A new method for the detection of Pneumocystis jirovecii using flow cytometry

Pneumocystis jirovecii is an opportunistic pathogen responsible for severe pneumonia in immunocompromised patients. Its diagnosis has been based upon direct microscopy either by classic staining (Gomori Grocott) or by epifluorescence microscopy (immunofluorescence staining, IFS), both of which are time-consuming and low on sensitivity. Our aim was to develop a flow cytometric (FC) protocol for the detection of P. jirovecii on respiratory samples. In our study, 420 respiratory samples were analysed in parallel by IFS and FC, and compared from clinical diagnosis to its resolution upon specific anti-Pneumocystis therapy. The optimum specific antibody concentration for FC analysis was determined to be 10 microg/ml, without any cross-reactions to bacteria or fungi. All positive cases detected by IFS were positive by FC; however, FC classified eight samples to be positive which were classified as negative by routine technique. These samples were obtained from patients with respiratory symptoms who responded favourably to Pneumocystis-specific therapy and were subsequently considered to be true-positives. Using clinical diagnosis as a reference method, FC showed 100% sensitivity and specificity, whereas IFS showed 90.9% sensitivity and 100% specificity. According to our results, a new diagnostic approach is now available to detect P. jirovecii in respiratory samples.