Evaluation of the BACTEC MGIT 960 system and the resazurin microtiter assay for susceptibility testing of Mycobacterium tuberculosis to second-line drugs.

Drug resistance in tuberculosis is a major threat to public health and control of the disease worldwide. Given the need of a rapid and accurate detection of Mycobacterium tuberculosis resistance to second-line drugs, this study evaluated the performance of the BACTEC MGIT 960 for second-line, drug susceptibility testing in comparison with the resazurin microtiter assay (REMA), in order to implement the automated methodology in the diagnostic routine of a reference laboratory. Drug susceptibility testing (DST) for second-line drugs of 151 MDR M. tuberculosis clinical isolates was performed by both BACTEC MGIT 960 and REMA, and a panel of 26 M. tuberculosis reference isolates from a proficiency test was tested by the BACTEC MGIT 960. DST for second-line drugs by the BACTEC MGIT 960 system was more rapid, highly reproducible and showed 100% of proficiency. After these results, this methodology was successfully implemented in our diagnostic routine for all MDR-TB patients.

Hepatitis B virus infection in children, adolescents, and their relatives: genotype distribution and precore and core gene mutations.

INTRODUCTION: The objectives of this study were evaluate hepatitis B virus (HBV) serological markers in children and adolescents followed up at the Child Institute of the Hospital das Clínicas, Faculdade de Medicina de São Paulo, Universidade de São Paulo; identify chronic HBV carriers and susceptible individuals in the intrafamilial environment; characterize HBV genotypes; and identify mutations in the patients and household contacts. METHODS: Ninety-five hepatitis B surface antigen-positive children aged <19 years and 118 household contacts were enrolled in this study. Commercial kits were used for the detection of serological markers, and PCR was used for genotyping. RESULTS: Hepatitis B e antigen (HBeAg) was detected in 66.3% (63/95) of cases. Three of the 30 HBeAg-negative and anti-HBeAg-positive patients presented with precore mutations and 11 presented with mutations in the basal core promoter (BCP). Genotype A was identified in 39 (43.8%) patients, genotype D in 45 (50.6%), and genotype C in 5 (5.6%). Of the 118 relatives, 40 were chronic HBV carriers, 52 presented with the anti-HBc marker, 19 were vaccinated, and 7 were susceptible. Among the relatives, genotypes A, D, and C were the most frequent. One parent presented with a precore mutation and 4 presented with BCP mutations. CONCLUSIONS: Genotypes A and D were the most frequent among children, adolescents, and their relatives. The high prevalence of HBV in the families showed the possibility of its intrafamilial transmission.

Hepatitis B virus infection in children, adolescents, and their relatives: genotype distribution and precore and core gene mutations / Infecção pelo vírus da hepatite B em crianças, adolescentes e seus familiares: distribuição dos genótipos e mutações no gene pré-core e core

INTRODUCTION:The objectives of this study were evaluate hepatitis B virus (HBV) serological markers in children and adolescents followed up at the Child Institute of the Hospital das Clínicas, Faculdade de Medicina de São Paulo, Universidade de São Paulo; identify chronic HBV carriers and susceptible individuals in the intrafamilial environment; characterize HBV genotypes; and identify mutations in the patients and household contacts. METHODS: Ninety-five hepatitis B surface antigen-positive children aged <19 years and 118 household contacts were enrolled in this study. Commercial kits were used for the detection of serological markers, and PCR was used for genotyping. RESULTS: Hepatitis B e antigen (HBeAg) was detected in 66.3% (63/95) of cases. Three of the 30 HBeAg-negative and anti-HBeAg-positive patients presented with precore mutations and 11 presented with mutations in the basal core promoter (BCP). Genotype A was identified in 39 (43.8%) patients, genotype D in 45 (50.6%), and genotype C in 5 (5.6%). Of the 118 relatives, 40 were chronic HBV carriers, 52 presented with the anti-HBc marker, 19 were vaccinated, and 7 were susceptible. Among the relatives, genotypes A, D, and C were the most frequent. One parent presented with a precore mutation and 4 presented with BCP mutations. CONCLUSIONS: Genotypes A and D were the most frequent among children, adolescents, and their relatives. The high prevalence of HBV in the families showed the possibility of its intrafamilial transmission.

INTRODUÇÃO: Os objetivos deste estudo foram: avaliar os marcadores sorológicos nas crianças e adolescentes acompanhadas no Instituto da Criança do Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP); identificar portadores crônicos do VHB e indivíduos suscetíveis no ambiente intrafamiliar; caracterizar o genótipo do VHB; observar a presença de cepas mutantes entre os pacientes e familiares estudados. MÉTODOS: Noventa e cinco crianças e adolescentes positivas para o antígeno de superfície do vírus da Hepatite B (AgHBs), menores de 19 anos, e 118 familiares foram envolvidos neste estudo. Foram utilizados kits comerciais para a pesquisa dos marcadores sorológicos e a PCR foi utilizada para genotipagem. RESULTADOS: O antígeno e do vírus da hepatite B (AgHBe) foi detectado em 66,3% (63/95) dos casos. Três dos 30 pacientes AgHBe negativo e anti-HBe positivo apresentaram mutação na região pré-core e 11 na região BCP. Em 39 (43,8%) pacientes, foi identificado o genótipo A, 45 (50,6%)o genótipo D e cinco (5,6%) o genótipo C. Dos 118 familiares estudados, 40 eram portadores crônicos do VHB, 52 tinham marcador sorológico de contato prévio e sete eram suscetíveis. Dentre os familiares AgHBs positivos, os genótipos A, D e C foram os mais frequentes. Um familiar apresentou mutação na região pré-core e quatro apresentaram mutação na região do BCP. CONCLUSÕES: Os genótipos A e D foram os mais frequentes dentre as crianças adolescentes e seus familiares. Alta frequência do VHB nos familiares mostrou a possibilidade de transmissão intrafamiliar.

Hepatitis B virus genotypes from European origin explains the high endemicity found in some areas from southern Brazil.

Southern Brazil is considered an area of low Hepatitis B endemicity, but some areas of higher endemicity have been described in the Southwest of Paraná and Santa Catarina states. The aim of this study was to evaluate viral genotypes circulating throughout Paraná state. PCR amplification and partial sequencing of the S gene was carried out in 228 samples from HBsAg positive candidate blood donors. Samples have been collected in seven different counties (Cascavel, Curitiba, Foz do Iguaçu, Francisco Beltrão, Maringá, Londrina and Paranaguá). The most common HBV genotype in Paraná state was D (82.9%; 189/228), followed by A (14.1%; 32/228). Genotypes F (1.3%; 3/228), C (1.3%; 3/228) and H (0.4%; 1/228) were also found. Distribution of genotypes was different in the studied counties, but genotype D was the most frequent in all of them. In Francisco Beltrão, all studied samples belonged to genotype D. The high prevalence of HBV genotype D in South of Brazil is explained by the intense migration of settlers from Europeans countries. Subgenotypes A1 and A2 were identified circulating in all cities where HBV/A was found. As observed in other areas of Brazil, HBV/A1 is more frequent than the HBV/A2 in Paraná state and its presence was significantly larger in black and mulatto individuals. Genotype C was found only in individuals with Asian ancestry from Londrina and Maringá. Most HBV/F sequences identified in this study were classified as subgenotype F2a that was previously described in Brazil. The sole case of subgenotype F4 was from Foz do Iguaçu city, near to Northern Argentina, where F4 is highly prevalent. The single genotype H sample was from Curitiba. This is the first case of this genotype described in Brazil. Further studies should be carried out to determine if more genotype H samples can be found in other populations from Brazil.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance.

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

HBV markers in haemodialysis Brazilian patients: a prospective 12-month follow-up.

The aim of this study was to determine the prevalence and the incidence of hepatitis B virus (HBV) among haemodialysis (HD) subjects and to evaluate whether testing for serological markers at the time of admission is suitable for HBV screening in this population. One hundred twenty-three patients belonging to two HD centres from São Paulo, Brazil, were tested prospectively. HBV DNA was detected by polymerase chain reaction (PCR) in each of the prospective subjects (n = 123) during one year. Additionally, all samples (n = 1,476) were analysed for HBV serological markers. The prevalence of hepatitis B core antibody (anti-HBc), hepatitis B surface antigen (HBsAg) and HBV DNA were 34.1%, 15.4% and 8.1%, respectively, while the incidence was null. Fluctuation in HBV serology was observed in one patient. Only 37.8% (17/45) of cases responded to the HBV vaccine. Our results suggest that employing more than one HBV marker and repeated follow-up evaluations may improve HBV screening in HD units.

HBV markers in haemodialysis Brazilian patients: a prospective 12-month follow-up

The aim of this study was to determine the prevalence and the incidence of hepatitis B virus (HBV) among haemodialysis (HD) subjects and to evaluate whether testing for serological markers at the time of admission is suitable for HBV screening in this population. One hundred twenty-three patients belonging to two HD centres from São Paulo, Brazil, were tested prospectively. HBV DNA was detected by polymerase chain reaction (PCR) in each of the prospective subjects (n = 123) during one year. Additionally, all samples (n = 1,476) were analysed for HBV serological markers. The prevalence of hepatitis B core antibody (anti-HBc), hepatitis B surface antigen (HBsAg) and HBV DNA were 34.1 percent, 15.4 percent and 8.1 percent, respectively, while the incidence was null. Fluctuation in HBV serology was observed in one patient. Only 37.8 percent (17/45) of cases responded to the HBV vaccine. Our results suggest that employing more than one HBV marker and repeated follow-up evaluations may improve HBV screening in HD units.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance / Caracterização de uma cepa de hepatite por vírus B no sudoeste do Paraná, Brasil, apresentando mutações previamente associadas à resistência anti-HBs

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36 percent of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

O presente estudo investigou se mutantes do vírus da hepatite B (HBV) circulam na região Sudoeste do Estado do Paraná, Brasil, analisando amostras de crianças que receberam a imunoprofilaxia por terem nascido de mães portadoras do HBV. Amostras de 25 crianças foram analisadas para os marcadores sorológicos do HBV e para o DNA-HBV por PCR. Somente uma amostra foi positiva para AgHBs, anti-HBs e DNA-HBV, apesar da criança ter sido vacinada. Análises da seqüência do gene S desta amostra mostrou a presença de uma prolina na posição 105, uma serina na posição 114, três treoninas nas posições 115, 116 e 140, e uma glutamina na posição 129. A presença destes aminoácidos, exceto para Serina na posição 114, tem sido relacionada a terapia monoclonal ou policlonal com anti-HBs após transplante de fígado, enquanto a presença da treonina na posição 116 tem sido descrita em crianças imunizadas de Singapura. Este achado demonstra a possível circulação de cepas do HBV resistentes a imunoprofilaxia para hepatite B no Sudoeste do Paraná, Brasil. O genótipo da amostra foi identificado como genótipo D, o qual é frequentemente encontrado na região estudada. Desde que 36 por cento das crianças tinham recebido incompleta ou nenhuma imunoprofilaxia, um seguimento mais intensivo das crianças nascidas de mães AgHBs positivo é necessário.

Soroprevalência da hepatite B e avaliação da resposta imunológica à vacinação contra a hepatite B por via intramuscular e intradérmica em profissionais de um laboratório de saúde pública / Hepatitis B seroprevalence and evaluation of immune response to hepatitis B vaccination using intramuscular and intradermal routes in public health laboratory employees

OBJETIVOS: Determinar a prevalência dos marcadores da hepatite B (HBsAg e anti-HBs) e avaliar a resposta à vacinação contra hepatite B por via intradérmica (ID) em profissionais de saúde que não responderam à vacinação por via intramuscular (IM). MÉTODO: Todos os funcionários do Instituto Adolfo Lutz (IAL) foram convidados a participar do estudo. Amostras de soro foram colhidas no momento da administração da primeira dose de vacina (Engerix® B) e o HBsAg e o anti-HBs foram pesquisados, utilizando-se kits comerciais (Laboratórios Abbott®). Aos funcionários que não responderam à vacinação convencional (três doses por via IM) foram oferecidas doses de 5µg da mesma vacina por via ID. RESULTADOS: Foram envolvidos nesse estudo 404 funcionários do IAL, dos quais dois (0,5 por cento) eram HBsAg e 42 (10,5 por cento), anti-HBs reagentes. Dos 360 voluntários com sorologia negativa, 316 (87,8 por cento) receberam três doses de vacina (IM) e, desses, 259 colheram soros para avaliação pós-vacinal. Do total, 242 (93,4 por cento) apresentaram anticorpos acima de 10 UI/L após completarem o esquema inicial. Foram administradas duas doses de reforço, porém sete funcionários permaneceram sem resposta imunológica. A vacinação intradérmica foi realizada em cinco voluntários, e todos produziram anticorpos após a utilização dessa via de administração. CONCLUSÕES: A prevalência da hepatite B não foi maior nessa população do que na população geral. A vacinação por via intradérmica pode ser uma boa alternativa na imunização de pessoas que não respondem ao esquema convencional.

OBJECTIVES: To determine the prevalence of HBsAg and anti-HBs and to evaluate the response of intradermal hepatitis vaccination in healthcare workers non-responsive to previous repeated intramuscular vaccination. MATERIAL AND METHOD: All of the employees from Instituto Adolfo Lutz were invited to participate on this study. Serum samples were obtained and HBsAg and anti-HBs were detected using commercial kits (Abbott® Laboratories). Employees were submitted to the conventional three-dose vaccination by intramuscular route. To those employees who did not respond to intramuscular vaccination, 5 µg doses of Engerix® B were then administered by intradermal route up to nine doses. RESULTS: Four hundred and four healthcare workers were enrolled in this study. Initially, two (0.5 percent) and 42 (10.4 percent) were HBsAg and anti-HBs reagent, respectively. Among the 360 negative volunteers, 316 (87.8 percent) received three vaccine doses and in 259 of them, serum samples were collected to evaluate vaccine efficacy. Among them, 242 (93.4 percent) showed antibodies titer higher than 10 UI/l. Intradermal vaccination was carried out in five volunteers and all of them responded to this vaccine administration route. CONCLUSION: The prevalence of hepatitis B was not higher than in general population. Intradermal vaccine administration could be a good alternative in people that did not respond to previous intramuscular route.

Prospective study of hepatitis C virus infection in hemodialysis patients by monthly analysis of HCV RNA and antibodies.

The aims of this study were to (i) evaluate the prevalence and the incidence of hepatitis C virus (HCV) infection in hemodialysis patients in two different centers in São Paulo (Brazil), (ii) determine the time required to detect HCV infection among these patients by serology or PCR, (iii) establish the importance of alanine aminotransferase determination as a marker of HCV infection, and (iv) identify the HCV genotypes in this population. Serum samples were collected monthly for 1 year from 281 patients admitted to hospital for hemodialysis. Out of 281 patients, 41 patients (14.6%) were HCV positive; six patients seroconverted during this study (incidence = 3.1/1000 person-month). In 1.8% (5/281) of cases, RNA was detected before the appearance of antibodies (up to 5 months), and in 1.1% (3/281) of cases, RNA was the unique marker of HCV infection. The genotypes found were 1a, 1b, 3a, and 4a. The presence of genotype 4a is noteworthy, since it is a rare genotype in Brazil. These data pointed out the high prevalence and incidence of HCV infection at hemodialysis centers in Brazil and showed that routine PCR is fundamental for improving the detection of HCV carriers among patients undergoing hemodialysis.

Vigilância das hepatites virais: a experiência de Vargem Grande Paulista, 1997-1999 / Surveillance viral hepatitis: an experience of Vargem Grande Paulista, Säo Paulo State, Brazil, from 1997 to 1999

Analisa aspectos da operacionalizaçäo de um sistema de vigilância nas atuais condiçöes de trabalho da Rede Pública de Saúde e sua potencialidade em descrever o compartimento das hepatites nessa comunidade para oferecer subsídios para a elaboraçäo e aprimoramento de estratégicas de controle. O sistema inclui a análise de dados obtidos a partir de notificaçäo de casos suspeitos hepatite A, B, C e E entre residentes no município, assim como dados de soroprevalência de macadores de infecçäo para esses mesmos vírus numa populaçäo formada pelas gestantes inscritas no Serviço Pré-natal do Município. A confirmaçäo dos casos foi realizada pela identificaçäo dos marcadores sorológicos das hepatites A, B, C e E. Foram identificados 125 casos suspeitos, dos quais 41 (32,8 por cento) foram confirmados como hepatite A, B, C e E. A incidência de hepatite A foi 21,1/100.000 hab., 69,3/100.000 hab. e 9,3/100.000 hab. para os anos de 1997, 1998 e 1999, respectivamente. A incidência de hepatite B foi de 3,5/100.000 hab. e 9,9/100.000 hab. para os anos de 1997 e 1998 respectivamente. Näo foi identificado nenhum caso em 1999. A prevalência de hepatite C foi 3,5/100.000 hab. em 1997 e 9,9/100.000 hab. em 1998. A incidência de hepatite E foi 3,5/100.000 hab., 3,3/100.000 hab. e 3,1/100.000 hab. para 1997, 1998 e 1999. Entre as 793 gestantes que participaram do estudo, a prevalência de anti-VHA foi de 94,7 por cento, de anti-HBc 4,9 por cento, de HbsAg 0,1 por cento, de anti-VHC 0,6 por cento, e anti-VHE 0,8 por cento. Os resultados indicam que Vargem Grande Paulista apresentou alta endemicidade para hepatite A e baixa endemicidade para hepatite B. A prevalência de hepatite C foi semelhante à encontrada em outro estudos. A prevalência de hepatite C foi semelhante à encontrada em outros estudos. A prevalência e incidência da hepatite E mostrou que o vírus circulou na regiäo. Os dados demonstram que o sistema de vigilância pode contribuir com informaçöes importantes no comportamento das hepatites virais no município, oferecendo subsídios para a elaboraçäo de estratégia de prevençäo e controle dessas infecçöes

Detection of hepatitis A antibodies by ELISA using saliva as clinical samples

The possibility of detecting acute infection and immunity using body fluids that are easier to collect than blood, mainly in children, would facilitate the investigation and follow-up of outbreaks of hepatitis A (HAV). Our study was carried out to evaluate the detection of anti-HAV IgM, IgA and total antibodies in saliva using serum samples as reference. Forty three paired serum and saliva samples were analyzed. From this total, 24 samples were obtained from children and 1 from one adult during the course of acute hepatitis A; an additional 18 samples were obtained from health professionals from Adolfo Lutz Institute. The sensitivity to detect anti-HAV IgM was 100 per cent (95 per cent CI: 79.1 to 100.0 per cent), employing saliva as clinical samples. In detecting anti-HAV IgA, the sensitivity was 80.8 per cent (95 per cent CI: 60.0 to 92.7 per cent) and for the total antibodies was 82.1 per cent (95 per cent CI: 62.4 to 93.2 per cent). The specificity was 100 per cent for each. The rate of agreement was high comparing the results of serum and saliva samples for detecting HAV antibodies. We conclude that saliva is an acceptable alternative specimen for diagnosing acute hepatitis A infection, and for screening individuals to receive hepatitis A vaccine or immunoglobulin.