Cell-associated HIV-1-DNA quantitation after highly active antiretroviral therapy-treated primary infection in patients with persistently undetectable plasma HIV-1 RNA.

OBJECTIVE: To determine the usefulness of cell-associated HIV-1-DNA quantification during the follow-up of highly active antiretroviral therapy (HAART)-treated primary-infected patients with persistently undetectable plasma RNA loads. PATIENTS AND METHODS: In 27 patients given HAART within a median of 24 days after symptomatic primary HIV infection, plasma and peripheral blood mononuclear cell (PBMC) HIV-1 RNA were less than 50 copies/ml and less than 50 copies/10(6) cells after 18 months of treatment. HIV-1 RNA and DNA were quantified every 6 months in PBMC in these 27 patients, 14 of whom accepted excision lymph node biopsy after month 18 for HIV-1-RNA and -DNA quantification in lymph node mononuclear cells (LNMC). RESULTS: The median decreases in plasma HIV-1 RNA, PBMC HIV-1 RNA and DNA over the 18 months of follow-up were 3.6 log (P< 0.005), 1.1 log (P< 0.05), and 1.0 log (P<0.001), respectively. HIV-1 DNA was detected in 92.3% of PBMC samples at baseline and at month 18. In LNMC, 100% of samples were detectable for HIV-1 DNA. CONCLUSION: In this highly selected population of patients with excellent plasma virological response under HAART, HIV-1 DNA showed a progressive decrease but was still detectable in 92.3% of samples at month 18, whereas all LNMC samples tested scored positive for HIV-1 DNA. The utility of proviral HIV-1-DNA monitoring was not clearly demonstrated in this 18-month follow-up of HAART-treated primary-infected patients. However, this finding could be reconsidered when using other therapeutic strategies such as structured treatment interruptions, reinforced treatment or additive immunotherapy.

HIV RNA and HIV DNA in peripheral blood mononuclear cells are consistent markers for estimating viral load in patients undergoing long-term potent treatment.

The aim of this study was to evaluate residual viral replication by assessing the HIV load of circulating infected cells in patients given an effective antiprotease-containing treatment for 1 year. PBMC HIV RNA and HIV DNA was quantified by techniques standardized and evaluated by interlaboratory quality control testing. Viral markers identified in a multicenter study were validated in a cross-sectional study of 121 patients beginning treatment. A longitudinal study of 3 viral markers was carried out in 18 patients, each of whom had fewer than 200 copies of HIV RNA per milliliter of plasma after 12 months of treatment. The cross-sectional study showed that viral replication in PBMCs was correlated with the number of circulating infected cells (Spearman rank correlation; p = 0.0001, r = 0.35) and the concentration of virus particles in the plasma (Spearman; p = 0.0001, r = 0.54). The longitudinal study showed that the decrease in HIV RNA levels was smaller in PBMCs than in the plasma. The largest decrease in HIV DNA levels after 12 months of treatment was recorded in patients with low levels of intracellular replication (Spearman; p = 0.005, r = 0.69). PBMC HIV RNA and HIV DNA levels were very informative markers, complementary to plasma HIV RNA levels. They should be used in future trials evaluating the long-term efficacy of new associations of highly active antiretroviral treatments.

Evaluation of the Amplicor Chlamydia trachomatis test versus culture in genital samples in various prevalence populations.

OBJECTIVE: To evaluate a newly developed polymerase chain reaction (PCR) assay, Amplicor C trachomatis for the detection of C trachomatis in genital samples using cell culture for comparison. SUBJECTS: 501 patients (431 women and 70 men) attending an STD clinic in Hôpital Pellegrin (high-risk population) and gynaecological clinics (low-risk population) in Bordeaux, France. METHODS: The genital samples (cervical and urethral) were tested for the presence of C trachomatis using the Amplicor test and using standard cell culture identified by the immunofluorescence test using a monoclonal antibody to C trachomatis. Discrepancies between the results of culture and Amplicor were further analysed by major outer membrane protein gene (omp1)-PCR of the specimens taken in transport media and by direct fluorescent antibody (DFA) staining of elementary bodies in culture transport tubes. RESULTS: After analysis of discrepancies, the revised sensitivity and specificity of PCR were 95.3% and 100% and the positive and negative predictive values were 100% and 99.5%, respectively. CONCLUSION: The present results indicate that the Amplicor assay is rapid, specific and more sensitive than the culture method. This test provides an excellent non-culture method for the detection of C trachomatis in various prevalence populations.

Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells.

Retroviral reverse transcriptase (RT) is involved in the selection of a specific tRNA primer which initiates proviral DNA minus-strand synthesis. Studies of the interactions between human immunodeficiency virus type 1 (HIV-1) RT and primer tRNALys3 have shown that the dihydrouridine (diHU), anticodon, and pseudouridine regions of tRNA are highly protected in the RT-tRNA complex. The CCA 3' end of tRNA is also in close contact with the enzyme during the cDNA initiation step. Using synthetic oligoribonucleotides corresponding to the anticodon and diHU regions, we have previously shown a low but significant inhibition of HIV-1 RT activity. We extend this observation and show that primer tRNA-derived oligodeoxynucleotides (ODNs) carrying a phosphorothioate (PS) modification are strong inhibitors of HIV-1 RT. The affinity of PS-ODNs for the enzyme was monitored by gel mobility shift electrophoresis. Experiments with HIV-1-infected human cells (MT-2 cells) were performed with the latter ODNs. A PS-ODN corresponding to the 3' end of tRNALys3 (acceptor stem [AS]) was able to inhibit HIV-1 replication. No effect of the other modified ODNs was observed in infected cells. The analysis of HIV-1 RNase H activity in a cell-free system strongly suggests that the inhibitory effect of the PS-AS may be mediated via both a sense and an antisense mechanism.

Acquired immunodeficiency syndrome-associated Kaposi's sarcoma and human herpesvirus 8 DNA detection in serial peripheral blood mononuclear cell samples.

Human herpesvirus 8 (HHV8) DNA was amplified from peripheral blood mononuclear cells (PBMCs) using PCR in 120 HIV-seropositive in- and outpatients who were enrolled in a cohort study between January 1994 and June 1995. Risk factors for HIV infection were homosexuality/bisexuality alone in 64 cases (30 with Kaposi's sarcoma (KS) and 34 without KS, 4 of whom had KS lesions that appeared during follow-up in the cohort), heterosexual contact alone in 32 cases (among whom 1 woman with KS who was the spouse of a bisexual with KS), and transfusion of blood or blood products alone in 24 cases. Three blood samples at 3-4-month intervals were scheduled for each patient. Twenty-five HIV1-seronegative patients served as controls. A total of 47.1% of homo- or bisexual males with KS and 26.7% of homo- or bisexual males without KS had positive HHV8 DNA detection as compared with 21.9% of patients contaminated by heterosexual contact, 8.3% of blood product recipients and 0% of controls. HHV8 DNA detection was intermittent in all but 3 patients according to sequential sampling. Multivariate analysis showed that AIDS-KS was associated with sexual transmission, mainly homo- or bisexual practices and with HHV8 infection assessed by PCR in PBMCs.

Comparison of the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of levels of human immunodeficiency virus type 1 RNA in plasma of patients receiving stavudine-didanosine combination therapy.

We compared the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma in the Stadi trail, which evaluated a stavudine plus didanosine combination therapy in 52 patients. HIV-1 RNA baseline values measured with AMPLICOR HIV-1 MONITOR 1.0 were significantly higher than those measured with QUANTIPLEX HIV-1 RNA 2.0, and decreases in HIV-1 RNA levels from baseline were also found to be significantly higher when measured with the AMPLICOR HIV-1 MONITOR 1.0 assay. The frequency of HIV-1 RNA levels below the lower limit of quantitation was significantly higher with QUANTIPLEX HIV-1 RNA 2.0 than with AMPLICOR HIV-1 MONITOR 1.0. Reanalysis of these results by an ultrasensitive procedure of AMPLICOR HIV-1 MONITOR 1.0 or by a modified version of the test that included additional primers adapted for non-B HIV-1 clades yielded greater differences between the QUANTIPLEX HIV-1 RNA 2.0 assay and the AMPLICOR HIV-1 MONITOR 1.0 assay. Our results indicate that a valid comparison of the virological efficacies obtained with different antiretroviral drug regimens requires the use of the same viral load quantitation procedure; further standardization between the different HIV-1 RNA quantitation kits is therefore needed.

Evolution of cytotoxic T lymphocyte responses to human immunodeficiency virus type 1 in patients with symptomatic primary infection receiving antiretroviral triple therapy.

The impact of highly active antiretroviral treatment (HAART) on anti-human immunodeficiency virus (HIV) cytotoxic T lymphocytes (CTL) was studied in 17 patients with recent symptomatic HIV-1 primary infection receiving triple combination therapy. Anti-HIV CTL were initially detected in 15 patients. In 6, CTL disappeared rapidly and persistently after initiation of therapy. Most of them had a rapid and sustained decrease in plasma HIV RNA to undetectable levels. Conversely, in 6 other patients, CTL remained detectable, which was associated with a less efficient control of viral replication. In 3 others, CTL disappeared only transiently, without clear correlation with the virologic profile. Altogether, despite individual variations, there was a positive correlation between viral replication and anti-HIV-1 cytotoxicity in most subjects, suggesting that the persistence of viral antigens is the main determinant for the maintenance of CTL activity. This raises the question of the potential benefit of anti-HIV CTL induction by immunotherapy in acute seroconverters treated by HAART.

Highly active antiretroviral treatment initiated early in the course of symptomatic primary HIV-1 infection: results of the ANRS 053 trial.

Highly active antiretroviral treatment (HAART) was given early to 64 patients with symptomatic primary human immunodeficiency virus (HIV)-1 infection. At the time of analysis, patients had been followed up for 9-21 months. No patient had died or developed an AIDS-defining event. Survival analysis showed that by month 21 the proportion of patients with plasma HIV-1 RNA <50 copies/mL was 72% (95% confidence interval, 58%-95%) in intention-to-treat analysis. After 18 months of treatment, 50% of the patients with undetectable plasma HIV-1 RNA also had undetectable HIV-1 RNA in peripheral blood mononuclear cells (PBMC). Only 1 of 3 patients had undetectable HIV-1 RNA in lymphoid tissue, while all patients had quantifiable HIV-1 DNA both in PBMC and lymphoid tissue. The median CD4 lymphocyte increase from baseline was 230 cells/microL. These preliminary results support the use of HAART in patients with primary HIV-1 infection.