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HIV Resistance Testing: An
UpdateBen J. Barnett, MD
University of Texas Health Science Center, Houston
May 26, 2010
Outline
Overview of HIV resistance Resistance testing
Types of tests available Indications for use Test limitations
Interpretation of resistance tests New drugs and new assays
Case
54 year old man from Houston, diagnosed HIV positive 12/09 Found by “universal screening” in HCHD
ER Presenting syndrome: pneumonia
PMHx GERD, psoriasis, depression
Social Hx HIV risk: MSM Not in relationship now, casual partners
in past
Initial Evaluation
Clinically appeared well except for wasting syndrome and oral thrush
Initial laboratory tests Blood counts, liver, kidney tests normal Hepatitis B and C negative CD4 = 16 (5%) HIV viral load 650,000 copies/mL (5.81
log)
Some Considerations in starting HAART
Patient adherence potential Comorbid conditions that complicate
ARV treatment Potential drug interactions Potential adverse effects Convenience Results of genotypic drug resistance
testing Do we need to be concerned about
resistance in this case?Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.
Initial Genotype
“Transmitted” HIV drug resistance
Definition The presence of a resistant strain of HIV in a
person who has never taken antiretroviral drugs
Transmission Drug resistant HIV can be transmitted to a
previously HIV negative person by any way HIV is usually transmitted
Also can occur by “super-infection” but this is rare
The only way to know if someone has been infected with a resistant strain is to do a resistance test on the newly diagnosed person Called “Baseline” resistance testing
Baseline Genotypes, Thomas Street clinic1999 2003-04
Sample size 44 40CD4 288 (6-904) 274 (9-727)Time since HIV diagnosis (mo.)
17 (0-144) 39 (1-236)
NRTI mutations
2 (4.5) 2 (5)
NNRTI mutants
1 (2.3) 2 (5)
PI 1 (2.3) 1 (2.5)Total mutants
4 (9.1) 5 (12.5)
Increasing Primary Drug Resistance: CDC Survey
1. Bennett D, et al. CROI 2002. Abstract 372. 2. Bennett D, et al. CROI 2005. Abstract 674.
Prevalence of Drug Resistance, %
1998[1] (n = 257)
1999[1
] (n = 239)
2000[1]
(n = 299)
2003-2004[2]
(n = 787)
Any drug 5.5 8.8 10.7 14.5
NRTI 5.1 7.1 7.7 7.1
NNRTI 0.4 2.1 1.7 8.4
PI 0 0.8 3.0 2.8
≥ 2 drug class
0 1.3 1.3 3.1
Transmitted Drug Resistance in
US: Newly Diagnosed 2007 CDC surveillance
for transmitted drug resistance (TDR) 10,496 with new HIV
Dx 2,480 with genotype
TDR detected in 16% of patients with new HIV diagnosis Most common: NNRTI 83% had single
mutation No demographic risks
factors identified
HIV TDR Surveillance Areas (2007)Seattle-King
County
HIV TDR (2007)
Per
cent
with
TD
R50% K103N
Kim D, et al. 17th CROI; San Francisco CA USA; February 16-19, 2010. Abst. 580.
What Is HIV Resistance?
The ability of HIV to multiply in the presence of suppressive levels of antiretroviral drugs
Does not make HIV more
pathogenic Perhaps less pathogenic?
How Does Resistance Occur?
Primary transmission of resistant virus (TDR)
Sub-suppressive levels of anti-viral drugs and then natural selection of mutant strains
Partial adherence to regimen
Sub-optimal dosing of drugs
Drug interactions
Incomplete absorption in intestinal tract
Hirsch. JAMA 1998;279:1984.
Viral Resistance is the Outcome of Viral Replication, Mutations and
Selection Pressure
Viral Resistance is the Outcome of Viral Replication, Mutations and
Selection Pressure
Original VirusOriginal VirusQuasispeciesQuasispecies
Selection PressureSelection Pressureexerted by Drugsexerted by Drugs
HIV
RN
A L
evel
New VirusNew VirusQuasispeciesQuasispecies
Resistant cloneResistant cloneResistantResistant clonesclones
Time
Resistance Testing
Methods to measure resistance
Genotype Direct sequencing of viral genes reverse
transcriptase and protease, less commonly integrase and envelope
Resistance to specific drugs is predicted based on known mutations
Requires some knowledge of which mutations affect which drugs
Phenotype Grow virus in culture with various amounts of
drugs added Direct measure of viral resistance Does not explore the underlying mutations, just
their affect on the ability of the drug to stop the virus
Genotype reporting Sequencing of various HIV genes and
comparing result to reference strain Example: M 184 V in reverse
transcriptase 184 refers to amino acid position 184 in
enzyme M (methionine) is the “wild-type” amino
acid V (valine) is the “mutant” amino acid
“Mixtures” are when both WT and mutant amino acids are detected M 184 M/V
Hirsch. JAMA 1998;279:1984.
Genotypic Assays
Relative Advantages
Wide Availability Results available
in days Less technically
demanding Less expensive FDA approved
kits
Relative Limitations Indirect measure
of susceptibility
Expert interpretation required
Unable to detect linkage of multiple mutations or the overall affect of many mutations
Example of Genotype Report
Phenotypic Assays
Direct measure of viral susceptibility to individual drugs, reported as “fold change”
Drug Concentration
Inh
ibit
ion
of
Vir
al
Re
plic
ati
on
(%
) 100
0
ICIC5050
50
ICIC5050
Fold Fold ResistanceResistance
Wild-type strain
Mutant strain
Reviewed in Wilson. AIDS Read 2000;10:469.
Phenotypic AssaysRelative Advantages Direct measure
of susceptibility Fold change
in IC50
May account for effects of multiple mutations at different sites
Relative Limitations Restricted availability Results may take 2-3
weeks Technically demanding Clinically significant
cut-off values notdefined for many drugs
More expensive
Hirsch. JAMA 1998;279:1984.
Example of Phenotype Report
General Limitations of Resistance Assays
Viral load must be at least 500 - 1000 copies
Mutations are detected only if mutant virus is at least 10-20% of virus population Viral “mixtures” or minor variants can be
missed by genotypes, and not accounted for by phenotypes.
Resistance tests are most accurate in assessing the current regimen If resistance has ever been detected, then
archived mutations exist If no drug pressure exists, “wild type” virus
will often overgrow the mutant strains
Discordance between GT and PT
Incomplete genotypic algorithms (rules) Novel mutations discovered Improper weighting of mutations in
algorithms (both over- and under-weighted) Non-B subtype resistance patterns
Mixtures are present (some wild type, some mutant)
Suppressive mutations or “re-sensitization” caused by specific mutations (e.g. 184V)
Discordance due to mixtures
Result: Phenotype reads “sensitive” because of presence of wild-type virus in the mixture But Genotype predicts “resistant” due to presence of mutant virus in the mixture
Incomplete Rules may lead to discordance
Result: Genotype predicts resistance based on rules-based algorithm, but Phenotype reads “sensitive” due to interactions of complex mutation patterns
PT GT NET
“Virtual” Phenotype
27
DHHS Guidelines 12/09 Recommended in acute HIV
infection In chronic infection, recommended
for all patients on entry into care, regardless of treatment plan If therapy deferred, consider retesting
on treatment initiation Recommended to assist in selecting
active drugs for patients with viral failure and HIV RNA > 1000 copies Consider for viral load > 500 copies
Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.
DHHS Guidelines 12/09
Should be performed when managing suboptimal viral load reduction
In the setting of viral failure, testing should be done while the patient is on therapy, or within 4 weeks of stopping
Should be done for all pregnant women prior to therapy, or for those entering pregnancy with a detectable HIV viral load on therapy
Which Resistance Test When? (DHHS)
Genotype preferred due to faster result, lower cost and enhances sensitivity for detecting “mixtures” In anti-retroviral naïve patients In patients with sub-optimal viral response on therapy Virologic failure on a first or second regimen
Phenotype “Addition of phenotypic testing to genotypic testing is
generally preferred for persons with known or suspected complex drug resistance mutation patterns, particularly to protease inhibitors”
Virtual Phenotype (not specifically stated in DHHS) As a substitute when actual phenotype not available
Interpretation of Resistance Tests
Genotype interpretation
Memorize common mutations Pattern recognition Most assays come with an “expert”
interpretation included, but be careful http://hivdb.stanford.edu is a reliable resource
Limited by complex interactions of multiple mutations, especially for protease inhibitors
Newer drugs come with “prediction rules”
Mutations in Reverse Transcriptase
Mutations in Reverse Transcriptase
NucleosideMutation Sites
Non-NucleosideMutation Sites
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIs
Abacavir
Didanosine
Emtricitabine
Lamivudine
Stavudine
Tenofovir
Zidovudine
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIsMutations Selected by nRTIs
Multi-nRTI Resistance: 69 Insertion Complex (affects all nRTIs currently approved by the US FDA)
Multi-nRTI Resistance: 151 Complex (affects all nRTIs currently approved by the US FDA except tenofovir)
Multi-nRTI Resistance: Thymidine Analogue-Associated Mutations (TAMs; affect all nRTIs currently approved by the US FDA)
Major nRTI mutations M184V
Resistance to lamivudine and emtricitabine Some resistance to didanosine and abacavir May restore some activity to zidovudine and
tenofovir K65R
Broad resistance to all nRTI except zidovudine
L74V Resistance to abacavir and didanosine
Thymidine analogue mutations (TAMS) Affect all nRTI to a variable degree
Even with nRTI resistance, most nRTI retain some activity and may help suppress virus with other agents
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIs
Efavirenz
Etravirine
Nevirapine
Major NNRTI mutations K103N
Most common NNRTI mutation High level resistance to efavirenz and
nevirapine but not etravirine Y181C
High level resistance to nevirapine and intermediate efavirenz resistance
Some etravirine resistance, but provides a mutational foundation for development of higher levels of resistance
Broad cross resistance with nevirapine and efavirenz with low genetic barriers Single mutation can eliminate activity of EFV
or NVP No impact of NNRTI mutations on viral
replication ability, so continued use of NNRTI in the face of resistance adds nothing
Resistance Mutations in Protease
8282
8484
9090
4848
5454
3030
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by PIsMutations Selected by PIs
Atazanavir +/-ritonavir
Darunavir/ritonavir
Fosamprenavir/ritonavir
Indinavir/ritonavir
Lopinavir/ritonavir
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by PIs (cont)Mutations Selected by PIs (cont)
Nelfinavir
Saquinavir/ritonavir
Tipranavir/ritonavir
Major protease mutations
“Signature” mutations for non-boosted PI D30N: nelfinavir I50L: atazanavir I50V: fosamprenavir
Boosted PIs usually do not select for mutations if used as first PI with nucleosides Otherwise have broad general cross
resistance Gets very complicated very quickly
Newer protease “mutation score”
Daruavir is a newer PI with activity against resistant HIV, approved June 2006
POWER studies showed patients treated with darunavir and optimized background meds had VL < 50 copies/mL greater than for comparator PIs
Response to darunavir was found to be dependent on 11 PI mutations at baseline
Darunavir response by DRV score
0-2 mutations 3 mutations 4 mutations
50%
22%
10%Pati
en
ts (
%)
wit
h H
IV-1
R
NA
<
50
cop
ies/
mL
at
Week
24
Number of Darunavir mutations at baseline
A wrinkle: The common PI mutation at 82A may actually have a positive effect on viral response to darunavir
Pro
bab
ilit
y of
resp
on
se
Fold Change
Lower clinical cutoffResponse is significantly reduced
Upper clinical cutoffResponse is unlikely
“Zone of Intermediate Response”
Interpreting PhenotypesCutoffs differ for each drug
Newer drugs and assays
Etravirine “Second generation” NNRTI
Raltegravir Integrase inhibitor
Entry inhibitors Enfuvirtide Maraviroc
Etravirine
Second generation NNRTI FDA approved January 2008
May retain activity against HIV with NNRTI resistance from NVP or EVF K103N alone does not affect etravirine
Has a higher “genetic barrier” than other NNRTI, and therefore a “mutation score” has been developed similar to PIs
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIsMutations Selected by NNRTIs
Efavirenz
Etravirine
Nevirapine
TTCA0066-07332-29UN
Impact of BL Resistance in DUET: The Number of Baseline ETV RAMs Correlated with the Virologic Response (<50 copies/mL)
Number of ETV RAMs present at baseline
Placebo + OBT (n=414)
ETV + OBT (n=406)
0 2 3 41
7/28 3/18
25
17
25
41
6/2413/32
25
58
17/6837/64
38
59/15773/121
60
64/147
121/161
75
44
Pati
en
ts w
ith
con
firm
ed
vi
ral
load
<
50 H
IV-1
RN
A c
op
ies/
mL
(%
)
0
20
40
60
80
Vingerhoets J, et al. 11th EACS 2007. Abstract P7.3/05
7/28 3/186/2413/3217/6837/6459/15773/12164/147
121/161
3 or more ETV associated mutations give a reduced response to ETV
TTCA0100-08173-8UN
aMedian (Q1–Q3) FC for all isolates was 3.0 (1.1–9.3);bV179F was never present as single INTELENCE RAM (always with Y181C)
Median Q1–Q3 n
Y181I 1.5 42.0 23.2–129.7 34 12.5 High 3Y181V 0.9 10.4 3.9–60.6 28 17.4 High 3K101P 2.6 22.3 5.6–42.9 65 6.2 High 2.5L100I 8.4 6.7 2.7–17 264 1.8 Medium 2.5Y181C 32.0 4.4 2.1–11.6 552 3.9 Medium 2.5M230L 1.1 4.3 2.7–10.5 20 3.4 High 2.5E138A 2.5 2.9 1.4–10.6 44 2.0 Medium 1.5V106I 4.4 2.6 1.4–5.2 63 NA Low 1.5G190S 3.7 0.8 0.6–1.7 32 0.2 Low 1.5V179Fb 0.7 – – 0 0.1 Medium 1.5V90I 6.8 2.0 0.8–3.6 97 1.5 Low 1
V179D 2.1 1.7 1.0–4.7 33 2.6 Low 1K101E 9.9 1.5 0.8–2.5 24 1.7 Low 1K101H 2.2 1.1 0.6–2.8 8 1.3 Low 1A98G 9.5 1.0 0.5–1.9 127 2.5 Low 1V179T 0.6 0.9 0.7–1.2 2 0.8 Low 1G190A 23.3 0.8 0.5–1.5 226 0.8 Low 1
Effect on FC in linear model
Weight factor
INTELENCE FC in the subset of HIV-1 clinical isolates with 1 INTELENCE RAM
(n=1,619), regardless of the presence of other NRTI or
NNRTI RAMsa
Prevalence (%) in the panel of
4,248 HIV-1 clinical isolatesMutation
INTELENCE FC in
a single SDM
Updated List of INTELENCE RAMs: Weight Factors for 2008 INTELENCE RAMs
Vingerhoets J, et al. IHDRW 2008; Abstract 24Mutation not in table is scored 0; RAMs, resistance-associated mutations; FC, fold change
Raltegravir
First HIV “integrase” inhibitor FDA approved October 2007
Should be very little transmitted resistance as it is the first of a new class of agents
Genotype assays for integrase are now available, but clinical significance undefined
Phenotypic assay for raltegravir resistance also now available
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations in the Integrase Gene Associated Mutations in the Integrase Gene Associated With Resistance to Integrase InhibitorsWith Resistance to Integrase InhibitorsMutations in the Integrase Gene Associated Mutations in the Integrase Gene Associated With Resistance to Integrase InhibitorsWith Resistance to Integrase Inhibitors
Raltegravir
Raltegravir resistance Raltegravir failure is associated with
integrase mutations in at least 3 genetic pathways defined by at least 2 mutations Major mutation at 148, 155, or 143 One or more additional minor mutations
Most common and highest degree of resistance is Q148H plus G140S
Continued raltegravir in the presence of viral failure and resistance is not recommended
HIV-1 Entry Inhibitors
Virus core
Enfuvirtide
TNX-355Maraviroc
Enfuvirtide First HIV “fusion” inhibitor
FDA approved March 2003 Patients who have not taken it before
generally have virus susceptible to it Mutations in “envelope” gene region
gp41 have been seen in viruses exposed to ENF, but these mutations do not easily predict loss of anti-viral effect
Phenotypic assay is available but its clinical utility is not well defined
© 2010. The International AIDS Society–USA© 2010. The International AIDS Society–USA Johnson et al. Topics HIV Med. December 2009.
Updates available at www.iasusa.org.
Mutations in the Envelope Gene Associated Mutations in the Envelope Gene Associated With Resistance to Entry InhibitorsWith Resistance to Entry InhibitorsMutations in the Envelope Gene Associated Mutations in the Envelope Gene Associated With Resistance to Entry InhibitorsWith Resistance to Entry Inhibitors
Enfuvirtide
Maraviroc
0.001 0.01 0.1 1.0 10.0
Normalized IC50 (g/mL)
ENF Naive
ENF Failure
0.001 0.01 0.1 1.0 10.0
Enfuvirtide PT Susceptibility During TORO 1 & 2
Biological Cutoff
Maraviroc
First HIV “CCR5 inhibitor” FDA approved in August 2007
Only effective against strains that use the CCR5 co-receptor for cell entry Little effect on viruses which use CXCR4
co-receptor, or which use both Activity of maraviroc is tested by co-
receptor “tropism” test, which is not a resistance test
HIV tropism defined R5 HIV: uses only CCR5 co-receptor X4 HIV: uses only CXCR4 co-receptor Dual-tropic HIV: uses either co-
receptor Mixed tropism: some R5 and some X4
virus present in the same sample
Maraviroc binds to the co-receptor, which is a HUMAN protein that cannot “mutate” in a given individual patient
Maraviroc resistance?? Emergence of X4 Virus
Switch of virus tropism from R5 to X4: unusual
Outgrowth of Dual/Mixed or X4 virus from pre-existing minority population present at levels too low to be detected by current tropism assays
Virus remains R5, with true resistance Mutations in HIV gp120 that allow HIV to bind
to CCR5 even though maraviroc is also bound Most mutations are in the V3 loop, but no
consensus yet on the specific mutations required
DHHS on New Classes The clinical utility of testing for integrase
and fusion inhibitor resistance is limited Lack of availability of second-line drugs in
class In patients failing integrase or fusion
inhibitor treatment, resistance testing may be helpful to determine whether to include these drugs in subsequent regimens, or in preparation for newer drugs in these classes
In naïve patients, there is no general recommendation to test integrase, but this may become more common in the future As yet, no documented cases of integrase TDR
Management of a treatment experienced patient
The goal of treatment for patients with prior drug exposure and resistance is to re-establish maximal viral suppression (<50 copies/mL)
Treatment history and past and current resistance tests should be used to identify fully active agents for the new regimen This is complex, and expert advice is
criticalPanel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. DHHS. December 1, 2009; 1-161.
Management of a treatment experienced patient
Adding at least two (preferably three) fully active agents to an optimized background ARV regimen can provide significant activity
For some highly treatment experienced patients, maximal viral suppression is not possible. In this case, ARV therapy should be continued with regimens designed to minimize toxicity, preserve CD4 cell counts and avoid clinical progression Expert advice is again essential
Case Follow up
Patient was started on anti-retrovirals BEFORE results of GT were returned Baseline VL was 650,000 Initial regimen was 2 nRTI and boosted-
PI
Resistance tests repeated Genotype, virtual phenotype, tropsim test
DATE Viral Load CD4 Meds
1/14/10 650,000 16 Naïve
2/5/10 3,230 2nRTI, PI/r
2/22/10 595,000 175 Same
Resistance tests for Case
Case follow up
Regimen was then changed to Tenofovir/emtricitabine Darunavir/r 600/100 mg BID
(resistance dose) Added raltegravir Added etravirineDATE Viral Load CD4 Meds
1/14/10 650,000 16 Naïve
2/5/10 3,230 2nRTI, PI/r
2/22/10 595,000 175 Same
4/14/10 1,150 193 Above, one month
Conclusions Resistance can occur in patients new to
ARV Resistance testing can be used to optimize
an antiretroviral regimen Must use in context of treatment history and
results of all prior resistance tests Goal for all HIV infected patients is HIV RNA <
50 Factors other than resistance may
cause regimen failure Resistance testing is reliable and cost-
effective but must be interpreted in context and may require expert advice
More information IAS-USA mutation cards and notes
http://www.iasusa.org Update of the Drug Resistance
Mutations in HIV-1: December 2009 Topics HIV Med. 17(5):138-145.
Stanford resistance database http://hivdb.stanford.edu
DHHS guidelines for HIV http://www.aidsinfo.nih.gov/guidelines/