phosphorus-radioisotopes and HIV-Infections

3'-Azido-3'-deoxythymidine (AZT, Zidovudine) has been effectively used for HIV infection treatment. It inhibits virus reproduction through viral reverse transcriptase inhibition. However, the side effects of this anti-retroviral drug might be cumulative, particularly in its effects on the patients' DNA. As a nucleoside analogue, AZT might incorporate into hosts' DNA, and then form DNA adducts. This may result in potential long-term risks of mutagenesis in AIDS patients who received therapy. In this feasibility study, a (32)P-post-labeling thin-layer chromatography (TLC) assay is successfully used to measure AZT-DNA analogue and adducts formed in peripheral blood leukocytes of AZT treated patients. There are DNA analogue/adducts measured in all four AZT treated patients' DNA specimens. This assay is reliable with the significant coefficient of correlation in both intra-assay (r = 0.8761, P = 0.0001) and inter-assay (r = 0.8761, P = 0.0001).

Topics: Animals; Autoradiography; Cattle; Chromatography, Thin Layer; Densitometry; DNA Adducts; DNA Fingerprinting; DNA Repair; Feasibility Studies; HIV Infections; Humans; Isotope Labeling; Lasers; Leukocytes; Phosphorus Radioisotopes; Zidovudine

A quantitative isotopic competitive PCR (icPCR) assay was established using 32P-labeled primers targeting the HIV-1 gag gene followed by quantification using a phosphoimager. The detection limit varied from 3 to 10 molecules of DNA and 10 to 100 molecules of RNA per reaction. The icPCR quantification of HIV-1 DNA copies correlated well with the cell number of 8E5/LAV cells bearing a single provirus (r2 = 0.95). Provirus quantification was applied to overnight infected donor PBMCs, thereby determining infectious virus titres in culture supernatants as a rapid alternative to limiting dilution culture. Parallel quantification of the HIV-1 RNA indicated the infectious virus fraction to be 0.3%. In 39 HIV-1-infected patients with clinical stages A (n = 17), B (n = 15), and C (n = 7), the HIV-1 RNA in the plasma was determined ranging from 100 to 90600 RNA copies/ml. The results of icPCR and a commercial assay (ROCHE Amplicor HIV-1 Monitor) correlated well (r = 0.97). In 13 additional patients, the plasma viral load per ml was compared with the proviral load per 10(6) PBMC showing a viral excess of 10-1000-fold (mean of 85, r = 0.7, P < 0.01). It is concluded that icPCR is suitable for the measurement of proviral and viral load in experimental and clinical settings.

Topics: DNA, Viral; Genes, gag; HIV Infections; HIV-1; Humans; Leukocytes, Mononuclear; Phosphorus Radioisotopes; Polymerase Chain Reaction; Proviruses; RNA, Viral; Sensitivity and Specificity; Viral Load