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Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

ABSTRACT: The widely used antimalarial combination therapy dihydroartemisinin?+?piperaquine (DHA?+?PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA?+?PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from resistant Cambodian isolates, we show that the H97Y, F145I, M343L, or G353V mutations each confer resistance to PPQ, albeit with fitness costs for all but M343L. These mutations sensitize Dd2 parasites to chloroquine, amodiaquine, and quinine. In Dd2 parasites, multicopy plasmepsin 2, a candidate molecular marker, is not necessary for PPQ resistance. Distended digestive vacuoles were observed in pfcrt-edited Dd2 parasites but not in Cambodian isolates. Our findings provide compelling evidence that emerging mutations in PfCRT can serve as a molecular marker and mediator of PPQ resistance.

SUBMITTER: Ross LS

PROVIDER: S-EPMC6095916 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

Ross Leila S LS Dhingra Satish K SK Mok Sachel S Yeo Tomas T Wicht Kathryn J KJ Kümpornsin Krittikorn K Takala-Harrison Shannon S Witkowski Benoit B Fairhurst Rick M RM Ariey Frederic F Menard Didier D Fidock David A DA

Nature communications 20180817 1

The widely used antimalarial combination therapy dihydroartemisinin + piperaquine (DHA + PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA + PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from r ...[more]

PMID: 30115924

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Project description:Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials. Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined. The PPQ median IC50 interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC50s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC50s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC50 and Pfcrt K76T allele (P=0.0026). Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.

Project description:BackgroundDue to the widespread resistance of Plasmodium falciparum to chloroquine drug, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment. This study aims to evaluate the extent of chloroquine resistance in P. falciparum infection after the introduction of ACT. This study was carried out based on the mutation analysis in P. falciparum chloroquine resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes. Identification of these molecular markers plays a significant role in monitoring and assessment of drug resistance as well as in designing an effective antimalarial drug policy in India.MethodsSixty blood samples were collected from patients infected with P. falciparum from JIPMER, Puducherry and MKCG Medical College, Odisha. Polymerase chain reaction-restriction fragment length polymorphism was performed, targeting the point mutation of K76T in pfcrt and N86Y in pfmdr1 gene. The PCR products were sequenced, genotyped and further analysed for amino acid changes in these codons.ResultsThe frequency of pfcrt mutation at 76th position was dominant for mutant T allele with 56.7% and wild type K, 43.3%. Majority of pfmdr1 86 allele were wild type, with N (90%) and mutant, Y (10%). Additionally, we found three haplotypes for CQ resistance, SVMNT, CVIET and CVIKT in association with the pfcrt gene. However, a poorly studied SNP in pfmdr1 gene (Y184F) associated with CQ resistance showed high frequency (70%) in P. falciparum isolates.ConclusionsThe point mutation K76T of pfcrt is high in P. falciparum suggesting a sustained high CQ resistance even after five years of the introduction of ACTs for antimalarial therapy. The present study suggests a strong association of CQ resistance with pfcrt T76, but not with the pfmdr1 Y86 mutation. However, sequence analysis showed that Y184F mutation on pfmdr1 gene was found to be associated with high resistance. Also, a new pfcrt haplotype 'CVIKT' associated with CQ resistance was found to be present in Indian strains of P. falciparum. The data obtained from this study helps in continuous monitoring of drug resistance in malaria and also suggests the need for careful usage of CQ in Plasmodium vivax malarial treatment.

Dataset Information

Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

Publications

Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.

Similar Datasets

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