We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from as few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.
Two of the nonculture approaches to the diagnosis of DC, enzymatic-fluorometric determination of serum D-arabinitol and detection of marker antigens in antigenemia (enolase and CWMP), have been commercialized and have shown promise in limited clinical trials. These approaches are not new but are the culmination of efforts made over 10 or more years. Clearly, further fine-tuning of both metabolite and antigen detection is needed to simplify the methods and to improve their sensitivity and specificity so that they will be valuable in guiding clinical treatment decisions. An alternative approach, detection of DC by DNA amplification methods such as PCR, is a special case of a compelling technology and one that is capable of standardization across microbial genera. The availability of simplified PCR diagnostic methods for DC remains a tantalizing prospect. Nevertheless, the development of methods to release DNA from very small numbers of Candida organisms in the blood in a form that is sufficiently free of inhibitors of PCR will require further intensive effort. The maturation of these converging laboratory approaches to nonculture diagnosis of DC leads to more optimism about the eventual use of these methods in clinical laboratories.
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