. 40 1 INTRODUCTION Warts are papulonod ular epidermal lesions with a horny or papillomatous surface caused due to infection by human papillomavirus (HPV) . 1 Cutaneous warts occur in 7% to 10% of the general population, with a maximum incidence betwee n the ages of 12 and 16 years.2 Warts can persist as it is for years and then spontaneously clear at any time from a few mo nths to a few years . In children , clearance can occu r even after only a few months, with 50% at 1 year and about 65% –78% by 2 years . 2,3 The rate of clearance is influenced by factors like virus strain , host immune status, extent, and duration of warts 4 HPVs are ub iquitous, epitheliotropic non -enveloped sm all double -stranded DNA viruses.5 Over 150 types of papillomavirus have been identified based on DNA studies and serological detection of type -specific antibodies against HPV capsi d antigens .1,6 Among them v erruca vulgaris or common w arts are usually caused by Human Papilloma Virus (HPV) types 1, 2, 4, 27 or 57, and plane warts by HPV types 3 or 10.2 The t ransmission of warts occ urs from direct contact or indirectly via fomites. Usually , warts spread in s wimming pools and bathrooms as the skin is mace rated and is more prone to m inor abrasions and infections thus serving as conduits for HPV to the basal keratinocytes, the primary targets for HPV infection. 2,6 There they undergo a latent phase of slow replication and then induce epidermal hyperplasia and hyperkeratosis as the epidermis grows superficially. 7 2 The destructive therapies in treatment of wart include salicylic acid, trichloroacetic acid, cryotherapy, silver nitrate, phenol, cantharidin , surgical interventions and laser s; antiproliferative agents include bleomycin, vitamin D analogs, podophyllin, podophyllotoxin, and 5 -fluorouracil ; antiviral agents include cidofovir a nd retinoids .2 Other modalities include hypnotherapy, acupuncture, local hyperthermia, therapeutic vaccination, and combinations of the previous agents.8 Destructive therapies are usually uncomfortable, require multiple sessions and individual treatment of each wart, and are often associated with variable efficacy, high recurrence, and significant adverse effects such as scarring .9 Hence immunotherapy is becoming more and more popular, especially in the treatment of refractory cutaneous and gen ital warts. These include various topical, intralesional (IL), and systemic agents.10 Immunotherapy is defined as a type of biological therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer, infection, and other diseases. Immunotherapy can affect the immune system in general or can be cell -specific . 10 Immunotherapeutic agents include: 1. Prophylactic vaccination using viral antigens 2. Therapeutic vaccination using viral antigens 3. Pro-inflammatory cytokines and tumor destroyers (i nterferon) 4. Immune enhancement a. Topical (imiquimod) b. Systemic (cimetidine, levamisole , zinc sulfate ) 3 5. Induction of delayed (cellular) hypersensitivity a. Intradermal (tuberculin, candidin, trichophyton , mumps antigen, BCG , Vitamin D ) b. Topical (DNCB, DPCP, SAD BE, TEIB) 6. Combination treatment 11,12 The exa ct mechanism of immunotherapy is not yet known but it is believed that the injection to the HPV -infected tissue induces a strong nonspecific pro - inflammatory signal and attracts th e antigen -presenting cells. This is associate d with the release of cytokines such as IL -2, IL-8, IL-12, IL-I8, tumor necrosis factor – α, and interfer on-γ.13,14 This then promotes a Th1 cyto kine response which leads to the activation of cytotoxic T cells and natural killer cells i.e. delayed -type hypersensitivity reaction leading to the eradication of the HPV -infected cells.15,16 Furthermore, the trauma of the injection may also cause a resolution in previously sensitized individuals.17 Since intralesional antigen immunotherapy enhances recognition of the vi rus by the immune system it causes clearance of both treated and untreated lesions and helps to prevent future clinical infection through induction of long -term acquired immunity to HPV.13 Measles, Mumps Rubella ( MMR ) vaccine is included in immunization schedule so pre-sensitization skin test is not needed as all patients are expected to be immune.5 In 198 9, monovalent measles vaccine wa s introduced in Nepal for vaccination of infants at age nine months . In 2013, measles -rubella (MR) vaccine was introduced into the national routin e immunization schedule and replaced monovalent measles vaccine.18 The presence of three different antigens in MMR 4 increases the sensitivity to the injected antigen and decreases the likelihood of anergy.8 Side effects of MMR include pain during injection , flu-like symptoms , pruritus and burning sensation .19,20 Intrales ional (IL) vitamin D is a novel addition to therapies in warts as it is a simple, effective, well -tolerable, and inexpensive method with negligible local and systemic side effects21,22 Side effects of Vitamin D3 include transient mild to moderate pain, edema at the site of injec tion, resolving within 1 week m ild eryt hema.22 Further it would be safer to measure serum vitamin D and calcium levels before and after intralesional vitamin D treat ment to prevent possible hypervitaminosis D.23 Vitamin D3 acts on the principle of immunotherapy by stimulating cell -mediated immunity21 In addition, it is also claimed to regulate epidermal cell differentiation and proliferation and may modulate cytokine production through its action upon vitamin D receptors (VDRs).22 VDRs are present in the keratinocy tes, melanocytes, fibroblasts, and immune system cells of the skin. Activation of Toll - like receptor (TL R) of human macrophages up -regulates the expression of vitamin D receptor and vitamin D1 - hydroxylase genes, leading to induction of the antimicrobial p eptide like thymic stromal, lymphopoietin and cathelecidin. 24,12 It also reduces the synthesis of IL 1a and IL6 resulting in decreased inflammation. VDR activators (VDRAs) have been shown to inhibit cell replication and have immunomodulatory properties.25 5 It is suppose d that combining intrale sional immunotherapy with a destructive treatment method might enhance the efficacy, shorten the t reatment duration, and reduce the possible side effects.7 6 RATIONALE OF THE STUDY Intralesional Immunotherapy is a emerging method of treatment of wart in which injection in a single wart causes the resolu tion of distant warts as well. Compared to other destructive modalities, it is mor e effective and comfortable for the patient as it obviates the need for individual treatment of each wart. In addition it has a lower rate of recurrence, avoids adve rse effects such as scarring, is more cost - effective in multiple warts and decreases the ti me a physician has to spend on each patient. Further both injection MMR and Vitamin D3 are readily available as compared to other immunotherapeutic agen ts like PPD and candida antigen . IL MMR vaccine has been used for a longer time than IL v itamin D and there are more comparative studies show ing efficacy of MMR vaccine with rates of clearance ranging from 70.4% to 82.4%.26,27 The clearance rate s for IL vitamin D ranges from 40% to 90%.22,28 As per Committee to Review Adverse Effects of Vaccines; Institute of Medicine (IOE) , there is evidence of causal relati onship between MMR vaccine and measles inclusion body encephalitis in individuals with demonstrated immunodeficiencies.29 Hence immunodeficiency is one the exclusion criterias for MMR administration. The advantages of vitamin D over MMR vaccine are cost-effective ness , non- requi rement of maintenance of cold chain, easy availability , and feasibility of use in immunosuppressed patients. As per Institute of Medicine (IOM), the tolerable upper intake of vitamin D is 4000 IU/day for anyone older than 9 years such that annual maximum d ose will be 1,460,000 IU.30 The total amount of vitamin D that we will be administering will be 7 1,500,000 IU. Furthermore, since we are giving it intralesionally systemic absorption is likely to be low although data on exact amount of IL vitamin D that gets absorbed systemically i s not available. In other studies, patients have been evaluated clinically for signs and symptoms of hypervitaminosis D however, no signs of toxicity were observed . 12,21,22,23,28,31 As per my literature search, 1. No previous studies on immunotherapy in wart have been conducted in our country 2. No comparative studies have been done to compare IL MMR and IL vitamin D3 in treatment of wart hence we aim to establish efficacy of each and compare the two . 8 REVIEW OF LITERATURE Warts are com mon skin conditions resulting from infection of keratinocytes by human papillomavirus (HPV)1. The development of e pidermal thickening and hyperk eratinizat ion occurs following infection at the basal layer and clonal proliferation, which eventually results in a visible wart, weeks or even months later.2 Many studies have documented the prevalence of cutaneous warts in children 9– 13, ran ging widely from 3.3% in the USA to 33% in the Netherlands .32 In a study conducted by. Liu et al it was found that 1.4% of college students were affected with warts on their hands and/or feet. 33 This frequency is estimated to be 7 to 10% of the population in Europe a nd in the United States.34 According to age group, the prevalence of warts is highest in school -aged children, followed by adults then preschool -aged c hildren . 1 In Great Britain , 6.5% of the school children had plantar warts, and 9.5% had warts at other sites.34 Most large studies have found no evidence of a sex difference in wart prevalence. However , a female preponderance is seen in the frequency of plantar warts.34 In the East Anglia survey, 8.4% of females had plantar warts compared to 4.9% of males.1 The use of swimming pools, sports clubs, gymnasiums, or public baths increases the risk of cont racting plantar warts. School children and young adults constitute a high epidemiologic risk population and an important source of the dissemination of cutaneous warts since their f amilies are more often infected up to 50% of c ases. 9 Among b utchers and work ers in the meat -handling industry, the prevalence of warts on the hands is much higher than in the general population as their hands are subjected to many traumatisms. Other factors thought to be responsible are cold, permanently humid conditions, in which workers often use the same tools and working tables.34 Classification of w arts35 Cutaneous warts can be classified in relation to the clinical morphology and the type of infecting HPV as follows: 1. Common wart (Verruca Vulgaris) a. typical or exophytic b. mosaic c. endophytic, d. papillomatous or filiform 2. Plane and intermediate warts 3. Myrmecia 4. Pigmented plaques . 1. Typical common warts are exophytic, i.e. elevated. Their surface is rough irregular, hyperkeratotic, with min ute papillary projections. 2. Mosaic warts appear usually in plantar location s. They are superficial, only slightly raised above the skin level, hyperkeratotic, multiple, confluent with polygonal outlines, and painless . 10 3. Endophytic common warts appear characte ristically in palmar and plantar localizations. Deep plantar common warts are most often multiple, painless, with a slightly raised, hyperkeratotic surface. Even for experienced dermatologists, the differentiation between myrmecias and deep common warts is difficult. 4. Papillomatous common warts are usually less typical, resembling papillomas, localized most often on the face, head, neck, in the folds. 5. Plane warts are slightly raised above the skin level, smaller than common warts, with flatter, smoother surf aces and irregular outline. Intermediate warts, a term applied to lesions that cannot be classified as common or plane warts, combine clinical features of both. 6. Myrmecia warts, if in plantar location, are often referred to as deep plantar warts. They may a lso be palmar and the warts are endophytic, deep, and more often single. Characteristic findings include small bleeding points and punctate dots appearing after trimming off the hard horny covering . A few atypical warts, e xample , pigmented plaques in immunosuppressed patients, remain unclassified until more is known about the incidence and natural history.35 Intralesional MMR vaccine The mech anism of action of intralesional MMR immunotherapy is still an enigma. Some authors hypothesize that it acts through the induction of a strong nonspecific inflammatory response against the HPV -infected cells.19,20 It has also been suggested that the trauma itself may cause wart clearance in previously 11 sensitized individuals.15 The cytokines produced by immune system such as interleukin (IL) -2, IL -4, IL -5, IL -8, interferon (IFN) -γ and t umor necrosis factor (TNF) -α stimulate a strong immune response against H PV may be another possible mechanism of action.21 Another author reports that the response to antigen injection was associated with the proliferation of peripheral blood mononuclear cells that promotes Th1 cytokines including IFN -γ and IL 2, which further activate cytotoxic T cells and natural killer (NK) cells that eradicate HPV - infected cells.17,26 Subjects can be tested for existing immunity by intrademal injection of 0.1 mL of MMR vaccine into the skin of the forearm. Determina tion of a positive reaction requires erythema and induration of at least 5 mm in diameter within 48 –72 hours. Patients reactive to the skin t est are labelled responders and IL MMR is more effective in them.20 Nofal A et al conducted a randomized controlled trial (RCT) with 135 patients of which 85 patients received the MMR vaccine and 50 received Normal saline as control. Both treatment groups were injected in single largest wart at 2 weekly interval s until complete clearance for a maximum of 5 treatment and were followed up bimonthly for 6 months . In the MMR group, complete response was achieved in 80% and 84.6% of patients presenting with recalcitrant and multiple warts respectively. No recurrence w as observed in the MMR group and side effects included pain during injection and flu -like symptoms. 20 12 Author Setting Type of Study Sample size Type of wart Percentage of complete clearance Nofal A and Nofal E, 201020 Zagazig University , Egypt Randomize d controlled trial 135 Common warts Recalcitrant wart - 80% Multiple Wart -84.6% Nofal A et al. 20158 Zagazig U niversity, Egypt Prospective 65 recalcitran t wart 63% Naseem R and Aamir S, 201336 Federal postgraduate medical Institute & Sheikh Zayed Hospital, Lahore, Pakistan Prospective 170 Common warts 81.3% Mohamad NS et al, 201337 Alexandria Main University Hospital, Egypt Quasi experimenta l trial 100 Plantar wart 82% Shah A et al, 201638 NHL Municipal Medical College, Ahmedabad, Gujarat, India Prospective study 50 Common warts 72% Raju J et al, 201639 Mysore Medical College & Research Institute, Mysore , India Prospective study 27 Common warts 70.4% Rohit V et al, 201740 Chalmeda Anand Rao Institute of Medical Sciences, Telangana, India. Case - control study 50 Common warts 72% Chauhan PS et al, 201727 Dr.R.P. Govt. Medical College, Kangra (Tanda), Himachal Pradesh, India Prospective study 51 Common warts 82.4% Table 1: C omparison of different studies on IL MMR in warts 13 Again in 2015 , Nofal et al conducted a similar study in recalcitrant wart where they included sixty -five patients . Recalcitrant warts were defined as warts persistent for more than two years despite treatment with at least two different moda lities. Distant warts were defined as warts at different anatomic sites away from the treated wart. Complete clearance of the lesions was observed in 41 patients (63%), partial response in 15 patients (23%), and no response in nine patients (14%). Complete response was demonstrated in 74.5% of those presenting with distant warts. Side effects were mild and insignificant in the form of pain during injection, itching, erythema, and edema at the site of injection and flu-like symptoms. Recurrence was detected in two patients only8 Similarly i n a study conducted by Naseem, A amir in 2013 t here were total 170 patients and final efficacy came as 81.3% whi ch shows that a large number (122) of patients show complete response to the MMR vaccination.36 Nagat Sobhy Mohamad, et al observed that the response of the target wart, MMR- treated group showed a significantly higher rate of complete clearance compared with the control group (82% versus 0% respectively). The rate of partial response was 6% versus 30%, and the rate of no response was 12% versus 70%, respectively. Regarding the response of untreated distant warts, the MMR -treated group showed 86.9% complete and 13.1% partial clearance of the warts whereas the control group showed 100% no response .37 In a prospective study conducted in the Dermatology outpatient department of V.S Hospital, Ahmedabad by Shah A et al over a period of one year patients received 14 intralesional MMR vac cine 0.5ml into a single wart or the largest wart in case of multiple lesions at an interval of two weeks for three treatments. The response was evaluated as 0 -49% as no response, 50 -99% as partial response and 100% as complete response. Follow up was made every 02 weeks for 06 weeks and then monthly for 06 months to detect any recurrence. They found c omplete response was seen in 36 (72%), partial response in 08 (16%) and no response in 06 (12%) patients. No recurrence was observed. Pain at the site of inje ction in 18 (36%) and the flu like symptoms in 02 (04%) patients were observed. 38 In 2016 J Raju et al injected MMR vaccine into the largest single wart intralesionally and gave subsequent injections every 2 wee ks apart for about 3 to 5 times and obtaine d complete remission of warts in 70.4% of patients, partial remission in 22.2% and no response in 7.4% of patients. 39 Vontela R et al observed complete clearance in 72% patients and partial clearance in 16% of patients receiving the MMR vaccine. No response is seen in 12% of patients. The recurrence rate during the 6 month s follow up period is 12%. Pain during injection was noted in 60% of patients without any other adverse effects in the treated patients. The mean duration taken to show the complete clearance of the lesions is 9 weeks40 In 2017 Chauhan et al injected 0.25 mL MMR vaccine intralesionally in the largest wart and repeated at 2 -week interval until complete clearance or a maximum of five doses. The y evaluated outcome as complete clearance, excellent, good, or unsatisfactory response on a visual analog scale at every visit 15 and at 4 and 8 weeks, thereafte r by comparing baseline clinical photogr aphs. They used a Likert scale for patient satisfaction level assessment similarly. Out of 51 patients who completed the study 42 (82.4%) of them showed complete clearance of warts and 9 (17.6%) patients showed good or unsatisfactory response. In 4 (7.8%) patients, warts subsided completely after one dose itself. The four patients showing an excellent response after five doses initially also continued to improve during a follow‑up period of 8 weeks. Except for injection site pain, no adverse effects were noted. There was no recurrence of warts among them.27 Intralesional Vitamin D3 Vitamin D3 acts on the principle of immunotherapy by stimulating cell -mediated immunity21 Vitamin D is also clai med to regulate epidermal cell differentiation and proliferation and may modulate cytokine production through its action upon vitamin D receptors.22 The vitamin D receptors are present in the keratinocytes, melanocytes, fibr oblasts, and immune system cells of the skin. Activation of Toll - like receptor (TLR) of human macrophages up -regulates the expression of vitamin D receptor and vitamin D1 - hydroxylase genes, leading to induction of the antimicrobial peptide 24 like thymic stromal, lymphopoietin and cathelecidin. It also reduces the synthesis of IL 1a and IL6 resulting in decreased inflammation. VDR activators (VDRAs) have been shown to inhibit cell replication and have immunomodulatory properties. 25 16 Author Setting Type of study Sample size Type of wart Percentage of complete clearance Recurre nce Aktaş et al. 201623 Different hospitals of Turkey Prospective study 20 Common warts 80% No Jakhar, Kaur, and Misri 201821 NDMC Medical College and Hindu Rao Hospital, Delhi case report 1 Periungal wart - Not mentione d Raghukumar et al. 201722 Hassan Institute of Medical Sciences, Hassan, Karnataka state, India Prosp ective study 64 recalcitrant warts 90% 2(3.7%) Kareem et al. 201912 Al-Azhar University, Cairo, Egypt RCT 50 Common warts 40% Not mentione d El-Taweel, Salem, and Allam 201928 Benha University, Egypt Prospective study 20 Common warts 40% No Manjunath K et al. 201931 Mandya Institute of Medical Sciences, Mandya, Karnataka, India Prospective study 42 Multiple warts 78.57% 1(5.26%) Table no. 2: Comparison of studies on IL Vitamin D3 in Warts 17 Aktas et al conducted study with 20 patients with single or multiple plantar warts. Vitamin D3 (0.2 mL, 7.5 mg/mL) was injected into the base of the warts after prilocaine (0.1 mL, 20 mg/mL) injection. A maximum of 5 warts were treated in 1 session, with a maximum 2 injections performed at 4 -week intervals. In total, 16 of 20 patients (80%) showed complete resolution of warts, and 1 patient showed partial resolution. Three patients failed to show any response. No recurrence or serious adverse effects were observed. 23 In a case report d one by Jakhar, Kaur, and Misri in 2018, the injection was given at a dose of 0.1 mL/cm2 just beneath the wart. A maximum of 0.4 mL was used in a single session in cases of mu ltiple warts. The session was repeated at 2 -week intervals for a maximum of 4 sess ions or complete resolution of warts, whichever is earlier. The r esolution of warts typically started in 7 to 10 days, and warts are shed spontaneously within 4 to 6 weeks. The only side effect was pain during injection, which was minimized with a dose of lidocaine before injection with vitamin D3. Two to 4 sessions are usually required on average for complete cure. 21 Raghukumar et al. conducted a study in 2017 in 64 patients with recalcitran t warts . Complete response with IL vitamin D was seen in 54 of 60 (90%), partial response in 4 of 60 (6.66%), and no response in 2 of 60 (3.33%). The average number of injections required to achieve a complete resolution was 3.66. Complete resolution of distant warts was noticed in all patients.22 18 Kareem et al. conducted RCT in 50 p atients who were divided into two groups: thirty patient s as cases group who received an intralesional injection of 0.2 ml of vitam in D3 (300.000 IU) and another twenty patients as a control group who were injected with normal saline solution. Standardized photographs were taken before, one month and thr ee months after the procedure. The degree of the response was classified into complete, partial and no response. C omplete clearance of th e target injected warts was seen in 40% of patients in the cases group and 5% of patients in the control group (P≤0.001 ) that was statistically significant.12 Simila rly, in a study conducted by El-Taweel et al. 20 patients with verruca vulga ris and deep palmoplantar warts were included. Forty percent of the lesions treated with IL vitamin D3 showed complete clearance and the rate of distant wart response was 17.65%. A mong different demographic and clinical variables in the studied patients, smoking and older age seemed to decrease the therapeutic response 28 In a prospective study conducted by Shilpa et al on 2019, 33 of 42 patients (78.57%) showed a complete response, 6 patients (14.28%) showed moderate response and three patients (7.14%) showed mild response with IL vitamin D3 . Recurrence was observed in one patient with the palmoplantar wart. No serious adverse effects were reported.31 19 OBJECTIVES Primary Objective s: 1. To determine the efficacy of IL MMR vaccine in the treatment of Wart 2. To determine the efficacy of IL vitamin D in the treatment of wart 3. To compare the efficacy of IL MMR vaccine and IL Vitamin D Secondary Objective: 1. To determine the clinical and demographic profile of patients with different types of wart s visiting Dermatology outpatient department of BPKIHS 2. To determine the side effects of IL MMR vaccine and IL Vitamin D in treatment of wart 3. To determine Dermatology life quality index (DLQI) in wart patients. 20 RESEARCH HYPOTHESIS Null Hypotheses (H0) : IL MMR vaccine is not better than IL Vitamin D in the treatment of wart Alternative hypothesis: IL MMR vaccine is better than IL Vitamin D in the treatment of wart Operational definition Wart – clinical diagnosis made by a dermatologist Grades of clinic al improvement Definition VAS score Complete response Complete disappearance of warts including distant ones and skin texture at the site is restored to normal 100% Excellent response Reduction in size and number including distant ones and few residual w arts still visible 75–99% Good response Some reduction in size only including that of distant ones but no decrease in number of warts 50-74% Poor or no response No significant change in size and number of warts 0–49% Recurrence Recurrence during the st udy period Table no. 3 : Patient and physician global assessment using visual analog scale score27 21 MATERIALS AND METHODOLOGY Materials: The s tudy population will be all patients clinically diagnosed with warts visiting the Dermatology outpa tient department of BPKIHS, Dharan . Study design: A Prospective comparativ e longitudinal study Study Period : The probable duration of the study will be one year after approval fr om th e Institutional Review C ommittee and Nepal Health Research Council (NHRC) Ethical Clearance Ethical clearance will be taken from Institutional Review Committee (IRC), BPKIHS and Nepal Health Research Council (NHRC). Conflict of interest : None Sampling T echnique : Census method with all consecutive patients meeting inclusion criteria for initial 6 months Inclusion criteria 1. Clinically diagnosed patients who have more than three warts or single wart in difficult to treat sites (periungual, palms and soles) 2. Age >/= 12 years 22 Exclusion criteria 1. Patients under any systemic or topical treat ment of warts for the last 4 weeks 2. Patients with a past history of an allergic response to MMR or any other vaccine or Vitamin D 3. Patients with current acute febrile illness or any bacterial infection 4. Immunosuppressed patients 5. Pregnant or lactating women 6. Patients having a past history of asthma, allergic skin disorders or convulsions 7. Patients with keloidal tendency 8. Patient r efusal for consent 9. Treating physician’s decision to giv e other modality of treatment 10. Patients with hypervitaminosis D, muscle weakness, bone pain, altered sensorium Sample size The study considers a 95% confidence interval, 80% power to estimate sample size. According to the literature review, it was found th at 84.6% and 40% improvement were seen with IL MMR and IL Vitamin D respectively.20,12 Now using the sample size estimation formula for 2 proportion Now using the sample size estimation formula for 2 proportion n = 2p(1 -p) (Zβ + Zα/2)2 (p1-p2) 2 n= sample size for each group p1 = 0.846 23 p2 = 0.40 p= (p 1 +p2)/2 Zα/2=1.96 at 95% CI Zβ = 0.842 at 80% power Using above formula, n= 18.54 Considering loss of follow up, sample size in each group= 30 Total sample size= 60 Methodol ogy Based on the computer generated random number table, patients will be assigned to either Group A or Group B once they come to OPD. Informed consent will be taken.