Changes in susceptibility ofPlasmodium falciparumto antimalarial drugs in Uganda over time: 2019-2024

Changes in susceptibility of Plasmodium falciparum to antimalarial drugs in Uganda over time: 2019-2024 | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Changes in susceptibility of Plasmodium falciparum to antimalarial drugs in Uganda over time: 2019-2024 Martin Okitwi , Stephen Orena , Patrick K. Tumwebaze , Thomas Katairo , Yoweri Taremwa , Oswald Byaruhanga , Stephen Tukwasibwe , Samuel L. Nsobya , Jennifer Legac , Roland A. Cooper , View ORCID Profile Melissa D. Conrad , View ORCID Profile Philip J. Rosenthal doi: https://doi.org/10.1101/2024.12.31.24319821 Martin Okitwi 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen Orena 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Patrick K. Tumwebaze 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas Katairo 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yoweri Taremwa 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Oswald Byaruhanga 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen Tukwasibwe 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Samuel L. Nsobya 1 Infectious Diseases Research Collaboration , Kampala, Uganda ; PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Legac 2 Department of Medicine, University of California , San Francisco, CA, USA ; BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Roland A. Cooper 3 Department of Natural Sciences and Mathematics, Dominican University of California , San Rafael, CA, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Melissa D. Conrad 2 Department of Medicine, University of California , San Francisco, CA, USA ; PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Melissa D. Conrad For correspondence: philip.rosenthal{at}ucsf.edu melissa.conrad{at}ucsf.edu Philip J. Rosenthal 2 Department of Medicine, University of California , San Francisco, CA, USA ; MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philip J. Rosenthal For correspondence: philip.rosenthal{at}ucsf.edu melissa.conrad{at}ucsf.edu Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Background The treatment and control of malaria in Africa is challenged by drug resistance, including Plasmodium falciparum transporter, folate pathway, and PfK13 mutations that mediate resistance to aminoquinolines, antifolates, and artemisinins, respectively. Characterization of drug susceptibility informs optimal control strategies. Methods We characterized ex vivo susceptibilities to nine drugs of isolates collected from individuals presenting with uncomplicated falciparum malaria in eastern (2019-2024) and northern (2021-2024) Uganda using a growth inhibition assay and the dihydroartemisinin (DHA) ring survival assay (RSA). Genetic polymorphisms were characterized by molecular inversion probe and dideoxy sequencing. We assessed drug susceptibilities over time and evaluated associations between susceptibilities and potential resistance markers for samples studied since 2016. Results Of 1,297 collected, 724/828 eastern and 390/469 northern Uganda isolates were successfully evaluated for ex vivo drug susceptibilities. Median half-maximal inhibitory concentrations (IC 50 s) were low-nanomolar for chloroquine, monodesethylamodiaquine, piperaquine, pyronaridine, lumefantrine, mefloquine, and DHA, but higher for quinine and pyrimethamine. Over time, susceptibilities improved for chloroquine, decreased for lumefantrine, mefloquine, and DHA, and were unchanged for other drugs. Changes in prevalences of known markers of altered drug susceptibility followed the same patterns. Genotypes associated with drug susceptibility were those previously identified for aminoquinolines and pyrimethamine. For lumefantrine, susceptibility was decreased with wild-type PfCRT K76T or PfMDR1 N86Y, mutant PfK13 C469Y or A675V, the newly identified PfCARL D611N mutation, which increased in prevalence over time, and a number of other polymorphisms. For DHA, RSA results were not associated with PfK13 mutations, but susceptibilities based on IC 50 s were decreased in parasites with the PfK13 C469Y or A675V mutations and the newly identified PfMDR1 Y500N mutation. Interpretation Susceptibilities to antimalarial drugs were mostly excellent, but decreased activities of lumefantrine and DHA over time suggest potential loss of efficacies of leading regimens. Funding National Institutes of Health, Medicines for Malaria Venture, Gates Foundation. Evidence before this study We searched PubMed for combinations of the terms “antimalarial resistance”, “malaria”, “ Plasmodium ”, “Africa”, “ex vivo”, “pfmdr1”, “pfcrt”, “kelch”, or “K13” and identified papers published between Jan 1, 2020, and Dec 30, 2024 on antimalarial drug sensitivity and resistance in Africa. A prior identical search was conducted for papers published from Jan 1, 2000 to Dec 31, 2020 in preparation for an earlier publication. We reviewed and included any relevant articles cited in those references. Our search identified many studies on antimalarial drugs and molecular markers of resistance, but few combining ex vivo drug susceptibility with genotyping results. Added value of this study This study provides a comprehensive assessment of ex vivo susceptibility of Ugandan Plasmodium falciparum parasites to nine antimalarial drugs from July, 2019 to June, 2024. It also characterized genotype-phenotype associations based on these ex vivo data and sequencing of 80 genes identified as potential resistance mediators. Our findings add value to the existing literature by providing comprehensive data on antimalarial drug susceptibility in Uganda, including ex vivo drug susceptibilities for >1100 isolates from two regions of the country, description of changes in drug susceptibilities over time, and characterisation of genotype-phenotype associations, considering genetic polymorphisms previously associated with resistance to various antimalarials and potential novel resistance mediators. Implication of all the available evidence Malaria parasites circulating in eastern and northern Uganda over the past five years were mostly sensitive to commonly used antimalarial drugs. However, parasite genotypes and phenotypes have changed over time. Most importantly, susceptibilities to dihydroartemisinin and lumefantrine, the components of the first-line antimalarial therapy in Uganda, have decreased over time, although the magnitudes of these decreases are modest, and the clinical implications of the results are uncertain. Continued performance of parasitological and genomic surveillance for evidence of antimalarial drug resistance and institution of policy changes to limit resistance selection and treatment failure should be high priorities. Introduction After good progress early this century, the control of malaria in Africa, the region responsible for ∼95% of malaria morbidity and mortality, has stalled. 1 A challenge to malaria control and eventual elimination is increasing resistance of malaria parasites, in particular Plasmodium falciparum , to available drugs. 2 Of particular concern is potential resistance to artemisinin-based combination therapies (ACTs), including artemether-lumefantrine and artesunate-amodiaquine, the most widely used therapies for uncomplicated malaria in Africa, and the alternative ACTs dihydroartemisinin (DHA)-piperaquine, artesunate-pyronaridine, and artesunate-mefloquine. In addition, sulfadoxine-pyrimethamine (SP) has an important role in malaria control, including intermittent preventive therapy in pregnant women, perennial malaria chemoprevention in infants, and, in combination with amodiaquine, seasonal malaria chemoprevention in children. 3 Increasing resistance to artemisinins, ACT partner drugs, and SP threatens effective treatment and prevention of malaria across Africa. Three categories of antimalarial drug resistance are highly relevant for Africa. 4 , 5 First, resistance to chloroquine and amodiaquine is mediated principally by the PfCRT K76T and PfMDR1 N86Y mutations, which alter drug transport. 6 The prevalence of these mutations was previously high across Africa, but it has decreased greatly in many areas. 4 An opposite effect is seen with lumefantrine and mefloquine, with the PfCRT K76T and PfMDR1 N86Y wild-type alleles and pfmdr1 amplification associated with decreased susceptibility. Different mutations in PfCRT and amplification of plasmepsin genes have been associated with resistance to piperaquine, a related aminoquinoline, but to date only in southeast Asia 5 and South America. 7 Second, partial resistance to artemisinins (ART-R) became well established in the Greater Mekong Subregion of southeast Asia early this century. 8 ART-R manifests as delayed parasite clearance after therapy with artemisinins and enhanced parasite survival after in vitro exposure to DHA, and is mediated principally by any of ∼20 mutations in the P. falciparum kelch (PfK13) protein propeller domain. 9 Multiple PfK13 mutations previously validated as mediators of ART-R have emerged in Africa, including R561H in Rwanda, western Tanzania, and southwestern Uganda; C469Y and A675V in northern Uganda; and R622I in Eritrea and Ethiopia. However, these mutations have not been clearly linked to decreased clinical efficacy of ACTs in Africa, and associations between PfK13 mutations and resistance phenotypes do not appear to be as straightforward in Africa as in southeast Asia. 9 Third, resistance to SP, mediated principally by mutations in the target enzymes dihydrofolate reductase (PfDHFR N51I, C59R, S108N) and dihydropteroate synthase (PfDHPS A437G, K540E), is common in much of Africa, with two additional mutations, PfDHFR I164L and PfDHPS A581G, which mediate high-level SP resistance, 10 spreading across parts of east Africa. 4 , 11 In Uganda, serial surveillance has shown loss of the PfCRT and PfMDR1 mutations that mediate resistance to chloroquine and amodiaquine, emergence of five validated or candidate PfK13 ART-R mutations in different parts of the country, and increasing prevalence of the PfDHFR I164L and PfDHPS A437G SP-resistance mutations. 11 , 12 Prior ex vivo studies in Tororo, in eastern Uganda, showed generally good activities of studied antimalarials, except for the PfDHFR inhibitor pyrimethamine. 13 , 14 However, modest decreases in susceptibility to DHA and lumefantrine, which may challenge the efficacy of artemether-lumefantrine, the first-line malaria therapy, were recently seen in isolates from northern Uganda. 15 Most clinical trials have shown excellent efficacies of leading ACTs in Uganda, 16 but a recent trial at a site without known ART-R at the time of the study reported <90% genotype-corrected efficacy for artemether-lumefantrine, 17 although genotyping to assign outcomes is challenging in such high transmission regions. Considering the dynamic nature of antimalarial drug sensitivity in Africa, it is important to maintain surveillance for emerging drug resistance. For this reason, we have regularly assessed ex vivo drug susceptibilities and genotypes of P. falciparum causing malaria in eastern Uganda since 2010 and in northern Uganda since 2021, and we report here results of surveillance conducted over the last five years. Methods Isolates for study We collected blood from patients diagnosed with uncomplicated malaria between July, 2019 and June, 2024 at three sites in eastern Uganda (Tororo District Hospital, Tororo District; Masafu General Hospital, Busia District; and Busiu Health Centre IV, Mbale District) and assessed drug susceptibilities at our laboratory in Tororo ( Figure 1 ). We added collection of samples from patients with uncomplicated malaria in Agago District, in northern Uganda, first with transport of samples from Patongo Health Centre IV to our Tororo laboratory from May to July, 2021 and April to July, 2022, and since January, 2023 with samples from Patongo Health Centre IV and Dr. Ambrosoli Memorial Hospital, Kalongo assessed in a new laboratory at the hospital. To increase sample size, for some comparisons over time and genotype-phenotype association studies we extended our analyses to include 1668 samples collected since 2016; results for samples collected from June, 2016 to July, 2019 were published previously. 14 Patients reporting antimalarial treatment within the previous 30 days or infected with non-falciparum species were excluded. Written informed consent was obtained from adults and parents or guardians of children <18 years; children aged 8–17 years provided assent. Venous blood (2-5 ml) was collected in a heparin tube, after which participants were treated with artemether-lumefantrine, following national guidelines. The study was approved by the Makerere University School of Biomedical Sciences Research and Ethics Committee, the Uganda National Council for Science and Technology, and the University of California, San Francisco Committee on Human Research. Download figure Open in new tab Figure 1. Study sites. Samples were collected at the indicated health facilities and at clinics adjacent to the two indicated laboratories. Procedures Samples containing only P. falciparum and at least 0·2% parasitaemia by Giemsa-stained thin smear were analysed. Samples were centrifuged, buffy coats removed, and erythrocyte pellets washed 3X with wash medium (RPMI 1640 with 25 mM HEPES, 24 mM NaHCO 3 , 10 μg/mL gentamicin) and resuspended in complete culture medium (RPMI 1640 with 25 mM HEPES, 24 mM NaHCO 3 , 0·1 mM hypoxanthine, 10 μg/mL gentamicin, and 0·5% AlbuMAX II (Thermo Fisher Scientific)) to produce a haematocrit of 50%, and aliquots were spotted onto filter paper for molecular analysis. Drug susceptibilities were assessed using a 72 h growth inhibition assay with SYBR Green detection. 14 Briefly, study compounds (chloroquine, monodesethylamodiaquine (MDAQ, the active metabolite of amodiaquine), piperaquine, pyronaridine, mefloquine, lumefantrine, DHA, quinine, and pyrimethamine), supplied by Medicines for Malaria Venture, were dissolved in dimethyl sulfoxide (water for chloroquine) as 10 mM stocks (50 mM for pyrimethamine) stored at -20°C, and three-fold serial dilutions in complete medium were placed in 96-well microplates (50 μL per well), including drug-free and parasite-free controls. Cultures were diluted with uninfected erythrocytes to 200 μL per well at 0·2% parasitaemia and 2% haematocrit. Plates were maintained at 5% CO 2 , 5% O 2 , and 90% N 2 for 72 h at 37°C in a humidified modular incubator. After 72 h, 100 μL resuspended culture per well was transferred to black 96-well plates containing 100 μL SYBR Green lysis buffer (20 mM Tris, 5 mM EDTA, 0·008% saponin, 0·08% Triton X-100, and 0·2 μL/mL SYBR Green I) per well and mixed, plates were incubated for 1 h in the dark at room temperature, and fluorescence (485 nm excitation and 530 nm emission) was measured. IC 50 values were derived from plots of fluorescence intensity vs. log drug concentration and fit to non-linear curves using a four-parameter Hill equation in Prism. Z factors to assess well-to-well variability and signal-to-noise ratios were calculated as previously described. 14 When steep slopes resulted in poor curve fit, slopes were fixed to a constant value of -6. For results with incomplete curves at low drug concentrations but at least 50% of the curve present, the upper plateau was constrained to that of drug-free wells on the same plate. Control P. falciparum Dd2 (MRA-156) and 3D7 (MRA-102) strains from MR4/BEI Resources were maintained in culture, synchronised with a magnetic column, and assayed, beginning at the ring-stage, approximately monthly. The ex-vivo ring-stage survival assay, which entails comparing parasitaemias 66 h after a 6 h incubation with 700 nM DHA with that of untreated controls, was performed as previously described. 14 For genotyping, parasite DNA was extracted from filter paper blood spots using Chelex-100 and analysed by molecular inversion probe (MIP) capture and deep sequencing, using a MIP panel with probes targeting 80 genes of interest, as previously described. 11 To resolve ambiguities, some PfK13 sequences were additionally analyzed by dideoxy sequencing. Sequencing reads are available in the National Center for Biotechnology Information Archive (BioProject PRJNA850445). Raw sequencing data were analysed using MIPTools. Copy numbers were estimated based on sample and probe normalised depth of coverage from 31 unique probes for pfmdr1 and 21 probes for plasmepsin 2/3 ; copy number >1.7 was considered multiple. The Dd2 strain, which has amplified pfmdr1 and is single copy for plasmepsin 2/3 , and the G8 clone of Cambodian isolate KH001 _ 053, with one copy of pfmdr1 and two copies of plasmepsin 2/3 , were included as controls. Statistical analysis All statistical tests were done in R Studio (version 2024.09.1+394). Baseline characteristics of participants and isolates were computed as frequencies, medians with interquartile ranges (IQR), or means with standard deviations. Summary statistics for ex vivo susceptibilities were median IC 50 with IQR. The Mann-Kendall non-parametric test was used to detect monotonic trends (change over time in a consistent positive or negative direction) in drug susceptibilities with the Kendall package, using months as categorical variables. Genotype-phenotype associations for known resistance markers were evaluated with pairwise Wilcoxon texts. Potential novel markers were assessed for all SNPs with data for >50% of isolates available and at least one wild-type and mutant sample identified. The Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons was used to identify loci with different median drug susceptibilities between wild-type, mixed, and mutant isolates, followed by pairwise-Wilcoxon tests with Benjamini-Hochberg correction for multiple comparisons. For loci with at least 20 samples per category, a significant difference between IC 50 s for wild type and mutants was required. Statistical tests were two-tailed, and significance was considered p ≤0·05. Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Results Of 1,297 P. falciparum isolates collected, 724/828 in eastern Uganda and 390/469 in northern Uganda were successfully evaluated for ex vivo drug susceptibilities. Baseline characteristics of subjects were similar over time, although parasitemias were lower and participant ages higher in northern Uganda ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Characteristics of study subjects and samples studied by ex vivo analysis We measured ex vivo susceptibilities of all isolates to nine standard antimalarials. For successful assays, the mean Z factor was 0·75 (SD ± 0·26), indicating robust assays. Median IC 50 values were at low nanomolar levels for chloroquine (12·6 nM), MDAQ (7·8 nM), piperaquine (5·4 nM), DHA (2·9 nM), lumefantrine (11·3), mefloquine (15·2 nM), and pyronaridine (1·5 nM), consistent with potent activity, and higher for quinine (115 nM), which is typically less potent than the other studied compounds, and pyrimethamine (35,100 nM), against which resistance is well-established ( Table 2 ; Figure 2 ). From 2016 to 2024, marked changes in susceptibilities (Mann-Kendall Tau ≥±0·2) were seen for four drugs, with increasing susceptibility for chloroquine and decreasing susceptibilities for DHA, lumefantrine, and mefloquine ( Supplementary Table 1 ). The more marked changes in susceptibilities occurred in eastern Uganda, consistent with a longer period of sample collection in that area and, as suggested by our earlier comparison of the sites, likely decreases in susceptibilities to key drugs in northern Uganda before initiation of studies in that region ( Supplementary Figures 1 and 2 ). 15 Susceptibilities of Dd2 and 3D7 laboratory reference strains yielded IC 50 values similar to those reported previously ( Supplementary Table 2 ). Comparing median values for five drugs studied in both 2010-13 13 and the current study, susceptibilities increased for chloroquine (IC 50 248 to 12·6 nM), MDAQ (76·9 to 7·8 nM), and piperaquine (19·1 to 5·4 nM) and decreased for DHA (1·4 to 2·9 nM) and lumefantrine (3·0 to 11·3 nM; p <0·001 for all comparisons). View this table: View inline View popup Download powerpoint Supplementary Table 1. Statistical analysis of changes in drug susceptibilities over time (2016-2024) View this table: View inline View popup Download powerpoint Supplementary Table 2. Susceptibilities of P. falciparum control strains to antimalarials Download figure Open in new tab Supplementary Figure 1. Ex vivo drug susceptibilities in eastern Uganda over time. Data for samples collected in eastern Uganda are displayed as described for Figure 2 . Download figure Open in new tab Supplementary Figure 2. Ex vivo drug susceptibilities in northern Uganda over time. Data for samples collected in northern Uganda are displayed as described for Figure 2 . Download figure Open in new tab Figure 2. Ex vivo drug susceptibilities in Uganda over time. Paired plots present the distribution of susceptibilities for assayed isolates (left; median values shown) and results over time (right; median values shown for each year). Each dot represents an isolate. Boxes show the first to third quartiles, and whiskers extend to the largest values no more than 1.5X the interquartile ranges. The curves were generated using loess smoothing implemented by geom_smooth; the gray bands represent the 95% confidence intervals. View this table: View inline View popup Download powerpoint Table 2. Summary of drug susceptibility data (2019-2024) RSAs were performed on a subset of isolates, including 126 from eastern and 314 from northern Uganda. Consistent with other recent results from Uganda, 15 but in contrast to results from before 2020 in Uganda 14 , 18 and from southeast Asia, 19 many isolates had survival above previously-established ART-R cut-offs ( Figure 2 ). Overall, 78·2% of isolates had 72-hour survival >1%, and 35·8% survival >5% of control values. RSA survival increased over time, but with the significance criterion of Mann-Kendall Tau ≥±0·2 the increase was significant only in eastern Uganda. Of the 1,113 isolates with ex vivo results, we characterized sequences of 1,070 for known markers of altered drug susceptibility. Prevalences of the PfCRT K76T and PfMDR1 N86Y mutations, which are associated with resistance to chloroquine and amodiaquine, 6 have been decreasing, 11 and were very low in recent years in our studied isolates ( Figure 3 ; Supplementary Table 3 ). Prevalences of two other common PfMDR1 mutations were similar to those reported previously, 11 with stable prevalence of the Y184F mutation, which has generally not been associated with drug susceptibility, but may impact on parasite fitness, 20 and low and decreasing prevalence of the D1246Y mutation. Mutations associated with aminoquinoline resistance in Southeast Asia (PfCRT H97Y, F145I, M343L, G353V 21 ) or South America (PfCRT C350R 7 ) were not detected in any isolates. Increased copy number of pfmdr1 22 or plasmepsin 2/3, 7 , 23 which has been associated with decreased susceptibility to lumefantrine and mefloquine or piperaquine, respectively, was not observed ( Supplementary Table 4 ). Prevalences of five mutations associated with resistance to SP (PfDHFR N51I, C59R, S108N; PfDHPS A437G, K540E) were very high, as has been the case in Uganda for at least two decades, 24 and two additional mutations associated with higher level resistance (PfDHFR I164L, PfDHPS A581G 10 ), and seen in recent years at increasing prevalence in western Uganda, 11 had modest prevalence in both eastern and northern Uganda ( Figure 3 ; Supplementary Table 3 ). The PfK13 C469Y and A675V mutations were first identified in southeast Asia and more recently validated as markers of ART-R in northern Uganda. 9 , 15 , 25 These mutations were at moderate prevalence in northern Uganda at the time of our first collections in 2021 (combined prevalence 30·5%), with stable prevalence since that time. In eastern Uganda, the mutations were at very low prevalence until 2021, with increasing prevalence since that time. Other PfK13 validated or candidate ART-R mutations that have been seen elsewhere in eastern Africa (P441L, C469F, R561H, R622I) were not seen. View this table: View inline View popup Supplementary Table 3. Prevalence of genetic polymorphisms associated with altered drug susceptibility over time at sites in eastern and northern Uganda. View this table: View inline View popup Download powerpoint Supplementary Table 4. Copy number data for genes of interest Download figure Open in new tab Figure 3. Prevalence of genetic polymorphisms associated with altered drug susceptibility over time at sites in eastern and northern Uganda. We searched for associations between genotypes identified by sequencing of 80 candidate genes and drug susceptibility phenotypes, considering available data from 2016-2024, including older results published previously, 15 and we included strict criteria for significant associations. Of greatest interest were results for DHA and lumefantrine. Considering PfK13 mutations previously associated with ART-R, the C469Y and A675V mutations were associated with decreased activity (based on IC 50 s) for DHA and lumefantrine ( Figure 4 , Supplementary Tables 5 and 6 ). Interestingly, these mutations were not associated with RSA results. As described previously 26 the PfMDR1 N86Y wild-type allele was associated with decreased susceptibility to lumefantrine and mefloquine and the PfCRT K76T wild-type allele with decreased susceptibility to lumefantrine, although analyses were limited by low prevalence of mutant genotypes. Multiple other polymorphisms were associated with susceptibilities to DHA and lumefantrine ( Supplementary Tables 5 and 6 ). Strong associations included, for lumefantrine, PfCARL D611N (IC 50 7.5 nM for wild type, 20.2 for mixed, and 44.3 for mutant), which increased in prevalence over time, and, for DHA, PfMDR1 Y500N (IC 50 1.9 nM for wild type, 2.6 for mixed, and 5.4 for mutant; Figure 4 , Supplementary Tables 5 and 6 ). View this table: View inline View popup Download powerpoint Supplementary Table 5. Genotype-phenotype associations for lumefantrine. View this table: View inline View popup Download powerpoint Supplementary Table 6. Genotype-phenotype associations for DHA. Download figure Open in new tab Figure 4. Associations between genotypes of interest and ex vivo drug susceptibility. Drug susceptibility for wild-type (WT), mixed WT/mutant, and pure mutant (Mut) isolates are shown for selected drugs and polymorphisms of interest. Benjamini-Hochberg corrected p-values for pairwise-Wilcoxon tests are indicated. Additional associations are shown in Supplementary Tables 5 - 7 . View this table: View inline View popup Download powerpoint Supplementary Table 7. Genotype-phenotype associations for pyremethamine, mefloquine, chloroquine, and MDAQ. For chloroquine and amodiaquine, consistent with earlier results, the PfCRT K76T mutation ( Figure 4 , Supplementary Table 7 ) and other PfCRT mutations that usually form a haplotype, were associated with decreased activity. For pyrimethamine, susceptibilities were poor with the PfDHFR C59R mutation (IC 50 37,800 nM) and even poorer with the I164L mutation (86,800 nM; Figure 4 , Supplementary Table 7 ). No significant associations were seen between studied genotypes and the ex vivo activities of piperaquine, pyronaridine, or quinine, or DHA activity based on the RSA. Discussion In the face of changing drug susceptibilities, it is critical that we understand the antimalarial efficacies of key drugs in Africa. We have studied the ex vivo activities of antimalarial drugs against freshly isolated P. falciparum in Uganda since 2010. This report offers data collected over the last five years. Commonly used drugs, including DHA, the active metabolite of all clinically relevant artemisinins, and all widely used ACT partner drugs (except SP, which is little used for treatment in Africa) remained active against P. falciparum in standard assays at low nM concentrations, a reassuring result. However, important changes in susceptibilities were seen. Specifically, susceptibility to chloroquine increased, suceptibilities to most other tested drugs remained stable, but susceptibilities to both DHA and the ACT partner drug lumefantrine decreased over time. Absolute changes in susceptibilities to DHA and lumefantrine were modest, and the clinical consequences of these changes are uncertain. Overall, our results offer reassurance that the activities of widely used antimalarials generally remain good, but also raise concern that the efficacies of leading ACTs may be decreasing. Coincident with the emergence and spread of PfK13 mutations previously validated as markers of ART-R, susceptibility to DHA has decreased. However, significant decreases and significant correlations between PfK13 mutations and DHA susceptibility were seen only with the standard DHA growth inhibition assay and not with the RSA, which includes a 6 h incubation with DHA to mimic the short clinical exposure to artemisinins, and was a better indicator of ART-R than DHA IC 50 measurements in southeast Asia. 19 Factors contributing to the lack of significant correlation between PfK13 mutations and RSA survival may have included the polyclonal nature of most Ugandan isolates, technical differences between the ex vivo RSA (which does not include parasite synchronization) and assays with synchronized culture adapted parasites, and our relatively small sample size for RSAs. Of note, enhanced parasite survival in the ex vivo RSA was more common than in studies of Ugandan isolates collected from 2015-20 using the same assay, 18 , 26 suggesting loss of DHA activity over time, and that mediators in addition to PfK13 mutations impact parasite clearance. However, the clinical relevance of these results, in particular modest increases in DHA IC 50 s and enhanced ex vivo RSA survival in the absence of known markers of ART-R, is unclear. Lumefantrine, the partner drug for the most widely used ACT, has demonstrated potent activity and a strong barrier to resistance since initiation of widespread use in Africa about two decades ago. However, lumefantrine susceptibility was lower in isolates collected in 2021 from northern, compared to eastern Uganda, and susceptibility was tightly correlated with prevalence of the PfK13 C469Y and A675V mutations, which at that time had high prevalence in northern, but not eastern Uganda. 15 Since that time, prevalence of the C469Y and A675V mutations has increased and lumefantrine susceptibility has decreased in eastern Uganda, 12 and prevalence of these mutations was again tightly correlated with lumefantrine susceptibility. Of interest, a P. falciparum infection acquired in Uganda in 2022 was noteworthy for carriage of the PfK13 A675V mutation, decreased in vitro activity of lumefantrine, enhanced RSA survival, and multiple failures of therapy with artemether-lumefantrine. 27 There is no direct evidence that decreased lumefantrine susceptibility is mediated by PfK13 mutations, but susceptibility has clearly decreased coincident with emergence and spread of ART-R. We assessed associations between polymorphisms in 80 genes known or suspected to be linked to antimalarial drug resistance and ex vivo drug susceptibility. We confirmed previously identified associations for chloroquine and MDAQ (decreased susceptibility with the PfCRT K76T mutation), lumefantrine and mefloquine (decreased susceptibility with the wild-type PfMDR1 N86Y allele), and pyrimethamine (stepwise decreased susceptibility with the PfDHFR C59R and I164L mutations). These results suggest improved activity of ASAQ, consistent with recent therapeutic efficacy studies, modest decreases in lumefantrine activity, and minimal activity for pyrimethamine as a component of SP. As shown previously, the PfK13 C469Y and A675V mutations were associated with decreased susceptibility to both DHA and lumefantrine. 15 Among other studied polymorphisms, multiple associations were seen, and of interest were strong associations for the PfCARL D611N mutation for lumefantrine and the PfMDR1 Y500N mutation for DHA; whether these polymorphisms directly impact drug susceptibility is unknown. Our new data offer insights into mediators of drug susceptibility and the consequences of changing P. falciparum genetics in Uganda over time. First, for chloroquine and amodiaquine, drug susceptibility remains strongly correlated with the primary resistance mediator, the PfCRT K76T mutation, but this mutation is now very uncommon in Uganda. Second, susceptibility to DHA and lumefantrine was significantly decreased in parasites with the PfK13 C469Y or A675V mutations, which now have combined prevalences approaching 50% in much of Uganda. 12 Third, lumefantrine susceptibility was also modestly, but significantly decreased in parasites with the wild-type PfMDR1 N86Y sequence, which is now nearly universal in Uganda. Fourth, other polymorphisms associated with susceptibility to DHA and lumefantrine were identified, highlighting additional potential resistance mediators. Fifth, pyrimethamine susceptibility was poor for isolates containing three common PfDHFR mutations and even worse for those that also contained the I164L mutation, suggesting that, consistent with recent studies from Uganda, 28 the antimalarial protective efficacy of SP will be poor. Taken together, our results indicate a number of polymorphisms with clear impacts on P. falciparum drug susceptibility and evidence that susceptibilities to the components of artemether-lumefantrine have decreased. Thus, continued performance of parasitological and genomic surveillance for evidence of antimalarial drug resistance and institution of policy changes to limit resistance selection and treatment failure are high priorities. 29 Data Availability All data produced in the present study are available upon reasonable request to the authors. https://github.com/bailey-lab Contributors MO, SO, PKT, TK, YT, OB, JL, and MDC performed parasitology and genomics experiments and archived data. JL provided administrative and logistical support. ST, SLN, RAC, MDC, and PJR provided oversight for all experiments. MDC led analysis of the data. All authors contributed to the writing of the manuscript. All authors had full access to all the data and the corresponding authors had final responsibility for the decision to submit for publication. Declaration of interests We declare no competing interests. Data sharing Raw sequencing reads for PfK13 are available in the NCBI Sequence Read Archive (BioProject PRJNA850445). MIP probes and PCR primers used in this study are listed in appendix 2 (pp 2–3) of reference 14. MIPWrangler and MIPTools software is available on GitHub ( https://github.com/bailey-lab ). All additional data are available by contacting the corresponding authors. Acknowledgments The study was funded by the National Institutes of Health (R01AI075045, R01AI173557, U19AI089674, RO1AI117001, and R01AI139179), the Medicines for Malaria Venture (RD/15/0001), and the Gates Foundation (INV-035751). We thank study participants and staff members of the clinics where samples were collected. We thank Patrick Angutoko, Jackson Asiimwe, Evans Muhanguzi, Solomon Opio, Innocent Tibagambirwa, Frida G. Ceja, Shreeya Garg, and Sevil Chelebieva for performance of laboratory studies in Uganda, Bienvenu Nsengimaana, David Giesbrecht, Rebecca Crudale, Alfred Simkin, Oriana Kreutzfeld, and Jeffrey A. Bailey for assistance with deep sequencing and genomic analyses, and Selina Bopp for the gift of the control KH001 _ 053 clone. References 1. ↵ World Health Organization . World Malaria Report . Geneva : World Health Organization ; 2024 . 2. ↵ Duffey M , Shafer RW , Timm J , et al. Combating antimicrobial resistance in malaria, HIV and tuberculosis . Nat Rev Drug Discov 2024 ; 23 : 461 – 79 . OpenUrl PubMed 3. ↵ Plowe CV . Malaria chemoprevention and drug resistance: a review of the literature and policy implications . Malar J 2022 ; 21 : 104 . 4. ↵ Conrad MD , Rosenthal PJ . Antimalarial drug resistance in Africa: the calm before the storm? Lancet Infect Dis 2019 ; 19 : e338 – e51 . 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