1) Primer pairs that permit detection and discrimination of HIV-1 and HIV-2 by PCR have been designed for use with clinical samples. We have also optimized a multiplex PCR assay for the detection of HIV-1, -2, HTLV-I, -II. The performance of the assay has been evaluated on clinical samples from patients with co-infections. 2) A sensitive RNA-PCR assay was developed for the detection of viral RNA in body fluids. and applied to the detection and quantitation of virus in body fluids and infected cells from patients. This method was used to assess the presence of HIV in saliva of infected individuals and in commercial products. 3) A novel, viral capture assay based on magnetic beads coated with HIV env antibodies has been developed for detection of viral RNA in body fluids. The assay has demonstrated high sensitivity in HIV positive individuals. A non isotopic format has also been optimized. Future applications include studies on the infectious """"""""window"""""""" period, a primary concern to blood safety and virus load assessment in HIV disease and antiviral therapy involving patients. One of these studies involves an evaluation of primary infection and progressive vs. non progressive infection where virus will be quantitated by PCR and characterized by nucleotide sequencing of virus isolates and phenotypic culture assays. A multiplex format of the assay will also be optimized for HIV and HCV RNA for detection in the window phase. Since multiplex formats may be the cost-effective approach to implementation of these techniques for blood screening, these studies will allow us to be familiar with issues in optimization and standardization of such assays. 4) Efforts are in progress to generate standards for quantitation of HIV RNA in plasma. QC-PCR and Q-PCR are being evaluated using virus stocks containing known amounts of HIV p24 antigen spiked into HIV negative plasma. These reagents will be assessed in collaboration with the International Working Group on Gene Amplification techniques and used to quantitate HIV RNA in plasma (Ag+ve/Ab+ve or -ve) from HIV positive individuals.
