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

Author:

Vera-Arias Claudia A1,Holzschuh Aurel12ORCID,Oduma Colins O34,Badu Kingsley5ORCID,Abdul-Hakim Mutala5,Yukich Joshua6ORCID,Hetzel Manuel W27,Fakih Bakar S278,Ali Abdullah9,Ferreira Marcelo U10ORCID,Ladeia-Andrade Simone11,Sáenz Fabián E12,Afrane Yaw13,Zemene Endalew14,Yewhalaw Delenasaw14,Kazura James W15,Yan Guiyun16,Koepfli Cristian1ORCID

Affiliation:

1. University of Notre Dame

2. Swiss Tropical and Public Health Institute

3. Kenya Medical Research Institute-Centre for Global Health Research

4. Department of Biochemistry and Molecular Biology, Egerton University

5. Kwame Nkrumah University of Science and Technology

6. Tulane University

7. University of Basel

8. Ifakara Health Institute

9. Zanzibar Malaria Elimination Programme, Zanzibar

10. University of São Paulo

11. Laboratory of Parasitic Diseases, Fiocruz

12. Centro de Investigación para la Salud en América Latina, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador

13. Department of Medical Microbiology, University of Ghana

14. Tropical and Infectious Diseases Research Center, Jimma University

15. Case Western Reserve University

16. Program in Public Health, University of California, Irvine

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.

Funder

National Institutes of Health

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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