Influenza A(H1N1)pdm09 infection and viral load analysis in patients with different clinical presentations

BACKGROUND Influenza viral load (VL) can be a decisive factor in determining the antiviral efficacy in viral clearance. OBJECTIVE This study aimed to evaluate the rate of infection and the role of influenza VL on the clinical spectrum of illnesses among different patient groups attended at a tertiary hospital in Brazil. METHODS Samples were collected from patients presenting acute respiratory infection from 2009 to 2013. Overall, 2262 samples were analysed and distributed into three groups: (i) asymptomatic (AS); (ii) symptomatic outpatients (OP); and (iii) hospitalised patients (HP). VL (expressed in Log10 RNA copies/mL) was calculated through a quantitative real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) assay aimed at the M gene, with human RNAseP target as internal control and normalising gene of threshold cycle values. FINDINGS A total of 162 (7.16%) H1N1pdm09 positive samples were analysed. Patients aged from 0.08 to 77 years old [median ± standard deviation (SD): 12.5 ± 20.54]. Children with 5 to 11 years old presented the highest detection (p < 0.0001). AS patients had the lowest VL, with a significant difference when compared with symptomatic patients (p = 0.0003). A higher VL was observed within two days of disease onset. Ten patients (HP group) received antiviral treatment and were followed up and presented a mean initial VL of 6.64 ± 1.82. A complete viral clearance for 50% of these patients was reached after 12 days of treatment. MAIN CONCLUSIONS It is important to evaluate AS patients as potential spreaders, as viral shedding was still present, even at lower VL. Our results suggest that patients with underlying diseases and severe clinical symptoms may be considered for prolonged viral treatment.

Genotyping Influenza Virus by Next-Generation Deep Sequencing in Clinical Specimens

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

Dinâmica molecular dos vírus Influenza A (H1N1) pandêmico em cinco anos de circulação no Brasil / Molecular dynamic of influenza a (h1n1) pandemic viruses five years of circulation in Brazil

A primeira detecção do vírus Influenza A (H1N1)pdm09 no Brasil aconteceu em maio de2009, e foi seguida de uma extensa disseminação por toda a população brasileira, com grandeimpacto em morbidade e mortalidade. Para entender a dinâmica molecular do Influenza A(H1N1)pdm09 no país, a presente tese reuniu sete trabalhos que abordaram a análise filogenéticadeste agente viral durante e após o período pandêmico (2009 a 2014) e buscou indentificarpolimorfismos virais associados à virulência e à resistência ao antiviral Oseltamivir (OST). Para isso,as metodologias realizadas foram o sequenciamento dos genes de hemaglutinina (HA) eneuraminidase (NA) utilizando a metodologia de Sanger e a metodologia de pirosequenciamento paradetectar polimorfismos de base única (SNPs).Nossos resultados revelaram a circulação de nove grupos filogenéticos ao longo dos cincoanos do estudo, indicando uma substituição temporal dos grupos e ocasionalmente umaestratificação geográfica. No entanto, nenhum dos grupos filogenéticos identificados foramassociados com um pior prognóstico da infecção por influenza. Ao contrário do que foi observado emestudos anteriores, as mutações K-15E e Q310H no gene HA não se associaram ao aumento devirulência, mesmo na infecção de indivíduos imunocomprometidos. Por outro lado, polimorfismos noresíduo 222 da HA, que caracterizaram a presença de quasispecies virais, mostraram uma forteassociação com a gravidade da infecção, especialmente em gestantes. Nesta tese, tambémrealizamos a vigilância de marcadores de resistência no gene NA. Entre as amostras analisadasencontramos sete vírus com a mutação H275Y e dois com S247N, esses marcadores estãorelacionados com a diminuição de sensibilidade ao antiviral OST. Entre as amostras resistentes, agrande maioria foi detectada na região Sul do Brasil, em pacientes que não receberam OST. Istosugere uma possível transmissão sustentada do vírus resistentes no país...

The Influenza A (H1N1)pdm09 virus was first detected in May 2009 in Brazil and later resultedin an extensive spread throughout the Brazilian population with a severe impact on morbidity andmortality. To understand the molecular dynamic of (H1N1)pdm09 virus in Brazil this thesis groupedseven papers which approached the phylogenetic reconstruction of the virus during and after thepandemic period (2009 to 2014) and the genomic identification of viral polymorphisms associated withvirulence or antiviral resistance to Oseltamivir (OST). For this, we performed genome sequencing,focusing especially on the hemagglutinin (HA) and neuraminidase (NA) genes using conventionalSanger sequencing and PyroMark 96ID to detect single nucleotide polymorphisms (SNPs).Our results showed that in Brazil nine (H1N1)pdm09 phylogenetic groups circulated along thefive years of the study, indicating a temporal replacement of groups and ocasionally a geographicstratification. However, no phylogenetic group seemed to be associated with a worse clinical outcome.The increased virulence observed in previous studies with a 2009 group bearing the genetic markersK-15E and Q310H was not confirmed in our analyses, even evaluating an immunocompromisedpopulation. On the other hand, polymorphysms at position 222 of HA gene, which characterized thepresence of viral quasispecies, showed an association with increased virulence in brazilian samples,especially in pregnant women. In this study we also performed surveillance of resistance markers atthe NA gene. From the analysed samples we found seven viruses with H275Y and two with S247Nmutation, that diminish the sensibility to oseltamivir (OST). Among the resistant samples, the largemajority was detected in the Southern region of Brazil in patients that did not receive OST. Thissuggests a possible sustained transmission of resistant virus in the country...

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Clinical profile of pneumonia and its association with rain wetting in patients admitted at a tertiary care institute during pandemic of influenza A (H1N1) pdm09 virus infection.

Background. Influenza pneumonia often occurs as epidemics in the Asian countries and have significant impact on the health of world population. Methods. We studied the association of rain-wetting with occurrence of pneumonia during the outbreak of the influenza A (H1N1) pdm09 virus infection. All patients admitted with community-acquired pneumonia during the period 13th September to 10th October 2010 were recruited in the present study. The diagnosis of influenza was established by realtime polymerase chain reaction (RT-PCR). The demographic data and clinical profile of the patients were recorded with a special consideration to record of possible risk factors. Results. Of the 123 patients studied, 39 (32%) patients had tested positive for influenza A (H1N1) pdm09; 12 (10%) tested positive for influenza A and remaining 72 (58%) patients were negative for influenza virus. Pattern of illness was almost identical in H1N1-positive and-negative groups. History of rain-wetting was present in 48 patients (39%) preceding the onset of illness. Getting wet in the rain was significantly higher in patients with pneumonia than control subjects [odds ratio 2.53, 95% confidence intervals (CI) 1.301 - 4.91; p=0.009)]. The number of pneumonia patients was also higher on rainy days and the numbers started declining a week later. Conclusion. More pneumonia patients are admitted during the periods of greater rainfall and rain-wetting may be an important risk factor for the occurrence of pneumonia.

Genetic analysis of HA gene of pandemic H1N1 2009 influenza viruses circulating in India.

The H1N1 2009 influenza pandemic took the health care workers by surprise in spite of warning about influenza pandemic. Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes by shift or drift. Thus, understanding the evolution of the viruses in human is important for the surveillance and the selection of vaccine strains. A total of 23 pandemic A/H1N1 2009 viral HA gene sequences were downloaded from NCBI submitted during March and May 2010 by NIV and were analysed. Along with that the vaccine strain A/California/07/2009 was also downloaded from NCBI. All the sequences were used to analyse the evolution of the haemagglutinin (HA) by phylogenetic analysis. The HA gene could be divided into four groups with shift from 1 to lV revealing that the HA genes of the influenza A viruses evolved in a sequential way, in comparison to vaccine strain A/California/07/2009. Amino acid sequence analysis of the HA genes of the A/H1N1 2009 isolates, revealed mutations at positions 100, 220 and additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285 .The mutations identified showed the adaptation of the new virus to the host that could lead to genetic changes inherent to the virus resulting in a reassortant which could be catastrophic, hence continuous monitoring of strains is mandatory.

La primera pandemia del siglo XXI: infección por virus de influenza a (H1N1) / The first pandemic of the 21st century: infection with influenza A (H1N1) virus

El 2009 marcó el reinicio de las sempiternas luchas universales contra las enfermedades I infecciosas; el aparecimiento del Virus de Influenza A H1N1 ha desatado una tarea global para su rápido e i eficiente control, minimizando con el esfuerzo común los daños a la población general y las economías locales y regionales. Este artículo es una revisión rápida a s u historia, características microblológicas, comportamiento epidemiológico, estrategias de control y controversias...(AU)

Circulación de los virus influenza durante la primera ola pandémica en Chile / Circulation of influenza virus during the first pandemic wave in Chile

Se caracteriza el comportamiento de la circulación de los virus respiratorios durante la primera ola del virus influenza pandémico A (H1N1) en 2009 en Chile, a partir de datos del sistema de vigilancia centinela en el sector público como a partir de consultas ambulatorias en el sector privado. La influenza A estacional tuvo escasa circulación durante el período estudiado, lo cual permitió plantear que la influenza A sin tipificar podría corresponder mayoritariamente a influenza pandémica A (H1N1). La importancia relativa del virus pandémico mostró un comportamiento que varió con la edad, afectando en forma más marcada a niños escolares entre 5 y 14 años y adultos jóvenes, y en menor proporción a los niños menores de 5 años, entre los cuales predominó como agente etiológico principal el virus respiratorio sincicial.

Circulation of respiratory viruses during the first wave of pandemic influenza virus A (H1N1) in 2009 in Chile isdescribed, from data extracted from the sentinel surveillance system in the public sector and from outpatient clinics from the private sector. Seasonal influenza A had little circulation during the period studied, which allowed us to suggest that the influenza A without classification could be mainly pandemic influenza A (H1N1). The relative importance of the pandemic virus varied with age, affecting more markedly school children between 5 and 14 years and young adults, and to a lesser extent, children under 5 years, among which the predominant primary etiologic agent was respiratory syncytial virus.