Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

10.21203/rs.3.rs-103611/v1 ◽

2020 ◽

Author(s):

Suttipat Srisut ◽

Kanokon Suwannasin ◽

Rungirun Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Antimalarial Drug Resistance ◽

Quantification Limit ◽

Field Samples

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial 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 antimalarial drugs.Methods: The ddPCR assays were developed to detect the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes. 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 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: 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. The accuracy and %RSD value of the duplex ddPCR assay were 94.88% and 3.71, 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). 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 antimalarial drug resistance.

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No evidence of amplified Plasmodium falciparum plasmepsin II gene copy number in an area with artemisinin-resistant malaria along the China–Myanmar border

Malaria Journal ◽

10.1186/s12936-020-03410-6 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Fang Huang ◽

Biraj Shrestha ◽

Hui Liu ◽

Lin-Hua Tang ◽

Shui-Sen Zhou ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Molecular Markers ◽

Therapeutic Efficacy ◽

Copy Number ◽

Copy Number Variants ◽

Gene Copy Number ◽

Artemisinin Resistance ◽

World Health ◽

Gene Copy ◽

Synonymous Mutations

Abstract Background The emergence and spread of artemisinin resistance in Plasmodium falciparum poses a threat to malaria eradication, including China’s plan to eliminate malaria by 2020. Piperaquine (PPQ) resistance has emerged in Cambodia, compromising an important partner drug that is widely used in China in the form of dihydroartemisinin (DHA)-PPQ. Several mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 (k13) are associated with artemisinin resistance and have arisen spread in the Great Mekong subregion, including the China–Myanmar border. Multiple copies of the plasmepsin II/III (pm2/3) genes, located on chromosome 14, have been shown to be associated with PPQ resistance. Methods The therapeutic efficacy of DHA-PPQ for the treatment of uncomplicated P. falciparum was evaluated along the China–Myanmar border from 2010 to 2014. The dry blood spots samples collected in the efficacy study prior DHA-PPQ treatment and from the local hospital by passive detection were used to amplify k13 and pm2. Polymorphisms within k13 were genotyped by capillary sequencing and pm2 copy number was quantified by relative-quantitative real-time polymerase chain reaction. Treatment outcome was evaluated with the World Health Organization protocol. A linear regression model was used to estimate the association between the day 3 positive rate and k13 mutation and the relationship of the pm2 copy number variants and k13 mutations. Results DHA-PPQ was effective for uncomplicated P. falciparum infection in Yunnan Province with cure rates > 95%. Twelve non synonymous mutations in the k13 domain were observed among the 268 samples with the prevalence of 44.0% and the predominant mutation was F446I with a prevalence of 32.8%. Only one sample was observed with multi-copies of pm2, including parasites with and without k13 mutations. The therapeutic efficacy of DHA-PPQ was > 95% along the China–Myanmar border, consistent with the lack of amplification of pm2. Conclusion DHA-PPQ for uncomplicated P. falciparum infection still showed efficacy in an area with artemisinin-resistant malaria along the China–Myanmar border. There was no evidence to show PPQ resistance by clinical study and molecular markers survey. Continued monitoring of the parasite population using molecular markers will be important to track emergence and spread of resistance in this region.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

Malaria Journal ◽

10.1186/s12936-021-03659-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Suttipat Srisutham ◽

Kanokon Suwannasin ◽

Rungniran Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Quantification Limit ◽

Field Samples ◽

The Cost

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.

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