Brendan Jacka

Phylogenetic clustering of hepatitis C virus among people who inject drugs in Vancouver, Canada

Little is known about factors associated with hepatitis C virus (HCV) transmission among people who inject drugs (PWID). Phylogenetic clustering and associated factors were evaluated among PWID in Vancouver, Canada. Data were derived from the Vancouver Injection Drug Users Study. Participants who were HCV antibody‐positive at enrolment and those with HCV antibody seroconversion during follow‐up (1996 to 2012) were tested for HCV RNA and sequenced (Core‐E2 region). Phylogenetic trees were inferred using maximum likelihood analysis and clusters were identified using ClusterPicker (90% bootstrap threshold, 0.05 genetic distance threshold). Factors associated with clustering were assessed using logistic regression. Among 655 eligible participants, HCV genotype prevalence was: G1a: 48% (n = 313), G1b: 6% (n = 41), G2a: 3% (n = 20), G2b: 7% (n = 46), G3a: 33% (n = 213), G4a: <1% (n = 4), G6a: 1% (n = 8), G6e: <1% (n = 1), and unclassifiable: 1% (n = 9). The mean age was 36 years, 162 (25%) were female, and 164 (25%) were HIV+. Among 501 participants with HCV G1a and G3a, 31% (n = 156) were in a pair/cluster. Factors independently associated with phylogenetic clustering included: age <40 (versus age ≥40, adjusted odds ratio [AOR] = 1.64; 95% confidence interval [CI] 1.03, 2.63), human immunodeficiency virus (HIV) infection (AOR = 1.82; 95% CI 1.18, 2.81), HCV seroconversion (AOR = 3.05; 95% CI 1.40, 6.66), and recent syringe borrowing (AOR 1.59; 95% CI 1.07, 2.36). Conclusion: In this sample of PWID, one‐third demonstrated phylogenetic clustering. Factors independently associated with phylogenetic clustering included younger age, recent HCV seroconversion, prevalent HIV infection, and recent syringe borrowing. Strategies to enhance the delivery of prevention and/or treatment strategies to those with HIV and recent HCV seroconversion should be explored, given an increased likelihood of HCV transmission in these subpopulations. (Hepatology 2014;60:1571–1580)

The Influence of Hepatitis C Virus Genetic Region on Phylogenetic Clustering Analysis

Sequencing is important for understanding the molecular epidemiology and viral evolution of hepatitis C virus (HCV) infection. To date, there is little standardisation among sequencing protocols, in-part due to the high genetic diversity that is observed within HCV. This study aimed to develop a novel, practical sequencing protocol that covered both conserved and variable regions of the viral genome and assess the influence of each subregion, sequence concatenation and unrelated reference sequences on phylogenetic clustering analysis. The Core to the hypervariable region 1 (HVR1) of envelope-2 (E2) and non-structural-5B (NS5B) regions of the HCV genome were amplified and sequenced from participants from the Australian Trial in Acute Hepatitis C (ATAHC), a prospective study of the natural history and treatment of recent HCV infection. Phylogenetic trees were constructed using a general time-reversible substitution model and sensitivity analyses were completed for every subregion. Pairwise distance, genetic distance and bootstrap support were computed to assess the impact of HCV region on clustering results as measured by the identification and percentage of participants falling within all clusters, cluster size, average patristic distance, and bootstrap value. The Robinson-Foulds metrics was also used to compare phylogenetic trees among the different HCV regions. Our results demonstrated that the genomic region of HCV analysed influenced phylogenetic tree topology and clustering results. The HCV Core region alone was not suitable for clustering analysis; NS5B concatenation, the inclusion of reference sequences and removal of HVR1 all influenced clustering outcome. The Core-E2 region, which represented the highest genetic diversity and longest sequence length in this study, provides an ideal method for clustering analysis to address a range of molecular epidemiological questions.

Sequencing of the Hepatitis C Virus: A Systematic Review

Since the identification of hepatitis C virus (HCV), viral sequencing has been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of certain regions of the virus, however there has been little standardisation of protocols. This systematic review was undertaken to map the location and frequency of sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of sequencing primers and amplicons to inform future research. Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (9 terms) or HCV (3 terms), plus “primer” as a general search term. Exclusion criteria included non-HCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. A total of 450 studies were accepted from the 2099 identified, with 629 HCV sequencing amplicons identified and mapped on the HCV genome. The most commonly sequenced region was the HVR-1 region, often utilised for studies of natural history, clustering/transmission, evolution and treatment response. Studies related to genotyping/classification or epidemiology of HCV genotype generally targeted the 5′UTR, Core and NS5B regions, while treatment response/resistance was assessed mainly in the NS3–NS5B region with emphasis on the Interferon sensitivity determining region (ISDR) region of NS5A. While the sequencing of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing, providing a comprehensive database of previously published primer sets to be utilised in future sequencing studies.

A molecular phylogenetics-based approach for identifying recent hepatitis C virus transmission events.

UNLABELLED Improved surveillance methods are needed to better understand the current hepatitis C virus (HCV) disease burden and to monitor the impact of prevention and treatment interventions on HCV transmission dynamics. Sanger sequencing (HCV NS5B, HVR1 and Core-E1-HVR1) and phylogenetics were applied to samples from individuals diagnosed with HCV in British Columbia, Canada in 2011. This included individuals with two or three sequential samples collected <1 year apart. Patristic distances between sequential samples were used to set cutoffs to identify recent transmission clusters. Factors associated with transmission clustering were analyzed using logistic regression. From 618 individuals, 646 sequences were obtained. Depending on the cutoff used, 63 (10%) to 92 (15%) unique individuals were identified within transmission clusters of predicted recent origin. Clustered individuals were more likely to be <40 years old (Adjusted Odds Ratio (AOR) 2.12, 95% CI 1.21-3.73), infected with genotype 1a (AOR 6.60, 95% CI 1.98-41.0), and to be seroconverters with estimated infection duration of <1 year (AOR 3.13, 95% CI 1.29-7.36) or >1 year (AOR 2.19, 95% CI 1.22-3.97). CONCLUSION Systematic application of molecular phylogenetics may be used to enhance traditional surveillance methods through identification of recent transmission clusters.

Transmission of hepatitis C virus infection among younger and older people who inject drugs in Vancouver, Canada

BACKGROUND & AIMS Understanding HCV transmission among people who inject drugs (PWID) is important for designing prevention strategies. This study investigated whether HCV infection among younger injectors occurs from few or many transmission events from older injectors to younger injectors among PWID in Vancouver, Canada. METHODS HCV antibody positive participants at enrolment or follow-up (1996-2012) were tested for HCV RNA and sequenced (Core-E2). Time-stamped phylogenetic trees were inferred using Bayesian Evolutionary Analysis Sampling Trees (BEAST). Association of age with phylogeny was tested using statistics implemented in the software Bayesian Tip Significance (BaTS) testing. Factors associated with clustering (maximum cluster age: five years) were identified using logistic regression. RESULTS Among 699 participants with HCV subtype 1a, 1b, 2b and 3a infection (26% female, 24% HIV+): 21% were younger (<27years), and 10% had recent HCV seroconversion. When inferred cluster age was limited to <5years, 15% (n=108) were in clusters/pairs. Although a moderate degree of segregation was observed between younger and older participants, there was also transmission between age groups. Younger age (<27 vs. >40, AOR: 3.14; 95% CI: 1.54, 6.39), HIV (AOR: 1.97; 95% CI: 1.22, 3.18) and subtype 3a (AOR: 2.12; 95% CI: 1.33, 3.38) were independently associated with clustering. CONCLUSIONS In this population of PWID from Vancouver, HCV among young injectors was seeded from many transmission events between HCV-infected older and younger injectors. Phylogenetic clustering was associated with younger age and HIV. These data suggest that HCV transmission among PWID is complex, with transmission occurring between and among older and younger PWID.

Methamphetamine injecting is associated with phylogenetic clustering of hepatitis C virus infection among street-involved youth in Vancouver, Canada

BACKGROUND Among prospective cohorts of people who inject drugs (PWID), phylogenetic clustering of HCV infection has been observed. However, the majority of studies have included older PWID, representing distant transmission events. The aim of this study was to investigate phylogenetic clustering of HCV infection among a cohort of street-involved youth. METHODS Data were derived from a prospective cohort of street-involved youth aged 14-26 recruited between 2005 and 2012 in Vancouver, Canada (At Risk Youth Study, ARYS). HCV RNA testing and sequencing (Core-E2) were performed on HCV positive participants. Phylogenetic trees were inferred using maximum likelihood methods and clusters were identified using ClusterPicker (Core-E2 without HVR1, 90% bootstrap threshold, 0.05 genetic distance threshold). RESULTS Among 945 individuals enrolled in ARYS, 16% (n=149, 100% recent injectors) were HCV antibody positive at baseline interview (n=86) or seroconverted during follow-up (n=63). Among HCV antibody positive participants with available samples (n=131), 75% (n=98) had detectable HCV RNA and 66% (n=65, mean age 23, 58% with recent methamphetamine injection, 31% female, 3% HIV+) had available Core-E2 sequences. Of those with Core-E2 sequence, 14% (n=9) were in a cluster (one cluster of three) or pair (two pairs), with all reporting recent methamphetamine injection. Recent methamphetamine injection was associated with membership in a cluster or pair (P=0.009). CONCLUSION In this study of street-involved youth with HCV infection and recent injecting, 14% demonstrated phylogenetic clustering. Phylogenetic clustering was associated with recent methamphetamine injection, suggesting that methamphetamine drug injection may play an important role in networks of HCV transmission.

Sequencing of Hepatitis C Virus for Detection of Resistance to Direct-Acting Antiviral Therapy: A Systematic Review

The significance of the clinical impact of direct‐acting antiviral (DAA) resistance‐associated substitutions (RASs) in hepatitis C virus (HCV) on treatment failure is unclear. No standardized methods or guidelines for detection of DAA RASs in HCV exist. To facilitate further evaluations of the impact of DAA RASs in HCV, we conducted a systematic review of RAS sequencing protocols, compiled a comprehensive public library of sequencing primers, and provided expert guidance on the most appropriate methods to screen and identify RASs. The development of standardized RAS sequencing protocols is complicated due to a high genetic variability and the need for genotype‐ and subtype‐specific protocols for multiple regions. We have identified several limitations of the available methods and have highlighted areas requiring further research and development. The development, validation, and sharing of standardized methods for all genotypes and subtypes should be a priority. (Hepatology Communications 2017;1:379–390)

Deep sequencing increases hepatitis C virus phylogenetic cluster detection compared to Sanger sequencing

Effective surveillance and treatment strategies are required to control the hepatitis C virus (HCV) epidemic. Phylogenetic analyses are powerful tools for reconstructing the evolutionary history of viral outbreaks and identifying transmission clusters. These studies often rely on Sanger sequencing which typically generates a single consensus sequence for each infected individual. For rapidly mutating viruses such as HCV, consensus sequencing underestimates the complexity of the viral quasispecies population and could therefore generate different phylogenetic tree topologies. Although deep sequencing provides a more detailed quasispecies characterization, in-depth phylogenetic analyses are challenging due to dataset complexity and computational limitations. Here, we apply deep sequencing to a characterized population to assess its ability to identify phylogenetic clusters compared with consensus Sanger sequencing. For deep sequencing, a sample specific threshold determined by the 50th percentile of the patristic distance distribution for all variants within each individual was used to identify clusters. Among seven patristic distance thresholds tested for the Sanger sequence phylogeny ranging from 0.005-0.06, a threshold of 0.03 was found to provide the maximum balance between positive agreement (samples in a cluster) and negative agreement (samples not in a cluster) relative to the deep sequencing dataset. From 77 HCV seroconverters, 10 individuals were identified in phylogenetic clusters using both methods. Deep sequencing analysis identified an additional 4 individuals and excluded 8 other individuals relative to Sanger sequencing. The application of this deep sequencing approach could be a more effective tool to understand onward HCV transmission dynamics compared with Sanger sequencing, since the incorporation of minority sequence variants improves the discrimination of phylogenetically linked clusters.

HIV infection and hepatitis C virus genotype 1a are associated with phylogenetic clustering among people with recently acquired hepatitis C virus infection.

The aim of this study was to identify factors associated with phylogenetic clustering among people with recently acquired hepatitis C virus (HCV) infection. Participants with available sample at time of HCV detection were selected from three studies; the Australian Trial in Acute Hepatitis C, the Hepatitis C Incidence and Transmission Study - Prison and Community. HCV RNA was extracted and Core to E2 region of HCV sequenced. Clusters were identified from maximum likelihood trees with 1000 bootstrap replicates using 90% bootstrap and 5% genetic distance threshold. Among 225 participants with available Core-E2 sequence (ATAHC, n=113; HITS-p, n=90; and HITS-c, n=22), HCV genotype prevalence was: G1a: 38% (n=86), G1b: 5% (n=12), G2a: 1% (n=2), G2b: 5% (n=11), G3a: 48% (n=109), G6a: 1% (n=2) and G6l 1% (n=3). Of participants included in phylogenetic trees, 22% of participants were in a pair/cluster (G1a-35%, 30/85, mean maximum genetic distance=0.031; G3a-11%, 12/106, mean maximum genetic distance=0.021; other genotypes-21%, 6/28, mean maximum genetic distance=0.023). Among HCV/HIV co-infected participants, 50% (18/36) were in a pair/cluster, compared to 16% (30/183) with HCV mono-infection (P=<0.001). Factors independently associated with phylogenetic clustering were HIV co-infection [vs. HCV mono-infection; adjusted odds ratio (AOR) 4.24; 95%CI 1.91, 9.39], and HCV G1a infection (vs. other HCV genotypes; AOR 3.33, 95%CI 0.14, 0.61).HCV treatment and prevention strategies, including enhanced antiviral therapy, should be optimised. The impact of targeting of HCV treatment as prevention to populations with higher phylogenetic clustering, such as those with HIV co-infection, could be explored through mathematical modelling.

Venue-Based Networks May Underpin HCV Transmissions amongst HIV-Infected Gay and Bisexual Men

Background This study aimed to investigate the potential influence of venue-based networks on HCV transmission in HIV-positive gay and bisexual men (GBM). Methods This was a prospectively recruited cohort of HIV-infected GBM with recently-acquired HCV infection resident in Melbourne and Sydney. Clinical and demographic data were collected together with blood samples for HCV sequencing. Phylogenies were inferred and clusters of individuals infected with HCV with genetic sequence homology were identified. Venues used for sourcing sexual partners were identified; sourcing partners from the same venue was considered a potential social link. Using the Jaccard similarity coefficient, associations were identified between the network of sites where men sourced sex partners and transmission relationships as defined by phylogenetic clustering. Results Forty individuals were recruited, of whom 62.5% were considered to have sexually- and 37.5% IDU-acquired HCV. Venue use was consistent with men being members of a more sexually adventurous gay community subculture. Six phylogenetically-determined pairs or clusters were identified, comprising fifteen (15/28, 53.6%) individuals. Participants belonging to phylogenetic clusters were observed within the same networks. There was a significant correlation between the network and phylogenetic clustering when both cities were considered simultaneously (p = 0.005), raising the possibility that social connections may be important for HCV transmissions. Conclusions Venue-based network elicitation is a promising approach for elucidating HCV transmissions amongst HIV-infected GBM. Public health approaches targeting individuals and venues prominent within networks may reduce onward HCV transmission.