Clinical Performance of FXG^TM: RESP (Asp +) assay for Aspergillus on respiratory specimens

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Objectives: Early diagnosis of invasive aspergillosis (IA) is essential to survival. Classical microbiological methods are slow and insensitive. Real-Time PCR offers the prospect of both faster and more sensitive microbiological confirmation of IA. FXG : RESP (Asp +) is a new test kit that detects both Aspergillus spp. and Pneumocystis jirovecii, utilising molecular beacons. This ready to use PCR assay consists of an extraction kit plus PCR assay and is CE marked. In this report we focus on the clinical performance for Aspergillus spp.
Methods: The FXG : RESP (Asp +) real-time PCR kit was tested on 198 respiratory specimens from a wide variety of patient groups, collected from 4 European hospitals. Analysis was carried out using an AB7500 thermocycler. Samples of BAL were mostly stored at -20 to -80C prior to DNA extraction using the MycXtra^TM fungal DNA extraction kit,. Some sputum samples were also processed. The FXG : RESP (Asp +) results were compared with culture for Aspergillus spp. The whole assay time including extraction is <4 hours from BAL and <5 hours from sputum. The limit of detection was ~1 genome for A. fumigatus (as assessed by purified Af293 DNA), and this was used as the clinical cut-off. An internal amplification control reaction within the kit detects inhibitors that might affect the PCR reaction, although positive results in the presence of inhibition may be reported.
Results: Overall, for all 198 respiratory samples the sensitivity was 74% and specificity 93% with positive and negative predictive values 76% and 92% respectively. On 75 freshly collected sputum and other lower respiratory tract samples the sensitivity and specificity were 79% and 88% respectively. The FXG : RESP (Asp +) assay also detects Penicillium spp. as the target sequence in this fungus is identical. 3 samples were culture positive for Penicillium spp, and positive for FXG : RESP (Asp +) and recorded as 'false positives'. The FXG : RESP (Asp +) does not detect Zygomycetes or Candida spp., and this was confirmed in the clinical study.
Conclusions: Overall the speed of detection and sensitivity of the FXG : RESP (Asp +) assay will bring considerable clinical benefits. Additional prospective and supportive clinical trials are ongoing