Background: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials. Methods: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019–2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined. Results: The PPQ median IC₅₀ interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2–45.6) in 2008 and 27.30 nM (IQR 6.9–52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0–5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC₅₀s rose significantly to 62.40 nM (IQR 26.9–100.8) (P = 0.0201); 7.00 nM (IQR 2.4–13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1–64.3); and 2.70 nM (IQR 1.3–10.4) in 2008, respectively. Conversely, chloroquine median IC₅₀s decreased significantly to 10.30 nM (IQR 7.2–20.9) in 2021 from 15.30 nM (IQR 7.6–30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC₅₀ and Pfcrt K76T allele (P=0.0026). Conclusions: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum multi-drug resistance protein 1 (Pfmrp1) gene have previously been reported to confer resistance to Artemisinin-based Combination Therapies (ACTs) in Southeast Asia. A total of 300 samples collected from six sites between 2008 and 2019 under an ongoing malaria drug sensitivity patterns in Kenya study were evaluated for the presence of SNPs at Pfmrp1 gene codons: H191Y, S437A, I876V, and F1390I using the Agena MassARRAY® platform. Each isolate was further tested against artemisinin (ART), lumefantrine (LU), amodiaquine (AQ), mefloquine (MQ), quinine (QN), and chloroquine (CQ) using malaria the SYBR Green I-based method to determine their in vitro drug sensitivity. Of the samples genotyped, polymorphism at Pfmrp1 codon I876V was the most frequent, with 59.3% (163/275) mutants, followed by F1390I, 7.2% (20/278), H191Y, 4.0% (6/151), and S437A, 3.3% (9/274). A significant decrease in median 50% inhibition concentrations (IC₅₀s) and interquartile range (IQR) was noted; AQ from 2.996 ng/ml [IQR = 2.604–4.747, n = 51] in 2008 to 1.495 ng/ml [IQR = 0.7134–3.318, n = 40] (P<0.001) in 2019, QN from 59.64 ng/ml [IQR = 29.88–80.89, n = 51] in 2008 to 18.10 ng/ml [IQR = 11.81–26.92, n = 42] (P<0.001) in 2019, CQ from 35.19 ng/ml [IQR = 16.99–71.20, n = 30] in 2008 to 6.699 ng/ml [IQR = 4.976–9.875, n = 37] (P<0.001) in 2019, and ART from 2.680 ng/ml [IQR = 1.608–4.857, n = 57] in 2008 to 2.105 ng/ml [IQR = 1.266–3.267, n = 47] (P = 0.0012) in 2019, implying increasing parasite sensitivity to the drugs over time. However, no significant variations were observed in LU (P = 0.2692) and MQ (P = 0.0939) respectively, suggesting stable parasite responses over time. There was no statistical significance between the mutation at 876 and parasite sensitivity to selected antimalarials tested, suggesting stable sensitivity for the parasites with 876V mutations. These findings show that Kenyan parasite strains are still sensitive to AQ, QN, CQ, ART, LU, and MQ. Despite the presence of Pfmrp1 mutations in parasites among the population.

High malaria mortality coupled with increased emergence of resistant multi-drug resistant strains of Plasmodium parasite, warrants the development of new and effective antimalarial drugs. However, drug design and discovery are costly and time-consuming with many active antimalarial compounds failing to get approved due to safety reasons. To address these challenges, the current study aimed at testing the antiplasmodial activities of approved drugs that were predicted using a target-similarity approach. This approach is based on the fact that if an approved drug used to treat another disease targets a protein similar to Plasmodium falciparum protein, then the drug will have a comparable effect on P. falciparum. In a previous study, in vitro antiplasmodial activities of 10 approved drugs was reported of the total 28 approved drugs. In this study, six out of 18 drugs that were previously not tested, namely epirubicin, irinotecan, venlafaxine, palbociclib, pelitinib, and PD153035 were tested for antiplasmodial activity. The drug susceptibility in vitro assays against five P. falciparum reference strains (D6, 3D7, W2, DD2, and F32 ART) and ex vivo assays against fresh clinical isolates were done using the malaria SYBR Green I assay. Standard antimalarial drugs were included as controls. Epirubicin and irinotecan showed excellent antiplasmodial ex vivo activity against field isolates with mean IC₅₀ values of 0.044 ± 0.033 μM and 0.085 ± 0.055 μM, respectively. Similar activity was observed against W2 strain where epirubicin had an IC₅₀ value of 0.004 ± 0.0009 μM, palbociclib 0.056 ± 0.006 μM, and pelinitib 0.057 ± 0.013 μM. For the DD2 strain, epirubicin, irinotecan and PD 153035 displayed potent antiplasmodial activity (IC₅₀ < 1 μM). Epirubicin and irinotecan showed potent antiplasmodial activities (IC₅₀ < 1 μM) against DD2, D6, 3D7, and F32 ART strains and field isolates. This shows the potential use of these drugs as antimalarials. All the tested drugs showed antiplasmodial activities with IC₅₀ values below 20 μM, which suggests that our target similarity-based strategy is successful at predicting antiplasmodial activity of compounds thereby circumventing challenges in antimalarial drug discovery.

The molecular mechanism underlying Plasmodium falciparum's persistence in the asymptomatic phase of infection remains largely unknown. However, large-scale shifts in the parasites' gene expression during asymptomatic infections may enhance phenotypic plasticity, maximizing their fitness and leading to the persistence of the asymptomatic infections. To uncover these mechanisms, we aimed to identify parasite genetic factors implicated in asymptomatic infections through whole transcriptome analysis. We analyzed publicly available transcriptome datasets containing asymptomatic malaria (ASM), uncomplicated malaria (SM), and malaria-naïve (NSM) samples from 35 subjects for differentially expressed genes (DEGs) and long noncoding RNAs. Our analysis identified 755 and 1773 DEGs in ASM vs SM and NSM, respectively. These DEGs revealed sets of genes coding for proteins of unknown functions (PUFs) upregulated in ASM vs SM and ASM, suggesting their role in underlying fundamental molecular mechanisms during asymptomatic infections. Upregulated genes in ASM vs SM revealed a subset of 24 clonal variant genes (CVGs) involved in host-parasite and symbiotic interactions and modulation of the symbiont of host erythrocyte aggregation pathways. Moreover, we identified 237 differentially expressed noncoding RNAs in ASM vs SM, of which 11 were found to interact with CVGs, suggesting their possible role in regulating the expression of CVGs. Our results suggest that P. falciparum utilizes phenotypic plasticity as an adaptive mechanism during asymptomatic infections by upregulating clonal variant genes, with long noncoding RNAs possibly playing a crucial role in their regulation. Thus, our study provides insights into the parasites' genetic factors that confer a fitness advantage during asymptomatic infections.

Background: As studies continue to confirm the rising frequency of mixed Plasmodium species infections, there is little data on the response of non-falciparum species to the Artemisinin Combination Therapies (ACTs) which is a critical tool for malaria elimination. This study compared the therapeutic response of P. falciparum single infection versus mixed Plasmodium species to ACTs. Methods: A total of 1211 blood samples collected on days 0, 7, 14, 21, 28, 35, and day 42 from 173 individuals out of 236 enrolled in an ACT efficacy study between 2013 and 2015 were diagnosed for malaria using the 18s rRNA-based real-time PCR. All recurrent parasitaemia were also characterized for species composition; Plasmodium falciparum (Pf), Plasmodium malariae (Pm), Plasmodium ovale curtisi (Poc), and Plasmodium ovale wallikeri (Pow). ACTs treatment outcome was monitored during the 42-day follow-up period. Findings: Overall, 71•1% (116/163) of the day zero samples were single Pf infections while 28•2% (46/163) were co-infections. 54.0% (88/163) of individuals tested positive for infection at least once between days 7 - 42 after treatment. 19•3% (17/88) of individuals with recurrent infections were infected with a different Plasmodium species than observed at day 0, with 76•5% (13/17) of these “hidden” infections appearing after clearing P. falciparum present at day 0. Artesunate-mefloquine (ASMQ) (16•4) and dihydroartemisinin-piperaquine (DHA-PPQ) (17•6) had the increased clearance rates over artemether-lumefantrine (AL) (21•0) with DHA-PPQ exhibiting the longest duration of reinfection prophylaxis based on day 28 positivity. Interpretation: Analysis based on species composition revealed minor difference in clearance rates depending on whether the infection contained other species other than P. falciparum. Trial Registration: This study was registered at ClinicalTrials.gov Identifier: NCT01976780. Funding: AFHSB and its GEIS Section. Schmidt Science Fellows in partnership with the Rhodes Trust Declaration of Interest: We GCC, BMA, IAO, DWJ, BHO, ROO, JAJ, EWM, JAG, DMW, ACC, RAY, ALR, BRO, TEE, ECG, EK OW and HMA declare no competing interests Ethical Approval: Samples analyzed by this study were collected under two sequential randomized, two-arm open-label clinical efficacy trials under the approval of the Kenya Medical Research Institute (KEMRI), Scientific and Ethics Review Unit (SERU) and Walter Reed Army Institute of Research (WRAIR) institutional review boards. The first study was entitled, “Therapeutic efficacy of artesunate-mefloquine and artemether-lumefantrine for the treatment of uncomplicated malaria in Kisumu County, Western Kenya,” protocol numbers: WRAIR#1935, SSC#2518 conducted between 2013 and 2014. The second study was entitled, “Therapeutic efficacy of ihydroartemisinin-piperaquine and artemether-lumefantrine for the treatment of uncomplicated malaria in Kisumu County, Western Kenya,” protocol numbers: WRAIR#1935A, SSC#2722 between 2014 and 2015.