Genotypic analysis of HIV-1 drug resistance mutations

Sven Thamm looks at a fully integrated and complete solution for detecting and reporting HIV-1 resistance to specific anti-retroviral therapy.

Drug treatment of HIV infected patients includes use of multiple antiretroviral drugs in a strategy known as anti-retroviral therapy (ART). ART usually includes adherence of three different drugs at several time points per day.

Currently, anti-retroviral therapeutics are used for inhibiting the activity of the protease and reverse transcriptase (RT) and thereby inhibiting the replication of the virus.

However, during the replication of the virus the reverse transcriptase often makes mistakes that may lead to mutations that cause resistance against the drug. This is often the cause of viral rebound and failure of the therapy.

Drug identification

Identification of the presence of patient-specific drug resistant virus populations by genotypic resistance tests, can help the clinician to select an effective combination of drugs that are likely to suppress HIV-1 replication sufficiently.

International guidelines are established that recommend genotyping for naïve HIV-infected patients or patients experiencing ART failures. These guidelines also suggest genotyping of infected mothers and their newborns.

The ViroSeq HIV-1 Genotyping System, from Abbott GmbH, is a fully integrated and complete solution for detecting and reporting HIV-1 resistance to specific ART used in therapeutic management of HIV-1 positive patients.

ViroSeq detects mutations in the RT and protease genes of the pol region and provides the physician with a report indicating genetic evidence of viral resistance to known ART. The ViroSeq HIV-1 Genotyping System consists of different modules for sample preparation, reverse transcription, polymerase chain reaction (RT-PCR), cycle sequencing, automated electrophoresis and detection and analysis, and results interpretation.

Genetic analysers

Applied Biosystems, Foster City, CA, offers a variety in throughput of fully automated walk-away capillary-based genetic analysers (ABI PRISM 310, 3100-Avant, 3100, 3700, and 3730 Genetic Analysers) for the detection and analysis of cycle sequencing products. The variation in throughput serves diagnostic laboratories in low, medium, and high throughput segments.

The ViroSeq HIV-1 Genotyping System has also been validated for use with the ABI PRISM 377 DNA sequencer which is a slab gel-based instrument. u Sample preparation to amplification: The virus particles are isolated by centrifugation of the patient's plasma. This is followed by a lysis and precipitation to isolate the RNA. HIV-1 RNA isolation, RT-PCR, and PCR use standard techniques and procedures. In the one-tube amplification steps a 1.8 kb DNA fragment of the HIV-1 pol region, spanning the entire protease and two third of virus RT gene, is created (Fig. 1).

A built in contamination control protects carryover from other samples by using Uracil-N-glycosylase. Due to the incorporation of dUTPs into the PCR product, cross contaminations from previously amplified patient samples will be digested by Uracil-N-glycosylase. This digestion is initiated before the first cycle of the PCR.

The PCR product is finally purified and checked for yield on a 1 per cent agarose gel. u Cycle sequencing: Cycle sequencing is using BigDye Terminator chemistry providing different fluorescent dyes for each of the four different dye terminators that are used to identify the A, C, G, and T extension/termination reactions.

This approach enables a one-tube sequencing reaction and in contrast to other methods there is no need to split samples into four different reactions. Four forward and three reverse primers are used to sequence the entire protease gene from codon 1-99 and the adjacent reverse transcriptase gene from codon 1­335.

By using this seven primers approach, the vast majority of the generated sequence is available as double stranded at minimum. u Automated detection: Cycle sequencing products are cleaned up by precipitation or by column purification in order to remove the unincorporated BigDye Terminators.

After denaturation samples are placed into the instrument to start electrophoresis. For using the 377 sequencer samples have to be loaded manually on the gel. u Software analysis: The ViroSeq HIV-1 Genotyping System software combines the sequence data obtained with the seven primers of each patient sample into a single project (Fig. 2).

The seven sequences are assembled into a consensus sequence that is compared to the HXB2 HIV-1 reference strain. The vast majority of the sequence information is available from both strands for optimal reliability of the sequence.

Results reporting

Finally, the ViroSeq HIV-1 Genotyping System software offers three options for reporting the results of a project.

(I) An Antiretroviral Drug Resistance Report, providing information's whether mutations constitute evidence of viral resistance to known ART (Fig. 3).

(II) A text mutation report showing a list of mutations found and categorised as known or unknown.

(III) A FASTA sequence that can be used to obtain drug resistance information from other drug interpretation tools.

Performance characteristics:

* Sensitivity of 1000 copies/mL.

* Superior peak resolution, long sequence read and reliable consensus sequence by sequencing both strands using BigDye Terminator chemistry.

* No gaps in sequence and RT sequence information up to codon 335.

* Good performance on non-subtype B HIV-1 strains and recombinants.

* Mixed populations detectable with a threshold of 20 per cent on the 3100 Genetic Analyser.

Taken together, ViroSeq in conjunction with Applied Biosystems' DNA sequencers is the state of the art for detecting and reporting HIV-1 resistance to specific ART used in therapeutic management of HIV infection.

Enquiry No 58

Sven Thamm, PhD, is Scientific Advisor Infectious Diseases, Medical & Scientific Development Molecular Diagnostics, Europe, Middle East & Africa, Abbott GmbH & Co. KG, Wiesbaden-Delkenheim, Germany. www.abbott.com

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