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Resistance Testing – Where Do I Start?

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1 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

2 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.

3 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 Present objectives for the module.

4 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.

5 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.

6 If you have developed resistance to a drug, does that mean that you are resistant to ALL the drugs in the same class? True False False

7 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

8 Selective Pressures of Therapy
Drug-susceptible quasispecies Drug-resistant quasispecies Treatment begins Selection of resistant quasispecies Viral load Incomplete suppression Inadequate potency Inadequate drug levels Inadequate adherence Pre-existing resistance Time

9 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.

10 Wild Virus vs Mutant virus
Why is it important to measure drug resistance? Wild Virus vs Mutant virus

11 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

12 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)

13 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

14 What is the Impact of HIV Mutations?
To the virus, mutations can be: 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:

15 Single-base Mutations May Confer Antiretroviral Resistance
3 nucleotides specify an amino acid HIV RNA wild type Single-base mutation HIV RNA mutant Drug resistant SPEAKING POINTS: Sets of 3 nucleotides in the HIV RNA, which code for a single amino acid of a protein are referred to as a codon Single-base mutations to the codon may result in coding for a different amino acid, which may result in drug resistance Such resistance mutations occur in regions of the HIV RNA specific to the antiretroviral drug class (eg, PI, NNRTI, NRTI) and are named according to: A letter to the left that designates the amino acid in the wild type virus A number in the center that designates the location of the amino acid impacted by the mutation A letter to the right that designates the amino acid in the drug-resistant virus due to the mutation Note to Speaker: The components of this slide build in the following order: HIV RNA, wild type Single base mutation HIV RNA drug resistant mutant AA position, 123 AA wild type, X AA mutation, Y 123 X Y Wild-type AA: X Position AA: 123 Mutant AA: Y AA=amino acid Hoffman C et al. HIV Medicine th ed. Paris, France: Flying Publisher

16 Resistance Profile and Potential for Cross Resistance
Region of HIV RNA associated with antiretroviral drug class specific resistance HIV RNA Mutant Drug A Drug B Drug C SPEAKING POINTS: Single-point mutations associated with resistance to a given antiretroviral drug may also be associated with cross resistance to other antiretroviral drugs This schematic shows how mutations that cause resistance to hypothetical antiretroviral drugs A and B can have cross resistance to drugs C and D Drugs A and B share no cross-resistance mutations and are therefore unlikely to have any impact on efficacy when used sequentially, following virologic failure Drug C can similarly be used sequentially after Drug A, but will be cross resistant to Drug B and likely demonstrate reduced efficacy if used sequentially Drug D is cross resistant to Drugs A and B and C Note to Speaker: The components of this slide build in the following order: Region of HIV RNA associated with ARV drug class specific resistance Drug A associated resistance mutations Drug B associated resistance mutations Drug C associated resistance mutations Highlight cross resistance of Drug C with Drug B Drug D associated resistance mutations Highlight cross resistance of Drug D with Drugs A, B, C B cross resistance Drug D A & B & C cross resistance Adapted from: Paredes R, et al. Antiviral Res Jan;85(1):

17 How is resistance measured?
By Genotype Amino acid substitution on chain By phenotype (IC50) IC50 The minimum concentration of a drug needed to inhibit the growth of the virus by 50% in vitro IC50 is analogous to MIC90 IC90 is not sufficiently reproducible for routine clinical use The lower the IC50, the more potent the drug

18 Resistance mutations per HIV class therapy

19 Available Antiretroviral Agents
Nucleoside Reverse Transcriptase Inhibitors (RTIs) Zidovudine (ZDV) Didanosine (ddI) Zalcitabine (ddC) Stavudine (d4T) Lamivudine (3TC) Abacavir (ABC) Emtricitabine (FTC) Tenofovir DF (TDF) Nonnucleos(t)ide RTIs Nevirapine (NVP) Delavirdine (DLV) Efavirenz (EFV) Etravirine (ETR) Rilpivirine (RPV) Protease Inhibitors Saquinavir (SQV) Ritonavir (RTV) Indinavir (IDV) Nelfinavir (NFV) Amprenavir (APV) Lopinavir/r (LPV/r) Atazanavir (ATV) Fosamprenavir (Fos-APV) Tipranavir (TPV) Darunavir (DRV) Integrase Inhibitors Raltegravir (RAL) Elvitegravir(ELV)* Dolutegravir (DTG)* Boosters Ritonavir (RTV) Cobicistat (cobi) Fusion Inhibitor Enfuvirtide (T-20) CCR5 Antagonist Maraviroc (MVC) * In expanded access August 28, 2012

20 Nucleoside Reverse Transcriptase Inhibitors (NRTI’s)
Zidovudine (ZDV) Didanosine (ddI) Stavudine (d4T) Lamivudine (3TC) Abacavir (ABC) Tenofovir (TDF) Emtricitabine (FTC) Structure of a covalently trapped catalytic complex of HIV-1 RT published by Huang H et al, Science Accessed Sept 3, 2012

21 NRTI’s Resistance Mutations
CLASSIFICATION OF MAJOR DRUG RESISTANCE MUTATIONS: NRTI-resistance mutations are classified into those that inhibit NRTI incorporation into the HIV-1 primer DNA strand ('Discriminatory' Mutations) and those that promote excision of chain-terminating NRTIs via ATP-mediated pyrophosphorolysis ('Excision' Mutations). Excision mutations are usually called TAMs (Thymidine Analog Mutations) because they are selected by AZT and d4T. Two mutation complexes are associated with high-levels of multi-NNRTI resistance: (i) A 2-amino acid insertion at codon 69, which nearly always occurs in combination with multiple TAMS; and (ii) Q151M which usually occurs with several otherwise uncommon accessory mutations: A62V, V75I, F77L, and F116Y. Red: Primary Mutations to confer resistance Gray: Secondary Mutations ***: Increase susceptibility Adapted from: Accessed Sept 3, 2012

22 NRTI’s Signature Mutations
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)

23 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.

24 Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI’s)
Nevirapine (NVP) Efavirenz (EFV) Etravirine (ETR) Rilpivirine (RPV) The NNRTIs inhibit HIV-1 RT allosterically by binding to a hydrophobic pocket about 10 angstroms under the active site. The above figure shows the 5’ polymerase-coding region of HIV-1 RT encompassing the fingers, palm, and thumb subdomains of the p66 subunit bound to the NNRTI nevirapine (Kohlstaedt) PDB #. Positions associated with NNRTI resistance that make up the central NNRTI binding pocket are shown: L100, K101, K103, V106, V108, V179, Y181, Y188, G190, F227. Additional positions that make up the pocket include E138 which is contributed by the p51 subunit) not shown and M230, L234, P236, K238, and L318 which form part of an extended pocket. Additional accessory NNRTI-resistance abutting positions that form the NNRTI binding pocket include A98 and P225. Non-nucleoside RT inhibitor (NNRTI)-resistance mutations Accessed Sept 3, 2012

25 NRTI’s Resistance Mutations
MAJOR MUTATIONS: Mutations that are associated with high levels of phenotpyic resistance or clinical evidence for reduced virological response. Those in bold red are associated with the highest levels of phenotypic resistance or with the strongest clinical evidence for reduced virological response. Mutations in bold grey are intermediate to those in bold red vs. those in plain text. The mutations shown at positions 103, 106, 181, 188, and 190 are generally primary because they generally occur before the mutations L100I and K101EP (Bacheler 2000, Koval 2006, Reuman 2010). M230L is uncommon and may occur alone or with other mutations (Huang 2000, Vingerhoets 2005, RPV Prescribing Information) Position 138 is included because E138K is the most commonly occurring mutation to develop in patients receiving RPV (Rimsky 2011). V106M is more common than V106A in subtype C viruses. In contrast to V106A, V106M causes high-level EFV resistance (Brenner 2003, Grossman 2004, Cane 2004) Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: Accessed Sept 3, 2012

26 NNRTI’s Signature Mutations
Medication with decrease susceptibility K103N Efavirenz (EFV) and Nevirapine (NVP) Y181I/V Etravirine (ETR) and Rilpivirine (RPV) Y188L Rilpivirine (RPV) K101P ALL NNRTI

27 Protease Inhibitors (PI’s)
Saquinavir (SQV) Ritonavir (RTV) Indinavir (IDV) Nelfinavir (NFV) Lopinavir/r (LPV/r) Atazanavir (ATV) Fosamprenavir (FPV) Tipranavir (TPV) Darunavir (DRV) Major protease inhibitor (PI)-resistance mutations Accessed Sept 3, 2012

28 PI’s Resistance Mutations
MAJOR MUTATIONS: Mutations that are associated with high levels of phenotypic resistance or clinical evidence for reduced virological response. Those in bold red are associated with the highest levels of phenotypic resistance or with the strongest clinical evidence for reduced virological response. Underlined mutations are potential contraindications to the use of the indicated PIs when other alternatives are available. Positions 30, 32, 46, 47, 48, 50, 54, 82, and 84 are in or near (positions 46, 47, and 54) the substrate cleft. (Jacobsen 95; Partaledis 95; Condra 96; Carrillo 98; Patick 98; Schapiro 99; Atkinson 00; Gong 00; Scott 00; Maguire 02a; Parkin 03; Colonno 04; Doyon 05; Kagan 05; Vermeiren 07; De Meyer 08a; Marcelin 08; Sista 08; Van Marck 09; Llibre 10; Rhee 10; Schapiro 10). Positions 76, 88, and 90 interfere with PI susceptibility indirectly (Patick 98; Atkinson 00; Mahalingam 01; Malan 08; Rhee 10; Louis 11). Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: Accessed Sept 3, 2012

29 PI’s Signature Mutations
Medication with decrease susceptibility D30N Nelfinavir (NFV) I50L Atazanavir (ATV) I47V ALL except Saquinavir (SQV) I54V ALL except Darunavir (DRV) I84V

30 Integrase Inhibitors (INI)
Raltegravir (RAL) Elvitegravir(ELV) Dolutegravir(DTG)* * Investigational Accessed 06/OCT/12

31 INI Resistance Mutations
MAJOR MUTATIONS: Mutations that cause high-levels of decreased susceptibility of strong clinical evidence for contributing to virological failure. Those in bold red are associated with the highest levels of phenotypic resistance or with the strongest clinical evidence for reduced virological response. Mutations in bold grey are intermediate to those in bold red vs. those in plain text. Although G140SA do not decrease INI susceptibility, they occur almost uniformly in combination with the highly potent Q148 INI-resistance mutations. The major mutations shown here have been selected in persons receiving RAL (Cooper 2008, Malet 2008, Sichtig 2009, Wittkp 2009, Canducci 2009, Baldanti 2010, Reuman 2010, Hatano 2010), or ELV (McColl HIVDRW 2007, Zolopa JID 2010) and characterized for in vitro susceptibility (Shimura 2007, Goethals 2008, Kobayashi 2008, Fransen 2009, Jones 2009, da Silva 2009, Reuman 2010, Goethals 2010, Kobayashi 2011). Mutations in bold red are associated with >5-10 fold decreased susceptibility. Although E138 and G140 mutations alone do not decrease INI susceptibility that greatly contributed to decreased susceptibility in combination with Q148 mutations. Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: Accessed Sept 3, 2012

32 INI Signature Mutations
Medication with decrease susceptibility Q148H/K/R Raltegravir (RAL) and Elvitegravir(ELV) * In expanded access

33 Fusion Inhibitors (FI)
Enfuvirtide (T-20) Accessed Sept 3, 2012

34 Fusion Inhibitor Resistance Mutations
Gray: Intermediate level of resistance Adapted from: Accessed Sept 3, 2012

35 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: Accessed Sept 3, 2012

36 CCR5 Antagonist Resistance is Associated with Amino Acid Changes in the V3 Loop of gp120*
G/A 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 Tip Tip Stem Stem Base Base *Substitutions outside the V3 loop of gp120 may also contribute to reduced susceptibility to Maraviroc Change Maraviroc-resistant Insertion Deletion Adapted from Data on file. ViiV Healthcare, RTP, NC.

37 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. 16th IHIVDRW. Barbados, 2007. Abstract 10.

38 Recommendations for drug resistance testing

39 In which one of the following situations would HIV resistance testing NOT usually be recommended?
Acute HIV infection, regardless of whether treatment is to be started Chronic HIV infection, at entry into care After discontinuation (>4 weeks) of ARVs Suboptimal suppression of viral load after starting antiretroviral therapy (ART) Answer: C

40 Trends of Phenotypic 1-, 2-, and 3-Class Resistance
60 60 60 PI PI and NRTI NNRTI PI and NNRTI NRTI NRTI and NNRTI 50 50 50 40 40 40 Resistant Samples (%) 30 30 30 20 20 20 10 10 10 Speaker Notes: The study examined 68,587 resistant samples submitted for routine phenotypic and genotypic patient testing that showed phenotypic resistance to at least one drug within the ARV classes of PIs, NRTIs, and NNRTIs. Each bar represents the percentage of samples that exhibited reduced phenotypic susceptibility to either one, two, or three drug classes (NRTI, NNRTI, PI) compared to the sum total of all samples that exhibited reduced susceptibility to any drug class (NRTI, NNRTI, or PI). 2003 2004 2005 2006 2007 2008 2009 2010 2003 2004 2005 2006 2007 2008 2009 2010 2003 2004 2005 2006 2007 2008 2009 2010 NNRTI=non-nucleoside reverse transcriptase inhibitor; NRTI=nucleoside reverse transcriptase inhibitor; PI=protease inhibitor. Adapted from Paquet A et al. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2011; Chicago, Illinois. Abstract H2-800. ARV=antiretroviral; NNRTI=non-nucleoside reverse transcriptase inhibitor; NRTI=nucleoside reverse transcriptase inhibitor; PI=protease inhibitor. Paquet A et al. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2011; Chicago, Illinois. Abstract H2-800.

41 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 Accessed Sept 03, 2012.

42 Drug Resistance Testing: Recommendations
RECOMMENDED COMMENT 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.

43 Drug Resistance Testing: Recommendations(2)
RECOMMENDED COMMENT Virologic failure during ART To assist in selecting active drugs for a new regimen. Genotype preferred if patient on 1st or 2nd 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 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.

44 Drug Resistance Testing: Recommendations(3)
RECOMMENDED COMMENT 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. 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.

45 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.

46 Available Resistance test
Accessed 06/OCT/12

47 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

48 Available Tests NRTI, NNRTI AND PI ENTRY INHIBITORS
GENOTYPE Virtual Phenotype PHENOTYPE ENTRY INHIBITORS INTEGRASE INHIBITORS Co-Receptor Tropism Assay RNA DNA

49 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 Resistance studies share certain limitations, including the lack of uniform quality controls across different laboratories, a high cost compared with other tests routinely done in HIV care, the fact that the tests cannot be reliably performed with an HIV RNA count less than copies/mL, and that they may not be able to detect minority populations which account for < 20% of the sample. In addition, resistance tests cannot detect HIV strains in unsampled viral reservoirs.

50 Reversion to Predominant Wild-Type Virus After Discontinuing ART
It is clear, however, that patients who develop resistance while on ART, then discontinue ART, tend to experience a reversion of their virus to wild-type (though again, NNRTI resistance mutations may persist as these are generally not associated with a significant loss in viral fitness). 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. Illustration by David Spach, MD

51 genotype

52 Advantages and Disadvantages of Genotype Testing
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 Each type of resistance testing has advantages and disadvantages. Advantages of the genotype test include rapid turnaround time, lower cost, potential detection of mutations prior to phenotypic resistance, wide availability, and higher sensitivity than phenotype testing for detecting mixtures of resistant and wild‑type virus. One disadvantage of genotype testing is that it is an indirect measure of resistance. Another is the difficulty in determining the relevance of certain mutations without phenotypic correlates. And, as with all resistance tests, genotype testing is unable to detect minority variants comprising less than 20% to 25% of viral samples. The most significant disadvantage of genotype testing is the difficulty of interpretation when there are many mutations or highly complex patterns.

53 Genotype Report 53

54 Genotype & Phenotype Data
The Virtual Phenotype Genotype Access Data HIV RT Protease Genotype & Phenotype Data Virtual Phenotype Wild-type HIV The virtual phenotype uses a combination of the genotypic and phenotypic techologies. Virologic, the company that offers this test, has a database containing over ten thousand samples of patient serum on which both genotypic and phenotypic assays were performed. When one submits a sample to Virologic for a virtual phenotype, a genotype is performed. From the database of paired genotype and phenotype results, patient samples having the same genotype as that obtained on the sample you submitted are drawn. The corresponding phenotypes for those samples are then analyzed, averaged, and reported to the ordering clinician as a ‘virtual phenotype’, i.e. the phenotype that might be expected if one actually performed a phenotypic assay on that sample. Potential advantages of this approach include the lower cost and quicker turn around time associated with the genotype, but the easy-to-interpret results of a phenotype. However, one should keep in mind that a phenotypic assay on the sample you submit is never actually performed, and that the clinical utility of this approach is not as clearly established as it is for the genotypic assay. 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. Illustration by David Spach, MD 1

55 Advantages and Disadvantages of “Virtual” Phenotype Testing
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 CCO, clinical cutoff. “Virtual” phenotype testing has similar advantages to genotype testing in terms of turnaround time, cost, and sensitivity. Again, 2 different CCOs define the spectrum of resistance: CCO1 is the value below which response is expected to be comparable to wild‑type virus. CCO2 is the value above which most virologic activity would be lost. As a result, the virtual phenotype test can also identify drugs with partial activity. Disadvantages of virtual phenotyping include availability from a single vendor and its higher cost compared with genotype testing. Also, the methodology for linking genotype results to the phenotypic database uses a proprietary “virtual phenotype linear regression model engine,” the derivation of which is not always intuitively obvious to the user.

56 Phenotype

57 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. Drug susceptibility assays are not designed to determine the exact amount of drug required to inhibit virus replication in vivo but rather to identify differences in the drug concentration required to inhibit a fixed inoculum of a virus relative to the concentrations required to inhibit wild-type viruses. https://www.23andme.com/health/Resistance-to-HIV-AIDS/ Accessed 06/OCT/12

58 Advantages and Disadvantages of Phenotype Testing
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 Phenotype testing is significantly different from genotype testing. Its primary advantage is that it provides a direct and quantitative measure of resistance and, as such, the methodology can be applied to any antiretroviral agent, including new drugs, for which genotype correlates of resistance are unclear. Two clinical cutoffs (CCO) derived from clinical cohorts are used to define the spectrum of resistance. The lower cutoff, CCO1, is the fold change in susceptibility below which reduced virologic response is likely, whereas the higher cutoff, CCO2, is the fold change above which a virologic response is unlikely. In addition, phenotype resistance testing can assess interactions among mutations and has been shown to be accurate in non‑B clade HIV subtypes. Disadvantages of phenotype testing include variability between assays and that not all drugs have defined susceptibility cutoffs. As with genotype testing, phenotype testing may be unable to detect minority variants of some mutations. This is a complex technology with a turnaround time of approximately 3 weeks. It is also considerably more expensive than genotyping—approximately twice as much in the United States.

59 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 IC50 fold change values reported for site-directed mutants FC, fold change; RAL, raltegravir. For more information, go online to: Fransen S, et al. ICAAC/IDSA Abstract Accessed SEP 3, 2012

60 Tropisms test

61 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 Slide: Typical HIV Co-Receptor Usage Patterns HIV-1 viruses can be characterized into four broad classifications based on their tropism status.1 CCR5-tropic viruses or virus populations can use only the CCR5 chemokine co-receptor to infect CD4 cells. This is the most prevalent in early disease and predominates throughout HIV disease.1 CXCR4-tropic viruses or virus populations can use only the CXCR4 chemokine co-receptor to infect CD4+ cells. These populations of virus usually emerge in later stages of disease and are associated with an accelerated CD4 cell decline and disease progression.1 Dual (D)-tropic viruses or virus populations can use either the CCR5 or CXCR4 co-receptors to infect CD4+ cells.1 Mixed (M)-tropic virus populations may contain various combinations of R5 virus, X4 virus, and/or dual-tropic viruses.1 Reference Tsibris AMN, Kuritzkes DR. Chemokine antagonists as therapeutics: focus on HIV-1. Annu Rev Med. 2007;58: 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:

62 No light generated No CXCR4 Use Light generated CCR5 Use
Demonstration of R5 virus No light generated No CXCR4 Use Not an X4 Virus Virus Virus Light generated CCR5 Use R5 Virus Virus Example of CCR5 virus entering R5 cell line and generating RLU (relative light units) in the Trofile assay CCR5 CXCR4

63

64 Demonstration of dual virus
Light is generated on both CCR5 and CXCR4 cell lines This is a DUAL virus Virus Virus CCR5 CXCR4 Example of CCR5 virus entering R5 cell line and generating RLU (relative light units) in the Trofile assay

65 Demonstration of mixed virus population
This population shows CCR5 AND CXCR4 co-receptor use This is a mixed population CCR5 CXCR4 Example of CCR5 virus entering R5 cell line and generating RLU (relative light units) in the Trofile assay

66

67 Virologic Failure With Resistance
Virologic failure is defined as the inability of ARV regimen to achieve virologic suppression or occurrence of virologic rebound1 A confirmed viral load >200 copies/mL can be considered virologic failure2 Resistant Virus Viral Load Time on Antiviral Therapy Speaker Notes: Virologic failure is defined as the inability of ARV regimen to achieve virologic suppression or occurrence of virologic rebound1 The DHHS Guidelines consider a confirmed viral load >200 copies/mL as a virologic failure2 Virologic suppression to <50 copies/mL is a marker for the suppression of viral replication3 Based on Clavel et al.3 AIDSinfo. Glossary of HIV/AIDS-related terms. 7th ed Available at Accessed November 18, 2011 DHHS Guidelines. Accessed November 18, 2011. Clavel F et al. N Engl J Med. 2004;350: ARV=antiretroviral; DHHS=Department of Health and Human Services. 1. AIDSinfo. Glossary of HIV/AIDS-related terms. 7th ed Available at Accessed November 18, 2011 2. DHHS Guidelines. GL.pdf. Accessed November 18, 2011. 3. Podsadecki TJ et al. J Infect Dis. 2007;196:

68

69 Case presentation

70 The follow factors leads to developing drug-resistance EXCEPT…?
Poor drug absorption Poor adherence High drug levels Pre-existing resistance Interactions with other drugs, supplements or recreational drugs Answer: C

71 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

72 Case 1: 42 y/o AA female Date VL CD4 11/06 <400 400 05/07 <50 480 05/08 600 05/09 680 11/09 30,000 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

73 Case 1: 42 y/o AA female What would be your next step?
Enforce patient adherence and continue with same regimen. Perform Genotype test and keep patient on the same regiment until genotype results. Perform a tropism and Phenotype test and remove patient from medicines because there are toxic. Change patient regiment without any resistance test performed.

74 Case 1: 42 y/o AA female With this genotype result, is Efavirenz still available?
Answer: B Yes No

75 Case 1: 42 y/o AA female Resistance testing result.
January 2010 patient was place on EPZ/ATV Labs 04/10 CD4: 700 cells/mm3 VL: <50 copies Pt. continue with non-detectable (ND) viral load until 04/12 CD4 800 cells/mm3 VL: 15,000 copies Resistance testing result. NRTI’s: M184V NNTRI’s: K103N PI’s: I50L

76 Based on new resistance pattern (NRTI’s: M184V NNTRI’s: K103N; PI’s: I50L) which statement is true?
All NNRT’s are available to use Atazanavir still available for use 3Tc is a full active drug Patient loss susceptibility to rilpivirine Keeping patient on efavirenz will increase the amount of NNRTI mutations and reduce the susceptibility to all other NRTI’s Answer: E

77 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.

78 Ritonavir-boosted darunavir Ritonavir-boosted tipranavir
The next BEST antiretroviral regimen for this patient would include which of the following? Enfuvirtide Maraviroc Etravirine Ritonavir-boosted darunavir Ritonavir-boosted tipranavir Response: C

79 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.

80 Which regimen would be the BEST for him?
Raltegravir, etravirine, and atazanavir/ritonavir Raltegravir, etravirine, and darunavir/ritonavir Raltegravir, etravirine, and fosamprenavir/ritonavir Raltegravir, etravirine, and indinavir/ritonavir Raltegravir, etravirine, and maraviroc Response: B

81 Reference http://hivdb.stanford.edu/
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 Accessed Sept 03, 2012. 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. Accessed Sept 03, 2012


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