Asian Pacific Journal of Tropical Medicine (2012)514-517 514 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: Molecular detection of Leishmania isolated from Cutaneous leishmaniasis patients in Jask County, Hormozgan Province, Southern Iran, 2008 Koroush Azizi1, Aboozar Soltani2*, Hamzeh Alipour1 1Department of Medical Entomology, Schools of Health and Nutrition, Shiraz University of Medical Sciences, Shiraz, Iran 2Deparment of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran ARTICLE INFO ABSTRACT Article history: Objective: Methods: To investigate on patients leishmanial infections in Jask County. Received 10 November 2011 Impression smears were prepared from patients in 2008, all, were chequed for leishmanial Received in revised form 15 January 2012 infection by microscopy and molecular assays. Whole DNA was extracted using Proteinase K and Accepted 15 March 2012 Phenol/Chloroform/Isoamyl alcohol method. The variable segment on minicircles of kinetoplast Available online 20 July 2012 DNA wasResults: amplified via a Nested-PCR technique using species-specific primers (LIN R4-LIN 17 -Lin 19). A total of 40 smears were prepared from 20 patients, from which, eight samples Keywords: 18 (90%) (40%) were positive for leishman body by microscopicLeishmania method, major while, (L. major) samplesConclusions: were positive, molecularly. The parasite was identified as . LeishmaniaZoonotic Cutaneous major Leishmaniasis L.Zoonotic major or Rural cutaneous leishmaniasis is endemic in Jask County whose pathogen is Nested PCR . Molecular assays using specific primers are very accurate and more sensitive and Jask specific than microscopy which is time consuming and needs master microscopists. Iran 1. Introduction leave severe and permanently disfiguring scars. The cutaneous form may produce up to 200 lesions and lead to disability. The patient is left permanently scarred, Leishmaniases are diseases caused by protozoan parasites and so may become socially stigmatized[4]. Cutaneous transmitted via bites of infected sand flies. The differing leishmaniasis (CL) continues to be an increasing public- manifestations of theLeishmania disease arise from infections with health problem in Iran. This form can be seen in zoonotic different species’ of . Leishmaniasis is still one (ZCL) or anthroponotic (ACL) forms. It is endemic in half of of the world s most important neglected diseases, mainly the 30 Iranian provinces. ZCL is widespread in the central, affecting the poor caste of the developing communities[1]. I Leishmaniasouthern and major easternL. major). provinces of ran, mostly caused by 350 million people in 88 countries of developing countries of ( four continents are considered to be at the risk of infection, In the recent years several new endemic foci have been two million new cases occur yearly, of which an estimated reported, indicating the potential spread of disease in the 500 000 are visceral and 1.5 million cases are cutaneous country. During 2001-2008, the number of CL cases has (90% of them occurring in Afghanistan, Algeria, Brazil, the been progressively increased from 11 505 to 26 824 in the Islamic Republic of Iran, Peru, Saudi Arabia, Sudan and country[5]. Jask County which is located in south eastern the Syrian Arab Republic)[2]. Since 1993, the distribution of part of Iran has recently been introduced as the most leishmaniases has expanded, and there has been a sharp important endemic focus of cutaneous leishmaniasis in increase in the number of cases recorded[3]. Hormozgan province (245 and 195 cases in 2007 and 2008, The cutaneous form is more common. It usually causes respectively) [6]. ulcers on the face, arms and legs. Although the ulcers PCR-based techniques now days are routinely used heal spontaneously, they cause serious disability and for detection of Leishmania infections, provides a rapid, sensitive, and specific alternative for previous traditional Leishmania *Corresponding author: Aboozar Soltani. Department of Medical Entomology & techniques. Moreover, diagnosis of infection Vector Control, School of Public Health & National Institute of Health Research, and species identification is done, simultaneously[7-10]. Tehran University of Medical Sciences, P. O. Box: 6446 Tehran 14155, Iran. E-mail: [email protected]; [email protected] This study was designed to investigate on human infections Koroush Azizi et al./Asian Pacific Journal of Tropical Medicine (2012)514-517 515 in this new-emerged focus because any effective control phenol:chloroform before the DNA was precipitated with programmes should be based on the accurate baseline ethanol, resuspended in 100 mL double-distilled water and information about disease form and pathogen species. stored at 4 曟[10, 12]. 2.4. Detection and identification of Leishmania species 2. Materials and methods 2.1. Study area The variable segment on minicircles of kinetoplast DNA ( DNA) N PCR k was amplified in a two-rounds, ested- ’ 2 (LINR4 5 Hormozgan Province covers an area of 71 139.62 km . It technique using species-specific’ primers : ’- I P G GGGGTTGGTGTAAAATAGGG-3 as the forward; LIN17: 5 - is located in south≈ of ran 2and north of the ersian ulf. ’ ( ) TTTGAACGGGATTTCTG-3 as the first-step reverse and The Jask County 154 km is a wideº ’aeolic ºplain’ on the ’ ’ northern coastline of Oman Sea (25 38 N, 57 46 E, at an LIN19: 5 -CAGAACGCCCCTACCCG-3 as the second-step 4 8 ) ( 1) 30 reverse). The Nested-PCR was carried out in two steps, both altitude of . m a.s.l. Figure . A roughly km littoral et al plain belt leads to hilly regions. It is characterized by long in the same tube, as described by Aransay [13]. The first- dry summers and has a hot humid climate over most of its round reaction mixture contained 250 毺M deoxynucleoside arid and semi-arid regions with sandy hills. The average of triphosphate (dNTP), 1.5 毺M MgCl2, 1 U Taq polymerase, total annual rainfall, mean relative humidity and the mean 1 毺M LINR4, 1 毺M LIN17 and 5 毺L DNA extract in daily temperature were 136.4 mm, 69.5% and 27.3 曟 during × 1 PCR buffer in a final volume of 25 毺L. This mixture was ( ) 2004-2008, respectively. www.weather.ir . incubated in a CG1-96 thermocycler set to run for 5 min at 94 曟, followed by 30 cycles each of 30 s at 95 曟, 1 min at 52 曟, 1 min at 72 曟 and a final extension at 72 曟 for 10 min and kept at 4 曟. The first-round product (2 毺L of a 4:1 dilution in ddH2O) was used as template for the second round, in a total volume of 20 毺L and under similar conditions to those for the first round but using LINR4 and LIN19 as the primers in 33 cycles[10,14,15]. This PCR was able to identify promastigote infection of sand flies or amastigote infection in clinical samples and Leishmania shows specific bands for each species. The L. major Leishmania band size was 560 and 720 bp for and tropica (L. tropica) [13,15] Leishmania Figure 1. , respectively . species Location of sampling zones identification was done comparing with standard strains after 2.2. Sampling electrophoresis of PCR products. In order to comparison and Leishmania confirmation of identified species, three strains (Leishmania infantum (L. infantum) : MCAN/IR/96/Lon 49, The present study was descriptive; cross sectional has been L. tropica L. major MHOM IR 89 ARD 2 MHOM IR 54 done in 2008. Sampling was achieved from 20 patients (about : / / / , : / / / LV 39) L 10 percent of patients) with acute cutaneous ulcers according which were kept in the eishmaniasis laboratory of P D T S to the data of Jask health center. Seven villages containing arasitology epartment at ehran and hiraz university, Lyrdaf, Soorak, Negar, Zikdaf, Koohert, Shemshi and Kangan were used as the standard strains. which had been previously reported as infected areas were considered as sampling zones. Impression smears were 3. Results prepared on two microscopic slides for each patient. These slides were investigated for leishman body (leptomonad) A 40 20 after fixation by Methanol and Giemsa staining. total of smears were prepared from patients. S mears were examined by two diagnostic techniques, direct 2.3. DNA extraction (microscopic) examination and a species-specific Nested- PCR. Eight out of 20 samples (40%) were positive for leishman 18 (90%) All microscopically positive and negative slides were then body by microscopic examination and samples were shown to be positive for kDNA by molecular assay. chequed for Leishmania kDNA by molecular assays. Each L. major L dry smear was scraped off the slide with a sterile scalpel and eishman body of observed in eight prepared smears (Figure 2). Infective parasite was identified as the scrapings transferred to a 1.5 mL reaction tube[11]. L. major PCR T The smear scrapings were added to 150 mL lysis buffer [50 using the nested method. he size of all amplified products was about 560 bp equal to the band size mm Tris-HCL (pH 7.6), 1 mm EDTA (pH 8.0), 1% Tween 20, L. major (F 3) and 8.5 m proteinase K solution (19 mg/mL] and incubated of standard strain igure . for 2 h at 55 曟. The lysate was then extracted twice with Koroush Azizi et al./Asian Pacific Journal of Tropical Medicine (2012)514-517 516 by microscopicLeishmania examination of clinical prepared smears, however, all species are very similar and their morphological species identification is so difficult[23]. ( Traditional diagnostic techniques directLeishmania examination and culture) can not differentiate among species and [24,25] their sensitivity is low . Although isoenzymLeishmania analysis is a gold standard for differentiation of species but this technique is almost expensive, time consuming and [19] usually requires in vitro cultivationLeishmania of parasite .