Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Zinke, S.
Evaluation of a new mutiplex qPCR-based assay for detection of clinically relevant Aspergillus species
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2017. pp. 36
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Kessler Harald
- Prattes Jürgen
- Altmetrics:
- Abstract:
- Background: Recently, the AspID®(OLM Diagnostics, Newcastle, UK) has been introduced. This assay is based on multiplex real-time PCR (qPCR) for detection of clinically relevant Aspergillus spp. Objectives: The analytical performance of the AspID® was evaluated by using reference material, the diagnostic performance by testing bronchoalveolar lavage fluid (BALF) specimens from patients with invasive pulmonary aspergillosis (IPA) and those with no evidence for IPA. Materials and Methods: The analytical performance of the AspID® was evaluated utilizing the Quality Control for Molecular Diagnostics (QCMD) 2016 Aspergillus spp. DNA External Quality Assurance Program which consisted of 9 sample members. The clinical performance of the AspID® was evaluated with 36 BAL specimens obtained from 18 patients with IPA and 18 with no evidence for IPA. Patients were classified as having IPA if the BALF galactomannan (GM) concentration yielded an optical density index (ODI) >3.0 and patients had clinical and radiological findings compatible with IPA. Those without IPA had a BALF GM ODI <0.5 and no clinical and radiological findings compatible with IPA. Patients with and without IPA were matched 1:1 regarding underlying diseases and ICU admission. For detection of Aspergillus DNA, samples were extracted using the specific B protocol of the NucliSENS easyMAG instrument (bioMérieux, Marcy-l’Etoile, France).The input volume was 400 µl and the elution volume 40 µl. After the lysis step, 4µl of internal extraction control included in the AspID® assay was added. Amplification and detection were performed on the LC 480 II instrument (Roche Diagnostics, Rotkreuz, Switzerland). Results: When the analytical performance was evaluated, 5 out of 6Aspergillus fumigatus positive samples were identified as positive; however, the assay was not able to detect one panel member correctly that contained Aspergillus fumigatus DNA. All Aspergillus negative samples were correctly identified as negative. When the clinical performance was evaluated, 20 BALF samples were found to be positive and 14 negative. Two samples (one from a patient with IPA and one from a patient without IPA) showed inhibition and were excluded from analysis. When AspID® results were compared to those obtained from GM determination, 29 were found to be concordant and 5 discordant (4 AspID®-positives in patients without IPA and one AspID®-negative in a patient with IPA). Sensitivity and specificity for AspID® including 95% confidence intervals (CI) were 94.1% (95%CI 73.3 – 99.9) and 76.5% (95% CI 50.1 – 93.2), respectively. Conclusions: Detection of clinically relevant Aspergillus spp. in BALF specimens with the AspID® seems to be a promising diagnostic approach in patients at risk for IPA. It may allow early diagnosis and rapid initiation of anti-mold therapy