Comparison of changes in viral load measurements
As expected, viral load levels in the SMH cohort decreased steeply, owing to SCART initiation, from seroconversion up to a period of 6 months, coinciding with the end of SCART. Thereafter, there was a steep increase followed by a stabilization at approximately 4 log10 copies/ml. Viral load levels of CASCADE controls showed a gradual decrease during the first year after seroconversion followed by approximately stable levels thereafter. At a mean of 6 months (SD, 1.5) after the end of SCART, the median viral load of 81 SMH individuals was 4.36 log10 copies/ml (IQR, 3.60–5.00). At approximately the same time from seroconversion, the median viral load level for 130 CASCADE individuals with available measurements at that period was 4.46 log10 copies/ml (IQR, 3.92–5.00). This difference was not statistically significant (Mann–Whitney U-test, P = 0.171).
Mean estimated viral load set-point levels were 4.19 log10 copies/ml (SD, 0.095) and 4.25 log10 copies/ml (SD, 0.093) for the 81 SMH and 97 CASCADE individuals, respectively. The difference (0.06 log10 copies/ml) was not statistically significant (two-sample t-test, P = 0.684). Controlling for potential confounding effect of sex, exposure category, seroconversion calendar year and age at seroconversion, the estimated difference was 0.111 log10 copies/ml but remained not significant (P = 0.415).
Average modelled viral load trends are shown in Fig. 2. Estimated mean viral load levels for the SMH and CASCADE groups, respectively, were 4.16 log10 copies/ml (95% CI, 3.99–4.33) and 4.40 log10 copies/ml (95% CI, 4.28–4.53) at 1 year from seroconversion; 4.31 log10 copies/ml (95% CI, 4.14–4.48) and 4.47 log10 copies/ml (95% CI, 4.28–4.66) at 2 years; and 4.09 log10 copies/ml (95% CI, 3.83–4.35) and 4.53 log10 copies/ml (95% CI, 4.23–4.83) at 3 years. These differences are only marginally significant. These results are in agreement with those found earlier with simpler methods and indicate that SCART may have some small effect on lowering the viral load set-point. Furthermore, the rate of change in viral load appeared to increase for the CASCADE group following the set-point being reached [mean annual rate of change, 0.064 log10 copies/ml (95% CI, −0.064 to 0.192)], but this was not significant, whereas for the treated SMH group, the viral load levels appeared to fall slowly but significantly after the initial steep increase that followed the end of SCART [mean annual rate of change, −0.246 log10 copies/ml (95% CI, −0.079 to −0.412)]. This did not result in significant differences in viral load between the two groups for at least up to 2 years after seroconversion. However, estimated viral load levels at 3 years after seroconversion differed significantly, although it should be noted that the 75th percentile of analyses follow-up time for CASCADE was 22.05 and 22.04 months for CD4 cell and HIV RNA data, respectively (Table 1). Results beyond 2 years are, therefore, largely based on extrapolated data.
Time to the initiation of clinically indicated therapy or reaching a CD4 cell count < 350 cells/μl
Clinically indicated ART was started in 26 (29.2%) SMH individuals and 51 (28.5%) CASCADE individuals. A CD4 cell count < 350 cells/μl was achieved by 51 of 89 (57.3%) SMH and 80 of 179 (44.7%) CASCADE individuals. Incidence rates for reaching this level did not appear to differ significantly between the two groups (0.308 and 0.383 events/person-year for SMH and CASCADE, respectively; P = 0.3538). However, given that the median and last CD4 cell measurements prior to ART initiation was significantly higher for the CASCADE group (330 cells/μl; IQR, 236–420) than the SMH group (225 cells/μl; IQR, 180–290) (P = 0.0014), time to CD4 cell count < 350 cells/μl with the initiation of clinically indicated ART was considered as a competing risk. In the CASCADE controls, 79 (44.1%) reached this CD4 cell count before ART initiation (or did not start ART during follow-up) and 14 (7.8%) initiated ART without reaching this CD4 cell count level. The corresponding figures for the SMH population were 51 (57.3%) and none, respectively. Hazard ratios, using competing risks methodology, could not be estimated because there were no SMH individuals initiating ART with a CD4 cell count > 350 cells/μl. The estimated hazard ratio for the combined event ‘ART initiation or CD4 < 350 cells/μl’ was 1.447 (95% CI, 1.020–2.054; P = 0.039). Figure 3 shows Kaplan–Meier curves for the cumulative probability of the combined event by group.
Average CD4 cell count trajectories for the SMH population estimated by the linear and nonlinear model almost coincided after the first year following seroconversion.
Estimating viral load set-point by utilizing only measurements taken within the first 2.5 years after seroconversion, but after the first year following seroconversion, did not significantly affect these estimates or differences between the two groups, even after adjusting for confounders. Treating clinically indicated ART as an informative censoring event and analysing CD4 cell count using models with appropriate adjustments affected mainly estimates of the rate of decline of CD4 cells. The estimated CD4 cell slopes for SMH individuals and especially CASCADE individuals (where the rate of censoring was higher, owing to higher rates of ART initiation) were steeper. As changes in the estimations of slopes were towards the same direction and more pronounced in the CASCADE population, the difference between the two groups in CD4 cell rate of decline became larger and remained significant. Adjusted rate of CD4 cell decline per year on the square root scale were 1.42 (95% CI, 0.89–1.94) and 2.66 (95% CI, 2.23–3.10) for SMH and CASCADE individuals, respectively (P < 0.001).
As the last update of the SMH cohort was almost a year after the last update of the CASCADE database, censoring SMH individuals’ follow up at an arbitrary date (1 May 2004), the total follow-up time (i.e., irrespective of initiation of clinically indicated ART) for both groups became comparable. Results regarding CD4 cell evolution and viral load set-point remained almost the same as those reported in the main analysis. These results also remained virtually unchanged when the number of CD4 cell and viral load measurements in the SMH population was randomly halved, and when those identified through the detuned assay were excluded from the analysis.
This is the first report showing an apparently significant alteration in the rate of CD4 cell decline in persons treated with SCART at PHI when compared with a matched untreated population, albeit for the 3 year duration of follow up included in these analyses. Given that the comparison between the two groups was not performed within the context of a randomized clinical trial, analyses were subject to bias owing to unmeasured confounders as well as confounding by indication [19,20]. These observational data cannot, therefore, be used to infer treatment efficacy but clearly signal the need for a randomized comparison.
There are several factors that could account for the observed differences in CD4 cell decline. First, technical differences in the measurement of CD4 cell counts between the two groups could contribute to the observed differences; all SMH samples were measured in one laboratory while CASCADE included measurements from several different sites. However, all quantification of CD4 cell subsets performed during the study period were in accordance with standardized laboratory codes of practice using well-established techniques , making this explanation unlikely. Furthermore, a simulation exercise assuming a CD4 cell assay cohort effect in the CASCADE data was undertaken and found that any such assay differences did not affect differences in the two slopes. Second, the broad definition of PHI using the detuned assay as evidence of seroconversion in the SMH cohort could have influenced the observations. Only six subjects entered the SMH cohort on this criterion, and were they not true seroconverters but individuals with advanced HIV infection, their inclusion would be anticipated to increase the observed rate of CD4 cell decline in the SMH group rather than delay it. In any case, their exclusion was evaluated using sensitivity analysis and found to have virtually no effect on the main findings.
We observed an average increase of 41 cells/μl per month in the SMH group during the 3 month period on SCART, which, it may be argued, would inevitably delay the time to CD4 cell count < 350 cells/μl for that group, at least by an interval equivalent to the time spent on SCART. To acknowledge this, CD4 cell data were censored in the SMH cohort until 6 months after the end of SCART, at which time median CD4 cell counts were similar in the two groups. Importantly, we observed not only a significant difference in the overall time taken to reach CD4 cell count < 350 cells/μl between the two groups (allowing for ART initiation as a competing risk), but also that the rate of decline over the subsequent years appeared to have been delayed.
Despite the more rapid rate of CD4 cell decline observed in the CASCADE untreated population, there was no statistically significant difference in plasma viral load or viral set-point between the two groups at 2 years. The lack of effect on viral set-point concurs with previous data from other groups, who have similarly failed to demonstrate an impact on subsequent long-term viral control following ART intervention in PHI [11,22]. This finding is interesting but difficult to explain as it questions the mechanisms underlying the observed effect. The accepted paradigm of HIV pathogenesis dictates a clear relationship between plasma viraemia [23,24], CD4 cell decay and consequent disease progression. Establishment of an inaccessible latent HIV reservoir occurs early in the course of HIV infection . It is plausible, that early treatment may limit the size of the latent HIV infected reservoir in the treated patients [26–28], with consequent reduction in CD4 cell destruction but with minimal effect on plasma viral set-point. Alternatively, it may be that we failed to detect the true difference between the two groups where one exists because the number of participants in the risk set decreased over follow-up time. One other variable often cited is the correlation between the initial plasma HIV RNA levels at seroconversion and disease progression. It has been reported that plasma HIV-1 RNA level > 1 × 105 copies/ml at seroconversion is the most powerful predictor of AIDS (odds ratio, 10.8; P = 0.01) . However, there was no such difference between the baseline plasma viral loads of the two populations studied here that would account for the observed differences in CD4 cell decline.
A number of other factors may have acted as confounders for which we were unable to control in analyses. Clearance of virus is a dynamic process that involves both the adaptive and innate immune systems [4,5,29,30]; these, in turn, are influenced by genetic factors [31–33]. In particular, host chemokine genotypes  as well as HLA genotypes  are known to influence the development, and breadth of the immune responses generated . It is well established that HLA haplotype influences the rate of HIV-1 disease progression and time to AIDS [37–39], probably through immunological responses affecting the rate of HIV-1 replication . In addition, chemokine genotypes influence rates of disease progression . As neither the HLA haplotype nor chemokine receptor usage was available for the CASCADE population, we were unable to include these variables in our analyses. However, HLA data from the SMH cohort did not demonstrate an overrepresentation in White men of the HLA types known to confer enhanced outcomes nor a high prevalence of CCR5 heterozygotes (data not shown).
Despite these confounders, this is the first suggestion that even a short course of HAART implemented in PHI may have some influence on long-term clinical outcome. Given that we are unable to account for possible sources of bias, however, we cannot conclude that a 3 month short course of HAART at PHI is superior to no therapy. Our findings may at least indicate that there is a transient beneficial effect over a period longer than has hitherto been described through other studies, and they raise the question as to whether a longer course may have provided greater benefit of longer duration and an improved clinical outcome. Our research has highlighted the urgent need for a randomized clinical trial in acute and early HIV disease. This is currently underway though the SPARTAC trial, in the UK, Ireland, South Africa, Australia, Italy, Brazil and Uganda (http://www.ctu.mrc.ac.uk/studies/spartac.asp).
The authors are grateful to Dr Sarah Walker for insightful comments in analyses and interpretation of findings. We would also like to thank all study participants particularly those who took part in the SMH open study.
Sponsorhsip: The SMH study was funded by a Wellcome Trust grant. CASCADE has been funded through grants from the European Union BMH4-CT97-2550, QLK2-2000-01431, QLRT-2001-01708 and LSHP-CT-2006-018949
CASCADE Collaboration: Francois Dabis, Rodolphe Thiébaut, Geneviève Chêne, Sylvie Lawson-Ayayi (Aquitaine cohort, France); Laurence Meyer, Faroudy Boufassa (SEROCO cohort, France); Osamah Hamouda, Claudia Kücherer (German cohort, Germany); Benedetta Longo, Patrizio Pezzotti, Giovanni Rezza, Maria Dorrucci (Italian Seroconversion Study, Italy); Giota Touloumi, Angelos Hatzakis, Anastasia Karafoulidou (Greek Haemophilia cohort, Greece); Ray Brettle (Edinburgh Hospital cohort, UK); Julia Del Amo, Jorge del Romero (Madrid cohort, Spain); Liselotte van Asten, Akke van der Bij, Ronald Geskus, Maria Prins, Roel Coutinho (Amsterdam Cohort Studies among Homosexual Men and Drug Users, the Netherlands); Niels Obel, Court Pedersen, Claus Nielsen, Louise Bruun Jorgensen (Danish HIV cohort, Denmark); Ildefonso Hernández Aguado, Santiago Pérez-Hoyos (Valencia IDU cohort, Spain); Anne Eskild, Oddbjorn Brubakk, Mette Sannes (Oslo and Ulleval Hospital cohorts, Norway); Caroline Sabin, Christine Lee (Royal Free Haemophilia cohort, UK); Anne M. Johnson, Andrew N. Phillips, Abdel Babiker, Janet H. Darbyshire, Noël Gill, Kholoud Porter (UK Register of HIV Seroconverters, UK); Patrick Francioli, Heiner Bucher, Martin Rickenbach (Swiss HIV cohort, Switzerland); David Cooper, John Kaldor, Tony Kelleher (Sydney AIDS Prospective Study, Australia); David Cooper, John Kaldor, Tim Ramacciotti, Don Smith (Sydney Primary HIV Infection cohort, Australia); Roberto Muga, Jordi Tor (Badalona IDU hospital cohort, Spain); Philippe Vanhems (Lyon Primary Infection cohort, France); John Gill (South Alberta Clinic, Canada); Joan Cayla, Patricia Garcia de Olalla (Barcelona IDU cohort, Spain). Steering Committee: Valerie Beral, Roel Coutinho, Janet Darbyshire (Project Leader), Julia Del Amo, Noël Gill (Chairman), Christine Lee, Laurence Meyer, Giovanni Rezza. CASCADE Coordinating Centre: Kholoud Porter (Scientific Coordinator), Abdel Babiker, A. Sarah Walker, Janet Darbyshire, Krishnan Bhaskaran.
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