key: cord-0961530-vgo80lm0 authors: Mäkelä, H.; Poukka, E.; Hagberg, L.; Vo, T.; Nohynek, H.; Ikonen, N.; Liitsola, K.; Helve, O.; Savolainen-Kopra, C.; Dub, T. title: Detection of SARS-CoV-2 infection in gargle, spit and sputum specimens date: 2021-05-03 journal: nan DOI: 10.1101/2021.05.02.21255857 sha: f5dc9348e41d8efed94d3410d8f1aa0fffca0e59 doc_id: 961530 cord_uid: vgo80lm0 The gold standard for SARS-CoV-2 infection diagnosis is RT-PCR from nasopharyngeal specimen (NPS). Its collection involves a close contact between patients and healthcare workers requiring a significant amount of workforce and putting them at risk of infection. We evaluated self-collection of alternative specimens and compared their sensitivity and Ct values to NPS. We visited acute COVID-19 outpatients to collect concomitant nasopharyngeal and gargle specimens and had patients self-collect a gargle and either sputum or spit specimens on the next morning. We included 40 patients and collected 40 concomitant nasopharyngeal and gargle specimens, as well as 40 gargle, 22 spit and 16 sputum specimens on the next day, as 2 patients could not produce sputum. All specimens were as sensitive as NPS. Gargle specimens had a sensitivity of 0.97 (CI 95% 0.92-1,00), whether collected concomitantly to NPS or on the next morning. Next morning spit and sputum specimens showed a sensitivity of 1.00 CI (95% 1.00-1.00) and 0.94 (CI 95% 0.87-1.00), respectively. The gargle specimens had a significantly higher mean cycle threshold (Ct) values, 29.89 (SD 4.63) (p-value <0.001) and 29.25 (SD 3.99) (p-value <0.001) when collected concomitantly and on the next morning compared to NPS (22.07, SD 4.63). Ct value obtained with spit (23.51, SD 4.57, p-value 0.11) and sputum (25.82, SD 9.21, p-value 0.28) specimens were close to NPS. All alternative specimen collection methods were as sensitive as NPS, but spit collection appeared more promising, with a low Ct value and ease of collection. Our findings warrant further investigation. In December 2019, SARS-CoV-2 emerged from the Chinese city of Wuhan (1) . The disease spread 36 into countries outside China and was declared a global pandemic in March 2020 (2) . Within a year, 37 more than 115 million COVID-19 cases were confirmed, including two and a half million deaths 38 (3). 39 The SARS-CoV-2 pandemic control relies on a test-trace-isolate strategy with early diagnosis and 40 isolation of infected individuals and identification of their contacts (4) which has led to an initial 41 shortage of personal protective and sampling equipment, as well as increasing healthcare workers' 42 workload (6,7). Alone in Finland, a country with approximately 5 500 000 inhabitants, by the 20 th 43 . CC-BY-NC-ND 4.0 International license It is made available under a 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 May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint 3 of March 2021 over 3 700 000 tests, of which 145 000 during the first week of March 2021, were 44 conducted and analyzed nationwide during the COVID-19 pandemic (5,6). 45 Collection of nasopharyngeal specimen (NPS) is the gold standard for SARS-CoV-2 infection 46 diagnosis (7) . However, it is an unpleasant procedure requiring a close contact with HCW with a 47 risk of discomfort and in worst cases, epistaxis for the patient, and infections exposure for the 48 HCW (8, 9) . The use of an alternative specimen collection method could increase specimen 49 collection and testing capacities as well as decrease HCWs' workload and risk for infection (8) . 50 There has been several studies evaluating alternative specimen collection methods but none of 51 the specimen collection methods have yet superseded NPS (10-13), even though one Finnish 52 private healthcare provider now offers asymptomatic patients the possibility to self-collect a 53 gargle specimen as an alternative to NPS (14). 54 We evaluated and compared three alternative specimen collection methods that would not 55 require close contact to a HCW and compared their sensitivity and Ct values to NPS. 56 57 Materials and methods 58 We contacted confirmed COVID-19 outpatients who had been diagnosed with SARS-CoV-2 59 infection by RT-PCR (reverse transcription polymerase chain reaction) on NPS a few days earlier. 60 Children under 2 years old were not eligible for participation. After calling the patient for 61 recruitment, we visited them on the same and following day. 62 During the first home visit, we gathered informed consent, gave participants a link to an online 63 symptom-questionnaire and collected a NPS and gargle specimen (gargle 1). We also gave them 64 instructions and containers for collection of gargle (gargle 2) and, depending on the recruitment 65 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) week, sputum or spit specimen on the next morning. On the following day, the patients collected 66 themselves the second gargle (gargle 2) and either spit or sputum specimens. The next day the 67 self-collected specimens were collected. The timeline of the study is presented in Figure 1 . 68 All alternative specimens were collected into a 70 ml plastic container. To collect the gargle 69 specimens, the patients were asked to have sip of water, and gargle it for 5-20 seconds before 70 spitting it in the container. For spit collection, the patients were asked to spit continuously into the 71 container until filled to half its volume, and with sputum specimen they were asked to cough 72 sputum deep from their lungs and then spit it into the container. Both the spit and sputum 73 specimens were advised to be collected in the morning before the patients ate, drunk or brushed 74 their teeth. 75 All specimens were transported at room temperature and analyzed on the same day in Expert CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) used to assess the differences in sensitivity and specificity. 96 We calculated Cohen's kappa coefficient to evaluate the agreement between the reference and 97 the index tests. Area under a receiver operating characteristic curve (AUC) and 95% CI were 98 reported. Because of the imperfect reference test, latent class analysis was used as a correction 99 method to adjust the estimated sensitivity and specificity of the index tests based on the existing 100 sensitivity and specificity of the reference test. Model selection was based on Bayesian 101 information criterion (BIC). Data analysis was performed using R software (version. 3.6.0). We enrolled 40 patients with a mean age of 38.7 years old (SD: 12.6) including 21 (53%) females. 105 Enrolment was done as soon as they received the positive testing results, either one day (n=27/40, 106 67.5%) or two days (n=13/40, 32.5%) after they had been NPS sampled for COVID-19 diagnosis. 107 Thirty-one patients had been symptomatic since disease onset (supplementary table 1) with most 108 prevalent symptoms being fatigue (86%), headache (81%) and cough (79%). At the time of 109 . CC-BY-NC-ND 4.0 International license It is made available under a 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 May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint 6 specimen collection, only 24 patients were symptomatic with the most prevalent being cough 110 (44%), anosmia (42%) and headache (40%). 111 We collected 40 concomitant nasopharyngeal and gargle specimens on recruitment day, as well as 112 40 next morning gargle specimens. Out of 22 patients assigned to the spit specimen collection 113 group, all of them gave back specimens, while out of 18 patients assigned to the sputum group, 114 two patients could not produce sputum. 115 All specimens were generally as sensitive as NPS to diagnose SARS-CoV-2 infection in our study 116 population. The spit specimen showed the highest sensitivity (sensitivity 1.00 CI 95 % 1.00-1.00), 117 followed by gargle specimens, regardless of when they were collected: 0.97 (CI 95 % 0.92-1,00). 118 The sputum specimen had the lowest sensitivity (0.94 CI 95 % 0.87-1.00). 119 We compared Ct values obtained from alternative specimens to NPS ( Discussion 126 We evaluated self-collection of alternative specimens: gargle, spit and sputum and compared their 127 sensitivity and Ct values to NPS specimens as alternative methods for SARS-CoV-2 infection 128 diagnosis. All specimen collection methods were as sensitive as NPS, with sensitivities exceeding 129 90 %. In comparison with NPS, the gargle specimens had higher Ct values, likely due to dilution by 130 gargling water. Therefore in milder cases with low viral load, it might not be as sensitive as NPS. . CC-BY-NC-ND 4.0 International license It is made available under a 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 May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint 7 Spit and sputum specimens collected on the following day had a higher though not significantly 132 different Ct value compared to NPS, however, sputum appeared more challenging to collect in 133 patients with milder symptoms. Hence, spit would appear as the most suitable alternative 134 specimen to NPS for SARS-CoV-2 infection diagnosis. 135 Since the beginning of the COVID-19 pandemic, several studies or meta-analysis have investigated 136 the potential use of saliva specimen for SARS-CoV-2 infection diagnosis among both symptomatic 137 (8, 9, 11, (16) (17) (18) (19) (20) (21) and asymptomatic patients (8, 9, 11, (16) (17) (18) 21, 22) . ECDC and CDC have approved . CC-BY-NC-ND 4.0 International license It is made available under a 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 May 3, 2021. Alternative specimens' collection main benefit compared to NPS would be to avoid close contact 159 with a HCW, to allow participants to collect the specimen themselves and to avoid an unpleasant 160 procedure. Overall, it would also increase the willingness to apply for SARS-CoV-2 testing and 161 allocation of current resources in HCWs. 162 The main limitations of our work are that we did not analyse whether delayed transport or 163 extended storage before analysis could hamper sensitivity as all samples were transported and 164 processed on the very same day and our low sample size (40 participants in total), however we 165 conducted this study as an exploratory assessment of alternative specimen collection. We focused 166 on patients with the most common clinical picture of COVID-19: mild symptoms, as they are the 167 ones that would most benefit from non-invasive alternative specimen collection. An additional 168 strength was that all samples were collected within 1-2 days after diagnosis, while patients were 169 still at the acute phase of the disease. CC-BY-NC-ND 4.0 International license It is made available under a 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 May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint 9 larger study population. We will consider offering the possibility to patients with mild symptoms 177 seeking diagnosis in a pilot testing centre, the possibility to enroll in a study assessing whether in 178 general population, spit collected on the next morning has the same performance as in our 179 exploratory sample. If so, in the long run, we could offer patients with mild symptoms to choose 180 between at home self-collection of spit versus nasopharyngeal specimen collection at a testing 181 centre. Not only would it decrease discomfort, but also decrease HCWs' exposure and burden. 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 May 3, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 3, 2021. ; https://doi.org/10.1101/2021.05.02.21255857 doi: medRxiv preprint A Novel Coronavirus from Patients with 199 Pneumonia in China Director-General's opening remarks at the media briefing on COVID-19 -11 COVID-19) Dashboard European Centre for Disease Prevention and Control. Contact tracing: public health 207 management of persons, including healthcare workers, who have had contact with COVID-19 208 cases in the European Union -third update. ECDC Finnish Institute for Health and Welfare. COVID-19-epidemian hybridistrategian seuranta -210 tilannearvioraportti 10.3.2021. 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