key: cord-0890223-fwg96sqr authors: Yokota, Isao; Hattori, Takeshi; Shane, Peter Y.; Konno, Satoshi; Nagasaka, Atsushi; Takeyabu, Kimihiro; Fujisawa, Shinichi; Nishida, Mutsumi; Teshima, Takanori title: Equivalent SARS-CoV-2 viral loads by PCR between nasopharyngeal swab and saliva in symptomatic patients date: 2021-02-24 journal: Sci Rep DOI: 10.1038/s41598-021-84059-2 sha: 67c80144b561b6d1ee2e748cca3486dca2360771 doc_id: 890223 cord_uid: fwg96sqr Emerging evidences have shown the utility of saliva for the detection of SARS-CoV-2 by PCR as alternative to nasopharyngeal swab (NPS). However, conflicting results have been reported regarding viral loads between NPS and saliva. We conducted a study to compare the viral loads between NPS and saliva in 42 COVID-19 patients. Viral loads were estimated by the cycle threshold (Ct) values. SARS-CoV-2 was detected in 34 (81%) using NPS with median Ct value of 27.4, and 38 (90%) using saliva with median Ct value of 28.9 (P = 0.79). Kendall’s W was 0.82, showing a high degree of agreement, indicating equivalent viral loads in NPS and saliva. After symptom onset, the Ct values of both NPS and saliva continued to increase over time, with no substantial difference. Self-collected saliva has a detection sensitivity comparable to that of NPS and is a useful diagnostic tool with mitigating uncomfortable process and the risk of aerosol transmission to healthcare workers. 17 female (40%) and 25 male (60%) patients participated in the study. Median age of the patients was 73 years-old (range 27 to 93) and specimens were obtained at a median of 6 days (range 1-12) after symptom onset. SARS-CoV-2 was detected in NPS and saliva in 81% (34/42) and 90% (38/42) of the patients, respectively ( (Fig. 1D ). In this study in symptomatic inpatients with COVID-19, SARS-CoV-2 was detected in saliva in 90% of the patients compared to 81% in NPS, with equivalent viral loads in the two specimens. Although there have been conflicting results reported to date 6,7 , our study was designed to have significant advantages over previous studies; the number of patients was relatively large, paired samples were simultaneously collected, and qRT-PCR was performed at an independent central laboratory. In our study, viral loads were roughly estimated by the Ct values. Our results demonstrate that self-collected saliva is a useful alternative to NPS for the diagnosis of COVID-19. Furthermore, we recently reported equivalent sensitivity and specificity of qRT-PCR using saliva and NPS, again with equivalent viral loads in a large number of asymptomatic individuals in the setting of mass-screening 8 . www.nature.com/scientificreports/ Taken together, self-collected saliva provides highly accurate results and should be considered as an easier and cost-efficient alternative for the detection of SARS-CoV-2 in both symptomatic and asymptomatic individuals. In summary, self-collected saliva is a useful alternative to NPS as a specimen for detecting SARS-CoV-2 nucleic acids. The methodology of self-collection carries significant logistical and cost advantages over NPS by mitigating the risk of aerosol transmission to healthcare workers and obviating the need for full protective suits. Forty-two patients diagnosed with COVID-19 by positive qRT-PCR of NPS were enrolled in this study. Paired NPS and saliva samples were simultaneously collected from all patients upon hospital admission between June 12, 2020 and August 6, 2020. This study was approved by the Institutional Ethics Board (Hokkaido University Hospital Division of Clinical Research Administration Number: 020-0116) and informed consent was obtained from all patients. All the procedures are carried out according to relevant guidelines. qRT-PCR was performed at a central laboratory (SRL, Tokyo, Japan). Self-collected saliva was diluted fourfold with phosphate buffered saline and centrifuged at 2000 × g for 5 min to remove cells and debris. RNA was extracted from 200 µL of the supernatant or nasopharyngeal swab samples using QIAsymphony DSP Virus/Pathogen kit and QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). qRT-PCR tests were performed, according to the manual by the National Institute of Infectious Diseases (NIID, https ://www.niid.go.jp/niid/image s/epi/coron a/2019-nCoVm anual 20200 217-en.pdf). Briefly, 5uL of the extracted RNA was used to perform one step qRT-PCR using Thunderbird Probe One-step qRT-PCR Kit (Toyobo, Osaka, Japan) and 7500 Real-time PCR Systems (Thermo Fisher Scientific, Waltham, USA). The Ct values were obtained by using N1 primers (N_Sarbeco_F1, N_Sarbeco_R1) with N1 probe (N_Sarbeco_P1) and by using N2 primers (NIID_2019-nCOV_N_F2, NIID_2019-nCOV_N_R2) with N2 probe (NIID_2019-nCOV_N_P2), as described 9 . Ct values of qRT-PCR using NPS and saliva were expressed as scatter plots with Kendall's coefficient of concordance W as nonparametric intraclass correlation coefficient. Scatter plots of Ct values and days from symptom onset for each type of specimen were also provided to examine the relationship between disease course and viral load. To find the longitudinal trends, we performed a median spline regression using "qsreg" function with the default parameters in R. Statistical analysis was conducted by R 4.0.2. All analyzed data were distributed in Supplement. Detection of SARS-CoV-2 in different types of clinical specimens Consistent detection of 2019 novel coronavirus in saliva Saliva is a reliable tool to detect SARS-CoV-2 Swabs collected by patients or health care workers for SARS-CoV-2 testing Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva Saliva as a noninvasive specimen for detection of SARS-CoV-2 Saliva or nasopharyngeal swab specimens for detection of SARS-CoV-2 Mass screening of asymptomatic persons for SARS-CoV-2 using saliva Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19 This study was funded by Health, Labour and Welfare Policy Research Grants 20HA2002. I.Y., T.T., designed study and wrote paper; T.H., P.Y.S., S.K., A.N., K.T.; collected samples; S.F., M.N., designed study. The authors declare no competing interests. Supplementary Information The online version contains supplementary material available at https ://doi. org/10.1038/s4159 8-021-84059 -2.Correspondence and requests for materials should be addressed to T.T. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.