[version 4; peer review: 2 approved, 3 not approved]
No competing interests were disclosed.
Dermatophytes are the keratinophilic fungi which infect humans and is the most recurring type of disease. The high level of transmissibility creates an epidemiological risk and emphasises the significance of these illnesses. However, a growing number of reports describing dermatophytes can cause deep infections in diabetic and immunocompromised patients, by invading deep layers like the dermis and hypodermis. Despite the prevalence and significance of dermatophytes in clinical mycology, it is not always possible to accurately diagnose this specific infection due to its overlapping structures among species of dermatophytes. Since it is difficult to identify species that exhibit weak characteristics in the morphological highlights, identification of the dermatophyte is often relied on its morphological analysis, which is a laborious process and demands skill. The massive shift in genetic variation, the source of infection, and epidemiological research can be discovered using molecular approaches. Therefore, the development of an accurate laboratory test for dermatophyte species identification is essential for the prevention and efficient management of dermatophytoses. One such methodology allows use of PCR technology which has many methods for molecular level characterization which is rapid, efficient, and capable of producing DNA polymorphisms specific to various dermatophyte species based on distinctive band patterns seen by agarose gel electrophoresis. The RAPD-PCR approach will be used in this study protocol to molecularly characterize the dermatophytes for precise speciation of the sample. In addition to improving knowledge of fungal biology and pathology with a focus on adaptive mechanisms to combat difficult conditions from host counteractions, there is a need to improve awareness of the importance of these diseases through accurate epidemiological data. The advantages of molecular approaches for characterizing objects over traditional methods are their sensitivity and specificity.
The previously published version described an initial study protocol while the investigation was ongoing and the proposed methodology was under validation; therefore, detailed procedural information was limited. In the present updated version (Version 3), the Materials and Methods section has been revised and expanded to include methodological details and clarified study processes based on protocol validation. These updates improve clarity of the protocol. The overall study objectives and design remain unchanged. As this manuscript represents a study protocol, the revisions reflect methodological refinement.
Dermatophytosis has emerged as a major public health problem in India over the last decade with the increased prevalence and transmission.
Molecular-based methods rely on identifying genotypic variations in pathogenic organisms.
Study participants: 100 clinically diagnosed cases of dermatophytes will be included in the study.
Samples will be collected from the patients visiting the Dermatology OPD, Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe), Wardha. Samples will be processed in the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha. Method of selection of participants: Patients having skin lesions will be included for taking the skin scrapings. Patients with hair infections positive for fungal infection will be included for hair plucking. Patients with nail infections will be considered for nail clippings.
All the samples will be collected on a sterile black paper and stored in sterile container with proper patients details for further processing and identification.
Clinically diagnosed cases of dermatophytes will be included in the study.
Samples will be collected directly from the patients and reports will be obtained from the microbiology laboratory. Data of all patients will be entered in
Sensitivity formula for calculating the sample size.
Alpha (α) 0.05
Estimated sensitivity (Sens)
Prevalence of disease (Prev)
Estimated error (d)
Minimum number of diseases needed: 73
Minimum total sample size needed: 93
Culture media Sabouraud Dextrose Agar (SDA) Culture media Sabouraud Chloramphenicol + Cycloheximide Agar Culture media Dermatophyte Agar Base Dermato Supplement Culture Media Corn meal agar Distilled Water Potassium Hydroxide Pellets Normal Saline 10% Glycerol Lactophenol Cotton Blue Stain Polyvinyl Alcohol Ethyl Alcohol Sodium Hypochlorite Solution dNTP mix, 40mM Hi-Temp PCR Master mix Primers ZymoResearch Fungal DNA isolation kit PCR Block Plates Pipette Pipette tips (10μl, 20μl, 100μl, 1000μl) Microscopic glass slides Coverslip Inoculating loop Teasing needle Petri dishes Forceps Glass Beakers Flasks Glass rod Bunsen Burner Gel electrophoresis chamber
Light Microscope Autoclave PCR Biosafety Cabinet Biological Oxygen Demand (BOD) Incubator Electronic Weighing Machine
The area of sample collection will be cleaned with 70% alcohol. For skin samples, the lesion will be scraped with a sterile scalpel. For nail samples, the nail clippings will be collected using a nail cutter in a sterile container For hair samples, the hair will be plucked from the shaft of the hair.
The skin scrapings will be dissolved in 10% KOH and nail clippings will be dissolved in 40% KOH for microscopic observation. Samples will be inoculated on Dermatophyte Test Medium (DTM) as well as Sabouraud’s dextrose agar (SDA+CC) containing chloramphenicol and cycloheximide and incubated in the BOD incubator (25°C) for observation of macroscopic features. Microscopic examination will be performed using Lactophenol cotton blue stain and the slide culture technique to observe the undisturbed morphology of the isolates.
DNA extraction of the growth isolates will be done using ZymoResearch fungal DNA isolation kit. Standardization of molecular assay will be done using the standard strains of Trichophyton (D15P127, CBS 118892, UCMS-IGIB-CI11), Microsporum (ATCC 36299), and laboratory-confirmed strains. The primer required for the species-specific identification of dermatophytes by real time PCR reaction will be synthesized by Beacon designer probe/primer designer software from GeneX India Biosciences Velachery, Chennai, Tamil Nadu. PCR assay mixture, reaction buffer, dNTPs, standardized primer set novel primer designed by targeting the Internal Transcribed region (ITS), DNA template, using these PCR reaction cycles will be carried out.
(39 cycles) Denaturation at 93° - 1 minute, Annealing step at 55° - 1 minute, Extension step at 72°- 1 minute Final-extension step at 72°- 7 min Final PCR products will be separated in 0.5X (Tris Borate-Ethylene diamine tetraacetic acid) Buffer and 1% Agarose and stained with the Ethidium-Bromide solution and then the image will be obtained. Interspecies and intraspecies patterns and polymorphism for strains will be studied as there is resistance among the dermatophytes species, thus species level identification is important.
All the results will be calculated using R version 4.3.2. Patients enrolled in the study sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) resulting from molecular methods used in comparison to the conventional method (gold standard).
Categorical variable will be summarized by the samples positive for fungal hyphae in KOH and culture on SDA (Sabouraud Dextrose Agar) and Dermatophyte Test Medium (DTM).
The percentage of agreement (overall, positive, and negative) between the two methods will be calculated using agreement analysis (primary and secondary endpoints), along with the Kappa coefficient, p-value, and 95% confidence interval.
Papers will be presented at relevant conferences and related studies will be published in indexed journals.
This research is ongoing. Using previously used primers, we are validating the protocol. The instruments, reagents, enzymes, and primers are set up. Clinical sample collection and processing of the sample by conventional methods and standardization of molecular methods for the identification of species is ongoing. We are focusing on the species-level identification for the accurate diagnosis of dermatophyte species.
This study has been granted by the ethics committee of Datta Meghe Institute of Higher Education and Research, Sawangi Meghe, Wardha with the approval number: DMIMS (DU)/IEC/2022/851, dated: 05/04/2022.
Written informed consent will be obtained from the study participants for participation in the study and publication of their data.
Fungal infections in humans are becoming more common, especially in immunocompromised people, which has made them a global public health concern. The course of the disease, which can range from mild cutaneous or subcutaneous infections to invasive, widespread, and potentially fatal infections, is determined by the immunological health of the host.
No data are associated with this article.
We would like to acknowledge the Department of Microbiology, JNMC and Central Research Laboratory (CRL) for their support. Also, we would acknowledge the tool ChatGPT for giving the outline for the rationale.
The protocol in the current form may be approved.
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
clinical dermatology, dermatomycology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Accurate identification of dermatophtosis species is an interesting topic. Currently, it is particularly important to detect Trichophyton indotineae, which frequently exhibits resistance to terbinafine. However, the rationale for this protocol and the proposed method are not suitable. For example, it states that rapid identification is desirable, but the use of a RAPD-PCR method, as proposed by the authors, will not allow for faster identification. It is also noted that the prevalence of the disease in the studied population will be 0.79, which is undoubtedly excessive. No reference method is proposed in this protocol, so the robustness of the results obtained cannot be assessed. Finally, RAPD-PCR methods are now abandoned, as many authors consider them non-reproducible. The use of MALDI-TOF mass spectrometry or targeted PCR followed by sequencing should be preferred.
Is the study design appropriate for the research question?
Partly
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Partly
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Medical Mycology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
Dear Editor in Chief,
There was no proof of that the authors have performed RAPD-PCR and other phenotypic tests to confirm the fungal isolates.
DTM is not mentioned in the Materials & Methods.
The data sets are not provided in the manuscript and it is hard to review this manuscript
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
No
Are sufficient details of the methods provided to allow replication by others?
No
Are the datasets clearly presented in a useable and accessible format?
No
Reviewer Expertise:
Zoonotic diseases (Bacterial, fungal), sexually transmitted infections, Diabetic foot infections, Antimicrobial resistance
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
Respected Sir/Madam,
Thank you for the comments on the study protocol. I want to answer the comments below in points.
Concern 1:
The present manuscript is submitted as a study protocol, not as a completed original research article. As clearly stated under the
DTM is listed under the reagents required section as
As this manuscript is a study protocol, no datasets are generated or analyzed at this stage. This is explicitly stated in the Data availability section, which notes that
I request to consider the protocol further for any review. Thank you
We thank the reviewer for this important observation. In the revised version of the manuscript (Version 3), we have explicitly clarified that species specific primers will be designed targeting ITS region and identification will be done by Real time PCR, and phenotypic identification procedures will be performed on all culture-positive isolates. The phenotypic confirmation will be done by macroscopic colony morphology and microscopic features on lactophenol cotton blue mount. Details regarding DNA extraction, primer usage, amplification conditions, and gel electrophoresis have now been clearly described in the Materials and Methods section. Representative workflow descriptions have also been added to support methodological verification.
The use of Dermatophyte Test Medium (DTM) as a screening medium for presumptive identification of dermatophytes has now been explicitly included in the Materials and Methods section, thereby ensuring completeness of the diagnostic workflow.
We appreciate the reviewer’s comment regarding data availability. As this manuscript is a
-Clearly stated the data collection framework and outcome variables
-Included a description of how datasets will be generated, stored, and analyzed
-Added a data availability statement, indicating that datasets will be made available upon study completion. This clarification ensures integrity of an ongoing study.
The current state of knowledge clearly indicates
Please explain how you conceive the results of the study to translate into clinical practice: how will the application of the proposed method help in identifying resistant cases (e.g., to terbinafine), modifying treatment, or monitoring the effectiveness of therapy? Do you plan to collect data on the course of treatment and clinical response to link them to the species result?
RAPD is a technique developed many years ago, burdened with well-documented problems with repeatability and limited ability to differentiate between closely related species. Currently, the standard in dermatophyte diagnostics is to base identification on sequences (at least ITS, often also additional genes) and/or methods such as specific qPCR or MALDI-TOF MS. Why are you choosing RAPD as your primary species identification tool in 2025, rather than methods based directly on sequencing or modern molecular tests? Can you clearly compare the anticipated cost, time and technical requirements of RAPD with ITS sequencing (± other loci) in your conditions?
To assign an isolate not only to a ‘complex’ but to a specific species, the current literature emphasises the need to analyse the ITS sequence and, often, additional markers. With this assumption, RAPD serves at best as a support technique for typing, rather than as a standalone species identification tool. How do you intend to assign isolates to specific species, especially within the
Anyone who has ever worked with RAPD in practice knows how difficult and time-consuming it is to establish stable PCR parameters (ionic conditions, annealing temperature, number of cycles) and electrophoresis conditions (agarose concentration, time, voltage) to obtain a reproducible and readable number of bands. Please describe how you planned the RAPD optimisation stage in detail: how many pilot rounds were conducted, how pattern stability was assessed, and what criteria must the conditions meet to be considered stable?
Please explain how you intend to ensure the reproducibility of RAPD profiles in practice: will each sample be tested at least twice (on different days or by other operators), or do you plan to formally assess pattern consistency (e.g. similarity coefficient, cluster analysis)? How will it be documented that the banding pattern for a given isolate is stable and not a random effect of a single run?
Comparing RAPD profiles between unknown isolates is methodologically difficult: there are no standardised reference databases, and assessing ‘similarity’ can be subjective. How do you intend to objectify the comparison of patterns: is the use of specialised software for gel image analysis and similarity coefficient calculation envisaged, or will the study be carried out exclusively ‘manually’ based on visual assessment? What similarity thresholds do you consider sufficient to conclude that two isolates belong to the same species?
Please specify the exact composition of the panel of reference strains used for RAPD standardisation: which species does it include, how many strains per species, and does sequencing confirm any T. indotineae isolates among them? What if the RAPD profile of the tested isolate is not similar to any of the standards in the panel – how will it be classified then?
Please clearly define what the ‘gold standard’ is in your study: is it only culture and classical morphological identification, or a combination of KOH and culture? If you consider only a situation where both KOH and culture are positive to be a ‘true case’, please explain why such a restrictive criterion was adopted and whether you are concerned about omitting clinically typical scenarios (KOH−/culture+ or KOH+/culture−).
What specifically will you do in the event of a discrepancy between morphological identification and RAPD results (e.g., morphologically
In the statistical section, you report the calculations of sensitivity, specificity, PPV, NPV, and the kappa coefficient. Do you plan to report 95% confidence intervals for these parameters and possibly use the McNemar test to compare pairs of methods on the same samples? How will you deal with ambiguous cases (contaminated culture, no growth, illegible RAPD profile) – will they be excluded from the analysis or assigned to specific categories, and how will this decision affect the sensitivity/specificity estimates?
Have you considered referring to existing standards and recommendations in the text (e.g. CLSI documents, ESCMID/ECMM recommendations, ISHAM comments on
Since the starting point is the problem of chronic, resistant infections, why does the protocol not provide for any form of drug susceptibility testing (MIC tests, even for selected isolates) or at least systematic archiving of isolates for later analysis of resistance genes? Does this not significantly limit the practical value of species identification, since it is not accompanied by information on potential resistance?
Please specify exactly what quality control elements will be used in the molecular part: will each RAPD series include a positive control, a negative control and an extraction control; will each sample be analysed in duplicate or triplicate; how will you document the absence of contamination and the stability of the profiles?
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
No
Are the datasets clearly presented in a useable and accessible format?
Partly
Reviewer Expertise:
mycology, veterinary mycology, micorbiology, resistance
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
I thank the authors for their response. My comments on the current version are as follows:
1. While I appreciate the deletion of the characterization of dermatophytosis in the study as 'chronic and recurring', the subtitle "an observational study" is neither here nor there. While the earlier description of the study design as 'case-control' was plainly incorrect, the correct description would have been "a cross-sectional study". It is to be noted that 'case control' as well as 'cross-sectional' studies are all observational ones.
2. The reference given for the calculation of sample size is of a review article published in 2008. Other than being outdated, it is not possible to decipher exactly which Indian study has been taken as the prevalence reference.
3. The rationale of the study still remains inadequately addressed. One of the two references cited as explanation of the current epidemic was published in 1995!!!!
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Dermatology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
Respected Sir/Madam,
Thank you for reviewing the protocol. I would like to answer the comments given below and request to kindly reconsider reviewing the protocol.
We agree with the reviewer that the subtitle
We acknowledge that the reference used for sample size calculation was outdated and insufficiently transparent. In the revised version, the sample size calculation is based on recent, original Indian epidemiological studies, and the exact source of the prevalence data is clearly cited to ensure methodological clarity and reproducibility.
3.
We agree that the study rationale requires strengthening. The 1995 reference was cited only to provide historical background on dermatophyte biology and diagnostics and was not intended to explain the current epidemic. However, we accept that this was inadequate. The Introduction is revised to explicitly address the contemporary Indian dermatophytosis epidemic, supported by recent Indian and regional studies describing the rising burden, changing species distribution, and associated diagnostic challenges. This revision will clearly contextualize the need for the present study within current epidemiological realities.
The respected authors responded clearly to the previously addressed comments and I don't have any further comments at this stage.
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
Yes
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Mycology, dermatophytosis, and mycological diagnostic techniques are my major research areas
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an interesting and relevant protocol, given that there is an epidemic of chronic and recurrent dermatophytosis that is currently ongoing in our country as well as in several other countries and regions in the world. However, the protocol suffers from some serious lapses:
1. The protocol is titled as 'Study of molecular characterization for diagnosis of chronic and recurrent dermatophytosis'. However, the sampling methods and selection criteria of the cases do not mention these conditions at all!
2. Study design is mentioned as 'case control'. However, there is no description of the control group. The description matches that of a cross-sectional study, which is what it should be.
3. In sample size estimation, there is no reference to the prevalence value that has been taken, thus making the whole exercise infructuous.
4. The basic rationale of the study has not been mentioned - the current epidemic of recalcitrant dermatophytosis is due to the emergence of a new species called
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Dermatology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Respected Madam,
Thank you for considering our manuscript and suggesting the following changes to be processed for further publication in F1000 research subject to a satisfactory revision as per your suggestions. We have made the changes in our manuscript. Please consider the following changes.
This is an interesting and relevant protocol, given that there is an epidemic of chronic and recurrent dermatophytosis that is currently ongoing in our country as well as in several other countries and regions in the world. However, the protocol suffers from some serious lapses:
1. The protocol is titled as 'Study of molecular characterization for diagnosis of chronic and recurrent dermatophytosis'. However, the sampling methods and selection criteria of the cases do not mention these conditions at all!
2. Study design is mentioned as 'case-control'. However, there is no description of the control group. The description matches that of a cross-sectional study, which is what it should be.
3. In sample size estimation, there is no reference to the prevalence value taken, thus making the whole exercise infructuous.
4. The basic rationale of the study has not been mentioned - the current epidemic of recalcitrant dermatophytosis is due to the emergence of a new species called
The current work describes a protocol for an observational study through collecting samples from dermatophytes-suffering patients followed by conventional and molecular diagnostic process, the obtained data will be very useful in improving and updating the epidemiological data in this field.
While the research focuses on an encouraging area of research and addresses an area of study with minimal existing research, it requires substantial editing of vocabulary in English especially in the following sections; abstract, introduction, and discussion.
- The title needs certain modifications to be more representable for the designed study. Therefore, I suggest "Diagnosis and molecular characterization of dermatophytosis: an observational study"
- The abstract, introduction, and methodological protocol sections are well designed, structured, and described.
- The discussion is missing certain necessary references to support the flow of the work and the researcher point of view in the following points;
- The suggested [Ref 2]
- The suggested [Ref 3]
- The suggested [Ref 1] with all respect of the cited study as Higashi
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
Yes
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Mycology, dermatophytosis, and mycological diagnostic techniques are my major research areas
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Respected sir,
Thank you for considering our manuscript and suggesting the following changes to be processed for further publication in F1000 research subject to a satisfactory revision as per your suggestions. We have made the changes in our manuscript as per the suggestions given. Please consider the following changes
The title needs certain modifications to be more representable for the designed study. Therefore, I suggest "Diagnosis and molecular characterization of dermatophytosis: an observational study"
Thank you for the suggestion, sir. I agree with the changes suggested and would like to change the title to "Diagnosis and Molecular Characterization of Dermatophytosis: An Observational Study"
- The abstract, introduction, and methodological protocol sections are well-designed, structured, and described.
- The discussion is missing certain necessary references to support the flow of the work and the researcher's point of view in the following points;
- The suggested [Ref 2]
- The suggested [Ref 3]
- The suggested [Ref 1] with all respect of the cited study as Higashi