Time to Detection of Hepatitis C Virus Infection With the Xpert HCV Viral Load Fingerstick Point-of-Care Assay: Facilitating a More Rapid Time to Diagnosis.

BACKGROUND: Xpert HCV Viral Load Fingerstick assay (Xpert HCV VL FS) is a point-of-care test quantifying HCV RNA in <1 hour, enabling same-visit diagnosis and treatment. METHODS: This study evaluated time to HCV RNA detection using the Xpert HCV VL FS assay. Fingerstick whole-blood samples were collected from participants in an observational cohort in Australia. RESULTS: In May 2018-2019, 1468 participants were enrolled, 1426 had Xpert HCV VL FS testing performed, and 1386 had a valid result. HCV RNA was detected in 23% (325/1386). Among people with undetectable HCV RNA (n = 1061), median time to result was 57 minutes. Among people with detectable HCV RNA (n = 325), median time to HCV RNA detection was 32 minutes and 80% (261/325) had a detectable HCV RNA result in ≤40 minutes. Median time to HCV RNA detection was dependent on HCV RNA level. CONCLUSIONS: A quicker HCV diagnosis could be achieved by monitoring the time when HCV RNA is first detected with the Xpert HCV VL FS test, rather than HCV RNA quantification, although the current platform does not allow for this. These findings could facilitate new strategies to reduce waiting times for an HCV diagnosis and improve linkage to treatment.

Patterns and correlates of hepatitis C virus phylogenetic clustering among people living with HIV in Australia in the direct-acting antiviral era: A molecular epidemiology study among participants in the CEASE cohort.

Background and Aims: In moving towards the elimination of hepatitis C virus (HCV) infection among people living with HIV, understanding HCV transmission patterns may provide insights to guide and evaluate interventions. In this study, we evaluated patterns of, and factors associated with HCV phylogenetic clustering among people living with HIV/HCV co-infection in Australia in the direct-acting antiviral era. Methods: HCV RNA was extracted from dried blood spot (DBS) samples collected between 2014 and 2018 in the CEASE cohort study. The HCV Core-E2 region was amplified by a polymerase chain reaction and Sanger sequenced. Maximum likelihood phylogenetic trees (1000 bootstrap replicates) were used to identify patterns of clustering (3% genetic distance threshold). Mixed-effects logistic regression was used to determine correlates of phylogenetic clustering. Factors assessed were sexual risk behavior, education, injecting drug use, housing, employment, HIV viral load, age, sex, and sexuality. Results: Phylogenetic trees were reconstructed for HCV subtype 1a (n = 139) and 3a (n = 63) sequences, with 29% (58/202) in a pair or cluster. Overall (n = 202), phylogenetic clustering was positively associated with younger age (under 40; adjusted odds ratio [aOR] 2.52, 95% confidence interval [CI] 1.20-5.29), and among gay and bisexual men (n = 168), was positively associated with younger age (aOR 2.61, 95% CI 1.10-6.19), higher education (aOR 2.58, 95% CI 1.09-6.13), and reporting high-risk sexual behavior (aOR 3.94, 95% CI 1.31-11.84). During follow-up, five reinfections were observed, but none were in phylogenetic clusters. Conclusion: This study found a high proportion of phylogenetic relatedness, predominantly among younger people and gay and bisexual men reporting high-risk sexual behavior. Despite this, few reinfections were observed, and reinfections demonstrated little relationship with known clusters. These findings highlight the importance of rapid HCV treatment initiation, together with monitoring of the phylogeny.

Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing.

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct < 30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis.

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.