Investigating the optimum sample type and target genes for SARS-CoV-2 detection
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Abstract
Aims
The cycle threshold (Ct) value for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection is important because of the criteria for quarantine management, including release from it, which are defined in Guidelines on the Novel Coronavirus-Infected Pneumonia Diagnosis and Treatment (Provisional 9th Edition, China). As this is also currently relevant because of the recent SARS-CoV-2 epidemic in Shanghai, we discuss the SARS-CoV-2 nucleic acid detection and its problems. We focus on the gene fragments and sample types involved in nucleic acid detection and their effect on the latest criteria for release from quarantine.
Methods
A total of 215 patients with SARS-CoV-2 infection were included. Pharyngeal swabs (nasopharyngeal swabs plus oropharyngeal swabs) were collected in the early stage of the disease, and pharyngeal swabs, sputum samples, and anal swabs were collected both in the middle and advanced stages of the disease. The Open reading frame 1ab (ORF lab) gene, Nucleocapsid (N) gene and Envelop (E) gene of each sample were quantitatively analyzed using fluorescence qPCR technique.
Results
Exclusion of the E gene detection results had no significant effect on the interpretation of the nucleic acid Ct value of 35, with a positive concordance rate of 98.7% (95% CI 86.0%–100%) and an overall concordance rate of 99.7% (95% CI 92.9%–100%). The kappa coefficient was 0.99 (95% CI 0.92–1.00). Compared with nucleic acid detection using both pharyngeal swab and sputum sample, the positive concordance rate of the detection using pharyngeal swab alone was 47.6% (95% CI 27.8%–99.3%). The kappa coefficient was 0.63 (95% CI 0.53–0.75), and the consistency was not ideal.
Conclusions
Nucleic acid detection using the ORF 1ab gene and the N gene can achieve the purpose of SARS-CoV-2 detection. Nucleic acid detection using sputum samples is significant in the determination of Ct values and its significance in the development of the criteria for release from quarantine needs to be taken into account. It is suggested that to increase the accuracy of nucleic acid detection, instead of unilaterally pursuing increasing the number of target genes for amplification and improving PCR techniques, more attention should be paid to sampling and sample reliability, as well as strict quality control of the detection process.
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SciScore for 10.1101/2022.05.25.22275564: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The trial was approved by the ethics committee of Changzheng Hospital, and the patients signed informed consent forms on admission and agreed to anonymously authorize their clinical data for academic use.
Consent: The trial was approved by the ethics committee of Changzheng Hospital, and the patients signed informed consent forms on admission and agreed to anonymously authorize their clinical data for academic use.Sex as a biological variable Sample types based on source: A total of 213 patients (all male, age: 22.4±3.2 years) with COVID-19 who were admitted to a designated hospital in Shanghai on April 11, 2022 were included. Randomization not detected. Blinding not detected. Power Analysis n… SciScore for 10.1101/2022.05.25.22275564: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The trial was approved by the ethics committee of Changzheng Hospital, and the patients signed informed consent forms on admission and agreed to anonymously authorize their clinical data for academic use.
Consent: The trial was approved by the ethics committee of Changzheng Hospital, and the patients signed informed consent forms on admission and agreed to anonymously authorize their clinical data for academic use.Sex as a biological variable Sample types based on source: A total of 213 patients (all male, age: 22.4±3.2 years) with COVID-19 who were admitted to a designated hospital in Shanghai on April 11, 2022 were included. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Statistical methods: Using SPSS 26.0 (IBM) software and a four-grid table, the Kappa coefficient was used to measure the agreement between the interpretation methods using different sample types, which were evaluated using the concordance rate and the Kappa coefficient. SPSSsuggested: (SPSS, RRID:SCR_002865)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:However, there are still some limitations in this study. First, a cut-off Ct value of 35 was adopted, however, some cellular experiments have now confirmed that an excreted viral load in the environment with Ct values of less than 35 does not result in effective infection. The relationship between a Ct value of 35 and infectivity needs to be supported by more genuine clinical data. Second, although all patients enrolled in this study had asymptomatic infections and mild disease, the inconsistent test results between sputum sample and pharyngeal swab cannot be ruled out as being partially related to differences in patient disease status and distribution of the virus. Third, sputum samples are relatively more accurate in the determination of Ct values, but they are obtained by induced nebulization. Whether nebulization represents the normal state of virus excretion and its relationship with infectivity needs to be further investigated. Nevertheless, the large sample of cases with the same exposure period and similar clinical characteristics and the analysis of multiple synchronous triple-sample collections of pharyngeal swabs, sputum samples, and anal swabs showed that the combined sputum sample detection has an important role in determining the criteria for release from quarantine.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a protocol registration statement.
Results from scite Reference Check: We found no unreliable references.
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