1. Antiretroviral Resistance Testing in the Management of HIV-infected Patients Christopher Behrens, MD Amy Kindrick, MD Robert Harrington, MD

2. Overview of Antiretroviral Resistance Testing How does resistance develop? What is the relationship between adherence and resistance? How much resistance is out there? How do we test for resistance? How do we interpret the results of a resistance test? Does resistance testing improve care? When should you order resistance tests? Can a single dose of nevirapine for Prevention of Mother to Child Transmission (PMTCT) result in clinically significant resistance?

3. How Does HIV Develop Resistance to Antiretrovirals?

4. HIV Life Cycle RNA DNA HIV Nucleus Host Cell Nucleoside Analogues (NRTIs) Non-Nucleosides (NNRTIs)Protease Inhibitors (PIs) Reverse Transcriptase

5. How does HIV Develop Resistance to Antiretrovirals? HIV reverse transcriptase is a low-fidelity enzyme, i.e., transcription mistakes are common Mistakes (mutations) lead to mutant strains of HIV Most mutations are inconsequential or result in incompetent strains of HIV, but certain mutations confer resistance to currently available antiretroviral drugs (ARVs) Administration of antiretrovirals in an insufficiently potent manner exerts reproductive pressure that selects for resistance-bearing strains which then become the majority strain of HIV in that patient

6. How drug resistance arises. Richman, DD. Scientific American, July 1998 How Drug Resistance Arises

7. How does resistance develop? Continuation of a failing ART regimen after early resistance has developed selects for expansion of resistance

8. How does resistance develop? Poor Adherence Drug Resistance Regimen Failure

9. How does resistance develop? Insufficient drug level Viral replication in the presence of drug Resistant virus Poor adherence Social/personal issues Regimen issues Toxicities Poor potency Wrong dose Host genetics Poor absorption Rapid clearance Poor activation Drug interactions

10. True or False? The patients with the lowest levels of adherence are the most likely to develop resistance to their ARVs

11. What is the relationship between adherence and resistance?

12. Harrigan, JID, 2005 Prospective, observational study N = 1191 Predictors of resistance –High baseline VL –Good (not great) adherence What is the relationship between adherence and resistance?

13. Is Resistance Becoming More Common? JAMA. 2002 Jul 10;288(2):181-8. % of resistant isolates Prevalence of resistance among recently-infected patients at San Francisco General Hospital

14. Is Resistance Becoming More Common? Little SJ, Holte S, Routy JP, et al. N Engl J Med. 2002;347:385-94 Recently Infected, ART Naïve, United States

15. How do we test for resistance? 1. Genotype 2. Phenotype 3. Virtual Phenotype

16. HIV Life Cycle RNA DNA HIV Nucleus Host Cell Nucleoside Analogues (NRTIs) Non-Nucleosides (NNRTIs)Protease Inhibitors (PIs) Reverse Transcriptase

17. Genotypic Resistance Assay Sequences relevant portions of the HIV genome coding for Reverse Transcriptase and Protease enzymes Detects and reports variations in the sequences of these genes that are known or suspected to confer antiretroviral resistance AAA GAC AGT AAA AAC AGC LysLys AspAspSerSer LysLys AsnAsnSerSer Codon Silent Mutation Mutation Silent Mutation Adapted from Winters. Reviewed in Wilson. AIDS Read 2000;10:469.

18. M184V M = Methionine 184 = the codon # V = Valine A mutation at codon #184 in the gene Reverse Transcriptase codes for a Valine residue where normally a Methionine residue is found.

19. Adapted from D’Aquila. Topics in HIV Medicine 2001;9(2):31. L N R W YF Q 4167 70 560 AZT L T (Wild Type) 1 (Mutant) 219 215 210 K MDK L M K ddI ddI 6574 184 V V R ddC T D 65 74 184 69 4167 70 219 215 210 d4T 75 V TMSA MutationSelected in vitro ABC 74115 Y F V 219 184 215210 41 67 70 65 3TC 3TC 184 E 118 V I 44 D VI Clinical significance under investigation Reverse Transcriptase Mutations Selected by NRTIs

20. SA L K 100 181 100 NVP DLV EFV V V 103 106 108 188 190 Y G I N A IC LH A L 103 108 181 Y CI 236 P C 188 L 190 225 P H 560 1 Adapted from D’Aquila. Topics in HIV Medicine 2001;9(2):31. Mutation Reverse Transcriptase Mutations Selected by NNRTIs

21. KV L 1 99 10 L IDV 202432 L L M 46 I 54 71 I V G A 828490 5471828490 104854 71 828490 7177828890 VLILF G V D 363046 N 84 I 46 I 10 RTV SQV NFV APV 46 32 FIRV 54 47 50 I V 10 FI IL 84 AFTS 77 73 SA 77 36 73 77 M V PrimarySecondary M V AFTS I V SAVTII IIIRV MR 20 323336 ND VVM 84 46 10 LPV/RTV 54 53 202471829063 L P F L 10 Adapted from D’Aquila. Topics in HIV Medicine 2001;9:31. Protease Mutations Selected by PIs

22. Interpretation of the results: what are the clinical implications of these mutations in terms of resistance to antiretroviral agents?

23. Interpretation of the Genotypic Resistance Assay The genotype report typically includes an interpretation of the clinical implications of the identified mutations However: –The exact significance of many mutations remains controversial –Interactions between mutations further complicate estimation of the clinical impact of a given set of mutations –Interpretation of genotypic resistance assays is not standardized across different laboratories –Assays will not detect minority resistant strains (less than 10- 20% of the viral population)

30. Phenotypic Resistance Testing Tests viability of a synthetic version of the patient’s HIV in the presence of antiretroviral agents Similar to traditional bacterial antibiotic susceptibility assays Results reported as fold- change in susceptibility to antiretroviral agents

31. Drug Concentration Inhibition of Viral Replication (%) 100 0 IC 50 50 IC 50 Fold Resistance Wild-type strain Mutant strain Reviewed in Wilson. AIDS Read 2000;10:469. Phenotype Resistance Testing

32. PhenoSense HIV Patient Report

33. PhenoSense™ HIV Report

34. Which Resistance Assay is Better? ProsCons Genotypic assay More clinical experience and evidence of clinical utility Less expensive (~$400) Results available in 1-2 weeks Results difficult to interpret Does not directly measure net effect of multiple mutations Phenotypic assay Simpler to interpret More directly estimates net effect of multiple mutations Clinically relevant thresholds of resistance not yet determined for many agents Less evidence of clinical utility More expensive (~$1000) Results in 3-4 weeks

35. Does the use of resistance assays improve clinical results?

36. Published Randomized Controlled Trials of Resistance Testing

37. % of Patients with HIV-1 RNA <400 copies/mL wk 12 NO G, NO Expert (N = 77)36.4% NO G, Expert (N = 67) 49.3% G, NO Expert (N = 69)46.4% G, Expert (N = 65) 69.2% 0 20 40 60 80 100 No G No Expert Op. No G Expert Op. G No Expert Op. G Expert Op. P =.0206P =.00132 (ITT) Tural. 40th IACAC; 2000; Toronto. Presentation LB-10. BLwk 24 Havana Results:

38. Expert Consultation Resources National Clinicians’ Telephone Consultation Service (Warmline): 800-933-3413 Internet: –http://hivdb.stanford.edu –www.hivresistance.com Others?

39. The Virtual Phenotype Genotype Proteas e RTHIV Access Data Genotype & Phenotype Data Virtual Phenotype Wild-type HIV Resistant HIV Illustration by David Spach, MD

40. The Virtual Phenotype Sample report

41. When Should a Resistance Assay be Ordered?

42. Antiretroviral Resistance Testing: Guidelines for Implementation Clinical Setting/ Recommendation Rationale Recommended: Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Chronic HIV infection before starting ART Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted; select regimen accordingly Assays may not detect minor resistant species, but some resistance mutations may persist for years. Consider testing early after diagnosis of HIV infection. Usually not recommended: After discontinuation of drugs Plasma VL <1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low Adapted from DHHS, Antiretroviral Guidelines, October 6, 2005

43. Antiretroviral Therapy: Virologic Failure Medications Started 50 Illustration by David Spach, MD Time

44. Antiretroviral Therapy: Failure to Suppress Medications Started 50 Illustration by David Spach, MD Time

45. Time Trends in Primary HIV-1 Genotypic Drug Resistance Among Recently Infected Persons JAMA. 2002 Jul 10;288(2):181-8. % of resistant isolates

46. Antiretroviral Resistance Testing: Guidelines for Implementation DHHS. Antiretroviral Guidelines, July 14, 2003, Table 3.

47. Antiretroviral Resistance Testing: Guidelines for Implementation Clinical Setting/ Recommendation Rationale Recommended: Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted; select regimen accordingly Consider: Chronic HIV infection before starting ART Assays may not detect minor resistant species, but consider if significant probability of transmitted drug- resistant virus Usually not recommended: After discontinuation of drugs Plasma VL <1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low Adapted from DHHS, Antiretroviral Guidelines, October 6, 2005

48. Reversion to Predominant Wild-Type Virus After Discontinuing ART Illustration by David Spach, MD

50. Drug resistance is Significantly Correlated with Reduction in Replication Capacity Wrin T, et al. 5th International Workshop on HIV Drug Resistance and Treatment Strategies. Scottsdale, AZ: June 2001 (Abstract 24)

51. What is the relationship between adherence and resistance?

52. Antiretroviral Resistance Testing: Guidelines for Implementation Clinical Setting/ Recommendation Rationale Recommended: Virologic failure during ART Suboptimal suppression of viral load (VL) after initiation of ART Acute (primary) HIV infection Chronic HIV infection before starting ART Determine role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine the role of resistance in drug failure and maximize the number of active drugs in the new regimen Determine if resistant virus was transmitted; select regimen accordingly Assays may not detect minor resistant species, but some transmitted resistance mutations may persist for years. Consider testing early in the course of HIV infection. Usually not recommended: After discontinuation of drugs Plasma VL <1,000 copies/mL Resistance mutations may become minor species in the absence of selective drug pressure Resistance assays unreliable if VL is low Adapted from DHHS, Antiretroviral Guidelines, May 4, 2006

53. Persistence of Resistant Strains Following Primary HIV Infection 11 subjects with primary HIV infection who deferred ART and who had at least one major drug resistance mutation identified at presentation, followed with serial resistance assays. –7 subjects with NNRTI resistance –2 with NRTI and PI resistance –1 with NNRTI and PI resistance –1 with resistance to all three classes of drugs NNRTI resistance was lost slowly: the average time to reversion of 103N variants to mixed 103N/K populations was 196 days following the estimated date of infection (153 to 238 days, 95%CI). PI resistance was not lost at all: In the 4 patients with protease resistance mutations, no reversion was detected at 64, 191, 327, and 342 days after infection. Complete reversion of genotypic resistance was observed in only one patient, at 1019 days after infection. Little SJ. 11 th CROI, February 2004, Abstract 36LB

54. Barbour JD et al. AIDS: Volume 18(12) 20 August 2004 pp 1683-1689 Persistence of Resistant Strains Following Primary HIV Infection N = 6 patients infected with resistant strains of HIV; none reverted to wild-type over the course of several months of observation

55. Persistence of Resistant Strains after Primary HIV Infection? Illustration by David Spach, MD

56. Persistence of Some Resistant Strains after Primary HIV Infection ? Illustration by David Spach, MD

57. Testing for Antiretroviral Drug Resistance: Conclusions The proportion of new HIV infections that involve resistant strains tends to increase with increasing availability of ART Initial ART is more likely to fail in patients with a resistant strain In patients treated with ART, resistance mutations, especially those affecting the NNRTIs and PIs, have been found to persist for up to two years after discontinuation of ART Resistance testing is becoming more common in chronically- infected patients in North America who acquired their infection in the past few years

58. What if you cannot obtain a resistance assay for your patient who is failing therapy? Empiric sequencing of ART regimens

59. Empiric design of salvage regimens for patients failing ART: key considerations The genotypic barrier to resistance varies across different antiretroviral agents –For some ARVs, a single mutation can induce high-level resistance (e.g., lamivudine, efavirenz, nevirapine) –For other ARVs, resistance generally does not develop until multiple mutations accumulate (eg, AZT, stavudine, tenofovir)

60. The phenotypic barrier to resistance can vary for different ARVs as well –High serum levels can help to prevent or even overcome resistance mutations Ritonavir boosting of protease inhibitors will increase their phenotypic barrier to resistance –PIs, like many medications, are metabolized in the liver by the cytochrome P450 enzyme complex –Ritonavir inhibits this complex, thereby boosting serum levels of co-administered PIs –Low doses of ritonavir can be used to increase the potency and simplify the dosing of PI-based regimens Empiric design of salvage regimens for patients failing ART: key considerations

61. Time after dose (hours) 024681012 100 1,000 10,000 An Example of Ritonavir Boosting: Indinavir/Ritonavir BID PK Study IDV/RTV q12h: 800/200 High-fat Meal 800/100 High-fat Meal 400/400 High-fat Meal IDV q8h: 800 mg Fasted Indinavir Plasma Concentration (nM) 6th Conference on Retroviruses and Opportunistic Infections; 1999. Abstract 362.

62. Implications of varying genotypic and phenotypic barriers to resistance Resistance develops initially to NNRTIs (efavirenz, nevirapine) and lamivudine If treatment is continued, resistance can subsequently develop to other NRTIs such as AZT, stavudine, didanosine, abacavir, tenofovir Protease inhibitors: –Variable, but generally resistance develops more slowly than to lamivudine and NNRTIs –Ritonavir boosting significantly delays development of resistance to protease inhibitors

63. % of Patients with HIV-1 RNA <400 copies/mL wk 12 NO G, NO Expert (N = 77)36.4% NO G, Expert (N = 67) 49.3% G, NO Expert (N = 69)46.4% G, Expert (N = 65) 69.2% 0 20 40 60 80 100 No G No Expert Op. No G Expert Op. G No Expert Op. G Expert Op. P =.0206P =.00132 (ITT) Tural. 40th IACAC; 2000; Toronto. Presentation LB-10. BLwk 24 Havana Results:

64. Should we discuss resistance with patients?

65. Factors Associated with Higher Levels of Adherence Twice-daily or once-daily regimens 1,4 Belief in own ability to adhere to regimen 1 Not living alone 2 Dependent on a significant other for support 2 History of opportunistic infection or advanced HIV disease 3 1. Eldred L, et al, J Acquir Immune Defic Syndr Hum Retrovirol 1998;18:117-125. 2. Morse EV et al, Soc Sci Med 1991;32:1161-1167. 3. Singh N, et al, AIDS Care 1996;8:261-269. 4. Stone VE, et al. JAIDS 2001; 28:124-131.

66. Factors Associated with Higher Levels of Adherence Belief in efficacy of antiretroviral therapy Belief that non-adherence will lead to viral resistance Wenger N, et al. 6th Conference on Retroviruses and Opportunistic Infections, 1999; Chicago. Abstract 98.

67. Teaching the concept of resistance to patients A cartoon metaphor

68. How Resistance Develops to HIV  This is the virus known as HIV. The only thing that matters to him in his short, nasty life is to destroy T-Cells. To do this, he must somehow get over this wall.  The wall is created by taking anti-HIV medications. When the medicines are taken correctly, the virus is unable to climb over the wall to get to your T-cells.

69. Sometimes the Wall Comes Down  When you forget to take your evening dose, or only take 2 of your anti-HIV medicines, the strong wall comes down.  The virus breaks free and is able to get over the wall.  When he gets to the other side, he discovers a way to get over the wall in the future. This is called resistance. He finds a spring that will give him a little more bounce.

70. The Wall Goes Back Up  When you start taking the medicine regularly again, the wall goes back up.  Sometimes, it’s too late and the virus uses the spring to jump over the wall. At this point, it is a resistant virus The drugs may not be able to keep the wall high enough to stop the springing virus.

71. Lessons to Be Learned  It is better to not take anti-HIV drugs at all than to take them only some of the time.  If you think you may be missing doses often, please tell your health care provider or pharmacist! We promise not to tell your mother.

72. Antiretroviral Resistance Summary & Conclusions

73. Resistance develops in the setting of an inadequately suppressive ART regimen Educating patients about resistance may promote better adherence For the patient who is failing therapy: –Revisit adherence issues –Consider obtaining a resistance assay –Resistance assay results need to be interpreted with caution, and ideally with expert assistance –Salvage regimens can be designed empirically without the assistance of a resistance assay

74. Cases

75. In which of these situations is resistance testing clearly indicated? A 28 yo male just diagnosed with acute HIV infection A 38 yo woman on d4T/3TC/indinavir who had enjoyed full virologic suppression but whose last two HIV viral loads were 72 and 110 copies/mL A 41 yo man on AZT/3TC/nelfinavir whose last three viral loads were 256, 865, and 1838 copies/mL A 35 yo woman with a history of spotty adherence and a viral load of 20,000 copies/mL while on d4T/3TC/efavirenz one year ago. She discontinued all antiretrovirals shortly thereafter, but now wants to restart ART and appears highly motivated.

76. In which of these situations is resistance testing clearly indicated? A 28 yo male just diagnosed with acute HIV infection A 38 yo woman on d4T/3TC/indinavir who had enjoyed full virologic suppression but whose last two HIV viral loads were 72 and 110 copies/mL A 41 yo man on AZT/3TC/nelfinavir whose last three viral loads were 256, 865, and 1838 copies/mL A 35 yo woman with a history of spotty adherence and a viral load of 20,000 copies/mL while on d4T/3TC/Efavirenz one year ago. She discontinued all antiretrovirals shortly thereafter, but now wants to restart HAART and appears highly motivated.

77. Case 1 A 33 yo woman with a baseline CD4 count of 260 cells/mm³ and a viral load of 90,000 copies/mL initiates ART with a regimen of d4T/ddI/nelfinavir. She achieves virologic control with a viral load <50 copies/mL and her CD4 count rises to 420. 6 months later, she develops pancreatitis; 3TC is substituted for ddI, and her viral load remains <50 copies/mL on d4T/3TC/nelfinavir 4 months later her viral load rises to 25,000 copies/mL, and her CD4 count drops to 320.

78. Case 1: Figure Initiate ART Pancreatitis; lamivudine substituted for didanosine Genotype ordered 50 Viral Load (copies/mL)

79. Case 1 continued You order a genotypic resistance assay, which reveals the following mutations: –Reverse transcriptase: M184V –Protease: D30N Which of the following regimens is/are reasonable options for this patient? –ddI/abacavir/efavirenz –AZT/3TC/nevirapine –d4T/abacavir/ritonavir/saquinavir –d4T/tenofovir/efavirenz

80. Case 1 continued You order a genotypic resistance assay, which reveals the following mutations: –Reverse transcriptase: M184V –Protease: D30N Which of the following regimens is/are reasonable options for this patient? –ddI/abacavir/efavirenz –AZT/3TC/nevirapine –d4T/abacavir/ritonavir/saquinavir –d4T/tenofovir/efavirenz

81. Case 2 A 37 yo male initiated ART 5 years ago Initial regimen: AZT/ddI/nevirapine Responded well initially with VL drop to undetectable, rise in CD4 from 240 to 400 cells/mm³ However, experienced virologic failure within one year with rise in viral load to 12,000 copies/mL Regimen changed to d4T/3TC/indinavir; change made without using a resistance assay

82. Case 2 continued He again achieved an undetectable viral load, on his new regimen of d4T/3TC/indinavir 6 months ago lost to follow-up One month ago returned to clinic, describing recent depression and spotty adherence (both of which improved in past month) labs reveal CD4=320, viral load=10,000.

83. Case 2 continued You order a genotypic resistance assay while he is still taking d4T/3TC/indinavir, which reveals: –Reverse transcriptase: M41L, M184V, T215Y –Protease: I84V Which regimen(s) would you recommend? –ddI/nevirapine/nelfinavir –AZT/3TC/tenofovir/lopinavir+ritonavir –AZT/tenofovir/efavirenz –d4T/abacavir/ritonavir/saquinavir –AZT/d4T/lopinavir+ritonavir Failed AZT/ddI/nevirapine in remote past

84. Case 2 continued You order a genotypic resistance assay while he is still taking d4T/3TC/indinavir, which reveals: –Reverse Transcriptase: M41L, M184V, T215Y –Protease: I84V Which regimen(s) would you recommend? –ddI/nevirapine/nelfinavir –AZT/3TC/tenofovir/lopinavir+ritonavir –AZT/tenofovir/efavirenz –d4T/abacavir/ritonavir/saquinavir –AZT/d4T/lopinavir+ritonavir

85. Extra slides

86. REACH study: NNRTIs vs PIs in indigent/homeless in SF (n=108) Unboosted PI or NNRTI x >6 months What is the relationship between adherence and resistance? Viral suppression better with NNRTI vs PI Resistance more common with NNRTIs at low adherence, but declines with higher adherence; trend is opposite with PIs 0-53% 54-79% 80-94% 95-100% n=29 n=28 n=25 n=26 % Resistant 100 90 80 70 60 50 40 30 20 10 0 NNRTI p=0.03 Chi trend PI p=0.47 Chi trend Adherence Quartile p=0.01 Bangsberg DR et al. XV Int AIDS Conf; July 2004, Bangkok. #5820.

87. How much resistance is out there? 89 diagnostic and clinical sites in 6 U.S. states 828 newly diagnosed patients, 95% genotyped Overall prevalence of resistance was 14.5% Underwood M et al. 12th CROI; 2005, Boston. #674. Prevalence of resistance among new 787 HIV diagnostic specimens from 899 sites in six states CategoriesParticipants with HIVDR Any drug class: RTI or primary PI114 (14.5%) NRTI56 (7.1%) NNRTI66 (8.4%) PI22 (2.8%) Two or more drug classes24 (3.1%)