High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy
- University of Notre Dame, United States
- Kenya Medical Research Institute, Kenya
- Kwame Nkrumah University of Science and Technology, Ghana
- Tulane University, United States
- Swiss Tropical and Public Health Institute, Switzerland
- Ifakara Health Institute, United Republic of Tanzania
- Ministry of Health - Zanzibar, United Republic of Tanzania
- University of São Paulo, Brazil
- Laboratory of Parasitic Diseases, Fiocruz, Brazil
- Pontificia Universidad Católica del Ecuador, Ecuador
- University of Ghana, Ghana
- Jimma University, Ethiopia
- Case Western Reserve University, United States
- 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
Marcelo U Ferreira
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.
Reviewing Editor
- Bavesh D Kana, University of the Witwatersrand, South Africa
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).
Version history
- Preprint posted: June 4, 2021 (view preprint)
- Received: July 9, 2021
- Accepted: June 5, 2022
- Accepted Manuscript published: June 28, 2022 (version 1)
- Version of Record published: June 30, 2022 (version 2)
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.
Metrics
-
- 2,994
- views
-
- 632
- downloads
-
- 23
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Microbiology and Infectious Disease
The microbiota is a key determinant of the physiology and immunity of animal hosts. The factors governing the transmissibility of viruses between susceptible hosts are incompletely understood. Bacteria serve as food for Caenorhabditis elegans and represent an integral part of the natural environment of C. elegans. We determined the effects of bacteria isolated with C. elegans from its natural environment on the transmission of Orsay virus in C. elegans using quantitative virus transmission and host susceptibility assays. We observed that Ochrobactrum species promoted Orsay virus transmission, whereas Pseudomonas lurida MYb11 attenuated virus transmission relative to the standard laboratory bacterial food Escherichia coli OP50. We found that pathogenic Pseudomonas aeruginosa strains PA01 and PA14 further attenuated virus transmission. We determined that the amount of Orsay virus required to infect 50% of a C. elegans population on P. lurida MYb11 compared with Ochrobactrum vermis MYb71 was dramatically increased, over three orders of magnitude. Host susceptibility was attenuated even further in the presence of P. aeruginosa PA14. Genetic analysis of the determinants of P. aeruginosa required for attenuation of C. elegans susceptibility to Orsay virus infection revealed a role for regulators of quorum sensing. Our data suggest that distinct constituents of the C. elegans microbiota and potential pathogens can have widely divergent effects on Orsay virus transmission, such that associated bacteria can effectively determine host susceptibility versus resistance to viral infection. Our study provides quantitative evidence for a critical role for tripartite host-virus-bacteria interactions in determining the transmissibility of viruses among susceptible hosts.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.
