Highly active antiretroviral (ARV) therapy (HAART) has dramatically

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1 ORIGINAL STUDIES Pattern and Predictors of Immunologic Recovery in Human Immunodeficiency Virus-Infected Children Receiving Non-Nucleoside Reverse Transcriptase Inhibitor-Based Highly Active Antiretroviral Therapy Thanyawee Puthanakit, MD,* Stephen J. Kerr, PhD, Jintanat Ananworanich, MD, PhD, Torsak Bunupuradah, MD, Pitch Boonrak, MSc, and Virat Sirisanthana, MD Background: Non-nucleoside reverse transcription inhibitor (NNRTI)- based highly active antiretroviral therapy (HAART) is the recommended first-line regimen for children in Thailand. This study was aimed to assess pattern and predictors of immune recovery in antiretroviral-naive Thai children starting NNRTI-based HAART. Methods: Records were extracted from clinical databases of 2 treatment cohorts in Thailand. The inclusion criteria were HIV-infected naive children who initiated NNRTI-based HAART when CD4 25%. Immune recovery was defined as achieving a target CD4% of 25. The impact of age, gender, baseline clinical category, CD4 and HIV RNA titer, and regimen on immune recovery to weeks 96 was assessed using multiple logistic regression. Results: There were 274 patients (52% females) with a median baseline age of 7 (Interquartile range IQR : 4 9) years and a median CD4% of 5 (IQR: 1 12) who started treatment with nevirapine (66%) or efavirenz (34%) based HAART. Median duration of follow-up was 168 (IQR: ) weeks. The median CD4% increase from baseline was 7% (IQR: 5 11) and 18% (IQR: 12 23) at weeks 24 and 96, respectively. The probability of reaching target CD4% was 51% (95% confidence interval: 45% 57%) by week 96. The predictors of immune recovery at week 96 were younger age, female gender, higher baseline CD4%, and sustained virologic suppression after week 24. Conclusion: In this cohort of children with low baseline CD4, half achieved immune recovery after 96 weeks of HAART. The predictors for immune recovery are younger children, female gender, high baseline CD4%, and long-term virologic suppression. Key Words: antiretroviral therapy, CD4, NNRTI, resource-limited settings, pediatrics (Pediatr Infect Dis J 2009;28: ) Highly active antiretroviral (ARV) therapy (HAART) has dramatically improved the prognosis for HIV-infected children in western, 1,2 and developing countries. 3 7 As a result, there have been clinically significant reductions in morbidity and mortality, Accepted for publication November 12, From the *Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand; HIV Netherlands Australia Thailand Research Collaboration, Bangkok, Thailand; National Centre in HIV Epidemiology and Clinical Research, Sydney Australia; South East Asia Research Collaboration with Hawaii (SEARCH) Bangkok, Thailand; and Department of Pediatrics, Chiang Mai University, Chiang Mai, Thailand. Address for correspondence: Thanyawee Puthanakit, MD, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand. thanyawee@rihes-cmu.org. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal s Web site ( Copyright 2009 by Lippincott Williams & Wilkins ISSN: /09/ DOI: /INF.0b013e318194eea6 converting the infection into a chronic condition in persons who receive HAART. There is a balance regarding the optimal time to commence HAART, a compromise between maximizing immunologic responses and minimizing long-term toxicities, resistance, and cost, while judging the right time to intervene before the patient develops life-threatening immunodeficiency. The current World Health Organization guideline 8 recommend initiating HAART when CD4 20% in children 1 to 5 years, and CD4 15% or 200 cells/mm 3 in children older than 5 years. Although, the 2008 US guideline 9 recommend initiating HAART at higher CD4 value, using criteria of CD4 25% in children 1 to 5 years of age, and CD4 350 cells/mm 3 in children greater than 5 years of age. The goal of HAART is to suppress HIV viral replication and restore immune function. There is controversy regarding the ability to restore immune function to near normal values (CD4 25%) among children who initiate treatment in severe immunodeficiency stage. It is unclear in these children whether the CD4% can recover back to normal values or whether it would reach a less-thannormal plateau. Some studies in which the majority of children are treated with protease inhibitor (PI)-based HAART, indicate that younger age and higher baseline CD4 values were associated with better chance to reach near normal CD4% values Other studies have reported that children can restore their CD4 T cell values independent of age and baseline CD4% values. 13 Data from several HIV-infected adult cohorts have shown that the recovery of CD4 reaches a plateau stage after 4 to 5 years of HAART despite complete viral suppression In the past few years, ARV therapy has been more widely available in resource-limited settings using non-nucleoside reverse transcription inhibitor (NNRTI)-based HAART as a first-line regimen. To describe the immunologic response in children who initiate HAART at different baseline CD4 levels, we evaluated the pattern of CD4 cell recovery in Thai children and also the predictors of successful immune recovery. This information will be useful for clinicians who work in resource-limited settings to have a pattern of immune recovery after HAART of ARV naive children who access to treatment while in states of moderate to severe immunosuppression. METHODS Patient Population We combined data of HIV-infected ARV naive children who were starting treatment with NNRTI-based HAART regimens from 2 sites in Thailand. The first site was the Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai, which serves as a tertiary-care referral center located in the Northern part of Thailand. The second site was the HIV Netherlands Australia Thailand Research Collaboration in Bangkok, which is a research center where patients are treated in an out-patient setting. The The Pediatric Infectious Disease Journal Volume 28, Number 6, June 2009

2 The Pediatric Infectious Disease Journal Volume 28, Number 6, June 2009 Immunologic Response to HAART inclusion criteria were (1) HIV-infected children aged less than 18 years (2) starting treatment with NNRTI-based HAART, defined as nevirapine (NVP) or efavirenz (EFV) plus 2 NRTI and (3) a baseline CD4% 25%. Demographic information, CD4 counts, HIV RNA levels (HIV viral load), and ARV treatment regimens were extracted from clinical databases of children who initiated NNRTI-based HAART between December 2001 and May Laboratory tests were done locally. CD4 cell counts were based on flow cytometry. HIV RNA levels were assessed by the Roche Amplicor Monitor Test (version 1.0 and 1.5; Roche Diagnostic Systems). Statistical Methods We assessed short-term and long-term immunologic response to HAART at 24 weeks and 96 weeks, respectively. Because of the natural decline in CD4 counts in uninfected children in the first years of life, immunologic response was assessed by CD4%. The majority of children had CD4% measured at 24 week intervals, so we used a 24 week visit schedule for all patients for this analysis. If patients had more than one visit in the 24 week period, the date closest to the scheduled 24 week visit was selected. Since, the CD4% of 25 is defined as no immunologic suppression, it was therefore chosen as a value of target CD4% (TCD4%) representing immune recovery. Short-term immunologic response at weeks 24 were increase of CD4% by 10 percent and increase of CD4% to the target value of 25%. Long-term immunologic response at weeks 96 was increase of CD4% to the target value. We assessed the impact of gender, age, pre-haart CD4% and HIV-RNA, AIDS diagnosis, and type of NNRTI used in the regimen on the immunologic response to HAART, using a logistic regression model. Variables with P 0.15 in univariate analysis were entered into multivariate models. Patients who died before reaching target endpoints were imputed as immunologic failures in logistic regression models. Patients who changed to PI-based regimens because of intolerance or immunologic/virologic failure were censored at the time that NNRTIs were ceased. Patients who were intolerant to NNRTI and changed treatment before week 24 were excluded from the analysis. Because our study was conducted in a resource limited setting, not all children had access to regular monitoring of plasma HIV RNA. We, therefore, included an additional variable in the 96 week logistic regression modeling. This was a categorical variable describing whether the patient had sustained virologic suppression after initial suppression (defined as plasma HIV RNA 1000 copies/ml after initial suppression), or whether the patient had ongoing viral replication (defined as HIV RNA in plasma greater than 1000 copies/ml on 2 consecutive visits after week 24) or whether this was unknown. Lastly, we used time to event methodology in the form of Kaplan-Meier technique to assess the cumulative probability of achieving a TCD4% over time. In this analysis, patients were censored at their last follow-up visit or when they ceased NNRTIbased HAART, and 15 patients who died without reaching TCD4 cell percentage were carried forward as failures to week 192. We examined the effect of same variables as were used in logistic regression models, using the log-rank test to compare probability of achieving TCD4% over time in different strata. In rare cases when there was a missing CD4 result, we used the previous observation carried forward. All statistical analyses were done with SAS, Version 9.1 (SAS Institute Inc, Cary, NC) and Stata Version 10 (StataCorpLP, College Station, TX). RESULTS Two hundred seventy-four patients were included in the analysis, and the baseline characteristics are shown in Table 1. Children in Chiang Mai had lower baseline CD4% (median: CD4% 4, Interquartile range IQR : 1 10 cells/mm 3 ) and higher viral loads (5.47, IQR: log 10 copies/ml) compared with children in Bangkok (median: CD4% 9, IQR: 3 16 cells/mm 3 ; median viral load 5.06, IQR: log 10 copies/ml). The median duration of follow-up was 144 weeks (IQR: ) and the total duration of follow-up was 714 patient years. The proportion of children followed for 48, 96, 144, and 192 weeks was 93%, 80%, 57%, and 30%, respectively. Fifteen patients (5.5%) died, all before reaching TCD4%. The median CD4% at baseline was 2% (IQR: 1 6) and the median time to death was 9 weeks (IQR: 6 52). Twenty patients (NVP 18, EFV 2) changed from NNRTI-based regimens to PI-based therapy. Two patients changed because of hypersensitivity reactions to NVP after 5 and 11 weeks of treatment, respectively, so were excluded from the analysis. Eight patients changed because of virologic failure and 10 patients changed because of immunologic TABLE 1. Baseline Characteristics for 274 Children Starting NNRTI-Based HAART Characteristic Chiang Mai N 203 Bangkok N 71 Total N 274 Age yr, median (IQR) 7 (5 9) 5 (3 8) 7 (4 9) Gender, n (%) Female 103 (51%) 39 (55%) 142 (52%) Male 100 (49%) 32 (45%) 132 (48%) CDC classification, n (%) CDC N 27 (13%) 1 (1%) 28 (10%) CDC A 32 (16%) 34 (48%) 66 (25%) CDC B 47 (23%) 21 (30%) 68 (25%) CDC C 97 (48%) 15 (21%) 112 (40%) Immunologic status CD4%, median (IQR) 4 (1 10) 9 (3 16) 5 (1 12) CD4 count, median (IQR) cells/mm 3 65 (19 307) 208 (36 555) 88 (22 346) Virologic status* Viral load, median (log 10 copies/ml IQR ) 5.47 ( ) 5.06 ( ) 5.36 ( ) HAART regimen NNRTI used, n (%) NVP 126 (62%) 54 (76%) 180 (66%) EFV 77 (38%) 17 (24%) 94 (34%) NRTI backbone, n (%) Stavudine/lamivudine 195 (96%) 48 (68%) 243 (89%) Zidovudine/lamivudine 8 (4%) 23 (32%) 31 (11%) *Twenty-two patients did not have a baseline viral load result available Lippincott Williams & Wilkins 489

3 Puthanakit et al The Pediatric Infectious Disease Journal Volume 28, Number 6, June 2009 failure. The median times to changing regimen because of virologic or immunologic failure were 82 (64 102) weeks and 70 (62 92) weeks, respectively. The median changes in CD4% from baseline at each study visit up to week 192 are shown in Table 2 (Supplemental Digital Content 1, Short-Term CD4 Response to HAART At 24 weeks, 252 (92%) children had a higher CD4% than at baseline. The median CD4 increase was 7% (IQR: 5% 11%). Ninety (33%) patients had CD4% increases of at least 10%. In univariate analysis and multivariate analysis (Table 3), younger age (odds ratio OR : 0.91, 95% confidence interval 95% CI : ) and female gender (OR: 2.05, 95% CI: ) were predictors of CD4% increases of at least 10%. Neither baseline CD4% nor baseline HIV-1 RNA were important predictors in univariate or multivariate models. At 24 weeks, 39 (14%) children had reached TCD4%. Predictors of CD4% increase to at least 25% or more in both univariate and multivariate models were of younger age (OR: 0.80, 95% CI: ) and higher baseline CD4% (OR: 3.46, 95% CI: ). Female gender had a higher chance of achieving CD4% of 25%, but this did not reach statistical significance in univariate or multivariate models. Baseline HIV-1 RNA did not influence the chance of achieving a TCD4% at 6 months. In univariate models, treatment with NVP was associated with a higher chance of reaching CD4%, reflecting the higher proportion of NVP use in Bangkok (77% vs. 62%) where children had higher baseline CD4%. CDC category A, B, and N patients also had a higher chance of achieving TCD4% at 24 weeks compared with CDC category C patients in univariate models. Neither NVP treatment nor CDC category C were significant after adjusting for other covariates in multivariate models. Short-Term Virologic Response to HAART In 223 (81%) children with a viral load available at baseline and 24 weeks, the median reduction in HIV-1 RNA after 24 weeks of HAART was 3.43 (IQR: 3.86 to 2.89) log 10 copies/ml. Two hundred thirty-eight children had a viral load result available at 24 weeks (including 10 children who died before week 24 and considered virologic failures), and of these, 195 (79%) children were suppressed at 400 copies/ml and 137 (55%) were suppressed at 50 copies/ml. Long-Term CD4 Response to HAART Two hundred eighteen children had follow-up data available at 96 weeks. An additional 14 who died before week 96 were included as immunologic failures in logistic regression models. The median CD4% increase at 96 weeks was 18 (IQR: 12 23) percent. Of the 232 children 107 (46%) were at TCD4%. In both univariate and multivariate models (Table 4), younger age (OR: 0.86, 95% CI: ), higher baseline CD4% (OR: 2.15, 95% TABLE 3. Predictors of CD4 Response to HAART at Week 24 in Children With Baseline CD4% 25% at Initiation of HAART Using Multivariate Logistic Regression Model CD4% Increase by 10% CD4% Increase to 25% OR (95% CI) P* OR (95% CI) P* Age per yr 0.91 ( ) ( ) Baseline CD4% per 5% 1.07 ( ) ( ) Baseline HIV RNA per 1.48 ( ) 0.16 NS NS 1 log 10 copies/ml Female vs. male 2.05 ( ) ( ) 0.07 CDC N, A, B vs. CDC C 1.51 ( ) ( ) 0.95 Regimen NVP vs. EFV NS NS 1.74 ( ) 0.38 *Multivariate models including all variables significant in univariate models at P Variables with P NS indicates not significant in univariate model. TABLE 4. Univariate and Multivariate Predictors of Reaching CD4% of 25% at Week 96 in 232* Children With Baseline CD4% 25% at Initiation of HAART Univariate Model Multivariate Model OR (95% CI) P* OR (95% CI) P* Age per yr 0.83 ( ) ( ) Baseline CD4% per 5% 2.27 ( ) ( ) Baseline HIV RNA per ( ) 0.87 log 10 copies/ml Female vs. male 2.37 ( ) ( ) CDC N, A, B, vs. CDC C 1.98 ( ) ( ) 0.58 Regimen NVP vs. EFV 0.78 ( ) 0.36 Virologic suppression over all follow-up Ongoing viral 1.00 (ref) (ref) 0.04 replication Virologic suppression 3.95 ( ) 3.74 ( ) Unknown 2.01 ( ) 3.34 ( ) *This includes 14 children who died before week 96 and were imputed as immunological failures. Variables with P Ongoing viral replication defined as plasma HIV RNA 1000 copies/ml on 2 consecutive visits after week 24. Virologic suppression defined as plasma HIV RNA 1000 copies/ml after initial suppression Lippincott Williams & Wilkins

4 The Pediatric Infectious Disease Journal Volume 28, Number 6, June 2009 Immunologic Response to HAART CI: ), female gender (OR: 2.71, 95% CI: ), and being virologically suppressed after week 24 (OR: 3.74, 95% CI: ) were associated with reaching TCD4%. Kaplan-Meier Estimates of Time to Reach TCD4 Cell Percent of 25% We used the entire cohort to assess the probability of reaching CD4% target over time. The overall probability of reaching TCD4% was 51% (95% CI: 45% 57%) by week 96 and 75% (95% CI: 69% 81%) by week 192. Kaplan-Meier curves of the probability of reaching TCD4% with time stratify by baseline CD4, gender, age, and degree of viral suppression are shown in Figure 1 (Supplemental Digital Content 2, In these analyses, children with the highest probabilities of achieving TCD4% with time were those with higher baseline CD4% (Logrank ; P ; Fig. 1A, Supplemental Digital Content 2, females (Logrank ; P ; Fig. 1B, Supplemental Digital Content 2, and younger children (Logrank ; P ; Fig. 1C, Supplemental Digital Content 2, The probability of children with a baseline CD4% of 0% to 5% and 6% to 14% achieving TCD4% by week 192 was 65% (95% CI: 56% 74%) and 80% (95% CI: 69% 88%), respectively; the probability of children with baseline CD4% of 15% to 24% reaching TCD4% by week 168 was 98% (95% CI: 89% 99.8%). We also conducted a sensitivity analysis comparing probability of achieving TCD4 count in children who always had undetectable viral load compared with those who had ongoing viral replication. Children who had viral load that was always undetectable after week 24 had a significantly higher probably of reaching TCD4 cell count (Logrank ; P ; Fig. 1D, Supplemental Digital Content 2, This was also true after stratifying for baseline CD4 count (Stratified Logrank ; P ), however within stratum tests only showed a significant difference in those with baseline CD4% between 0% and 5%. Because the proportional hazards assumption did not hold, we were unable to perform a multivariate survival analysis with these data. DISCUSSION This study demonstrates the efficacy of NNRTI-based HAART regimens in ARV-naive Thai children. We found the probability of immune recovery, defined as reaching CD4% of 25% was 51% and 75% by week 96 and 192, respectively. Predictors of successful immune recovery were younger age, female gender, a higher baseline CD4%, and sustained virologic suppression. Immune recovery was not related to pre-haart viral load or clinical status. The magnitude of improvement in CD4% in our study is better than that in pretreated children reported from western countries. In our study, the probability of achieving CD4% 25 during 192 weeks of follow-up was 65%, 80% and 98% in children with baseline CD4 5%, 6% to 14% and 15% to 24%, respectively. In contrast, in the Pediatric AIDS Clinical Trials Group 219 study of 702 pretreated children who received PI-based HAART, only 33%, 26%, and 49% of children with pre-pi CD4 of 5%, 5% to 14%, and 15% to 24%, respectively, achieved CD4% of 25% after 3 years of HAART. 10,16 However, our results are similar to another study conducted by Medecins Sas Frontieres (MSF) in a resource limited setting which showed that 62% of children younger than 5 years of age achieved a CD4 cell percentage 25% after 1 year of NNRTI-based HAART. The immune recovery rate was dependent upon baseline CD4; 32%, 63%, and 82% of children with baseline CD4 of 5%, 5% to 15%, and 15% achieved near normal CD4 value, respectively. 6 The time to reach TCD4 from the MSF cohort was shorter than this report, which might be explained by the younger age and higher baseline CD4% at initiation of HAART. The information from our study and from MSF cohort shows the great potential for immune recovery after initiation of NNRTI-based HAART in children. The association between younger age and successful immune recovery demonstrated in our study is similar to previous reports both in pediatric, 10,11 and adult cohorts. 15,16 This might be explained by a more preserved thymus gland in repopulating the T lymphocyte after HAART in young children. However, the gender difference on response to HAART that we found is controversial in adults and no data exist in children. A large Italian cohort of over 2400 adults who received PI-based HAART showed no difference in terms of proportion of patients achieving viral suppression at 12 month between female and male patient (HR 1.04, 95% CI: ) and also CD4 response after HAART. 17 Another study in 450 adults showed that women achieved viral suppression at a faster rate than men with an adjusted HR of 1.46 (95% CI: ). 18 The study from Brazil where the access to ARV therapy is widely available, there was an evidence that women had higher risk of AIDS-related death compare with men with HR of 1.86 (95% CI: ) after controlling for prescribed ARV regimen. 19 In our cohort, female children had a better immunologic and virologic response than males. The reasons for this gender difference in our study remains unclear, but might be related to confounding factors, for example, adherence to treatment. Further studies are needed to confirm whether gender differences exist. Our finding that higher baseline CD4 value predicts immunologic recovery is supported by several other cohorts. A large cohort from 861 HIV-infected adults in Spain with median follow-up time of 42 months showed that patients with baseline CD4 count of 200 and of 201 to 350 cells/mm 3 had a significantly lower chance of reaching CD4 500 cells/mm 3 compared with patients with baseline CD4 350 cells/mm 3 (OR 0.32 and 0.69, respectively). 14 A cohort of 655 adults from Johns Hopkins HIV clinic showed that the CD4 recovery reached a plateau by the fourth year of sustained viral suppression in all CD4 cell count strata. The percentage of patients who achieved a CD4 cell count of 500 cells/ mm 3 after receiving 6 years of suppressive HAART were 42%, 66%, and 85% for those who started HAART with a CD4 cell count of 200, 201 to 359, and 350 cells/mm 3, respectively. 16 Although we found that children treated by NNRTI-based HAART have a good potential for immune recovery, the probability of reaching CD4% of 25% in children with baseline CD4% 5% was only 65% by week 192. This finding is supported by Soh et al 10 who reported that children starting HAART with CD4 5% are less likely to attain values approaching the normal range at the median follow-up time of 3 years. The recent published data of Pediatric AIDS Clinical Trials 219 study, which included data of 1236 children receiving HAART showed that only 36% of children with CD4 15% at baseline achieved CD4 25% at 5 years after HAART initiation compared with 59% of children with baseline CD4% of 15% to 24%. 20 This raises the importance of early diagnosis and treatment for HIV-infected children before they reach a stage of profound immunodeficiency. The strength of our study is that it is based on the prospective data collection of a large cohort of children from resourcelimited settings with long follow-up to 4 years, which allow us to assess the probability to achieve immune recovery. Many countries that lack routine viral load monitoring follow the World Health Organization or their country guidelines immunologic failure criteria in defining treatment failure to HAART. Adult studies have 2009 Lippincott Williams & Wilkins 491

5 Puthanakit et al The Pediatric Infectious Disease Journal Volume 28, Number 6, June 2009 shown that this could incorrectly diagnose treatment failure in patients who have viral load suppression but have a slow CD4 recovery due to their low baseline CD4. 15,16,21 Our results may help to guide clinicians in assessing immunologic response to NNRTI-based HAART in children of different age groups and degrees of immunosuppression. Nevertheless, there are several limitations. First, viral load testing was not performed routinely on all children. Secondly, we did not have data on the adherence to therapy, which is important in assessing treatment success. In conclusion, this study demonstrates that in resourcelimited settings, administration of NNRTI-based HAART to children with moderate to severe immunosuppression can have good outcomes; however, the CD4 recovery was limited by older age, lower baseline CD4%, and HIV viremia. 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