1. Background Surveillance data indicate a decline in the prevalence of antiretroviral drug resistance among treated patients. Improved treatment strategies and prompt detection and management of virological failure may contribute to reduce the emergence of resistance. A VL >1000 cp/ml is the recommended optimal threshold for resistance testing. Genotypic resistance assays however can be adapted to perform well at low VL, although some debate ensues as to whether results are fully representative of the dominant quasispecies. Resistance testing at low VL is often required in clinical practice to allow a timely and optimised therapeutic change, and many centres perform resistance tests at VL <1000 cp/ml as part of routine care 1,2. Yet, the yield and clinical utility of testing at low VL remain uncertain. Aims: To characterise the population undergoing genotypic resistance testing at VL <1000 cp/ml, describe their genotypic resistance profiles, and identify factors associated with the detection of resistance mutations among treated patients. Methods Resistance results from the UK HIV Drug Resistance Database were linked to clinical data from UK CHIC. The analysis considered all resistance tests performed after the start of HAART, with a matching VL measured within the previous 4 wks. For patients with multiple tests, all results were included. Major resistance mutations were scored by the IAS-USA list (Sept-Oct 2007). Virological failure of a drug was defined by VL >400 cp/ml after >4 months of continuous use of the drug. Sequencing success rate in centres providing specialised testing at VL <1000 cp/ml was 85%. Generalized linear models with log link and Poisson error (using GEE) were used to assess multivariable (adjusted) relative risks (SAS 9.1). Prevalence and Patterns of Antiretroviral Drug Resistance at Low Plasma HIV RNA Load Levels Geretti AM 1, Phillips A 1, Kaye S 2, Booth C 1, Garcia A 1, Mackie N 2, on behalf of the UK HIV Drug Resistance Database and CHIC Study. 1 Royal Free & University College Medical School, 2 Imperial College Healthcare NHS Trust, London, UK Cohort (%)High-VL (%)Low-VL (%)P* GenderMale6199 (78.7)5444 (79.2)755 (75.4)0.0244 Female1679 (21.3)1433 (20.8)246 (24.6) Age<30 671 (8.5) 604 (8.8) 67 (6.7)0.0002 30-455425 (68.9)4764 (69.3)661 (66.0) >451783 (22.6)1510 (22.0)273 (27.3) HIV exposure MSM4771 (60.6)4200 (61.1)571 (57.0)0.0029 Hetero2275 (28.9)1956 (28.4)319 (31.9) IDU 355 (4.5) 321 (4.7) 34 (3.4) Other/Unk 478 (6.1) 401 (5.8) 77 (7.7) Current regimen NNRTI + NRTIs1856 (23.6)1572 (22.9)284 (28.4)<0.0001 PI/r + NRTIs1798 (22.8)1469 (21.4)329 (32.9) PI + NRTIs 950 (12.1) 850 (12.4)100 (10.0) NRTIs only1124 (14.3) 967 (14.1)157 (15.7) Other 539 (6.8) 481 (7.0) 58 (5.8) Off treatment1612 (20.5)1539 (22.4) 73 (7.3) Number of drugs previously failed 01014 (12.9) 831 (12.1)183 (18.3)<0.0001 1-32517 (31.9)2244 (32.6)273 (27.3) 4-62471 (31.4)2166 (31.5)305 (30.5) 7-91377 (17.5)1194 (17.4)183 (18.3) > 10 500 (6.3) 443 (6.4) 57 (5.7) Time since start of HAART 0-3 years2792 (35.4)2462 (35.8)330 (33.0)<0.0001 3-6 years2407 (30.5)2123 (30.9)284 (28.4) 6-9 years1621 (20.6)1416 (20.6)205 (20.5) > 9 years1059 (13.4) 877 (12.7)182 (18.2) VL ever <50 cp/ml Yes4499 (57.1)3733 (54.3)766 (76.5)<0.0001 No3380 (42.9)3145 (45.7)235 (23.5) Table 1. Cohort undergoing resistance testing Prevalence (%) of resistance Proportion (%) of total tests Whole cohortNN (%) with RAMsRR (95% CI) <300449 270 (60)0.94 (0.87-1.01) 300-999552 399 (72)0.99 (0.94-1.04) 1000-29991120 851 (76)1 3000-999913121015 (77)1.01 (0.97-1.05) 10000-299991326 891 (67)0.91 (0.87-0.95) 30000-999991438 863 (60)0.84 (0.80-0.88) 1000001682 816 (49)0.70 (0.66-0.74) NRTIsNN (%) with NAMsRR (95% CI) <300410219 (53)0.89 (0.81-0.98) 300-999508345 (68)1.00 (0.94-1.07) 1000-2999992713 (72)1 3000-99991135851 (75)1.01 (0.96-1.06) 10000-299991006703 (70)0.93 (0.88-0.98) 30000-999991019599 (59)0.79 (0.74-0.84) 1000001082488 (45)0.62 (0.57-0.67) NRTIs+NNRTINN (%) with nNAMsRR (95% CI) <300 124 59 (48)0.86 (0.72-1.02) 300-999 160123 (77)1.06 (0.97-1.17) 1000-2999 313241 (77)1 3000-9999 362277 (77)1.02 (0.94-1.10) 10000-29999 294216 (73)0.97 (0.89-1.06) 30000-99999 283170 (60)0.84 (0.75-0.94) 100000 320154 (48)0.68 (0.60-0.77) NRTIs+PI*NN (%) with PRAMsRR (95% CI) <300 193 55 (29)0.85 (0.70-1.04) 300-999 236 89 (38)1.00 (0.86-1.16) 1000-2999 424182 (43)1 3000-9999 479222 (46)0.89 (0.79-1.01) 10,000-29999 452198 (44)0.88 (0.78-1.00) 30,000-99999 459190 (41)0.85 (0.75-0.96) 100,000 505157 (31)0.66 (0.55-0.75) Table 3. Multivariate relative risk (RR) of detecting resistance according to the VL in the whole cohort, and while on NRTIs, NRTIs+NNRTI, or NRTIs+PI* *chi-square test Conclusions Testing at VL <1000 cp/ml is as likely to detect resistance as testing at higher levels. The yield of testing declines progressively at VL 10000 cp/ml for NAMs and 30000 for nNAMS and PRAMs. Common (>5%) mutations at VL<1000 cp/ml include the NAMs K65R, M184V, and TAMs, the nNAMs K103N, Y181C and G190A, and the PRAMs M46I, V82A, and L90M. Testing at low-VL does not explain the observed decline in the prevalence of antiretroviral drug resistance among treated patients. The findings rather indicate a protective role of newer treatment regimens and use of PI/r. *Includes both PI and PI/r RR (95% CI)P Calendar yr of testPer yr more recent 0.96(0.95-0.96)<0.0001 Current regimenNNRTI+NRTIs1.00-<0.0001 NRTIs+PI0.91(0.87-0.95) NRTIs+PI/r 0.73(0.70-0.77) NRTIs0.95(0.91-0.99) Other0.90(0.86-0.95) Off0.54(0.50-0.58) Number of drugs previously failed 00.50(0.44-0.56) 1-30.70(0.65-0.76) 4-60.79(0.74-0.85) 7-90.89(0.83-0.95) >101.00-<0.0001 Time since start of HAARTPer yr greater1.001.00-1.010.02 VL ever <50 cp/mlYes0.90(0.87-0.94) No1.00-<0.0001 Table 2. Independent predictors of resistance Results The dataset comprised 7879 resistance test results, from 3795 patients with 1 test, 1817 with 2 tests and 2267 with 3 tests. Most patients were receiving HAART with 2 NRTIs plus either NNRTI or PI/r. There were 1621 tests done off treatment, after patients had discontinued HAART for median 542 days (IQR 236-1099) (Table 1). 1001/7879 (12.7%) tests were performed at VL 1000 cp/ml (high-VL). The total number of tests remained fairly stable through the years, while the proportion at low- VL increased significantly, from 3.4% before 1999 to 21.8% in 2006 (chi- square test, p<0.0001) (Fig 1). In the whole cohort, the overall prevalence of major resistance- associated mutations (RAMs) declined over time (Fig 2). Factors independently associated with the detection of resistance in the multivariate analysis are indicated in Table 2. There was no significant effect of gender, age, risk group, and only a marginal effect of time since start of HAART. In the multivariate analysis, the relative risk (RR) of detecting any resistance was highest at VL 1000 and <10000 cp/ml and overall similar at VL<1000 cp/ml (Table 3). The frequency of RT and PR mutations in the whole cohort, stratified by VL, is shown in Fig 3. Among low-VL patients on PIs at the time of testing, mutations detected at a frequency 5% included D30N (5%), M46I (10%), V82A (11%), I84V (5%) and L90M (14%) (not shown). Fig 3. Frequency of RT and PR mutations % NRTIs+PI/rNN (%) with PRAMsRR (95% CI) <30015939 (24)0.84 (0.68-1.04) 300-99917051 (30)0.98 (0.78-1.22) 1000-2999272100 (37)1 3000-9999292116 (40)0.94 (0.80-1.10) 10000-29999276105 (38)0.93 (0.80-1.07) 30000-99999283106 (37)0.83 (0.70-0.99) 10000034695 (27)0.70 (0.59-0.84) References: 1. Mackie et al, J Virol Methods 2004; 2. Cane et al, HIV Med 2008 RAMS: Resistance-associated mutations; NAMs: NRTI RAMs; nNAMs: NNRTI RAMs; PRAMs: PI RAMs.