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.

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. .

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors.

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Human platelet antigen genotype is associated with progression of fibrosis in chronic hepatitis C.

Although progression of fibrosis in the chronic hepatitis C depends on environmental, viral, and host factors, genetic polymorphisms have been associated recently with this progression, including the expression of integrins, adhesion proteins. Some integrins expressed on the platelet membrane show polymorphic antigenic determinants called human platelet antigens (HPA), where the major ones are HPA-1, -3, -5. The association between HCV infection and HPA-5b has been demonstrated. Similarly, the HPA profile could determine if HPA is related to progression of fibrosis. The goal of this study was to evaluate the association between the frequencies of HPA-1, -3, and -5 and degree of fibrosis in HCV-infected patients. Genomic DNA from 143 HCV-infected patients was used as the source for HPA genotyping by PCR-SSP or PCR-RFLP. Progression of fibrosis was evaluated using the METAVIR scoring system, and the patients were grouped according to degree of fibrosis into G1 (n = 81, with F1, portal fibrosis without septa or F2, few septa) and G2 (n = 62, with F3, numerous septa, or F4, cirrhosis). Statistical analysis was performed using the proportional odds model. The genotypic frequency of HPA-1a/1b was significantly higher in the patients in G2. To evaluate the influence of the time of infection to the development of fibrosis and its effect on the genetic factor HPA-1, 96 patients from 143 studied were evaluated considering the time of HCV infection, and these results suggest that the HPA-1a/1b genotype promotes the development of fibrosis in HCV infection with time.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report.

Amino acid insertions in the protease have rarely been described in HIV-infected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report / Primeiro caso de inserção de aminoácidos no codon 35 da protease em uma sequência de HIV-1 do subtipo B detectada na região de Bauru, estado de São Paulo, Brasil: relato de caso

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

[Reverse hybridization and sequencing for genotyping the hepatitis C virus]. / Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C.

INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA(R) v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.

Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C / Reverse hybridization and sequencing for genotyping the hepatitis C virus

INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.

INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA® v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.

Using pegylated interferon alfa-2b and ribavirin to treat chronic hepatitis patients infected with hepatitis c virus genotype 1: are nonresponders and relapsers different populations?

The combination of pegylated interferon (PEG-INF) and ribavirin is currently the best treatment for chronic hepatitis C, providing a sustained virological response (SVR) in 54 percent-63 percent of patients. In patients infected with hepatitis C virus (HCV) genotype 1, the SVR rate is 42 percent-52 percent. To evaluate the treatment efficacy of this drug combination, we conducted an open, prospective study of 58 consecutive treatment-naïve patients infected with HCV genotype 1 and treated at a university hospital, comparing those presenting an SVR (SVRs), nonresponders (NRs), and relapsers (RELs). Among the intent-to-treat patients, an end-of-treatment virological response was achieved in 69 percent of the sample as a whole and in 52 percent of the SVRs. We found that being an SVR was significantly associated with mild fibrosis (p = 0.04) and with undetectable HCV RNA at weeks 12 and 24 of treatment (p < 0.0001). Comparing the SVR and REL groups, we observed that being older than 40 was significantly associated with being a REL (p = 0.04). Being an NR was found to be associated with severe fibrosis and moderate inflammatory infiltrates (portal or periportal). In the polytomous logistic regression, no independent factors were associated with the REL group when compared with the SVR group. We conclude that RELs and NRs differ in comparison with SVRs. The RELs accounted for 17 percent of the sample. The HCV RNA test results at weeks 12 and 24 of treatment, although independent predictors of non-response (OR: 4.8 and 8.2, respectively), did not differ between SVRs and RELs.

Using pegylated interferon alfa-2b and ribavirin to treat chronic hepatitis patients infected with hepatitis c virus genotype 1: are nonresponders and relapsers different populations?

The combination of pegylated interferon (PEG-INF) and ribavirin is currently the best treatment for chronic hepatitis C, providing a sustained virological response (SVR) in 54%-63% of patients. In patients infected with hepatitis C virus (HCV) genotype 1, the SVR rate is 42%-52%. To evaluate the treatment efficacy of this drug combination, we conducted an open, prospective study of 58 consecutive treatment-naïve patients infected with HCV genotype 1 and treated at a university hospital, comparing those presenting an SVR (SVRs), nonresponders (NRs), and relapsers (RELs). Among the intent-to-treat patients, an end-of-treatment virological response was achieved in 69% of the sample as a whole and in 52% of the SVRs. We found that being an SVR was significantly associated with mild fibrosis (p = 0.04) and with undetectable HCV RNA at weeks 12 and 24 of treatment (p < 0.0001). Comparing the SVR and REL groups, we observed that being older than 40 was significantly associated with being a REL (p = 0.04). Being an NR was found to be associated with severe fibrosis and moderate inflammatory infiltrates (portal or periportal). In the polytomous logistic regression, no independent factors were associated with the REL group when compared with the SVR group. We conclude that RELs and NRs differ in comparison with SVRs. The RELs accounted for 17% of the sample. The HCV RNA test results at weeks 12 and 24 of treatment, although independent predictors of non-response (OR: 4.8 and 8.2, respectively), did not differ between SVRs and RELs.