Toxoplasma gondii is an opportunistic parasite which has recently increased a significance due to its frequent infection of patients with Acquired Immunodeficiency Syndrome (AIDS). Current methods to diagnose this parasite in humans generally rely on serological procedures which are unreliable in AIDS patients due to their immune dysfunction. Instead, for such patients, the costly (and risk-associated) alternatives of CT scan and brain biopsy are used. In this proposal, we describe the application of recently developed technology to the diagnosis of infection through detection of parasite nucleic acid. Although DNA probes have been used in the diagnosis of other pathogens (e.g., bacteria in stool samples), the numbers of T. gondii parasites obtainable from clinical specimens from AIDS patients is insufficient for simple detection schemes. We describe here preliminary results using the """"""""Polymerase Chain Reaction"""""""" (recently described by Ehrlich and co-workers) for the amplication of parasite DNA. Using this procedure we can specifically amplify an already repetitive gene in the genome of T. gondii such that a discrete signal can be obtained starting with DNA from as few as two parasites. We also demonstrate that the PCR method can be radically improved in its speed, cost and sensitivity through the use of alternating cycles of amplification and transcription (using AMV reverse transcriptase and T7 RNA polymerase, respectively). This application requests funds to pursue this work to develop these procedures into a routine diagnostic method. To do this, we will optimize the reaction conditions, first under ideal laboratory conditions using purified DNA and then under conditions increasingly representative of clinical specimens (cerebrospinal fluid and blood). In addition to the diagnosis of toxoplasmosis, this procedure could be readily adapted to diagnosis of other opportunistic infections of AIDS patients, including those where direct diagnosis is currently difficult due to the small numbers of microorganisms obtainable.
