Halliday CL, Hoile R, Sorrell TCS, James G, Shaw P, Meyer W, Chen SCA

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The high mortality rate associated with invasive aspergillosis (IA) in recipients of haematological stem cell transplants (HSCT) highlights the need for rapid and accurate diagnosis. Many PCR-based assays have been developed to detect Aspergillus DNA in clinical specimens, but these are not standardised and most have had limited evaluation in a clinical setting. We developed a DNA extraction method for use in a nested PCR assay that targets a conserved part of the 18S rDNA gene in Aspergillus spp. The assay detects 10 cfu/mL of Aspergillus conidia and fails to amplify DNA from other common clinically significant fungi. A two-step PCR assay was used to improve sensitivity and specificity. Negative controls were included during both DNA extraction and PCR amplification for each specimen tested. We evaluated the PCR using whole blood and/or tissue specimens from 10 patients with definite or probable aspergillosis according to the European Organisation for Research and Treatment of Cancer (EORTC) definitions. In all cases of definite IA (n=5), there was 100% concordance between culture and/or histology and PCR results. Two patients had probable IA and PCR was +ve in both. PCR performed on sinus tissue obtained from a patient with radiological findings consistent with fungal sinusitis, who responded to antifungal therapy, was also +ve. Two patients with suspected IA were PCR -ve and had no evidence of IA at autopsy. The clinical usefulness of the assay for early diagnosis of IA was evaluated prospectively by testing blood drawn twice weekly from HSCT recipients and patients undergoing chemotherapy. Fifty patients had at least 6 samples tested; 32 were PCR -ve, 5 had 1 +ve PCR result, 9 had single intermittently +ve results, and 4 had at least 2 consecutively +ve results. One PCR -ve patient (3%), 1 patient with a single +ve result (20%), 1 patient with intermittently +ve results (11%), and 4 patients with at least 2 consecutively +ve PCR results (100%) had probable IA. The results suggest that consecutively positive PCR results have a higher positive predictive value. We are currently developing a real-time PCR assay to detect and quantify A. fumigatus in whole blood specimens. Prospective studies are underway to develop effective screening protocols and determine if screening by PCR influences the outcome of IA.

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The 15th Congress of the International Society for Human and Animal Mycology
    • ISHAM 15th (2003)