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. 2023 Jul 20;18(7):e0288877. doi: 10.1371/journal.pone.0288877

Bone mineral density among children living with HIV failing first-line anti-retroviral therapy in Uganda: A sub-study of the CHAPAS-4 trial

Eva Natukunda 1,*, Alex Szubert 2, Caroline Otike 1, Imerida Namyalo 1, Esther Nambi 1, Alasdair Bamford 2, Katja Doerholt 2, Diana M Gibb 2, Victor Musiime 1,3, Phillipa Musoke 3,4
Editor: Chika Kingsley Onwuamah5
PMCID: PMC10359007  PMID: 37471330

Abstract

Background

Children living with perinatally acquired HIV (CLWH) survive into adulthood on antiretroviral therapy (ART). HIV, ART, and malnutrition can all lead to low bone mineral density (BMD). Few studies have described bone health among CLWH in Sub-Saharan Africa. We determined the prevalence and factors associated with low BMD among CLWH switching to second-line ART in the CHAPAS-4 trial (ISRCTN22964075) in Uganda.

Methods

BMD was determined using dual-energy X-ray Absorptiometry (DXA). BMD Z-scores were adjusted for age, sex, height and race. Demographic characteristics were summarized using median interquartile range (IQR) for continuous variables and proportions for categorical variables. Logistic regression was used to determine the associations between each variable and low BMD.

Results

A total of 159 children were enrolled (50% male) with median age (IQR) 10 (7–12) years, median duration of first -line ART 5.2(3.3–6.8) years; CD4 count 774 (528–1083) cells/mm3, weight—for–age Z-score -1.36 (-2.19, -0.65) and body mass index Z-score (BMIZ) -1.31 (-2.06, -0.6). Low (Z-score≤ -2) total body less head (TBLH) BMD was observed in 28 (18%) children, 21(13%) had low lumbar spine (LS) BMD, and15 (9%) had both. Low TBLH BMD was associated with increasing age (adjusted odds ratio [aOR] 1.37; 95% CI: 1.13–1.65, p = 0.001), female sex (aOR: 3.8; 95% CL: 1.31–10.81, p = 0.014), low BMI (aOR 0.36:95% CI: 0.21–0.61, p<0.001), and first-line zidovudine exposure (aOR: 3.68; 95% CI: 1.25–10.8, p = 0.018). CD4 count, viral load and first- line ART duration were not associated with TBLH BMD. Low LS BMD was associated with increasing age (aOR 1.42; 95% CI: 1.16–1.74, p = 0.001) and female sex: (aOR 3.41; 95% CI: 1.18–9.8, p = 0.023).

Conclusion

Nearly 20% CLWH failing first-line ART had low BMD which was associated with female sex, older age, first-line ZDV exposure, and low BMI. Prevention, monitoring, and implications following transition to adult care should be prioritized to identify poor bone health in HIV+adolescents entering adulthood.

Introduction

Globally, 38.4 million people are living with HIV (PLWH), with 1.7 million children aged 0–14 years. There were 160,000 new paediatric infections in 2021 with 90% living in Sub-Saharan Africa. Among children living with HIV (CLWH), 52% have access to antiretroviral therapy (ART) [1]. The World Health Organization (WHO) recommends that ART should be is initiated as soon as the HIV diagnosis is made in infants, children and young people [2]. Children now live longer into adulthood because of early ART initiation hence, the relative impact of long-term HIV and ART- related comorbidities, such as low bone mineral density (BMD) is becoming much more relevant. Low childhood BMD may result in suboptimal peak bone mass in adulthood and may predispose to osteoporosis and fracture risk in adulthood [3].

Low BMD has been previously described in PLWH and CLWH but most studies have been conducted in high-income countries [4, 5]. The prevalence of low BMD ranges from 20% to 80% in adults living with HIV [6, 7], and 4–32% in CLWH [8–10]. Few studies have been conducted in resource-limited settings where the majority of CLWH reside and where malnutrition, which also contributes to low BMD, is common [8, 10–12].

The aetiology of low BMD in the context of HIV infection is multifactorial. Relevant factors include uncontrolled HIV with a high viral load which favors osteoclast recruitment, low Vitamin D levels, low body mass index (BMI) and physical inactivity [13, 14]. Some antiretroviral drugs, including protease inhibitors, tenofovir disoproxil fumarate (TDF), and zidovudine (ZDV) contribute to low BMD while nevirapine (NVP) may be protective [8, 13]. Initiation of ART in treatment naïve adults or switching treatment due to virologic failure has also been found to be associated with reduced BMD in the first year followed by stabilization [10, 11, 13].

Bone health has not been widely studied in resource-limited settings yet it has the potential to be a long-term complication in PLWH. Under recognition is in part due to limited access to diagnostic dual-energy X-ray absorptiometry (DXA) facilities that detect low BMD before fracture occurrence.

In this sub-study of the CHAPAS-4 trial, we describe the baseline characteristics of a cohort of patients enrolled in the toxicity sub-study of the CHAPAS 4 trial in addition to reporting the bone health status of children switching to second-line ART at enrolment. We also examined the prevalence and risk factors associated with low BMD among children switching to second-line ART.

Materials and methods

We conducted a cross-sectional study using baseline data from the toxicity sub-study of the CHAPAS 4 clinical trial, an ongoing open-label trial evaluating the virologic response to alternative second line antiretroviral therapy in CLWH.

In CHAPAS 4, 919 children aged 3–15 years failing on their 2NRTI+NNRTI- based first-line regimen with a viral load of 400copies/ml or more were randomized to receive tenofovir alafenamide/emtricitabine (TAF/FTC) or standard of care (abacavir/lamivudine [ABC/3TC] or zidovudine/lamivudine [ZDV/3TC], depending on the first-line treatment. Children were also randomized in a factorial design to dolutegravir (DTG) or darunavir/ritonavir (DRV/r) or atazanavir/ritonavir (ATV/r) or lopinavir/ritonavir (LPV/r). They are being followed for 96 weeks. In the toxicity sub-study, 444 children from study sites in Zimbabwe and Uganda were simultaneously enrolled to evaluate the potential renal and bone effects of TAF. Of these, 159 participants aged 5 years and above were enrolled at Joint Clinical Research Centre (JCRC) and consented to a DXA scan. The Centre recruited study participants from the paediatric clinic in the facility and neighbouring public health facilities in Wakiso, Mpigi, and Mukono districts. Reference BMD Z-scores were available for children who were ≥ 5 years and hence children who were 5 years and above were included for this study [15].

Measurements

Data were collected at enrolment into the CHAPAS 4 trial from January 2019 to March 2021. A case report form was used to collect sociodemographic and clinical data. These included sex, age, and WHO classification of HIV disease [16], first-line ART and duration on treatment. Weight was measured using a Seca®weight scale, height was measured by wall mounted Seca® 206 stadiometer. BMI was calculated as weight (kg)/height (m2). Weight, height and BMI Z-scores were measured using the British 1990 reference data [17]. Blood samples were collected to determine the viral load and CD4 cell counts by real-time HIV-RNA (COBAS Ampliprep/Taqman 96 analyzer-detection range of 20–10,000,000 copies per ml) and BD FACS Calibur respectively.

BMD was measured using one DXA scan (Hologic Discovery Wi Apex 13.5, Hologic Bedford, MA USA) for all participants. DXA scans were conducted by an experienced technician while the participant was lying still and flat on an examination table. A posterior–anterior scan was obtained for four lumbar spines (L1-L4) and the whole body in the array mode. The lumbar spine (LS) BMD assesses trabecular bone density while the whole- body is largely cortical. The head was excluded from the analysis as recommended by International Society for Clinical Densitometry 2019 (ISCD) [18]. BMD was measured in grams per square centimetre. Normative values for sex, age, race and height adjusted Z-scores were obtained from bone mineral density children study (BMDCS) reference data [15]. Low BMD was defined as a LS or total body less head (TBLH) BMD Z-score of less than or equal to -2 according to the ISCD [18].

Quality control

A standard operating procedure was developed for participant preparation for BMD determination. The DXA scanner was calibrated by the technician daily for accuracy of the spine BMD determination with a phantom scan, standardization of the whole-body BMD determination was performed annually and a uniformity test was performed weekly.

Ethical review

The main CHAPAS 4 trial was approved by the JCRC institutional review board (reference–JC 1417).

Caretakers of the enrolled children gave written informed consent; children aged eight years and above gave written informed assent as appropriate and according to the knowledge of their HIV status.

Statistical analysis

Data were exported to STATA (StataCorp Texas USA) for analysis. Participant characteristics were summarized using median with interquartile range (IQR) for numerical data. Categorical data were summarized using frequencies and proportions. Low BMD was defined as either a height adjusted TBLH BMD or LS BMD z-score less than or equal to -2.

To measure the association of low BMD with other exposure variables, a logistic regression model was used. Bivariate analysis was performed for each of the independent variables to determine whether they were independently associated with low BMD using odds ratios and p-values. In multivariate analysis, stepwise backward elimination method was used to identify the independent variables with P-values <0.05 which were considered statistically significant. Confounding was assessed for all the non-significant independent variables and a relative difference of 10% or more between the crude and adjusted OR was considered as confounding.

Results

A total of 159 CLWH failing their first-line ART regimen were enrolled. The median age was 10.0 years (IQR 7.0, 12.0) and 79/159 (49%) participants were male. The median BMI z-score was -1.31 (IQR-2.06, -0.60). The median age at ART initiation and duration of first-line ART were 4.0 (IQR 2.0, 7.0) and 5.2 (IQR 3.3, 6.8) years, respectively. The median CD4 count was 774 (528, 1083) cells/ul and all children had viral loads ≥400 copies/ml, as per the main trial inclusion criteria. The majority of the participants were stable: 144 (90.6%) had WHO clinical stage 1 and 2, with only 15 (9.45%) ever having stage 3 and 4 (Table 1).

Table 1. Characteristics of study participants, N = 159.

Characteristic Measure
Age: median (IQR) 10 (7,12)
Male sex (n/%) 79 (49.7)
Anthropometry
Weight-for-age-Z-score (WAZ): median (IQR) -1.36 (-2.19, -0.65)
Height-for-age-Z-score (HAZ): median (IQR) -1.12 (-1.58, -0.10)
BMI- for- age-Z-score: median (IQR) -1.31 (-2.06, -0.60)
BMI- for- age-Z-score:<-2 (%) 18 (11.3)
HIV characteristics
CD4 count (cells/ul): median (IQR) 774 (528, 1083)
CD4%, median (IQR) 31 (22, 37)
Viral load (log10copies/ml): median (IQR) 4.25 (3.73, 4.85)
WHO Stage (n/N)
Stage 1&2 144 (90.6%)
Stage 3&4 15 (9.4%)
First- line ART regimen (n/N)
ABC- based 75 (47.2%)
ZDV- based 83 (52.2%)
EFV- based 80 (50.3%)
NVP- based 79 (49.7%)
Age at First-line ART initiation: median (IQR) 4 (2,7)
First- line ART duration in years: median (IQR) 5.21 (3.33, 6.84)
Bone mineral density measures
Lumbar spine
BMD g/cm3: median (IQR) 0.545 (0.487–0.648)
LSBMDHAZ: median (IQR) -0.99 (-1.46, -0.34)
Low lumbar BMD n/N (%) 21 (13.2)
TBLH
BMDg/cm3: median (IQR) 0.656 (0.577, 0.732)
TBLH HAZ: median (IQR) -1.37 (-1.87, -0.81)
Low TBLH BMD n/N (%) 28 (17.6)
Low LSBMD with low TBLH BMD 15 (9.4)
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Abbreviations: IQR = interquartile range, BMI = body mass index, LSBMD = lumbar spine bone mineral density, TBLH BMD = total body less head bone mineral density, HAZ = height adjusted Z- score; ZDV, zidovudine; NVP = nevirapine; EFV = efavirenz, ABC = abacavir

Associations with low TBLH BMD are summarised in Table 2. In the bivariate analysis, participants exposed to ZDV-based first-line therapy were more likely to have low TBLH BMD compared to those on ABC crude OR [cOR] = 3.2, p = 0.012). Increasing age (cOR = 1.33 per year older p < 0.001) and BMI (cOR = 0.43 per unit increase, p<0.001) were associated with low TBLH BMD. There was no significant association between low TBLH BMD and absolute CD4 count, viral load, time on first- line ART or height.

Table 2. Bivariate total body less head BMD analysis, N = 159.

Characteristic Abnormal BMD Normal BMD cOR 95%CI P-Value
N (%) N (%)
Sex
Male 10 (12.7) 69 (87.3) 1
Female 18 (22.5) 62 (77.5) 2.00 0.86–4.7 0.107
WHO stage
Stage 1&2 24 (16.7) 120 (83.3) 1
Stage 3&4 4 (26.7) 11 (73.3) 1.82 0.53–6.19 0.339
Regimen
ABC- based 7 (9.3) 68 (90.7) 1
ZDV- based 21 (25.0) 63 (75.0) 3.2 1.29–8.14 0.012
EFV- based 14 (17.5) 66 (82.5) 1
NVP- based 14 (17.7) 65 (82.3) 1.01 0.45–2.29 0.97
Median (IQR) Median (IQR)
Age 12 (10,14) 9 (7,12) 1.33 1.13–1.57 0.001
BMI-Z -2.0 (-3.0, -1.0) -1.0 (-2.0, -1.0) 0.43 0.28–0.66 <0.001
Abs CD4 count 738 (564.5,1052) 774 (524,1083) 1.00 0.99–1.00 0.953
CD4% 31 (23,35.5) 30 (22,37) 1.00 0.96–1.03 0.948
HIV RNA log 10 copies per ml 4.33 (3.87,4.94) 4.24 (3.73,4.84) 1.05 0.62–1.78 0.86
Time on first-line ART 6.0 (4.0,7.0) 5.0 (3.0,7.0) 1.06 0.91–1.25 0.439
Age at first-line ART start 6.0 (4.0,8.0) 4.0 (2.0,6.0) 1.22 1.06–1.399 0.05
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Abbreviations: IQR = interquartile range, BMI = body mass index, LSBMD = lumbar spine bone mineral density, TBLH BMD = total body less head bone mineral density, HAZ = height adjusted Z- score; ZDV = zidovudine, EFV = efavirenz, NVP = nevirapine, ABC = abacavir, ART = antiretroviral therapy, BMD = bone mineral density (BMD).

Associations with low LS BMD at bivariate analysis are presented in Table 3. Females were more likely to have low LS BMD compared to males (cOR = 2.8, p = 0.04). Older age was also associated with low LS BMD (cOR = 1.38 per year older p = 0.001), as was older age at first line ART initiation (cOR = 1.17, p = 0.037).

Table 3. Bivariate Lumbar spine bone mineral density analysis, N = 159.

Characteristics Abnormal Normal cOR 95%CI P-Value
N (%) N (%)
Sex
Male 6 (7.59) 73 (92.41)
Female 15 (18.75) 65 (81.25) 2.8 1.03–7.67 0.04
WHO stage
Stage 1&2 19 (13.19) 125 (86.81) 1
Stage 3&4 2 (13.3) 13 (86.67) 1.01 0.21–4.84 0.988
Regimen
ABC- based 7 (9.33) 68 (90.7) 1
ZDV- based 21 (25) 63 (75) 1.94 0.74–5.11 0.18
EFV- based 11 (13.75) 69 (86.25) 1
NVP- based 10(12.7) 69(87.34) 0.91 0.36–2.27 0.84
Median (IQR) Median (IQR)
Age 13 (11,14) 9.5 (7.0,12.0) 1.38 1.14–1.68 0.001
BMIZ -2.0 (-2.0, -1.0) -1.0 (-2.0, -1.0) 0.80 0.54–1.19 0.277
Weightz -2.0 (2.0) -1.0 (2.0) 1.09 0.78–1.55 0.591
Abs CD4 808 (615,970) 770 (505,1083) 1.00 0.99–1.00 0.782
CD4% 32 (26,37) 30 (22,37) 1.025 0.98–1.07 0.251
HIV RNA 3.97(3.49,4.54) 4.54(3.85,4.886) 0.56 0.30–1.05 0.07
Time on first-line ART 6.0 (5.0,8.0) 5.0 (3.0,7.0) 1.149 0.96–1.37 0.119
Age at first-line ART start 6.0 (4.0,8.0) 4.0 (2.0,6.0) 1.17 1.01–1.37 0.037
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Abbreviations: IQR = interquartile range, BMIZ = body mass index Z-score, Weightz = weight Z score, HIV RNA = HIV viral load, ART = anti-retroviral therapy, EFV = efavirenz, NVP = nevirapine, ABC = abacavir, ZDV = zidovudine

The results of multivariate analysis are summarized in Table 4.

Table 4. Multivariate analysis of baseline characteristics and bone mineral density, N = 159.

TBLH BMD
Characteristic aOR 95%CI P-Value
Age (/year older) 1.37 1.13–1.65 0.001
Female sex 3.80 1.31–10.81 0.014
ZDV compared to ABC based regimen 3.68 1.2510.80 0.018
BMIZ (/unit increase) 0.36 0.21–0.61 <0.001
LS BMD
Age (/year older) 1.42 1.16–1.74 0.001
Female Sex 3.41 1.18–9.8 0.023
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Abbreviations: aOR = adjusted odds ratio, BMIZ = body mass index Z-score, LS BMD = lumbar spine bone mineral density, TBLH BMD = total body less head bone mineral density, ZDV = zidovudine, ABC = abacavir

In the multivariate analysis of TBLH BMD, first-line ZDV exposure (aOR = 3.68, p = 0.018), older age (aOR = 1.37 per year older, p = 0.001), female sex (aOR = 3.8, p = 0.014) and lower BMI (aOR = 0.36 per unit higher, p<0.001) were independently associated with low TBLH BMD.

Females were more likely to have a lower LS BMD compared to males (aOR = 3.41, p = 0.023), and older age (aOR = 1.42 per year older, p<0.001) was independently associated with low LS BMD.

Discussion

The overall prevalence of low BMD in our study was high at 21%, with higher detection by TBLH (18%) compared to the LS (13%), whereas 9% children had both. The factors associated with low TBLH BMD were age, sex and low BMI, which is consistent with the results of other studies. In our study, ZDV exposure was the only drug-related factor associated with low BMD. However, since this was a cross-sectional study, associations do not imply causality.

The prevalence of low BMD in this study is comparable to that in other studies that have described a high prevalence of low BMD in CLWH [8, 19]. However, a prevalence rate as low as 4–7% has been reported in well-resourced countries [20–22]. Associated inadequate nutrition may have contributed to the higher prevalence of low BMD, given that our study population resides in a resource-limited setting, where balanced diets are not readily available [23]. In addition, half of the participants had prolonged exposure to efavirenz (EFV), which disrupts calcium and vitamin D metabolism, that are essential for bone development [24]. Low vitamin D status has been described among CLWH in Africa with a prevalence of 17.3% [25]. Although it was not assessed in this study, a recent study among Ugandan CLWH found an association between EFV use and vitamin D deficiency [26].

Increasing age was independently associated with low BMD, which may be explained by the delayed puberty that has been observed in CLWH and is associated with delayed skeletal growth [19, 27]. More females in this study had low BMD compared to males in both TBLH and LS which is consistent with other studies [8]. Sex differences in pre-pubertal BMD are conflicting. However, females tend to have smaller bones compared to males. BMD is a two- dimensional measurement that tends to underestimate the BMD for smaller bones [28]. The females in the study population may have smaller bones compared to the reference population that had height- for- age Z-scores ranging from 0.2 to 0.45 [29].

Low BMD was associated with low BMI in this study similar to the results of other studies where adequate BMI- for- age was correlated with increased bone mass. Bones adapt to mechanical loading and an increase in load results in strengthening of bone by remodeling itself to accommodate the increased load, while a decrease in load causes bone weakening [6, 30, 31].

Data regarding the effect of ART on bone are variable. In previous studies, use of ZDV or EFV was associated with low BMD, which is consistent with our findings where low BMD was associated with ZDV use. Kim et al found bone loss in Korean adults who were exposed to ZDV for over a year [32]. Similarly in a longitudinal study, patients who switched to ZDV had a small but significant decrease in BMD compared to those taking ABC who remained stable [33]. This is also consistent with the in vitro observation that ZDV stimulates osteoclast activity in mice [34]. Paediatric data regarding ZDV and bone density is limited, although Bunders et al found no association between ZDV and low spinal BMD. Similarly, there was no association of ZDV with spinal BMD in our study [20]. There was also no significant association between EFV and BMD in our study. Adult studies have associated EFV with reduced BMD but there is limited data regarding BMD and EFV in the paediatric population [35]. However there was bone accrual in South African children who switched from lopinavir/ritonavir based ART to EFV [36]. This finding may have been due to change to a drug with a better bone safety profile but is difficult to compare with our study findings. Bunders et al found no association between low BMD and EFV in children which is similar to our findings [13, 19, 29, 30, 32, 33]. The mechanism for no association is unclear but may be due to the difference in the pharmacokinetics of EFV in children that are distinct from adults. Plasma clearance of EFV in children is faster than in adults and hence may have a less significant effect on bone health [37, 38].

The mechanisms through which HIV causes low BMD include osteoclast activation by the HIV viral protein gp120 and production of pro-inflammatory cytokine TNFα and interleukin 6 that increase bone resorption. There was no association between HIV viral load and BMD in our study. Viral load at a single time point may not be an accurate measure of lifetime HIV virological control. Different studies have reported inconsistent results. Dimeglio et al did not adjust for height hence they found that a high viral load was associated with low LS BMD. However, other paediatric studies found no association after height adjustment for BMD was done similar in our study where [21, 39, 40] height adjustment for BMD Z- score was performed.

There are several potential limitations of this study. The DXA scanner does not measure volumetric density but only measures areal density, therefore underestimates the BMD for smaller bones.

There are no local or African BMD reference data for children. The reference data used for determining BMD Z scores were obtained from the BMDCS that used a healthy black American population. It is possible that we may have overestimated low BMD due to the geographical, environmental and dietary differences between our study population and the reference data [15]. Thirdly there was no HIV negative control group from a similar environment. This was a single-site study which limits the generalizability.

Conclusion

Low BMD is prevalent among Ugandan CLWH with virologic failure following first-line ART. Low TBLH BMD was associated with older age, female sex, low BMI and first- line ZDV exposure.

Health intervention programs should promote improved nutrition and also minimize the use of regimens associated with low BMD to prevent potential future fracture risk in low-income settings. Follow-up of these children in the CHAPAS-4 trial to a minimum of 96 weeks will enable us to evaluate the long-term effects of second-line ART on BMD. Future research should focus on therapeutic and preventive interventions and on monitoring the impact of these findings on bone health following transition to adult care.

Acknowledgments

This work was done as part of the CHAPAS 4 trial. The authors would like to thank the children and their caretakers who volunteered to participate in this study.

We thank the data collection team at the Joint Clinical Research Centre Kampala.

We thank Gilead sciences, CIPLA, ViiV healthcare and Janssen for providing the trial drugs.

Data Availability

The data underlying the results presented in the study are available from the institution (jcrc@jcrc.org.ug). Per the Joint clinical research centre data policy,the data is provided on request from the institution.

Funding Statement

The funding was obtained from EDCTP, Gilead Sciences and Janssen Pharmaceuticals and was received by Diana M Gibb. The funds for conference attendance were provided by Viatris and received by Diana M Gibb.

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Decision Letter 0

Chika Kingsley Onwuamah

10 Apr 2023

PONE-D-23-05357Bone mineral density among children living with HIV failing first-line antiretroviral therapy in Uganda. A sub-study of the CHAPAS-4 trial.PLOS ONE

Dear Dr. Natukunda,

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Chika Kingsley Onwuamah, Ph.D.

Academic Editor

PLOS ONE

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"This work was done as part of the CHAPAS 4 trial. The authors would like to thank the children and their caretakers who volunteered to participate in this study.

We thank all the data collection team at the Joint Clinical Research Centre Kampala

CHAPAS 4 trial is mainly funded by the European and Developing Countries Clinical Trials Partnership (grant number TRIA2015-1078) and Medical Research Council (MRC)

Additional support was received from: Gilead Sciences, Janssen Pharmaceuticals, ViiV Health care and CIPLA"

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"DMG received the award from European and Developing Countries Clinical Trials Partnership (EDCTP) grant number TRIA2015-1078.https://www.edctp.org

Additional support was provided by Gilead sciences , https://www.gilead.com,Jannsen Pharmaceuticals,https://www.janssen.comViiV Health care,https://viivhealthcare.com

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study that highlights the prevalence of co-morbidities such as low BMD in CLWH, especially pertaining to the cohort that is switching treatment due to virologic failure.

These observations are even more relevant in populations that are resource-limited and do not have access to a balanced diet.

The manuscript if re-structured in the context of the larger question addressed in the CHAPAS4 trial will have significant implications.

1. Although this study does an excellent job characterizing the BMD, LSBMD, and TBLH in CLWH, it is unclear how these findings address the questions asked in the CHAPAS4 trial and specifically pertaining to the toxicity sub-study.

2. The authors have not been able to tease out the impact of the high viral load and the first-line ART (BAC-based, ZDV based, etc.) on BMD.

3. Did the authors normalize or control for low BMI as a confounder to low BMD, since the study was conducted in a low-resource environment?

4. It is unclear from the results and the lack of description in the manuscript, the characteristics (if any) of the age-matched controls or if the "normal" BMD is that of controls.

5. There should be 2 separate control cohorts (if feasible) included in this study

a. Uninfected controls with normal BMD

b. CLWH that responded to the first line of ART

6. There is no graphical representation of the data.

7. Details of the DXA and parameters of the scans and the analyses need to be included. Was the same DXA machine used for performing scans on all participants?

8. The interquartile range is a useful measure of statistical dispersion because it is less sensitive to outliers. It will be useful to compare the median to that of control subjects to identify any potential outliers.

Reviewer #2: Thank you for a well written paper on the prevalence and associated factors of low BMD in your study population of children switching to second line ART due to virological failure. The findings showed a high prevalence of low BMD and several associated factors, including age, sex, BMI and AZT use. I think highlighting the need for following up these children/adolescents as they transition into adult care and assessing the long term outcomes, in terms of possible fracture risk, is very important as this is not a risk that is frequently monitored/assessed in the resource-limited settings where these adolescents are being cared for. I recommend that the article is accepted for publication after minor revisions.

Major Revisions

Nil

Minor Revisions

Abstract Results: Please review minor grammar, capitals and spaces before/after brackets as needed

Abstract Conclusion: “and development of cost-effective screening tools to identify poor bone health in HIV+ adolescents entering adulthood.” Development of screening tools is not mentioned in your main text. Either add this into your main text (because it is very important) or adjust your abstract conclusion to reflect your main text conclusion

Introduction: “Because of early ART initiation…” Suggest rephrasing this sentence so that it does not begin with “Because”

Introduction: “ART-related comorbidities including low bone mineral density (BMD) are becoming…” Suggest adding commas to read “comorbidities, including low bone mineral density (BMD), …”

Introduction: “where the majority of children” Should this read “where the majority of CLWH reside”

Introduction: “and where malnutrition that also contributes to low BMD is common” Suggest adding commas to read “and where malnutrition, which also contributes to low BMD, is common”

Introduction: “also contributes to low BMD is common. [8, 10-12] However BMD

is higher in the black population compared” The link between these two sentences needs to be more clearly explained

Introduction: “zidovudine (ZDV), contribute to” Suggest removing comma here

Introduction “also been documented to be associated” Suggest removing “as documented” from this sentence to make it easier to read

Introduction: “BMD is determined by z-scores in adolescents and children according to the International Society for Clinical Densitometry 2019(ISCD).[16] Low BMD is defined as z--scores less or equal to -2 defines low BMD.[16]. Suggest moving this paragraph to methods. The second sentence of this paragraph has repetition and needs to be reviewed.

Methods: “lamivudine(ZDV/3TC), depending on first-line). Children” Suggest removing bracket at the end of the sentence.

Methods: “Zimbabwe and Uganda were consecutively enrolled” Should this say simultaneously instead of consecutively?

Methods: “Reference BMD Z-scores were available for children who were ≥ 5 years.” If this is the reason only children > 5 were enrolled suggest making this more clear

Methods: “and classification of HIV disease[18], first” Please clarify what classification was used in the text

Methods: “The LS BMD assesses trabecular” This is the first time this abbreviation is used, please put lumbar spine (LS)

Methods: “children aged eight years and above gave written informed assent according to and as appropriate based on, knowledge of HIV status.” This sentence is not clear, please review (possibly remove the comma and say knowledge of their HIV status)

Methods: “or (LS) BMD z-score less than or equal to -2.” Remove brackets around LS

Results: “with only 15(9.45%) ever had stage 3 and 4.” Suggest changing to “ever having”

Results: “Associations with low total body less head (TBLH) BMD are summarised” Can just use TBLH here as abbreviation explained previously in the text. It will make it easier to read

Results: “Increasing age was associated with low TBLH BMD (cOR=1.33 per year older=0.001). Low BMI was also associated with low BMD (cOR=0.43 per unit increase. p<0.001).” Suggest making this into one sentence.

Results: “Associations with low lumbar spine (LS) BMD” Can just use LS here as abbreviation explained previously

Results: “LSBMD” separate into LS BMD throughout text

Results: “Multivariate analysis is summarized in Table 4,” Is this part of the next paragraph? Not clear

All tables: all abbreviations used in the table must be shown below the table, including ARVs. Please check this with all your tables

Discussion: “Paediatric data regarding ZDV and bone density is limited though in one study bunders et al found no association with spinal BMD.Similarly,there was no association with spinal BMD in our study [22]” Capitalise Bunders. Please clarify these two sentences…no association with low or worsening BMD?

Discussion: “HIV viral protein gp120 causes osteoclast activation while pro-inflammatory cytokine TNFα and interleukin 6 increase bone resorption. There was no association between HIV viral load and BMD in our study. Viral load at a single time point may not be an accurate measure of lifetime HIV virological control. Different studies reported inconsistent results. Dimeglio et al found high viral load was associated with low LSBMD while other paediatric studies found no association after height adjustment for BMD was done as in our study. In our study height adjustment for BMD Z- score was done.[23, 41, 42]

It would be useful to link the first sentence into the rest of the paragraph more, by making it clear that those are potential ways HIV causes low BMD. Did Dimeglio et al not adjust for height and that is why they potentially found a low LSBMD compared to other studies that did height adjust? This is not clear in this paragraph. Your references at the end should be after the second to last sentence where the studies are mentioned.

Discussion: “Thirdly there was no control group from a similar environment.” Suggest adding in what sort of control group may have improved the study…HIV negative children from the same environment? HIV positive children who are virally suppressed?

Discussion: “And this was a single- site study limiting generalizability.” Please rephrase so that the sentence does not begin with And.

Discussion: “Vitamin D is essential for bone formation. Low Vitamin D status has been described among children living in Africa with a prevalence of 17.3%.[43] We did not assess vitamin D status but our study participants were exposed to EFV that may interfere with vitamin D metabolism.” This is already mentioned in the second paragraph of your discussion. Suggest merging this paragraph into that second paragraph. It is a bit repetitive and out of place after the limitations.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

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Decision Letter 1

Chika Kingsley Onwuamah

6 Jul 2023

Bone mineral density among children living with HIV failing first-line antiretroviral therapy in Uganda. A sub-study of the CHAPAS-4 trial.

PONE-D-23-05357R1

Dear Dr. Natukunda,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Chika Kingsley Onwuamah, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please add the the parameters set in the statistical codes for variables that are potentially confounding in the results section. Refer to the author response to comment #3 made by the reviewer.

Reviewer #2: Thank you for your revisions made to the paper and I look forward to seeing the results from the follow up study that will assess the changes in BMD on second line regimens. I recommend that the article is accepted for publication. I have a few suggestions below. The article does not need further review if these changes are made.

Introduction line 1: Suggest removing the abbreviation PLWH from this sentence as in this sentence it reads “people are living with HIV” rather than people living with HIV. Suggest putting it into the first sentence of the second paragraph to read “Low BMD has been previously described in people living with HIV (PLWH) and

Introduction: “recommends that ART should be is initiated”: Remove “is”

Discussion: “Similarly, there was no association of ZDV with spinal BMD in our study” Suggest changing to read: “Similarly, there was no association of ZDV with low spinal BMD in our study”

Discussion: “height adjustment for BMD was done similar as in our study” Suggest changing to read “height adjustment for BMD was done, similar to our study

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Chika Kingsley Onwuamah

12 Jul 2023

PONE-D-23-05357R1

Bone mineral density among children living with HIV failing first-line anti-retroviral therapy in Uganda: A sub-study of the CHAPAS-4 trial.

Dear Dr. Natukunda:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

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    Data Availability Statement

    The data underlying the results presented in the study are available from the institution (jcrc@jcrc.org.ug). Per the Joint clinical research centre data policy,the data is provided on request from the institution.

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