Investigation of single nucleotide polymorphisms in MRPA and AQP-1 genes of Leishmania donovani as resistance markers in visceral leishmaniasis in Kenya

Abstract

Visceral Leishmaniasis (VL) remains a major public health problem mainly affecting the poorest populations across Asia, Africa, Middle East, Europe, Southern and Central America. For seven-decade now, the first-line drug of choice for leishmaniasis has been pentavalent antimonials. However, the clinical value of these drugs is threatened by the emergence of drug-resistant parasites. Clinical resistance to sodium stibogluconate (pentostam) has been a challenge in the Indian subcontinent, raising concerns for the endemic countries in Africa. This study aimed to identify and describe Single Nucleotide Polymorphism (SNPs) in gene markers associated with drug resistance among the clinical samples. The study was an experimental laboratory investigation on Dry Blood Spots (DBS). DNA was extracted from 18 VL positive samples, and Internal Transcribed Spacer-1 Polymerase Chain Reaction confirmed the positivity. Two target resistance markers, aquaglyceroporin 1 ( AQP-1 ) and the Multi-Drug Resistant Protein A ( MRPA ), were PCR-amplified and resulting amplicons sequenced using the Sanger sequencing platform. Multiple sequence alignments were performed using ClustalW, and the phylogenetic tree was constructed in MegaX using the Maximum Likelihood method. A total of 84 SNPs in the AQP-1 gene were identified from six clinical samples. Fifty-nine of the SNPs (70.2%) were non-synonymous, while 25 (29.8%) were synonymous. Among the non-synonymous SNPs, three (5.1%) were nonsense, and 56 (94.9%) were missense point mutations. Two missense SNPs A188T and E185A in S17608 reported to be associated with drug resistance phenotype were observed. The study describes the resistance associated with the pentostam uptake by Leishmania donovani .