Ulf Herbers

The rtA194T polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis B e antigen–positive and hepatitis B e antigen–negative hepatitis B virus strains

Tenofovir is a new effective treatment option for patients with chronic hepatitis B, but could be potentially hampered by mutations in the hepatitis B virus (HBV) polymerase conferring drug resistance. Drug resistance may occur preferentially if long‐term administration is required, for example, in patients with hepatitis B e antigen (HBeAg)‐negative HBV infection bearing precore (PC) and basal core promoter (BCP) mutations. The rtA194T polymerase mutation has been found in HBV/HIV coinfected patients during tenofovir treatment and may be associated with tenofovir resistance. We generated replication‐competent HBV constructs harboring rtA194T alone or in addition to lamivudine (LAM) resistance (rt180M + rtM204V), PC mutations, and BCP mutations and assessed their replicative capacity after transient transfection in human hepatoma cells. The rtA194T polymerase mutation alone or in conjunction with LAM resistance reduced the replication efficiency as compared with wild‐type (WT) HBV. In contrast, combination of rtA194T (± LAM resistance) with HBeAg‐negative PC or BCP mutants increased the replication capacity of the drug‐resistant polymerase mutants, thereby restoring the viral replication to similar levels as WT clones. Clones harboring rtA194T showed partial resistance to tenofovir in vitro and also to LAM but remained susceptible to telbivudine and entecavir. Conclusion: The rtA194T polymerase mutation is associated with partial tenofovir drug resistance and negatively impacts replication competence of HBV constructs. Viral replication, however, can be restored to WT levels, if these polymerase mutations occur together with precore or basic core promoter substitutions as found in HBeAg‐negative hepatitis B. Patients with HBeAg‐negative chronic HBV infection may therefore be at particular risk when developing drug resistance to tenofovir. Telbivudine or entecavir should be considered as effective alternative treatment options for these patients. (HEPATOLOGY 2009.)

Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome.

BACKGROUND/AIMS The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. METHODS The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/75 females, 194 treatment-nai ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. RESULTS In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-nai ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. CONCLUSIONS Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications.

Molecular and clinical aspects of hepatitis D virus infections

Hepatitis D virus (HDV) is a defective virus with circular, single-stranded genomic RNA which needs hepatitis B virus (HBV) as a helper virus for virion assembly and infectivity. HDV virions are composed of a circular shape HDV RNA and two types of viral proteins, small and large HDAgs, surrounded by HBV surface antigen (HBsAg). The RNA polymerase II from infected hepatocytes is responsible for synthesizing RNAs with positive and negative polarities for HDV, as the virus does not code any enzyme to replicate its genome. HDV occurs as co-infection or super-infection in up to 5% of HBsAg carriers. A recent multi-center study highlighted that pegylated interferon α-2a (PEG-IFN) is currently the only treatment option for delta hepatitis. Nucleotide/nucleoside analogues, which are effective against HBV, have no relevant effects on HDV. However, additional clinical trials combining PEG-IFN and tenofovir are currently ongoing. The molecular interactions between HDV and HBV are incompletely understood. Despite fluctuating patterns of HBV viral load in the presence of HDV in patients, several observations indicate that HDV has suppressive effects on HBV replication, and even in triple infections with HDV, HBV and HCV, replication of both concomitant viruses can be reduced. Additional molecular virology studies are warranted to clarify how HDV interacts with the helper virus and which key cellular pathways are used by both viruses. Further clinical trials are underway to optimize treatment strategies for delta hepatitis.

Gastrointestinale Blutungen als Komplikation von Patienten mit Leberzirrhose auf der Intensivstation

Due to portal hypertension and bleeding disorders, patients with liver cirrhosis are at increased risk for severe gastrointestinal bleedings (GIB), commonly requiring therapy at the intensive care unit (ICU). In order to identify epidemiological and prognostic factors for GIB in cirrhotic patients, we retrospectively analysed patients from our medical ICU from 1999 to 2010. Among 7376 critically ill patients, 650 (8.8 %) were diagnosed with liver cirrhosis. Hepatic cirrhosis was frequently found in ICU patients admitted due to severe GIB (23.2 % of 711 patients had cirrhosis). Moreover, patients with cirrhosis were at increased risk to develop severe GIB during intensive care treatment (40.9 % of 44 patients with GIB during ICU stay had cirrhosis). Besides the high rate of variceal bleedings (64.4 %) in cirrhotic patients, non-variceal haemorrhages were also common (28.5 %). We identified the MELD score and necessity of mechanical ventilation as independent risk factors for mortality in cirrhotic patients with severe GIB. Patients with liver cirrhosis and severe GIB had significantly impaired prognosis (case-related fatality rate of 26.1 % with cirrhosis vs. 6.8 % without cirrhosis), especially in cases of newly developed GIB during ICU therapy. Advanced therapeutic approaches and novel strategies are warranted to improve the critical prognosis of these high-risk patients.

Impact of hepatitis B e antigen-suppressing mutations on the replication efficiency of entecavir-resistant hepatitis B virus strains.

Summary.  Hepatitis B e antigen (HBeAg)‐negative hepatitis B commonly requires long‐term treatment with nucleos(t)ide analogues aiming at persistently suppressing hepatitis B virus (HBV) replication to halt progression of liver disease and prevent complications. Entecavir (ETV) is widely used in HBeAg‐negative hepatitis B, but distinct HBV polymerase mutations can confer resistance against ETV, in conjunction with lamivudine resistance. Precore (PC) and basal core promoter (BCP) mutations that underlie HBeAg‐negativity enhance replication of lamivudine‐resistant mutants. To comprehensively analyse the impact of PC or BCP mutations on viral replication of ETV‐resistant HBV mutants, replication‐competent HBV constructs were generated harbouring lamivudine resistance (rtM204V/rtL180M, rtM204I) plus ETV resistance (rtS202G, rtS202I or rtT184G) on wild‐type (WT)‐, PC‐ and BCP‐backgrounds. Functional consequences on viral fitness and susceptibility to antivirals were assessed in vitro. The presence of any ETV resistance drastically reduced viral replication when compared to WT HBV. In rtS202G mutants (plus lamivudine resistance), addition of either PC or BCP mutations moderately enhanced the reduced replication, without reaching WT HBV levels. In rtS202I or rtT184G mutants, PC and BCP mutations did not significantly improve viral fitness. All ETV‐resistant constructs, independently of PC or BCP mutations, showed resistance towards ETV and lamivudine, but remained susceptible to tenofovir. Our data demonstrate that HBeAg‐suppressing PC or BCP mutations cannot restore the strongly reduced replicative capacity of ETV‐resistant HBV mutants to WT level, although they moderately increase replication of rtS202G combination mutants. ETV resistance thereby differs from lamivudine resistance alone, corroborating that ETV is in short term a safe option for HBeAg‐negative patients.

Hepatitis B e antigen-suppressing mutations enhance the replication efficiency of adefovir-resistant hepatitis B virus strains

Summary.  Hepatitis B e antigen (HBeAg) negative hepatitis B virus (HBV) infections caused by precore (PC) or basal core promoter (BCP) mutations are associated with disease progression and complications. PC or BCP mutations may enhance the replication capacity of distinct drug‐resistance‐associated polymerase mutations, but their effect on adefovir‐resistant HBV mutants is unclear. Importantly, BCP mutations were an independent risk factor for virological breakthrough in lamivudine‐resistant patients treated with adefovir. We aimed at addressing the functional consequences of PC and BCP mutations on the replication and drug susceptibility of adefovir‐resistant HBV mutants. Therefore, HBV constructs with wild type (WT) or adefovir‐resistant rtN236T, rtA181V and rtA181T mutations, with or without concomitant PC or BCP mutations, were analysed in vitro using molecular assays. The adefovir‐resistant polymerase mutations rtN236T, rtA181V and rtA181T showed a drastically reduced viral replication compared with WT. Interestingly, additional PC or BCP mutations enhanced the reduced replication efficacy of adefovir‐resistant constructs and restored HBV replication to WT level. HBV rtA181T mutants displayed abolished hepatitis B surface antigen (HBsAg) secretion, owing to a sW172* stop codon in the overlapping envelope gene. All rtN236T‐ or rtA181V/T‐containing constructs, regardless of concomitant PC or BCP mutations, were resistant to adefovir, but remained susceptible to telbivudine, entecavir and tenofovir. In conclusion, adefovir drug resistance mutations reduced viral replication, which can be significantly increased by additional HBeAg‐suppressing PC or BCP mutations. Because increased HBV replication in HBeAg‐negative patients has been associated with an unfavourable clinical course, close monitoring appears indispensable during adefovir treatment in HBeAg‐negative patients.

Growth Differentiation Factor-15 Is a Predictor of Mortality in Critically Ill Patients with Sepsis

Growth differentiation factor-15 (GDF-15) is a member of the transforming growth factor-β superfamily related to inflammation and macrophage activation. Serum concentrations of GDF-15 can predict poor survival in chronic diseases, but its role in sepsis is obscure. Therefore, we investigated GDF-15 as a prognostic biomarker in critically ill patients. We measured GDF-15 levels in 219 critically ill patients (146 with sepsis, 73 without sepsis) upon admission to the intensive care unit (ICU), in comparison to 66 healthy controls. GDF-15 levels were significantly increased in ICU patients compared to controls. GDF-15 was further increased in sepsis and showed a strong association with organ dysfunction (kidney, liver and lactate) and disease severity (APACHE II and SOFA score). High GDF-15 concentrations at admission independently predicted ICU (HR 3.42; 95% CI 1.33–8.78) and overall mortality (HR 2.02, 95% CI 1.02–3.88) in all ICU critically ill patients as well as in a large subgroup of sepsis patients (ICU mortality: HR 3.16; 95% CI 1.10–9.07; overall mortality: HR 2.62; 95% CI 1.14–6.02). Collectively, serum GDF-15 levels are significantly increased in critically ill patients, associated with sepsis, organ failure, and disease severity. High GDF-15 levels at ICU admission predict short- and long-term mortality risk.

High mobility group box 1 as a biomarker in critically ill patients

Extracellular release of high mobility group box 1 (HMGB1) acts as a danger‐associated molecular pattern, thereby “alarming” the immune system and promoting systemic inflammation. We investigated plasma HMGB1 concentrations as a potential diagnostic and prognostic biomarker in critical illness.

Clinical relevance of copeptin plasma levels as a biomarker of disease severity and mortality in critically ill patients

Copeptin, also termed C‐terminal pre‐pro‐vasopressin or CTproAVP, mirrors endogenous vasopressin (anti‐diuretic hormone, ADH) activity and might thereby serve as a biomarker reflecting the biological stress level. We therefore hypothesized that copeptin plasma concentrations are associated with disease severity in critically ill patients and could predict mortality.

Visfatin Serum Levels Predict Mortality in Critically Ill Patients

The adipokine visfatin, also termed pre-B-cell colony-enhancing factor (PBEF), is mainly derived from adipose tissue but has been implicated in the regulation of innate immune responses. We hypothesized that visfatin could be a potential circulating biomarker in critical illness and sepsis. We therefore measured serum levels of visfatin in a cohort of 229 critically ill medical patients upon admission to the intensive care unit (ICU). In comparison to 53 healthy controls, visfatin levels were significantly elevated in medical ICU patients, especially in patients with sepsis. Visfatin serum concentrations were strongly associated with disease severity and organ failure but did not differ between patients with or without obesity or type 2 diabetes. Visfatin levels correlated with biomarkers of renal failure, liver dysfunction, and other adipokines (e.g., resistin, leptin, and adiponectin) in critically ill patients. High visfatin levels at ICU admission indicated an increased mortality, both at the ICU and during long-term follow-up of approximately two years. Our data therefore demonstrate that circulating visfatin is a valuable biomarker for risk and prognosis assessment in critically ill patients. Furthermore, visfatin seems to be involved in the pathogenesis of excessive systemic inflammation, supporting further research on visfatin as a therapeutic target.