Direct detection of Aspergillus and azole resistance of Aspergillus fumigatus on broncho-alveolar lavage fluid. Validation of a new Aspergillus real-time PCR

Introduction
Azole resistance in Aspergillus fumigatus is increasingly reported. We describe the validation of
AsperGenius® , a new multiplex real-time polymerase chain reaction (PCR) assay consisting of two multiplex real-time PCRs: one which identifies the clinically relevant Aspergillus species, and one which detects the TR34, L98H, T289A, Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains.
Methods
The diagnostic performance was tested on 37 bronchoalveolar lavage (BAL) samples from
haematology patients and on 40 BAL samples from intensive care unit (ICU) patients using BAL
galactomannan ≥1.0 or positive culture as the gold standard for the presence of Aspergillus.
Results
In the haematology and ICU groups combined, there were 22 BAL samples with IA (2 proven, 9
probable and 11 non-classifiable). Nineteen of the 22 BAL samples were positive according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus). This resulted in a sensitivity, specificity, positive and negative predictive value of 88.9%, 89.3%, 72.7% and 96.2% for the haematology group and 80.0%, 93.3%, 80.0% and 93.3% in the ICU group, respectively. The CYP51A real-time PCR confirmed 12 wildtype and 2 resistant strains (1 TR34/L98H and 1 TR46/Y121F/T289A mutant).
Conclusion
The AsperGenius® multiplex real-time PCR allows for a sensitive and fast detection of Aspergillus species directly in BAL samples. More importantly, this assay detects and differentiates wildtype from resistant strains even if BAL cultures remained negative.