Transcript Poster

Detection of clinically relevant antiretroviral drug resistance mutations among treated patients
undergoing testing at low levels of viremia
AM Geretti1, AN Phillips1, S Kaye2, C Booth1 and NE Mackie2 on behalf of the UK HIV Drug Resistance Database and the UK Collaborative HIV Cohort (CHIC) Study
Free Hampstead NHS Trust & UCL Medical School and 2Imperial College Healthcare NHS Trust, London, UK
755 (75.4)
0.03
246 (24.6)
<30
30-45
>45
671 (8.5)
604 (69.3)
5415 (68.9) 4754 (69.3)
1775 (22.6) 1502 (21.9)
67 (6.7)
661 (66.0) 0.0002
273 (27.3)
MSM
Hetero
IDU
Other/UK
4755 (60.5) 4184 (61.0)
2273 (28.9) 1954 (28.5)
355 (4.5)
321 (4.7)
478 (6.1)
401 (5.8)
571 (57.0)
319 (31.9)
0.003
34 (3.4)
77 (7.7)
ART regimen
at time of testing
NNRTI+NRTIs
PI/r+NRTIs
PI+NRTIs
NRTIs only
Other
None
1864 (23.7) 1577 (23.0)
1816 (23.1) 1482 (21.6)
943 (12.0) 847 (12.4)
1131 (14.4) 974 (14.2)
496 (6.3)
442 (6.4)
1611 (20.5) 1538 (22.4)
287 (28.7)
334 (33.3)
157 (15.7)
<0.0001
96 (9.6)
54 (5.4)
73 (7.3)
Number of drugs
previously failed
0
1-3
4-6
7-9
>10
1008 (12.8) 826 (12.0)
2514 (32.0) 2240 (32.7)
2464 (31.3) 2159 (31.5)
1372 (17.5) 1189 (17.3)
503 (6.4)
446 (6.5)
182 (18.2)
274 (27.4)
305 (30.5) <0.0001
183 (18.3)
57 (5.7)
0-3
3-6
6-9
≥9
Yes
No
2783 (35.4)
2402 (30.6)
1617 (20.6)
1059 (13.4)
4484 (57.0)
3377 (43.0)
330 (33.0)
284 (28.4)
<0.0001
205 (20.5)
182 (18.2)
766 (76.5)
<0.0001
235 (23.5)
Gender
Age (yrs)
Risk group
 Data analysis. The analysis considered the relationship between VL at the time of the
resistance test and the probability of detection of RAMs. Virological failure of a drug was
defined by a VL >400 copies/ml after >4 months of continuous use of the drug. Generalized
linear models with log link and Poisson error (using generalised estimating equations) were
used to assess multivariable (adjusted) relative risks (RR) for the association between various
covariates and a risk of a RAM being present (SAS 9.1). The same approach was used for
analyses of detection of resistance to any drug class and class-specific. All analyses were
repeated excluding and including those off ART at the time of the resistance test.
Aim
To characterize the population undergoing genotypic resistance testing at VL <1000 copies/ml,
describe their resistance profiles, identify factors associated with the detection of RAMs
according to the VL level, and specifically compare the numbers and patterns of RAMs
detected at VL above and below 1000 copies/ml.
Figure 2. Median number of RAMs detected
according to VL, in tests with ≥1 RAM*
7
6
5
4
3
2
1
0
4
3
3
99
 Study population. Genotypic resistance test results were obtained from the UK HIV Drug
Resistance Database, which collates results of tests performed at most centres in the UK11.
Results are provided in the form of plasma pol sequences, and amino acid sequences and
mutations (relative to HXB2) are derived via the Stanford Algorithm web service (Sierra).
Resistance test results were linked to clinical data from the UK CHIC study, an observational
cohort collating data from 10 clinics12. Patients eligible for inclusion in this analysis had a
resistance test performed after the start of antiretroviral therapy (ART) and underwent a VL
test within 2 weeks before to 4 weeks after the date of the resistance test. If patients had >1
resistance test performed after the start of ART all tests were included. RAMs were scored
according to the IAS-USA list (Oct. 2007). The majority (856/1001, 86%) of tests performed at
VL <1000 copies/ml came from 3 centres where either the VircoType (Virco, Belgium) or inhouse methodologies including a nested PCR step were used. The sequencing success rates
in these 3 centres were 70% for the VircoType13 and 85-92% for the in-house methodologies
(Imperial College Healthcare NHS Trust and Royal Free Hampstead NHS Trust).
 Patient characteristics are shown in Table 1. At the time of testing, 1611
(20.5%) patients were not on ART having discontinued therapy a median of 542
(IQR 236-1099) days previously. Excluding these patients from the analyses
did not significantly alter the findings.
 The total number of tests per year was broadly constant: <1999 787, 1999
813, 2000 874, 2001 897, 2002 1032, 2003 963, 2004 778, 2005 831, and
2006 813. Overall 1001 (12.7%) tests were performed at low VL, and their
number as a proportion of all tests increased over time (Fig 1).
 Factors associated with undergoing testing at low VL comprised centre of
care, more recent calendar year of testing, a previous undetectable VL, no
previous virological failure, and receiving PI/r+NRTIs (p<0.0001 for all).
25
20
15
10
5
0
<1999 1999 2000 2001 2002 2003 2004 2005 2006
Year of test
Table 2. Prevalence and relative risk (RR) of detection of RAMs according to VL and ART regimen,
following adjustment for variables shown in Table 1
Viral load
copies/ml
<300
300-999
1000-2999
3000-9999
10000-29999
30000-99999
≥100000
Whole cohort
no
no (%)
with RAM
RR (95% CI)
on NRTIs
no
449 270 (60) 0.94 (0.87-1.01) 410
552 399 (72) 0.99 (0.94-1.04) 508
1119 850 (76)
1
994
1311 1014 (77) 1.01 (0.97-1.05) 1135
1323 888 (67) 0.90 (0.86-0.95) 1003
1433 858 (60) 0.84 (0.80-0.88) 1014
1674 809 (48) 0.69 (0.65-0.74) 1076
On NNRTIs
On PIs
no (%)
with NAMs
RR (95% CI)
no
no (%)
with nNAMs
RR (95% CI)
no
no (%)
with PRAMs
RR (95% CI)
219 (53)
345 (68)
712 (72)
850 (75)
700 (70)
595 (59)
482 (45)
0.89 (0.81-0.98)
1.01 (0.94-1.07)
1
1.01 (0.96-1.06)
0.92 (0.88-0.97)
0.79 (0.74-0.84)
0.61 (0.57-0.66)
126
161
315
363
298
282
319
61 (48)
124 (77)
242 (77)
278 (77)
218 (73)
169 (60)
153 (48)
0.87 (073-1.02)
1.07 (0.97-1.18)
1
1.02 (0.94 -1.01)
0.97 (0.89-1.06)
0.84 (0.75-0.94)
0.68 (0.59-0.77)
193
237
423
483
454
462
507
55 (29)
90 (38)
182 (43)
224 (46)
198 (44)
192 (42)
157 (31)
0.85 (0.70-1.04)
1.00 (0.87-1.16)
1
0.89 (0.79-1.01)
0.87 (0.77-0.99)
0.85 (0.75-0.96)
0.64 (0.55-0.75)
NAMs = NRTI RAMs; nNAMs = NNRTI RAMs; PRAMs = PI RAMs
Results-2
30
09
Methods
MSM = Men who have sex with men; Hetero = heterosexual; IDU = injecting drug user; UK = unknown
<3
00
 Current genotypic resistance assays are validated for a VL >1000 copies/ml and both
treatment guidelines1,4 and assay manufacturers recommend this as the optimal threshold for
testing. Routine genotypic assays can however be adapted to perform well at lower levels of
viremia, with high (>75%) levels of success5,6. Although some debate ensues as to whether
the sequences obtained are fully representative of the dominant virus species7, many centres
perform resistance tests at VL levels <1000 copies/ml as part of routine care6. There are
limited data however supporting the tool of drug-resistance testing as an aid to selecting active
drugs at VL <1000 copies/ml8-10.
VL ever <50
copies/ml
Median no of RAMs (IQR)
 In treated patients with detectable viremia and remaining therapy options, switching therapy
is more likely to be successful when the CD4 count is higher and the VL is lower. The
importance of detecting drug-resistance may therefore be paramount in patients with low-level
viremia in order to allow a timely and optimized therapeutic change.
Time since start
of ART (yrs)
2453 (35.8)
2118 (30.9)
1412 (20.6)
877 (12.7)
3718 (54.2)
3142 (45.8)
 There were 7861 resistance tests from 3791 patients with 1 test, 1814
patients with 2 tests and 2256 patients with ≥3 tests. For 5738 (73%) tests, the
VL was measured on the same day of the test, for 865 (11%) within the
previous 2 weeks and for 1258 (16%) within the subsequent 4 weeks.
Figure 1. Resistance tests performed at
VL <1000 copies/ml as a proportion of all tests
Proportion (%) of tests
6182 (78.6) 5426 (79.1)
1679 (21.4) 1433 (20.9)
Background
 Current treatment guidelines recommend that virological failure should be managed
promptly by the design of a new regimen containing ≥2, and ideally 3, fully active drugs, as
guided by resistance testing and treatment history1-3.
P
Male
Female
3
3
Viral load (copies/ml)
*Global test for differences p=0.015 (Kruskall Wallis test)
3
=>
10
00
00
CONCLUSIONS: Several clinically relevant mutations can be detected at high frequency at low levels of
viremia. Genotypic resistance testing at low VL is informative and can guide a timely and optimized therapeutic
change in patients failing antiretroviral therapy (ART).
7861
10
00
029
99
9
30
00
099
99
9
RESULTS: Overall 5088/7861 (65%) resistance tests showed ≥1 RAM. Independent predictors of the
detection of resistance included earlier calendar year of testing, use of NNRTI-containing regimens, increasing
numbers of previously failed drugs, and never having achieved a VL <50 copies/ml (P<0.0001). In patients
with ≥1 RAM, the median (IQR) number of mutations was 3 (1–5), 3 (2–6), 3 (2–5), 3 (2–6), 4 (2–6), 3 (2–6)
and 3 (1–6) for the VL strata <300, 300–999, 1000–2999, 3000–9999, 10000–29999, 30000–99999 and
≥100000 copies/ml, respectively (P=0.015). Among 6136 patients experiencing NRTI failure, the most common
RAMs were the TAMs M41L, D67N, K70R, L210W, T215Y/F, K219Q/E; M184V; K65R; and L74V; only M41L,
L210W, T215Y, and L74V were significantly less prevalent at VL <1000 copies/ml than at higher levels. Among
1864 patients experiencing NNRTI failure, the most common RAMs were K103N, Y181C, G190A and V108I,
with no significant difference in prevalence according to VL. Among 2759 patients experiencing PI failure (66%
on a PI/r), the most common mutations were L90M, V82A, M46I, I84V and D30N; only I84V and L90M were
significantly less prevalent at VL <1000 copies/ml than at higher levels.
Total no of tests
99
99
METHODS: Genotypic resistance results with linked clinical data were obtained from the UK HIV Drug
Resistance Database and CHIC Study, including 1001/7861 (12.7%) performed at VL <1000 copies/ml.
Treatment regimens comprised ≥2 NRTIs with either an NNRTI (29.8%), a ritonavir-boosted PI (PI/r; 29.1%), a
third NRTI (18.1%) or a non-boosted PI (15.1%), or other combinations (7.9%).
Viral load copies/ml
>1000
<1000
no (%)
no (%)
6860
1001
Cohort (%)
30
00
-
OBJECTIVES: We previously reported that among patients undergoing routine genotypic resistance testing,
the detection of ≥1 drug-resistance mutation (RAM), both overall and drug class-specific, was most frequent at
300–10000 and declined >10000 HIV-1 RNA copies/ml. Here we compared the number and patterns of RAMs
in patients undergoing testing at viral load (VL) above or below 1000 copies/ml, the recommended threshold
for resistance testing in routine practice.
[email protected]
Results-1
Table 1. Cohort undergoing resistance testing at VL above and
below 1000 copies/ml
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99
Abstract
10
00
-
1Royal
3
 Overall 5088/7861 (65%) tests showed ≥1 RAM. Independent predictors of
the detection of RAMs comprised earlier calendar year of testing, receiving
NNRTI-based ART, increasing numbers of previously failed drugs, no previous
undetectable VL (p<0.0001 for all), and, marginally, longer time since starting
ART (p=0.02). The prevalence of RAMs varied according to the VL, but testing
at low VL did not significantly reduce the likelihood of detecting resistance
compared to testing at higher levels (Table 2). There was also no convincing
evidence of a significant difference in the number of RAMs detected according
to the VL, among those with ≥1 RAM (Fig 2).
 Table 3 shows the prevalence of RAMs stratified by VL for RAMs occurring
at a prevalence >5%, and stratified by class of drugs received at the time of
testing. In 6136 tests performed at NRTI failure, the most common NAMs were
the TAMs, M184V, K65R and L74V; the prevalence of pathway 1 TAMs (M41L,
L210W and T215Y) and L74V was significantly lower at low VL than at higher
levels, but there were no significant difference in the prevalence of other NAMs.
In 1864 tests performed at NNRTI failure, the most common nNAMs were
K103N, V108I, Y181C and G190A, with no significant difference in prevalence
according to VL. In Of 2759 tests performed at PI failure (66% PI/r, mostly
lopinavir/r), the most common PRAMs were D30N, M46I, V82A, I84V, and
L90M; of these, I84V and L90M were less prevalent at low VL than at higher VL
levels. The significant differences persisted when PI/r regimens were analysed
separately from overall PI regimens.
Conclusions
 This study provides substantive evidence that several clinically relevant RAMs are as likely to be detected at VL <1000 copies/ml as above this level. These include
the NAMs K65R, M184V and pathway 2 TAMs, major nNAMs, and the PRAMs D30N, M46I, and V82A. Consistent with this high detection yield, there has been
increased uptake of resistance testing at VL <1000 copies/ml over recent years in the UK. Nearly a quarter of all resistance tests performed in routine care in 2006
were at low VL.
 The prevalence of ≥1 RAM was 65% overall. The highest detection rates were observed at VL between 300 and 10,000 copies/ml. The prevalence of resistance
declined progressively when the VL exceeded 10,000 copies/ml for NAMs and PRAMs, and 30,000 copies/ml for nNAMs. This is likely to reflect the effect of
declining levels of adherence, although this is difficult to assess because of the lack of formally collected adherence data in our cohort.
 The higher frequency of TAMs at VL >1000 copies/ml may be expected as TAMs will accumulate with prolonged virological failure. However it is unclear why our
observation was only relevant to TAM 1 pathway mutations. We were unable to infer from this analysis whether the presence of RAMs at low VL should be
interpreted as the early emergence of these mutations within the quasispecies, or rather as a possible effect on VL of a reduced viral fitness. Whereas significant
fitness effects have been reported for several NAMs including K65R and M184V, nNAMs such as K103N do not appear to diminish viral fitness.
 Overall, our data indicate that whilst overall rates of drug-resistance are declining amongst treatment-experienced patients undergoing resistance testing in routine
practice, genotypic resistance testing at VL <1000 copies/ml did not significantly reduce the likelihood of detecting resistance compared to testing at higher levels.
The finding of clinically significant RAMs supports the practice of genotypic resistance testing at VL <1000 copies/ml in order to guide the choice of an effective
alternative regimen in patients experiencing treatment failure. Although data do not yet exist regarding the utility of resistance testing at low VL in terms of clinical
outcomes, guidelines exist which recommend prompt switching in patients with detectable viremia. The use of genotypic resistance testing at low VL may be helpful
in clinical practice to allow a timely and optimised therapeutic change , and may improve outcomes.
Table 3. Prevalence of individual RAMs
according to VL and ART regimen*
RAM and ART
regimen
On NRTIs
M41L
K65R
D67N
K70R
L74V
M184V
L210W
T215Y
T215F
K219Q
K219E
On NNRTI
K103N
V108I
Y181C
G190A
On PI/PI/r
D30N
M46I
V82A
I84V
L90M
Viral load (copies/ml)
P
<1000
>1000
20.7
5.34
21.5
17.1
3.27
38.8
12.2
19.1
6.2
7.0
6.2
27.0
4.18
23.3
16.1
6.50
39.3
16.0
25.6
7.6
7.7
5.7
<0.0001
0.11
0.24
0.43
0.0001
0.75
0.003
<0.0001
0.14
0.44
0.55
38.0
5.9
15.7
12.2
35.6
5.7
19.7
15.2
0.45
0.88
0.11
0.19
5.4
12.3
10.7
5.4
14.0
6.1
10.5
11.7
11.2
21.2
0.57
0.28
0.54
0.0002
0.0006
* Mutations with prevalence >5% in either or both VL groups
References
1. Hammer SM, et al. JAMA 2008; 2. Panel on Antiretroviral Guidelines for
Adult and Adolescents. USA Department of Health and Human Sciences
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Ther 2004; 5. Mackie N, et al. J Virol Meth 2004; 6. Cane PA, et al. HIV Med
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Monte Carlo, Monaco. March 2006. [Abstract 63]; 8. Parkin NT, et al. AIDS
2000; 9. Aleman S, et al. AIDS 2002; 10. Nettles RE, et al. Clin Infect Dis
2004; 11. The UK Drug Resistance Database. Available at
www.hivrdb.org.uk; 12. The UK Collaborative HIV Cohort Steering
Committee. HIV Med 2004; 13. Waters L, et al. AIDS 2006.