Microsatellites reveal high polymorphism and high potential for use in antimalarial efficacy studies in areas with different transmission intensities in mainland Tanzania

Abstract

BackgroundTanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization’s (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of antimalarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes (msp1, msp2, andglurp) and highly polymorphic neutral microsatellites inPlasmodium falciparumto adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which are routinely performed in Tanzania.MethodsP. falciparumsamples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro), and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was performed using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and 2490) by the capillary method, and the data were analyzed to determine the extent of polymorphisms and genetic diversity at the four sites.ResultsOverall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥50.0% of the markers), and >50.0% (range = 47.6-59.1%) were polyclonal, with the mean multiplicity of infection ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range= 7.27- 8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He= 0.83, range= 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not detect any population structure, and isolates clustered independently of geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while 2490 was the least polymorphic.ConclusionMicrosatellite genotyping revealed high polyclonality and genetic diversity but without any significant population structure. Poly-α, C2M34 and TA1 were the top polymorphic markers and could be adopted for use in TES in Tanzania.