J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 124 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Molecular Identification of Leishmania Species in a Re-Emerged Focus of Cutaneous Leishmaniasis in Varamin District, Iran Mahmoodreza Behravan 1, *Vahideh Moin-Vaziri 1, Ali Haghighi 1, Nourina Rahbarian 1, 2, Niloofar Taghipour 1, Alireza Abadi 3, Homa Hajjaran 4 1Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Department of Social Medicine and Health, School of Public Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 28 Feb 2015; accepted 9 Apr 2016) Abstract Background: Cutaneous leishmaniasis (CL) is one of the most important neglected tropical diseases and a major public health challenge in Iran caused by Leishmania spp and transmitted by phlebotomine sand flies. The number of CL cases has shown an increasing pattern all over the country, including the district of Varamin, southeast of Tehran, Iran. This study aimed to identify the Leishmania spp isolated from CL patients using molecular methods in Varamin during 2012–2013. Methods: Exudate materials collected from the swollen edge of the skin lesions of 44 parasitological positive CL patients by disposable lancet. They were referred to Varamin Health Center by physician. The samples were sub- jected to molecular method for Leishmania species identification. Results: The digestion pattern of restriction enzyme revealed that 37 (84.1%) CL patients were infected with L. ma- jor and 7 (15.9%) were infected with L. tropica. They were mostly male than female. More than half of the patients (58%) had multiple lesions, and they were mostly observed on extremities, 34.1% on legs and 29.5% on hands. Le- sions were mostly of wet ulcerative type. Conclusion: Dominancy of L. major provides more evidence that Varamin District probably could be considered as Zoonotic Cutaneous Leishmaniasis (ZCL) areas. More investigation on other epidemiological aspects of disease is needed. Keywords: Cutaneous leishmaniasis, PCR-RFLP, Leishmania tropica, Leishmania major, Iran Introduction Leishmaniasis is one of the most devastat- ing neglected tropical diseases with a com- plicated ecology (Alvar et al. 2012). Disease is a sand fly borne infection caused by the blood parasite Leishmania, more than 20 spe- cies of parasites are transmitted via the infec- tive bites of different species of sand flies (Sub- family Phlebotominae) (Alvar et al. 2012). Leishmaniasis has been clinically categorized into three main forms: visceral (VL), cutaneous (CL), and mucocutaneous (MCL) (Reithing- er et al. 2007). Endemic transmission of the disease has been reported from different trop- ical and subtropical countries with 350 mil- lion people at risk, the overall prevalence is about 12 million worldwide with yearly esti- mated incidence of 0.2–0.4 million cases of VL and 0.7–1.2 million cases of CL (Alvar et al. 2012). Iran is a main endemic region of CL in the *Corresponding author: Dr Vahideh Moin-Vaziri, E-mail: vmvaziri@gmail.com J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 125 http://jad.tums.ac.ir Published Online: March 14, 2017 Middle East and North Africa (McDowell et al. 2011). Cutaneous leishmaniasis is endem- ic in two forms, Anthroponotic Cutaneous Leishmaniasis (ACL) and Zoonotic Cutane- ous Leishmaniasis (ZCL). Anthroponotic form is still of great importance in many parts of the country, including some large and me- dium sized cities such as Tehran, Mashhad, Neishabur and Sabzevar in the north-east, Shiraz in the south, Kerman and Bam in the southeast, Yazd, Kashan and parts of the city of Esfahan in the central region (Yaghoobi- Ershadi et al. 2010, Yaghoobi-Ershadi 2012). The parasite is L. tropica and the vector is supposed to be Phlebotomus (Paraphleboto- mous) sergenti Parrot 1917. The main reser- voir host is human but dogs have a role as animal reservoir host (Yaghoobi-Ershadi 2012). Zoonotic form is endemic in many rural are- as of 17 out of 31 provinces of Iran (Yaghoobi- Ershadi 2012). Rodents are considered as the main reservoirs of disease, Rhombomys opi- mus is the main animal reservoir in foci lo- cated in the north-east and central part of the country, Meriones libycus in some parts of central and south of the country, Tatera in- dica in the southeast and M. hurrianae in southeastern part of Baluchistan, neighbor- ing Pakistan (Akhavan et al. 2010, Azizi et al. 2012, Yaghoobi-Ershadi 2012, Hajjaran et al. 2014). Phlebotomus papatasi Scopoli 1786, the most prevalent species among Phlebotomus genus, is the only known vector (Yaghoobi-Ershadi 2012). From 1983 to 2012, cases of all types of leishmaniasis in Iran reached to 569164, which 99.5% of that was CL. In 2012, totally 20947 cases were recorded all through the country and highest incidence rate was observed in Ilam, Fars and Khorassan Razavi provinces in that year (Shirzadi et al. 2015). Official reports showed an increasing trend in number of CL cases in Varamin Dis- trict, from 18 cases in 2010 to 32 cases in 2011 (Communicable disease report, unpublished data). Varamin is located 35km, southeast of Tehran, Iran. A purely parasitological study was done in Varamin by the same authors (Behravan et al. 2015), but parasite identifi- cation at species level was not done. Leish- mania species are not morphologically dis- tinguishable. Identification of Leishmania parasites is essential for precise prognosis of the disease as well as making proper decision regarding control and probably treatment (Hajjaran et al. 2014). Therefore, this study was conduct- ed to characterize the causative agents of disease by molecular tools. In recent years, different molecular methods with different genetic markers have been developed for parasite identification (Schonian et al. 2003, Parvizi et al. 2008, Parvizi and Ready 2008, Azizi et al. 2012). Internal Transcribed Spac- er-1 (ITS1) of the small subunit ribosomal DNA was known as a reliable marker for de- tection of Leishmania parasite. Different di- gestion patterns which were produced by HaeIII (BsuRI) enzyme can clearly differ- entiate the parasite (Hajjaran et al. 2011, Al- Nahhas and Kaldas 2013, Hajjaran et al. 2013). This method also was used in current study to identify Leishmania species in CL suspect- ed patients in Varamin district. Materials and Methods Study Area The present study was conducted in Var- amin (35°19′27″N, 51°38′45″E), located 35 km southeast of Tehran, Iran (Fig. 1). Sampling and DNA extraction This study was done on CL suspected pa- tients who were referred to Varamin Health Center laboratory during 2012–2013 from 11 different areas mentioned in Figure and Ta- ble 1. Patient’s information (age, sex, living area, ulcer duration and number and site of lesions) was recorded by a special question- naire. J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 126 http://jad.tums.ac.ir Published Online: March 14, 2017 Ethic clearance was obtained from Re- search Ethical Committee of Shahid Beheshti University of Medical Sciences (Approval Number: sbmu.rec.1392.253). Smears were prepared from exudate ma- terials of swollen edge of lesions of patients collected by sterile lancet, then fixed in meth- anol, stained by Giemsa and examined under a light microscope for the presence of amastigotes. Grading of Leishmania parasites was obtained by average parasite density as follows: 4+ (1–10 parasites/fields), +3 (1–10 parasites/10 fields), 2+ (1–10 parasites/100 fields), 1+ (1–10 parasites/1000 field) ac- cording to WHO protocols (WHO 1991). Exudate materials from ulcer of CL suspect- ed patients also were used for molecular identification. DNA was extracted by using the Bioneer DNA Extraction Kit (Bioneer, Republic of Korea) in accordance with the manufacturer’s instructions. DNA extracts were stored at -20 ˚C until being used. ITS1 amplification and enzymatic digestion ITS1 was amplified by using specific pri- mers, LITSR (forward: 5′-CTGGATCATTTT CCGATG-3′) and L5.8S: (reverse: 5′-TGA TACCACTTATCGCACTT-3′). The ampli- fication was carried out by using the PCR- Ready premix (Roche, Germany) in 25µ l to- tal reactions comprising 10 µ l premix, 2µ l forward and reverse primers (10 pmol), 1µ l DNA template and 13µ l double distilled wa- ter. Iranian reference strains of L. major (Accession Number: JN860745) and L. trop- ica (Accession. Noumber: EF653267) were used as positive standard controls to monitor the reactions. The PCR conditions consisted of one initial denaturing cycle at 95 ˚C for 5 min, followed by 35 cycles of 94 ˚C for 30s, 47 ˚C for 30 s, 72 ˚C for 45 s. This was fol- lowed by a final extension cycle at 72 ˚C for 7min. These set of primers amplify a fragment of about 300–360bp of Leishmania genome (Schonian et al. 2003, Hajjaran et al. 2013). The PCR products were visualized after stain- ing with ethidium bromide by 1.2% agarose gel electrophoresis in UV transluminator. Molecular identification of Leishmania spe- cies was achieved by RFLP analysis. Ten microliters of the PCR product were added to 2µ l of the enzyme buffer and 1µ l of the HaeIII (BsuRI) Enzyme (Fermentas, Life Sci- ences, Germany), this mixture was incubated at 37 ˚C for 10 minutes, as recommended by the manufacturer. The cut site of enzyme was GG↓CC, producing different patterns based on Leishmania species. Digestion prod- ucts were separated by using 3% agarose gels and visualized after staining by ethidi- um bromide. Results Totally, 44CL parasitologically positive patients were selected for molecular exami- nation. The positive microscopic slides were scored for Leishman Body. The grading of Leishmania amastigotes numbers were as fol- low, 8% (1+), 42% (2+), 42% (3+) and finally 8% (4+). As Table 1 indicates the patients affected by CL were mostly from Bagher- Abad (27.3%) and Kheir-Abad (22.7%), nearly located in north-west of Varamin dis- trict near to Pakdasht. Among the patients, 63.6% were male. Patients ranged in age from 1 to 75yr old, the parasite infected all age groups except children under 1yr old and only 3 were above 65yr. The most highly infected age group was 25–39yr old (29.5%). Most of the patients (71%) had multiple le- sions over exposed parts of body, even with 20 lesions in one case. Hands, legs and facial areas were the most affected parts of the body with 34.1%, 29.5% and 15.9%, respec- tively. Lesions were mostly of wet ulcerative type. The size of the most lesions was more than 1cm (Fig. 2). The average duration of lesions was 5 months; most of patients (99%) had no history of treatment. J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 127 http://jad.tums.ac.ir Published Online: March 14, 2017 Molecular outcomes Using the LITSR/L5.8S primers set, a single product (300–360bp) has been ampli- fied in all 44 samples (100%) (Fig. 3), the same as what observed for standard strains of parasite. After using the restriction en- zyme, totally two bands of 220bp and 140bp were observed in 37 (84.1%) samples which was similar to what expected for L. major as it could be seen also in standard sample. They were mostly from Bagher-Abad (27.3%), Kheir-Abad (22.7%) and Varamin (18.2%) and the rest was scattered among 8 remained areas (Table 1). Besides, in 7 cases (15.9%), three bands of 200, 60 and 40bp were ob- served, comparing to standard strains, they are representative of L. tropica (Fig. 4). The- se cases were distributed in different areas as follows, 2 cases in Bagher-Abad, 2 in Kheir- Abad, 2 in Varamin and just one case in Rey- han-Abad. Table 1 showed more detailed in formation for Leishmania spp which were identified by PCR-RFLP. Table 1. Number of cases and distribution of cuta- neous leishmaniasis agents based on study areas, Var- amin District, Iran, 2012–2013 Study area Cases No. (%) PCR-RFLP results No. L. major L. tropica Bagher-Abad 12(27.3) 10 2 Kheir-Abad 10(22.7) 8 2 Varamin 8(18.2) 6 2 Deh-Sharifa 3(6.8) 3 0 Ahmad-Abad 2(4.5) 2 0 Vali-Abad 2(4.5) 2 0 Mohammad- Abad 2(4.5) 2 0 Omr-Abad 2(4.5) 2 0 Reyhan-Abad 1(2.3) 0 1 Charm-Shahr 1(2.3) 1 0 Javad-Abad 1(2.3) 1 0 Total 44 (100) 37 7 Fig. 1. Geographical location of Varamin, (inset is map of Iran), showing the study area and distribution of Leish- mania major and Leishmania tropica isolated from patients during 2012–2013. Numbers on the map refer to the vil- lages of: 1: Bagher-Abad, 2: Kheir-Abad, 3: Varamin, 4: Deh-Sharifa, 5: Ahmad-Abad, 6: Vali-Abad, 7: Moham- mad-Abad, 8: Omr-Abad, 9: Reyhan-Abad, 10: Charm-Shahr, 11: Javad-Abad J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 128 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 2. Representative pictures of skin lesions caused by Leishmania major in cutaneous leishmaniasis pa- tients, Varamin, Iran, 2011–2012. Fig. 3. Electrophoresis of Leishmania DNA ampli- fied with primers LITSR and L5.8S from exudate materials of lesions of suspected patients to cutaneous leishmaniasis, Varamin District, Iran, 2011–2012, Line 1–9: samples of patients with CL, line 10: L. major (Accession Number: JN860745), line 11: 100bp ladder marker (Fermentas) and line 12 nega- tive control. Fig. 4. Digestion pattern of ITS1 amplicons of Leishmania spp of cutaneous leishmaniasis patients of Varamin District, by using HaeIII enzyme Line 1: 50 bp ladder marker, Line 2: sample of pa- tients (L. tropica), Line 3: L. tropica (Accession. Noumber: EF653267), Line 4: L. major (Accession. Noumber: JN860745), Line 5 samples of patients (L. major), Line 6: 100 bp ladder marker Discussion Recently, the reported cases especially that of CL due to L. major have increased (Yaghoobi-Ershadi et al. 2010, Yaghoobi- Ershadi 2012, Hajjaran et al. 2014), and they were reported in areas known to be non-en- demic or regions with decreasing trend over the passing years. Although Varamin is one of the old known foci of CL (Ardehali et al. 1994), but comprehensive data on CL was not available. A preliminary and purely par- asitological study was done by the same au- thors who reported confirmed parasitological cases during 2012–2013 (Behravan et al. 2015). The evidence obtained by mentioned study indicates that Varamin district potentially en- countered with ZCL. Therefore, ITS1-PCR- RFLP was conducted to identify the parasite species in patients. Entomological survey for vector incrimination was done in studied ar- ea by the same authors (Data under assay). J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 129 http://jad.tums.ac.ir Published Online: March 14, 2017 Totally among 44 CL patients, frequency of male was higher than of female. Some other endemic regions of the country have the same pattern, such as Damghan and Kashan (Rafati et al. 2007, Talari et al. 2006, Khosravi et al. 2013). It could be due to more contact of men with vectors based on the type and time of their work compared with women. Conversely, in Kerman Province, CL was dis- tributed more significantly in females (Shari- fi et al. 2008). Some studies have shown an equal distribution of ZCL infection among two sexes (Fakoorziba et al. 2011). Although most age groups were at risk of the disease, but based on obtained results, the prevalence of CL was higher in age group 25–39yr old. In known ZCL endemic regions of Iran, the highest risk group is often children less than 15yr old, in Kerman (< 10yr old), Fars Prov- ince (≤ 10 yr old), Damghan (10–19 yr) and part of Isfahan ( under 1 yr of age) (Rafati et al. 2007, Sharifi et al. 2008, Nateghi Ros- tami et al. 2013). The effect of age might be actually influenced by disease endemicity and immune reactions of the host. In this study, most of the lesions appeared on the extremities, this pattern is common in ZCL foci such as Damghan (Rafati et al. 2007) and Orzoieh district (Sharifi et al. 2008). Over half of the patients in current study had more than one lesion similar to what observed by other researchers in ZCL foci in Iran (Rafati et al. 2007, Sharifi et al. 2008). In CL due to L. major, multiple lesions are common (Yaghoobi- Ershadi et al. 2003). It could be because sand flies usually have a discontinuous blood-suck- ing habit, may bite several times and cause the development of several lesions on the skin (Yaghoobi-Ershadi et al. 2003, Yaghoobi- Ershadi et al. 2010, Nateghi Rostami et al. 2013). All 44 available exudate materials which were confirmed positive by microscopic meth- od were positive also by molecular tools (100%), which could be dedicated that the applied genetic marker and molecular meth- od is quit useful, although it was not the aim of this study. Leishmania major as the main causative agent of ZCL was identified in most lesions (84.1%), which was not far from our expectation. About 80% of cases reported in the country are of ZCL form (Yaghoobi-Ershadi 2012). Same observations were reported from neighboring provinces of Varamin such as Semnan and Qom, which L. major was identified as the dominant causa- tive agents of leishmaniasis in both areas (Ra- fati et al. 2007, Nateghi Rostami et al. 2013). One the most important factors in leish- maniasis spreading are migration and travel- ling (Yaghoobi-Ershadi 2012). Varamin is the passage-way to the several large cities, espe- cially religious city which caused a lot of peo- ple travel around. As a matter of fact, Vara- min located very close to the capital of Iran, is one of the most important agricultural and animal husbandry axis, which caused it to be a good site for job seekers and consequently a lot of migration happen which increase the potential risk of disease. Conclusion The achieved results, specifically dominancy of L. major, indicate that Varamin district could be regarded as one of the ZCL region in Iran, but to identify all epidemiological aspects of disease, a comprehensive epide- miological study is highly recommended. Acknowledgements This study was funded by the Research Council of Shahid Beheshti University of Medical Sciences (Grant No. 11119-91-1- 1392). The authors have great thanks to Health center staff of Varamin, Mr Amini and Mr Hoobar and Mr Farnoosh. The authors high- ly appreciate the participated patients to the current study. The authors declare that there is no conflict of interest. J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 130 http://jad.tums.ac.ir Published Online: March 14, 2017 References Akhavan AA, Yaghoobi-Ershadi MR, Khame- sipour A, Mirhendi H, Alimohammadian MH, Rassi Y, Arandian MH, Jafari R, Abdoli H, Shareghi N, Ghanei M, Jalali-zand N (2010) Dynamics of Leish- mania infection rates in Rhombomys opimus (Rodentia: Gerbillinae) popu- lation of an endemic focus of zoonotic cutaneous leishmaniasis in Iran. Bull Soc Pathol Exot. 103: 84–89. Al-Nahhas SA, Kaldas RM (2013) Charac- terization of Leishmania species iso- lated from cutaneous human samples from central region of Syria by RFLP analysis. ISRN Parasitol. 3: 5–11. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7:e35671. Ardahali S, Rezaei HR, Nadim A (1994) Leishmania and leishmaniases. 2nd ed. Markaze Nashr Daneshgah Press, Tehran. Azizi K, Moemenbellah-Fard MD, Kalantari M, Fakoorziba MR (2012) Molecular detection of Leishmania major kDNA from wild rodents in a new focus of zoonotic cutaneous leishmaniasis in an oriental region of Iran. Vector Born Zoontic Dis. 12(10): 844–850. Behravan MR, Hajjaran H, Abadi A, Haghi- ghi A, Rahbarian N, Amini A , Hoo- bar MR, Khajeh- Farokhi M, Moin- Vaziri V (2015) Cutaneous leishman- iasis in suspected refereed patients to health centers of Varamin and deter- mination of sand flies species during 2012–2013. Med J Tabriz Uni Med Sci Health Services. 37(2): 6–11. Fakoorziba MR, Baseri A, Eghbal F, Rezaee S, Azizi K, Moemenbellah-Fard MD (2011) Post-earthquake outbreak of cu- taneous leishmaniasis in a rural region of southern Iran. Ann Trop Med Parasi- tol. 105(3): 217–224. Hajjaran H, Mohebali M, Teimouri A, Oshaghi MA, Mirjalali H, Kazemi-Rad, Shiee MR, Naddaf SR (2014) Identification and phylogenetic relationship of Ira- nian strains of various Leishmania spe- cies isolated from cutaneous and vi- sceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate trans- ferase gene. Infect Genet Evol. 26: 203– 212. Hajjaran H, Vasigheh F, Mohebali M, Rezaei S, Mamishi S, Charedar S (2011) Di- rect diagnosis of Leishmania species on serosity materials punctured from cuta- neous leishmaniasis patients using PCR- RFLP. J Clin Lab Anal. 25: 20–24. Hajjaran H, Mohebali M, Mamishi S, Vasigheh F, Naddaf SR, Teimouri A, Edrissian GH, Zarei Z (2013) Molecular identi- fication and polymorphism determina- tion of cutaneous and visceral leish- maniasis agents isolated from human and animal hosts in Iran. BioMed Res Int. pp. 1–7. Khosravi A, Sharifi I, Dortaj E, Aghaei Afshar A, Mostafavi M (2013) The present status of cutaneous leishmaniasis in a recently emerged focus in south- west of Kerman Province, Iran. Iran J Public Health. 42: 182–187. McDowell MA, Rafati S, Ramalho-Ortigao M, Salah AB (2011) Leishmaniasis: Middle East and North Africa research and development priorities. PLoS Negl Trop Dis. 5:e1219. Nateghi Rostami M, Saghafipour A, Vesali E (2013) A newly emerged cutaneous leishmaniasis focus in central Iran. Int J of Inf Dis. 17. e1198–e1206. Parvizi P, Moradi G, Akbari G, Farahmand M, Ready P, Piazak N, Assmar M, Amirkhani A (2008) PCR detection and sequencing of parasite ITS-rDNA gene J Arthropod-Borne Dis, March 2017, 11(1): 124–131 M Behravan et al.: Molecular Identification … 131 http://jad.tums.ac.ir Published Online: March 14, 2017 from reservoirs host of zoonotic cuta- neous leishmaniasis in central Iran. Parasitol Res. 103: 1273–1278. Parvizi P, Ready P (2008) Nested PCRs and sequencing of nuclear ITS-rDNA frag- ments detect three Leishmania species of gerbils in sandflies from Iranian foci of zoonotic cutaneous leishmaniasis. Trop Med Int Health. 13: 1159–1171. Rafati N, Shaporimoghadem A, Ghorbani R (2007) Epidemiological study of cuta- neous leishmaniasis in Damghan (2000– 2006). J Semnan Univ Med Sci. 24: 250–251. Reithinger R, Dujardin J-C, Louzir H, Pirmez C, Alexander B, Brooker S (2007) Cutaneous leishmaniasis. Lancet Infect Dis 7: 581–596. Schonian 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 Microbiol Infect Dis. 47: 349–358. Sharifi I, Zamani F, Aflatoonian MR, Fekri AR (2008) An epidemic of cutaneous leishmaniasis in Baft District in Kerman Province and its probable causative risk factors. Iran J Epidemiol. 4: 53–8. Shirzadi MR, Esfahani B, Mohebali M, Yaghoobi Ershadi MR, Gharachorlo F, Razavi MR, Postigo JAR (2015) Epide- miological status of leishmaniasis in the Islamic Republic of Iran, 1983– 2012. East Mediterr Health J. 21(10): 736–742. Talari SA, Talaei R, Shajari G, Vakili Z, Taghaviardakani A (2006) Childhood cutaneous leishmaniasis: report of 117 cases from Iran. Korean J Parasitol. 44: 355–60. WHO (1991) Basic laboratory methods in medical parasitology. Available at: www.who.int/malaria/publications/ato z/9241544104_part1/en. Yaghoobi-Ershadi M, Akhavan A, Zahraei- Ramazani A, Abai M, Ebrahimi B, Vafaei-Nezhad R, Hanafi-Bojd AA, Jafari R (2003) Epidemiological study in a new focus of cutaneous leishma- niasis in the Islamic Republic of Iran. East Mediterr Health J. 9: 816–826. Yaghoobi-Ershadi MR, Hakimiparizi M, Zahraei-Ramazani AR, Abdoli H, Akhavan AA, Aghasi M, Arandian M, Ranjbar A (2010) Sand fly surveillance within an emerging epidemic focus of cutaneous leishmaniasis in southeast- ern Iran. Iran J Arthropod Borne Dis. 4(1): 17–23. Yaghoobi-Ershadi MR (2012) Phlebotomine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania transmi- ssion. J Arthropod Borne Dis. 6(1): 1–17.