Effective highly active antiretroviral therapy (HAART) suppresses viremia to levels below the limit of detection of commercial assays (<50 copies/ml) in individuals infected with human immunodeficiency virus-1 (HIV) [1,2]. However, several studies demonstrate that a significant proportion of infected individuals receiving apparently suppressive HAART still maintain residual plasma viremia (1–49 copies/ml) [3–5]. This residual viremia is also detectable in HIV-infected individuals who have received effective HAART for almost a decade [6,7]. The source of residual viremia in individuals receiving HAART is unclear, but it may originate from infected cells supporting ongoing viral replication, release of virus from stable reservoirs upon sporadic activation, cell-to-cell spread, and/or a combination of these [4,8–12]. If so, then the addition of potent new therapeutics may further reduce low-level viremia in individuals taking standard HAART and constitute a key step towards clearing HIV infection [9,13].
The emergence of new HIV antiviral agents such as raltegravir, an integrase inhibitor, presents a novel way to potentially target residual virus. When added to a standard HAART regimen, raltegravir intensification has shown promise in reducing the time to attainment of an undetectable blood viral load [14–16]. In addition, mathematical models predict that raltegravir treatment intensification may result in reduction of residual low-level viremia . Despite these theoretical benefits, recent studies have not shown any impact of raltegravir intensification on residual plasma viremia or the proviral load in blood [17–20]. However, little has been done to characterize of the impact of raltegravir intensification on HIV proviral levels in the gut mucosa, which is believed to be a potentially important reservoir of latent virus [6,21–23]. In this regard, a recent promising study by Yukl et al.  demonstrated that raltegravir intensification could reduce the amount of unspliced HIV RNA in the terminal ilium mucosa. However, this was a short-term, open-label pilot study that had enrolled participants receiving a variety of other antiretroviral agents in addition to raltegravir .
Therefore, to better ascertain the impact of raltegravir-only intensification on the viral reservoir in both the blood and gastrointestinal tissues there is a need for a long-term randomized, placebo-controlled trial (RCT). We hypothesized that prolonged raltegravir intensification in this context would be associated with a decrease in HIV proviral DNA in both blood and gut CD4+ T cells, and conducted a prospective, double-blind, RCT to assess this.
Materials and methods
All participants provided written informed consent, and the study protocol was reviewed and approved by the Research Ethics Boards at St. Michael's Hospital and the University of Toronto, Toronto, Ontario, Canada.
Participant recruitment and study design
Twenty-four participants were enrolled through the Maple Leaf Medical Clinic in Toronto, Canada, and consisted of HIV-infected men on HAART with an undetectable HIV plasma viral loads (<50 copies/ml) for at least 4 years. Enrolled participants were required to be on their first standard HAART regimen with 2–3 nucleoside reverse transcriptase inhibitors (NRTIs), and 1–2 protease inhibitors (PIs) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) for at least 4 years. Participants were excluded if they had an AIDS-defining illness in the 6-months preceding recruitment or had taken mono or dual HAART in the past. An a priori sample size calculation identified that 12 participants were required per group to detect a 0.4 log10 difference in the change from baseline to 48 weeks in plasma proviral HIV DNA levels between treatment groups with 80% power and P-value less than 0.05. Blocked randomization was carried out using variable block sizes. Randomization and concealment allocation was organized by the study statistician (J.R.). Once enrolled, each participant was required to complete a baseline questionnaire, which included demographic, clinical and laboratory characteristics.
In this double-blind RCT, enrolled participants were randomly assigned in a 1 : 1 fashion to receive either raltegravir (400 mg twice a day) or placebo for 48 weeks. At week 48 all participants were unblinded and those receiving the placebo were rolled over to the intervention arm and all patients were treated with raltegravir to week 96.
The primary objective of this study was to evaluate the change of proviral HIV-1 DNA in total CD4+ T cells from baseline to week 48 in participants randomized to the raltegravir arm (400 mg raltegravir) for 48 weeks in addition to their current standard combination antiretroviral regimen versus the control arm, who remained on their current standard combination antiretroviral regimen. As a secondary objective we also evaluated the effect of raltegravir intensification on blood CD4+ T-cell populations. At week 48, a post-hoc phase II analysis of our study was conducted in which raltegravir therapy was added to the previous placebo group and independently analysed to confirm our previous week 0–48 results in our raltegravir-only group. In addition, in participants who received raltegravir for 96 weeks (raltegravir-arm) were independently evaluated to determine if prolonged raltegravir intensification had any long-term effects on proviral HIV DNA and CD4+ T-cell populations in the blood and gut.
To evaluate clinical and laboratory parameters, blood draws were performed on all participants at approximately 4-week intervals from baseline to week 96, and sigmoid biopsies were obtained at baseline, week 48 and at week 96 for all participants. This study summarizes results for the MK-0518 (raltegravir) viral decay study registered at ClinicalTrials.gov (NCT#:NCT00520897).
Processing of peripheral blood and sigmoid biopsies
Blood was collected by venipuncture into Ethylenediaminetetraacetic acid (EDTA) tubes (BD Bioscience, La Jolla, California, USA) and peripheral blood mononuclear cells (PBMCs) were then isolated via Ficoll-hypaque density centrifugation as previously described . Sigmoidoscopy samples were obtained approximately 25–30 cm from the anal verge, and were incubated first in a 0.5 μg/ml and then a 1 μg/ml collagenase solution on a shaking heating block at 37°C for 30 min each. After obtaining single cell suspension, CD8+ T cells were depleted using anti-CD8 magnetic beads (StemCell Technologies, Vancouver, British Columbia, Canada).
Measurement of HIV proviral DNA
CD4+ T cells were enriched from PBMC using a column-based cell separation technique (StemCell Technologies). Realtime PCR was carried out on genomic DNA isolated from 1–2 × 106 purified resting or activated CD4+ T cells using the Puregene DNA isolation kit (Gentra, Minneapolis, Minnesota, USA) in accordance with the manufacturer's specifications. One microgram of DNA was then used as a template for PCR in an iCycler (Bio-Rad, Hercules, California, USA). The amplification reaction was carried out in triplicate using 0.5 μmol/l primers, 0.2 μmol/l fluorescent probe, 0.8 mmol/l dNTPs, 5 μmmol/l MgCl2, and 2.5 U Platinum Taq Polymerase (Invitrogen) in 50 μl total volume. The following primers were used: 5′ GGTCTCTCTGGTTAGACCAGAT-3′ (5′ primer) and 5′-CTGCTAGAGATTTTCCACACTG-3′ (3′ primer) along with the fluorescent probe 5′-6FAM-AGTAGTGTGTGCCCGTCTGTT-TAMRA-3′. PCR conditions consisted of a denaturation step at 95°C for 3 min, followed by 45 cycles of 15 s at 95°C and 1 min at 59°C. Serially diluted ACH-2DNA (40 000, 8000, 1600, 320, 64, 12.8, 2.56, and 0.56 cell equivalents per well in triplicates) was also subjected to the PCR conditions above to obtain standard curves. The detection limit of the assay was 2.56 copies of HIV DNA. After endoscopic terminal ileum biopsies, tissue samples were incubated with 0.5 mg/ml collagenase (Type II-S; Sigma Aldrich, St Louis, Missouri, USA) in Roswell Park Memorial Institute medium containing 5% fetal bovine serum, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and pen-strep at 37°C for 30 min. After frequent pipetting and vortexing, cells were washed and stored on ice, and the remaining undigested tissue was treated with 1 mg/ml collagenase for an additional 30 min. The frequency of CD3+CD4+ T cells was determined by fluorescence-activated cell sorter analysis (FACS; FACSCanto; BD Biosciences La Jolla, California, USA). A fraction of the cells were subjected to CD8 depletion (Invitrogen-Dynal, Carlsbad, California, USA) and the percentage of CD3+CD4+ T cells was determined by FACS. Approximately, 200 000 CD8-depleted cells were lysed in 10 mmol/l Tris-HCl at pH 8 that contained 100 μg/ml proteinase K (Roche Applied Science) for 1 h at 56°C, followed by heat inactivation of the enzyme. PCR specific for human β-actin DNA (Applied Biosystems, Foster City, California, USA) was carried out on the cell lysates described above to determine the exact copy number of cells per microlitre of cell lysate. Serially diluted ACH-2cell lysates were prepared to obtain standard curves. Finally, PCR specific for HIV DNA was carried out as described above and the number of copies of HIV DNA per 1 × 106 CD4+ T cells was calculated on the basis of results obtained from the FACS and PCR experiments.
Antiretroviral drug concentration measurement
To assess adherence, plasma drug concentrations were analysed in plasma samples from a randomly selected subset of participants (three in the raltegravir group at week 48, four in the raltegravir group at week 96, and three in the placebo group that had previously received placebo then later received 48 weeks of raltegravir therapy and sampled at study week 96). Plasma drug concentrations of raltegravir were determined by using a validated liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) method. A 200 μl plasma sample was spiked with 50 μl 6,7-dimethyl-2,3-di(2-pyridyl)-quinoxaline (Aldrich, Milwaukee, Wisconsin, USA) as internal standard and subjected to protein precipitation with acetonitrile (1 : 3) followed by centrifugation at 2000 × g for 5 min. The LC-MS/MS system consisted of a HP1100 LC system (Agilent Technologies, Wilmington, Delaware, USA) with a SupelcosilTM ABZ+ (15 cm × 4.6 mm, 3 μm) C18 column (Supelco, Bellefonte, Pennsylvania, USA) coupled to an API-2000 mass spectrometer (AB/MDS/Sciex, Concord, Ontario, Canada) with a turbo ion spray source. LC was performed at 40°C with a gradient elution of acetonitrile-0.1% (v/v) formic acid in water at a flow rate of 1 ml/min. Mass was quantified using electrospray multiple reaction monitoring (MRM) in positive mode and the MRM transitions were m/z 445–109 and m/z 313–246.4 for raltegravir and the internal standard, respectively. The absolute recoveries were 93–100% and raltegravir was stable for 24 h at 4°C after sample preparation and during three freeze-thaw cycles. The effective linear range was 22.5–4500 ng/ml. Interbatch precision varied between 6.1 and 12.7% (CV%) and intrabatch accuracy varied between 98.8 and 102.8%. In addition, as all enrolled study participants were receiving standard HAART, the concentrations of PIs and NNRTIs in the plasma samples were also tested in those patients in whom raltegravir levels were also assayed. These drug concentrations were determined using a validated LC-MS/MS assay as previously published .
Baseline demographic and clinical characteristics were summarized within treatment groups using median and interquartile ranges (IQR) for continuous variables, and frequencies and percentages for categorical variables, and were compared using Wilcoxon rank sum tests for continuous variables and χ 2 tests or Fisher's exact test for categorical variables. Wilcoxon signed-rank tests were used to compare HIV proviral DNA or CD4+ cell counts between visits within groups. Analysis of covariance (ANCOVA) was used to assess the effect of treatment between groups at week 48 by adjusting for baseline values.
Twenty-four HIV-infected men who had maintained undetectable HIV viral loads (<50 copies/ml) for at least 4 years were recruited into the study. Enrolled participants were randomized to receive raltegravir intensification (n = 12) or placebo (n = 12) in addition to their standard HAART regimen.
At baseline, the two study groups had comparable demographic and clinical variables including age, CD4+ and nadir T cell counts, blood and sigmoid proviral levels (Table 1). Participants in the placebo and raltegravir-intensified groups were all receiving standard HAART and had a comparable distribution of those taking NNRTI, PI or boosted PI-based therapies (Table 1). One participant in the placebo group dropped out at week 6 before the study completion due to mild self-reported adverse events (insomnia, dizziness, nausea).
Effect of HAART intensification with raltegravir on blood HIV DNA proviral load
To observe the effect of raltegravir-intensified therapy over time, blood HIV DNA proviral levels in CD4+ T cells were assayed in all enrolled participants (Fig. 1). Compared with baseline levels, a minor increase in blood DNA proviral level was observed in the raltegravir-intensified group (median + 0.06 log10 /1 × 106 CD4+ T cells; Wilcoxon signed-rank test P = 0.04) and a similar but nonsignificant trend to increased proviral levels was also observed in the placebo group (median + 0.06; P = 0.11). However, there was no difference in HIV DNA proviral levels between the two groups following 48 weeks of raltegravir intensification (ANCOVA P = 0.62).
Effect of raltegravir therapy on CD4+ T-cell counts
After 48 weeks of raltegravir intensification, there was a slight but nonsignificant decrease in CD4+ T-cell counts in the raltegravir-intensified group (median −20 cells/μl; Wilcoxon signed-rank test P = 0.26) and in the placebo group (median −10 cells/μl, P = 0.68). In addition, blood CD4+ T-cell counts did not differ at week 48 between groups (ANCOVA P = 0.25; Fig. 2).
Effect of raltegravir therapy on HIV proviral loads in the sigmoid colon
Proviral HIV DNA loads in the sigmoid colon were assayed and evaluated at baseline and week 48. The raltegravir-intensified group had a slight, and not statistically significant, decrease in sigmoid proviral levels/1 × 106 CD4+ T cells (median −0.06 log10 copies, Wilcoxon signed-rank test P = 0.08). However, despite an even greater decrease in proviral levels in the placebo group relative to the raltegravir group, this change did not attain significance in the placebo group (median −0.13 log10; P = 0.13). Nonetheless, raltegravir intensification did not decrease sigmoid proviral levels more than the placebo group (ANCOVA P = 0.74; Fig. 3).
Effect of prolonged raltegravir therapy intensification in the blood and sigmoid
It is possible that the 48-week duration of raltegravir intensification was insufficient to establish the efficacy of raltegravir intensification. To assess this point, enrolled study participants were followed for another 48 weeks out to week 96 and a post-hoc analysis of blood and sigmoid DNA proviral levels and blood CD4+ T-cell counts was then performed. Similar to week 48, there was a slight but significant increase in blood HIV DNA proviral loads in participants intensified with raltegravir for 96 weeks compared with baseline levels (median + 0.10 log10 copies/1 × 106 CD4+ T cells, Wilcoxon signed-rank test P = 0.01). This increase in blood proviral levels at week 96 was concurrent with a significant decrease in blood CD4+ T-cell counts compared with baseline (median −115 cells/μl; Wilcoxon signed-rank test P = 0.05). However, no significant changes in the gut mucosa proviral levels were apparent at week 96 compared with baseline levels in the raltegravir-intensified group (median + 0.03 log10 copies/1 × 106 CD4+ T cells; Wilcoxon signed-rank test P = 0.58). For those participants at week 48 who were switched from placebo to raltegravir-intensified therapy and followed up to week 96, a similar analysis was carried out using preintensification values at week 48 as the new baseline. After 48 weeks of raltegravir intensification, there was a slight increase in the HIV proviral DNA load (median + 0.06 log10 copies/1 × 106 CD4+ T cells, Wilcoxon signed-rank test P = 0.01), whereas no change was observed in the blood CD4+ T-cell counts within this group compared to baseline levels (median −20 CD4+ T cells/μl, Wilcoxon signed-rank test P = 0.41). In the sigmoid mucosa, proviral HIV DNA loads remained unaltered compared with week-48 baseline (+0.04 log10 copies/1 × 106 CD4+ T cells; Wilcoxon signed-rank test P = 0.48).
Plasma levels of raltegravir and other antiretrovirals
To ascertain treatment adherence of study participants, raltegravir concentrations were analysed in plasma samples collected from a randomly selected subset (∼25–33%) of participants in each study arm (see methods for breakdown). All available raltegravir levels were higher than the 95% inhibitory concentration of 33 nmol/l (≈0.0146 mg/l) , and are summarized in Table 2 . In addition, the concentrations of PIs and NNRTIs in the plasma samples of this subset of patients were consistent with good treatment adherence since all levels were in the therapeutic range (data not shown).
HIV eradication may be hindered by the inability of standard HAART to abrogate low-level residual virus replication [3,4,6,7,27]. We hypothesized that prolonged therapy intensification with raltegravir would further reduce the HIV proviral DNA load in CD4+ T cells. However, we found no evidence that 48 weeks of raltegravir intensification in the context of long-term HIV suppression on standard HAART regimens reduced blood or gut HIV CD4+ T cells carrying HIV proviral DNA, or improved blood CD4+ T-cell counts. Furthermore, unblinded continuation of raltegravir intensification for an additional 48 weeks (out to 96 weeks in total) had no impact on these parameters, despite attainment of therapeutic plasma levels of raltegravir and other antiretroviral agents.
Following HAART initiation, the kinetics of viral decay are typically defined by a significant decrease in plasma virus levels and the subsequent clearance of HIV-infected cells in three distinct phases characterized by short (1 day), intermediate (14 days) and extended (39 weeks) half-lives [1,2,27,28]. However, other studies suggest that an even longer lived stable reservoir of T cells is maintained for at least 7–10 years after the initiation of therapy characterizing a fourth phase of viral decay [6,29]. These latent reservoirs are thought to have a potential ability to replenish their levels despite effective HAART [6,29]. Thus, the absence of decreased HIV DNA in raltegravir-treated participants suggests that this may represent an already pre-established long-lived viral reservoir, which is not significantly impacted by new infectious events that may be prevented while on intensified effective HAART .
Our findings are consistent with other recent studies that have found no effect of raltegravir intensification on residual HIV RNA and proviral DNA loads in the blood and also the gut [17–20]. However, not all mucosal reservoirs of HIV appear recalcitrant to raltegravir therapy. A recent report by Yukl et al.  demonstrated that the basal cell-associated unspliced HIV RNA loads in the terminal ileum were higher than that in other gut sites, and that raltegravir intensification significantly reduced the unspliced HIV RNA burden in the ileum but not in the rest of the gut. However, in our study, we only sampled the sigmoid mucosa, and we measured proviral HIV DNA rather than cell-associated unspliced HIV RNA. Although we are unable to comment on the possible effects of raltegravir intensification on this parameter, it should be noted that Yukl et al.  also found no decrease in HIV proviral DNA in the ileum or elsewhere in the gut, and in the blood , which is consistent with our findings.
There are some potential limitations to our study. First, low-level residual viremia (<50 copies/ml) was not assessed, although other studies have reported that raltegravir intensification did not affect residual viremia [18–20,24]. Furthermore, the clinical significance of this residual viremia remains to be clearly understood. In addition, we measured the HIV DNA proviral load, which also serves as a useful estimation of the persistence of HIV in viral reservoirs . Second, our sample size was small and our findings may need to be confirmed in larger studies. Lastly, raltegravir-driven integrase mutations may occur in individuals receiving combination therapy , and this may be an important aspect to explore in future studies. Nevertheless, raltegravir-induced mutations in the integrase gene have mainly been reported in individuals with a history of treatment failure [31–33] and are rare in individuals with successful viral suppression .
In summary, we found that raltegravir intensification in long-term supressed individuals on standard HAART had no impact on HIV DNA proviral levels in the blood or the sigmoid colon. Therefore, potent additional therapeutics will need to be developed, either to prevent repletion of the residual HIV-latent reservoir or to reach the ultimate goal of HIV eradication.
We would like to thank Dr Tae-Wook Chun (NIH/NIAD) for his technical assistance in the HIV DNA proviral assays, Roberta Halpenny (CIRC research manager), all study participants and CIRC research staff at the Maple Leaf Clinic in Toronto, Ontario Canada and the Leukopherisis lab at St. Michaels Hospital, Toronto, Ontario, Canada. The Canadian Institute of Health Research (CIHR) Banting and Best Scholarship (D.C. salary), in part supported this work. We would also like to thank Merck Frosst Canada Ltd. for funding this research. Study sponsors played no role in study design, collection or analysis of data, interpretation of results, writing of the manuscript, or decision to submit for publication.
Conflicts of interest
D.C., C.K., C.L.P., M.O., R.K. M.R.L. conceived and designed the experiments. C.K., J.B., M.R.L. recruited patients. D.C., C.L.P., G.K., C.J.K., P.M.S. performed the experiments. D.C., J.R., D.S., M.R.L. analyzed the data. C.K., C.L.P., M.O., J.R., R.K., M.R.L. contributed reagents/materials/analysis tools. D.C., C.L.P., D.S., M.R.L. wrote the article.
Some of the authors listed above have acted as speakers (M.R.L.), advisors (C.K.) for Merck Frosst Canada Ltd. This project was funded by a research grant by Merck Frosst Canada Ltd. Study sponsors played no role in study design, collection or analysis of data, interpretation of results, writing of the manuscript, or decision to submit for publication.
1. Perelson AS, Essunger P, Cao Y, Vesanen M, Hurley A, Saksela K, et al. Decay characteristics of HIV-1-infected compartments during combination therapy
2. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection
3. Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia
4. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy
. Proc Natl Acad Sci U S A
5. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy
6. Chun TW, Nickle DC, Justement JS, Meyers JH, Roby G, Hallahan CW, et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy
. J Infect Dis
7. Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells
. Nature Medicine
8. Tobin NH, Learn GH, Holte SE, Wang Y, Melvin AJ, McKernan JL, et al. Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus
. J Virology
9. Coiras M, Lopez-Huertas MR, Perez-Olmeda M, Alcami J. Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs. Nature reviews
10. Ramratnam B, Ribeiro R, He T, Chung C, Simon V, Vanderhoeven J, et al. Intensification of antiretroviral therapy accelerates the decay of the HIV-1 latent reservoir and decreases, but does not eliminate, ongoing virus replication
. J Acquir Immune Defic Syndr
11. Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy
. Nat Med
12. Sigal A, Kim JT, Balazs AB, Dekel E, Mayo A, Milo R, et al. Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy
13. Chun TW, Justement JS, Moir S, Hallahan CW, Maenza J, Mullins JI, et al. Decay of the HIV reservoir in patients receiving antiretroviral therapy for extended periods: implications for eradication of virus
. J Infect Dis
14. Lennox JL, DeJesus E, Lazzarin A, Pollard RB, Madruga JV, Berger DS, et al. Safety and efficacy of raltegravir-based versus efavirenz-based combination therapy in treatment-naive patients with HIV-1 infection: a multicentre, double-blind randomised controlled trial
15. Markowitz M, Nguyen BY, Gotuzzo E, Mendo F, Ratanasuwan W, Kovacs C, et al. Rapid and durable antiretroviral effect of the HIV-1 Integrase inhibitor raltegravir as part of combination therapy in treatment-naive patients with HIV-1 infection: results of a 48-week controlled study
. J Acquir Immune Deficy Syndr
16. Murray JM, Emery S, Kelleher AD, Law M, Chen J, Hazuda DJ, et al. Antiretroviral therapy with the integrase inhibitor raltegravir alters decay kinetics of HIV, significantly reducing the second phase
17. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects
. Nat Med
18. McMahon D, Jones J, Wiegand A, Gange SJ, Kearney M, Palmer S, et al. Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy
. Clin Infect Dis
19. Gandhi RT, Zheng L, Bosch RJ, Chan ES, Margolis DM, Read S, et al. The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial
. PLoS Med
20. Hatano H, Hayes TL, Dahl V, Sinclair E, Lee TH, Hoh R, et al. A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response
. J Infect Dis
21. Reinhart TA, Rogan MJ, Huddleston D, Rausch DM, Eiden LE, Haase AT. Simian immunodeficiency virus burden in tissues and cellular compartments during clinical latency and AIDS
. J Infect Dis
22. Veazey RS, DeMaria M, Chalifoux LV, Shvetz DE, Pauley DR, Knight HL, et al. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection
23. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, McNeil A, et al. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy
. J Virol
24. Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, Hare CB, et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy
25. Chege D, Sheth PM, Kain T, Kim CJ, Kovacs C, Loutfy M, et al. Sigmoid Th17 populations, the HIV latent reservoir, and microbial translocation in men on long-term antiretroviral therapy
26. Lorello G, la Porte C, Pilon R, Zhang G, Karnauchow T, MacPherson P. Discordance in HIV-1 viral loads and antiretroviral drug concentrations comparing semen and blood plasma
. HIV Med
27. Di Mascio M, Dornadula G, Zhang H, Sullivan J, Xu Y, Kulkosky J, et al. In a subset of subjects on highly active antiretroviral therapy, human immunodeficiency virus type 1 RNA in plasma decays from 50 to <5 copies per milliliter, with a half-life of 6 months
. J Virol
28. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, et al. Viral dynamics in human immunodeficiency virus type 1 infection
29. Palmer S, Maldarelli F, Wiegand A, Bernstein B, Hanna GJ, Brun SC, et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy
. Proc Nat Acad Sci U S A
30. Croxtall JD, Keam SJ. Raltegravir: a review of its use in the management of HIV infection in treatment-experienced patients
31. Cooper DA, Steigbigel RT, Gatell JM, Rockstroh JK, Katlama C, Yeni P, et al. Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection
. N Engl J Med
32. Canducci F, Sampaolo M, Marinozzi MC, Boeri E, Spagnuolo V, Galli A, et al. Dynamic patterns of human immunodeficiency virus type 1 integrase gene evolution in patients failing raltegravir-based salvage therapies
33. Charpentier C, Karmochkine M, Laureillard D, Tisserand P, Belec L, Weiss L, et al. Drug resistance profiles for the HIV integrase gene in patients failing raltegravir salvage therapy
. HIV Med
34. Ferns RB, Kirk S, Bennett J, Williams I, Edwards S, Pillay D. The dynamics of appearance and disappearance of HIV-1 integrase mutations during and after withdrawal of raltegravir therapy