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Background Globally, an estimated 20 million Hepatitis E infections occur every year. The course of viremia and antibody response has been investigated in patients with symptomatic hepatitis E. However, the majority of HEV infections in industrialized countries take a subclinical course. Objectives To investigate the course of HEV viremia and epitope specific anti-HEV IgM/IgG response in asymptomatic blood donors in order to understand the immune response and viral clearance in asymptomatic blood donors with HEV infections. Methods In this study 27 HEV viremic donors were identified by HEV-PCR during routine screening of blood donors and the course of anti-HEV IgM/IgG and HEV-RNA was retrospectively studied using RT-PCR and a commercial immunoblot (Mikrogen®) allowing classification of the antibody response according to HEV epitopes. Results At time of donation, serological testing failed to identify viremic donors as 70.4% had no detectable antibody response. Anti-HEV IgM could be detected in 22.2% of viremic donors while anti-HEV IgG could be found in 7.4%. At least three donors experienced prolonged viremia beyond 100 days. Spontaneous HEV-RNA clearance within a median time span of 57 days was observed in all 27 donors. In all donors anti-HEV IgG specific for the immunogenic viral epitope O2C could be detected in close temporal association with viral clearance. Conclusion Serological testing is inappropriate for identifying HEV-viremic blood donors. Acute HEV infection in asymptomatic blood donors can persist for more than 100 days. HEV-RNA clearance coincided with the appearance of anti-HEV IgM/IgG confirming the importance of a B-cell mediated response in clearing acute infections. Anti-HEV IgM and IgG specific for the epitope O2C are associated with the clearance of HEV-viremia.

Background Hepatitis C virus (HCV) infection is associated with a particularly poor outcome after liver transplantation. In December 2014, sofosbuvir/ledipasvir (SOF/LDV) fixed-dose combination (FDC) was approved for HCV genotype 1 and 4 in Europe. In orthotopic liver transplantation (OLT) recipients, the interferon-free treatment of HCV re-infection with novel direct-acting antivirals has been demonstrated to be safe and effective in clinical trials, but real-world data are missing. The aim of this study was to investigate the safety and efficacy of SOF/LDV FDC in OLT recipients in the real-life setting. Methods All consecutive OLT patients started on SOF/LDV FDC for 12 or 24 weeks at the University Medical Center Hamburg-Eppendorf and Medical School Hannover between October 2014 and August 2015 were retrospectively analyzed (n = 30). The primary efficacy endpoint was sustained virological response (SVR), i.e., absence of viremia 12 weeks after end of treatment (SVR 12). Liver function tests, creatinine, blood count, and HCV RNA (by polymerase chain reaction assay) were determined at each visit. Results SVR was achieved in 29/30 patients (96.67%) treated with SOF/LDV ± ribavirin (RBV) for 12 (n = 4) or 24 weeks (n = 25). Twenty-five patients (86.2%) received RBV. However, in 15 of the 25 patients, RBV administration had to be discontinued because of severe anemia (57.7%). One RBV-treated patient died of a myocardial infarction during antiviral therapy; this event was most likely not directly related to SOF/LDV. Aside from RBV-associated anemia, no severe side effects of the antiviral regimen were observed. Conclusion Antiviral treatment with SOF/LDV is highly effective, safe, and well tolerated in OLT recipients. The addition of RBV often results in severe anemia, requiring dose reduction or discontinuation.

Background & Aims Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. Methods UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. Results Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. Conclusion We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.

BACKGROUND Hepatitis C virus (HCV) infection is associated with reduced graft survival in orthotopic liver transplant recipients. Treatment with the new direct-acting antivirals (DAAs) is safe and efficient, but no reliable predictive factors for sustained virologic response (SVR) have been identified so far. The HCV core antigen assay (HCV-core-Ag) is a new, inexpensive, and efficient method to detect viral antigens, but the value of this technique to predict treatment response in orthotopic liver transplantation (OLT) patients is still unclear. METHODS All OLT patients who were treated with a sofosbuvir-based antiviral regimen at our center between March 2014 and August 2014 were included in the analysis (n = 20). HCV-core-Ag and HCV RNA (polymerase chain reaction [PCR]) were determined at each visit. Primary endpoints of this study were SVR at 4 or 12 weeks after end of treatment (SVR 4 and SVR 12). RESULTS HCV-core-Ag tested negative after a median of 2 weeks (range 1-16 weeks) while PCR tests became negative after a median of 4 weeks (range 2-12 weeks). Time until PCR negativity and until HCV-core-Ag negativity showed a good correlation (R = 0.711, P < 0.001, Fig. ). Seventeen of 20 patients (85%) achieved SVR 12. SVR 12 was associated with a short time interval between treatment start and HCV PCR negativity (P = 0.005) or HCV-core-Ag negativity (P = 0.003, Mann-Whitney test). No severe side effects were observed. CONCLUSIONS DAA treatment is safe and well tolerated in OLT. The time points of HCV-core-Ag loss and PCR negativity were predictors of SVR 12.

Background: Hepatitis A and hepatitis E are not limited to tropical countries but are also present in industrialized countries. Both infections share similar clinical features. There is no comparative study evaluating the clinical parameters of autochthonous and imported hepatitis A virus and hepatitis E virus infections. Aims: The aim of this study was to determine differences between autochthonous and imported hepatitis A virus (HAV) and hepatitis E virus (HEV) infections. Methods: Medical charts of all patients at our center with acute HAV and HEV infections were analyzed retrospectively (n = 50, study period 01/2009 – 08/2013). Results: Peak bilirubin (median 8.6 vs. 4.4 mg/dL, p = 0.008) and ALT levels (median 2998 vs. 1666 IU/mL, p = 0.04) were higher in patients with hepatitis A compared to hepatitis E. In comparison to autochthones hepatitis E cases, patients with imported infections had significantly higher peak values for AST, ALT, bilirubin and INR (p = 0.009, p = 0.002, p = 0.04 and p = 0.049, respectively). In HAV infection, AST levels tended to be higher in imported infections (p = 0.08). Conclusions: (i) It is not possible to differentiate certainly between acute HAV and HEV infections by clinical or biochemical parameters, however, HAV infections might be associated with more cholestasis and higher ALT values. (ii) Imported HEV infections are associated with higher transaminases, INR and bilirubin levels compared to autochthonous cases and (iii) imported HAV infections tend to be associated with higher transaminases in comparison to autochthonous cases.

Several test systems are available for the diagnosis of infection with the hepatitis B virus (HBV). In most cases, reliable results lead to the detection of acute infection or chronic HBV infection or immunity due to past infection or active immunization. In some cases, however, results may be difficult to interpret or even misleading and special assays are necessary to answer more specific questions. During acute or chronic HBV infection, not only the qualitative detection of HBsAg is necessary, but also the quantitative determination may allow for the diagnosis of activity of the disease. In most cases, HBeAg closely correlates to viraemia and infectivity, but mutations within the pre-core region of the HBV genome may lead to a loss of HBeAg and to seroconversion to anti-HBe while the virus concentration in serum is high and patients are severely ill. Anti-HBc assays often give false positive test results which can be ruled out by re-testing a serum dilution. IgM antibodies against HBcAg are not only found during acute infection but may become positive again during reactivation in chronic virus carriers. Different genotypes of the virus are prevalent in different parts of the world and multiple infections are possible; these may lead to the simultaneous detection of HBsAg and anti-HBs. Viral DNA can be detected by several methods with different lower detection limits. Screening of blood donors to rule out HBsAg-negative virus carriers is done by polymerase chain reaction, since this method has the highest sensitivity. Infectivity of HBsAg-positive carriers may also be determined by other assays such as branched DNA or molecular hybridization. Patients on treatment with nucleoside analogs may show an increase in DNA concentration after an initial decrease or even loss. This may be due to mutation within the YMDD-motif of the DNA polymerase which can be detected by sequence analysis. In this overview, hints will be given on how to evaluate positive results in the different assays. In addition, it gives advice on how to establish a suitable diagnosis for a variety of clinical questions. Several suspicious constellations are discussed as well as the possibilities to clarify them.

The diagnosis of HCV infection is often hampered by false positive reactivities in antibody screening assays. Therefore, confirmatory assays are necessary. Since most unspecific reactivities are only slightly above the cut-off value, the intensity of the reactivity should always be considered. Particularly in groups with low HCV risk such as blood donors, positive reactivities obtained by one test system can often be ruled out by re-testing with another format (e.g. an enzyme immunoassay instead of a microparticle enzyme immunoassay). Most confirmatory assays are based on a recombinant immunoblot assay (RIBA); these tests should always be done in patients diagnosed as HCV positive for the first time. Additionally, in many cases, tests for HCV RNA are necessary. Based on these results, an evaluation of the risk of transmission to household contacts is possible and in patients receiving antiviral treatment, the success can be monitored. A negative PCR result in patients showing high positive antibody reactivities does not necessarily represent loss of the virus since even in chronic carriers, viral replication can be intermittently very low. Immuno-compromised patients often show prolonged seroconversion so that antibody screening assays remain negative for a long time while the patients have high level viraemia. Therefore, we recommend regular RT-PCR in populations at risk such as patients on chronic dialysis. To evaluate patients for antiviral treatment, the determination of the genotype is necessary. The HCV genotype also has an influence on the course of liver disease in chronic carriers. Possible transmission of HCV, e.g. by blood products, can be elucidated by sequence analysis of the high variance region (HVR) of the virus.

A new virus named hepatitis G virus (HGV) has been detected recently. Until now, no assays for the detection of antibodies against different HGV proteins have been commercially available. Therefore, a strip immunoblot assay has been established to investigate seroreactivity against recombinant structural (core) and nonstructural proteins (NS3 and NS4) of HGV produced in Escherichia coli. Seropositivity for HGV was evaluated and concordanced with HGV polymerase chain reaction (PCR) results in 709 subjects. These individuals were classified into a nonrisk or a risk group, on the basis of infection with human immunodeficiency virus (HIV) or hepatitis C virus (HCV) or frequent parenteral exposure, including hemophilia, intravenous drug addiction, receipt of blood transfusion, or hemodialysis. The nonrisk group consisted of 257 healthy blood donors with normal alanine transaminase (ALT) levels (ALT30 U/L) and 154 patients with suspected non-A-E hepatitis (ALT45 U/L). In the group of healthy blood donors, 1.9% (5 of 257) had detectable HGV viremia and 15.9% (41 of 257) showed antibody response to HGV. In the collective of patients with suspected non-A-E hepatitis, results from 1.9% of patients (3 of 154) were positive by HGV PCR, and 15.6% of patients (24 of 154) showed seropositivity against the recombinant HGV proteins. In six groups of patients (n = 298) with different risk factors, the prevalence of both HGV viremia (V) and serological reactivity (SR) was higher compared with that of the nonrisk group: V, 6.80%-35.2%; serological reactivity (SR), 25.4%-52.9%. The following conclusions can be derived from our data. HGV infection is widespread in the general population. The prevalence of antibodies against HGV or detectable HGV viremia is higher in patients with risk factors for parenteral viral transmission than in those without risk factors. The majority of HGV infections (70.2%) is self-limiting and not persistent in our collective of patients. We found no correlation between HGV viremia and clinical or biochemical signs of hepatitis in individuals without risk factors for acquiring parenterally transmitted agents.

The distribution of hepatitis C virus (HCV) genotypes was determined in isolates of 447 chronically HCV-infected German patients by nucleotide sequencing. Of these, 206 (46.1%) were infected with the subtype 1a, 215 (48.1%) with subtype 1b, 2 (0.4%) with subtype 1c, 9 (2.0%) with subtype 3a, and 15 (3.4%) with subtype 4a. Subtype 1a was predominant in those40 years old (62.6%) and was associated with the risk factor of intravenous drug addiction and with shorter duration of disease. Conversely, subtype 1b was more frequent in patients50 years old (84.7%; P.001) and was associated with the risk factor of blood transfusions and with longer duration of disease. These data suggest that a shift from subtype 1b to subtype 1a occurred in the population studied. An increase in HCV infection with subtype 1a and a diminution of subtype 1b in the future can be expected.

Objective: Patients on maintenance hemodialysis are known to have an elevated risk of acquiring hepatitis C virus (HCV) infection. Therefore, a reliable diagnosis of HCV infection is essential in order to prevent the spread of the disease in dialysis units. However, whether PCR examination is dispensable in hemodialysis patients has been debated. Methods: From 1995 to 2002, serum samples from all hemodialysis patients at our hospital (n = 1,774) were screened by serological assays and by polymerase chain reaction (PCR). Results: In 25 of these patients acute HCV infection was observed and in 11 patients HCV seroconversion was delayed for 3–16 months. During this time the infection was exclusively detectable by PCR. Conclusion: Despite the growing demand for cost-effectiveness in the health system, HCV PCR examination must remain an essential part of the routine screening in hemodialysis patients.

To evaluate the risk of hepatitis C virus (HCV) transmission via breast milk, we collected 76 samples of breast milk from 73 chronically HCV-infected women and serum samples from their 76 perinatally HCV-exposed children. Enzyme immunoassay and strip immunoblot assay were used for detection of antibodies to HCV, and reverse transcriptase-polymerase chain reaction analysis was used for detection of HCV RNA. None of the 76 samples of breast milk contained HCV RNA, whereas 37 (59.7%) of 62 mothers tested for HCV RNA had HCV viremia. Only 1 of the 76 breast-fed infants had evidence of HCV infection. Because HCV infection in this child was detected 1 month after birth, it seems unlikely that it was transmitted by breast-feeding. These results indicate that HCV infection in pregnant women should not be a contra-indication for breast-feeding.

Spinal cord chimeras were produced by replacing a small fragment of neural tube of a 2-day-old White Leghorn chicken embryo with a similar fragment from a Japanese quail embryo. The embryo mortality w

Needle sharing among drug addicts leads to the transmission of infectious diseases such as hepatitis B and AIDS. After development of a test system based on gene technology against the hepatitis C virus (HCV), drug addicts have been regarded as an important reservoir for hepatitis C. In our study 113 (40.1%) out of 282 addicts who died from drug abuse in Hamburg between 1988 and 1990 had antibodies against HCV (anti-HCV). The prevalence of anti-HCV differed in various age groups; the highest prevalence was found in addicts aged 30-34 years. Co-infections with hepatitis B and hepatitis C virus were found in 57 drug addicts (59.4%) out of 96 deceased with antibodies to hepatitis B (anti-HBc), whereas only 8 out of 23 HIV-infected were anti-HCV positive (34.8%).

In 1989 blood samples from 2581 fatalities investigated at the Institute of Forensic Medicine in Hamburg were screened for HIV-1-antibodies. Sera from 13 corpses were confirmed positive for HIV-1 (prevalence rate approx 0.5 %).-Viable HIV was found in blood cultures of 4 cadavers stored under non-refrigerated conditions up to 36 hours after death.

Bovine hepacivirus (BovHepV) is a recently added member to the growing genus Hepacivirus within the family Flaviviridae. Animal hepaciviruses are rarely characterized so far. Apart from norway rat hepacivirus which represents a promising HCV surrogate model, only equine hepaciviruses have been studied to some extent. BovHepV has been initially identified in bovine samples and was shown to establish persistent infections in cattle. However, consequences of those chronic infections, humoral immune response and the possibility of an extended host spectrum have not been explored so far. Therefore, we here investigated (a) the presence of anti-NS3-antibodies and viral RNA in cattle herds in Germany, (b) the course of infection in cattle, and (c) the host tropism including zoonotic potential of bovine hepaciviruses. Our results show that 19.9% of investigated bovine serum samples had antibodies against BovHepV. In 8.2% of investigated samples, viral RNA was detected. Subsequent genetic analysis revealed a novel genetic cluster of BovHepV variants. For 25 selected cattle in a BovHepV positive herd the presence of viral genomic RNA was monitored over one year in two to three months intervals by RT-PCR in order to discriminate acute versus persistent infection. In persistently infected animals, no serum antibodies were detected. Biochemical analyses could not establish a link between BovHepV infection and liver injury. Apart from a single sample of a pig providing a positive reaction in the antibody test, neither BovHepV-specific antibodies nor viral RNA were detected in porcine, equine or human samples implying a strict host specificity of BovHepV.