RSV adults 2023.pdf

Infect Dis Ther (2023) 12:1593–1603 https://doi.org/10.1007/s40121-023-00805-1 ORIGINAL RESEARCH Diagnosis of Respiratory Syncytial Virus in Adults Substantially Increases When Adding Sputum, Saliva, and Serology Testing to Nasopharyngeal Swab RT–PCR Julio Ramirez. Ruth Carrico. Ashley Wilde. Ala...

Infect Dis Ther (2023) 12:1593–1603 https://doi.org/10.1007/s40121-023-00805-1 ORIGINAL RESEARCH Diagnosis of Respiratory Syncytial Virus in Adults Substantially Increases When Adding Sputum, Saliva, and Serology Testing to Nasopharyngeal Swab RT–PCR Julio Ramirez. Ruth Carrico. Ashley Wilde. Alan Junkins. Stephen Furmanek. Thomas Chandler. Paul Schulz. Robin Hubler. Paula Peyrani. Qing Liu. Sonali Trivedi. Sonal Uppal. Warren V. Kalina. Ann R. Falsey. Edward E. Walsh. Kari Yacisin. Luis Jodar. Bradford D. Gessner. Elizabeth Begier Received: January 18, 2023 / Accepted: April 6, 2023 / Published online: May 6, 2023 Ó Pfizer Inc 2023 ABSTRACT Methods: This was a prospective cohort study over two study periods (27 December 2021 to 1 Introduction: Nearly all existing respiratory April 2022 and 22 August 2022 to 11 November syncytial virus (RSV) incidence estimates are 2022) of patients aged C 40 years hospitalized based on real-time polymerase chain reaction for acute respiratory illness (ARI) in Louisville, (RT–PCR) testing of nasal or nasopharyngeal KY. NP swab, saliva, and sputum specimens (NP) swabs. Adding testing of additional speci- were collected at enrollment and PCR tested men types to NP swab RT–PCR increases RSV (Luminex ARIES platform). Serology specimens detection. However, prior studies only made were obtained at acute and convalescent time- pairwise comparisons and the synergistic effect points (enrollment and 30–60-day visit). RSV of adding multiple specimen types has not been detection rate was calculated for NP swab alone quantified. We compared RSV diagnosis by NP and for NP swab plus all other specimen type/ swab RT–PCR alone versus NP swab plus saliva, test. sputum, and serology. Results: Among 1766 patients enrolled, 100% had NP swab, 99% saliva, 34% sputum, and 21% paired serology specimens. RSV was diagnosed Supplementary Information The online version in 56 (3.2%) patients by NP swab alone, and in contains supplementary material available at https:// 109 (6.2%) patients by NP swab plus additional doi.org/10.1007/s40121-023-00805-1. specimens, corresponding to a 1.95 times higher rate [95% confidence interval (CI) 1.62, J. Ramirez (&)
R. Carrico
A. Wilde
A. Junkins
2.34]. Limiting the comparison to the 150 sub- S. Furmanek
T. Chandler
P. Schulz Norton Infectious Diseases Institute, Norton jects with all four specimen types available (i.e., Healthcare, 601 S Floyd St, Louisville, KY 40202, NP swab, saliva, sputum, and serology), there USA was a 2.60-fold increase (95% CI 1.31, 5.17) e-mail: [email protected] compared to NP swab alone (3.3% versus 8.7%). R. Hubler
P. Peyrani
Q. Liu
S. Trivedi
Sensitivities by specimen type were: NP swab S. Uppal
W. V. Kalina
K. Yacisin
L. Jodar
51%, saliva 70%, sputum 72%, and serology B. D. Gessner
E. Begier 79%. Pfizer Inc, Collegeville, PA, USA Conclusions: Diagnosis of RSV in adults was A. R. Falsey
E. E. Walsh several-fold greater when additional specimen Department of Medicine, Infectious Diseases types were added to NP swab, even with a rela- Division, University of Rochester Medical Center, tively low percentage of subjects with sputum Rochester, NY 14642, USA 1594 Infect Dis Ther (2023) 12:1593–1603 and serology results available. Hospitalized RSV pulmonary, or immune systems most at risk of ARI burden estimates in adults based solely on severe disease [1–5]. Although underrecognized, NP swab RT–PCR should be adjusted for estimated RSV disease burden is comparable to underestimation. the burden of influenza in older adults, with both viruses contributing to a similar number of Keywords: Respiratory syncytial virus; Acute symptomatic illnesses, hospitalizations, and respiratory illness; Polymerase chain reaction; death overall, despite substantial variability in Disease diagnosis the relative burden of the two viruses from year to year. Due to the nonspecific clinical manifestations of RSV, which often overlap Key Summary Points with those of other viral and bacterial causes of acute respiratory illness (ARI), and can con- Why carry out this study? tribute to exacerbations of common illnesses Adding the collection and testing of such as COPD or CHF, laboratory testing is additional specimen types to NP swab required for confirmation of RSV infection. RT–PCR increases RSV detection, but prior Published incidence estimates of RSV disease studies only made pairwise comparisons in adult patients hospitalized with ARI have and the synergistic effect of adding primarily relied on reverse transcription poly- multiple specimen types has not yet been merase chain reaction (RT–PCR) testing of NP quantified. swabs [8–10]. However, the results of upper respiratory tract testing using NP swabs in We sought to compare RSV diagnosis by adults may be discordant with positive lower PCR testing of NP swab alone versus NP respiratory tract (LRT) testing. Possible swab plus saliva, sputum, and serology. explanations for this finding include: (1) a What was learned from the study? decreased viral concentration in the nasophar- ynx due to sampling late in the infection at a RSV was diagnosed in 56 (3.2%) patients time when virus may still be present at higher by NP swab alone, and in 109 (6.2%) concentrations in the lower respiratory tract patients by NP swab plus additional , (2) lower viral concentrations in adult specimens, corresponding to a 1.95 times nasal secretions when compared with children higher diagnosis rate (95% CI 1.62, 2.34). , and (3) inadequate NP swab samples due to Approximately half of identified positives dry nasal mucosa and operational reasons. were missed by NP swab testing, even with Adding the collection and testing of an a relatively low percentage of subjects additional specimen type to NP/nasal swab with sputum and serology results RT–PCR has been documented to increase RSV available. detection in pairwise comparisons. A recent metaanalysis quantified the percent increase in Hospitalized RSV ARI burden estimates in RSV diagnosis by specimen type added: 52% adults based solely on NP swab RT–PCR increase for sputum RT–PCR, 44% for paired should be adjusted for underestimation. serology testing, and 28% for oropharyngeal swab RT–PCR. However, the synergistic effect of adding multiple specimen types to NP swab testing has not yet been quantified. Fur- thermore, saliva has recently been shown to be INTRODUCTION a high yield specimen for severe acute respira- tory syndrome coronavirus 2 (SARS-CoV-2) Respiratory Syncytial Virus (RSV) is a leading RT–PCR testing , but it has not been evalu- cause of respiratory illness in adults, with older ated directly head-to-head against NP or nasal adults and those with compromised cardiac, swab RT–PCR for RSV testing. Infect Dis Ther (2023) 12:1593–1603 1595 The quantification of RSV underestimation of symptoms more than 21 days before hospital associated with sole use of NP swab for diag- admission, or were previously enrolled in this nosis will allow for adjustment of published RSV study within the 45 days prior of their current incidence rates to estimate the true burden of admission. RSV disease. These more accurate burden of This study was approved (#21-N0325) by disease estimates will facilitate appropriate WCG IRB. This study was performed in accor- decision making regarding the use of RSV dis- dance with the Helsinki Declaration of 1964 ease preventive interventions, such as vaccina- and its later amendments. After informed con- tion. The objective of this study was to define sent was obtained, we sought to collect NP the underestimation in RSV diagnosis by com- swab, saliva, sputum, and an acute blood spec- paring RSV diagnosis rates with NP swab imen on all subjects. Respiratory samples on RT–PCR alone to RSV diagnosis rates with the any given subject were collected on the day of addition of saliva, sputum, and/or serology enrollment. Patients were scheduled for a follow testing. up between 30 and 60 days to collect convales- cent blood specimens. In subjects that were unable to produce saliva, a saline mouth wash METHODS was obtained. Study Design, Setting, and Patients Study Definitions This was a prospective cohort study of patients RSV Diagnosis hospitalized with ARI in four adult acute care RSV detection by RT–PCR from NP swab, saliva, hospitals in Louisville, KY during two study and sputum specimens was defined as a positive periods from 27 December 2021 to 1 April 2022, RSV case. Evidence of concurrent RSV infection and 22 August 2022 to 11 November 2022. was defined as a four-fold increase between Study periods were chosen to align with real- acute and convalescent paired blood specimens time RSV activity in Louisville, KY. Patients in antibodies to any of four RSV antigens tested, were eligible for inclusion if they (1) were aged consistent with previously published RSV 40 years or older, (2) were hospitalized with an serology studies. ARI, defined as the presence of at least one of the following: (a) new onset or increase from baseline in any of following nine signs and RSV Diagnosis Rates symptoms—nasal congestion, rhinorrhea, sore throat, hoarseness, cough, sputum production, RSV diagnosis rate from NP swab alone was dyspnea, wheezing, hypoxemia, or (b) admit- calculated as the number of subjects with RSV ting diagnosis suggestive of ARI or (c) exacerba- detected from NP swab specimens divided by tion of underlying cardiopulmonary disease the number of subjects in the study. RSV diag- involving acute respiratory symptoms, and (3) nosis rate from NP swab plus other specimens consented to have NP swab plus at least one was calculated as the number of subjects with other specimen obtained. These criteria for ARI RSV diagnosed by any specimen divided by the are consistent with previous prospective inci- number of patients in the study. dence studies [2, 5]. The age cutoff was selected to include older RSV diagnosis rate increased using additional adults, as well as some middle-aged adults, with specimens a higher likelihood of having underlying con- The ratio of RSV diagnosis was calculated by ditions such as cardiopulmonary disease, whose dividing the proportion of RSV diagnosis from prevalence increases with advancing age. NP swab plus other specimens by the propor- Patients were excluded from the study if they tion of RSV diagnosis from NP swab alone. The developed signs and symptoms of ARI after inverse of this ratio was used to determine the being hospitalized for 48 h or more, had onset underestimation of RSV diagnosis. Additionally, 1596 Infect Dis Ther (2023) 12:1593–1603 the ratio of RSV diagnosis was calculated for (Cambridge Research Biochemicals). Anti- each combination of sample types in addition gen specific antibodies were detected with a to NP swab. This was calculated for the study goat-anti-human total Ig Phycoerythrin labeled population overall, regardless of specific results antibody (Southern Biotech). Fluorescence available, and for each subset of the study was expressed as median fluorescence intensity population with specific results available to and results are calculated using a serum refer- illustrate what the increase in diagnosis might ence standard. Persons who received intra- be in ideal conditions with high levels of sample venous immunoglobulin (IVIG) treatment with collection. positive serology results were considered false positives and removed from analysis. Study Variables Data on demographics, social, and medical history, vaccination status, Statistical Analysis standard of care SARS-CoV-2 testing, clinical diagnosis, as well as hospital course, including Only patients with an NP swab and at least one length of stay and patient mortality, were col- other specimen result were included in this lected from patient questionnaires and from analysis. Patient characteristics were reported as electronic medical records. medians and interquartile ranges (for continu- ous data) and frequencies and percentages (for Specimen Processing and Testing categorical data). Venn and Euler diagrams were produced to show RSV diagnosis by specimen NP swab NP swab specimens were tested using type. RSV diagnosis rates were calculated and the ARIES Luminex FluA/B/RSV panel and pro- reported as percentage positive. The RSV cessed in accordance with standard operating detection rate by NP swab alone was used as the procedures. baseline RSV rate for comparison, and RSV diagnosis ratios were calculated as percent Sputum Sputum specimens were processed increase from baseline, with 95% confidence and tested using the ARIES Luminex FluA/B/ intervals (CIs) calculated. The sensitivity of each RSV platform. Briefly, sputum specimens were assay for diagnosing RSV was calculated for each diluted to 50% water solution, and mixed by sample type and combination, with 95% confi- vortex. A swab of this solution was mixed into dence intervals calculated as well. P-values less 700 uL of sterile water and mixed by vortex a than 0.05 were considered statistically signifi- second time. From this mixed solution, 200 uL cant. Sensitivity calculations for each specimen was pipetted into the Luminex cartridge vial. type were limited to subjects that had results for Saliva Saliva specimens were processed and tested using the ARIES Luminex FluA/B/RSV platform. Briefly, saliva was handled separately depending on viscosity. For normal saliva, the specimen was mixed by vortex and 200 uL was pipetted into the Luminex cartridge vial. Saliva that was too thick or viscous to pipette was processed the same way as sputum (above). Serology Serology was performed by Pfizer central laboratory using Luminex-based total antibody RSV assays. Briefly, the 4-plex assay included spectrally distinct magnetic micro- spheres coated with recombinant matrix pro- tein, nucleoprotein, and peptide sequences unique to the G protein for RSVA and RSVB Fig. 1 Study flowchart  Infect Dis Ther (2023) 12:1593–1603 1597 that specimen type. The proportion of all posi- *Sensitivity is limited to those with results with that specimen type, i.e., count of positives by that specimen type divided by count of all positives from any source 42.0–60.8% 61.4–78.8% 60.3–83.6% 64.6–94.1% tives detected by a given specimen type was also Sensitivity* 95% CI calculated to capture the real-world benefit of N/A adding a given specimen type, given its opera- tional feasibility and sensitivity. All analysis was performed using R version 4.1.2 and SAS 51.4% version 9.4 (SAS 9.4, Cary, NC. SAS Institute 70.1% 71.9% 79.3% N/A Inc.). any source among patients RESULTS with results by that RSV positives from Patient Population specimen type A total of 1766 participants provided informed consent and enrolled in the study. Figure 1 109 42.0–60.8% 109 60.1–77.5% 107 depicts the study flowchart. Among enrolled 28.5–46.7% 57 29 participants hospitalized for ARI, most were female (55%), white (70%), and were commu- 28.8% 95% CI nity-dwelling (95%). Diabetes mellitus and 13.4%- N/A chronic obstructive pulmonary disease were the Table 1 Study specimen collection, RSV prevalence results, and sensitivity by specimen type most frequent comorbidities (39% and 38% of subjects, respectively), and 28% (n = 494) of participants were immunocompromised. **In subjects that were unable to produce saliva, a saline mouth wash was obtained Table 1 depicts patient characteristics for study specimen overall identified by this Percentage of all participants. specimen type RSV Diagnosis positive positives 51.4% 68.8% 37.6% 21.1% N/A Among 1766 participants enrolled, 100% had NP swab (n = 1766), 99% saliva (n = 1740), 34% 6.2% 3.2% 4.3% 6.8% 6.3% n positive % among those with results by that specimen type 109 56 75 41 23 by this specimen Percentage 98.5% 34.3% 20.8% 1766 100.0% 1766 100.0% with type 1740 Fig. 2 Respiratory syncytial virus (RSV) diagnosis by 606 367 N specimen type: all specimen types contribute unique positives. A (left) Venn diagram of nasopharyngeal (NP) specimens swab, saliva, and sputum specimens detecting RSV from NP swab Serology Sputum Saliva** Overall RT–PCR diagnostic testing. B (right) Euler diagram of Study NP swab, saliva, sputum, and serology specimens diagnos- ing RSV 1598 Infect Dis Ther (2023) 12:1593–1603 sputum (n = 606), and 21% (n = 367) paired study population, saliva detected the most serology specimens tested. Overall, RSV was positives (75/109, 69%), followed by NP swab diagnosed in 109 participants using any/all (56/109, 51%), sputum (41/109, 38%), and specimens. Figure 2 depicts RSV detection by serology (23/109, 21%). We examined each sample type for the entire study population for test’s sensitivity by limiting to those with that respiratory specimens alone (panel A) and all result type, and sensitivity ranged from 51% for four specimen types (panel B). While there was NP swab to 79% for serology. some overlap in positives for each specimen type, all specimens contributed unique posi- RSV Diagnosis Rates by Sample Type tives, with the greatest number of unique posi- Combinations tives contributed by saliva and serology. A total of 56 among 109 participants (51%) had RSV For the entire study population, regardless of detected from NP swabs, while 53 participants specific results available, the percent increase in had RSV diagnosed only by other specimen RSV diagnosis when adding results from addi- types (i.e., negative by NP swab), corresponding tional sample types, relative to using NP swab to 49% of positives missed by NP swab testing alone (reference value), is depicted in Fig. 3 for alone. Table 1 depicts specimen collection fre- each combination of sample types. As noted, NP quency, percent RSV diagnosis by sample type swab alone diagnosed RSV in 56 (3.2% of par- for the entire study population, as well as test ticipants). Adding results from other specimen sensitivity estimates. Although sputum and types, RSV diagnoses increased to 109 (6.2% of serology were not available in all participants, participants), corresponding to a 1.95-fold these samples yielded the highest percent of increase (95% CI 1.62, 2.34-fold increase) in RSV diagnoses when a sample was available detection, over NP swab results alone. The (sputum n = 41 of 606 participants, 6.8%, and number of participants with each of the various serology n = 23 of 367 specimens, 6.3%). The combinations of specimen types tested and the percent detection of saliva and NP swabs was percent increases in RSV diagnosis for each less, but the overall number of positives greater: combination of sample types, relative to using saliva (n = 75 of 1740 specimens, 4.3%), and NP NP swab alone (reference value), limiting to swabs (n = 56 of 1766, 3.2%). In the overall those participants with the specific results available in the comparison, are depicted in Table 2. When limiting analysis to the 150 participants with all four specimen types avail- able, RSV was diagnosed in 5 participants (3.3%) by NP swab alone and in 13 participants (8.67%) when all results were used. A 3.20-fold increase in detection (95% CI 1.83, 5.78) was seen add- ing saliva and serum results to NP among 363 participants with those three specimen types tested. For the 580 subjects with available NP swab, saliva, and sputum samples, there was a 1.63-fold increase in detection (95% CI 1.31, 2.04) when adding the saliva and sputum results. Participant Characteristics by Specimen Type Positive Fig. 3 The percent increase in respiratory syncytial virus (RSV) diagnosis when adding additional specimen types in Characteristics for RSV positive participants by the analysis, over using nasopharyngeal (NP) specimens specimen type positive are depicted in Table 2. alone Infect Dis Ther (2023) 12:1593–1603 1599 Table 2 Increase in RSV detection associated with testing additional specimen types, beyond NP swab, for populations with specific sample results available Groups by N Count of Detection rate Count of Detection rate Detection 95% CI of available patients by with NP patients by with any rate ratio detection specimens NP swab swab positive any listed listed (any listed/ rate ratio positive (per 100 specimen specimen NP swab) patients) positive positive (per 100 patients) All subjects All four specimens 150 5 3.33 13 8.67 2.60 1.31, 5.17 Three specimens NP swab/saliva/ 580 30 5.17 49 8.45 1.63 1.31, 2.04 sputum NP swab/saliva/ 363 8 2.20 26 7.16 3.25 1.83, 5.78 serum NP swab/sputum/ 154 5 3.25 14 9.09 2.80 1.39, 5.65 serum Two specimens NP swab/saliva 1740 55 3.16 85 4.89 1.55 1.32, 1.81 NP swab/sputum 606 31 5.12 43 7.10 1.39 1.15, 1.67 NP swab/serum 367 8 2.18 24 6.54 3.00 1.70, 5.28 Overall, participants with RSV identified by NP fold or more in adult patients hospitalized with swab (regardless of other results) had a similar ARI. Even though RT–PCR of NP swab is the time from symptom onset to specimen collec- most commonly used test to detect RSV in tion than subjects exclusively positive by other hospitalized patients, our study indicates that it non-NP respiratory specimen types (median of will miss a substantial percentage of patients 4 days versus 3 days, respectively), but subjects hospitalized with RSV-associated ARI. Prior lit- that were NP negative and serology positive had erature has reported increased detection associ- a longer median duration of 6 days for symp- ated with adding sputum or serology to NP swab tom onset. Thirty percent of RSV-diagnosed , but this is the first study utilizing a wide subjects were immunocompromised, 23% of variety of specimen types, including saliva, and participants with RSV identified by NP swab assessing their synergistic effects for RSV were immunocompromised compared to 38% diagnosis. of subjects detected by non-NP swab specimen Our data indicate that a more accurate bur- types. den of RSV disease in future studies can be achieved by testing multiple specimen types, or adjusting for underestimation associated with DISCUSSION use of limited specimen types. Furthermore, our study suggests that vaccine studies evaluating Our study indicates that the inclusion of saliva, efficacy or effectiveness of an RSV vaccine sputum, and serology to RT–PCR of NP swab should include multiple specimens for diagno- increased the diagnostic yield for RSV by two- sis of disease. In a Centers for Disease Control 1600 Infect Dis Ther (2023) 12:1593–1603 and Prevention (CDC) meeting of experts for the difficulty of taking a sufficient sample and the purpose of identifying gaps in the epi- nasal dryness, potentially from nasal oxygen demiology of RSV, the experts noted a need to use , diuretic administration, or dry indoor document potential underestimation of disease air. The lower positivity rate of NP swab testing burden due to testing behaviors. In a recent may also be due in part to a prolonged time metaanalysis of RSV incidence among older from symptom onset to hospitalization and adults in the USA, an adjustment factor of 1.5 swab collection, such that at the time of hos- was included to account for diagnostic testing pitalization, the viral titers in nasal secretion under-ascertainment when only RT–PCR was may have dropped and RSV may no longer be used. This correction factor was based on detectable in the nasopharynx. Nasal swabs pairwise comparisons of different specimen are more likely to be positive in persons that type results from the literature that did not still have upper respiratory symptoms. account for synergistic effects of multiple spec- Patients with RSV detected by serology speci- imen use. Our results indicate that a correction mens only, had a longer duration of symptoms factor greater than 2 may be more appropriate, at the time of sampling (median 6 days versus as indicated by the 2.6-fold increase in yield 4 days), but further study is needed to better among those with all four specimen types. characterize the differences in the cases detec- Notably, saliva specimens yielded the high- ted by each specimen type. est number of RSV detections among respiratory In patients with a productive cough, sputum specimens if used alone (n = 75) and added an was a useful specimen for RSV identification. additional 30 unique RSV cases to the 56 diag- Sputum has been shown to have higher RSV nosed with NP samples. Since saliva is readily titers than nasal swabs , allowing for obtained from most subjects, as shown in our increased detection of RSV when this specimen data, the simple addition of this sample to NP is available , which is consistent with results swabs may provide much more accurate esti- from other respiratory viruses such as influenza mates of RSV incidence in this population. and SARS-CoV-2 [13, 19–21]. In our study, we There are several potential reasons for this found seven patients with RSV that were NP finding. RSV may replicate in the primary sali- swab negative and diagnosed by sputum alone vary glands such as parotid, submandibular, (Fig. 1), all but one of whom had lower respi- and sublingual glands, producing a constant ratory tract illness diagnosis. This corresponds flow of the virus or viral genetic material into to a 39% increase in RSV detection over NP the saliva. In addition, saliva may also serve as swab alone among subjects with both specimen reservoir of pooled secretions from the types, comparable to published paired assess- nasopharynx. Saliva has emerged as a sensitive ments of adding sputum to NP swab testing and reliable specimen type for SARS-CoV-2 [pooled percent increase from recent meta- testing, with one study finding that saliva has analysis: 52% (95% CI 15, 101)]. higher viral titers than NP swab and is a more Serology testing does not impact the clinical consistent specimen, such that no instances management of a hospitalized patient; how- were seen of a negative result followed by a ever, it represents an important epidemiological positive result. We did not attempt to tool to define the burden of disease and can be measure viral load in the saliva in comparison used in vaccine efficacy studies to augment RSV to NP swabs. In our study, it is notable that diagnosis end points when feasible. A recent saliva (or normal saline mouth wash) was metaanalysis reported a 42% increase in detec- available in nearly all study subjects. It is pos- tion (95% CI 19, 70) over NP/nasal RT–PCR sible that saliva may be a more desirable diag- swab alone. Analyses limited to older adults nostic sample for the diagnosis of respiratory (more comparable to our study population) viruses, both for better yield and tolerability to reported a detection increase of 50% to 64% patients.. This higher detection rate by serology Among hospitalized adults, material cap- among older adults may be due to their higher tured with NP or nasal swabs can be limited by serum IgG responses following RSV infection Infect Dis Ther (2023) 12:1593–1603 1601 compared to younger adults, possibly related to In conclusion, our study found that RSV their higher RSV nasal titers and longer viral detection increases several-fold with the addi- shedding. This longer viral shedding cor- tion of testing from other specimen types relates with persistent secretion of antibody by besides NP swab, especially saliva. Future studies plasma cells and is presumed due to diminished assessing the RSV burden should consider cellular immunity associated with immune-se- additional testing of saliva, sputum, and serol- nescence. One potential limitation of ogy to adequately detect RSV-positive patients. serology is being certain that the rise in IgG Burden of disease estimates based solely on NP clearly brackets an identifiable illness. It is pos- swab RT–PCR should be adjusted for underesti- sible that a rise is RSV specific IgG could be mation, as should metaanalyses of existing RSV related to an illness that occurred after hospital incidence estimates. discharge. To mitigate this, we collected NP swabs at convalescent visits from anyone with intercurrent ARI symptoms; we did not have ACKNOWLEDGEMENTS any positive results suggesting intercurrent RSV was not an important contributor to infections, We would like to thank the NIDI RSV study identified by a four-fold rise in serology. group (see supplementary materials) for per- One strength of our study was that all res- forming the study. Additionally, we would like piratory specimens were collected on the same to thank the nurses at Norton Healthcare for day, at time of enrollment. Additionally, all their collaboration with the study. collected specimens had RT–PCR tests per- formed on the same platform. Furthermore, we Funding This work was supported by Pfizer collected sputum in 95% of the 646 patients Inc., including supporting the journal’s rapid producing sputum in our study population. service fee. This study was conducted as a col- The primary limitation of our study was the laboration between Norton Infectious Diseases low number of subjects with serology results Institute, Norton Healthcare, and Pfizer. Norton available, thus diminishing the number of sub- Infectious Diseases Institute, Norton Healthcare jects with all 4 sample types for analysis. Con- is the study sponsor. sequently, our estimate of the increase in Author Contributions Study concept and detection of RSV may be too conservative. design was carried out by Elizabeth Begier, Another limitation of our study is that with Robin Hubler, Julio Ramirez, Ruth Carrico, only 109 patients with RSV detected, we were Ashley Wilde, Paula Peyrani, Ann R. Falsey, unable to perform analysis in subgroups such as Edward Walsh, Luis Jodar, and Bradford D. the immunocompromised patients. Further Gessner. Data acquisition was performed by study is required to improve precision regarding Julio Ramirez, Ruth Carrico, Ashley Wilde, Alan the level of RSV underestimation within specific Junkins, Stephen Furmanek, Thomas Chandler, subgroups. Lastly, another potential limitation and Warren V Kalina. Data interpretation and is that we did not assess if nasal swab or analysis was performed by all authors. Writing oropharyngeal swab may increase RSV detection the first draft was performed by Julio Ramirez when compared with RT–PCR of NP swab alone, and Stephen Furmanek. Participation in draft- because of its dominance in RSV incidence ing or revision involved all authors. studies [9, 13]. Nasal and oropharyngeal swab likely have substantial overlap regarding mate- Disclosures Julio Ramirez, Ruth Carrico, Ash- rial collected with other specimens included in ley Wilde, Alan Junkins, Stephen Furmanek, the study, namely NP swab and saliva, respec- Thomas Chandler, and Paul Schulz are tively. Finally, RT–PCR positive results may employees of Norton, which received fees from uncommonly reflect a prior infection with Pfizer in relation to this study. Robin Hubler, residual viral RNA in the nasopharynx, partic- Paula Peyrani, Qing Liu, Sonali Trivedi, Sonal ularly among immunocompromised Uppal, Warren V Kalina, Kari Yacisin, Luis individuals. 1602 Infect Dis Ther (2023) 12:1593–1603 Jodar, Bradford D. Gessner, and Elizabeth Begier REFERENCES are employees of Pfizer and may hold Pfizer stock and/or stock options. Ann R. Falsey has 1. Ivey KS, Edwards KM, Talbot HK. Respiratory syn- research grants from Pfizer, Janssen, CyanVac, cytial virus and associations with cardiovascular and BioFire Diagnostics, served on the Data and disease in adults. J Am Coll Cardiol. 2018;71(14): Safety Monitoring Board for Novavax, and 1574–83. consulted for Arrowhead Pharmaceuticals. 2. Falsey AR, et al. Respiratory syncytial virus infection Edward E. 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