Point-of-care testing for COVID-19: a simple two-step molecular diagnostic development and validation during the SARS-CoV-2 pandemic

• Published in: Memórias do Instituto Oswaldo Cruz 2024, Vol.119, p.e230236
• Main Authors: Yoshikawa, Andre Akira Gonzaga, Cardoso, Sabrina Fernandes, Eslabão, Lívia Budziarek, Pinheiro, Iara Carolini, Valverde, Priscila, Caminha, Gisele, Romero, Oscar Bruna, Medeiros, Leandro, Rona, Luísa Damazio Pitaluga, Pitaluga, André Nóbrega
• Format: Article
• Language: English
• Subjects: Brazil; COVID-19 - diagnosis; COVID-19 Nucleic Acid Testing - methods; COVID-19 Testing - methods; Humans; Molecular Diagnostic Techniques - methods; Nasopharynx - virology; Nucleic Acid Amplification Techniques - methods; Pandemics; PARASITOLOGY; Point-of-Care Testing; Reproducibility of Results; RNA, Viral - analysis; RNA, Viral - isolation & purification; Saliva - virology; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Sensitivity and Specificity; TROPICAL MEDICINE
• ISSN: 0074-0276; 1678-8060; 1678-8060
• DOI: 10.1590/0074-02760230236

During the coronavirus disease 19 (COVID-19) pandemic, diagnostic testing of the general population proved challenging due to limitations of the gold-standard diagnostic procedure using reverse transcription real-time polymerase chain reaction (RT-qPCR) for large-scale testing on the centralised model, especially in low-resource areas. To address this, a point-of-care (PoC) diagnostic protocol for COVID-19 was developed, providing fast, reliable, and affordable testing, particularly for low-mid develop areas. The PoC diagnostic process combines a simple paper-based RNA extraction method housed within a 3D-printed plastic device with a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Nasopharyngeal/oropharyngeal swabs (NOS) and saliva samples were tested between 2020 and 2021, with the assistance of Santa Catarina's State Health Secretary, Brazil. The developed diagnostic protocol showed a limit of detection of 9,900 copies and an overall diagnostic specificity of 98% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 1,348 clinical analysed samples. The diagnostic sensitivity was 95% for NOS samples, 85% for early morning saliva, and 69% for indiscriminate saliva. In conclusion, the developed device successfully extracted SARS-CoV-2 viral RNA from swabs and saliva clinical samples. When combined with colorimetric RT-LAMP, it provides results within 45 min using minimal resources, thus delivering a diagnostic kit protocol that is applicable in large-scale sampling.