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Does the introduction explain the objective of the research presented in the preprint?

Yes

It clearly explains the objective. However it is important to acknowledge that diagnostic performance assessment was conducted in comparison with culture methods and qPCR.

Are the methods well-suited for this research?

Highly appropriate

They follow best practices throughout the research. They are rigorously executed and provide a robust foundation for drawing valid conclusions. The authors have explicitly clarified the implications of the different specimen collection methods -sputum, endotracheal aspirates, bronchoalveolar lavage fluids, bronchial washings, protected brush specimens and lung aspirates- in the identification of S. pneumoniae, as well as their differential impact on the sensitivity and specificity analysis of this new diagnostic approach compared with traditional methods. It is important to highlight that the methodology employed in cell cultures, qPCR and fluorescent antibody detection are thoroughly explained and highly aligned with the research objectives by the authors. Because we are not experts in this field and this would be our own limitation as reviewers, we encourage the consultation of an expert in identification of infectious pathogens using confocal microscopy methods to ensure the accuracy of the results presented, concerning the confocal microscopy methods.

Are the conclusions supported by the data?

Somewhat supported

The conclusions are mostly, but not always, thorough. They provide a reasonable interpretation of the data without overreaching or adding interpretations not reflected in the results. The authors acknowledge some of the potential limitations of the study. They state that “fluorescence antibody-based detection suitable for resource-limited regions, as DNA extraction, reaction mixtures, and real-time systems are not necessary, and minimal training is required”. However, this might not apply in health care facilities in low income countries where the availability of trained personnel is scarce, therefore, the generalizability is compromised. All in all, while the timing of Spn-Fluo makes it a quick diagnostic test, it is not necessarily straightforward, mainly because of the need of trained personnel -with expertise in confocal microscopy detection of pathogens- and comprenhensive laboratory infrastructure that are necessary for the implementation of the test.

Are the data presentations, including visualizations, well-suited to represent the data?

Somewhat appropriate and clear

They follow accessibility best practices and well communicate the results and main patterns in the data, making it easy to comprehend or interpret the data effectively. Since the preprint focuses on sensitivity and specificity analysis of this new diagnostic approach compared with cultured methods, incorporating a ROC curve would provide a better visualization of the diagnostic performance of the test compared with conventional methods. The “+++” grading mechanism used may be unclear, as it may be better to simplify the grading to a numeric scale. Wasting data is also a concern, as Figure 2 “B” may have benefitted from additional data collected. The confocal microscopy methodology and results are clearly explained and appropriate for identification of specific pathogens, however, we encourage that someone with expertise in microbial identification and antibody-labeling methods review the interpretation of the images and graphic analysis.

How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research?

Very clearly

They demonstrate clarity, depth, and insight in their discussion, explanation, and interpretation of their findings and potential next steps. They highlight the relevance and novelty of this study by confirming that Spn-FLUO is a rapid diagnostic test, while also acknowledging the implications of using a fluorescence microscope. They are also detailed in explaining their main limitations, without forgetting to emphasize the importance as a precedent for the investigation of aggregates and ultrastructure of streptococcus pneumoniae.

Is the preprint likely to advance academic knowledge?

Highly likely

The study goes into great detail regarding their methodology and analysis, being very specific about what was done in their experiment. Since SpnFLUO is a relatively new method in detecting Spn in human sputum, it was appropriate to see that the authors had compared it to conventional methods, including culture and qPCR. Evaluations were done on both mice and human specimens (patients with pneumococcal pneumonia), which is important when assessing the performance of this diagnostic test.

Would it benefit from language editing?

No

There are minor language issues, but they do not impact the clarity or understanding of the study.

Would you recommend this preprint to others?

Yes, it’s of high quality

Yes, it is a solid and well thought work that shows a new technique to identify Streptococcus pneumoniae infection. This new approach might be of practical knowledge for experts who specialize in identification of infectious pathogens using confocal and/or fluorescence microscopy. Moreover, this new diagnostic approach opens not only the discussion about the performance of the test, but also it opens the pathway to new research in the area.

Is it ready for attention from an editor, publisher or broader audience?

Yes, after minor changes

After clarifying the aforementioned details. It would also be beneficial for an expert to give their insight on identification of infectious pathogens using confocal microscopy methods.

Competing interests

The authors declare that they have no competing interests.