A prospective randomized controlled study of Mycobacterium Indicus Pranii vaccine, Measles Mumps Rubella vaccine and Vitamin D3 in extragenital cutaneous warts

Interventional, prospective, four arm randomized control.


| INTRODUC TI ON
Cutaneous warts (CW) are hyperkeratotic benign papilloma's. The prevalence rate among adults varies from 1.2% (reported in UK) to 3.5% (reported in the United States). The usual sites involved are the hands and feet. 1 CW may resolve spontaneously in 15%-63% of the cases. [2][3][4][5][6] Age, stress, sleep quality, socio-economic status, and immune system of an individual are the factors affecting the spontaneous resolution. 1,7 The mechanism of CW primarily involves derangement of the immune mechanisms, that is, inhibition of helper T cells stimulated release of interleukins, which is similar to various viral infections such as human immunodeficiency virus (HIV) and human T lymphotropic virus-4. [8][9][10] The treatment of CW has been debated for a long time.
Destructive therapies like electrocautery, cryotherapy and many are ineffective for distant lesions and often need multiple, often painful sessions with a risk of scarring. Evidence showing that cellular immune responses play a critical role in wart clearance that has inspired the development of topical and intralesional immunotherapy regimens for patients with multiple and/or persistent warts. 11 Various systemic immunotherapies like intralesional vaccines (BCG vaccine, measles, mumps, rubella virus or MMR vaccine, and Mycobacterium indicus pranii-MIP) and antiproliferative agents (Vitamin D) have been tried for the treatment of common warts with promising results. 8,[12][13][14][15][16][17][18][19][20][21] Their mechanism is based on the local stimulation of cellmediated and humoral immunity using different antigens to clear HPV and the host's infected cells. 22,23 However, selection among existing immunotherapeutic modalities remains challenging as none of them have proven 100% efficacious.
Literature review of the primary researches 8,13,[15][16][17] and a metaanalysis 23 shows that the MMR vaccine is one of the most effective modalities for CW clearance at primary and distant sites.
Though BCG vaccine (live attenuated) has also been found to be equally effective as MMR, 8 lately the use of Mycobacterium indicus pranii (MIP or Mw) vaccine has been found to be more promising due to its strong antigenic nature. 12,13 It differs from the vaccine as it is a killed vaccine derived from atypical Mycobacterium. 24 Vitamin D is an anti-proliferative agent with immunomodulatory functions, which has proven efficacy similar to MIP and MMR. 19 However, till date, no randomized comparative trial has been done on the comparison of the vaccines with anti-proliferative agents.
Thus, we conducted this randomized trial to compare the safety and efficacy of MIP, MMR, and vitamin D3 among themselves and in comparison with control.

| ME THODS
An interventional four arm randomized controlled trial was conducted in the Department of Dermatology, Venereology, and Leprology at a tertiary care hospital at Jodhpur, India, from January Furthermore, the patients were randomized (via computer generated randomization method with a table of random numbers) to four groups. Only the assessor (SS) was blinded, the patients and the primary investigator (UL) were treatment aware. The study flow chart has been shown in Figure 1 as a CONSORT diagram.
The study subjects were allocated to one of the 4 groups, namely placebo (saline, group A), vitamin D 3 (group B), MIP (group C), and MMR (group D).The volume injected in the largest wart at each visit was 0.5 ml each in grp A and D, 0.2 ml in grp B, and 0.1 ml in grp C.
In each patient, the largest wart was injected with the appropriate intervention every 2 weeks until response to treatment was investigated by the decrease in the wart number and photographic comparison. Complete response was defined as the disappearance of the wart(s) and the return of normal skin markings; good response was defined as the regression in the wart number or by photographic comparison to 76%-99%; moderate response as 51%-75% regression; mild response as 25%-50% regression; and poor response as <25% decreased in number of warts by photographic comparison.
The immediate and late adverse effects of the all vaccines were evaluated after each treatment session. Post-treatment follow-up was performed for all the study participants to compare the side effects, probable relapse, and maintenance of therapeutic outcomes.

| Statistical analysis
The comparison of the variables which were quantitative and normally distributed like age, baseline mean number of warts, mean time (week) for complete clearance were analyzed using Independent t test (for two groups) and ANOVA (for more than two groups). The comparison of the variables which were qualitative in nature like treatment response, percentage improvement according to foot, pain, erythema, and swelling at injection site were analyzed using chi-square test. Post-treatment wart clearance, family history, scalp as site of wart, type of wart, percentage improvement according to type of wart and wart site except for foot, fever, and grade of response had an expected value of <5 so Fisher's exact test was used.
Kaplan-Meier survival analysis curve of complete clearance in injected wart was plotted, and comparison between placebo, Vit D 3 , MIP, and MMR was done using log rank test.

| RE SULTS
We randomized 200 patients out of which 3 patients did not receive treatment at initial visit. So 197 patients were analyzed among which 135 completed all the 7 visits.
The mean age of patients was 24.94 + 8.17 years (13-60 years), and the mean duration of illness was 13.21 + 17.97 months (2 weeks-96 months). Male-to-female ratio was 3.6:1. Past history of treatment for warts was present in 4.5% of patients, and positive family history for warts was found in 2.5% in our study population.
All groups were comparable (p > 0.05) in terms of demography and basic clinical details. (Table 1). Patients with grade 1 response were highest in MMR group (31%), whereas those with grade 2 response were highest in MIP group (11.6%). Lack of response was not seen in any patients. The overall grades of wart clearance are shown in Figure 3, both as per ITT and PP analysis.
According to intention to treat analysis, out of all four groups, patients in MIP group took lesser mean time (7.1 ± 3.1 weeks) to achieve grade 5 response whereas placebo group took maximum mean time to achieve the same (7.8 ± 2.7 weeks). However, the difference among all four groups was not significant (p = 0.736)

| DISCUSS ION
Multiple topical and systemic therapeutic modalities have been used to treat cutaneous warts ( Table 2). Previous studies involving immunotherpy in cutaneous warts have been summarized in Table 3.   As for the mechanism in the placebo injections where the function of antigen presentation (as done by immunotherapy) is bypassed, the local trauma of the injection stands enough to induce a sufficient immune response. 27 injections. 18,20,34,35 The effect of vitamin D on the distant warts was also adequate making this therapy a novel one to be used in the current practice in substitution of MIP and MMR.
A recent meta-analysis of intralesional therapies had reported an overall response rate of 60.6% in the injected or adjacent warts.
This was comparable to the efficacy rates of 66%, 58%, and 55%, Overall, since placebo injections with normal saline also achieved similar efficacy as immunotherapies with minimal side effects, the role of local injection trauma needs to be further addressed in future randomized case control trials. We also hypothesize that the healing with placebo have a link with psychological aspect of the patient since Cw is a viral phenomenon linked with altered immunity and stress. [37][38][39] The few limitations of our study include loss to follow-up (31.1%, owing to covid situation), requirement of longer follow up in immunotherapies as sometimes even responses may be delayed and finally inability to perform HPV serotyping and histopathology for better understanding of the virus-host response. These lacunae could be subjects for future research.

| CON CLUS ION
In conclusion, the efficacy of immunotherapies was comparable to placebo with minimal side effects. Further studies with larger sample size are required to conclude and include intralesional immunotherapies for warts in daily practice.

AUTH O R CO NTR I B UTI O N S
U.L. and S.S., performed the research. U.L., S.S., A.B., and A.BU. designed the research study. N.C. and V.R. contributed essential reagents or tools. U.L., S.S., and S.SI analyzed the data. U.L., S.S., and N.C wrote the paper.

CO N FLI C T O F I NTER E S T S TATEM ENT
Nil for all author.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.