Student Number : 9903082V - MSc (Med) dissertation - Faculty of Health Sciences / Chronic infection with the hepatitis B virus (HBV) is endemic to sub-Saharan Africa and south-east Asia where it is a major risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). Currently available therapy is only effective in a small subset of chronic carriers. The development of novel treatment modalities for the management of HBV therefore remains an important global medical objective. Sequence plasticity of the HBV genome is limited by its small size and the overlapping nature of its open reading frames (ORFs). These features make HBV an ideal target for therapy based on nucleic acid hybridization. The use of ribozymes (RNA enzymes) and antisense molecules to inhibit gene expression is well documented. The recent discovery of RNA interference (RNAi) has added to the arsenal of therapy based on nucleic acid hybridization. RNAi is the process whereby short RNA duplexes (called short interfering RNA or siRNA) mediate the sequence-specific post-transcriptional silencing of genes homologous in sequence to the siRNA. siRNA function by guiding a protein complex (RNA Induced Silencing Complex or RISC) to target mRNA for degradation or translational repression. The protein X ORF (HBx ORF) is a conserved region of the HBV genome and is common to all viral transcripts. HBx is required for infection by the virus and plays an important role in the establishment of chronic infections in vivo as well as in the development of HCC. RNAi targeted against the HBx ORF may therefore prove useful as treatment of chronic HBV infection. Plasmid based expression cassettes capable of endogenously generating short hairpin RNA (shRNA) targeted to the HBx ORF were constructed. The shRNA function as substrates for the RNAi machinery and are processed into siRNA. The ability of the expression cassettes to knockdown markers of HBV gene expression was tested in a human hepatoma cell line. A panel of 10 U6 promoter-driven shRNA expression vectors was generated. The U6 promoter (an RNA polymerase III promoter) is normally involved in the transcription of small nuclear RNA and as such is ideal for the generation of shRNA of precisely defined length. Three cytomegalovirus (CMV) promoter-driven shRNA expression cassettes incorporating ribozymes that produce defined hairpin sequences were also generated. The CMV promoter (an RNA polymerase II) promoter is involved in the transcription of large messenger RNA. Two hammerhead ribozymes lying 5’ and 3’ of the shRNA encoding sequence were incorporated into the cassette. Cis-cleavage by the ribozymes releases a shRNA of defined length thereby overcoming the limitations imposed by extraneous sequences from CMV promoter-driven transcription. U6 promoter-driven shRNA expression vectors efficiently knocked down markers of HBV replication in liver cells. The CMV promoter-driven expression vectors were incapable of inhibiting HBV gene expression; however shRNA generated in vitro from these vectors mediated efficient knockdown of HBV replication. shRNA-mediated inhibition of gene expression therefore holds promise as a novel treatment strategy for the management of HBV and other mobile genetic elements.

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy

Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery

Contexte : Environ 257 millions de personnes dans le monde sont infectées de manière chronique par le virus de l'hépatite B (VHB). La transmission mère-enfant représente la majorité des nouveaux porteurs chroniques du VHB, en particulier en Asie. Le VHB peut être transmis in utero, lors de l'accouchement ou pendant la petite enfance voire plus tard. Environ 80 à 90% des nourrissons infectés à la naissance développent une infection chronique par le VHB avec le risque élevé de développer de graves complications, notamment une fibrose du foie, une cirrhose, un carcinome hépatocellulaire jusqu’à un décès lié au foie à l’âge adulte. Nous avons évalué la proportion des enfants immunisés avec succès dans deux grands hôpitaux de Vientiane, en République démocratique populaire lao, où l’immunoglobuline HB (HBIg) n’est pas disponible. Méthodes : Nous avons étudié une cohorte prospective de femmes enceintes infectées par le VHB et de leurs enfants jusqu'à six mois après la naissance de janvier 2015 à mars 2017. Tous les nourrissons ont reçu le vaccin anti-HB à la naissance et six, 10 et 14 semaines après la naissance. Le statut d’infection par le VHB chez l’enfant a été évalué à l’âge de 6 mois. Le séquençage du gène de surface du VHB a été effectué chez des couples mère-enfant infectés. Résultats : Sur 153 mères ayant été dépistées positives pour l'antigène de surface HB (AgHBs), 60 (39%) avaient l'antigène HBe (AgHBe) positif. Les femmes enceintes ayant AgHBe-positif étaient plus jeunes que les femmes ayant d’AgHBe négatif ((âge médian 26 versus 28 ans; p = 0,02). et avaient une charge virale du VHB significativement plus élevée à l'accouchement (médiane 8,0 versus 4,0 log10 UI / mL, p <0,001). Au total, 141 nouveau-nés, dont une paire de jumeaux, sont nés pendant l'étude. Parmi les 112 nouveau-nés pour lesquels l’information concernant le délai entre la naissance et l’heure d’administration du vaccin contre le VHB était disponible, 110 (98%) ont reçu le vaccin dans les 24 heures suivant la naissance. Le délai médian entre la naissance et l'administration du vaccin était de 6 heures (EI 3 à 13), avec 95 (72%) dans les 12 heures suivant la naissance. Un nouveau-né a reçu le vaccin 26 heures après la naissance car le vaccin n'était pas disponible dans la salle d'accouchement et un autre nouveau-né a reçu le vaccin 3 jours après la naissance car, en raison d'une détresse respiratoire sévère à la naissance, le vaccin a été considéré comme contre-indiqué. Parmi les 120 enfants évalués à l'âge de 6 mois, 5 (4%) étaient positifs pour AgHBs et présentaient une charge virale détectable pour le VHB par réaction en chaîne avec une polymérase. Tous sont nés de mères ayant l’AgHBe positif et une charge virale> 8,5 log10 UI / mL. Cependant, seulement quatre enfants (3,3%, IC à 95%, 0,5% à 7,0%) avaient une souche virale étroitement apparentée à celle de leur mère. Des mutations du gène de surface du VHB (G145G/R, G145G/A, M133T, M133I) ont été détectées chez 4 des 5 enfants infectés. Le taux d'anticorps anti-HBs était supérieur à 10 UI / L chez 105 nourrissons (88%) à l'âge de 6 mois. Conclusions : La transmission de la mère à l'enfant s'est produite moins souvent que ce que nous avions prévu en absence de l'utilisation de HBIg. L’ajout d’une prophylaxie par HBIg et / ou antivirale maternelle aurait pu prévenir certaines de ces infections. L'observation du taux d'anticorps anti-HBs non satisfait chez 9% des enfants non infectés à 6 mois souligne la nécessité d'améliorer les procédures d'immunisation universelles / Background: An estimated 257 million people are chronically infected with the hepatitis B virus (HBV) worldwide. Mother-to-child transmission accounts for the majority of new chronic HBV carriers, especially in Asia. HBV can be transmitted in utero, during delivery or during infancy and later. About 80–90% of infants infected at birth will develop a chronic HBV infection, and will have a high risk of developing serious complications including liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and liver-related death during adult age. We aimed at assessing the percentage of infants successfully immunized in two major hospitals in Vientiane, Lao People's Democratic Republic (Lao PDR) where HB immune globulin (HBIg) is not available. Methods: We studied a prospective cohort of chronically HBV infected pregnant women and their infants until 6 months post-partum from January 2015 to March 2017. All infants received the HB vaccine at birth and 6, 10 and 14 weeks thereafter, and their HBV status was assessed at 6 months of age. HBV surface gene sequencing was performed in infected mother-infant pairs.Results: Of 153 mothers with HB surface antigen (HBsAg), 60 (39%) had detectable serum HBe antigen (HBeAg). HBeAg positive pregnant women were younger than those negative (median age 26 versus 28 years; p=0.02) and had a significantly higher HBV viral load at delivery (median 8.0 versus 4.0 log10 IU/mL, p <0.001). A total of 141 infants including a pair of twins were included in the study and information at the time of vaccine administration after birth was available for 112 newborns. Of these, 110 (98%) received the HepB-BD within 24 hours after birth. One newborn received the vaccine 26 hours after birth because the vaccine was not available at the delivery room, and another newborn 3 days after birth due to fetal distress, which was erroneously considered to be a vaccine contra-indication. Among the 120 infants assessed at 6 months of age, 5 (4%) were positive for HBsAg and had a detectable HBV viral load by polymerase chain reaction. All were born to mothers with HBeAg and a viral load >8.5 log10 IU/mL. However, only four (3.3%, 95% CI 0.5% to 7.0 %) had a virus strain closely related to their mother’s strain. HBV surface gene mutations were detected in 4 of the 5 infected infants (G145G/R, G145G/A, M133T, M133I). Anti-HBs antibody level was above 10 IU/L in 105 (88%) infants at 6 months of age. Conclusions: Mother-to-child transmission occurred less frequently than expected without the use of HBIg. Adding HBIg and/or maternal antiviral prophylaxis may have prevented some of these infections. The observation of unsatisfactory levels of anti-HBs antibodies in 9% of the uninfected infants at 6 months highlights the need for improvement of the universal immunization procedures

Os exames laboratoriais são fundamentais como ferramentas para elucidar o diagnóstico e avaliar prognóstico na prática médica. Novos exames são constantemente adicionados ao elenco já existente na clínica, o que pode resultar em elevação dos custos, nem sempre com o retorno esperado. A padronização da solicitação de exames tem sido estimulada, tanto na saúde pública quanto na saúde suplementar considerando os impactos clínicos, financeiros e ambientais de solicitações desnecessárias. O Hospital das Clínicas de Ribeirão Preto (HCRP) tem revisto protocolos e criado uma hierarquização para solicitação de testes complementares. Este estudo justifica-se por avaliar uma ação que visou adequar a solicitação de marcadores sorológicos de hepatite B, ajustando-os às normas preconizadas por resolução da Secretaria da Saúde do Estado de São Paulo. Objetivo: Avaliar a adequação das requisições médicas dos marcadores sorológicos para hepatite B enviadas ao laboratório de sorologia do HCRP e o impacto da triagem (\"reflective testing\"/\"reflex testing\") instituída pelo laboratório. Métodos: Estudo retrospectivo, de caráter descritivo, com dados coletados através de 24.649 solicitações médicas de marcadores de hepatite B recebidas em dois períodos: Fase 1 - sete meses entre 2012 e 2013; Fase 2 - nove meses entre 2014 e 2015. Avaliamos os marcadores solicitados e a hipótese diagnóstica informada pelo médico. Foram consideradas adequadas as solicitações de rastreio para HBV com os marcadores HBsAg e Anti-HBcAg Ig-total isolados ou em conjunto. A rotina de adequação incluiu a exclusão de marcadores inadequados ou a complementação dos marcadores sorológicos indicados, sempre que o HBsAg era positivo, independente da solicitação médica. Uma análise de custos foi realizada, considerando os exames solicitados e aqueles que foram efetivamente realizados. Resultados: Fase 1 - das 11.167 solicitações avaliadas, 1.143 (10,23%) foram consideradas inadequadas. Fase 2 - das 13.482 solicitações analisadas, 251 (1,86%) foram consideradas inadequadas. Com o processo de triagem realizado pelo laboratório foi possível economizar respectivamente R$ 19.617,25 e R$ 1.683,80 nos dois períodos analisados. Estas informações servirão de base para estruturação de uma proposta de orientação para solicitação dos marcadores sorológicos da hepatite B, junto às equipes onde identificamos maior frequencia de inadequação. Conclusão: Os processos de triagem manual (\"reflective testing\") e eletrônico (\"reflex testing\") realizados pelo laboratório demonstraram ser custo-efetivos e aceleram a liberação de resultados relevantes para a tomada de decisão clínica / Laboratory tests are critical as tools to elucidate the diagnosis and assess prognosis in medical practice. New tests are constantly added to the list already exists in the clinic, which can result in higher costs, not always with the expected return. The standardization of test ordering has been stimulated both in public health and in health insurance considering the clinical, financial and environmental impacts of unnecessary requests. The Hospital of the Faculty of Medicine of Ribeirão Preto (HCRP) has revised protocols and created a hierarchy to request additional tests. This study is justified by assessing an action that aimed to suit the request of serological markers of hepatitis B, adjusting them to the standards established by resolution of the São Paulo State Health Department. Objective: To evaluate the adequacy of medical requests of serological markers for hepatitis B sent to the serology lab HCRP and the impact of screening (\"reflective testing\" / \"reflex testing\") established by the laboratory. Methods: Retrospective study of a descriptive nature, with data collected through 24,649 medical claims of hepatitis B markers received in two periods: Phase 1 - seven months between 2012 and 2013; Phase 2 - nine months between 2014 and 2015. We evaluated the requested markers and the diagnosis reported by the physician. Tracing requests for HBV with HBsAg and anti-HBcAg alone or together were considered adequate. The adjustment routine includes the exclusion of inappropriate markers or serological markers of the indicated complementation, where the HBsAg was positive, whether for medical application. A cost analysis was performed, considering the requested examinations and those who have been realized. Results: Phase 1 - 11,167 of assessed requests, 1,143 (10.23%) were considered inadequate. Phase 2 - the 13,482 requests analyzed, 251 (1.86%) were considered inadequate. With the screening process carried out by the laboratory was possible to save respectively R $ 19,617.25 and R $ 1,683.80 in both periods analyzed. This information will form the basis for structuring a proposal to request guidance of serological markers of hepatitis B, with teams where we identify higher frequency of inadequacy. Conclusion: The manual sorting processes (\"reflective testing\") and electronic (\"reflex testing\") performed by the laboratory have shown to be cost-effective and accelerate the release of relevant results for clinical decision making

O objetivo deste estudo foi avaliar a distribuição genotípica e mutações no genoma do vírus da hepatite B (VHB) em um grupo de pacientes co-infectados pelo VHB e vírus da imunodeficiência humana (HIV). Foram incluídos pacientes AgHBs +/HIV+ , atendidos em um ambulatório de referência para pacientes infectados pelo HIV, na cidade de São Paulo. Para a detecção dos marcadores sorológicos para infecção pelo VHB utilizou-se técnica de ELISA através de kits comerciais. A detecção do DNA-VHB foi realizada através de nested-PCR e sua quantificação foi realizada por COBAS AMPLICOR. De acordo com a literatura, a infecção pelo VHB no Brasil varia de 0,4 a 8,5%. Os genótipos de VHB, as mutações na região do core, BCP, pré-core e na região da polimerase foram determinados por seqüenciamento. Cinqüenta e nove pacientes foram incluídos neste estudo e cinqüenta e seis pacientes relatavam uso prévio de lamivudina ou tenofovir. A presença do DNA-VHB foi detectada em 22 pacientes AgHBs positivos. A identificação dos genótipos foi realizada em 16 pacientes e a distribuição dos genótipos do VHB foi: A (12-75%); G (2-13%), D (1-6%) e F (1-6%). Em 10 dos pacientes com viremia presente para DNA-VHB, foram observadas mutações na região da polimerase (rtL180M + rtM204V, rtV173L + rtL180M + rtM204V) e no gene do envelope (sI195M, sW196L, sI195M/sE164D). Mutações na região do BCP (A1762T, G1764A) e do pré-core (G1896A) foram identificados em quatro pacientes. Em conclusão, entre os pacientes analisados observou-se uma alta prevalência de mutações associadas a resistência à lamivudina e associadas a resistência a anti-HBs. O genótipo G, raramente descrito em nosso meio, foi também observado nesse grupo de pacientes. / The objective of this study was to evaluate the genotype distribution and genomic mutations of hepatitis B virus (HBV) among a group of HIVHBV co-infected patients from an AIDS outpatient clinic in São Paulo. HBV serological markers were detected by commercially available enzyme immunoassay kits. HBV DNA was detected by using an in-house nested PCR and quantified by COBAS AMPLICOR. HBV genotypes, basal core promoter (BCP) / pre-core / core region and surface / polymerase genes mutations were determined by sequencing. Among the 59 patients included in this study, 56 reported previous use of lamivudine or tenofovir. According to the literature HBV infection in Brazil varies from 0,4 to 8,5%. HBV DNA was detected in 16/22 patients and the genotypes distribution was A (n=12, 75%); G (n=2, 13%); D (n=1, 6%), and F (n=1, 6%). In 10 patients with viremia, lamivudine-resistance mutations in the polymerase gene (rtL180M + rtM204V, rtV173L + rtL180M + rtM204V) were found, accompanied by changes in the envelope gene (sI195M, sW196L, and sI195M/sE164D). Mutations in the BCP and pre-core regions were identified in 4 patients. In conclusion, genotype G, rarely seen in Brazil, was observed in this group of patients. A high prevalence of mutations associated with lamivudine-resistance accompanied by mutations associated with anti-HBs resistance was also found among these patients.

INTRODUÇÃO: Hepatite B é uma das doenças infecciosas mais prevalentes no mundo, controlada em países onde a vacinação foi implantada, porém permanece alta em populações de risco e em países onde a transmissão vertical e horizontal intradomiciliar ainda persiste. O Estado de Santa Catarina é considerado hoje uma região de alta endemicidade para o VHB, tem uma prevalência estimada de AgHBs em doadores de sangue de 1,14%, o que é consideravelmente maior do que aquela estimada para o Brasil (0,61%). Em Chapecó, oeste de Santa Catarina, a prevalência do AgHBs em doadores de sangue foi de 3,2% em 1999, 1,63% em 2000 e 1,54% em 2001, significativamente maior do que em outras cidades do Estado. OBJETIVOS: Estudar os portadores de hepatite B crônica no município de Chapecó, possibilitando o conhecimento das características epidemiológicas, clínicas e laboratoriais do vírus nesta região de alta endemicidade. MÉTODOS: Estudou-se dois grupos de portadores de hepatite B crônica naquela região. O primeiro grupo (Grupo A) foi composto pelos casos notificados no ano de 1996 e o segundo grupo (Grupo B) foi composto pelos casos notificados no ano de 2006. RESULTADOS: Dos 352 pacientes notificados nos anos de 1996 e 2006 como portadores de hepatite B crônica no Município de Chapecó, 150 pacientes foram elegíveis após a aplicação dos critérios de inclusão e exclusão. A média de idade na época da notificação do grupo A foi de 29,9 anos (± 10,3) e do grupo B foi de 34,9 anos (± 11,9). O principal meio de diagnóstico foi após doação de sangue, sendo 53,6% pacientes no grupo A e 34,8% pacientes no grupo B. A maioria nasceu na região Oeste de Santa Catarina, sendo a cidade de Chapecó a mais freqüente. Seus familiares são naturais, em sua maioria, do Estado do Rio Grande do Sul. 96,4% dos pacientes do grupo A e 86,4% do grupo B apresentam AgHBe negativo. A maioria dos pacientes nos dois períodos estudados apresentava valores de ALT até 2x o LSN. 60,2% dos pacientes AgHBe negativos do grupo A apresentava carga viral <= 2.000 UI/mL, enquanto no grupo B 78,3% apresentava carga viral > 2.000 UI/mL. Todos os 105 pacientes que tiveram o VHB genotipado, apresentaram VHB genótipo D. CONCLUSÕES: A maioria dos pacientes é natural do Oeste de Santa Catarina e seus familiares do Estado do Rio Grande do Sul e Região do Mediterrâneo. Aminotransferases próximas aos limites da normalidade, AgHBe negativo e VHB genótipo D caracterizam os portadores de hepatite B crônica nessa região. A maioria dos pacientes do grupo A apresentou carga viral <= 2.000 UI/mL e no grupo B carga viral > 2.000 UI/mL. / INTRODUCTION: Viral hepatitis B is one of the most prevalent infectious diseases in the world. It remains under control in countries where vaccination programs have been established; however, its prevalence is still high in atrisk populations and in countries where vertical and horizontal intra domestic transmission persists. Santa Catarina province is currently considered a region of high endemicity for hepatitis B virus (HBV), with an estimated prevalence of 1.14% HBsAg in blood donors, which is considerably higher than the estimated prevalence for the country (0.61%). In Chapecó, a city located in the western region of the province, HBsAg prevalence in blood donors was 3.2% in 1999, 1.63% in 2000 and 1.54% in 2001, values that were significantly higher than in other cities of the province. OBJECTIVES: To study the carriers of chronic hepatitis B in the city of Chapecó in order to determine the epidemiological, clinical, and laboratorial aspects of HBV infection in this high endemicity region. METHODS: Two groups of carriers in that region were studied. The first group, designated Group A, was composed of cases that had been notified in the year 1996; while the second group, designated Group B, comprised cases that had been notified in the year 2006. RESULTS: From the 352 patients that had been notified as chronic hepatitis B carriers in the years 1996 and 2006 in Chapecó, 150 were eligible for inclusion in the study according to the inclusion and exclusion criteria. The mean age at the time of notification for Group A was 29.9 yearsold (±10.3) and for Group B was 34.9 years-old (± 11.9). The main reason leading to the diagnosis was routine screening of blood donors, which was responsible for 53.6% of diagnoses in Group A and 34.8% of diagnoses in Group B. The majority of patients were born in western Santa Catarina province, most frequently in the city of Chapecó. Their relatives were mostly from Rio Grande do Sul province. HBeAg was negative in 96.4% of Group A patients and 86.4% of Group B patients. The majority of patients from both periods studied had ALT values up to twice the upper normal limit (UNL). From HBeAg negative patients of Group A, 60.2% had a viral load <= 2.000 UI/mL; while from those of Group B, 78.3 % had viral loads > 2.000 UI/mL. All of the 105 cases in which the genotype of HBV has been determined were infected by genotype D HBV. CONCLUSIONS: The majority of patients were born in western Santa Catarina, and their relatives were from Rio Grande do Sul province or from Europe Mediterranean countries. Viral hepatitis B carriers from this region characteristically had aminotransferases close to the normal limits, negative HBeAg and were infected with genotype D HBV. Most of the patients from Group A had viral loads <= 2.000 UI/mL, while most of the patients from Group B had viral loads > 2.000 UI/mL.

As hepatites virais continuam sendo uma preocupação em nível de saúde pública no Brasil e no Mundo, tanto pelo número de indivíduos atingidos, como pela possibilidade de complicação das formas agudas e crônicas. Segundo a Organização Mundial de Saúde (OMS), 170 milhões de pessoas são portadoras crônicas de hepatite C e 350 milhões portadoras crônicas de hepatite B. No Brasil, a estimativa de portadores de hepatite B crônica é de aproximadamente 600 mil pessoas e de hepatite C crônica, 1,5 milhão. Quanto à hepatite aguda A foram confirmados no país, em 2010, 5943 casos. O objetivo deste estudo foi conhecer a prevalência de marcadores sorológicos dos vírus das hepatites A, B e C em crianças e adolescentes matriculados em creches e escolas de ensino infantil e fundamental da rede municipal na cidade de Santos; conhecer os aspectos moleculares dos vírus das hepatites B e C, identificando o genótipo dos dois agentes e estudar modo de aquisição nos casos com sorologias positivas. Tratou-se de um estudo transversal realizado no período de 28 de Junho a 14 de Dezembro de 2007 onde foram coletadas 4680 amostras de sangue colhidas através de punção capilar e ao mesmo tempo aplicado um questionário nos familiares das crianças e adolescentes. Os exames sorológicos foram realizados utilizando a técnica de ELISA. O estudo molecular foi realizado pela técnica de reação em cadeia de polimerase \"in House\". A idade da população estudada variou de 7 meses a 18 anos e 1 m. A prevalência geral do anti-HVA IgG reagente foi de 9,7% e desses 74,6% foi anti-HVA IgM reagente. A prevalência de anti-HVA IgG foi maior entre as crianças mais velhas, meninas, aquelas que brincavam em córregos, sem esgoto em sua moradia, de pais com baixa instrução, de baixa renda familiar e aquelas que não eram moradoras da Orla. A prevalência de anti-HVA IgM, não foi diferente entre as diferentes categorias, exceção feita à faixa etária (pico no primeiros anos e posterior queda) e morro e Zona Noroeste foi mais baixa. A prevalência geral do anti-HBc reagente foi de 0,1%, do AgHBs de 0,02% e do anti-HVC foi de 0,02%. Conclui-se que a prevalência geral em crianças dos marcadores sorológicos para hepatites A, B e C na cidade de Santos foi baixa, quando comparada com os dados de literatura. Apesar dos nossos dados confirmarem uma mudança no perfil epidemiológico da hepatite A, as medidas preventivas atuais quanto ao saneamento, grau de instrução, habitação, ainda permanece com uma deficiência em Santos, cidade balneária com o maior porto do Brasil. A vacinação para hepatite B foi altamente eficaz com a baixa prevalência encontrada dos marcadores sorológicos. A utilização do papel de filtro em estudos epidemiológicos para hepatite A foi eficaz. Entretanto para o vírus da hepatite C ainda necessita de estudos comparativos utilizando sangue venoso, uma vez que a prevalência de crianças infectadas com hepatite C foi muito baixa na cidade de Santos. / Viral hepatitis are still a concern in the public health level in Brazil and around the Word, due both to the number of affected subjects and the possibility of complication in the acute and chronic forms. According to the World Health organization (WHO), 170 million people are chronic carriers of hepatitis C and 350 million chronic carriers of hepatitis B. In Brasil, the estimate of people with chronic hepatitis B is approximately 600 thousand people and chronic hepatitis C, 1,5 million. It has been confirmed in the country, in 2010, 5943 cases of acute hepatitis A. The The aim of this study was to learn the prevalence of serological markers of hepatitis A, B and C virus in children and teenagers enrolled at the municipal education network in the city of Santos, to learn molecular aspects of hepatitis B and C, identifying the genotype of the two agents and to study the acquisition mode in cases with positive serology. Cross-sectional study carried out over the period from June 28 to December 14, 2007, in which 4680 fingerprick blood samples were collected; at the same time, a survey questionnaire was applied to the family members of the children and teenagers. The serological tests were performed using the ELISA technique. The molecular analysis was performed using the technique of polymerase chain reaction \"in House\". Age of the population studied ranged from 7 months to 18 years and 1 month. . The general prevalence of serological markers anti-HAV IgG reagent was 9.7% and between them 74,7% was anti-HAV IgM reagent. There was higher prevalence among older children, females, those who used to play in streams near their home, the absence of a sewage system in home, parents with low education, low household income and among those who did not live in the seashore. The prevalence of anti-HAV IgM was not different between the categories, except for the age (peak in the early years and subsequent fall) and lower on the Hills and Northweast Zone. The general prevalence of anti-HBc reagent was 0,1%, AgHBs was 0,02% and anti-HCV was 0,02%. It is concluded that, in children, the general prevalence of serological markers for hepatitis A, B and C in the city of Santos was low when compared with literature data. Although our data confirm a change in the epidemiological profile of hepatitis A, the current preventive measures regarding sanitation, education level and housing still remain with a deficiency in Santos, the coastal city with the largest harbor in Brazil. Vaccination for hepatitis B was highly effective because it found a low prevalence of serological markers. The use of filter paper in epidemiological studies for hepatitis A was effective. However for the vírus of hepatitis C still requires comparative studies using venous blood because the prevalence of infected children was very low in the city of Santos.

Foi realizada uma pesquisa com cirurgiões-dentistas (CDs) e auxiliares odontológicos (AOs) das redes pública e privada, no município de Ribeirão Preto,SP – Brasil, com os objetivos de: 1. avaliar a cobertura vacinal e a efetividade da vacinação contra a hepatite B; 2. verificar o uso de equipamentos de proteção individual (EPIs) na rotina de trabalho; 3. verificar a prevalência de marcadores sorológicos do HBV; 4. analisar possíveis associações entre marcadores sorológicos do HBV e alguns fatores de risco de infecção pelo vírus. A pesquisa contou com 338 participantes, de consultórios e clínicas odontológicas – 201 CDs(26 da rede pública e 175 da rede privada) e 137 AOs (23 da rede pública e 114 da rede privada). Foi aplicado um questionário individual para obter informações referentes a características dos participantes, vacinação contra a hepatite B e fatores de risco relacionados à infecção pelo HBV. Simultaneamente, coletou-se uma amostra de 8 ml de sangue para a pesquisa dos seguintes marcadores sorológicos do HBV: HBsAg, anti-HBc e anti-HBs. Todos os exames foram feitos, através de técnicas imunoenzimáticas, no Laboratório de Sorologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP. Estavam vacinados com três doses ou mais da vacina contra a hepatite B 80,6% dos CDs (80,8% da rede pública e 80,6% da rede privada) e 24,8% dos AOS(82,6% da rede pública e 13,2% da rede privada). A prevalência de anti-HBs entre os vacinados com três doses ou mais da vacina foi de 84,3% - 84,6% entre CDs e 82,8% entre AOs. A prevalência dos marcadores sorológicos do HBV ficou assim distribuída: HBsAg, 0,5% entre CDs, não encontrado entre AOs; anti-HBc, 9,5% entre CDs (IC 95%: 5,4% - 13,5%) e 2,9% entre AOs (IC 95%: 0,1% - 5,7%);anti-HBs, 78,1% entre CDs (IC 95%: 72,3% - 83,8%) e 29,9% entre AOs (IC 95%: 22,2 - 37,6%). Os EPIs mais utilizados pelos CDs eram as luvas, com percentual de 98,0%, e a máscara, com 96,5%, no atendimento de todos os pacientes. Os CDs da rede privada utilizavam os seguintes EPIs: luvas, 97,7%; máscara, 96,0%; óculos de proteção, 70,3%; avental, 55,4%; gorro 32,6%. Os CDs da rede pública usavam os seguintes EPIs: luvas e máscaras, 100,0%; avental, 88,5%; óculos de proteção, 65,4%; gorro, 30,8%. O EPI mais utilizado pelos AOs eram as luvas, com percentual de 97,1%. Os AOs da rede pública utilizavam: luvas, 100,0%; avental, 95,7%; máscara, 78,3%; óculos de proteção; 34,8%; gorro, 17,4%. Os AOs da rede privada utilizavam: luvas, 96,5%; máscara, 37,7%; avental, 28,1%; óculos de proteção, 11,4%; gorro, 10,5%. Dos AOs da rede privada, 3,5% referiram não usar nenhum EPI. O estudo mostrou que a prevalência dos marcadores sorológicos do HBV entre CDs e AOs é semelhante à da população de pacientes de Unidades de Saúde de Ribeirão Preto e que parte dos profissionais, especialmente os AOs, negligenciam a proteção pela imunização ativa e uso de EPIs. / A survey was carried out among dentists (D) and dental assistants (DA) of the public and the private sectors in the city of Ribeirão Preto, São Paulo, Brazil, in order to a) evaluate the vaccine coverage as well as the effectiveness of the vaccination against hepatitis B; b) check for the presence of serological markers of the hepatitis B virus(HBV)infection; assess the use of individual protection equipments (IPE). The survey,involving 338 participants working in dentist private offices or in dental clinics, comprised 201 D (26 of the public sector and 175 of the private sector) and 137 DA(23 of the public sector and 114 of private practices). Individual survey questionnaires were used to gather information regarding the participants, the vaccination against hepatitis B, and the risk factors related to the HBV infection. Blood samples (8 ml) were collected to assess the following serological markers for the HBV infections: HBsAg, Anti-HBc, and Anti-HBs. All essays were done with the immunoenzimatic technique by the Serological Laboratory of the Hospital das Clinicas of the University of São Paulo Medical School, at Ribeirão Preto. A higher proportion of the D compared to the DA were vaccinated with three or more doses of the hepatitis B: 80.6% of the D (80.8% of the public sector and 80.6% of the private sector) but only 24,8% of the DA (82.6% of the public sector and 13,2% of the private sector). Among those vaccinated with three or more doses 84.3% (84.6% for D and 82.8% for DA) presented positive tests for the anti-HBs marker. Considering the whole population evaluated, the occurrence of serological markers for HBV infection was distributed as follows: HBsAg, 0.5% in D and zero in DA; anti-HBc, 9.5% in D (95% CI: 5.4% to 13.5%) and 2.9% in DA (95% CI: 0.1% to 5.7%); and anti-HBs, 78.1% in D (95% CI: 72.3% to 83.8%) and 29.9% in DA (95% CI: 22.2% to 37.6%). Most dentists make use of some IPE when treating patients: 98.0% wore gloves and 96.5% face masks. There was little difference in use of IPE between dentists working in the private and the public sectors: for the former 97.7% wore gloves, 96.0% face masks, 70.3% protective eyewear, 55.4% protective uniform, and 32.6% caps; for the later, 100% wore gloves and face masks, 88.5% protective uniform, 65.4% protective eyewear, and 30.8% caps. A high percentage of the DA also wore IPEs, although the numbers were lower for those of the private sector. Among the former group 100% wore gloves, 95.7% protective uniform, 78.3% face masks, 34.8% protective eyewear, and 17.4% caps. The corresponding figures for the DA of the private dental offices and clinics were: 96.5% wore gloves, 37.7% face masks, 28.1% protective uniform, 11.4% protective eyewear, and 10.5% caps. A small percentage (3.5%) of the DA working in the private sector did not report the use of any IPE. The study showed that the occurrence of serological markers for infection with HBV in dental professionals is similar to that of the population as a whole, and that part of the professionals, especially the DAs, neglect to secure protection by active immunization and use of IPEs.

Introdução. A infecção causada pelo vírus da hepatite B (HBV) é um dos agravos de maior prevalência no mundial. Segundo a Organização Mundial da Saúde (OMS) existem mais de 350 milhões de portadores crônicos da doença. A infecção pelo HBV é rotineiramente identificada quando há a presença do HBsAg circulante. Entretanto, em alguns casos o HBV-DNA tem sido detectado em indivíduos HBsAg negativos, positivos ou negativos para anti-HBc e anti-HBs. Essa apresentação sorológica e molecular é denominada infecção oculta pelo HBV (OBI). Geralmente a concentração do HBV-DNA no soro será abaixo de 200 UI/mL. Objetivo: Determinar a prevalência da OBI, em doadores de sangue do Estado do Amapá no ano de 2014. Material e Métodos. Foram analisadas um total de 62 amostras de doadores de sangue do estado do Amapá, no ano de 2014, que apresentavam o perfil sorológico: HBsAg negativo, anti-HBc positivo e com anti-HBs negativo ou positivo. Os marcadores sorológicos HBsAg e anti-HBc foram determinados através do imunoensaio quimioluminescente. As amostras selecionadas para a detecção do HBV-DNA foram testadas utilizando as metodologias de Real-Time PCR Kit NAT HIV/HCV/HBV (Bio-Manguinhos®) e PCR \"in house\". Resultados. Do total de 13.261 doadores triados para infecções transmissíveis pelo sangue, 283 apresentaram resultados reagentes para os marcadores da hepatite B, nos quais: 35 (0,3%) foram HBsAg e 248 (1,9%) para anti-HBc reagentes. Um total de 62 amostras foram testadas pelos métodos moleculares. Todas as amostras apresentaram resultado HBV-DNA não-detectável. Conclusão: O índice de inaptidão sorológica no ano de 2014 no HEMOAP foi de 1,9% para anti-HBc e 0,3% para HBsAg. A população estudada foi constituída predominantemente por adultos com idade entre 29-65 anos, do sexo masculino, casados e naturais de municípios do estado do Amapá / Introduction. The infection caused by the hepatitis B virus (HBV) is one of the most prevalent diseases in the world. According to the World Health Organization (WHO) there are more than 350 million chronic carriers of the disease. HBV infection is routinely identified when there is presence of circulating HBsAg. However, in some cases HBV-DNA has been detected in HBsAg negative individuals, positive or negative for anti-HBc and anti-HBs. This serological and molecular presentation is termed HBV-occult infection (OBI). Usually the concentration of HBV-DNA in serum will be below 200 IU / mL.Objective: To determine the prevalence of OBI in blood donors, in the State of Amapá, in the 2014. Material and methods. A total of 62 samples of blood donors from the State of Amapá in the year 2014 were analyzed, presenting the serological profile: HBsAg negative, anti-HBc positive and with negative or positive anti-HBs. Serum markers HBsAg and anti-HBc were determined by the chemiluminescent immunoassay. Samples selected for HBV-DNA detection were tested using the Real-Time PCR Kit Kit HIV / HCV / HBV (Bio-Manguinhos®) and in-house PCR. Results. Of the total of 13,261 donors screened for blood-borne infections, 283 presented reactive results for hepatitis B markers, in which: 35 (0.3%) were HBsAg and 248 (1.9%) for anti-HBc reagents. All samples showed nondetectable HBV-DNA result. Conclusion: The serological disability index in the year 2014 in HEMOAP was 1.9% for anti-HBc and 0.3% for HBsAg. The studied population consisted predominantly of adults aged 29-65 years, males, married and natural of cities of the state of Amapá