Leishmaniasis in the Sudan: Parasite Characterization and Phylogeny

Abstract

ntrated mainly on characterization of Sudanese Leishmania isolates; and the phylogenetic relation between isolates from different clinical forms in addition to the relation between these isolates and reference leishmania strains. The study also included attempts to identify parasite factors associated with PKDL in Sudan. In order to achieve the mentioned aims, more than 120 Sudanese leishmania isolates were cultured from different clinical forms of the disease. Isolated parasites were primarily characterized by their isoenzyme profiles compared with reference strains.64 isolates were characterized using isoenzyme: 8 were identified as L.donovani, 9 as L.infantum, 44as L.archibaldi and 3 as L.major. Interestingly L.archibaldi was strongly associated with VL and was the main parasite isolated from PKDL. Total genomic DNA was extracted and kDNA PCR and genomic PCR were performed. The kDNA was performed using species-specific primers followed by restriction fragment length polymorphism (RFLP) with AluI restriction enzyme. The total genomic DNA was analysed using 2 sets of primers; RH1, RH2 and SG1, SG2. The first set was used to amplify parasite sequences by PCR while the second set was used to amplify the gp63 genes then followed by RFLP and hybridisation technique. Radiolabeled probes were used to hybridize the digested genomic DNA in 2 ways; after gp63 amplification and restriction enzyme digestion, and after restriction enzyme digestion without gene amplification. Gp63 PCR analysis of selected 29 isolates identified 26 as L.archibaldi and 3 as L.major. Differential display technique was used for RNA in order to determine the differentially expressed candidate genes between VL/PKDL paired isolates. Cloning and sequencing were used to achieve full descriptions of these differentially expressed genes and northern blot was done to ensure that genes were not false expressed genes. The results obtained in this study suggest that L.archibaldi is the major cause of VL, PKDL and ML and also associated with CL infection. Another important finding in this study is that the PKDL infection is caused by the same parasite that caused the VL form. The high incidence of PKDL in Sudan might be L.archibaldi infection.