Lesion Aspirate Isolation, Culture, Diagnosis and Molecular Identification of Cutaneous Leishmaniasis in District Kohat, Khyber Pakhtunkhwa, Pakistan

Abstract

Leishmaniasis is a poverty-associated zoonotic tropical disease transmitted by the bite of infected female sand fly. Diagnosis and recognition of clinical manifestation and morphological features of Leishmania parasites involved in the spread of disease is mandatory to design appropriate strategies for ecologic control and preventive plans. Although clinical investigations are necessary to estimate and help define about epidemiological findings but they are not sufficient for identification and characterization of the particular parasite, as the transmission of the disease especially in endemic areas is manifested by various Leishmania species. Samples from 41 patients of both gender (22 males and 19 females) with variable age groups (median age was 27) irrespective of location and immune status suggested with suspected cutaneous leishmaniais lesions fulfilling the inclusion criteria were enrolled and examined. Aspirate smears and skin scrapings collected from the active edge of the lesion were stained with Giemsa and confirmed via microscopy using fine needle aspirate biopsy and/or lancet. Productivity and potential isolates of Leishmania samples inoculated in Biphasic Novy-MacNeal- Nicolle (NNN) and cultured in Monophasic Roswell Park Memorial Institute (RPMI) 1640 media were used in evaluating the efficiency for each sample under study. In order to identify Leishmania specie a definite molecular technique, Polymerase Chain Reaction (PCR) product analyzed by gel electrophoresis using 2% agarose was adopted. Genomic DNA was extracted using proteinase k and amplified by specific primers of kinetoplast DNA. Gel staining was performed using ethidium bromide. The presence of 186bp fragment indicated L. tropica confirming it as the prevalent species in Khyber Pakhtunkhwa, Pakistan, which has immense importance from an epidemiological, transmission and treatment point of view. PCR is a very reliable and sensitive method to detect Leishmania DNA as compared to Giemsa and culture techniques.