anti-HCV Reagent Research Articles

ANÁLISE COMPARATIVA DE MÉTODOS PARA TRIAGEM DE HCV EM SERVIÇOS DE HEMOTERAPIA

Dentre as hepatites virais, a hepatite C (HCV) constitui um significativo problema de saúde pública, havendo a necessidade de testes de triagem cada vez mais eficazes na detecção do HCV, a fim de aumentar a segurança do sangue doado. Esta pesquisa teve por objetivo comparar a eficácia da triagem para HCV entre as metodologias utilizadas pelo hemocentro, verificando, ainda, a possível ocorrência de redução da janela imunológica após a introdução da metodologia de teste de ácidos nucleicos (NAT). O estudo foi realizado por meio de avaliação retrospectiva de planilhas de registro sorológico de 12.443 doadores de sangue triados no período de julho de 2014 a junho de 2015 para o vírus HCV no Hemocentro Regional de Passo Fundo – Hemopasso. Dos possíveis doadores de sangue avaliados, 45 indivíduos (0,36%) apresentaram anti-HCV CMIA inconclusivo com resultado do NAT indetectável. Apenas 7 (0,05%) dos candidatos à doação de sangue apresentaram anti-HCV reagente pela metodologia CMIA e NAT detectável. Não foi observada qualquer detecção no período de janela imunológica do HCV pelo teste NAT. Não houve nenhum resultado falso-positivo pela metodologia CMIA. Assim, as metodologias CMIA e NAT utilizadas em associação são de extrema importância para o esclarecimento de possíveis falsos resultados, essenciais para a diminuição do risco de contaminações pós-transfusionais.

Reavaliação laboratorial da infecção pelo vírus da hepatite C em dialisados atendidos na década de 1990, em Belém, estado do Pará, Brasil

ABSTRACT OBJECTIVE: To describe aspects of laboratory serological and molecular diagnosis of hepatitis C virus (HCV) infection in patients undergoing treatment at a dialysis clinic in Belem, Para State, Brazil, from 1990 to 1993. MATERIALS AND METHODS: Retrospective study for reassessment of serum samples from dialysed patients using serological tests for the detection of HCV (anti-HCV and Ag-Ab HCV) and HCV-RNA by molecular RT-PCR. RESULTS: A total of 115 dialysed patients with serum samples collected during the study period were included. Analysis of these samples showed a 16.5% prevalence of anti-HCV reagent, among which 4.3% showed an indicator of active viral replication (HCV-RNA reagent). Seroconversion rate was detected in 55.2% of patients in the analyzed period. The anti-HCV group, Ag-Ab HCV and HCV-RNA reagent were the most prevalent (41.7%). For non-reactive anti-HCV samples, viral replication with HCV-RNA reagent was 22.7%. Genotypes 1 and 3 were detected. Genotype 1 was prevalent in 73.0% of the samples. CONCLUSION: When laboratory screening for HCV (anti-HCV) was introduced in the dialysis clinic, it was possible to observe: high prevalence of HCV infection among patients under treatment; high rates of seroconversion; equation between serological results using second and third generation ELISA methods; presence of viral replication among nonreactive anti-HCV patients; and higher prevalence of HCV genotype 1. Keywords: Hepatitis C Virus; Serological Tests; Hemodialysis

Prevalência sorológica e intensidade da resposta humoral entre indivíduos infectados com Vírus da Hepatite C

A infecção crônica pelo vírus da Hepatite C (HCV) é considerada grave, podendo progredir para cirrose e descompensação hepática. Objetivos: Verificar a prevalência da infecção pelo HCV entre os indivíduos triados, entre setembro de 2007 e setembro de 2009 determinando a correlação dos níveis séricos de anticorpos com presença do RNA viral. Métodos: Os resultados sorológicos da pesquisa de anticorpos anti HCV pelo método imunoenzimático ELISA (BioMérieux®) de 4536 indivíduos foram analisados retrospectivamente. O resultado da pesquisa molecular (COBAS AMPLICOR HCV Roche Diagnostics®) de 79 pacientes foi obtido através de registros médicos, sendo os genótipos 1 e 3 os mais frequentes. A probabilidade do resultado sorológico estar relacionado com a pesquisa molecular foi verificada através de curva ROC, construída para diferentes valores de Índex (DO amostra/DO ponto de corte do ELISA). Resultados: Das amostras para as quais foi realizado o teste molecular, 82,3% confirmaram presença de RNA HCV. Os Valores preditivos positivos (VPP) e Valores preditivos negativos (VPN) para presença de viremia, considerando valores de Índex 4,0 3,0 e 2,0 foram: para Índex 4,0 sensibilidade (S) de 62%, especificidade (E) 64%, VPP de 89%, VPN de 27%. Para Índex 3,0 S=93,0%; E=36,0%, VPP 87%, VPN 55%. Para Índex 2,0 S=100%, E=21%, VPP 85%; VPN 100%. Conclusão: Pode-se inferir que há grande probabilidade de pacientes com reação sorológica para pesquisa de anti-HCV reagente e Índex menor que 2,0, terem suprimido a infecção pelo HCV, e com Índex superior a 4,0 apresentarem viremia presente.

A sero-epidemiological study on hepatitis C in China

To better understand and measure the status of hepatitis C virus (HCV) infection, we conducted a sero-epidemiological study using the remaining blood samples and data of the nationwide survey of hepatitis B in Chinese residents which was carried out in 2006. The anti-HCV reagent was screened out from the reagents by the HCV infection blood serum plate with anti-HCV positives or negatives. This plate recognized the Murex 3.0 and Ortho 3.0 reagents as gold standards. Anti-HCV in the blood samples were tested using this reagent and confirmed by Chiron HCV RIBA 3.0 reagents. Among the population aged 1 year to 59 year-olds, the overall prevalence rate of anti-HCV was 0.43% (95%CI: 0.33% - 0.53%), with the rates of anti-HCV among males and females as 0.46% and 0.40%, respectively. The prevalence rate of anti-HCV in urban area was 0.43%, and in rural area it was 0.43%. The prevalence rate of anti-HCV in the Eastern, Middle and Western areas were 0.37% (95%CI: 0.21% - 0.53%), 0.67% (95%CI: 0.40% - 0.94%) and 0.31% (95%CI: 0.20% - 0.42%) respectively. The prevalence rates of anti-HCV for the three areas did not show significant differences, statistically. The prevalence rate of anti-HCV in the South and North areas were 0.29% (95%CI: 0.21% - 0.52%) and 0.53% (95%CI: 0.38% - 0.64%) respectively. Our data revealed that China was in the low prevalence area for hepatitis C infection and the results also suggested that the comprehensive measures for HCV control and prevention had been successfully achieved in the country.

Hydroxyurea as an inhibitor of hepatitis C virus RNA replication

Hepatitis C virus (HCV) is the main causative agent of chronic liver disease, which may develop into liver cirrhosis and hepatocellular carcinoma. By using a recently developed reporter assay system in which genome-length HCV RNA replicates efficiently, we found that hydroxyurea (HU), a DNA synthesis inhibitor, inhibited HCV RNA replication. Moreover, we demonstrated that the anti-HCV activity of the combination of IFN-alpha and HU was higher than that of IFN-alpha alone. These results suggest that HU may be an effective anti-HCV reagent that can be used not only singly but also in combination with IFN-alpha to treat chronic hepatitis C.

Suppression of Hepatitis C Virus (HCV) Replication with Serine Palmitoyltransferase Inhibitor

Hepatitis C virus (HCV) persists chronically in most infected patients, eventually causing chronic hepatitis, liver cirrhosis, and in some cases hepatocellular carcinoma. The combination therapy of PEG-IFN and ribavirin improves efficacy in many patients, although it does not lead to sufficient achievements in genotype 1b patients. To aid in invention of new anti-HCV reagents, we focused on host factors that contributed to HCV lifecycle. We identified serine palmitoyltransferase inhibitor as an anti-HCV reagent through high-throughput screening using HCV replicon cells. We investigated the mechanism of anti-HCV effect of SPT inhibitor. It has been reported that sphingolipids and cholesterol compose the lipid raft where replication of HCV occurs. We investigated the influence of SPT inhibitor to lipid rafts by analyzing the detergent-resistant membrane (DRM). The analysis showed that SPT inhibitor moved HCV RNA-dependent RNA polymerase (NS5B) to detergent-soluble fraction from DRM, and Biacore analysis indicated binding of sphingomyelin to NS5B. These results suggest that SPT inhibitor disrupts the interaction between NS5B and sphingomyelin. Moreover, we evaluated the anti-HCV effect of SPT inhibitor in vivo with humanized chimeric mice. SPT inhibitor led to rapid decline in serum HCV-RNA of about 1-2 log within 8 days. Furthermore, combination therapy of SPT inhibitor and PEG-IFN achieved about 3 log reduction in serum HCV-RNA.

HCV genotype 1b chimeric replicon with NS5B of JFH-1 exhibited resistance to cyclosporine A

Cyclosporine A (CsA) is a well-characterized anti-HCV reagent. Recently it was reported that the genotype 2a JFH-1 strain was more resistant than genotype 1 HCV strains to CsA in a cell culture system. However, the JFH-1 responsible region for the resistance to CsA remains unclear. It was also demonstrated that in genotype 1b HCVs, NS5B interacts with cyclophilin (CyP). To clarify whether or not NS5B of JFH-1 is significant for CsA resistance, we developed a chimeric replicon with NS5B of JFH-1 in the genotype 1b backbone. The chimeric replicon was more resistant to CsA than the parental genotype 1b replicon. Furthermore, reduction of CyPA had a greater effect on HCV RNA replication and sensitivity to CsA than reduction of CyPB. Here, we demonstrated that NS5B of JFH-1 contributed to this strain's CsA-resistant phenotype. NS5B and CyPA are significant for determining HCV's sensitivity to CsA.

Efficient replication systems for hepatitis C virus using a new human hepatoma cell line

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23's cDNA expression profile differed from HuH-7's, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets.

Oncostatin M synergistically inhibits HCV RNA replication in combination with interferon-α

Oncostatin M (OSM), a member of the interleukin-6 family, possesses various functions, including hepatocyte differentiation and suppression of melanoma cell growth. Here, we report anti-hepatitis C virus (HCV) activity of OSM as a new function of this cytokine. OSM possessed marked anti-HCV activity (50% effective concentration: 0.71 ng/ml) in an HCV RNA replication cell culture system. The most striking finding is that OSM exhibited synergistic inhibitory activity on interferon (IFN)-α even at a low concentration with weak anti-HCV activity, such as 25 pg/ml. OSM is a candidate anti-HCV reagent and may improve the current IFN therapy for patients with chronic hepatitis C.

Suppression of hepatitis C virus with the reagent targetting host factors

Hepatitis C virus (HCV) develops persistent infection in most infected patients, and eventually cause chronic hepatitis, liver cirrhosis and then hepatocellular carcinoma. The combination therapy of PEG-IFN and ribavirin improves the efficacy in many patients, while it does not lead to sufficient achievements in genotype1b patients. To invent new anti-HCV reagent, we focused on host factors which HCV take advantage of in its life-cycle. We identified serine palmitoyltransferase inhibitor as anti-HCV reagent through high-through put screenig using HCV replicon cells. Moreover, we evaluate the anti-HCV effect of SPT-inhibitor in vivo with humanized chimeric mice. SPT-inhibitor led to rapid decline in serum HCV-RNA of about 1-2log within 8 day, futhermore the combination therapy of SPT-inhibitor and PEG-IFN achieved about 3log reduction in serum HCV-RNA. At last, we investigated the mechanism of anti-HCV effect of SPT-inhibitor. It has been reported that sphingolipids and cholesterol compose the lipid raft, in which the replication of HCV occur. We investigated the influence of SPT-inhibitor to lipid rafts by analysing the detergent resistant membrane (DRM). The analysis proved that SPT inhibitor got HCV RNA dependent RNA polymerase (NS5B) to move to detergent soluble fraction from DRM, and Biacore analysis indicated the binding of sphingomyelin to NS5B. These results suggested SPT inhibitor got NS5B to release from replication complex.

Effects of bezafibrate in patients with chronic hepatitis C virus infection: combination with interferon and ribavirin

An association of hepatitis C virus (HCV) with low-density lipoproteins (LDL) in serum of patients with chronic hepatitis C (CHC) has been suggested. We conducted a prospective study in CHC patients complicated with hyperlipidaemia, to examine whether bezafibrate, which is commonly used for treatment of hyperlipidaemia, reduces serum HCV-RNA titre and improves liver dysfunction. Fifteen patients received daily oral bezafibrate treatment (400 mg/day) for 8 weeks, and its effects on serum lipids, transaminases, HCV-RNA titres, and HCV-RNA titres bound to LDL were evaluated. Fifteen untreated patients with CHC and hyperlipidaemia were used as controls. The mean serum alanine aminotransferase levels and HCV-RNA titres significantly decreased at the end of bezafibrate therapy in the treated group (105 +/- 34 to 80 +/- 32 IU/L, P = 0.02 and 2.23 +/- 2.71 to 1.78 +/- 2.38 x 10(7) copies/mL, P < 0.01 respectively), but no changes were observed in the control group. Serum HCV-RNA titres bound to LDL, as quantified by immunoprecipitation using anti-LDL antibody, also decreased in all 15 treated patients [5.55 +/- 6.59 to 1.07 +/- 1.58 x 10(6) copies/ml, P < 0.01 (mean reduction rate was -78.5 +/- 17.0%)]. Sucrose density-gradient ultracentrifugation study revealed that HCV-RNA-decreased density fractions after the bezafibrate were identical to LDL-density fractions (1.015-1.062 g/mL). Eight CHC patients were treated with bezafibrate, interferon, and ribavirin triple therapy for 32 weeks, and four patients achieved sustained virological response to therapy. This pilot study provides further evidence of an association between HCV and LDL in serum and suggests the potential usefulness of bezafibrate as an anti-HCV reagent for the treatment of CHC patients.

Mizoribine inhibits hepatitis C virus RNA replication: Effect of combination with interferon-α

Interferon (IFN)-α monotherapy, as well as the more effective combination therapy of IFN-α and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side effects such as anemia due to the administration of ribavirin present a problem for patients who are advanced in years. Using a recently developed reporter assay system in which genome-length dicistronic HCV RNA encoding Renilla luciferase gene was found to replicate efficiently, we found that mizoribine, an imidazole nucleoside, inhibited HCV RNA replication. The anti-HCV activity of mizoribine (IC 50: approximately 100 μM) was similar to that of ribavirin. Using this genome-length HCV RNA replication monitor system, we were the first to demonstrate that the combination of IFN-α and ribavirin exhibited more effective anti-HCV activity than the use of IFN-α alone. Moreover, we found that the anti-HCV activity of mizoribine in co-treatment with IFN-α was at least equivalent to that of ribavirin. This effect was apparent in the presence of at least 5 μM mizoribine. Since mizoribine is currently used in several clinical applications and has not been associated with severe side effects, mizoribine is considered to be of potential use as a new anti-HCV reagent in combination with IFN-α.

A clinical, epidemological, laboratorial, histological and ultrasonographical evaluation of anti-HCV EIA-2 positive blood donors.

Between 1992 and 1997, 790 blood donors with anti-HCV EIA-2 strongly reagent (relationship between the sample optical density/cut-off > 3) detected at the blood bank serological screening, were evaluated in ambulatory environment. They were all negative for Chagas disease, syphilis, hepatitis B (HBsAg) and AIDS. Blood samples were collected at the first ambulatorial evaluation, for hemogram, biochemical tests and new serological tests for HCV (anti-HCV EIA-2). In blood samples of 226 repeatedly reagent anti-HCV EIA-2 blood donors, supplementary "immunoblot" test for HCV (RIBA-2) was used. In 209 donors, the presence of HCV-RNA was investigated by the PCR test. The abdominal ultrasonography was realized in 366 donors. In 269 patients liver biopsy was performed for the histopathological study. The follow-up of blood donors showed that 95.6% were repeatedly EIA-2 reagent, 94% were symptomless and denied any hepatitis history, with only 2% mentioning previous jaundice. In 47% of this population at least one risk factor has been detected for the HCV transmission, the use of intravenous drugs being the main one (27.8%). Blood transfusion was the second factor for HCV transmission (27.2%). Hepatomegaly was detected in 54% of the cases. Splenomegaly and signs of portal hypertension have seldom been found in the physical examination, indicating a low degree of hepatic compromising in HCV. Abdominal ultrasound showed alterations in 65% of the subjects, being the steatosis the most frequent (50%). In 83. 5% of the donors submitted to the liver biopsy, the histopathological exam showed the presence of chronic hepatitis, usually classified as active (89%) with mild or moderate grade in most of the cases (99.5%). The histopathological exam of the liver was normal in 1.5% of blood donors. The RIBA-2 test and the HCV-RNA investigation by PCR were positive in respectively 91.6 and 75% of the anti-HCV EIA-2 reagent donors. The HCV-RNA research was positive in 82% of the RIBA-2 positive subjects, in 37.5% of the indeterminate RIBA-2 donors and in 9% of the negative RIBA-2 donors. Chronic hepatitis has also been observed in 50% of the histopathological exams of the anti-HCV EIA-2 reagent donors which were indeterminate RIBA-2. Among 18 blood donors with minimal changes histopathological exam 11 (61%) were HCV-RNA positive. Our blood donors anti-HCV reagent generally had clinical, laboratorial and histopathological features observed in patients with chronic HCV hepatitis and a high proportion could be identified in interviews and medical evaluation realized in blood blanks. Generally, these HCV infected donors are identified and discharged only by the serological tests results.

Lactoferrin Markedly Inhibits Hepatitis C Virus Infection in Cultured Human Hepatocytes

We found that bovine lactoferrin (bLF), a milk protein belonging to the iron transporter family, effectively prevented hepatitis C virus (HCV) infection in cultured human hepatocytes (PH5CH8), a cell line susceptible to HCV infection and supportive of HCV replication. Because preincubation of HCV with bLF was required to prevent the infection of HCV to the cells, and preincubation of bLF with the cells showed no inhibitory effect on HCV infection, we demonstrated that the anti-HCV activity of bLF was due to the interaction of bLF with HCV, but not due to the interaction of bLF with the cells. We further found that human lactoferrin also had anti-HCV activity, but bovine transferrin, the other member of the iron transporter family, did not have anti-HCV activity. Our findings suggest that lactoferrin is one of candidates for an anti-HCV reagent that will be well-tolerated and effective in the treatment of patients with chronic hepatitis.