key: cord-0822556-8jylnyfq
authors: Cheuk, Sally; WONG, Ying; TSE, Herman; SIU, Hon Kei; KWONG, Tsz Shan; Chu, Man Yee; YAU, Felix Yat Sun; CHEUNG, Ingrid Yu Ying; TSE, Cindy Wing Sze; POON, Kin Chiu; CHEUNG, Kwok Chi; WU, Tak Chiu; CHAN, Johnny Wai Man; CHEUK, Wah; LUNG, David Christopher
title: Posterior oropharyngeal saliva for the detection of SARS-CoV-2
date: 2020-06-21
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa797
sha: 1861b6734864dbb23d79ee884da307cbfe61a870
doc_id: 822556
cord_uid: 8jylnyfq

BACKGROUND: The coronavirus disease-2019 (COVID-19) pandemic has put tremendous pressure on the healthcare system worldwide. Diagnostic testing remained one of the limiting factors for early identification and isolation of infected patients. This study aimed to evaluate posterior oropharyngeal saliva (POPS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection among patients with confirmed or suspected COVID-19. METHODS: The laboratory information system was searched retrospectively for all respiratory specimens and POPS requested for SARS-CoV-2 RNA detection between Feb 1, 2020 and Apr 15, 2020. The agreement and diagnostic performance of POPS against NPsp were evaluated. RESULTS: A total of 13772 specimens were identified during the study period, including 2130 POPS and 8438 NPsp. Two hundred and twenty-nine same-day POPS-NPsp paired were identified with POPS and NPsp positivity of 61.5% (95% CI [55.1–67.6%]) and 53.3% (95% CI [46.8–59.6%]). The overall, negative and positive percent agreement were 76.0% (95% CI [70.2–80.9%]), 65.4% (95% CI [55.5–74.2%]), 85.2% (95% CI [77.4–90.8%]). Better positive percent agreement was observed in POPS-NPsp obtained within seven days (96.6%, 95% CI [87.3–99.4%]) compared with after seven days of symptom onset (75.0%, 95% CI [61.4–85.2%)). Among the 104 positive pairs, the mean difference in Cp value was 0.26 (range: 12.63 to -14.74), with an overall higher Cp value in NPsp (Pearson coefficient 0.579). No significant temporal variation was noted between the two specimen types. CONCLUSIONS: POPS is an acceptable alternative specimen to nasopharyngeal specimen for the detection of SARS-CoV-2.

Nasopharyngeal specimens (NPsp), e.g. nasopharyngeal swabs (NPS) and aspirates (NPA) are the recommended specimen types for the investigation of viral respiratory infections [1] . Currently, the World Health Organization and Centers for Disease Control and Prevention recommended an upper respiratory specimen (NPA or NPS with throat swab) and/or a lower respiratory specimen for the diagnosis of coronavirus disease-2019 (COVID-19) [2, 3] . However, the collection of NPsp is relatively invasive with significant patient discomfort, and is contraindicated in patients with recent nasal trauma or surgery, or severe thrombocytopenia [4] . In addition, obtaining NPsp required trained healthcare workers (HCW) and has infection control implications mandating use of personal protective equipment (PPE), including N95 respirator or equivalent, gloves, faceshield/eye protection, and gown [3, 4] . The procedure should also be performed in a well-ventilated room, or a negative pressure airborne infection isolation room (AIIR) due to the potential generation of aerosols.

Hence, these requirements might become limiting factors in providing adequate capacity for diagnosing infections during the COVID-19 pandemic. Moreover, there is a surge in global demand for flocked swabs and viral transport medium (VTM) used for collecting NPsp, resulting in substantial pressure and uncertainty over the reliable supply of these essential material [5, 6] .

A c c e p t e d M a n u s c r i p t Saliva has previously been shown to be a useful specimen type for other respiratory virus detections, such as influenza and human metapneumovirus, with comparable sensitivity [7] and agreement [8] [9] [10] [11] with NPsp. Preliminary studies showed persistent detectable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in saliva from patients with confirmed COVID-19 [12, 13] . Viral load was highest in posterior oropharyngeal saliva (POPS) during the first week of symptom onset, with median viral load of first available saliva specimen being around 10 6 copies/mL [14] .

On April 13, 2020, the Food and Drug Administration granted accelerated emergency use authorization for the use of saliva, in addition to other respiratory specimen types, in a SARS-CoV-2 assay to facilitate mass screening, based on an evaluation of 60 specimens obtained with the Spectrum Solutions LLC SDNA-1000 Saliva Collection Device [15] . Thus far, studies on saliva as diagnostic specimens for SARS-CoV-2 were small and varied in saliva specimen collection methods (Supplementary Table   1 ) [16] [17] [18] [19] [20] . Hence, to understand the performance of saliva specimens in a field situation, we performed a retrospective comparison of saliva against NPsp in a larger population of patients with suspected or confirmed COVID-19.

A c c e p t e d M a n u s c r i p t

Since the announcement of clustered pneumonia of unknown origin by the Wuhan c c e p t e d M a n u s c r i p t various types were taken from these patients during the study period to assess if they could be discharged from AIIR.

For the collection of POPS, standard instructions prepared by HA were given to patients (https://www.ha.org.hk/haho/ho/cc/Information_sheet_en_txt.pdf), an online video was also available (https://www.youtube.com/watch?v=rZ3oNsJGBxo). In brief, patients were asked to clear saliva from back of throat into a sterile container as soon as possible after waking up, before any eating, drinking or teeth brushing. For NPsp, patients were instructed to blow their nose to clear the nostril and both NPS and NPA were collected by a HCW wearing full PPE. NPS was collected by insertion of a flock swab into the nostril parallel to the palate with a rotatory motion to a depth equal to the distance from the nostril to the tragus [4] . The flock swab was left in the position for a few seconds before removal with a rotatory motion. The swab was then placed in 1 mL of VTM for transportation. NPS was collected using a catheter connected one end to a mucus trap and the other end to a vacuum source, which is then inserted into the nasopharynx similar to NPS to the nasopharynx for aspirate nasopharyngeal secretion into the mucus trap. One mL of VTM was added to the secretion before transportation. If POPS and NPSp were taken at the same time, POPS was always obtained before NPsp. Lower respiratory samples were always preserved in 1 mL of A c c e p t e d M a n u s c r i p t VTM. Other conventional respiratory specimens such as sputum, tracheal aspirate and broncheoalveolar lavage were collected following usual practice.

Upper respiratory tract specimens (e.g. NPA or NPS with or without pooled throat swabs), lower respiratory tract specimens (e.g. sputum, tracheal aspirate or bronchoalveolar lavage) and POPS were acceptable specimen types for the detection of SARS-CoV-2 RNA in our laboratory. All respiratory specimens, and POPS from inpatients, were received in 1 mL of VTM. POPS collected from outpatients were sent to our laboratory as neat, and 1 mL of VTM was added by the laboratory staff if the specimen was too viscous for accurate pipetting. All specimens were processed within c c e p t e d M a n u s c r i p t conditions: RT step at 55°C for 5 minutes and 95°C for 5 minutes, then 45 thermal cycling at 95°C for 3 seconds, 60°C for 12 seconds and 72°C for 3 seconds, followed by cooling at 40°C for 30 seconds. SARS-CoV-2 RNA was considered present if the Cp value of the reaction was ≤ 40 and a sigmoidal amplification curve was observed.

Test data for SARS-CoV-2 RNA detection in all respiratory and POPS specimens between Feb 1, 2020 and Apr 15, 2020 were retrieved from the laboratory information Analyses of diagnostic performance, agreement and temporal variation of result discordance were performed.

Sample size for the study was calculated to detect the difference between a true kappa A c c e p t e d M a n u s c r i p t of 0.5 and a kappa of 0.7 under the null hypothesis, at α=0.05 and power=0.80, assuming positivity rates for the two sample types are 0.5 and 0. 6 A c c e p t e d M a n u s c r i p t

A total of 13772 POPS and respiratory specimens, from 8596 patients, were received for SARS-CoV-2 RNA detection between Feb 1, 2020 and Apr 15, 2020. Of which 12700 were performed for diagnostic purpose, with an overall positive rate of 1.55%.

In particular, the positive rate of specimens sent from 'Tier 1' patients and testing centers for symptomatic returned travelers were 8.34% and 6.18%, respectively. The number of specimens per patient ranged from 1 to 41, with a median of 1. In additional to monitoring in COVID-19 patients, more than one specimen was sent from some of the patients due to the paucity of data on the optimal specimen type, and concerns with inter-specimen variation. The calculated sample size required for the study was 109 pairs, and we identified 229 same-day POPS-NPsp from 95 patients, 

Of the 229 POPS-NPsp identified, majority (70.3%) of the NPsp were NPS (Figure 1 ).

The results of the POP-NPsp are shown in Table 2 

Of the 229 POPS-NPsp, 161 were collected from 44 symptomatic COVID-19 patients. 

Only 21 POPS-NPsp from 7 pediatric patients were identified (age range: 4-18 years) (Supplementary Table 3 c c e p t e d M a n u s c r i p t have higher sensitivity [12] [13] [14] . Secondly, POPS collected in this study were not routinely mixed with VTM, unlike NPsp which are transported in VTM, which minimized any potential dilution effect. Thirdly, epithelial cells lining salivary gland ducts were previously shown to be an early target for SARS-CoV in a rhesus macaque model [29] , and a more recent study demonstrated high expression of ACE2 receptors, a receptor used by SARS-CoV-2 and SARS-CoV, on the mucosa of oral cavity [30] , thus POPS might theoretically contain more SARS-CoV-2 virions during early illness.

As expected, the percent agreement of POPS-NPsp is higher during the first 7 days of illness [12, 14, 31, 32] which adds to the support that POPS is suitable for the diagnosis of patients presenting during early infection. Moreover, no difference in categorical agreement was noted between POPS collected during early morning compared with other times of the day, implying that 'early morning' collection of POPS may not be mandatory. However, it must be noted that all patients included had refrained from eating, drinking and teeth brushing for at least two hours before obtaining POPS regardless of actual collection time. Finally, POPS may also be an acceptable alternative specimen type for SARS-CoV-2 RNA detection in children as fair categorical concordance among POPS-NPsp were also seen in this group.

However, more data is required to confirm our finding due to the small number of pediatric patients in our cohort.

A c c e p t e d M a n u s c r i p t There are several limitations in our study. Firstly, specimen subtypes among NPsp were rather heterogeneous and it could not be excluded that one or more NPsp subtype would be superior to POPS for SARS-CoV-2 RNA detection. However, this candidly reflects the logistic difficulties in standardizing the specimen collection protocol among inpatients, ambulatory patients and asymptomatic patients from different surveillance programs and settings, particularly during the present pandemic.

Secondly, not all POPS were mixed with VMT, which could affect the overall sensitivity of POPS due to dilution effect. However, this should only lead to a bias against POPS and would not change the overall conclusion in favor of POPS specimens in the present study. Finally, due to the retrospective nature of the study, there is considerable heterogeneity in sampling of the patient population, as both suspected and confirmed cases were included and tested at different frequencies. This is further complicated by the absence of a diagnostic gold standard, which precluded the calculation of diagnostic performance like test sensitivity and specificity. Hence, our results may not apply in other settings with a different mix of patients or disease prevalence.

Our result suggested that POPS is a satisfactory alternative specimen type for SARS-CoV-2 RNA detection, with acceptable agreement of test results when compared with NPsp. In addition to the ease of collection and lesser patient A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 

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We thank the Molecular Laboratory of Department of Pathology, Queen Elizabeth Hospital for their tireless support in performing molecular testing for SARS-CoV-2 RNA for patients with suspected or confirmed COVID-19.

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

None.

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