Ultrasensitive ELISA for Accurate Detection of Plasmodium falciparum Infection.

Background: Rapid and accurate diagnostic methods are crucial for curbing the spread of malaria. Antigen-based rapid diagnostic tests are highly valued for their simplicity, but improved sensitivity is needed for more accurate detection. Furthermore, cases of infection with HRP2-deficient parasites are evading testing, meaning patients are not receiving treatment. Methods: We developed an ultrasensitive protein detection system for Plasmodium falciparum (Pf) histidine-rich protein 2 (PfHRP2) and pan-lactate dehydrogenase (pLDH) by combining an ELISA and a thio-NAD cycling (TN-cyclon™). The reason for attempting to detect pLDH is to accommodate HRP2-deficient strains. The samples measured included recombinant protein in BSA buffer, Pf-parasitized erythrocytes in BSA buffer, and Pf-parasitized erythrocytes in non-infected human whole blood. Results: Our technology detected recombinant PfHRP2 and pLDH at concentrations as low as 0.782 pg/mL and 1.33 pg/mL, respectively. This system detected Plasmodium falciparum parasitemia in in vitro culture at levels as low as 2.57 × 10-5% for PfHRP2 and 2.41 × 10-5% for pLDH. We then mixed purified parasitized erythrocytes with human whole blood to mimic whole blood conditions. Parasitemia of Pf-parasitized erythrocytes in human whole blood was successfully detected at levels as low as 0.5 parasites/μL for both PfHRP2 and pLDH. Conclusions: The detection sensitivity of our system is approximately 10 times greater than that of the latest ultrasensitive antigen detection kits. Our results demonstrate that our developed assay requires no additional processing to separate parasitized erythrocytes from whole blood samples and that the pfhrp2-gene deletion strain can be successfully detected using the pLDH-based TN-cyclon™ test.