In HIV-1-infected children, disease progression and response to antiretroviral therapy (ART) have traditionally been monitored using CD4 cell percentage (i.e. the number of CD4 cells expressed as a percentage of total lymphocytes) rather than the absolute CD4 cell count, the marker generally used in adults. This is primarily because of the large natural decline in CD4 cell count in early life . In line with clinical practice, most paediatric studies of prognostic markers for clinical HIV disease progression have focussed on CD4 cell percentage, and comparatively little information is available on CD4 cell count . It is important to fill this gap in the current knowledge for two reasons. First, many of the cheaper CD4 cell assays being developed for use in resource-limited settings estimate the absolute CD4 cell count but not the CD4 cell percentage [3,4]. Paediatricians in these settings may therefore have no choice but to monitor HIV infection with the CD4 cell count, or else to embark on a more complex course involving the separate measurement of differential white blood cell counts and deriving the CD4 cell percentage from the CD4 cell count and total lymphocyte count. Second, in Europe and the United States increasing numbers of perinatally infected children are surviving into late childhood and adolescence [5,6]. Treatment guidelines suggest that CD4 cell count as well as CD4 cell percentage should be considered when deciding when to initiate ART in older children or adolescents [7,8]. However, it has not been established whether the CD4 cell count levels for initiating ART in adults are also appropriate for older children, nor, if this is confirmed, the age from which they should be used. To inform these issues we have examined the relationship between absolute CD4 cell count and the short-term risk of disease progression in a large group of prospectively followed HIV-infected children.
The organizers of all major European and USA cohort studies and trials of HIV-1-infected children with data before the introduction of combination ART were invited to participate in the HIV Paediatric Prognostic Markers Collaborative Study (HPPMCS). Longitudinal data were received and pooled from a total of 17 studies (five birth cohort studies, three general cohort studies, nine randomized trials), covering the period 1983–2002. Full details of the individual studies have been described elsewhere . Variables provided by the studies included dates of initial AIDS diagnosis, last clinical examination, death, and when last known to be alive; dates when zidovudine and any other antiretroviral drugs were first started; and CD4 cell count and percentage. Details of immunological methods used were not available, but it is presumed that most or all CD4 cell counts were derived from dual-platform methodology, i.e. multiplying CD4 cell percentage obtained from flow cytometry and total lymphocyte count obtained from a haemocytometer.
Separate analyses were conducted to estimate the 12-month risk of progression to death and the 12-month risk of progression to AIDS or death in the absence of effective ART. To minimize presentation bias, children in non-birth cohort studies were excluded from the analysis if they experienced an endpoint within one month of diagnosis of HIV infection. To take account of the multiple CD4 cell measurements per child, each value contributed a unit of observation to a survival analysis, i.e. the timescale was reset to 0 at each new measurement, with current age and CD4 cell count defined as the baseline covariates. Follow-up was censored at the earliest of the following: date of last clinical visit (for analyses of AIDS) or date last known to be alive (for analyses of death); 12 months after the last measurement of CD4 cell count; 6 months after starting any antiretroviral drug other than zidovudine monotherapy, which has a marginal clinical effect . The rationale for the 6-month extension was to reduce bias resulting from the selective use of combination therapy in children with a poor prognosis .
Disease progression estimates were derived from exponential survival models with a hazard rate λ = a + b.exp(−k.CD4). The parameters a, b, and k were allowed to depend on age: loge (a) = a1 + (a2.age), loge (b) = b1 + (b2.age), loge (k) = k1 + (k2.age). The fit of the model was significantly improved by the age transformation: 4 + 0.1*(age − 4) if ≥ 4 years. Model validity was assessed through plots comparing cumulative numbers (by CD4 cell count) of predicted and observed events. Models that also included study as a factor showed that this variable did not confound the relationship between disease progression risk and the joint effects of age and CD4 cell count (not shown).
A total of 3944 children with a median of six [interquartile range (IQR) three to 10] CD4 cell count measurements per child were included; the median interval between successive measurements was 2.8 months (IQR 1.8–4.6). A total of 566 deaths were observed over 9128 person-years of follow-up (47% during zidovudine monotherapy), and 992 children progressed to AIDS or death over 7309 person-years of follow-up (44% during zidovudine monotherapy). Data after the age of 10 years were infrequent, comprising approximately 5% of total follow-up. The distribution of the CD4 cell count was highly age-specific: the proportion of measurements less than 1000 cells/μl increased from 28% at 6 months, to 34% at one year, 48% at 2 years, 79% at 5 years, and 97% at 10 years; the corresponding proportions less than 500 cells/μl were 13, 16, 21, 30, and 69%.
The estimated 12-month risks of death and AIDS according to current CD4 cell count and age shows two main features (Fig. 1a,b). First, younger children experience a greater risk of disease progression than older children at the same level of CD4 cell count, although this effect is much less pronounced after approximately 4 years of age, particularly for mortality. Poisson regression models restricted to this older age group, adjusting for study and calendar year, showed that the effect of age was statistically non-significant for progression to death (P = 0.8) and for progression to AIDS (P = 0.2), although the power of this analysis is limited by the relatively small number of events (147 deaths and 269 AIDS diagnoses after age 4 years, of which only 20 and 141, respectively, were immediately preceded by a CD4 cell count above 100 cells/μl). Second, the increase in the risk of disease progression with declining CD4 cell count occurs much more sharply for older children than for younger children, the former having a low and stable risk above a CD4 cell count of 200–300 cells/μl. For example, the estimated 12-month risk of AIDS for a 10-year-old child increases from 2.9% at 400 cells/μl, 3.3% at 300 cells/μl, 5.3% at 200 cells/μl, and 14.9% at 100 cells/μl. The corresponding values for the 12-month risk of death are 0.2, 0.3, 1.0, and 5.3%.
The Centers for Disease Control and Prevention have defined severe immune suppression in HIV-1-infected children in terms of the CD4 cell percentage (< 15% at all ages) and CD4 cell count (< 750 cells/μl for < 1 year, < 500 cells/μl for 1–5 years, < 200 cells/μl for 6–12 years) . Figure 2 shows the estimated 12-month risk of death at these levels of CD4 cell count and CD4 cell percentage. In infancy, the pattern is similar for both markers, a very high risk after birth, declining rapidly to a moderately high risk (12%) at the age of one year. Between 6 and 12 years of age, a CD4 cell count of 200 cells/μl predicts a 12-month mortality risk of 0.7–2.1%; the risk for a CD4 cell percentage of 15% is slightly lower, ranging from 0.5 to 1.4%. At intermediate ages (1–5 years) the age gradient is much steeper for a fixed level of CD4 cell count (500 cells/μl) than for the CD4 cell percentage (15%); a similar pattern is seen if alternative CD4 cell count/percentage values are used. This difference between the two markers can be explained, at least partly, by the much larger decline in the absolute CD4 cell count than in the CD4 cell percentage in young normal children .
Another way of showing these data, which has been used in guidelines to justify the timing of therapy initiation, is to compare the estimated values of CD4 cell percentage and CD4 cell count that relate to a specified level of risk . Table 1 shows the 12-month risk of death of 2 and 5% and of AIDS of 5 and 10%, and further illustrates the strong influence of age on these values. Changes are particularly striking for the CD4 cell count at younger ages. For example, for a 5% risk of death, the CD4 cell count threshold would need to change from 1162 cells/μl at one year to 295 cells/μl at 3 years, a fourfold reduction. In contrast, the reduction for the CD4 cell percentage would be only 1.8-fold, from 24 to 13%. The change would be similar for the two markers between the ages of 4 and 10 years (1.5-fold reduction for CD4 cell count from 153 to 103 cells/μl; 1.6-fold reduction for CD4 cell percentage from 11 to 7%). It should be noted that at certain ages the specified level of risk is not attainable at any value of CD4 cell count or percentage, for example, a 5% risk of AIDS below the age of 2 years. This is a consequence of the intrinsically weak prognostic value of both markers in infancy.
Treatment guidelines for HIV-1-infected adults currently recommend that ART should be deferred in asymptomatic individuals until the CD4 cell count declines to 200–350 cells/μl [12,13]. The rationale for this recommendation is the very low short-term risk of AIDS above this range of values in untreated individuals, coupled with increasing concerns about the long-term toxicity of ART . A key finding from our analysis is the demonstration of a similar CD4 cell count threshold effect in children older than 4 or 5 years, consistent with the results of a previous smaller study that found a threshold of 200 cells/μl in children older than 6 years . Although different AIDS case definitions apply for HIV-1-infected adults and children, it is nonetheless of interest to compare its frequency of occurence between the two groups. An annual AIDS incidence of approximately 4% among untreated adults with a CD4 cell count of 200–350 cells/mm3 was reported from a large cohort of seroconverters . This is comparable to the level observed among older children in the present study; for example, the estimated annual incidence for a 10-year-old child is 5.3%, and 3.0% at a CD4 cell count of 200 cells/μl and 350 cells/μl, respectively.
Evidence from this study therefore suggests that it may be appropriate to extend CD4 cell count criteria for initiating ART in HIV-1-infected adults to children, possibly as young as 4 or 5 years of age, i.e. considerably earlier than currently suggested by European and USA paediatric guidelines [7,8]. There was some indication that the risk of disease progression at a given level of CD4 cell count may continue to decline in older children, but this was not conclusive. In adults, the risk increases rather than decreases with age; controlling for the CD4 cell count, AIDS incidence is approximately twice as high at age 55 years than at age 25 years . However, it is noteworthy that adult guidelines do not emphasize this substantial age gradient in adulthood.
One approach in deciding marker thresholds for ART initiation is to choose values that predict a specified level of risk of disease progression. As indicated by Fig. 2 and Table 1, this would require, at minimum, a lowering of the CD4 cell count threshold at each birthday in the first few years of life. This is likely to be too complex for routine clinical practice, particularly in resource-limited settings. Monitoring with the CD4 cell percentage allows the use of wider age bands because its association with disease progression risk is less age dependent. The interpretation of longitudinal measurements within an individual child is also more problematical for CD4 cell count because of the large natural decline in this parameter over the first 5 years of life .
Standard technology for CD4 cell count measurements currently remains impractical in many resource-limited settings because of issues of cost and technical expertise. Given that many alternative low-cost assays enumerate the CD4 cell count but not the CD4 cell percentage [3,4], the risk estimates presented here provide a context for deciding when to initiate ART when only CD4 cell count measurements are available. A research priority is to validate CD4 cell count levels for initiating ART using data from cohorts of untreated HIV-1-infected children in resource-limited settings. Finally, an unresolved question for developed countries is whether CD4 cell count or CD4 cell percentage should be the preferred marker for monitoring, particularly in older children, or whether it may be advantageous to consider both markers in conjunction. The observation that the total lymphocyte count is highly prognostic suggests that the CD4 cell count may be the more powerful marker , and this is currently being investigated in further analyses.
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Appendix: Steering Committee
D.T. Dunn, D.M. Gibb, T. Duong, A.G. Babiker (Medical Research Council Clinical Trials Unit, London, UK); J.P. Aboulker (INSERM SC10, Villejuif, France); M. Bulterys (Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA; Perinatal AIDS Collaborative Transmission Study); M. Cortina-Borja (Institute of Child Health, University College London, London, UK; European Collaborative Study); C. Gabiano (Department of Pediatrics, University of Turin, Turin, Italy; Italian Register for HIV Infection in Children); L. Galli (Department of Pediatrics, University of Florence, Florence, Italy; Italian Register for HIV Infection in Children); C. Giaquinto (Department of Pediatrics, University of Padova, Padova, Italy; Paediatric European Network for Treatment of AIDS; PENTA); D.R. Harris (Westat, Rockville, MD, USA; National Institute of Child Health and Human Development; NICHD Intravenous Immunoglobulin Study Group); M. Hughes (Harvard School of Public Health, Boston, MA, USA; Pediatric AIDS Clinical Trials Group; PACTG); R. McKinney (Duke University Medical Center, Durham, NC, USA; PACTG); L. Mofenson (National Institute of Child Health and Human Development (NICHD), National Institutes of Health, Rockville, MD, USA; NICHD Intravenous Immunoglobulin Study Group); J. Moye (NICHD; Women and Infants Transmission Study); M.L. Newell (Institute of Child Health, University College London, London, UK; European Collaborative Study); S. Pahwa (North Shore–LIJ Research Institute, Manhasset, NY, USA; PACTG); P. Palumbo (UMDNJ Medical School, Newark, NJ, USA; Perinatal AIDS Collaborative Transmission Study); C. Rudin (University Children's Hospital, Basel, Switzerland; Swiss Mother and Child HIV Cohort Study); M. Sharland (St George's Hospital Medical School, London, UK; Collaborative HIV Paediatric Study [CHIPS] of UK and Ireland); W. Shearer (Baylor College of Medicine, Houston, TX, USA; Pediatric Pulmonary and Cardiovascular Complications of HIV Infection Study); B. Thompson (Clinical Trials and Surveys Corp, Baltimore, MD, USA; Women and Infants Transmission Study); P. Tookey (Institute of Child Health, University College London, London, UK; CHIPS).
16. HIV Paediatric Prognostic Markers Collaborative Study. Use of total lymphocyte count for informing when to start antiretroviral therapy in HIV-infected children: a meta-analysis of longitudinal data