A comparative clinico- dermoscopic study of intralesional injection of combined digoxin and furosemide, Candida antigen, and vitamin D3 for multiple warts

Background: Immunostimulatory and antiproliferative therapies have been widely used for the treatment of multiple warts. Recently, anti- HPV activity of ionic contra viral therapy (ICVT) which is comprised of combined digoxin and furosemide has been demonstrated. Aim: To evaluate and compare the effectiveness and safety of intralesional injection of Candida antigen, vitamin D3


| INTRODUC TI ON
Warts are common benign proliferations of the skin and mucosae caused by human papilloma virus (HPV) infection. In spite of the existence of multiple therapeutic regimens, treatment of warts still represents a real challenge. Treatment options for warts could be classified into destructive and nondestructive therapies. Destructive therapies are associated with scarring, act only on the treated warts and not the remote warts, and are not associated with enhanced cell-mediated immune (CMI) response. 1 Many immunotherapeutic agents have been used for the treatment of warts to overcome the challenges associated with the use of destructive therapies. Among these agents is the intralesional Candida antigen immunotherapy that basically stimulates the immunologic response to HPV that not only clears the treated warts but also the distant warts. 2 Intralesional Candida antigen immunotherapy has shown a promising efficacy and safety in the treatment of different types of warts, because of the high prevalence of immunity to Candida in the general population. 3 Different antiproliferative therapies have been successfully used for the management of warts. Among these agents is the intralesional injection of vitamin D3 that has been successfully evaluated for the treatment of different types of warts by regulating the proliferation and differentiation of epidermal cells as well as modulating the cytokine production. 4 On the contrary, recent studies have demonstrated anti-HPV activity of ionic contra viral therapy (ICVT), which is comprised of combined digoxin and furosemide. 5,6 Ionic contra viral therapy is based on blocking of intracellular potassium (K) influx needed for HPV replication, that in turn impairs viral replication. 5 Hence, we decided to evaluate and compare the efficacy and safety of intralesional combined digoxin and furosemide, intralesional Candida antigen, and intralesional vitamin D3 in the treatment of multiple warts.

| Patients
• This study included 75 adult patients (>18 years of age) with multiple (≥3 warts) warts of different types, sites, sizes, and duration with or without distant warts, after approval by the institutional review board (IRB) of Medical School at Zagazig University, Egypt.
Prior to enrollment, each patient provided an informed consent.
Patients were excluded from the study if they had acute febrile illness, past history of asthma, severe allergic skin disorders, immunodeficiency, autoimmune disease, meningitis, or convulsions.
Pregnant or lactating women, those who had any other form of wart management in the month preceding enrollment, and patients who have clinically relevant abnormal laboratory results (increased K level), ECG, vital signs, or physical findings at screening were also excluded.
• All patients were subjected to history taking, general and dermatological examination, ECG, serum K level measurement, and dermoscopic evaluation using Dermlite II Pro HR "USA" dermoscope at baseline and at each visit to assess decrease in wart size and disappearance of dermoscopic features such as red dots with treatment. This study was performed in accordance with the Helsinki Declaration of 1975 and its later amendments.

| Methods
Seventy-five patients were allocated in a random fashion using simple random sampling method to one of three groups (25 patients Group (C): received intralesional injection of 0.1 ml containing 0.0008 mg of combined digoxin and furosemide into the base of each wart with a maximum of five injected warts (a total of 0.5 ml per session). Digoxin used for injection is available as ampoules containing 0.5 mg/2 ml (Lanoxin, GlaxoSmithKline). Injectable furosemide is available as vials with a 20 mg/2 ml dosage (Lasix, Sanofi Aventis).
In each of the three study groups, injections into the wart were made, without presensitization testing, using an insulin syringe at 2-week intervals until full clearance, or for a total of five sessions.

| Calculation of the digoxin and furosemide dose
As there are no standard guidelines of intralesional digoxin and furosemide dosage in wart therapy, this dose was extrapolated from the study conducted by Fathy et al. 6 who in turn adopted the dose from a previous study on a topical gel comprising 0.125 (w/w) each of digoxin and furosemide, with concentrations of 0.125% mg/ml, applied for common warts for 7 days. 7 The dose for an injection is divided by 3, since only a third of a topical concentration is absorbed through the skin.

| Evaluation of the clinical response
The size and number of warts were assessed, both clinically and dermoscopically, at baseline and at each visit to determine response to therapy.
Complete response of warts was recorded if there was complete disappearance of the warts and return of normal skin markings; partial response if the warts have regressed in size by 50%-99%; and no response with less than 50% decrease in wart size. 3 Following each treatment session, adverse effects were examined both immediately and later.

| Follow-up
Every month for 6 months, a follow-up was performed to detect any wart recurrence.

| Statistical analysis
Data were entered, checked, and analyzed using SPSS (statistical package for social science) version 24. Data were expressed as mean + standard deviation (SD) for quantitative variables and number and percentage for qualitative variables. ANOVA (F-test), Chisquared test (X2), and Kruskal-Wallis test were used as appropriate.

| RE SULTS
The study and the follow-up period were completed by all patients.
The patients were 41 males and 34 females who varied in age from 19 to 47 years (average of 28.4 years), and warts lasted between 2 and 36 months. The number of lesions varied between 3 and 10.
A total of 21 palmoplantar warts, 16 common warts, 9 periungual warts, 8 filiform warts, 15 plane warts, and 6 genital warts were injected. The studied groups did not show any significant differences regarding baseline characteristics (Table 1).
In the ICVT group, 7 patients (28%) experienced complete clearance, none showed partial response (0%), and 18 patients (72%) had no response ( Figure 3). A statistically significant difference in the therapeutic response was noted among the three treatment groups in favor of the Candida antigen group followed by the vitamin D group and the ICVT group (p=0.02). Also, a statistically significant difference was noted between the Candida antigen group and the ICVT group (p=0.03) and between the vitamin D3 group and the ICVT group (p=0.02). However, a nonsignificant difference was observed in the therapeutic response between both Candida antigen and vitamin D groups (p=0.59). The rate of wart clearance was significantly faster in the Candida antigen group (a median of 2.5 weeks) as compared to the vitamin D3 (3.5 weeks) and ICVT groups (5 weeks) (p=0.02) ( Table 2).

| Distant warts
Complete response of the distant warts was noted in 33.3%, 0%, and 50% of the Candida antigen, vitamin D3, and ICVT groups, respectively, with no overall statistically significant difference among the studied groups (p = 0.45) ( Table 2).

| Relationship between therapeutic response and different clinical variables
No statistically significant relationship was found between therapeutic response and the different clinical variables, including age and sex of patients, types, size, number, and duration of warts in any of the studied groups.

| Adverse effects
The most common side effect was pain during injection, which was present in all cases. While edema and erythema occurred more frequently among patients of the Candida antigen group (52% and 28%, respectively) and vitamin D3 group (40% and 16%, respectively), with statistically significant difference as compared to the ICVT group (p = 0.003 & 0.04, respectively). Vasovagal attack was reported in one patient injected with vitamin D3 (Table 3).

| Recurrence
No statistically significant difference in the recurrence rate was observed among the studied groups after the 6-month-follow-up period, where recurrence was reported in 6.7%, 25% and 0% of the Candida, the vitamin D3 and the ICVT groups, respectively.

| DISCUSS ION
The idea of ICVT was conceptualized in 2006 by Hartley et al. 5 who demonstrated the anti-HPV activity of digoxin and furosemide that was more potent when both molecules were combined in an in vitro study and paved the way for further studies on ICVT, whether TA B L E 1 Baseline characteristics of the patients in the 3 studied groups.  In the present study, no statistically significant relationship was found between the therapeutic response to intralesional In the intralesional digoxin and furosemide group, there was no systemic exposure regarding the cardiac system (by ECG and serum K), just as stated in the study conducted by Fathy et al. 6 denoting that intralesional ICVT is safe and tolerable treatment modality for warts.
In the current study, no statistically significant difference in the recurrence rate was reported among all the studied groups.
Recurrence was noted in 1/15 patients treated with Candida antigen (6.7%), and 3/12 patients treated with Vitamin D3 (25%), while no recurrence was noted in the intralesional ICVT group. A similar finding of low rates of recurrence with intralesional Candida antigen immunotherapy has also been reported by similar related studies. 18,22,24 This observation has been explained by the acquisition of sustained long-term immunity to HPV by the immunomodulatory effects of Candida antigen. However, regarding intralesional vitamin D3, the recurrence rate was higher than many of related studies that showed no or lower recurrence rates. 17,18,21,22 This may be attributed to the different baseline characteristics, injected dose of vitamin D, and sample size. Concerning the lack of wart recurrence with the intralesional ICVT group, no prior study of either intralesional or topical ICVT had published any data on the recurrence rate. [6][7][8][9] In the present work, we used dermoscopy in assessment of treat- et al. 24 who have also shown a higher, nonsignificant superiority of Candida antigen (45%) as compared to vitamin D3 (40%) in the treatment of plantar warts, and Abdel Razik et al. 18 who have demonstrated a higher significant superiority of Candida antigen (76.7%) as compared to vitamin D3 (20%) in the treatment of multiple common warts. On the contrary, Fathy et al. 22 have reported a higher efficacy of vitamin D3 (70%) than Candida antigen (25%) in the treatment of plantar warts.
Given the low success rate of intralesional ICVT, we do not recommend it as a routine use in the treatment of warts, especially with the potential cardiotoxicity that may theoretically complicate ionic therapy, even though it was found safe and well-tolerated in the current study. However, further studies on larger population are recommended to prove or disprove this low success rate.
In conclusion, intralesional Candida antigen immunotherapy and antiproliferative therapy with intralesional vitamin D3 are significantly more effective than, and as safe as, intralesional ICVT comprised of digoxin and furosemide.

AUTH O R CO NTR I B UTI O N S
Nasr M. and Elkholy B involved in conceptualisation, writing-original draft, methodology, writing-review and editing, and supervision.
Abdelaty S involved in investigation and resources. All authors contributed to formal analysis, acquisition, visualization, validation, project administration, and interpretation of data, gave final approval of the manuscript, and agree to be accountable for all aspects of work ensuring integrity and accuracy.

FU N D I N G I N FO R M ATI O N
None.

CO N FLI C T O F I NTE R E S T
None.

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.

E TH I C A L A PPROVA L
The protocol of the study was approved by IRB at Zagazig university, Egypt. IRB# 9117-30-11-2021. Prior to enrollment, each patient provided an informed consent.