Changing Molecular Markers of Antimalarial Drug Sensitivity across Uganda

Abstract

The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda

“…Taken together, these results strongly suggest that neither back mutation nor additional mutations in pfcrt was associated with the observed recovery of chloroquine sensitivity in the study area. It is of note that recovery of chloroquine sensitivity after its withdrawal occurred much earlier in Gulu than in other regions in Uganda [9,[22][23][24]41]. In 2013, as much as 65% of parasites displayed ex vivo chloroquine resistance [24] and 60-80% carried K76T allele in Tororo, Eastern Uganda [42].…”

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

“…It is of note that recovery of chloroquine sensitivity after its withdrawal occurred much earlier in Gulu than in other regions in Uganda [9,[22][23][24]41]. In 2013, as much as 65% of parasites displayed ex vivo chloroquine resistance [24] and 60-80% carried K76T allele in Tororo, Eastern Uganda [42].…”

The Diversity of the Plasmodium falciparum K13 Propeller Domain Did Not Increase after Implementation of Artemisinin-Based Combination Therapy in Uganda

“…However, the pressure of millions of doses of ACTs offers continued risk of resistance selection. Polymorphisms associated with resistance in Southeast Asia (1, 10) have already been reported at low frequencies in African P. falciparum populations (12,18,21,34,38,(42)(43)(44)(45)(46)(47)(48). It is likely only a matter of time before a favorable combination of variants allows resistant parasites to gain a foothold in Africa, and continued surveillance and work to improve tools to detect early indicators of resistance will be essential.…”

“…Samples. Samples were collected in clinical studies conducted at multiple sites across Uganda from 1999 to 2016 (17,18,22,25,26,(49)(50)(51)(52). For this study, pre-ACT samples were randomly selected from available DNA samples with the goal of obtaining 100 K13PD sequences from each site, with the exception of Arua, where only 92 samples were available.…”

“…In Uganda, the standard therapy for uncomplicated malaria changed from chloroquine to chloroquine plus sulfadoxine-pyrimethamine (SP) in 2000 and then to the ACT artemether-lumefantrine (AL) in 2006 (16). In recent studies, K13PD mutations (17)(18)(19)(20)(21), delayed clearance after ACT therapy (22)(23)(24), and persistent parasitemia following DHA exposure in ring survival assays (25,26) have all been uncommon in Uganda. However, it is unclear whether changes in the prevalence of K13PD mutations occurred after the institution of treatment with ACTs.…”