ORIGINAL ARTICLE

An investigation into the inhibitory effect of ultravioletradiation on Trichophyton rubrum

Leah J. Cronin & Richard P. Mildren & Michelle Moffitt &Antonio Lauto & C. Oliver Morton & Colin M. Stack

Received: 7 August 2012 /Accepted: 8 February 2013 /Published online: 23 March 2013# Springer-Verlag London 2013

Abstract Fungal infection of nails, onychomycosis, is pre-dominantly caused by Trichophyton rubrum. This infection isan important public health concern due to its persistent natureand high recurrence rates. Alternative treatments are urgentlyrequired. One such alternative is phototherapy involving theaction of photothermal or photochemical processes. The aimof this novel study was to assess which wavelengths withinthe ultraviolet (UV) spectrum were inhibitory and equallyimportant nail transmissible. Initial irradiations of T. rubrumspore suspensions were carried out using a tunable wave-length lamp system (fluence ≤3.1 J/cm2) at wavelengths be-tween 280 and 400 nm (UVC to UVA) to evaluate whichwavelengths prevented fungal growth. Light-emitting diodes(LEDs) of defined wavelengths were subsequently chosenwith a view to evaluate and potentially implement this tech-nology as a low-cost “in-home” treatment. Our experimentsdemonstrated that exposure at 280 nm using an LED with afluence as low as 0.5 J/cm2 was inhibitory, i.e., no growthfollowing a 2-week incubation (p<0.05; one-way ANOVA),while exposure to longer wavelengths was not. A key

requirement for the use of phototherapy in the treatment ofonychomycosis is that it must be nail transmissible. Our re-sults indicate that the treatment with UVC is not feasible giventhat there is no overlap between the antifungal activity ob-served at 280 nm and transmission through the nail plate.However, a potential indirect application of this technologycould be the decontamination of reservoirs of infection such asthe shoes of infected individuals, thus preventing reinfection.

Keywords Onychomycosis . Trichophyton rubrum . LED .

UVC . Infection . Treatment

Introduction

Dermatophytoses or cutaneous mycoses are superficial in-fections of the skin and related structures (nails and hair)usually caused by three medically important genera of fungi,Epidermophytum, Microsporum, and Trichophyton. Whilethese infections are not lethal, they do have a profoundeffect on an individual’s quality of life and may persist forprolonged periods [1].

Trichophyton rubrum is the primary fungal pathogen (80–90% of cases) responsible for onychomycosis (chronic fungalinfection of the nail) [2, 3]. Onychomycosis affects 50 % ofthe population over 70 years of age and causes significantmorbidity in this cohort [4, 5]. The biochemical and mechan-ical factors involved in the pathogenesis of onychomycosisproduce a condition that can be difficult to treat using current-ly available antifungal therapies. Treatment of onychomycosisrequires the use of topical or oral antifungals or a combinationof both. The choice of therapy is dependent on a number offactors such as patient history (age, recurrent infection anddrug interactions), likelihood of compliance, and cost [6].Compliance is a major issue given that treatments can requireup to 12 months for clinical cure despite the additional ad-ministration of oral antifungal agents. Even so, the actual cure

L. J. Cronin :M. Moffitt :A. Lauto :C. O. Morton :C. M. StackSchool of Science and Health, University of Western Sydney,Campbelltown, NSW 2560, Australia

R. P. MildrenMQ Photonics Research Centre, Department of Physics andAstronomy, Macquarie University, North Ryde,Sydney, NSW 2109, Australia

A. LautoThe Bioelectronics and Neuroscience (BENS) Research Group,University of Western Sydney, Campbelltown,NSW 2560, Australia

C. M. Stack (*)School of Science and Health, University of Western Sydney,Locked Bag 1797,Penrith, NSW 2571, Australiae-mail: c.stack@uws.edu.au

Lasers Med Sci (2014) 29:157–163DOI 10.1007/s10103-013-1287-4

rates vary dramatically from study to study [7]. If these treat-ments fail to work, the only alternative is chemical or surgicalavulsion (removal) of the affected nail [8, 9].

The difficulties and costs of current treatments haveresulted in an increase in the number of studies using photo-therapy as an alternative fungicidal treatment for infectionsinvolving T. rubrum [10]. Evidence of the antimicrobial ef-fectiveness of phototherapy has resulted in “light” treatmentsin the form of expensive lamps or lasers (including wave-lengths within the infrared, visible, and ultraviolet (UV) spec-trum) being installed within various settings, such asventilation systems and water treatment plants. Dai etal. [11] have successfully demonstrated the potentialapplication of UVC in the treatment of onychomycosisusing a germicidal lamp (254 nm; fluence 120 mJ/cm2)and have been strong advocates of phototherapy to treatthis condition.

Lasers have been suggested as alternative light sources;however, this not only requires specialized and costly equip-ment, but also application of photosensitizers. To providenovel low-cost treatments for onychomycosis in a clinical or“in-home” setting, the research presented in this study ex-amined the application of phototherapy using versatile light-emitting diodes (LEDs). The benefits of using LEDs includethe lack of toxic mercury, long lifetimes, flexibility and easeof use due to their small size, and lower costs. To date, thisarea has received little attention, particularly LEDs withinthe UVA, UVB, and UVC spectra, with respect to infectionsinvolving T. rubrum. The only other report employing anLED to target T. rubrum viability involved photodynamictherapy (the use of an LED in conjunction with the photo-sensitizer toluidine blue O) [12]. Therefore, the aim of thisstudy was to investigate whether eradication of T. rubrumusing cost-effective light therapies (LEDs) is possible withinclinical and other settings.

Materials and methods

Preparation of T. rubrum spore suspension

T. rubrum 09-043-3609 (Westmead Hospital, NSWAustralia) cultures were inoculated onto Sabouraud dextroseagar (SDA) (Oxoid, Hants, England) plates and incubatedfor 14 days at 30 °C. Following the incubation period, 10 mlof a 0.05 % Tween 80 solution (Sigma-Aldrich, NSW,Australia) was added to the surface of the plate and theculture was brushed with a sterile glass rod to produce aspore suspension. The resulting arthroconidial spore suspen-sion was passed through an 8-μm filter (Millipore, MA,USA) and adjusted with phosphate-buffered saline(Oxoid, Hants, England) (pH 7.4) to an absorbancereading A300 of 0.6.

Transmission of UV and visible light through the toenail

Four samples of toenails were obtained to cover a 5-mmaperture of a UV–Visible spectrophotometer (Cary 5000,USA). The nails were washed with 70 % ethanol and anyresidual epithelium was removed. The thickness of eachsample was measured using a digital micrometer. Each nailwas fixed onto the aperture and transmission of UV andvisible light through the toenails was measured between 200and 700 nm.

UV deuterium lamp irradiation

UV irradiation was performed using a deuterium lamp(Hamamatsu, Japan), which was filtered using a 0.3-m grat-ing monochromator (Spex, USA) to produce narrowbandoutput (full width at half maximum 17 nm) at selectedwavelengths in the range of 280 to 400 nm. The lampemitted a beam spot size of approximately 0.78 cm2 withan irradiance of 17.7, 7.7, 6.5, 6.1, 6.1, 2.7, and 1.3 μW/cm2

at wavelengths of 280, 300, 320, 340, 360, 380, and400 nm, respectively.

Two individual aliquots (10 μl) of the T. rubrum sporesuspension (A300 0.6) were pipetted onto two separate areas(0.80 cm2) marked on SDA plates and allowed to absorb for15 min at room temperature. The plate was wrapped with aclear film (cling wrap previously sterilized with 70 % etha-nol) that was UV transmissible and placed in an invertedposition on top of the deuterium lamp. The sample to beirradiated was aligned with the light beam. The second spotarea of the spore suspension served as a control and waspositioned away from the UV illumination spot (Fig. 1). Thesample being irradiated was then covered with a dark box toprevent light from scattering. After 48 h of irradiation, theplate was removed from the lamp and incubated for 7 daysat 30 °C to measure inhibition of fungal growth. Followingthe incubation period, images of the agar plates wereobtained and the surface area of the fungal growth wasmeasured using the open source ImageJ software [13]. Thediameter of the Petri dish (88 mm) was used as the referenceto calculate the surface area of the fungal growth. Allwavelengths tested using the tunable lamp were performedin triplicate (n=3). Results were analyzed with one-wayANOVA, Tukey’s posttest, and paired t test, using a p valuewith a significance level of 0.05.

In vitro LED irradiations

Five top ball lens LEDs (Sensor Electronic Technology Inc.,SC, USA) with wavelengths of 280, 320, 330, 350, and365 nm and irradiances of 1.8, 0.5, 0.5, 0.8, and1.0 mW/cm2, respectively (Table 1), were used to irradiateindividual aliquots of the T. rubrum spore suspension. The

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UV fluences were determined from the LED manufacturerspecifications and demonstrated to be consistent with mea-surements using a calibrated UV detector (Model AXUV,International Radiation Detectors Inc., USA). Each LEDwas mounted onto a retort stand and was installed with“spacers” to ensure the spore suspension was a defineddistance to produce a spot size the same size as the well(∼0.28 cm2; Fig. 1b).

Two aliquots of 100 μl of the spore suspension (A300 0.6)were pipetted into two wells of a 96-well plate. The UV LEDswere aligned above one of the single wells and the sporesuspension was irradiated. The second well that contained theremaining spore suspension was spatially separated from theUV LED and served as the control. Following irradiationtreatments at various specified time periods ranging from 30 sto 8 h (Table 1), 10-μl samples of the irradiated and controlspore suspension were removed and plated onto SDA platesand incubated as previously described (7 days at 30 °C,followed by a further week of incubation). Measurements of

the surface area of the fungal growth were performed as men-tioned above. Percentage growth of the irradiated samples wasthen calculated relative to the surface area of the control. Resultswere analyzed with one-way ANOVA, Tukey’s posttest, andpaired t test, using a p value with a significance level of 0.05.

Results

Transmission of UV and visible light through the toenail

The transmission of UVand visible light through toenails (n=4)with a mean thickness of 0.8 mm was shown to be blockedbetween 200 and 317 nm. As the wavelengths became longer,the light transmission increased (Fig. 2). Only wavelengthslonger than 317 nm were transmitted through the toenail.Note that the nails cause significant scatter, particularly forshort wavelengths, which is not collected by the spectrometerdetector. As a result, the spectrometer values represent lower

Fig. 1 Schematic diagram of athe deuterium lamp used inlamp irradiations of T. rubrum.The monochromaticwavelength passes through theslit onto the aliquot to beirradiated. b LED irradiationsetup. “Spacers” were used asguides to ensure the irradiationwas directed into the well andthe spot size (0.28 cm2)remained consistent

Table 1 In vitro LED irradia-tion of T. rubrum experimentalresults

The parameters for the LED ir-radiations over a 0.28-cm2 areaare: irradiance (power over ar-ea), estimated irradiance;fluence, irradiance multiplied byexposure time (average LEDfluence); percentage growth,percentage of growth relative tothe surface area growth of thecontrol (mean±standarddeviation)

*p value<0.05

Wavelength (nm) n Irradiance (mW/cm2) Fluence (J/cm2) Percentage growth (%)

280±2 3 1.8 0.05 94±32

280±2 3 1.8 0.1 64±25

280±2 4 1.8 0.2 39±24*

280±2 3 1.8 0.5 0*

280±2 3 1.8 1.1 0*

280±2 3 1.8 2.2 0*

280±2 3 1.8 3.2 0*

320±2 30 0.5 ≤15.6 90±9

330±2 33 0.5 ≤14.1 84±5

350±2 33 0.8 ≤23.9 87±12

365±2 30 1.0 ≤29.1 102±9

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bounds on the actual light transmitted by the nail. Nevertheless,the significant decrease in transmission at wavelengths less than320 nm was attributed to strong absorption by the nail.

Irradiation treatment

Deuterium lamp UV radiation

In terms of defining antifungal activity, following our variousirradiation treatment regimes, we refer to inhibitory/antifungalactivity as >99.9 % kill after incubation for 7 days (and nogrowth following a further 7-day incubation at 30 °C), whilenon-inhibitory irradiations led to fungal growth after 7 days ofincubation. Inhibitory activity was observed only for the sam-ples irradiated at a wavelength of 280 nm with a fluence of3.1 J/cm2. In these samples, no fungal growth was observedwhile T. rubrum colonized the plate in the controls (paired ttest, p=0.027, n=4). No inhibitory effect on fungal growthwas induced in the samples irradiated at 300 to 400 nm, whencompared to the controls (paired t test, p>0.05, n=3; Fig. 3).

In vitro UV LED irradiation

Wavelengths 280, 320, 330, 350, and 365 nm were selectedbased on either the results from the initial deuterium lampirradiations or the ability of the wavelength to penetrate thenail and their availability as an LED. The ratio between thefungal growth area of irradiated and nonirradiated sampleswas considered for statistical comparison. Results from theLED irradiations demonstrated that none of the longer wave-lengths (320–365 nm) that could transmit through the nailwere inhibitory under the conditions tested (with a maximumfluence of 15.6, 14.1, 23.9, and 29.1 J/cm2, respectively (one-

way ANOVA, p>0.05)). Our results also show that the inhib-itory effect of using an LEDwith a wavelength of 280 nmwasdose dependent. The lower fluences between 0.05 and0.2 J/cm2 were not inhibitory as regrowth occurred duringthe 7-day incubation period at 30 °C. Although irradiancewith a fluence of 0.2 J/cm2 highlighted a dose-dependentantifungal effect as compared to the controls (39±24 %), thiseffect was likely fungistatic at best and merely resulted in aslower regrowth. However, irradiations with fluences greaterthan 0.5 J/cm2 had a profound effect on the viability of T.rubrumwith 100% inhibition of regrowth following a 2-weekincubation at 30 °C. These results are summarized in Table 1.Figure 4 demonstrates that significant growth inhibition wasachieved between 5 and 30 min irradiance (fluence, 0.5 to3.2 J/cm2) (n=3; one-way ANOVA, p<0.0001). Figure 5shows a pictorial representation of the inhibitory activityachieved following irradiation with a fluence of 0.5 J/cm2;this corresponds to a treatment time of just 5 min.

Discussion

To our knowledge, this is the first study to investigate theeffects of wavelengths across the UVC to UVA spectrum onthe growth of T. rubrum. The results from our tunable lampsystem provide important insights regarding toxicity versuswavelength with respect to nail transmission. Whenattempting to use “light therapy” to treat onychomycosis,the extent to which a given wavelength of light can pene-trate the nail is a critical consideration. The longerwavelengths, 320 to 365 nm, were of particular interestto us given their ability to penetrate the nail (Fig. 2)[14, 15]. However, our results using the tunable lamp

Fig. 2 Transmission of UV andvisible light through uninfectedtoenails with a mean width of0.8 mm. Wavelengths longerthan 317 nm transmittedthrough the toenail

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system and the LEDs emitting UVA wavelengths of 320to 365 nm clearly demonstrated that these key wave-lengths were not inhibitory at the fluences tested(320 nm≤15.6 J/cm2; 330 nm≤14.1 J/cm2; 350 nm≤23.9 J/cm2; 365 nm≤29.1 J/cm2) (Table 1). These find-ings are consistent with current literature [16]. Thelongest exposure to UVA irradiation we employed was8 h, after which no inhibitory effect was evident.

The results from both the tunable lamp and LED irradi-ations clearly demonstrated that a wavelength of 280 nm hasa direct inhibitory effect on T. rubrum spore suspensions invitro (Figs. 3 and 5); with antifungal activity occurring atfluences as low as 0.5 J/cm2 (Table 1). No regrowth wasevident on SDA plates following a 2-week incubation peri-od. The mechanism of germicidal activity of UVC is wellestablished and involves the inactivation of DNA throughthe absorption of photons, preventing DNA replication andcell division [17–19]. Unfortunately, we found no overlap intoxicity between these wavelength ranges and their ability totransmit through a healthy toenail. Based on our findings,we did not investigate further whether the affect of UVCwas fungicidal versus fungistatic to T. rubrum as we believethat the use of UVC to treat these and other fungal nailinfections is not feasible [20].

Our findings contradict those of Dai et al. [10] who, usingan ex vivo model of T. rubrum infecting porcine hoof andhuman toenail clippings, demonstrated that UVC irradiation(254 nm; fluence 36 to 576 J/cm2) was able to kill T. rubruminside the toenail. They noted that depending on a number offactors such as thickness, cracking, and discoloration, the

Fig. 3 Irradiation of T. rubrum using the tunable lamp system: Sam-ples were irradiated for 48 h and incubated for 7 days at 30 °C to assessviability. a 280 nm irradiation inhibited fungal growth, b 300 nm, c320 nm, d 340 nm, e 360 nm, f 380 nm, and g 400 nm had no effect onfungal growth. C control, I irradiated samples (n=3)

Fig. 4 Growth percentage of T. rubrum following 280 nm LEDirradiation. Fluences greater than 0.2 J/cm2 significantly inhibited thefungal growth of T. rubrum (*p value<0.05)

Fig. 5 Irradiation of T. rubrum with 280 nm LED: fluence ∼0.5 J/cm2.Areas labeled 1, 2, and 3 were plated with aliquots of the irradiatedsample and produced no growth, while the nonirradiated sample C(control) grew confluent (n=3)

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radiant exposure of UVC needed for complete sterilizationwas usually in the order of tens to hundreds of joule per squarecentimeter. These workers alluded to the completion of aphase I clinical trial of UVC irradiation for onychomycosis;however, no information pertaining to this work could befound in the most recent scientific literature.

There appears to be some conjecture in the scientificliterature regarding the transmission of UV radiationthrough nails [14, 15]. An early study by Parker andDiffey [14], investigating the transmission of optical radia-tion (300 to 600 nm), through human cadaver toenails,found that 22 % of UVA and little UVB transmitted throughthe nail plate to reach the nail bed and concluded that thenail plate is in fact a “very efficient sunscreen.” A recentstudy by Stern et al. [15] found transmission to be even lesswith UVB transmission completely blocked while the meanpenetration of UVAwas found to be 1.65 %. The findings ofour study are in agreement with those of Stern et al. [22] aswe too found the nail plate to be very efficient at blockingUVB and UVC transmission.

A study by Watanabe et al. [21] demonstrated the poten-tial of photodynamic therapy (PDT) in treating two clinicalcases of onychomycosis using the photosensitizer 5-aminolevulinic acid (ALA). However, this treatment re-quired a 10-h pretreatment with 20 % urea (to enhance nailtransmission), 5-h pre-irradiation treatment with a topicalapplication of 20 % ALA cream, followed by irradiation of100 J/cm2 using a laser at 630 nm. A similar study bySotiriou et al. [22] treated the onychomycotic nail plates of30 patients, with treatment repeated three times at 2-weeklyintervals, and resulted in a 43 % cure rate after 12 months.Both of these studies highlight a number of important is-sues: (1) transmission through the nail plate is a criticalrequirement for treatment and (2) these types of infectionsare inherently difficult to cure.

Given the need for any potential application of photo-therapy to penetrate the nail plate, we recently investigatedthe use of longer transmissible wavelengths in combinationwith PDT in reducing the viability of T. rubrum sporesuspensions in vitro. In contrast to our experimental modeloutlined here, we employed a two-step in vitro approachrequiring a 30-min exposure to the nontoxic photosensitizer,rose bengal (RB), and subsequent illumination using a greenlaser (λ=532 nm). The activation of RB using fluences of133 and 228 J/cm2 resulted in significant inhibition of T.rubrum (75 % that of controls following a single 5 min ofexposure). Based on our current observations of trans-mission through a healthy toenail, the minimum trans-mission achievable using a green laser would be at least2 % without any manipulation of the nail to increaseoptical transmission. Further studies are currently under-way to establish the in vivo validity of this treatmentapproach [23].

We chose T. rubrum as the test fungus because it is theleading cause of onychomycosis [1, 3, 24]. The fact that ourin vitro treatment was inhibitory at fluences as low as0.5 J/cm2 is significant. Unfortunately, from a clinical per-spective, this wavelength is not transmissible through thenail and is thus not a viable treatment option. However, apotential indirect application of this methodology could bethe in-home decontamination of reservoirs of reinfectionsuch as the footwear of patients suffering onychomycosis,particularly those sensitive to current antifungal powdersused to decontaminate footwear.

Acknowledgments This project was supported by the University ofWestern Sydney Honors Scholarship. Many thanks to Sue Sleimanfrom the Centre for Infectious Diseases and Microbiology, WestmeadHospital for donating the T. rubrum sample.

Conflict of interest The authors have no conflicts of interest regard-ing any information presented in this paper.

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