Revisions for Diagnostics manuscript

[agitter]

Here are the first round reviews from the Diagnostics manuscript as an itemized list and the full text:

• Reviewer 1.1 --> Figure for Diagnostics Manuscript #1173
• Reviewer 1.2 (this is a long one, we could split it up)
• Reviewer 1.3
• Reviewer 1.4
• Reviewer 1.5
• Reviewer 1.6
• Reviewer 1.7
• Reviewer 1.8
• Reviewer 1.9
• Reviewer 1.10
• Reviewer 1.11
• Reviewer 2.1
• Reviewer 2.2
• Reviewer 2.3
• Reviewer 2.4
• Reviewer 2.5

Reviewer 1

1.1 The authors present an overview of molecular and serologic testing options and technologies for SARS-CoV-2. Overall, it is well written although additional editing could streamline the text, and a high quality figure to illustrate the different molecular assays and another to illustrate the different serologic testing strategies would certainly make things easier on the reader.

1.2 The authors acknowledge this fact in a couple of areas of the paper (e.g.: ""Generally, patient specimens tend to not contain culturable virus past day 5 of symptom onset [157,158]. However, due to their sensitivity to post-infectious viral RNA in specimens, PCR-based methods may mislead individuals to believe that they are still infectious several days after symptom onset [131]. Furthermore, detection of viral RNA can occur days and weeks after an active infection due to the sensitivity of PCR-based methods [18,159,160]." and "Positive PCR results have also been reported for extended periods of time from symptom onset and/or the first positive PCR test [21], meaning that in some cases, a positive PCR may not indicate that someone is contagious [1]."

Despite this, they make a number of misleading statements implying that a positive RT-PCR is equivalent to diagnosing an active infection. For example, "Molecular tests, which can diagnose an active infection by identifying the presence of SARS-CoV-2" AND "Tests that identify SARS-CoV-2 using molecular technologies will identify only individuals with current infections and are not appropriate for identifying individuals who have recovered from a previous infection." AND "and therefore the amount of virus present can be indirectly measured using the cycle
threshold (Ct) determined by qPCR." AND "A lower Ct corresponds to fewer qPCR cycles needed to reach a detectable level, indicating that higher amounts of virus were present in the initial reaction."

All these statements are inaccurate. RT-PCR does NOT detect virus, it detects viral RNA. Therefore, it also does not detect "active infection". The authors need to be consistent on this point throughout their paper and state, not just at the end, but at the beginning that only a plaque assay performed on cultured virus can determine the amount of virus in a patient.

Similarly, the authors state that "detect the presence of antibodies in blood plasma samples or other biological samples, providing insight into whether an individual has acquired immunity against SARS-CoV-2." A positive antibody test is NOT equivalent to knowing whether someone has acquired immunity to a virus for a whole host of reasons (e.g., you don't know anything about the quality of the antibodies, don't know anything about the T cell response, etc)

For the study cited by the authors in this statement: "This assay was tested on samples from two COVID-19 patients and a panel of positive and negative controls consisting of RNA extracted from several cultured viruses." -> wouldn't the positive controls be the 2 covid patients? what are the other positive controls?

1.3 "SARS-CoV-2 are the only sarbecoviruses currently known to infect humans, a positive test can be assumed to indicate that the patient is infected with SARS-CoV-2, although this test is not able to discriminate the genetics of viruses within the sarbecovirus clade. The fact that the
targets are so conserved offers the advantage of reduced concern about sensitivity in light of the evolution of SARS-CoV-2." -> Do the authors know whether these regions of the genome were conserved amongst all subsequent SARS-CoV-2 variants? given their later statement that "Additionally, the impact of viral evolution on RT-PCR sensitivity is a concern [26,27].'

1.4 In the ddPCR section, it is confusing to end one paragraph with "While dPCR equipment is not yet as common as that for qPCR, dPCR for DNA targets generally achieves higher sensitivity than other PCR technologies while maintaining high specificity, though sensitivity is slightly lower for RNA targets [30]." And then in the next paragraph state "Thus, this study suggests that ddPCR provides an extremely sensitive molecular test that is able to detect SARS-CoV-2 even at very low viral loads."

1.5 The sequencing section of the review is too short and vague. What types of sequencing are we talking about here? There is no mention of the importance of Sanger sequencing in the PCR section as a way to confirm that the PCR amplicon isn't a false positive. Presumably in this section the authors want to talk about next-generation sequencing approaches, like the ARTIC protocol for capturing emerging SARS-CoV-2 variants as well as hypothesis free metagenomic next-generation sequencing to capture both SARS-CoV-2 and other potential co-infections or alternate infection that might mimic COVID-19 (including host response data that are generated from these assays)

1.6 The authors note the "Pooled and Automated PCR Testing" approach - important to note that this is a cost-effective approach when the burden of infections is relatively low

1.7 A couple of helpful citations for real world, rigorous benchmarking of antigen testing vs RT-qPCR - PMID 33367619 and 33394052

1.8 An important serologic assay for low resource settings - PMID 33767397

1.9 Important to note that the importance of the specificity of an assay in determining the meaning of a positive result is dependent on the burden of the infection in the population. For example, if no one has been infected with SARS-CoV-2 in a population, then even an assay with a specificity of 99% will generate only false positives in that population.

1.10 The "Possible Alternatives to Current Diagnostic Practices" section is a bit out of place and incomplete and can probably be deleted

1.11 PMID 32855547 - important study early in the pandemic on the inconsistency of many serologic assays

Reviewer 2

This review by Rando et al. analyzes and discusses the different categories of tests that were developed for the detection of the SARS-CoV-2 virus or antibodies against the virus and their role in the COVID-19 pandemic.
Overall, this manuscript is well written and thorough. The message conveyed by this meticulous review is clear and important.

I only have the following comments:

2.1 Introduction, second to last paragraph: "Thus, serological tests might be useful to address population-level questions, such as the percent of cases that manifest as severe versus mild...". Could the authors please explain how serological tests can identify the percentage of cases that had severe versus mild illness? I would suggest to rephrase this sentence.

2.2 Molecular Tests to Identify SARS-CoV-2, first paragraph: "and they thus can be used to diagnose an active viral infection...". As stated later in the manuscript, positive PCR-based tests do not always mean active infection and viral replication ("detection of viral RNA can occur days and weeks after an active infection due to the sensitivity of PCR-based methods"). Therefore it might be better to substitute the term "active" with "current" or something similar.

2.3 Lateral Flow Immunoassay: This section has been virtually restricted only to a test from one specific company. Several other LFIAs are commercially available. I suggets to elaborate more on this section

2.4 In serologic tests I suggest to add a paragraph for their role post-vaccination and whether they can be useful in differentiating between infection, re-infection and vaccination.

2.5 Figure: Similar (and maybe more informative) figures have been published in (several) other manuscripts as well. Unfortunately, it is not novel and does not add any important information to the manuscript. I suggest the authors to generate a new figure, e.g. one that may summarize the content of the manuscript.

[agitter]

@rando2 @RLordan @cgreene we received reviews on the diagnostics manuscript almost a year ago. Because it gets harder to revise as time passes, should we decide what we'd like to do with this manuscript?

A few options come to mind:

• recruit new or existing collaborators to address the comments above and resubmit without changing the scope of the manuscript
• recruit new or existing collaborators to address the comments above and update the manuscript to account for major changes in the COVID-19 diagnostics landscape since April 2022
• decide to leave this as a preprint, optionally make any updates we feel like only in the Manubot version

Any other outcomes I'm missing?

[RLordan]

Hi @agitter, I'm on board to help. I think we can do option 2 without too much additional information needed. However, I am happy to go with general consensus. You are right though, it will get harder the longer we leave it.

[cbrueffer]

Thanks @agitter for the ping; I've been meaning to get back to this. Happy to contribute so we can get this out again! As to options you outlined, I agree with Ronan that 2 seems to most sensible; I think the scope is still fine, the content just needs updating.

[agitter]

@RLordan and @cbrueffer that's great to hear you are interested in helping and supportive of option 2. Looking at the reviewer comments above, do you think you have bandwidth and expertise to handle most of them? Otherwise I'd like to start rallying other new or existing contributors to help.

@rando2 I could help manage the general Manubot workflow and organize pull requests but don't feel well-qualified to review the scientific content here. If you are busy, we may be able to manage a lot of the revisions with me and 2-3 contributors who know the diagnostics material.

[cbrueffer]

Yes, I'll have bandwidth from late June onwards.

One of the reviewers mentioned adding more figures to highlight key aspects of the review; that would indeed be great and help is probably needed with that. Maybe @nilswellhausen is up this again if we agree on what exactly to do?

[nilswellhausen]

Hi everyone! Yes, I actually have the bandwidth to make two figures once we agree on what exactly we want to illustrate.

[RLordan]

Hi Gang, sure I can help out at weekends with this, also free more next week.

[rando2]

Hi everyone! I'm so sorry I haven't been seeing these, I got behind on my spam folder! I'm so excited to hear there is interest in getting these out. I am transitioning to a computer science role as of July 1, so biology will be taking a backseat in my life, but I would definitely be happy to help push things along to get these finished! Please tag me on any PRs you need reviewed, and I am more than happy to help with getting everything built and ready to ship out, since I've done that many times now!

[agitter]

I'm returning to this to see if we can make a plan to get started on the revisions. I'll have more time to help organize and review pull requests in August and a lot less once our fall semester begins.

Because @RLordan and @cbrueffer are our primarily volunteers, could the two of you please look at the numbered reviewer comments above and respond here with a list of the numbered comments you believe you could address? That will help us identify if there is a gap.

@nilswellhausen that is excellent you're willing to help with a figure. I'm not sure what we want to illustrate. If none of us have ideas, we could start by creating a new issue to brainstorm ideas and search for related figures for inspiration.

[RLordan]

I'm returning to this to see if we can make a plan to get started on the revisions. I'll have more time to help organize and review pull requests in August and a lot less once our fall semester begins.

Because @RLordan and @cbrueffer are our primarily volunteers, could the two of you please look at the numbered reviewer comments above and respond here with a list of the numbered comments you believe you could address? That will help us identify if there is a gap.

@nilswellhausen that is excellent you're willing to help with a figure. I'm not sure what we want to illustrate. If none of us have ideas, we could start by creating a new issue to brainstorm ideas and search for related figures for inspiration.

Will do this week, sorry for delay.

[cbrueffer]

Ditto, "slightly" behind schedule.