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Review 2: "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer"

This preprint offers a novel diagnostic platform that uses off-the-shelf glucometers to detect SARS-CoV-2 antigens in saliva. The current manuscript offers rigorous validation of the diagnostic technology, but further experiments should be performed before clinical use.

Published onOct 30, 2020
Review 2: "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer"
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Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer
Description

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of effective antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones providing an opportunity to integrate with contract tracing apps, medical providers, and electronic medical records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within one hour, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach can provide an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.

RR:C19 Evidence Scale rating by reviewer:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

The authors developed a novel antigen assay for detecting SARS-CoV-2 virus. This review was divided into two parts. The first part was the review of principles and validation of the assay, the second part was the clinical application of the assay. 

1.        Principles and validation of the assay

1.1 Principles of the assay

The assay was based on an aptamer-based competitive approach using a glucometer as a device to read the signals. Aptamer refers to the molecules that bind to a specific target molecule. The SARS-CoV-2 specific aptamer was utilized in this assay. This aptamer was conjugated to magnetic bead while the aptamer was also pre-hybridized with an oligonucleotide strand. This oligonucleotide strand was covalently linked to an invertase enzyme.

In the presence of SARS-CoV-2 antigen/protein, a complex was formed between the SARS-CoV-2 antigen/protein and the aptamer. The low affinity oligonucleotide strand will be released. This released strand will convert sucrose to glucose and the results will be read by the glucometer.

In the absence of SARS-CoV-2 antigen/protein, the aptamer remains hybridized with the oligonucleotide strand, sucrose will not convert to glucose. No glucose signals were read by the glucometer.

A series of experiments were performed to establish the assay and confirmed the above principles.

1.2 Validation of the assay

Two aptamers were evaluated, they were SARS-CoV-2 specific S protein aptamer and N protein aptamer.

The assay specificity was confirmed by testing against influenza A (H1N1) and MERS-CoV proteins. The signals for these two proteins were <300% less than the SARS-CoV-2 specific proteins.

The assay sensitivity was confirmed by testing seven saliva samples, three of them were SARS-CoV-2 patients confirmed by RT-PCR. Although the glucose signals for all of the seven samples were read by the glucometer, the signals from the three SARS-CoV-2 patients (positive signals) were higher than the four non-SARS-CoV-2 patients (noise signals). When comparing between S protein aptamer and N protein aptamer, the ratio between the two signals (positive and noise) were higher for the former one. It means that the S protein aptamer had a higher signal to noise ratio than the N protein aptamer. 

The S protein aptamer was chosen for evaluating another set of 24 saliva samples which comprised 16 SARS-CoV-2 patients and 8 healthy individuals. When the cut-off 52 mg/dl was set, the assay was capable of distinguishing between positive and negative samples. Positive samples ranged 68-404 mg/dl while negative samples ranged 14-37 mg/dl.

2.        Clinical application of the assay 

2.1 Technical requirements

The aptamer-based assay required more technical steps when comparing with lateral flow antigen detection assays. The aptamer-based assay was not easy to execute. It is recommended that a pre-training course should be provided for individuals to perform the assay. 

The turnaround time for the aptamer-based assay was 60 minutes which was longer than the lateral flow antigen detection assays. For lateral flow antigen detection assays, results were read by the operator within 10 to 30 minutes [1].

2.2. Sensitivity 

In the ‘Introduction’ section, the authors mixed up the lateral flow antigen detection assays with lateral flow antibody detection assays. For the lateral flow antigen detection assays, data on the specificity were consistently reported to be high (>97%) while the sensitivity were highly variable between different studies and different brands [1].

In the ‘Discussion’ section, the authors compared the aptamer-based assay with other assays. The information for the test ‘Respi-Strip (Coris Bio)’ listed in ‘Table 1’ was incorrect. The turnaround time should be 15 minutes but not 30 minutes.

‘Table 1’ showed that the ‘Respi-Strip (Coris Bio)’ test shared similar limit of detection with the aptamer-based assay. Different studies evaluated the performance of the ‘Respi-Strip (Coris Bio)’ test [2-5], the authors are recommended to compare the clinical sensitivity between the ‘Respi-Strip (Coris Bio)’ test and the aptamer-based assay.

‘Table 1’ showed that the sensitivity of the aptamer-based assay was 100%. Recently, numerous lateral flow antigen detection tests for diagnosing COVID-19 patients are available in the market and evaluated in different studies. Irrespective of the days after symptom onset, the clinical sensitivity of these tests ranged from 70.6% to 76.3% [6-8]. Some of them were placed in the list of the ‘WHO Emergency Use Listing for In vitro diagnostics (IVDs) Detecting SARS-CoV-2’ [9]. The authors are recommended to comment the clinical sensitivity between these tests with the aptamer-based assay.

2.3 Specificity 

False positive issue has been reported for the antigen assay in Japan. This antigen assay is a quantitative test which required setting cutoff value to distinguish positive and negative cases. The dilemma for this test is the occurrence of false positive cases and false negative cases by adjusting the cutoff value. High cutoff value can prevent false positive cases, however, sensitivity is lower. Low cutoff value can increase sensitivity, however, false positive cases will be increased [10].

The aptamer-based assay was also a quantitative test and required setting a cutoff value. The authors are recommended to comment on this issue.

3.        Conflicts of interest 

None.

4.        References 

1.     WHO. Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immunoassays. Interim guidance, 11 September 2020. Available at: https://www.who.int/publications/i/item/antigen-detection-in-the-diagnosis-of-sars-cov-2infection-using-rapid-immunoassays (Accessed 20 October 2020).

2.     Lambert-Niclot S, Cuffel A, Le Pape S, et al. Evaluation of a Rapid Diagnostic Assay for Detection of SARS-CoV-2 Antigen in Nasopharyngeal Swabs. J Clin Microbiol. 2020;58(8):e00977-20. Published 2020 Jul 23. doi:10.1128/JCM.00977-20.

3.     Scohy A, Anantharajah A, Bodéus M, et al. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J Clin Virol. 2020;129:104455. doi:10.1016/j.jcv.2020.104455.

4.     Blairon L, Wilmet A, Beukinga I, et al. Implementation of rapid SARS-CoV-2 antigenic testing in a laboratory without access to molecular methods: Experiences of a general hospital. J Clin Virol. 2020;129:104472. doi:10.1016/j.jcv.2020.104472.

5.     van Beek J, Igloi Z, Boelsums T, et al. From more testing to smart testing: data-guided SARS-CoV-2 testing choices. Preprints (2020), https://doi.org/10.1101/2020.10.13.20211524

6.     Cerutti F, Burdino E, Milia MG, et al. Urgent need of rapid tests for SARS CoV-2 antigen detection: evaluation of the SD-Biosensor antigen test for SARS-CoV-2. J Clin Virol. available online 29 Sep 2020. doi:10.1016/j.jcv.2020.104654

7.     Young S, Taylor SN, Cammarata CL, et al. Clinical evaluation of BD Veritor SARS-CoV-2 point-of-care test performance compared to PCR-based testing and versus the Sofia 2 SARS Antigen point-of-care test. J Clin Microbiol. 2020 Oct 6:JCM.02338-20. doi: 10.1128/JCM.02338-20. Epub ahead of print. PMID: 33023911.

8.     Linares M, P´erez-Tanoira R, Carrero A, et al. Panbio antigen rapid test is reliable to diagnose SARS-CoV-2 infection in the first 7 days after the onset of symptoms. J Clin Virol. available online 16 Oct 2020. https://doi.org/10.1016/j.jcv.2020.104659

9.     WHO. WHO Emergency Use Listing for In vitro diagnostics (IVDs) Detecting SARS-CoV-2 (last updated: 2 October 2020). Available at: https://www.who.int/diagnostics_laboratory/201002_eul_sars_cov2_product_list.pdf?ua=1 (Accessed 20 October 2020)

10.  Ogawa T, Fukumori T, Nishihara Y, et al. Another false-positive problem for a SARS-CoV-2 antigen test in Japan. J Clin Virol. 2020 Oct;131:104612. doi: 10.1016/j.jcv.2020.104612. Epub 2020 Aug 25. PMID: 32871543; PMCID: PMC7445490.

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