RESUMO
BACKGROUND: SARS-CoV-2 infection is associated with myocardial injury, but there is a paucity of experimental platforms for the condition.MethodsâandâResults:Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected by SARS-CoV-2 for 3 days ceased beating and exhibited cytopathogenic changes with reduced viability. Active viral replication was evidenced by an increase in supernatant SARS-CoV-2 and the presence of SARS-CoV-2 nucleocaspid protein within hiPSC-CMs. Expressions of BNP, CXCL1, CXCL2, IL-6, IL-8 and TNF-α were upregulated, while ACE2 was downregulated. CONCLUSIONS: Our hiPSC-CM-based in-vitro SARS-CoV-2 myocarditis model recapitulated the cytopathogenic effects and cytokine/chemokine response. It could be exploited as a drug screening platform.
Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/complicações , Células-Tronco Pluripotentes Induzidas/virologia , Miocardite/complicações , Miócitos Cardíacos/virologia , Pneumonia Viral/complicações , Enzima de Conversão de Angiotensina 2 , Betacoronavirus/genética , COVID-19 , Sobrevivência Celular , Células Cultivadas , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus , Citocinas/metabolismo , Efeito Citopatogênico Viral , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocardite/metabolismo , Miocardite/virologia , Miócitos Cardíacos/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Fosfoproteínas , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Replicação ViralRESUMO
To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.
Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , COVID-19 , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , SARS-CoV-2 , Carga ViralRESUMO
IMPORTANCE: We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.