key: cord-0984340-tm43ilr1
authors: Le Goff, J.; Kerneis, S.; Elie, C.; Mercier Delarue, S.; Gastli, N.; Choupeaux, L.; Fourgeaud, J.; Alby, M.-L.; Quentin, P.; Pavie, J.; Brazille, P.; Nere, M.-L.; Gabassi, A.; Minier, M.; Leroy, C.; Parfait, B.; Treluyer, J.-M.; Delaugerre, C.
title: Evaluation of saliva molecular point of care for detection of SARS-CoV-2 in ambulatory care
date: 2021-06-16
journal: nan
DOI: 10.1101/2021.06.12.21258811
sha: 18d0fc06be93d0e0db22fc5bd450f224210cff65
doc_id: 984340
cord_uid: tm43ilr1

Background: The rapid identification of SARS-CoV-2 infected individuals is a cornerstone in strategies for the control of virus spread. The sensitivity of SARS-CoV-2 RNA detection by RT-PCR is similar in saliva and nasopharyngeal swab. Rapid molecular point-of-care tests in saliva could facilitate, broaden and speed up the diagnosis. Objectives and methods: We conducted a prospective study in two community COVID-19 screening centers to evaluate the performances of a CE-marked RT-LAMP assay (EasyCoV) specifically designed for the detection of SARS-CoV-2 RNA from fresh saliva samples, compared to nasopharyngeal RT-PCR as reference test, saliva RT-PCR and nasopharyngeal antigen testing. Results: Overall, 117 of the 1718 participants (7%) were tested positive with nasopharyngeal RT-PCR. Sensitivities of saliva RT-PCR and nasopharyngeal antigen test were 93% (95% Confidence Interval (95%CI): 86-97) and 85% (95%CI: 77-91), respectively. The sensitivity and specificity of the RT-LAMP assay in saliva were 34% (95%CI: 26-44) and 97% (95%CI: 96-98). The performance was similar in symptomatic and asymptomatic participants and whatever the reference standard considered. Ct values of nasopharyngeal RT-PCR were significantly lower in the 40 true positive subjects with saliva RT-LAMP (Ct 25.9) than in the 48 false negative subjects with saliva RT-LAMP (Ct 28.4) (p=0.028). Conclusion: In the ambulatory setting, the detection of SARS-CoV-2 from crude saliva samples with the RT-LAMP assay had a lower sensitivity than nasopharyngeal RT-PCR, saliva RT-PCR and nasopharyngeal antigen testing.

Coronavirus disease 2019 (COVID- 19) pandemic has caused significant impact on the healthcare system and socioeconomic activity. Early diagnosis is critical for prompt actions on patient management, infection control, and public health control measures (1) . Since transmission can occur from asymptomatic or pre-symptomatic patients, mass testing, together with rigorous contact tracing and isolation, has been recommended to control the pandemic (2) (3) (4) . This strategy implies rapid and reliable testing methods. Although molecular detection of SARS-CoV-2 RNA in nasopharyngeal swabs is considered as the "gold standard" for identifying infected individuals (1, 5) , nasopharyngeal sampling requires specific sampling equipment and trained personnel and may be difficult in some patients.

Mass RT-PCR testing is carried out in specialized laboratories and needs several hours before results release. Altogether, these constrains restrain access to massive testing, increase time-to-result and consequently delay isolation of contagious individuals (6) .

Rapid antigen point-of-care (Ag) testing allows to overcome the drawback of RT-PCR time-to-result but still requires nasopharyngeal sampling. Sensitivity of Ag tests was estimated at 50-90% and specificity at 90-100% as compared to nasopharyngeal RT-PCR (7, 8) . Recently self-anterior nasal sampling has been tested to reduce patient discomfort and avoid requirements for nasopharyngeal swabbing (9,10).

Self-collected saliva is non-invasive and easy to collect and thus more suitable for mass-screening than nasopharyngeal sampling (11) (12) (13) (14) . Recent meta-analyses assessed performances of saliva RT-PCR tests for the diagnosis of COVID-19 (15) (16) (17) (18) and we recently confirmed in a large prospective study the excellent sensitivity of saliva RT-PCR, as compared to nasopharyngeal RT-PCR, for the detection of SARS-CoV-2 in community screening centers (19) .

The combination of saliva sampling with rapid point-of-care testing could facilitate screening and isolation of infected individuals. Rapid single use RT-PCR assays for SARS-CoV2 RNA detection are available but were validated mainly on nasopharyngeal samples (20, 21) and rarely in saliva (22) , require sophisticated equipment and remain expensive. Nucleic acid detections based on isothermal amplification, such as loop-mediated isothermal amplification (LAMP), are interesting approaches as . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint they simplify the analytical process, reduce the cost and enable to speed up the diagnosis. The sensitivity of RT-LAMP directly from NPS samples varies from 65% to 87 % compared to RT-PCR. Few studies tested RT-LAMP on self-collected saliva without RNA extraction. Sensitivities ranged from 45 to 85%, results being better after RNA purification than from crude samples (23) (24) (25) (26) . No studies estimated the performances of RT-LAMP on saliva samples as point-of-care systems directly in screening centers.

We conducted a prospective study in two community COVID-19 screening centers to evaluate the performances of a CE-marked RT-LAMP assay specifically designed for the detection of SARS-CoV2 RNA from fresh saliva samples compared to nasopharyngeal RT-PCR, saliva RT-PCR and nasopharyngeal Ag tests.

All adults and children, either symptomatic or asymptomatic, referred to the two participating COVISAN centers, Paris, France, were eligible as described previously (19) . In accordance with EasyCoV® assay manufacturer instructions of (SkillCell-Alcen, Jarry, France), performances of saliva RT-LAMP were estimated on saliva tested in screening centers, immediately after collection (< 5 minutes) or stored immediately at 4°C and then tested within a maximal 90 minutes interval after collection. In addition, patients should have a valid nasopharyngeal SARS-CoV-2 RT-PCR test. Eligible persons received oral and written detailed information, adapted to their age. Data on sociodemographics, past medical history, presence of symptoms, consumption of alcohol, coffee, food, smoking and teeth brushing in the hours before testing were collected. The NPS was sent to the APHP high throughput platform for RT-PCR as part of routine care (reference method). Participants were asked to self-collect saliva sample after salivating 30 second in their mouth. Saliva were tested directly in the screening center (see below) and then centralized for RT-PCR testing and frozen at -80°C within 24 hours.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) (27) . Ct values equal or higher than 32 corresponded to low viral loads.

Saliva RT-PCR Saliva samples were tested at the APHP high throughput platform with RT-PCR on MGI instrument as described previously ((19) , appendix). A 300 µl aliquot of saliva was mixed with 300 µl of NucliSENS® lysis buffer (Biomerieux, Marcy l'Etoile, France). Nucleic acid extraction and SARS Cov-2 RNA amplification were performed with the same procedure used for nasopharyngeal RT-PCR.

The test EasyCov® (SkillCell-Alcen, Jarry, France) is a CE-marked extraction-free RT-LAMP test specifically developed for saliva samples as point of care (saliva POC-LAMP). Detection of SARS-CoV2 was carried out according manufacturer's instructions (EasyCOV®, SkillCell) (appendix). The procedure includes a step of virus inactivation and lysis at 80 ° C for 10 minutes and a step of viral genome amplification at 65 ° C for 30 minutes. The two steps take place in the Easyvid® system. After amplification, a reagent sensitive to pH is added to reveal the amplification. The result is immediately . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Quantitative data were expressed as median [interquartile range], and qualitative data as counts (percentages). Diagnostic accuracy of the index tests was evaluated by calculating sensitivity and specificity. Confidence intervals were calculated by the exact binomial method. Subgroups analyses were performed according to: i) the presence of symptoms on day of testing, ii) the Ct value of the nasopharyngeal RT-PCR, expressed as low (at least one of the 3 targets with Ct ≤ 28, i.e. high viral . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint shedding), or high (all 3 targets with Ct > 28, i.e. low viral shedding), and iii) to the consumption of alcohol, coffee, food, and smoking or teeth brushing before sample collection.

Sensitivity analyses were performed considering 6 alternate criteria for positivity for the reference standard: i) ≥ 2 positive targets with nasopharyngeal RT-PCR, ii) ≥ 1 positive target with nasopharyngeal RT-PCR and at least one of the 3 targets with Ct < 32, iii) ≥ 1 positive target with saliva RT-PCR, iv) ≥ 1 positive target with either the nasopharyngeal or saliva RT-PCR, v) ≥ 1 positive target with either the nasopharyngeal or saliva RT-PCR and at least one of the 3 targets with Ct < 32, and vi) NPS antigen test.

Quantitative variables were compared with Wilcoxon's test, with a significance level of 5%. The statistical analysis was performed using R software (http://cran.r-project.org/). Reporting of results followed the Standards for Reporting Diagnostic accuracy studies (STARD 2015) guideline (28) .

The funding sources had no role in the study's design, conduct and reporting.

The IRB Ile-de France III approved the study protocol prior to data collection (approval number 3840-NI) and all subsequent amendments.

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Between November 4 th 2020 and February 15 th 2021, 1718 participants were enrolled with a nasopagryngeal sampling for RT-PCR and saliva sampling for RT-LAMP assay. Details of samples collected and tests performed for nasopharyngeal antigen assay and saliva RT-PCR are detailed in Figure 1 . Median age of study participants was of 37 years and 55% were females (Table 1) .

Indications for testing and clinical symptoms reported on day of inclusion are detailed in table 1. One to three symptoms were observed in 530/1712 (31%) participants.

Overall, 117/1718 (7%) tested positive on nasopharyngeal RT-PCR: 78/691 (11%) in symptomatic and 39/1027 (4%) in asymptomatic participants (Table 2) . Detection rates were of 2%, 9% and 7% for saliva RT-LAMP, saliva RT-PCR and nasopharyngeal Ag test, respectively.

Diagnostic accuracy of the two methods on saliva and the nasopharyngeal Ag test are presented in Table 3 . Compared to RT-PCR on NPS, the sensitivity of saliva RT-LAMP was 34% (95% Confidence Interval (95%CI): 26-44). The sensitivity of saliva RT-PCR was 93% (95%CI: 86-97) and those of nasopharyngeal Ag test was 85% (95%CI: 77-91). The sensitivity and specificity of saliva RT-LAMP were similar in symptomatic and asymptomatic participants. Sensitivity analyses of saliva RT-LAMP according to six references (Table 4) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint

In this large prospective controlled study, the performance of a rapid RT-LAMP assay performed with crude saliva samples directly after saliva collection was analyzed. We used a CE marked assay specifically designed for saliva samples and for a point-of-care use. The test was authorized in France on November 2020 in symptomatic individuals for whom nasopharyngeal sampling was impossible or difficult. Our results showed, in a real-life rigorous evaluation, a low sensitivity of this method (34%) compared to nasopharyngeal RT-PCR. Its sensitivity remained low whatever the reference test considered (saliva RT-PCR, nasopharyngeal Ag test), ranging between 28 to 37%.

Our results differed strongly with the sensitivity of 86% (95CI 78%-94%) reported in Santos Schneider et al. study (29) , and whatever the reference test used, nasopharyngeal RT-PCR or any other composite reference test including saliva RT-PCR and antigen test. In Santos Schneider et al. study, the authors evaluated the EasyCOV® assay in a central laboratory and tested each sample in triplicate. A sample was considered positive if at least two replicates out of three were positive. In our study, we tested all samples once directly in screening centers and according to manufacturer instructions, and to its expected use in routine conditions.

No difference in RT-LAMP sensitivity between symptomatic or asymptomatic participants was reported. Median time of testing was 3 days after symptom onset or 6 days after last contact of confirmed case. In 103 subjects already diagnosed for COVID-19, Nagura-Ikeda et al. reported, with another RT-LAMP assay, sensitivity results on saliva that differed according to the clinical state (23) .

The RT-LAMP assay was performed with nucleic acid extract of saliva instead of crude saliva. Overall sensitivity was of 71% compared to nasopharyngeal RT-PCR, with a higher sensitivity (85%) in patients tested within 9 days after symptom onset than after 9 days (44%). In asymptomatic individuals, the sensitivity was 60%.

According to other studies evaluating RT-LAMP tests, the critical step for sensitivity seemed to be the RNA extraction (13, 23, 25, 30) . A high level of concordance between RT-PCR on nasopharyngeal samples and RT-LAMP on saliva was observed when an automated extraction step (i.e. Qiasymphony RNA kit)

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The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint was used. In a limited series of 34 positive samples (17 nasopharyngeal swabs and 17 saliva) tested by RT-PCR, Taki et al. reported a sensitivity of a RT-fluorescence LAMP assay performed with nucleic acid extracts of 97% and 100% in nasopharyngeal and saliva samples, respectively (25) . Without RNA extraction on the same samples sensitivities decreased respectively to 71% and 47%, suggesting that RNA extraction process may be critical for the SARS-CoV-2 RNA detection by RT-LAMP especially for saliva samples. In our study, the RT-LAMP assay is an extraction-free test based on a 10 minutes heating at 80°C for virus inactivation and viral RNA release. This quick step suitable for a point-of-care test might be not optimal for RT-LAMP reaction with saliva samples and results may depend on miscellaneous factors according to the quality of saliva (volume, pH, viscosity, food by-products). The participants did not drink, eat or smoke within 30 minutes before saliva sampling. In addition, we did not find any significant effect of cigarettes or alcohol consumption within 2 or 24 hours. Another Finally, our study confirmed, as previously (19) , the good performance of saliva RT-PCR and nasopharyngeal antigen testing as reliable alternative strategies to detect SARS-CoV-2 in both symptomatic and asymptomatic individuals in the ambulatory setting. Further work is needed to optimize an assay combining collected saliva and rapid point-of-care isothermal detection of SARS-CoV-2 RNA.

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The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint Chest pain 50 (3) Smoking in the last 24 hours 331 (19) Consumption of alcohol in the last 24 hours 371 (22) Consumption of coffee in the last hour 347 (20) Teeth brushing in the last 2 hours 736 (43) Mouth washing in the last 2 hours 61 (4)

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The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint Figure 1 . Study flowchart.

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The copyright holder for this preprint this version posted June 16, 2021. ; https://doi.org/10.1101/2021.06.12.21258811 doi: medRxiv preprint within a maximal 90 minutes interval after collection. Briefly, 200 μl of saliva was mixed with the pretreatment buffer in tube 1 placed into the Easyvid® system for automated inactivation and lysis step (heating at 80 ° C for 10 minutes). Tube 1 is then taken out of Easyvid® and left to stand for 1 minute at room temperature. Three microliters of pretreated saliva sample are introduced into tube 2 containing RT-LAMP reaction mix. Tube 2 is incubated at 65 ° C for 30 minutes in dedicated positions of the Easyvid® system for viral RNA amplification. Once the 30 minutes have elapsed, tube 2 is taken out of the Easyvid® system and left to stand for 1 minute at room temperature. One microliter of revelation reagent is introduced in tube 2. The result is immediately read by visual observation. The color turns yellow for a sample positive for SARS-CoV2 RNA and remains orange for a sample negative for SARS-CoV2 RNA.

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The authors thank the following members of the scientific committee for insightful comments on the 

Saliva aliquots, stored frozen at minus 80°C, were thawed, equilibrated to room-temperature and then homogenized with a vortex for five seconds, and the 300 µl was mixed with 300 µl of NucliSENS® lysis buffer (Biomerieux, Marcy l'Etoile, France) and then extracted with the same procedure used for the nasopharyngeal samples. Saliva nucleic acids extracts were tested with the same RT-PCR procedure than for SARS-CoV-2 RT-PCR on nasopharyngeal samples.

The test EasyCov® (SkillCell-Alcen, Jarry, France) is a CE-marked extraction-free RT-LAMP test specifically developed for saliva samples. Detection of SARS-CoV-2 was carried out according manufacturer's instructions (EasyCOV®, SkillCell) (appendix). In each screening center, saliva samples were tested immediately after collection (<5 minute) or stored immediately at 4°C and then tested