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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. Richman, DD. Scientific American , July 1998

How drug resistance arises

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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 client’s 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 = 10M

Concentration of Indinavir required to inhibit growth of the test strain = 100M

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 client’s 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 < 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