Performance of Loop-Mediated Isothermal Amplification (LAMP) Targeting the Nucleocapsid (N) Gene of SARS-CoV-2 for Rapid Diagnosis of COVID-19: Systematic Review and Meta-Analysis

Round 1

Reviewer 1 Report

See the document

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 comments:

• Elaborate a bit more on why LAMP is chosen as amplification method of interest. What are the main advantages and the drawbacks of LAMP (compared to RT-PCR).

Answear: We added an extra phrase R54, but we thing the other advantages are included throughout the text.

• My main point of feedback is that the article search and selection strategy is restricted to articles that are published until July 2021, while it already is May/June 2022. This is a big gap and I would suggest to broaden the search and including more recent publications (if they are suitable).

Answear: This is a pertinent observation. We did searched again for more recent articles, but we couldn't find any other recent publication relevant to this review. So, we decided to keep it with the articles found during the indicated period of time.

• Only scientific articles/publications are mentioned in this review. However, some commercial devices also make use of the LAMP technique for amplification (e.g. Lucira and Talis). How is there performance compared to these scientific publications?

Answear: Thank you for bringing this point of commercial devices. Since the application of commercial devices were not in the scope of this review, we decided not to include  and no articles related those were found during our initial search.

• Elaborate a bit more on the (analytical and clinical) sensitivity and the selectivity of LAMP compared to PCR. Is the sensitivity and selectivity sufficient?

Answear: We believe this is cover trhoughout the text. Sensitivity is important, however, clinical sensitivity may be more important, since detecting low levels or SARS-CoV-2 RNA may not correlate with symptoms or spread of the virus to others. What is the best clinical sensitivity for molecular tests still remains to be determined by concomitant viral culture to demonstrate its dissemination potential.

 

• How is the sensitivity and selectivity compared to other genes/targets? Is the N-gene indeed the best option?

Answer: Our initial search, when we were deciding on what target to use, indicated that the N region was the most stable region of the vital genome and, probably due to that, the majority of articles were on this gene as target for LAMP. To compare the N to other viral regions we would have to do a similar review using articles related to non-N target, which was not the scope of this review.  

 

Lay-out and textual:
• R15: Please rephrase this sentence.

Answear: We tried to improve the sentence

• R37: Golden standard

Answear:  Corrected! Thank you

• R44: This is a very long sentence, I would recommend to split it up.

Answear: Corrected. Thank you

• R105: The reference to the figure is ‘Fig. x’, while in R108 the reference is ‘Table x’. This is not consistent. Check the guidelines how to refer to figures and tables.
• Figure 1: The use of a spaces is not consistent. And the ‘spell check markings’ are not removed.

Answear: Thank you for this comment! We improved the Tables and Figures

• The resolution of both Figure 1 and Table 1 are bad. They seem to be a print screen.

Answear: We tried to improve the quality of the figures

• Table 1: There is space between Manzano and Roumani.

Answear: Corrected. Thank you!

• Table 1: Something is going wrong with the caption. Part is missing and underneath the table there is the word ‘caption’.

Answear: We noted that, it seems it was a problem during automatic manuscript format in the platform. We tried to correct that.

• Figure 2: This figure also has a very poor resolution.

Answear: Thank you! We tried to improve the figure quality

• R177: The word ‘use’ is missing in this sentence.

Answear: added! Thank you

• R186: Sample (the s is missing)

Answear: Added, Thank you!

Introduction:
• R49: What are ‘RNA-type nucleic acids’? Can it be changed to ‘RNA’?

Answear: Indeed! it was changed as suggested

• R49: ‘Several pathogens’ is claimed, but there is only 1 reference. Include more references to strengthen this claim.

Answear: Added another reference

Results:
• Table 1: What does ‘No’ mean underneath the ‘target gene’?

Answear: Yes, that was corrected

• Table 1: Add the confidence intervals for the sensitivity and specificity.

Answear: Added

• Elaborate a bit more on how to read a forest graph plot.

Answear: It was added as suggested

Discussion
• R127: What is meant with the ‘analytical sensitivity’? Usually this is expressed in terms of an limit of detection, e.g. copies/µL.

Answear: The authors of the selected articles did not mentioned copies/ul, only L.O.D

• R138: Elaborate a bit more on the statement that some articles claim a more sensitive than specific test.

Answear: Thank you! We tried to improve that

Conclusion
• The first paragraph is not a conclusion, but new information (which would better suit in the discussion section).

Answear: Changed to reflect that

Reviewer 2 Report

Date: May 23, 2022

Title: Performance of loop-mediated isothermal amplification (LAMP) targeting the nucleocapsid (N) gene of SARS-CoV-1 for rapid diagnosis of covid-19: Systemic review and meta-analysis

Authors:  Hoffmann et al.,

Journal: COVID

Critiques:

This purpose of this manuscript was to evaluate the specificity and sensitivity of “the loo-mediated isothermal amplification (LAMP)” methodology in diagnosis of COVID-19 cases through a systematic review and meta-analysis of the literature.

Every molecular biology technique has advantages and disadvantages. It is already known that LAMP is a relatively cost-effective and a rapid diagnostic technique compared to the “gold standard” RT-PCT technology. However, the main disadvantage of this technique is its relatively low sensitivity towards low copy number samples. Although this point was addressed in this manuscript, this reviewer recommends that it should be more clearly highlighted for this sake of the reliability of the data resulting from the use of this technique.

In addition, this method produces a long chain of DNA expanding every cycle of the DNA amplification process, which is not suitable for subcloning and thus for obtaining more definitive diagnostics. This point should also be re-emphasized in the manuscript.

Author Response

Comments to reviewer 2:

Every molecular biology technique has advantages and disadvantages. It is already known that LAMP is a relatively cost-effective and a rapid diagnostic technique compared to the “gold standard” RT-PCT technology. However, the main disadvantage of this technique is its relatively low sensitivity towards low copy number samples. Although this point was addressed in this manuscript, this reviewer recommends that it should be more clearly highlighted for this sake of the reliability of the data resulting from the use of this technique.

 

Answer: LAMP sensitivity, in general, seems to vary greatly, with some authors claiming it to be more sensitive than PCR, while others have found the opposite results. This is probably the result of different sample volume, extraction versus non extraction prior to use in the reaction and detection methods. The big question, regarding SARS-CoV-2 detection, is how this is going to affect patient care, sice low levels of viral RNA may simply indicate a mild or cured status. Epidemiologically it may be relevant, since false-negative results are not accounted for. We agree with your comment, every molecular methodology has advantages and disadvantages, and understanding its individual limitations is a crucial point for final decisions, especially, in this case, the need of retesting negative samples with another methodology when patient conditions is consistent with the disease.

In addition, this method produces a long chain of DNA expanding every cycle of the DNA amplification process, which is not suitable for subcloning and thus for obtaining more definitive diagnostics. This point should also be re-emphasized in the manuscript.

Answer: Indeed, this  may be a disadvantage to LAMP, but usually, when positive samples are submitted to sequencing, it is the original sample that is (re)processed especifically for NGS, and it is not the PCR (or LAMP) products that is going to be used directly for sequencing.