FormalPara Key Summary Points

Why carry out this study?

Despite the existence of numerous immunotherapeutic modalities, treatment of warts remains challenging and an optimal treatment is yet to be developed. In fact, no treatment is 100% effective for multiple warts.

No studies to date have objectively compared the safety and efficacy of a triple combination immunotherapy composed of purified protein derivative (PPD), Candida antigen, and measles–mumps–rubella (MMR) vaccine, versus each monoantigen alone, in the treatment of multiple recalcitrant warts.

What was learned from the study?

Triple immunotherapy is a safe and effective therapeutic option for multiple recalcitrant warts, and can be added to the armamentarium against recalcitrant human papilloma virus (HPV) infections.

Intralesional Candida antigen seems to be more favorable in patients with plantar warts, whereas the triple immunotherapy was superior in common warts.

Introduction

Warts are very common benign tumors caused by human papilloma virus (HPV) infection. They are classified according to their appearance or site into different types, including common warts, plane warts, plantar warts, and genital warts [1].

Many destructive and immunotherapeutic modalities have been used in the treatment of warts. Destructive therapies are commonly associated with significant pain, tissue destruction with subsequent scarring, and high recurrence rate. Additionally, these modalities are not practical for patients presenting with numerous warts [2].

On the other hand, several immunotherapeutic agents have been introduced for the treatment of warts to overcome the challenges associated with destructive therapies. Among these immunotherapeutic agents is intralesional antigen immunotherapy, which stimulates cell-mediated immunity (CMI) against HPV to clear not only the injected warts but also distant lesions and has shown promising efficacy and safety in the treatment of different wart types [3].

Despite the existence of numerous immunotherapeutic modalities, treatment of warts remains challenging and an optimal treatment is yet to be developed. In fact, no treatment is 100% effective for multiple warts [4].

The idea of combining different antigens was first tested in 2004 by Johnson and Horn [5], who assessed the efficacy of intralesional injection of a combination of Trichophyton, mumps, and Candida antigen in the treatment of warts in an open-label single-arm study and paved the way for further studies on combining different antigens, in an alternating or in a concomitant manner. This concept was also suggested and proposed by other authors in the following years [6, 7].

In this study, we compared the efficacy and safety of intralesional triple-antigen immunotherapy composed of purified protein derivative (PPD), Candida antigen, and measles–mumps–rubella vaccine (MMR) with that of each monoantigen alone in the management of multiple resistant extragenital warts.

Methods

Patients

This study included 160 patients with multiple (three or more warts) recalcitrant (warts of at least 6 months duration that did not respond to at least two treatment modalities) extragenital warts of different types, sites, sizes, and durations 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 informed consent. This study was performed in accordance with the Helsinki Declaration of 1964 and its later amendments. 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 and those who had any other form of wart management in the month preceding enrollment were also excluded.

Methods

In total, 160 patients were allocated in a random fashion to one of four groups (40 patients each).

Group A: intralesional injection of 0.3 ml of PPD.

Group B: intralesional injection of 0.3 ml of 1/100 solution of Candida antigen.

Group C: intralesional injection of 0.3 ml of MMR.

Group D: intralesional injection of 0.3 ml of PPD, Candida antigen, and MMR, 0.1 ml each, combined in the same syringe.

In each of the four study groups, injections into the largest wart were made, without presensitization testing, using an insulin syringe at 2-week intervals until full clearance, or for a maximum of five sessions.

Evaluation of the clinical response

The size and number of warts were assessed 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 regressed in size by 50–99%; and no response if there was less than 50% decrease in wart size. 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 identify possible recurrence of warts.

Statistical analysis

SPSS version 24 was used to enter, check, and analyze data. For quantitative data, mean and standard deviation (SD) were used; for qualitative variables, number and percentage were used. As appropriate, ANOVA (F test), χ2 test, and Kruskal–Wallis test were employed. P values less than 0.05 were considered significant.

Results

The study and the follow-up period were completed by all patients. The patients were 71 males and 89 females who varied in age from 12 to 67 years (average 27.15 years), with warts lasting between 6 and 72 months. The number of lesions varied between 3 and 40, and the majority of patients had warts greater than 1 cm in diameter (n = 102 patients). Among the studied groups, distant wart prevalence showed no statistically significant difference. Cryotherapy was the most often utilized therapeutic method prior to the study. A total of 72 palmoplantar warts, 68 common warts, 17 subungual/periungual warts, 2 filiform warts, and 1 plane wart underwent injection treatment. The studied groups did not show any significant differences in baseline characteristics (Table 1).

Table 1 Baseline characteristics of the studied patients
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Therapeutic response

1. Treated warts


In triple immunotherapy group, complete response rate was observed in 77.5%, partial response in 7.5%, and no response in 15%.

In the PPD group, 57.5% of patients had complete response, while partial response was noted in 15%, and no response in 27.5%.

In the Candida antigen group, complete clearance was achieved in 72.5%, partial response in 10%, and no response in 17.5%.

In the MMR group, 62.5% experienced complete clearance, 17.5% showed partial response, and 20% had no response.

The complete response rate was higher in the triple intralesional immunotherapy group than in the other three groups; however, the difference was not statistically significant. Similarly, there was no statistically significant difference in the rate of wart clearance (time to complete response) (Figures 1, 2, 3, 4; Table 2).

Fig. 1
figure 1

a Multiple plantar and common warts on the left foot, b Complete clearance after only one session of triple immunotherapy

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Fig. 2
figure 2

a A single palmar wart injected on the left hand. b Multiple distant common warts on the dorsum of the right hand, c and d Complete response of all the warts after five sessions of intralesional PPD injection

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Fig. 3
figure 3

a Multiple plantar warts on the sole of the left foot. b Complete clearance of all warts after five sessions of intralesional Candida antigen injection

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Fig. 4
figure 4

a Multiple common warts on the dorsum of the right foot. b Complete clearance of all warts after five sessions of intralesional MMR

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Table 2 Therapeutic response among the studied patients
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2. Distant warts


Complete response of the distant warts was noted in 48%, 61.5%, 55.5%, and 65% of the PPD, Candida antigen, MMR, and triple immunotherapy groups respectively, with no statistically significant difference among the studied groups.

Relationship between therapeutic response and different clinical variables

No significant relationship was found between the therapeutic response and the different clinical variables, including type, size, number, and duration of warts, in any of the four groups.

Complete cure of common warts was noted in 6/11 (54.5%), 7/13 (53.8%), 10/20 (50%), and 20/24 (83.3%) in the PPD, Candida antigen, MMR, and triple immunotherapy groups, respectively. Palmoplantar warts were cleared in 15/22 (68.2%), 20/22 (90.9%), 11/15 (73.3%), and 10/13 (76.9%) in the PPD, Candida antigen, MMR, and triple immunotherapy groups, respectively. For subungual/periungual warts, complete response was achieved in 2/5 (40%), 2/4 (50%), 4/5 (80%), and 1/3 (33.3%) in the PPD, Candida antigen, MMR, and triple immunotherapy groups, respectively.

Adverse effects

There was no statistically significant difference in adverse effects among groups. The most common side effect was pain during injection, which was present in all cases, followed by edema and erythema (Table 3).

Table 3 Adverse effects among the studied groups
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Recurrence

No statistically significant difference in the recurrence rate after the 6-month follow-up period was observed between the studied groups, where recurrence was reported in 8.7%, 13.8%, 12%, and 6.45% of the PPD, Candida, MMR, and triple immunotherapy groups, respectively.

Discussion

The idea of combining different antigens was conceptualized in 2004 by Johnson and Horn [5], who assessed the efficacy of intralesional injection of a combination of Trichophyton, mumps, and Candida antigen in the treatment of warts and paved the way for further studies on combining different antigens, in an alternating or concomitant manner.

Moreover, Aldahan et al. [6] and Marei et al. [7] have proposed that the combination of antigens with different modes of action might improve the efficacy, and decrease the adverse effects and recurrence potential, by targeting multiple immune pathways simultaneously.

On the basis of these observations, we decided to evaluate the efficacy and safety of a triple intralesional immunotherapy combination composed of PPD, Candida antigen, and MMR, versus each agent alone, in the treatment of multiple recalcitrant warts.

In the triple intralesional immunotherapy group, complete clearance of warts was observed in 31 patients (77.5%) (5 palmar warts, 5 plantar warts, 20 common warts, and 1 subungual wart). To the best of our knowledge, this triple combination was not previously studied. However, Johnson and Horn [5] used another combination of Trichophyton, mumps, and Candida antigen (0.1 ml each; combined in one syringe) and observed a very similar response rate (70.9% complete clearance). .

Furthermore, Horn et al. [8] compared a combination of single-agent antigen (mumps, Candida, or Trichophyton) plus interferon alpha-2b (IFNα-2b) versus single-agent intralesional antigen and intralesional IFNα-2b. The superiority was in favor of the combined therapy (68%) followed by the individual antigen (54%) and intralesional IFNα-2b (26%); however, the differences were not statistically significant. Adding IFNα-2b did not improve response compared with injection of antigen alone. Notably, the combined group reported more side effects (73%), compared with only 13% in the antigen alone group. These results suggest a slightly increased immunogenic effect of combination therapy at the expense of greatly increased adverse effects.

King et al. [9] further tested the idea of antigen combination immunotherapy for the treatment of genital warts. Patients received either single-agent treatment (mumps, Candida, or Trichophyton) or combination treatment with mumps, Candida, and Trichophyton skin test antigens (combination 1), or with Candida, and Trichophyton skin test antigens (combination 2). Complete clearance of genital warts (50%) was observed only in those receiving the combination therapies.

Combined immunotherapy, in an alternating manner, was also tried by Nofal et al. [10], who compared an alternating therapy of PPD and Candida antigen versus therapy of each individual antigen. A statistically significant difference in the therapeutic response was found between the alternating therapy group (complete clearance in 70.6%) and the individual antigen groups (61.1% of the PPD group and 36.8% of the Candida antigen group). Adverse effects were insignificant in the studied groups.

Moreover, a statistically significant difference in favor of a combination immunotherapy (70% cure rate) composed of bivalent HPV vaccine (injected intramuscularly) and Candida antigen (injected intralesionally) versus Candida antigen monotherapy was noted in a study conducted by Marei et al. [7]. A summary of the studies concerned with combination intralesional antigen immunotherapy is presented in Table 4.

Table 4 Summary of studies on combination intralesional antigen immunotherapy for the treatment of warts
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In the PPD group of the current study, 57.5% of the 40 studied patients showed complete response (23 patients; nine palmar warts, six plantar warts, six common warts, and two subungual warts). Our results for PPD were very similar to those reported by Kaimal et al. [11] (55%), Shaheen et al. [12] (60%), Nofal et al. [10] (61.1%), and Mohammed et al. [13] (60%).

On the other hand, the clearance rate with intralesional PPD in the present study was much lower than that reported by Wananukul et al. [14] (93%), Elela et al. [15] (94.1%), Abd-Elazeim et al. [16] (75%), Amirnia et al. [17] (77.1%), and Saoji et al. [18] (76%). This difference may be explained by the lower number of treatment sessions and injection of PPD in the largest wart only in our study compared with a higher number of treatment sessions and injection of multiple warts in other related studies.

In the Candida antigen group, complete clearance was achieved in 72.5% of the studied patients (29 patients; 1 palmar wart, 19 plantar warts, 7 common warts, and 2 subungual warts). This clearance rate was much lower than that reported by Perman et al. [19] (100%), Maronn et al. [20] (87%), and Kim et al. [21] (82%), but similar to those reported by Phillips et al. [22] (72%), Johnson et al. [23] (74%), and Johnson and Horn [5] (71%).

Our clearance rate (72.5%) was much higher than that reported by King et al. [9] (50%), Horn et al. [8] (54%), Majid and Imran [3] (56%), Alikhan et al. [24] (39%), Nofal et al. [25] (33.3%), Marei et al. [7] (40%), and Eldahshan et al. [26] (43.33%). Our higher response rate may be attributed to the injection of a higher dose of Candida antigen (0.3 ml) compared with doses (0.1 ml) injected in many of the previously mentioned studies.

In the MMR group, 62.5% of the treated 40 patients showed complete clearance (25 patients; eight palmar warts, three plantar warts, ten common warts, and four subungual warts). This response rate was very similar to that reported by Nofal et al. [27] (63%) and Fawzy et al. [28] (62.5%), higher than that reported by Shaker et al. [29] (30% cure rate), and lower than that reported by Eldahshan et al. [26] (73.33%) and Mohammed et al. [13] and Shaheen et al. [12] (80%). The higher clearance rate in these studies might be attributed to the higher reactivity to MMR and the higher number of the treatment sessions.

In the present study, no statistically significant relationship was found between the therapeutic response to intralesional PPD, Candida antigen, MMR, and combined therapy, and the different clinical variables. This was in agreement with many of the related studies that also reported absence of significant associations between the therapeutic response to the injected antigen and most of the clinical variables [12, 23, 28].

Adverse effects were mild and insignificant in all the groups, as was the case in many of the previous trials of intralesional antigen immunotherapy [5, 23, 30]. It is worth noting that the adverse effects were not statistically higher in the combination therapy group than in the other monotherapy groups, indicating that combining more than one antigen is still associated with high safety profile.

In the current study, no statistically significant difference in the recurrence rate was reported between the studied groups. Recurrence was noted in 2/23 patients treated with PPD (8.7%), 4/29 patients treated with Candida Ag (13.8%), 3/25 cases treated with MMR (12%), and 2/31 patients injected with triple immunotherapy (6.45%). This recurrence rate is relatively higher than that observed in most of the related studies. [12, 23, 28]. This may be attributed to the different baseline characteristics and the recalcitrant nature of warts in this study.

It worth noting the heterogeneity of results of intralesional antigen immunotherapy, which is mostly attributed to the variable baseline characteristics of patients, and the different features and nature of warts. Studies of Candida antigen, MMR, and PPD provided comparable complete response rates of injected warts, ranging from 39% to 88% for Candida, 26.5% to 92% for MMR, and 23.3% to 94.4% for PPD [12, 31].

One of the important observations in the current work is that intralesional Candida antigen seems to be more favorable in patients with plantar warts, whereas the triple immunotherapy was superior in common warts. Thus, Candida antigen may be recommended as immunotherapy for plantar warts and the triple immunotherapy, when available, for common warts.

Ultimately, it seems that triple intralesional immunotherapy has its pros and cons. The advantages are that triple immunotherapy was superior to, and as safe as, monoantigen immunotherapy in recalcitrant warts, which may be attributed to more robust stimulation of CMI by induction of diverse immune pathways by various microbial antigens and differential nature of vaccines, each acting on a specific arm of the immune response, resulting in optimization of results.

Moreover, since most people are reactive to at least one skin test antigen, there is no need to conduct a skin test when administering three skin test antigens for warts. For physicians who prefer to routinely adopt presensitization tests, eliminating skin tests enhances the cost-effectiveness of triple therapy and saves time as well as an extra-office visit for the intradermal test.

The main disadvantage is that it is difficult to have all three antigens available at the same time. Nevertheless, when available simultaneously, the combination therapy could be tried in warts recalcitrant to monoantigen injection. Moreover, the difference between triple immunotherapy and monoantigen immunotherapy was not statistically significant; however, this needs to be confirmed by further studies on larger populations. Therefore, we do not recommend triple immunotherapy as a routine therapeutic modality.

Spontaneous regression of warts is a well-recognized phenomenon; however, in the present study, the long duration of warts (lowest median was 20 months) that failed to respond to at least two treatment modalities makes it unlikely that the complete resolution reported in our patients was spontaneous. The clearance of the distant, non-injected warts, a well-established effect of the intralesional antigen immunotherapy, in a significant proportion of patients in the four studied groups further confirms that the wart regression was not spontaneous.

Limitations of the present study include the relatively small sample size and the short follow-up duration. Therefore, further studies on larger populations with long term follow-up of the patients are highly warranted to assess the long-term efficacy of each therapeutic modality in prevention of recurrence.

Conclusion

In conclusion, triple immunotherapy is a safe and effective therapeutic option for multiple recalcitrant warts, and can be added to the armamentarium against recalcitrant HPV infections.