PONE-D-24-24002Diagnostic value of urinary and serum IgG antibodies in evaluating drug treatment response in strongyloidiasis assessed by fecal examination and digital droplet PCRPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: No

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: Diagnostic value of urinary and serum IgG antibodies in evaluating drug treatment response in strongyloidiasis assessed by fecal examination and digital droplet PCR

A lot of data make it a bit difficult to follow.

I would like to see the results in the same order as in the M&M section with the same paragraph haedings.

Line 258 - 265 please provide the actual numbers for the ddPCR, APCT and composite standard as well likewise as in table 1 for FECT

Line 233 The IgG cutoff values (antibody units/ml) were 140.7 in urine and 174.4 in serum. Seems in contrast with the cut-off line in figures 3C and 3D just above 2

Comparing fig3C and fig3D, it seems to me that serum IgG follows a more logical decline in time as compared to urine IgG

Reviewer #2: This manuscript reports the results of study conducted in a cohort of participants with and without strongyloidiasis to compare the performance of an in-house ELISA developed (and previously published) that utilises Strongyloides ratti larval antigens to detect IgG in urine or serum. The authors have used, as a composite diagnostic reference test, a combination of microscopy + agar culture and/or ddPCR detection of parasite DNA in stools. The study population, residents of a region endemic for strongyloidiasis, with a previously reported prevalence of >20%, included sub-groups of ‘proven’ Strongyloides-infected, Strongyloides-uninfected but infected with other helminth/non-helminth parasites, and uninfected individuals. In addition to a direct comparison of the three diagnostic tests in baseline (pre-treatment) samples, a sub study compared decline in the Strongyloides-specific IgG levels in urine and serum at 5 pre-selected timepoints post treatment with a single dose of ivermectin.

The experimental design and study populations are credible, and the study appears to have been conducted in a planned and methodical manner. The key findings of the study are, first, a high prevalence of strongyloidiasis (48%) in the screened, selected population, which may be explained by the selection criteria used in enrolment of the study populations. Second, the sensitivity of the ELISA when applied to urine was high (91%), essentially equivalent or higher than that of the serum assay (88%), as was the accuracy (measured by AUC of ROC: ~0.66 for urine and ~0.73 for serum) when compared with the reference composite tests. Third, there was a moderate-strong correlation between urine and serum IgG positivity. Fourth, the results of the post-treatment follow up is in line with previous studies showing a decline in IgG to Strongyloides Ags by 3 months post treatment.

These findings are remarkable and portend a useful role for the urine assay in the diagnostic “toolbox” for strongyloidiasis, but these preliminary findings in a selected population will need confirmation in a less selected populations, including in populations with lower prevalence rates of Strongyloides infection and those with other helminth infections known to cross react with Strongyloides Ags (primarily other soil-transmitted helminths and tapeworms).

Specific comments

Methods

- Ethics: were the participants in the treatment-follow up cohorts blinded to the category to which they were assigned? Could this knowledge affect their exposure to re-infection (e.g. by changing behaviour that risks exposure)?

- The term ‘proven’ positive/negative is somewhat misleading – it presumably refers to the reference test positive individuals – samples that were positive on either /and culture + microscopy or PCR, i.e., parasite-detection tests. I would recommend using ‘reference positive’ or ‘Strongyloides positive by reference test’

- ‘Negativization’ is an awkward and grammatically unconventional term – consider a better term, maybe something like ‘Conversion to negative’ or ‘reversion to negative’.

- Statistical analysis: although most readers will assume that corrections for multiple comparisons are incorporated into the p-values shown, it should be stated specifically whether this type of correction was performed in the analyses

Results

- Table 1 and lines 250-256: the authors have listed the numbers of those infected with S. stercorales (Ss) and with other parasites. Do those that are indicated as having Ss infection have only Ss in their stool? How many had both Ss and other parasites?

- Since the denominator used to calculate the percentages for detection rates (Table 2) and sensitivity (Table 3) is the number positive by the reference test (i.e., ‘True positives’), to avoid confusing readers who are used to the conventional 2 x2 cross-tabulation format of gold-standard test and experimental test, this should be so stated in the text or legend to the two tables.

- Table 3: Please check the means for sensitivity for all the cells – e.g, the mean for urine (91.1) falls outside the CI95 (98.8-95)

- Figures 3 and 4: The legend should include a description of what was the definition of ‘positive’ and ‘negative’ for urine and serum. In Figure 3/4, the X-axis title could be better expressed as ‘Interval post-treatment with ivermectin’

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Reviewer #1: No

Reviewer #2: No

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Diagnostic value of urinary and serum IgG antibodies in evaluating drug treatment response in strongyloidiasis assessed by fecal examination and digital droplet PCR

PONE-D-24-24002R1

Dear Dr. Sithithaworn,

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

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David Joseph Diemert, M.D.

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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: PONE-D-24-24002 Diagnostic value of urinary and serum IgG antibodies in evaluating drug treatment response in strongyloidiasis assessed by fecal examination and digital droplet PCR

Well written and very elaborate work, three things that would have added value

- A figure showing the positive findings in either FECT, APCT and/or ddPCR

- Quantification with FECT is given, please also give quantification with ddPCR

- Figures for urine and serum IgG like S3fig but all time points in one figure can replace S3fig

Reviewer #2: The revisions to the manuscript addressed my questions adequately and I believe that the changes have improved it.

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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: No

Reviewer #2: No

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