Temporal changes in prevalence of molecular markers mediating antimalarial drug resistance in a high malaria transmission setting in Uganda.

Standard therapy for malaria in Uganda changed from chloroquine to chloroquine + sulfadoxine-pyrimethamine in 2000, and artemether-lumefantrine in 2004, although implementation of each change was slow. Plasmodium falciparum genetic polymorphisms are associated with alterations in drug sensitivity. We followed the prevalence of drug resistance-mediating P. falciparum polymorphisms in 982 samples from Tororo, a region of high transmission intensity, collected from three successive treatment trials conducted during 2003-2012, excluding samples with known recent prior treatment. Considering transporter mutations, prevalence of the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles decreased over time. Considering antifolate mutations, the prevalence of pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E were consistently high; pfdhfr 164L and pfdhps 581G were uncommon, but most prevalent during 2008-2010. Our data suggest sequential selective pressures as different treatments were implemented, and they highlight the importance of genetic surveillance as treatment policies change over time

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. METHODS: Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. RESULTS: Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. CONCLUSIONS: These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum

Field evaluation of the Bioline Malaria Ag P.f/Pan rapid diagnostic test: causes of microscopy discordance and performance in Uganda

Background Histidine Rich Protein 2 (HRP2)/pan-Lactate Dehydrogenase (pLDH) combination rapid diagnostic tests (RDTs) may address the shortcomings of RDTs that detect HRP2 alone. However, the relative contribution of the possible causes of discordant results (RDT-negative and microscopy-positive) and performance in field settings across Uganda are poorly quantified. Methods This study utilized samples from two cross-sectional surveys conducted in 32 districts at 64 sites across Uganda between November 2021 and March 2023 that enrolled 6354 febrile participants  ≥ two years of age. Discordant samples (negative by HRP2/pLDH RDT and positive by microscopy) underwent quantitative PCR (qPCR) to detect and quantify parasitaemia. Those confirmed to be positive for Plasmodium falciparum at > 1 parasites/microlitre (p/µL) were tested for pfhrp2 and pfhrp3 deletions using digital PCR. Those that were negative or had P. falciparum detected at ≤ 1 p/µL underwent Plasmodium species testing using nested PCR. The performance of the Bioline Malaria Ag P.f/Pan combination RDT was evaluated by comparison with microscopy and qPCR. Results There were 166 (8.4%) discordant samples out of 1988 microscopy positive samples. Of these, 90/166 (54.2%) were confirmed to contain P. falciparum at levels > 1 p/µL, whereas 76/166 (45.8%) were negative or had P. falciparum levels ≤ 1 p/µL. Only one P. falciparum positive sample was confirmed to have a deletion in pfhrp3. The primary reasons for RDT-negative, microscopy-positive discordance in samples testing negative for P. falciparum by PCR were non-falciparum species (37/76, 48.7%) or false positives by microscopy (31/76, 40.8%). The sensitivity of the Bioline Malaria Ag P.f/Pan combination RDT was high (> 91%) using either microscopy or qPCR as the gold standard. However, specificity was low (56.7%) when microscopy was used as the gold standard; it improved to 64.0% when qPCR was used as the gold standard. Conclusion The Bioline Malaria Ag P.f/Pan combination RDT was found to be highly sensitive in Uganda and reliable for ruling out malaria. False negative RDT results were primarily due to low density P. falciparum infections, non-falciparum infections, or incorrect microscopy results. In contrast, false positive RDT results were common, most likely due to persistent HRP2 antigenaemia in this high transmission setting though causes of false positive RDTs were not investigated. The low specificity of HRP2-based RDTs may result in overuse of anti-malarial drugs and missed diagnoses of non-malarial febrile illnesses

The cost‐effectiveness of prophylaxis strategies for individuals with advanced HIV starting treatment in Africa

Introduction Many HIV‐positive individuals in Africa have advanced disease when initiating antiretroviral therapy (ART) so have high risks of opportunistic infections and death. The REALITY trial found that an enhanced‐prophylaxis package including fluconazole reduced mortality by 27% in individuals starting ART with CD4 <100 cells/mm3. We investigated the cost‐effectiveness of this enhanced‐prophylaxis package versus other strategies, including using cryptococcal antigen (CrAg) testing, in individuals with CD4 <200 cells/mm3 or <100 cells/mm3 at ART initiation and all individuals regardless of CD4 count. Methods The REALITY trial enrolled from June 2013 to April 2015. A decision‐analytic model was developed to estimate the cost‐effectiveness of six management strategies in individuals initiating ART in the REALITY trial countries. Strategies included standard‐prophylaxis, enhanced‐prophylaxis, standard‐prophylaxis with fluconazole; and three CrAg testing strategies, the first stratifying individuals to enhanced‐prophylaxis (CrAg‐positive) or standard‐prophylaxis (CrAg‐negative), the second to enhanced‐prophylaxis (CrAg‐positive) or enhanced‐prophylaxis without fluconazole (CrAg‐negative) and the third to standard‐prophylaxis with fluconazole (CrAg‐positive) or without fluconazole (CrAg‐negative). The model estimated costs, life‐years and quality‐adjusted life‐years (QALY) over 48 weeks using three competing mortality risks: cryptococcal meningitis; tuberculosis, serious bacterial infection or other known cause; and unknown cause. Results Enhanced‐prophylaxis was cost‐effective at cost‐effectiveness thresholds of US$300 and US$500 per QALY with an incremental cost‐effectiveness ratio (ICER) of US$157 per QALY in the CD4 <200 cells/mm3 population providing enhanced‐prophylaxis components are sourced at lowest available prices. The ICER reduced in more severely immunosuppressed individuals (US$113 per QALY in the CD4 <100 cells/mm3 population) and increased in all individuals regardless of CD4 count (US$722 per QALY). Results were sensitive to prices of the enhanced‐prophylaxis components. Enhanced‐prophylaxis was more effective and less costly than all CrAg testing strategies as enhanced‐prophylaxis still conveyed health gains in CrAg‐negative patients and savings from targeting prophylaxis based on CrAg status did not compensate for costs of CrAg testing. CrAg testing strategies did not become cost‐effective unless the price of CrAg testing fell below US$2.30. Conclusions The REALITY enhanced‐prophylaxis package in individuals with advanced HIV starting ART reduces morbidity and mortality, is practical to administer and is cost‐effective. Efforts should continue to ensure that components are accessed at lowest available prices

Late presentation with HIV in Africa : phenotypes, risk, and risk stratification in the REALITY trial

REALITY was funded by the Joint Global Health Trials Scheme (JGHTS) of the UK Department for International Development, the Wellcome Trust, and Medical Research Council (MRC) (grant number G1100693). Additional funding support was provided by the PENTA Foundation and core support to the MRC Clinical Trials Unit at University College London (grant numbers MC_UU_12023/23 and MC_UU_12023/26). Cipla Ltd, Gilead Sciences, ViiV Healthcare/GlaxoSmithKline, and Merck Sharp & Dohme donated drugs for REALITY, and ready-to-use supplementary food was purchased from Valid International. A. J. P. is funded by the Wellcome Trust (grant number 108065/Z/15/Z). J. A. B. is funded by the JGHTS (grant number MR/M007367/1). The Malawi-Liverpool–Wellcome Trust Clinical Research Programme, University of Malawi College of Medicine (grant number 101113/Z/13/Z) and the Kenya Medical Research Institute (KEMRI)/Wellcome Trust Research Programme, Kilifi (grant number 203077/Z/16/Z) are supported by strategic awards from the Wellcome Trust, United Kingdom. Permission to publish was granted by the Director of KEMRI. This supplement was supported by funds from the Bill & Melinda Gates Foundation.Background. Severely immunocompromised human immunodefciency virus (HIV)-infected individuals have high mortality shortly afer starting antiretroviral therapy (ART). We investigated predictors of early mortality and "late presenter" phenotypes. Methods. Te Reduction of EArly MortaLITY (REALITY) trial enrolled ART-naive adults and children =5 years of age with CD4 counts .1). Results. Among 1711 included participants, 203 (12%) died. Mortality was independently higher with older age; lower CD4 count, albumin, hemoglobin, and grip strength; presence of World Health Organization stage 3/4 weight loss, fever, or vomiting; and problems with mobility or self-care at baseline (all P <.04). Receiving enhanced antimicrobial prophylaxis independently reduced mortality (P =.02). Of fve late-presenter phenotypes, Group 1 (n = 355) had highest mortality (25%; median CD4 count, 28 cells/μL), with high symptom burden, weight loss, poor mobility, and low albumin and hemoglobin. Group 2 (n = 394; 11% mortality; 43 cells/μL) also had weight loss, with high white cell, platelet, and neutrophil counts suggesting underlying inflammation/infection. Group 3 (n = 218; 10% mortality) had low CD4 counts (27 cells/μL), but low symptom burden and maintained fat mass. Te remaining groups had 4%-6% mortality. Conclusions. Clinical and laboratory features identifed groups with highest mortality following ART initiation. A screening tool could identify patients with low CD4 counts for prioritizing same-day ART initiation, enhanced prophylaxis, and intensive follow-up.Peer reviewe

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum

Abstract Background Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. Methods Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. Results Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. Conclusions These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum. </jats:sec

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BackgroundAnti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1.MethodsImpacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted.ResultsCo-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184&nbsp;F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact.ConclusionsThese results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum

Impact of Intermittent Preventive Treatment During Pregnancy on Plasmodium falciparum Drug Resistance-Mediating Polymorphisms in Uganda.

BackgroundIn a recent trial of intermittent preventive treatment in pregnancy (IPTp) in Uganda, dihydroartemisinin-piperaquine (DP) was superior to sulfadoxine-pyrimethamine (SP) in preventing maternal and placental malaria.MethodsWe compared genotypes using sequencing, fluorescent microsphere, and quantitative polymerase chain reaction assays at loci associated with drug resistance in Plasmodium falciparum isolated from subjects receiving DP or SP.ResultsConsidering aminoquinoline resistance, DP was associated with increased prevalences of mutations at pfmdr1 N86Y, pfmdr1 Y184F, and pfcrt K76T compared to SP (64.6% vs 27.4%, P &lt; .001; 93.9% vs 59.2%, P &lt; .001; and 87.7% vs 75.4%, P = .03, respectively). Increasing plasma piperaquine concentration at the time of parasitemia was associated with increasing pfmdr1 86Y prevalence; no infections with the N86 genotype occurred with piperaquine &gt;2.75 ng/mL. pfkelch13 propeller domain polymorphisms previously associated with artemisinin resistance were not identified. Recently identified markers of piperaquine resistance were uncommon and not associated with DP. Considering antifolate resistance, SP was associated with increased prevalence of a 5-mutation haplotype (pfdhfr 51I, 59R, and 108N; pfdhps 437G and 581G) compared to DP (90.8% vs 60.0%, P = .001).ConclusionsIPTp selected for genotypes associated with decreased sensitivity to treatment regimens, but genotypes associated with clinically relevant DP resistance in Asia have not emerged in Uganda

Performance of molecular inversion probe DR23K and Paragon MAD4HatTeR Amplicon sequencing panels for detection of Plasmodium falciparum mutations associated with antimalarial drug resistance

Abstract Background Molecular surveillance of drug-resistant Plasmodium falciparum is crucial for malaria control in endemic regions. Two targeted-resequencing tools, the Molecular Inversion Probe (MIP) drug resistance panel DR23K and the Multiplexed Amplicons for Drugs, Diagnostics, Diversity, and Differentiation using High-Throughput Targeted Resequencing (MAD4HatTeR) panel, are widely used to detect resistance genotypes. However, comparisons of their performance for genotyping drug resistance polymorphisms in malaria parasites and their comparative utility for other use cases is lacking. Methods To compare the performance of DR23K and MAD4HatTeR in terms of sequencing depth, sensitivity to minor alleles, and precision, each platform was used to evaluate SNP alleles and microhaplotypes in double- and triple-strain mixtures of well-characterized laboratory parasites at densities of 10, 100, 1000, and 10,000 parasites/μL. In addition, 67 Ugandan field samples collected in 2022 were genotyped using each platform to assess performance and concordance. Results Across the four parasite densities of 10, 100, 1000, and 10,000 parasites/μL, MAD4HatTeR exhibited superior sequencing depth (mean reads per locus: 144, 992, 1153, and 1300) compared to DR23K (mean unique molecular identifiers [UMIs] per locus: 1, 4, 49, and 364). For SNP detection, MAD4HatTeR achieved 100% sensitivity at 2% within-sample allele frequency (WSAF) at 1000 and 10,000 parasites/μL, whereas DR23K achieved 100% sensitivity at 40% and 5% WSAF at these densities, respectively. Microhaplotype sensitivity was lower for both assays; MAD4HatTeR reached 69% sensitivity at 10 parasites/μL when WSAF was ≥ 10%, increasing to 100% sensitivity at 2% WSAF and 100 parasites/μL. DR23K had < 50% sensitivity at 10 and 100 parasites/μL. In field samples, which commonly contain polyclonal infections, high concordance was observed between the two methods for all SNPs (94%, 1848/1969) and polymorphic SNPs (88%, 898/1019). All discrepancies were attributed to varied detection of minority alleles in mixed genotype infections. Conclusions MAD4HatTeR demonstrated higher sensitivity than DR23K, particularly at low parasite densities. Both assays showed strong concordance for genotyping key resistance mutations in field samples, supporting their reliability. These findings suggest MAD4HatTeR as the preferred assay for low-density parasite studies and microhaplotype analysis, while DR23K may be appropriate for specific applications with high-parasite density samples, where detection of minority alleles is not prioritized, or when more comprehensive genome coverage is required

Natural killer cell antibody‐dependent cellular cytotoxicity to <i>Plasmodium falciparum</i> is impacted by cellular phenotypes, erythrocyte polymorphisms, parasite diversity and intensity of transmission

Publication status: PublishedFunder: Stanford University Maternal Child Health Research InstituteAbstractObjectivesNatural killer (NK) cells make important contributions to anti‐malarial immunity through antibody‐dependent cellular cytotoxicity (ADCC), but the role of different components of this pathway in promoting NK cell activation remains unclear.MethodsWe compared the functions and phenotypes of NK cells from malaria‐exposed and malaria‐naive donors, and then varied the erythrocyte genetic background, Plasmodium falciparum strain and opsonising plasma used in ADCC to observe their impacts on NK cell degranulation as measured by CD107a mobilisation.ResultsNatural killer cells from malaria‐exposed adult Ugandan donors had enhanced ADCC, but an impaired pro‐inflammatory response to cytokine stimulation, compared to NK cells obtained from malaria‐naive adult North American donors. Cellular phenotypes from malaria‐exposed donors reflected this specialisation for ADCC, with a compartment‐wide downregulation of the Fc receptor γ‐chain and enrichment of highly differentiated CD56dim and CD56neg populations. NK cell degranulation was enhanced in response to opsonised P. falciparum schizonts cultured in sickle cell heterozygous erythrocytes relative to wild‐type erythrocytes, and when using opsonising plasma collected from donors living in a high transmission area compared to a lower transmission area despite similar levels of 3D7 schizont‐specific IgG levels. However, degranulation was lowered in response to opsonised field isolate P. falciparum schizonts isolated from clinical malaria infections, compared to the 3D7 laboratory strain typically used in these assays.ConclusionThis work highlights important host and parasite factors that contribute to ADCC efficacy that should be considered in the design of ADCC assays.</jats:sec