Abstract
Background:
In British Columbia (BC), self-collected saline gargle (SG) is the only alternative to health care provider (HCP)-collected nasopharyngeal (NP) swabs to detect SARS-CoV-2 in an outpatient setting by polymerase chain reaction (PCR). However, some individuals cannot perform a SG. Our study aimed to assess combined throat-bilateral nares (TN) swabbing as a swab-based alternative.
Methods:
Symptomatic individuals greater than 12 years of age seeking a COVID-19 PCR test at one of two COVID-19 collection centres in Metro Vancouver were asked to participate in this study. Participants provided a HCP-collected NP sample and a self-collected SG and TN sample for PCR testing, which were either HCP observed or unobserved.
Results:
Three-hundred and eleven individuals underwent all three collections. Compared against HCP-NP, SG was 99% sensitive and 98% specific (kappa 0.97) and TN was 99% sensitive and 99% specific (kappa 0.98). Using the final clinical test interpretation as the reference standard, NP was 98% sensitive and 100% specific (kappa 0.98), and both SG and TN were 99% sensitive and 100% specific (both kappa 0.99). Mean cycle threshold values for each viral target were higher in SG specimens compared to the other sample types; however, this did not significantly impact the clinical performance, because the positivity rates were similar. The clinical performance of all specimen types was comparable within the first 7 days of symptom onset, regardless of the observation method. SG self-collections were rated the most acceptable, followed by TN.
Conclusions:
TN provides another less invasive self-collection modality for symptomatic outpatient SARS-CoV-2 PCR testing.
Keywords: COVID-19, diagnostics, NAT, OMICRON, outpatient SARS-CoV-2, PCR, saline gargle, self-collection, throat-bilateral nares
Abstract
Historique :
En Colombie-Britannique (C.-B.), l’autoprélèvement de gargarisme d’eau saline (GS) est la seule alternative aux écouvillons nasopharyngés (NP) prélevés par un professionnel de la santé (PdS) pour déceler le SRAS-CoV-2 par test PCR en milieu ambulatoire. Cependant, certaines personnes ne peuvent pas effectuer de GS. La présente étude visait évaluer l’écouvillonnage de la gorge et des deux narines (GN) pour remplacer le GS.
Méthodologie :
Les personnes symptomatiques de plus de 12 ans qui demandaient un test PCR de la COVID-19 à l’un des deux centres de dépistage de la COVID-19 du Grand Vancouver ont été invitées à participer à la présente étude. Les participants ont fourni un prélèvement NP recueilli par un PdS ainsi qu’un autoprélèvement de GS et GN en vue d’un test PCR, observés ou non par un PdS.
Résultats :
Au total, 311 personnes ont participé aux trois prélèvements. Par rapport au prélèvement NP-PdS, le GS avait une sensibilité de 99 % et une spécificité de 98 % (kappa 0,97) et le prélèvement GN, une sensibilité de 99 % et une spécificité de 99 % (kappa 0, 98). À l’aide de l’interprétation définitive du test clinique comme norme de référence, le prélèvement NP avait une sensibilité de 98 % et une spécificité de 100 % (kappa 0,98) et tant le GS que le prélèvement GN avaient une sensibilité de 99 % et une spécificité de 100 % (deux kappa 0,99). Les valeurs seuils du cycle moyen de chaque cible virale étaient plus élevées dans les échantillons de GS quand dans les autres types d’échantillons, mais n’avaient pas d’effet significatif sur le rendement clinique, puisque les taux de positivité étaient semblables. Le rendement clinique de tous les types d’échantillons était comparable dans les sept premiers jours suivant l’apparition de la maladie, quel que soit le mode d’observation. L’autoprélèvement de GS a été classé comme le plus acceptable, suivi du prélèvement GN.
Conclusions :
Le prélèvement GN est un autre mode d’autoprélèvement moins invasif chez les patients ambulatoires symptomatiques qui effectuent un test PCR du SRAS-CoV-2.
Mots-Clés : autoprélèvement, COVID-19, diagnostics, gargarisme d’eau salée, gorge et deux narines, Omicron, PCR, SRAS-CoV-2 ambulatoire, TAAN
Introduction
Testing for SARS-CoV-2 nucleic acids by laboratory polymerase chain reaction (PCR) (nucleic acid testing [NAT]) is the diagnostic gold standard for COVID-19. While NAT generally suffers from higher turnaround times and more expensive reagent costs over other testing modalities (eg, COVID-19 rapid antigen tests), its higher diagnostic sensitivity remains critical for facilitating public health surveillance, infection control, and treatment as suggested by several groups including the World Health Organization (WHO), the Government of Canada, and others (1–7).
In many jurisdictions, health care providers (HCPs) collect clinical specimens using nasopharyngeal (NP) swabs, which are commonly tested in the laboratory by NAT methods. British Columbia (BC) was one of the first jurisdictions to implement the saline swish and gargle (also known as saline gargle [SG]) collection system in September 2020 as part of the back to school initiative for school-aged children, in which the individual swishes and gargles saline in their mouth three times before spitting it into a tube for NAT. The use of this testing modality enabled the province to overcome supply chain challenges associated with the procurement of NP swabs during the pandemic and offered patients a less invasive collection technique than NP swabs (8). Unlike saliva samples, these samples are machine-ready and do not require any additional handling before testing. SG performed similarly compared to HCP-collected NP swabs in outpatients. The use of SG also increased throughput of patient sample collections at community testing centres (9). Similar performance between HCP-observed and unobserved self-collected SGs has been demonstrated previously, which made self-collection without any HCP involvement possible (10).
In BC, SG remains the only less invasive alternative to HCP-collected NP swabs in the outpatient setting. Some individuals, however, cannot perform a SG (ie, they are unable to gargle), arguing for the availability of other less invasive and accurate sample collection techniques for NAT. While the favourable performance of combined-, self-collected throat-bilateral nares (TN) swabs has been demonstrated for rapid antigen tests for SARS-CoV-2 (11), studies assessing the use of self-collected TN for SARS-CoV-2 NAT have had small sample sizes, and the sample collection techniques used have been variable, thereby limiting generalizability (12–14). Consequently, the WHO recommends to conduct local validation work to determine performance, appropriateness, and use-case scenarios before implementing (15).
In this study, the performance of self-collected TN swabbing was compared against HCP-collected NP swabbing, as well as self-collected SG for SARS-CoV-2 NAT. The goal of this study was to assess the performance of TN swabbing relative to the other sample collection techniques, and to establish guidelines and operational considerations on how to properly collect samples for testing, either unobserved, or under the observation of a trained HCP.
Methods
Recruitment
From April 21 to May 12, 2022, individuals from the Greater Vancouver Metropolitan Area seeking a COVID-19 PCR test at one of two COVID-19 outpatient-testing centres in the Fraser Health Region of Metro Vancouver were invited to participate. Individuals were eligible if they met B.C.'s PCR testing guidelines for COVID-19 as well as the SG pre-test criteria (both in Supplemental Material). Eligible individuals needed to be greater than 12 years of age, speak fluent English to provide informed verbal consent, and have a valid B.C. personal health number (PHN). Participation was voluntary. Verbal assent was also received from those aged 12–18 years old. Basic demographic and clinical information were collected including the participant's name, eligibility group for testing (ie, health care worker [HCW], immunocompromised, pregnant, or other [participants that are eligible for testing but chose not to identify with any of the previously mentioned categories]), age, PHN, symptoms, and date of symptom onset. Following consent, individuals provided three samples including a HCP-collected NP swab, a self-collected TN swab, and a self-collected SG specimen.
Specimen collections
Due to ongoing supply chain shortages associated with COPAN NP flocked swabs, YOCON NP flocked swabs, a Health Canada approved equivalent, were used (Virus Sampling Kit, MT0301, YOCON, Beijing, China). Trained HCPs collected NP swab specimens using routine clinical procedures with YOCON NP flocked swabs (16). Participants collected their own SGs by swishing and gargling a 5 mL aliquot of sterile 0.9% saline (Teleflex Medical, Research Triangle Park, North Carolina) three times and expelling it into a 10 mL sterile polypropylene collection tube provided by Columbia Plastics (Columbia Plastics Ltd., Surrey, Canada). Participants also self-collected TN samples using a dedicated COPAN throat flocked swab with a breakpoint beyond the anatomical site of collection (CA502CS01, Copan Diagnostics Inc., USA), by swabbing the posterior oropharynx and around the areas of the tonsils five times back-and-forth before inserting the swab into the nose and collecting a sample from both nostrils. A mirror was provided to assist with the collection. The swab was then placed into a YOCON viral transport medium (VTM) for transport. In the event that the participant could not collect a TN sample, the participant was asked to self-collect a sample from the buccal area instead with a fresh COPAN throat swab, before collecting a bilateral-nares sample. This was termed a buccal-bilateral nares collection (BN). Instructions for all self-collections were provided (Supplemental Material). The order of sample collections was altered depending on the day, with NP specimen being collected first on even numbered days followed by TN collections. This order was reversed on odd numbered days. SG specimens were always collected last. All study samples were collected during a single clinical encounter (Figure 1).
Figure 1:
Operational workflow at participating COVID-19 collection centres in the Fraser Health region of Metro Vancouver.
On even days, eligible individuals providing verbal consent were assigned to the first sample group. Individuals provided a health care provider (HCP) collected NP sample first, followed by a self-collected throat-bilateral nares (TN) or buccal-bilateral nares (BN) sample and finally a self-collected saline gargle (SG). On odd days, the order of HCP-collected NP and TN (or BN) was reversed
Observed versus unobserved collections
Self-collections were either observed or unobserved. In observed collections, HCPs observed the self-collection and intervened in case of incorrect technique. For unobserved self-collections, HCPs neither observed nor assisted the participant's collection. TN self-collections were observed during the first week of the study from April 21 to April 28, and unobserved during the remaining second and third weeks of the study, from April 28 to May 12. SG collections were observed during the first 2 weeks of the study, from April 21 to May 5, and switched to unobserved collections in the last week until May 12. Both switches were staggered to allow the collection centres to adapt to the new workflow.
User-acceptability rating
Following all three sample collections, participants were asked to rate the acceptability of each technique separately, using a 5-point Likert scale with ‘1’ being ‘totally unacceptable,’ ‘2’ being ‘slightly unacceptable,’ ‘3’ being ‘neutral,’ ‘4’ being ‘slightly acceptable,’ and ‘5’ being ‘perfectly acceptable.’
Laboratory workflow and testing
All samples were stored at room temperature after collection and couriered to the BC Centre for Disease Control Public Health Laboratory (BCCDC PHL) for routine SARS-CoV-2 NAT. Samples were extracted (input volume = 200 μL, eluate volume = 60 μL) on the MagMax Viral RNA Isolation kit (AM1836) and tested via the validated-, laboratory developed pentaplex reverse transcription-qPCR (RT-qPCR) assay. The assay detects the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene, envelope (E) gene, and human ribonuclease P (RNaseP) (endogenous control), as well as influenza A (M Segment), influenza B (NS Segment), and respiratory syncytial virus (RSV) A and B (Matrix gene) using a selection of primers and probes, and following a standard operating procedure (Supplemental Material). Nucleic acid targets detected with a cycle threshold (Ct) ≤35 were reported as positive, indeterminate when between Ct >35 and <40, and negative when they were not detected within 40 cycles or more. SARS-CoV-2 positive NP samples were whole genome sequenced (WGS) for variant determination following a standardized protocol (17). Participants were classified as either COVID-19 positive or negative based on their laboratory test results of the NP swab. Alternatively the participant was considered SARS-CoV-2 positive if any of the three specimen types tested positive for both targets, E gene and RdRp gene, with a Ct value of ≤35 on the BCCDC PHL laboratory developed test (LDT), and presented with COVID-19 symptoms. This was termed the final clinical test interpretation. Indeterminate or discrepant NP swab results were rerun if the other two sample types were concordant positive or negative, or upon the request of the ordering physician (Supplemental Material).
Calculations and statistical analysis
Descriptive statistics were presented as means or medians as appropriate for continuous variables and proportions for categorical variables. For the comparison between two independent groups (e.g., collection order or observed versus unobserved), the t-test or Mann–Whitney U test were used for numeric or ranked variables as appropriate, and the chi-square or Fisher exact test were used for categorical variables, as appropriate. To evaluate the sensitivity, specificity, and concordance of self-collected SG, TN, or BN, either the HCP-collected NP swab was used as the reference standard or the final clinical test interpretation (18). The McNemar exact test was used for this comparison and the Kappa value was used to indicate the level of agreement (19). Based on the positivity rate and testing volume at the participating collection centres, this study was intended to run for 3 weeks until at least 60 SARS-CoV-2 positive cases were detected via HCP-collected NP swab specimen, to get at least 75% power at 0.05 significance level with two-sides (9,20–22). The NAT Ct values obtained with baseline testing using all sample types were compared with a repeated-measures analysis of variance (ANOVA) using Box’s conservative correction factor, with post hoc significant pairwise differences determined using Tukey’s honestly significant difference (HSD). This methodology was also be used to compare acceptability ratings of all sample types (23,24). Statistical significance was set as a p-value of <0.05. All statistical analysis was done using SAS 9.4 (SAS Institute Inc., Cary, North Carolina).
Ethics
This project was reviewed by the Fraser Health Research Ethics Board and deemed a quality improvement (QI)/quality assurance (QA) activity.
Results
In total, there were 311 individuals who agreed to participate in this evaluation. One hundred and forty-four participants (46%) were positive for COVID-19 using the HCP-collected NP swab as the reference standard compared to 151 (48%) based on the final clinical test interpretation criteria. One hundred and five participants were HCWs, 26 were immunocompromised, 1 was pregnant, 173 individuals who did not identify with any of the previously mentioned categories, and for 2, information was unavailable. Participants presented on average with five symptoms and were on the third day since their symptom onset. The leading symptoms were cough (81%), sore throat (76%), fever/chills (65%), body/muscle ache (64%), headache (56%), and runny nose (57%). Fifty-one percent of sample collections were conducted with the HCP-collected NP swab first followed by the TN swab, while 48% were conducted with the TN swab first followed by HCP-collected NP swab. SG was collected last for all participants. Forty-eight percent of TN sample collections were observed by a HCP, compared to 52% that were unobserved. Eighty-one percent of SG collections were observed by a HCP, compared to 19% that were unobserved. WGS results showed the leading lineages were BA 2 (44%), BA 2.12 (30%), and BA 2.3 (11%) (Table 1). The youngest participant was 13 years and the oldest 80 years old, with the average age being 43. Fourteen individuals opted for BN collections in favor of TN.
Table 1:
Description of categorical variables in groups with or without SARS-CoV-2 positive results according to the final clinical test interpretation definition
| Final interpretation negative (N = 160) | Final interpretation positive (N = 151) | Total (N = 311) | p-value | ||
|---|---|---|---|---|---|
| Collection site | Coquitlam | 32 (20.0%) | 30 (19.9%) | 62 (19.9%) | 0.98 |
| Surrey | 128 (80.0%) | 121 (80.1%) | 249 (79.8%) | ||
| Collection order | Sample Group 1 | 77 (48.1%) | 83 (55.0%) | 160 (51.3%) | 0.23 |
| Sample Group 2 | 83 (51.9%) | 68 (45.0%) | 151 (48.4%) | ||
| TN/BN observed vs. unobserved | Observed | 77 (48.1%) | 72 (47.7%) | 149 (47.8%) | 0.94 |
| Unobserved | 83 (51.9%) | 79 (52.3%) | 162 (51.9%) | ||
| SG observed vs. unobserved | Observed | 124 (77.5%) | 128 (84.8%) | 252 (80.8%) | 0.1 |
| Unobserved | 36 (22.5%) | 23 (15.2%) | 59 (18.9%) | ||
| Final interpretation influenza A | Negative | 153 (95.6%) | 151 (100.0%) | 304 (97.4%) | 0.01 |
| Positive | 7 (4.4%) | 7 (2.2%) | |||
| Lineages* | BA.2 | 67 (44.4%) | 67 (21.5%) | n/a | |
| BA.2.12 | 45 (29.8%) | 45 (14.4%) | |||
| BA.2.12.1 | 5 (3.3%) | 5 (1.6%) | |||
| BA.2.20 | 2 (1.3%) | 2 (0.6%) | |||
| BA.2.23 | 2 (1.3%) | 2 (0.6%) | |||
| BA.2.3 | 16 (10.6%) | 16 (5.1%) | |||
| Unassigned | 8 (5.3%) | 8 (2.6%) | |||
| Eligibility group* | HCW | 53 (33.1%) | 52 (34.4%) | 105 (33.7%) | 0.29 |
| Immuno-compromised | 10 (6.3%) | 16 (10.6%) | 26 (8.3%) | ||
| Other | 92 (57.5%) | 81 (53.6%) | 173 (55.4%) | ||
| Pregnant | 1 (0.6%) | 1 (0.3%) | |||
| Unavailable | 2 (1.3%) | 2 (0.6%) |
Missing values are not included
When the NP swab result was taken as the reference standard, the clinical performance of self-collected SG and TN was concordant with the HCP-collected NP results and compared to each other. The sensitivity and specificity of SG was 99% and 98%, respectively (kappa 0.97). TN sample collections were 99% sensitive and 99% specific (kappa 0.98). When the final clinical test interpretation was used as the reference standard, the sensitivity was 98% for HCP-collected NP (kappa 0.98), 99% for SG (kappa 0.99), and 99% for TN (kappa 0.99). The specificity was 100% for all (Table 2). In terms of the analytical performance, it varied between sample types (Figure 2a). SG specimens had a significantly lower mean RNase P Ct value compared to NP specimens (28.7 versus 29.8, p < 0.0001), while the mean of NP specimens was significantly lower compared to TN specimens (29.8 versus 30.7, p < 0.0001). The mean E gene Ct value was significantly lower in NP specimens than TN (20.05 versus 21.54, p < 0.0008) and SG (20.05 versus 24.50, p < 0.0001). Similarly, the mean RdRP gene Ct value was significantly lower in NP specimens than TN (20.0 versus 21.8, p < 0.0001) and SG (20.0 versus 24.4, p < 0.0001) (Table 3). Ct values of RNaseP, E gene, and RdRp gene targets in SARS-CoV-2 positive NP specimens were significantly-, positively correlated with SG and TN (Figure 2b). The collection order did not significantly affect the clinical or analytical sensitivity (Tables 4 and 5). When comparing HCP-observed versus unobserved TN and SG self-collections, the clinical and analytical performance of TN was not significantly different. For SG specimens, however, while the clinical performance did not change significantly, the Ct values for RNase P, E gene, and RdRp gene were marginally or significantly lower in observed specimens (Tables 6 and 7). Out of the 311 participants, five tested indeterminate by HCP-collected NP, four by self-collected SG, and four by self-collected TN. Rerunning the indeterminate HCP-collected NP samples did not change the original result.
Table 2:
Sensitivity and specificity results for assessed sample collection techniques based on the reference standard and observation method
| Sample type | Reference standard | No. | Sensitivity | Specificity | Kappa (95% CI) | p-value from McNemar’s test | |
|---|---|---|---|---|---|---|---|
| SG | Total | NP | 299 | 0.99 (137/138) | 0.98 (158/161) | 0.97 (0.95–1.00) | 0.32 |
| Observed | NP | 242 | 0.99 (116/117) | 0.98 (123/125) | 0.98 (0.95–1.00) | 0.56 | |
| Unobserved | NP | 57 | 1 (21/21) | 0.97 (35/36) | 0.96 (0.89–1.00) | 0.32 | |
| TN | Total | NP | 288 | 0.99 (138/139) | 0.99 (147/149) | 0.98 (0.96–1.00) | 0.56 |
| Observed | NP | 139 | 0.99 (66/67) | 1 (72/72) | 0.99 (0.96–1.00) | 0.32 | |
| Unobserved | NP | 149 | 1 (72/72) | 0.97 (75/77) | 0.97 (0.94–1.00) | 0.16 | |
| NP | Observed | FCIP | 306 | 0.98 (144/147) | 1 (159/159) | 0.98 (0.96–1.00) | 0.08 |
| SG | Total | FCIP | 304 | 0.99 (144/145) | 1 (159/159) | 0.99 (0.98–1.00) | 0.32 |
| Observed | FCIP | 247 | 0.99 (122/123) | 1 (124/124) | 0.99 (0.98–1.00) | 0.32 | |
| Unobserved | FCIP | 57 | 1 (22/22) | 1 (35/35) | 1 | N/A | |
| TN | Total | FCIP | 293 | 0.99 (144/145) | 1 (148/148) | 0.99 (0.98–1.00) | 0.32 |
| Observed | FCIP | 143 | 0.99 (69/70) | 1 (73/73) | 0.99 (0.96–1.00) | 0.32 | |
| Unobserved | FCIP | 150 | 1 (75/75) | 1 (75/75) | 1 | N/A | |
Note: Indeterminate results were not included in the sensitivity and specificity analysis. Buccal-bilateral nares collections are not captured in this table due to low sample sizes
NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares; FCIP = final clinical test interpretation
Figure 2:
(a) Box graph of cycle threshold (Ct) values of RNaseP, E gene, RdRp, and influenza A plotted by sample type. The dark line in the box displays the median and the box extends to the 25th and 75th percentiles.
The whiskers extend to minimum and maximum values and the dots represent SARS-CoV-2 positive samples based on the final clinical test interpretation (NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares; BN = buccal-bilateral nares). All samples were tested on BCCDC PHL’s Pentaplex assay with a Ct cut-off at ≤Ct 35 (N = 437; NP = 144; SG = 144; TN = 144; BN = 5). Lower Cts correspond to more target. Mean E gene Ct values are lowest for NP swab (Ct 20.1), followed by TN (Ct 21.5), BN (Ct 22.1), and SG (Ct 24.5). Similarly, mean RdRP Ct values were lowest in NP swab (Ct 20.0), followed by TN (Ct 21.8), BN (Ct 22.1), and SG (Ct 24.4). Differences between mean RNaseP Ct values were within two Cts across sample types. Nine samples tested positive for influenza A, none for influenza B, and one for RSV (not shown) with Ct values 27.59 for TN and for 30.42 SG, respectively. The NP swab result for this participant was negative. (b) Correlation graphs show RNaseP, E gene, and RdRp gene cycle threshold (Ct) values from self-collected saline gargle (SG) and throat-bilateral nares swabs (TN) plotted against health care provider (HCP)-collected nasopharyngeal (NP) swabs. Ct values are significantly positively correlated between HCP-NP swab specimens and respective self-collected SG and TN specimens for SARS-CoV-2 positive specimens, based on the final clinical test interpretation criteria (N = 432)
NP = Nasopharyngeal swab; SG = Saline gargle; TN = Throat-bilateral nares; BN = Buccal-bilateral nares
Table 3:
Analytical performance results for assessed sample collection techniques
| Target | N | Min. | Q1 | Mean | STD. | Median | Q3 | Max. | |
|---|---|---|---|---|---|---|---|---|---|
| RNase P | NP | 311 | 21.12 | 28.75 | 29.80 | 1.90 | 29.95 | 30.86 | 35.28 |
| SG | 308 | 24.00 | 27.74 | 28.71 | 1.73 | 28.67 | 29.90 | 33.44 | |
| TN | 297 | 25.06 | 29.11 | 30.65 | 2.27 | 30.74 | 32.17 | 38.29 | |
| NP vs. SG Mean Ct Difference | 1.0921 | p-value | <0.0001 | ||||||
| NP vs. TN Mean Ct Difference | -0.8580 | p-value | <0.0001 | ||||||
| E gene | NP | 148 | 11.88 | 15.81 | 20.05 | 5.89 | 18.22 | 22.35 | 39.00 |
| SG | 148 | 14.64 | 20.84 | 24.50 | 4.80 | 24.32 | 26.76 | 38.17 | |
| TN | 148 | 15.25 | 18.26 | 21.54 | 4.55 | 20.06 | 23.79 | 36.15 | |
| NP vs. SG Mean Ct Difference | −4.4047 | p-value | <0.0001 | ||||||
| NP vs. TN Mean Ct Difference | −1.5001 | p-value | 0.0008 | ||||||
| RdRp gene | NP | 145 | 13.0 | 16.2 | 20.0 | 5.2 | 18.2 | 22.4 | 39.7 |
| SG | 144 | 14.8 | 21.1 | 24.4 | 4.1 | 24.5 | 27.0 | 33.1 | |
| TN | 144 | 15.5 | 18.7 | 21.8 | 4.4 | 20.4 | 24.3 | 35.2 | |
| NP vs. SG Mean Ct Difference | -4.4163 | p-value | <0.0001 | ||||||
| NP vs. TN Mean Ct Difference | -1.7492 | p-value | <0.0001 | ||||||
Note: Buccal-bilateral nares collections are not captured in this table due to low sample sizes
NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares
Table 4:
Impact of collection order on the clinical performance for assessed sample collection techniques
| SARS-CoV-2 positivity rate according to | Collection order | ||
|---|---|---|---|
| NP -> TN -> SG (number of positives/total) | TN -> NP -> SG (number of positives/total) | p-value | |
| NP | 77/157 (49.04%) | 67/149 (44.97%) | 0.475 |
| SG | 79/155 (50.97%) | 65/149 (43.62%) | 0.199 |
| TN | 78/146 (53.42%) | 66/147 (44.90%) | 0.144 |
| FCIP | 83/160 (51.88%) | 68/151 (45.03%) | 0.228 |
Note: Buccal-bilateral nares collections are not captured in this table due to low sample sizes
NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares; FCIP = final clinical test interpretation
Table 5:
Impact of collection order on the analytical performance for assessed sample collection techniques
| Mean Ct | |||||
|---|---|---|---|---|---|
| RNase P | E gene | RdRP gene | |||
| NP | SARS-CoV-2 positive specimens | NP -> TN -> SG | 29.49 | 19.12 | 19.45 |
| TN -> NP -> SG | 30.02 | 20.09 | 20.39 | ||
| P value | 0.107 | 0.266 | 0.256 | ||
| All | NP -> TN -> SG | 29.69 | N.A. | N.A. | |
| TN -> NP -> SG | 29.92 | N.A. | N.A. | ||
| P value | 0.307 | N.A. | N.A. | ||
| SG | SARS-CoV-2 positive specimens | NP -> TN -> SG | 28.72 | 24.51 | 24.71 |
| TN -> NP -> SG | 28.59 | 23.68 | 23.96 | ||
| P value | 0.638 | 0.252 | 0.276 | ||
| All | NP -> TN -> SG | 28.62 | N.A. | N.A. | |
| TN -> NP -> SG | 28.81 | N.A. | N.A. | ||
| P value | 0.324 | N.A. | N.A. | ||
| TN | SARS-CoV-2 positive specimens | NP -> TN -> SG | 30.72 | 21.56 | 21.84 |
| TN -> NP -> SG | 30.28 | 21.51 | 21.83 | ||
| P value | 0.711 | 0.941 | 0.989 | ||
| All | NP -> TN -> SG | 30.69 | N.A. | N.A. | |
| TN -> NP -> SG | 30.61 | N.A. | N.A. | ||
| P value | 0.753 | N.A. | N.A. | ||
Note: Buccal-bilateral nares collections are not captured in this table due to low sample sizes
NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares
Table 6:
Impact of the observation method on the clinical performance for assessed self-collection techniques
| TN | SG | |||||
|---|---|---|---|---|---|---|
| SARS-CoV-2 positivity rate according to | Observed | Unobserved* | Observed | Unobserved* | ||
| (Number of positives/total) | p-value | (Number of positives/total) | p-value | |||
| SG | N.A. | N.A. | N.A. | 122/247 (49.39%) | 22/57 (38.60%) | 0.141 |
| TN | 69/145 (47.59%) | 75/161 (46.58%) | 0.861 | N.A. | N.A. | N.A. |
| FCIP | 72/149 (48.32%) | 79/162 (48.77%) | 0.938 | 128/252 (50.79%) | 23/59 (38.98%) | 0.102 |
Note: Buccal-bilateral nares collections are not captured in this table due to low sample sizes
There were 59 participants in the final week of the evaluation when both SG and TN were collected unobserved. After excluding one participant who provided no SG, one with an indeterminate SG, and 12 who collected an unobserved BN instead of a TN, there were 45 participants left. For those 45 participants, the positivity rate of SG was the same as TN (18/45 or 40%)
SG = saline gargle; TN = throat-bilateral nares; FCIP = final clinical test interpretation
Table 7:
Impact of the observation method on the analytical performance for assessed self-collection techniques
| Mean Ct | |||||
|---|---|---|---|---|---|
| RNase P | E gene | RdRP gene | |||
| SG | SARS-CoV-2 positive specimens | Observed | 28.52 | 23.84 | 24.06 |
| Unobserved | 29.50 | 25.77 | 26.09 | ||
| p-value | 0.018 | 0.055 | 0.033 | ||
| All | Observed | 28.58 | N.A. | N.A. | |
| Unobserved | 29.29 | N.A. | N.A. | ||
| p-value | 0.004 | N.A. | N.A. | ||
| TN | SARS-CoV-2 positive specimens | Observed | 30.75 | 21.84 | 22.14 |
| Unobserved | 30.56 | 21.26 | 21.55 | ||
| p-value | 0.618 | 0.442 | 0.424 | ||
| All | Observed | 30.55 | N.A. | N.A. | |
| Unobserved | 30.75 | N.A. | N.A. | ||
| p-value | 0.439 | N.A. | N.A. | ||
Note: Buccal-bilateral nares collections are not captured in this table due to low sample sizes
SG = saline gargle; TN = throat-bilateral nares
Detection of SARS-CoV-2 positive individuals was comparable across sample types within the first seven days after symptom onset. According to the HCP-collected NP swab, 137 participants tested positive for COVID-19 within seven days of experiencing symptoms. In contrast, when considering the final clinical interpretation criteria, 144 tested positive within the same time frame (Figure 3). Participants rated SG as the most acceptable sample collection technique, followed by TN and finally HCP-collected NP swab (Figure 4). Nine cases of influenza A and one RSV case were detected. The small number of identified cases was too low to make any specific conclusions.
Figure 3:
Bar graph showing the number of SARS-CoV-2 positive participants identified by sample type and days after symptom onset.
Day ‘0’ indicates participants that tested positive on the same day they first developed symptoms (NP = nasopharyngeal swab; SG = saline gargle; TN = throat-bilateral nares; BN = buccal-bilateral nares)
Figure 4:
Five-point Likert scale showing the relative sample acceptability score by collection technique, throat-bilateral nares (TN), saline gargle (SG), health care provider (HCP)-collected nasopharyngeal (NP) swab, and buccal-bilateral nares (BN) (N = 918; NP = 308; SG = 301; TN = 295; BN = 14). Totally unacceptable = 1; slightly unacceptable = 2; neutral = 3; slightly acceptable = 4; perfectly acceptable = 5
Discussion
As the public health response against the SARS-CoV-2 virus continues to evolve, the diagnostic testing landscape follows suit. Self-collection options are increasingly popular to spare vital health care resources, minimize the economic burden related to the pandemic response, and enable access to testing where appropriate, while not compromising on quality, accuracy, or safety (25–27).
This study aimed to evaluate combined TN sample collections as another self-collection option for COVID-19 symptomatic outpatients besides HCP-collected NP swabs and self-collected SG. RNaseP, a housekeeping gene confirming sample collection and quality, was assessed for all samples types in accordance with other studies (28,29). The mean RNaseP Ct was lowest in SG with the least amount of spread (ie, standard deviation), indicating a more consistent and reproducible sampling approach relative to the other sample types. However, mean E gene and RdRP gene Ct values, were significantly lower in HCP-collected NP and self-collected TN specimens than SG, which may be attributed to the dilution of the targets by the saline in SG. Despite the observed analytical performance differences, all three sample types, self-collected TN, SG, and HCP-collected NP demonstrated comparable clinical sensitivity and specificity results within the first 7 days of symptom onset, regardless of whether collections were observed or unobserved, because the positivity rate was similar. Proportionally, the number of indeterminate results was also similar across sample types with no significant differences.
While SG had highest user acceptability, TN self-collections were also rated higher than HCP-collected NP swabs with only a few participants opting for the BN collection instead. The user acceptability ratings reflect a similar pattern observed in the evaluation ‘Acceptability of self-collected samples for COVID-19 diagnosis in outpatients’, by Finlayson-Trick and colleagues conducted at B.C. Children’s Hospital (unpublished). Nasal swabbing alone was not assessed in this study, as TN offers sampling from two regions thereby improving sensitivity from sampling from only one area (30,31).
The clinical sensitivity and specificity findings from our study are comparable to several other publications (31–34). Kandel and colleagues evaluated several self-collection techniques against HCP-collected NP swabs on symptomatic and asymptomatic individuals. They reported an 85% sensitivity and 99% specificity of oral-nasal swabs compared to NP, while SG was 89% sensitive and 100% specificity. Similarly, Gertler and colleagues chose to evaluate a ‘multi swabbing’ technique in which a sample is collected from a combination of the tongue, inner cheek, and both nasal vestibules. They achieved a sensitivity of 95.2% with this multi-swab technique and SG samples (ie, gargled water) were 88.7% sensitive. Methodological variations concerning the collection techniques may account for the observed clinical performance differences. Kandel and colleagues performed the SG by swishing and gargling three times with 3 mL of 0.9% normal saline instead of 5 mL as was done in this evaluation. Gertler and colleagues used 10 mL tap water without any apparent gargling and swishing intervals. Viral load dynamics associated with the underlying variant and the sampling location could offer another explanation (35,36). These studies also reported differences in Ct values with self-collection techniques averaging higher Ct values for viral targets compared to HCP-collected NP swabs. This was especially noticeable in the self-collected SG specimens, which others attributed in their studies to the gargling and rinsing process involved in the collection and the subsequent dilution of the virus in the medium (ie, gargled water or saline) (10,37). While higher Ct values in SG and TN specimen did not significantly impact the clinical interpretation in this study, they could cause indeterminate results which would require follow-up consultation and additional testing. From an epidemiological perspective, the relationship between Ct values and the level of infectiousness may require future consideration of the sample type, or a broadening of the infectiousness classification based on Ct ranges to accommodate the observed differences (38–40).
This study had a few limitations. All samples were run on BCCDC PHL's LDT exclusively, which may not reflect the Ct performance results of other PCR NAT platforms (41,42). However, this approach also enabled a more valid comparison of Ct values between targets. While different platforms may yield different Ct values depending on the chosen target, this would reflect the performance of the assay itself and not the sample collection technique. In addition, while HCPs were instructed to intervene when they observed an incorrect collection from the throat during the observed phase of the evaluation, given the logistical challenges observing a sample collection at a drive-through collection centre the RNaseP Ct values were the only way to provide a consistent indication of sampling adequacy. Other limitations of this study include that only symptomatic outpatients were evaluated. Future studies might evaluate the performance of TN in a severe inpatient setting and replicate testing on different LDTs and platforms to expand generalizability (43); potentially even in non-laboratory (ie, community-based) settings.
The strengths of this study included a relatively large number of participants providing all three sample collection types for analysis and comparison with more participants participating in the study than initially anticipated presumably given the opportunity to be tested for multiple infectious pathogens concurrently. Other strengths include the assessment of each sample type performance across the date of symptom onset, as well as documenting standardized steps to perform the TN self-collection. The latter is especially important given that self-collections rely on untrained individuals performing their own collection and procedural errors during the collection can contribute to more false-negative results (32,34).
Conclusions
Combined TN swabbing represents another effective less-invasive way to collect an appropriate sample for SARS-CoV-2 NAT. For individuals seeking to collect a sample, but are unable to gargle, this technique can serve as a viable alternative to SG. Caution should be warranted to use swabs with a breakpoint beyond the anatomical site of collection, such as the posterior oropharynx and nares, to minimize the risk of these swabs being a choking hazard and getting caught in the nose. BN samples showed promising results too, although there is less certainty in these results given the small number of samples included in this study. The ability of TN to be self-collected with or without the observation of a trained HCP can help improve testing access by offering another less-invasive testing modality that does not require HCP involvement, thereby sparing vital health care staffing resources. The future application of these sampling techniques can be further expanded by validating the performance of TN and SG self-collections for other respiratory viruses such as influenza A, B, and RSV (25,28,30).
Acknowledgements:
The authors thank all members of the Fraser Health Operations Team who supported the study, including nurses, operations team members and management, and staff. We also give thanks to the BCCDC Virology Department and laboratory technologists for their tremendous support in setting up the laboratory workflow and data capture, the BCCDC Knowledge Translation team in developing the sampling instructions, and PHSA Supply Chain in providing the equipment. They give credit to Public Health Ontario for their image demonstrating a throat-bilateral nares and buccal-bilateral nares collection. Finally, we would like to thank all participants in this study who set time aside to support the COVID-19 pandemic response in B.C.
Contributors:
Conceptualization, EM Hempel, A Bharmal, G Li, LMN Hoang; Data curation, EM Hempel, G Li, A Minhas, R Manan, B Cheung, M Chan, K Gunadasa, R Chow; Formal analysis, EM Hempel, A Bharmal, G. Li, LMN Hoang; Investigation, EM Hempel, A Bharmal, LMN Hoang; Methodology, EM Hempel, A Bharmal, G Li, L Hamilton, B Cheung, M Chan, K Gunadasa, R Chow, F Tsang, M Krajden, A Jassem, LMN Hoang; Software, G Li; Project administration, EM Hempel, A Minhas, R Manan; Resources, EM Hempel, K Doull, F Tsang, K Mooder, A Jassem; Visualization, EM Hempel, G Li; Supervision, EM Hempel, A Bharmal, T Kassan, LMN Hoang; Validation, EM Hempel, A Bharmal, A Minhas, R Manan, T Lee, N Prystajecky, A Jassem, LMN Hoang; Visualization, EM Hempel, G Li; Supervision, EM Hempel, A Bharmal, T Kassan, LMN Hoang; Validation, EM Hempel, A Bharmal, A Minhas, R Manan, T Lee, N Prystajecky, A Jassem, LMN Hoang; Writing - original draft, EM Hempel, A Bharmal, G Li, LMN Hoang; Writing - review & editing, EM Hempel, A Bharmal, G Li, M Krajden, K Mooder, T Kassan, A Jassem, and LMN Hoang.
Data Availability:
All raw data and analysis code are available upon a request to the corresponding author.
Ethics Approval:
This study was approved by the Fraser Health Research Ethics Board, Surrey, British Columbia, Canada, on March 28, 2022.
Informed Consent:
Informed consent has been obtained from patients.
Registry and the Registration No. of the Study/Trial:
N/A
Funding:
No funding was received for this work.
Disclosures:
The authors have nothing to disclose.
Peer Review:
This manuscript has been peer reviewed.
Animal Studies:
N/A
Supplemental Material
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All raw data and analysis code are available upon a request to the corresponding author.