HIV Drug Resistance Training
Module 1:
Introduction to HIV
Drug Resistance
1
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


2
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.
3
impact of drug resistance
Why is it important to measure drug resistance?
How does it impact the success of large-scale treatment programs?
4
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
5
Adults and Children Estimated to be
Living with HIV, 2007
Western & Eastern Europe
Central Europe & Central Asia
730 000
North America
1.2 million
[760 000 – 2.0 million]
Caribbean
230 000
1.5 million
[580 000 – 1.0 million] [1.1 – 1.9 million] East Asia
Middle East & North
Africa
[210 000 – 270 000]
380 000
[280 000 – 510 000]
Sub-Saharan Africa
Latin America
1.7 million
[1.5 – 2.1 million]
22.0 million
[20.5 – 23.6 million]
740 000
[480 000 – 1.1 million]
South & South-East
Asia
4.2 million
[3.5Oceania
– 5.3 million]
74 000
[66 000 – 93 000]
Total: 33 million (30 – 36 million)
6
Number of People Receiving ARV Therapy In
Low- and Middle-income Countries 2002-2007
7
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008
Median Price (USD) of First-line ARV Regimens in
Low-income Countries, 2004-2007
51%
12%
14%
9%
8
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008
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
9
Towards Universal Access – Scaling up priority HIV/AIDS interventions in the health sector. WHO/UNAIDS/UNICEF, June 2008
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
10
HIV DR Testing in Resource Rich Settings
Prevalence of HIVDR at
baseline
Resistance developing on
therapy
Utility of resistance testing
before initiating therapy
Resistance testing before
switching therapy (SOC)
Individualization of 1st line
regimen
Individualization of 2nd line
and subsequent regimens
24+ drugs from 6 classes
11
HIV DR Testing in Resource Limited
Settings
Prevalence of HIVDR at
baseline
Resistance developing on
therapy
Utility
Willof
standard
resistance
1st line
testing
before
regimens
initiating
be effective?
therapy
Prevalence
Resistance and
testing
patterns
before
of
resistance
switching therapy
in population
(SOC)
Individualization
Determination of of
standard
1st line
1st line
regimen
regimen
Individualization
Determination ofof
standard
2nd line
and 2nd
subsequent
line regimens
regimens
24+
drugs from
from 36 classes
classes
~6 drugs
12
Need for Population-based Therapies
1
2
• Need for rapid scale-up
• Limitations in health infrastructure, trained
personnel, facilities, lab capacity, drug
transport and storage
• Need for standardized simplified treatment
protocols
• Regimen selection not by clinicians but by
national policy—first-line and second-line
regimens
13
Need to Maintain Effectiveness
1
2
• Limited number of
regimens available
• Need to minimize drug
resistance
14
Drug Resistance and HIV
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 reemerge if same drugs are stopped and restarted
(even if they are not detected by standard
resistance assays).
15
Review


Why is it important to measure drug resistance?
How does it impact the success of large-scale
treatment programs?
16
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?
17
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
18
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



19
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
20
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!
21
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


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22
Discussion



What regimens influence drug resistance?
What patient factors influence drug resistance?
What public health approaches influence drug
resistance?
23
Reflection


What regimens do we use in our country?
What systematic and programmatic challenges do
we face?
24
how to minimize drug resistance
What can countries do to minimize or suppress drug resistance?
25
Elements of a National HIVDR Prevention and
Assessment Strategy
A.
B.
C.
D.
E.
F.
G.
H.
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
26
Bennett, Bertagnolio, Sutherland and Gilks, Antiviral Therapy 13 Suppl 2:1–13, 2008
Relationship of Different Assessment
Factors
Transmission of DR HIV
to uninfected individuals
Emergence of
HIVDR in treated
patients
ART site factors
associated with minimizing
HIVDR
Threshold Surveys
(Surveillance)
Sentinel ART Site
Monitoring Surveys*
Early Warning Indicators
Genotyping
Laboratory
Genotyping, VL
Laboratory
Non-Laboratory
Data collection
DR HIV prevalence < or
≥ 5%, 15%
To which drugs?
HIVDR Prevention at 12
months; prevalence and
patterns of DR; associated
factors
Areas to directly target for
improvement
PUBLIC HEALTH ACTION
Database,
analysis
27
*Surveys to monitor HIV DR prevention and associated factors in sentinel ARV treatment sites
General WHO Strategies (1 of 2)




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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
28
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

29
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
30
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
31
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
32
HIVDR Early Warning Indicators (EWI)
Proportion lost to
follow-up during the
first 12 months of ART
Patient retention
on first-line ART
Prescribing
practices
Drug supply
continuity
Viral load
suppression
@12 months
Site-level ART
Program
Function
ART
appointmentkeeping
On-time ARV
drug pick up
Pill count/
adherence
33
HIVDR EWI Site-Based Report Example
Site
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
12
104/104 (100%)
10/99 (10%)
891/993 (90%)
483/508 (95%)
5
12
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
12
98/98 (100%)
9/88 (10%)
602/683 (88%)
369/402 (95%)
8
12
203/203 (100%)
43 /195 (22%)
292/356 (82%)
254/284 (86%)
9
12
304/305 (99.7%)
117/260 (45%)
753/1506 (50%)
829/1202 (69%)
10...
12
94/94 (100%)
12/90 (13%)
271/305 (89%)
269/290 (93%)
152
12
33/33(100%)
4/31 (13%)
147/180 (82%)
143/159 (90%)
153
10
26/34 (76%)
7/35 (20%)
148/224 (66%)
129/182 (71%)
154
12
73/73(100%)
9/69 (16%)
178/203 (87% )
146/154 (95%)
34
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 2:113.
35
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:
Countries implementing or planning a national HIVDR strategy
– Africa (3), Asia, Caribbean



22 accredited HIVDR Genotyping labs
HIVDR laboratory training package
Annual external Quality Assurance (supported by NIH,
through the VQA)
Countries with a national HIVDR working group implementing > 1 assessment element as of 2/2009
Countries setting up a working group and planning to implement > 1 assessment element in 2009
36
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

37
Discussion

What can countries do to minimize drug
resistance?
38
Reflection

What are our highest-priority “next steps” for
minimizing drug resistance?
39
how to respond to detection of
drug resistance
What can be done if widespread drug resistance is found?
40
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

41
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.

42
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?
43
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

45
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
46
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
47
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



48
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?

49
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
50
Genotype Assays: Generic Procedure
Patient virus
RT-PCR
PR-RT DNA
Sequencing
Protein Sequence
Selection
Resistance Mutations
Interpretation
Prediction of Drug
Susceptibility
51
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
52
Data Analysis (TruGene)
53
Genotype Report (TruGene)
54
Genotype Report (ViroSeq)
55
Genotyping: Pros and Cons
Advantages
Can be performed rapidly
(days)
 Relatively inexpensive
($100 - $400)
 Available in many labs

Disadvantages
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

56
Phenotype Assays: Generic Procedure
Patient virus
RT-PCR
PR-RT DNA
(Vector Assembly)
(Resistance Test Vector)
Transfection
Recombinant Virus
Infection
Measure of Drug
Susceptibility
57
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)
– Homologous recombination used to generate vector
– Uses VSV-G protein for virus entry
– Replication limited to single cycle
58
Phenotype Report (Monogram)
59
Phenotype Report (Virco)
60
Phenotyping: Pros and Cons
Advantages
Direct measure of drug
susceptibility
 Quantitative
 Can immediately test new
RT and PR inhibitors

Disadvantages
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

61
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
Phenotypic testing
Utility
Genotypic testing
Treatment rounds
62
Phenotype/Genotype Discordance
Genotype
Phenotype
Patient virus
Patient virus
RT-PCR
RT-PCR
PR-RT DNA
PR-RT DNA
(Vector Assembly)
Sequencing
Protein Sequence
(Resistance Test Vector)
Transfection
Selection
Resistance Mutations
Recombinant Virus
Infection
Interpretation
Prediction of Drug
Susceptibility
?
Measure of Drug
Susceptibility
63
PhenoSense GT Report Form (Monogram)
64
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 ("PTR, GT-S")
65
Parkin et al. JAIDS 2002
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
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?


67
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?

68
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


69