Background In Uganda artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine (SP) for chemoprevention during pregnancy, but drug resistance may limit efficacies. Methods Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and qPCR assays. Results Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of PfCRT 76T decreased, but varied markedly between sites (0-48% in 2018; 0-22% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For PfMDR1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14/16 sites, and gene amplification was not seen. Considering mutations associated with high level SP resistance, prevalences of PfDHFR 164L (up to 80%) and PfDHPS 581G (up to 67%) were high at multiple sites. Considering PfK13 propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 40%, respectively). Conclusions We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

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.

Tumwebaze, P.; Tukwasibwe, S.; Taylor, A.; Conrad, M.; Ruhamyankaka, E.; Asua, V.; Walakira, A.; Nankabirwa, J.; Yeka, A.; Staedke, S.G.; Greenhouse, B.; Nsobya, S.L.; Kamya, M.R.; Dorsey, G.; Rosenthal, P.J. (2016) We assessed Plasmodium falciparum drug resistance markers in parasites collected in 2012, 40

Background Treatment and control of malaria depends on artemisinin-based combination therapies (ACTs) and is challenged by drug resistance, but thus far resistance to artemisinins and partner drugs has primarily occurred in southeast Asia. The aim of this study was to characterise antimalarial drug susceptibility of Plasmodium falciparum isolates from Tororo and Busia districts in Uganda.Methods In this prospective longitudinal study, P falciparum isolates were collected from patients aged 6 months or older presenting at the Tororo District Hospital (Tororo district, a site with relatively low malaria incidence) or Masafu General Hospital (Busia district, a high-incidence site) in eastern Uganda with clinical symptoms of malaria, a positive Giemsa-stained blood film for P falciparum, and no signs of severe disease. Ex-vivo susceptibilities to ten antimalarial drugs were measured using a 72-h microplate growth inhibition assay with SYBR Green detection. Relevant P falciparum genetic polymorphisms were characterised by molecular methods. We compared results with those from earlier studies in this region and searched for associations between drug susceptibility and parasite genotypes.

Contributions of species other than to human malaria in sub-Saharan Africa are uncertain. We collected blood from children aged 6 months to 10 years diagnosed with malaria by Giemsa-stained blood smears (176 subjects) or histidine rich protein-2-based rapid diagnostic tests (323 subjects) in 2016; 50 samples from each of 10 sites across Uganda were studied to identify infecting species. Of 499 available samples, 474 demonstrated plasmodial infection by polymerase chain reaction amplification of 18S ribosomal RNA genes, including in 472, in 22, in 15, and in four; 435 were pure, two did not contain , and the remainder were mixed infections including. The prevalence of nonfalciparum species varied geographically. Stratifying based on recent history of indoor residual spraying (IRS) of insecticides, nonfalciparum infections were seen in 27/189 (14.8%) samples from sites that received and 13/285 (4.6%) samples from sites that did not receive IRS since 2010 ( = 0.0013). Overall, 39/474 (8.2%) samples from individuals diagnosed with malaria included nonfalciparum infections. Thus, a substantial proportion of episodes of malaria in Uganda include infections with plasmodial species other than .