K. Rajender Reddy

Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation

BACKGROUNDnOrthotopic liver transplantation for end-stage hepatitis-B-virus (HBV) infection is commonly complicated by recurrence of HBV. Lamivudine, a cytosine nucleoside analogue, has been shown to suppress HBV infection. We report the development of resistance to lamivudine in three patients who underwent transplantation for end-stage liver disease secondary to hepatitis B.nnnMETHODSnTwo of the patients received lamivudine for recurrent HBV infection after transplantation, whereas the third patient began treatment 1 month before transplantation in an attempt to prevent HBV recurrence after transplantation. The three patients initially responded well to treatment, but viral recurrence occurred after 9-10 months of treatment in all patients. HBV DNA was amplified from serum and sequenced through a conserved polymerase domain-the tyrosine, methionine, aspartate, aspartate (YMDD) locus. We assessed the susceptibility of HBV to lamivudine by infecting primary human hepatocytes with serum taken before the start of treatment and after recurrence in varying concentrations of lamivudine.nnnFINDINGSnDNA sequencing showed a common mutation within the YMDD locus of the HBV polymerase gene in all patients during lamivudine treatment. In hepatocyte cultures infected with pretreatment serum, HBV DNA concentrations were reduced to less than 6% of those in control cultures by addition of lamivudine in concentrations as low as 0.03 mumol/L. By contrast, in cultures treated with serum taken after recurrence, HBV DNA concentrations did not fall below 20% of control values, even with lamivudine at 30 mumol/L.nnnINTERPRETATIONnResistance to lamivudine has been reported in HIV patients with mutations in the YMDD locus of the polymerase gene. Our findings indicate a common mechanism of lamivudine resistance for HIV and HBV that involves similar point mutations in homologous domains of the viral polymerases.

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

Hepatic iron concentration as a predictor of response to interferon alfa therapy in chronic hepatitis C

BACKGROUND/AIMSnIt has been reported that hepatic iron concentration (HIC) may influence response to therapy in chronic viral hepatitis. The aim of this study was to determine the relationship between HIC and response to interferon alfa therapy in patients with chronic hepatitis C.nnnMETHODSnHIC was measured in liver biopsy specimens from 58 patients with chronic hepatitis C treated at three centers. Three patients had mild chronic hepatitis C, 35 had moderate to severe chronic hepatitis C, and 20 had active cirrhosis. Serum ferritin levels were measured in 51 of these 58 patients. Response to therapy was defined as normalization of alanine aminotransferase levels at the end of treatment.nnnRESULTSnTwenty-four patients (41%) responded to therapy. HICs were generally within the normal range (< 1500 micrograms/g). The mean HIC in nonresponders (860 +/- 100 micrograms/g; range, 116-2296 micrograms/g) was significantly higher than in responders (548 +/- 85 micrograms/g; range, 29-1870 micrograms/g) (P < 0.05). Eighty-eight percent of patients with an HIC of > 1100 micrograms/g and 87% of patients with an elevated serum ferritin concentration did not respond to interferon alfa therapy.nnnCONCLUSIONSnHIC seems to influence response to interferon alfa therapy among patients with chronic hepatitis C. A subgroup of patients with chronic hepatitis C has been identified for which an HIC of > 1100 micrograms/g predicted nonresponse in 88% of patients.

Controlled-Release, Pegylation, Liposomal Formulations: New Mechanisms in the Delivery of Injectable Drugs

OBJECTIVE: To review recent developments in novel injectable drug delivery mechanisms and outline the advantages and disadvantages of each. DATA SOURCES: A MEDLINE (1995–January 2000) search using the terms polyethylene glycol, liposomes, polymers, polylactic acid, and controlled release was conducted. Additional references were identified by scanning bibliographies. STUDY SELECTION AND DATA EXTRACTION: All articles were considered for inclusion. Abstracts were included only if they were judged to add critical information not otherwise available in the medical literature. DATA SYNTHESIS: A number of systems that alter the delivery of injectable drugs have been developed in attempts to improve pharmacodynamic and pharmacokinetic properties of therapeutic agents. New drug delivery systems can be produced either through a change in formulation (e.g., continuous-release products, liposomes) or an addition to the drug molecule (e.g., pegylation). Potential advantages of new delivery mechanisms include an increased or prolonged duration of pharmacologic activity, a decrease in adverse effects, and increased patient compliance and quality of life. Injectable continuous-release systems deliver drugs in a controlled, predetermined fashion and are particularly appropriate when it is important to avoid large fluctuations in plasma drug concentrations. Encapsulating a drug within a liposome can produce a prolonged half-life and a shift of distribution toward tissues with increased capillary permeability (e.g., tumors, infected tissue). Pegylation provides a method for modification of therapeutic proteins to minimize many of the limitations (e.g., poor stability, short half-life, immunogenicity) associated with these agents. CONCLUSIONS: Pegylation of therapeutic proteins is an established process with new applications. However, not all pegylated proteins are alike, and each requires optimization on a protein-by-protein basis to derive maximum clinical benefit. The language required to describe each pegylated therapeutic protein must be more precise to accurately distinguish each proteins differential pharmacologic properties.

Portal vein thrombosis: a concise review

Portal vein thrombosis (PVT) is an uncommon cause for presinusoidal portal hypertension. Although several predisposing conditions are known to exist in the background of PVT, there still remains a proportion of patients in whom the etiology is not known and the pathogenesis is unclear. In this review we summarize the literature on PVT and present the current knowledge about the precipitating factors of PVT. Further, we discuss the advances in the radiological diagnosis that have improved diagnostic accuracy and are noninvasive. Finally, we discuss the treatment options for patients who have varying extents of thrombosis in the portal vein and specifically focus on PVT that is encountered before and after liver transplantation.

Prognostic factors and early predictability of sustained viral response with peginterferon alfa-2a (40KD)

BACKGROUND/AIMSnBaseline factors and early decline in serum hepatitis C virus RNA are predictive of sustained virological response to interferon therapy in patients with chronic hepatitis C. We evaluated the prognostic value of baseline factors and early viral RNA among patients treated with peginterferon alfa-2a (40KD).nnnMETHODSnData were pooled from three randomized trials involving 814 patients treated with peginterferon alfa-2a (40KD) (90, 135, or 180 mirog). Stepwise and multiple logistic regression identified independent baseline factors associated with response. Receiver operating characteristic curves for both absolute values and log(10) decline in viral RNA at 4, 8, 12 and 24 weeks of therapy were created.nnnRESULTSnIndependent prognostic factors for sustained virological response included viral genotype non-1, low pretreatment viral load, age (<40 years), no cirrhosis and body weight (<85 kg). In addition, alanine aminotransferase quotient (>3) and histological activity index score (>10) were also independently prognostic. Receiver operating characteristic curves showed that detectable or less than 2-log(10) decline in viral RNA at week 12 predicted sustained virological non-response (negative predictive value is 98%) .nnnCONCLUSIONSnIn patients with chronic hepatitis C treated with peginterferon alfa-2a (40KD), the decision to continue or stop treatment can be made as early as week 12.

Prolonged Intrahepatic Cholestasis Secondary to Acute Hepatitis A

Viral hepatitis characterized by prolonged cholestasis has not been associated with a specific serologic marker. We report the cases of six patients presenting with a clinical syndrome typical of cholestatic hepatitis who were subsequently found to have acute hepatitis A. Usual features include pruritus, fever, diarrhea, and weight loss with serum bilirubin levels greater than 10 mg/dL, and a clinical course lasting at least 12 weeks. All patients recovered completely without sequelae. Knowledge of this unusual manifestation of hepatitis A may help avoid potentially invasive procedures involved in the evaluation of suspected obstructive jaundice and facilitate appropriate immunoprophylactic measures.

Comparison of tacrolimus with microemulsion cyclosporine as primary immunosuppression in hepatitis C patients after liver transplantation

BACKGROUNDnImmunosuppression in patients with hepatitis C virus (HCV) following orthotopic liver transplantation can lead to significant increases in serum viral loads. Our aim was to analyze the effect of a randomized study of two immunosuppressive agents (tacrolimus vs. microemulsion cyclosporine) on the outcome of HCV patients following orthotopic liver transplantation.nnnMETHODSnFrom December 1995 to September 1996, 50 adult patients transplanted for HCV cirrhosis were randomly assigned to receive tacrolimus (Prograf) (group 1, 25 patients) or microemulsion cyclosporine (Neoral) (group 2, 24 patients). All patients received alpha-interferon after transplantation, and the overall steroid doses were no different between the groups. Serum RNA levels were measured by signal amplification of Chiron. Biopsies were taken when transaminases were greater than 2x base line or when there was an inappropriate response to alterations in immunosuppression regimens.nnnRESULTSnThere were more episodes of rejection in the Neoral group, but there were no differences in bacterial and viral infections, nor in the rate of HCV recurrence between the two groups. There were seven deaths in group 1 and eight in group 2. Overall patient and graft survival rates in the Prograf and Neoral groups at 18 months were 72 and 68% and 67 and 64%, respectively.nnnCONCLUSIONSn(a) Both immunosuppression regimens had similar HCV recurrence rates; (b) there were no differences in bacterial or opportunistic infections; and (c) patient and graft survival was similar between the two groups.

Large cystic lesions of the liver in adults: a 15-year experience in a tertiary center

BACKGROUNDnCystic lesions of the liver consist of a heterogeneous group of disorders and may present a diagnostic and therapeutic challenge. Large hepatic cysts tend to be symptomatic and can cause complications more often than smaller ones.nnnSTUDY DESIGNnWe performed a retrospective review of adults diagnosed with large (> or = 4 cm) hepatic cystic lesions at our center, over a period of 15 years. Polycystic disease and abscesses were not included.nnnRESULTSnSeventy-eight patients were identified. In 57 the lesions were simple cysts, in 8 echinococcal cysts, in 8 hepatobiliary cystadenomas, and in 1 hepatobiliary cystadenocarcinoma. In four patients, the precise diagnosis could not be ascertained. Mean size was 12.1 cm (range, 4 to 30 cm). Most simple cysts were found in women (F:M, 49:8). Bleeding into a cyst (two patients) and infection (one patient) were rare manifestations. Percutaneous aspiration of 28 simple cysts resulted in recurrence in 100% of the cases within 3 weeks to 9 months (mean 4(1/2) months). Forty-eight patients were treated surgically by wide unroofing or resection (laparoscopically in 18), which resulted in low recurrence rates (11% for laparoscopy and 13% for open unroofing). Four of the eight patients with echinococcal cysts were symptomatic. All were treated by open resection after irrigation of the cavity with hypertonic saline. There was no recurrence during a followup period of 2 to 14 years. Hepatobiliary cystadenomas occurred more commonly in women (F:M, 7:1) and in the left hepatic lobe (left:right, 8:0). Seven were multiloculated. All were treated by open resection, with no recurrence, and none had malignant changes. Cystadenocarcinoma was diagnosed in a 77-year-old man, and was treated by left hepatic lobectomy.nnnCONCLUSIONSnLarge symptomatic simple cysts invariably recur after percutaneous aspiration. Laparoscopic unroofing can be successfully undertaken, with a low recurrence rate. Open resection after irrigation with hypertonic saline is a safe and effective treatment for echinococcal cysts. Hepatobiliary cystadenomas have predilection for women and for the left hepatic lobe. Malignant transformation is an uncommon but real risk. Open resection is a safe and effective treatment for hepatobiliary cystadenoma, and is associated with a low recurrence rate.

First phase viral kinetic parameters as predictors of treatment response and their influence on the second phase viral decline.

summary.u2002It has recently been shown that upon initiation of interferon (IFN) treatment there is a biphasic decline in hepatitis C virus (HCV) RNA levels. In preliminary results, the rate of second phase viral decline has been shown to be an excellent predictor of treatment response. In this analysis, we determined whether the first phase viral kinetic parameters affected the rate of second phase viral decline. We also assessed whether first phase viral kinetic parameters could be used to predict treatment response within 24 h of initiating treatment. This study is a retrospective analysis of two completed studies from which detailed kinetic data were obtained in patients infected with genotype 1 HCV. In both studies, viral levels were measured frequently over the first 24u2003h, allowing the determination of IFNs effectiveness in blocking viral production and the viral load at the end of the first phase (v1). The second phase decline slope was calculated by log‐linear regression on measurements of serum HCV RNA during days 2, 7 and 14. In study one, sustained viral response (SVR) rates were obtained, allowing the determination of the first phases predictive power for SVR. Logistic regression and fisher exact tests were used to analyse data. In study one, no patient achieved SVR without an IFN effectiveness greater than 98% and a V1 less than 250u2003000 copies/mL. When V1 and IFN effectiveness werecombined to predict SVR, a negative predictive value= 100%, positive predictive value=71% and accuracy of 95% was obtained after only 24 h of IFN treatment. Both studies illustrated strong correlations for both IFN effectiveness and V1 with the rate of 2nd phase slope (Pu2003<u20030.001). V1 also correlated significantly with a calculation of infected cell loss (delta), which is a major determinant of the second phase viral decline. These results suggest that early viral kinetics may predict lack of response after only 24u2003h of treatment initiation and indicate a strong link between the degree of viral load reduction during the first phase, and the subsequent 2nd phase decline slope. This might be explained by a viral dynamics model assuming a jump‐start of the immune response when viral loads are reduced below a threshold, subsequently giving rise to a faster 2nd phase decline slope.