Resistance Testing: What is it What does it mean

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Resistance Testing What is it? What does it
mean?

• How does drug resistance emerge?
• Overview of methods
• Advantages and disadvantages
• Current recommendations for use
• Jill Taylor, Ph.D.
• Director
• Viral Genotyping Laboratory
• Wadsworth Center
• New York State Department of Health

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Why do mutations occur in the HIV genome?

• HIV makes copies of itself very rapidly
• 1-10 billion new virus particles/day
• During its replication, HIV is prone to make
  errors when copying itself
• This results in mutations or errors in the
  genetic material of the virus which make the
  structure of the offspring virus slightly
  different to that of the parent virus
• Some of these mutations will result in an
  increased ability of the virus to grow in the
  presence of antiretroviral drugs

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How drug resistance arises
How drug resistance arises. Richman, DD.
Scientific American , July 1998
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How does this lead to drug resistance?

• When HIV replication is not completely blocked
• Sub-optimal therapy regimens e.g. monotherapy
• Adherence problems
• Pharmacokinetic problems poor drug absorption,
  inadequate dosing or drug-drug interactions
• These conditions can allow drug-resistant virus,
  already present in the population to dominate

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Situations in which to consider drug resistance
testing

• Patients on HAART showing inadequate viral load
  suppression or viral load increase after previous
  suppression
• Acute infection - possibility of transmission of
  resistant virus
• HIV-infected pregnant women
• BUT
• Other factors to consider before resorting to
  resistance testing adherence profile, toxicity,
  pharmacokinetic issues

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How do you measure drug resistance?

• Phenotyping
• Direct assay Measures the ability of the virus
  to grow in various concentrations of
  antiretroviral drugs.
• Genotyping
• Indirect assay Detects drug resistance
  mutations that are present in the relevant virus
  genes.
• Both assays focus on the reverse transcriptase
  (RT) and protease genes of the virus where many
  of the mutations occur

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Basis of phenotyping assays

• HIV circulating in the clients plasma is
  isolated and the RT and protease genes are
  copied. The copies are inserted into another
  strain of HIV which scientists are able to grow
  in the laboratory. This is now called a
  recombinant virus.
• The recombinant virus is then grown in presence
  of known concentrations of antiretroviral drugs
  and its ability to grow in the presence of these
  drugs is compared to that of a reference strain
  of HIV that is known to be sensitive to the drugs.

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Basis of phenotyping assays (cont.)

• The concentrations of drug required to inhibit
  the replication of the test and the reference
  virus are calculated. For example
• Concentration of Indinavir required to inhibit
  growth of the reference strain 10?M
• Concentration of Indinavir required to inhibit
  growth of the test strain 100?M
• Test virus is ten-fold less sensitive to
  Indinavir than reference strain
• A report is generated documenting the decreased
  sensitivity to particular drugs
• 13-15 drugs are assayed, cost 880-955
• Turnaround time is 2 to 3 weeks

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Phenotyping Advantages

• Provides resistance information on each drug
  regardless of the presence of multiple mutations
• Interpretation may be more intuitive than for
  genotype assay
• Very useful in patients with complex drug history
  and complicated mutation profile
• Very useful for deciphering cross-resistance
• May be more useful than genotyping for new drugs
  until appropriate mutations are established by
  clinical data

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Phenotyping Disadvantages

• If drug resistant population is minor the
  phenotypic effect may not be detected
• Current limitation of use is that viral load
  needs to ? 1000 copies/ml - need greater
  sensitivity
• Very expensive and time-consuming
• The relevance of small changes in drug
  sensitivity not yet fully determined - drugs to
  which patient is actually still sensitive may be
  unnecessarily eliminated

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Sequence-based genotyping assays

• HIV circulating in the clients plasma is
  isolated. The RT and protease genes are copied,
  amplified and sequenced
• The test sequence is then compared to the
  sequence of a reference HIV strain and all
  changes (mutations) documented.
• Computer software then compares these changes to
  a list of the mutations known to be associated
  with drug resistance.
• Based on the presence or absence of particular
  mutations, a report is produced documenting the
  mutations and (optimally) the antiretroviral
  drugs to which the patient is sensitive or
  resistant

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Genotyping Advantages

• Identification of all nucleotides, amino acid
  differences, deletions insertions
• Genotyping has the ability to detect resistant
  virus that constitutes only a small proportion
  (about 20) of the viral population.
• This can provide predictive early warning of
  developing resistance before full resistance
  develops
• Faster and less expensive than phenotype assay -
  cost is 400-500 and turnaround time lt 2weeks

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Genotyping Disadvantages

• Reports may be difficult to interpret unless
  clinician is very experienced
• Labs use different software programs to predict
  resistance - need a consensus on which mutations
  are important
• There is a lot of variation in the quality of the
  product from different laboratories especially
  in the ability to detect minority species in the
  population
• Current limitation of use is that viral load
  needs to ? 1000 copies/ml - need greater
  sensitivity

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Current recommendations for use of resistance
tests

• Guidelines for use of antiretroviral agents in
  HIV-infected adults and adolescents. DHHS Panel
  on Clinical Practices for Treatment of HIV
  Infection and Kaiser Family Foundation.
• http//www.hivatis.org
• Antiretroviral Therapy in Adults. Updated
  recommendations of the International AIDS
  Society-USA Panel. JAMA May 10, 2000 283(18)
  2417-2426

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Current recommendations (DHHS)

• Useful for selection of active drugs when
  changing antiretroviral regimens if viral load
  does not decrease
• Both phenotyping and genotyping may be useful in
  patients with complex prior treatment history
• Resistance testing may be useful in cases of
  acute infection
• Resistance testing not yet recommended at
  initiation of therapy in treatment-naïve
  individuals until more information available on
  prevalence
• Do recommend use of resistance testing in cases
  where viral load suppression is sub-optimal in
  initial regimen
• Recommendations are the same for pregnant and
  non-pregnant patients

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What is the benefit for the client?

• If a client is failing on his current drug
  regimen these tests can help the physician
  determine whether the cause of treatment failure
  is the presence of drug resistant virus or some
  other reason e.g. adherence problems, toxicity
  problems
• If there is resistant virus present, these tests
  help the physician understand which of the drugs
  the client is resistant to. The physician can
  then substitute drugs to which the client is
  sensitive.
• Resistance testing may allow the physician to
  detect emerging drug resistance early and change
  the drug regimen before it becomes a big problem.
  This also saves drugs for potential later use.

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Useful websites for further information

• www.HIVresistance.com
• www.medscape.com
• www.hivatis.com
• www.virologic.com
• www.vircolab.com
• www.visgen.com

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For more HIV-related resources, please visit
www.hivguidelines.org