Temporal and spatial trends of plasmodium falciparum multi-drug resistance protein 1 gene mutations during implementation of artemisinin combination therapies between 2008 and 2019 in Kenya

Abstract

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum multi-drug resistance protein 1 (Pfmrp1) gene have previously been associated with conferring resistance against artemisinin and its partner drugs in Southeast Asia (SEA) and there are concerns of resistance spreading to Africa as the previous patterns. With no suitable replacement for artemisinin combination therapies (ACTs), establishing the frequency of these polymorphisms contributing to impaired response to ACTs is key for continued drug resistance surveillance in Africa, where the few putative Kelch 13 propeller polymorphisms reported in Rwanda and the horn of Africa have not affected ACTs tested and used there. Apart from Kelch 13, Pfmrp1 gene is also a potential drug resistance marker neglected in Kenya as compared to chloroquine (CQ) and sulfadoxine pyrimethamine (SP) targets. The frequency of Pfmrp1 SNPs associated with anti-malarial resistance and correlation with in vitro drug sensitivities from Kenyan parasite isolates between 2008 and 2019 is not known. Of particular interest between 2008 and 2019 was a representation of the transition period and post-ACTs timelines of the study period, to compare the trends over time to help understand the prevalence of resistance. Therefore the general objective of this study was to investigate the temporal and spatial trends of the Pfmrp1 gene mutations during the implementation of ACTs between 2008 and 2019 in Kenya. The specific objectives were to determine the frequency of SNPs of Pfmrp1 gene, determine in vitro Plasmodium falciparum (P.f.) drug response patterns and establish the correlation between the polymorphic versus wild-type and in vitro anti-malarial response profiles for the Kenyan field isolates collected between 2008 and 2019. In a cross-sectional retrospective study of 6 months and older participants, 300 samples collected from 6 sites across Kenya namely; Kisumu, Kombewa, Malindi, Marigat, Kisii and Kericho between 2008 and 2019 under an ongoing, epidemiology of malaria and drug resistance patterns in Kenya study, were assayed for SNPs in Pfmrp1 gene codons; H191Y, S437A, I876V and F1390I using Agena MassARRAY platform. Field isolates were also tested against standard antimalarials selected; artemisinin (ART), lumefantrine (LU), amodiaquine (AQ), mefloquine (MQ), quinine (QN) and CQ to determine their in vitro drug sensitivity using the malaria SYBR Green I-based fluorescence assay. Categorical data was analyzed as proportions, while the continous data was reported as median IC50 values. Of the 300 samples typed, polymorphisms at Pfmrp1 codon I876V was the most frequent at 58.9% (162/275) mutants followed by F1390I, 7.1% (19/267) and S437A, 3.3% (9/274) while H191Y was the least at 3.1% (5/151). The antimalarial sensitivity patterns of AQ and QN were shown to have median IC50s that increased over time between 2008 and 2019 from 2.959ng/ml [IQR=2.453-4.189, n=47] and 1.967ng/ml [IQR=1.332-3.243, n=54] to 7.111ng/ml [IQR=6.562-9.054, n=11] and 3.046ng/ml [IQR=2.178-6.175, n=20] respectively. MQ appeared to be undergoing positive selection, it was shown to have median IC50s that increased resistance over time from 16.59ng/ml [IQR=12.13-35.96, n=16] to 20.32ng/ml [IQR=12.54-23.12, n=5] and it increased significantly (P<0.0001) during the same period. However, LU showed contradicting results of increased sensitivity and resistance over time during the study period. The correlation of infections with mutation at codon I876V were associated with higher QN and LU with 50% inhibition concentration during in vitro tests, suggesting reduced sensitivity. Identifying these markers are critical to understanding and tracking other rising prevalence of ACTs resistance within this region after its implementation besides, is essential as prerequisites for any control and elimination programs. Study findings showed early indicators signaling resistance patterns to ACTs and selection should be tracked. Pfmrp1 gene mutations should also be tracked as an important candidate gene for monitoring drug resistance in Kenya.