During the course of diagnosis, indication and subsequent treatment of hepatitis C, there are three types of molecular tests that we can consider: (i) detection and quantification of HCV RNA, (ii) genotyping and (iii) resistance. Before deciding on what to use, we need to think of the practical use of each test in patient screening, diagnosis and monitoring.

The first step in HCV diagnosis is antibody testing followed by the confirmation of active infection in form of HCV RNA testing. There are two dominant, accurate and reliable Real Time PCR molecular diagnostic platforms from Roche and Abbott; however there are other tests in development stage. These real time PCR assays are very sensitive and they are appropriate for confirmation of infection.

The presence of HCV RNA is an indication for therapy as all clinical practice guidelines advise to treat patients with “active infection”. Although there are issues with drug cost and patient prioritization, eventually any patient with detectable HCV RNA should qualify for treatment.

When a patient is diagnosed as HCV RNA positive, the next step is to determine HCV Genotype. Genotyping is important as it helps the clinician to decide on choice of drugs and the duration of therapy.

Even though we are through with INF based regimens it is still a dilemma we face today in the era of DAAs (direct acting antivirals) when making a clinical choice on the course of treatment?

Genotyping can be based on a number of different platforms. One is direct sequence analysis. Often this is done using homemade tests but these have less reliability. The best performing technology is the second-generation line probe assay. i.e. Inno-LiPA HCV II assay, which not only identifies the genotypes but also accurately identifies the subtypes i.e. 1a and 1b. Identification of subtypes is extremely useful as the treatment is different and it can help decide whether to use Ribavirin regimens and on the duration of therapy with new DAA-based regimens.

The Inno-liPA assay is the most accurate one however other assays based on sequencing analysis and real time PCR with specific probes exist too.

In the future, we will have pan-genotypic drug combinations that work equally on all genotypes and we will be able to treat without the need to identify the genotypes which in itself will improve access to care.

In the past, we were using HCV RNA viral load monitoring in order to characterize the response to therapy and guide treatment decisions (so called response guided therapy) and we were adapting treatment duration to the in vivo response based on HCV viral load measurement.

However, in the new era of DAAs and INF free treatment, viral load measurement as a tool for response guided therapy is going to disappear.

We do not have lots of data on on-treatment kinetics, however, recent experience, especially in France, shows that with Sofosbuvir + daclatasvir and Sofosbuvir + Simeprevir drug combinations, the viral kinetics are no longer predictive of the virologic outcome of therapy. Some of the patients become undetectable very early on treatment, but relapse after treatment and conversely, we have patients who are still positive at week 4 or even up to end-of-treatment, but eventually will achieve sustained virological response (SVR).

Therefore, quantification/measurement of VL is no longer useful except in one indication, which is testing at week 2 to see whether VL is going down as a measure of checking patient compliance.

Otherwise it is not recommended to do HCV RNA viral load test to make treatment decisions. RNA will be used again at the end of treatment and 12 weeks later. If the VL is negative 12 weeks after the end of treatment, the patient has achieved SVR and is cured.

Another test is done six months after achieving SVR followed by another one after a year to make sure a relapse does not happen. After two years and another test you may inform the patient that he/she is cured.

Patients who turn positive in later stage are actually often re-infected and mostly belong to high risk groups.

In new era of DAAs the SVR rates can be as high as 90%; however there are patients who fail on treatment. Most of them relapse and there are few with virological breakthrough. Patients who relapse generally have a dominant resistant viral population at the time of relapse. The virus is different and it is resistant to drugs administered during therapy.

The important question here is, whether we should use resistance testing to make treatment decisions. To do this we have three different options:

(i) To use it as baseline test to look for resistant mutations and if present, help make appropriate decisions prior to starting treatment. We should not do this, as for the simple reason that many resistant strains can be defeated with new drugs. In the majority of cases i.e. more than 90% patients carrying resistant strains can be cured with new drugs, hence, paying for test is a waste of money.

(ii) Do resistance testing at the time of treatment failure? Again, the answer is no as it’s known that the drug resistant population has been enriched. It does not give us any additional vital information for decision making as it’s already late, as the treatment has failed and there is nothing we can do or achieve by doing resistance testing.

(iii) Do resistance testing at the time of retreatment after failure. We know that viruses which are resistant to protease inhibitors and to non-nucleoside inhibitors disappear progressively within a year or so after treatment. This is not the case for NS5A resistant viruses. At present it is not clear as to what is the influence of these persistent resistant viruses and whether and when to make a decision for retreatment.

In my opinion, we could take benefit of resistance testing at the time of retreatment if the patient fails on DAA regime but the problem is that at the moment we do not know how to use this information so further studies are needed.

We need these resistance based retreatment studies to make more informed treatment decisions. There is however, still a position to do some resistance testing especially when re-treating patients who have been exposed to DAAs and when the treatment has failed.

It is possible to reduce the absolute number of test for HCV. In developing countries, where they have access to drugs at more affordable prices (subject to compliance), they only need to do a viral load measurement at baseline and even this can be replaced with HCV core antigen testing as it is a surrogate test for micro-active replication. This is completed with one more test 12 weeks after the end of treatment. This can be modulated onsite in line with the resources available.