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The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC50 values

We measured susceptibilities of Ugandan Plasmodium falciparum isolates assayed on the day of collection or after storage at 4°C. Samples were incubated with serial dilutions of 8 antimalarials, and susceptibilities were determined from 72-h growth inhibition assays. Storage was associated with decreased growth and lower 50% inhibitory concentration values, but differences between assays beginning on day 0 or after 1 or 2 days of storage were modest, indicating that short-term storage before drug susceptibility determination is feasible.

BackgroundThe Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited.MethodsWe studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes.ResultsMedian half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites.ConclusionsResistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Artemisinin partial resistance may facilitate selection of Plasmodium falciparum resistant to combination therapy partner drugs. We evaluated 99 P. falciparum isolates collected in 2021 from northern Uganda, where resistance-associated PfK13 C469Y and A675V mutations have emerged, and eastern Uganda, where these mutations are uncommon. With the ex vivo ring survival assay, isolates with the 469Y mutation (median survival 7.3% for mutant, 2.5% mixed, and 1.4% wild type) and/or mutations in Pfcoronin or falcipain-2a, had significantly greater survival; all isolates with survival >5% had mutations in at least one of these proteins. With ex vivo growth inhibition assays, susceptibility to lumefantrine (median IC50 14.6 vs. 6.9 nM, p  20 nM (p = 0.0002). Targeted sequencing of 819 isolates from 2015-21 identified multiple polymorphisms associated with altered drug susceptibility, notably PfK13 469Y with decreased susceptibility to lumefantrine (p = 6 × 10-8) and PfCRT mutations with chloroquine resistance (p = 1 × 10-20). Our results raise concern regarding activity of artemether-lumefantrine, the first-line antimalarial in Uganda.

Febrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. In this retrospective exploratory study, we used metagenomic next-generation sequencing (mNGS) to evaluate serum, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda. The most common microbes identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from serum; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. We also report the near complete genome of a highly divergent orthobunyavirus, tentatively named Nyangole virus, identified from the serum of a child diagnosed with malaria and pneumonia, a Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis, and the genomes of two novel human rhinovirus C species. In this retrospective exploratory study, mNGS identified multiple potential pathogens, including 3 new viral species, associated with fever in Ugandan children.

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.

Polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene impact sensitivity to multiple antimalarials. In Africa, polymorphisms at N86Y and D1246Y are common and have various impacts on sensitivity to different drugs. To gain insight into the fitness consequences of these polymorphisms, we cultured parasites isolated from children with malaria in Tororo, Uganda, where the multiplicity of infection is high, and used pyrosequencing to follow polymorphism prevalences in culture over time. Of 71 cultures, parasites in 69 were successfully analyzed at N86Y and parasites in 68 were successfully analyzed at D1246Y over 3 to 36 days of culture. For position 86, the sequences of 39/69 (56.5%) parasites remained stable (>90% prevalence over 2 to 17 time points), with 82.1% of these being stable for the 86Y mutation. For position 1246, the sequences of 31/68 (45.6%) parasites remained stable, with 64.5% of these being stable for the wild-type D1246 sequence (P = 0.0002 for comparison of stable mutant genotypes for the two alleles). Defining allele selection as a ≥15% change in prevalence between the first and last samples assessed, for position 86, 11 samples showed selection, with selection toward 86Y occurring in 72.7% of alleles; for position 1246, 14 samples showed selection, with selection toward D1246 occurring in 64.3% of alleles (P = 0.11 for comparison of selection of mutations at the two alleles). Among the 7 samples with selection at both alleles, 5 showed selection for both 86Y and D1246. Overall, consistent trends in the direction of selection were seen, although differences were not statistically significant. Our results suggest fitness advantages for parasites with the pfmdr1 86Y mutation and wild-type D1246, highlighting the complex interplay between drug resistance and fitness in malaria parasites. (This study has been registered at ClinicalTrials.gov under registration no. NCT00948896 and NCT00993031.).

Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of Plasmodium falciparum to artemisinins. The microscopy-based ring-stage survival assay (RSA) provides a laboratory correlate of ART-R but is limited by low throughput and subjectivity of microscopic counts of viable parasites. The extended recovery ring-stage survival assay (eRRSA) replaces microscopy with efficient quantitative PCR (qPCR) readouts but has been studied only with culture-adapted P. falciparum clones. We measured susceptibility to dihydroartemisinin (DHA) after a 6-h incubation with 700-nM DHA, followed by culture without drug, by comparing survival with that of untreated controls by microscopy (the RSA) or qPCR (the eRRSA) and also performed standard growth inhibition (half-maximal inhibitory concentration [IC 50 ]) assays for 122 P. falciparum isolates freshly collected in eastern and northern Uganda from March to July 2022. The median values for RSA survival, eRRSA fold change, and DHA IC 50 were 3.0%, 46.2, and 3.2 nM, respectively. RSA percent survival and eRRSA fold changes correlated strongly (Spearman correlation coefficient [ r s ] = -0.7411, P < 0.0001), with modest associations between the presence of validated P. falciparum Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, P = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, P = 0.003) results. Significant correlations were also observed between DHA IC 50 values and both RSA percent survival ( r s = 0.4235, P < 0.0001) and eRRSA fold changes ( r s = -0.4116, P < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh P. falciparum isolates, providing similar results with improved throughput.

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P . falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC 50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC 50 s (31/750 with IC 50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility., Competing Interests: Benjamin Blasco, Maelle Duffey, and Melanie Rouillier were employed by MMV, who funded part of the study.