HIV Resistance Testing Clinical Implications Cyril K. Goshima, M.D. Director, AIDS Education Project June, 2009

HIV Resistance TestingHIV Resistance TestingClinical ImplicationsClinical Implications

Cyril K. Goshima, M.D.Cyril K. Goshima, M.D.

Director, AIDS Education Director, AIDS Education ProjectProject

June, 2009June, 2009

I am a?I am a?

1.1. PhysicianPhysician

2.2. NurseNurse

3.3. PharmacistPharmacist

4.4. DentistDentist

5.5. StudentStudent

6.6. PatientPatient

7.7. OtherOther

Resistance Testing will tell us what Resistance Testing will tell us what meds will work for a patient?meds will work for a patient?

1.1. TrueTrue

2.2. FalseFalse

Resistance Testing is Resistance Testing is recommended before starting recommended before starting

HAART.HAART.

1.1. TrueTrue

2.2. FalseFalse

Resistance Testing can be done on Resistance Testing can be done on patients with viral load less than patients with viral load less than

500.500.

1.1. True True

2.2. FalseFalse

The K103N mutation is an NNRTI The K103N mutation is an NNRTI MutationMutation

1.1. TrueTrue

2.2. FalseFalse

M184V is a common PI MutationM184V is a common PI Mutation

1.1. True True

2.2. FalseFalse

When to Use Resistance TestingWhen to Use Resistance Testing

DHHS GuidelinesDHHS Guidelines– Recommend testing: acute infection, Recommend testing: acute infection,

suboptimal virologic suppression after suboptimal virologic suppression after treatment is initiated, treatment failure, prior to treatment is initiated, treatment failure, prior to the initiation of therapy.the initiation of therapy.

– Consider: chronic infection < 2 yrs.Consider: chronic infection < 2 yrs.– Which test is not recommendedWhich test is not recommended

Resistance TestingResistance Testing

Genotypic Resistance TestingGenotypic Resistance TestingPhenotypic Resistance TestingPhenotypic Resistance TestingCombined Geno/PhenoCombined Geno/Pheno““Virtual Phenotype” TestingVirtual Phenotype” TestingTrofile (HIV Tropism Assay)Trofile (HIV Tropism Assay)PhenoSense Entry (FI Resistance Testing)PhenoSense Entry (FI Resistance Testing)Integrase Resistance TestingIntegrase Resistance TestingReplication CapacityReplication Capacity

Requirements for Resistance Requirements for Resistance TestingTesting

Viral Load must be greater than 500 for Viral Load must be greater than 500 for genotype & phenotype resistance testing, genotype & phenotype resistance testing, integrase resistance testingintegrase resistance testing

Viral load must be greater than 1000 for Viral load must be greater than 1000 for Trofile and PhenoSense EntryTrofile and PhenoSense Entry

Genotypic Resistance TestingGenotypic Resistance Testing

Detects mutations in the HIV genome associated Detects mutations in the HIV genome associated with resistance to specific drugs.with resistance to specific drugs.

AdvantagesAdvantages– Adequate turn-around time (1-2 wks)Adequate turn-around time (1-2 wks)– Less expensiveLess expensive– Detect mutations that may precede phenotypic Detect mutations that may precede phenotypic

resistanceresistance– Widely availableWidely available– More sensitive in detecting mixtures of resistant and More sensitive in detecting mixtures of resistant and

wild type viruseswild type viruses

Genotypic Resistance TestingGenotypic Resistance Testing

DisadvantagesDisadvantages– Indirect measure of resistanceIndirect measure of resistance– Relevance of some mutations unclearRelevance of some mutations unclear– Unable to detect minority variants (<20 – 25% Unable to detect minority variants (<20 – 25%

of viral sample)of viral sample)– Complex patterns may be difficult to interpretComplex patterns may be difficult to interpret– Genotypic correlates of resistance not well Genotypic correlates of resistance not well

defined for non-B subtypes.defined for non-B subtypes.

Phenotypic Resistance TestingPhenotypic Resistance Testing

Measures the patient’s HIV isolates ability Measures the patient’s HIV isolates ability to replicate in the presence of varying to replicate in the presence of varying concentration of specific drugs.concentration of specific drugs.AdvantagesAdvantages– Direct and quantitative measure of resistanceDirect and quantitative measure of resistance– Method can be applied to any agent incl. new Method can be applied to any agent incl. new

where genotypic correlates are unclearwhere genotypic correlates are unclear– Can assess interactions among mutationsCan assess interactions among mutations– Accurate with non-B HIV subtypes.Accurate with non-B HIV subtypes.

Phenotypic Resistance TestingPhenotypic Resistance Testing

DisadvantagesDisadvantages– Susceptibility cut-offs not standard between Susceptibility cut-offs not standard between

assaysassays– Clinical cut-offs not defined for some drugsClinical cut-offs not defined for some drugs– Unable to detect minority speciesUnable to detect minority species– Complex technologyComplex technology– More expensiveMore expensive– Longer turn-around time.Longer turn-around time.

Other TestsOther Tests

Geno/Phenotype Resistance TestingGeno/Phenotype Resistance Testing– e.g. Phenosense GT from Monograme.g. Phenosense GT from Monogram– Both tests are performedBoth tests are performed– The discordance is reportedThe discordance is reported

““Virtual” PhenotypeVirtual” Phenotype– Genotyping is performed and the phenotype Genotyping is performed and the phenotype

is determined by looking at all the matched is determined by looking at all the matched pairs of genotype with phenotype in a data set pairs of genotype with phenotype in a data set to give the best estimateto give the best estimate

Other TestsOther Tests

Fusion Inhibitor Resistance TestingFusion Inhibitor Resistance Testing– Resistance to EnfuvirtideResistance to Enfuvirtide

Replication CapacityReplication Capacity– How weak is your patient’s virus?How weak is your patient’s virus?

Chemokine Receptor IdentificationChemokine Receptor Identification– CCR5, CXCR4, or mixed virus presentCCR5, CXCR4, or mixed virus present

Integrase Inhibitor Resistance TestingIntegrase Inhibitor Resistance Testing

How We Identify a MutationHow We Identify a Mutation

How do we identify a resistance mutation?How do we identify a resistance mutation?

“M” is the “wild type” amino acid

“184” is the codon

position

M 184 M

How We Identify a MutationHow We Identify a Mutation

How do we identify a resistance mutation?How do we identify a resistance mutation?



position

“V” is the mutant amino

acid

M 184 V

How We Identify a MixtureHow We Identify a Mixture



position

“M/V” is the mixture of wild type & mutant

amino acid

M 184 M/V

Definitions for Phenotypic Definitions for Phenotypic Resistance TestingResistance Testing

IC50 = Concentration of drug required to IC50 = Concentration of drug required to inhibit replication by 50%inhibit replication by 50%Fold Change = IC50 pt./IC50 reference Fold Change = IC50 pt./IC50 reference Cut Off = Fold change or concentration Cut Off = Fold change or concentration below which the virus is considered below which the virus is considered susceptible, above which non-susceptiblesusceptible, above which non-susceptibleBiological Cut Off = Fold change based on Biological Cut Off = Fold change based on variations in clinical samples from variations in clinical samples from treatment naïve individuals.treatment naïve individuals.

Definitions for Phenotypic Definitions for Phenotypic Resistance TestingResistance Testing

Clinical Cut Off = Fold change based on Clinical Cut Off = Fold change based on virologic response to ARV in Clinical Trialsvirologic response to ARV in Clinical Trials

Replication Capacity: The ability of a pt’s Replication Capacity: The ability of a pt’s virus to replicate in the absence of drugvirus to replicate in the absence of drug

NRTIsNRTIs

NRTI MutationsNRTI Mutations

Single point mutation can result in high level Single point mutation can result in high level resistance e.g. M184V (3TC, FTC), K65R (TDF)resistance e.g. M184V (3TC, FTC), K65R (TDF)

TAMS pattern of mutations e.g. codons 41, 67, TAMS pattern of mutations e.g. codons 41, 67, 70, 210, 215, 219 (AZT, D4T)70, 210, 215, 219 (AZT, D4T)

2 other patterns that are selected for by AZT/DDI 2 other patterns that are selected for by AZT/DDI & DDI/D4T& DDI/D4T– Q151M:resist. all NRTI except TDFQ151M:resist. all NRTI except TDF– T69insertion + 1 or more TAMS @ 41, 210, 215: T69insertion + 1 or more TAMS @ 41, 210, 215:

resist. all NRTIresist. all NRTI

Common Mutations: NRTIsCommon Mutations: NRTIs

TAMS = thymidine analog mutations (aka ZDV TAMS = thymidine analog mutations (aka ZDV mutations): M41L, D67N, K70R, L210W, T215F/Y, mutations): M41L, D67N, K70R, L210W, T215F/Y, K219E/QK219E/Q

NAMS = nucleoside analog mutations: TAMS plus NAMS = nucleoside analog mutations: TAMS plus E44A/D, A62V*, K65R, T69D, T69ins, L74I/V, E44A/D, A62V*, K65R, T69D, T69ins, L74I/V, V75A/I*/M/S/T, V77L*, Y115F, F116Y*, V118I, Q151M, V75A/I*/M/S/T, V77L*, Y115F, F116Y*, V118I, Q151M, M184I/VM184I/V

*Secondary mutations seen with Q151M*Secondary mutations seen with Q151M

NRTI Signature MutationsNRTI Signature Mutations

*TAMS=Thymidine analog mutations.

Position Drug74, 65, TAMS ddI

(41, 67, 70, 210, 215, 219),* 333 ZDV

184 (TAMS, 44, 65,118) 3TC/FTC

75, TAMS d4T

184 + TAMs or 65, 74, 115 ABC

65; 41, 210, 215Y TDF

NNRTIs NNRTIs

Common Mutations: NNRTIsCommon Mutations: NNRTIs

Delavirdine (DLV)Delavirdine (DLV)– L100I, K103N, V106M, Y181C, I; Y188L, L100I, K103N, V106M, Y181C, I; Y188L,

G190E/QG190E/Q– P236L(rare), Y318F P236L(rare), Y318F

Efavirenz (EFV)Efavirenz (EFV)– L100I, K103N, V106M, Y181C, I; Y188L, L100I, K103N, V106M, Y181C, I; Y188L,

G190A, S, E, Q…; P225HG190A, S, E, Q…; P225H

Nevirapine (NVP)Nevirapine (NVP)– L100I, K103N, V106A, M; Y181C, I; Y188C, L, L100I, K103N, V106A, M; Y181C, I; Y188C, L,

H; G190A, E, S, Q…,F227LH; G190A, E, S, Q…,F227L

NNRTI Multi-Drug ResistanceNNRTI Multi-Drug ResistanceClass ResistanceClass Resistance– L100I, K101E or P, K103N or S, V106A or M, L100I, K101E or P, K103N or S, V106A or M,

Y188C, H, or L, M230LY188C, H, or L, M230L

Resistance to one NNRTI usually confers Resistance to one NNRTI usually confers cross resistance to all other agents cross resistance to all other agents (exceptions: 181 and EFV, 190A/S and DLV)(exceptions: 181 and EFV, 190A/S and DLV)Continued viral replication in the presence of Continued viral replication in the presence of NNRTI results in accumulation of additional NNRTI results in accumulation of additional resistance mutationsresistance mutations– May impact clinical utility of future NNRTIsMay impact clinical utility of future NNRTIs

NNRTI Novel MutationsNNRTI Novel Mutations

Those exhibiting a > 10 fold change:Those exhibiting a > 10 fold change:

– K103R and V179D (in combination)K103R and V179D (in combination)

– K101PK101P

NNRTI: EtravirineNNRTI: Etravirine

K103N NNRTI mutation is not associated K103N NNRTI mutation is not associated with resistance to Etravirinewith resistance to Etravirine

Multiple Resistance Associated Mutations Multiple Resistance Associated Mutations (RAMS)(RAMS)

Scoring of the number of RAMS Scoring of the number of RAMS determines resistance to Etravirine determines resistance to Etravirine

Similar to Protease Inhibitor ScoringSimilar to Protease Inhibitor Scoring

PIsPIs

PI ResistancePI Resistance

Cross resistance is commonCross resistance is commonPI mutations are uncommon in boosted PI PI mutations are uncommon in boosted PI regimensregimensMulticlass experienced pts. may have Multiclass experienced pts. may have been exposed to unboosted regimensbeen exposed to unboosted regimensThe number of primary PI mutations may The number of primary PI mutations may predict the response to therapy e.g. TPV predict the response to therapy e.g. TPV score 0-3 good, 4-7 intermediate, >8 poor score 0-3 good, 4-7 intermediate, >8 poor or Kaletraor Kaletra

PI Common MutationsPI Common MutationsLopinavir V32I, M46I/V, I47A/V, G48V, I50V, I54A/L/M/S/T/V,

V82A/F/S/T, I84V, L90M (need several of these)

FosAmprenavir V32I, M46I/V,I47A/V, I50V, I54A/L/M/S/T, I84A/C/V, V82F, L33F, M46I/L (in certain combinations)?

Nelfinavir D30N, V82A/F/S/T*, N88D/S/T, I84A/C/V, L90M

Saquinavir G48V, V82A/F/S/T*, I84A/C/V, L90M*

Indinavir M46I/L, V82A/F/S/T*, N88S/T, I84A/C/V, L90M*

Ritonavir M46I/L, V82A/F/S/T*, N88S/T, I84A/C/V, L90M*

Tipranavir 10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V

Darunavir V11I, V32I, L33F, I47V, I50V, I54M/L, G73S, L76V, I84V, L89V

Atazanavir Needs 5 or more from list. Following exposure to ATV: I50L

HypersusceptibilityHypersusceptibility

NRTI Increased SusceptibilityNRTI Increased Susceptibility

Mutation Selected By… Increases Susceptibility

K65R TDF, ABC, ddI ZDV

L74I or V ddI, ABC ZDV, TDF

L100I NNRTI ZDV, TDF

Y181C, I, or V NVP, DLV ZDV, TDF

M184V 3TC, FTC, ABC, ddI ZDV, TDF, d4T

NNRTI Increased SusceptibilityNNRTI Increased Susceptibility

Mutation Selected by… Increases Susceptibility

G190A, C, S, T, or V

EFV, NVP DLV

P225H EFV, NVP? DLV

F227L EFV, NVP DLV

L100I NNRTI ZDV, TDF

Y181C, I, or V NVP, DLV ZDV, TDF

PI Increased SusceptibilityPI Increased Susceptibility

Mutation Selected by…

Increases Susceptibility

N88S NFV, IDV, ATV

APV

I50L ATV RTV, LPV, SQV, other PIs?

PI HypersusceptibilityPI Hypersusceptibility

Mutation I50V, selected by LPVr and APV, Mutation I50V, selected by LPVr and APV, increased susceptibility to ATV, TPV.increased susceptibility to ATV, TPV.

Integrase and Entry Inhibitor Integrase and Entry Inhibitor ResistanceResistance

Resistance has been seen against the Resistance has been seen against the Entry Inhibitors and Integrase Inhibitors Entry Inhibitors and Integrase Inhibitors

There are resistance tests that can be There are resistance tests that can be orderedordered

For CCR5 it may just be a repeat of the For CCR5 it may just be a repeat of the Trophile test to determine change to mixed Trophile test to determine change to mixed or dual tropic virusesor dual tropic viruses

Case DiscussionCase Discussion

Patient CB, 42 y/o, homosexual malePatient CB, 42 y/o, homosexual male

Current Regimen (05/31/06): CBV/TDF/EFVCurrent Regimen (05/31/06): CBV/TDF/EFV

Past Drugs: CBV/ IDV, CBV/NFVPast Drugs: CBV/ IDV, CBV/NFV

CD4/VLCD4/VL– Date: 09/08/05 349/8,810Date: 09/08/05 349/8,810– Date: 03/07/06 192/10,300Date: 03/07/06 192/10,300– Date: 06/02/06 186/9,400Date: 06/02/06 186/9,400– Date: 09/18/06 92/6,610Date: 09/18/06 92/6,610– Date: 10/17/06 /12,000Date: 10/17/06 /12,000


NRTI NRTI – M184V present (3TC/FTC resist, TDF hs)M184V present (3TC/FTC resist, TDF hs)– Multiple TAMs Multiple TAMs – No K65R (TDF sens despite 41 & 215 mut)No K65R (TDF sens despite 41 & 215 mut)

NNRTINNRTI– No significant mutationsNo significant mutations

PIPI– 4 TPV assoc mut (intermediate response)4 TPV assoc mut (intermediate response)– DRV sensDRV sens


Was the CBV/TDF/EFV regimen a Was the CBV/TDF/EFV regimen a reasonable one?reasonable one?

There has been no response to this There has been no response to this therapy after 3 mos.therapy after 3 mos.

What should you do?What should you do?

Any suggestions on a possible new Any suggestions on a possible new regimen?regimen?

DiscordanceDiscordance

Inaccurate genotype interpretation Inaccurate genotype interpretation algorithm that does not account for novel algorithm that does not account for novel or previously unknown mutation effector previously unknown mutation effectMixtures of wild type and resistant strains. Mixtures of wild type and resistant strains. Phenotype underestimates resistancePhenotype underestimates resistanceVariability in phenotypic susceptibility with Variability in phenotypic susceptibility with specific mutationsspecific mutationsBelieve the genotype. Genotypic change Believe the genotype. Genotypic change may precede phenotypic resistance.may precede phenotypic resistance.

Clinical ImplicationsClinical Implications

Is there evidence for sequencing of Is there evidence for sequencing of NRTIs?NRTIs?

Should the initial regimen be a boosted PI Should the initial regimen be a boosted PI or a NNRTI?or a NNRTI?

Is 3TC = FTC as far as resistance is Is 3TC = FTC as far as resistance is concerned?concerned?


Try to use at least 2 new potent agents to switch Try to use at least 2 new potent agents to switch from a failing regimen.from a failing regimen.The longer a failing regimen is continued, the The longer a failing regimen is continued, the more mutations accumulate. If there is no new more mutations accumulate. If there is no new agent, better to cont. the same regimen unless agent, better to cont. the same regimen unless compelled to do otherwise.compelled to do otherwise.Resistance is relative. 3TC cont. to have Resistance is relative. 3TC cont. to have virological effect despite M184V mutation. virological effect despite M184V mutation. Boosted PIs may have more of a response than Boosted PIs may have more of a response than an unboosted PI evidenced by a lower fold an unboosted PI evidenced by a lower fold change. change.


NRTINRTI– TAMs can prevent K65R mutation. K65R is TAMs can prevent K65R mutation. K65R is

associated with multiple NRTI resistance and associated with multiple NRTI resistance and TDF resistance. ? Add ZDV to failing regimenTDF resistance. ? Add ZDV to failing regimen

– Continue 3TC or FTC despite a M184V Continue 3TC or FTC despite a M184V mutation (hypersusc. ZDV, TDF, D4T; RC)mutation (hypersusc. ZDV, TDF, D4T; RC)

NNRTINNRTI– DC NNRTI as soon as mutations develop. DC NNRTI as soon as mutations develop.

There is no virological or RC advantage. There is no virological or RC advantage.


PIPI– Never use an unboosted PI.Never use an unboosted PI.

Antiretroviral susceptibility is on a Antiretroviral susceptibility is on a continuum. Using drugs with the most continuum. Using drugs with the most activity (lower fold change) is a reasonable activity (lower fold change) is a reasonable choice. choice.


In initial therapy, a boosted PI regimen In initial therapy, a boosted PI regimen may have an advantage over a NNRTI may have an advantage over a NNRTI regimen because of fewer HIV mutations. regimen because of fewer HIV mutations. ((J. Bartlett, et al, JAIDS, 4(3): 323-331; Swiss HIV J. Bartlett, et al, JAIDS, 4(3): 323-331; Swiss HIV Cohort Study, oral abstract 72, XV International HIV Cohort Study, oral abstract 72, XV International HIV

Drug Resistance Workshop)Drug Resistance Workshop) Possible Possible explanations maybe lower genetic barrier explanations maybe lower genetic barrier and pharmacokinetics with missed doses.and pharmacokinetics with missed doses.


Replication CapacityReplication Capacity– Lower RC with certain NRTI (3TC) and PI Lower RC with certain NRTI (3TC) and PI

(NFV).(NFV).– No change in RC with NNRTINo change in RC with NNRTI

Resistance Testing will tell us what Resistance Testing will tell us what meds will work for a patient?meds will work for a patient?

Tru

e

Fal

se

50%50%1.1. TrueTrue

2.2. FalseFalse

Resistance Testing is Resistance Testing is recommended before starting recommended before starting

HAART.HAART.

Tru

e

Fal

se

50%50%1.1. TrueTrue

2.2. FalseFalse

Resistance Testing can be done on Resistance Testing can be done on patients with viral load less than patients with viral load less than

500.500.

Tru

e

Fal

se

50%50%

1.1. True True

2.2. FalseFalse

The K103N mutation is an NNRTI The K103N mutation is an NNRTI MutationMutation

Tru

e

Fal

se

50%50%1.1. TrueTrue

2.2. FalseFalse

M184V is a common PI MutationM184V is a common PI Mutation

Tru

e

Fal

se

50%50%1.1. True True

2.2. FalseFalse

AcknowledgementsAcknowledgements

Monogram Bioscience, Sharon Martens, Monogram Bioscience, Sharon Martens, MN, ARNP/FNPMN, ARNP/FNP

Dr. Joel Gallant, MD, MPH from Clinical Dr. Joel Gallant, MD, MPH from Clinical Care Options HIV LLC, “Use and Care Options HIV LLC, “Use and Interpretation of Resistance Tests in Multi-Interpretation of Resistance Tests in Multi-Class Experienced Patients,” September Class Experienced Patients,” September 2, 2005.2, 2005.

Thank YouThank You

Questions?Questions?

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HIV Resistance Testing Clinical Implications Cyril K. Goshima, M.D. Director, AIDS Education Project June, 2009