Presentation on theme: "Resistance Testing – Where Do I Start? Iván Meléndez-Rivera, MD Fellow, American Academy of Family Physicians Assistant Professor of Family Medicine Ponce."— Presentation transcript:
Resistance Testing – Where Do I Start? Iván Meléndez-Rivera, MD Fellow, American Academy of Family Physicians Assistant Professor of Family Medicine Ponce School of Medicine, Ponce PR, USA Board Member, American Academy of HIV Medicine Faculty, Florida/Caribbean AETC Medical Director, Centro Ararat, Inc Ponce PR, USA
Disclosures of Financial Relationships This speaker has significant financial relationships with the following commercial entities to disclose: Advisory Board or Panel: Gilead, Merck Consultant: Bristol-Myers Squibb Grants/Research Support: Abbott, Boehringer, GlaxoSmithKline, Napo, Pfizer Speakers Bureau: Abbott, Boehringer, Bristol-Myers Squibb, GlaxoSmithKline, Merck, Pfizer, Tibotec This speaker will not discuss any off-label use or investigational product during the program. This slide set has been peer-reviewed to ensure that there are no conflicts of interest represented in the presentation.
Objectives Introduce major resistance mutations for each class of HIV therapy Order currently available resistance tests Present illustrative case(s) of diagnosing resistance pattern resulting in treatment plan adjustment
Definition of Antiviral Drug’s Resistance Presence of viral mutations that reduce drug susceptibility compared with the susceptibility of wild-type viruses. Can directly or indirectly interfere with a drug's activity. Should be distinguished from other causes of drug failure such as: –Non-adherence –Insufficient drug levels –Drug regimens with intrinsically weak antiviral activity.
Why does Resistance Develop? HIV can develop resistance quickly because: –HIV reverse transcriptase makes errors in matching bases when converting HIV RNA to DNA –HIV replicates at a rapid rate: 1 to 10 billion viral particles produced daily –In an untreated infected host, every possible mutation occurs at least once every 24 hours, some of which may impart drug resistance.
If you have developed resistance to a drug, does that mean that you are resistant to ALL the drugs in the same class? A.True B.False
When does resistance occur? Resistance occurs when the virus has an opportunity to replicate in the presence of sub- inhibitory concentrations of drugs. –Treatment with <3 active drugs –Inappropriate selection of drugs Pharmacokinetics or drug-drug interactions lead to inadequate drug exposures –Non-adherence to the treatment regimen Interruption of treatment (even for a few days) –Adding one drug to a failing regimen –Prolonging a failing regimen
Selective Pressures of Therapy Selection of resistant quasispecies Incomplete suppression Inadequate potency Inadequate drug levels Inadequate adherence Pre-existing resistance Viral load Time Drug-susceptible quasispecies Drug-resistant quasispecies Treatment begins
What is viral resistance? A continuum of reduced susceptibility of HIV to the inhibitory effects of drugs More Susceptible More Resistant The terms "drug resistance" and "reduced drug susceptibility" have similar meanings, provided that each term is viewed as representing a continuum between susceptible and highly resistant. Because antiretroviral drugs differ in their potencies, reductions in susceptibility must be related to the activity of the drug against wild-type viruses.
WILD VIRUS VS MUTANT VIRUS Why is it important to measure drug resistance?
What is wild-type virus? HIV virus that has not been exposed to drug therapy The predominant sequence of nucleotide bases in a heterogeneous mixture of virions It is the most fit form of HIV in the absence of drug
Identifying Mutations How do we identify a resistance mutation? M 184 M “184 is the codon position” M is the “wild-type” amino acid (AA)
Identifying Mutations How do we identify a resistance mutation? M 184 V “184 is the codon position” V is the mutant AA M is the “wild-type” amino acid
To the virus, mutations can be: What is the Impact of HIV Mutations? Decreasing the virus’ ability to survive and/or replicate (viral fitness) or may make the virus hypersusceptible to certain antiretrovirals (ARVs) No effect on virus function Changing the structure of the virus to evade antiretroviral treatment. These mutations may or may not affect viral fitness Johnson VA, et al. Topics in HIV Medicine. 2009;17:
HIV RNA mutant Drug resistant Single-base Mutations May Confer Antiretroviral Resistance 123 Position AA: 123 Mutant AA: Y Y Wild-type AA: X X 3 nucleotides specify an amino acid Single-base mutation HIV RNA wild type Hoffman C et al. HIV Medicine th ed. Paris, France: Flying Publisher AA=amino acid
Resistance Profile and Potential for Cross Resistance HIV RNA Mutant Drug A Drug B Drug C Drug D B cross resistance A & B & C cross resistance Adapted from: Paredes R, et al. Antiviral Res Jan;85(1): Region of HIV RNA associated with antiretroviral drug class specific resistance
How is resistance measured? By Genotype Amino acid substitution on chain By phenotype (IC 50 ) IC 50 –The minimum concentration of a drug needed to inhibit the growth of the virus by 50% in vitro IC 50 is analogous to MIC 90 –IC 90 is not sufficiently reproducible for routine clinical use The lower the IC 50, the more potent the drug
NRTI’s Signature Mutations Mutation Medication with decrease susceptibility M184V Lamivudine (3TC) and Emtricitabine (FTC) K65R Tenofovir (TDF) L74V Abacavir (ABC) and Didanosine (ddI) Y215F Zidovudine (ZDV) and Stavudine (d4T) T69ins or Q151M All except, Lamivudine (3TC) and Emtricitabine (FTC)
Not ALL mutations are bad M184V/I cause high-level resistance to 3TC and FTC and low-level resistance to ddI and ABC. M184V/I are not contraindications to continued treatment with 3TC or FTC because they increase susceptibility to AZT, TDF, and d4T and are associated with clinically significant decreased HIV-1 replication.
CCR5 Antagonist Maraviroc (MVC) Moore J, et al. Proc Natl Acad Sci USA. 2003;100: ; Yost R, et al. Am J Health-Syst Pharm. 2009;66: Moore J, et al. Proc Natl Acad Sci USA. 2003;100: ; Yost R, et al. Am J Health-Syst Pharm. 2009;66: Accessed Sept 3, 2012
CCR5 Antagonist Resistance is Associated with Amino Acid Changes in the V3 Loop of gp120* Variable pattern of Maraviroc resistance-associated amino acid substitutions No signature mutations have been identified Currently, there is no assay available to assess Maraviroc resistance Change Maraviroc-resistant Base Tip Stem Tip Base Deletion Insertion G/A *Substitutions outside the V3 loop of gp120 may also contribute to reduced susceptibility to Maraviroc Adapted from Data on file. ViiV Healthcare, RTP, NC.
CCR5-Resistant Virus Recognizes Drug- Bound Receptors Mutated gp120 recognizes CCR5 differently MVCres Virus MVCres gp120 Binding site NOT disrupted by maraviroc Entry Mori J, et al. 16 th IHIVDRW. Barbados, Abstract 10.
RECOMMENDATIONS FOR DRUG RESISTANCE TESTING
In which one of the following situations would HIV resistance testing NOT usually be recommended? A.Acute HIV infection, regardless of whether treatment is to be started B.Chronic HIV infection, at entry into care C.After discontinuation (>4 weeks) of ARVs D.Suboptimal suppression of viral load after starting antiretroviral therapy (ART)
Trends of Phenotypic 1-, 2-, and 3-Class Resistance NNRTI=non-nucleoside reverse transcriptase inhibitor; NRTI=nucleoside reverse transcriptase inhibitor; PI=protease inhibitor Class Resistance3-Class Resistance Class Resistance Resistant Samples (%) PI NNRTI NRTI PI and NRTI PI and NNRTI NRTI and NNRTI Adapted from Paquet A et al. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2011; Chicago, Illinois. Abstract H2-800.
Testing for Drug Resistance Before initiation of ART: –Transmitted resistance in 6-16% of HIV-infected patients –In absence of therapy, resistance mutations may decline over time and become undetectable by current assays, but may persist and cause treatment failure when ART is started –Identification of resistance mutations may optimize treatment outcomes –Resistance testing (genotype) recommended for all at entry to care –Recommended for all pregnant women Patients with virologic failure: –Perform while patient is taking ART, or ≤4 weeks after discontinuing therapy –Interpret in combination with history of ARV exposure and ARV adherence Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at display&resource=etres-6. Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres- display&resource=etres-6
Drug Resistance Testing: Recommendations RECOMMENDEDCOMMENT Acute HIV infection, regardless of whether treatment is to be started To determine if resistant virus was transmitted; guide treatment decisions. If treatment is deferred, consider repeat testing at time of ART initiation. Genotype preferred. Chronic HIV infection, at entry into care Transmitted drug-resistant virus is common in some areas; is more likely to be detected earlier in the course of HIV infection. If treatment is deferred, consider repeat testing at time of ART initiation. Genotype preferred to phenotype. Consider integrase genotypic resistance assay if integrase inhibitor resistance is a concern. Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6
RECOMMENDEDCOMMENT Virologic failure during ART To assist in selecting active drugs for a new regimen. Genotype preferred if patient on 1 st or 2 nd regimen; add phenotype if known or suspected complex drug resistance pattern. If virologic failure on integrase inhibitor or fusion inhibitor, consider specific genotypic testing for resistance to these to determine whether to continue them. (Coreceptor tropism assay if considering use of CCR5 antagonist; consider if virologic failure on CCR5 antagonist.) Suboptimal suppression of viral load after starting ART To assist in selecting active drugs for a new regimen. Drug Resistance Testing: Recommendations (2) Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6
RECOMMENDEDCOMMENT Pregnancy Recommended before initiation of ART or prophylaxis. Recommended for all on ART with detectable HIV RNA levels. Genotype usually preferred; add phenotype if complex drug resistance mutation pattern. Drug Resistance Testing: Recommendations (3) Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6
Drug Resistance Testing: Recommendations (4) NOT USUALLY RECOMMENDED COMMENT After discontinuation (>4 weeks) of ARVs Resistance mutations may become minor species in the absence of selective drug pressure Plasma HIV RNA <500 copies/mL Resistance assays cannot consistently be performed if HIV RNA is low Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6
AVAILABLE RESISTANCE TEST Accessed 06/OCT/12
Genotypic and Phenotypic Resistance Tests Translation into linear string of amino acids Folding into functional protein HIV RNA Phenotypic assays test this Genotypic assays test this
Available Tests NRTI, NNRTI AND PI –GENOTYPE –Virtual Phenotype –PHENOTYPE ENTRY INHIBITORS –PHENOTYPE INTEGRASE INHIBITORS –GENOTYPE –PHENOTYPE Co-Receptor Tropism Assay –RNA –DNA
General Limitations of Resistance Testing Lack of uniform quality controls across different laboratories High cost compared with other tests routinely done in HIV care Cannot be reliably performed with HIV RNA < copies/mL May not be able to detect minority populations of resistant virus if they account for < 20% of the sample—especially common after drug discontinuation Resistant strains that are in viral reservoirs also not detected
Reversion to Predominant Wild-Type Virus After Discontinuing ART Behrens C, Kindrick A, Harrington R. Antiretroviral Resistance Testing in the Management of HIV-Infected Patients [PowerPoint]. Northwest AIDS Education and Training Center; July Available at Accessed SEP 3, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-9 Illustration by David Spach, MD
Advantages and Disadvantages of Genotype Testing Advantages Rapid turnaround (1-2 wks) Less expensive than phenotyping Detection of mutations may precede phenotypic resistance Widely available More sensitive than phenotype for detecting mixtures of resistant and wild-type virus Disadvantages Indirect measure of resistance Relevance of some mutations unclear Unable to detect minority variants (< 20% to 25% of viral sample) Complex mutational patterns may be difficult to interpret
The Virtual Phenotype Genotype ProteaseRTHIV Access Data Genotype & Phenotype Data Virtual Phenotype Wild-type HIV Resistant HIV Behrens C, Kindrick A, Harrington R. Antiretroviral Resistance Testing in the Management of HIV-Infected Patients [PowerPoint]. Northwest AIDS Education and Training Center; July Available at Accessed SEP 3, 2012.http://aidsetc.org/aidsetc?page=etres-display&resource=etres-9 Illustration by David Spach, MD
Advantages and Disadvantages of “Virtual” Phenotype Testing Advantages Similar advantages to genotype (turnaround time, cost, sensitivity) Defines resistance based on database of in vivo responses in treated patients Uses 2 clinical cutoffs (CCO) to define spectrum of resistance –CCO1: value below which response expected to be comparable to wild type –CCO2: value above which most virologic response would be lost Indicates which drugs have partial activity Disadvantages Is an estimated phenotype based on the patient’s genotype, not an actual measured phenotype Reliability will depend on the accuracy of the genotype Available only from 1 vendor More expensive than genotype alone Methodology of linking genotype to phenotypic database not intuitively obvious—uses a proprietary “virtual phenotype linear regression
Drug Susceptibility Testing Involves culturing a fixed inoculum of HIV-1 in the presence of serial dilutions of an inhibitory drug. –The concentrations of drug required to inhibit virus replication by 50% (IC50) or 90% (IC90) Drug susceptibility results depend on: –inoculum size of virus tested –cells used for virus replication –the means of assessing virus replication. https://www.23andme.com/health/Resistance-to-HIV-AIDS/https://www.23andme.com/health/Resistance-to-HIV-AIDS/ Accessed 06/OCT/12
Advantages and Disadvantages of Phenotype Testing Advantages: Provides direct and quantitative measure of resistance Methodology can be applied to any antiretroviral agent, including new drugs, for which genotypic correlates of resistance are unclear Uses 2 clinical cutoffs (CCO) derived from clinical cohorts to define spectrum of resistance –CCO1: value below which reduced virologic response is likely –CCO2: value above which a virologic response is unlikely Indicates which drugs have partial activity Can assess interactions among mutations Accurate with non-B HIV subtypes Disadvantages: Susceptibility cutoffs not standardized between assays Clinical cutoffs not defined for some agents May be unable to detect minority variants for some mutations (< 20% to 25% of viral sample) Complex technology with longer turnaround (~ 3 wks) More expensive than genotyping
Integrase Phenotype Phenotypic integrase resistance assay is commercially available –Amplification threshold: HIV-1 RNA > 500 copies/mL –Biological cutoff for raltegravir is fold change (FC) > 1.5 –Clinical cutoff not yet determined –Report does not detail genotypic mutations High assay accuracy demonstrated by IC 50 fold change values reported for site-directed mutants Fransen S, et al. ICAAC/IDSA Abstract Accessed SEP 3, 2012
Typical HIV Co-Receptor Usage Patterns R5 Viruses Use only the CCR5 co-receptor Most prevalent in early disease Predominate throughout infection X4 Viruses Use only the CXCR4 co-receptor Emerge in later disease Associated with accelerated CD4+ cell decline and disease progression Dual-Tropic Viruses Use either the CCR5 or the CXCR4 co-receptor Mixed-Tropic Virus Population Tsibris AMN, Kuritzkes DR. Annu Rev Med. 2007;58:
Light generated CCR5 Use R5 Virus No light generated No CXCR4 Use Not an X4 Virus Demonstration of R5 virus CCR5 CXCR4 Virus
Light is generated on both CCR5 and CXCR4 cell lines This is a DUAL virus Demonstration of dual virus CCR5 CXCR4 Virus
This population shows CCR5 AND CXCR4 co-receptor use This is a mixed population Demonstration of mixed virus population CCR5 CXCR4
Virologic Failure With Resistance Virologic failure is defined as the inability of ARV regimen to achieve virologic suppression or occurrence of virologic rebound 1 A confirmed viral load >200 copies/mL can be considered virologic failure 2 1.AIDSinfo. Glossary of HIV/AIDS-related terms. 7th ed Available at Accessed November 18, DHHS Guidelines. Accessed November 18, Clavel F et al. N Engl J Med. 2004;350: Based on Clavel et al. 3
The follow factors leads to developing drug- resistance EXCEPT…? A.Poor drug absorption B.Poor adherence C.High drug levels D.Pre-existing resistance E.Interactions with other drugs, supplements or recreational drugs
Case 1: 42 y/o AA female Dx in 2006 Current Medical Hx –Diabetes –Hyperlipidemia –Hypertension Baseline Labs (1/06) –VL: 230,000 –CD4: 320 cells/mm3 Baseline Genotype –Wild-Type Pt request a once a day pill ARV and start 5/06 on EFV/TDF/ETC FDC Viral Load quickly becomes undetectable and CD4+ counts raises over time
Case 1: 42 y/o AA female DateVLCD4 11/06< /07< /08< /09< /0930, Patient experiences virologic failure on Nov 2009 while taking EVF/TDF/FTC Pt. reports occasional poor adherence due to occasional sleep disturbances and concerns about lipid problems
Case 1: 42 y/o AA female What would be your next step? A.Enforce patient adherence and continue with same regimen. B.Perform Genotype test and keep patient on the same regiment until genotype results. C.Perform a tropism and Phenotype test and remove patient from medicines because there are toxic. D.Change patient regiment without any resistance test performed.
Case 1: 42 y/o AA female With this genotype result, is Efavirenz still available? A.Yes B.No
Case 1: 42 y/o AA female January 2010 patient was place on EPZ/ATV Labs 04/10 –CD4: 700 cells/mm 3 –VL: <50 copies Pt. continue with non-detectable (ND) viral load until 04/12 –CD4 800 cells/mm 3 –VL: 15,000 copies Resistance testing result. –NRTI’s: M184V –NNTRI’s: K103N –PI’s: I50L
Based on new resistance pattern (NRTI’s: M184V NNTRI’s: K103N; PI’s: I50L) which statement is true? A.All NNRT’s are available to use B.Atazanavir still available for use C.3Tc is a full active drug D.Patient loss susceptibility to rilpivirine E.Keeping patient on efavirenz will increase the amount of NNRTI mutations and reduce the susceptibility to all other NRTI’s
Case 2 A 45 year-old man who is highly treatment- experienced with antiretroviral therapy has virologic failure on tenofovir/emtricitabine and lopinavir/ritonavir. His enhanced Trofile assay reveals a dual/mixed- tropic virus. His current genotype reveals reverse transcriptase mutations M41l, V90I, K103N, M184V, L210W, T215Y and protease inhibitors mutations V32I, I47V, I54A, V82L, I84V, and L90M. In addition, a 2006 genotype also reveled the envelope glycoprotein 41 mutation G36D.
The next BEST antiretroviral regimen for this patient would include which of the following? A.Enfuvirtide B.Maraviroc C.Etravirine D.Ritonavir-boosted darunavir E.Ritonavir-boosted tipranavir
Bonus Case A 21 years-old man who acquired HIV through vertical transmission presents for routine care. Recent genotypic analysis reveals a 69 insertion complex and K103N reverse transcriptase mutations and L10I, K20M, M46I, G48V, I50V, N88S and L90M protease mutations.
Which regimen would be the BEST for him? A.Raltegravir, etravirine, and atazanavir/ritonavir B.Raltegravir, etravirine, and darunavir/ritonavir C.Raltegravir, etravirine, and fosamprenavir/ritonavir D.Raltegravir, etravirine, and indinavir/ritonavir E.Raltegravir, etravirine, and maraviroc
Reference https://www.iasusa.org/content/hiv-drug-resistance-mutations Behrens C, Kindrick A, Harrington R. Antiretroviral Resistance Testing in the Management of HIV-Infected Patients [PowerPoint]. Northwest AIDS Education and Training Center; July Available at display&resource=etres-9. Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres- display&resource=etres-9 Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March Available at display&resource=etres-6. Accessed Sept 03, 2012.http://aidsetc.org/aidsetc?page=etres- display&resource=etres-6 between-treatment-and-prevention. Accessed Sept 03, 2012http://www.targethiv.org/library/hiv-resistance-intersection- between-treatment-and-prevention