Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

Restructuring the neuronal stress response with anti-glucocorticoid gene delivery.

Glucocorticoids, the adrenal steroids released during stress, compromise the ability of neurons to survive neurological injury. In contrast, estrogen protects neurons against such injuries. We designed three genetic interventions to manipulate the actions of glucocorticoids, which reduced their deleterious effects in both in vitro and in vivo rat models. The most effective of these interventions created a chimeric receptor combining the ligand-binding domain of the glucocorticoid receptor and the DNA-binding domain of the estrogen receptor. Expression of this chimeric receptor reduced hippocampal lesion size after neurological damage by 63% and reversed the outcome of the stress response by rendering glucocorticoids protective rather than destructive. Our findings elucidate three principal steps in the neuronal stress-response pathway, all of which are amenable to therapeutic intervention.

A 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors.

While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.

Clinical cross-resistance between the HIV-1 protease inhibitors saquinavir and indinavir and correlations with genotypic mutations.

OBJECTIVES: To determine the clinical efficacy of the HIV-1 protease inhibitor indinavir (IDV) in saquinavir (SQV)-experienced patients and delineate the developing drug-resistance patterns. DESIGN: Open-label prospective clinical trial. SETTING: University hospital research center. PATIENTS: Ten patients who had completed a SQV monotherapy study in which they had received SQV at a dose of 3600 or 7200 mg daily (two and fourfold the standard dose). INTERVENTIONS: At enrollment patients received IDV for 4 weeks as monotherapy, after which zidovudine (ZDV) and lamivudine (3TC) were added to their drug regimen. Patients then received combination therapy (IDV-ZDV-3TC) for an additional 20 weeks to complete a total of 24 weeks of therapy. MAIN OUTCOME MEASURES: Plasma HIV RNA viral load and CD4+ T-cell counts were monitored. Sequencing of the HIV protease gene was performed to determine the development of resistance mutations. Plasma samples for sequencing were taken before initial SQV therapy, after SQV therapy before starting IDV, and after 24 weeks of IDV therapy. RESULTS: The average duration of high-dose SQV before starting IDV was 58+/-29.2 weeks. A 0.58 log10 RNA copies/ml increase was noted during the 3-week washout phase followed by a mean reduction in plasma HIV RNA viral load of 1.2 log10 RNA copies/ml after 4 weeks of IDV. After the addition of ZDV and 3TC at week 4, HIV RNA continued to fall reaching a mean reduction of 1.96 log10 RNA copies/ml at week 24. Plasma HIV RNA was below 400 RNA copies/ml in six out of nine patients at week 24. CD4+ T-cell counts showed a gradual rise from 328 x 10(6)/l to 453 x 10(6)/l by week 24. SQV therapy had resulted in multiple mutations in the protease gene. Six of the patients had developed five or more mutations: L90M in two, G48V in four (of which three also contained L101), and V82A in three. Patients in whom plasma HIV RNA was not durably suppressed by subsequent IDV combination therapy developed multiple (up to four) additional mutations within 24 weeks, including codons 54, 82 and 93 amongst others. No clear correlation was found between the mutations that had developed in individual patients after SQV and the subsequent efficacy of IDV. CONCLUSION: Prolonged use of SQV at potent doses in the presence of elevated viral load levels resulted in the development of multiple resistance mutations. Individual resistance patterns varied greatly between patients, as did their virological response to therapy. Resistance assays may be useful in identifying which patients will benefit from salvage therapy with a second protease inhibitor.

The prognostic significance of serum viral load, codon 215 reverse transcriptase mutation and CD4+ T cells on progression of HIV disease in a double-blind study of thymopentin.

OBJECTIVE: To determine in asymptomatic HIV-infected subjects the prognostic value of virion reverse transcriptase (RT) codon 215 mutation, serum HIV RNA level, CD4+ T-cell count and immune complex dissociated (ICD) p24 level. The retrospective evaluation of thymopentin treatment effect on subjects in high risk groups for progression was a secondary objective. PARTICIPANTS: Zidovudine (ZDV)-experienced asymptomatic HIV-infected subjects (n = 352) who had been enrolled in a 48-week placebo-controlled double-blind trial of thymopentin treatment were studied. METHODS: Post hoc analyses were conducted to determine which subjects at study entry were at greater risk for progression to AIDS-related complex (ARC), AIDS or death, and to determine the effect of treatment on these subjects. Four potential prognostic variables (virion RT codon 215 mutation, circulating HIV virion RNA copies, CD4+ T-cell count, and ICD p24) were evaluated by dichotomizing subjects for each variable based on the median of the observed values. CD4+ T-cell count was evaluated prospectively, whereas frozen samples were evaluated under blinded conditions for the other variables after the study was completed. RESULTS: The presence of the codon 215 mutation [P = 0.044; relative hazard (RH), 2.6], > or = 20,000 HIV RNA copies/ml (P = 0.002; RH, 5.5), and < 350 CD4+ cells 10(6)/l (P = 0.042; RH, 2.2) were prognostic factors, and > or = 30 pg/ml ICD p24 level (P = 0.52; RH, 1.4) was not a prognostic factor in predicting progression. Subjects were prestratified by previous ZDV use (< or = 6 or > 6 months). Across both strata thymopentin delayed treatment progression to ARC, AIDS, or death (P = 0.015; RH, 3.0). This effect was magnified in the ZDV-experienced subjects at greater risk, where thymopentin delayed progression compared to placebo in the presence of the codon 215 mutation (P = 0.007; RH, 10.1), > or = 20,000 RNA copies/ml (P = 0.012; RH, 8.9), and CD4+ T-cell count < 350 x 10(6)/l (P = 0.005; RH, 10.4). CONCLUSIONS: Codon 215 mutation, serum HIV RNA and CD4 T-cell count are independent predictors of progression in ZDV-experienced asymptomatic subjects. Furthermore, thymopentin delays HIV disease progression in the presence of a key ZDV resistance mutation as well as high viral load and low CD4+ T-cell counts.

A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS.

OBJECTIVE: To determine the short-term effects of using genotypic antiretroviral resistance testing (GART) with expert advice in the management of patients failing on a protease inhibitor and two nucleoside reverse transcriptase inhibitors. DESIGN: Prospective randomized controlled trial. SETTING: Multicenter community-based clinical trials network. PATIENTS: One-hundred and fifty-three HIV-infected adults with a threefold or greater rise in plasma HIV-1 RNA on at least 16 weeks of combination antiretroviral therapy. INTERVENTIONS: Randomization was either to a GART group, where genotype interpretation and suggested regimens were provided to clinicians, or to a no-GART group, where treatment choices were made without such input. MAIN OUTCOMES MEASURES: Plasma HIV-1 RNA levels and CD4 cell counts were measured at 4, 8, and 12 weeks following randomization. The primary endpoint was change in HIV-1 RNA levels from baseline to the average of the 4 and 8 week levels. RESULTS: The average baseline CD4 cell count was 230 x 10(6) cells/l and the median HIV-1 RNA was 28,085 copies/ml. At entry, 82 patients were failing on regimens containing indinavir, 51 on nelfinavir, 11 on ritonavir, and nine on saquinavir. HIV-1 RNA, averaged at 4 and 8 weeks, decreased by 1.19 log10 for the 78 GART patients and -0.61 log10 for the 75 no-GART patients (treatment difference: -0.53 log, 95% confidence interval, -0.77 to -0.29; P = 0.00001). Overall, the best virologic responses occurred in patients who received three or more drugs to which their HIV-1 appeared to be susceptible. CONCLUSION: In patients failing triple drug therapy, GART with expert advice was superior to no-GART as measured by short-term viral load responses.

HIV-1 syncytium-inducing phenotype, virus burden, codon 215 reverse transcriptase mutation and CD4 cell decline in zidovudine-treated patients.

The variable rate of disease progression in HIV-1-infected patients treated with zidovudine may be related to certain viral characteristics, such as, antiviral drug resistance, virus burden, and viral syncytium-inducing (SI) capacity. Thirty-two HIV-1-infected patients treated with zidovudine (mean of 34 months) were studied to determine the relationship of SI phenotype and the codon 215 pol gene mutation (a marker of zidovudine resistance) to virus burden and CD4 cell decline. Patients with SI strains and the codon 215 mutation in their proviral DNA had a 54% decline in CD4 cells and a virus burden of 21,480 proviral DNA copies/10(6) CD4 cells. In contrast, patients with non-SI (NSI) strains and wild-type at codon 215 had a 10% increase in CD4 cells and had a viral burden 1/46 that of patients with SI and the 215 mutation. Among patients with NSI strains, changes in CD4 cells depended on the presence of the codon 215 mutation (-160 CD4 cells/microliters), compared with those wild-type at codon 215 (+28 CD4 cells/microliters) (p < 0.01). There was a concordant rise in virus burden between proviral DNA and plasma HIV RNA depending on HIV phenotype and genotype. Using multiple linear regression, SI phenotype and the codon 215 mutation were found to independently predict CD4 cell decline and increased virus burden in zidovudine-treated patients.

Frequency of antiretroviral drug resistance mutations in HIV-1 strains from patients failing triple drug regimens. The Terry Beirn Community Programs for Clinical Research on AIDS.

The frequency of protease and reverse transcriptase (RT) gene mutations was determined in HIV-1 strains from 153 patients entering the CPCRA 046 (GART) study who were failing triple-drug regimens consisting of one protease inhibitor (PI) and two RT inhibitors. Population-based sequence analyses showed that nearly all patients had similar RT gene mutations regardless of prior drug exposure, although the M184V mutation was significantly less prevalent in patients not recently treated with lamivudine. Whilst typical inhibitor-specific ('signature') protease gene mutations were found in patients failing their first PI, these mutations were significantly less likely to be found in patients exposed to two or more PIs. Protease gene mutations associated with multi-PI resistance were more likely to be observed in patients treated with more than one PI. These results suggest sequential treatment with PIs select for a relatively limited number of protease gene mutations that likely originated during early PI therapy. These protease gene mutations and a similarly limited set of RT gene mutations appear to be responsible for treatment failure in antiretroviral therapy.

Detection and quantification of gene amplification products by a nonisotopic automated system.

We describe in this report the ability to determine human immunodeficiency virus proviral copy number by an automated nonisotopic method. Our system utilizes a FACStarPLUS cell sorter, the GeneAmp PCR System 9600 and a Biomek 1000 robotic workstation. Linking these three machines allows cell populations to be sorted and the DNA amplified and quantitated with minimal technical effort. We have developed this system to quantitate proviral DNA copy number in sorted subpopulations of peripheral blood cells in one day.

Continuous presence of CD4-PE40 is required for antiviral activity against single-passage HIV isolates and infected peripheral blood mononuclear cells.

CD4-PE40, a recombinant protein consisting of a portion of human CD4 linked to Pseudomonas aeruginosa exotoxin, was studied in vitro to assess its ability to inhibit the replication of primary isolates of HIV. CD4-PE40 was added to cultures of phytohemagglutin (PHA)-stimulated normal peripheral blood mononuclear cells (PBMCs) infected either with the laboratory strain HIVIIIb or single-passage virus stocks derived from patient PBMCs. Results showed that the replication of HIVIIIb was inhibited by a single pulse of CD4-PE40 and, more significantly, by continuous exposure to the drug. The replication of primary virus isolates, however, was inhibited only by continuous exposure to CD4-PE40. Cultures of freshly isolated PBMCs from HIV-seropositive individuals that were directly treated with CD4-PE40 before culture also required the continuous presence of drug to demonstrate inhibition of HIV replication. These results suggest that continuous administration of CD4-PE40 may be required to produce a significant anti-HIV effect in vivo.

Changes in CD4+ and CD8+ T cell subsets in response to highly active antiretroviral therapy in HIV type 1-infected patients with prior protease inhibitor experience.

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.

Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals.

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discordances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consensus sequence), the proportion of sequences having a mutation when at least two sequences from that sample had the same mutation were examined. There was a median of 13 protease and 18 RT mutations per sample for a total of 3126 mutations; 95% of these mutations were detected. However, sequencing of multiple clones from two samples demonstrated that those mutations present in a minority of clones were often not detected by population-based sequencing. These results suggest that HIV-1 protease and RT sequencing of circulating plasma virus is highly reproducible but that the sensitivity at detecting mutations may be low if those mutations are present as minor variants.

Precipitation of proteins in supercritical carbon dioxide.

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.

Rapid method for epidemiological evaluation of gram-positive cocci by field inversion gel electrophoresis.

We report a rapid method for the isolation of intact chromosomal DNA from gram-positive cocci that is suitable for in situ restriction endonuclease digestion in agarose blocks. When combined with a rapid field inversion gel electrophoresis protocol, this approach allows the preparation and electrophoretic analysis of chromosomal restriction fragments produced by rare-cutting enzymes in a total time period of 2 days from start to finish. The utility of the method is demonstrated in the epidemiological evaluation of Staphylococcus epidermidis clusters from two hospitals as well as of additional representative staphylococci and enterococci.

Induction of antigen-specific immunity and tolerance to Mycobacterium leprae in Lewis rats.

Intradermal (i.d.) immunization of Lewis rats with autoclaved Mycobacterium leprae resulted in antigen-specific proliferation responses and interleukin-2 release from spleen and lymph node cells that were detectable as early as 21 days, persisted for at least 9 months, and were dependent on the dose of antigen administered. Immunized animals were also completely resistant to a footpad challenge with viable M. leprae. In contrast, intravenous (i.v.) administration of at least 10(8) irradiated M. leprae isolates induced a state of nonresponsiveness characterized by the absence of proliferation and interleukin-2 release by antigen-stimulated lymphoid cell cultures; however, in vitro responses to mitogenic stimulation and in vivo responses to keyhole limpet hemocyanin and Listeria monocytogenes were normal. Animals that received an i.v. injection of M. leprae remained nonresponsive to M. leprae antigens even after a subsequent i.d. immunization. This state of nonresponsiveness persisted for at least 6 months after induction. Results of footpad challenge experiments showed that the ability of animals rendered nonresponsive by an i.v. injection of M. leprae to control the growth of viable M. leprae in the footpad was not different from that of untreated rats. In addition, animals receiving an initial i.v. injection and a subsequent i.d. immunization with M. leprae were not protected from a viable challenge, as were rats that received only i.d. immunization. These results suggest that i.v. administration of a large dose of M. leprae to rats induces a state of nonresponsiveness to M. leprae antigens that may be similar to that seen in lepromatous leprosy patients.

Variants other than aspartic acid at codon 69 of the human immunodeficiency virus type 1 reverse transcriptase gene affect susceptibility to nuleoside analogs.

The T69D mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) gene has been associated with reduced susceptibility to dideoxycytosine (ddC); however, several other mutations at codon 69 have been observed in antiretroviral drug-treated patients. The Stanford HIV RT and Protease Sequence Database was interrogated and showed that 23% of patients treated with nucleoside RT inhibitors (NRTI) had mutations at codon 69. These variants included T69N, -S, -A, -G, -E, -I, and -K mutations that were present in patients treated with NRTI but not in drug-naive patients. Treatment history information showed that a substantial percentage of these codon 69 changes occurred in patients administered non-ddC-containing regimens. Different and specific patterns of other RT gene mutations were associated with the various codon 69 mutations. Drug susceptibility assays showed that viral constructs containing codon 69 variants could have reduced susceptibility to ddC and other RT inhibitors. These results suggest that the T69D mutation is not the only codon 69 variant associated with drug resistance and that ddC is not the only drug affected.

Detection of human immunodeficiency virus type 1 in semen: effects of disease stage and nucleoside therapy.

The effects of clinical stage of infection and antiviral therapy on the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in semen were investigated by the polymerase chain reaction. HIV-1 was detected in 45 (87%) of 52 semen specimens from 29 (81%) of 36 men. Seventeen (77%) of 22 stage II or III subjects and 12 (86%) of 14 stage IV subjects had positive specimens. The CD4+ lymphocyte count was not significantly different comparing subjects with positive and negative semen. Moreover, 6 (67%) of 9 untreated men had positive specimens compared with 23 (85%) of 27 men treated with zidovudine, 2',3'-dideoxyinosine, or both for a mean of 20 months. Thus, the detection of HIV-1 in semen was independent of both stage of infection and long-term treatment. In a semiquantitative analysis of 6 men followed for 8 weeks after the start of nucleoside therapy, a decrease in HIV-1 RNA in seminal plasma was demonstrated in 2.

Quantitative RNA and DNA gene amplification can rapidly monitor HIV infection and antiviral activity in cell cultures.

We have developed a quantitative gene amplification procedure to assess the replication of human immunodeficiency virus (HIV) in cell cultures and evaluate the effect of drugs on viral replication. Increases in HIV gag RNA and DNA in phytohemagglutinin-stimulated normal peri-pheral blood mononuclear cells (PBMC) infected with HIV at very low multiplicity of infection paralleled the production of HIV p24 antigen in culture supernatants. Quantitative gene amplification was able to monitor the accumulation of viral nucleic acids in control cultures and demonstrate the effect of various concentrations of azidothymidine (AZT) on the replication of both AZT-sensitive and -resistant strains of HIV. The sensitivity of patient-derived virus strains to AZT could also be successfully measured by these procedures. The results of our studies suggest that quantitative measurement of HIV gag RNA and DNA can be used to monitor the kinetics of viral replication, antiviral activity, viral drug resistance, and mechanism of drug action.

Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction.

Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in the plasma of seropositive individuals was performed by using an external control assay with techniques to standardize and control each measurement. Rigorous study of the variability of the assay showed that the median intraassay reproducibility was log10 0.15 RNA copies per ml of plasma, while the median interassay reproducibility on replicate plasma samples was log10 0.25 copies perml. Specimen stability studies showed reproducible recovery of RNA from plasma stored at -70 degrees C for up to 12 months. In clinically stable patients who were either untreated or taking zidovudine, the average week-to-week variation in plasma RNA levels, measured in real time, was log10 0.30 RNA copies per ml. In contrast, patients either initiating or changing antiretroviral therapy showed a fall of log10 0.8 to log10 2.0 copies per ml in plasma RNA levels. Overall, 105 of 110 (96%) HIV-1-seropositive individuals with CD4 counts of 36 to 868 cells per mm3 had quantifiable HIV-1 RNA over a range of log10 2.70 to log10 6.23 RNA copies per ml, including 81% (13 of 16) of the individuals with greater than 500 CD4 cells per mm3. Accurate and reproducible quantitation of plasma viremia in real time by reverse transcriptase polymerase chain reaction, particularly in asymptomatic HIV-1-infected individuals with high CD4 counts, provides a basis for the use of this virologic measure to monitor the short- and long-term effects of early intervention therapeutic strategies on viral burden.