Abstract
BackgroundDue 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.MethodsSerially 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.ResultsAs 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).ConclusionFLISA has a keen and high performance to detect malaria antigen, suggesting a potential assay as malaria immunodiagnostic.
Highlights
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
Coumarin-derived dendrimer was chosen as labelling fluorophore, which was confirmed to have good spectroscopic properties in previous study [13]
Two newly developed monoclonal antibodies (mAbs) targeting pLDH were used for fluorescence-linked immunosorbent assay (FLISA) instead of commercialized mAbs of pLDH, and performance in FLISA was defined to determine antigen-antibody reactivity
Summary
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. The establishment of accurate diagnostics of malaria based on quantification of malaria parasite number with high sensitivity and specificity is the top priority for the management of malaria. As an alternative quantitative diagnosis of malaria, over the last few decades, numerous polymerase chain reactions (PCR)-based diagnostic tests targeting RNA or DNA have been developed to confirm the malaria infection in addition to microscopic observation [7,8,9]. Due to the limitation of PCR, different immunological assays that use antibodies to detect parasites have been developed with greater potential for adaption to field application than previous approaches, even though immune assays provide sensitivity issues [11]. Resulting devices for the diagnosis of malaria based on malariaspecific antigens, such as histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH), have been developed as alternative diagnostics to microscopy and PCR [14]
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