Sensitive and rapid RT-qPCR quantification of pathogenic Candida species in human blood

Abstract For accurate diagnosis and appropriate treatment of candidiasis, we developed a highly sensitive quantitative RT-PCR (RT-qPCR) system for five Candida species that have been reported to be the major causes of bloodstream fungal infection ( Candida albicans , Candida glabrata , Candida tropicalis , Candida parapsilosis , and Candida krusei ), together with a system for all pathogenic Candida species. Cells of each fungal species spiked into human peripheral blood (PB) were specifically detected at a lower detection limit of 10 0  cell/1 mL PB by this system using the newly developed specific primer sets targeting 18S or 26S rRNA of the five Candida species, together with the existing group primer set. The total count of the five Candida spp. as the sum of those obtained by using the five species primer sets was equivalent to the count obtained by using the group primer set, indicating that the group set covered the major five Candida spp. in human blood with the same degree of accuracy as the species primer sets. The RT-qPCR counts of the Candida species were in good agreement with CFU counts obtained by their culture on CHROMagar™, with a lower detection limit of 10 0  cell/mL of PB. Candida rRNA molecules were stably stored for at least 7 days at 4 °C by keeping the blood specimens in an RNA stabilizing reagent. These results strongly suggest that this sensitive system is useful for accurate and rapid diagnosis of Candida bloodstream infections.