Virus isolation and genotype identification of human respiratory syncytial virus in Guizhou Province, China

ABSTRACT To investigate the genetic variation and molecular epidemiology characteristics of Human Respiratory Syncytial Virus (HRSV) in Guizhou Province, nasopharyngeal aspirates were collected from patients with acute respiratory infection (ARI) in Guizhou Provincial People's Hospital, from December 2017 to March 2018, and inoculated to Hep-2 cells to isolate HRSV. Cells that showed cytopathic effect (CPE) were then confirmed by indirect immunofluorescence assay and reverse transcription. The sequence of the PCR products was determined for HRSV isolates, and the genetic variation was analyzed. Out of 196 nasopharyngeal aspirate samples, HRSV were isolated in 39. The second hypervariable region at the 3' terminal of glycoprotein gene (HVR2) sequence analysis showed that subgroup A was dominant. Seventy-nine percent of the isolates belonged to subgroup A, ON1 genotype, and 21 % belonged to subgroup B, BA9 genotype, which indicates that the dominant HRSV circulating in Guizhou Province was subgroup A, genotype ON1, co-circulating with a less prevalent subgroup B, genotype BA9.

Evaluación de un método de amplificación isotérmica mediado por bucle para la detección rápida del virus sincicial respiratorio en niños con infección respiratoria aguda / Evaluation of a loop-mediated isothermal amplification method for the rapid detection of human respiratory syncytial virus in children with acute respiratory infection

Resumen Introducción. El virus sincicial respiratorio humano (hRSV) es la causa más frecuente de infección respiratoria aguda de las vías respiratorias inferiores en niños menores de cinco años. El desarrollo de técnicas moleculares para identificarlo es uno de los retos actuales en el campo de la investigación clínica. Objetivo. Evaluar un método de amplificación isotérmica para la detección rápida del hRSV en niños con infección respiratoria aguda. Materiales y métodos. Se extrajo el ARN viral de 304 muestras de hisopado nasal en niños con síntomas de infección respiratoria aguda atendidos en el servicio de urgencias del Hospital de la Universidad del Norte en Barranquilla entre abril del 2016 y julio del 2017. Se evaluó la prueba de amplificación isotérmica mediada por bucle mediante transcriptasa inversa de la proteína de la matriz (M) (Reverse Transcription Loop-Mediated Isothermal Amplification, RT-LAMP) comparada con técnicas moleculares como la reacción en cadena de la polimerasa mediante transcriptasa inversa múltiple anidada (Reverse Transcription- Polymerase Chain Reaction, RT-PCR), la cual se empleó como la prueba estándar, la PCR en tiempo real (quantitative PCR, qPCR) y la RT-LAMP de la proteína L (L) para la detección rápida del virus sincicial respiratorio (VSR), subtipo A y subtipo B. Resultados. La prueba de RT-LAMP (M) tuvo una sensibilidad de 93,59 %, una especificidad de 92,92 % y una concordancia de 0,83 ± 0,036 comparada con la prueba de RT-PCR anidada. El índice kappa del RT-LAMP (M) fue superior, y los valores del RT-LAMP (L) y la qPCR concordaron (0,75 ± 0,043 y 0,71 ± 0,045, respectivamente). Conclusiones. Estos resultados indican que la prueba RT-LAMP (M) puede considerarse como una herramienta de utilidad clínica para detectar el hSRVA, dado que el tiempo requerido para la obtención de resultados, así como los costos, es menor, y su desempeño es mejor que el de las otras pruebas moleculares evaluadas.

Abstract Introduction: Human respiratory syncytial virus (hRSV) is the most frequent cause of acute respiratory infection of the lower respiratory tract in children under the age of five. The development of molecular techniques able to identify hRSV is one of the current challenges in the field of clinical research. Objective: To evaluate the ability of an isothermal amplification method to rapidly detect hRSV in children with acute respiratory infection. Materials and methods: We collected 304 nasopharyngeal swab samples from children with symptoms of acute respiratory infection who attended the emergency unit at Hospital de la Universidad del Norte in Barranquilla from April, 2016, to July, 2017. After extracting viral RNA from the samples, we evaluated the ability of the reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) M assay to rapidly detect hRSVA and hRSVB compared to other molecular techniques: quantitative PCR (qPCR), reverse transcriptase-LAMP L assay, and as a standard, the multiplex nested reverse transcriptase polymerase chain reaction (nested RT-PCR). Results: The RT-LAMP M assay had a sensitivity of 93.59% and a specificity of 92.92%, and a concordance of 0.83 ± 0.036 as compared with the nested RT-PCR test. While the Kappa index of the RT-LAMP M assay was higher than the values for the RT-LAMP L assay and the qPCR, the values of the latter two methods were in agreement (0.75 ± 0.043 and 0.71 ± 0.045, respectively). Conclusion: Due to the shorter running times, lower costs and better performance of the RT-LAMP M assay, it can be considered as a useful clinical tool for the detection of RSVA.

Human metapneumovirus in Southern Brazil

INTRODUCTION Infections caused by respiratory viruses are important problems worldwide, especially in children. Human metapneumovirus (hMPV) is a respiratory pathogen and causes severe infections with nonspecific symptoms. This study reports the hMPV occurrence and dissemination in southern Brazil and compares the frequency of occurrence of this virus and the human respiratory syncytial virus (hRSV) in the epidemiological weeks in a three-year period (2009-2011). METHODS: In total, 545 nasopharyngeal (NP) specimens from individuals with Severe Acute Respiratory Syndrome (SARS) who were negative for other seven respiratory viruses were analyzed for the presence of hMPV. Human metapneumovirus was detected by direct immunofluorescence and real-time reverse transcription polymerase chain reaction. RESULTS: hMPV was detected in 109 patients from the main geographic regions of the southernmost state of Brazil, presenting similar overall prevalence in males (46.8%) and females (53.2%). Among children who were less than six years old, hMPV was detected in 99 samples of all age groups, with a higher frequency in infants who were less than one year old (45.7%) compared to all other age groups until six years. hMPV and hRSV infection occurred in almost the same epidemiological weeks (EWs) of each year, with peaks of incidence between EW 31/37 and EW 26/38 for the years 2009 and 2011, respectively. hMPV was further detected in several cases of SARS and it was the only virus detected in three deaths. CONCLUSIONS These findings indicate that hMPV is in circulation in southern Brazil and highlight the importance of diagnosing hMPV for influenza-like illness in the population. (AU)

El virus respiratorio sincicial: patógeno de niños... y de grandes / Respiratory syncytial virus: a pathogen for the small ones and the big ones

Desde el descubrimiento del virus respiratorio sincicial (VRS) en 1956, se ha demostrado en todo el mundo su impacto como el principal causante de infecciones respiratorias agudas bajas (IRAB) que requieren hospitalización en el lactante. Posteriormente se ha descrito que una inadecuada respuesta inmune favorece reinfecciones en la infancia. Más recientemente, numerosos trabajos epidemiológicos lo han asociado a IRAB en adultos, especialmente de tercera edad y en ciertos pacientes inmunocomprometidos. Se ha avanzado en el conocimiento de la estructura y función de los diferentes componentes del VRS, lo que ha permitido facilitar su diagnóstico y avanzar en estrategias de desarrollo de antivirales y vacunas. En efecto, el diagnóstico de laboratorio de VRS es muy simple en niños, por su alta excreción viral, pero para demostrar su participación en adultos se requieren técnicas de alta sensibilidad. La patogenia de la infección es muy compleja y muchos aspectos todavía no se han aclarado. Intervienen factores dependientes del virus -cepa, dosis infectiva, capacidad del virus de inhibir la respuesta inmune- y del hospedero humano, como edad, enfermedades concomitantes, integridad del aparato inmune y otros. Se menciona que otros factores como frío, humedad ambiental, contaminación aérea, hacinamiento, también actuarían en combinación con los inicialmente mencionados. Es necesario conocer los mecanismos responsables de la adquisición de inmunidad contra el VRS para entender las estrategias usadas en el intento de desarrollar vacunas, cuyos esfuerzos son todavía infructuosos. Actualmente se conoce bastante del VRS como patógeno de niños. Sin embargo, cada día se documenta más su participación en enfermedades de adultos, por lo que haremos un resumen para promover su consideración como posible patógeno respiratorio.

Since respiratory syncytial virus (RSV) was identified in 1956, its impact as the main cause of severe acute lower respiratory infections in infants has been shown. Studies about RSV immunopathogenesis have demonstrated that the host immune response is important in protecting from re-infections. The presence of RSV in exacerbation of chronic diseases as COPD and bronchial asthma in adults and its severity in cases with immunodeficiency has been also related to an inadequate response. The actual knowledge on the molecular structure and functions of the virus has allowed to improve diagnosis and to develop new strategies for vaccines and antiviral drugs. The etiologic diagnosis in children is easier than in adults due to the higher viral shedding; therefore techniques based on antibody reactions (immunofluorescence, immunocromatography, etc) are good enough in this group. By contrast, in adults, highly sensitive molecular techniques are needed. Although the advances in understanding the pathogenesis process in neonates and infants, many pathogenic factors still need to be elucidated. The virus strains, viral loads and immune response have been described as important players; however, the changes on the host immunity to RSV according to age and co-morbidities associated to severity of illness needs to be explored. RSV has been known as a children pathogen, nowadays this agent is being recognized as an important agent in adults, especially in those with chronic diseases, immunodeficiency and in immune-senescence.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool / Teste rápido de detecção de antígenos para o diagnóstico do Vírus Sincicial Respiratório como ferramenta de diagnóstico

Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

Resumo Objetivo: Avaliar o teste QuickVue® RSV Test Kit (QUIDEL Corp, CA, EUA) para o diagnóstico rápido do vírus sincicial respiratório em crianças com doença respiratória aguda, comparandoo com a imunofluorescência indireta como padrão ouro. Visto que, no Brasil, testes rápidos para detecção de antígenos para vírus sincicial respiratório não são rotineiramente utilizados como ferramenta de diagnóstico, exceto para Dengue e Influenza. Métodos: Um total de 486 amostras de aspirado de nasofaringe de crianças menores de 5 anos com doença respiratória aguda, coletadas entre dezembro de 2013 e agosto de 2014, foram analisadas por imunofluorescência e pelo teste QuickVue®. Amostras com resultados discordantes entre os métodos foram submetidas a PCR em tempo real e sequenciamento. Resultados: Das 313 amostras positivas por IFI, 282 foram positivas no teste rápido (90%), 2 amostras foram positivas apenas no teste rápido (0.6%), 33 apenas na imunofluorescência (10.5%) e 171 foram negativas em ambos os métodos. As 35 amostras com resultados discordantes foram testadas por PCR em tempo real, sendo que duas que foram positivas apenas no teste rápido e 5 apenas na imunofluorescência confirmaram-se positivas. Não houve relação entre a ausência de positividade no teste QuickVue® com a carga ou com a cepa viral. O teste QuickVue® mostrou sensibilidade de 90.1%, especificidade 98.9%, valor preditivo positivo 99.3%, valor preditivo negativo de 94.6%, acurácia de 93.2% e índice de concordância de 0.85 em comparação à imunofluorescência. Conclusões: Nosso estudo demonstrou que o teste QuickVue® RSV pode ser efetivo na detecção precoce do vírus sincicial respiratório em amostras de aspirado de nasofaringe e é confiável como uma ferramenta de diagnósticos em pediatria.

Prevalence of rhinovirus in wheezing children: a comparison with respiratory syncytial virus wheezing

Abstract Objective To explore the distribution and clinical manifestations of rhinovirus infection in wheezing children, and compare the clinical differences between rhinovirus- and respiratory syncytial virus-induced wheezing. Materials and methods This prospective cohort study was carried out in Children's Hospital of Soochow University from Dec 2012 to Nov 2014. We enrolled consecutive hospitalized children <60 months of age presented with wheezing. Clinical data including cough, fever, dyspnea, crackles were recorded by pediatricians on the first day of admission. Meanwhile, nasopharyngeal aspirates were obtained to test for respiratory viruses, by using polymerase chain reaction method for rhinovirus, human bocavirus, and human metapneumovirus, and direct immunofluorescence assay to test for respiratory syncytial virus, adenovirus, parainfluenza virus types 1–3, and influenza virus types A and B. Results Rhinovirus was a main causative agent isolated in 14.7% of the hospitalized wheezing children in Suzhou, China, being second to respiratory syncytial virus (21.0%). Different from respiratory syncytial virus infection, which peaked in winter months, rhinovirus could be detected all year round, peaked between July and September, and in November. Children with rhinovirus infection were older and presented with more often allergic sensitizations, blood eosinophilia, and leukocytosis than those of respiratory syncytial virus infection. Logistic regression analysis revealed that rhinovirus-infected children experienced earlier wheezing more often than respiratory syncytial virus children (odds ratio, 3.441; 95% confidence interval, 1.187–9.979; p = 0.023). Conclusion Rhinovirus was a main viral pathogen in wheezing children, especially in summer time. Rhinovirus-induced wheezing was different from respiratory syncytial virus, apart from seasonal epidemics; these two groups differed with regard to age, allergic sensitizations, laboratory test, and history of wheezing episodes.

Parainfluenza virus as a cause of acute respiratory infection in hospitalized childrens

Background: Human parainfluenza viruses account for a significant proportion of lower respiratory tract infections in children.Objective: To assess the prevalence of Human parainfluenza viruses as a cause of acute respiratory infection and to compare clinical data for this infection against those of the human respiratory syncytial virus.Methods: A prospective study in children younger than five years with acute respiratory infection was conducted. Detection of respiratory viruses in nasopharyngeal aspirate samples was performed using the indirect immunofluorescence reaction. Length of hospital stay, age, clinical history and physical exam, clinical diagnoses, and evolution (admission to Intensive Care Unit or general ward, discharge or death) were assessed. Past personal (premature birth and cardiopathy) as well as family (smoking and atopy) medical factors were also assessed.Results: A total of 585 patients were included with a median age of 7.9 months and median hospital stay of six days. No difference between the HRSV+ and HPIV+ groups was found in terms of age, gender or length of hospital stay. The HRSV+ group had more fever and cough. Need for admission to the Intensive Care Unit was similar for both groups but more deaths were recorded in the HPIV+ group. The occurrence of parainfluenza peaked during the autumn in the first two years of the study.Conclusion: Parainfluenza was responsible for significant morbidity, proving to be the second-most prevalent viral agent in this population after respiratory syncytial virus. No difference in clinical presentation was found between the two groups, but mortality was higher in the HPIV+ group.

Clinical and epidemiological aspects related to the detection of adenovirus or respiratory syncytial virus in infants hospitalized for acute lower respiratory tract infection / Aspectos clínicos e epidemiológicos relacionados à detecção de adenovírus ou vírus sincicial respiratório em crianças hospitalizadas por doença aguda do trato respiratório inferior

OBJECTIVE: To characterize and compare clinical, epidemiological, and laboratory aspects ofinfants with acute lower respiratory infection (ALRI) associated with the detection of adenovirus(ADV) or respiratory syncytial virus (RSV). METHODS: A preliminary respiratory infection surveillance study collected samples of nasopharyngeal aspirate (NPA) for viral research, linked to the completion of a standard protocol, from children younger than two years admitted to a university hospital with ALRI, between March of 2008 and August of 2011. Polymerase chain reaction (PCR) was used for eight viruses: ADV, RSV, metapneumovirus, Parainfluenza 1, 2, and 3, and Influenza A and B. Cases with NPA collectedduring the first 24 hours of admission, negative results of blood culture, and exclusive detection of ADV (Gadv group) or RSV (Grsv group) were selected for comparisons. RESULTS: The preliminary study included collection of 1,121 samples of NPA, 813 collected in thefirst 24 hours of admission, of which 50.3% were positive for at least one virus; RSV was identifiedin 27.3% of cases surveyed, and ADV was identified in 15.8%. Among the aspects analyzed inthe Gadv (n = 58) and Grsv (n = 134) groups, the following are noteworthy: the higher meanage, more frequent prescription of antibiotics, and the highest median of total white blood cellcount and C-reactive protein values in Gadv. CONCLUSIONS: PCR can detect persistent/latent forms of ADV, an aspect to be considered wheninterpreting results. Additional studies with quantitative diagnostic techniques could elucidatethe importance of the high frequency observed. .

OBJETIVO: Caracterizar e comparar aspectos clínicos, epidemiológicos e laboratoriais delactentes com evidências de infecção aguda do trato respiratório inferior (IATRI) associada à detecção do adenovírus (ADV) ou do vírus sincicial respiratório (VSR). MÉTODOS: Um estudo preliminar de vigilância de infecções respiratórias desenvolveu coleta de aspirado nasofaríngeo (ANF) para pesquisa viral, vinculada ao preenchimento de protocolo padrão, de menores de dois anos internados com quadro de IATRI em hospital universitário, entre março de 2008 e agosto de 2011. Utilizou-se técnica da reação em cadeia da polimerase (PCR) para oito vírus: ADV, VSR, metapneumovírus, parainfluenza 1, 2 e 3 e influenza A e B. Foram selecionados para comparações os casos com ANF coletado nas primeiras 24 horas da admissão, resultado de hemocultura negativo e detecção exclusiva de ADV (grupo Gadv) ou VSR (grupo Gvsr). RESULTADOS: O estudo preliminar incluiu coleta de 1.121 amostras de ANF, sendo 813 coletadas nas primeiras 24 h da admissão, das quais 50,3% foram positivas para ao menos um dos vírus, com VSR em primeiro lugar, em 27,3%, e ADV em segundo, em 15,8% dos casos pesquisados. Dentre os aspectos analisados nos grupos Gadv (n = 58) e Gvsr (n = 134), destacaram-se a média da idade mais elevada, maior frequência da prescrição de antibióticos e medianas mais elevadas para contagem total de leucócitos e valores da proteína C-reativa no Gadv. CONCLUSÕES: A PCR utilizada pode detectar formas persistentes/latentes de ADV, aspecto aser considerado ao interpretar os resultados. Estudos complementares com técnicas diagnósticas quantitativas, por exemplo, poderiam evidenciar a importância da elevada frequência verificada. .

Aumento de interleuquinas proinflamatorias y de cortisol plasmático en bronquiolitis por virus respiratorio sincicial: relación con la gravedad de la infección / Levels of inflammatory cytokines and plasma cortisol in respiratory syncytial virus bronchiolitis

Background: An increased inflammatory innate response may play a role in pathogenesis of respiratory syncytial virus (RSV) infection. Aim: To quantify pro-inflammatory cytokines (IL-6-IL-8, ÍL-2-P and TNF-a) in nasopharyngeal aspirate (NPA) and plasma, and plasma cortisol in previously healthy infants with RSV bronchiolitis. Patients and Methods: We studied 49 infants aged less than one year of age with RSV bronchiolitis and 25 healthy controls. Severity was defined using a previously described modified score. We quantified interleukins in NPA and plasma by flow cytometry and plasma cortisol by radioimmunoanalysis. Results: Among patients with RSV bronchiolitis, 25 were classified as severe and 24 as moderate or mild. Significantly higher levels ofIL-6 and IL-8 in NPA and plasma and IL-lfi in NPA were found in children classified as severe, when compared to those with moderate or mild disease and controls. There was a positive correlation between IL-6 and cortisol in plasma (r = 0,55; p < 0,0001) and both were correlated with the severity of the disease. Conclusions: RSV bronchiolitis severity was associated with higher levéis of inflammatory interleukins and plasma cortisol.

Sentinel surveillance of influenza and other respiratory viruses, Brazil, 2000-2010

There are scanty data on the epidemiology of influenza and other respiratory viruses in South America and Brazil. The aim of this study was to summarize the data from the Brazilian surveillance system of influenza and other respiratory viruses and discuss the patterns of viral circulation. The system is based on detecting cases of influenza-like illness in sentinel sites and weekly collection of five nasopharyngeal secretions samples, which are processed in state public health laboratories for respiratory viruses by indirect immunofluorescence assay. Data from 2000 to 2010 were described over time, by region, gender, and age group, and an analysis of Spearman correlation was performed between monthly influenza detection and rainfall and temperature data in two state capitals with the highest number of positive samples, one from the northeast region (Maceió) and other from the southern region (Curitiba). There were 3,291,946 visits for influenza-like illness; of these, 37,120 had samples collected and 6421 tested positive: 1690 (26%) influenza A, 567 (9%) influenza B, 277 (4%) parainfluenza 1, 571 (9%) parainfluenza 2, 589 (9%) parainfluenza 3, 742 (12%) adenovirus, and 1985 (31%) respiratory syncytial virus. Overall, increased activity of respiratory syncytial virus was observed from March to June, preceding the peak of influenza activity, from May to August, but with regional differences. In Maceió, there was a weak correlation between temperature and influenza detection (ρ = 0.05), but a moderate positive correlation between rainfall and influenza detection (ρ = 0.36). In Curitiba, a high correlation was observed between the decrease in temperature and rainfall and the increase in influenza detection (ρ = -0.83 and -0.78 respectively). These data are important to guide public health control measures as the best time for influenza vaccination and use of antivirals.

Gravidade das coinfecções virais em lactentes hospitalizados com infecção por vírus sincicial respiratório / Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection

OBJETIVO: Comparar a gravidade de infecções causadas por um único vírus (VSR) com a gravidade de coinfecções. MÉTODOS: Este estudo avaliou uma coorte histórica de lactentes com infecção aguda por VSR. Secreção de nasofaringe foi coletada de todos os pacientes rotineiramente para pesquisa viral usando técnicas de biologia molecular. Os seguintes desfechos foram analisados: tempo total de internação, duração da oxigenioterapia, admissão em unidade de terapia intensiva e uso de ventilação mecânica. Os resultados foram ajustados para os fatores confundidores (prematuridade, idade e aleitamento materno). RESULTADOS: Foram incluídos no estudo 176 lactentes com idade média de 4,5 meses e diagnósticos de bronquiolite e/ou pneumonia. Cento e vinte e um tinham infecção única por VSR, e 55 tinham coinfecções (24 VSR + adenovírus, 16 VSR + metapneumovírus humano e 15 outras associações menos frequentes). Os quatro desfechos de gravidade avaliados foram semelhantes entre o grupo com infecção única por VSR e os grupos com coinfecções, independente do tipo de vírus associado com o VSR. CONCLUSÃO: As coinfecções virais não parecem alterar o prognóstico de lactentes hospitalizados com infecção aguda por VSR.

OBJECTIVE: To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS: A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS: A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION: Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

Vírus respiratórios e ventilação mecânica em lactentes brasileiros / Respiratory virus and mechanical ventilation in Brazilian infants

Introdução: Entre lactentes, Vírus Sincicial Respiratório e Metapneumovírus são agentes importantes de infecção respiratória baixa com necessidade de ventilação mecânica. Índice de Ventilação e Relação PaO2/FiO2 podem ser fatores prognósticos do tempo de ventilação mecânica nestes casos. Métodos: Dentre 284 lactentes (zero a 12 meses), hospitalizados por infecção respiratória aguda baixa em 2004, 2007 e 2008, foram avaliados 20 que necessitaram de ventilação mecânica. Análise da secreção nas ofaríngea para vírus por Polimerase Chain Reaction foi realizada; o Índice de Ventilação Mecânica e a Relação PaO2/FiO2 foram obtidos nos primeiros cinco dias de ventilação mecânica; tempo prolongado de ventilação pulmonar mecânica foi considerado sete dias ou mais. Resultados: Dez em vinte lactentes foram identificados com Vírus Sincial Respiratório; 0/20 foram positivos para Metapneumovírus. A análise estatística comparativa não mostrou diferenças entre Índice de Ventilação Mecânica e Relação PaO2/FiO2 e tempo de ventilação pulmonar prolongada entre os grupos Vírus Sincicial Respiratório positivo e negativo. A identificação do genótipo foi realizada em 6 de 10 Vírus Sincicial Respiratórios encontrados; o pequeno número de casos não permitiu relacionar a apresentação clínica com as características virais (subgrupos e genótipos). Conclusão: Índice de Ventilação Mecânica e Relação PaO2/FiO2 não foram úteis como fatores prognósticos de tempo de ventilação mecânica prolongada para este grupo. De maneira ideal, estudo com maior número de lactentes, teste para vários vírus, e testes para a imunidade inata e adaptativa, poderia mostrar o impacto destes fatores na evolução dos lactentes em ventilação pulmonar mecânica. Infelizmente, recursos para pesquisas como esta não estão facilmente disponíveis.

Background: Among infants, respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are important agents of lower respiratory tract infection requiring mechanical ventilation. Ventilation index and PaO2/FiO2 ratio may be prognostic factors for duration of mechanical ventilation in these cases. Methods: From a population of 284 infants (aged zero to 12 months) hospitalized with lower respiratory tract infection in 2004, 2007, and 2008, 20 infants requiring mechanical ventilation were evaluated. Nasopharyngeal secretions were analyzed for virus detection using a polymerase chain reaction (PCR) test. Ventilation index and PaO2/FiO2 ratio were obtained during the first five days of mechanical ventilation. Prolonged mechanical ventilation was defined as required ventilatory support for 7 days or longer. Results: Out of 20 infants assessed, 10 were positive for RSV and none for HMPV. Comparative statistical analysis showed no difference in ventilation index, PaO2/FiO2 ratio, and prolonged mechanical ventilation between RSV-positive and –negative groups. Genotypic identification was performed in 6 of 10 RSV-positive infants. The small number of cases did not allow a statistical correlation between clinical presentation and viral characteristics (subgroups and genotypes). Conclusion: Ventilation index and PaO2/FiO2 were not useful as prognostic factors for prolonged mechanical ventilation in this group. Ideally, studies involving more infants and including tests for several viruses and for innate and adaptive immunity should be conducted to further evaluate the impact of these factors on the outcome of infants requiring mechanical ventilation. Unfortunately, resources for this type of research are not readily available.

Genotypes and clinical data of respiratory syncytial virus and metapneumovirus in brazilian infants: a new perspective

The aim of this study was to determine if there was a correlation between respiratory syncytial virus (RSV) and metapneumovirus (MPV) genotypes and clinical data of Brazilian infants hospitalized for acute lower respiratory infection. The viruses in the patients' nasopharyngeal secretions were studied using the polymerase chain reaction and phylogenetic analysis. The study assessed 144 infants; 31.9 percent were RSV positive and 5.6 percent were MPV positive. Statistical analysis was performed using the chi-squared test, Fisher's test, Odds ratio, univariate logistic regression, non-conditional multivariate logistic regression and the forward - stepwise method. Multivariate analysis confirmed a significant relationship between a positive PCR test for RSV and hospitalization during the month of May and with pulse oximetry less than 90 percent. The phylogenetic analysis indicated the genotypes GA2, GA5, SAA1 (Group A), SAB1, SAB3 and BA (Group B) for RSV and Group B, subgroup B1, for MPV.

Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil

Respiratory syncytial virus (RSV) is well recognized as the most important pathogen causing acute respiratory disease in infants and young children, mainly in the form of bronchiolitis and pneumonia. Two major antigenic groups, A and B, have been identified; however, there is disagreement about the severity of the diseases caused by these two types. This study investigated a possible association between RSV groups and severity of disease. Reverse transcription-polymerase chain reaction was used to characterize 128 RSV nasopharyngeal specimens from children less than five years old experiencing acute respiratory disease. A total of 82 of 128 samples (64.1 percent) could be typed, and, of these, 78 percent were group A, and 22 percent were group B. Severity was measured by clinical evaluation associated with demographic factors: for RSV A-infected patients, 53.1 percent were hospitalized, whereas for RSV B patients, 27.8 percent were hospitalized (p = 0.07). Around 35.0 percent of the patients presented risk factors for severity (e.g., prematurity). For those without risk factors, the hospitalization occurred in 47.6 percent of patients infected with RSV A and in 18.2 percent infected with RSV B. There was a trend for RSV B infections to be milder than those of RSV A. Even though RSV A-infected patients, including cases without underlying condition and prematurity, were more likely to require hospitalization than those infected by RSV B, the disease severity could not to be attributed to the RSV groups.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants / Comparação entre imunofluorescência direta, cultura convencional de células e reação em cadeia de polimerase para detectar vírus respiratório sincicial em lavados de nasofaringe de crianças

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.

Utilidad de la reacción de polimerasa en cadena en tiempo real en el diagnóstico de infecciones por virus respiratorio sincicial en adultos / Utility of real time polymerase chain reaction in the diagnosis of respiratory syncytial virus infection among adult patients

Introducción: Las infecciones respiratorias virales (IRV) son causa importante de morbilidad en adultos. Virus respiratorio sincicial (VRS) causa hasta 20 por ciento de las IRV en esta edad; sin embargo, su diagnóstico es subestimado debido a una menor sensibilidad de las técnicas diagnosticas convencionales (IF y ELISA). Objetivos: Evaluar el impacto del uso de reacción de la polimerasa en cadena en tiempo real (TR-RPC en tiempo real) en el diagnóstico de IRV por VRS en adultos y caracterizar su perfil clínico. Pacientes y Métodos: Durante ocho semanas del año 2005, los adultos hospitalizados en Hospital Clínico de la Universidad Católica con sospecha de IRV, e IFD negativa para VRS, FLU-A, -B, paraFLU-1, 2, 3 y ADV de muestra de hisopado nasofaríngeo, fueron sometidos a detección de VRS por TR-RPC en tiempo real. Se confeccionó una base de datos con los antecedentes clínicos, laboratorio y evolución de cada paciente. Resultados: De 114 pacientes con IFD negativa en 17 (14,9 por ciento) se detectó VRS. Fiebre, congestión faríngea, tos y signos de obstrucción bronquial, configuraron en más de 80 por ciento de los casos el perfil clínico de los pacientes. Treinta por ciento presentaba enfermedad crónica y 47 por ciento eran inmunocomprometidos. Tres de 17 (18 por ciento) presentaron descompensación de la enfermedad de base y 1/17 (6 por ciento) requirió ventilación mecánica. No hubo mortalidad asociada. Conclusiones: El uso de TR-RPC en tiempo real permitió duplicar la detección de infecciones por VRS en adultos hospitalizados respecto a las diagnosticadas por IFD. Se recomienda considerar el empleo la técnica de TR-RPC en tiempo real en aquellos pacientes con sospecha clínica de VRS durante la temporada de VRS y estudio viro lógico negativo por métodos convencionales.

Introduction: Viral respiratory infections (VRI) are a frequent cause of morbidity among adult population. Respiratory syncytial virus (RSV) produces 20 percent> of VRI, however diagnosis is limited for a low sensitivity of conventional (FDA and ELISA) tests. Aim: To assess the impact of real time reverse transcriptase-polymerase chain reaction (real time RT-PCR) technique in RSV diagnosis in adult hospitalized patients; to characterize RSV infection among these patients. Patients and Methods: All adults hospitalized in Hospital Clínico Universidad Católica during 8 weeks of winter season, with clinical picture of VRI, and with negative DFA for influenza A and B, parainfluenza 1, 2, 3 and adenovirus were included. Real time RT-PCR was performed from nasopharyngeal sample. Clinical information, general laboratory exams and chest X ray reports were collected. Results: Out of 114 patients with negative DFA, 17 (14.9 percento) Debe decir: RSV cases were demonstrated using real time RT- PCR. Fever, pharyngeal congestion, cough and bronchial obstruction were present in 80 percent> of patients. Thirty percent of them had a baseline chronic disease and 47 percent> were immunocompromised. One out of 17 patients (6 percent) required mechanical ventilation. No mortality was observed. Conclusions: Use of RT-PCR allowed increasing detection of RSV infection over 100 percent> among adults with VRI without virological diagnosis with conventional techniques. It is necessary to consider RSV RT-PCR test among patients with clinical picture of VRI during RSV season, with negative virological screening tests.

Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20°C from one to fifteen months after collection

Cell culture isolation is used for recovering respiratory syncytial virus (RSV) from respiratory specimens. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. The isolation rate of RSV from nasopharyngeal aspirates (NPA) stored at 20°C for one to 15 months after collection was investigated. A total of 126 samples considered positive for RSV by indirect fluorescence-antibody were tested by virus isolation in HEp-2 cell culture. RSV was isolated from 47/126 specimens (37.3 percent). These results show that RSV may be recovered from NPA stored at 20°C by cell culture.