935 resultados para Hepatitis C virus
Resumo:
We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4%) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America.
Resumo:
Few data are available in the literature concerning the efficacy of standard hysteroscope disinfection procedures to prevent hepatitis B transmission. The aim of the present study was to determine the risk of hepatitis B virus (HBV) transmission during hysteroscopy among anti-HBc-seropositive women. Serum and hysteroscopic samples were collected from 62 women after diagnostic hysteroscopy. All samples were tested for serologic HBV markers. Polymerase chain reactions (PCR) were carried out to amplify regions C and S of the viral genome and only samples amplified by both pairs of primers were considered to be positive. Anti-HBc was repeatedly reactive in 48 (77%) of 62 serum samples, and HBsAg was detected in 8 (13%). At least one HBV serologic marker was found in 49 (79%) samples. Only one sample was HBsAg positive and anti-HBc negative. HBV-DNA was detected by PCR in 7 serum samples but in only 3 hysteroscopic samples obtained just after hysteroscopy. It is noteworthy that high levels of anti-HBc IgM were detected in one HBsAg-negative patient who showed an HBV-DNA-positive hysteroscopic sample. An elevated sample/cut-off ratio for anti-HBc IgM suggests recent infection and reinforces the need for testing for HBsAg and anti-HBc before hysteroscopy, since acute hepatitis B can be clinically asymptomatic. Viral DNA was not detected in any hysteroscopic samples collected after washing and disinfecting procedures with glutaraldehyde. We conclude that HBV-DNA can be found in the hysteroscope soon after hysteroscopy, but standard disinfecting procedures are effective in viral removal.
Resumo:
Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).
Resumo:
The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5%. HAV-RNA was detected in 46% IgM-positive serum samples and in 16% stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90% of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.
Resumo:
The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8%) than in family members of Western origin (36.5%). HBsAg was also more frequent among brothers (79.6 vs 8.5%; P < 0.0001), children (37.9 vs 3.3%; P < 0.0001) and other family members (33.9 vs 16.7%; P < 0.0007) of Asian than Western origin, respectivelly. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7%) and 23/26 (88.5%) of the Asian and Western origin, respectively.
Resumo:
The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.
Resumo:
The prevalence of hepatitis B virus (HBV) in Brazil increases from South to North but moderate to elevated prevalence has been detected in the Southwest of Paran State. The prevalence of serological markers of HBV was evaluated in 3188 pregnant women from different counties in Paran State and relevant epidemiological features were described. The prevalence of HBV markers in pregnant women for the state as a whole was 18.5% (95% CI = 17.2-19.9), ranging from 7.2% in Curitiba to 38.5% in Francisco Beltro. The endemicity of HBV marker prevalence in pregnant women was intermediate in Cascavel, Foz do Iguau, and Francisco Beltro, and low in Curitiba, Londrina, Maring, and Paranagu. Multiple logistic regression showed that HBV marker prevalence increased with age, was higher among black women, among women of Italian and German descent, and among women who had family members in neighboring Rio Grande do Sul State. Univariate analysis showed that HBV marker prevalence was also higher among women with no education or only primary education, with a lower family income and whose families originated from the South Region of Brazil. Pregnant women not having positive HBV markers (anti-HBc, HBsAg or anti-HBs detected by ELISA) corresponded to 73.7% of the population studied, implying that HBV vaccination needs to be reinforced in Paran State. The highest prevalence was found in three counties that received the largest number of families from Santa Catarina and Rio Grande do Sul, where most immigrants were of German or Italian ascendance. This finding probably indicates that immigrants that came to this area brought HBV infection to Southwestern Paran State.
Resumo:
Genotype E of hepatitis B virus (HBV) has not been described in Brazil and is found mainly in Africa. Genotype A is the most prevalent in Brazil, and genotypes B, C, D, and F have already been reported. We report here an HBV genotype E-infected patient and some characterization of surface (S) protein, DNA polymerase (P) and precore/core (preC/C) coding regions based on the viral genome. The patient is a 31-year-old black man with chronic hepatitis B who was born and raised in Angola. He has been followed by a hepatologist in So Paulo, Brazil, since November 2003, and he is a frequent traveler to Latin America, Africa, and Europe. In 2003, he was diagnosed with HBV infection and started treatment with lamivudine with the later addition of adefovir dipivoxil. No known risk factor was identified. Serologically, he is HBsAg and anti-HBe positive, but HBeAg and anti-HBs negative. DNA sequence analysis of the S/P region confirmed that this patient is infected with genotype E, subtype ayw4. The preC/C region showed G1896A and G1899A mutations but no mutations in the basal core promoter. Nucleotide substitutions common in genotype E were also observed (C1772, T1858 and A1757). Although this is not an autochthonous case and there is no evidence of further spread, the description of this case in Brazil highlights the current risk of viral genotypes spreading with unprecedented speed due to constant travel around the world.
Resumo:
The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.
Resumo:
Estimates of occult hepatitis B virus (HBV) infection prevalence varies among different studies depending on the prevalence of HBV infection in the study population and on the sensitivity of the assay used to detect HBV DNA. We investigated the prevalence of occult HBV infection in cirrhotic patients undergoing liver transplantation in a Brazilian referral center. Frozen liver samples from 68 adults were analyzed using a nested polymerase chain reaction assay for HBV DNA. The specificity of the amplified HBV sequences was confirmed by direct sequencing of the amplicons. The patient population comprised 49 (72.1%) males and 19 (27.9%) females with a median age of 53 years (range=18-67 years). Occult HBV infection was diagnosed in three (4.4%) patients. The etiologies of the underlying chronic liver disease in these cases were alcohol abuse, HBV infection, and cryptogenic cirrhosis. Two of the patients with cryptic HBV infection also presented hepatocellular carcinoma. Markers of previous HBV infection were available in two patients with occult HBV infection and were negative in both. In conclusion, using a sensitive nested polymerase chain reaction assay to detect HBV DNA in frozen liver tissue, we found a low prevalence of occult HBV infection in cirrhotic patients undergoing liver transplant, probably due to the low prevalence of HBV infection in our population.
Resumo:
Linterfron- pegyl en combinaison avec la ribavirin est le seul traitement approuv pour le traitement de linfection au virus de lhpatite C (VHC). Lefficacit est de 50-75%, la thrapie est coteuse et induit beaucoup deffets secondaires. Il est impratif davoir une meilleure comprhension de la pathogense du VHC afin de dvelopper des traitements plus efficaces ou un vaccin. cette fin, notre approche est de caractriser la rponse immunitaire cellulaire induite par ARFP, un antigne nouveau et conserv chez le VHC, et de cartographier les pitopes de la rponse immunitaire cellulaire dun patient infect au gnotype 3a ayant rsolu spontanment. Le gnotype 3a, tant prvalant chez les utilisateurs de drogues intraveineuses (IDUs) constitue 60% des nouvelles infections. Peu dpitopes furent identifis auparavant pour ce gnotype, ce qui rend ltude de la rponse immunitaire difficile chez cette population. Dans cette tude, pour la rponse immunitaire cellulaire dirige contre ARFP, nous navons pas observ de diffrence significative entre les patients ayant rsolu spontanment comparativement avec ceux ayant dvelopp une infection persistante. Ceci suggre fortement que ARFP ne joue pas un rle majeur lors de la rsolution de linfection aigue au VHC. Pour la caractrisation de la rponse immunitaire cellulaire chez un des patients infects au gnotype 3a, nous avons identifi et caractris 5 pitopes spcifiquement reconnus par des lymphocytes T, CD3+, CD4+ et CD8- : E2504-521, NS31064-1081, NS4b1759-1776, NS5a2074-2091, NS5b2421-2436. Nous avons compar avec ceux connus pour le gnotype 1a. Nous avons identifi 4 nouveaux pitopes. Enfin, lpitope NS4b1759-1776, identifi auparavant, pourrait savrer tre un candidat intressant dans la mise au point dun vaccin base de peptides immunogniques contre le VHC.
Resumo:
L'hpatite C pose un problme de sant publique majeur, dans la mesure o le risque de dvelopper une infection chronique est relativement lev (40 60%) et o la rsistance au traitement de choix - linterfron alpha pgyl et la ribavirine - touche prs de la moiti des patients. Cette persistence virale repose avant tout sur de puissantes stratgies dvasion du systme immunitaire inn de lhte par le virus. Dans ce projet, nous nous sommes intresss la caractrisation de la rponse antivirale dans des hpatocytes primaires humains normaux et chroniquement infects avec le VHC, un domaine encore largement inconnu d la difficult dobtenir ce type de matriel primaire. Nous avons tudi la fonctionnalit de deux voies majeures de dtection des pathognes viraux suite lexposition dhpatocytes primaires humains de lARNdb intracellulaire, via le rcepteur et adaptateur RIG-I/MDA5-CARDIF, et extracellulaire via TLR3-TRIF, mimant ainsi les tapes prcoces de la dtection dun virus par la cellule hte. Nous avons tabli par RT-PCR quantitatif et analyse transcriptomique par microarray, que ces deux voies de stimulation sont fonctionnelles dans des hpatocytes primaires normaux et que leur activation entrane la fois lexpression de gnes antiviraux communs (ISG56, ISG15, CXCL10, ) mais aussi spcifiques avec les gnes IL28A, IL28B et IL29 qui sont une signature de lactivation de la voie de dtection de lARNdb intracellulaire. La protine virale NS3/4A joue un rle majeur la fois dans le clivage de la polyprotine virale initiale, mais aussi en interfrant avec les cascades de signalisation engages suite la dtection par la cellule hte de lARN du VHC. Plus particulirement, nous avons dmontr que lexpression ectopique de NS3/4A dans des hpatocytes primaires humains normaux entrane une diminution significative de linduction des gnes antiviraux de au clivage de CARDIF au cours de lactivation de la voie de signalisation mdie par RIG-I. Nous avons galement dmontr que lexpression de la NS3/4A entrane des modifications de lexpression de gnes-cl impliqus dans la rgulation de lapoptose et du programme de mort cellulaire, en particulier lorsque la voie TLR3 est induite. Lensemble de ces effets sont abolis en prsence de BILN2061, inhibiteur spcifique de NS3/4A. Malgr les stratgies de subversion de limmunit inne par le VHC, nous avons dmontr linduction significative de plusieurs ISGs et chemokines dans des hepatocytes primaires provenant de patients chroniquement infects avec le VHC, sans toutefois dtecter dinterfrons de type I, III ou certains gnes antiviraux prcoces comme CCL5. Ces observations, concomitantes avec une diminution de lexpression de CARDIF et une correlation inverse entre les niveaux dARNm des ISGs et lARN viral rvlent une rponse antivirale partielle de des mcanismes interfrents sous-jacents. Cette rponse antivirale dtectable mais inefficace est mettre en lien avec lchec du traitement classique PEG-IFN-ribavirine chez la moiti des patients traits, mais aussi en lien avec linflammation chronique et les dommages hpatiques qui mnent ultimement au dveloppement dune fibrose puis dune cirrhose chez une grande proportion de patients chroniquement infects.
Resumo:
La majorit des individus exposs au virus de lhpatite C (VHC) dveloppent une infection chronique. Une rponse immunitaire adaptative forte et soutenue est associe avec la gurison spontane du VHC, mais les mcanismes sous-jacents demeurent mal dfinis. Le rle des cellules NK et des cellules dendritiques (DC) dans la gurison spontane du VHC est encore mconnu. Les cellules NK sont la population effectrice la plus importante de limmunit inne car elles tuent les cellules infectes et scrtent diverses cytokines. Les DC reconnaissent des agents infectieux et elles sont les premires initier et rguler limmunit adaptative. Les cellules NK et les DC interagissent galement entre elles afin de rguler limmunit inne et adaptative. Lhypothse du projet de doctorat est que l'activit des cellules NK pendant la phase aigu de l'infection par le VHC module la fonction des DC afin que ces dernires puissent gnrer une rponse immunitaire adaptative capable d'liminer le VHC. Le premier objectif tait dtablir une corrlation entre l'activit des cellules NK et l'volution de l'infection au VHC. Nous avons observ une augmentation de la cytotoxicit, mais une diminution de la scrtion de cytokines par les cellules NK chez les patients chroniques et qui ont rsolu spontanment pendant la phase aigu en comparaison aux contrles non infects, dmontrant alors une dissociation entre ces deux fonctions. Nos rsultats suggrent que les cellules NK sont actives pendant la phase aigu indpendamment de lvolution de linfection. Le deuxime objectif tait dtablir une corrlation entre le phnotype et la fonction des DC, et l'volution de l'infection. Nous avons dabord observ que les DC plasmacytodes de tous les patients infects ont un phnotype plus immature que les contrles, et que ce phnotype est plus prononc chez les patients ayant rsolu spontanment. De plus, en rponse des stimulations, nous avons observ que pendant la phase aigu prcoce, les DC mylodes (mDC) de tous les patients infects indpendamment de lvolution de linfection produisent davantage de cytokines en comparaison aux contrles. Cependant, cette hyperractivit nest pas soutenue au cours de lvolution chronique. Le troisime objectif tait dtablir une corrlation entre les interactions NK/DC et lvolution de linfection. Nous avons tudi la capacit des cellules NK lyser les DC potentiellement tolrogniques, ainsi que la capacit des DC matures activer les cellules NK, et nous avons observ aucune diffrence entre les patients infects et les contrles. Finalement, nous avons dmontr pour la premire fois la capacit des DC immatures inhiber la fonction des cellules NK. En conclusion, nous avons dmontr que les cellules NK sont actives pendant la phase aigu de linfection par le VHC indpendamment de lvolution de linfection. De plus, la capacit des cellules NK liminer les DC potentiellement tolrogniques est intacte. Finalement, les mDC sont hyperractives pendant la phase aigu de linfection, mais cette hyperractivit nest pas soutenue avec la persistance de linfection. Cette perte dhyperractivit des mDC ne semble pas affecter la capacit des DC activer les cellules NK, mais elle pourrait jouer un rle dans linefficacit de limmunit adaptative liminer le VHC.
Resumo:
La protine core du virus de lhpatite C (VHC) serait responsable des principaux effets pathognes du VHC, dont le dveloppement de fibrose, statose, cirrhose et carcinome hpatocellulaire. Un cadre de lecture alternatif existe dans le gne de core, permettant la synthse dune autre protine appele ARFP (pour alternatate reading frame protein) ou protine F (pour frameshift), dont le rle reste encore mal compris. La prsence de la protine F lors de ltude des fonctions biologiques de core ne pouvant tre exclue, il est possible que certains rles attribus core refltent en ralit lactivit de la protine F. Afin de dterminer les fonctions biologiques de la protine F dans les hpatocytes et son influence dans la pathogense associe au VHC, nous avons gnr des lignes transgniques de poissons zbrs (Danio rerio) dans lesquelles lexpression de deux versions de la protine F (AF11opti et AUG26opti) a t cible au foie par lutilisation du promoteur de la liver fatty acid binding protein (L-FABP). Le phnotype des poissons transgniques de gnration F2 a t analys au niveau morphologique, histologique et microscopique afin de rechercher des signes de pathologie hpatique. Nos rsultats ont dmontr limplication de la protine F dans le dveloppement de statose hpatique chez les deux lignes transgniques, mais aucun signe de fibrose ou doncogense na t dtect. Lidentification des mcanismes cellulaires et molculaires responsables de laccumulation lipidique induite par la protine F pourrait permettre de mieux comprendre son rle dans la pathogense du VHC, et mener au dveloppement de nouvelles stratgies antivirales.
Resumo:
La replication et lassemblage du virus de lhepatite C (VHC) sont regules finement dans le temps et lespace par les interactions proteiques entre le virus avec lhote. La comprehension de la biologie du virus ainsi que sa pathogenicite passe par les connaissances relatives aux interactions virus/hote. Afin didentifier ces interactions, nous avons exploite une approche dimmunoprecipitation (IP) couplee a une detection par spectrometrie de masse (MS), pour ensuite evaluer le role des proteines identifiees dans le cycle viral par une technique de silencage genique. Les proteines virales Core, NS2, NS3/4A, NS4B, NS5A et NS5B ont ete exprimees individuellement dans les cellules humaines 293T et immunoprecipitees afin disoler des complexes proteiques qui ont ete soumis a lanalyse MS. Ainsi, 98 proteines de lhote ont ete identifiees avec un enrichissement significatif et illustrant une specificite dinteraction. Lenrichissement de proteines connues dans la litterature a demontre la force de lapproche, ainsi que la validation de 6 nouvelles interactions virus/hote. Enfin, le role de ces interactants sur la replication virale a ete evalue dans un criblage genomique par ARN interferant (ARNi). Deux systemes rapporteurs de la replication virale ont ete utilises : le systeme de replicon sous-genomique (Huh7-Con1-Fluc) et le systeme infectieux (J6/JFH-1/p7Rluc2a), ainsi quun essai de toxicite cellulaire (Alamar Blue). Parmi les proteines de lhote interagissant avec le VHC, 28 proteines ont demontre un effet significatif sans effet de toxicite cellulaire, suggerant fortement un role dans la replication du VHC. Globalement, letude a mene a lidentification de nouvelles interactions virus/hote et lidentification de nouvelles cibles therapeutiques potentielles.
