Quantitation of HIV-1 RNA in Plasma Predicts Outcome after 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 ( −70°C) 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 β₂-Microglobulin and Neopterin Assays

We measured serum β₂-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, β₂-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 during the 38 to 74 months of follow-up (Figure 1). In contrast, HIV-1 RNA levels in the progressors were greater than 1 × 10⁴ Eq/mL at the initial time point (3 of 5 patients) or increased to greater than 1 × 10⁴ Eq/mL (2 of 5 patients) before AIDS developed. Plasma HIV-1 RNA levels increased 30 to 50 months before the diagnosis of AIDS, suggesting that plasma HIV-1 RNA could be an early predictor of AIDS (Figure 1).

We compared the results of the bDNA assay with those of a semi-quantitative PCR assay of HIV-1 gag RNA in stored peripheral blood mononuclear cell samples obtained at the same times as the plasma samples (Table 1). Despite the differences in sample types and quantitative methods, the levels of HIV-1 RNA in plasma and peripheral blood mononuclear cells were strongly correlated (Spearman rank-order correlation coefficient, 0.66; P < 0.001). Cellular HIV-1 gag RNA levels remained low throughout follow-up in nonprogressors but increased in progressors as AIDS developed.

Study of HIV-1 Seroconversion

The findings of the pilot study led to a larger evaluation of plasma HIV-1 RNA quantitation in 62 men with known dates of HIV-1 seroconversion. Eighteen of the men (29%) progressed to AIDS after a median of 3.8 years (maximum, 6.5 years). Twenty-one patients (34%) had a significant decline in the CD4+ T-cell count but did not develop AIDS (CD4-decline group), and 23 (37%) had stable CD4+ T-cell counts without symptoms (stable-CD4 group). The median duration of follow-up for the CD4-decline and stable-CD4 groups were similar (5.3 and 5.5 years, respectively). We compared the ability of plasma HIV-1 RNA with that of serum neopterin, β₂-microglobulin, and ICD p24 antigen to discriminate between the outcome groups.

Serum Neopterin and β₂-Microglobulin

The median longitudinal neopterin and β₂-microglobulin levels in the three outcome groups are shown in (Figure 2). Overall, the median neopterin and β₂-microglobulin levels in the outcome groups did not change significantly over time. Median neopterin levels for the AIDS group, the CD4-decline group, and the stable-CD4 group ranged from 17.8 to 21.0 nmol/L, 11.9 to 18.0 nmol/L, and 9.3 to 14.0 nmol/L, respectively. Corresponding values for β₂-microglobulin levels were 3.0 to 3.6 µg/L, 2.1 to 3.5 µg/L, and 2.0 to 2.5 µg/L, respectively. Neopterin and β₂-microglobulin levels were significantly higher in the AIDS group than in the other groups (P < 0.05; Wilcoxon test). However, neopterin or β₂-microglobulin levels in the CD4-decline group and the stable-CD4 group did not significantly differ at any time point after seroconversion. In fact, the overall median neopterin and β₂-microglobulin levels for the stable-CD4 group were higher than those for the CD4-decline group (13.7 nmol/L compared with 11.9 nmol/L and 2.5 µg/L compared with 2.3 µg/L, respectively).

Serum Immune Complex Dissociated p24 Antigen and Plasma HIV-1 RNA Levels in Patients with HIV-1 Seroconversion

In Figure 3, the proportion of samples for each outcome group that had detectable serum ICD p24 antigen (>12 pg/mL) is shown as a function of time after seroconversion. In the first year after seroconversion, 36.3% of samples in the AIDS group were positive for p24 antigen compared with 4% to 9% of the samples in the other groups (P < 0.001; Fisher exact test). The proportion of samples for the AIDS group that were positive for p24 antigen increased to 66.7% by the fifth year after seroconversion. In the first 4 years after seroconversion, 0% to 22.7% of samples in the stable-CD4 and CD4-decline groups were positive for p24 antigen. No significant differences in p24 antigen positivity were noted between the stable-CD4 and CD4-decline groups in this interval (Fisher exact test).

The proportion of plasma samples in each outcome group that had detectable HIV-1 RNA (>1 × 10⁴ Eq/mL) is shown in Figure 3. In the AIDS group, 72.7% of samples had detectable plasma HIV-1 RNA within 1 year of seroconversion, which is twice the proportion that were positive for p24 antigen (36.3%). We observed significant differences in the proportion of the three outcome groups that had detectable plasma HIV-1 RNA levels in the first year of seroconversion (P = 0.01; chi-square test). The proportion of samples in the AIDS and CD4-decline groups that had detectable HIV-1 RNA levels increased with time; in the fifth year after seroconversion, 100% of samples in the AIDS group and 71% in the CD4-decline group were positive for HIV-1 RNA. In contrast, the proportion of the stable-CD4 group that was positive for HIV-1 RNA decreased from 27% at seroconversion to 0% by the end of the fourth year. Overall, the detection of HIV-1 RNA in all or most of the plasma samples after seroconversion was strongly associated with the AIDS (16 of 18 patients) and CD4-decline groups (13 of 21 patients) compared with the stable-CD4 group (4 of 23 patients; P < 0.001; Fisher exact test). Conversely, the absence of detectable HIV-1 RNA in most or all of the plasma samples was strongly associated with the stable-CD4 group (19 of 23 patients) and a lower risk for AIDS or decline in the CD4 count (10 of 39 patients; P < 0.001; Fisher exact test). A subgroup of 6 patients had evidence of clearance of detectable HIV-1 RNA from plasma after one or two initial positive samples. None of these patients progressed to AIDS.

Risk for AIDS Development

Table 2 shows the proportion of men developing AIDS as a function of HIV-1 RNA positivity, stratified by the CD4+ T-cell count at seroconversion. In patients with no detectable HIV-1 RNA in plasma for the first 2 years after seroconversion, the proportion developing AIDS was 6% or less. In contrast, when more than one plasma sample was positive for HIV-1 RNA within the first 2 years of seroconversion, the proportion of patients developing AIDS ranged from 45% to 86%, depending on whether the initial CD4+ T-cell count was less than 500/mm³ (86%) or 500/mm³ or more (45%). In addition, the mean plasma HIV-1 RNA level at the seroconversion visit was significantly higher in patients who developed AIDS than in those who did not (74.1 × 10³ Eq/mL compared with 18.9 × 10³ Eq/mL; P < 0.001; Wilcoxon test).

We did a logistic regression analysis to determine the strength and independence of the laboratory variables as predictors of AIDS development. All laboratory measurements from the seroconversion visit, including CD4+ T-cell counts and levels of HIV-1 RNA, ICD p24 antigen, neopterin, and β₂-microglobulin, were eligible for entry into the model. In this analysis, the quantity of HIV-1 RNA in plasma was categorized into three strata: greater than 1 × 10⁵ Eq/mL; 1 × 10⁴ to 10⁵ Eq/mL; or less than 1 × 10⁴ Eq/mL.

Plasma HIV-1 RNA was the first and only variable that was entered into the stepwise model; no other marker significantly improved the model fit when forced in after HIV-1 RNA. A plasma HIV-1 RNA level greater than 1 × 10⁵ Eq/mL at the seroconversion visit was associated with an odds ratio for AIDS development of 10.8 (P = 0.01).

Discussion

Our study is the largest to date to compare the predictive value of plasma HIV-1 RNA with that of other surrogate markers in a well-characterized cohort of patients with HIV-1 seroconversion. Of the markers evaluated, plasma HIV-1 RNA was the earliest and most powerful predictor of outcome after seroconversion. The persistence of detectable HIV-1 RNA in plasma (>1 × 10⁴ Eq/mL) after seroconversion led to the identification of groups at high risk for AIDS or a significant decline in the CD4+ T-cell count. The risk associated with persistent viremia was independent of the CD4+ T-cell count at the time of seroconversion. An initial plasma HIV-1 RNA level of greater than 1 × 10⁵ Eq/mL at the seroconversion visit was associated with a more than 10-fold increase in the risk for AIDS development. In multivariate modeling, the CD4+ T-cell count, ICD p24 antigen, and β₂-microglobulin or neopterin levels at seroconversion did not add predictive power beyond that of plasma HIV-1 RNA.

These findings provide strong additional evidence that the course of HIV-1 infection parallels that of HIV-1 viremia. Failure of host immune mechanisms to adequately suppress viremia during the earliest stages of infection appears to be a critical factor in rapid progression to AIDS. Clearance of HIV-1 RNA to levels less than 1 × 10⁴ Eq/mL after seroconversion indicates a better prognosis because this was observed only in persons who did not develop AIDS during follow-up.

These finding are in agreement with those in the recent report by Jurriaans and colleagues [21] that serum HIV-1 RNA levels 3 years after seroconversion, measured by a quantitative nucleic acid sequence-based amplification technique, were significantly lower in persons who did not progress to AIDS within 5 years than in those who did. However, Jurriaans and colleagues [21] did not observe a significant difference in serum HIV-1 RNA levels at seroconversion between progressors and nonprogressors. This discordance with our observation may be related to differences between the two studies in the interval after seroconversion at which initial samples were obtained, as well as differences in the type of sample tested (serum compared with plasma), the method of HIV-1 RNA quantitation (quantitative nucleic acid sequence-based amplification technique compared with bDNA), and the patient cohort studied.

Serum ICD p24 antigenemia was also associated with AIDS development, but only in univariate analyses. Immune complex dissociated p24 antigenemia was a less sensitive marker than plasma HIV-1 RNA; only 36% of samples from persons developing AIDS had detectable serum ICD p24 antigen within the first year of infection compared with 73% for HIV-1 RNA. In addition, ICD p24 antigen could not discriminate persons at risk for a significant decline in the CD4+ T-cell count from those in whom CD4+ T-cell counts remained stable.

Serum β₂-microglobulin and neopterin levels were significantly elevated in the AIDS group within the first year of seroconversion. This is consistent with findings in previous reports that β₂-microglobulin and neopterin are associated with AIDS development [9]. The differences in median values between the outcome groups were small, however, and did not increase with time. Most important, neither marker could discriminate the CD4-decline group from the stable-CD4 group. Median β₂-microglobulin and neopterin levels were actually higher in the stable CD4 group than in the CD4-decline group.

In the initial pilot study of seroprevalent men, we compared the bDNA assay with a semi-quantitative PCR assay of cellular HIV-1 gag RNA. Both assays gave similar findings (Table 1). When plasma HIV-1 RNA levels were below the detection limit of the bDNA assay, fewer than 100 copies of HIV-1 gag RNA per million CD4+ T cells were detected by PCR (mean, 19.9 copies/10⁶ CD4+ T cells). Similarly, when plasma HIV-1 RNA was detectable by bDNA, much higher levels of cellular HIV-1 RNA were detected by PCR (mean, 2813 copies/10⁶ CD4 cells). Increases in HIV-1 RNA occurring before clinical disease progression were also reflected in both assays. Thus, in this study, the plasma level of HIV-1 RNA correlated with intracellular expression of HIV-1 gag RNA in circulating lymphocytes (Spearman r = 0.66; P < 0.001).

Although PCR-based detection methods have greater sensitivity than bDNA technology for detection of HIV-1 RNA, the bDNA assay can be done in standard clinical laboratories without the need for special facilities or procedures to avoid carry-over contamination of amplification reactions with PCR product. In addition, the assay is reproducible (interassay coefficient of variation, 11.2%) and rapid; 42 samples can be tested in duplicate in only 36 hours. These characteristics make the bDNA assay a suitable candidate for use in clinical practice.

In summary, the quantity of HIV-1 RNA in plasma was a strong, CD4+ T-cell-independent predictor of a rapid progression to AIDS after HIV-1 seroconversion in homosexual men. Measurement of plasma HIV-1 RNA may substantially improve prognostic accuracy in HIV-1 infection, which would be advantageous for clinical management of infected persons.