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Evaluative Assay of Nuclear and Mitochondrial Genes to Diagnose Leishmania Species in Clinical Specimens.

Background: Leishmaniasis as an emerging and reemerging disease is increasing worldwide with high prevalence and new incidence in recent years. For epidemiological investigation and accurate identification of Leishmania species, three nuclear and mitochondrial genes (ITS-rDNA, Hsp70, and Cyt b) were employed and analyzed from clinical samples in three important Zoonotic Cutaneous Leishmaniasis (ZCL) foci of Iran. Methods: In this cross-sectional/descriptive study conducted in 2014–15, serous smears of lesions were directly prepared from suspected patients of ZCL in Turkmen in northeast, Abarkouh in center and Shush district in southwest of Iran. They were directly prepared from suspected patients and DNA was extracted. Two nuclear genes of ITS-rDNA, Hsp70 and one mitochondrial gene of Cyt b within Leishmania parasites were amplified. RFLP was performed on PCR-positive samples. PCR products were sequenced, aligned and edited with sequencher 4.1.4 and phylogenic analyses performed using MEGA 5.05 software. Results: Overall, 203 out of 360 clinical samples from suspected patients were Leishmania positive using routine laboratory methods and 231 samples were positive by molecular techniques. L. major L. tropica, and L. turanica were firmly identified by employing different molecular genes and phylogenic analyses. Conclusion: By combining different molecular genes, Leishmania parasites were identified accurately. The sensitivity and specificity three genes were evaluated and had more advantages to compare routine laboratory methods. ITS-rDNA gene is more appropriate for firm identification of Leishmania species.


Introduction
Leishmaniasis is one of the most important human protozoan parasitic diseases worldwide by increasing the prevalence and incidence rates in recent years (1)(2)(3). From three types of leishmaniasis in Iran, Zoonotic Cutaneous Leishmaniasis (ZCL) has a great distribution reported from more than half of Iranian provinces (4,5). ZCL is a single cell parasitic disease that considered as a major health problem in many areas of Iran.
Rodents and other mammals are reservoir hosts and human infects the causative agent of Leishmania major accidentally by biting female sand flies (6,7). The presence of ZCL in Turkmen Sahara (Golestan), Abarkouh (Yazd), and Shush (Khuzestan) has been demonstrated in addition new ZCL foci in some locations located in border of Iran and Iraq (7,8). L. major, L. turanica, L. jerbilli and a new Leishmania close to L. Jerbilli have been reported in ZCL foci but L. major is the principal agent of ZCL in Iran (3,9,10). Phlebotomus papatasi is the main vector and the most important reservoir hosts are Rhombomys opimus and Meriones libycus in Turkmen Sahara and Abarkouh, and Tateraindica in Shush (8,(11)(12)(13). ZCL represents a typical model of emerging and reemerging zoonosis disease (14). ZCL can cause substantial morbidity because of the presence of a chronic skin ulcer and the psychological effect of disfigurement (15). There are no proper vaccines to protect people against the parasites. Although, epidemiological investigation and finding Leishmania species are helpful in the diagnosis of ZCL; however they are not sufficient methods to identify firmly the agent of the disease. Accurate identification of the causative agent of Leishmania species is essential to give us knowledge of the Leishmania species in the endemic and specific geographical area, and better approach in control measurements and treatment of disease (16). Hence, firmly identification and determination of Leishmania parasites are indispensable advanced molecular methods for human cases because of being deficient information for routine laboratory techniques.
For this investigation, three nuclear and mitochondrial genes (ITS-rDNA, Hsp70, and Cyt b) of Leishmania were employed, compared and designed to identify the Leishmania species parasites circulating in suspected patients of ZCL in different regions in Northeast, Central and Southwest to Iran.

Materials and Methods
In this cross-sectional/descriptive study conducted in 2014-2015, serous smears of lesions were directly prepared from suspected patients of Human samples were collected from urban and rural areas surrounding Shush, Turkmen Sahara and Abarkouh districts of Iranian provinces. The personal information, lesion duration, type and number of lesion, ulcer(s)' location, patients' traveling to endemic regions and the grading numbers of amastigotes were recorded individually and kept confidential ( Fig. 1).  (Table 1). DNA was extracted from the slides using Phenol-Chlorophorme method with minor modification (3). Two nuclear genes of ITS-rDNA (480 bp), Hsp70 (750 bp) and one mitochondrial gene of Cyt b (880 bp) within Leishmania parasites were amplified to detect any Leishmania infection among samples from suspected patients following the primers and protocols (16). To select suitable restriction enzyme, CLC DNA workbench 5.2 software (CLC bio A/S, Aarhus, Denmark) In Silico was employed. By choosing sequences of different Leishmania species regis-tered in GenBank; enzyme BsuRI (HaeIII) for ITS-rDNA and SspI enzyme for Cyt b gene were selected (Fig. 2). Hsp70 gene was not used for RFLP. Selected enzyme had different cut sites at GG↓CC in various species of Leishmania as a proper enzyme for PCR product digestion. RFLP was performed on PCR-positive samples for identification of Leishmania species. A master mix containing enzyme, Buffer and PCR product was prepared and stored at 37 ºC for 4 hours. Moreover, for accurate identification and confirmation of the specific PCR products were sequenced, aligned and edited with sequencher 4.1.4 software and phylogenic analyses was done using MEGA 5.05 software (Fig. 3).

Results
The serous smears of lesions from 360-suspected patients (clinical samples) were sampled. Using routine laboratory methods, 203 samples from suspected patients were identified positive (Table  1). By employing molecular tools, using three different genes, 231 samples were Leishmania posi-tive ( Table 2, Fig. 2). After RFLP and/or sequencing; L. major L. tropica and L. turanica were firmly identified (Table 2). Shush district in south of Iran had higher infections than two other location where were sampled (87/360). The highest infection was in age group 10-25 yr (40/360) and male had more positive than females (Table 1). Seventy-nine percent of the lesions were wet, 40% of them were in the hands, 42% of patients had single lesions and 32% of the slides were 4+, which was the highest velocity among the surveyed categories (Table 1). Overall, 231 (64.1%) of samples were confirmed to be Leishmania positive via molecular methods (Table 2), of which 46.7%, 23.8%, and 28.5% were tested positive with by ITS-rDNA, Hsp70 and Cyt b genes, respectively ( Table 2). After RFLP and/or sequencing; from 231 Leishmania positive samples 217 (93.9%), 4 (1.73%) and 2 (0.86%) were indefinitely identified as L. major L. tropica, and L. turanica, respectively. In addition, 8 (3.46%) of the samples were unable to be accurately confirmed due to lack of PCR product and/or bad sequence reads (Table 2). In this investigation, L. major, L. tropica and L. turanica were unambiguously typed and identified after analyzing and sequencing with molecular software with comparison to those sequences which have already been registered in GenBank in case of any similarity and homology. The obtained sequences of this investigation were homolog with Leishmania species after direct sequencing, editing, aligning and comparing with the sequences submitted to GenBank using Sequencher TM 4.1.4.
In the present work, we have compared the specificity and sensitivity of three nuclear and mitochondrial genes, we discovered that sensitivity of ITS-rDNA was more than Cyt b and Hsp70 and ITS-rDNA show the highest specificity for Leishmania species.

Discussion
In this investigation, L. major, L. tropica and L. turanica among suspected patients were firmly identified in three endemic ZCL foci, Iran. Three well-known molecular markers ITS-rDNA, Hsp70 and Cyt b genes were employed (3). Three genes were applied concurrently to compare specificity, sensitivity and to increase the chance of detecting any Leishmania parasites even in low concentrations (17). ITS-rDNA gene is a nuclear, liner, homogenous and conserves gene with low intracellular polymorphism and readable sequences. ITS-rDNA is ideal for phylogenic analysis (12). Cyt b as a mitochondrial marker can identify the novel nucleotide variations (haplotype) superior to ITS-rDNA (nucleus gene), this is associated with highly being conserved and high copy numbers of Cyt b per cell (20-50 maxi circles in 30 Kbp) (Fig. 2) (5, 18, 19). ITS-rDNA shows high sensitivity because of approximately 20-400 copies of gene for Leishmania but not for differentiate Leishmania species. Cyt b as an evolutionary mitogenome marker has its semi conserved structure and low copy number could able to utilize in the discrimination of new mutants, whereas no significant mutant was observed in ITS-rDNA sequences (7). By analyzing of CLC DNA workbench software, digestion sites of different enzymes were recognized on ITS-rDNA, Hsp70 and Cyt b genes of Leishmania species (Fig. 2). BusRI enzyme has one digestion site in L. major for ITS-rDNA gene that gives two fragments (120bp and 300 bp), three digestion sites for Hsp70 gene. SspI enzyme has one digestion site (~ 500bp) for Cyt b gene in L. major. The different and variation fragments by digesting site make RFLP method, which generated species-specific patterns of bands visualized in agarose gels, a useful technique for accurate determination of Leishmania species. Only amplifying DNA by PCR and observation of relevant band in agarose gels without sequences, molecular and phylogenetic analysis could not be effective and trusted firmly identification of Leishmania species (20). Regarding data of this investigation, males are at higher risk than females for Leishmania infection in Iran because of wearing Hejab, covering skin by females, preventing sand fly bites and decreasing the risk for leishmaniasis. Moreover, men usually work in farms, fields and mostly sleep outside during the adult sand flies activity; these provide a good source of blood meals and transferring Leishmania parasites in ZCL foci (21). These also could be reason for high rate of infection in 10-25 age groups.

Conclusion
Different genes combined for accurate identifications of clinical samples of Leishmania parasites sampled in three well-known ZCL foci in Iran. To find standardized, sensitive, specific, practical and reproducible genes for molecular identification and typing Leishmania species were evaluated and compared using three genes in this investigation. Molecular tools are more trustable than routine laboratory methods, and ITS-rDNA gene is more appropriate for accurate identification of Leishmania species.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.