Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors of this manuscript have attempted the tricky goal of both detecting and serotyping Dengue virus (DENV) and additionally Chikungunya virus (CHIKV) in a single reaction. This is not necessarily novel. Indeed the authors highlight multiple manuscripts that have designed assays to achieve the same aim. Overall it is a nice manuscript. However, I do have some questions and there are areas in which I feel it can be improved.

1) the authors simply state that their sample set was positive for DENV or CHIKV; how was this infection status defined? A gold-standard real-time RT-PCR? This should be clarified in the materials and methods.

2) How many representative sequences were used for each serotype?

3) why did the authors feel it necessary to develop another DENV-3 assay for RT-qPCR?

4) The amplicons are very long for RT-PCR diagnostic targets. It would be useful for the authors to comment on this.

5) the authors refer to 1kb ladder. However, it is impossible to tell from this information what each band represents in the gels. Rather than have arrows pointing to the target amplicons, I feel that it would be much clearer to simply label the size of the marker bands.

6) The authors claim that this assay is an improvement on previously published assays. However, without testing the assays side-by-side using the identical samples I feel that this is an impossible claim, in which case it should be removed. Without these data the authors cannot claim that the assay is an improvement.

7) There is a lack of data demonstrating specificity for the target viruses. This is crucial when evaluation a test, particularly considering related viruses, e.g. Zika co-circulate.

8) The authors state 10-2 copies per ul. Could this please be clarified? The authors state the addition of 4 ul of RNA template, so is this in fact 4x10-2?

9) In my opinion Fig. 4 is meaningless. It might be useful if it is used to demonstrate differentiation between melt curves of the different viruses, but currently it served no purpose.

Overall, whilst the manuscript holds promise, I think these are major issues that need addressing.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments on manuscript cimb-2799665 "Development of Multiplex Molecular Assay for Simultaneous Detection of Dengue serotypes and Chikungunya virus for ar-bovirus surveillance"

The study aimed to develop a multiplex molecular test for simultaneous detection of dengue virus and chikungunya virus serotypes, using serum samples from India and Burkina Faso. The researchers designed specific primers for conserved regions of the viruses' genomes and validated the multiplex RT-PCR and RT-qPCR methods using characterized serum samples. The developed assay demonstrated high sensitivity and specificity, offering a promising tool for enhanced surveillance and diagnosis of dengue and chikungunya viruses in endemic regions.

I have 5 major comments for the authors NEED to address as follows:

1) The authors miss some current similar papers and need to discuss and compare the results between this study and similar papers:
-A Novel Multiplex RT-PCR for Simultaneous Detection of Malaria, Chikungunya and Dengue Infection (MCD-RT-PCR)

2) As the authors mentioned that there are several similar papers reporting same methods, but the authors did not clarify why this study is needed. Why do you just use previous methods?

3) The authors need detail the source of 130 human serum samples. It is better to show them in a flow chart. It is confusing because the sum of 16 + 16 + 32 is < 130.

4) Where were the experiments performed? How did the authors bring human samples from another country?
Did both research institutes approve the IRB?

5) The manuscript has a serious level of plagiarism, many sentences were copied from other papers. For example, this is a 100% similarity: The specificity of the assay was evaluated using 70 negative control samples, and the assay showed that none of them were detected by the assay, leading to a conclusion that the both conventional multiplex RT-PCR and SYBR Green RT-PCR assays were 100% specific.

Minor:
1-Do not capitalize in the middle of a sentence (Sequence Analysis)

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Firstly, congratulations to the authors for a rapid turnaround and addressing some of the original comments. However, I still find that there are issues with this manuscript.

In my original report I commented upon the labelling of marker bands rather than amplicon sizes; however despite the authors stating that this has been addressed it appears not to have been.

There is no description regarding how the authors generated synthetic RNA transcripts. How can the authors define copy number if they do not synthesise RNA transcripts of known concentration? Regardless of what has been undertaken in other publications, in my opinion the authors need to either confirm that they have used in vitro transcribed RNA to define copy numbers, or remove the concept of copy number from their LOD experiments.

Unfortunately, demonstrating specificity of primers against each other does not validate their specificity relative to other pathogens. Whilst P. falciparum is a good addition, other pathogens should also be tested. 

Lastly, I still don't see a convincing explanation as to why new molecular assays are required when others have been published.

It is noticeable that the English in the edited manuscript is of poorer quality than the original manuscript; perhaps it has been somewhat rushed.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors did not fully address two major comments:

Previous comment number:

2) As the authors mentioned that there are several similar papers reporting same methods, but the authors did not clarify why this study is needed. Why do you just use previous methods?
The authors need to provide more rationale in the introduction not discussion. Why you conduct this research instead of using previous published methods.

4) Where were the experiments performed? How did the authors bring human samples from another country?
You collected samples from 2 countries, then perform a Multiplex Molecular Assay for all samples. Did you ship the sample from Burkina Faso to India and conduct the assay in India. You need to clarify it in the methods.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Thankyou for the authors for addressing the comments. I still feel that a line or statement acknowledging that there is a need to test genetically and symptomatically similar viruses to evaluate specificity. 

Author Response

Reviewer 2

Comments and Suggestions for Authors:

Thank you for the authors for addressing the comments. I still feel that a line or statement acknowledging that there is a need to test genetically and symptomatically similar viruses to evaluate specificity. 

Response: We have now included a statement in the introduction in the revised manuscript in page 2, line number 77-82 and in the result in page 5, line number 221-227.