A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread
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Abstract
Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/μL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.
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SciScore for 10.1101/2021.06.07.21258288: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Subjects: This project was approved by the Ethics Committee of Instituto de Biociências, Universidade de São Paulo, Brazil (accession number 31655320.0.0000.5464), and involved the collaboration with several groups in order to gain access to anonymized clinical samples from individuals with respiratory symptoms: Saliva collection: Briefly, individuals were asked not to eat for at least 30 minutes before collection of 3 mL of saliva in sterile, nuclease-free 15 mL conical tubes. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We …
SciScore for 10.1101/2021.06.07.21258288: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Subjects: This project was approved by the Ethics Committee of Instituto de Biociências, Universidade de São Paulo, Brazil (accession number 31655320.0.0000.5464), and involved the collaboration with several groups in order to gain access to anonymized clinical samples from individuals with respiratory symptoms: Saliva collection: Briefly, individuals were asked not to eat for at least 30 minutes before collection of 3 mL of saliva in sterile, nuclease-free 15 mL conical tubes. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
No key resources detected.
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:We also characterized saliva and NOP swab specimens according to viral load, gender, age, and time from symptom onset, providing more insight into the advantages and limitations of salivary SARS-CoV-2 diagnostics. In simulated saliva, we observed that heat treatment with PK-based formulations is insufficient to stabilize viral RNA, contrasting previous reports using samples containing SARS-CoV-2 virions (7,13–15). This discrepancy could be explained by the fact that the spiked RNA in our simulated samples is not protected by the nucleocapsid and other structural viral proteins, being readily digested by any active nucleases left after sample processing. This indicates that PK did not sufficiently inactivate salivary nucleases under the conditions examined here. Thus, care must be taken when using PK and other agents to process specimens, especially when combined with additional viral lysis methods, which may expose viral RNA to digestion. Accordingly, although SARS-CoV-2 remains stable in crude saliva (16), diagnostic sensitivity may drop depending on the method employed to inactivate/process samples, such as heat inactivation, inclusion of detergents, and other factors (17,18), making the RNA protection provided by DGS nevertheless critical. In clinical saliva, DGS heat treatment improves detection of SARS-CoV-2 compared to heating without DGS. The active ingredients in DGS act by reducing and denaturing salivary extracellular ribonucleases and other inhibitors, and may also...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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