Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.Aspergillus species cause life-threatening acute invasive disease in immunocompromised patients. The incidence of invasive aspergillosis has been steadily increasing over the last few decades due to an increasing number of patients undergoing chemotherapy or bone marrow and solid organ transplantation and to an improved survival rate among patients with AIDS. The incidence is estimated to be up to 25% and the mortality is up to 90% in patients with acute leukemia (5). Early diagnosis is essential for appropriate and successful antifungal therapy. However, conventional tests for the detection of Aspergillus spp., such as blood culture and serology, have limited sensitivity and specificity (5). Diagnosis by biopsy or histopathology is difficult due to the severity of illness in these patients. Recently developed methods such as antigen detection and PCR-based assays may provide sensitive tools for the diagnosis of invasive aspergillosis.Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification technology which specifically amplifies RNA sequences in a DNA background by using T7 RNA polymerase (2). NASBA-based assays have been described for the detection of Candida spp. (19), Salmonella spp. (15), human immunodeficiency virus (14), cytomegalovirus (1), and hepatitis C virus (4) RNA in clinical specimens. However, to our knowledge, NASBA protocols have not yet been applied to the detection of Aspergillus RNA.The objective of our study was to establish and to evaluate a NASBA-based assay for a sensitive and specific extraction, amplification, and detection of RNA from different Aspergillus species in blood and to compare these data with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene (11).For the determination of the lower detection limit of the NASBA assay, as well as for RNA degradation experiments, blood from healthy volunteers was spiked with Aspergillus fumigatus conidia (10 6 to 10 0 /ml, in serial dilution). Additionally, 77 blood specimens from neutropenic patients after allogeneic bone marrow transplantation (n ϭ 20) were analyzed in parallel by real-time PCR and NASBA-based assays for the presence of Aspergillus nucleic acid.RNA was...

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