Consistency of influenza A test results across respiratory specimen collection methods using real time reverse 1 transcription polymerase chain reaction 2 3 Downloaded from Downloaded from Acknowledgments 93

Abstract 19 In our prospective cohort study, we compared the performance of nasopharyngeal, oropharyngeal, and 20 nasal swabs for the detection of influenza using real-time reverse transcription polymerase chain reaction assay. 21 Joint consideration of results from oropharyngeal and nasal swabs performed as well as nasopharyngeal alone 22 as measured by sensitivity and noninferiority analysis. Downloaded from In a prospective cohort study of 1781 healthcare personnel (HCP) at two medical centers during the 26 2010-11 influenza season, which is described elsewhere (1), we collected three respiratory specimen types for 27 acute respiratory illnesses (ARI) (with fever and cough) within 8 days of onset and tested them separately using 28 real-time reverse transcription polymerase chain reaction (rRT-PCR) assay. Each swab was placed in separate 29 viral transport tubes and shipped frozen to a CDC reference laboratory for testing. Influenza A positives (CT 30 values of < 40.0) identified by any swab type were considered true positives. 31 During the surveillance period, 268 HCP reported one or more ARI and provided all three upper 32 respiratory tract specimens (NP=nasopharyngeal; OP=oropharyngeal; NS=nasal swabs). The mean age of 33 participants was 42 years old, and most were female (87%), white (75%), in very-good or excellent self-rated-34 health (77%), and received the 2010-11 seasonal influenza vaccine (82%) (descriptive supplemental Negative predictive value was similar (> 95%) across swabs considered alone or in pairs The mean CT value was lower, indicating more viral RNA, among the 33 consistently influenza A positive 48 specimens compared with inconsistent positives; this was statistically significant for NP and OP swabs (p's < .01; 49 data not shown). 50 Inconsistent influenza results across swab types were associated with younger age, more days between 51 illness onset and swab collection, and taking antiviral medication prior to swab collection (p's < .01; 52 supplemental Table B available from authors). The association between age and inconsistency remained 53 significant after controlling for days since onset (p < .05). Illnesses with consistent vs. inconsistent swab results 54 did not differ in duration of illness, subjective ratings of illness severity, vaccination status, or other 55 characteristics we examined (data not shown). 56 In sum, we found that two-thirds (62%) of the influenza A positives were identified consistently across 57 swab types. Given the high sensitivity of the rRT-PCR assay (3) and the use of specific symptom criteria (i.e., 58 fever and cough) associated with influenza illness (4), we are confident that an influenza positive case identified 59 on October 6, 2016 by PENN STATE UNIV http://jcm.asm.org/ Downloaded from for any swab reflects a true influenza virus infection. Therefore, we were surprised that one in three (38%) 60 influenza A positives was not detected by one or more swab type if considered alone. Nonetheless, our finding 61 fits with previous studies showing that a modest number of influenza cases are missed if only one specimen type 62 is examined We also observed that joint consideration of results from more than one swab resulted in higher 64 sensitivity; though these differences were not statistically significant, the direction of the effect was consistent 65 with prior studies showing increased sensitivity with combinations of specimens Clinicians and researchers concerned about the discomfort associated with NP swab collection will be 73 reassured by our finding that joint consideration of NS and OP swabs resulted in equivalent sensitivity with 74 statistically noninferior results and fewer undetected cases than consideration of NP swabs alone. Our results 75 indicate that joint consideration of NS+OP swab results is not inferior to the NP-only method. 76 Prior studies have observed that the sensitivity of rRT-PCR results declines for specimens collected from 77 adults 5-days (9) or 7-days after illness onset (10), which is consistent with observations that virus shedding in 78 adults typically peaks during the first 24-72 hours of illness and lasts for about 5-7 days (11). Our findings 79 complement previous research by noting that the consistency of influenza positive results using rRT-PCR across 80 swab types also declines with greater days since illness onset. We also found that consistency increased with 81 older age in our cohort of adults aged 18-64 years, and this trend was independent of days since illness onset. 82 Although prior studies have noted higher rRT-PCR sensitivity for specimens collected from children compared to 83 adults (9), we are not aware of research showing differences in sensitivity or consistency of results with age 84 among adults. 85 Among the study's strengths are its prospective design, weekly surveillance of ARI, and focus on HCP 86 who are at increased risk of influenza exposure. Among the study's limitations is the small number of influenza 87 cases identified, which reduced statistical power and resulted in wide confidence intervals for sensitivity 88 calculations. The generalizability of our findings to studies using combined specimens is also limited because we 89 examined specimens stored and tested separately