Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran

Ameneh Karimi; Elham Jahanifard; Mohammad Reza Abai; Yavar Rassi; Arshad Veysi; Ahmad Ali Hanafi-Bojd; Mostafa Hosseini; Zahra Saeidi; Mahdieh Tavakoli; Mehdi Maleki; Reza Jafari; Abdul Rahman Hejazi; Mohammad Reza Yaghoobi-Ershadi; Amir Ahmad Akhavan

doi:10.4103/0972-9062.310861

RESEARCH ARTICLE

Year : 2020 | Volume : 57 | Issue : 2 | Page : 121-127

Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran

Ameneh Karimi¹, Elham Jahanifard², Mohammad Reza Abai¹, Yavar Rassi¹, Arshad Veysi³, Ahmad Ali Hanafi-Bojd¹, Mostafa Hosseini⁴, Zahra Saeidi¹, Mahdieh Tavakoli⁵, Mehdi Maleki⁵, Reza Jafari⁶, Abdul Rahman Hejazi⁵, Mohammad Reza Yaghoobi-Ershadi¹, Amir Ahmad Akhavan¹
¹ Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
² Department of Medical Entomology and Vector Control, School of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
³ Zoonoses Research Center, Research Institute for Health Development, Kurdistan University of Medical sciences, Sanandaj, Iran
⁴ Department of Epidemiology and biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
⁵ Ahvaz Health Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
⁶ Esfahan Health Research Station, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Date of Submission	08-May-2018
Date of Acceptance	09-Jan-2020
Date of Web Publication	14-Jul-2021

Correspondence Address:
Dr. Amir Ahmad Akhavan
Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran
Iran
Prof. Mohammad Reza Yaghoobi-Ershadi
Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran
Iran

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-9062.310861

Abstract

Background & objectives: Cutaneous leishmaniasis (CL) is still a public health threat in many rural areas of Iran. It causes considerable morbidity among a huge number of people in the endemic areas. Khuzestan is considered as a CL focus in Iran and recently several cases have been reported from Shush County. The aim of the current study was to determine different aspects of CL epidemiology in this County.
Methods: The study was conducted in four villages in the rural district of Shush County. In order to survey the scar and acute rate of the disease, 100 inhabitants were questioned in each village. In addition, to determine the reservoir host and vector of the disease, rodents were captured using Sherman traps, and sandflies were collected from around the rodent burrows using sticky paper traps.
Results: The scar and acute lesion rates were reported as 5.26 and 0.57% among inhabitants, respectively. Fifty-four rodents including Tatera indica (55.5%), Nesokia indica (33.3%), Mus musculus (9.3%) and Rattus norvegicus (1.9%) were captured and identified. Nested PCR showed 3 out of 54 rodents infected with Leishmania major. In total, 1122 sandflies were collected, and Leishmania major detected in Phlebotomus papatasi (4.35%), Sergentomyia dentata (23%), and Sergentomyia clydei (10%).
Interpretation & conclusion: Zoonotic cutaneous leishmaniasis due to L. major prevailed in this area. The common and the main vector was Ph. papatasi and, T. indica seems to be the main while N. indica as a secondary reservoir host of the disease in this area.

Keywords: Epidemiology; Iran; leishmaniasis; southwestern Iran

How to cite this article:
Karimi A, Jahanifard E, Abai MR, Rassi Y, Veysi A, Hanafi-Bojd AA, Hosseini M, Saeidi Z, Tavakoli M, Maleki M, Jafari R, Hejazi AR, Yaghoobi-Ershadi MR, Akhavan AA. Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran. J Vector Borne Dis 2020;57:121-7

How to cite this URL:
Karimi A, Jahanifard E, Abai MR, Rassi Y, Veysi A, Hanafi-Bojd AA, Hosseini M, Saeidi Z, Tavakoli M, Maleki M, Jafari R, Hejazi AR, Yaghoobi-Ershadi MR, Akhavan AA. Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran. J Vector Borne Dis [serial online] 2020 [cited 2023 Mar 31];57:121-7. Available from: http://www.jvbd.org//text.asp?2020/57/2/121/310861

Introduction

Zoonotic cutaneous leishmaniasis (ZCL) is still a neglected tropical disease in many rural areas of Iran including 17 out of 31 provinces^[1]. It causes considerable morbidity among a huge number of people in the endemic areas, and is manifested as skin lesions followed by permanent scars and tissue deformation^[2]. Recently, the number of reported cases of cutaneous leishmaniasis (CL) increased due to several factors, including migration of non-immune individuals to the disease foci, ecological and/or biological changes of the vectors and reservoirs, reducing the use of residual insecticides to control malaria, improving the disease surveillance and reporting of positive cases, expansion of residential suburbs areas and drug resistance^[3]. Annually, around 20,000 new cases of leishmaniasis are reported in Iran; it seems that the real cases are 4–5 folds^[4]. Phlebotomus papatasi known as the main vector of ZCL and Leishmania major is the causative agent of the disease in the country. The parasite has been isolated and identified from naturally infected Ph. papatasi, Phlebotomus caucasicus, Rhombomys opimus, Meriones libycus, and humans inside the country^[4]. Rodents in Gerbillinae subfamily are known as the main reservoir hosts for ZCL in Iran^[5]. The distribution and the role of the rodents as ZCL reservoir hosts is geographically specific in Iran as in the south and the south west of the country, including Iran-Iraq border; the reservoir host is Tatera indica (Cricetidae: Gerbillinae)^[6]. Since the last two decades, several measures including spraying of rodent burrows with DDT, poisoning the rodent reservoir hosts and using deltamethrin-impregnated bed nets and curtains have been accomplished to control ZCL in the country^[7],[8],[9]. Furthermore, until now there is no licensed or registered vaccine against any form of leishmaniasis. An experience has shown a successful prevention of ZCL through leishmanization in Iran, but it has been stopped due to rare unusual form of non-healing lesions; although it is still recommended for the military personnel in high risks areas^[2].

Khuzestan is a province with political and economic importance and known as a CL focus in the country, but there is not enough information in this regard. Previously, to identify sandfly fauna and Leishmania infections, entomological studies were begun in 1962 and continued till 1971 in this province^[10].

In 1980, due to increased new cases of CL among military forces, researchers were invited to conduct an epidemiological survey to determine the reservoirs, vector(s) and, the causative agent of the disease in several areas of the province. In this survey, T. indica and Ph. papatasi were incriminated as the main reservoir host and the main vector of CL respectively, in this area^[11]. Most of the previous studies were limited to passive surveillance, and L. major has been the predominant agent in the suspected cases^[12].

In recent years, number of ZCL cases were increased and this highlighted the importance of the disease in this endemic area. Recently, most cases have been reported from Shush County (Shush Health Center, unpublished data), considered as the most important cities of the province and have several leishmaniasis foci. Based on the health authorities report, Shush County had the most cases of CL between 2006 to 2010. Due to vast ecological changes and necessity for updating the epidemiological data^[4], researchers of the current study decided to update data of ZCL epidemiology in this county. The aim of the current study was to determine different aspects of ZCL epidemiology with the aim to propose control measures for the disease in some rural areas of Shush County, Khuzestan Province, Iran.

Material & Methods

Study area

The study was accomplished in Shush County, Khuzestan province (32°11’37”N, 48°16’10”E), located in the southwest of Iran, bordering Iraq and the Persian Gulf. Khuzestan is the fifth most populated province of the country with huge reserves of oil and gas. At the 2011 census, the county’s population was 202,762 in 47,317 households^[13].

The study was conducted in four villages including Malehe, Shavoor, Abazar-Ghafari, and, Shahid-Danesh, in the rural district of Shush County [Figure 1]. The climate of the area is very hot and dry; the temperature reaches to 50°C during summer. The total annual rainfall was 215 mm, and there was no recorded rainfall during summer of 2012 (Khuzestan Meteorological Department 2012).

Figure 1: Map of studied villages in Shush County, Khuzestan province, Iran.

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Human infection

In December 2012, in order to survey the scar and acute lesion rate of the disease, 100 households were randomly questioned in each selected village. The data of each individual such as; age, gender, the presence of acute lesions or scars were recorded in the questionnaire. To prepare microscopic slides from the acute lesions, they were initially disinfected with ethanol 70%, then the edge of lesion was scraped aseptically, and transferred on the slides as well as to alcohol 96% for molecular parasite detection.

Statistical analysis

Fisher’s exact test was used to compare scar rate and/ or ulcer rate differences between male and female or age groups.

Rodents collection

In June, September, October, November and, December 2012, rodents were collected by means of 30 Sherman traps, baited with cucumber and/or bread. The traps were set before sunset and collected on the following sunrise. Captured rodents were identified using valid identification keys^[14]. After recording the morphological characteristics, impression smears were prepared from rodent ear-lobes for microscopic identification; and then, the whole ear lobes were cut, preserved in 96% ethanol, and stored in a -20°C freezer for further molecular examination.

Sandfly collection

Sandflies were collected around rodent burrows using 30 sticky traps (white papers coated with castor oil) in June, September and, October 2012. Collected sandflies were washed in absolute acetone and preserved in 70% ethanol till use. The head and the last two segments of the abdomens of sandflies were cut and mounted in Puri’s medium, and the rest of the body kept in alcohol 96%; stored in a -20°C freezer for molecular assay. Species were identified using valid morphological keys^[15].

Ethical statement

Animal experiments were approved by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Iran.

DNA extraction

Phenol-chloroform protocol was used for genomic DNA extraction from both acute lesions and rodent tissues with some modifications. Initially, 200μL of lysis buffer (100 mM Tris-HCl, pH 8; 10 mM EDTA, pH 8; 1% SDS;100 mM NaCl; 2% Triton X-100) was added to 200 μL of the disrupted tissue sample in a microtube, and then the prepared suspension was incubated at 60°C with proteinase K (100 mg/mL) for 1 h. Following, 400 μL of phenol–chloroform (1:1) was added, vortexed and, centrifuged at 2236 RCF for 5 min, and the supernatant was carefully transferred into a 1.5 mL tube. The chloroform extraction was performed two times. After that, the supernatant was transferred to a new microtube containing an equal volume of isopropanol and 1/10 volume of 3 M sodium acetate (pH 5.2), and then centrifuged at maximum speed of 15115 RCF for 10 min. The pellet was washed with 70% ethanol and centrifuged at 5000 rpm for 5 min. Following, the dried pellet was re-suspended in 30 μL of distilled water and stored at -20^oC. Genomic DNA of collected sandflies was extracted by GeneAll® Exgene^TM Tissue kit (Cat. No: 109-152), according to the manufacture’s instruction.

Polymerase chain reaction (PCR) assay

Nested PCR assay was employed to amplify targeted sequences of the Leishmania parasite DNA. External primers including Leish out F (50-AAA CTC CTC TCT GGT GCT TGC-30) and Leish out R (50-AAA CAAAGG TTG TCG GGG G-30) and internal primers including Leish in F (50-AAT TCA ACT TCG CGT TGG CC-30) and Leish in R (50-CCT CTC TTT TTT CTC TGT GC-30) were used to amplify ITS2 region of ribosomal DNA of the parasite. The initial PCR contained 0.6 μM of each forward (Leish out F) and reverse (Leish out R) external primers, 12.5 μL Taq DNA polymerase, 2X Master Mix Red (Amplicon, Denmark) and, sterile distilled water to a final volume of 25 μL. The first round of PCR condition was as follows: an initial denaturation step at 95°C for 5 min, was followed by 30 cycles of: denaturation at 94°C for 30 sec, annealing at 60°C for 45 sec and extension at 72°C for 1 min, with a final extension step of 72°C for 5 min. The second-round nested PCR was performed in a final volume of 20 μL containing 1 μL of a 1:10 dilution in distilled water of the first-round PCR product as a template DNA, 0.3 μM of each forward (Leish in F) and reverse (Leish in R) internal primers, 10 μL of Taq DNA polymerase and 2X Master mix Red. The reactions were cycled under the following conditions: 95°C for 2 min, 25 cycles of 94°C for 15 sec, 62°C for 30 sec, 72°C for 45 sec followed by 72°C for 5 min^[16].

Restriction fragment length polymorphism (RFLP)

To distinguish leishmania parasite species, restriction fragment length polymorphism (RFLP) was employed using Rapid Digest Mnl1 (Cat. No: RD 1191). PCR-RFLP was conducted in a final volume of 30 μL, containing 10μL PCR products of the nestedPCR, 2 μL Buffer, 1 μL Mnl1 and, 17μL sterile distilled water. The resulted mixture was incubated at 37°C for 1 h and then the products were loaded onto 2.5% (w/v) agarose gel electrophoresis in TBE buffer. Finally, gels were stained by ethidiumbromide (0.5 μg/mL), and photographed.

Results

Active case finding of leishmaniasis among 400 households with a total population of 1746 (890 males and 856 females) in four villages of Shush showed 5.26% scar rate and 0.57% acute lesion rate. Acute lesions were diagnosed in 10 cases (7 females and 3 males) and the highest infected age group for acute lesions was 0–4 year (1.87%). No individual with acute lesion was seen in 20–24 age group [Table 1]. Frequency of people with one, two and three lesions were 70, 10 and, 20%, respectively. Face and hands were the most affected parts of the patients’ body. Ninety-two individuals had scars, of which 65.2, 18.5, 6.5 and 9.8% of them had one, two, three and more than three scars on their bodies, respectively. Results of nested PCR experiment re-confirmed L. major infection among individual with active lesion(s) [Figure 2] and [Figure 3].

Figure 2: Nested PCR amplification of DNA extracted from sandflies, rodents and human 1: Leishmania major from Sergentomyia dentata. 2, 3 and 6: L. major from Phlebotomus papatasi. 4 and 10: 50 bp DNA ladder (Fermentas). 5: L. major from Sergentomyia clydei. 7: Leishmania major from Nesokia indica. 8 and 9: L. major from Tatera indica. 11: Negative control. 12: L. major positive control. 13 and 15: L. major from human.

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Figure 3: RFLP products of nested PCR amplicons after digestion with Mnl1. 1 and 15: 50 bp DNA ladder (Fermentas). 2: Leishmania major from Nesokia indica. 3, 5, 7, 9 and 10: Leishmania major from Phlebotomus papatasi. 4, 8 and 11: Leishmania major from Sergentomyia dentata. 6: Leishmania major from Sergentomyia clydei. 12 and 13: Leishmania major from Tatera indica 14: Leishmania major from human.

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Table 1: The scar rate and ulcer rate among the households in the studied villages, Shush, Khuzestan Province, Iran in 2012

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Fifty-four rodents were captured and four species including Tatera indica (55.5%), Nesokia indica (33.3%), Mus musculus (9.3%) and, Rattus norvegicus (1.9%) were identified. For detection and identification of Leishmania infection, captured rodents were examined both by microscopic and molecular diagnostic techniques. Direct examination revealed no positive infection in all examined rodents, although nested PCR showed that 3 out of 54 rodents were infected with L. major (6.7% of T. indica and 5.5% of N. indica) [Table 2] and [Figure 2]. The most collected species in some rural areas was N. indica, and taken together, T. indica was the dominant species in the studied villages.

Table 2: Natural Leishmania infection rate among captured rodents by nested PCR in the rural areas of Shush, Khuzestan Province, Iran in 2012

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During June, September, and October 2012, a total of 1122 sandflies were collected from rodents burrows and the following species including Ph. papatasi 52%, Ph. alexandri 0.4% belonging to Phlebotomus genus and Se. sintoni 31.6%, Se. dentata 9.5%, Se. iranica 0.3%, Se. tiberiadis 1.4%, Se. theodori 0.6%, Se. squamipleuris 0.5%, Se. clydei 3.6% and Se. christophersi 0.1% belonging to Sergentomiya genus, were identified. The common and dominant species was Ph. papatasi, and the sex ratio for this species (number of males per 100 females) was calculated at 389.

To detect Leishmania infection, all of the Ph. papatasi females and some species more frequently, including Se. sintoni, Se. clydei, Se. dentata and Se. clydei were examined by nested-PCR. The results of nested PCR showed 4.35, 23 and 10% L. major infection in Ph. papatasi, Se. dentata and Se. clydei, respectively. [Table 3] and [Figure 2].

Table 3: Natural Leishmania major infection in sandflies collected from rodent burrows, Shush rural district, Khuzestan Province, 2012

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Results of RFLP test re-confirmed L. major infection in all of the infected samples [Figure 3]. Surprisingly, results of the sequencing revealed that amplified region of the parasites’ DNA from sandflies was 12 nucleotide shorter, in comparison to amplified DNA extracted from rodents and human samples [Figure 4].

Figure 4: Comparing the aligned-sequences of partial ITS2 region of parasite ribosomal DNA, extracted from sandflies, rodents and human samples (1, 3, 5, 6, 9 and 10) to the sequences deposited in gene bank using ClustalW software.

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Discussion

In recent years, due to increasing Leishmaniasis cases, researchers have focused to identify different aspects of CL epidemiology in some rural areas of Shush in Khuzestan Province. Active case finding survey showed a background of Leishmania infection in all age groups. Fishers exact test showed significant statistical differences in the group with over “15-year old” with “under-15 year old” regarding acute lesions (P=0.003). This indicates that most of the new ZCL cases occurred in less than 15-yr old group. Nevertheless, no significant difference was seen in the scar rates of these two groups. In both men and women, there was no significant difference in the scar rate. Based on these findings, it seems that we are facing an increasing trend of ZCL cases in the region. Similar to the results of the present study, researchers have previously introduced L. major as the dominant parasite that causes ZCL in the region^[12].

Based on the results of the current study, T. indica is a predominant species in six villages and Nesokia indica is ranked in the second place. Furthermore, L. major infection was detected in T. indica (6.7%) and N. indica (5.5%). During 1987–1995, investigations accomplished in Ilam and Khuzestan showed 12.5 and 9% Leishmania infection in T. indica, respectively^[6],[17]. Predominant rodent species in these provinces was T. indica and its infection rate for L. major in Ilam and Khuzestan was calculated to be at 21 and 27% in 2013, consequently^[18].

Nested PCR showed 8.3% L. major infection in T. indica in Rofae of Khuzestan and recently 17% infection rate was reported for this species in Khuzestan as well^[19]. In southeastern foci of ZCL in the country, N. indica beside T. indica consider as a secondary reservoir of ZCL^[17]. Another study in Khuzestan showed 17% infection rate to Leishmania for N. indica^[18].

In the current study, 10 sandfly species are identified and Sergentomyia christophersi reported for the first time in the region while pervious entomological study reported 15 species of sandflies from Shush County^[20]. Phlebotomus papatasi was the common and dominant species and L. major infection of this species was identified by using nested PCR. Natural Leishmania infection in Ph. papatasi has also been reported from other ZCL foci such as Sabzevar^[21], Shahrood^[22] and similar to the current study, L. major infection has been reported from Orzoieh^[23] and Sarakhs^[24] using nested PCR.

During this study, only 2 male specimens of Ph. al-exandri were collected from some villages and it is unexpected to incriminate this species as a main vector of the disease in this area. However, previous study reported natural Leishmania infantum and L. major infection in Ph. alexandri from Fars province^[25] and Sarakhs^[24], respectively. The competency of Ph. alexandri to transmit either visceral or cutaneous leishmaniasis is expected but it needs further investigation.

The current study reports 4.35% L. major infection rate in Ph. papatasi species using molecular technique. Previous studies in northwestern Khuzestan showed Leishmania infection rate in Ph. papatasi, Ph. alexandri and Se. sintoni, as 1.9, 2.3 and 2.2% through direct examination, respectively^[10]; the highest Ph. papatasi infection rate (37.8%) was observed during epidemic situation in Badrood, Esfahan Province^[26].

Sandflies belonging to Sergentomyia genus including Se. dentata, Se. clydei and Se. sintoni were tested to find trace of parasite infection using PCR technique. Leishmania major infection rate was reported as 23 and 10% in Se. dentata and Se. clydei respectively. Other studies have confirmed Leishmania infections in some species of Sergentomiya genus including: Se. darlingi, Se. babu, Se. garnhami, Se. sintoni, Se. minuta and Se. gemmea in Mali, India, Kenya, Iran, Portugal and Thailand respectively^{[27],[28],[29],[30]}. These consecutive reports of infection by Leishmania parasites among Sergentomiya genus raise concerns about the possibility of this group of sandflies in leishmaniasis transmission among human population in the diseases foci.

In the current study all of the Leishmania infections were identified as L. major. Nevertheless, there were extra 12 nucleotides in amplified sequences of Leishmania DNA isolated from human and rodents, comparing to sand fly isolates. Observation of the sequences in the Gene Bank database revealed that the difference was also seen in some isolated parasites samples from human lesions. This could be due to the presence of at least two haplotype of the parasite in the study area. Further study is needed to confirm this hypothesis.

Conclusion

It is concluded that the Cutaneous Leishmaniasis due to L. major prevails in Shush County, Khuzestan province, Iran; the main vector of the disease is Ph. papatasi, also, T. indica and N. indica act as primary and secondary reservoir hosts of CL, respectively. Regarding the epidemiological cycle of the disease, rodent control operation could be an effective measure to control ZCL in this foci^[8],[9],[31], integrated with other measures such as impregnated bed nets and curtains and health education^[7],[32].

Conflict of interest: None

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