Antiretroviral Resistance Testing in the Management of HIV-infected Patients

1
Antiretroviral Resistance Testing in the
Management of HIV-infected Patients
0

• Christopher Behrens, MD
• Amy Kindrick, MD
• Robert Harrington, MD

2
Overview of Antiretroviral Resistance Testing
0

• How does resistance develop?
• What is the relationship between adherence and
  resistance?
• How much resistance is out there?
• How do we test for resistance?
• How do we interpret the results of a resistance
  test?
• Does resistance testing improve care?
• When should you order resistance tests?
• Can a single dose of nevirapine for Prevention of
  Mother to Child Transmission (PMTCT) result in
  clinically significant resistance?

3
How Does HIV Develop Resistance to
Antiretrovirals?
0
4
HIV Life Cycle
0
Nucleoside Analogues (NRTIs)
HIV
Reverse Transcriptase
RNA
DNA
Nucleus
Host Cell
Non-Nucleosides (NNRTIs)
Protease Inhibitors (PIs)
5
How does HIV Develop Resistance to
Antiretrovirals?
0

• HIV reverse transcriptase is a low-fidelity
  enzyme, i.e., transcription mistakes are common
• Mistakes (mutations) lead to mutant strains of
  HIV
• Most mutations are inconsequential or result in
  incompetent strains of HIV, but certain mutations
  confer resistance to currently available
  antiretroviral drugs (ARVs)
• Administration of antiretrovirals in an
  insufficiently potent manner exerts reproductive
  pressure that selects for resistance-bearing
  strains which then become the majority strain of
  HIV in that patient

6
How Drug Resistance Arises
0
How drug resistance arises. Richman, DD.
Scientific American , July 1998
7
How does resistance develop?
0
Continuation of a failing ART regimen after early
resistance has developed selects for expansion of
resistance
8
How does resistance develop?
0

• Poor Adherence
• Drug Resistance
• Regimen Failure

9
How does resistance develop?
0
Social/personal issues
Regimen issues
Poor potency
Toxicities
Wrong dose
Poor adherence
Host genetics
Poor absorption
Insufficient drug level
Rapid clearance
Viral replication in the presence of drug
Poor activation
Resistant virus
Drug interactions
10
True or False?
0

• The patients with the lowest levels of adherence
  are the most likely to develop resistance to
  their ARVs

11
What is the relationship between adherence and
resistance?
0
12
What is the relationship between adherence and
resistance?
0

• Harrigan, JID, 2005
• Prospective, observational study
• N 1191
• Predictors of resistance
• High baseline VL
• Good (not great) adherence

13
Is Resistance Becoming More Common?
0
Prevalence of resistance among recently-infected
patients at San Francisco General Hospital
of resistant isolates
JAMA. 2002 Jul 10288(2)181-8.
14
Is Resistance Becoming More Common?
0
Recently Infected, ART Naïve, United States
Little SJ, Holte S, Routy JP, et al. N Engl J
Med. 2002347385-94
15
How do we test for resistance?
0

• 1. Genotype
• 2. Phenotype
• 3. Virtual Phenotype

16
HIV Life Cycle
0
Nucleoside Analogues (NRTIs)
HIV
Reverse Transcriptase
RNA
DNA
Nucleus
Host Cell
Non-Nucleosides (NNRTIs)
Protease Inhibitors (PIs)
17
Genotypic Resistance Assay
0

• Sequences relevant portions of the HIV genome
  coding for Reverse Transcriptase and Protease
  enzymes
• Detects and reports variations in the sequences
  of these genes that are known or suspected to
  confer antiretroviral resistance

Codon
Mutation
Silent Mutation
AAA GAC AGT
AAA AAC AGC
Lys
Lys
Asp
Ser
Asn
Ser
Adapted from Winters. Reviewed in Wilson. AIDS
Read 200010469.
18
0
M184V
M Methionine 184 the codon V Valine A
mutation at codon 184 in the gene Reverse
Transcriptase codes for a Valine residue where
normally a Methionine residue is found.
19
Reverse Transcriptase Mutations Selected by NRTIs
0
L T
(Wild Type)
M
D
K
K
AZT
41
67
70
219
215
210
1 (Mutant)
L N R
W YF
Q
560
M
K
L
T
ddC
184
65
74
69
D
V
E
3TC
44
184
118
I
D
VI
V
d4T
41
67
70
215
210
75
219
TMSA
Y
ABC
74
115
219
215
210
67
70
65
184
41
F
V
Clinical significance under investigation
Mutation
Selected in vitro
Adapted from DAquila. Topics in HIV Medicine
20019(2)31.
20
Reverse Transcriptase Mutations Selected by NNRTIs
0
L K
V
V
Y
G
Y
106 108
NVP
100
188
181
103
190
I N A I
C LH A
CI
560
1
P
DLV
181
103
236
L
C
P
EFV
100
108
188
190
225
103
SA
L
H
Adapted from DAquila. Topics in HIV Medicine
20019(2)31.
21
Protease Mutations Selected by PIs
0
K
V
L
L
L
M
I
I
V
G
A
M
V
10
20
24
32
46
54
71
82
84
90
36
73
77
IDV
1
99
M
V
AFTS
I
V
SA
VT
I
I
I
I
IRV
MR
L
54
71
82
84
90
46
77
32
33
36
20
10
RTV
VL
IL
F
G
10
48
54
71
82
84
90
77
73
SQV
V
S
A
D
N
NFV
71
77
82
88
90
36
30
46
10
84
IL
FI
AFTS
N
D
I
I
APV
84
46
10
32
54
47
50
FIRV
V
VM
I V
L
F
LPV/RTV
84
46
10
54
53
20
24
71
82
90
63
P
L
Adapted from DAquila. Topics in HIV Medicine
2001931.
22
0
Interpretation of the results what are the
clinical implications of these mutations in terms
of resistance to antiretroviral agents?
23
Interpretation of the Genotypic Resistance Assay
0

• The genotype report typically includes an
  interpretation of the clinical implications of
  the identified mutations
• However
• The exact significance of many mutations remains
  controversial
• Interactions between mutations further complicate
  estimation of the clinical impact of a given set
  of mutations
• Interpretation of genotypic resistance assays is
  not standardized across different laboratories
• Assays will not detect minority resistant strains
  (less than 10-20 of the viral population)

24
0
25
0
26
0
27
0
28
0
29
0
30
Phenotypic Resistance Testing
0

• Tests viability of a synthetic version of the
  patients HIV in the presence of antiretroviral
  agents
• Similar to traditional bacterial antibiotic
  susceptibility assays
• Results reported as fold-change in susceptibility
  to antiretroviral agents

31
Phenotype Resistance Testing
0
100
Fold Resistance
Inhibition of Viral Replication ()
50
0
IC50
IC50
Drug Concentration
Reviewed in Wilson. AIDS Read 200010469.
32
PhenoSense HIV Patient Report
0
33
PhenoSense HIV Report
0
34
Which Resistance Assay is Better?
0
Pros Cons
Genotypic assay More clinical experience and evidence of clinical utility Less expensive (400) Results available in 1-2 weeks Results difficult to interpret Does not directly measure net effect of multiple mutations
Phenotypic assay Simpler to interpret More directly estimates net effect of multiple mutations Clinically relevant thresholds of resistance not yet determined for many agents Less evidence of clinical utility More expensive (1000) Results in 3-4 weeks
35
Does the use of resistance assays improve
clinical results?
0
36
Published Randomized Controlled Trials of
Resistance Testing
0
37
Havana Results
0
of Patients with HIV-1 RNA lt400 copies/mL
(ITT)
100
No G No Expert Op.
P .0206
P .00132
80
No G Expert Op.
60
G No Expert Op.
G Expert Op.
40
20
0
wk 12
BL
wk 24
NO G, NO Expert (N 77) 36.4 NO G, Expert (N
67) 49.3 G, NO Expert (N 69) 46.4 G,
Expert (N 65) 69.2
Tural. 40th IACAC 2000 Toronto. Presentation
LB-10.
38
Expert Consultation Resources
0

• National Clinicians Telephone Consultation
  Service (Warmline) 800-933-3413
• Internet
• http//hivdb.stanford.edu
• www.hivresistance.com
• Others?

39
The Virtual Phenotype
0
Genotype
Access Data
Protease
RT
HIV
Genotype Phenotype Data
Virtual Phenotype
Wild-type HIV
Resistant HIV
Illustration by David Spach, MD
40
0
The Virtual Phenotype Sample report
41
When Should a Resistance Assay be Ordered?
0
42
Antiretroviral Resistance Testing Guidelines for
Implementation
Clinical Setting/ Recommendation Rationale
Recommended Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Chronic HIV infection before starting ART Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted select regimen accordingly Assays may not detect minor resistant species, but some resistance mutations may persist for years. Consider testing early after diagnosis of HIV infection.
Usually not recommended After discontinuation of drugs Plasma VL lt1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low
Adapted from DHHS, Antiretroviral Guidelines,
October 6, 2005
43
Antiretroviral Therapy Virologic Failure
0
Medications Started
50
50
Time
Illustration by David Spach, MD
44
Antiretroviral Therapy Failure to Suppress
0
Medications Started
50
50
Time
Illustration by David Spach, MD
45
Time Trends in Primary HIV-1 Genotypic Drug
Resistance Among Recently Infected Persons
0
of resistant isolates
JAMA. 2002 Jul 10288(2)181-8.
46
Antiretroviral Resistance Testing Guidelines for
Implementation
0
DHHS. Antiretroviral Guidelines, July 14, 2003,
Table 3.
47
Antiretroviral Resistance Testing Guidelines for
Implementation
Clinical Setting/ Recommendation Rationale
Recommended Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted select regimen accordingly
Consider Chronic HIV infection before starting ART Assays may not detect minor resistant species, but consider if significant probability of transmitted drug-resistant virus
Usually not recommended After discontinuation of drugs Plasma VL lt1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low
Adapted from DHHS, Antiretroviral Guidelines,
October 6, 2005
48
Reversion to Predominant Wild-Type Virus After
Discontinuing ART
0
Illustration by David Spach, MD
49
0
50
Drug resistance is Significantly Correlated with
Reduction in Replication Capacity
0
Wrin T, et al. 5th International Workshop on HIV
Drug Resistance and Treatment Strategies.
Scottsdale, AZ June 2001 (Abstract 24)
51
What is the relationship between adherence and
resistance?
0
52
Antiretroviral Resistance Testing Guidelines for
Implementation
Clinical Setting/ Recommendation Rationale
Recommended Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Chronic HIV infection before starting ART Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted select regimen accordingly Assays may not detect minor resistant species, but some transmitted resistance mutations may persist for years. Consider testing early in the course of HIV infection.
Usually not recommended After discontinuation of drugs Plasma VL lt1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low
Adapted from DHHS, Antiretroviral Guidelines,
May 4, 2006
53
Persistence of Resistant Strains Following
Primary HIV Infection

• 11 subjects with primary HIV infection who
  deferred ART and who had at least one major drug
  resistance mutation identified at presentation,
  followed with serial resistance assays.
• 7 subjects with NNRTI resistance
• 2 with NRTI and PI resistance
• 1 with NNRTI and PI resistance
• 1 with resistance to all three classes of drugs
• NNRTI resistance was lost slowly the average
  time to reversion of 103N variants to mixed
  103N/K populations was 196 days following the
  estimated date of infection (153 to 238 days,
  95CI).
• PI resistance was not lost at all In the 4
  patients with protease resistance mutations, no
  reversion was detected at 64, 191, 327, and 342
  days after infection.
• Complete reversion of genotypic resistance was
  observed in only one patient, at 1019 days after
  infection.

Little SJ. 11th CROI, February 2004, Abstract
36LB
54
Persistence of Resistant Strains Following
Primary HIV Infection
0
N 6 patients infected with resistant strains of
HIV none reverted to wild-type over the course
of several months of observation
Barbour JD et al. AIDS Volume 18(12) 20 August
2004 pp 1683-1689
55
Persistence of Resistant Strains after Primary
HIV Infection?
0
Illustration by David Spach, MD
56
Persistence of Some Resistant Strains after
Primary HIV Infection?
0
Illustration by David Spach, MD
57
Testing for Antiretroviral Drug Resistance
Conclusions
0

• The proportion of new HIV infections that involve
  resistant strains tends to increase with
  increasing availability of ART
• Initial ART is more likely to fail in patients
  with a resistant strain
• In patients treated with ART, resistance
  mutations, especially those affecting the NNRTIs
  and PIs, have been found to persist for up to two
  years after discontinuation of ART
• Resistance testing is becoming more common in
  chronically-infected patients in North America
  who acquired their infection in the past few years

58
What if you cannot obtain a resistance assay for
your patient who is failing therapy?
0

• Empiric sequencing of ART regimens

59
Empiric design of salvage regimens for patients
failing ART key considerations
0

• The genotypic barrier to resistance varies across
  different antiretroviral agents
• For some ARVs, a single mutation can induce
  high-level resistance (e.g., lamivudine,
  efavirenz, nevirapine)
• For other ARVs, resistance generally does not
  develop until multiple mutations accumulate (eg,
  AZT, stavudine, tenofovir)

60
Empiric design of salvage regimens for patients
failing ART key considerations
0

• The phenotypic barrier to resistance can vary for
  different ARVs as well
• High serum levels can help to prevent or even
  overcome resistance mutations
• Ritonavir boosting of protease inhibitors will
  increase their phenotypic barrier to resistance
• PIs, like many medications, are metabolized in
  the liver by the cytochrome P450 enzyme complex
• Ritonavir inhibits this complex, thereby boosting
  serum levels of co-administered PIs
• Low doses of ritonavir can be used to increase
  the potency and simplify the dosing of PI-based
  regimens

61
An Example of Ritonavir BoostingIndinavir/Ritona
vir BID PK Study
0
10,000
IDV/RTV q12h 800/200 High-fat Meal
800/100 High-fat Meal 400/400 High-fat
Meal IDV q8h 800 mg Fasted
IndinavirPlasmaConcentration(nM)
1,000
100
0
2
4
6
8
10
12
Time after dose (hours)
6th Conference on Retroviruses and Opportunistic
Infections 1999. Abstract 362.
62
Implications of varying genotypic and phenotypic
barriers to resistance
0

• Resistance develops initially to NNRTIs
  (efavirenz, nevirapine) and lamivudine
• If treatment is continued, resistance can
  subsequently develop to other NRTIs such as AZT,
  stavudine, didanosine, abacavir, tenofovir
• Protease inhibitors
• Variable, but generally resistance develops more
  slowly than to lamivudine and NNRTIs
• Ritonavir boosting significantly delays
  development of resistance to protease inhibitors

63
Havana Results
0
of Patients with HIV-1 RNA lt400 copies/mL
(ITT)
100
No G No Expert Op.
P .0206
P .00132
80
No G Expert Op.
60
G No Expert Op.
G Expert Op.
40
20
0
wk 12
BL
wk 24
NO G, NO Expert (N 77) 36.4 NO G, Expert (N
67) 49.3 G, NO Expert (N 69) 46.4 G,
Expert (N 65) 69.2
Tural. 40th IACAC 2000 Toronto. Presentation
LB-10.
64
Should we discuss resistance with patients?
0
65
Factors Associated with Higher Levels of Adherence
0

• Twice-daily or once-daily regimens1,4
• Belief in own ability to adhere to regimen1
• Not living alone2
• Dependent on a significant other for support2
• History of opportunistic infection or advanced
  HIV disease3

1. Eldred L, et al, J Acquir Immune Defic Syndr
Hum Retrovirol 199818117-125. 2. Morse EV et
al, Soc Sci Med 1991321161-1167. 3. Singh N,
et al, AIDS Care 19968261-269. 4. Stone VE, et
al. JAIDS 2001 28124-131.
66
Factors Associated with Higher Levels of Adherence
0

• Belief in efficacy of antiretroviral therapy
• Belief that non-adherence will lead to viral
  resistance

Wenger N, et al. 6th Conference on Retroviruses
and Opportunistic Infections, 1999 Chicago.
Abstract 98.
67
Teaching the concept of resistance to patients
0

• A cartoon metaphor

68
How Resistance Develops to HIV
0

• This is the virus known as HIV. The only thing
  that matters to him in his short, nasty life is
  to destroy T-Cells. To do this, he must somehow
  get over this wall.
• The wall is created by taking anti-HIV
  medications. When the medicines are taken
  correctly, the virus is unable to climb over the
  wall to get to your T-cells.

69
Sometimes the Wall Comes Down
0

• When you forget to take your evening dose, or
  only take 2 of your anti-HIV medicines, the
  strong wall comes down.
• The virus breaks free and is able to get over the
  wall.
• When he gets to the other side, he discovers a
  way to get over the wall in the future. This is
  called resistance. He finds a spring that will
  give him a little more bounce.

70
The Wall Goes Back Up
0

• When you start taking the medicine regularly
  again, the wall goes back up.
• Sometimes, its too late and the virus uses the
  spring to jump over the wall. At this point, it
  is a resistant virus The drugs may not be able to
  keep the wall high enough to stop the springing
  virus.

71
Lessons to Be Learned
0

• It is better to not take anti-HIV drugs at all
  than to take them only some of the time.
• If you think you may be missing doses often,
  please tell your health care provider or
  pharmacist! We promise not to tell your mother.

72
Antiretroviral Resistance
0

• Summary Conclusions

73
Summary Conclusions
0

• Resistance develops in the setting of an
  inadequately suppressive ART regimen
• Educating patients about resistance may promote
  better adherence
• For the patient who is failing therapy
• Revisit adherence issues
• Consider obtaining a resistance assay
• Resistance assay results need to be interpreted
  with caution, and ideally with expert assistance
• Salvage regimens can be designed empirically
  without the assistance of a resistance assay

74
Cases
0
75
In which of these situations is resistance
testing clearly indicated?

• A 28 yo male just diagnosed with acute HIV
  infection
• A 38 yo woman on d4T/3TC/indinavir who had
  enjoyed full virologic suppression but whose last
  two HIV viral loads were 72 and 110 copies/mL
• A 41 yo man on AZT/3TC/nelfinavir whose last
  three viral loads were 256, 865, and 1838
  copies/mL
• A 35 yo woman with a history of spotty adherence
  and a viral load of 20,000 copies/mL while on
  d4T/3TC/efavirenz one year ago. She discontinued
  all antiretrovirals shortly thereafter, but now
  wants to restart ART and appears highly motivated.

76
In which of these situations is resistance
testing clearly indicated?

• A 28 yo male just diagnosed with acute HIV
  infection
• A 38 yo woman on d4T/3TC/indinavir who had
  enjoyed full virologic suppression but whose last
  two HIV viral loads were 72 and 110 copies/mL
• A 41 yo man on AZT/3TC/nelfinavir whose last
  three viral loads were 256, 865, and 1838
  copies/mL
• A 35 yo woman with a history of spotty adherence
  and a viral load of 20,000 copies/mL while on
  d4T/3TC/Efavirenz one year ago. She discontinued
  all antiretrovirals shortly thereafter, but now
  wants to restart HAART and appears highly
  motivated.

77
Case 1

• A 33 yo woman with a baseline CD4 count of 260
  cells/mm³ and a viral load of 90,000 copies/mL
  initiates ART with a regimen of
  d4T/ddI/nelfinavir. She achieves virologic
  control with a viral load lt50 copies/mL and her
  CD4 count rises to 420.
• 6 months later, she develops pancreatitis 3TC is
  substituted for ddI, and her viral load remains
  lt50 copies/mL on d4T/3TC/nelfinavir
• 4 months later her viral load rises to 25,000
  copies/mL, and her CD4 count drops to 320.

78
Case 1 Figure
Initiate ART
Genotype ordered
Pancreatitis lamivudine substituted
for didanosine
Viral Load (copies/mL)
50
79
Case 1 continued

• You order a genotypic resistance assay, which
  reveals the following mutations
• Reverse transcriptase M184V
• Protease D30N
• Which of the following regimens is/are reasonable
  options for this patient?
• ddI/abacavir/efavirenz
• AZT/3TC/nevirapine
• d4T/abacavir/ritonavir/saquinavir
• d4T/tenofovir/efavirenz

80
Case 1 continued

• You order a genotypic resistance assay, which
  reveals the following mutations
• Reverse transcriptase M184V
• Protease D30N
• Which of the following regimens is/are reasonable
  options for this patient?
• ddI/abacavir/efavirenz
• AZT/3TC/nevirapine
• d4T/abacavir/ritonavir/saquinavir
• d4T/tenofovir/efavirenz

81
Case 2

• A 37 yo male initiated ART 5 years ago
• Initial regimen AZT/ddI/nevirapine
• Responded well initially with VL drop to
  undetectable, rise in CD4 from 240 to 400
  cells/mm³
• However, experienced virologic failure within one
  year with rise in viral load to 12,000 copies/mL
• Regimen changed to d4T/3TC/indinavir change made
  without using a resistance assay

82
Case 2 continued

• He again achieved an undetectable viral load, on
  his new regimen of d4T/3TC/indinavir
• 6 months ago lost to follow-up
• One month ago returned to clinic, describing
  recent depression and spotty adherence (both of
  which improved in past month)
• labs reveal CD4320, viral load10,000.

83
Case 2 continued
Failed AZT/ddI/nevirapine in remote past

• You order a genotypic resistance assay while he
  is still taking d4T/3TC/indinavir, which reveals
• Reverse transcriptase M41L, M184V, T215Y
• Protease I84V
• Which regimen(s) would you recommend?
• ddI/nevirapine/nelfinavir
• AZT/3TC/tenofovir/lopinavirritonavir
• AZT/tenofovir/efavirenz
• d4T/abacavir/ritonavir/saquinavir
• AZT/d4T/lopinavirritonavir

84
Case 2 continued

• You order a genotypic resistance assay while he
  is still taking d4T/3TC/indinavir, which reveals
• Reverse Transcriptase M41L, M184V, T215Y
• Protease I84V
• Which regimen(s) would you recommend?
• ddI/nevirapine/nelfinavir
• AZT/3TC/tenofovir/lopinavirritonavir
• AZT/tenofovir/efavirenz
• d4T/abacavir/ritonavir/saquinavir
• AZT/d4T/lopinavirritonavir

85
Extra slides
86
What is the relationship between adherence and
resistance?

• REACH study NNRTIs vs PIs in indigent/homeless
  in SF (n108)
• Unboosted PI or NNRTI x gt6 months

100 90 80 70 60 50 40 30 20 10 0
NNRTI p0.03 Chi trend PI p0.47 Chi trend
p0.01
Resistant
0-53 54-79 80-94 95-100 n29 n28 n25
n26
Adherence Quartile

• Viral suppression better with NNRTI vs PI
• Resistance more common with NNRTIs at low
  adherence, but declines with higher adherence
  trend is opposite with PIs

Bangsberg DR et al. XV Int AIDS Conf July 2004,
Bangkok. 5820.
87
How much resistance is out there?
0

• 89 diagnostic and clinical sites in 6 U.S. states
• 828 newly diagnosed patients, 95 genotyped
• Overall prevalence of resistance was 14.5

Prevalence of resistance among new 787 HIV diagnostic specimens from 899 sites in six states Prevalence of resistance among new 787 HIV diagnostic specimens from 899 sites in six states
Categories Participants with HIVDR
Any drug class RTI or primary PI 114 (14.5)
NRTI 56 (7.1)
NNRTI 66 (8.4)
PI 22 (2.8)
Two or more drug classes 24 (3.1)
Underwood M et al. 12th CROI 2005, Boston. 674.