Molecular characterization of Leishmania infection in sand flies from Al-madinah Al-munawarah province, western Saudi Arabia

Abstract

Cutaneous leishmaniasis (CL) is caused by various species of the genus Leishmania. The disease is considered a major health problem in different areas of Saudi Arabia including Al-madinah Al-munawarah province. We aimed to identify Leishmania species isolated from sand fly vectors by molecular analysis. Sand fly sampling was carried out from May 2010 to October 2010 in province of Al-madinah Al-munawarah from four different localities. Female sand flies collected were subjected to DNA extraction followed by molecular analysis using the semi-nested PCR and conventional PCR protocols, respectively, against minicircle kDNA and ribosomal internal transcribed spacer 1 (ITS1-rDNA). The PCR positive specimens against ITS1-rDNA locus were digested for further confirmation of species identification. A total of 2910 sand flies were collected. Phlebotomus papatasi accounted for 93.8% (1673 males and 1057 females), however, the number of Phlebotomus sergenti was only 180 (109 males and 71 females). Sixty-two out of 250 (23.7%) female P. papatasi tested for Leishmania parasite were positive for Leishmania major using the semi-nested PCR method against kDNA. All of the 62 positive specimens produced a band size 650 bp. A 31% of female P. sergenti were positive against kDNA of Leishmania tropica and produced a 720 bp band. These positive P. sergenti for L. tropica DNA produced ITS1-PCR-RFLP profile showed two bands of ∼200 bp and 57 bp which are specific for L. tropica, confirming the presence of L. tropica in P. sergenti. However, the ITS1-PCR-RFLP profile showed two bands of ∼203 bp and 132 bp which are specific for L. major in P. papatasi. We concluded that, the semi-nested PCR method against kDNA and the ITS1-PCR-RFLP analysis are useful tools for molecular identification of both L. major and L. tropica. A multicenter study is necessary in order to evaluate the extent of the disease and functional analysis of new Leishmania genes.