Journal of Dermatological Treatment, 2012; Early Online: 1–4© 2012 Informa Healthcare USA on behalf of Informa UK Ltd.ISSN: 0954-6634 print / 1471-1753 onlineDOI: 10.3109/09546634.2012.714455

1 ORIGINAL ARTICLE

2 Antifungal effects of a 1444-nm neodymium:Yttrium–aluminum–garnet3 laser on onychomycosis: a pilot study

4 Min Ju Choi1, Zhenlong Zheng1,2, Boncheol Goo3 & Sung Bin Cho1

5 1Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea, 2Department of6 Dermatology, Yanbian University Hospital, Yanji, China and 3Clinic L, Goyang, Korea

789 Investigations of laser- or light-assisted antibacterial and antifungal10 treatments have been introduced. In the present study, we11 investigated the antifungal activities of 1444-nm Nd:YAG lasers12 against onychomycosis by microbiologic analysis and scanning13 electron microscopy. Scraped toenails from 20 participants with14 mycologically confirmed onychomycosis were prepared on15 polystyrene weighing dishes and treated with a 1444-nm Nd:16 YAG laser. The samples were analyzed for the presence of17 colony-forming units (CFUs) and scanning electron microscopy18 was performed using an toenail treated with the 1444-nm Nd:19 YAG laser. The mean reduction rate achieved by treatment with a20 total energyof 300 Jwas 75.9% (range: 33.3–100), andby treatment21 with 450 J was 85.5% (range: 66.7–100). However, the difference in22 CFUreduction ratesbetween the laser settingsof300 Jand450Jwas23 not significant. Analysis by scanning electron microscope revealed24 numerous disintegrated spores on the lower portions of the nail25 plate treatedwith the 1444-nm laser,while the upper portion of the26 nail plate presented only a few small and greatly disintegrated27 fungal spores. Our results suggest that a Nd:YAG laser with a28 wavelength of 1444 nm has antifungal effects on onychomycosis.29 However, further investigations should be performed to determine30 the long-term clinical and microbiologic effects of this treatment.3132 Key words: onychomycosis, 1444-nm Nd:YAG laser, antifungal,33 colony-forming unit, scanning electron microscope

34 Introduction35 Currently available treatment modalities for onychomycosis36 include systemic and topical antifungal agents and surgical nail37 treatment. Selection of treatment modalities mainly depends on38 the severity of onychomycosis and the causative organisms. In39 addition, systemic adverse reactions and drug interactions of the40 oral antifungal agents may occur. Therefore, many patients with41 systemic diseases, especially hepatic or renal disorders, have no42 choice but to undertake monotherapy with a topical antifungal43 agent (1,2).44 Recently, investigations of laser- or light-assisted antifungal45 treatments have been increasing. Examples include studies of46 870- and 930-nm light sources, photodynamic therapy, titanium47 sapphire laser, and the 1064-nm neodymium:yttrium–aluminum–48 garnet (Nd:YAG) laser for onychomycosis and erbium, chromium:49 yttrium–scandium–gallium–garnet (Er,Cr:YSGG) laser for dental

50candidiasis (3–8). Laser energies of specific wavelengths have been51suggested to interact with plasma and mitochondrial membranes52of fungi. The production of endogenous radical oxygen species is53also postulated to damage and inactivate fungi.54The use of 1444-nm Nd:YAG lasers to treat various dermato-55logic conditions has been investigated, especially for laser lipolysis56and skin tightening. When compared with the 1064-nm and571320-nm wavelengths of the Nd:YAG lasers, the 1444-nm wave-58length has higher absorption coefficients for fat and water (9,10).59In this report, we investigated the antifungal activities of the601444-nm Nd:YAG laser for onychomycosis by microbiologic61analysis and scanning electron microscopy.

62Materials and methods63Treatment protocol64This study was approved by the Institutional Review Board of65Severance Hospital, Yonsei University College of Medicine, Seoul,66Korea. A 1444-nm Nd:YAG laser (AccuSculpt�, Lutronic Corp.,67Goyang, Korea) was used in this study to investigate the antifungal68effects of a 1444-nm wavelength laser on onychomycosis. Because69the laser energy of this device is delivered by a fiber cannula into70subcutaneous fat tissues for interstitial lipolysis, we additionally71equipped the laser with an external hand-piece for this study72(Figure 1A). The settings of the 1444-nm laser were as follows: 5W73of power, a pulse width of 100 ms, pulse energy of 250 mJ, and a74pulse rate of 20 Hz. To determine the appropriate total energies,75total energies of 150 J, 300 J, and 450J were delivered on 100 mm2

76of the thin plastic weighing dishes (AccuResearch Korea,77Seoul, Korea).78The toenails of 20 participants who had been diagnosed with79mycologically confirmed onychomycosis were scraped with sterile80blades. An equal amount of scraped toenails of each patient were81prepared on two plastic weighing dishes. One dish was untreated82and the other dish was treated with a 1444-nm laser delivering83total energies of 300 J (n = 10) and 450 J (n = 10). In order to84reduce the excessive thermal effects, the plastic weighing dishes85were cooled with ice cubes every 150 J.

86Fungus culture87After the treatment protocol, scraped toenails that did or did not88receive the laser treatment in each dish were transferred to89Sabouraud’s liquid medium and incubated at 37.8�C for 24 hours90as described in a previous report (8). An equal amount (100 ml) of

Correspondence: Sung Bin Cho, MD, PhD, Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine,50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. E-mail: sbcho@yuhs.ac

(Received 22 May 2012; accepted 24 June 2012)

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91 Sabouraud’s liquid medium from each sample was then inocu-92 lated onto Sabouraud’s dextrose agar. After 5 days of incubation,93 the colony forming units (CFUs) were counted on Sabouraud’s94 dextrose agar and divided by the amount of scraped toenails as95 presented with CFU/ml/mg.

96 Scanning electron microscope97 An extracted toenail, which was diagnosed with mycologically98 confirmed onychomycosis, was obtained and longitudinally cut99 into halves. One half of the toenail was untreated and the other100 half was treated with the 1444-nm laser delivering a total energy of101 450 J at 5 W of power, pulse width of 100 ms, pulse energy of102 250 mJ, and pulse rate of 20 Hz. For scanning electron micros-103 copy, the specimens were fixed in 50 mM sodium cacodylate104 buffer (pH 7.4) that contained 2% glutaraldehyde in paraformal-105 dehyde for 30 min at 32�C, post-fixed in 1% osmium tetraoxide106 for 2 hours at 4�C, and dehydrated by treatment with a graded107 ethanol series (5 minutes each in 50, 60, 70, 80, 90, and 95%108 ethanol, and finally, twice in 100% ethanol). A scanning electron109 microscope (Hitachi, S-800, FESEM, Tokyo, Japan) was used with110 magnifications of 100–20 000.

111 Statistical analysis112 We analyzed the antifungal effects of the laser according to total113 energies using the nonparametric Mann-Whitney U test with the114 Statistical Package for the Social Sciences version 18.0 (SPSS Inc.,115 Chicago, IL, USA). Differences were considered statistically sig-116 nificant when p values were less than 0.05.

117 Results118 Energy settings119 Each plastic weighing dish (47.7 mm � 47.7 mm) was made of120 polystyrene and was 0.2 mm thick. The settings of the 1444-nm121 laser (5W of power, pulse width of 100 ms, pulse energy of 250 mJ,122 and 150 J of total energy resulted in no remarkable morphologic123 changes to the plastic weighing dishes with the temperature124 estimated to be 39.8�C (Figure 1B). The dishes showed minimal125 changes with a total energy of 300 J (Figure 1C), whereas marked126 disfigurements on the dishes were found at treatment settings of127 450 J (Figure 1D).

128 Mycologic evaluation129 The mean number of CFUs of 20 untreated toenails with ony-130 chomycosis was 109 CFU/ml/mg (range: 13–442). For the 10131 toenails treated with a total energy of 300 J, the figure was reduced

132to 28 CFU/ml/mg (range: 0–115) (Figure 2), and for the13310 toenails treated with 450 J, the figure was further reduced134to 10 CFU/ml/mg (range: 0–42) (Figure 3). The reduction rate was135calculated by comparing the CFUs of each group. The mean136reduction rate achieved by treatment with a total energy of 300 J137was 75.9% (range: 33.3–100), and by treatment with 450 J was13885.5% (range: 66.7–100). The number of CFUs in toenails treated139with the 1444-nm laser with total energies of 300 J and 450 J was140significantly reduced compared to the controls (p = 0.016 and141p = 0.002, respectively). However, there was no significant142difference in the CFU reduction rates between the laser settings143of 300 J and 450 J (p > 0.05).

A B

C D

Figure 2. Sabouraud’s dextrose agar plates after seven days of incubationwith scraped toenails (A, case 6; C, case 7) with or (B, case 6; D, case 7)without a 1444-nm Nd:YAG laser treatment at a total energy of 300 J.

A B

C D

Figure 3. Sabouraud’s dextrose agar plates after seven days of incubationwith scraped toenails (A, case 14; C, case 17) with or (B, case 14; D, case 17)without a 1444-nm Nd:YAG laser treatment at a total energy of 450 J.

A

B C D

Figure 1. (A) A hand-piece equipped with a 1444-nm laser for external energydelivery. Effects of the 1444-nm laser with total energies of (B) 150 J/100 cm2,(C) 300 J/100 cm2, and (D) 450 J/100 cm2 were delivered on thin plasticweighing dishes.

M. J. Choi et al.

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144 Scanning electron microscopy145 The scanning electronmicroscopic evaluation of the toenail without146 laser treatment revealed numerous spores between tile-like struc-147 tures (Figure 4A). In a higher magnification, the densely accumu-148 lated spores were uniformly large and spherical (Figure 4B).149 After 1444-nm laser treatment at a total energy of 450 J, the150 lower portions of the toenail demonstrated numerous spores151 similar to that shown in the control nail plate (Figure 4C);152 however, the spores showed various patterns in size and shape153 and most of them were disintegrated compared with the fungal154 structures in the control nail plate (Figure 4D). In the upper155 portion of the nail plate, only a few small, greatly disintegrated156 structures that were suggestive of fungal spores were found157 (Figures 4E, F). In addition, the tile-like structures of the toenail

158seemed to be preserved in the lower parts of the toenail after the159laser treatment, whereas the structures were disintegrated in the160upper parts of the toenail.

161Discussion162The germicidal properties of ultraviolet light on bacteria and163fungus without the aid of a photosensitizer have been shown164(11,12). However, the photocarcinogenic effects of the ultraviolet165light limit its usage on the treatment of human prokaryotic and166eukaryotic pathogens (11). Neuman et al. (13) demonstrated that167cellular damage by a near-infrared wavelength could be achieved168through absorption by unidentified endogenous chromophores in169both pathogens and human cells. In addition, Bornstein et al. (11)

A B

C D

E F

Figure 4. Numerous spherical spores between tile-like structures of the nail plate without the 1444-nm laser treatment (A,�5000; B,�10 000). Disintegratedspores in the lower portion of the nail plate with the 1444-nm laser treatment (C, �5000; D,�10 000). Only a few disintegrated and melted spores present inthe upper portion of the nail plate with the laser treatment (E, �5000; F, �10 000).

Antifungal effects of 1444-nm Nd:YAG laser

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170 suggested that the photoinactivating effects of multiplexed near-171 infrared wavelengths (870 nm and 930 nm) on microorganisms172 could be achieved by a decrease in transmembrane potentials as173 well as an increase in the production of reactive oxygen species at174 physiologic temperatures.175 The clinical investigation of a 0.65-ms pulsed 1064-nm laser176 treatment for onychomycosis was performed on eight patients,177 who were treated two to three sessions with at least 3-week178 intervals (4). Post-treatment mycologic evaluation presented179 that seven out of eight patients had negative results in fungus180 culture (4). In addition, 26 toes with onychomycosis, which were181 treated with 4-minute exposure to 870- and 930-nm wavelengths182 followed by 2-minute exposure to 930-nm wavelength, demon-183 strated negative results with fungus culture and periodic acid-184 Schiff in 30% at 180 days (3). According to the previous reports,185 the subjects with onychomycosis were recommended to use186 topical antifungal agents to prevent re-infection and filing of187 toenails to keep them short and thin (3,4). We suggest that fungal188 spores and hyphae in the lower portions of toenails can be189 effectively treated by debridement and trimming of the nails as190 well as repeated laser delivery.191 In the pilot study described here, we evaluated the antifungal192 effects of the 1444-nm Nd:YAG laser on onychomycosis. Until193 recently, most of the studies for investigating antifungal effects of194 laser or light treatment on onychomycosis have been performed195 on toenails in situ and with subsequent scraping for mycologic196 evaluation (3,4,11). However, we scraped toenails before treat-197 ment and divided them equally among the control and treatment198 groups to obtain samples of toenails at identical sites and amounts199 in each patient. Laser energies were then delivered to the scraped200 toenails and mycologic evaluation was performed on the treated201 samples and controls. Among the controls, the number of CFUs202 was highly variable, perhaps due to such factors as disease203 duration, extent of onychomycosis, collection site, and prior204 treatment modalities. To minimize the effects of these factors,205 patients with treatment histories of onycomycosis within six206 months were excluded, all of the toenails were collected by a207 single dermatologist, and the scraped toenails were subjected to208 laser treatment within six hours from collection.209 Because both pathogens and human cells are mainly composed210 of lipids and water, we hypothesized that the 1444-nm Nd:YAG211 laser with high absorption coefficients to these components could212 provide non-specific tissue damage. The differences between213 fungal and human cells with regard to the absorptiveness of214 the chromophores as well as the vulnerability to the 1444-nm215 wavelength energies, may have resulted in the significant reduc-216 tion of CFUs and remarkable disintegration of the fungal spores.217 Scanning electron microscope examinations demonstrated that218 the 1444-nm Nd:YAG laser caused remarkable morphologic219 changes to the fungal spores at the lower portions of the toenail.220 The upper portion of the nail plate, which was affected by the221 stronger laser fluencies than the lower portion, showed greatly222 disintegrated structures of both fungus and toenail. We suggest223 that the results of the mycologic analyses are correlated to the224 morphologic findings by scanning electron microscope analysis.225 In addition, the risk of unwanted photothermal damage to the226 human cells should be considered although more apparent anti-227 fungal effects can be expected with higher laser fluencies.

228It appeared that a total energy of 450 J was more effective than229that of 300 J for reducing CFUs, although the results were not230statistically significant. Out of 20 participants, three cases dem-231onstrated a reduction rate of 100% in CFUs after the laser232treatment and among them, one was treated with a total energy233of 300 J and two were treated with 450 J. However, we were unable234to conclude that the higher energy delivery was superior for235onychomycosis when considering the risk of photothermal dam-236age to the surrounding human tissues. Rather, we suggest multiple237sessions of treatments with lower fluencies at certain intervals as a238practical alternative.239In conclusion, our data suggest that the use of a Nd:YAG laser240with a wavelength of 1444 nm can be used to obtain antifungal241effects against onychomycosis. However, to minimize the danger242of excessive photothermal effects on human tissues and maximize243the antifungal effects of the 1444-nm Nd:YAG lasers, further244investigations should be performed.

245Declaration of interest: The authors report no conflicts of246interest. The authors alone are responsible for the content and247writing of the paper.

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