Método de reacción en cadena de la polimerasa para determinar la replicación del virus de la hepatitis B / Polymerase chain reaction method to determine replication of hepatitis B virus

Objetivo: evaluar el desempeño clínico de un método de reacción en cadena de la polimerasa con modificaciones para la detección de la replicación del virus de la hepatitis B en pacientes infectados. Métodos: se estudiaron 266 muestras de suero de pacientes provenientes de los servicios de Gastroenterología, Trasplante, Hemodiálisis y Hematología del Hospital Clínicoquirúrgico Hermanos Ameijeiras, con el antígeno de superficie (HBsAg) positivo y altos valores de enzimas hepáticas (aspartatoaminotransferasa, aspartatoaminotransglutaminasa y ganmaglutamiltrasferasa), así como 5 muestras de individuos clínicamente sanos. El ADN se extrajo mediante el método del fenol-cloroformo. Se amplificó un fragmento de la región Pre S del virus de la hepatitis B y un fragmento del gen de la ß-globina como control interno de la reacción. Resultados: El fragmento del gen de la ß-globina se verificó en las 266 muestras estudiadas. La región Pre S del virus de la hepatitis B en cambio, fue detectada en 103 de ellas, siendo informadas como positivas para la replicación viral. En la muestra se obtuvo una prevalencia 98,15 por ciento para la replicación del virus de la hepatitis B. La especificidad clínica del ensayo fue del 100 por ciento, la sensibilidad clínica del 38 por ciento, el valor predictivo positivo del 100 por ciento y el valor predictivo negativo del 64 por ciento. El análisis de concordancia no mostró acuerdo (κ= 0,022 para una p= 0,07) entre las enzimas hepáticas y la reacción en cadena de la polimerasa. Los ensayos de repetitividad y de reproducibilidad mostraron que los resultados son reproducibles. El mayor porcentaje de positividad se encontró en los pacientes remitidos por el Servicio de Hematología (66,7 por ciento). Conclusiones: el método de reacción en cadena de la polimerasa evaluado constituye una herramienta certera para el monitoreo de la replicación del virus de la hepatitis B. Contar con su implementación y aplicación en la institución que se efectuó el estudio reviste gran importancia para el sistema de salud del país(AU)

Objective: evaluate the clinical performance of a modified polymerase chain reaction method to detect replication of the hepatitis B virus in infected patients. Methods: a study was conducted of 266 serum samples from patients cared for at gastroenterology, transplantation, hemodialysis and hematology services of Hermanos Ameijeiras Clinical Surgical Hospital with positive surface antigen HBsAg and high liver enzyme values (aspartate aminotransferase, aspartate aminotransglutaminase and gamma-glutamyltransferase), and 5 samples from clinically healthy individuals. The DNA was extracted by the phenol-chloroform method. Amplification was performed of a fragment of the Pre S region of the hepatitis B virus and a fragment of the ß-globin gene as internal control of the reaction. Results: the ß-globin gene fragment was found in the 266 samples studied. The Pre S region of the hepatitis B virus, however, was detected in 103 of them. These were reported as positive for viral replication. The sample exhibited a prevalence of 98.15 percent for replication of the hepatitis B virus. Clinical specificity of the assay was 100 percent, clinical sensitivity was 38 percent, positive predictive value was 100 percent and negative predictive value was 64 percent. Concordance analysis did not reveal any agreement (κ= 0.022 for p= 0.07) between the liver enzymes and the polymerase chain reaction. Repeatability and reproducibility assays showed that the results are reproducible. The highest positivity percentage was found in patients referred from the Hematology Service (66.7 percent). Conclusions: the polymerase chain reaction method evaluated is an accurate tool to monitor replication of the hepatitis B virus. The possibility of its implementation and application at the institution where the study was conducted is very important for the Cuban health system(AU)

functional E1-E2 heterodimer HCV glycoproteins

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. Methods and pathways: After their synthesis, HCV glycoproteins E1 and E2 associate as a non covalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1-E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1-E2 has been proposed to be essential for HCV entry. Results and However, for a long time, HCV entry studies have been limited by the lack of a robust cell culture system for HCV replication and viral particle production. Recently, a model mimicking the entry process of HCV lifecycle has been developed by pseudo typing retroviral particles with native HCV envelope glycoproteins, allowing the characterization of functional E1-E2 envelope glycoproteins., we review our understanding to date on the assembly of the functional HCV glycoprotein heterodimer

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

Introduction Despite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus. Methods Platelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR. Results After incubation in the presence of virus, all samples of platelets showed HCV RNA. Conclusions The results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association. .

Hepatitis C virus: Molecular biology & current therapeutic options.

Hepatitis C virus (HCV) is a small (~55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent development of a robust cell culture system for HCV propagation. HCV proteins assemble and form replication complexes on modified host membranes, called as membranous webs. Even though HCV is detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments; and the viral population is known to rapidly change during the course of a natural infection thereby escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission. The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins during HCV lifecycle is vital for developing a successful cure. This review deals with current status of efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and rapidly changing hepatitis C virus.

Characteristics of dual infection of hepatitis B and C viruses among patients with chronic liver disease: a study from tertiary care hospital.

The major causes of chronic liver disease (CLD) are infection with Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) either alone or together. The clinical course of the disease varies in cases ofcoinfection with HCV and HBV as compared to single infection. The present study was carried out to determine the occurrence of coinfection of HCV with HBV in CLD patients and to look for the presence of suppressive effect of the two viruses on each other. The severity of liver disease was also assessed and correlated with biochemical profiles. Sera from 150 patients of CLD were tested serologically for the presence of HBsAg, IgG anti HBc and anti-HCV antibodies. HBV DNA and HCV RNA were also detected by amplifying surface region and 5' noncoding-core region respectively by polymerase chain reaction. Forty-seven (31.3%) cases showed the presence of HBsAg or anti IgG-HBc or HBV DNA either alone or together (Group A). Thirty-nine (26%) cases were found to be positive for HCV by detecting either anti-HCV antibodies or HCV RNA (Group B). Coinfection ofHCV with HBV (Group C) could be detected in twenty-four (16%) cases, of these twenty-one cases (87.5%) were positive both for HCV RNA and IgG anti-HBc without the presence of HBV DNA whereas in none of the cases could HBV DNA be detected in the absence of HCV RNA. Forty (26.6%) cases had neither HCV or HBV related CLD. Amongst, the biochemical parameters, the liver function test profiles were altered and found to be statistically significantly in HCV positive cases (Group B) when compared to the negative ones while in case of HBV (Group A) and coinfected (Group C) cases none of the parameters was statistically significant when compared with non-HBV and non-coinfected cases respectively. Thus, coinfection of HCV with HBV is seen in a substantial number of CLD cases. It is also revealed from the present study that HCV infection has a suppressive effect on the replication of HBV as seen by the loss of replicative markers like HBV DNA.

The relationship between virological characteristics of hepatitis C virus (HCV) and reactivity to the regional specific proteins of HCV

BACKGROUND: Although the polyproteins of hepatitis C virus(HCV) are processed and formed in nearly equimolar amounts, individual functional proteins have a discrepancy in their time of appearance following HCV infection and eliciting immune response. This study was conducted to compare the reactivity toward regional specific HCV protein in relation to virological characteristics, including HCV genotype and HCV replication. METHODS: Sera from forty-five patients with chronic HCV infection were analyzed through the experiments of the recombinant immunoblot assay(RIBA-2), HCV genotyping and HCV RNA quantitation. RESULTS: The frequencies of seropositivity to C22-3, C33C, C100-3 and 5-1-1 proteins were 91.1+ACU-, 91.1+ACU-, 64.4+ACU- and 53.3+ACU-, respectively, of all the patients, and thus the antibodies to C22-3 and C33C proteins were found more frequently (p +ADw- 0.05). The antibody responses between core or NS3 proteins and NS4 proteins showed more discrepancy in the HCC group than that in the CH group, implying a possibility of oncogenic potential of core or NS3 gene in hepatocarcinogenesis. The detection rate of antibodies to C22-3 and C33C, in accordance with serum HCV RNA levels, was significantly higher in highly viremic patients than that in low viremic patients (p +ADw- 0.05). Antibodies to C22-3, C33C, C100-3 and 5-1-1 were also found more frequently in patients with HCV genotype 1b, compared to those with HCV genotype 2a (p +ADw- 0.05). CONCLUSION: These results suggest that antibody detection of HCV may depend on the virological characteristics of HCV, the levels of HCV replication and HCV genotype and, therefore, HCV RNA detection using RT-PCR technique is essential for confirmatory diagnosis for HCV infection. Furthermore, the HCV core or NS3 Protein may play important role in hepatocarcinogenesis.

Determinación del RNA genómico y replicativo del virus de la hepatitis C en pacientes tratados con interferón / Determination of genomic RNA and replicative hepatitis C viruses in patients treated with interferon

En este ensayo se trató de correlacionar la evolución clínica e histológica de un grupo de pacientes tratados con IFN, con la presencia del RNA genómico y replicativo del HCV en plasma y tejido hepático. Se estudiaron 11 pacientes con hepatitis crónica "C" diagnosticada por elevación de transaminasas durante los últimos 6 meses, anti-HCV positivo "(Elisa II)", y cuadro histológico compatible. En todos ellos se efectuó biopsia hepática y extracción de sangre en paralelo, inmediatamente antes y después de finalizar el tratamiento con 4.5 millones de IFN Alfa2a administrado 3 veces por semana, durante 6 meses. El tecijo hepático se almacenó a-80§c y el plasma a-20§c hasta su procesamiento. La extracción de RNA se realizó a partir de 100microliter de plasma y de 20mg de tejido hepático con isotiocianato de guanidinio (Chomcznski). La transcripción reversa se llevó a cabo "primers" sense o antisense para detectar la hélice replicativa (-) o genómica (+) respectivamente. El cDNA de la región 5' no codificante fue amplificado por el sistema "nested". Antes del tratamiento los 11 pacientes evidenciaron la presencia de la hélice genómica en tecijo hepático y plasma. En cambio la hélice replicativa se detectó en 5 casos en hígado, 7 pacientes se revelaron como respondedores y 4 como no respondedores. En los pacientes respondedores las hélices genómica y replicativa en hígado desaparecieron en un 43 por ciento y 57 por ciento respectivamente, y en plasma se observó un descenco del 71 por ciento para la hélice genómica y de un 100 por ciento para la hélice replicativa. En los 4 pacientes no respondedores la hélice genómica permaneció en tecijo hepático y plasma, en cambio la replicativa se mantuvo en tejido hepático y se negativizó en un 75 por ciento en plasma. El índice de Knodell, que determina el estadío histológico, disminuyó en 5 de lo 7 respondedores y permaneció igual en 3 de los 4 no respondedores...

Virus de la hepatitis C y hepatocarcinoma

Se peresenta el caso de un paciente de sexo masculino de 56 años de edad con antecedentes de hemofilia B, que recibió suplencia de factor IX para un procedimiento quirúrgico y diez años más tarde se encuentra con cuadro de hepatitis crónica por virus C, cirrosis secundaria asociada a una masa hepática compatible con hepatocarcinoma comprobada por ultrasonografía, tomografía axial computarizada,arteriografía hepática, y por los valores anormalmente altos de alfafetoproteina. El virus de la hepatitis C recientemente identificado, es responsable de al menos 85 por ciento de la llamadas hepatits No-A-No-B y puede ser factor desencadenante de cirrosis y hepatocarcinoma, por lo cual es indispensable su identificación y prevención adecuadas.