With the success of highly active antiretroviral regimens, a significant number of patients have no detectable plasma viral RNA by the most sensitive assays available. These patients however, have persistent, replication competent virus as determined by in vitro culture as well as detectable viral DNA. To gain a clearer understanding of the state of the virus in these patients, we examined PBMC for the presence of closed circular forms (cc) of extrachromosomal HIV DNA. While high levels of cc DNA are seen in acutely infected PBMC they rapidly decline with a short half-life, therefore, detection would be suggestive of ongoing new infection in vivo.

DNA was extracted from the PBMC isolated from 35 patients with plasma viral loads of <50 copies HIV RNA/ml of blood. All of the patients were on multi-drug antiretroviral regimens. The mean CD4+ cell count was 493 cells/mm3 (33-984) and the mean duration of viral suppression was 13 months (3-27.5 mths). Low molecular weight (LMW) DNA was specifically enriched from the cell pellet during extraction. The PCR reaction was a modification of that previously described ( Cara A. et al, Virology 208, 242-248) with the addition of an initial round of amplification using external primers to enhance sensitivity. A total of 70% of the patients had detectable cc DNA using the nested amplification while only 7% were positive using a single round of amplification.

While the presence of cc DNA in the majority of patients with no detectable plasma RNA suggests that there is ongoing de novo infection, further evaluation of the stability of this form of extrachromsomal DNA in other viral reservoirs is needed. Longitudinal studies are required to determine whether the presence of cc DNA is predictive of duration of viral suppression (or viral breakthrough).