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. 2017 Apr 24;61(5):e00110-17.
doi: 10.1128/AAC.00110-17. Print 2017 May.

The Malaria TaqMan Array Card Includes 87 Assays for Plasmodium falciparum Drug Resistance, Identification of Species, and Genotyping in a Single Reaction

Suporn Pholwat  1 , Jie Liu  1 , Suzanne Stroup  1 , Shevin T Jacob  2 , Patrick Banura  3 , Christopher C Moore  1 , Fang Huang  4   5 , Miriam K Laufer  4 , Eric Houpt  1 , Jennifer L Guler  6   7
Affiliations

The Malaria TaqMan Array Card Includes 87 Assays for Plasmodium falciparum Drug Resistance, Identification of Species, and Genotyping in a Single Reaction

Suporn Pholwat et al. Antimicrob Agents Chemother. .

Abstract

Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in countries of malaria endemicity, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TaqMan array card, TAC). We included 87 assays for species identification, for the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and for neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1 to 10 pg of DNA and yielded 100% accuracy compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world, and the malaria TAC once again achieved 100% accuracy compared to sequencing and in addition detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria TAC should be a useful tool for large-scale antimalarial resistance surveillance.

Keywords: TaqMan PCR; antimalarial resistance; malaria; mutation; surveillance tool.

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Figures

FIG 1
FIG 1
Malaria TAC design. The antimalarial resistance-focused TaqMan array card (malaria TAC) includes eight ports/card (one port is shown), and each port is connected to 48 assay wells. Each assay well contains prespotted primers and probes and is configured as shown on the basis of gene, codon, and wild-type/mutant-specific loci. The antimalarial to which resistance is conferred is listed next to each group of targets (MDR, multidrug resistance; Sulf, sulfadoxine; Pyr, pyrimethamine; Ato, atovaquone; CQ, chloroquine; Art, artemisinin). For genotyping, the SNPs are identified as follows: SNP1, Pf_01_000130573; SNP2, Pf_01_000539044; SNP3, Pf_02_000842803; SNP6, Pf_06_000145472; SNP7, Pf_06_000937750; SNP8, to Pf_07_000277104. Plasmodium species-specific probes are included to confirm the presence of P. falciparum DNA in each sample.
FIG 2
FIG 2
Examples of heteroresistance and mixed infection as determined by the malaria TAC. The DB133 and DB009 samples were detected as mixed alleles by the malaria TAC and then sequence confirmed. (A) TaqMan probe-based amplification plots are shown for the wild-type probe (pfMDR1 184Y) and mutant probe (pfMDR1 184F); Sanger sequencing shows mixed T/A residues (arrow), indicating mixed F(TTT) and Y(TAT) alleles for the DB133 sample (bottom panel). (B) TaqMan probe-based amplification plots are shown for the major and minor allele probes for SNP3 (Pf_02_000842803 (T) and Pf_02_000842803 (C); Sanger sequencing shows mixed T/C residues for the DB009 sample (arrow).
See this image and copyright information in PMC

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