Patients and Methods

PatientsEuroSIDA is a prospective cohort study of patients with HIV infection in 52 centers across Europe and Israel [5]. Each center provided data on consecutive outpatients until a predetermined number was enrolled. Three waves of enrollment constituted the EuroSIDA I cohort (started May 1994, 3120 patients), the EuroSIDA II cohort (started December 1995, 1367 patients), and the EuroSIDA III cohort (started April 1997, 2846 patients). Information from up to 11, 8, and 5 follow-up visits was available for cohorts I, II, and III, respectively. The present analysis included all follow-up visits to February 2000

EuroSIDA study patients had ⩽500×10⁶ CD4 cells/L in the 4 months before enrollment in the study and were ⩾16 years old at enrollment. Information was collected from patient case notes onto a standardized collection form at baseline and every 6 months. At each visit, the most recent weight and hemoglobin level and all CD4 cell count and virus load measurements since the last follow-up were requested. Dates of starting and stopping each antiretroviral drug and prophylaxis against opportunistic infections were recorded. Dates of diagnosis of AIDS-defining diseases, as defined by the Centers for Disease Control and Prevention (CDC) 1993 clinical guidelines [6], were recorded. Members of the coordinating office visited all centers to ensure correct patient selection and accurate data collection

Statistical methodsThis study included all EuroSIDA patients who started a HAART regimen of 2 nucleoside reverse-transcriptase inhibitors plus either 1 or 2 protease inhibitor(s) (PIs) or a nonnucleoside reverse-transcriptase inhibitor (NNRTI) during follow-up, who had CD4 cell counts measured in the 3 months before starting HAART and at least once after, and who had ⩾12 months of follow-up after starting HAART. Patients were split into 4 age groups according to the age quartiles at the start of HAART. Statistical comparisons between age groups and baseline data or demographic data were done by Wilcoxon or χ² tests. Changes in CD4 cell count after starting HAART were estimated by linear interpolation at 3 monthly intervals until <100 patients remained in any age group. For analysis of peak CD4 lymphocyte response, values for CD4 cell count were log transformed, and univariate and multivariate linear regression analyses were used to determine the factors related to peak CD4 cell increase

Variables included in the univariate analysis were demographic (sex, race, risk factor for HIV infection, and region of Europe), clinical and biologic (CDC disease stage, CD4 cell count at HAART initiation, and CD4 cell nadir), and therapeutic (date of start of HAART, treatment-naive status, PI- or NNRTI-based regimen, whether the regimen included a double-PI association, number of new drugs started, and time on treatment to achieve maximum response). The multivariate analysis included variables significant in the univariate analysis. An additional analysis was adjusted for plasma virus load at HAART initiation for patients for whom this information was available. We used Kaplan-Meier survival curves to assess the time to an increase in CD4 cell count of >200×10⁶ cells/L. We used Cox proportional hazards models to determine factors associated with this CD4 cell increase. The same variables as above were entered for univariate analysis. Any variable significant in univariate analysis at P<.1 was included in the multivariate models: One model was based on these variables; another was also adjusted for the most recent available value for plasma virus load. All models were stratified by center

All statistical analyses were performed on an intent-to-treat basis, and no adjustments were made for patients stopping or changing regimens. All analyses were performed with SAS software (version 6.12; SAS Institute). P<.05 was considered significant

Results

Characteristics of patients and treatment historyIn total, 1956 patients (median age, 37.2 years; interquartile range [IQR], 32.7–44.5 years) fulfilled inclusion criteria. There were no significant differences among age quartiles in terms of CD4 nadir (median, 142×10⁶ cells/L; IQR, 60–234×10⁶ cells/L; P=.18), CD4 cell count at start of HAART (median, 192×10⁶ cells/L; IQR, 88–305×10⁶ cells/L; P=.33), time from CD4 cell nadir to HAART initiation (median, 7 months; IQR, 2–16 months; P=.18), date of HAART initiation (median, March 1997; IQR, September 1996–September 1997; P=.065), length of follow-up (median, 31 months; IQR, 24–36 months; P=.19), number of CD4 cell measurements while on HAART (median, 10; IQR, 7–13; P=.41), or interval between measurements (median, 3 months; IQR, 2–4 months; P=.91). A total of 1344 (68.7%) patients had plasma virus load measured before or at the start of HAART. These measurements were not distributed differently by age group (P=.80). Baseline plasma virus load did not differ by age quartile (median, 4.28 log₁₀ RNA copies/mL; IQR, 3.48–4.94 log₁₀ RNA copies/mL; P=.53); 135 patients had initial virus loads of <2.7 log₁₀ copies/mL (no difference between age groups; P=.08). All patients had ⩾1 virus load measurement after starting HAART

Of the subjects, 261 (13.3%) were antiretroviral naive, 371 (19.0%) started a regimen of ⩾4 antiretrovirals, 123 (6.3%) started an NNRTI-based regimen, 101 (5.2%) started HAART with 2 PIs, and 779 (39.8%) started 1 antiretroviral drug they had never taken before, 573 (29.3%) started 2 new drugs, and 604 (30.9%) started ⩾3 new drugs. There were no significant differences between age quartiles in the distribution of these variables. There were also no differences in terms of individual drugs used in the 4 age groups (data not shown)

CD4 cell response to HAARTThe median increase in CD4 lymphocytes after initiation of HAART at 3 monthly intervals was estimated and stratified by age group. At each time point from 3 to 36 months, younger age groups had significantly greater median CD4 cell increases (P=.023)

For each patient we determined the maximum CD4 cell count and maximum CD4 cell gain after starting HAART. Older patients had poorer responses for these variables: In the 4 groups, median maximum CD4 cell counts (by increasing age quartile) were 500, 448, 430, and 419×10⁶ cells/L (P=10^-4), and maximum CD4 cell increases (by increasing age quartile) were 251, 246, 212, and 213×10⁶ cells/L (P=.0003), respectively. Furthermore, the proportion of patients whose maximum response was >100×10⁶ or >200×10⁶ CD4 cells/L significantly decreased with increasing age: 81.4%, 85.9%, 77.5%, and 79.7% (P=.007) and 61.3%, 58.7%, 52.7%, and 54.3% (P=.024), respectively

Multivariate linear regression analysis showed that patients who started a PI-including regimen had greater median maximum CD4 cell changes than persons receiving an NNRTI (237 vs. 164×10⁶ cells/L; P=.026). There was a strong relationship between time to response and maximum response (P<10^-4): Subjects who had been on HAART longer had higher maximum CD4 cell increases. While CD4 cell number at the start of HAART was not related to maximum CD4 cell increase (P=.58), a higher CD4 cell nadir was associated with greater maximum increase (P<10^-4). After adjustment for all these factors, age was still significantly related to maximum CD4 cell response (P=.023). Similar results were obtained after the model was adjusted for virus load at start of HAART (P=.047), for the subset of patients for whom these data were available

An increase of ⩾200×10⁶ CD4 cells/L was observed in 1109 patients (56.7%). Kaplan-Meier curves (figure 1) indicate that younger patients required less time to reach this end point. The median times to response in the 4 groups (by increasing age quartile) were 21 months (95% confidence interval [CI], 19–24), 22 months (95% CI, 20–25), 26 months (95% CI, 23–30), and 27 months (95% CI, 23–31) (P=.0026). Table 1 shows the results of univariate and multivariate analyses of time to achieve the responses. The multivariate model led to similar conclusions. After adjustment, patients in the third and fourth age quartiles were significantly less likely to experience an increase of ⩾200×10⁶ CD4 cells/L than were the youngest age groups. Similar results were obtained after the model was adjusted for the most recent virus load measurement, for the subset of patients for whom these data were available (P<10^-4). As expected, patients with higher virus loads were less likely to reach this end point than were patients with virus loads of <2.7 log₁₀ RNA copies/mL

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Figure 1

Kaplan-Meier curves of time to increase in CD4 cell count of ⩾200×10⁶cells/L after start of HAART in subjects in 4 age quartiles

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Table 1

Influence of age on reaching an increase in CD4 cell count of ⩾200×106cells/L after starting highly active antiretroviral therapy (HAART)

Discussion

Quantitative CD4 cell response to HAART is variable and not fully explained. Among possible genetic factors, the heterozygous state for the Δ32 deletion in the CCR-5 HIV coreceptor gene was recently shown to favorably influence response to treatment [7], and several predictors of virologic successful response to HAART have been described [8, 9]. These predictors were confirmed in this study in terms of CD4 cell recovery

In our study, time on treatment was strongly correlated with the CD4 cell response in all age groups. However, younger age was still associated both with a higher absolute CD4 cell gain and a shorter time to maximum response independent of baseline CD4 cell and plasma HIV RNA levels and of other variables that influence CD4 cell response, whereas therapeutic regimens did not differ between age groups. The median age in our study was 37.2 years, and older age has predicted good virologic response to HAART, possibly because of more-favorable socioeconomic conditions [9]. Therefore, differences in CD4 cell response reaching statistical significance inside the relatively narrow age range of this study appear to be all the more relevant

Older age at seroconversion unfavorably influences the course of untreated HIV infection [10], which may reflect the more pronounced inability of an aging immune system to counterbalance CD4 cell loss. The favorable impact of younger age on quantitative CD4 T cell recovery while a patient is receiving HAART is a new finding and could reflect the state of thymic function. Indeed, CD4 cell recovery after intensive chemotherapy and hematopoietic stem cell transplantation is inversely correlated with age and is thymus dependent [11, 12]. In treated HIV infection, CD4 cell recovery is biphasic and involves first memory cells and then naive cells

The first rapid phase probably depends on cell redistribution from lymphoid tissue and may also result to some degree from cell proliferation in the periphery [13, 14]. HAART seems to restore normal T cell production rate and half-life within the first 3 months [15] of treatment. The second phase is slower, and the question of whether these cells originate from thymopoiesis has been addressed. First, the number of naive CD4 cells in blood has been correlated with the size of the thymus in untreated HIV-positive adults [1]. Furthermore, the extent of naive CD4 cell recovery on treatment in adults and children [3, 4] and the correction of T cell half-life [15] may be associated with an increase in thymic volume. Finally, by using T cell receptor rearrangement excision circles (TRECs) as a quantification method for cells produced by the thymus, Douek et al. [2] showed that such cells persist in adulthood and slowly decrease with age in healthy subjects but are absent in athymic persons. In addition, they showed that the number of TRECs in naive CD4 T cells increases in HAART-exposed HIV patients with undetectable plasma virus load [2]. HAART also normalizes the function of progenitor cells in HIV infection and thus might allow de novo production of T lymphocytes [16]. Therefore, we suggest that the favorable influence of younger age on CD4 cell recovery while a patient is receiving HAART may be explained by more-effective thymic function

The present findings confirm that renewal of CD4 cells is a long-term process and demonstrate that age is inversely correlated with the magnitude and speed of CD4 cell recovery in actively treated HIV infection. These are additional arguments for the involvement of renewed lymphopoiesis in CD4 cell reconstitution

EuroSIDA Study Group Members and Centers

Study group members and centersThe study group members are listed by country, and the names of the national coordinators are in italics. Austria: N. Vetter (Pulmologisches Zentrum der Stadt Wien, Vienna). Belgium: N. Clumeck P. Hermans, and B. Sommereijns (Saint-Pierre Hospital, Brussels); and R. Colebunders (Institute of Tropical Medicine, Antwerp). Czech Republic: L. Machala and H. Rozsypal (Faculty Hospital Bulovka, Prague). Denmark: J. Nielsen J. Lundgren, T. Benfield, and O. Kirk (Hvidovre Hospital, Copenhagen); J. Gerstoft, T. Katzenstein, B. Røge, and P. Skinhøj (Rigshospitalet, Copenhagen); and C. Pedersen (Odense University Hospital, Odense). France: C. Katlama and C. Rivière (Hôpital de la Pitié-Salpétière, Paris); J.-P. Viard (Hôpital Necker-Enfants Malades, Paris); T. Saint-Marc (Hôpital Edouard Herriot, Lyon); P. Vanhems (Université Claude Bernard, Lyon); and C. Pradier (Hôpital de l'Archet, Nice). Germany: M. Dietrich and C. Manegold (Bernhard-Nocht-Institut for Tropical Medicine, Hamburg); J. van Lunzen (Eppendorf Medizinische Kernklinik, Hamburg); V. Miller and S. Staszewski (J. W. Goethe University Hospital, Frankfurt); F.-D. Goebel (Medizinische Poliklinik, Munich); Bernd Salzberger (Universität Köln, Cologne); and J. Rockstroh (Universitäts Klinik, Bonn). Greece: J. Kosmidis P. Gargalianos, and H. Sambatakou (Athens General Hospital); and G. Panos and M. Astriti (1st IKA Hospital, Athens). Hungary: D. Banhegyi (Szent Lásló Hospital, Budapest). Ireland: F. Mulcahy (St. James's Hospital, Dublin). Israel: I. Yust and D. Turner (Ichilov Hospital, Tel Aviv); S. Pollack and Z. Ben-Ishai (Rambam Medical Center, Haifa); Z. Bentwich (Kaplan Hospital, Rehovot); and S. Maayan (Hadassah University Hospital, Jerusalem). Italy: S. Vella and A. Chiesi (Istituto Superiore di Sanita, Rome); F. Suter and A. Cremaschi (Ospedale Riuniti, Bergamo); R. Pristerá (Ospedale Generale Regionale, Bolzano); F. Mazzotta and F. Vichi (Ospedale S. Maria Annunziata, Florence); B. DeRienzo and A. Bedini (Università di Modena); A. Chirianni and E. Montesarchio (Presidio Ospedaliero A. D. Cotugno, Naples); V. Vullo and P. Santopadre (Università di Roma La Sapienza, Rome); C. Arici, P. Franci, P. Narciso, A. Antinori, and M. Zaccarelli (Ospedale Spallanzani, Rome); A. Lazzarin and R. Finazzi (Ospedale San Raffaele, Milan); and A. D'Arminio Monforte (Ospedale L. Sacco, Milan). Luxembourg: R. Hemmer and T. Staub (Centre Hospitalier, Luxembourg). The Netherlands: P. Reiss (Academisch Medisch Centrum bij de Universiteit van Amsterdam). Norway: J. Bruun and A. Maeland (Ullevål Hospital, Oslo). Poland: B. Knysz and J. Gasiorowski (Medical University, Wroclaw); A. Horban (Centrum Diagnostyki i Terapii AIDS, Warsaw); R. Rogowska-Szadkowska (Medical University, Bialystok); A. Boron-Kaczmarska (Medical Univesity, Szczecin); M. Beniowski (Osrodek Diagnostyki i Terapii AIDS, Chorzow); and H. Trocha (Medical University, Gdansk). Portugal: F. Antunes (Hospital Santa Maria, Lisbon); K. Mansinho (Hospital de Egas Moniz, Lisbon); and R. Proenca (Hospital Curry Cabral, Lisbon). Spain: J. González-Lahoz R. Polo, and V. Soriano (Hospital Carlos III, Madrid); B. Clotet, A. Jou, J. Conejero, and C. Tural (Hospital Germans Trias i Pujol, Badalona); and J. M. Gatell and J. M. Miró (Hospital Clinic i Provincial, Barcelona). Sweden: A. Blaxhult (Karolinska Hospital, Stockholm); B. Heidemann (Södersjukhuset, Stockholm); and P. Pehrson (Huddinge Sjukhus, Stockholm). Switzerland: B. Ledergerber and R. Weber (University Hospital, Zurich); P. Francioli and A. Telenti (Centre Hospitalier Universitaire Vaudois, Lausanne); and B. Hirschel and V. Soravia-Dunand (Hôpital Cantonal Universitaire de Genève, Geneva). United Kingdom: S. Barton (St. Stephen's Clinic, Chelsea, and Westminster Hospital, London); A. M. Johnson and D. Mercey (Royal Free and University College Medical School, London [University College Campus]); A. Phillips, C. Loveday, M. A. Johnson, and A. Mocroft (Royal Free and University College Medical School, London [Royal Free Campus]); A. Pinching and J. Parkin (Medical College of Saint Bartholomew's Hospital, London); J. Weber and G. Scullard (Imperial College School of Medicine at St. Mary's, London); M. Fisher (Royal Sussex County Hospital, Brighton); and R. Brettle (City Hospital, Edinburgh)

Steering committeeJ. Nielsen (chair), N. Clumeck, M. Dietrich, J. M. Gatell, A. Horban, A. M. Johnson, C. Katlama, B. Ledergerber, C. Loveday, A. Phillips, P. Reiss, and S. Vella

Coordinating center staffJ. Lundgren (project leader), I. Gjørup, T. Benfield, O. Kirk, A. Mocroft, D. Mollerup, M. Nielsen, A. Sørensen, H. Buch, and L. Teglbjaerg