Sensitive and reproducible quantitation of mucosal HIV-1 RNA and DNA viralburden in patients with detectable and undetectable plasma viral HIV-1 RNAusing endoscopic biopsies

Mucosal tissue is the main portal of entry for HIV-1 infection and, in maca ques. has been demonstrated to be a significant compartment for viral repli cation and CD4(+) T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this s tudy was to develop reliable, reproducible, and sensitive assays to quantit ate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acqui red. rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polyme rase chain reaction (PCR) assay in the: HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and D NA can be reproducibly quantified from a single rectosigmoid biopsy with mi nimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately refl ecting in vivo levels. The techniques outlined differ from currently availa ble approaches by incorporating control standards to identify loss or degra dation of RNA and DNA from acquisition through the in vitro assay and permi t extraction with high yields of RNA and DNA from the same tissue sample. ( C) 2001 Elsevier Science B.V. All rights reserved.