Chuyu FU Miaomiao LI Fanfan CHEN Houyu HE Jia LIU Chunjun YANG Shenghai HUANG

Abstract Balanitis and scrotal dermatitis are common skin diseases of the male external genitalia， causing agony and poor quality of life in patients. Conventional drug therapy is often unsuccessful and has a high recurrence rate. Cold atmospheric plasma （CAP1） is a novel treatment method that is effective in various skin disorders. Herein， we aim to investigate the clinical potential of CAP in treating these two diseases. A total of 17 patients were included （10 with balanitis and 7 with scrotal dermatitis）. All patients received CAP treatment， and we recorded symptomatic changes and graded them accordingly. Our results revealed that 9 patients with balanitis and 6 with scrotal dermatitis were sensitive to CAP treatment. Only one patient relapsed after 6 months of follow-up， while 2 patients showed resistance to CAP treatment. Apart from tingling and itchiness， no other adverse reactions were observed. Overall， we show that CAP is an effective treatment， providing a new treatment avenue for male genitourinary skin disorders.

Key Words： cold atmospheric plasma; balanitis; scrotal dermatitis; skin lesion; wound healing; innovative treatment

Introduction

Cold atmospheric plasma is an ionized gas composed of electrons， positive and negative ions， UV radiation， visible light， thermal radiation and free radicals. Plasma is considered to be the fourth state of matter and is generally electrically neutral 1. With the development of plasma medicine in recent years， researchers uncovered CAP's capacity to kill pathogenic microorganisms 2， accelerate blood coagulation and promote wound healing 3. In vitro experiments demonstrated that CAP could promote stem cell differentiation and induce apoptosis in tumor cells 4.Oxidative stress could trigger the generation of active products in the whole procedure of CAP exposure， mainly reactive oxygen species （ROS2） and reactive nitrogen species （RNS3）， which induce a series of biological effects. Since the temperature of CAP is close to that of the human skin surface （35°C-45°C）， its potential application in the treatment of skin diseases has recently attracted the attention of many researchers. CAP can rapidly inactivate common pathogens on the body surface， such as Staphylococcus aureus， by various mechanisms， and more importantly， it is not limited by drug resistance. In addition， the active molecules induced by CAP treatment can affect cell signal transduction， impacting the body's immune response by regulating related molecules （4）. By down-regulating the expression of related cytokines， CAP can effectively inhibit the progression of psoriasis and improve skin lesions 5. The outer layer of skin is covered with a hydrolipid film composed of glandular secretions to form an epidermal barrier to resist various harmful external stimuli. The changes in its physical and chemical properties result in the occurrence and development of many diseases. The pH value of the hydrolipid film is reduced after CAP exposure， indicating a potential value for the treatment of ichthyosis and atopic dermatitis 6. Given the ability of CAP to kill pathogenic microorganisms， regulate cell signals and change the property of skin barrier， researchers have formerly used CAP clinically to treat dermatoses， such as acne 7， verruca plantaris 8， haily-haily disease 9， diaper dermatitis 10 and superficial mycosis infection 11.64EA0DC8-EA4E-497B-A985-46AD3E31303F

Balanitis and scrotal dermatitis are common conditions of male external genital dermatosis. Due to its anatomical location， the male external genital area is covered in a moist and warm environment for a long time. The infection and inflammation of local skin originate from the growth of pathogenic microbes， mechanical friction， urine residue on the underwear， and chemical irritation. There are multiple risk factors involved in the development of balanitis and scrotal dermatitis， which complicates treatment selection. On the one hand， single-drug treatment is often insufficient for disease management， easily inducing drug resistance leading to recurrence of the disease while combined application of multiple drugs will increase the incidence of adverse reactions; on the other hand， topical drugs themselves are chemical skin irritants， which could aggravate the lesions. According to recent researches， CAP exhibits pronounced bactericidal and anti-inflammatory effects in the treatment of dermatosis. Thus， we believed that CAP could be used to effectively treat clinically refractory balanitis and scrotal dermatitis.

Materials and Methods

CAP device

Our equipment has been improved according to the results of in vitro experiments， animal models and common clinical situations， whereby the plasma jet device was used as a prototype. The equipment is divided into two major modules： the control system and the application system. The control system is an electronic computer equipped with a screen display to regulate the output power. The application system consists of a treatment handle with an electrode sheet connected to the negative pole of the power supply， as shown in Fig. 1a. The end of the treatment handle is designed as a replaceable treatment terminal. The combination of different numbers and arrangement of electrodes ensure that the copper electrode array at the tip， which is further covered by an outer shell of ABS plastic material， can properly deal with skin lesions of various shapes and sizes （Fig. 1b）. The electrode sheet is then connected to the human body to form a closed loop. Plasma is generated between the treatment terminal and the skin by high-voltage electrode discharge. The discharged voltage was set at an adjustable range of 9～15kV， and the ballast resistor was set at 10MΩ to limit the generation of arcs. During the whole process， the distance between the tips of the multi-electrodes and the target area of the skin was 1cm when the long axis of handle was held perpendicular to the lesion. The real-time energy output during the treatment was maintained at about 0.8～1.0j/s.64EA0DC8-EA4E-497B-A985-46AD3E31303F

Recruitment of study samples

Patients were recruited from the Department of Dermatology， the Second Affiliated Hospital， Anhui Medical University， from March 2020 to March 2021. Inclusion criteria were as follows： male patients who have been diagnosed with balanitis or scrotal dermatitis by an outpatient doctor， receiving routine treatment for more than one month with symptoms persisting or recurring episodes. The exclusion criteria were patients with organic heart disease， history of metal implant， and patients who cannot cooperate with treatment and follow-up post-treatment. A total of 17 patients were included in our study， ranging from 23 to 80 years old. All demographic characteristics and therapeutic backgrounds of these patients are shown in the table below （Table Ⅰ）. All procedures performed in this study were in accordance with the Second Affiliated Hospital of Anhui Medical University Institutional Ethical Review Board and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All recruits were notified of the risks of CAP treatment and signed the informed consent forms.

Treatment methods

During the plasma treatment， patients took the supine position with the perineum fully exposed by flexing the hip and knees and separating their lower limbs. The physicians prepared the skin before the operation to avoid discomfort caused by the discharge between the treatment terminal and hair on the skin. A sterile cotton swab dipped in physiological saline was then used to wipe the local skin area to be treated with CAP， removing secretions and scurf on the surface， which enhanced extent of local skin hydration that contribute to the generation of CAP efflux. There are two types of electrodes tips available for treatment： point electrode （6 copper needles） and flat electrode （14 copper needles）， which can be replaced depending on the lesion's area and diameter. The skin of the genital area， especially the scrotum composed of a thinner horny layer， is more sensitive to external stimuli than other regions. Therefore， we limited the exposure time to plasma to about 2 minutes for each area， which could be adjusted based on the individuals pain tolerance. After starting the CAP device， the clinicians slowly guided the electrode along the edge of the lesion and towards the center area until the whole lesion was covered. Clinicians need to pay more attention to the display panel during treatment to stabilize energy output by keeping a steady distance between the electrode and skin. Patients were asked to keep the local area dry and to avoid hot-water baths after treatment. Treatments were conducted once every 2 days; in the initial stage， around 5 rounds were used， while appropriate prolonged intervals were used for the intensive stage. Patients received a six months follow-up after CAP treatment.64EA0DC8-EA4E-497B-A985-46AD3E31303F

Evaluation criteria

The patient's subjective feelings， pruritus and pain， were measured using a Numerical Rating Scale （NRS4）. The treatment effect was recorded and evaluated at 3-time points， baseline， after 2 rounds of treatment and at the end of treatment. Objective signs were assessed by comparing skin lesions before and after treatment， focusing on improving erythema and exudation. All adverse reactions that occurred during the treatment period were included in the evaluation of the efficacy. The treatment endpoint is the patient's subjective symptoms achieving effective remission （with the score decreasing to 0 or by 3 levels） and significant improvement of the skin lesions. Another important index is no recurrence in the next 6-month during follow-up.

Results

A total of 17 people were included in this study. CAP treatment was effective for 15 patients with an effective rate of 88.2%， one patient relapsed， with a recurrence rate of 6.7%.

The symptoms between patients with scrotal dermatitis and balanitis at the baseline were significantly different. Balanitis patients mainly presented with pruritus， with 6 patients having pain in the skin lesions of the glans， 5 had itchy skin lesions， and 1 patient suffered from a combination of both symptoms. In contrast， only 2 patients with scrotal dermatitis had pain from their skin lesions， with all 7 patients having itching symptoms， while two had both symptoms （Table Ⅱ）. After 2 rounds of treatment， the pruritus of balanitis patients improved significantly， 5 patients felt less pain， and 2 achieved complete remission. At the treatment endpoint， 9 patients achieved complete symptom relief， but 1 showed significant resistance to treatment （Fig. 2a and Fig. 2b）. Patients with scrotal dermatitis were more sensitive to CAP treatment， with 6 patients achieving complete symptomatic relief from pruritus after 2 rounds of treatment. At the treatment endpoint， all 6 scrotal dermatitis patients achieved complete relief from pain and more importantly， no recurrence was observed. There was also 1 scrotal dermatitis patient who showed resistance to CAP treatment （Fig. 2c and Fig. 2d）

After a complete CAP treatment course， the remission rate of erythema in patients with balanitis was 88.9% （8/9）， while the remission rate from exudation was 83.3% （5/6）. The remission rate of the two indexes was 100% in patients with scrotal dermatitis （3/ 3） （Table Ⅱ）. Interestingly， we observed that during the CAP treatment course， patients suffering from balanitis showed a quicker relief from symptoms compared to the resolution of their skin lesions （Fig. 3）. On the other hand， for patients with scrotal dermatitis， the symptoms and lesions improved at the same pace （Fig. 4）.64EA0DC8-EA4E-497B-A985-46AD3E31303F

Two patients with balanitis received holmium laser treatment for condyloma acuminatum before CAP treatment. A number of wounds on the glans and erosion in the inner plate of prepuce appeared after holmium laser treatment， and visible improvement could be observed in the next week after receiving several rounds of CAP exposure （Fig. 5）.

Two scrotal dermatitis patients suffered from concurrent tinea cruris with erythema and desquamation covering the bilateral groin area， receiving significant improvement of the lesions after CAP treatment （Fig. 6）.

The majority of subjects expressed mild discomfort during the treatment （12 patients）， and the most common was a transient sting sensation （9 patients） and a burning sensation in the treated area （3 patients）. All discomfort disappeared when treatment ended. Considering the particularity of the treated area， we paid special attention to whether the patient had any changes in urination habits before and after treatment， but no abnormalities were reported. Fifteen CAP-sensitive patients were followed up to 6 months after effective treatment; only one patient relapsed， with a total recurrence rate of 6.7%.

Discussion

A wide range of conditions could lead to balanitis， and pathogenic microorganism infection is the most common etiology 12. A retrospective analysis found that fungal infections， especially Candida albicans， were the most frequent cause of infectious balanitis， accounting for 32% of the total reported cases 13. Scholars have found that patients with diabetes are at an increased risk of acquiring candidal balanitis 13，14. Interestingly， previous in vitro experiments have shown that CAP has a powerful killing effect on various fungi. In our study， 2 out 3 diabetic patients who previously received standardized hypoglycemic therapy were completely cured after CAP treatment. Due to the lack of bacterial culture results， we assumed that CAP might treat topical inflammation by eliminating local foci of Candida albicans. It was reported that more than half of patients with onychomycosis treated with CAP had achieved clinical cure 15， which is in line with our opinion. Overall， ninety percent of patients with balanitis were cured in our study， but there were exceptions. Case A8 used topical antifungal drugs and systemic antihistamines at treatment baseline， but erythema and pain of the glans lesions remained unchanged. After 18 rounds of CAP treatment， little improvement was observed， and the treatment was deemed a failure despite a treatment course of more than 2 months. By comparing demographic characteristics， we found that this patient was the oldest of all subjects， which indicates that age may have an impact on the efficacy of CAP.64EA0DC8-EA4E-497B-A985-46AD3E31303F

In fact， approximately 3%～11% of males are affected by balanitis in their lifetime. On the one hand， recurrent balanitis causes symptoms such as itching， urinary urgency and dysuria， which seriously affect the patients sexual function and quality of life; on the other hand， irregular treatment leads to prolonged disease and the occurrence of various complications， such as ulcers， phimosis and cancer. Balanitis with foreskin involvement can cause paraphimosis， and if the circulation block in the glans is not relieved in time， this can result in necrosis of the penis， which is a life-threatening complication. At present， the clinical treatment of balanitis is mainly based on topical medication 14. Although circumcision is recommended as the most effective prophylaxis to balanitis 12，16， it cannot prevent the risks of bleeding， infection， and poor incision healing. In addition， some scholars believe that circumcision may lead to a serious complication of meatal stenosis 16. There is no record of serious adverse reactions caused by CAP treatment to human bodies in the currently reported cases. Friedman et al. used CAP to treat palmoplantar warts in children and achieved good results 17， which caused less pain than conventional methods such as salicylic acid and liquid nitrogen freezing. This team also conducted a study on actinic keratosis （AK5）， and the results showed that most of the AK lesions were eliminated or effectively relieved after a month of CAP treatment 18，19. There is no reported case of balanitis treatment using CAP to this date， and our team used a customized plasma device to treat 10 patients with refractory balanitis. A total of 8 males recovered after treatment in our study， with a curative rate of 80%， suggesting that CAP could effectively treat balanitis. Scrotal dermatitis is not currently considered as an independent disease entity but as a condition resembling contact dermatitis occurring elsewhere， although this is up for debate.

Scrotal dermatitis is not currently considered as an independent disease entity but as a condition resembling contact dermatitis occurring elsewhere， although this is still up for debate 20. Only a few pieces of literature have been published on scrotal dermatitis， despite being a quite common outpatient occurrence with patients complaining of itching of the scrotum with exudation and desquamation. An 18-month study conducted in India showed that scrotal dermatitis accounted for the highest proportion out of 242 patients treated for non-STD6 genital diseases， exceeding more than 19% 21. Scrotal dermatitis is characterized by severe pruritus. As a result， patients will repeatedly scratch the local skin unconsciously， bringing about social embarrassment and poor sleep quality. In general， if primary scrotal dermatitis is actively managed， the symptoms will improve quite rapidly. However， if allowed to progress secondary to a systemic disease， such as diabetes and chronic renal failure， patients often sustain recurrent scrotal dermatitis， which is often insensitive to conventional medications. In this study， patients with scrotal dermatitis exposed to CAP achieved quick symptom relief after several rounds of treatment. In the pathogenesis of atopic dermatitis （AD7） and prurigo nodularis， local pathogen colonization leads to aggravation of itching 22. Therefore， it is believed that CAP can alleviate itching by killing the colonizing bacteria. In the mouse AD model， the inhibition of infiltrating mast cells and eosinophils in the dermis was observed in the skin tissue following exposure to CAP 23. Furthermore， Gao et al. showed that using a modified CAP device to treat a patient with eczema and pruritus could improve lesion healing compared to traditional drug treatment 24.64EA0DC8-EA4E-497B-A985-46AD3E31303F

In addition， CAP is also effective in treating combined dermatosis. Studies have pointed out that CAP can be utilized for viral warts treatment8，17，25. Our study revealed that CAP could effectively promote the healing of wounds without recurrence in patients with condyloma acuminatum after holmium laser treatment， which is consistent with the results of Gao et al 24. Furthermore， when treating scrotal dermatitis patients with concomitant tinea cruris， we found that the skin lesions in the groin and scrotum areas improved simultaneously.

CAP is deemed to be a safe treatment method. In a CO2 laser-induced wound test， compared with the control group， patients treated with CAP showed no significant differences in local microcirculatory parameters， dermatology life quality index， and scar assessment scale， and no malignancy was found 26. In our study， some patients reported local transient stinging or burning sensations， but all disappeared quickly. No urination habits change was observed in all participants.

However， our study has some shortcomings. We did not design an effective control group， our sample size was quite small， and we did not perform histopathological and pathogenic examinations. This research is only a preliminary exploration of the effect of CAP in the clinical treatment of balanitis and scrotal dermatitis. Future RCT tests with larger samples are needed to further verify whether our conclusion has?general?applicability. Based on our preliminary results and previous studies， we believe that CAP has the notable advantages of wide adaptation， fewer side effects， and is not limited by drug resistance in treating refractory external genital inflammation. Therefore this warrants further exploration and clinical application.

The results of our preliminary research reveal that CAP could be used as a non-invasive， safe and effective treatment strategy for balanitis and scrotal dermatitis patients that had a poor initial response to drug therapy. CAP is quite effective because it can kill various pathogens， prevent bacterial colonization on the skin surface and inhibit local inflammation. Given the incidence of male external genital skin diseases such as balanitis and scrotal dermatitis， CAP could prove to be a potentially effective and safe treatment option in the future.

The authors have no conflicts of interest to declare.

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