key: cord-0847076-pue4mp4o
authors: Gobeille Paré, Sarah; Bestman‐Smith, Julie; Fafard, Judith; Doualla‐Bell, Florence; Jacob‐Wagner, Mariève; Lavallée, Christian; Charest, Hugues; Beauchemin, Stéphanie; Coutlée, François; Dumaresq, Jeannot; Busque, Lambert; St‐Hilaire, Manon; Lépine, Guylaine; Boucher, Valérie; Desforges, Marc; Goupil‐Sormany, Isabelle; Labbé, Annie‐Claude
title: Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS‐CoV‐2 detection using a laboratory‐developed test
date: 2021-11-03
journal: J Med Virol
DOI: 10.1002/jmv.27407
sha: dd4f23498114461cd338c034c8cd3fdb0425a0a8
doc_id: 847076
cord_uid: pue4mp4o

The objective of this study was to validate the use of spring water gargle (SWG) as an alternative to oral and nasopharyngeal swab (ONPS) for SARS‐CoV‐2 detection with a laboratory‐developed test. Healthcare workers and adults from the general population, presenting to one of two COVID‐19 screening clinics in Montréal and Québec City, were prospectively recruited to provide a gargle sample in addition to the standard ONPS. The paired specimens were analyzed using thermal lysis followed by a laboratory‐developed nucleic acid amplification test (LD‐NAAT) to detect SARS‐CoV‐2, and comparative performance analysis was performed. An individual was considered infected if a positive result was obtained on either sample. A total of 1297 adult participants were recruited. Invalid results (n = 18) were excluded from the analysis. SARS‐CoV‐2 was detected in 144/1279 (11.3%) participants: 126 from both samples, 15 only from ONPS, and 3 only from SWG. Overall, the sensitivity was 97.9% (95% CI: 93.7–99.3) for ONPS and 89.6% (95% CI: 83.4–93.6; p = 0.005) for SWG. The mean ONPS cycle threshold (C(t)) value was significantly lower for the concordant paired samples as compared to discordant ones (22.9 vs. 32.1; p < 0.001). In conclusion, using an LD‐NAAT with thermal lysis, SWG is a less sensitive sampling method than the ONPS. However, the higher acceptability of SWG might enable a higher rate of detection from a population‐based perspective. Nonetheless, in patients with a high clinical suspicion of COVID‐19, a repeated analysis with ONPS should be considered. The sensitivity of SWG using NAAT preceded by chemical extraction should be evaluated.

The SARS-CoV-2 epidemic that reached the province of Québec at the end of February 2020 mobilized the healthcare network in an unprecedented way. The Laboratoire de santé publique du Québec (LSPQ) rapid development of an in-house nucleic acid amplification test (NAAT) based on Corman and colleagues, 1 followed by the deployment of a dozen of commercial platforms allowed Québec's laboratory network to offer over 39 000 daily tests during the study period. 2 The exponential increase of COVID-19 cases during the second and third epidemic waves has put a strain on the healthcare system.

To meet the increased testing demands, a number of strategies had to be implemented to maintain mass-scale testing. These strategies had to consider the acceptability and cost of the sampling methods, and especially the performance of self-collected samples compared to collection methods by professionals.

The oral and nasopharyngeal swab (ONPS), the most recommended sample collection method for the detection of SARS-CoV-2, remains invasive and requires trained professionals. 3 Less invasive and self-collected specimens, such as saliva or gargle, have been proposed for COVID-19 diagnostic purposes. Systematic reviews on the sensitivity of SARS-CoV-2 testing using saliva revealed a good sensitivity as well as important drawbacks limiting its use, such as specimen viscosity and the inability of some individuals to produce sufficient quantity for testing. [4] [5] [6] There have been less publications using gargle as an alternative specimen for SARS-CoV-2 diagnostic. [7] [8] [9] [10] [11] [12] Gargles has some advantages over saliva for people who cannot produce or spit out saliva and for the processing of viscous saliva samples that require dilution before processing. However, the limited number of gargle samples studied, and the exclusive use of a saline solution for gargling, a compound incompatible with at least one commercial assay performed in our jurisdiction, 13 required further investigations before large-scale use.

To compare the sensitivity of the SARS-CoV-2 LSPQ-developed NAAT, preceded by thermal lysis, using spring water gargle (SWG) and ONPS.

The present study was part of the G-SPIT multicenter project comparing the performance of SWG for SARS-CoV-2 detection on various NAAT platforms, in a population at high risk to be infected with SARS-CoV-2. In this substudy, adults presenting at two COVID-19

walk-in clinics in Québec city (healthcare workers; HCW) and Montréal (HCW and general population) for routine testing were eligible to participate if they had a recent contact or symptoms compatible with COVID-19.

First, an ONPS was collected by a trained HCW by swabbing the posterior oropharynx and inserting the same flexible swab through one nostril, and rotating for 5-10 s before removing. 14, 15 The swab was transported in 3 ml of molecular water (RNase/DNase free).

Participants were asked not to eat, drink, or smoke for 15 min before collecting the SWG. They were handed a flexible cup with 5 ml of natural spring water (ESKA ® ) and were told to rinse their mouth and their throat for a total of 20 s (5 s in the mouth, 5 s in the throat, 5 s in the mouth, 5 s in the throat) and then to spit as much as possible in the initial cup. The content of the cup was emptied into a 15 ml conical tube and sent with the ONPS to the designated laboratory for processing and testing: in Québec City, at the CHU de Québec-Université Laval (CHUQ); in Montréal, at the Hôpital Maisonneuve-Rosemont (HMR). The samples were stored at 4°C and tested within 24-48 h according to the regular laboratory workflow.

SARS-CoV-2 testing was performed using an in-house laboratorydeveloped (LD)-NAAT test targeting the structural protein envelope E gene. 1, 16 Although the same SARS-CoV-2 primers and probes were used (TaqPath 1-Step Multiplex NO ROX; ThermoFisher Scientific cat no. A28523), the protocols in both laboratories differed in some aspects. At the CHUQ, 25 µl of the sample were first diluted with 25 µl of RNase-free water containing proteinase K (PK) (Qiagen cat n°19133; 200 µg/ml final concentration) and heated at 56°C for 10 min; at HMR, 50 µl of undiluted sample was used directly. Thermal lysis was performed on a thermal cycler: 90°C for 2 min (CHUQ) or 1 min (HMR), after which microwell plates were placed on a cooling bloc for at least 2 min. Five microliters were then added to 15 µl of SARS-CoV-2 master mixture including human internal controls pri- 

Participants were asked to answer few questions: age, contact with COVID-19, presence, and duration of COVID-19 symptoms.

In the absence of a gold standard for SARS-CoV-2 NAAT, data were first analyzed using a contingency table to assess the positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA), and κ statistics, 18 

The study received ethical approval from the review boards of each participating institution. Verbal informed consent was obtained from each participant.

Between November 19 and December 14, 1297 participants provided paired samples. As shown in Table 1 

As illustrated in Figure 1 

This study demonstrates that SWG is a less sensitive sampling method than ONPS to diagnose SARS-CoV-2 when used with thermal lysis and LD-NAAT in a COVID-19 high-risk population. When symptomatic participants were stratified according to the duration of their symptoms, the difference in sensitivity between the SWG and [25] [26] [27] The larger difference in sensitivity observed in Montréal (9.8%) than in Québec city (3.1%) is intriguing. It could be due, at least in part, to differences in positivity rate and characteristics of the study population. For example, the symptomatic HCW recruited in Québec city had symptoms for a shorter period of time before sampling compared to symptomatic participants in Montréal (Table 2) . Another possibility would be the use of proteinase K (PK) in the CHUQ protocol. Indeed, some studies suggested that pretreatment of samples with PK increased the sensitivity of direct rRT-PCR for SARS-CoV-2 detection, 28, 29 particularly in low positive samples with high C t values. 13 It has been suggested that PK could degrade or inactivate RNAses, allowing to purify a better quality of RNA. 30 Another study showed that sputum specimens pretreated with PK for homogenization before nucleic acid extraction for RT-PCR had a higher T A B L E 4 Agreement between the thermal lysis LD-NAAT and other assays, by sample type detection rate than those pretreated with saline only. The authors suggested this could be because PK is able to digest mucous protein, resulting in an increased concentration of extracted RNA. 31 Although SWG specimens are less mucous than sputum, they may have benefited from PK pretreatment.

Although the ΔC t between the ONPS and the SWG was variable among the concordant positive samples, it was mostly in favor of the ONPS. Again, this suggests a lower amount of SARS-CoV-2 RNA in SWG compared to ONPS, which would explain the loss of clinical sensitivity of SWG among samples with a low viral load. Indeed, the vast majority of false-negative SWG were paired with high C t -value ONPS samples (Figure 2 ). The analysis of SWG samples using more sensitive NAAT platforms or using several primer pairs might mitigate Participants were asked to use 5 ml of spring water to gargle, which is slightly more than the amount of molecular water (3 ml) used to transport the ONPS. This volume was chosen to allow participants to have enough liquid in their mouth to gargle effectively and is 

The authors declare that there are no conflict of interests.

Investigation, formal analysis, data curation, data interpretation, visua- 

Data are available from the corresponding author upon reasonable request.

François Coutlée http://orcid.org/0000-0002-2025-8928

Annie-Claude Labbé http://orcid.org/0000-0001-8936-3307

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Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS-CoV-2 detection using a laboratory-developed test