Evaluation of the therapeutic response of hepatitis C in coinfected patients (HIV/HCV): a study of cases from a hospital for chronic liver diseases in the Eastern Brazilian Amazon

INTRODUCTION: The aim of this study was to evaluate the therapeutic response of hepatitis C in patients coinfected with human immunodeficiency virus (HIV-1). METHODS: A retrospective study of 20 patients coinfected with HIV-1/HCV who were treated in the outpatient liver clinic at the Sacred House of Mercy Foundation Hospital of Pará (Fundação Santa Casa de Misericórdia do Pará - FSCMPA) from April 2004 to June 2009. Patients were treated with 180µg PEG interferon-α2a in combination with ribavirin (1,000 to 1,250mg/day) for 48 weeks. The end point was the sustained virological response (SVR) rate (HCV RNA negative 24 weeks after completing treatment). RESULTS: The mean age of the patients was 40±9.5 years, of which 89% (n=17) were male, and the HCV genotypes were genotype 1 (55%, n=11/20), genotype 2 (10%, n=2/20) and genotype 3 (35%, n=7/20). The mean CD4+ lymphocyte count was 507.8, and the liver fibrosis stages were (METAVIR) F1 (25%), F2 (55%), F3 (10%) and F4 (10%). The early virological response (EVR) was 60%, the end-of-treatment virological response (EOTVR) was 45% and the SVR was 45%. CONCLUSIONS: The median HCV viral load was high, and in 85% of cases in which highly active antiretroviral therapy (HAART) was used, none of the patients with F3-F4 fibrosis responded to treatment. Of the twenty patients treated, 45% achieved SVR and 45% achieved EOTVR. Studies that include cases from a wider region are needed to better evaluate these findings.

Estudo in vitro do efeito da interferência por RNA (RNAi) na replicação do vírus da hepatite C

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Incidence and risk factors for chronic elevation of alanine aminotransferase levels in HIV-infected persons without hepatitis b or c virus co-infection

Chronic liver disease in human immunodeficiency virus (HIV)-infected patients is mostly caused by hepatitis virus co-infection. Other reasons for chronic alanine aminotransferase (ALT) elevation are more difficult to diagnose.

Molecular evolution of primate immunodeficiency viruses and hepatitis delta virus

Primate immunodeficiency viruses, or lentiviruses (HIV-1, HIV-2, and SIV), and hepatitis delta virus (HDV) are RNA viruses characterized by rapid evolution. Infection by primate immunodeficiency viruses usually results in the development of acquired immunodeficiency syndrome (AIDS) in humans and AIDS-like illnesses in Asian macaques. Similarly, hepatitis delta virus infection causes hepatitis and liver cancer in humans. These viruses are heterogeneous within an infected patient and among individuals. Substitution rates in the virus genomes are high and vary in different lineages and among sites. Methods of phylogenetic analysis were applied to study the evolution of primate lentiviruses and the hepatitis delta virus. The following results have been obtained: (1) The substitution rate varies among sites of primate lentivirus genes according to the two parameter gamma distribution, with the shape parameter $\alpha$ being close to 1. (2) Primate immunodeficiency viruses fall into species-specific lineages. Therefore, viral transmissions across primate species are not as frequent as suggested by previous authors. (3) Primate lentiviruses have acquired or lost their pathogenicity several times in the course of evolution. (4) Evidence was provided for multiple infections of a North American patient by distinct HIV-1 strains of the B subtype. (5) Computer simulations indicate that the probability of committing an error in testing HIV transmission depends on the number of virus sequences and their length, the divergence times among sequences, and the model of nucleotide substitution. (6) For future investigations of HIV-1 transmissions, using longer virus sequences and avoiding the use of distant outgroups is recommended. (7) Hepatitis delta virus strains are usually related according to the geographic region of isolation. (8) Evolution of HDV is characterized by the rate of synonymous substitution being lower than the nonsynonymous substitution rate and the rate of evolution of the noncoding region. (9) There is a strong preference for G and C nucleotides at the third codon positions of the HDV coding region. ^

RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.

Virus de la hepatitis C: variabiliad en las regiones del CORE y NS5A y su impacto en la infección por el genotipo 1b

La infección por el virus de la hepatitis C (VHC) tiene un gran impacto a nivel mundial, considerándose una de las principales causas de hepatitis crónica, enfermedad hepática terminal y hepatocarcinoma. Hasta la incorporación de los nuevos agentes antivirales, el genotipo 1b era el que tenía tasas más bajas de respuesta viral sostenida (RVS); y a su vez, el de mayor prevalencia en nuestro medio. Lo que ha llevado a la búsqueda de marcadores predictores de respuesta, con el fin de identificar a aquellos pacientes que pudieran beneficiarse del tratamiento. Estudios previos han mostrado que la variabilidad genómica en la región codificante de la proteína C del Core y en la región comprendida entre los aminoácidos 2209 y 2248 (región determinante de la sensibilidad al IFN: ISDR) de la región que codifica la proteína no-­‐estructural 5A (NS5A) están relacionados con la respuesta al tratamiento con biterapia. Es por ello, que el objetivo de nuestro estudio es caracterizar a nivel genómico ambas regiones y analizar su impacto en pacientes con infección crónica por el genotipo 1b. Se analizan, además, otros factores basales implicados en la respuesta: sexo, edad y carga viral basal...

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

Efecto del tratamiento antirretroviral en la respuesta virológica a interferón pegilado y ribavirina en pacientes coinfectados por el virus de la inmunodeficiencia humana y el virus de la hepatitis C

Introducción: En los pacientes coinfectados VIH/VHC, la influencia del tratamiento antirretroviral sobre la respuesta al tratamiento de la hepatitis C con interferón pegilado y ribavirina no se conoce bien. El objetivo de este estudio es investigar el efecto del tratamiento antirretroviral sobre la respuesta al tratamiento en los pacientes coinfectados. Métodos: Los pacientes se seleccionaron de dos cohortes de pacientes coinfectados por VIH y VHC de GESIDA y tratados con interferón pegilado y ribavirina entre 2001 y 2007. La respuesta viral sostenida se definió como una carga viral indetectable a las 24 semanas de haber finalizado el tratamiento. Se utilizaron métodos de regresión logística para estudiar posibles asociaciones entre la ausencia de respuesta y las características basales, incluyendo los fármacos antirretrovirales acompañantes. Resultados: Se incluyeron 1701 pacientes: el 63% tenía el genotipo 1 o 4 del VHC y el 88% estaba tomando TARGA. Los factores asociados de forma independiente con mayor probabilidad de RVS fueron el genotipo 2 o 3, la concentración de ARN-VHC<500000 UI/ml y la categoría clínica A o B. Se realizó un análisis ajustado a los factores pronósticos y la dosis de ribavirina /kg de peso, obteniendo una OR ajustada de RVS en los pacientes sin TARGA de 1,31 (IC95% 0,91-1,88, p=0,144). Tomando la pauta de TDF+FTC/3TC como referencia, se vio que solamente el AZT se asoció a una menor probabilidad de RVS, con una OR ajustada de RVS de 0,65 (IC95% 0,46-0,93, p=0,017). Conclusiones: El hecho de estar recibiendo TAR no tuvo ningún efecto sobre la respuesta al tratamiento de la hepatitis C. El único fármaco antirretroviral que se asoció de forma significativa con menor probabilidad de RVS fue el AZT.

RNA Virus Evolution: a Cross-scale, Disease Dynamic Perspective

RNA viruses are an important cause of global morbidity and mortality. The rapid evolutionary rates of RNA virus pathogens, caused by high replication rates and error-prone polymerases, can make the pathogens difficult to control. RNA viruses can undergo immune escape within their hosts and develop resistance to the treatment and vaccines we design to fight them. Understanding the spread and evolution of RNA pathogens is essential for reducing human suffering. In this dissertation, I make use of the rapid evolutionary rate of viral pathogens to answer several questions about how RNA viruses spread and evolve. To address each of the questions, I link mathematical techniques for modeling viral population dynamics with phylogenetic and coalescent techniques for analyzing and modeling viral genetic sequences and evolution. The first project uses multi-scale mechanistic modeling to show that decreases in viral substitution rates over the course of an acute infection, combined with the timing of infectious hosts transmitting new infections to susceptible individuals, can account for discrepancies in viral substitution rates in different host populations. The second project combines coalescent models with within-host mathematical models to identify driving evolutionary forces in chronic hepatitis C virus infection. The third project compares the effects of intrinsic and extrinsic viral transmission rate variation on viral phylogenies.

Hepatitis B and C prevalence in Portugal: disparity between the general population and high-risk groups

Background and aims: The prevalence of anti-HCV and HBsAg in Portugal has been shown to be elevated in high-risk groups, such as intravenous drug-users and incarcerated individuals. However, in the general population, prevalence remains largely unknown. The aims of this study were to estimate the prevalence of anti-HCV and HBsAg in the general Portuguese population and identify associated risk factors. Materials and methods: We carried out a nationwide, population-based cross-sectional study of adults resident in mainland Portugal. Serology for HBsAg, anti-HBc, anti-HBs, and anti-HCV was performed. Anti-HCV-positive individuals were tested for HCV RNA by PCR. Results: Of 1685 participants, 50.6% were men, mean age 50.2±18.3 years. In terms of hepatitis C, the prevalence of anti-HCV was 0.54% [95% confidence interval (CI): 0.2–0.9] and 0.12% (95% CI: 0.0–0.3) were viremic, with peak prevalence among individuals 35–64 years of age (0.8%), men (0.8%), and individuals from Lisbon and Tagus Valley region (1.9%). In terms of hepatitis B, the estimated prevalence of HBsAg was 1.45% (95% CI: 0.9–2.0). A higher prevalence was found in individuals who were 35–64 years old (2.2%), in men (2.5%), and in the Northern region (2.6%). The presence of positive serological markers of hepatitis C virus and hepatitis B virus infection did not correlate with elevated aminotransferases, race, place of birth, and alcohol consumption. Conclusion: These results suggest a low endemicity for both hepatitis B and hepatitis C in the general population, in contrast to a very high prevalence in risk groups, thus suggesting that targeted screening to high-risk groups may be more cost-effective than general population screening.

Viral hepatitis in patients infected with human immunodeficiency virus

From 1992 to 1995 we studied 232 (69% male, 87% Caucasian) anti-human immunodeficiency virus (anti-HIV) positive Brazilian patients, through a questionnaire; HIV had been acquired sexually by 50%, from blood by 32%, sexually and/or from blood by 16.4% and by an unknown route by 1.7%. Intravenous drug use was reported by 29%; it was the most important risk factor for HIV transmission. The alanine aminotransferase quotient (qALT) was >1 for 40% of the patients, 93.6% had anti-hepatitis A virus antibody, 5.3% presented hepatitis B surface antigen, 44% were anti-hepatitis B core antigen positive and 53.8% were anti-hepatitis C virus (anti-HCV) positive. The anti-HCV test showed a significant association with qALT>1. Patients for whom the probable HIV transmission route was blood had a 10.8 times greater risk of being anti-HCV positive than patients infected by other routes. Among 30 patients submitted to liver biopsy, 18 presented chronic hepatitis.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and high plasma homocysteine in chronic hepatitis C (CHC) infected patients from the Northeast of Brazil

Background/Aim: Hyperhomocysteinemia due to Methylenetetrahydrofolate Reductase (MTHFR) gene, in particular the C677T (Ala222Val) polymorphism were recently associated to steatosis and fibrosis. We analyzed the frequency of MTHFR gene in a cross-sectional study of patients affected by Chronic Hepatitis C (CHC) from Northeast of Brazil. Method: One hundred seven-four untreated patients with CHC were genotyped for the C677T MTHFR. Genomic DNA was extracted from peripheral blood cells and the C677T MTHFR polymorphism was identified by PCR-RFLP. The homocysteine (Hcy) levels were determined by chemiluminescence method. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and have current and past daily alcohol intake less than 100 g/week. Results: Among subjects infected with CHC genotype non-1 the frequency of MTHFR genotypes TT was 9.8% versus 4.4% genotype 1 (p = 0.01). Nevertheless, association was found between the MTHFR genotype TT x CT/CC polymorphism and the degree of steatosis and fibrosis in both hepatitis C genotype (p < 0.05). A significant difference was found on plasma Hcy levels in patients with steatosis regardless of HCV genotype (p = 0.03). Conclusion: Our results indicate that plasma Hcy levels is highly prevalent in subjects with chronic hepatits C with steatosis regardless of HCV genotype and vitamin deficiency. The presence of genotype TT of MTHFR C677T polymorphism was more common in CHC genotype non-1 infected patient regardless of histopathological classification and genotype TT+CT frequencies were significant in the presence of fibrosis grade 1+2 and of steatosis in CHC infected patients from the northeast of Brazil regardless of HCV genotype. The genetic susceptibility of MTHFR C677T polymorphism should be confirmed in a large population.

Prognostic factors for progression of liver structural lesions in chronic hepatitis C patients

AIM: To evaluate the epidemiological, clinical, laboratory and histological variables capable of predicting the progression of hepatic structural disturbances in chronic hepatitis C patients during the time interval between two liver biopsies. METHODS: Clinical charts of 112 chronic hepatitis C patients were retrospectively analyzed, whereas liver biopsies were revised. Immunohistochemical detection of interferon receptor was based on the Envision-Peroxidase System. RESULTS: In the multivariate analysis, the variables in the age at first biopsy, ALT levels, presence of lymphoid aggregates and siderosis were the determinants of the best model for predicting the severity of the disease. The direct progression rate of hepatic structural lesions was significantly higher in untreated patients, intermediate in treated non-responders and lower in treated responders to antiviral therapy (non-treated vs responders, 0.22 +/- 0.50 vs -0.15 +/- 0.46, P = 0.0053). Immuno-expression of interferon receptor is not a relevant factor. CONCLUSION: The best predictors of the progression of fibrosis are age at the first liver biopsy, extent of ALT elevation, inflammation at liver histology and hepatic siderosis. Antiviral treatment is effective in preventing the progression of liver structural lesions in chronic hepatitis C patients. (C) 2008 WJG. All rights reserved.