MOLECULAR APPROACHES TO THE IDENTIFICATION OF UNCULTURABLE INFECTIOUSAGENTS

New molecular biologic techniques, particularly representational diffe rence analysis, consensus sequence-based polymerase chain reaction, an d complementary DNA library screening, have led to the identification of several previously unculturable infectious agents. New agents have been found in tissues from patients with Kaposi's sarcoma, non-A, non- B hepatitis, hantavirus pulmonary syndrome, bacillary angiomatosis, an d Whipple's disease by using these techniques without direct culture. The new methods rely on identifying subgenomic fragments from the susp ected agent. After a unique nucleic acid fragment belonging to an agen t is isolated from diseased tissues, the fragment can be sequenced and used as a probe to identify additional infected tissues or obtain ext ended portions of the agent's genome. For agents that cannot be cultur ed by standard techniques, these approaches have proved invaluable for identification and characterization studies. Applying these technique s to other human diseases of suspected infectious etiology may rapidly elucidate novel candidate pathogens.