Determination of Leishmania Species Using Molecular Biology Techniques

Abstract

This study was carried out on 117 samples, collection from cutaneous leishmaniasis patients and 47 samples collected from visceral leishmaniasis patients. Fifty three skin samples and one bone marrow aspirate were collected from Karachi, Sind. We received 59 samples. From Balochistan, 56 of these were skin aspirates and three were bone marrow samples. Fifty one samples were collected from Northern areas, these included eight skin aspirate, 18 blood samples and 25 bone marrow smears. From Sind, 38 skin samples and one bone-marrow samples were cultured in RPMI medium. Of the skin samples 23, showed growth of Leishmania parasite, 15 showed no growth of the parasite or any other contaminant while the bone-marrow sample showed parasitic growth. Twenty five skin samples grew fungus and 21 samples were culture positive. The parasitic DNA isolated from skin sample received from Karachi, Balochistan and Northern Areas were subjected to PCR based detection of the parasite. Out of these forty six skin samples from Sind were found to be PCR positive and seven skin samples showed no amplification. Eighteen blood samples, three bone marrow aspirate and 26 bone marrow smears from visceral leishmaniasis patient were also analysed. Further identification of species was carried out by PCR based assays. We were able to identify 27 samples which showed specific bands for L. donovani. When blood samples collected from Northern Areas were subjected to PCR, 12 samples were found positive for L . donovani while 6 samples show no band for Leishmania specie. Two samples were also found culture negative. Two other culture negative samples. Were PCR positive. L donovani can be detected from dried bone marrow samples. The PCR analysis of cutaneous leishmaniasis indicate the presence of both L.major and l.tropica in Pakistan On the basis of our over observations and results we concluded that the method of collection of skin or bone marrow samples for culture of Leishmania is critical. The PCR assay results of our study indicate that identification of Leishmania parasite from both cutaneous (skin biopsy aspirates) and visceral (peripheral blood and bone marrow) leishmaniasis patients can become a valuable tool for clinical laboratories.