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Highlights
•
Saliva has been recommended as an alternative sample for detection of SARS CoV-2 infection,
but data are limited.
•
In this study, SARS CoV-2 was detected by RT-PCR in 35 of 124 patients, 30 (85.7 %)
by saliva and 33 (94.3 %) by NP swab.
•
The median cycle threshold value was significantly lower for NPS than for saliva.
•
A third of pure saliva samples were difficult to pipet, which slowed processing.
Abstract
Background
A major expansion in SARS CoV-2 testing is urgently needed. Saliva is an attractive
option as an alternative for nasopharyngeal swabs (NPS), since saliva can be self-collected,
is non-invasive, and sample quality is not dependent on the expertise of the collector.
Objective
To compare SARS CoV-2 positivity on paired NPS and saliva samples.
Study design
NPS and paired saliva samples were prospectively collected from symptomatic outpatients
suspected of having COVID-19 and were tested by real-time RT-PCR.
Results
In total, 35/124 (26.6 %) samples were RT-PCR positive, with 33/35 positive by NPS
(sensitivity = 94.3 % (95 % CI 81.4%–99.0%)) and 30/35 by pure saliva (sensitivity = 85.7
% (95 % CI 70.6%–93.7%)), for an overall agreement of 117/124 (94.4 %). The median
cycle threshold value was significantly lower for NPS than for saliva (p = 0.0331).
A third or more of pure saliva samples from symptomatic patients were thick, stringy,
and difficult to pipet.
Conclusions
Real-time RT-PCR of pure saliva had an overall sensitivity for SARS CoV-2 RNA detection
of 85.7 % when compared to simultaneously collected NPS. Our study highlighted the
need to optimize collection and processing before saliva can be used for high volume
testing.
Summary Background Coronavirus disease 2019 (COVID-19) causes severe community and nosocomial outbreaks. Comprehensive data for serial respiratory viral load and serum antibody responses from patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not yet available. Nasopharyngeal and throat swabs are usually obtained for serial viral load monitoring of respiratory infections but gathering these specimens can cause discomfort for patients and put health-care workers at risk. We aimed to ascertain the serial respiratory viral load of SARS-CoV-2 in posterior oropharyngeal (deep throat) saliva samples from patients with COVID-19, and serum antibody responses. Methods We did a cohort study at two hospitals in Hong Kong. We included patients with laboratory-confirmed COVID-19. We obtained samples of blood, urine, posterior oropharyngeal saliva, and rectal swabs. Serial viral load was ascertained by reverse transcriptase quantitative PCR (RT-qPCR). Antibody levels against the SARS-CoV-2 internal nucleoprotein (NP) and surface spike protein receptor binding domain (RBD) were measured using EIA. Whole-genome sequencing was done to identify possible mutations arising during infection. Findings Between Jan 22, 2020, and Feb 12, 2020, 30 patients were screened for inclusion, of whom 23 were included (median age 62 years [range 37–75]). The median viral load in posterior oropharyngeal saliva or other respiratory specimens at presentation was 5·2 log10 copies per mL (IQR 4·1–7·0). Salivary viral load was highest during the first week after symptom onset and subsequently declined with time (slope −0·15, 95% CI −0·19 to −0·11; R 2=0·71). In one patient, viral RNA was detected 25 days after symptom onset. Older age was correlated with higher viral load (Spearman's ρ=0·48, 95% CI 0·074–0·75; p=0·020). For 16 patients with serum samples available 14 days or longer after symptom onset, rates of seropositivity were 94% for anti-NP IgG (n=15), 88% for anti-NP IgM (n=14), 100% for anti-RBD IgG (n=16), and 94% for anti-RBD IgM (n=15). Anti-SARS-CoV-2-NP or anti-SARS-CoV-2-RBD IgG levels correlated with virus neutralisation titre (R 2>0·9). No genome mutations were detected on serial samples. Interpretation Posterior oropharyngeal saliva samples are a non-invasive specimen more acceptable to patients and health-care workers. Unlike severe acute respiratory syndrome, patients with COVID-19 had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. This finding emphasises the importance of stringent infection control and early use of potent antiviral agents, alone or in combination, for high-risk individuals. Serological assay can complement RT-qPCR for diagnosis. Funding Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, and Sanming Project of Medicine.
Highlights • Saliva is a reliable tool to detect SARS-Cov-2 by RT-rPCR analysis. • Saliva may provide information about the clinical evolution of the disease. • Saliva could represent a valid instrument in COVID-19 diagnosis. • Patients should be checked for salivary viral load at hospital discharge.
LETTER Diagnostic testing for COVID-19 is central to controlling the global pandemic. Recently, To and colleagues reported that 20 of 23 (87%) patients who had SARS-CoV-2 detected by reverse transcriptase PCR (RT-PCR) in nasopharyngeal swabs (NPS) or sputum also had SARS-CoV-2 detectable in saliva (1). The use of saliva has several advantages compared to collection of NPS. In particular, the close contact involved in swab collection poses a risk to health care workers, and collection of saliva may reduce this risk. Further, saliva collection does not require specialized consumables, causes less patient discomfort, and may be a useful sample for self-collection (2). We further investigated the feasibility and utility of saliva collection from ambulatory patients presenting to a dedicated COVID-19 screening clinic at the Royal Melbourne Hospital (RMH), Melbourne, Australia. Between 25 March and 1 April 2020, 622 patients were tested for COVID-19 through the screening clinic. All patients had NPS, and 522/622 (83.9%) patients also provided saliva. Patients were asked to pool saliva in their mouth for 1 to 2 min prior to collection and gently spit 1 to 2 ml of saliva into a 25-ml collection pot. Neat saliva specimens were transported to the laboratory where an approximate 1:1 ratio of liquid Amies medium was immediately added. We specifically chose to use liquid Amies medium in order to (i) evaluate the use of an alternative transport medium in the face of global shortages of viral transport medium (VTM) and (ii) to preserve VTM in our own laboratory. The median time from sample collection to addition of medium was 180 min (range, 55 to 537 min). NPS and saliva specimens underwent nucleic acid extraction on the Qiagen EZ1 platform (Qiagen, Hilden, Germany). An extraction volume of 200 μl of the sample was used, with RNA eluted in 60 μl. Reverse transcriptase PCR (RT-PCR) testing was performed using a multiplex RT-PCR test for SARS-CoV-2 and other seasonal coronaviruses (coronavirus typing [8-well] assay; AusDiagnostics, Mascot, Australia). All NPS samples positive for SARS-CoV-2 underwent confirmatory testing at a local reference laboratory (the Victorian Infectious Diseases Reference Laboratory) using previously published primers (3). Overall, 39/622 (6.3%; 95% confidence interval [CI], 4.6% to 8.5%) patients had PCR-positive NPS, and 33/39 patients (84.6%; 95% CI, 70.0% to 93.1%) had SARS-CoV-2 detected in saliva. The median cycle threshold (CT ) value was significantly lower in NPS than saliva (Fig. 1A), suggestive of higher viral loads in NPS, and in both samples, there was a correlation between CT value and days from symptom onset (Fig. 1B). To assess specificity, a subset of saliva specimens from 50 patients with PCR-negative swabs was also tested. Of note, SARS-CoV-2 was detected in 1/50 (2%; 95% CI, 0.1% to 11.5%) of these saliva samples, which may reflect differing quality of NPS collection. FIG 1 (A) Median cycle threshold (CT ) value in nasopharyngeal swabs and saliva specimens positive for SARS-CoV-2. NPS, nasopharyngeal swab. (B) Median cycle threshold (CT ) value and days from symptom onset in nasopharyngeal swabs and saliva specimens positive for SARS-CoV-2. Data points represent the median CT value from patient samples, and bars represent the interquartile range. The slope represents the line of best fit. To date, studies assessing the utility of different patient samples for the diagnosis of COVID-19 have largely been conducted on inpatients with known COVID-19 infection (1, 4). Here, we demonstrate the feasibility, acceptability, and scalability of prospectively collecting saliva from ambulatory patients in a busy screening clinic and further demonstrate the value of saliva as a noninvasive specimen for the detection of SARS-CoV-2. Although the sensitivity of saliva as a diagnostic specimen is less than NPS, saliva testing may be a suitable alternative first-line screening test in several environments, including low-resource settings, with NPS reserved for patients with an ongoing high clinical index of suspicion. These findings are highly relevant in the face of shortages of both swabs and personal protective equipment in many settings (5).
[a
]Clinical Virology Laboratory, Yale New Haven Hospital, New Haven, Connecticut, United
States
[b
]Department of Laboratory Medicine, Yale University School of Medicine, New Haven,
Connecticut, 06520, United States
[c
]Department of Medicine, Yale University School of Medicine, New Haven, Connecticut,
06520, United States
Author notes
[*
]Corresponding author at: Yale University School of Medicine, P.O. Box 208035, New
Haven, CT, 06520-8035, United States.
marie.landry@
123456yale.edu
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