Review Reports - Histopathological and Immunohistochemical Findings in Postmortem Lungs from Mexican Patients with Severe COVID-19

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a descriptive histopathology and immunohistochemistry (IHC) analysis of nine post-mortem lung samples from qRT-PCR-confirmed severe COVID-19 cases, reporting diffuse alveolar damage in all cases and IHC detection of Spike (S) and Nucleocapsid (N) in multiple cell types and intravascular/intraalveolar material. Please find below my comments:

-The manuscript states nine deceased patients with confirmed SARS-CoV-2 diagnosis, but provides no structured clinical table (age, sex, BMI, comorbidities, smoking history, etc....). Without these, it is hard to interpret variability (e.g., why samples 3–5 show “higher variability” of lesions) and impossible to assess confounding.

-Histopathology is not scored, and some terms need operational definitions. In histopathology, reproducibility requires either quantitative measurements or standardized semi-quantitative scoring.

-Cell-type attribution on H&E alone is overconfident. In severely damaged lungs (detachment, necrosis, inflammatory influx), morphology can be misleading.

-Several references and citations appear incorrect or mismatched.

Minor comments:

-Table 1 is dense and uses symbols that are easy to misread. Please consider a cleaner scoring format and consistent denominators.

-Terms like “leukocytosis” in a histology table are confusing. If this refers to clinical blood counts, it should be moved to the clinical table, not the tissue finding table.

-In the figures: Captions are extremely long and color-coded with many shapes. This makes them hard to read and can feel like over-annotation.

Author Response

Major comments:

Comments 1: The manuscript states nine deceased patients with confirmed SARS-CoV-2 diagnosis, but provides no structured clinical table (age, sex, BMI, comorbidities, smoking history, etc....). Without these, it is hard to interpret variability (e.g., why samples 3–5 show “higher variability” of lesions) and impossible to assess confounding.
Response 1: The authors appreciate the reviewer’s comments. The authors would like to share with the reviewer some important points related to these considerations:

The study is a retrospective and descriptive analysis based on paraffin embedded postmortem lung samples from patients with COVID-19 confirmed. These samples were donated to our laboratory to find or identify the SARS CoV-2, which had not been carried out at that time. All the samples were collected by the authorized personal (Pathologist), under the Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas” (National Institute of Respiratory Diseases “Ismael Cosío Villegas” or INER) approval number B09-20, at Mexico City. All their legal guardians provided written informed consent in accordance with the Declaration of Helsinki for Human Research. Clinical samples were managed according to mexican norm NOM-012-SSA3-2012, which establishes the criteria for the execution of clinical investigations in humans. It is important to mention that we did not have any access to patients and much less obtain lung samples from patients with other types of pathologies, this is restricted to the pathologist of INER.
During the period that the samples were collected, there were serious restrictions to take the samples from patients who died because of COVID-19, so we are not capacitated to do this job. Therefore, the pathologist responsible and his research group collaborated with us through the donation of the samples, and we made the rest of the analysis (H&E and SARSCoV-2 immune detection). The rules of the institution are very clear: Informed consents are part of the institutional documentation of INER and are subject to internal confidentiality policies, as well as current ethical and legal regulations. Therefore, any review or release cannot be carried out on an individual basis but must be processed and authorized by the corresponding institutional authorities.

Comments 2: Histopathology is not scored, and some terms need operational definitions. In histopathology, reproducibility requires either quantitative measurements or standardized semi-quantitative scoring.

Response 2: The authors appreciate the comments, and they modified the text of the manuscript adding the percentage as a scoring for the histological findings taken into consideration the nine samples as 100 % and the remaining percentages were established based on this 100%. From this 100% (9/9), the percentage of findings was established based on the number of samples that presented one or more of them. The modifications are from line 99 to line 193.
Line 101: 100% of samples (9/9)
Line 102: 100% of samples (9/9 samples),
Line 103: (22.2% of samples) resulted in acute inflammatory infiltration (mainly neutrophils)
Line 104-105: the rest of the samples (77.7%; samples 1, 3- 7, 9) resulted in chronic inflammatory infiltration (mainly mononuclear cells).
Line 105: (9/9; 100% of samples)
Line 106: 100% of
Line 107: (9/9)
Line 109: were the hyperplasia and vacuolization of
Line 110: 66.6% of samples (6/9;
Line 112-113: 88.8% of samples (8/9;
Line 114: air and country
Line 116: in 100% of samples. and 44.4% of samples; samples
Line 118: (33.3% of samples)
Line 120: was identified in 66.6% of samples (6/9; samples 2, 6-9)
Line 121-123: were present in 88.8% of samples (only sample 4 did not show these findings), pulmonary and intravascular dead cells were observed in samples 3 and 5 (2/9, 22.2% of samples).
Line 123: binucleated and
Line 123-124: in 100% of samples
Line 124-125: in 44.4% of simples
Line 125: only in 11.1% of samples, Clots and intravascular
Line 126: (fibrin) and 77.7% of samples (7/9; samples)
Line 180-181: 88.8% of the samples (8/9)
Line 188: 77.7% of samples (7/9).

Comments 3: Cell-type attribution on H&E alone is overconfident. In severely damaged lungs (detachment, necrosis, inflammatory influx), morphology can be misleading.

Response 3: The findings reported in this manuscript correspond to those obtained in routine histopathological work with paraffin-embedded sections stained with H&E. Certainly, specific markers could be used for type I pneumocytes (T1-alpha (Podoplanin/Glycoprotein 38) and Aquaporin 5 (AQP5)); type II pneumocytes (Surfactant Protein B (SP-B) and Surfactant Protein C (SP-C)); and alveolar macrophages (CD11c, CD64, and MerTK) (The Journal of Infectious Diseases® 2023;227:1245–54 https://doi.org/10.1093/infdis/jiad056; PLoS Pathog 21(9): e1013469. https://doi.org/10.1371/journal.ppat.1013469). However, SARS-CoV-2 infection can significantly alter the expression of these markers, also limiting the reliability of the histopathological study. However, type II pneumocytes can be identified with immunohistochemistry since they express Thyroid transcription factor 1 (TTF1) in the nucleus as was recently demonstrated in lung from infant with lethal COVID-19 (Gulubova et al. Italian Journal of Pediatrics (2025) 51:174 https://doi.org/10.1186/s13052-025-01984-y)

Comments 4: Several references and citations appear incorrect or mismatched.

Response 4: The authors thank the reviewer for the observations. The authors have corrected the references.
Line 416-417. Reference 3. The link has been corrected.
Line 424, Reference 6. The link has been corrected.

Minor comments:

Comments 5: Table 1 is dense and uses symbols that are easy to misread. Please consider a cleaner scoring format and consistent denominators.

Response 5: Line 129-136.The authors appreciate the reviewer’s comments. Table 1. The authors modified the symbols to avoid misreading.

Comments 6: Terms like “leukocytosis” in a histology table are confusing. If this refers to clinical blood counts, it should be moved to the clinical table, not the tissue finding table.

Response 6: The authors appreciate the comment, and they modified the term as the reviewer suggested. The term leukocytosis has been changed to “leukocyte infiltration” and remains in the table of histopathological findings.

Comments 7: In the figures: Captions are extremely long and color-coded with many shapes. This makes them hard to read and can feel like over-annotation.

Response 7: The authors appreciate the observations, and they have done the modifications over the figures, so these changes modified the number of the lines in which each figure previously appeared.

Line 152: Figure 1

Line 153-156: of alveolar fibrosis, capillary distension and focal leukocytes infiltration in the histopathological analysis of lung postmortem samples of patients with severe COVID-19. Alveolar wall fibrosis (Fig. 1A-1C, red circle), capillary dilation (Fig. 1D, orange circle), leukocyte infiltration (Fig. 1E, 1F, yellow circle).

Line 157: Figure 2

Line 158-161: multinucleated and cytoplasmic/nuclear eosinophilic inclusion bodies cells in histopathological analysis of lung postmortem samples of patients with severe COVID-19. Multinucleated cells (Fig. 2A-2H, dark arrow), carbon inclusions (Fig. 2D, 2G, 2I, yellow arrow) and cells with cytoplasmic/nuclear inclusion (Fig. 2E-2I, cyan arrow).

Line 163: Figure 3

Line 164-167: of hyaline membrane and pleural fibrosis in histopathological analysis of lung postmortem samples of patients with severe COVID-19. Hyaline membrane (Fig. 3A-3C, cyan circle), pleural fibrosis (Fig 3D-3F, red circle) and clot/fibrin protein inside vessels (Fig. 3F, blue asterisk).

Line 168: Figure 4

Line 169-171: perivascular damage in histopathological analysis of lung postmortem samples of patients with severe COVID-19. Perivascular damage (Fig. 4A-4F, green circle), carbon inclusions (Fig. 4E-4F, yellow arrow).

Line 197: Figure 5

Line 203: perivascular (Fig. 5D, 5G, red)

Line 206: Figure 6

Line 209: perivascular (Fig. 6H, red)

Line 212: Figure 7

Line 215: perivascular fibrosis (Fig. 7A-7C, red)

The authors thank all the observations and comments from the reviewer; The authors modified the text. All the changes appear in pink.

Materials and methods

Line 278-381: Developed methodology

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your efforts on the manuscript. Upon reviewing the current version, I would like to suggest some revisions and supplementary additions to improve clarity and completeness.

Comments for author File: Comments.pdf

Author Response

Major comments:

Comments 1: The study includes only nine postmortem lung samples. Although the limited availability of autopsy material during the early COVID-19 pandemic—when autopsies were prohibited mainly in many regions—partially explains the low sample size, this remains a major limitation that limits the generalizability of the findings. Furthermore, the analyses are exclusively qualitative or semi-structured. No statistical analysis is presented. Even with a limited sample size, the authors should incorporate quantitative or semi-quantitative approaches, such as scoring systems for histopathological features, cell counts, or the percentage of immunoreactive cells.

Response 1: The authors thank the reviewer’s comments. This study is limited by the small number of post-mortem lung samples analyzed (n = 9). While the limited availability of autopsy material during the early phase of the COVID-19 pandemic explains this limitation, the sample size restricts the generalization of the findings and prevents robust statistical analyses. Therefore, this work should be interpreted as a descriptive and exploratory study rather than a quantitative or inferential analysis.
Limitations: This study was designed as a descriptive pathological analysis of post-mortem lung samples. Given the limited number of cases and the absence of control tissues, the findings should be interpreted as observational and hypothesis-generating, rather than inferential.

Comments 2: Alternatively, the authors should explicitly acknowledge the study's descriptive nature and revise the conclusions accordingly to avoid overinterpretation.

Response 2: The authors appreciate the comments of the reviewer. Thus, considering their observations about the conclusion, the authors modified the text as follows:
Lines 271-276
“In conclusion, this descriptive study reports the histopathological findings in a small series of post-mortem lung samples from patients with severe COVID-19, including changes compatible with different stages of diffuse alveolar damage and detectable immunoreactivity to SARS-CoV-2 S and N proteins, without indicating disease specificity or causal associations, which are consistent with two other studies previously published in Mexico”.
Instead of “In conclusion, our study is the third report in our country that demonstrated lung alterations in postmortem samples characterized by the exudative (early) phases and the proliferative or organizational (late) phase of DAD, associated with immunoreactivity to S and N proteins of SARS-CoV-2.”

Comments 3: Although Figure 7 serves as an appropriate technical negative control by omitting the primary antibody, a significant limitation of the manuscript is the absence of biological control groups.

Response 3: The authors thank the observation. The study is a retrospective and descriptive analysis based on the postmortem samples that were paraffin embedded. Especially the lung samples were donated to our laboratory to find or identify the SARS CoV-2. We did not have access to any types of postmortem samples, including a normal lung. Recently, we could access a normal lung, but not from the same Institution and not of the same time, so we consider that it is not an adequate control because of the conditions are not the same to be contemplate as the reviewer suggest “biological control groups”.

Comments 4: The study does not include lung tissue from COVID-19–negative individuals, non-COVID ARDS cases, other viral pneumonias, or non-infectious diffuse alveolar damage, which precludes proper assessment of the specificity of the observed immunoreactivity to COVID-19–related pathology. Immunohistochemical staining of lung tissue from non-COVID control patients would be necessary to support claims of disease specificity. If such control samples are unavailable, this limitation should be explicitly acknowledged and discussed in greater depth, and conclusions implying COVID-19 specificity should be avoided.

Response 4: The authors appreciate the reviewer’s comments. The authors would like to share with the reviewer some important points related to these considerations:

The study is a retrospective and descriptive analysis based on paraffin embedded postmortem lung samples from patients with COVID-19 confirmed. These samples were donated to our laboratory to find or identify the SARS CoV-2, which had not been carried out at that time. All the samples were collected by the authorized personal (Pathologist), under the Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas” (National Institute of Respiratory Diseases “Ismael Cosío Villegas” or INER) approval number B09-20, at Mexico City. All their legal guardians provided written informed consent in accordance with the Declaration of Helsinki for Human Research. Clinical samples were managed according to the Mexican Constitution law NOM-012-SSA3-2012, which establishes the criteria for the execution of clinical investigations in humans. It is important to mention that we did not have any access to patients and much less obtain lung samples from patients with other types of pathologies, this is restricted to the pathologist of INER.
During the period that the samples were collected, there were serious restrictions to take the samples from patients who died because of COVID-19, so we are not capacitated to do this job. Therefore, the pathologist responsible and his research group collaborated with us through the donation of the samples, and we made the rest of the analysis (H&E and SARSCoV-2 immune detection). The rules of the institution are very clear: Informed consents are part of the institutional documentation of INER and are subject to internal confidentiality policies, as well as current ethical and legal regulations. Therefore, any review or release cannot be carried out on an individual basis but must be processed and authorized by the corresponding institutional authorities.

Comments 5: Eosinophilic cytoplasmic and nuclear inclusion bodies are interpreted by the authors as cytopathic effects of SARS-CoV-2 infection. However, such inclusions are not specific to COVID-19 and may also be observed in other viral infections, degenerative processes, or conditions associated with severe cellular stress. Inclusion of control lung samples from patients with other viral pneumonias would be particularly important to assess the specificity of these findings.

Response 5: The authors appreciate the observations of the reviewer. The authors completely agree with the comments about the inclusion bodies. Certainly, the inclusion bodies are associated with viral infections but not exclusively, their presence could be associated with non-infectious pathologies, including lung diseases (for example Asthma, Eosinophilic Pneumonias, Acute Respiratory Distress Syndrome (ARDS), Organizing Pneumonia (COP/BOOP). However, as previously mentioned, the authors did not have the possibility to access to samples of healthy lung or lungs associated with other pathologies (infectious or noninfectious), this was a problem, but the results of the immunohistochemistry assay showed the presence of SARS CoV-2 immunorreactivity. Detached epithelial cells, some cytoplasmatic inclusion bodies, endothelial cells were positively stained with anti-Spike protein, mainly). With this data, the authors only “suggested” (line 247), that the inclusion bodies could be associated with the viral presence.

Comments 6: The presence of carbon inclusions in macrophages is highlighted as a novel observation and linked by the authors to environmental exposure, COPD, and increased COVID-19 severity; however, it is unclear whether detailed clinical data for these patients were available, particularly regarding smoking status, occupational exposure, or environmental risk factors, which would be essential to support such associations.

Response 6: The authors point out in the paragraph (lines 236-248), the different factors that may be involved in the development of COPD, and the relevance of COPD as a risk factor in severe COVID leading to death. According to the observation of the reviewer, the authors consider adding two more references: Clin. Med. 2023, 12, 3791. https://doi.org/10.3390/jcm12113791 (line 454-456, ref 20); Finnerty et al. BMC Pulmonary Medicine https://doi.org/10.1186/s12890-023-02761-5 (line457-458. ref 21).
Line 247: The authors change “without a doubt by could”
Line; 236-248

Minor comments:

Comments 7: Minor typographical issues were noted in the manuscript. In lines 160, 169, 184, 220, 229, and 236, the term “cloth/fibrin” appears to be a typographical error and should be corrected to “clot/fibrin.” In addition, in lines 201 and 225, “figure 5” and “figure 6” should be capitalized as “Figure 5” and “Figure 6,” respectively.

Response 7: All the mistakes have been corrected. The changes are in pink.

The authors thank all the observations and comments from the reviewer; The authors modified the text. All the changes appear in pink.
Materials and methods
Line 278-381: Developed methodology