Transmitted drug resistance

Pat Cane

Questions

• What is the level of TDR and is it changing?

• Are we measuring TDR accurately?

• Are more sensitive methods of detecting TDR useful?

• What are the consequences of TDR?

ART status by year of sample

0

2000

4000

6000

8000

10000

12000

1997-9 2000 2001 2002 2003 2004 2005 2006 2007 2008

Year of sample

Num

ber

of te

sts

naive experienced unclassified

Linking to HPA databases(Sam Lattimore’s work)

• 64,888 records in HIVDR database had sufficient information for linkage– Clinic ID & Region– Soundex, DOB & region– Clinic ID (modified), site, DOB– Clinic ID alone

• 40,609 (62.6%) linked to new dx database• 48,826 (75.3%) linked to SOPHID (accessing treatment + care)• Overall, 51,033 records linked to either new dx or SOPHID

New dx2,207

SOPHID10,424

SOPHID & New dx38,402

0

20

40

60

80

2000 2001 2002 2003 2004 2005 2006 2007 2008

Year of sample

Per

cen

tag

e o

f tes

ts

NRTI

PI

nNRTI

any class

Prevalence of HIV drug resistance in ART-experienced patients (IAS 2009)

0

2

4

6

8

10

12

14

2000 2001 2002 2003 2004 2005 2006 2007 2008

Year of sample

Per

cen

tag

e o

f tes

ts

NRTI

PI

nNRTI

any class

Prevalence of HIV drug resistance in ART-naive patients (WHO list)

Misclassification of TDR(Hannah Castro’s work)

• Estimates of TDR will be distorted if some ART-experienced patients are misclassified as ART-naïve

• Aim: to quantify the potential extent of misclassification of treatment status bias

• The effect of misclassification depends on the number of naïve tests relative to the number of experienced tests, as well as the rate of resistance

• Explore possibility of distinguishing primary (in ART-naïve patients) and secondary (in ART-experienced patients) resistance based on patterns of mutations.

Number of observed naïve tests

326 365 409 537 622 617 994 1867 3268 3520

Number of observed experienced tests

Ratio of observed naive to experienced tests

113 214 568 847 819 1004 1163 1253 1550 1712

2.88 1.71 0.72 0.63 0.77 0.61 0.85 1.49 2.11 2.06

0.0

2.0

4.0

6.0

8.1

.12

.14

.16

Rat

e of

TH

DR

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

Observed THDR Adjusted THDR m = 0.02Adjusted THDR m = 0.05 Adjusted THDR m = 0.07

Year

Number of observed naïve tests

326 365 409 537 622 617 994 1867 3268 3520

Number of observed experienced tests

Ratio of observed naive to experienced tests

113 214 568 847 819 1004 1163 1253 1550 1712

2.88 1.71 0.72 0.63 0.77 0.61 0.85 1.49 2.11 2.06

0.0

2.0

4.0

6.0

8.1

.12

.14

.16

Rat

e of

TH

DR

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006

Observed THDR Adjusted THDR m = 0.02Adjusted THDR m = 0.05 Adjusted THDR m = 0.07

Year

Trend in TDR over time in UK

Effect of TDR on pre-therapy viral load(Linda Harrison’s work)

• In vitro studies indicate that most drug resistance mutations reduce replication capacity of HIV

• Patients with TDR might be expected to have lower HIV RNA viral load (VL) than patients infected with wild-type virus

• Epidemiological studies to date have been inconclusive (patients with TDR: n=9-77)

Viral load by class of TDR

Group No. (%) Mean log10 VL (SD)

Adjusted* difference

P

No TDR 7285 (91) 4.60 (0.82) REF

Any TDR 709 (9) 4.58 (0.83) -0.04 0.12

NRTI only

NNRTI only

PI only

≥2 classes

350 (4)

164 (2)

90 (1)

105 (1)

4.60 (0.85)

4.59 (0.86)

4.71 (0.75)

4.44 (0.86)

-0.05

0.00

0.09

-0.21

0.21

0.96

0.27

0.004

Adjusted for: CD4 count, viral subtype, ethnicity, exposure group, sex, age, calendar year, clinical centre, VL assay

other PI (n=67)V82A/L/T/F (n=39)

M46L/I (n=48)PI L90M (n=55)

other NNRTI (n=35)K101E/P (n=23)G190A/E (n=37)Y181C/V (n=48)

NNRTI K103N/S (n=154)

other NRTI (n=46)K70R/E (n=33)

T215Y/F (n=42)L210W (n=42)

M184V/I (n=61*)D67N/G/E (n=73)

K219N/Q/E/R (n=112)M41L (n=152)

NRTI 215 variants (n=250)

-.5 -.4 -.3 -.2 -.1 0 .1 .2 .3 .4 .5Difference in HIV RNA log10(c/ml)

*55V, 4I, 2V/I mixture

Eurocord analysis of impact of TDR on virological response up to 16 months

(Linda Wittcop)

TDR categories(WHO and Stanford)

N* %

2: at least one mutation of the WHO and resistant to at least one of their prescribed drugs (classified as 3,4,5 using Stanford)

477 4.6

1: at least one mutation of the WHO list but no drug resistance to their prescribed regimen (classified 1,2 using Stanford)

476 4.5

0: no detected mutations of the WHO list 2009 9505 90.9

* UK provided 22% of data

VF according to TDR categories

%

VF

months

Group 2: at least one mutation of WHO and receiving at least one resistant drug (3,4,5)Group 1: at least one mutation of WHO but receiving a fully active treatment (1,2)Group 0: no mutations on WHO list

Summary of Eurocord study

• Treatment response is poorer in patients having transmitted rug resistance mutations only when the ARV regimen is based upon drugs to which the virus has lost susceptibility– At least intermediate/low-level resistance to at least one

drug– Gradient effect among intermediate/low (Stanford 3+4)

and fully resistant (Stanford 5) levels– Less than three active drugs

• Having transmitted drug resistance mutations is not predictive of failure when the regimen has not lost susceptibility– Exception patients receiving 2NRTI + 1NNRTI (just sig.)

Minority TDR testing

• Allele specific PCR and deep sequencing can detect mutations present as minority populations.

CAVEATS!• Sensitivity: only to ~1% reliably due to error rates in

enzymes used in testing process (Shafer et al.)• Background incidence of each mutation in vivo (due to

inherent variability of quasispecies due to RT and RNA polymerase errors) can be assumed to be about 0.01-0.1%.

• Sensitivity cannot exceed input number of molecules ie need 1 ml with a viral load of ~10,000 cp/ml to get a sensitivity of 1% due to inefficiencies of RNA extraction etc.

Prevalence of drug resistance mutations in undiagnosed MSM

K103N by 30% (p=0.25, 95% CI: 1.2-67%)

-2

0

2

4

6

8

10

12

14

K103N Y181C M184V

Drug Resistance Mutation

% PrevalenceStandard Genotyping

minority species

M184V by 13-fold (p=0.0005, 95% CI: 2-85-fold increase) (Buckton et al.)

SENSE StudyTim Conibear’s (RF) data

Study Design• Several study sites with patients in Europe and Russia• Baseline plasma screened for drug resistance mutations by

population sequencing – all positive patients excluded

• 157 ART drug naïve HIV-1 infected patients eligible for study• Randomised into EFV or ETV + 2 NRTIs• Low-frequency drug resistance mutations to be detected in:

– plasma RNA by sensitive real-time PCR (CDC method)

Target codons: 100I, 101E, 103N, 181C, 184V, 188L, and 190A

– matched PBMC DNA pol gene sequences using both bulk and low-frequency assay

SENSE Study: ResultsResults from sensitive mutation detection (detection limit ranges: 0.1-10% mutant virus)

• ≥1 drug resistance mutation (101E/103N/181C/184V/190A) in 5.1% (8/157) patients• One patient showed both Y181 and M184 mutation, and no patients showed 100I or 188L.• All borderline result amplicons also sequenced – all non-significant mutations• All PBMC samples sequenced. No significant mutations detected.

Conclusions• Population sequencing on PBMC does not appear to increase detection of resistance

relative to plasma sequencing• Clinical significance not yet known (study still blinded)• Cutoffs are optimised for surveillance. Adjustment for clinical significance considered.

HIV-1 Subtype

n157

100I 101E 103N 181C 184V 188L 190A

B 99 0 2 1 0 1 0 1

Non-B 58 N/A N/A 0 1 3 N/A 0

% 0 2 1 0.6 2.5 0 0.6

CHAIN proposal

Clinical multicenter study

“Virological efficacy of first-line NNRTI-based

antiretroviral therapy in antiretroviral-naïve

subjects with minority HIV-1 mutants

resistant to reverse transcriptase inhibitors”

Roger Paredes, Karin Metzner

Objectives of the minority study

• To assess the risk of virological failure to first-line NNRTI-including ART in antiretroviral naïve HIV-1-infected subjects with pre-existing minority NNRTI-resistant HIV-1 variants

• To establish a clinically relevant pre-ART threshold of minority NNRTI-resistant HIV-1 variants

• To investigate if linkage of NNRTI-resistant mutations improves the prediction of virological failure in antiretroviral-naïve HIV-1-infected subjects initiating NNRTI-based ART

Study design

• Case control study within observational multi-cohort studies with retrospective testing of plasma samples collected prior to first-line ART

• To achieve 80% power 211 patients failing first-line NNRTI-containing ART and 633 matched controls will be included

Inclusion criteria

• Adult patients (defined as age ≥ 18 years)• Chronic HIV-1 infection • Initiation of first-line cART with a NNRTI plus at least 2

NRTIs• Available resistance test (population sequencing): No

evidence for NNRTI-resistance• Available clinical data (e.g., CD4 count, viral load, ART

changes) during follow-up• Pre-treatment plasma sample available for 454 ultra-deep

sequencing fulfilling the following criteria– Collected within 6 months before ART initiation– HIV-1 RNA levels ≥10.000 copies/mL plasma– 1 mL plasma available for UDS testing

Definition of cases and controls

• Cases = Patients who experienced virological failure • Controls = Patients of the same cohort/clinical center (i.e.,

cases extracted from EuroSIDA will be matched with controls selected from EuroSIDA, etc) who did not experienced virological failure

• Three controls per case will be identified• At analysis time (i.e., the time point of failure (or matching

time for controls)) patients have to be still receiving a first-line NNRTI, while patients are allowed to switch NRTIs

Research questions

• What is the impact of M184V present as a minority on treatment outcomes?

• Should treatment where any TDR detected by standard methods include a boosted PI?

• How long does it take majority mutations to go to minority?

• Can minority TDR be onward transmitted?• If a significant proportion of treated patients who show

no resistance mutations when failing boosted PIs are phenotypically resistant, will this resistance be transmitted?