JG DISERENS¹, A CROXATTO¹, R BROUILLET¹, P.M HAUSER¹, G GREUB¹, K JATON<sup>1

1</sup>Institute of Microbiology, University Hospital and University of Lausanne, 1011 Lausanne, Switzerland

BACKGROUND: Pneumocystis jirovecii (PCP) is an opportunistic pathogen causing infection in both AIDS and immunocompromised patients. The examination of respiratory sample for the detection of PCP is conventionally performed by microscopy. However, PCR exhibits a higher sensitivity than microscopy and allows accurate quantification. Thus, a quantitative real-time Taqman PCR was developed in our platform with a time to results of 4 hours, but this test is only available from Monday to Friday. Therefore, we decided to transfer our Taqman PCR to a rapid PCR format i.e. the BD MAXTM system (BD Diagnostics) that provides results in about 100 minutes without laborious hands-on work.

METHODS: The BD MAX system was compared with automatic MagNA Pure DNA extraction and conventional real-time PCR using 200µl of BAL. The same primers and probe, with different concentrations, and the same master mix (Applied Biosystem) were used with the two systems. Intra- and inter- run assays were performed to test the robustness of the PCR and various concentration of plasmids carrying the PCP DNA sequence were used to measure the quantification accuracy. A total of 25 frozen BAL (15 positive and 10 negative) were analysed with both molecular assays in a retrospective study. The Ct were compared to quantify the results.

RESULTS: For the BD MAX, optimal concentrations of 0.6μM of primers and 0.3μM of probe were used and allowed a detection of 100% of the 2E5 and 2E4 copies/ml of plasmidic PCP DNA and 60% of 2E3 copies/ml. In the retrospective study, the BD MAX system showed a 100% qualitative correlation with the conventional TaqMan assay. For positive results, a good quantification correlation of the Ct values between the two systems was observed with an average of 1 Ct difference. Moreover, no cross contamination were observed with the BD MAX.

CONCLUSION: The transfer of a home-made PCR on the BD MAX system was relatively easy to perform and allows the detection and quantification of Pneumocystis DNA. The new rapid molecular BD MAX test showed equal performance and good quantitative correlation with our home-made real-time PCR allowing a significant decrease of the time to results.