High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy

Abstract

Most rapid diagnostic tests for <i>Plasmodium falciparum</i> malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the <i>hrp2</i> and <i>hrp3</i> genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of <i>hrp2</i>/<i>hrp3</i> deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies <i>hrp2</i>, <i>hrp3</i>, and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and <i>hrp2</i>-deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR <i>hrp2</i> deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more <i>hrp3</i> than <i>hrp2</i> deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., <i>hrp2</i> deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.