key: cord-0732197-0lzoxvr6
authors: Laferl, Hermann; Seitz, Tamara; Baier-Grabner, Sebastian; Kelani, Hasan; Scholz, Elisabeth; Heger, Florian; Götzinger, Florian; Frischer, Thomas; Wenisch, Christoph; Allerberger, Franz
title: Evaluation of RT-qPCR of mouthwash and buccal swabs for detection of SARS-CoV-2 in children and adults
date: 2021-10-28
journal: Am J Infect Control
DOI: 10.1016/j.ajic.2021.10.019
sha: 882f48edfaf3e522e1728d851d4ee2609d96b1bc
doc_id: 732197
cord_uid: 0lzoxvr6

BACKGROUND: The use of nasopharyngeal (NP) swabs as a specimen collection method to diagnose SARS-CoV-2 infection is frequently perceived as uncomfortable by patients and requires trained personnel. In this study, detection rate of SARS-CoV-2 in mouthwash samples and buccal swabs were compared in both children and adults. MATERIAL AND METHODS: In patients admitted to hospital with confirmed COVID-19 within the previous 72 hours, NP and buccal swabs as well as mouthwash samples were collected. RT-qPCR was performed on all samples. RESULTS: In total, 170 samples were collected from 155 patients (137 adults and 18 children). Approximately 91.7% of the collected NP swabs were positive in RT-PCR compared to 63.1% of mouthwash samples and 42.4% of buccal swabs. Compared to NP swabs, the sensitivity of using mouthwash was 96.3% and 65.4% for buccal swabs in NP swab samples with a CT value <25. With increasing CT values, sensitivity decreased in both mouthwash and buccal swabs. The virus load was highest during the first week of infection, with a continuous decline observed in all three collection methods over time. DISCUSSION: Mouthwash presents an alternative collection method for detecting SARS-CoV-2 in the case of unfeasible NP swab sampling. Buccal swabs should not be used due to their low sensitivity.

Infection rates with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and subsequently morbidity and mortality remain high. Rapid identification and isolation of infected individuals are crucial for targeted infection control and mitigation measures. The current gold standard to diagnose early infection is the nucleic acid-based polymerase chain reaction (PCR) performed on naso-oropharyngeal swabs (NP). However, NP swab sampling is accompanied by several disadvantages. It is frequently perceived as uncomfortable by patients and is therefore not well tolerated particularly by children [1] [2] [3] . Furthermore, trained healthcare personnel with protective equipment are required for sampling [4, 5] . If sampling is not performed adequately its sensitivity declines [6] . In patients with complex anatomy or cases of improper sampling, adverse events, such as cerebrospinal fluid leakage, , temporomandibular joint dislocation and swab breakage in the nasopharyngeal area, may occur [7, 8] . Furthermore, especially in children, patients with mental disability or preexisting naso-or pharyngeal disease, NP swabs might not be obtainable.

Additionally, sneezing or coughing during NP swab sampling might generate aerosols which again could increase SARS-CoV-2 transmission.

Since viral replication of SARS-CoV-2 has been shown to be highest in the upper respiratory tract tissues [9] , the collection and evaluation of other respiratory specimens for SARS-CoV-2 detection should be considered. In a recent study, a high viral load of SARS-CoV-2 in sputum and saliva was reported [10] . However, critically ill adults or children are often unable to spontaneously produce sufficient amounts of saliva or sputum [10] . Findings of previous studies concerned with detection of SARS-CoV-2 in saliva specimens were limited by small sample size and diverse methodology [11] [12] [13] [14] . Whether PCR from saliva mouthwash is an accurate method to detect SARS-CoV-2 in symptomatic patients remains, therefore, yet to be determined.

This study aims to compare the sensitivity and specificity of saliva samples collected as mouthwash and buccal swabs (using a standardized collection method) with the gold standard, NP swabs, in adults and children. Mouthwash and buccal swabs facilitate producing and collecting saliva and avoid discomfort for the patient, with the potential for self-collection.

The study took place at two different hospitals in Vienna, Austria. Pediatric and adolescent patients were recruited at one site (Clinic Ottakring, Vienna), adult patients at another (Clinic Favoriten, Vienna). The study was approved by the local ethics committee of the Vienna Health Care Group (EK-20-190-0820).

The Saliva Collection System from Greiner Bio-One [15] was used to collect saliva samples. Collection occurred through thorough rinsing of the oral cavity for 2 minutes using the saliva extraction solution Balanced-Salt-Solution buffer. Subsequently, the solution was expelled into the small empty collection beaker of the Greiner Bio-One system.

Buccal swab sampling was performed on both buccal sides. NP swab sampling was performed on both nostrils and oropharynx sampling using a single swab. FLOQSwab (Modell) swab preserved in universal transport medium (Copan UTM system, both, CA, USA) was used.

Medicine, Clinic Favoriten due to COVID-19 (confirmed by RT-qPCR within the previous 72 hours) were asked to participate in the study. After obtaining written consent, mouthwash samples were collected by the patient under supervision by a heathcare personel. NP and buccal swab sampling were performed by a trained health care staff member.

At the second site, the Department of Pediatrics and Adolescent Medicine, Clinic Ottakring, patients above the age of 7 years were asked to participate in the trial.

Written informed consent was obtained by patients (age 14 to 18 years) or by patients and legal guardians (age 7 to 13 years). Due to small number of eligible pediatric inpatients with COVID-19, specimens were obtained either from pediatric in-and outpatient to facilitate recruitment. All inpatients were previously tested positive for SARS-CoV-2 by NP swab within 72 hours. Outpatients eligible for study entry either had to have contact with a known COVID-19 case and symptoms of any kind or signs and symptoms highly suspicious of SARS-CoV-2 infection (such as anosmia or atypical pneumonia) without known contact. Only outpatients who were tested positive for SARS-CoV-2 by in-house PCR at study entry, were included in data evaluation.

The NP and buccal swabs were stored in virus transport medium. All sample types were immediately sent to the laboratory, where RT-qPCR was performed within 24 hours after specimen collection. The reference method for comparing cycle threshold variables. For small sample numbers (n < 5), Fisher's exact test was used. The sensitivity and specificity were calculated using a 2x2 table. Pearson's correlation calculation was used to evaluate the correlation between cycle threshold (CT value) and time since symptom onset or outcome (death in hospital vs. discharge). A p-value < 0.05 was considered statistically significant. IBM SPSS Statistics 26 was used for statistical analysis.

At total of 170 samples were collected in 155 patients (137 adult and 18 pediatric patients). One patient refused a NP swab, in two patients mouthwash samples were not collected, two pediatric patients only tolerated oropharyngeal swab. In general, the sample collection method mouthwash and buccal swab was tolerated well from all participants.

The basic demographics and outcome parameter are listed in table 1. Mouthwash was shown to be significantly more sensitive compared to buccal swabs regarding the overall sensitivity (p < 0.001) and in NP swab samples with a CT value less than 30 (p = 0.021). As seen in Figure 1 mouthwash and buccal swab samples (p < 0.001).

A significant correlation between time from symptom onset and CT value was calculated using NP swabs (r = 0.420, p < 0.001), mouthwash (r = 0.442, p < 0.001), and buccal swabs (r = 0.315, p = 0.009). As depicted in Figure 2 , there is a peak at the start of symptoms, followed by a continuous decline. Compared to NP swabs, the sensitivity of mouthwash and buccal swab samples is highest during the first week since symptom onset. Subsequently, a strong decline in the sensitivity is observed for buccal swabs. The difference is demonstrated to be significant on days 7−12 (p = 0.057) and day > 13 (p = 0.039) since symptom onset.

The difference at up to day six since symptom onset was not observed to be significant (p = 0.057); however, a tendency was found.

The CT value in NP swab (r = -0.288, p = 0.004) and mouthwash samples (r = -0.249, p = 0.010) were demonstrated to correlate negatively with hospital mortality. There was no correlation found using buccal swabs (r = -0.128, p = 0.284).

Our data demonstrate that sample collection via mouthwash is a potential method that can be used if NP swabbing is not possible. Although CT values were shown to be higher in mouthwash samples and the overall sensitivity was significantly lower than in NP swab samples, the sensitivity in patients with high virus load (CT value ≤ 25) is 95.5% compared to NP swabs. In three patients, mouthwash samples were demonstrated to have a higher sensitivity than NP swabs.

Regarding buccal swabs, our findings are consistent with other studies [3, 16] that demonstrate the detection of SARS-CoV-2 via RT-qPCR in buccal specimens and a high viral load during the first week of disease or diagnosis. Similar to our findings, the authors report significantly higher CT values in buccal swab and lower sensitivity compared to NP swab.

Other reports on the use of saliva for SARS-CoV-2 detection emerged in the last months [11, 14] . However, these were criticized for the small sample size and that, unlike sampling with nasopharyngeal swabs, collection and processing methods for saliva specimens differed broadly among studies.

Mouthwash, that we used in our study, using the Saliva Collection System from Greiner Bio-One and following the given construction, enables standardized collection.

Interestingly, not only does the viral load influences the sensitivity (the higher the CT value, the less sensitivity), but the time from symptom onset was also found to play a role. The highest sensitivity was observed during the first week, correlating with the highest detected viral load in mouthwash samples.

Up to now, especially data from pediatric patients on SARS-CoV-2-detection in saliva specimens are scarce [17] . In recent trial, authors report a sensitivity of 87.7%

when compared to NP swabs in children presenting for COVID-19 screening [18] .

Guzmán-Ortiz et al. [19] report a sensitivity of 82.3% in saliva specimens compared to NP swabs from pediatric patients with COVID-19. Sensitivity was lower in this trial, which might be attributable to different methodology and a high-rate of asymptomatic patients in our pediatric population. In contrast to our study, in these studies saliva was collected by spitting in a collection tube with and without gargeling first.

Caulley et al. demonstrated that in paired NP and saliva samples taken from adults, in 20% of patients SARS-CoV-2-PCR was detected only in saliva specimens [20] . Cases of children that test positive for SARS-CoV-2 by PCR only in saliva samples have been reported in other trials [18, 19] No adverse events (such as aspiration, nausea, or vomiting) or abortion of mouth rinsing was observed during the study period. This study demonstrates that using mouth washes to detect SARS-CoV-2 could be both save and feasible, even in patients with dyspnea or children and adolescents with limited compliance.

Although several studies have evaluated saliva or sputum for SARS-CoV-2 detection, this is the first known trial utilizing mouthwash samples without gargling. The strength of the present study is the inclusion of both minors and adults, as well as symptomatic and asymptomatic individuals. However, the rate of asymptomatic patients was only 5.8%, therefore, data validity in this group is limited. Further studies are required to evaluate the sensitivity of mouthwash samples in individuals with asymptomatic or mild COVID-19. We included a high number of adults, but only 18 pediatric patients participated in the trial. Many children and/or legal guardians denied study participation due to fear of additional NP sampling. Another strength of the study is the standardized sample collection using a collection system.

Similar to previous studies, our data shows that detection of SARS-CoV-2 in saliva specimens is possible. However, due to the low sensitivity and higher CT values compared to the NP swabs, buccal swabbing does not represent a suitable screening modality for COVID-19.

Though, RT-qPCR of mouthwash samples appears to be a reliable, easy-to-use collection method for the detection of SARS-CoV-2. Despite a lower sensitivity compared to NP swabs overall, especially in the first week of symptom onset, in low resource settings (i.e. few NP sampling equipment, shortage of trained personnel), and patients, where NP sampling is not feasible (i.e. children, reduced collaboration, underlying medical conditions) detection of SARS-CoV-2 in mouthwash samples is a suitable alternative.

Perceived discomfort levels in healthy children participating in vaccine research

Epub 2013/08/13

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Validation of self-collected buccal swab and saliva as a diagnostic tool for COVID-19

Coronavirus disease 2019 (COVID-19): current status and future perspectives

How to Obtain a Nasopharyngeal Swab Specimen

SARS-CoV-2 Nasopharyngeal Swab Testing-False-Negative Results From a Pervasive Anatomical Misconception

Cerebrospinal Fluid Leak After Nasal Swab Testing for Coronavirus Disease

PubMed Central PMCID: PMCPMC7736753 Borsche has nothing to disclose. Conflict of interest: A. Balck has nothing to disclose. Conflict of interest: S. Taube has nothing to disclose. Conflict of interest: J. Rupp has nothing to disclose. Conflict of interest: C. Klein reports personal fees from Centogene GmbH, outside the submitted work

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Are saliva and deep throat sputum as reliable as common respiratory specimens for SARS-CoV-2 detection? A systematic review and meta-analysis

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Buccal swabs as non-invasive specimens for detection of severe acute respiratory syndrome coronavirus-2

Saliva as a gold-standard sample for SARS

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Clinical Utility of Buccal Swabs for Severe Acute Respiratory Syndrome Coronavirus 2 Detection in Coronavirus Disease 2019-Infected Children

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Pediatric Patients With Concurrent Conditions

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