Edward L. Krawitt

Simplified criteria for the diagnosis of autoimmune hepatitis

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life‐saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobulins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff ≥6) and 81% sensitivity and 99% specificity (cutoff ≥7) in the validation set. Conclusion: A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher. (HEPATOLOGY 2008.)

Diagnosis and management of autoimmune hepatitis

Clinical practice guidelines are defined as ‘‘systematically developed statements to assist practitioner and patient decisions about appropriate heath care for specific clinical circumstances.’’ (All references are available in the Supporting Information.) These guidelines on autoimmune hepatitis provide a data-supported approach to the diagnosis and management of this disease. They are based on the following: (1) formal review and analysis of the recently-published world literature on the topic [Medline search]; (2) American College of Physicians Manual for Assessing Health Practices and Designing Practice Guidelines; (3) guideline policies, including the AASLD Policy on the Development and Use of Practice Guidelines and the American Gastroenterological Association Policy Statement on Guidelines; and (4) the experience of the authors in the specified topic. These recommendations, intended for use by physicians, suggest preferred approaches to the diagnostic, therapeutic and preventive aspects of care. They are intended to be flexible, in contrast to standards of care, which are inflexible policies to be followed in every case. Specific recommendations are based on relevant published information. To more fully characterize the quality of evidence supporting the recommendations, the Practice Guidelines Committee of the AASLD requires a class (reflecting benefit versus risk) and level (assessing strength or certainty) of evidence to be assigned and reported with each recommendation. The grading system applied to the recommendations has been adapted from the American College of Cardiology and the American Heart Association Practice Guidelines, and it is given below (Table 1).

Double blind, placebo controlled trial of metronidazole in Crohn's disease.

A double blind study compared the efficacy of metronidazole in two doses (20 mg/kg, 10 mg/kg) with placebo in patients with Crohns disease. One hundred and five patients participated but only 56 completed the 16 week study -21 were withdrawn for deterioration of symptoms, 17 for adverse experiences, and 11 for protocol violation. Significant improvement in disease activity as measured by the Crohns disease activity index (metronidazole 20 mg/kg, 97 units; metronidazole 10 mg/kg, 67 units; placebo -1 unit, p = 0.002) and serum orosomucoid (metronidazole 20 mg/kg/day, 49; 10 mg/kg/day, 38; placebo, -9, p = 0.001)) were detected. Changes in C reactive protein concentrations did not achieve significance when all three groups were considered but were significant when all metronidazole treated patients were grouped and compared with the placebo treated patients (0.8 v -0.9, p less than 0.05). Although patients receiving metronidazole 20 mg/kg/day had a greater improvement in disease activity than those receiving 10 mg/kg/day (difference 30 units (95% confidence intervals -27-87), the small sample size may have precluded the detection of statistical significance. Preliminary analysis suggests that metronidazole was more effective in patients with disease confined to the large intestine or affecting both small and large bowel than in those with small bowel disease only. There were no differences in remission rates between metronidazole and placebo treated patients. We conclude that metronidazole warrants further assessment in the treatment of patients with active Crohns disease.

Hepatitis C Virus Genotypes in the United States: Epidemiology, Pathogenicity, and Response to Interferon Therapy

Infection with hepatitis C virus (HCV) has been identified as the major cause of post-transfusion non-A, non-B hepatitis [1]. Chronic liver disease occurs in at least 50% of patients with acute HCV infection, and cirrhosis develops in 20% of these patients [2]. The virus has a single-stranded RNA genome that is approximately 10 Kbp long. A comparison of HCV genomic sequences from around the world has shown substantial heterogeneity of nucleotide sequences within several regions of the viral genome [3]. Hepatitis C virus has been classified into multiple strains or genotypes on the basis of the identification of these genomic differences. It has been suggested that the heterogeneity in sequence seen among HCV genotypes may be associated with variant antigenic and biological properties [4]. In addition, outcome of liver disease and rates of response to interferon therapy may vary according to HCV genotype [5, 6]. Therefore, understanding the distribution and properties of HCV genotypes may have important implications for prognosis and therapy. We evaluated the distribution of HCV genotypes in distinct geographic regions of the United States and determined the clinical characteristics of and response to interferon therapy in patients with one of several HCV genotypes. We used the classification system developed by Simmonds and colleagues [7] because it was recently adopted by consensus at the Second International Conference of HCV and Related Viruses (August 1994, San Diego, California). In this system, HCV genotypes are classified into six major genotypes (1 to 6, ordered according to when they were discovered) and 11 subtypes (1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6a). Methods Serum Samples We analyzed serum samples of 208 patients who were positive for antibody to HCV and had chronic liver disease. The samples were retrospectively obtained from four tertiary referral centers in the United States (59 consecutive samples from the Mayo Clinic, Rochester, Minnesota; 48 consecutive samples from the University of Vermont, Burlington, Vermont; 49 consecutive samples from the University of Miami, Miami, Florida; and 52 consecutive samples from the University of Washington Virology Laboratory, Seattle, Washington [this last center provided samples from Washington State, Idaho, Utah, Oregon, and California]). Twenty-nine patients were excluded from the study: Nineteen had no detectable products for DNA sequencing, and 10 had ambiguous sequencing results. The remaining 179 samples were the focus of this study. No clinical information was available on the patients whose samples were obtained from the University of Washington Virology Laboratory; thus, these samples were used only to study the geographic distribution of HCV genotypes. Data on interferon treatment were available for 78 patients from the Mayo Clinic and the University of Vermont. Samples from these two institutions were obtained from patients who had agreed to participate in trials of interferon treatment. Reverse Transcriptase and Polymerase Chain Reaction We selected the direct sequencing technique because it remains the gold standard and the only way to definitively identify all HCV genotypes and subtypes. Hepatitis C virus RNA was extracted from 100-L aliquots of serum after the addition of 1 mL of RNAzol B solution (Biotecx Laboratories, Houston, Texas) (2 mol of guanidinium thiocyanate per L, 12.5 mol of sodium citrate per L, 0.25% N-laroylsarcosine, 0.05 mol of 2-mercaptoethanol per L, 100 mmol of sodium acetate per L, and 50% water-saturated phenol). After the addition of 100 L of chloroform, samples were spun for 15 minutes at 14 000 g and the aqueous phase was extracted. Total RNA was precipitated by the addition of isopropanol and 2 L of glycogen and incubation at 4 C for 45 minutes. An RNA pellet was recovered by centrifugation at 14 0006 g, washed in 1 mL of 70% ethanol solution, dried, and resuspended in 10 L of RNAase-free water (Promega, Madison, Wisconsin). Ribonucleic acid was reverse-transcribed into complementary DNA by using reverse transcriptase and an antisense oligonucleotide primer (5-CGCGGAATTCCTGGTCATAGCCTCCGTGAA-3) in the presence of reverse-transcriptase buffer (100 mmol of tris-HCl per L, 500 mmol of KCl per L, 1% Triton X-100, and a pH of 8.6 at 25 C) (Promega) and 3.0 mmol of magnesium per L. Hepatitis C virus complementary DNA was amplified by polymerase chain reaction (PCR) in the presence of the sense oligonucleotide primer (5-TGGGGATCCCGTATGATACCCGCTGCTTTGA-3), PCR buffer (500 mmol of KCl per L, 100 mmol of tris-HCl per L, and a pH of 8.3) (Perkins-Elmer-Cetus, Norwalk, New Jersey), 2.0 mmol of magnesium per L, and Amplitaq DNA polymerase (Perkins-Elmer-Cetus). The PCR assay was done in a DNA thermal cycler for 50 cycles (94 C for 1 minute, 58 C for 1 minute, and 72 C for 5 minutes). Products of the PCR assay were analyzed by gel electrophoresis in 3% agarose gel that was stained with ethidium bromide. The appearance of a band 401-base pair was considered a positive result. To avoid and monitor for possible contamination with exogenous sequences during extraction or amplification, extraction of nucleic acid and genomic amplification steps were done in separate laboratories. Ribonucleic acid samples from at least one negative and one positive sample were extracted, subjected to reverse transcription, and amplified in each batch of samples tested by PCR. No false-positive results were obtained in any of the negative controls. Sequencing and Genotyping Each fragment of the PCR product, which was approximately 401 base pairs long, was desalted before undergoing sequencing with a direct column-purification method (Wizard PCR Preps DNA Purification System, Promega). Automated sequencing was done by using a standard Sanger procedure, which involved the incorporation of fluorescein-labeled dideoxynucleotides and detection on an acrylamide gel (ABI model 373 A, Applied Biosystems, Hercules, California). Nucleotide sequences were aligned and compiled with the previously reported sequences by using the Pileup program (Wisconsin Genetic Computer Group, Madison, Wisconsin)[8]. Cluster analysis was done by using the unweighted-pair group mean average, which was included in the program. These methods allowed comparison of a 222-base pair fragment of DNA that was homologous to nucleotide positions 7975 to 8196 in the NS5 region of the prototype virus. Collection of Epidemiologic Data We studied the geographic distribution of the HCV genotypes identified in the blood samples. Data from all samples were combined to define the prevalence of the HCV genotypes in patients with chronic hepatitis C in the United States. When available, age, sex, risk factors for HCV acquisition, and liver histologic findings at the time of presentation were recorded for each patient. Risk factors for acquiring HCV included history of blood transfusion, history of injection drug use, and employment at a health care facility. Liver histologic findings were classified into three groups: mildly active hepatitis (portal inflammation without substantial hepatocyte necrosis), moderately active hepatitis (inflammation with hepatocyte necrosis), and liver cirrhosis. Accurate history of alcohol consumption was not available for many of these patients and thus was not included in the analysis. The investigator who did the genotyping was blinded to the clinical data of patients at the time of analysis. Liver biopsy specimens were independently interpreted at each center. Pathogenicity of Hepatitis C Virus Genotypes To study the possible differences in the pathogenicity of HCV genotypes, we divided patients into two groups: patients with mild hepatitis and patients with severe hepatitis. Mild hepatitis was defined as 1) pretreatment alanine aminotransferase levels that were less than three times the normal level and 2) no cirrhosis seen during examination of the liver biopsy specimen obtained before treatment. Severe hepatitis was defined as pretreatment alanine aminotransferase levels greater than three times the normal level or the presence of liver cirrhosis on pretreatment biopsy. Response to Interferon Seventy-eight patients received an average dose of 3 million U of interferon (interferon- or consensus interferon) for 6 months. Response to interferon was defined as the normalization of alanine aminotransferase levels at the end of therapy. Partial response to interferon (defined as decreased but not completely normal alanine aminotransferase levels) was considered to be a treatment failure. Sustained biochemical response was defined as a normal alanine aminotransferase level 6 months after the discontinuation of interferon treatment. Statistical Analysis We used the rank-sum and Kruskal-Wallis tests to compare continuous variables (such as age) between groups, and we used the Fisher exact test to assess associations in tabular data. Because few patients had genotype 2a, 3, or 4, all tests of association between genotype and other factors are based on data that were collapsed into four groups: genotype 1a, genotype 1b, genotypes 2a and 2b, and genotypes 3 and 4. Logistic regression was used to evaluate the association between response to interferon and the combined predictors of cirrhosis and genotype. We used the SAS statistical analysis package (SAS Institute, Cary, North Carolina) for all calculations. Results Geographic Distribution of Hepatitis C Virus Genotypes Hepatitis C virus genotype 1a was present in 104 of 179 (58%) patients with chronic HCV infection; genotype 1b was the second most common genotype encountered (38 of 179 patients [21%]). Genotype 2b was present in 23 patients (13%), and genotype 3a was present in 8 patients (5%). Four patients (2%) had HCV genotype 2a, and 2 (1%) had genotype 4a. Geographic region and distribution of genotypes were not significantly associated (P = 0.18). However, samples obtained from the western United States conta

Mesalamine capsules for the treatment of active Crohn's disease: Results of a 16-week trial

BACKGROUND Mesalamine is released from sulfasalazine in the colon and benefits colonic Crohns disease. The mesalamine used in this study releases the drug throughout the small bowel and colon. Therefore, this study was designed to detect benefit for Crohns disease involving the small bowel alone or both the colon and small bowel. METHODS This double-blind, randomized, multicenter prospective controlled trial compared placebo and three daily doses of mesalamine in 310 patients. The primary outcome criterion was change in the Crohns Disease Activity Index (CDAI) from baseline to final study visit. RESULTS Patients taking 4 g/day mesalamine experienced a decrease of 72 CDAI points compared with 21 points in the placebo group (P < 0.01). Remission occurred in 43% of the 4-g group and 18% of the placebo group. Patients with ileum-only disease showed a 93-point improvement on 4 g mesalamine, compared with a 2-point improvement in similar patients on placebo. Mesalamine in this trial was not associated with clinically significant toxicity. CONCLUSIONS This controlled-release mesalamine preparation is safe and effective at 4 g/day as a single agent in treatment of active Crohns disease of the ileum and colon.

PBC Screen: An IgG/IgA dual isotype ELISA detecting multiple mitochondrial and nuclear autoantibodies specific for primary biliary cirrhosis

A dual isotype (IgG, IgA) enzyme-linked immunosorbent assay (ELISA) designed to provide enhanced detection of primary biliary cirrhosis (PBC)-specific autoantibodies against both major mitochondrial and nuclear antigens has been developed and recently become commercially available. The assay (PBC Screen) simultaneously detects IgG and IgA autoantibodies to the immunodominant portions of the 3 major mitochondrial (MIT3) and nuclear (gp210, and sp100) antigens. The aim of this study was to compare the performance of the PBC Screen to the combined performance obtained with individual IgG ELISAs to MIT3, gp210, and sp100 on a large group of selected patients from multiple centers. A total of 1175 patients with PBC and 1232 subjects without PBC were evaluated. Non-PBC groups included healthy controls (624) as well as individuals with autoimmune hepatitis (281), primary sclerosing cholangitis (77), viral hepatitis (91 hepatitis B and 98 hepatitis C), other liver diseases (31), and other infectious or autoimmune diseases (30). The PBC Screen at the receiver operator characteristic optimized cutoff of 27.8 units, had an overall sensitivity of 83.8%, specificity of 94.7% and area under curve of 0.9212. This was similar to the specificity of 96.1% obtained by the combined results of individual MIT3, sp100, and gp210 IgG ELISAs (kappa index at 0.898). Of the 253 PBC patients without AMA detectable by immunofluorescence, 113 (44.7%) were interpreted as positive for PBC-specific autoantibodies. In conclusion, the PBC Screen is an appropriate first-line test for the diagnosis of PBC, including for patients negative for markers assessed using conventional methods.

Demographics of anti-asialoglycoprotein receptor autoantibodies in autoimmune hepatitis

BACKGROUND/AIMS The asialoglycoprotein receptor (ASGPR) is an established, liver-specific autoantigen. This multicenter study investigated the specificity of anti-ASGPR autoantibodies for autoimmune hepatitis (AIH) in different ethnic groups. METHODS Nine hundred fourteen sera from European, Japanese, and North American (U.S.) patients with chronic inflammatory liver disorders were tested. An enzyme-immunoassay using human ASGPR and a radioimmunoassay against rabbit ASGPR, performed independently on coded sera, were compared. RESULTS The highest frequency (76%) of anti-human ASGPR was found in AIH patients (11/24 U.S.; 21/25 European; 28/30 Japanese), particularly in those with active disease before treatment (53/62, 85%), and decreased in titer with response to immunosuppressive therapy. These antibodies were found at low titers in 43 (11%) of 385 patients with viral hepatitis and in 25 (7.6%) of 328 patients with other chronic inflammatory liver disorders (P < 0.0005 compared with all AIH patients). Twenty of 37 sera tested by enzyme-immunoassay and radioimmunoassay were positive, and nine were negative for anti-ASGPR by both assays (78% concordance); six sera were exclusively positive on human substrate. CONCLUSIONS Circulating anti-ASGPR autoantibodies are closely associated with autoimmune hepatitis independent of geographic or ethnic criteria. Two anti-ASGPR assays currently in use show high reliability.

Treatment challenges and investigational opportunities in autoimmune hepatitis

New drugs and advances in molecular biology afford opportunities to upgrade the treatment of autoimmune hepatitis. The aims of this study were to define treatment problems, identify possible solutions, and stimulate investigations to improve patient care. A clinical subcommittee of the International Autoimmune Hepatitis Group reviewed current management difficulties and proposed corrective actions. The assessment of new front‐line and salvage therapies for adults and children were given top priority. Cyclosporine and mycophenolate mofetil were endorsed as drugs worthy of rigorous study in severe disease, and budesonide was endorsed for study as front‐line therapy in mild disease. Diagnostic criteria and treatment regimens for children required codification, and pharmacokinetic studies were encouraged to develop optimal dosing schedules based on therapeutic ranges. Collaborative efforts were proposed to help understand racial, geographical, and genetic factors affecting outcome and to establish definitions and therapies for variant syndromes and graft dysfunction after transplantation. The development of experimental animal models was deemed essential for the study of site‐specific molecular interventions, and gene therapy was endorsed as a means of bolstering reparative processes. In conclusion, evolving pharmacological and technical advances promise to improve the treatment of autoimmune hepatitis, and investigations of these advances are timely, feasible, and necessary. (HEPATOLOGY 2005;41:207–215.)

Variability of grade and stage in simultaneous paired liver biopsies in patients with hepatitis C

Background: Grading and staging of liver biopsies in patients with chronic hepatitis remains an inexact “gold standard” that is influenced by variabilities in scoring systems, sampling, observer agreement and expertise. Spatial disease variability relative to markers of the adequacy of biopsy has not been studied previously. Methods: Paired liver biopsy specimens were obtained from the right and left hepatic lobes of 60 patients with chronic hepatitis C. Histological grade and disease stage were assessed according to the Ludwig scoring system, and scores were evaluated in relation to differences in size and number of portal tracts in all paired samples. Results: The relative difference (%) in aggregate biopsy size and number of portal tracts was similar between paired samples with and without a difference in grade. Paired samples with a difference in stage showed a larger relative difference in biopsy size (p = 0.09) and in the number of portal tracts (p = 0.016). Conclusions: Our study shows a difference of one grade or one stage in 30% of paired liver biopsies, due to a combination of sampling variability and observer variability. Acknowledgment of “built-in” variability in grading and staging chronic hepatitis C by both clinicians and pathologists is essential for managing the individual patient with chronic hepatitis C.

Calcium Absorption in Crohn's Disease

Calcium absorption and endogenous loss of calcium were measured in a group of patients with Crohns disease, using a simultaneous metabolic balance and calcium isotope regimen. Calcium malabsorption resulting in negative calcium balance was found in only 4 of 31 patients with Crohns disease. No elevation of endogenous fecal calcium or total secreted intestinal calcium was observed in 10 patients studied, regardless of the level of net or true calcium absorption. Correlation between calcium balance and serum protein loss was observed, but no association was noted with intestinal fat excretion, d-xylose absorption, bacterial colonization of the jejunum, or glucocorticosteroid therapy. The results indicate that in this group of patients with Crohns disease involving different areas of the intestine, calcium malabsorption occurred infrequently and that the levels of calcium excretion correlated best with enteric protein loss.