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Most rapid diagnostic tests for  Plasmodium falciparum  malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the  hrp2  and  hrp3  genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of  hrp2 / hrp3  deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies  hrp2 ,  hrp3 , 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  hrp2 -deleted parasites at a density of &gt;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  hrp2  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  hrp3  than  hrp2  deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e.,  hrp2  deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.

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