Evaluation of MycAssayAspergillus molecular diagnostic test and automated EZ1 DNA extraction for detecting Aspergillus spp. DNA in bronchoalveolar samples of patients with risk factors for pulmonary invasive aspergillosis
Abstract number: P1942
Roselló E., Culebras M., de Gracia J., Codina G., Alvarez A., Ruiz I., Figueras C., Moreno A., Torrent A., Díaz de Heredia C., Andreu A.
Objectives: Evaluation of a new real time PCR technique (MycAssay, Myconostica) combined with automated DNA extraction (EZ1, Qiagen) to detect Aspergillus DNA in BAL samples of patients with pulmonary Aspergillosis (PA) risk factors in comparison with the Aspergillus galactomannan antigen (AGA) detection and conventional culture.
Methods: 53 BAL samples belonging to 42 patients with PA risk factors were analyzed: 29 solid organ transplants, 9 with haematologic diseases and 4 with primary or secondary immunodeficiencies. After vortexing the sample, three aliquots were prepared to perform: AGA detection (Platelia Aspergillus®, Bio-Rad); conventional culture and automated EZ1 DNA extraction (Qiagen®) following the 2.0 virus protocol with 400 microlitres of sample. All PCR (two per sample) were performed with the same real time thermocycler (Cepheid SmartCycler system). The PA criteria to classify cases of aspergillosis were those of the European Organization for Research and Treatment of Cancer.
Results: Seven probable cases of PA were diagnosed in 5 lung transplantation and 2 haematologic patients. Conventional culture was positive in 75% of their samples. If detection of AGA in BAL is not taken into account as a micological criterion, the sensitivity and specificity of this technique cut off for positive >1(OD) was 85.7 and 91% respectively. PCR technique with EZ1 DNA extraction system showed a 100% sensitivity and 93.3% specificity. The extraction method did not include any steps for fungal cell wall lysis (for example bead beating) and was rapid (45 mins) giving patient results in <3 hrs. In four false positive AGA cases, the PCR was negative. During the study period one case of zygomycosis and one of fusariosis were diagnosed by conventional culture and no PCR cross reactions were detected.
Conclusion: The MycAssayAspergillus molecular diagnostic test performed in BAL samples combined with the EZ1 automated DNA extraction system provides greater sensitivity and specificity in diagnosing PA than AGA detection or conventional culture. Automated EZ1 DNA extraction system is fast, simple and sensitive, despite not including any fungal cell wall disruption step.
|Session name:||Abstracts of 21st ECCMID / 27th ICC|
|Location:||Milan, Italy, 7 - 10 May 2011|
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