Persistent Detection and Infectious Potential of SARS-CoV-2 Virus in Clinical Specimens from COVID-19 Patients

Round 1

Reviewer 1 Report

The review topic is relevant. The paper synthesize very well the information regarding SARS-CoV-2 shedding and potential factors involved in viral persistence. Additionally, the results are very well discussed and interpreted by the author. The author describes the implications of viral shedding for disease control measures and provide readers with a useful overview of knowledge about SARS-CoV-2 shedding.

Author Response

Thank you for your thoughtful review of my paper. No recommended revisions were noted. 

Reviewer 2 Report

The manuscript "Prolonged Viral Shedding by Individuals Infected with the SARS-CoV-2 Virus" by Michael Zapor is a review describing some features of SARS-CoV-2, focusing on "shedding". My main concern is that the use of the term "shedding" is very misleading and confusing in this study. In this context "viral shedding" should refer to the step of releasing newly-formed virions outside the cell membrane, as hinted by the inclusion of the non-original Figure 1 from the Springer publication "Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)". However, the author the author seems to use the term "shedding" as a fully interchangeable synonym of "spreading" or "propagate" (see e.g. Paragraph 5, Paragraph 9 and Table 2), to indicate the period in which the patient is actively producing and releasing new viral particles. Obviously, viral shedding (in the molecular sense) and shedding of viruses by patients (in the clinical sense) are related by the same underlying mechanism, but, in order to transform to review to a sound scientific manuscript, I urge a full redefinition of the nomenclature and a review by a specialist in Clinical Virology. Also, a major concern is the inclusion of two clinical cases without indicated patient consent and without a clear connection to the rest of the manuscript, except as anecdotal elements. I would re-review this manuscript once it has been rewritten in order to avoid any potential confusion and with more clarity, since the topic of differential infectivity of SARS-CoV-2 (in different age ranges, population groups, and compared to other coronaviruses) is of interest.

Other points:

1) I would significantly restructure the abstract, which focuses extensively on the history of SARS-CoV-2 and very little on the actual focus of the paper, i.e. how the specific features of SARS-CoV-2 shedding are associated to its atypical properties.

2) The author enunciates many correct facts in the Introduction, however without proper referencing them. E.g. the first citation comes only at page 1, line 41, and specifically for an animal coronavirus case. The author should provide either a more systematic review on coronavirus or specific publications supporting claims such as "Coronaviruses are ubiquitous" and the general classification of human coronaviruses. The rest of the introduction (starting at page 2, line 52) is a good example of proper citations.

3) On line 63, "Viet Nam" (which should properly spelled "Vietnam") is referred to as one of the geographical areas where the virus was first reported, together with United States of America and Europe. It would be better to indicate the virus spreading by continents: North America, Europe and Southeast Asia, since Vietnam is not at all a peculiar country in the Eastern spread of the pandemic.

4) No introduction to the Clinical Vignettes is written. Case 1 and Case 2 appear to be reported by the author as individuals analyzed by the author, since no reference is provided. The author simply shifts from a general description of SARS-CoV-2 history and general distribution to reporting two clinical cases. What is the connection here? Are these reported as examples to what? Why these two in particular?

5) Reporting novel clinical information requires evaluation of consent of the patients: the author should provide the appropriate paperwork as requested by the journal.

6) What are the parameters of positivity of the PCR test performed on the two patients? I.e. what is the ct? Which primers were used in the Abbott RealTime Assay? What is the viral strain reported for the two patients?

7) It is unclear why the two clinical cases are reported, given the fact that they have no clear connection to viral shedding properties or to any clinical feature that would make SARS-CoV-2 appear as different from SARS-CoV in terms of etiology (which is one of the focuses of the paper, according to the abstract).

8) Figure 1 is a copy of Figure 3.3 from Springer book "Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)" by Kumar and colleagues, as indicated by the author. Being the focus of this paper on viral shedding, I would have expected the figure to be focusing on the discussed topic, and perhaps its associated systemic effects and propagation properties.

9) A comparison with other viruses is required (e.g. as a Table), indicating clinical parameters such as Median Duration in Respiratory samples, in Fecal Samples, Positivity in Serum (IgM and IgG) and any parameter associated to viral persistence. Otherwise, any claim about "atypical properties" of SARS-CoV-2 vs. SARS-CoV and other coronaviruses or RNA viruses should be removed from the text (which would negatively impact the relevance of the study).

Author Response

The manuscript "Prolonged Viral Shedding by Individuals Infected with the SARS-CoV-2 Virus" by Michael Zapor is a review describing some features of SARS-CoV-2, focusing on "shedding". My main concern is that the use of the term "shedding" is very misleading and confusing in this study. In this context "viral shedding" should refer to the step of releasing newly-formed virions outside the cell membrane, as hinted by the inclusion of the non-original Figure 1 from the Springer publication "Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)". However, the author the author seems to use the term "shedding" as a fully interchangeable synonym of "spreading" or "propagate" (see e.g. Paragraph 5, Paragraph 9 and Table 2), to indicate the period in which the patient is actively producing and releasing new viral particles. Obviously, viral shedding (in the molecular sense) and shedding of viruses by patients (in the clinical sense) are related by the same underlying mechanism, but, in order to transform to review to a sound scientific manuscript, I urge a full redefinition of the nomenclature and a review by a specialist in Clinical Virology. Also, a major concern is the inclusion of two clinical cases without indicated patient consent and without a clear connection to the rest of the manuscript, except as anecdotal elements. I would re-review this manuscript once it has been rewritten in order to avoid any potential confusion and with more clarity, since the topic of differential infectivity of SARS-CoV-2 (in different age ranges, population groups, and compared to other coronaviruses) is of interest.

Wherever practical, I have replaced the term “shedding” with a term such as “detection of viral RNA by polymerase chain reaction amplification”.

With respect to the clinical vignettes, I will point out that when I was invited to submit this review article, it was suggested that as a physician I include some case presentations to illustrate the phenomenon of persistent detection of virus in the clinical specimens of some of my COVID-19 patients. That was my purpose in including the two de-identified clinical vignettes. Nonetheless, I have replaced them with a brief discussion in the introduction of clinical, radiographical, and laboratory findings common to COVID-19 patients. The latter includes mention of persistent viral RNA in body fluids and feces.

Other points:

1) I would significantly restructure the abstract, which focuses extensively on the history of SARS-CoV-2 and very little on the actual focus of the paper, i.e. how the specific features of SARS-CoV-2 shedding are associated to its atypical properties.

I have restructured the abstract, as requested, to include less historical background and more focus on the main topic of the paper.

2) The author enunciates many correct facts in the Introduction, however without proper referencing them. E.g. the first citation comes only at page 1, line 41, and specifically for an animal coronavirus case. The author should provide either a more systematic review on coronavirus or specific publications supporting claims such as "Coronaviruses are ubiquitous" and the general classification of human coronaviruses. The rest of the introduction (starting at page 2, line 52) is a good example of proper citations.

I have cited additional references, as requested, in the opening parts of the introduction.

3) On line 63, "Viet Nam" (which should properly spelled "Vietnam") is referred to as one of the geographical areas where the virus was first reported, together with United States of America and Europe. It would be better to indicate the virus spreading by continents: North America, Europe and Southeast Asia, since Vietnam is not at all a peculiar country in the Eastern spread of the pandemic.

Although “Vietnam” is the spelling most commonly used in the U.S. nowadays, the country was historically referred to as Viet Nam, in keeping with the Vietnamese Việt Nam, and both are considered correct. Nonetheless, I have replaced the word with “Southeast Asia”, as requested.

4) No introduction to the Clinical Vignettes is written. Case 1 and Case 2 appear to be reported by the author as individuals analyzed by the author, since no reference is provided. The author simply shifts from a general description of SARS-CoV-2 history and general distribution to reporting two clinical cases. What is the connection here? Are these reported as examples to what? Why these two in particular?

As mentioned above, I have replaced the clinical vignettes with a brief discussion in the introduction of the clinical, radiographical, and laboratory findings common to COVID-19 patients. The latter includes mention of persistent viral RNA in body fluids and feces.

5) Reporting novel clinical information requires evaluation of consent of the patients: the author should provide the appropriate paperwork as requested by the journal.

As mentioned above, I have replaced the clinical vignettes with a brief discussion in the introduction of the clinical, radiographical, and laboratory findings common to COVID-19 patients. The latter includes mention of persistent viral RNA in body fluids and feces.

6) What are the parameters of positivity of the PCR test performed on the two patients? I.e. what is the ct? Which primers were used in the Abbott RealTime Assay? What is the viral strain reported for the two patients?

Because I have removed the clinical vignettes, clarification of the parameters of the assay used to diagnose their infection is no longer relevant.

7) It is unclear why the two clinical cases are reported, given the fact that they have no clear connection to viral shedding properties or to any clinical feature that would make SARS-CoV-2 appear as different from SARS-CoV in terms of etiology (which is one of the focuses of the paper, according to the abstract).

As mentioned above, I have replaced the clinical vignettes with a brief discussion in the introduction of the clinical, radiographical, and laboratory findings common to COVID-19 patients. The latter includes mention of persistent viral RNA in body fluids and feces.

8) Figure 1 is a copy of Figure 3.3 from Springer book "Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)" by Kumar and colleagues, as indicated by the author. Being the focus of this paper on viral shedding, I would have expected the figure to be focusing on the discussed topic, and perhaps its associated systemic effects and propagation properties.

As I was writing about SARS-CoV-2 entry and replication, it occurred to me that the process is complicated and, anticipating a suggestion from a reviewer to do so, I included a diagram. Because our hospital does not have a medical illustrator, I reached out to Dr. Saxena to reproduce his picture. I have previously forwarded his written permission to do so.

I do not know what other figure would be more appropriate, as the process of binding, entry, replication, and release is fairly similar regardless of the body fluid/material into which the virus is shed.

9) A comparison with other viruses is required (e.g. as a Table), indicating clinical parameters such as Median Duration in Respiratory samples, in Fecal Samples, Positivity in Serum (IgM and IgG) and any parameter associated to viral persistence. Otherwise, any claim about "atypical properties" of SARS-CoV-2 vs. SARS-CoV and other coronaviruses or RNA viruses should be removed from the text (which would negatively impact the relevance of the study).

I have removed comments about SARS-CoV-2 being unlike SARS-CoV or other coronaviruses.

Reviewer 3 Report

The topic of this review is extremely important. Understanding the duration of viral shedding of the SARS-CoV-2, as well as how it relates to a positive or negative PCR test, is crucial to the implementation of effective public health efforts aimed towards controlling the spread of this virus. The review is carefully prepared and to the best of my knowledge is not biased to any prejustices. My only concern is that Discussion section is too short. I would like to see it four times longer or, at least twice longer as it is. I propose to discuss "limited evidence that children actually pay a prominent role in propagating COVID-19". Especially interesting should be comparison between conclusions that were drawn in Hua CZ, et al. (2020) Epidemiological features and viral shedding in children with SARS-CoV-2 infection. J Med Virol., de Ioris et al. [66], Zhu et al. [70] and Posfay Barbe et al. [71]. I would propose to extend Discussion in the direction of "Persistent Shedding, Relapse, and Reinfection" as well.

Author Response

The topic of this review is extremely important. Understanding the duration of viral shedding of the SARS-CoV-2, as well as how it relates to a positive or negative PCR test, is crucial to the implementation of effective public health efforts aimed towards controlling the spread of this virus. The review is carefully prepared and to the best of my knowledge is not biased to any prejustices. My only concern is that Discussion section is too short. I would like to see it four times longer or, at least twice longer as it is. I propose to discuss "limited evidence that children actually pay a prominent role in propagating COVID-19". Especially interesting should be comparison between conclusions that were drawn in Hua CZ, et al. (2020) Epidemiological features and viral shedding in children with SARS-CoV-2 infection. J Med Virol., de Ioris et al. [66], Zhu et al. [70] and Posfay Barbe et al. [71]. I would propose to extend Discussion in the direction of "Persistent Shedding, Relapse, and Reinfection" as well.

Thank you for reviewing my paper. Per your request, I have fleshed out the Discussion section and it is now twice as long as the original submission.

Reviewer 4 Report

The article is well written, with comprehensive review of COVID-19 and SARS-CoV-2. A minor revision is recommended. Below are specific suggestions for consideration:

Lines 2-3: The title doesn’t reflect the contents which are much broader than prolonged viral shedding. I would suggest replacing the word prolonged to avoid mistaking it as prolonged COVID-19 diseases.

Line 48-49: Since China was the first country COVID-19 happened, the disease transmission history was unclear and somehow controversial. I would suggest providing solid references or removing the statement.

Line 65-66: suggest listing dates when COVID-19 cases reached milestones, such as 1, 5, 10, 15, 20 mil cases.

Line 67: the section can be placed before Discussion with more comparisons with what are described in the review.

Line 343: specify the date

Author Response

The article is well written, with comprehensive review of COVID-19 and SARS-CoV-2. A minor revision is recommended. Below are specific suggestions for consideration:

Lines 2-3: The title doesn’t reflect the contents which are much broader than prolonged viral shedding. I would suggest replacing the word prolonged to avoid mistaking it as prolonged COVID-19 diseases.

I have changed the title to reflect the focus of the paper on both the persistence of virus in clinical specimens as well as the infectious potential.

Line 48-49: Since China was the first country COVID-19 happened, the disease transmission history was unclear and somehow controversial. I would suggest providing solid references or removing the statement.

I have added a reference that describes the emergence and spread of the virus in Wuhan, China in December 2019.

Line 65-66: suggest listing dates when COVID-19 cases reached milestones, such as 1, 5, 10, 15, 20 mil cases.

I have added the milestones as requested.

Line 67: the section can be placed before Discussion with more comparisons with what are described in the review.

Because of comments made by another reviewer, I removed the clinical vignettes and replaced them with a brief synopsis of the distinctive clinical, radiographic, and laboratory findings associated with severe COVID-19 infection.

Line 343: specify the date

Done, as requested.

Round 2

Reviewer 2 Report

I believe the author, Michael Zapor, has put considerable effort in addressing my comments and in smoothing out the major defects of this manuscript. I think the current version of the manuscript has improved significantly from the first draft.

A doubt remains about Figure 1, which is an exact copy of a previously published figure. I believe it is not entirely fitting to the topic (detection and infectious potential of the virus), but it is describing the molecular events of viral entry and release. The combination of not-being-original and not-being-relevant is, I believe, problematic.

I put myself in the author's shoes, and specifically with his remark "I do not know what other figure would be more appropriate". In my opinion, it would be necessary to include a figure that describes the main topic of the paper, i.e. the current knowledge we have on average times of incubation, infectious potential, and detectability. Possibly with graphs, or infographics, highlighting differences observed across geographic areas, or genders, or age groups, or different studies (e.g. those reported in the main Tables). I do not believe that every figure in a scientific paper needs a professional scientific illustrator with 3D and shading effects: this is not a book chapter after all.

Author Response

I prefer to keep Figure 1, as I think it clarifies the complicated viral replicative cycle described in the text. Nonetheless, I added three additional figures. Two of these (Figures 2 and 4) depict the relative mean duration of clinical shedding in the adult (Figure 2) and pediatric studies (Figure 4) cited in the review. Moreover, I added a figure (Figure 3) that depicts the CDC's move from a test-based to a symptom-based approach for discontinuing transmission precautions. I think this figure is relevant because it reflects the CDC's gradual recognition that persistent clinical shedding of inert virus is common and poses problems for providers.