Raboud, Janet M.1; Montaner, Julio S.G.1,8; Conway, Brian1; Rae, Sandra1; Reiss, Peter2; Vella, Stephano3; Cooper, David4; Lange, Joep5; Harris, Marianne1; Wainberg, Mark A.6; Robinson, Patrick7; Myers, Maureen7; Hall, David7
1Canadian HIV Trials Network and Centre for Excellence in HIV/AIDS, St Paul's Hospital/University of British Columbia, Vancouver, Canada
2Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands
3Laboratory of Virology, Istituto Superiore di Sanita, Rome, Italy
4National Centre in HIV Epidemiology and Clinical Research, St. Vincent's Hospital Medical Centre, Darlinghurst, Sydney, Australia
5Department of Internal Medicine and Human Retrovirus Laboratory, Academic Medical Centre, Amsterdam, The Netherlands
6McGill University AIDS Centre, Jewish General Hospital, Montreal, Canada
7Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, Connecticut, USA.
8Requests for reprints to: J.S.G. Montaner, St. Paul's Hospital/University of British Columbia, 667–1081 Burrard St., Vancouver, Canada, V6Z 1Y6.
Note: This paper was presented at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, September, 1997 in Toronto, Ontario, Canada and at the 6th European Conference on Clinical Aspects and Treatment of HIV Infection, October 1997, Hamburg, Germany.
Sponsorship: this project was funded partially by the Canadian HIV Trials Network and Boehringer Ingelheim Pharmaceuticals Inc.
Date of receipt: 7 April 1998; revised: 27 May 1998; accepted 3 June 1998
Serial plasma viral load monitoring has been accepted as the principal means of evaluating antiretroviral drug efficacy. In fact, the very goal of therapy has been redefined as the achievement of maximal reduction of plasma viral load for as long as possible [1,2]. As more sensitive assays have become available, it has become possible to quantitate plasma HIV to lower levels than previously possible. However, it is not known what degree of virologic suppression is necessary to ensure a long-term response, or whether certain baseline characteristics could help predict the likelihood of achieving long-term suppression of viral replication.
In a controlled randomized trial of combinations of zidovudine (ZDV), didanosine (ddl) and nevirapine (NVP), we have shown that the combination of all three drugs reduced plasma viral load (pVL) to a greater extent than either of the combinations of ZDV and ddl or of ZDV and NVP, using an assay able to quantitate plasma HIV RNA to 20 copies/ml . Using data from this trial, predictors of achieving and maintaining high level pVL suppression have been have examined.
Participants and treatment regimens
The INCAS trial, a randomized double-blind trial of ZDV/NVP, ZDV/ddl and ZDV/ddl/NVP, was a multinational study conducted in Italy, the Netherlands, Canada, and Australia. At enrolment, patients had CD4+ cell counts of between 200 and 600 × 106 cells/l, were naive to antiretroviral therapy and had not been previously diagnosed with AIDS . CD4+ counts and pVL were measured at a screening visit within 1 month of starting therapy, at baseline, at weeks 1, 2, 4, and every 4 weeks until all participants had completed 52 weeks of follow-up. Study medications were prescribed orally as currently recommended (NVP, 200 mg once a day for 2 weeks and 200 mg twice a day thereafter; ZDV, 200 mg three times a day and ddl mint flavoured tablets, 125 or 200 mg according to body weight twice a day). The primary endpoint of the study was the effect of study treatments on plasma viral RNA load and CD4+ cell count over time. Non-compliance was defined as failure to take one or more of the active medications for more than 28 days within the first year of follow-up, as assessed by review of dose adjustment records and verified by pill counts.
Plasma viral RNA load
Plasma viral RNA load was measured at the BC Centre for Excellence in HIV/AIDS, St. Paul's Hospital, University of British Columbia (Vancouver, British Columbia, Canada). Assays were performed using the Amplicor HIV Monitor Assay (Roche Diagnostics, Mississauga, Ontario, Canada) . Samples with pVL less than 500 copies/ml were retested using the prototype Ultra Direct Assay with a limit of quantitation of 20 copies/ml (Roche Molecular Systems, Alameda, California, USA) . All plasma samples were separated within 2 h and stored at −70°C in 0.5 ml aliquots. Plasma viral RNA load results were transformed to log10 values. The plasma viral load nadir was defined to be the lowest plasma viral load level recorded during the study period.
A virologic failure was defined to be a rise in pVL above 500 copies/ml subsequent to having reached the nadir. The duration of virologic response was defined as the time between the visit at which an individual's pVL fell below 500 copies/ml and the visit at which the pVL rose above 500 copies/ml . If the pVL fell below 500 copies/ml during two or more periods, the duration of response was defined as the longer time period. Patients whose final pVL was below 500 copies/ml were censored at their last study visit. To be included in analyses of time to pVL above 500 copies/ml, participants had to have a pVL at baseline above 500 copies/ml and a pVL nadir less than 500 copies/ml. The relationships between the duration of response and baseline pVL, baseline CD4+ cell count, pVL nadir and time to reach pVL nadir were examined with scatterplots and quantified with Spearman's correlation coefficient. The relative risks of virologic failure associated with treatment assignment, compliance, baseline pVL, pVL nadir, and baseline CD4+ cell count were determined with proportional hazards models. The relationships of these variables and the time that pVL remained below 5000 copies/ml were also examined. To be included in analyses of the time to pVL above 5000 copies/ml, participants had to have pVL above 5000 copies/ml at baseline and a pVL nadir less than 5000 copies/ml.
The baseline characteristics of participants whose pVL nadir was less than or equal to 20 copies/ml were compared with those of participants whose nadir was above 20 copies/ml using the two sample t test for continuous variables, the Wilcoxon rank sum test for ordinal and skewed data and the χ2 test for categorical data. Logistic regression models were used to determine predictors of achieving a nadir below 20 copies/ml.
Among participants whose pVL nadir was 20 copies/ml or less, proportional hazards models were used to determine relative risks of the pVL rising above 20 copies/ml associated with treatment assignment, compliance, baseline pVL, baseline CD4+ cell count and maximum CD4+ cell count during the study period.
One hundred and fifty-one participants were enrolled into the three arms of the trial: ZDV/NVP (n = 47), ZDV/ddl (n = 53) and ZDV/ddl/NVP (n = 51). One patient on the ZDV/NVP arm was excluded because he did not have any pVL measurements. The median pVL at baseline was 4.5 log10 copies/ml (range, 2.5–5.9 log10 copies/ml) and the CD4+ cell count at baseline was 370 × 106 cells/l (range, 145–755 × 106 cells/l). The median duration of follow-up was 54 weeks (range, 0–84 weeks) and the median number of pVL measurements per participant was 17 (range, 3–20). Previously, it has been demonstrated that ZDV/ddl/ NVP drug therapy reduced plasma viral load to a statistically greater extent than either of the two-drug regimens. At 8 weeks of follow-up, plasma viral load decreased by 0.90 log10, 1.55 log10 and 2.18 log10 in the ZDV/NVP, ZDV/ddl and ZDV/ddl/NVP arms respectively. The proportions of patients with plasma viral load below 20 copies/ml at all times during the 40–52 week interval were 0%, 6% and 45% in the ZDV/NVP, ZDV/ddl and ZDV/ddl/NVP arms respectively (P < 0.001) .
One hundred and four patients from all three treatment groups had pVL > 500 copies/ml at baseline and a pVL nadir of less than 500 copies/ml. Among these patients, the median baseline pVL was 4.3 log10 copies/ml (range, 2.8–5.6), the median baseline CD4+ cell count was 379 × 106 cells/l (range, 145–755 × 106 cells/l), and the median pVL nadir was 1.3 1og10 copies/ml (range, 1.3–2.7 log10 copies/ml). The median time to pVL < 500 copies/ml and the median duration of pVL < 500 copies/ml were 14 days (range, 7–195 days) and 85 days (range, 1–577 days), respectively, Fifty-six patients had a pVL nadir ≤ 20 copies/ml. Among these patients, the median time to pVL ≤ 20 copies/ml and the median duration of pVL ≤ 20 copies/ml were 84 days (range, 7–252 days) and 137 days (range, 1–547 days), respectively.
The plasma viral load of 77 patients rose above 500 copies/ml during the trial. The relationship between the duration of response and pVL nadir are shown in Fig. 1, for all patients. It should be noted that the duration of virologic response is limited by the follow-up time in this trial. Patients whose last pVL was below 500 copies/ml were right-censored in these analyses. A more prolonged virologic response was correlated with a lower pVL nadir (Spearman's correlation coefficient = −0.73; P < 0.001). Fig. 1 demonstrates that, almost exclusively, the only subjects whose viral load stayed below 500 copies/ml for an extended period of time were those whose nadir was below the limit of quantitation of the Ultradirect assay (20 copies/ml). This relationship is most dramatic for patients randomized to the ZDV/ddl/NVP group. The duration of response was not statistically significantly correlated with the baseline CD4+ cell count (Spearman's correlation coefficient = −0.12; P = 0.23), the baseline pVL (Spearman's correlation coefficient = −0.11; P = 0.26) or with the number of days to attain a pVL below 500 copies/ml (Spearman's correlation coefficient = 0.05; P = 0.61).
Table 1 shows the results of proportional hazards models of the time taken for pVL to increase to more than 500 copies/ml. A higher baseline pVL was associated with an increased risk of virologic failure [relative risk (RR) = 1.63; P = 0.01], after adjusting for treatment assignment. A pVL nadir below 20 copies/ml was strongly associated with a decreased risk of virologic failure (RR = 0.11; P = 0.0001). Non-compliers had an increased risk of virologic failure (RR = 1.64; P = 0.06). Baseline CD4+ cell count was not predictive of virologic failure. In a multivariate model in which both the pVL nadir and the baseline pVL were included as covariates, a pVL nadir of ≤ 20 copies/ml remained highly statistically significantly associated with delaying virologic failure (RR = 0.12; P = 0.0001) and the baseline pVL was no longer predictive (RR = 1.10; P = 0.66). In a second multivariate model in which the pVL nadir, compliance and treatment assignment were included as covariates, the pVL nadir ≤ 20 copies/ml remained highly statistically significantly associated with delaying virologic failure (RR = 0.12; P = 0.0001) whereas compliance was no longer predictive (RR = 1.30; P = 0.31). Analyses that standardized the duration of suppression for the length of follow-up and time to attain pVL nadir yielded similar results.
One hundred and twenty-five patients from all three treatment groups had pVL > 5000 copies/ml at baseline and a pVL nadir below 5000 copies/ml. For these patients, the RR of pVL rising above 5000 copies/ml associated with a pVL nadir ≤ 20 copies/ml and a pVL nadir of between 21 and 400 copies/ml were RR = 0.05 (95% CI, 0.02–0.12; P = 0.0001) and RR = 0.37 (95% CI, 0.23–0.61; P = 0.0001) according to a proportional hazards model. Individuals with a pVL nadir ≤ 20 copies/ml were at a significantly lower risk of virologic failure than individuals with a pVL nadir of between 20 and 400 copies/ml (P = 0.0001). A Kaplan–Meier curve of time to pVL > 5000 as a function of pVL nadir shows this relationship graphically (Fig. 2).
Table 2 compares the baseline characteristics of participants who achieved a nadir of 20 copies/ml (n = 56) with those of participants whose nadir was greater than 20 copies/ml (n = 48). Participants with a nadir of ≤ 20 copies/ml had lower pVL at baseline (4.11 versus 4.46 log10 copies/ml; P = 0.01) but there was no significant difference in CD4+ cell counts at baseline (390 versus 358 × 106 cells/l; P = 0.28). Participants with a nadir of ≤ 20 copies/ml were more likely to have been assigned to the ZDV/ddl/NVP arm (P = 0.001) but were similar with regard to compliance (75% versus 65%; P = 0.25). The median durations of pVL suppression were 285 days and 42 days for participants whose nadir was ≤ 20 copies/ml and > 20 copies/ml respectively (P = 0.0001).
Multivariate logistic regression models were used to estimate odds ratios (OR) associated with achieving a nadir ≤ 20 copies/ml. Patients with higher pVL at baseline were less likely to have a nadir ≤ 20 copies/ml (OR, 0.19; 95% CI, 0.07–0.51; P = 0.0009) as were non-compliant patients (OR, 0.22; 95% CI, 0.07–0.70; P = 0.01). Compared with patients on the ZDV/ddl/NVP arm, patients assigned to double therapy arms were less likely to have a nadir ≤ 20 copies/ml. The odds ratios for the ZDV/NVP and ZDV/ddl treatment arms were OR, 0.03 (95% CI, 0.01–0.12); P = 0.0001 and OR, 0.26 (95% CI, 0.08–0.86); P = 0.03, respectively.
Of the 56 participants whose nadir was below 20 copies/ml, 40 had pVL above 20 copies/ml at a later visit. Proportional hazards models restricting the analysis to these 56 participants showed that treatment assignment was the strongest predictor of an increase in pVL above 20 copies/ml, with patients assigned to double therapy being at increased risk compared with patients assigned to ZDV/ddl/NVP (Table 3). Participants with higher baseline pVL were at a higher risk of an increase in pVL above 20 copies/ml (RR, 2.07; P = 0.02). The time for pVL to reach the detection limit, the baseline CD4+ cell count and compliance during the study period were not predictive of an increase in pVL above 20 copies/ml after controlling for treatment assignment. of subsequent virologic failure. Similar preliminary results have been shown when protease inhibitor-containing regimens were evaluated, indicating that this is a widespread phenomenon [10–13]. In view of this information, the management of antiviral therapy should incoporate the most sensitive tests of viral load if we are to make optimal use of the available drugs. Our data strongly support the need for a revision of the current guidelines for the management of HIV infection.
Our results clearly illustrate the importance of achieving a high level of pVL suppression in order to maximize the durability of the treatment effect. The risk of virologic failure was reduced by a factor of almost 10 for participants whose nadir was below 20 copies/ml. After controlling for treatment assignment and whether or not a participant's pVL nadir was above or below 20 copies/ml, baseline pVL, baseline CD4+ cell count and compliance were not associated with virologic failure. Our results remained consistent when failure was defined to be an increase in pVL of 0.5 log10 above the nadir or a return to the baseline pVL.
When virologic failure was defined to be an increase in pVL above 5000 copies/ml, we were able to compare rates of failure by pVL nadir ≤ 20 copies/ml, 21–400 copies/ml. Whereas attaining a pVL nadir of below 400 copies/ml reduced the risk of virologic failure, it was interesting that individuals whose nadir was ≤ 20 copies/ml were at substantially reduced risk of virologic failure compared with those with a pVL nadir between 21 and 400 copies/ml.
Among participants whose nadir was below 20 copies/ml, treatment assignment was the strongest predictor of a later rise in pVL above 20 copies/ml: patients assigned to the ZDV/ddl or ZDV/NVP arms were at an increased risk compared with patients assigned to the ZDV/ddl/NVP arm. Higher baseline pVL was associated with increased risk of virologic failure whereas the CD4+ cell count at baseline and time to nadir were not associated significantly with time to virologic failure.
Baseline CD4+ cell count and pVL may not have been shown to be predictive of pVL response in our study because of the relatively narrow range of these values due to the inclusion criteria. However, Vella et al. found that the baseline CD4+ cell count and pVL levels were not predictive of virologic failure even in a population with a wider range of these values at baseline, thus supporting our findings .
Current therapeutic guidelines recommend that combinations of regimens be used to decrease plasma viral load to below 400 copies/ml. Reductions in HIV RNA have been shown to correlate with increasing CD4+ cell counts, decreased morbidity and increased survival among HIV-infected individuals [7–9]. In this report, we have shown that reducing plasma viral load below the limit of quantitation of the most sensitive assay currently available (20 copies/ml) resulted in a reduced risk
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