Our study is one of the first to examine the association of low level viremia with inflammatory biomarkers and mortality in a large, nationally representative cohort of HIV-infected adults which allowed for a fully developed multivariable analysis. Contrary to our hypothesis, we found little association of low level viremia with levels of CRP, IL-6 and fibrinogen. Rather, there was little association of any HIV RNA level with CRP, and only HIV RNA level ≥10,000 copies/ml had a relatively strong association with IL-6 and fibrinogen levels.
We made several other noteworthy observations. First, we found that increasing HIV RNA was progressively associated with higher fibrinogen levels, whereas those with HIV RNA level <10,000 copies/ml did not appear to have progressively higher IL-6 levels. Second, the association of increasing viremia with higher fibrinogen did not appear to be attenuated by immune status, whereas the association with IL-6 was strongly attenuated by CD4+ cell count. These findings suggest that the mechanisms by which HIV affects inflammatory and coagulation markers are different. Finally, after adjustment for CD4+ cell count, and inflammatory and coagulation markers, HIV RNA did not appear to be associated with mortality. A recent study from the ATHENA cohort found that low level (HIV RNA 50–400 copies/ml) and high level viremia (HIV RNA >400 copies/ml) were associated with 1.6-fold and 1.5-fold higher risks of death (respectively) compared with undetectable viremia, but the associations did not reach statistical significance, and the study did not adjust for inflammatory and coagulation markers 
Our finding that low level viremia had little association with CRP, fibrinogen, and IL-6 was somewhat unexpected. Data from small studies of specific populations of HIV-infected patients suggest a possible association based on other indices of inflammation 
. In one study of 23 patients on combination ART for a median of 84 months 
, residual HIV replication <50 copies/ml was observed, and in those with poor immunologic response to ART (i.e. gain of <200 CD4 cells), the residual viremia was associated with activation of CD4+ and CD8+ T-cells. In two other small studies 
, HIV controllers (i.e., those with undetectable virus using conventional assays despite the absence of ART) demonstrated abnormal immune responses. In one, cellular HIV RNA and DNA levels were associated with greater %CD38+ HLA-DR+ CD4+ and HIV gag
responsive T cells17
. In the other, low level viremia was associated with higher HIV-1- neutralizing antibody levels19
. By contrast, a recent study of 127 HIV-infected persons found that low level viremia (<50 copies/ml) had little association with markers of immune activation, and had no statistically significant association with CRP, IL-6 or D-dimer 
We observed a significant association of viremia with fibrinogen and IL-6 at levels well above the lower limit of detection for conventional HIV RNA assays. Our findings should be compared to data from the SMART trial 
. In that study, changes in IL-6 and D-dimer levels were measured in 132 previously suppressed subjects, stratified by HIV RNA category one month after discontinuing ART (<400, 401–10,000, 10,000–50,000, and ≥50,000 copies/ml). That study found significant increases in IL-6 and D-dimer with increasing HIV RNA level. However, only the increase in patients whose HIV RNA rose above 50,000 copies/ml one month after discontinuation appeared appreciably greater than those who remained suppressed (<400 copies/ml).
Our study found that the association of HIV RNA with IL-6 was strongly attenuated after adjustment for CD4+ cell count suggesting that immunosuppression is an important determinant of IL-6 levels. Those with both high viremia and low CD4+ cell count were most likely to have high IL-6 levels. In the SMART trial, IL-6 levels were a strong predictor of mortality. By contrast, the association of HIV RNA with fibrinogen showed little attenuation after adjusting for CD4+ cell count, suggesting that immunosuppression does not significantly affect fibrinogen levels. We previously found that the association of fibrinogen with increased mortality risk was also independent of CD4+ cell count 
. It is therefore of interest that we observed that greater HIV RNA levels did not predict substantially increased mortality in models that included both CD4+ cell count and fibrinogen.
Finally, the reasons for the lack of a significant association of HIV RNA with CRP are unclear. We previously reported that the median CRP in our cohort 
was well below 3 mg/L. In the general population, CRP levels above 3 mg/L are considered high risk for cardiovascular disease 
. HIV-infected participants in our cohort had higher median CRP and fibrinogen levels than controls 
. Similar to a recent study that compared HIV-infected participants from the SMART trial with control participants from the Coronary Artery Risk Development in Young Adults (CARDIA) Study 
, we found that median IL-6 levels were higher in HIV-infected than control participants (1.11 vs. 0.88 pg/ml).
There are limitations to our study. First, because of the nature of our cohort study, we were not able to assess low level viremia from a very large volume of plasma or using a single copy assay, and therefore the reported values at ranges <20 copies/ml may not be accurate. However, this new assay was strongly correlated with the COBAS® AMPLICOR HIV-1 Monitor Test (lower limit of detection: 400 copies/ml) originally used in our cohort. Furthermore, the distribution of clinical characteristics by HIV RNA category (even at very low levels of virus) was in the expected direction. Second, our study was limited by the frequency of sampling, as viremia was assessed at only two timepoints, and by the limited number of inflammation and coagulation markers studied (although the select markers studied allowed comparison with similar markers examined in prior studies). Third, IL-6 was not measured at the first FRAM exam, and so analyses of IL-6 are cross-sectional and limited to participants who enrolled in FRAM 2. Fourth, death and loss- to-follow up after the first FRAM exam may have contributed to bias in the participants enrolled in the second FRAM exam. However, we used inverse probability weighting to mitigate any potential bias from those who did not enroll in the second exam. Fifth, we did not have information for the cause of death. Finally, as with all observational studies, our findings are subject to possible unmeasured confounding.
We conclude that there is little association of low level viremia with levels of CRP, fibrinogen, and IL-6. Our finding that there was little association of any level of HIV RNA with CRP raises the possibility that CRP may not be a reliable marker of inflammation that is caused by ongoing HIV replication or persistence. However, HIV RNA levels ≥10,000 copies/ml were strongly associated with fibrinogen and IL-6. The relationship of HIV RNA with IL-6 (but not fibrinogen) was strongly affected by CD4 depletion. After adjustment for CD4+ cell count and inflammation, there was no apparent association of HIV RNA level with mortality. Our findings suggest that the mechanisms by which HIV infection induces inflammation, influences coagulation, and causes mortality are complex. Additional study of mechanistic pathways including markers of microbial translocation is needed.