application abstract): This proposed two-year program is to survey the microbial constituents of pulmonary granulomas associated with human tuberculosis, using recently developed, ribosomal RNA (rRNA) gene-based technology that does not require cultivation of microbes for their detection and identification. Although Mycobacterium tuberculosis is the primary causative organism in tuberculosis, it is possible that other organisms, perhaps indigenous and so far undiscovered, participate in or accompany the disease process. The program will be conducted collaboratively with clinicians and tuberculosis experimentalists who will provide materials for molecular analyses. DNA from human pulmonary tubercular granulomas of different types (e.g., encapsulated, proliferative, etc.) and locations (e.g. central airway proximal and distal) will be subjected to polymerase chain reaction using primers that amplify all (`universal`) or selected (e.g., bacterial, archaeal, mycobacterial, fungal) rRNA genes. Mixed-species amplification products will be separated by a cloning step, sorted by restriction analysis and unique types sequenced. Phylogenetic analysis of the sequences will identify organisms present in the original granulomas. Fluorescence in situ hybridization analyses will be used to confirm selected potential co-infecting organisms and to study distributions of organisms in tissues. It is estimated that they can process and analyze approximately 100 tissue samples during the course of the two-year survey. The results will prove the monotypic etiology of tuberculosis, or alternatively, implicate novel potential participants. Finally, as a minor effort and as tissues are available, the microbial constituents of granulomas associated with human pulmonary sarcoidosis will be determined. The results may resolve the question of the bacterial etiology of sarcoidosis, identify the causative organism(s), if any, and provide for a sequence-based diagnostic test.
