Dataset Information

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

ABSTRACT: The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct-acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8%; however, this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (P < .05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR next-generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the nonquantitative nature of the technique and despite the designation of false-positive thresholds based on negative controls.

SUBMITTER: McNaughton AL

PROVIDER: S-EPMC5947153 | biostudies-other | 2018 May

REPOSITORIES: biostudies-other

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Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

McNaughton A L AL Sreenu V B VB Wilkie G G Gunson R R Templeton K K Leitch E C M ECM

Journal of viral hepatitis 20180221 5

The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct-acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen ...[more]

PMID: 29274184

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Project description:BackgroundShort read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.ResultsTotal substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations.ConclusionsQuantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Dataset Information

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

Publications

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

Similar Datasets

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