Evaluation of the ImmuView RSV Test for Rapid Detection of Respiratory Syncytial Virus in Adult Patients with Influenza-Like Symptoms

ABSTRACT Rapid antigen tests may enhance the diagnostic yield of respiratory syncytial virus (RSV) infections, but studies have shown low sensitivity in adults. We evaluated the novel ImmuView RSV test in adult patients with influenza-like symptoms who were prospectively enrolled at three emergency departments in two Swedish hospitals during two influenza seasons, 2017 to 2018 and 2018 to 2019. The ImmuView RSV test was performed on nasopharyngeal swabs and results were compared to those of the BinaxNOW RSV test. In the first season, tests were performed on frozen samples, while unfrozen samples were used in the second season. For comparison, tests were also performed on selected samples from children. Of 333 included adult patients, the sensitivity of ImmuView and BinaxNOW was 27% for both tests and specificities were 98% and 100%, respectively. The interassay agreement was good (κ = 0.61). There was no significant difference in test performance between frozen and unfrozen samples. In samples from children, the sensitivities of ImmuView and BinaxNOW were 67% and 70%, respectively. In conclusion, the ImmuView RSV test showed low sensitivity and high specificity for identifying RSV in adult patients with influenza-like symptoms, comparable with the BinaxNOW RSV test. Rapid RSV testing is of limited value for diagnosing RSV infection in adults. IMPORTANCE By timely RSV diagnosis among patients with influenza-like symptoms, especially when influenza diagnostics turn negative, it is possible to prevent unnecessary antibiotic usage as well as reduce diagnostic testing, nosocomial transmission, and hospital stay. Previous rapid RSV tests have demonstrated poor sensitivity in adults, and we could demonstrate that the novel ImmuView RSV test similarly showed limited value for diagnosing RSV infection in adult patients. However, in contrast to many other studies, we investigated patient characteristics in cases with false-positive tests and we compared the performance between unfrozen and frozen samples. Thus, our results are important, as they generate new knowledge about rapid antigen tests.

In the article by Larsson et al, the authors evaluated the ImmuView RSV antigen test relative to the Alere BinaxNOW RSV antigen test, using RT-PCR testing as the reference method. Overall the authors data demonstrates poor sensitivity for the ImmuView RSV and BinaxNOW RSV antigen tests in adults. Both assays performed better (sensitivity of 66 and 70%) in pediatric patients but still not equivalent to RT-PCR. Finally, there were 5 false-positive ImmuView RSV results, resulting in a specificity of 98% versus the 100% specificity for the BinaxNOW RSV.
A. On line 44, it states that "influenza diagnostics turn negative". It would be better to say "influenza testing was negative" or something similar.
B. On line 68, it states patients got "cultures of NP swabs". What pathogens were cultured for from NP swabs? C. In the materials and methods section, please include a description of how the BinaxNOW RSV testing was performed, as was done with the ImmuView RSV. Per the package inserts for these tests, is freezing and then thawing or refrigerating specimens up 7 days before testing acceptable? Please add this information to the Discussion section. D. On line 125, the BinaxNOW sensitivity should be 66% (6/9) not 60%).
E. On lines 160-162, it is noted that "repeated test procedures on these samples did not alter the results" in regards to the false positive RSV results from the ImmuView RSV test. Did the authors repeat the antigen testing for these false-positive specimens for both assays and they RT-PCR? Please clarify.
Reviewer #2 (Comments for the Author): Manuscript ID: Spectrum 00937-21 Manuscript title: Evaluation of the ImmuView RSV test for rapid detection of respiratory syncytial virus in adult patients with influenza-like symptoms Summary and reviewer general comment: The manuscript is well written and the study design reasonable (the number of RSV PCR positive adults tested is low, but this is as acknowledged by the authors). The findings are not new, but confirmatory, as antigen detection insensitivity in adults is well recognized for most respiratory viruses.
Specific reviewer comments: 1) Introduction, Line 49. Change PCR to RT-PCR here and throughout. 2) Methods, Line 80, 82. Does "liquid medium" mean commercial viral transport media? 3) Results, Line 113. I would consider changing the term "good" to "moderate to substantial" agreement for a kappa of 0.61. 4) Table 2 is not essential and could be removed. It should be noted that the possibility that another viral/bacterial etiology might be responsible for the 5 false positive ImmuView test results, but could not be determined here as only influenza A/B was tested for by RT-PCR. 5) A more comprehensive assessment of the false negative test results of both antigen assays would have examined the raw RT-PCR data (Ct values) to see if low virus loads account for antigen detection failures.

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Response to Reviewers
Please address all reviewer comments below. In addition, please consider including the data for children in a table.
Response: Thank you for this suggestion. However, we consider it is better not to include children´s data in existing tables of adults since the main comparison is between adults during two seasons and the number of children is small. Also, existing data only require a small space as written in text and would need to be added as footnotes in a table. Therefore, after we have tried to merge the data in tables in different ways, we would prefer to keep it as it is if possible.
Reviewer #1 (Comments for the Author): In the article by Larsson et al, the authors evaluated the ImmuView RSV antigen test relative to the Alere BinaxNOW RSV antigen test, using RT-PCR testing as the reference method. Overall the authors data demonstrates poor sensitivity for the ImmuView RSV and BinaxNOW RSV antigen tests in adults. Both assays performed better (sensitivity of 66 and 70%) in pediatric patients but still not equivalent to RT-PCR. Finally, there were 5 false-positive ImmuView RSV results, resulting in a specificity of 98% versus the 100% specificity for the BinaxNOW RSV.
A. On line 44, it states that "influenza diagnostics turn negative". It would be better to say "influenza testing was negative" or something similar.
Response: The text was changed.
B. On line 68, it states patients got "cultures of NP swabs". What pathogens were cultured for from NP swabs?
Response: Cultures of NP swabs, sputum and blood were performed in clinical routine. For NP swabs, identification of common airway pathogens causing lower and upper airway infections was performed, such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and hemolytic streptococci. Other species, such as Staphylococcus aureus, Pseudomonas aeruginosa, Neisseria meningitides etc are reported depending on quantity and reported medical conditions, such as immunosuppression, cystic fibrosis.
C. In the materials and methods section, please include a description of how the BinaxNOW RSV testing was performed, as was done with the ImmuView RSV. Per the package inserts for these tests, is freezing and then thawing or refrigerating specimens up 7 days before testing acceptable? Please add this information to the Discussion section.
Response: This was added to the material and method section and to the discussion section, respectively.
Response: The sensitivity was changed.
E. On lines 160-162, it is noted that "repeated test procedures on these samples did not alter the results" in regards to the false positive RSV results from the ImmuView RSV test. Did the authors repeat the antigen testing for these false-positive specimens for both assays and they RT-PCR? Please clarify.
Response: We repeated the rapid antigen test procedure for ImmuVIew only. This was clarified in the text.
Reviewer #2 (Comments for the Author): Manuscript ID: Spectrum 00937-21 Manuscript title: Evaluation of the ImmuView RSV test for rapid detection of respiratory syncytial virus in adult patients with influenza-like symptoms Summary and reviewer general comment: The manuscript is well written and the study design reasonable (the number of RSV PCR positive adults tested is low, but this is as acknowledged by the authors). The findings are not new, but confirmatory, as antigen detection insensitivity in adults is well recognized for most respiratory viruses.
Specific reviewer comments: 1) Introduction, Line 49. Change PCR to RT-PCR here and throughout.
Response: This was corrected throughout the manuscript.
Response: Yes, this was clarified in the text.
3) Results, Line 113. I would consider changing the term "good" to "moderate to substantial" agreement for a kappa of 0.61.
Response: This was changed according to suggested definition. Table 2 is not essential and could be removed. It should be noted that the possibility that another viral/bacterial etiology might be responsible for the 5 false positive ImmuView test results, but could not be determined here as only influenza A/B was tested for by RT-PCR.

4)
Response: Thank you for the recommendation. We are aware of that extended methodology needs to be performed in order to cover for the most common etiologies to influenza-like illness, including wide panels for viral and bacterial identification. However, data on false-positive test results are rarely presented to let the reader objectively assess the clinical picture of patients tested. Therefore, we wish to present these data to extend the information on cases responsible for false-positive test results by ImmuView. 5) A more comprehensive assessment of the false negative test results of both antigen assays would have examined the raw RT-PCR data (Ct values) to see if low virus loads account for antigen detection failures.
Response: Thank you for this suggestion, we agree that Ct values may have strengthened the discussion regarding false-positive and false-negative results. However, Ct values were not available in this study. Thank you for submitting your manuscript to Microbiology Spectrum. I have included comments below that require further clarification for this manuscript. Please submit a revised version addressing these questions. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed information on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Line 29: I believe this also needs to be changed from 60% to 67%.
Line 44: "was negative" should be changed to "is negative". Apologies for not seeing this on the first draft.
Line 69: "from clinical routine" is unclear. Please clarify.
Line 80: Following up on original reviewer comments (reviewer 2, comment #2)---there are a number of liquid transport media available. What was acceptable for samples to arrive in? Saline? UTM? VTM? Something else? Please specify which type(s) of liquid transport media. Line 166-7: As a follow up to original reviewer comments (reviewer 2, comment #4) ---authors mention "no other method supported true positivity". Any additional methods used here should be included in the methods section. Was RT-PCR repeated? If so, that should be stated. If not, what are the "other methods" referenced here?
Reviewer 2, comment #5: regarding false negative test results and CT values. Would authors be able to comment on the limit of detection (LOD) for the antigen assays vs the RT PCR assay? Although CT values are not available, having an understanding of the LODs for each assay may help make a similar point if the RT PCR assay has a much lower LOD.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.