Optimized molecular diagnosis of invasive aspergillosis in patients after allogeneic stem cell transplantation - a second con64257; rmatory assay is crucial

J. Springer (1), M. Paholcsek (2), M. Alzheimer (1), W. Heinz (1), H. Schloßnagel (1), H. Einsele (1), J. Löf64258; er (1)

Author address: 

(1)University of Würzburg (Würzburg, DE); (2)University of Debrecen (Debrecen, HU)

Abstract: 

Objectives: Invasive aspergillosis (IA) is still a major complication in immunocompromised patients after allogeneic stem cell and solid organ transplantation. Mortality rate is high and reaches up to 100% in cerebral IA. Diagnosis of IA remains diffi cult because of unspecifi c clinical signs and insensitivity of conventional fungal diagnosis. Therefore, early and sensitive molecular assays are highly warranted. In our study, EDTA whole blood and serum specimens were collected twice weekly from October 2008 until October 2010 for comparative analyses, followed by defi ned DNA extraction and PCR detection protocols. Comparison of inter-assay performances, sensitivities and variability leads to optimization of molecular systems used to detect IA. Methods: Patients after allogeneic stem cell transplantation were categorized according to the criteria for IA of the European Organization for Research and Treatment of Cancer/ Mycoses Study Group. Whole blood samples were prospectively analyzed for the presence of Aspergillus-DNA by an inhouse DNA extraction method followed by qPCR (ITS) and in parallel by Platelia Aspergillus EIA (Biorad; GM). Additionally, selected sera from patients with probable IA (n=10) and control episodes (n=9) were extracted using the QIAamp UltraSens Virus kit (Qiagen), followed by our in-house PCR assay (SEP). Results: We collected 3595 blood samples from 280 high-risk patients. From these, 19 patient episodes were selected and analyzed again by ITS, GM and SEP in parallel. Single positive results in controls were obtained by ITS, GM and SEP in 67%, 11% and 33% (197 samples), and in 100%, 90% and 90% of the probable IA patients (189 samples), respectively. Reanalysis of the data (a fi rst positive result had to be confi rmed by a second positive within 10 days) revealed that the combinations GM/SEP, ITS/GM and ITS alone ranked best detecting 90%, 80% and 70% of the probable IA cases, respectively. By ITS, we revealed a positive PCR result in 6/10 patients prior to the EORTC criteria for probable IA, whereas this occurred in only 4/10 cases by GM and SEP, respectively. Conclusions: Whole blood analysis by ITS showed high sensitivity, but low specifi city. Therefore, we recommend performing GM or SEP in parallel allowing confi rmation of a single positive result by a second diagnostic assay within 10 days, resulting in a signifi cant increase in sensitivity and specifi city
2011

abstract No: 

P765

Full conference title: 

Annual Meeting of the EBMT, 37th
    • EBMT 37th (2011)