B-cell characteristics define HCV reinfection outcome.

BACKGROUND & AIMS: In individuals highly exposed to HCV, reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. Strong evidence of antigen imprinting was found, and the B-cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single-cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.

'Where is the diversity in this facility?' Experiences of emergency care among visible minority individuals in Kingston, Ontario.

INTRODUCTION: Visible minorities, a growing segment of Canada's population, have voiced concerns about experiencing racism while receiving care in the emergency department (ED). Understanding the ED care experiences of visible minorities is crucial to improving care and reducing health disparities. METHODS: From June to August 2021, we collected data from participants in Kingston, Ontario using a sensemaking approach. Individuals who had accessed emergency care or accompanied someone else to the ED in the prior 24 months were eligible to participate. After sharing a brief narrative about their care experience, participants interpreted the experience by plotting their perspectives on a variety of pre-determined questions. Here, we conducted a thematic analysis of narratives involving patients who identified as visible minorities and complemented it with quantitative analysis of the participants' interpretative responses. This mixed-methods approach highlighted the distinct experiences of visible minority participants in relation to a comparison group. RESULTS: Of the 1973 unique participants, 117 identified as a visible minority and 949 participants did not identify with an equity-deserving group (comparison group). Visible minority participants were more likely to report that too little attention was paid to their identity and more likely to express a desire for a balance between receiving the best medical care and being treated with kindness and respect. Visible minorities' ED experiences were also more likely to be impacted by how emergency staff behaved. Qualitative analysis revealed negative experiences of feeling uninformed and disempowered, facing judgement and discrimination, and experiencing language barriers. Positive experiences of receiving compassionate care from staff also emerged. CONCLUSION: Visible minority perceptions of ED care were often negative and mainly focused on staff treatment. Cultural competency and language translation services are key areas for improvement to make ED care more accessible and equitable.

Incidence and risk factors of post-stroke seizure among ischemic stroke patients.

BACKGROUND: Post-stroke seizure (PSS) increases morbidity and mortality after ischemic stroke, but a comprehensive understanding of its incidence and risk factors is lacking. We report the rate and risk factors of PSS at a single institution. METHODS: A retrospective cohort study of adult acute ischemic stroke patients between 2018 and 2022 at a comprehensive stroke center was conducted. Patients with a history of seizures, additional stroke during index admission, or death within 7 days of stroke onset were excluded. Early PSS was defined as a new seizure occurring ≤7 days after stroke onset, while late PSS occurred >7 days after stroke onset. Multivariable logistic regression and cox proportional hazard analysis was conducted. RESULTS: 1211 participants met inclusion criteria. Patients were a mean age of 67.82 and were primarily male (58.7 %), white (72.6 %), and non-Hispanic (91.9 %). Incidence of PSS was 8.8 % (n = 106), of which 53.8 % (n = 57) were early and 46.2 % (n = 49) were late. Bivariate analysis identified younger age, diabetes, baseline National Institutes of Health Stroke Scale (NIHSS), Alberta Stroke Program Early Computed Tomography Score ≤5, cortical involvement, and hemorrhagic transformation as significant in the development of PSS. Multivariable cox proportional hazard analysis identified cortical involvement (hazard ratio [HR]: 2.31, 95 % confidence interval [CI] [1,29, 4.14]), NIHSS ≥ 21 (HR: 1.82, 95 % CI [1.02, 3.22]),and younger age (HR: 0.97, 95 % CI [0.96, 0.98]) as significant PSS predictors. CONCLUSION: PSS occurred in 8.8 % of patients presenting with ischemic stroke. Hemorrhagic transformation, cortical involvement, high NIHSS, and younger age were significant predictors of PSS.

Emergency department care experiences among people who use substances: a qualitative study.

BACKGROUND: People who use substances (PWUS) encounter significant barriers to accessing care for their complex health needs. As a result, emergency departments (EDs) often become the first point of healthcare access for many PWUS and are a crucial setting for the study of health inequities. This study aimed to understand the ED healthcare experiences of PWUS with the intent of informing ways of improving the delivery of equitable care. METHODS: This qualitative study was part of a larger cross-sectional, mixed-methods study that examined ED experiences among diverse underserved and equity-deserving groups (EDGs) within Kingston, Ontario, Canada. Participants shared and self-interpreted a story about a memorable ED or UCC visit within the preceding 24 months. Data from participants who self-identified as having substance use experiences was analyzed through inductive thematic analysis. RESULTS: Of the 1973 unique participants who completed the survey, 246 participants self-identified as PWUS and were included in the analysis. Most participants were < 45 years of age (61%), male (53%), and white/European (57%). 45% identified as a person with a disability and 39% frequently struggled to make ends meet. Themes were determined at the patient, provider, and system levels. PATIENT: history of substance use and experience of intersectionality negatively influenced participants' anticipation and perception of care. Provider: negative experiences were linked to assumption making, feelings of stigma and discrimination, and negative perceptions of provider care. Whereas positive experiences were linked to positive perceptions of provider care. System: timeliness of care and the perception of inadequate mental health resources negatively impacted participants' care experience. Overall, these themes shaped participants' trust of ED staff, their desire to seek care, and their perception of the care quality received. CONCLUSIONS: PWUS face significant challenges when seeking care in the ED. Given that EDs are a main site of healthcare utilization, there is an urgent need to better support staff in the ED to improve care experiences among PWUS. Based on the findings, three recommendations are proposed: (1) Integration of an equity-oriented approach into the ED, (2) Widespread training on substance use, and (3) Investment in expert resources and services to support PWUS.

Urgent care-seeking and injury severity for intimate partner violence during COVID-19: a Canadian retrospective chart review.

BACKGROUND: Early reports raised alarms that intimate partner violence (IPV) increased during the COVID-19 pandemic, but initial studies showed that visits to emergency departments (EDs) decreased. This study assessed the impact of the prolonged pandemic and its associated restrictions on both rates of urgent care-seeking and injury severity for IPV. METHODS: Data from the Kingston Health Sciences Centre's (KHSC) ED were utilized to compare IPV presentations during 'Pre-COVID' (December 17, 2018 - March 16, 2020) and 'COVID' (March 17, 2020 - June 16, 2021), as well as three periods of heightened local restrictions: 'Lockdown-1' (March 17 - June 12, 2020), 'Lockdown-2' (December 26, 2020 - February 10, 2021) and 'Lockdown-3' (April 8 - June 2, 2021). The primary outcomes were incidence rate of IPV visits and injury severity, which was assessed using the Clinical Injury Extent Score (CIES) and Injury Severity Score (ISS). RESULTS: A total of 128 individuals were included. This sample had mean age of 34 years, was comprised of mostly women (97%), and represented a variety of intimate relationship types. Some individuals presented multiple times, resulting in a total of 139 acute IPV presentations. The frequency of IPV visits during COVID was similar to the Pre-COVID time period (67 vs. 72; p = 0.67). Incidence rate was 13% higher during COVID, though this difference was non-significant (6.66 vs. 5.90; p = 0.47). IPV visit frequency varied across lockdown periods (11 in Lockdown-1, 12 in Lockdown-2 and 6 in Lockdown-3), with the highest incidence rate during Lockdown-2 (12.71). There were more moderate and severe injuries during COVID compared to Pre-COVID, but mean CIES was not statistically significantly different (1.91 vs. 1.69; p = 0.29), nor was mean ISS (11.88 vs. 12.52; p = 0.73). CONCLUSIONS: During the 15-months following the start of COVID-19, there were small, but non-significant increases in both incidence rate and severity of IPV presentations to the KHSC ED. This may reflect escalation of violence as pandemic restrictions persisted and requires further investigation.

Lesbian, gay, bisexual, transgender and or queer patient experiences in Canadian primary care and emergency departments: a literature review.

This literature review synthesises existing evidence and offers a thematic analysis of primary care and emergency department experiences of lesbian, gay, bisexual, transgender, queer and/or any other sexual or gender minority (LGBTQ+) individuals in Canada. Articles detailing first-person primary or emergency care experiences of LGBTQ + patients were included from EMBASE, MEDLINE, PsycINFO and CINHAL. Studies published before 2011, focused on the COVID-19 pandemic, unavailable in English, non-Canadian, specific to other healthcare settings, and/or only discussing healthcare provider experiences were excluded. Critical appraisal was performed following title/abstract screening and full-text review by three reviewers. Of sixteen articles, half were classified as general LGBTQ + experiences and half as trans-specific experiences. Three overarching themes were identified: discomfort/disclosure concerns, lack of positive space signalling, and lack of healthcare provider knowledge. Heteronormative assumptions were a key theme among general LGBTQ + experiences. Trans-specific themes included barriers to accessing care, the need for self-advocacy, care avoidance, and disrespectful communication. Only one study reported positive interactions. LGBTQ + patients continue to have negative experiences within Canadian primary and emergency care - at the provider level and due to system constraints. Increasing culturally competent care, healthcare provider knowledge, positive space signals, and decreasing barriers to care can improve LGBTQ + experiences.

Emergency department care experiences among members of equity-deserving groups: quantitative results from a cross-sectional mixed methods study.

BACKGROUND: Emergency departments (EDs) serve an integral role in healthcare, particularly for vulnerable populations. However, marginalized groups often report negative ED experiences, including stigmatizing attitudes and behaviours. We engaged with historically marginalized patients to better understand their ED care experiences. METHOD: Participants were invited to complete an anonymous mixed-methods survey about a previous ED experience. We analysed quantitative data including controls and equity-deserving groups (EDGs) - those who self-identified as: (a) Indigenous; (b) having a disability; (c) experiencing mental health issues; (d) a person who uses substances; (e) a sexual and gender minority; (f) a visible minority; (g) experiencing violence; and/or (h) facing homelessness - to identify differences in their perspectives. Differences between EDGs and controls were calculated with chi squared tests, geometric means with confidence ellipses, and the Kruskal-Wallis H test. RESULTS: We collected a total of 2114 surveys from 1973 unique participants, 949 controls and 994 who identified as equity-deserving. Members of EDGs were more likely to attribute negative feelings to their ED experience (p < 0.001), to indicate that their identity impacted the care received (p < 0.001), and that they felt disrespected and/or judged while in the ED (p < 0.001). Members of EDGs were also more likely to indicate that they had little control over healthcare decisions (p < 0.001) and that it was more important to be treated with kindness/respect than to receive the best possible care (p < 0.001). CONCLUSION: Members of EDGs were more likely to report negative ED care experiences. Equity-deserving individuals felt judged and disrespected by ED staff and felt disempowered to make decisions about their care. Next steps will include contextualizing findings using participants' qualitative data and identifying how to improve ED care experiences among EDGs to make it more inclusive and better able to meet their healthcare needs.

International controlled study of revascularization and outcomes following COVID-positive mechanical thrombectomy.

BACKGROUND AND PURPOSE: Previous studies suggest that mechanisms and outcomes in patients with COVID-19-associated stroke differ from those in patients with non-COVID-19-associated strokes, but there is limited comparative evidence focusing on these populations. The aim of this study, therefore, was to determine if a significant association exists between COVID-19 status with revascularization and functional outcomes following thrombectomy for large vessel occlusion (LVO), after adjustment for potential confounding factors. METHODS: A cross-sectional, international multicenter retrospective study was conducted in consecutively admitted COVID-19 patients with concomitant acute LVO, compared to a control group without COVID-19. Data collected included age, gender, comorbidities, clinical characteristics, details of the involved vessels, procedural technique, and various outcomes. A multivariable-adjusted analysis was conducted. RESULTS: In this cohort of 697 patients with acute LVO, 302 had COVID-19 while 395 patients did not. There was a significant difference (p < 0.001) in the mean age (in years) and gender of patients, with younger patients and more males in the COVID-19 group. In terms of favorable revascularization (modified Thrombolysis in Cerebral Infarction [mTICI] grade 3), COVID-19 was associated with lower odds of complete revascularization (odds ratio 0.33, 95% confidence interval [CI] 0.23-0.48; p < 0.001), which persisted on multivariable modeling with adjustment for other predictors (adjusted odds ratio 0.30, 95% CI 0.12-0.77; p = 0.012). Moreover, endovascular complications, in-hospital mortality, and length of hospital stay were significantly higher among COVID-19 patients (p < 0.001). CONCLUSION: COVID-19 was an independent predictor of incomplete revascularization and poor functional outcome in patients with stroke due to LVO. Furthermore, COVID-19 patients with LVO were more often younger and had higher morbidity/mortality rates.

Alveolar progenitor cells in the mammary gland are dependent on the ß4 integrin.

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6ß4 integrin (ß4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that ß4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.

Real-time imaging of integrin ß4 dynamics using a reporter cell line generated by Crispr/Cas9 genome editing.

The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the ß4 subunit of the α6ß4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin ß4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin ß4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin ß4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin ß4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin ß4 expression in cells proximal to the wound edge was evident, and a population of integrin ß4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin ß4 dynamics during invasion and metastasis. Moreover, these integrin ß4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer.This article has an associated First Person interview with the first author of the paper.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

Relationship between women's decision-making power over their own health care and use of modern contraception in the Democratic Republic of the Congo: a cross-sectional secondary data analysis.

BACKGROUND: In sub-Saharan Africa, the use of modern contraception (MC) is a critical intervention aimed at reducing mortality rates associated with unintended, high-risk pregnancies. However, among Congolese women aged 15-49, the prevalence of MC use is low. Research suggests that women's general participation in decision-making is important in increasing MC use. However, little is known about the specific role of women's decision-making power over their own health care and how it relates to MC use. Thus, this study aimed to investigate the relationship between women's decision-making power over their own health care and use of MC. METHODS: A cross-sectional secondary data analysis was conducted using the most recent data from the 2013-2014 Democratic Republic of the Congo (DRC) Demographic and Health Survey. Women who were considered in need of contraception based on their family planning preferences were included in the study population (N = 6422). Multivariate logistic regression was used to determine whether women's decision-making power over their own health care was associated with the use of MC. RESULTS: Only one in ten women reported using a modern method of contraception. Logistic regression showed that women who made decisions alone regarding their own health care were more likely to use MC than women who had no say in these decisions, even after controlling for important covariates (OR 1.48; 95% CI 1.00, 2.17). CONCLUSION: The results of this study lend further support that promoting women's autonomy and right to independently make decisions regarding their own health may be important in increasing the use of MC in the DRC. However, in order to effectively empower women to negotiate for the use of MC, qualitative research is needed to further assess the relationship between decision-making power and MC use.

Challenging the Ischemic Core Concept in Acute Ischemic Stroke Imaging.

Endovascular treatment is a highly effective therapy for acute ischemic stroke due to large vessel occlusion and has recently revolutionized stroke care. Oftentimes, ischemic core extent on baseline imaging is used to determine endovascular treatment-eligibility. There are, however, 3 fundamental issues with the core concept: First, computed tomography and magnetic resonance imaging, which are mostly used in the acute stroke setting, are not able to precisely determine whether and to what extent brain tissue is infarcted (core) or still viable, due to variability in tissue vulnerability, the phenomenon of selective neuronal loss and lack of a reliable gold standard. Second, treatment decision-making in acute stroke is multifactorial, and as such, the relative importance of single variables, including imaging factors, is reduced. Third, there are often discrepancies between core volume and clinical outcome. This review will address the uncertainty in terminology and proposes a direction towards more clarity. This theoretical exercise needs empirical data that clarify the definitions further and prove its value.

B cell immunodominance in primary hepatitis C virus infection.

BACKGROUND & AIMS: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. METHODS: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. RESULTS: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. CONCLUSIONS: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. LAY SUMMARY: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.

Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection.

In rare cases, individuals with a history of long-term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high-risk behaviour. We describe anti-HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus-neutralizing activity was assessed on HCV pseudoparticles, and HCV E-specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti-E, predominantly in the IgG2 subclass, which was not found in the pre-infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV-specific B cells detected significantly more than Incident cases pre-infection. In conclusion, the HESN phenotype is associated with IgG2 anti-E antibodies, neutralization activity and HCV E-specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune-mediated mechanisms.

Association Between a Dietary Inflammatory Index and Prostate Cancer Risk in Ontario, Canada.

BACKGROUND: Evidence exists showing that various aspects of diet are implicated in the etiology of prostate cancer, although results across studies remain inconsistent. METHODS: We examined the ability of the dietary inflammatory index (DII) to predict prostate cancer in a case-control study conducted in Kingston, Ontario, Canada, between 1997 and 1999. The study included 72 cases of incident primary prostate cancer patients and 302 controls of urology clinic patients who had prostate conditions other than prostate cancer. The DII was computed based on intake of 18 nutrients assessed using a 67-item food frequency questionnaire. Univariate and multivariate logistic regression models were used to estimate odds ratios (ORs). RESULTS: Men with higher DII scores were at increased risk of prostate cancer using DII score fit both as a continuous [OR = 1.58, 95% confidence interval (CI) 1.05-2.38] and categorical variable [compared to men in the lowest DII quartile, men in the highest quartile were at elevated risk (OR = 3.50, 95% CI 1.25-9.80; ptrend = 0.02)]. There was no significant heterogeneity by weight status, but stronger association was observed in men with body mass index >25 kg/m2 versus <25 kg/m2. CONCLUSION: These findings suggest that a proinflammatory diet, as indicated by increasing DII score, is a risk factor for prostate cancer.

A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

Incident Hepatitis C Virus Genotype Distribution and Multiple Infection in Australian Prisons.

Hepatitis C virus (HCV) is a highly diverse pathogen that is classified into seven distinct genotypes. Simultaneous or sequential reinfection with multiple HCV genotypes is recognized in high-risk populations, such as injecting drug users (IDUs). Multiple infection is of clinical concern as different genotypes have various sensitivities to current antiviral therapies. Therefore, a better understanding of the frequency of multiple infection and of the genotypes currently being transmitted is clinically relevant. An Australian cohort of IDUs (n = 123), identified with primary incident infection, was followed for multiple infection by regular HCV RNA testing between 2005 and 2013. A total of 354 samples were tested. Sequencing of primary incident infections revealed that genotype 3a was the most common circulating genotype, followed by genotype 1a. Examination of longitudinally collected samples identified complex patterns of multiple infection, including reinfection and superinfection. In those with multiple infection, there was no apparent evidence of homotypic immunity conferring protection against reinfection of the same subtype. This study revealed frequent multiple infection in a high-risk prisoner cohort, illustrating the complex nature of HCV infection and reinfection and highlighting the need for pan-genotypic antiviral therapies.

Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection.

UNLABELLED: The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8(+) T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance (n = 1) or replacement with viral variants leading to establishment of chronic infection (n = 2). CD8(+) T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8(+) T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8(+) T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8(+) T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of individual epitopes may be required. IMPORTANCE: A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic samples were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.