Review Reports - From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion<sup>®</sup> Open Access™

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the development and extensive analytical and clinical validation of a fully automated quadruplex RT-qPCR assay for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV on the Panther Fusion® Open Access™ platform. The study is technically rigorous, with comprehensive assessment of analytical sensitivity, specificity, precision, commutability, and mixed-infection detection using a large set of clinical samples and appropriate commercial comparator assays.

While the work is methodologically strong and the results are robust, the manuscript is overly long - especially in the Introduction and Discussion - and would benefit from substantial condensation, particularly in the Introduction and Discussion sections, which are overly long and repetitive relative to the core technical contribution.

Minor comments

Lines 112–114: Please specify more clearly the exclusion criteria applied according to the laboratory SOPs. The current description is too generic and does not allow the reader to fully assess how samples were excluded during the pre-analytical phase.
Introduction: it is excessively long and repetitive, particularly in: epidemiological background (influenza, RSV, SARS-CoV-2 burden); justification for multiplex testing; general statements on NAAT superiority. Much of this material is well known to the target readership and could be substantially reduced without loss of scientific context.
The Discussion is technically correct but overly exhaustive, reading more like a regulatory validation report than a scientific synthesis. Too much space is devoted to reiterating Results rather than interpreting them; comparative discussions with prior assays are extremely detailed (e.g. RSV oligo design comparisons); confirmations that the assay “performs well” is redundant as it is already evident from the data.
The take-home messages are buried under methodological commentary.

Author Response

Author’s Reply to Reviewer 1

Manuscript ID: microorganisms-4068209

From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion® Open Access™

Comment 1:

While the work is methodologically strong and the results are robust, the manuscript is overly long - especially in the Introduction and Discussion - and would benefit from substantial condensation, particularly in the Introduction and Discussion sections, which are overly long and repetitive relative to the core technical contribution.

Response 1:

Thank you for pointing this out. We agree with this comment. Therefore, we have carefully revised the manuscript and supplementary materials to improve clarity and readability. We substantially condensed the manuscript. Key reductions are summarized below:

Introduction: 789 → 316 words (−9%)
Materials & Methods: 4957 → 3778 words (−23.9%)
Results: 3235 → 2847 words (−11.9%)
Discussion: 2491 → 1572 words (−36.9%)
References: 64 → 55 references (−14.1%)
Total pages: 32 → 26 pages (−18.8%)
Total words: 15249 → 11974 words (−21.5%)
Tables: Tables 2 and 4 were moved to the supplementary material.
Figures: Figure 3 was moved to the supplementary material.

Revised text:

All changes made to the manuscript can be tracked using Microsoft Word’s Track Changes feature.

Location in revised manuscript:

Throughout the manuscript.

Comment 2:

Lines 112–114: Please specify more clearly the exclusion criteria applied according to the laboratory SOPs. The current description is too generic and does not allow the reader to fully assess how samples were excluded during the pre-analytical phase.

Response 2:

Thank you for pointing this out. We agree with this comment. We have clarified the sample acceptance/rejection criteria applied before NAAT testing. We now explicitly state that no demographic eligibility criteria were applied and provide a structured list of pre-analytical rejection reasons (e.g., labeling/traceability issues, incorrect transport medium, leakage, insufficient volume, improper transport/storage conditions, visible contamination, or invalid prescription).

Revised text:

Pre-analytical acceptance/rejection criteria were applied according to the laboratory standard operating procedures (SOPs) for respiratory NAAT testing. Specimens were rejected if they: (i) were mislabeled or had mismatched identifiers; (ii) were collected in an inappropriate tube/transport medium; (iii) were in a leaking or broken container; (iv) had insufficient specimen volume; (v) were stored or transported outside SOP specifications; (vi) had gross specimen quality issues likely to compromise NAAT performance (e.g., obvious contamination); or (vii) had no or invalid test prescription. All residual clinical samples included in this evaluation satisfied the laboratory’s acceptance criteria. No demographic eligibility criteria were applied. Residual specimens were stored at −70 °C immediately after routine testing.

Location in revised manuscript:

Page 2, paragraph 4, lines 73-82.

Comment 3:

Introduction: it is excessively long and repetitive, particularly in: epidemiological background (influenza, RSV, SARS-CoV-2 burden); justification for multiplex testing; general statements on NAAT superiority. Much of this material is well known to the target readership and could be substantially reduced without loss of scientific context.

Response 3:

Thank you for pointing this out. We agree with this comment. We have substantially streamlined the Introduction to focus on post-pandemic co-circulation and diagnostic uncertainty and to state the study aim clearly. Redundant background statements and repeated assertions of NAAT superiority were removed, and the reference list was updated accordingly. The Introduction was reduced from 789 to 316 words (−59.9%).

Revised text (excerpt):

The coronavirus disease 2019 pandemic (COVID-19) has dramatically altered the epidemiology of other respiratory viruses. Between 2020 and 2021, the traditionally endemic influenza A (IAV), influenza B (IBV), and respiratory syncytial virus (RSV) were virtually absent due to public health measures aimed at containing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SC2) [1,2]. In the post-pandemic period, as previously anticipated [3], influenza, SC2, and RSV infections represent a global public health threat due to their high transmissibility, morbidity, recurrent seasonal waves, and economic burden [4,5].

…

Location in revised manuscript:

Page 1, paragraph 3, lines 34-61.

Comment 4:

The Discussion is technically correct but overly exhaustive, reading more like a regulatory validation report than a scientific synthesis. Too much space is devoted to reiterating Results rather than interpreting them; comparative discussions with prior assays are extremely detailed (e.g. RSV oligo design comparisons); confirmations that the assay “performs well” is redundant as it is already evident from the data.

The take-home messages are buried under methodological commentary.

Response 4:

Thank you for pointing this out. We agree with this comment. We have revised the Discussion to eliminate repetition, reduce methodological re-description, and foreground the main take-home messages. Specifically, we tightened the interpretation of analytical performance (LoD, specificity, precision), clarified how to interpret “100% accuracy” under a majority-rule consensus, and consolidated explanations of discordances at high Ct values into a single focused paragraph. The Discussion was reduced from 2491 to 1572 words (−36.9%), improving readability while ensuring the revised reference list remains appropriate to the shortened text.

Revised text (excerpt):

This work designs an oligo set for detection of RSV A/B and validates a laboratory-developed respiratory assay (LDRA), resulting from combining the LDT RSV with the CDC Flu-SC2 [19]. The new quadruplex enables fully automated, multiplex RT-qPCR detection of IAV, IBV, SC2, and RSV on the Panther Fusion® Open Access™ platform. In the current post-pandemic setting—where influenza, RSV, and SC2 can co-circulate and produce clinically indistinguishable syndromes [2,20]—such syndromic molecular testing can improve time-to-diagnosis and strengthen infection control and surveillance workflows [12]. In our proposal, using of human RNase P as an IC instead of IC-S adds a safety element because it ensures adequate sample collection. Assays, that rely only on process controls, such as the PFRA comparator used in this study, introduce an additional risk of false negatives when clinical samples have not been collected effectively [21].

…

Location in revised manuscript:

Page 18, paragraph 2, lines 521-651.

Sincerely,
The Authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe a trial to enhance the CDC Respiratory 2 qPCR Assay by incorporating RSV detection on the Panther Fusion® Open Access™ platform. Overall, the manuscript is well written, comprehensive, and addresses an important diagnostic need. My comments are as follows.

Abbreviations should be defined at their first occurrence and then used consistently throughout the manuscript. At present, the full names of viruses and their abbreviations are repeatedly written, which could affect readability.
In the ethical approval section, the authors state that ethical review and approval were waived. It would be helpful to clarify explicitly by whom this waiver was granted.
In the limitations section, the authors indicate that oligonucleotide designs target single genomic regions with high genetic variability, particularly for influenza A virus and SARS-CoV-2. However, according to the methodology, the selected targets are described are conserved regions, with the possible exception of the NS2 gene of influenza B virus. Please revise.
The limitations of the study should also address inherent constraints of probe-based qPCR methodologies compared with alternative approaches, such as longer assay development time, the need for specialized excitation and detection instruments, and higher costs. The authors may also consider mentioning isothermal amplification methods and microfluidic-based platforms as potential future directions.
Including a figure that illustrates amplification curve profiles for a single-virus–positive sample, dual infection (e.g., SARS-CoV-2 and RSV), triple infection, and quadruple infection would strengthen the manuscript. In addition, demonstrating the quantitative performance of the assay using clinical samples would be valuable, as the amplification curves currently provided in the supplementary material are difficult to interpret.
Finally, for improved readability, the authors may consider moving some tables and figures to the supplementary material, like Tables 2, 4, and 8, and Figure 3.

Author Response

Author’s Reply to Reviewer 2

Manuscript ID: microorganisms-4068209

From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion® Open Access™

Comment 1:

Abbreviations should be defined at their first occurrence and then used consistently throughout the manuscript. At present, the full names of viruses and their abbreviations are repeatedly written, which could affect readability.

Response 1:

Thank you for pointing this out. We agree with this comment. We have revised the manuscript to define abbreviations at first mention and to use abbreviations consistently thereafter. This includes common pathogens (IAV, IBV, SC2, RSV), assay names, and key performance metrics. Repeated full-form restatements were removed where they did not add clarity, while retaining precise assay identifiers when needed for unambiguous interpretation. Some abbreviations that were mentioned only once were removed from the text and from the Abbreviations list.

Revised text:

Throughout the manuscript, first definitions occur mainly in the Introduction and Methods. All changes made to the manuscript can be tracked using Microsoft Word’s Track Changes feature.

Location in revised manuscript:

Throughout the manuscript, particularly in the Introduction, Materials and Methods, Results, and the Abbreviations list at the end of the manuscript.

Comment 2:

In the ethical approval section, the authors state that ethical review and approval were waived. It would be helpful to clarify explicitly by whom this waiver was granted.

Response 2:

Thank you for pointing this out. We agree with this comment. We have now explicitly stated which body granted the waiver. We also clarified that the study used de-identified remnant specimens collected for routine diagnostic purposes, and that informed consent was obtained under the approved framework.

Revised text:

Institutional Review Board Statement: Ethical review and approval were waived by the Scientific Committee of Referencia Laboratorio Clínico, as the study exclusively used remnant clinical specimens originally collected for routine diagnostic testing under medical prescription. All samples were handled in compliance with the clinical laboratory’s confidentiality and informed consent policies. In short, all patients expressly agreed to the possibility of using the remnants of their clinical samples (residual clinical samples) in research and development, and to the possibility of publishing the results obtained in the research, provided that the privacy of patient data is protected.

Location in revised manuscript:

Page 20, paragraph 7, lines 678-684.

Comment 3:

In the limitations section, the authors indicate that oligonucleotide designs target single genomic regions with high genetic variability, particularly for influenza A virus and SARS-CoV-2. However, according to the methodology, the selected targets are described are conserved regions, with the possible exception of the NS2 gene of influenza B virus. Please revise.

Response 3:

Thank you for pointing this out. We agree with this comment. We decided to remove the sentence indicating that oligonucleotide designs target single genomic regions to avoid confusion with the design principles that underpin the strong performance discussed in the manuscript.

Revised text:

Finally, several limitations should be acknowledged. This was a single-center study, which may limit extrapolation to other regions and epidemiological contexts. Although samples were collected over an extended period, the overall cohort size was modest, and no demographic, epidemiological, or clinical data were available; therefore, potential associations with patient factors or outcomes could not be explored. Because specimens were de-identified residual samples stored retrospectively, the observed detection frequencies reflect only the analyzed cohort and should not be interpreted as population prevalence. Moreover, the analytical sensitivity estimates were generated using synthetic evaluation materials with target concentrations not traceable to a certified reference standard; thus, LoD values may not be directly comparable across studies and may not fully reflect performance in clinical matrices. Finally, discordant results were not adjudicated using an independent orthogonal method (e.g., sequencing or an additional reference assay), which limits definitive attribution of assay-specific errors. Despite these limitations, the study provides a technically relevant analytical and clinical performance assessment of the proposed quadruplex approach.

Location in revised manuscript:

Page 20, paragraph 2, lines 637-651.

Comment 4:

The limitations of the study should also address inherent constraints of probe-based qPCR methodologies compared with alternative approaches, such as longer assay development time, the need for specialized excitation and detection instruments, and higher costs. The authors may also consider mentioning isothermal amplification methods and microfluidic-based platforms as potential future directions.

Response 4:

Thank you for pointing this out. We respectfully disagree with this comment. We appreciate this suggestion and agree that isothermal amplification and microfluidic platforms are important and rapidly evolving. However, the scope of the present study is the analytical and diagnostic validation of a probe-based multiplex RT-qPCR assay (LDRA) on the Panther Fusion® Open Access™ system. A balanced discussion of “inherent constraints” of probe-based RT-qPCR relative to alternative technologies—especially with respect to workflow complexity, turnaround time, instrumentation requirements, and cost—would require additional data and/or a structured comparative analysis that is not within the scope of our study design and would be speculative in this context. In line with the reviewers’ requests to tighten the manuscript, we therefore kept the Limitations focused on factors that directly affect interpretation of our validation results (e.g., single-center design, lack of clinical metadata, retrospective remnant specimens, and LoD estimation using synthetic materials). We would be pleased to address broader cross-technology comparisons in a dedicated follow-up study where appropriate evaluation metrics can be systematically assessed.

Revised text:

Location in revised manuscript:

Page 20, paragraph 2, lines 637-651.

Comment 5:

Including a figure that illustrates amplification curve profiles for a single-virus–positive sample, dual infection (e.g., SARS-CoV-2 and RSV), triple infection, and quadruple infection would strengthen the manuscript. In addition, demonstrating the quantitative performance of the assay using clinical samples would be valuable, as the amplification curves currently provided in the supplementary material are difficult to interpret.

Response 5:

Thank you for pointing this out. We agree with this comment, at least in part. We agree that interpretability of amplification curves is important, but we believe their main value is limited to assessing assay suitability based on empirical, experience-driven criteria, which can be inherently subjective. Therefore, we prefer to place amplification-curve images in the Supplementary Material, limiting them to those that can provide relevant information—such as amplification-efficiency curves—because efficiency directly reflects prior optimization. We revised the supplementary material to improve clarity and presentation of amplification-curve information of the amplification efficiency and multiplex compatibility results. In particular, we cite the updated multiplex compatibility figure in the Results and ensured the caption and labeling more clearly describe the meaning of the images and their layout.

Revised text:

Figure S5. Multiplex compatibility analysis: The top row shows fluorescence amplification curves obtained with the CFX96-IVD analyzer during the multiplex compatibility experiment; light-colored curves correspond to the multiplex format, whereas dark-colored curves correspond to the monoplex format of the LDRA assay. The middle row presents fluorescence amplification curves generated on the Panther Fusion® system during verification of the amplification efficiency of the quadruplex LDRA assay; curve colors in this row have no analytical significance. The bottom row shows the linear regression of Ct values versus log₁₀(C_i) derived from the middle-row data, including error bars representing the standard deviation at each concentration level, used for amplification efficiency estimation. Panels correspond to: (a) influenza A virus; (b) influenza B virus; (c) SARS-CoV-2; (d) respiratory syncytial virus, type A; (e) respiratory syncytial virus, type B.

Location in revised supplementary material:

Page 10, paragraph 1, lines 131-137.

Comment 6:

Finally, for improved readability, the authors may consider moving some tables and figures to the supplementary material, like Tables 2, 4, and 8, and Figure 3.

Response 6:

Thank you for pointing this out. We agree with this comment, at least in part. We agree with the goal of improving readability. Therefore, we have carefully revised the manuscript and supplementary materials to improve clarity and readability. We substantially condensed the manuscript. Key reductions are summarized below:

Introduction: 789 → 316 words (−9%)
Materials & Methods: 4957 → 3778 words (−23.9%)
Results: 3235 → 2847 words (−11.9%)
Discussion: 2491 → 1572 words (−36.9%)
References: 64 → 55 references (−14.1%)
Total pages: 32 → 26 pages (−18.8%)
Total words: 15249 → 11974 words (−21.5%)
Tables: Tables 2 and 4 were moved to the Supplementary Material.
Figures: Figure 3 was moved to the Supplementary Material.

In addition to the above, we moved Tables 2 and 4 and Figure 3 to the Supplementary Material, as recommended (now, Tables S3 and S5, and Figure S6, respectively). Nevertheless, We have retained Table 8 (now, Table 6) in the main text because it reports core analytical performance metrics necessary for most readers to evaluate the work without repeatedly consulting supplementary files.

Revised text:

Supplementary Table S3 summarizes the optimized PPR composition for the quadruplex LDRA.

Absolute and relative hit frequencies by target and dilution level are shown in Supplementary Table S5.

According to the probit regression, the LDRA C₉₅ LoDs were: 37.8 copies/reaction (95% CI: 29.5–58.0) for IAV; 13.9 copies/reaction (95% CI: 11.0–21.1) for IBV; 9.6 copies/reaction (95% CI: 7.4–14.6) for SC2; 12.3 copies/reaction (95% CI: 9.6–18.0) for RSV A; and 25.1 copies/reaction (95% CI: 20.4–34.4) for RSV B (Table 3 and Supplementary Figure S6).

Location in revised manuscript:

Page 5, paragraph 7, lines 187-188.

Page 11, paragraph 1, lines 410-411.

Page 11, paragraph 2, lines 413-414.

Location in revised supplementary material:

Page 5, paragraph 2, lines 54-58.

Page 11, paragraph 2, lines 144-148.

Page 11, paragraph 3, lines 149-153.

Sincerely,
The Authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors presented the validation of a novel laboratory-developed respiratory assay (LDRA) targeting several major respiratory viruses. Moreover, these data indicated good analytical performance, particularly 100% diagnostic sensitivity and specificity for all targets compared to a consensus reference. I think it's a valuable contribution to the field of multiplex molecular diagnostics, while I have several issues left to be addressed.

The manuscript states that LDRA results "agreed with the consensus" and uses this as the gold standard. However, the nature of this "consensus" is not defined in the provided text. I think the authors should explain this to make this better understood. This is very important for interpreting the claimed 100% accuracy of the LDRA. If the consensus includes the LDRA itself, this could introduce circularity.

In the manuscipt, while the LDRA showed no false positives/negatives, the comparator assays (PFRA and APRA) did. The manuscript notes no statistically significant differences (p>0.125), which is expected given the small number of errors. However, it doesn't investigate why these discrepancies occurred. I think it is better to address this.

While I found the provided text focused on diagnostic accuracy using clinical samples, it lacked details on the analytical sensitivity (LoD). So I recommend to include a dedicated LoD study for each target in the LDRA. It is advisable to determine the LoD using standardized protocols and compare it directly to the LoDs of the PFRA and APRA assays.

Additionally, it states "APRA produced three (0.74%) false-negative results: one for IAV (0.25%), one for IBV (0.25%) and one for RSV (0.25%)". The percentages appear to be the FN rate for that specific virus (1/405 ≈ 0.25%), while 0.74% is the overall FN rate for APRA across all targets (3/405). This could be slightly confusing. So I think the authors should clarify this.

Author Response

Author’s Reply to Reviewer 3

Manuscript ID: microorganisms-4068209

From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion® Open Access™

Comment 1:

Response 1:

Thank you for pointing this out. We agree with this comment. We had conceptualized this in Section 2.4, Diagnostic Performance; we apologize if this was not sufficiently evident. Nevertheless, We have clarified the consensus-reference approach in the revised manuscript as majority agreement among the three assays. In addition, we have added a clear caution that LDRA’s “100% accuracy” should be interpreted as complete agreement with the majority-rule consensus rather than infallibility against an independent gold standard.

Regarding the possibility of introducing circularity, we respectfully disagree. In this context, including the index test within the consensus is not new. Several authors have used this approach when evaluating diagnostic performance of molecular assays in the absence of, or when facing major difficulties with, an external reference comparator, or even in the presence of a reference comparator with suboptimal performance (e.g., culture) [1-5]. A consensus approach is often the best available approximation to the true result. This can also be viewed from another angle: external quality assessment (EQA) providers, such as CAP. In these providers’ assessment reports, consensus is formed from all participating laboratories, which necessarily includes the laboratory being assessed (comment based on experience).

Revised text (excerpt):

Diagnostic sensitivity (dSens), diagnostic specificity (dSpec), positive predictive value (PPV), and negative predictive value (NPV) for LDRA, PFRA and APRA were calculated using 2 × 2 contingency tables against an expected result defined as the per-target consensus across the three assays. Consensus rules were: A) “positive” if at least two of the three assays were positive for the same target; and B) “negative” if at least two of the three assays were negative.

…Against the study’s consensus reference standard (majority agreement among the three assays), LDRA showed 100% dSens and dSpec for each target. Importantly, because the consensus is derived from the three assays, LDRA’s “100% accuracy” should be interpreted as complete agreement with the majority-rule consensus rather than as proof of infallibility against an independent gold standard…

Location in revised manuscript:

Page 9, paragraph 4, lines 487-494.

Page 19, paragraph 1, lines 586-591.

Comment 2:

Response 2:

Thank you for pointing this out. We agree with this comment. Therefore, we have expanded the Discussion to explain that discordances clustered at high Ct values are expected near the LoD due to stochastic sampling effects, as well as platform- and assay-specific factors (e.g., thresholding algorithms and cross-channel fluorescence bleed-through). We also reference previously reported PFRA SC2 false positives associated with platform software/crosstalk correction, and we describe replicate re-analysis behavior for discordant specimens.

Revised text:

Discordant results in the comparator assays clustered at high Ct values, consistent with low-titer specimens in which stochastic sampling near the LoD can yield intermittent detection and increase the likelihood of apparent false negatives on repeat aliquots. In addition, multiplex assays may differ in target selection, chemistry, and signal processing; minor cross-channel fluorescence bleed-through (“crosstalk”) or differences in thresholding algorithms could contribute to occasional false-positive calls on specific platforms. In this dataset, the pattern of PFRA false positives (notably for SC2 and RSV) and APRA false negatives (one each for IAV, IBV, and RSV) is consistent with rare, low-frequency events expected near decision thresholds rather than with systematic assay failure, supported by the reproducibility of discordant calls upon reanalysis and by the observation that at least one SC2 specimen showed variable hit rates across replicate testing. Notably, SC2 false positives with PFRA—the most frequent discordance in this study—have been previously reported [16]. These false positives (two coincided with IAV co-detection) have been at least partially associated with software version 7.2.7 of the Panther Fusion® platform or the crosstalk correction algorithm [44]. Although the manufacturer has recently implemented an adaptive crosstalk correction to mitigate inter-channel artifacts in high viral-load samples, this study was performed using software version 7.2.7 [45]. In the SC2-positive agreed specimen that showed variable detection across six replicates, APRA detected only the SC2 S-gene target; this pattern may reflect target-specific performance, as RdRp detection sensitivity for that assay has been reported to exceed S-gene sensitivity [42]. This observation supports the added robustness of multitarget detection strategies for high-mutation RNA respiratory viruses.

Location in revised manuscript:

Page 19, paragraph 2, lines 597-618.

Comment 3:

Response 3:

Thank you for pointing this out. We agree with this comment, that analytical sensitivity is essential but, the methodological approach, results, and discussion for per-target LoD assessment of the quadruplex assay were already included in the manuscript. We apologize if this was not sufficiently evident. Anyway, We have revised the LoD discussion to present the C₉₅ LoD values per target and to explicitly note the limitations of direct LoD comparisons across assays due to differences in reference materials, matrices, and reporting units. Where comparator LoD claims are not reported in directly comparable units, we avoid over-interpreting cross-assay LoD differences and instead contextualize the observed LoDs against published benchmarks and the CDC Flu-SC2 pre-defined acceptability criteria.

Revised text (excerpt):

The limit of detection (LoD) was estimated using dilution panels prepared from the five synthetic Ultramer™ duplex controls. Starting from stock solutions at 2.41 × 10⁷ copies/mL, each control was first diluted to a working concentration of 6.4 × 10³ copies/mL in a UTM/Panther Fusion® Specimen Lysis Tube (SLM) medium matrix. UTM from negative nasopharyngeal swabs was mixed with SLM (Hologic Inc., CA, USA) at the manufacturer-recommended proportion (0.5 mL UTM per 0.71 mL SLM) to generate the UTM/SLM.

…

A dedicated analytical sensitivity study was performed for each target using standardized replicate testing and probit analysis. The LDRA assay achieved C₉₅ LoDs in the low tens of copies per reaction, corresponding to approximately 10³–10⁴ copies/mL, depending on the target. Direct LoD comparisons across candidate (LDRA) and comparator (PFRA and APRA) assays are inherently constrained by differences in sample materials, matrices, and reporting units. However, compared with the CDC Flu-SC2 assay, the quadruplex configuration showed slightly higher LoDs for influenza and SC2 (9.6–37.8 copies/reaction vs 5 copies/reaction) [19]. This difference may reflect the lower effective input volume (approx. 15 µL) dictated by the platform workflow. Overall, the LoDs observed for all four targets align with analytical sensitivity benchmarks commonly reported for contemporary multiplex RT-qPCR assays, particularly when assays are capable of detecting ≤ 10² copies/reaction (≤ 10⁴ copies/mL) [3,5,19,37-40].

Location in revised manuscript:

Page 6, paragraph 7, lines 220-248.

Page 11, paragraph 3, lines 413-416.

Page 18, paragraph 5, lines 555-566.

Comment 4:

Additionally, it states "APRA produced three (0.74%) false-negative results: one for IAV (0.25%), one for IBV (0.25%) and one for RSV (0.25%)". The percentages appear to be the FN rate for that specific virus (1/405 ≈ 0.25%), while 0.74% is the overall FN rate for APRA across all targets (3/405). This could be slightly confusing. So I think the authors should clarify this.

Response 4:

Thank you for pointing this out. We agree with this comment. We have revised the sentences describing the discordant results to improve clarity.

Revised text (excerpt):

… Relative to PFRA, four SC2 specimens and one RSV specimen were PFRA-positive/LDRA-negative (0.99% and 0.25%, respectively; 1.23% overall). Relative to APRA, one specimen each for IAV, IBV, and RSV was LDRA-positive/APRA-negative (0.25% each; 0.74% overall)…

Location in revised manuscript:

Page 15, paragraph 2, lines 461-464.

Sincerely,
The Authors

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