Non-AIDS Deaths In HIV Positive Patients Linked To Inflammation And Coagulation Markers...and ART interruption
21 Oct 2008 - 0:00 PDT
"Following ART interruption, biomarker increases were greater for those with higher HIV-RNA levels at 1 mo.....ART interruption resulted in increases in IL-6 and D-dimer......Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality......ongoing activation of these pathways may exist even in the presence of effective ART, consistent with data demonstrating ongoing viral replication, even in patients with HIV-RNA of less than 50 copies/ml . Considering our finding that ART is associated with lower levels of these biomarkers, more aggressive ART that might lower IL-6 and D-dimer may warrant further investigation......Increases in hsCRP, IL-6, and D-dimer from study entry to the visit preceding the death were associated with an increased risk of death. Whether the activation of tissue factors secondary to inflammation is the key event is likely but unproven in this study. Several alternative hypotheses are possible.....Specific therapies that reduce the inflammatory response to HIV and decrease hsCRP, IL-6, and D-dimer levels may warrant investigation as an approach for reducing risk of death among HIV-infected individuals"
Metabolic Complications: A CROI 2008 Summary - Written by David Alain Wohl, MD - The University of North Carolina - (04/03/08)
What does this all mean? These results provide strong support for the hypothesis that discontinuation of HIV therapy leads to inflammatory and endovascular reactions and that these in turn are associated with all-cause mortality. Further, if one was not already convinced of the hazards of treatment interruption these studies demonstrate concerning evidence that the resurgence of HIV viremia leads to the expression of evil humors. For the audience of infectious diseases and HIV specialists, the presenter of the SMART Study data, Dr. Kuller, an epidemiologist from the University of Pittsburg, framed the significance of the strength of the relationship between D-dimer and IL-6 and death by calling the findings "extraordinary" and of a much greater magnitude than that seen in HIV-uninfected cohorts. He raised the interesting question of whether, given these data, IL-6 and D-dimer levels or other markers should be monitored among individuals with HIV infection. Application of interventions that aim to reduce inflammation are already under investigation and one small proof of concept study examining pentoxifylline, a TNF-alpha antagonist, was presented at CROI and found reductions in serum VCAM-1 and CXCL10 (a interferon-gamma induced protein), as well as improvement in flow mediated vasodiliation - a marker of endothelial function (6).
Certain biomarker for inflammation and coagulation in HIV positive patients have been associated with increased risk of death from non-AIDS diseases, such as cardiovascular disease, according to an article published in the open-access journal PLoS Medicine onOctober 20, 2008.
This study, called the Strategies for Management of Anti-Retroviral Therapy (SMART) trial, was performed under the auspices of the International Network for Strategic Initiatives in Global HIV Trials. The SMART examined approaches to treatment of HIV with Anti-Retroviral Treatment (ART): one with continuous treatment and one with intermediate treatment. In the intermediate treatment, ART was discontinued, except when CD4 immune cell count dropped to a certain range of concentration (between 250 and 350 cells per microliter). The continuous treatment is the current standard, but if intermediate treatment is possible, it may help conserve drug cost.
However, though the study initially aimed to contrast these two treatment methods for AIDS, more subjects in the intermittent treatment group died from diseases not associated with AIDS, and the study was terminated ahead of schedule. To follow-up on this study, James Neaton of the University of Minnesota and colleagues reexamined the data to investigate the possibility that this death was due to an inflammatory response caused by the higher HIV levels during periods of ART interruption.
Blood samples were analyzed from people who had died in the study (85 subjects), and for each of these patients, blood samples from two matched controls who survived were analyzed and compared (170 subjects). Of the 85 deaths, 55 had been assigned intermediate ART and 30 had been assigned continuous ART. The samples were analyzed for biomarkers specific to inflammation or coagulation of blood.
Three of these biomarkers were associated with increased risk of death across both treatment groups: high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL-6), and D-dimer. Further, throughout the entire trial base, IL-6 and D-dimer levels were higher in the intermittently treated patients in the first month of the trial, while patients receiving continuous ART treatment had unchanged levels of these substances.
The researchers conclude that this association between inflammatory biomarkers and AIDS death risk from non-AIDS related diseases is important. gThe magnitude of the association between these biomarkers and mortality is clinically relevant and reasons for it require further study,h they say. They note that, because the sub-study's size was relatively small, it is important that further research be performed before clinical recommendations are made for patients on continuous ART treatments. However, they point out, this study brings forth the possibility for development of therapies capitalizing on inflammation and coagulation pathways.
Inflammatory and Coagulation Biomarkers and Mortality in Patients with HIV Infection
Lewis H. Kuller1, Russell Tracy2, Waldo Belloso3, Stephane De Wit4, Fraser Drummond5, H. Clifford Lane6, Bruno Ledergerber7, Jens Lundgren8, Jacqueline Neuhaus9, Daniel Nixon10, Nicholas I. Paton11, James D. Neaton9*, for the INSIGHT SMART Study Group
1 University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 University of Vermont, Burlington, Vermont, United States of America, 3 Hospital Italiano de Buenos Aires, Buenos Aires, Argentina, 4 Saint-Pierre Hospital, Brussels, Belgium, 5 National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia, 6 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 7 University Hospital, Zurich, Switzerland, 8 University of Copenhagen, Copenhagen, Denmark, 9 University of Minnesota, Minneapolis, Minnesota, United States of America, 10 Virginia Commonwealth University, Richmond, Virginia, United States of America, 11 Medical Research Council Clinical Trials Unit, London, United Kingdom
In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]).
We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis.
Methods and Findings
Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case-control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0-4.1; p = 0.05), 8.3 (95% CI, 3.3-20.8; p < 0.0001), and 12.4 (95% CI, 4.2-37.0; p < 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case-control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1-3.1; p = 0.02) to 1.5 (95% CI, 0.8-2.8) and 1.4 (95% CI, 0.8-2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively.
IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation.
The Strategies for Management of Anti-Retroviral Therapy (SMART) trial compared episodic use of antiretroviral treatment (ART) guided by CD4+ count with the current practice of continuous ART. Risk of opportunistic disease (OD) or death was more than twice as great for those in the episodic compared to the continuous ART group (hazard ratio = 2.6; p < 0.001). The episodic ART strategy was also associated with an 84% (p = 0.007) increased risk of all-cause mortality. Most of the deaths that occurred were not attributable to AIDS-defining conditions .
Several studies have shown that HIV replication is an important determinant of endothelial dysfunction [2-5]. As a consequence of impaired endothelial function, HIV-infected patients may be in a hypercoagulable state . In cross-sectional studies, higher levels of IL-6, an inflammatory cytokine, have been found for HIV-infected compared to uninfected individuals [7,8]. IL-6 has also been shown to be correlated with HIV-RNA levels among patients with advanced HIV . C-reactive protein, a proinflammatory marker, increases over time for patients with HIV, and individuals who progress to AIDS have greater increases .
We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis, and that these inflammatory changes were associated with an increased risk of all-cause mortality.
Prior studies of inflammatory and coagulation markers in HIV-infected patients have not compared those using ART and not using ART in a randomized trial, thereby controlling known and unknown confounding variables. To our knowledge, this is the first planned investigation of inflammatory and coagulation markers with all-cause mortality in HIV-infected patients.
Elevated levels of either IL-6 or D-dimer at study entry were strongly related to all-cause mortality in the case-control study. In the random sample, both D-dimer and IL-6 increased in the DC group compared to the VS group, particularly in the large subgroup on ART at entry with a suppressed HIV-RNA level. Increases in both markers in the DC group were related to the level of HIV-RNA after 1 mo. Finally, increases in these markers following randomization were associated with mortality. Taken together, these findings suggest that HIV-induced activation of inflammatory and coagulation pathways has an adverse effect on all-cause mortality among patients with relatively preserved CD4+ counts, and that interrupting ART may further increase this risk by raising IL-6 and D-dimer levels. Further research on the relationship of these biomarkers with mortality and morbidity in treated and untreated HIV-infected individuals is warranted.
The associations between IL-6 and D-dimer levels at study entry with all-cause mortality were much stronger than in previous studies of non-HIV-infected populations that usually focused on CVD morbidity and mortality [13,14,16-18,20-22,31]. While these strong associations persisted in a number of different analyses, including subgroups defined by hepatitis co-infection, baseline CD4+ cell count, and prior disease history, the relatively small number of deaths considered here compared to studies in the general population suggest that the results should be interpreted with caution and require confirmation. Findings for deaths attributed to CVD or unwitnessed deaths were consistent with those for all-cause mortality, although the number of these deaths was small. The relationship of these biomarkers with CVD morbidity and mortality will be the subject of a separate report.
Patients in SMART were relatively healthy and did not have advanced HIV disease. Deaths were attributed to a variety of causes and were largely not due to AIDS, as would be expected in a cohort of patients with CD4+ counts that averaged about 600 cells/mm3 at entry (Table S1) . Associations of D-dimer and IL-6 with all-cause mortality have been reported in other studies that included non-HIV-infected participants [15,23,33].
Relationships between elevated IL-6 and D-dimer levels and all-cause mortality were strong in both DC and VS patients. However, our findings for patients on continuous ART (VS group) require validation, as mortality rates were low. However, if substantiated, and because most patients in the VS group had HIV-RNA levels of 400 copies/ml or less at study entry, ongoing activation of these pathways may exist even in the presence of effective ART, consistent with data demonstrating ongoing viral replication, even in patients with HIV-RNA of less than 50 copies/ml . Considering our finding that ART is associated with lower levels of these biomarkers, more aggressive ART that might lower IL-6 and D-dimer may warrant further investigation.
Increases in hsCRP, IL-6, and D-dimer from study entry to the visit preceding the death were associated with an increased risk of death. Whether the activation of tissue factors secondary to inflammation is the key event is likely but unproven in this study. Several alternative hypotheses are possible. One study showed that tissue factor, an initiator of coagulation, is generated by HIV-related proteins and could have pathologic effects . Another study found that HIV-infected leukocytes transmigrate across the endothelium , and this dissemination of virus could result in damage to multiple organs. Alternatively, circulating lipopolysaccharide, which has been shown to be higher in HIV-infected compared to uninfected individuals , induces tissue factor transcription, which in turn decreases F1.2 and soluble fibrin, resulting in fibrin split products such as D-dimer . Lipopolysaccharide also activates monocytes to produce inflammatory cytokines, including IL-6 . It is also possible that elevations of inflammatory markers, such as hsCRP and IL-6, and of D-dimer are independent events. In analyses that considered the joint influence of IL-6 and D-dimer, each remained strongly associated with all-cause mortality. Specific therapies that reduce the inflammatory response to HIV and decrease hsCRP, IL-6, and D-dimer levels may warrant investigation as an approach for reducing risk of death among HIV-infected individuals [40-42].
ART interruption resulted in increases in IL-6 and D-dimer, therefore the effect of these treatment differences on the DC/VS OR for mortality was explored. Adjustment for follow-up (latest) levels of IL-6 and D-dimer resulted in a modest reduction of the DC/VS OR for mortality, supporting the hypothesis that the excess risk of death in the ART interruption group may be explained by biological mechanisms for which IL-6 and D-dimer are markers.
There are some limitations in our work. Confidence intervals for ORs were wide due to the small number of deaths. While we selected six biomarkers from a large number of possible markers based on previous work in non-HIV-infected populations, some associations may have resulted from chance. This is less likely for the associations of IL-6 and D-dimer levels at study entry with all-cause mortality, which were highly significant. Cost considerations limited the number of controls for each death for which we could assess biomarkers. This reduced the statistical power to detect associations with mortality. The limited matching carried out, particularly for the expanded case-control study with four controls, may have resulted in incomplete control of confounding factors. However, regression adjustment for a number of covariates did not alter our overall conclusions. We measured latest levels at the visit immediately prior to the event of interest. For biomarker levels proximal to death, it is possible that reverse causality (i.e., an already present disease process caused the increase in the biomarker instead of vice versa) may explain these findings. In addition, follow-up specimens were not available for 11 deaths and 29 controls, and this limited our ability to assess the prognostic importance of biomarker changes on mortality. Finally, while we did not find evidence for different associations with mortality for DC compared to VS patients, the number of events experienced by VS patients was small, limiting the power for those comparisons. Similarly, power for other subgroup analyses considered was also low.
In summary, elevated levels of D-dimer and IL-6 identify HIV-infected patients at high risk of death. The magnitude of the association is clinically relevant and reasons for it require further study.
Case-Control Sample: Baseline Biomarkers and All-Cause Mortality
Most of the deaths (79 of 85) occurred in the US. Sites in the US began enrollment 2 to 3 y before most sites in other countries and accounted for 55% of the randomized participants. In univariate analyses, cases and controls differed with respect to age (p = 0.007), baseline CD4+ count (p = 0.03), co-infection with hepatitis B or C (p = 0.0008), smoking status (p = 0.0001), diabetes (p = 0.03), use of BP-lowering treatment (p = 0.02), and prior CVD (p = 0.04) (Table 1). In multivariate analyses that included the baseline covariates described in Methods for adjusted analyses, age (p = 0.02), smoking (p = 0.01), prior CVD (p = 0.04), co-infection with hepatitis B or C (p = 0.03), and baseline CD4+ cell count (p = 0.10) were associated (p = 0.10 or lower) with mortality.
With the exception of amyloid P, levels of the inflammatory markers were higher in deaths than matched controls (Table 2, gstudy entryh). Differences between deaths and controls for IL-6 and D-dimer were highly significant (p < 0.0001); hsCRP was also significantly higher among deaths than controls (p = 0.005).
Figures S2-S7 display biomarker levels for each triad (death and two matched controls) ordered by the biomarker level for the death. For IL-6 (Figure S5) and D-dimer (Figure S6), and to a lesser extent hsCRP (Figure S2), there is a shift in the distribution for deaths (red circles) compared to controls (blue circles). For other biomarkers, control levels are lower than deaths with high levels and higher than deaths with low levels (in part, a reflection of the regression to the mean phenomenon).
For both IL-6 and D-dimer, the discordance of cases and matched controls in the lower quartile (<25th percentile) and upper quartile (>75th percentile) was striking. For D-dimer, there were 23 controls with levels in the lowest quartile (<0.18 νg/ml) that were matched with deaths that had levels in the upper quartile. In contrast, there were only two controls with D-dimer levels in the upper quartile matched with deaths in the lowest quartile. For IL-6, there were 25 controls with levels in the lowest quartile (<1.6 pg/ml) matched with deaths that had levels in the upper quartile, and three controls with IL-6 in the upper quartile matched with deaths in the lowest quartile.
Strong risk gradients with mortality were evident for both IL-6 and D-dimer (Table 3). For IL-6, unadjusted ORs (for each of the three upper quartiles versus lowest) were 8.3 (95% CI, 3.3-20.8), 3.2 (95% CI, 1.3-7.9), and 1.3 (95% CI, 0.5-3.6). For D-dimer corresponding ORs were 12.4 (95% CI, 4.2-37.0), 4.0 (95% CI, 1.3-12.3), and 3.2 (95% CI, 1.1-9.0). In models that considered these biomarkers as continuous variables after log10 transformation, a difference corresponding to the IQR was associated with an OR of 3.4 (95% CI, 2.2-5.4) for IL-6 and 3.9 (95% CI, 2.3-6.6) for D-dimer. Covariate adjustment tended to strengthen these associations (Table 3), and sensitivity analyses yielded consistent findings (Table S2). Similar analyses with the 1:4 matching yielded results with reduced, but still very large, ORs for IL-6 and D-dimer (Table S3). For example, unadjusted ORs (upper quartile versus lowest) were 6.1 (95% CI, 2.7-13.6) and 6.6 (95% CI, 2.9-14.9) for IL-6 and D-dimer, respectively.
Significant associations between study entry levels of hsCRP and amyloid P and mortality were also evident. Risk of death increased with increasing levels of hsCRP but not as strongly as for IL-6 and D-dimer. The unadjusted OR (upper versus lower quartile of hsCRP) was 2.0 (95% CI, 1.0-4.1). Higher levels of amyloid P were associated with a lower risk of death in analyses based on the continuous biomarker level, but no apparent trend by quartile was evident. This association was only of borderline significance after covariate adjustment and may be due in part to outliers. After excluding outliers (ten deaths and seven controls), the OR associated with a one IQR higher level of amyloid P was 1.1 (95% CI, 0.7-1.7) (Table S2).
When IL-6 and D-dimer were considered together in the same model they each remained significantly associated with all-cause mortality. Unadjusted ORs for the highest versus lowest quartile were 4.7 (95% CI, 1.7-12.7; p = 0.002) for IL-6 and 6.1 (95% CI, 2.0-18.6; p = 0.001) for D-dimer. ORs for a difference corresponding to the IQR were 2.7 (95% CI, 1.6-4.4; p < 0.0001) for IL-6 and 2.6 (95% CI, 1.5-4.6; p = 0.001) for D-dimer.
In the analyses described above, the DC and VS groups were combined. In separate analyses for each group (Table 4), associations between biomarker levels at baseline and mortality were similar (Table 3, note last two columns for comparison to models that considered both treatment groups combined). For DC participants, median levels (expressed as deaths, controls) were 4.49, 1.78 νg/ml for hsCRP; 3.85, 2.24 pg/ml for IL-6; and 0.63, 0.22 νg/ml for D-dimer. For VS participants, these median levels were 3.60, 3.07 νg/ml for hsCRP; 3.78, 2.43 pg/ml for IL-6; and 0.37, 0.29 νg/ml for D-dimer.
Likewise, associations were similar for those with prior cancer, CVD, renal or liver disease (31% of deaths and 16% of controls), and those with no history of these conditions (p = 0.33 for hsCRP, p = 0.88 for IL-6, and p = 0.74 for D-dimer interactions); for those co-infected with hepatitis or not (p = 0.37 for hsCRP, p = 0.41 for IL-6, and p = 0.76 for D-dimer interactions); and for those with a baseline CD4+ cell less than 600 cells/mm3 (approximate median) and 600 cells/mm3 or more(p = 0.85 for hsCRP, p = 0.21 for IL-6, and p = 0.41 for D-dimer interactions).
Associations with hsCRP, Il-6, and D-dimer were also similar for deaths in the first year (38 deaths) and after the first year (47 deaths). During the first year, the OR corresponding to the IQR for hsCRP, IL-6, and D-dimer were 1.5 (95% CI, 0.9-2.4; p = 0.16), 2.5 (95% CI, 1.4-4.3; p = 0.002), and 3.9 (95% CI, 1.9-8.0; p = 0.0003), respectively. For deaths occurring after the first year, ORs for hsCRP, IL-6, and D-dimer were 1.9 (95% CI, 1.1-3.2; p = 0.01), 5.0 (95% CI, 2.4-10.4; p < 0.0001), and 3.9 (95% CI, 1.8-8.4; p = 0.0005).
Deaths due to substance abuse (eight deaths) or to accidents, violence, or suicide (seven deaths) could attenuate the associations between the biomarkers and mortality. Thus, analyses were carried out excluding these 15 deaths. Unadjusted ORs corresponding to a difference equal to the IQR were 1.9 (95% CI, 1.2-2.8) for hsCRP, 3.7 (95% CI, 2.2-6.3) for IL-6, and 4.0 (95% CI, 2.1-7.4) for D-dimer. Each of these ORs is larger by a small amount compared to the corresponding estimates in Table 3. Twenty-one deaths were classified as CVD or were unwitnessed. Unadjusted ORs of CVD or unwitnessed death corresponding to a difference equal to the IQR were 2.3 (95% CI, 1.0-5.0; p = 0.04) for hsCRP, 3.2 (95% CI, 1.2-8.4; p = 0.02) for IL-6, and 3.2 (95% CI, 1.1-9.3; p = 0.04) for D-dimer.
Case-Control Sample: Change in Biomarkers and All-Cause Mortality
Table 2 (glatest levelh) compares latest levels of each biomarker for deaths and matched controls (Table S4 gives similar results for the 1:4 matching). In these univariate analyses, significant differences were observed for all of the biomarkers except F1.2. Deaths had higher latest levels than controls, except amyloid P for which latest levels were lower for deaths than controls. Findings were strongest for hsCRP, IL-6, and D-dimer. Average differences between deaths and controls were greater for latest levels than for study entry levels (Table 2 gstudy entryh and Table S4).
Table 5 gives adjusted ORs for latest levels of each biomarker. Adjusted ORs corresponding to a difference equal to the IQR were 2.4 (95% CI, 1.4-4.2) for hsCRP, 2.0 (95% CI, 1.2-3.1) for IL-6, and 2.2 (95% CI, 1.1-4.1) for D-dimer. These models also included study entry levels of each biomarker. Study entry levels of IL-6 and D-dimer remained significantly associated with all-cause mortality after consideration of latest levels (p = 0.0008 for IL-6 and p = 0.003 for D-dimer). After considering latest level of hsCRP, the study entry level was not significant (p = 0.07). In models that also included latest levels of HIV-RNA and CD4+ cell count, neither of which were significantly associated with all-cause mortality, these associations were diminished slightly but remained significant. For latest levels of hsCRP, IL-6, and D-dimer, the ORs were 2.2 (p = 0.009), 1.7 (p = 0.04), and 2.0 (p = 0.04).
Random Sample: Associations at Baseline
As previously reported, the majority of patients in SMART were using ART at entry . In the random sample, 74% were using ART; among those using ART, 71% had an HIV-RNA level 400 copies/ml or lower. Approximately 6% of patients had not previously used ART; the remainder of those not using ART had discontinued it prior to enrolling in SMART. In the random sample, treatment groups were well balanced (Table 6).
Multiple regression analyses of each biomarker (after log10 transformation) on baseline covariates were performed. The covariates used in the regression analyses were the same as those used in the adjusted case-control analysis. An exception was history of CVD since no one in the random sample had a history of CVD (Figure 2). D-dimer was significantly higher for those not on ART than for those on ART with an HIV-RNA level 400 copies/ml or lower (0.15 on log10 scale; p = 0.0007) and for those on ART with an HIV-RNA over 400 copies/ml (0.12; p = 0.02). Other biomarkers did not vary significantly according to use of ART and HIV-RNA level at study entry.
Smoking and co-infection with hepatitis B or C, which were both significantly related to mortality, were not significantly associated with either IL-6 (p = 0.19 for smoking and p = 0.16 for co-infection) or D-dimer (p = 0.64 for smoking and p = 0.39 for co-infection) at baseline. Smoking was not significantly associated with any of the biomarkers. hsCRP was lower by 0.217 νg/ml after log10 transformation (p = 0.0001) and amyloid P was lower by 0.068 (p = 0.0005) for those who were co-infected with hepatitis; co-infection was not associated with the other biomarkers.
Significant predictors of log10 IL-6 were age (0.069 higher with each 10 y in age; p < 0.0001) and BMI (0.009 higher with each kg/m2 greater BMI; p = 0.0004). For log10 D-dimer, in addition to ART and HIV-RNA level, levels were greater among older participants (0.062 higher with each 10 y; p = 0.002), for black participants (0.123; p = 0.0009), for participants with diabetes (0.161; p = 0.01), and for those with greater BMI (0.008 per unit higher; p = 0.02). D-dimer levels were lower for men (_0.151; p = 0.0003) and for those with higher CD4+ cell counts (_0.023 per 100 cells/mm3 higher; p = 0.002). To put these changes in perspective, the IQRs on the log10 scale for IL-6 and D-dimer were 0.39 pg/ml and 0.58 νg/ml, respectively. Differences as large as the IQR for each marker were associated with 3- to 4-fold greater risks of all-cause mortality.
Random Sample: Treatment Differences at 1 mo
Table 7 shows average changes in log10 transformed biomarker levels 1 mo after randomization. IL-6 and D-dimer increased significantly (p = 0.0005 and p < 0.0001, respectively) from study entry to 1 mo in the DC group compared to the VS group (p < 0.0001). Considering the non-transformed levels, the median increase in D-dimer for DC patients was 0.05 νg/ml (a 16% increase); IL-6 increased by 0.60 pg/ml in the DC group (a 30% increase). For VS patients, the median increases were 0.0 νg/ml and 0.12 pg/ml (a 5% increase) for D-dimer and IL-6, respectively. Changes in hsCRP and amyloid A were in the same direction-greater increases for DC compared to VS patients-but did not differ significantly between treatment groups.
For both IL-6 and D-dimer, treatment differences were greater for patients who were on ART at entry and had HIV-RNA levels 400 copies/ml or below. For this subgroup (52% of patients), D-dimer increased in the DC group by 0.07 νg/ml (a 27% increase) and declined in the VS group by _0.02 νg/ml (p < 0.0001 for treatment difference). Similarly, for IL-6, the median increases for DC and VS patients were 0.98 (a 43% increase) and 0.08 pg/ml, respectively (p < 0.0001 for difference).
The changes in IL-6 and D-dimer for the subgroup of DC patients with HIV-RNA levels 400 copies/ml or below were further examined according to HIV-RNA levels at 1 mo. Following ART interruption, biomarker increases were greater for those with higher HIV-RNA levels at 1 mo (Figures 3 and 4).
Results in Table 5 were used to estimate the potential impact of treatment differences on mortality. For IL-6, the DC/VS difference after 1 mo on the log10 scale was 0.08 pg/ml. Based on the regression analysis cited in Table 5, a difference of this magnitude is associated with a 16% increased risk of death (95% CI, 10%-25%). Similarly, the 0.11 log10 higher level of D-dimer for DC compared to VS participants is associated with a 24% (95% CI, 13%-46%) increased risk of death.
Impact of Adjustment for Latest Levels of IL-6 and D-dimer on OR for DC Versus VS for All-Cause Mortality
Matched logistic models were used to assess the effect of adjusting for latest levels of IL-6 and D-dimer on the DC/VS OR for death. In the model with two controls per case, the unadjusted OR for the DC versus the VS group was 1.3 (95% CI, 0.8-2.2). Because this OR was considerably lower than the hazard ratio previously reported for all-cause mortality , we explored reasons for it and created an expanded case-control data set. A chance imbalance in the number of DC and VS participants selected as controls is the reason the OR was lower. Among the 170 controls, 99 were in the DC group and 71 were in the VS group. The expected number was 85 in each group. With the expanded case-control study (four controls for each death), the unadjusted DC/VS OR for all-cause mortality was 1.8 (95% CI, 1.1-3.1). This estimate is identical to that previously reported . With adjustment for latest level of IL-6, the OR was 1.5 (95% CI, 0.8-2.8); with adjustment for latest level of D-dimer the OR was 1.4 (95% CI, 0.8-2.5).
We also considered the effect of adjusting for both study entry and latest levels of IL-6 and D-dimer and of adjusting for HIV- RNA and CD4+ cell count on the DC/VS OR (Table S5). Similar to an earlier report, adjustment for CD4+ cell count had a greater effect on the OR (DC/VS) for mortality (OR = 1.2; 95% CI, 0.7-2.2) than adjustment for latest HIV-RNA levels (OR = 1.6; 95% CI, 0.9-2.9) . With adjustment for latest levels of IL-6, D-dimer, CD4+ cell count, and HIV-RNA, the OR (DC/VS) was 1.3 (95% CI, 0.6-2.6).