key: cord-0777234-3tnmk78l
authors: Sutjipto, Stephanie; Lee, Pei Hua; Tay, Jun Yang; Mendis, Shehara M; Abdad, Mohammad Yazid; Marimuthu, Kalisvar; Ng, Oon Tek; Lin, Cui; Chan, Monica; Soon, Margaret; Lin, Raymond T P; Leo, Yee-sin; De, Partha P; Barkham, Timothy; Vasoo, Shawn
title: The effect of sample site, illness duration and the presence of pneumonia on the detection of SARS-CoV-2 by real-time reverse-transcription PCR
date: 2020-08-03
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa335
sha: a6ab0edf70aa344fd32f78af4d2a6caf79e08194
doc_id: 777234
cord_uid: 3tnmk78l

BACKGROUND: The performance of rRT-PCR for SARS-CoV-2 varies with sampling site(s), illness stage and infection site. METHODS: Unilateral nasopharyngeal, nasal mid-turbinate, throat swabs, and saliva were simultaneously sampled for SARS-CoV-2 rRT-PCR from suspect or confirmed cases of COVID-19.True positives were defined as patients with at least one SARS-CoV-2 detected by rRT-PCR from any site on the evaluation day or at any time point thereafter, till discharge. Diagnostic performance was assessed and extrapolated for site combinations. RESULTS: We evaluated 105 patients; 73 had active SARS-CoV-2 infection. Overall, nasopharyngeal specimens had the highest clinical sensitivity at 85%, followed by throat, 80%, mid-turbinate, 62%, and saliva, 38-52%. Clinical sensitivity for nasopharyngeal, throat, mid-turbinate and saliva was 95%, 88%, 72%, and 44-56% if taken ≤7 days from onset of illness, and 70%, 67%, 47%, 28-44% if >7 days of illness. Comparing patients with URTI vs. pneumonia, clinical sensitivity for nasopharyngeal, throat, mid-turbinate and saliva was 92% vs 70%, 88% vs 61%, 70% vs 44%, 43-54% vs 26-45%. A combination of nasopharyngeal plus throat or mid-turbinate plus throat specimen afforded overall clinical sensitivities of 89-92%, this rose to 96% for persons with URTI and 98% for persons <7 days from illness onset. CONCLUSION: Nasopharyngeal followed by throat specimens offer the highest clinical sensitivity for COVID-19 diagnosis in early illness. Clinical sensitivity improves and is similar when either mid-turbinate or nasopharyngeal specimens are combined with throat specimens. Upper respiratory specimens perform poorly if taken after the first week of illness or if there is pneumonia.

Since its emergence in December 2019, Coronavirus disease 2019 (COVID-19) has infected over 14 million people across 213 countries and territories as of mid-July 2020. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) offers definitive diagnosis for COVID-19, but the diagnostic yield of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 may vary with sampling site(s), stage of illness and whether disease involves predominantly the upper or lower respiratory tract.

Nasopharyngeal specimens comprising of flocked swabs, washes or aspirates are generally considered as optimal specimen type for the diagnosis of respiratory virus infections [1] , but compared to oropharyngeal or nasal swabs these are more technically complex to perform and often unpleasant for patients [2] .

While SARS-CoV-2 shedding from various sites may be prolonged, it is maximal in the upper respiratory tract in the first week of illness, as evidenced by studies on viral kinetics [3] [4] [5] [6] .

Besides more conventional respiratory specimens (nasopharyngeal, oropharyngeal or throat swabs), recent reports also indicate that SARS-CoV-2 can also be detected in saliva, which has been suggested as an alternate specimen for diagnostics [7] [8] [9] [10] [11] [12] . Limitations of available data include a lack of simultaneous sampling, smaller study numbers and variability in collection techniques.

The National Centre for Infectious Diseases (NCID) is a 330-bed facility (able to ramp up to 586 beds) which has admitted the majority of COVID-19 patients in Singapore. From the start of the COVID-19 outbreak, bilateral nasopharyngeal swabs were the primary specimen type used for SARS-CoV-2 diagnostics at our facility. Given the limited comparative data on simultaneously obtained clinical specimens, we sought to assess the clinical sensitivity of various specimen types for the diagnosis of SARS-CoV-2, in relation to duration of onset of illness and the presence of pneumonia.

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As part of a clinical audit, specimens from multiple sites (unilateral nasopharyngeal, midturbinate, throat swabs and saliva) were simultaneously taken on a single audit day for rRT-PCR for SARS-CoV-2, prospectively from a convenience sample of suspect or confirmed cases of COVID-19 admitted to the National Centre for Infectious Diseases, Singapore, from Besides the samples taken on the day of clinical audit, a standard bilateral nasopharyngeal swab was done upon admission as per protocol for all admitted for suspected COVID-19, and this was repeated 24 hours later if initial testing was negative [13] . If SARS-CoV-2 was detected (confirmed case of COVID-19), rRT-PCR was performed daily upon clinical recovery to ascertain virologic clearance (clinical recovery was defined as being afebrile for at least 24 hours, with improvement of clinical symptoms and being deemed medically fit for discharge by managing clinicians). Two negative PCR results 24 hours apart were required as a prerequisite for discharge.

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The nasopharyngeal swab was collected using a flexible minitip flocked swab (220252, Copan Diagnostics), inserted half the distance from the nares to the base of the ear, or to a depth of approximately 5 centimetres, and only unilateral swabs were sampled. Both midturbinate and throat swabs were done using a regular flocked swab (502C201, Copan Diagnostics). For mid-turbinate sampling, the swab was inserted approximately 1-2.5 centimetres, rubbed along the septum of the contralateral nostril for 3 to 5 seconds around the area of the middle turbinate, and withdrawn [14] . For throat (oropharyngeal) swabs, the posterior oropharynx was swabbed under direct vision. Each specimen was collected in an individual universal transport medium (UTM-RT, Copan Diagnostics).To collect saliva samples, patients were asked to rinse their mouth with plain water at least 30 minutes postmeal and 10 minutes pre-collection, to remove residual food debris. Two millilitres of fresh salivary sample was then spit out (drooling method) by the patient into a sterile container containing an equal amount of nucleic acid stabilisation formula (SF, Institute of Bioengineering and Nanotechnology) and this was mixed after capping, by gently inverting the container five times. All specimens were obtained by trained nurses. and processed within 24 hours.

The A*STAR Fortitude Kit (Accelerate Technologies, Singapore) was used for all samples after extraction (NucliSens EasyMAG, Biomerieux), as previously described [15] .

Additionally, saliva samples were tested with a second rRT-PCR assay targeting the N and ORF1ab genes, after extraction (EZ1 virus mini kit v2.0,Qiagen) [15] . and P values <0.05 were considered statistically significant.

This audit was performed as part of clinical operations at the National Centre for Infectious Diseases and results were used as part of routine clinical care. The reporting of this audit with waiver of written informed consent was approved by the institutional review board (National Healthcare Group Domain Specific Review Board reference number 2020/00338).

A c c e p t e d M a n u s c r i p t 9 guidelines (Supplementary material, https://www.equator-network.org/reportingguidelines/stard/).

A total of 581 patients were potentially eligible (171 with COVID-19). We included 105 patients in this evaluation, 32 patients who were tested negative for SARS-CoV-2 (11 patients recovered from COVID-19 and 21 had alternate diagnoses), and 73 with active SARS-CoV-2 infection (Table 1 , STARD flow diagram in Supplementary Figure S1 ). Twentyeight (27%) patients had pneumonia as evidenced by radiological changes, while 77 (73%) had upper respiratory tract infection (URTI) with normal radiological results. Overall, the median duration from onset of illness to the day of audit was 7 days (IQR: 3-10 days). One saliva sample was unavailable for analysis with the Fortitude kit, and seven for second assay targeting the N and ORF1ab genes, leaving 104 and 98 samples, respectively, for analysis for this particular sample type.

Data analysis of clinical sensitivity and specificity of individual sample sites and combination testing is summarised in Table 2 . Overall, nasopharyngeal specimens were found to have the highest clinical sensitivity at 85%, followed by throat, 80%, mid-turbinate, 62%, and saliva, 38-52%. There was no statistically significant difference between the nasopharyngeal and throat site for rRT-PCR overall, but the nasopharyngeal site was found to be more sensitive compared to mid-turbinate or saliva (either assay) (P <0.01, Supplementary table S1).

Clinical sensitivity of combined sites was extrapolated from results of individual sites. While the mid-turbinate site alone was less sensitive compared to nasopharyngeal, when either A c c e p t e d M a n u s c r i p t 10 was combined with throat swabs, Clinical sensitivity was similar at 89% vs 92%, respectively (P = 0.5); either combination offered the highest yield compared to others.

We performed a sub-group analysis of data based on the presence or absence of radiologic evidence of pneumonia and day of illness (Tables 2 and 3 ). For patients with URTI who by definition had normal radiological findings (n=77), a similar pattern was found with nasopharyngeal swabs having the highest clinical sensitivity (92%), followed by throat (88%), mid-turbinate (70%). Saliva had poor clinical sensitivity (43-54%). Combined nasopharyngeal and throat, or mid-turbinate and throat swabs showed the best performance at 96% and 94%, respectively (P =1.00). Nasopharyngeal specimens were found to be more sensitive than both mid-turbinate or saliva in patients with URTI (P = 0.003 and P <0.001, respectively, supplementary Table S1 ).

For patients with radiographic evidence of pneumonia (n=28), performance of upper respiratory tract samples was overall much less sensitive, with nasopharyngeal swabs having the highest clinical sensitivity but only at 70%, and the other upper respiratory samples 26-61%. Testing with a nasopharyngeal or mid-turbinate swabs, combined with throat swab improved clinical sensitivity to 82% and 78%, respectively. Nasopharyngeal specimens were found to be more sensitive than saliva, but not mid-turbinate specimens in patients with pneumonia (P = 0.002 and P=0.11, respectively, supplementary Table S1 ).

A c c e p t e d M a n u s c r i p t 11

In patients with onset of symptoms of ≤7 days (n=57), unilateral nasopharyngeal swab showed highest clinical sensitivity for COVID-19 diagnosis (95%), followed by throat swab (88%) and mid-turbinate swabs (72%) (nasopharyngeal versus throat P = 0.25; nasopharyngeal vs mid-turbinate, P = 0.006) ( Table 4 and supplementary table S1).

Combination testing from patients in their first week of COVID-19 showed the best performance, with a clinical sensitivity of 98% for either mid-turbinate or nasopharyngeal swabs combined with throat swabs.

As the illness progresses to day 8 and beyond, the clinical sensitivity of individual sample sites decreased significantly and ranged from 28% to 70%, with nasopharyngeal swabs still showing the best clinical sensitivity amongst the upper respiratory specimens. Notably, among the true positives in this audit, three were detected on post-audit lower respiratory specimens (two rRT-PCR positive by sputum, and another via endotracheal aspirate).

The performance of each sampled site and their associated rRT-PCR cycle threshold (Ct) values (Fortitude kit) with days of illness, is summarised in Figure 1 . Nasopharyngeal and throat swabs showed generally good performance results across the first week of illness up to day 10 but of symptoms onset but saliva samples, however, did not yield consistent results even in the first week of illness, in our study. These findings were corroborated by lower Ct values (a surrogate marker for higher viral loads) in the corresponding sampling sites demonstrating higher sensitivity, in earlier illness (≤7 days), as opposed to later illness (>7 days). The median Ct values for nasopharyngeal, mid-turbinate, throat and saliva A c c e p t e d M a n u s c r i p t 

Although the audit was not designed to address this primarily, we also examined the performance of bilateral nasopharyngeal swabs done on the pre-audit day (24-hours prior, with likely higher viral loads) as compared to the unilateral nasopharyngeal swabs done on the audit day. Given that viral shedding from upper respiratory tract is highest in the first week of illness, we limited this sub-analysis to patients with ≤7 days from onset of symptoms (n = 57 (54%)). We found that the clinical sensitivity of bilateral nasopharyngeal swab to be 98% (95% CI:87%-100%) versus unilateral nasopharyngeal swab 89% (95% CI:76%-96%), however this difference was not statistically significantly (P = 0.13).

Our study highlights several important findings and provides clinicians practical guidance as to optimal sampling sites for SARS-CoV-2 detection. Firstly, consistent with reports on higher viral shedding in early COVID-19 [4, 9, [16] [17] [18] , the clinical sensitivity of PCR diagnostics is highest across upper respiratory specimen types in early illness (≤ 7 days from symptoms onset), compared to later illness. Given that pneumonia occurs in later illness, the sensitivity of PCR was also found to be correspondingly diminished when upper respiratory tract specimens were sampled. This phenomenon is not peculiar to SARS-CoV-2 [19] , as viral loads in various anatomic sites are related to pathology at the site and degree of viral replication. Specifically to SARS-CoV-2 this is likely related to distribution of ACE2 (the receptor for viral entry) in the respiratory tract and the stage of disease [20, 21] .

A c c e p t e d M a n u s c r i p t 13 Secondly, among upper respiratory specimens, nasopharyngeal swabs were found to offer the best clinical sensitivity, followed by oropharyngeal specimens, although statistically significant differences between both sites were not found overall, or in sub-analyses for early illness or the presence of pneumonia. Mid-turbinate swabs were less sensitive than either naso-or oropharyngeal swabs. From clinical sensitivities extrapolated from the performance of individual sites, a nasopharyngeal or mid-turbinate swabs combined throat swab offered the highest, and similar clinical sensitivities. Mid-turbinate swabs may be less uncomfortable or invasive compared to nasopharyngeal swabs [2] , and if used, should be combined with oropharyngeal sampling.

Besides clinical sensitivity, the choice of sampling site may also depend on other factors including familiarity and training of staff who are obtaining samples, patient preference comfort, and adequate supply of requisites (such as the appropriate swabs). In the face of increased global demand on available trained staff and in areas with limited supply of personal protective equipment, patient-collected mid-turbinate sample may be considered as it offered up to 96% clinical sensitivity for the detection of SARS-CoV-2 when compared to health care worker-collected NP samples, as suggested in a study by Tu et al [22] , although based on our findings we suggest that this be combined with a oropharyngeal specimen to increase sensitivity.

Third, although saliva was previously reported as a promising sample and more convenient diagnostic sample for COVID-19, our data suggests otherwise. Several factors may account for this. Studies reporting higher sensitivities had small numbers, ranging from 12-39 SARS-CoV-2 positive patients studied [7] [8] [9] [10] [11] [12] . [7] . Also, the severity of illness differed in some studies. For example, Azzi et al studied 25 patients who had severe COVID-19, collecting salivary specimens by the 'drool technique' or pipetting pooled oral secretions, and found SARS-CoV-2 by PCR in all patients studied. This group also found that higher LDH levels (indicative of tissue damage) were correlated with lower Ct values with salivary samples. Another reason for differences in our study for saliva was that sensitivity could also have been influenced by pre-rinsing, as instructed by the developer of the transport medium. Nonetheless, given the findings of the various studies on saliva as a diagnostic for COVID-19, and older supportive data for saliva as a diagnostic specimen in humans and a rhesus macaque infection model [23, 24] for SARS-CoV-1 (which also uses ACE2 as its receptor), further standardization of methods for salivary diagnostics and larger confirmatory studies are needed.

Strengths of our study include its large sample size, and simultaneous sampling from multiple sites of each patient. Limitations of our study were that results from combination of sites were extrapolated from results of each site. We were also not able to systematically study lower respiratory tract or non-respiratory samples and are thus not able to assess the comparative performance of these. It is noted, however, that not all patients with COVID-19 may have a productive cough (and thus are unable to produce sputum) or are ill enough to warrant more invasive sampling (e.g. endotracheal aspirates or bronchoalveolar lavage).

Stool shedding, on the other hand, has been reported to persist for as long as 5 weeks in some patients and SARS-CoV-2 may be detected in stool beyond the duration respiratory shedding, and could be an alternate diagnostic specimen if upper respiratory samples are negative, although shedding may be not be detectable in all patients or may be intermittent [25] [26] [27] . Anecdotally, in our institution, we have diagnosed several cases of COVID-19 in the pneumonic phase, with stool being the only positive sample by PCR. Stool PCR, along with A c c e p t e d M a n u s c r i p t 15 other adjuncts to diagnosis such as serology or computed tomography (which may have characteristic findings, albeit not specific), may assist clinicians in the diagnosis of COVID-19. Another limitation is although one nasopharyngeal swab is sufficient for the detection of SARS-CoV-2, two swabs from each nostril are recommended for mid-turbinate swabs [28] .

This was not performed in our audit so as to minimise discomfort and trauma from repetitive sampling of the same nostrils, as sampling of all four sites (nasopharyngeal, mid-turbinate, throat and saliva) were done in the same setting during audit day. Lastly, the design of our audit did not allow us to fully compare the relative performance of unilateral versus bilateral nasopharyngeal swabs. Although the clinical sensitivity of bilateral nasopharyngeal swabs was found to be slightly higher than unilateral in early illness, our comparison was biased towards bilateral nasopharyngeal swabs (as these were obtained ~24 hours prior to the unilateral swab). Reassuringly, these differences were not statistically different.

In summary, nasopharyngeal and throat specimens offer the best clinical sensitivity for the molecular diagnosis of COVID-19 in early illness. More data and standardization of collection techniques is required to assess the utility of saliva as a diagnostic specimen. Although the sensitivity of mid-turbinate specimens was lower than nasopharyngeal and throat specimens, test sensitivities improve and are similar when mid-turbinate or nasopharyngeal are combined with throat specimens. Mid-turbinate combined with throat swabs may thus be an alternative to nasopharyngeal swabs which are more invasive and may cause more patient discomfort. In the pneumonic stage or later disease (>8 days), upper respiratory specimens perform poorly, and clinicians should be aware and seek alternate specimen types and other adjuncts to support a diagnosis of COVID-19. M a n u s c r i p t 23 1 All samples (unless otherwise stated) were tested with the A*STAR Fortitude Kit (Accelerate Technologies, Singapore). 2 A second PCR assay targeting the N and ORF1ab genes was used for saliva samples. 3 For data for sample site combinations, data from the A*STAR Fortitude Kit was used for saliva.

A c c e p t e d M a n u s c r i p t 24 

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We thank all clinical and nursing staff who provided care for the patients at National Centre for Infectious Diseases, Singapore ; and staff at the Department of Laboratory Medicine, Tan Tock Seng Hospital and the National Public Health Laboratory for their excellent work in this pandemic. We also thank Dr Tan Min Han (Institute of Bioengineering and Nanotechnology, A*STAR), for kindly supplying the saliva nucleic acid stabilization formula used for this study.Funding: None.

Diagnostica. Dr Barkham is a co-inventor of the Fortitude Kit, with a patent 'DETECTING A VIRUS' and royalties paid to Tan Tock Seng Hospital.

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 21