Pfmdr1 N86Y Research Articles

Changes in susceptibility of Plasmodium falciparum to antimalarial drugs in Uganda over time: 2019-2024.

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. 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. 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. Susceptibilities to antimalarial drugs were mostly excellent, but decreased activities of lumefantrine and DHA over time suggest potential loss of efficacies of leading regimens. 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.

Trend of N86Y and Y184F Mutations in Pfmdr1 Gene in Children Under Seasonal Malaria Chemoprevention Coverage in Nanoro, Burkina Faso

BackgroundSeasonal malaria chemoprevention (SMC) is an effective malaria preventive intervention in sub-Sahara Africa. However, as with any other drug-based intervention, the large-scale deployment of this strategy could lead to Amodiaquine plus Sulfadoxine-Pyrimethamine (AQSP) drug pressure on the circulating parasites population with selection for specific alleles that could compromise the impact of the intervention in the near future. This study aimed to assess the distribution of the Pfmdr1 mutation involved in resistance to AQ before and after the annual campaign of SMC in the health district of Nanoro.MethodsRandomly selected dried blood spots collected prior (n = 100) and after (n = 100) the 2021 SMC campaign were used for the detection of mutation in codons 86 and 184 of the Pfmdr1 gene using a nested PCR with restriction fragment length polymorphism approach.ResultsNo significant change in the prevalence of Pfmdr1 N86Y mutation was observed before and after the SMC campaign (p = 0.28). The mutant allele 86Y was observed at low prevalences, representing only 2.17% and 6.12%, respectively, before and after the SMC campaign. Patients harboring the mutant Pfmdr1 86Y allele exhibited higher parasite densities compared to patients with the wild-type Pfmdr1 N86 allele (p = 0.04). A significant increase in the prevalence of the mutant allele 184 F was observed in the period before and after the SMC campaign (p = 0.03).ConclusionThis selective pressure needs to be closely monitored in order to preserve the efficacy of this intervention for a long-term period in Burkina Faso.

Varied Prevalence of Antimalarial Drug Resistance Markers in Different Populations of Newly Arrived Refugees in Uganda.

Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Coinfection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G, K540E, and A581G) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in 2 previously little-studied countries.

Ex vivo drug susceptibility and resistance mediating genetic polymorphisms of Plasmodium falciparum in Bobo-Dioulasso, Burkina Faso.

Malaria remains a leading cause of morbidity and mortality in Burkina Faso, which utilizes artemether-lumefantrine as the principal therapy to treat uncomplicated malaria and seasonal malaria chemoprevention with monthly sulfadoxine-pyrimethamine plus amodiaquine in children during the transmission season. Monitoring the activities of available antimalarial drugs is a high priority. We assessed the ex vivo susceptibility of Plasmodium falciparum to 11 drugs in isolates from patients presenting with uncomplicated malaria in Bobo-Dioulasso in 2021 and 2022. IC50 values were derived using a standard 72 h growth inhibition assay. Parasite DNA was sequenced to characterize known drug resistance-mediating polymorphisms. Isolates were generally susceptible, with IC50 values in the low-nM range, to chloroquine (median IC5010 nM, IQR 7.9-24), monodesethylamodiaquine (22, 14-46) piperaquine (6.1, 3.6-9.2), pyronaridine (3.0, 1.3-5.5), quinine (50, 30-75), mefloquine (7.1, 3.7-10), lumefantrine (7.1, 4.5-12), dihydroartemisinin (3.7, 2.2-5.5), and atovaquone (0.2, 0.1-0.3) and mostly resistant to cycloguanil (850, 543-1,290) and pyrimethamine (33,200, 18,400-54,200), although a small number of outliers were seen. Considering genetic markers of resistance to aminoquinolines, most samples had wild-type PfCRT K76T (87%) and PfMDR1 N86Y (95%) sequences. For markers of resistance to antifolates, established PfDHFR and PfDHPS mutations were highly prevalent, the PfDHPS A613S mutation was seen in 19% of samples, and key markers of high-level resistance (PfDHFR I164L; PfDHPS K540E) were absent or rare (A581G). Mutations in the PfK13 propeller domain known to mediate artemisinin partial resistance were not detected. Overall, our results suggest excellent susceptibilities to drugs now used to treat malaria and moderate, but stable, resistance to antifolates used to prevent malaria.

Increase of Plasmodium falciparum parasites carrying lumefantrine-tolerance molecular markers and lack of South East Asian pfk13 artemisinin-resistance mutations in samples collected from 2013 to 2016 in Côte d’Ivoire

One of the major obstacles to malaria elimination in the world is the resistance in Plasmodium falciparum to most antimalarial drugs. This study aimed to estimate the prevalence of molecular markers of antimalarial drugs resistance in Côte d’Ivoire. Samples were collected from 2013 to 2016 from asymptomatic and symptomatic subjects in Abengourou, Abidjan, Grand Bassam, and San Pedro. A total of 704 participants aged between 1 year and 65 years (Mean age: 9 years ± 7.7) were enrolled. All the dried filter paper blood spots were genotyped by sequencing. Plasmodium falciparum kelch propeller domain 13 (pfk13) gene were analyzed for all the samples, while 344 samples were examined for Plasmodium falciparum multi-drug resistance 1 (pfmdr1). Overall, the success rate of molecular tests was 98.8% (340/344), 99.1% (341/344), and 94.3% (664/704) for pfmdr1 N86Y, pfmdr1 Y184F, and pfk13 genes respectively. Molecular analysis revealed twenty (5.9%; 20/340) and 219 (64.2%; 219/341) mutant alleles for pfmdr1 86Y and pfmdr1 184 F, respectively. Twenty-nine mutations in pfk13 gene (4.4%; 29/664) with 2.7% (18/664) of non-synonymous mutations was found. None of the mutations previously described in South East Asia (SEA) involved in P. falciparum resistance to artemisinin derivatives were observed in this study. According to year of collection, a decrease of the prevalence of pfk13 mutation (from 3.6 to 1.8%) and pfmdr1 N86Y mutation (from 8.5 to 4.5%) and an increase of mutant allele of pfmdr1 Y184F proportion (from 39.8 to 66.4%) were found. Comparing to previous studies in the country, this study showed an increase in lumefantrine tolerance of P. falciparum strains. This demonstrates the importance of establishing a strong system for molecular surveillance of malaria in Côte d’Ivoire.

Plasmodium falciparum Multidrug Resistance-1 (pfmdr1) Gene Mutation in Adults Malaria Patients Attending Murtala Muhammad Specialist Hospital Kano, Northwest Nigeria

Suspicion of failure in the effectiveness of artemisinin-based combination therapies (currently the first-line treatment of malaria, worldwide) is leading to the unofficial use of alternative antimalarials, including chloroquine and sulfadoxine/pyrimethamine in Kano state northwestern Nigeria. To facilitate evidence-based resistance management, antimalarial resistance mutations were investigated in Plasmodium falciparum multidrug resistance-1 (pfmdr1) gene. Hundred adult patients comprising 43 males and 57 females were recruited for the study. The mean age of participants is 36.4 years, minimum and maximum ages were 16 and 60 years respectively, while 41% of them fall within the range of 16 to 30 years. Blood isolates were then analyzed for the presence of malaria parasite using microscopy, the results show a high prevalence of P. falciparum infection in the subject (30%). Pfmdr1 gene, a molecular marker of artemisinin resistance, was successfully sequenced in 21 out of 100 P. falciparum isolates collected from recruited participants. Pfmdr1 mutations were found in 19.5% (4/33) of the samples isolated. The prevalence of the Pfmdr1 N86Y allele was found in 4 samples whilst Y184F and D1246Y were not detected. A total of 4 non-synonymous mutations at codon N86Y were detected. The presence of these mutations highlights the challenges for malaria treatment in Kano state, northwestern Nigeria using antimalarials such as artemether lumefantrine, mefloquine, amodiaquine quinine and lumefantrine.

Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

In vitro susceptibility profile of Plasmodium falciparum clinical isolates from Ghana to antimalarial drugs and polymorphisms in resistance markers.

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016–2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.

High prevalence of Pfcrt 76T and Pfmdr1 N86 genotypes in malaria infected patients attending health facilities in East Shewa zone, Oromia Regional State, Ethiopia

BackgroundPlasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine–pyrimethamine (SP) and subsequently in 2004 from SP to artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia.MethodsFinger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively.ResultsOf 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86).ConclusionAlthough CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria.

Pcr-rflp genotyping of pfcrt and pfmdr1 in plasmodium falciparum isolates from children in Vatomandry, Madagascar

RésuméContexteLa surveillance de l'efficacité thérapeutique et des marqueurs génétiques de la résistance aux antipaludiques est primordiale afin de détecter le plus tôt possible l’émergence des parasites potentiellement résistants. Dans ce contexte, notre étude a pour objectif de réaliser le typage du gène Plasmodium falciparum chloroquine resistance transporter ou Pfcrt et Plasmodium falciparum multidrug resistant gene 1 ou Pfmdr1 sur des isolats provenant des enfants du district de Vatomandry.MéthodologiesAu total, 142 isolats de P. falciparum collectés lors d'un dépistage actif du paludisme chez des enfants âgés de moins de 15 ans, entre février et mars des années 2016 et 2017 à Vatomandry, ont été analysés. Le typage du codon K76T du gène Pfcrt et du codon N86Y du gène Pfmdr1 a été ensuite effectué par la technique de polymérisation en chaîne suivie de digestion enzymatique (restriction fragment length polymorphism) ou PCR-RFLP.RésultatsLe taux de succès d'amplification des gènes Pfcrt et Pfmdr1 était faible, de l'ordre de 27 % et 39 % respectivement. La prévalence des isolats mutants pour le codon K76T de Pfcrt était de 2,6 % [intervalle de confiance à 95 % (IC95%) : 0,1 - 15,0 %] et de 36 % [IC95% : 23,7 - 49,7 %] pour le codon N86Y du gène Pfmdr1.ConclusionNotre étude a mis en évidence la présence d'isolats portant à la fois la mutation au niveau du codon K76T de Pfcrt et N86Y de Pfmdr1. Bien que le taux de mutation que nous avons observé soit faible, d'autres études méritent d’être effectuées afin de suivre l’évolution de ces marqueurs dans le temps et dans l'espace à Madagascar.

Identification of polymorphisms in genes associated with drug resistance in Plasmodium falciparum isolates from school-age children in Kinshasa, Democratic Republic of Congo.

The emergence and spread of Plasmodium falciparum parasites resistant to antimalarial drugs constitutes an obstacle to malaria control and elimination. This study aimed to identify the prevalence of polymorphisms in pfk13, pfmdr1, pfdhfr, pfdhps and pfcrt genes in isolates from asymptomatic and symptomatic school-age children in Kinshasa. Nested-PCR followed by sequencing was performed for the detection of pfk13, pfmdr1, pfdhfr, pfdhps and pfcrt polymorphisms. Two mutations in pfk13, C532S and Q613E were identified in the Democratic Republic of Congo for the first time. The prevalence of the drug-resistance associated mutations pfcrt K76T, pfdhps K540E and pfmdr1 N86Y was low, being 27%, 20% and 9%, respectively. We found a low prevalence of genetic markers associated with chloroquine and sulfadoxine-pyrimethamine resistance in Kinshasa. Furthermore, no mutations previously associated with resistance against artemisinin and its derivatives were observed in the pfK13 gene. These findings support the continued use of ACTs and IPTp-SP. Continuous molecular monitoring of antimalarial resistance markers is recommended.

Plasmodium falciparum Drug Resistance Genes pfmdr1 and pfcrt In Vivo Co-Expression During Artemether-Lumefantrine Therapy.

Background: Artemisinin-based combination therapies (ACTs) are the global mainstay treatment of uncomplicated Plasmodium falciparum infections. PfMDR1 and PfCRT are two transmembrane transporters, associated with sensitivity to several antimalarials, found in the parasite food vacuole. Herein, we explore if their relatedness extends to overlapping patterns of gene transcriptional activity before and during ACT administration. Methods: In a clinical trial performed in Tanzania, we explored the pfmdr1 and pfcrt transcription levels from 48 patients with uncomplicated P. falciparum malaria infections who underwent treatment with artemether-lumefantrine (AL). Samples analyzed were collected before treatment initiation and during the first 24 h of treatment. The frequency of PfMDR1 N86Y and PfCRT K76T was determined through PCR-RFLP or direct amplicon sequencing. Gene expression was analyzed by real-time quantitative PCR. Results: A wide range of pre-treatment expression levels was observed for both genes, approximately 10-fold for pfcrt and 50-fold for pfmdr1. In addition, a significant positive correlation demonstrates pfmdr1 and pfcrt co-expression. After AL treatment initiation, pfmdr1 and pfcrt maintained the positive co-expression correlation, with mild downregulation throughout the 24 h post-treatment. Additionally, a trend was observed for PfMDR1 N86 alleles and higher expression before treatment initiation. Conclusion: pfmdr1 and pfcrt showed significant co-expression patterns in vivo, which were generally maintained during ACT treatment. This observation points to relevant related roles in the normal parasite physiology, which seem essential to be maintained when the parasite is exposed to drug stress. In addition, keeping the simultaneous expression of both transporters might be advantageous for responding to the drug action.

Effect of three years\u2019 seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali

BackgroundIn 2012, seasonal malaria chemoprevention (SMC) was recommended as policy for malaria control by the World Health Organization (WHO) in areas of highly seasonal malaria transmission across the Sahel sub-region in Africa along with monitoring of drug resistance. We assessed the long-term impact of SMC on Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) over a 3-year period of SMC implementation in the health district of Ouelessebougou, Mali.MethodsIn 8 randomly selected sub-districts of Ouelessebougou, Mali, children aged 0–5 years were randomly selected during cross-sectional surveys at baseline (August 2014) and 1, 2 and 3 years post-SMC, at the beginning and end of the malaria transmission season. Blood smears and blood spots on filter paper were obtained and frequencies of mutation in P. falciparum genes related to resistance to SP and AQ (Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt) were assessed by PCR amplification on individual samples and PCR amplification followed by deep sequencing on pooled (by site and year) samples.ResultsAt each survey, approximately 50–100 individual samples were analysed by PCR amplification and a total of 1,164 samples were analysed by deep sequencing with an average read depth of 18,018–36,918 after pooling by site and year. Most molecular markers of resistance did not increase in frequency over the period of study (2014–2016). After 3 years of SMC, the frequencies of Pfdhps 540E, Pfdhps 437G and Pfcrt K76T remained similar compared to baseline (4.0 vs 1.4%, p = 0.41; 74.5 vs 64.6%, p = 0.22; 71.3 vs 67.4%, p = 0.69). Nearly all samples tested carried Pfdhfr 59R, and this proportion remained similar 3 years after SMC implementation (98.8 vs 100%, p = 1). The frequency of Pfmdr1 N86Y increased significantly over time from 5.6% at baseline to 18.6% after 3 years of SMC (p = 0.016). Results of pooled analysis using deep sequencing were consistent with those by individual analysis with standard PCR, but also indicated for the first time the presence of mutations at the Pfdhps A581G allele at a frequency of 11.7% after 2 years of SMC, as well as the Pfdhps I431V allele at frequencies of 1.6–9.3% following 1 and 2 years of SMC, respectively.ConclusionTwo and 3 years of SMC implementation were associated with increased frequency of the Pfmdr1 N86Y mutation but not Pfdhps 540E, Pfdhps 437G and Pfcrt K76T. The first-time detection of the Pfdhps haplotype bearing the I431V and A581G mutations in Mali, even at low frequency, warrants further long-term surveillance.

Analysis of drug resistance marker genes of Plasmodium falciparum after implementation of artemisinin-based combination therapy in Pune district, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is a major problem in malaria control and elimination. In this study, samples from Pune district were characterized to determine prevalence of molecular markers of resistance to chloroquine (pfcrt codons C72S, M74I, N75E, K76T and pfmdr-1 N86Y, Y184F), pyrimethamine (pfdhfr C50R, N51I, C59R, S108N), sulfadoxine (pfdhps, S436A, A437G, K540E, A581G), and artemisinin (pfkelch13, C580Y, R539T). The pfcrt K76T mutation was found in 78% samples as CVMNT, SVMNT and CVIET haplotype. The pfmdr-1 N86Y and Y184F mutations were found in 54% of samples. The pfdhfr double mutation C59R + S108N was present in 67% of samples, while the pfdhfr triple mutation (N51I + C59R + S108N) was not detected. The pfdhps mutations A437G and K540E were found in 67% of samples. Single mutants of pfdhps were rare, with K540E detected in only 6 patient samples. Similarly, pfdhps A581G was found in 13 of the isolates. The molecular markers associated with artemisinin resistance (mutations in pfkelch13 C580Y, R539T) were not detected in any of the isolates. These results suggest an emerging problem with multidrug-resistant P. falciparum. Though the genotype conventionally associated with artemisinin resistance was not observed, chloroquine-resistant genotype has reached complete fixation in the population. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, with the presence of the quadruple mutant, indicates that continued monitoring is required to assess whether sulfadoxine-pyrimethamine can be used efficiently as a partner drug for artemisinin for the treatment of P. falciparum.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia

BackgroundIn 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether–lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure.MethodsSamples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed.ResultsAmong the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N.ConclusionThis study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.

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 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.

Polymorphism Analysis of pfmdr1 and pfcrt from Plasmodium falciparum Isolates in Northwestern Nigeria Revealed the Major Markers Associated with Antimalarial Resistance.

Suspicion of failure in the effectiveness of artemisinin-based combination therapies (currently the first-line treatment of malaria, worldwide) is leading to the unofficial use of alternative antimalarials, including chloroquine and sulfadoxine/pyrimethamine, across northern Nigeria. To facilitate evidence-based resistance management, antimalarial resistance mutations were investigated in Plasmodium falciparum multidrug resistance-1 (pfmdr1) and chloroquine resistance transporter (pfcrt), in isolates from Kano, northwestern Nigeria. Out of the 88 samples genotyped for pfmdr1 N86Y mutation using PCR/restriction fragment length polymorphism, one sample contained the 86Y mutation (86Yfrequency = 1.14%). The analysis of 610 bp fragments of pfmdr1 from 16 isolates revealed two polymorphic sites and low haplotype diversity (Hd = 0.492), with only 86 Y mutations in one isolate, and 184 F replacements in five isolates (184Ffrequency = 31.25%). The analysis of 267 bp fragments of pfcrt isolates revealed high polymorphism (Hd = 0.719), with six haplotypes and seven non-synonymous polymorphic sites. Eleven isolates (61.11%) were chloroquine-resistant, CQR (C72V73I74E75T76 haplotype), two of which had an additional mutation, D57E. An additional sequence was CQR, but of the C72V73M74E75T76 haplotype, while the rest of the sequences (33.33%) were chloroquine susceptible (C72V73M74N75K76 haplotype). The findings of these well characterized resistance markers should be considered when designing resistance management strategies in the northwestern Nigeria.

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan.

ABSTRACTThis molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan

This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Prevalence and factors associated with carriage of Pfmdr1 polymorphisms among pregnant women receiving intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and artemether-lumefantrine for malaria treatment in Burkina Faso

BackgroundSingle nucleotide polymorphisms occurring in the Plasmodium falciparum multidrug resistant gene 1 (pfmdr1) are known to be associated with aminoquinoline resistance and, therefore, represent key P. falciparum markers for monitoring resistance both in susceptible groups (children under 5 years old and pregnant women) and in the general population. This study aimed to determine prevalence and factors associated with the carriage of pfmdr1 N86Y, Y184F and D1246Y polymorphisms among pregnant women in a setting of high malaria transmission in Burkina Faso.MethodsPlasmodium falciparum isolates were collected at the first antenatal care visit (ANC-1) as well as at delivery from pregnant women participating in the COSMIC trial (NTC01941264), which assessed malaria preventive interventions during pregnancy in the Nanoro Health District. Here, pregnant women received intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and malaria infections and/or diseases were treated using artemether-lumefantrine (AL) during the trial. Parasite DNA was extracted from dried blood spots and the presence of pfmdr1 mutations at positions 86, 184 and 1246 was determined using nested PCR, followed by restriction fragment length polymorphism (RFLP) analysis.ResultsA prevalence of 13.2% (20/151) and 12.1% (14/116) of the pfmdr1 86Y mutant allele was found at ANC-1 and at delivery, respectively, while no mutant allele was observed for Y184F and D1246Y codons at both ANC-1 and at delivery. There were no significant factors associated with pfmdr1 86Y mutant allele carriage at ANC-1. However, malaria infections at delivery with a parasite density above the median (2237.2 (IQR: 613.5–11,425.7) parasites/µl) was associated with an increase risk of pfmdr1 86Y mutant allele carriage (AOR = 5.5 (95% CI 1.07–28.0); P = 0.04). In contrast, both three or more IPTp-SP doses (AOR = 0.25 (95% CI 0.07–0.92); P = 0.04) and one or more AL treatment (AOR = 0.25 (95% CI 0.07–0.89); P = 0.03) during pregnancy were associated with a significant reduce risk of pfmdr1 86Y mutant allele carriage at delivery.ConclusionThese findings suggest that both high coverage of IPTp-SP and the use of AL for the treatment of malaria infection/disease during pregnancy select for pfmdr1 N86 wild-type allele at delivery.