key: cord-0919769-5kcgsb0v
authors: Kim, Seong Eun; Lee, Ji Yeon; Lee, Ahrang; Kim, Soosung; Park, Kyung-Hwa; Jung, Sook-In; Kang, Seung-Ji; Oh, Tae Hoon; Kim, Uh Jin; Lee, Seung Yeob; Kee, Seung-Jung; Jang, Hee-Chang
title: Viral Load Kinetics of SARS-CoV-2 Infection in Saliva in Korean Patients: a Prospective Multi-center Comparative Study
date: 2020-08-05
journal: J Korean Med Sci
DOI: 10.3346/jkms.2020.35.e287
sha: c7dd9e6d51e8158eb70c533bea799406c966c95a
doc_id: 919769
cord_uid: 5kcgsb0v

BACKGROUND: This study was performed to compare the viral load and kinetics of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in saliva with those in standard nasopharyngeal/oropharyngeal (NP/OP) swabs. METHODS: Fifteen patients with SARS-CoV-2 infection from four hospitals were prospectively enrolled and matched samples of nasopharyngeal/oropharyngeal swabs and saliva were collected at Day 1 of admission and every other day till consequently negative for two times. Real-time reverse transcription polymerase chain reaction (rRT-PCR) was performed to detect the envelope (E) and RNA-dependent RNA polymerase (RdRP) genes. RESULTS: The cycle threshold values of saliva were comparable to those of NP/OP swabs overall (P = 0.720, Mann–Whitney U test). However, the overall sensitivity of rRT-PCR using saliva was 64% (34/53), which is lower than the 77% (41/53) using NP/OP swabs. The sensitivity of rRT-PCR using saliva was especially lower in early stage of symptom onset (1–5 days; 8/15; 53%) and in patients who did not have sputum (12/22; 55%). CONCLUSION: Saliva sample itself is not appropriate for initial diagnosis of coronavirus disease 2019 (COVID-19) to replace NP/OP swabs, especially for the person who does not produce sputum. COVID-19 cannot be excluded when the test using saliva is negative, and it is necessary to retest using NP/OP swabs.

Saliva samples have merits compared with nasopharyngeal/oropharyngeal (NP/OP) swabs for developing a rapid self-diagnosis kit, and tests in resource-poor countries or overwhelming outbreak settings lacking well-trained healthcare workers (HCW). No HCW or personal protection equipment is needed to collect saliva, which can reduce the infection risk, time, cost and effort of HCW. Therefore, the US Food and Drug Administration (FDA) authorized a saliva test for emergency use for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mid-April 2020 based on limited data. 1 Regarding the diagnostic value of saliva samples for SARS-CoV-2 infection, few clinical data are available and the results are inconsistent 2 -8 ; moreover, the viral load and kinetics of SARS-CoV-2 in saliva are not clear, because recent comparative studies of the viral kinetics at multiple body sites did not include the oral cavity or saliva. 9, 10 This study was performed to evaluate the viral load in saliva and to compare the viral load and kinetics of SARS-CoV-2 in saliva with those in standard NP/OP swabs.

Fifteen patients with SARS-CoV-2 infection who were admitted to Chonnam National University (CNU) Hospital (3 patients), CNU Hwasun Hospital (2 patients), CNU Bitgoeul Hospital (7 patients), and Keimyung University Dongsan Hospital (3 patients) from March 19 to April 10, 2020 in Korea were enrolled prospectively.

Matched samples of NP/OP swab, saliva, sputum (if the patient had sputum) were collected at Day 1 of admission and every other day till consequently negative for two times. Saliva was collected by asking the patient to spit from the oral cavity in accordance with the recent study approved by FDA. 1 Sputum was collected by asking the patient to cough from the throat. NP/OP swabs were collected in the same tube containing 2.5 mL of universal transport medium (Asan Pharmaceutical, Seoul, Korea). In patient's room, 1-2 mL of saliva or sputum was collected in sterile containers, and then diluted using same volume of sterile saline or universal transport medium in the Laboratory.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) was performed at laboratory department of CNU hospital. First, 200 μL was taken from each sample and RNA was extracted using an automated nucleic acid extraction system (AdvanSure™ E3 System; LG Chem, Seoul, Korea). The extracted RNA was amplified using a commercial rRT-PCR kit (PowerChek™ 2019-nCoV Real-time PCR Kit; Kogenebiotech, Seoul, Korea) and detection system (CFX96™ Real-time PCR detection system; Bio-Rad, Hercules, CA, USA) to detect the envelope (E) and RNA-dependent RNA polymerase (RdRP) genes. Forty cycles of PCR were performed and cycle threshold (Ct) values were determined for each gene. A positive rRT-PCR result was defined when the Ct values of both the E and RdRP genes of SARS-CoV-2 were 35 or less. The Ct value of RdRP of each sample was used for further statistical analyses.

Continuous variables were expressed as means ± standard errors of the means or median and interquartile range (IQR). Mann-Whitney U test or Kruskal-Wallis test were used to compare statistical differences in continuous variables and χ 2 test was used in categorical variables. All tests of significance were two-tailed, and P values ≤ 0.05 were deemed to indicate statistical significance. SPSS (version 26.0; IBM Corporation, Armonk, NY, USA) and GraphPad Prism (version 8.4.3; GraphPad software, San Diego, CA, USA) were used for statistical analyses.

The study was approved by the Institutional Review Board (IRB) of CNU Hospital (IRB no. CNUH-2020-149) and Keimyung University Dongsan Hospital (IRB No. 2020-03-027). CNU Hwasun Hospital (2 patients) and CNU Bitgoeul Hospital (7 patients) belong to the same group as CNU Hospital. Informed consent was waived for observational study design. IRB approval for these two hospitals was jointly conducted by IRB of CNU Hospital. Informed consent was waived for the observational study design.

Median age of 15 patients was 59 (range, 17-91; IQR, 25-62) years and 5 (33%) were male. Two (11%) patients were asymptomatic, 8 (53%) patients had upper respiratory symptoms without pneumonia, and 7 (47%) patients had pneumonia. Three (20%) patients required oxygen therapy.

The overall sensitivity of rRT-PCR using saliva was 64% (34/53), which is lower than the 77% (41/53) using NP/OP swabs ( Table 1 ). The sensitivities of rRT-PCR using NP/OP swabs, sputum, and saliva were 74% (23/31), 68% (21/23) and 71% (22/31) in patients with sputum. The sensitivity of rRT-PCR using saliva (8/15, 53%) was especially significantly lower than that using the NP/OP swab specimen (14/15, 93%) in early stage (1-5 days after symptom onset; P = 0.013) ( Table 1 ). The sensitivity of rRT-PCR using saliva was especially lower in patients without sputum (12/22, 55%) ( Table 1) .

The Ct values of saliva (median, 32; IQR, 28-38) were comparable to those of NP/OP swabs (median, 33; IQR, 27-35) (P = 0.753) (Fig. 1A) in overall 53 samples from 15 patients. In 8 patients with sputum, the Ct values of saliva (32; IQR, 29-37) were also not different to those of NP/OP swabs (33; IQR, 26-36) and sputum (29; IQR, 24-38: P = 0.664) (Fig. 1B) . (33; IQR, 24-40) was higher in early stage compared to NP/OP swabs (32; IQR, 21-35, P = 0.267, Fig. 2A) .

The viral load kinetics of SARS-CoV-2 in each samples were shown in Fig. 3 . Ct values of SARS-CoV-2 in saliva seemed to be higher in early phase compared to those in NP/OP swabs, however, the slope was not statistically significant between the two (Fig. 3A) . Viral load kinetics are similar between saliva and NP/OP swabs in patients who had sputum (Fig. 2B) ; however, Ct values of SARS-CoV-2 in saliva seem higher in early phase and lower in late phase compared to those in NP/OP swabs in patients without sputum (Fig. 3, Supplementary Figs. 1 and 2) .

The detection rates of virus particle in saliva and in NP/OP swabs differ according to the respiratory virus species; influenza virus was better detected in NP/OP swabs, but adenovirus was better detected in saliva, compared to other respiratory viruses. Recent studies by one investigator group suggested that SARS-CoV-2 is detected consistently in samples of posterior oropharyngeal secretion coughed by patients. 12 -16 However, the samples are similar to pharyngeal secretions or sputum rather than saliva considering the anatomical site and method of collection.

Several studies evaluated the diagnostic value of saliva samples for SARS-CoV-2 infection; however, the results are inconsistent. In SARS-CoV-2 assay by Rutgers Clinical Genomics Laboratory, 1 there was 100% positive and negative agreement between the results obtained from testing of saliva and those obtained from NP/OP swabs, suggesting that saliva samples can completely replace NP/OP swabs. Additional studies showed that SARS-CoV-2 is also detected consistently in saliva samples, similar to the NP/OP samples in three peer-reviewed studies with limited sample sizes and two non-peer reviewed studies. 2 -6 In contrast, lower sensitivity of rRT-PCR using saliva sample compared to NP/OP swabs were reported in other studies; one peer reviewed 8 and one non-peer reviewed. A recent study suggested that the positive rate rRT-PCR using NP swab was 6% higher than that of using saliva in the first week from symptom onset and 20% higher in the second week 8 . In contrast to the study, the sensitivity of rRT-PCR using saliva was significantly lower than that using NP/OP swabs in 1-5 days from symptom onset in this study. Several studies have shown that even in the case of saliva, a large amount of virus was detected in the early stages and gradually decreased over time. However, in these studies on SARS-CoV-2 kinetics of saliva, the posterior oropharyngeal saliva sample, which is easy to mix with sputum, was used to increase sensitivity. There is a study in Korea that reported SARS-CoV-2 kinetics of saliva in two cases, but the sample size are not enough to make a conclusion and they were patients who had sputum. The viral load kinetics of saliva in patients who had sputum was similar to NP/OP swabs in this study; however, the viral load kinetics of saliva in patients who did not have sputum was rather similar to viral kinetics of samples from gastrointestinal tract including stool 17 than respiratory specimens in this study; Our data suggest that passive contamination of sputum affect the kinetics of saliva. Additional studies in patients who do not have productive sputum or pharyngeal secretion are needed to make a conclusion on the viral load kinetics of SARS-COV-2 in saliva.

The Ct value of the NP/OP and sputum in this study was lowest in the early stage of disease and it was reached around cut-off value within 2 weeks which was similar to previous Korean cases. 18, 19 The positive rate of NP/OP swab was significantly higher than that of saliva in early stage of disease and the difference was disappeared in the mid and late stage of disease.

Our study suggests that saliva samples can be used for a rapid screening test or a complementary test to NP/OP swabs. However, it has been shown in this study that saliva samples cannot replace NP/OP swabs collected by HCW especially in early stage and in patients without sputum.

Our study have several limitations. First, saliva was not collected under direct observation, although it was collected from every admitted patient after education to minimize contamination of sputum. Second, in this study, no mechanism studies have been conducted on how viruses are detected in saliva. Additional study is needed to evaluate whether SARS-CoV-2 is secreted from oral mucosa or salivary glands or passively contaminated by pharyngeal secretion. 20

In conclusion, saliva sample itself is not appropriate for initial diagnosis of COVID-19 to replace NP/OP swabs, especially for the person who does not produce sputum. COVID-19 cannot be excluded when the test using saliva is negative, and it is necessary to retest using NP/OP swabs.

Supplementary Fig. 1 Comparison of Ct value of RdRP gene between NP/OP swabs, sputum and saliva according to the time period after symptom onset in patients who had sputum. Lines are the mean ± standard error of the mean. P = 0.261 (A), P = 0.003 (B), and P = 0.580 (C) each by Kruskal-Wallis test.

Accelerated emergency use authorization (EUA) summary. SARS-CoV-2 assay

Saliva is a reliable tool to detect SARS-CoV-2

Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

Patient-collected tongue, nasal, and mid-turbinate swabs for SARS-CoV-2 yield equivalent sensitivity to health care worker collected nasopharyngeal swabs

Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs

Saliva is less sensitive than nasopharyngeal swabs for COVID-19 detection in the community setting

Clinical significance of a high SARS-CoV-2 viral load in the saliva

Sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Detection of SARS-CoV-2 in different types of clinical specimens

Viral load of SARS-CoV-2 in clinical samples

Comparison between saliva and nasopharyngeal swab specimens for detection of respiratory viruses by multiplex reverse transcription-PCR

Saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Consistent detection of 2019 novel coronavirus in saliva

Saliva as a non-invasive specimen for detection of SARS-CoV-2

Evaluating the use of posterior oropharyngeal saliva in a point-of-care assay for the detection of SARS-CoV-2

Virological assessment of hospitalized patients with COVID-2019

Viral load kinetics of SARS-CoV-2 infection in first two patients in Korea

Clinical course and outcomes of patients with severe acute respiratory syndrome coronavirus 2 infection: a preliminary report of the first 28 patients from the Korean cohort study on COVID-19

Comparison of Ct value of RdRP gene between NP/OP swabs and saliva according to the time period after symptom onset in patients who did not have sputum. Lines are the mean ± standard error of the mean. P = 0.353 (A), P = 0.796 (B), and P = 0.667 (C) each by Mann-Whitney U-test.