Development of a highly sensitive, quantitative, and rapid detection system for Plasmodium falciparum-infected red blood cells using a fluorescent blue-ray optical system

A highly sensitive diagnostic system for determining low-density infections that are missed by conventional methods is necessary to detect the carriers of Plasmodium falciparum. A fluorescent blue-ray optical system with a polycarbonate scan disc was developed to detect P. falciparum-infected red blood cells (Pf-iRBCs), and nine samples could be analyzed simultaneously. The cultured P. falciparum strain 3D7 was used to examine the potential of the system for diagnosing malaria. After an RBC suspension had been applied to the disc, the cells were dispersed on the disc by rotation. During the 10 min standing period to allow the RBCs to settle on the disc surface, the cells were simultaneously stained with nuclear fluorescence staining dye Hoechst 34580, which was previously adsorbed on the disc surface. RBCs were arranged on the disc surface as a monolayer by removing excess cells through momentary rotation. Over 1.1 million RBCs remained on the disc for fluorescence analysis. A portable, battery-driven fluorescence image reader was employed to detect fluorescence-positive RBCs for approximately 40 min. A good correlation between examination of Giemsa-stained RBCs by light microscopy and the developed system was demonstrated in the parasitemia range of 0.0001-1.0% by linear regression analysis (R2 = 0.99993). The limit of detection of 0.00020% and good reproducibility for parasitemia determination were observed. The ability of the developed system to detect sub-microscopic low-density Pf-iRBCs and provide accurate quantitative evaluation with easy operation was demonstrated.