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 × 106 TCID50/mL, titre 1.5 × 106 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.

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

OBJETIVOS: Identificar e caracterizar os vírus respiratórios que acometeram crianças de creche que apresentavam sintomas de infecção respiratória e avaliar a associação dos dados clínicos e epidemiológicos da doença com os vírus identificados. MÉTODOS: O estudo foi realizado entre os anos de 2003 e 2005 com 176 crianças que apresentavam sintomas de infecção respiratória e frequentavam uma creche municipal. Amostras de secreção nasofaríngea foram testadas por meio da técnica de reação em cadeia da polimerase com transcrição reversa e as amostras positivas para picornavírus foram sequenciadas. RESULTADOS: Todas as 782 amostras coletadas foram analisadas, e 31,8% foram positivas para algum dos vírus respiratórios estudados. As infecções respiratórias foram caracterizadas pela presença de sintomas moderados do trato respiratório superior, sendo os mais comuns coriza e tosse. Em 2 anos de estudo, a maioria dos casos de infecção ocorreram no outono e no inverno, mas vírus respiratórios foram detectados ao longo de todo o período de estudo. CONCLUSÕES: Vírus respiratórios e as infecções respiratórias causadas por eles fazem parte do cotidiano de crianças que frequentam creches. Nossos resultados mostram o grande impacto gerado pelas infecções respiratórias nessas crianças e sugere que uma maior atenção deve ser dispensada aos patógenos virais.

OBJECTIVES: To identify and characterize respiratory viruses that infect children from daycare centers with symptoms of respiratory infection and to evaluate the association of clinical and epidemiological disease data with the identified virus. METHODS: We conducted a study between 2003 and 2005 in 176 children with respiratory infection symptoms attending a municipal daycare center. Samples from nasopharyngeal secretion were tested by reverse transcriptase polymerase chain reaction and positive samples for picornavirus were sequenced. RESULTS: All 782 collected samples were analyzed and 31.8% were positive for at least one of the studied respiratory viruses. Respiratory infections were characterized by the presence of mild symptoms of the upper respiratory tract, the most common of which were runny nose and cough. In the 2 years of study, most cases of infection occurred in autumn and winter, but respiratory viruses were detected throughout all the study period. CONCLUSIONS: Respiratory viruses and respiratory infections caused by them are part of the daily life of children attending daycare centers. Our results show the great impact that respiratory infections have on these children and suggest that more attention must be paid to viral pathogens.

OBJETIVO: Determinar a prevalência, distribuição etária, sazonalidade, características clínicas da doença Lyme-símile em menores de 15 anos. MÉTODOS: De julho/1998 a dezembro/2000 foi conduzido um estudo transversal em 333 pacientes, com exantema e febre. Foram coletadas amostras pareadas de sangue para a identificação de patógenos. Somente em 193 amostras, negativas aos outros patógenos (Parvovirus B19, Herpesvírus 6 humano, Sarampo, Rubéola, Dengue, Escarlatina e Enterovírus), foram realizadas a pesquisa da borreliose pelos métodos de Enzyme-Linked Immunosorbent Assay e Western-blotting. Outras variáveis clínicas, socioeconômicas, demográficas e climáticas foram estudadas. RESULTADOS: A prevalência da doença foi de 6,2%(12/193). Das variáveis estudadas, houve predomínio em <6anos(83,2%); sexo feminino (66,7%); procedência da cidade de Franco da Rocha (58,3%); com sazonalidade no outono-verão. O intervalo de atendimento foi de quatro dias. Sinais e sintomas com significância estatística: prurido, ausência da fissura labial e bom estado clínico. Outros dados presentes foram: irritabilidade (80%); febre (?38ºC) (58,3%) com duração de um a três dias. O exantema foi do tipo máculo-papular (33,3%), urticariforme (25%) e escarlatiniforme (16,7%); predominando em tronco (60%). Não houve apresentação clínica característica para diagnóstico da doença de Lyme-símile nestes pacientes. A sensibilidade e especificidade para o diagnóstico clínico contraposta com o diagnóstico laboratorial foi zero. O acompanhamento de 10 casos durante dois anos não evidenciou complicações cardiológicas ou neurológicas. Este é o primeiro estudo desta doença em crianças brasileiras. CONCLUSÃO: A prevalência da doença Lyme-símile foi baixa, não tendo sido lembrada no diagnóstico inicial dos exantemas, mas seu conhecimento é necessário, necessitando maior atenção médica.

BACKGROUND: To determine the prevalence, age distribution, seasonality and clinical characteristics of Lyme-simile disease in Brazilians less than 15 years of age. METHODS. From July, 1998 to November, 2000, a cross-sectional study was conducted in 333 patients with skin rash and fever. Paired blood samples were collected for identification of the pathogens. Only 193 samples which were negative for other pathogens (Parvovirus B19 Human, Herpesvirus 6 Human, Measles, Rubella, Dengue, Scarlet fever and Enterovirus), were tested for borreliosis by Enzyme-Linked Immunosorbent Assay and Western-blotting. Other clinical, socioeconomic, demographic and climatic variables were studied. RESULTS: Prevalence of the disease was 6.2%(12/193). Of the variables studied, there was predominance in: <6 years old (83.2%); females (66.7%); being from the city of Franco da Rocha (58.3 %); and a summer/fall seasonality. The duration of care was 4 days. Signs and symptoms with statistical significance were itching; absence of lip notch and ocular pain; irritability and good clinical condition. Other clinical data presented were: pruritus (90%), irritability (80%) and fever (?38ºC) (58.3%) with a duration of 1 to 3 days. Erythema was maculo-papular (40%), urticaria-like (25%) and scarlatiniform (16.7%), occurring predominately on the trunk (60%). There were no primary clinical evidences of Lyme-simile disease in the patients under study. The sensitivity and specificity of the clinical diagnosis as opposed to the laboratory diagnosis was zero. There was no initial clinical suspicion of the disease in the 10 cases studied and followed up for two years that showed no evidence of cardiologic or neurological complications. This is the first study of Lyme-simile in Brazilian children. CONCLUSION: Prevalence of Lyme-simile disease was low, and it was not remembered at the initial diagnosis of those with skin rash. However, practical knowledge is necessary, demanding increased medical attention.

Aspergillus flavus is a very important toxigenic fungus that produces aflatoxins, a group of extremely toxic substances to man and animals. Toxigenic fungi can grow in feed crops, such as maize, peanuts, and soybeans, being thus of high concern for public health. There are toxigenic and non-toxigenic A. flavus variants, but the necessary conditions for expressing the toxigenic potential are not fully understood. Therefore, we have studied total-DNA polymorphism from toxigenic and non toxigenic A. flavus strains isolated from maize crops and soil at two geographic locations, 300 km apart, in the Southeast region of Brazil. Total DNA from each A. flavus isolate was extracted and subjected to polymerase chain reaction amplification with five randomic primers through the RAPD (random amplified polymorphic DNA) technique. Phenetic and cladistic analyses of the data, based on bootstrap analyses, led us to conclude that RAPD was not suitable to discriminate toxigenic from non toxigenic strains. But the present results support the use of RAPD for strain characterization, especially for preliminary evaluation over extensive collections.

Desenvolveu-se uma metodologia própria ("in-house") baseada em RT-PCR, que permite detectar simultaneamente o RNA do vírus HCV e de um RNA artificial empregado como controle externo. As amostras são analisadas em pools de 6-12 doações, cada doação sendo incluída em dois pools diferentes, um horizontal e um vertical, permitindo a identificação imediata de uma doação reativa, sem a necessidade de desmembrar-se um pool reativo. O processo todo consumiu de 6-8 horas diárias e os resultados foram emitidos em paralelo à sorologia. O método detectou os seis genótipos de HCV, com um limite de sensibilidade de 500 UI/mL (95% hit rate). Até julho de 2005 haviam sido testadas 139.678 doações com a detecção de 315 (0,23%) doações reativas para HCV-RNA. Exceto cinco falso-positivas, todas estas doações também apresentavam o respectivo anticorpo, portanto não se detectou nenhuma doação em janela imunológica. A especificidade foi de 99,83%. A detecção de amostra em janela imunológica, nesta população de doadores, provavelmente demandará a análise de um número maior de doações, espelhando-se na experiência internacional que tem mostrado a detecção de amostras HCV-RNA isoladas em 1:200.000 - 1:500.000 doações.

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

O uso de testes de ácidos nucleicos (NAT) na rotina de triagem de doadores de sangue tornou-se uma realidade ao final da década de 1990. Descreve-se aqui uma metodologia de RT-PCR multiplex "in-house" que permite a detecção simultânea dos RNAs dos vírus HIV e HCV além de uma molécula artificial de RNA usada como controle externo. O método detecta todos os subtipos de HIV do grupo M e também do grupo N e O, com uma sensibilidade de 500 UI/mL. Após validação, este teste substituiu o do antígeno p24, até então na rotina de triagem em nosso laboratório, desde 1996. De julho de 2001 a fevereiro de 2006 foram testadas 102.469 doações e 41 (0.04%) foram NAT reativas. Uma doação NAT isoladamente reativa (anticorpo não-reativa) foi detectada com soroconversão subseqüente do doador, portanto, o rendimento do NAT nesta população até o presente momento é de 1:102.469. Este número contrasta com a experiência obtida internacionalmente, onde taxas de 1:600.000 - 1:3.100.000 foram descritas.

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.