Contacts To order tests or for more information:
ViroLogic Customer Service: (800) 777-0177
ViroLogic has developed both a genotypic (GeneSeq™ HIV) and a phenotypic (PhenoSense™ HIV) assay for resistance testing.
GENERAL INFORMATION
Assay Name
Genotype
GeneSeq™ HIV
Phenotype
PhenoSense™ HIV
Collaborators/Distributors
American Medical Laboratories
ARUP
Laboratory Corporation of America
Mayo Medical Laboratories
Quest Diagnostics
Specialty Laboratories
Availability
Commercially available for research only.
Regulatory Status
CLIA certified and CAP, New York State and Maryland accredited. The assays are non-kit-based. ** The FDA does not currently regulate non-kit-based tests through published regulations or site inspections.
Proprietary/Non-kit-based assays are sometimes referred to as "home brew" assays. See the background article regarding FDA regulation of kit- vs. non-kit-based assays.
ASSAY INFORMATION
Genotypic
Phenotypic
Codons Interrogated
GeneSeq™ HIV
PhenoSense™ HIV
RT
PI
Full sequence assay
Recombinant
1–305
1–99
Turnaround time
Sensitivity
GeneSeq™ HIV
PhenoSense™ HIV
GeneSeq™ HIV
PhenoSense™ HIV
14 days
14 days
500 RNA copies/mL
500 RNA copies/mL
METHODOLOGY
Genotype GeneSeq™ HIV is a full sequence genotyping assay, using Applied Biosystems’s sequencing methods and in-house primers. The genotype of patient viruses are evaluated using a reaction chemistry based on the extension of sequence specific primers and random chain termination (Sanger). The amino acid sequences of viral genes encoding drug targets are derived from the same RTV used in the PhenoSense™ assay in order to relate patient virus genotype directly to phenotype. Sequence analysis is performed using a reaction chemistry that incorporates fluorescent dye labeled nucleotides into the reaction products.
Phenotype
PhenoSense™ HIV uses nucleic acid amplification to derive HIV protease and reverse transcriptase sequences from plasma. These patient-derived segments are incorporated into a viral vector to construct a resistance test vector (RTV). The viral vector also contains an indicator gene, luciferase, inserted within a deleted portion of the HIV envelope gene. The assay is performed by introducing the RTV into host cells, collecting virus particles after transfection, and using the particles to infect target cells. Drug susceptibility is measured by comparing luciferase activity produced in the presence and absence of drugs. The data are analyzed by plotting the percent inhibition of viral replication, as measured by luciferase activity, against the log10 concentration of drug for the patient virus and a drug sensitive reference virus. The resulting drug susceptibility curve is used to calculate the concentration of drug required to inhibit viral replication by 50% (IC50). A shift in the patient inhibition curve toward a higher drug concentration as compared to the curve of the drug-sensitive reference virus is interpreted as reduced drug susceptibility.
REPORT INFORMATION
Report
Drug Resistance Interpretation
Genotype
Yes
For the final interpretation of the GeneSeq™ HIV assay, amino acid sequences of patient viruses are compared to reference virus strains to identify mutations associated with alterations in drug susceptibility. Resistance mutations are classified with an algorithm based on the consensus table established by the Resistance Collaborative Group (Chair: D.D. Richman).
Phenotype
Yes
The final interpretation for the PhenoSense™ assay indicating resistance is provided. PhenoSense™ HIV reports generated after Jan 22, 2000 include clinically relevant cutoffs* for d4T, ddI, abacavir and Kaletra (lopinavir/ritonavir). Clinical cutoffs for the remaining HIV drugs will be added as additional clinical trial data become available. For drugs without established clinical cutoffs, laboratory cutoffs of 2.5-fold are used to indicate reduced susceptibility, based on the fold reduction in susceptibility that is significantly different from a standard drug sensitive virus.
Clinical cutoffs are defined by the drug susceptibility level at which a patient's probability of treatment failure with a particular drug significantly decreases. These cutoffs are determined by correlating clinical outcome data with changes in p[phenotypic drug susceptibility.
500 RNA copies/mL