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Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring.

OBJECTIVES: Accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex qPCR and conventional PCR (cPCR) using existing samples with clonality previously determined by microsatellite genotyping.

METHODS: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital PCR. Sample classification was compared with cPCR, and ROC analysis used to determine the optimal ΔCq threshold that aligned results of the two assays.

RESULTS: qPCR classified 75% (637/849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. Sample classification agreement between cPCR and qPCR was 75.1% (95% CI 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ=0.804; 95% CI 0.714-0.895) and substantial agreement (κ=0.717; 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching assay results was ΔCq=3.

CONCLUSION: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate, but qPCR provides higher estimates where multiclonal infections are common.

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