Development and Performance of Conventional HIV-1 Phenotyping (Antivirogram®) and Genotype-Based Calculated Phenotyping Assay (virco®TYPE HIV-1) on Protease and Reverse Transcriptase Genes to Evaluate Drug Resistance

Abstract

Objectives: A wide array of monitoring tests is commercially available to gauge HIV-1 disease progression and the overall health status of an HIV-1-infected patient. Viral load tests provide a picture of viral activity, while CD4 cell counts shed light on the immune status and can help physicians to prevent the development of opportunistic infections in patients. On the other hand, genotypic and phenotypic resistance testing and therapeutic drug monitoring help to optimize HIV-1 antiretroviral therapy. Resistance testing is currently recommended within the standard of care guidelines to aid the choice of new drug regimens following treatment failure(s). Methods: Genotypic testing described here is based on the amplification and sequencing of an HIV-1 protease (PR) and reverse transcriptase (RT) region from a patient sample to identify resistance mutations associated with PR and RT inhibitor resistance. A genotypic test takes a week to perform and the results are reported as a list of detected mutations. The virco®TYPE HIV-1 report uses genotypic data to predict phenotypic susceptibility by linear regression modeling that uses a large correlative database of genotype-phenotype pairs. Phenotypic testing measures the ability of the virus to replicate in the presence of a drug and provides a direct measurement of drug susceptibility in vitro. Since phenotypic analysis is laborious and time consuming (28 days), genotypic resistance testing is currently the standard reference method used for HIV-1 resistance testing. However, a phenotypic test is important when a patient harbors virus with complex genetic patterns, or when the mutational resistance profile for a particular drug is not well-characterized. Results and Conclusions: Some of the currently used resistance tests are partially automated enabling laboratories to increase overall efficiency. However, maximum automation and standardization of the process, instruments and software that we have described here can overcome many of the problems encountered with current tests and aims at having a compliant, high-throughput, diagnostic laboratory, which can guarantee sample integrity from sample reception to result reporting. We also describe in detail the development and performance of virco®TYPE HIV-1 (genotype) and Antivirogram® (phenotype) assay on PR and RT genes to evaluate antiretroviral resistance.

Characterization of the Drug Resistance Profiles of Patients Infected with CRF07_BC Using Phenotypic Assay and Ultra-Deep Pyrosequencing

“…Each experiment used the NL4.3 HIV-1 strain as control. For comparison, an in vitro phenotypic resistance assay that measures the level of resistance of recombinant HIV-1 from plasma samples was performed using Antivirogram assay [11]. …”

“…In contrast, phenotypic assays measure HIV-1 viral replication in cells cultured in different drug concentrations. There are two types of phenotypic assays: commercially available phenotypic assays generate chimeric viruses by homologous recombination of PCR-derived sequences and then culture with cells in different drug concentrations [10, 11] and in-house phenotypic assay use peripheral blood mononuclear cells (PBMCs) to isolate HIV-1 and then incubate them in target cells (MAGIC-5 cells) with different drug concentrations [12, 13]. It has been reported that phenotypic drug resistance using recombinant virus assay was limited to detect low-frequency viral quasispecies below than 50% [14].…”

Current Assays for HIV-1 Diagnostics and Antiretroviral Therapy Monitoring: Challenges and Possibilities

“…No molecular cloning step is involved in this phenotyping assay, and a panel of recombinant strains is created to reflect the diversified viral population circulating in the patients. The assay compares the replicative capacity between the wild-type virus and the constructed virus to determine the IC 50 of the antiretroviral drugs [59]. Both assays can readily assess the drug susceptibility of a patient with viral load over 500 copies/ml.…”

“…In combining the advantages of both genotyping and phenotyping, a third hybrid approach, the VircoTYPE™ HIV-1 test (Virco BVBA, Beerse, Belgium), comprises not only genotyping technique but also phenotypic ana lysis. It is a modified version of VirtualPhenotype-LM and uses linear regression modeling with over 80,000 pairs of correlated genotypic and phenotypic samples for accurate drug susceptibility prediction [59]. This approach provides a third option for drug resistance monitoring by obtaining phenotypic information from genotyping only.…”

The Evolution of Drug Resistance Interpretation Algorithms: ANRS, REGA and Extension of Resistance Analysis to HIV-1 Group O and HIV-2

“…org), see also Thielen and Lengauer [5] and Lengauer [6] , Virco (http://vircolab.com/) in Pattery et al [7] and HIV-GRADE (http://www.hiv-grade.de/cms/grade/) in Obermeier et al [8] .…”