J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 198 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Molecular Identification of Species Caused Cutaneous Leishmaniasis in Southern Zone of Iran *Afshin Barazesh1; Mohammad Hossein Motazedian2; Moradali Fouladvand3; Gholamreza Hatam2; Saeed Tajbakhsh3; Sepideh Ebrahimi2; Danial Purkamal4 1Department of Microbiology and Parasitology, Faculty of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran 2Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 3The Persian Gulf Marine Biotechnology Research Center, Bushehr University of Medical Sciences, Bushehr, Iran 4Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz University of Medical Sciences, Shiraz, Iran (Received 14 Apr 2018; accepted 30 Apr 2019) Abstract Background: Leishmania major and Leishmania tropica are two main species causing cutaneous leishmaniasis (CL) in Iran. Recently, Crithidia spp. has also been reported in the wound of patients with CL. In this study, we determined the species causing CL in the southern of Iran and the role of Crithidia spp. in creating skin ulcers. Methods: In this cross-sectional study from Apr to Sep 2016, 66 patients with CL referred to Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran, were selected. After DNA extraction from the Giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate Leishmania species and then to identify Crithidia spp. Results: Two species L. major and L. tropica were responsible for 60 and 6 cases, respectively. Moreover, in two patients, mixed infection with Crithidia was confirmed. In mix infection cases, the morphology of the cutaneous ul- cers was not different from the wounds of other patients. Conclusion: Leishmania major is responsible for the most common CL in southern Iran. In addition, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed. The potential role of Crithidia as the main factor for CL and the probability of this parasite to have synergistic effects on Leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon. Keywords: Cutaneous leishmaniasis; Leishmania; Crithidia; Iran Introduction Cutaneous leishmaniasis (CL) is one of the six important diseases in tropical and subtrop- ical regions which WHO has recommended and supported researches on its various aspects (1, 2). The factor causing CL is various species of bloody-tissue protozoan parasites called Leish- mania (3). This disease has distributed to all continents of the world except Australia and it can be seen in 88 countries. Annually, 1–1.5 million people in the world become infected with Leishmania and a population of about 350 million people are at risk for this disease (4, 5). About 90% of CL cases worldwide are re- ported from seven countries: Afghanistan, Al- geria, Brazil, Iran, Peru, Saudi Arabia and Syr- ia (6) which among these countries, Iran and Saudi Arabia have the highest incidence of the disease (7). About 20,000 CL cases are reported annu- ally from different parts of Iran estimated that the real rate is several times higher (8). In *Corresponding author: Dr Afshin Barazesh, E- mail: afshin914@gmail.com mailto:afshin914@gmail.com J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 199 http://jad.tums.ac.ir Published Online: June 24, 2019 recent years, the prevalence CL in Iran, espe- cially in the southern regions, has been grow- ing gradually and alarmingly and is endemic in 17 out of 31 provinces of Iran (9), as the number of positive cases has increased from 1560 in 1991 to 3861 in 2001 (10, 11). In an epidemiological survey about CL in Iran during 2013–2015, CL is an endemic disease in Fars Province, and it annually infects numerous peo- ple (12). In general, provinces such as Fars and Isfahan with an incidence of 1.66 cases per 1,000 people, have the highest incidence of the disease in the country (13). Leishmania major and L. tropica are two main species causing CL in Iran (9), which have a different distribution in different parts of the country (14). In recent studies, besides these two spe- cies, some trypanosomatidal protozoa includ- ing Herpetomonas spp., Leptomonas spp. and Crithidia spp. have also been reported in the culture of cutaneous ulcers (15-17). Crithid- ia spp. are classified in Kinetoplastida order and Mastigophora subphylum and are often referred to as insects’ parasites (16). Two spe- cies (Crithidia lucilia and Crithidia fasciculata) are also considered to be infectious agents of the Leishmania culture medium (18, 19). How- ever, the interesting thing is that the patients who the Crithidia spp. have been isolated from their wounds are often drug resistant to vari- ous drugs, and their cutaneous ulcers some- times remain for 2–3yr and become chronic (17), which is probably due to the presence of Crithidia alone or simultaneously with Leish- mania spp. as mix-infection in these groups. In Iran, Crithidia spp. has been reported fre- quently from patients suspected of drug-re- sistant cutaneous leishmaniasis and the ge- nomic sequence of the ribosomal region has proven its belonging to the genus Crithidia (17). In another study, Crithidia was isolated from the spleen and foot-pad of Tetera in- dica in Fars Province, Iran which considers the presence and role of this protozoon in infecting vertebrate hosts (20). Crithidia spp. are able to present simultaneously with some trypanosomatidal parasites such as Leishma- nia spp. and be transferred to different hosts (21). For the first time reported that C. deanei has the ability to infect mouse fibroblast cells (22). In the following in 2010, it concluded the ability of this species to cause infections in BALB/c (23). Since the establishment of a specific spe- cies of Leishmania parasite in a geographical region depends on several factors such as the presence of a specific vector and a suitable reservoir (24), the determination of the dom- inant species in an endemic or hyper-endem- ic region can be important for adopting an appropriate strategy to control vectors and res- ervoirs. Furthermore, identification of species is essential for more effective treatment of the patients due to the fact that the response to treatment varies in different species (13). There- fore, considering the high incidence of CL and its health importance, we decided to de- termine the species causing CL in the south- ern region of Iran and the role of Crithidia spp. in creating skin ulcers by using the mo- lecular methods. Materials and Methods Sample Preparation This study was approved by the Ethics Committee of the Bushehr University of Medical Sciences (Ethics Committee code: IR.BPUMS.REC.1395.125) and informed con- sent was signed by each of the patients. In this cross-sectional study, in a period of 6 months from Apr to Sep 2016, patients with cutaneous ulcers referred to the Diag- nostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran were tested. Smears of them from exudates of margin of the wounds were prepared and stained with Giemsa. Fi- nally 66 patients with CL were selected. Then a questionnaire containing demographic in- formation and some of the variables such as J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 200 http://jad.tums.ac.ir Published Online: June 24, 2019 age, sex, etc. were completed separately for each patient. DNA Extraction DNA was extracted from the Giemsa stained smears using the commercial kit (Fa- vorgen Biotech Corp, Cat No. FABGK001, Taiwan). Briefly, the smears on the slides were scratched and collected by lysis buffer into the microtubes and after the addition of pro- teinase K, incubated initially for 1 h at 60 ºC and then 10min at 70 ºC. By addition of ab- solute ethanol and transferring the samples to the column, the rest of the process was carried out according to the kit's manufactur- er's protocol. PCR and Gel Electrophoresis All extracted DNA samples were PCR- amplified in two separate steps. First, a pair of primers LIN4R and LIN17 were used to detect and differentiate of three species L. ma- jor, L. infantum and L. tropica, and in the next step, in order to identify Crithidia spp., CRF and CRR primers were used in this purpose. Further details about the sequence of primers used and the PCR programs adjusted for the amplification of both genomic pieces, has been presented in Table 1. Statistical analysis The results and data of the questionnaires were analyzed using SPSS software version 18 and chi-square test and P-values. At lev- els < .05, the P-values were considered as statistically significant. Results Of the 66 patients, L. major and L. tropica were responsible for 60 and 6 cases, respec- tively (Fig. 1). Moreover, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed (Fig. 2). There was no significant relationship be- tween the disease-causing species and the fac- tors such as the number, appearance, and lo- cation of the wounds in the body, the resi- dence as well as the age and gender of the patients (Table 2). In two patients with mix infection (Leish- mania and Crithidia), the morphology of the cutaneous ulcers was not different from the wounds of the other patients. Table 1. Characteristics of primers and temperature patterns used in PCR tests Genus Primers Sequence Program Processes Cycles Temp Time Leishmania LIN4R (F) 5‘- GGG GTT GGT GTA AAA TAG GG -3‘ P. denaturation 1 95 ⁰C 5min Denaturation 35 94 ⁰C 30sec Annealing 52 ⁰C 30sec LIN17 (R) 5‘- TTT GAA CGG GAT TTC TG -3‘ Extension 72 ⁰C 45sec Final Extension 1 72 ⁰C 8min Crithidia FCR (F) 5‘- TCC ATG TGC GAG GAC AAC GTG CT -3‘ P. denaturation 1 94 ⁰C 3min Denaturation 30 95 ⁰C 30sec Annealing 62 ⁰C 30sec RCR (F) 3‘- CGC GTC GTT GAT GAA GTC GCT -5‘ Extension 72 ⁰C 45sec Final Extension 1 72 ⁰C 5min PCR products were separated on 1.2% agarose gel and TAE (Tris, Acetate, and EDTA) buffer, and the obtained bands were detected by a UV detector (Bio-Rad, USA) J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 201 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 1. Electrophoresis of PCR products using LIN4R and LIN17 primers on agarose gel 1.2% to differentiate Leishmania species. Lane 1: Molecular weight marker, Lane 2: Positive control for L. major, Lane 3: Positive control for L. tropica, Lane 4: Negative control, Lane 5, 6: Positive samples for L. major, Lane 7, 8: Positive samples for L. tropica Fig. 2. Electrophoresis of PCR products using CRF and CRR primers on agarose gel 1.2% to identify Crithidia spp. Lane 1: Molecular weight marker, Lane 2: Positive control for Crithidia spp, Lane 3: Negative control, Lane 4, 5: Positive samples for Crithidia spp., Lane 6: Molecular weight marker J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 202 http://jad.tums.ac.ir Published Online: June 24, 2019 Table 2. Demographic features of the patients with CL and the relationship of disease-causing species and some variables Variables Species P-value L. major n (%) L. tropica n (%) Crithidia+Leishmania n (%) Sex Male Female 33 (50) 26 (39.4) 2 (3) 3 (4.5) 1 (1.5) 1 (1.5) 0.78 Age (yr) < 10 10–20 20–30 30–40 > 40 6 (9.1) 9 (13.6) 9 (13.6) 11 (16.7) 24 (36.4) 1 (1.5) 0 (0) 1 (1.5) 2 (3) 1 (1.5) 0 (0) 0 (0) 1 (1.5) 0 (0) 1 (1.5) 0.73 Residence City Village 46 (69.7) 13 (19.7) 5 (7.6) 0 (0) 1 (1.5) 1 (1.5) 0.30 City Shiraz Marvdasht Daryun Kharameh Tiun Meimand Maharlou Sharifabad 41 (62.1) 1 (1.5) 7 (10.6) 1 (1.5) 2 (3) 2 (3) 1 (1.5) 4 (6.1) 5 (7.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.99 Number of wounds 1 2–5 > 5 23 (34.8) 22 (33.3) 14 (21.2) 3 (4.5) 2 (3) 0 (0) 1 (1.5) 0 (0) 1 (1.5) 0.53 Duration the advent of the wounds 1–4 wk 4–8 wk > 8 wk 22 (33.3) 24 (36.4) 13 (19.7) 1 (1.5) 2 (3) 2 (3) 1 (1.5) 0 (0) 1 (1.5) 0.64 Discussion Cutaneous leishmaniasis is one of the vec- tor-borne diseases and is caused by various spe- cies of Leishmania spp. (3, 25). Establishment of a specific species of this parasite in any ge- ographical region depends on several factors such as the presence of appropriate reservoir and vector, and determining the dominant species in each area can be very important in controlling the life cycle as well as the more effective treatment of the disease (13). The isolation and mass cultivation of par- asite in order to determine the species is not only very time-consuming but also is expen- sive (26, 27), therefore, in the present study, Giemsa stained slides were used for this pur- pose to reduce the costs and overcome the problem. The success of DNA extraction from these smears has already been studied (26). Further- more, the primers used for PCR in various studies were able to identify the genus, so to determine the parasite species, the research- ers had to develop advanced molecular meth- ods, such as PCR-RFLP or sequencing. In this J Arthropod-Borne Dis, June 2019, 13(2): 198–205 A Barazesh et al.: Molecular Identification of … 203 http://jad.tums.ac.ir Published Online: June 24, 2019 study, we used the primers previously de- signed based on the variable part of DNA minicircles in kinatoplast (13, 28, 29). These primers have the ability to identify three spe- cies so that by electrophoresis of the PCR prod- uct, L. major, L. infantum and L. tropica create a specific band in the 650, 720 and 760 bases, respectively. The specificity of 100% has been reported for PCR tests using primers LIN4R and LIN17 to detect Leishmania in atypical cases (13, 30). In some endemic and hyper-endemic re- gions of Iran L. major and in other areas, L. tropica plays a role in causing disease, alt- hough, in southern regions such as Fars Prov- ince, considered as one of the most important foci in Iran, both species are listed as endem- ic factors (24, 31). Recently, alongside these two dominant parasites, Crithidia spp. has been reported from cultivation of cutaneous ulcers of patients (15, 16). In this study, in order to determine the role of Crithidia spp. in cutaneous lesions, sam- ples were again amplified with two specific FCR and PCR primers. In two CL cases, there was a mix infection by Leishmania and Crithid- ia and, as shown in Fig. 2, a band in 850 ba- ses characterizes this parasite (Fig. 2). Crithid- ia spp. are classified in flagellate subphylum and are often considered as insect parasites (16). Since the sequence of their rRNA (Ri- bosomal RNA) gene is very close to Leish- mania, they are usually associated with to- gether (16, 18). Crithidia spp. are considered as infectious agents of the Leishmania culture medium. Crithidia lucilia and C. fasciculata are two important species of this genus (18). Over the past 20 years, various methods have been developed to determine the differ- ent subspecies of Leishmania and Crithidia, as well as to study the molecular diversity and the interaction between this parasite and the host (32-34). In recent reports, besides two L. major and L. tropica species, Crithidia spp. has also been proven in the wound of patients with CL, and interestingly, even in some of these reports, Crithidia has been reported as the only factor isolated from the wound of men- tioned patients. In a study to investigate the pol- ymorphism of two species L. tropica and L. major in the central and desert regions of Iran, Crithidia has been isolated from 6 of 215 and 3 of 125 patients with cutaneous ulcers in Is- fahan and Bam, respectively. Molecular anal- ysis of Crithidia-positive samples using BLAST software has shown that their sequence is 97 % similar to C. fasciculata and 90% to C. lu- cilia (15). 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