At Tanner 1–2, there were no significant differences in any outcome between HIV-pos and HIV-neg. At Tanner 3–4, BMC was significantly lower in HIV-pos. At Tanner 5, total body and spinal BMD and total body BMC were all significantly lower in HIV-pos relative to HIV-neg (unadjusted). In the partially adjusted linear regression model, there were at least marginally significant interactions between HIV and Tanner group for all outcomes (total BMD: P = 0.065; spinal BMD: P = 0.018; total BMC: P = 0.068). In fully adjusted models, the interaction term between HIV and Tanner group was statistically significant (P < 0.011) for all outcomes. Similar to the unadjusted differences, HIV-pos males had significantly lower scores than HIV-neg males at Tanner 5 for total body and spinal BMD and total BMC in fully adjusted models. For BMC, HIV-pos also had significantly lower predicted mean outcomes than HIV-neg at Tanner 3–4. There were no differences at Tanner 1–2. For all three outcomes, the magnitude of the observed and estimated differences between HIV-pos and HIV-neg increased with Tanner group.
No outcomes differed significantly between HIV-pos and HIV-neg in any Tanner group in unadjusted comparisons. In partially adjusted regression models there were no statistically significant interactions between HIV and Tanner group (P > 0.113) for any outcome. Total BMC was marginally significantly lower in HIV-pos (P = 0.045). In fully adjusted models, there was a marginally significant interaction of HIV and Tanner group for spinal BMD (P = 0.078), but no significant interaction in models for total BMC and BMD. In fully adjusted models with no interaction term, HIV was not a significant predictor of any outcome. As with males, the magnitude of the estimated differences between HIV-pos and HIV-neg increased with Tanner group, but the magnitude of predicted difference at Tanner 3–4 and 5 was greater in males than females.
Addition of variables for adiposity did not change the results among males or females and were not included in any of the final models. In addition, spinal BMAD yielded the same conclusions as spinal BMD. These results are not shown.
In models with an indicator variable for each antiretroviral class, NNRTI use was significantly associated with higher BMC (62 g; 95% CI 1–122; P = 0.047) and higher spinal BMD (0.039 g/cm2; 95% CI 0.006–0.072; P = 0.021) compared to no NNRTI use. Protease inhibitors and NRTIs were not associated with any outcome at P < 0.10. In the final models (Table 4) evaluating the effect of individual antiretroviral drugs, Kaletra/ritonavir was associated with lower BMC and total body and spinal BMD. ZDV was also associated with marginally lower BMC but not other outcomes. In contrast, NVP was associated with higher BMC and spinal BMD but not total BMD. The results were consistent when antiretroviral class and individual agents were modeled as cumulative time on treatment. There was no strong trend for greater spinal BMD or total BMC with increasing time on NNRTIs.
Highly active antiretroviral therapy (HAART) has improved the health and survival of perinatally HIV-infected children, thus many are now entering adolescence. Despite improved general health, HIV-infected children are likely to have persistent deficits in growth and be at risk for delayed puberty  and decreased bone mass [2,4,26,27]. The timing of these deficits during puberty has not been well studied, especially in the HAART era. We evaluated bone mass across stages of puberty in a randomly selected group of perinatally HIV-infected compared to uninfected children/youth of similar Tanner stage and sociodemographic status. Our most striking finding was that HIV-infected boys had significantly lower spinal and total body bone mass relative to uninfected boys and that difference was more pronounced with advancing puberty. The trend was similar in girls, but the difference between the infected and uninfected was smaller and not statistically significant. A secondary finding was that those receiving ritonavir, with or without lopinavir, had lower bone mass at the spine and total body and those receiving ZDV had lower total body bone mass. In contrast, nevirapine users tended to have higher bone mass at both sites.
To our knowledge, ours is the first study to report differences in bone mass between HIV-infected and uninfected children/youth across Tanner stage and by sex. Others adjusted for these factors. Our findings are consistent with dimorphic trends in bone density, structure and strength observed in healthy adolescent girls and boys . As long bones increase in length, bone formation beneath the outer envelope (or periosteum) widens the skeletal shaft. Simultaneously, removal and replacement of bone along the inner envelope (endocortical component) establishes a medullary canal. Periosteal apposition generally exceeds endocortical resorption in young children and enlarging long bones thereby develop an increasingly thick cortex. This remodeling is similar in both sexes until puberty when sexual dimorphism occurs [28,29]. In girls, estrogen inhibits periosteal bone formation and limits growth of the skeletal diameter and promotes bone formation along the endocortical surface, ultimately narrowing a bone's inner diameter . In boys, androgens secreted during puberty increase periosteal apposition, bone diameter, and cortical thickness. As a result, men have much larger, denser bones on average than women. Although speculative, in the current study, hormonal changes could have exaggerated the dimorphic changes in bone density that we observed.
Interestingly, dimorphic differences in bone loss are observed in HIV-infected adults. Two studies reported a higher prevalence of osteopenia in HIV-infected men compared with HIV-infected women [31,32]. Jacobson et al.  observed that HIV-infected adult men tended to have greater loss in bone mass over follow-up compared to premenopausal women, whereas postmenopausal women had greater losses than both men and premenopausal women. The reasons for this apparent sexual dimorphism have yet to be identified.
In our study, HIV-pos and HIV-neg boys and girls at Tanner stages 1–2 did not differ significantly for any skeletal outcome. This contrasts with the findings of Arpadi et al.  in which prepubertal perinatally HIV-infected children had lower total body BMC compared to uninfected children of similar age and sex, but different race/ethnicity. There are two potential reasons why our findings may differ. By design our comparison group was not matched on age but selected within the same Tanner groups as the HIV-pos. In fact, within Tanner 1–2, our HIV-infected were somewhat older than the uninfected (approximately 2 years for boys and more than 1½ years older for girls) and may reflect delayed puberty [9,34]. Thus, prior to the adolescent growth spurt, our HIV-infected boys and girls may have acquired a similar amount of bone mass as the uninfected, but at a later age. Also, we enrolled participants in later years than Arpadi et al. (2004–2005 versus 1995–2000). Our HIV-infected children may have been treated with antiretroviral drugs younger, which may have had a positive impact on bone acquisition.
Whereas we did not observe differences in bone mass at Tanner 1–2 in either boys or girls, there were pronounced differences at later Tanner stages in boys. A few studies observed greater differences between HIV-infected and uninfected children/youth in older versus younger children while not specifically looking by sex or Tanner stage. Among HIV-infected girls aged 5–15 years old, O'Brien et al.  noted that total body BMC was further below the normal curve in older compared to younger children. Mora et al.  found lower spinal and total body BMD in the HIV-infected children compared to uninfected children aged 6.3–17.7 years. The groups were of similar age within each Tanner stage. Over a 12-month follow-up, the actual rate of increase in spinal BMD among the HIV-infected was similar to that estimated for HIV-uninfected. In contrast, the rate of increase in total body BMD was slower in the HIV-infected.
The effect of antiretroviral drugs on bone loss is inconsistent across studies. In our study, children receiving ritonavir had lower BMD and BMC. In a meta-analysis of adult cross-sectional studies , protease inhibitor-treated patients had a higher prevalence of low BMD (osteopenia or osteoporosis) than non-protease inhibitor-treated. In contrast, several longitudinal studies have not demonstrated greater losses in bone mass over time in protease inhibitor-treated patients. [36–40]. However, individual protease inhibitors may differ in their effect. In vitro, ritonavir was an inhibitor of osteoclast differentiation , which would be expected to protect bone mass . We observed somewhat lower total body BMC among participants receiving ZDV. Jacobson et al.  observed greater total body bone loss in HIV-infected adults on ddI. Pan et al.  reported that ZDV was associated with increased osteoclastogenesis in vitro and decreased BMD in mice, and van Vonderen et al.  observed greater loss of BMD in the spine and femur in adult men receiving ZDV and 3TC compared to nevirapine, with a backbone of Kaletra/ritonavir in both groups. Other studies support our finding of NNRTI and especially nevirapine use and greater bone mass in perinatally infected children . Nevirapine may have protective effects on bone, be a marker for better control of underlying disease or mitigate the effects of protease inhibitor treatment . Only 5.9% of children/youth in our study were receiving tenofovir, a drug associated with bone loss in adults [33,45] and children .
Dual-energy X-ray absorptiometry is the standard clinical method to screen for low bone mass in children, adolescents, and adults . DXA is preferred over axial computed tomography (CT) because it delivers lower radiation and well established normative data exist for children over 4 years up to adulthood . DXA measures BMC and the projected surface area from which areal bone density, a two-dimensional measure, is calculated (in g/cm2), whereas CT provides a three-dimensional, volumetric bone density measure in g/cm3. A limitation of DXA is that measurements may be confounded by bone size as the anteroposterior diameter of bone is not evaluated, resulting in systemic underestimation of volumetric BMD for age in children with impaired growth and pubertal development who may have smaller bones . By DXA, Pitukcheewanont et al.  found lower total body and spinal BMC and BMD in HIV-infected compared with matched uninfected children. In contrast, using CT these two groups did not differ on bone measures, but vertebral cross-sectional area, height and volume were lower in the HIV-infected. Thus, our finding of lower BMC and BMD during puberty in our HIV-infected compared to uninfected may be somewhat exaggerated on DXA because we could not account for bone size. However, we carefully adjusted for height and LBM which could partially explain body size and are positively correlated with bone mass. In addition, we evaluated differences across Tanner stages, not assuming a similar age between HIV groups at each Tanner stage. Finally, we repeated our analysis using spinal BMAD to account for bone size and our findings did not change .
Our HIV-infected and uninfected were well matched by sex, race and Tanner stage and sociodemographics which may decrease unmeasured confounding. However, our results may not represent all perinatally infected children and our estimates may be biased because some children/youth chose not to enroll, although we found no systematic reason for nonparticipation . In the future, use of peripheral computed tomography (pQCT), which measures volumetric BMD and assesses skeletal dimensions , will improve our understanding of skeletal sequalae of this disease and associated therapies.
Future studies are needed to understand the cause of differences in HIV-infected compared to uninfected children/youth with measurement of hormones and other circulating mediators. Longitudinal studies that examine changes within individuals will help to clarify the effect of puberty and sex on bone acquisition and inform the design of interventions to improve bone accrual and prevent skeletal losses in HIV-infected patients.
All authors were involved in the design phase of the study and contributed intellectually to writing the manuscript. J.C.L., D.L.J., G.M.A. K.M. and C.M.G. participated during the analytic phase. This work was supported by 5U01A1068616 (D.L.J., J.C.L.) and 1 U01 AI068632-01 (G.M.A.).
Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) was provided by the National Institute of Allergy and Infectious Diseases (NIAID) (U01 AI068632), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Mental Health (NIMH) (AI068632). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was supported by the Statistical and Data Analysis Center at Harvard School of Public Health, under the National Institute of Allergy and Infectious Diseases cooperative agreement #5 U01 AI41110 with the Pediatric AIDS Clinical Trials Group (PACTG) and #1 U01 AI068616 with the IMPAACT Group. Support of the sites was provided by the National Institute of Allergy and Infectious Diseases (NIAID) the NICHD International and Domestic Pediatric and Maternal HIV Clinical Trials Network funded by NICHD (contract number N01-DK-9-001/HHSN267200800001C).
The authors would like to thank the children who participated in this study, their families and the entire protocol 1045 team for their contributions and support. We would also like to thank Barb Heckman for outstanding support. The following sites and individuals have contributed to this study: Children's Hospital of Chicago; Tulane University School of Medicine, Charity Hospital of New Orleans: M. Silio, T. Alchediak, C. Borne, S. Bradford; SUNY Health Science Center, Stony Brook: S. Nachman, D. Ferraro, J. Perillo, S. Muniz; University of Puerto Rico; Harlem Hospital; NYU Medical Center/Bellevue; City Hospital at San Juan; New Jersey Medical School; Jacobi Medical Center; St. Jude Children's Hospital: M. Donohoe, N. Patel, S. Kaste, J. Utech; Boston Children's Hospital; University of North Carolina at Chapel Hill; University of South Florida Physicians Group; The Children's Hospital, University of Colorado, Denver: E. Barr, J. Maes, B. McFarland, S. Paul, Grant Number MO1 RR00069, GCRC Program, National Center for Research Resources, NIH; Medical College of Georgia; Duke University Medical Center: J. Hurwitz, J. Simonetti, M. Donnelly, C. Mathison; Texas Children's Hospital/Baylor; University of California San Francisco Medical Center and PCRC (RR001271); Yale University School of Medicine: W. Andiman, L. Hurst, S. Romano; Los Angeles County Medical/University of Southern California; Lincoln Medical and Mental Health; University of Rochester: G. Weinberg, B. Murante, S. Laverty; Metropolitan Hospital Center; Long Beach Memorial Medical Center; Johns Hopkins University Hospital; UCSD Medical Center; Children's Hospital at SUNY Downstate; Bronx-Lebanon Hospital; Harbor General – UCLA Medical Center; Children's National Medical Center – D.C.: D. Dobbins, T. Peron, D. Wimbley, H. Spiegel; Children's Diagnostic and Treatment Center of South Florida; Robert Wood Johnson University Hospital; Howard University Hospital; University of Florida at Gainesville; Mt. Sinai Hospital Medical Center, Women's and Children's HIV Program; SUNY Health Science Center, Syracuse; University of Alabama, Birmingham School of Medicine: R. Pass, M. Crain, N. Beatty, H. Charlton.
The data were presented at the 10th Workshop on Adverse Drug Reactions and Lipodystrophy. London, England, November 2008 (oral abstract).
The study was supported by the National Institutes of Health (NIH).
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