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Abstract 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 K76T mutation in the Pfcrt gene, N86Y and Y184F 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 Pfmdr1gene, and Pfk13 gene were sequenced. The prevalences of K76T, N86Y, Y184F 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 K76T, N86Y, Y184F 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.

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. [ABSTRACT FROM AUTHOR]

Abdalmoneim M Magboul,1 Bakri YM Nour,2 Abdelhakam G Tamomh,1 Rashad Abdul-Ghani,3,4 Sayed Mustafa Albushra,5 Hanan Babiker Eltahir6 1Department of Parasitology & Medical Entomology, Faculty of Medical Laboratory Sciences, University of El Imam El Mahdi, Kosti, Sudan; 2Department of Parasitology, Faculty of Medical Laboratory Sciences, University of Gezira, Wad Madani, Sudan; 3Department of Medical Parasitology, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen; 4Tropical Disease Research Center, Faculty of Medicine and Health Sciences, University of Science and Technology, Sana’a, Yemen; 5Department of Internal Medicine, Faculty of Medicine, University of Gezira, Wad Madani, Sudan; 6Department of Biochemistry, Faculty of Medicine, University of El Imam El Mahdi, Kosti, SudanCorrespondence: Abdelhakam G Tamomh, Email abdelhakam738@gmail.com; abdelhakam738@mahdi.edu.sdBackground: Due to the increasing resistance of Plasmodium falciparum to chloroquine (CQ) in Sudan, a shift from CQ to artesunate combined with sulfadoxine/pyrimethamine as a first-line treatment for uncomplicated falciparum malaria was adopted in 2004. This study aimed to determine the frequency distribution of K76T and N86Y mutations in P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes as key markers of resistance to CQ among P. falciparum isolates from patients in Nyala district of South Darfur state, west of Sudan.Methods: A descriptive, cross-sectional study was conducted among 75 P. falciparum isolates from Sudanese patients diagnosed with falciparum malaria mono-infection. Parasite DNA was extracted from dried blood spots and amplified using a nested polymerase chain reaction (PCR). Then, restriction fragment length polymorphism (RFLP) was used to detect the genetic polymorphisms in codons 76 of pfcrt and 86 of pfmdr1. PCR-RFLP products were analyzed using 1.5% gel electrophoresis to identify the genetic polymorphisms in the studied codons. The wild-type (pfcrt K76 and pfmdr1 N86), mutant (pfcrt 76T and pfmdr1 86Y) and mixed-type (pfcrt K76T and pfmdr1 N86Y) alleles were expressed as frequencies and proportions.Results: The wild-type pfcrt K76 allele was observed among 34.7% of isolates and the mutant 76T allele among 20% of isolates, while the mixed-type K76T allele was observed among 45.3% of isolates. On the other hand, 54.7% of isolates harbored the wild-type pfmdr1 N86 allele and 5.3% of isolates had the mutant 86Y allele, while the mixed-type N86Y allele was observed among 40% of isolates.Conclusion: The key molecular markers associated with CQ resistance (pfcrt 76T and pfmdr1 86Y) are still circulating in high frequency among P. falciparum isolates in South Darfur state, about twelve years after the official withdrawal of the drug as a treatment for uncomplicated falciparum malaria.Keywords: chloroquine, drug resistance, molecular markers, pfcrt, pfmdr1, Sudan

Abstract Background Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine–pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. Methods Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. Results A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. Conclusion The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Background: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine–pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. Methods: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. Results: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. Conclusion: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites. [ABSTRACT FROM AUTHOR]

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

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.

Abstract Background Dihydroartemisinin–piperaquine has been Indonesia’s first-line anti-malarial treatment since 2008. Annual therapeutic efficacy studies (TES) done in the last 12 years showed continued high treatment efficacy in uncomplicated Plasmodium falciparum malaria. Although these studies did not show evidence for artemisinin resistance, a slight increase in Late Treatment Failure was observed over time. It is highlight to explore the evolution of genetic markers for ACT partner drug resistance since adopting DHA–PPQ. Methods Dry blood spots were identified from a mass blood survey of uncomplicated falciparum malaria patients (N = 50) in Sumba from 2010 to 2018. Analysis of genotypic profile (N = 51) and a Therapeutic Efficacy Study (TES) from Papua (N = 142) from 2020 to 2021, 42-day follow-up. PCR correction using msp1, msp2, and glurp was used to distinguish recrudescence and reinfection. Parasite DNA from DBSs was used for genotyping molecular markers for antimalaria drug resistance, including in Pfk13, pfcrt, and pfmdr1, as well as gene copy number variation in pfpm2/3 and pfmdr1. Results The study revealed the absence of SNPs associated with ART resistance and several novel SNPs such as L396F, I526V, M579I and N537S (4.25%). In Sumba, the mutant haplotype SDD of pfmdr1 was found in one-third of the isolates, while only 8.9% in Papua. None of the pfcrt mutations linked to piperaquine resistance were observed, but 71% of isolates had pfcrt I356L. Amplification of the pfpm2/3 genes was in Sumba (17.02%) and Papua (13.7%), while pfmdr1 copy number prevalence was low (3.8%) in both areas. For the TES study, ten recurrences of infection were observed on days 28, 35, and 42. Late parasitological failure (LPF) was observed in 10/117 (8.5%) subjects by microscopy. PCR correction revealed that all nine cases were re-infections and one was confirmed as recrudescence. Conclusion This study revealed that DHA–PPQ is still highly effective against P. falciparum. The genetic architecture of the parasite P. falciparum isolates during 2010–2021 revealed single copy of Pfpm2 and pfmdr1 were highly prevalent. The slight increase in DHA–PPQ LTF alerts researchers to start testing other ACTs as alternatives to DHA–PPQ for baseline data in order to get a chance of achieving malaria elimination wants by 2030. Graphical Abstract

Abstract 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.

BackgroundIn Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies.MethodsGenetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays.ResultsConsidering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively).ConclusionsWe demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

Background: Dihydroartemisinin–piperaquine has been Indonesia's first-line anti-malarial treatment since 2008. Annual therapeutic efficacy studies (TES) done in the last 12 years showed continued high treatment efficacy in uncomplicated Plasmodium falciparum malaria. Although these studies did not show evidence for artemisinin resistance, a slight increase in Late Treatment Failure was observed over time. It is highlight to explore the evolution of genetic markers for ACT partner drug resistance since adopting DHA–PPQ. Methods: Dry blood spots were identified from a mass blood survey of uncomplicated falciparum malaria patients (N = 50) in Sumba from 2010 to 2018. Analysis of genotypic profile (N = 51) and a Therapeutic Efficacy Study (TES) from Papua (N = 142) from 2020 to 2021, 42-day follow-up. PCR correction using msp1, msp2, and glurp was used to distinguish recrudescence and reinfection. Parasite DNA from DBSs was used for genotyping molecular markers for antimalaria drug resistance, including in Pfk13, pfcrt, and pfmdr1, as well as gene copy number variation in pfpm2/3 and pfmdr1. Results: The study revealed the absence of SNPs associated with ART resistance and several novel SNPs such as L396F, I526V, M579I and N537S (4.25%). In Sumba, the mutant haplotype SDD of pfmdr1 was found in one-third of the isolates, while only 8.9% in Papua. None of the pfcrt mutations linked to piperaquine resistance were observed, but 71% of isolates had pfcrt I356L. Amplification of the pfpm2/3 genes was in Sumba (17.02%) and Papua (13.7%), while pfmdr1 copy number prevalence was low (3.8%) in both areas. For the TES study, ten recurrences of infection were observed on days 28, 35, and 42. Late parasitological failure (LPF) was observed in 10/117 (8.5%) subjects by microscopy. PCR correction revealed that all nine cases were re-infections and one was confirmed as recrudescence. Conclusion: This study revealed that DHA–PPQ is still highly effective against P. falciparum. The genetic architecture of the parasite P. falciparum isolates during 2010–2021 revealed single copy of Pfpm2 and pfmdr1 were highly prevalent. The slight increase in DHA–PPQ LTF alerts researchers to start testing other ACTs as alternatives to DHA–PPQ for baseline data in order to get a chance of achieving malaria elimination wants by 2030. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium 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. Methods Finger-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. Results Of 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). Conclusion Although 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.

Abstract Background Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. Methods Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. Results The prevalence of SP dhfr/dhps quintuple mutant haplotype C50 I 51 R 59 N 108I164/S436 G 437 E 540A581A613 increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C50 I 51 R 59 N 108I164/H 436 G 437 E 540A581A613 containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N86 F 184S1034N1042D1246 increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. Conclusion This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N86 F 184S1034N1042D1246 was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation.

Background: A wide spread of chloroquine resistance prompted its discontinued use for treatment of uncomplicated malaria in several African countries. However, disappearances of chloroquine-resistant parasites have been reported in areas with restricted use of chloroquine. This review reports the current prevalence of chloroquine-resistant Plasmodium falciparum using Pfcrt K76T and Pfmdr1 N86Y genotypes. Methods: A PROSPERO registered systematic review searched evidence from PubMed/MEDLINE, Science Direct and Google Scholar. The search included studies on chloroquine-resistant/ susceptible P. falciparum in humans between January 1st, 2000 and May 15th, 2020. The search was conducted on 15th of May, 2020. Results: Out of 519 searched records, 15 studies qualified for final analysis with 8040 samples genotyped for Pfcrt K76T. Of 8040, 43.6% (837/1572; 95%CI: −0.9 to 88.1%) carried resistant genotypes versus 23.0% (1477/6468; 95%CI: 15.7–30.2%) while for 4698 samples analyzed for Pfmdr1 N86Y, 52.4% (592/1090; 95%CI: 42.3–62.5%) had resistant genotypes versus 25.9% (1314/3608; 95%CI: 5.8–46.0%), before and after chloroquine withdrawal, respectively. The median time since chloroquine withdrawal to data collection was 7.0 (interquartile range: 4.5–13.5) years. Low prevalence of resistant genotypes (Pfcrt K76T) was reported in Zambia (0%) in 2013, Malawi (0.1%) in 2009, Tanzania (0.2%) in 2018 and Madagascar (0.3%) in 2007 with significant variations in the included studies. Conclusions: Chloroquine-resistant P. falciparum continues to disappear in countries with withdrawal of chloroquine. Areas with significant susceptible parasites, reintroduction of chloroquine can be considered, preferably in combination with other safe and affordable antimalarials.

Abstract 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.

Introduction: Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods: P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results: The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions: This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

ABSTRACT Since single nucleotide polymorphisms (SNPs) have attracted attention, there have been many explorations and improvements in screening and detection methods for SNPs. Traditional methods are complex and time-consuming and rely on expensive instruments. Therefore, there is an urgent need for a low-cost, simple, and accurate method that is convenient for use in resource-poor areas. Thus, a platform based on allele-specific PCR (AS-PCR) and a gold nanoparticle-based lateral flow assay (LFA) was developed, optimized, and used to detect the SNPs of the drug resistance gene pfmdr1. Subsequently, the system was assessed on clinical isolates and compared with nested PCR followed by Sanger sequencing. The sensitivity and specificity of the AS-PCR-LFA platform were up to 99.43% and 100%, respectively, based on the clinical isolates. The limit of detection is approximately 150 fg/μL for plasmid DNA as the template and 50 parasites/μL for dried filter blood spots from clinical isolates. The established and optimized AS-PCR-LFA system is more adaptable and rapidly translated to SNP analysis of other drug resistance genes and genetic diseases. In addition, while actively responding to the point-of-care testing policy, it also contributes to the Global Malaria Eradication Program. IMPORTANCE Rapid detection of single nucleotide polymorphisms (SNPs) is essential for malaria treatment. Based on the techniques of allele-specific PCR (AS-PCR) and lateral flow assay (LFA), an accurate and powerful platform for SNP detection of pfmdr1 was developed and evaluated with plasmid and clinical isolates. It offers a useful tool to identify antimalarial drug resistance and can support the effort to eliminate malaria globally.

Background: Plasmodium 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. Methods: Finger-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. Results: Of 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). Conclusion: Although 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. [ABSTRACT FROM AUTHOR]

Background: Over the last two decades, the scale-up of vector control and changes in the first-line anti-malarial, from chloroquine (CQ) to sulfadoxine-pyrimethamine (SP) and then to artemether-lumefantrine (AL), have resulted in significant decreases in malaria burden in western Kenya. This study evaluated the long-term effects of control interventions on molecular markers of Plasmodium falciparum drug resistance using parasites obtained from humans and mosquitoes at discrete time points. Methods: Dried blood spot samples collected in 2012 and 2017 community surveys in Asembo, Kenya were genotyped by Sanger sequencing for markers associated with resistance to SP (Pfdhfr, Pfdhps), CQ, AQ, lumefantrine (Pfcrt, Pfmdr1) and artemisinin (Pfk13). Temporal trends in the prevalence of these markers, including data from 2012 to 2017 as well as published data from 1996, 2001, 2007 from same area, were analysed. The same markers from mosquito oocysts collected in 2012 were compared with results from human blood samples. Results: The prevalence of SP dhfr/dhps quintuple mutant haplotype C50I51R59N108I164/S436G437E540A581A613 increased from 19.7% in 1996 to 86.0% in 2012, while an increase in the sextuple mutant haplotype C50I51R59N108I164/H436G437E540A581A613 containing Pfdhps-436H was found from 10.5% in 2012 to 34.6% in 2017. Resistant Pfcrt-76 T declined from 94.6% in 2007 to 18.3% in 2012 and 0.9% in 2017. Mutant Pfmdr1-86Y decreased across years from 74.8% in 1996 to zero in 2017, mutant Pfmdr1-184F and wild Pfmdr1-D1246 increased from 17.9% to 58.9% in 2007 to 55.9% and 90.1% in 2017, respectively. Pfmdr1 haplotype N86F184S1034N1042D1246 increased from 11.0% in 2007 to 49.6% in 2017. No resistant mutations in Pfk13 were found. Prevalence of Pfdhps-436H was lower while prevalence of Pfcrt-76 T was higher in mosquitoes than in human blood samples. Conclusion: This study showed an increased prevalence of dhfr/dhps resistant markers over 20 years with the emergence of Pfdhps-436H mutant a decade ago in Asembo. The reversal of Pfcrt from CQ-resistant to CQ-sensitive genotype occurred following 19 years of CQ withdrawal. No Pfk13 markers associated with artemisinin resistance were detected, but the increased haplotype of Pfmdr1 N86F184S1034N1042D1246 was observed. The differences in prevalence of Pfdhps-436H and Pfcrt-76 T SNPs between two hosts and the role of mosquitoes in the transmission of drug resistant parasites require further investigation. [ABSTRACT FROM AUTHOR]

Abstract Background The return of chloroquine-sensitive Plasmodium falciparum in sub-Saharan Africa countries offers the opportunity for the reintroduction of chloroquine (CQ) either in combination with other drugs or as a single therapy for the management of malaria. This study assesses the influence of individual study sites on the selection of CQ sensitive P. falciparum markers in the Central region of Ghana. Methods Genomic DNA was extracted from an archived filter paper blood blot from Cape Coast, Elmina, Assin Fosu, and Twifo Praso using the Chelex DNA extraction method. The age metadata of the patients from whom the blood spots were taken was collected. The prevalence of CQ-sensitive markers of pfcrt K76 and pfmdr1 N86 was performed using nested PCR and RFLP. The data were analysed using Chi-square and Odd ratio. Results The overall prevalence of CQ-sensitive P. falciparum markers, pfcrt K76 and pfmdr1 N86 in the Central Region of Ghana were 142 out of 184 (77.17%) and 180 out of 184 (97.83%), respectively. The distribution of pfcrt K76 was assessed among the age groups per the individual study sites. 12 out of 33 (36.36%), 8 out of 33 (24.24%) and 6 out of 33 (18.18%) of pfcrt K76 CQ-sensitive marker were isolated from age 0 to 5 years, 16 to 30 years and 31 to 45 years old respectively at Cape Coast. Assin Fosu and Twifo Praso had the highest pfcrt K76 prevalence in 0–5 years, followed by 16–30 years and 6–15 years of age. The results showed that there was a significant prevalence of pfcrt K76 in all study sites; Cape Coast (χ2 = 26.48, p

Abstract Background Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated. Methods Children aged 6–59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing. Results No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72–76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons. Conclusion In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique.

Objectives: This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. Methods: Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. Results: A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7–5.2%) in Sarangani, 10% (95% CI: 7.7–12.7%) in South Cotabato, and 4.2% (95% CI: 3.2–5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72–76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. Conclusions: Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

The emergence and spread of Plasmodium falciparum (P.f) drug resistance is still a major concern in Sub-Saharan Africa and warrants that its evolution be monitored continuously. The present study aimed at determining the distribution of key P.f drug resistance-mediating alleles in circulating malaria parasites in the West region of Cameroon. A cross sectional hospital-based study was conducted in Dschang and Ngounso in the West region of Cameroon. The Pfcrt, Pfmdr1, and the Pfdhps genes were amplified through nested PCR in 208 malaria-infected samples of the 301 febrile outpatients enrolled. The presence or absence of mutations in the K76T, N86Y, A437G and A581G codons of these P.f. genes respectively were determined through restriction digestion analysis. The proportion of different alleles were estimated as percentages and compared between two study sites using the Chi square test. A p value

Steede Seinnat Ontoua,1,&ast; Lady Charlene Kouna,1,&ast; Sandrine Lydie Oyegue-Liabagui,1,2 Dominique Fatima Voumbo-Matoumona,3 Diamella Nancy Moukodoum,1 Romeo Karl Imboumy-Limoukou,1 Jean Bernard Lekana-Douki1,4 1Unité d’Evolution, Epidémiologie et Résistance Parasitaire (UNEEREP), Centre Interdisciplinaire des Recherches Médicales de Franceville (CIRMF), Franceville, BP 769, Gabon; 2Ecole Doctorale Régionale d’Afrique Centrale en Infectiologie Tropicale (ECODRAC), Université de Sciences et Techniques de Masuku (USTM), Franceville, BP 876, Gabon; 3Départements des Masters/Licences, Parcours-Types des Sciences Biologiques, Faculté des Sciences et Techniques, Université Marien Ngouabi, Brazzaville, BP 69, Congo; 4Département de Parasitologie-Mycologie Médecine Tropicale, Faculté de Médecine, Université des Sciences de la Santé (USS), Libreville, BP 4009, Gabon&ast;These authors contributed equally to this workCorrespondence: Jean Bernard Lekana-DoukiUNEEREP-CIRMF, Franceville, B.P. 769, GabonTel +241 62521250; +241 6259590Email lekana_jb@yahoo.frPurpose: Plasmodium falciparum malaria remains a major public health challenge in sub-Saharan Africa. Plasmodium falciparum drug resistance mediated by polymorphisms in the Pfmdr1 gene contributes to the persistence of the disease on the African continent. This study investigated P. falciparum infection features and differences in the Pfmdr1 genotypes between symptomatic and asymptomatic malaria cases in a rural area in east-central Gabon.Patients and Methods: A total of 875 children aged from 5 to 185 months were screened for P falciparum infection using Optima-IT® rapid diagnostic tests and standard microscopy. Pfmdr1 polymorphisms at codons 86, 184 and 1246 were investigated using PCR-RFLP.Results: Among the 448 P. falciparum-infected children, 57.08% (n=250) were symptomatic and 42.92% (n=198) were asymptomatic (p < 0.0001). In a sub-set of 79 isolates, the Pfmdr1 wild-type N86 was more prevalent in symptomatic (100%) than in asymptomatic infections (70.7%) (p=0.007). The mutant 86Y and mixed 86N/Y genotypes were observed only in asymptomatic infections. The Y184 and 184F genotype prevalences (39.1% vs 19.4% and 60.9% vs 80.6%, respectively) were not significantly different between the two groups (p=0.097). The prevalence of the wild-type D1246 differed significantly between symptomatic (10.3%) and asymptomatic (100%) (p < 0.0001). The NFD and YFD haplotypes were more prevalent in asymptomatic than in symptomatic infections [(61.9% vs 31%; p=0.005) and (16.7% vs 0.0%; p=0.01)], whereas the NYD and YYD haplotypes were not significantly different between the two groups [(21.4% vs 14.3%, p=0.39) and (0.0% vs 7.1%, p=0.24)].Conclusion: Our results confirm a high transmission of P. falciparum infection in rural Gabon, with a high prevalence of asymptomatic carriage. The higher prevalences of wild-type N86 in symptomatic infections and of D1246 in asymptomatic infections suggest a pathogenicity associated with polymorphisms in Pfmdr1. These results highlight the need to monitor the efficacy of artemisinin-based combination therapies in Gabon.Keywords: symptomatic malaria, asymptomatic malaria, Pfmdr1, drug resistance, rural Gabon

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.

We assessed Plasmodium falciparum drug resistance markers in parasites collected in 2012, 2013, and 2015 at 3 sites in Uganda. The prevalence and frequency of parasites with mutations in putative transporters previously associated with resistance to aminoquinolines, but increased sensitivity to lumefantrine (pfcrt 76T; pfmdr1 86Y and 1246Y), decreased markedly at all sites. Antifolate resistance mutations were common, with apparent emergence of mutations (pfdhfr 164L; pfdhps 581G) associated with high-level resistance. K13 mutations linked to artemisinin resistance were uncommon and did not increase over time. Changing malaria treatment practices have been accompanied by profound changes in markers of resistance.

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

A wide spread of chloroquine resistance prompted its discontinued use for treatment of uncomplicated malaria in several African countries. However, disappearances of chloroquine-resistant parasites have been reported in areas with restricted use of chloroquine. This review reports the current prevalence of chloroquine-resistant Plasmodium falciparum using Pfcrt K76T and Pfmdr1 N86Y genotypes. A PROSPERO registered systematic review searched evidence from PubMed/MEDLINE, Science Direct and Google Scholar. The search included studies on chloroquine-resistant/ susceptible P. falciparum in humans between January 1st, 2000 and May 15th, 2020. The search was conducted on 15th of May, 2020. Out of 519 searched records, 15 studies qualified for final analysis with 8040 samples genotyped for Pfcrt K76T. Of 8040, 43.6% (837/1572; 95%CI: −0.9 to 88.1%) carried resistant genotypes versus 23.0% (1477/6468; 95%CI: 15.7–30.2%) while for 4698 samples analyzed for Pfmdr1 N86Y, 52.4% (592/1090; 95%CI: 42.3–62.5%) had resistant genotypes versus 25.9% (1314/3608; 95%CI: 5.8–46.0%), before and after chloroquine withdrawal, respectively. The median time since chloroquine withdrawal to data collection was 7.0 (interquartile range: 4.5–13.5) years. Low prevalence of resistant genotypes (Pfcrt K76T) was reported in Zambia (0%) in 2013, Malawi (0.1%) in 2009, Tanzania (0.2%) in 2018 and Madagascar (0.3%) in 2007 with significant variations in the included studies. Chloroquine-resistant P. falciparum continues to disappear in countries with withdrawal of chloroquine. Areas with significant susceptible parasites, reintroduction of chloroquine can be considered, preferably in combination with other safe and affordable antimalarials. [ABSTRACT FROM AUTHOR]

Abstract 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: 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. [ABSTRACT FROM AUTHOR]

Abstract Background The analysis of single nucleotide polymorphism (SNPs) in drug-resistance associated genes is a commonly used strategy for the surveillance of anti-malarial drug resistance in populations of parasites. The present study was designed and performed to provide genetic epidemiological data of the prevalence of N86Y-Y184F-D1246Y SNPs in Plasmodium falciparum multidrug resistance 1 (pfmdr1) in the malaria hotspot of Northern Nigeria. Methods Plasmodium falciparum-positive blood samples on Whatman-3MM filter papers were collected from 750 symptomatic patients from four states (Kano, Kaduna, Yobe and Adamawa) in Northern Nigeria, and genotyped via BigDye (v3.1) terminator cycle sequencing for the presence of three SNPs in pfmdr1. SNPs in pfmdr1 were used to construct NYD, NYY, NFY, NFD, YYY, YYD, YFD and YFY haplotypes, and all data were analysed using Pearson Chi square and Fisher’s exact (FE) tests. Results The prevalence of the pfmdr1 86Y allele was highest in Kaduna (12.50%, 2 = 10.50, P = 0.02), whilst the 184F allele was highest in Kano (73.10%, 2 = 13.20, P = 0.00), and the pfmdr1 1246Y allele was highest in Yobe (5.26%, 2 = 9.20, P = 0.03). The NFD haplotype had the highest prevalence of 69.81% in Kano (2 = 36.10, P = 0.00), followed by NYD with a prevalence of 49.00% in Adamawa, then YFD with prevalence of 11.46% in Kaduna. The YYY haplotype was not observed in any of the studied states. Conclusion The present study suggests that strains of P. falciparum with reduced sensitivity to the lumefantrine component of AL exist in Northern Nigeria and predominate in the North-West region.

Abstract Background Chile is one of the South American countries certified as malaria-free since 1945. However, the recent increase of imported malaria cases and the presence of the vector Anopheles pseudopunctipennis in previously endemic areas in Chile require an active malaria surveillance programme. Methods Specimens from 268 suspected malaria cases—all imported—collected between 2015 and 2018 at the Public Health Institute of Chile (ISP), were diagnosed by microscopy and positive cases were included for epidemiological analysis. A photo-induced electron transfer fluorogenic primer real-time PCR (PET-PCR) was used to confirm the presence of malaria parasites in available blood samples. Sanger sequencing of drug resistance molecular markers (pfk13, pfcrt and pfmdr1) and microsatellite (MS) analysis were performed in confirmed Plasmodium falciparum samples and results were related to origin of infection. Results Out of the 268 suspected cases, 65 were Plasmodium spp. positive by microscopy. A total of 63% of the malaria patients were male and 37% were female; 43/65 of the patients acquired infections in South American endemic countries. Species confirmation of available blood samples by PET-PCR revealed that 15 samples were positive for P. falciparum, 27 for Plasmodium vivax and 4 were mixed infections. The P. falciparum samples sequenced contained four mutant pfcrt genotypes (CVMNT, CVMET, CVIET and SVMNT) and three mutant pfmdr1 genotypes (Y184F/S1034C/N1042D/D1246Y, Y184F/N1042D/D1246Y and Y184F). MS analysis confirmed that all P. falciparum samples presented different haplotypes according to the suspected country of origin. Four patients with P. vivax infection returned to the health facilities due to relapses. Conclusion The timely detection of polymorphisms associated with drug resistance will contribute to understanding if current drug policies in the country are appropriate for treatment of imported malaria cases and provide information about the most frequent resistant genotypes entering Chile.

Abstract Background Currently, artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment in malaria-endemic areas. However, resistance in Plasmodium falciparum to artemisinin-based combinations emerging in the Greater Mekong Sub-region is a major problem hindering malaria elimination. To continuously monitor the potential spread of ACT-resistant parasites, this study assessed the efficacy of artemether-lumefantrine (AL) for falciparum malaria in western Myanmar. Methods Ninety-five patients with malaria symptoms from Paletwa Township, Chin State, Myanmar were screened for P. falciparum infections in 2015. After excluding six patients with a parasite density below 100 or over 150,000/µL, 41 P. falciparum patients were treated with AL and followed for 28 days. Molecular markers associated with resistance to 4-amino-quinoline drugs (pfcrt and pfmdr1), antifolate drugs (pfdhps and pfdhfr) and artemisinin (pfk13) were genotyped to determine the prevalence of mutations associated with anti-malarial drug resistance. Results For the 41 P. falciparum patients (27 children and 14 adults), the 28-day AL therapeutic efficacy was 100%, but five cases (12.2%) were parasite positive on day 3 by microscopy. For the pfk13 gene, the frequency of NN insert after the position 136 was 100% in the day-3 parasite-positive group as compared to 50.0% in the day-3 parasite-negative group, albeit the difference was not statistically significant (P = 0.113). The pfk13 K189T mutation (10.0%) was found in Myanmar for the first time. The pfcrt K76T and A220S mutations were all fixed in the parasite population. In pfmdr1, the Y184F mutation was present in 23.3% of the parasite population, and found in both day-3 parasite-positive and -negative parasites. The G968A mutation of pfmdr1 gene was first reported in Myanmar. Prevalence of all the mutations in pfdhfr and pfdhps genes assessed was over 70%, with the exception of the pfdhps A581G mutation, which was 3.3%. Conclusions AL remained highly efficacious in western Myanmar. Pfk13 mutations associated with artemisinin resistance were not found. The high prevalence of mutations in pfcrt, pfdhfr and pfdhps suggests high-degree resistance to chloroquine and antifolate drugs. The pfmdr1 N86/184F/D1246 haplotype associated with selection by AL in Africa reached > 20% in this study. The detection of > 10% patients who were day-3 parasite-positive after AL treatment emphasizes the necessity of continuously monitoring ACT efficacy in western Myanmar.

Abstract Background Anti-malarial drug resistance remains a major threat to global malaria control efforts. In Africa, Plasmodium falciparum remains susceptible to artemisinin-based combination therapy (ACT), but the emergence of resistant parasites in multiple countries in Southeast Asia and concerns over emergence and/or spread of resistant parasites in Africa warrants continuous monitoring. The World Health Organization recommends that surveillance for molecular markers of resistance be included within therapeutic efficacy studies (TES). The current study assessed molecular markers associated with resistance to Artemether−lumefantrine (AL) and Dihydroartemisinin−piperaquine (DP) from samples collected from children aged 6–59 months enrolled in a TES conducted in Siaya County, western Kenya from 2016 to 2017. Methods Three hundred and twenty-three samples collected pre-treatment (day-0) and 110 samples collected at the day of recurrent parasitaemia (up to day 42) were tested for the presence of drug resistance markers in the Pfk13 propeller domain, and the Pfmdr1 and Pfcrt genes by Sanger sequencing. Additionally, the Pfpm2 gene copy number was assessed by real-time polymerase chain reaction. Results No mutations previously associated with artemisinin resistance were detected in the Pfk13 propeller region. However, other non-synonymous mutations in the Pfk13 propeller region were detected. The most common mutation found on day-0 and at day of recurrence in the Pfmdr1 multidrug resistance marker was at codon 184F. Very few mutations were found in the Pfcrt marker (

Abstract Background Artesunate plus sulfadoxine–pyrimethamine (ASP) is first-line treatment for uncomplicated Plasmodium falciparum malaria in most of India, except for six North-eastern provinces where treatment failure rates were high. In Ujjain, central India, the frequency of mutations associated with increased drug tolerance, but not overt resistance to sulfadoxine and pyrimethamine were 9% and > 80%, respectively, in 2009 and 2010, just prior to the introduction of ASP. The frequency of drug resistance associated mutations in Ujjain in 2015–2016 after 3–4 years of ASP use, are reported. Methods Blood samples from patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons pfdhfr 16–185, pfdhps 436–632 and K13 407–689 were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism. Results Sulfadoxine–pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 100/104 (96%) and 87/91 (96%) samples, respectively. Pfdhps 437G was found in 10/105 (10%) samples. Double mutant pfdhfr 59R + 108 N were found in 75/81 (93%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/78 (8%) samples. Chloroquine-resistance-associated pfcrt 76T was found in 102/102 (100%). Pfmdr1 N86 and 86Y were identified in 83/115 (72%) and 32/115 (28%) samples, respectively. Conclusion The frequency of P. falciparum with reduced susceptibility to sulfadoxine–pyrimethamine remained high, but did not appear to have increased significantly since the introduction of ASP. No polymorphisms in K13 associated with decreased artemisinin susceptibility were found. ASP probably remained effective, supporting continued ASP use.

Abstract Background Malaria in pregnancy is associated with considerable morbidity and mortality. Regular surveillance of artemisinin-based combination therapy tolerance, or molecular makers of resistance, is vital for effective malaria treatment, control and eradication programmes. Plasmodium falciparum multiple drug resistance-1 gene (Pfmdr1) N86Y mutation is associated with reduced susceptibility to lumefantrine. This study assessed the prevalence of Pfmdr1 N86Y in Brazzaville, Republic of Congo. Methods A total 1001 of P. falciparum-infected blood samples obtained from asymptomatic malaria pregnant women having a normal child delivery at the Madibou Integrated Health Centre were analysed. Pfmdr1 N86Y genotyping was conducted using PCR-restriction fragment length polymorphism. Results The wild type Pfmdr1 N86 allele was predominant (> 68%) in this study, whereas a few isolates carrying the either the mutant allele (Pfmdr1 86Y) alone or both alleles (mixed genotype). The dominance of the wildtype allele (pfmdr1 N86) indicates the plausible decline P. falciparum susceptibility to lumefantrine. Conclusion This study gives an update on the prevalence of Pfmdr1 N86Y alleles in Brazzaville, Republic of Congo. It also raises concern on the imminent emergence of resistance against artemether–lumefantrine in this setting. This study underscores the importance to regular artemether–lumefantrine efficacy monitoring to inform the malaria control programme of the Republic of Congo.

Abstract Background Mizoram, a northeastern state in India, shares international borders with Myanmar and Bangladesh and is considered to be one of the key routes through which drug-resistant parasites of Southeast Asia enter mainland India. Despite its strategic location and importance, malaria epidemiology and molecular status of chloroquine resistance had not been well documented, and since chloroquine (CQ), as the first-line treatment in Plasmodium falciparum infection was discontinued since 2008, it was expected that CQ-sensitive haplotype would be more abundant. Methods Malaria epidemiology data for the period 2010 to 2018 was collected from the office of State Vector Disease Control Programme. Plasmodium falciparum-positive blood samples were collected from government district hospitals, community health centres, primary health centres, sub-centres, and diagnostic centres from six malaria-prone districts. The samples were processed and analysed using genes–P. falciparum chloroquine-resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) via sequencing of PCR amplicon from 2015 to 2017. Results Malaria occurred throughout the year and P. falciparum accounted for > 89% of total malaria cases. During 2010–2018, the highest number of malaria incidence was recorded in Lawngtlai (36% of total malaria cases; average API2010–2018 of 34.8) while Champhai remained consistently low (0.4%; average API2010–2018 of 0.04). Males of ≥ 15 years old contributed maximum (35.7%) among gender and age malarial distribution recorded during 2014–2018. Death due to malaria gradually decreased over the years. A higher abundance of mutated pfcrt (58.5% of the total sample analysed) and a lower prevalence of mutated pfmdr1 (48.7%) were observed. All mutations identified for pfcrt belong to the Southeast Asian CVIET haplotype. Only a single point mutation was observed at 86 (N → Y) position in pfmdr1 (48.7%). The key N86Y mutation in pfmdr1 that had been shown to modulate CQR was found in 67.1% of the samples positive for the CVIET haplotype. Conclusions This is the first report that details malaria epidemiology and also the molecular status of CQ-resistance in P. falciparum population of the region. The efforts of the State Vector Borne Disease Control Programme have proved to be quite effective in controlling the malaria burden in the state. Despite the discontinuation of CQ for a decade, local P. falciparum is observed with decreased CQ-sensitive haplotype. It is believed that the present findings will form a basis for further studies on genetic diversity in P. falciparum, which could confer better understanding of the complexity of the disease in Southeast Asia.

Abstract Background Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether–lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. Methods The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART‐R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). Results Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P

Background: The return of chloroquine-sensitive Plasmodium falciparum in sub-Saharan Africa countries offers the opportunity for the reintroduction of chloroquine (CQ) either in combination with other drugs or as a single therapy for the management of malaria. This study assesses the influence of individual study sites on the selection of CQ sensitive P. falciparum markers in the Central region of Ghana. Methods: Genomic DNA was extracted from an archived filter paper blood blot from Cape Coast, Elmina, Assin Fosu, and Twifo Praso using the Chelex DNA extraction method. The age metadata of the patients from whom the blood spots were taken was collected. The prevalence of CQ-sensitive markers of pfcrt K76 and pfmdr1 N86 was performed using nested PCR and RFLP. The data were analysed using Chi-square and Odd ratio. Results: The overall prevalence of CQ-sensitive P. falciparum markers, pfcrt K76 and pfmdr1 N86 in the Central Region of Ghana were 142 out of 184 (77.17%) and 180 out of 184 (97.83%), respectively. The distribution of pfcrt K76 was assessed among the age groups per the individual study sites. 12 out of 33 (36.36%), 8 out of 33 (24.24%) and 6 out of 33 (18.18%) of pfcrt K76 CQ-sensitive marker were isolated from age 0 to 5 years, 16 to 30 years and 31 to 45 years old respectively at Cape Coast. Assin Fosu and Twifo Praso had the highest pfcrt K76 prevalence in 0–5 years, followed by 16–30 years and 6–15 years of age. The results showed that there was a significant prevalence of pfcrt K76 in all study sites; Cape Coast (χ2 = 26.48, p < 0.0001), Assin Fosu (χ2 = 37.67, p < 0.0001), Twifo Praso (χ2 = 32.25, p < 0.0001) and Elmina (χ2 = 17.88, p < 0.0001). Again, the likelihood to detect pfcrt K76 (OR (95% CI) was 7.105 (3.118–17.14), p < 0.0001 and pfmdr1 (2.028 (1.065–3.790), p < 0.001) among P. falciparum isolates from Cape Coast to be seven times and two times, respectively. Conclusion: The study showed a significant selection and expansion of chloroquine-sensitive P. falciparum markers in all the selected study areas in the Central region. This finding has a significant implication for the future treatment, management, and control of P. falciparum malaria. [ABSTRACT FROM AUTHOR]

Background: The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG. Methods: In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016–2018. Results: A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10–3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10–16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10–6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia. Conclusions: Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important. [ABSTRACT FROM AUTHOR]

Background: Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated. Methods: Children aged 6–59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing. Results: No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72–76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons. Conclusion: In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique. [ABSTRACT FROM AUTHOR]

Background: The spread of drug resistance has seriously impacted the effective treatment of infection with the malaria parasite, Plasmodium falciparum. Continuous monitoring of molecular marker polymorphisms associated with drug resistance in parasites is essential for malaria control and elimination efforts. Our study describes mutations observed in the resistance genes Pfkelch13, Pfcrt , and Pfmdr1 in imported malaria and identifies additional potential drug resistance-associated molecular markers. Methods: Chinese patients infected in Africa with P. falciparum were treated with intravenous (IV) injections of artesunate 240–360 mg for 3–5 days while hospitalized and treated with oral dihydroartemisinin-piperaquine (DHP) for 3 days after hospital discharge. Blood samples were collected and PCR sequencing performed on genes Pfkelch13, Pfcrt , and Pfmdr1 from all isolates. Results: We analyzed a total of 225 patients from Guangxi, China with P. falciparum malaria acquired in Africa between 2016 and 2018. All patients were cured completely after treatment. The F446I mutation of the Pfkelch13 gene was detected for the first time from samples of West African P. falciparum , with a frequency of 1.0%. Five haplotypes of Pfcrt that encode residues 72–76 were found, with the wild-type CVMNK sequence predominating (80.8% of samples), suggesting that the parasites might be chloroquine sensitive. For Pfmdr1 , N86 Y (13.1%) and Y184 F (58.8%) were the most prevalent, suggesting that artemether-lumefantrine may not, in general, be a suitable treatment for the group. Conclusions: For the first time, this study detected the F446I mutation of the Pfkelch13 gene from Africa parasites that lacked clinical evidence of resistance. This study provides the latest data for molecular marker surveillance related to antimalarial drug resistance genes Pfkelch13, Pfcrt , and Pfmdr1 imported from Africa, in Guangxi, China from Chinese migrate workers. Clinical Trial Registration: ChiCTROPC17013106. [ABSTRACT FROM AUTHOR]

• A total of 142 Plasmodium spp infections were detected by PCR, all but two of which were asymptomatic. • The results showed poor diagnostic accuracy of microscopy and RDT for the detection of asymptomatic malaria. • There was a higher prevalence of P. vivax than expected, based on figures for Mindanao. • Drug-resistant gene alleles occurred in both P. falciparum and P. vivax. This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7–5.2%) in Sarangani, 10% (95% CI: 7.7–12.7%) in South Cotabato, and 4.2% (95% CI: 3.2–5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72–76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level. [ABSTRACT FROM AUTHOR]

Background: The spread of drug resistance has seriously impacted the effective treatment of infection with the malaria parasite, Plasmodium falciparum. Continuous monitoring of molecular marker polymorphisms associated with drug resistance in parasites is essential for malaria control and elimination efforts. Our study describes mutations observed in the resistance genes Pfkelch13, Pfcrt, and Pfmdr1 in imported malaria and identifies additional potential drug resistance-associated molecular markers.Methods: Chinese patients infected in Africa with P. falciparum were treated with intravenous (IV) injections of artesunate 240–360 mg for 3–5 days while hospitalized and treated with oral dihydroartemisinin-piperaquine (DHP) for 3 days after hospital discharge. Blood samples were collected and PCR sequencing performed on genes Pfkelch13, Pfcrt, and Pfmdr1 from all isolates.Results: We analyzed a total of 225 patients from Guangxi, China with P. falciparum malaria acquired in Africa between 2016 and 2018. All patients were cured completely after treatment. The F446I mutation of the Pfkelch13 gene was detected for the first time from samples of West African P. falciparum, with a frequency of 1.0%. Five haplotypes of Pfcrt that encode residues 72–76 were found, with the wild-type CVMNK sequence predominating (80.8% of samples), suggesting that the parasites might be chloroquine sensitive. For Pfmdr1, N86Y (13.1%) and Y184F (58.8%) were the most prevalent, suggesting that artemether-lumefantrine may not, in general, be a suitable treatment for the group.Conclusions: For the first time, this study detected the F446I mutation of the Pfkelch13 gene from Africa parasites that lacked clinical evidence of resistance. This study provides the latest data for molecular marker surveillance related to antimalarial drug resistance genes Pfkelch13, Pfcrt, and Pfmdr1 imported from Africa, in Guangxi, China from Chinese migrate workers.Clinical Trial Registration: ChiCTROPC17013106.

Emergence and spread of resistance in Plasmodium falciparum to the frontline treatment artemisinin-based combination therapies (ACTs) in the epicenter of multidrug resistance of Southeast Asia threaten global malaria control and elimination. Artemisinin (ART) resistance (or tolerance) is defined clinically as delayed parasite clearance after treatment with an ART drug. The resistance phenotype is restricted to the early ring stage and can be measured in vitro using a ring-stage survival assay. ART resistance is associated with mutations in the propeller domain of the Kelch family protein K13. As a pro-drug, ART is activated primarily by heme, which is mainly derived from hemoglobin digestion in the food vacuole. Activated ARTs can react promiscuously with a wide range of cellular targets, disrupting cellular protein homeostasis. Consistent with this mode of action for ARTs, the molecular mechanisms of K13-mediated ART resistance involve reduced hemoglobin uptake/digestion and increased cellular stress response. Mutations in other genes such as AP-2μ (adaptor protein-2 μ subunit), UBP-1 (ubiquitin-binding protein-1), and Falcipain 2a that interfere with hemoglobin uptake and digestion also increase resistance to ARTs. ART resistance has facilitated the development of resistance to the partner drugs, resulting in rapidly declining ACT efficacies. The molecular markers for resistance to the partner drugs are mostly associated with point mutations in the two food vacuole membrane transporters PfCRT and PfMDR1, and amplification of pfmdr1 and the two aspartic protease genes plasmepsin 2 and 3. It has been observed that mutations in these genes can have opposing effects on sensitivities to different partner drugs, which serve as the principle for designing triple ACTs and drug rotation. Although clinical ACT resistance is restricted to Southeast Asia, surveillance for drug resistance using in vivo clinical efficacy, in vitro assays, and molecular approaches is required to prevent or slow down the spread of resistant parasites. [Display omitted] [ABSTRACT FROM AUTHOR]