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. M alaria, in particular disease caused by Plasmodium falciparum, remains an overwhelming problem in most of subSaharan Africa (1, 2). Malaria control was greatly limited by resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP), leading to adoption of artemisinin-based combination therapy (ACT) as the standard treatment for uncomplicated falciparum malaria in the last decade (3). ACT consists of a rapid-acting artemisinin derivative plus a longer-acting partner drug that clears parasites not eliminated by the artemisinin component and limits selection of artemisinin resistance (4, 5). In nearly all countries in sub-Saharan Africa, either artemether-lumefantrine (AL) or artesunate-amodiaquine (AS-AQ) is recommended to treat uncomplicated malaria (6). Other ACTs are dihydroartemisinin (DHA)-piperaquine (DP), a first-line therapy in some countries in Asia, with particular promise for malaria prevention due to the extended halflife of piperaquine (7), and artesunate-mefloquine (AS-MQ), which is used in some countries in Asia and South America. In Uganda, AL was named the national ma...

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.

Dihydroartemisinin-piperaquine (DP) has demonstrated excellent efficacy for the treatment and prevention of malaria in Uganda. However, resistance to both components of this regimen has emerged in Southeast Asia. The efficacy of artemether-lumefantrine, the first-line regimen to treat malaria in Uganda, has also been excellent, but continued pressure may select for parasites with decreased sensitivity to lumefantrine. To gain insight into current drug sensitivity patterns, ex vivo sensitivities were assessed and genotypes previously associated with altered drug sensitivity were characterized for 58 isolates collected in Tororo, Uganda, from subjects presenting in 2016 with malaria from the community or as part of a clinical trial comparing DP chemoprevention regimens. Compared to community isolates, those from trial subjects had lower sensitivities to the aminoquinolines chloroquine, monodesethyl amodiaquine, and piperaquine and greater sensitivities to lumefantrine and mefloquine, an observation consistent with DP selection pressure. Compared to results for isolates from 2010 to 2013, the sensitivities of 2016 community isolates to chloroquine, amodiaquine, and piperaquine improved (geometric mean 50% inhibitory concentrations [IC 50 ] ϭ 248, 76.9, and 19.1 nM in 2010 to 2013 and 33.4, 14.9, and 7.5 nM in 2016, respectively [P Ͻ 0.001 for all comparisons]), the sensitivity to lumefantrine decreased (IC 50 ϭ 3.0 nM in 2010 to 2013 and 5.4 nM in 2016 [P Ͻ 0.001]), and the sensitivity to dihydroartemisinin was unchanged (IC 50 ϭ 1.4 nM). These changes were accompanied by decreased prevalence of transporter mutations associated with aminoquinoline resistance and low prevalence of polymorphisms recently associated with resistance to artemisinins or piperaquine. Antimalarial drug sensitivities are changing in Uganda, but novel genotypes associated with DP treatment failure in Asia are not prevalent.

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.

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 < 0.0001) and dihydroartemisinin (2.3 vs. 1.5 nM, p = 0.003) was decreased in northern vs. eastern Uganda; 14/49 northern vs. 0/38 eastern isolates had lumefantrine IC50 > 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.

bPolymorphisms 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.)

44Background: Febrile illness is a major burden in African children, and non-malarial causes of 45 fever are uncertain. We built and employed IDseq, a cloud-based, open access, bioinformatics 46 platform and service to identify microbes from metagenomic next-generation sequencing of 47 tissue samples. In this pilot study, we evaluated blood, nasopharyngeal, and stool specimens 48 from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, 49Uganda. 50 51 Results: The most common pathogens identified were Plasmodium falciparum (51.1% of 52 samples) and parvovirus B19 (4.4%) from blood; human rhinoviruses A and C (40%), 53 respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; 54 and rotavirus A (50% of those with diarrhea) from stool. Among other potential pathogens, we 55 identified one novel orthobunyavirus, tentatively named Nyangole virus, from the blood of a 56 child diagnosed with malaria and pneumonia, and Bwamba orthobunyavirus in the nasopharynx 57 of a child with rash and sepsis. We also identified two novel human rhinovirus C species. 58 59 Conclusions: This exploratory pilot study demonstrates the utility of mNGS and the IDseq 60 platform for defining the molecular landscape of febrile infectious diseases in resource limited 61 areas. These methods, supported by a robust data analysis and sharing platform, offer a new tool 62 for the surveillance, diagnosis, and ultimately treatment and prevention of infectious diseases. 63 64To address these challenges, we developed IDseq, a cloud-based open-source 90 bioinformatics platform and service for detection of microbial pathogens from metagenomic 91 next-generation sequencing (mNGS) data. IDseq requires minimal computational hardware and 92 is designed to enhance accessibility and build informatics capacity in resource limited regions. 93Here, we leveraged IDseq and mNGS data to perform an exploratory proof-of-concept molecular 94 survey of febrile children in rural Uganda to characterize pathogens associated with fever, 95 including both well-recognized and novel causes of illness. 96 97 Analyses/Results 98

In many malaria‐endemic regions, current detection tools are inadequate in diagnostic accuracy and accessibility. To meet the need for direct, phenotypic, and automated malaria parasite detection in field settings, a portable platform to process, image, and analyze whole blood to detect Plasmodium falciparum parasites, is developed. The liberated parasites from lysed red blood cells suspended in a magnetic field are accurately detected using this cellphone‐interfaced, battery‐operated imaging platform. A validation study is conducted at Ugandan clinics, processing 45 malaria‐negative and 36 malaria‐positive clinical samples without external infrastructure. Texture and morphology features are extracted from the sample images, and a random forest classifier is trained to assess infection status, achieving 100% sensitivity and 91% specificity against gold‐standard measurements (microscopy and polymerase chain reaction), and limit of detection of 31 parasites per µL. This rapid and user‐friendly platform enables portable parasite detection and can support malaria diagnostics, surveillance, and research in resource‐constrained environments.