antiretroviral resistance testing in the management of hiv-infected

Antiretroviral Resistance Testing in the Management of HIV-infected

Patients

Christopher Behrens, MDAmy Kindrick, MD

Robert Harrington, MD

Overview of Antiretroviral Resistance Testing

• 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?

How Does HIV Develop Resistance to Antiretrovirals?

HIV Life Cycle

RNA DNA

HIV

Nucleus

Host Cell

Nucleoside Analogues (NRTIs)

Non-Nucleosides (NNRTIs) Protease Inhibitors (PIs)

Reverse Transcriptase

How does HIV Develop Resistance to Antiretrovirals?

• 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

How drug resistance arises. Richman, DD. Scientific American , July 1998

How Drug Resistance Arises

How does resistance develop?

Continuation of a failing ART regimen after early resistance has developed selects for expansion of resistance


Poor Adherence

Drug Resistance

Regimen Failure


Insufficient drug level

Viral replication in the presence of drug

Resistant virus

Poor adherence

Social/personal issuesRegimen issues

ToxicitiesPoor potency

Wrong dose

Host genetics

Poor absorption

Rapid clearance

Poor activation

Drug interactions

True or False?

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

What is the relationship between adherence and resistance?

00.10.20.30.40.50.60.70.80.9

1

0 10 20 30 40 50 60 70 75 80 85 90 95 100

Adherence

Res

ista

nce

Harrigan, JID, 2005• Prospective, observational

study• N = 1191• Predictors of resistance

– High baseline VL– Good (not great) adherence


0

5

10

15

20

25

1996-97 1998-99 2000-01

NRTI anyNNRTI anyPI any primarytwo classesthree classes

Is Resistance Becoming More Common?

JAMA. 2002 Jul 10;288(2):181-8.

% o

f res

ista

nt is

olat

es

Prevalence of resistance among recently-infected patients at San Francisco General Hospital

Is Resistance Becoming More Common?

Little SJ, Holte S, Routy JP, et al. N Engl J Med. 2002;347:385-94

Recently Infected, ART Naïve, United States

How do we test for resistance?

1. Genotype2. Phenotype3. Virtual Phenotype

HIV Life Cycle

RNA DNA

HIV

Nucleus

Host Cell

Nucleoside Analogues (NRTIs)

Non-Nucleosides (NNRTIs) Protease Inhibitors (PIs)

Reverse Transcriptase

Genotypic Resistance Assay• 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

AAA GAC AGTAAA GAC AGT AAAAAA AAACAC AGAGCC

LysLys AspAsp SerSer LysLys AsnAsn SerSer

Codon Silent MutationMutation Silent Mutation

Adapted from Winters. Reviewed in Wilson. AIDS Read 2000;10:469.

M184V

M = Methionine184 = 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.

Adapted from D’Aquila. Topics in HIV Medicine 2001;9(2):31.

L N R W YF Q

41 67 70

560560AZTAZT

L T(Wild Type)

1 (Mutant)219215210

KM D K

L M K

ddIddI 65 74 184V V R

ddCddCT

D

65 74 18469

41 67 70 219215210d4Td4T 75

V

TMSA

Mutation Selected in vitro

ABCABC 74 115Y

F V219184 21521041 67 7065

3TC3TC 184

E

118

V

I44

D VI

Clinical significance under investigation

Reverse Transcriptase Mutations Selected by NRTIs

SA

L K100

181

100

NVP

DLV

EFV

V V

103

106108

188 190

Y G

I N A I C LH A

L

103

103 108

181 Y

CI

236 P

C

188

L

190 225 P

H

56056011

Adapted from D’Aquila. Topics in HIV Medicine 2001;9(2):31.

Mutation

Reverse Transcriptase Mutations Selected by NNRTIs

K V L

1 99

10

L

IDV 20 24 32

L L M

46

I

54 71

I V G A

82 84 90

54 71 82 84 90

10 48 54 71 82 84 90

71 77 82 88 90

VLILF G

V D

3630 46

N

84

I

46

I

10

RTV

SQV

NFV

APV

46

32

FIRV

5447 50

I V

10

FI IL

84

AFTS

7773S A

77

36 73 77

M V

Primary Secondary

M V AFTS IV SAVTIII IIRV MR

20 32 33 36

N D

V VM

844610LPV/RTV 545320 24 71 82 9063

L

P

F

L

10

Adapted from D’Aquila. Topics in HIV Medicine 2001;9:31.

Protease Mutations Selected by PIs

Interpretation of the results: what are the clinical implications of these mutations in terms of resistance to antiretroviral agents?

Interpretation of the Genotypic Resistance Assay

• 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)

Phenotypic Resistance

Testing• Tests viability of a synthetic

version of the patient’s HIV in the presence of antiretroviral agents

• Similar to traditional bacterial antibiotic susceptibility assays

• Results reported as fold-change in susceptibility to antiretroviral agents

Drug Concentration

Inhi

bitio

n of

Vira

l R

eplic

atio

n (%

) 100

0

ICIC5050

50

ICIC5050

Fold Fold ResistanceResistance

Wild-type strainMutant strain

Reviewed in Wilson. AIDS Read 2000;10:469.

Phenotype Resistance Testing

PhenoSense HIV Patient Report

PhenoSense™ HIV ReportPhenoSense™ HIV Report

Which Resistance Assay is Better?Pros Cons

Genotypicassay

• 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

Does the use of resistance assays improve clinical results?

Study (year published)

Study Arms Conclusions

VIRADAPT (1999)

Genotype + expert advice vs

usual care (no expert advice)

Genotype with expert advice better than usual care without expert advice

CPCRA 046 (2000)



Genotype with expert advice better than usual care without expert advice

Havana (2002)

Genotype +/- expert advice vs

usual care (+/- expert advice)

Genotype and expert advice each helpful; genotype with expert advice resulted in best clinical outcomes

ARGENTA (2002)


usual care (+ expert advice)

Genotype with expert advice not significantly better than standard of care + expert advice

VIRA3001 (2002)

Phenotype (no expert advice) vs


Phenotype without expert advice better than usual care without expert advice

Narval (2002)

Genotype (no expert advice) vs

phenotype (no expert advice) vs


No signficant advantage of genotype or phenotype over usual care

Published Randomized Controlled Trials of Resistance Testing

% of Patients with HIV-1 RNA <400 copies/mL

wk 12NO 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%

0

20

40

60

80

100No G No Expert Op.

No G Expert Op.G No Expert Op.G Expert Op.

P = .0206 P = .00132

(ITT)

Tural. 40th IACAC; 2000; Toronto. Presentation LB-10.

BL wk 24

Havana Results:

Expert Consultation Resources

• National Clinicians’ Telephone Consultation Service (Warmline): 800-933-3413

• Internet:– http://hivdb.stanford.edu– www.hivresistance.com

• Others?

The Virtual Phenotype

Genotype

ProteaseRTHIV

Access Data

Genotype & Phenotype DataVirtual Phenotype

Wild-type HIV

Resistant HIV

Illustration by David Spach, MD

The Virtual PhenotypeSample report

When Should a Resistance Assay be Ordered?

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 regimenDetermine the role of resistance in drug failure and maximize the number of active drugs in the new regimenDetermine if resistant virus was transmitted; select regimen accordinglyAssays 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 <1,000 copies/mL

Resistance mutations may become minor species in the absence of selective drug pressureResistance assays unreliable if VL is low

Adapted from DHHS, Antiretroviral Guidelines, October 6, 2005

Antiretroviral Therapy: Virologic Failure

10

100

1000

10000

100000

HIV

RN

A

Medications Started

50 50


Time

Antiretroviral Therapy: Failure to Suppress

10

100

1000

10000

100000

HIV

RN

A

Medications Started

50 50


Time

0

5

10

15

20

25

1996-97 1998-99 2000-01

NRTI anyNNRTI anyPI any primarytwo classesthree classes

Time Trends in Primary HIV-1 Genotypic Drug Resistance Among

Recently Infected Persons

JAMA. 2002 Jul 10;288(2):181-8.

% o

f res

ista

nt is

olat

es


Clinical Setting/Recommendation Rationale

Recommended Virologic failure during HAART Suboptimal suppression of viral load after initiation of HAART Acute (Primary) HIV Infection

Determine the role of resistance in drug failure and maximize number of active drugs in a new regimen if indicated Determine the role of resistance and maximize number of active drugs in new regimen Determine if drug resistant virus was transmitted and design initial regimen accordingly

Consider Chronic HIV infection prior to initiation of HAART

Minor drug-resistant species might not be detected, but consider testing if significant probability that patient may have been infected with a drug-resistant virus

Not Generally Recommended After discontinuation of drugs Plasma viral load < 1000 copies/mL

Resistant quasispecies tend to become minor species in the absence of selective drug pressure, making detection by current assays unlikely Current assays unreliable at low viral loads

DHHS. Antiretroviral Guidelines, July 14, 2003, Table 3.



Rationale



Determine role of resistance in drug failure and maximize the number of active drugs in the new regimenDetermine the role of resistance in drug failure and maximize the number of active drugs in the new regimenDetermine 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




Adapted from DHHS, Antiretroviral Guidelines, October 6, 2005

Reversion to Predominant Wild-Type Virus After Discontinuing ART


Drug resistance is Significantly Correlated with Reduction in Replication Capacity

Wrin T, et al. 5th International Workshop on HIV Drug Resistance and Treatment Strategies. Scottsdale, AZ: June 2001 (Abstract 24)


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Adherence

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Rationale



•Chronic HIV infection before starting ART

Determine role of resistance in drug failure and maximize the number of active drugs in the new regimenDetermine the role of resistance in drug failure and maximize the number of active drugs in the new regimenDetermine if resistant virus was transmitted; select regimen accordinglyAssays may not detect minor resistant species, but some transmitted resistance mutations may persist for years. Consider testing early in the course of HIV infection.




Adapted from DHHS, Antiretroviral Guidelines, May 4, 2006

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, 95%CI).

• 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

Barbour JD et al. AIDS: Volume 18(12) 20 August 2004 pp 1683-1689

Persistence of Resistant Strains

Following Primary HIV Infection

N = 6 patients infected with resistant strains of HIV; none reverted to wild-type over the course of several months of observation

Persistence of Resistant Strains after Primary HIV Infection?


Persistence of Some Resistant Strains after Primary HIV Infection?


Testing for Antiretroviral Drug Resistance: Conclusions

• 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

What if you cannot obtain a resistance assay for your patient who

is failing therapy?

Empiric sequencing of ART regimens

Empiric design of salvage regimens for patients failing ART: key considerations

• 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)

• 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

Empiric design of salvage regimens for patients failing ART: key considerations

Time after dose (hours)

0 2 4 6 8 10 12100

1,000

10,000

An Example of Ritonavir Boosting:Indinavir/Ritonavir BID PK Study

IDV/RTV q12h:

800/200 High-fat Meal

800/100 High-fat Meal 400/400 High-fat Meal

IDV q8h: 800 mg Fasted

IndinavirPlasma

Concentration(nM)

6th Conference on Retroviruses and Opportunistic Infections; 1999. Abstract 362.

Implications of varying genotypic and phenotypic barriers to resistance

• 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

% of Patients with HIV-1 RNA <400 copies/mL

wk 12NO 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%

0

20

40

60

80

100No G No Expert Op.

No G Expert Op.G No Expert Op.G Expert Op.

P = .0206 P = .00132

(ITT)

Tural. 40th IACAC; 2000; Toronto. Presentation LB-10.

BL wk 24

Havana Results:

Should we discuss resistance with patients?

Factors Associated with Higher Levels of Adherence

• 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 1998;18:117-125.

2. Morse EV et al, Soc Sci Med 1991;32:1161-1167. 3. Singh N, et al, AIDS Care 1996;8:261-269. 4. Stone VE, et al. JAIDS 2001; 28:124-131.

Factors Associated with Higher Levels of Adherence

• 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.

Teaching the concept of resistance to patients

A cartoon metaphor

How Resistance Develops to HIV

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.

Sometimes the Wall Comes Down

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.

The Wall Goes Back Up When you start taking the medicine

regularly again, the wall goes back up.

Sometimes, it’s 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.

Lessons to Be Learned

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.

Antiretroviral Resistance

Summary & Conclusions

Summary & Conclusions• 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

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.

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.

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 <50 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 <50 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.

Case 1: Figure

0100002000030000400005000060000700008000090000

100000

30 mosago

24 mosago

18 mosago

12 mosago

6 mosago

1 moago

Viral Load

Initiate ARTPancreatitis;lamivudine substituted fordidanosine

Genotype ordered

50

Vira

l Loa

d (c

opie

s/m

L)

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

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

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 CD4=320, viral load=10,000.

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/lopinavir+ritonavir– AZT/tenofovir/efavirenz– d4T/abacavir/ritonavir/saquinavir– AZT/d4T/lopinavir+ritonavir

Failed AZT/ddI/nevirapine in remote past

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/lopinavir+ritonavir– AZT/tenofovir/efavirenz– d4T/abacavir/ritonavir/saquinavir– AZT/d4T/lopinavir+ritonavir

Extra slides

How much resistance is out there?

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

Underwood M et al. 12th CROI; 2005, Boston. #674.

Prevalence of resistance among new 787 HIV diagnostic specimens from 899 sites in six states

Categories Participants with HIVDRAny 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%)

antiretroviral resistance testing in the management of hiv-infected

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