Summarizing Study Characteristics and Diagnostic Performance of Commercially Available Tests for Respiratory Syncytial Virus: A Scoping Literature Review in the COVID-19 Era

Abstract Background Nonpharmaceutical interventions to prevent the spread of coronavirus disease 2019 also decreased the spread of respiratory syncytial virus (RSV) and influenza. Viral diagnostic testing in patients with respiratory tract infections (RTI) is a necessary tool for patient management; therefore, sensitive and specific tests are required. This scoping literature review aimed to summarize the study characteristics of commercially available sample-to-answer RSV tests. Content PubMed and Embase were queried for studies reporting on the diagnostic performance of tests for RSV in patients with RTI (published January 2005–January 2021). Information on study design, patient and setting characteristics, and published diagnostic performance of RSV tests were extracted from 77 studies that met predefined inclusion criteria. A literature gap was identified for studies of RSV tests conducted in adult-only populations (5.3% of total subrecords) and in outpatient (7.5%) or household (0.8%) settings. Overall, RSV tests with analytical time >30 min had higher published sensitivity (62.5%–100%) vs RSV tests with analytical time ≤30 min (25.7%–100%); this sensitivity range could be partially attributed to the different modalities (antigen vs molecular) used. Molecular-based rapid RSV tests had higher published sensitivity (66.7%–100%) and specificity (94.3%–100%) than antigen-based RSV tests (sensitivity: 25.7%–100%; specificity:80.3%–100%). Summary This scoping review reveals a paucity of literature on studies of RSV tests in specific populations and settings, highlighting the need for further assessments. Considering the implications of these results in the current pandemic landscape, the authors preliminarily suggest adopting molecular-based RSV tests for first-line use in these settings.


INTRODUCTION
Respiratory syncytial virus (RSV) infection is responsible for a significant proportion of outpatient visits and hospitalizations in children <5 years old, and is associated with substantial clinical and economic burden (1). Once considered to be a disease of childhood, there is increasing recognition of the prevalence of RSV infection in the community-dwelling (2) and hospitalized adult populations (3)(4)(5). Both adult and pediatric patients infected with RSV often present with nonspecific, overlapping symptoms that can lead to difficulty in distinguishing it from influenza, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or other respiratory illnesses (6). Thus, empiric diagnosis is often insufficient and should be supported by viral diagnostic testing to facilitate appropriate treatment, improved surveillance, and timely infection control (7).
Historically, viral culture was the gold standard technique for diagnosis of a productive RSV infection; however, it does not provide timely results to inform clinical management (8). Therefore, real-time reverse transcription-polymerase chain reaction (rRT-PCR), which detects the presence of the virus (active or inactive) with equal or greater sensitivity than viral culture, is often referred to as the reference/gold standard for RSV diagnosis in clinical laboratories (8). There are further modalities available for the detection of RSV with variable diagnostic performance; for example, antigen-based testing is sensitive for detecting RSV in young children but is not sensitive enough for use in older children or adults, as per the CDC guidance, due to lower viral loads in the respiratory specimens of this group (9). Thus, rRT-PCR testing is recommended for adults with suspected RSV infection (9).
Most RSV testing takes place in hospitalized patients (10) where selection bias exists toward more severe cases and pediatric patients, the age group most likely to be hospitalized due to RSV infection (11). Testing for RSV in adults by internists and general practitioners is rare, partially due to lack of awareness (12). A recent international study conducted across 15 countries reported that cases of RSV in adults ≥65 years old were notably underrepresented in national surveillance programs (13). RSV testing is also limited in the younger population as shown by a prospective study in pediatric patients (≤18 years old) in Germany, which revealed that only 8.7% of patients presenting with symptoms of a respiratory tract infection (RTI) underwent viral diagnostic testing during standard-of-care procedures (14). The lack of routine testing for RSV may contribute to the underestimation of disease prevalence, and this has practical implications. In one study based in the emergency department of a US hospital, patients IMPACT STATEMENT Viral diagnostic testing in patients with respiratory tract infection is a powerful tool for patient management.
This scoping literature review included 77 studies reporting diagnostic performance of commercially available respiratory syncytial virus (RSV) tests (published January 2005-January 2021) and summarized the characteristics of such studies. The collated data suggest that molecular-based RSV tests have higher published sensitivity and specificity than antigen-based tests and thus should be considered for first-line use for timely diagnosis and to detect infections in adults with a low-level viral load. Future studies should investigate the diagnostic performance of RSV tests in adults and in outpatient/household settings.

REVIEW
Diagnostic Performance of RSV Tests aged 6 to 21 years old accounted for 8.7% of the total number of RSV-positive tests, whereas patients aged 22 to 59 years old, and those aged ≥60 years old accounted for 14.0% and 10.5%, respectively (15). Viral diagnostic testing in pediatric and adult populations helps to tailor patient management and the implementation of hospital infection prevention policies as well as reduce the inappropriate use of antibiotics (16).
Nonpharmaceutical interventions implemented to prevent the spread of SARS-CoV-2 have also impacted the spread of RSV and influenza virus, resulting in a larger population of potential immune-naïve populations, which could lead to an increase in disease burden for future respiratory virus seasons. As a result, models predict sporadic outbreaks and an increase in the prevalence of these diseases (17). Indeed, the CDC recently released a health advisory notice warning of increased interseasonal RSV activity across the southern United States (18), and a similar interseasonal resurgence of RSV has been reported in pediatric populations in another area of the United States (19), Switzerland (20), and Australia (21). Viral diagnostic testing in patients presenting with symptoms of an RTI is a powerful tool for surveillance and patient management during such periods of interseasonal resurgence and for future respiratory virus seasons.
Reducing the time needed to diagnose RSV has been shown to be beneficial in adult and pediatric populations. The duration of time to RSV diagnosis, from the point of hospital admission to test result, is positively correlated with length of hospital stay and antibiotic use in hospitalized adults (10). Additionally, the use of point-of-care (POC) testing for RSV in pediatric patients has been associated with a reduction in the use of antibiotic treatment, the need for further clinical investigations, and time spent in the emergency department (22).
In summary, there is a need to test for RSV in patients with RTIs, which has been enhanced by the current COVID-19 pandemic. There are several commercially available tests for the diagnosis of RSV, but there are no publications that comprehensively discuss the study characteristics and reported diagnostic performance of such tests in patients with acute RTI. As study characteristics have been known to impact diagnostic performance, summarizing the literature will empower healthcare professionals to make decisions about the diagnostic modalities that are optimal for their patient population. The objective of this scoping literature review was to provide a high-level qualitative overview of the literature on commercially available sample-to-answer diagnostic tests for RSV in patients with acute RTI by summarizing the characteristics of published studies, including a preliminary data synthesis on reported diagnostic performance.

Scoping Review Design, Data Sources, and Search Strategy
This scoping literature review was conducted using the scoping review methodology as described by Peters et al. (23) and the guidance provided in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (24). PubMed (https:// pubmed.ncbi.nlm.nih.gov/) and Embase (https:// www.embase.com/) were searched on January 21, 2021 using the search terms and criteria in online Supplemental Table 1 to identify studies reporting on the sensitivity and specificity of commercially available sample-to-answer tests for RSV in patients with acute respiratory infection published between January 2005 and January 2021. Database searches were supplemented by manual searches and references, as appropriate. Duplicate articles and ineligible publication types (narrative reviews, editorials, case reports, addresses, biographies, comments, directories, Festschrifts, interviews, lectures, legal cases, legislation, news, newspaper articles, patient education Diagnostic Performance of RSV Tests REVIEW 2022 | 00:0 | 1-19 | JALM handouts, popular works) were excluded. The titles and abstracts of the remaining articles were reviewed by 2 independent reviewers in parallel, and discrepancies were resolved through discussion. Full-text articles were then obtained, and a second round of screening was conducted by 2 reviewers working in parallel, with adjudication through discussion. Articles not meeting the inclusion criteria were excluded as necessary.

Study Selection
This scoping literature review included any peerreviewed studies in the English language providing original data on the sensitivity and specificity of a commercially available sample-to-answer test for RSV (using any molecular or nonmolecular diagnostic tools) relative to an in-house or commercial rRT-PCR, viral culture, and/or immunofluorescence assay as the reference standard in patients of any age with symptoms of an RTI in any setting. Original research articles, systematic reviews, and meta-analyses were included in the review. Studies that used a noncommercial RSV test, studies where the RSV and reference test were not carried out in the same samples, studies in immunocompromised patients, or studies not otherwise meeting the inclusion criteria were excluded. Health economic analyses and preclinical research studies were also excluded.

Data Extraction
The following information was extracted, where available: RSV test sensitivity, RSV test specificity, commercial brand of RSV test, data collection (prospective vs retrospective), industry sponsorship, age group of study population (adults were defined as patients ≥18 years old), majority (>75%) specimen type, majority (>75%) setting of patient recruitment, and setting where the RSV test was performed. Any missing data were recorded as "not reported" and included in the data synthesis. The analytical time for each test was taken from its respective manufacturer's data sheet. Rapid tests, including those that were suitable for use at the POC, were defined as having an analytical time ≤30 min. Where one article reported several relevant sensitivity and specificity values (e.g., when more than one RSV test was studied or if there was a prospective and retrospective arm of the study), then each test or study arm was extracted as a subrecord. Following data extraction, analysis of discordant results between the 2 reviewers was conducted by a third independent party, and discrepancies were resolved through discussion.

Data Reporting
All data handling was carried out using Microsoft Excel 365 (Microsoft Corporation). The range of sensitivity and specificity values and summary statistics were recorded including the lowest and highest value quoted for a particular RSV test from the relevant subrecords. Sensitivity and specificity ranges were not reported for RSV tests with <3 supporting subrecords.

Literature Search Outcome
Following screening of titles and abstracts, 200 articles were subject to full-text screening, and 77 studies were eventually included in the qualitative synthesis ( Fig. 1). In studies reporting several relevant sensitivity and specificity values (e.g., when more than one RSV test was studied), each test was extracted as a subrecord. The 77 included studies corresponded to 133 included subrecords. Overall, the literature search detected 39 different commercially available RSV tests from 27 manufacturers, which represented a variety of technologies and analytical times (Supplemental Table 2).

Characteristics of All Included Studies and Gap Analysis
Most studies examined RSV tests with analytical time ≤30 min relative to RSV tests with analytical REVIEW Diagnostic Performance of RSV Tests time >30 min (66.2% vs 33.8% of included subrecords) ( Table 1). The analytical times taken from the manufacturer's data sheet for each test are shown in Supplemental Table 2. Most studies assessed RSV tests in mixed (49.6% of included subrecords) or pediatric (38.3%) populations, were prospective in design (62.4% of included subrecords), used rRT-PCR as the reference standard (68.4%), and were industry sponsored (65.4%) (Fig. 2). In all studies evaluated, a nasopharyngeal swab was the specimen type most used (48.9% of included subrecords) (Fig. 2). Most patients were recruited when they were admitted to the hospital (42.1%) or from mixed (34.6%) settings (Fig. 2). Flow chart summarizing data sources and study selection. Duplicate articles and ineligible publication types were excluded at the screening step (n = 1297). Where 1 article reported several relevant sensitivity and specificity values (e.g., when more than 1 RSV test was studied), then each test was extracted as a subrecord. The 77 included studies corresponded to 133 included subrecords.

Diagnostic Performance of RSV Tests
For RSV tests with analytical time ≤30 min, most were performed at the POC (52.3%), whereas RSV tests with analytical time >30 min were predominantly conducted in the clinical laboratory (64.4%) ( Table 1). Regarding knowledge gaps in the literature, there was a notably small percentage of studies conducted in adult-only patients (5.3%), few conducted in outpatient settings (7.5%), and only one study was conducted in a household setting (Table 1).

Published Sensitivity and Specificity of Antigen-vs Molecular-Based RSV Tests
RSV tests with analytical time ≤30 min had a greater variability in published sensitivity values (25.7%-100%) ( Table 2) relative to RSV tests with analytical time >30 min (62.5%-100%) ( Table 2); this is partially reflective of the different assays (antigen and molecular) used for RSV tests with analytical time ≤30 min. The range of published specificity values was similar for RSV tests with analytical time ≤30 min (80.3%-100%) relative to RSV tests with analytical time >30 min (77.0%-100%) ( Table 2).
Of RSV tests with analytical time ≤30 min, 70.0% (14/20) were antigen-based, and 30.0% (6/20) were molecular tests (Supplemental Table 2). Overall, molecular RSV tests with analytical time ≤30 min had a higher published sensitivity (66.7%-100%) and specificity (94.3%-100%), relative to antigen-based RSV tests with analytical time ≤30 min (reported sensitivity 25.7%-100% and specificity 80.3%-100%) ( Table 2). This trend for higher published diagnostic performance in molecular-vs antigen-based RSV tests with analytical time ≤30 min was preserved in all but one of the categories (specificity in the emergency department/outpatient setting) when the reported sensitivity and specificity ranges were broken down by patient age and setting in which the test was carried out ( Table 2).
The published sensitivity values of molecularbased tests was highest for those that detected RSV only (93%-100%), followed by RSV and influenza (66.7%-100%), and then multiplex (≥3 viruses detected) platforms (62.5%-100%) (see Supplemental Table 3). Such summary statistics should be interpreted with caution given the differences in reported sensitivity between different tests with similar modalities and analytical times. For example, the cobas ® Liat ® Influenza A/B & RSV Assay (Roche Diagnostics International Ltd) and

Diagnostic Performance of RSV Tests
the Xpert Xpress Flu/RSV (Cepheid) are both molecular-based tests that detect RSV and influenza in ≤30 min; however, the sensitivity range reported in the literature for each test is 94.2% to 100.0% and 66.7% to 98.1%, respectively (Supplemental Table 4).

Published Sensitivity and Specificity of CLIA-Waived Tests for RSV
There was a variety of published sensitivity and specificity ranges for the 14 RSV tests included in this review that were assessed under CLIA guidance (Fig. 3). Reported sensitivity and specificity values for all RSV tests included in the review are shown in Supplemental

DISCUSSION
This scoping literature review summarized the study characteristics and diagnostic performance reported in the peer-reviewed literature for commercially available sample-to-answer tests for RSV. We identified a knowledge gap for studies of RSV tests conducted in adult-only populations or in outpatient or household settings. Overall, RSV tests with analytical time ≤30 min had a greater variability in published sensitivity values relative to RSV tests with analytical time >30 min, which could be partially attributed to the different diagnostic tools (antigen vs molecular) used. Molecular-based rapid RSV tests had higher published sensitivity and specificity ranges than antigen-based RSV tests, which aligns with CDC guidance to use molecular testing for RSV where available (9).
The results from this scoping literature review showed a notable gap in studies of diagnostic performance of RSV tests in adults. Utilizing viral diagnostic testing in adult patients presenting with symptoms of acute respiratory infection would improve current surveillance efforts and allow for efficient triage and treatment decisions (e.g., local infection control guidance could be followed in a timely manner). This could be particularly important for elderly patients who are at a higher risk of hospitalization and death from RSV infection compared with younger adults (79). Testing strategies for SARS-CoV-2 developed in response to the global COVID-19 pandemic have undoubtedly brought diagnostics closer to the patient. With respect to RSV, this review identified few published studies available on the diagnostic performance of tests in outpatient or household settings. The nasopharyngeal shedding of RSV rapidly decreases 1 to 3 days after the onset of symptoms (80); therefore, accessible at-home or POC testing could be a valuable tool in timely infection control.
Approximately half of all studies included in this review used a nasopharyngeal swab as the majority specimen type. Notably, it has been shown that the diagnostic performance of some types of RSV tests is dependent upon sample type. The sensitivity and specificity of immunofluorescence-based RSV tests is higher in nasopharyngeal aspirates, relative to nasal swabs (81,82). However, there is no difference in test performance between aspirate and swab specimens when using molecular-based RSV testing (81,83). Additionally, mid-turbinate nasal swabs have been shown to have a comparative viral load to nasopharyngeal swabs in infants <2 years old (84) and are equally sensitive for the diagnosis of multiple respiratory viruses in adults (85). The advantages of using a nasal swab rather than a nasopharyngeal aspirate/swab are that it is less invasive for the patient and easier for clinical staff to transport (81,83).
The gold standard for RSV testing, rRT-PCR, was the most used comparator assay across all the studies included in this review. The use of different reference standards has been shown to affect the calculated sensitivity and specificity value of an index test; for example, a significant increase in test sensitivity has been reported in the literature when immunofluorescence is used as the reference standard compared with rRT-PCR (32).
A systematic review and meta-analysis of the sensitivity and specificity of RSV rapid antigenbased tests by Chartrand et al. reported a pooled sensitivity and specificity of 80% (95% CI, [76][77][78][79][80][81][82][83] and 97% (95% CI, 96-98), respectively (32). In addition, there was a large disparity observed in sensitivity of RSV tests between studies in pediatric patients (81% [95% CI, [78][79][80][81][82][83][84]) and in adults (29% [95% CI, ). In contrast, a systematic review by Bruning et al. reported that age did not affect diagnostic performance of RSV tests; however, this analysis only focused on 3 rapid RSV tests (BD Veritor System RSV, Sofia RSV FIA, and Alere BinaxNOW RSV) (33). Furthermore, while RSV rapid antigen-based tests are thought to be useful for diagnosis in infants, sensitivity values as low as 7.6% have been reported for a particular brand of rapid antigen-based test in this age group (86). Fig. 3. Published sensitivity and specificity of RSV tests under CLIA guidance. The published sensitivity, specificity, and analytical time are shown for the RSV tests included in this review that were CLIA-waived (A) and classed as moderate/high complexity (B). To support interpretation of the diagnostic performance data, the number of studies that were used to extract the published sensitivity and specificity values can be found in Table 3.   (87). In addition to considering time to result for multiplex tests, users should also pay close attention to hands-on time when implementing a new assay. Multiplex RSV tests such as the BioFire FilmArray Respiratory 2.1 Panel and the ePlex Respiratory Pathogen Panel, included in this review, can detect >20 infectious respiratory pathogens; however, these tests are not CLIA-waived and have a longer turnaround time but may be extremely valuable in patients with severe disease where rapid identification of the causative agent(s) in a simultaneous manner may be beneficial. In addition, discrepancies in sensitivity between multiplex and RSV-only rRT-PCR tests have been reported in the literature; this could result in varying thresholds for different respiratory viruses between different brands of multiplex rRT-PCR tests (14). The findings from our review also showed that there are differences in the published sensitivity and specificity values for tests for RSV only relative to multiplex tests.

REVIEW
The evidence outlined in this study highlights the need for healthcare professionals to consider the spectrum of respiratory disease, not just SARS-CoV-2 or influenza, and consider how viral diagnostic testing could inform their patient management and treatment decisions. If clinicians do not test for RSV, it leads to selection bias and potentially an underestimation of the prevalence of the virus. Most importantly, it could lead to inappropriate treatment for the patient. Healthcare professionals should assess the benefits and drawbacks of each RSV testing method and decide which would be most appropriate in their practice. Factors to consider include the site of testing, the location of the testing instrument, the age and immune status of the individual being tested, end user of the test, where the test results will be analyzed, the clinical significance of the results, implications for infection control, and the added value of a combination test result (87,88).
One strength of this scoping review is its comprehensive and structured search strategy, which has maximized the capture of relevant information. In addition, this review has considered a broad spectrum of molecular and nonmolecular RSV tests with different analytical times. The purpose of this scoping review was to summarize the published data available. An inherent limitation of scoping reviews is that the data synthesis is based on the values extracted from any given study; therefore, results may not be comparable in terms of methodology. Any observations related to differences in the published sensitivity and specificity values between RSV tests from different studies should be interpreted with this limitation in mind.
Further research is needed to carry out the statistical analysis required for a full systematic review and/or meta-analysis. The sensitivity and specificity of RSV tests in adult populations and in outpatient and household settings should be assessed. In addition, studies should control for selection bias and adjust for differences in settings where the RSV test was performed, seasonality, and staff utilization of RSV tests. The use of POC testing for influenza and RSV across 4 centers in Denmark resulted in a significant reduction in antibiotic prescription and median hospitalization time in adults (44.3 h) and children (14.2 h); there was also an increase in the use of antiviral treatment in adults only (16). These positive results indicate that further studies are warranted to explore the effects of testing for RSV on patient outcomes (89).
In conclusion, different clinical situations (e.g., the clinical laboratory of a large hospital vs an outpatient clinic) will require different diagnostic solutions. Given the higher published sensitivity and specificity of molecular-based testing over antigen-based modalities for RSV infection, rRT-PCR tests should be considered for first-line use when possible. By summarizing the reported sensitivity and specificity data available in the peer-reviewed literature for commercially available RSV tests, this review might provide an initial reference point for healthcare professionals to further investigate which test is suitable for their practice. Presently, there are several monoclonal antibodies and vaccines in development for RSV prevention and some promising antiviral therapeutic agents for RSV treatment (90). The concurrent use of viral diagnostic testing will be become increasingly important to identify the effectiveness and appropriateness of these products in the future.

SUPPLEMENTAL MATERIAL
Supplemental material is available at The Journal of Applied Laboratory Medicine online. Data Availability: The data supporting this review were derived from publicly available databases.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
for the development of this manuscript, under the direction of the authors, was provided by Heather Small, PhD, of Ashfield MedComms, Macclesfield, UK, an Inizio company, and was funded by Roche Diagnostics International Ltd, Rotkreuz, Switzerland. COBAS and LIAT are trademarks of Roche. All other product names and trademarks are the property of their respective owners.