DEVELOPMENT OF METHODS FOR EPIDEMIOLOGIC TYPING OF PNEUMOCYSTIS CARINII

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The inability to culture Pneumocystis carinii has proven to be a major stumbling block in efforts to understand the epidemiology of disease caused by this organism. Much remains to be resolved concerning transmission, communicability of infection, and whether disease results from de novo infection of susceptible individuals or reactivation of latent infection during immunosuppression.. These issues cannot be addressed completely until methods for 'typing' isolates of P. carinii are available. Advancements in molecular technology make it possible to investigate the genetic relatedness of organisms by amplification of nucleic acid even when the organism has not been cultivated, and we intend to use thesE approaches to study P. carinii. The first stage of this project is currently underway, in which regions of the P. carinii genome that potentially could be sites for inter-strain variability are being investigated. Of particular interest are regions of the ribosomal RNA gene cluster that have proven useful for similar investigations in other organisms. These regions will be amplified by PCR using P. carinii-specific primers and the products analyzed electrophoretically. If necessary, restriction endonuclease analysis or sequence determination will be used to increase discriminatory power.