Plasmodium falciparum hrp2 and hrp3 gene deletion status in Africa and South America by highly sensitive and specific digital PCR

Background: The most commonly used Plasmodium falciparum rapid diagnostic tests target the Histidine-Rich Proteins 2 and 3 (HRP2, HRP3). An increasing number of countries report parasites that carry hpr2 and/or hrp3 gene deletions, resulting in false negative test results. Molecular surveillance of hrp2 and hrp3 deletions is crucial but adequate protocols have been lacking. Methods and Findings: We have developed novel assays for deletion typing based on droplet digital PCR (ddPCR), targeting hrp2 exon1, hrp2 exon 2, and hrp3. In the ddPCR assay, hrp2 or hrp3 and a control gene were quantified with very high accuracy in a single tube. The theoretical limit of detection of the ddPCR assay was 0.33 parasites/uL, and thus well suited for typing of low-density asymptomatic infections. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites when the proportion of parasites carrying the deletion was >40%. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples from asymptomatic individuals from western Kenya were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR no band for hrp2 was observed in 8% of samples. The ddPCR assay was applied to screen 777 samples from six countries in Africa and South America. No deletions were observed in Kenya (n=241) and Zanzibar/Tanzania (n=91), and very few in Ghana (3/170 hrp3 deletions). In southwestern Ethiopia, 1/47 (2.1%) samples carried hrp2 deletion, and 35/47 (74.5%) hrp3 deletions. In Brazil, 87/187 (46.5%) samples carried hrp2 deletions, and 116/187 (62%) hrp3 deletions. In Ecuador, no hrp2 deletions were observed, but 22/41 (53.7%) samples carried hrp3 deletions. Conclusions: Compared to nPCR, the ddPCR assay minimizes the risk of false-negative results (i.e. hrp2 deletion observed when the sample is wild type), increase sensitivity, and greatly reduces the number of reactions that need to be run. Pronounced differences in the prevalence of deletion were observed among sites, with more hrp3 than hrp2 deletions.