Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis.

The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis

ABSTRACT The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Hepatitis B: Prevalence and occult infection in HIV-infected patients.

INTRODUCTION: HBV and HIV have identical transmission routes. The aim of this study was to determine the prevalence of HBV in HIV patients and to detect the presence of occult HBV infection. METHODS: All samples were tested for serology markers and using qPCR. RESULTS: This study included 232 individuals, out of which 36.6% presented with HBV markers and 11.8% presented with HBsAg or HBV-DNA, including 3 patients that showed OBI. CONCLUSIONS: We observed a high prevalence of HBV among HIV patients. In addition, the results suggest that OBI can occur in patients with serological profiles that are indicative of past infection. Therefore, the application of molecular tests may enable the identification of infections that are not evident solely based on serology.

Hepatitis B: Prevalence and occult infection in HIV-infected patients

Abstract INTRODUCTION: HBV and HIV have identical transmission routes. The aim of this study was to determine the prevalence of HBV in HIV patients and to detect the presence of occult HBV infection. METHODS: All samples were tested for serology markers and using qPCR. RESULTS: This study included 232 individuals, out of which 36.6% presented with HBV markers and 11.8% presented with HBsAg or HBV-DNA, including 3 patients that showed OBI. CONCLUSIONS: We observed a high prevalence of HBV among HIV patients. In addition, the results suggest that OBI can occur in patients with serological profiles that are indicative of past infection. Therefore, the application of molecular tests may enable the identification of infections that are not evident solely based on serology.

Caracterização de mutações de resistência primária para as drogas antivirais de ação direta (DAA) para o vírus da Hepatite C / Characterization of primary resistance mutations for direct-acting antiviral drugs (DAA) for Hepatitis C virus

A Hepatite C constitui grave problema de saúde pública em todo mundo. Estima-se que, atualmente, em todo o mundo, cerca de 71 milhões de indivíduos estejam infectados por esse vírus. Na atualidade destacam-se os inibidores de polimerase (NS5B) e de N5A, como classes de medicamentos utilizados no tratamento, e todos esses estão disponíveis no SUS. O alto nível de replicação do HCV e sua falta de mecanismos de correção pós transcricionais resultam na rápida emergência de variantes virais no nível de quasispécies, determinando não somente polimorfismos virais, bem como variantes que albergam substituições associadas à resistência e/ou redução de suscetibilidade aos novos antivirais. A região com maior barreira genética é a NS5B, porém estudos devem ser realizados para avaliar a população de nosso país. O objetivo principal desse trabalho foi o mapeamento de polimorfismos e mutações de resistência às novas drogas destinadas ao tratamento da Hepatite C crônica, disponíveis em diferentes centros de referência para tratamento da Hepatite C e acompanhados laboratorialmente pelo Instituto Adolfo Lutz em São Paulo. Foram analisadas amostras de soros de pacientes que realizaram os testes de carga viral de rotina no laboratório de Hepatites do IAL, sendo distribuídas da seguinte maneira: para as regiões NS5A e NS5B 996 do GT1a e 1b. Além dessas amostras, foram analisadas 227 amostras do GT3, também para as regiões NS5A e NS5B. Para a região NS5A GT1, a prevalência de mutações de resistência foi de 4,5%, sendo as principais RAS encontradas: Y93H, L31M, Q30R e Y93N e para o GT3 foi de 8,8%, sendo as principais RAS encontradas: A30K, A62S e Y93H, para todos os medicamentos analisados e para a região NS5B não foram observados substituições de resistência em nenhum dos GT. Nossos achados são importantes para as avaliações clínicas, econômicas e para novas propostas de retratamento da Hepatite C crônica em nosso meio.

Fluctuations in serological hepatitis C virus levels in HIV patients.

INTRODUCTION: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) have identical transmission routes, explaining the high prevalence of coinfections. The main aim of this study was to detect fluctuations in serological HCV levels in HIV patients. METHODS: We analyzed samples of 147 patients who attended an outpatient service that supports HIV/AIDS patients in São Paulo city. We also recruited 22 HCV-monoinfected patients who attended the Instituto Adolfo Lutz Laboratory in São Paulo city, to compare the test results. Serological testing of the blood samples was performed for the detection of HCV antibodies. The samples were then analyzed using real-time PCR for RNA viral quantification and sequencing. RESULTS: We found that 13.6% of the study population was coinfected with HIV and HCV. In 20% of coinfected patients, fluctuations in serology results were detected in samples collected during the follow-up. No changes in anti-HCV serological markers were observed in HCV-monoinfected patients. An HCV viral load was detected in 9,5% of the samples collected from HIV patients. CONCLUSIONS: Our findings provide important clinical data to public health professionals and highlight the importance of periodic monitoring of HCV/HIV coinfected patients.

Fluctuations in serological hepatitis C virus levels in HIV patients

Abstract INTRODUCTION: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) have identical transmission routes, explaining the high prevalence of coinfections. The main aim of this study was to detect fluctuations in serological HCV levels in HIV patients. METHODS: We analyzed samples of 147 patients who attended an outpatient service that supports HIV/AIDS patients in São Paulo city. We also recruited 22 HCV-monoinfected patients who attended the Instituto Adolfo Lutz Laboratory in São Paulo city, to compare the test results. Serological testing of the blood samples was performed for the detection of HCV antibodies. The samples were then analyzed using real-time PCR for RNA viral quantification and sequencing. RESULTS We found that 13.6% of the study population was coinfected with HIV and HCV. In 20% of coinfected patients, fluctuations in serology results were detected in samples collected during the follow-up. No changes in anti-HCV serological markers were observed in HCV-monoinfected patients. An HCV viral load was detected in 9,5% of the samples collected from HIV patients. CONCLUSIONS: Our findings provide important clinical data to public health professionals and highlight the importance of periodic monitoring of HCV/HIV coinfected patients.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA.

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Identification of hepatitis B virus genotypes in the state of São Paulo / Identificação dos genótipos do vírus da hepatite B no Estado de São Paulo

Objective: the aim of this study was to identify HBV genotypes in serum samples from patients from the state of São Paulo, received by the viral hepatitis laboratory, at the Virology Centre of Instituto Adolfo Lutz, from various municipalities. Methods: a total of 94 serum samples were randomly analyzed. Genotyping was performed using nested PCR for amplification of S and Pol regions from viral genome. Genotypes were identified comparing the sequences obtained with the sequences deposited in GenBank. Results: we were able to determine the genotype of 91 (97%) samples, as follows: genotype A (55.3%), D (32%), F (5.3%), C (3.2%) and G (1%). There are few data on the epidemiology of genotype G. This genotype has been detected in restricted areas around the world. Frequently, the genotype G infection occurs in HIV-positive male patients. In our case, the sample identified as G was also positive for HIV but in a female patient, which is an uncommon finding in the scientific literature. Conclusion: in this work, we identified the most frequent genotypes in São Paulo as well as the genotype G, rare among the genotypes found in our environment. .

Objetivo: o objetivo deste estudo foi identificar os genótipos do HBV nas amostras de soros recebidas pelo Laboratório de Hepatites do Centro de Virologia do Instituto Adolfo Lutz. Métodos: foram analisadas aleatoriamente 94 amostras de soropositivas, provenientes de diversos municípios do Estado de São Paulo. Para determinação dos genótipos, foi realizada Nested-PCR das regiões S e Pol do HBV. Os genótipos foram identificados comparando os resultados amplificados com as sequências depositadas no GenBank. Resultados: foi possível determinar o genótipo de 91 (97%) amostras do total analisado e os genótipos identificados foram: genótipos A (55,3%), D (32%), F (5,3%), C (3,2%) e G (1%). Há poucos dados a respeito da epidemiologia do genótipo G. Esse genótipo tem sido detectado em áreas restritas do mundo. Geralmente, a infecção pelo genótipo G ocorre em indivíduos HIV positivos do sexo masculino. Neste trabalho, a amostra identificada como G foi também positiva para HIV e era de uma paciente do sexo feminino, dado raro na literatura científica. Conclusão: neste trabalho, foram identificados os genótipos mais frequentes, assim como uma cepa do genótipo G, rara entre os encontrados em nosso meio. .

Padronização da PCR em Tempo Real para o Diagnóstico da Hepatite B / Standardization of real-time PCR for the hepatitis B diagnosis

A hepatite B é uma doença de interesse em saúde pública, acometendo cerca de 2 bilhões de pessoas no mundo, das quais aproximadamente 350 milhões tornam-se portadoras crônicas. Os ensaios quantitativos de detecção do DNA viral são empregados para avaliar a infectividade do vírus e para o monitoramento do tratamento, sendo importante para avaliar a progressão da doença. O objetivo deste estudo foi padronizar a técnica de amplificação quantitativa em tempo real (PCR em tempo real) do DNA do vírus da Hepatite B e comparar a técnica com métodos comerciais (COBAS AmpliPrep/COBAS TaqMan® e COBAS AmpliPrep/COBAS TaqMan®). A amostragem foi constituída por 397 amostras de soro ou plasma recebidas e estocadas no laboratório de hepatites virais do Centro de Virologia do Instituto Adolfo Lutz, no período de 2009 a 2011. Do total de amostras, 345 foram utilizadas para a comparação com os testes comerciais disponíveis e 52 amostras foram utilizadas para o teste de especificidade, sendo 27 amostras HIV positivas e 25 amostras HCV positivas, todas com AgHBS e Anti-HBc total não reagentes. Estas amostras foram inicialmente analisadas por testes comerciais e posteriormente pela técnica padronizada. Para o controle da quantificação, uma curva padrão foi construída em todas as reações, utilizando-se padrões internacionais produzidos pela Organização Mundial de Saúde, em diluições seriadas. Para a extração do DNA das amostras e padrão, foi utilizado kit comercial (QIAGEN®), seguindo os procedimentos descritos pelo fabricante. Os testes foram realizados empregando o método do TaqMan – PCR, em um equipamento ABI 7300 (Applied Biosystems, Foster City, CA). Os “primers” e sondas utilizadas para a padronização foram selecionados a partir da literatura e os que apresentaram melhores resultados nos testes iniciais foram escolhidos para o estudo. A técnica de sequenciamento foi realizada nas amostras com carga viral quantificável...

Hepatitis B is a disease of public health concern. Worldwide, it affects approximately 2 billion people, of whom approximately 350 million become chronic carriers. Quantitative assays for the detection of viral DNA are used to evaluate the infectivity of the virus and to monitor treatment, which is crucial to evaluate the progression of the disease. The aim of this study was to standardize real-time (real-time PCR) quantitative amplification of the DNA of hepatitis B and to compare this technique with commercial methods (COBAS AmpliPrep / COBAS ® TaqMan and COBAS AmpliPrep / COBAS TaqMan ®). The sample consisted of 397 serum or plasma samples received and stored at the laboratory of viral hepatitis from the Center of Virology, Instituto Adolfo Lutz, from 2009 to 2011. Of the total samples, 345 were used for comparison with the available commercial tests and 52 samples were used for the specificity test. Of these, 27 were HIV positive and 25, HCV positive, and all were HBsAg and anti- HBc nonreactive. These samples were initially analyzed by commercial tests and later by the standardized technique. To control the quantification, a standard curve was constructed in all reactions using international standards established by the World Health Organization, in serial dilutions. For the extraction of DNA samples and standard, a commercial kit used was (QIAGEN ®) according to the manufacturer’s instructions. The tests were performed using the TaqMan method - ABI 7300 PCR system (Applied Biosystems, Foster City, CA). Primers and probes used for standardization were selected from the literature and those presenting better results in initial tests were chosen for the study. Sequencing technique was performed on samples with measurable viral load. The standardized real-time PCR technique yielded satisfactory results...

Echovírus 6 associado à doença exantemática / Echovirus 6 associated with exanthematic disease

Exantema viral é considerado problema comum em regiões tropicais, afetando principalmente crianças. Diversos exantemas cutâneos estão associados a infecções por Enterovirus. Amostras biológicas provenientes de uma criança apresentando exantema generalizado foram enviadas ao Laboratório de Vírus Entéricos do Instituto Adolfo Lutz para a realização do diagnóstico laboratorial. Amostra viral isolada em RD (human rhabdomyosarcoma cells) foi submetida à reação em cadeia pela polimerase apresentando um produto de 437 pares de base, característico de gênero Enterovirus. O sorotipo echovirus 6 (E-6) foi identificado por ensaio de imunofluorescência indireta. Em adição, as amostras pareadas de soro apresentaram soroconversão para E-6. Até o momento, não há relatos do envolvimento de E-6 associado a doenças exantemáticas no Brasil, enfatizando a importância da vigilância epidemiológica para essas doenças e suas complicações.

Viral exanthems are a common problem in tropical regions, particularly affecting children. Various skin rashes have been reported in acute infections caused by Enterovirus. Biological samples from a child who presented generalized rashes were sent to the Enteric Virus Laboratory of the Adolfo Lutz Institute for laboratory diagnosis to be performed. A viral sample isolated from RD (human rhabdomyosarcoma cells) was subjected to the polymerase chain reaction and showed a 437-base pair product that was characteristic of the Enterovirus genus. Echovirus 6 (E-6) serotype was identified using the indirect immunofluorescence test. In addition, paired serum samples presented seroconversion to E-6. So far, there have not been any reports of E-6 involvement in exanthematic diseases in Brazil. Thus, the importance of epidemiological surveillance for these diseases and their complications is emphasized.

[Echovirus 6 associated with exanthematic disease]. / Echovírus 6 associado à doença exantemática.

Viral exanthems are a common problem in tropical regions, particularly affecting children. Various skin rashes have been reported in acute infections caused by Enterovirus. Biological samples from a child who presented generalized rashes were sent to the Enteric Virus Laboratory of the Adolfo Lutz Institute for laboratory diagnosis to be performed. A viral sample isolated from RD (human rhabdomyosarcoma cells) was subjected to the polymerase chain reaction and showed a 437-base pair product that was characteristic of the Enterovirus genus. Echovirus 6 (E-6) serotype was identified using the indirect immunofluorescence test. In addition, paired serum samples presented seroconversion to E-6. So far, there have not been any reports of E-6 involvement in exanthematic diseases in Brazil. Thus, the importance of epidemiological surveillance for these diseases and their complications is emphasized.