Epidemiology of Onychomycosis in the United States Characterized Using Molecular Methods, 2015–2024

Round 1

Reviewer 1 Report

It's interesting in this work the data on NDM, as well as, the relationship between different fungi and specific nail disorders. Although not all laboratories perform PCR for identification, it is important to know the frequency of NDM and take it into account for the diagnosis of onychomycosis.

There are only a few suggestions.

-            Figure 1, Line 86. Indicates that images A and C correspond to subungual infection, change A and D.

-            Correct the title of the Y axis in figures 3-4, it does not correspond to what is indicated in the figure caption and the text of the manuscript.

-            Could the authors indicate the software they used to perform the statistics analysis of the data?

-            The authors mention that in the case of NDM, they used samples with abundant fungal structures in the histopathology. PCR is being validated to identify the fungi that cause onychomycosis.

• • What happens when the presence of the fungus is rare or less than the value set as rare?
  • The authors found a relationship in the positivity associated with their classification of the amount of fungus in the tissue. Would it be possible for the authors to explain something about this in their discussions or limitations?

Author Response

Major comments

It's interesting in this work the data on NDM, as well as, the relationship between different fungi and specific nail disorders. Although not all laboratories perform PCR for identification, it is important to know the frequency of NDM and take it into account for the diagnosis of onychomycosis.

Authors: Thank you for taking the time to review our work. We hope this work will help us gain a more comprehensive understanding of the pathogens involved in onychomycosis.

 

Detailed comments

There are only a few suggestions.

• Figure 1, Line 86. Indicates that images A and C correspond to subungual infection, change A and D.

Authors: Thank you for spotting this error. We have corrected the figure legend (line 88).

 

• Correct the title of the Y axis in figures 3-4, it does not correspond to what is indicated in the figure caption and the text of the manuscript.

Authors: Thank you for your comment. We have corrected the Y-axis of Figures 3-4.

 

• Could the authors indicate the software they used to perform the statistics analysis of the data?

Authors: Yes, the odds ratios and the 95% CI were calculated using Microsoft Excel (line 113); 2-sided p-values were calculated as described by Altman and Bland (PMID: 22803193).

 

• The authors mention that in the case of NDM, they used samples with abundant fungal structures in the histopathology. PCR is being validated to identify the fungi that cause onychomycosis.
  • What happens when the presence of the fungus is rare or less than the value set as rare?

Authors: Thank you your comment. The proprietary primer probe sets used in the multiplex qPCR assay reported in this study are highly sensitive and specific for the respective targets and validated by Sanger sequencing. The Ct cut-offs for each target was selected via ROC analysis and taken in consideration of the limit of blank (LoB) using healthy control individuals. Within this context, a “detected” call by PCR, defined as a Ct below the critical cut-off, would denote a pathogen load above that typically associated with commensal or environmental contaminants. However, based on the sensitivity of PCR and the increased likelihood of rare or minimal subungual growth of an NDM identified on histopathology (see Figure 1) representing an environmental contaminate, these cases were excluded from the data set. For the purposes of this analysis, only cases with histopathologically defined disease showing moderate-to-florid subungual growth of an NDM were included, samples showing rare-to-minimal subungual growth of an NDM were excluded (see lines 188-193).

If we do not consider fungal element quantities, most if not all the NDMs exhibited higher likelihoods of causing a subungual infection than dermatophytes likely due to the higher prevalence of commensals/contaminants in the dataset. The same is seen if we look at subungual patterned infections with rare-to-minimal quantities. However, in contrast, if we restrict the dataset to moderate-to-florid fungal element quantities, all NDMs exhibited a lower likelihood of causing a subungual infection reflecting a lower prevalence of commensals/contaminants; in this analysis (Figure 3), Aspergillus, Fusarium, Scopulariopsis and Neoscytalidium exhibiting the same or similar likelihood as dermatophytes are more likely to be representing ‘true infections’ with clinical significance as opposed to Acremonium, Alternaria and Curvularia.   

The same cannot be said for superficial or dystrophic patterned infections, since we directly observe fungal elements within the nail plate, which indicates keratinolytic activities from a dermatophytic pathogen irrespective of fungal element quantities (see Figure 1). When rare or minimal fungal elemental quantities are detected, this can partially be explained by sampling artifacts (i.e., the portion of the nail plate that was sampled represent an infection area with less fungal elements than the rest of the affected nail plate).

 

• The authors found a relationship in the positivity associated with their classification of the amount of fungus in the tissue. Would it be possible for the authors to explain something about this in their discussions or limitations?

Authors: We agree that this point should be expanded for clarity (lines 320-343).

Reviewer 2 Report

The MS entitle"Epidemiology of Onychomycosis in the United States Characterized using Molecular Methods, 2015-2024 provide valuable information regarding Epidemiology of onycomycosis in US.Below some modification/editions need to be performed to develop the quality of MS and increase the scientific value of this research.

 If this research had been conducted across multiple centers, it would be worthwhile to reflect that in the title.

It would be valuable to include relevant reports on the epidemiology of onychomycosis in the US and other countries to compare how the causative agents have changed over time.

 

Line 61-63 need to rewrite as this current aim don't elucidate the aim of study.

Line 68-69 is not the inclusion criteria, it's the techniques you used in this study to identify the fungi.

Methods: for Real-tie PCR did you design the primers to identify the fungi or just used from published data?if so, need to include the references.

Please notify the Name of Real-time Machine(Brand, country).

Histopathology grading in line 97-100 needs a relevant reference.

In the methods section, the authors should specify that all samples were previously confirmed to contain fungi through direct microscopic examination and/or positive culture.

Result:What was the reason for including only three demographic characteristics (sex, age range, and regions) in the results table? Consideration of other clinical presentations, such different types of nail infection, would also be valuable.

Categorise result in tree Table including Yeast , Dermatophyte and Non- Dermatophate , much easier to read the result. include P value for those variable that shown significant difference with clinical presentation.

Discussion:line 264 is vague and need to explain more to show this effect.

This section should take into account previous reports on the prevalence of yeast, dermatophyte, and non-dermatophyte species from the US, if such reports are available.

Since the results section does not address the immune status of the patients, it is not reasonable to discuss immune host factors in the discussion section.

In the last paragraph of the discussion, it should be clarified that in some developing countries, performing PCR to identify the causative agents of onychomycosis may not be feasible. In these cases, direct examination and culture remain the gold standard for diagnosis. 

Please refer to comments above.

Author Response

The MS entitle “Epidemiology of Onychomycosis in the United States Characterized using Molecular Methods, 2015-2024 provide valuable information regarding Epidemiology of onychomycosis in US. Below some modification/editions need to be performed to develop the quality of MS and increase the scientific value of this research.

• If this research had been conducted across multiple centers, it would be worthwhile to reflect that in the title.

Authors: Thank you for your comment. We consider our work to be a single-center study since we reviewed data from one molecular diagnostic lab (lines 66-67).

 

• It would be valuable to include relevant reports on the epidemiology of onychomycosis in the US and other countries to compare how the causative agents have changed over time.

Authors: Thank you for your valued suggestion. Recent studies on the epidemiology of onychomycosis are not directly comparable to our findings due to the differences in mycology testing. Even through a repeated isolation on fungal culture, spaced ≥1 week apart, is recommended to confirm NDM onychomycosis, this approach is not uniformly adopted across studies (PMID: 38606891). Analysis of any temporal trends is also complicated by differences in the patient population and sampling bias. We have referenced other recent epidemiological surveys for discussion (lines 245-253).

 

• Line 61-63 need to rewrite as this current aim don't elucidate the aim of study.

Authors: Thank you for the feedback. We have revised our aim for clarity (lines 61-64).

 

• Line 68-69 is not the inclusion criteria, it's the techniques you used in this study to identify the fungi.

Authors: Thank you for your comment. We wish to clarify that in this study, we reviewed records of nail specimens tested at a single U.S. laboratory. We included specimens only if they were tested by both multiplex real-time PCR and histopathologic examination as per physician’s order. In other words, samples tested by multiplex PCR only, histopathologic examination only, or fungal culture only, were excluded (lines 69-71).

 

• Methods: for real-time PCR did you design the primers to identify the fungi or just used from published data? If so, need to include the references.

Authors: Thank you for the comment. The multiplex qPCR primers/probes were internally developed at Bako Diagnostics and subjected to in silico analysis and wet testing to ensure appropriate specificity. The primers/probes were optimized by testing combinations of various concentrations and annealing temperatures. PCR amplification conditions were concurrently optimized and validated during these experiments. Additionally, the assay was validated against clinical samples by comparing PCR results with an established comparator method (PMID: 37236595). Accuracy for the qPCR assay utilized in this study was determined to be 88.5-100%. This assay validation is in concordance with both CLIA (Clinical Laboratory Improvement Amendments) and NYS DOH (New York State Department of Health) standards.

 

• Please notify the Name of Real-time Machine (Brand, country).

Authors: Thank you for the suggestion. The assay was developed using a Life Technologies Quantstudio^TM 6 Flex (USA) and associated software (lines 79-80). All developmental work was performed in a CLIA-certified molecular diagnostic laboratory (Bako Diagnostics, GA, USA).

 

• Histopathology grading in line 97-100 needs a relevant reference.

Authors: Thank you for your suggestion. We have added a reference (line 103).

 

• In the methods section, the authors should specify that all samples were previously confirmed to contain fungi through direct microscopic examination and/or positive culture.

Authors: Thank you for the comment. The specimens analyzed in the present study were not tested by direct microscopic examination and/or fungal culture. Mycology tests were conducted as per physician’s order, and we only included specimens that were tested by multiplex qPCR and histopathologic examination.

 

• Result: What was the reason for including only three demographic characteristics (sex, age range, and regions) in the results table? Consideration of other clinical presentations, such different types of nail infection, would also be valuable.

Authors: Thank you for the valued feedback. Unfortunately, our data collection is limited by what physicians submit to the diagnostic lab since we do not have access to patient electronic health records. The type of nail infection (subungual, superficial, dystrophic) was determined by a dermatopathologist which doesn’t necessarily reflect the clinical observation made by the treating physician. The association between different histopathologic findings and pathogen identification results are shown in Figures 3-5.

 

• Categorise result in three tables including Yeast, Dermatophyte and Non- Dermatophyte, much easier to read the result. include P value for those variables that shown significant difference with clinical presentation.

Authors: Thank you for your feedback. We can reaffirm that the results shown in Tables 1-3 have been categorized in Section 3.1. (sex differences), Section 3.2. (age differences) and Section 3.3 (regional differences). The p-values for individual OR (95% CI) have been provided in the text of the respective sections. In this study, due to large sample sizes, a statistical significance could be detected across multiple parameters. However, statistical significance does not necessarily indicate clinical significance (e.g., an OR of 0.9 [95% CI: 0.9, 0.9] may be statistically significant, but not clinically significant especially when comparing to an OR of 3.9 [95% CI: 3.7, 4.1]), hence we chose to highlight ORs ≤0.5 or ≥1.5 in Tables 1-3 as we judge these differences to be more clinically relevant.

 

• Discussion: line 264 is vague and need to explain more to show this effect.

Authors: Thank you for the comment. We agree that this statement is too speculative and have revised it (line 280-282).

 

• This section should take into account previous reports on the prevalence of yeast, dermatophyte, and non-dermatophyte species from the US, if such reports are available.

Authors: Thank you for your valued feedback. We have referenced two previous epidemiology studies conducted in North America (lines 242-244)

 

• Since the results section does not address the immune status of the patients, it is not reasonable to discuss immune host factors in the discussion section.

Authors: We agree that any discussions on host immune factors are not relevant to our findings. We have revised the discussion section accordingly (lines 283-285).

 

• In the last paragraph of the discussion, it should be clarified that in some developing countries, performing PCR to identify the causative agents of onychomycosis may not be feasible. In these cases, direct examination and culture remain the gold standard for diagnosis.

Authors: We are in full agreement that PCR testing for onychomycosis is difficult to access especially for developing countries. Even in Canada, PCR testing for onychomycosis is currently not available. We have added this point to the discussion (lines 316-319).