Inflammatory Skin Lesions in Three SARS-CoV-2 Swab-Negative Adolescents: A Possible COVID-19 Sneaky Manifestation?

Round 1

Reviewer 1 Report

There are several reports described that granuloma annulare and chilblains were triggered by SARS‐CoV‐2 infection. Although, SARS-CoV2 swab-negative and the absence of flu-like symptoms of those cases in this study. However, they had histological findings of chronic immune-mediated inflammation and immunohistochemical evidence of SARS-CoV-2 spike glycoprotein in endothelial cells and eccrine sweat glands. The authors must be citing those well.  

Author Response

Dear Reviewer,

We appreciate your helpful comment.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

Review n’1: “There are several reports described that granuloma annulare and chilblains were triggered by SARS‐CoV‐2 infection. Although, SARS-CoV2 swab-negative and the absence of flu-like symptoms of those cases in this study. However, they had histological findings of chronic immune-mediated inflammation and immunohistochemical evidence of SARS-CoV-2 spike glycoprotein in endothelial cells and eccrine sweat glands. The authors must be citing those well. The authors must be citing those well”.

Answer: Thank you very much, we have proceeded to add some quotes that are related to the topics you mentioned.

Reviewer 2 Report

Interesting article nicely illustrated.

Extensive language revision needed.

Consistency of the term :SARS-COV-2/SARS-COV2

More/better argumentation required in the conclusion;

Interpretation of histology (legend of the figures 2a,2b,2c more in detail)

fig 3. not convincing 

Author Response

Dear Reviewer,

We appreciate your helpful comments; we believe that our manuscript has been improved greatly through the incorporation of your suggestions.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

Review n’2: “Extensive language revision needed”.

Answer: Done, Thank you very much. The whole paper has been thoroughly checked for grammar, style, typos and syntax mistakes.

Review n’2: “Consistency of the term: SARS-COV-2/SARS-COV2”.

Answer: We have standardized the diction in SARS-CoV-2.

Review n’2: “More/better argumentation required in the conclusion”.

Answer: We have proceeded to better argue the conclusions.

Review n’2: “Interpretation of histology (legend of the figures 2a,2b,2c more in detail)”

Answer: Done, Thank you very much.

Review n’2: “fig 3. not convincing”.

Answer: Thank you very much, we have eliminated the figure and renumbered the remaining ones.

Round 2

Reviewer 2 Report

language much better but can still be improved.

no consistency: Erythema Multiforme vs erythema multiforme

terminology: urticaroid erythema (?); mucinosis phenomena (?)

Orceina method vs orcein staining

some references are incomplete  ex.13,14

check references for accuracy

Author Response

Dear Reviewer,

We appreciate your helpful comments.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

Review: language much better but can still be improved.

Answer 1: Thank you very much, we have taken steps to further implement the English language.

Review: no consistency: Erythema Multiforme vs erythema multiforme.

Answer 2: Thank you very much, we have arranged according to the right observation.

Review: terminology: urticaroid erythema (?); mucinosis phenomena (?)

Answer 3: We have proceeded to correct. Thank you very much.

Review: Orceina method vs orcein staining.

Answer 4: We have proceeded to correct. Thank you very much.

Review: some references are incomplete ex.13,14. check references for accuracy.

Answer 5: We have proceeded to correct. Thank you very much.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dear authors 

I have read with interest your article. The issue of COVID-19 related skin lesions is certainly very challenging and interesting and merits attention. Unfortunately, after having read the paper I have more questions than answers. You report 3 cases of uncertain diagnosis (‘granuloma-annulare-like lesions’) in three children with unconfirmed COVID-19 infection (SARS-CoV-2 negative test) and claim to have found SARS-CoV-2 in the lesions with techniques that are not convincing to me. Indeed, the reliability of the antibody you used for SARS-CoV-2 detection is uncertain; is this monoclonal or polyclonal (monoclonal antibodies are notoriously known to produce cross-reactivities with irrelevant antigens) ? Most importantly, you have not provided appropriate positive (and negative) controls, i.e., tissues from patients with confirmed, severe COVID-19where SARS-CoV2 has been detected with certainty. The results you present are not described appropriately, you describe reactivity in ‘sweat glands’ – this is too imprecise - where exactly? Cytoplasmic, nuclear? In the secretory, or excretory segment? if in the secretory part, clear cells, dark cells or myoepithelial cells? The figures you provide seem to show nuclear staining but in the EM study you describe cytoplasmic presence of virus-like particles, which is contradictory. If the immunohistochemical staining you show were specific, you should have observed hundreds-to-thousands of virions in the cells but your figures only show few virus-like particles. Of note, even if these are consistent with viruses, there is no formal proof - you should provide a positive PCR (or in situ hybridization) in the skin to convince the readers that the particles shown are SARS-CoV-2 (the particles are consistent also with cytoplasmic organelles, pinocytotic vesicles, lysosomes etc). The role of EM in this setting is supportive but is no proof. In the past, EM was used to claim the viral origin of diseases (such as lupus erythematosus on the basis of paramyxovirus-like particles in endothelial cells, which proved to be metabolic by-products of the cells – this example just to highlight the fact that EM observations should be supported by additional convincing methods of virus detection, namely molecular biology). In this respect, I consider that describing in the figure legends your findings as ‘coronaviruses’ is an overstatement, and the title itself ‘’…a possible COVID-19 sneaky manifestation’ deceptive as you bring no proof of COVID-19 infection in these patients (PCR negative, no symptoms) .

Additional, more or less minor issues that need improvement:

- the diagnosis of the skin lesions needs to be better substantiated (although in the light of the above considerations this becomes not so important). Mucin deposits should be confirmed with alcian blue staining and elastic fiber changes with orcein staining. As you state, the diagnosis of GA is doubtful since you have not seen infiltrating histiocytes, which are one of the hallmarks of GA. Before publishing a case, one shoud propose a convincing diagnosis;

- your conclusion is difficult to understand; on the one hand you claim to have shown SARS-CoV-2 in the lesions and on the other you state that the lesions may be ‘more related to the immune responses secondary to viral infection than to the direct cytopathic effect of the virus’

- The English needs some revision for typos

Author Response

Dear Reviewer,

We appreciate your helpful comments; we believe that our manuscript has been improved greatly through the incorporation of your suggestions.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

Point 1: “the reliability of the antibody you used for SARS-CoV-2 detection is uncertain; is this monoclonal or polyclonal (monoclonal antibodies are notoriously known to produce cross-reactivities with irrelevant antigens)?”.

Answer: Thank you very much, in our laboratory we have used SARS-CoV-2 Spike Protein S1 Antibody (MA5-36247) in IHC (P), rabbit monoclonal to SARS-CoV-2 spike glycoprotein – Coronavirus; suitable for: IHC-P, ELISA, ICC and WB, isotype: IgG. In the technical data sheet of the company, this antibody is considered appropriate for use for immunohistochemistry on paraffin, and covered by the ThermoFisher guarantee, as it is directly tested according to the company's internal protocols. The company provides in the datasheets a very high specificity towards SARS-CoV-2 and defines this antibody as "recombinant" as further proof of reliability. Only after acquiring this information, we bought it. (https://www.thermofisher.com/antibody/product/SARS-CoV-2-Spike-Protein-S1-Antibody-clone-HL6-Monoclonal/MA5-36247?imageId=733489). There was an error in the initial version of the manuscript as the clone used was not from ABCAM!

 

Point 2: “Most importantly, you have not provided appropriate positive (and negative) controls, i.e., tissues from patients with confirmed, severe COVID-19 where SARS-CoV2 has been detected with certainty”.

Answer: Thank you very much, just for reasons of space, we have not reported the images of the positive controls, which of course were conducted on lung tissues of patients who died from SARS-CoV-2 and with positive swab. Conversely, paraffin embedded tissue (lung and skin samples) of five years ago was totally negative for the immunohistochemical reaction. We have faithfully followed the firm's recommendations. (https://www.thermofisher.com/antibody/product/SARS-CoV-2-Spike-Protein-S1-Antibody-clone-HL6-Monoclonal/MA5-36247).

 

Point 3: “the results you present are not described appropriately; you describe reactivity in ‘sweat glands’ – this is too imprecise - where exactly? Cytoplasmic, nuclear? In the secretory, or excretory segment? if in the secretory part, clear cells, dark cells or myoepithelial cells?”

Answer: The immunoreactivity is cytoplasmic granular in the secretory portion of eccrine units.

 

Point 4: “Of note, even if these are consistent with viruses, there is no formal proof - you should provide a positive PCR (or in situ hybridization) in the skin to convince the readers that the particles shown are SARS-CoV-2 (the particles are consistent also with cytoplasmic organelles, pinocytotic vesicles, lysosomes etc.)”.

Answer: Thank you very much for your observation, we were unable to provide the data on the PCR as it was unfortunately not possible to carry out this investigation, but in an attempt to respond to your correct considerations, we were able to get in touch with the parents of 2/3 cases described that provided us with information: the serological examination carried out after 3 and a half months and 4 months respectively was positive for IgG. We don't know if this provides definitive proof but we wanted to make an effort to fully understand it!

 

Point 5: “The role of EM in this setting is supportive but is no proof. In the past, EM was used to claim the viral origin of diseases (such as lupus erythematosus on the basis of paramyxovirus-like particles in endothelial cells, which proved to be metabolic by-products of the cells – this example just to highlight the fact that EM observations should be supported by additional convincing methods of virus detection, namely molecular biology). In this respect, I consider that describing in the figure legends your findings as ‘coronaviruses’ is an overstatement, and the title itself ‘’…a possible COVID-19 sneaky manifestation’ deceptive as you bring no proof of COVID-19 infection in these patients (PCR negative, no symptoms)”.

Answer: Thank you so much for the stark observation. In reality, we weren't going to write something "deceptive" as much as simply to bring our experience to the attention of readers. We understand very well that electron microscopy cannot be a proof, but only "something more" in the context of an immunohistochemical investigation and a serological positivity that we have now acquired as data. We have revised the title to avoid any misunderstanding.

 

Point 6: “the diagnosis of the skin lesions needs to be better substantiated (although in the light of the above considerations this becomes not so important). Mucin deposits should be confirmed with Alcian blue staining and elastic fiber changes with orcein staining. As you state, the diagnosis of GA is doubtful since you have not seen infiltrating histiocytes, which are one of the hallmarks of GA. Before publishing a case, one should propose a convincing diagnosis”.

Answer: Thank you very much, we have added an image with the Orcein method to document the presence of fragmentation of the elastic fibers and an image with the Alcian Blue method to document the deposition of mucin. Regarding the diagnosis, we ourselves have indicated how these features CLINICALLY suggested an annular granuloma, but histologically it did not have all the characteristics. Precisely for this reason we have decided to leave the diagnosis "open".

 

Point 7: “your conclusion is difficult to understand; on the one hand you claim to have shown SARS-CoV-2 in the lesions and on the other you state that the lesions may be ‘more related to the immune responses secondary to viral infection than to the direct cytopathic effect of the virus”.

Answer: Thank you very much, we wanted to state that we are not sure that the lesions are directly related to the cytopathic effect of the virus but rather to the "immune surge" secondary to the presence of the virions, although we have tried to demonstrate their presence in the skin. Also, in this case it was our intention to leave the conclusions "open" given the multitude of reports that are often apparently "conflicting" that are present in the Literature.

 

Point 8: The English needs some revision for typos.

Answer: Thank you so much for letting us know. The whole paper has been checked for typos and syntax mistakes.

Reviewer 2 Report

Clinical figure 1 shows nodules with accentuated skin markings. This is a sign of chronic rubbing and/scratching the lesions, as also evidenced in Figure 2a (acanthosis and hyperkeratosis).  What about symptoms? Duration? 

Concerning figure 2a, in my view, there are no clear signs of vacuolization of basal keratinocytes in the Fig 2.  I would substitute this figure with a lower power image, to better show the mucin deposits throughout the dermis.

Fig 2b e 2c. Elastic fibers fragmentation cannot be appreciated by H-E stain!! 

Discussion. Delete the first 7 lines (lines 168-173).  They are unnecessary and reduntant.

 

Discussion. Lines 182-184. The authors refers the publication by Colmenero et al. However, they should also mention the criticism about the electron microscopy findings by  Brealey and Miller in a letter published on Br J Dermatol 2020.  SARS‐CoV‐2 has not been detected directly by electron microscopy in the endothelium of chilblain lesions.

 

As far as electron microscopy is concerned, the criticism is that the magnifications lack any indication of the length of the nanometer bar and above all that the magnification in Fig. 3 [which is twice as high (89,000) as that in Fig. 4 (44,000)] shows much smaller particles and so something is wrong... Furthermore, the red arrow points to an image that is not convincing for virions information.

Author Response

Dear Reviewer,

We appreciate your helpful comments; we believe that our manuscript has been improved greatly through the incorporation of your suggestions.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

Point 1: Clinical figure 1 shows nodules with accentuated skin markings. This is a sign of chronic rubbing and/scratching the lesions, as also evidenced in Figure 2a (acanthosis and hyperkeratosis).  What about symptoms? Duration?

Answer: Thank you very much, in all three cases, the boys showed no particular symptoms, except a slight itchy feeling. These lesions were approximately 20 days old from the time they were evaluated by the paediatric dermatologist. We have proceeded to add this element in the text.

 

Point 2: Concerning figure 2a, in my view, there are no clear signs of vacuolization of basal keratinocytes in the Fig 2.  I would substitute this figure with a lower power image, to better show the mucin deposits throughout the dermis.

Answer: Thank you very much for your right observation. We proceeded to modify the image with one that showed the mucinosis as indicated by you and furthermore we decided not to give so much weight to the "vacuolization of the keratinocytes of the basal layer" as to the deposition, in fact, of mucin.

 

Point 3: “Fig 2b e 2c. Elastic fibres fragmentation cannot be appreciated by H-E stain!!”

Answer: Thank you very much, we proceeded to provide an image with the Orcein stain to demonstrate the fragmentation of the elastic fibres.

 

Point 4: “Discussion. Delete the first 7 lines (lines 168-173).  They are unnecessary and redundant”.

Answer: Thank you very much, Done.

 

Point 5: “Discussion. Lines 182-184. The authors refer the publication by Colmenero et al. However, they should also mention the criticism about the electron microscopy findings by Brealey and Miller in a letter published on Br J Dermatol 2020.  SARS‐CoV‐2 has not been detected directly by electron microscopy in the endothelium of chilblain lesions”.

Answer: Thank you very much, we have revised this part of the discussion and added the quote you provided which leaves the situation relating to electron microscopy at stake.

 

Point 6: “As far as electron microscopy is concerned, the criticism is that the magnifications lack any indication of the length of the nanometer bar and above all that the magnification in Fig. 3 [which is twice as high (89,000) as that in Fig. 4 (44,000)] shows much smaller particles and so something is wrong... Furthermore, the red arrow points to an image that is not convincing for virions information”.

Answer: Thank you very much for the right observation, we have eliminated the red arrow, and double-checked the original enlargement of the images of ME which had suffered from a small imperfection.

Round 2

Reviewer 1 Report

Dear authors 

You have replied to my criticisms in the rebuttal letter but not so in the body of the article:

 - please mention in the article the precise antibody you used; the new version still mentions the 'Abcam' antibody, which you mentioned by mistake, according to your reply;

- please add a photomicrograph from positive (endothelial or other cell) immunostaining with the antibody in tissue from a COVIC-19-confirmed case. You can delete figure 5a which is low magnification and does not show clearly the labelling. Furthemore, in your reply you state that the immunolabelling for SARS-CoV-2 was 'granular cytoplasmic' but figures 5b and 5c show rather a nuclear labelling (clearly stronger than any possible cytoplasmic staining). This is also contradictory with the fact that the viral-like particles you found by EM are in the cytoplasm (as one would expect from figures 5b and 5C) 

- please add the serological data of the patients. 

Author Response

Dear Reviewer,

We appreciate your helpful comments.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

Review: “please mention in the article the precise antibody you used; the new version still mentions the 'Abcam' antibody, which you mentioned by mistake, according to your reply”. Answer: Done, Thank you very much. Sorry for the inconvenience.
Review: “please add a photomicrograph from positive (endothelial or other cell) immunostaining with the antibody in tissue from a COVIC-19-confirmed case. You can delete figure 5a which is low magnification and does not show clearly the labelling. Furthemore, in your reply you state that the immunolabelling for SARS-CoV-2 was 'granular cytoplasmic' but figures 5b and 5c show rather a nuclear labelling (clearly stronger than any possible cytoplasmic staining). This is also contradictory with the fact that the viral-like particles you found by EM are in the cytoplasm (as one would expect from figures 5b and 5c)”. Answer: Thanks a lot for your advice. We removed figure 5a as it was inconclusive and modified images 5b and 5c to better show granular cytoplasmic positivity.
Review: “please add the serological data of the patients”. Answer: Done, Thank you very much.

Reviewer 2 Report

The authors answered to all the suggestions.

In the present form, the manuscript is fine.

Author Response

Dear Reviewer,

Thank you very much.

Best Regards

Giuseppe Ingravallo

On behalf of the co-authors

 

 

Round 3

Reviewer 1 Report

Dear authors

You have not provided photomicrographs of adequate  positive and negative immunohistochemical controls 

Please complete ref. 21 which is incomplete