Supplementary Online Content

Transcription

1 Supplementary Online Content Rodger AJ, Cambiano V, Bruun T, et al; PARTNER Study Group. Sexual activity without condoms and risk of HIV transmission in serodifferent couples when the HIVpositive partner is using suppressive antiretroviral therapy: the PARTNER Study. JAMA. doi:./jama emethods etable. Partners Sample Type (Either Plasma or PBMCs) and pol HIV- Subtype etable 2. Pairwise Genetic Distances of pol and env Sequences From Partners and Positive Controls (Comprising Replicate Partners Sequences and Sequences From Confirmed Transmission Pairs Obtained in a Separate Study [ref: Beloukas Virus Res 22]) efigure. Phylogenetic Tree of pol Sequences From Couples, Including Couple 5 and Couple 6 With Subtype-Discordant Infection efigure 2. Phylogenetic Tree of pol Sequences From Nine Couples With Subtype B Infection etable 3. Sensitivity Analysis Including and Excluding Follow-up Time in Which the HIV-RNA was Suppressed at the Beginning of the Period but Became Elevated etable 4. Hierarchical Approach to Classifying Types of Sex etable 5. Baseline Characteristics in Participants Who Contributed to the Eligible Couple-Years of Follow-up and Participants Who Did Not (Either Because They Dropped Out or Because the Couple-Years of Follow-up They Contributed Were Not Eligible) 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

2 emethods Virological Methods. HIV- pol and env sequencing and analysis Viral sequences were obtained from either HIV- RNA recovered from plasma (n=4) or HIV- DNA recovered from peripheral blood mononuclear cells (n=3) (Supplementary Table ). HIV- RNA was extracted with NucliSENS easymag (biomérieux, France), reverse transcribed with Invitrogen SuperScript III First-Strand Synthesis SuperMix (Life Technologies, CA, USA), and amplified by nested PCR with Invitrogen Platinum PCR SuperMix High Fidelity (Life Technologies). HIV- DNA was extracted with QIAamp DNA Blood Mini Kit (Qiagen, Netherlands), and input into a first round PCR with Invitrogen Platinum Taq DNA Polymerase High Fidelity (Life Technologies) followed by nested PCR with Invitrogen Platinum PCR SuperMix High Fidelity (Life Technologies). Sequencing was carried out with the BigDye Terminator v3. Cycle Sequencing Kit (Life Technologies) on the ABI 373xl DNA analyzer (Applied Biosystems, CA, USA). Sequences spanning amino acids -44 of pol (HXB nt) and of env (HXB nt) were aligned and manually edited using MUSCLE ( and MEGA 6. software [ref: Tamura, 23], respectively. Subtyping of pol sequences was performed using the online automated subtyping tool COMET (Context-based Modeling for Expeditious Typing) [ref: Struck, 24] and the REGA HIV- Subtyping Tool v3. [ref: Pineda-Pena, 23]. Automated subtyping results were confirmed by phylogenetic analysis with 26 reference sequences representing pure HIV- subtypes (A D, F H, J, and K) and 49 circulating recombinant forms (CRFs), as previously described [ref: Paraskevis, 25]. 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

3 For the analysis of linkage between the partners viral sequences, phylogenetic analyses included either all available sequences (regardless of HIV- subtype) or only those sequences that were typed as subtype B. Codons associated with antiretroviral drug resistance [ref: Wensing, 24] were removed from the partners alignments prior to analysis. jmodeltest2 [ref: Darriba, 22] was used to identify the best fitted substitution model under both AICc and BIC criteria, allowing searches across the three main substitution models and all possible Generalised Time Reversible (GTR) submatrices. GTR+I+G and 222+I+G+F were identified as the best fitted models. Maximumlikelihood (ML) estimates with bootstrap values were obtained with RaxML-HCP2 v8 using GTR+I+G [Ref: Stamatakis,24]. Bayesian Markov Chain Monte-Carlo (MCMC) inference with posterior probabilities was obtained with MrBayes v3.2.6 [ref: Ronquist, 22] using GTR+I+G and 222+I+G+F, for 7 generations sampling every th tree, as previously described [Beloukas, 22]. Convergence of the MCMC procedure was assessed by calculating the effective sampling size (ESS) with Tracer v..6 [Rambaut, 24], with a target ESS >. Analyses showing an initial ESS < were expanded (up to 5 7 generations) until efficient mixing of the Markovian chains was achieved. Each run consensus tree was implied and the Tree visualization and annotation was performed using FigTree v.4. Pairwise genetic distances were calculated using MEGA 6. under the Maximum Composite Likelihood model and the rate variation among sites was modeled with a gamma distribution (shape=4) [ref: Tamura, 23]. Analyses were run on the CIPRES Science Gateway [Ref: Miller 2] 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

4 References for Virological Methods. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.. Mol Biol Evol 23;3: Struck D, Lawyer G, Ternes AM, Schmit JC, Bercoff DP. COMET: adaptive contextbased modeling for ultrafast HIV- subtype identification. Nucleic Acids Res 24;42: e Pineda-Pena AC, Faria NR, Imbrechts S, et al. Automated subtyping of HIV- genetic sequences for clinical and surveillance purposes: performance evaluation of the new REGA version 3 and seven other tools. Infect Genet Evol 23;9: Paraskevis D, Kostaki E, Beloukas A, et al. Molecular characterization of HIV- infection in Northwest Spain (29-23): Investigation of the subtype F outbreak. Infect Genet Evol 25;3: Wensing AM, Calvez V, Gunthard HF, Johnson VA, Paredes R, Pillay D, et al. 24 Update of the drug resistance mutations in HIV-. Top Antivir Med 24,22: Darriba D, Taboada GL, Doallo R, Posada D. jmodeltest 2: more models, new heuristics and parallel computing. Nat Methods 22;9: Stamatakis A. RaxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 24;3: Ronquist F, Teslenko M, van der Mark P, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. System Biol 22;6: American Medical Association. All rights reserved. Downloaded From: on 6/8/28

5 9. Beloukas A, Magiorkinis E, Magiorkinis G, Zavitsanou A, Karamitros T, Hatzakis A, Paraskevis D. Assessment of phylogenetic sensitivity for reconstructing HIV- epidemiological relationships. Virus Res. 22;66: Rambaut A, Suchard M, Xie D, Drummond A. Tracer v.6. Available from Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE); 2; New Orleans, LA; 2. Pp American Medical Association. All rights reserved. Downloaded From: on 6/8/28

6 Calculation of Number of HIV Transmission Expected in the absence of ART We calculated the number of HIV transmissions that we would have expected in this study if the positive index partners were not on ART, based on the numbers of condomless sex acts reported during follow-up. The assumptions regarding the probability of HIV transmission per condomless sex act (in the absence of ART) come from two meta-analyses: Boily MC, Baggaley RF, Wang L, Masse B, White RG, Hayes RJ, et al. Heterosexual risk of HIV- infection per sexual act: systematic review and metaanalysis of observational studies. Lancet Infect Dis. 29;9(2):8-29. Baggaley RF, White RG, Boily MC. HIV transmission risk through anal intercourse: systematic review, met a-analysis and implications for HIV prevention. Int J Epidemiol. 2;39(4):48-63 The first (Boily 29) estimated the rate of transmission per heterosexual sex act in high income countries to be.8 (95% CI:.6-.) for male to female and.4 (95% CI:.-.4) for female to male. The second (Baggaley 2) estimated the probability of transmission per receptive anal sex act to be.4 (95%CI:.2-.25). No separate estimates for anal and vaginal sex were available for heterosexuals. For MSM, we conservatively only accounted for sex acts in which the negative partner was receptive. In order to estimate the number of HIV transmission expected to observe in absence of ART, we calculate separately for heterosexual couples with a male index partner, couples 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

7 with a female index partner and MSM couples, the cumulative probability of HIV transmission (CP) for each couple in the group was calculated, based on the number of sex acts the HIV-negative partner reported, using the formula below: CCCC = (( pp) nn ) where: n is the number of condomless sex acts reported by the negative partner sex act) p is the probability of transmission per condomless sex act (specific to the type of The cumulative probability of HIV transmission was summed across all couples to determine the number of sex acts across all couples in the study. These estimates suggest that we would have expected over transmissions in the MSM group alone if the positive partner had not been on ART. These estimates should be considered with caution because the sexual behaviour could have been different if the positive partner was not on ART and the number of sex acts since the last visit was collected as a categorical variable (,, 2-, -2, 2-4, >4) and we used the mid-point in the category they indicated (for the highest category we used a value of 5). Sensitivity Analysis Sensitivity analysis was conducted including the period of follow-up time in which the HIV-RNA was suppressed at the beginning of the period, but during which became elevated (etable 3). If we include the periods of follow-up time (defined between two 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

8 consecutive HIV tests), in which the HIV-RNA was suppressed at the beginning of the period but during which the HIV-RNA became elevated, the number of within-couple transmissions is still zero, therefore the estimated rate is zero. The estimates / confidence intervals included in the manuscript and the alternative described above are shown in the table below. In a further sensitivity analysis we calculated the rate and confidence interval taking a hierarchical approach to classifying types of sex (etable 4). Having defined such a hierarchy of risk, in referring to a specific sex act, we estimated the upper limit of the rate if this type of sex is the highest risk sex being performed. With the hierarchy (from the highest to the lowest) being: receptive anal sex with ejaculation, receptive anal sex without ejaculation, insertive anal sex, vaginal sex with ejaculation and vaginal sex without ejaculation. Those not indicated in the table below will remain invariate using the hierarchical approach. When considering a hierarchical approach (i.e. the act-specific rate were restricted to person-years of follow-up where that type of act was associated with the highest risk of acquisition), the 95% upper limit of the confidence interval increases: for receptive anal sex without ejaculation it increased from 8.4 to.95 per couple-years-of-follow up for heterosexual women, and from.68 to 3.6 per couple-years-of-follow up for MSM, while for vaginal sex it increased from.59 to.69 per couple-years-offollow up (Heterosexual men and women combined). 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

9 etable. Partners Sample Type (Either Plasma or PBMCs) and pol HIV- Subtype Couple no Initial HIV status Sample HIV- subtype NEGATIVE Plasma B POSITIVE Plasma B 2 NEGATIVE Plasma B 3 NEGATIVE Plasma B 4 NEGATIVE Plasma B 5 NEGATIVE Plasma A 6 NEGATIVE Plasma CRF 4_BG 7 NEGATIVE Plasma B POSITIVE Plasma B 8 NEGATIVE Plasma B 9 NEGATIVE PBMC B NEGATIVE PBMC B 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

10 NEGATIVE PBMC B NEGATIVE Plasma B NEGATIVE Plasma B POSITIVE Plasma B Footnotes: PBMC= Peripheral blood mononuclear cells; CRF = circulating recombinant form. HIV- subtype refers to the HIV positive partner initially and the previously HIV negative partner at follow-up after seroconversion 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

11 etable 2. Pairwise Genetic Distances of pol and env Sequences From Partners and Positive Controls (Comprising Replicate Partners Sequences and Sequences From Confirmed Transmission Pairs Obtained in a Separate Study [ref: Beloukas Virus Res 22]) Source Sequence (subtype) Median genetic distance (IQR) Partners pol (all).7 (.56,.79) Partners pol (B).68 (.56,.76) Partners env (all).28 (.,.43) Partners env (B).25 (.98,.37) Partner replicates pol. (<.,.3) Partner replicates env.24 (.2,.37) Separate study pol.8 (.6,.9) All positive controls pol.4 (<.,.8) IQR= Interquartile range 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

12 efigure. Phylogenetic Tree of pol Sequences From Couples, Including Couple 5 and Couple 6 With Subtype-Discordant Infection. Bayesian Markov Chain Monte-Carlo (MCMC) inference (222+I+G+F model). Branch line weight is proportional to the posterior probability. Partners sequences are in blue; N indicates the initially HIVnegative partner whereas P indicates the initially HIV-positive partner. Control sequences comprised the closest sequences identified through BLAST searches of GenBank. Positive control sequences comprised replicate sequences from study partners (in red) and sequences from confirmed transmission pairs obtained in a separate study (in orange) [Ref: Beloukas, Virus Res, 22]. *Sequences 9N2 and 9N3 were obtained from the same sample in two separate experiments. 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

13 AY6655 AY AY 679 AY GQ GQ GQ HM Couple_9N Couple_9N3.97 Couple_9N EU EU EU AY 6652 AY EU67338 FJ HQ76.9 Couple_5N GQ4235 Couple_6N HM3548 DQ Couple_N EF36323 JN94 EF JN833 Couple_8N JQ65835 JQ6565 HQ2658 EU69373 D2. JN55.54 JN355 AY JQ HM46462 GU3449 GQ39995 GQ39977 FJ64745 EU EF Couple_8P K C6977 JQ65838 JN837 HM46474 K C23842 JQ65848 U M KF379 AY GQ39967 HM35483 HM4649 HQ AF42.59 AF AF AY U U AY U Couple_N.74 AY 9424 AY Couple_P Couple_P HIV HX B2 AF42 JF EF EF36324 EF JN AY HQ26534 JQ363 D HQ26584 JQ3628 K J JQ39523 K J426 AY AB AY AF FJ JQ434 JQ58577 JQ KJ AY9423 AY 9423 GU8755 Couple_9P Couple_9P2 GU EF HQ768 GQ Couple_3P Couple_7P2 Couple_7P EF54993 Couple_5P.67 Couple_4N K C EU8779 EU8772 Couple_7N Couple_6P AF FJ AF3389 EF K3455. K J76758 M992.2 JN97 EU8778 FJ22836 FJ2288 JN7 JN995 GQ432 GQ39953 EU8777 FJ2288 JN58 K C EU8769 Couple_2P Couple_4P EU24853 AY AY M AY DQ GQ33845 AY DQ97739 AY AY AY AY AY AY K C AY 362 JX EF36755 AY 3256 AY AY AF AJ49469 GQ39927 AJ AJ9793 AF46728 Couple_N JN6382 AF Couple_3N JQ L GU8756 K 23. FJ65959 FJ D8669. FJ K 283. JN HM45325 U255. KC34798 K C3477 K C AY 368 AY 3679 AY3678 FJ JQ K F38489 AY FJ GU26438 Couple_2N Couple_9N2 EU24855 EU EU EU Couple_P2 Couple_P GU2646 EU25539 GQ4455 EU24859 EU EU24844 EU24845 EU American Medical Association. All rights reserved. Downloaded From: on 6/8/28

14 efigure 2. Phylogenetic Tree of pol Sequences From Nine Couples With Subtype B Infection. Maximum-likelihood (ML) inference (GTR+I+G model) from 9 couples with subtype B infection. Branch length is proportional to the genetic distance and line weight is proportional to the posterior probability. Partners sequences are in blue; N indicates the initially negative partner whereas P indicates the initially positive partner. Control sequences comprised the closest sequences identified through BLAST searches of GenBank. Positive control sequences comprised replicate samples from study partners (in red) and sequences from confirmed transmission pairs obtained in a separate study (in orange)[ref: Beloukas, Virus Res, 22]. *Sequences 9N2 and 9N3 were obtained from the same sample in two separate experiments. 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

15 JQ65838 K C AY JN58 3 FJ Couple_4P FJ GU2646 AY 368 GU26438 JQ65744 Couple_2N EF EF JN6439 Couple_N Couple_P AY Couple_N Couple_P2 Couple_P EF36323 Couple_5P Couple_3P EF JQ65747 Couple_6P EU GU EU AY3256 GU3449 EU24845 EU EU2484 GQ EU EU EU EU EU EU EU D2. EU69373 JQ6565 JQ65797 JQ65835 KC AY Couple_P AY AF AF42 9 AF42 79 U U U AY9424 JF EF36324 AY AF56824 AY HQ26534 JQ KJ JQ39523 HQ K J JQ3628 AY 9423 HQ2658 AY9423 GU8755 Couple_9P2 Couple_9P KC EF54993 AF D AY AY HM AY AB K J AF JQ58577 AY 336 U FJ JQ434 Couple_9N3 Couple_9N2 Couple_9N4 JX 6277 EF36755 FJ86827 K F AY AF25949 M M992.2 JQ K J Couple_9N GQ39927 JN837 AY 3848 HIVHXB 2 FJ65959 FJ6595 K 283. D8669. FJ65957 K3455. Couple_7P JQ65848 AJ9793 GU8756 L JN HM U AF EF JN FJ Couple_7P2 AY AY GQ33845 K23. K F AY FJ AY AY AY HM46474 DQ DQ M AY EU AJ49469 AF K C6977 GQ AJ GQ39995 JN JN JN995 JN94 EU8779 JN7 GQ39953 EU EU8772 EU8778 JN97 EU8777 GQ432 Couple_2P K C3429 AF46728 EF EU Couple_7N AY 3679 AY 3678 FJ4885 K C34467 Couple_N EF JN833 Couple_8N Couple_8P GQ FJ2288 Couple_3N HQ768 Couple_4N AY K C34798 EU24853 K C American Medical Association. All rights reserved. Downloaded From: on 6/8/28

16 etable 3. Sensitivity Analysis Including and Excluding Follow-up Time in Which the HIV-RNA was Suppressed at the Beginning of the Period but Became Elevated Rate of within couple transmission (per Upper 95% confidence Couple years of follow-up couple years of follow-up) limit Results presented in the All any sex manuscript HTW any sex HTM any sex MSM any sex Including the periods within which the most recent HIV- RNA load at the beginning of the period was <2 copies/ml, but was subsequently measured as >2 copies/ml during the period All any sex HTM: heterosexual men; HTW: heterosexual women, MSM: men having sex with men; 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

17 etable 4. Hierarchical Approach to Classifying Types of Sex Approach used in the Hierarchical approach manuscript Upper 95% confidence Couple years of Upper 95% confidence Couple years of follow-up limit follow-up limit All vaginal sex All insertive anal sex HTW vaginal sex with ejaculation HTW vaginal sex without ejaculation HTW receptive anal sex without ejaculation HTM vaginal sex MSM insertive anal sex MSM receptive anal sex without ejaculation HTM: heterosexual men; HTW: heterosexual women, MSM: men having sex with men; 26 American Medical Association. All rights reserved. Downloaded From: on 6/8/28

18 etable 5. Baseline Characteristics in Participants Who Contributed to the Eligible Couple-Years of Follow-up and Participants Who Did Not (Either Because They Dropped Out or Because the Couple-Years of Follow-up They Contributed Were Not Eligible) Heterosexuals MSM p-value* Contributed to eligible Contributed to eligible (HT) couple years of follow-up couple years of follow-up Yes (n=96) Age, median (IQR) [ missing] 42.7 ( ) No (n=252) 4.7 ( ) Yes (n=68) 4. ( ) No (n=34) 37.6 ( ) p-value* (MSM).334 <. Ethnicity, n (%) White 837 (78%) 73 (74%) 66 (9%) 245 (9%) Education level, n (%) Black 4 (3%) 37 (6%) 6 (%) 5 (2%) Asian 39 (4%) 5 (2%) 8 (3%) (<%) Other 6 (6%) 2 (9%) 42 (6%) 22 (8%) Missing High school or less 458 (43%) 4 (49%) 4 (2%) 56 (2%) Vocational education College or university 36 (3%) 49 (2%) 9 (28%) 7 (26%) 29 (27%) 7 (3%) 339 (5%) 42(53%) Missing HIV Acquisition Heterosexual 285 (53%) 7 (6) (%) (%) American Medical Association. All rights reserved. Downloaded From: on 6/8/28

19 route, n (%) [applies only to HIV+] Years of CL sex, median(iqr) Years on ART, median(iqr) [applies only to HIV+] Self-reported ART adherence >9%, n (%) [applies only to HIV+] Informed their partner if they missed doses of ART, n (%) [applies only to HIV+] MSM 23 (4%) (%) 324 (97%) 24 (93%) Shared needles or other injection equipment 97 (8%) 9 (6%) (%) (%) Other 3 (24%) 27 (23%) (3%) (7%) Missing (.7-9.4) 3.4 (.5-.2).5 (.5-4.) Missing ( ) 8. ( ) 4.8 (.9 -.4) Missing (.3-2.5) (.6-9.8) Yes 477 (94%) 98 (89%) 39 (97%) 25 (98%) Missing No 4 (7%) 9 (8%) 3 (4%) 6 (5%) Yes 256 (48%) 6 (52%) 33 (4%) 57 (43%) Did not miss doses 239 (45%) 47 (4%) 9 (57%) 7 (53%) Missing Self-reported Yes 45 (85%) 97 (87%) 39 (94%) (84%) American Medical Association. All rights reserved. Downloaded From: on 6/8/28

20 undetectable HIV- RNA load, n (%) [applies only to HIV+] CD4 count >35 mm 3, n (%) [applies only to HIV+; missing] Missing Yes 478 (87%) (8%) 39 (9%) 34 (89%) American Medical Association. All rights reserved. Downloaded From: on 6/8/28