Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Bernardino JI, Mocroft A, Mallon PW, et al. 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. Lancet HIV 2015; published online Sept 30. http://dx.doi.org/10.1016/S2352-3018(15)00181-2.

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NEAT 001/ANRS 143 Study Group

Asterisk (*) indicates staff who left during the trial. Trial Development Team (TDT):

Belgium: Nikos Dedes (Brussels) France: Genevieve Chene, Laura Richert (Bordeaux), Clotilde Allavena, Francois Raffi (Nantes) and Brigitte Autran (Paris) Italy: Andrea Antinori, Raff aella Bucciardini and Stefano Vella (Rome) Poland: Andrzej Horban (Warsaw) Spain: Jose Arribas (Madrid) UK: Abdel G Babiker, Marta Boffito, Deenan Pillay and Anton Pozniak (London)

Trial Steering Committee (TSC):

Belgium: Xavier Franquet* and Siegfried Schwarze (Brussels) Denmark: Jesper Grarup (Copenhagen) France: Genevieve Chene, Aurelie Fischer*, Laura Richert, Cedrick Wallet (Bordeaux), Francois Raffi (Nantes), Alpha Diallo, Jean-

Michel Molina, and Juliette Saillard (Paris) Germany: Christiane Moecklinghoff (Janssen Pharmaceuticals; Freiburg) and

Hans-Jurgen Stellbrink (Hamburg) Italy: Stefano Vella (Rome) Netherlands: Remko Van Leeuwen (Amsterdam) Spain: Jose Gatell (Barcelona) Sweden: Eric Sandstrom (Stockholm) Switzerland: Markus Flepp (Zurich) UK: Abdel G Babiker, Fiona Ewings*, Elizabeth C George, Fleur Hudson, and Anton Pozniak (London) USA: Gillian Pearce*, Romina Quercia*, Felipe Rogatto (Gilead Sciences; Foster City, CA), Randi Leavitt, and Bach-Yen Nguyen*

(Merck Laboratories; Whitehouse Station, NJ). Independent Data Monitoring Committee (IDMC):

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Germany: Frank Goebel (Munich) Italy: Simone Marcotullio (Rome) UK: Abdel Babiker, Fiona Ewings*, Elizabeth C George, Fleur Hudson*, Navrup Kaur, Peter Sasieni, Christina Spencer-Drake*

(London) and Tim Peto (Oxford) USA: Veronica Miller (Washington DC).

Trial Management Team (TMT):

France: Clotilde Allavena and François Raffi (Nantes) Italy: Stefano Vella (Rome) UK: Anton Pozniak (London)

CMG-EC, INSERM U897 Coordinating Unit, Bordeaux, France:

Geneviève Chêne, Head of coordinating CTU, Member, Bordeaux, France Fabien Arnault*, Coordinating CTU representative, Member, Bordeaux, France Céline Boucherie*, Bordeaux CTU representative, Observer, Bordeaux, France Aurélie Fischer*, Coordinating CTU representative, Member, Bordeaux, France Delphine Jean*, Bordeaux CTU representative, Observer, Bordeaux, France Virginie Paniego*, Coordinating CTU representative, Member, Bordeaux, France Felasoa Paraina, Bordeaux CTU representative, Observer, Bordeaux, France Laura Richert, Coordinating CTU representative, Member, Bordeaux, France Elodie Rouch*, Bordeaux CTU representative, Observer, Bordeaux, France Christine Schwimmer, Coordinating CTU representative, Member, Bordeaux, France Malika Soussi*, Bordeaux CTU representative, Observer, Bordeaux, France Audrey Taieb*, Bordeaux CTU representative, Observer, Bordeaux, France Monique Termote, Coordinating CTU representative, Member, Bordeaux, France Guillaume Touzeau*, Coordinating CTU representative, Member, Bordeaux, France Cédrick Wallet, Bordeaux CTU representative, Member, Bordeaux, France

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MRC Clinical Trials Coordinating Unit, London, UK:

Abdel G Babiker, Trial Statistician, Member, London, UK Adam Cursley, MRC CTU representative, Observer, London, UK Wendy Dodds*, MRC CTU representative, Member, London, UK Fiona Ewings*, Trial Statistician, Member, London, UK Elizabeth C George, Trial Statistician, Member, London, UK Anne Hoppe*, MRC CTU representative, Observer, London, UK Fleur Hudson, MRC CTU representative, Member, London, UK Ischa Kummeling*, MRC CTU representative, Observer, London, UK Filippo Pacciarini*, MRC CTU representative, Observer, London, UK Nick Paton*, MRC CTU representative, Observer, London, UK Charlotte Russell, MRC CTU representative, Observer, London, UK Kay Taylor*, MRC CTU representative, Observer, London, UK Denise Ward, MRC CTU representative, Observer, London, UK

CHIP Coordinating Unit, Copenhagen, Denmark:

Bitten Aagaard*, CHIP CTU representative, Observer, Copenhagen, Denmark Marius Eid, CHIP CTU representative, Observer, Copenhagen, Denmark Daniela Gey*, CHIP CTU representative, Member, Copenhagen, Denmark Birgitte Gram Jensen*, CHIP CTU representative, Observer, Copenhagen, Denmark Jesper Grarup, CHIP CTU representative, Member, Copenhagen, Denmark Marie-Louise Jakobsen*, CHIP CTU representative, Observer, Copenhagen, Denmark Per O. Jansson, CHIP CTU representative, Member, Copenhagen, Denmark Karoline Jensen*, CHIP CTU representative, Member, Copenhagen, Denmark Zillah Maria Joensen, CHIP CTU representative, Observer, Copenhagen, Denmark Ellen Moseholm Larsen*, CHIP CTU representative, Observer, Copenhagen, Denmark Christiane Pahl*, CHIP CTU representative, Observer, Copenhagen, Denmark

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Mary Pearson*, CHIP CTU representative, Member, Copenhagen, Denmark Birgit Riis Nielsen, CHIP CTU representative, Observer, Copenhagen, Denmark Søren Stentoft Reilev*, CHIP CTU representative, Observer, Copenhagen, Denmark

Amsterdam Medical Center Coordinating Unit, Amsterdam, The Netherlands:

Ilse Christ, AMC CTU representative, Observer, Amsterdam, The Netherlands Desiree Lathouwers*, AMC CTU representative, Member, Amsterdam, The Netherlands Corry Manting, AMC CTU representative, Member, Amsterdam, The Netherlands Remko Van Leeuwen, AMC CTU representative, Member, Amsterdam, The Netherlands

ANRS, Paris, France: Alpha Diallo, Pharmacovigilance representative, Member, Paris, France Bienvenu Yves Mendy*, Pharmacovigilance representative, Member, Paris, France Annie Metro*, Pharmacovigilance representative, Member, Paris, France Juliette Saillard, Sponsor representative, Member, Paris, France Sandrine Couffin-Cadiergues, Sponsor representative, Observer, Paris, France

ISS, Rome, Italy: Anne-Laure Knellwolf*, NEAT management representative, Observer, Rome, ltaly Lucia Palmisiano, NEAT management representative, Member, Rome, ltaly Local CTUs:

GESIDA, Madrid, Spain:

Esther Aznar, Cristina Barea*, Manuel Cotarelo*, Herminia Esteban, Iciar Girbau*, Beatriz Moyano, Miriam Ramirez*, Carmen Saiz, Isabel Sanchez, Maria Yllescas

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ISS, Rome, Italy:

Andrea Binelli, Valentina Colasanti, Maurizio Massella, Lucia Palmisiano.

University of Athens Medical School, Greece:

Olga Anagnostou, Vicky Gioukari, Giota Touloumi. Study Investigators:

Austria: Brigitte Schmied (National Coordinating Investigator), Armin Rieger, Norbert Vetter

Belgium: Stephane De Wit (National Coordinating Investigator), Eric Florence, Linos Vandekerckhove

Denmark: Jan Gerstoft (National Coordinating Investigator), Lars Mathiesen

France: Christine Katlama (National Coordinating Investigator), Andre Cabie, Antoine Cheret, Michel Dupon, Jade Ghosn*, Pierre-Marie Girard, Cécile Goujard, Yves Lévy, Jean-Michel Molina, Philippe Morlat, Didier Neau, Martine Obadia, Philippe Perre, Lionel Piroth, Jacques Reynes, Pierre Tattevin, Francois Raffi, Jean Marie Ragnaud*, Laurence Weiss, Yazdanpanah Yazdan*, Patrick Yeni, David Zucman

Germany: Georg Behrens (National Coordinating Investigator), Stefan Esser, Gerd Fätkenheuer, Christian Hoffmann, Heiko Jessen, Jürgen Rockstroh, Reinhold Schmidt, Christoph Stephan, Stefan Unger

Greece: Angelos Hatzakis (National Coordinating Investigator), George L Daikos, Antonios Papadopoulos, Athamasios Skoutelis

Hungary: Denes Banhegyi (National Coordinating Investigator)

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Ireland: Paddy Mallon (National Coordinating Investigator), Fiona Mulcahy

Italy: Andrea Antinori (National Coordinating Investigator), Massimo Andreoni, Stefano Bonora, Francesco Castelli, Antonella D’Arminio Monforte, Giovanni Di Perri, Massimo Galli, Adriano Lazzarin, Francesco Mazzotta, Torti Carlo*, Vincenzo Vullo

The Netherlands: Jan Prins (National Coordinating Investigator), Clemens Richter, Dominique Verhagen, Arne Van Eeden*

Poland: Andrzej Horban (National Coordinating Investigator)

Portugal: Manuela Doroana (National Coordinating Investigator), Francisco Antunes*, Fernando Maltez, Rui Sarmento-Castro,

Spain: Juan Gonzalez Garcia (National Coordinating Investigator), José López Aldeguer, Bonaventura Clotet, Pere Domingo, Jose M Gatell, Hernando Knobel, Manuel Marquez, Martin Pilar Miralles, Joaquin Portilla, Vicente Soriano, Maria-Jesus Tellez

Sweden: Anders Thalme (National Coordinating Investigator), Anders Blaxhult, Magnus Gisslen

UK: Alan Winston (National Coordinating Investigator), Julie Fox, Mark Gompels, Elbushra Herieka, Margaret Johnson, Clifford Leen, Anton Pozniak, Alastair Teague, Ian Williams

Endpoint Review Committee (ERC):

Australia: Mark Alastair Boyd, (Sydney) Denmark: Jesper Grarup, Per O Jansson, Nina Friis Møller, and Ellen Frøsig

Moseholm Larsen (Copenhagen) France: Philippe Morlat (Bordeaux), Lionel Piroth (Dijon), and Vincent Le Moing (Montpellier) Netherlands: Ferdinand W N M Wit, chair (Amsterdam) Poland: Justyna Kowalska (Warsaw) Spain: Juan Berenguer and Santiago Moreno (Madrid) Switzerland: Nicolas J Müller (Zurich)

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UK: Estée Török (Cambridge), Frank Post (London), and Brian Angus (Oxford) Sub-study working groups:

Virology working group: Vincent Calvez (coordinator), Charles Boucher, Simon Collins, David Dunn (statistician), Sidonie Lambert, Anne-Geneviève Marcelin, Carlo Federico Perno, Deenan Pillay, Ellen White (statistician)

Pharmacology and adherence working group: Marta Boffito (coordinator), Adriana Ammassari, Andrea Antinori, Wolgang Stoehr (statistician)

Immunology working group: Brigitte Autran (coordinator), Reinhold Ernst Schmidt, Michal Odermarsky, Colette Smith, Rodolphe Thiébaut (statistician)

Toxicity, including co-infection working group: Jose Arribas (coordinator), Jose Ignacio Bernardino De La Serna, Antonella Castagna, Stephane De Wit, Xavier Franquet, Hans-Jackob Furrer, Christine Katlama, Amanda Mocroft (statistician), Peter Reiss

Quality of life working group: Raffaella Bucciardini (coordinator), Nikos Dedes, Vincenzo Fragola, Elizabeth C George (statistician), Marco Lauriola, Rita Murri, Pythia Nieuwkerk, Bruno Spire, Alain Volny-Anne, Brian West

Neurocognitive function working group: Hélène Amieva (coordinator), Andrea Antinori, Josep Maria Llibre Codina, Laura Richert, Wolgang Stoehr (statistician), Alan Winston

Pharmaco-economics working group: Francesco Castelli (coordinator), Marco Braggion (statistician), Emanuele Focà

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Additional information about bone and inflammatory biomarkers The following markers were measured by automated chemiluminescence immunoassay

(CLIA): bone specific alkaline phosphatase (reference ranges 5.7-32.9 µg/L for men

and 4.7-27 µg/L for premenopausal women, intra-assay coefficient of variation (CV)

1.5%), c-terminal collagen crosslinks (CTX-1) (reference ranges 0.15-0.75 ng/mL for

men and 0.11-0.74 ng/mL for post-menopausal women, intra-assay CV 3.2%), intact N-

terminal pro-peptide of type 1 procollagen (P1NP) reference ranges 27.7-127.6 ng/mL,

intra-assay CV 3%) osteocalcin (OC) (reference ranges 10.4-45.6 ng/mL, intra-assay

CV 2.2%), and 25-OH-vitamin D (intra-assay CV 5.5%; intact PTH intra-assay CV

2.6%). RANKL was measured by enzyme-immunoanalysis with an intra-assay CV 8%.

The following markers were analysed by Multiplex enzyme-immunoanalysis

(HBNMAG-51K): osteoprotegerin (OPG) (reference ranges 130-350 pg/mL, intra-assay

CV 3%), osteopontin (OPN) (reference ranges 2008-20631 pg/mL, intra-assay CV 3%),

IL-6 (reference ranges 5-7 pg/mL, intra-assay CV 5%), IL-1 (reference ranges 5-10

pg/mL, intra-assay CV 6%) and TNF- (reference ranges 1-3 pg/mL, intra-assay CV

4%).

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Table 1. Bone Mineral Density and DEXA parameters at randomisation in the three anatomical sites DRV/r + RAL

(N = 65) DRV/r + TDF/FTC

(N = 68) Total

(N = 133) P value*

Lumbar spine

BMD (g/cm2): median (IQR) 1.10 (1.0 - 1.22) 1.16 (1.02 - 1.23) 1.13 (1.01 - 1.23) 0.26

Z-score: median (IQR) -0.4 (-1.1 - 0.3) -0.2 (-0.9 - 0.3) -0.2 (-1.0 - 0.3) 0.43

T-score: median (IQR) -0.5 (-1.2 - 0.3) -0.2 (-0.9 - 0.4) -0.3 (-1.1 - 0.4) 0.31

Osteoporosis: n (%) 4 (6.6) 1 (1.5) 5 (3.9) 0.14

Osteopenia: n (%) 14 (23) 13 (19.1) 27 (20.9) 0.59

Z score < -2: n (%) 6 (9.8) 3 (4.4) 9 (7.0) 0.23

Femoral neck

BMD (g/cm2): median (IQR) 0.93 (0.86 - 1.06) 0.98 (0.89 - 1.11) 0.96 (0.87 - 1.08) 0.13

Z-score: median (IQR) -0.2 (-0.8 - 0.6) 0.2 (-0.3 - 0.7) 0 (-0.6 - 0.6) 0.05

T-score: median (IQR) -0.6 (-0.9 - 0.4) -0.1 (-0.8 - 0.6) -0.2 (-0.8 - 0.5) 0.16

Osteoporosis: n (%) 0 (0) 1 (1.5) 1 (0.8) 0.35

Osteopenia: n (%) 10 (16.7) 11 (15.9) 21 (16.3) 0.91

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Z score < -2: n (%) 0(0) 0(0) 0(0) -

Total hip

BMD (g/cm2): median (IQR) 1.01 (0.93 - 1.11) 1.07 (0.97 - 1.16) 1.04 (1.00 - 1.10) 0.032

Z-score: median (IQR) -0.1 (-0.7 - 0.7) 0.3 (-0.3 - 0.8) 0.1 (-0.4 - 0.7) 0.05

T-score: median (IQR) -0.2 (-0.9 - 0.5) 0.2 (-0.5 - 0.8) 0 (-0.7 - 0.6) 0.048

Osteoporosis: n (%) 0 (0) 1 (1.5) 1 (0.8) 0.35

Osteopenia: n (%) 12 (19.7) 9 (13) 21 (16.2) 0.31

Z score < -2: n (%) 0 (0) 1 (1.5) 1 (0.8) 0.34

*P value from Kruskall-Wallis test for continuous variables and chi-squared test for categorical variables Bone mineral density data was not available for all patients at baseline.

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Table 2. New cases of osteoporosis, osteopenia and Z score < -2 during the trial

RAL + DRV/r TDF/FTC + DRV/r OR (95% CI)*

Week 48 Osteoporosis Osteopenia Z < -2.0 Osteoporosis Osteopenia Z < -2.0

Lumbar spine 0 4 1 1 7 2 1.42 (0.39-5.26)

Femoral neck 0 6 0 0 3 0 0.37 (0.087-1.57)

Total hip 1 2 1 0 5 0 2.03 (0.38-10.98)

Week 96

Lumbar spine 0 4 1 2 4 2 0.81 (0.19-3.47)

Femoral neck 1 8 0 1 7 1 0.67 (0.22-2.04)

Total hip 1 2 1 0 2 0 0.79 (0.11-5.88)

* OR for Osteopenia (TDF/FTC + DRV/r vs. RAL + DRV/r)

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Table 3. Absolute values for bone and inflammatory biomarkers at randomisation.

Biomarker N DRV/r + RAL N DRV/r + TDF/FTC P-value Osteocalcin ng/mL 48 11.6 (10.2, 13) 56 11.7 (10.3, 13.1) 0.88 BSAP µg/L 48 12.5 (11.7, 13.3) 55 13.5 (12.5, 14.5) 0.17 P1NP ng/mL 48 44.3 (38.4, 50.6) 56 45.1 (40, 50.2) 0.83 Osteoprotegerin pg/mL 47 299 (265.9, 352.1) 56 338 (289.2, 386.8) 0.22 Serum CTX-1 ng/mL 48 0.4 (0.35, 0.5) 55 0.4 (0.3, 0.5) 0.4 RANKL pmol/L 20 0.59 (0.22, 0.96) 31 0.52 (0.28, 0.76) 0.72 Osteopontin pg/mL 47 5421 (4317, 6526) 56 5425 (4145, 6705) 0.99 Urine CTX-1 /creatinine ratio 65 195.4 (163.5, 227.3) 68 206.8 (180.6, 232) 0.6 25-vit D ng/mL 47 19.2 (16.5, 21.9) 53 20.6 (17.5, 23.5) 0.49 iPTH pg/mL 47 31.9 (27.2, 36.6) 56 32.2 (28.5, 35.9) 0.91 IL-1 pg/mL 46 0.4 (0.3, 0.5) 55 0.4 (0.3, 0.5) 0.22 IL-6 pg/mL 47 1.2 (0.0, 2.4) 56 1.4 (0.6, 2.2) 0.80

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TNF- pg/mL 44 4.1 (2.7, 5.5) 56 4 (3.6, 4.4) 0.77

Note: BSAP: Bone specific Alkaline Phosphatase. P1NP: Pro-collagen type 1 N-pro-peptide. CTX-1: Type 1 C terminal collagen crosslinks. RANKL: Receptor activator of NF- ligand. CRLP: C-terminal collagen crosslinks. iPTH: intact-parathyroid hormone.

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Figure2.Boxplotofboneandinflammatorybiomarkersbytreatment2a.Formationmarkers

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