Detection of Cutaneous Leishmaniasis Based on ITS1 Gene by PCR-RFLP Technique

Abstract

Background: Cutaneous leishmaniasis is a vector-borne disease transmitted by biting of the sandfly, it is a severe health problem in many countries and endemic in most regions of Iraq. Objectives: This study was conducted to find the best method for diagnosis of cutaneous leishmaniasis, detect the genotypes of Leishmania tropica and Leishmania major in Ramadi (Iraq) by PCR-RFLP technique. Materials &methods: One hundred twenty-two patients 68 were males while the females gender were 54 with age ranged 1-68 years, CL who attended to Department of Dermatology in Al-Ramadi Teaching Hospital and dermatology Private clinics, during the period between November 2017 to April 2018. The Molecular study was carried out to detect the ITS1 gene by (PCR). The restriction fragment length polymorphism (RFLP) was adopted on ITS1-PCR product and after HaeIII digestion at 37Co for 2 hours. Results: Laboratory examination of 122 cases showed 62 infection cases in Cutaneous leishmaniasis by using PCR technique and in infection proportion reaches at 51% out of the total number of the cutaneous cases which are similar to leishmaniasis during the months of the study. The demographic study dealt with age, gender, number of lesions and body site of infection, demonstrated that the majority of patients at the age of 1-10 years with percent 28.7%. Also Males (55.7%) had higher infection than females (44.3%), upper limbs had the highest percentage (48%) when compared with other sites of infection, single lesion was documented in 55% of patients, while two lesions were observed in 25% and multiple (3-10) lesions were observed in 20%. Different techniques were used for diagnosis of CL including routine method performed by direct microscopic smear from lesion which showed amastigotes in the macrophage in 50 (41%) positive case. The Molecular study was carried out to detect the ITS1 gene (internal transcribed spacer1) by (PCR). DNA extracted from 122 samples showed 62 (51%) were positive for (ITS1)gene, The restriction fragment length polymorphism (RFLP) was adopted on ITS1-PCR product and after HaeIII digestion at 37Co for 2 hours obtained two fragments of 60 and 200 bp 42 as L.tropica, and two fragments of 140 and 210 bp were identified 20 as L.major, genotype techniques were performed for all positive samples. Conclusion: CL is highly spread with single lesions more than multiple lesions and molecular detection showed that L.tropica more common than L.major.