Onychomycosis is the most common infectious nail disorder, with a reported prevalence of approximately 5.5% in the general population¹. This disease is commonly caused by fungal infections, dermatophytes, especially Trichophyton (T.) rubrum, nondermatophyte molds (NDMs), and yeasts. Recently, bacteria-fungal coinfections and fungal biofilms in onychomycosis have been discovered, leading to difficulty in treating onychomycosis^2-5. It has also been reported that resistance to antifungal agents is increasing⁶.

Plasma is the fourth state of matter comprising electro- magnetic radiation, excited gas, ions, reactive species, and free radicals. One of the well-known effects of plasma is its ability to eliminate bacteria, affording it antimicrobial effects⁷. However, previous studies have shown that plasma also has antifungal properties^8-11. In particular, the nonthermal atmo- spheric plasma (NTAP) has demonstrated its ability to kill clinically important fungal species like T. rubrum, Microsporum canis, and Candida albicans in vitro⁸. Recent studies have also shown the effectiveness of NTAP in treating fungal infections using a human nail model. In this study, we aim to evaluate the efficacy of NTAP in treating T. rubrum and analyze the ultrastructural changes in the fungus before and after NTAP treatment^11,12.

In vitro experiments were conducted to assess the effect- iveness of NTAP against fungal infections and to determine the optimal duration of fungal inhibition required for ex vivo experiments.

1. T. rubrum strain and culturing in vitro

In this in vitro study, a fungal strain called T. rubrum was isolated from patients who visited our clinic and gave informed consent. The patients were diagnosed with onychomycosis, tinea pedis, or tinea cruris through mycological examination, such as potassium hydroxide (KOH) smear, fungal culture, and periodic acid-Schiff staining. The strain was confirmed to be T. rubrum through fungal culture. The isolate was then inoculated on Sabouraud dextrose agar (SDA) plates and incubated at 30℃ for a week.

Twelve samples were taken using an 8 mm punch biopsy instrument and transplanted into four new SDA plates, with three colonies per plate. The colonies were then exposed to NTAP jet plasma for 0, 10, and 30 minutes (as shown in Fig. 1), and photographs were taken daily with a digital camera (Canon EOS 80D). The colony sizes were measured using Image J software to assess their growth.

Figure 1. Preparation of T. rubrum discs in vitro. T. rubrum discs were obtained using an 8-mm punch and moved to a new agar plate for plasma exposure.

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2. Ex vivo nail specimens

Nail samples were collected from twelve patients with in- grown nails or onychogryphosis combined with onychomycosis. After obtaining informed consent from the patients, the nails were partially removed for medical purposes (Institutional Review Board of KHUH 2020-09-034). The nails were exam- ined through mycological studies using techniques like KOH smear and fungal culture. The samples infected with T. rubrum were included in the study. Each nail sample was divided into two parts. One part was left untreated, while the other was exposed to plasma for 30 minutes.

3. NTAP irradiation

We utilized NTAP generated using argon gas with 99.99% purity at 40℃. The argon gas flow rate was a constant 1.5 liters per minute (lpm) during NTAP generation. The specimens were placed 3 mm away from the bottom of the NTAP flame and were directly exposed for either 10 or 30 minutes.

4. Scanning electron microscopy (SEM)

To analyze the morphological changes in fungi, the samples of fungal colonies and nail specimens were prepared as done in previous studies^13,14. The samples were fixed with 4% paraformaldehyde and 2.5% glutaraldehyde at 4℃. They were then washed with 0.1 M sodium cacodylate buffer (pH 7.4) for 10 min. The subsequent procedures were performed using an automatic tissue processing device. Specimens were fixed with osmium tetroxide for 2 h, cleaned in distilled water for 2 × 10 min, and dehydrated subsequently in 50%, 70%, 90%, and 100% ethanol for 10 min each, followed by washing in solutions with 1:1, 3:7, and 1:9 ratios of alcohol: acetic acid for 10 min each, pure isoamyl acetate for 10 min, and subsequently dried in hexamethyldisilane amine dioxane. The samples were observed under a field-emission scanning electron microscope (Inspect F, FEI, USA) before and after NTAP.

5. Reflectance confocal microscopic (RCM) examination

Ex vivo morphological changes of fungal nail specimens were analyzed with RCM (VivaScope○R 1500, Lucid Inc., Rochester, NY, USA) before and after NTAP irradiation at a laser power below 5.8~6.0 watts.

6. Statistical analysis

The colony sizes obtained from the in vitro study were analyzed using the Kruskal-Wallis and Mann-Whitney tests with SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA) at a significance level of p<0.05.

1. In vitro: The change of colony sizes after NTAP irradiation in vitro

The study's results are presented in Fig. 2, showing the median colony sizes between days 0 and 7 after exposure to NTAP irradiation for different durations. The initial colony size was approximately 0.503 cm2. Compared to the control group that was not irradiated, the size of the colonies exposed to 10 minutes of irradiation was reduced to 0.49 cm2 on day 2. Still, the colony sizes gradually increased after day 2. Colonies exposed to 30 minutes of irradiation demonstrated progressive reductions in size from days 1 to 7, resulting in significantly reduced sizes on day 7 (0.421 cm2). When com- paring the macroscopic morphologies of colonies, it was observed that the peripheral growth zone was destroyed in the 30 min-irradiated colonies. In contrast, the peripheral zone was visible in the control colony (Fig. 2).

Figure 2. Growth of T. rubrum colonies irradiated with different plasma exposure times. From days 1 to 7, the area of non-exposed colonies consecutively increased, but 10-minute exposed colonies decreased until day 2, followed by an increase. The area of 30-minute exposed colonies was significantly smaller than that of the other colonies.

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The structure of T. rubrum was examined using SEM, as shown in Fig. 3. In the untreated T. rubrum sample, smooth-surfaced, long, and branching hyphae were observed, along with a biofilm covering the hyphae (Fig. 3A). The hyphae of the T. rubrum sample treated with NTAP shrank and cracked (Fig. 3B). After 30 minutes of NTAP irradiation, the hyphae structures appeared severely damaged; no smooth-surfaced, long hyphae or biofilm structures were detected (Fig. 3C).

Figure 3. Representative scanning electron microscopy images of T. rubrum colony in vitro. (A) Without plasma exposure, intact T. rubrum was observed as a network of hyphae. Yellow arrows denote the exopolymeric matrix of the biofilm covering the hyphae. (B) The hyphae of T. rubrum turned rough and shrunken after plasma exposure for 10 minutes. (C) Intact hyphae structures were rarely observed after plasma treatment for 30 minutes (×5,000).

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2. Ex vivo: Effects of NTAP irradiation on the ultrastructure of fungal infected nails by using SEM

SEM was used to examine nail specimens before and after NTAP irradiation. A total of 362 images from 12 infected nails were analyzed. Fig. 4A and D show images of untreated nails. Long and intact hyphae structures, along with numerous rod-shaped bacteria, were observed (Fig. 4A). Fungal spores with smooth globular structures were also observed (Fig. 4D). After NTAP exposure for 30 minutes, the structure of hyphae changed to rough and short, and the rod-shaped bacteria around the hyphae disappeared (Fig. 4B and C). Several fungal spores ruptured and collapsed (Fig. 4E).

Figure 4. Scanning electron microscopy images of T. rubrum and infected nails ex vivo. SEM images of infected nails before (A, D) and after (B, C, and E) the plasma irradiation (×5,000). (A) The hyphae were straight and smooth in shape before the irradiation. (B) The hyphae became rough and broken, and numerous surrounding rod-shaped microorganisms disappeared. (C) The hyphae had rough and irregular surfaces after plasma irradiation. (D) The spores were smooth and round before the irradiation. (E) Several ruptured spores (yellow arrows) were observed after the irradiation.

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The cross-section of the nail plate under SEM revealed that the porosity increased after NTAP exposure (Fig. 5B) compared to the untreated nail (Fig. 5A).

Figure 5. Scanning electron microscopy images of the nail cross-section ex vivo. (A) Before the irradiation, the nail showed several small holes. (B) After the irradiation, the nail plate showed increased porosity and bigger lacunae. The SEM images of the nail cross-section before and after the plasma exposure are shown in Fig. 5.

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3. Use of RCM to evaluate the effects of NTAP irradiation on nail ultrastructure in nails with fungal infections

Long filamentous hyphal structures and spherical fungal spores were identified in the infected nails (as seen in Fig. 6A and C). Post-NTAP irradiation, the hyphal structures shortened and burst, and grouped fungal spores scattered (as seen in Fig. 6B and D).

Figure 6. Reflective confocal microscopic images of infected nails ex vivo. (A, C) The long hyphae and grouped fungal spores (yellow arrows) were shown before irradiation. (B, D) The hyphae burst (red arrow) and broke, and fungal spores scattered after irradiation. The RCM images of the infected nails before and after the plasma exposure are shown in Fig. 6.

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NTAP has attracted increasing interest as a possible treat- ment for infectious diseases^15,16. We applied NTAP to T. rubrum, the most common cause of fungal skin infections. The results showed that NTAP inhibited fungal growth in vitro and induced morphological changes in the fungus in vitro and ex vivo.

Importantly, fungal growth inhibition depended on plasma exposure duration. Although a 10-minute exposure initially proved sufficient to prevent growth, after 7 days, NTAP ex- posure for 10 minutes had a similar effect to that of the untreated control. In contrast, a 30-minute exposure demon- strated a significant fungistatic effect. Moreover, the dose-dependent fungistatic effect was confirmed by the structural changes observed under SEM in vitro (as shown in Fig. 4).

Further examination of T. rubrum or nail specimens in vivo and ex vivo, respectively, under SEM and RCM after NTAP irradiation, showed that the hyphae changed from long and smooth to rough and bursting, the spores ruptured, and the fungal biofilm was destroyed. This suggests that NTAP has a fungistatic and even fungicidal effect in vitro and ex vivo.

Although there have been many hypotheses about the mechanisms of biological effects of NTAP, some researchers have postulated an association with oxidative damage caused by reactive species, such as reactive oxygen or nitrogen species, generated as a product of plasma interactions with the surrounding media^15,17-19. Therefore, it is hypothesized that the antifungal effects of NTAP are synergistically attributed to (1) oxidative damage caused by reactive species and (2) partial mechanical damage to fungus, based on the micro- scopic changes in fungal structures we observed.

NTAP can enhance drug permeation, as shown in the larger size and number of pores observed in the plasma-exposed nails (Fig. 5). Nails are composed of keratins with disulfide bridges that act as major barriers against drug per- meation^20,21. A study by Gómez and colleagues revealed that nails affected by onychomycosis had increased porosity and decreased disulfide bonds compared to healthy nails²⁰. Fig. 5 of the study displayed untreated nails with several pores in the cross-section, while nail plate porosity increased after NTAP irradiation20. Borges et al. analyzed the impact of NTAP on the keratin structure of nail surfaces using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR)¹¹. Plasma exposure increases porosity, improving drug delivery. NTAP aids percutaneous drug absorption^22,23. although the exact mechanism remains unknown. However, research on skin porosity after plasma exposure suggests that pore formation is similar to electroporation or results from changes in lipid structures under an electric field after plasma. The pores generated can facilitate drug permeation. There- fore, NTAP may also affect topical antifungal and systemic treatments.

NTAP could be safer than systemic treatments for treating onychomycosis, which can cause liver failure and compliance issues in some patients, including the elderly¹.

In a pilot study conducted by Lipner et al.²⁴, NTAP was used to treat onychomycosis. The study showed that NTAP was relatively safe and effective, with a clinical cure rate of 53.8% observed in 13 patients. A few studies used NTAP to treat oral Candida species. Zamperini et al.²⁵ demonstrated that plasma exposure could modify the surface of the resin and reduce Candida glabrata adhesion in vitro. Preissner et al.²⁶ conducted a pilot study that found NTAP could be used as an adjuvant antifungal therapy for oral candidiasis. There- fore, the antifungal effects of NTAP are demonstrated against onychomycosis by T. rubrum and onychomycosis caused by other fungal pathogens. Additionally, NTAP destroyed bacteria and fungi, making it helpful in treating onychomycosis with bacterial coinfection³.

This research was conducted to validate the effectiveness of NTAP against T. rubrum, a dermatophyte. However, further studies on other causal species are required. Nonetheless, SEM and RCM allowed for observing structural changes in real-time, confirming that NTAP causes physical damage to the fungal surfaces and spores. NTAP can eliminate fungal biofilms known to cause stubborn onychomycosis. Biofilms are organized microbial communities that adhere to surfaces and each other through an extracellular polymeric matrix and can be verified by SEM images²⁷. Several studies have demon- strated the antibiofilm properties of NTAP for bacteria^28,29, and this research has shown efficacy against fungus.

In summary, NTAP irradiation inhibited the growth of T. rubrum, and structural damage worsened as the dose in- creased. NTAP may be an alternative therapy to treating drug-resistant infections and enhancing the therapeutic effects of antifungal drugs.