In the present study, we determined that biomarkers measured in ART-naive patients immediately prior to ART initiation and 1 month after are associated with adverse outcomes (AIDS events or death) during the first year of ART. Our results suggest that D-dimer, CRP, IL-6, and hyaluronic acid could be useful in identifying ART-naive patients at higher risk of AIDS or death after ART initiation. In addition, levels of TNF-α prior to ART and levels of CRP, IFN-γ, IL-8, and IL-6 at 1 month after ART initiation could be helpful in recognizing a subgroup of patients at risk for IRIS events.
This study builds on the emerging field of biomarkers, which is now being explored in order to improve the care of HIV-infected individuals. Data from the SMART study suggested that markers of activation of the coagulation pathway (D-dimer) and IL-6 were the strongest predictors of all-cause mortality after controlling for CD4 T-cell counts and HIV viral load [14
]. Most of the events that occurred in the SMART study were noninfectious, which suggests that the chronic inflammation and activation of coagulation may be driving some of these events independent of the degree of immunodeficiency. In a separate nested case-control study of AIDS events in SMART, CRP was a stronger predictor than were IL-6 and D-dimer [16
]. IL-6 generally induces hepatic CRP production; however, IL-6 and CRP do not always correlate, because IL-6 induces CRP production only in the absence of IFN-α [25
], which may be induced during viral diseases. Yet even in the absence of clinical illness or any effective CD4 T-cell recognition, antigen-presenting cells produce IL-6 and TNF-α in response to foreign antigen. Thus, TNF-α, IL-6, and downstream CRP may serve as biomarkers for occult infections that are unmasked during immune reconstitution with ART (ie, unmasking IRIS). Our findings are thus consistent with data published from the SMART study and more recently from 2 other studies showing that markers of acute inflammation (such as soluble TNF receptor 1, sCD27, and sCD40L) appear to be important prognostic indicators in ART-naive patients upon therapy initiation [14
]. In contrast to a recent SMART case-control study that found a strong association between mortality and sCD14 [15
], an association of events with sCD14 was not observed in this study. This could be due to the fact that only a small number of deaths were evaluated here and most events were AIDS events occurring shortly after ART initiation.
Many studies have now shown that tissue fibrosis (eg, lymphatics, liver, gut, bone marrow) occurs in untreated HIV infection and may both hamper immune restoration and lead to organ dysfunction [27
]. Hyaluronic acid, a polysaccharide component of the extracellular matrix, provides cellular support for adhesion, orientation, migration, and differentiation of cells. Hyaluronic acid accumulation in plasma reflects a loss of balance between biosynthesis and degradation and thereby is a marker of tissue fibrosis that has been shown in chronic liver disease to predict liver-related mortality with and without HIV [28
]. Our data suggest that hyaluronic acid may be useful before the initiation of ART to identify patients at risk for AIDS events or death, even in the absence of chronic liver disease. It is unclear whether the high hyaluronic acid levels in chronic HIV infection represent higher production or decreased metabolism and clearance. Plasma levels of inflammatory cytokines, such as IFN-γ, TNF-α, interleukin 1, tissue growth factor (TGF)-β, and lipopolysaccharide, are increased in untreated HIV infection, and each of these is known to stimulate hyaluronic acid production [29
In our study, we also considered the subgroup of case patients identified as experiencing possible IRIS by means of retrospective record review. The pathogenesis of IRIS remains unclear, with some data pointing to the possibility of exuberant Th1 responses [31
]. Regardless of the underlying pathogen, it is thought that unmasking IRIS happens in the context of subclinical occult infections or neoplasms. It is thus plausible that patients with biomarker abnormalities could be targeted for further screening evaluation to investigate for subclinical disease. This concept is supported in other reversible immunosuppressant conditions, such as severe malnutrition, where CRP is useful for predicting the unmasking of subclinical infections during refeeding [33
]. Our data support a Th1-bias and proinflammatory component being associated with IRIS with higher levels of IFN-γ, IL-6, and downstream Th1 inflammatory chemokines, such as CXCL10, which has been reported as elevated in tuberculosis-IRIS, cryptococcal IRIS, and hepatitis B IRIS events [34
]. In addition, pre-ART TNF-α levels were higher in those who developed IRIS. This is in agreement with a recent study showing that soluble TNF receptor 1 was higher in ART-naive patients who went on to develop AIDS events soon after ART initiation [26
] and with an older study that described high serum TNFα and CRP levels in HIV-infected patients with pulmonary mycobacterial infection [37
]. With the exception of TNF-α, the distinction of IRIS predilection became more evident at 1 month of ART when many of the inflammatory indices started decreasing in patients with uneventful immune recovery while remaining elevated or increasing in patients with impending IRIS events. It also appears that the level of CRP at 1 month may be a useful clinical marker for upcoming IRIS events, since only 2 possible IRIS case patients out of the 28 had levels below the sampled population's median of 2.1 mg/L. The elevated 1-month inflammatory biomarker levels could be a consequence of an event pathogenesis already in development but still represent an early indicator preceding the clinical event. Whereas the AIDS events and deaths were prospectively identified, the retrospective evaluation of IRIS limited our ability to recognize all cases of, particularly, paradoxical IRIS events, which typically lack microbiologic confirmation. Thus, the cases identified represent severe manifestations associated with AIDS events and could only be classified as possible IRIS cases. This limitation, however, may have enhanced the specificity of our findings. Because this was a case-control study, determining specific cut points for risk was not feasible to determine the diagnostic performance (eg, sensitivity and positive predictive value) in the overall cohort; this should be explored in future studies.
In summary, our data show that pre-ART plasma levels of CRP, IL-6, D-dimer, and hyaluronic acid may be useful in risk stratification of patients who are about to initiate ART. The presence of elevated levels of these biomarkers at baseline, as well as persistently high or increasing levels after 1 month of ART, could assist in selecting patients who could benefit from closer monitoring or additional workup to unveil occult infections. In addition, these biomarkers support a central role for inflammation, coagulation pathways, and tissue fibrosis in HIV pathogenesis.