HCV genotyping and genotypic drug resistance testing

Scope of the tests

The use of some HCV directly acting agents (DAA) is restricted to certain HCV genotypes. Under these circumstances, it is important to determine the HCV genotype in advance of DAA therapy for the benefit of the patient and to prevent non-response to very costly treatment. Additionally, decreased susceptibility of HCV to DAAs is usually associated with sequence changes in the viral genome (genotypic drug resistance). Detection of these mutations in the viral genome can be used to predict the susceptibility of the virus to currently licensed DAAs and thus can be used to optimise the use of DAA treatment on an individual patient basis. Currently, the Molecular Diagnostics Unit (MDU) offers routine sequencing of the NS3 (protease) NS5a and NS5b (polymerase) genes of HCV from plasma viral RNA.

Test details

HCV genotype is determined by sequencing of the NS5b gene of the virus. The routine test generates a sequence covering 293 codons (879 bases) of the gene. This sequence also covers the major described DAA resistance mutations to the NS5b inhibitor sofosbuvir. Resistance to NS3 inhibitors is predicted from a sequence of the first 237 codons (711 bases) of the gene, including the Q80K mutation coding for resistance to simeprevir, and resistance to NS5a inhibitors from the whole of the gene (448 Codons, 1344 bases). The methods are fully validated in-house methods. Summaries of the validations are available from the Unit Manager.

Reporting results

The genotyping and resistance assays produce an edited nucleotide sequence which is used to generate a report by submission to an online algorithm 'Geno2pheno' hosted by the Max Planck Institute. This interpretation is included in the final MDU report. If the user requires the sequence file ('FASTA' file) or the sequencing chromatogram these can be supplied by arrangement with the Unit Manager. MDU does not provide additional clinical interpretation beyond that provided by the Geno2pheno algorithm.

Turnaround time

We aim to issue reports within two weeks (ten working days) of receiving a sample. However, as we offer a bespoke service and make rigorous efforts to produce a result from any sample, turnaround times may be extended if repeat testing is required.

Complaints

If you are unhappy with the service provided by MDU or if you wish to make suggestions on how our service can be improved, please contact the Unit Manager, Simon Dustan (s.dustan@imperial.ac.uk).

User instructions

For routine testing, the required sample is either two 1ml vials of frozen plasma, prepared by your local virology or microbiology department or two 4.5ml EDTA Vacutainers of whole blood. If plasma is prepared locally you should contact the laboratory for their sample requirements but the anticoagulant must be EDTA and the plasma should be stored at -70°C or lower prior to shipping. Each sample must be accompanied by a request form. This can either be the form provided by MDU (HCV Request Form Word Document) or your own form. If it is the latter it must contain the following details: patient clinic number (not the patient’s name), date of birth, sample date, most recent virus load and HCV genotype if known. Other details, such as reason for test, current and previous antiviral therapy, are useful but not essential.

Only one sample is required for three sequences but the required sequences (NS3, NS5a, NS5b) must be indicated on the request form. If genotyping only is required please tick the NS5b box.

Whole blood samples should be couriered to MDU the same day if possible. However, MDU closes at 17:00 so if a sample cannot be guaranteed to arrive before that time (from a late afternoon or evening clinic) it can be kept in the refrigerator (not freezer) and shipped the next day or can be shipped the following Monday if taken late on Friday. Plasma samples can be shipped at ambient temperature if they are guaranteed to arrive the next day.

Each request accepted by the laboratory for examination(s) shall be considered an agreement.