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A quantitative reverse transcriptase PCR assay with automated detection by nonradioactive hybridization was developed for the determination of human immunodeficiency virus (HIV) type 1 RNA levels. This assay is based on the use of an external standard curve with an internal standard. The accuracy of quantification was verified by comparison with reference commercial tests, the Chiron branched-DNA and Roche AMPLICOR HIV MONITOR assays. This assay was able to quantify viremia in patients with CD4 cell numbers below and above 500/mm3 and to quantify some HIV strains which could not be titrated by the MONITOR assay.

Changes in HIV replication during progression of HIV infection were assessed by estimation of the number of peripheral blood mononuclear cells (PBMC) harboring HIV proviral sequences. Samples from 23 patients at different stages of HIV infection were analyzed by quantitative polymerase chain reaction (PCR) using GAG primers. HIV titers in PBMC were also determined by serial dilutions of cells in coculture with phytohemagglutinin-activated normal PBMC. A positive correlation was observed between the number of HIV DNA copies and the HIV titer in PBMC. The PCR test was more sensitive than the coculture technique. The number of HIV copies detected by PCR ranged from 50 to 10,500 per 10(6) PBMC: assuming one copy per cell this implies a frequency of proviral HIV-containing cells of one per 100 to one per 20,000 mononuclear cells. The mean number of HIV DNA sequences in PBMC was significantly lower in asymptomatic patients than in AIDS patients and patients with AIDS-related complex (ARC). In patients who progressed from asymptomatic infection to AIDS, the number of HIV DNA copies in PBMC rose, indicating an increase of HIV replication. These results show that the number of infected PBMC increases during clinical progression. However, some asymptomatic patients had a higher number of HIV DNA copies in their PBMCs suggesting that increased HIV replication precedes the appearance of clinical symptoms.

Two hundred and twenty-eight blood samples from 154 HIV-seropositive subjects were investigated for the presence of HIV in peripheral blood mononuclear cells (PBMC) and plasma (without prior ultracentrifugation or filtration), using normal PBMC as the target for replication. HIV was recovered from PBMC and plasma in 80.5 and 19.5% of the patients, respectively. Plasma viraemia was significantly associated with clinical manifestations of HIV infection, indicating that HIV replication increased as disease progressed. This was confirmed by the statistically significant correlations between plasma viraemia and low CD4 cell counts (P less than 0.01) and low anti-p24 antibody titers (P less than 0.01). On patient follow-up, detection of HIV in plasma was transient. p24 antigenaemia was only detected in 13.6% of cases and was also associated with advanced clinical stages of the disease. When HIV RNA detection by polymerase chain reaction (PCR) was compared with plasma viraemia, HIV RNA was detected in plasma in all symptomatic cases and in 53.8% (seven out of 13) of asymptomatic patients [Centers for Disease Control (CDC) stages II and III], confirming that PCR was far more sensitive than plasma culture. These results indicate that cell-free virus production is associated with the clinical stage of HIV infection and may serve as a marker for disease progression. Detection of HIV RNA by PCR appears to be the most sensitive method to detect viraemia.

An early and accurate diagnosis of HIV infection is needed in the offspring of seropositive mothers. To this end, we have used two techniques for the direct detection of HIV in 12 newborns tested within 2 weeks after birth and 12 children. HIV isolation was carried out in lymphocyte cocultures and compared with detection of DNA and RNA sequences by molecular amplification using the polymerase chain reaction (PCR). In lymphocyte cocultures, HIV was isolated in 8 of 24 cases (33%), including 3 newborns, 3 symptomatic children, and 2 asymptomatic ones. HIV DNA was detected by PCR in twice as many cases, i.e., in 16/24 cases (66%), including 7/12 newborns, 4/4 symptomatic children, and 5/8 asymptomatic ones, 2 of whom became seronegative, HIV RNA was detected in 10 of 16 cases (60%) with detectable HIV DNA, including all of the cases who had a positive HIV isolation. Only children with clinical or biological signs of HIV infection were positive for HIV RNA. Furthermore, signs of HIV infection appeared within 6 months in three of the four newborns who were positive for HIV RNA at birth. These results indicate that HIV DNA detection by PCR is far more sensitive than HIV isolation in culture for the early diagnosis of HIV infection in offspring of seropositive mothers. HIV RNA detection appears to be a useful prognostic marker since it does correlate with disease progression and may serve as a clue for HIV replication in vivo.

To define the characteristics of 1899 patients diagnosed with AIDS at Lyon University Hospitals (LUH) across four time periods corresponding to different antiretroviral eras, and to analyse the evolution of specific AIDS-defining illnesses (ADIs) with time. The decline in the incidence of AIDS with the advent of HAART was confirmed in our hospital cohort. The gradual increase in the proportion of NHL among ADIs underscores the long latency period between infection with HIV and the achievement of an effect of HAART on HIV-associated lymphomagenesis. [ABSTRACT FROM AUTHOR]