J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 301 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Original Article Accurate Identification of Leishmania Parasites in Sand Flies by Polymorphism Analysis of Cytochrome Oxidase Subunit 2 Gene Using Polymerase Chain Reaction and Quantitative PCR-High Resolution Melting Techniques in Iranian Border with Iraq Seyedeh Maryam Ghafari1,2, Reza Fotouhi- Ardakani1,2, *Parviz Parvizi1 1Molecular systematics Laboratory, Parasitology Department, Pasteur institute of Iran, Tehran, Iran 2Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran *Corresponding author: Dr Parviz Parvizi, E-mail: parp@pasteur.ac.ir (Received 08 Nov 2020; accepted 03 Sep 2022) Abstract Background: Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. Methods: Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sand flies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concen- tration was measured at 260 and 280nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Results: Among about 3000 collected sand flies, 89 female Ph. papatasi were identified and two with L. major. In am- plified fragment of COII gene among 611bp, 452bp had no genetic variations with low polymorphic sites (P= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). Leishmania major was discriminated in Ph. papatasi with 0.84 °C melting temperature (Tm) and unique curve based on thermodynamic differences was an im- portant criterion using HRM technique. Conclusion: Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accu- rate diagnostic procedures for leishmaniasis control. Keywords: Cloning; Cytochrome oxidase II; qPCR-HRM Introduction Phlebotomine sand flies (Diptera: Psycho- didae, Phlebotominae) are the only proven nat- ural vectors of Leishmania species, causative agents of a neglected tropical disease, leish- maniasis (1-3). Leishmania is a digenetic par- asite with the extracellular stage within an in- vertebrate vector which called promastigote and the intracellular stage within a vertebrate hosts and reservoirs which called amastigote (4). In- crimination and identification of Leishmania parasite, has been developed and performed using different molecular methodologies and analyses for more than 30 years (2, 5-7). Genetic analysis of mitochondrial and nuclear genes has commonly been employed for Leishmania species identification considering their sensi- tivity, simplicity, reliability and specificity in epidemiological studies (2, 8). High Resolution Melting (HRM) method is an efficient and cost-effective method that elim- inates the risk of laboratory contaminations. In recent years, quantitative PCR using SYBR Green or TaqMan chemistries have been de- veloped and evaluated for detection, quantifi- cation, identification and Leishmania species differentiation in human samples, a few in res- Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:parp@pasteur.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 302 http://jad.tums.ac.ir Published Online: Dec 31, 2022 ervoir hosts and few in sand flies (9-13). Suitable methodology helps to distinguish among Leishmania species especially in tiny insect of female sand flies as natural vectors of leishmaniasis. This is crucial and essential in epidemiological studies to determine appro- priate procedures (14). In this research for typ- ing at intra-species level of Leishmania species, new discriminative molecular markers have been applied (2, 8). qPCR-HRM, may answer the ep- idemiological questions of ZCL in the region of border between Iran and Iraq such as transmis- sion cycles of animal reservoirs and transmit- ting of the vectors, infection’s origin, model- ing of spreading in this border and disease dis- tinction of imported cases. The objectives of this investigation were to determine Leishma- nia species and genotyping of Leishmania par- asites in sand flies of Ilam Province. Materials and Methods Locations, sand flies trapping and morpho- logical identification Sand flies were captured from gerbil bur- rows and nearby domestic animal shelters in two locations of Ilam Province, border with Iraq where Ph. papatasi is proven vector of Zoonotic Cutaneous Leishmaniasis (ZCL). Ac- cordingly, Mehran and Dehloran were consid- ered (31°, 58’ to 34°, 15’ N and 45°, 24’ to 48°, 10’ E) in West of Iran, with altitude of 2740 meters above sea level (masl) (Fig. 1 and Table 1). The sticky papers, CDC miniature light traps (with the white light bulb 1–2m above ground level) which were set overnight to sam- ple sand flies in domestic animal shelters and inside houses in the morning and manual as- pirators were applied for sand flies sampling by expert field collectors. Besides, funnel traps were used to sample from rodent borrows. The collected specimens were put in microtubes without ethanol and then were frozen. All fe- male sand flies were identified by morphologi- cal characters of the head and terminal genitalia, following a dissection of fed females with ster- ilized forceps and microneedles and mounted in Berlese fluid (15)( Fig. 2). DNA extraction, PCR and sequencing DNA from thorax and abdomen of sand flies were extracted using ISH-Horovize meth- od with minor modifications and also using Genet Bio kits (Genet Bio, Daejeon, Korea) (5, 15). Total DNA was extracted from the dis- sected thorax and attached anterior abdomen of individual females of Ph. papatasi. Each sample contained the midgut, the location of most L. major promastigotes, and was homog- enized in a 1.5ml microfuge tube using a dis- posable plastic tip of a micropipette. Follow- ing ethanol precipitation, the DNA was dis- solved in 15µl 1×TE (10mM Tris-HCL, 1mM EDTA pH 8.0), to give a concentration of 5– 10ng/µl, and stored at −20 °C (5, 15). Cyto- chrome oxidase subunit II (COII) as an enzy- matic gene was amplified to detect any Leish- mania infection among sand flies (2, 8). New sensitive and specific primers were designed and employed to amplify COII gene to detect Leishmania parasite in sand flies. PCR was performed on every female Ph. papatasi using COII specific new designed for- ward primer, COII F (5′-ATGGCTTTTATA TTATCATTTTG-3′) and reverse primer, COII R (5′- GGCATAAATCCATGTAAGAC-3′). The amplification reaction was carried out in a total of 20µl containing 1× Taq polymer- ase buffer (Promega), 1.5mM MgCl2, 60µM of each dNTP, 1µM both forward and reverse primers, 1µM primer unit Taq polymerase (Promega) and 1.5µl of DNA (5–10ng/µl) ex- tracted from individual wild caught sand flies. The mixture was incubated in a PE GeneAmp ®PCR Thermocycler 9700 (0.2ml block) at 94 °C for 3min followed by 37 cycles, each con- sisting of 30s at 94 °C, 30s at 58 °C and 90s at 72 °C. After the last cycle, the extension was continued for a further 10min then held at 4 °C (16). PCR products were subjected to electropho- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 303 http://jad.tums.ac.ir Published Online: Dec 31, 2022 resis on 1.5% agarose gel and Leishmania pos- itive PCR products were used for sequencing by ABI PRISMTM 310 automated sequencer (Applied Biosystems, Thermo Fisher Scientific, Foster City, USA) in order to accurate iden- tification of species and haplotypes (2, 8). Se- quences of COII gene was compared in differ- ent Leishmania species to determine conserved areas in sand flies of Ilam. The amplified se- quences with those from GenBank, were com- pared and analyzed in aspect of their phyloge- netic and polymorphism. Cloning In this study, in order to prepare standards for qPCR-HRM and besides for re-sequencing of ambiguous sites, PCR products were cloned directly into pTG19-T vector using SinaClon PCR TA Cloning kit (SinaClon Bioscience, Tehran, Iran). To confirm cloning results, col- ony PCR with vector specific primers was per- formed (17). Plasmid was extracted from trans- formed bacterias, using SinaClon Plasmid Iso- lation Kit (SinaClon Bioscience, Tehran, Iran). Purified plasmid concentration was measured at 260 and 280nm wavelength using NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). In accordance with the following formula DNA copy number was calculated. Then, seven serial dilutions of plas- mids were prepared in the ratio of one to ten (Fig. 3). DNA (copy number)= [6.02× 1023 (copy/ mol)× DNA amount (g)]/ DNA length (dp)× 660 (g/mol/dp) Quantitative PCR-High Resolution Melting High Resolution Melting (HRM) analysis were carried out by Corbett Rotor-Gene 6000 HRM Real Time PCR instrument (Corbett Life Science, Sydney, Australia). A part of COII gene was amplified using designed CO.A. 470.2 forward and reverse primers in the pres- ence of Evagreen dye (Fig. 4a). In this meth- od, the observed differences in the melting temperature were noticed and the use of HRM method was considered for separation of dif- ferent species. HRM procedure was designed and per- formed in 20µl containing 2µl of DNA or plasmid (10 ng/reaction), 0.7µl of each primer (10pmol/µl or 10µmol/l) and 4µl of 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis Bio Byne, Tartu, Estonia). Then, 12.6µl of PCR- grade H2O was added to the volume. The in- itial denaturation for 1 cycle was 15 minutes at 95 °C then it was followed by 45 cycles, 15 seconds in each cycle for denaturation; 30 seconds at 55 °C for annealing, 30 seconds at 72 °C for extension and final extension was 5 minutes at 72 °C (1 cycle) and then stored for 10 minutes at 4 °C. The qPCR-HRM products were followed by a conventional melting step: melting curve was performed from 65 to 92 °C, with an increasing slope of 0.1 °C each step, with 2 seconds rest at each step afterwards. Finally, to determine the average Tm for each Leishmania spp., the melting curve plots were generated and analyzed using HRM software (Corbett Life Science, Sydney, Australia). Statistical analysis DNA sequences were edited and aligned using Sequencher 3.1.1 and CLC Main Work- bench 5.5 softwares. Polymorphism and phylo- genetic analyses of our sequences with stand- ard sequences from GenBank were carried out using MEGA 6 and DnaSP5.10.01 softwares (18). In order to compare the median and Tm, the MedCalc® version 13.3.3 software and Mann-Whitney U test were used. The calcula- tion of diversity between sequences and neu- trality were performed by DnaSP 5.10.01 soft- ware. The ratio of non-synonymous substitu- tion (dN) to synonymous substitution (dS) was assessed using DnaSP 5.10.01 software (19). Results Sand flies sampling and preparation Out of more than 3000 collected sand flies, 234 sand flies were dissected, mounted and five http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 304 http://jad.tums.ac.ir Published Online: Dec 31, 2022 species were identified (Table 1). More than collected samples were male sand flies and more of the sand flies were not Ph. papatasi so 234 were female Ph. papatasi. Eighty nine out of 234 were fed which were screened in order to detect Leishmania infection. Leish- mania major was found at least in two Ph. papatasi out of 89 female sand flies from two locations (Mehran and Dehloran) (Table 1). Polymorphism analysis From 89 female Ph. papatasi only two were infected with Leishmania which identified and sequenced. The monomorphic and polymor- phic positions of COII were compared using Chi-square test. Of 611bp nucleotides, 452bp had no genetic variations. Also, the number of polymorphic sites were significantly lower than that of monomorphic sites sequences (P= 0.001). For informative and non-informative sequenc- es in the evaluated gene, genetic variation oc- curred in 46 nucleotide positions. Bioinformat- ics analyses were performed by DnaSP5.10.01 software and number of synonymous sites was significantly greater than non-synonymous sites (P= 0.00). In the expression gene (COII), among 26% segregating sites in nucleic acid varia- tion areas, 20.2% sites were non-synonymous mutations also, more mutations might have occurred in the negative selection side. Based on the findings by DnaSP5.10.01 software, dS/dN ratio for COII had the low amino acid changes (dS: Synonymous mutations in silent sites, dN: Non-synonymous mutations in re- placement sites). According to Tajima's D In- dex, dN/dS ratio was calculated to be 0.14 (in the positive direction) so COII gene has pro- duced through evolutionary process (Table 2) (20). A natural evolution was reported, based on the comparison of mean nucleotide diversity (π= 0.075) and expected number of mutations (Θ= 41.51) in each sequence of gene. The study of different COII sites of Leishmania species showed no significant difference in nucleotide diversity (π) or expected number of mutations (Θ) (P= 0.86). The comparison of synonymous mutations in silent sites (dS) and non-synony- mous mutations in replacement sites (dN) in COII expression gene showed a significant dif- ference between two Leishmania species (P= 0.001). The number of dS (79.8%) was great- er than dN (20.2%). The greatest dissimilarities in dS site were detected in the comparison of L. major and L. tropica populations (P= 0.019). Among 12 se- quences of L. major, 19 nucleotide positions (13 dS and 6 dN) were different in terms of gene expression. Also, among four sequences of L. tropica species, 27 nucleotide positions (7 dS and 20 dN) varied in terms of gene ex- pression. Haplotype diversity was analyzed for L. major and L. tropica and was observed 81.35% and 28.38%, in L. tropica and L. ma- jor populations respectively. The average, stand- ard deviation (SD), and coefficient variation (CV) showed low to high haplotype diversity (P= 0.000). To determine the extent of natural selection in Leishmania species, the average number of nucleotide differences (k) and the number of expected genetic differences in the whole sequence (Θ) were compared, using Ta- jima’s D index. Results showed that evolution was positive in COII gene (Table 2). Genetic evaluation and phylogenetic analysis 30 Leishmania sequences were employed. These species are included two L. major (two haplotype were found in two Ph. papatasi in Ilam) and 28 Leishmania species reference strains: 10 L. major, four L. tropica, three L. donovani, and three L. infantum from Old World and five from New World: two L. mexicana and two L. tarentolae and one L. amazonen- sis. They were employed to evaluate the sen- sitivity and specificity of COII gene for accu- rate identification of Leishmania species (Fig. 5 and Table 3). To determine the extent of natural selec- tion in Leishmania species, the average num- ber of nucleotide differences (k) and the num- ber of expected genetic differences in the whole http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 305 http://jad.tums.ac.ir Published Online: Dec 31, 2022 sequence (Θ) were compared, using Tajima’s D Index between L. major and L. tropica spe- cies. Based on the findings, COII gene be- longs to L. tropica species has been produced through evolutionary processes (Tajima’s D= 2.85, P< 0.01). This indicates that evolution was positive in COII gene. Molecular phylogenetic analysis of COII gene is presented in Figure 5 based on the Maximum Liklihood (ML) method. It presents the concatenated tree of COII gene. Figure 5a was drawn, based on the nucleotide sequenc- es. Kimura’s two-parameter method was used for 30 nucleotide sequences. The topology of the tree revealed that this mitochondrial gene caused the separation of Leishmania species, whereas this was unable to separate subspe- cies or determine haplotype diversity. By ap- plying the ML method, 1326 sites were ana- lyzed. Also, under similar conditions, the phy- logenetic tree of COII gene based on the mi- tochondrial genetic amino acid codes, showed a similar topology by ML model (Fig. 5b). Quantitative PCR-High Resolution Melting Different dilutions of DNA template were prepared for validation, accuracy and sensitiv- ity of HRM assay (R2= 0.94, efficiency= 4.49). High sensitivity of HRM could pull out mini- mum amount of DNA to draw appropriate melt- ing curves. Serial dilutions of specimens were experienced on three consecutive days with similar reactions. Identical DNA concentrations were repeated with COII gene. Melting curves for serial dilutions were compared. Identical DNA were analyzed and melt patterns were appropriated for both species on different days. According to results, two common Leishma- nia species in Ilam (L. major and L. tropica) were discriminated with 0.84 °C difference in Tm, using HRM technique (mean Tm were 74.8 °C and 75.64 °C for L. major and L. tropica respectively (Fig. 6, 7 and Table 4). Fig. 1. Geographical location of Ilam Province, and sampling sites Table 1. Sampled sand flies collected in June to August 2016, from Ilam Province has shown base on gender, species and locations http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 306 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Species Sergentomia clydei Sergentomia dentata Sergentomia sintoni Phlebotomus mongolensis Phlebotomus papatasi Gender F M F M F M F M F M Location In RB ASH In RB ASH In RB ASH In RB ASH In RB ASH Mehran Dehlouran Nirogah Bargh 0 0 1 0 0 0 1 0 0 0 1 3 0 0 0 0 0 9* 2 38 Imamzadeh Hasan 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 2 11 0 4 56 Dasht Akbar 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 2 0 0 15 7 Chehmeh gheer 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 10 Janbazan 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 12 Roosta Ali 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 6 0 33* 2 1 1 1 0 6 4 0 7 89 125 Total 234 (97F+137M) In: Indoor, ASH: Animal shelter, RB: Rodent burrow, F: Female, M: Male, *Leishmania positive Fig. 2. Phlebotomus papatasi female, A: unfed, B: Fed, C: Gravid, D: Semi gravid Fig. 3. A: Two Leishmania infection were detected by PCR. The sequences were identified as L. major based on se- quencing. -ve: Negative control, +ve: Positive control, M: Markers (100bp left). B: COII gene was cloned in the pTG19-T vector. Plasmids were extracted and serial dilution of them were prepared. M: Markers (1000bp right, 100bp left). 1: PCR products with COII primers for plasmid including COII in L. tropica with 611bp, 2: PCR products with COII primers for plasmid including COII in L. major with 607bp, 3: PCR products with M13 primers for plasmid in- cluding COII in L. major with 800bp, 4: Plasmid extraction products contains COII gene in L. major with 3500bp, 5: Plasmid extraction products contains COII gene in L. tropica with 3500bp, 6: Plasmid without pTG19-T vector with 2880bp Table 2. Polymorphism analysis of COII sequences from Leishmania species by DnaSP5.10.01 software http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 307 http://jad.tums.ac.ir Published Online: Dec 31, 2022 A Gene No. Seq. No. Nucleotide (bp) V (%) Singleton variable (%) Parsimony variable (%) Synonymous/replacement change 2 V a r ia n ts 3 V a r ia n ts 4 V a r ia n ts T o ta l 2 V a r ia n ts 3 V a r ia n ts 4 V a r ia n ts T o ta l C .C d S (% ) d N (% ) d N /d S r a tio COII 29 611 24 0 0 24 114 21 1 136 15 147 (87) 21 (12) 0.14 B Gene No. Seq. No. Nucleotide (bp) S Eta K π Ө per site Ө per seq. Tajima’s D H Hd COII 29 611 149 183 45.97 0.075 0.067 41.51 0.42 27 1 A: V: Variable (polymorphic) nucleotide site, C·C: Total number of sites in other codons (complex codons), which were not analyzed because of their highly variable regions, dS: Synonymous mutations in silent sites, dN: Non-synonymous mutations in replacement sites B: S: segregation site, Eta: Total number of mutations, K: Average number of nucleotide differences between pairs of sequences, π: Mean nucleotide diversity, Θ per site: Expected number of mutations per a site, Θ per seq: Expected number of mutations per a sequence, Tajimaʼs D: the statistical test proposed by Tajima (1996), H: No. of haplotypes, HD: Haplotype diversity Fig. 4. A: Based on obtained sequences and polymorphism analysis results, forward and reverse primers were designed for qPCR of COII gene. CO.A.R470.2 (F: TGGAGAAACAACAATATTTAGTAA, R: CCTAAACTTGAAATT- GCAAATG) B: Schematic illustration of COII gene, amplified with specific primers (small black arrows) for Leishma- nia species using CLC bioinformatics software (8) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 308 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Table 3. Details of COII sequences of Leishmania parasites from Ilam and GenBank sequences for constructing phy- logenetic tree The name of species Accession No. Origin Source Reference L. major **KU680818 **KU680819 KU680820 AF287688 EU140338 EF633106 KF815208 KF815210 KF815211 MH443402 MH443403 MH443404 Soviet Union Iran Italy Sudan Japan Not found Sudan “ “ “ “ “ “ Not found Not found Not found Ulcers of patients Ulcers of patients Ulcers of patients Not found Ulcers of patients Not found Clinical sam- ples:HB,HBM,HLN* “ “ “ “ “ “ Not found Not found Not found This study This study 8 21 22 21 23 23 23 Direct submission (Aghai Maybodi et al. 2018), Unpublished Direct submission (Eslami et al. 2019), Unpublished Direct submission (Aghai Maybodi et al. 2018), Unpublished L. donovani FJ416603 KF815198 AY660023 US Sudan “ “ “ Cultivated parasites Ulcers of patients Clinical sam- ples:HB,HBM,HLN Nebohacova et al. 2009 23 23 L. infantum KF815207 KF815206 KF302727 “ “ “ “ “ “ Not found “ “ “ “ “ “ Not found 23 23 Direct submission (Soares, 2013), Unpublished L. tarentolae KU680825 L07544 Germany USA Ulcers of patients Cultured parasites 8 25 L. amasonensis HQ586836 China “ “ “ 26 L. mexicana HQ586845 KU680824 “ “ “ Brazil “ “ “ Cultured parasites 26 8 L. tropica KF302720 KU680821 HQ586846 HQ586847 Brazil Iran Shandong, China Soviet union Not found Ulcers of patients Cultured parasites Cultured parasites Direct submission (Soares, 2013), Unpublished 8 26 26 26 L. sp. HQ586841 HQ586843 China Jiangsu, China Cultivated parasites “ “ “ 26 26 HB: blood, HBM: bone marrow, HLN: lymph node, **: Identified L. major in this study, “ “ “: Same as above http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 309 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Fig. 5. Phylogenetic analyses discriminated Leishmania species well. A: Phylogenetic tree based on the mitochondrial nucleotides (COII gene) drawn by Maximum Likelihood method. B: Phylogenetic tree based on the mitochondrial ge- netic amino acid codes, drown by Maximum Likelihood method Fig. 6. Specifications performance of Leishmania detection by qPCR validation. Amplification plots derived from seri- al dilutions of cultured parasites (L. major and L. tropica standards with R and F CO.A.470.2 primers), ranging from 20–2× 107 copies / reaction plasmids by qPCR http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 310 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Table 4. Quantitative PCR-High Resolution Melting analysis of COII gene for Leishmania major and L. tropica in different plasmid copy number Fig. 7. Melting temperatures and normalized melting profiles obtained with the HRM assays for L. major and L. tropi- ca. A: Derivative melt curves, B: Aligned and normalized melt curves, C: Differences melt curves Discussion For this investigation, blood fed female sand flies were screened for detecting Leishmania in- fections from two locations in Ilam Province (Mehran and Dehlouran) in Iranian border with Leishmania species Plasmid copy number qPCR HRM Tm mean Efficiency R2 Tm L. major 104 4.49 4.49 0.94 0.94 74.60 74.8 104 74.83 105 74.65 105 74.58 106 74.55 106 74.63 107 74.63 107 74.85 L. tropica 104 75.08 75.64 104 75.22 105 75.00 105 74.98 106 75.50 106 75.42 107 74.25 107 74.22 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 311 http://jad.tums.ac.ir Published Online: Dec 31, 2022 Iraq where the endemic foci of ZCL places in these two countries. In these locations, Ph. papatasi is proven and main vector of L. ma- jor (27). We were not surprised to find only two Ph. papatasi which carried L. major out of 89 sampled in our preliminary screen. The infec- tion rates of Ph. papatasi can be low even in well studied in different ZCL foci in the world while infection rates can be high in human and reservoir hosts (5, 28-29). In about 20 past years, the sensitivity and specificity of molec- ular techniques was compared targeting dif- ferent mitochondrial and nuclear genes for iden- tifying L. major (2, 8). Now, we have tried to take an effective step with a new modified mo- lecular method and genetic analysis for detec- tion and identification of infection in native Ph. papatasi samples, in Ilam Province. Rapid, sensitive and accurate diagnostic pro- cedures are crucial for detecting and charac- terizing of Leishmania parasites in sand flies, in order to provide accurate treatment, precise prognosis and appropriate control measurements. For this research a new technique of HRM was employed for fast detecting of Leishmania spe- cies and mix infections targeting COII gene in sand flies. COII gene is an expression gene. The si- lent site (dS) and replacement site (dN) of L. major and L. tropica were significantly differ- ent (P= 0.001). Nucleotides were 611 positions in the final dataset and 452 out of 611bp had no genetic variations. Evolutionary relationships of taxa for COII maxicircle mitochondrial were analyzed using Molecular Phylogenetic of Max- imum Likelihood method. The nucleotide's substitution in COII gene in L. major had 86% similarity with sequenc- es available in the GenBank. COII is a mito- chondrial gene and all mitochondrial genes have more mutations to compare with nuclear genes. When COII gene compared with other polymorphic genes in different Leishmania species has less diversity. Although Boite and colleagues (2012) mentioned that COII was less capable for discriminating and distinguish- ing different Leishmania species but this re- search and other investigations showed that the phylogenetic analyses and trees of this gene is able to identify Leishmania strains at the spe- cies level (8, 30). COII gene is conserve enough to discriminate Leishmania parasites and has polymorphism sites to discriminate some spe- cies, more over it has 20–50 copy numbers (8, 26). According to previous findings, COII is the most sensitive as compared with ITS- rDNA, HSP70, nagt and Cyt b genes that were tested (2, 8). In this research new primers were designed to detect Leishmania species and strains. The prediction’s results were analyzed using CLC software and followed by HRM method. The altered regions could make a desirable tem- perature difference in species of Leishmania parasites (31). According to polymorphism and phylogenetic analyses of COII gene, L. major was discriminated in Ph. papatasi from Ilam Province with 0.84 °C Tm using HRM tech- nique. Determining unique curves based on ther- modynamic differences is the important crite- rion of HRM method. HRM can detect single nucleotide polymorphisms based on small dif- ferences in the nucleotide composition for any suitable mitochondrial and nuclear genes (6, 32). HRM to compare with conventional PCR is more sensitive, specific, simpler, less ex- pensive and faster. DNA can be extracted di- rectly from samples, blood and other tissues. HRM results can be obtained without addi- tional post-PCR processing in less than 2.5 hours (33). So far, many effective and effi- cient studies have been conducted to isolate Leishmania parasite from sand flies (9, 33-35). Because of eight years-imposed war be- tween Iraq and Iran subsequently Kuwait then long time Iraq occupation by USA following ISIS, much transmission of L. major occur in locations in both sides of Iranian border with Iraq. These situations have provided refuges for many sand fly species as well as reservoir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 312 http://jad.tums.ac.ir Published Online: Dec 31, 2022 hosts to make a high risk habitat for transmis- sion of parasites (5, 36). Conclusions The transmission cycles of L. major might depend on many criteria including diversity of Leishmania species and intraspecific strains, sand fly species, reservoir hosts, locations and migration of two countries people. Different parasite species interact to maintain L. major infections in reservoir hosts and people. Un- derstanding the roles of Ph. papatasi as a proven vector which transmits L. major to the reservoir hosts and people, is important; also it is equally important to discover the com- plementary roles of rapid, sensitive and accu- rate diagnostic procedures for leishmaniasis in order to disease control and treatment in Ira- nian border with Iraq. Acknowledgements The authors wish to thank the healthcare authorities of Ilam Province as well as the staff of Molecular Systematics Laboratory of Pasteur institute for their assistance. There are no disputes over the ownership of the data pre- sented in the paper. This work was supported by Pasteur Institute of Iran (grant number 735) as well as the National Institute for Medical Research Development (NIMAD) (grant num- ber 973166) awarded to Prof Parvizi. Ethical considerations This work was supported by Pasteur Insti- tute of Iran (grant number 735) as well as the National Institute for Medical Research De- velopment (NIMAD) (grant number 973166). Conflict of interest statement The authors declare there is no conflict of interests. References 1. Bates PA, Depaquit J, Galati EA, Kamhawi S, Maroli M, McDowell MA, Picado A, Ready PD, Salomón D, Shaw JJ, Traub- Csekö YM, Warburg A (2015) Recent advances in phlebotomine sand fly re- search related to leishmaniasis control. Parasit Vectors. 8: 131. 2. Ghafari SM, Fotouhi-Ardakani R, Parvizi P (2020a) Designing and developing a high- resolution melting technique for accu- rate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real- time PCR and in silico genetic evalua- tion. Acta Trop. 211: e105626. 3. Ghafari SM, Ebrahimi S, Nateghi-Rostami M, Bordbar A, Parvizi P (2020b) Com- parative evaluation of salivary glands pro- teomes from wild Phlebotomus papatasi proven vector of zoonotic cutaneous leishmaniasis. Vet Med Sci. 00: 1–8. 4. Dostálová A, Volf P (2012) Leishmania de- velopment in sand flies: parasite-vector interactions overview. Parasit Vectors. 5: 276. 5. Parvizi P, Ready PD (2008) Nested PCRs and sequencing of nuclear ITS-rDNA frag- ments detect three Leishmania species of gerbils in sand flies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 13(9): 1159–1171. 6. Müller KE, Zampieri RA, Aoki JI, Muxel SM, Nerland AH, Floeter-Winter LM (2018) Amino acid permease 3 (aap3) Coding sequence as a target for Leish- mania identification and diagnosis of leish- maniases using high resolution Melting analysis. Parasit Vectors. 11: 421. 7. Asfaram S, Fakhar M, Mirani N, De- rakhshani‑niya M, Valadan R, Ziaei Hez- arjaribi H, Emadi SN (2019) HRM–PCR is an accurate and sensitive technique for the diagnosis of cutaneous leishmani- asis as compared with conventional PCR. Acta Parasitol. 65(2): 310–316. http://jad.tums.ac.ir/ https://pubmed.ncbi.nlm.nih.gov/25885217/ https://pubmed.ncbi.nlm.nih.gov/25885217/ https://pubmed.ncbi.nlm.nih.gov/25885217/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 313 http://jad.tums.ac.ir Published Online: Dec 31, 2022 8. Fotouhi-Ardakani R, Dabiri S, Ajdari S, Alimohammadian MH, AlaeeNovin E, Taleshi N, Parvizi P (2016) Assessment of nuclear and mitochondrial genes in precise identification and analysis of ge- netic polymorphisms for the evaluation of Leishmania parasites. Infect Genet Evol. 46: 33–41. 9. Karaku ŞM, Pekag Irba ŞM, Demir S, Eren H, Toz S, Özbel Y (2017) Molecular screening of Leishmania spp. infection and blood meals in sand flies from a leish- maniasis focus in southwestern Turkey. Med Vet Entomol. 31: 224–229. 10. Antonia AL, Wang L, Ko DC (2018) A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis. Peer J. 6: e5905. 11. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M (2018) Real time PCR applications for diagnosis of leishmaniasis. Parasit Vectors. 11: 273. 12. Rojas-Jaimes J, Rojas-Palomino N, Pence J, Lescano AG (2019) Leishmania spe- cies in biopsies of patients with different clinical manifestations identified by high resolution melting and nested PCR in an Endemic district in Peru. Parasite Epi- demiol Control. 3: e 00095. 13. Ahuja K, Vats A, Beg MA, Kariyawasam K, Chaudhury A, Chatterjee M, Karun- aweera ND, Selvapandiyan A (2020) High resolution melting based method for rap- id discriminatory diagnosis of Frotorco- infecting Leptomonas seymouri in Leish- mania donovani-induced leishmaniasis. Parasitol Int. 75: e102047. 14. Schönian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, Jaffe CL (2003) PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Mi- crobiol Infect Dis. 47(1): 349 ̶ 358. 15. Parvizi P, Benlarbi M, Ready PD (2003) Mitochondrial and Wolbachia markers for the sand fly Phlebotomus papatasi: little population differentiation between peridomestic sites and gerbil burrows in Isfahan Province, Iran. Med Vet Entomol. 17: 351–362. 16. Parvizi P, Mauricio I, Aransaya AM, Miles MA, Ready PD (2005) First de- tection of Leishmania major in perido- mestic Phlebotomus papatasi from Isfa- han Province, Iran: comparison of nest- ed PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle ki- netoplast DNA. Acta Trop. 93: 75–83. 17. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA. 18. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 30(12): 2725–2729. 19. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformat- ics. 25(11): 1451–1452. 20. Tajima F (1996) The amount of DNA polymorphism maintained in a finite pop- ulation when the neutral mutation rate var- ies among sites. Genetics. 143: 1457–1465. 21. Ibrahim ME, Barker DC (2001) The origin and evolution of the Leishmania donovani complex as inferred from a mitochondri- al cytochrome oxidase II gene sequence. Infect Genet Evol. 1(1): 61–68. 22. Yatawara L, Le TH, Wickramasinghe S, Agatsuma T (2008) Maxicircle (mitochon- drial) genome sequence (partial) of Leish- mania major: gene content, arrangement and composition compared with Leish- mania tarentolae. Gene. 424(1–2): 80– 86. 23. Babiker AM, Ravagnan S, Fusaro A, Has- san MM, Bakheit SM, Mukhtar MM, Cattoli G, Capelli G (2014) Concomi- tant infection with Leishmania donovani and L. major in single ulcers of Cutane- ous Leishmaniasis patients from Sudan. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, Dec 2022, 16(4): 301–314 SM Ghafari et al.: Accurate Identification of … 314 http://jad.tums.ac.ir Published Online: Dec 31, 2022 J Trop Med. e170859. 24. Nebohacova M, Kim CE, Maslov DA (2009) RNA editing and mitochondrial activity in promastigotes and amastigotes of Leish- mania donovani. Int J Parasitol. 39: 635– 644. 25. Shaw JM, Campbell D, Simpson L (1989) Internal frameshifts within the mito- chondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA. Proc Natl Acad Sci USA. 86(16): 6220–6224. 26. Cao DP, Guo XG, Chen DL, Chen JP (2011) Species delimitation and phylo- genetic relationships of Chinese Leish- mania isolates reexamined using kineto- plast cytochrome oxidase II gene sequenc- es. Parasitol Res. 109: 163–173. 27. Mirzaei A, Schweynoch C, Rouhani S, Parvizi P, Scho¨nian G (2014) Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leish- maniasis in Iran. Trans R Soc Trop Med Hyg. 108: 502–512. 28. Haydon DT, Cleaveland S, Taylor LH, Lau- renson MK (2002) Identifying reservoirs of infection: A conceptual and practical challenge. Emerg Infect Dis. 8(12): 1468– 1473. 29. Yaghoobi-Ershadi MR (2012) Phlebotom- ine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania trans- mission. J Arthropod Borne Dis. 6(1): 1. 30. Boite MC, Mauricio IL, Miles MA, Cu- polillo E (2012) New insights on taxon- omy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis. 6: e1888. 31. Hernandez C, Alvarez C, Gonzalez C, Aya- la MS, León CM, Ramírez JD (2014) Identification of six New World Leish- mania species through the implementa- tion of a High-Resolution Melting (HRM) genotyping assay. Parasite Vectors. 7: 501–508. 32. Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floe- ter-Winter LM (2016) High resolution melting analysis targeting hsp 70 as a fast and efficient method for the discrim- ination of Leishmania species. PLoS Negl Trop Dis. 10(2): e0004485. 33. Słomka M, Sobalska-Kwapis M, Wachulec M, Bartosz G, Strapagiel D (2017) High Resolution Melting (HRM) for High- Throughput Genotyping-Limitations and Caveats in Practical Case Studies. J Clin Child Adolesc Psychol. 18: 2316–2337. 34. Bezerra-Vasconcelos DR, Melo LM, Al- buquerque ÉS, Luciano MCS, Bevilaqua CML (2011) Real-time PCR to assess the Leishmania load in Lutzomyia long- ipalpis sand flies: screening of target genes and assessment of quantitative methods. Exp Parasitol. 129: 234–239. 35. González E, Álvarez A, Ruiz S, Molina R, Jiménez M (2017) Detection of high Leishmania infantum loads in Phleboto- mus perniciosus captured in the leish- maniasis focus of southwestern Madrid region (Spain) by real time PCR. Acta Trop. 171: 68–73. 36. Strelkova MV, Eliseev LN, Ponirovsky EN, Dergacheva TI, Evans DA (2001) Mixed leishmanial infections in Rhom- bomys optimus: a key to the persistence of Leishmania major from one transmis- sion season to the next. Ann Trop Med Parasitol. 95: 811–819. http://jad.tums.ac.ir/