Development and Evaluation of a Flocked Nasal Mid-Turbinate Swab. for Self-Collected Respiratory Virus Diagnostic Testing

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1 JCM Accepts, published online ahead of print on July 0 J. Clin. Microbiol. doi:./jcm.0-0 Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Development and Evaluation of a Flocked Nasal Mid-Turbinate Swab for Self-Collected Respiratory Virus Diagnostic Testing Marek Smieja* 1,,,, MD PhD Santina Castriciano, BSc, ART Susan Carruthers, MLT Geoffrey So, BSc Sylvia Chong, BSc Kathy Luinstra, BSc James B. Mahony 1,,, PhD Astrid Petrich 1,,, PhD Max Chernesky, PhD Mario Savarese, PhD Daniele Triva, P. Eng. 1 Dept. of Pathology & Molecular Medicine, McMaster University, Hamilton ON Canada Hamilton Regional Laboratory Medicine Programme, Hamilton ON Canada Dept. of Clinical Epidemiology & Biostatistics, McMaster University Institute of Infectious Diseases Research, McMaster University Copan Italia SpA, Brescia, Italy *Correspondence: Marek Smieja, MD PhD L St. Joseph s Hospital 0 Charlton Ave. E. Hamilton ON LN A Canada Telephone: FAX: smiejam@mcmaster.ca Word count: abstract 0; text: 1 Keywords: diagnosis, specimen, respiratory virus Smieja et al. Nasal flocked swab Page 1

2 Abstract We developed and evaluated flocked nasal mid-turbinate swabs in asymptomatic and symptomatic volunteers. Self-collected swabs in asymptomatic volunteers yielded comparable numbers of respiratory epithelial cells versus staff-collected nasal (n=) or nasopharyngeal swabs (n=0). Self-collected swabs in symptomatic volunteers detected a specific virus by multiplex PCR in / (.%). Smieja et al. Nasal flocked swab Page

3 We report herein the design and evaluation of a novel self-collected nasal mid-turbinate swab for respiratory virus diagnosis. We previously demonstrated that Copan flocked nasopharyngeal swabs (NPS) collected significantly more respiratory epithelial cells than conventional rayon swabs, and improved sample collection for direct fluorescent antibody (DFA) testing of respiratory viruses[1]. We observed that cell yield from sampling the nose using a flocked swab designed for nasopharyngeal sampling was equivalent to nasopharyngeal sampling with rayon NPS. This led us to hypothesize that a flocked nasal swab designed to contact a greater nasal surface area would further improve cell sampling, and enable self-collection. We measured the nasal passages of adult white cadavers at the Michael DeGroote School of Medicine anatomy laboratory, McMaster University, and designed a tapered cone-shaped swab with greater length and diameter of flocked nylon (Figure). A collar was added at. cm as a guide to maximum insertion depth for adults. The nasal swab samples a large surface area of respiratory mucosa covering the inferior and middle turbinate bones, and is now commercially available in pediatric and adult sizes (Copan FLOQSwabs TM, Copan SpA, Brescia, Italy). Our primary study objectives were to determine the feasibility, acceptability, and performance characteristics of self-sampled nasal mid-turbinate swabs. We tested the adequacy of self-collected flocked nasal swabs; the equivalence of nasal and nasopharyngeal sampling; and the diagnostic yield for specific respiratory viruses by multiplex PCR. The study protocol was approved by the Research Ethics Board at St. Joseph s Healthcare, Hamilton, Canada. To examine the adequacy of respiratory specimen self-collection, healthy asymptomatic adult volunteers were recruited from hospital staff and visitors. After signed informed consent, volunteers self-collected two nasal flocked swabs, following printed instructions with illustrations. Each swab was inserted up to. cm, as tolerated, into the same Smieja et al. Nasal flocked swab Page

4 nostril of their choice. An experienced staff member then collected two additional nasal swabs, using the opposite nostril: a flocked mid-turbinate nasal and a rayon pernasal swab, in randomized order. A self-administered questionnaire assessed ease of self-collection, discomfort, and swab preferences. All four nasal swabs were placed into universal transport medium (UTM, Copan Italia SpA), and coded to maintain blinding. Samples were processed identically, as for current DFA protocols. Briefly, specimens were vortexed, centrifuged, and the pellet resuspended in buffered saline. µl of suspension was added to glass slide wells, air dried, fixed, and stained with the negative control for DFA (Diagnostic Hybrids, Athens OH). Respiratory epithelial cells were counted, and averaged over four high powered fields (hpf) at 00X magnification[1]. No viral diagnostics were performed on swabs from asymptomatic volunteers. As shown in the Table, cell yields varied significantly between the four nasal swabs collected (one-way ANOVA, P<0.001). The first and second self-collected nasal swabs yielded a mean±sd epithelial cell count of ± and ± cells/hpf, respectively (P=0.001, Dunnett s post-hoc test). An adequate specimen, defined as > cells/hpf [], was obtained in / (.%) of first self-collected swabs and / (.%) of second self-collected swabs. Staff-collected flocked nasal and rayon pernasal swabs yielded 1±1 and ± cells/hpf, respectively (P<0.001). The first self-collected nasal swab was superior to staff-collected rayon (P=0.001), but collected fewer cells than either the second self-collected swab (P<0.001), or the staff-collected nasal flocked swab (P<0.001). There was no significant difference between the second self-collected flocked nasal swab, and the staff collected flocked nasal swab (mean difference: -1 cells/hpf, %CI: -, +1, P=0.0). Smieja et al. Nasal flocked swab Page

5 To directly compare self- and staff-collected nasal swabs versus NPS, 0 of the asymptomatic volunteers consented to staff-collected NPS (see Table). Immediately after obtaining the two self-collected and two staff-collected nasal swabs, staff collected two additional swabs: a flocked pernasal NPS, and a rayon pernasal NPS, in randomized order. Selfcollected nasal swabs were done with one nostril, and all four staff-collected specimens used the opposite nostril. Amongst these 0 subjects, cell yields were significantly different between the swabs (one-way ANOVA, P<0.001). The mean±sd cell yield for the first and second selfcollected flocked mid-turbinate nasal swabs was ± and 1± (P<0.001); 1± and ± for flocked or rayon staff-collected nasal swabs (P<0.001); and 1± and ±0 cells/hpf for flocked and rayon NPS (P<0.001). The first self-collected flocked swab cell yield was higher than rayon nasal swab (P=0.001) and comparable to rayon NPS (P=0.), but collected fewer cells than the second self-collected flocked nasal swab, or the flocked NPS (all P=0.00). The second self-collected flocked nasal swab was superior to rayon NPS (P<0.001) and equivalent to flocked NPS (P=0.). The higher yield of the second self-collected swab, compared with the first, may have reflected an increase in confidence of self-collection, or perhaps an effect of cleaning from the first swab. As an additional measure of sample adequacy, we quantitated beta-actin gene DNA by PCR [] in the 0 volunteers undergoing concurrent nasal and nasopharyngeal sampling (see Table). Epithelial cell counts were highly correlated with log-transformed beta-actin copy numbers (R=0., P<0.001). Beta-actin concentrations were similar in self-or staff-collected flocked nasal swabs, and flocked NPS, but lower in rayon nasal or rayon NPS swabs (P<0.001). Sixty-five percent of volunteers reported no or mild discomfort from self-swabbing, with the remainder reporting moderate (1%) or severe (%) discomfort. Eighty-seven percent Smieja et al. Nasal flocked swab Page

6 reported little or no difficulty in self-swabbing. Only % preferred staff collection, 0% stated a preference for self-swabbing, and % expressed no preference. To evaluate the self-collected flocked nasal swab for diagnosing viral infections, 0 hospital volunteers were given a kit consisting of two flocked nasal swabs, UTM, and printed instructions. Volunteers self-swabbed twice using the same nostril, and placed swabs into appropriately-labeled UTM transport media. Over the following year, symptomatic volunteers self-swabbed within days of acute respiratory infection symptoms, and returned the swabs within days. We previously demonstrated that swabs in UTM are suitable for PCR diagnosis for 1 days at room temperature (Luinstra K, 00, unpublished). Specimens were batch extracted with NucliSENS easymag (biomérieux, Marcy l Etoile, France) and tested with the xtag Respiratory Virus Panel (Luminex, Austin TX), which detects 1 virus types and subtypes.[]. Forty-two of (.%) symptomatic volunteers had a virus detected in their nasal swab, including rhino/enterovirus, influenza A (one seasonal H1, one seasonal H and one pandemic H1N1 A/swine/California/0/00), influenza B, metapneumovirus, coronaviruses (two E, 1 HK-1, and NL), and 1 each of respiratory syncytial virus type A, parainflueza, parainfluenza, and adenovirus. Amongst the volunteers diagnosed with a respiratory virus, submitted two swabs, and submitted a single swab. Concordant virus infections were detected with both swabs in of (.%, P= 1.0, McNemar test). In volunteers with discrepant swab results, were positive in the first swab (one rhino/enterovirus, one RSV A and one pandemic H1N1), while were positive in the second swab (all rhino/enterovirus). Thus, the first and second self-collected swabs were equivalent for diagnosing respiratory virus infections by multiplex PCR, despite the Smieja et al. Nasal flocked swab Page

7 lower cell yields of first versus second self-collected swabs in asymptomatic volunteers. We did not perform antigen testing on nasal swabs, and the adequacy of first and second nasal swabs for antigen-based testing requires further research. In summary, self-collected nasal flocked swabs were equivalent to staff-collected nasal or NPS, and superior to rayon NPS the traditional reference standard using respiratory epithelial cell yield as a proxy for specimen adequacy. Self-swabbing was feasible without previous training. Coupled with multiplex PCR, self-collection enabled early diagnosis of many symptomatic volunteers with common respiratory viruses. These findings expand on previous studies of nasal sampling in children [-] and have implications for the diagnosis and study of respiratory viruses. First, nasal self-sampling was simple, preferred to staff collection, and may enable testing more people earlier in the course of their illness. Earlier testing may facilitate timely anti-viral treatment for influenza, and reduce influenza-associated complications and hospitalizations[]. Second, self-testing eliminates any biohazard to clinical staff from specimen collection, potentially reducing transmission to health care personnel. Third, as nasal swabs are minimally invasive, serial sampling may be feasible for outbreak investigation or for studies of transmission or response to therapy. We did not compare diagnostic yield of nasal swabs versus NPS amongst hospitalized patients, and do not recommend them for such patients without further study. In conclusion, the new flocked nasal mid-turbinate swab enabled self-collection of highquality samples, and facilitated diagnosis of respiratory virus infections by PCR. Acknowledgements. Funded by the Canadian Institutes for Health Research (CIHR). We thank our volunteers for participating, and Copan Italia, Brescia, Italy for contributing all swabs and media. Smieja et al. Nasal flocked swab Page

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9 References 1. Daley, P., Castriciano, S., Chernesky, M., Smieja, M. 00. Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients. J Clin Microbiol,, -.. Landry, ML., Ferguson, D. 00. Suboptimal Detection of Influenza Virus in Adults by the Directigen Flu A+B Enzyme Immunoassay and Correlation of Results with the Number of Antigen-Positive Cells Detected by Cytospin Immunofluorescence. J Clin Microbiol, 1(): Jobin, C., S. Haskill, L. Mayer, A. Panja and R. B. Sartor. 1. Evidence of altered regulation of I kappa B alpha degradation in human colonic epithelial cells. J. Immunology 1: -.. Mahony, J., Chong, S., Merante, F., Yaghoubian, S., Sinha, T., Lisle, C., Janeczko, R. 00. Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay. J Clin Microbiol :-0.. Heikkinen T, Salmi AA, Ruuskanen O Comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza. BMJ ; :1. Heikkinen T. Marttila J, Salmi AA, Ruuskanen O. 00. Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses. J Clin Microbiol; 0:-. Macfarlane P, Denham J, Assous J, Hughes C. 00. RSV testing in bronchiolitis: which nasal sampling method is best? Arch Dis Child; 0:-. Abu-Diab, A., M. Azzeh, R. Ghneim, R. Ghneim, M. Zoughbi, S. Turkuman, N. Rishmawi, A.E.R. Issa, I. Siriani, R. Dauodi, R. Kattan, and M.Y. Hindiyeh. 00. Comparison between Smieja et al. Nasal flocked swab Page

10 pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children. J Clin Microbiol :1-1.. Spyridaki, I.S., I. Christodoulou, L. de Beer, V. Hovland, M. Kurowski et al. 00. Comparison of four nasal sampling methods for detection of viral pathogens by RT-PCR-A GA() LEN project. J. Virol. Methods; Dec 1 Epub.. Harper SA, Bradley JS, Englund JA, File TM, Gravenstein S et al. 00. Seasonal influenza in adults and children--diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis : Smieja et al. Nasal flocked swab Page

11 Table. Mean Respiratory Epithelial Cell Yield amd Bet-Actin Levels Amongst Volunteers Sampled by Self- or Staff-Collected Flocked Nasal Swabs, Rayon Pernasal Swabs, or Nasopharyngeal Swabs (NPS) Mean ± SD Number of Cells/High Powered Field Self-Collected Nasal Staff-Collected Nasal Staff-Collected NPS* First Flocked Second Flocked Flocked Rayon Flocked Rayon N= ± ± 1±1 ± - - N=0* ± 1± 1± ± 1± ±0 Mean (SD) Log () Beta-Actin DNA Copies/Reaction N=0*.±0..±0..±0..0±0..±0.1.±0. *0 volunteers from the total of consented to NPS. Smieja et al. Nasal flocked swab Page

12 Figure. Flocked nasal mid-turbinate swab (upper) compared with flocked nasopharyngeal (middle) and rayon nasopharyngeal swabs used for both nasal and nasopharyngeal sampling (lower) Smieja et al. Nasal flocked swab Page 1

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