Lipodystrophy develops in the course of HIV infection under intensive treatment , such as highly active antiretroviral therapy (HAART). Fat redistribution, including peripheral atrophy and central hypertrophy, was first described in adults but has also been reported in children .1-11 children presenting with lipodystrophy is variable (10%-33%) in the series of patients under study. As in adults, the assessment of lipodystrophy is hampered by the lack of a consensus on the definition.
Several serious metabolic complications have been described in association with fat redistribution, such as insulin resistance and dyslipidemia. The mechanisms underlying these changes are not yet elucidated. Although drug toxicity has been strongly suspected and can be influenced by individual susceptibility, the role of the virus itself has also been suggested. For several authors, these complications are mostly viewed as adverse effects of HAART. Protease inhibitors (PIs) have been strongly suspected in the development of insulin resistance and hypertriglyceridemia, and it has been shown that short-term administration of PIs in healthy volunteers can indeed induce insulin resistance .12 treatment and quality of life.
In children , there are few reports documenting these complications of HIV infection. The frequency and severity of lipodystrophy and metabolic disturbances are still poorly understood in the pediatric population. It seems that lipodystrophy is indeed detectable in children , although the clinical picture of fat redistribution may be more subtle and more difficult to identify than in adults. If the definition of lipodystrophy is still controversial in adults, it is even more so in children , where patients are less numerous and adiposity itself is changing with physiologic growth. Moreover, the rate of progression and the factors associated with the evolution of these complications need to be documented.
The aim of the present study was first to assess the rate of progression of lipodystrophy and the associated metabolic disturbances based on a prospective and standardized evaluation over a 2-year period in a large multicenter sample of HIV -infected children . The second aim was to assess risk factors associated with lipodystrophy and metabolic disturbances.
PATIENTS AND METHODS
Patients
Patients were enrolled in this study from December 2000 to April 2002 in the Clinical Immunology Departments of 3 different university children 's hospitals in Paris. Eligibility criteria were proven HIV infection, age from 2 to 18 years, and not receiving treatment of any other chronic disease. Exclusion criteria were a severe clinical illness in the weeks before the observation and administration of steroids.
A total of 130 children were included, and 89 were seen for a new evaluation 24.7 ± 1.7 months after the first observation.
Children were included after receipt of informed parental consent, and the study protocol was approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale (CCPPRB) Paris/St. Louis (Paris, France). Consent from the children was also obtained whenever possible.
Protocol
At each visit, the children were evaluated at the time of an outpatient medical visit. All patients were seen by the same investigator at each center.
Puberty was rated using Tanner stages. To describe fat distribution, 3 parameters were used: body mass assessed by body mass index (BMI), percent fat assessed by bioimpedancemetry for the overall fat mass, and skinfold thickness for the distribution of adiposity. BMI was calculated as the weight (kg)/height2 (m2 ) ratio and was expressed as a z -score according to reference values established in French children .13
Percent of body fat mass was measured using bioelectrical impedance measurement at 50 kHz and standard electrode positions (RJL Systems, Clinton Township, NJ), and calculations were made using the equation by Schwenk et al14 children . An independent investigator blinded to the children 's treatment performed evaluation of the lipodystrophy . Skinfold thicknesses (biceps, triceps, suprailiac, and subscapular) were measured twice by Harpenden skinfold calipers (Chambly-Medical, Chambly, France) at 5-minute intervals at each site. Subsequently, the 2 individual values for each site were averaged, and results were expressed as a z -score of the normal distribution for gender and age according to the French growth standard curve for each site.15 z -score.
Waist circumference was measured as the smallest circumference at the level of the umbilicus and was expressed as a z -score for gender and age.
Serum samples were collected after a 10-hour fast, and total cholesterol, triglycerides, free thyroxine (T4 ), thyroid stimulating hormone (TSH), and cortisol concentrations were measured. Some children underwent an oral glucose tolerance test (OGTT) according to the World Health Organization's guidelines for children . Plasma glucose and serum insulin concentrations were measured at fasting and 30 and 120 minutes after loading. Results were interpreted according to the Word Health Organization's criteria,16 insulin resistance .
Antiretroviral treatment (medication and dosage) was recorded from the medical charts of the children from 4 years before inclusion onward. This time lag covers the whole period of prescription of PIs widely used since 1996 but not the period of prescription for all antiretroviral drugs in the study population; indeed, in most children , treatment had been given for much longer than 4 years.
Nadir values of CD4 T-lymphocyte percentage and highest plasma HIV RNA levels were recorded from the chart, and current values were measured at the time of observations. Severity of the disease was classified according to the Centers for Disease Control and Prevention's (CDC) revised pediatric classification17
Analytic Methods
Glucose, cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured by automated enzymatic methods.
Serum insulin concentrations were measured using an immunoradiometric method (Bi-Insulin IRMA; Sanofi-Diagnostic Pasteur, Nanterre, France). Cross-reactivity with proinsulin and derived metabolites was less than 1%.
Serum free T4 , TSH, and cortisol concentrations were measured using an automated chemoluminescence system (ACS 180 Chiron Diagnostic; Bayer Diagnostic, Cergy-Pontoise, France).
Values for HIV RNA and CD4 T-lymphocyte percentages were taken from the medical charts of the children .
Statistical Analysis
Definition of Lipodystrophy
The definition of lipodystrophy was based on skinfold thickness measurements. Values obtained for each skinfold were transformed into z -scores for gender and age.
Hypertrophic lipodystrophy is made up of accumulation of fat in the central truncal region. To identify the redistribution of fat depots, this lipodystrophy was defined by at least 1 abnormal central skinfold measurement (>3 standard deviations [SDs] for age and gender), together with an increased ratio of central/peripheral skinfolds (>3 SDs) with no major evidence of obesity (BMI <3 SDs).
Atrophic lipodystrophy is made up of loss of limb tissue and peripheral subcutaneous tissue. It was defined as sunken cheeks and/or any thin peripheral skinfold (<−3 SDs) with BMI in the normal range for age and gender (between −2 SDs and +2 SDs) so as to exclude “wasting syndrome.”
Biologic Values
Triglyceride and cholesterol values were transformed into z -scores according to the reference distributions for age, gender, and ethnicity.18
Hyperinsulinemia was defined as a fasting value >7 mU/L in prepubertal children and >11 mU/L in pubertal children . These values correspond to the 95th percentile of the distribution observed in 105 and 115 normoglycemic healthy children in the 2 groups, respectively.
Statistical Analyses
All data were entered and analyzed on the SAS 8.2 statistical package (SAS-Abacus, Meylan, France). Results are expressed as medians and interquartile ranges. Plasma insulin values were log-transformed before statistical analyses.
Because most of the variables vary with age, gender, and puberty , crude values have been translated into z -scores to allow for statistical comparisons. The z -score for a variable indicates how far and in what direction this variable deviates from its distribution's mean, expressed in units of its distribution's SD. The mathematics of the z -score transformation are such that if every item in a distribution is converted to its z -score, the transformed scores necessarily have a mean of 0 and an SD of 1. The z -score is a measure of the distance of a given value from the mean in SDs of the sample and is calculated as: (x − m)/SD.
Principal component factor analysis was used to investigate the relations among the risk factors for the metabolic disturbances and fat redistribution.
Statistically significant differences between groups were tested using a χ2 test for qualitative variables and the Mann-Whitney U test for quantitative variables. The paired Kruskal-Wallis test or paired χ2 test was used for the comparisons between baseline and follow-up.
Multivariate logistic regression was performed to identify the risk factors significantly associated with fat redistribution and metabolic disturbances. Results are given as odds ratios (ORs) with 95% confidence intervals.
A P value ≤0.05 was considered statistically significant.
RESULTS
Proportions of Children Affected With Lipodystrophy and Metabolic Changes at Inclusion
A total of 130 children (64 boys and 66 girls) were included, with a median age of 10.0 years and for whom baseline characteristics are given in Table 1 . Most children had acquired HIV infection through mother-to-child transmission. At the time of observation, 14 children were not receiving any treatment , among whom 5 had never been previously treated. The remaining children were being administered various HAART regimens.
TABLE 1: Characteristics of the Study Population (n = 130)
According to the definitions given previously, 32 (24.6%) children presented with evidence of fat redistribution at the time of the first observation: 14 with peripheral atrophy and 18 with central hypertrophy (Table 2 ). As shown in Table 3 , BMI and waist circumferences, expressed as z -scores for age and gender, significantly differed between children with atrophy, hypertrophy, or no lipodystrophy ; in contrast, percentage of fat mass did not differ between the 3 groups, attesting to a clear fat redistribution without any drastic change in fat mass.
TABLE 2: Fat Redistribution and Metabolic Changes at Baseline and 2 Years Later
TABLE 3: Adiposity According to the Type of Lipodystrophy
Twenty-three (18.9%) children had HDL values less than 1 mmol/L; 29 (22.3%) and 20 (15.4%) children had values greater than 2 SDs for age, gender, and ethnicity for cholesterol and triglycerides, respectively. None of the children had diabetes or impaired fasting glycemia; among 32 children who underwent an OGTT, 1 (3%) was found with impaired glucose tolerance. Sixteen children (13.2%) had signs of insulin resistance as evidenced by fasting hyperinsulinemia. Fifty-two children (42.7%) showed at least 1 of these biologic disturbances.
Longitudinal Evaluation and Proportions of Children Affected With Lipodystrophy and Metabolic Changes After 2 Years of Prospective Observation
Eighty-nine of the children were seen for a second observation after 2 years (median follow-up = 24.7 months.). The children who did not participate in this second observation did not significantly differ from the rest of the group in terms of gender, ethnicity, or nadir CD4 T-lymphocyte percentage. They had a shorter infection duration (8 ± 3 vs. 10.5 ± 2 years; P = 0.03). The proportion of fat redistribution in this subgroup was not statistically different at baseline than that in the children who maintained subsequent follow-up (21.9% vs. 25.8%; P = 0.63).
As indicated in Table 2 , there was no significant change in the proportions of children with fat redistribution or any of the biologic disturbances over the 2-year period, except a doubling of the proportion of children showing insulin resistance (25.6% vs. 13.2%; P = 0.01).
Five children had never received any antiretroviral treatment . None of these children showed any sign of fat redistribution at inclusion or follow-up. One child had elevated triglycerides at inclusion and remained the same at follow-up, with an additional high cholesterol value.
Risk Factors for Lipodystrophy and Associated Metabolic Changes
Principal component factor analysis was used to investigate the relations among the risk factors for the metabolic disturbances and fat redistribution and was performed on the study population at inclusion (n = 130). Five variables were introduced in the model: hypercholesterolemia, low HDL cholesterol, hypertriglyceridemia, fasting hyperinsulinemia, and lipodystrophy . Three components explain 86.6% of the variance. The first component explains 38.1% of the variance and encompasses 3 variables: cholesterol, triglycerides, and insulinemia. A second component explains 28.7% of the variance and is represented by the variable HDL cholesterol, and a third component explains 19.8% of the variance and is represented by lipodystrophy .
Subsequently, bivariate analyses for risk factors were conducted with these 3 variables: “HDL” for the risk of having a low HDL cholesterol value; “HIns/dyslipidemia” for the risk of having a high value of any of the 3 variables of cholesterol, triglycerides, and fasting insulinemia; and lipodystrophy for the risk of having any type of fat redistribution. Results are shown in Tables 4 and 5 .
TABLE 4: Bivariate Analysis Between Children With and Without Lipodystrophy
TABLE 5: Bivariate Analysis Between Children With and Without Metabolic Changes
TABLE 5: (continued) Bivariate Analysis Between Children With and Without Metabolic Changes
Risk Factors for Lipodystrophy (bivariate analysis)
Thirty-two children presented with any type of lipodystrophy . In our population, 2 factors were found as significant predictors of lipodystrophy . Being of African origin was associated with a reduced risk in comparison to being white (OR = 0.19; P = 0.001). A longer NRTI treatment during the 4 years preceding the observation was associated with an increased risk of lipodystrophy (OR = 1.8 per year; P = 0.001). Although not statistically significant, a longer duration of the disease was also associated with an increased risk of lipodystrophy (OR = 1.1; P = 0.07).
Risk Factors for Metabolic Changes (bivariate analysis)
Two factors were significantly associated with a reduced risk of having a low HDL cholesterol value: African origin (OR = 0.38; P = 0.004) and NRTI treatment (OR = 0.3; P = 0.03); additionally, there was a borderline reduction of the risk with the duration of NRTI administration (OR = 0.6 per year; P = 0.06).
For the HIns/dyslipidemia variable, complete development of puberty was found to be significantly associated with such metabolic disturbances (OR = 3.3; P = 0.003) as well as treatment with PIs (OR = 2.4; P = 0.002) and previous severe clinical symptoms (CDC B or C classification) (OR = 2.27; P = 0.03).
Multivariate Analyses
Multivariate models of logistic regression were further tested to identify the independent effect of the different covariates and are presented in Table 6 . Ethnicity, previous severe clinical condition, duration of HIV infection, and NRTI treatment remained significantly associated with lipodystrophy . Ethnicity and duration of NRTI treatment remained significantly associated with low HDL cholesterol values. For the HIns/dyslipidemia variable, Tanner stage V of puberty , severe clinical symptoms, and PI treatment remained independently associated with the risk of metabolic disturbances.
TABLE 6: Models of Multivariate Logistic Regression
DISCUSSION
Lipodystrophy and metabolic disturbances, such as dyslipidemia and insulin resistance , have been reported in HIV -infected children receiving intensive antiretroviral therapy, but the natural history of these complications is still poorly known. Using an objective assessment based on the measurement of 4 skinfold thicknesses, we observed a proportion of 25% of children being affected by lipodystrophy . This finding is in agreement with a previous observation reported by our group, where the frequency of lipodystrophy was evaluated to be 33% in a smaller independent group of children using the same methodology.6 children , where fat redistribution was based on clinical observation.5
A recent study aimed at introducing an objective and sensitive definition of lipodystrophy .19 children , however. The study showed that the definition of lipodystrophy was more sensitive when including an imaging measurement of fat mass by biphotonic absorptiometry. In our study, dual-energy x-ray absorptiometry (DEXA) could not be used for 2 reasons: restricted availability of the tool and the lack of reference values in children for the measurement of fat compartments. Instead, we used another objective measurement of fat mass based on the measurement of skinfold. For this parameter, French reference values for gender and age were available in children , allowing each value to be quantified calculating a z -score. The fact that no case of fat redistribution was observed in the small group of children (n = 5) who had never received any antiretroviral therapy points to the relevance of the measurement of skinfold thickness in the detection of fat redistribution in children . The extensive use of this method in clinical practice is hindered by the fact that it is tedious, however. The question of measurement of fat mass is crucial in children , because adiposity naturally changes with growth in terms of mass and distribution. It may be difficult to distinguish between natural changes or changes indicative of lipodystrophy using only clinical observation.
By contrast, we found a much larger proportion of children presenting with one of the metabolic disturbances (42%) already described as HAART associated. Usually, dyslipidemia is the most frequently documented complication in children , although HDL cholesterol is not frequently measured in children . We found a significant proportion of children (19%) with low HDL cholesterol values, however, indicating that here also the metabolic disturbances induced in children may not be different from those reported in adults. In the same line of evidence, fasting insulin levels, interpreted with respect to the physiologic insulin resistance induced by puberty ,20 children were indeed insulin resistant. When the OGTT was performed, only a few children were identified as having impaired glucose tolerance, whereas none of the 130 children showed overt diabetes. These results indicate that the complications of glucose metabolism may be rare but do exist in HIV -infected children .
The remarkable observation in this study is the great stability over 2 years of fat redistribution and metabolic disorders in HIV -infected children on HAART, except for insulin resistance (the proportion of which doubled over 2 years). This observation can be interpreted as an increase of the physiologic insulin resistance induced by puberty itself and amplified in this case by the interaction with the drugs.
Although based on cross-sectional observation at baseline, the present study identified factors associated with lipodystrophy or metabolic disturbances similar to those already reported in adults and children : severity of HIV infection, duration of treatment , NRTI use for lipodystrophy , and PI use for metabolic changes. We found a lesser proportion of African children affected by lipodystrophy , and they did not seem to progress rapidly. A large European pediatric study that included African children did not find ethnicity to be a risk factor of lipodystrophy as assessed by clinical observation.5 lipodystrophy .
Our observations have 2 major clinical implications. First, puberty seems to be the time when children are most likely to develop lipodystrophy and metabolic complications, especially in children with severe underlying HIV infection; consequently, screening for metabolic changes should be reinforced at this age. Second, once developed, lipodystrophy and metabolic changes seem to be extremely stable over time. A better understanding of the pathogenesis of these disorders is necessary, however, so as to propose interventions to reduce the impact of these complications in HIV -infected children .
ACKNOWLEDGMENTS
The authors are grateful to the skillful assistance of the nursing and medical staff of the Centre d'Investigation Clinique at the Robert Debré Hospital; the Centre d'Investigation Clinique at the Enfants Malades Hospital; and the Daycare Unit at the Trousseau Hospital, most particularly G. Vaudre .
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