key: cord-0428777-cx46pwqt
authors: Saluzzo, F.; Mantegani, P.; Poletti de Chaurand, V.; Cugnata, F.; Rovere-Querini, P.; Cilla, M.; Erba, P.; Racca, S.; Tresoldi, C.; Uberti-Foppa, C.; Di Serio, C.; Cirillo, D. M.
title: Salivary molecular testing for SARS-CoV-2: simplifying the diagnosis without losing accuracy
date: 2021-07-22
journal: nan
DOI: 10.1101/2021.07.18.21260706
sha: 02678008edcb4e2076250683ebf92343fcf61883
doc_id: 428777
cord_uid: cx46pwqt

Background Quantitative RT-PCR on NasoPharyngeal Swab (NPS) is still considered the standard for the diagnosis of SARS-CoV-2 infection, even if saliva has been evaluated in several studies as a possible alternative. The use of point of care (POC) platforms, providing highly specific results performed on saliva could simplify the diagnosis of COVID-19 and contribute to contain the spreading of SARS-CoV-2. Methods We assess the sensitivity and specificity of molecular testing performed on saliva in comparison to NPS using two different POC platforms (DiaSorin Simplexa and Cepheid Xpert (R)). NPS and saliva were collected prospectically from asymptomatic health care workers and mildly symptomatic patients. Moreover, the stability of saliva samples after storage at -80 C {degrees} for up to 45 days was tested. Results The obtained results in comparison to NPS demonstrated for both DiaSorin Simplexa and Xpert (R) Xpress a specificity of 100% and a sensitivity of 90.24%. The overall agreement between the tests performed on saliva was 98%. A positive correlation in Ct values detected on saliva and on NPS was identified for all the targets shared by the tests in analysis (Orf1ab, E and N2). Both S Ct values and Orf1ab Ct values were not significantly different before and after the freezing in the tested saliva samples. Conclusion The obtained results demonstrated an overall performance of saliva comparable to NPS, confirming that RT-PCR performed using POCs on saliva could represent a valid public health solution for controlling SARS-CoV-2 pandemic.

stability of saliva samples after storage at -80°C for up to 45 days was tested. 23

The obtained results in comparison to NPS demonstrated for both DiaSorin Simplexa™ and Xpert® Xpress a 25 specificity of 100% and a sensitivity of 90.24%. The overall agreement between the tests performed on saliva 26 was 98%. A positive correlation in Ct values detected on saliva and on NPS was identified for all the targets 27 shared by the tests in analysis (Orf1ab, E and N2). Both S Ct values and Orf1ab Ct values were not significantly 28 different before and after the freezing in the tested saliva samples. 29

The obtained results demonstrated an overall performance of saliva comparable to NPS, confirming that RT-31 PCR performed using POCs on saliva could represent a valid public health solution for controlling SARS-32

CoV-2 pandemic. 33 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 July 22, 2021.

The possibility to rely on rapid and accurate diagnostic techniques has proved itself crucial during the last year 35 to contain the spreading of SARS-CoV-2 infection 1 

NasoPharyngeal Swab (NPS) is still considered the standard for the diagnosis of COVID19, saliva has been 37 evaluated in several studies as a possible alternative to NPS and it is now extensively utilized in South Korea, 38

Germany, and Japan 2,3 . Nonetheless, the use of saliva is still debated, and a rigorous standardization of the 39 analysis protocol is deeply needed 4-6 . The application of point of care (POC) technologies on saliva, capable 40 of rapidly performing high specific and sensitive molecular testing, could prove invaluable to allow the 41 diagnosis even in challenging and remote settings, simplifying and speeding up the diagnostic process 1 . 42

To assess the sensitivity and specificity of molecular testing performed on saliva in comparison to NPS using 44 two different POC platforms (DiaSorin Simplexa™ and Cepheid Xpert®), a total 129 individuals were 45 enrolled into the study. Samples were collected prospectically, from January 2021 to May 2021, from 21 46 asymptomatic health care workers, who took part to the screening campaign, and from 79 outpatients who had 47 developed mild symptoms consistent with COVID19 up to 10 days before accessing the Preventive Medicine 48

Hospital, Milan. Moreover, samples from 29 patients, hospitalized for COVID19 in March 2020, were 50 retrieved by San Raffaele Hospital Biobank. For each patient, a self-collected saliva sample and an NPS, 51 collected at the same timepoint by a healthcare worker, were analysed. 52

With the exclusion of the samples collected in March 2020, that were stored at -80°C immediately after the 53 sampling, all samples were preserved at 4°C and analysed within 24 hours from the collection. The DiaSorin 54

Simplexa™ COVID-19 Direct tests (Simplexa™) were performed on saliva diluted 1:1 with saline as per the 55 instruction for use, and the same condition was used off label for the Xpert® Xpress SARS-CoV-2 kit 56 (Xpert®). NPS were analysed with Xpert® Xpress SARS-COV-2 or Roche Cobas® SARS-CoV-2 test 57 (Cobas®), as by manufacturers' instructions. 58

The results obtained on saliva samples collected prospectively in the first months of 2021, in comparison to 60 NPS demonstrated for both Simplexa™ and Xpert® a specificity of 100% (95% CI: 93.94%-100%) and a 61 sensitivity of 90.24% (95% CI: 76.87%-97.28%). The overall agreement between the two tests performed on 62 saliva was of 98%. 63

Since the two kits employed on saliva, as well as the ones utilized on NPS, adopted different target genes 64 (Simplexa™: Orf1ab and S; Xpert®: N2 and E; Cobas®: E and Orf1ab) an analysis to evaluate the correlation 65 between Cycle threshold (Ct) values detected respectively on saliva and on NPS was performed only for the 66 shared targets. 67 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. of Xpert® performed on saliva and on NPS (Fig.1 A) . 71

Hence, when compared to NPS, both tests on saliva freshly collected appeared to have good sensitivity and 72 specificity as well as a positive correlation in Ct values detected for the shared targets. (which was not certified by peer review) 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 July 22, 2021. ; https://doi.org/10.1101/2021.07.18.21260706 doi: medRxiv preprint

Commercialized diagnostic technologies to combat SARS-CoV2: Advantages 128 and disadvantages

Saliva as a gold-standard sample for SARS-131

CoV-2 detection

Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2

Exploring salivary 144 diagnostics in COVID-19: a scoping review and research suggestions

Saliva as an Alternate Specimen Source for Detection of SARS-CoV-2 in Symptomatic Patients Using 148

Cepheid Xpert Xpress SARS-CoV-2

Evaluation on testing of deep throat saliva and 151 lower respiratory tract specimens with Xpert Xpress SARS-CoV-2 assay

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) 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 July 22, 2021. ; https://doi.org/10.1101/2021.07.18.21260706 doi: medRxiv preprint Both Xpert® and Simplexa™ platforms proved themselves practical and easy to use on saliva and the obtained 101 results demonstrated an overall performance comparable to NPS, with a specificity of 100% and a sensitivity 102 higher than 90% for freshly collected samples and higher than 87% for the ones stored at -80°C, demonstrating 103 the possibility to perform these tests also on frozen samples with only a minimal loss in sensitivity. It is 104 interesting to notice that the samples contained all the different SARS-CoV-2 variants of concern currently 105 represented in Italy, but both kits' performance was not compromised by this factor. 106The tests employed exhibited an overall excellent level of agreement, even considering the differences 107 identified once the Biobank samples have been included into the analysis. 108As the pandemic evolves, the implementation of a testing strategy based on POCs widespread on the territory 109 in a capillary manner, could help to guarantee a prompt on-site diagnosis, allowing the rapid identification and 110 control of clusters and outbreaks, finally protecting the community from the transmission. Moreover, if this 111 new diagnostic plan would involve the use of self-collecting highly reliable samples, as saliva, directly at 112 patients' homes, we would reduce the burden on healthcare workers, and the costs related to the use of NPS 113 with specific transport medium. This approach would also contribute to drastically decreasing the number of 114 possible infective individuals moving to reach the sampling hubs, who could represent a major public health 115

This diagnostic approach could be easily implemented also in Low Middle Income Countries, where POCs 117 platforms are already widely employed for the diagnosis of other illnesses, as Tuberculosis, HIV and viral 118

In conclusion, our findings support the use of saliva on POCs technologies as a valid solution to simplify, 120 speed up and widespread the diagnostic process for the control of COVID19 epidemic. 121 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) 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 July 22, 2021. ; https://doi.org/10.1101/2021.07.18.21260706 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) 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 July 22, 2021. ; https://doi.org/10.1101/2021.07.18.21260706 doi: medRxiv preprint