Dengue virus 1 in Buenos Aires from 1999 to 2010: towards local spread.

Dengue virus (DENV) is a public health problem representing the most important arthropod-borne viral disease in humans. In Argentina, Northern provinces have reported autochthonous cases since 1997, though these outbreaks have originated in bordering countries, where co-circulation of more than one serotype has been reported. In the last decade, imported dengue cases have been reported in Buenos Aires, the urban area of Argentina with the highest population density. In 2009, a dengue outbreak affected Buenos Aires and, for the first time, local transmission was detected. All cases of this outbreak were caused by DENV-1. In this report, we present the full-length sequences of 27 DENV-1 isolates, corresponding to imported cases of 1999-2000, as well as local and imported cases of the 2009 and 2010 outbreaks. We analyzed their phylogenetic and phylodynamic relationships and their global and local spread. Additionally, we characterized their genomic and phenotypic features. All cases belonged to DENV-1 genotype V. The most recent ancestor for this genotype was dated ∼1934, whereas that for the 2009 outbreak was dated ∼2007. The mean rates of nucleotide substitution were 4.98E-4 and 8.53E-4 subs./site/yr, respectively. We inferred an introduction from Paraguay in 1999-2000 and mainly from Venezuela during 2009-2010. Overall, the number of synonymous substitutions per synonymous site significantly exceeded the number of non-synonymous substitutions per site and 12 positively selected sites were detected. These analyses could contribute to a better understanding regarding spread and evolution of this pathogen in the Southern Cone of South America.

Molecular evidence of St. Louis encephalitis virus infection in patients in Buenos Aires, Argentina.

We report two cases of St. Louis encephalitis where the virus was detected in patients' sera directly by molecular techniques allowing subsequent typing. Phylogenetic analysis of both samples showed that NS5 sequences clustered with viruses previously classified as genotype III.

Emergence of intratreatment resistance to oseltamivir in pandemic influenza A H1N1 2009 virus.

BACKGROUND: Pandemic influenza A H1N1 2009 virus presents a new challenge to health authorities and communities worldwide. In Argentina, the outbreak was at its peak by the end of June 2009, during the southern winter. A systematic analysis of samples from patients with pandemic H1N1 2009 studied in our laboratory (Virology Laboratory, Hospital de Niños R Gutiérrez, Buenos Aires, Argentina) detected two patients presenting intratreatment emergence of the H275Y neuraminidase mutation, which confers resistance to oseltamivir. METHODS: Complementary DNAs, including the 275 codon, were obtained by reverse transcriptase PCR using viral RNAs extracted from nasopharyngeal or tracheal aspirates. Conventional sequencing and pyrosequencing were performed on each sample. In order to measure the virus susceptibility to oseltamivir, 50% inhibitory concentration determinations were performed by chemiluminescence. RESULTS: Sequential samples of two paediatric patients under oseltamivir treatment were analysed. Pretreatment samples were composed of 100% oseltamivir-sensitive variants. In case 1, the oseltamivir-resistant variant was found 8 days after the beginning of treatment. In case 2, the viral population became resistant on the second day of treatment, with 83% of the viral population bearing the mutation and this reached 100% on the seventh day. CONCLUSIONS: We describe the intratreatment emergence of oseltamivir resistance in two paediatric patients. Pyrosequencing allowed us to detect variant mixtures, showing the transition of the viral population from sensitive to resistant.

Viruses and atypical bacteria associated with asthma exacerbations in hospitalized children.

UNLABELLED: OBJECTIVES AND WORKING HYPOTHESIS: To evaluate the prevalence of respiratory viruses Mycoplasma pneumoniae and Chlamydophila pneumoniae and gain insight into their seasonal circulation pattern in children with acute asthma exacerbations in a temperate southern hemisphere region. STUDY DESIGN: Patients hospitalized between 3 months and 16 years of age were included in a 1-year prospective, observational, cross-sectional study. Respiratory secretions were collected and the presence of different viruses and atypical bacteria analyzed by immunofluorescence and polymerase chain reaction. RESULTS: Two hundred nine patients (118 females) aged (mean +/- SD) 4.4 +/- 4 years were included. A potential causative agent was detected in 78% of the patients. The most frequently detected viruses were respiratory syncytial virus (HRSV) (n = 85; 40%) and rhinovirus (HRV) (n = 52; 24.5%); M. pneumoniae and C. pneumoniae were detected in 4.5% and 2% of the cases, respectively. Patients with HRSV (vs. HRV) were hospitalized for a longer time (6.7 vs. 5.2 days, P = 0.012), required more days of oxygen supply (5.1 vs. 3.4, P = 0.005), had a longer duration of the exacerbation before hospitalization (3.6 vs. 1.9 days, P = 0.001) and were younger (3.7 vs. 5.1 years, P = 0.012). Three peaks of admissions were observed. A first peak (early autumn) caused by HRV, a second peak (winter) caused mainly by HRSV and a third one (spring), caused by HRSV, an increase in HMPV together with a second outbreak of HRV. CONCLUSIONS: Children with an acute asthma exacerbation presented a high prevalence of respiratory viruses. Most hospitalizations corresponded to seasonal increases in prevalence of HRV and HRSV.

Phylogenetic analysis of rubella viruses isolated in 2008 outbreak in Argentina.

BACKGROUND: A rubella outbreak was recorded in Buenos Aires during 2008. OBJECTIVES: The objective of this communication is to present the genetic and phylogenetic analyses of wild-type RUBV circulating in Buenos Aires during the 2008 outbreak. STUDY DESIGN: Throat swab samples collected from patients diagnosed with rubella between June 2008 and December 2008 were inoculated in cell culture and 23 isolates were sequenced. RESULTS: Phylogenetic analysis of the WHO-recommended window (nt 8731-9469) of the E1 envelope glycoprotein was performed and all isolates clustered with the 2B genotype. CONCLUSIONS: Genotype 2B seems to be endemically circulating in the Southern cone of Latin America, thus causing recent outbreaks.

[New respiratory viruses in children 2 months to 3 years old with recurrent wheeze]. / Nuevos virus respiratorios en niños de 2 meses a 3 años con sibilancias recurrentes.

INTRODUCTION: Respiratory viruses are associated with respiratory exacerbations, more frequently Respiratory Syncytial Virus in infants and Rhinovirus in children. OBJECTIVE: To evaluate the prevalence and epidemiological features of newer and traditional respiratory viruses in infants and young children with recurrent wheeze. MATERIAL AND METHODS: Cross sectional, prospective and descriptive study. Patients with recurrent wheeze and risk factors for asthma, age 2 months to 3 years, hospitalized with bronchial obstruction were included. On admission a respiratory sample was obtained through a nasopharyngeal aspirate. Immunofluorescence was performed to detect Respiratory Syncytial Virus, Adenovirus, Parainfluenza 1, 2, 3 and Influenza A and B. Polymerase Chain Reaction was used to detect Rhinovirus, Enterovirus, Metapneumovirus, Bocavirus, Adenovirus and Coronavirus. RESULTS: 119 patients (61 female), age (x E DS) 1.5 E 0.9 years were included. Days on admission and on oxygen requirement were, respectively (x E DS): 6.3 E 2.9 y 4.4 E 2.7. One hundred and two (86%) positive cases were diagnosed. Fifty five percent of the viruses were detected by Immunofluorescence and 45% by Polymerase Chain Reaction. A single virus was present in 75% of the samples, 22% had a double co-infection and 3% a triple virus co-infection. Overall, the prevalence of detected respiratory viruses was: Respiratory Syncytial Virus 55 (43%); Rhinovirus 30 (23%); Metapneumovirus 13 (10%); Influenza A 8 (6%); Enterovirus 6 (5%); Bocavirus 6 (5%); Adenovirus 4 (3%); Coronavirus 3 (2%); Parainfluenza 1: 2 (1%); Influenza B, 1 (1%) and Parainfluenza 3: 1 (1%). CONCLUSIONS: Infants and young children with recurrent wheeze and risk factors for asthma hospitalized for bronchial obstruction present a high prevalence of respiratory viruses. Hospital admissions were more frequent during months of higher respiratory circulation.

Nuevos virus respiratorios en niños de 2 meses a 3 años con sibilancias recurrentes / New respiratory viruses in children 2 months to 3 years old with recurrent wheeze

Introducción. Los virus respiratorios son los agentes que con más frecuencia desencadenan sibilancias, especialmente, el virus sincicial respiratorio en los lactantes y los rinovirus en niños mayores.Objetivos. Conocer la prevalencia y la circulación estacional de los virus respiratorios nuevos y tradicionalesen lactantes y niños pequeños con sibilancias recurrentes.Material y métodos. Estudio de corte transversal, prospectivo y descriptivo. Se incluyeron pacientes de 2 meses a 3 años con sibilancias recurrentes yfactores de riesgo para desarrollar asma hospitalizados por obstrucción bronquial. Se obtuvo una muestra de secreciones respiratorias por aspiradonasofaríngeo y se utilizó la técnica de inmunofluorescencia para detectar Virus Sincicial Respiratorio, Adenovirus, Parainfluenza 1, 2 y 3 e Influenza A y B,y la Reacción en Cadena de la olimerasa para determinar Rinovirus, Enterovirus, Virus Sincicial Respiratorio, Bocavirus, Adenovirus y Coronavirus.Resultados. Se evaluaron 119 pacientes (61 femeninos), edad (x más menos DE) 1,5 más menos 0,9 años. Los días de internación y de requerimientos de oxígeno fueron(x más menos DE): 6,3 más menos 2,9 y 4,4 más menos 2,7 respectivamente. Se hallaron 102 (86 por ciento) casos positivos. El 55 por ciento de los virus se detectó por Inmunofluorescencia y el 45 por cientopor Reacción en Cadena de la polimerasa. El 75 por ciento delas muestras respiratorias presentó un solo agente viral, el 22 por ciento una coinfección doble y el 3 por ciento unacoinfección triple. Las prevalencias de los virus respiratorios detectados fueron: Virus Sincicial Respiratorio55 (43 por ciento); Rinovirus 30 (23 por ciento); Metapneumovirus13 (10 por ciento); Influenza A 8 (6 por ciento), Enterovirus 6 (5 por ciento); Bocavirus 6 (5 por ciento); Adenovirus 4 (3 por ciento); Coronavirus3 (2 por ciento); Parainfluenza 1: 2 (1 por ciento); Influenza B, 1(1 por ciento) y Parainfluenza 3: 1 (1 por ciento).

Respiratory viruses seasonality in children under five years of age in Buenos Aires, Argentina: a five-year analysis.

OBJECTIVES: During the winter months there is a remarkable increase in paediatric hospitalisations due to viral acute lower respiratory infections (ALRI). We aimed to perform a five-year retrospective analysis (1998-2002) of ALRI viral aetiology in children under 5 years of age admitted to public hospitals in Buenos Aires city to evaluate its seasonality. MATERIALS AND METHODS: Nasopharyngeal aspirates (NPA) were analysed by indirect immunofluorescence to determine viral aetiology. A Spearman's rank correlation test between meteorological parameters and viral frequencies was performed. RESULTS: Viruses were identified in 6083 (32.8%) of 18,561 NPA tested. Among the former 4796 (78.8%) were RSV, 508 (8.3%) IA, 473 (7.8%) AV, 293 (4.8%) PIV and 13 (0.2%) IB. RSV and IA peaked during the coldest and dampest months, whereas PIV did so in early spring and AV lasted throughout the year. For RSV and IA an inverse correlation with mean monthly temperature (r = -0.9 and r = -0.87, respectively, p<0.0001) and solar UVB radiation (r = -0.92 and r = -0.80, respectively, p<0.0001) was detected, while it was positive when relative humidity was considered (r = 0.6, p<0.0001 and r = 0.47, p=0.0068, respectively). CONCLUSIONS: This study highlights the seasonal variation of ALRI and allows the implementation of adequate healthcare strategies and practice guidelines.

Complete genome sequencing of dengue virus type 1 isolated in Buenos Aires, Argentina.

Dengue (DEN) constitutes a major viral arthropod-borne human illness. South America was last considered free of dengue two decades ago when a dramatic increase in the number of dengue fever and hemorrhagic dengue cases had been reported. Five viruses were isolated in Buenos Aires City from the 1999-2000 Paraguay outbreak. RT-PCRs obtained directly from plasma were cloned into pGemT vectors and sequences of the structural genes and NS1 were analyzed. Three viruses were full-length sequenced from RT-PCR obtained from cell-culture isolates. Excess of synonymous over non-synonymous mutations suggested that the structural proteins were under strong functional constraints while a weak purifying selection was operating in the whole polyprotein. Sequence diversity and selective pressures varied among patients but results were significantly above the procedure threshold. One sample showed small-plaque phenotype and impaired growth coupled to 3'untranslated region mutations. Phylogenetic analysis of full-length sequences split Buenos Aires isolates into two clusters within American DEN-1 genotype V: Clade I was phylogenetically linked to Brazilian samples and Clade II with samples from Paraguay and Northeastern Argentina. In Buenos Aires City, only dengue virus serotype 1 imported from Paraguay has been detected, though without evidence of local transmission.