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

University of Notre Dame, Notre Dame, United States.
University of Notre Dame, Notre Dame, United States / Swiss Tropical and Public Health Institute, Allschwil, Switzerland.
Kenya Medical Research Institute-Centre for Global Health Research, Kisumu, Kenya / Department of Biochemistry and Molecular Biology, Egerton University, Nakuru, Kenya.
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Tulane University, New Orleans, United States.
Swiss Tropical and Public Health Institute, Allschwil, Switzerland / University of Basel, Basel, Switzerland.
Swiss Tropical and Public Health Institute, Allschwil, Switzerland / University of Basel, Basel, Switzerland / Ifakara Health Institute, Dar es Salaam, United Republic of Tanzania.
Zanzibar Malaria Elimination Programme, Zanzibar, Zanzibar, United Republic of Tanzania.
Universidade de São Paulo. São Paulo, SP, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ, Brasil.
Centro de Investigación para la Salud en América Latina, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador.
Department of Medical Microbiology, University of Ghana, Accra, Ghana.
Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia.
Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia.
Case Western Reserve University, Cleveland, United States.
Program in Public Health, University of California, Irvine, Irvine, United States.
University of Notre Dame, Notre Dame, United States.

Abstract

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.

Keywords in Portuguese

Plasmodium falciparum
Alto rendimento
Tipagem de deleção dos genes hrp2 e hrp3
PCR digital
Eficácia do teste de diagnóstico
Para monitorar a malária rapidamente

Keywords

Plasmodium falciparum
High-throughput
Hrp2 and hrp3 gene deletion typing
Digital PCR
To monitor malaria rapid
Diagnostic test efficacy

Publisher

Bavesh D Kana, University of the Witwatersrand, South Africa

Citation

VERA-ARIAS, Claudia A. et al. High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy. eLife, v.11, e72083, p. 1 - 16, June 2022.

DOI

10.7554/eLife.72083

ISSN

2050-084X

Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/56029

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Open access

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