The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in immunocompromised patients. Therapeutic success depends on an early diagnosis and the early initiation of antifungal therapy (5). The rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of IPA. Molecular diagnostics have emerged as promising methods for the diagnosis of a variety of infectious diseases, including fungal infections (22). Given the advantages of detection sensitivity and speed, real-time quantitative PCR (qPCR) has been extensively studied and explored as a tool for use for the detection and identification of Aspergillus and other pathogenic fungi in clinical samples (9-12, 31). However, a lack of standardization often makes it difficult to compare results between laboratories, which is critical for the ultimate approval of a test as a standard diagnostic test for IPA (2).Nucleic acid sequence-based amplification (NASBA) is an isothermal amplification process that specifically amplifies RNA even in the presence of genomic DNA (30). The amplification is highly robust, yielding Ͼ10 12 amplicons in 30 min (4). The amplified single-stranded RNA can easily be detected in real time by the use of molecular beacon (MB) probes, which are self-reporting, hairpin-structured, single-stranded nucleic acid (NA) probes that brightly fluoresce when they are bound to their targets (33). NASBA-based assays for the detection of Aspergillus have been described by a few study groups (14, 36). However, to our knowledge, real-time NASBA has not been applied to the detection of Aspergillus fumigatus from an animal model of IPA.The object of the study described here was to evaluate a highly sensitive real-time NASBA assay for its ability to detect Aspergillus burdens in an inhalational rat model of IPA at selected time points in the course of infection and to compare the diagnostic yield with the yields obtained by culture and qPCR. MATERIALS AND METHODSAnimal model. Thirty male Sprague-Dawley rats (Charles River, Margate, United Kingdom) weighing 200 to 250 g were used in the experiment, after being allowed 7 days to acclimatize. The rats were housed in cages provided with HEPA filters and were allowed free access to food and water. The rats were immunosuppressed with cyclophosphamide (75 mg/kg of body weight intraperitoneally twice) plus prednisolone (Depo-medrone; 16 mg/kg intramuscularly once) 2 days before infection. The animals received eurofloxacin (Baytril; 10 mg/kg subcutaneously daily) from day 2 preinfection to prevent secondary bacterial infections. A frozen glycerol stock of A. fumigatus A1163 was cultured into Sabouraud dextrose agar (SAB) for 10 days. The spores were harvested by the use of phosphate-buffered saline (PBS) plus 0.05% Tween 80 and counted with a hemocytometer. The rats were infected with 4 ϫ 10 8 /ml spores in an inhalation acrylic chamber in a class II hood for an hour (28). The rats (n ϭ 5) were checked for symptoms such as body weight loss, dyspnea, and le...

Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.