Evaluation of a new commercial real time PCR for the detection of Aspergillus spp. in serum and respiratory samples

Objectives. Diagnosis of invasive aspergillosis is still disappointing and often delayed because of the lack of sensitivity of diagnostic tools. DNA detection based-methods have been developed, but differ widely and comparisons are difficult to assess. The objective of the study is to compare a new commercial real-time PCR kit, affigene® Aspergillus tracer assay, with an in house nested PCR targeting 18S rRNA Aspergillus sp. gene.
Methods. Twelve patients at risk for invasive aspergillosis were included in the study. They were classified to have possible (5 cases), probable (1 case) or proven (6 cases) invasive aspergillosis following E.O.R.T.C. criteria. Fifteen serum and respiratory paired samples were collected. The DNA extraction was performed by using the QIAmp DNA mini kit® (Qiagen, Germany). All samples were tested by both PCR assays and respiratory samples were cultured.
Results. Respiratory samples. A. fumigatus, A. niger and A. flavus were isolated from 10/15 samples; both PCR methods were positive for these samples except one that was positive for affigene® and equivocal for the nested PCR. The real-time PCR assay reported cycle thresholds ranging from 25 to 38. Three of the five culture-negative samples were negative by both PCR methods; one of three was negative in affigene® assay and equivocal by nested PCR; the last sample was positive in affigene® assay and negative by nested PCR. Serum. Thirteen of fifteen blood samples were negative by both PCR methods. One sample was equivocal by nested PCR and was inhibited in affigene® assay despite a culture-positive paired respiratory sample. The last case was inhibited by the real-time PCR assay and negative by nested PCR. Nor the nested PCR, nor affigene® assay could detect any Aspergillus DNA in serum. In total, there was 93% of agreement between the two PCR assays.
Conclusion. Both methods are in good agreement and can detect at least three different species of Aspergillus. However, the sensitivity of both assays does not permit the detection of Aspergillus DNA in serum. affigene® assay can easy replace the “in house” assay: it allows a fast and standardized detection of Aspergillus sp. DNA in respiratory samples without inconvenient due to the handling of PCR products.