PONE-D-22-14092Transcriptome analysis in nasopharyngeal samples reveals increased abundance and diversity of opportunistic fungal pathogens in COVID-19 patientsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well-designed study with a good representation of data. However, there are some concerns and issues that need to be addressed by the authors before considering for publication in PLOS One.

Abstract

Rephrase the first sentence of the abstract and remove “we previously reported”.

Line 45: Surprising to see S. cerevisiae (88.62%) and Phaffia rhodozyma (10.30%) comprised 99% total species where authors reported 863 species.

Introduction:

1. Clinical trials and high throughput sequencing ………..respiratory viral pathogens [14], and bacteria and/or fungi [11, 13, 15, 16], make no sense. Rephrase the sentence.

2. Similarly, Fungal infections are known…….patients admitted to intensive care units with ARDS [11]. Revise the sentence to make it meaningful to the readers.

3. The hypothesis is time demanding and carries significant importance. However, the big sentence representing the hypothesis is very difficult to follow and coordinate. Please rephrase it to make a simpler and reader-friendly statement.

Method

1. The samples were collected more than two years ago. In the meantime, the variants of COVID have been changed a couple of times. In that case, what approach should be used by the authors to address concerns like currently circulating COVID strains?

Results

1. Our primary microbiome compositional analysis….. 11 species as differentially abundant across Healthy, COVID-19 and Recovered metagenomes. Make no sense. Rephrase it.

2. What is the necessity of mentioning major fungal species detected (Fig 5) and the top twelve fungal species detected (Fig 6) in two different figures? Isn’t it an exaggeration? Keep a single figure to represent major or top abundant fungal species and the rest may be replaced in the supplementary figures.

3. The Figures 5 and 6 legends for species names should be italic.

Discussion

1. Needs to correlate fungal infection with SARS-CoV2 infection in the discussion section.

2. Is there any report of secondary infection of fungus after COVID-19 infection?

3. Discuss fungal opportunistic pathogenic character in COVID-19 cases.

Reviewer #2: Summary

This study compared the nasopharyngeal fungal microbiome of 15 people: Eight COVID-19 patients, and seven healthy controls. Seven of the COVID-19 patients were subsequently included in the recovered group after tested negative and recovered from COVID-19.

The authors found that the three groups had unique fungal microbiome: 37% of fungal species were exclusively associated with SARS-CoV-2, with Saccharomyces cerevisiae and Phaffia rhodozyma being the two species with the highest abundance. The recovered patients’ fungal microbiome was dominated by several species of the Aspergillus genus, including A. penicillioides, A. keveii, A. oryzae, and A. pseudoglaucus.; Healthy controls had high abundance of Nannochloroopsis oceanica and Saccharomyces pastoriaus. Another main finding of the current study was that there was an increase in the alpha diversity of fungal microbiome in COVID-19 patients. The authors went on to perform metabolic functions analysis and postulated on the possible role of dysbiosis in the role of COVID-19 pathogenesis.

Overall Comment

This is an important study to allow deeper understanding of nasopharyngeal microbiome in COVID-19 patients. Extensive bioinformation analysis work was performed with interesting findings.

The description of patient recruitment could be made clearer. It took me some time to understand the 7 subjects in the recovered group were the sample subjects from the COVID-19 group, after they have recovered. The authors should also explain why only 7 of the 8 COVID-19 patients were included in the recovered group.

There was inconsistency in the terminology used for the three groups: For example: line 129 “Recovered”, line 133 “Recovered humans”, line 134 “Recovered subjects; Another example: line 138 “Healthy people”, line 136 “Healthy control subjects”.

The difference between dysbiosis and clinical infection was not clear throughout the manuscript. For example, in Lines 110-115, the authors stated the importance of understanding fungal microbiome in COVID-19 patients, however, in the next sentence, the authors advocated a timely diagnosis of fungal co-infections to limit the overuse of antimicrobial agents.

The authors included subject information in Table S1 with a column stated whether the subjects received “COVID-19 medicine”. Specific information on antibiotics used would be helpful to the interpretation of the study results: One major finding of the current study was that COVID-19 patients exhibited higher fungal microbiome diversities than those of recovered human and healthy controls, could it be due to effect of antibiotics, killing off most bacteria that allow the blooming of fungal organisms? Another major finding of the current study was that various species in the Aspergillus genus were over-represented in the recovered group. Aspergillus is well known to be present in the healthcare environment [1]. Could the observation be explained by nasopharynx flora being colonized by hospital molds with the aid of antibiotics therapy used?

Specific Comments

Lines 63 - 65: Redundance, can omit the second “SARS-CoV-2” of the sentence

Lines 65 – 68: The sentence seemed to suggest that all COVID-19 infections will result in ARDS. Suggest rephrasing.

Line 71: What is the meaning of “resilient microbiomes”?

Line 88: Mucormycosis is well studied and should not be considered as “mysterious fungal infection”

Lines 95-98: broad spectrum antibiotics are for treatment of bacterial coinfections, dexamethasone and immunosuppressive therapies are for immune modulation for treatment of immune dysregulation such as ARDS.

Lines 111: what is the meaning of “migration, propagation and immune response”?

Lines 250-251: “….which probably due to the opportunistic inclusion during the pathogenesis of SARS-CoV-2 infection” should be moved to discussion section

Line 314: Can consider delete the word “However”

Line 427: Typo: “COVI1D-9”

Line 488: redundance: “…..with SARS-CoV-2 infections in COVID-19 patients”

Line 502-505: Different species of aspergillus has different pathogenic potential. As the authors rightly pointed out A. fumigatus, and A. flavus to a lesser extent, are associated with clinical important aspergillus infections, but not the A. penicilliodes and A. keveii that is found in abundance in recovered patients. Suggest discussing pathogenic and non-pathogenic aspergillus species separately.

Line 570-572: was there any specific impairment of antiviral activities that was mentioned in the text?

Figures: some figures placed healthy control on the left side of the charts (e.g. Fig 1A, Fig 4), while others were in the opposite order, with COVID-19 on left side (e.g. Fig 3, Fig 5, and 6)

Figure 5: the color used for different species are similar to each other.

Table S1: Inconsistency noted: there were information included for 7 COVID-19 patients, 7 recovered patients, and 7 healthy controls. However, there were 8 COVID patients recruited in the study (line 37).

References

David J. Weber, MD, Amanda Peppercorn, Melissa B. Miller, Emily Sickbert-Benett, William A. Rutala, Preventing healthcare-associated Aspergillus infections: review of recent CDC/HICPAC recommendations, Medical Mycology, Volume 47, Issue Supplement_1, 2009, Pages S199–S209, https://doi.org/10.1080/13693780802709073

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Reviewer #1: No

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Transcriptome analysis in nasopharyngeal samples reveals increased abundance and diversity of opportunistic fungal pathogens in COVID-19 patients

PONE-D-22-14092R1

Dear Dr. Islam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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David M. Ojcius

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors addressed my comments and concerns with sufficient details. Though there are some limitations including sampling time (two-years back), however the findings are interesting and significant in terms of fungal pathogens in COVID19 infection.

Reviewer #2: The authors had adequately addressed all the comments and concerns I raised previously. The revised manuscript has improved significantly and is good for publication.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

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