In most HIV-infected infants, children and adults, combination antiretroviral therapy (cART) results in suppression of plasma viral load and an increase in peripheral CD4+ T-lymphocyte cell counts [1,2]. In the United States and Western Europe, the availability of cART has been associated with a marked reduction in HIV-related mortality attributable to perinatal HIV infection [3–5]. These successes are being recapitulated in resource-limited settings [3,6–9].
Unfortunately, a discordant treatment response is seen in some paediatric patients in whom immunologic reconstitution does not occur despite virologic suppression [10–18]. This immunological failure (IF) phenotype has not been rigidly defined, but in a child age 5 years or older at baseline, it may be defined as a failure to achieve or maintain a CD4+ T-cell count above the level associated with severe immune suppression (CD4+ cell count <200 cells/μl) .
A variety of explanations could account for this discordant immunological failure–virologic suppression phenotype, including the antiretroviral agents being used, depletion of bone marrow precursor cells that must undergo thymic differentiation into T cells, presence of active coinfections, malnutrition, failure of HIV RNA assays to detect the genetic subtype of HIV-1 with which the child is infected or laboratory error . Previous reports suggest that IF despite virologic suppression (VS) is more common in children with a lower nadir CD4+ T-cell count and older age, but conflicting data have been reported [6,19]. In all of these reports, the number of children with the immunological failure–virologic suppression phenotype appears to be small, and, consequently, the frequency and clinical significance of immunological failure among children with prolonged virologic suppression has remained unclear. In one recent study of adults with persistently low CD4+ T-cell counts during virologically successful therapy , the incidence rate of new AIDS events was higher in the first 6 months after virologic suppression was achieved than in later periods of follow-up. After 2 years of successful suppression, no new AIDS-defining illnesses were seen, despite persistent severe CD4+ lymphocytopenia (<200 cells/μl). No comparable data are available to inform the management of children and adolescents whose CD4+ T-cells remain abnormal despite successful suppression of HIV plasma viremia by antiretroviral therapy (ART).
We examined the frequency and clinical significance of the immunological failure–virologic suppression phenotype in perinatally HIV-infected patients to enhance our understanding of immune reconstitution in HIV-infected children and the risks of continuing a cART regimen that has failed to achieve substantial improvement in CD4+ T-cell counts. We hypothesized that children and adolescents with incomplete immune reconstitution in the setting of sustained virologic suppression are at a greater risk of new HIV/AIDS-related clinical events than individuals whose CD4+ T-cell counts improve or remain above levels indicative of immune suppression.
Materials and methods
The source populations for this study were the Adolescent Master Protocol (AMP) of the Pediatric HIV/AIDS Cohort Study (PHACS), the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Protocol 219C (219C) and the NICHD International Site Development Initiative (NISDI) [3,21,22]. These prospective cohort studies were designed to evaluate the impact of HIV infection and ART on HIV-infected children and adolescents with perinatal infection and enrolled over 3700 perinatally HIV-1 infected children and adolescents in the USA, Latin America and the Caribbean. The protocols were approved by human individuals review boards at each participating institution and written informed consent was obtained from each child's parent or legal guardian. The final study population for our analysis was restricted to perinatally HIV-1-infected children only and included those with a documented period of virologic suppression lasting at least 1 year in duration beginning at or after age 5 years with available CD4+ T-cell counts at the start of the ART that led to virologic suppression, the start of virologic suppression and at 1 year into the period of virologic suppression. Periods of virologic suppression were restricted to those starting after age 5 years to discount the variability in CD4+ T-cell count prior to 5 years of age . For children with multiple periods of virologic suppression meeting the above eligibility criteria, their first period of virologic suppression was chosen for analyses.
Periods of virologic suppression were defined as beginning with two consecutive HIV viral load measures less than 400 copies/ml of plasma and maintaining subsequent measurements below 400 copies/ml, but allowing for isolated (single) measurements of at least 400 copies/ml (i.e. intermittent viremia). The period of virologic suppression was said to have ended at either the final less than 400 readings that preceded two consecutive at least 400 readings or the final viral load less than 400 copies/ml during follow-up if virologic rebound did not occur. If the last measurement during follow-up was at least 400 copies/ml and was preceded by a qualifying suppression period, the suppression period was defined to have ended at the time of the preceding viral load measurement of less than 400 copies/ml. A cART regimen was defined as at least three antiretroviral drugs from at least two different drug classes. Diagnoses with onset during the first 3 years of virologic suppression were reviewed and classified as meeting the definition of an AIDS-defining condition on the basis of the age-appropriate Centers for Disease Control and Prevention (CDC) classification system (i.e. meeting the definition of a CDC class C event) [24,25]. Other clinical outcomes of interest during the first 3 years of virologic suppression included bacterial meningitis, sepsis, cardiomyopathy, cervical dysplasia, mucocutaneous herpes simplex virus (HSV) infections, herpes zoster, lymphoid interstitial pneumonia, nephropathy, nocardiosis, oropharyngeal candidiasis, pneumonia and disseminated varicella.
CD4+ T-cell count trajectories during virologic suppression were examined by plotting mean CD4+ T-cell counts during the study period of virologic suppression by CD4+ cell count at the start of virologic suppression and fitting piece-wise linear regression models to estimate the observed slopes. CD4+ T-cell counts at 6 months, 1 year and 2 years after virologic suppression were also compared relative to counts at the start of virologic suppression. Kaplan–Meier survival curves were used to estimate the median time to achieve CD4+ T-cell counts at least 500 cells/μl, relative to CD4+ T-cell count categories at the beginning of virologic suppression. Kaplan–Meier survival curves were also used to estimate time to first sustained measures of CD4+ cell count less than 200, less than 350 and less than 500 cells/μl among those with CD4+ cell count at least 500 cells/μl at baseline to inform CD4+ cell count monitoring strategies. To evaluate the clinical significance of incomplete immune reconstitution during virologic suppression, incidence rates for first AIDS-defining event during the first 3 years of follow-up were calculated by CD4+ T-cell count at the time of the event and CD4+ T-cell count at baseline. Events were also plotted by time since virologic suppression and CD4+ cell count at the time of the event. Similar analyses were conducted for the other clinical outcomes of interest. Sensitivity analyses excluding those with intermittent (single) viral loads at least 10 000 copies/ml were also conducted for the evaluation of incidence of all clinical events. All analyses were conducted using SAS version 9.2 (SAS Institute, Cary, North Carolina, USA).
Records were available for 3784 children with perinatal HIV enrolled in the AMP and 219C cohorts in the U.S. and Puerto Rico and NISDI sites in Central and South America (Brazil, Mexico, Argentina, Puerto Rico and Peru). Among these, CD4+ T-cell counts and plasma HIV viral load data needed for analysis were available for 933 children who experienced at least 1 year of virologic suppression at 5 years of age or more after starting a cART regimen (Supplemental Table 1, http://links.lww.com/QAD/A645). Most were seen at centres in the U.S. (87%) (Table 1), with the majority of the AMP/219C cohort born prior to 1996 (83%). By contrast, the majority of the NISDI cohort (72%) was born after 1996. Prior to the initiation of a suppressive cART regimen, many had only been previously treated with a non-cART regimen (32% of children from the United States and Puerto Rico, 15% from NISDI sites). At the time of first suppressive cART, participants had a median age of 8.8 years. Virologic suppression was achieved in most cases with cART that employed an HIV protease inhibitor as well as nucleoside/nucleotide reverse transcriptase inhibitors (nRTIs) (52%), a nonnucleoside reverse transcriptase inhibitor as well as nRTIs (12%), or both protease inhibitor and nonnucleoside reverse transcriptase inhibitor in combination with nRTIs (21%) (Table 1).
Immunological changes in response to combination antiretroviral therapy
At the time of initiation of cART that resulted in virologic suppression, 92 children (10%) had CD4+ T-cell counts below 200 cells/μl, while 118 (13%) had 200–349 CD4+ T cells/μl and 138 (15%) had 350–499 CD4+ T cells/μl (Table 2). Early improvements in CD4+ T-cell counts were commonly seen, and by the time virologic suppression was achieved, the number of children with CD4 T-cell counts under 500 cells/μl had decreased from 348 to 206 (37% to 22% of the total cohort) and only 29 (3%) children still had a CD4+ T-cell count of less than 200 cells/μl (Table 2). In children whose CD4+ T-cell counts were initially less than 500 cells/μl, a biphasic response was seen with CD4+ T-cell numbers rising more rapidly during the first 6 months after cART initiation than in subsequent months (Fig. 1a). The rate of initial rise was twice as great in children with baseline CD4+ T-cell counts less than 200 cells/μl than in children with 350–499 CD4+ T cells (556 vs. 258 cells/year). (Supplemental Table 2, http://links.lww.com/QAD/A645). The time until the first CD4+ T-cell count met or exceeded 500 cells/μl was strongly and inversely related to the baseline CD4+ cell count values (Fig. 1b): the median time to reach this value was 1.29, 0.78 and 0.46 years for children with CD4+ T cells at baseline of less than 200, 200 to less than 350 and 350–499 cells/μl, respectively. The proportion of children with CD4+ T cell counts less than 500 cells/μl progressively declined during virologic suppression, but CD4+ cell counts below this threshold were observed in 14% after 1 year and 8% at 2 years (Supplemental Table 3, http://links.lww.com/QAD/A645).
CD4+ T-cell counts were very stable among the 727 children (78% of the total study population) whose counts were 500 cells/μl or higher at the time that virologic suppression was confirmed: fewer than 10% experienced sustained periods in which CD4+ T-cell counts dropped below thresholds of either 350 or 200 cells/μl (Fig. 2a). This was also true for the NISDI cohort when examined separately from children in the United States or Puerto Rico (Supplemental Figure 1, http://links.lww.com/QAD/A645).
These data indicate that durable increases in CD4+ T-cell counts occur in children with perinatal HIV infection, although maximal improvement in the T-cell count may not occur for years after virologic suppression has occurred. However, prolonged CD4+ lymphocytopenia is seen in some individuals even after prolonged virologic suppression (Supplemental Table 3, http://links.lww.com/QAD/A645).
Clinical events occurring during virologic suppression
To assess the clinical impact of low CD4+ T-cell counts at the beginning of and during virologic suppression, we examined the incidence of new clinical events indicative of immune suppression. As outlined in Table 3, nine children experienced one or more new CDC C events during the first 3 years following virologic suppression, including four in the first 6 months following virologic suppression and five at a time when CD4+ T-cell counts were 500 cells/μl or more. Incidence rates for the first CDC C event during this follow-up period did not statistically differ by person-time spent with CD4+ T-cell counts less than 500 cells/μl or by CD4+ T-cell counts at the start of virologic suppression (data not shown).
We also examined reports of non-AIDS-defining clinical events of interest. Ninety individuals experienced a CDC C event and/or a non-AIDS defining clinical event of interest throughout the first 3 years of virologic suppression; these events largely occurred at a time when CD4 counts were at least 500 cells/μl (Fig. 2b and Supplemental Table 4, http://links.lww.com/QAD/A645). Similar to the CDC C events, incidence rates for these events did not statistically differ by person-time spent with CD4+ T-cell counts less than 500 cells/μl or by CD4+ T-cell counts at the start of virologic suppression. We performed a similar analysis with data related to the CDC C and the other clinical events combined, with similar findings: the incidence rates of these events were not influenced by these CD4+ T-cell parameters. Twenty-three individuals experienced recurrent events (CDC C and/or non-AIDS defining) during the first 3 years of follow-up; 14 of these individuals experienced one or more episodes of pneumonia after an earlier significant clinical event (Supplemental Table 5, http://links.lww.com/QAD/A645).
In adults, discordant virologic and immunological responses to ART have been associated with a higher risk of death from both AIDS-defining and non-AIDS defining causes among those with persistently low CD4+ lymphocyte counts [20,26–28]. A failure of cART to increase CD4+ T-cell counts, despite suppression of HIV-1 plasma viremia, also occurs in infants, children and adolescents, but the frequency and clinical significance of this phenomenon have been unclear. In this multinational, large paediatric observational study, we found that a failure to achieve or maintain a CD4+ T-cell count above 200 cells/μl in the setting of virologic suppression appears to be rare: at 2 years into virologic suppression, only four children had CD4+ cell counts recorded below this threshold. Subsequent sustained periods of severe CD4+ lymphocytopenia (<200 cells/μl) also appeared to be very uncommon during prolonged follow-up, occurring in only three of 727 (0.4%) individuals with data extending to 15 years of follow-up (Fig. 2a). These data contrast with studies of adults, in whom failure to achieve full CD4+ T-cell count reconstitution appears to be more common; this likely reflects residual thymic function generally seen in children and adolescents, including those with histories of AIDS-defining conditions [27,29].
However, immune reconstitution was protracted in some children. In agreement with smaller paediatric studies [15,30–35], the time to achieve a maximal CD4+ T-cell count was highly dependent on the CD4+ cell count at the beginning of the cART regimen that resulted in virologic suppression. We found this to be true when data from the smaller NISDI and the larger AMP/219c cohorts were examined separately, or when the data were examined together. Children with the lowest CD4+ T-cell counts showed the greatest relative and absolute initial improvements in CD4+ T-cell concentration, but took the longest to achieve a normal CD4+ cell count threshold. These data are consistent with adult cohort studies [26–27]. Data from this study also suggest that clinicians should anticipate that the maximal improvement in CD4+ T-cell count may not occur for several years after virologic suppression has occurred.
The clinical significance of having low CD4+ T-cell counts during virologic suppression has been evaluated in a limited number of studies of adults. Zoufaly et al. examined clinical outcomes of HIV-infected adults with discordant virologic and immunological responses to ART. New AIDS-defining events occurred most frequently during the initial 6 months of complete virologic suppression and decreased significantly thereafter. We attempted to replicate this analysis but found that AIDS-defining illnesses were uncommon in the paediatric cohorts studied: only nine study participants experienced one or more new CDC C events during the first 2 years of virologic suppression. This likely reflects the fact that only 10% of individuals studied had CD4+ T-cell counts below 200 cells/μl at the beginning of cART. It may also reflect the fact that majority of children were receiving some form of ART when the regimens resulting in virologic suppression were begun, as immunologic improvements may occur even if HIV viremia is not fully suppressed. However, we also noted a high frequency of serious, albeit non-AIDS defining, illnesses among children in this cohort (Supplemental Table 5, http://links.lww.com/QAD/A645). The occurrence of these events throughout the follow-up period was independent of CD4+ T-cell count and is indicative of ongoing immunologic deficits. Nonetheless, incidence rates for CDC C defining and non-AIDS defining clinical events did not statistically differ by person-time spent with CD4+ T-cell counts less than 500 cells/μl (compared with time above this threshold) or by CD4+ T-cell counts at start of virologic suppression, serving as a reminder that CD4+ T-cell counts are an imperfect indicator of immune function .
The strengths of this study include its multinational composition, prolonged data collection period and study population that reflects the racial and ethnic diversity of communities most affected by paediatric HIV in the western hemisphere. However, this analysis has several limitations. First, it focused on children 5 years of age or more. In contrast to this older population, disease progression is more rapid among infants and younger children, in whom AIDS-defining illnesses often occur at CD4+ T-cell counts that are well above normal range for adolescents and adults. In addition, the spacing of viral load measurements from some individual children may have been broad enough to miss periods of HIV viremia that had deleterious effects on immune reconstitution. We used less than 400 copies/ml as the threshold for defining virologic suppression, as most data in the cohorts studied were accumulated prior to widespread use of HIV-1 RNA monitoring with greater levels of sensitivity. It is possible that lower level viremia, or transient increases in HIV viral load, may have interfered with or disrupted immune reconstitution in some children. In an effort to determine whether individuals with high transient increases in viral load might have significantly influenced our results, we performed an additional analysis restricted to individuals who did not experience a viral load at or above 10 000 copies/ml during the first 3 years of follow-up. Removing these 34 individuals from the original set of 933 did not alter our estimates of the rates of CDC C or other clinical events or the impact of baseline CD4+ T-cell counts on CD4+ cell reconstitution (data not shown).
Although not the primary focus of this study, we also examined the stability of CD4+ T-cell counts in children who had counts of 500 cells/μl or more at the time that virologic suppression was confirmed. It was very uncommon for these children to have subsequent reductions in CD4+ T-cell counts to below 500 cells/μl, lending support to the recent suggestion that infrequent CD4+ cell count monitoring may be reasonable during long periods of virologic suppression .
Overall, the vast majority of children with perinatal HIV infection in this study who began a cART regimen associated with virologic suppression experienced significant improvements in CD4+ T-cell counts. This immunological improvement was not maximized in some individuals for several years. In addition, a small, but significant proportion of children (8%) did not reach a CD4+ T-cell count of 500 cells/μl or more during virologic suppression during the periods of observation. The continued occurrence of clinically important diagnoses despite CD4+ cell reconstitution in most individuals highlights the need for improved measures of immune function in children.
We thank the children and families for their participation in PHACS, and the individuals and institutions involved in the conduct of PHACS. The study was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development with cofunding from the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Diseases, the Office of AIDS Research, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, the National Institute on Deafness and Other Communication Disorders, the National Heart Lung and Blood Institute, the National Institute of Dental and Craniofacial Research, and the National Institute on Alcohol Abuse and Alcoholism, through cooperative agreements with the Harvard University School of Public Health (HD052102) (Principal Investigator: George Seage; Project Director: Julie Alperen) and the Tulane University School of Medicine (HD052104) (Principal Investigator: Russell Van Dyke; Co-Principal Investigator: Kenneth Rich; Project Director: Patrick Davis). Data management services were provided by Frontier Science and Technology Research Foundation (PI: Suzanne Siminski), and regulatory services and logistical support were provided by Westat, Inc (PI: Julie Davidson).
The following institutions participated in conducting PHACS AMP in 2013, in an alphabetical order: Ann & Robert H. Lurie Children's Hospital of Chicago, Baylor College of Medicine, Bronx Lebanon Hospital Center, Children's Diagnostic & Treatment Center, Children's Hospital, Boston, Jacobi Medical Center, Rutgers – New Jersey Medical School, St. Christopher's Hospital for Children, St. Jude Children's Research Hospital, San Juan Hospital/Department of Pediatrics, Tulane University Health Sciences Center, University of California, San Diego, and the University of Colorado Denver Health Sciences Center.
The following sites participated in PACTG 219/219C: University of New Jersey Medical and Dental School – Department of Pediatrics, Division of Allergy, Immunology & Infectious Diseases, Boston Medical Center, Division of Pediatric Infectious Diseases, Med, Children's Hospital LA – Department of Pediatrics, Division of Clinical Immunology & Allergy, Long Beach Memorial Medical Center, Miller Children's Hospital, Harbor - UCLA Medical Center - Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins Hospital & Health System – Department of Pediatrics, Division of Infectious Diseases, University of Maryland Medical Center, Division of Pediatric Immunology & Rheumatology, Texas Children's Hospital, Allergy & Immunology Clinic, Cook County Hospital, Children's Hospital of Columbus, Ohio, University of Miami Miller School of Medicine, Division of Pediatric Immunology & Infectious Disease, University of California San Francisco School of Medicine, Department of Pediatrics, Children's Hospital & Research Center Oakland, Pediatric Clinical Research Center & Research Lab, University of California San Diego Mother, Child & Adolescent HIV Program, Duke University School of Medicine - Department of Pediatrics, Children's Health Center, University of North Carolina at Chapel Hill School of Medicine - Department of Pediatrics, Division of Immunology and Infectious Diseases, Schneider Children's Hospital, Harlem Hospital Center, New York University School of Medicine, Division of Pediatric Infectious Diseases, Children's National Medical Center, ACT, University of Washington School of Medicine - Children's Hospital and Regional Medical Center, University of Illinois College of Medicine at Chicago, Department of Pediatrics, Yale University School of Medicine - Department of Pediatrics, Division of Infectious Disease, SUNY at Stony Brook School of Medicine, Division of Pediatric Infectious Diseases, Howard University Hospital, Department of Pediatrics & Child Health, LA County/University of Southern California Medical Center, University of Florida Health Science Center Jacksonville, Division of Pediatric Infectious Disease & Immunology, North Broward Hospital District, Children's Diagnostic & Treatment Center, University of Rochester Medical Center, Golisano Children's Hospital, Medical College of Virginia, St. Jude Children's Research Hospital, Department of Infectious Diseases, University of Puerto Rico, U. Children's Hospital AIDS, Children's Hospital of Philadelphia, Center for Pediatric & Adolescent AIDS, St. Christopher's Hospital for Children/Drexel University College of Medicine, Bronx-Lebanon Hospital Center, Infectious Diseases, New York Medical College/Metropolitan Hospital Center, University of Massachusetts Memorial Children's Medical School, Department of Pediatrics, Baystate Health, Baystate Medical Center, Connecticut Children's Medical Center, Medical College of Georgia School of Medicine, Department of Pediatrics, Division of Infectious Disease, University of South Alabama College of Medicine, Southeast Pediatric ACTU, LSU Health Sciences Center, Tulane University Health Sciences Center, St. Josephs Hospital and Medical Center, Cooper University Hospital - Children's Hospital Boston, Division of Infectious Diseases, David Geffen School of Medicine at UCLA - Department of Pediatrics, Division of Infectious Diseases, Children's Hospital of Orange County, Children's Memorial Hospital - Department of Pediatrics, Division of Infectious Disease, University of Chicago - Department of Pediatrics, Division of Infectious Disease, Mt. Sinai Hospital Medical Center - Chicago, Women's & Children's HIV Program, Columbia University Medical Center, Pediatric ACTU, Incarnation Children's Center, Cornell University, Division of Pediatric Infectious Diseases & Immunology, University of Miami Miller School of Medicine - Jackson Memorial Hospital, Bellevue Hospital (Pediatric), San Francisco General (Pediatric), Phoenix Children's Hospital, Metropolitan Hospital Center (N.Y.), University of Cincinnati, SUNY Downstate Medical Center, Children's Hospital at Downstate, North Shore University Hospital, Jacobi Medical Center, University of South Florida - Department of Pediatrics, Division of Infectious Diseases, Cornell University, Oregon Health & Science University - Department of Pediatrics, Division of Infectious Diseases, Children's Hospital of the King's Daughters, Infectious Disease, Lincoln Medical & Mental Health Center, Mt. Sinai School of Medicine, Division of Pediatric Infectious Diseases, Emory University Hospital, San Juan City Hospital, UMDNJ - Robert Wood Johnson, Ramon Ruiz Arnau University Hospital, Medical University of South Carolina, SUNY Upstate Medical University, Department of Pediatrics, Wayne State University School of Medicine, Children's Hospital of Michigan, Children's Hospital at Albany Medical Center, Children's Medical Center of Dallas, Children's Hospital - University of Colorado at Denver and Health Sciences, Center, Pediatric Infectious Diseases, Columbus Children's Hospital, University of Florida College of Medicine - Department of Pediatrics, Division of Immunology, Infectious Diseases & Allergy, University of Mississippi Medical Center, Palm Beach County Health Department, Children's Hospital LA - Department of Pediatrics, Division of Adolescent Medicine, Vanderbilt University Medical Center, Division of Pediatric Infectious Diseases, Washington University School of Medicine at St. Louis, St. Louis Children's Hospital, Children's Hospital & Medical Center, Seattle ACTU, Oregon Health Sciences University, St. Luke's-Roosevelt Hospital Center, Montefiore Medical Center - Albert Einstein College of Medicine, Children's Hospital, Washington, D.C., Children's Hospital of the King's Daughters, University of Alabama at Birmingham, Department of Pediatrics, Division of Infectious Diseases, Columbus Regional HealthCare System, The Medical Center, Sacred Heart Children's Hospital/CMS of Florida, Bronx Municipal Hospital Center/Jacobi Medical Center.
The conclusions and opinions expressed in this article are those of the authors and do not necessarily reflect those of the National Institutes of Health or U.S. Department of Health and Human Services.
P.K., R.B.V.D., K.P., R.H., M.J.A., J.O., G.R.S., P.L.W., W.B., A.W. were involved in study design. K.P. and B.K. performed the data acquisition and analysis, and participated in drafting and revision of manuscript with P.K. and R.V.D., with input from all authors. R.V.D., R.H. and J.P. were responsible for oversight of the study for the contributing PHACS and NISDI networks. All authors played a role in review in review of the final manuscript.
Conflicts of interest
There are no conflicts of interest.
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