Hepatitis B Virus Serum DNA andRNA Levels in Nucleos(t)ide Analog-Treated or Untreated Patients During Chronic and Acute Infection.

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.

High frequency of genotype D and spontaneous hepatitis B virus genomic mutations among Haitians in a multiethnic North American population.

GOALS/BACKGROUND: The importance of hepatitis B virus (HBV) genotype and mutations has been increasingly recognized. We aimed to determine HBV genotype, precore (PC), and basal core promoter region (BCP) mutations in a HBV multiethnic South Florida population. STUDY: Samples from 213 patients were tested for HBV-DNA using Abbott RealTime HBV IUO assay, and for mutations using INNO-LiPA assay. RESULTS: Patients were predominantly male (67%); 61 (31%) were African American, 60 (28%) Hispanic, 37 (17%) Haitian, 27 (19%) white non-Hispanic, and 14 (6.6%) Asian. Genotype A was found in 101 (69%), D in 25 (17%), F in 9 (6%), G in 7 (5%), C and E in 6 (4%) each, B in 4 (3%), and H in 2 (1%) patients. Mixed genotypes were detected in 11 patients. Genotype A was more prevalent in all ethnicities except for Asian. Among hepatitis B e antigen (HBeAg)-negative patients (59%), BCP, PC, and combined BCP/PC mutations were found in 30 (37.5%), 13 (16.3%), and 14 (17.5%), respectively. Genotype D was associated with higher frequency of HBeAg-negative status [18/24 (75%) vs. 62/121 (51%) P=0.03] and mutations [16/19 (84%) vs. 40/67 (60%) P=0.04] compared with others. Genotype A was negatively associated with mutations [26/31 (84%) vs. 30/55 (55%), P=0.009]. PC mutations were more common in genotype D (14/19, 73%) compared with genotype A (7/54, 13%, P<0.0001). One-hundred percent and 79% of Asians and Haitians had spontaneous mutations, respectively. All Haitians with genotype D had PC mutations and 3 (50%) had BCP/PC. CONCLUSIONS: The present study shows that HBeAg-negative status and spontaneous mutations were more common with genotype D; the presence of genotype D in Haitians was always associated with spontaneous mutations.

Improved detection of hepatitis C virus infection by transcription-mediated amplification technology in dialysis population.

BACKGROUND: Hepatitis C virus (HCV) infection remains common among patients undergoing maintenance dialysis and plays an adverse effect on survival in this population. Accurate detection of HCV viremia (HCV RNA) in dialysis patients requires a sensitive and specific diagnostic test. METHODS: The Versant HCV RNA Qualitative Assay, based on transcription-mediated amplification (TMA) technique, was prospectively evaluated in 112 dialysis patients. Performance characteristics of the Versant HCV TMA Assay were evaluated in comparison to the Amplicor((R)) 2.0 HCV test based on polymerase chain reaction (PCR) technique. In addition, anti-HCV serologic tests including third-generation enzyme immunoassay and Recombinant Immunoblot Assay were performed. RESULTS: Of the 112 specimens tested, 29 were reactive by Versant HCV TMA Assay, yielding an overall prevalence of HCV viremia of 25.9%. The concordance between TMA and PCR techniques was excellent [91% (101/112)]. Eleven specimens (10%) were invalid or equivocal by PCR due to interference phenomena; all 11 specimens had valid TMA results (2 patients being TMA reactive and 9 nonreactive). Four specimens [3.6% (4/112)] that tested PCR-negative and HCV TMA nonreactive were anti-HCV seropositive, consistent with resolved HCV infection. In the group of seronegative samples, one was reactive by TMA Assay [1.25% (1/80)]. CONCLUSIONS: The HCV TMA technology seems a highly sensitive tool for detecting HCV RNA in the dialysis population, with no evidence of specimen interference. One EIA-negative but HCV-RNA-positive patient by Versant HCV TMA Assay was identified. Prospective clinical trials are under way to assess the clinical impact related to the use of HCV TMA technology in dialysis population.

Multicenter evaluation of the Bayer ADVIA Centaur HIV 1/O/2 enhanced (EHIV) assay.

BACKGROUND: It is important that serological assays detect antibodies to human immunodeficiency virus (HIV) in all infected individuals, including those infected with less prevalent, more diverse subtypes. METHODS: Performance of the ADVIA Centaur HIV 1/O/2 Enhanced (EHIV) Assay was tested on 1344 samples from HIV-positive subjects, 6061 samples from groups at low-risk for HIV infection, and 1042 samples from groups at high-risk for HIV-1 and HIV-2 infection. Results were compared with those of an FDA-approved predicate assay. RESULTS: The ADVIA Centaur EHIV Assay showed good precision with a diagnostic specificity of 99.9% and diagnostic sensitivity of 100%. HIV seroconversion was detected earlier in 6 panels, at the same time in 13 panels and later in only 1 of the panels when compared to the predicate assay, thereby narrowing the window period between infection and antibody detection. Of clinical significance, a blood donor sample that was indeterminate by HIV-1 Western blot and non-reactive by the predicate assay was repeatedly reactive in the ADVIA Centaur Assay and confirmed as positive by HIV-2 immunoblot. CONCLUSIONS: The ADVIA Centaur EHIV Assay is useful as an aid in the diagnosis of individuals infected with HIV-1 and/or HIV-2.

Treatment of established recurrent hepatitis C in liver-transplant recipients with pegylated interferon-alfa-2b and ribavirin therapy.

INTRODUCTION: The management issues of transplant patients with hepatitis C virus (HCV) are complex, and interferon therapy is often ineffective. We present data from a retrospective review in liver-transplant recipients suffering from HCV recurrence that were treated with pegylated alpha-2b interferon and ribavirin. METHODS: A retrospective review of transplant recipients that received combination pegylated alpha-2b interferon (1.5 mcg/kg/wk) and ribavirin (400-600 mg/day) therapy intended for at least 48 weeks. Complications were recorded and included neutropenia (<750 cells), anemia (hemoglobin <8 g) with and without treatment consisting of blood transfusions, erythropoietin, or dose reduction of ribavirin, and depression. The diagnosis of HCV recurrence was determined by an increase in liver chemistries, histopathologic findings with inflammation along with viral recurrence using the COBAS AMPLICOR HCV test. RESULTS: Fifty-seven liver-transplant recipients were included, 29 naive (group 1) to therapy and 28 nonresponders (group 2) to at least 6 months of interferon and ribavirin therapy. Eight (27.6%) patients in group 1 and six (21%) patients in group 2 were HCV nondetectable at the end of 48 weeks of therapy. Ribavirin therapy was decreased in 13 of 29 (45%) for group 1 and 11 of 28 (39%) in group 2. Therapeutic interventions were 4 of 57 (7%) blood transfusions, 23 of 57 (40%) erythropoietin, and 17 of 57 (30%) filgrastim. CONCLUSION: Combination pegylated interferon with ribavirin appears to effective therapy in HCV recurrence and in HCV nonresponsive to interferon and ribavirin. This data reveals the difficulty and caution that must be taken when treating HCV-R liver-transplant recipients with combination pegylated alpha-2b interferon and ribavirin therapy.

Comparison of detection and quantification of HBV DNA in chronic HBeAg negative and positive patients by Abbott RealTime HBV and Roche Cobas TaqMan HBV assays.

Monitoring the serum hepatitis B viral DNA levels with sensitive realtime PCR assays is strongly recommended for the management of patients with chronic HBV infection. This study compares the performance of two realtime HBV quantitative PCR assays with samples from chronic HBeAg(+) and HBeAg(-) patients.

Absence of Hepatitis B Resistance Mutants before Introduction of Oral Antiviral Therapy.

Introduction. The aim of this study was to assess whether hepatitis B virus drug resistant mutations antedated the widespread use of nucleos(t)ide analogues in treatment naïve patients. A number of reports have suggested that drug resistant mutants can be detected in apparently treatment naïve patients. Study. Fifty deidentified serum samples collected from 1986 to 1992 from patients with replicative chronic HBV infection at the University of Miami were genotyped and tested for resistance mutations using a line probe assay InnoLiPA HBV DR v2/v3. Serum HBV DNA was measured. All patients had documented chronic HBV infection with a detectable viral load, HBeAg seropositivity, and absence of HIV infection. Results. Of the 50 individuals included, 86% were male, mean age was 40 ± 12 years, and mostly genotype A. The mean HBV DNA was 126 pg/mL (range 6.4 to 557.0). No mutations were identified. Conclusions. The absence of drug induced mutations in these sera collected several years prior to the introduction of oral antiviral therapy suggests that these mutations do not occur in treatment naïve populations. Detection of drug resistance in an apparently treatment naïve subject suggests either unrecognized prior antiviral therapy or infection by an inoculum from a treatment experienced patient.

Screening for hepatitis B virus and hepatitis C virus at a community fair: a single-center experience.

Despite recommendations for screening for hepatitis B virus (HBV) and hepatitis C virus (HCV), most individuals are still unaware of their infection status. The disparities in screening for HBV and HCV can be attributed to lack of awareness, language barriers, and difficulty in accessing healthcare. To address these issues, an exhibit booth was set up at an annual cultural festival to promote awareness about HBV and HCV and also provide free screening for a local Floridian community. Recruitment was conducted in various languages by physicians and nurses who specialize in hepatology. All materials associated with the screening process were sponsored by the Schiff Center for Liver Diseases, which is located at the University of Miami Miller School of Medicine in Florida. In the first year of the screening initiative, 173 of 11,000 fair attendees were screened for HBV. Twenty-nine (17%) of those screened tested positive for antibodies to hepatitis B core antigen (anti-HBc), and only 1 individual tested positive for chronic HBV, with positive hepatitis B surface antigen (HBsAg). Screening for HCV and an extended patient questionnaire were added to the screening program in the second year of the initiative. A total 231 of 9,000 fair attendees volunteered to be screened for both HBV and HCV. Twenty-nine (13%) of these people tested positive for anti-HBc, and 3 tested positive for HBsAg. Only 1 person tested positive for anti-HCV, but this individual had undetectable HCV RNA levels. Our single-center experience illustrates that, despite efforts to improve access to screening, only 2-3% of attendees at a cultural fair embraced the screening efforts. Other strategies will be required to enhance participation in screening programs for viral hepatitis.

Correlation of Laparoscopic Liver Biopsy to Elasticity Measurements (FibroScan) in Patients With Chronic Liver Disease.

BACKGROUND: Elastography is a noninvasive method to assess liver fibrosis by measuring liver stiffness. Studies have compared elas-tography to percutaneous biopsy. Laparoscopic biopsy is associated with decreased sampling error compared to percutaneous biopsy, as laparoscopic biopsies are obtained from both liver lobes and gross nodu-larity can be visualized. METHODS: Patients undergoing laparoscopic liver biopsy were enrolled. Gross liver appearance was assessed, and biopsy specimens were blindly evaluated by a pathologist. Elastography (FibroScan) was used to measure liver stiffness. RESULTS: 101 patients were examined. Fibrosis was related to elasticity (Spearman correlation r=0.63; P<.0001). Elasticity was strongly associated with advanced stages of fibrosis (stages 3 and 4; Spearman correlation r(2)=0.44; P<.001). Significant fibrosis was associated with an irregular liver surface, nodularity, and thickened edge (multiple regression r(2)=0.41; P<.001). Increased elasticity was associated with a fatty-appearing liver, irregular surface, firmness, and nodularity (multiple regression r(2)=0.46; P<.001). Receiver operating characteristic curve for elasticity for identifying patients with a liver fibrosis stage of at least 3 or of 4 had an area under the curve (AUC) of 0.85 or 0.86, respectively. AUC was 0.857 when gross nodularity was used as the gold standard for cirrhosis and 0.875 when nodularity/histology were used. Elasticity of at least 7 kPa, at least 9.5 kPa, and at least 11.8 kPa had the highest accuracy for identifying patients with a fibrosis stage of at least 2, at least 3, and 4, respectively. In hepatitis C patients, AUC was 0.921, 0.882, and 0.925 when histology, gross nodularity, and nodularity/histology, respectively, were used as the gold standard for cirrhosis. CONCLUSION: FibroScan could be useful for detecting advanced stages of fibrosis when validated against laparoscopic liver biopsy.

Prospective Evaluation of FIBROSpect II for Fibrosis Detection in Hepatitis C and B Patients Undergoing Laparoscopic Biopsy.

Serum markers of liver fibrosis are difficult to validate, due to the sampling error and observer variability associated with percutaneous liver biopsies. Laparoscopic biopsy decreases sampling error and increases the reliability of histopathologic assessment. We prospectively evaluated the FIBROSpect(SM) II serum marker test for viral liver fibrosis against laparoscopic biopsies by studying 145 patients with chronic hepatitis B or C who underwent laparoscopy in a tertiary care setting. Serum samples obtained at biopsy were tested with FIBROSpect II to assess the degree of fibrosis. Multiple biopsies were obtained from each patient and scored blindly using the Batts-Ludwig system. An average biopsy stage was calculated and the performance of the test panel assessed. FIBROSpect II was able to rule in significant fibrosis (stages 2-4), with a likelihood ratio of 2.6. It correctly indicated absence of disease in 74% of stages 0-1 patients and correctly predicted significant disease in 67% of stages 2-4 patients. Test correlation was highest with Batts-Ludwig stages 3 (77%) and 4 (96%) and lowest with stage 2 (43%). Multiple biopsies from 52% of patients differed by at least 1 stage. In 13 patients (9%), cirrhosis was detected by laparoscopy but not histologically; in 4 (3%), a stage of 4 was obtained, but cirrhosis was not evident by laparoscopy. FIBROSpect II provided valuable additional information for assessing fibrosis. The discordance in fibrosis stage seen in multiple biopsies from the same patient underscores the need to consider all available information when assessing fibrosis. This study confirms and extends results of previous studies evaluating FIBROSpect II using percutaneous liver biopsy.

Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? Interim results of a large case-control study.

BACKGROUND: The role of the heterozygous PiZ state of alpha-1 antitrypsin deficiency (alpha1ATD) in the pathogenesis of chronic liver disease (LD) is still a matter of controversy. AIM: To determine the prevalence of alpha1ATD heterozygote states in a large population of patients with established LD compared with individuals with no LD, and to determine whether the prevalence of PiZ is increased in patients with more severe LD. METHODS: A cross sectional case-control study among patients with and without LD. Blood samples were tested for alpha1AT levels and alpha1AT phenotype. The severity of LD was determined by clinical evaluation, lab tests, imaging studies and histopathology. RESULTS: In total, 1405 patients were enrolled; 651 with, and 754 without LD. Out of them, 173 patients had decompensated cirrhosis requiring liver transplantation. PiMZ was significantly more prevalent in White patients (3.5%) compared with Hispanics (1.7%; P = 0.029). There was no difference in PiMZ prevalence between the total LD group and the group with no LD (2.1% vs. 1.7%; P = 0.64). Within the LD group, 5.7% of 173 patients with decompensated LD, listed for liver transplantation, had PiMZ, compared with 2.1% of 478 patients with less severe LD (P = 0.016). Similarly, there was a disproportionately higher prevalence of PiZ among hepatitis C virus (HCV) patients (5.6%) and patients with nonalcoholic fatty liver disease (NAFLD) (5.0%) with decompensated LD, compared with HCV patients (1.2%) and NAFLD patients (1.9%) with less severe LD (P = 0.044 and 0.017, respectively). Patients with cryptogenic cirrhosis, who were not considered NAFLD patients, did not have a higher prevalence of PiMZ compared with patients with LD of known etiologies (1.9% vs. 2.3%; P = 0.12). CONCLUSIONS: We found no association between the heterozygous PiZ state of alpha1ATD and the presence of chronic LD in-general or the presence of cryptogenic cirrhosis. In contrast, patients with decompensated LD of any etiology had a significantly higher prevalence of PiMZ compared with patients with compensated LD. Furthermore, in patients with chronic LD due to HCV or NAFLD there was a significant association between the PiMZ heterozygous state and increased severity of LD and the need for liver transplantation. These interim results suggest that the PiMZ alpha1ATD heterozygous state may have a role in worsening LD due to HCV or NAFLD.

Outcomes in liver transplant recipients with hepatitis B virus: resistance and recurrence patterns from a large transplant center over the last decade.

Hepatitis B virus (HBV) recurrence following liver transplantation (LTx) has been controllable primarily with the use of hepatitis B immune globulin (HBIg) and lamivudine (LAM). However, HBV resistance to LAM and/or HBIg has become an increasing problem prompting the use of newer antiviral agents. The purpose of our study was to investigate the association between therapy, HBV breakthrough, and allograft / patient survival in HBV-positive liver transplant recipients. We performed a retrospective review of the medical records of patients that were transplanted for HBV from June 1994 to May 2003. A total of 92 patients, positive for either hepatitis B surface antigen (HBsAg) or HBV deoxyribonucleic acid (DNA) pretransplant, received LAM monotherapy or HBIg (6 months) plus LAM therapy post-liver transplant. HBV breakthrough post-LTx was noted in 14 patients. All patients had detectable HBV DNA prior to liver transplantation; none of the patients that were HBV DNA negative prior to transplant had detectable HBV DNA posttransplant. Of these 14, 9 patients (64%) were switched from LAM to adefovir dipivoxil (ADF) and 5 patients (36%) to tenofovir disoproxil fumarate (TNV). In conclusion, pre-LTx HBV viremia should be considered in planning post-LTx prophylaxis. Trials to evaluate oral antiviral agents in combination with or without HBIg therapy are needed.

Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa-2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing.

The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha-2b interferon (1.5 mcg/kg) and ribavirin (200-600 mg/d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT-PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT-PCR while 3/10 had nondetectable HCV RNA in their liver by RT-PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT-PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum.

Comparison of three commercially available assays for HCV RNA using the international unit standard: implications for management of patients with chronic hepatitis C virus infection in clinical practice.

OBJECTIVES: The present study was performed to evaluate the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. METHODS: The three assays used were Amplicor Monitor PCR, the National Genetics Institute PCR assay, and branched chain DNA. HCV RNA was measured at four time points (baseline, 3 months after the start of therapy, at the end of treatment, and 6 months after discontinuation of therapy) in 106 consecutive patients who received interferon and ribavirin for chronic HCV. RESULTS: The mean age of the patients was 44 yr. Of the patients, 62% were male, 24% were African American, 38% had bridging fibrosis or cirrhosis, and 75% were HCV genotype 1. Of the 424 samples analyzed, 82-89% of values were within 1 log unit and 85-92% were within 2 log units by the various assays. This variability was not dependent upon HCV genotype. HCV RNA was undetectable in 1.4-6.8% of samples when virus was detected by another assay. The mean HCV RNA in these discordant samples was 1.47-6.33 log IU/ml (30-2,100,000 IU/ml). CONCLUSIONS: These data demonstrate that approximately 90% of serum values for HCV RNA were within 1 log unit by the international unit standard regardless of which virological assay was used. However, false positive and false negative results as well as variations in the HCV RNA level of more than 1 to 2 log units can occur with any of the assays, and these results may have an impact upon the management of patients receiving interferon therapy. It is therefore unwise in clinical practice to base important treatment decisions upon a single HCV RNA determination.

Nutrición preventiva: hipertensión; diagnóstico preliminar de la situación de la hipertensión en Costa Rica y factores nutricionales asociados

Se realizó un diagnóstico preliminar de la situación de la hipertensión en Costa Rica y los factores nutricionales asociados. La información se recolectó de diferentes fuentes tales como: documentos escritos, numericos, datos previamente recolectados, entrevistas a funcionarios y encuestas a proveedores y usuarios de los servicios de salud. Se analizó lainformación clasificandola en tres rubros: Morbi-mortalidad, aspectos de dieta y atención en salud. Aunque no se contó con información directa sobre la morbi-mortalidad por hipertensión, se obtuvieron datos que indirectamente nos mostraron que la prevalencia es de aproximadamente 7.5% en 1985, y la tasa de mortalidad proporcional por hipertensión fue de 3.6 en ese mismo año. Al analizar los aspectos de dieta tales como la ingesta calórica, de proteinas, carbohidratos, vitaminas y minerales, se encontró que posiblemente la deficiencia de ácido linoléico en las grasas de mayor consumo en el pais, es el factor nutricional que contribuye en mayor forma a la presencia de esta enferemdad; como factores asociados se encontraron los siguientes: alto consumo de sodio a través del pan blanco (francés), y de alcohol en la población mayor de 15 años. Contribuye también las ingestas moderadamente bajas de potasio, calcio y magnesio a través de los alimentos y en las aguas que se consumen, sobre todo las áreas que consumen aguas suaves que a la vez son las que presentan mayor cantidad de personas hipertensas. Se encontró que embutidos, productos e industrializados, que son los alimentos con mayor contenido de sodio, tienen un consumo muy bajo. Solamente dos a cinco por ciento de la población los consume a diario