PNEUMOCYSTIS CARINII IN CONTINOUS AXENIC CULTURE

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DESCRIPTION (Adapted from Investigator's Abstract) Pneumocystis carinii is also a very poorly understood fungal pathogen and P. carinii pneumonia (PcP) remains one of the most common opportunistic infections associated with AIDS despite effective anti-retroviral therapy, improved PcP treatment protocols and widespread PcP prophylaxis. The greatest obstacle in P. carinii research has been the inability to culture this organism other than in short term systems, generally with a co-cultured mammalian cell line. This laboratory has obtained continuous axenic growth of both rat- and human-derived P. carinii.The overall goal of this project is to develop this culture system further and use it to study P. carinii. Conditions will be established allowing for optimal growth in culture. Temperature and pH optima will be determined. Validation will be sought for the supplements now added to the culture medium and other supplements will be tested. Supplement concentrations will be optimized. Minimum medium exchange rates for optimal growth will be determined. A method of growing a P. carinii culture from a single cell will be developed. Cultured P. carinii will be characterized with reference to morphology, movement, and aspects of cell physiology such as respiration and polyamine metabolism. A library of 50 isolates of human-derived P. carinii will be obtained using bronchoalveolar lavage fluid from patients with P. carinii pneumonia. These isolates will be studied in vitro for sensitivity to the combination of trimethoprim + sulfamethoxazole (the mainstay for both treatment of and prophylaxis against P. carinii pneumonia) and to atovaquone (a commonly used alternative drug). In these isolates, we will determine critical sequences in the sulfamethoxazole target enzyme (dihydropteroate synthase) gene and the atovaquone target enzyme (cytochrome b) gene. An attempt will be made to correlate drug sensitivity, gene sequence and patient response to determine if there is resistance to this drug combination in human P. carinii.