CanID-PCR: a quick and low-cost PCR tool to identify <i>Candida</i> species on gDNA directly extracted from positive blood bottles

<i>Candida</i> bloodstream infection carries high mortality, and antifungal-resistant species, such as <i>Candida auris</i> and <i>C. glabrata</i>, are increasingly common. Delays in species identification can adversely affect patient outcomes. Conventional workflows by clinical microbiology laboratories require ~24 hours for <i>Candida</i> speciation to allow isolate growth on agar plates. We developed CanID-PCR, a simple, inexpensive PCR-based tool for rapid <i>Candida</i> identification directly from positive blood culture bottles. The assay targets the <i>ACT1</i> gene, which contains a variable-length intron enabling discrimination among nine common <i>Candida</i> species. CanID-PCR was optimized for genomic DNA extracted directly from positive blood culture bottles and tested on 64 patient samples. Overall, species identification matched the clinical microbiology laboratory with 99% of concordance. One limitation was the inability to distinguish <i>C. parapsilosis</i> from <i>C. metapsilosis</i>, which are highly similar members of the <i>C. parapsilosis</i> complex. The unique discrepancy involved a failure to detect <i>C. tropicalis</i> in a mixed infection with <i>C. glabrata</i>. Among a total of seven samples with two species, one sample had a second species: <i>C. fabianii</i> (<i>Cyberlindnera fabianii</i>), detected only by CanID-PCR, but was missed by the clinical laboratory. These findings demonstrate that CanID-PCR can reveal polymicrobial <i>Candida</i> infections often overlooked when only a few selected colonies are analyzed. CanID-PCR provides rapid, accurate <i>Candida</i> speciation without the need for additional culture, requiring only standard laboratory equipment. This low-cost, straightforward approach is suitable for research laboratories, resource-limited clinical settings, or scenarios where timely species-level identification is critical.IMPORTANCEIdentifying species in <i>Candida</i> infections is of utmost importance to clinicians and researchers alike. Here, we describe CanID-PCR: a quick and low-cost PCR tool to identify <i>Candida</i> species on genomic DNA directly extracted from positive blood cultures. This technique uses basic laboratory equipment, and there is no need for high investment in special equipment. The tool allows identification of the nine most prevalent species in candidiasis. The technique is simple and can be used without the need for an additional culture to isolate the cells. It is noteworthy to mention that this tool can detect polymicrobial infections caused by more than one <i>Candida</i> species.