The benefits of highly active antiretroviral therapy (HAART) is now well established [1–3]. Among patients seen in clinic cohorts, between 50 and 80% of patients who start HAART respond in terms of achieving a viral load level below the limit of detection [4–9]. Although there is a considerable rate of success, a substantial proportion of patients fail to suppress viraemia to below the limit of detection, and between 20 and 40% of patients are estimated to fail due to subsequent virological rebound within 12 months of initial virological success [4,5,7,10]. Continuing a HAART regimen with a low but detectable level of viraemia is associated with virus evolution and the development of resistance, but this must be balanced against premature treatment changes limiting future treatment options . Patients who fail their initial regimen may be switched to second-line regimens. Cross resistance among antiretroviral drugs is common and failure of one regimen may reduce the efficacy of subsequent regimens. In addition, It is unclear at what level of virological failure treatment changes are required, although there may be a better response to second-line regimens if patients are switched sooner rather than later [11–13].
There have been several relatively small studies which consider response to second-line regimens [12–19]. These studies typically report patients changing from a single protease inhibitor (PI)-containing regimen to a dual PI regimen as second-line treatment, with or without the addition or substitution of new nucleosides or a new class of drugs. In general, they report a poor response to second-line treatment, which may be improved with the addition of a new class of drugs [12,16,18], or commencing the second-line treatment at lower levels of viraemia [12,13]. To date, there have been few reports of the virological outcome after initiation of second-line PI-containing regimens among large series of patients. The aims of this study were therefore to investigate the virological and immunological outcome among 984 patients initiating a second-line PI-containing regimen in the EuroSIDA study.
The EuroSIDA study is a prospective, European study of over 8500 patients from four cohorts with HIV infection from 63 centres across Europe (including Israel - see Appendix). Details of the study have been published . Three regions of Europe were defined as described previously: the South: Greece, Israel, Italy, Portugal, and Spain; Central: Belgium, France, south Germany, Luxembourg, Switzerland; and North: Denmark, Ireland, north Germany, Netherlands, Norway, Sweden, United Kingdom. Patients were aged older than 16 years at the time of enrolment. Information was collected from patient case notes onto a standardized data collection form at baseline and every 6 months thereafter. At each follow-up visit, details are collected on all CD4 lymphocyte counts and viral loads measured since last follow-up, including the method used and the lower limit of detection for the latter. For each patient, the date of starting and stopping each antiretroviral drug is recorded, as is the use of drugs for prophylaxis against opportunistic infections. Dates of diagnosis of all AIDS-defining diseases are also recorded, using the 1993 clinical definition of AIDS from the Centers for Disease Control and Prevention . Members of the co-ordinating office visited all centres to ensure correct patient selection and accurate data collection. At site visits, patient notes are checked against the recorded information for a proportion of patients to verify the accuracy of the data abstracted from medical charts. Information from up to eleven follow-up visits is available from cohort I, up to eight visits for cohort II, and five for cohort III. Cohort IV was excluded from analyses as there is currently no follow-up data available. The analyses presented here include all data collected by February 2000.
For the present analysis, eligible patients were those starting a second PI during prospective follow-up with a viral load > 1000 copies/ml and at least 16 weeks after initiating their first PI regimen, limited to those starting ritonavir, saquinavir hard gel capsule (HGC) or indinavir as a first-line regimen. Patients were required to have both a viral load and CD4 lymphocyte count record in the 6 months prior to starting the second-line regimen and at least one measure of each subsequently. Patients who started non-nucleoside reverse transcriptase inhibitors (NNRTI) prior to, or at the date of starting the second PI were excluded from the analyses.
The characteristics of patients starting second-line PI-containing regimens were compared using χ2 tests for categorical variables and non-parametric methods for continuous variables.
Kaplan–Meier survival curves were used to determine the proportion of patients who responded with a viral load of below 500 copies/ml or had a 1 log drop in viral load within 6 months of starting the second-line regimen. Patient follow-up was measured from the date of starting the second-line regimen to the date of virological response (first viral load < 500 copies/ml), to 6 months follow-up or the last measured viral load. Patients were therefore censored at 6 months of follow-up or their last viral load measurement if this occurred prior to 6 months. Cox proportional hazards models were used to investigate the factors associated with virological success. Several models were constructed adjusting for factors such as the CD4 cell count and viral load at initiation of the second-line regimen, whether a patient responded to their first-line regimen, the number of new nucleosides added to the second-line regimen, the PI used in the first and second regimens, and nucleoside combinations. All analyses of response to the second-line regimen were based on the intent-to-treat principle in that no adjustments have been made for modification or discontinuation of the second-line regimen. Models constructed included factors associated with response among all patients who initiated a second-line regimen, a model stratified by whether the first PI regimen was successful or not, a model confined to those who stayed on a single PI as their second-line regimen and a model using viral load as a categorical variable to describe virological success according to different levels of viraemia at starting the second-line regimen. Similar methods were used to investigate the factors associated with immunological response, measured as an increase in CD4 lymphocyte count of 50 × 106 cells/l or more.
All statistical analyses were performed using SAS statistical software (SAS Inc., Cary, North Carolina, USA). All tests of significance were two-sided and P-values of less than 0.05 were considered significant. There was no evidence that the proportional hazards assumption was violated in any of the Cox models constructed.
The patients included in this study are described in Table 1. The initial PI regimen was saquinavir (HGC) in 443 patients (45.0%), indinavir in 349 patients (35.5%) and ritonavir in 192 patients (19.5%). Just under half of the patients (444, 45.1%) started a dual PI regimen as second-line treatment; the most common combinations were ritonavir/saquinavir (HGC) in patients (47.3%), nelfinavir/saquinavir (HGC) in 75 patients (16.9%) and indinavir/saquinavir (HGC) in 54 patients (12.2%). Among those patients who stayed on a single PI regimen, indinavir and nelfinavir were the most common PIs used. There were few differences between the second-line regimens according to demographic characteristics. Notably, most patients from Southern Europe were more likely to be using a single PI as the second-line regimen. The median viral load at starting the second-line regimen was 4.45 log copies/ml [interquartile range (IQR), 3.85–5.00] and median CD4 cell count was 171 × 106 cells/l (IQR, 88–293 × 106 cells/l), both measured at a median of 1 month prior to starting the second-line PI regimen (IQR, 0–2 months). The nadir CD4 cell count since recruitment to the EuroSIDA study was low at 89 × 106 cells/l (IQR, 26–160 × 106 cells/l). The median date of starting the second-line regimen was October 1997 (IQR, May 1999 – May 1998), which followed a median of 12 months after the first PI-containing regimen (IQR, 8–17 months). Both viral loads and CD4 lymphocyte counts were measured regularly during follow-up, with a median of eight measurements per patient at a median frequency of 2 months apart.
A total of 441 patients took two PIs as their second-line PI regimen whereas 540 stayed on a single PI. Of those patients who stayed on a single PI as their second-line PI regimen , the most common changes were saquinavir (HGC) to indinavir in 171 patients (31.7%), indinavir to nelfinavir in 100 patients (18.5%) and ritonavir to indinavir in 57 patients (10.6%). The majority of patients were taking three-drug regimens (418, 42.5%), with 267 patients taking four-drug regimens (27.1%) and 299 patients taking five or more drug regimens (30.4%). Over half of the patients did not add any new nucleosides to their second-line regimen (538, 54.7%), that is, nucleosides to which they were previously treatment naive. However, 313 patients (31.8%) were able to add one new nucleoside and 133 patients (13.5%) added two. A small proportion of patients, 74 (7.5%), subsequently started a NNRTI in the 6 months after starting the second-line PI regimen. This did not differ according to the second-line PI regimen started (P = 0.16, χ2 test). The most common nucleoside combinations were lamivudine/stavudine, used by 468 patients (47.6%), didanosine/stavudine used by 234 patients (23.8%) and zidovudine/lamivudine, used by 137 patients (13.9%).
Immunological and virological responses on second-line PI regimens
The virological and immunological responses are shown in Figure 1. In the first 6 months of treatment, 419 patients achieved a viral load below 500 copies/ml (42.6%) and 534 patients had at least 1 log drop in viral load (54.3%). Using Kaplan–Meier estimation, at 6 months 41.0% of patients were estimated to have achieved a viral load below 500 copies/ml [95% confidence interval (CI), 37.9–44.1%] and 52.7% were estimated to have experienced a 1 log drop in viral load (95% CI, 49.5–55.9%). The rise in CD4 cell count was around 40 × 106 cells/l at 6 months, with approximately 27% of patients achieving a CD4 cell count increase of 100 × 106 cells/l at 6 months and 46% achieving a 50 × 106 cells/l increase.
Factors associated with virological response on second-line PI regimens (viral load < 500 copies/ml)
Table 2 describes the factors associated with achieving a viral load below 500 copies/ml after starting a second-line PI regimen. Demographic factors, age, and clinical status were not found to be related to virological success and were excluded from the multivariate model. Similarly, calendar quartile of starting the second-line regimen, CD4 nadir, and starting a NNRTI (modelled as a time-updated covariate) were excluded from the final model. As shown in Table 2, patients who started the second-line PI regimen at higher viral loads were less likely to respond, whereas patients who achieved a viral load of less than 500 copies/ml on their initial regimen were 65% more likely to respond to the second-line regimen. In addition, patients with a higher CD4 cell count were more likely to respond, as were patients who added at least two new nucleosides to their second-line regimen. Patients who started saquinavir (HGC) as their first-line regimen were almost 59% more likely to respond to their second-line regimen. There were no significant differences in virological success between different second-line regimens or according to nucleoside combinations used in the second-line regimen. The length of time on the first PI regimen was not related to virological success, nor was the time since failure of the first-line regimen.
The factors related to virological success among patients starting their second-line PI regimen were very consistent between different Cox models. In particular, in models stratified by whether a patient had responded to their initial PI regimen, among those patients who stayed on a single PI regimen as second-line treatment, and after stratification by the initial PI regimen. Consistent results were also obtained when the virological response was measured in terms of a 1 log drop in viral load (data not shown).
Factors associated with immunological response on second-line PI regimens (CD4 cell count rise of 50 × 106 cells/l)
The factors related to immunological response (CD4 cell count rise of 50 × 106 cells/l or more) are presented in Table 3. In this multivariate model, achieving a viral load of less than 500 copies/ml on the second-line regimen was included as a time-dependent covariate. The strongest factors related to immunological response was the viral load falling below 500 copies/ml on the second PI regimen and virological success on the first PI regimen. The CD4 cell count at starting the second-line PI regimen and whether the CD4 cell count responded to the initial PI regimen were not related to immunological success. There were no differences in immunological response between different first- or second-line regimens or according to nucleoside combinations used.
This study has described the virological and immunological response to second-line PI-containing antiretroviral regimens among almost 1000 HIV-infected patients from across Europe. Both the immunological and virological response to second-line regimens was generally poorer than that of first-line regimens reported from observational studies, although by definition this was a selected group of patients who failed an initial regimen. The virological response depended on the first PI used, the CD4 cell count and the viral load, with those who switched at lower viral load more likely to achieve a viral load < 500 copies/ml. In addition, those patients who were able to start a second-line regimen with nucleosides to which they were previously naive were more likely to respond virologically. The immunological response was strongly linked to virological response, with those patients who responded virologically being much more likely to respond immunologically.
Previous studies considering second-line treatment regimens tend to be based on small series of patients with response rates of up to 70%[12–19]. Such studies tend to be those in which a new class of drugs, such as the NNRTIs were added after failure of the first PI regimen [14,15,18]. This study is considerably larger than previously published work and specifically considers patients who were starting a second-line PI-containing regimen, rather than starting a new class of antiretrovirals as second-line therapy. Patients who started a new class of drug as a second-line regimen were not included in the present analysis in order to specifically address the response to exposure to a second protease after failure of the first, although some patients may have started an NNRTI after starting the second-line PI regimen. In the multivariate analyses, subsequently starting a NNRTI was not associated with either virological or immunological response, although the power to detect this was limited due to the small numbers who started a NNRTI in the 6 months after starting a second-line PI regimen. In agreement with previous work we found that patients who started their second-line regimen at lower levels of viraemia were more likely to respond [11,14,17]. Delaying the switch of a failing regimen may allow continuing viral replication and the accumulation of resistance mutations [12,22]. Those that started at a higher CD4 cell count were more likely to respond, supporting the concept of changing treatment regimens earlier rather than later. Further follow-up is needed to describe the long-term response to second-line PI regimens, toxicities and further salvage regimens that may be undertaken.
Patients who were able to add one or two nucleosides to which they were previously naive were also more likely to respond, although it should be noted that this was the minority of patients by the time of starting the second-line PI-regimen. The benefits of adding new nucleosides to an initial PI regimen has been shown from several observational studies [4,5,9]. The addition of as many new antiretrovirals as possible to a regimen is recommended [11,23], as it reduces the number of antiretrovirals to which the patient may have accumulated resistance, permitting the best virological outcome .
Patients who started saquinavir (HGC) in their first-line PI regimen were more likely to respond virologically to their second-line PI regimen. Response to hard gel saquinavir in an initial HAART regimen has been shown to be poor [4-5,7], and may be due to limited bioavailability , and thus patients who fail their first PI-containing regimen within the EuroSIDA study may be more likely to have started saquinavir (HGC). A previous study suggested prior exposure to saquinavir (HGC) may blunt the subsequent virological response to treatment with indinavir or nelfinavir . The present results cannot be explained by a shorter duration of treatment with saquinavir (HGC) as first-line PI regimen compared with ritonavir or indinavir, as duration of first-line PI treatment was similar between the proteases and adjusted for in the multivariate analyses. Mutations of HIV occur when viral suppression is incomplete, but in the case of saquinavir (HGC), which has a low bioavailability, there may not be sufficient selection pressure for virus with mutations to survive and accumulate, and this may lead to a better response to a second-line PI regimen that is more virologically active in vivo. Craig et al. suggested that the majority of patients treated with saquinavir (HGC) retained sensitivity to at least one other protease inhibitor . Drug resistance is only one component of failure of a treatment regimen, and the role of adherence and compliance will also be crucial [26,27]. Information on adherence is not available within this study, although information on reasons for changing treatment regimens is now being collected prospectively. Soft-gel saquinavir, which has a better bioavailability [24,28], has only recently been introduced into Europe and longer follow-up is needed to determine response to second-line regimens when saquinavir soft gel is used as part of an initial HAART regimen.
There was a poor immunological response, with less than 50% of patients having a 50 × 106 cells/l increase in the CD4 cell count. There are few reports of the immunological response to second-line regimens, and few clinical trials available for comparison. It was clear that the immunological response depended heavily on virological success. Those who responded virologically with either the first- or second-line regimen were significantly more likely to achieve an immunological response. This study did not specifically address clinical outcome due to the limited follow-up currently available. To date, little is known about the relationship between virological failure, immunological failure and clinical progression, or if this sequence of events always occurs, or the role that levels of CD4 lymphocyte count and viral load will play in determining risk of clinical failure.
There are several limitations to this study which deserve attention. We have concentrated on second-line PI regimens among those that failed an initial regimen, and by definition, this may have selected patients who were not able to respond to therapy or who started regimens that are now known to be less potent (saquinavir (HGC)). We do not know why patients were switched from their first PI regimen, contributory factors may have been poor virological response, a rise in viral load, toxicities or intolerance. We were not able to distinguish between dual PI-containing regimens where both PI components were used for antiretroviral efficacy and dual PI regimens where one of the PIs was used to boost pharmacokinetics. In addition, no information was available on genotypic or phenotypic resistance, which is increasingly being used to guide treatment options, although the long-term clinical benefit of resistance testing is not yet known. Further work is being undertaken to evaluate the relationship between drug resistance and virological, immunological and clinical outcome among treated patients in the EuroSIDA study.
The patients included in this analyses were typically treated with saquinavir HGC as their first-line PI regimen, although in previous analyses of the response to a first HAART regimen the use of saquinavir HGC was less common . Further, we have no data on compliance with either the first- or second-line PI regimen, seen to be essential components of a successful HAART regimen [26,27]. However, to our knowledge, this is the largest study to date of response to second-line regimens among a heterogeneous group of patients. The response to an initial HAART regimen has improved over time, mainly due to changes in the way HAART is started and better management of side effects and toxicities . There was no evidence from these analyses of a change over time in response to second-line regimens, but with further follow-up and improvement in initial HAART regimens the initially poor response seen to second-line PI regimens may also improve.
In summary, we have observed a moderate virological response to second-line PI-containing regimens which depended on the success of the first-line regimen, the PI used as first-line, the ability to add new nucleosides and the level of viraemia and CD4 lymphocyte counts at which therapy started. The relationship between cross-resistance and adherence will undoubtedly also play a role. The immunological response was dependent on a good virological response.
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The EuroSIDA Study Group
The multicentre study group on EuroSIDA (names of national coordinators in parenthesis)
Austria (N Vetter) Pulmologisches Zentrum der Stadt Wien, Vienna. Belgium (N Clumeck) P Hermans, B Sommereijns, Saint-Pierre Hospital, Brussels; R Colebunders, Institute of Tropical Medicine Antwerp. Czech Republic (L Machala) H Rozsypal, Faculty Hospital Bulovka, Prague. Denmark (J Nielsen) J Lundgren,T Benfield, O Kirk, Hvidovre Hospital, Copenhagen; J Gerstoft, T Katzenstein, B Røge, P Skinhøj, Rigshospitalet, Copenhagen; C Pedersen, Odense University Hospital, Odense. France (C Katlama) C Rivière, Hôpital de la Pitié-Salpétière, Paris; J-P Viard, Hôpital Necker-Enfants Malades, Paris; T Saint-Marc, Hôpital Edouard Herriot, P Vanhems, University Claude Bernard, Lyon; C Pradier, Hôpital de l'Archet, Nice. Germany (M Dietrich) C Manegold, Bernhard-Nocht-Institut for Tropical Medicine, Hamburg; J van Lunzen, Eppendorf Medizinische Kernklinik, Hamburg; V Miller, S Staszewski, JW Goethe University Hospital, Frankfurt; F-D Goebel, Medizinische Poliklinik, Munich; Bernd Salzberger, Universität Köln, Cologne; J Rockstroh, Universitäts Klinik Bonn. Greece (J Kosmidis) P Gargalianos, H Sambatakou, J Perdios, Athens General Hospital, Athens; G Panos, M Astriti, 1st IKA Hospital, Athens. Hungary (D Banhegyi) Szent Lásló Hospital, Budapest. Ireland (F Mulcahy) St. James's Hospital, Dublin. Israel (I Yust) D Turner, Ichilov Hospital, Tel Aviv; S Pollack, Z Ben-Ishai, Rambam Medical Center, Haifa: Z Bentwich, Kaplan Hospital, Rehovot; S Maayan, Hadassah University Hospital, Jerusalem. Italy (S Vella, A Chiesi) Istituto Superiore di Sanita, Rome; F Suter, A Cremaschi, Ospedale Riuniti, Bergamo; R Pristerá, Ospedale Generale Regionale, Bolzano; F Mazzotta, F.Vichi, Ospedale S. Maria Annunziata, Florence; B DeRienzo, A Bedini, Università di Modena, Modena; A Chirianni, E Montesarchio, Presidio Ospedaliero AD. Cotugno, Naples; V Vullo, P Santopadre, Università di Roma La Sapienza, Rome; C Arici, P Franci, P Narciso, A Antinori, M Zaccarelli, Ospedale Spallanzani, Rome; A Lazzarin, R Finazzi, Ospedale San Raffaele, Milan; A D'Arminio Monforte, Osp. L. Sacco, Milan. Luxembourg (R Hemmer), T Staub, Centre Hospitalier, Luxembourg. Netherlands (P Reiss) Academisch Medisch Centrum bij de Universiteit van Amsterdam, Amsterdam. Norway (J Bruun) A Maeland, Ullevål Hospital, Oslo. Poland (B Knysz) J Gasiorowski, Medical University, Wroslaw; 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; H Trocha, Medical University, Gdansk. Portugal (F Antunes) Hospital Santa Maria, Lisbon; K Mansinho, Hospital de Egas Moniz, Lisbon; R Proenca, Hospital Curry Cabral, Lisbon. Spain (J González-Lahoz) R Polo, V Soriano, Hospital Carlos III, Madrid; B Clotet, A Jou, J Conejero, C Tural, Hospital Germans Trias i Pujol, Badalona; JM Gatell, JM Miró, Hospital Clinic i Provincial, Barcelona. Sweden (A Blaxhult) Karolinska Hospital; B Heidemann, Södersjukhuset; P Pehrson, Huddinge Sjukhus, Stockholm. Switzerland (B Ledergerber) R Weber, University Hospital, Zürich; P Francioli, A Telenti, Centre Hospitalier Universitaire Vaudois, Lausanne; B Hirschel, V Soravia-Dunand, Hospital Cantonal Universitaire de Geneve, Geneve. United Kingdom (S Barton) St. Stephen's Clinic, Chelsea and Westminster Hospital, London; AM Johnson, D Mercey, Royal Free and University College London Medical School, London (University College Campus); A Phillips, C Loveday, MA Johnson, A Mocroft, Royal Free and University College Medical School, London (Royal Free Campus); A Pinching, J Parkin, Medical College of Saint Bartholomew's Hospital, London; J Weber, G Scullard, Imperial College School of Medicine at St. Mary's, London; M Fisher, Royal Sussex County Hospital, Brighton; R Brettle, City Hospital, Edinburgh.
Steering committee: J Nielsen (chair), N Clumeck, M Dietrich, JM Gatell, A Horban, AM Johnson, C Katlama, B Ledergerber, C Loveday, A Phillips, P Reiss, S Vella.
Coordinating centre staff: J Lundgren (project leader), I Gjørup, T Benfield, O Kirk, A Mocroft, D Mollerup, M Nielsen, A Sørensen, H Buch, L Madsen, L Teglbjærg.