HIV Resistance TestingClinical Implications Cyril K. Goshima, M.D. Director, AIDS Education Project June, 2009
I am a? • Physician • Nurse • Pharmacist • Dentist • Student • Patient • Other
Resistance Testing will tell us what meds will work for a patient? • True • False
Resistance Testing is recommended before starting HAART. • True • False
Resistance Testing can be done on patients with viral load less than 500. • True • False
The K103N mutation is an NNRTI Mutation • True • False
M184V is a common PI Mutation • True • False
When to Use Resistance Testing • DHHS Guidelines • Recommend testing: acute infection, suboptimal virologic suppression after treatment is initiated, treatment failure, prior to the initiation of therapy. • Consider: chronic infection < 2 yrs. • Which test is not recommended
Resistance Testing • Genotypic Resistance Testing • Phenotypic Resistance Testing • Combined Geno/Pheno • “Virtual Phenotype” Testing • Trofile (HIV Tropism Assay) • PhenoSense Entry (FI Resistance Testing) • Integrase Resistance Testing • Replication Capacity
Requirements for Resistance Testing • Viral Load must be greater than 500 for genotype & phenotype resistance testing, integrase resistance testing • Viral load must be greater than 1000 for Trofile and PhenoSense Entry
Genotypic Resistance Testing • Detects mutations in the HIV genome associated with resistance to specific drugs. • Advantages • Adequate turn-around time (1-2 wks) • Less expensive • Detect mutations that may precede phenotypic resistance • Widely available • More sensitive in detecting mixtures of resistant and wild type viruses
Genotypic Resistance Testing • Disadvantages • Indirect measure of resistance • Relevance of some mutations unclear • Unable to detect minority variants (<20 – 25% of viral sample) • Complex patterns may be difficult to interpret • Genotypic correlates of resistance not well defined for non-B subtypes.
Phenotypic Resistance Testing • Measures the patient’s HIV isolates ability to replicate in the presence of varying concentration of specific drugs. • Advantages • Direct and quantitative measure of resistance • Method can be applied to any agent incl. new where genotypic correlates are unclear • Can assess interactions among mutations • Accurate with non-B HIV subtypes.
Phenotypic Resistance Testing • Disadvantages • Susceptibility cut-offs not standard between assays • Clinical cut-offs not defined for some drugs • Unable to detect minority species • Complex technology • More expensive • Longer turn-around time.
Other Tests • Geno/Phenotype Resistance Testing • e.g. Phenosense GT from Monogram • Both tests are performed • The discordance is reported • “Virtual” Phenotype • Genotyping is performed and the phenotype is determined by looking at all the matched pairs of genotype with phenotype in a data set to give the best estimate
Other Tests • Fusion Inhibitor Resistance Testing • Resistance to Enfuvirtide • Replication Capacity • How weak is your patient’s virus? • Chemokine Receptor Identification • CCR5, CXCR4, or mixed virus present • Integrase Inhibitor Resistance Testing
How We Identify a Mutation • How do we identify a resistance mutation? M 184 M “M” is the “wild type” amino acid “184” is the codon position
How We Identify a Mutation • How do we identify a resistance mutation? M 184 V “M” is the “wild type” amino acid “184” is the codon position “V” is the mutant amino acid
How We Identify a Mixture M 184 M/V “M” is the “wild type” amino acid “184” is the codon position “M/V” is the mixture of wild type & mutant amino acid
Definitions for Phenotypic Resistance Testing • IC50 = Concentration of drug required to inhibit replication by 50% • Fold Change = IC50 pt./IC50 reference • Cut Off = Fold change or concentration below which the virus is considered susceptible, above which non-susceptible • Biological Cut Off = Fold change based on variations in clinical samples from treatment naïve individuals.
Definitions for Phenotypic Resistance Testing • Clinical Cut Off = Fold change based on virologic response to ARV in Clinical Trials • Replication Capacity: The ability of a pt’s virus to replicate in the absence of drug
NRTI Mutations • Single point mutation can result in high level resistance e.g. M184V (3TC, FTC), K65R (TDF) • TAMS pattern of mutations e.g. codons 41, 67, 70, 210, 215, 219 (AZT, D4T) • 2 other patterns that are selected for by AZT/DDI & DDI/D4T • Q151M:resist. all NRTI except TDF • T69insertion + 1 or more TAMS @ 41, 210, 215: resist. all NRTI
Common Mutations: NRTIs • TAMS = thymidine analog mutations (aka ZDV mutations): M41L, D67N, K70R, L210W, T215F/Y, K219E/Q • NAMS = nucleoside analog mutations: TAMS plus E44A/D, A62V*, K65R, T69D, T69ins, L74I/V, V75A/I*/M/S/T, V77L*, Y115F, F116Y*, V118I, Q151M, M184I/V *Secondary mutations seen with Q151M
NRTI Signature Mutations *TAMS=Thymidine analog mutations.
Common Mutations: NNRTIs • Delavirdine (DLV) • L100I, K103N, V106M, Y181C, I; Y188L, G190E/Q • P236L(rare), Y318F • Efavirenz (EFV) • L100I, K103N, V106M, Y181C, I; Y188L, G190A, S, E, Q…; P225H • Nevirapine (NVP) • L100I, K103N, V106A, M; Y181C, I; Y188C, L, H; G190A, E, S, Q…,F227L
NNRTI Multi-Drug Resistance Class Resistance • L100I, K101E or P, K103N or S, V106A or M, Y188C, H, or L, M230L • Resistance to one NNRTI usually confers cross resistance to all other agents (exceptions: 181 and EFV, 190A/S and DLV) • Continued viral replication in the presence of NNRTI results in accumulation of additional resistance mutations • May impact clinical utility of future NNRTIs
NNRTI Novel Mutations • Those exhibiting a > 10 fold change: • K103R and V179D (in combination) • K101P
NNRTI: Etravirine • K103N NNRTI mutation is not associated with resistance to Etravirine • Multiple Resistance Associated Mutations (RAMS) • Scoring of the number of RAMS determines resistance to Etravirine • Similar to Protease Inhibitor Scoring
PI Resistance • Cross resistance is common • PI mutations are uncommon in boosted PI regimens • Multiclass experienced pts. may have been exposed to unboosted regimens • The number of primary PI mutations may predict the response to therapy e.g. TPV score 0-3 good, 4-7 intermediate, >8 poor or Kaletra
PI Hypersusceptibility • Mutation I50V, selected by LPVr and APV, increased susceptibility to ATV, TPV.
Integrase and Entry Inhibitor Resistance • Resistance has been seen against the Entry Inhibitors and Integrase Inhibitors • There are resistance tests that can be ordered • For CCR5 it may just be a repeat of the Trophile test to determine change to mixed or dual tropic viruses
Case Discussion • Patient CB, 42 y/o, homosexual male • Current Regimen (05/31/06): CBV/TDF/EFV • Past Drugs: CBV/ IDV, CBV/NFV • CD4/VL • Date: 09/08/05 349/8,810 • Date: 03/07/06 192/10,300 • Date: 06/02/06 186/9,400 • Date: 09/18/06 92/6,610 • Date: 10/17/06 /12,000
Case Discussion • NRTI • M184V present (3TC/FTC resist, TDF hs) • Multiple TAMs • No K65R (TDF sens despite 41 & 215 mut) • NNRTI • No significant mutations • PI • 4 TPV assoc mut (intermediate response) • DRV sens
Case Discussion • Was the CBV/TDF/EFV regimen a reasonable one? • There has been no response to this therapy after 3 mos. • What should you do? • Any suggestions on a possible new regimen?
Discordance • Inaccurate genotype interpretation algorithm that does not account for novel or previously unknown mutation effect • Mixtures of wild type and resistant strains. Phenotype underestimates resistance • Variability in phenotypic susceptibility with specific mutations • Believe the genotype. Genotypic change may precede phenotypic resistance.
Clinical Implications • Is there evidence for sequencing of NRTIs? • Should the initial regimen be a boosted PI or a NNRTI? • Is 3TC = FTC as far as resistance is concerned?
Clinical Implications • Try to use at least 2 new potent agents to switch from a failing regimen. • The longer a failing regimen is continued, the more mutations accumulate. If there is no new agent, better to cont. the same regimen unless compelled to do otherwise. • Resistance is relative. 3TC cont. to have virological effect despite M184V mutation. Boosted PIs may have more of a response than an unboosted PI evidenced by a lower fold change.
Clinical Implications • NRTI • TAMs can prevent K65R mutation. K65R is associated with multiple NRTI resistance and TDF resistance. ? Add ZDV to failing regimen • Continue 3TC or FTC despite a M184V mutation (hypersusc. ZDV, TDF, D4T; RC) • NNRTI • DC NNRTI as soon as mutations develop. There is no virological or RC advantage.