Molecular Boards: Id

Front 1

Why obtain an HIV viral load (RNA)?

Back 1

It is a better predictor of the rate of progression to AIDS than CD4+ count (if greater than 10,000). Those with counts less than 1,000 do not readily progress.

Also can monitor response to therapy

Also can aid in Dx if recent exposure before seroconversion when ELISA and Westerns are still negative (during acute infection)

Front 2

When should HIV viral load testing be performed?

1.
2.
3.

Back 2

When should HIV viral load testing be performed?

1. At baseline- before therapy
2. immediately before therapy
3. 2 to 8 weeks after therapy start
4. after 3 to 4 months

any increase triggers repeat testing

Front 3

Why is monotherapy ineffective in HIV?

Back 3

as HIV is an RNA virus and replicates with RT (reverse transcriptase), is has no error proofing and about 1 error/division occurs. That means a lot of mutations and variants under selective pressure.

Front 4

What are the 4 classes of HIV medication?

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2.
3.
4.

Back 4

What are the 4 classes of HIV medication?

1. nucleoside RT inhibitors (NRTIs)
2. non-nucleoside RT inhibitors (NNRTIs)
3. protease inhibitors (PIs)
4. fusion inhibitirs

Front 5

What are the differences in viral load testing assays from different manufacturers?

Back 5

Typically range in copy numbers and methods. Roche has to Amplicor assays, on with low range (sensitive- 50-100K), and one with high range (400-750K).
Bayer (Versant) has a branched DNA test with a range of 176-3.4M copies

Front 6

How is the limit of quantification defined in a molecular test?

Back 6

the amount of DNA that can be detected in 95% of replicate samples.

Front 7

What is the most common subtype of HIV in the US, and how do different systems identify other variant strains?

Back 7

subtype B is the most common in NA and Europe. there are variances among the different platforms for identification of Strains A and O.

Front 8

Are viral loads from different HIV platforms comparable?

Back 8

No. They use different technologies. Amplicor RT-PCR results are often twice what the Versant assay gets.

Front 9

How is molecular drug resistance to HIV drugs tested?

1.
2.

Back 9

1. Genotyping assays looking for specific mutations known to confer resistance to specific drugs. This can be done by sequencing or binding of genotype-specific probes (GeneChip).
2. Phenotyping assays look at viral replication in response to specific drugs. This is measured as IC50 score- the inhibitory concentration of a drug that reduces replication by 50%.

Front 10

What is the difference between primary and secondary mutations in HIV testing?

Back 10

The terms “primary” and “secondary” may be used when referring to resistance mutations. Primary mutations are relatively drug specific and may decrease viral susceptibility to the drug. Primary mutations may lower virus fitness and replication compared to the wild-type virus.
Secondary mutations are not drug-specific and reflect mutations that increase virus fitness.

Front 11

Examples of HIV drug resistance:
1- NRTIs
2. NNRTIs
3. Protease inhibitors

Back 11

1. Zidovudine and Lamivudine-resistance with D67N, K70R, M184V, K219E
2. NNRTIs- Nevirapine, Delavirdine, and Efavirenz with K103N
3.Protease inhib- Nelfinavir with D30N

Front 12

What are the differences between the Hepatitis B and C viruses?

1.
2.
3.

Back 12

Hep B &C

1. Hep B is a DNA virus that replicates via reverse transcriptase; hep C is an RNA virus
2. B has a nucleocapsid,
3. Hep C has 6 major genotypes and 12 variants, B has 7 genotypes. Groups A and D are most Common in the US
4. Hep C is much larger than B

Front 13

Treatments for Hep B and C?

Back 13

Hep B- interferon-a with Lamivudine

Hep C- interferon-a with ribavirin

Front 14

When is HBV DNA level testing performed?

A. pretreatment
B. after initial response to therapy
C. for maintenance or flare ups

Back 14

When is HBV DNA level testing performed?

A and B. C is done with following serum ALT. treatment is based on rising ALT, liver biopsy and possibly DNA levels. High serum ALT and low virus levels means more likely to respond to INF vs. Lamivudine therapy (a nucleoside analog- NRT).

Front 15

1. How efficient is Hep B treatment with Lamivudine?

Back 15

limited-
Patients develop drug resistant mutants- 15-25% after 1 year of treatment, 49% after 3 years.

The goal of therapy is to clear HBeAg, and lower to develop anti- HBeAg antibodies. (thus preventing future liver damage). Patients do not totally clear DNA levels, but at a certain point the disease does not progress (only 10% of infected patients develop liver disease).

There are also genotype variants that are resistant to Lamivudine therapy

Front 16

What are the tests available for HBV DNA-quantity detection?

Back 16

1. Versant (same as HIV, by Bayer)
2. Amplicor (same as HIV, by Roche)
3. Taqman assay
4. DiGene standard and ultra-sensitive hybrid capture

Front 17

When is the HCV RNA assay performed?

What if the test is negative?

Back 17

As soon as infection is suspected. Elevations in HCV come much earlier than liver damage and serology. It can also identify active vs, resolved infection in the presence of anti-HCV antibodies (80% progress to liver disease).

If negative, f/u with RIBA (recombinant immunoblot assay)

Front 18

How is active HCV treated?

Back 18

ribavirin, and IFN if ribavirin is not tolerated. If the patient has a large decrease in HCV levels post treatment, they are likely to have a sustained response.

Front 19

What is HCV viral load testing good (and not good) for?

Back 19

good- pretreatment evaluation for therapy- low viral levels = likely to respond

not good- measuring disease progression.

Front 20

What is the strongest predictor of response to treatment for HCV?

Back 20

HCV genotype. Types 2 and 3 have much better response than type 1. algorithms have been developed for duration of treatment based on genotype.

Front 21

When is CMV molecular testing utilized?

1.
2.

Back 21

1. In organ and bone marrow transplant patients- in patients at high risk for infection for initiation of preemptive therapy.
2. to monitor for relapsing infection in positive patients. rate of decline of CMV load with treatment predicts recurrence.
3. individualize treatment length- treat with IVIG until viral load is undetectable.

Front 22

How is CMV tested for drug resistance?

1.
2.

Back 22

1. phenotypic (growth in response to treatment)
2. genotypic- mutations in the UL97 and DNA polymerase genes

Front 23

What are CMV treatments?

Do patients develop resistance?

Back 23

1. prophylactic ganciclovir (GCV)
2. Foscarnet (FOS)
3. cidofovir (CDV)

Not usually- CMV is a DNA virus

Front 24

What causes PTLD (post-transplant lymphoproliferative disease)?

2. is it more common in children or adults?
3. how is it tested in the molecular lab?

Back 24

EBV!

2. children
3. QPCR in serum

Front 25

Why is molecular testing for the etiology of CNS viral infection so important?

How are they tested?

Back 25

eliminates the need for brain biopsy!

2. Mostly by PCR or RT-PCR based methods (or TaqMan).

Front 26

Molecular testing is available (DNA amplification based) for what STDs?

Back 26

Chlamydia, Gonorrhea, HPV and trichomonas