Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples

Abstract

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...

Semiquantitative Detection by Real-Time PCR ofAspergillus fumigatusin Bronchoalveolar Lavage Fluids and Tissue Biopsy Specimens from Patients with Invasive Aspergillosis

“…Recently, they developed a LightCycler assay which successfully detected A. fumigatus DNA in all 12 tested BAL fluids but only in 6 of 14 blood samples from neutropenic patients with proven or probable IPA, which had all been positive by the previously described nested PCR assay (30). Another German group has been concentrating on the detection of Aspergillus DNA in whole blood by 18S rDNA PCR with panfungal primers and species-specific probes (8,19,21,22) or, more recently, on the detection of rRNA by nucleic acid sequence-based amplification (20). Two recent studies evaluated a PCR-ELISA in screening patients at high risk for invasive aspergillosis.…”

Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

“…and Aspergillus spp. (7,29,46), and no real-time NASBA methods had been reported for BSIs. Conversely, during the past decade, many PCR-based assays were developed and dominated the methods used for the identification of microorganisms and the diagnosis of infections.…”

Clinical significance of non‐Candida fungal blood isolation in patients undergoing high‐risk allogeneic hematopoietic stem cell transplantation (1993–2001)

“…27 These methods also may improve the ability of clinicians to make informed, evidence-based decisions when considering treatment options for immunosuppressed cancer patients or stem cell transplantation recipients with positive blood cultures for a non-Candida fungal species. 28,29 Detection of fungal cell wall components 22 as well as organism-specific and classspecific nucleic acid amplification assays 30 appears promising in diagnosis of clinically significant fungemia and pseudofungemia. We recognize that a critical evaluation of the new and old diagnostic laboratory tests is needed to establish a comprehensive protocol that can be applied with relative ease in hospitalized patients to improve the accuracy of antemortem diagnoses of invasive fungal infections.…”