Background: Due to limitation of conventional malaria diagnostics, including microscopy, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), alternative accurate diagnostics have been demanded for improvement of sensitivity and specificity.
Methods: Serially diluted Plasmodium LDH antigens, Plasmodium falciparum-infected human red blood cells (RBC) derived from in vitro culture or patient's samples were used for evaluation of the performance of fluorescence-linked immunosorbent assay (FLISA). Microscopic examination was used to determine parasite density and the performance of FLISA was compared to ELISA. Finally, sensitivity and specificity of FLISA was determined by human specimens infected with P. falciparum, Plasmodium vivax, Toxoplasma gondii, and amoebae.
Results: As a result of FLISA, the fluorescent intensity was highly correlated with antigen amount and FLISA was more sensitive than ELISA. FLISA detected at least 0.01 ng/ml of pLDH antigen, which showed 1,000-fold higher sensitivity than ELISA. In vitro-cultured P. falciparum was detected up to 20 parasite number/μL in FLISA but 5120 parasite number/μLin sandwich ELISA. In vitro P. falciparum-infected RBC number was highly correlated with fluorescent intensity (R2 = 0.979), showing that FLISA was reliable for detection of P. falciparum and available for quantification of parasite numbers. Furthermore, eighteen patient samples infected with P. falciparum (n = 9) and P. vivax (n = 9) showed 100% of sensitivity (18/18). FLISA showed 96.3% of specificity (26/27) because one sample of patient blood infected with T. gondii gave a false positive reactivity among healthy donors (n = 9), T. gondii-infected patients (n = 9), and amoeba-infected patients (n = 9).
Conclusion: FLISA has a keen and high performance to detect malaria antigen, suggesting a potential assay as malaria immunodiagnostic.