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Type III interferons (IFN-λ) are part of the innate immune response to hepatitis C virus (HCV) infection however the specific role of IFN-λ4 and the nature of the viral adaption to this pressure have not been defined. Here we use paired genome-wide human and viral genetic data in 485 patients infected with HCV genotype 3a to explore the role of IFN-λ4 on HCV evolution during chronic infection. We show that genetic variations within the host IFNL4 locus have a broad and systematic impact on HCV amino acid diversity. We also demonstrate that this impact is larger in patients producing a more active form of IFN-λ4 protein compared to the less active form. A similar observation was noted for viral load. We conclude that IFN-λ4 protein is a likely causal agent driving widespread HCV amino acid changes and associated with viral load and possibly other clinical and biological outcomes of HCV infection.

Hepatitis C virus (HCV) genotype (gt) 3 is highly prevalent globally, with non-gt3a subtypes common in Southeast Asia. Resistance-associated substitutions (RASs) have been shown to play a role in treatment failure. However, the role of RASs in gt3 is not well understood. We report the prevalence of RASs in a cohort of direct-acting antiviral treatment-naive, gt3-infected patients, including those with rarer subtypes, and evaluate the effect of these RASs on direct-acting antivirals in vitro. Baseline samples from 496 gt3 patients enrolled in the BOSON clinical trial were analyzed by next-generation sequencing after probe-based enrichment for HCV. Whole viral genomes were analyzed for the presence of RASs to approved direct-acting antivirals. The resistance phenotype of RASs in combination with daclatasvir, velpatasvir, pibrentasvir, elbasvir, and sofosbuvir was measured using the S52 ΔN gt3a replicon model. The nonstructural protein 5A A30K and Y93H substitutions were the most common at 8.9% (n = 44) and 12.3% (n = 61), respectively, and showed a 10-fold and 11-fold increase in 50% effect concentration for daclatasvir compared to the unmodified replicon. Paired RASs (A30K + L31M and A30K + Y93H) were identified in 18 patients (9 of each pair); these combinations were shown to be highly resistant to daclatasvir, velpatasvir, elbasvir, and pibrentasvir. The A30K + L31M combination was found in all gt3b and gt3g samples. Conclusion: Our study reveals high frequencies of RASs to nonstructural protein 5A inhibitors in gt3 HCV; the paired A30K + L31M substitutions occur in all patients with gt3b and gt3g virus, and in vitro analysis suggests that these subtypes may be inherently resistant to all approved nonstructural protein 5A inhibitors for gt3 HCV. (Hepatology 2018).

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

Objectives To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management. Design The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Setting Isolates were obtained from potential outbreaks associated with three UK hospitals. Participants Isolates were sequenced from a cluster of eight MRSA carriers and an associated bacteraemia case in an intensive care unit, another MRSA cluster of six cases and two clusters of C difficile. Additionally, all C difficile isolates from cases over 6 weeks in a single hospital were rapidly sequenced and compared with local strain sequences obtained in the preceding 3 years. Main outcome measure Whole-genome genetic relatedness of the isolates within each epidemiological cluster. Results Twenty-six MRSA and 15 C difficile isolates were successfully sequenced and analysed within 5 days of culture. Both MRSA clusters were identified as outbreaks, with most sequences in each cluster indistinguishable and all within three single nucleotide variants (SNVs). Epidemiologically unrelated isolates of the same spa-type were genetically distinct (≥21 SNVs). In both C difficile clusters, closely epidemiologically linked cases (in one case sharing the same strain type) were shown to be genetically distinct (≥144 SNVs). A reconstruction applying rapid sequencing in C difficile surveillance provided early outbreak detection and identified previously undetected probable community transmission. Conclusions This benchtop sequencing technology is widely generalisable to human bacterial pathogens. The findings provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection and therefore improve hospital infection control and patient outcomes in routine clinical practice., Article summary Article focus To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. In particular to investigate the potential of such technology for identification of transmission events of healthcare-associated pathogens. Key messages We demonstrate benchtop sequencing can enhance hospital infection control through high precision support and rejection of transmission using genetic data. Whole-genome data provided additional genetic resolution over existing genetic typing strategies. We also show this technology offers turnaround times of under a week in a format that, in contrast to molecular typing, is organism independent. Strengths and limitations of this study The case studies presented provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection. Given this is a pilot study, further evaluations of the impact of this technology on hospital infection control are required. However, this study provides a clear rationale for future work undertaking formal comparisons of benchtop sequencing with existing local and national typing schemes.

BACKGROUND: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions. METHODS: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location. RESULTS: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile. CONCLUSIONS: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.)

Affordable next generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting anti-viral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV viral sequence datasets and evaluated their utility for diagnostics and clinical assessment.NGS methods using (1) unselected HCV RNA (metagenomic); (2) pre-enrichment of HCV RNA by probe capture and (3) HCV pre-amplification by PCR implemented in four UK centres were compared. Metrics of sequence coverage and depth, quasispecies diversity and detection of DAA-resistance associated variants (RAVs), mixed HCV genotype and other co-infections were compared using a panel of samples of varying viral load, genotype and mixed HCV geno(sub)types. Each NGS method generated near complete genome sequences from over 90% of samples. Enrichment methods and PCR pre-amplification generated greater sequence depth and were more effective for low viral load samples. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus co-infections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing genotype and information on potential DAA resistance.