Viral replication and immune activation in HIV-1 infection induce a pro-inflammatory state associated with raised levels of inflammatory and coagulation biomarkers. Two such biomarkers, interleukin-6 (IL-6) and D-dimer, have been shown to predict clinical disease outcome when measured in chronic HIV-1 infection. In the SMART study, which investigated interruption of antiretroviral therapy (ART) in individuals with chronic HIV infection, IL-6 and D-dimer levels were strongly predictive of all-cause mortality . IL-6 levels also predicted development of opportunistic infections  and were associated with an increased cancer risk . Discontinuation of ART is associated with significant rebound in IL-6 and D-dimer levels, providing an explanation for adverse outcomes seen in those interrupting treatment in chronic infection . IL-6 and D-dimer are associated with an increased risk of cardiovascular disease and other non-AIDS comorbidities in HIV-infected adults, even in those receiving effective ART [4,5]. In advanced HIV disease, elevated pretreatment levels of IL-6 and D-dimer have been shown to be predictive of early mortality and AIDS events after commencing ART [6,7].
Many studies have focused on the measurement of IL-6 and D-dimer in chronic infection and the relationship between these markers and ART [1,8,9]. Little is known, however, about their relationship with other routinely measured disease markers or their predictive value in primary HIV-1 infection (PHI), a time of intense viral replication associated with high levels of immune activation and inflammation. We describe IL-6 and D-dimer levels up to 6 months from HIV seroconversion in individuals enrolled in the SPARTAC (Short Pulse Anti-Retroviral Therapy At HIV Seroconversion) trial  and examine whether IL-6 and D-dimer levels at that time are predictive of HIV disease progression.
Materials and methods
SPARTAC is an international multicentre randomized controlled trial that compared two intervention strategies using combination ART in PHI with standard care of no therapy. Participants (N = 366) were randomized within 6 months of seroconversion to no therapy (standard of care arm, SOC), 12-week (ART12) or 48-week ART (ART48). The primary endpoint was time to CD4+ cell count less than 350 cells/μl or initiation of long-term ART .
Plasma IL-6 and D-dimer were analysed from baseline samples using Quantikine HS IL-6 immunoassay (R&D Systems, Minneapolis, Minnesota, USA) and Innovance D-dimer (Siemens Healthcare Diagnostics, Tarrytown, New York, USA), in a subset of participants from sites in Australia, Brazil, UK and Italy (N = 229). We evaluated age, sex/HIV risk group (sex between men, male: sex with women, and female: sex with men), time since estimated HIV seroconversion, baseline HIV-RNA, CD4+ cell count and BMI as possible predictors of baseline levels of IL-6 and D-dimer using multivariable linear regression, adjusting for country of enrolment. Log10 transformations were used to approximate normal distribution for both markers. For participants who did not start ART (72 SOC participants and one individual randomized to ART48 who did not start), further samples from weeks 12, 16, 48, 52, 60 and 108 were analysed to establish the natural history of IL-6 and D-dimer.
For participants who did not start ART, we further evaluated whether baseline IL-6 and D-dimer levels predicted time to reaching the trial primary endpoint in univariable and multivariable Cox proportional hazards models adjusting for the factors listed above. Fractional polynomials were used to allow for simple nonlinearity of effects. As this population was relatively small compared with the number of variables involved, we performed a number of sensitivity analyses: models including only covariates that were significant at a 0.1 level in multivariable analysis of the primary endpoint (age, CD4+ cell count and RNA at baseline), models including only one of the two immune markers at a time and models after multiple imputation of missing variables (D-dimer: n = 4; BMI: n = 2). Statistical analyses were performed using Stata (Version 12.1; Stat Corp., College Station, Texas, USA).
Of 229 SPARTAC participants, plasma samples were available for 200 individuals infected between 2003 and 2007, 191 (96%) were men, 182 (91%) had B clade infection and median (IQR) age was 34 (29–42) years. The predominant risk factor was sex between men in 179 (90%) individuals. Median time from estimated seroconversion was (IQR) 76 days (50–98). Median (IQR) CD4+ T-cell count and HIV-RNA levels were 559 (443–700) cells/μl and 4.66 (3.84–5.25) log10 copies/ ml, respectively. Median (IQR) BMI was 23.5 (21.7–25.4) kg/m2.
Median overall (IQR) baseline IL-6 and D-dimer levels were 1.45 (0.88–2.41) pg/ml and 0.34 (0.20–0.50) μg/l, respectively. D-dimer levels were missing due to assay failure (automated analyser unable to recognize sample) in nine out of 200 participants. Biomarker levels were correlated with one another: r = 0.31 for IL-6 and D-dimer (P < 0.001; log-transformed).
Predictors of interleukin-6 and D-dimer levels at HIV seroconversion
In multivariable models, levels of both markers were higher with older age, equivalent to a 16% [95% confidence interval (CI) 5–29] and 19% (95% CI 5–35) increase per 10-year increase in age for D-dimer and IL-6, respectively (Table 1). D-dimer levels were also significantly associated with baseline HIV-RNA, equivalent to a 21% (9–34) increase per 1 log10 increase in RNA. There was no evidence of an association with sex/risk group, time since estimated HIV seroconversion, baseline CD4+ cell count or BMI for either marker.
Evolution of markers
For the 73 participants who did not initiate ART, median (IQR) follow-up was 225 (191 262) weeks. IL-6 levels remained stable throughout ART-free follow-up. D-dimer levels significantly rose at 12 weeks (P = 0.002) and remained stable thereafter.
Baseline predictors of time to primary endpoint
During follow-up, 48 reached the trial primary endpoint (CD4+ cell count <350 cells/μl or long-term ART initiation). In unadjusted analyses, higher baseline IL-6 was associated with a shorter time to the primary endpoint (hazard ratio 1.41, 1.20–1.65). There was a similar trend for D-dimer (3.12, 0.95–10.29). As expected, older age, higher HIV-RNA and lower CD4+ cell count values at study inclusion were all associated with an increased risk of reaching the primary end point (Table 2).
In multivariable analysis, the association between higher baseline IL-6 and time to endpoint (hazard ratio 1.38, 1.09–1.75) remained (Table 2). In contrast, there was no evidence of an independent association with baseline D-dimer levels, with the hazard ratio falling considerably after adjusting for age, RNA and IL-6 levels (Table 2).
In all sensitivity analyses, the association between higher baseline IL-6 levels and time to primary endpoint remained, as did the lack of an independent association with baseline D-dimer levels (results not shown).
Recent studies have shown that IL-6 and D-dimer may be useful biomarkers for predicting outcome in chronic HIV-1 infection, either when measured individually or in combination [4,11]. This is the first study to examine the predictive value of these biomarkers early in the course of infection on long-term HIV disease progression, defined here as time to CD4+ cell count less than 350 cells/μl or initiation of long-term ART. We demonstrate that levels of IL-6, a pro-inflammatory cytokine, independently predict HIV disease progression, even after controlling for age, HIV RNA and CD4+ cell count levels at baseline. Whether IL-6 is directly involved in disease progression, or whether the findings are indicative of a more generalized immune activation is not known, and warrants further investigation. We found no such evidence of a relationship between levels of D-dimer, a fibrin degradation product and marker of procoagulant activity, and HIV disease progression. Both D-dimer and IL-6 have been shown to predict AIDS events in individuals with advanced HIV infection [6,7]; however in the SMART study, in which patients were relatively healthy with less advanced disease, IL-6 but not D-dimer was predictive of development of opportunistic infection . It is possible that D-dimer is a less important marker for HIV progression earlier in the disease process and that the consequences of activated coagulation and inflammatory pathways differ during early stages of HIV infection. The association between D-dimer with mortality in some studies including SMART has been postulated to reflect non-HIV associated disease including cardiovascular disease . However, we cannot completely exclude a relationship between D-dimer and HIV disease progression due to the small numbers in the longitudinal analysis.
In chronic HIV-1 infection, both IL-6 and D-dimer levels have been independently associated with mortality and non-HIV related clinical endpoints, including cardiovascular, liver and renal disease [1,4,5,12]. We could not examine the association between IL-6 and D-dimer levels in PHI and such clinical endpoints due to the low overall number of such events occurring in the SPARTAC study .
During PHI, massive viral replication is accompanied by high levels of immune activation and raised levels of circulating cytokines and other pro-inflammatory markers [13,14]. The level of T-cell activation measured in early infection is a strong independent predictor of the rate of CD4+ cell count decline , and measurement of certain cytokines in acute infection, measured at a median of 6 weeks postinfection, has been shown to predict viral load set point or CD4+ cell count loss at approximately 1 year postinfection . Stacey et al.  showed that elevations in plasma cytokines and chemokines during PHI are of much greater magnitude than those observed in acute hepatitis B or C infection, and suggested that the systemic cytokine response in PHI is not a prerequisite for viral clearance, but may instead promote ongoing immune activation, viral replication and CD4+ cell count loss. We cannot speculate from our data whether IL-6 is directly involved in HIV-1 disease pathogenesis, leading to faster disease progression, or whether it is a consequence of more generalized inflammatory process and immune activation.
High HIV-1 RNA was associated with higher levels of D-dimer at HIV-1 seroconversion even after accounting for duration of HIV-1 infection. This is consistent with SMART study data showing correlation between IL-6 and D-dimer with HIV RNA levels in chronic infection . Both IL-6 and D-dimer in PHI were associated with age, with increased age being associated with higher biomarker levels. Again, these data are consistent with observations in chronic infection . It is well established that older individuals experience faster rates of CD4+ cell count decline  and high levels of inflammation and immune activation, independent of HIV RNA levels, may additionally contribute to the more rapid disease progression experienced by older individuals.
Although it is known that those with severe clinical manifestations during acute infection experience faster progression to AIDS [18,19], for most individuals, it is difficult to predict the rate of disease progression at the time of presentation. Development of a predictive algorithm, combining indicators such as CD4+ cell count, HIV RNA, age and biomarker results, could provide a useful tool for managing PHI. The clinically relevant definition of PHI used in this study, as well as clinically relevant endpoints, indicate that measurement of IL-6 during PHI is feasible and could provide additional information to facilitate management decisions in early infection.
In summary, we have demonstrated an independent relationship between IL-6 levels, measured within 6 months of HIV-1 infection, and HIV disease progression. D-dimer levels were not independently associated with prognosis. Measurement of IL-6 during PHI may provide additional prognostic information to identify individuals at a high risk of HIV-1 disease progression.
E.H., K.P., A.B., J.W., J.M., S.F. designed the study. D.C., G.T., M.S., S.F. did the enrolment of participants. W.S. performed the statistical analyses E.H. and M.M. did the laboratory analyses. E.H., W.S. and K.P. wrote the first draft of the article. All authors contributed to subsequent drafts and approved the final version. J.W. was Principle Investigator for the SPARTAC study.
We thank all the participants and staff at all the sites participating in the SPARTAC trial. SPARTAC was funded by Wellcome Trust grants WT069598MA and 069598/Z/02/B. The Biomedical Research Centre provided staff and infrastructure support in the UK. Abbott Laboratories provided Kaletra/Aluvia (lopinavir and low-dose ritonavir) for the African sites.
SPARTAC Trial Investigators
Trial Steering Committee (TSC) Independent Members: A. Breckenridge (Chair), P. Clayden, C. Conlon, F. Conradie, J. Kaldor*, F. Maggiolo, F. Ssali Country Principal Investigators: D.A. Cooper, P. Kaleebu, G. Ramjee, M. Schechter, G. Tambussi, J.M. Miro, J. Weber Trial Physician S. Fidler Trial Statistician A. Babiker Data and Safety Monitoring Committee (DSMC) T. Peto (Chair) A. McLaren (in memoriam), V. Beral, G. Chene, J. Hakim Co-ordinating Trial Centre MRC Clinical Trials Unit at UCL, London (A. Babiker, K. Porter, M. Thomason, F. Ewings, M. Gabriel, D. Johnson, K. Thompson, A. Cursley*, K. Donegan*, E. Fossey*, P. Kelleher*, K. Lee*, B. Murphy*, D. Nock*) Central Immunology Laboratories and Repositories The Peter Medawar Building for Pathogen Research, University of Oxford, UK (R. Phillips, J. Frater, L. Ohm Laursen*, N. Robinson, P. Goulder, H. Brown) Central Virology Laboratories and Repositories Jefferiss Trust Laboratories, Imperial College, London, UK (M. McClure, D. Bonsall*, O. Erlwein*, A. Helander*, S. Kaye, M. Robinson, L. Cook*, G. Adcock*, P. Ahmed*) Clinical Endpoint Review Committee N Paton, S Fidler Investigators and Staff at Participating Sites Australia: St Vincent's Hospital, Sydney (A Kelleher), Northside Clinic, Melbourne (R Moore), East Sydney Doctors, Sydney, (R McFarlane), Prahran Market Clinic, Melbourne (N Roth), Taylor Square Private Clinic, Sydney (R Finlayson), The Centre Clinic, Melbourne (B Kiem Tee), Sexual Health Centre, Melbourne (T Read), AIDS Medical Unit, Brisbane (M Kelly), Burwood Rd Practice, Sydney (N Doong) Holdsworth House Medical Practice, Sydney (M Bloch) Aids Research Initiative, Sydney (C Workman) Coordinating centre in Australia: Kirby Institute University of New South Wales, Sydney (P Grey, DA Cooper, A Kelleher, M Law) Brazil: Projeto Praça Onze, Hospital Escola São Francisco de Assis, Universidade federal do Rio de Janeiro, Rio de Janeiro (M Schechter, P Gama, M Mercon*, M Barbosa de Souza, C Beppu Yoshida, JR Grangeiro da Silva, A Sampaio Amaral, D Fernandes de Aguiar, M de Fátima Melo, R Quaresma Garrido) Italy: Ospedale San Raffaele, Milan (G Tambussi, S Nozza, M Pogliaghi, S Chiappetta, L Della Torre, E Gasparotto,), Ospedale Lazzaro Spallanzani, Roma (G D’Offizi, C Vlassi, A Corpolongo) South Africa: Cape Town: Desmond Tutu HIV Centre, Institute of Infectious Diseases, Cape Town (R Wood, J Pitt, C Orrell, F Cilliers, R Croxford, K Middelkoop, LG Bekker, C Heiberg, J Aploon, N Killa, E Fielder, T Buhler) Johannesburg: The Wits Reproductive Health and HIV Institute, University of Witswatersrand, Hillbrow Health Precinct, Johannesburg. (H Rees, F Venter, T Palanee), Contract Laboratory Services, Johannesburg Hospital, Johannesburg (W Stevens, C Ingram, M Majam, M Papathanasopoulos) Kwazulu-Natal: HIV Prevention Unit, Medical Research Council, Durban (G Ramjee, S Gappoo, J Moodley, A Premrajh, L Zako) Uganda: MRC/Uganda Virus Research Institute, Entebbe (H Grosskurth, A Kamali, P Kaleebu, U Bahemuka, J Mugisha*, H F Njaj*) Spain: Hospital Clinic-IDIBAPS, University of Barcelona, Barcelona (JM Miro, M López-Dieguez*, C Manzardo, JA Arnaiz, T Pumarola, M Plana, M Tuset, MC Ligero, MT García, T Gallart, JM Gatell) UK and Ireland: Royal Sussex County Hospital, Brighton (M Fisher, K Hobbs, N Perry, D Pao, D Maitland, L Heald), St James's Hospital, Dublin (F Mulcahy, G Courtney, S O’Dea, D Reidy), Regional Infectious Diseases Unit, Western General Hospital and Genitourinary Dept, Royal Infirmary of Edinburgh, Edinburgh (C Leen, G Scott, L Ellis, S Morris, P Simmonds), Chelsea and Westminster Hospital, London (B Gazzard, D Hawkins, C Higgs), Homerton Hospital, London (J Anderson, S Mguni), Mortimer Market Centre, London (I Williams, N De Esteban, P Pellegrino, A Arenas-Pinto, D Cornforth*, J Turner*) North Middlesex Hospital (J Ainsworth, A Waters), Royal Free Hospital, London (M Johnson, S Kinloch, A Carroll, P Byrne, Z Cuthbertson), Barts & the London NHS Trust, London (C Orkin, J Hand, C De Souza), St Mary's Hospital, London (J. Weber, S. Fidler, E. Hamlyn, E. Thomson*, J. Fox*, K. Legg, S. Mullaney*, A. Winston, S. Wilson, P. Ambrose), Birmingham Heartlands Hospital, Birmingham (S. Taylor, G. Gilleran) Imperial College Trial & DSMC Secretariat S. Keeling, A. Becker Imperial College DSMC Secretariat C. Boocock.(*left the study team before the trial ended).
The SPARTAC trial was funded by Wellcome Trust grants WT069598MA and 069598/Z/02/B.
Conflicts of interest
There are no conflicts of interest.
This study was presented at the 19th Conference on Retroviruses and Opportunistic Infections; 2012; Seattle, Washington, USA.
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