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Deep sequencing creates distinction between acute and chronic HCV

Deep sequencing accurately differentiated between acute and chronic hepatitis C virus infection, according to published study data.

“Deep sequencing studies have demonstrated improved sensitivity for detection of minor sequence variants, compared to clonal sequencing, enabling highly sensitive, specific differentiation of acute and chronic HIV infection, but similar differentiation analyses have heretofore not been applied to HCV,” the researchers wrote. “In this study, amplicons from the HCV nonstructural 5B region from 77 individuals with documented seroconversion time frames were deep sequenced to assess its potential to differentiate acute from chronic infection.” 

For the study, 94 HCV-positive nonstructural 5B (NS5B) amplicons were collected from 77 patients from British Columbia, Canada. Of these patients, 13 had acute and 64 had chronic HCV infections.

“Amplicons were deep sequenced and HCV genomic diversity was measured using Shannon entropy and a single nucleotide variant analysis,” researchers wrote.

To measure the relationship between diversity and days from infection diagnosis, linear mixed models were used, and each measure used to differentiate the acute from chronic infections was assessed using generalized estimating equations, according to the researchers.

Overall, Shannon entropy (SE) and single nucleotide variant (SNV) measures were different for acute and chronic infections (P < .009), with SE outperforming SNV based on area under the receiver operating characteristic curves (AUROC). However, the SNV analysis outperformed SE without the removal of sequencing errors, leading researchers to suggest that this error removal method could be removing “true” HCV sequence variants.

“These findings highlight some of the challenges in evaluating the utility of sequence diversity measures where the sequencing technology itself can introduce random and nonrandom errors,” the researchers wrote.

In patients with an unknown duration of infection (n = 39), the AUROC was 0.96 for SE and 0.98 for SNV. The researchers observed AUROCs were lower when all data was included for all the patients (0.86 for SE; 0.8 for SNV), but they were still considered high.

Over 78% of the genetic diversity in the samples was due to synonymous mutations. NS5B nucleotide diversity increased up until at least 3 years post-infection.

“NS5B diversity measures derived from deep sequencing are significantly different for acute vs. chronic HCV infections,” the researchers concluded. “This information could, in turn, help guide prevention, care and treatment programming. Further studies with larger numbers of acute and chronic infections, together with sequential samples from individual patients, will be required to validate the findings of this study.” – by Melinda Stevens

Disclosures: Montoya reports no relevant financial disclosures. Please see the study for a full list of all other authors’ relevant financial disclosures.