key: cord-0741041-19rkzb8b
authors: Labbé, Annie‐Claude; Benoit, Patrick; Gobeille Paré, Sarah; Coutlée, François; Lévesque, Simon; Bestman‐Smith, Julie; Dumaresq, Jeannot; Lavallée, Christian; Houle, Claudia; Martin, Philippe; Mak, Anton; Gervais, Philippe; Langevin, Stéphanie; Jacob‐Wagner, Mariève; Gagnon, Simon; St‐Hilaire, Manon; Lussier, Nathalie; Yechouron, Ariane; Roy, David; Roger, Michel; Fafard, Judith
title: Comparison of saliva with oral and nasopharyngeal swabs for SARS‐CoV‐2 detection on various commercial and laboratory‐developed assays
date: 2021-05-03
journal: J Med Virol
DOI: 10.1002/jmv.27026
sha: 1bfa854ecf25a4b0938ff269a57898916d4a0b31
doc_id: 741041
cord_uid: 19rkzb8b

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial element in the fight against coronavirus disease 2019 (COVID‐19). Reverse transcription‐polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS‐CoV‐2 on various commercial and laboratory‐developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS‐CoV‐2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS‐CoV‐2, Cobas® SARS‐CoV‐2, Simplexa™ COVID‐19 Direct, Allplex™ 2019‐nCoV, RIDA®GENE SARS‐CoV‐2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C (t)) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C (t) was 2.0, while individual ∆C (t) varied importantly from −17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS‐CoV‐2 by various assays.

Strategies for the prevention of transmission and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on accurate and timely diagnosis of the infection. The gold standard for the diagnosis of coronavirus disease 2019 (COVID-19) from the outset of the pandemic has been a reverse transcription-polymerase chain reaction (RT-PCR) test on a combined oral and nasopharyngeal swab (ONPS). ONPS is considered to have the highest sensitivity but suffers from several limitations. 1 First, a trained professional is required to obtain a specimen, which represents a substantial strain on human resources and poses a risk of infection transmission. Second, flocked swabs and transport media are prone to shortages in the context of the high number of tests. Third, this invasive sample collection is associated with significant discomfort which may impact on acceptability, particularly in repeated screening settings. This has motivated the search for new specimens, such as saliva, that yield acceptable results on commercial platforms and laboratorydeveloped tests (LDTs).

Salivary specimens have the advantage of being easily selfcollected and more acceptable by the patient than an ONPS. 2,3 A recent meta-analysis of 16 pooled studies using different protocols showed a similar sensitivity for SARS-CoV-2 detection when saliva and nasopharyngeal swab (NPS) specimens were compared. 4 Large unpooled studies are required to confirm these findings. Since most studies have been conducted using varying testing platforms, it remains unknown whether a specific nucleic acid amplification test (NAAT) protocol is more suitable for saliva than others.

The Laboratoire de Santé Publique du Québec (LSPQ), a provincial public health laboratory, therefore undertook a multicentric study in eight hospital laboratories to compare saliva samples to ONPS for SARS-CoV-2 detection. A secondary objective was to compare the difference in cycle threshold (C t ) values between saliva and ONPS samples. Individuals were eligible if they were ≥18 years old, and had SARS-CoV-2 RNA detected from an ONPS recently collected using a NAAT method. They were recruited between May 12, 2020 and June 25, 2020, if they accepted to provide another ONPS at the same time of a saliva sample. The time elapsed between the first positive test and the inclusion in the study was not recorded. This study was approved by the Provincial Public Health Authority and was conducted following the principles set out in the Helsinki Accord. Free and informed consent was obtained from the subjects. Samples were anonymized to maintain the confidentiality of participants and their utilization was restricted to SARS-CoV-2 nucleic acid detection only. Demographic characteristics and clinical information of participants were not collected.

Saliva was collected before ONPS samples at the same visit. Subjects were told to avoid smoking, drinking, eating, brushing teeth, or chewing gum at least 30 min before sample collection. Saliva samples were collected into 50 ml conical Falcon tubes or in 80 ml sterile plastic urine containers. Participants were instructed to spit repeatedly until 2-10 ml of saliva was obtained.

The ONPS was obtained by first sampling the oropharynx with a flocked swab and then the nasopharynx by inserting the same swab in the nostril to reach the nasopharyngeal cavity and rotating it a few times. Flocked swabs were placed into one of the following transport media: Modified Hanks Balanced Salt Solution, molecular biology grade water, or 0.9% saline.

All samples were sent to one of the eight participant laboratories on icepacks, where they were refrigerated between 2°C and 8°C until processing.

Saliva and ONPS were tested in parallel in the aforementioned laboratories using in-house or commercial SARS-CoV-2 NAAT. Since some laboratories had more than one platform, the 125 specimen pairs were tested with up to four different NAATs, for a total of 320 distinct tests. The volume of saliva was recorded and, when sufficient volume was available (n = 24), 1 ml of saliva was aliquoted to test the undiluted sample in parallel with the diluted sample which was processed as follows: An equal volume of molecular biology grade water was mixed with the remaining saliva sample (dilution 1:1) to decrease the viscosity. All saliva samples (undiluted and diluted) were vortexed using biosafety work practices and when deemed required by the technician, highly viscous saliva samples were cen- Pure platform or the Cobas ® 4800 system (Roche) as previously described. 5 For thermal lysis, 20-25 µl of the sample was diluted 1:1 in molecular grade water, heated at 90°C for 2 min and then cooled to 4°C for 3 min. Five microlitres were taken directly as a template for RT-PCR (LSPQ in-house protocol). None of the specimens were inactivated with an external lysis buffer before being processed on any of the platforms mentioned.

Diagnostics of SARS-CoV-2 viral infection were conducted based on an RT-PCR approach. All procedures were conducted according to the different manufacturer's recommendations. RT-PCR targets are listed in Table 1 .

All recruited individuals (n = 125) had at least one positive NAAT result either on saliva or ONPS. Thus, in the calculations of specimen performance, all individuals were considered to be SARS-CoV-2 positive. The sensitivity of assays and concordance between assays was calculated with a 95% confidence interval. Differences in continuous variables were assessed using the two-sample Wilcoxon rank-sum (Mann-Whitney) test. Statistical analyses were done using STATA v16.1 (College Station). The secondary objective of the study was to compare C t in both specimen types. The mean difference in C t when NAAT was positive for both samples was slightly (but not significantly) in favor of the ONPS for all NAATs, except for LDT with RNA extraction with the Cobas ® 4800

( Table 2 ). The maximum mean ΔC t was 2.0, while individual ΔC t varied importantly from −17.5 to 12.4. When discordant results were analyzed (ONPS was positive and saliva negative), the mean C t on ONPS tended to be higher than when both results were found to be positive (Table 3) .

Those mean C t differences were statistically significant for the Abbott When a sample was tested on different platforms, the concordance was high, as only eight saliva samples (6.4%) were found to be discordant (data not shown). As reported by Barat et al., 6 individuals with a false negative result on saliva samples were associated with a higher C t value on ONPS. As higher C t values are associated with lower viral loads, as occurs with resolved infections, the public health and clinical consequences of a false-negative result in this context would probably be diminished. 7 High C t values can also occur when a person is tested early in the disease course (and therefore infectious); to lessen the consequences of a false-negative result, 8 it is crucial to consider the epidemiological and clinical con- collect and manipulate. 13 Future studies will assess if this type of specimen is easier to process and if sensitivity is decreased by diluting saliva in water.

This study shows that saliva is a suitable sample for the detection of SARS-CoV-2 by RT-PCR, with a similar sensitivity to ONPS on multiple available assays. The nonstatistically significant trend toward a higher sensitivity of the ONPS was driven by some false-negative results on saliva samples when high C t was obtained on ONPS paired samples. The clinical consequences of a false negative result in a low viral load infection are still unclear.

Sarah Gobeille Paré: Investigation, data interpretation, writing -original draft. François Coutlée: Conceptualization, methodology, data interpretation, writing -review and editing. Simon Lévesque: Data interpretation, writing -review and editing. Julie Bestman-Smith: Conceptualization, methodology, writing -review and editing

Ariane Yechouron: Investigation, formal analysis, writing -review and editing. David Roy: Data interpretation, writing -original draft. Michel Roger: Project administration, writing -review and editing. Judith Fafard: Conceptualization, methodology, data curation, formal analysis, data interpretation

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The authors are thankful to all study participants and laboratory technicians.

The authors declare that there are no conflict of interests.

https://orcid.org/0000-0001-8936-3307François Coutlée https://orcid.org/0000-0002-2025-8928