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AAbbssttrraaccttss EEppiiddeemmiioollooggyy aanndd MMooddeelliinngg Ana E Arango, MSc Associated investigator, Universidad de Antioquia, Colombia aearangor@gmail.com Translated to English using Google Translate: Ana E. Arango Restrepo1, Eric Halsey2, Sergio Jaramillo V3, David Espinal3, Jorge Donado3, Jane Ríos2, Alberto Laguna-Torres2. Sentinel surveillance of influenza-like illness (ILI) in a hospital in Medellin, Colombia. 1. Universidad de Antioquia, Grupo Inmunovirología, Medellín, Colombia. Introduction: Viruses are the main agents that cause acute respiratory disease. Best known for their pathogenicity in the respiratory tract are influenza A and B, respiratory syncytial virus (RSV), adenovirus, parainfluenza viruses 1, 2 and 3, and human metapneumovirus (hMPV). Objective: Contribute to the knowledge of local (Medellín and its metropolitan area), national (Colombia), and South American region epidemiology, with the diagnosis of respiratory infections who attended the HPTU between December 2006 and December 2010. Methods: Anyone arriving at HPTU with influenza-like illness who voluntarily participated in the study. Each patient had a history filled and two samples were taken: a nasal swab for influenza rapid testing (Quick Vue) and a throat swab for viral isolation. The patient was immediately informed of the the rapid testresults, and the other sample was stored at -70 ° C until shipment to NAMRU-6 in Lima for cultivation in different cell types. The culture results were sent to to HPTU and INS in Bogota. Results: We present the results obtained from a total of 1,592 patients. 723 were female (45.41%), 1,146 were children under 5 years (71.98%), and 92.9% came from the Medellín metropolitan area. The predominant symptoms were cough (87.56%), malaise (74.94%), fever (74.72%) and rhinorrhea (55.46%). 316 (19.85%) patients were hospitalized. The positivity found for respiratory virus was 34.23%. Viral agents most frequently isolated viruses were influenza A (14.1%), adenovirus (11.9%), influenza B (5.5%), VRS (1.26%). Mortality in two patients (0.13%) was probably due to other causes. The isolated viruses were circulating throughout the year, except for influenza B outbreaks presented in 2008 and 2010. For the first time, hMPV was described in Colombia. Conclusions: Our results are consistent with the literature; however, they are an important contribution to the knowledge of the local epidemiology of viral respiratory infections because there are few studies of their own. Some viruses circulate throughout the year in the city and others do so intermittently. It highlights the movement of some viruses that are often ignored in the medical consultation. To strengthen the viral diagnosis of TSIs to rationalize the use of antibiotics.

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En español: VIGILANCIA SENTINELA DE LA ENFERMEDAD TIPO INFLUENZA (ETI) EN UN HOSPITAL DE MEDELLÍN, COLOMBIA. Ana E. Arango Restrepo1, Eric Halsey2, Sergio Jaramillo V3, David Espinal3, Jorge Donado3, Jane Ríos2, Alberto Laguna-Torres2 1. Universidad de Antioquia, Grupo Inmunovirología, Medellín, Colombia. INTRODUCCIÓN Los virus son los principales agentes productores de enfermedad respiratoria aguda. Los más conocidos por su patogenicidad en el tracto respiratorio, son los virus de Influenza A y B, el Virus Respiratorio Sincitial (VRS), los Adenovirus, los virus Parainfluenza 1, 2 y 3 y el Metaneumovirus humano (hMPV) recientemente descrito. OBJETIVO GENERAL Contribuir al conocimiento de la epidemiología local (Medellín y su Área Metropolitana), y nacional (Colombia), así como en la región suramericana, mediante el diagnóstico de los casos de infección respiratoria que acudieron al HPTU entre diciembre de 2006 y diciembre de 2010. METODOLOGÍA Cualquier persona que consulte al HPTU con enfermedad tipo influenza y que voluntariamente participe en el estudio. A cada paciente se le llenó una historia clínica y se le tomaron dos muestras: hisopado nasal para prueba rápida de Influenza (Quick Vue) y un escobillón faríngeo para aislamiento viral. El resultado de la prueba rápida se le informó al paciente inmediatamente y la otra muestra se conservó a -70ºC hasta su envío a NAMRU-6 en Lima para su cultivo en diferentes tipos celulares. Los resultados de los cultivos fueron enviados a su recibo al HPTU y al INS en Bogotá. RESULTADOS Se presentan los resultados obtenidos de un total de 1.592 pacientes. 723 fueron de sexo femenino (45.41%). 1.146 eran menores de 5 años (71.98%). El 92.9% provenían de Medellín y su área metropolitana. La sintomatología predominante fue tos (87.56%), malestar general (74.94%), fiebre (74.72%) y rinorrea (55.46%). 316 (19.85%) pacientes fueron hospitalizados. La positividad encontrada para los virus estudiados fue del 34.23%. Los agentes virales aislados con mayor frecuencia fueron los virus de Influenza A (14.1%), adenovirus (11.9%), influenza B (5.5%), VRS (1.26%). La mortalidad, de dos pacientes, (0.13%) fue probablemente debida a otras causas. Los virus aislados se encontraron circulando durante todo el año, excepto influenza B que presentó brotes epidémicos en 2008 y 2010. Por primera vez se describe hMPV en Colombia. CONCLUSIONES Nuestros resultados coinciden con la literatura, sin embargo, son una contribución muy importante al conocimiento de la epidemiología local de las infecciones respiratorias virales, ya que no hay muchos estudios propios. Algunos virus circulan durante todo el año en la ciudad y otros lo hacen de manera intermitente. Se destaca la circulación de algunos virus que son ignorados frecuentemente en la consulta médica. Se debe reforzar el diagnóstico viral de las ETI para racionalizar el uso de antibióticos.

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Alfredo Bruno Investigador , Insituto Nacional de Higiene, Ecuador alfredobrunoc@yahoo.es Translated to English using Google Translate: Bruno A, de Mora D, Gonzalez M. Surveillance of influenza virus and other causes of SARI in Ecuador. Instituto Nacional de Higiene y Medicina Tropical LIP The geographic conditions of Ecuador directly affect the behavior of respiratory infections due to a number of factors, including its tropical location, divided by ridges of the Andes into three natural regions (Costa, Sierra and Oriente) and the continuous flow of warm and cold ocean currents. Despite being a small country with an area of 256,000,370 km, these factors contribute to a variety of climates that experience sudden changes in temperature and humidity. Coupled with intense internal and external population migration, these factors can facilitate the movement of a variety of respiratory viruses. Due to these characteristics and the global alert system created by WHO / PAHO for H5N1 influenza cases from Asia, health authorities in Ecuador have decided since 2006 to resume monitoring respiratory viruses in the National Influenza Center ( NIC) located at the headquarters of the National Institute of Hygiene in Guayaquil, capturing TSI level cases and processing the samples for identification by immunofluorescence, RT-PCR in real time, and conventional virus isolation in MDCK cells and embryonated chicken eggs. Since March 2011, the Ministry of Public Health designated the Expanded Program on Immunization (EPI) responsible for monitoring SARI, having as components the establishment of sentinel sites in three major cities ( Quito, Guayaquil, and Cuenca), responsible for the uptake of cases, the epidemiology tab, and the discharge status of patients in relation to virological investigation by the National Institute of Health. The NIH is responsible for the identification and molecular characterization of virus influenza, as well as other respiratory virus detection by IIF technique. Another strength of the monitoring program is online computer software designed by the MSP, which allows us to have a wide gamma of epidemiological variables, including general patient data, co-morbidities, date of collection of cases, application of vaccine, laboratory results, epidemiological week, and discharge status, among others. Within the jurisdiction of the NIC is the technology transfer for the decentralization of the diagnosis, monitoring, technical assistance, and quality control on a quarterly basis for the results obtained from the regional laboratories of the INH-MT Quito and Cuenca. Among the prospects for this year is the implementation of molecular biology techniques for the identification of other respiratory viruses, sequencing studies for drug resistance, and phylogenetics. En español: VIGILANCIA DE INFLUENZA Y OTROS VIRUS RESPIRATORIOS CAUSANTES DE IRAG EN EL ECUADOR. Med/Vet Alfredo Bruno, Blga. Domenica de Mora, Dr. Manuel Gonzalez Instituto Nacional de Higiene y Medicina Tropical LIP

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Las condiciones geográficas del Ecuador incide directamente en el comportamiento de las infecciones respiratorias debido a una serie de factores: estar ubicado en la zona tropical, ser dividido por las cordilleras de los Andes en tres regiones naturales, Costa, Sierra y Oriente, el continuo flujo de corrientes marinas cálidas y frías; todo esto permite que a pesar de ser un país pequeño de 256.000.370Km de superficie, cuente con una gran variedad de climas que experimentan cambios bruscos de temperatura y humedad. A esto se suma una intensa migración tanto interna como externa de la población que puede facilitar la circulación de una gran diversidad de virus respiratorios. Debido a estas características y ante la alerta mundial por parte de OMS/OPS de los casos de Influenza H5N1 procedentes del continente asiático, las autoridades de salud del Ecuador desde el año 2006 decidieron retomar la vigilancia de virus respiratorios en el Centro Nacional de Influenza (NIC) ubicado en la sede del Instituto Nacional de Higiene de la ciudad de Guayaquil, captando casos a nivel de ETI y procesando las muestras para la identificación mediante técnicas de inmunofluorescencia, RT-PCR en tiempo real y convencional; aislamiento viral en células MDCK y huevos embrionados de gallina. A partir del mes de Marzo del año 2011 el Ministerio de Salud Publica designo al Programa Ampliado de Inmunizaciones (PAI) como responsable de la vigilancia de las IRAG; teniendo como componentes la conformación de los puestos centinelas en las tres ciudades mas importantes del país (Quito, Guayaquil y Cuenca); responsables de la captación del caso, llenado de la ficha epidemiologia y condición de egreso del paciente; en lo relacionado a la investigación virológica el Instituto Nacional de Higiene es el responsable de la identificación, caracterización molecular del virus de Influenza, al igual que la detección de otros virus respiratorios por la técnica de IFI. Otra fortaleza del programa de vigilancia es el software informático on-line diseñado por el MSP, el cual nos permite disponer de una amplia gamma de variables epidemiológicas que incluyen datos generales del paciente, comorbilidades, fecha de captación de caso, aplicación de vacuna, resultado de laboratorio, semana epidemiológica, condición de egreso, entre otros. Dentro de las competencias del NIC esta la transferencia tecnológica para la descentralización del diagnostico; supervisión, asesoría técnica, control de calidad de manera trimestral para el aseguramiento de la calidad de los resultados obtenidos de los laboratorios regionales del INH-MT Quito y Cuenca. Entre las perspectivas para este año esta la implementación de técnicas de biología molecular para la identificación de otros virus respiratorios, estudios de secuenciación para la fármaco resistencia y filogenéticos. Wilson Chicaiza, MD Director of Medical Education and Research, Hospital Vozandes Quito, Ecuador wchicaiza@hcjb.org Wilson Chicaiza-Ayala1, 3; Betzabé Tello1 ; V. Alberto Laguna-Torres 2; Julia S. Ampuero 2;. Richard W Douce1, Eric Halsey2. Surveillance of Influenza in an Emergency Department in Ecuador, 2008-2011. 1. Hospital Vozandes Quito, Ecuador 2. U.S. Naval Medical Research Center Detachment Lima- Perú 3. Universidad de las Américas The goal of this influenza surveillance study is to correlate syndromic surveillance with virologic surveillance and climatological data in Quito. This surveillance will be supported with data of virological

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characterization of influenza-like illness (ILI) detected in the emergency room of Hospital Vozandes Quito (HVQ) and, on the other hand, data of syndromic definition of acute respiratory infection (ARI) based on clinical diagnosis reported daily. In temperate climates, increases in the number of persons seen in the emergency department (ED) with fever and respiratory complaints has been correlated to seasonal outbreaks of respiratory syncytial virus (RSV) and influenza A and B. The incidence of acute respiratory infection has been proposed as a less restrictive criterion for monitoring influenza activity. There is data that support that temperature and absolute humidity have been correlated with seasonal outbreaks. The epidemiologic behavior of influenza in tropical climates where there is no well defined influenza season is less well studied. Several climatological variables will be described in order to find correlation with the emergence of respiratory infections in Quito. Expected results are to find correlation between climatological changes in Quito, influenza outbreaks, and acute respiratory disease reported in the emergency room. These findings could suggest changing the epidemiologic way of assessment of outbreaks of acute respiratory disease based on syndromic surveillance supported with laboratory confirmation. Gerardo Chowell, PhD Associate Professor, Arizona State University & Fogarty International Center, USA gchowell82@gmail.com G. Chowell et al. Dynamics of the 2009 A/H1N1 influenza pandemic using high resolution surveillance data. Arizona State University and Fogarty International Center Detailed surveillance data on the 2009 A/H1N1 influenza pandemic are crucial to quantify the spatial and temporal characteristics of pandemic influenza. We provide a quantitative description of the age-specific and regional 2009 A/H1N1 pandemic incidence patterns in Mexico and two southern hemisphere countries, Peru and Chile. We used daily cases of influenza-like-illness, tests for A/H1N1 influenza virus infections, and laboratory-confirmed A/H1N1 influenza cases to analyze the geographic spread of the pandemic waves and their association with the winter school closing periods, and demographic, geographic, and climatic factors. We also estimated the reproduction number and quantified the association between school closing periods and the age distribution of cases. Our results indicate substantial regional variation in pandemic patterns, highlight the importance of demographic and geographic factors and the role of school cycles in shaping the transmission dynamics of this pandemic influenza strain, and suggests that school closure and other mitigation measures could be useful to mitigate future influenza pandemics. Andreu Comas-Garcia, MD MHS student, Instituto Nacional de Salud Publica, Mexico andreucomasg@gmail.com Comas-García Andreu, Noyola Daniel E, Christen-García J. Andres, Velasco-Hernández Jorge X., Yarza-Acuña Sergio, Hernández-Quintero Angélica, Morales-Bárcenas J. Héctor, Capistran-Ocampo Marcos A. Model for early detection of influenza outbreaks based on the weekly report of acute respiratory infections.

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Instituto Nacional de Salud Pública, Universidad Autónoma de San Luis Potosí, Instituto Mexicano del Petróleo, Universidad Autónoma de Nayarit, Centro de Investigaciones en Matemáticas, A. C. Project description: The application of mathematical and statistical modeling to epidemiology has a long standing tradition, and it remains an active research area. In this project, we propose to use novel methods of uncertainty quantification and inference in stochastic epidemic models. Our methodology includes phylodynamics of influenza aimed at inferring past population dynamics from reconstructed genealogies. Acute respiratory infections (ARI) are among the leading causes of both morbidity and mortality worldwide. In this scenario, the principal causative agents are influenza and respiratory syncytial virus (RSV). The impact of influenza can be measured by time series of excess of cases and/or deaths related to pneumonia and excess of respiratory/cardiovascular disease. This time series contains information of: 1) timing of the epidemic, 2) the spatial dispersion of the outbreak, and 3) incidence. ARI time series, together with records of confirmed cases of influenza, may constitute sufficient information to identify influenza epidemics in a population. Hypothesis: Using the weekly record of ARI reported for different age groups a combined integration of mathematical, statistical, and epidemiological approaches will be able to predict and estimate the onset of Influenza epidemics in a population at an early phase. Goals: To characterize early demographic mechanisms in the dynamic of an influenza outbreak using weekly reports of ARI. Analyze by numerical simulations, Bayesian inference and MCMC the effect of Influenza vaccination to targeting different age groups on the outcome of an outbreak. To characterize the contribution of phylogenetic differences between influenza viruses from different periods in the evolution of the ARI epidemics. To use the predictive power of a mathematical and statistical model using epidemiological and virological information collected prospectively for in two different states of Mexico. Methodology: 1) Time series analysis of the prevalence of ARI disease with wavelet decomposition, and also by nonlinear methods. 2) Bayesian inference with stochastic processes. 3) MCMC to fit stochastic, nonlinear mechanistic models for complex population dynamics to gene genealogies and time series data in a Bayesian framework. 4) Classical epidemiological analysis of ARI: We will use the weekly ARI registry, divided into different age groups, for San Luis Potosi and Guanajuato. 5) Detection of Influenza and RSV associated with ARI: We will use the information generated at the Universidad Autónoma de San Luis Potosí. Diego Garcilazo Medical Doctor, Instituto Nacional de Enfermedades Respiratorias, Argentina diegogarcilazo@hotmail.com Garcilazo DA, Bossio JC, Fernandez H, Arias SJ, Landra FO. Time series analysis about pneumonia and influenza mortality: Argentina, 1980 - 2010 Instituto Nacional de Enfermedades Respiratorias INER - CONI - ANLIS Objectives: To describe trends in historical pneumonia and influenza mortality data in Argentina since 1980 to 2010.

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Methods: Data about deaths notified in Argentina was compiled from information reported on the Certificate of Death provided by the national official agency Dirección de Estadísticas e Información en Salud (DEIS). The data include causes of death, group of age and sex since 1980 to 2010. Data of monthly influenza-classed death were obtained since 2002 to 2010. Data was classified according to ICD9 (1980 - 1996) and ICD10 (1997-2010) as death due to: pneumonia and influenza, respiratory system, cardiovascular system, chronic respiratory diseases and all-cause death. Mortality rates, annual percent change (APC), and breakpoints over trends were calculated. The software R project 2.13 was used for computing data and Joinpoint 3.5 to analyze breakpoints models. Results: A considerable increase (+2.91% IC[ +2.26 to +3.57]) in influenza-classed mortality rate was observed during the period 1980-2010, when rates raised from 28.5 to 48.2 cases per 100,000 population. The increment was especially pronounced since 1986 (+4.1% IC[+3.5 to +4.7]). Some peaks turned the trend irregular on years 1999, 2003, 2007 and 2009. A similar pattern was observed for death due to Chapter of Diseases of the respiratory system, when since 1989 the trend keeps growing up (+4.9% IC[+4.1 to +5.8]). By contrast, the section Chronic lower respiratory diseases rates showed an significant increment (+3.1% IC[+2.1 to +4.2]) until 1995, then rates started to go down; a similar pattern drew the death due to cardiovascular system diseases. All death rates showed a decrease (-0.8% IC[ -1.1 to -0.4]) until 1994, after which the trend showed an insignificant increment. The death due to pneumonia and influenza rates by age revealed an important increment for group of age 20 to 64 years (+8.1% IC[+4.6 to +11.7]) since 2001 (peak 2009) and 65 to 74 years (+3.5% IC[+2.8 to +4.1]) since 1986, but an important decrease for group of age less than 5 years (-6.1% IC[ -6.8 to -5.4]). Monthly data revealed that the highest numbers of death occurred during winter months. The death rates compared by sex didn’t show a difference between males and females, but women showed less risk than men. Conclusion: The historical dataset about mortality, extracted from certificate of death, suggests an increment of death due to pneumonia and influenza in adult people in Argentine, death occurs specifically on winter months. Probably several factors should be taken into account despite of public health and ecological factors to evaluate the role of them in influenza mortality risk. Eric Halsey, MD Head, Virology Lab, NAMRU-6, Peru eric.halsey@med.navy.mil Halsey E. NAMRU-6 Passive Respiratory and Febrile Disease Surveillance Network The NAMRU-6 Virology Department oversees a vast respiratory and febrile disease network with over 40 sites in South and Central America. Annually, we examine, interview, and collect a respiratory sample from over 5000 people. Our Lima-based laboratory investigates for the presence of influenza virus in all samples and looks for a wide array of other pathogens on a selective basis. In addition, our febrile surveillance network annually collects samples and data from roughly 3000 people, both in the acute and convalescent periods. These samples are also processed in our Lima-based laboratory, an undertaking that has resulted in the identification of multiple new viruses (five in 2011) as well as the molecular, epidemiological, and clinical characterization of the main causes of febrile disease in the region. Our data has been incorporated into multiple manuscripts (see accompanying list of manuscripts from the past 12 months). I predict that the burgeoning NIH-NAMRU6 collaboration will result in tangible benefits for both parties. NAMRU-6 provides a unique and extensive surveillance network along with highly-trained laboratory

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personnel, a large bank of well-preserved samples, researchers familiar with the local epidemiology, and contacts within various ministries throughout Latin America. NIH provides a fresh perspective on data analysis and new skills that may be implemented to enhance the way we report our results. Sergio Jaramillo Head Laboratory, Hospital Pablo Tobón Uribe, Colombia sjaramillo@hptu.org.co Sergio Jaramillo V1, Ana E. Arango Restrepo2, Isabel Cristina Hurtado4, David Espinal1, Josefina García3, Alberto Laguna-Torres3 . Metapneumovirus humano en niños en un hospital de Medellín, Colombia 1. Hospital Pablo Tobón Uribe (HPTU), Medellín, Colombia. Introduction: Human metapneumovirus (HMPV) is a new pathogen associated with respiratory infections mainly in children, producing a clinical syndrome similar to bronchiolitis caused by other viruses and can range from mild to severe, diagnosis is difficult until the chain reaction with reverse transcription polymerase and cell culture methods are used for diagnosis. Materials and methods: UdeA HPTU and participate in a network to determine the etiology of influenza-like viral disease, this included patients with fever and respiratory symptoms who were sampled pharyngeal secretion which was sent to NAMRU-6 in Peru, where cell lines were seeded for culture, if positive isolates, confirmation of the virus was performed using the RT-PCR, using specific DNA probes. Results: We report 8 patients between two and nine years with HMPV isolation, male: female ratio 1:1, 7 came from Medellin, all presented with fever (mean 38.9 ° C), dry cough and malaise, four of they had nasal symptoms and abnormalities on lung auscultation, especially wheezing, dyspnea or tachypnea three and two of them epiphora or conjunctival injection. There was no contact with animals and travel in the previous 15 days. The initial diagnosis was bronchiolitis, pneumonia, or IRA. Two required hospitalization (both coinfected), both marked respiratory distress, none died. Two patients had chronic lung disease (asthma and bronchopulmonary dysplasia). Coinfection was found in two patients, one with Mycoplasma pneumoniae, and one with adenovirus and RSV. One patient had a history of cardiovascular HTP VSD. The rapid influenza test was negative for the 8 Discussion: We found 8 patients who had lower respiratory tract symptoms associated with fever, which agrees broadly with the symptoms and differential diagnoses reported in the literature. There are multiple reports of viral and bacterial coinfections. The diagnosis was made by cell culture, which takes a long time though useful, but has the option early to assess the cytopathic effect of the virus on cells of the crop. It is important to future studies to know where the real prevalence of HMPV in our country and where rapid diagnostic methods are validated and cost-effective to help clarify the etiology and guide treatment of our patients.

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Iveth Lorenzana, Msc Professor, UNAH, Honduras ivettelorenzana@yahoo.com Ivette Lorenzana de Rivera1, Victor A. Laguna-Torres2, Maribel Rivera3, Cynthia Rodríguez1, Erick S. Halsey2, Tadeusz J. Kochel2. Surveillance of Viral Respiratory Pathogens in Tegucigalpa, Honduras 2006-2009. 1Universidad Nacional Autónoma de Honduras,Tegucigalpa, Honduras,2. US Naval Medical Research Center Detachment, Lima, Perú.3Instituto Hondureño de Seguridad Social.Tegucigalpa, Honduras. Background: Acute respiratory infections (ARI) constitute one of the principal causes of morbidity and mortality especially in developing countries. The disease burden is estimated to be 94 037 000 DALYs and 3.9 million deaths worldwide [2002]. In Honduras ARI is the number one cause of morbidity in children less than 5 years old. ARI clinical presentation varies from mild to severe illness and could be life threatening. Aims: The purpose of the study was to asses the viral etiological agent’s associated to ARI in participants enrolled at Hospital de Espacialidades del Seguro Social in Tegucigalpa, Honduras collected in a surveillance basis during these years. Materials and Methods: From 2006 to 2009, pharyngeal swabs were collected from consented participants. They were frozen at -80C and sent off regularly on Dry Ice to Lima, Peru; were they were cultured on cell monolayers, and viral antigens were detected by immunofluorescence staining. The participant’s age ranged from 1 to 68 years old. Genotyping of the Influenza A isolates was done by sequencing analysis of the Hemagglutinin gene. Results: A total of 719 samples were analyzed, of whom 271(37%) were positive by any viral respiratory agent. A 48% of the cases were in children between 1-5 years of age. A third of the patients took antibiotics unnecessarily. The positivity by year range from: 23% to 46%, with the highest incidence observed in 2009. The most common viruses isolated was Influenza A (175; 67% of cases) Adenovirus (33;12.5%),Parainfluenza (22,8.3%), Influenza B (17; 6.5%), Herpes simplex (10; 3.8%), RSV (4; 1.5%) and Human Metapneumovirus (2;0.7%). Dual infections were detected in 2.3% of the total of cases. Discussion: A significantly higher positivity was found in age group 1-5 years old. The higher prevalence by viral etiological agent found was for Influenza A with a significant number of Adenoviruses which is in concordance with what has been reported previously in the country. The prevalence of RSV was low. The higher incidence observed in 2009 was due to worldwide epidemic of Influenza A H1N1. Genetic analysis of influenza isolates showed 15 different genotypes all coming from the Americans region. Conclusion: The data demonstrates that surveillance studies such as this are important, to be able to report that are several viral agents that are associated with IRA in Tegucigalpa, Honduras and to show seasonality, yearly peaks of incidence as well its association with age group and severity of the disease.

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Aba Mahamat, MD, PhD Infectious diseases Specialist, Cayenne General Hospital, Cayenne, French Guiana mahamataba@gmail.com A. Mahamat*1, P Dussart2, A. Bouix3, F. Eltges4, L. Carvalho5, P. Quenel5, W.J. Alonso1, M.A. Miller1, C. Viboud1. Climatic drivers of influenza seasonality in French Guiana, 2006-2010 1National Institues of Health, Fogarty International Center, Bethesda, USA. 2Pasteur Institute of Guyane, Virology, Cayenne, French Guiana. 3Sentinel Practitioner Network, Cayenne, French Guiana. 4Regional Agency of Health, Cayenne, French Guiana. 5Region Introduction. Data on seasonal influenza in tropical regions remain scarce compared with that in temperate region. Our objectives in this study were to describe the pattern of the seasonal epidemic of influenza in French Guiana, a tropical region of South America and to analyze the role of climatic factors on the epidemiology of influenza transmission. Material and Methods: Weekly Influenza-like illnesses (ILI) were recorded from the sentinel GP network whereas isolation and identification of influenza virus from 2006 to 2010 were performed at the Pasteur Institute laboratory of virology. Climatic parameters (rainfall, temperature and relative humidity) were obtained from the regional office of Météo France. Times series analysis (ARIMA models) and dynamic regression models were used to investigate relationship between ILI incidence and climatic parameters. The pandemic period was excluded from the analysis. Results. From January 2006 to Decmber 2010, a total of 1,533 viruses were isolated from the sentinel surveillance.The univariate analysis of the ILI incidence showed a seasonal autoregressive variation with a mean of 81 ILI/100,000 inhabitants. In bivariate analysis, we observed that including rainfall or specific humidity as input series result in models with better performance than the univariate one where the ILI incidence series depend only on its past values and error signal. Using multivariate dynamic regression analysis, we estimated that an increase of 1 mm of precipitation induced in an average delay of 1 week, an increase of 0.33% in ILI incidence (p < 0.01); while an increase of 1kg/kg for specific humidity resulted with an average delay of 3 weeks, in a significant decrease of 11% in the ILI incidence rate. Conclusion. Rainfall and specific humidity played significant roles on the transmission of seasonal influenza in French Guiana. Bibligraphic citations: Update of the poster presented at the ESWIG 2011.

Edwin Miranda MD Medico Infectologo, INSN, Peru emirandach@gmail.com Translated to English using Google Translate: Analysis of the severity of pneumonia in children hospitalized for the AH1N1/2009 influenza pandemic, Peru Objective: To analyze the clinical and laboratory severity of pneumonia in children hospitalized for the 2009 AH1N1 influenza pandemic in pediatric patients in the INSN.

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Methods: Retrospective case series of children hospitalized with pneumonia and pandemic influenza AH1N1/2009. We reviewed the medical records between the months of June to September 2009. After virological confirmation, we analyzed the conditions of severity and compared cases of pneumonia associated with pandemic influenza AH1N1/2009. Results: We studied 74 children, of which 24 cases were nosocomial and 16 required mechanical ventilation. There were 12 deaths, all with pre-existing conditions. Being an inpatient case was associated with higher mortality. In-hospital cases under 6 years of age were 72%. The median disease duration was five days. The most common symptoms were fever, cough, and runny nose. 82% received oseltamivir. In chest radiography, 48% of cases showed patchyinfiltrates and 44% interstitial infiltrate. C-reactive protein over 10 mg / L was associated with respiratory failure. In the multivariate analysis, we found that fever and lymphopenia-associated factors of pneumonia are influenza pandemic AH1N1/2009 compared with negative pneumonias. Conclusion: We found that in-hospital cases that had poor prognoses also had the highest PCR and were those with pre-existing conditions. The clinical and laboratory tests can help differentiate pneumonia from influenza pandemic AH1N1/2009 and other pneumonias. En español: Análisis de la severidad de neumonía en niños hospitalizados por Influenza AH1N1/2009 pandémica, Perú Objetivo: Analizar las características clínicas y laboratorios de severidad de neumonía en niños hospitalizados por influenza AH1N1/2009 pandémica en pacientes pediátricos en el INSN. Métodos: Estudio retrospectivo de serie de casos de niños hospitalizados con neumonía por la Influenza AH1N1/2009 pandémica. Se revisaron las historias clínicas entre los meses de Junio a Setiembre 2009. Todos con confirmación virológica, analizamos las condiciones de severidad. Se comparó con los casos de neumonía no asociada a influenza AH1N1/2009 pandémica. Resultados: Se estudiaron 74 niños, de los cuales 24 casos son intrahospitalarios, 16 requirieron ventilación mecánica. Fallecieron 12, todos con condición preexistentes. Ser caso intrahospitalario estaba asociado a mayor mortalidad. En los casos extrahospitalarios, los menores de 6 años son el 72%. La mediana de tiempo de enfermedad fue de 5 días. Los síntomas más frecuentes fueron fiebre, tos, rinorrea. Recibieron oseltamivir el 82%. En la radiografía de tórax el 48% de los casos presentó infiltrado en parches y el 44% infiltrado intersticial. La Proteína C Reactiva más de 10mg/L se asoció con insuficiencia respiratoria. En el análisis multivariable se hallaron que fiebre y linfopenia son factores asociados de neumonía de influenza AH1N1/2009 pandémica en comparación con las neumonías negativas para neumonía de influenza AH1N1/2009 pandémica Conclusiones: Encontramos casos intrahospitalarios quienes tuvieron mal pronóstico, también los que presentaron el PCR elevado y los que presentaron condición preexistente. La clínica y exámenes de laboratorios pueden ayudar a diferenciar los casos de neumonía por influenza AH1N1/2009 pandémica de las otras neumonías. Palabras Claves: influenza AH1N1/2009 pandémica, neumonía, niños

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Fernanda Moura, PhD Professor, Universidade Federal do Ceará, Brasil fernandaedna@terra.com.br Fernanda EA Moura (1); Luciano M Thomazelli (2); Caroline MGD Florencio (1); Samuel AR Pereira (3); Francisco MS Oliveira (3); Alice A Alves (3); Crister J Ocadaque (3); Daniela B Oliveira (2); Edison L Durigon (2). Influenza in an equatorial city: Something else about the more recent influenza pandemic in Brazil. (1) Universidade Federal do Ceará, Fortaleza, CE, Brazil; (2) Universidade de São Paulo, São Paulo, Brazil; Faculdde de Tecnologia Intensiva, Fortaleza, CE, Brazil Most information about the latest pandemic influenza in Brazil came from the south and southeast regions of the country. It is known that seasonal influenza presents seasonally variable in some regions of Brazil. In Fortaleza, a city in northeastern Brazil, a continuous monitoring of respiratory viruses has been held since 2001. Clinical respiratory samples from children attending at Hospital Infantil Albert Sabin due to acute respiratory infection were analyzed initially by immunofluorescence for detection of influenza and other respiratory viruses. The qPCR was used to identify seasonal influenza A seasonal / pandemic. Objectives: (1) present the circulation pattern of influenza virus (A seasonal / pandemic / B) during the period January 2009 to July 2011 (2) describe the clinical epidemiology of influenza. Results: Influenza viruses were detected in 130 of 2503 samples analyzed. Influenza A H1n1 pandemic corresponded to 32.3% of influenza viruses detected. The circulation of pandemic virus was limited to the period from October 2009 to February 2010, outside of the period of higher impact of pandemic influenza in Brazil. Maria de Lourdes Oliveira Senior Technologist in Public Health, Oswaldo Cruz Institute, National Reference Laboratory for Influenza, Brazil mlaoliveira@fiocruz.br Maria de Lourdes Aguiar Oliveira1; Valdiza Valente2; Renato Rodrigues Medeiros1; Roger Rohloff3; Jaline Alves Cabral da Costa1; Sharon Marie Carney1; Valeria Saraceni2; Glaucia Paranhos-Baccala4; Marilda Mendonça Siqueira1. Influenza A(H1N1)pdm09 and other viral respiratory infections among fatal cases in pregnant and non-pregnant women – Preliminary findings Respiratory Viruses Laboratory, the National Reference Laboratory for Influenza, Oswaldo Cruz Foundation, Brazil; 2Health Secretariat from Rio de Janeiro; 3Municipal Hospital Fernando Magalhães, RJ; 4Emergent Pathogens Laboratory, Merieux Foundation. During 2009 pandemics, mortality in Brazil was estimated as 1.1/100,000 habitants, with higher incidence in the South/Southeast regions. Pregnant women compose a relevant population due to the elevated chance of clinical progression to severe disease/death and neonatal complications. Although influenza virus is the main etiology of acute respiratory infections (ARI), they can also be caused by other pathogens. We investigated the epidemiological and virological features of fatal cases among pregnant and non-pregnant women from Rio de Janeiro (June/2009-june/2011), information still scarce in Brazil. The study population was composed by 741 women at reproductive age (15-44 years), from which 38 progressed to death. Demographic/clinical/epidemiological information was assessed by the

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Epidemiological Surveillance team/Rio de Janeiro, using a nationally standardized questionnaire. After RNA extraction (QIAmp viral RNA and QIAmp viral MinElute spin kit, QIAGEN), Influenza A and other respiratory pathogens detection were carried out using real time RT-PCR (CDC protocol and FTD Respiratory Pathogens 21plus, Fast-Track Diagnostics, respectively). Descriptive, bivariate analyses (chi-square/Fisher’s exact test and t-test for means) and multiple logistic regressions (considering variables of epidemiological relevance/plausibility) were carried out and considered significant when p< 0.05. Influenza A(H1N1)pdm09 was detected in 23 (60.5%) and 300 (42.7%) from fatal and non-fatal cases, respectively (p=.024). Infected pregnant women were about 3 times more likely to progress to death than their counterparts (OR=3.01). Among confirmed Influenza A(H1N1)pdm09 cases, those who died presented lower per capita income (p<.001), higher frequencies of dyspnea (p=.003), hospitalization (p<.001) and co-morbidities (p=.025), as a higher interval between the 1st. symptoms and hospitalization (p<.001). The last, a significant risk determinant for deceases among infected pregnant women (5.1±3.9 vs. 2.5±3.2 days, p=.012). Among infected non-pregnant women, the presence of any co-morbidity was identified as an independent risk factor for death (AOR=18.5). From 34 fatal cases, 4 samples remained negative for all tested pathogens. Among 15 Influenza A(H1N1)pdm09 negatives, other pathogen was detected in 13 samples. Detected mono-infections were as follows: Influenza A(H1N1)pdm09 (9), followed by FluA (2); Bocavirus (2); Rhinovirus (1) and Coronavirus OC43 (1). Bacterial infections were found in 10 fatal cases. Finally, 5 cases of multiple infection were found (3 Influenza A(H1N1)pdm09 + Bocavirus; 1 Influenza A(H1N1)pdm09 + Adenovirus and 1 Influenza A(H1N1)pdm09 + Bocavirus + RSV). These preliminary findings reinforce the fatal role of Flu infections, especially among pregnancy and highlight the usefulness of new molecular tests for elucidation of SARI/fatal cases. Financial support: Science and Technology Department (DECIT), Ministry of Health and National Counsel of Technological and Scientific Development (CNPq), Brazil

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Maria de Lourdes Aguiar Oliveira1; Renato Rodrigues Medeiros1; Valdiza Valente2; Roger Rohloff3; Valeria Saraceni2; Sharon Marie Carney1; Marilda Mendonça Siqueira1. Influenza A(H1N1)pdm09 infections among pregnant and non-pregnant women from Rio de Janeiro, 2009-2011 1Respiratory Viruses Laboratory, the Brazilian National Referece Laboratory for Influenza and National Influenza Centre/WHO, Oswaldo Cruz Foundation, Fiocruz, RJ, Brazil; 2Epidemiological Surveillance team, Health Secretariat from Rio de Janeiro, Brazil; 3Municipal Hospital Fernando Magalhães, RJ, Brazil Influenza infections are a major public health issue, given their relevant morbid- mortality patterns and economical impact. Some exposure categories are under a higher risk for further complications, severe clinical presentations and death, such as pregnant women. However, this scenario is still poorly explored in Brazil. In this investigation, we assessed the clinical and epidemiological features of Influenza A(H1N1)pdm09 infections among women at reproducing age from Rio de Janeiro city, from june/2009 to june/2011. This is a descriptive cross-sectional study conducted among 845 women at reproductive age (15-44 years) from Rio de Janeiro, during June/2009-june/2011. Demographic, clinical and epidemiological information were assessed by the Epidemiological Surveillance team/RJ, using a nationally standardized questionnaire. Suspected cases were defined according to MoH and WHO. Nasopharyngeal swabs or aspirates were collected after the interview. After RNA extraction (QIAmp viral RNA kit, QIAGEN), Influenza A detection was carried out using real time RT-PCR (CDC protocol). Descriptive and Contingency table statistics (chi-square/Fisher’s exact test and t-test for means) were employed to assess putative associations between variables. Multiple logistic regressions were carried out, considering variables of epidemiological relevance and plausibility, significant at the level of p < 0.05 in

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bivariate analyses. Influenza A(H1N1)pdm09 was detected in 43.1% (n=326; 185 pregnant and 141 non-pregnant women). Among the first, 173 (95.6%) has information about gestational trimester: 42.2% were at the 3rd, followed 36.4% at the 2nd and 21.4% at the 1st. Pregnant women presented significantly higher frequencies of dyspnea (65.9% vs. 52.4%, p=.006), SARI (56.9% vs. 45.7%, p=.030) and hospitalization (82.7% vs. 47.4%,p<.001), suggesting a more severe clinical presentation and a higher demand on health services (OR=5.3; 95%CI 3.1-8.8), when compared to their counterparts. Infected pregnant were younger (p<.001) and presented lower education (p<.001) and per capita income (BRL, p<.001). An epidemic peak was observed at epidemiological week 30/2009 after which cases declined significantly, with only 1 confirmed case in 2010 (non-pregnant woman). Among pregnant, progression to death (AOR=3.7; 95%CI 1.1-12.3); tabagism (AOR=3.1; 95%CI 1.2-7.6), fever (AOR=2.2; 95%CI1.1-4.4) and older age (0.96; 95%CI 0.93-0.99) were independently associated with Flu A(H1N1)pdm09 infection. Our results corroborate previous studies on the impact of Influenza infections during pregnancy, highlighting the benefits of vaccination within this population. This is pivotal information to evaluate/tailor/improve health policies directed to prevention and control of Influenza infections among pregnancy, not only in Rio de Janeiro (RJ), but in the scope of the National Program for Influenza Surveillance. Financial support:Science and Technology Department (DECIT), Ministry of Health and National Counsel of Technological and Scientific Development (CNPq), Brazil

Victoria Pando-Robles, PhD Researcher, Instituto Nacional de Salud, Mexico victoria.pando@insp.mx Respiratory pathogens detection in samples collected during pandemic outbreak of respiratory illness associated to influenza A (H1N1 pdm), by RT-PCR and Mass-tag Multiplex PCR Victoria Pando-Robles1, Ian W. Lipkin2, Rafal Tokarz2, Pablo Cruz1, Victor Bermudez1, Rosa E. Gomez1, Cesar Fuentes1, Rosa M. Medina3, Lourdes García-García1. 1Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública.2Center for Infection and Immunity, Mailman School of Public Health, Columbia University, NY-USA. 3Laboratorio Estatal. Morelos. México. Background: Acute respiratory infections are a leading cause of morbidity and mortality worldwide. However, in approximately 50% of cases, the etiologic agent is not identified. During the outbreak of pandemic influenza A (H1N1) 2009, we investigated other respiratory pathogens associated with influenza-like illnesses (ILIs). Methods: From May to December 2009, respiratory samples from patients with ILIs were submitted to the Public Health Laboratory of Morelos State-Mexico. 1042 samples were evaluated for the presence of H1N1pdm by real time PCR, and then 562 positive and negative samples were selected using random stratified test to analyze 22 respiratory pathogens, including influenza B (Flu B); parainfluenza 1, 2, 3, and 4 (HPIV); metapneumovirus (MPV), enterovirus (EV), coronavirus(OC-43), respiratory syncitial virus A and B (RSV), adenovirus (Ad), bordetella pertussis (BPER), chlamydia pneumoniae (CPNE), haemophilus influenzae (HINF), legionella pneumoniae (LPNE), moraxella catarrhalis (MCAT), mycoplasma pneumoniae (MPNE), mycobacterium tuberculosis (MTUB), nisseria meningitidis (NMEN), pneumocystis jirovecii (PJIR), and streptococcus pneumoniae (SPNE) by MassTag Multiplex PCR. Results: We detected 513 (49%) samples positives to influenza H1N1 pdm, 53 (5%) positives to Influenza

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A, and 476 (46%) negatives to influenza by RT-PCR. In the 562 selected samples, the most frequently detected pathogens were: enterovirus 16.2%; haemophilus influenza 15.5%, streptococcus pneumonia 13.5%, and pneumocystis jiroveci 5.87%. Co-infections were detected in 19.6% of samples. In 56.9% of the 562 samples, only viruses were identified, and in 0.4%, only bacteria. 9.3% (24/257) of samples negative for H1N1 pdm were identified with bacterial organisms susceptible to antibiotic treatment. Conclusion: The rapid detection of multiple pathogens by Mass-tag Multiplex PCR allows to improve the epidemiology of infectious diseases, affect patient managements decisions and lead to improved patient outcomes, more appropriate use of antivirals and antibiotics, and/or more cost-effective delivery of care. Isaias Raw MD, PhD Pres.Countil of R&D, Butantan, Brazil iraw@butantan.gov.br Kobrusly F et al. Development in influenza production Butantan is the sole influenza vaccine producer in LA. The factory is designed to supply 20 million doses for >60 years. After the A/H1N1 Pandemic, we had to provide about 100 million doses, that include children, young adults, > 60 years and heath care personnel. This was achieved with the development of an adjuvant-containing B. pertussis MPLA that decreased the amount of vaccine four-fold/dose (Vaccine 28:2505, 2009) and the use of whole virus with an increase of doses per egg by 2-5 fold, depending on the serotype (Plos one 2010) and the production of Northern vaccine half of the year. We tested in 2004 a suspension of vitamin A, to supply months the recommended dose. Recent results showed that vitamins A, D, and E shortage are necessary for T-cell maturation, indicating that vitamin shortage may affect influenza immunization. A combination vitamin A, D, E, and MPLA is being tested in mice and later in children. Yeny Tinoco, DVM Research Scientist, NAMRU-6, Peru yeny.tinoco@med.navy.mil Tinoco Y(1,5), Razuri H(1), Romero C(1), Ghersi B(1), Guezala M(1), Silva M(1), Halsey E(1), Laguna-Torres A(1), Gomez J(2), Widdowson MA(3), Azizz-Baumgartner E(3), Uyeki TM(3), Gonzalez A(4), Gilman RH(5), Montgomery JM(7), Bausch GD(1,6). A multidisciplinary approach for the study of influenza epidemiology in Peru (1)U.S. Naval Medical Research Unit 6, Lima, Peru. (2)General Directorate of Epidemiology, Ministry of Health, Lima, Peru. (3)Influenza Division, Centers for Disease Control and Prevention, Atlanta, USA. (4)San Marcos University, Veterinary School, Lima, Peru. Influenza causes approximately 28,000 to 111,500 annual deaths globally, most of them in developing countries. Until now, very little was known about the burden of influenza among persons living in different climatic regions and in urban/rural settings in developing countries of Latin America. Measures of prevention and control of influenza outbreaks require the understanding of the epidemiology and ecology of the disease in both human and animals. Therefore, research with a multidisciplinary approach (medicine, veterinary, biology) is required. We at the US Naval Medical Research Unit Six’s Emerging

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Infectious Department have developed a multidisciplinary research program integrating different stakeholders and institutions comprising the human and animal health sectors. This presentation will provide an overview of the interactions among different studies combining diverse approaches: an active community-based household surveillance in four ecologically distinct regions of Peru, surveillance in live bird markets, surveillance of wild birds in the Peruvian coast, and swine surveillance in both the community and slaughterhouses. In addition, last year, we implemented a prospective surveillance of people highly exposed to zoonotic influenza in order to understand better the human-animal interface. Results of these studies will contribute to the understanding of the dynamics of influenza virus transmission and help designing effective preventive and control measures. Bibliographic References: 1. Tinoco Y., Razuri, H., Ortiz E., Romero C., Morales M., Breña J, Estela A., Reaves E., Halsey E., Laguna-Torres A., Gomez J., Widdowson MA., Azizz-Baumgartner E., Uyeki T., Gilman R., Bausch G.D., Montgomery JM. Active Household-Based Surveillance and Regional Variation in Incidence of Influenza in Peru. Abstract submitted to the 62th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, November, 2012. 2. Razuri, H., Romero C., Ortiz E., Estela A., Morales M., Breña J, Guevara C., Williams M., Gomez J., Reaves E., Uyeki T., Widdowson MA., Bausch D., Montgomery J.M. Mild and Asymptomatic Transmission of Influenza Virus A in Peru. Abstract submitted to the 62th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, November, 2012. 3. Tinoco Y, Montgomery JM, Gilman RH, Razuri H, Guezala MC, Ghersi B, Segovia K, Ayvar V4 Barnes J, Bausch DG, Gonzalez AE. Cross-species transmission of pandemic influenza (H1N1pdm09) virus in backyard pig farms in semirural communities in Tumbes, Peru. Oral presentation at the XIV International Symposium on Respiratory Viral Infections. Istanbul. Turkey. 23 - 26 March 2012. 4. Razuri, H., Romero C., Tinoco Y., Guezala MC., Ortiz E., Silva M., Reaves E., Williams M., Laguna-Torres A., Halsey E., Gomez J., Azizz-Baumgartner E., Widdowson MA., Bresee J., Moen A., Uyeki T., Bennett A., Montgomery J.M, Bausch D. Population based active surveillance cohort studies for influenza: lessons from Peru. Bull World Health Organ 2012;90:318-320. 5. Ghersi BM, Sovero MM, Icochea E, Gonzalez RI, Blazes DL, Gonzalez AE, Montgomery JM. Isolation of low-pathogenic H7N3 avian influenza from wild birds in Peru. J Wildl Dis. 2011 Jul;47(3):792-5. 6. Ortiz EJ., Razuri H., Tinoco Y., Romero C., Estela A., Morales M., Breña J., Gonzaga V., Guezala M, Kochel T., Laguna-Torres A., Uyeki T., Widdowson MA.,Gomez J., Gilman R., Montgomery JM. Economic impact of influenza pH1N1 in Peru. Results from a population based study. Poster presentation at the XII International Symposium on Respiratory Viral Infections, Chinese Taipei, March 2010. 7. Tinoco Y, Razuri H, Ortiz E.J., Gomez J, Widdowson M.A, Uyeki T, Laguna-Torres V.A, Kochel T.J, Gilman R.H and Montgomery J.M. for the Peru Influenza Working Group. 2009. Preliminary population-based epidemiological and clinical data on pandemic H1N1 influenza (pH1N1) from Lima, Peru. Influenza Other Respi Viruses, 3(6): 253-6. Epub 2009 Oct 22. 8. Ghersi B, Blazes D, Icochea E, Gonzalez R, Kochel T, Tinoco Y, Sovero M, Lindstrom S, Shu B, Klimov A, Gonzalez A, Montgomery J. 2009. Avian Influenza in wild birds from the central coast of Peru. Emerg Infect Dis, 2009. 15(6): 935-8

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Elsa Baumeister, PhD Director Argentina National Influenza Centre, INEI-ANLIS Carlos G Malbran, Argentina ebaumeister@anlis.gov.ar M Russo, A Pontoriero, A Campos, E Baumeister*. Sequencing and phylogenetic analysis of influenza virus A(H3N2) and (H1N1)pdm09 viruses in Argentina, 2010-2011. Argentina National Influenza Centre PAHO/WHO, Servicio de Virosis Respiratorias, Instituto Nacional de Enfermedades Infecciosas, ANLIS Carlos G. Malbrán. Introduction: Genetic analysis of circulating influenza strains is essential for influenza surveillance, not only by contributing to a better understanding of molecular epidemiology and evolutionary relations of influenza virus circulation, but also predicting virus evolution for influenza vaccine optimisation. Objective: To monitor the genetic drift in influenza A viruses circulating in Argentina during the period 2010-2011 by sequencing the genes coding for the surface glycoproteins of representative strains. Materials and Methods: Nucleotide sequences of partial HA (H3N2: 986 nt and H1N1: 775nt) and NA (H3N2: 1052 nt and H1N1: 525 nt) amplicons were obtained from clinical specimen and isolates of influenza A(H3N2) and A(H1N1)pdm09 viruses after RT-PCR (H3N2) and RT-nested PCR (H1N1)pdm09 amplification. In total, 40 HA segments (9 from 2010 and 31 from 2011) and 32 NA segments (8 from 2010 and 24 from 2011) for influenza A(H3N2) viruses and 22 HA segments (1 from 2010 and 21 from 2011) and 11 NA segments (1 from 2010 and 10 from 2011) for influenza A(H1N1)pdm viruses were studied. Sequences were analyzed using the BioEdit program and the phylogenetic trees were constructed using Mega program (version 5) by the Neighbour-Joining algorithm. Results: The phylogenetic tree based on the nucleotide sequence of influenza A(H3N2) HA gene revealed that the majority of the Argentinean strains studied fell within the clade represented by A/Victoria/208/09 virus, only one strain grouped in the clade represented by A/Perth/16/2009 strain. Tree based on NA sequences showed a similar pattern to that of HA strains with the exception of 9 strains isolated in 2011 that presented an additional change S315R. Influenza A(H1N1)pdm09 phylogenetic analysis showed that the Argentinean viruses studied have 6 substitutions with respect to the vaccine strain A/California/7/2009 H1N1pdm: S203T, T209K, R223Q, I321V, S185T and D97N. In this case, the topology of the phylogenetic tree based on the NA sequences is different from the HA tree. Most sequenced strains from Argentina are grouped together in one clade sharing an amino acid change at position I188F. Conclusion: Although both subtypes of circulating influenza viruses isolated in 2010 and 2011 showed genetic variation, antigenic studies performed by the WHO CCs show that both subtypes remained similar to their corresponding vaccine components.

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José Correa-Basurto, PhD Dr/Research, ESM-IPN, Mexico corrjose@gmail.com Tolentino-Lopez LE1, Aldo Segura-Cabrera2, Reyes-Loyola Paola1, Mirko Zimic3, Quiliano Miguel3, Verónica Briz4, Ma Angeles Muñoz-Fernández4, Ilizaliturri-Flores I,1 Correa-Basurto J*1 . Outside-binding site mutations modify the active site shapes from neuraminidase of influenza A H1N1. 1Laboratorio de Modelado Molecular y Bioinformática, Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, 11340, México City, México. corrjose@gmail.com 2Laboratorio After the influenza A H1N1 pandemic occurred recently in Mexico and around the world, the number of clinical cases resistance to oseltamivir (Tamiflu) is greater than the limited number of neuraminidase (NA) mutations (H275Y, N294S and I223R) located at the active site. In this work, we have focused on studying a set of NAs with the most representative mutations located outside of the active site and one NA that contains a H275Y mutation. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as wild-type structure. After selecting the target NA sequences, their three-dimensional (3-D) structure was built using 3NSS as template. Then, each built 3-D NA structure was refined by molecular dynamics (MD) simulations during 50 ns. The refined models were used to perform a docking study using oseltamivir as a ligand. The analysis of results from the all MD simulations showed that NA models have reached convergence during the first 10 ns. However, the visual inspection showed that the NA active sites have different shape. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The docking and MM-PBSA results from the complexes showed different binding poses and free energy values. These results suggest that the mutations located outside of the active site of NA could be considered as a new source of resistance to oseltamivir, which would agree with reports in the clinical literature. Juan Cristina, PhD Professor, Facultad de Ciencias, Uruguay juan.cristinag@gmail.com Juan Cristina, Natalia Goñi, Gonzalo Moratorio, Viviana Ramas, Leticia Coppola and Hector Chiparelli. Phylogenetic analysis of pandemic 2009 influenza A virus circulating in the South American region: genetic relations and vaccine strain match. Archives of Virology (2011) 156, 87-94. DOI 10.1007/ s00705-010-0825-7 Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República and Centro Nacional de Referencia de Influenza, MSP, Uruguay The first influenza pandemic of this century was declared in April of 2009, with the emergence of a novel H1N1 influenza A virus strain (H1N1pdm). Understanding the evolution of H1N1pdm strains within the South American region is essential for studying global diversification, emergence, and resistance, as well as determining vaccine efficacy. In order to gain insight into these matters, phylogenetic analysis was performed using 29 hemagglutinin (HA) gene sequences from H1N1pdm strains isolated in South America. The results of these studies revealed that clade 7 was the dominant H1N1pdm lineage in South America. None of the strains isolated in South America cluster together with the 2010 H1 vaccine strain. Amino acid substitutions P100S, S220T and I338V were found in almost all HAs of South American H1N1pdm strains.

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Paola Cristina Resende, MSc Doctoral Student, FIOCRUZ, Brasil paolabmrj@gmail.com Resende PC (1), Motta FC (1), Souza TML (1), Born PS (1), Oliveira MLA (1), Lima CHA (1), Pinhão A (1), Andrade T (1), Gregianini TS (2), Fernandes SB (3), Oliveira ICD (4), Rosa MCD (5), Siqueira MM (1). Molecular analyses of Influenza A H1N1pdm09 viruses at the hemagglutinin gene in mild, severe and death cases in Brazil from 2009 to 2011 (1) Laboratory of Respiratory and Measles Viruses, Oswaldo Cruz Institute – FIOCRUZ, RJ, Brazil; (2) LACEN-RS, (3) LACEN-SC, (4) LACEN-MG, (5) LACEN-PR Brazil Influenza A is the main cause of tract respiratory infection and leads to significant impact in public health and economic burden [1]. This was highlighted by the emergence of the pandemics in 2009 by H1N1pdm09 virus [2]. New variants of influenza A viruses are constantly emerging due to accumulation of mutations during replication (antigenic drift), which requires annual vaccine reformulation and intense surveillance to detect the emergence of variants resistant to antiviral drugs [3]. The aim of this study was evaluated the drift dynamic in hemagglutinin (HA) gene of H1N1pdm09 viruses comparing mild influenza cases (MIC), severe influenza cases (SIC), and death caused by influenza illness in Brazil from 2009 to 2011. The samples analyzed were 54 nasopharyngeal aspirates collected during the epidemiological weeks 21 from 2009 to 23 from 2011. Seventeen samples were from the Northeast (3 deaths; 9 SICs; and 5 MICs), 14 samples were from the Southeast (10 deaths; 1 SICs; and 3 MICs), and 23 samples were from the South (7 deaths; 4 SICs; and 12 MICs). The median age of these individuals was 28 years-old (range: 0-72 years-old), and 61% of patients were female. The phylogenetic analysis demonstrated a close correlation between influenza circulating samples and the vaccine prototype A/CA/7/2009. Recently, attention has been directed to explain the severity of influenza illness, especially in patients at higher risk for complications by the influenza infection. In our sequences, we found mainly at the 222 residue of HA gene an important detection of polymorphisms especially in death and SIC. The HA residue 222 is located within the receptor binding domain and changes (D222G) in this residue can alter viral tropism of sialic acids with &#945;2,3-linkage to &#945;2,6-linkage [4,5,6]. This change has been associated with disease severity [7,8,9]. We observed that 44 (9 deaths; 13 SICs; and 20 MICs) viruses present the amino acid D222; however the other 12 (11 deaths and 1 SICs) viruses from nasopharyngeal aspirates showed polymorphisms in this potential virulence marker. Two samples presented the G222 in 100% of population, however the large majority of these samples showed a mixed population with two (N/G222 in 6 samples or D/G222 in 2 samples) or three (D/G/N222 in 2 samples) different amino acids to this residue. Further analyses need to be conducted; however, our conclusions indicate that viral populations with mixed polymorphisms may have a challenger profile to interpret the biological role of influenza HA in death or SIC. 1. Carrat F, Flahault A (2007) Influenza vaccine: the challenge of antigenic drift. Vaccine 25: 6852-6862. 2. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, et al. (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325: 197-201. 3. Nelson MI, Holmes EC (2007) The evolution of epidemic influenza. Nat Rev Genet 8: 196-205. 4. Tse H, Kao RY, Wu WL, Lim WW, Chen H, et al. (2011) Structural basis and sequence co-evolution analysis of the hemagglutinin protein of pandemic influenza A/H1N1 (2009) virus. Exp Biol Med (Maywood) 236: 915-925. 5. Yang H, Carney P, Stevens J (2010) Structure and Receptor binding properties of a pandemic H1N1 virus hemagglutinin. PLoS Curr 2: RRN1152. 6. Liu Y, Childs RA, Matrosovich T, Wharton S, Palma AS, et al. (2010) Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol 84: 12069-12074. 7. Kilander A, Rykkvin R, Dudman SG, Hungnes O (2010) Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010. Euro Surveill 15.

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8. Chutinimitkul S, Herfst S, Steel J, Lowen AC, Ye J, et al. (2010) Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding. J Virol 84: 11802-11813. 9. World Health Organization (2009) Preliminary review of D222G amino acid substitution in the haemagglutinin of pandemic influenza A (H1N1) 2009 viruses. Wkly Epidemiol Rec 85: 21-22. Marina Escalera Zamudio, M.Sc Student, IBT-UNAM, Mexico marinaescalera@gmail.com Marina Escalera-Zamudio1, Irma López Martinez2, Susana López1, Carlos F. Arias1 y Pavel Isa1*. Evolutionary Dynamics of H3N2 Influenza Viruses in Mexico. 1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelos, México; 2Instituto de Diagnóstico y Referencia Epidemiológicos, México D.F., México, *e-mail: pavel@ibt.unam.mx. The mechanism underlying how H3N2 viruses spread worldwide during each flu season is not yet fully understood. A fundamental question behind such a paradigm is whether the evolution of H3N2 viruses is influenced by the persistence of different viral lineages that change both by genetic shift and genetic drift, or if yearly epidemics originate exclusively from seed strains changing mainly through genetic drift and that are eliminated at the end of each season. In order to address this problem, we analyzed the evolutionary dynamics of twenty-one Mexican influenza A/H3N2 viruses that circulated from 2003 to 2009 on a genomic scale. Our findings point out that the evolution of H3N2 influenza is influenced by the local persistence of distinct lineages and by the occurrence of genetic shift events that influence directly the long-term evolution of these viruses. Our results demonstrate the existence of a novel viral lineage that had not been previously described, the Korea Clade, (KC). We also found evidence of persistence and co-circulation of three different viral lineages within the same human population in the defined geographical region that comprises Mexico and detected genetic shift events between viruses of different human lineages. Bibliographic Citations VII National Virology Congress. Chiapas, Mexico. Oral and poster presentation (26-30 September 2011). Eric Halsey, MD Head, Virology Lab, NAMRU-6, Peru eric.halsey@med.navy.mil Halsey E. NAMRU-6 Passive Respiratory and Febrile Disease Surveillance Network The NAMRU-6 Virology Department oversees a vast respiratory and febrile disease network with over 40 sites in South and Central America. Annually, we examine, interview, and collect a respiratory sample from over 5000 people. Our Lima-based laboratory investigates for the presence of influenza virus in all samples and looks for a wide array of other pathogens on a selective basis. In addition, our febrile surveillance network annually collects samples and data from roughly 3000 people, both in the acute and convalescent periods. These samples are also processed in our Lima-based laboratory, an undertaking that has resulted in the identification of multiple new viruses (five in 2011) as well as the molecular, epidemiological, and clinical characterization of the main causes of febrile disease in the region. Our data has been incorporated into multiple manuscripts (see accompanying list of manuscripts from the past 12

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months). I predict that the burgeoning NIH-NAMRU6 collaboration will result in tangible benefits for both parties. NAMRU-6 provides a unique and extensive surveillance network along with highly-trained laboratory personnel, a large bank of well-preserved samples, researchers familiar with the local epidemiology, and contacts within various ministries throughout Latin America. NIH provides a fresh perspective on data analysis and new skills that may be implemented to enhance the way we report our results. Although there is limited space available for the workshop, I would like to reserve seats for Brett Forshey, myself, and one or two other members of our department (Virology). I foresee that the NAMRU-6 Emerging Infections Department will want to have one or two members attend, as well. In addition, these two departments are willing to provide lectures on various topics such as: 1) Overview of our influenza passive surveillance network, 2) Overview of our five Peruvian influenza cohorts, 3) Phylogenetic findings from our respiratory surveillance network (influenza and/or other respiratory viruses), 4) Epidemiological findings from our respiratory surveillance network (influenza and/or other respiratory viruses). Maria de Lourdes Oliveira Senior Technologist in Public Health, Oswaldo Cruz Institute, National Reference Laboratory for Influenza, Brazil mlaoliveira@fiocruz.br Maria de Lourdes A. Oliveira1, Fernando C. Motta1, Priscila S.Born1, Paola C. Resende1, Thiago M. L. Souza1, Carlos H.Azeredo-Lima1, Milene Mesquita1, Xenia Lemos, Suelen S.Silva, Tânia Andrade, Tatiana Gregianini2, Sandra Fernandes3, Imaculada Oliveira4,. Epidemiological and molecular patterns of Influenza A(H1N1)pdm09 infections in Brazil: findings from the pandemic and post-pandemic periods. 1National Reference Laboratory for Influenza, FIOCRUZ, Rio de Janeiro, Brazil; 2LACEN-RS, 3LACEN-SC; 4LACEN-MG; 5LACEN-PR; Brazil. Influenza infections are an ongoing public health concern, due to the socioeconomic impact associated with morbid-mortality patterns of viral infection. In 2009, a new influenza virus – the product of a triple rearrangement between North-American and Eurasian swine viruses – grounded the first flu pandemic in the 21th century. In this study, epidemiological and molecular pattern of Influenza infections was assessed. Our study comprised 15490 suspected cases with Acute Respiratory Infection (ARI), collected in the South, Southeast (except SP), and Northeast (AL, SE, BA) regions of Brazil, between April/2009 and August/2011. The clinical samples were collected by the Vigilance Surveillance teams and screened by real-time RT-PCR (CDC protocol). FluA(H1N1)pdm09 haemagglutinin 1701pb (N=67) was further sequenced in both directions by automated sequencing. Phylogenetic trees were reconstructed using the Neighbor-Joining method, with bootstrap re-sampling (1000 replicates). Evolutionary distances were computed using the Kimura 2-parameter method. The mean age (±SD) of the studied sample was 26.4 (±19.7) and 56.4% were females. About 40.0% reported any co-morbidity such as cardio-respiratory/renal/hematologic diseases or pregnancy and 66.0% of subjects reported hospitalization. The total prevalence of FluA(H1N1)pdm09, seasonal Flu A and B infections were respectively 37.5%, 12.1% and 0.4%. Different temporal and geographical patterns of viral circulation were observed. Resembling other countries in the Americas, after 2009 the incidence of FluA(H1N1)pdm09 infections decreased in most regions - especially in the Northeast and Southeast states – followed by alternating periods of prevailing A/H3 or Flu B infections. Under the molecular perspective, although nucleotide/amino acid substitutions were identified, none relevant antigenic mutation was found among FluA(H1N1)pdm09

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sequences. The high incidence of FluA(H1N1)pdm09 infections during the influenza pandemics, associated with massive vaccination – especially among high-risk groups – seem to have contributed to this declining scenario. Moreover, our findings suggest that, in the absence of major antigenic changes, FluA(H1N1)pdm09 tends to circulate much like the other seasonal viruses, after the pandemic period. Financial support: Science and Technology Department (DECIT), Ministry of Health and National Counsel of Technological and Scientific Development (CNPq), Brazil Bibliographic Citations Information has been presented at the XIV International Symposium on Respiratory Viral Infections, Turkey, 2012.