1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.12 Drug resistance testing
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Gag Protein Gag amino acid substitutions and their location Reference Associated with PI resistance Associated with PI exposure
P6 P453A/L/T (312, 314, 315, 323, 341, 343,
344, 351, 352)
P6 E468K (310)
P6 Q474L (16)
P6 V484G/I/P/S (338)
P6 A487S (16)
P6 P497L (16)
All amino acid substitutions are expressed relative to HIV-1 subtype B consensus HXB2 (GenBank accession number: K03455).
50 1.12.1 Genotypic drug resistance testing
Most laboratories utilize either in-house developed methods or commercially available kits for standard genotyping of the HIV-1 PR and RT genes. There are currently three commercially available genotyping kits including: ViroSeq (Applied Biosystems, California), GeneSeq (Virologic, South San Fanscisco, CA) and TruGene (Bayer, Pittsburgh, PA) used for genotyping the PR and RT genes. All three of these kits require: viral RNA isolation from plasma of an infected individual, reverse transcription of RNA to cDNA and amplification of cDNA by PCR in order to produce adequate quantities of DNA for Dideoxynucleotide sequencing (Sanger sequencing) (297, 353)
As part of Sanger sequencing, amplicons are combined with a mixture of dNTPs, dideoxynucleotide triphosphates (ddNTPs), DNA polymerase and region specific primers.
This mixture is subject to several thermal cycling steps to facilitate amplicon denaturation, primer annealing and dNTP incorporation into the growing strand. The incorporation of a ddNTP, in the place of a dNTP, results in chain termination and production of several strands of DNA of varying lengths. These strands are then sequenced using an automated sequencer based on a fluorometric method dependent upon labelling of either the primer or ddNTPs (355).
Sanger sequencing generates a consensus nucleotide sequence based on the most prevalent viral strain within a patient sample. This sequence is translated into a corresponding aa sequence and aligned to a reference sequence (i.e. WT strain, most commonly HXB2) (355). The input of the aligned sequence into one of several interpretation databases including: the Stanford HIV Drug Resistance Database (HIVdb), the Rega database (RegaDB) and the French AIDS research agency database (ANRS) produces a list of RAMs present in each sample and a score which predicts the level of drug susceptibility based on mutational patterns (356).
Whilst Sanger sequencing is widely used, it lacks the ability to detect DRMs at frequencies below 15 – 20% (357, 358). Several studies have shown that mutations occurring at frequencies as low as 1% can impact the clinical outcomes of a patient, as such various sequencing approaches have been developed to allow the detection of low frequency mutations (i.e. minority variants) (359-367). These include: point mutation assays (i.e.
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allele specific PCR and oligonucleotide ligation assays) (364, 368), single genome amplification (SGA) assays (358) and next generation sequencing (369).
Point mutation assays generally employ specific probes/primers and/or labelled oligonucleotides to detect specific mutations of interest. Whilst the assay is very sensitive and can detect mutations present at frequencies between 0.1-1%, its major limit is only a few RAMs are detected in each run, rendering this method impractical for clinical settings where simultaneous detection of several RAMs is required per run. Additionally, the assay can be compromised by insensitivity of the template to primers and the large number of sequences which need to be analysed to detect low frequency mutations can be cumbersome (368, 370).
Single genome amplification (also known as limiting dilution PCR) involves the generation of cDNA from a patient sample, dilution of cDNA to one copy, amplification of that single template by PCR and sequencing of amplicons representing only one viral strain (369).
This method avoids the preferential amplification of the dominant viral strain and reduces polymerase induced recombination artefacts commonly seen in bulk sequencing. The pitfalls however are that SGA is expensive, labour-intensive and several SGA’s have to be conducted in order to achieve the same depth as that of multiple bulk PCRs (371).
Ultra-deep pyrosequencing (UDPS) is a parallel sequencing approach which allows for the sequencing of a mixed sample at a coverage of more than 1,000 reads per base. As part of UDPS viral RNA is extracted from plasma, purified and quantified. This is followed by generation of cDNA and the amplification of cDNA which is tagged with multiplex identifiers (MIDs). Tagged amplicons are purified, quantified, normalized and pooled. The sample pool is clonally amplified and sequenced. The three most commonly used deep sequencing platforms include: Roche 454 (GS Junior and GS-FLX), Ion Torrent PGM and Illumina Miseq (372). Deep sequencing offers extremely high throughput and requires expertise in bioinformatics to manage and interpret the data. Platforms such as the Roche 454 GS Junior and FLX are accompanied with built in bioinformatics tools which allows for user friendly data management and interpretation. A major benefit of UDPS is that all mutations in a region of interest can be identified and quantified in a single run making it suitable for use in clinical settings. Its major limitation however, particularly in resource limited settings, is its high cost (369, 370, 372).
52 1.12.2 Phenotypic drug resistance testing
Phenotyping assays measure the susceptibility of a clinical HIV-1 isolate to ARVs of interest by comparing the concentration of ARV required to inhibit the clinical sample to that of an HIV-1 WT/reference strain.
Similarly to conventional sequencing, phenotypic assays utilize viral RNA extracted from patient plasma for PCR in order to generate amplicons of the gene of interest. Amplicons are then used to generate recombinant viruses in a recombinant virus construct which has the analogous sequence deleted. A standard inoculum of recombinant virus is then used to infect a relevant cell line in the presence of varying concentrations of ARVs. The proliferation of the recombinant construct in the presence/absence of ARVs can be measured using either a single cycle phenotypic assay or a multiple cycle phenotypic assay. Results are obtained between –10 days and are reported as a fold change (FC) in drug susceptibility of the test sample in comparison to the reference strain (373).
As the name suggests, a single round infectivity assay is based on a single round of viral infection. A replication defective resistance test vector (RTV) is formed by cloning the region of interest from a patient sample into an HIV-1 expression vector which lacks the analogous region. Thereafter a cell line is co-transfected with a total of three plasmids: the RTV (comprising of the patient derived sequence), a vesicular stomatitis G protein expressing vector (this provides the Env region to the virus) and a reporter vector (this vector expresses luciferase which is used as a marker of viral replication and it also contains the HIV packaging sequence). The cell-line in conjunction with the three plasmids are exposed to varying concentrations of ARVs. The measurement of luciferase production in test samples versus the reference strain and drug control yields insight into viral replicative capacity and drug susceptibility.
For a multiple cycle assay, replication competent virus is produced via homologous recombination in cell culture to incorporate a patient derived sequence into a molecular HIV clone (typically NL43 which lacks the analogous region). A standardized inoculum of virus is then added to an appropriate cell line and the drug susceptibility is measured via luciferase production or expression of a reporter gene such as 3-(4, 5-dimethylthiazol)—2, 5 –diphenyltetrazolium bromide (MTT).
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Whilst single cycle assays are performed in a shorter time and offer the benefit of accurate representation of the original virus, multiple cycle assays mimic in vivo conditions more closely and thus provide more accurate results.
Both the single and multiple cycle assays measure the change in IC50 of an ARV required to inhibit 50% of viral growth and report variations in drug susceptibility as FC. The FC is calculated by dividing the IC50 of the test sample by the IC50 of the reference strain.
There are currently two commercially available phenotyping kits: Antivirogram (Tibotec- Virco, Mechelen, Belgium) and Phenosense (Virologic, South San Francisco, California) that are used to measure variations in drug susceptibility of the PR, RT and a portion of the Gag gene. Antivirogram is a multiple cycle assay, whilst Phenosense is a single cycle assay (373).