Mutations Identified in the Hepatitis C Virus (HCV) Polymerase of Patients with Chronic HCV Treated with Ribavirin Cause Resistance and Affect Viral Replication Fidelity.

Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.

Ribavirin inhibition of cell-culture infectious hepatitis C genotype 1-3 viruses is strain-dependent.

Ribavirin remains relevant for successful treatment of chronic hepatitis C virus (HCV) infections in low-income settings, as well as for therapy of difficult-to-treat HCV patients. We studied the effect of ribavirin against cell-culture adapted HCV of genotypes 1, 2 and 3, representing ~80% of global infections. TNcc(1a) was the most sensitive to ribavirin, while J6/JFH1(2a) was the most resistant. EC50s ranged from 21 µM (95%CI: 20-22 µM) to 189 µM (95%CI: 173-207 µM). Substitutions at position 415 of NS5B resulted in little or no change to ribavirin sensitivity (0.7-0.9 fold) but conferred moderate drug resistance during extended treatment of genotype 1 (1.8-fold). NS5A and NS5B sequences could alter ribavirin sensitivity 2-4-fold, although their contribution was not simply additive. Finally, we detected limited accumulation of mutations associated with ribavirin treatment. Our findings show that the antiviral effect of ribavirin on HCV is strain-dependent and is influenced by the specific sequence of multiple HCV nonstructural proteins.

Prevalence of anti-hepatitis E virus immunoglobulin G in HIV-infected individuals over three decades.

BACKGROUND: Hepatitis E virus (HEV) genotype 3 is endemic in Europe, and the infection is mostly subclinical or acute and self-limiting. However, persistent infection is described among HIV-infected individuals. The prevalence of antibodies against HEV (anti-HEV) among HIV-infected persons varies geographically and is unknown in Denmark. Rates of co-infection with HEV among HIV-infected individuals in Denmark over three decades, from the early 1980s to 2013, were investigated. METHODS: A total of 2506 HIV-infected persons were investigated from two cohorts followed at Hvidovre Hospital, Denmark. Blood samples were tested retrospectively for anti-HEV, including samples from 2216 persons who were enrolled in a prospective clinical cohort and followed between 1995 and 2013, as well as samples from 290 persons from a historical cohort followed between 1980 and 1994. For anti-HEV seroconverting individuals, serial samples were tested for HEV RNA. Factors associated with anti-HEV status were explored using multivariable logistic regression analysis. RESULTS: The overall HEV seroprevalence rates were stable during the 1980s, 1990s, and 2000-2013 (23.1%, 22.9%, and 23.7%, respectively). In all decades, rates of anti-HEV increased with older age, and anti-HEV seropositivity was associated with older generations, HIV risk group, and geographic origin. Persistent HEV infection was not detected in any of 57 individuals with anti-HEV seroconversion. CONCLUSIONS: HEV seroprevalence rates were stable in HIV-infected individuals from the early 1980s to 2013. Rates increased with age. No evidence of persistent HEV infection was detected. Infection with HEV is frequent, but persistent HEV infection is rare among HIV-infected individuals.

Increased risk of diabetes mellitus five years after an episode of Staphylococcus aureus bacteraemia.

Background: Diabetes mellitus is a risk factor for infection with Staphylococcus aureus, but it is unclear whether S. aureus infection is a prediabetic condition. Methods: Nationwide population-based matched cohort study. Incidence rate and ratio with 95% confidence interval of diabetes were estimated by negative binomial regression. Results: Of 19,988 individuals with S. aureus bacteraemia and 185,579 population comparators, 667 and 4974 had a primary diagnose of diabetes within five years after discharge of S. aureus bacteraemia corresponding to a more than double risk of diabetes (adjusted incidence rate ratio 2.28 (95% confidence interval: 2.10-2.46)). Other factors associated with an increased risk of diabetes during follow-up were male sex, increasing age and level of comorbidity. Of the S. aureus bacteraemia and population cohort, 422 (2.11%) and 4048 (2.18%), respectively, developed diabetes without complications, while 245 (1.23%) and 926 (0.50%), respectively, developed diabetes with complications. Rates of diabetes without complication were increased for individuals in the S. aureus bacteraemia cohort compared to the population cohort within the first two years after which rates were comparable while rates of diabetes with complications remained higher throughout the five year follow-up period compared to the population cohort. Conclusions: The risk of diabetes was markedly increased up to five years after S. aureus bacteraemia compared to a population cohort. In addition to screening for diabetes during hospital admittance, screening cases of S. aureus bacteraemia for diabetes in the years following S. aureus bacteraemia may allow for earlier detection of diabetes.

Ribavirin-induced mutagenesis across the complete open reading frame of hepatitis C virus genotypes 1a and 3a.

Ribavirin (RBV) has been used for the last 20 years to treat patients with chronic hepatitis C virus (HCV) infection. This pluripotent drug is believed to induce mutagenesis in HCV RNA. However, for cell-cultured HCV (HCVcc) this phenomenon has only been investigated in genotype 2a recombinants. Here we studied the mutations that developed in HCVcc of genotypes 1a and 3a treated with RBV or ribavirin triphosphate (RBV-TP) compared to non-treated controls. Analysis was performed on the amplified full-length open reading frame (ORF) of recovered viruses following next-generation sequencing and clonal analyses. Compared to non-treated controls, the spread of TNcc(1a) and DBN3acc(3a) HCVcc was delayed by RBV and RBV-TP at concentrations of 40 µM or higher. The delay in HCVcc spread was associated with increased new single-nucleotide polymorphisms (SNP). Significantly higher numbers of new SNP were observed in TNcc(1a) viruses treated with RBV or RBV-TP compared to matched non-treated controls. RBV or RBV-TP treatment led to significantly increased proportions of new G-to-A and C-to-U SNP compared to non-treated TNcc(1a). Clonal analyses confirmed a significantly increased mutation rate in RBV-treated TNcc(1a). Synonymous pairwise distances increased in both viruses across the complete ORF under RBV and RBV-TP treatment compared to controls. Consensus-shifts in single samples of RBV- or RBV-TP-treated TNcc(1a) viruses occurred in proteins E1, p7, NS3 and NS4B. No non-synonymous consensus changes were observed in DBN3acc(3a). This study supports a biased G-to-A and C-to-U mutagenic effect of RBV and RBV-TP throughout the entire ORF of HCV genotypes 1a and 3a.

Soluble Urokinase Plasminogen Activator Receptor Is a Predictor of Incident Non-AIDS Comorbidity and All-Cause Mortality in Human Immunodeficiency Virus Type 1 Infection.

Persistent inflammation and immune activation have been associated with non-AIDS comorbidity and mortality in human immunodeficiency virus (HIV) infection. We aimed to investigate the potential association between soluble urokinase plasminogen activator receptor (suPAR) and incident non-AIDS comorbidity and all-cause mortality in a well-treated HIV-infected population. suPAR was measured by enzyme-linked immunosorbent assay, and events of comorbidity and mortality were ascertained by registry linkage. The study showed an independent association between a high suPAR level at baseline and increased hazard rates for both non-AIDS comorbidities (cardiovascular disease, chronic kidney disease, chronic lung disease, liver disease, and cancer) and all-cause mortality.

Diabetes increases the risk of disease and death due to Staphylococcus aureus bacteremia. A matched case-control and cohort study.

BACKGROUND: This study investigated the effect and influence of diabetes severity on susceptibility and 30-day mortality of Staphylococcus aureus bacteremia (SAB). METHODS: Nationwide population-based study of individuals with SAB and matched population controls. Diabetes severity was categorized based on International Classification of Diseases codes and the odds ratio (OR) with 95% confidence intervals (CI) of SAB associated with diabetes was estimated by conditional logistic regression analysis. Hazard ratios (HR) were analyzed by Cox proportional regression. Analyses were adjusted for age, sex, comorbidity, hospital contact and diabetes duration. RESULTS: Of 25,855 SAB cases, 2797 (10.8%) had diabetes and 2913 (11.3%) had diabetes with complications compared to 14,189 (5.5%) and 5499 (2.1%) of 258,547 controls. This corresponded to an increased risk of SAB associated with diabetes without complications (OR 1.83 (95% CI 1.74-1.92)) and of diabetes with complications (OR 3.62 (95% CI 3.43-3.81) compared to no diabetes. The risk of SAB was highest within the first year of diabetes. Diabetes without complications was associated with an increased risk of 30-day mortality (HR 1.62 (95% CI 1.01-2.60)) compared to no diabetes. Diabetes with complications was overall not associated with increased 30-d mortality (HR 1.36 (95% CI 0.84-2.20)) except for individuals with ketoacidosis/coma (HR 2.01 (95% 1.17-3.45)). CONCLUSIONS: Diabetes, particularly, diabetes with complications significantly increased the risk of SAB. In contrast, there was an increased risk of 30-day mortality after SAB for diabetes without complications but not for diabetes with complications overall. Diabetes with ketoacidosis/coma conferred the highest relative risk of 30-day mortality.

Soluble CD163 predicts incident chronic lung, kidney and liver disease in HIV infection.

OBJECTIVE: To examine if monocyte and macrophage activity may be on the mechanistic pathway to non-AIDS comorbidity by investigating the associations between plasma-soluble CD163 (sCD163) and incident non-AIDS comorbidities in well treated HIV-infected individuals. DESIGN: Prospective single-center cohort study. METHODS: Plasma sCD163 was quantified by ELISA technique at study entry in 2004/2005, and non-AIDS comorbidity was identified by International Classification of Disease Tenth revision diagnosis codes and registry linkage in 2015. Associations between sCD163 and incident comorbidity was examined using multivariable Cox proportional hazards models adjusted for pertinent covariates. RESULTS: In HIV-1-infected individuals (n = 799), the highest quartile of plasma sCD163 was associated with incident chronic lung disease [adjusted hazard ratio (aHR), 3.2; 95% confidence interval (CI): 1.34; 7.46] and incident chronic kidney disease (aHR, 10.94; 95% CI: 2.32; 51.35), when compared with lowest quartiles. Further, (every 1 mg) increase in plasma sCD163 was positively correlated with incident liver disease (aHR, 1.12; 95% CI: 1.05; 1.19). The sCD163 level was not associated with incident cancer, cardiovascular disease or diabetes mellitus. CONCLUSION: sCD163 was independently associated with incident chronic kidney disease, chronic lung disease and liver disease in treated HIV-1-infected individuals, suggesting that monocyte/macrophage activation may be involved in the pathogenesis of non-AIDS comorbidity and a potential target for therapeutic intervention.

Stable incidence and continued improvement in short term mortality of Staphylococcus aureus bacteraemia between 1995 and 2008.

BACKGROUND: The objective of this study was to assess temporal changes in incidence and short term mortality of Staphylococcus aureus bacteraemia (SAB) from 1995 through 2008. METHODS: The study was conducted as a nation-wide observational cohort study with matched population controls. The setting was hospitalized patients in Denmark 1995-2008. Uni- and multivariate analyses were used to analyze the hazard of death within 30 days from SAB. RESULTS: A total of 16 330 cases of SAB were identified: 57% were hospital-associated (HA), 31% were community-acquired (CA) and 13% were of undetermined acquisition. The overall adjusted incidence rate remained stable at 23 per 100 000 population but the proportion of SAB cases older than 75 years increased significantly. Comorbidity in the cohort as measured by Charlson comorbidity index (CCI) score and alcohol-related diagnoses increased over the study period. In contrast, among the population controls the CCI remained stable and alcohol-related diagnoses increased slightly. For HA SAB crude 30-day mortality decreased from 27.8% to 21.8% (22% reduction) whereas the change for CA SAB was small (26.5% to 25.8%). By multivariate Cox regression, age, female sex, time period, CCI score and alcohol-related diagnoses were associated with increased mortality regardless of mode of acquisition. CONCLUSIONS: Throughout a 14-year period the overall incidence of SAB remained stable while the overall short term prognosis continued to improve despite increased age and accumulation of comorbidity in the cohort. However, age and comorbidity were strong prognostic indicators for short term mortality.