Human papilloma virus (HPV) is a double-stranded DNA virus that causes cutaneous viral warts, most commonly located on the skin and genitalia 1. Plantar warts are painful, endophytic papules that have deeply penetrating sloping sides and a central depression 2. Numerous coalesced warts on the plantar surface will form a tile-like pattern known as mosaic warts 3.
Currently, there is no cure for HPV infection, and therapy does not affect transmissibility 1. Recurrence after clinical cure is often owing to latent virus versus re-infection 4. The presence of HPV-DNA in subclinical or latent forms can be detected by nested PCR and hybridization 4,5. Current therapies aim at eliminating signs and symptoms 6. Currently available HPV treatments can be grouped into destructive (e.g. cantharidin and salicylic acid), virucidal (e.g. cidofovir and interferon-α), antimitotic (e.g. bleomycin and podophyllotoxin, 5-fluorouracil), immunotherapy (e.g. Candida antigen, contact allergen immunotherapy, and imiquimod), or miscellaneous (e.g. trichloroacetic acid and polyphenon E) 7.
Formaldehyde and liquid nitrogen cryotherapy are some of the agents that are used for treatment of warts. Formaldehyde is a virucidal agent and works by disrupting the upper layer of epidermal cells and possibly damaging the virions 4, whereas liquid nitrogen crytherapy does not kill the virus, rather its effect on wart clearance may be through necrotic destruction of HPV-infected keratinocytes or by inducing local inflammation that triggers an effective cell-mediated response 6.
The aim of the current study was to compare the clinical, histopathologic, and viral load changes [in-situ hybridization (ISH)] in multiple plantar warts following two different therapeutic modalities: topical 10% formaldehyde soaks and liquid nitrogen cryotherapy spray technique.
Patients and methods
The present study included 28 patients with clinically and histopathologically diagnosed plantar warts. Patients were recruited from those attending the outpatient clinic of Dermatology Department, Minia University Hospital, Al-Minya, Egypt. The study was approved by institutional Research Ethics Committee of Faculty of Medicine, Minia University. Each patient or guardian signed a written consent to be involved in the study. Patients included in the study were those with plantar warts (single or multiple) or mosaic warts with no history of prior treatment, at least for 1 month before enrollment in the study. The exclusion criteria were pregnant or lactating females, children younger than 12 years, patients with secondarily infected warts, those with a known hypersensitivity to formaldehyde, and those with cold urticaria.
Patients were randomized into two groups: group I (13 patients) and group II (15 patients) that received topical 10% formaldehyde soaks and cryotherapy, respectively. Restricted randomization method ‘adaptive biased-coin randomization’ was used 8.
- Group I: patients in this group were treated using topical 10% formaldehyde soaks for 5 min that were applied by the patients at home twice daily. A barrier of soft paraffin was applied to the surrounding areas. The patient used a brush for application, with no occlusion. The patients waited for a few minutes until the skin was thoroughly dry. The treatment continued for a period of 3 months or until complete clinical clearance of the lesions, whichever was shorter (the end point).
- Group II: patients in this group were treated with cryotherapy spray technique using a hand-held device (Brymill’s Cry-Ac; Brymill Cryogenics Systems, Ellington, USA). Two freeze-thaw cycles, 15 s each, were applied by the physician every 2 weeks for 3 months or until complete clinical clearance of the lesions, whichever was shorter (the end point).
Clinical status and improvement regarding changes in the size and number of plantar warts were evaluated and rated by two independent dermatologists before treatment and every 2 weeks for 3 months or until complete clinical clearance of the lesions, whichever is shorter. The efficacy of the treatment was evaluated by considering a complete response, when there is a total disappearance of the warts; a partial response, when there is clinical reduction in the number and/or size of the warts (>50%); and no response, when no reduction was seen in the number and/or size of the warts at the end of treatment period. All patients in two treatment groups were followed up monthly for another 3 months after the end of treatment period to report any recurrences. Data were recorded regarding recurrences and adverse effects that appeared in relation to the treatment during the study period.
Histopathologic and in-situ hybridization analysis
Punch skin biopsies were obtained from one untreated wart in every patient (to confirm clinical diagnosis) and from the site of resolved lesion or from an unresolved lesion at the end of the study duration (3 months) to detect histopathologic changes after treatment and for ISH.
Sections were subjected to ISH technique to detect the viral load changes before and after treatment. ISH was done using the following: (a) Digene HPV tissue hybridization kit (Cat# 4206 0100) and (b) Digene HPV omni probe set (Cat# 4206 1032) (Digene Diagnostic Inc., Ellington, USA). Digene HPV tissue hybridization kit is based on colorimetric ISH techniques, which detect the presence of nucleic acid in cells or tissues that appear as a purplish blue precipitate at the sites of probe hybridization to HPV-DNA. The Digene HPV omni probe set is the biotinylated nucleic acid probe for the detection of HPV in neutral, buffered formaldehyde fixed, paraffin-embedded skin biopsy. Positive cells were counted in each section using ×400 magnification. Quantitative counting of positive cells was done in five high-power fields with measurement of the mean value for each biopsy. Evaluation was done by two independent pathologists blinded to the treatment protocol.
Data were analyzed using Excel (Microsoft, One Microsoft Way, Redmond, Washington, USA) for Windows. Descriptive statistics in the form of the percentage, mean, and SD were calculated and were expressed in the form of mean±SD. Comparison between the two groups of patients was done using χ2 test for qualitative data, and Wilcoxon’s signed ranks test and Mann–Whitney test for quantitative data. Significance was expressed in terms of P value, which was considered significant when it was less than or equal to 0.05.
The present study included 28 patients, 22 males and six females, with clinically and histopathologically diagnosed plantar warts. The age of patients ranged from 12 to 55 years (24.5±9.39 years). Group I (formaldehyde group) included 10 males and three females, whereas group II (cryotherapy group) included 12 males and three females. The demographic data of both groups were statistically comparable with no statistically significant difference regarding age, number, and duration of lesions between the two groups (Table 1).
The difference in mean number of warts between two groups before treatment was statistically insignificant. After treatment, the mean number of lesions decreased significantly in both groups (P=0.007). When the mean number of warts in two groups was compared after treatment, it was statistically insignificant (P=0.827) (Table 2).
Collectively, in group I, six (46.2%) patients of 13 treated with formaldehyde soaks achieved complete clinical cure (Fig. 1a–c), one (7.7%) patient achieved partial improvement, and failure of treatment was reported in six (46.2%) patients by the end of 3 months. Complete cure was reported in one patient during follow-up period. No recurrences were reported by patients by the end of the follow-up period.
In group II, seven (46.7%) patients of 15 patients treated with liquid nitrogen cryotherapy achieved complete clinical cure (Fig. 2 a and b). Partial improvement was achieved in five (33.3%) patients, and failure of treatment was noted in three (20%) patients by the end of 3 months, with no further healing or recurrences being reported during the follow-up period. On comparing both groups, there was no significant difference regarding clinical outcome (P=0.164) (Table 3). Collectively, the treatment was more clinically effective in those patients having small warts (<0.5 cm) than those having medium (0.5–1 cm) and large (>1 cm) warts. Accordingly, warts of short duration gave a faster response than chronic lesions (>6 months) in both groups, regardless of the method used for therapy.
Histopathological diagnosis of lesions was based on the identification of the hallmarks of warts in hematoxylin and eosin-stained sections under microscope (obtained from lesions before treatment) (Figs 3a and 4a). After treatment, sections obtained from group I showed sheets of epidermal cell necrosis that involve the entire thickness of the epidermis in warts that showed complete clinical cure, whereas areas of upper epidermal necrosis only were detected in cases of partial clinical improvement (Fig. 3b). In group II, after treatment, sections from completely cured lesions demonstrated complete absence of parakeratosis with foci of epidermal necrosis (Fig. 4b) and disappearance of mononuclear cells in dermis. Sections from lesions of partial clinical response showed decrease in size and number of vacuolated keratinocytes with decrease in clumps of keratohyaline granules and decreased mononuclear cells in dermis when compared with untreated lesions.
Human papilloma virus-DNA in-situ hybridization (viral load)
Before treatment, there was no significant difference between the mean number of cells with positive staining for HPV-DNA in both groups (P=0.763) (Figs 3c and 4c). After treatment, the mean number of positive cells decreased significantly in both groups (P=0.001) (Figs 3d and 4d). When post-treatment results of both groups were compared, no statistically significant difference was found in the reduction of the number of cells with positive HPV-DNA staining (P=0.622) (Table 2).
In both groups, post-treatment biopsies obtained from cases that achieved complete clinical cure were completely negative for HPV-DNA staining. However, biopsies obtained from cases that achieved ‘partial clinical improvement’ still showed positive HPV-DNA staining, with a lesser number of positive cells.
Safety, tolerance, and compliance
Among patients of group I, the main adverse effect reported by some patients was dryness and discoloration of plantar skin (Fig. 1b) that started to appear few days after commencing treatment; none of the patients complained of allergic contact dermatitis owing to formalin application. On the contrary, among the patients of group II, the main adverse effect reported by all patients was pain during and for few hours after the procedure, and scarring in one patient. However, in both treatment groups, the adverse effects were well tolerated and accepted by patients. Participants’ compliance in both treatment groups was high; none of the patients left the study because of adverse effects.
HPVs are small DNA viruses of the papovavirus family, with more than 100 types already described. Plantar warts are more commonly associated with HPV 1 and sometimes HPV 4. Other rare types include HPV 57, 60, 63, 65, and 66. Although there are various therapies available, these types of viral warts are notoriously difficult to treat 9.
Formaldehyde is a virucidal agent and works by disrupting the upper layer of epidermal cells and possibly damaging the virions 4. Despite its viricidal action, formaldehyde as a treatment of viral warts was only the subject of few studies. One controlled trial had used formaldehyde soaks 3% to treat plantar warts in 192 participants and reported a cure rate of 61% 10. Our results showed that six (46.2%) of 13 patients treated with formaldehyde soaks (group I) achieved complete clinical cure by the end of treatment period. Our findings are consistent with the study of Jennings et al.11, who conducted a clinical trial using the same concentration of formaldehyde 10% alone versus monochloroacetic acid and 10% formaldehyde, and they reported that the overall cure rate was 61.4%, with no statistically significant difference in the cure rate between treatment groups. The only adverse effect noticed by some patients in our study was dryness and discoloration of treated areas; none of our patients complained of irritation or allergic reaction owing to formalin application, suggesting a resonable safety profile that could encourage further studies on a larger number of cases to confirm its effecacy and safety.
Our results showed that HPV-DNA viral load following treatment with formaldehyde soaks decreased significantly and had become totally negative in those cases who achieved complete clinical cure; however, only partial decline was detected in viral load in those who achieved partial clinical cure or even failed cases. This suggests that formaldehyde’s beneficial effect on clearing viral warts might be related to a possible in-vivo viricidal effect towards HPV; however, this needs further investigation. To the best of our knowledge, our work is the first to explore viral load changes in cutaneous warts following treatment with formaldehyde soaks.
The most commonly used cryogen to treat warts is liquid nitrogen with a temperature of −196°C. Cure rates for cryotherapy vary widely, depending on the treatment regimen 12. Traditionally, cryotherapy is applied until the wart has a 2-mm white halo around it. An aggressive or longer freeze maintains a white halo for 5–20 s 4; which is more effective than the traditional method 13. It was also reported that the double freeze-thaw technique is more effective than a single freeze technique for plantar warts 14 and that 2-week interval is optimal for cryotherapy treatment for plantar warts 15. In the present study, we have used the aggressive cryospary technique in a double freeze-thaw cycle once every 2 weeks. Using this cryotherapy technique, we were able to achive clinical clearance of plantar warts in 46.7% of patients included in group II. Previous studies using cryospary gun had reported cure rates ranging from 44 15 to 52% 10. The results of cryotherpay techniques used in our work falls within the same range.
Furthermore, our results showed that HPV-DNA viral load after cryotherapy was significantly decreased and had become totally negative in those cases that achieved complete clinical cure, though only partial decline in viral load in those who achieved partial clinical cure or even failed cases. A previous study exploring changes in HPV-DNA viral load following liquid nitrogen cryotherapy for cutaneous warts had shown results ranging from only 14% HPV-DNA clearing for plantar warts to 18% HPV-DNA clearing for genital warts 16. This suggests that the technique that we have used in our work was more superior in clearing the HPV-DNA than previous reports.
The clinical cure rates as well as the reduction in HPV-DNA viral load of the two treatment modalities used in the present work are quite similar as there was no statistical significant difference in either clinical outcome or in viral load changes after treatment in both groups. This suggests that both treatments are equally effective. Our results are consistent with the study of Banihashemi et al.17, who indicated that 80% phenol and cryotherapy are equally effective and simple treatments for common warts of hands. However, as our cure rates revolve around 50% in both groups, we suggest that these treatment modalities may not be considered as a first-line treatment for plantar warts. This view may be supported by the Cochrane review, which had reported that cryotherapy is no more effective than simple topical treatments for nongenital cutaneous warts 18. Cryotherapy is often used for the treatment of warts, but it is less convenient, more painful, and also more expensive. Aggressive cryotherapy appears to be more effective than gentle cryotherapy, but with an increased risk of adverse effects, such as pain, blistering, scarring, and postinflammatory pigment alternation (hyperpigmentation or hypopigmentation) 19. Local anesthesia (topical or injected) might be needed to facilitate therapy if warts are present in many areas or if the area of warts is large 20. In addition, multiple treatment visits are needed for complete cure by liquid nitrogen, a process that can take more than 2 months. Furthermore, it may be unsuitable for children because they cannot be still during treatment.
In conclusion, the results of the present study suggested that treatment of multiple plantar warts using either formaldehyde soaks or liquid nitrogen cryotherapy yields almost similar cure rates as well as similar reduction in HPV-DNA viral load. However, as formaldehyde soaks are equally effective and less costly, it may be more suitable for patients of low economic standard. Moreover, there is no pain associated with application of formalin soaks like that may be experienced with cryotherapy, with no risk of scaring, and it treats the entire plantar skin all together and is used by the patients at home, with no need for multiple visits for clinic; these may favor the use of formalin soaks over cryoherapy as a treatment for multiple and extensive plantar warts. Irritation from formalin is temporary and reversible. Furthermore, its possible in-vivo HPV viricidal effect should direct the attention for further research utilizing this remedy. One of the main shortcomings of the present work is the small number of cases included in each treatment group. As our study had utilized safely formaldehyde 10% soaks twice/day, we recommend home treatment of warts with formalin soaks, unless contraindicated. We also suggest that further researches should focus on the study of its safety and efficacy in a larger number of patients to support these findings.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2018 Egyptian Women's Dermatologic Society
formaldehyde; human papilloma virus-DNA viral load; in-situ hybridization; liquid nitrogen cryotherapy; plantar warts