J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 82 http://jad.tums.ac.ir Published Online: March 31, 2021 Original Article Comparative Testing of Susceptibility Levels of Phlebotomus sergenti, the Main Vector of Anthroponotic Cutaneous Leishmaniasis, to Conventional Insecticides Using Two Capture Methods in Kerman City, Southeastern Iran Yavar Rassi 1 ; Shahla Ebrahimi 1 ; *Mohammad Reza Abai 1,2 ; *Hassan Vatandoost 1,2 ; Amir Ahmad Akhavan 1,2 ; Abass Aghaie Afshar 3 1 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 3 Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran *Corresponding authors: Mr Mohammad Reza Abai, E-mail: abaimr@tums.ac.ir, abaiemr@gmail.com, Prof Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, vatando@tums.ac.ir (Received 01 Aug 2020; accepted 13 Mar 2021) Abstract Background: Collecting live sand flies from indoor sites is a major challenge for researchers in large cities due to the reluctance of families to survey their homes. This study was conducted to assess the efficacy of two methods for col- lecting sand flies for use in susceptibility tests in the urban area of Kerman, southeastern Iran. Methods: Sandflies were mainly collected using both baited traps and hand catch methods from outdoor and indoor sites. Susceptibility tests were separately done according to the standard World Health Organization testing protocol on Phlebotomus sergenti, including 60-minute exposure to DDT 4.0%, propoxur 0.1%, deltamethrin 0.05%, and malathion 5.0%. Results: During this research, the natural habitats and suitable indoor sites were selected to predict the density of live sand fly with perfect accuracy. The number of live Ph. sergenti caught by hand catch and baited traps methods was 42 and 361 in indoor and outdoor sites, respectively. The mortality rate of Ph. sergenti exposed to DDT 4%, deltamethrin 0.05, malathion 5%, and propoxur was 100%. Conclusion: The baited traps showed a significant efficiency compared to hand catch for collecting live Ph. sergenti for use in susceptibility tests in urban areas. The Ph. sergenti collected from both indoor and outdoor sites were susceptible to all insecticides. Keywords: Phlebotomus sergenti; Susceptibility; Insecticides; Baited traps; Hand catch Introduction Anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica is trans- mitted by Phlebotomus sergenti (1). In the ACL foci, the humans could act as a L. tropica res- ervoir for sand fly infection during the year. In this regard, patients with acute ulcers (2), chronic diffuse ulcers, lupoid or tuberculous ul- cers (3), non-healing ulcers (4), recurrent ul- cers (5) and ulcers resistant to pentavalent an- timonial drugs could provide enough parasitic reservoirs for establishing an ACL transmission cycle by sandflies in the area (6). Dogs are con- sidered as an accidental host (7) in which the lesions usually appear on the snout, interdigit, and sometimes on the corner of the eyelids (1, 8-9). The majority of ACL cases in the world occur in Morocco (10-11), Algeria (12), Libya (13), Tunisia (14-15), Afghanistan (16-21), Iran (22), Pakistan (23), Saudi Arabia (24), Syria (25), Jordan (26-27), Iraq (27), Israel (28), and Tur- key (29). Anthroponotic cutaneous leishmani- asis is a well-known disease in densely popu- Copyright © 2020 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. mailto:abaiemr@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 83 http://jad.tums.ac.ir Published Online: March 31, 2021 lated cities in Iran and is predominantly endem- ic in urban and peri-urban areas including Teh- ran (30), Mashhad (31), Neishabour (32), Shi- raz (33), Kerman (34-35), Bam (37-40), Isfahan (41), and Jiroft (42). Phlebotomus sergenti is a proven vector of ACL (43). Dissection of many specimens of this species in Tehran failed to show the Leishmania (1), while there are sev- eral reports of the leptomonad of L. tropica in dissected Ph. sergenti in Mashhad (44). More- over, Leishmania parasites have been found in Kerman (45-46) and Shiraz using restriction fragment length polymorphism (RFLP) (47-49). Leptomonad infections of Ph. sergenti have al- so been reported from Afghanistan, Saudi Ara- bia (50) and Tunisia (51). In some urban foci, Ph. caucasicus is suspected as a secondary vec- tor (1). In the ACL foci located in the plains, the behavior of Ph. sergenti is completely domestic with more density found at indoor sites ver- sus the mountainous ACL foci. This species is found in moderate density in caves and crev- ices of mountains and foothills where the birds act as a host for blood feeding. The presence of light or other avian hosts attracts the sand- flies to the houses located adjacent to the foot- hills or mountains (43). In southern ACL foci, the activity of Ph. sergenti begins in late June and ends in mid-September (52). In the cen- tral ACL foci, the activity starts in early April and ends in mid-November with two peaks at the end of April and in mid-September (53). Some ecological aspects and surveillance of Ph. sergenti have been studied in endemic foci of ACL (54-57). The ACL occurrence and emer- gence are characterized by several factors such as environmental changes, poor housing con- ditions, unprotected people from sand fly bites, resettlement of non-immune people, deterio- ration of the infrastructure and migration (58- 62), natural disasters like the earthquake (63- 65), displacement of people in borderlines (66), civil wars (67-69), and refugee camps (70-76). At present, spraying operations are limited to the residual foci of malaria in the country (79) and this method is not applied for controlling the ACL vectors. Based on the endophilic hab- its of Ph. sergenti, some attempts have been made to reduce the density of sandflies (78-81) using other insecticide-based vector control measures such as insecticide impregnation of the bed nets (ITNs) (82-86), curtains (ITCs) (87-88), tents (ITTs) (89). Moreover, integrated vector control methods (89-90) have also been applied to control Ph. sergenti population in ep- idemic conditions. In order to analyze the sus- ceptibility tests on sand flies, testing of insec- ticides at discriminative doses has been used for ranking the susceptibility level and as a cri- terion for interpreting the results. It is difficult to collect live sandflies from indoor places in large cities. Therefore, the present study was conducted to determine the most convenient capture method for collecting live sandflies from indoor and outdoor sites and to use the collected sand flies for determining their susceptibility to conventional insecticides in the ACL foci in Ker- man, southeastern Iran, where there is scarce data in this regard. Materials and Methods Study area The study area was located in Kerman, south- eastern Iran. The city borders Khorasan and Yazd to the north, the Lut Desert to the east, Bam to the south, Sirjan to the west, and Raf- sanjan to the northwest. Kerman is elevated 1793 meters above the sea level and the main coor- dinates of the study locations were between 30°06'32"N and 57°06'27.6"E. The study area was a wide plain surrounded by stony moun- tains with a poor vegetation. The studied lo- calities for collecting the adult sand flies were Sarasiyab, Masjed-e-Sahebzaman, Shahzadeh- Mohammad, Safa Cave, Allahabad, Shahrak- Sanaati, Sarbaz Mountains and Shahrak-e- Almahdi (Fig. 1). The entomological operations were carried out from July to September 2019. Sand fly collection Live sand flies were collected using an as- J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 84 http://jad.tums.ac.ir Published Online: March 31, 2021 pirator from the homes located in the periph- ery of the mountains in the early morning. The collected sand flies were released into paper cups and transferred to the laboratory for test- ing. The baited traps were equipped with mini gas lamps, and the netted tents were installed close to the caves or crevices near the moun- tains around Kerman City (Fig. 2), where the presence of adult sand flies was already prov- en during pretest trials using sticky paper traps. The collected live sand flies were also released into paper cups and transferred in a box under a wet towel. Sucrose solution was provided on the top of cups as a cotton pad soaked in su- crose solution 10%. Tested insecticides The insecticide-impregnated papers were pur- chased from the WHO Representative in Pe- nang, Malaysia. The papers were impregnated with DDT 4.0% (batch number DD 265, ex- pire date: July 2022), deltamethrin 0.05% (batch number DE 527, expire date: August 2019), mal- athion 5.0% (batch number MA 234, expire date: July 2020), and propoxur 0.1%, (batch number PR, 123 expire date: August 2020) and tested using the WHO’s testing kit. Susceptibility tests Susceptibility tests were carried out on sand flies collected from both indoor and outdoor sites. The collected sandflies were tested sep- arately according to the capture method using the World Health Organization (WHO) proto- cols (91-93). The mortality rates were deter- mined at discriminative doses for 60 minutes using the WHO test tubes for the adult sand flies. At each replicate, 20–25 sand flies were tested. A 10% sucrose solution was provided during the recovery period. The mortality was recorded after 24 hours of the recovery period. Both dead and live sandflies were preserved in separate tubes containing 70% ethanol accord- ing to tested insecticides and collecting meth- ods until microscopic slides were prepared us- ing the Puri’s medium at the appropriate time. The mounted sandflies were identified using valid identification keys (94-96). Data Analysis The susceptibility data including the number of live, dead, and total sandflies were ranked according to sex (male and female) and physi- ological condition (blood-fed, unfed and gravid) separately in two treatment groups compared to the control group. The charts of mortality rate were drawn with standard errors to show any significant differences using the Microsoft Ex- cel 2010 software. The independent t-test was applied to compare the abundance and sex ra- tio of Ph. sergenti between hand-catch and bait- ed trap methods using the IBM SPSS software version (25). Results Efficiency of collection methods The efficiency the baited traps for collect- ing live Ph. sergenti from outdoor was 370 compared to 45 sand flies collected using hand- catch from indoor sites (Table 1) indicating an significant difference (t= 7.214, df= 9, p= 0.001) (Fig. 3). The sex ratio of Ph. sergenti was 1.0 at indoor compared to 1.2 at outdoor places (Ta- ble 1) showing no significance difference (t= 2.574, df= 8, p= 0.07) (Fig. 4). Susceptibility of Phlebotomus sergenti col- lected from indoors The mortality rate of Ph. sergenti collect- ed from indoor places using hand-catch meth- od was 100% after exposure to DDT and mal- athion. The mortality rate was 0.0% in the con- trol group (Fig. 5). Susceptibility of Phlebotomus sergenti col- lected from outdoors The mortality rate of Ph. sergenti collect- ed from outdoor sites using baited traps was 100 %. After exposed to DDT, propoxur, del- tamethrin, and malathion. The mortality rate was 0.0% in the control group (Fig. 6). J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 85 http://jad.tums.ac.ir Published Online: March 31, 2021 Fig. 1. Map showing Kerman City and the locations studied the collecting methods of live sand flies used for suscep- tibility tests, Kerman Province, 2019 Fig. 2. Using the baited traps for collection of live Phlebotomus sergenti in the mountainous areas around Kerman City, southeastern Iran, Kerman Province, 2019 J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 86 http://jad.tums.ac.ir Published Online: March 31, 2021 Table 1. Statistical comparison of efficiency of two collection methods used for live collection of Phlebotomus ser- genti, Kerman City, southeastern Iran, July–Sep 2019 Capture methods Capture place Sessions of collection Total collected Capture Rate (%) No. male No. female Sex ratio Physiologic conditions Environmental condition No. fed No. unfed No. gravid Temp °C Relative humidity (%) Baited traps Outdoor 9 370 89.2* 198 172 1.2† 29 (16.8%) 112 (65.1%) 31 (18.0%) 28–30 <40 Hand- catch Indoor 5 45 10.8* 22 23 1.0† 3 (17.4%) 17 (73.9%) 2 (8.7%) 26–28 <40 T=-7.214, df= 9, p= 0.001 † t=-2.574, df= 8, p= 0.07 Fig. 3. Statistical comparison of abundance of Phlebotomus sergenti collected using hand-catch and baited traps from indoor and outdoor places, Kerman City, southeastern Iran, 2019 Fig. 4. Statistical comparison of sex ratio of Phlebotomus sergenti collected using hand-catch and baited traps from indoor and outdoor places, Kerman City, southeastern Iran, 2019 J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 87 http://jad.tums.ac.ir Published Online: March 31, 2021 Fig. 5. Susceptibility of Phlebotomus sergenti to DDT and malathion, collected using hand-catch from indoor places, Kerman City, southeastern Iran, 2019 Fig. 6. Susceptibility of Phlebotomus sergenti to DDT, deltamethrin, malathion and propoxur collected using baited traps from outdoor places, Kerman City, southeastern Iran, 2019 J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 88 http://jad.tums.ac.ir Published Online: March 31, 2021 Discussion Despite the implementation of the national leishmaniasis surveillance program and the ef- forts for adopting different control methods dur- ing past decades, ACL is still a major health challenge with increasing trends in many parts of Iran, particularly Kerman Province. This study was conducted to investigate the susceptibility level of Ph. sergenti to conventional insecti- cides according to the WHO instruction for the phlebotomine susceptibility test. Furthermore, there is an operational gap for employing a cap- ture method for adequate yielding of live Ph. sergenti, which is necessary for the standard sample sizes used in susceptibility tests. In this study, efficiency of baited traps showed the more yield of live Ph. sergenti compared to hand- catch method from indoor places (t= 7.214, df= 9, p= 0.001) with no significant difference for the sex ratio. The most important obstacle for collecting sand flies from indoor places using the hand-catch method is the lack of coopera- tion and willingness of the households to check their houses for the presence of sand flies. In addition, in urban areas, due to the application of aerosol sprays or using cooling systems in the houses, the abundance of sand flies is usu- ally lower than the suburbs. In the other study, different collecting methods were assessed for live sand flies with a total of 122 live Ph. ser- genti collected at outdoor sites in the moun- tainous parts of Kashan District. The compar- ison of different traps showed the most effi- ciency (59.8%) for the black Shannon traps during the summer (97). In other attempts made in the northeastern parts of Iran as well as in Cukurova Plain, southern Anatolia, Turkey, the CO2 CDC light traps showed the most efficien- cy with 43.2% and 75.0 percent for collecting live Ph. sergenti with a total abundance of 2521 and 4 from outdoor sites (98-99). In an- other testing, different commercial traps were assessed for collecting live Ph. sergenti in the north of Aswan, southern Egypt which the BGS traps showed more efficacy (35.5%) (100). The results of susceptibility tests on Ph. sergenti collected from outdoor sites showed 100% mor- tality exposed to DDT 4.0%, deltamethrin 0.05%, malathion 5.0%, and propoxur 0.1%, indicat- ing the complete susceptibility. The Ph. ser- genti caught in the houses located adjacent to the mountains using hand-catch method revealed 100% mortality when exposed to DDT 4.0% and malathion 5.0%. Due to the obvious dif- ferences in the biology and behavior of Ph. ser- genti with other species of sand flies, the find- ing of the present study was compared with other studies if the tests were only carried out on Ph. sergenti. The number of reports on the sus- ceptibility level of Ph. sergenti to insecticides is limited in the world. During the years 1971, 1976, and 1998, three trials were carried out in the ACL foci in the large cities located in the northeast including Mashhad (31) and Neisha- bur (32) as well as Kerman (101) located in the southeast of Iran. The findings revealed the com- plete susceptibility of this species to the DDT. Moreover, there are three other published rec- ords for assessing the susceptibility status of Ph. sergenti in Mashhad, northeast of Iran (102) and Dehbakri County, southeast of Iran (103) in 2007 and 2011, which indicated the complete susceptibility of Ph. sergenti to DDT 4.0% and deltamethrin 0.05%. In a more recent study car- ried out in Maneh and Samalqan County, north- eastern Iran, all sand flies were collected using CDC light traps from outdoor and tested with papers impregnated with DDT 4.0%, bendio- carb 0.1%, and permethrin 0.75%. The results showed mortality rates of 89.8±1.4, 93.6±1.4, and 95.6±1.7% respectively, indicating the re- sistance of Ph. sergenti to DDT (104). A study carried out in North Africa and the Middle East in 2007 found the high susceptibility of Ph. sergenti to malathion, DDT, cyfluthrin, bendi- ocarb, permethrin, and resmethrin (105). A sus- ceptibility test conducted in Morocco showed the full susceptibility of wild populations of Ph. sergenti to DDT, lambdacyhalothrin, and malathion (106). The finding of the present re- search showed the susceptibility of Ph. sergenti J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 89 http://jad.tums.ac.ir Published Online: March 31, 2021 to DDT, malathion, deltamethrin, and propoxur in indoor and outdoor populations collected in the ACL foci of Kerman. It is expected that application of the insecticide-based control strat- egies for sand flies, for example IRS, ITNs, ITC and ITT, could be an effective method for con- trolling Ph. sergenti in indoor and outdoor plac- es if the measures are performed correctly. Conclusion Anthroponotic cutaneous leishmaniasis is still a major health problem in Kerman, south- east of Iran. This research showed the high sus- ceptible status of Ph. sergenti in both the in- door and outdoor populations against all test- ed insecticides. The research-based evidence in- dicates the success of future insecticide-based vector control methods for controlling sand flies in the ACL foci. Acknowledgements The authors are grateful to Mrs M Mozafa- ri due to her sincere assistance in providing the data of the ACL cases in Kerman. This study was part of a MSc dissertation funded and sup- ported by Tehran University of Medical Sci- ences (TUMS) [grant no. 98-01-27-41998, re- search ethics approval ID: IR.TUMS.SPH.REC. 1398.079]. The authors declare that there is no conflict of interest. References 1. Javadian E (2008) Epidemiology of cutane- ous leishmaniasis in Iran. In: Nadim A, Javadian E, Momeni A (Eds) Leishmania and leishmaniasis. Iran University Press 1295, Tehran, pp. 191–211. 2. Seyedi-Rashti MA, Keighobadi K, Nadim A (1984) Urban cutaneous leishmaniasis in Kerman, southeast Iran. Bull Soc Pathol Exot. 77: 312–319. 3. Jafari S, Hajiabdolbaghi M, Mohebali M, Haj- jaran H, Hashemian H (2010) Dissemi- nated leishmaniasis caused by Leishma- nia tropica in HIV-positive patients in the Islamic Republic of Iran. East Medi- terr Health J. 16: 340–343. 4. Bamorovat M, Sharifi I, Mohammadi MA, Eybpoosh S, Nasibi S, Aflatoonian MR, Khosravi A (2018) Leishmania tropica isolates from non-healed and healed pa- tients in Iran: A molecular typing and phylogenetic analysis. Microb Pathog. 1 (116): 124–129. 5. Sharifi I, Fekri AR, Aflatoonian MR, Khamesipour A, Mahboudi F, Dowlati Y, Nadim A, Modabber F (2010) Leishman- iasis recidivans among school children in Bam, Southeast Iran, 1994–2006. Intern J Dermatol. 49: 557–561. 6. Bamorovat M, Sharifi I, Aflatoonian MR, Sharifi H, Karamoozian A, Sharifi F, Kar- amoozian A, Sharifi F, Khosravi A, Has- sanzadeh S (2018) Risk factors for an- throponotic cutaneous leishmaniasis in unresponsive and responsive patients in a major focus, southeast of Iran. PloS One. 13: e0192236. 7. Bamorovat M, Sharifi I, Dabiri S, Moham- madi MA, Fasihi Harandi M, Mohebali M, Aflatoonian MR, Keyhani A (2015) Leishmania tropica in stray dogs in south- east Iran. Iran J Public Health. 44(10): 1359–1366. 8. Mohebali M, Malmasi A, Hajjaran H, Jam- shidi S, Akhoundi B, Rezaei M, Janita- bar S, Zarei H, Charehdar S (2011) Dis- seminated leishmaniasis caused by Leish- mania tropica in a puppy from Karaj, cen- tral Iran. Iran J Parasitol. 6: 69–73. 9. Dereure J, Rioux JA, Gallego M, Perieres J, Pratlong F, Mahjour J, Saddiki H (1991) Leishmania tropica in Morocco: infec- tion in dogs. Trans R Soc Trop Med Hyg. 85(5): 595–599. 10. Guilvard E, Rioux JA, Gallego M, Pratlong F, Mahjour J, Martinez-Ortega E, Dereure J, Saddiki A, Martini A (1991) Leishma- J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 90 http://jad.tums.ac.ir Published Online: March 31, 2021 nia tropica in Morocco. III identification of 89 isolates from the vector Phleboto- mus sergenti. Ann Parasitol Hum Comp. 66: 96–99. 11. Yahia H, Ready PD, Hamdani A, Testa JM, Guessous N (2004) Regional differenti- ation of Phlebotomus sergenti in three Moroccan foci of cutaneous leishmania- sis cause by Leishmania tropica. Parasite. 11: 189–199. 12. Izri A, Bendjaballah A, Andriantsoanirina V, Durand R (2014) Cutaneous leishman- iasis caused by Leishmania killicki, Al- geria. Emerg Infect Dis. 20(3): 502–504. 13. Amro A, Gashout A, Al-Dwibe H, Zahangir Alam M, Annajar B, Hamarsheh O (2012) First Molecular Epidemiological Study of Cutaneous Leishmaniasis in Libya. PLoS Negl Trop Dis. 6(6): e1700. 14. Tabbabi A (2019) Review of Leishmania- sis in the Middle East and North Africa. Afr Health Sci. 19(1): 1329–1337. 15. Tabbabi A, Ghrab J, Aoun K, Ready PD, Bouratbine A (2011) Habitats of the sand- fly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leish- maniasis in South East Tunisia. Acta Trop. 119: 131–137. 16. Nadim A, Rostami GS (1974) Epidemiol- ogy of cutaneous leishmaniasis in Kabul, Afghanistan. Bull World Health Org. 51: 45–49. 17. Nadim A, Javadian E, Noushin MK, Nayil AK (1979) Epidemiology of cutaneous leishmaniasis in Afghanistan. Part II. An- throponotic cutaneous leishmaniasis. Bull Soc Pathol Exot Filiales. 72: 461–466. 18. Javadian E, Nadim A, Nayil AK (1982) Ep- idemiology of cutaneous leishmaniasis in Afghanistan. Part III. Notes on sandflies of Afghanistan. Bull Soc Pathol Exot Fil- iales. 75: 284–290. 19. Reithinger R, Mohsen M, Aadil K, Sidiqi M, Erasmus P, Coleman PG (2003) Anthro- ponotic cutaneous leishmaniasis, Kabul, Afghanistan. Emerg Infect Dis. 9: 727–729. 20. Hewitt S, Reyburn H, Ashford R, Rowland M (1998) Anthroponotic cutaneous leish- maniasis in Kabul, Afghanistan: vertical distribution of cases in apartment blocks. Trans R Soc Trop Med Hyg. 92: 273–274. 21. Reyburn H, Rowland M, Mohsen M, Khan B, Davies C (2003) The prolonged epi- demic of anthroponotic leishmaniasis in Kabul, Afghanistan: ‘bringing down the neighborhood'. Trans R Soc Trop Med Hyg. 97: 170–176. 22. Shirzadi MR, Gouya MM (2010) National Guidelines for Cutaneous Leishmaniasis Surveillance in Iran. Ministry of Health and Medical Education, Zoonoses Con- trol Department, Tehran, Iran. 23. Hussain M, Munir S, Jamal MA, Ayaz S, Akhoundi M, Mohamed K (2017) Epi- demic outbreak of anthroponotic cutane- ous leishmaniasis in Kohat District, Khy- ber Pakhtunkhwa, Pakistan. Acta Trop. 172: 147–155. 24. El-Beshbishy HA, Al-Ali KH, El-Badry AA (2013) Molecular characterization of Leishmania infection in sand flies from Al-Madinah Al-Munawarah Province, Western Saudi Arabia. Exp Parasitol. 134: 211–215. 25. Muhjazi G, Gabrielli AF, Ruiz-Postigo JA, Atta H, Osman M, Bashour H, Al Tawil A, Husseiny H, Allahham R, Allan R (2019) Cutaneous leishmaniasis in Syr- ia: A review of available data during the war years: 2011–2018. PLoS Negl Trop Dis. 13(12): e0007827. 26. Kamhawi S, Abdel-Hafez SK, Arbagi A (1995) A new focus of cutaneous leish- maniasis caused by Leishmania tropica in northern Jordan. Trans R Soc Trop Med Hyg. 89(3): 255–257. 27. Salam N, Al-Shaqha WM, Azzi A (2014) Leishmaniasis in the Middle East: inci- dence and epidemiology. PLoS Negl Trop Dis. 8(10): e3208. 28. Svobodova M, Votypka J, Peckova J, Dvo- rak V, Nasereddin A, Baneth G, Sztern J, J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 91 http://jad.tums.ac.ir Published Online: March 31, 2021 Kravchenko V, Orr A, Meir D, Schnur LF, Volf P, Warburg A (2006) Distinct transmission cycles of Leishmania trop- ica in 2 adjacent foci. Emerg Infect Dis. 12: 1860– 1868. 29. Zeyrek FY, Korkmaz M, Ozbel Y (2007) Serodiagnosis of anthroponotic cutaneous leishmaniasis (ACL) caused by Leishma- nia tropica in Sanliurfa Province, Tur- key, where ACL is highly endemic. Clin Vaccine Immunol. 14: 1409–14015. 30. Nadim A, Javadian E, Seyedi- Rashti MA (1994) Epidemiology of leishmaniasis in Iran. In: Ardehali S, Rezaei R, Nadim A (Eds) Leishmania Parasite and leishman- iasis. University Publishing Center, Teh- ran, pp. 176–208. 31. Nadim A, Seyedi-Rashti MA, Mesghali A (1971) Epidemiology of cutaneous leish- maniasis in Iran. B. Khorassan. Part IV. Distribution of sand flies. Bull Soc Path Exot. 64(6): 865–870. 32. Nadim A, Tahvildari-Bidruni GH (1977) Epidemiology of cutaneous leishmania- sis in Iran: B. Khorassan, Part VI: Cuta- neous leishmaniasis in Neishabur, Iran. Bull Soc Pathol Exot. 70: 171–177. 33. Ghatee MA, Mirhendi H, Marashifard M, Kanannejad Z, Taylor WR, Sharifi I (2018) Population structure of Leishma- nia tropica causing anthroponotic cuta- neous leishmaniasis in southern Iran by PCR-RFLP of kinetoplastid DNA. Bi- omed Res Int. 2018: 6049198. 34. Yaghoobi-Ershadi MR (1977) Studies on Cutaneous Leishmaniasis in the City of Kerman. [MSPH Thesis]. University of Tehran, Iran. 35. Sharifi I, Aflatoonian MR, Fekri AR, Ha- kimi Parizi M, Aghaei-Afshar A, Khosra- vi A (2015) A comprehensive review of cutaneous leishmaniasis in Kerman Prov- ince, southeastern Iran. Narrative review article. Iran J Public Health. 44: 299–307. 36. Yaghoobi-Ershadi MR, Hanafi-Bojd AA, Javadian E, Jafari R, Zahraei-Ramazani AR, Mohebali M (2002) A new focus of cutaneous leishmaniasis caused by Leish- mania tropica. Saudi Med J. 23: 291–294. 37. Nadim A, Aflatoonian MR (1995) Anthro- ponotic cutaneous leishmaniasis in Bam, southeast Iran. Iran J Public Health. 24: 15–24. 38. Aflatoonian MR, Sharifi I, Nadim A, Af- latoonian B (2014) Comparison of the pe- riod prevalence of urban cutaneous leish- maniasis (CL) in Bam in two time peri- ods of 1990–1992 and 2010–2012. Iran J Public Health. 9: 32–39. 39. Sharifi I, Fekri AR, Aflatoonian MR, Nadim A, Nikian Y, Khamesipour A (1998) Cu- taneous leishmaniasis in primary school children in the southeastern Iranian city of Bam, 1994–1995. Bull World Health Org. 76: 289–293. 40. Sharifi I, Nakhaei N, Aflatoonian MR, Ha- kimi Parizi M, Fekri AR, Safizadeh H, Shirzadi MR, Goya MM, Khamesipour A (2011) Cutaneous leishmaniasis in Bam: A comparative evaluation of pre- and post- earthquake years (1999–2008). Iran J Public Health. 40: 49–56. 41. Zahraei-Ramazani A, Yaghoobi-Ershadi MR, Mokhtari A, Akhavan AA, Abdoli H, Arandian M (2007) Anthroponotic cu- taneous leishmaniasis in nonendemic quar- ters of a central city in Iran. Iran J Pub- lic Health. 36(2): 7–11. 42. Mirzaei M, Sharifi I, Poursmaelian S (2012) A new focus of anthroponotic cutaneous leishmaniasis and identification of para- site species by nested PCR in Jiroft, Iran. Comp Clin Pathol. 21(5): 1071–1075. 43. 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): 1–17. 44. Mesghali A, Seyedi-Rashti MA, Nadim A (1967) Epidemiology of cutaneous leish- maniasis in Iran. II. Natural leptomonad infection of sand flies in the Mashhad and J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 92 http://jad.tums.ac.ir Published Online: March 31, 2021 Lotfabad areas. Bull Soc Pathol Exot. 60: 514–517. 45. Mozafary M, Dayer MS, Aghaei-Afshar A, Mollaie HR (2016) Epidemiology of cutaneous leishmaniasis and molecular characterization of its causative agents in naturally infected sand flies in endemic foci of Kerman City, southeastern Iran. Asian Pac J Trop Dis. 6(3): 188–192. 46. Aghaei Afshar A, Rassi Y, Sharifi I, Vatan- doost H, Mollaie HR, Oshaghi MA, Abai MR, Rafizadeh S (2014) First report on natural Leishmania infection of Phleboto- mus sergenti due Leishmania tropica by high resolution melting curve method in southeastern Iran. Asian Pac J Trop Med. 7(2): 93–96. 47. Azizi K, Askari MB, Kalantari M, Moemenbellah-Fard MD (2016) Molec- ular detection of Leishmania parasites and host blood meal identification in wild sand flies from a new endemic rural region, south of Iran. Pathog Glob Health. 110: 303–309. 48. Moemenbellah-Fard MD, Ahmadyousefi- Sarhadi M, Azizi K, Fakoorziba MR, Kalantari M, Amin M (2015) Faunal iden- tification and frequency distribution of wild sand flies infected with Leishmania tropica. Asian Pac J Trop Dis. 5(10): 792– 797. 49. Oshaghi MA, Rasolian M, Shirzadi MR, Mohtarami F, Doosti S (2010) First re- port on isolation of Leishmania tropica from sand flies of a classical urban cu- taneous leishmaniasis focus in southern Iran. Exp Parasitol. 126: 445–450. 50. Al-Zahrani MA, Peters W, Evans DA, Ching C, Smith IV, Lane RP (1988) Phleboto- mus sergenti, a vector of Leishmania trop- ica in Saudi Arabia. Trans R Soc Trop Med Hyg. 82: 416. 51. Tabbabi A, Bousslimi N, Rhim A, Aoun K, Bouratbine A (2011) First report on nat- ural infection of Phlebotomus sergenti with Leishmania promastigotes in the cutane- ous leishmaniasis focus in southeastern Tunisia. Am J Trop Med Hyg. 85: 646– 647. 52. Aghasi M, Sharifi I (2003) Survey of the fauna and monthly activity of the sand fly as the vectors of the cutaneous leish- maniasis in the city of Bam. J Kerman Univ Med Sci. 10(2): 85–91. 53. Mirhoseini M, Salehzadeh A, Ramazan Ja- maat S, Zahirnia A H, Rahmanzadeh N (2017) Distribution and seasonal activ- ity of Phlebotominae sand flies in Yazd and its outskirts, center of Iran. Sci World J. 1486845. 54. Nadim A, Seyedi-Rashti MA (1991) Some aspects to the ecology of Phlebotomus sergenti in Iran. First International Sym- posium on Phlebotomine Sand flies Rome, Italy, p. 79. 55. Fakoorziba MR, Nazari M (2006) Ento- mological studies of Phlebotomus papa- tasi and Ph. sergenti (Diptera: Psycho- didae) as vectors of cutaneous leishman- iasis in Shiraz, Iran. Southeast Asian J Trop Med Public Health. 37 Suppl 3: 115– 117. 56. Yaghoobi-Ershadi MR, Hakimiparizi M, Zahraei-Ramazani AR, Abdoli H, Akha- van AA, Aghasi M, Arandian MH, Ranjbar AA (2010) Sand fly surveillance within an emerging epidemic focus of cu- taneous leishmaniasis in southeastern Iran. Iran J Arthropod Borne Dis. 4(1): 17–23. 57. Zahraei-Ramazani AR, Akhavan AA, Abdoli H, Jafari R, Jalalizand AR, Arandian MH, Shareghi N, Ghanei M (2008) Some eco- logical aspects of phlebotominae sandflies (Diptera: Psychodidae) in an endemic fo- cus of anthroponotic cutaneous leishman- iasis of Iran. J Entomol. 5 (1): 17–23. 58. Hmamouch A, El Alem MM, Hakkour M, Amarir F, Daghbach H, Habbari K, Fel- lah H, Bekhti K, Sebti F (2017) Circu- lating species of Leishmania at microcli- mate area of Boulemane Province, Mo- rocco: impact of environmental and hu- J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 93 http://jad.tums.ac.ir Published Online: March 31, 2021 man factors. Parasite Vectors. 10(1): 100. 59. Ullah K, Khan NH, Sepúlveda N, Munir A, Wahid S (2016) Assessing incidence patterns and risk factors for cutaneous leishmaniasis in Peshawar region, Khy- ber Pakhtunkhwa, Pakistan. J Parasitol. 102: 501–506. 60. Aflatoonian MR, Sharifi I, Parizi MH, Fekri AR, Aflatoonian B, Sharifi M, Khosravi A, Khamesipour A, Sharifi H (2014) A prospective cohort study of cutaneous leishmaniasis risk and opium addiction in south eastern Iran. PLoS One. PMID: 24586494. 61. Desjeux P (2001) The increase in risk fac- tors of leishmaniasis worldwide. Trans R Soc Trop Med Hyg. 95: 239–245. 62. Dujardin JC (2006) Risk factors in the spread of leishmaniases: towards integrated mon- itoring? Trends Parasitol. 22: 4–6. 63. Aflatoonian MR, Sharifi I, Poursmaelian S, Hakimi-Parizi M, Ziaali N (2013) The emergence of anthroponotic cutaneous leishmaniasis following the earthquake in southern villages of Bam District, south- eastern Iran, 2010. J Arthropod Borne Dis. 7(1): 8–14. 64. Aflatonian MR, Montazeri F, Jalali M, Nadim A, Sharifi F (2006) The status of the disease in Bam in 2005 years and the comparison with the last five years (be- fore and after the earthquake). J Kerman Uni Med Sci. 13: 37–43. 65. Sharifi I, Poursmaelian S, Aflatoonian MR, Fotouhi Ardakani R, Mirzaei M, Fekri AR, Khamesipour A, Hakimi Parizi M, Fasihi Harandi M (2011) Emergence of a new focus of anthroponotic cutaneous leish- maniasis due to Leishmania tropica in ru- ral communities of Bam District after the earthquake, Iran. Trop Med Intern Health. 16(4): 510–513. 66. Fazaeli A, Fouladi B, Sharifi I (2009) Emer- gence of cutaneous leishmaniasis in a bor- der area at south-east of Iran: an epide- miological survey. J Vector Borne Dis. 46: 36–42. 67. Alawieh A, Musharrafieh U, Jaber A, Ber- ry A, Ghosn N, Bizri AR (2014) Revis- iting leishmaniasis in the time of war: the Syrian conflict and the Lebanese out- break. Int J Infect Dis. 29: 115–119. 68. Alasaad S (2013) War diseases revealed by the social media: massive leishmani- asis outbreak in the Syrian Spring. Para- site Vectors. 6: 94. 69. Inci R, Ozturk P, Mulayim MK, Ozyurt K, Alatas ET, Inci MF (2015) Effect of the Syrian civil war on prevalence of cuta- neous leishmaniasis in southeastern An- atolia, Turkey. Medical science monitor. Intl Med J Exp Clin Res. 21: 2100–2104. 70. Kolaczinski J, Brooker S, Reyburn H, Row- land M (2004) Epidemiology of anthro- ponotic cutaneous leishmaniasis in Af- ghan refugee camps in northwest Paki- stan. Tran Roy Soc Trop Med Hyg. 98: 373–378. 71. Rowland M, Munir A, Durrani N, Noyes H, Reyburn H (1999) An outbreak of cu- taneous leishmaniasis in an Afghan ref- ugee settlement in north-west Pakistan. Trans R Soc Trop Med Hyg. 93: 133–136. 72. Saroufim M, Charafeddine K, Issa G (2014) Ongoing epidemic of cutaneous leishman- iasis among Syrian refugees, Lebanon. Emerg Infect Dis. 20(10): 1712–1715. 73. Brooker S, Mohammed N, Adil K, Agha S, Reithinger R, Rowland M, Ali I, Ko- laczinski J (2004) Leishmaniasis in ref- ugee and local Pakistani populations. Emerg Infect Dis. 10(9): 1681–1684. 74. His Excellency, Murshidi MM, Hijjawi MQ, Jeriesat S, Eltom A (2013) Syrian refu- gees and Jordan's health sector. Lancet. 382(9888): 206–207. 75. Du R, Hotez PJ, Al-Salem WS, Acosta-Ser- rano A (2016) Old world cutaneous leish- maniasis and refugee crises in the Middle East and North Africa. PLoS Negl Trop Dis. 10(5): e0004545. 76. Saroufim M, Charafeddine K, Issa G, Kha- J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 94 http://jad.tums.ac.ir Published Online: March 31, 2021 lifeh H, Habib RH, Berry A, Ghosn N, Rady A, Khalifeh I (2014) Ongoing ep- idemic of cutaneous leishmaniasis among Syrian refugees, Lebanon. Emerg Infect Dis. 20(10): 1712–1715. 77. Seyedi-Rashti MA, Nadim A (1975) Re- establishment of cutaneous leishmania- sis after cessation of anti-malaria spray- ing. Trop Geogr Med. 27(1): 79–82. 78. World Health Organization (2010) Control of the Leishmaniasis. Report of a meeting of the WHO export committee on the con- trol of leishmaniasis, WHO Technical Re- port Series 949, Geneva. 79. Yaghoobi-Ershadi MR (2016) Control of phlebotomine sand flies in Iran: a review article. J Arthropod Borne Dis. 10(4): 429– 444. 80. González U, Pinart M, Sinclair D (2015) Vector and reservoir control for prevent- ing leishmaniasis. Cochrane Database Syst Rev. published by John Wiley and Sons, Ltd. on behalf of The Cochrane Collab- oration. p. 80. 81. Maroli M, Khoury C (2004) Prevention and control of leishmaniasis vectors: current approaches. Parasitologia. 46: 211–215. 82. Nadim A, Motabar M, Houshmand B, Key- ghobadi K, Aflatoonian MR (1995) Eval- uation of Pyrethroid Impregnated Bed Nets for Control of Anthroponotic Cuta-neous Leishmaniasis in Bam (Islamic Re-public of Iran). WHO, Geneva, Switzerland. Available at: https://apps.who.int/iris/handle/10665/6 1138. 83. Yaghoobi-Ershadi MR, Moosa-Kazemi SH, Zahraei-Ramazani AR, Jalaizand AR, Akhavan AA, Arandian MH (2006) Eval- uation of deltamethrin impregnated bed nets and curtains for control of zoonotic cutaneous leishmaniasis in hyperendemic area of Iran. Bull Soc Pathol Exot. 99(1): 43–48. 84. Kolaczinski JH, Muhammad N, Khan QS, Jan Z, Rehman N (2004) Subsidized sales of insecticide-treated nets in Afghan ref- ugee camps demonstrate the feasibility of a transition from humanitarian aid to- wards sustainability. Malar J. 3: 15. 85. Courtenay O, Gillingwater K, Gomes PA, Garcez LM, Davies CR (2007) Deltame- thrin-impregnated bednets reduce human landing rates of sandfly vector Lutzomy- ia longipalpis in Amazon households. Med Vet Entomol. 21(2): 168–176. 86. Jalouk L, Al Ahmed M, Gradoni L, Maro- li M (2007) Insecticide-treated bed nets to prevent anthroponotic cutaneous leish- maniasis in Aleppo Governorate, Syria: re- sults from two trials. Trans R Soc Trop Med Hyg. 101(4): 360–367. 87. Kroeger A, Avila EV, Morison L (2002) In- secticide impregnated curtains to control domestic transmission of cutaneous leish- maniasis in Venezuela: cluster randomized trial. BMJ. 325(7368): 810–813. 88. Moosa-Kazemi SH, Yaghoobi-Ershadir MR, Akhavan AA, Abdoli H, Zahraei-Ram- azani AR (2007) Deltamethrin-impreg- nated bed nets and curtains in an anthro- ponotic cutaneous leishmaniasis control program in northeastern Iran. Ann Saudi Med. 27: 6–12. 89. Reyburn H, Ashford R, Mohsen M, Hewitt S, Rowland M (2000) A randomized con- trolled trial of insecticide-treated bednets and chaddars or top sheets, and residual spraying of interior rooms for the pre- vention of cutaneous leishmaniasis in Ka- bul, Afghanistan. Trans R Soc Trop Med Hyg. 94: 361–366. 90. Aghaei-Afshar A, Vatandoost H, Sharifi I, Mollaie H, Oshaghi MA (2013) First de- termination of impact and outcome indi- cators following indoor residual spray- ing (IRS) with deltamethrin in a new fo- cus of Anthroponotic Cutaneous Leish- maniasis (ACL) in Iran. Asian Pac J Trop Dis. 3: 5–9. 91. World Health Organization (1981) Instruc- tions for determining the susceptibility J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 95 http://jad.tums.ac.ir Published Online: March 31, 2021 or resistance of blackflies, sand flies and biting midges to insecticides. WHO/VBC/ 81.810. 92. World Health Organization (1986) Re- sistance of vectors and reservoirs of dis- ease to pesticides. 10 th report of the WHO Expert committee. Vector Biology and Control. Tech Rep Ser, p. 737. 93. World Health Organization (2016) Test procedures for insecticide resistance moni- toring in malaria vector mosquitoes, II, WHO, Geneva. Available at: http://apps.who.int/iris/bitstream/handle/ 10665/250677/9789241511575- eng.pdf;jsessionid=B32ACC064A1217 C97C2142CEBC2FA440?sequence=1 94. Nadim A, Javadian E (1976) Key for spe- cies identification of sandflies (Phlebotom- inae: Diptera) of Iran. Iran J Public Health. 5: 33–44. 95. Javadian E, Mesghali A (1975) Checklist of phlebotomine sand flies (Diptera: Psy- chodidae) of Iran. Bull Soc Pathol Exot. 68: 207–209. 96. Rassi Y, Hanafi-Bojd AA (2006) Phlebotom- ine Sand Flies, Vectors of Leishmaniasis: Morphology, biology, ecology, and field and laboratory methods with pictorial key of Iranian sand flies. Noavaran-Elm Pub- lication, Tehran, Iran. 97. Hesam-Mohammadi M, Rassi Y, Abai MR, Akhavan AA, Karimi F, Rafizadeh S, Sanei-Dehkordi A, Sharafkhah M (2014) Efficacy of different sampling methods of sand flies (Diptera: Psychodidae) in en- demic focus of cutaneous leishmaniasis in Kashan District, Isfahan Province, Iran. J Arthropod Borne Dis. 8(2): 156–162. 98. Arzamani K, Rassi Y, Vatandoost H, Akha- van AA, Abai MR, Alavinia M, Akbar- zadeh K, Mohebali M, Rafizadeh S, Karimian F, Badakhshan M, Absavaran A (2019) Comparative Performance of different traps for collection of phlebotom- inae sand flies and estimation of biodi- versity indices in three endemic leishman- iasis foci in North Khorasan Province, Northeast of Iran. J Arthropod Borne Dis. 13(4): 399–406. 99. Kasap ÖE, Belen A, Kaynas S, Simsek FM, Biler L, Ata N, Alten B (2009) Activity patterns of sand fly (Diptera: Psychodi- dae) species and comparative performance of different traps in an endemic cutane- ous leishmaniasis focus in Cukurova Plain, Southern Anatolia, Turkey. Acta Vector Borne. 78(2): 327–335. 100. Hoel DF, Kline DL, Hogsette JA, Bernier UR, El-Hossary SS, Hanafi HA, Watany N, Fawaz EY, Furman BD, Obenauer PJ, Szumlas DE (2010) Efficacy of com- mercial mosquito traps in capturing Phlebotomine sand Flies (Diptera: Psy- chodidae) in Egypt. J Med Entomol. 47 (6): 1179–1184. 101. Aghasi M (1996) Present Status of An- throponotic Cutaneous Leishmaniasis in Kerman, Southeast Iran. [MSPH thesis]. School of Public Health, Tehran Uni- versity of Medical Sciences, Tehran, Iran. 102. Moosa-Kazemi SH, Yaghoobi-Ershadi MR, Akhavan AA, Abdoli H, Zahraei-Rama- zani AR, Jafari R, Houshmand B, Nadim A, Hosseini M (2007) Deltamethrin-im- pregnated bednets and curtains in an an- throponotic cutaneous leishmaniasis con- trol program in northeastern Iran. Ann Saudi Med. 27(1): 6–12. 103. Afshar AA, Rassi Y, Sharifi I, Abai MR, Oshaghi M, Yaghoobi-Ershadi MR, Vatandoost H (2011) Susceptibility sta- tus of Phlebotomus papatasi and Ph. sergenti (Diptera: Psychodidae) to DDT and deltamethrin in a focus of cutane- ous leishmaniasis after earthquake strike in Bam, Iran. J Arthropod Borne Dis. 5(2): 32–41. 104. Arzamani K, Vatandoost H, Rassi Y, Abai MR, Akhavan AA, Alavinia M, Akbar- zadeh K, Mohebali M, Rafizadeh S (2017) Susceptibility status of wild pop- J Arthropod-Borne Dis, March 2021, 15(1): 82–96 Y Rassi et al.: Comparative Testing of … 96 http://jad.tums.ac.ir Published Online: March 31, 2021 ulation of Phlebotomus sergenti (Dip- tera: Psychodidae) to different imagi- cides in a endemic focus of cutaneous leishmaniasis in northeast of Iran. J Vector Borne Dis. 54: 282–286. 105. Tetreault GE, Zayed AEBB, Hanafi-Bojd AA, Beavers GM, Zeichner BC (2001) Susceptibility of sandflies to selected in- secticides in North Africa and the Mid- dle East. J Am Mosq Control Assoc. 17 (1): 23–27. 106. Faraj C, Ouahabi S, Adlaoui EB, El Elkohli M, Lakraa L, El Rhazi M, Ameur B (2012) Insecticide susceptibility status of Phlebotomus (Paraphlebotomus) ser- genti and Phlebotomus (Phlebotomus) papatasi in endemic foci of cutaneous leishmaniasis in Morocco. Parasite Vec- tors. 5: 51.