Abstract: Transient low-level viremia (TLLV) of 50–400 HIV RNA copies per milliliter is common during antiretroviral therapy, but its pathogenesis, consequences, and optimal management are unclear. Heightened immune activation is associated with detrimental outcomes, including impaired CD4+ T-cell reconstitution. Using CD38/HLA-DR expression on CD8+ T cells measured in 2 large studies, we determined associations between TLLV and immune activation levels before, during, and after TLLV. We found that TLLV does not significantly change CD8+ T-cell activation and that higher CD8+ T-cell activation during viral suppression <50 copies per milliliter is associated with a modest increase in the risk of a subsequent TLLV.
*Division of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL;
†San Francisco General Hospital, University of California, San Francisco, CA;
‡Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA;
§Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Charlestown, MA;
‖Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, MA;
¶Division of Infectious Diseases and HIV Medicine, Drexel University College of Medicine, Philadelphia, PA; and
#Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, OH.
Correspondence to: Babafemi Taiwo, MBBS, Division of Infectious Diseases, Northwestern University, 645 N Michigan Avenue, Suite 900, Chicago, IL 60611 (e-mail: email@example.com).
Supported by award number U01AI068636 and U01AI68634 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. The authors would like to thank the chairs and teams of the contributing AIDS Clinical Trials Group studies and the study volunteers for their time and effort.
Presented at the 19th Conference of Retroviruses and Opportunistic Infections, March 5–8, 2012, Washington, DC.
The authors have no conflicts of interest to disclose.
Received November 23, 2012
Accepted January 22, 2013
Transient low-level viremia (TLLV) of 50–400 copies per milliliter is common after plasma HIV RNA suppression to <50 copies per milliliter during antiretroviral therapy (ART),1–4 but its pathogenesis, consequences, and optimal management are unclear. Occurrence of TLLV is sometimes linked to lapses in adherence.5 It is uncertain whether TLLV reflects release of archived viruses from reservoirs and/or bursts of viral replication. Random HIV RNA variation around a mean value <50 copies per milliliter3 and laboratory assay artifacts6 are other potential explanations. Detection of HIV RNA below 50 copies per milliliter may also be a risk factor for TLLV because detection of HIV RNA below 50 copies per milliliter correlates with subsequent virologic rebound.7 Compared with transient viremia above 400 copies per milliliter, TLLV is less likely to increase the risk of virologic failure or antiretroviral drug resistance,1,3,4,8 although some studies suggest that TLLV may be important clinically.7,9,10
The dominant source of immune activation in untreated HIV infection is antigenic stimulation, whether by HIV, copathogens, or products of bacterial translocation from the gut.11–13 Markers of immune activation decline during ART; however, the levels remain abnormally elevated after ART-mediated viral suppression.14–16 It is unknown whether TLLV causes recrudescence of HIV antigenic stimulation and immune activation or whether elevated immune activation causes TLLV. Understanding these issues is important because heightened immune activation has been linked to detrimental outcomes such as blunted CD4+ T-cell recovery.15,17 If TLLV elevates immune activation, one would expect that an episode of TLLV would precede an increase in activation. If, alternatively, heightened immune activation triggers transient release of HIV from reservoirs and results in TLLV, then an episode of TLLV would be preceded by an increase in immune activation.
To dissect these issues, we determined within-subject changes in immune activation as measured by CD38/HLA-DR expression on CD8+ T cells before, during, and after TLLV. We also conducted between-groups analyses, comparing immune activation levels during viral suppression <50 copies per milliliter between subjects who subsequently experienced TLLV versus those who maintained HIV RNA <50 copies per milliliter. In an additional between-groups analysis, we explored whether having detectable HIV RNA below 50 copies per milliliter at a pre-TLLV time point is predictive of a subsequent TLLV.
We analyzed data from AIDS Clinical Trials Group (ACTG) study A384, a randomized trial in treatment-naive HIV-infected patients18,19 and ACTG longitudinal linked randomized trials (ALLRT), an observational cohort of HIV-infected patients who initiated ART in randomized clinical trials.20 CD38/HLA-DR expression on CD8+ T cells was measured on fresh peripheral blood mononuclear cell samples using ACTG consensus methods every 16–24 weeks in 623 subjects in ACTG A38416,17 and in 2391 subjects during 2000–2003 in ALLRT (every 16 weeks in 2000–2002 and every 48 weeks in 2002–2003). HIV RNA quantification in ACTG A384 and ALLRT was performed using the Roche Amplicor assay v1.0 or v1.5 (Roche Diagnostic Systems, Branchburg, NJ). HIV RNA from 1997 to 2012 was examined for TLLV (measured every 8 weeks after week 24 in A384 and every 16 weeks in ALLRT through 2006, then twice every 48 weeks). Samples with <50 HIV RNA copies per milliliter were qualitatively subclassified into HIV RNA undetected or detected based on optical density readings <0.2 or ≥0.2 in the Roche assay.21 Institutional review boards at participating sites approved the protocols; subjects provided written informed consent.
We defined TLLV as an isolated HIV RNA 50–400 copies per milliliter that was preceded and followed by values <50 copies per milliliter with no more than 6 months between measurements and no change in the ART regimen. We included subjects who (1) experienced TLLV after at least 6 months of HIV RNA <50 copies per milliliter and (2) had immune activation data available before, during, and after an episode of TLLV (aim 1) and at one time point of interest for aim 2 (pre-TLLV). In subjects who had more than 1 episode of TLLV, we only analyzed the first occurrence (along with immune activation data around the time of that event).
For aim 1 (longitudinal within-subject analysis), each eligible subject served as his/her own control. CD8+ T-cell activation was compared between the TLLV time point and the pre- and post-TLLV time points using repeated measures analysis (generalized estimating equations with identity link). In supplemental analyses, we compared just the pre-TLLV and TLLV time points and the TLLV and post-TLLV time points. Sensitivity analyses examined the subset of subjects whose TLLV amplitudes were 50–200 copies per milliliter versus 201–400 copies per milliliter. Variability (SD) in the change in CD8+ T-cell activation over short time frames during viral suppression was estimated to be 8.5% over 48 weeks in ACTG 384. Using SD = 8.5%, a sample size of 66 subjects was estimated to provide statistical power >80% to detect a 3.0 percentage-point within-subject increase in immune activation.
For aim 2 (cross-sectional between-groups analysis), we employed a case–control design and conditional logistic regression to compare CD8+ T-cell activation during viral suppression (HIV RNA <50 copies per milliliter) between subjects who subsequently experienced TLLV (cases) and those whose HIV RNA remained <50 copies per milliliter (controls). Case and control subjects were matched by parent study, sex, age (±5 years), duration of HIV RNA suppression (±6 months), and duration of ART (±6 months).
Finally, in an exploratory analysis, we focused on subjects with information on whether HIV RNA was qualitatively detected or undetected when HIV RNA was <50 copies per milliliter; conditional logistic regression evaluated whether detection versus nondetection of HIV RNA at the pre-TLLV time point with HIV RNA <50 copies per milliliter was predictive of a subsequent TLLV.
Population With TLLV in A384 and ALLRT
A total of 1861 of 5042 (37%) subjects experienced TLLV. For those with TLLV, there was a median of 6.0 person-years of follow-up and a median of 31 HIV RNA measurements per subject.
No Significant Within-Subject Change in CD8+ T-Cell Activation Pre-, During, and Post-TLLV
There were 64 subjects who had TLLV and CD8+ T-cell activation data available at the pre-TLLV, TLLV, and post-TLLV time points; these subjects formed the population for the within-subject longitudinal analysis. They were 88% male with a median age of 39 years pre-TLLV. The median HIV RNA during TLLV was 94 (range: 50–400) copies per milliliter. The median (25th–75th percentiles) CD4+ T-cell count before TLLV was 539 (348–759) cells per cubic millimeter. Median (25th–75th percentiles) CD8+ T-cell activation before, during, and after TLLV was 21% (10–33%), 22% (12–33%), and 20% (12–31%), respectively. The pre-TLLV time point was a median (25th–75th percentiles) of 3.7 (3.4–3.9) months before the TLLV event and the post-TLLV time point was 3.7 (3.6–4.0) months after the TLLV event. We did not detect a significant association between TLLV and CD8+ T-cell activation (95% CI: −1.4 to 1.6 percentage-points, comparing pre- and post-TLLV with the TLLV time point; P = 0.88) nor in CD8+ T-cell activation changes between the pre-TLLV and the TLLV time points (P = 0.65) or between the TLLV and the post-TLLV time points (P = 0.86). Results were similar in a sensitivity analysis restricted to 58 subjects with HIV RNA of 50–200 copies per milliliter at TLLV (95% CI: −1.8 to 1.5 percentage-points; P = 0.84).
Higher CD8+ T-Cell Activation During Viral Suppression Is Associated With Increased Risk of Subsequent TLLV
In a cross-sectional between-groups analysis to examine whether CD8+ T-cell activation was associated with a subsequent episode of TLLV, cases were 123 subjects with subsequent TLLV (89% male; median age = 42 years; median CD4 count = 547 cells/mm3; median CD8+ T-cell activation = 20%). CD8+ T-cell activation was measured a median (25th–75th) 1.9 (1.8–3.5) months before TLLV. There were 629 matched controls who maintained HIV RNA <50 copies per milliliter (88% male; median age = 40 years; median CD4 count = 490 cells/mm3; median CD8+ T-cell activation = 18%). Figure 1 shows slightly higher CD8+ T-cell activation in cases versus controls. CD8+ T-cell activation at the time of viral suppression was significantly associated with increased risk of a subsequent TLLV (odds ratio = 1.19 per 10 percentage-points higher CD8+ T-cell activation; 95% CI: 1.01 to 1.41; P = 0.034). Neither CD4 cell count at the time of viral suppression nor nadir CD4 count was associated with risk of TLLV (P = 0.20 and 0.27, respectively).
We conducted a similar case–control analysis using 118 case and 569 control subjects with information on whether HIV RNA was qualitatively detected when HIV RNA was <50 copies per milliliter. HIV RNA was detected in 10.2% of samples with <50 HIV RNA copies per milliliter. A trend toward increased risk of TLLV in the group with detected HIV RNA was found, but this did not reach significance (odds ratio = 1.79; 95% CI: 0.96 to 3.35, P = 0.07).
We investigated associations between TLLV and immune activation using CD38/HLA-DR expression on CD8+ T cells measured as part of 2 ACTG studies. In a longitudinal within-subject analysis, with each patient serving as his/her own control, we found no significant change in CD8+ T-cell activation during TLLV, consistent with results of a smaller study (N = 15) that used a broader definition of low-level viremia (50–1000 HIV RNA copies/mL).22 However, another study reported increased T-cell activation during transient viremia above 200 copies per milliliter, although no association was found when viremia was <200 copies per milliliter.23 In cross-sectional between-groups case–control analyses, higher CD8+ T-cell activation during viral suppression (HIV RNA <50 copies/mL) was associated with an increased risk of a subsequent TLLV, though this effect seems to be of limited magnitude.
The clinical ramifications of TLLV are unknown and most clinicians manage these episodes with adherence reinforcement. There have been signals, however, that TLLV may not be innocuous. Each episode lasts approximately 22 days2 and increased TLLV frequency is associated with slower decay of latently infected cells.24 Because heightened immune activation during ART may be detrimental, the absence of a significant increase in immune activation with TLLV in our study provides reassurance that TLLV is unlikely to cause clinical harm through this mechanism.
Although we found an association between higher CD8+ T-cell activation during viral suppression and increased risk of subsequent TLLV, there is no proof that higher immune activation is the cause of TLLV. Another potential explanation for the observed association is that increased CD8+ T-cell activation is related to as yet unidentified factors that drive TLLV. Alternatively, CD8+ T-cell activation and TLLV may be driven by common factors, such as incomplete adherence or larger virus reservoirs.
In an exploratory analysis, we found a trend toward an association between qualitative detection of HIV RNA below 50 copies per milliliter and subsequent TLLV, but this did not reach statistical significance, perhaps due to small sample size. The fact that only 10% of subjects with HIV RNA <50 copies per milliliter in our study had detectable HIV RNA raises questions about the sensitivity of the test; approximately two-thirds have detectable virus using single-copy assays.25 Other studies have reported a relationship between HIV detection below 50 copies per milliliter and subsequent confirmed viral rebound.7,9
Our study has a number of limitations. First, some antiretroviral drugs used in ALLRT and A384 (such as unboosted protease inhibitors) are no longer recommended due partly to inferior virologic efficacy. We addressed this by focusing on patients who attained HIV RNA <50 copies per milliliter. Second, only 1 marker of immune activation (ie, CD38/HLA-DR expression on CD8+ T cells) was evaluated, constraining on our ability to address the full breath of immune activation and inflammation associated with HIV and ART. Potential coinfections on which we lacked information (such as herpesviruses) may have confounded our within-patient analysis. Hypothetically, coinfections can trigger immune activation, increase target T-cell availability, and cause transient viremia.26 The follow-up time points were over 3 months after the TLLV episodes and it is possible for an increase in T-cell activation because of the TLLV to have occurred sooner and waned by the follow-up time point. Finally, the relatively small number of patients in the analysis may limit the generalizability of our results.
In conclusion, TLLV during suppressive ART (HIV RNA <50 copies/mL) is unlikely to worsen CD8+ T-cell activation, whereas higher CD8+ T-cell activation during viral suppression is associated with a modest increase in the risk of a subsequent TLLV. Future studies should further characterize the association between CD8+ T-cell activation milieu during viral suppression and subsequent TLLV and probe the role of potential nonimmunologic precursors of TLLV including residual viremia measured with single-copy HIV RNA assay.
1. Sungkanuparph S, Overton ET, Seyfried W, et al.. Intermittent episodes of detectable HIV viremia in patients receiving nonnucleoside reverse-transcriptase inhibitor-based or protease inhibitor-based highly active antiretroviral therapy regimens are equivalent in incidence and prognosis. Clin Infect Dis. 2005;41:1326–1332.
2. Di Mascio M, Markowitz M, Louie M, et al.. Dynamics of intermittent viremia during highly active antiretroviral therapy in patients who initiate therapy during chronic versus acute and early HIV type 1 infection. J Virol. 2004;78:10566–10573.
3. Nettles RE, Kieffer TL, Kwon P, et al.. Intermittent HIV-1 viremia (blips) and drug resistance in patients receiving HAART. JAMA. 2005;293:817–829.
4. Grennan JT, Loutfy MR, Su D, et al.. The magnitude of virologic blips is associated with a higher risk for virologic rebound in HIV-infected individuals: a recurrent events analysis. J Infect Dis. 2012;205:1230–1238.
5. Miller LG, Golin CE, Liu H, et al.. No evidence of an association between transient HIV viremia (blips) and lower adherence to the antiretroviral medication regimen. J Infect Dis. 2004;189:1487–1496.
6. Stosor V, Palella FJ, Berzins B, et al.. Transient viremia in HIV infected patients and use of plasma preparation tubes. Clin Infect Dis. 2005;41:1671–1674.
7. Doyle T, Smith C, Vitiello P, et al.. Plasma HIV-1 RNA detection below 50 copies/mL and risk of virologic rebound in patients receiving highly active antiretroviral therapy. Clin Infect Dis. 2012;54:729–737.
8. Easterbrook PJ, Ives N, Waters A, et al.. The natural history and clinical significance of intermittent viraemia in patients with initial viral suppression to < 400 copies/ml. AIDS. 2002;16:1521–1527.
9. Maggiolo F, Callegaro A, Cologni G, et al.. Ultrasensitive assessment of residual low-level HIV viremia in HAART-treated patients and risk of virological failure. J Acquir Immune Defic Syndr. 2012;60:473–482.
10. Li JZ, Gallien S, Do TD, et al.. Prevalence and significance of HIV-1 drug resistance mutations among patients on antiretroviral therapy with detectable low-level viremia. Antimicrob Agents Chemother. 2012;56:5998–6000.
11. Cohen Stuart JWT, Hazebergh MD, Hamman D, et al.. The dominant source of CD4+ and CD8+ T-cell activation in HIV infection is antigenic stimulation. J Acquire Immune Defic Syndr. 2000;25:203–211.
12. Plaeger SF, Collins BS, Musib R, et al.. Immune activation in the pathogenesis of treated chronic HIV disease: a workshop summary. AIDS Res Hum Retroviruses. 2012;28:469–477.
13. Sandler NG, Douek DC. Microbial translocation in HIV infection: causes, consequences and treatment opportunities. Nat Rev Microbiol. 2012;10:655–666.
14. French MA, King MS, Tschampa JM, et al.. Serum immune activation markers are persistently increased in patients with HIV infection after 6 years of antiretroviral therapy despite suppression of viral replication and reconstitution of CD4+ T-cells. J Infect Dis. 2009;200:1212–1215.
15. Hunt PW, Martin JN, Sinclair E, et al.. T-cell activation is associated with lower CD4+ T-cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis. 2003;187:1534–1543.
16. Robbins GK, Spritzler JG, Chan ES, et al.. Incomplete reconstitution of T-cell subsets on combination antiretroviral therapy in the AIDS Clinical Trials Group protocol 384. Clin Infect Dis. 2009;48:350–361.
17. Gandhi RT, Spritzler J, Chan E, et al.. Effect of baseline- and treatment-related factors on immunologic recovery after initiation of antiretroviral therapy in HIV-1–positive subjects: results from ACTG 384. J Acquir Immune Defic Syndr. 2006;42:426–434.
18. Robbins GK, De Gruttola V, Shafer RW, et al.. Comparison of sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med. 2003;349:2304–2315.
19. Shafer RW, Smeaton LM, Robbins GK, et al.. Comparisons of four-drug regimens and pairs of sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med. 2003;349:2304–2315.
20. Smurzynski M, Collier AC, Koletar SL, et al.. AIDS clinical trials group longitudinal linked randomized trials (ALLRT): rationale, design, and baseline characteristics. HIV Clin Trials. 2008;9:269–282.
21. Flynn PM, Rudy BJ, Douglas SD, et al.. Virologic and immunologic outcomes after 24 weeks in HIV type-1 infected adolescents receiving highly active antiretroviral therapy. J Infect Dis. 2004;190:271–279.
22. Karlsson AC, Younger SR, Martin JN, et al.. Immunologic and virologic evolution during periods of intermittent and persistent low-level viremia. AIDS. 2004;18:981–989.
23. Castro P, Plana M, Gonzalez R, et al.. Influence of episodes of intermittent viremia)blips) on immune responses and viral load rebound in successfully treated HIV-infected patients. AIDS Res Hum Retroviruses. 2013;29:68–76.
24. Ramratnam B, Mittler JE, Zhang L, et al.. The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy. Nat Med. 2000;6:82–85.
25. Chun T, Murray D, Justement JS, et al.. Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy. J Infect Dis. 2011;204:135–138.
26. Jones LE, Parelson AS. Transient viremia, plasma viral load and reservoir replenishment in HIV-infected patients on antiretroviral therapy. J Acquir Immune Defic Syndr. 2007;45:483–493.