Clinical performance of FXG: RESP (Asp+) assay for Pneumocystis jirovecii on respiratory specimens

Objectives: Pneumocystis pneumonia continues to be a common infection in HIV patients and also occurs in other immunocompromised patients. Early diagnosis is known to improve outcome, and specifically, exclusion of the diagnosis, reduces the need for toxic empirical high dose cotrimoxazole therapy. Real-Time PCR offers the prospect of faster and highly sensitive detection of P. jirovecii. FXG : RESP (Asp +) [Myconostica, UK] is a test kit that detects both P. jirovecii and Aspergillus spp., utilising molecular beacons. In this report we focus on the clinical performance for P. jirovecii. Methods: The FXG : RESP (Asp +) real-time PCR kit utilises the large subunit mtRNA gene as a target to detect P. jirovecii and the assay is highly sensitive, being able to detect 6 target copies. The assay appears to be specific for Pneumocystis spp, with the possible exception of Fusarium solani. It was tested blindly on 196 BAL samples, collected from 4 European hospitals. All results were compared to microscopy, usually Calcofluor or Gomori methanamine silver stains, performed shortly after the sample was collected, and in non-AIDS patients whether patient was treated for PCP. Most HIV/AIDS samples had been previously extracted and stored frozen as DNA; the remainder were extracted with the MycXtra™ fungal DNA extraction kit, having been stored frozen unprocessed. Results: The kit contains an internal amplification control to detect potential inhibition of the PCR reaction and 6 (3%) of the clinical samples showed evidence of inhibition. These results were excluded from analysis, although 5 were positive by both microscopy and the FXG : RESP (Asp +) assay, and would be reported clinically. 42 samples were from HIV/AIDS patients and 148 from other patients, mostly with leukaemia. With respect to Pneumocystis detection, the FXG : RESP (Asp +) assay had good performance with a sensitivity of 97.4%, specificity of 92.9%, positive and negative predictive values of 90.4% and 98.1%. 8 samples had negative microscopy but very high FXG assay signals, suggesting that the microscopy was falsely negative, as reported in prior literature. These were reported as false positives Conclusions: The clinical performance of the FXG : RESP (Asp +) assay for the diagnosis of Pneumocystis pneumonia is superb. Overall the speed of detection and sensitivity of the FXG : RESP (Asp +) assay should bring substantial clinical benefits. Prospective and supportive clinical trials are ongoing.status: publishe