Multiplex Immunoassays Enable Quantification of Malaria Antigens

The standard of care for malaria diagnosis is blood smear microscopy and antigen detection through rapid diagnostic test (RDT). Microscopy has limitations in terms of difficulty in identifying mixed infections and RDTs are more amenable for the diagnosis of malaria in settings with limited laboratory infrastructure.

Immunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. The gold standard for malaria detection is confirmation of the presence of parasite DNA or RNA in whole blood by polymerase chain reaction (PCR) testing.

A team of medical scientists led by those at the Diagnostic Group, PATH (Seattle, WA, USA) studied the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species.

The team used a 77-member panel of specimens composed of the World Health Organization (WHO, Geneva, Switzerland) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens.

Assays for HRP2, P. falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex (Quansys Biosciences, Logan, UT, USA) and the xMAP platforms (Luminex, Austin, TX, USA) were evaluated with these panels.

The investigators reported that the xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH and PvLDH, moderate positive correlation for HRP2, and poor correlation for PanLDH. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS.

The authors concluded that Q-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform. The study was published on June 7, 2022 in the Malaria Journal.

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