key: cord-0857767-elzamh1y
authors: MARQUETTE, C. H.; Boutros, J.; Benzaquen, J.; Selva, E.; Labaky, M.; Benchetrit, D.; Lavrut, T.; Leroy, S.; Chemla, R.; Carles, M.; Tanga, V.; Maniel, C.; Bordone, O.; Allegra, M.; Lespinet, V.; Fayada, J.; Griffonnet, J.; Hofman, V.; Hofman, P.
title: Clinical performance of oral sponge sampling for detection by RT-PCR of SARS-CoV-2
date: 2021-02-19
journal: nan
DOI: 10.1101/2021.02.17.21251556
sha: 1f82a883a868eb84df5c4473d99496ca43438b68
doc_id: 857767
cord_uid: elzamh1y

Background: The current standard for coronavirus 2019 disease (COVID-19) diagnosis is reverse transcriptase-polymerase chain reaction (RT-PCR) testing of naso-pharyngeal swabs (NPS), Sampling with NPS is invasive and requires specialized and trained personnel, which limits rapid and repeated screening for the disease. A less invasive and possibly self-administered sampling method may increase the capacity for testing and be more effective in identifying, isolating, and filtering out currently infected persons. Methods: Over a period of three months, we included volunteers presenting with recent symptoms suggestive of a SARS-CoV-2 infection at a free COVID-19 screening center in the city of Nice, France. NPS as well as nasal and oral sponges were collected in parallel and analyzed by RT-PCR for SARS-CoV-2. Results: One hundred and forty-seven subjects were included, of whom, 41.5% were diag-nosed with COVID-19 using NPS RT-PCR. RT-PCR on nasal and oral sponges showed a sensitivity of 87 to 98% and 72 to 87%, respectively for diagnosis of COVID-19 in symptomat-ic subjects, depending on the type of RT-PCR technique used. The specificity was 100% whatever the RT-PCR test. The viral load determined with the oral samples was significantly lower than with NPS. Conclusion: Taken together, these results demonstrated that the oral sponge sampling method can be standardized, is easy to use and cheap. The acceptability makes it a repeata-ble test, notably for elderly people or children. It may become a high-frequency - low analyti-cal sensitive testing strategy.

To date, reverse transcriptase-polymerase chain reaction (RT-PCR) testing of naso-pharyngeal swab (NPS) specimens is the gold standard for diagnosis of coronavirus 2019 disease [1] [2] [3] [4] . While its specificity is 100%, its sensitivity depends on the operator and on the moment the sampling is performed during the course of the infection which is 80-90% during the first 10 days for a COVID-19 positive subject [5, 6] . RT-PCR with, NPS, known as a "high analytical sensitivity" technique, is particularly well suited for symptomatic patients. However, in the context of a pandemic it is not very well suited to the management of contact tracing of the general population. Not only does it require dedicated trained staff, its acceptability by the population is limited due to its unpleasant or even painful nature, especially when it involves repeating samples. When repeated testing is required, as for residents of nursing homes and health care workers, or in cases of massive testing of university students or of the population of a town or region for the purpose of monitoring lockdown/lockdown easing, the use of NPS for RT-PCR is not appropriate [7] .

Antigen-detecting rapid diagnostic tests (Ag-RDT) are quick, simple, inexpensive and allow the decentralization of testing of symptomatic people at the point of care. As for a conventional RT-PCR test, they are performed with a NPS and thus need to be done by a trained operator [8] . Their sensitivity varies considerably (60 to 95%) [8] [9] [10] and their invasiveness makes Ag-RDT poorly suited for repeated testing of populations.

Sampling of saliva offers a promising alternative to NPS. [11] Various pathophysiological pathways explain the presence of the SARS-CoV-2 in saliva of patients with COVID-19 [12] [13] [14] [15] [16] [17] .

Tests with saliva involve amplification of viral RNA by RT-PCR or by Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP). They are non-invasive, easily repeatable and can be performed without the need of trained staff to collect the sample. Although saliva testing has been the subject of many studies, routine application is still rare due to the lack of well-standardized clinically validated protocols [11, 12, [18] [19] [20] [21] [22] p. 3

Based on our clinical experience on the management of COVID-19 patients and our research on the respiratory epithelium and on we aimed to evaluate non-invasive alternatives to NPS for RT-PCR for the diagnosis of COVID-19.

We investigated in 31 healthy volunteers (unpublished data) sampling with small hydroxylated polyvinyl acetate (PVA) sponges (Merocel ® Standard Dressing, réf 400400, Medtronic), used as haemostatic material in the management of acute epistaxis, to collect nasal secretions and saliva. As show, in figures 1S and 2S (supplementary appendix) the sponges were placed in the nasal cavity (between the nasal septum and the inferior turbinate or in the mouth [28] ). To facilitate the collection of nasal secretions or saliva, each sponge was placed in a tube containing 2 ml of 0.9 % saline (figure 3S). This medium does not interfere with the techniques of gene amplification by RT-PCR [29] . The nasal secretions and saliva were then obtained by squeezing the sponge (figure 4S). The sponges recovered on average 0.56 ml (range 0.06 -2.74 ml) of nasal secretions and 2.09 ml (range 0.54 -5.74 ml) of oral secretions, respectively.

Considering the dilution factor linked to the addition of physiological serum (a factor of 1/5 for nasal secretions and 1/2 for saliva), we showed that the collected nasal secretions and saliva had a cellularity of 279 ± 467 and 109 ± 183 cells / µl, respectively. Nasal secretions were rich in ciliated epithelial cells and poor in squamous cells. Saliva contained exclusively squamous cells (data not shown).

After signing agreement to participate, all participants were interviewed and then four samples were taken: bilateral NPS, nasal sponge (NS) and oral sponge (OS) were administered in a random order. The NPS and NS sampling was performed by the same practitioner (CHM). OS were placed in the mouth by the subjects themselves, taken out after one minute and inserted into the collection tube (Figures 3S to 5S in the appendix). One NPS was transported within 4 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

RT-PCR with NPS using the Idylla platform has been described previously (Hofman et al, submitted). RT-PCR with NS and OS was performed upon arrival of the tubes at the LPCE.

After homogenization by pipetting from top to bottom of the eluate of the nasal and oral sponges soaked in saline (using D. Dutscher pipettes, reference 134000), they were placed in the Idylla ™ cartridge for SARS-CoV-2 (Biocartis, Belgium, reference A1042/6 and A1043/6) detection, which was performed on a fully automated system for extraction, amplification and detection of nucleic acids using a single-use cartridge (see processing of the sponges in the appendix). After treatment, the residual volume of media was immediately aliquoted and stored at -80°C in the Nice COVID-19 biobank [25] . According to the manufacturer, the Idylla SARS-CoV-2 test provides a qualitative result for the presence or absence of SARS-CoV-2 RNA with a corresponding quality status. The Biocartis SARS-CoV-2 test included 2 genes (N, Orf1b) covered by 5 PCR targets (2 N targets and 3 Orf1b targets). A positive result required at least 2 amplified N targets [by setting a quantification cycle (Cq) of 41.9] and/or at least one or more amplified Orf1b targets. As Orf1b is highly specific no threshold was required for this gene.

RT-PCR with NPS, at the Synlab Barla laboratory, was routinely performed with the DAAgene Kit (NE / CA09 / 170 / D01 / IVD / 016-03) (Da An Gene Co., Ltd. Sun Yat-sen University, All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

In the absence of data into the sensitivity of the technique, we calculated the sensitivity after the first 40 samples, which was 85%. Given this estimate, and a desired lower bound of the 95% confidence interval for sensitivity of at least 80%, 144 volunteers were needed to complete the study. To deal with the dropout risk we decided to include 149 volunteers.

The result of RT-PCR with NPS was the reference (standard of care) to determine the clinical performance of RT-PCR on the NS and the OS (index tests). Continuous variables are presented as means (± SD), and categorical variables as numbers and percentages. Baseline characteristics between patients with and without COVID-19 were compared using the Student's t-test or Wilcoxon -Mann Whitney for quantitative variables based on the normality of the distribution of parameters or using the Chi-Square test for qualitative variables.

The promoter of the study was the Center Hospitalier Universitaire de Nice. The agreement for the study of the Institutional review board Sud Méditerranée V was obtained on April 22, 2020 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in (registration # 20.04014.35208). SHAM liability insurance (n ° 159087). The study is registered in ClinicalTrial.gov (NCT04418206).

The organizations that supported this study played no role in its design, patient selection, data collection, analysis or interpretation, report writing, or decision to submit the document for publication. Authors had full access to all data and responsibility of submission for publication.

One hundred and forty-nine subjects with COVID-19 symptoms were included during the 15- week study period (September 21, 2020 to January 6, 2021). Two subjects declined to continue sampling after the NPS was performed and were excluded from subsequent analyses.

One had a positive NPS RT-PCR, and the other was negative. The study therefore covers the remaining 147 participants.

The interval between symptom onset and testing was 3.6 ± 2.6 days and most participants (107/147 [72.8%]) were sampled at the early stage of the disease, i.e., within 4 days of symptom onset. Participants were predominantly women 86/147 (58.5%). The mean age was 40 ± 15 years. Of these 147 subjects, 61 (41.5%) had a positive RT-PCR with NPS and were therefore diagnosed with COVID-19. No clinical symptoms were distinctive between RT-PCR positive and negative subjects (Figure 1) , with the exception of anosmia and dysgeusia, which were more frequent in RT-PCR positive subjects (42% vs. 10%, p <0.001 and 38 vs. 16%, p = 0.004) and sore throat which was significantly more common in RT-PCR negative subjects (36 vs. 10%, p = 0.001). There was no significant difference between the oxygen saturation levels measured by pulse oximetry between RT-PCR positive (SpO2 = 98.3% ± 1.88%) and negative subjects (SpO2 = 97.7 ± 1.89%) (p = 0.17).

NS sampling, although significantly more comfortable than NPS sampling, required an experienced operator to be properly positioned between the nasal septum and the inferior turbinate. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Since our primary goal was to develop a self-sampling technique, we abandoned nasal sponge sampling after the 101 st volunteer.

Oral sampling using OS was obtained from all 147 volunteers. In two cases, external validation by the Synlab Barla laboratory was not possible since there was not enough residual eluate.

Comparison of viral loads between each kind of sampling method, as assessed by the Ct for the Orf1b and the N gene showed a statistically significant gradient between the oral cavity and the nasopharynx and between the nose and the nasopharynx (Figure 2 ). NS RT-PCR showed a sensitivity of 78 to 98% depending on the RT-PCR technique used and the positivity threshold selected (Table 1) . OS RT-PCR showed a sensitivity of 57 to 87% depending on the RT-PCR technique and the positivity threshold used ( Table 1 ). The specificity was 100%, whatever the technique and whatever the threshold used.

Our results showed that the respective sensitivities of RT-PCR for diagnosing COVID-19 in symptomatic subjects using NS and OS were 98 and 87%, respectively on the Idylla platform and 87 and 72% on the Synlab Barla platform, with a specificity of 100% for both.

During the early phase of the COVID-19 pandemic, many molecular tests and immunoassays were rapidly developed and validated on archived biological samples of known virological status, albeit many still await clinical validation [22] . The present study was carried out according to the standards recommended by the French Haute Autorité de Santé for evaluating the clinical performance of diagnostic tests for COVID-19 [8] . i.e. a prospective comparative clinical study relating to a series of individuals of unknown COVID-19 status, recruited consecutively. We found a significantly different viral load between the nasopharynx, the nasal and the oral cavities ( Figure 2) . This gradient has a pathophysiological origin, since it is known that the nasopharynx is rich in ciliated epithelial cells and, as we showed, the genes associated with viral entry are highly expressed in these cells, unlike in the oral cavity [26, 27] . Such a All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in gradient in viral load between NPS and OS has already been reported [2, [31] [32] [33] and explains why the diagnostic yield of NPS is considered to be higher than that of throat swabs [32, 33] and why RT-PCR with NPS specimens became the gold standard for diagnosis of COVID-19 in symptomatic subjects. Because of this gradient, the positivity threshold recommended by the French Society of Microbiology cannot be used for OS, for which a threshold of 40 or more is more appropriate.

The external validation of our approach in a private laboratory (Synlab Barla platform) showed a sensitivity of 10 to 30% lower than that obtained with the Idylla platform (Table 1) . This discrepancy may be due to at least three factors. The first is the cut-off used by the private laboratory (Ct ≤ 36) which, as discussed above, is lower. The second is the delay in performing the RT-PCR on the Synlab Barla platform with biobanked samples. This factor probably plays only a minor role since the preservation conditions (-80 ° C in an experienced biobank) do not alter the nucleic acids. The third may be related to the fact that the Idylla platform uses 3 Orf1 and 2 N targets while the Synlab Barla platform uses only one Orf1 and one N target. Other factors that may have contributed are differences in algorithms for Ct value determination, overall test design, extraction efficiencies, amount of sample equivalent tested per PCR reaction, and workflow integration.

The OS method is fairly well standardized. It does not need sialagogical substances, nor clearing the throat or spitting effort, nor particular constraints such as early morning samples before tooth brushing and breakfast, avoiding eating, drinking, gum chewing, smoking, or vaping. [34] [35] [36] It is cheap and simple since there is no need for viral transport medium to be added to the saliva nor for dedicated trained nursing staff. Lastly, its acceptability makes it possible to consider repeating the test even on institutionalized elderly people or on children.

The question as to whether a 72-87% sensitivity (OS sampling method) is good enough to identify SARS-CoV-2 infected people has been recently debated by Mina et al. in a landmark position paper [7] in which low-frequency testing with a high analytical sensitivity was compared to high-frequency testing with a low analytical sensitivity. RT-PCR with NPS belongs to the former category and is well suited to diagnosis of COVID-19 in symptomatic subjects. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Figure 1S : naso-pharyngeal swab vs nasal sponge vs oral sponge.

: The oral sponge is introduced into the mouth by the subject. The subject reopens the mouth after one minute, as soon as he/she feels that the sponge has swollen.

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