Absence of Chronic Human Immunodeficiency Virus Infection without Seroconversion in Intravenous Drug Users: A Prospective and Retrospective Study

Quantitation of Human Immunodeficiency Virus Provirus and Circulating Virus: Relationship with Immunologic Parameters

“…To relate the number of proviral copies to 10 6 CD4 cells, the number ofcopies measured in I ILg of DNA corresponding to 150,000 cells was multiplied by a correcting factor (6.66) and further divided by the ratio of CD4 cells per microliter to total lymphocytes per microliter. We have shown previously that the sensitivity of proviral HIV DNA detection is on the order of one proviral copy per sample [12,13]. The one-copy samples were detected in 6 of 14 experiments and three copies in at least one of the two duplicate amplifications in all assay runs.…”

“…The choice of a pol gene segment for HIV DNA amplification was based on the low number of base substitution among HIV isolates in these parts of the pol gene [12,13]. A method based on immunocapture of the virus rather than RNA purification was selected for the detection ofcirculating HIV.…”

“…The absorption step is convenient in terms of rapidity, simplicity, specificity (only the virus is absorbed), and suitability for quantitation by limiting dilutions. The main limitation ofthe quantitation of RNA is linked to the RT step, whose efficacy is --5% [16] (in contrast, DNA amplification can detect a single copy of provirus [12,17,18]). For this reason, it may be more appropriate to express the results of circulating RNA in arbitrary units rather than in number of RNA molecules per milliliter.…”

“…The LAV-8E5 cell line (National Institute ofAllergy and Infectious Diseases, AIDS Research and Reference Reagent Program, Bethesda, MD) containing one copy of HIV -I provirus per cell was grown in culture as recommended [II]. DNA was extracted from a pellet of 50 X 10 6 cells and serially diluted in HIV-negative human genomic DNA to obtain the equivalent of 1-1000 LAV-8E5 cells in I ILg of human genomic DNA/ I0 ILL. For provirus amplification, a 238-bp segment of a conserved region of the HIV-I pol gene was selected and amplified [12]. All samples were tested in duplicate using I ILg ofgenomic DNA corresponding to 150,000 cells/test.…”

“…The reaction mixture of 100 ILl containing I ILg of genomic DNA and 2.5 units of DNA polymerase (Amplitaq; PerkinElmer Cetus, Norwalk, CT) was adjusted to 10 mM TRIS-HCl (pH 8.3),50 mMKCl, 2.5 mM MgC I 2 , 0.5 mM ofeach deoxynucleoside triphosphate (dNTP), 0.5 ILM of POL I and POL 2 primers (POL 1,5'-TGGGTACCAGCACACAAAGG, 3734-3753 in HIV BRU; POL 2,5'-ACTTGTCCATGCATGGC-TTC, 3953-3972 in HIV BRU) [13]. The reaction was cycled 37 times (94°C, I min; 56°C, 1.5 min; 72°C, I min) using a DNA thermal cycler (model 480; Perkin-Elmer Cetus) [12]. Five samples of human genomic DNA containing 1,3, 10, 100, and 1000 copies of HI V provirus from the LAV-8E5 cells were included in each experiment.…”