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

  1. Claudia A Vera-Arias
  2. Aurel Holzschuh
  3. Colins O Oduma
  4. Kingsley Badu
  5. Mutala Abdul-Hakim
  6. Joshua Yukich
  7. Manuel W Hetzel
  8. Bakar S Fakih
  9. Abdullah Ali
  10. Marcelo U Ferreira
  11. Simone Ladeia-Andrade
  12. Fabián E Sáenz
  13. Yaw Afrane
  14. Endalew Zemene
  15. Delenasaw Yewhalaw
  16. James W Kazura
  17. Guiyun Yan
  18. Cristian Koepfli  Is a corresponding author
  1. University of Notre Dame, United States
  2. Kenya Medical Research Institute, Kenya
  3. Kwame Nkrumah University of Science and Technology, Ghana
  4. Tulane University, United States
  5. Swiss Tropical and Public Health Institute, Switzerland
  6. Ifakara Health Institute, United Republic of Tanzania
  7. Ministry of Health - Zanzibar, United Republic of Tanzania
  8. University of São Paulo, Brazil
  9. Laboratory of Parasitic Diseases, Fiocruz, Brazil
  10. Pontificia Universidad Católica del Ecuador, Ecuador
  11. University of Ghana, Ghana
  12. Jimma University, Ethiopia
  13. Case Western Reserve University, United States
  14. University of California, Irvine, United States

Abstract

Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2, 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.

Data availability

All data is provided in supplementary file S3.

Article and author information

Author details

  1. Claudia A Vera-Arias

    Biological Sciences, University of Notre Dame, Notre Dame, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Aurel Holzschuh

    Biological Sciences, University of Notre Dame, Notre Dame, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2681-1114
  3. Colins O Oduma

    Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya
    Competing interests
    The authors declare that no competing interests exist.
  4. Kingsley Badu

    Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7886-5528
  5. Mutala Abdul-Hakim

    Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
    Competing interests
    The authors declare that no competing interests exist.
  6. Joshua Yukich

    Department of Global Health Systems and Development, Tulane University, New Orleans, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6160-5295
  7. Manuel W Hetzel

    Swiss Tropical and Public Health Institute, Allschwil, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Bakar S Fakih

    Ifakara Health Institute, Dar es Salaam, United Republic of Tanzania
    Competing interests
    The authors declare that no competing interests exist.
  9. Abdullah Ali

    Zanzibar Malaria Elimination Programme, Ministry of Health - Zanzibar, Zanzibar, United Republic of Tanzania
    Competing interests
    The authors declare that no competing interests exist.
  10. Marcelo U Ferreira

    Departamento de Parasitologia, ICB II, University of São Paulo, São Paulo, Brazil
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5293-9090
  11. Simone Ladeia-Andrade

    Laboratory of Parasitic Diseases, Fiocruz, Rio de Janeiro, Brazil
    Competing interests
    The authors declare that no competing interests exist.
  12. Fabián E Sáenz

    Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Quito, Ecuador
    Competing interests
    The authors declare that no competing interests exist.
  13. Yaw Afrane

    Department of Medical Microbiology, University of Ghana, Accra, Ghana
    Competing interests
    The authors declare that no competing interests exist.
  14. Endalew Zemene

    Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia
    Competing interests
    The authors declare that no competing interests exist.
  15. Delenasaw Yewhalaw

    Tropical and Infectious Diseases Research Center, Jimma University, Jimma, Ethiopia
    Competing interests
    The authors declare that no competing interests exist.
  16. James W Kazura

    Case Western Reserve University, Cleveland, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Guiyun Yan

    Program in Public Health, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Cristian Koepfli

    Biological Sciences, University of Notre Dame, Notre Dame, United States
    For correspondence
    ckoepfli@nd.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9354-0414

Funding

National Institutes of Health (R21 AI137891)

  • Cristian Koepfli

National Institutes of Health (D43 TW001505)

  • Guiyun Yan

National Institutes of Health (U19 AI129326)

  • Guiyun Yan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: Informed written consent was obtained from all study participants or their parents or legal guardians prior to sample collection. The study was approved by the University of Notre Dame Institutional Review Board (approvals 18-08-4803, 19-04-5321, 18-12-5029), the Institutional Scientific and Ethical Review boards of the Noguchi Memorial Institute of Medical Research, University of Ghana, the Committee on Human Research, Publication and Ethics, School of Medical Science, Kwame Nkrumah University of Science and Technology, Kumasi (CHRPE/AP/375/20), the Zanzibar Medical Research Ethics Committee (ZAMREC/0001/Feb/17), the Institutional Review Board of Tulane University (17-993573), the Institutional Review Board of the Ifakara Health Institute (003-2017), the Ethics Commission of North-western and Central Switzerland (Req-2017-00162), the Institutional Review Board of Institute of Health, Jimma University, Ethiopia (RPGC/486/06), Maseno University Ethics Review Committee (MUERC protocol number 00456), the Ethics Committee for Research in Human Beings of the Pontificia Universidad Católica del Ecuador (CEISH-571-2018), the Ministry of Public Health of Ecuador (MSP-DIS-2019-004-O), and the institutional review board of Oswaldo Cruz Foundation, Brazil (no. 022/2009).

Copyright

© 2022, Vera-Arias et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Claudia A Vera-Arias
  2. Aurel Holzschuh
  3. Colins O Oduma
  4. Kingsley Badu
  5. Mutala Abdul-Hakim
  6. Joshua Yukich
  7. Manuel W Hetzel
  8. Bakar S Fakih
  9. Abdullah Ali
  10. Marcelo U Ferreira
  11. Simone Ladeia-Andrade
  12. Fabián E Sáenz
  13. Yaw Afrane
  14. Endalew Zemene
  15. Delenasaw Yewhalaw
  16. James W Kazura
  17. Guiyun Yan
  18. Cristian Koepfli
(2022)
High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy
eLife 11:e72083.
https://doi.org/10.7554/eLife.72083

Share this article

https://doi.org/10.7554/eLife.72083

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