John A. Todd

Prognosis in HIV-1 Infection Predicted by the Quantity of Virus in Plasma

The relation between viremia and clinical outcome in individuals infected with human immunodeficiency virus-type 1 (HIV-1) has important implications for therapeutic research and clinical care. HIV-1 RNA in plasma was quantified with a branched-DNA signal amplification assay as a measure of viral load in a cohort of 180 seropositive men studied for more than 10 years. The risk of acquired immunodeficiency syndrome (AIDS) and death in study subjects, including those with normal numbers of CD4+ T cells, was directly related to plasma viral load at study entry. Plasma viral load was a better predictor of progression to AIDS and death than was the number of CD4+ T cells.

Quantitation of HIV-1 RNA in Plasma Predicts Outcome after Seroconversion

The course of infection with human immunodeficiency virus type 1 (HIV-1) varies considerably. Although the median interval between HIV-1 infection and the development of the acquired immunodeficiency syndrome (AIDS) in adults is 10 to 11 years [1], some infected persons rapidly progress to AIDS in less than 5 years [2]. Still others remain asymptomatic without evidence of immunologic decline for more than 6 years [3]. The biological basis of this variability is unknown, but differences in viral strains, host immune responses [4], and exposure to microbial [5] or environmental cofactors probably contribute. The variable course of HIV-1 infection causes uncertainty for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Many clinical and laboratory markers have been used to estimate prognosis in patients with HIV-1 infection [6]. Markers of AIDS development include HIV-related symptoms [7, 8], depletion of CD4+ T cells [9], cutaneous anergy [7, 10], elevated serum 2-microglobulin and neopterin levels [9], HIV-1 p24 (core) antigenemia [11, 12], and syncytium-inducing HIV-1 phenotype [13]. None of these markers is ideal; all have limitations in sensitivity, specificity, or predictive power. The single best predictor of AIDS onset identified thus far is the percentage or absolute number of circulating CD4+ T cells [9], but less variable and earlier markers of risk for AIDS are needed. Several new methods have been developed to directly measure HIV-1 nucleic acid in body fluids. One of these technologies is the branched-DNA (bDNA) signal amplification method for quantitating HIV-1 RNA in plasma [14]. Although less sensitive than RNA detection by the polymerase chain reaction (PCR), the bDNA method has the advantage of large sample capacity, speed, reproducibility, and a format similar to an enzyme-linked immunosorbent assay. The ability of the bDNA assay or other HIV-1 RNA detection methods to predict clinical outcome in HIV-1 infection has not been clearly defined in appropriate cohorts or been compared with the ability of other predictive markers. Previous studies have shown a strong correlation between disease stage and the amount of circulating HIV-1, whether measured as cell-free infectious virus [15, 16], viral proteins [11, 12], or RNA [17, 18]. Recent studies have shown that an increase in HIV-1 expression in peripheral blood mononuclear cells can precede immunologic deterioration by 1 to 2 years [17, 18]. Our objective, therefore, was to compare plasma HIV-1 RNA with determinations of serum p24 antigen, neopterin, and 2-microglobulin levels and CD4+ T-cell counts as predictors of outcome in a cohort of homosexual men with documented HIV-1 seroconversion. Methods Study Populations The initial pilot study population consisted of 10 seroprevalent men (unknown date of seroconversion) enrolled in the Pittsburgh portion of the Multicenter AIDS Cohort Study (MACS). Five of these men developed AIDS (Centers for Disease Control and Prevention [CDC] 1987 definition) after 35 to 74 months of follow-up (median, 59 months), and five remained asymptomatic with stable CD4+ T-cell counts after a similar follow-up interval (median, 56 months). The second study population consisted of 62 homosexual men enrolled in the MACS who had documented seroconversion (change from negativity for HIV-1 antibody to positivity). Eighteen of these men progressed to AIDS (CDC 1987 definition) by a median of 3.8 years after seroconversion (maximum, 6.5 years), and 44 did not develop AIDS after a median follow-up of 5.4 years (maximum, 8.3 years). Details about the recruitment and characteristics of the MACS cohort have been described previously [19]. All participants gave written informed consent, and the MACS protocol was approved by the Internal Review Board of the University of Pittsburgh. Study Samples The study samples were selected from stored ( 70C) longitudinal plasma and serum samples obtained from enrollees at 6-month intervals as part of the MACS protocol. In patients who developed AIDS, the samples tested were obtained from the seroconversion visit (first visit at which the patient was positive for the HIV-1 antibody), the most recent visit before AIDS diagnosis, and equally spaced visits in between. In patients without AIDS, the samples tested were obtained from the seroconversion visit; visits 1, 2, and 3 years after seroconversion; and the last available visit, which occurred as long as 8.3 years after seroconversion. Definition of Outcomes Study patients were classified into one of three outcome groups: 1) AIDS; 2) decline in the CD4 count; and 3) stable CD4 count. Patients in the AIDS outcome group (n = 18) met the CDC 1987 case definition for AIDS. For each patient who had seroconversion and did not develop AIDS, we used linear regression to fit a line through prospective CD4+ T-cell measurements (minimum of three measurements per patient). We calculated the slope of each line and determined the statistical significance of the negative slopes. Patients with declining CD4 counts (n = 21) had statistically significant (P < 0.05) negative slopes but did not develop AIDS during follow-up. Patients with stable CD4 counts (n = 23) had no significant decline in the CD4+ T-cell count during follow-up, and 6 of 23 patients (26.1%) had a positive slope, that is, an increasing linear trend in the number of CD4+ T cells. Measurement of T-Lymphocyte Subsets We measured T-lymphocyte subsets in whole blood by staining them with fluorescent dye-conjugated monoclonal antibodies specific for CD3, CD4, and CD8 (Becton Dickinson, Mountain View, California) as previously described [20]. The total number of CD4+ T cells was determined by multiplying the percentage of lymphocytes that were CD4+ T cells by the total lymphocyte count. Serum 2-Microglobulin and Neopterin Assays We measured serum 2-microglobulin (Kabi Pharmacia, Uppsala, Sweden) and serum neopterin levels (Henning, Berlin, Germany) with commercial radioimmunoassays and standards provided by the manufacturers. Four replicates of normal control serum were included in each assay to assess variability. The coefficient of variation for control samples was 15% or less. Serum Immune Complex Dissociated p24 Assay Immune complex dissociated (ICD) p24 antigen levels were measured with a commercial enzyme immunoassay (Dupont, NEN Products, Wilmington, Delaware). The ICD p24 antigen levels in serum were interpolated from a standard curve provided by the manufacturer. The assay has a sensitivity of 12 pg of p24 antigen/mL. The interassay coefficient of variation for the p24 standards was less than 10%. Plasma and Cellular HIV-1 RNA Assays Levels of HIV-1 RNA in plasma samples were quantitated with the Quantiplex HIV-1 RNA assay, which is based on bDNA signal amplification technology (Chiron Corp., Emeryville, California). This assay measures HIV-1 RNA associated with viral particles that are pelleted from 1.0-mL plasma samples (23 500 g for 1 hour at 4 C). The assay has a quantitation limit of 1 104 HIV-1 genome equivalents per mL of plasma (Eq/mL) and is linear at levels as high as 1.6 106 Eq/mL. For this study, the interassay coefficient of variation for the positive control samples run with each assay was 11.2%. Additional details about the assay procedure and its performance characteristics have been described previously [14]. We categorized longitudinal plasma HIV-1 RNA results from individual patients into one of four groups: 1) detection of HIV-1 RNA (>1 104 Eq/mL) in all samples tested [n = 9]; 2) detection in most ( 50%) samples [n = 24; mean percentage of positive samples, 67.3%]; 3) detection in fewer than 50% of samples [n = 16; mean percentage of positive samples, 29.3%]; and 4) detection in none of the samples tested (n = 13). We identified an additional subgroup (n = 6) that showed evidence for clearance of detectable HIV-1 RNA from plasma, that is, two or more consecutive negative samples and no further positive samples after one or two initial positive samples. Assays for neopterin, 2-microglobulin, ICD p24, and HIV-1 RNA were done in duplicate on coded serum or plasma samples. Samples from a given patient were batch-tested to minimize the potential effect of interassay variability. Semi-quantitative PCR-based assays for cellular HIV-1 gag RNA were done on stored peripheral blood mononuclear cell samples as described previously [17]. Statistical Analyses The pilot study data are shown in the tables and figures to familiarize the reader with the raw data obtained from the bDNA assay. All cellular PCR results were adjusted per million CD4+ T cells. Analyses of the data set from patients with HIV-1 seroconversion were similarly stratified by outcome group. The Fisher exact test, chi-square test, and Wilcoxon rank-sum test were done where noted in the text. We estimated the association between progression to AIDS and laboratory covariates at seroconversion by multiple logistic regression analysis using BMDP statistical software (BMDP Statistical Software, Inc., Los Angeles, California). Results HIV-1 Quantitation by Branched DNA and Polymerase Chain Reaction in Seroprevalent Patients An initial pilot study of plasma HIV-1 RNA quantitation was done in 10 seroprevalent men enrolled in the MACS. Five of the men developed AIDS after 35 to 64 months of follow-up (progressors), and 5 remained asymptomatic with stable CD4+ T-cell counts (nonprogressors) after 38 to 74 months of follow-up. The median duration of follow-up for progressors and nonprogressors was similar (59 and 56 months, respectively). The bDNA assay was done on stored longitudinal plasma samples from 4 to 6 time points for each patient. Figure 1 shows the plasma HIV-1 RNA levels in the nonprogressors and progressors. Levels of HIV-1 RNA in all five nonprogressors were less than the limit of quantitation (<1 104 Eq/mL) at each time point dur

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AimTo determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. MethodTwo HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. ResultsIn one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 105 copies/ml, and the branched DNA results ranged from 2.6 x 104 to 4.2 x 104 HIV RNA equivalents/ml. In the other sample the corresponding figures were 6.3 x 104 to 5.5 x 105 copies/ml and 5.7 x 104 to 7.5 x 104 HIV RNA equivalents/ml. ConclusionIn contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Quantitation of human immunodeficiency virus plasma RNA by branched DNA and reverse transcription coupled polymerase chain reaction assay methods: A critical evaluation of accuracy and reproducibility

Abstract The present study was designed to evaluate the utility of two assays, reverse transcription coupled polymerase chain reaction (RT-PCR) and branched DNA (bDNA), to accurately and reproducibly quantitate plasma human immunodeficiency virus (HIV) RNA levels. The bDNA assay quantitated RNA transcripts, prepared from different HIV-1 subtypes (A-E), within 1.5-fold. Similarly, the bDNA assay, standardized to subtype B, was used to quantitate cultured isolates from subtypes A, C-F within 2-fold; however, the RT-PCR assay displayed a 904-fold range. Reproducibility studies demonstrated that the bDNA and RT-PCR assays could be used statistically (P

Correlation of Virus Load in Plasma and Lymph Node Tissue in Human Immunodeficiency Virus Infection

The impact of long-term changes in plasma viremia, produced by effective combination antiretroviral therapy, on human immunodeficiency virus (HIV) burden within tissue reservoirs is unknown. Fifteen patients who had received at least 1 year of therapy with two or three drug combinations of zidovudine, didanosine, and nevirapine had suitable samples of lymph node tissue obtained by ultrasound-guided core needle biopsy. HIV RNA was extracted from homogenized tissue samples and quantitated using a modified branched DNA assay. Results were correlated with antiretroviral treatment effect on the basis of plasma virus load measurements over the preceding 12-18 months. A statistically significant negative correlation was observed between magnitude of treatment effect on plasma viremia and lymph node virus load. These data suggest that combinations of antiretroviral drugs that produce sustained suppression of plasma HIV RNA may also be able to reduce the virus burden in lymphoid tissues.