SYTO-13, a Viability Marker as a New Tool to Monitor In Vitro Pharmacodynamic Parameters of Anti-Pneumocystis Drugs.

While Pneumocystis pneumonia (PcP) still impacts the AIDS patients, it has a growing importance in immunosuppressed HIV-negative patients. To determine the anti-Pneumocystis therapeutic efficacy of new compounds, animal and in vitro models have been developed. Indeed, well-designed mouse or rat experimental models of pneumocystosis can be used to describe the in vivo anti-Pneumocystis activity of new drugs. In vitro models, which enable the screening of a large panel of new molecules, have been developed using axenic cultures or co-culture with feeder cells; but no universally accepted standard method is currently available to evaluate anti-Pneumocystis molecules in vitro. Thus, we chose to explore the use of the SYTO-13 dye, as a new indicator of Pneumocystis viability. In the present work, we established the experimental conditions to define the in vitro pharmacodynamic parameters (EC50, Emax) of marketed compounds (trimethoprim/sulfamethoxazole, pentamidine, atovaquone) in order to specifically measure the intrinsic activity of these anti-P. carinii molecules using the SYTO-13 dye for the first time. Co-labelling the fungal organisms with anti-P. carinii specific antibodies enabled the measurement of viability of Pneumocystis organisms while excluding host debris from the analysis. Moreover, contrary to microscopic observation, large numbers of fungal cells can be analyzed by flow cytometry, thus increasing statistical significance and avoiding misreading during fastidious quantitation of stained organisms. In conclusion, the SYTO-13 dye allowed us to show a reproducible dose/effect relationship for the tested anti-Pneumocystis drugs.