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Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms. Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia. IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

Malaria parasite resistant to drugs has been a major barrier to effective treatment of malaria. Therefore, the study aimed to evaluate the distribution resistant Kelch protein gene on chromosome 13 (Kelch 13) and multidrug resistant ( ) mutant genes among children aged five years and below who attended Mother and Child Hospital, Akure, Nigeria. Thin and thick smears were prepared from the blood collected aseptically through venepuncture from five hundred (500) children. Structured questionnaires were used to obtain demographic data from the respondents. Two hundred malaria positive samples were randomly selected from the 500 samples for PCR analysis to detect and mutant genes. The results showed that of the 500 respondents, 288 (57.6%) were males while 21 (42.4%) were females. Pfmdr1distribution include: mixed group (mutant/wild) 38.5%, mutant gene 35.5%, wild gene 20.5% and the resistant genes were absent in 5.5% of the infected children. The mixed group of gene was higher among infants (51.9%), children with birth order 4 (60.0%) and children that have blood group B (51.3%), however, there is no significant difference in the distribution of between gender (χ2 = 0.634, df = 1,  > 0.05). There was a point mutation in the codon position 557 where the amino acid Alanine was replaced by Serine in the The presence of mutant genes and point mutation in the gene of among children, calls for development of innovative drugs targeted on these resistant strains.

This study aimed to analyze polymorphisms in Pfcrt , Pfmdr1 , and Pfk13 genes’ markers of resistance to Artemisinin-based combination therapy (ACT), in Plasmodium falciparum isolates from southern Brazzaville, 15 years after the adoption of ACT in the Republic of Congo. A total of 369 microscopy-confirmed malaria-infected individuals were enrolled from March to October 2021 in the community and in health facilities during a cross-sectional study. The K76 T mutation in the Pfcrt gene, N86 Y and Y184 F mutations in the Pfmdr1 gene were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while the codons region (1005–1300) of the Pfmdr1 gene, and Pfk13 gene were sequenced. The prevalences of K76 T , N86 Y , Y184 F mutations were 26.0%, 6.8%, and 27.7%, respectively. However, no mutations were detected in codons 1034, 1042, and 1246 of the Pfmdr1 gene. None of the mutations previously associated with artemisinin-based resistance were detected in the Pfk13 gene. The results reveal a significant decrease in the prevalence of K76 T , N86 Y , Y184 F mutations, in Plasmodium falciparum isolates following the change of therapeutic policy. As artemisinin resistance is emerging throughout Africa, continued surveillance for early detection of these mutations and relevant partner markers of drug resistance are recommended in the Republic of Congo.

Background. Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. Methods. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. Results. Polymerase chain reaction (PCR)—adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. Conclusions. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border.

Background Thanks to the scale up of malaria control interventions, the malaria burden in Senegal has decreased substantially to the point that the National Malaria Control Programme plans to achieve malaria elimination by 2030. To guide such efforts, measuring and monitoring parasite population evolution and anti-malarial drugs resistance is extremely important. Information on the prevalence of parasite mutations related to drug resistance can provide a first signal of emergence, introduction and selection that can help with refining drug interventions. The aim of this study was to analyse the prevalence of anti-malarial drug resistance-associated markers before and after the implementation of artemisinin-based combination therapy (ACT) from 2005 to 2014 in Dielmo, a model site for malaria intervention studies in Senegal. Methods Samples from both malaria patients and Plasmodium falciparum asymptomatic carriers were analysed with high resolution melting (HRM) technique to genotype P. falciparum chloroquine resistance transporter ( Pfcrt ) gene haplotypes and multidrug-resistant protein 1 ( Pfmdr1 ) gene at codons N86 and Y184. Results Among the 539 samples analysed, 474, 486, and 511 were successfully genotyped for Pfmdr1 N86, Y184, and Pfcrt , respectively. The prevalence of drug resistance markers was high, particularly during the malaria upsurges. Following the scale-up in bed net distribution, only the mutant (86F-like) variant of Pfmdr1 86 was present while during the malaria upsurges the predominance of two types 86Y-86N (43%) and 86F-like (56%) were observed. Most infections (87%) carried the wild type Y-allele at Pfmdr1  184 during the period of nets scale-up while during the malaria upsurges only 16% of infections had wild type and 79% of infections had mixed (mutant/wild) type. The frequency of the mixed genotypes SVMNT-like_CVMNK and SVMNT-like_CVIET within Pfcrt gene was particularly low during bednet scale up. Their frequency increased significantly (P < 0.001) during the malaria upsurges. Conclusion This data demonstrated the effect of multiple interventions on the dynamics of drug resistance-associated mutations in the main malaria parasite P. falciparum in an endemic village in Senegal. Monitoring drug resistance markers should be conducted periodically to detect threats of emergence or resurgence that could compromise the efficacy of anti-malarial drugs.

Background Pfcrt gene has been associated with chloroquine resistance and the pfmdr1 gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether–lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria, pfcrt haplotype, and pfmdr1 single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission. Methods 230 microscopically confirmed P. falciparum isolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes and pfmdr1 SNPs. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A P -value of less or equal to 0.05 was considered significant. Results Of the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM for pfcrt haplotype, pfmdr1 -86, pfmdr1 -184, pfmdr1 -1042 and pfmdr1 -1246, respectively. The mutant pfcrt haplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively. Plasmodium falciparum with chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4, P  = 0.00). Pfmdr1 -N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at the pfmdr1- 1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codons N86Y–Y184F–D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution of pfmdr1 SNPs, haplotypes, and CNV between the two study sites ( P  > 0.05). Conclusion Plasmodium falciparum with the pfcrt wild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y–Y184F–D1246Y. A continuous investigation is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT.

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13- propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2 , earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

Background Central America and the island of Hispaniola have set out to eliminate malaria by 2030. However, since 2014 a notable upturn in the number of cases has been reported in the Mosquitia region shared by Nicaragua and Honduras. In addition, the proportion of Plasmodium falciparum malaria cases has increased significantly relative to vivax malaria. Chloroquine continues to be the first-line drug to treat uncomplicated malaria in the region. The objective of this study was to evaluate the emergence of chloroquine resistant strains of P. falciparum using a genetic approach. Plasmodium vivax populations are not analysed in this study. Methods 205 blood samples from patients infected with P. falciparum between 2018 and 2021 were analysed. The pfcrt gene fragment encompassing codons 72–76 was analysed. Likewise, three fragments of the pfmdr1 gene were analysed in 51 samples by nested PCR and sequencing. Results All samples revealed the CVMNK wild phenotype for the pfcrt gene and the N86, Y184F, S1034C, N1042D, D1246 phenotype for the pfmdr1 gene. Conclusions The increase in falciparum malaria cases in Nicaragua and Honduras cannot be attributed to the emergence of chloroquine-resistant mutants. Other possibilities should be investigated further. This is the first study to report the genotype of pfmdr1 for five loci of interest in Central America.

Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 ( pfmdr1 ), P. falciparum plasmepsin2 ( pfplasmepsin2 ) and P. falciparum GTP cyclohydrolase 1 ( pfgch1 ) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2 , was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes ( p  = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene ( p  = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1 , pfplasmepsin2 and pfgch1 genes of P. falciparum . The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

Background: The proliferation of Plasmodium parasites resistant to antimalarial drugs poses a serious threat to human life and remains an obstacle to managing and eradicating Plasmodium falciparum. The surveillance of molecular markers has become necessary to monitor the spread of resistant haplotypes and discover emerging mutations. Objective: This molecular epidemiological study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt, Pfmdr1, Pfdhfr and Pfdhps in the Central Region of Ghana. Design: A multi-centre cross-sectional study. Methods: This prospective study utilised dried blood spots from individuals with P. falciparum-infection from five districts in the Central Region of Ghana. Selective Whole Genome Amplification (sWGA) and Single Nucleotide Polymorphisms (SNPs) in P. falciparum chloroquine transporter genes (Pfcrt), P. falciparum multidrug resistance 1 (Pfmdr1), P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) were analysed. Results: Whole genome sequencing was carried out on 522 samples. Of these, 409 (78%) samples were successfully sequenced. Six (6) of the sequenced samples were of co-infection of other parasite species with P. falciparum and excluded from the analysis. Analysis of the Pfcrt gene revealed 0.5% were CVIET (C72, V73, M74I, N75E, K76T) while the Pfcrt CVMNK (C72, V73, M74, N75, K76) wild-type haplotypes were 97% with (2.5%) (CV[M/I][N/E][K/T]) being mixed haplotypes. In the Pfmdr1 gene, monoclonal haplotypes; NFD (N86, Y184F, D1246) and YFN (N86Y, Y184F, D1246N) occurred at 44% and 9.8%, respectively, whereas mixed- haplotypes (N[Y/F]D and [N/Y][Y/F]D) were 23.5% and 0.3%, respectively. Combined Pfdhfr/Pfdhps genes yielded about 88% Pfdhfr IRNI (N51I, C59R, S108N, I164) + Pfdhps A437G haplotypes (conferring partial resistance to Sulphadoxine-Pyrimethamine (SP)) while 9% of the parasites had Pfhdfr IRNI + Pfdhps A437G + K540E haplotypes (conferring full resistance to SP). The wild-type haplotype, Pfdhfr (N51, C59, S108, I164) and Pfdhps (S436, A437, K540, A581, A613) was not observed. Conclusion: The findings show a low prevalence of CVIET and relatively higher rates for Pfmdr1 NFD and parasites with Pfdhfr IRNI (N51I, C59R, S108N, I164) + Pfdhps A437G haplotypes. These observations advocate for enhanced surveillance which is inimical to malaria management in an endemic area.