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MISMS Latin America Influenza Research Workshop
Lima, Peru • June 25-28, 2012
Abstracts
Epidemiology and Modeling
Ana E Arango, MSc
Associated investigator, Universidad de Antioquia, Colombia
[email protected]
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
[email protected]
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, comorbidities, 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
[email protected]
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, 20082011.
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
[email protected]
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
[email protected]
Comas-García Andreu, Noyola Daniel E, Christen-García J. Andres, Velasco-Hernández Jorge X., YarzaAcuña 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
[email protected]
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
[email protected]
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
[email protected]
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 influenzalike 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
[email protected]
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
[email protected]
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
[email protected]
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 lymphopeniaassociated 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
11
Fernanda Moura, PhD
Professor, Universidade Federal do Ceará, Brasil
[email protected]
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
[email protected]
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
12
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 (chisquare/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
-------------------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
1
Respiratory 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
13
bivariate analyses. Influenza A(H1N1)pdm09 was detected in 43.1% (n=326; 185 pregnant and 141 nonpregnant 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
[email protected]
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
14
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
[email protected]
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
[email protected]
Tinoco Y(1,5), Razuri H(1), Romero C(1), Ghersi B(1), Guezala M(1), Silva M(1), Halsey E(1), LagunaTorres 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
15
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., LagunaTorres 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
16
Phylogenetics and evolution
Elsa Baumeister, PhD
Director Argentina National Influenza Centre, INEI-ANLIS Carlos G Malbran, Argentina
[email protected]
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.
17
José Correa-Basurto, PhD
Dr/Research, ESM-IPN, Mexico
[email protected]
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 . Outsidebinding 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. [email protected] 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
[email protected]
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.
18
Paola Cristina Resende, MSc
Doctoral Student, FIOCRUZ, Brasil
[email protected]
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
[email protected]
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:
[email protected].
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
[email protected]
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
[email protected]
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 postpandemic 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 cardiorespiratory/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.
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