Articles. Published online September 30,

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1 Bone mineral density and inflammatory and bone biomarkers after darunavir ritonavir combined with either raltegravir or tenofovir emtricitabine in antiretroviral-naive adults with HIV-1: a substudy of the NEAT001/ANRS143 randomised trial Jose I Bernardino, Amanda Mocroft, Patrick W Mallon, Cedrick Wallet, Jan Gerstoft, Charlotte Russell, Peter Reiss, Christine Katlama, Stephane De Wit, Laura Richert, Abdel Babiker, Antonio Buño, Antonella Castagna, Pierre-Marie Girard, Genevieve Chene, Francois Raffi, Jose R Arribas, for the NEAT001/ANRS143 Study Group* Summary Background Osteopenia, osteoporosis, and low bone mineral density are frequent in patients with HIV. We assessed the 96 week loss of bone mineral density associated with a nucleoside or nucleotide reverse transcriptase inhibitor (NtRTI)-sparing regimen. Methods Antiretroviral-naive adults with HIV were enrolled in 78 clinical sites in 15 European countries into a randomised (1:1), open-label, non-inferiority trial (NEAT001/ANRS143) assessing the efficacy and safety of darunavir (800 mg once per day) and ritonavir (100 mg once per day) plus either raltegravir (400 mg twice per day; NtRTI-sparing regimen) or tenofovir (245 mg once per day) and emtricitabine (200 mg once per day; standard regimen). For this bone-health substudy, 20 of the original sites in six countries participated, and any patient enrolled at one of these sites who met the following criteria was eligible: plasma viral loads greater than 1000 HIV RNA copies per ml and CD4 cell counts of fewer than 500 cells per μl, except in those with symptomatic HIV infection. Exclusion criteria included treatment for malignant disease, testing positive for hepatitis B virus surface antigen, pregnancy, creatinine clearance less than 60 ml per min, treatment for osteoporosis, systemic steroids, or oestrogen-replacement therapy. The two primary endpoints were the mean percentage changes in lumbar spine and total hip bone mineral density at week 48, assessed by dual energy x-ray absorptiometry (DXA) scans. We did the analysis with an intention-to-treatexposed approach with antiretroviral modifications ignored. The parent trial is registered with ClinicalTrials.gov, number NCT , and is closed to new participants. Findings Between Aug 2, 2010, and April 18, 2011, we recruited 146 patients to the substudy, 70 assigned to the NtRTI-sparing regimen and 76 to the standard regimen. DXA data were available for 129, 121 and 107 patients at baseline, 48 and 96 weeks respectively. At week 48, the mean percentage loss in bone mineral density in the lumbar spine was greater in the standard group than in the NtRTI-sparing group (mean percentage change 2 49% vs 1 00%, mean percentage difference 1 49, 95% CI 2 94 to 0 04; p=0 046). Total hip bone mineral density loss was similarly greater at week 48 in the standard group than in the NtRTI-sparing group (mean percentage change 3 30% vs 0 73%; mean percentage difference 2 57, 95% CI 3 75 to 1 35; p<0 0001). Seven new fractures occurred during the trial (two in the NtRTI-sparing group and five in the standard group). Interpretation A raltegravir-based regimen was associated with significantly less loss of bone mineral density than a standard regimen containing tenofovir disoproxil fumarate, and might be a treatment option for patients at high risk of osteopenia or osteoporosis who are not suitable for NtRTIs such as abacavir or tenofovir alafenamide. Funding The European Union Sixth Framework Programme, Inserm-ANRS, Ministerio de Sanidad y Asuntos Sociales de España, Gilead Sciences, Janssen Pharmaceuticals, and Merck Laboratories. Introduction Antiretroviral therapy (ART) combinations are highly effective in controlling HIV replication, with around 90% of patients achieving maximum viral suppression within the first year of first-line therapy. However, in the absence of strategies capable of inducing HIV cure, lifelong ART is needed. In this scenario of continuous ART and an ageing HIV-infected population, non-aids comorbidities have become an issue of increasing concern. Among these comorbidities, metabolic bone disease is one of the most important. Findings from cross-sectional and longitudinal studies have shown that patients infected with HIV have a higher prevalence of low bone mineral density, osteoporosis, 1 4 and fractures 5 7 than in the general population. Some reports even suggest that fragility fractures could occur at a younger age in people with HIV. 8 Lancet HIV 2015 Published Online September 30, S (15) See Online/Comment S (15) *Members listed in appendix Department of Internal Medicine, Hospital Universitario La Paz, Idipaz, Madrid, Spain (J I Bernadino MD, J R Arribas MD); University College London, London, UK (A Mocroft PhD); School of Medicine, University College Dublin, Dublin, Ireland (P W Mallon PhD); University Bordeaux, ISPED, Centre Inserm U897-Epidemiologie- Biostatistique, Bordeaux, France (C Wallet MSc, L Richert PhD, Prof G Chene PhD); Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark (Prof J Gerstoff MD); Medical Research Council Clinical Trials Unit at University College London, London, UK (C Russell BSc, Prof A Babiker PhD); Department of Global Health and Division of Infectious Diseases, Academic Medical Centre, Amsterdam, Netherlands (Prof P Reiss MD); Hospital Pitie-Salpetriere UPMC University Paris 06, Institut Pierre Louis d Epidémiologie et de Santé Publique, Paris, France (C Katlama MD); Saint Pierre University Hospital Brussels, Brussels, Belgium (S De Wit MD); Laboratory Medicine Department, Hospital Universitario La Paz, Madrid, Spain (A Buño MD); Vita-Salute San Raffaele Scientific Institute, Milan, Italy (A Castagna MD); Published online September 30,

2 Department of Infectious and Tropical Diseases, Hôpital Saint-Antoine, AP-HP and INSERM UMR_S 1136, Paris, France (P-M Girard MD); and College of Professors of Infectious and Tropical Diseases, Paris, France (Prof F Raffi MD) Correspondence to: Dr Jose I Bernardino, HIV Unit, Internal Medicine Department, Hospital La Paz, Idipaz, Madrid 28046, Spain jose.bernardino@salud. madrid.org See Online for appendix Research in context Evidence before this study We reviewed meta-analyses and a review that put into perspective the effects of different antiretroviral therapy regimens on bone. Low bone mineral density is associated with HIV infection in both cross-sectional and longitudinal studies and many cohorts have shown a high prevalence of osteopenia or osteoporosis, and fragility fractures in patients infected with HIV compared with non-infected cohorts. We then searched the PubMed database with the keywords antiretroviral therapy, clinical trial, HIV protease inhibitors, HIV integrase inhibitor, and nucleoside sparing, for reports published in English between Jan 1, 2006, and July 15, We identified some reports of the effect of a nucleoside or nucleotide reverse transcriptase inhibitor (NtRTI)-sparing dual therapy on bone mineral density and markers of bone turnover, but only two trials included a dual therapy consisting of a protease inhibitor plus an integrase strand-transfer inhibitor. PROGRESS, was a randomised, open-label trial comparing lopinavir ritonavir plus raltegravir with lopinavir ritonavir plus tenofovir emtricitabine. In this trial, the NtRTI-sparing group had a significantly lower decrease in total body bone mineral density at 96 weeks than the tenofovir-containing (0 68% vs 2 48%). RADAR compared an NtRTI-sparing regimen of ritonavir boosted darunavir plus raltegravir with boosted darunavir plus tenofovir with emtricitabine. Changes in absolute total bone mineral density at 48 weeks were more favourable with the NtRTI-sparing regimen than with the tenofovir-containing regimen (11 3 g per cm² vs 6 9 g per cm²; p=0 013). In both studies, exploratory analyses showed an association between early changes in bone biomarkers and loss of bone mineral density. These studies were not fully powered to identify differences in virological endpoints and did not explore changes in bone mineral density at different anatomical sites. Added value of this study The parent study NEAT 001/ANRS143 was the first fully powered trial to show non-inferiority of a NtRTI-sparing regimen composed of darunavir ritonavir plus raltegravir compared with a standard regimen of darunavir ritonavir plus tenofovir emtricitabine in antiretroviral-naive patients. Our substudy of a patient cohort of this trial is also the first NtRTI-sparing study examining changes in bone mineral density at three different anatomical sites: lumbar spine, femoral neck, and total hip. The results from our substudy show a significantly greater loss of bone mineral density at these three sites with the tenofovir-containing regimen, with a prominent loss at the femoral neck at 96 weeks. Greater percentage changes in bone mineral density were associated with the tenofovir-containing regimen and higher baseline viral load. These differences were associated with greater increases in biomarkers of bone turnover in the tenofovir-containing group. The findings from the large number of patients assessed with complete data from dual energy x-ray absorptiometry scans over 96 weeks and biomarkers over 48 weeks further support the association between changes in bone biomarkers and subsequent changes in bone mineral density. Implications of all the available evidence The results of our study and others suggest that an NtRTI-sparing ART regimen has a less pronounced negative effect on bone mineral density than a standard tenofovir-containing regimen, with stabilisation over time for at least the first 2 years of therapy. In an ageing population infected with HIV, our NtRTI-sparing regimen might have a role in specific populations at high risk of osteopenia, osteoporosis, or fragility fractures. Although more evidence of longitudinal changes is needed, use of bone biomarkers, especially in high-risk populations, could help to identify patients who would benefit most from NtRTI-sparing regimens. The results from several randomised clinical trials have shown a 2 6% decrease in bone mineral density after ART initiation in ART-naive patients Although this accelerated loss tends to stabilise over time, 12 its magnitude could be important because an increased risk of fractures during the first 2 years after ART initiation has been reported. 13 Use of tenofovir disoproxil fumarate has been associated with reductions in bone mineral density, 9,10 increased concentrations of biomarkers of bone turnover, 9,14 16 and increased risk of fragility fractures. 17 During the past years, efforts have been made to identify alternative ART combinations and, particularly, nucleoside or nucleotide reverse transcriptase inhibitor (NtRTI)-sparing regimens, to avoid toxic effects. In the RADAR 18 and PROGRESS 19 studies, a combination of a protease inhibitor (darunavir with ritonavir or lopinavir with ritonavir) plus raltegravir led to smaller changes in total bone mineral density than the protease inhibitor plus tenofovir with emtricitabine, although effects at different anatomical sites such as total hip, femoral neck, and lumbar spine were not reported. Bone turnover biomarkers increase in concentration after ART initiation in patients with HIV. 20 Significant reductions in concentrations of bone turnover biomarkers were noted after ART interruption in the intermittent therapy group of the SMART trial, 21 which suggest that inflammatory and immune-activation biomarkers related to uncontrolled HIV infection or combination ART, or both, could also play a part in changes in bone mineral density. 22 The effects of different ART regimens on bone biomarkers are unclear, because the findings from some studies report similar increases inde pendently of the antiretroviral drug used 23 and findings from other studies show greater increases in biomarkers of bone resorption with regimens based on tenofovir disoproxil fumarate. 9,19, Published online September 30,

3 No clinical trial comparing an NtRTI-sparing regimen with a regimen containing tenofovir disoproxil fumarate in treatment-naive patients has investigated the effect of the type of ART on changes in bone mineral density at different anatomical sites (total hip, femoral neck, and lumbar spine). In the NEAT 001/ANRS143 trial, 27 a regimen of ritonavir-boosted darunavir plus raltegravir (NtRTI-sparing) showed noninferior antiviral efficacy in comparison with ritonavirboosted darunavir plus tenofovir with emtricitabine (standard regimen), although the nucleoside-sparing combination did not meet non-inferiority in patients with baseline viral loads greater than copies per ml or a CD4 cell count of less than 200 cells per μl. 27 Because this study compared an NtRTI-sparing regimen with a tenofovir-containing regimen, we did a substudy with a cohort of these patients and assessed longitudinal changes in bone mineral density at three anatomical sites, and changes in biomarkers of bone turnover and inflammation. We postulated that the NtRTI-sparing regimen would have less effect on these variables. Methods Study design and participants NEAT001/ANRS143 was a randomised, open-label, 96 week, non-inferiority trial done in 78 clinical sites in 15 European countries between Aug 2, 2010, and April 18, Ethics committee approval was obtained from all participating centres, in accordance with the principles of the Declaration of Helsinki. Eligible patients had to be ART-naive, with a viral load greater than 1000 HIV RNA copies per ml and a CD4 cell count of fewer than 500 cells per μl (the full study design and patient population have been previously described). 27 For this substudy, any patient enrolling in the NEAT001/ANRS143 parent study in a site participating in the bone substudy (20 centres from six European countries: France, Belgium, Italy, Denmark, Spain, and Netherlands) and meeting the substudy eligibility criteria was suitable for enrolment. The eligibility criteria were the same as those for the parent study: 27 plasma viral loads greater than 1000 HIV RNA copies per ml and no evidence of major International Antiviral Society USA resistance mutations. Participants had to have a CD4 cell count of fewer than 500 cells per μl, except in those with symptomatic HIV infection. We excluded patients if they were receiving treatment for malignant disease, tested positive for hepatitis B virus surface antigen, were pregnant, had relevant laboratory abnormalities, and had an estimated creatinine clearance of less than 60 ml per min. Additional exclusion criteria for this substudy were use of treatment for osteoporosis (bisphosphonates, teriparatide, stron tium ranelate, raloxifen, or calcitonin) and concomitant use of systemic corticosteroids or oestrogen-replacement therapy at screening. Calcium and vitamin supplements and oestrogen as oral contraception were allowed. We obtained written informed consent from all participants for the parent study and this bone substudy at the same time. Randomisation and masking We generated a randomisation list before the start of the study, using permuted blocks of randomly varying sizes and stratified by country and participation in the viral and immunological dynamics and inflammation substudy. 27 We randomly assigned patients (1:1) to receive oral treatment with either 800 mg darunavir and 100 mg ritonavir once per day plus 400 mg raltegravir twice daily (NtRTI-sparing regimen) or 800 mg darunavir and 100 mg ritonavir plus tenofovir 245 mg and 200 mg emtricitabine in a fixed-dose combination once per day (standard regimen). Tenofovir emtricitabine was provided by Gilead Sciences, darunavir by Janssen Pharmaceuticals, and raltegravir by Merck Laboratories. Procedures Participants attended study centres at screening, baseline, weeks 2, 4, 8, 12, 18, 24, 32, 48, 64, 80, and 96, and every weeks thereafter. We recorded patients full history including occurrence of fractures, family history of fractures, bone-related diseases (such as hypogonadism, rheumatoid arthritis, metabolic bone diseases, hyperthyroidism, anorexia nervosa, inflam matory bowel disease, malabsorption, and Cushing s syndrome), and physical activity. Patients gave fasting serum and second morning urine void samples, which were stored at 80 C at baseline and week 48 until analysis. We did dual energy x-ray absorptiometry (DXA) scans at baseline, and weeks 48 and 96 to assess total body, total hip, femoral neck, and lumbar spine (L1 L4) bone mineral density. We used both Hologic (Hologic, Bedford, MA, USA) and GE Lunar (GE Healthcare Lunar, Madison, WI, USA) devices and no central reading was done. We used standard scanning protocols based on each manufacturer s specifications across all sites. Technicians, who were masked to study clinical information, were instructed to use the same device on the same patient and scan the same hip during the trial. We analysed known and potential (ie, osteopontin) biomarkers of inflammation and bone turnover in batches of stored samples from baseline and week 48 at the end of the trial in the Department of Laboratory Medicine at Hospital Universitario La Paz (La Paz, Madrid, Spain). Laboratory personnel, masked to randomised treatment allocation, measured the following biomarkers: bone formation biomarkers (bone specific alkaline phosphatase, intact N-terminal propeptide of type 1 procollagen [P1NP], and osteocalcin), bone turnover regulators (25-hydroxy-vitamin D, intact parathyroid hormone, receptor activator of NFκb Published online September 30,

4 805 included in NEAT001/ANRS143 study 401 assigned to RAL plus DRV/r 404 assigned to TDF FTC plus DRV/r 65 any bone biomarker measurement 70 assigned to the bone substudy 76 assigned to the bone substudy 68 any bone biomarker measurement 61 had DXA at baseline 68 had DXA at baseline 54 had DXA at week had DXA at week had DXA at week had DXA at week 96 Figure 1: Trial profile Numbers indicate patients who had a full dataset for DXA for femoral neck, hip, and lumbar spine. 47 patients in the RAL plus DRV/r group, and 57 in the TDF FTC plus DRV/r group, had DXA data for all three timepoints. RAL=raltegravir. DRV/r=darunavir plus ritonavir. TDF FTC=tenofovir plus emtricitabine. DXA=dual energy x-ray absorptiometry. RAL plus DRV/r (n=70) TDF FTC plus DRV/r (n=76) Total (n=146) Male 62 (89%) 72 (95%) 134 (92%) Age (years) 39 (30 45) 40 (32 45) 40 (31 45) Ethnic origin White 57 (81%) 63 (83%) 120 (82%) Other 13 (19%) 13 (17%) 26 (18%) HIV risk group Homosexual 48 (69%) 52 (69%) 100 (69%) Heterosexual 15 (22%) 19 (25%) 34 (23%) Other 7 (10%) 5 (7%) 12 (8%) Current smoker 26 (37%) 34 (45%) 60 (41%) Current alcohol user 3 (4%) 2 (3%) 5 (4%) Body-mass index (kg per m²) 22 5 ( ) 23 3 ( ) 23 ( ) Baseline HIV-1 RNA concentration (log 10 copies 4 8 ( ) 4 7 ( ) 4 7 ( ) per ml) Baseline CD4 cell count (cells per μl) 344 ( ) 333 ( ) 338 ( ) CD4 cell count nadir (cells per μl) 327 ( ) 327 ( ) 327 ( ) Presence of HCV antibody 2 (3%) 1 (1%) 3 (2%) History of fracture 8 (12%) 15 (20%) 23 (16%) Family history of fracture 9 (13%) 7 (9%) 16 (11%) Walking hours per week 4 (2 6) 4 (2 7) 4 (2 7) Current use of calcium or vitamin D supplements 14 (20%) 10 (13%) 24 (17%) Data are n (%) or median (IQR). RAL=raltegravir. DRV/r=darunavir plus ritonavir. TDF FTC=tenofovir plus emtricitabine. HCV=hepatitis C virus. Table 1: Baseline characteristics (RANKL), and osteoprotegerin), bone resorption bio markers (type 1 C-terminal collagen crosslinks [CTX-1] and osteopontin), and biomarkers of inflammation (interleukin 6, interleukin 1b, and tumour necrosis factor [TNF] α; for reference values and coefficients of variation see appendix, p 8). Outcomes The two primary endpoints of the bone substudy were the mean percentage change from baseline to week 48 in bone mineral density at the lumbar spine and total hip. Secondary endpoints were the mean percentage change from baseline in bone mineral density at the lumbar spine and total hip at week 96, the proportion of patients meeting WHO criteria for osteoporosis and osteopenia at weeks 48 and 96, the proportion of patients with a Z score of less than 2 at weeks 48 and 96, occurrence of fractures, changes from baseline to week 48 in biomarkers of bone turnover and inflammation, and the proportion of patients with vitamin D deficiency and secondary hyperparathyroidism at weeks 48 and 96. Statistical analysis We calculated that 64 patients per group would provide 80% power to detect a treatment difference of 2 5% in bone mineral density in mean lumbar spine change between groups, assuming an SD of 5% and a two-sided t test with α of Assuming a 10% dropout rate, we planned a total sample size of 142 patients. We did the analysis with an intention-to-treat approach with antiretroviral treatment modifications ignored. Patients without DXA scans were not included in the analysis. We used descriptive statistics to summarise patients baseline characteristics in each treatment group, which we compared with χ² tests, unpaired t tests, and non-parametric Wilcoxon tests. We assessed and compared changes in bone mineral density (total spine, lumbar hip, or femoral neck) from baseline to week 48 and week 96 between treatment groups using linear regression. We did multivariate linear regression analysis to explore factors associated with the percentage change in bone mineral density at week Published online September 30,

5 Covariates included in the analysis were age, sex, ethnic origin, smoking, body-mass index (BMI), log 10 HIV-1 RNA and baseline bone mineral density. We adjusted multivariate models for variables that were associated with changes in univariate analyses with p less than 0 1. Models were confirmed with forward and backward selection and logistic regression to compare the proportion with a greater change than the median in bone mineral density at each site or the proportion with a loss greater than 5% in bone mineral density. We selected this cutoff, deemed clinically relevant, to enable comparison with previous studies. We used similar methods to assess the biomarkers at baseline and over time. All tests of significance were two-sided. An independent data monitoring committee reviewed data from the main parent trial 27 and this substudy. We did the statistical analyses using Statistical Analysis Software version 9.3. NEAT001/ANRS143 is registered with ClinicalTrials.gov, number NCT , and is closed to new participants. Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. Results Between Aug 2, 2010, and April 18, 2011, we recruited 146 patients to the substudy, 70 assigned to the NtRTI-sparing regimen and 76 to the standard regimen. DXA data were available for 129 patients, 61 in the NtRTI-sparing group and 68 in the standard group (figure 1). The proportions with missing data were similar in both groups. Baseline characteristics were well balanced between groups (table 1), with no notable differences from the main trial population (data not shown). Apart from lower total hip bone mineral density in the NtRTI-sparing group compared with the standard group (median 1 01 g/cm² [IQR ] vs 1 07 g/cm² [ ]; p=0 032), groups were well matched for other baseline measures of bone mineral density (appendix, pp 9 and 10). Biomarker concentrations did not differ between groups at baseline (appendix, pp 12 and 13). Of the 146 patients included, 122 had total hip and spine DXA at week 48 and 107 patients at week 96. After 48 and 96 weeks of antiretroviral therapy both groups had a reduction in total hip, lumbar spine, and femoral neck bone mineral density (figure 2). The decrease in total hip bone mineral density from baseline was greater in the standard group than in the NtRTI-sparing group at 48 weeks (mean percentage change 3 30% vs 0 73%, p<0 0001; table 2). Similarly, the loss in bone mineral density of the lumbar spine at week 48 was greater in the standard group than in the NtRTI-sparing group (mean percentage change 2 49% vs 1 00%, p=0 046). Intragroup differences in mean percentage change in total hip bone mineral density were significant in both groups except for the change in total hip at week 48 in the NtRTI-sparing group (p=0 17; figure 2). For the mean Change (%) Patients at visit RAL plus DRV/r TDF FTC plus DRV/r Change (%) Patients at visit RAL plus DRV/r TDF FTC plus DRV/r Change (%) A *Mean values between groups p= Baseline B Baseline Figure 2: Difference in mean percentage change in bone mineral density Intragroup differences from baseline: (A) lumbar spine at week 48, standard group, p<0 0001; NtRTI-sparing group, p=0 05; week 96 standard group, p<0 0001; NtRTI-sparing group, p=0 44; (B) total hip at week 48, standard group, p<0 0001; NtRTI-sparing group, p=0 17; week 96 standard group, p<0 0001; NtRTI-sparing group, p=0 016; (C) femoral neck at week 48, standard group, p<0 0001; NtRTI-sparing group, p=0 0005; week 96 standard group, p=0 02; NtRTI-sparing group, p= RAL=raltegravir. DRV/r=darunavir plus ritonavir. TDF FTC=tenofovir plus emtricitabine * Mean values between groups p= * *Mean values between groups Mean values between groups p< p= C RAL plus DRV/r TDF FTC plus DRV/r 5 *Mean values between groups Mean values between groups 5 99 p= p= Time (weeks) Patients at visit RAL plus DRV/r TDF FTC plus DRV/r Baseline * Published online September 30,

6 Univariate Parameter estimate of mean percentage difference Multivariate 95% CI p value Parameter estimate of mean percentage difference 95% CI p value Total hip Treatment RAL plus DRV/r 1 1 TDF FTC plus DRV/r to 1 35 < to Any effect of sport versus no effect to to Body-mass index (per kg/m² higher) to to Viral load at baseline (per log 10 copies per ml higher) to to Lumbar spine Treatment RAL plus DRV/r 1 1 TDF FTC plus DRV/r to to Any effect of sport versus no effect to to Body-mass index (per kg/m² higher) to to Viral load at baseline (per log 10 copies per ml higher) to to Femoral neck Treatment RAL plus DRV/r 1 1 TDF FTC plus DRV/r to to Any effect of sport versus no effect to to Body-mass index (per kg/m² higher) to to Viral load at baseline (per log 10 copies per ml higher) to to RAL=raltegravir. DRV/r=darunavir plus ritonavir. TDF FTC=tenofovir plus emtricitabine. Table 2: Covariates associated with percentage change in bone mineral density at 48 weeks percentage change in lumbar spine bone mineral density, intragroup differences were significant in the standard group at week 48 (figure 2). The loss in bone mineral density was still greater in the standard group than in the NtRTI group at 96 weeks for total hip (mean percentage differences 2 29; 95% CI 3 78 to 0 80; p=0 003) and lumbar spine ( 2 37; 4 00 to 0 78; p=0 005). Intragroup differences in total hip bone mineral density were significant in both the standard and NtRTI-sparing groups at 96 weeks (p< and p=0 016, respectively), but only significant at this timepoint in the standard group for lumbar spine bone mineral density. The reduction in bone mineral density at the femoral neck was similarly greater in the standard group than in the NtRTI-sparing group at 48 weeks (table 2); this loss was even greater at 96 weeks (mean percentage difference 4 25 [95% CI 7 92 to 0 58]; p=0 025]. Intragroup differences in mean percentage change at the femoral neck were significant in both groups at weeks 48 and 96 weeks (figure 2). We noted no between-group differences in occurrence of new cases of osteopenia, osteoporosis, or patients with a Z score of less than 2 (appendix, p 11). At both weeks 48 and 96, the proportion of patients with a loss of bone mineral density of at least 5% at any site was greater in the standard group than in the NtRTI-sparing group: 29 (43%) of 68 compared with nine (14%) of 65 at week 48 (p=0 0002) and 34 (51%) of 68 compared with 13 (20%) of 65 at week 96 (p=0 0003). Four new fractures were reported between baseline and week 48 (one in the NtRTI-sparing group and three in the standard group), and three more between weeks 48 and 96 (one in the NtRTI-sparing group and two in the standard group). However, the mechanism and localisation of fractures were not recorded. In multivariate analysis, being assigned standard treatment was significantly associated with greater loss of bone mineral density at both the femoral neck and total hip, and baseline viral load was associated with a percentage change in lumbar spine bone mineral density (table 2). At week 96, standard treatment remained significantly associated with lumbar spine and total hip bone-mineral-density loss. Samples were available from 133 patients for biomarker analysis. Absolute baseline values for bone turnover and inflammatory biomarkers were similar between treatment groups (appendix, pp 12 and 13). We noted increases in the absolute value of most bone turnover biomarkers at week 48, except for decreases in RANKL in both groups, a decrease in osteoprotegerin in the standard group, and a decrease in osteopontin in the NtRTI-sparing group (table 3). Generally, changes in bone turnover biomarkers were significantly greater in the standard group 6 Published online September 30,

7 RAL plus DRV/r TDF FTC plus DRV/r Mean difference between groups (95% CI) p value n Mean absolute change (95% CI) n Mean absolute change (95% CI) Bone formation markers Osteocalcin (ng/ml) (2 3 to 7 3) (6 6 to 10 6) 3 7 (0 6 to 6 8) 0 02 BSAP (μg/l) (0 8 to 4 0) (5 1 to 8 3) 4 3 (1 9 to 6 7) P1NP (ng/ml) (3 8 to 20 2) (20 1 to 31 5) 13 7 (4 1 to 23 3) Bone resorption markers Serum CTX-1 (ng/ml) (0 0 to 0 2) (0 20 to 0 40) 0 23 (0 11 to 0 35) Osteopontin (pg/ml) ( 1117 to 1098) ( 983 to 1884) 460 ( 1411 to 2331) 0 6 Urine CTX-1/creatinine (26 1 to 91 9) (78 4 to 145 4) 52 9 (5 9 to 99 9) 0 03 Bone turnover regulators Osteoprotegerin (pg/ml) (17 3 to 109 9) ( 81 8 to 22 8) 93 0 ( to 21 7) 0 01 RANKL (pmol/l) ( 0 79 to 0 23) ( 0 37 to 0 07) 0 13 ( 0 40 to 0 66) hydroxy vitamin D (ng/ml) (0 9 to 7 9) (2 4 to 9 8) 1 7 ( 3 4 to 6 8) 0 5 Intact PTH (pg/ml) ( 4 9 to 5 9) (3 2 to 11 4) 6 8 (0 1 to 13 5) Inflammatory markers Interleukin-1b (pg/ml) (0 01 to 0 21) (0 02 to 0 18) 0 03 ( 0 17 to 0 12) 0 7 Interleukin-6 (pg/ml) ( 2 0 to 1 6) ( 0 9 to 1 3) 0 4 ( 1 6 to 2 4) 0 7 TNFα (pg/ml) ( 0 2 to 3 8) (1 2 to 2 4) 0 3 ( 1 4 to 1 9) 0 7 RAL=raltegravir. DRV/r=darunavir plus ritonavir. TDF FTC=tenofovir plus emtricitabine. BSAP=bone-specific alkaline phosphatase. P1NP=procollagen type 1 N-propeptide. CTX-1=type 1 C terminal collagen crosslinks. RANKL=receptor activator of NF-kb ligand. CRLP=C-terminal collagen crosslinks. PTH=parathyroid hormone. TNF=tumour necrosis factor. Table 3: Changes in bone and inflammatory biomarkers from baseline to week 48 (appendix, pp 14 26). We noted no difference in changes in inflammatory biomarkers between groups. No differences in phosphate excretion at 48 weeks were noted between the groups. Proximal tubular dysfunction was present in three (4%) of 80 patients who had serum and urine measurements at 48 weeks (none in the NtRTI-sparing group and three in the standard group; p=0 25 Fisher s exact test). Although intact parathyroid hormone significantly increased in the standard group at week 48 (p=0 045), no betweengroup differences in the proportion of patients with 25-hydroxy-vitamin D insufficiency and hyperparathyroidism were noted. In view of their large variations, biomarker values were classified as greater or lower than their baseline median value. In the multivariate analysis, a baseline P1NP concentration of 44 7 ng/ml or less, and a baseline osteopontin concentration of 5423 pg/ml or less, were significantly associated with a loss in bone mineral density of at least 5% at different anatomical sites at weeks 48 and 96 (table 4). Discussion NEAT001/ANRS was the first fully powered trial showing non-inferiority of an NtRTI-sparing regimen of ritonavir boosted darunavir plus raltegravir compared with a standard regimen of boosted darunavir plus tenofovir with emtricitabine in antiretroviral-naive patients with HIV. Here, we showed that significantly Univariate Multivariate Odds ratio (95% CI) p value Odds ratio (95% CI) p value Spine 48 weeks BSAP (μg/l) > ( ) ( )* Spine 96 weeks Osteopontin (pg/ml) > ( ) ( ) 0 03 Femoral neck 96 weeks P1NP (ng/ml) > ( ) ( ) 0 03 Total hip 48 weeks P1NP (ng/ml) > ( ) ( ) 0 02 Each biomarker was categorised as greater than baseline median value versus equal or less than median value because of wide variation in laboratory values. Data adjusted for baseline viral load, sports, treatment group, and body-mass index.*not adjusted for sports because model did not converge. BSAP=bone-specific alkaline phosphatase. P1NP=procollagen type 1 N-propeptide. Table 4: Association between baseline biomarkers and changes in bone mineral density of at least 5% greater loss of bone mineral density occurred in the lumbar spine, total hip, and femoral neck at 48 weeks in the group assigned the standard regimen, with a further Published online September 30,

8 loss at the femoral neck at 96 weeks. Differences in bone turnover biomarkers were also greater in the tenofovir-containing group. Baseline bone biomarkers were associated with changes in bone mineral density, irrespective of treatment group. To our knowledge, this is the first report of changes in bone mineral density at three anatomical sites after initiation of an NtRTI-sparing regimen with a combination of integrase strand-transfer inhibitor and boosted protease inhibitor in antiretroviral-naive patients. Two other studies 18,19 have reported total changes in bone mineral density after initiation of an integrase strand-transfer inhibitor plus a boosted protease inhibitor. PROGRESS 18 change in total body bone-mineral density at 96 weeks was significantly different in antiretroviralnaive patients given a combination of ritonavir boosted lopinavir plus raltegravir (increase of 0 68%) and those receiving boosted lopinavir plus tenofovir with emtricitabine (decrease of 2 48%). However, this study did not report specific anatomical site changes in body mineral density, such as at the lumbar spine, total hip, or femoral neck. In RADAR, 19 changes in absolute total body bone mineral density at 48 weeks were more favourable with ritonavir boosted darunavir plus raltegravir than with boosted darunavir plus tenofovir with emtricitabine (11 3 g/cm²; p=0 013), but again no site-specific data were provided. The mean percentage change in bone mineral density in the NtRTI-sparing group of our study at week 96 ( 0 43% in the lumbar spine, 1 74% at the femoral neck, and 1 57% at the total hip) substantiates that an NtRTI-sparing regimen with an integrase strand-transfer inhibitor plus a boosted protease inhibitor regimen has a slight effect on bone mineral density, especially on the spine trabecular bone. Bone mineral density loss after ART initiation tends to stabilise after the initial decreases at weeks By contrast, an additional decrease of 2 5% occurred in femoral neck bone mineral density between 48 and 96 weeks in the standard group. Different population characteristics could possibly account for the progressive reduction in our study. Patients enrolled in the parent study 27 tended to be old, with low baseline BMI. In addition to traditional risk factors for osteoporosis, factors associated with low bone mineral density in populations infected with HIV, apart from ART initiation, include duration of HIV infection, increased plasma viral load, lower nadir CD4 cell counts, and use of tenofovir with protease inhibitors. 11,28 31 In our substudy, the covariate that was independently associated with loss of bone density at both the femoral neck and total hip at week 48 was allocation of the standard regimen of ritonavir boosted darunavir plus tenofovir with emtricitabine. For lumbar spine, the only variable significantly associated with loss of bone mineral density was baseline viral load. In the ACTG5224 study, 10 a higher baseline viral load was also associated with greater declines in lumbar spine bone mineral density, whereas higher baseline CD4 cell counts were associated with greater increases in bone mineral density in the lumbar spine. In our analysis, CD4 cell count was moderately associated with baseline viral load. Because viral load was the strongest factor in the univariate analysis, it was the only factor that we included in the model. Few prospective data exist about the clinical usefulness of bone biomarkers to predict changes in bone mineral density after initiation of ART. How useful bone turnover biomarkers are as predictors of bone mineral density loss is controversial, and their use is limited by their lack of reproducibility and standardisation. 32,33 Data for the general population suggest that men older than 60 years with concentrations of CTX-1 and P1NP in the upper quintile have a greater risk of accelerated loss of hipbone. 34 In our study, increases in bone biomarkers were greater in the standard treatment group than in the NtRTI sparing group. Other studies have also reported that tenofovir with emtricitabine has a large effect on bone biomarkers. 9,19,35 In the ASSERT study, 9 although bone resorption biomarkers increased after 24 weeks of therapy independently of treatment group, increases were greatest in patients given tenofovir with emtricitabine. CTX-1 and osteocalcin concentrations rose significantly more in patients taking tenofovir with emtricitabine than in those taking raltegravir over 96 weeks in the PROGRESS study. 35 In the RADAR study, 19 CTX-1 and P1NP changes at week 48 were highest in the group given ritonavir-boosted darunavir plus tenofovir with emtricitabine. Several studies have investigated the predictive value of bone turnover biomarkers in ART-naive patients after initiation of ART. In ASSERT, 9 changes in biomarkers were negatively correlated with changes in bone mineral density in both total hip and lumbar spine. Changes in the same biomarkers at week 16 were associated with changes in total body bone mineral density minus that of head at 48 weeks. In PROGRESS, 35 high baseline concentrations and greater changes at week 4 of CTX-1, osteocalcin, and P1NP were all associated with a loss of bone mineral density of at least 5% at 96 weeks. In our study, baseline osteopontin and P1NP concentrations were associated with a change in bone mineral density of more than 5% at the lumbar spine, femoral neck, and total hip. Larger longitudinal analyses are needed in the future to substantiate the clinical predictive value of these biomarkers. The long-term relevance of differences in changes in bone mineral density in our study is unknown. Although bone mineral density is a well established surrogate biomarker for fragility fractures in the general population, associations between bone mineral density, fracture rates, and specific ART regimens have not been clearly established. A population-based Spanish study showed that the risk of hip fractures is increased by almost five times in patients infected with HIV, and 8 Published online September 30,

9 findings from other studies have shown a greater risk of fractures during the first 2 years of ART. 5,13 An independent association has been reported between cumulative use of tenofovir disoproxil fumarate, protease inhibitors, and osteoporotic fracture. 17 In our study, we noted no differences in cases of osteopenia or osteoporosis, or a Z score of less than 2 or bone fractures between groups, but the study was not powered to detect differences in these endpoints. Studies with a longer follow-up, preferentially in an older population, are needed to clarify the association of low bone mineral density, fractures, and ART in populations infected with HIV. The clinical usefulness of NtRTI-sparing regimens is unclear, especially after new tenofovir combinations, including tenofovir alafenamide, become available. Compared with tenofovir disoproxil fumarate, tenofovir alafenamide has less effect on glomerular function, tubular function, and bone mineral density. 36 The increased safety of tenofovir alafenamide is encouraging, but whether these differences would translate into real clinical benefit, although highly probable, remains to be proven. Until this combination becomes widely available, we think that NtRTI-sparing regimens could be of use, especially for populations with a high risk of bone disease after initiation of ART. Our study had several limitations. Although DXA scans were locally calibrated according to the manufacturer, no central calibration or central reading of DXA measurements took place. Additionally, the methods of analysis of bone resorption (preanalytical and analytical variation and an absence of standardisation) make comparisons of studies almost impossible. 17 patients had missing data or did not have all necessary DXA data or biological samples, which reduced the sample size for analysis. This small sample size limited our power to find differences between the treatment groups in measurements of bone and inflammatory biomarkers. No adjustments were made for repeated statistical testing; however, the difference between groups was higher than expected and highly significant. Additionally, we did not record other variables that affect bone mineral density such as use of recreational drugs and statins, and concentrations of sexual hormones. Such caveats are noted in other studies on this topic. In conclusion, after 48 and 96 weeks, an NtRTI-sparing regimen of ritonavir boosted darunavir plus raltegravir was associated with significantly less decrease in bone mineral density at three different anatomical sites (lumbar spine, femoral neck, and total hip) than a standard regimen of boosted darunavir plus tenofovir with emtricitabine. This difference was associated with greater increases in bone turnover biomarkers in the tenofovirwiith emtricitabine group. Baseline concentrations of bone biomarkers were associated with large changes in bone mineral density irrespective of the ART regimen used. Contributors JIB, AM, CW, PR, PWM, LR, AC, SDW, CK, and FR reviewed and interpreted the data analyses, and designed and oversaw the study. JIB and JRA wrote the first draft of this report; all authors enrolled patients, acquired data, reviewed and amended the draft report, and approved the final version. Declaration of interests JIB reports grants from European Commission and Ministerio de Sanidad y Asuntos Sociales de España to the author s institution, during the conduct of the study; personal fees from Bristol-Myers Squibb, Gilead Sciences, ViiV Healthcare, Merck, Janssen, and Abbvie, outside the submitted work. AM reports speaker fees, honoraria, travel support, and consultancy fees from Bristol-Myers Squibb, Boehringer Ingelheim, Gilead, Merck, Pfizer, and Wragge LLC, outside the submitted work. PWM reports grants and personal fees from Gilead Sciences, Bristol-Myers Squibb, Janssen, and ViiV Healthcare, outside the submitted work. CW reports grants from European Commission, non-financial support from Gilead, grants and non-financial support from Janssen Pharmaceuticals and Merck, and grants from ANRS, during the conduct of the study. JG reports grants, personal fees, and non-financial support from Gilead, ViiV, Abbvie, Merck, Bristol-Myers Squibb, Medivir, and Janssen, outside the submitted work. PR reports grants from Gilead Sciences, ViiV Healthcare, Janssen Pharmaceutica, Bristol-Myers Squibb, Merck, and ViiV Healthcare, outside the submitted work. CK reports grants from Giléad and Bristol-Myers Squibb, outside the submitted work. SDW reports grants from MSD and Janssen, and grants and non-financial support from Gilead, outside the submitted work. LR reports grants from European Commission, non-financial support from Gilead, grants and non-financial support from Janssen Pharmaceuticals and Merck, and grants from ANRS, during the conduct of the study; grants from Abbott, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Janssen Pharmaceuticals, MSD, Pfizer, Roche, Tibotec, and ViiV Healthcare to the author s institution, outside the submitted work. ABa reports grants from EU (NEAT Foundation), grants from ANRS and UK MRC, during the conduct of the study. AC received grants from ViiV Healthcare, Bristol-Myers Squibb, MSD, Abbvie, Janssen, outside this work. PMG reports grants and personal fees from Bristol-Myers Squibb, grants from Janssen, personal fees from Gilead, ViiV Healthcare, and Abbvie, outside the submitted work. GC reports grants from European Commission, non-financial support from Gilead, grants and non-financial support from Janssen Pharmaceuticals and Merck, grants from ANRS, during the conduct of the study; grants from Gilead, Tibotec-Janssen, Roche, MSD, Janssen, Boehringer Ingelheim, Bristol-Myers Squibb, GlaxoSmithKline, ViiV Healthcare, Mylan, Abbvie, Abbott, Pfizer, and non-financial support from Lundbeck: Academy on Neurology of Human Behavior, outside the submitted work. FR received research funding, honoraria, or consulted for Abbvie, Bristol-Myers Squibb, Gilead Sciences, Janssen, MSD, and ViiV Healthcare. JRA reports personal fees from ViiV Healthcare, Janssen, Abbvie, Bristol-Myers Squibb, Gilead, and MSD, outside the submitted work. ABu and CR declare no competing interests. Acknowledgments Funding was provided to the Instituto Superiore di Sanita-Rome by the European Commission (project number LSHP-CT ). Bone biomarkers analysis was funded by a grant from the Ministerio de Sanidad y Asuntos Sociales de España (2009/TRA-054). The trial drugs were supplied by Gilead Sciences, Janssen Pharmaceuticals, and Merck Laboratories. The French National Institute for Health and Medical Research-France Recherche Nord&Sud SIDA-HIV Hépatites (Inserm-ANRS) is the sponsor and a funder of the trial. We thank the study participants and their partners, families, and caregivers, local investigators, and study coordinators from all the centres taking part in the trial; the HIV CTU, INSERM U897 Coordinating Unit (Bordeaux, France); MRC Clinical Trials Unit (University College London, London, UK); CHIP Coordinating Unit (Copenhagen, Denmark); Amsterdam Institute for Global Health and Development (which served as the Academic Medical Centre Coordinating Unit, Amsterdam, Netherlands); Istituto Superiore di Sanità (Rome, Italy); Gesida (Madrid, Spain); University of Athens Medical School (Athens, Greece); and especially Antonio Buño and his laboratory staff at the biochemistry department of Hospital Universitario La Paz (La Paz, Spain) for analysing the bone and inflammatory biomarkers. Published online September 30,

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