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

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.

Keywords: Plasmodium falciparum; ddPCR; pfgch1; pfmdr1; pfplasmepsin2.

Fig. 1

Two dimensional ddPCR amplitude plots of multiplex ddPCR assays. Heat map shows 8 clusters of droplets (a) including, droplets contain pfmdr1, pfplasmepsin2, and pfbtubulin (cluster 1), droplets contain both pfplasmepsin2 and pf-β-tubulin (cluster 2), droplets contain both pfmdr1 and pf-β-tubulin (cluster 3), droplets with at least one copy of pf-β-tubulin (cluster 4), droplets contain both pfmdr1 and pf-β-tubulin (cluster 5), droplets with at least one copy of pfplasmepsin2 (cluster 6), droplets with at least one copy of pfmdr1 (cluster 7), Empty droplets, no DNA target (cluster 8). Classification cluster of droplets for pfmdr1 copy number detection (b). Classification cluster of droplets for pfplasmepsin2 copy number detection (c)

Fig. 2

Limitation of gene quantification, accuracy and precision of ddPCR assays for pfmdr1 copy number detection (a), pfplasmepsin2 copy number detection (b), and pfgch1 copy number detection (c)

Fig. 3

Whisker plots show median, maximum, and minimum of estimated pfmdr1 (a), pfplasmepsin2 (b), and pfgch1 (c) copy number

Fig. 4

Genes copy number of P. falciparum mdr1 (a), plasmepsin2 (b), and gch1 of reference strains estimated by ddPCR in replicates

Fig. 5

Prevalence of pfmdr1, pfplasmepsin2, and pfgch1 gene amplification isolated from Ubon Ratchathani and Yala

Fig. 6

A standardized analytical workflow of ddPCR analysis used for genes copy number quantification