RESUMO
We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.
Assuntos
Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/epidemiologia , Hepatite C Crônica/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Epidemiológicos , Feminino , Técnicas de Genotipagem , Hepacivirus/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogeografia , Prevalência , Estudos Retrospectivos , Espanha/epidemiologiaRESUMO
AIMS: To investigate an optimal long-term prophylactic strategy for prevention of hepatitis B virus (HBV) recurrence after liver transplantation, we conducted a randomized study of 29 transplant recipients receiving a short course of hepatitis B immune globulin (HBIg) + lamivudine (LAM), followed by randomization to long-term prophylaxis with LAM with or without HBIg. METHODS: The efficacy and safety, and impact on survival and HBV recurrence of these 2 prophylactic regimens were compared over a mean period of 10 years. In patients with viral recurrence, the HBV quasispecies in the surface/polymerase region were studied by ultra-deep pyrosequencing (UDPS). RESULTS: The 10-year survival rate was 76% and was not affected by the type of prophylaxis. Four patients had hepatitis B surface antigen (HBsAg) recurrence within the first 48 months after orthotopic liver transplantation (OLT). HBsAg-positive and -negative patients showed similar mean survival times, with no differences between the 2 regimens. Low HBV DNA levels were transiently detected in 32% of HBsAg-negative patients. UDPS showed major changes after OLT in the HBV quasispecies of patients with viral recurrence, which may be explained by a "bottleneck" effect of OLT together with prophylactic therapy. CONCLUSION: Long-term survival after OLT in end-stage chronic hepatitis B patients was good with both prophylactic strategies. However, low, transient HBV DNA levels were detected even in the absence of HBsAg, showing the importance of continuing HBV prophylaxis.
Assuntos
DNA Viral/sangue , Doença Hepática Terminal/cirurgia , Vírus da Hepatite B/genética , Hepatite B Crônica/prevenção & controle , Imunoglobulinas/uso terapêutico , Lamivudina/uso terapêutico , Transplante de Fígado , Inibidores da Transcriptase Reversa/uso terapêutico , Doença Hepática Terminal/virologia , Evolução Molecular , Feminino , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Prevenção Secundária , Análise de Sequência de DNA , Resultado do TratamentoRESUMO
The success of current antiviral treatment for hepatitis C virus (HCV) recurrence in liver transplant (LT) recipients remains limited. We aimed at evaluating the value of IL28B genotype and early viral kinetics to predict response to standard treatment in the transplant setting. We retrospectively evaluated 104 LT recipients treated for HCV genotype 1 recurrence between 2001 and 2010. Baseline variables, including IL28B genotype, and early viral kinetics were compared among patients who did or did not achieve a sustained virological response (SVR). Logistic regression analyses of candidate variables were conducted to generate a reliable predictive model based on the minimum set of variables. Twenty-nine (28%) achieved an SVR. On multivariate analysis, the magnitude of HCV RNA decline at 4 weeks (OR: 3.74, 95% CI: 1.64-9.39; P = 0.003) and treatment compliance (OR: 35.27, 95% CI: 3.35-365.54; P = 0.003) were the only independent predictors of SVR. Favourable recipient IL28B genotype significantly correlates with virological response at week 4 (OR 3.23; 95% CI, 1.12-9.15; P = 0.03). By logistic regression analysis, a model including donor age, recipient rs12979860 genotype and viral load at 4 weeks showed the best predictive value for SVR with an area under the receiver operating curve of 0.861. Favourable recipient IL28B genotype strongly correlates with the viral response at week 4 which is the strongest predictor of response. The combination of recipient IL28B genotype and donor age with the week 4 response reliably estimates the probability of SVR early on-treatment and may facilitate therapeutic strategies incorporating new antiviral agents.
Assuntos
Antivirais/uso terapêutico , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Transplante de Fígado , Transplantados , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Interferons , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Recidiva , Estudos Retrospectivos , Resultado do Tratamento , Carga Viral , Adulto JovemRESUMO
BACKGROUND: Obesity is a growing global health problem, and currently, bariatric surgery (BS) is the best solution in terms of sustained total weight loss (TWL). However, a significant number of patients present weight regain (WR) in time. There is a lack of biomarkers predicting the response to BS and WR during the follow-up. Plasma SHBG levels, which are low in obesity, increase 1 month after BS but there is no data of plasma SHBG levels at long term. We performed the present study aimed at exploring the SHBG role in predicting TWL and WR after BS. METHODS: Prospective study including 62 patients with obesity undergoing BS. Anthropometric and biochemical variables, including SHBG were analyzed at baseline, 1, 6, 12, and 24 months; TWL ≥ 25% was considered as good BS response. RESULTS: Weight loss nadir was achieved at 12 months post-BS where maximum SHBG increase was reached. Greater than or equal to 25% TWL patients presented significantly higher SHBG increases at the first and sixth months of follow-up with respect to baseline (100% and 150% respectively, p = 0.025), than < 25% TWL patients (40% and 50% respectively, p = 0.03). Also, these presented 6.6% WR after 24 months. The first month SHBG increase predicted BS response at 24 months (OR = 2.71; 95%CI = [1.11-6.60]; p = 0.028) and TWL in the 12th month (r = 0.330, p = 0.012) and the WR in the 24th (r = - 0.301, p = 0.028). CONCLUSIONS: Our results showed for the first time that increase in plasma SHBG levels within the first month after BS is a good predictor of TWL and WR response after 2 years.
Assuntos
Cirurgia Bariátrica , Obesidade Mórbida , Humanos , Obesidade Mórbida/cirurgia , Estudos Prospectivos , Obesidade/cirurgia , Redução de Peso/fisiologia , Estudos RetrospectivosRESUMO
OBJECTIVES: The aim of the study was to assess the seroprevalence of hepatitis E virus (HEV) infection in an HIV-infected population, as determined by HEV immunoglobulin G (IgG) antibodies (anti-HEV). METHODS: The design of the study was cross-sectional. Serum anti-HEV IgG was determined by enzyme immunoassay in 238 HIV-infected patients consecutively attending our out-patient clinic between April and May 2011. In HEV-seropositive patients, HEV RNA was analysed by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Associations between anti-HEV and liver cirrhosis, route of HIV infection, hepatitis B virus (HBV) and hepatitis C virus (HCV) serological markers, age, sex and alanine aminotransferase (ALT) levels were examined by univariate and multivariate analysis. RESULTS: One hundred and forty patients (59%) had chronic liver disease (99% were HBV- and/or HCV-coinfected). Liver cirrhosis was detected in 44 individuals (19%). Two hundred and twelve patients (89%) were on antiretroviral treatment; the median CD4 T-cell count was 483 cells/µL [interquartile range (IQR) 313-662 cells/µL] and the HIV viral load was <25 HIV-1 RNA copies/mL. Overall, 22 patients (9%) were anti-HEV positive. Liver cirrhosis was the only factor independently associated with the presence of anti-HEV, which was documented in 23% of patients with cirrhosis and 6% of patients without cirrhosis (P=0.002; odds ratio 5.77). HEV RNA was detected in three seropositive patients (14%), two of whom had liver cirrhosis. CONCLUSIONS: Our findings show a high prevalence of anti-HEV in HIV-infected patients, strongly associated with liver cirrhosis. Chronic HEV infection was detected in a significant number of HEV-seropositive patients. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection and to assess the role of chronic HEV infection in the pathogeneses of cirrhosis in this population.
Assuntos
Anticorpos Antivirais/sangue , Soropositividade para HIV/epidemiologia , Vírus da Hepatite E/imunologia , Hepatite E/epidemiologia , Cirrose Hepática/epidemiologia , Adulto , Contagem de Linfócito CD4 , Feminino , Soropositividade para HIV/genética , Soropositividade para HIV/imunologia , Hepatite E/genética , Hepatite E/imunologia , Vírus da Hepatite E/genética , Humanos , Imunoglobulina G/sangue , Cirrose Hepática/genética , Cirrose Hepática/imunologia , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espanha/epidemiologiaRESUMO
Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4(+) T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-γ ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4-25.3, P < 0.001). HCV-specific CD4(+) T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4(+) T-cell responses towards NS3 were only evident among those with non-CC haplotypes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Variação Genética , Hepacivirus/imunologia , Hepatite C/imunologia , Interleucinas/genética , Interleucinas/imunologia , Adulto , Antígenos Virais/imunologia , Doadores de Sangue , ELISPOT , Feminino , Haplótipos , Humanos , Interferon gama/metabolismo , Interferons , Masculino , Pessoa de Meia-Idade , Espanha , Adulto JovemRESUMO
Long-term changes in the frequency and outcome of hepatitis delta virus (HDV) infection have seldom been analysed. This retrospective, longitudinal study includes 398 consecutive hepatitis B surface antigen (HBsAg)-positive patients with anti-HDV antibodies who attended our institution between 1983 and 2008. At enrolment, 182 patients had acute and 216 chronic hepatitis. Patients were grouped into two periods. Those who attended between 1983 and 1995 and those between 1996 and 2008. The former group was significantly younger, mainly intravenous drugs users, and had a greater incidence of acute HDV and HIV and HCV coinfection. Patients with acute HBV/HDV coinfection cleared both infections in 90% of cases, while all patients with HDV superinfection evolved to chronic disease. One hundred and fifty-eight patients with chronic HDV were followed for a median period of 158months. Seventy-two per cent of the patients remained stable, 18% had hepatic decompensation, 3% developed hepatocellular carcinoma, and 8% cleared HBsAg. Liver-related death was observed in 13% of patients and mainly occurred in patients from the first period (P=0.012). These results indicate an outbreak of HDV at the end of the 1980s and the beginning of the 1990s, with a large number of acute HDV cases affecting predominately young, male intravenous drug users. Currently, patients with chronic HDV disease are older, and factors associated with worse prognosis include the presence of cirrhosis and age at the time of diagnosis.
Assuntos
Hepatite D Crônica/epidemiologia , Doença Aguda , Adolescente , Adulto , Alanina Transaminase , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/virologia , Surtos de Doenças , Usuários de Drogas , Feminino , Seguimentos , HIV , Infecções por HIV/complicações , Hepatite B/complicações , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B , Hepatite D Crônica/complicações , Hepatite D Crônica/diagnóstico , Hepatite D Crônica/imunologia , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Humanos , Cirrose Hepática/virologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/virologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Viral/análise , Estudos Retrospectivos , Superinfecção/complicações , Superinfecção/virologia , Adulto JovemRESUMO
alpha(1)-Antitrypsin (AT) deficiency is a hereditary disorder that may lead to early-onset emphysema, and chronic liver disease later in life. Although there are validated methods for testing, the vast majority of alpha(1)-AT-deficient individuals remain undiagnosed. Recommendations have been published for the testing and diagnosis of alpha( 1)-AT deficiency; however, guidelines on best practice are not well established. In our article, we review the developments in diagnostic techniques that have taken place in recent years, and describe the practices used in our three European centres. The determination of the level of alpha(1)-AT and genotyping are reported as the main diagnostic steps, whereas isoelectric focusing (also referred to as phenotyping) is reserved for confirmatory analysis. The following recommendations for best practice are put forward: detection of all PiZZ and other severe deficiency individuals; automated genotyping; preparation of reference standards; quality control programmes; development of standard operating procedure documents; and standardised methods for the collection of dried blood samples. Closer cooperation between laboratories and the sharing of knowledge are recommended, with the objectives of improving the efficiency of the diagnosis of severe alpha(1)-AT deficiency, increasing the numbers of individuals who are detected with the disorder, and assisting the establishment of new patient identification programmes.
Assuntos
Testes Genéticos/métodos , Hepatopatias/sangue , Enfisema Pulmonar/sangue , Deficiência de alfa 1-Antitripsina/sangue , Algoritmos , Coleta de Amostras Sanguíneas , Doença Crônica , Alemanha/epidemiologia , Humanos , Focalização Isoelétrica , Itália/epidemiologia , Hepatopatias/epidemiologia , Hepatopatias/genética , Fenótipo , Guias de Prática Clínica como Assunto , Enfisema Pulmonar/epidemiologia , Enfisema Pulmonar/genética , Espanha/epidemiologia , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/epidemiologia , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
Accurate subtyping of hepatitis C virus genotype 1 (HCV-1) remains clinically and epidemiologically relevant. The Abbott HCV Genotype Plus RUO (GT Plus) assay, targeting the core region, was evaluated as a reflex test to resolve ambiguous HCV-1 results in a challenging sample collection. 198 HCV-1 specimens were analysed with GT Plus (38 specimens with and 160 without subtype assigned by the Abbott RealTime Genotype II (GT II) assay targeting the 5'NC and NS5B regions). Sanger sequencing of the core and/or NS5B regions were performed in 127 specimens without subtype assignment by GT II, with "not detected" results by GT Plus, or with mixed genotypes/subtypes. The remaining GT Plus results were compared to LiPA 2.0 (n = 45) or just to GT II results if concordant (n = 26). GT Plus successfully assigned the subtype in 142/160 (88.8%) samples. "Not detected" results indicated other HCV-1 subtypes/genotypes or mismatches in the core region in subtype 1b. The subtyping concordance between GT Plus and either sequencing or LiPA was 98.6% (140/142). Therefore, combined use of GT II and GT Plus assays represents a reliable and simple approach which considerably reduced the number of ambiguous HCV-1 results and enabled a successful subtyping of 98.9% of all HCV-1 samples.
Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Regiões 5' não Traduzidas , Genótipo , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Israel , Filogenia , Espanha , Proteínas não Estruturais Virais/genéticaRESUMO
OBJECTIVES: To investigate whether hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels are useful to identify inactive carriers among HBeAg-negative patients infected by different hepatitis B virus (HBV) genotypes. METHODS: In all, 202 consecutive HBeAg-negative patients with chronic hepatitis B, 135 inactive carriers and 67 with HBV activity, were prospectively followed for 1 year. RESULTS: In HBeAg-negative patients, HBsAg levels differed across the different genotypes (p <0.001). The highest levels were observed in genotypes F or H (4.2 ± 0.6 logIU/mL), followed by genotype E (3.4 ± 1.1 logIU/mL), genotype A (3.4 ± 0.8 logIU/mL), and the lowest in genotype D (2.7 ± 1.1 logIU/mL). Variations in HBsAg levels were similar in inactive carriers and patients with HBV activity. HBsAg <3 logIU/mL showed good performance for identifying genotype D inactive carriers: 76% of genotype D inactive carriers met this cut-off versus ≤31% for genotypes A, E, F or H. However, in patients with genotype A, HBsAg levels ≤3.7 logIU/mL better classified inactive carriers. The combination of a single measurement of HBcrAg ≤3 logU/mL plus HBV DNA ≤2000 IU/mL yielded a positive predictive value and diagnostic accuracy >85% in all HBV genotypes, except genotype H or F, with values of 62.5% and 72.7%, respectively, for the two parameters. CONCLUSIONS: HBsAg levels varied across genotypes in HBeAg-negative patients. HBsAg levels <3 logIU/mL were only useful for identifying genotype D inactive carriers. A single HBcrAg measurement ≤3 logU/mL plus HBV DNA ≤2000 IU/mL was highly accurate for identifying inactive carriers, regardless of their HBV genotype.
Assuntos
Portador Sadio/diagnóstico , Portador Sadio/epidemiologia , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/epidemiologia , Adulto , Portador Sadio/sangue , Portador Sadio/virologia , Estudos de Coortes , Feminino , Genótipo , Vírus da Hepatite B/genética , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Hepatitis is a general term meaning inflammation of the liver, which can be caused by a variety of viruses. However, a substantial number of cases remain with unknown aetiology. We analysed the serum of patients with clinical signs of hepatitis using a metagenomics approach to characterize their viral species composition. Four pools of patients with hepatitis without identified aetiological agents were evaluated. Additionally, one pool of patients with hepatitis E (HEV) and pools of healthy volunteers were included as controls. A high diversity of anelloviruses, including novel sequences, was found in pools from patients with hepatitis of unknown aetiology. Moreover, viruses recently associated with gastroenteritis as sapovirus GV.2 and astrovirus VA3 were also detected only in those pools. Besides, most of the HEV genome was recovered from the HEV pool. Finally, GB virus C and human endogenous retrovirus were found in the HEV and healthy pools. Our study provides an overview of the virome in serum from hepatitis patients suggesting a potential role of these viruses not previously described in cases of hepatitis. However, further epidemiologic studies are necessary to confirm their contribution to the development of hepatitis.
Assuntos
Anelloviridae/isolamento & purificação , Hepatite Viral Humana/virologia , Mamastrovirus/isolamento & purificação , Sapovirus/isolamento & purificação , Viremia/sangue , Doença Aguda , Anelloviridae/classificação , Estudos de Casos e Controles , Hepatite Viral Humana/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mamastrovirus/classificação , Filogenia , Viremia/classificaçãoRESUMO
OBJECTIVES: This study aimed to characterize the chronically infected general hepatitis C virus (HCV) population in Barcelona using a highly sensitive subtyping method that can identify the 67 recognized HCV subtypes and diagnose mixed infection by various genotypes/subtypes in a single individual. The resulting information has implications for selecting optimal direct-acting antiviral (DAA) treatment for each patient and establishing public healthcare policies in our setting. METHODS: Consecutive HCV patients (treatment-naïve or interferon-based failures) attending Vall d'Hebron Hospital outpatient clinics from February 2015 to May 2016 (N=1473) were included in the study. Patient samples were characterized using HCV subtyping by next-generation ultra-deep pyrosequencing. RESULTS: The following genotypes (G) were found: G1 (1126/1473 (76.4%)), G4 (145/1473 (9.8%)), G3 (135/1473 (9.2%)), G2 (51/1473 (3.5%)), and G5 (1/1473 (0.1%)). Twenty-two subtypes were seen: 1b (790/1473 (53.6%)), 1a (332/1473 (22.5%)), 3a (133/1473 (9.0%)), 4d (105/1473 (7.1%)), 4a (29/1473 (2.0%)), and 2c (25/1473 (1.7%)), with 16 low-prevalence subtypes accounting for the remaining 3.0% (44/1473). There was a worrisome 1.0% (15/1473) of mixed infections. G2 (51/1473 (3.5%)) showed a high level of heterogeneity. Analyses by age groups showed a predominance of G1b over G1a (428/506 (84.6%) vs. 24/506 (4.7%)) in patients born before 1950 (N=506/1473), and similar percentages of these subtypes in those born between 1951 and 1975 (N=834/1473) (315/834, 37.8% vs. 266/834, 31.9%) and after 1976 (N=133/1473) (47/133, 35.3% vs. 42/133, 31.6%). CONCLUSIONS: Subtype distribution showed a higher level of heterogeneity than was expected, particularly for G2. Prevalence of mixed infections was around 1%. HCV subtype distribution related to patient age group suggested that patients born from 1936 to 1975 in our setting should undergo screening for the infection. Next-generation sequencing enabled better classification of candidates for DAA-based treatment.
Assuntos
Variação Genética , Genótipo , Técnicas de Genotipagem/métodos , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/virologia , Adolescente , Adulto , Idoso , Coinfecção/epidemiologia , Coinfecção/virologia , Feminino , Hepacivirus/isolamento & purificação , Hepatite C Crônica/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Prevalência , Espanha/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Lamivudine therapy for chronic hepatitis B has been associated with changes in different regions of the hepatitis B virus nucleotide sequence. AIM: To study changes in the sequences of polymerase and precore/core promoter regions of hepatitis B virus, before and during 5 years of therapy with lamivudine. METHODS: Eighty consecutive samples were taken from 10 chronic hepatitis B 'e' antigen-negative patients. RESULTS: Nine patients carried hepatitis B virus precore mutations during the study. Before therapy, wild type was replaced by A1896 in two (20%) cases. During treatment, A1896 reverted transitory to wild type in five cases (50%) and in one case wild type was replaced by A1896. The continuous detection of precore mutations during therapy was associated with a lower response rate. YMDD mutations were observed in nine cases and both, L180M and M204V/I mutations were simultaneously detected in six cases. About 75% of the patients with M204V mutations were responders and none with M204I or mixed pattern sustained response. CONCLUSION: Hepatitis B 'e' antigen-negative patients exhibit changes in the precore regions both spontaneously and under lamivudine therapy, the transitory reversion to wild type being most frequently witnessed. Patients carrying M204V mutations are more likely to respond to therapy. If, in further studies, these results are confirmed some patients with YMDD mutations could benefit from prolonging the duration of lamivudine therapy.
Assuntos
Antígenos E da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Lamivudina/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Adulto , DNA Viral/análise , Feminino , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Humanos , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Mutação/genética , Regiões Promotoras GenéticasRESUMO
In order to study the prevalence of hepatitis E virus (HEV) infection in developed countries, IgG and IgM anti-HEV were determined in serum samples from 382 patients with acute viral hepatitis (244 hepatitis A, 48 hepatitis B and/or D, and 90 non-A, non-B, non-C hepatitis), 76 healthy subjects, 55 hemophiliacs and 50 patients on hemodialysis. IgG anti-HEV antibodies were detected and confirmed by a synthetic peptide-based EIA in 5 (5.6%) non-A, non-B, non-C acute hepatitis, in 3 (6.5%) B and D acute hepatitis, in 10 (4%) acute A hepatitis, in 3 (5.5%) of 54 healthy adults in none of the hemophiliacs and in 3 (6%) patients on hemodialysis. IgM anti-HEV antibodies were only detected in two cases of acute hepatitis B and/or D. Analysis of serial serum samples demonstrated IgG anti-HEV seroconversion in 3 of the 18 confirmed cases; one of them was also positive for IgM anti-HEV. All 3 acute anti-HEV-positive hepatitis cases occurred in adults, were community-acquired (two of them were intravenous drug addicts) and had a self-limited course. These results demonstrate that HEV is a minor cause of acute hepatitis in Spain. A similar low rate of IgG anti-HEV antibodies was detected in patients with different diseases, suggesting that HEV has a very low epidemiological impact. An apparent association of HEV infection with hepatitis B and D suggests a possible parenteral transmission of a mainly enteral pathogen.
Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Feminino , Hemofilia A/imunologia , Hemofilia A/virologia , Hepatite E/epidemiologia , Hepatite E/imunologia , Vírus da Hepatite E/imunologia , Humanos , Lactente , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prevalência , Diálise Renal , Estudos Retrospectivos , Espanha/epidemiologiaRESUMO
The amino acid substitution from methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HBV polymerase gene is the main mutation responsible for resistance to lamivudine treatment. Detection of emerging HBV variants by direct sequencing of the HBV genome is excessively time-consuming for studying large numbers of clinical samples. The aim of the study was to analyse the emergence of lamivudine-resistant HBV genotypes by means of restriction fragment length polymorphism (RFLP) of the PCR product generated from a fragment of domain C of the polymerase gene, in clinical samples from patients receiving treatment. The results with this method were compared with those obtained with a direct sequencing technique. In total, 139 serum samples were studied from 37 patients with chronic hepatitis B, obtained at pre-treatment and at 9, 12, 18 and 24 months of treatment. Variants were detected by cleavage of the products of the three PCRs with the following enzymes: FokI (identifies the normal variant, YMDD, and the mutant variant YVDD), SspI (identifies the mutant variant, YIDD) and Alw44I (identifies the variant, YVDD). The digested fragments were separated by electrophoresis in 3% agarose gel. Of the 139 serum samples analysed, the wild-type YMDD sequence was detected in 106 (76%), the YVDD variant in 20 (15%) and the YIDD variant in 13 (9%) cases. The non-mutated variant, YMDD, was detected in all the pre-treatment samples. After 9 months of treatment the mutant variant was detected in four of 37 serum samples analysed (11%) (two YVDD and two YIDD). At 12 months, 12 of the 37 serum samples (32%) showed mutations in the YMDD sequence (seven YVDD and five YIDD). Among the 16 serum samples obtained at 18 months, nine had the YMDD variant (56%) (seven YVDD and two YIDD). At 24 months, variants in the YMDD sequence were detected in eight of the 12 patients treated (66%) (four YVDD and four YIDD). HBV genotypes were confirmed by direct sequencing, with consistent results. In 45% of cases, the emergence of HBV variants was not associated with ALT breakthrough. The PCR-RFLP assay used in this study, in perfect concordance with direct sequencing, is an accurate method for genotyping lamivudine-resistant HBV variants. Since it is a rapid low-cost technique, PCR- RFLP is suitable for large-scale screening of these polymorphisms in clinical samples.
Assuntos
Antivirais/farmacologia , DNA Polimerase Dirigida por DNA/genética , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Lamivudina/farmacologia , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Sequência de Bases , Primers do DNA/genética , DNA Viral/sangue , DNA Viral/genética , Resistência Microbiana a Medicamentos/genética , Genes Virais , Variação Genética , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/enzimologia , Humanos , Polimorfismo de Fragmento de Restrição , Fatores de TempoRESUMO
Severe alpha1-anti-trypsin (AAT) deficiency implies a high risk of pulmonary emphysema development. The possible relationship between partial deficiencies of this enzyme and bronchial asthma remains controversial. The objective of this study was to ascertain the distribution of AAT phenotypes in a non-selected asthmatic patient population. Across-sectional study on a sample of 111 patients with asthma was carried out. Demographic and clinical variables were collected with serum IgE concentrations, plasma eosinophil number and serum AAT concentrations determined, together with the Pi phenotype. Asthma was mild in 36 (32.4%) patients, moderate in 45 (40.5%) and severe in 30 (27%). No differences were observed in eosinophil count or serum IgE or AAT concentrations among patients with different degrees of severity. Twenty-two (19.8%) asthmatics with deficient phenotypes for AAT were identified, distributed equally in all severity stages of the disease. No significant differences were found in clinical and functional characteristics, or in asthma morbidity between PiMM and PiMS patients or the heterozygote group (PiMS and PiMZ). Eosinophil count and IgE concentrations did not differ significantly between asthmatics with normal phenotype and heterozygotes. In conclusion, the distribution of AAT phenotypes in asthmatic patients did not differ from that found in the general population. Heterozygote phenotypes for the deficiency do not appear to confer greater severity or different clinical expression of asthma in adults.
Assuntos
Asma/complicações , Deficiência de alfa 1-Antitripsina/complicações , Adulto , Análise de Variância , Asma/imunologia , Estudos Transversais , Eosinófilos/imunologia , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Fenótipo , Risco , Deficiência de alfa 1-Antitripsina/imunologiaRESUMO
BACKGROUND: Population studies indicate that alpha-1-antitrypsin (AAT) deficiency is an under diagnosed disease. Although alpha-1 serum protein is widely known to accompany AAT deficiency, the diagnostic utility of measuring the alpha-1 band to screen for this condition has not been assessed in the literature. SUBJECTS AND METHOD: Electropherograms with alpha-1 band widths under the reference values were collected over a period of 3 months. The Pi phenotype of AAT was identified for these sera by isoelectric point determination. The phenotypes were compared to those obtained for the population of the same geographic area (n = 440). The alpha-1 band reference values were obtained from 73 healthy individuals with no Pi phenotype deficiency. Moreover, the alpha-1 band was also measured for a group of 17 PiZZ deficient patients. RESULTS: We analyzed 7,305 electropherograms. One hundred four individuals (1.4%) without hypoproteinemia had alpha-1 readings below reference (set at 2.3%). The phenotypes in this group were 25 PiMM (24%), 52 PiMS (54%), 13 PiMZ (12.5%) and 5 PiSS 5 (5%). The odds ratios (CI 95%) in comparison with the normal population were, respectively, 0.10 (0.16-0.06); 4.58 (2.97-7.04); 4.35 (2.09-9.04) and 5.51 (1.66-18.16) (p < 10-5 in all cases except PiSS, which was p < 0.05). The levels for PiZZ patients were 1.4% +/- 0.3% (range 1.0%-2.1%). CONCLUSIONS: Three times fewer subjects with a normal PiMM phenotypes are found among individuals with low alpha-1 band serum protein levels, and many more of such individuals are carriers of Z allele heterozygotes. Alpha-1 band readings in patients with AAT deficiency (PiZZ phenotype) have alpha-1 values below reference. Measuring alpha-1 protein is an easy technique, within the expertise of any laboratory, and may be very useful for screening for AAT deficiency in patients with chronic respiratory diseases.
Assuntos
Deficiência de alfa 1-Antitripsina/diagnóstico , alfa 1-Antitripsina/análise , Humanos , Valores de Referência , Deficiência de alfa 1-Antitripsina/sangueRESUMO
Alpha-1 antitrypsin (AAT) deficiency is an under-diagnosed disease and screening programs have therefore been recommended for patients with chronic obstructive pulmonary disease (COPD). We present the results of the pilot phase of a screening program for AAT deficiency in order to evaluate the technique used, the procedures for transporting samples and the results obtained. Over a period of one month, five centers collected samples from all COPD patients for whom plasma concentrations of AAT or Pi phenotype had not yet been determined. Capillary blood spots were dried on filter paper and then sent by surface mail to a central laboratory for study. An immunonephelometric assay was used to determine AAT and DNA phenotyping was done by use of a Light Cycler. Samples were analyzed from 86 COPD patients (76 men, 10 women) with a mean age of 68.2 years. AAT deficiency was ruled out for 74 patients (86%) who had concentrations above the cutoff established, although one of them was MZ heterozygote by genotype. Among the 12 remaining patients (13.9%), only two also had a Z allele. The rest were individuals with concentrations below the established threshold and no evidence of a Z allele (10 patients, 11.6%). The Z allele frequency observed (3/172; 1.74%) was very similar to that found in the general population. The results of this pilot study allowed us to confirm that the method used to collect samples worked well. The sampling method is applicable, easy and well-accepted by participating physicians. It allowed AAT concentrations and Z allele deficiency to be determined. The method correlates well with standard techniques used for samples in whole blood.
Assuntos
Programas de Rastreamento/métodos , Doença Pulmonar Obstrutiva Crônica/sangue , Deficiência de alfa 1-Antitripsina/sangue , Idoso , Alelos , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/genética , Espanha , alfa 1-Antitripsina/genéticaRESUMO
BACKGROUND: Alpha 1 antitrypsin (AAT) is a highly polymorphic protein, having more than 75 different variants. In this work two rare AAT deficient variants were characterized by DNA study. PATIENTS AND METHODS: Members of three generations of two separate families were studied. In family 1, the index case was affected with pulmonary emphysema and presented AAT deficiency (23 mg/dl). In family 2, the index case had a normal pulmonary function, an AAT serum level of 72 mg/dl and a phenotype heterozygous for an AAT variant migrating in the P variant region. The AAT variants were characterized by polymerase chain reaction amplification of the coding exons and direct sequencing of the amplification products. RESULTS: Direct DNA sequencing from a member of family 1 demonstrates that in the exon II of the normal M1 (Val213) allele there was a 3-bp deletion (TTC), corresponding to Phe51 or Phe52. This mutation is characteristic of the Pl Mpalermo variant. In our study, Pl Mpalermo was detected in six members of three generations of this same family. Sequencing of exon III in a member of family 2, identified in the common M1 (Val213) allele a single base substitution of GAT-GTT, with the resulting amino acid change Asp256 for Val256. This mutation characterizes the Pl Plovel allele. The Pl Plovel was also detected in nine members of five others independent families. All of them have AAT serum levels between 80 and 102 mg/dl. None of the studied subjects had clinical evidence of lung disease. CONCLUSIONS: The results of our study show the presence of the two AAT deficient variants in Spain and suggest that the Pl Plovel variant might be more common than expected.
Assuntos
Alelos , Enfisema Pulmonar/genética , Deficiência de alfa 1-Antitripsina/genética , Adulto , Éxons , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Mutação Puntual/genética , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Severe alpha-1-antitrypsin (AAT) deficiency is caused by homozygous inheritance of gene Z, and is associated with a high risk of developing pulmonary emphysema. Determination of frequencies of different genes associated with the deficiency (especially S and Z) gives a clue to estimate the number of individuals homozygous PiZZ, carrying a high risk for pulmonary disease, in any given population. PATIENTS AND METHODS: Pi phenotypes of 440 healthy individuals were determined by means of isoelectrofocusing in polyacrylamide gel. Seric values of AAT were determined by immunonephelometry. Mean age of participants was 30 years (range 18-49 yrs.). Results are compared with other published series. RESULTS: Distribution of phenotypes was: PiMM 333 individuals (75%), PiMS 84 (19%), PiMZ 14 (3%), PiSS 4 (0.9%), PiM 3 (0.6%), PiMF 1 (0.2%), PiMP 1 (0.2%). The corresponding gene frequencies were Pi*M 87%, Pi*S 10.4%, and Pi*Z 1.5%. Normal values of AAT (phenotype PiMM) established in our laboratory were 116-232 mg/dl (21-41 micromol/I) (mean +/- 2 SD). According to Hardy-Weinberger equation, expected frequency of PiZZ individuals in our area would be 225 per million. CONCLUSIONS: The frequency of Z gen individuals observed in our study is one of the highest in the Iberian Peninsula, but lower than the frequency in northern Europe. According to these results, AAT deficiency (PiZZ) is not a rare condition in contrast with the small number of patients diagnosed. The gen frequency of the S variant is higher than that of the rest of Europe, and similar to others found in some Spanish populations.