PONE-D-19-23307

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Jasper et al. describes the optimisation and validation of a LAMP assay for wAlbB and Aedes aegypti, using primers previously published. The assay was able to detect both targets in samples from laboratory and the field, even in some simulated extreme conditions. The present study brings new findings regarding the ability to detect Wolbachia but I have some comments that must be considered, as follow:

1) The number of mosquitoes tested in each of the extreme conditions (i.e. 30 d old vs 7d old; immediately or 24hs after bloodmeal) was really low, being only 6 (Lane 137). Why was it not tested in a larger number of samples? Those extreme conditions that may happen in the field are so hard to reproduce in the lab. I would suggest to test more mosquitoes.

2) In methods, for different storage conditions, the number of mosquitoes tested should be mentioned. (lane 137-142).

3) The description of figures 1A, 1B, S1A and S1B are confusing along the manuscript. The use of two different LAMP reactions (5 or 6 primers) should be better described in methods and results and discussion at “primer validation and characterization”. It is not clear if supplementary figures were used with 5 or 6 primers. This information should be added to the legend of each figure to make it easier to be interpreted.

4) Are there figures to confirm the findings presented in lanes 235-239 (the results when 1 infected mosquito was tested with 99 uninfected and also the absence of amplification over 30min of reaction)? If so, the figure should be mentioned or I would recommend to say the data was not shown.

Also, the use of a pool of 99 uninfected mosquitoes with 1 infected should be better addressed along the manuscript.

5) The data obtained when performing the qLAMP with pooled DNA was too variable within the groups. i.e. when you have 8 infected samples, some of the pools have similar concentration when you have 12 or 15 infected out of 20. The number of samples tested was very low (n= 3), so I believe this big variation might be due to it. I would suggest running with a bigger number of samples.

Would you consider using pools in large scale releases for Wolbachia monitoring? How would you discriminate if you have 8 or 15 mosquitoes infected in a pool? An accurate method to calculate Wolbachia frequency is essential to plan releases in order to reach a population 100% Wolb-infected. I would suggest to mention this limitation in the discussion.

6)In the discussion, the LAMP assay optimised in this study was considered low cost and fast (lane 332). What is the cost per sample if you run both genes by qPCR?

Reviewer #2: This study describes the validation of two previously published sets of LAMP primers, one specific to wAlbB (and wPip) Wolbachia wsp gene and the other specific for Aedes aegypti ITS gene. While the authors did not modify the published ITS primer set, they modified the published Wolbachia assay by not using the probe that was used in the original paper for accurate visual ‘yes/no’ assay readout. Instead the authors designed a second loop primer to bind to the loop region used by the probe in order to speed up the LAMP reaction. In very well thought out experiments, the authors then validated performance characteristics of the primer sets with mosquitoes that were reared and stored in the lab under different conditions designed to mimic scenarios that would likely be encountered with field collected mosquitoes. They also directly compared performance with qPCR using field collected mosquitoes. This work would be of value to the research community because such independent validation of LAMP assays reported in literature provides necessary confirmation and confidence that these assays would indeed be successful diagnostic tools for vector surveillance under a variety of application conditions and variables.

The paper is well written and documented, however a few questions should be addressed:

1. The authors state that their aim was to improve the previously reported Wolbachia assay and that they do so by removing the sequence-specific probe used in the original assay and replacing it with a second loop primer, which as expected increases the speed of the reaction by ~5-10 min. It is debatable that this change can be considered an improvement. While assay speed was modestly increased, readout mode was changed from a sequence-specific signal (hallmark of most gold standard diagnostic applications, for instance TaqMan qPCR) to a non-sequence-specific signaling method. Moreover, this change results in an overall increase in assay cost probably due to the use of proprietary LAMP master mixes and a precision Genie II machine that costs close to $18,000. The authors should refer to their alteration of the Wolbachia assay only as a modification and not as an improvement.

2. Although the authors mention that they are using previously published LAMP assays either directly (ITS) or with a slight modification of adding one loop primer (wsp), their statements later on in the manuscript, such as “The LAMP primer set we have developed” in line 244 and “The LAMP assay we have developed” in line 290 appear misleading. These statements should be modified appropriately to indicate that in the current work the authors have validated previously published assays and made modifications that can increase speed, albeit at the cost of eliminating sequence-specific visual readout.

3. In Figure 1 the authors should indicate how many samples were tested for each DNA concentration. It appears that although there is a general trend between decreasing template amount and increasing Tp, at many positions in the standard curves lower template concentrations are being amplified faster than higher template concentrations. The authors should address how this lack of exact correlation between template amount and Tp, which is quite common for continuous amplification methods such as LAMP, would affect the accuracy of any qLAMP-based quantitations.

4. One of the most interesting notions in the manuscript was the attempt to standardize determination of Wolbachia infection frequency in a population of mosquitoes by comparing concentrations of mosquito-specific marker (ITS) with that of the Wolbachia marker. However, as shown in Figure 2, there is considerable variation. This might partly be due to the inherent inaccuracy in qLAMP quantitation. It could also be, as the authors suggest, individual to individual variation. The authors should test more mosquitoes in each of their low, mid, and high infection groups to see if this variation can be reduced to achieve more accurate prediction.

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Reviewer #1: No

Reviewer #2: No

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A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

PONE-D-19-23307R1

Dear Dr. Jasper,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Luciano Andrade Moreira, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Daniela da Silva Goncalves

Reviewer #2: No