Ongoing Gaps in the Hepatitis C Care Cascade during the Direct-Acting Antiviral Era in a Large Retrospective Cohort in Canada: A Population-Based Study.

To achieve hepatitis C virus (HCV) elimination, high uptake along the care cascade steps for all will be necessary. We mapped engagement with the care cascade overall and among priority groups in the post-direct-acting antivirals (DAAs) period and assessed if this changed relative to pre-DAAs. We created a population-based cohort of all reported HCV diagnoses in Quebec (1990-2018) and constructed the care cascade [antibody diagnosed, RNA tested, RNA positive, genotyped, treated, sustained virologic response (SVR)] in 2013 and 2018. Characteristics associated with RNA testing and treatment initiation were investigated using marginal logistic models via generalized estimating equations. Of the 31,439 individuals HCV-diagnosed in Quebec since 1990 and alive as of 2018, there was significant progress in engagement with the care cascade post- vs. pre-DAAs; 86% vs. 77% were RNA-tested, and 64% vs. 40% initiated treatment. As of 2018, a higher risk of not being RNA-tested or treated was observed among individuals born <1945 vs. >1965 [hazard ratio (HR); 95% CI; 1.35 (1.16-1.57)], those with material and social deprivation [1.21 (1.06-1.38)], and those with alcohol use disorder [1.21 (1.08-1.360]. Overall, non-immigrants had lower rates of RNA testing [0.76 (0.67-0.85)] and treatment initiation [0.63 (0.57-0.70)] than immigrants. As of 2018, PWID had a lower risk of not being RNA tested [0.67 (0.61-0.85)] but a similar risk of not being treated, compared to non-PWID. Engagement in the HCV care cascade have improved in the post-DAA era, but inequities remain. Vulnerable subgroups, including certain older immigrants, were less likely to have received RNA testing or treatment as of 2018 and would benefit from focused interventions to strengthen these steps.

Hepatitis B virus genotype surveillance in Canadian blood donors and a referred patient population, 2016-2021.

BACKGROUND AND OBJECTIVES: Hepatitis B virus (HBV) genotypes (A-H) have a distinct geographic distribution and are highly associated with the country of birth. Canada has experienced increased immigration over the past decade, primarily from regions where HBV is endemic. This study investigated the proportions and trends of HBV genotypes within blood donor and clinical populations of Canada over the period 2016-2021. MATERIALS AND METHODS: Study samples involved two cohorts: (1) Canadian blood donors (n = 246) deferred from donation due to HBV test positivity and (2) chronic HBV patients from across Canada (clinically referred population, n = 3539). Plasma or serum was extracted, and the surface antigen and/or polymerase-coding region was amplified and sequenced to determine genotype by phylogenetic analysis. RESULTS: Six (A-E, G) and eight (A-H) HBV genotypes were detected among deferred blood donors and the clinically referred population, respectively. Differences in HBV genotype proportions between the two cohorts were observed across Canada. Males comprised most of the referred population among genotypes A-E (p < 0.0001), except for genotypes B and C. The median age was younger among blood donors (36 years [range 17-72]) compared with the referred population (41 years [range 0-99]). Distinct trends of increasing (E, referred; B, blood donor) and decreasing genotype prevalence were observed over the study period. CONCLUSION: HBV genotypes in Canada are highly diverse, suggesting a large immigrant population. Observed trends in genotype prevalence and proportional differences among cohorts imply shifts among the HBV-infected population of Canada, which warrants continued surveillance.

A population-based study of reported hepatitis C diagnoses from 1998 to 2018 in immigrants and nonimmigrants in Quebec, Canada.

Immigrants living in low hepatitis C (HCV) prevalence countries bear a disproportionate HCV burden, but there are limited HCV population-based studies focussed on this population. We estimated rates and trends of reported HCV diagnoses over a 20-year period in Quebec, Canada, to investigate subgroups with the highest rates and changes over time. A population-based cohort of all reported HCV diagnoses in Quebec (1998-2018) linked to health administrative and immigration databases. HCV rates, rate ratios (RR) and trends overall and stratified by immigrant status and country of birth were estimated using Poisson regression. Among 38,348 HCV diagnoses, 14% occurred in immigrants, a median of 7.5 years after arrival. The average annual HCV rate/100,000 decreased for immigrants and nonimmigrants, but the risk (RR) among immigrants increased over the study period [35.7 vs. 34.5 (RR = 1.03) and 18.4 vs. 12.7 (1.45) between 1998-2008 and 2009-2018]. Immigrants from middle-income Europe & Central Asia [55.8 (RR = 4.39)], sub-Saharan Africa [51.7 (RR = 4.06)] and South Asia [32.8 (RR = 2.58)] had the highest rates between 2009 and 2018. Annual HCV rates decreased more slowly among immigrants vs. nonimmigrants (-5.9% vs. -8.9%, p < 0.001), resulting in a 2.5-fold (9%-21%) increase in the proportion of HCV diagnoses among immigrants (1998-2018). The slower decline in HCV rates among immigrants over the study period highlights the need for targeted screening for this population, particularly those from sub-Saharan Africa, Asia and middle-income Europe. These data can inform micro-elimination efforts in Canada and other low-HCV-prevalence countries.

Highly Diverse Hepatitis C Strains Detected in Sub-Saharan Africa Have Unknown Susceptibility to Direct-Acting Antiviral Treatments.

The global plan to eradicate hepatitis C virus (HCV) led by the World Health Organization outlines the use of highly effective direct-acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus in sub-Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population-based, nested case-control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC) to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterize genetic diversity of the virus. Using next-generation and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. A total of 7,751 Ugandan patients were initially screened for HCV, and 20 PCR-positive samples were obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotype (g) 4k, g4p, g4q, and g4s and a newly identified unassigned g7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full open reading frame sequence). These g4 and g7 strains contain nonstructural (ns) protein 3 and 5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: Although HCV prevalence and genotypes have been well characterized in patients in well-resourced countries, clinical trials are urgently required in SSA, where highly diverse g4 and g7 strains circulate.

Acute hepatitis B virus infection with delayed appearance of hepatitis B core antibody in an immunocompromised patient: a case report.

BACKGROUND: Despite the introduction of universal hepatitis B immunization programs worldwide, outbreaks of acute infection still occur in unimmunized individuals. A timely diagnosis of hepatitis B is necessary to ensure adequate clinical care and public health interventions that will reduce transmission. Yet, interpretation of hepatitis B serological markers can be complex. We present a case of hepatitis B with atypical markers, including delayed appearance of hepatitis B core antibody. CASE PRESENTATION: A 62-year-old white woman was identified as a sexual contact of a male individual with acute hepatitis B virus infection. She had a history of recurrent low-grade non-Hodgkin lymphoma and had recently received immunosuppressive therapy. At baseline she had a negative serology and received three double doses (40 µg) of Engerix-B vaccine (hepatitis B vaccine) with a 0-month, 1-month, and 6-month schedule. One month following the last dose, hepatitis B surface antigen was positive in the absence of hepatitis B core antibody. The only sign of infection was a slight elevation of alanine aminotransferase enzymes a few months after first sexual contacts with the male individual. Hepatitis B virus infection was later confirmed despite the absence of hepatitis B core antibody. The development of hepatitis B core antibody was finally noted more than 6 months after the first positive hepatitis B surface antigen and more than 12 months after elevation of alanine aminotransferase enzymes. Immunosuppression including rituximab treatment was the most likely explanation for this serological profile. On her last medical assessment, she had not developed HBeAg seroconversion despite lower hepatitis B virus deoxyribonucleic acid levels with tenofovir treatment. CONCLUSIONS: When confronted with positive hepatitis B surface antigen in the absence of hepatitis B core antibody, consideration should be given to the possibility of both acute and persistent infection particularly in the setting of immunosuppression so that appropriate clinical management and public health interventions can take place. Given the increasing use of biologicals such as anti-tumor necrosis factor therapies either alone or with other immunosuppressive agents, this phenomenon may be encountered more frequently.

Analysis of HCV-6 isolates among Asian-born immigrants in North America reveals their high genetic diversity and a new subtype.

We characterized full-length genomes for 15 HCV-6 isolates, all from Asian immigrants living in North America. Among these isolates, nine were novel variants showing >15% nucleotide differences from their nearest relatives, representing lineages distinct from known subtypes. The other six were classified into subtypes 6c, 6h, 6q, 6r, and 6s. The partial sequences were also determined for five additional HCV-6 isolates, three from the US and two from Canada. The latter two were assigned to new subtype 6xf as they were found to classify with two other isolates for which we recently reported their full-length genomes. We further analyzed partial Core-E1 sequences of 100 HCV-6 isolates sampled in North America, seven from the US and 93 from Canada and all from Asian immigrants except for four from Caucasians. These 100 isolates belonged to 20 assigned subtypes and 16 unclassified lineages showing great genetic diversity and enhanced significance to public health.

The full-length genome sequences of nine HCV genotype 4 variants representing a new subtype 4s and eight unclassified lineages.

We characterized the full-length genomes for nine novel variants of HCV genotype 4 (HCV-4), representing a new subtype 4s and eight unclassified lineages. They were obtained from patients who resided in Canada but all had origins in Africa. An extended maximum clade credibility (MCC) tree was reconstructed after the inclusion of 30 reference sequences. It differentiated 18 assigned subtypes and 10 unclassified lineages within HCV-4. Similar analysis of 102 partial NS5B sequences resulted in another MCC tree that revealed 22 assigned subtypes (4a-4t, 4w, and 4v) and 30 unclassified lineages at the subtype level. Our study shows that HCV-4 is taxonomically complex and it displays high genetic diversity to support an African origin.

Hepatitis C virus genotype 7, a new genotype originating from central Africa.

We report a new hepatitis C virus (HCV) genotype identified in patients originating from the Democratic Republic of Congo. The prototype QC69 virus is shown to be a new lineage distinct from genotypes 1 to 6. Three additional patients were also found to be infected by a virus from this lineage, confirming its circulation in humans. We propose that these viruses be classified into HCV genotype 7.

A significant reduction in the frequency of HIV-1 drug resistance in Québec from 2001 to 2011 is associated with a decrease in the monitored viral load.

BACKGROUND: HIV drug resistance represents a major threat for effective treatment. We assessed the trends in the frequency of drug resistance mutations and the monitored viral load (VL) in treatment-naïve (TN) and treatment-experienced (TE) individuals infected with HIV-1 in Québec, Canada, between 2001 and 2011. METHODS AND FINDINGS: Resistance data were obtained from 4,105 and 5,086 genotypic tests performed on TN and TE patients, respectively. Concomitantly, 274,161 VL tests were carried out in the Province. Changes over time in drug resistance frequency and in different categories of VL were assessed using univariate logistic regression. Multiple logistic regression was used to evaluate associations between the rates of certain mutations and antiretroviral prescriptions. From 2001 to 2011, the proportion of undetectable VL test results continually increased, from 42.1% to 75.9%, while a significant decrease in the frequency of resistance mutations associated with protease inhibitors [PI (from 54% to 16%)], nucleoside [NRTI (from 78% to 37%) and non-nucleoside reverse transcriptase inhibitors [NNRTI (from 44% to 31%)] was observed in TE patients. In TN individuals, the overall frequency of transmitted drug resistance was 13.1%. A multiple logistic regression analysis indicated that the introduction of co-formulated emtricitabine/tenofovir or emtricitabine/tenofovir/efavirenz was positively associated with the decrease of the frequency of the M184I/V mutations observed overtime (p = 0.0004). CONCLUSIONS: We observed a significant decrease in the frequency of drug resistance mutations in TE patients, concomitant with a decrease in the proportion of patients with detectable viremia. These findings may be related to both the increased potencies and adherence to therapy associated with newer antiretroviral regimens. Nevertheless, our data demonstrate that broad use of antiretrovirals does not increase the level of circulating drug resistant variants.

Origin of hepatitis C virus genotype 3 in Africa as estimated through an evolutionary analysis of the full-length genomes of nine subtypes, including the newly sequenced 3d and 3e.

We characterized the full-length genomes of nine hepatitis C virus genotype 3 (HCV-3) isolates: QC7, QC8, QC9, QC10, QC34, QC88, NE145, NE274 and 811. To the best of our knowledge, NE274 and NE145 were the first full-length genomes for confirming the provisionally assigned subtypes 3d and 3e, respectively, whereas 811 represented the first HCV-3 isolate that had its extreme 3' UTR terminus sequenced. Based on these full-length genomes, together with 42 references representing eight assigned subtypes and an unclassified variant of HCV-3, and 10 sequences of six other genotypes, a timescaled phylogenetic tree was reconstructed after an evolutionary analysis using a coalescent Bayesian procedure. The results indicated that subtypes 3a, 3d and 3e formed a subset with a common ancestor dated to ~202.89 [95% highest posterior density (HPD): 160.11, 264.6] years ago. The analysis of all of the HCV-3 sequences as a single lineage resulted in the dating of the divergence time to ~457.81 (95% HPD: 350.62, 587.53) years ago, whereas the common ancestor of all of the seven HCV genotypes dated to ~780.86 (95% HPD: 592.15, 1021.34) years ago. As subtype 3h and the unclassified variant were relatives, and represented the oldest HCV-3 lineages with origins in Africa and the Middle East, these findings may indicate the ancestral origin of HCV-3 in Africa. We speculate that the ancestral HCV-3 strains may have been brought to South Asia from Africa by land and/or across the sea to result in its indigenous circulation in that region. The spread was estimated to have occurred in the era after Vasco da Gama had completed his expeditions by sailing along the eastern coast of Africa to India. However, before this era, Arabians had practised slave trading from Africa to the Middle East and South Asia for centuries, which may have mediated the earliest spread of HCV-3.

Full-length genomes of 16 hepatitis C virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes.

We characterized the full-length genomes of 16 distinct hepatitis C virus genotype 1 (HCV-1) isolates. Among them, four represented the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four corresponded to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 were assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152 and QC180) did not classify among the established subtypes and corresponded to four new lineages. Subtypes 1j, 1k, 1m, 1n and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtype 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages, indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV-1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b diverged from its nearest relatives 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent with the time period when the transatlantic slave trade was active. When taking all the HCV-1 isolates as a single lineage, the divergence time was 326 years ago. This analysis suggested the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d was also implied.

Full-length genome sequences of five hepatitis C virus isolates representing subtypes 3g, 3h, 3i and 3k, and a unique genotype 3 variant.

We characterized the full-length genomes of five distinct hepatitis C virus (HCV)-3 isolates. These represent the first complete genomes for subtypes 3g and 3h, the second such genomes for 3k and 3i, and of one novel variant presently not assigned to a subtype. Each genome was determined from 18-25 overlapping fragments. They had lengths of 9579-9660 nt and each contained a single ORF encoding 3020-3025 aa. They were isolated from five patients residing in Canada; four were of Asian origin and one was of Somali origin. Phylogenetic analysis using 64 partial NS5B sequences differentiated 10 assigned subtypes, 3a-3i and 3k, and two additional lineages within genotype 3. From the data of this study, HCV-3 full-length sequences are now available for six of the assigned subtypes and one unassigned. Our findings should add insights to HCV evolutionary studies and clinical applications.

Whole genome pyrosequencing of rare hepatitis C virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants.

BACKGROUND: Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. METHODS: Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. RESULTS: Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. CONCLUSIONS: These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistance.

Can healing be taught?

The recent explosion of opportunities and interest in learning to heal has not been accompanied by sufficiently convincing empirical data to show that healing is teachable. We explore selected examples of teaching modalities and outline their general ethos. Five empirical criteria necessary to demonstrate teachability are outlined. We suggest that no research to date, including a previous claim by one of us (W.F.B.), has surmounted the difficult obstacles that need to be overcome to make such a claim. Some scientific and social implications of the teachability of healing are discussed.

Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences.

Nucleotide sequence analysis of the NS5B region was performed to identify genotypes of 8,479 hepatitis C virus (HCV) RNA-positive patient samples collected in the Canadian province of Quebec. Genotypes could be determined for 97.3% of patients. Genotypes 1 to 6 were found in 59.4, 9.0, 25.7, 3.6, 0.6, and 1.8% of patients, respectively. Two isolates did not classify within the six genotypes. The subtype 1 distribution was 76.7% 1a, 22.6% 1b, and 0.7% others, while the subtype 2 distribution was 31.8% 2a, 47.6% 2b, 10.9% 2c, 4.1% 2i, and 5.6% others. Subtype 3a accounted for 99.1% of genotype 3 strains, while all genotype 5 samples were of subtype 5a. The subtype 4 distribution was 39.2% 4a, 15.4% 4k, 11.6% 4d, 10.2% 4r, and 23.6% others. The subtype 6 distribution was 40.4% 6e, 20.5% 6a, and 39.1% others. The 5' untranslated region (5'UTR) sequences of subtype 6e were indistinguishable from those of genotype 1. All samples that did not classify within the established subtypes were also sequenced in C/E1 and 5'UTR. C/E1 phylogenetic reconstructions were analogous to those of NS5B. The sequences identified in this study allowed the provisional assignments of subtypes 1j, 1k, 2m, 2r, 3i, 4q, 6q, 6r, and 6s. Sixty-four (0.8%) isolates classifying within genotypes 1 to 6 could not be assigned to one of the recognized subtypes. Our results show that genotyping of HCV by nucleotide sequence analysis of NS5B is efficient, allows the accurate discrimination of subtypes, and is an effective tool for studying the molecular epidemiology of HCV.

Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes.

International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV-related liver disease and important biological and antigenic differences that exist between variants. A group of scientists expert in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and Los Alamos (United States), met to re-examine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name re-assignments to create consistency in nomenclature. The paper also contains consensus proposals for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. A proposal was made that HCV variants be classified into 6 genotypes (representing the 6 genetic groups defined by phylogenetic analysis). Subtype name assignment will be either confirmed or provisional, depending on the availability of complete or partial nucleotide sequence data, or remain unassigned where fewer than 3 examples of a new subtype have been described. In conclusion, these proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future.