Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds.

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days.

Clinical laser treatment of toenail onychomycoses.

Onychomycoses are fungal infections of the fingernails or toenails having a prevalence of 3% among adults and accounts for 50% of nail infections. It is caused by dermatophytes, non-dermatophyte filamentous fungi, and yeasts. Compressions and microtraumas significantly contribute to onychomycosis. Laser and photodynamic therapies are being proposed to treat onychomycosis. Laser light (1064 nm) was used to treat onychomycosis in 156 affected toenails. Patients were clinically followed up for 9 months after treatment. Microbiological detection of fungal presence in lesions was accomplished. A total of 116 samples allowed the isolation of at least a fungus. Most of nails were affected in more than two thirds surface (some of them in the full surface). In 85% of cases, after 18 months of the onset of treatment, culture turned negative. After 3 months months, only five patients were completely symptom-free with negative culture. In 25 patients, only after 6 months, the absence of symptoms was achieved and the cultures negativized; in 29 patients, 9 months were required. No noticeable adverse effects were reported. This study reinforces previous works suggesting the applicability of laser therapies to treat toenail onychomycosis.

In vitro fungicidal effects of methylene blue at 625-nm.

The aim of the study is to confirm the effectiveness of photodynamic therapy (PDT) as a significant inhibitor of Trichophyton rubrum (T. rubrum) and to determine the most appropriate dose and rate of delivery. Trichophyton rubrum is the most common dermatophyte worldwide, responsible for the majority of superficial fungal infections. The traditional treatment of T. rubrum has known adverse effects. An alternative treatment is warranted. Photosensitised T. rubrum specimens were treated with 625-nm light at doses of 3, 12, 24, 40 and 60 J/cm2 . Colony counts were performed and compared to untreated controls. Doses of 24, 40 and 60 J/cm2 all produced kill rates of over 94%. A lower rate of delivery (7.80 mW/cm2 ) was shown to be a greater inhibitor of T. rubrum than a higher rate of delivery (120 mW/cm2 ). Photodynamic therapy with methylene blue (MB) at 625 nm using a low rate of delivery at doses of 24, 40 and 60 J/cm2 is an effective inhibitor of T. rubrum. A rate of delivery of 7.80 mW/cm2 is a significantly greater inhibitor of T. rubrum than a rate of 120 mW/cm2 when applying 625-nm light in PDT using MB.

Comparison of photoinactivation of T. rubrum by new methylene blue (NMB) and indocyanine green (EmunDo®).

BACKGROUND: Superficial mycotic skin infections which are predominantly caused by Trichophyton rubrum, poorly responsd to conventional therapies. A great amount of attention has focused on finding more effective treatments. The current work is aimed to compare the effectiveness of phoinactivation of Trichophyton rubrum by two relatively new photosensitizers: a phenothiazinium dye(New methylene blue) and Indocyanine green (EmunDo®). MATERIALS AND METHODS: A Final inoculum of T. rubrum which corresponded to 106 colony forming unit per milliliter (CFUml-1) was prepared. Antimicrobial Photodynamic treatment (aPDT) of T. rubrum was carried out by either EmunDo® (1mg/ml, Infra-red laser (IRL, λ=810nm, Energy Density 55J/cm2)) or NMB (10µM, Red laser (RL), λ=630nm, Energy Density of 5J/cm2). The suspensions thereafter were subcultured on Sabouraud dextrose agar (SDA) and were counted on due time. based on colony-forming unit per milliliter (CFU/ml). RESULTS: aPDT with either EmunDo® (E) or NMB (N) considerably diminished the viability of inoculated T. rubrum with respective reduction of 0.64 log and 0.4 log compared to the control group (P<0.001). No significant difference was found between two laser only groups (P=0.79) and two aPDT groups (P=0.73), however significant reduction of T. rubrum in red laser only group (P=0.04) and EmunDo® only group (P=0.04) was found as compared to the control group (P<0.05). CONCLUSION: The study provides evidence regarding satisfactory photodynamic inactivation of T. rubrum with EmunDo® or NMB as photosensitizers. Irradiation by only red laser source was found superior to only infra-red laser source. Dark toxicity of EmunDo® was more successful than new methylene blue dye.

In Vitro Antimicrobial Photodynamic Therapy Against Trichophyton mentagrophytes Using New Methylene Blue as the Photosensitizer. / Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador.

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620-645nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50µM for a fluence of 81J/cm2 after a contact time of 10minutes with the photosensitizing-agent. Increasing the concentration to 100µM allowed the fluence to be decreased to 9J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is strain-dependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte.

Comparative Sensitivity of Trichophyton and Aspergillus Conidia to Inactivation by Violet-Blue Light Exposure.

OBJECTIVE: To investigate the use of 405 nm light for inhibiting the growth of selected species of dermatophytic and saprophytic fungi. BACKGROUND DATA: The increasing incidence and resilience of dermatophytic fungal infections is a major issue, and alternative treatment methods are being sought. METHODS: The sensitivity of the dermatophytic fungi Trichophyton rubrum and Trichophyton mentagrophytes to 405 nm violet-blue light exposure was investigated, and the results compared with those obtained with the saprophytic fungus Aspergillus niger. Microconidia of T. rubrum and T. mentagrophytes and conidia of A. niger were seeded onto Sabauroud dextrose agar plates and irradiated with 405 nm light from an indium-gallium-nitride 99-DIE light-emitting diode (LED) array and the extent of inhibition was measured. RESULTS: Germination of the microconidia of the Trichophyton species was completely inhibited using an irradiance of 35 mW/cm(2) for 4 h (dose of 504 J/cm(2)). A. niger conidia showed greater resistance, and colonial growth developed after light exposure. In liquid suspension tests, 405 nm light dose levels of 360, 720, and 1440 J/cm(2) resulted in complete inactivation of T. rubrum microconidia, whereas A. niger showed greater resistance, and at the highest dose level applied (1440 J/cm(2)) although A niger hyphae were completely inactivated, only a 3-log10 reduction of a 5-log10 conidial suspension was achieved. CONCLUSIONS: The study results demonstrate the relatively high sensitivity of Trichophyton microconidia to 405 nm violet-blue light, and this is may be of potential interest regarding the control and treatment of dermatophyte infections.

In vitro combination therapy using low dose clotrimazole and photodynamic therapy leads to enhanced killing of the dermatophyte Trichophyton rubrum.

BACKGROUND: Superficial infections of the skin and mucous membranes caused by dermatophyte fungi are amongst the most common and challenging infections to treat. Previously we demonstrated the phototoxic effects of photodynamic therapy (PDT) towards Trichophyton rubrum, using a green laser to photoactivate Rose Bengal (RB). The aim of this study was to evaluate whether we could; (1) achieve a similar effect using an inexpensive light-emitting diode (LED) to photoactivate RB and (2) to evaluate whether our PDT regime could be combined with standard antifungal drug therapy and increase its effectiveness. METHODS: We designed and built our own inexpensive green (530 nm) LED source and tested its efficacy as part our RB-PDT regime in vitro against T. rubrum. We also examined the potential benefits of incorporating PDT as part of combination therapy and whether the order in which this was done had an impact. First we subjected spore suspensions to sub-inhibitory concentrations of a number of antifungal agents (CLT, MCZ and TRB) for 72 hours followed by RB-PDT. Secondly we subjected spore suspensions to sub-inhibitory PDT followed by drug treatment and evaluated if there were any changes to the minimum inhibitory concentrations (MICs) of the drugs tested. RESULTS: The optimal conditions for photoinactivation of T. rubrum using RB-PDT alone were 140 µM of RB and 24 J/cm2 of LED (equating to a 30-minute exposure). These parameters also caused a 100% reduction in the viability of the pathogenic yeast Candida albicans and the model fungus Saccharomyces cerevisiae. By combining our RB-PDT regime as an adjunct to antifungal drugs we were able to dramatically reduce the exposure times. Treatment of spore suspensions using a sub-inhibitory dose of clotrimazole (CLT) followed by RB-PDT, this order was critical, significantly reduced the exposure times required to achieve 100% inhibition of T. rubrum to 15 minutes as compared to RB-PDT alone. CONCLUSIONS: The combination of antifungal drug and RB-PDT represents an attractive alternative to the current antifungal therapies used to treat superficial fungal diseases. Our approach has the potential to reduce treatment times and drug dosages which can also reduce drug toxicity and improve patient compliance.

In vitro fungicidal photodynamic effect of hypericin on Trichophyton spp.

Hypericin is a natural photosensitizer used in photodynamic therapy (PDT), which has shown in vitro antifungal effect against Candida spp. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin-PDT on dermatophytes. Trichophyton rubrum and Trichophyton mentagrophytes strains were incubated with different concentrations of hypericin for different times and exposed to light-emitting diode lamp (602 ± 10 nm, 10.3 mW cm(-2), and fluence 37 J cm(-2)). Using the optimal incubation time, 60 min, a 3-log fungicidal effect was achieved with hypericin concentration ranges of 10-20 µM for T. rubrum and 20-50 µM for T. mentagrophytes (p = 0.95). Confocal fluorescence microscopy showed the localization of hypericin inside the dermatophytes diffusely distributed in the cytoplasm of conidia and hyphae and outside the nucleus. In conclusion, hypericin-PDT has a fungicidal effect in vitro on dermatophytes. Hypericin seems to be a promising photosensitizer to treat localized dermatophytic infections such as tinea pedis and onychomycosis.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 µM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

An investigation into the inhibitory effect of ultraviolet radiation on Trichophyton rubrum.

Fungal infection of nails, onychomycosis, is predominantly caused by Trichophyton rubrum. This infection is an important public health concern due to its persistent nature and high recurrence rates. Alternative treatments are urgently required. One such alternative is phototherapy involving the action of photothermal or photochemical processes. The aim of this novel study was to assess which wavelengths within the ultraviolet (UV) spectrum were inhibitory and equally important nail transmissible. Initial irradiations of T. rubrum spore suspensions were carried out using a tunable wavelength lamp system (fluence ≤3.1 J/cm(2)) at wavelengths between 280 and 400 nm (UVC to UVA) to evaluate which wavelengths prevented fungal growth. Light-emitting diodes (LEDs) of defined wavelengths were subsequently chosen with a view to evaluate and potentially implement this technology as a low-cost "in-home" treatment. Our experiments demonstrated that exposure at 280 nm using an LED with a fluence as low as 0.5 J/cm(2) was inhibitory, i.e., no growth following 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 of onychomycosis is that it must be nail transmissible. Our results indicate that the treatment with UVC is not feasible given that there is no overlap between the antifungal activity observed at 280 nm and transmission through the nail plate. However, a potential indirect application of this technology could be the decontamination of reservoirs of infection such as the shoes of infected individuals, thus preventing reinfection.

Inactivation of pathogenic dermatophytes by ultraviolet irradiation in swimming pool thermal water.

The effect of ultraviolet (UV) radiation from low-pressure mercury lamp against some pathogenic dermatophytes species such as Epidermophyton floccosum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans and Trichophyton violaceum suspended in thermal water was evaluated in laboratory-scale condition at various times. The main results showed that within 120 s of exposure, all species of dermatophytes are completely inactivated, which was evidenced by the absence of fungal regrowth, while after 60 s only T. tonsurans was recovered, with a reduction of 3.28 log. Shorter exposure times were not enough to completely inactivate all dermatophytes species. The samples treated with UV radiation for 120 s did not give evidence of fungal regrowth indicating that this disinfectant action is persistent over time. In conclusion, UV radiation can be proposed to reduce the risk of infection by dermatophytes eventually present in swimming pools that use thermal water.

Antifungal efficacy of lasers against dermatophytes and yeasts in vitro.

PURPOSE: Approximately 2-13% of the world population suffers from onychomycosis. Recently, lasers have been introduced for treatment. However, no effect was found with in vitro laser irradiation of pathogens on agar plates. This study aimed to investigate the efficacy of laser irradiation against fungi using an alternative in vitro approach. MATERIALS AND METHODS: Lasers of 808, 980 and 1064 nm were used to heat cell culture media and a nail clipping. Trichophyton rubrum. T. interdigitale. Microsporum gypseum. Candida albicans. C. parapsilosis, and C. guilliermondii species were subcultured and subjected to laser treatments (808/980 nm: 9-27 J/cm(2), 6 ms, 12 × 12 or 12 × 50 mm and 1064 nm: 50-240 J/cm(2), 90 ms, 5-10 mm). After irradiation, the fungal elements were transferred onto agar plates using conventional and Drigalski spatulas and were incubated for 6 days. RESULTS: The highest increase in temperature was found using a 980-nm laser with a pulse duration of 6 ms and a fluence of 27 J/cm(2). The histology work-up revealed a dissection of the nail plate from the nail bed tissue after laser irradiation. Growth inhibition was only found for C. guilliermondii and T. interdigitale. All other pathogens presented only reduced growth, and C. albicans growth was unaffected. CONCLUSIONS: This study demonstrates a clear thermal effect for linear scanning 980-nm and long-pulsed 1064-nm laser systems on either nail clippings or cell culture media. Complete pathogen growth impairment was achieved if temperatures were measured above 50 °C. The results for the 1064-nm system were almost comparable to 980 nm results.

Treatment of onychomycosis using a submillisecond 1064-nm neodymium:yttrium-aluminum-garnet laser.

BACKGROUND: Laser treatment has emerged as a novel treatment modality for onychomycosis. OBJECTIVE: We sought to determine thermal response and optical effects of a submillisecond neodymium:yttrium-aluminum-garnet (Nd:YAG) 1064-nm laser on common fungal nail pathogens, and the clinical efficacy and safety of the Nd:YAG 1064-nm laser on onychomycotic toenails. METHODS: A 4-part in vitro and in vivo study was conducted using a Nd:YAG 1064-nm laser. The first portion evaluated 3 different nail pathogens in suspension at 7 heat and time exposures. The second and third parts of the study irradiated pure fungal colonies. The final portion involved an in vivo treatment of toenails over 5 treatment sessions. RESULTS: A fungicidal effect for Trichophyton rubrum was seen at 50°C after 15 minutes, and for Epidermophyton floccosum at 50°C after 10 minutes. Limited growth of Scytalidium was seen at 55°C after 5 minutes. No inhibition was observed after laser treatment of fungal colonies or suspensions. In vivo treatment of toenails showed no improvement in Onychomycosis Severity Index score. LIMITATIONS: The Nd:YAG 1064-nm laser was the only laser tested. CONCLUSIONS: Laser treatment of onychomycosis was not related to thermal damage or direct laser effects. In vivo treatment did not result in onychomycosis cure.

Laser treatment of onychomycosis: an in vitro pilot study.

BACKGROUND: Laser treatment of onychomycosis is the object of considerable interest. Laser therapy could be a safe and cost-effective treatment modality without the disadvantages of drugs. Some studies have described the inhibitory effects of lasers on the growth of fungal colonies. We therefore examined the effects of various laser wavelengths, which have previously shown inhibitory potential, on the fungal isolate Trichophyton rubrum. PATIENTS AND METHODS: Isolates of fungal colonies were placed clockwise on culture plates. Each culture plate was irradiated on one half with one of the following treatment regimens: 1064 nm-Q-switched Nd:YAG laser at 4 J/cm(2) and 8 J/cm(2) ; 532 nm-Q-switched Nd:YAG laser at 8 J/cm(2) ; 1064 nm-long-pulsed Nd:YAG laser at 45 J/cm(2) or 100 J/cm(2) . The other half remained untreated. Standardized photographs were taken and areas of treated and untreated colonies were compared for growth inhibition. RESULTS: There was no inhibition of fungal growth in any of the treated plates. Differences in size between treated and untreated colonies were not significant (p > 0.10). CONCLUSIONS: In this in vitro study Nd:YAG laser treatment of Trichophyton rubrum colonies failed to inhibit fungal growth. Nevertheless there might be an effectiveness in vivo which has to be clarified by clinical studies.

Phototoxic action of light emitting diode in the in vitro viability of Trichophyton rubrum.

BACKGROUND: Trichophyton rubrum is the most common agent of superficial mycosis of the skin and nails causing long lasting infections and high recurrence rates. Current treatment drawbacks involve topical medications not being able to reach the nail bed at therapeutic concentrations, systemic antifungal drugs failing to eradicate the fungus before the nails are renewed, severe side effects and selection of resistant fungal isolates. Photodynamic therapy (PDT) has been a promising alternative to conventional treatments. OBJECTIVES: This study evaluated the in vitro effectiveness of toluidine blue O (TBO) irradiated by Light emitting diode (LED) in the reduction of T. rubrum viability. METHODS: The fungal inoculums' was prepared and exposed to different TBO concentrations and energy densities of Light emitting diode for evaluate the T. rubrum sensibility to PDT and production effect fungicidal after photodynamic treatment. In addition, the profiles of the area and volume of the irradiated fungal suspensions were also investigated. RESULTS: A small reduction, in vitro, of fungal cells was observed after exposition to 100 µM toluidine blue O irradiated by 18 J/cm² Light emitting diode. Fungicidal effect occurred after 25 µM toluidine blue O irradiation by Light emitting diode with energy density of 72 J/cm². The analysis showed that the area and volume irradiated by the Light emitting diode were 52.2 mm² and 413.70 mm³, respectively. CONCLUSION: The results allowed to conclude that Photodynamic therapy using Light emitting diode under these experimental conditions is a possible alternative approach to inhibit in vitro T. rubrum and may be a promising new treatment for dermatophytosis caused by this fungus.

Phototoxic action of light emitting diode in the in vitro viability of Trichophyton rubrum / Ação fototóxica do diodo emissor de luz na viabilidade de Trichophyton rubrum in vitro

BACKGROUND: Trichophyton rubrum is the most common agent of superficial mycosis of the skin and nails causing long lasting infections and high recurrence rates. Current treatment drawbacks involve topical medications not being able to reach the nail bed at therapeutic concentrations, systemic antifungal drugs failing to eradicate the fungus before the nails are renewed, severe side effects and selection of resistant fungal isolates. Photodynamic therapy (PDT) has been a promising alternative to conventional treatments. OBJECTIVES: This study evaluated the in vitro effectiveness of toluidine blue O (TBO) irradiated by Light emitting diode (LED) in the reduction of T. rubrum viability. METHODS: The fungal inoculums' was prepared and exposed to different TBO concentrations and energy densities of Light emitting diode for evaluate the T. rubrum sensibility to PDT and production effect fungicidal after photodynamic treatment. In addition, the profiles of the area and volume of the irradiated fungal suspensions were also investigated. RESULTS: A small reduction, in vitro, of fungal cells was observed after exposition to 100 µM toluidine blue O irradiated by 18 J/cm² Light emitting diode. Fungicidal effect occurred after 25 µM toluidine blue O irradiation by Light emitting diode with energy density of 72 J/cm². The analysis showed that the area and volume irradiated by the Light emitting diode were 52.2 mm² and 413.70 mm³, respectively. CONCLUSION: The results allowed to conclude that Photodynamic therapy using Light emitting diode under these experimental conditions is a possible alternative approach to inhibit in vitro T. rubrum and may be a promising new treatment for dermatophytosis caused by this fungus.

FUNDAMENTOS: Trichophyton rubrum é o agente mais comum das micoses superficiais de pele e unhas causando infecções de longa duração e altas taxas de recidiva. As desvantagens do tratamento atual envolvem medicações tópicas as quais não são capazes de alcançar o leito ungueal em concentrações terapêuticas, antifúngicos sistêmicos que não erradicam o fungo antes das unhas serem renovadas, efeitos colaterais graves e seleção de isolados fúngicos resistentes. A terapia fotodinâmica tem sido uma alternativa promissora aos tratamentos convencionais. OBJETIVOS: Este estudo avaliou a eficácia, in vitro, de azul de orto-toluidina irradiado por diodo emissor de luz na redução da viabilidade de T. rubrum. MÉTODOS: O inóculo fúngico foi preparado e exposto a diferentes concentrações de azul de orto-toluidina e densidades de energia do diodo emissor de luz, para avaliar a sensibilidade de T. rubrum e o efeito fungicida, após terapia fotodinâmica. Além disso, os perfis da área e volume das suspensões fúngicas irradiados também foram investigados. RESULTADOS: Uma pequena redução, in vitro, de células fúngicas foi observada após a exposição a 100 mM azul de orto-toluidina irradiados por diodo emissor de luz a 18 J/cm². Efeito fungicida ocorreu após irradiação 25 µM orto-toluidina por diodo emissor de luz com densidade de energia de 72 J/cm². A análise mostrou que a área e o volume irradiados pelo diodo emissor de luz foram 52,2 mm² e 413,70 mm³, respectivamente. CONCLUSÕES: Os resultados permitiram concluir que a terapia fotodinâmica com diodo emissor de luz, nas condições experimentais é uma abordagem alternativa para inibir, in vitro, T. rubrum e pode ser um tratamento promissor para as dermatofitoses causadas por este fungo.

Direct antifungal effect of femtosecond laser on Trichophyton rubrum onychomycosis.

Onychomycosis is caused by dermatophyte infection of the nail. Though laser energy has been shown to eliminate dermatophytes in vitro, direct laser elimination of onychomycosis is not successful due to difficulties in selectively delivering laser energy to the deeper levels of the nail plate without collateral damage. Femtosecond (fsec) infrared titanium sapphire lasers circumvent this problem by the nonlinear interactions of these lasers with biological media. This quality, combined with the deeply penetrating nature of the near-infrared radiation, allows elimination of deeply seeded nail dermatopytes without associated collateral damage. Nail cuttings obtained from patients with onychomycosis caused by Trichophyton rubrum underwent fsec laser irradiation using increasing laser intensities with the focus scanned throughout the whole thickness of the nail specimen. The efficacy of the laser treatment was evaluated by subculture. Scanning electron microscopy was used to determine fsec laser-induced collateral damage. We found that a fsec laser fluence of 7 x 10(31) photons m(-2) s(-1) or above successfully inhibited the growth of the fungus in all samples examined, whereas laser intensities above 1.7 x 10(32) photons m(-2) s(-1) affected the structure of the nail plate. Our findings suggest that T. rubrum-mediated onychomycosis may be treated by fsec laser technology.

The effects of laser irradiation on Trichophyton rubrum growth.

The effects of various laser wavelengths and fluences on the fungal isolate, Trichophyton rubrum, were examined in vitro. Standard-size isolates of T. rubrum were irradiated by using various laser systems. Colony areas were compared for growth inhibition on days 1, 3, and 6 after laser irradiation. Statistically significant growth inhibition of T. rubrum was detected in colonies treated with the 1,064-nm Q-switched Nd:YAG laser at 4 and 8 J/cm(2) and 532-nm Q-switched Nd:YAG laser at 8 J/cm(2). Q-switched Nd:YAG laser at 532- and 1,064-nm wavelengths produced significant inhibitory effect upon the fungal isolate T. rubrum in this in vitro study. However, more in vitro and in vivo studies are necessary to investigate if lasers would have a potential use in the treatment of fungal infections of skin and its adnexa.

Bioadhesive patch-based delivery of 5-aminolevulinic acid to the nail for photodynamic therapy of onychomycosis.

The in vitro penetration of 5-aminolevulinic acid (ALA) across human nail and into neonate porcine hoof when released from a novel bioadhesive patch containing 50 mg cm(-2) ALA is described. ALA is a naturally occurring precursor of the photosensitiser protoporphyrin IX (PpIX). Topical application of excess ALA bypasses negative feedback inhibition and yields photosensitising concentrations of PpIX at the application site. ALA-based photodynamic therapy (PDT) has been extensively investigated in the topical treatment of various skin neoplasias. Recently, its use has been extended to the microbiological field. If sufficient concentrations of ALA could be achieved within the nail matrix, and at the nail bed, PDT may prove to be a useful treatment for onychomycosis. Patch application for 24 h allowed an ALA concentration of 2.8 mM to be achieved on the ventral side of excised human nail. Application for 48 h induced a concentration of 6.9 mM. Application time had no significant effect on the ALA concentration at mean depths of 2.375 mm in neonate porcine, with application times of 24, 48 and 72 h all producing concentrations of 0.1 mM. Incubation of Candida albicans and Trichophyton interdigitale with ALA concentrations of 10.0 mM for 30 min and 6 h, respectively, caused reductions in viability of 87% and 42%, respectively, following irradiation with red light. Incubation with 0.1 mM ALA for 30 min and 6 h, respectively, caused reductions in viability of 32% for Candida albicans and 6% for Trichophyton interdigitale, following irradiation. Drug penetration across nail may be improved using penetration enhancers, or by filing of the impenetrable dorsal surface of the nail. Moreover, iron chelators can be used to increase PpIX production for a given ALA dose. Therefore, with suitable modifications, ALA-PDT may prove to be a viable alternative in the treatment of onychomycosis.

Photodynamic inactivation of the dermatophyte Trichophyton rubrum.

Dermatophytes are fungi that can cause infections (known as tinea) of the skin, hair and nails because of their ability to use keratin. Superficial mycoses are probably the most prevalent of infectious diseases worldwide. One of the most distinct limitations of the current therapeutic options is the recurrence of the infection and duration of treatment. The present study shows that Trichophyton rubrum in suspension culture is susceptible to photodynamic treatment (PDT), a completely new application in this area. T. rubrum could be effectively killed with the use of the light-activated porphyrins deuteroporphyrin monomethylester (DP mme) and 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B). The photodynamic efficacy was compared with that of some other photosensitizers that are well known in the field of PDT: the porphyrins deuteroporphyrin and hematoporphyrin, the drug Photofrin and several phthalocyanines. It was demonstrated that with the use of broadband white light, the phthalocyanines and Photofrin displayed a fungistatic effect for about 1 week, whereas all the porphyrins caused photodynamic killing of the dermatophyte. Sylsens B was the most effective sensitizer and showed no dark toxicity; therefore, in an appropriate formulation, it could be a promising candidate for the treatment of various forms of tinea. For Sylsens B and DP mme, which displayed the best results, a concentration-dependent uptake by T. rubrum was established.