Diagnosis of Invasive Mold Infection by a Quantitative Real-Time PCR

Audrey S. Pham, Jeffrey J. Tarrand, Gregory S. May, Ming S. Lee, Issam I. Raad, Dimitrios P. Kontoyiannis, Xiang Y. Han

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Background: Invasive mold infection (IMI) in immunocompromized patients may present a diagnostic challenge due to associated risks of tissue sampling. Non-invasive diagnostic methods are needed clinically. Methods: We developed a quantitative real-time PCR assay to detect the 5.8S ribosomal RNA gene of Aspergillus and other molds in the serum. A total of 559 serum samples from 106 patients, with or without suspected IMI, were tested. Serum DNA was extracted using a silica-based method. Results: This assay was able to detect 110 fg or 3 genomes of mold DNA in the serum and showed logarithmic linearity upwards to 10 ng. Purified human or candidal DNA was not amplified. Specificity was also demonstrated by negative results in all 35 patients (76 sera) with unlikely IMI at the cutoff of 110 fg. For patients with possible, probable and documented IMI that were diagnosed by a combination of clinical, microbiological and histological criteria (EORTC), this real-time PCR showed positivity in 40% (12 of 30), 68% (19 of 28) and 85% (11 of 13) cases respectively upon testing of multiple serum samples. The overall serum positive rate for these IMI patients was 15% (73 of 483). Quantitative analysis of the positive sera estimated the bodily circulating mold burden to be 1.6 x 105 genomes or 5.3 ng (geometric mean) with 4.2 x 107 genomes (1,400 ng) being the highest. Conclusions: These results suggest that, for definitive, tissue-based diagnosis of IMI, this real-time PCR may be a promising alternative to other invasive methods. Further evaluation is in progress.

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    • ICAAC 42nd