This work was developed based on the notion that direct examination of postmortem human brain by extremely sensitive techniques would be important to determine persistent reservoirs of active/restricted infection HIV-1. In this way the true potential of highly active anti- retroviral therapies (HAART) can be determined. The hope is that these therapies will, inevitably, completely eliminate HIV from infected patients. As correctly stated by the investigators there are currently no reliable, consistent, and sensitive techniques for in situ identification of HIV in postmortem brain. The technique being developed in the application is the ramification amplification assay (RAM). This is a newly invented, solution-phase, isothermal procedure that utilizes a unique DNA polymerize to generate large, multimeric molecules from circular probes that are covalently locked on to their RNA targets. In solution phase, the technique appears to be more sensitive than single round (35 cycle) polymerase chain reaction (PCR). Dr. Morgello's proposes that this technique would be ideally suited for the sensitive in situ detection of active and restricted HIV infection in human postmortem brain. The questions being answered are : (1) What is the cellular reservoir/distribution of HIV in human brain? (2) Does this change with histologic evidence of inflammatory disease? (3) What is the proportion of productive, restricted and/or latent HIV infection in the brain?
