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Sofie Hallager,1 Steen Ladelund,2 Peer Brehm Christensen,3 Mette Kjær,4,5 Birgit Thorup Roege,6 Karin Elmegaard Grønbæk,7 Erika Belard,8 Toke S Barfod,9 Lone Galmstrup Madsen,10 Jan Gerstoft,11 Britta Tarp,12 Henrik Bygum Krarup,13 Nina Weis,1,5 1Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, 2Clinical Research Center, Copenhagen University Hospital, Hvidovre, 3Department of Infectious Diseases and Clinical Institute, Odense University Hospital, University of Southern Denmark, Odense, 4Department of Hepatology, Copenhagen University Hospital, Rigshospitalet, 5Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, 6Department of Internal Medicine, Kolding Hospital, Kolding, 7Department of Gastroenterology, Copenhagen University Hospital, Hvidovre, 8Department of Gastroenterology, Copenhagen University Hospital, Herlev, 9Department of Internal Medicine, Zealand University Hospital, Roskilde, 10Department of Gastroenterology, Zealand University Hospital, Køge, 11Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, 12Diagnostic Centre, University Research Clinic for Innovative Patient Pathways, Silkeborg Regional Hospital, Silkeborg, 13Section of Molecular Diagnostics, Clinical Biochemistry and Department of Medical Gastroenterology, Aalborg University Hospital, Aalborg, Denmark Background: Chronic hepatitis C (CHC) causes liver cirrhosis in 5%–20% of patients, leading to increased morbidity and mortality. This study aimed to estimate liver-related morbidity and mortality among patients with CHC and cirrhosis in Denmark with and without antiviral treatment and sustained virologic response (SVR). Furthermore we aimed to estimate the rate of hepatocellular carcinoma (HCC) and decompensation associated with certain prognostic factors.Materials and methods: Patients with CHC and cirrhosis registered in the Danish Database for Hepatitis B and C were eligible. Cirrhosis was based on liver biopsy, transient elastography, and clinical cirrhosis. Data were extracted from nationwide registries. The study period was from 2002 until 2013.Results: Of 1,038 patients included, 716 (69%) were male and the median age was 52 years. Median follow-up was 3.8 years, 360 patients died, and 233 of 519 treated patients achieved SVR. Alcohol overuse and hepatitis C virus genotype 3 were associated with an increased incidence rate (IR) of HCC, whereas diabetes and alcohol overuse were associated with increased IRs of decompensation. Achieving SVR reduced all-cause mortality (adjusted mortality rate ratio 0.68 [95% CI 0.43–1.09]) and liver-related mortality (mortality rate ratio 0.6 [95% CI 0.36–1]), as well as liver-related morbidity with adjusted IR ratios of 0.37 (95% CI 0.22–0.62) for HCC and 0.31 (95% CI 0.17–0.57) for decompensation. The IRs of HCC and decompensation remained elevated in patients with alcohol overuse after SVR.Conclusion: Alcohol overuse, hepatitis C genotype 3, and diabetes were associated with liver-related morbidity in patients with CHC and cirrhosis. SVR markedly reduced liver-related morbidity and mortality; however, special attention to patients with alcohol overuse should continue after SVR. Keywords: chronic hepatitis C, cirrhosis, liver-related morbidity, cohort study, sustained virologic response

Background: Today, the prognostic tools available at the time of diagnosis in colorectal cancer (CRC) are limited. Better prognostic tools are a prerequisite for personalised treatment. This study aimed to investigate whether circulating tumour DNA (ctDNA) markers found in plasma before clinical diagnosis of CRC could contribute to the prediction of poor prognosis., Methods: This observational cohort study included patients diagnosed with CRC stage I-III within 24 months following participation in the Trøndelag Health Study (n = 85). Known methylated ctDNA biomarkers of CRC were analysed by PCR in plasma. Outcomes were overall survival (OS), recurrence-free survival (RFS) and poor prognosis (PP). Candidate clinical and methylated ctDNA predictors of the outcomes were identified by Cox regression analyses., Results: Methylated GRIA4 (HR 1.96 (1.06-3.63)), RARB (HR 9.48 (3.00-30.00)), SLC8A1 (HR 1.97 (1.03-3.77)), VIM (HR 2.95 (1.22-7.14)) and WNT5A (HR 5.83 (2.33-14.56)) were independent predictors of OS, methylated RARB (HR 9.67 (2.54-36.81)), SDC2 (HR 3.38 (1.07-10.66)), SLC8A1 (HR 2.93 (1.01-8.51)) and WNT5A (HR 6.95 (1.81-26.68)) were independent predictors of RFS and methylated RARB (HR 6.11 (1.69-22.18)), SDC2 (HR 2.79 (1.20-6.49)) and WNT5A (HR 5.57 (3.04-15.26)) were independent predictors of PP (p < 0.05)., Conclusions: Prediagnostic ctDNA markers are promising contributors to predicting poor prognosis in CRC, potentially becoming one of the tools guiding more personalised treatment., (© 2024. The Author(s).)

Background: Gaming has become an integrated part of life for children and adults worldwide. Previous studies on the impact of gaming on biochemical parameters have primarily addressed the acute effects of gaming. The literature is limited, and the study designs are very diverse. The parameters that have been investigated most thoroughly are blood glucose and cortisol., Objective: This exploratory study is the first to investigate the effects of long gaming sessions on the biochemical parameters of healthy male adults. The extensive testing allowed us to observe short-term changes (within 6 hours), long-term changes during the duration of the gaming sessions, and follow-up after 1 week to determine whether any changes were longer lasting., Methods: In total, 9 experienced gamers completed 2 back-to-back 18-hour gaming sessions interspersed with a 6-hour rest period. All participants adhered to a structured sleep pattern due to daytime employment or attending university. Blood, saliva, and urine samples were collected from the participants every 6 hours. Linear mixed-effect models were used to analyze the repeated-measures data accumulated during the study. A total of 51 biochemical parameters were investigated., Results: In total, 12 of the 51 biochemical parameters significantly changed during the study: alkaline phosphatase, aspartate aminotransferase, bilirubin, chloride, creatinine, glucose, hemoglobin, immature reticulocyte fraction, lactate, methemoglobin, sodium, and thrombocytes. All changes were within the normal range. The mean glucose level of the participants was 4.39 (SD 0.07) mmol/L at baseline, which increased significantly by 0.24 (SD 0.07) mmol/L per 6 hours during the first period and by 0.38 (SD 0.07) mmol/L per 6 hours in the second period (P<.001). The glucose levels during the second session increased even though the participants had little energy intake. Cortisol levels did not change significantly, although the cortisol pattern deviated from the typical circadian rhythm. During both gaming sessions, we observed increasing cortisol levels from 6 AM until noon. The participants were relatively dehydrated at the start of the study. The patients were asked to fast before the first blood sampling. Within the first 6 hours of the study, the participants rehydrated, followed by relative dehydration during the remainder of the study. This pattern was identified using the following parameters: albumin, creatinine, hemoglobin, erythrocytes, potassium, and platelets., Conclusions: This study is the first of its kind, and many of the analyses in the study yielded novel results. The study was designed to emulate the behavior of gamers during the weekend and other long gaming sessions. At this point, we are not able to determine the difference between the effects of gaming and behavior during gaming. Regardless, the results of this study suggest that healthy gamers can partake in long gaming sessions, with ample amounts of unhealthy foods and little rest, without acute impacts on health., (©Kasper Bygum Krarup, Johannes Riis, Morten Mørk, Hien Thi Thu Nguyen, Inge Søkilde Pedersen, Søren Risom Kristensen, Aase Handberg, Henrik Bygum Krarup. Originally published in the Interactive Journal of Medical Research (https://www.i-jmr.org/), 08.07.2024.)

During the last two decades, several cases of venous thrombosis (VTE) after a prolonged period at a computer have been described, denominated as "eThrombosis". Video gaming on a computer has become very popular and can be a social activity where several players gather to play against each other or in a virtual environment for several days ("LAN (i.e., L ocal A rea N etwork) parties") where the participants are sedentary and consuming calorie-rich food items. The aim of this study was to investigate potential coagulation activation during a 42 h LAN party. Nine male gamers volunteered for the LAN party. Citrated blood was sampled before and every 6 h, and plasma was analyzed for thrombin generation, thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), and D-dimer. Thrombin generation increased slightly but not significantly during the LAN party, whereas the coagulation activation markers were unchanged. These results do not indicate that the coagulation system is activated significantly during 42 h of gaming with minimal physical activity. Although increased activity cannot be excluded, it does not directly indicate a risk of VTE in general., Competing Interests: The authors declare no conflicts of interest.

Homologous recombination deficiency (HRD) can arise from germline or somatic pathogenic variants as well as other genomic damage and epigenetic alterations in the HR repair pathway. Patients with tumors presenting with an HRD phenotype can show sensitivity to Poly (ADP-ribose) polymerase inhibitors (PARPis). Several promising tests to detect HRD have been developed based on different HRD definitions, biomarkers, and algorithms. However, no consensus on a gold standard HRD test has been established. In this systematic review, a comprehensive list of tests for the detection of HRD was identified and compared regarding HRD definition, biomarkers, and algorithms. PubMed's Medline and Elsevier's Embase were systematically searched, resulting in 27 eligible articles meeting the inclusion criteria. The primary challenge when comparing HRD tests lies in the lack of a consensus definition of HRD, as the HRD definition influences the proportion of samples being classified as HRD and impacts the classification performance. This systematic review provides an overview of available HRD tests that can inspire other researchers in searching for a gold standard HRD definition and highlights the importance of the factors that should be considered when choosing an HRD definition and tests for future planning of clinical trials and studies.

Background: Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis., Methods: In this case-control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study., Results: The most specific single markers were BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7-43.4) and the CRC specificity was 86% (95% CI 85.7-86.2). The findings were reproduced in an independent validation set of samples., Conclusions: Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme., (© 2023. The Author(s).)

Background: Pancreatic ductal adenocarcinoma remains one of the major causes of cancer-related mortality globally. Unfortunately, current prognostic biomarkers are limited, and no predictive biomarkers exist. This study examined promoter hypermethylation of secreted frizzled-related protein 1 (phSFRP1) in cfDNA as a prognostic biomarker and predictor of treatment effect in patients with metastatic FOLFIRINOX-treated PDAC and locally advanced PDAC., Methods: We performed methylation-specific PCR of the SFRP1 genes' promoter region, based on bisulfite treatment. Survival was assessed as time-to-event data using the pseudo-observation method and analyzed with Kaplan-Meier curves and generalized linear regressions., Results: The study included 52 patients with FOLFIRINOX-treated metastatic PDAC. Patients with unmethylated (um) SFRP1 (n = 29) had a longer median overall survival (15.7 months) than those with phSFRP1 (6.8 months). In crude regression, phSFRP1 was associated with an increased risk of death of 36.9% (95% CI 12.0%-61.7%) and 19.8% (95% CI 1.9-37.6) at 12 and 24-months, respectively. In supplementary regression analysis, interaction terms between SFRP1 methylation status and treatment were significant, indicating reduced benefit of chemotherapy. Forty-four patients with locally advanced PDAC were included. phSFRP1 was associated with an increased risk of death at 24-months CONCLUSIONS: This indicates that phSFRP1 is a clinically useful prognostic biomarker in metastatic PDAC and possibly in locally advanced PDAC. Together with existing literature, results could indicate the value of cfDNA-measured phSFRP1 as a predictive biomarker of standard palliative chemotherapy in patients with metastatic PDAC. This could facilitate personalized treatment of patients with metastatic PDAC., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Current prognostic blood-based biomarkers for pancreatic adenocarcinoma (PDAC) are limited. Recently, promoter hypermethylation of SFRP1 (phSFRP1) has been linked to poor prognosis in patients with gemcitabine-treated stage IV PDAC. This study explores the effects of phSFRP1 in patients with lower stage PDAC., Methods: Based on a bisulfite treatment process, the promoter region of the SFRP1 gene was analyzed with methylation-specific PCR. Kaplan-Meier curves, log-rank tests, and generalized linear regression analysis were used to assess restricted mean survival time survival at 12 and 24 months., Results: The study included 211 patients with stage I-II PDAC. The median overall survival of patients with phSFRP1 was 13.1 months, compared to 19.6 months in patients with unmethylated SFRP1 (umSFRP1). In adjusted analysis, phSFRP1 was associated with a loss of 1.15 months (95%CI -2.11, -0.20) and 2.71 months (95%CI -2.71, -0.45) of life at 12 and 24 months, respectively. There was no significant effect of phSFRP1 on disease-free or progression-free survival. In stage I-II PDAC, patients with phSFRP1 have worse prognoses than patients with umSFRP1., Discussion: Results could indicate that the poor prognosis may be caused by reduced benefit from adjuvant chemotherapy. SFRP1 may help guide the clinician and be a possible target for epigenetically modifying drugs., Competing Interests: SH, PM, OT-U, and HK declare that they have a patent on a diagnostic panel including the promoter hypermethylation status of 8 genes. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stubbe, Larsen, Madsen, Krarup, Pedersen, Lundbye-Christensen, Hansen, Hasselby, Johansen, Thorlacius-Ussing, Johansen and Henriksen.)

Background: Accurate prognosis is important either after acute infection or during long-term follow-up of patients infected by severe acute respiratory syndrome coronavirus 2. This study aims to predict coronavirus disease 2019 (COVID-19) severity based on clinical and biological indicators, and to identify biomarkers for prognostic assessment., Methods: We included 261 Vietnamese COVID-19 patients, who were classified into moderate and severe groups. Disease severity prediction based on biomarkers and clinical parameters was performed by applying machine learning and statistical methods using the combination of clinical and biological data., Results: The random forest model could predict with 97% accuracy the likelihood of COVID-19 patients who subsequently worsened to the severe condition. The most important indicators were interleukin (IL)-6, ferritin and D-dimer. The model could still predict with 92% accuracy after removing IL-6 from the analysis to generalise the applicability of the model to hospitals with limited capacity for IL-6 testing. The five most effective indicators were C-reactive protein (CRP), D-dimer, IL-6, ferritin and dyspnoea. Two different sets of biomarkers (D-dimer, IL-6 and ferritin, and CRP, D-dimer and IL-6) are applicable for the assessment of disease severity and prognosis. The two biomarker sets were further tested through machine learning algorithms and relatively validated on two Danish COVID-19 patient groups (n=32 and n=100). The results indicated that various biomarker sets combined with clinical data can be used for detection of the potential to develop the severe condition., Conclusion: This study provided a simple and reliable model using two different sets of biomarkers to assess disease severity and predict clinical outcomes in COVID-19 patients in Vietnam., Competing Interests: Conflict of interest: No conflicts of interest exist for any of the authors., (Copyright ©The authors 2023.)

Background: The study aims to estimate the prevalence of lactase non-persistence (LNP) among Greenlandic Inuit and Scandinavians living in Nuuk and East Greenland. The C to T transition in LCT - 13910 (rs4988235) is an autosomal inherited variant that provides the ability to lifelong lactase production, necessary to digest milk. The transition is very common in North European populations. However, LNP has only been sparsely studied in Greenland and never in Eastern Greenland, and genotype data has not previously been reported., Methods: Whole blood samples were collected from 535 participants, and rs4988235 was typed using a PCR-based method. Ethnicity was defined by parents' place of birth. Results were compared between East and West Greenland and Inuit and Scandinavians using Pearson's Chi 2 test., Results: 82.2% of the participants were Inuit, and 17.8% were of Scandinavian ancestry. Among Inuit, 88.5% had LNP compared to 7.5% among Scandinavians ( p  < 0.001). The prevalence of LNP in Inuit varied significantly between East and West Greenland ( p  < 0.001). In the capital, 67.6% of Inuit had LNP compared to 98.6% in Tasiilaq and 100% in the villages around Tasiilaq., Discussion: The difference in LNP between East and West Greenland and the Inuit and Scandinavian population found in our study suggests that the original Inuit population was lactose maldigesters. Our findings suggest that the -13910 T allele was introduced into the original Inuit population by the Danes.

The effect of long gaming sessions on energy intake, caffeine intake, blood pressure, heart rate, heart rate variability, and biochemical cardiac injury markers is unknown. The objective of this exploratory study was to investigate the changes in healthy male adults during two consecutive 18-hour sedentary video gaming sessions. Nine participants were enrolled in the study. Energy intake was noted in food diaries. Heart rate variability was monitored continuously; blood pressure and cardiac injury markers were measured every three to six hours. During the 42-hour study, the participants had an energy and caffeine intake of 8004.9 kcal and 1354.4 mg, respectively. The participants had a significant decrease in energy intake in the second session (p=0.01). A strong, negative correlation was found between body mass index and total energy intake (R=-0.84, p=0.005) and waist circumference and total energy intake (R=-0.70, p=0.036) in the first session. No nightly dip in blood pressure or heart rate was observed. Based on this study, long-term adverse effects of gaming cannot be ruled out. The non-dip of HR and BP suggests that long gaming sessions could be detrimental to cardiovascular health long term., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

In-depth reviewing of all medical records and clinical databases concluded a 7-year shorter lifespan among Greenlanders infected with hepatitis B virus (HBV) compared with non-infected. Mortality did not associate with liver disease or any other specific disease entity. A possible mechanism for the reduced lifespan is subclinical inflammation that may be augmented by chronic viral infection. We hypothesized that chronic HBV infection contributes to this process causing a reduced life span. We added measurement of two markers of inflammation to the 10-year follow-up on our study of HBV among 50- through 69-years-old subjects in Greenland. The markers were YKL40 related to liver disease and hsCRP as a global marker of inflammation. Survival was evaluated using Cox regression with time until death entered as dependent variable and age, sex, smoking, alcohol intake, BMI, the presence of HBsAg and one marker of inflammation as explanatory variables. Forty-eight percent of participants with chronic HBV infection were alive after 10 years compared with 65% of participants without infection (p = 0.003). Survival associated with age (p < 0.001), BMI (p = 0.003) and both YKL40 and hsCRP (both, p < 0.001). Harbouring HBV influenced 10-year survival in the Cox regression after adjusting for age, sex, BMI, smoking, alcohol intake and inflammation. In conclusion, chronic low-grade inflammation and being infected with HBV were independent markers of mortality in otherwise healthy subjects. Thus, the 7-year shorter lifespan among Greenlanders with chronic HBV infection seems related to the long-lasting infection. Our findings call for caution in perceiving a chronic infection as benign., (© 2022 The Authors. Journal of Viral Hepatitis published by John Wiley & Sons Ltd.)

Background: Approximately 71 million people are still in need of direct-acting antiviral agents (DAAs). To achieve the World Health Organization Hepatitis C elimination goals, insight into the prevalence and influence of resistance associated substitutions (RAS) is of importance. Collaboration is key since DAA failure is rare and real-life data are scattered. We have established a European collaboration, HepCare, to perform in-depth analysis regarding RAS prevalence, patterns, and multiclass occurrence. Methods: Data were extracted from the HepCare cohort of patients who previously failed DAA therapy. Geno-and subtypes were provided by submitters and mostly based on in-house assays. They were reassessed using the Comet HCV subtyping tool. We considered RAS to be relevant if they were associated with DAA failure in vivo previously reported in literature. Results: We analyzed 938 patients who failed DAA therapy from ten different European countries. There were 239 genotypes (GT) 1a, 380 GT1b, 19 GT2c, 205 GT3a, 14 GT4a, and 68 GT4d infections. Several unusual subtypes (n = 15) (GT1b/g/l, GT3b, GT4k/n/r/t) were present. RAS appeared in over 80% of failures and over a quarter had three or more RAS. Multiclass RAS varied over target region and genotype between 0-48%. RAS patterns such as the Q30R + L31M and Q30R + Y93H in GT1a, the L31V + Y93H and L31V + Y93H for GT1b, and A30K + L31M and A30K/V + Y93H for GT3a all occurred with a prevalence below 5%. Conclusion: RAS occur frequently after DAA failures and follow a specific genotype and drug related pattern. Interpretation of the influence of RAS on retreatment is challenging due to various patterns, patients' characteristics, and previous treatment history. Moving towards HCV elimination, an ongoing resistance surveillance is essential to track the presence of RAS, RAS patterns and gather data for a re-treatment algorithm.

No reliable predictive blood-based biomarkers are available for determining survival from pancreatic adenocarcinoma (PDAC). This combined discovery and validation study examines promoter hypermethylation (ph) of secreted frizzled-related protein 1 (SFRP1) in plasma-derived cell-free DNA as an independent prognostic marker for survival and Gemcitabine effectiveness in patients with stage IV PDAC. We conducted methylation-specific polymerase chain reaction analysis of the promoter region of the SFRP1 gene, based on bisulfite treatment. Survival was analyzed with Kaplan-Meier curves, log-rank test, and Cox regression. The discovery cohort included 40 patients, 25 receiving Gem. Gem-treated patients with phSFRP1 had a shorter median overall survival (mOS) (4.4 months) than unmethylated patients (11.6 months). Adjusted Cox-regression yielded a hazard rate (HR) of 3.48 (1.39-8.70). The validation cohort included 58 Gem-treated patients. Patients with phSFRP1 had a shorter mOS (3.2 months) than unmethylated patients (6.3 months). Adjusted Cox regression yielded an HR of 3.53 (1.85-6.74). In both cohorts, phSFRP1 was associated with poorer survival in Gem-treated patients. This may indicate that tumors with phSFRP1 are more aggressive and less sensitive to Gem treatment. This knowledge may facilitate tailored treatment of patients with stage IV PDAC. Further studies are planned to examine phSFRP1 in more intensive chemotherapy regimens.

The purpose of this study was to characterize the diagnostic performance of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of SARS-CoV-2 nucleocapsid protein (NP) in blood. Blood samples were collected during hospitalization of 165 inpatients with PCR-confirmed SARS-CoV-2 infection and from 505 outpatients predominantly with relevant symptoms of COVID-19 simultaneously with PCR testing. For the 143 inpatients who had their first blood sample collected within 2 weeks after PCR-confirmed infection, the diagnostic sensitivity of the ELISA was 91.6%. The mean NP concentration of the 131 ELISA-positive blood samples was 1,734 pg/ml (range, 10 to 3,840 pg/ml). An exponential decline in NP concentration was observed for 368 blood samples collected over the first 4 weeks after PCR-confirmed SARS-CoV-2 infection, and all blood samples taken later had an NP concentration below the 10-pg/ml diagnostic cutoff. The diagnostic sensitivity of the ELISA was 81.4% for the 43 blood samples collected from outpatients with a simultaneous positive PCR test, and the mean NP concentration of the 35 ELISA-positive samples was 157 pg/ml (range, 10 to 1,377 pg/ml). For the 462 outpatients with a simultaneous negative PCR test, the diagnostic specificity of the ELISA was 99.8%. In conclusion, the SARS-CoV-2 NP ELISA is a suitable laboratory diagnostic test for COVID-19, particularly for hospitals, where blood samples are readily available and screening of serum or plasma by ELISA can facilitate prevention of nosocomial infections and reduce the requirement for laborious swab sampling and subsequent PCR analysis to confirmatory tests only.

Direct-acting antivirals (DAAs) have proven highly effective against chronic hepatitis C virus (HCV) infection. However, some patients experience treatment failure, associated with resistance-associated substitutions (RASs). Our aim was to investigate the complete viral coding sequence in hepatitis C patients treated with DAAs to identify RASs and the effects of treatment on the viral population. We selected 22 HCV patients with sustained virologic response (SVR) to match 21 treatment-failure patients in relation to HCV genotype, DAA regimen, liver cirrhosis and previous treatment experience. Viral-titre data were compared between the two patient groups, and HCV full-length open reading frame deep-sequencing was performed. The proportion of HCV NS5A-RASs at baseline was higher in treatment-failure (82%) than matched SVR patients (25%) (p = .0063). Also, treatment failure was associated with slower declines in viraemia titres. Viral population diversity did not differ at baseline between SVR and treatment-failure patients, but failure was associated with decreased diversity probably caused by selection for RAS. The NS5B-substitution 150V was associated with sofosbuvir treatment failure in genotype 3a. Further, mutations identified in NS2, NS3-helicase and NS5A-domain-III were associated with DAA treatment failure in genotype 1a patients. Six retreated HCV patients (35%) experienced 2nd treatment failure; RASs were present in 67% compared to 11% with SVR. In conclusion, baseline RASs to NS5A inhibitors, but not virus population diversity, and lower viral titre decline predicted HCV treatment failure. Mutations outside of the DAA targets can be associated with DAA treatment failure. Successful DAA retreatment in patients with treatment failure was hampered by previously selected RASs., (© 2020 John Wiley & Sons Ltd.)

Introduction: Chronic hepatitis B (CHB) affects 257 million individuals worldwide with an annual estimated mortality rate of 880,000 individuals. Accurate diagnosis of the stage of disease is difficult, and there is considerable uncertainty concerning the optimal point in time, when treatment should be started., Objectives: By analyzing and comparing the metabolomes of patients at different stages of CHB and comparing them to healthy individuals, we want to determine the metabolic signature of disease progression and develop a more accurate metabolome-based method for diagnosis of disease progression ultimately giving a better basis for treatment decisions., Methods: In this study, we used the combination of transient elastography and serum metabolomics of 307 serum samples from a group of 90 patients with CHB before and under treatment (with a follow-up time up to 10 years) at different progression stages over the clinical phases and 43 healthy controls.., Results: Our data show that the metabolomics approach can successfully discover CHB changing from the immune tolerance to the immune clearance phase and show distinctive metabolomes from different medical treatment stages. Perturbations in ammonia detoxification, glutamine and glutamate metabolism, methionine metabolism, dysregulation of branched-chain amino acids, and the tricarboxylic acid (TCA) cycle are the main factors involved in the progression of the disease. Fluctuations increasing in aspartate, glutamate, glutamine, methionine and 13 other metabolites are fingerprints of progression., Conclusions: The metabolomics approach may expand the diagnostic armamentarium for patients with CHB. This method can provide a more detailed decision basis for starting medical treatment.

Prevalence of hepatitis B virus (HBV) infections varies markedly with geography and is endemic in the Arctic. Travel and migration have increased markedly while the influence of migration to high endemic areas remains unknown. We surveyed subjects migrating from an area with a low prevalence of chronic HBV infection (Denmark, 0.01%) to an endemic HBV area (West- and East Greenland, 3% and 29%) in order to describe the prevalence of HBV exposure among migrants. We included 198 Caucasian Danes that had migrated to Greenland and repeated the cross-sectional investigation after 10 years. We performed thorough serological testing for HBV. None had ongoing HBV infection. Migrants to East Greenland were more frequently exposed to HBV than those in West Greenland (34.3% vs 10.3%; p < 0.01). This difference was reduced at 10-year follow-up (8.1% vs 5.7%; ns) and the overall number of participants with past HBV infection decreased over the 10-year period from 19.4% to 6.9% (p = 0.02). In conclusion, migration from very low prevalence to endemic HBV areas associated with a markedly increased risk of exposure to HBV. Lack of vaccination among migrants from Denmark to Greenland was frequent and it poses a continuing risk. All who migrate from low to high endemic HBV areas should be vaccinated., Abbreviations: HBV: Hepatitis B virus; HBV-DNA: Hepatitis B virus deoxyribonucleic acid; HBsAg: Hepatitis B surface antigen; Anti-HBs: Antibodies against hepatitis B surface antigen; Anti-HBc: Antibodies against hepatitis B core antigen; BMI: Body mass index.

Background: Chronic hepatitis C (CHC) can be eliminated as a public health threat by meeting the WHO targets: 90% of patients diagnosed and 80% treated by 2030. To achieve and monitor progress towards elimination, an updated estimate of the size of the CHC population is needed, but Denmark has no complete national CHC register. By combining existing registers in 2007, we estimated the population living with CHC to be 16,888 (0.38% of the adult population)., Aim: To estimate the population living with diagnosed and undiagnosed CHC in Denmark on 31 December 2016. Among additional aims were to estimate the proportion of patients attending specialised clinical care., Methods: People with diagnosed CHC were identified from four national registers. The total diagnosed population was estimated by capture-recapture analysis. The undiagnosed population was estimated by comparing the register data with data from two cross-sectional surveys., Results: The population living with diagnosed CHC in Denmark was 7,581 persons (95%CI: 7,416-12,661) of which 6,116 (81%) were identified in the four registers. The estimated undiagnosed fraction was 24%, so the total CHC infected population was 9,975 corresponding to 0.21% of the adult population (95%CI: 9,758-16,659; 0.21%-0.36%). Only 48% of diagnosed patients had received specialised clinical care., Conclusion: CHC prevalence in Denmark is declining and 76% of patients have been diagnosed. Linking diagnosed patients to care and increasing efforts to test people with former or current drug use will be necessary to achieve CHC elimination., Competing Interests: Names of funding sources: MSD Denmark, (Merck Sharp & Dohme) The funder had no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication None of the authors has served or currently serve on the editorial board of PlosOne None of the authors has acted as an expert witness in relevant legal proceedings None of the authors has sat or currently sit on a committee for an organization that may benefit from publication of the paper The funding from MSD was used for dataextraction, datacleaning, and statistical expertise during the analysis of data. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: Lactose intolerance (LI) may be considered in patients with unspecific gastrointestinal symptoms, but there is no clear consensus on when and how to diagnose the disorder. The LCT -13910 CC genotype is associated with acquired primary lactase deficiency (adult-type hypolactasia; ATH). We aimed to describe the number of tests and test results in the North Denmark Region considering patient age, geographical origin and repeated testing., Methods: Retrospective evaluation of the polymerase chain reaction-based LCT -13910 genotype tests registered in the clinical laboratory information system (LABKA II) with data linkage to Danish nationwide registers., Results: Between 18 May 2007 and 31 December 2018, a total of 23,560 individuals were tested. There was a sevenfold increase in the number of tests performed during the study period. About 9.8% of the tests performed in 2018 were repeated testing in the same individuals. Overall, 8.8% of tested individuals were younger than 5 years, 90.7% were of Danish origin and 5.5% originated from outside of Europe. The LCT -13910 CC genotype was identified in 13.3% of all tested individuals, in 16.0% of children younger than 5 years, in 6.8% of Danish individuals and in 90.9% originating from outside of Europe., Conclusions: In the North Denmark Region, a marked increase in the use of genetic testing for hypolactasia was observed and repeated testing was frequent. Furthermore, the use of the test and the test results were dependent on patient age and geographical origin. Results inform the debate on when and how to use genetic testing in the diagnosing of LI.

Patients with blood transfusion-dependent anemias develop transfusional iron overload (TIO), which may cause cardiosiderosis. In patients with an ineffective erythropoiesis, such as thalassemia major, common transfusion regimes aim at suppression of erythropoiesis and of enteral iron loading. Recent data suggest that maintaining residual, ineffective erythropoiesis may protect from cardiosiderosis. We investigated the common consequences of TIO, including cardiosiderosis, in a minipig model of iron overload with normal erythropoiesis. TIO was mimicked by long-term, weekly iron-dextran injections. Iron-dextran loading for around one year induced very high liver iron concentrations, but extrahepatic iron loading, and iron-induced toxicities were mild and did not include fibrosis. Iron deposits were primarily in reticuloendothelial cells, and parenchymal cardiac iron loading was mild. Compared to non-thalassemic patients with TIO, comparable cardiosiderosis in minipigs required about 4-fold greater body iron loads. It is suggested that this resistance against extrahepatic iron loading and toxicity in minipigs may at least in part be explained by a protective effect of the normal erythropoiesis, and additionally by a larger total iron storage capacity of RES than in patients with TIO. Parenteral iron-dextran loading of minipigs is a promising and feasible large-animal model of iron overload, that may mimic TIO in non-thalassemic patients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests, and there are no conflicts of interest to disclose., (Copyright © 2020. Published by Elsevier Inc.)

Background: Recreational, seated video gaming (gaming) has become a favorite pastime of children, adolescents, and adults (gamers) in developed countries. Some engage in gaming behavior for more than 6 h daily, which can subsequently lead to less time spent being physically active. Gaming can potentially have a serious impact on the physiology and biochemistry of gamers and can influence both short-term and long-term health. The aim of this review was to provide an overview of what is known about how gaming affects physiological and biochemical parameters in the human body and how studies have previously been designed and to discuss how studies can be designed moving forward., Methods: The literature search included material from three scientific databases (PubMed, EMBASE, and Web of Science) using a two-block search strategy. To be included in this review, studies had to investigate a biochemical or physiological aspect of sedentary, video game-related activities. Studies that investigated neurological, psychologic or musculoskeletal outcomes along with physiological or biochemical outcomes in gaming were eligible for inclusion. Studies regarding psychiatric conditions were excluded as this subject was outside the scope of this review. Additionally, non-English language articles were excluded., Results: A total of 5417 articles were screened, 138 studies from the literature search and 4 studies from reference lists were selected for further evaluation. The studies were evaluated based on their abstracts or full texts, and 51 studies were eventually included in the review. Thirty-seven studies included physiological results, seven studies included biochemical results, and seven studies included both. Several outcomes such as heart rate, blood pressure, blood glucose levels, and cortisol levels, were the subjects of a large number of investigations., Conclusion: This field is heterogenic and does not lend itself to firm conclusions. Tentatively, it seems reasonable to conclude that heart rate variability studies show that gaming increases activity in the sympathetic nervous system. More high-quality studies are required, and the lack of studies using uniform, standardized designs and realistic gaming sessions (i.e., longer than 30 min) limits our current knowledge., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: One out of seven women will develop a state of chronic postoperative pain following robot-assisted hysterectomy for endometrial cancer. Recently, metabolic studies have indicated that circulating lipids and lipoproteins could act as nociceptive modulators and thereby influence the induction and perpetuation of pain. The objectives of this explorative study were (1) to examine the preoperative serologic variations in concentrations of lipids, lipoproteins, and various low-molecular metabolites in patients with and without chronic postoperative pain after robot-assisted hysterectomy and (2) to explore if any of these serological biomarkers were predictive for development of chronic postoperative pain., Materials and Methods: The study was designed as a nested case-control study within a cohort of women treated for endometrial cancer with robot-assisted laparoscopic hysterectomy. Twenty-six women with chronic postoperative pain were matched on age and body mass index with fifty-two controls without chronic postoperative pain, and metabolic profiling of preoperatively drawn blood samples from a biobank was performed by means of nuclear magnetic resonance spectroscopy., Results: Nineteen metabolites, including cholesterol, cholesteryl ester, linoleic acid, phospholipids, lipids, and triglycerides had statistically significant higher concentrations in a subgroup of patients who would develop chronic postoperative pain on a later stage compared to the group of patients who would not develop chronic postoperative pain ( p  <   0.05). A sparse Partial Least Squares-Discriminant Analysis model explained 38.1% of the variance and had a predictive accuracy of 73.1%., Conclusions: This explorative study substantiates the hypothesis that certain lipids, lipoproteins, and fatty acids are associated with chronic postoperative pain.

Background and Aims: Hereditary nonpolyposis colon cancer (HNPCC) and Lynch syndrome (LS) are characterized by defects in the mismatch repair (MMR) system, which protects the integrity of the genome. Pathogenic variants in four MMR genes ( MLH1 , MSH2 , MSH6 , and PMS2 ) are responsible for LS, an autosomal, dominant hereditary disease that occurs with a frequency of 2-5% among all colorectal cancer cases. It has been estimated that ∼2-5% of all pathogenic variants found in the four MMR genes in LS cases are detected in the PMS2 gene. An overview of detected variants is presented here. Materials and Methods: Long-range (LR) PMS2 polymerase chain reaction (PCR) and PMS2 multiplex ligation probe amplification (MLPA) assays were used to detect PMS2 variants in ∼1500 probands. In a subset of the probands, pathogenic PMS2 variants were detected by next-generation sequencing, and all detected variants were confirmed by LR-PCR combined with an MLPA assay. Results: A summary of PMS2 mutation analyses performed on colon cancer patients from molecular diagnostic laboratories in Denmark and Sweden is presented. By screening ∼1500 HNPCC probands, a total of 40 different PMS2 variants were detected in 71 probands (5%); 20 variants were classified as pathogenic (C5), 2 variants as likely pathogenic (C4), 15 variants as variants of unknown significance (VUSs) (C3), 1 variant as likely benign (C2), and 2 variants as benign (C1). In total, 22/71 (31%) of the probands carried a pathogenic sequence variant. Among the probands with isolated loss of pPMS2 expression, the fraction of pathogenic variants was 20/35 (55%). Conclusions: Approximately 5% of the probands found in the Danish and Swedish populations presented here carried a PMS2 variant. In this study, six novel pathogenic variants and seven VUSs are reported.

Next-generation sequencing is a powerful diagnostic tool, and even though it is still of limited use in clinical practice, genome sequencing will be increasingly applied. Transition to wider genetic screening methods as clinical exome or genome sequencing has the potential to detect more variants of unknown significance as well as secondary findings. It calls for close cooperation between the laboratory geneticist and the medical geneticist. Pre- and post-test genetic counselling must be offered systematically, and always when there is a high risk of finding germ line variants.

Background: Gene expression profiles of normal and tumor tissue reflect both differences in biological processes taking place in vivo and differences in response to stress during surgery and sample handling. The effect of cold (room temperature) ischemia in the time interval between surgical removal of the specimen and freezing is described in a few studies. However, not much is known about the effect of warm (body temperature) ischemia during surgery., Methods: Three women with primary operable breast cancer underwent in situ biopsies from normal breast and tumor tissue prior to radical mastectomy. Ex vivo biopsies from normal and tumor tissue were collected immediately after surgical excision. The putative effects on gene expression of malignancy (tumor versus normal), surgical manipulation (post- versus pre-surgical) and interaction between the two (differences in effect of surgical manipulation on tumor and normal samples) were investigated simultaneously by Generalized Estimating Equation (GEE) analysis in this self-matched study., Results: Gene set enrichment analysis (GSEA) demonstrates a marked difference in effect of surgical manipulation on tumor compared to normal tissue. Interestingly, a large proportion of pathways affected by ischemia especially in tumor tissue are pathways considered to be specifically up regulated in tumor tissue compared to normal., Conclusion: The results of this study suggest that a large contribution to this differential expression originates from altered response to stress in tumor cells rather than merely representing in vivo differences. It is important to bear this in mind when using gene-expression analysis to deduce biological function, and when collecting material for gene expression profiling.

Background: There is little information about serum phosphate levels among patients with pulmonary tuberculosis (TB) and HIV infection., Objective: We aimed to assess the role of TB, HIV, inflammation and other correlates on serum phosphate levels., Methods: A cross-sectional study was conducted among TB patients and age- and sex-matched non-TB controls. Pulmonary TB patients were categorized as sputum -negative and -positive, based on culture. Age- and sex-matched non-TB controls were randomly selected among neighbours to sputum-positive TB patients. Data on age, sex, alcohol and smoking habits were obtained. HIV status, serum phosphate, and the acute phase reactants C-reactive protein (serum CRP) and α 1 -acid glycoprotein (serum AGP) were determined. Linear regression analysis was used to identify correlates of serum phosphate., Results: Of 1605 participants, 355 (22.1%) were controls and 1250 (77.9%) TB patients, of which 9.9% and 50.4% were HIV-infected. Serum phosphate was determined before start of TB treatment in 44%, and 1-14 days after start of treatment in 56%. Serum phosphate was up to 0.10 mmol/L higher 1-3 days after start of TB treatment, and lowest 4 days after treatment, after which it increased. In multivariable analysis, TB patients had 0.09 (95% CI: 0.05; 0.13) mmol/L higher serum phosphate than controls, and those with HIV had 0.05 (95% CI: 0.01; 0.08) mmol/L higher levels than those without. Smoking was also a positive correlate of serum phosphate, whereas male sex and age were negative correlates., Conclusion: While HIV and TB are associated with higher serum phosphate, our data suggest that TB treatment is followed by transient reductions in serum phosphate, which may reflect hypophosphataemia in some patients., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Background: With the introduction of direct acting antivirals, treatment of hepatitis C virus (HCV) in hard-to-reach populations is now feasible. Therefore, new cost-effective and reliable test methods are needed. Determination of HCV antibodies and HCV-RNA from dried blood spots samples could represent one such method. Here we examined whether anti-HCV could be detected-and HCV-RNA quantified-from dried blood spots, sent by regular mail. We also investigated, if IP-10 determined from dried blood spots correlated with fibrosis progression appraised by transient elastography., Method: Forty chronic HCV infected patients were consecutively enrolled. At baseline and after six months, dried blood spots were prepared from blood collected by venous puncture, dried for 4-6 hours, then stored in gas-impermeable plastic bags with a desiccator, before being sent by regular mail. At each visit, approximately six months apart, paired venous samples was obtained and analyzed for anti-HCV, HCV-RNA and IP-10., Results: Anti-HCV was found in 66/67 of the dried blood spots. Sixty-six paired samples were available for HCV-RNA analysis. A statistically significant correlation was found between log HCV-RNA concentrations in plasma, and log HCV-RNA obtained from (P < 0.0001, Pearson's R 0.6788, R2 0.4607). HCV-RNA, derived from DBS samples, was lower than the corresponding plasma concentration, reflected by a Bland-Altman bias of 3 with SD of bias ± 0.6472. We found no correlation between IP-10 and fibrosis progression., Conclusions: We identified anti-HCV in 66/67samples, and quantified IP-10 and HCV-RNA from dried blood spots, dried at room temperature and sent by regular mail. HCV-RNA concentrations from the dried blood spots were lower than corresponding plasma values; a probable result of heparin coated test tubes. We found no correlation between IP-10 and fibrosis progression. Overall, dried blood spots could be a cost-effective and easy-to-use alternative to standard tests for the diagnosis of HCV infections., Competing Interests: At initial submission we had noted that co-author Morten Ruhwald was registered as inventor on a patent application disclosing IP-10 based liver fibrosis monitoring, using DBS, which could be viewed as a conflict-of-interest. However, this application was dropped prior to submission. Thus, it does not constitute a conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objectives: We describe factors associated with and barriers to initiation of Direct Acting Antiviral (DAA) treatment in patients with chronic hepatitis C, who fulfill national fibrosis treatment guidelines in Denmark., Materials and Methods: In this nationwide cohort study, we included patients with chronic hepatitis C from The Danish Database for Hepatitis B and C (DANHEP) who fulfilled fibrosis treatment criteria. Factors associated with treatment initiation and treatment failure were determined by logistic regression analyses. Medical records were reviewed from patients who fulfilled fibrosis treatment criteria, but did not initiate DAA treatment to determine the cause., Results: In 344 (49%) of 700 patients, who fulfilled treatment criteria, factors associated with DAA treatment initiation were transmission by other routes than injecting drug use odds ratio (OR) 2.13 (CI: 1.38-3.28), previous treatment failure OR 2.58 (CI: 1.84-3.61) and ALT above upper limit of normal OR 1.60 (CI: 1.18-2.17). The most frequent reasons for not starting treatment among 356 (51%) patients were non-adherence to medical appointments (n = 107/30%) and ongoing substance use (n = 61/17%). Treatment failure with viral relapse occurred in 19 (5.5%) patients, who were more likely to have failed previous treatment OR 4.53 (CI: 1.59-12.91)., Conclusions: In this nationwide cohort study, we found non-adherence to medical appointments and active substance use to be major obstacles for DAA treatment initiation. Our findings highlight the need for interventions that can overcome these barriers and increase the number of patients who can initiate and benefit from curative DAA treatment.

Epigenetic alterations in colorectal cancer (CRC) cause important differences in the underlying tumor biology and aggressiveness. DNA hypermethylation is central for the development of CRC but the prognostic impact remains elusive. We aimed to assess the association between cell-free hypermethylated DNA and stage and survival in colorectal cancer (CRC). We analyzed pre-treatment plasma samples from 193 patients with CRC. Thirty gene-promoter regions were analyzed using methylation specific PCR. We compared the median number (range) of hypermethylated promoter regions with CRC stage, and constructed a multivariable Cox-regression model adjusted for stage, to evaluate the added prognostic information. The median number of hypermethylated promoter regions was nine (0-28) in patients with distant metastasis compared to five (0-19) in patients without metastatic disease (p < 0.0001). The majority of the hypermethylated promoter regions inferred a poor prognosis. Cox-regression analysis adjusted for patient age, sex, pre-treatment CEA-levels, and disease stage, showed that RARB (HR = 1.99, 95% CI [1.07, 3.72]) and RASSF1A (HR = 3.35, 95% CI [1.76, 6.38]) hypermethylation inferred a significant effect on survival. The risk of metastasis increase with the number of cell-free hypermethylated promoter regions. The presence of RARB and RASSF1A hypermethylation indicated aggressive disease, regardless of stage at the time of diagnosis., Competing Interests: CONFLICTS OF INTEREST The authors have none to disclose.

Cirrhosis in patients with chronic hepatitis C increases the risk of hepatocellular carcinoma (HCC), and surveillance with ultrasound (US) and alpha-fetoprotein (AFP) is recommended. This study aimed to estimate changes in the HCC incidence rate (IR) over time, HCC stage and prognosis, and AFP and US performed in patients with hepatitis C and cirrhosis. Eligible patients were identified in the Danish Database for Hepatitis B and C, and data from national health registries and patient charts were obtained. Tumour stage was based on Barcelona-Clinic Liver Cancer stage, TNM classification and size and number of lesions combined into stages 0-3. We included 1075 patients with hepatitis C and cirrhosis, free of HCC and liver transplant at baseline. During 4988 person years (PY), 115 HCC cases were diagnosed. The HCC incidence rate increased from 0.8/100 PY [CI95% 0.4-1.5] in 2002-2003 to 2.9/100 PY [2.4-3.4] in 2012-2013. One-year cumulative incidence of at least one AFP or US was 53% among all patients. The positive predictive value of an AFP ≥ 20 ng mL -1 was 17%. Twenty-three (21%) patients were diagnosed with early-stage HCC (stage 0/1) and 84 (79%) with late stage. Median survival after HCC for early-stage HCC disease was 30.1 months and 7.4 months for advanced HCC (stage 2/3). The incidence rate of HCC increased over time among patients with hepatitis C and cirrhosis in Denmark. Application of AFP and US was suboptimal, and most patients were diagnosed with advanced HCC with a poor prognosis., (© 2017 John Wiley & Sons Ltd.)

Background: Colorectal cancer (CRC) is one of the most common cancers in the western world. Screening is an efficient method of reducing cancer-related mortality. Molecular biomarkers for cancer in general and CRC in particular have been proposed, and hypermethylated DNA from stool or blood samples are already implemented as biomarkers for CRC screening. We aimed to evaluate the performance of proven hypermethylated DNA promoter regions as plasma based biomarkers for CRC detection., Methods: We conducted a cross-sectional case-control study of 193 CRC patients and 102 colonoscopy-verified healthy controls. Using methylation specific polymerase chain reaction, we evaluated 30 DNA promoter regions previously found to be CRC specific. We used multivariable logistic regression with stepwise backwards selection, and subsequent leave-pair-out cross validation, to calculate the optimism corrected area under the receiver operating characteristics curve (AUC) for all stage as well as early stage CRC., Results: None of the individual DNA promoter regions provided an overall sensitivity above 30% at a reasonable specificity. However, seven hypermethylated promoter regions (ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, and VIM) along with the covariates sex and age yielded an optimism corrected AUC of 0.86 for all stage CRC and 0.85 for early stage CRC. Overall sensitivity for CRC detection was 90.7% at 72.5% specificity using a cut point value of 0.5., Conclusions: Individual hypermethylated DNA promoter regions have limited value as CRC screening markers. However, a panel of seven hypermethylated promoter regions show great promise as a model for CRC detection.

Objectives: Surviving long lasting cardiac arrest following accidental hypothermia has been reported after treatment with extra corporeal life support (ECLS), but there is a risk of neurologic injury. Most surviving hypothermia patients have a prolonged stay in the intensive care unit, where most patients experience polyneuropathy. Theoretically, accidental hypothermic cardiac arrest may in itself contribute to polyneuropathy. This study was designed to examine the impact of three hours of cardiac arrest at a core temperature of 20°C followed by reanimation of peripheral nerve function., Methods: Seven pigs were cannulated for ECLS and cooled to a core temperature of 20°C followed by three hours of circulatory arrest where the extremities were packed with ice. After three hours, ECLS was started and rewarming was performed. During the process, neural testing of the ulnar nerve (a somatic nerve) and of the vagus nerve (an autonomic nerve) were performed and blood was drawn for analysis of p-potassium, serum-neuron-specific enolase, and S100b protein., Results: The ulnar nerve was cooled from 34.9±1.6°C to 12.8±3.8°C and the vagus nerve from 36.2±1.2°C to 15.4±1.4°C. Physiologic function of both somatic and autonomic nerves were strongly affected by cooling, but recovered to almost normal levels during rewarming, even after three hours of hypothermic cardiac arrest. P-potassium rose from 3.9 (3.6-4.6)mmol/l to 8.1 (7.2-9.1)mmol/l after three hours of cardiac arrest, but normalized after recirculation. There was no rise in serum-neuron-specific enolase, but a slight rise in S100b protein during three hours of hypothermic cardiac arrest was observed. All pigs obtained return of spontaneous circulation (ROSC)., Conclusions: Reanimation after three hours of hypothermic cardiac arrest using ECLS was possible with no or, if present, minor damage to the two nerves tested., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: Improved methods for early detection of colorectal cancer (CRC) are essential for increasing survival. Hypermethylated DNA in blood or stool has been proposed as a biomarker for CRC. Biochemical methods have improved in recent years, and several hypermethylated genes that are sensitive and specific for CRC have been proposed. Articles describing the use of hypermethylated promoter regions in blood or stool as biomarkers for CRC were systematically reviewed., Method: A systematic literature search was performed using the Medline, Web of Science and Embase databases. Studies were included if they analysed hypermethylated genes from stool or blood samples in correlation with CRC. Studies in languages other than English and those based on animal models or cell lines were excluded., Results: The literature search yielded 74 articles, including 43 addressing blood samples and 31 addressing stool samples. In blood samples, hypermethylated ALX4, FBN2, HLTF, P16, TMEFF1 and VIM were associated with poor prognosis, hypermethylated APC, NEUROG1, RASSF1A, RASSF2A, SDC2, SEPT9, TAC1 and THBD were detected in early stage CRC and hypermethylated P16 and TFPI2 were associated with CRC recurrence. In stool samples, hypermethylated BMP3, PHACTR3, SFRP2, SPG20, TFPI2 and TMEFF2 were associated with early stage CRC., Conclusion: Hypermethylation of the promoters of specific genes measured in blood or stool samples could be used as a CRC biomarker and provide prognostic information. The majority of studies, however, include only a few patients with poorly defined control groups. Further studies are therefore needed before hypermethylated DNA can be widely applied as a clinical biomarker for CRC detection and prognosis., (Colorectal Disease © 2016 The Association of Coloproctology of Great Britain and Ireland.)

Background: Hepatitis B virus (HBV) infection is common in Arctic populations and high alcohol intake has been associated with an increased risk of a number of diseases. Yet, a description of the influence of alcohol intake in persons with HBV infection on liver biochemistry is lacking., Objective: We aimed to describe the association between reported alcohol intake and liver biochemistry taking into account also HBV infection, ethnicity, Inuit diet, body mass index (BMI), gender and age in an Arctic population., Design and Methods: Population-based investigation of Inuit (n=441) and non-Inuit (94) in Greenland and Inuit living in Denmark (n=136). Participants filled in a questionnaire on alcohol intake and other life style factors. Blood samples were tested for aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), bilirubin, albumin, hepatitis B surface antigen, hepatitis B surface antibody and hepatitis B core antibody. We also performed physical examinations., Results: Participation rate was 95% in Greenland and 52% in Denmark. An alcohol intake above the recommended level was reported by 12.9% of non-Inuit in Greenland, 9.1% of Inuit in East Greenland, 6.1% of Inuit migrants and 3.4% of Inuit in the capital of Greenland (p=0.035). Alcohol intake was associated with AST (p<0.001) and GGT (p=0.001), and HBV infection was associated with ALP (p=0.001) but not with AST, GGT, bilirubin or albumin in the adjusted analysis. Inuit had higher AST (p<0.001), GGT (p<0.001) and ALP (p=0.001) values than non-Inuit after adjustment for alcohol, diet, BMI and HBV exposure. Ethnic origin modified the association between alcohol and AST, while HBV infection did not modify the associations between alcohol and liver biochemistry., Conclusions: Non-Inuit in Greenland reported a higher alcohol intake than Inuit. Ethnic origin was more markedly associated with liver biochemistry than was alcohol intake, and Greenlandic ethnicity modified the effect of alcohol intake on AST. HBV infection was slightly associated with ALP but not with other liver biochemistry parameters.

Hepatitis B virus (HBV) infection is a disease with a highly variable course. Chronic HBV infection may cause end-stage liver disease including cirrhosis and hepatocellular carcinoma, which is the 3(rd) most common cause of cancer related death due to the poor prognosis. The prevalence of HBV infection is low in many countries. Still, it remains important due to the potential consequences of the disease. HBV is endemic in the Arctic with serologic markers of chronic HBV infection in up to 29% of the population in some areas in Greenland. Interestingly, Inuit populations rarely show signs of liver disease despite the fact that around half of all Inuit has been exposed to HBV and around 8% of Inuit are chronically infected with HBV. These findings have been consistent in surveys conducted for more than four decades among Arctic Inuit. We thus review HBV infection in the Arctic with focus on Greenland Inuit and compared with Inuit in Canada, Alaska and Siberia. The aspects described include epidemiology and monitoring of the disease, as well as treatment and the risk of liver cancer.

Genetic variation upstream of the apoptosis pathway has been associated with outcome of hepatitis C virus (HCV) infection. We investigated genetic polymorphisms in the intrinsic apoptosis pathway to assess their influence on sustained virological response (SVR) to pegylated interferon-α and ribavirin (pegIFN/RBV) treatment of HCV genotypes 1 and 3 infections. We conducted a candidate gene association study in a prospective cohort of 201 chronic HCV-infected individuals undergoing treatment with pegIFN/RBV. Differences between groups were compared in logistic regression adjusted for age, HCV viral load and interleukin 28B genotypes. Four single nucleotide polymorphisms (SNPs) located in the B-cell lymphoma 2-like 1 (BCL2L1) gene were significantly associated with SVR. SVR rates were significantly higher for carriers of the beneficial rs1484994 CC genotypes. In multivariate logistic regression, the rs1484994 SNP combined CC+TC genotypes were associated with a 3.4 higher odds ratio (OR) in SVR for the HCV genotype 3 (p=0.02). The effect estimate was similar for genotype 1, but the association did not reach statistical significance. In conclusion, anti-apoptotic SNPs in the BCL2L1 gene were predictive of SVR to pegIFN/RBV treatment in HCV genotypes 1 and 3 infected individuals. These SNPs may be used in prediction of SVR, but further studies are needed.

Background: Treponema pallidum, the causative agent of syphilis, elicits a vigorous immune response in the infected host. This study sought to describe the impact of syphilis infection on hepatitis C virus (HCV) RNA levels in patients with HIV and chronic HCV infection., Methods: Patients with chronic HIV/HCV and syphilis co-infection were identified by their treating physicians from 1 October 2010 to 31 December 2013. Stored plasma samples obtained before, during, and after syphilis infection were analysed for interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and IFN-γ-inducible protein 10 kDa (IP-10)., Results: Undetectable HCV RNA at the time of early latent syphilis infection was observed in 2 patients with HIV and chronic HCV infection. After treatment of the syphilis infection, HCV RNA levels increased again in patient 1, whereas patient 2 initiated HCV therapy and remained HCV RNA-negative. Available plasma samples obtained before and after the episode with undetectable HCV RNA were phylogenetically identical, making the possibility of spontaneous clearance and HCV reinfection less likely. The IL-10, TNF-α, and IP-10 levels increased at the time of syphilis diagnosis in patient 1 and decreased again after treatment of the syphilis infection., Conclusions: We propose that T. pallidum-induced cytokine secretion resulted in an immune response hindering HCV replication during syphilis infection. We suggest that HIV/HCV-co-infected patients with unexpected undetectable HCV RNA are tested for syphilis infection and that the serological tests include both non-treponemal and treponemal tests to avoid false-positive results caused by HCV.

Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistance in vitro. Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-α resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-α therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-α resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-α resistance. While we established a close link between viral fitness and IFN-α resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.

Introduction: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant condition accounting for 2% to 4% of all colorectal cancer cases worldwide. Families with germ line mutations in 1 of 6 mismatch repair genes are known as Lynch syndrome families. The largest number of mutations has been detected in the mismatch repair genes MLH1 and MSH2, but several mutations in MSH6 have also been demonstrated., Aim: : Whether HNPCC families are screened for mutations in mismatch repair genes often relies on their immunohistochemical profile. The aim of the present study was to evaluate this approach in Lynch families carrying mutations in MSH6., Materials and Methods: Results of the screening of the MSH6 gene in HNPCC families were compared with those obtained on immunohistochemical protein analysis., Results: In 56 (7%) of 815 families, at least 1 MSH6 mutation, 23 definitively pathogenic mutations and 38 missense mutations or unclassified variants, and several polymorphisms in the MSH6 gene were detected. In families carrying a pathogenic MSH6 mutation, 69.6% of 23 colon adenocarcinomas showed absence of pMSH6 in tumor tissue by immunohistochemical analysis. In 34.5%, all proteins could be detected, whereas in 34.5% pMSH6 was present and pMLH1/pPMS2 was absent., Conclusions: If genetic screening of HNPCC families depended on immunohistochemical results, a substantial number of families harboring a pathogenic mutation in MSH6 and the vast majority of families harboring an MSH6 unclassified variant would not be detected.

Objective: The aim of this study was to examine the early viral kinetics as predictor for sustained virological response (SVR) during hepatitis C treatment., Materials and Methods: We included patients with biopsy-proven chronic hepatitis C and ALT above the upper limit of normal, who received a standard treatment of pegylated interferon alfa-2a and ribavirin. The HCV-RNA concentration (limit of detection 20 IU/mL) was determined at days 0, 1, 2, 3, 4, 7, 14, 21 and monthly thereafter., Results: Among 46 patients who completed the trial, 30 (65%) had SVR. Low baseline viral load, IL28B genotype CC and absence of cirrhosis were statistically associated with SVR. In multivariate analysis only absence of cirrhosis and HCV-RNA negativity at day 14 were independent predictors for SVR. Eight patients who became HCV-RNA negative on day 14 as well as 13 of 14 patients (93%) with HCV-RNA levels of <1000 IU/mL at day 7 obtained a SVR. Among 8 of 18 (44%) genotype 1 and 4 patients with more than a one log drop in HCV-RNA titer at day 7, 75% achieved SVR., Conclusions: We observed a correlation between low HCV-RNA titers in week 2 and SVR during pegylated interferon/ribavirin-based treatment. This may help identify a group of patients for whom SVR may be obtained without the addition of directly acting antivirals, and thereby save the patients for unnecessary side effects and the health care system for additional costs.

Background: A national survey for chronic hepatitis C has not been performed in Denmark and the prevalence is unknown. Our aim was to estimate the prevalence of chronic hepatitis C from public registers and the proportion of these patients who received specialized healthcare., Methods: Patients with a diagnosis of chronic hepatitis C were identified from four national registers: a laboratory register, the Hospital Discharge Register, a clinical database of chronic viral hepatitis and the Register of Communicable Diseases. The total population diagnosed with hepatitis C was estimated by capture-recapture analysis. The population with undiagnosed hepatitis C was derived from the national register of drug users by comparing diagnosed and tested persons., Results: A total of 6,935 patients diagnosed with chronic hepatitis C were identified in the four registers and the estimated population diagnosed with the disease was 9,166 persons (95% C.I. interval 8,973 - 9,877), corresponding to 0.21% (95% CI 0.21%-0.23%) of the Danish population over 15 years of age. The prevalence was highest among persons 40-49 years old (0.39%) and males (0.28%). It was estimated that 40% of the diagnosed patients lived in the capital region, and 33.5% had attended specialised healthcare. It was estimated that 46% of hepatitis C patients had not been diagnosed and the total population with chronic hepatitis C in Denmark was 16,888 (95% C.I. 16,474-18,287), corresponding to 0.38% (95% CI 0.37-0.42) of the population over 15 years of age., Conclusions: The estimated prevalence of chronic hepatitis C in Denmark was 0.38%. Less than half of the patients with chronic hepatitis C in Denmark have been identified and among these patients, one in three has attended specialised care.

Objective: Infection with hepatitis B virus (HBV) is endemic among Arctic populations where it may have a benign course. However, the relation of HBV to migration to low endemic areas is unknown, as it is for hepatitis D and C, and details on the influence of delta virus at a population level are lacking., Material and Methods: Population-based investigation of Greenlanders living in Denmark (n = 136) and in Greenland (n = 441). We tested for HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe, HBV-DNA, HBV genotypes, anti-HDV, HDV-RNA, anti-HCV, HCV-Elisa test, HCV-RNA, aspartate aminotransferase, gamma-glutamyl transferase, bilirubin, and albumin, and performed a physical examination., Results: Participation rate was 52/95% in Denmark/Greenland. Half of participants in Denmark had lived more than half of their lives in Denmark, and 54.5% had been exposed to HBV. This was similar to 53% among Greenlanders living in West Greenland (p = 0.76). HBsAg was positive in 4.4% of Greenlanders in Denmark (n = 6), who all were anti-HBe positive and had low viral load. Serological signs of HBV infection associated with having both parents born in Greenland (p = 0.007) and with IV drug use (p = 0.03). We found serological signs of HDV exposure among participants in Denmark/Greenland in 0.7/1.1% (n = 1/5) and HCV exposure in 1.5/0.0% (n = 2/0). Liver biochemistry was elevated in Greenlanders exposed to HDV., Conclusions: Hepatitis B, D, and C occurrences among Greenlanders in Denmark mirrored that of Greenland. Importantly, previously undetected exposure to delta virus associated with elevated liver biochemistry, and the introduction of delta virus is a liability to Greenlanders and to Greenland.

Background: Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amounts of cell-free DNA in plasma, analytical sensitivity is of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulfite-mediated deamination of cytosine. Cytosine is rapidly converted to uracil during bisulfite treatment, whereas 5-methylcytosine is only slowly converted. Hence, bisulfite treatment converts an epigenetic modification into a difference in sequence, amenable to analysis either by sequencing or PCR based methods. However, the recovery of bisulfite-converted DNA is very poor., Results: Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours., Conclusions: The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.

In Denmark selective screening programs of pregnant women for hepatitis B missed 30-50% of high-risk groups and in late 2005 a universal screening of pregnant women for HBsAg was implemented. During a 2-year period a prospective enhanced surveillance of the universal screening was performed to examine the effectiveness of universal HBV-screening of pregnant women and HBV-immunizations of their newborn, and to provide a prevalence-estimate for HBV in Denmark. On a opt out basis all women in Denmark attending antenatal care were tested for hepatitis B serology. Vaccination data of the newborns and households of HBsAg positive pregnant women were assembled. Among 140,376 HBsAg tests of pregnant women, 371 (0.26%) were positive. The prevalence among women of Danish origin was 0.012% and 2.74% among foreign born women, highest for women from Southeast Asia (14.5%). Genotype C was the most prevalent (37%) and 13% had a HBVDNA ≥10(8) IU/ml. The prevalence estimate of chronic hepatitis B in Denmark was 0.2-0.3% in the general population. Among children born within the project period, 96% received vaccination at birth compared to 50% of siblings born prior to universal screening. During 3 years of passive follow-up two transmissions (0.5%) have been notified. Among children born of the positive mothers prior to the trial-period 7.3% had been notified. Thus the prevalence of HBV positive mothers has more than doubled in Denmark over the last 40 years, but among women of Danish origin it has decreased 10-fold. By replacing selective screening with universal, identification of newborns in need of HBV-immunization was increased from 50% to almost complete coverage, and also identifies mothers with high viral load for evaluation of pre-term treatment to interrupt in utero transmission., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Differences in the genetic architecture of inflammatory bowel disease between different European countries and ethnicities have previously been reported. In the present study, we wanted to assess the role of 11 newly identified UC risk variants, derived from a recent European UC genome wide association study (GWAS) (Franke et al., 2010), for 1) association with UC in the Nordic countries, 2) for population heterogeneity between the Nordic countries and the rest of Europe, and, 3) eventually, to drive some of the previous findings towards overall genome-wide significance., Methods: Eleven SNPs were replicated in a Danish sample consisting of 560 UC patients and 796 controls and nine missing SNPs of the German GWAS study were successfully genotyped in the Baltic sample comprising 441 UC cases and 1156 controls. The independent replication data was then jointly analysed with the original data and systematic comparisons of the findings between ethnicities were made. Pearson's χ2, Breslow-Day (BD) and Cochran-Mantel-Haenszel (CMH) tests were used for association analyses and heterogeneity testing., Results: The rs5771069 (IL17REL) SNP was not associated with UC in the Danish panel. The rs5771069 (IL17REL) SNP was significantly associated with UC in the combined Baltic, Danish and Norwegian UC study sample driven by the Norwegian panel (OR = 0.89, 95% CI: 0.79-0.98, P = 0.02). No association was found between rs7809799 (SMURF1/KPNA7) and UC (OR = 1.20, 95% CI: 0.95-1.52, P = 0.10) or between UC and all other remaining SNPs. We had 94% chance of detecting an association for rs7809799 (SMURF1/KPNA7) in the combined replication sample, whereas the power were 55% or lower for the remaining SNPs.Statistically significant PBD was found for OR heterogeneity between the combined Baltic, Danish, and Norwegian panel versus the combined German, British, Belgian, and Greek panel (rs7520292 (P = 0.001), rs12518307 (P = 0.007), and rs2395609 (TCP11) (P = 0.01), respectively).No SNP reached genome-wide significance in the combined analyses of all the panels., Conclusions: This replication study supports an important role for the studied rs5771069 (IL17REL) SNP, but not for rs7809799 (SMURF1/KPNA7), in UC etiology in the Danish, Baltic, and Norwegian populations. Significant genetic heterogeneity was suggested for rs7520292, rs12518307, and rs2395609 (TCP11) in UC etiology between the Nordic and the other European populations.

Background: The effect of peginterferon and ribavirin treatment on chronic hepatitis C virus (HCV) infection has been established in several controlled clinical studies. However, the effectiveness of treatment and predictors of treatment success in routine clinical practice remains to be established. Our aim was to estimate the effectiveness of peginterferon and ribavirin treatment in unselected HCV patients handled in routine clinical practice. The endpoint was sustained virological response (SVR), determined by the absence of HCV RNA 24 weeks after the end of treatment., Methods: We determined the proportion of SVR in a nationwide, population-based cohort of 432 patients with chronic HCV infection who were starting treatment, and analyzed the impact of known covariates on SVR by using a logistic regression analysis., Results: The majority of treated patients had genotype 1 (133 patients) and genotype 2/3 (285 patients) infections, with 44% and 72%, respectively, obtaining SVR. Other than genotype, the predictors of SVR were age≤45 years at the start of treatment, completion of unmodified treatment, the absence of cirrhosis and non-European origin., Conclusions: The effectiveness of peginterferon and ribavirin treatment for chronic hepatitis C in a routine clinical practice is comparable to that observed in controlled clinical trials, with a higher SVR rate in genotype 2 and 3 patients compared to genotype 1 patients. Our data further indicate that age at start of treatment is a strong predictor of SVR irrespective of HCV genotype, with patients 45 years or younger having a higher SVR rate.

Background: Inflammatory bowel diseases (IBDs) are a result of interactions between luminal pathogens and the intestinal immune response. Cyclooxygenase-2 (COX-2) plays a key role in the regulation of the inflammatory response upon stimulation by luminal pathogens via Toll-like receptors., Methods: Genotypes of the COX-2/PTGS2/PGHS2 A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) polymorphisms were assessed in a Scottish and Danish case-control study including 732 Crohn's disease (CD) cases, 973 ulcerative colitis (UC) cases, and 1157 healthy controls using logistic regression., Results: Carriers of the COX-2 A-1195G variant allele had increased risk of UC (odds ratio [OR], 95% confidence interval [CI] = 1.25 [1.02-1.54], P = 0.03) and of both UC and IBD among never smokers (OR [95% CI] = 1.47 [1.11-1.96], P = 0.01 and OR [95% CI] = 1.37 [1.06-1.77], P = 0.02, respectively). Furthermore, this variant genotype was associated with increased risk of diagnosis of UC before age 40 years and with extensive UC (OR [95% CI] = 1.34 [1.11-1.62], P = 0.002 and OR [95% CI] = 1.32 [1.03-1.69], P = 0.03, respectively)., Conclusions: COX-2 A-1195G polymorphism was associated with the risk of UC, especially among never-smokers, suggesting that low activity of COX-2 may predispose to UC. Our results suggest that inclusion of smoking status may be essential for the evaluation of the role of genetic predisposition to IBD., (Copyright © 2010 Crohn's & Crohn's & Colitis Foundation of America, Inc.)