Detection of clinically relevant antiretroviral drug resistance mutations among treated patients und

1
Detection of clinically relevant antiretroviral
drug resistance mutations among treated
patientsundergoing 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 1Royal Free Hampstead
NHS Trust UCL Medical School and 2Imperial
College Healthcare NHS Trust, London, UK
a.geretti_at_medsch.ucl.ac.uk

• Results-1
• 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.
  
• 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 lt1999 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/rNRTIs (plt0.0001 for all).

Table 1. Cohort undergoing resistance testing at
VL above and below 1000 copies/ml
Figure 1. Resistance tests performed at VL lt1000
copies/ml as a proportion of all tests
Abstract 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
30010000 and declined gt10000 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. 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 lt1000 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). 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 lt50 copies/ml
(Plt0.0001). In patients with 1 RAM, the median
(IQR) number of mutations was 3 (15), 3 (26), 3
(25), 3 (26), 4 (26), 3 (26) and 3 (16) for
the VL strata lt300, 300999, 10002999,
30009999, 1000029999, 3000099999 and 100000
copies/ml, respectively (P0.015). 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 lt1000
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 lt1000 copies/ml than at higher
levels. 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).
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

• 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.
• 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.  
• Current genotypic resistance assays are
  validated for a VL gt1000 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 (gt75) 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 lt1000 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
  lt1000 copies/ml8-10.

MSM Men who have sex with men Hetero
heterosexual IDU injecting drug user UK
unknown
NAMs NRTI RAMs nNAMs NNRTI RAMs PRAMs PI
RAMs

• Results-2
• 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 (plt0.0001 for all), and, marginally, longer
  time since starting ART (p0.02). 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 gt5, 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.

Figure 2. Median number of RAMs detected
according to VL, in tests with 1 RAM
Table 3. Prevalence of individual RAMs according
to VL and ART regimen
Median no of RAMs (IQR)

• Methods
• 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 gt1 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 lt1000 copies/ml
  came from 3 centres where either the VircoType
  (Virco, Belgium) or in-house 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).
•  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 gt400 copies/ml after gt4 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.

Global test for differences p0.015 (Kruskall
Wallis test)

• Conclusions
• This study provides substantive evidence that
  several clinically relevant RAMs are as likely to
  be detected at VL lt1000 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 lt1000 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 gt1000
  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 lt1000 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 lt1000 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.

Mutations with prevalence gt5 in either or both
VL groups
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Mackie N, et al. J Virol Meth 2004 6. Cane PA,
et al. HIV Med 2008 7. Stone C, et al, 4th
European HIV Drug Resistance Workshop. 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.
Aim To characterize the population undergoing
genotypic resistance testing at VL lt1000
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.