Combination of vial culture and broad-range PCR for the diagnosis of spontaneous bacterial peritonitis: experience in a Greek tertiary care hospital.

Spontaneous bacterial peritonitis (SBP) is often difficult to diagnose because bacteria in ascites cannot be detected accurately by conventional culture. In this study, we evaluated the use of broad-range 16S rRNA PCR, applied either directly to a total of 32 ascitic fluids (AFs) or to the AF vial cultures, after a long incubation of 14 days; the results were compared with those of AF vial cultures. Escherichia coli was isolated in four of 32 AF vial cultures (12.5%). The application of 16S rRNA PCR directly to AF detected only one of the four positive samples (sensitivity 25%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value (NPV) 90.32%). However, the application of 16S rRNA PCR to AF vial cultures after 14 days of incubation correctly identified all the positive samples, including one more that was positive for Brucella mellitensis (sensitivity 100%, specificity 80%, PPV 80%, NPV 100%). The elongation of the incubation period of the AF vial cultures, combined with the use of 16S rRNA in negative vials, increases the possibility of identifying the causative agents of SBP and could be applied in the clinical laboratory.

Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification.

OBJECTIVES: Sepsis-3 definitions generated controversies regarding their general applicability. The Sepsis-3 Task Force outlined the need for validation with emphasis on the quick Sequential Organ Failure Assessment (qSOFA) score. This was done in a prospective cohort from a different healthcare setting. METHODS: Patients with infections and at least two signs of systemic inflammatory response syndrome (SIRS) were analysed. Sepsis was defined as total SOFA ≥2 outside the intensive care unit (ICU) or as an increase of ICU admission SOFA ≥2. The primary endpoints were the sensitivity of qSOFA outside the ICU and sepsis definition both outside and within the ICU to predict mortality. RESULTS: In all, 3346 infections outside the ICU and 1058 infections in the ICU were analysed. Outside the ICU, respective mortality with ≥2 SIRS and qSOFA ≥2 was 25.3% and 41.2% (p <0.0001); the sensitivities of qSOFA and of sepsis definition to predict death were 60.8% and 87.2%, respectively. This was 95.9% for sepsis definition in the ICU. The sensitivity of qSOFA and of ≥3 SIRS criteria for organ dysfunction outside the ICU was 48.7% and 72.5%, respectively (p <0.0001). Misclassification outside the ICU with the 1991 and Sepsis-3 definitions into stages of lower severity was 21.4% and 3.7%, respectively (p <0.0001) and 14.9% and 3.7%, respectively, in the ICU (p <0.0001). Adding arterial pH ≤7.30 to qSOFA increased sensitivity for prediction of death to 67.5% (p 0.004). CONCLUSIONS: Our analysis positively validated the use of SOFA score to predict unfavourable outcome and to limit misclassification into lower severity. However, qSOFA score had inadequate sensitivity for early risk assessment.

Expert clinical management of autoimmune hepatitis in the real world.

BACKGROUND: High-quality data on the management of autoimmune hepatitis (AIH) are scarce. Despite published guidelines, management of AIH is still expert based rather than evidence based. AIM: To survey expert hepatologists, asking each to describe their practices in the management of patients with AIH. METHODS: A survey questionnaire was distributed to members of the International AIH Group. The questionnaire consisted of four clinical scenarios on different presentations of AIH. RESULTS: Sixty surveys were sent, out of which 37 were returned. None reported budesonide as a first line induction agent for the acute presentation of AIH. Five (14%) participants reported using thiopurine S-methyltransferase measurements before commencement of thiopurine maintenance therapy. Thirteen (35%) routinely perform liver biopsy at 2 years of biochemical remission. If histological inflammatory activity is absent, four (11%) participants reduced azathioprine, whereas 10 (27%) attempted withdrawal altogether. Regarding the management of difficult-to-treat patients, mycophenolate mofetil is the most widely used second-line agent (n = ~450 in 28 centres), whereas tacrolimus (n = ~115 in 21 centres) and ciclosporin (n = ~112 in 18 centres) are less often reported. One centre reported considerable experience with infliximab, while rescue therapy with rituximab has been tried in seven centres. CONCLUSIONS: There is a wide variation in the management of patients with autoimmune hepatitis even among the most expert in the field. Although good quality evidence is lacking, there is considerable experience with second-line therapies. Future prospective studies should address these issues, so that we move from an expert- to an evidence- and personalised-based care in autoimmune hepatitis.

A real-world study focused on the long-term efficacy of mycophenolate mofetil as first-line treatment of autoimmune hepatitis.

BACKGROUND: Front-line therapy with mycophenolate mofetil (MMF) in autoimmune hepatitis (AIH) has shown high on-treatment remission rates. AIM: To study prospectively in a real-world fashion the long-term outcome of a large group of consecutive treatment-naïve AIH patients. METHODS: Between 2000 and 2014, 158 patients were recruited but only 131 were eligible for treatment (109 MMF/prednisolone; 22 prednisolone ± azathioprine). Long-term data on outcome after drug withdrawal were evaluated. Patients stopped treatment after having achieved complete response (normal transaminases and IgG) for at least the last 2 years. RESULTS: At diagnosis, 31.6% of patients had cirrhosis and 72.8% insidious presentation. A total of 102 of 109 (93.6%) responded initially to MMF within 2 (1-18) months. A total of 78 of 109 (71.6%) had complete response on treatment and 61 of 78 (78.2%) maintained remission off prednisolone. MMF-treated patients had increased probability of complete response compared to those receiving azathioprine (P = 0.03). Independent predictors of complete response were lower ALT at 6 months (P = 0.001) and acute presentation (P = 0.03). So far, treatment withdrawal was feasible in 40/109 patients and 30 (75%) are still in remission after 24 (2-129) months. Remission maintenance was associated with longer MMF treatment (P = 0.005), higher baseline ALT (P < 0.02), lower IgG on 6 months (P = 0.004) and histological improvement. CONCLUSIONS: Mycophenolate mofetil proved to be an efficient first-line treatment for AIH, achieving so far the highest rates of remission maintenance off treatment (75%) ever published for at least a median of 2 years, although the remission criteria used were strict. However, the risk of potential bias and overestimation of intervention benefits from MMF cannot be completely excluded as this is a real world and not a randomised controlled trial.

Addressing barriers to the prevention, diagnosis and treatment of hepatitis B and C in the face of persisting fiscal constraints in Europe: report from a high level conference.

In the WHO-EURO region, around 28 million people are currently living with chronic viral hepatitis, and 120,000 people die every year because of it. Lack of awareness and understanding combined with the social stigma and discrimination exacerbate barriers related to access to prevention, diagnosis and treatment services for those most in need. In addition, the persisting economic crisis has impacted on public health spending, thus posing challenges on the sustainable investment in promotion, primary and secondary prevention, diagnosis and treatment of viral hepatitis across European countries. The Hepatitis B and C Public Policy Association in cooperation with the Hellenic Center for Disease Prevention and Control together with 10 partner organizations discussed at the Athens High Level Meeting held in June 2014 recent policy developments, persisting and emerging challenges related to the prevention and management of viral hepatitis and the need for a de minimis framework of urgent priorities for action, reflected in a Call to Action (Appendix S1). The discussion confirmed that persisting barriers do not allow the full realisation of the public health potential of diagnosing and preventing hepatitis B and C, treating hepatitis B and curing hepatitis C. Such barriers are related to (a) lack of evidence-based knowledge of hepatitis B and C, (b) limited access to prevention, diagnosis and treatment services with poor patient pathways, (c) declining resources and (d) the presence of social stigma and discrimination. The discussion also confirmed the emerging importance of fiscal constraints on the ability of policymakers to adequately address viral hepatitis challenges, particularly through increasing coverage of newer therapies. In Europe, it is critical that public policy bodies urgently agree on a conceptual framework for addressing the existing and emerging barriers to managing viral hepatitis. Such a framework would ensure all health systems share a common understanding of definitions and indicators and look to integrate their responses to manage policy spillovers in the most cost-effective manner, while forging wide partnerships to sustainably and successfully address viral hepatitis.

Association of antiviral therapy with reduced disease progression in chronic Hepatitis B patients: Results from a nation-wide cohort study.

BACKGROUND AND AIMS: Although effective treatment in terms of inducing virological and biochemical response for chronic hepatitis B (CHB) is available, its effect on the clinical course of the disease has not yet been accurately estimated. Objective of this study was to evaluate the effect of antiviral therapy and its type [interferon +/- nucleos(t)ide analogs (NAs) vs. NAs] on the occurrence of a clinical event (liver decompensation, liver transplant, hepatocellular carcinoma and death from a liver-related cause) in CHB patients. METHODS: The study population was derived from the HEPNET-Greece, a nationwide cohort study aimed to evaluate the current epidemiological course of viral hepatitis. To account for time-dependent confounding, Cox marginal structural models were used to analyze data. RESULTS: Thirty out of 2,125 eligible patients experienced a clinical event during their follow-up. When comparing treated to untreated individuals, the hazard ratio (HR) for a clinical event was 0.39 (95% CI: 0.16-0.98; p =0.044) in the whole sample, whereas there were indications of a more intense effect in the subgroup of patients with cirrhosis at presentation (HR =0.16, 95% CI: 0.02-1.21; p =0.075). The effect of Interferon initiated treatment was not significantly different of that of NAs. There was some evidence, albeit not statistically significant, of a protective treatment effect on hepatocellular carcinoma development (HCC). CONCLUSIONS: Data from observational studies can provide useful inference, provided they are analyzed appropriately. The current study has shown that the available treatment options for CHB offer a significant clinical benefit to CHB infected individuals. Hippokratia 2016, 20(3): 214-221.

Rapid dissemination of colistin and carbapenem resistant Acinetobacter baumannii in Central Greece: mechanisms of resistance, molecular identification and epidemiological data.

BACKGROUND: Colistin-resistant/carbapenem-resistant Acinetobacter baumannii is a significant challenge for antibiotic treatment and infection control policies. Since 2012, in Central Greece an increase of colistin/pan- resistant A. baumannii has occurred, indicating the need for further analysis. METHODS: A total of 86 colistin-resistant/carbapenem-resistant out of 1228 A. baumannii clinical isolates, consecutively collected between 2012 and 2014 in a tertiary Greek hospital of Central Greece, as well as one environmental isolate from surveillance cultures were studied. Molecular typing and mechanisms of resistance to colistin and to carbapenems were assessed, whereas, epidemiological and clinical data of the patients were reviewed. RESULTS: During the study period, the rate of colistin resistance gradually increased and reached 21.1 % in 2014. All colistin-resistant/carbapenem-resistant A. baumannii belonged to 3LST ST101 clone that corresponds to the international clonal lineage II. Carbapenem resistance was associated with the presence of bla oxa-23-like, while resistance to colistin probably correlated with G54E and R109H amino acid substitutions in PmrA and PmrC, respectively. CONCLUSIONS: Epidemiological data of the patients indicated that the first detection of colistin-resistant/carbapenem-resistant ST101 clone in the University Hospital of Larissa (UHL) was associated with a patient who previously had received colistin, while, the movement of the infected patients into the hospital probably resulted to its spread.

Hepatocellular carcinoma risk in HBeAg-negative chronic hepatitis B patients with or without cirrhosis treated with entecavir: HepNet.Greece cohort.

Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients treated with lamivudine. Whether HCC rates are comparable in patients treated with the current first-line antivirals remains uncertain. We estimated the incidence and evaluated predictors of HCC in a large nationwide prospective cohort (HepNet.Greece) of HBeAg-negative CHB patients treated with entecavir. HBeAg-negative CHB patients from the same cohort who were initially treated with lamivudine were used as controls. We included 321 patients treated with entecavir for a median of 40 months and 818 patients treated initially with lamivudine for a median of 60 months. In the entecavir group, HCC developed in 4 of 321 (1.2%) patients at a median of 1.5 (range: 1.0-4.5) years, while the cumulative HCC incidence was significantly higher in cirrhotics than noncirrhotics (1, 3, 5 years: 0%, 3%, 9% vs 1%, 1%, 1%; P = 0.024) and in older patients (P = 0.026). Entecavir compared with lamivudine group patients had lower HCC incidence (1, 3, 5 years: 0.3%, 1.2%, 2.8% vs 0.7%, 3.8%, 5.6%; P = 0.024). However, in multivariable Cox regression analysis, the HCC risk was independently associated with older age (P < 0.001), male gender (P = 0.011) and cirrhosis (P = 0.025), but not with the initial agent. In conclusion, our large nationwide study indicates that the HCC risk remains increased in entecavir-treated HBeAg-negative CHB patients with cirrhosis, particularly of older age, at least for the first 5 years. The HCC risk does not seem to be significantly reduced with entecavir compared with antiviral therapy starting with lamivudine.

Genotype 4 HCV infection is difficult to cure with pegylated interferon and ribavirin. Results from a Greek Nationwide Cohort Study.

BACKGROUND AND AIM: Patients with genotype 4 (G4) chronic hepatitis C (CHC) are considered a difficult to treat population, although current data on G4 treatment responsiveness and duration are controversial. Greece represents a country with an intermediate prevalence of G4 infections, offering an opportunity to compare treatment outcomes by genotype and to identify potential prognostic factors for sustained virologic response (SVR). METHODS: All CHC patients from the HepNet.Greece, an ongoing nationwide cohort study on viral hepatitis, with known hepatitis C virus (HCV) genotype who received treatment with Peg-IFNa and ribavirin were analyzed. RESULTS: From 4443 patients, 951 (61.7% males, 78.4% Greeks, median age 40.6 years, 10% cirrhosis) fulfilled the inclusion criteria. G4 was found in 125 (13.1%) patients. Genotype distribution was not significantly different between Greeks and immigrants. Patients with G4 had similar odds of SVR compared to G1 but significantly lower compared to G2/G3. Age, treatment discontinuation, presence of cirrhosis and previous history of HCV-treatment were associated with lower probabilities of SVR. Ethnicity did not affect SVR for all genotypes while response to treatment was similar between Greek and Egyptian patients groups (35.7% vs 40.9%, p=0.660%) with G4 infection. The relation between SVR and genotype did not substantially change after adjustment for age, gender, cirrhosis, treatment interruption and history of HCV-treatment. CONCLUSIONS: The findings of this large cohort of CHC patients with a well balanced genotype distribution further supports the idea of considering G4 as a difficult to treat genotype. Further investigation is needed to identify genotype specific prognostic factors.

Cluster of Fusarium verticillioides bloodstream infections among immunocompetent patients in an internal medicine department after reconstruction works in Larissa, Central Greece.

BACKGROUND: Fusarium spp. can cause disseminated infections, particularly in immunocompromised patients. Fusarium verticillioides is a human pathogen, and sporadic cases of fusariosis have been reported. AIM: To report a nosocomial cluster of F. verticillioides bloodstream infections among seven immunocompetent inpatients following reconstruction works. METHODS: Identification was performed using macroscopic and microscopic morphology, and molecular assays (sequencing the nuclear ribosomal internal transcribed spacer region and translation elongation factor-1α gene). Susceptibility testing was performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute. Environmental surveillance specimens were taken and cultured on Sabouraud dextrose agar plates. FINDINGS: In total, 16 blood cultures obtained from the seven patients were positive for F. verticillioides. All surveillance cultures were negative. CONCLUSIONS: In order to prevent fungaemia, it is important to implement effective infection control measures, before, during and after demolition and construction activities in healthcare settings.

Review article: autoimmune hepatitis -- current management and challenges.

BACKGROUND: Autoimmune hepatitis (AIH) is a disease of unknown aetiology characterised by interface hepatitis, hypergammaglobulinaemia, circulating autoantibodies and a favourable response to immunosuppression. AIM: To review recent advancements in understanding aetiopathogenesis, clinical, serological and histological features, diagnostic criteria and treatment strategies of AIH. METHODS: Published studies on AIH extracted mainly from PubMed during the last 15 years. RESULTS: Autoimmune hepatitis has a global distribution affecting any age, both sexes and all ethnic groups. Clinical manifestations are variable ranging from no symptoms to severe acute hepatitis and only seldom to fulminant hepatic failure. Autoimmune attack is perpetuated, possibly via molecular mimicry mechanisms, and favoured by the impaired control of regulatory T-cells. A typical laboratory finding is hypergammaglobulinaemia with selective elevation of IgG, although in 15-25% of patients - particularly children, elderly and acute cases - IgG levels are normal. Liver histology and autoantibodies, although not pathognomonic, still remain the hallmark for diagnosis. Immunosuppressive treatment is mandatory and life-saving; however, to meet strict response criteria, the conventional therapy with prednisolone with or without azathioprine is far from ideal. CONCLUSIONS: Autoimmune hepatitis remains a major diagnostic and therapeutic challenge. The clinician, the hepato-pathologist and the laboratory personnel need to become more familiar with different expressions of the disease, interpretation of liver histology and autoimmune serology. According to the strict definition of treatment response issued by the 2010 AASLD guidelines, many patients are nonresponders to conventional treatment. Newer immunosuppressive agents targeting pathogenetic mechanisms can improve patient management, which needs to be tailored on a case-by-case basis.

Renal function during treatment with adefovir plus peginterferon alfa-2a vs either drug alone in hepatitis B/D co-infection.

Long-term safety of treatment with hepatitis B virus (HBV) polymerase inhibitors is a concern. Adefovir dipivoxil (ADV) therapy has previously been associated with impairment of renal function. Limited data are available on the safety of combination therapy with nucleos(t)ide analogues and interferon alfa (IFNα). The aim of this analysis was to assess the renal function during combination therapy with peginterferon alfa-2a (PegIFNα-2a) plus ADV vs either drug alone in patients with hepatitis B/D co-infection. We performed a retrospective analysis of renal function data of patients treated in the Hep-Net/International Delta Hepatitis Intervention Trial 1(HIDIT-1-trial), a European multicenter study to investigate the efficacy of 48 weeks of therapy with PegIFNα-2a+ADV vs either drug alone in 90 patients with chronic hepatitis B/D co-infection. Glomerular filtration rates (GFR) were calculated by Cockcroft-Gault (CG), abbreviated Modification of Diet in Renal Disease (MDRD) study and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. After 48 weeks of therapy GFR values were significantly lower in patients receiving adefovir-containing treatment vs PegIFNα-2a alone [mean difference 16.1 mL/min (CG) and 10.2 mL/min (MDRD), respectively, P < 0.05] while no differences were observed between patients receiving adefovir alone vs combination treatment. Twenty-four weeks after treatment GFR values did not differ between treatment arms. A decrease in GFR ≥ 20% was observed more often in patients during adefovir-containing treatment vs PegIFNα-2a alone (P < 0.05) which was confirmed by Kaplan-Meier analysis. Adefovir-containing but not PegIFNα-2a treatment was associated with a decrease in GFR values in about one-fifth of patients. Combination treatment of PegIFNα-2a+ADV in chronic hepatitis B/D co-infection did not lead to any further impairment of kidney function.

Anti-α actinin antibodies as new predictors of response to treatment in autoimmune hepatitis type 1.

BACKGROUND: We reported that combined presence of autoantibodies (Abs) against filamentous-actin (AFA) and α-actinin are specific for autoimmune hepatitis type 1 (AIH-1) diagnosis. AIM: To explore our data and assess whether anti-α-actinin and AFA Abs could be used as indicators of response to treatment and predictors of AIH-1 flares in a large cohort of AIH-1 patients. METHODS: Seven hundred and sixty-four serial serum samples of 86 consecutive AIH-1 patients, 509 pathological and 110 normal controls were tested for the presence of anti-α-actinin and AFA Abs by an in-house IgG-specific ELISA and a standardised commercially available ELISA respectively. Patients sera were divided into baseline group (active disease before treatment initiation, n = 86) and then according to treatment response into group A-responders (n = 40 patients), group B-relapsers/incomplete responders (n = 37 patients) and group C-not-treated (n = 9 patients). RESULTS: Anti-α-actinin and AFA levels were significantly higher at baseline. Double reactivity against α-actinin and AFA was associated with disease activity (OR 4.9; 95% CI: 2.7-9). Anti-α-actinin optical densities (ODs) before treatment decreased significantly at first remission (P < 0.05). Treatment response was associated with anti-α-actinin Abs negativity before treatment (OR 3.4; 95% CI: 1.3-8.9) and absence of double positivity for anti-α-actinin and AFA Abs before treatment (OR 3.8; 95% CI: 1.4-10.4). Responders had lower baseline levels of anti-α-actinin than relapsers and/or incomplete responders (P = 0.002). Binary logistic regression revealed lower levels of anti-α-actinin as the only independent predictors of response (P = 0.05). CONCLUSIONS: Anti-α-actinin Abs at baseline appear to predict treatment response and therefore they might be used for monitoring treatment outcome in AIH-1.

Multiple autoimmune propensity and B-non-hodgkin lymphoma: cause or effect?

We report a case of multiple autoimmunity consisting of the presence of autoimmune haemolytic anaemia (AIHA), antimitochondrial antibodies (AMAs), and antiphospholipid antibodies (APLAbs) as the presenting manifestations of an extrahepatic B-non-Hodgkin lymphoma (B-NHL) in a 63-year-old woman. The patient presented with fatigue attributed to severe AIHA. Due to increased serum IgM and γ-GT levels, an investigation for AMA was performed, which proved positive with anti-M2 specificity. A prolongation of activated partial thromboplastin time (aPTT) led to the determination of APLAbs (lupus anticoagulant and other APLAbs) which were also positive. Bone marrow biopsy in combination with immmunohistochemical studies established the diagnosis of lymphoplasmacytic B-NHL. Ten months later, B-NHL was in remission while AMA and APLAbs were still positive. In conclusion, we documented the coexistence of multiple autoimmune reactions together with B-NHL highlighting the possible common pathogenetic pathways of the two entities.

A case of membranous nephropathy associated with Sjögren syndrome, polymyositis and autoimmune hepatitis.

Sjögren syndrome (SS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration of exocrine glands, especially lacrimal and salivary. The immunologic process which occurs in this syndrome is B cell hyperactivity, which results in production of autoantibodies and immune complexes. SS can exist as a primary disorder or in association with other autoimmune processes. A usually mild, proximal and insidious inflammatory myopathy can occur in patients with SS with a broad clinical and pathological spectrum. Interstitial nephritis with mild proteinuria and tubular dysfunction is the most common renal manifestation of SS, but glomerular involvement due to immune complex deposition may also rarely occur [Goules et al. 2000]. There is an association of SS with hepatic abnormalities, as evidenced by abnormal liver biochemical tests or histological characteristics of primary biliary cirrhosis (PBC), portal tract fibrosis, or autoimmune hepatitis [Abraham et al. 2004]. The pathogenetic mechanism of liver involvement in SS is not clear, but it is possible that hepatic and salivary gland damage share a similar pathology. The combination of Sjögren syndrome with kidney, liver and muscle involvement in one entity is extremely rare and data in the literature are remarkably sparse. We present a case of a 43-year-old female patient suffering from SS accompanied by polymyositis, membranous nephropathy and autoimmune hepatitis.

Enzymes as target antigens of liver-specific autoimmunity: the case of cytochromes P450s.

Characterization of liver-specific autoantigens has given a fresh impetus to research in the pathogenesis of autoimmune liver diseases, viral-triggered and drug-induced autoimmunity affecting the liver. Intriguing is the fact that most of the liver-specific autoantigens are enzymes of key importance for cell's homeostasis. Detection of autoantibodies against the respective antigens is carried out for diagnostic and research purposes using indirect immunofluorescence, immunoblotting, enzyme-linked immunosorbent assays, radioimmunoassay, immunoprecipitation or assays determining inhibition of enzyme activity. In patients with autoimmune hepatitis, a liver disorder of unknown etiology and pathogenesis, disease-specific autoantibodies are frequently directed against drug metabolizing enzymes of phase 1, namely cytochrome P450 2D6 (CYP2D6). The same and other members of these families of enzymes (CYPs) have also been described as targets of liver-specific autoimmunity in chronic hepatitis C virus (HCV)-infected patients, patients with autoimmune hepatitis as part of the autoimmune polyglandular syndrome type-1 (APS-1) and drug-induced autoimmunity. How these enzymes become 'self targets' is not yet established. An antigen release following hepatocyte injury could provide the stimulus for an immune response towards epitopes on these enzymes but the highly-specific, antigen-restricted initiation of the observed autoimmune response is against such an explanation. Accordingly, in this review we will focus on the pathogenic role -if any- of autoimmune responses against liver-related CYPs in autoimmune hepatitis, HCV infection, APS-1 and drug-induced autoimmunity. Learning more about the specificity of antibody responses against these enzymes may help us better understand the mechanisms underlying liver autoimmunity and may facilitate the development of therapeutic and preventive interventions.

Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073.

The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.