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        Development of a Simple DNA Extraction Method and Diagnosis of Candidemia

        Separa GVS vials in a row.

        Candida species are among the top five infectious bloodstream pathogens and remain the most common cause of invasive fungal infections, a early diagnosis is critical for appropriate patient management and for improving the outcomes of candidemia.

        To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary.

        To shorten the time for diagnosis, fast nucleic acid extraction from Candida in blood is needed. The sensitivity of any molecular diagnostic method for the detection of fungal pathogens depends on the lysis efficiency of fungal cells from blood samples and purification of DNA without PCR inhibitors. Current fungal DNA extraction protocols involve enzymatic, chemical or physical disruption steps, bead beating using glass, or ceramic beads to disrupt the fungal cell wall. Unlike nucleic acid extraction of animal cells or viruses, the additional cell wall disruption step makes rapid nucleic acid extraction from fungi more difficult. In the study, it was developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min - the filtration process was performed using a SEPARA® tube (GVS, Bologna, Italy). The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method.

        The Candida Pan/IC LAMP assay followed by Chelex-100/boilingmediated DNA extraction showed high sensitivity and specificity against clinical samples infected with Candida. To optimize the Chelex-100/boiling method, different concentrations of the Chelex 100 Resin solutions (0%, 5% and 10%) were tested using C. albicans real-time PCR and the Candida Pan/IC LAMP assay for C. albicans DNA extracted from the whole blood samples spiked with Candida cells.

        In this study, it was developed a fast candidemia detection system including Chelex100/boiling DNA extraction and the Candida Pan/IC LAMP assay, which is capable of diagnosing Candida species in blood within 1 h. In a sensitivity test with Candida clinical samples, the Candida Pan/IC LAMP assay showed superior performance to the two reference qPCRs. Thus, Chelex-100/boiling DNA extraction followed by the Candida Pan/IC LAMP assay could serve as a useful fast molecular diagnostic test for Candida spp. in blood. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.

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