Journal of Antimicrobial Chemotherapy Advance Access published May 15, 2015

Journal of Antimicrobial Chemotherapy Advance Access published May 15, 2015 J Antimicrob Chemother doi:10.1093/jac/dkv120 Trends in use of genotypic resistance testing and frequency of major drug resistance among antiretroviral-naive persons in the HIV Outpatient Study, 1999 2011 Kate Buchacz 1 *, Benjamin Young 2,3, Frank J. Palella Jr 4, Carl Armon 5 and John T. Brooks 1 on behalf of the HIV Outpatient Study (HOPS) investigators 1 Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA; 2 APEX Family Medicine, Denver, CO, USA; 3 International Association of Providers of AIDS Care, Washington, DC, USA; 4 Northwestern University, Chicago, IL, USA; 5 Cerner Corporation, Vienna, VA, USA *Corresponding author. Tel: +1-404-639-5167; Fax: +1-404-639-6127; E-mail: acu7@cdc.gov HOPS investigators and sites are listed in the Acknowledgements section. Received 11 December 2014; returned 6 February 2015; revised 9 April 2015; accepted 9 April 2015 Background: Monitoring antiretroviral drug resistance can inform treatment recommendations; however, there are few such data from US patients before they initiate ART. Methods: We analysed data from HIV Outpatient Study (HOPS) participants from nine US HIV clinics who were diagnosed with HIV infection during 1999 2011. Using the IAS-USA December 2010 guidelines, we assessed the frequency of major drug resistance mutations (mdrms) related to antiretroviral agents in viral isolates from patients who underwent commercial genotypic testing (GT) for resistance before initiating ART. We employed general linear regression models to assess factors associated with having undergone GT, and then factors associated with having mdrm. Results: Among 1531 eligible patients, 758 (49.5%) underwent GT before first ART, increasing from 15.5% in 1999 2002 to 75.9% in 2009 11 (P). GT was carried out a median of 1.2 months after the diagnosis of HIV. In adjusted regression analyses, patients with pre-art CD4+ T lymphocyte counts 200 cells/mm 3 or with HIV RNA levels.5.0 log 10 copies/ml and those with a first HOPS visit in 2006 or later were significantly (P, 0.05) more likely to have undergone GT. Of the 758 patients, 114 (15.0%) had mdrms; mutations relating to NRTIs, NNRTIs and PIs were present in 8.0%, 7.1% and 2.6%, respectively. There was no temporal change in the frequency of mdrm, and mdrms were associated with an HIV RNA level,4.0 log 10 copies/ml. Conclusions: During 1999 2011, GT use among antiretroviral-naive patients became more common, but a quarter of patients in recent years remained untested. The frequency of mdrms remained stable over time at about 15%. Keywords: HIV infection, genotype, mutation, epidemiology, primary, transmitted Introduction Within HIV transmission networks, individuals who have never been exposed to ART may become infected with HIV viral strains that already carry mutations associated with resistance to antiretroviral agents. 1 The screening of antiretroviral-naive patients for HIV drug resistance has been recommended by the US Department of Health and Human Services (DHHS) treatment guidelines since 2006. 2 Studies of population-based and convenience samples of HIV-infected individuals in the USA during the past decade suggest a frequency of primary or transmitted drug resistance of about 10% 24%, 3 14 with higher rates among those with acute or recent HIV infection rather than chronic HIV infection 6,8,15 and higher rates in persons from certain HIV risk and demographic subgroups, such as MSM and white individuals. 5,13,16 The synthesis and interpretation of data from disparate studies in the USA poses challenges because of the variability in study design and methodology, including the patient inclusion criteria and the mutations surveyed. 3,4,9,17 Stable or possibly increasing rates of transmitted drug resistance have been noted in prior population-based studies in the USA 6,8,18 and in some parts of Europe and Canada, 17,19 23 except for the notable decreases that were seen in a recent European analysis; 24 however, data on temporal patterns from large multisite clinical HIV Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US. 1of10

Buchacz et al. cohorts in the USA are few. Monitoring for drug resistance among antiretroviral-naive persons is important because patients who have viral isolates with resistance mutations and who receive antiretroviral drugs to which their virus is resistant experience higher rates of virological non-suppression. 11,25 Previously, we reported that acquired HIV drug resistance was common, but declined in frequency among HIV isolates from antiretroviral-experienced patients followed in the US HIV Outpatient Study (HOPS) during 1999 2008. 26 In the present report, we explore temporal trends and sociodemographic disparities in the frequency of genotypic testing (GT) for resistance and of major drug resistance mutations (mdrms) among HIV-infected antiretroviral-naive HOPS participants seen in routine HIV clinical practice. Methods HOPS HOPS is an ongoing longitudinal open cohort study that has prospectively followed a cohort of HIV-infected adults recruited from 1993 until the present day. The cross-sectional analyses described in this paper focus on baseline data from antiretroviral-naive HOPS patients attending nine university-based, public and private clinics in six cities (Chicago, IL, USA; Denver, CO, USA; Stony Brook, NY, USA; Philadelphia, PA, USA; Tampa, FL, USA; and Washington, DC, USA) after 1 January 1999. Patient data, including demographic and social characteristics, symptoms, diagnoses, prescribed medications (including dose and duration) and laboratory values, including antiretroviral resistance mutations reported on commercial genotypic tests, were abstracted from the medical records and entered by trained staff into a single database. These data were reviewed for quality and analysed centrally. Data quality assurance measures included supervisory reviews of randomly selected charts to ascertain the accuracy and completeness of abstracted data, and centralized checks of data files to resolve discrepancies. Annually, the institutional review boards of the CDC (Atlanta, GA, USA), Cerner Corporation (Vienna, VA, USA) and each local site reviewed and approved the HOPS protocol and informed consent forms. The study protocol conforms to the guidelines of the US DHHS for the protection of human participants in research. Study design and population We selected patients who had their first HOPS visit (baseline visit) between 1999 and 2011 and were antiretroviral-naive at baseline. We performed cross-sectional analyses of patients who underwent GT for the presence of antiretroviral resistance mutations prior to first ART use. In 2013, we validated the completeness of the GT results in the HOPS database through 2011. The present analyses are based on the HOPS dataset updated as of 30 June 2013. Outcome variables First, we assessed the cumulative frequency of undergoing GT among patients who were antiretroviral-naive and under HOPS observation, according to the calendar period of their first (baseline) visit for HOPS: 1999 2002, 2003 05, 2006 08 and 2009 11. Next, we assessed the frequency of mdrms in viruses isolated from patients who underwent commercial GT before initiating ART; mdrms were defined as per the IAS-USA December 2010 guidelines. 27 We summarized the percentage of patients whose standard GT reports indicated that they had HIV isolates with at least one mdrm, according to the calendar period during which their first documented GT was performed. We determined the most frequent mdrm by antiretroviral drug class. Finally, we repeated analyses by calendar period applying the definition for transmitted drug resistance mutations using the Stanford HIV database criteria published in 2009. 28 Independent variables: definitions for analysis For these analyses, we included as independent variables the following measured as of the baseline visit: age, sex, race/ethnicity, HIV risk group, insurance status, year of first HOPS visit, CD4+ T lymphocyte (CD4) counts and plasma HIV RNA viral loads. For HIV laboratory markers, baseline values were assigned using data collected from any time before and up to the first 6 months after the baseline date as long as the patients remained antiretroviral-naive. Statistical analyses We evaluated the trends over time in GT use and the frequency of mdrms using the Cochran Armitage test for trend for categorical variables. General linear models were used to estimate relative risks (RRs) and their associated 95% CIs for factors associated with having undergone GT and with harbouring one or more mdrms. We employed this method instead of standard logistic regression because ORs overestimate RRs when outcomes are more common. We used Fisher s exact test or Yates corrected x 2 test to compare the frequencies of antiretroviral drug class resistance across the time periods. All analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC, USA). All statistical tests were carried out at the 5% level of significance. Results Among the 10333 patients in the HOPS database as of 30 June 2013, 4283 had their first HOPS visit during 1999 2011, and among these 2444 were also diagnosedwithhivinfectionin 1999 or later. We excluded 913 patients who either were antiretroviral-experienced at HOPS entry or did not have a complete record of their antiretroviral history, leaving 1531 patients who were antiretroviral-naive at baseline and eligible for this analysis. Of the 1531 antiretroviral-naive patients studied, 59% were aged 35 years or older at their first HOPS visit, 76% were male, 42% were non-hispanic black, 41% were non-hispanic white and over half were privately insured. The median year of HIV diagnosis was 2004, the median CD4 cell count was 331 cells/mm 3 and the median plasma HIV RNA level closest to the first HOPS visit was 4.6 log 10 copies/ml (Table 1). About three-quarters of patients had their first HOPS visit within 3 months of their HIV diagnosis (median interval 32 days; IQR 12 99 days). The bulk of patients in our clinical cohort had an established/chronic HIV infection rather than a recent/acute HIV infection upon their initial HIV diagnosis, consistent with the characteristics of the cohort (percentages with AIDS and low CD4 cell counts) and the prevailing methods of diagnosing HIV infection in the period of the study. Further, because most patients are either already ARTexperienced or prescribed ART promptly 29 upon entering care at the HOPS clinics, patients eligible for this analysis comprised a minority of the entire HOPS cohort: among 3000 HOPS patients under observation each year, on average only 5.0% were ARTnaive at the mid-point of the year during 1999 2011. A total of 758 (49.5%) patients underwent GT during 1999 2011 and before starting ART. For these patients, the median time from HIV diagnosis to undergoing GT was 1.2 months (IQR 0.5 5.5 months). The median time between the first HOPS visit 2of10

Antiretroviral resistance in HIV-infected patients JAC Table 1. Baseline characteristics of antiretroviral-naive patients who did and did not undergo GT: HOPS, 1999 2011 Characteristic a n (%) of total Not tested, n (%) Tested, n (%) Percentage tested P Total patients 1531 (100.0) 773 (100.0) 758 (100.0) 49.5 Age, years 0.30,35 634 (41.4) 308 (39.8) 326 (43.0) 51.4 35 49 695 (45.4) 366 (47.3) 329 (43.4) 47.3 50 202 (13.2) 99 (12.8) 103 (13.6) 51.0 Sex male 1163 (76.0) 535 (69.2) 628 (82.8) 54.0 female 368 (24.0) 238 (30.8) 130 (17.2) 35.3 Race/ethnicity white, non-hispanic/latino 627 (41.0) 275 (35.6) 352 (46.4) 56.1 black, non-hispanic/latino 643 (42.0) 364 (47.1) 279 (36.8) 43.4 Hispanic/Latino 198 (12.9) 97 (12.5) 101 (13.3) 51.0 other/unknown 63 (4.1) 37 (4.8) 26 (3.4) 41.3 HIV transmission risk MSM 826 (54.0) 354 (45.8) 472 (62.3) 57.1 injection drug use 84 (5.5) 56 (7.2) 28 (3.7) 33.3 heterosexual 533 (34.8) 315 (40.8) 218 (28.8) 40.9 male 233 (43.7) 129 (41.0) 104 (47.7) 44.6 female 300 (56.3) 186 (59.0) 114 (52.3) 38.0 other/unknown 88 (5.7) 48 (6.2) 40 (5.3) 45.5 Payer 0.003 private insurance 835 (54.5) 392 (50.7) 443 (58.4) 53.1 public insurance 477 (31.2) 251 (32.5) 226 (29.8) 47.4 other/unknown 219 (14.3) 130 (16.8) 89 (11.7) 40.6 Median year of first HOPS visit 2004 2002 2006 Median year of HIV diagnosis 2004 2002 2005 AIDS diagnosis no 1006 (65.7) 472 (61.1) 534 (70.4) 53.1 yes 525 (34.3) 301 (38.9) 224 (29.6) 42.7 Median log 10 HIV RNA (copies/ml) (IQR) b 4.6 (3.9 5.1) 4.6 (3.6 5.1) 4.6 (4.0 5.2) 0.005 CD4 cell count (cells/mm 3 ),100 286 (18.7) 172 (22.3) 114 (15.0) 39.9 100 199 166 (10.8) 90 (11.6) 76 (10.0) 45.8 200 349 300 (19.6) 134 (17.3) 166 (21.9) 55.3 350 499 280 (18.3) 113 (14.6) 167 (22.0) 59.6 500 385 (25.1) 185 (23.9) 200 (26.4) 51.9 unknown 114 (7.4) 79 (10.2) 35 (4.6) 30.7 Year of first HOPS visit 1999 2002 516 (33.7) 426 (55.1) 90 (11.9) 17.4 2003 05 406 (26.5) 164 (21.2) 242 (31.9) 59.6 2006 08 328 (21.4) 112 (14.5) 216 (28.5) 65.9 2009 11 281 (18.4) 71 (9.2) 210 (27.7) 74.7 Year of first GT 1999 2002 78 (10.3) 2003 05 241 (31.8) 2006 08 215 (28.4) 2009 11 224 (29.6) a All characteristics reported as of first HOPS visit. b At or after first HOPS visit; pre-art HIV RNA levels and CD4 cell counts were available for 1369 and 1417 of the 1531 patients, respectively. 3of10

Buchacz et al. 100% GT, ART start GT, no ART start No GT, ART start No GT, no ART start 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1999 2011 (n = 1531) 1999 2002 (n = 516) Median (IQR) CD4 count at ART initiation: GT, ART start (solid black): 272 (93 459) No GT, ART start (solid grey): 251 (102 380) Wilcoxon rank-sum P value: 0.38 and the date of the GT was 0 days (IQR 0 7 days). One hundred and thirty-five (17.8%) patients underwent their first GT prior to their first HOPS visit (median 31 days before; IQR 11 77 days before), 389 (51.3%) on the same day as their first HOPS visit, 111 (14.6%) 1 30 days later, 75 (9.9%) 31 288 days later and 48 (6.3%).288 days later. The patterns of utilization of GT for the antiretroviral-naive patients evolved in HOPS over time (Figure 1). Whereas over 80% of patients had their first ART initiation documented during the available HOPS observation in all periods (the sum of the black and the grey groups in Figure 1), a substantial subset of patients did not undergo GT, even in the most recent calendar period. Overall, the percentage of patients who underwent GT while they were antiretroviral-naive increased over time, from 17.4% among patients with a baseline visit during 1999 2002 to 74.7% among patients with a baseline visit during 2009 11 (P value for trend across the four time periods ) (Figure 1; the black and hatched groups combined). When we stratified the results for the most recent time period (2009 11) by HOPS clinic site, the percentage of patients who underwent GT while antiretroviral-naive ranged from 55% to 100% across the study sites. The majority of patients who received GT underwent such testing within 1 month after their first HOPS visit (75% 85% of patients for all time periods). For the subset of patients who underwent GT followed by ART initiation while under observation in HOPS (depicted in black in Figure 1), the median time between these events shortened from 93 days in 1999 2002 to 39 days in 2009 11. Patients who underwent GT differed from patients who did not in terms of most of the demographic and clinical factors examined, with the exception of age at baseline (Table 1). In multivariable analyses over the entire observation period (1999 2011), patients whose first HOPS visit occurred in 2006 11 (after the release of guidelines 2 advocating the consideration of routine 2003 2005 (n = 406) 251 (78 386) 275 (143 384) 0.21 2006 2008 (n = 328) 288 (125 432) 322 (180 400) 0.40 2009 2011 (n = 281) 289 (147 485) 337 (205 503) 0.54 Figure 1. Percentage of antiretroviral-naive patients receiving GT and subsequently initiating ART, by year of first HOPS visit, 1999 2011. GT for all antiretroviral-naive patients prior to ART initiation) were 1.8 times as likely to undergo GT as patients whose first HOPS visit occurred before 2006. Women (RR 0.75) and patients with other or unknown insurance (RR 0.78, compared with private) were less likely to undergo GT (Table 2). Patients with CD4 cell counts between 200 and 349 cells/mm 3 and between 350 and 499 cells/mm 3 were 1.3 times as likely to undergo GT as those with CD4 cell counts,200 cells/mm 3, and patients with an HIV RNA level.5.0 log 10 copies/ml were more likely to undergo GT compared with those with HIV RNA values,4.0 log 10 copies/ ml. However, in analyses limited to 295 persons who had their first baseline visit in HOPS during 2009 11, of whom 224 (75.9%) underwent GT, neither female sex nor other demographic or clinical factors were significantly associated with the receipt of GT (see Table S1, available as Supplementary data at JAC Online). Among the 758 antiretroviral-naive patients who underwent GT while antiretroviral-naive, 114 (15.0%) had HIV isolates with mdrms: 61 (8.0%) with mutation(s) associated with NRTI resistance,54(7.1%)withnnrtiresistanceand20(2.6%)withpi resistance (Table 3). While the percentage of patients tested increased over 4-fold during the study period, there was no statistically significant change in the frequency of resistance overall or by antiretroviral drug class across the four time periods (P¼0.51, 0.96 and 0.14 for NRTIs, NNRTIs and PIs, respectively) (Figure 2). Overall, 94 (12.4%) patients had viral isolates with mutations associated with resistance to at least one antiretroviral drug class, 18 (2.4%) with resistance to two antiretroviral drug classes and 2 (0.3%) with resistance to all three main antiretroviral-drug classes; there were no statistically significant changes in the frequency of multi-antiretroviral drug class resistance over time (data not shown). For the 758 GT results, the five most frequent resistance mutations by antiretroviral drug class were: for NRTIs, A62V (n ¼22), M41L (n¼18), K70R (n¼13), K219Q (n¼10) and D67N (n¼9); 4of10

Antiretroviral resistance in HIV-infected patients JAC Table 2. Factors associated with undergoing GT among antiretroviral-naive patients: HOPS, 1999 2011 (n¼1531; 758 patients were tested) Univariate Full multivariable model Parsimonious multivariable model Characteristic a RR (95% CI) P RR (95% CI) P RR (95% CI) P Age, years,50 0.97 (0.79 1.19) 0.75 0.93 (0.75 1.16) 0.53 50 referent referent Sex male referent referent referent female 0.65 (0.54 0.79) 0.79 (0.62 1.01) 0.06 0.75 (0.61 0.91) 0.005 Race/ethnicity white, non-hispanic/latino referent referent black, non-hispanic/latino 0.77 (0.66 0.90) 0.001 0.94 (0.77 1.14) 0.52 Hispanic/Latino 0.91 (0.73 1.13) 0.40 0.98 (0.78 1.25) 0.89 other/unknown 0.74 (0.49 1.10) 0.13 0.82 (0.55 1.23) 0.33 HIV transmission risk MSM referent referent injection drug use 0.58 (0.40 0.85) 0.006 0.82 (0.55 1.23) 0.33 heterosexual 0.72 (0.61 0.84) 0.93 (0.74 1.17) 0.52 other/unknown 0.80 (0.58 1.10) 0.16 0.97 (0.69 1.37) 0.87 Payer private insurance referent referent referent public insurance 0.89 (0.76 1.05) 0.17 1.07 (0.88 1.31) 0.49 1.03 (0.86 1.22) 0.77 other/unknown 0.77 (0.61 0.96) 0.022 0.80 (0.63 1.01) 0.06 0.78 (0.62 0.98) 0.032 AIDS diagnosis no referent referent yes 0.80 (0.69 0.94) 0.006 0.96 (0.76 1.22) 0.74 CD4 cell count (cells/mm 3 ),200 referent referent referent 200 349 1.32 (1.07 1.62) 0.010 1.23 (0.94 1.60) 0.13 1.27 (1.02 1.57) 0.031 350 499 1.42 (1.15 1.75) 0.001 1.21 (0.90 1.61) 0.20 1.27 (1.02 1.58) 0.030 500 1.24 (1.01 1.51) 0.037 1.13 (0.84 1.52) 0.42 1.20 (0.97 1.48) 0.10 unknown 0.73 (0.51 1.05) 0.09 0.86 (0.57 1.28) 0.45 0.87 (0.60 1.25) 0.44 HIV RNA (log 10 copies/ml),4.0 referent referent referent 4.0 5.0 1.19 (0.99 1.42) 0.07 1.19 (0.98 1.44) 0.08 1.18 (0.98 1.42) 0.09.5.0 1.18 (0.98 1.43) 0.09 1.24 (1.01 1.54) 0.043 1.25 (1.02 1.54) 0.033 unknown or missing 0.27 (0.17 0.43) 0.36 (0.23 0.58) 0.36 (0.23 0.57) Time span between HIV diagnosis and first HOPS visit (months),3 referent referent 3 to 6 1.08 (0.83 1.41) 0.57 1.16 (0.89 1.51) 0.28.6 1.16 (0.97 1.39) 0.11 1.06 (0.88 1.27) 0.56 Year of first HOPS visit 1999 2005 referent referent referent 2006 11 1.94 (1.68 2.24) 1.81 (1.56 2.09) 1.83 (1.58 2.11) a All characteristics reported as of first HOPS visit. for NNRTIs, K103N (n¼55), P225H (n¼8), V108I (n¼7), K101E (n¼7) and Y181C (n¼3); and for PIs, M46I (n¼8), I84V (n¼7), L90M (n¼5), D30N (n¼3), M48L (n¼2). Three patients (0.4%) had viral isolates that possessed the K65R reverse transcriptase mutation, and six patients (0.8%) had isolates that possessed the M184V reverse transcriptase mutation. The three isolates 5of10

Buchacz et al. Table 3. Frequency of major IAS-USA genotypic resistance mutations among antiretroviral-naive patients: HOPS, 1999 2011 n (column %) Any major IAS mutation, n (row %) Mutation indicating NRTI resistance, n (row %) Mutation indicating NNRTI resistance, n (row %) Mutation indicating PI resistance, n (row %) Total with GT 758 (100.0) 114 (15.0) 61 (8.0) 54 (7.1) 20 (2.6) Age (at first HOPS visit), years,35 326 (43.0) 55 (16.9) 22 (6.7) 27 (8.3) 10 (3.1) 35 49 329 (43.4) 49 (14.9) 29 (8.8) 22 (6.7) 9 (2.7) 50 103 (13.6) 10 (9.7) 10 (9.7) 5 (4.9) 1 (1.0) Sex male 628 (82.8) 91 (14.5) 47 (7.5) 41 (6.5) 18 (2.9) female 130 (17.2) 23 (17.7) 14 (10.8) 13 (10.0) 2 (1.5) Race/ethnicity white, non-hispanic/latino 352 (46.4) 49 (13.9) 22 (6.3) 22 (6.3) 12 (3.4) black, non-hispanic/latino 279 (36.8) 45 (16.1) 24 (8.6) 25 (9.0) 6 (2.2) Hispanic/Latino 101 (13.3) 18 (17.8) 13 (12.9) 7 (6.9) 2 (2.0) other/unknown 26 (3.4) 2 (7.7) 2 (7.7) 0 (0.0) 0 (0.0) HIV transmission risk MSM 472 (62.3) 71 (15.0) 37 (7.8) 29 (6.1) 16 (3.4) injection drug use 28 (3.7) 4 (14.3) 3 (10.7) 2 (7.1) 0 (0.0) heterosexual 218 (28.8) 36 (16.5) 19 (8.7) 22 (10.1) 3 (1.4) male 104 (47.7) 15 (14.4) 7 (6.7) 11 (10.6) 1 (1.0) female 114 (52.3) 21 (18.4) 12 (10.5) 11 (9.6) 2 (1.8) other/unknown 40 (5.3) 3 (7.5) 2 (5.0) 1 (2.5) 1 (2.5) Payer private insurance 443 (58.4) 63 (14.2) 30 (6.8) 27 (6.1) 15 (3.4) public insurance 226 (29.8) 37 (16.4) 24 (10.6) 19 (8.4) 2 (0.9) other/unknown 89 (11.7) 14 (15.7) 7 (7.9) 8 (9.0) 3 (3.4) CD4 cell count (cells/mm 3 ) a,100 118 (15.6) 16 (13.6) 8 (6.8) 10 (8.5) 2 (1.7) 100 199 78 (10.3) 10 (12.8) 5 (6.4) 6 (7.7) 2 (2.6) 200 349 172 (22.7) 19 (11.0) 12 (7.0) 9 (5.2) 4 (2.3) 350 499 167 (22.0) 28 (16.8) 14 (8.4) 11 (6.6) 4 (2.4) 500 193 (25.5) 33 (17.1) 16 (8.3) 15 (7.8) 8 (4.1) unknown 30 (4.0) 8 (26.7) 6 (20.0) 3 (10.0) 0 (0.0) HIV RNA (log 10 copies/ml) a,4.0 164 (21.6) 38 (23.2) 26 (15.9) 15 (9.1) 5 (3.0) 4.0 5.0 324 (42.7) 43 (13.3) 21 (6.5) 19 (5.9) 12 (3.7).5.0 249 (32.8) 31 (12.4) 12 (4.8) 20 (8.0) 3 (1.2) unknown or missing 21 (2.8) 2 (9.5) 2 (9.5) 0 (0.0) 0 (0.0) Year of first GT 1999 2002 78 (10.3) 11 (14.1) 7 (9.0) 1 (1.3) 5 (6.4) 2003 05 241 (31.8) 34 (14.1) 15 (6.2) 24 (10.0) 4 (1.7) 2006 08 215 (28.4) 36 (16.7) 19 (8.8) 15 (7.0) 8 (3.7) 2009 11 224 (29.6) 33 (14.7) 20 (8.9) 14 (6.3) 3 (1.3) Time span between first HOPS visit and GT (months),1 634 (83.6) 90 (14.2) 45 (7.1) 43 (6.8) 16 (2.5) 1to,6 62 (8.2) 11 (17.7) 8 (12.9) 7 (11.3) 2 (3.2) 6 62 (8.2) 13 (21.0) 8 (12.9) 4 (6.5) 2 (3.2) Only one patient had resistance to fusion inhibitors: in 2006. a Closest to the date of GT. 6of10

Antiretroviral resistance in HIV-infected patients JAC 20 % Any major IAS mutation % NRTI mutation % NNRTI mutation % PI mutation Percentage of patients with HIV isolates having a mutation 18 16 14 12 10 8 6 4 2 0 14.1% 9.0% 1.3% 6.4% 14.1% 6.2% 10.0% 1.7% with the K65R mutation were tested for resistance in 2003, 2004 and 2011; the six isolates with M184V were tested for resistance in 2003 (n¼3), 2005 (n¼2) and 2008 (n¼1). In univariate analyses (as well as in multivariable analyses; data not shown), the only factor of those assessed that was associated with having any mdrm was an HIV RNA level,4.0 log 10 copies/ml closest to the date of GT (RR 1.86, 95% CI 1.16 2.99 compared with HIV RNA.5.0 log 10 copies/ml); the frequency of mdrms was not associated with any demographic characteristics (see Table S2). In further analyses restricted to the 439 patients who underwent GTs during 2006 11 and who had been diagnosed with HIV infection up to 1 year earlier, the findings were qualitatively similar. Sixty-nine (15.7%) had any mdrms; in 39 patients (8.9%) mdrms were associated with NRTI resistance, in 29 (6.6%) with NNRTI resistance and in 11 (2.5%) with PI resistance. Furthermore, when we conducted alternate analyses using the surveillance definition for transmitted drug resistance developed using the Stanford HIV database and published in 2009, 28 the estimates for transmitted drug resistance were 18.0% for 1999 2002, 13.7% for 2003 05, 14.4% for 2006 08 and 12.5% for 2009 11, qualitatively similar to the frequencies of any major IAS-USA mutation that were found for the same periods: 14.1%, 14.1%, 16.7% and 14.7%, respectively (Figure 2). Using the Stanford definition for GT results during 2009 11, 5.8% of the isolates had mutations associated with NRTI resistance, 5.8% with NNRTI resistance and 1.8% with PI resistance. 16.7% 8.8% Discussion 7.0% 3.7% 14.7% 1999 2002 (n = 78) 2003 2005 (n = 241) 2006 2008 (n = 215) 2009 2011 (n = 224) Calendar year of GT Figure 2. Percentage of antiretroviral-naive patients with major IAS-USA mutations, by year of GT, HOPS, 1999 2011. 8.9% 6.3% 1.3% Although baseline GT for antiretroviral-naive HIV-infected patients became more common over time in our demographically diverse US-based clinical cohort, it was not obtained for about 25% of patients diagnosed with HIV during the most recent years. Recommendations from the DHSS to consider conducting baseline GT for antiretroviral-naive patients, even if initial ART was being deferred, have been in place since May 2006. 2 In December 2007, the panel recommended performing GT for all treatmentnaive patients entering into clinical care, regardless of whether antiretroviral therapy was to be initiated. 30 Over the entire study period (1999 2011), patients with pre-art CD4 cell counts 200 cells/mm 3 or with HIV RNA levels.5.0 log 10 copies/ml and those diagnosed with HIV in later calendar years were significantly more likely to undergo GT; women and patients with an other or unknown insurance payer were less likely to undergo GT before ART initiation over the entire study period; however, none of these associations persisted from 2009 onwards, after GT utilization became more widespread. Overall, 15.0% of patients had at least one major IAS-USA mutation, and there was no evidence of a statistically significant change in the frequency of mdrms over time. We studied the frequency of resistance testing and patterns of major IAS-USA resistance mutations among antiretroviral-naive patients in order to focus on clinically relevant resistance mutations that could influence treatment outcomes among these 7of10

Buchacz et al. individuals. Although close to one-third of antiretroviral-naive patients had been diagnosed with AIDS prior to their baseline HOPS visit, about three-quarters had been diagnosed with HIV within the prior 3 months. In addition, persons who underwent GT had such testing performed a median of 1.2 months after their HIV diagnosis; thus, our study population largely met the proposed US surveillance criteria for ascertaining transmitted drug resistance (GT within 3 months of HIV diagnosis). 6,8,12 Although our definition of mdrm included some naturally occurring polymorphic mutations (e.g. V118I) and excluded some minor resistance mutations captured by one proposed transmitted drug resistance surveillance definition, 28 the frequency of resistance estimated by the two methods did not differ markedly within our population. Recent population-based studies have found incomplete adherence to DHHS recommendations for conducting baseline GT before ART initiation, although such routine testing has been recommended in the USA since 2006. 2 A recent study from the New York State HIV surveillance programme found that only 60% of newly diagnosed patients with HIV had had a laboratoryreported GT performed within 3 months of their HIV diagnosis in 2013, and that overall 18.7% had surveillance resistance mutations, 7 with a somewhat higher prevalence of resistance among a minority of patients with recent HIV infections (24%) than those with established HIV infections (18%) as determined by the BED assay. 18 Although we did not collect data on reasons for the lack of GT among antiretroviral-naive patients, we found that GT was obtained most frequently for patients with baseline CD4 cell counts between 200 and 499 cells/mm 3,butlessfrequently for patients with CD4 counts,200 cells/mm 3,agroup for whom immediate ART has been uniformly indicated over time, and less frequently for those with CD4 counts 500 cells/ mm 3, individuals for whom ART may have been deferred according to prevailing DHHS guidelines. 31 Patients with baseline HIV RNA values.5.0 log 10 copies/ml were more likely to have undergone GT than those with HIV RNA,4.0 log 10 copies/ml, possibly indicating that clinicians were more inclined to order GT among the most viraemic patients, a group for whom the selection of fully suppressive ART regimens can be more challenging. However, we found no evidence that clinicians were more or less likely to obtain GT for antiretroviral-naive patients who hadaidsorhadbeendiagnosedwithhivinfectionforlonger periods of time. We also made what we believe to be a novel observation, namely that having lower pre-art HIV RNA values (,4.0 log 10 copies/ml) was associated with having mdrms. The finding that patients with lower pre-treatment HIV RNA levels were more likely to harbour resistance mutations may support the assertion that antiretroviral-resistant HIV strains often have lower replicative capacity, 32 but we did not conduct virological substudies to explore this further. Reassuringly for future HIV prevention efforts, despite the fact that tenofovir is among the most widely prescribed antiretroviral drugs in the USA (prescribed for 72% of HOPS patients receiving ART in 2011), we observed a very low frequency of transmission of the K65R reverse transcriptase mutation, which confers resistance to tenofovir. Our estimate of 0.4% was similar to the estimate of 0.5% from an earlier study. 8 Tenofovir in combination with emtricitabine is currently approved for pre-exposure prophylaxis against HIV in the USA. 33 Since other antivirals may eventually be used for both pre-exposure prophylaxis and empirical post-exposure prophylaxis, monitoring the frequency of transmitted tenofovir resistance as well as mdrms may be warranted, if feasible. Our findings are subject to several caveats and limitations. During 1999 2011, fewer than half of all antiretroviral-naive HOPS patients underwent GT and therefore our resistance estimates may not reflect the true underlying frequency of mdrms among all the antiretroviral-naive patients in HOPS. If patients referred by their physicians for GT also had a higher risk of drug resistance (e.g. from residing in communities with more circulating drug-resistant viral strains), this would lead to overestimates of the prevalence of mdrms in the antiretroviral-naive HOPS population as a whole. However, given that we found no patient demographic factors to be associated with mdrms, we believe this bias to be unlikely. Although most of the individuals undergoing GT in this analysis had been diagnosed with HIV in the prior 3 6 months, their time from acquiring HIV infection to undergoing GT was unknown, as is usually the case in routine clinical HIV practice. 18 Thus, for at least some patients with mdrms, mutations may become archived in the absence of antiretroviral drug pressure and are thus no longer apparent on the initial GT for antiretroviral-naive persons. 15,18,34 However, data from a recent Canadian study revealed that results from pre-art genotypes obtained in clinical practice may be valid proxies for those obtained shortly after HIV infection. 35 Temporal trends in the frequency of mdrms may be further affected by two factors: (i) the use of an increasingly diverse formulary of antiretrovirals available for treatment in the USA and the duration for which they have been on the market, which may modify the risk of acquiring an HIV strain that possesses an mdrm; and (ii) the recognition of and availability of commercial GT screening for an increasing number of clinically relevant drug resistance mutations over time, which may enable the detection of mdrms in later years that may not have been recognized as clinically relevant in earlier years. In addition, due to the potentially variable rates of archiving different transmitted minority HIV variants that possess resistance to various antiretroviral drugs and classes, frequency estimates of mdrms related to different antiretroviral classes may not be directly comparable. Finally, although cases of transmitted integrase strand transfer inhibitor (INSTI) resistance have been reported, 36 commercially available genotypic resistance assays during the period of our study did not routinely assess for resistance mutations in the integrase gene, limiting our ability to detect INSTI mdrms. In summary, we observed that 15% of antiretroviral-naive patients entering HOPS care carried viral strains that exhibited clinically relevant IAS-USA mdrms. Our observations corroborateestimatesreportedfromthefewexistinglargemultisite US studies 3,6,12 and extend these findings by documenting stable temporal trends over more than a decade (1999 2011) of observation. Although transmitted antiretroviral resistance may be more completely captured in research cohorts of seroconverters and acutely infected individuals, 10,37 there are few such cohorts, especially ones that are representative of the diverse population of HIV-infected adults in the USA. Our results reflect observations from patients who, in the course of standard clinical practice, have typically undergone GT shortly after their HIV diagnosis while they are antiretroviral-naive and suggest that major drug resistance persists and warrants continued monitoring as per guidelines 2 to optimize health outcomes for patients starting ART. 8of10

Antiretroviral resistance in HIV-infected patients JAC Acknowledgements HOPS investigators and sites Kate Buchacz, John T. Brooks, Marcus D. Durham, Division of HIV/AIDS Prevention, National Center for HIV, STD, Viral Hepatitis and TB Prevention (NCHHSTP), Centers for Disease Control and Prevention (CDC), Atlanta, GA; Harlen Hays, Kathleen C. Wood, Darlene Hankerson, Rachel Hart, Thilakavathy Subramanian, Carl Armon, Bonnie Dean, Dana Franklin, Cerner Corporation, Vienna, VA; Frank J. Palella, Joan S. Chmiel, Saira Jahangir, Conor Daniel Flaherty, Jerian Denise Dixon-Evans, Feinberg School of Medicine, Northwestern University, Chicago, IL; Kenneth A. Lichtenstein, Cheryl Stewart, National Jewish Health, Denver, CO; John Hammer, Kenneth S. Greenberg, Barbara Widick, Rosa Franklin, Rose Medical Center, Denver, CO; Bienvenido G. Yangco, Kalliope Chagaris, Infectious Disease Research Institute, Tampa, FL; Doug Ward, Troy Thomas Dupont Circle Physicians Group, Washington, DC; Jack Fuhrer, Linda Ording-Bauer, Rita Kelly, Jane Esteves, State University of New York (SUNY), Stony Brook, NY; Ellen M. Tedaldi, Ramona A. Christian, Faye Ruley, Dania Beadle, Princess Graham, Temple University School of Medicine, Philadelphia, PA; Richard M. Novak, Andrea Wendrow, Renata Smith, University of Illinois at Chicago, Chicago, IL; Benjamin Young, Barbara Widick, Mia Scott, APEX Family Medicine, Denver, CO. Funding The work was supported by the US Centers for Disease Control and Prevention (contract numbers 200-2001-00133, 200-2006-18797 and 200-2011-41872). Transparency declarations B. Y. has received research grants from Bristol-Myers Squibb, Cerner Corporation, Gilead Sciences, Merck, Roche and GlaxoSmithKline, and/or is a member of advisory boards for Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Merck and ViiV Healthcare. F. J. P. has received consulting or speaking fees from Bristol-Myers Squibb, Gilead Sciences, Janssen Pharmaceuticals and Merck & Co., and has received research funding from Gilead Sciences. All other authors: none to declare. Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Supplementary data Tables S1 and S2 are available as Supplementary data at JAC Online (http:// jac.oxfordjournals.org/). References 1 Booth CL, Geretti AM. Prevalence and determinants of transmitted antiretroviral drug resistance in HIV-1 infection. 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