Comparison of serum vitamin D level and vitamin D receptor gene <i>FokI</i> polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study

Paulus Anthony Halim; Sondang P. Sirait; Eliza Miranda; Yulia Ariani; Wresti Indriatmi; Hok Bing Thio

doi:10.12688/f1000research.157869.3

Home Browse Comparison of serum vitamin D level and vitamin D receptor gene FokI...

ALL Metrics

Views

Downloads

Get PDF

Get XML

Export

▬

✚

Study Protocol

Revised

Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study

[version 3; peer review: 2 approved with reservations]

Paulus Anthony Halim¹, Sondang P. Sirait¹, Eliza Miranda¹, Yulia Ariani^2,3, Wresti Indriatmi¹, Hok Bing Thio⁴

Paulus Anthony Halim¹, Sondang P. Sirait¹, [...] Eliza Miranda¹, Yulia Ariani^2,3, Wresti Indriatmi¹, Hok Bing Thio⁴

PUBLISHED 23 Oct 2025

Author details Author details

¹ Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia
² Department of Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
³ Human Genetic Research Center, Indonesian Medical Education and Research Institute (IMERI), Universitas Indonesia, Jakarta, 10430, Indonesia
⁴ Department of Dermatology, Erasmus University Medical Center, Rotterdam, The Netherlands

Paulus Anthony Halim
Roles: Conceptualization, Funding Acquisition, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Sondang P. Sirait
Roles: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing – Review & Editing

Eliza Miranda
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Yulia Ariani
Roles: Conceptualization, Methodology, Supervision

Wresti Indriatmi
Roles: Methodology, Writing – Review & Editing

Hok Bing Thio
Roles: Funding Acquisition, Methodology, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Neglected Tropical Diseases collection.

Abstract

Background

Leprosy, or Hansen’s disease, is a chronic infectious disease that primarily affects the skin and peripheral nervous system, often leading to disabilities, including trophic ulcers (TU). The role of vitamin D and vitamin D receptor (VDR) gene polymorphisms in the development of TU among leprosy patients remain unclear.

Objective

This study aims to investigate the differences in serum 25-hydroxyvitamin D [25(OH)D] levels and the frequency of VDR gene FokI polymorphism between leprosy patients with and without TU, and to evaluate their association with the occurrence of TU.

Methods

This Institutional Review Board-approved, single-center, observational, analytic case-control study will enroll adult leprosy patients from the Dermatology and Venereology Clinic at Dr. Cipto Mangunkusumo Hospital in Jakarta, Indonesia. Participants will be divided into cases (patients with TU) and controls (patients without TU). Serum 25(OH)D levels will be measured using a chemiluminescence immunoassay, and FokI polymorphism detection is conducted through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. A total of 82 participants (41 cases and 41 controls) will be recruited. Statistical analysis will be performed to evaluate the differences and associations.

Results

Data collection commenced in March 2024 and was completed in September 2024, during the Indonesia’s dry season. Data analysis and reporting are expected to be finalized by January 2025.

Discussion

The findings of this study will enhance the current understanding of the role of the vitamin D axis in the development of TU among leprosy patients. This could lead to new therapeutic strategies, such as vitamin D supplementation or personalized medicine based on genetic profiling, potentially reducing the burden of TU among leprosy patients.

Keywords

trophic ulcer, leprosy, vitamin D, vitamin D receptor gene polymorphisms

Corresponding authors: Sondang P. Sirait, Eliza Miranda

Competing interests: No competing interests were disclosed.

Grant information: The study is funded by the Directorate of Research and Development, Universitas Indonesia under the Hibah PUTI 2023 scheme (Grant No. NKB-689/UN2.RST/HKP.05.00/2023).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 Halim PA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Halim PA, Sirait SP, Miranda E et al. Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study [version 3; peer review: 2 approved with reservations]. F1000Research 2025, 14:57 (https://doi.org/10.12688/f1000research.157869.3) First published: 09 Jan 2025, 14:57 (https://doi.org/10.12688/f1000research.157869.1) Latest published: 23 Oct 2025, 14:57 (https://doi.org/10.12688/f1000research.157869.3)

Revised Amendments from Version 2

In response to Reviewer 2's comments, we revised the manuscript to improve clarity and methodological transparency. We detailed how potential confounding variables such as neuropathy, deformities, nutritional status, and sun exposure are assessed and included in multivariate analysis. We clarified that participant recruitment was conducted during the Indonesian dry season (March to September 2024) to minimize variability in vitamin D levels. The rationale for using PCR-RFLP for genotyping was explained based on feasibility, simplicity of the FokI polymorphism, and cost-effectiveness. We acknowledged the potentially optimistic sample size for the FokI polymorphism analysis assumption as a limitation in the discussion. Ulcer documentation procedures were strengthened by clarifying that ulcer assessments were conducted by a senior dermatology resident under consultant dermatologist supervision using standardized PUSH criteria, supported by clinical photographs for verification. Ethical safeguards for image capture and storage were described. Additional minor revisions included stating the planned sample size in the abstract, adding gene-environment interaction to the objectives, outlining missing data handling strategies, and clarifying covariate selection criteria. A note was also added that a participant flow diagram will be included in the future publication of study results. We added a paragraph of study limitations on the Discussion section.

See the authors' detailed response to the review by Khairuddin Djawad
See the authors' detailed response to the review by Ravindra Turankar

Introduction

Background

Leprosy, also known as Hansen’s disease (HD), is a chronic infectious disease caused by the bacteria Mycobacterium leprae and Mycobacterium lepromatosis. This disease primarily affects the skin and peripheral nervous system, leading to disabilities. Leprosy remains endemic in many parts of the world, with Brazil, India, and Indonesia collectively accounting for 80% of new cases globally.¹ In 2019, the Indonesian Ministry of Health reported 17,439 new cases of HD, while in 2020, only 11,173 new cases were identified.^2,3 The COVID-19 pandemic has impacted early detection efforts due to social restrictions and the reallocation of healthcare resources, potentially increasing the incidence of disabilities resulting from delayed diagnosis and treatment.⁴

Peripheral nerve damage secondary to leprosy can lead to various types of disabilities. Ulcers are a common complication and are classified as grade 2 disabilities.⁵ The rate of grade 2 disabilities is a key indicator in the World Health Organization’s (WHO) 2021-2030 leprosy eradication strategy. In 2021, the rate of grade 2 disabilities in Indonesia was 2.48 cases per 1,000,000 people, whereas the WHO’s 2030 target is 0.12 cases per 1,000,000 people.² Disabilities associated with leprosy create social stigma and lead to significant economic losses, including healthcare costs and reduced productivity.⁶

Ulcers occur in 10–20% of HD patients and are mostly classified as trophic ulcers (TU).⁵ Most ulcers in HD patients occur in the lower extremities, particularly on the plantar surface of the feet.^5,7 Static and dynamic deformities of the feet, coupled with reduced sensitivity, lead to recurring wounds that do not heal and become chronic ulcers.^5,7 The occurrence of TU in leprosy patients can be influenced by several factors, including neuropathy, physical deformities, nutritional deficiencies, and other systemic comorbidities. In leprosy patients, ulcers are often difficult to treat and frequently recur. If not properly managed, ulcers can become secondarily infected and may even undergo malignant transformation.⁸

Vitamin D is a steroid hormone that plays a crucial role as an immune system modulator.^9,10 Over the past decade, studies have reported the role of vitamin D in wound healing.^9,11–13 Vitamin D enhances the differentiation of fibroblasts and keratinocytes, mainly through the modulation of growth factors and cytokine production.^14,15 In vivo, vitamin D is converted to its active form, 25-hydroxyvitamin D [25(OH)D], by the hepatic hydroxylase enzymes, which is an indicator of vitamin D status. In the innate immune system, 25(OH)D can trigger the production of antimicrobial peptides (AMPs) by monocytes and macrophages.¹⁰ Additionally, 25(OH)D reduces the production of proinflammatory cytokines and increases anti-inflammatory responses.¹⁶ The kidneys or injured tissues can convert 25(OH)D into 1,25-dihydroxy vitamin D3 or calcitriol, which regulates the expression of cathelicidin and defensins.^15,17 These AMPs can kill intracellular bacteria and play a significant role in wound healing.^10,15

The biological effects of vitamin D are mediated by the vitamin D receptor (VDR), which is encoded by the VDR gene on chromosome 12. Animal studies have reported that loss of VDR inhibits tissue re-epithelialization and inflammatory response in wound healing.^18,19 Various single nucleotide polymorphisms (SNPs) can affect the structure and function of VDR. The FokI polymorphism (rs2228570), which involves a nucleotide substitution from thymine (T) to cytosine (C) at the start codon of the VDR gene, is the only polymorphism known to structurally alter the VDR protein. The mutant T allele (f allele) yields a receptor with a longer amino acid chain compared to the wild-type C allele (F allele).²⁰ The F allele produces a VDR with more optimal gene transcription effects, thereby triggering increased immune responses and wound healing.^20,21 A study in Medan, Indonesia reported that the frequency of homozygous mutant subjects for the FokI polymorphism was 19.2% in multibacillary HD patients and 13.7% in healthy household contacts of HD patients.²²

Previous studies have shown that serum 25(OH)D levels in patients with other chronic ulcers (e.g., venous or diabetic ulcers) are decreased,^23,24 and vitamin D supplementation can improve wound healing in these conditions.^12,13 The role of vitamin D in leprosy is increasingly investigated, with previous studies reporting varied results.^25–27 However, to the best of our knowledge, no research has compared vitamin D levels and the polymorphisms of its receptor in leprosy patients with and without TU.

Aim

The aim of this study is to investigate whether vitamin D and the polymorphisms of its receptor (VDR) play a role in the development of TU in leprosy patients. The primary and secondary objectives of the study are displayed in Table 1. An exploratory objective is to assess potential interactions between serum vitamin D levels and FokI polymorphism in relation to TU development.

Table 1. Primary and secondary objectives.

Objective type

Description

Primary

• To analyze the differences of 25-hydroxyvitamin D [25(OH)D] levels in leprosy patients with and without trophic ulcers.
• To analyze the proportion difference of FokI polymorphism in leprosy patients with and without trophic ulcers.
• To evaluate whether 25(OH)D levels and FokI polymorphism are associated with the occurrence of trophic ulcers in leprosy patients.

Secondary

To determine whether there is any correlation between 25(OH)D levels and the severity of trophic ulcers.

Protocol

Study design

This study is a single-center, observational, analytic case-control study.

Site and population

Leprosy patients with and without TU who are attending the Dermatology and Venereology Clinic at Dr. Cipto Mangunkusumo Hospital in Jakarta, Indonesia will be recruited. Serum 25(OH)D levels will be analyzed at the Clinical Pathology Laboratory of the same hospital. Examination of the FokI polymorphism will be conducted at the Laboratory of the Medical Biology Department, Faculty of Medicine, Universitas Indonesia, Jakarta.

Case and control definitions

Both cases and controls include adult leprosy patients diagnosed according to the cardinal signs defined by WHO. Patients who are newly diagnosed, currently undergoing treatment, or have been released from treatment are eligible for inclusion. The inclusion and exclusion criteria are detailed in Table 2. Screening for excluded systemic conditions was conducted through structured clinical interviews and review of the patients’ medical records, including relevant laboratory and radiologic data when available. The control group will consist of leprosy patients without TU, who meet the same inclusion and exclusion criteria as the case group.

Table 2. Inclusion and exclusion criteria.

Criteria

Description

Inclusion

• Patients willing to participate in the study by completing and signing an informed consent form.
• Male or female patients aged ≥18 years old.
• Patients who meet the World Health Organization (WHO) diagnostic criteria for leprosy, including those who are newly diagnosed, currently undergoing treatment, or have been released from treatment.
• Leprosy patients with trophic ulcers (case group), adhering to the following criteria:
- ○ Ulcer duration of ≥4 weeks, with no history of sharp debridement or callus thinning during this period.
- ○ Wound depth not exceeding the subcutaneous layer.
- ○ Ulcer not secondarily infected or only mildly infected (characterized by erythema <2 cm around the ulcer, potential signs of edema, pain, minimal purulent secretions, or warmth on palpation).

Exclusion

• Patients who have consumed vitamin D supplementation within the past 4 weeks.
• Patients exhibiting signs and symptoms of leprosy reactions.
• Patients with concurrent systemic infections, diabetes mellitus, severe liver dysfunction, end-stage kidney disease, malignancy, and autoimmune diseases.

Subject recruitment

Subjects will be recruited consecutively based on the eligibility criteria. All leprosy patients attending the Dermatology and Venereology Clinic of Dr. Cipto Mangunkusumo Hospital will be given the opportunity to participate in this study and will be screened according to the inclusion and exclusion criteria. Participant recruitment is conducted during Indonesia’s dry season (March to September 2024). Before further examination, each potential subject will be informed about the purpose and methods of the study, as well as the benefits and potential disadvantages of participating. Written informed consent will be obtained from each subject. A participant flow diagram will be included during reporting of study results.

Procedures and examinations

History taking and physical examination will be conducted in a structured manner using a standardized study data collection form. Anamnesis will include identity, sociodemographic information (e.g., education level, occupation, and household income), leprosy history (duration, treatment, and reaction history), sun exposure duration, smoking status, and ulcer history. Sun exposure will be estimated based on the number of hours spent outdoors per week as reported by the subject.

Clinical data related to leprosy, including disease duration, WHO and Ridley-Jopling classification, treatment status, and history of reactions, will be obtained through patient interviews and verified from medical records where available. Physical examination will include vital signs, anthropometric measurements, dermatologic status of the ulcer, presence of non-ulcer deformities (e.g., claw hand, foot drop, amputation). Sensory function will be evaluated using Semmes-Weinstein monofilaments (0.2 g for hands, 2 g for feet) to clinically assess peripheral neuropathy. Nutritional status will be assessed using body mass index (BMI), and categorized based on the Asia-Pacific classification.²⁸

Clinical photographs of the subjects’ hands and feet are collected, clearly showing the ulcers if present. If ulcers occur on other areas of the extremities (e.g., knee or elbow), documentation will also be performed on these areas. Photos are taken using an iPhone 12 camera (12 megapixel, aperture size F1.6, focal length 26 mm, sensor size 1/2.55", pixel size 1.7 μm) from a distance of 15 cm. A blue cloth will be used as the background during photo taking.

The dimensions of the ulcer are measured using a digital caliper, with the ulcer area calculated by the multiplying the largest vertical and horizontal lengths that intersect perpendicularly. Ulcer depth was assessed based on clinical examination of the wound bed and further categorized using the Pressure Ulcer Scoring System (grades I to III). In cases where necrotic tissue or slough prevented adequate visualization, sharp debridement was performed as part of standard clinical care to facilitate accurate depth assessment. Only patients with ulcers confined to the subcutaneous layer were included, while those with wounds exposing deeper structures such as fascia, tendon, or bone were excluded.

Ulcer severity will be assessed using the Pressure Ulcer Scale for Healing (PUSH) criteria, which encompasses ulcer area, exudate amount, and wound tissue type ( Table 3). This scoring system is chosen for its validated and standardized criteria. All ulcer assessments are performed by the first author (PAH), who at time of the study is a third-year dermatology resident, and directly supervised by two board-certified consultant dermatologists (SPS and EM) to ensure consistency and reduce inter-observer variability. Clinical photographs of each ulcer will be reviewed for retrospective verification, if required. In patients with multiple ulcers, the largest ulcer by area will be selected for reporting and further statistical analysis.

Table 3. Pressure Ulcer Scale for Healing (PUSH).

Characteristics	Score
Length × Width (in cm²)	0 0	1 <0.3	2 0.3–0.6	3 0.7–1.0	4 1.1–2.0	5 2.1–3.0
		6 3.1–4.0	7 4.1–8.0	8 8.1–12.0	9 12.1–24.0	10 >24.0
Exudate amount	0 None	1 Light	2 Moderate	3 Heavy
Tissue type	0 Closed	1 Epithelial Tissue	2 Granulation Tissue	3 Slough	4 Necrotic tissue
						Total:

Laboratory prodecures

Blood samples totaling 6 mL will be collected from each subjects into plain (red-capped) and K₃ EDTA (purple-capped) tubes and temporarily stored in a cool box at 2–8°C. The blood samples will be sent to the laboratories within 2 hours of collection.

25(OH)D Level quantification

Blood collected in a plain tube will be analyzed for 25(OH)D levels using the Architect 25(OH)D vitamin kit (Abbott Diagnostics, Lake Forest, IL, USA) based on the chemiluminescence immunoassay method. Vitamin D status will be classified under four categories: severe deficiency (<10 ng/mL), deficiency (10-20 ng/mL), insufficiency (20-30 ng/mL), and sufficiency (>30 ng/mL).²⁹

FokI polymorphism detection

The anticoagulated blood will be used for FokI polymorphism detection, which consists of DNA extraction, subsequent amplification using polymerase chain reaction (PCR), and polymorphism detection using the restriction fragment length polymorphism (RFLP) method. DNA extraction from EDTA blood samples will be performed using the salting out method.³⁰ Extracted genetic materials will be stored on a -80°C freezer for subsequent analysis.

Amplification of the genetic materials are performed using PCR. The primers for the reaction are based on a previous study by Soroush et al.,²⁰ with a forward primer 5′-CACTGACTCTGGCTCTGACCGT-3′ and a reverse primer 5′-AACACCTTGCTTCTTCTCCCTCC-3′. Initial denaturation of the genetic material will be performed at 95°C for 5 minutes. Amplification will be conducted for 35 cycles with denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The final extension will be performed at 72°C for 7 minutes, resulting in a nucleic acid product of 250 base pairs (bp) in length.

The RFLP will be conducted using the FokI restriction enzyme (New England Biolabs, Ipwich, MA, USA). After incubation, the results will be visualized with 2% agarose gel electrophoresis. The genetic material fractions that will be detected are 192 and 58 bp in homozygous ff genotype samples, 250 bp, 192 bp, and 58 bp in heterozygous Ff genotype samples, and an intact 250 bp fragment in FF genotype samples.

Sample size estimates

The calculation of sample size is carried out using a type I error probability of 0.05 and type II error of 0.20, corresponding to a power of 80%.

To test the difference in mean 25(OH)D levels, sample size calculation for the difference between the mean values of two independent groups is performed using the following formula³¹:

n_{1} = n_{2} = 2 {[\frac{(z_{\propto} + z_{β}) s}{(x_{1} - x_{2})}]}^{2}

where n₁ = n₂ = minimum sample size for each group; z_α = z-score corresponding to the type I error probability, which is 1.96; z_β = z-score corresponding to the type II error probability (β = 0.20), which is 0.84); s = standard deviation; and (x₁ − x₂) = clinically significant mean difference. The clinically significant mean difference is determined to be 5 ng/mL. Since there has been no studies reporting the difference in 25(OH)D levels between leprosy groups with and without TU, the standard deviation value used is based on a previous study in general leprosy patients, which is 5.42 ng/mL.²⁷ Thus, the sample size needed to detect a difference in 25(OH)D levels in leprosy patients with and without TU is 19 per group.

To test the effect of FokI polymorphism on the occurrence of TU in leprosy patients, the sample size for an unmatched case-control study is calculated using the formula³¹:

n_{1} = n_{2} = \frac{{(z_{α} \sqrt{2 PQ} + z_{β} \sqrt{P_{1} Q_{1} + P_{2} Q_{2}})}^{2}}{{(P_{1} - P_{2})}^{2}}

where n₁ = n₂ = minimum sample size for each group; z_α = 1.96; z_β = 0.84; P₁ = estimated proportion of effect in the case group calculated using

P_{1} = \frac{OR \times P_{2}}{(1 - P_{2}) + (OR \times P_{2})}

; OR = estimated odds ratio; P₂ = proportion of effect in the control group; P = ½ (P₁ + P₂); and Q = 1 – P. The proportion of the homozygous ff genotype in leprosy patients based on a previous study by Lubis et al.²² is 19.2%. Assuming an estimated odds ratio (OR) of 3.9 as the effect size of FokI polymorphism on TU occurrence in leprosy patients, the formula corresponds to a sample size of 41 for each of the case and control groups. While this assumption may be optimistic, it was deemed acceptable given the exploratory nature of this study and the limited prior data available in this population. The final analysis will interpret findings in light of this assumption, acknowledging that smaller true effect sizes may reduce the statistical power.

Finally, to test the correlation between 25(OH)D levels and ulcer severity, sample size calculation for correlation is conducted using the following formula³¹:

n = {[\frac{(z_{\propto} + z_{β})}{0.5 ln (\frac{1 + r}{1 - r})}]}^{2} + 3

where n = minimum sample size for the group; z_α = 1.96; z_β = 0.84; and r = estimated correlation coefficient. A moderate correlation (r = 0.5–0.7) is estimated between ulcer severity score and serum 25(OH)D levels. Thus, setting the correlation coefficient at 0.5, the minimal sample size is 29 for this analysis. The overall sample size for this study is determined based on the largest minimum sample size required to test all outcomes, which is 41 for each group of leprosy with and without TU, totaling 82 subjects.

Data analysis

All data obtained from anamnesis, physical examinations, and laboratory tests are recorded in the research file. The collected data will be reviewed, coded, and then entered into a master table in Excel version 16 (Microsoft Corporation, Washington, USA). Data analysis will be conducted using STATA version 16.0 (StataCorp LLC, College Station, TX, USA) and the online software SNPStats (www.snpstats.net, Catalan Institute of Oncology, Spain).

Sociodemographic and clinical characteristics data will be processed descriptively and presented in tables to understand the distribution of data across groups. Clinical characteristics of TU will also be described and presented in a table. Data normality will be tested using the Kolmogorov-Smirnov test. Numerical data will be presented as mean and standard deviation (SD), or as median and interquartile range (IQR) if not normally distributed.

A significance level of p < 0.05 will be used throughout the study. The comparison of means between two groups will be conducted using the independent t-test for normally distributed data or the Mann-Whitney U test if the data are not normally distributed. Relationship between two categorical variables will be analyzed using the chi-square or Fisher’s exact test, as appropriate. Correlations between two numeric variables will be calculated using the Pearson’s correlation coefficient for normally distributed data or Spearman’s rank correlation coefficient if the data are not normally distributed.

The Hardy-Weinberg equilibrium will be assessed to determine if the observed genotype frequencies are consistent with those expected in a population for both case and control groups. Different genetic models (i.e. codominant, dominant. recessive, overdominant, and log-additive) for the FokI polymorphism will be evaluated. Both the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) will be used to compare these models. The genetic model yielding the lowest AIC and BIC values will be considered the best-fitting model. As only one genetic variant (FokI) is analyzed, correction for multiple testing is not required.

Finally, multivariate logistic regression analyses will be conducted to identify factors independently associated with the occurrence of TU in leprosy patients. The variables may include vitamin D level, FokI genotype, and relevant sociodemographic and clinical factors such as neuropathy, non-ulcer deformities, smoking, and leprosy classification. Variables with a p-value < 0.25 in bivariate analysis will be considered for inclusion in the multivariate model. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) will be reported for the variables included in the final model.

As this is a prospective study, missing data are expected to be minimal. Any missing values will be transparently reported. Where applicable, sensitivity analyses will be performed using either complete-case analysis or multiple imputation methods to assess the robustness of the findings.

Ethics and consent

This study protocol has received ethical clearance from the Health Research Ethics Committee –Faculty of Medicine Universitas Indonesia and Dr. Cipto Mangunkusumo Hospital (HREC FMUI-CMH), with the approval letter number KET-82/UN2.F1/ETIK/PPM.00.02/2024, dated January 15, 2024.

We will explain to potential subjects that participation is voluntary and does not affect the management provided at the hospital. Written informed consent will be obtained from each subject. Clinical photographs will be taken only following written consent, primarily focusing on distal extremities where most ulcers occur. Any identifiable features, such as tattoos or nevi, will be digitally blurred. All image files will be stored on a password-protected USB drive accessible only to study investigators.

Dissemination

The results of the study will be disseminated through peer-reviewed publications and will be made available in an open-access format. Any significant changes to the protocol will be communicated directly to HREC FMUI-CMH. We also plan to publish any modifications made to this protocol during publication of the study results.

Study status

Subject recruitment, assessment, and laboratory work for 25(OH)D level measurement and FokI polymorphism detection have been completed. Formal data analysis is currently ongoing.

Discussion

Summary

This study aims to investigate the association between serum vitamin D levels and FokI polymorphism of VDR in leprosy patients, comparing those with and without TU. Leprosy often leads to peripheral nerve damage, with TU as a common complication. We hypothesize that vitamin D and its receptor polymorphisms, particularly FokI, might influence ulcer development in these patients.

Our study is a single-center, case-control study involving adult leprosy patients with and without TU. Serum vitamin D levels are quantified using chemiluminescence immunoassay, while the FokI polymorphism of the VDR gene is detected through PCR-RFLP method. The primary objectives are to analyze differences in 25(OH)D levels and the proportion of FokI polymorphism between the two groups, and to evaluate their association with TU occurrence. Secondary objectives include examining the correlation between vitamin D level and the severity of TU.

In this study, we utilize the PCR-RFLP method to detect the FokI polymorphism. While newer techniques such as TaqMan assays and next generation sequencing offer higher throughput and potentially greater precision, PCR-RFLP remains a widely accepted method for analysing well-characterized SNP with known restriction sites. Furthermore, given that only one SNP (FokI) is being investigated in this study, the use of multiplex or sequencing platforms was not warranted. PCR-RFLP was selected based on considerations of cost-effectiveness, simplicity, and compatibility with the technical capacity of our local laboratory setting.

Perspective and clinical implications

Trophic ulcers may still occur in leprosy patients long after treatment completion. As the world approaches the leprosy post-elimination era, TU will continue to cause significant morbidity. Therefore, the study’s focus on the potential influence of vitamin D and its receptor polymorphisms on TU development in leprosy is of significant clinical importance.

If substantial evidence is found, this could pave the way for new treatment strategies, such as vitamin D supplementation or personalized therapies based on genetic profiles in leprosy patients. This could enhance patient outcomes, reduce the incidence of trophic ulcers, and consequently diminish the healthcare and social burdens associated with leprosy-related disabilities. Additionally, the findings could contribute to a better understanding of the role of the vitamin D axis in the pathophysiology of TU and other chronic ulcers in general.

Limitations

This study has several limitations. First, the single-center design may limit generalizability to broader populations. Second, the observational nature of a case–control study restricts the ability to infer causality. Third, although multivariable analyses will be conducted, the potential for residual confounding remains. Finally, the sample size was calculated based on an estimated odds ratio that may be optimistic due to limited prior data on the association between FokI polymorphism and trophic ulcer risk in leprosy. As such, the findings will be interpreted with caution and should be validated in larger, multi-center studies.

Data availability

Underlying data

No data is associated with this article.

Reporting guidelines

The final publication of the study results will adhere to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.

References

1. World Health Organization: Weekly epidemiological report (WER) 10 September 2021. Weekly Epidemiological Report. 2021; 96(36): 421–444.
2. Kementerian Kesehatan Republik Indonesia: Profil Kesehatan Indonesia Tahun 2020. Kementerian Kesehatan RI; 2021.
3. Kementerian Kesehatan Republik Indonesia: Profil Kesehatan Indonesia Tahun 2021. Kementerian Kesehatan RI; 2022.
4. Chen X, Liu HB, Shui TJ, et al.: Risk factors for physical disability in patients with leprosy disease in Yunnan, China: Evidence from a retrospective observational study. PLoS Negl. Trop. Dis. 2021; 15(11): e0009923. PubMed Abstract | Publisher Full Text | Free Full Text
5. Upputuri B, Srikantam A, Mamidi RS: Comorbidities associated with non-healing of plantar ulcers in leprosy patients. PLoS Negl. Trop. Dis. 2020; 14(6): e0008393–e0008312. PubMed Abstract | Publisher Full Text | Free Full Text
6. Xiong M, Li M, Zheng D, et al.: Evaluation of the economic burden of leprosy among migrant and resident patients in Guangdong Province, China. BMC Infect. Dis. 2017; 17(1): 760. PubMed Abstract | Publisher Full Text | Free Full Text
7. Barreto JG, Salgado CG: Clinic-epidemiological evaluation of ulcers in patients with leprosy sequelae and the effect of low level laser therapy on wound healing: A randomized clinical trial. BMC Infect. Dis. 2010; 10: 237. PubMed Abstract | Publisher Full Text | Free Full Text
8. Karthikeyan K, Thappa DM: Squamous cell carcinoma in plantar ulcers in leprosy: a study of 11 cases. Indian J. Lepr. 2003; 75(3): 219–224. PubMed Abstract
9. Bikle D, Christakos S: New aspects of vitamin D metabolism and action — addressing the skin as source and target. Nat. Rev. Endocrinol. 2020; 16(4): 234–252. PubMed Abstract | Publisher Full Text
10. Schwalfenberg GK: A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency. Mol. Nutr. Food Res. 2011; 55(1): 96–108. PubMed Abstract | Publisher Full Text
11. Razzaghi R, Pourbagheri H, Momen-Heravi M, et al.: The effects of vitamin D supplementation on wound healing and metabolic status in patients with diabetic foot ulcer: A randomized, double-blind, placebo-controlled trial. J. Diabetes Complicat. 2017; 31(4): 766–772. PubMed Abstract | Publisher Full Text
12. Burkiewicz CJCC, Guadagnin FA, Skare TL, et al.: Vitamin D and skin repair: a prospective, double-blind and placebo controlled study in the healing of leg ulcers. Rev. Col. Bras. Cir. 2012; 39(5): 401–407. PubMed Abstract | Publisher Full Text
13. Dai J, Yu M, Chen H, et al.: Association between serum 25-OH-vitamin D and diabetic foot ulcer in patients with type 2 diabetes. Front. Nutr. 2020; 7: 7. PubMed Abstract | Publisher Full Text | Free Full Text
14. Ding J, Kwan P, Ma Z, et al.: Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing. Burns. 2016; 42(6): 1277–1286. PubMed Abstract | Publisher Full Text
15. Oda Y, Tu CL, Menendez A, et al.: Vitamin D and calcium regulation of epidermal wound healing. J. Steroid Biochem. Mol. Biol. 2016; 164: 379–385. PubMed Abstract | Publisher Full Text | Free Full Text
16. Afarideh M, Ghanbari P, Noshad S, et al.: Raised serum 25-hydroxyvitamin D levels in patients with active diabetic foot ulcers. Br. J. Nutr. 2016; 115(11): 1938–1946. PubMed Abstract | Publisher Full Text
17. Segaert S: Vitamin D regulation of cathelicidin in the skin: Toward a renaissance of vitamin D in dermatology? J. Invest. Dermatol. 2008; 128(4): 773–775. PubMed Abstract | Publisher Full Text
18. Oda Y, Hu L, Nguyen T, et al.: Vitamin D receptor is required for proliferation, migration, and differentiation of epidermal stem cells and progeny during cutaneous wound repair. J. Invest. Dermatol. 2018; 138(11): 2423–2431. PubMed Abstract | Publisher Full Text | Free Full Text
19. Song L, Papaioannou G, Zhao H, et al.: The Vitamin D receptor regulates tissue resident macrophage response to injury. Endocrinology. 2016; 157(10): 4066–4075. PubMed Abstract | Publisher Full Text | Free Full Text
20. Soroush N, Radfar M, Hamidi AK, et al.: Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress. Gene. 2017; 599: 87–91. PubMed Abstract | Publisher Full Text
21. Usategui-Martín R, De Luis-Román DA, Fernández-Gómez JM, et al.: Vitamin D Receptor (VDR) gene polymorphisms modify the response to vitamin D supplementation: A systematic review and meta-analysis. Nutrients. 2022; 14(2). PubMed Abstract | Publisher Full Text | Free Full Text
22. Lubis RD, Roesyanto-Mahadi ID, Siregar Y, et al.: FokI Vitamin D receptor gene polymorphism in leprosy household contacts. Malay J. Med. Health Sci. 2021; 17: 113–116.
23. Priyanto MH, Legiawati L, Saldi SRF, et al.: Comparison of vitamin D levels in diabetes mellitus patients with and without diabetic foot ulcers: An analytical observational study in Jakarta, Indonesia. Int. Wound J. 2023; 20: 2028–2036. PubMed Abstract | Publisher Full Text | Free Full Text
24. Agius C, Micallef D, Brincat I, et al.: Plasma total ascorbic acid and serum 25-hydroxy-vitamin-D status in patients with venous leg ulcers: a case–control study. Int. J. Lower Extremities Wound. 2021; 23: 421–427. PubMed Abstract | Publisher Full Text
25. Luong KVQ, Nguyen LTH: Role of the vitamin D in leprosy. Am. J. Med. Sci. 2012; 343(6): 471–482. PubMed Abstract | Publisher Full Text
26. Dennison CL, de Oliveira LB , Fraga LA d O, et al.: Mycobacterium leprae–helminth co-infections and vitamin D deficiency as potential risk factors for leprosy: A case–control study in south-eastern Brazil. Int. J. Infect. Dis. 2021; 105: 261–266. PubMed Abstract | Publisher Full Text
27. Darus NIM, Lubis RD, Jusuf NK: Analysis of serum vitamin D level in leprosy patients. Bali Med. J. 2019; 8(3): 795–799. Publisher Full Text
28. Pan WH, Yeh WT: How to define obesity? Evidence-based multiple action points for public awareness, screening, and treatment: an extension of Asian-Pacific recommendations. Asia Pac. J. Clin. Nutr. 2008; 17(3): 370–374. PubMed Abstract
29. Avci E, Demir S, Aslan D, et al.: Assessment of Abbott Architect 25-OH vitamin D assay in different levels of vitamin D. J. Med. Biochem. 2020; 39(1): 100–107. PubMed Abstract | Publisher Full Text | Free Full Text
30. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16(3): 1215. PubMed Abstract | Publisher Full Text | Free Full Text
31. Madiyono B, Sastroasmoro S, Budiman I, et al.: Perkiraan besar sampel.Sastroasmoro S, Ismael S, editors. Dasar-Dasar Metodologi Penelitian Klinis. 5th ed.Sagung Seto; 2014; pp. 352–387.
32. Gardner SE, Frantz RA, Bergquist S, et al.: A prospective study of the pressure ulcer scale for healing (PUSH). J. Gerontol. 2005; 60(1): 93–97. Publisher Full Text

Comments on this article Comments (0)

Version 3

VERSION 3 PUBLISHED 09 Jan 2025

Author details Author details

Paulus Anthony Halim
Roles: Conceptualization, Funding Acquisition, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Sondang P. Sirait
Roles: Conceptualization, Funding Acquisition, Methodology, Supervision, Writing – Review & Editing

Eliza Miranda
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Yulia Ariani
Roles: Conceptualization, Methodology, Supervision

Wresti Indriatmi
Roles: Methodology, Writing – Review & Editing

Hok Bing Thio
Roles: Funding Acquisition, Methodology, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The study is funded by the Directorate of Research and Development, Universitas Indonesia under the Hibah PUTI 2023 scheme (Grant No. NKB-689/UN2.RST/HKP.05.00/2023).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (3)

version 3

Revised

Published: 23 Oct 2025, 14:57

https://doi.org/10.12688/f1000research.157869.3

version 2

Revised

Published: 05 Sep 2025, 14:57

https://doi.org/10.12688/f1000research.157869.2

version 1

Published: 09 Jan 2025, 14:57

https://doi.org/10.12688/f1000research.157869.1

© 2025 Halim PA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

SEE MORE DETAILS

CITE

how to cite this article

Halim PA, Sirait SP, Miranda E et al. Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study [version 3; peer review: 2 approved with reservations]. F1000Research 2025, 14:57 (https://doi.org/10.12688/f1000research.157869.3)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Version 2

VERSION 2

PUBLISHED 05 Sep 2025

Revised

Views

Reviewer Report 07 Oct 2025

Ravindra Turankar, Stanley Browne Research Laboratory, The Leprosy Mission Trust India, New Delhi, New Delhi, India

Approved with Reservations

https://doi.org/10.5256/f1000research.187546.r415296

COMMENTS ON THE STUDY PROTOCOL ENTITLED, "Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study".

This is a well-designed and relevant protocol that explores vitamin D status and the VDR FokI polymorphism in leprosy patients with and without trophic ulcers. The study addresses a significant gap with potential implications for clinical management and public health. I have a few points for clarification and improvement.
The following points are being made as follows:

Major Comments:

Confounders:
- The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.
Vitamin D and Seasonality:
- Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.
Laboratory Methods:
- While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.
Sample Size Assumptions:
- The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.
Ulcer Documentation:
- Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.
Ethical Safeguards:
- Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.

Minor Points

Include the total planned sample size in the abstract.
Add an exploratory objective concerning potential gene–environment interaction (vitamin D and VDR polymorphism).
Explain how missing data will be managed.
Incorporate a flow diagram of participant recruitment to enhance clarity.
Clearly specify which covariates will be adjusted for in the multivariate analysis.
Indicate if correction for multiple testing will be used for genetic model comparisons.
Clearly state limitations, such as the single-centre design, the inability of a case-control study to establish causality, and the potential for residual confounding.

These points aim to enhance the clarity, reliability, and ethical standards of the study protocol, ensuring it makes a substantial contribution to the field.

Are sufficient details of the methods provided to allow replication by others?
Partly – The methods are thorough overall, but some areas need more clarification to ensure full reproducibility. These include details on controlling confounders, inter-observer reliability in ulcer grading, the rationale for choosing PCR-RFLP over other genotyping techniques, and how seasonal variation in vitamin D was managed.
Are the datasets clearly presented in a useable and accessible format?
❌ Not applicable – This is a study protocol; no datasets are presented yet.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
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

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Dr. Ravindra P. Turankar is a researcher in leprosy and neglected tropical diseases, with expertise in Mycobacterium leprae transmission, host susceptibility, and environmental surveillance. His work spans molecular diagnostics, CRISPR-based tools, cytokine profiling, and One Health approaches. He emphasizes community engagement, WASH interventions, and AI-powered surveillance to advance early diagnosis, disability prevention, and climate-resilient strategies for leprosy elimination.

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.

CITE

Report a concern

Author Response 23 Oct 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

23 Oct 2025

Author Response
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major ... Continue reading
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major Comments
1. Confounders:
Reviewer comment: “The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.”
Response: We have expanded the “Procedures and examinations” section to provide detailed descriptions of how each of the relevant factors is assessed, including structured history taking, Semmes-Weinstein monofilament testing for neuropathy, physical examination for deformities, BMI classification for nutritional status, and sun exposure estimation. Footwear habits are not formally assessed, but are indirectly represented by socioeconomic status and presence of deformities. Given that this study is conducted in an urban setting, regular footwear use is generally expected among participants. These variables will also be incorporated into bivariate and multivariate analyses as described in the “Data analysis” section.

2. Vitamin D and Seasonality:
Reviewer comment: “Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.”
Response: We acknowledge the important influence of seasonality on vitamin D levels. To minimize this variability, participant recruitment was planned and completed during the Indonesian dry season (March to September 2024), which provides stable sun exposure. This has been explicitly stated in the Abstract and the “Subject recruitment” section.

3. Laboratory Methods (PCR-RFLP vs. Other Genotyping Methods):
Reviewer comment: “While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.”
Response: We agree with the reviewer that PCR-RFLP may offer lower precision than newer genotyping techniques. However, the FokI polymorphism is a well-characterized variant with a simple restriction site, and PCR-RFLP remains a well-accepted method in these settings. As we are analyzing only one SNP, the higher-throughput capacity of TaqMan or next-generation sequencing is not required for this study. Additionally, PCR-RFLP was chosen based on cost-effectiveness and existing laboratory capacity. This rationale has been included in the “Discussion” section.

4. Sample Size Assumptions:
Reviewer comment: “The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.”
Response: We acknowledge that the assumed odds ratio of 3.9 for FokI polymorphism may be optimistic, as it is based on preliminary data in a different leprosy population. The “Sample size” section now includes a statement acknowledging this assumption and its limitations. A note has also been added to the “Discussion” section to encourage cautious interpretation and future validation in larger studies.

5. Ulcer Documentation and Inter-Rater Reliability:
Reviewer comment: “Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.”
Response: All ulcer scoring is conducted by the first author (a senior dermatology resident), directly supervised by two consultant dermatologists to ensure consistency. The use of the validated PUSH tool, as well as ulcer photography for retrospective verification, has been clarified in the “Procedures and examinations” section.

6. Ethical Safeguards for Photographs:
Reviewer comment: “Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.”
Response: Thank you for highlighting this. Clinical photographs are taken only following written consent. Photographs primarily involve distal extremities where the ulcers are located, which limit the likelihood of revealing identifiable features. In cases where identifying marks such as tattoos or moles are visible, they will be digitally blurred. All images are stored in an encrypted, password-protected USB flash drive accessible only to the study investigators. These details have been added to the “Ethics and Consent” section.

Minor Points

Planned sample size added to Abstract.

Exploratory gene-environment interaction (vitamin D and FokI genotype) is now mentioned as an exploratory analysis in the Objectives section.

Missing data:
As this is a prospective study, we anticipate complete data collection. However, if missing data arise, complete-case analysis or multiple imputation will be used if necessary. This has been added to the “Data Analysis” section.

Flow diagram of participant recruitment will be developed during study conduct and included in the final results publication. This has now been mentioned in the manuscript.

Specify covariates for multivariate analysis:
All collected sociodemographic and clinical variables may be included in the multivariate model if their bivariate p-value is <0.25, as outlined in the “Data Analysis” section.

Correction for multiple testing in genetic model comparisons:
As only one SNP (FokI) is analyzed, correction for multiple testing is not deemed necessary, as now clarified in the “Data analysis” section

State limitations (single-centre, causality, residual confounding):
Limitations regarding the single-centre setting, observational design, potential residual confounding, and potentially optimistic effect size for FokI polymorphism analysis have been added to the Discussion section.
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major Comments
1. Confounders:
Reviewer comment: “The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.”
Response: We have expanded the “Procedures and examinations” section to provide detailed descriptions of how each of the relevant factors is assessed, including structured history taking, Semmes-Weinstein monofilament testing for neuropathy, physical examination for deformities, BMI classification for nutritional status, and sun exposure estimation. Footwear habits are not formally assessed, but are indirectly represented by socioeconomic status and presence of deformities. Given that this study is conducted in an urban setting, regular footwear use is generally expected among participants. These variables will also be incorporated into bivariate and multivariate analyses as described in the “Data analysis” section.

2. Vitamin D and Seasonality:
Reviewer comment: “Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.”
Response: We acknowledge the important influence of seasonality on vitamin D levels. To minimize this variability, participant recruitment was planned and completed during the Indonesian dry season (March to September 2024), which provides stable sun exposure. This has been explicitly stated in the Abstract and the “Subject recruitment” section.

3. Laboratory Methods (PCR-RFLP vs. Other Genotyping Methods):
Reviewer comment: “While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.”
Response: We agree with the reviewer that PCR-RFLP may offer lower precision than newer genotyping techniques. However, the FokI polymorphism is a well-characterized variant with a simple restriction site, and PCR-RFLP remains a well-accepted method in these settings. As we are analyzing only one SNP, the higher-throughput capacity of TaqMan or next-generation sequencing is not required for this study. Additionally, PCR-RFLP was chosen based on cost-effectiveness and existing laboratory capacity. This rationale has been included in the “Discussion” section.

4. Sample Size Assumptions:
Reviewer comment: “The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.”
Response: We acknowledge that the assumed odds ratio of 3.9 for FokI polymorphism may be optimistic, as it is based on preliminary data in a different leprosy population. The “Sample size” section now includes a statement acknowledging this assumption and its limitations. A note has also been added to the “Discussion” section to encourage cautious interpretation and future validation in larger studies.

5. Ulcer Documentation and Inter-Rater Reliability:
Reviewer comment: “Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.”
Response: All ulcer scoring is conducted by the first author (a senior dermatology resident), directly supervised by two consultant dermatologists to ensure consistency. The use of the validated PUSH tool, as well as ulcer photography for retrospective verification, has been clarified in the “Procedures and examinations” section.

6. Ethical Safeguards for Photographs:
Reviewer comment: “Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.”
Response: Thank you for highlighting this. Clinical photographs are taken only following written consent. Photographs primarily involve distal extremities where the ulcers are located, which limit the likelihood of revealing identifiable features. In cases where identifying marks such as tattoos or moles are visible, they will be digitally blurred. All images are stored in an encrypted, password-protected USB flash drive accessible only to the study investigators. These details have been added to the “Ethics and Consent” section.

Minor Points

Planned sample size added to Abstract.

Exploratory gene-environment interaction (vitamin D and FokI genotype) is now mentioned as an exploratory analysis in the Objectives section.

Missing data:
As this is a prospective study, we anticipate complete data collection. However, if missing data arise, complete-case analysis or multiple imputation will be used if necessary. This has been added to the “Data Analysis” section.

Flow diagram of participant recruitment will be developed during study conduct and included in the final results publication. This has now been mentioned in the manuscript.

Specify covariates for multivariate analysis:
All collected sociodemographic and clinical variables may be included in the multivariate model if their bivariate p-value is <0.25, as outlined in the “Data Analysis” section.

Correction for multiple testing in genetic model comparisons:
As only one SNP (FokI) is analyzed, correction for multiple testing is not deemed necessary, as now clarified in the “Data analysis” section

State limitations (single-centre, causality, residual confounding):
Limitations regarding the single-centre setting, observational design, potential residual confounding, and potentially optimistic effect size for FokI polymorphism analysis have been added to the Discussion section.
Competing Interests: None. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 23 Oct 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

23 Oct 2025

Author Response
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major ... Continue reading
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major Comments
1. Confounders:
Reviewer comment: “The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.”
Response: We have expanded the “Procedures and examinations” section to provide detailed descriptions of how each of the relevant factors is assessed, including structured history taking, Semmes-Weinstein monofilament testing for neuropathy, physical examination for deformities, BMI classification for nutritional status, and sun exposure estimation. Footwear habits are not formally assessed, but are indirectly represented by socioeconomic status and presence of deformities. Given that this study is conducted in an urban setting, regular footwear use is generally expected among participants. These variables will also be incorporated into bivariate and multivariate analyses as described in the “Data analysis” section.

2. Vitamin D and Seasonality:
Reviewer comment: “Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.”
Response: We acknowledge the important influence of seasonality on vitamin D levels. To minimize this variability, participant recruitment was planned and completed during the Indonesian dry season (March to September 2024), which provides stable sun exposure. This has been explicitly stated in the Abstract and the “Subject recruitment” section.

3. Laboratory Methods (PCR-RFLP vs. Other Genotyping Methods):
Reviewer comment: “While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.”
Response: We agree with the reviewer that PCR-RFLP may offer lower precision than newer genotyping techniques. However, the FokI polymorphism is a well-characterized variant with a simple restriction site, and PCR-RFLP remains a well-accepted method in these settings. As we are analyzing only one SNP, the higher-throughput capacity of TaqMan or next-generation sequencing is not required for this study. Additionally, PCR-RFLP was chosen based on cost-effectiveness and existing laboratory capacity. This rationale has been included in the “Discussion” section.

4. Sample Size Assumptions:
Reviewer comment: “The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.”
Response: We acknowledge that the assumed odds ratio of 3.9 for FokI polymorphism may be optimistic, as it is based on preliminary data in a different leprosy population. The “Sample size” section now includes a statement acknowledging this assumption and its limitations. A note has also been added to the “Discussion” section to encourage cautious interpretation and future validation in larger studies.

5. Ulcer Documentation and Inter-Rater Reliability:
Reviewer comment: “Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.”
Response: All ulcer scoring is conducted by the first author (a senior dermatology resident), directly supervised by two consultant dermatologists to ensure consistency. The use of the validated PUSH tool, as well as ulcer photography for retrospective verification, has been clarified in the “Procedures and examinations” section.

6. Ethical Safeguards for Photographs:
Reviewer comment: “Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.”
Response: Thank you for highlighting this. Clinical photographs are taken only following written consent. Photographs primarily involve distal extremities where the ulcers are located, which limit the likelihood of revealing identifiable features. In cases where identifying marks such as tattoos or moles are visible, they will be digitally blurred. All images are stored in an encrypted, password-protected USB flash drive accessible only to the study investigators. These details have been added to the “Ethics and Consent” section.

Minor Points

Planned sample size added to Abstract.

Exploratory gene-environment interaction (vitamin D and FokI genotype) is now mentioned as an exploratory analysis in the Objectives section.

Missing data:
As this is a prospective study, we anticipate complete data collection. However, if missing data arise, complete-case analysis or multiple imputation will be used if necessary. This has been added to the “Data Analysis” section.

Flow diagram of participant recruitment will be developed during study conduct and included in the final results publication. This has now been mentioned in the manuscript.

Specify covariates for multivariate analysis:
All collected sociodemographic and clinical variables may be included in the multivariate model if their bivariate p-value is <0.25, as outlined in the “Data Analysis” section.

Correction for multiple testing in genetic model comparisons:
As only one SNP (FokI) is analyzed, correction for multiple testing is not deemed necessary, as now clarified in the “Data analysis” section

State limitations (single-centre, causality, residual confounding):
Limitations regarding the single-centre setting, observational design, potential residual confounding, and potentially optimistic effect size for FokI polymorphism analysis have been added to the Discussion section.
We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major Comments
1. Confounders:
Reviewer comment: “The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.”
Response: We have expanded the “Procedures and examinations” section to provide detailed descriptions of how each of the relevant factors is assessed, including structured history taking, Semmes-Weinstein monofilament testing for neuropathy, physical examination for deformities, BMI classification for nutritional status, and sun exposure estimation. Footwear habits are not formally assessed, but are indirectly represented by socioeconomic status and presence of deformities. Given that this study is conducted in an urban setting, regular footwear use is generally expected among participants. These variables will also be incorporated into bivariate and multivariate analyses as described in the “Data analysis” section.

2. Vitamin D and Seasonality:
Reviewer comment: “Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.”
Response: We acknowledge the important influence of seasonality on vitamin D levels. To minimize this variability, participant recruitment was planned and completed during the Indonesian dry season (March to September 2024), which provides stable sun exposure. This has been explicitly stated in the Abstract and the “Subject recruitment” section.

3. Laboratory Methods (PCR-RFLP vs. Other Genotyping Methods):
Reviewer comment: “While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.”
Response: We agree with the reviewer that PCR-RFLP may offer lower precision than newer genotyping techniques. However, the FokI polymorphism is a well-characterized variant with a simple restriction site, and PCR-RFLP remains a well-accepted method in these settings. As we are analyzing only one SNP, the higher-throughput capacity of TaqMan or next-generation sequencing is not required for this study. Additionally, PCR-RFLP was chosen based on cost-effectiveness and existing laboratory capacity. This rationale has been included in the “Discussion” section.

4. Sample Size Assumptions:
Reviewer comment: “The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.”
Response: We acknowledge that the assumed odds ratio of 3.9 for FokI polymorphism may be optimistic, as it is based on preliminary data in a different leprosy population. The “Sample size” section now includes a statement acknowledging this assumption and its limitations. A note has also been added to the “Discussion” section to encourage cautious interpretation and future validation in larger studies.

5. Ulcer Documentation and Inter-Rater Reliability:
Reviewer comment: “Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.”
Response: All ulcer scoring is conducted by the first author (a senior dermatology resident), directly supervised by two consultant dermatologists to ensure consistency. The use of the validated PUSH tool, as well as ulcer photography for retrospective verification, has been clarified in the “Procedures and examinations” section.

6. Ethical Safeguards for Photographs:
Reviewer comment: “Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.”
Response: Thank you for highlighting this. Clinical photographs are taken only following written consent. Photographs primarily involve distal extremities where the ulcers are located, which limit the likelihood of revealing identifiable features. In cases where identifying marks such as tattoos or moles are visible, they will be digitally blurred. All images are stored in an encrypted, password-protected USB flash drive accessible only to the study investigators. These details have been added to the “Ethics and Consent” section.

Minor Points

Planned sample size added to Abstract.

Exploratory gene-environment interaction (vitamin D and FokI genotype) is now mentioned as an exploratory analysis in the Objectives section.

Missing data:
As this is a prospective study, we anticipate complete data collection. However, if missing data arise, complete-case analysis or multiple imputation will be used if necessary. This has been added to the “Data Analysis” section.

Flow diagram of participant recruitment will be developed during study conduct and included in the final results publication. This has now been mentioned in the manuscript.

Specify covariates for multivariate analysis:
All collected sociodemographic and clinical variables may be included in the multivariate model if their bivariate p-value is <0.25, as outlined in the “Data Analysis” section.

Correction for multiple testing in genetic model comparisons:
As only one SNP (FokI) is analyzed, correction for multiple testing is not deemed necessary, as now clarified in the “Data analysis” section

State limitations (single-centre, causality, residual confounding):
Limitations regarding the single-centre setting, observational design, potential residual confounding, and potentially optimistic effect size for FokI polymorphism analysis have been added to the Discussion section.
Competing Interests: None. Close
Report a concern

Version 1

VERSION 1

PUBLISHED 09 Jan 2025

Views

Reviewer Report 29 Jul 2025

Khairuddin Djawad, dermatovenereologi, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia; Wahidin Sudirohusodo General Hospital, Makassar, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.173385.r392598

This research has novel value regarding the polymorphism of the fokI gene in ulcers and non-ulcers in leprosy sufferers, but it is necessary to exclude possible confounding factors such as the possibility of other systemic diseases accompanying leprosy such as osteophorosis, cardiovascular disease, other chronic infectious diseases, etc. Information is also needed on what tests you use to exclude the disease. This examination needs to be disclosed so that this research can be repeated by other researchers. How do you ensure that the wound does not pass through the subcutis? What tests do you use? However, what needs to be taken into account is that wounds in leprosy patients are closely related to factors such as the severity of the leprosy, the patient's environment and the patient's lifestyle which have not been taken into account in this research protocol.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

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?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: dermatologyst

CITE

Report a concern

Author Response 11 Sep 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

11 Sep 2025

Author Response

We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
... Continue reading We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
Thank you for your thoughtful comment. We agree that addressing potential confounding factors is critical to ensure internal validity. In designing the exclusion criteria (Table 2), we focused on systemic conditions that are known to significantly alter vitamin D metabolism or immune function, including diabetes mellitus, end-stage kidney disease, severe liver dysfunction, malignancy, autoimmune disorders, and systemic infections. To operationalize this, we screen participants using a combination of structured clinical interviews and medical record reviews, including relevant laboratory and radiologic findings when available. We have clarified these procedures in the revised manuscript (“Case and control definitions” section).

2. Regarding methods to ensure wound depth:
We appreciate this important point. As stated in the inclusion criteria, eligible wounds must not extend beyond the subcutaneous layer. Wound depth is primarily assessed through clinical inspection of the ulcer base. In cases where slough or necrotic tissue obscures the wound base, we would perform sharp debridement as part of standard clinical practice to allow proper visualization. Patients are eligible for inclusion if, following debridement, the wound bed is confined to the subcutaneous layer. Any wound showing evidence of deeper tissue involvement, such as fascia, tendon, or bone exposure, would be excluded. This has been clarified in the revised manuscript (“Procedures and examinations” section ) to support reproducibility.

3. Regarding other factors that might influence the presence of trophic ulceration:
We thank the reviewer for this important observation. We fully agree that trophic ulceration in leprosy is multifactorial. In this study, we collect detailed sociodemographic data (including occupation, education level, and household income) as well as clinical variables (nutritional status, smoking history, disease duration, WHO classification, Ridley-Jopling classification, history of leprosy reactions, presence of peripheral neuropathy, and other deformities). These variables serve as proxies for disease severity, environmental conditions, and lifestyle-related factors. All these factors are incorporated into bivariate and multivariate analyses to assess their potential as confounding factors. We have clarified this approach in both the “Procedures and Examinations” and “Data Analysis”.
We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
Thank you for your thoughtful comment. We agree that addressing potential confounding factors is critical to ensure internal validity. In designing the exclusion criteria (Table 2), we focused on systemic conditions that are known to significantly alter vitamin D metabolism or immune function, including diabetes mellitus, end-stage kidney disease, severe liver dysfunction, malignancy, autoimmune disorders, and systemic infections. To operationalize this, we screen participants using a combination of structured clinical interviews and medical record reviews, including relevant laboratory and radiologic findings when available. We have clarified these procedures in the revised manuscript (“Case and control definitions” section).

2. Regarding methods to ensure wound depth:
We appreciate this important point. As stated in the inclusion criteria, eligible wounds must not extend beyond the subcutaneous layer. Wound depth is primarily assessed through clinical inspection of the ulcer base. In cases where slough or necrotic tissue obscures the wound base, we would perform sharp debridement as part of standard clinical practice to allow proper visualization. Patients are eligible for inclusion if, following debridement, the wound bed is confined to the subcutaneous layer. Any wound showing evidence of deeper tissue involvement, such as fascia, tendon, or bone exposure, would be excluded. This has been clarified in the revised manuscript (“Procedures and examinations” section ) to support reproducibility.

3. Regarding other factors that might influence the presence of trophic ulceration:
We thank the reviewer for this important observation. We fully agree that trophic ulceration in leprosy is multifactorial. In this study, we collect detailed sociodemographic data (including occupation, education level, and household income) as well as clinical variables (nutritional status, smoking history, disease duration, WHO classification, Ridley-Jopling classification, history of leprosy reactions, presence of peripheral neuropathy, and other deformities). These variables serve as proxies for disease severity, environmental conditions, and lifestyle-related factors. All these factors are incorporated into bivariate and multivariate analyses to assess their potential as confounding factors. We have clarified this approach in both the “Procedures and Examinations” and “Data Analysis”.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 11 Sep 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

11 Sep 2025

Author Response

We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
... Continue reading We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
Thank you for your thoughtful comment. We agree that addressing potential confounding factors is critical to ensure internal validity. In designing the exclusion criteria (Table 2), we focused on systemic conditions that are known to significantly alter vitamin D metabolism or immune function, including diabetes mellitus, end-stage kidney disease, severe liver dysfunction, malignancy, autoimmune disorders, and systemic infections. To operationalize this, we screen participants using a combination of structured clinical interviews and medical record reviews, including relevant laboratory and radiologic findings when available. We have clarified these procedures in the revised manuscript (“Case and control definitions” section).

2. Regarding methods to ensure wound depth:
We appreciate this important point. As stated in the inclusion criteria, eligible wounds must not extend beyond the subcutaneous layer. Wound depth is primarily assessed through clinical inspection of the ulcer base. In cases where slough or necrotic tissue obscures the wound base, we would perform sharp debridement as part of standard clinical practice to allow proper visualization. Patients are eligible for inclusion if, following debridement, the wound bed is confined to the subcutaneous layer. Any wound showing evidence of deeper tissue involvement, such as fascia, tendon, or bone exposure, would be excluded. This has been clarified in the revised manuscript (“Procedures and examinations” section ) to support reproducibility.

3. Regarding other factors that might influence the presence of trophic ulceration:
We thank the reviewer for this important observation. We fully agree that trophic ulceration in leprosy is multifactorial. In this study, we collect detailed sociodemographic data (including occupation, education level, and household income) as well as clinical variables (nutritional status, smoking history, disease duration, WHO classification, Ridley-Jopling classification, history of leprosy reactions, presence of peripheral neuropathy, and other deformities). These variables serve as proxies for disease severity, environmental conditions, and lifestyle-related factors. All these factors are incorporated into bivariate and multivariate analyses to assess their potential as confounding factors. We have clarified this approach in both the “Procedures and Examinations” and “Data Analysis”.
We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
Thank you for your thoughtful comment. We agree that addressing potential confounding factors is critical to ensure internal validity. In designing the exclusion criteria (Table 2), we focused on systemic conditions that are known to significantly alter vitamin D metabolism or immune function, including diabetes mellitus, end-stage kidney disease, severe liver dysfunction, malignancy, autoimmune disorders, and systemic infections. To operationalize this, we screen participants using a combination of structured clinical interviews and medical record reviews, including relevant laboratory and radiologic findings when available. We have clarified these procedures in the revised manuscript (“Case and control definitions” section).

2. Regarding methods to ensure wound depth:
We appreciate this important point. As stated in the inclusion criteria, eligible wounds must not extend beyond the subcutaneous layer. Wound depth is primarily assessed through clinical inspection of the ulcer base. In cases where slough or necrotic tissue obscures the wound base, we would perform sharp debridement as part of standard clinical practice to allow proper visualization. Patients are eligible for inclusion if, following debridement, the wound bed is confined to the subcutaneous layer. Any wound showing evidence of deeper tissue involvement, such as fascia, tendon, or bone exposure, would be excluded. This has been clarified in the revised manuscript (“Procedures and examinations” section ) to support reproducibility.

3. Regarding other factors that might influence the presence of trophic ulceration:
We thank the reviewer for this important observation. We fully agree that trophic ulceration in leprosy is multifactorial. In this study, we collect detailed sociodemographic data (including occupation, education level, and household income) as well as clinical variables (nutritional status, smoking history, disease duration, WHO classification, Ridley-Jopling classification, history of leprosy reactions, presence of peripheral neuropathy, and other deformities). These variables serve as proxies for disease severity, environmental conditions, and lifestyle-related factors. All these factors are incorporated into bivariate and multivariate analyses to assess their potential as confounding factors. We have clarified this approach in both the “Procedures and Examinations” and “Data Analysis”.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (0)

Version 3

VERSION 3 PUBLISHED 09 Jan 2025

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2
Version 3 (revision) 23 Oct 25
Version 2 (revision) 05 Sep 25		read
Version 1 09 Jan 25	read

Khairuddin Djawad, Hasanuddin University (Ringgold ID: 64739), Makassar, Indonesia; Wahidin Sudirohusodo General Hospital, Makassar, Indonesia
Ravindra Turankar, The Leprosy Mission Trust India, New Delhi, India

Comments on this article

All Comments(0)

Add a comment

Browse by related subjects

Back to all reports

Reviewer Report

11 Views

07 Oct 2025 | for Version 2

Ravindra Turankar, Stanley Browne Research Laboratory, The Leprosy Mission Trust India, New Delhi, New Delhi, India

11 Views Cite this report Responses(1)

Approved With Reservations

Confounders:
- The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.
Vitamin D and Seasonality:
- Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.
Laboratory Methods:
- While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.
Sample Size Assumptions:
- The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.
Ulcer Documentation:
- Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.
Ethical Safeguards:
- Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.

Minor Points

Include the total planned sample size in the abstract.
Add an exploratory objective concerning potential gene–environment interaction (vitamin D and VDR polymorphism).
Explain how missing data will be managed.
Incorporate a flow diagram of participant recruitment to enhance clarity.
Clearly specify which covariates will be adjusted for in the multivariate analysis.
Indicate if correction for multiple testing will be used for genetic model comparisons.
Clearly state limitations, such as the single-centre design, the inability of a case-control study to establish causality, and the potential for residual confounding.

These points aim to enhance the clarity, reliability, and ethical standards of the study protocol, ensuring it makes a substantial contribution to the field.

Are sufficient details of the methods provided to allow replication by others?
Partly – The methods are thorough overall, but some areas need more clarification to ensure full reproducibility. These include details on controlling confounders, inter-observer reliability in ulcer grading, the rationale for choosing PCR-RFLP over other genotyping techniques, and how seasonal variation in vitamin D was managed.
Are the datasets clearly presented in a useable and accessible format?
❌ Not applicable – This is a study protocol; no datasets are presented yet.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
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

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Dr. Ravindra P. Turankar is a researcher in leprosy and neglected tropical diseases, with expertise in Mycobacterium leprae transmission, host susceptibility, and environmental surveillance. His work spans molecular diagnostics, CRISPR-based tools, cytokine profiling, and One Health approaches. He emphasizes community engagement, WASH interventions, and AI-powered surveillance to advance early diagnosis, disability prevention, and climate-resilient strategies for leprosy elimination.

Respond to this report

Responses (1)

Author Response

23 Oct 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

We thank the reviewer for the thoughtful and encouraging comments regarding the significance and methodological design of our study. Below, we respond to each of the points raised.

Major Comments
1. Confounders:
Reviewer comment: “The multifaceted factors leading to ulceration in leprosy encompass neuropathy, deformities, nutrition, footwear, and sun exposure. It is crucial to specify the methods used to measure and control these variables during analysis to guarantee the reliability of the study outcomes.”
Response: We have expanded the “Procedures and examinations” section to provide detailed descriptions of how each of the relevant factors is assessed, including structured history taking, Semmes-Weinstein monofilament testing for neuropathy, physical examination for deformities, BMI classification for nutritional status, and sun exposure estimation. Footwear habits are not formally assessed, but are indirectly represented by socioeconomic status and presence of deformities. Given that this study is conducted in an urban setting, regular footwear use is generally expected among participants. These variables will also be incorporated into bivariate and multivariate analyses as described in the “Data analysis” section.

2. Vitamin D and Seasonality:
Reviewer comment: “Since vitamin D levels fluctuate considerably with seasons and sunlight exposure, it is crucial to determine whether participant recruitment was evenly distributed throughout different seasons or if this variability might introduce bias into the study.”
Response: We acknowledge the important influence of seasonality on vitamin D levels. To minimize this variability, participant recruitment was planned and completed during the Indonesian dry season (March to September 2024), which provides stable sun exposure. This has been explicitly stated in the Abstract and the “Subject recruitment” section.

3. Laboratory Methods (PCR-RFLP vs. Other Genotyping Methods):
Reviewer comment: “While PCR-RFLP is a viable method for analyzing the VDR FokI polymorphism, it may be less precise than sequencing or TaqMan assays. Please explain why this approach was chosen, considering factors such as cost, feasibility, and resource availability in local laboratories.”
Response: We agree with the reviewer that PCR-RFLP may offer lower precision than newer genotyping techniques. However, the FokI polymorphism is a well-characterized variant with a simple restriction site, and PCR-RFLP remains a well-accepted method in these settings. As we are analyzing only one SNP, the higher-throughput capacity of TaqMan or next-generation sequencing is not required for this study. Additionally, PCR-RFLP was chosen based on cost-effectiveness and existing laboratory capacity. This rationale has been included in the “Discussion” section.

4. Sample Size Assumptions:
Reviewer comment: “The assumed odds ratio for the FokI polymorphism effect (around 3.9) might be an overly optimistic estimate. Performing a sensitivity analysis with different effect sizes could support the justification for the chosen sample size.”
Response: We acknowledge that the assumed odds ratio of 3.9 for FokI polymorphism may be optimistic, as it is based on preliminary data in a different leprosy population. The “Sample size” section now includes a statement acknowledging this assumption and its limitations. A note has also been added to the “Discussion” section to encourage cautious interpretation and future validation in larger studies.

5. Ulcer Documentation and Inter-Rater Reliability:
Reviewer comment: “Using PUSH scoring and photography for ulcer documentation is suitable; however, it's important to specify measures like staff training, double scoring, or adjudication to minimize inter-observer variability.”
Response: All ulcer scoring is conducted by the first author (a senior dermatology resident), directly supervised by two consultant dermatologists to ensure consistency. The use of the validated PUSH tool, as well as ulcer photography for retrospective verification, has been clarified in the “Procedures and examinations” section.

6. Ethical Safeguards for Photographs:
Reviewer comment: “Since the study involves collecting patient photographs, it is essential to outline the methods for anonymization, secure storage, and obtaining informed consent for possible publication, ensuring compliance with ethical research standards.”
Response: Thank you for highlighting this. Clinical photographs are taken only following written consent. Photographs primarily involve distal extremities where the ulcers are located, which limit the likelihood of revealing identifiable features. In cases where identifying marks such as tattoos or moles are visible, they will be digitally blurred. All images are stored in an encrypted, password-protected USB flash drive accessible only to the study investigators. These details have been added to the “Ethics and Consent” section.

Minor Points

Planned sample size added to Abstract.
Exploratory gene-environment interaction (vitamin D and FokI genotype) is now mentioned as an exploratory analysis in the Objectives section.
Missing data:
As this is a prospective study, we anticipate complete data collection. However, if missing data arise, complete-case analysis or multiple imputation will be used if necessary. This has been added to the “Data Analysis” section.
Flow diagram of participant recruitment will be developed during study conduct and included in the final results publication. This has now been mentioned in the manuscript.
Specify covariates for multivariate analysis:
All collected sociodemographic and clinical variables may be included in the multivariate model if their bivariate p-value is <0.25, as outlined in the “Data Analysis” section.
Correction for multiple testing in genetic model comparisons:
As only one SNP (FokI) is analyzed, correction for multiple testing is not deemed necessary, as now clarified in the “Data analysis” section
State limitations (single-centre, causality, residual confounding):
Limitations regarding the single-centre setting, observational design, potential residual confounding, and potentially optimistic effect size for FokI polymorphism analysis have been added to the Discussion section.

View more View less

Competing Interests

None.

Back to all reports

Reviewer Report

24 Views

29 Jul 2025 | for Version 1

Khairuddin Djawad, dermatovenereologi, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia; Wahidin Sudirohusodo General Hospital, Makassar, Indonesia

24 Views Cite this report Responses(1)

Approved With Reservations

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

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?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

dermatologyst

Respond to this report

Responses (1)

Author Response

11 Sep 2025

P. Anthony Halim, Department of Dermatology and Venereology, Faculty of Medicine Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, 10430, Indonesia

We thank the reviewer for the constructive feedback on our work. Below, we provide point-by-point responses to each of the concerns raised.

1. Regarding potential confounders and inclusion/exclusion criteria:
Thank you for your thoughtful comment. We agree that addressing potential confounding factors is critical to ensure internal validity. In designing the exclusion criteria (Table 2), we focused on systemic conditions that are known to significantly alter vitamin D metabolism or immune function, including diabetes mellitus, end-stage kidney disease, severe liver dysfunction, malignancy, autoimmune disorders, and systemic infections. To operationalize this, we screen participants using a combination of structured clinical interviews and medical record reviews, including relevant laboratory and radiologic findings when available. We have clarified these procedures in the revised manuscript (“Case and control definitions” section).

2. Regarding methods to ensure wound depth:
We appreciate this important point. As stated in the inclusion criteria, eligible wounds must not extend beyond the subcutaneous layer. Wound depth is primarily assessed through clinical inspection of the ulcer base. In cases where slough or necrotic tissue obscures the wound base, we would perform sharp debridement as part of standard clinical practice to allow proper visualization. Patients are eligible for inclusion if, following debridement, the wound bed is confined to the subcutaneous layer. Any wound showing evidence of deeper tissue involvement, such as fascia, tendon, or bone exposure, would be excluded. This has been clarified in the revised manuscript (“Procedures and examinations” section ) to support reproducibility.

3. Regarding other factors that might influence the presence of trophic ulceration:
We thank the reviewer for this important observation. We fully agree that trophic ulceration in leprosy is multifactorial. In this study, we collect detailed sociodemographic data (including occupation, education level, and household income) as well as clinical variables (nutritional status, smoking history, disease duration, WHO classification, Ridley-Jopling classification, history of leprosy reactions, presence of peripheral neuropathy, and other deformities). These variables serve as proxies for disease severity, environmental conditions, and lifestyle-related factors. All these factors are incorporated into bivariate and multivariate analyses to assess their potential as confounding factors. We have clarified this approach in both the “Procedures and Examinations” and “Data Analysis”.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. World Health Organization: Weekly epidemiological report (WER) 10 September 2021. Weekly Epidemiological Report. 2021; 96(36): 421–444.

[2] 2. Kementerian Kesehatan Republik Indonesia: Profil Kesehatan Indonesia Tahun 2020. Kementerian Kesehatan RI; 2021.

[3] 3. Kementerian Kesehatan Republik Indonesia: Profil Kesehatan Indonesia Tahun 2021. Kementerian Kesehatan RI; 2022.

[4] 4. Chen X, Liu HB, Shui TJ, et al.: Risk factors for physical disability in patients with leprosy disease in Yunnan, China: Evidence from a retrospective observational study. PLoS Negl. Trop. Dis. 2021; 15(11): e0009923. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Upputuri B, Srikantam A, Mamidi RS: Comorbidities associated with non-healing of plantar ulcers in leprosy patients. PLoS Negl. Trop. Dis. 2020; 14(6): e0008393–e0008312. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Xiong M, Li M, Zheng D, et al.: Evaluation of the economic burden of leprosy among migrant and resident patients in Guangdong Province, China. BMC Infect. Dis. 2017; 17(1): 760. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Barreto JG, Salgado CG: Clinic-epidemiological evaluation of ulcers in patients with leprosy sequelae and the effect of low level laser therapy on wound healing: A randomized clinical trial. BMC Infect. Dis. 2010; 10: 237. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Karthikeyan K, Thappa DM: Squamous cell carcinoma in plantar ulcers in leprosy: a study of 11 cases. Indian J. Lepr. 2003; 75(3): 219–224. PubMed Abstract

[9] 9. Bikle D, Christakos S: New aspects of vitamin D metabolism and action — addressing the skin as source and target. Nat. Rev. Endocrinol. 2020; 16(4): 234–252. PubMed Abstract | Publisher Full Text

[10] 10. Schwalfenberg GK: A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency. Mol. Nutr. Food Res. 2011; 55(1): 96–108. PubMed Abstract | Publisher Full Text

[11] 11. Razzaghi R, Pourbagheri H, Momen-Heravi M, et al.: The effects of vitamin D supplementation on wound healing and metabolic status in patients with diabetic foot ulcer: A randomized, double-blind, placebo-controlled trial. J. Diabetes Complicat. 2017; 31(4): 766–772. PubMed Abstract | Publisher Full Text

[12] 12. Burkiewicz CJCC, Guadagnin FA, Skare TL, et al.: Vitamin D and skin repair: a prospective, double-blind and placebo controlled study in the healing of leg ulcers. Rev. Col. Bras. Cir. 2012; 39(5): 401–407. PubMed Abstract | Publisher Full Text

[13] 13. Dai J, Yu M, Chen H, et al.: Association between serum 25-OH-vitamin D and diabetic foot ulcer in patients with type 2 diabetes. Front. Nutr. 2020; 7: 7. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Ding J, Kwan P, Ma Z, et al.: Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing. Burns. 2016; 42(6): 1277–1286. PubMed Abstract | Publisher Full Text

[15] 15. Oda Y, Tu CL, Menendez A, et al.: Vitamin D and calcium regulation of epidermal wound healing. J. Steroid Biochem. Mol. Biol. 2016; 164: 379–385. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Afarideh M, Ghanbari P, Noshad S, et al.: Raised serum 25-hydroxyvitamin D levels in patients with active diabetic foot ulcers. Br. J. Nutr. 2016; 115(11): 1938–1946. PubMed Abstract | Publisher Full Text

[17] 17. Segaert S: Vitamin D regulation of cathelicidin in the skin: Toward a renaissance of vitamin D in dermatology? J. Invest. Dermatol. 2008; 128(4): 773–775. PubMed Abstract | Publisher Full Text

[18] 18. Oda Y, Hu L, Nguyen T, et al.: Vitamin D receptor is required for proliferation, migration, and differentiation of epidermal stem cells and progeny during cutaneous wound repair. J. Invest. Dermatol. 2018; 138(11): 2423–2431. PubMed Abstract | Publisher Full Text | Free Full Text

[19] 19. Song L, Papaioannou G, Zhao H, et al.: The Vitamin D receptor regulates tissue resident macrophage response to injury. Endocrinology. 2016; 157(10): 4066–4075. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Soroush N, Radfar M, Hamidi AK, et al.: Vitamin D receptor gene FokI variant in diabetic foot ulcer and its relation with oxidative stress. Gene. 2017; 599: 87–91. PubMed Abstract | Publisher Full Text

[21] 21. Usategui-Martín R, De Luis-Román DA, Fernández-Gómez JM, et al.: Vitamin D Receptor (VDR) gene polymorphisms modify the response to vitamin D supplementation: A systematic review and meta-analysis. Nutrients. 2022; 14(2). PubMed Abstract | Publisher Full Text | Free Full Text

[22] 22. Lubis RD, Roesyanto-Mahadi ID, Siregar Y, et al.: FokI Vitamin D receptor gene polymorphism in leprosy household contacts. Malay J. Med. Health Sci. 2021; 17: 113–116.

[23] 23. Priyanto MH, Legiawati L, Saldi SRF, et al.: Comparison of vitamin D levels in diabetes mellitus patients with and without diabetic foot ulcers: An analytical observational study in Jakarta, Indonesia. Int. Wound J. 2023; 20: 2028–2036. PubMed Abstract | Publisher Full Text | Free Full Text

[24] 24. Agius C, Micallef D, Brincat I, et al.: Plasma total ascorbic acid and serum 25-hydroxy-vitamin-D status in patients with venous leg ulcers: a case–control study. Int. J. Lower Extremities Wound. 2021; 23: 421–427. PubMed Abstract | Publisher Full Text

[25] 25. Luong KVQ, Nguyen LTH: Role of the vitamin D in leprosy. Am. J. Med. Sci. 2012; 343(6): 471–482. PubMed Abstract | Publisher Full Text

[26] 26. Dennison CL, de Oliveira LB , Fraga LA d O, et al.: Mycobacterium leprae–helminth co-infections and vitamin D deficiency as potential risk factors for leprosy: A case–control study in south-eastern Brazil. Int. J. Infect. Dis. 2021; 105: 261–266. PubMed Abstract | Publisher Full Text

[27] 27. Darus NIM, Lubis RD, Jusuf NK: Analysis of serum vitamin D level in leprosy patients. Bali Med. J. 2019; 8(3): 795–799. Publisher Full Text

[28] 28. Pan WH, Yeh WT: How to define obesity? Evidence-based multiple action points for public awareness, screening, and treatment: an extension of Asian-Pacific recommendations. Asia Pac. J. Clin. Nutr. 2008; 17(3): 370–374. PubMed Abstract

[29] 29. Avci E, Demir S, Aslan D, et al.: Assessment of Abbott Architect 25-OH vitamin D assay in different levels of vitamin D. J. Med. Biochem. 2020; 39(1): 100–107. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16(3): 1215. PubMed Abstract | Publisher Full Text | Free Full Text

[31] 31. Madiyono B, Sastroasmoro S, Budiman I, et al.: Perkiraan besar sampel.Sastroasmoro S, Ismael S, editors. Dasar-Dasar Metodologi Penelitian Klinis. 5th ed.Sagung Seto; 2014; pp. 352–387.

[32] 32. Gardner SE, Frantz RA, Bergquist S, et al.: A prospective study of the pressure ulcer scale for healing (PUSH). J. Gerontol. 2005; 60(1): 93–97. Publisher Full Text

Comparison of serum vitamin D level and vitamin D receptor gene FokI polymorphism in leprosy patients with and without trophic ulcers: protocol for a case-control study

Abstract

Background

Objective

Methods

Results

Discussion

Keywords

Revised Amendments from Version 2

Introduction

Background

Aim

Table 1. Primary and secondary objectives.

Protocol

Study design

Site and population

Case and control definitions

Table 2. Inclusion and exclusion criteria.

Subject recruitment

Procedures and examinations

Table 3. Pressure Ulcer Scale for Healing (PUSH).

Laboratory prodecures

25(OH)D Level quantification

FokI polymorphism detection

Sample size estimates

Data analysis

Discussion

Summary

Perspective and clinical implications

Limitations

Data availability

Underlying data

Reporting guidelines

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated