1. HIV Drug Resistance Training
Module 1:
Introduction to HIV Drug Resistance
Present: This module will focus on understanding the basics of HIV drug resistance and its importance to maintaining effective treatment regimens for a population.

2. Topics Impact of HIV Drug Resistance
Factors that Influence Development of Drug Resistance
How to Minimize Drug Resistance
How to Respond to Detection of Drug Resistance
Drug Resistance Assays
Present an overview of the topics in this module:
First, we’ll look at how HIV drug resistance can affect local and national efforts to treat HIV.
Then, we’ll look at how drug resistance develops; that is, what factors influence it. Make no mistake; resistance is unavoidable, but our goal is to minimize and delay it.
We’ll explore the basic principles behind DR assays.
Then we’ll look at some general strategies for minimizing drug resistance in a population, and
finally, we’ll discuss some options in the face of widespread drug resistance.

3. Objectives
Understand importance of measuring drug resistance for large-scale treatment programs.
Identify factors that influence drug resistance.
Understand the methods available for the measuring HIV drug resistance.
Identify strategies for minimizing development of drug resistance.
Identify strategies for responding to detection of moderate to high levels of drug resistant HIV.
Present objectives for the module.

4. impact of drug resistance
Why is it important to measure drug resistance?
How does it impact the success of large-scale treatment programs?
Transition to next section. Note that we will try to answer these questions.

5. What is Antiviral Drug Resistance?
Drugs no longer block virus replication
Cause:
Mutations in the viral genome
One or more:
Specific for an antiviral drug OR
Affecting related drugs (cross-resistance)
How much resistance? Which drugs?
Depends on type and number of mutations
Present definition of drug resistance and its causes:
Reduced susceptibility of the virus to a specific antiviral drug (drug is less effective in blocking virus replication)
Caused by mutations in the viral genome, generally located in the gene encoding the protein targeted by the antiviral drug
Mutations can be specific for an antiviral drug or lead to reduced susceptibility to related drugs (cross-resistance)
Magnitude of resistance imparted depends on mutations
In some cases (e.g. 3TC or nevirapine) a single mutation may result in a completely resistant virus (low genetic barrier drugs)
In some cases multiple mutations are needed (e.g. lopinavir or darunavir) to reduce drug activity (high genetic barrier drugs)
Accumulation of mutations may result in increase in the level of resistance (AZT or PIs)

6. Adults and Children Estimated to be Living with HIV, 2007
Western &
Central Europe
[ – 1.0 million]
Eastern Europe & Central Asia
1.5 million
[1.1 – 1.9 million]
North America
1.2 million
[ – 2.0 million]
East Asia
[ – 1.1 million]
Middle East & North Africa
[ – ]
Caribbean
[ – ]
South & South-East Asia
4.2 million
[3.5 – 5.3 million]
Sub-Saharan Africa
22.0 million
[20.5 – 23.6 million]
Latin America
1.7 million
[1.5 – 2.1 million]
Oceania
74 000
[ – ]
Present information on prevalence of HIV.
Total: 33 million (30 – 36 million)

7. Number of People Receiving ARV Therapy In Low- and Middle-income Countries 2002-2007
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008

8. Median Price (USD) of First-line ARV Regimens in Low-income Countries, 2004-2007
51% d4T/3TC/NVP; 14% AZT/3TC/NVP; 12% d4T/3TC/EFV; 9% AZT/3TC/EFV
51% 12% 14% 9%
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008

9. Median Price (USD) of second-line ARV regimens in Low-income Countries, 2004-2007
AZT/3TC/EFV
AZT/3TC/NVP
d4T/3TC/EFV
d4T/3TC/NVP
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008

10. HIV Drug Resistance is Inevitable
HIV DR is an inevitable consequence of ART, influenced by:
Ability of regimens to suppress replication completely
Adherence and tolerability of regimens
"Genetic barrier" to resistance
Relative fitness of resistant variant(s)
Pharmacokinetics (IQ)
Availability/continuity of drug supply
Removal of barriers to access to care
Therefore, efforts to minimize HIVDR should be focused on these factors
Emphasize that while it is impossible to eliminate or completely prevent drug resistance, it is possible and necessary to minimize drug resistance. Barriers = transportation, costs.

11. HIV DR Testing in Resource Rich Settings
Prevalence of HIVDR at baseline
Utility of resistance testing before initiating therapy
Individualization of 1st line regimen
Resistance developing on therapy
Resistance testing before switching therapy (SOC)
Individualization of 2nd line and subsequent regimens
Trainer’s Note: This slide animates upon mouse clicks: (1) left column of boxes, (2) right column of boxes, and (3) box at bottom.
Explain how HIV DR testing occurs in resource rich settings, emphasizing the variety of different regimens available and the individualization of regimens.
24+ drugs from 6 classes

12. HIV DR Testing in Resource Limited Settings
Prevalence of HIVDR at baseline
Will standard 1st line regimens be effective?
Determination of standard 1st line regimen
Prevalence of HIVDR at baseline
Utility of resistance testing before initiating therapy
Individualization of 1st line regimen
Resistance developing on therapy
Prevalence and patterns of resistance in population
Determination of standard 2nd line regimens
Resistance developing on therapy
Resistance testing before switching therapy (SOC)
Individualization of 2nd line and subsequent regimens
Trainer’s Note: This slide animates upon mouse clicks: (1) box at bottom, (2) left column of boxes , and (3) right column of boxes.
Explain how HIV DR testing occurs in resource limited settings, emphasizing the reduced number of different regimens available and the need to have standardized regimens.
24+ drugs from 6 classes
~6 drugs from 3 classes

13. Need for Population-based Therapies1
Need for rapid scale-up
Limitations in health infrastructure, trained personnel, facilities, lab capacity, drug transport and storage 2
Need for standardized simplified treatment protocols
Regimen selection not by clinicians but by national policy—first-line and second-line regimens
Present information on the need to have an effective but limited set of treatment options when resources are scarce:
Rapid scale-up of antiretroviral therapy is an international priority.
Many countries have targeted coverage for 80% of individuals in need of treatment.
Making this scale-up more challenging are infrastructure limitations.
Given these constraints, the public health approach to extend ART rapidly to the maximum number of people relies on standardized simplified treatment protocols, standardized management approaches, and decentralized service delivery.
The decision on an effective first-line regimen (usually one NNRTI and two NRTIs) and a second-line regimen (usually a PI boosted by ritonavir and two NRTIs) is made at the national level, because there is not the money, equipment, facilities, or people to support individually-prescribed regimens. Regimen selection is based on the unique challenges (availability, cost) of that country.

14. Need to Maintain Effectiveness1
Limited number of regimens available 2
Need to minimize drug resistance
Present the importance of minimizing drug resistance given the factors previously described: With standardized simplified treatment protocols across a large number of people, the emergence and spread of drug resistance can have extensive and costly consequences. At the national level, you can minimize drug resistance by carefully monitoring it and by promoting adherence to the recommended regimen.

15. Drug Resistance and HIV
evolves rapidly within human body
has a high replication rate
has a high mutation rate 
Resistant strains can emerge within days if drug pressure is not sufficient to suppress replication.
Resistant strains persist indefinitely and can re-emerge if same drugs are stopped and restarted (even if they are not detected by standard resistance assays).
Present factors related to this virus in particular that lead to drug resistance in a population.

16. Review Why is it important to measure drug resistance?
How does it impact the success of large-scale treatment programs?
Facilitate a group discussion of the questions that were presented at the beginning of the section, as a review and to make sure participants understand the key points.

17. factors that influence development of drug resistance
What regimens influence drug resistance?
What patient factors influence drug resistance?
What public health approaches influence drug resistance?
Transition to next section. Note that we will try to answer these questions.

18. In Which Conditions is DR More Likely?
Treatment with <3 drugs
Inappropriate selection of drugs
Adding one drug to a failing regimen
Interruption of treatment (even for a few days)
Prolonging a failing regimen
Present information on factors that are associated with drug resistance.
An example of inappropriate drug selection would be AZT plus d4T or the use of an NNRTI in a patient co-infected with HIV-2

19. In Which Conditions is DR Less Likely? Medication Factors
All patients treated with 3 or more drugs
Use of appropriate drug regimens
Can reliably suppress HIV replication to levels of <50 copies/ml
Use of fixed-dose combinations to support adherence
Present information on factors that are associated with avoiding widespread drug resistance.

20. In Which Conditions is DR Less Likely? Systems Factors
Limited number of regimens
Trained personnel, low turnover
Supervision and monitoring
Adequate lab services
Drug supply and delivery systems
Present information on factors that are associated with avoiding widespread drug resistance.

21. In Which Conditions is DR Less Likely? Patient Factors
Adherence to treatment regimen
Avoiding interruption of treatment, even if only a few days
Regular follow-up (going to clinic)
Staying on uninterrupted first-line ART as long as possible
CONTINUITY!
Present information on factors that are associated with avoiding widespread drug resistance.

22. Programmatic Factors Affecting Patient Compliance
Cost of treatment to patient
Distance patient must travel to get treatment
Supply interruptions
Availability of second-line regimens for patients whose first-line regimens fail
Timing of use of second-line regimens
Present information on factors that are associated with avoiding widespread drug resistance.

23. Discussion What regimens influence drug resistance?
What patient factors influence drug resistance?
What public health approaches influence drug resistance?
Facilitate a group discussion of the questions that were presented at the beginning of the section, as a review and to make sure participants understand the key points.

24. Reflection What regimens do we use in our country?
What systematic and programmatic challenges do we face?
Ask participants to write their own answers to these questions in the blank space on their handouts.

25. how to minimize drug resistance
What can countries do to minimize or suppress drug resistance?
Transition to next section. Note that we will try to answer these questions.

26. Elements of a National HIVDR Prevention and Assessment Strategy
Development of a national HIVDR Working Group, five year plan and budget
Regular assessment of HIVDR early warning indicators (EWI) from all ART sites (or representative sites)
Surveys to monitor HIVDR and associated factors in sentinel ART sites
HIVDR transmission threshold surveys in geographic areas where ART has been widespread for > 3 years
HIVDR database development
Designation of an in-country or regional WHO-accredited HIVDR genotyping laboratory
HIVDR minimization activities review and support
Preparation of annual HIVDR report and recommendations; use of data for ART and planning
Present an overview of the strategies.
Bennett, Bertagnolio, Sutherland and Gilks, Antiviral Therapy 13 Suppl 2:1–13, 2008

27. Relationship of Different Assessment Factors
Transmission of DR HIV to uninfected individuals
Threshold Surveys
(Surveillance)
Genotyping Laboratory
DR HIV prevalence < or ≥ 5%, 15%
To which drugs?
Emergence of HIVDR in treated patients
Sentinel ART Site Monitoring Surveys*
Genotyping, VL Laboratory
HIVDR Prevention at 12 months; prevalence and patterns of DR; associated factors
ART site factors
associated with minimizing HIVDR
Early Warning Indicators
Areas to directly target for improvement
PUBLIC HEALTH ACTION
Non-Laboratory Data collection
Present: This slide shows how the bullets on the previous slide relate to each other, as they relate to assessment. (Click to show each column in turn.)
Emphasize:
Depending on what is going on with patients, you have different sources of data. Genotyping information can help inform the public health action. (For example, if you have DR of 15%+ to a given regimen, may need to change that regimen.)
Specific ART programme issues are known to be associated with the emergence of HIVDR
HIVDR EWI collection should start even before surveys that include genotyping are implemented
Surveys indicating the emergence of resistance are of limited use without ART programme information on which to base public health action
Database, analysis
*Surveys to monitor HIV DR prevention and associated factors in sentinel ARV treatment sites

28. General WHO Strategies (1 of 2)
Strengthen existing programmes that minimize HIVDR
Form national HIV drug resistance working group
Monitor Early Warning Indicators
Survey to monitor HIVDR prevention and associated factors in sentinel ART sites
Watch for transmitted HIVDR in recently infected individuals
Present: These are general strategies recommended by the World Health Organization. We’ll explore each in a little more detail in the following slides.
Each country should judge the priorities of the different elements of the overall strategy (EWI, monitoring and transmission surveys) in the context of their ART treatment programmes and resources; in general the order listed is the preferred order of priorities

29. General Strategies (2 of 2)
Designate one or more genotyping labs for HIVDR surveillance and monitoring
Maintain a national database to hold the HIV sequences collected through surveillance, monitoring, and research projects
Produce an annual report summarizing results from all the above
(continued from previous)

30. Strengthen Existing Programmes That Minimize HIVDR
Support for adherence and follow-up
Removal of barriers to ART access
Drug supply continuity at the individual, ART site, and national levels
Present information on this strategy.

31. Form National HIV Drug Resistance Working Group
Develop HIVDR prevention, surveillance and monitoring plan
Make evidence-based recommendations for HIVDR prevention
Includes epidemiologists, clinicians, researchers, and laboratorians
Collect indicators and implement surveys
Develop evaluation strategies
Present information on this strategy.

32. Monitor Early Warning Indicators
Routine collection of medical and pharmacy records
Monitor for factors associated with HIVDR prevention or emergence
Extent to which prescribing practices meet national and international guidelines
% of patients still on first-line; % lost to follow-up
% patients with timely medication pick up and clinical follow-up
Drug supply continuity at site
Adherence and viral load, if feasible
Present information on this strategy.

33. HIVDR Early Warning Indicators (EWI)
Proportion lost to follow-up during the first 12 months of ART
Prescribing practices
Patient retention on first-line ART
Site-level ART Program Function
Drug supply continuity
On-time ARV drug pick up
Present: This side shows ix different recommended EWIs (in black) and two additional EWIs (in blue). These blue ones are good, but only cost effective if there are already infrastructures in place to gather the data.
Emphasize:
On time pickup: % of ART patients picking up prescribed ARV drugs on time (before previous drugs run out) Suggested target: ≥ 90%
Drug supply: ART stops, substitutions, and switches due to ARV shortages during a specified period Suggested target: 0%
OR % of months during a year with no antiretroviral drug stock outages Suggested target: 100%
Viral load months
ART appointment-keeping
Pill count/ adherence

34. HIVDR EWI Site-Based Report Example
Months with no ARV drug stockouts
Target: 12
% appropriate Initial ART regimen prescriptions
Target: 100%
% starting first line ART lost to follow up at 12 months
Target: < 20%
% on ART keeping all clinical appointments on time
Target: > 80%
% on ART picking up all ART drugs on time
Target: ≥ 90% 1 12
94/94 (100%)
4/96 (4%)
182/209 (87%)
184/192 (96%) 2 10
81/81 (100%)
9/74 (12%)
342/402 (85%)
176/220 (80%) 3 9
31/40 (78%)
12/37 (32%)
122/244 (50%)
144/206 (70%) 4
104/104 (100%)
10/99 (10%)
891/993 (90%)
483/508 (95%) 5
112/112(100%)
13/105 (12%)
262/305 (85%)
184/202 (91%) 6 11
98/101 (97%)
2/90 (2%)
416/442 (95%)
254/359 (71%) 7
98/98 (100%)
9/88 (10%)
602/683 (88%)
369/402 (95%) 8
203/203 (100%)
43 /195 (22%)
292/356 (82%)
254/284 (86%)
304/305 (99.7%)
117/260 (45%)
753/1506 (50%)
829/1202 (69%)
10...
12/90 (13%)
271/305 (89%)
269/290 (93%)
152
33/33(100%)
4/31 (13%)
147/180 (82%)
143/159 (90%)
153
26/34 (76%)
7/35 (20%)
148/224 (66%)
129/182 (71%)
154
73/73(100%)
9/69 (16%)
178/203 (87% )
146/154 (95%)
Present: This is what the results look like when gathering EWI data. The ones in red show targets not met. Look for patterns across indicators (rows) and across labs in country (columns).
Discuss what patterns they see in this chart (good and bad).

35. For More Information
See Bennett, Bertagnolio, Sutherland and Gilks (2008). The World Health Organization’s global strategy for prevention and assessment of HIV drug resistance. Antiviral Therapy 13 Suppl 2:1-13.
Distribute the article and point out that it explains a lot of the information in this manual as well as listing the Early Warning Indicators. This article can serve as a resource, for later reading.

36. Implementation of the WHO HIVDR Strategy – June 2009
Implementation of at least 1 component of the HIVDR strategy in 41 countries
HIVDR transmission surveys completed in 11 countries
EWI piloted in 23 countries
HIVDR training workshops:
Africa (3), Asia, Caribbean
22 accredited HIVDR Genotyping labs
HIVDR laboratory training package
Annual external Quality Assurance (supported by NIH, through the VQA)
EWIs have been piloted in 19 countries, Number of ART sites monitored range from 2-89 sites
EWI 1: Few countries < 100% appropriate prescriptions in all sites; Inappropriate prescribing often associated with drug stock-outs
EWI 2: % sites meeting < 20% target for LTFU range from 40% - 95%
EWI 3: Still on first-line ART at 12 months: % sites meeting >70% target range from 50% - 93%
EWI 4: On time drug pick-up: % of sites meeting > 90% target range from 0% to 85%
EWI 5: On time clinic appointment keeping: % of sites meeting > 80% target range from 20% - 95%
EWI 6: Drug Stockouts: Over 30% of countries reported some first-line regimen stockouts. % of sites meeting 100% target range from 60% to 100%.

37. Other
Designate one or more genotyping labs for HIVDR surveillance and monitoring
The WHO offers accreditation, support, and training through the WHO Global HIVDR laboratory network
Maintain a national database to hold the HIV sequences collected through surveillance, monitoring, and research projects
Produce an annual report summarizing results from all the above

38. Discussion What can countries do to minimize drug resistance?
Facilitate discussion:
Ask participants to work in table teams (two or four people per team).
Ask them to go over the list of strategies just presented and discuss how each can be put in place in their locales.
Allow about 10 minutes.
Ask each group to report out one strength and one challenge facing them at the national level.

39. Reflection
What are our highest-priority “next steps” for minimizing drug resistance?
Ask participants to write their own answers to this question in the blank space on their handouts.

40. how to respond to detection of drug resistance
What can be done if widespread drug resistance is found?
Transition to next section. Note that we will try to answer this question.

41. Response to Detection of Transmitted Drug Resistance
If the prevalence of transmitted resistance to important drugs or drug classes is 5-15% or > 15%, surveys should be repeated in the original areas annually and extended to additional areas
Consider changing standard 1st line regimen
Monitor Early Warning Indicators for clues about what can be improved at a programmatic level
Present: Minimizing drug resistance is the most important step, because once drug resistance is widespread, the only alternative is to adopt a different standard regimen (national treatment regimen) using drugs to which the current mutations are not resistant. However, this is a very costly step, so again, prevention is the key!

42. Response to Results from Monitoring Surveys
If the patterns of resistance indicate reduced susceptibility to 2nd line drugs, surveys should be repeated in the original areas annually and extended to additional areas.
Consider changing standard 2nd line regimen.
Monitor Early Warning Indicators for clues about what can be improved at a programmatic level.
Present: Minimizing drug resistance is the most important step, because once drug resistance is widespread, the only alternative is to adopt a different standard regimen (national treatment regimen) using drugs to which the current mutations are not resistant. However, this is a very costly step, so again, prevention is the key! 42

43. hiv drug resistance assays
What are the different types of resistance assays?
What are the advantages and limitations of these assays?
What results can we expect from these tests?
What if the results from one type of test are inconsistent with results from another?
Transition to next section. Note that we will try to answer these questions. 43

44. Advantages of Drug Resistance Testing
Shows which drugs not to use
Save costs associated with switching drugs too early or using drugs that are no longer effective
Avoid toxicity of inactive drugs
May avoid further development of resistance
Improved short term response in several prospective studies
Present information on advantages of DR testing. 44

45. Clinical Use of Resistance Data
Resistance tests are most accurate in assessing resistance to current regimen
Absence of resistance to previously used drug does not rule out reservoirs of resistant virus that might emerge after re-initiation of that drug
If resistance to given drug has ever been detected, that drug should probably not be used again, even if current test results suggest viral susceptibility
Present the importance of drug resistant testing. 45

46. Conditions for Drug Resistance Testing: Individual Patient Management
When to use:
When there are treatment options
To indicate drug exposure
While patient is on therapy or before starting for the 1st time
What to consider:
Treatment history, other reasons for therapy failure
Lab-to-lab variability of results and interpretations
Requires expert advice for optimal use
Only test single agents, not combinations
Present information on when drug resistance testing is helpful along with factors that may influence results. 46

47. Conditions for Drug Resistance Testing: Public Health Applications
When to use:
When making decisions for national treatment guidelines
1st line regimens (based on results of surveillance for transmitted drug resistant HIV)
2nd line regimens (based on patterns of resistance in treated patients)
To monitor program effectiveness minimizing HIVDR
What to consider:
Early Warning Indicators, programme and site factors
Treatment history, other reasons for therapy failure
Lab-to-lab variability of results and interpretations
Present information on when drug resistance testing is helpful along with factors that may influence results. 47

48. Limitations of Resistance Testing:
Only possible in case of a detectable viral load
Lack of consistent quality control (see ENVA)
Current assays do not pick up “minority species;” information is given on predominant strain
Assays have mostly been studied in “late” failures; what is their value in early failures?
Susceptibility is not equal to activity (clinical efficacy)
Clinical validation: more data necessary
Present limitations on DR testing.
Discuss: Based on the pros and cons of resistance testing, what value do you see to DR testing in your country? What potential benefits do you hope to see? What are some of the most important obstacles to getting those benefits? 48

49. Reflection
What benefits do I hope to see through DR testing in our country?
What are my concerns right now about this type of testing?
Ask participants to write their own answers to these questions in the blank space on their handouts. 49

50. Types of Resistance Assays
Genotypic Testing: Prediction of phenotype based on sequence
Phenotypic Testing: Measure of susceptibility to specific drugs
Recombinant Assays: RT/PCR portion of patient virus and transfer into a vector
Several different versions commercialized, automated and regulated
PBMC Assay: Culture virus from patient
Largely replaced by recombinant assays due to difficulties in reproducibility and throughput
Present:
There are two types of tests which may be performed.
Genotyping is more common in resource-limited countries, as it is less expensive and less time consuming. 50

51. Genotype Assays: Generic Procedure
Patient virus
PR-RT DNA
RT-PCR
Protein Sequence
Sequencing
Resistance Mutations
Selection
Present:
This shows the basic sequence of genotypic assays.
We’ll explain this in more detail in later modules.
Right now the important thing is to see that it is a multi-step procedure.
Sources of subjectivity/variability include:
Detection of mixtures in the sequence data
Selection of which mutations are considered to be relevant for each drug
The algorithm used to interpret each set of mutations
Prediction of Drug Susceptibility
Interpretation 51

52. Sources and Types of Genotype Assays
Reference Labs (North America and Europe)
Send blood sample in, receive results on report
LabCorp, Quest, AML, Specialty, Mayo, etc.
Monogram Biosciences, Virco
Commercial kits
Can be performed on site (in laboratory)
TruGene (Siemens)
ViroSeq (Abbott/Celera)
In-house assays
"home brew" assay performed at one site (clinical, hospital or research laboratory)
Must be validated and approved if used for patient management
Present information on the pros and cons of different sources of genotype assays.
Emphasize: Validation and approval of in house assays is dependent on the regulatory authority (e.g., in the US, CLIA and CAP. Kits are FDA-regulated, both kits listed here are approved.) 52

53. Data Analysis (TruGene)
Present: This is a sample of the data one sees at the “sequence” step.
Explain that the patterns in the chromatogram can be examined to identify mixtures.
Ask participants to identify any mixtures they see on the chromatogram. 53

54. Genotype Report (TruGene)
Present: This is an example of the report that is generated as a result of the genotyping assay. Although the reports are slightly different from one source to another, they all contain the same basic information.
Explain how to read this report. 54

55. Genotype Report (ViroSeq)
Present: This is another example of a genotype report.
Explain how to read this report. 55

56. Genotyping: Pros and Cons
Advantages
Disadvantages
Can be performed rapidly (days)
Relatively inexpensive ($100 - $400)
Available in many labs
Does not directly measure susceptibility
Sometimes difficult to interpret results
Not all patterns of resistance mutations are known (esp. for new drugs and combinations)
Generally qualitative
Subjectivity in mixture detection
Present the pros and cons of genotyping. 56

57. Phenotype Assays: Generic Procedure
Patient virus
RT-PCR
PR-RT DNA
(Resistance Test Vector)
(Vector Assembly)
Recombinant Virus
Transfection
Present: An alternative to genotyping is phenotyping.
Present a general overview of the steps in phenotyping.
Measure of Drug Susceptibility
Infection 57

58. Commercially Available Phenotypic Assays
All are recombinant virus phenotypic assays
Antivirogram (Virco)
Homologous recombination used to generate vector
Replication occurs over multiple cycles
PhenoSense (Monogram)
Vector generated directly by digestion and ligation
Replication limited to single cycle
High precision and reproducibility
Phenoscript (VIRalliance)
Uses VSV-G protein for virus entry
Compare and contrast the different types of commercially available phenotypic assays. 58

59. Phenotype Report (Monogram)
Present: This is one example of a type of report you would get by sending a sample to a testing company for phenotyping.
Explain how to read the report. 59

60. Phenotype Report (Virco)
Present: This is another example of a type of report you would get by sending a sample to a testing company for phenotyping.
Explain how to read the report. 60

61. Phenotyping: Pros and Cons
Advantages
Disadvantages
Direct measure of drug susceptibility
Quantitative
Can immediately test new RT and PR inhibitors
Longer time to obtain results (weeks)
Relatively complex technology
More expensive than genotypic assays
Available in fewer labs
Clinical cutoff values for drug resistance not clearly defined for all drugs
Present: As you might expect, the pros and cons for phenotyping are the opposite as for genotyping; that is, it directly measures drug susceptibility, but it is more complex and expensive.
Because of its disadvantages, phenotyping is generally done only at central reference labs. 61

62. GENO vs PHENO? A gross oversimplification:
Utility of genotypic testing greatest earlier in treatment continuum
Utility of phenotypic testing increases with subsequent treatment rounds
Increasing Genetic Complexity
Present: Another way of comparing genotyping to phenotyping is to look at WHEN each one is most useful.
Present information on slide. Emphasize that this is a generalization.
Genotypic testing
Phenotypic testing
Utility
Treatment rounds 62

63. Phenotype/Genotype Discordance
Patient virus
Patient virus
PR-RT DNA
RT-PCR
RT-PCR
PR-RT DNA
Protein Sequence
Sequencing
Resistance Mutations
Selection
Prediction of Drug Susceptibility
Interpretation
(Resistance Test Vector)
(Vector Assembly)
Recombinant Virus
Transfection
Measure of Drug Susceptibility
Infection
Present:
As we presented earlier, genotyping and phenotyping lead to different types of results. Genotyping offers a prediction of drug susceptibility, while phenotyping actually measures it.
Prediction of susceptibility to a given drug(s) based on genotype is dependent on complete understanding of the complex relationship between patterns of mutations and viral phenotype.
Early in a drug’s development, knowledge of the effects of various mutations is gathered from in vitro studies and early clinical trials (usually does not include all possible “real-world” cases).
Therefore, there may be a discordance, or inconsistency, between results of the two types of tests. ? 63

64. PhenoSense GT Report Form (Monogram)
??? 64

65. 3 Types of Phenotype-Genotype Discordance
Genotype predicts resistance, not reflected in phenotype ("PT-S, GT-R"); mixtures of resistant and sensitive viruses are present in the specimen
Genotype predicts resistance, not reflected in phenotype ("PT-S, GT-R"); not explained by mixtures
Genotype predicts susceptibility, but phenotype detects reduced susceptibility or resistance ("PT-R, GT-S")
Present the three types of discordance.
PT-S = phenotype is “sensitive”
PT-R = phenotype is “resistant”
GT-S = genotype is “susceptible”
GT-R = genotype is “resistant”
Parkin et al. JAIDS 2002 65

66. Genotype/Phenotype “Discordance”
Previously unrecognized cross-resistance (or lack of expected cross-resistance).
Previously unrecognized resistance-associated mutations (“RAMs”).
New amino acids at known positions.
e.g. N88S, I84A, K103S, I47A, K101P, V106M…
Effect of recognized RAMs tempered by other mutations.
e.g. L90M, V82A, K103N, suppression of ZDV by 184, etc.
Particular combinations of polymorphisms or “secondary” mutations
e.g. K103R/V179D 66

67. Discussion What are the different types of resistance assays?
What are the advantages and limitations of these assays?
What results can we expect from these tests?
What if the results from one type of test are inconsistent with results from another?
Facilitate a group discussion of the questions that were presented at the beginning of the section, as a review and to make sure participants understand the key points. 67

68. Reflection
What type of DR assay is most appropriate for our situation?
What results can we expect?
What factors should we keep in mind about this type of assay?
Ask participants to write their own answers to these questions in the blank space on their handouts. 68

69. Summary Impact of HIV Drug Resistance
Factors that Influence Development of Drug Resistance
How to Minimize Drug Resistance
How to Respond to Detection of Drug Resistance
Drug Resistance Assays
Ask select participants to summarize, in one or two sentences, the key points they learned in this module.