key: cord-259558-remrzrq1
authors: LeBlanc, Jason J.; Heinstein, Charles; MacDonald, Jimmy; Pettipas, Janice; Hatchette, Todd F; Patriquin, Glenn
title: A combined oropharyngeal/nares swab is a suitable alternative to nasopharyngeal swabs for the detection of SARS-CoV-2
date: 2020-05-16
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104442
sha: 
doc_id: 259558
cord_uid: remrzrq1

Given the global shortage of nasopharyngeal (NP) swabs typically used for respiratory virus detection, alternative collection methods were evaluated during the COVID-19 pandemic. This study showed that a combined oropharyngeal/nares swab is a suitable alternative to NP swabs for the detection of SARS-CoV-2, with sensitivities of 91.7% and 94.4%, respectively.

The first reports of 2019 novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from China in December, but quickly spread as a pandemic. [1] [2] [3] Laboratory testing for SARS-CoV-2 plays an essential role in infection control and public health mitigation strategies; however, testing has been hampered by global supply chain shortage nasopharyngeal (NP) swabs and universal transport medium (UTM). As such, alternative collection methods were rapidly evaluated, including nasal swabs, oropharyngeal (OP) swabs, throat washings, and saliva. [4] [5] [6] [7] [8] While NP swabs in UTM are the specimen of choice for respiratory virus testing, a recent study demonstrated the feasibility of COVID-19 testing from nasal sample collected with a swab typically used for chlamydia and gonorrhea testing: the Aptima Multitest swab (Hologic, Inc.) and its accompanying specimen transport medium (STM). [9] This study sought to further validate the Aptima swab/STM collection kit for the detection of SARS-CoV-2 using a single swab approach to sample the oropharynx and anterior nares (OP/Na).

In community assessment centers prioritizing areas with suspected community spread of SARS-CoV-2, specimens were collected for COVID-19 testing from 190 individuals using two different collection devices: a flocked NP swab in 3 ml UTM (Copan Diagnostics Inc., Murrieta, CA) and combined OP/Na sampling using the Aptima Multitest swab in 2.9 ml of STM (Hologic, Inc., San Diego CA), according to an accompanying instructional video (https://vimeo.com/397169241). Each specimen was stored at 4C until testing, and an aliquot was stored at -80C. Both swabs were in parallel within 12h of collection using two molecular methods. First, the SARS-CoV-2 assay, was performed on a Cobas 6800 system Amplification was performed on an Applied BioSystems 7500 Fast system (Thermo Fisher Scientific), and threshold cycles (Ct) values were determined by the manufacturer software. Results for each instrument were classified as positive or negative, and specimens yielding discrepant results were subjected to testing using the Xpert Xpress SARS-CoV-2 assay (Cepheid). Each test was compared to a modified reference standard defined as concordant results from at least two methods with different genetic targets. Sensitivity and specificity were calculated from 2 × 2 contingency tables with 95% confidence intervals (CI) for each collection (NP or OP/Na swabs) and instrument (LDT and commercial assay) using J o u r n a l P r e -p r o o f online software (https://www.medcalc.org/calc/diagnostic_test.php). A Fisher exact test was used to assess differences and P ≤ 0.05 was considered statistically significant.

The limited and unpredictable supply of NP swabs during the COVID-19 pandemic prompted the evaluation of swabs that were readily available and commonly used for sexually transmitted infections. OP/Na was lower than NP swabs using the LDT or commercial assays, no significant differences were observed (P = 0.679 and 0.115, respectively).

Patients with discrepant NP and OP/Na results are summarized in (Table 1) . With the exception of patient 4, the other five patients with discrepant NP and OP/Na results had specimens with low viral loads (Table 1) . Low viral loads are known to occur in the early and late stages of COVID-19 illness [4] [5] [6] [11] [12] [13] [14] [15] [16] [17] [18] [19] , and false negative results can arise from differences in analytical sensitivity between methods (Table S1 ) [20, 21] , the variability in specimen collection, or factors influencing specimen stability or recovery of SARS-CoV-2 RNA during specimen transport, storage or processing. [4, 13] For example, three different SARS-CoV-2 targets were detected between the various PCR methods used for testing of J o u r n a l P r e -p r o o f specimens from patient 1, yet high Ct values were observed for these targets (Table 1) . High Ct values are suggestive of low viral loads, and it is known that detection of PCR targets near the limit of detection lacks reproducibility. [20, 21] Therefore, low viral loads and differences in analytical sensitivity of the various molecular methods could explain differences in SARS-CoV-2 detection between the NP and OP/Na collections (Table S1 ). Similar arguments could be made for patients 2 to 4, who were either asymptomatic or in the pre-symptomatic stage of infection where low viral loads can occur. [4] [5] [6] [11] [12] [13] [14] [15] [16] [17] [18] [19] Discrepant results for patients 5 and 6 were in the setting of known positive cases, with symptoms predating their sample collection by 14 and 18 days, respectively. Waning viral loads over time in the upper respiratory tract are well documented for SARS-CoV-2; however, discrepant NP and OP/Na results from sampling in the later stages of illness may be of little clinical significance, as detection of SARS-CoV-2 RNA does not imply infectivity. [4, 6, 11, 19] Further analyses are underway to correlate SARS-CoV-2 detection, and better understand viral shedding from various anatomical sites in patients stratified by disease onset, clinical presentation, and outcomes. Interestingly, patient 4 had a positive NP swab with low Ct values (i.e. high viral load) by three different methods, but the OP/Na on the same patient was negative. The exogenous internal control in the commercial assay was amplified from the OP/Na specimen (arguing against the presence of PCR inhibitors); however, the LDT endogenous control in the OP/Na reaction was near the cutoff (Ct value of 34.9). While an unlikely alternative explanation could be a false-positive result for the NP swab, it is more likely that there were collection or transport deficiencies for the OP/Na specimen.

The data obtained from this study represents a relatively short time period in a community setting with a mixed population of asymptomatic and mildly-symptomatic patients. While OP/Na swabs collection showed excellent performance for the detection of SARS-CoV-2, as previously shown for nasal sampling [9] , one should exercise caution in applying these findings to other patient populations, collection *Discrepant analysis using Xpert testing was only performed on nasopharyngeal swabs in UTM, as the OP/Na showed reduced sensitivity for this assay (Table S1 ). Abbreviations: Threshold cycle (Ct), envelope (E); laboratory-developed test (LDT); nucleoprotein (N2); not available (N/A); J o u r n a l P r e -p r o o f

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on behalf of the COVID-19 Pandemic Diagnostics Investigation Team of the Canadian Public Health Laboratory Network (CPHLN) Respiratory Virus Working Group