Ayoade Babatunde Olanrewaju¹, Charles Obirinanwa², Ochuele Dominic Agida²,³, Moses Adondua Abah 3,4, Micheal Oladosu Abimbola4,5 and Jabir Hassan Abdullahi³
¹Department of Biology and Environmental Science, College of Arts and Sciences, University of New Haven, West Haven, Connecticut, USA
²Stockton University, New Jersey, United States.
³Department of Biochemistry, Faculty of Biosciences, Federal University Wukari, Taraba State, Nigeria
4ResearchHub Nexus Institute, Nigeria
5Department of Chemical Sciences, Faculty of Science, Anchor University, Ayobo, Lagos State, Nigeria
(✉) Corresponding Author: Ayoade Babatunde Olanrewaju olanrewajuayoade75@gmail.com
Received: July 22, 2026/ Revised: Sept 10, 2026/Accepted: Sept 15, 2026
Highlights
- HRP2-based RDTs are widely used for P. falciparum malaria diagnosis.
- pfhrp2/pfhrp3 gene deletions can cause false-negative malaria RDT results.
- Digital PCR provides sensitive and quantitative detection of parasite gene deletions.
- dPCR can help characterize complete and partial pfhrp2/pfhrp3 deletions.
- Variation in deletion prevalence highlights the need for standardized molecular surveillance.
- Molecular monitoring can improve detection of diagnostic escape variants and strengthen malaria surveillance.
Abstract
Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are widely used for detecting Plasmodium falciparum malaria, particularly in settings where microscopy and molecular diagnostics are limited. However, deletions in the pfhrp2 and pfhrp3 genes can result in false-negative RDT results, undermining malaria case detection and surveillance. Digital polymerase chain reaction (dPCR) provides highly sensitive and quantitative molecular detection that may improve characterization of these genetic deletions. This systematic review evaluates the application of dPCR for detecting and quantifying pfhrp2/pfhrp3 gene deletions among RDT-negative P. falciparum infections and examines their diagnostic and epidemiological implications. Relevant studies were systematically identified through electronic database searches using predefined eligibility criteria. Studies investigating pfhrp2, pfhrp3, or dual-gene deletions in RDT-negative or suspected RDT-missed P. falciparum infections using dPCR or related quantitative molecular approaches were considered. Data on study populations, molecular targets, assay characteristics, deletion frequencies, and diagnostic findings were extracted and critically assessed. Evidence was synthesized narratively, with quantitative analysis considered where methodological and clinical homogeneity permitted. The review synthesizes evidence on the detection and quantitative characterization of pfhrp2/pfhrp3 deletions, their association with RDT-negative infections, and their geographic distribution. It further evaluates how dPCR can distinguish complete and partial deletions and provide quantitative evidence relevant to parasite detection and diagnostic failure. Variation in deletion prevalence and methodological approaches across settings highlights the importance of standardized molecular surveillance. dPCR represents a valuable tool for investigating pfhrp2/pfhrp3 deletions and clarifying their contribution to false-negative malaria RDT results. Its integration into molecular surveillance could strengthen detection of diagnostic escape variants and inform evidence-based malaria diagnostic strategies.
Keywords: Digital PCR, Plasmodium falciparum, pfhrp2, pfhrp3, Malaria rapid diagnostic tests and Gene deletions.
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How to cite this article
Olanrewaju, A. B., Obirinanya, C., Agida, O. D., Abah, M. A., Abimbola, M. O., & Hassan, A. J. (2026). Digital PCR quantification of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in malaria RDT-negative patients. Microbial Science Archives, 6(3), 104–115. https://doi.org/10.47587/MSA.2026.6304
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