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.

Evaluation of microbiological and physical parameters of water from dental clinics in a brazilian university / Evaluación de parámetros microbiológicos y físicos del água de clínicas odontológicas en una universidad brasileña / Avaliação de parâmetros biológicos e físicas da água de clínicas odontológicas de uma universidade brasileira

Abstract Introduction and objective : Contamination of the water used in health care units can induce adverse individual and collective consequences. The objective of this study was to evaluate the water quality in dental clinics of a dentistry program of a university in Brazil, calculating the total coliforms, thermotolerant coliforms, heterotrophic bacteria and pH. Materials and methods: In each clinic, water samples were collected for analysis at different points: external cistern, clinic faucet, dental chair tank and triple syringe. After sample collection, analysis was performed: the presence of total coliforms and thermotolerant coliforms was determined by chromogenic substrate technique, the presence or absence of Escherichia coli by fluorescence in ultraviolet light, the count of heterotrophic bacteria through the number of colonies and the determination of pH. Results: In all studied sites, the presence of total coliforms, Escherichia coli and thermotolerant coliforms was observed in at least 26% of the samples. In relation to CFU / ml, all mean values were much higher than the maximum established by legislation (500 CFU / ml). It was verified a statistically significant difference was found in the external cistern and tap when compared to the chair tank and triple syringe (p < 0.05). All pH samples were within the standards. Conclusion : The global analysis showed that 100% of the samples were inadequate, which classified the water potability as nonstandard in regard to the current Brazilian legislation.

Resumen Introducción y objetivo : La contaminación del agua utilizada en las unidades de salud puede inducir malas consecuencias individuales y colectivas. El objetivo de esta investigación fue evaluar la calidad del agua en clínicas odontológicas de una universidad en Brasil, a través del análisis de coliformes totales, coliformes termotolerantes, bacterias heterotróficas y pH. Materiales y métodos: En cada clínica, las muestras de agua fueron recolectadas para análisis en diferentes puntos: cisterna externa, grifo clínico, reservatorio de la silla odontológica y jeringa tríplice. Después de la recolección de las muestras, el análisis fue realizado: la presencia de coliformes totales y coliformes termotolerantes fue determinada por la técnica del sustrato cromogénico; la presencia o ausencia de Escherichia coli por fluorescencia en luz ultravioleta; el recuento de bacterias heterotróficas a través del número de colonias y de la determinación del pH. Resultados: En todos los puntos estudiados, se observó la presencia de coliformes totales, Escherichia coli y coliformes termotolerantes en al menos el 26% de las muestras. Respecto a la UFC / ml, todos los valores medios fueron muy superiores al máximo establecido por la legislación (500 UFC / ml). Todas las muestras de pH estaban dentro de los patrones. Conclusión : El análisis global mostró que el 100% de las muestras fueron inadecuadas, lo que clasificó la capacidad de agua como fuera del estándar en relación a la legislación brasileña actual.

Resumo Introdução e objetivo: A contaminação da água utilizada nas unidades de saúde pode induzir consequências adversas individuais e coletivas. O objetivo deste estudo foi avaliar a qualidade da água em clínicas odontológicas de um curso de odontologia de uma universidade no Brasil, através da análise de coliformes totais, coliformes termotolerantes, bactérias heterotróficas e pH. Materiais e métodos: Em cada clínica, amostras de água foram coletadas para análise em diferentes pontos: cisterna externa, torneira clínica, reservatório da cadeira odontológica e seringa tríplice. Após a coleta das amostras, a análise foi realizada: a presença de coliformes totais e coliformes termotolerantes foi determinada pela técnica do substrato cromogênico; a presença ou ausência de Escherichia coli por fluorescência em luz ultravioleta; a contagem de bactérias heterotróficas através do número de colônias e a determinação de pH. Resultados : Em todos os pontos estudados, a presença de coliformes totais, Escherichia coli e coliformes termotolerantes foi observada em pelo menos 26% das amostras. Em relação à UFC/ml, todos os valores médios foram muito superiores ao máximo estabelecido pela legislação (500 UFC/ml). Todas as amostras de pH estavam dentro dos padrões. Conclusão: A análise global mostrou que 100% das amostras foram inadequadas, o que classificou a capacidade de água como fora do padrão em relação à legislação brasileira atual.

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

Prediction equation for the mini-mental state examination: influence of education, age, and sex / Equação de predição para o miniexame do estado mental: influência da educação, idade e sexo / Ecuación de predicción para el mini-examen del estado mental: influencia de la educación, edad y sexo

ABSTRACT The mini-mental state examination (MMSE) is a screening test used worldwide for identifying changes in the scope of cognition. Studies have shown the influence of education, age and sex in the MMSE score. However, in Brazil, the studies consider only one factor to score it. The aim of this study was to establish a prediction equation for the MMSE. An exploratory cross-sectional study was developed and trained researchers examined participants at the community. The volunteers were evaluated by the MMSE and also by the Geriatric Depression Scale (GDS). The MMSE score was the dependent variable. Age, educational level, sex, and GDS score were the independent variables. Multivariate regression analysis was used to determine the model of best prediction value for MMSE scores. A total of 250 participants aged 20-99 years, without cognitive impairment, were assessed. The educational level, age, and sex explained 38% of the total variance of the MMSE score (p<0.0001) and resulted in the following equation: MMSE=23.350+0.265(years of schooling)-0.042(age)+1.323(sex), in which female=1 and male=2. The MMSE scores can be better explained and predicted when educational level, age, and sex are considered. These results enhance the knowledge regarding the variables that influence the MMSE score, as well as provide a way to consider all of them in the test score, providing a better screening of these patients.

RESUMO O mini-exame do estado mental (MEEM) é um teste de rastreio mundialmente utilizado para identificar alterações no âmbito da cognição. Estudos têm demonstrado a influência da educação, idade e gênero na pontuação do MEEM. No entanto, no Brasil, os estudos consideram apenas um fator para a pontuação no teste. O objetivo do estudo foi estabelecer uma equação preditiva para o MEEM. Um estudo transversal exploratório foi desenvolvido e examinadores treinados avaliaram participantes da comunidade. Os voluntários foram avaliados pelo MEEM e pela Escala de Depressão Geriátrica (EDG). A pontuação do MEEM foi a variável dependente. A idade, nível educacional, gênero e pontuação na EDG foram as variáveis independentes. A análise de regressão multivariada foi utilizada para determinar o modelo de melhor valor preditivo para os escores do MEEM. Foram avaliados 250 indivíduos entre 20 e 99 anos, sem comprometimento cognitivo. O nível educacional, a idade e o sexo explicaram 38% da variância total da pontuação do MEEM (p<0,0001) e resultaram na equação: MEEM=23,350+0,265(anos de escolaridade)-0,042(idade)+1,323(gênero), em que mulher=1 e homem=2. A pontuação do MEEM pode ser melhor explicada e predita quando o nível educacional, idade e gênero são considerados. Os resultados contribuem para o conhecimento sobre as variáveis que influenciam o escore do MEEM, bem como fornece uma maneira de considerá-las na pontuação do teste, proporcionando uma melhor triagem desses pacientes.

RESUMEN El mini-examen del estado mental (MEEM) es una prueba de rastreo mundialmente utilizada para identificar alteraciones en el ámbito de la cognición. Los estudios han demostrado la influencia de la educación, la edad y el sexo en la puntuación del MEEM. Sin embargo, en Brasil, los estudios consideran sólo un factor para la puntuación en la prueba. El objetivo del estudio fue establecer una ecuación predictiva para el MEEM. Un estudio transversal exploratorio fue desarrollado y examinadores entrenados evaluaron a participantes de la comunidad. Los participantes fueron evaluados por el MEEM y la Escala de Depresión Geriátrica (EDG). La puntuación del MEEM fue la variable dependiente. La edad, nivel educativo, sexo y puntuación en la EDG fueron las variables independientes. El análisis de regresión multivariada fue utilizado para determinar el modelo de mejor valor predictivo para los escores del MEEM. Se evaluaron 250 individuos entre 20 y 99 años, sin comprometimiento cognitivo. El nivel educativo, la edad y el sexo explicaron el 38% de la varianza total de la puntuación del MEEM (p <0,0001) y resultaron en la ecuación: MEEM=23,350+0,265(años de escolaridad)-0,042 (edad)+1,323 (sexo), en que mujer = 1 y hombre = 2. La puntuación del MEEM puede ser mejor explicada y predecible cuando se considera el nivel educativo, la edad y el sexo. Los resultados contribuyen para el conocimiento sobre las variables que influencian el score del MEEM, así como proporciona una manera de considerar las variables en la puntuación de la prueba, proporcionando una mejor forma de triar a estos pacientes.

Lipase and factor V (but not viral load) are prognostic factors for the evolution of severe yellow fever cases

BACKGROUND Despite a highly efficacious vaccine, yellow fever (YF) is still a major threat in developing countries and a cause of outbreaks. In 2018, the Brazilian state of São Paulo witnessed a new YF outbreak in areas where the virus has not been detected before. OBJECTIVE The aim is to describe the clinical and laboratorial characteristics of severe cases of YF, evaluate viral to determine markers associated with fatal outcome. METHODS Acute severe YF cases (n = 62) were admitted to the Intensive Care Unit of a reference hospital and submitted to routine laboratorial evaluation on admission. YFV-RNA was detected in serum and urine by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and then sequenced. Patients were classified in two groups: survival or death. FINDINGS In the univariate analysis the following variables were associated with outcome: alanin aminotransferase (ALT), aspartat aminotransferase (AST), AST/ALT ratio, total bilirubin (TB), chronic kidney disease epidemiology collaboration (CKD-EPI), ammonia, lipase, factor V, international normalised ratio (INR), lactate and bicarbonate. Logistic regression model showed two independent variables associated with death: lipase [odds ratio (OR) 1.018, 95% confidence interval (CI) 1.007 to 1.030, p = 0.002], and factor V (OR -0.955, 95% CI 0.929 to 0.982, p = 0.001). The estimated lipase and factor V cut-off values that maximised sensitivity and specificity for death prediction were 147.5 U/L [area under the curve (AUC) = 0.879], and 56.5% (AUC = 0.913). MAIN CONCLUSIONS YF acute severe cases show a generalised involvement of different organs (liver, spleen, heart, kidneys, intestines and pancreas), and different parameters were related to outcome. Factor V and lipase are independent variables associated with death, reinforcing the importance of hemorrhagic events due to fulminant liver failure and pointing to pancreatitis as a relevant event in the outcome of the disease.

Índice de vulnerabilidade socioambiental: uma proposta metodológica utilizando o caso do Rio de Janeiro, Brasil / Socio-environmental vulnerability index: a methodological proposal based on the case of Rio de Janeiro, Brazil

Resumo O termo vulnerabilidade tem sido utilizado com mais frequência, visando uma melhor compreensão acerca dos diferentes grupos populacionais, suas especificidades e necessidades. O objetivo deste estudo foi identificar, caracterizar e analisar populações em situação de vulnerabilidade socioambiental no município do Rio de Janeiro, consolidando indicadores sociais, econômicos, ambientais, de saúde e de segurança pública, em um índice de síntese - o Índice de Vulnerabilidade Socioambiental. A metodologia foi baseada em análise multicritério de apoio à decisão integrada a um Sistema de Informação Geográfica. As fontes de dados utilizadas foram o Censo Demográfico do IBGE-2010, Fundação Geo-Rio e Instituto de Segurança Pública do estado do Rio de Janeiro. Os resultados demonstram que a vulnerabilidade socioambiental no Rio de Janeiro é agravada por situações de risco e degradação ambiental. Estes aspectos são acentuados pelo adensamento das áreas faveladas, onde os estratos mais vulneráveis residem, em um processo de exclusão ambiental e urbana. O estudo permite localizar espacialmente áreas mais vulneráveis, enfatizando a importância destas ferramentas para guiar alocação de recursos, formulação e implementação de políticas públicas mais adequadas.

Abstract The term vulnerability has been more frequently used in several studies, striving to better understand the specificities and needs of different population groups. The scope of this study was to identify, characterize and analyze populations in situations of socio-environmental vulnerability in Rio de Janeiro city, consolidating social, economic, environmental, health and public security indicators in a synthesis index - the Socio-Environmental Vulnerability Index. The data sources used were the IBGE-2010 Demographic Census, the Geo-Rio Foundation and the Public Security Institute of the state of Rio de Janeiro. The methodology integrated Multicriteria Decision Analysis into a Geographic Information System. According to our results, the socio-environmental vulnerability in Rio de Janeiro city is aggravated by risk situations and environmental degradation. Those aspects are accentuated by the population density in shantytown areas, where the most disadvantaged strata exist in a process of environmental and urban exclusion. The study makes it possible to locate spatially vulnerable areas, emphasizing the importance of these tools to guide resource allocation, formulation and implementation of more effective public policies.

Basal core promoter and precore mutations among hepatitis B virus circulating in Brazil and its association with severe forms of hepatic diseases

BACKGROUND In Brazil, few studies have investigated the prevalence of infection with the precore (PC) and basal core promoter (BCP) mutants of the hepatitis B virus (HBV). OBJECTIVES This study aimed to analyse the frequency of PC and BCP mutations among patients infected with HBV and to evaluate the association between the variants and advanced hepatic disease. METHODS A total of 161 patients infected with HBV were studied. To identify PC and BCP mutations, a 501-bp fragment of HBV DNA was amplified and sequenced. FINDINGS PC and BCP regions from HBV strains were successfully amplified and sequenced in 129 and 118 cases, respectively. PC and BCP mutations were detected in 61.0% and 80.6% of the cases, respectively. The A1762T/G1764A variant was identified in 36.7% of the patients with grade 1 and 2 liver fibrosis (29/79) and in 81.8% of the patients with grade 3 and 4 liver fibrosis (9/11) (p < 0.01); in 76.9% of the patients with cirrhosis (10/13) and in 38.1% of the patients without cirrhosis (40/105) (p = 0.01); and in 77.8% of the patients with hepatocellular carcinoma (HCC) (7/9) and in 39.4% of the patients without HCC (43/109) (p = 0.03). MAIN CONCLUSIONS A high prevalence of HBV PC and BCP mutants was found. The A1762T/G1764A variant was independently associated with advanced forms of liver fibrosis, hepatic cirrhosis, and HCC.

Distribution of HBV subgenotypes in Ribeirão Preto, Southeastern Brazil: a region with history of intense Italian immigration

Abstract Hepatitis B virus (HBV) is distributed worldwide, with geographical variations regarding prevalence of the different genotypes. The aim of this study was to determine the HBV genotypes and subgenotypes circulating in Southeast Brazil and compare the genetic sequences found with HBV sequences previously described in the world. Sequences from 166 chronic HBV carriers were analyzed using the fragment constituted by 1306 base pairs comprising surface and polymerase regions of the HBV genome. The sequences obtained were submitted to phylogenetic analysis. HBV subgenotypes A1, A2, D1-D4, F2a, and F4 were found. HBV genotype D was the most frequent, found in 99 patients (58.4%). Within this group, subgenotype D3 was the most prevalent, in 73 patients (42.9%). HBV genotype A was identified in 58 (36%) patients, subgenotype A1, in 48 (29.8%) subjects. Genotype F was identified in 9 (5.4%). According to the phylogenetic analysis, the sequences found were grouped with sequences from Europe, Asia and Middle East (subgenotypes D1, D2, D3) and sequences from Latin America and Africa (subgenotype A1). HBV D3 grouped in different clusters inside D3 clade, several of them with sequences isolated in Italy. We also identified eight families whose relatives were infected with the same HBV subgenotype, most with high similarity between sequences. In conclusion, the distribution of the HBV sequences obtained interweaved with sequences from other continents, corresponding to regions from where many immigrants came to this region, in accordance to the hypothesis that the HBV detected over there were brought during the colonization times.

Hepatitis C among blood donors: cascade of care and predictors of loss to follow-up

ABSTRACT OBJECTIVE To investigate the HCV cascade of care and to identify the factors associated with loss or absence to follow-up of patients identified as infected with hepatitis C through blood donation. METHODS Blood donors from 1994 to 2012, identified with positive anti- HCV by enzyme immunoassay and immunoblot tests were invited to participate in the study, through letters or phone calls. Patients who agreed to participate were interviewed and their blood samples were collected for further testing. The following variables were investigated: demographic data, data on comorbidities and history concerning monitoring of hepatitis C. Multiple regression analysis by Poisson regression model was used to investigate the factors associated with non-referral for consultation or loss of follow-up. RESULTS Of the 2,952 HCV-infected blood donors, 22.8% agreed to participate: 394 (58.2%) male, median age 48 years old and 364 (53.8%) Caucasian. Of the 676 participants, 39.7% did not receive proper follow-up or treatment after diagnosis: 45 patients referred not to be aware they were infected, 61 did not seek medical attention and 163 started a follow-up program, but were non-adherent. The main reasons for inadequate follow-up were not understanding the need for medical care (71%) and health care access difficulties (14%). The variables showing a significant association with inadequate follow-up after multiple regression analysis were male gender (PR = 1.40; 95%CI 1.15–1.71), age under or equal to 50 years (PR = 1.36; 95%CI 1.12–1.65) and non-Caucasians (PR = 1.53; 95%CI 1.27–1.84). CONCLUSIONS About 40.0% of patients did not receive appropriate follow-up. These data reinforce the need to establish strong links between primary care and reference centers and the need to improve access to specialists and treatments.

Myxovirus resistance, osteopontin and suppressor of cytokine signaling 3 polymorphisms predict hepatitis C virus therapy response in an admixed patient population: comparison with IL28B

OBJECTIVES: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin. METHOD: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers. RESULTS: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population. CONCLUSION: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus ...

Perfil de células natural killer e dendríticas em casos de soroconversão espontânea e infecção crônica pelo vírus da Hepatite C / Profile of natural killer and dendritic cells in cases of spontaneous clearance and chronic infection with Hepatitis C virus

INTRODUÇÃO: O fato do vírus da Hepatite C (HCV) estabelecer uma infecção crônica persistente, na maioria dos casos, mesmo sendo reconhecido e alvejado pelos sistemas imune inato e adaptativo sugere que o mesmo tenha desenvolvido estratégias eficazes para driblar a ação desses sistemas. O HCV interfere na fase inicial de ativação da resposta imune adaptativa alterando a função das células dendríticas (DCs), o que provavelmente leva a uma ativação deficiente das células natural killer (NKs) e de linfócitos T. Portanto, a realização de estudos sobre DCs e NKs na infecção pelo HCV se torna de fundamental importância para a compreensão da patogênese e persistência desta infecção. MÉTODOS: Foram selecionados indivíduos com resolução espontânea da infecção pelo HCV, indivíduos com infecção crônica e indivíduos saudáveis. A técnica de citometria de fluxo foi utilizada para a determinação da frequência e do fenótipo de células dendríticas e NKs nesses indivíduos. Além disso, foi avaliada a atividade citotóxica das células NKs sob estímulo de IL-12 e IL-18, e também da linhagem K-562. RESULTADOS: A frequência de DC mielóides (mDC) expressando CD86, nos indivíduos crônicos, foi elevada e uma correlação positiva com a carga viral foi observada. Na análise do ensaio funcional foi observado que as populações de células NKs CD7+ CD57+ apresentaram maior expressão da molécula CD107a e baixa produção de IFNy nos indivíduos com infecção crônica. A constante exposição das células imunes ao IFN-alfa, induzido durante a infecção pelo HCV, resulta na polarização do fenótipo citotóxico, caracterizado por células NK ativadas com elevado poder de degranulação, mas com deficiente produção de IFN-y. CONCLUSÕES: As frequências das células DCs e NKs eram semelhantes em todos os indivíduos. A expressão da molécula CD86 na superfície das mDCs pode ter sido induzida pela presença do HCV, uma vez que foi observada correlação positiva com a carga viral...

INTRODUCTION: Hepatitis C virus (HCV) develops a chronic persistent infection in most of the cases, even being recognized and targeted by the innate and adaptive immune systems, suggests that the virus have developed effective strategies to circumvent the action of these systems. HCV interferes in the initial activation of the adaptive immune response by altering the function of dendritic cells (DCs), which probably leads to a deficient activation of natural killer cells (NK) and T lymphocytes. Therefore, studies of DCs and NK in HCV infection are very important for understanding the pathogenesis and the persistence of this infection. METHODS: We selected subjects with spontaneous resolution of HCV infection, with chronic infection and healthy subjects. Flow Cytometry was used to determine the frequency and phenotype of dendritic cells and NK cells of these individuals. In addition, we evaluated the NK cell cytotoxic activity in response to stimulation of IL-12 and IL-18 and in co-cultivation with the cell line K-562. RESULTS: In individuals with chronic infection, the frequency of myeloid (m) DC cells expressing CD86 was elevated and a positive correlation between these cells and viral load was observed. It was observed in chronic infected individuals that NK cells co-expressing CD7 and CD57 showed higher expression of CD107a and low production of IFN gamma. The constant exposure of immune cells to IFN-alfa induced during HCV infection results in the polarization of cytotoxic phenotype characterized by activated NK cells with high power degranulation, but with impaired production of IFN-y. CONCLUSIONS: The frequency of DCs and NK cells were similar in all individuals. The expression of CD86 molecule on the surface of mDCs may have been induced by the presence of HCV, since a positive correlation was observed with viral load. Cytotoxic NK cells, highly differentiated and unable to produce IFN-y, were the most frequent in chronic HCV infection...

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.