key: cord-1025862-88kwhbf0
authors: Lantry, F Julian; Epsi, Nusrat; Pollett, Simon; Simons, Mark P; Lindholm, David A; Colombo, Rhonda E; Fries, Anthony C; Maves, Ryan C; Ganesan, Anuradha; Utz, Gregory C; Lalani, Tahaniyat; Smith, Alfred G; Mody, Rupal M; Colombo, Christopher J; Chi, Sharon W; Madar, Cristian; Huprikar, Nikhil; Larson, Derek T; Bazan, Samantha; Broder, Christopher; Laing, Eric; English, Caroline; Lanteri, Charlotte; Mende, Katrin; Tribble, David R; Agan, Brian K; Burgess, Timothy H; Richard, Stephanie A
title: Anatomical site, viral RNA abundance, and time of sampling correlate with molecular detection of SARS-CoV-2 during infection
date: 2021-12-08
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofab623
sha: 54ca6ab30ac8a1c8bc6cf24591ac090ffcdf24c2
doc_id: 1025862
cord_uid: 88kwhbf0

Background Nasopharyngeal (NP) swabs are the standard for SARS-CoV-2 diagnosis. If less invasive alternatives to NP swabs (eg, oropharyngeal [OP] or nasal swabs [NS]) are comparably sensitive, the use of these techniques may be preferable in terms of comfort, convenience, and safety. Methods This study compared the detection of SARS-CoV-2 in swab samples collected on the same day among participants with at least one positive PCR test. Results Overall, 755 participants had at least one set of paired swabs. Concordance between NP and other swab types was 75% (NS), 72% (OP), 54% (rectal swabs [RS]), and 78% (NS/OP combined). Kappa values were moderate for the NS, OP, and NS/OP comparisons (0.50, 0.45, and 0.54, respectively). Highest sensitivity relative to NP (0.87) was observed with a combination of NS/OP tests (positive if either NS or OP was positive). Sensitivity of the non-NP swab types was highest in the first week postsymptom onset and decreased thereafter. Similarly, virus RNA quantity was highest in the NP swabs as compared with NS, OP, and RS within two weeks postsymptom onset. OP and NS performance decreased as virus RNA quantity decreased. No differences were noted between NS specimens collected at home or in clinic. Conclusions NP swabs detected more SARS-CoV-2 cases than non-NP swabs, and the sensitivity of the non-NP swabs decreased with time postsymptom onset. While other swabs may be simpler to collect, NP swabs present the best chance of detecting SARS-CoV-2 RNA, which is essential for clinical care as well as genomic surveillance.

The rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a fundamental aspect of clinical care and population surveillance in order to limit its spread.

The Centers for Disease Control and Prevention (CDC) has recommended the use of swabs to collect upper respiratory specimens for the detection of SARS-CoV-2 [1] . Nasopharyngeal (NP) swabs have been regarded as the preferred collection device for SARS-CoV-2 detection [2, 3] . However, non-NP collection routes, such as anterior nasal swabs (NS), may be preferred based on patient comfort and potential decreased infection risk to the healthcare worker [2, 4] . The existing literature is mixed regarding the comparability of different sample collection methods for SARS-CoV-2 detection [2, 5] , including whether such differences change with time post-symptom onset or may simply be explained by differences in collection adequacy. Moreover, there are limited data on the performance of rectal swabs (RS) compared to upper respiratory specimens for detection of SARS-CoV-2 virus [6] .

The primary goal of this analysis was to compare NP swabs for the detection of SARS-CoV-2 with alternative collection types (NS, OP, and RS) using paired samples collected on the same day from participants enrolled in the Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) cohort study. Secondarily, the study compared time-varying viral RNA load estimates determined from quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) values among the different sample types. In addition to examining how anatomical site, RNA abundance, and illness time changed PCR sensitivity, we further investigated whether self-collection of NS specimens changed PCR assay sensitivity.

The EPICC study is a longitudinal cohort study investigating the risk factors associated with and the short-and long-term effects of SARS-CoV-2 infection in ten military treatment facilities (MTFs), nine of which provided data and samples for this analysis ( c c e p t e d M a n u s c r i p t EPICC study is enrolling participants who are military healthcare beneficiaries (active duty, dependents or retirees) who have confirmed SARS-CoV-2 infection, a COVID-like illness, a high-risk exposure to SARS-CoV-2, or who have been tested for or vaccinated against SARS-CoV-2 [7] .

Participants included in this analysis were required to have tested positive for SARS-CoV-2 infection (confirmed case of COVID-19 based on any PCR positive test within 7 days pre-to 21 days postsymptom onset), and have at least one pair of swabs from different sites collected on the same day within 30 days of symptom onset.

EPICC study personnel collected swab specimens using synthetic flocked swabs from outpatients at day 0 and 14 post-enrollment from March 2020 onward. Beginning in July 2020, participants were offered the option of at-home NS collection if they were unable to present for inperson collection. Inpatients had swabs collected on day 0, 3, 7 and then weekly thereafter, if they continued to be hospitalized. NS were self-collected, whereas OP and NP were collected by clinic staff, and RS were either self-or clinic staff-collected. As concurrent, standardized swab collections were required for comparison, we excluded results from clinical specimens collected prior to enrollment into this study. qPCR was performed on study samples using the SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay research use only kit (Cat. # 10006770, produced by Integrated DNA Technologies, Inc., Coralville, IA). Two regions of the nucleocapsid (N) gene are targeted with the assay (N1 and N2), and an additional primer/probe is included to detect the human Ribonuclease P gene (RP) in the samples as a quality control measure. Samples with cycle threshold (Ct) values of <40 for both N1 and N2 were considered to be SARS-CoV-2 positive consistent with the instructions for use. Viral RNA load (genome equivalents (GE)/reaction) were determined using standard curves specific to each assay plate. Each curve was calculated based on at least three dilutions of known SARS-CoV-2 gene copy numbers. These plate specific curves represent the relationship between Ct and viral copy number for each plate and then allowed for conversion of Ct values to GE/reaction for each swab specimen [8] .

To compare SARS-CoV-2 detection among NP, OP, NS, and RS samples collected on the same day from the same participant, we calculated positive result concordance, discordance, and A c c e p t e d M a n u s c r i p t associated Kappa (SE) values for NP:OP, NP:NS, NP:RS, and NP:OP+NS paired sets. For the OP+NS paired set, participants had NP, OP, and NS samples, and results from the OP and NS swab were combined; if either the OP or the NS sample was positive, the OP+NS category was positive.

We also determined the sensitivity of OP, NS, RS, and OP+NS samples as compared to the NP samples as the reference. For this initial analysis, we tested only the first paired sample set collected for each person for the analysis. We also compared the sensitivity in different categories of time since onset of symptoms (<7 days, 7-13 days, and 14-30 days) and age groups (18-44, 45-64, and 65+); for the time since onset analysis, we retained the first paired sample collected in each time period for each person. Viral load quantity (GE/reaction) and RP values (to estimate collection adequacy [9] ) were compared among swab sample types using paired T-tests for paired samples and standard T-tests for non-paired samples. Viral load quantity (GE/reaction) for the N1 and N2 nucleocapsid gene targets was log 10 transformed for the comparisons.

EPICC was conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines. Informed consent was obtained from all individual participants in the study and this study was approved by the Uniformed Services University Institutional Review Board (IDCRP-085).

In total, 755 SARS-CoV-2 positive participants were included in this analysis (Table 1) Overall concordance values ranged from 54% for the NP:RS pairs to 72% for the NP:OP pairs and 75% for the NP:NS pairs (Table 2) Table 2) . Rectal swabs had very low sensitivity (0.20). Sensitivity was higher for all non-NP (alternative) types in the first week following symptom onset and decreased thereafter ( Figure 1) . A sub-analysis considering performance of the alternative sample types by age category identified a trend toward lower sensitivity in all alternative sample types in the youngest age group (18-44, Supplementary Figure 1 ), but the differences were not statistically significant.

When we compared N1 and N2 RNA quantity (GE/reaction) between NP:OP, NP:NS, and NP:RS sample pairs, we found that the viral copy number was higher in the NP sample than it was in the OP sample (N1: mean log 10 difference=0. A c c e p t e d M a n u s c r i p t

This study was a sub-analysis within EPICC, enabled by the robust longitudinal collection of multiple sample types. Our findings demonstrate that NP swabs routinely yielded better detection of SARS-CoV-2 nucleic acid in SARS-CoV-2 positive participants than alternative swab types (OP, NS, RS) collected concurrently. NS and OP had relatively low sensitivity when compared with NP swabs, while kappa scores ranged from fair to moderate. RS had very low sensitivity and kappa, compared to NP swabs which is consistent with other studies [10, 11] . In addition, sampling time, but not age, was a predictor of diagnostic performance of the test with different sampling techniques in our study, as has been noted elsewhere [12] .

Our findings highlight the importance of early collection, as the sensitivity of OP and NS swabs compared to NP swabs was highest when the samples were collected within seven days postsymptom onset and decreased in later timepoints. These results are similar to those from a systematic review comparing alternative swab types to NP swabs [2] , which found that the alternative swabs identified a lower percent of positives than the NP swabs; however, the review noted some degree of publication bias, i.e., the published studies appeared to be biased toward those that demonstrate that alternative sample types perform well. RS samples did not perform well, relative to NP samples, although the numbers were limited.

The quantity of virus detected in the swab samples was significantly higher for NP swabs than for NS and OP swabs, despite higher RP values in the alternative sample types (RP values suggest adequate collection technique) [9] . These results would support a hypothesis that differences in detection for the NP swabs (compared to other swabs) are not explained by poor collection technique for NS and OP swabs. Our findings are consistent with prior studies showing a higher RNA abundance in NP swabs (compared to OP swabs) [3] , although our study also demonstrated this in comparison with nasal and rectal swabs. All of the EPICC participants included in this analysis were symptomatic; given that NP swabs are more sensitive than the alternative sample types, one might expect that NP would perform better in asymptomatic individuals with potentially lower viral loads, but further research is needed.

A c c e p t e d M a n u s c r i p t In some cases, NS samples may be preferred based on the potential for at-home collection of samples [9] which has the potential to increase enrollment in studies and facilitate expanded genomic surveillance. Importantly, no difference in virus quantity was observed between NS collected at home or in the clinic for these EPICC participants, indicating that self-collected NS specimens are a reasonable alternative when clinic visits are not possible, which is similar to previously published studies [4] . A previous study has demonstrated that combined oropharyngeal/nares swabs may be comparable to NP swabs [13] ; when we compared a combined OP+NS value with NP, sensitivity was much higher, indicating that a combined OP/NS strategy may be a good alternative when selfcollection at home is preferred.

EPICC is a large SARS-CoV-2 longitudinal cohort that has been recruiting since March 2020, and multiple swab types were collected in EPICC which allows for the exploration of different SARS-CoV-2 diagnostic testing methods. Yet, this analysis has some limitations. First, individuals were identified for recruitment into the cohort after an interaction with a healthcare system, sometimes weeks after developing symptoms, which resulted in relatively low sensitivity of all research sample types, particularly those collected beyond one-week post-symptom onset. However, this limitation allowed for the examination of swab performance across a wide time range relative to symptom onset.

Second, in the absence of a gold standard, the performance of alternative swab types was compared with results from NP swabs, which is an imperfect reference standard. In EPICC, NP swabs were found to perform best in detecting SARS-CoV-2 in the subset of SARS-CoV-2 positive participants with paired samples; therefore, NP swabs were used as the reference in this analysis. The viability of virus that was detected in the swabs was not determined for this analysis; it is possible that some of the positive tests detected dead virus particles and a positive test in this case does not identify infectivity.

The results of this study confirm NP swabs as the preferred collection route for detecting SARS-CoV-2, indicating that the convenience and comfort of OP and NS samples are offset by the lower sensitivity unless a combined OP+NS strategy is used. The study also confirms that NS collected at home are of equivalent quality to clinician-observed collected NS in diagnosing infection.

A c c e p t e d M a n u s c r i p t NP swabs present the best chance of detecting SARS-CoV-2 virus for clinical and public health indications, the latter of which is critical as variants of concern continue to emerge. 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 17 

Interim Guidelines for Collecting and Handling of Clinical Specimens for COVID-19 Testing

Performance of Saliva, Oropharyngeal Swabs, and Nasal Swabs for SARS-CoV-2 Molecular Detection: a Systematic Review and Meta-analysis

Nasopharyngeal Swabs Are More Sensitive Than Oropharyngeal Swabs for COVID-19 Diagnosis and Monitoring the SARS-CoV-2 Load

Self-Collected Oral Fluid and Nasal Swab Specimens Demonstrate Comparable Sensitivity to Clinician-Collected Nasopharyngeal Swab Specimens for the Detection of SARS-CoV-2. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

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Prolonged SARS-CoV-2 RNA detection in anal/rectal swabs and stool specimens in COVID-19 patients after negative conversion in nasopharyngeal RT-PCR test

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Different Types of Clinical Specimens

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A combined oropharyngeal/nares swab is a suitable alternative to nasopharyngeal swabs for the detection of SARS-CoV-2

A c c e p t e d M a n u s c r i p tWe appreciate the EPICC participants for their central role in this study. Many thanks to the IDCRP team at the clinical research sitesphysician/clinical investigators, site managers, regulatory staff, clinical research coordinators, and laboratory personnelfor their support of this study and contributions to its success under very challenging circumstances. The authors would like to thank Marietta Grother for her editorial assistance.We thank the members of the EPICC COVID-19 Cohort Study Group for their many contributions in conducting the study and ensuring effective protocol operations. The following members were all closely involved with the design, implementation, and/or oversight of the study and have met group authorship criteria for this manuscript: 

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