University of Notre Dame, Notre Dame, United States; Swiss Tropical and Public Health Institute, Allschwil, Switzerland
Colins O Oduma
Kenya Medical Research Institute-Centre for Global Health Research, Kisumu, Kenya; Department of Biochemistry and Molecular Biology, Egerton University, Nakuru, Kenya
Swiss Tropical and Public Health Institute, Allschwil, Switzerland; University of Basel, Basel, Switzerland
Bakar S Fakih
Swiss Tropical and Public Health Institute, Allschwil, Switzerland; University of Basel, Basel, Switzerland; Ifakara Health Institute, Dar es Salaam, United Republic of Tanzania
Abdullah Ali
Zanzibar Malaria Elimination Programme, Zanzibar, Zanzibar, United Republic of Tanzania
Laboratory of Parasitic Diseases, Fiocruz, Rio de Janeiro, Brazil
Fabián E Sáenz
Centro de Investigación para la Salud en América Latina, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador
Yaw Afrane
Department of Medical Microbiology, University of Ghana, Accra, Ghana
Endalew Zemene
Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia
Delenasaw Yewhalaw
Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia
James W Kazura
Case Western Reserve University, Cleveland, United States
Guiyun Yan
Program in Public Health, University of California, Irvine, Irvine, United States
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 >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.
