key: cord-0708362-1oyv4x1w authors: Kahamba, T. R.; Noble, L.; Scott, L.; Stevens, W. title: Comparison of three nasopharyngeal swab types and the impact of physiochemical properties for optimal SARS-CoV-2 detection date: 2020-10-23 journal: nan DOI: 10.1101/2020.10.21.20206078 sha: 7399b75974c883febeb2fc0be36189d9acccab7b doc_id: 708362 cord_uid: 1oyv4x1w Adequate swab specimen collection, release and detection of nucleic acids by molecular diagnostic assays is largely attributed to the physical and chemical characteristics of different swab types. We investigated properties of three types of commercial nasopharyngeal swabs (nylon flocked: Type 1-Media Merge; Type 2- Kang Jian Medical Apparatus, China and Type 3- Wuxi NEST Biotechnology Co. Ltd, China) used in clinical diagnostics with the aim to establish if different swab designs and configurations had any effect on swab performance. Properties investigated included viral absorption, release, capture, extraction and recovery efficiency from each swab for the detection of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). All swab types (n=18) were inoculated with different amounts of SARS-CoV-2 live viral cultures (1:10, 1:100 and 1:1000 copies/ml) and eluted in sterile phosphate buffer saline. RNA was extracted from all swab eluates using a fully automated system (BD MAX System) and cycle threshold (Ct) values were compared. RNA stability was also investigated after dry storage of swabs at room temperature for 72 hours. Statistically significant differences (p<0.05) were observed in the absorption and release capabilities between Type 1 and 3 as well as between Type 2 and 3 swabs, however, no significant difference was observed between Type 1 and 2. Ct values and extraction efficiency amounts of SARS-CoV-2 varied amongst the swab types. We conclude that in order to facilitate accurate SARS-CoV-2 diagnosis, assessment of NP swab characteristics is of importance before implementation for specimen collection in the clinical setting. In order to determine the absorption capacity and volume released of each swab, we adopted 80 methods and formulae for calculations from Zasada et al., and Wanke et al., (14, 15) with some 81 modifications. Briefly, swabs were immersed for 5-10s in pre-weighed 1.5ml eppendorf tubes that . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted October 23, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 (ng/µl) following formula: We performed the ANOVA statistical test using Stata/SE 16.0 (StataCorp LLC, USA) to determine 140 differences among means. When statistical significant differences were indicated (p<0.05), post-hoc 141 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. ; https://doi.org/10.1101/2020.10.21.20206078 doi: medRxiv preprint based on texture, odor and color of swab tip. Type 1 swab had the softest brush-like texture and was 147 given a score of 5. All swabs were odorless. Type 1 and type 3 swab tips appeared to be the whitest 148 in color (indicating high purity) and were therefore given a score of five. All shafts were made of 149 plastic, hence all swabs scored 5. Shaft properties included flexibility, brittleness and debris 150 production when broken beyond the breakpoint. All swab types were tough and did not produce any 151 debris when broken, however, types 1 and 2 appeared more flexible compared to type 3. Type 1 was 152 the shortest in length and types 2 and 3 were almost the same lengths. All swabs had good 153 breakpoints, but type 2 could not be snapped off and the breakpoint dismantled after a few twists. All 154 swabs were easy to use and did not disintegrate after vortexing/centrifugation (3000rpm, 5mins). Overall swab types 1 and 2 were given the highest scores. (Refer to supplementary table S1 for more 156 information). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. ; https://doi.org/10.1101/2020.10.21.20206078 doi: medRxiv preprint is crucial. The recommended shaft length is ~15cm and diameter of tip is ~1-3.2mm. Other important 218 properties include smoothness/softness, flexibility, ability to withstand torsion force, breakpoints, 219 durability, collection sufficiency and PCR compatibility (21, 23). In this study we performed a 220 qualitative analysis of the properties of three commercially available NP swabs. We observed some 221 variations in the length of swab tip, shaft and breakpoints which ranged from 16-27mm, 150-152mm 222 and 70-80mm respectively. All swabs had soft tips, which according to Copan Diagnostics facilitate 223 efficient sample release (24). The colour of the swab tip has also been highlighted to be an important 224 feature where generally a white swab tip indicates the absence of impurities/chemical 225 coating/treatment of the swab, hence will aid in pure sample collection (25, 26) . In the current study, 226 this was observed for swab types 1 and 3. The swab composition and structure have a significant impact on the main properties of the swab i.e. absorption, capture, extraction and recovery efficiency of collected sample (13, 14, 27) . In our study, 230 the three NP swabs revealed significant differences in volume absorption (the amount of fluid sample 231 absorbed) and volume released (volume of fluid sample released into buffer). These parameters were 232 inversely related to swab viral capture capacity (the amount of organisms removed from solution by 233 the swab) but strongly related to extraction efficiency (the effectiveness of sample transfer from the 234 swab tip to the extraction buffer) and recovery efficiency (the overall transfer effectiveness of swab 235 from the sampling site to the extraction buffer). Type 3 NP swabs had the highest absorbing and 236 releasing capacity compared to types 1 and 2 suggesting a direct relationship between the swab tip 237 length (i.e. high amount of nylon flock material on the tip (refer to supplementary table 2)) and these 238 properties. Less than 25%, 30% and 55% SARS-CoV-2 RNA was recoverable from swab types 1, 2 239 and 3 respectively, indicating that most of the viral particles either were left in buffer or were 240 entrapped by the swab material. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. ; https://doi.org/10. 1101 The quality of swab samples and other factors such as sampling procedures, time of sampling and 242 sample storage temperature contribute to adequate disease detection and monitoring of respiratory 243 infections (12, 28, 29) . We investigated storage of swabs over two time points (day 0 and day 3) at 244 room temperature. RNA was detected from all swabs at both time points without PCR 245 inhibition/interference but an increase in Ct values was observed at day 3 for all swab types, indicating lower viral loads detected. RNA stability was comparable for all swab types. Although there is a vast amount of NP swab validation studies (2, 6, 14, 27, (30) (31) (32) (33) (34) , this is, to our . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. ; nasopharyngeal swabs for influenza detection in adults. Clin Med Res 10:215-8. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. ; . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. . It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 23, 2020. Comparison of physical characteristics and 12 Interpreting a covid-19 test result The 15 Some are more equal-a comparative study on 308 swab uptake and release of bacterial suspensions Factors influencing recovery of microorganisms from surfaces by Collection and Diagnostics Effect of protein coating of flocked swabs on the collection 26 2012. eSwab flocked swabs unfit for viral culture Swab 337 sample transfer for point-of-care diagnostics: characterization of swab types and manual 338 agitation methods Sampling variability between two mid-turbinate swabs of the same patient has 341 implications for influenza viral load monitoring SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-344 analytics