Diagnosis and clinic-pathological findings of influenza virus infection in Brazilian pigs / Diagnóstico, achados clínicos e patológicos da infecção pelo vírus influenza em suínos no Brasil

Influenza A virus (IAV) is a respiratory pathogen of pigs and is associated with the porcine respiratory disease complex (PRDC), along with other respiratory infectious agents. The aim of this study was to diagnose and to perform a clinic-pathological characterization of influenza virus infection in Brazilian pigs. Lung samples from 86 pigs in 37 farrow-to-finish and two farrow-to-feeder operations located in the States of Minas Gerais, São Paulo, Paraná, Rio Grande do Sul, Santa Catarina, and Mato Grosso were studied. Virus detection was performed by virus isolation and quantitative real time reverse-transcription PCR (qRT-PCR). Pathologic examination and immunohistochemistry (IHC) were performed in 60 lung formalin-fixed paraffin-embedded tissue fragments. Affected animals showed coughing, sneezing, nasal discharge, hyperthermia, inactivity, apathy, anorexia, weight loss and growth delay, which lasted for five to 10 days. Influenza virus was isolated from 31 (36.0%) lung samples and 36 (41.9%) were positive for qRT-PCR. Thirty-eight (63.3%) lung samples were positive by IHC and the most frequent microscopic lesion observed was inflammatory infiltrate in the alveoli, bronchiole, or bronchi wall or lumen (76.7%). These results indicate that influenza virus is circulating and causing disease in pigs in several Brazilian states.

O vírus influenza A (IAV) é um patógeno respiratório comum de suínos e faz parte do complexo de doenças respiratórias do suíno (PRDC) junto com outros agentes infecciosos. O objetivo deste estudo foi diagnosticar e realizar a caracterização clínica e patológica de casos/surtos de influenza em suínos brasileiros. Foram utilizadas amostras de tecido pulmonar de 86 suínos de 37 granjas de ciclo completo e duas unidades produtoras de leitões localizadas em Minas Gerais, São Paulo, Paraná, Rio Grande do Sul, Santa Catarina e Mato Grosso. A detecção viral em fragmentos pulmonares frescos foi realizada através do isolamento viral e da transcrição reversa-PCR em tempo real quantitativa (qRT-PCR). Exame patológico e imuno-histoquímica (IHQ) foram realizados em 60 amostras de pulmão fixadas em formalina 10% e embebidas em parafina. As amostras eram de animais apresentando tosse, espirros, secreção nasal, hipertermia, prostração, apatia, anorexia, perda de peso e ganho de peso reduzido, com duração entre cinco e 10 dias. O vírus influenza foi isolado de 31 (36,0%) amostras e 36 (41,9%) foram positivas na qRT-PCR. Na IHQ, 38 (63,3%) amostras foram positivas e a lesão mais frequentemente observada foi a presença de infiltrado inflamatório na parede e lúmen de vias aéreas (76,7%). Estes resultados indicam que o vírus influenza está circulando e causando lesões e doença respiratória em suínos de diversos Estados do Brasil.

Orientações para o diagnóstico de influenza em suínos / Guidelines for diagnosis of swine influenza

Este trabalho descreve a colheita adequada de amostras, as técnicas/procedimentos disponíveis para o diagnóstico de influenza A em suínos, assim como os resultados e suas respectivas interpretações, para auxiliar médicos veterinários de campo na identificação dessa doença. Em suínos vivos, as amostras adequadas são: secreção nasal, fluido oral e sangue (soro). Para suínos mortos, colher preferencialmente amostras de pulmão com consolidação cranioventral. Secreção nasal e fragmentos de pulmão refrigerado são utilizados para detectar partícula viral viável (isolamento viral - IV) ou ácido nucleico viral (RT-PCR convencional e RT-PCR em tempo real). As amostras não devem ser congeladas, pois o vírus é inativado a -20°C. A caracterização molecular dos isolados é feita pela análise filogenética obtida pelo sequenciamento de DNA. O soro é utilizado para a detecção de anticorpos (Acs) por meio do teste da inibição da hemaglutinação e ELISA. O fluido oral pode ser utilizado para detecção de anticorpo (ELISA) ou de vírus. Fragmentos de pulmão fixados em formol a 10% são examinados microscopicamente para identificar pneumonia broncointersticial e para detecção de antígeno viral pela imuno-histoquímica (IHQ). Para o sucesso do diagnóstico, as amostras devem ser colhidas de suínos que estão preferencialmente na fase aguda da doença, para aumentar as chances de detecção viral. As melhores opções para o diagnóstico de influenza A em suínos vivos são RT-PCR e isolamento viral de amostras de swab nasal ou fluido oral. Pulmão para análise por RT-PCR, isolamento viral ou IHQ é a amostra de escolha em suínos mortos. Testes sorológicos têm valor diagnóstico limitado e são utilizados apenas para determinar o estado imune do rebanho, não indicando doença clínica, pois os Acs são detectados 7-10 dias pós-infecção (fase subaguda). O diagnóstico de influenza é importante para avaliar o envolvimento desse agente no complexo de doença respiratória suína. Além disso, o isolamento do vírus influenza é essencial para o monitoramento dos principais subtipos circulantes em uma determinada região ou país, assim como para a detecção de novos rearranjos virais, já que influenza é considerada uma zoonose.

This article is intended to describe the adequate sample collection, the laboratory procedures/techniques, the expected results and their interpretation for diagnosis of influenza infection in swine, serving as a support for field veterinarians. In live pigs, the samples to be taken are nasal secretions, oral fluids and blood. For dead pigs, preference should be given to samples of cranioventral lung consolidation. Nasal discharge and chilled lung fragments are used for detection of virus (virus isolation - VI) or viral nucleic acids (conventional RT-PCR and real-time RT-PCR). Samples should not be frozen, because the virus is inactivated at -20°C. Molecular characterization of isolates is performed by phylogenetic analysis of gene sequences obtained by DNA sequencing. Serum is used for the detection of antibodies using hemagglutination inhibition (HI) test and ELISA. Oral fluid may be used for either antibody (ELISA) or viral detection. Fragments of lung fixed in 10% formaldehyde are used for histopathological analysis to identify bronchointerstitial pneumonia, and for immunohistochemistry (IHC) for antigens. For a successful diagnosis, sampling should be preferably performed in the acute phase of the disease to improve chances of virus detection. The best options to perform the diagnosis of influenza A in a swine herd are RT-PCR and VI from nasal swabs or oral fluid in live pigs and/or lung tissue for RT-PCR, VI or IHC in dead pigs. Serological tests are of very limited diagnostic value and are useful only to determine the immune status of the herd, not indicating clinical disease, because antibodies are detected after 7-10 days post infection (subacute phase). The diagnosis of influenza is important to evaluate the involvement of this agent in the complex of respiratory diseases in pigs. Furthermore, the isolation of influenza virus is essential for monitoring the main subtypes circulating in a given region or country, as well as for the detection of potential new viral reassortants, because influenza is considered a zoonosis.

Impact of respiratory infections by influenza viruses A and B in pediatrics patients from Federal University of Paraná, Brazil

The objective of the present study was to determine the impact of influenza virus on pediatric hospitalized patients. We retrospectively reviewed records of children with laboratory diagnoses, by cell culture and/or indirect immunofluorescence assay, of influenza virus seen in a period of 6 years. A total of 1,033 samples were analyzed, 45 (4.3 percent) of them being reactive to influenza virus. Thirty-one samples were positive to influenza A virus and 14 to influenza B. The frequency of hospitalization in intensive care and medical emergency was found to be high. Three (8.6 percent) patients died, two of them due to respiratory failure. Low frequency of influenza virus infection was observed in the study. The data suggest the need of more efficient epidemiological surveillance measures in order to obtain reliable information to better assess the impact of the virus on our region and determine the need of preventive measures, such as immunization.

Directigen Flu A + B test versus viral culture and reverse transcription-PCR for diagnosis of influenza virus type A and type B

In our study, 60 sputum specimens were collected from patients suffering from upper and lower respiratory tract manifestations with a mean age of 44.5 years. Out of the 60 patients, 49 patients had developed pneumonia as a complication. In addition to 20 specimens were collected from apparently healthy subjects matching in age with the patient group. All specimens were subjected to Directigen Flu A+B assay, viral culture on Madin Darby canine kidney cell and reverse transcription-PCR [RT-PCR] for demonstration of influenza type A and B. Directigen Flu test had a detection rate of 18.3% for influenza type A and 26.6% for influenza B. Viral culture had a detection rate of 20% and 26.6% for influenza type A and influenza type B respectively. While RT-PCR had a detection rate of 16.6% and 25% for influenza virus type A and B respectively. The sensitivity, specificity and positive and negative predictive value was[66%, 0%, 72.7%, 0% for influenza A virus and 75%, 0%, 75%, 0% for influenza type B by Directigen Flu A + B test. While the resolved sensitivity, specificity and positive and negative predictive value of RT-PCR was 100%, 83.3%, 100%, 60% for influenza A virus and 100%, 93.7%, 100%, 80% for influenza type B. Directigen Flu test is rapid, easy, reliable test that can be used for diagnosis and discrimination between influenza type A and type B infections. But it lacks specificity in some patients, thus, it requires confirmation by tissue culture and RT-PCR to rule out false-positive results