Abstract

Leishmaniosis is one of the most important vector borne diseases. Among different forms of the disease, cutaneous leishmaniosis (CL) is the most common. Determining the method of definitive diagnosis for the disease has been the aim of various studies. Therefore this study afforded an opportunity to investigate this subject. To diagnose CL in 150 suspected patients referred to Mehran and Dehloran health centers during June 2018 to November 2019, two polymerase chain reaction (PCR) methods were performed and compared with the in vitro culture and microscopic evaluation of stained slides. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For semi-nested PCR and PCR-RFLP, the tissue and serosity from the lesions were used for DNA extraction. The semi-nested PCR technique using minicircle kDNA gene showed the highest positivity rates among all diagnostic assays with 114/150 (76) of the samples and was used as reference standard, followed by the PCR-RFLP test using ITS1 gene with 112/150, (74.7) positivity rates, microscopy with 101/150 (67.3) and then culture 72/150 (48). microscopy and culture methods together improved overall positivity rates to 68.7 (103/150). The all positive samples using molecular technique were identified as Leishmania major. The highest sensitivity (98.3), specificity (100), accuracy (98.8), negative predictive value (94.7) and kappa coefficient (0.96=almost perfect) was observed by comparing PCR-RFLP and semi-nested PCR. kDNA-semi-nested PCR and ITS1-PCR-RFLP presented an interesting alternative to conventional methods for the identification of CL and improved its diagnostic value significantly in suspected patients with negative direct smears.