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Laboratory Tests in the Management of HIV Infection

^{Plasma Viral RNA Load}

^{Not Just another Surrogate Marker}

• ^{Predictive of Clinical Events}

^{-Viral Load in Untreated Subjects}

^{-Extent of Viremia Decline with Therapy}

• ^{Unknown Predictive Value}

^{-Tissue Viral RNA Decay}

^{-Extent of Virologic Rebound on Therapy}

^{Ultrasensitive Viral Load Assays}

	Old	New
	>500 copies	-50,copies

Roche	Monitor	Ultrasensitive
Organon Teknika	NASBA	Nuclisens
Chiron	bDNA v.2	bDNA b.3

 

^{Viral Load Below 500 Predicts Duration of Virologic Response}

^{Definition of Terms}

^{-Limit of Quantitation}

^{-The minimum that can be precisely quantified · Limit of Detection}

^{-The minimum viral load that can be reliably distinguished from uninfected specimens}

^{Roche HIV-1 Monitor}

• ^{Old assay is FDA approved (LOQ 400) - Ultrasensitive modification (LOQ 50) is not}
• ^{PCR amplification of template}
• ^{Increased Sensitivity by Centrifugation - Concentrates Virus}
• ^{Uses 0.5 ml of plasma}
• ^{Limit of quantitation 50 Copies/ml}
• ^{Specificity 100% at 50 Copies/ml}

^{Chiron bDNA v.3}

^{bDNA amplification of signal}

• ^{Increased Sensitivity by using more probes}

^{- novel chemistry gives higher specificity}

• ^{Uses 1 mL of plasma}
• ^{Limit of quantitation 50 Copies/ml}
• ^{Specificity 95%}
• ^{Calibrated to be comparable with Monitor}

^{Organon Teknika Nuclisens}

• ^{RNA amplification}
• ^{Increased Sensitivity by increased volume}
• ^{Uses up to 2 ml of plasma}
• ^{Limit of quantitation -40-100 copies}
• ^{RNA extraction by "Boom" Method}

   ^{- Separates RNA from PCR inhibitors}

   ^{- Better quantitation in Tissues}

   ^{- Automated Extractor may improve throughput}

^{Comparison Ultrasensitive Assays}

^{Summary Ultrasensitive VL}

• ^{<50 RNA Copies predicts decreased risk of virologic failure relative to 50-400 Copies}
• ^{Intensification of therapy if viral load is between 50-400 copies is of unknown value}
• ^{3 assays are available, but none FDA approved, with comparable Limits of quantitation}
• ^{"Limit of Detection" should not be used}

^{The Value of Viral Culture}

• ^{Measures of Infectious Virus}
    ^{-99.99% of Viral RNA is associated with replication defective virions}

• ^{Best Correlate of Infectiousness in a couples study in Amsterdam}
• ^{Can Culture Virus in Subjects With "Undetectable" Plasma Viral RNA}
• ^{Still Too Difficult for Routine Clinical Use.}

^{Definition of Viral Drug Resistance}

• ^{Genotypic Resistance}

^{-Viral mutations associated phenotypic resistance}

• ^{Phenotypic Resistance}

^{-Able to replicate in culture with drug}


• ^{Virologic Failure}

^{-Able to replicate in people during therapy}


• ^{Clinical Failure}

^{-Able to cause disease during therapy}

^{Methods of Measuring Resistance}

• ^{Identifying Viral Genotype}

^{-Sequencing of PR and RT}

^{-Automated FIourescent Sequencing (ABI)}

-^{Affymetrix sequencing - Line Probe Assay (LiPA)}

    ^{-Chiron Probe Hybddization Method}

^{Mutation Nomenclature}

^{"A threonine replacement by tyrosine at the 215th amino acid in the HIV-1 reverse transcriptase gene."}

^{= HIV rt215 (Thr Tyr)}

^=T215K

^=215K

^{= Tyr215}

^{Genotypic Patterns of Resistance}

• ^{A single mutation may be sufficient}

    ^{-EG: rt M184V is sufficient for 3TC resistance}


• ^{Multiple mutations may be required}

    ^{-EG: rt41, 70, 210, 215, 219 and AZT resistance}


• ^{Resistance Mutations are not always known}

    ^{-EG: D4T}


• ^{One mutation may suppress the effects of others}

    ^{-EG: M184V re-sensitizes to HIV-1 to AZT}


• ^{Mutations to combinations may not be clear}

    ^{-EG: Resistance to both AZT and 3TC}

^{Comparison of Methods for Viral Resistance Genotype}

^{The Problem of the Minor Variant}

• ^{Drug resistant variants present as a small percentage of the virus population will replicate exponentially when drugs are started}

    ^{-EG: 1 vidon per plasma volume could replicate to >100,000 copies/mL in 40 days}


• ^{A problem in two clinical settings}

    ^{-when drugs are stopped}

    ^{-when resistant viruses are transmitted}

^{Methods of Measuring Resistance}

• ^{Measuring Phenotype}

    ^{-viral Isolates grown with drug (ICs0)}


• ^{ACTG Protocol}

^{-Viral "constructs" grown with drug (IC60) · Antivirogram (VIRCO) · ViroLogic}

^{-Reverse transcriptase assays with drug}


• ^Nevirapine

 

^{Comparison of Phenotyping Methods}

^Conclusions

• ^{Drug Resistance Assessment can tell you what not to do,}
• ^{Correlation between Genotypic and Phenotypic Assays and Virologic Response have been demonstrated.}
• ^{The best predictive model for virologic response included viral drug resistance assessment as well as clinical history}
• ^{Drug resistance testing in drug naive subjects in highly treated communities maybe warranted}