RESUMO
Host population size, density, immune status, age structure, and contact rates are critical elements of virus epidemiology. Slum populations stand out from other settings and may present differences in the epidemiology of acute viral infections. We collected nasopharyngeal specimens from 282 children aged ≤5 years with acute respiratory tract infection (ARI) during 2005 to 2006 in one of the largest Brazilian slums. We conducted real-time reverse transcription-polymerase chain reaction (RT-PCR) for 16 respiratory viruses, nested RT-PCR-based typing of rhinoviruses (HRVs), and collected clinical symptoms. Viruses were common causes of respiratory disease; with ≥1 virus being detected in 65.2% of patients. We detected 15 different viruses during 1 year with a predominance of HRV (33.0%) and human respiratory syncytial virus (hRSV, 12.1%) infections, and a high rate of viral coinfections (28.3%). We observed seasonality of hRSV, HRV and human coronavirus infections, more severe symptoms in hRSV and influenza virus (FLU) infections and prolonged circulation of seven HRV clusters likely representing distinct serotypes according to genomic sequence distances. Potentially unusual findings included the absence of human metapneumovirus detections and lack of typical FLU seasonal patterns, which may be linked to the population size and density of the slum. Nonetheless, most epidemiological patterns were similar to other studies globally, suggesting surprising similarities of virus-associated ARI across highly diverse settings and a complex impact of population characteristics on respiratory virus epidemiology.
Assuntos
Coinfecção/epidemiologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/transmissão , Viroses/epidemiologia , Viroses/transmissão , Brasil/epidemiologia , Criança , Pré-Escolar , Coronavirus/genética , Coronavirus/isolamento & purificação , Humanos , Lactente , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Densidade Demográfica , Áreas de Pobreza , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Viroses/virologiaRESUMO
AIDS-associated Kaposi's sarcoma (AIDS-KS) caused by human herpes virus 8 (HHV-8) is the most severe and resistant form of KS tumor. Our aim was to verify whether there is an association between HHV-8 variability and development of AIDS-KS in Brazil by comparing the HHV-8 variability between individuals without and with KS. Saliva samples and blood, when available, were analyzed by polymerase chain reaction (PCR) techniques for detection of the fragments of ORF K1 of HHV-8, which were then genotyped and analyzed regarding the genetic variability. Our study described 106 positive cases for HHV-8 in the saliva from 751 AIDS patients without previous KS. In addition, we performed a phylogenetic analysis of HHV-8 in 34 of the 106 AIDS patients without KS and in 33 of the 37 patients with active KS. The distribution of HHV-8 genotypes A, B, C, and F in AIDS individuals was indistinguishable by comparing non-KS and KS groups, as well as regarding ethnicity. Considering the KS group, genotype B was associated with better prognosis of KS tumor. Interestingly, we found a particular profile of diversity within clade C and 2 recombinant patterns of HHV-8 in the saliva of AIDS individuals without KS. We emphasize the need to achieve standard genotyping protocol for ORF K1 amplification, thus allowing for substantial detection of HHV-8 variants. Our findings can shed light on the role of HHV-8 variability in the pathogenesis of AIDS-KS.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Infecções Oportunistas Relacionadas com a AIDS/virologia , Síndrome da Imunodeficiência Adquirida/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Proteínas Virais/genética , Brasil , Estudos Transversais , DNA Viral/análise , Feminino , Genes Virais , Variação Genética , Genótipo , Humanos , Masculino , Fases de Leitura Aberta , Filogenia , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Saliva/virologiaRESUMO
Several efforts have been made to establish novel biomarkers with relevant predictive values to monitor HCV-infected patients under pegilated Interferon-α2A-(PEG-IFN-α2A)/ribavirin therapy. The aim of this study was to monitor the kinetics of HCV viral load, serum levels of pro-inflammatory/regulatory cytokines and leukocyte activation status before and after PEG-IFN-α2A/ribavirin therapy in 52 volunteers, including 12 chronic HCV patients and 40 controls. The HCV viral load, serum levels of cytokines (IL-8/IL-6/TNF-α/IL-12/IFN-γ/IL-4/IL-10) and the phenotype of peripheral blood leukocytes were evaluated before and after 4, 12 and 24 weeks following the PEG-IFN-α2A/ribavirin therapy. Our results demonstrated that sustained virological response-(SVR) is associated with early decrease in the viral load after 4 weeks of treatment. The presence of a modulated pro-inflammatory profile at baseline favors SVR, whereas a strong inflammatory response at baseline predisposes to therapeutic failure. Furthermore, a time-dependent increase on serum IL-12 levels in patients under treatment is critical to support the SVR, while the early predominance of IL-10 correlates to late virological relapse. On the other hand, a broad but unguided "cytokine storm" is observed in the non-responder HCV patients after 12 weeks of treatment. Corroborating these findings, monocyte/lymphocyte activation at baseline is associated with the non-responders to therapy whereas high CD8(+) T-cell numbers associate with SVR. All in all, these data suggest that the baseline pattern of serum pro-inflammatory/regulatory cytokines and the immunological activation status of chronic HCV patients undergoing PEG-IFN-α2A/ribavirin therapy are closely related with the therapeutic response.
Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/imunologia , Antivirais/administração & dosagem , Biomarcadores Farmacológicos/metabolismo , Células Cultivadas , Citocinas/sangue , Quimioterapia Combinada , Humanos , Imunofenotipagem , Interferon-alfa/administração & dosagem , Interleucina-12/uso terapêutico , Polietilenoglicóis/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Ribavirina/administração & dosagem , Falha de Tratamento , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
We investigated the clinical impact of human coronaviruses (HCoV) OC43, 229E, HKU1 and NL63 in pediatric patients with cystic fibrosis (CF) during routine and exacerbation visits. A total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. Samples positive for HCoV were submitted for nucleotide sequencing to determine the species. Nineteen samples (4.65%) were positive for HCoV, of which 8 were positive for NL63, 6 for OC43, 4 for HKU1, and 1 for 229E. Identification of HCoV was not associated with an increased rate of respiratory exacerbations, but NL63-positive patients had higher exacerbation rates than patients who were positive for other HCoV species.
Assuntos
Infecções por Coronavirus/complicações , Infecções por Coronavirus/virologia , Coronavirus/classificação , Fibrose Cística/complicações , Adolescente , Sequência de Bases , Criança , Pré-Escolar , Coronavirus/genética , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Nasofaringe/virologia , Filogenia , RNA Polimerase Dependente de RNA/genéticaRESUMO
INTRODUCTION: In the State of Amazonas, data regarding the prevalence of different genotypes of hepatitis C virus remains scarce. METHODS: The genotype of 69 HCV positive patients was determined. An in-house standardized nested-PCR was used to detect HCV RNA. Genotype assignment was based on type-specific motifs on the sequenced amplicons delimited by primers HC11/HC18 from the 5' untranslated region. RESULTS: Of the 69 patients studied, 65.2% were male and 34.8% were female. Genotype 1 showed the greatest prevalence, followed by 3 and 2. CONCLUSIONS: These data suggesting that Manaus is the point of arrival of HCV in the State of Amazonas.
Assuntos
Hepacivirus/genética , Hepatite C Crônica/virologia , RNA Viral/análise , Brasil , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
INTRODUÇÃO: No Estado do Amazonas, os dados sobre a prevalência dos genótipos do vírus da hepatite C ainda são escassos. MÉTODOS: Os genótipos do VHC foram determinados em 69 pacientes da Fundação de Medicina Tropical do Amazonas - FMT-AM. O RNA do VHC foi detectado pela técnica de RT-PCR, utilizando-se iniciadores HC11/HC18 para a região 5'não traduzida. RESULTADOS: Dos 69 pacientes, 65,2 por cento era do sexo masculino e 34,8 por cento do feminino. O genótipo 1 foi o mais prevalente, seguidos dos 3 e 2. CONCLUSÕES: Estes dados sugerem que Manaus é uma porta de entrada do vírus VHC no Estado do Amazonas.
INTRODUCTION: In the State of Amazonas, data regarding the prevalence of different genotypes of hepatitis C virus remains scarce. METHODS: The genotype of 69 HCV positive patients was determined. An in-house standardized nested-PCR was used to detect HCV RNA. Genotype assignment was based on type-specific motifs on the sequenced amplicons delimited by primers HC11/HC18 from the 5' untranslated region. RESULTS: Of the 69 patients studied, 65.2 percent were male and 34.8 percent were female. Genotype 1 showed the greatest prevalence, followed by 3 and 2. CONCLUSIONS: These data suggesting that Manaus is the point of arrival of HCV in the State of Amazonas.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hepacivirus/genética , Hepatite C Crônica/virologia , RNA Viral/análise , Brasil , Genótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Treatment of HIV-1 infection with highly active antiretroviral therapy has led to sustained viral suppression in the plasma in a large number of children. However, studies have suggested that the integrated provirus in resting CD4+ T lymphocytes could be a source of reactivatable virus and maintain drug-resistant virus. We evaluated the resistance-related mutations in children receiving antiretroviral therapy with prolonged viral suppression. Thirty-two peripheral blood mononuclear cell samples from 16 children with viral loads that had been below detection limits for at least 12 months were obtained at two different time points and the DNAs sequenced. The median CD4 cell count was 1,016 cells/mm³ (347-2,588) and 938 cells/mm³ (440-3,038) at the first and second time points, respectively. The median follow-up time was 15 months (9-27). Six (37.5%) and seven (43.75%) of the 16 patients showed at least one NRTI-associated mutation in the first and second samples, respectively. Two out of 16 (12.5%) had an NNRTI-associated mutation at the first time point and three out of 16 (18.75%) at the second. In addition, 14 out of 16 (87.5%) had at least one PI-associated mutation at both time points. Despite plasma HIV-1 RNA suppression for at least 12 months, resistance-related mutations from previous antiretroviral failures could still be detected in archival virus. Furthermore, viral evolution occurred at the reverse transcriptase region in spite of viral suppression to levels below 400 copies/mL. Persistence of archival resistant virus may be relevant when considering future treatment options.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1/genética , Mutação/genética , Contagem de Linfócito CD4 , Criança , Seguimentos , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/virologia , Carga Viral , Viremia/virologiaRESUMO
Treatment of HIV-1 infection with highly active antiretroviral therapy has led to sustained viral suppression in the plasma in a large number of children. However, studies have suggested that the integrated provirus in resting CD4+ T lymphocytes could be a source of reactivatable virus and maintain drug-resistant virus. We evaluated the resistance-related mutations in children receiving antiretroviral therapy with prolonged viral suppression. Thirty-two peripheral blood mononuclear cell samples from 16 children with viral loads that had been below detection limits for at least 12 months were obtained at two different time points and the DNAs sequenced. The median CD4 cell count was 1,016 cells/mm³ (347-2,588) and 938 cells/mm³ (440-3,038) at the first and second time points, respectively. The median follow-up time was 15 months (9-27). Six (37.5 percent) and seven (43.75 percent) of the 16 patients showed at least one NRTI-associated mutation in the first and second samples, respectively. Two out of 16 (12.5 percent) had an NNRTI-associated mutation at the first time point and three out of 16 (18.75 percent) at the second. In addition, 14 out of 16 (87.5 percent) had at least one PI-associated mutation at both time points. Despite plasma HIV-1 RNA suppression for at least 12 months, resistance-related mutations from previous antiretroviral failures could still be detected in archival virus. Furthermore, viral evolution occurred at the reverse transcriptase region in spite of viral suppression to levels below 400 copies/mL. Persistence of archival resistant virus may be relevant when considering future treatment options.
Assuntos
Criança , Humanos , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1 , Mutação/genética , Seguimentos , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1 , Leucócitos Mononucleares/virologia , Carga Viral , Viremia/virologiaRESUMO
To investigate a possible role for human rhinovirus C in respiratory exacerbations of children with cystic fibrosis, we conducted microbiologic testing on respiratory specimens from 103 such patients in São Paulo, Brazil, during 2006-2007. A significant association was found between the presence of human rhinovirus C and respiratory exacerbations.
Assuntos
Fibrose Cística/complicações , Infecções por Picornaviridae/etiologia , Infecções Respiratórias/etiologia , Rhinovirus/isolamento & purificação , Adolescente , Brasil/epidemiologia , Criança , Pré-Escolar , Fibrose Cística/epidemiologia , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Muco/virologia , Nasofaringe/virologia , Filogenia , Infecções por Picornaviridae/epidemiologia , RNA Viral/análise , RNA Viral/genética , Infecções Respiratórias/epidemiologia , Rhinovirus/genética , Análise de Sequência de RNA , Especificidade da Espécie , Escarro/virologiaRESUMO
BACKGROUND: JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is classified in 8 different genotypes. Previous reports have suggested a positive association between specific genotypes and PML. OBJECTIVE: To compare genotypes and adaptive mutations of JCV strains from Brazilian AIDS patients with and without PML. STUDY DESIGN: The VP1 region of JCV was amplified by polymerase chain reaction from cerebrospinal fluid samples from 51 patients with PML and from urine samples of 47 patients with AIDS without central nervous system disease. Genotyping was done by phylogenetic analysis. Amino acid replacement and selection pressures were also investigated. RESULTS: JCV genotype frequency distributions showed that genotypes 2 (32.7%), 1 (26.5%) and 3 (23.5%) were the most prevalent. Genotype 1 had a positive association (p<0.0001) and genotype 3 showed an inverse association (p<0.001) with PML. A previously undescribed point mutation at residue 91 (L/I or L/V) and (L/P), non-genotype-associated, was found in 5/49 (10.2%) and 2/47 (4.3%) JCV sequences from PML and non-PML patients, respectively. This mutation was under positive selection only in PML patients. A previously described substitution of T-A in position 128 showed a significant difference between PML and non-PML cases (70% versus 16%, respectively, p<0.0005). CONCLUSION: In Brazilian patients with AIDS, JCV genotype 1 showed a strong association with PML (p<0.0001) and JCV genotype 3 showed an inverse association with PML. The possible association of aminoacids substitution in residues 91 and 128 with PML in patients with AIDS must be further investigated.
Assuntos
Infecções por HIV/virologia , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/virologia , Substituição de Aminoácidos , Teorema de Bayes , Brasil , Proteínas do Capsídeo/genética , Distribuição de Qui-Quadrado , DNA Viral/análise , Genótipo , Infecções por HIV/complicações , Humanos , Leucoencefalopatia Multifocal Progressiva/líquido cefalorraquidiano , Leucoencefalopatia Multifocal Progressiva/complicações , Leucoencefalopatia Multifocal Progressiva/urina , Método de Monte Carlo , Filogenia , Análise de Sequência de DNAAssuntos
Doadores de Sangue , Doenças Transmissíveis Emergentes/epidemiologia , Hepatite C/epidemiologia , Adolescente , Adulto , Brasil/epidemiologia , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/virologia , Reações Falso-Negativas , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Hepatite C/virologia , Humanos , Immunoblotting , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/sangue , RNA Viral/genética , Adulto JovemRESUMO
Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were performed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.
Assuntos
Infecção Hospitalar/genética , Infecção Hospitalar/transmissão , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/transmissão , Vírus Sinciciais Respiratórios/genética , Assistência Ambulatorial , Infecção Hospitalar/epidemiologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Transmissão de Doença Infecciosa do Profissional para o Paciente/prevenção & controle , Masculino , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.
Assuntos
Líquido da Lavagem Nasal/virologia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sinciciais Respiratórios , Doença Aguda , Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Técnicas de Cultura de Células , Pré-Escolar , Estudos de Coortes , Técnica Direta de Fluorescência para Anticorpo , Humanos , Lactente , Recém-Nascido , Estudos Prospectivos , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/microL (equivalent to 2465.8 molecules/microL) to 10-9 (equivalent to 2.46 molecules/microL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/microL and for the nested PCR was 2.46 molecules/microL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.
Assuntos
Exantema Súbito/diagnóstico , Corantes Fluorescentes , Herpesvirus Humano 6/genética , Compostos Orgânicos , Reação em Cadeia da Polimerase/métodos , Benzotiazóis , DNA Viral/análise , Diaminas , Humanos , Quinolinas , Sensibilidade e EspecificidadeRESUMO
Burkholderia cepacia complex isolates obtained by microbiological culture of respiratory samples from Brazilian CF patients were studied by recA based PCR, screened by specific PCR for virulence markers and genotyped by RAPD. Forty-one isolates of B. cepacia complex were identified by culture and confirmation of identity and genomovar determination obtained in 32 isolates, with predominance of B. cenocepacia (53.1%). Virulence markers were not consistently found among isolates. Genotyping did not identify identical patterns among different patients. B. cenocepacia was the most prevalent B. cepacia complex member among our patients, and cross-infection does not seem to occur among them.
Assuntos
Infecções por Burkholderia/genética , Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/isolamento & purificação , Fibrose Cística/microbiologia , Brasil/epidemiologia , Infecções por Burkholderia/epidemiologia , Estudos de Coortes , Genótipo , Humanos , Prevalência , Recombinases Rec A/genética , Virulência/genéticaRESUMO
A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4 percent) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1 percent) of the samples, followed by direct immunofluorescence (25/316, 7.9 percent) and viral isolation (20/315, 6.3 percent) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4 percent) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.
Um total de 316 amostras de lavado de nasofaringe obtidas de crianças em acompanhamento ambulatorial com até dois anos de idade durante episódio de doença aguda do trato respiratório foram processadas para detecção do vírus sincicial respiratório (VSR) utilizando três diferentes técnicas: isolamento viral, imunofluorescência direta e reação em cadeia por polimerase (RT-PCR). Destas amostras, 36 (11,4 por cento) foram positivas para o VSR. A RT-PCR foi a técnica mais sensível, com positividade em 35 (11,1 por cento) das amostras, seguindo-se a imunofluorescência direta (25/316, 7,9 por cento) e o isolamento viral (20/315, 6,3 por cento) (p < 0,001). Uma amostra foi positiva pela imunofluorescência e negativa pela RT-PCR, e 11/36 (31,4 por cento) foram positivas somente pela RT-PCR. Concluímos que a RT-PCR é mais sensível que a imunofluorescência e o isolamento viral para detecção do VRS em amostras de aspirado de nasofaringe de recém-nascidos e lactentes.
Assuntos
Pré-Escolar , Humanos , Lactente , Recém-Nascido , Líquido da Lavagem Nasal/virologia , Vírus Sinciciais Respiratórios , Infecções por Vírus Respiratório Sincicial/diagnóstico , Doença Aguda , Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Técnicas de Cultura de Células , Estudos de Coortes , Técnica Direta de Fluorescência para Anticorpo , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.
HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL) até 10-9 (equivalente a 2,46 moleculas/µL). As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com primers HHV3 5' TTG TGC GGG TCC GTT CCC ATC ATA 3' e HHV4 5' TCG GGA TAG AAA AAC CTA ATC CCT 3' e pelo método convencional, PCR nested usando primers HHV1 (externo): 5' CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (externo): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3', HHV3 (interno) e HHV4 (interno): 5' TCG GGA TAG AAA AAC CTA ATC CCT 3'. O limite de detecção foi determinado pelas diluições seriadas do plasmídio contendo um fragmento de HHV6: para o ensaio com Sybr Green, foi de 24,6 moleculas/µL e para a PCR nested, 2,46 moleculas/µL. Elegemos o PCR em Tempo Real - Sybr Green como método diagnóstico e quantitativo do HHV-6 devido a sua boa sensibilidade e menor risco de contaminação.
Assuntos
Humanos , Exantema Súbito/diagnóstico , Corantes Fluorescentes , /genética , Compostos Orgânicos , Reação em Cadeia da Polimerase/métodos , DNA Viral/análise , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Human herpesvirus type 8 (HHV-8) is hyperendemic in Amerindian populations, but its modes of transmission are unknown. METHODS: Antibodies against either HHV-8 lytic antigen or HHV-8 latency-associated nuclear antigen (LANA) were detected, by immunofluorescence assays, in 339 Amerindians and 181 non-Amerindians from the Brazilian Amazon. Serological markers of oro-fecal (hepatitis A), parenteral (hepatitis B and C), and sexual (herpes simplex virus type 2 and syphilis) transmission were measured by specific ELISAs. Salivary HHV-8 DNA was detected by use of a nested polymerase chain reaction assay and was sequenced. RESULTS: Antibodies against either lytic antigen or LANA were detected in 79.1% of Amerindians and in 6.1% of non-Amerindians (adjusted seroprevalence ratio [SR], 12.63 [95% confidence interval {CI}, 7.1-22.4]; P<.0001). HHV-8 seroprevalence increased with age among Amerindians (P(Trend) < .001) and already had high prevalence in childhood but was not sex specific in either population. The 2 populations did not differ in seroprevalence of oro-fecal or parenteral markers, but seroprevalence of markers of sexual transmission was lower among Amerindians. HHV-8 DNA in saliva was detected in 47 (23.7%) of 198 HHV-8 seropositive Amerindians. Detection of HHV-8 DNA decreased with age (P(Trend) < .04) and was more common in men (SR, 2.14 [95% CI, 1.3-3.5]; P=.003). A total of 36 (76.6%) of the 47 saliva HHV-8 DNA samples were sequenced, and all clustered as subtype E. CONCLUSION: The data support the hypothesis of early acquisition and horizontal transmission, via saliva, of HHV-8 subtype E in Amerindian populations.
Assuntos
Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/classificação , Herpesvirus Humano 8/isolamento & purificação , Eliminação de Partículas Virais , Adolescente , Adulto , Fatores Etários , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Criança , Pré-Escolar , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Feminino , Imunofluorescência , Infecções por Herpesviridae/transmissão , Herpesvirus Humano 8/genética , Humanos , Indígenas Sul-Americanos , Masculino , Filogenia , Reação em Cadeia da Polimerase , População Rural , Saliva/virologia , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Fatores SexuaisRESUMO
BACKGROUND: Early diagnosis of Pseudomonas aeruginosa colonization/infection in patients with cystic fibrosis (CF) using microbiological culturing methods may be difficult. Serology and polymerase chain reaction (PCR) may be useful techniques for early detection of P. aeruginosa in children with CF. METHODS: A cross-sectional analysis comparing results obtained by three different methods for P. aeruginosa identification was performed in 87 CF patients with a mean age of 9.7 years. Microbiological culturing and PCR targeting the algD GDP mannose dehydrogenase gene of P. aeruginosa were performed in sputum or oropharyngeal swabs samples, and serum antibodies against three P. aeruginosa antigens (elastase, alkaline protease, and exotoxin A) were assessed once. RESULTS: It was possible to isolate P. aeruginosa by culture in samples from 42 patients (48.2%), while PCR was positive in 53 (60.9%) patients. Serology was positive in 38 patients (43.6%), with a higher positivity for elastase (37.9%), followed by alkaline protease (29.9%) and exotoxin A (19.5%). The difference among the three isolated methods was not statistically significant. The combination of PCR + serology was significantly superior to single methods, to PCR + culture and also to culture + serology. CONCLUSIONS: PCR identified a higher number of patients with P. aeruginosa than serology and conventional culture, but the difference did not reach statistical significance. Any of the combination methods that included PCR resulted in significantly statistical differences in relation to isolated microbiological or serology methods, but not to the PCR method alone, suggesting that PCR may be the main additive method for P. aeruginosa identification.
Assuntos
Fibrose Cística/microbiologia , Reação em Cadeia da Polimerase , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/isolamento & purificação , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Técnicas Bacteriológicas , Criança , Pré-Escolar , Estudos Transversais , Fibrose Cística/complicações , Feminino , Humanos , Lactente , Masculino , Infecções por Pseudomonas/complicações , Testes SorológicosRESUMO
BACKGROUND: Discrimination between primary and secondary dengue virus infection traditionally has been performed using the hemagglutination inhibition (HI) test. However, this test has practical limitations and disadvantages. OBJECTIVE: To evaluate the ability of three ELISA-based methods (IgG avidity test, IgM/IgG ratio and IgG titer) to discriminate primary from secondary dengue infection. STUDY DESIGN: Serum samples from convalescent-phase patients with confirmed acute, primary (n=46) or secondary (n=33) dengue virus infection were tested using three ELISA-based methods. A ROC curve was employed to establish the cut-off points and to evaluate the ability of the three methods to distinguish between acute, primary and secondary dengue virus infection. RESULTS: All three assays exhibited sensitivity and negative predictive values of 100% for defining secondary infection. The specificity and positive predictive values were respectively 97.8% and 93.7% for the IgG avidity test, 95.7% and 88.2% for the IgM/IgG ratio assays, and 97.8% and 93.7% for the IgG titer assay. CONCLUSION: All three ELISA-based assays proved reliable tools for discriminating between acute, primary and secondary dengue virus infection when using serum samples from convalescent-phase patients.