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Quantification of HIV-1 subtypes is essential for appropriate clinical management. Whereas viral load assays were initially developed to accurately quantify subtype B, the recent worldwide spread of non-B subtypes and the introduction of treatment programs in regions with non-B subtypes have prompted adaptations of these assays. The Bayer Versant HIV-1 RNA 3.0 Assay (branched DNA [bDNA] 3.0) and the Roche Amplicor HIV-1 Monitor version 1.5 (Amplicor 1.5) assays are reported to quantify all subtypes in group M; however, evaluation of performance characteristics remains limited. In this study, we evaluated the accuracy and reliability of bDNA 3.0 and Amplicor 1.5 on multiple serially diluted viral isolates from HIV-1 group M, subtypes A through F. Testing was conducted on both assay systems in two independent laboratories. Comparative pansubtype quantification from regression analysis showed that quantification by bDNA 3.0 was approximately 0.3 log-fold lower than that by Amplicor 1.5. Comparative pansubtype accuracy analysis showed data points more closely distributed about their respective regression lines and thus showing greater reliability by bDNA 3.0 than by Amplicor 1.5.

Objective: Emergence of human immunodeficiency virus type-1 (HIV-1) genotypic drug resistance is generally attributed to noncompliance, poorly absorbed drugs, or drug-to-drug interaction. Attempts to determine emerging genotypic drug resistance from study subjects on highly active antiretroviral therapy (HAART) relied on insensitive polymerase chain reaction (PCR) techniques, revealing wild type HIV-1 or precursor resistant genotypes from few plasma samples successfully amplified with <50 copies/mL., Study Design/methods: In this analysis, using Applied Biosystems' ViroSeq HIV-1 Genotyping Systems, Version 2.0 (Applied Biosystems, Foster City, CA, USA) and the supplemental, for research use only, nested PCR primers, genotypic drug resistance was determined in longitudinal plasma samples from 11 study subjects on HAART., Results: In 4 of 11 study subjects, newly emerging genotypic primary resistant mutations were detected in plasma samples with <50 copies/mL. Most of these primary drug-resistant mutations were detected in subsequent longitudinal samples with detectable viral load (viral breakthrough)., Conclusions: This analysis suggests sufficient viral replication <50 copies/mL to generate genotypic drug resistance in study subjects on suppressive HAART.

Coinfection with hepatitis C virus (HCV) and HIV-1 is common in patients with hemophilia and in intravenous drug users. Little, however, is known about the relation between HIV-1 and HCV coinfection and the effects on HCV clearance and pathogenesis. We examined data from 207 HIV-1-infected and 126 HIV-1-uninfected patients with hemophilia enrolled in the multicenter Hemophilia Growth and Development Study. Participants were observed during prospective follow-up for approximately 7 years with annual measurements of alanine aminotransferase (ALT), CD4+ cells, and HCV and HIV-1 RNA levels. Clearance of HCV was more likely to occur in those uninfected with HIV-1 (14.3 versus 2.5%; odds ratio [OR] 4.79; 95% confidence interval [CI], 1.63-14.08, p =.005) and was more common with decreasing age (OR, 1.23; 95% CI, 1.04-1.47; p =.017). HCV RNA levels were higher throughout the 7 years of follow-up in those HIV-1-infected (p <.001). In the HIV-1-infected participants, baseline CD4+ cells were inversely related to HCV RNA with every 100-cell increase associated with a 0.19 log10 copy/ml decrease in HCV RNA (p =.002), and HIV-1 and HCV RNA levels were directly related (p =.008). Increasing HCV RNA levels were also associated with significantly higher ALT levels regardless of HIV-1 infection status. These results demonstrate that HIV-1/HCV co-infection is associated with a reduced likelihood of HCV clearance and that higher levels of HCV RNA are associated with increased hepatic inflammation.

We have investigated the effects of plasma HIV RNA, CD4+ T lymphocytes and chemokine receptors CCR5 and CCR2b on HIV disease progression in hemophiliacs. We prospectively observed during follow-up 207 HIV-infected hemophiliacs in the Hemophilia Growth and Development Study. Plasma HIV RNA was measured on cryopreserved plasma from enrollment using the Chiron Corporation bDNA (version 2.0) assay. Genoytpe variants CCR2b-641 and CCR5-delta32 were detected using standard molecular techniques. Those with the mutant allele for CCR2b, and to a lesser extent CCR5, had lower plasma HIV RNA, and higher CD4+ T lymphocytes than did those without these genetic variants. After controlling for the effects of plasma HIV RNA and CD4+ T lymphocytes, those with the CCR2b mutant allele compared with those wild-type, had a trend toward a lower risk of progression to AIDS, adjusted relative hazard of 1.94 (95% confidence interval [CI], 0.9-4.18; p = .092), and AIDS-related death, relative hazard 1.97 (95% CI, 0.98-4.00; p = .059). We conclude that plasma HIV RNA, CD4+ T lymphocytes, and CCR genotypes are correlated, and the protective affect of CCR2b against HIV disease progression is not completely explained by plasma HIV RNA or CD4+ T-lymphocyte number.

We assessed the effect of influenza vaccination on plasma levels of human immunodeficiency virus type 1 (HIV-1) RNA and the impact of age, plasma HIV-1 RNA level, CD4 cell count, and anti-HIV therapy on immune response. Forty-nine adults (mean age, 38.7 years; mean CD4 cell count +/- SD, 190 +/- 169/mL; mean plasma HIV-1 RNA level +/- SD, 154,616 +/- 317,192 copies/mL) were immunized. Elevations of > or = 0.48 log in plasma HIV-1 RNA levels occurred in two (4%) of 49 subjects within 4 weeks of vaccination. A fourfold or greater increase in antibody titer occurred in 13 (45%) of 29 subjects, correlating directly with CD4 cell count (P = .002) and inversely with plasma HIV-1 RNA level (P = .034). By multivariate analysis, CD4 cell count was a stronger predictor of antibody response than was plasma HIV-1 RNA level. We conclude that increases in plasma HIV-1 RNA levels following influenza vaccination are rare and transient and that antibody response is impaired with CD4 cell counts of < 100/mL and plasma HIV-1 RNA levels of > 100,000 copies/mL. Prospective trials are needed to evaluate the impact of highly active therapy on immune response after vaccination.

We have developed small-volume (50 or 250 microl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.

The relationship between Mycobacterium avium complex (MAC) bacteremia and proinflammatory cytokine and human immunodeficiency virus type 1 (HIV-1) RNA levels in AIDS was investigated. During a prospective study, blood samples were drawn monthly for mycobacterial cultures. Sera were available at baseline and onset of MAC bacteremia from 20 cases and at corresponding times from 19 controls. Mean interleukin-6 (IL-6) levels were 154% greater at the time of MAC bacteremia in cases than in controls. The IL-6 levels correlated with body temperature, serum tumor necrosis factor (TNF-alpha) levels, and alkaline phosphatase levels (P < or = .004 for each). Although TNF-alpha levels tended to rise more in MAC patients than in controls, the difference was not significant. However, among both cases and controls, serum TNF-alpha levels rose significantly from baseline to the time of last sample, irrespective of MAC infection (P = .015). Bacteremia was not associated with increased serum HIV-1 RNA levels. Thus, early MAC bacteremia is associated with increases in serum IL-6 levels, while TNF-alpha levels rise over time during advanced AIDS.

Previous studies have shown that CD4-positive T cells vary in a predictable manner over 24 h. This diurnal variance has significant clinical implications. Recently, viral RNA measurements have been increasingly used as a standard marker in the management of human immunodeficiency virus (HIV)-infected patients. Little detailed analysis of the variability of this marker has been conducted. To define the variance of plasma HIV-1 RNA levels within days, 11 clinically stable patients with established HIV infection and a baseline viral RNA level >40,000 copies/mL were studied. Following the patients' admission to an inpatient research unit, plasma samples were obtained frequently over 48 h and analyzed for HIV-1 RNA levels by use of a quantitative branched chain DNA assay (bDNA). No diurnal pattern was detected. In these clinically stable patients, viral RNA levels exhibited a variance of approximately 0.4 log.

Objective: The present study was designed to determine the effect of immune activation, achieved by influenza vaccination, on plasma HIV RNA levels and immunological parameters including CD4 cell levels, antigen-stimulated T-cell function and apoptotic death of peripheral blood mononuclear cells., Design and Methods: Thirty-four HIV-infected individuals and nine uninfected controls were immunized with influenza vaccine and blood was collected at weeks 0, 2, 4 and 16. Plasma was isolated and used for HIV RNA and influenza-specific antibody qualifications. CD4 cell counts, activation and maturation markers of T-lymphocyte subsets were determined by flow cytometry. In vitro T-helper responses, spontaneous- and activation-induced cell death assays were also performed., Results: Influenza-specific humoral and cellular immune responses correlated with CD4 count. Only in patients with CD4 counts > 300 x 10(6)/l there was a modest increase in T-cell responses to influenza virus, which was less than control subjects, observed after vaccination. Immunization had no significant effect on CD4 counts or plasma viral levels in the HIV-positive patients. Baseline apoptosis inversely correlated with CD4 counts and directly correlated with viral load. Activation-induced apoptosis did not change appreciably after vaccination and spontaneous apoptosis increased only in the < 300 CD4 group., Conclusion: These results indicate that immune stimulation resulting from influenza vaccination did not significantly change the levels of plasma virus, CD4 cell counts, or activation-induced apoptosis in HIV-infected individuals, although an increase in the T-cell response to influenza and spontaneous apoptosis was observed in the > 300 and < 300 CD4 groups, respectively.

The natural variability of quantitative virologic measures among human immunodeficiency virus (HIV) type 1-infected persons was prospectively studied in 29 untreated persons with >600 CD4 cells/microL and in 15 persons receiving zidovudine monotherapy who had 400-550 CD4 cells/microL at study entry. Cell- and plasma-associated infectious HIV-1, provirus, and virion RNA were determined monthly as were numbers of CD4 and CD8 cells. HIV-1 replication varied widely among subjects with similar CD4 cell counts. The within-individual variability was significantly less than the variability between subjects for all virologic measures. Plasma virion HIV-1 RNA levels had the least variability. A mathematical model was devised to assess whether a potential therapeutic intervention significantly alters peripheral HIV-1 load. The model indicated that three measurements of plasma RNA would be outside the 95th percentile for the expected change in an individual due to natural variability. This approach can be used to accurately assess a therapeutic intervention among persons with low plasma HIV-1 titers.

We investigated the safety and immunogenicity of a candidate HIV-1 vaccine, Env 2-3 (Chiron Biocine Co.), in combination with an adjuvant emulsion, MF59, with or without an additional immune modulator, MTP-PE 78 healthy HIV-1-seronegative adults. Sixteen subjects participated in a dose escalation study of MTP-PE in MF59 without Env 2-3, given at 0 and 1 months; 48 subjects participated in a study of a fixed dose of 30 micrograms of Env 2-3 in MF59 with increasing doses of MTP-PE (0, 5, 10, 25, 50, and 100 micrograms), and 14 subjects participated in a study of 100 micrograms of Env 2-3 in MF59 without MTP-PE. Subjects were assigned to study groups under a randomized, double-blind allocation. Subjects received immunization at 0, 1, and 6 months, and had the option of receiving a fourth dose at 12-18 months. Env 2-3 in MTP-PE/MF59 was associated with significant reactogenicity, in that severe, although self-limited systemic and/or local reactions occurred in 15 of 30 vaccinees. In contrast, Env 2-3 in MF59 without MTP-PE was relatively well tolerated, and severe local and/or systemic reactions occurred in only 2 of 18 subjects. Env 2-3 stimulated serum antibodies to HIV-1 envelope protein (gp120) as detected by Western blot in 39 of 43 subjects and to HIV-1 virus lysate by EIA in 28 of 43 subjects after three injections. The majority of subjects also developed EIA antibodies to recombinant gp120 (SF-2), gp120 (LAI), and V3 peptide (SF-2). Neutralizing antibodies to the homologous SF-2 strain developed in 30 of 43 and 27 of 34 subjects, and fusion inhibition antibodies in 25 of 43 and 15 of 36 subjects after three and four injections, respectively. Lymphoproliferative responses to the immunogen, Env 2-3 were observed in over 80% of the vaccinees examined, and CD4+ cytotoxic T cell activity directed against HIV-1 was noted transiently in 2 of 20 vaccinees. Addition of MTP-PE to Env 2-3 or increasing the dose of Env 2-3 from 30 to 100 micrograms did not augment immunogenicity. Env 2-3 in MF59 was well tolerated and immunogenic in HIV-1-seronegative individuals. The addition of MTP-PE significantly increased reactogenicity, but had little, if any, effect on immunogenicity.

A vaccine breakthrough occurred in a phase 1 clinical trial of a human immunodeficiency virus (HIV) type 1 candidate subunit vaccine. The vaccine antigen, gp120SF2, is a fully glycosylated protein produced in mammalian cells from the HIVSF2 isolate. After 4 immunizations, the subject developed neutralizing antibodies and lymphoproliferative responses to the gp120 protein. About 18 weeks after the last immunization, the subject became HIV infected. During the acute phase of infection, there was high virus burden, a decline in CD4+ T lymphocytes, increases in rgp120SF2-binding antibodies and HIVSF2- and HIVMN-neutralizing antibodies, and transient lymphoproliferative responses to HIV-1 envelope and core proteins. The nucleotide sequence of the V3 loop from 2 virus isolations displayed close similarity to the V3 sequence of the vaccine antigen. Thus, the immunologic responses induced by the vaccine in this subject did not protect him from HIV-1 infection.

Highly sensitive assays that quantitate human immunodeficiency virus type 1 (HIV-1) RNA may be valuable for clinical research and the treatment of HIV-1-infected patients. In this study we evaluated the reproducibility and accuracy of the first-generation branched DNA (bDNA-1.0) signal amplification assay under conditions that are relevant to routine use in a clinical context. We show that the bDNA-1.0 assay was able to discern two- to three-fold changes in plasma HIV-1 RNA levels as significant. Reverse transcription coupled to polymerase chain reaction (RT-PCR) was less reproducible and required a 3.7- to 5.8-fold change in plasma HIV-1 RNA levels to be statistically significant. The accuracy of the bDNA-1.0 assay in RNA quantitation was not affected by HIV-1 genotypic variation or by the presence of hemoglobin, bilirubin, lipemia, or any of a dozen therapeutic drugs. Using the bDNA-1.0 assay, we show that HIV-1 RNA levels in plasma specimens were stable when stored at -80 degrees C and were able to withstand at least three freeze-thaw cycles without significant loss. We also examined the performance of an ultrasensitive bDNA assay with improvements to the signal amplification technology. The ultrasensitive bDNA assay displayed a quantitation limit of approximately 500 RNA Eq/ml, yet maintained a dynamic quantitation range up to 1.6 x 10(6) RNA Eq/ml. Like the bDNA-1.0 assay, the ultrasensitive bDNA assay was not affected by HIV-1 genotype variability.