Abstract
BackgroundViral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. However, unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic.MethodsWe compared four nucleic acid detection methods [two kinds of reverse transcription polymerase chain reactions (RT-PCR A: ORF1ab and N testing; RT-PCRB: only ORF1ab testing), reverse transcription recombinase aided amplification (RT-RAA) and droplet digital RT-PCR (dd-RT-PCR)] using 404 samples of 72 hospitalized COVID-19 patients, including oropharyngeal swab (OPS), nasopharyngeal swabs (NPS) and saliva after deep cough, to evaluate the best sample type and method for SARS-CoV-2 detection.ResultsAmong the four methods, dd-RT-PCR exhibited the highest positivity rate (93.0%), followed by RT-PCR B (91.2%) and RT-RAA (91.2%), while the positivity rate of RT-PCR A was only 71.9%. The viral load in OPS [24.90 copies/test (IQR 15.58-129.85)] was significantly lower than that in saliva [292.30 copies/test (IQR 20.20-8628.55)] and NPS [274.40 copies/test (IQR 33.10-2836.45)]. In addition, if OPS samples were tested alone by RT-PCR A, only 21.4% of the COVID-19 patients would be considered positive. The accuracy of all methods reached nearly 100% when saliva and NPS samples from the same patient were tested simultaneously.ConclusionsSARS-CoV-2 nucleic acid detection methods should be fully evaluated before use. High-positivity rate methods such as RT-RAA and dd-RT-PCR should be considered when possible. Furthermore, saliva after deep cough and NPS can greatly improve the accuracy of the diagnosis, and testing OPS alone is not recommended.
Highlights
Since the first emerging in late 2019, coronavirus disease 2019 (COVID-19) has caused a worldwide pandemic, with more than 157 million confirmed cases and 3 million deaths (WHO, 2021)
This study enrolled a total of 72 COVID-19 patients, who were admitted to the First Affiliated Hospital, Zhejiang University School of Medicine, from 19th Jan 2020 to 23rd Feb 2020
Fever (83.3%), cough (54.2%), and expectoration (30.6%) were the most common clinical manifestations at the time of admission. 16 patients were admitted to the ICU, and 4 of them were under mechanical ventilation. 88.9% of patients received oxygen supplement
Summary
Since the first emerging in late 2019, coronavirus disease 2019 (COVID-19) has caused a worldwide pandemic, with more than 157 million confirmed cases and 3 million deaths (WHO, 2021). Viral nucleic acid detection is still the most effective method to confirm SARS-CoV-2 infection. A variety of detection methods based on specific SARS-CoV-2 nucleotide sequences have been rapidly developed and used as emergency applications in the laboratory. National Medical Products Administration (NMPA China) has approved 22 SARS-CoV-2 nucleic acid detection reagents, most of which are reverse transcription polymerase chain reaction (RT-PCR) methods. Some other detection techniques are waiting for approval, such as reverse transcription recombinase aided amplification (RT-RAA) method and droplet digital RT-PCR (dd-RT-PCR) method. Multiple samples and repeated testing may be required to diagnose patients who are infected with SARS-CoV-2. Viral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. Unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic
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