Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Boonyalai, Nonlawat; Vesely, Brian; Thamnurak, Chatchadaporn; Praditpol, Chantida; Fagnark, Watcharintorn; Kirativanich, Kirakarn; Saingam, Piyaporn; Chaisatit, Chaiyaporn; Lertsethtakarn, Paphavee; Gosi, Panita; Kuntawunginn, Worachet; Vanachayangkul, Pattaraporn; Spring, Michele; Fukuda, Mark M.; Lon, Chanthap; Smith, Philip L.; Waters, Norman C.; Saunders, David; Wojnarski, Mariusz

doi:10.21203/rs.3.rs-22444/v3

Cited by 3 publications

(11 citation statements)

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“…1A). A pfexo E415G was e ciently installed onto the P. falciparum B5 background [24]. In parallel, a pfexo E415E transgenic line expressing wild-type E415 codon in the context of recodonization abating the Cas9 protospacer adjacent motif (PAM) site was also attempted to rule out a role for silent mutations in drug susceptibility.…”

Section: Resultsmentioning

confidence: 99%

“…CRISPR/Cas9 approaches have transformed the speed and scale with which Plasmodium genome editing can be achieved, and the approach has been employed to introduce point mutations into several P. falciparum genes [53]. In this study, instead of using P. falciparum 3D7 or Dd2 lines, the Cambodia adapted-B5 line [24] was employed with the hypothesis that the B5 line has a close genetic background to the currently circulating P. falciparum in Cambodia where both ART-and PPQ-resistance parasites have been detected. It has been previously shown that different phenotypes were observed when using different parasite backgrounds.…”

Section: Discussionmentioning

confidence: 99%

“…The nucleotide sequence of pfexo from 3D7 was obtained from PlasmoDB with the accession number PF3D7_1362500. The P. falciparum B5 line was obtained from the cloning of a Cambodian P. falciparum isolate [24]. This isolate is ART-and PPQ-sensitive, but CQ-resistant.…”

Section: Materials and Methods Pfexo Gene And Amino Acid Predictionmentioning

confidence: 99%

“…To determine a bimodal-dose response curve, the PPQ concentration (2.44 to 100,000 ng/mL) and the dilution series were increased from 8 to 24 points, according to previously published reports [16,24]. Culture-adapted clinical isolates or engineered parasites were prepared in the similar manner as in in vitro drug susceptibility testing.…”

Section: Bimodal Dose Response Curvementioning

confidence: 99%

“…While the implication of multiple copies of pfpm2 and novel PfCRT mutations on PPQ resistance have been elucidated [19,20,29], and the PPQresistance phenotype can be observed in the presence of novel PfCRT mutations with the PfEXO-E415G mutation [24], there is still a lack of con rmation as to whether the PfEXO-E415G mutation alone causes PPQ-resistance. To investigate whether the PfEXO-E415G is directly associated with PPQ resistance, in this study, we used CRISPR-Cas9 genome editing to generate P. falciparum Cambodian parasites harbouring PfEXO-E415G.…”

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confidence: 99%

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A Single Point Mutation in the Plasmodium Falciparum 3´-5´Exonuclease Does Not Alter Piperaquine Susceptibility

Boonyalai

Kirativanich

Thamnurak

et al. 2022

Preprint

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Background The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. Methods Transgenic parasite clones carrying the E415G mutation in pfexo of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. Results The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. Conclusions Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and % PPQ survival, suggesting that this mutation is not associated with PPQ resistance and unsuitable to be used as a molecular marker for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Materials and Methods Pfexo Gene And Amino Acid Predictionmentioning

confidence: 99%

Section: Bimodal Dose Response Curvementioning

confidence: 99%

mentioning

confidence: 99%

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A Single Point Mutation in the Plasmodium Falciparum 3´-5´Exonuclease Does Not Alter Piperaquine Susceptibility

Boonyalai

Kirativanich

Thamnurak

et al. 2022

Preprint

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Molecular surveillance of dihydroartemisinin-piperaquine resistance in northwestern Thailand

Win

Manopwisedjaroen

Phumchuea

et al. 2022

Preprint

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Background: Dihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. Our aim is to assess whether the resistance genotypes have arisen the western part of country.Methods: Fifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt.Results:Among the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance.Conclusions: Kelch13 mutation was common in Tak Province in 2013-2019. A new mutation G533S emerged in 2014 and rose to dominance, possibly fixation, in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.

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Molecular surveillance of artemisinin resistance-related pfK13 and pfcrt polymorphisms in imported Plasmodium falciparum isolates reported in eastern China from 2015–2019

Kong

Feng

et al. 2022

Preprint

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Background The artemisinin-based combination therapies (ACTs) was recommended as the first line drugs by the World Health Organization to treat uncomplicated Plasmodium falciparum. However, the emergence and spread of P. falciparum resistant to artemisinins and their partner drugs is a significant risk for the global effort to reduce disease burden facing the world. Method A retrospective study was conducted to explore the prevalence and spatial distribution of pfK13 and pfcrt polymorphisms among imported P. falciparum isolates in years 2015–2019 in Shandong Province in eastern China. Individual epidemiological information was collected from a web-based reporting system were reviewed and analysed. Results A total of 425 P. falciparum blood samples in 2015–2019 were included and we found that 7.3% (31/425) carried pfK13 mutations. Out of the isolates that carried K13 mutations, 54.8% (17/31) were nonsynonymous polymorphisms. The mutant allele A578S, Q613H, C469C, and S549S in pfK13 were the more frequently detected allele, the mutation rate was the same as 0.7% (3/425). Another allele pfK13C580Y, closely associated with artemisinin (ART) resistance, was found as 0.5% (2/425), which was found in Cambodia. About 45.2% (14/31) of the P. falciparum isolates had pfK13 mutations resembled those from Western Africa. For the pfcrt gene, T76T356 and T76 were more frequent in all 13 different haplotypes with 4.7% (20/425) and 4.2% (18/425) that identified in 77 isolates (18.1%, 77/425). The CVIET and CVIKT mutant at loci 72–76 have exhibited a prevalence of 3.5% and 0.7%, respectively. The CVIET were mainly distributed in Congo (5.2%, 4/77) and Mozambique (5.2%, 4/77). No mutations were found at loci 97, 101 and 145. For polymorphisms at locus 356, a total of 24 isolates were identified and mainly from Congo (29.2%, 7/24). Conclusion These findings indicate a low prevalence of pfK13 in the African isolates, while the mutations related to piperaquine (PPQ) resistance remain at a certain level. Therefore, continuous molecular surveillance of pfcrt mutations and in vitro susceptibility tests related to PPQ are necessary.

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Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Cited by 3 publications

References 41 publications

A Single Point Mutation in the Plasmodium Falciparum 3´-5´Exonuclease Does Not Alter Piperaquine Susceptibility

A Single Point Mutation in the Plasmodium Falciparum 3´-5´Exonuclease Does Not Alter Piperaquine Susceptibility

Molecular surveillance of dihydroartemisinin-piperaquine resistance in northwestern Thailand

Molecular surveillance of artemisinin resistance-related pfK13 and pfcrt polymorphisms in imported Plasmodium falciparum isolates reported in eastern China from 2015–2019

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