Full-length sequence analysis of hepatitis C virus genotype 3b strains and development of an in vivo infectious 3b cDNA clone.

IMPORTANCE: HCV genotype 3b is a difficult-to-treat subtype, associated with accelerated progression of liver disease and resistance to antivirals. Moreover, its prevalence has significantly increased among persons who inject drugs posing a serious risk of transmission in the general population. Thus, more genetic information and antiviral testing systems are required to develop novel therapeutic options for this genotype 3 subtype. We determined the complete genomic sequence and complexity of three genotype 3b isolates, which will be beneficial to study its biology and evolution. Furthermore, we developed a full-length in vivo infectious cDNA clone of genotype 3b and showed its robustness and genetic stability in human-liver chimeric mice. This is, to our knowledge the first reported infectious cDNA clone of HCV genotype 3b and will provide a valuable tool to evaluate antivirals and neutralizing antibodies in vivo, as well as in the development of infectious cell culture systems required for further research.

Soluble inflammatory mediators identify HCV patients who may be cured with four weeks of antiviral treatment.

Soluble inflammatory mediators (SIM) can be predictive of treatment outcome in antiviral treatment of chronic hepatitis C. Recently, it was shown that a subgroup of patients can be cured with four weeks of therapy. We here profiled patients for 70 SIM before and during treatment of hepatitis C with glecaprevir/pibrentasvir (GLE/PIB) +/- ribavirin. Proximity extension assay was performed in a total of 32 patients. Pre-treatment SIM profiles did not distinguish patients achieving an SVR (n = 21) from patients experiencing antiviral relapse (n = 11). However, after 4 weeks of therapy, eight markers were identified that could distinguish patients with SVR from the relapsed group, namely MMP-10, CCL20, CXCL11, FGF-23, TNF, MCP-2, IL-18R1 and CXCL10. Thus, this study shows that a distinct on-treatment immune profile is associated with cure of HCV infection after ultrashort treatment.

A multi-level intervention to eliminate hepatitis C from the Region of Southern Denmark: the C-Free-South project.

Denmark has signed the WHO strategy to eliminate hepatitis C virus (HCV). In the absence of a national strategy for elimination, a local action plan was developed in the Region of Southern Denmark (RSD). The aim of the strategy is to diagnose 90% of HCV-infected persons and treat 80% of those diagnosed by 2025. The strategy was developed by reviewing Danish data on HCV epidemiology and drug use to identify key populations for screening, linkage to care, and treatment. Based on available published data from 2016, an estimated 3028 persons in the RSD were HCV-RNA positive (population prevalence 0.21%). Of these, 1002 were attending clinical care, 1299 were diagnosed but not in clinical care, and 727 were undiagnosed. Three different interventions targeting the HCV-infected population and two interventions for HCV surveillance are planned to achieve elimination. The "C-Free-South" strategy aims to eliminate HCV in our region by identifying (90%) and treating (80%) of infected persons by the end of 2025, 5 years earlier than the WHO elimination target date.

Prolonged viral shedding of SARS-CoV-2 in two immunocompromised patients, a case report.

BACKGROUND: The duration of viable Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) shedding in immunocompromised patients is still unknown. This case report describes the duration of viable SARS-CoV-2 in two immunocompromised patients with completely different clinical courses and further addresses the immunological aspects. CASE PRESENTATIONS: Oropharyngeal swaps were collected continuously during hospitalization for two immunocompromised patients infected with SARS-CoV-2 and sent for analysis to real time reverse transcription polymerase chain reaction (RT-PCR), viral culture assessed by plaque assay and full genome sequencing. Blood samples for flow cytometry and further immunological analysis were taken once during admission. One patient was without symptoms of Coronavirus disease 2019 (COVID-19) whereas the other had severe respiratory symptoms requiring a stay at an intensive care unit (ICU) and treatment with remdesivir and dexamethasone. Despite their difference in clinical courses, they both continuously shed SARS-CoV-2 with high viral loads in culture. Both patients had undetectable anti SARS-CoV-2 IgG levels about 2 weeks after the first positive real time RT-PCR test of SARS-CoV-2, marked expansions of virus reactive CD8+ T cells but cellular markers indicative of attenuated humoral immunity. CONCLUSIONS: Our case illustrates the importance of distinguishing isolation guidelines for patients infected with SARS-CoV-2 according to their immunological status. Furthermore, it demonstrates the need for immune markers relating to viral shedding in immunocompromised patients.

Combining cross-sectional survey and register data improved the estimate of hepatitis C prevalence among patients attending a psychiatric emergency department in Denmark.

BACKGROUND: The prevalence of hepatitis C (HCV) among psychiatric patients is elevated compared to the background population in many studies, but the prevalence among Danish psychiatric patients is unknown. The aim of the study was to determine the HCV prevalence and the proportion of the psychiatric patient population that remains to be diagnosed and treated in a Danish setting. METHODS: During a 5-month period, patients attending the psychiatric emergency room in Vejle, Denmark, were offered point-of-care anti-HCV testing. Previous hepatitis C tests for all patients attending the Psychiatric Department in the study period were extracted from the national laboratory database (DANVIR). We combined the survey and register data in a capture-recapture estimate of undiagnosed patients with HCV. RESULTS: During the study 24.9% (589 of 2364) patients seen at the psychiatric department attended the emergency room. The prevalence of anti-HCV among those tested in the emergency room was 1.6%. The laboratory register identified 595/2364 patients previously tested for anti-HCV with a positive prevalence of 6.1%. The undiagnosed anti-HCV positives among the 1483 never tested was estimated to 1.1%. Thus the total estimated prevalence of anti-HCV was 2.3% (54/2364, 95% CI 1.7%-3.0%) in the population, of whom 70.4% had been diagnosed, and 72.2% of diagnosed patients had received treatment or cleared HCV. CONCLUSION: Combining survey and register data showed that the WHO target of 90% diagnosed and 80% treated was not met. To eliminate HCV in the psychiatric population, both undiagnosed and untreated patients must be targeted.

Development of antibody levels and subsequent decline in individuals with vaccine induced and hybrid immunity to SARS-CoV-2.

OBJECTIVES: This study aimed to compare antibody trajectories among individuals with SARS-CoV-2 hybrid and vaccine-induced immunity. METHODS: Danish adults receiving three doses of BTN162b2 or mRNA-1237 were included prior to first vaccination (Day 0). SARS-CoV-2 anti-spike IgG levels were assessed before each vaccine dose, at Day 90, Day 180, 28 days after 3rd vaccination (Day 251), Day 365, and prior to 4th vaccination (Day 535). SARS-CoV-2 PCR results were extracted from the national microbiology database. Mixed-effect multivariable linear regression investigated the impact of hybrid-immunity (stratified into 4 groups: no hybrid immunity, PCR+ prior to 3rd dose, PCR+ after 3rd dose and before Day 365, PCR+ after Day 365) on anti-spike IgG trajectories. RESULTS: A total of 4,936 individuals were included, 47% developed hybrid-immunity. Anti-spike IgG increases were observed in all groups at Day 251, with the highest levels in those PCR+ prior to 3rd dose (Geometric Mean; 535,647AU/mL vs. 374,665AU/mL with no hybrid-immunity, P<0.0001). Further increases were observed in participants who developed hybrid immunity after their 3rd dose. Anti-spike IgG levels declined from Day 251-535 in individuals without hybrid-immunity and in those who developed hybrid-immunity prior to their 3rd dose, with lower rate of decline in those with hybrid-immunity. CONCLUSION: Hybrid-immunity results in higher and more durable antibody trajectories in vaccinated individuals.

Impact of age and comorbidities on SARS-CoV-2 vaccine-induced T cell immunity.

BACKGROUND: Older age and chronic disease are important risk factors for developing severe COVID-19. At population level, vaccine-induced immunity substantially reduces the risk of severe COVID-19 disease and hospitalization. However, the relative impact of humoral and cellular immunity on protection from breakthrough infection and severe disease is not fully understood. METHODS: In a study cohort of 655 primarily older study participants (median of 63 years (IQR: 51-72)), we determined serum levels of Spike IgG antibodies using a Multiantigen Serological Assay and quantified the frequency of SARS-CoV-2 Spike-specific CD4 + and CD8 + T cells using activation induced marker assay. This enabled characterization of suboptimal vaccine-induced cellular immunity. The risk factors of being a cellular hypo responder were assessed using logistic regression. Further follow-up of study participants allowed for an evaluation of the impact of T cell immunity on breakthrough infections. RESULTS: We show reduced serological immunity and frequency of CD4 + Spike-specific T cells in the oldest age group (≥75 years) and higher Charlson Comorbidity Index (CCI) categories. Male sex, age group ≥75 years, and CCI > 0 is associated with an increased likelihood of being a cellular hypo-responder while vaccine type is a significant risk factor. Assessing breakthrough infections, no protective effect of T cell immunity is identified. CONCLUSIONS: SARS-CoV-2 Spike-specific immune responses in both the cellular and serological compartment of the adaptive immune system increase with each vaccine dose and are progressively lower with older age and higher prevalence of comorbidities. The findings contribute to the understanding of the vaccine response in individuals with increased risk of severe COVID-19 disease and hospitalization.

Vaccination has proven very effective in protecting against severe disease and hospitalization of people with COVID-19, the disease caused by SARS-CoV-2. It is still unclear, however, how the different components of the immune system respond to SARS-CoV-2 vaccination and protect from infection and severe disease. Two of the most predominant components of the immune system are specialized proteins and cells. The proteins circulate in the blood and help clear the virus by binding to it, while the cells either kill the virus or help other cells to produce more antibodies. Here, we examined the response of these two components to the SARS-CoV-2 vaccine in 655 Danish citizens. The response of both components was lower in people over 75 years old and with other diseases. These findings help in understanding the immune responses following SARS-CoV-2 vaccination in people at increased risk of severe symptoms of COVID-19.

Immunological Characteristics of Patients Receiving Ultra-Short Treatment for Chronic Hepatitis C.

Reducing the treatment duration for chronic hepatitis C could be an important tool in the effort to reach the elimination goals set by the World Health Organization. The current challenge is to predict the target group who will achieve sustained virological response at week 12 (SVR12) with shorter treatment duration. The aim of this exploratory study was to characterize immune subsets with focus on inhibitory receptors in patients who experienced SVR12 or virological relapse following four weeks treatment with glecaprevir/pibrentasvir with or without ribavirin. A total of 32 patients were included in this study of whom 21 achieved SVR12 and 11 had virological relapse. All available samples at baseline (n = 31) and end of treatment (EOT) (n = 30) were processed for flow cytometric analysis in order to measure the expression of PD-1, 2B4, BY55, CTLA-4, TIM-3 and LAG-3 on 12 distinct T cell subsets. At baseline, patients with SVR12 (n=21) had numerically lower frequencies of inhibitory receptors for 83% (60/72) of the investigated T-cell subtypes. The most significant difference observed between the two groups was a lower frequency of stem cell-like memory T-cells CD4+PD1+ in the SVR group (p = 0.007). Furthermore, we observed a significant positive correlation between baseline viral load and the expression of PD-1 on the total CD8+ T-cells and effector memory T-cells CD4+ and CD8+ for patients with virological relapse. This study suggests a measurable immunologic phenotype at baseline of patients achieving SVR12 after short treatment compared to patients with virological relapse.

Rectally shed SARS-CoV-2 in COVID-19 inpatients is consistently lower than respiratory shedding and lacks infectivity.

OBJECTIVES: Assessment of whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been propagated during intestinal passage and infectivity is conserved when shed rectally by hospitalized individuals. METHODS: An exploratory cohort study including 28 inpatients with coronavirus disease 2019 with estimation of RNA levels by RT-PCR and of viral infectivity by culturing of viral material sampled concomitantly and identically from pharynx and rectum. RESULTS: SARS-CoV-2 RNA was detected more frequently (91%, 30/33 versus 42%, 14/33, p <0.0001) and at higher concentrations (median levels 2 190 186 IU/mL versus 13 014 IU/mL, p <0.0001) in the pharyngeal swabs than in the rectal swabs. For all sample pairs (n = 33) the rectal swabs contained undetectable or lower SARS-CoV-2 RNA concentrations than their paired pharyngeal swabs. Replicative virus was found in 37% (11/30) of the PCR-positive pharyngeal swabs, whereas none of the PCR-positive rectal swabs could be cultured (0%, 0/14) despite containing SARS-CoV-2 RNA concentrations up to 1 544 691 IU/mL. CONCLUSIONS: Our data draw into question whether SARS-CoV-2 is transmitted readily from faeces.

Characteristics associated with serological COVID-19 vaccine response and durability in an older population with significant comorbidity: the Danish Nationwide ENFORCE Study.

OBJECTIVES: To identify individual characteristics associated with serological COVID-19 vaccine responsiveness and the durability of vaccine-induced antibodies. METHODS: Adults without history of SARS-CoV-2 infection from the Danish population scheduled for SARS-CoV-2 vaccination were enrolled in this parallel group, phase 4 study. SARS-CoV-2 Spike IgG and Spike-ACE2-receptor-blocking antibodies were measured at days 0, 21, 90, and 180. Vaccine responsiveness was categorized according to Spike IgG and Spike-ACE2-receptor-blocking levels at day 90 after first vaccination. Nondurable vaccine response was defined as day-90 responders who no longer had significant responses by day 180. RESULTS: Of 6544 participants completing two vaccine doses (median age 64 years; interquartile range: 54-75), 3654 (55.8%) received BTN162b2, 2472 (37.8%) mRNA-1273, and 418 (6.4%) ChAdOx1 followed by an mRNA vaccine. Levels of both types of antibodies increased from baseline to day 90 and then decreased to day 180. The decrease was more pronounced for levels of Spike-ACE2-receptor-blocking antibodies than for Spike IgG. Proportions with vaccine hyporesponsiveness and lack of durable response were 5.0% and 12.1% for Spike IgG and 12.7% and 39.6% for Spike-ACE2-receptor-blocking antibody levels, respectively. Male sex, vaccine type, and number of comorbidities were associated with all four outcomes. Additionally, age ≥75 years was associated with hyporesponsiveness for Spike-ACE2-receptor-blocking antibodies (adjusted odds ratio: 1.59; 95% confidence interval: 1.25-2.01) but not for Spike IgG. DISCUSSION: Comorbidity, male sex, and vaccine type were risk factors for hyporesponsiveness and nondurable response to COVID-19 vaccination. The functional activity of vaccine-induced antibodies declined with increasing age and had waned to pre-second-vaccination levels for most individuals after 6 months.

Levels of SARS-CoV-2 antibodies among fully vaccinated individuals with Delta or Omicron variant breakthrough infections.

SARS-CoV-2 variants of concern have continuously evolved and may erode vaccine induced immunity. In this observational cohort study, we determine the risk of breakthrough infection in a fully vaccinated cohort. SARS-CoV-2 anti-spike IgG levels were measured before first SARS-CoV-2 vaccination and at day 21-28, 90 and 180, as well as after booster vaccination. Breakthrough infections were captured through the Danish National Microbiology database. incidence rate ratio (IRR) for breakthrough infection at time-updated anti-spike IgG levels was determined using Poisson regression. Among 6076 participants, 127 and 364 breakthrough infections due to Delta and Omicron variants were observed. IRR was 0.29 (95% CI 0.15-0.56) for breakthrough infection with the Delta variant, comparing the highest and lowest quintiles of anti-spike IgG. For Omicron, no significant differences in IRR were observed. These results suggest that quantitative level of anti-spike IgG have limited impact on the risk of breakthrough infection with Omicron.

SARS-CoV-2 viral load as a predictor for disease severity in outpatients and hospitalised patients with COVID-19: A prospective cohort study.

INTRODUCTION: We aimed to examine if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) cycle quantification (Cq) value, as a surrogate for SARS-CoV-2 viral load, could predict hospitalisation and disease severity in adult patients with coronavirus disease 2019 (COVID-19). METHODS: We performed a prospective cohort study of adult patients with PCR positive SARS-CoV-2 airway samples including all out-patients registered at the Department of Infectious Diseases, Odense University Hospital (OUH) March 9-March 17 2020, and all hospitalised patients at OUH March 10-April 21 2020. To identify associations between Cq-values and a) hospital admission and b) a severe outcome, logistic regression analyses were used to compute odds ratios (OR) and 95% Confidence Intervals (CI), adjusting for confounding factors (aOR). RESULTS: We included 87 non-hospitalised and 82 hospitalised patients. The median baseline Cq-value was 25.5 (interquartile range 22.3-29.0). We found a significant association between increasing Cq-value and hospital-admission in univariate analysis (OR 1.11, 95% CI 1.04-1.19). However, this was due to an association between time from symptom onset to testing and Cq-values, and no association was found in the adjusted analysis (aOR 1.08, 95% CI 0.94-1.23). In hospitalised patients, a significant association between lower Cq-values and higher risk of severe disease was found (aOR 0.89, 95% CI 0.81-0.98), independent of timing of testing. CONCLUSIONS: SARS-CoV-2 PCR Cq-values in outpatients correlated with time after symptom onset, but was not a predictor of hospitalisation. However, in hospitalised patients lower Cq-values were associated with higher risk of severe disease.

Low mortality of hospitalised patients with COVID-19 in a tertiary Danish hospital setting.

OBJECTIVES: We aimed to describe clinical characteristics and outcomes of admitted COVID-19 patients in a Danish hospital setting where an early active government intervention was taken. METHODS: Prospective cohort study including all admitted patients to the COVID-19 unit at Odense University Hospital from March 10 to April 21, 2020. Patients were assessed by a multidisciplinary team at admission. Outcome parameters were development of acute respiratory distress syndrome (ARDS), intensive care unit (ICU) admission, death and admission time. RESULTS: We included 83 patients (median age 62 years, 62.7% male). At hospitalization, 31.3% needed oxygen supplementation and the median National Early Warning Score was four. Median admission time was 7 days (Interquartile ranges (IQR) 3-12). In total, ARDS was diagnosed in 33.7% (28/83) of the patients corresponding to an incidence rate of 7.1 per 100 person days (95% CI: 4.1-10.2). Overall 13 patients (15.7%) were transferred to the ICU of whom 11 (84.6%) received corticosteroids.. No patients died while admitted to the ICU. Four patients (4.8%) died during admission. CONCLUSION: Despite similar patient characteristics compared to those reported by others, we found a low overall mortality of < 5%.

Incidence, prevalence and risk factors for hepatitis C in Danish prisons.

Hepatitis C virus (HCV) infection is prevalent among people in prison and prisons could therefore represent a unique opportunity to test risk groups for HCV. The aim of this sero-epidemiological study was to determine the incidence and prevalence of HCV infection and the corresponding risk factors in Danish prisons. Participants, recruited from eight Danish prisons, were tested for HCV using dried blood spots and filled out a questionaire with demographic data and risk factors for HCV infection. In total, 76.9% (801/1041) of all eligible prisoners consented to participate. The prevalence of HCV RNA positive prisoners was 4.2% (34/801) and the in-prison incidence rate was 0.7-1.0 per 100PY overall and 18-24/100PY among PWIDs. Infected prisoners were older than the overall population with a mean age of 42 years and only 17.6% (6/34) were younger than 35 years. The prevalence of PWID was 8.5% (68/801) and only 3% (2/68) of PWID were younger than 25 years. Among the PWID, 85.3% (58/68) had ever received opioid substitution therapy (OST) and 47.1% (32/68) were currently receiving OST. Risk factors associated with HCV infection were intravenous drug use, age ≥ 40 years, and being incarcerated ≥ 10 years. In conclusion, the prevalence of PWID in Danish prisons is low, possibly reflecting a decrease in injecting among the younger generation. This together with OST coverage could explain the low prevalence of HCV infection. However among PWIDs in prison the incidence remains high, suggesting a need for improved HCV prevention in prison.

[Peritoneal tuberculosis in a patient with suspected disseminated ovarian cancer]. / Peritoneal tuberkulose hos patient med formodet dissemineret ovariecancer.

We present a case study of a 43 year-old female immigrant from Turkey with abdominal pain, ascites and elevated cancer antigen 125. The symptoms were similar to those of ovarian cancer, and imaging (computed tomography (CT), positron emission tomography/computed tomography and magnetic resonance imaging) supported this suspicion. Peritoneal biopsy from laparoscopy showed granulomas with central necrosis. Microscopy, culture and polymerase chain reaction from biopsy samples were negative for Mycobacterium tuberculosis. Follow-up with a CT scan after six months of full tuberculosis treatment showed normal conditions. Peritoneal tuberculosis is a diagnostic challenge, but should be considered in case of immigrants from high-risk areas.