In vitro induction/augmentation of unresponsiveness to sodium stibogluconate in Leishmania donovani isolates

Abstract

Visceral leishmaniasis is a major health problem in Sudan; recent reports highlighted the emergence of SSG-resistant strains. Thirty Leishmania donovani isolates were successfully grown and passed in increasing concentrations of sodium stibogluconate (SSG). The (IC50) and the (IC90) of the isolates were initially established and were found to be 0.4 ± 0.04 mg/ml and 0.8 ± 0.1 mg/ml respectively. Following successful induction of resistance in the isolates, the IC50 and the IC90 rose to 50 times and 35 folds higher than those of the wild type (before induction of resistance) respectively (p<0.05). The time and the number of passages required for the isolates to recover to early log phase following exposure to SSG were first increasing with increasing SSG concentrations and then began to decline despite continued increasing SSG concentrations. The isolates were characterized as L.donovani using kDNA-PCR. SDS-PAGE technique showed that the resistant isolates developed additional three bands in their protein band profiles. Random amplified polymorphic DNA (RAPD) analysis, using three arbitrary oligonucleotide primers confirmed that the wild isolates were genetically divergent with three primary genotypes. For each primer, the PCR-RAPD band patterns of the resistant isolates were found to be similar to each other but different from those of the wild isolates. The resistant isolates were closely similar in their band patterns to that of one of the primary genotypes of the wild isolates. In conclusion, in vitro resistance to SSG was successfully induced/augmented in L.donovani isolates as evidenced by the increase in IC50 and the IC90 of the strains following passages in increasing concentrations of SSG. The shortening of time and the number of passages for the isolates to recover following SSG pressure is an added proof of acquiring resistance to SSG. L.donovani isolates from Sudan are genetically diverse. Two possible explanations were presented to explain the uniformity of PCR-RAPD band patterns of the resistant isolates. Firstly, two or more different strains could be present from the start (SSG-sensitive/resistant), the drug sensitive one/s was eliminated by the drug, while the other recovered after removal of drug effect (genetic selection). Alternatively, the isolates changed their DNA (genetic mutation) to face the drug pressure. Further studies with large numbers of isolates using PCR-RAPD primers (A12, A13 & IL-0875) are needed.