|Year : 2018 | Volume
| Issue : 3 | Page : 72-75
Epidemiological and clinical study on the cutaneous leishmaniasis in Aran and Bidgol, center of Iran
Sima Rasti1, Mahdi Delavari1, Tayebeh Sadat Tekieh Arani2, Seyed Gholam Abbas Mousavi3
1 Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran
2 PHC Center of Aran and Bidgol, Kashan University of Medical Sciences, Kashan, Iran
3 Department of Statistics and Public Health, Faculty of Health, Kashan University of Medical Sciences, Kashan, Iran
|Date of Web Publication||5-Sep-2018|
Dr. Mahdi Delavari
Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan
Source of Support: None, Conflict of Interest: None
Aims: Cutaneous leishmaniasis (CL) is a skin infection that causes various forms of ulcers and also remains scars even after treatment. This disease is prevalent in many countries of Middle East including Iran. Since determining the species of the parasite is important for prevention and control programs, this study was conducted to identify Leishmania species in Aran and Bidgol, Isfahan province, center of Iran.
Materials and Methods: This cross-sectional study was carried out on 112 CL suspected patients who referred to health centers of Aran and Bidgol. Serosity of the wound was collected, and amastigote form was detected by microscopic method. After extraction of DNA from serosity, kDNA-polymerase chain reaction (PCR) was used to identify Leishmania species.
Results: Fifty-four of all suspected CL samples (48.2%) were positive microscopically, while 55 (49.1%) were positive using kDNA-PCR. The results of PCR revealed that 51 isolates (92.7%) were Leishmania major and 4 (7.3%) Leishmania tropica, respectively. The most lesion form caused by L. major was papular or volcanic-like, while all of wounds caused by L. tropica were papular/nodular forms.
Conclusion: The results of the study indicated that the predominant species was L. major and zoonotic CL is more prevalent in this region.
Keywords: Aran and Bidgol, cutaneous leishmaniasis, kDNA
|How to cite this article:|
Rasti S, Delavari M, Arani TS, Mousavi SG. Epidemiological and clinical study on the cutaneous leishmaniasis in Aran and Bidgol, center of Iran. Int Arch Health Sci 2018;5:72-5
|How to cite this URL:|
Rasti S, Delavari M, Arani TS, Mousavi SG. Epidemiological and clinical study on the cutaneous leishmaniasis in Aran and Bidgol, center of Iran. Int Arch Health Sci [serial online] 2018 [cited 2019 Mar 26];5:72-5. Available from: http://www.iahs.kaums.ac.ir/text.asp?2018/5/3/72/240627
| Introduction|| |
Leishmaniasis, as vector-borne disease has global prevalence. Despite scientific advances, various clinical manifestations of this disease have great importance in public health.,, Leishmaniasis is known as a neglected infectious disease and has been reported from almost 100 countries. The cutaneous leishmaniasis (CL) is the most common form of disease. Zoonotic CL (ZCL) and anthroponotic CL (ACL), which caused by Leishmania major and L. tropica, respectively, are the different forms of disease. Of all reported cases, more than 70% have been reported from Brazil, Costa Rica, Afghanistan, Ethiopia, the Islamic Republic of Iran, Peru, Algeria, Sudan, Colombia, and the Syrian Arab Republic. The rate of CL varies in different parts of Iran from 1.8% to 37.9%, the annual incidence of the disease in Iran is about 20,000 per year.,,, Fars, Isfahan, and Khuzestan provinces are the most important endemic area of ZCL in Iran.,,, Mashhad, Bam, and Khoram Abad city are the endemic area of anthroponotic CL in Iran.,,. Both forms of CL is reported in Kashan. Aran-o-Bidgol is situated in northeast part of Kashan city in Isfahan province, center of Iran. Mosquitoes Phlebotominae (sand fly) are known as vector of parasite. Rodents are a major reservoir of this disease., Leishmania diagnosis depends on clinical observations, region epidemiology, and laboratory methods. Direct smear is known as first and common method for the diagnosis of CL., Due to the accuracy and sensitivity of molecular techniques, they are the best diagnostic methods for detection of CL. In different studies, molecular methods, such as polymerase chain reaction (PCR), random amplified polymorphic DNA-PCR, restriction fragment length polymorphism, nested PCR, real-time PCR, and DNA sequencing have been used to diagnosis of leishmania.,,, Different molecular targets, including internal transcribed spacer1 (ITS1), ITS2, gp63 (63-kd glycoprotein), mini-exon, kDNA, small subunit ribosomal RNA genes have been used for the identification of parasites from various samples.,,,,, Although there is no standard target molecule for the diagnosis of leishmania, some researchers due to multiple copies per parasite have introduced kDNA gene as most sensitive molecules for diagnostic purposes., In the Aran-o-Bidgol region, reservoir and main vector of disease are Rhombomys opimus and Phlebotomus papatasi., The aim of this study was to evaluate the epidemiological and clinical situation of the CL in the Aran-o-Bidgol region.
| Materials and Methods|| |
This cross–sectional study was carried out on 112 patients suspected toCL, referred to Aran and Bidgol healthcare center, Isfahan province, Iran, during January 2014 until September 2015. The demographic, symptoms, and signs of patients were recorded in questioner's forms by interview. Skin scrapings from the ridge lesions were taken for microscopic study; furthermore, serosity added in 1.5 microtubes containing 0.5 ml of sterilized normal saline and stored in −20°C for DNA extraction. Slides were stained using Giemsa. All slides were viewed under oil immersion for confirming of amastigotes inside or outside macrophages.
The genomic DNA of samples was extracted by specific Kit (Bioneer; South Korea) according to the manufacturer's instruction and stored in −20°C.
Polymerase chain reaction amplification
The PCR was performed on 112 DNA samples. Species-specific primers, LINR4 forward (GGG GTT GGT GTA AAA TAGGG), and LIN17 reverse (TTT GAA CGG GAT TTC TG) were used by previous study to kDNA gene amplification. The expected bands were 650 bp and 760 bp for L. major and L. tropica, respectively. PCR was carried out in a 20 μl reaction mixture including 1.5 mM MgCl2 (Amplicon, Denmark), 10 pMol LINR4, 10 pMol LIN17, and 100 pg DNA (2 μl). The PCR cycling program was 95°C for 5 min, followed by 30 cycles of 94°C for 30 ss, 52°C for 30 s, and 72°C for 40 seconds, and then, a final extension at 72°C for 5 min in a thermocycler (Analytik Jena's FlexCycler2). Reference strains L. tropica (MHOM/IR/89/AR2) and L. major (MHOM/IR/54/LV39) and DW instead of DNA template included in all PCR runs as positive and negative controls, respectively. PCR products were analyzed on 1.2% agarose gel by electrophoresis and visualized by ultraviolet transilluminator after stained with 0.5 μg/mL ethidium bromide. Diagnosis of CL was based on microscopic and kDNA PCR results.
All the data were recorded in SPSS software version 16.5 (SPSS Inc., Chicago, IL, USA) and were analyzed using the Chi-square and Exact significant Chi-squared test.
| Results|| |
Out of 112 suspected CL cases, 48.2% (54/112) were positive by microscopic method. The overall infection rate was 49.1% (55/112) by amplification of Leishmania using kDNA primers [Table 1] and [Figure 1]. One sample was negative microscopically, but was positive by PCR. Leishmania species were identified in 51 isolates (92.7%) as L. major, followed by L. tropica in 4 isolates (7.3%), respectively [Figure 1] and [Table 1]. The rate of infection in female and male were 36.4% (20/55) and 63.6% (35/55), respectively (P = 0.88). The mean age of the patients was 40.3 ± 21.6. The highest rate of positive PCR (40%) was observed in age group 20–39 years, but no positive cases were seen in more than 80 age groups (P = 0.21). The rate of infection peaked in 2014, and was diminished in 2015. From the 55 positive PCR, the highest rate and the lowest rate of CL 47.3% (26/55) and 3.6% (2/55) were seen in individuals with elementary and college education, respectively (P = 0.6). The main characteristics of CL according to Leishmania species were shown in [Table 1].
|Table 1: The main characteristics of cutaneous leishmaniasis according to Leishmania species|
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|Figure 1: Electrophoretic patterns of KDNA-PCR products of Leishmania species in cutaneous leishmaniasis patients. Lanes 1–5 L. major, Lane 6: ladder markers 50 bp, Lanes 7–11: L. major, Lane 12: L. tropica, Lane13: Reference strains of L. major(650 bp), Lane 14: Reference strains of L. tropica(760 bp). Lane 15: Negative control. PCR: Polymerase chain reaction, L. major: Leishmania major, L. tropica: Leishmania tropica|
Click here to view
In regard to the form of CL lesion, out of 51 L.major, the most form was papular or volcanic-like and the lowest rate 3.9% ulcer, but in L. tropica, all of the four isolates were papular/nodular [Table 1] and [Figure 2] (P = 0.34). Distribution of CL according to season was shown in [Table 2].
|Figure 2: Some clinical forms in patients with cutaneous leishmaniasis. (a and b) Ulcer, (c) sporotrichoid|
Click here to view
| Discussion|| |
Leishmaniasis prevention and control in endemic areas are dependent on several factors, which determination of the dominant causal agent is one of the most important factors. The finding of this study revealed that the prevalent species of Leishmania was L. major, and this region is a ZCL focus. The previous epidemiological finding on vector and reservoirs indicated that CL in Aran and Bidgol was mainly zoonotic diseases., The results of the previous study in Kashan revealed that from 130 patients suspected for CL 71.4% and 26.6% were infected with L. tropica and L. major, respectively. The results of another study showed L. major was the main causative agent in Isfahan region. Based on the results of the previous study, the dominant species in Natanz and Isfahan and Ahvaz were L. major., The result of a study in Mashhad using ITS-PCR showed that L. tropica is predominant agent of CL.
Molecular method is suitable for CL accuracy diagnosis, as well as for species characterization, fast treatment and monitoring of relapses. The small number of parasites in the wound of ZCL may cause false results; however, kDNA PCR with 10,000 copies of gene and high sensitivity give positive results.,,, The sensitivity of kDNA-PCR, KDNA-nested PCR, and microscopic methods were 99%, 97%, and 87.9%, respectively. CL infection was more prevalent among males (63.6%) than female (36.4%), respectively (P = 0.88). Moreover, the highest rate of infection was seen in the age of 20–39 years because occupational situation they are more exposed to mosquito bites, which is agreement with previous report., The most rate of ZCL was observed in autumn 62.7%, but no case was seen in spring [Table 2] (P = 0.004), which statistically was significant. The most distribution of CL lesions caused by L. major was observed on hands 47.1%, feet 31.4%, and the lowest lesions seen in trunk 1.9%, respectively, [Table 1] (P = 0.57), which is agreement with results of previous studies.,L. major cause moist wound in body extremities and quickly becomes infected, which creates many problems such as movement limitation so that they should be hospitalized. Since this area due to beautiful deserts has the tourist attractions; therefore, to diminish incidence of this disease, effective control programs are need.
| Conclusion|| |
kDNA-PCR is one of the most sensitive methods for the diagnosis of CL. L. major is main causative agent of ZCL in Aran and Bidgol region. A remarkable point is that Kashan is an endemic area for ACL and ZCL, while Aran and Bidgol (a city in the vicinity of Kashan) ZCL is more prevalent. These results can be used in health control programs and treatment systems.
Financial support and sponsorship
This paper was financially supported by grant No. 92154 by Vice-chancellor of Kashan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ashford RW. The leishmaniases as emerging and reemerging zoonoses. Int J Parasitol 2000;30:1269-81.
Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al.
Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012;7:e35671.
Leishmaniasis in high-burden countries: An epidemiological update based on data reported in 2014. Wkly Epidemiol Rec 2016;91:287-96.
Desjeux P. Leishmaniasis: Current situation and new perspectives. Comp Immunol Microbiol Infect Dis 2004;27:305-18.
Yaghoobi-Ershadi MR, Hanafi-Bojd AA, Javadian E, Jafari R, Zahraei-Ramazani AR, Mohebali M, et al.
A new focus of Cutaneous leishmaniasis caused by Leishmania tropica
. Saudi Med J 2002;23:291-4.
Ershadi MR, Zahraei-Ramazani AR, Akhavan AA, Jalali-Zand AR, Abdoli H, Nadim A, et al.
Rodent control operations against zoonotic Cutaneous leishmaniasis in rural Iran. Ann Saudi Med 2005;25:309-12.
] [Full text]
Tabibian E, Hosseini Shokouh SJ, Rahimi Dehgolan SH, Dabbagh Moghaddam A, Tootoonchian M, Noorifard M. Recent epidemiological profile of Cutaneous leishmaniasis in Iranian military personnel. J Arch Mil Med 2014;2:e14473.
Pourmohammadi B, Motazedian M, Hatam G, Kalantari M, Habibi P, Sarkari B, et al.
Comparison of three methods for diagnosis of Cutaneous leishmaniasis. Iran J Parasitol 2010;5:1-8.
Karamian M, Motazedian MH, Fakhar M, Pakshir K, Jowkar F, Rezanezhad H, et al.
Atypical presentation of old-world Cutaneous leishmaniasis, diagnosis and species identification by PCR. J Eur Acad Dermatol Venereol 2008;22:958-62.
Izadi S, Mirhendi H, Jalalizand N, Khodadadi H, Mohebali M, Nekoeian S, et al.
Molecular epidemiological survey of Cutaneous leishmaniasis in two highly endemic metropolises of Iran, application of FTA cards for DNA extraction from giemsa-stained slides. Jundishapur J Microbiol 2016;9:e32885.
Ghasemian M, Maraghi S, Samarbafzadeh AR, Jelowdar A, Kalantari M. The PCR-based detection and identification of the parasites causing human Cutaneous leishmaniasis in the Iranian city of Ahvaz. Ann Trop Med Parasitol 2011;105:209-15.
Shahbazi F, Shahabi S, Kazemi B, Mohebali M, Abadi AR, Zare Z, et al.
Evaluation of PCR assay in diagnosis and identification of Cutaneous leishmaniasis: A comparison with the parasitological methods. Parasitol Res 2008;103:1159-62.
Aflatoonian MR, Sharifi I, Poursmaelian S, Hakimi-Parizi M, Ziaali N. The emergence of anthroponotic Cutaneous leishmaniasis following the earthquake in southern villages of Bam district, Southeastern Iran, 2010. J Arthropod Borne Dis 2013;7:8-14.
Kheirandish F, Sharafi AC, Kazemi B, Bandehpour M, Tarahi Mj, Khamesipour A, et al.
First molecular identification of Leishmania
species in a new endemic area of Cutaneous leishmaniasis in Lorestan, Iran. Asian Pac J Trop Med 2013;6:713-7.
Ghasemloo H, Rasti S, Delavari M, Doroodgar A. Molecular diagnosis of clinical isolates of Cutaneous leishmaniasis using ITS1 and KDNA genes and genetic polymorphism of Leishmania
in Kashan, Iran. Pak J Biol Sci 2016;19:136-42.
Parvizi P, Akhoundi M, Mirzaei H. Distribution, fauna and seasonal variation of sandflies, simultaneous detection of nuclear internal transcribed spacer ribosomal DNA gene of Leishmania
major in Rhombomys opimus
and Phlebotomus papatasi
, in Natanz district in central part of Iran. Iran Biomed J 2012;16:113-20.
Yaghoobi-Ershadi MR, Hanafi-Bojd AA, Akhavan AA, Zahrai-Ramazani AR, Mohebali M. Epidemiological study in a new focus of Cutaneous leishmaniosis due to Leishmania
major in Ardestan town, central Iran. Acta Trop 2001;79:115-21.
Pezeshkpoor F, Tohidi M, Layegh P, Shahabi M. Comparison of fine needle aspiration and direct skin smear in the diagnosis of acute Cutaneous leishmaniasis: Polymerase chain reaction as a reference method. Iran J Dermatol 2013;16:45-8.
Vega-López F. Diagnosis of Cutaneous leishmaniasis. Curr Opin Infect Dis 2003;16:97-101.
Khosravi S, Hejazi SH, Hashemzadeh M, Eslami G, Darani HY. Molecular diagnosis of old world leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania
spp. J Vector Borne Dis 2012;49:15-8. [Full text]
Hajjaran H, Vasigheh F, Mohebali M, Rezaei S, Mamishi S, Charedar S, et al.
Direct diagnosis of Leishmania
species on serosity materials punctured from Cutaneous leishmaniasis patients using PCR-RFLP. J Clin Lab Anal 2011;25:20-4.
Castilho TM, Shaw JJ, Floeter-Winter LM. New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania
species. J Clin Microbiol 2003;41:540-6.
Disch J, Pedras MJ, Orsini M, Pirmez C, de Oliveira MC, Castro M, et al. Leishmania
(Viannia) subgenus kDNA amplification for the diagnosis of mucosal leishmaniasis. Diagn Microbiol Infect Dis 2005;51:185-90.
Koltas IS, Eroglu F, Uzun S, Alabaz D. A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis. Exp Parasitol 2016;164:43-8.
Graça GC, Volpini AC, Romero GA, Oliveira Neto MP, Hueb M, Porrozzi R, et al.
Development and validation of PCR-based assays for diagnosis of American Cutaneous leishmaniasis and identification of the parasite species. Mem Inst Oswaldo Cruz 2012;107:664-74.
Nicolas L, Prina E, Lang T, Milon G. Real-time PCR for detection and quantitation of Leishmania
in mouse tissues. J Clin Microbiol 2002;40:1666-9.
Rasti S, Ghorbanzadeh B, Kheirandish F, Mousavi SG, Pirozmand A, Hooshyar H, et al
. Comparison of molecular, microscopic, and culture methods for diagnosis of Cutaneous leishmaniasis. J Clin Lab Anal 2016;30:610-5.
Doroodgar A, Rasi Y, Sadr F. Sandflies Fauna in Cities of Kash an and Aran va Bidgol, 1990 To 2003. ISOPS-V Proceedings of the Fifth International Symposium on Phlebotomine Sandflies Gammarth, Tunis, Tunisia; 2005.
Doroodgar A, Javadian E, Dehghani R, Hooshyar H, Sayyah M. Leishmanial Infection among rodents in Kashan (1995). KAUMS 1997;1:53-9.
Aransay AM, Scoulica E, Tselentis Y. Detection and identification of Leishmania
DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA. Appl Environ Microbiol 2000;66:1933-8.
Parvizi P, Baghban N, Novin EA, Absavaran A. Detection, identification and molecular typing of Leishmania
major in Phlebotomus papatasi
from a focus of zoonotic Cutaneous leishmaniasis in central of Iran. Exp Parasitol 2010;124:232-7.
de Paiva-Cavalcanti M, de Morais RC, Pessoa-E-Silva R, Trajano-Silva LA, Gonçalves-de-Albuquerque Sda C, Tavares Dde H, et al.
Leishmaniases diagnosis: An update on the use of immunological and molecular tools. Cell Biosci 2015;5:31.
El-Beshbishy HA, Al-Ali KH, El-Badry AA. Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province, Western Saudi Arabia. Int J Infect Dis 2013;17:e334-8.
Yehia L, Adib-Houreih M, Raslan WF, Kibbi AG, Loya A, Firooz A, et al.
Molecular diagnosis of Cutaneous leishmaniasis and species identification: Analysis of 122 biopsies with varied parasite index. J Cutan Pathol 2012;39:347-55.
Farahmand M, Nahrevanian H, Shirazi HA, Naeimi S, Farzanehnejad Z. An overview of a diagnostic and epidemiologic reappraisal of Cutaneous leishmaniasis in Iran. Braz J Infect Dis 2011;15:17-21.
Hijjawi N, Kanani KA, Rasheed M, Atoum M, Abdel-Dayem M, Irhimeh MR, et al.
Molecular diagnosis and identification of Leishmania
species in Jordan from saved dry samples. Biomed Res Int 2016;2016:6871739.
[Figure 1], [Figure 2]
[Table 1], [Table 2]