Globally, there are an estimated 2.1 million adolescents who were perinatally infected and living with HIV infection . Effective antiretroviral therapy (ART) has dramatically reduced morbidity and mortality for children with perinatal HIV infection, leading to most surviving into adolescence and adulthood . Adolescence is an important period during which a significant amount of bone development happens with more than half of adult bone growth occurring during this time . Lifetime exposure to both HIV infection and ART to perinatally HIV-infected adolescence (PHIVA) may impact on future osteoporosis and related complications [4,5]. However, there is limited information on PHIVA bone health especially in sub-Saharan Africa which has the largest proportion of HIV-infected adolescents.
Low bone mineral density (BMD) is a common finding in HIV-infected adults [6,7]. A meta-analysis showed that among HIV-infected adults, 52% had osteopenia and 15% had osteoporosis . The presence of low BMD is well documented in HIV-infected adults. Little data are available for PHIVA, during this period of changing bone physiology and growth. Most studies are from high-income countries and report low BMD among PHIVA compared with uninfected adolescents [9–11]. Therefore, there is a critical need for data on PHIVA bone health from sub-Saharan Africa.
HIV-infected individuals may be at risk for low BMD as a result of chronic inflammation and/or promotion of osteoblast apoptosis and osteoclast proliferation by the HIV envelope glycoprotein [12,13]. Other HIV-associated risk factors are uncontrolled viral load , duration of HIV infection [14–16] or use of specific ARTs such as protease inhibitors [6,17] or tenofovir disoproxil fumarate (TDF) . In addition, malnutrition, poor intake of calcium and vitamin D can also be a contributing factor .
One of the reasons for limited data on bone health from lower and middle-income countries is the difficulty of measuring BMD. Dual-energy X-ray absorptiometry (DXA) is the gold standard but it is expensive and it requires specialized equipment and resources. Quantitative ultrasound (QUS) is an alternative method for measuring bone health that is quick and affordable. Many studies have shown good correlation between use of QUS with DXA across all age groups [19–22]. We investigated bone health using QUS in PHIVA on ART and compared them with matched HIV-uninfected adolescents in a cohort study in Cape Town, South Africa to determine which factors, were associated with bone health outcomes in the PHIVA.
The Cape Town Adolescent Antiretroviral Cohort (CTAAC) is a South African prospective cohort study investigating the long-term health of PHIVA receiving ART for at least 6 months at the time of the enrollment. Enrollment occurred between July 2013 and March 2015. Participants were enrolled from eight sites in Cape Town together with age and sex frequency-matched HIV-uninfected adolescents (HIV−) as a comparison group.
Sociodemographic data were collected from participants and caregivers at the time of enrollment, and each participants’ clinical record was reviewed by a study clinician to record ART history, time of HIV diagnosis, WHO HIV staging at the time of diagnosis and other pertinent information.
Clinical examination included Tanner staging. Blood pressure and anthropometry were measured by a trained nurse. Three measurements for waist, mid-upper-arm and mid-thigh circumferences were taken using a vinyl tape and mean of these measurements were used. Weight was measured in kilograms (kg) on a Scales 2000 calibrated digital scale to the nearest 0.1 kg. The standing height was measured in centimeters (cm) using a calibrated stadiometer with a moveable headboard to the nearest 0.1 cm. BMI was calculated as weight in kilograms divided by height in meters squared (kg/m2), height and BMI was expressed in z-score based on WHO references .
Laboratory measures included viral load (Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test, version 2.0-standard technique; Roche, Basel, Switzerland) and CD4+ cell count (Beckman Coulter FC500 MPL analyzers; Beckman Coulter, Milan, Italy) using the Pan Leukocyte Gating method. Highly sensitive C-reactive protein was measured using the Roche Cobas Tina-quant system (range, 0.1–20 mg/l). Alkaline phosphatase (ALP) was processed on the Beckman AU 480; Beckman, Brea, California, USA) using kinetic color test principle. Fasting lipid sub fractions including total cholesterol (TC), triglycerides (TG), HDL and LDL were measured in all participants. Abnormal TC, HDL and LDL were defined as more than 200, less than 35 and more than 130 mg/dl, respectively. Abnormal TG were defined as more than 110 mg/dl if age less than 10 years or more than 150 mg/dl if age at least 10 years .
Bone health measurement
Bone health was evaluated on PHIVA and HIV− adolescents using an Achilles EXPII (GE Healthcare Chicago, Illinois, USA), a water-based calcaneal QUS device. Participants placed their foot on the foot pad of the device while in a seated position. Ultrasound waves were transmitted from the water-inflated transducer through the calcaneus to another transducer and the results were analyzed. Two measurements were taken of each foot. Between each measurement the foot was repositioned and the average of the two recordings was used in the analysis. The device generated three ultrasound parameters – speed of sound (SOS), broadband ultrasound attenuation (BUA) and calcaneal stiffness index. SOS is the time taken for ultrasound waves to travel through the calcaneus, whereas BUA is the slope of attenuation of the ultrasound signals. The stiffness index is a composite parameter [(0.67 × BUA) + (0.28 × SOS) − 420] that makes use of the SOS and BUA. Denser bone transmits ultrasound waves faster (indicated by a higher SOS value) and attenuates ultrasound signals at higher frequency (indicated by a higher BUA value), thus resulting in a higher stiffness index value which indicates better bone health. We only captured stiffness index as this is the composite of BUA and SOS which was calculated automatically by the ultrasound machine. All measurements were done by a single trained technician. Quality control calibration was performed at the beginning of each screening session.
Written informed consent was obtained from a legal guardian and written informed assent from each participant. The study was approved by the Human Research Ethics Committees (051/2013) of the University of Cape Town and Stellenbosch University. Approval was also obtained from the Western Cape Provincial Health Research Committee.
Baseline variables were compared between groups using t tests, Wilcoxon, and chi-square tests as appropriate. BMI for age (BMIZ) and height-for-age (HAZ) z-scores were calculated using WHO references . Tanner staging was categorized into three groups (Prepuberty = Tanner Stage I, Early Puberty = Tanner Stage II and III, Late Puberty = Tanner Stage IV and V). Study age and sex frequency-matched HIV− adolescents were used to create a reference stiffness index z-score. Bone health was measured by stiffness index. Low stiffness index in PHIVA was defined as a z-score less than −2 SDs and the coefficient of variance was 0.14. Among PHIVA, multiple logistic and linear regression was used to examine the adjusted association between low stiffness index and both HIV-related and traditional risk factors. All the analyses were performed using statistical software Stata 14.2 (Stata Corp LP, College Station, Texas, USA).
Overall, 499 adolescents (407 PHIVA and 92 HIV−) had stiffness index recorded and were included in this analysis. Median age and sex distribution were similar in both groups (14.0 vs. 13.7 years, P = 0.22 and female 50.4 vs. 54.4%, P = 0.49, respectively). Tanner Stage distribution between PHIVA and HIV− were 16.5 vs. 12.2% for prepubertal, 39.5 vs. 45.5% for early puberty and 44.1 vs. 42.2% for late puberty, respectively, P = 0.46.
Median BMIZ, HAZ, mid-thigh circumference and mid upper arm circumference were lower in PHIVA compared with HIV− (−0.19 vs. 0.43, P < 0.01; −1.31 vs. −0.71, P < 0.01; 39 vs. 42 cm, P < 0.01; 22 vs. 23 cm, P < 0.01, respectively). Median ALP was higher in PHIVA as compared with HIV− (283 vs. 229.5 U/l, P < 0.01) (Table 1).
The median age of ART initiation was 4.2 [interquartile range (IQR) 1.8–7.4] years and the median duration of ART was 9.8 (IQR 6.8–11.5) years, with 38% initiating ART at 2 years of age or less. Most PHIVA had well controlled HIV, with 317 (79%) having a CD4+ cell count more than 500 cells/μl, whereas only 16 (4%) had a CD4+ cell count less than 200 cells/μl (Table 2). One hundred and forty-eight (37.5%) had a viral load more than 50 copies/ml. There were 93 (23.7%) PHIVA who were WHO Stage IV at the time of HIV diagnosis and the start of ART.
The most common nucleoside reverse transcriptase inhibitors (NRTIs) were abacavir (ABC) and lamivudine (3TC) (68 and 84% of participants, respectively). Two hundred and sixty-five (65%) were on both ABC and 3TC; smaller proportions of adolescents were receiving tenofovir (TDF), zidovudine (ZDV), emtricitabine (FTC) and stavudine (D4T) (13, 19, 12 and 2%, respectively). Over half (55.5%) of PHIVA were on a non-NRTI (NNRTI) regimen with most (97%) being on efavirenz (EFV) and only 3% on nevirapine. Thirty-six percent were receiving protease inhibitors in the form of lopinavir/ritonavir (LPV/r) (Table 1).
Mean stiffness index were lower in PHIVA as compared with HIV− (99 vs. 105.1, P < 0.01) and 13% of PHIVA had low stiffness index. When stratified by Tanner Stage, the mean stiffness index between PHIVA and HIV− were similar (93 vs. 94, P = 0.832) in Tanner Stage I. However, during puberty, mean stiffness index increased with Tanner Stage in both PHIVA and HIV− but there was more significant increase among HIV−; Tanner Stage II–III (96 vs. 101, P = 0.009) and Tanner Stage IV–V (104 vs. 112, P = 0.001) (Table 2 and Fig. 1).
In males, no difference was found between the PHIVA and HIV− groups in prepuberty and early puberty. However, in late puberty, PHIVA males had low mean stiffness index as compared with HIV− in late puberty stage (103.9 vs. 113.7, P = 0.02). There were no HIV− females who were prepubertal. Among females in early puberty and late puberty, PHIVA had low mean stiffness index as compared with the HIV− group (93.5 vs. 103.5, P < 0.01 and 104.9 vs. 111.8, P = 0.03, respectively) (Table 2).
Among PHIVA 13% had low stiffness index (Supplementary Table, https://links.lww.com/QAD/B841). Table 3 shows the results of logistic regression modeling to identify predictors of low stiffness index in PHIVA children. Univariate analysis identified that having a viral load more than 50 copies/ml or current or ever use of LPV/r were significant risk factors. However, having high ALP, high CD4+ cell count or previous and current use of EFV was associated with better stiffness index.
In multivariate analysis after adjusting for age, sex and Tanner Stage, ever use of LPV/r [odds ratio (OR) = 2.31, P = 0.012] and viral load more than 50 copies/ml (OR = 2.06, P = 0.023) were associated with increased risk of low stiffness index. However, ever use of EFV (OR = 0.41, P = 0.009) and low ALP (OR = 0.99, P = 0.02) were associated with better stiffness index (Table 3).
After adjusting each model for age, sex, HAZ and Tanner Stage, ever use of LPV/r (OR = 2.18, P = 0.02) and viral load more than 50 copies/ml (OR = 1.96, P = 0.04) were associated with low stiffness index. However, ever use of EFV (OR = 0.40, P = 0.01), CD4+ cell count at least 500 cells/μl (OR = 0.51, P = 0.05) and low ALP (OR = 0.99, P = 0.02), were associated with better stiffness index (Table 3). Similar results were found when linear regression models were used (Table 4).
The current study has shown that PHIVA established on ART had lower stiffness index compared with HIV−. Among PHIVA, the use of protease inhibitors especially LPV/r and having uncontrolled HIV were associated with adverse bone health. This is one of the first studies to evaluate the prevalence and predictors of bone health among PHIVA on ART in sub-Saharan Africa.
We found low mean stiffness index among PHIVA compared with age and sex frequency-matched HIV−, similar to previous studies [25,26]. However, the differences between groups could be due to the influence of bone size on stiffness index measurement, as our PHIVA were shorter and thinner than the HIV−. Thinner adolescents may have lower muscle mass exerting force on bone, resulting in lower bone mass, since the difference was also arbitrated by weight . Differences in body weight in HIV-infected adults largely explain differences in their bone mass .
During puberty bone growth is at maximum, exposure to both HIV and ART, which may delay bone formation. Our findings of low stiffness index during puberty are similar to other studies from high-income countries , in which no difference between HIV-infected and HIV-uninfected adolescents at Tanner Stages I–II for any skeletal outcome were found but there were differences in late Tanner Stages. They found more pronounced and statistically significant differences in boys as compared with girls. In another study total body bone mineral content in 5–15-year-old HIV-infected girls was lower than age matched uninfected girls and the effect was more noticeable with increasing age . However, our findings are in contrast with Arpadi et al. in which prepubertal perinatally HIV-infected children had low stiffness index compared with uninfected children of similar age and sex, but of different ethnicity.
Association of low stiffness index with advanced or uncontrolled HIV is also consistent with studies that have shown that detectable viral load and low CD4+ cell count are independent risk factors for low stiffness index [26,31,32]. Our results indicate that when there is increased ALP there is decreased risk of low stiffness index. This is a natural phenomenon as bone growth raises serum ALP levels. Therefore, the level of serum ALP activity is 1.5–2.5 times higher in growing children than in normal adults . In general, decreased ALP activity has been observed in children with cessation of bone growth . ALP is often high in HIV-infected patients on ART; however, it is unclear whether it is the result of specific ART or related to comorbidities seen in HIV-infected individuals .
Many studies reported the association of protease inhibitors with increased bone turnover, accelerated bone loss and a higher prevalence of reduced BMD [6,35]. Our study also showed low stiffness index among those PHIVA who had used LPV/r. This is similar to comparison studies of protease inhibitors vs. NNRTIs which have shown the association of protease inhibitors with a greater loss of BMD [6,17]. However, there are other studies that have failed to show such a difference [36,37]. In addition, we found the association of EFV with better stiffness index, similar to a recent South African randomized trial study in which children who were switched to EFV had a higher BMD compared those who remained on LPV/r .
LPV/r is generally used in children as it is a highly effective inhibitor of HIV replication and an essential part of treatment in resource limited settings. LPV/r efficacy and safety has been proven by randomized trials among children . Therefore, LPV/r has been part of the first-line ART regimen recommended by the WHO for young children . Despite this, there are several known side effects from long-term use of LPV/r such as dyslipidemia and lipodystrophy . Our results suggest the need to limit the use of LPV/r once viral suppression has been achieved and encourage the use of alternate ART for maintenance [40,42,43].
Previous studies in HIV-infected adults and children have shown TDF was a risk factor for low BMD, but the effect in adolescence is unclear [18,35]. This was not observed in our cohort, because only 66 (13%) PHIVA had used TDF. This pattern of use is expected because many adolescents in this cohort are relatively young and had not reached the age at which guidelines routinely recommend the transition to TDF .
Our study shows the feasibility of performing QUS to measure stiffness index. Stiffness index is a good indicator of bone structure and several studies have proven high correlation between the stiffness index calculated by QUS and DXA measurements across all age groups from children to older adults [19–22]. Moreover, stiffness index is a better predictor of fracture risk than either BUA or sound of speed (SOS) alone . Stiffness index also showed better correlation with DXA than BUA and sound of speed . In a Chinese study, calcaneal stiffness index calculated by QUS correlated moderately well with total body BMD measured by DXA in both sexes . In addition, studies on HIV-infected adults [47,48] and children  also showed a good correlation between finding of QUS and DXA. Although DXA requires specialized equipment and expertise, stiffness index is relatively easy to measure and can be used longitudinally to evaluate bone development.
Limitations of this study include lack of standard reference ranges from Africa for stiffness index; we therefore derived, reference ranges from age and sex frequency matched HIV− adolescents from similar socioeconomic backgrounds. However, the sample size of HIV− adolescents was small. Although there is good correlation between QUS and DXA, it is possible for individuals with normal QUS to have low BMD when measured by DXA. Furthermore, we did not capture BUA and SOS measurements separately which makes comparison with other studies difficult, but stiffness index is a composite of these two measurements, so indirectly reflects these. Another limitation is that there were no recordings of bone health prior to ART initiation. We were unable to compare the bone health results obtained via QUS with DXA due to lack of access and cost of DXA. Lastly, this is a cross-sectional study and therefore causal inference cannot be established; longitudinal studies are needed.
Differences between PHIVA and HIV− were more pronounced at late puberty. Among PHIVA, detectable viral load and use of LPV/r were risk factors for low stiffness index. This reduction in bone density is a long-term health concern for PHIVA. Longitudinal studies of BMD in a PHIVA cohort are essential to clarify risk factors and periods of greatest risk for poor BMD.
Currently, there are no recommendations for routine measurement of BMD in PHIVA. Based on the results from CTAAC, routine evaluation of BMD in PHIVA is indicated. Noninvasive techniques such as QUS technology provide an easy to use and cost-effective alternative to DXA and allow for changes in bone health to be followed in patients to better evaluate the role of disease-related conditions or treatments which may interfere with bone health during growth.
We would like to thank the adolescents and their caregivers who participated in this study, as well as the study staff for their support of this research. We also wish to thank Red Cross War Memorial Children's Hospital for supporting the study.
H.J.Z. has received funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant # R01HD074051) and the South African Medical Research Council (SAMRC) to support this research.
Conflicts of interest
The authors have declared that they have no conflicts of interest.
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