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Development and evaluation of the Nuclisens Basic Kit NASBA for the detection of RNA from yeast frequently resistant to antifungal drugs

Development and evaluation of the Nuclisens Basic Kit NASBA for the detection of RNA from yeast frequently resistant to antifungal drugs

Abstracts of the Interscience Conference on Antimicrobial Agents & Chemotherapy 41: 385

Background: Invasive Candida infections are often life-threatening diseases with increasing incidence in immunosuppressed patients. Early diagnosis is mandatory for appropriate, successful therapy. The aim of this study was to develop and evaluate a NASBA protocol (Nucleic Acid Sequence Based Amplification) for the detection of RNA from yeasts which frequently show resistance to azoles (Candida krusei, Candida glabrata, Candida inconspicua, Candida dubliniensis, Candida norvegensis) or amphotericin B (Candida lusitaniae) and to compare this test with a PCR assay based on slot blot hybridization with species-specific probes. Methods: Serially diluted cells of C. krusei, C. glabrata C. inconspicua, C. dubliniensis, C. norvegensis and C. lusitaniae were used for development and evaluation of the new molecular-based method. Primers and specific probes for the 6 species were designed within the fungal 18S rRNA gene region. Blood samples from healthy donors (n=90) spiked with fungal cells or -RNA and specimens from bone marrow transplant recipients (n=24 patients) were compared subsequently by PCR and NASBA. Results: NASBA showed a detection limit of 1 CFU/ml blood (PCR: 10 CFU/ml). The 6 probes did not show any cross-reaction to mould-, viral- and human RNA. In the presence of a specific RNA protection buffer, fungal RNA was detectable up to 24 h post-spiking demonstrating the robustness of the assay. In clinical specimens, both assays showed identical positive and negative results demonstrating the feasibility of the assay for clinical routine. Conclusion: The NASBA Basic Kit technology is a valuable tool for sensitive, specific, fast and reliable detection of Candida-RNA with potential for routine diagnosis.

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