Assistant Professor of Medicine, Division of Infectious Diseases University of Texas, Southwestern Medical Center at Da

2. Disclosure of Financial Relationships This speaker has the following financial relationships with commercial entities to disclose: Speaker�s Bureau - Pfizer Use this slide if you have no significant financial relationships with any commercial entities. If you use this slide, please delete slide 3.Use this slide if you have no significant financial relationships with any commercial entities. If you use this slide, please delete slide 3.

3. Course Objectives At the conclusion of this conference, the participant will be able to: Understand basic principles of virus growth and evolution Appreciate advantages and limitations of different types of resistance tests Recognize the effect of specific mutation patterns on drug susceptibility Interpret reports from various resistance tests

4. Course Objectives, cont. Evaluate the possible uses for resistance testing in patient management Compare the advantages and disadvantages of each type of test Understand supporting clinical data relevant to RT usage

5. Resistance Definition Resistance refers to the reduced susceptibility of a patient�s viral isolate to suppression by an antiviral drug This is a relative phenomenon, dependent on the levels of drug that are achieved and host factors

6. Increase in HIV Drug Resistance Mutations

7. Viral Resistance is the Outcome of Viral Replication, Mutations and Selection Pressure

9. Guidelines: Treatment-Naive Patients

10. HIV Drug Resistance Broadly defined as any change that improves viral replication in the presence of an antiretroviral (ARV) drug Measured by Genotype Phenotype VircoTYPE Clinical/Virologic response HIV resistance is broadly defined as any change that improves viral replication in the presence of an antiretroviral drug Changes are due to an altered phenotype resulting from a change in the genotype, or in other words, a change in the sequence of the virus. Such changes occur primarily in the reverse transcriptase and protease genes. Changes in the virus are usually compared against a wild-type virus, a virus that does not have any drug-associated mutations. HIV resistance is rarely absolute; rather, the susceptibility of the virus for a particular drug or combination of drugs is reduced but not complete Resistance can be measured in a test tube (in vitro) using laboratory virus strains with or without HIV drug-associated mutations, or it can be measured in a subject (in vivo) by obtaining a blood sample containing virus and doing a resistance testHIV resistance is broadly defined as any change that improves viral replication in the presence of an antiretroviral drug Changes are due to an altered phenotype resulting from a change in the genotype, or in other words, a change in the sequence of the virus. Such changes occur primarily in the reverse transcriptase and protease genes. Changes in the virus are usually compared against a wild-type virus, a virus that does not have any drug-associated mutations. HIV resistance is rarely absolute; rather, the susceptibility of the virus for a particular drug or combination of drugs is reduced but not complete Resistance can be measured in a test tube (in vitro) using laboratory virus strains with or without HIV drug-associated mutations, or it can be measured in a subject (in vivo) by obtaining a blood sample containing virus and doing a resistance test

11. Genotype Refers to the sequence of bases (A,C,T,G) coding for amino acids that comprise viral proteins Typically expressed as the coded amino acid and position (eg, M184) If mutated, the change is indicated after the position (eg, M184V)

12. HIV Resistance and Mutation Definitions Primary mutations are selected early and usually decrease drug susceptibility Secondary mutations are selected after primary mutations and may have a limited effect on drug susceptibility Multidrug resistance mutations can contribute to resistance or decreased susceptibility to many or all drugs in a single class Changes are due to a change in the sequence of the virus Resistance can be measured either in vitro or in vivo After primary mutations are detected, they are studied in vitro to determine if they confer drug resistance If secondary mutations alone are studied in the laboratory, drug resistance will not be observed A few known mutations confer drug resistance to the entire class of reverse transcriptase inhibitors or protease inhibitors Changes are due to a change in the sequence of the virus Resistance can be measured either in vitro or in vivo After primary mutations are detected, they are studied in vitro to determine if they confer drug resistance If secondary mutations alone are studied in the laboratory, drug resistance will not be observed A few known mutations confer drug resistance to the entire class of reverse transcriptase inhibitors or protease inhibitors

13. Anonymous questionnaire at a meeting in 2001 128/421 providers, 100 had used resistance testingList of 16 resistance associated point mutations

14. Knowledge of Specific Mutations

15. Overview of Current HIV Resistance Testing Technologies Genotypic testing Capillary sequencing (ABI) or other dideoxy method High level of concordance between two commercial assay kits when performed by the same laboratory Results interpreted by individual judgment or computerized rules-based algorithms Variation can be seen between algorithms in classification of expected drug activity Requires regular updating Still must interpret meaning of report with regard to regimen selection

16. Interpretation of Genotypic Assays Expert advice (common in Europe) May not be available Experts� views may be inconsistent Rules-based algorithms (Bayer/VGI, others) Provided by most labs, third-party sites Need regular updating vircoTYPE HIV-1 (Virco) Database of matched genotypes and phenotypes

17. Genotypic Assays More rapid results (days) Less technically complex Proven value in predictingshort-term virologic outcome Mutations may precede phenotypic resistance Less expensive than phenotype Genotypic correlates of resistance increasingly well-defined Indirect measure of resistance May not correlate with phenotype Requires viral load > 1000 copies/mL May not detect minor species Interpretation required Cannot assess interactions between mutations

18. Genotype Examples

21. Defining Genotypic Determinants of Amprenavir Resistance

22. Overview of Current HIV Resistance Testing Technologies Standard phenotypic testing Results usually expressed as fold-change in susceptibility compared to a laboratory control isolate Interpretation of drug activity dependent on methodology used to define cutoffs (technical, biologic, clinical) Virtual phenotype testing Matches genotypic data against database of virus samples with paired GT and PT data Confidence level based on number of matching genotypes within the database

23. Phenotype Phenotype refers to the growth characteristics of the virus in vitro, which is largely determined by genotype Analogous to tube dilution bacterial sensitivity testing. Remember, cellular factors may modify the effect of phenotype in vivo.

24. Phenotypic Susceptibility: RelationshipBetween Drug Concentration and Viral Inhibition

25. Phenotypic Susceptibility: RelationshipBetween Drug Concentration and Viral Inhibition

26. Interpretation of Phenotypic Assays Results reported as IC50 of patient virus vs IC50 of wild-type virus Fold-change (FC) from IC50 of wild-type virus Individual results provided for each drug Thresholds to define reduced susceptibility Biologic cut-off Based on biologic variations in treatment-naive patients Usually 2 SD above median Clinical cut-off Based on patient virologic response, compared with suboptimal or non-response

27. Phenotypic Assays Direct measure of resistance Results simple to understand (eg, IC50) Can be used for any drug without requiring knowledge of genotypic correlates of resistance Assesses impact of interactions between mutations Less rapid results (weeks) Results too simple to understand? Thresholds for resistance not fully defined for all drugs Does not take into account activity of drugs in combination Require HIV RNA > 500-1000 May not reflect minor species More expensive than genotype

28. Viral Fitness NRTI-resistant viruses may display impaired viral fitness Impaired fitness = ? replication efficiency, ? growth Impairment can be caused by single mutations Particularly M184V, L74V and K65R Specific combinations of NRTI-resistance mutations can have additive effects on viral fitness Virus containing both M184V and K65R is �less fit� than virus with either mutation alone Secondary PI resistance mutations and cleavage site mutations may improve viral fitness in viruses with primary PI mutations Ed: The text refers to a figure 2. Do we have this? Also, wasn�t sure where to fit this in the order of the slides. Ed: The text refers to a figure 2. Do we have this? Also, wasn�t sure where to fit this in the order of the slides.

29. Phenotype Examples

30. Drug Susceptibility Reports

31. HIV phenotype from >1000 drug na�ve patients Samples from N America, Europe & S Africa Mean & standard deviation values were derived for each data set For each drug, the cut-off was set at 2 standard deviations above the mean 97.5% samples Within normal susceptible range 2.5% samples Above normal susceptible range Re-defining Phenotypic Cut-Offs Based on Biological Variation

32. Fold-change IC50 associated with treatment-dependent viral load change, more relevant Difficult to ascertain since treatment with 3-5 drugs, effect of single drugs not measured Probably will be able to modify cutoff values determined by biologic variability based on virologic change with therapy of resistant virus strains Re-defining Phenotypic Cut-Offs Based on Clinical Response

33. Phenotype/Genotype Discordance

34. Genotypic Resistance Interpretation Concordance: ANRS, HIVDB, Rega, VGI

35. Which resistance test and when?

38. NRTI Drug Resistance Pathways TAMs � 215, 41, 210, 67, 70, 219 Decrease susceptibility to all NRTIs, unblocking mechanism Alternate pathway � 65, 74 Abacavir, tenofovir, didanosine, (3TC) Hypersusceptibility to ZDV, sensitive to d4T M184V � mixed effects High level 3TC resistance, can affect abacavir Increased susceptibility to AZT, d4T, TDF Diminishes viral fitness

39. Multinucleoside and Nucleotide Resistance Multinucleoside resistance is typically associated with high level resistance to most nucleosides: Q151M complex (TDF susceptibility preserved) serine insertions - 69S(S,S) multiple NAMS, especially with 184V K65R � only ZDV reliably active; some d4T/TDF/ABC activity possible, but reduced Tenofovir resistance: K65R, 41/210/215Y, but may retain phenotypic (and clinical) activity

40. Non-Nucleoside RTI Resistance Mutations 2 clusters around K103 Y181 98/101/106/108/188/190 also important K103N is dominant, associated with class cross-resistance by itself Y181C may be seen without 103N although some efavirenz in vitro activity, not clinically durable Non-clade B, 106 mutations may be primary

41. Protease Inhibitor Resistance Mutations

42. Mutations Selected by PIs Many mutations in the protease gene, as shown in this recent representation,1 confer significant cross-resistance across the entire class. The mutations highlighted in yellow generally develop in patients who receive the individual PIs for the first time with or without nucleosides. Indinavir�46 and 82 Ritonavir�84 and 82 Saquinavir�48 and 90 Nelfinavir�30 and 90, occasionally 88 Amprenavir�50, 54, and 84 Lopinavir/ritonavir�It is unclear which mutation develops first in patients, but the mutations shown all contribute to lopinavir resistance, based on phenotypic and genotypic analyses of clinical isolates. It has been suggested that as few as 4 mutations may be associated with high-level resistance to lopinavir/ritonavir. Although L63P causes no appreciable increase in IC50, it is shown for only lopinavir/ritonavir because, along with other mutations, it predicts a lack of viral load response to regimens containing this agent.Many mutations in the protease gene, as shown in this recent representation,1 confer significant cross-resistance across the entire class. The mutations highlighted in yellow generally develop in patients who receive the individual PIs for the first time with or without nucleosides. Indinavir�46 and 82 Ritonavir�84 and 82 Saquinavir�48 and 90 Nelfinavir�30 and 90, occasionally 88 Amprenavir�50, 54, and 84 Lopinavir/ritonavir�It is unclear which mutation develops first in patients, but the mutations shown all contribute to lopinavir resistance, based on phenotypic and genotypic analyses of clinical isolates. It has been suggested that as few as 4 mutations may be associated with high-level resistance to lopinavir/ritonavir. Although L63P causes no appreciable increase in IC50, it is shown for only lopinavir/ritonavir because, along with other mutations, it predicts a lack of viral load response to regimens containing this agent.

43. Mutations Selected by PIs (cont) Many mutations in the protease gene, as shown in this recent representation,1 confer significant cross-resistance across the entire class. The mutations highlighted in yellow generally develop in patients who receive the individual PIs for the first time with or without nucleosides. Indinavir�46 and 82 Ritonavir�84 and 82 Saquinavir�48 and 90 Nelfinavir�30 and 90, occasionally 88 Amprenavir�50, 54, and 84 Lopinavir/ritonavir�It is unclear which mutation develops first in patients, but the mutations shown all contribute to lopinavir resistance, based on phenotypic and genotypic analyses of clinical isolates. It has been suggested that as few as 4 mutations may be associated with high-level resistance to lopinavir/ritonavir. Although L63P causes no appreciable increase in IC50, it is shown for only lopinavir/ritonavir because, along with other mutations, it predicts a lack of viral load response to regimens containing this agent.Many mutations in the protease gene, as shown in this recent representation,1 confer significant cross-resistance across the entire class. The mutations highlighted in yellow generally develop in patients who receive the individual PIs for the first time with or without nucleosides. Indinavir�46 and 82 Ritonavir�84 and 82 Saquinavir�48 and 90 Nelfinavir�30 and 90, occasionally 88 Amprenavir�50, 54, and 84 Lopinavir/ritonavir�It is unclear which mutation develops first in patients, but the mutations shown all contribute to lopinavir resistance, based on phenotypic and genotypic analyses of clinical isolates. It has been suggested that as few as 4 mutations may be associated with high-level resistance to lopinavir/ritonavir. Although L63P causes no appreciable increase in IC50, it is shown for only lopinavir/ritonavir because, along with other mutations, it predicts a lack of viral load response to regimens containing this agent.

44. Mutations in the gp41 env associated with Resistance to enfuvirtide This slide summarizes NNRTI resistance patterns. There is a clustering of mutations between codons 100 and 106 and codons 181 and 190, with several others occurring in the low 200s. There is broad cross-resistance in these patterns, although subtle differences can be seen.1 This slide summarizes NNRTI resistance patterns. There is a clustering of mutations between codons 100 and 106 and codons 181 and 190, with several others occurring in the low 200s. There is broad cross-resistance in these patterns, although subtle differences can be seen.1

45. HIV Drug Resistance Testing All Approaches have Strengths and Limitations: Strong positive correlation with resistance or decreased virologic response when present All commercial assays relatively insensitive Lack of detection does not mean resistance is not present; resistance from previous regimens or exposure may not be detected Moving on to some additional definitions, primary mutations are those that are selected early and usually result in decreased drug susceptibility. For the most part, primary mutations are either discovered in vitro or discovered after the drug has been given to patients and virologic failure has ensued. After primary mutations are discovered in a patient, they are studied in vitro in order to determine if they do confer drug resistance Secondary mutations are those that are selected after primary mutations have occurred. Secondary mutations likely have a very limited effect on drug susceptibility. In other words, if secondary mutations alone are studied in the laboratory, drug resistance will not be observed Multi-drug resistance mutations often confer resistance or decreased susceptibility to many or all drugs in a single class. A few known mutations confer drug resistance to the entire reverse transcriptase inhibitor or protease inhibitor classMoving on to some additional definitions, primary mutations are those that are selected early and usually result in decreased drug susceptibility. For the most part, primary mutations are either discovered in vitro or discovered after the drug has been given to patients and virologic failure has ensued. After primary mutations are discovered in a patient, they are studied in vitro in order to determine if they do confer drug resistance Secondary mutations are those that are selected after primary mutations have occurred. Secondary mutations likely have a very limited effect on drug susceptibility. In other words, if secondary mutations alone are studied in the laboratory, drug resistance will not be observed Multi-drug resistance mutations often confer resistance or decreased susceptibility to many or all drugs in a single class. A few known mutations confer drug resistance to the entire reverse transcriptase inhibitor or protease inhibitor class

46. HIV Drug Resistance Testing All Approaches have Strengths and Limitations: Complex mutation patterns should be more readily interpreted by phenotype, but this has not been demonstrated in clinical trials VirtualPhenotype offers sequence analysis and interpretation based on viral growth properties related to patient RT and protease Moving on to some additional definitions, primary mutations are those that are selected early and usually result in decreased drug susceptibility. For the most part, primary mutations are either discovered in vitro or discovered after the drug has been given to patients and virologic failure has ensued. After primary mutations are discovered in a patient, they are studied in vitro in order to determine if they do confer drug resistance Secondary mutations are those that are selected after primary mutations have occurred. Secondary mutations likely have a very limited effect on drug susceptibility. In other words, if secondary mutations alone are studied in the laboratory, drug resistance will not be observed Multi-drug resistance mutations often confer resistance or decreased susceptibility to many or all drugs in a single class. A few known mutations confer drug resistance to the entire reverse transcriptase inhibitor or protease inhibitor classMoving on to some additional definitions, primary mutations are those that are selected early and usually result in decreased drug susceptibility. For the most part, primary mutations are either discovered in vitro or discovered after the drug has been given to patients and virologic failure has ensued. After primary mutations are discovered in a patient, they are studied in vitro in order to determine if they do confer drug resistance Secondary mutations are those that are selected after primary mutations have occurred. Secondary mutations likely have a very limited effect on drug susceptibility. In other words, if secondary mutations alone are studied in the laboratory, drug resistance will not be observed Multi-drug resistance mutations often confer resistance or decreased susceptibility to many or all drugs in a single class. A few known mutations confer drug resistance to the entire reverse transcriptase inhibitor or protease inhibitor class

47. Part IIResistance TestingClinical Applications

48. What is Treatment Failure?* True definition encompasses virologic, immunologic, and/or clinical status Different types First or second regimen: Initial goal is an �undetectable� viral load �Failure� therefore defined as Inability to achieve a viral load below the limit of quantification Any sustained return of virus to above the target level

49. Treatment Failure II* Multiple regimen failures: Achieving an undetectable viral load may not be realistic Goal is to prevent clinical progression Deterioration of the CD4 count or Occurrence of an OI Lack of viable options suggest continuing the regimen in the face of on-going viral replication and presence of resistance

50. Causes of HIV Treatment Failure

51. Why Do We Need Antiretroviral Resistance Testing? To help clinicians make better treatment decisions Before beginning antiretroviral therapy Naive patients infected with resistant HIV When to change a regimen To help determine which drug(s) are not working To decrease use of ineffective and/or potentially toxic drugs In the setting of transmission Vertical After �accidental� exposure To aid in the development of better treatment guidelines To help pharmaceutical companies make better drugs

52. Antiretroviral Resistance Testing Is Part of the Recommended STANDARD of CARE in the Management of the HIV-Infected Individual!1,2 References: Panel on Clinical Practices for Treatment of HIV Infection for the Department of Health and Human Services and the Henry J Kaiser Family Foundation. Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents. Washington, DC: Department of Health and Human Services; February 4, 2002. Available at: http://www.aidsinfo.nih.gov/guidelines/adult/html_adult_02-04-02.html. Accessed February 23, 2002. Yeni PG, Hammer SM, Carpenter CCJ, Cooper DA, Fischl MA, Gattell JM, et al. Antiretroviral Treatment for Adult HIV Infection in 2002: Updated Recommendations of the International AIDS Society-USA Panel. JAMA. July 10, 2002;288:222-252.References: Panel on Clinical Practices for Treatment of HIV Infection for the Department of Health and Human Services and the Henry J Kaiser Family Foundation. Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents. Washington, DC: Department of Health and Human Services; February 4, 2002. Available at: http://www.aidsinfo.nih.gov/guidelines/adult/html_adult_02-04-02.html. Accessed February 23, 2002. Yeni PG, Hammer SM, Carpenter CCJ, Cooper DA, Fischl MA, Gattell JM, et al. Antiretroviral Treatment for Adult HIV Infection in 2002: Updated Recommendations of the International AIDS Society-USA Panel. JAMA. July 10, 2002;288:222-252.

53. General Limitations and Caveats of Resistance Testing Current assays requires 500*�1000 viral copies to isolate sufficient nucleic acid to amplify Insensitive to minor viral species If drug-resistant viruses <10-20% of the circulating viral population, may not be detected by the assay In the absence of drug pressure Wild type virus will typically be preferentially expressed Resistant clones will be �overgrown� and fade to undetectable However, resistance is a genetic characteristic. It is �archived� and can be re-expressed rapidly * ViroLogic assays are validated at 500 copies/mL

54. Therefore, the absence of resistance does not necessarily indicate susceptibility!

55. Genotyping

56. Genotyping

57. Genotyping

58. Genotyping Advantages Specifies the mutations in the HIV genome associated with resistance Sensitive for detection of emerging resistance (mixtures) May provide early warning signals to development of full resistance More rapid and less expensive than a phenotype Offered by many commercial laboratories Limitations Not a direct measure of viral resistance Increasingly complex mutational patterns Cannot predict interactions of mutations Presupposes knowledge of critical mutations Requires interpretation for prediction of resistance or susceptibility to drugs Variations in available sequencing methodologies No Clinical Cutoffs for genotype

59. Phenotyping

60. Phenotyping

61. Phenotyping Advantages Direct measure of resistance�measures the ability of the patient�s virus to grow in different concentrations of drug in vitro: the gold standard Accounts for net effect of any and all resistance mutations Prediction of resistance or susceptibility already built-in and does not require external interpretation Limitations Technically more complex than genotyping More expensive May longer turnaround time Establishment of accurate fold-resistance cut-off values critical for prediction of response Need for true �clinical cut-offs� Only looks at individual drugs and not PK-enhanced PIs

63. VirtualPhenotype� Advantages Report provides both sequence data (GENOTYPE) and prediction of phenotype: �2-for-1� test Assessment of drug resistance based on actual data from a growing set of samples with paired genotype/phenotype Updated in real-time Limitations Not a real phenotype Strength of correlation depends on number of matches Number of matches for new drugs may be limited

64. Combination Genotype/Phenotype Report

65. HIV-1 Drug Resistance TestingItems To Consider Genotyping Does the test cover all known mutations? Are there any �gaps� in the sequencing? How often is the library/database updated? What does the genotype tell the healthcare provider? Can healthcare providers make a clinical judgment based on the report? Phenotyping Are drug-specific clinical cut-offs available for each drug? Is the report easy to read?

66. Clinical Trials of Antiretroviral Resistance Testing

67. Published Prospective Clinical Trials I* References: Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet. June 26, 1999;353(9171):2195-2199. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. AIDS. June 16, 2000;14(9):F83-F93. Tural C, Ruiz L, Holtzer C, et al, and the Havana Study Group. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS. January 25, 2002;16(2):209-218. Cingolani A, Antinori A, Rizzo MG, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: a randomized study (ARGENTA). AIDS. February 15, 2002;16(3):369-379. Cohen CJ, Hunt S, Sension M, et al, and the VIRA3001 Study Team. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS. March 8, 2002;16(4):579-588. Meynard JL, Vray M, Morand-Joubert L, et al, and the Narval Trial Group. Phenotypic or genotypic resistance testing for choosing antiretroviral therapy after treatment failure: a randomized trial. AIDS. March 29, 2002;16(5):727-736. References: Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet. June 26, 1999;353(9171):2195-2199. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. AIDS. June 16, 2000;14(9):F83-F93. Tural C, Ruiz L, Holtzer C, et al, and the Havana Study Group. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS. January 25, 2002;16(2):209-218. Cingolani A, Antinori A, Rizzo MG, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: a randomized study (ARGENTA). AIDS. February 15, 2002;16(3):369-379. Cohen CJ, Hunt S, Sension M, et al, and the VIRA3001 Study Team. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS. March 8, 2002;16(4):579-588. Meynard JL, Vray M, Morand-Joubert L, et al, and the Narval Trial Group. Phenotypic or genotypic resistance testing for choosing antiretroviral therapy after treatment failure: a randomized trial. AIDS. March 29, 2002;16(5):727-736.

68. References: Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet. June 26, 1999;353(9171):2195-2199. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. AIDS. June 16, 2000;14(9):F83-F93. Tural C, Ruiz L, Holtzer C, et al, and the Havana Study Group. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS. January 25, 2002;16(2):209-218. Cingolani A, Antinori A, Rizzo MG, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: a randomized study (ARGENTA). AIDS. February 15, 2002;16(3):369-379. Cohen CJ, Hunt S, Sension M, et al, and the VIRA3001 Study Team. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS. March 8, 2002;16(4):579-588. Meynard JL, Vray M, Morand-Joubert L, et al, and the Narval Trial Group. Phenotypic or genotypic resistance testing for choosing antiretroviral therapy after treatment failure: a randomized trial. AIDS. March 29, 2002;16(5):727-736. References: Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet. June 26, 1999;353(9171):2195-2199. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. AIDS. June 16, 2000;14(9):F83-F93. Tural C, Ruiz L, Holtzer C, et al, and the Havana Study Group. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS. January 25, 2002;16(2):209-218. Cingolani A, Antinori A, Rizzo MG, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: a randomized study (ARGENTA). AIDS. February 15, 2002;16(3):369-379. Cohen CJ, Hunt S, Sension M, et al, and the VIRA3001 Study Team. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS. March 8, 2002;16(4):579-588. Meynard JL, Vray M, Morand-Joubert L, et al, and the Narval Trial Group. Phenotypic or genotypic resistance testing for choosing antiretroviral therapy after treatment failure: a randomized trial. AIDS. March 29, 2002;16(5):727-736.

69. Clinical TrialsSummary and Conclusions* Many abstracts, but few published studies Difficult to compare Interpretations are complicated Short term benefits demonstrated for both assays Evidence strongest for genotyping Insufficient evidence favoring 1 test type over another Complex situations: genotyping and phenotyping provide complementary information

70. Recent Applications of Antiretroviral Resistance Testing

71. Transmitted HIV Drug Resistance New York City MDR / Rapid Disease Progression Case

72. Follow-Up on the New York City MDR Case Blick et al performed an analysis on 135,000 HIV strains in the database Matched the NYC strain with a strain from a 52 yo Conn. man who began AZT in 1995 & HAART in 1997 This �Pt Zero� reported having unprotected sex with the NYC pt while on crystal methamphetamine in 10/04 Phylogenetic analysis confirmed that the 2 strains were identical ; both dual tropic with identical resistance patterns Pt Zero currently has CD4 of 180-262 & VL < 400; and he is clinically stable Refutes the concept of a �new virulent strain� & suggests that host factors might explain the rapid decline of the NYC pt (CD4 of 60 four to twenty months into infection)

73. Patient Zero? Transmission of MDR Resistant Virus

74. US surveillance of HIV drug resistance Drug resistance in 787 newly diagnosed ART-na�ve subjects from 89 sites in 6 states (2003�2004) Background/Methods: CDC re-analysis newly diagnosed but chronically infected between 2002�2004. First analysis of new data set; 2003�4. Rise in resistance across all classes with a 3.1% rise in >2 class resistance. Methods: In 2003�4, drug resistance surveillance began in 65 sites in 6 states (Illinois, Colorado, Washington, Virginia, Maryland, and Michigan). A total of 595 residual HIV diagnostic sera from drug-na�ve persons newly diagnosed with HIV were processed in the public health laboratories where routine HIV testing takes place. Sequencing was performed at Stanford University, Maryland State, and University of Washington laboratories. Results were available to providers within 30 days. Conclusions: In participating surveillance sites, as in other recent US studies, resistance to NNRTI was more prevalent than resistance to NRTI, although overall HIV drug resistance prevalence was lower than prevalence reported in other studies. Persons diagnosed in publicly funded sites may have a lower prevalence of drug resistance than those in specialists� care, possibly because persons who transmit HIV to persons diagnosed in specialist centers may, as a group, have more access to HIV treatment and therefore to ARV drugs. Representative drug resistance surveillance methods and sufficiently large numbers are needed to obtain accurate national estimates. The use of residual HIV diagnostic sera from counseling and testing sites as well as clinical sites can provide a representative sample of persons newly diagnosed with HIV for surveillance purposes. Limitations: Not all newly diagnosed in 2003�4 in participating sites included; specimens not available from all persons and unable to GT in 5% of specimens. Analysis of associated risk factors was limited for patients for whom demographic data are currently available; numbers may be too small to show differences among groups. Patients in private practice or non�public-funded centers may have a higher risk for HIV drug resistance than those included in this analysis (possibly due to better access to care and access to ARVs?). Background/Methods: CDC re-analysis newly diagnosed but chronically infected between 2002�2004. First analysis of new data set; 2003�4. Rise in resistance across all classes with a 3.1% rise in >2 class resistance. Methods: In 2003�4, drug resistance surveillance began in 65 sites in 6 states (Illinois, Colorado, Washington, Virginia, Maryland, and Michigan). A total of 595 residual HIV diagnostic sera from drug-na�ve persons newly diagnosed with HIV were processed in the public health laboratories where routine HIV testing takes place. Sequencing was performed at Stanford University, Maryland State, and University of Washington laboratories. Results were available to providers within 30 days. Conclusions: In participating surveillance sites, as in other recent US studies, resistance to NNRTI was more prevalent than resistance to NRTI, although overall HIV drug resistance prevalence was lower than prevalence reported in other studies. Persons diagnosed in publicly funded sites may have a lower prevalence of drug resistance than those in specialists� care, possibly because persons who transmit HIV to persons diagnosed in specialist centers may, as a group, have more access to HIV treatment and therefore to ARV drugs. Representative drug resistance surveillance methods and sufficiently large numbers are needed to obtain accurate national estimates. The use of residual HIV diagnostic sera from counseling and testing sites as well as clinical sites can provide a representative sample of persons newly diagnosed with HIV for surveillance purposes. Limitations: Not all newly diagnosed in 2003�4 in participating sites included; specimens not available from all persons and unable to GT in 5% of specimens. Analysis of associated risk factors was limited for patients for whom demographic data are currently available; numbers may be too small to show differences among groups. Patients in private practice or non�public-funded centers may have a higher risk for HIV drug resistance than those included in this analysis (possibly due to better access to care and access to ARVs?).

75. Drug resistance and its association with increased risk of death in pts on 1st HAART 1388 ART-na�ve Canadian pts Initiated HAART Aug 96�July 00 Primary endpoint: all-cause mortality 238 deaths observed (17.2%) Increased risk of death Increased age Decreased adherence Lower baseline CD4+ Higher baseline HIV RNA Decreased physician experience Emergence of ART resistance ABSTRACT: Resistance to any class of drug was 28.3% (n=393). COMMENTS: Manuscript in review; this is first data looking at resistance as related to risk of mortality. Dr Hogg is not a physician but a �demographer� and doing great work at describing patient cohorts. One of two things might be going on for NNRTI: NNRTI-resistant virus thought to be more pathogenic relative to other viruses; generally see full viral rebound when there is resistance alluding to relatively fit viruses; often see CD4+ decline in these patients. Low levels of adherence puts patients at risk for resistance; pharmacy refill data used for this cohort and in other studies; not as precise as could be, might be some uncontrolled confounding adherence-related factors; nonadherent group probably in ballpark of <50%, ie, NNRTI-resistant virus �picks out� nonadherent patients. FOR PI: both theories above possibly true in opposite direction� With PI-resistant virus: generally see partial viral rebound; sustained elevations in CD4+ suggests PI-resistant virus not �fully� pathogenic; does not explain protective effect, but probably not as pathogenic; a lot of single PIs in this study; early data. PI resistance is a marker of high levels of adherence among patients who are on single PIs (Bangsberg et al); some uncontrolled confounding; this group probably taking a lot of their meds (>90%). ABSTRACT: Resistance to any class of drug was 28.3% (n=393). COMMENTS: Manuscript in review; this is first data looking at resistance as related to risk of mortality. Dr Hogg is not a physician but a �demographer� and doing great work at describing patient cohorts. One of two things might be going on for NNRTI: NNRTI-resistant virus thought to be more pathogenic relative to other viruses; generally see full viral rebound when there is resistance alluding to relatively fit viruses; often see CD4+ decline in these patients. Low levels of adherence puts patients at risk for resistance; pharmacy refill data used for this cohort and in other studies; not as precise as could be, might be some uncontrolled confounding adherence-related factors; nonadherent group probably in ballpark of <50%, ie, NNRTI-resistant virus �picks out� nonadherent patients. FOR PI: both theories above possibly true in opposite direction� With PI-resistant virus: generally see partial viral rebound; sustained elevations in CD4+ suggests PI-resistant virus not �fully� pathogenic; does not explain protective effect, but probably not as pathogenic; a lot of single PIs in this study; early data. PI resistance is a marker of high levels of adherence among patients who are on single PIs (Bangsberg et al); some uncontrolled confounding; this group probably taking a lot of their meds (>90%).

76. Reduced Transmission of Drug-Resistant Strains Containing M184V & PI Mutations Compared genotypes among pts with primary HIV infection (PHI) and chronically infected patients (CIP) Found that the RR of transmitting HIV harboring M184V was less than the RR of transmitting HIV with TAMS or NNRTI mutations (P < 0.05) RR of transmitting strains harboring PI mutations was less than the RR of those without PI mutations (P<0.01)

77. Reduced Transmission of Drug-Resistant Strains Containing M184V & PI Mutations In chronic pts with TAMS or NNRTI mutations, the median VL was 35,000; while pts with M184V, median VL was 4252 In chronic pts, median VL of pts with no PI mutations was 24,104; while pts with PI mutations had a median VL of 11,800 Hypothesis: M184V & PI mutations ?�s RC, which ?�s VL, which reduces the likelihood of transmission during sex

78. Future Directions for Resistance Testing Importance of Secondary Protease Resistance Mutations

79. Reduced Susceptibility to PI�s in the Absence of Primary PI Resistance Mutations Parkin et al defined primary PI mutations as the following: any change from wt in 23,24,30,32,46,47,48,50,54,82,84,88 & 90 Excluded I54V & N88D (which are not reported without other primary mutations) and V82I (known polymorphism in PI naives) Determined PR genotype and PI susceptibility (FC in IC 50); also determined gag genotypes Found 125 pts with no primary mutations but at least one PI with FC >5

80. Reduced Susceptibility to PI�s in the Absence of Primary PI Resistance Mutations Compared the 125 pts to 3,956 samples with no PI mutations & all PI FC�s < 5; described PR mutations over-represented in samples with PI FC>5: L10IV, I13V, L19V, K20IMT, A22V, M36IV, N37D, I54V, H69R, A71TV, G73S, T74KS, V82I, N83D, N88D, & I93L In gag, found that K418ER, A431V, I437V, L449IP, P453L & E482G were also associated with PI FC > 5 in pts without primary PR mutations Concluded that accumulation of secondary mutations in PR and also in gag contribute to PI susceptibility

81. NNRTI Hypersusceptibility:Nucleoside mutations (TAMS) create HIV strains uniquely sensitive to NNRTI therapy 215Y, 41L, 210W, 118I and 208Y

82. NNRTI hypersusceptibility � a review If you do phenotypic resistance testing, you may have noticed in some of your patients that sometimes, instead of seeing a 10-fold reduction in susceptibility, the fold change reported is 0.3 or 0.2, suggesting that the virus is hypersusceptible to the drug. Does that really matter? Here is a pooled analysis of 444 samples from 5 ACTG studies that was presented by Nancy Shulman at Stanford and they were able to look at this phenomenon of hypersusceptibility and link it to specific mutations in reverse transcriptase that are associated with thymidine analogs. The 5 mutations listed, all of which seem to have some linkage to NNRTI hypersusceptibility, are induced by thymidines and may actually have a favorable outcome in terms of the efficacy of NNRTI-based regimens and persons who are NNRTI-na�ve. Lisa Demeter also examined response to a regimen of efavirenz, indinavir and abacavir in nucleoside-experienced patients. The genotypic score for efavirenz hypersusceptibility from ACTG368 was predictive of outcome in these patients. The specific mutations that were associated with hypersusceptibility are shown. The more of these mutations present, the lower likelihood of virologic failure. Previous studies show improved response associated with HS to EFV1, 2 and APV3. References Haubrich R, et al. AIDS 2002;16:33�40 2. Shulman N, et al. AIDS 2001; 15:1125�1132 3. Schooley R, et al. 10th CROI, Boston 2003, #143)If you do phenotypic resistance testing, you may have noticed in some of your patients that sometimes, instead of seeing a 10-fold reduction in susceptibility, the fold change reported is 0.3 or 0.2, suggesting that the virus is hypersusceptible to the drug. Does that really matter? Here is a pooled analysis of 444 samples from 5 ACTG studies that was presented by Nancy Shulman at Stanford and they were able to look at this phenomenon of hypersusceptibility and link it to specific mutations in reverse transcriptase that are associated with thymidine analogs. The 5 mutations listed, all of which seem to have some linkage to NNRTI hypersusceptibility, are induced by thymidines and may actually have a favorable outcome in terms of the efficacy of NNRTI-based regimens and persons who are NNRTI-na�ve. Lisa Demeter also examined response to a regimen of efavirenz, indinavir and abacavir in nucleoside-experienced patients. The genotypic score for efavirenz hypersusceptibility from ACTG368 was predictive of outcome in these patients. The specific mutations that were associated with hypersusceptibility are shown. The more of these mutations present, the lower likelihood of virologic failure. Previous studies show improved response associated with HS to EFV1, 2 and APV3. References Haubrich R, et al. AIDS 2002;16:33�40 2. Shulman N, et al. AIDS 2001; 15:1125�1132 3. Schooley R, et al. 10th CROI, Boston 2003, #143)

83. K65R Leads to NNRTI Hypersusceptibility Not only TAM�s lead to NNRTI HS, but also K65R & L74V Landman et al evaluated salvage tx for 22 pts with K65R from the TONUS & GS903 trials Median baseline VL was 6336 At 48 wks, 86% were < 50 copies; mean RC was 54% The K65R and M184V double mutants were HS to EFV, NVP & ZDV Concluded that NNRTI hypersusceptibility & impaired RC associated with K65R contributed to successful salvage with EFV & ZDV

84. Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents July 14, 2003

86. Recommendations for Resistance Testing References: Panel on Clinical Practices for Treatment of HIV Infection for the Department of Health and Human Services and the Henry J Kaiser Family Foundation. Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents. Washington, DC: Department of Health and Human Services; February 4, 2002. Available at: http://www.aidsinfo.nih.gov/guidelines/adult/html_adult_02-04-02.html. Accessed February 23, 2002. Hirsch MS, Brun-Vezinet F, D�Aquila RT, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel. JAMA. May 10, 2000;283(18):2417-2426. Yeni PG, Hammer SM, Carpenter CCJ, Cooper DA, Fischl MA, Gattell JM, et al. Antiretroviral Treatment for Adult HIV Infection in 2002: Updated Recommendations of the International AIDS Society-USA Panel. JAMA. July 10, 2002;288:222-252. References: Panel on Clinical Practices for Treatment of HIV Infection for the Department of Health and Human Services and the Henry J Kaiser Family Foundation. Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents. Washington, DC: Department of Health and Human Services; February 4, 2002. Available at: http://www.aidsinfo.nih.gov/guidelines/adult/html_adult_02-04-02.html. Accessed February 23, 2002. Hirsch MS, Brun-Vezinet F, D�Aquila RT, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel. JAMA. May 10, 2000;283(18):2417-2426. Yeni PG, Hammer SM, Carpenter CCJ, Cooper DA, Fischl MA, Gattell JM, et al. Antiretroviral Treatment for Adult HIV Infection in 2002: Updated Recommendations of the International AIDS Society-USA Panel. JAMA. July 10, 2002;288:222-252.

87. Guidelines Vary by StateGeneral Recommendations

88. Antiretroviral Resistance TestingStandards for Medicaid Reimbursement Most state Medicaid guidelines agree with the current DHHS Guidelines and recommend RT in the following clinical settings: Virologic failure while on recommended Antiretroviral Therapy (ART) Suboptimal virologic response after beginning ART Acute HIV infection (ie, those strongly documented to have been infected within the previous 6 months) Consider in ARV-na�ve patients if the rate of resistance in the community is substantial

89. Resistance Testing in Pregnancy DOH recommends RT for: All HIV + pregnant women with viral loads >1000/mm3 or greater than the documented statement of threshold accuracy by the laboratory Recommended independent of whether or not they are already being treated with combination therapy Testing should be done as near to the first prenatal visit as possible Antiretroviral therapy should be continued or begun empirically pending results, with treatment becoming more urgent the higher the viral load

90. Pediatric Resistance TestingRecommendations Resistance testing is recommended for newly infected infants, children, and adolescents When the HIV RNA does not decrease as expected within 4 to 6 months of initiating HAART, or When there is viral rebound

91. When NOT to Use Antiretroviral Resistance Testing After discontinuation of antiretroviral treatment for more than 2 weeks If plasma HIV RNA <1000 co/ml or less than the documented statement of threshold accuracy by the laboratory To document lack of adherence and in situations other than above

92. Which antiretroviral resistance tests should be used? Studies in patients failing a first antiretroviral regimen: genotypic ~ phenotypic resistance testing In patients with multiple ART failures May be advantageous to use phenotypic resistance testing Or concurrent use of both tests, to optimize therapy

93. Cost-Effectivenessand Pharmacoeconomics Genotyping ARV-experienced patients VIRADAPT: 1st study to suggest cost-effectiveness of resistance testing Weinstein et al. (Ann Intern Med. 2001;134(6):440-450) Model based on 2 prospective short term trials Suggests genotyping is as cost effective as HAART ARV-na�ve patients Sax et al. (XIV IAC. Barcelona, July 7-12, 2002. Ab#MoPeB3129) Simulation model based upon prospective clinical trials Depends upon prevalence of resistant virus in a particular area Phenotyping: studies underway

94. VIRADAPT Study Mean 12-Month Cost per Patient Clinical Care* ART Drugs Genotyping� Total Cost Reference: Chaix C, Grenier-Sennelier C, Clevenbergh P, et al. Economic evaluation of drug resistance genotyping for the adaptation of treatment in HIV-infected patients in the VIRADAPT study. J Acquir Immune Defic Syndr. July 1, 2000;24(3):227-231.Reference: Chaix C, Grenier-Sennelier C, Clevenbergh P, et al. Economic evaluation of drug resistance genotyping for the adaptation of treatment in HIV-infected patients in the VIRADAPT study. J Acquir Immune Defic Syndr. July 1, 2000;24(3):227-231.

95. Cost-Effectiveness of Genotypic Resistance Testing AfterHAART Failure1,2 Models derived from 2 prospective clinical trials QALY used to determine cost-benefit <$25,000/QALY �very cost-efficient� PCPP: $16,000/QALY MACP: $35,000-58,000/QALY Others References: Weinstein MC, Goldie SJ, Losina E, et al. Use of genotypic resistance testing to guide HIV therapy: clinical impact and cost-effectiveness. Ann Intern Med. March 20, 2001;134(6):440-450. Saag MS. HIV resistance testing in clinical practice: a QALY-fied success. Ann Intern Med. March 20, 2001;134(6):475-477.References: Weinstein MC, Goldie SJ, Losina E, et al. Use of genotypic resistance testing to guide HIV therapy: clinical impact and cost-effectiveness. Ann Intern Med. March 20, 2001;134(6):440-450. Saag MS. HIV resistance testing in clinical practice: a QALY-fied success. Ann Intern Med. March 20, 2001;134(6):475-477.

96. How frequently should resistance testing be ordered? Currently, there are no guidelines regarding the frequency of resistance testing However, based on the accepted time frames defining virologic failure as outlined in the DHHS guidelines, reimbursement in most states will be limited to no more than 2-3 tests in a 12-month period Exceptions to the 2-test limit may be requested

97. Thank you for your time and attention