Christoph Eisenbach

Pharmacologic cholinesterase inhibition improves survival in experimental sepsis

Objective: Lethal sepsis occurs when an excessive inflammatory response evolves that cannot be controlled by physiologic anti-inflammatory mechanisms, such as the recently described cholinergic anti-inflammatory pathway. Here we studied whether the cholinergic anti-inflammatory pathway can be activated by pharmacologic cholinesterase inhibition in vivo. Design: Prospective, randomized laboratory investigation that used an established murine sepsis model. Setting: Research laboratory in a university hospital. Subjects: Female C57BL/6 mice. Interventions: Sepsis in mice was induced by cecal ligation and puncture. Animals were treated immediately with intraperitoneal injections of nicotine (400 &mgr;g/kg), physostigmine (80 &mgr;g/kg), neostigmine (80 &mgr;g/kg), or solvent three times daily for 3 days. Measurements and Main Results: Treatment with physostigmine significantly reduced lethality (p ≤ .01) as efficiently as direct stimulation of the cholinergic anti-inflammatory pathway with nicotine (p ≤ .05). Administration of cholinesterase inhibitors significantly down-regulated the binding activity of nuclear factor-&kgr;B (p ≤ .05) and significantly reduced the concentration of circulating proinflammatory cytokines tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6 (p ≤ .001), and pulmonary neutrophil invasion (p ≤ .05). Animals treated with the peripheral cholinesterase inhibitor neostigmine showed no difference compared with physostigmine-treated animals. Conclusions: Our results demonstrate that cholinesterase inhibitors can be used successfully in the treatment of sepsis in a murine model and may be of interest for clinical use.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. Conclusion: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult‐to‐treat patient cohorts. (HEPATOLOGY 2013)

Risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit

Respiratory syncytial virus (RSV) usually causes self-limiting upper respiratory tract infections, but can be associated with severe lower respiratory tract infection disease (LRTID) in infants and in patients with hematologic malignancies. We have analyzed the risk factors and the measures for containment within an outbreak of nosocomial RSV infections in a hematology and SCT unit. A total of 56 patients were affected (53 RSV-A and 3 RSV-B) including 32 transplant patients (16 allogeneic and 16 autologous). Forty (71%) of the 56 patients suffered from LRTID and 14 (35%) of the patients with LRTID subsequently died. However, because of concomitant infections with fungal and bacterial pathogens, the impact of RSV on the fatal outcome was difficult to assess. Multivariate analysis showed that low levels of IgG were significantly associated with fatal outcome (P=0.007), treatment with oral ribavirin represented a protective factor (P=0.02). An extremely protracted viral shedding was observed in this cohort of patients (median=30.5 days, range: 1–162 days), especially pronounced in patients after allogeneic transplantation (P=0.002). Implementation of rigorous isolation and barrier measures, although challenged by long-term viral carriers, was effective in containment of the outbreak.

Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg-negative patients – FINITE study

BACKGROUND & AIMS There is currently no virological cure for chronic hepatitis B but successful nucleos(t)ide analogue (NA) therapy can suppress hepatitis B virus (HBV) DNA replication and, in some cases, result in HBsAg loss. Stopping NA therapy often leads to viral relapse and therefore life-long therapy is usually required. This study investigated the potential to discontinue tenofovir disoproxil fumarate (TDF) therapy in HBeAg-negative patients. METHODS Non-cirrhotic HBeAg-negative patients who had received TDF for ≥4years, with suppressed HBV DNA for ≥3.5years, were randomly assigned to either stop (n=21) or continue (n=21) TDF monotherapy. Standard laboratory tests including HBV DNA viral load, HBsAg and alanine aminotransferase (ALT) measurements, and adverse event reporting were carried out during treatment and post-treatment follow-up for 144weeks. RESULTS Of the patients who stopped TDF therapy, 62% (n=13) remained off-therapy to Week 144. Median HBsAg change in this group was -0.59log10IU/ml (range -4.49 to 0.02log10IU/ml) vs. 0.21log10IU/ml in patients who continued TDF therapy. Four patients (19%) achieved HBsAg loss. Patients stopping therapy had initial fluctuations in viral load and ALT; however, at Week 144, 43% (n=9) had either achieved HBsAg loss or had HBV DNA <2,000IU/ml. There were no unexpected safety issues identified with stopping TDF therapy. CONCLUSIONS This controlled study demonstrated the potential for HBsAg loss and/or sustained virological response in non-cirrhotic HBeAg-negative patients stopping long-term TDF therapy. Lay summary: Nucleos(t)ide analogue (NA) is usually a life-long therapy for HBV patients. This randomised controlled study investigated the discontinuation of tenofovir disoproxil fumarate (TDF) therapy in HBeAg-negative patients. Of the patients who stopped TDF therapy, 62% remained off-therapy to Week 144, of which 43% of patients had achieved either HBsAg loss or HBV DNA <2,000IU/ml. This offers a potential for long-term HBV-suppressed patients without cirrhosis to stop NA therapy under strict surveillance. Clinical trial number: NCT01320943.

Molecular Characterization of a Respiratory Syncytial Virus Outbreak in a Hematology Unit in Heidelberg, Germany

ABSTRACT In 2011 and 2012, a large outbreak of respiratory syncytial virus (RSV) infections affecting 57 laboratory-confirmed patients occurred in an adult hematology unit in Heidelberg, Germany. During the outbreak investigation, we performed molecular genotyping of RSV strains to differentiate between single versus multiple introductions of the virus into the unit. Furthermore, we assessed the time of viral shedding of consecutive samples from the patients in order to better understand the possible impact of prolonged shedding for outbreak control management. We used subtype-specific reverse transcription-PCR on nasopharyngeal and bronchoalveolar specimens for routine diagnostics and for measuring the viral shedding period. Samples of 47 RSV-infected patients involved in the outbreak were genotyped by sequence analysis and compared to samples from RSV-infected hospitalized children representing the timing of the annual RSV epidemic in the community. Molecular investigation of the virus strains from clinical samples revealed a unique cluster with identical nucleotide sequences of RSV type A (RSV A outbreak strain) for 41 patients, while 3 patients were infected with different RSV A (nonoutbreak) strains and three other patients with RSV type B. Outbreak strains were identified in samples from November 2011 until January 2012, while nonoutbreak strains were from samples coinciding with the community epidemic in February and March 2012. Median duration of viral shedding time was 24.5 days (range, 1 to 168 days) with no difference between outbreak and nonoutbreak strains (P = 0.45). Our investigation suggests a single introduction of the RSV A outbreak strain into the unit that spread among the immunocompromised patients. Prolonged viral shedding may have contributed to nosocomial transmission and should be taken into account in the infection control management of RSV outbreaks in settings with heavily immunosuppressed patients.

Serum ceruloplasmin oxidase activity is a sensitive and highly specific diagnostic marker for Wilson’s disease

BACKGROUND/AIMS A low serum ceruloplasmin concentration is considered diagnostic for Wilsons disease. We aimed to evaluate an enzymatic test for ceruloplasmin oxidase activity and to compare it with the routinely used immunological ceruloplasmin measurement. METHODS Serum ceruloplasmin was measured enzymatically with o-dianisidine dihydrochloride as substrate and immunologically. 110 Wilsons disease patients, 52 healthy controls, and 51 patients with impaired liver function not due to Wilsons disease were analyzed. Assay performance was tested by receiver operating characteristic curve analysis, McNemar test, and Spearmans rank correlation. RESULTS The greatest sum of sensitivity and specificity was seen for the enzymatic ceruloplasmin assay at a cut-off point of 55 U/L (93.6% and 100%, respectively) and for the immunologic assay at a cut-off point of 0.19 g/L (93.6% and 78.8%, respectively). For healthy controls, the differences in specificity between both assays were statistically significant (McNemar, p=0.02). When additionally including patients with impaired liver function into the control group the specificity declined to 84.5% for the enzymatic assay and to 68.9% for the immunologic assay. The correlation between the enzymatic and immunologic assay was high in healthy controls (r=0.94), but weaker in Wilsons disease patients (r=0.70) and patients with impaired liver function not due to Wilsons disease (r=0.65). CONCLUSIONS For the enzymatic assay the best cut-off point for predicting Wilsons disease was estimated to be 55 U/L. Our data suggest that the enzymatic ceruloplasmin assay is superior to the immunologic assay in diagnosing Wilsons disease and should become the preferred method.

Sensitivity and specificity of plasma disappearance rate of indocyanine green as a prognostic indicator in acute liver failure

BackgroundIn patients presenting with acute liver failure (ALF) prediction of prognosis is vital to determine the need of transplantation. Based on the evidence that plasma disappearance rate of indocyanine green (ICG-PDR) correlates with liver cell function, we evaluated the ability of ICG-PDR measured by pulse dye densitometry to predict outcome in patients with acute liver failure.MethodsProspectively markers of hepatocellular injury, synthesis and excretion, including ICG-PDR were measured daily until liver transplantation, death, discharge from intensive care unit, or up to 7 days in 25 patients with acute liver failure. Receiver operating curve (ROC) analysis was performed to assess the value of ICG-PDR to predict outcome in ALF.ResultsThe 25 patients analyzed included 18 that recovered spontaneously and 7 that underwent liver transplantation (n = 6) or died (n = 1). Causes of ALF included viral hepatitis (n = 4), toxic liver injury (n = 15), ischemic liver injury (n = 2), and cryptogenic liver failure (n = 4). Kings college criteria were fulfilled in 85.7% of patients not recovering spontaneously and in 16.7% of patients recovering spontaneously. The mean ICG-PDR measured on day 1 in patients recovering spontaneously was 12.0 ± 7.8%/min and in patients not recovering spontaneously 4.3 ± 2.0%/min (P = 0.002). By ROC analysis the sensitivity and specificity of an ICG-PDR value ≤ 6.3%/min on study day 1 were 85.7% and 88.9%, respectively, for predicting a non spontaneous outcome in ALF.ConclusionICG-PDR allows early and sensitive bedside assessment of liver dysfunction in ALF. Measurement of ICG-PDR might be helpful in predicting the outcome in acute liver failure.Trial registrationClinicaltrials.gov, NCT 00245310

Recurrent herpes simplex virus hepatitis after liver retransplantation despite acyclovir therapy

Herpes virus hepatitis (HSV) represents a form of acute necrotizing hepatitis, which most frequently develops in immunocompromised patients. Therapeutic options include high‐dose intravenous acyclovir and liver transplantation. We report the first case of recurrent HSV hepatitis after liver retransplantation, which occurred despite continuous administration of high‐dose intravenous antiviral therapy. Because explant histology pointed to initial therapy response, we thought that the reason for recurrence might be due to acyclovir resistance. Most acyclovir resistance is caused by inactivating mutations in the herpes virus thymidine kinase gene. HSV infection was detected by histology and proofed by immunohistochemistry. PCR amplification of the herpes virus thymidine kinase gene was performed on histology specimens to demonstrate the course of viral infection in liver tissue. Genotypic resistance testing of the herpes virus was performed by sequencing the thymidine kinase amplicon. In serial biopsy, HSV‐DNA sequences were only detectable when histology revealed herpes hepatitis. Whereas the primary explant exhibited the wild‐type thymidine kinase gene, a biopsy of the second graft one month after retransplantation, which showed recurrent herpes virus hepatitis, had a single base insertion within a homopolymeric cytosine stretch. This mutation causes a frame shift leading to a premature stop codon and results in a known acyclovir‐resistant herpes strain. In conclusion, we believe that testing for acyclovir‐resistant herpes strains should be considered in high‐risk patients in whom viral clearance is not achieved serologically to prevent fatal recurrence of disease by using antiviral drugs such as inhibitors of HSV‐DNA polymerase or viral helicase primase inhibitors. (Liver Transpl 2005;11:1289–1294.)

Chronic hepatitis E virus infection in a patient with leukemia and elevated transaminases: a case report

IntroductionAcute hepatitis E virus infection may cause mild, self-limiting hepatitis, either as epidemic outbreaks or sporadic cases, the latter of which have been reported in industrialized countries. Chronic infections are uncommon and have been reported in immunosuppressed patients, patients with human immunodeficiency virus infection, and patients with hematological malignancies.Case presentationA 46-year-old Caucasian man was admitted to the gastroenterology clinic with a history of increasing transaminases, persistent exhaustion, and occasional right-side abdominal pain over the course of a 6-month period. B-cell chronic lymphocytic leukemia had been diagnosed several years earlier, and the patient was treated with rituximab, pentostatin, and cyclophosphamide. A diagnostic workup ruled out autoimmune and metabolic liver disease, hepatitis A-C, and herpes virus infection. A physical examination revealed enlarged axillary lymph nodes. The results of an abdominal ultrasound examination were otherwise unremarkable. Hepatitis E virus infection was diagnosed by detection of hepatitis E virus-specific antibodies. Blood samples were positive for hepatitis E virus ribonucleic acid with high viral loads for at least 8 months, demonstrating a rare chronic hepatitis E virus infection. Sequencing and phylogenetic analysis revealed hepatitis E virus genotype 3c with homologies to other European isolates from humans and swine, indicating an autochthonous infection.ConclusionsUsually, hepatitis E virus infection appears as an acute infection; rare chronic infections have been reported for transplant patients, patients with human immunodeficiency virus, and patients with hematological malignancies. The chronic nature of hepatitis E infection in our patient was most likely induced by the immunosuppressive B-cell chronic lymphocytic leukemia treatment. The differential diagnosis in patients with unexplained hepatitis should include hepatitis E virus infection, and appropriate laboratory analyses should be considered.

Noninvasive indocyanine green plasma disappearance rate predicts early complications, graft failure or death after liver transplantation

BACKGROUND Early detection of graft malfunction or postoperative complications is essential to save patients and organs after orthotopic liver transplantation (OLT). Predictive tests for graft dysfunction are needed to enable earlier implementation of organ-saving interventions following transplantation. This study was undertaken to assess the value of indocyanine green plasma disappearance rates (ICG-PDRs) for predicting postoperative complications, graft dysfunction, and patient survival following OLT. METHODS Eighty-six patients undergoing OLT were included in this single-centre trial. ICG-PDR was assessed daily for the first 7 days following OLT. Endpoints were graft loss or death within 30 days and postoperative complications, graft loss, or death within 30 days. RESULTS Postoperative complications of 31 patients included deaths (12 patients) or graft losses. ICG-PDR was significantly different in patients whose endpoints were graft loss or death beginning from day 3 and in those whose endpoints were graft-loss, death, or postoperative complications beginning from day 4 after OLT. For day 7 measurements, receiver operating characteristic curve analysis revealed an ICG-PDR cut-off for predicting death or graft loss of 9.6% per min (a sensitivity of 75.0%, a specificity of 72.6%, positive predictive value 0.35, negative predictive value 0.94). For prediction of graft loss, death, or postoperative complications, the ICG-PDR cut-off was 12.3% per min (a sensitivity of 68.9%, a specificity of 66.7%, positive predictive value 0.57, negative predictive value 0.77). CONCLUSIONS ICG-PDR measurements on postoperative day 7 are predictive of early patient outcomes following OLT. The added value over that of routinely determined laboratory parameters is low.