A combined oro-nasopharyngeal swab is more sensitive than mouthwash in detecting SARS-CoV-2 by a high-throughput PCR assay

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The optimal diagnostic specimen to detect SARS -CoV-2 by PCR in the 22 upper respiratory tract is unclear. Mouthwash fluid has been reported as an alternative 23 to nasopharyngeal and oropharyngeal swabs. We compared mouthwash fluid with a 24 combined oro-nasopharyngeal swab regarding test performance. 25

We tested asymptomatic persons with a previous diagnosis of COVID -19 26 and their household contacts. First, a mouthwash (gargling for at least 5 sec) with 27 sterile water was performed. Then, with a si ngle flocked swab the back of the throat 28 and subsequently the nasopharynx were sampled. Samples were inactivated and 29 analysed on a Roche cobas 6800® system with the Roche SARS-CoV-2 test. 30

Of 76 persons, 39 (51%) tested positive for SARS -CoV-2 by oro-31 nasopharyngeal swab. Mouthwash detected 13 (17%) of these infections but did not 32 detect any additional infection. Samples that were positive in both tests, had lower 33 cycle threshold (Ct)-values for oro-nasopharyngeal samples, indicating a higher virus 34 concentration, compared to samples only positive in oro-nasopharyngeal swabs. 35

Mouthwash is not as sensitive as combined oro -nasopharyngeal swab 36 in detecting upper respiratory tract infection. 37 38 39 40 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 3

41 In December 2019, a new lung disease called Coronavirus Disease 2019 (COVID-19) 42 first appeared in Wuhan, China, and subsequently spread globally [1]. The causative 43 agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-44 CoV-2 is a n enveloped, single -stranded positive-sense RNA virus. To gether with 45 SARS-1 and MERS coronavirus it is classified in the Orthocoronaviridae subfamily, 46 genus Betacoronavirus [2]. 47 SARS-CoV-2 is efficiently transmitted from person to person by respiratory droplets [3, 48 4]. Rapid and accurate detection of the virus is essential to contain outbreaks. The 49 most suitable diagnostic specimen is still unclear, as the virus is detectable in different 50 respiratory specimens, urine, and stool [5, 6]. One recommended diagnostic specimen 51 for S ARS-CoV-2 detection is the nasopharyngeal swab [7], but combined naso -52 oropharyngeal swabs can increase the sensivity of SARS-CoV-2 detection [4]. A meta-53 analysis of different SARS -CoV-2 studies showed the highest detection rates in 54 sputum, followed by nasopharyngeal and then oropharyngeal swab samples [8]. In 55 severe cases of COVID-19 or at later stages in the disease , SARS -CoV-2 can be 56 detected in samples from the lower respiratory tract , such as sputum or bronchial 57 aspirate [9]. 58 Expected shortages of swabs led us to assess alternative diagnostic specimens. In 59 this study, we compared test performance when using mouthwash or a combined oro-60 nasopharyngeal swab. 61 62 63

64 Residents (age >6 years) from a refugee facility with a previous diagnosis of COVID -65 19 and their household contacts were prospectively tested for SARS -CoV-2 with 66 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 4 mouthwash and a combined oro-nasopharyngeal swab in controlled conditions on two 67 occasions in May 2020 during an outbreak in the facility. Symptoms of COVID-19 were 68 recorded using a standardized questionnaire. Samples were taken by previously 69 instructed medical personnel. A single flocked swab (eSwab™ Copan) was used to 70 sample the back of the throat and subsequently the deep nasopharynx. For the 71 mouthwash, residents were instructed to gargle the mouth with 10 ml sterile water for 72 at least 5 seconds. Samples were transported at room temperature and stored 73 overnight at 4°C. All samples were mixed 1:1 with ATL buffer and analysed with the 74 cobas® SARS -CoV-2 ass ay on the Roche cobas 6800 system according to the 75 manufacturer's instructions . Detection of the E-(envelope)-gene and Orf1/a (open 76 reading frame 1) or only E-gene or only Orf1/a were interpreted as confirmation of 77 SARS-CoV-2 infection. Cycle threshold ( Ct)-values above 40 were considered as 78 negative. 79 The study was performed according to the principles of the Declaration of Helsinki. 80 Approval was obtained from the ethics committee of the Medical Faculty of the Otto -81 von-Guericke University Magdeburg . Written informed consent was obtained by all 82 participants or their guardians. 83 84 85

86 Overall, 64 asymptomatic persons with a previous diagnosis of COVID -19 and their 87 household contacts were tested on two occasions. Age ranged from 7 to 59 years, with 88 an average age of 29 years. At the time of testing, no person showed symptoms of 89 COVID-19. Fifteen persons recollected symptoms compatible with COVID -19 in the 90 past three weeks: cough (6 persons), headache (4 persons), rhinitis (4 persons), loss 91 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 5 of taste (2 persons), fatigue (2 persons), fever, sneezing, aching limbs, diarrhoea, and 92 mild shortness of breath (one person each). No person was hospitalised. 93 94 In 39 of the 76 participants (51%), PCR of the combined oro-nasopharyngeal swab 95 confirmed SARS-CoV2 infection. In contrast, SARS -CoV-2 was detected in 13 96 mouthwashes (17 %) of which all were positive by the oro-nasopharyngeal swab (table 97 1). When considering the oro -nasopharyngeal swab as gold standard, the sensitivity 98 of mouthwash was 33%. 99 100 The cycle threshold (Ct)-value is a measure for the abundance of the transcript in the 101 sample and correlates with viral load. When comparing Ct -values of oro-102 nasopharyngeal swabs, specimens that were positive by mouthwash had lower Ct -103 values than specimens negative by mouthwash , indicating a lower viral load in 104 mouthwash (Figure 1a). Samples that were positive by both methods showed higher 105 Ct-values in the mouthwash (Figure 1b). All results are consistent with a lower 106 sensitivity of detection in mouthwash. 107 108 109

110 The shortage of swabs that are suitable for PCR diagnostics led us to explore the utility 111 of mouthwash in a controlled study. We found a very low sensitivity of mouthwash 112 (33%), when using oro-nasopharyngeal swabs as comparator. We speculate that this 113 striking difference in sensitivity is partly due to the dilution of the mouthwash sample. 114 Thus, mouthwash is not suitable for the reliable detection of SARS-CoV-2 infection. 115 116 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 6 The only published study reported that the rate of positivity for SARS-CoV-2 was higher 117 in self-collected throat washings with sterile nor mal saline than in nasopharyngeal 118 swabs [10]. However, the small sample size of eleven patients does not allow firm 119 conclusions. 120 121 Our study has several strengths: We conducted the study in a controlled setting with 122 specifically trained personnel. This allows for a more rigorously sampling than in an 123 observational study conducted in the clinical setting. As gold standard , we chose 124 combined oro-nasopharyngeal swabs. A systematic review that assessed the positivity 125 rate of different specimens found that nasopharyngeal swabs had a slightly higher 126 positivity rate than oropharyngeal swabs, with larger differences when sampling was 127 performed more than 14 days after symptom onset [8]. 128 129 Our study population were asymptomatic persons, with a median time after diagnosis 130 of 14 days, and their household contact. Since the viral load decreases over time, this 131 population is expected to have a low viral load and thus high Ct-values. Indeed, 34 of 132 38 (89%) samples had Ct-values above 30 for the E-gene, a value currently discussed 133 as a cut-off for infection. Thus, this study was designed to rigorously assess differences 134 in sensitivity. 135 136 Our study has also limitations. Mouthwash with gargling was performed as a self -137 administered procedure and we observed some variation in adherence to the protocol 138 regarding the duration and intensity of gargling, which may have influenced the results. 139 Furthermore, we did not compare different RNA extraction methods, which may show 140 a better performance with mouthwash specimens. 141 142 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 7 There is a high likelihood of aerosol formation during gargling. Thus, mouthwash 143 should be performed alone in a well -ventilated area. This may limit its use in patients 144 to minimize exposure of health -care personel. In conclusion, SARS-CoV2 detection 145 with mouthwash showed a low sensitivity compared to oro -nasopharyngeal swabs. 146 Thus, we do not recommend mouthwash performing combined oro-nasopharyngeal 147 swabs, especially in patients with no or mild symptoms. 148 149 Transparency declaration 150 JF reports personal fees from Biomé rieux and the Medical Association of Saxony -151 Anhalt, outside the submitted work; WM, MD, IT, JB and AJK report no conflicts of 152 interest. There was no specific funding for this study. 153 154 Contribution 155 AJK and IT designed the study; DM, JB, IT and AJK conducted the investigation; MW, 156 BJ, and AJK edited, reviewed and interpreted the data; WM and AJK wrote the original 157 draft and all authors approved the manuscript. 158 159

160 We thank the technical personnel of the Institute of Medical Microbiology and Hospital 161 Hygiene and the Institute of Transfusion Medicine, Medical Faculty, Otto-von-Guericke 162 University, for technical support. 163 164 165 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 8

166 [1] Zhu N, Zhang D, Wang W, et al. A Novel Coronavirus from Patients with 167 Pneumonia in China, 2019. N Engl J Med. 2020;382(8):727-733. 168 doi:10.1056/NEJMoa2001017 169 [2] Ludwig S, Zarbock A. Coronaviruses and SARS-CoV-2: A Brief Overview. 170 Anesth Analg. 2020;131(1):93-96. doi:10.1213/ANE.0000000000004845 171 [3] WHO. Scientific Brief: Transmission of SARS-CoV-2: implications for infection 172 prevention precautions. URL: https://www.who.int/news-173 room/commentaries/detail/transmission-of-sars-cov-2-implications-for-174 infection-prevention-precautions 175 [4] Yan Y, Chang L, Wang L. Laboratory testing of SARS-CoV, MERS-CoV, and 176 SARS-CoV-2 (2019-nCoV): Current status, challenges, and countermeasures. 177 Rev Med Virol. 2020;30(3):e2106. doi:10.1002/rmv.2106 178 [5] Sun J, Zhu A, Li H, et al. Isolation of infectious SARS-CoV-2 from urine of a 179 COVID-19 patient. 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CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 9 EBioMedicine, 2020, 102903, ISSN 2352-3964, 192 https://doi.org/10.1016/j.ebiom.2020.102903 193 [9] Wang K, Zhang X, Sun J, et al. Differences of Severe Acute Respiratory 194 Syndrome Coronavirus 2 Shedding Duration in Sputum and Nasopharyngeal 195 Swab Specimens Among Adult Inpatients With Coronavirus Disease 2019 196 [published online ahead of print, 2020 Jun 20]. Chest. 2020;S0012-197 3692(20)31718-9. doi:10.1016/j.chest.2020.06.015 198 [10] Guo WL, Jiang Q, Ye F, et al. Effect of throat washings on detection of 199 2019 novel coronavirus [published online ahead of print, 2020 Apr 9]. Clin 200 Infect Dis. 2020;ciaa416. doi:10.1093/cid/ciaa416 201 [11] Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in Different 202 Types of Clinical Specimens. JAMA. 2020;323(18):1843-1844. 203 doi:10.1001/jama.2020.3786 204 [12] Ek P, Böttiger B, Dahlman D, Hansen KB, Nyman M, Nilsson AC. A 205 combination of naso- and oropharyngeal swabs improves the diagnostic yield 206 of respiratory viruses in adult emergency department patients. Infect Dis 207 (Lond). 2019;51(4):241-248. doi:10.1080/23744235.2018.1546055 208 [13] Mawaddah A, Gendeh HS, Lum SG, Marina MB. Upper respiratory tract 209 sampling in COVID-19. Malays J Pathol. 2020;42(1):23-35 210 211 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 10 212 213 Table 1: Comparison of m outhwash and combined oro-nasopharyngeal swab in 214 detecting SARS-CoV-2. The sensitivity of mouthwash is 33% , the specificity 100% 215 when using the combined oro-nasopharyngeal swab as gold standard (McNemar test 216 p-value <0.001). 217 218 oro-nasopharyngeal swab positive oro-nasopharyngeal swab negative total mouthwash positive 13 0 13 mouthwash negative 26 37 63 total 39 37 76 219 220 221 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint 11 222 223 224 225 Figure 1: The combined oro-nasopharyngeal swab has a higher sensitivity than 226 mouthwash. a) Cycle threshold (CT) values for Orf1/a- and E-gene for oro-227 nasopharyngeal swabs in samples positive and negative in mouth wash. A lower CT -228 value indicates a higher viral load. (Mann-Whitney U -Test E-gene: p -value<0.001, 229 Orf1/a-gene: p-value=0.036) b) Ct-values for samples positive in both specimen types. 230 Ct-values for the mouthwash were higher than for the combined oro-nasopharyngeal 231 swabs, indicating a lower viral load in mouthwash . Only 12 paired samples were 232 shown, since one sample was positive in the E-gene and another in the Orf1/a-gene, 233 only (Wilcoxon signed rank test E-gene: p-value=0.007, Orf1/a-gene: p-value=0.037). 234 235 236 . CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 27, 2020. ; https://doi.org/10.1101/2020.09.25.20201541doi: medRxiv preprint