order leishman bodies' density from +1 to +6 [41]. Various morphometric shapes and the sizes of amastigotes of Leishmania species were accurately assessed (30 min. per slide) by ocular micrometer (×100 objective = 1 m per unit space) and Dino Capture 2.0.
Serous of some samples were subcutaneously inoculated into the base tail of the BALB/c and then was examined weekly with the intent of inspecting the appearance of lesion at the position of injection for 6 months. Some serous from active lesion of suspected patients were cultured in Novy-MacNeal-Nicolle (NNN) medium and incubated in 22 ∘ C for 6 weeks. The samples were subcultured weekly and checked regularly to monitor the growth and the presence of promastigotes. All experiments on the humans and animals were performed according to the guidelines of the Ethical Board of Pasteur Institute of Iran.

Extraction of Total
Genomic DNA. DNA of parasites was directly extracted from graded slides based on modified Phenol-chloroform protocol [42]. All Giemsa-stained slides were washed with ethanol and covered with 300 L lysis buffer (50 mM NaCl, 50 mM Tris, 10 mM EDTA, pH 7.4, 1% v/v Triton x-100). Giemsa-stained slides should be free from blood in order to prevent misdiagnosis of visualized amastigotes from platelets as well as adverse effects of protoporphyrin on amplified DNA by PCR. The extraction of the genomic DNA of each Giemsa-stained slide was followed by the protocol of Bordbar and Parvizi [29]. DNA was resuspended in 50 L 1X TE and stored at −20 ∘ C. The DNA concentration of each extraction was measured using a NanoDrop (Thermo Scientific Inc., Wilmington, DE). DNA extraction of Leishmania parasites was carried out in a molecular biology laboratory where amplified and cloned DNAs had never been performed [30].

PCR Amplification and RLFP for ITS-rDNA, Cyt b, and microsatellite Genes of Leishmania Parasites from Suspected
Patients. The standard PCR was employed to detect Leishmania parasites in suspected patients by targeting three genes, ITS-rDNA about 480 bp, Cyt b about 880 bp, and microsatellite about 180 bp. PCR products were subjected to electrophoresis in 1.5% agarose gel and were observed under ultraviolet light after staining for 15 min with (0.5 g/mL) ethidium bromide.
For digesting the PCR amplicons in RFLP, the suitable enzymes were selected. The sequences of ITS-rDNA and Cyt b genes of Old World Leishmania were retrieved (ITS-rDNA: L. major: EF413078, L. tropica: KC540906, and L. infantum: EU330402; Cyt b: L. major: AB095961, L. tropica: EF579904, and L. infantum: EF579895). To standardize the number and the size of DNA fragments for each Leishmania species, the retrieved sequences were designed by in-silico analysis software CLC DNA Workbench 5.2 (CLC bio A/S, Aarhus, Denmark) (Figure 2). Endonuclease reaction of ITS-rDNA and Cyt b genes was performed in a volume of 30 L containing 2 L of BsuRI (HaeIII) with cut site GG↓CC and 2 L of Ssp1 with cut site AAT↓ATT, 10 L of PCR products, 2 L of 10x buffer, and 16 L of distilled water for 4 h (delayed digestion) at 37 ∘ C for ITS-rDNA gene and 5 min. (rapid digestion) at 37 ∘ C for Cyt b gene. Standard strains of WHO for L. major (HOM/SU/73/5ASKH), L. infantum (MHOM/TN/80/IPT1), and L. tropica (MHOM/PS/2008/344Jn SF53) were used as positive controls. The negative controls were applied, one of which had no PCR product and the other one had no restriction enzyme. After digesting PCR products, the fragments were analyzed using electrophoresis on agarose gel 3% containing ethidium bromide and ladder of 50 bp (Fermentas) (Figure 3).   137 out of 150 samples (91.3%) were observed microscopically positive with Leishmania parasites and cultured in NNN medium (Table 1). Leishmania infections were found more in 15-25 years old (37/137: 27%) than other groups and in males (94/137: 68.6%). The grades of positive smears were arranged from +1 to +6 (WHO 1991). More infected patients with L. major had grade +4 (45/137: 32.8%). The average size of L. major amastigotes was in range 3-5 ± 0.31 m in length and 2.15 ± 0.21 m in width (Table 2). L. major amastigotes were observed with obvious vacuole and polymorphic shapes (regular, oval, and round; 74/90: 82.2%) ( Table 2). The irregular polymorphic shapes of L. major amastigotes were classified into Cigarette, Spindle, and Pear; 16/90: 17.7% and visualized in three lesion types of wet, dry, and mixed. Interestingly, the sizes of regular and irregular amastigote shapes were approximately 3-4 and 4-5 m, respectively (Figures 4(k)-4(o)). It is noteworthy that polymorphic amastigote shapes were observed only in one lesion form. The wet lesions were grouped in classic (volcanic sign: 56/70: 80%) and nonclassic (herpetiform, erythematous papulonodules, hyperkeratotic, eczematoid, zosteriform, and psoriasiform patterns: 17/70: 24.3%) (Figures 4(d)-4(i)). In contrast, L. tropica amastigotes were sized approximately 2-3 ± 0.23 m in length and 2.2 ± 0.11 m in width with amastigotes' shapes of round and/or oval within dry lesions (100%) (Figures 4(a), 4(b), and 4(j)). The grade of positive smears of L. tropica showed that all observed smears were +6.

Morphometric Characterization of Leishmania
Details of frequency of Leishmania lesions in suspected patients, lesion duration after adult sandflies' biting, lesion types, and lesions' numbers are shown in Table 1.

The Pixel Intensity of Leishmania by Targeting Three Genes of Cyt b, Microsatellite
, and ITS-rDNA. The pixel intensity of applied genes was examined. The PCR products that were tested in gel electrophoresis rely on their chromatogram length. The sensitivity of PCR bands showed that the Cyt b gene has more pixel intensity (140) than microsatellite (125) and ITS-rDNA (110) genes in a minimum dilution, 1 : 16 ( Figure 5(a)).
The average of different PCR dilutions was statistically analyzed and the mean of PCR bands demonstrated that Cyt b gene has more intensity (110) than microsatellite (90) and ITS-rDNA (82) (Figure 5(b)).

Sequencing and Phylogenetic Analyses of Leishmania
Species by Targeting ITS-rDNA and Cyt b Genes. Only 25 out of 90 L. major and all nine L. tropica were sequenced from both Cyt b and ITS-rDNA genes. All sequences of Leishmania parasites in this study were aligned, edited, analyzed, and compared with some Leishmania sequences of Cyt b and ITS-rDNA genes with those which had been previously submitted to the GenBank. L. tropica had more molecular variation than L. major (25 L. major sequences versus nine L. tropica). Cyt b had a little more variation in comparison with ITS-rDNA gene for both L. major and L. tropica. L. major had three haplotypes for Cyt b gene. One common haplotype was reported in 23 L. major sequences which were similar with haplotype KHUZ57 (GenBank Accession no. AB095961). Two other haplotypes (haplotypes KHUZ17 and KHUZ19) were novel and unique (KM393221 and KM393217). For ITS-rDNA gene, all 25 L. (L.) major sequences were similar (GenBank Accession nos. EF413075 and AJ300481) and no variation was found.
Of our nine L. tropica sequences, three haplotypes were identified distinctively including a common and two new haplotypes for Cyt b gene. The common haplotype included six L. tropica sequences which were similar with haplotype KHUZ22 (GenBank Accession no. EF579904). Two new haplotypes were seen in three remained sequences. Haplotype KHUZ02 had two similar sequences (KM393218) and the other one (haplotype KHUZ05) was unique (KM393219). For ITS-rDNA gene of L. tropica, two haplotypes were found. One common haplotype KHUZ11 had eight similar sequences   (GenBank Access no. FN677343) and one new haplotype KHUZ07 which was unique (KM393220). Three Leishmania parasites were unidentified because of not enough DNA for direct sequencing or unreadable sequences and/or mixed infections (Table 1). A coinfection was found from a man (age about 24), resident of Kotabdullah village (Ahvaz region) who had L. major in left hand, L. tropica in face, and a Crithidia in the lesion of right hand. This could be very interesting to notice when different parasites are existed in one patient; therefore, each lesion should be identified to species and examined separately.
Phylogenetic analyses were conducted using our new and common haplotypes of both genes and Leishmania species with those which were submitted in GenBank using MEGA v5.05. Trypanosoma brucei was considered as an out group branch (GenBank Accession nos. M94286 and JN673390) (Figures 6(a) and 6(b)).

Discussion
In this investigation, two Leishmania species (L. major and L. tropica) were unequivocally identified in suspected patients of the three different districts including Sosangerd, Dashte Azadegan, and Hoveyze in western bordering with Iraq, Ahvaz as capital city of Khuzestan province in central and also Ramhormoz and Ramshir in eastern Khuzestan province where all these three different regions are not considered widely as ZCL foci (Figure 1) [4]. In north of Khuzestan province, Shush is well known as an endemic focus of ZCL [44]. In our study, prevalence of ZCL in ignored locations of disease was unexpectedly high (17.1 per 10,000) to be compared with previous report, 8 per 10,000 [4]. Also, L. major and L. tropica were found in Sosangerd, Dashte Azadegan, Hoveyze, and Ahvaz, while L. major was only found in Ramhormoz and Ramshir. This maybe because of sandflies' species or reservoir hosts distribution in different regions. Phlebotomus papatasi and P. (Paraphlebotomus) sergenti were collected and identified in these two first locations (Sosangerd, Dashte Azadegan, and Ahvaz). Those are the vectors which circulate in these areas but only P. papatasi was found in the third region (Ramhormoz and Ramshir) whilst the rodent Tatera indica and humans/dogs are reservoir hosts of L. major and L. tropica, respectively in the region [45,46].
In this study, three different genes were employed to detect Leishmania species. The advantages of RFLP for Cyt gene were sharp PCR bands with high sensitivity in the shortest possible time (5 min., rapid digestion) compared with ITS-rDNA and microsatellite (Figures 3(a), 3(b), 5(a), and 5(b)). These could depend on high multicopy sequences of Cyt per cell (comprised of 20-50 maxicircles, 20-35 kb in size) and presence of an unusually long noncoding region These findings attenuate explicitly relationship between genotypic and phenotypic characters of CL in L. major and L. tropica, which potentially indicates that genetic variation aspects are not always able to influence the formation of morphometric features or even different clinical appearances [8,9,56,57].
A comprehensive analysis on limits of morphological diversity in any protozoan parasite showed that morphogenetic constraints and extrinsic selection pressures are associated with the full diversity of trypanosomatid morphology including juxtaform and liberform superclasses [19].
Several factors can separately affect the clinical features of L. major lesions. These are including immunosuppressive diseases, migration of nonindigenous populations, host factors, the number of the inoculated parasites, using oral steroids, the site of inoculation, and even wound contamination with inorganic ingredients [8,9]. Considering challenge on phenotypic and genotypic patterns of L. tropica in suspected patients in Khuzestan was that L. tropica had no polymorphic amastigotes but have more molecular variations with many differentiations in nucleotides of each sequences (haplotype) [10,17].
Some studies revealed molecular diversity in mini-exon gene of CL agents (L. major and L. tropica) in Khuzestan isolates but the heterogeneity was not coordinated with the size of lesions [22,23].
Most publications showed that causative agent of cutaneous leishmaniasis is L. major; however Maraghi et al. (2007) identified both L. major (90%) and L. tropica (10%) in this regions [12,44,58]. Hajjaran et al. (2013) using only ITS-rDNA gene showed L. tropica with 3.6% polymorphisms and L. major with 7.3% genetic variations in Iran [59], whilst we found more variations in L. tropica to be compared with L. major using Cyt b mitochondrial and ITS-rDNA nucleus genes which are able to draw a real conception of genetic diversity among Leishmania species.
Many studies were carrying out regarding identifications of Leishmania parasites in human, reservoirs hosts, and vectors in Iran and the world which some of them correlated the molecular variations and heterogeneity of Leishmania species with clinical signs [59][60][61][62].

Conclusions
In this study, L. major and L. tropica were firmly identified in suspected patients with a coinfection of L. major, L. tropica, and Crithidia and some unidentified Leishmania which could be mixed of these three or with the expectation of the existence of L. turanica which recently was reported from human in north of Iran [7]. It is crucial that simultaneous screening of both valuable mitochondrial (Cyt b) and nucleus (ITS-rDNA) genes with morphometric, molecular, and phylogenetic analyses can provide an appropriate approach and reliable methods to identify Leishmania haplotypes, strains, and species.

Ethical Approval
The ethical approval was received from the Research Council of Pasteur Institute of Iran, Tehran, Iran.