J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 Original Article The Emergence of Anthroponotic Cutaneous Leishmaniasis Following the Earthquake in Southern Villages of Bam District, Southeastern Iran, 2010 Mohammad Reza Aflatoonian 1, *Iraj Sharifi 1, Somayeh Poursmaelian 1, Maryam Hakimi-Parizi 1, Nasser Ziaali 2 1Leishmaniasis Research Centre, Kerman University of Medical Sciences, Kerman, Iran 2Department of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran (Received 16 Mar 2011; accepted 15 Apr 2012) Abstract Background: The objective of this study was to assess the epidemiological characteristics of a new emerging focus of cutaneous leishmaniasis (CL) in southern villages of Bam District, southeastern Iran, 2010. Methods: A house-to- house census survey of 5544 individuals were interviewed and physically examined for the presence of active lesions or scars. Diagnosis was confirmed by direct smears, cultures and identification by PCR. The data were entered into a computer and SPSS ver. 15. Results: Overall, 1.2% of the inhabitants were infected, 0.5% active and 0.7% scars and females were more signifi- cantly infected (1.7%) than males (0.8%), (P= 0.003). All age groups were equally affected. Most of the lesions were on the face and majority had single lesion. Most of the cases appeared from 2006 to 2008 during the CL epidemic in the city of Bam. PCR indicated L. tropica as the causative agent. Conclusion: The presence of non-immune individuals along with suitable ecological conditions could induce a new emerging focus of ACL in villages. Keywords: Anthroponotic cutaneous leishmaniasis, Leishmania tropica, Emergence, Iran Introduction Leishmaniasis consists of a spectrum of clinical manifestation ranging from simple self-limiting cutaneous lesions to disfiguring and fatal visceral forms (Postigo 2010). The disease endemicity extends to over 88 coun- tries, but its public health impact remains grossly neglected. The overall prevalence is estimated 12 million, with 2 million new cas- es occur annually and the population at risk is 350 million. Cutaneous leishmaniasis (CL) comprises 1.5 million new cases each year and 90% are limited to 8 countries including Af- ghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru (WHO 2009). Two epidemiological forms are present in Iran, anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is a well- known disease in Bam (Nadim and Aflatoonian 1995) where field trials of killed L. major against L. tropica have recently been conduct- ed (Sharifi et al. 1998b). In Bam district, most of the cases are confined to the city, the reservoir is human and the main vector is Phlebotomus sergenti. Zoonotic CL (ZCL) due to L. major presents in a frequency of 5 to 10% where gerbils are the main reservoir and P. papatasi, the vector (Nadim and Aflatoonian 1995, Sharifi et al. 1997, Aghasi and Sharifi, 2003, Oshaghi et al. 2008), although, ZCL cases have been reported predominantly from southern and central Iran (Akhavan et al. 2007, Rassi et al. 2008, Parvizi et al. 2010, Yaghoobi-Ershadi et al. 2010). Epidemics of CL have been occurred af- ter the earth quake of December 2003, where the number of recorded cases increased to *Corresponding author: Prof Iraj Sharifi, E-mail: iraj.sharifi@yahoo.com 8 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 almost 4- folds two years following the earth quake (Sharifi et al. 2011). Several risk fac- tors such as human behaviors, ecological dis- turbances, environmental changes, and popu- lation displacement, due to natural disasters and human intervention influence on the emer- gence and spread of ACL (Ashford 2000, Patz et al. 2000, Desjeux 2001). The incidence of CL cases has been sig- nificantly increased. Simultaneously, local health authorities detected CL cases in these villages of Bam district. The selection of the- ses villages was based on the newly reported CL cases by passive case detection in early 2010. There has not previously been report of any case of CL from these areas. During June to August in 2010 the CL cases have been occurred and a survey team was appointed to carry out an epidemiological investigation and to identify the causative agent. The objective of this study was to assess the magnitude of a newly emerged focus in southern villages of Bam District, Kerman Province, southeastern Iran. Due to different strategic measures between ACL and ZCL this study is highly needed for planning a suitable future control method. Materials and Methods Study area This study was carried out in Nezamshahr County, 50 km south to the city of Bam, the center of Bam District, Kerman Province, southeastern Iran. The altitude is 1032 meters above sea level and the main crops are palm trees, orchard of oranges and alfalfa. The coun- ty is hot in summer (40–45 ○C) and rather mod- erate in winter (15–25 ○C). The yearly rainfall is about 40mm with relative humidity of 25%. Sampling and population This work was carried out as descriptive cross-sectional study from June to August 2010. A census survey of 5544 individuals was carried out by house-to-house visits. Diagnosis and culturing A list of households was obtained from the Bam Health Center. A team including an experienced physician, two health assistants and a driver performed interviewing and phys- ical examination. A whole body was examined for the presence of active lesions or scars. Suspected active lesions were scraped with a sterile blade. A questionnaire was completed for each individual, recording age, sex, the lo- cation and number of the CL lesions, place of residence and the year of contraction. The sam- ples smeared on to glass slides, fixed in meth- anol, stained using Giemsa and examined by a light microscope for presence of amastigotes. At the same time, samples from 15 patients were inoculated into Novy–MacNeal–Nicolle (NNN) culture media, transported on ice to Kerman and Leishmaniasis Research Centre for further characterization of the isolates by PCR. The culture media were maintained at 24±1 ○C for 7days, then transferred into RPMI 1640 medium (Gibco, UK) containing 15% heat inactivated fetal calf serum (FCS), penicil- lin (200 units/ml) and streptomycin (200 mg/ ml) and incubated at 24±1 ○C. Cultures were checked weekly for the growth of promas- tigotes for a period of 4 weeks. Of 26 samples we could extract DNA from 15 isolates. The remaining 11 samples were ei- ther culture negative or lost their viability thro- ugh being transferred from the rural health clinic to School of Medicine in Kerman. Alt- hough, the direct smear preparations were available, no attempt was made to scrape and extract DNA from these smears, since we in- tended to identify only a representative sample. Oral consent of the inhabitants was ob- tained. The infected subjects received proper medication free of charge. The data were en- tered into a computer and SPSS ver. 15 and χ2- test were used to determine any signifi- cant difference between disease prevalence and demographic characteristics. Statistical signif- icance was at P< 0.05. 9 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 Molecular characterization DNA was prepared from 15 randomly se- lected clinical isolates. The extraction proto- col followed relatively the method described elsewhere (Mahboudi et al. 2001). A pair of pri- mers, upstream (5'TCGCAGAACGCCCCTAC C3') and downstream (5'AGGGGTTGGTGT AAAATAGGC3') specific for conserved se- quences of kDNA of Leishmania species was used. The differentiation was based on the size of the products. Two Leishmania species pro- vided amplified fragments of about 800 bp for L. tropica and 620 bp for L. major. L. tropica (MHOM/IR/02/Mash2) and L. major (MRHO/ IR/75/ER), kindly provided from Tehran Uni- versity of Medical Sciences were used as posi- tive and distilled water as negative controls. The PCR was carried out applying a step- up program as follows, initial denaturation for 3 min at 95 ○C, followed by 30 cycles of 94 ○C for 1 min, 62 ○C for 1 min at 72 ○C, with a final extension for 7 min at 72 ○C. Results A total of 5544 inhabitants aged 1–96 years (mean, 26.3 years, SD±18.3), comprising 2647 females (47.8%) and 2897 males (52.2%) were interviewed and physically examined for pres- ence of active lesions or scars (Table1). In both sexes most of the individuals (24.4%) were in the age group 21–30 years and the lowest (12.5%) in 31–40 years. Overall, 1.2% of the population had le- sions, 0.5% active and 0.7% scars (Table 2). There was a significant difference between females (1.7%) and males (0.8%) in terms of the prevalence of active lesions and scars (P< 0.005). The age distribution of the CL in- fection is presented in Fig. 1. All age groups were affected, although the number of af- fected subjects was generally low and some increase was observed in the age group 11-20 years, though with no significant difference. Most of the lesions were on the face (37%) fol- lowed by legs (31%), hands (27%) and others (5.0%). The majority had one lesion (88%), and 6% had 2 or ≥3 lesions each. The aver- age number of lesions was 1.2, equally dis- tributed among the sex. The households had frequent history of travelling to the city of Bam and the lesions were merely restricted to the endogenous inhabitants in this locality. There are 4 main villages in this county; Taraz was the most infected (29.3%), followed by Nezamabad (28.4%), Bagh-e-Balla (25.5%) and Momenabad (16.8%). The first case started as sporadic during the winter of 2005, how- ever, the cases increased significantly, there after. Random selection of 15 isolates by PCR method indicated L. tropica the sole causa- tive species (Fig. 2). Table 1. Distribution by sex and age of 5544 inhabitants examined for cutaneous leishmaniasis in southern villages of Bam, southeastern Iran, 2010 Age (yr) Female Male Total No. Percent No. Percent No. Percent ≤10 565 21.3 592 20.4 1157 20.9 11-20 586 22.1 630 21.7 1216 21.9 21-30 615 23.2 737 25.5 1352 24.4 31-40 325 12.3 370 12.8 695 12.5 >40 556 21.1 568 19.6 1124 20.3 Total 2647 100.0 2897 100.0 5544 100.0 10 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 Table 2. Distribution of anthroponotic cutaneous leishmaniasis by sex and type of lesion in southern villages of Bam, southeastern Iran, 2010 Active lesion Scar Sex No. / Percent No. (Percent) No. (Percent) Female 44/1.7 16(0.6) 28(1.1) Male 23/0.8 10(0.3) 13(0.5) Total 67/1.2 26(0.5) 41(0.7) Fig. 1. Distribution of anthroponotic cutaneous leishmaniasis by age in southern villages of Bam, southeastern Iran, 2010 Fig. 2. Gel electrophoresis of the Leishmania species- specific PCR products using kDNA marker extracted from promastigotes grown in culture media. Lane 1: 100bp ladder marker, Lane2: negative control, Lane3: Leishmania major positive control, Lane4: Leishmania tropica positive control, Lane 5, 6, 7, 8, 9: Leishmania tropica isolates obtained from the cases with anthroponotic cutaneous leishmaniasis in southern villages of Bam, southeastern Iran 11 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 Discussion Cutaneous leishmaniasis is still a major health problem in Iran, where mainly non- healing forms of leishmaniasis recidivans and non-responsive clinical forms to pentavalent animony drugs are present (Esfandiarpour et al. 2007, Sharifi et al. 2010). The disease has a dynamic epidemiology with a complex life cycle and diverse transmission pattern. It can change under the influence of climate, ecology, human behavior, presence of suitable vectors and reservoirs (Ashford 2000, Patz et al. 2000, Desjeux 2001). These factors singly or in com- bination have been accompanied by global in- creases in morbidity and mortality from emer- gent Leishmania species (Patz et al. 2000, Desjeux 2001). The prevalence data showed that all age groups were affected, which indicated that this emergence was a new occurrence among a non-immune population. In addition the data suggest that females were more than twice susceptible (odd ratio= 2.1) to the infection than males. The households, frequently trav- el to the city of Bam, where they contract the infection, however, there is no clear explana- tion for such a sex distribution. It might be due to individual risk factors and more expo- sure of females to the source of infection (Desjeux 2001, Fazaeli et al. 2009). The clinical features of the CL cases in terms of the number and the location of the le- sions observed in this study are consistent with those of ACL reported previously from the city of Bam (Nadim and Aflatoonian 1995, Sharifi et al. 1998a, Aflatoonian and Sharifi 2006) or elsewhere (Zahraei-Ramazani et al. 2007). More over molecular characterization of the PCR of a number of isolates proved that L. tropica was responsible for this emergence. The devastating earthquake of Bam in December 2003 demolished almost 90% of the health infrastructures, sacrificed 30000 and left million tons of construction and raw bricks around the houses. Although, the health conditions and infrastructures have been con- siderably improved, but various risk factors are still around, creating a suitable condition for vector breeding and transmission of the caus- ative agent. In spite of remarkable efforts and implementation of various approaches to re- duce transmission and control the disease un- fortunately, the CL cases have been increased to epidemic proportion after the earthquake es- pecially during 2006–2008 in the city of Bam. In Bam district, CL control has been inte- grated with that of other infectious diseases. The disease has not been reported from this county before. Occupation is mainly restrict- ed to farming and agricultural activities. Move- ment of the villagers, both men and women back and forth to the city of Bam, more fre- quently after the earth quake, where the dis- ease is highly endemic, for various purposes is common. Therefore, due to presence of a suitable anthropophyllic vector (Aghasi and Sharifi 2003), the villagers become a highly sensitive reservoir for transmission of ACL endemic to their former home grounds. The current expansion of villages and ur- banization constitute other contributory fac- tors (Patz et al. 2000, Desjeux 2001). Similar risk factors have contributed to the emergence of new ACL foci in Sudan (El-Safi and Pe- ters 1991), Morrocco (Guessous-Idrissi et al. 1997, Ramaoui et al. 2008), Israel (Jacobson et al. 2003), Pakistan (Kolczinski et al. 2004) and new ZCL foci in Iran and abroad (Ashford et al. 2000, Yaghoobi- Ershadi et al. 2001, Fazaeli et al. 2009, Razmjou et al. 2009, Sharifi et al. 2011). In conclusion, the presence of non-immune population along with suitable ecological con- ditions could induce a new emerging focus of ACL for human, beyond the original focus. Further epidemiological studies are required to identify the main vectors and strains of the Leishmania species involved in this focus. 12 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 Acknowledgements This work was approved by the Vice Chan- cellor for Research and received financial sup- port from the Kerman University of Medical Sciences and Health Services (Project no. 89/24). We are grateful to the health-worker team in Bam Health System for their help in carrying out this study. The authors declare that there is no conflict of interest. References Aflatoonian MR, Sharifi I (2006) Prevalence of cutaneous leishmaniasis in primary school children in Bam District, 2005. J Kerman Univ Med Sci. 14: 82–84. Aghasi M, Sharifi I (2003) Survey of the fauna and monthly activity of the sand fly as the vector of the cutaneous leish- maniasis in the city of Bam. J Kerman Univ Med Sci. 10(2): 85–91. Akhavan AA, Yaghoobi-Ershadi MR, Hasibi F, Jafari R, Abdoli H, Arandian MH, Soleimani H, Zahraei-Ramazani AR, Mohebali M, Hajjaran H (2007) Emer- gence of cutaneous leishmaniasis due to Leishmania major in a new focus of southern Iran. Iran J Arthropod-Borne Dis. 1: 1–8. Ashford RW (2000) The leishmaniasis as emerging and reemerging zoonoses. Int J Parasit. 30: 1269–1281. Desjeux P (2001) The increase in risk factors for the leishmaniasis worldwide. Tran Roy Soc Trop Med Hyg. 95: 239–243. El-Safi SH, Peters W (1991) Studies on the leishmaniasis in the Sudan. 1. Epidemic of cutaneous leishmaniasis in Khartoum. Tran Roy Soc Trop Med Hyg. 85: 44–47. Esfandiarpour I, Dabiri SH (2007) Treatment of cutaneous leishmaniasis recidivans with a combination of allopurinol and meglumine antimoniate: a clin ical and histologic study. Int J Dermatol 46: 848–852. Fazaeli A, Fouladi B, Sharifi I (2009) Emer- gence of cutaneous leishmaniasis in a border area at south-east of Iran: an ep- idemiological survey. J Vector Borne Dis. 46: 36–42. Guessous-Idrissi N, Hamdani A, Rhalem A, Riyad M, Sahibi H, Dehbi F, Bichichi M, Essari A, Berrag B (1997) Epidemiol- ogy of human visceral leishmaniasis in Taounate, a northern province of Mo- rocco. Parasite. 4(2): 181–185. Jacobson RL, Eisenberger CL, Svobodova M, Banesh G, Sztern J, Carvalho J, Na- sereddin A, El Fari M, Shalom U, Volf P, Votypka J, Dedet JP, Pratlong F, Scho- nian G, Schnur LF, Jaff CL, Warburg A (2003) Outbreak of cutaneous leish- maniasis in northern Israel. J Infect Dis. 188: 1065–1074. Kolaczinski J, Brooker S, Reyburn H, Rowland M (2004) Epidemiology of anthroponotic cutaneous leishmaniasis in Afghan refu- gee camps in northwest Pakistan. Tran Roy Soc Trop Med Hyg .98: 373–378. Mahboudi F, Abolhassan M, Yaran M, Mob- taker H, Azizi M (2001) Identification and differentiation of Iranianian Leish- mania species by PCR amplification of kDNA. Scand J Infect Dis. 33: 596–598. Nadim A, Aflatoonian MR (1995) Anth- roponotic cutaneous leishmaniasis in Bam, southeast Iran. Iranian J Publ Health. 24: 15–24. Oshaghi MA, Yaghoobi-Ershadi MR, Abbassi M, Parvizi P, Akhavan AA, Rahimi Fo- roshani A , Zahraei AR, Rassi Y, Moh- tarami F (2008) Detection of Leish- mania major in naturally infected sand flies using semi nested-PCR. Iranian J Publ Health. 37(4): 59–64. Parvizi P, Baghban N, Novin EA, Absavaran A (2010) Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in Cen- tral of Iran. Exp Parasitol. 124(2): 232– 13 J Arthropod-Borne Dis, June 2013, 7(1): 8–14 MR Aflatoonian et al.: The Emergence of … http://jad.tums.ac.ir Published Online: June 10, 2013 237. Patz JA, Graczyk TK, Geller N, Vittor AY (2000) Effects of environmental change on emerging parasitic diseases. Inter J Parasitol. 30: 1395–1405. Postigo JA (2010) Leishmaniasis in the World Health Organization Eastern Mediterra- nean Region. Int J Antimicrob Agents. 36(1): 62–65. Ramaoui K, Guernaoui S, Boumezzough A (2008) Entomological and epidemio- logical study of a new focus of cutane- ous leishmaniasis in Morocco. Parasitol Res. 103: 859–863. Rassi Y, Abai MR, Javadian E, Rafizadeh S, Imamian H, Mohebali M, Fateh M, Haj- jaran H, Ismaili K (2008) Molecular data on vectors and reservoir hosts of zoono- tic cutaneous leishmaniasis in central Iran. Bull Soc Path Exot. 101: 425–428. Razmjou S, Hejazy H, Motazedian MH, Ba- ghaei M, Emami M, Kalantary M (2009) A new focus of zoonotic cutaneous leishmaniasis in Shiraz, Iran. Tran Roy Soc Trop Med Hyg. 103: 727–730. Sharifi I, Ardehali S, Motazadian H, Afla- toonian MR (1997) Identification and characterization of Leishmania isolates in school children in Bam, southeast- ern Iran. Iran J Med Sci. 22: 82–88. Sharifi I, Fekri AR, Aflatoonian MR, Nadim A, Nikian Y, Khamesipour A (1998a) Cu- taneous leishmaniasis in primary school children in the southeastern Iranian city of Bam, 1994–1995. Bull WHO. 76: 289–293. Sharifi I, Fekri AR, Aflatoonian MR, Khame- sipour A, Nadim A, Ahmadi Mousavi MR, Momeni A, Dowlati Y, Godal T, Zicker F, Smith GP (1998b) Randomized vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet. 351: 1540–1544. Sharifi I, Fekri AR, Aflatoonian MR, Khame- sipour A, Mahboudi F, Dowlati Y, Nadim A, Modabber F (2010) Leish- maniasis recidivans among school chil- dren in Bam, South-east Iran, 1994– 2006. Intern J Dermatol. 49: 557–561. Sharifi I, Nakhaei N, Aflatoonian MR, Hakimi Parizi M, Fekri AR, Safizadeh H, Shir- zadi MR, Goya MM, Khamesipour A (2011) Cutaneous leishmaniasis in Bam: A comparative evaluation of pre- and post- earthquake years (1999–2008). Iranian J Publ Health. 40: 49–56. 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 leishmaniasis due to Leishmania tropica in rural communities of Bam district after the earthquake, Iran. Trop Med Intern Health.16 (4): 510–513. World Health Organization (WHO) (2009) Leishmaniasis: background information. A brief history of the disease. Available at: www.who.int /leishmaniasis/en/ Yaghoobi-Erashadi MR, Hanafi-Bojd AA, Akhavan AA, Zahrai Ramazani AR, Mohebali M (2001) Epidemiological study in a new focus of cutaneous leish- maniasis due to Leishmaniasis major in Ardestan town, central Iran. Acta Trop. 79: 115–121. Yaghoobi-Ershadi MR, Hakimiparizi M, Zah- raei-Ramazani AR, Abdoli H, Akhavan AA, Aghasi M, Arandian MH, Ranjbar AA (2010) Sand fly surveillance within an emerging epidemic focus of cutaneous leishmaniasis in southeastern Iran. Iran J Arthropod-Borne Dis. 4: 17–23. Zahraei-Ramazani AR, Yaghoobi-Ershadi MR, Mokhtari AR, Akhavan AA, Abdoli H, Arandian MH (2007) Anthroponotic cu- taneous Leishmaniasis in nonendemic quarters of a central city in Iran. Iranian J Publ Health. 36: 7–11. 14