Antigen testing for COVID-19 using image-based assessment of oral specimens

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   ScreenIT

   May 30, 2022

   SciScore for 10.1101/2022.05.27.22274752: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	Field Sample Permit: VeroE6/TMPRSS2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. IRB: Specimen collection: This study was approved by the ethics committee of Keio University (approval number: 20210081) and was conducted in accordance with the Declaration of Helsinki and Title 45, U.S. Code of Federal Regulations, Part 46, Protection of Human Subjects, effective December 13, 2001. Consent: All patients provided written informed consent.
   Sex as a biological variable	not detected.
   Randomization	Quantification of SAES-CoV-2 infection: First, multiple fields of view (FOVs) containing observed cells were randomly selected.
   Blinding	not detected.
   Power …

   SciScore for 10.1101/2022.05.27.22274752: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	Field Sample Permit: VeroE6/TMPRSS2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. IRB: Specimen collection: This study was approved by the ethics committee of Keio University (approval number: 20210081) and was conducted in accordance with the Declaration of Helsinki and Title 45, U.S. Code of Federal Regulations, Part 46, Protection of Human Subjects, effective December 13, 2001. Consent: All patients provided written informed consent.
   Sex as a biological variable	not detected.
   Randomization	Quantification of SAES-CoV-2 infection: First, multiple fields of view (FOVs) containing observed cells were randomly selected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Visualizing squamous epithelial cells using an antibody against pan-cytokeratin: After reaction with a fluorescent Nb, cells were incubated in 0.7 mL PBS containing 0.1% (wt/vol) Triton X-100 and 1.25 μg/mL anti–pan-cytokeratin mouse mAb (AE1/AE3) (BioLegend, 914204) at RT for 60 min.	anti–pan-cytokeratin suggested: (BioLegend Cat# 914204, RRID:AB_2616960)
   Delineating individual cells by immunostaining the plasma membrane: After reaction with a fluorescent Nb, cells were incubated in 0.7 mL PBS containing 0.1% (wt/vol) Triton X-100 and 3.3 μg/mL anti–pan-cadherin rabbit polyclonal antibody (abcam, ab16505) at RT for 60 min.	anti–pan-cadherin suggested: (Abcam Cat# ab16505, RRID:AB_443397)
   After washing with PBS twice, cells were incubated in a 0.7 mL PBS containing 0.1% (wt/vol) Triton X-100 and 2.0 μg/mL Alexa Fluor 546–labeled donkey anti–rabbit IgG (H+L) antibody (Thermo Fisher/Invitrogen, A10040) at RT for 60 min.	anti–rabbit IgG suggested: (Thermo Fisher Scientific Cat# A10040, RRID:AB_2534016)
   Experimental Models: Cell Lines
   Sentences	Resources
   VeroE6/TMPRSS2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank.	VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Recombinant DNA
   Sentences	Resources
   Gene construction (Nb-FP fusion): The K-874A, E9, or N10 gene was amplified using primers containing the 5’-BamHI and 3’-EcoRI sites, and the restricted products were cloned into the BamHI/EcoRI sites of pBS Coupler 4 (Shimozono and Miyawaki, 2008) to generate pBS/K-874A=, pBS/E9=, or pBS/N10=, respectively. ‘=’ denotes the “coupler linker,” a triple repeat of the amino acid linker Gly–Gly–Gly–Gly–Ser [(GGGGS)3].	pBS/E9= suggested: None pBS/N10= suggested: None
   The KikG gene was amplified using primers containing the 5’-HindIII and 3’-SalI sites, and the restricted product was cloned in-frame into the HindIII/SalI sites of pBS/K-874A=, pBS/E9=, and pBS/N10= to generate pBS/K-874A=KikG, pBS/E9=KikG, and pBS/N10=KikG, respectively.	pBS/K-874A=KikG suggested: None pBS/E9=KikG suggested: None pBS/N10=KikG suggested: None
   The Azalea gene was amplified using primers containing the 5’-HindIII and 3’-SalI sites, and the restricted product was cloned in-frame into the HindIII/SalI sites of pBS/K-874A=, pBS/E9=, and pBS/N10= to generate pBS/K-874A=Azalea, pBS/E9=Azalea, and pBS/N10=Azalea, respectively.	pBS/K-874A=Azalea suggested: None pBS/E9=Azalea suggested: None pBS/N10=Azalea suggested: None
   The EGFP or Achilles gene was amplified using primers containing the 5’-HindIII and 3’-SalI sites, and the restricted product was cloned in-frame into the HindIII/SalI sites of pBS/K-874A= to generate pBS/K-874A=EGFP or pBS/K-874A=Achilles, respectively.	pBS/K-874A= suggested: None pBS/K-874A=EGFP suggested: None pBS/K-874A=Achilles suggested: None
   In parallel, pRSETB was engineered to have a SalI site instead of a HindIII site.	pRSETB suggested: RRID:Addgene_89510)
   The resultant plasmid was named pRSETB(S).	pRSETB(S suggested: None
   The DNA fragments encoding K-874A=KikG, E9=KikG, N10=KikG, K-874A=Azalea, E9=Azalea, N10=Azalea, K-874A=EGFP, and K-874A=Achilles were cloned into the BamHI/SalI sites of pRSETB(S) to generate pRSETB(S)/K-874A=KikG, pRSETB(S)/E9=KikG, pRSETB(S)/N10=KikG, pRSETB(S)/K-874A=Azalea, pRSETB(S)/E9=Azalea, pRSETB(S)/N10=Azalea, pRSETB(S)/K-874A=EGFP, and pRSETB(S)/K-874A=Achilles, respectively.	pRSETB(S)/K-874A=KikG suggested: None pRSETB(S)/E9=KikG suggested: None pRSETB(S)/N10=KikG suggested: None pRSETB(S)/K-874A=Azalea suggested: None pRSETB(S)/E9=Azalea suggested: None pRSETB(S)/N10=Azalea suggested: None pRSETB(S)/K-874A=EGFP suggested: None pRSETB(S)/K-874A=Achilles suggested: None
   Software and Algorithms
   Sentences	Resources
   Second, individual cells were manually delineated using the “Freehand selections” tool (ImageJ) in each FOV.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Third, the average intensity and the texture of the E9=KikG fluorescence were extracted using a customized program written using C++ and OpenCV 3.4.1 (https://opencv.org).	https://opencv.org suggested: (OpenCV, RRID:SCR_015526)

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   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).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.05.27.22274752 on medRxiv

   May 27, 2022