Rapid Identification of Candida Species in Candidemia Directly from Blood Samples Using Imperfect Match Probes

Abstract

Candidemia is associated with a high mortality rate, and initial adequate antifungal therapy results in a significant decrease in the crude mortality. We herein report a rapid method that can identify eight Candida species in candidemia using imperfect match quenching probes (IM Q-probes) within three and a half hours of whole blood sample collection. Furthermore, employing the D value, which reflects the difference between the Tm signature from a clinical isolate and that registered in the database, it is possible to quickly identify samples suitable for IM Q-probe identification. We first evaluated the method using 34 Candida colonies collected from different patients, and 100% (34/34) of the identification results matched the preidentified Candida species. We then performed blind tests using eight whole blood samples artificially mixed with eight different Candida species respectively, and all identification results correctly matched the preidentified Candida species. Finally, using 16 whole blood samples collected from candidemia patients, we compared the IM Q-probe method with the culture/sequencing method. Of a total of 16 patient samples, 100% (16/16) matched the culture and sequencing results. The IM Q-probe method is expected to contribute not only to the life expectancy of candidemia patients but also to antifungal stewardship. Candida species are the most frequent organisms involved in invasive fungal infections and major causes of not only local mucous membrane infections but also widespread propagation with multisystem organ failure 1. Recently, due to advances in medicine, such as dialysis, major surgery and immunosuppressants, the rates of invasive candidiasis have been increasing. Indeed, in the United States, Candida species are the fourth-most common pathogen of nosocomial bloodstream infection 2,3. Candidemia has an associated mortality that is as high as 25%, and initial adequate antifungal therapy results in a significant decrease in the crude mortality 4-6. However, blood cultures usually take two to five days to finalize, which leads to a significant delay in the implementation of culture-driven therapy 1,7,8. In addition, although most episodes of candidemia are caused by Candida albicans, opportunistic infections due to non-C. albicans species have been reported with increasing frequency 9. Pfaller et al. reported that the trends in descending order of frequency of Candida species causing candidemia were C. albicans (42.1%), C. glabrata (26.7%), C. parapsilosis (15.9%), C. tropicalis (8.7%), C. krusei (3.4%), C. lusitaniae (1.1%), C. dubliniensis (0.9%) and C. guilliermondii (0.4%) 10. Because definitive therapy by selecting an appropriate anti-Candida agent should be based on the species of Candida, the rapid identification of Candida species is critical for the prompt initiation of appropriate therapy 1. Niimi et al. reported the novel "melting temperature mapping method" for rapidly identifying the dominant bacteria in a clinical sample (whole blood sample, etc.) from a sterile site 11...

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

“…Mutations that occur in the SARS-CoV-2 genome [31, 32] imply that the presence of mismatches between primer/probe and the template eventually become inevitable. On the other hand, it is known that mismatch presence may affect only the first few cycles of PCR [33, 34] and with proper design may even be advantageous [35]. Therefore, as a rule of thumb, the occurrence of single mismatches are admitted in the hope that they may not affect the detection of the target and its amplification [36].…”