The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

ResumoObjetivo: Detecção de oito vírus respiratórios mais comuns: vírus respiratório sincicial humano (VRSH), vírus influenza tipo A e B (IA e IB), vírus da parainfluenza 1, 2 e 3 (VPIH1, 2 e 3), adenovírus (Ad) e metapneumovírus humano (MPVH), a fim de estabelecer a etiologia das infecções respiratórias agudas (IRA) e a epidemiologia desses vírus em crianças pequenas atendidas no Hospital Universitário da Universidade de São Paulo, em São Paulo, Brasil, durante o ano de 2003. Métodos:A vigilância epidemiológica foi realizada em todas as crianças menores de 5 anos hospitalizadas por causa de doenças do trato respiratório inferior (DTRI) entre 1º de janeiro de 2003 e 20 de dezembro de 2003, no hospital universitário. Amostras coletadas de nasofaringe foram analisadas quanto à presença de vírus respiratórios através da reação em cadeia da polimerase e detectadas pelo programa GeneScan. (55.6%) were positive for at least one of the respiratory viruses studied. Of all the children, HRSV was identified in 24.1%, HMPV in 17.8%, HPIV3 in 8.3%, Ad in 6.8%, IA in 5%, HPIV1 in 0.6%, but no virus could be detected in 44.1%. Dual virus infections were detected in 7.1% of all samples (12.8% of positive samples). HPIV2 and IB were not detected in the present study. Resultados Conclusions:This study confirms that children younger than 5 years and particularly younger than 1 year have a high hospitalization rate due to HRSV, HMPV, HPIV, influenza and adenovirus. We were able to determine the etiology and epidemiology of most ARIs and trace the seasonal profile of the commonest respiratory viruses among young children. Os critérios de inclusão foram os seguintes: todas as crianças menores de 5 anos com DTRI apresentando um ou mais dos seguintes sintomas físicos: dispnéia (taxa de respiração > 50), retrações torácicas, sibilância, crepitação, estridor e cianose; além de alterações pulmonares no raio X (hiperinsuflação, condensação). Os critérios de exclusão foram: doença respiratória crônica (> 7 dias do início da DTRI); e pacientes atendidos no HU entre as 18h de sexta-feira e as 8h de segunda-feira, por razões operacionais. Coleta das amostrasO aspirado nasal foi obtido pela lavagem das narinas com solução fisiológica e pela coleta do mesmo em um frasco esté-ril até no máximo 24 h após o atendimento. Os protocolos e procedimentos para coleta das amostras foram aprovados pelo Comitê de Ética em Pesquisa do ICB-USP. Todas as amostras foram mantidas a 4 ºC e levadas ao laboratório em até 2 h após a coleta, para subseqüente extração. Prevenção de contaminação cruzada (carryover)Para reduzir a oportunidade de contaminação do fragmento amplificado, separamos o pré e os pós-ensaios em três salas diferentes, trocamos de luvas com freqüência, pré distribuímos os reagentes em alíquotas e usamos múltiplos controles em cada lote de amostras testadas. Ponteiras equipadas com filtros de vedação foram usadas para a pipetagem dos reagentes e todas as áreas e equipamentos foram descontaminados com hipoclorito de sódio antes e depois da pipetag...

The use of RT-LAMP (reverse transcriptase—loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 10 6 TCID50/mL, titre 1.5 × 10 6 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

Background: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) collection began in two Brazilian hospitals for treatment of severe/ critical patients. Methods and Materials: Mild/moderate COVID-19 convalescents were selected as CCP donors after reverse transcription polymerase chain reaction (RT-PCR) confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and absence of symptoms for ≥14 days plus (a) age (18-60 years), body weight greater than 55 kg; (b) immunohematological studies; (c) no infectious markers of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T-lymphotropic virus-1/2, Chagas and syphilis infection; (d) no HLA antibodies (multiparous); (e) second RT-PCR (nasopharyngeal swab and/or blood) negativity; (f) virus neutralization test (cytopathic effect-based virus neutralization test neutralizing antibody) and anti-nucleocapsid protein SARS-CoV-2 IgM, IgG, and IgA enzyme-linked immunosorbent assays. Results: Among 271 donors (41 females, 230 males), 250 presented with neutralizing antibodies. Final RT-PCR was negative on swab (77.0%) or blood (88.4%; P = .46). Final definition of RT-PCR was only defined at more than 28 days after full recovery in 59 of 174 (33.9%) RT-PCR-ve, and 25/69 RT-PCR +ve (36.2%;