Using Deep Sequencing Technology to Detect HCV Resistant Variants

Although direct-acting antivirals (DAAs) are the current standard of care for patients with chronic HCV, strains of HCV that are resistant to DAAs are emerging. To this end, researchers from the Netherlands have turned to using precise, deep sequencing technology, to detect HCV-resistant variants in plasma and liver tissue samples. They discuss the results of using this technology in a new

study

, published in the online journal, Scientific Reports.

Traditionally, clinicians and researchers have used population-based Sanger sequencing as a means of finding drug-resistant variants of HCV.

However, a research team—led by V. Stalin Raj of the Department of Viroscience at Erasmus Medical Center in Rotterdam, Netherlands—stated in their study that “the abundant HCV diversity in chronically-infected HCV-infected patients cannot be fully mapped [using the traditional model] as relevant proportions of minority variants can be missed.” Thus, the researchers turned their attention to deep sequencing (DPS), which has been used in the past to identify rare, minority, drug-resistant variants of HIV. Other studies have found that DPS can identify “minor drug-resistant variants in plasma of HCV-infected individuals” as well.

For their study, the researchers investigated, “the potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients.”

After optimizing their “DPS protocols, data cleaning, and error correction strategies using a reference plasmid as input,” the researchers found that, “the data indicated that the intra-assay precision was very high and that most sequencing errors were introduced by PCR and deep sequencing and not by reverse transcription.”

“

Although the average error rate after read cleaning was very low in the control experiments, ~0.01% and ~0.03% on the nucleotide and amino acid level, respectively, the range of the error rates per nucleotide or amino acid was 0—3%,” the researchers wrote.

Similar results were obtained in the analyses of HIV quasispecies.

The researchers concluded that each “nucleotide- or amino acid-specific position should be evaluated separately” in order to achieve results “for specific positions of special interest,” such as drug resistance positions.

To analyze the NS5B quasispecies in the liver and plasma sample, “haplotypes were reconstructed and based on the control experiments, a conservative cut-off was placed at a haplotype frequency of 1%,” the authors wrote.

Previous studies using Sanger sequencing analyses showed that mutations that allow for drug resistance were present in treatment-naive patients at a rate of 0.19-24%. However, in the present study, those mutations were found in 16 out of 18 patients (~89%), and were either dominant (50%) or minority haplotypes (39%).

In addition, they found “variants containing 2 mutations in the same genomic strand involved in drug resistance against different compounds.”

When the researchers analyzed the liver and plasma separately, they found that there were slightly more resistant variants in the liver than in the plasma. “Overall 22/198 variants were detected in the liver but not in the plasma whereas less unique variants were observed in the plasma (12/198).” Because the prevalence of resistant variants was “only somewhat higher” in the liver than in the plasma, the authors concluded that “the use of plasma is most likely sufficient to detect HCV quasispecies and drug-resistant associated variants.” However, they note that in order to understand the clinical implications of these findings, more research needs to be done using “large cohorts of paired samples, including an analysis of other genome regions targeted by” direct-acting antivirals. In addition, the researchers suggested using caution in interpreting the data, noting that a liver biopsy obtained by a needle may not be a representative sample of the whole liver.

“Overall, our data thus provide insight into the HCV Ns5B quasispecies population in liver and plasma in treatment-naive patients obtained through state-of-the-art sensitive sequencing technologies,” the authors concluded.