Intralesional Therapies For Cutaneous Viral Warts: A Comparative Analysis Of Vitamin D3 And Acyclovir
Phase: PHASE4
Conditions: Viral Wart, Plantar Wart, Common Warts (Verruca Vulgaris), Common Wart, Flat Wart, Warts of Foot, Warts Hand, Wart
Sponsor: Riphah International University

STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN
Intralesional Therapies for Cutaneous Viral Warts:
A Comparative Analysis of Vitamin D3 and Acyclovir
Official Title: Intralesional Therapies for Cutaneous Viral Warts: A Comparative
Analysis of Vitamin D3 and Acyclovir
NCT Number: Not yet assigned
Document Date: December 15, 2025
Multicenter Randomized Controlled Trial
Site 1 (Lead Site): Department of Dermatology, Pakistan Railway Hospital
Principal Investigator: Dr. Nadia Ghazanfar
Email: nadia.ghazanfar@riphah.edu.pk
Site 2: Riphah International Hospital, Sector A DHA Phase 5, Islamabad
Site Investigator / Co-PI: Dr. Fatima Sajid
Email: fatima.sajid@riphah.edu.pk

TABLE OF CONTENTS
1. Study Synopsis
2. Introduction and Background
3. Study Objectives
4. Study Design
5. Study Population
6. Interventions
7. Study Procedures and Schedule
8. Outcome Measures
9. Statistical Analysis Plan
10. Safety Monitoring
11. Study Organization and Responsibilities
12. Ethical Considerations
13. Data Management
14. References

1. Study Synopsis
Study Title
Intralesional Therapies for Cutaneous Viral Warts: A Comparative Analysis of Vitamin D3 an
Study Design
Prospective, single-blinded, multicenter, randomized controlled trial
Study Sites
Site 1: Dermatology Department, Pakistan Railway Hospital
Site 2: Riphah International Hospital, DHA Phase 5, Islamabad
Principal Investigator
Dr. Nadia Ghazanfar (Pakistan Railway Hospital)
Site Investigator / Co-PI
Dr. Fatima Sajid (Riphah International Hospital)
Study Duration
3 months follow-up period
Sample Size
40 participants (20 per group)
Population
Patients aged 12 years and above with clinically diagnosed cutaneous warts
Intervention
(Group A)
Intralesional Acyclovir (70 mg/mL)
Comparator
(Group B)
Intralesional Vitamin D3 (200,000 IU; 5 mg/mL)
Primary Outcome
Clinical response at week 8 (complete clearance, partial response, or no response)
Secondary Outcomes
Recurrence rate, adverse effects, number of sessions to clearance
Randomization
Simple randomization with sealed opaque envelopes
Blinding
Single-blind (outcome assessor blinded)

2. Introduction and Background
Cutaneous warts are benign epithelial proliferations caused by human papillomavirus (HPV)
infection. They are among the most common dermatological conditions, affecting individuals of all
ages. Warts can present as common (verruca vulgaris), plantar, flat, or periungual types, and
while often self-limiting, they can be persistent, recurrent, and cosmetically distressing.
Current treatment modalities include destructive therapies (cryotherapy, electrocautery, laser),
topical agents (salicylic acid, imiquimod), and intralesional therapies. However, no single treatment
offers a consistently high cure rate, and recurrence remains a significant clinical challenge.
Intralesional therapies have gained attention due to their potential to stimulate local and systemic
immune responses against HPV.
Intralesional vitamin D3 has been proposed as an immunomodulatory agent that can enhance the
host immune response against HPV-infected keratinocytes. Intralesional acyclovir, an antiviral
agent primarily used against herpes viruses, has also been explored for its potential efficacy
against cutaneous warts. This multicenter study aims to compare the effectiveness and safety of
these two intralesional therapies across two clinical sites.

3. Study Objectives
3.1 Primary Objective
To compare the clinical efficacy of intralesional acyclovir versus intralesional vitamin D3 in the
treatment of cutaneous viral warts at week 8.
3.2 Secondary Objectives
 To compare the recurrence rate between the two treatment groups
 To evaluate the safety profile and adverse effects of each treatment
 To compare the number of treatment sessions required for complete clearance
4. Study Design
This is a prospective, single-blinded, multicenter, randomized controlled trial with two parallel
arms. The study is conducted at two sites: the Dermatology Department of Pakistan Railway
Hospital (lead site) and Riphah International Hospital, DHA Phase 5, Islamabad. The follow-up
period is three months.
4.1 Randomization
Eligible participants at each site are randomly allocated into two equal groups (Group A and Group
B) using a simple randomization technique. Allocation concealment is ensured using sealed
opaque envelopes prepared centrally and distributed to both sites.
4.2 Blinding
The study is single-blinded. Outcome assessment is performed by an independent investigator
who is blinded to group allocation at each site. Participants and treating physicians are aware of
the assigned intervention.
4.3 Multicenter Coordination
Both sites follow an identical study protocol, including the same inclusion and exclusion criteria,
treatment procedures, dosing schedules, outcome assessments, and data collection instruments.
The principal investigator (Dr. Nadia Ghazanfar) oversees the overall conduct of the study, while
the site investigator (Dr. Fatima Sajid) is responsible for day-to-day operations at the second site.
Regular communication between sites ensures protocol adherence and data quality.

5. Study Population
5.1 Inclusion Criteria
 Age 12 years and above
 Clinically diagnosed cutaneous warts (common, plantar, flat, or periungual)
 Both treatment-naive patients and those with recalcitrant warts
 Willingness to comply with follow-up visits
5.2 Exclusion Criteria
 Pregnant or lactating women
 Immunocompromised patients
 History of hypersensitivity to vitamin D3 or acyclovir
 Receiving systemic immunosuppressive or antiviral therapy
 Secondary infection at injection site
 Any topical or destructive treatment for warts in the previous three months
5.3 Sample Size
A total of 40 participants will be enrolled across both sites, with 20 participants allocated to each
group.
6. Interventions
6.1 Group A: Intralesional Acyclovir
Patients in Group A receive intralesional acyclovir (70 mg/mL), prepared by reconstituting a 250
mg vial with 3.5 mL of distilled water.
6.2 Group B: Intralesional Vitamin D3
Patients in Group B receive intralesional vitamin D3 (200,000 IU; 5 mg/mL).
6.3 Dosing Protocol
Lesion Size
Injection Volume
£0.5 cm
0.2 mL
0.5  1 cm
0.3  0.5 mL
1  1.5 cm
0.5  1 mL

Injections are administered at baseline and repeated every 2 weeks for a maximum of four
sessions, with follow-up until three months. The same dosing protocol is followed at both study
sites.

7. Study Procedures and Schedule
7.1 Baseline Assessment
At baseline, detailed patient biodata is recorded, including age, sex, duration, type, number, size,
and site of lesions. Standardized digital photographs are taken before initiation of therapy.
7.2 Treatment Visits
Injections are administered at baseline (week 0), week 2, week 4, and week 6 (maximum four
sessions). At each follow-up visit, the following are assessed:
 Change in size and number of lesions
 Treatment response
 Occurrence of any adverse events
 Standardized digital photographs
7.3 Follow-Up
Patients are followed up until three months from baseline to monitor for recurrence and any late
adverse effects.
8. Outcome Measures
8.1 Primary Outcome
Clinical response at week 8, categorized as:
 Complete clearance: 100% resolution of the wart lesion
 Partial response: Reduction in size or number of lesions without complete clearance
 No response: No change or increase in size or number of lesions
8.2 Secondary Outcomes
 Early recurrence of warts in participants achieving complete clearance
 Adverse effects (pain, erythema, swelling, ulceration, scarring)
 Number of treatment sessions required for complete clearance

9. Statistical Analysis Plan
9.1 Software
All data will be analyzed using IBM SPSS Statistics version 26.
9.2 Descriptive Statistics
Normality of continuous variables will be assessed using the Shapiro-Wilk test. Normally
distributed variables will be expressed as mean +/- standard deviation, while non-normally
distributed variables will be summarized as median and interquartile range (IQR). Categorical
variables will be presented as frequencies and percentages.
9.3 Baseline Comparisons
Baseline continuous variables will be compared between groups using the independent t-test or
Mann-Whitney U test, as appropriate. Categorical variables will be compared using the Chi-square
test or Fisher's exact test.
9.4 Efficacy Analysis
Treatment response at week 8 will be analyzed using the Chi-square test. Efficacy will be
assessed by comparing complete response versus partial/no response between groups.
9.5 Secondary Analyses
Among patients achieving complete clearance, the number of treatment sessions required will be
compared using the Mann-Whitney U test.
9.6 Site Effect Analysis
To assess potential site effects in this multicenter trial, baseline characteristics and treatment
outcomes will be compared across sites. If significant differences are identified, site will be
included as a covariate in secondary analyses.
9.7 Significance Level
A p-value less than 0.05 will be considered statistically significant for all analyses.
10. Safety Monitoring
Patients at both sites will be monitored at each visit for adverse effects including pain, erythema,
swelling, ulceration, or scarring. All adverse events will be documented systematically using
standardized case report forms. Any serious adverse event will warrant discontinuation of
treatment and appropriate medical management. Serious adverse events will be reported to the
principal investigator and the ethics committee within 24 hours.

11. Study Organization and Responsibilities
11.1 Principal Investigator
Dr. Nadia Ghazanfar
Department of Dermatology, Pakistan Railway Hospital
Email: nadia.ghazanfar@riphah.edu.pk
Responsibilities: Overall study oversight, protocol development and amendments, regulatory
submissions, data integrity, final analysis and reporting, coordination between sites.
11.2 Site Investigator / Co-Principal Investigator
Dr. Fatima Sajid
Riphah International Hospital, Islamabad Expy, Sector A DHA Phase 5, Islamabad
Email: fatima.sajid@riphah.edu.pk
Responsibilities: Day-to-day conduct of the study at Site 2, participant recruitment and enrollment,
treatment administration, data collection, adverse event reporting, and ensuring protocol
adherence at the site.
11.3 Multicenter Coordination
Regular meetings (at least monthly) will be held between the principal investigator and site
investigator to review enrollment progress, protocol deviations, adverse events, and data quality.
Both sites will use identical case report forms, treatment protocols, and outcome assessment
procedures to ensure consistency.
12. Ethical Considerations
The study was approved by the institutional ethical review committee. This single ethics approval
covers both study sites. Written informed consent will be obtained from all participants prior to
enrollment. For participants aged 12-17 years, assent will be obtained from the participant and
written informed consent from a parent or legal guardian. Confidentiality of patient data will be
maintained throughout the study at both sites.
13. Data Management
All data will be collected on standardized case report forms at both sites. Digital photographs will
be stored securely with coded identifiers. Data from both sites will be entered into a centralized
SPSS database managed by the principal investigator and verified for accuracy. Patient identifiers
will be kept separate from study data to ensure confidentiality. Data from Site 2 will be transmitted
securely to the lead site for central analysis.

14. References
References to be listed as per the final published manuscript.