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Photodynamic inactivation of normal and antifungal resistant Candida species
Photodiagnosis and Photodynamic Therapy (2010) 7, 98—105
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Photodynamic inactivation of normal and antifungal resistant Candida species T.S. Mang MD, PhD ∗, L. Mikulski, R.E. Hall University at Buffalo, School of Dental Medicine, Department of Oral and Maxillofacial Surgery, Squire 112, 3435 Main St., Buffalo, NY 14214, United States Available online 8 April 2010
KEYWORDS PDT; Candida; Antifungal resistance; AIDS
Summary Background: Susceptibility of bacterial and fungal species to the photodynamic killing effects of various photosensitizing dyes has received increasing attention. In the oral cavity oral candidiasis is primarily caused by Candida albicans. Evidence suggests that Oropharyngeal Candidiasis, found frequently in patients with immunodeficiency, present with mixed Candida organisms and are more difficult to treat than those solely due to C. albicans. In the present study we demonstrate the ability to efficiently kill antifungal resistant isolates of Candida using Photofrin induced PDT. Methods: Candida strains from the ATCC as well as fluconazole and amphotericin B resistant and sensitive isolates from adults with AIDS were grown cultures and grown under standard conditions. Photofrin was added to appropriate cultures as dictated by experimental design. Light was delivered to assigned cultures using a 630 nm laser source at a power density of 150 mW/cm2 , for appropriate time to deliver 45—135 J/cm2 . Colony forming assays were used to determine survival. Results: After illumination cultures treated with Photofrin had significant reduction in colony forming ability at all light doses examined. Isolates from AIDS patients which had demonstrated antifungal resistance showed equivalent sensitivity to photodynamic killing as did control ATCC cultures of the same strain. Conclusion: This study demonstrates Photofrin induced PDT can eliminate Candida species with significant efficiency as revealed by colony forming ability. Further Candida isolates from AIDS patients that had demonstrated fluconazole and amphotericin B resistance were equally susceptible to photodynamic killing. © 2010 Elsevier B.V. All rights reserved.
Introduction/background Cancer patients receiving chemotherapy are at high risk of morbidity and mortality from disseminated fungal infection.
∗
Corresponding author. E-mail address: [email protected] (T.S. Mang).
Patients with lymphoreticular malignancies are especially vulnerable because of suppressed cellular immune function. Oral Candida infections are common opportunistic infections that have emerged as a major side effect in cancer patients receiving chemotherapy or radiation to the head and neck area and have been identified risk factor for Candida dissemination. Patients with hematological and head and neck malignancies are especially vulnerable because of
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Photodynamic inactivation of normal and anti-fungal resistant suppressed leukocyte function in the former and compromised salivary flow in the latter. Oral Candida infections cause inflammation of the oral mucosa, pain and dysglusia. It is a common source of distress in cancer patients and an identified risk factor for more invasive lesions. Yeasts are the only fungi commonly found in the oral cavity of healthy individuals. In a healthy individual the strain most prevalent is C. albicans and is found in about 60—70% of all isolates. In a review by Odds [1], a compilation of 32 papers on yeast isolations from the ‘‘normal’’ oral cavity showed the median carriage frequency was 34.4% for all yeasts and 17% for C. albicans alone. C albicans is followed in frequency by C. glabrata, C. krusei, C. tropicalis and other Candida species. Data obtained for the frequency of yeast in the oral cavity are dependent on isolation techniques and time of sampling. Often harmless in the normal human cavity, Candida species, especially C. albicans, can lead to mucosal infections. Oropharyngeal Candidiasis (OPC) is a frequently found infection in patients with immunodeficiency like HIV, nutritional deficiencies, metabolic disorders such as diabetes, malignancies, xerostomia (secondary to radiation therapy), medication side effects, aging, Sjorgrens syndrome, and denture prostheses. The development of Candidiasis greatly depends on the host defense system and is considered to be an independent predictor of immunodeficiency in patients with acquired immune deficiency syndrome (AIDS). C. albicans has been described as the predominant organism and still remains so. Studies have also described the emergence of yeast other than C. Albicans as the causative pathogens in OPC both as co-infecting organisms with C. albicans and as sole pathogens themselves [2,3]. A wide variety of Candida organisms have been shown to cause infection, with C. dubliensis, C. glabrata, C. krusei and C. tropicalis being the most commonly described [2]. C. glabrata is the second or third leading cause of Candidiasis comprising 5—8% of Candida isolates and reports of OPC sue solely to C. glabrata have been increasing. OPC with C. krusei is only rare and C. krusei is found mixed with C. albicans and in C. glabrata. C. tropicalis in OPC has been reported. Recent evidence suggests that OPC infections with mixed C. albicans and other Candida organisms in HIV patients present more severe symptoms and are more difficult to treat than those infections sue solely to C. albicans. Erthematous Candidiasis (EC) also known as denture stomatitis and denture sore mouth is a disease that involves pathologic changes in denture-bearing oral mucosa. Candida species can be isolated in up to 93% of patients with denture stomatitis and in 46—78% of healthy denture wearers. The mucosal changes can be numerous but symptoms infrequent. Treatment requires removal and disinfection of the prosthesis, use of antimycotic drugs and identification of factors that may have lowered host resistance. In a study by Crockett [4] at least 5 species of Candida were found in EC, with C. albicans being the most prevalent. It was also found that strains of C. albicans associated with EC are similar to those found in healthy subjects wearing full dentures [3]. The therapy of choice for oral candidiasis is a course of systemic antifungal agents such as the azole antifungal fluconazole or amphotericin B. These antifungal therapies are effective, but re-occurrence of candidiasis is common [5]. Maintenance fluconazole therapy is used to combat
99 recurrent infections. The concomitant risk of antifungal resistance, fluconazole interactions with other medical regimens and organ toxicity are potential adverse events. Azole resistant oropharyngeal and esophageal candidiasis is a refractory form of the opportunistic infection occurring particularly in HIV infected patients [6]. Amphotericin B is well known for severe and potentially lethal side effects when administered intravenously. Oral preparations of the drug are less toxic.
Photodynamic therapy Photodynamic therapy, PDT, is a therapeutic modality involving the use of a photosensitizing agent and the local application of visible light. A photosensitizing agent approved for this use is Photofrin (porfimer sodium) (Axcan Pharma, Mt. St. Hilaire, QC, Canada). PDT has been employed in a wide range of cutaneous malignancies with excellent clinical and cosmetic response [7,8]. Fundamentally PDT involves application of a photosensitizing agent which for various reasons accumulates preferentially in malignant tissue and/or clears faster from normal tissues. Subsequently the photosensitizer is activated by the appropriate wavelength of light that results in the cytotoxic photodynamic reaction, involving the production of singlet oxygen [9]. The emergence of porphyrin mixtures in the treatment of cancer was described in a series of studies by Dougherty [10]. These studies investigated the application of Hematoporphyrin derivative (HPD) induced PDT in a broad range of malignancies in pre-clinical and clinical trials. These studies were focused on determining the sites of action, toxicity, drug and light dosimetry and efficacy of HPD-PDT. Through their success, PDT has become an emerging modality for the treatment of cancer today.
PDT for microbial diseases In 1924 Chavarria and Clark [11] produced dermatophytic cutaneous lesions in guinea pigs with Trichophyton acuminatum and treated them with eosin and visible light. Guinea pigs treated with eosin and visible light were cured of cutaneous dermatophyte infections while the control groups showed slight (light alone) or no improvement. In 1962 Dickey [12] demonstrated the photodynamic fungicidal effects of neutral red, methylene blue and other dyes on Candida albicans and Trichophyton mentagrophytes. Studies conducted to determine the susceptibility of pathogenic Candida species to photodynamic effects of photosensitizing agents’ demonstrated unique sensitivity of various strains in vitro which showed organisms damaged in a drug dose and light dose-dependent manner. Subsequent studies conducted in biofilms and in vivo have likewise concluded that photodynamic therapy could potentially be used to treat patients with oral candidiasis [13—15]. Photodynamic inactivation of viruses, bacteria and fungi by photoactive dyes and visible light is a well-documented phenomenon. Analysis of the data demonstrates that S. mutans and Candida cells grown in planktonic cultures are highly susceptible to killing with minimal exposures to PDT. Although dye-light therapy has shown to cure superficial bac-
100 terial and fungal infections the treatment has lost out to modern-day nonstaining antibiotics and antifungals [16,17]. Systemic azole antifungals however, rarely achieve enduring responses and have resulted in growing antifungal drug resistance issues [6]. Several studies have demonstrated the utility of PDT, utilizing a growing number of photosensitizing agents and light activation sources, for the inactivation of Candida species [13—19]. Dovigo et al., [20] have also demonstrated that PDT using a porphyrin based photosensitizer, and activation wavelengths in the visible blue light region, is effective against C. albicans and C. glabrata. That study demonstrated that under the same conditions fluconazole resistant strains of C. albicans and C. glabrata show some resistance to the action of PDT as well. In the present study we have examined the ability of porfimer sodium mediated PDT to treat different species of Candida as well as fluconazole (FL) and amphotericin B (AP) resistant and sensitive isolates of the following; C. albicans, C. glabrata, C. guilliermindi, C. parapsilosis and C. krusei, from a collection of isolates from adults with AIDS.
Materials and methods Yeast strains and growth conditions Candida albicans ATCC (American Type Culture Collection, Rockville, MD, USA) 90028, C. glabrata ATCC 90030, C. parapsilosis 22019, C. krusei 6258, and C. tropicalis 42678 were used for direct comparison of susceptibility to killing by Photodynamic antimicrobial chemotherapy. Cells were transferred from a 16 h SBA plate (Difco Sabouraud Dextrose Agar, Becton Dickinson, Sparks, MD, USA) into RPMI medium 1640 (GIBCO Invitrogen, Grand Island, NY, USA) with the addition of 5% newborn calf serum (Crane Laboratories, Syracuse, NY, USA). Cells were grown with shaking at 37◦ until the log growth phase was reached. At this point the optical density at 600 nm was 0.30—0.40 after 3—4 h of growth. The cells were centrifuged for 2 min at 14,000 rpm, resuspended in O.85% NaCl and adjusted to an O.D. of 0.10 at 600 nm.
Yeast growth and concentration Growth curves of C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis were performed to determine the range of the log phase of growth. It was at this phase that the yeast was used for subsequent experiments. A fixed optical density was used in the vials during Photofrin and light exposure to ensure yeast concentrations were consistent. Various dilutions after the treatment process were used to determine an optimal number of organisms on the plates that could be easily counted. Yeast isolates Strains are from the oral mucosa of adults with Candida infections C. albicans, (1 FL resistant) C. glabrata, (1 FL and AP resistant; 1 FL resistant) C. guilliermondi, (1 AP resistant), C. parapsilosis (1 FL sensitive) and C. krusei, (1 FL and AP resistant). The strains were pulled from a collection of isolates from a previous study on adults with AIDS. Resis-
T.S. Mang et al. tance and sensitivity were determined by standard Etest (AB BIODISK, Solna Sweden) for antifungal susceptibility. Each isolate was grown on SDA (Sabouraud Dextrose Agar) in a humidified incubator at 32 ◦ C. Inoculums were prepared from individual colonies (≥1 mm diameter) on the SDA into 5 ml of sterile 0.85% saline to a density of 0.5 McFarland standards to equalize the yeast cell density. A 1:10 dilution of the same suspension was then made for photodynamic illuminations.
Photosensitizing agent Photofrin® porfimer solution, (Axcan Scandipharm, Inc., Birmingham, AL, USA) stock solution was adjusted to 7.5 mg/ml in 0.85% sterile NaCl and frozen at −50 ◦ C until use. The final media, concentration used for all experiments was 25 g/ml.
Photofrin exposure to yeast cells The adjusted cells were aliquoted into 4.0 ml sterile glass vials (Wheaton, Millville, NJ, USA). Each control and test condition was done in triplicate. The conditions were as follows: For the controls, NLNP (no light exposure, no porfimer sodium), NLP (no light exposure, porfimer sodium), LNP (light exposure, no porfimer sodium), and for the test condition, LP (light exposure, porfimer sodium). Under conditions of darkness, porfimer sodium was added at a concentration of 25 g/ml and sterile saline was added in the same amount to the vials without porfimer sodium. The drug was added to growing yeast cultures at either 24 or 1 h prior to aliquot preparation for illumination. All subsequent procedures were carried out in darkness to avoid ambient light activation of the drug in incubated samples.
Laser light exposure to yeast cells The light source for PDT is generally derived from a laser and delivered via a fiberoptic device designed for that specific application. Light was delivered using a KTP: YAGdye laser combination (Laserscope, CA) tuned to deliver light at 630 nm or a Diomed 630 nm PDT laser (Diomed Inc. Cambridge UK). These systems are equivalent and utilized interchangeably in pre-clinical and clinical studies. The light from the laser was focused on suspensions of yeast cells following incubation with either porfimer sodium using a lens fiberoptic. This fiberoptic focuses light in a homogenous field over diameters of 1 mm to 15 cm. Light was delivered using a power density of 150 mW/cm2 for varying light doses according to the experimental plan. The vials were exposed to laser light directed from the bottom of the vial at a light level of 45, 90, or 135 J/cm2 . All vials not being treated were kept in darkness for periods of time equivalent to light treatment durations.
Plating and enumeration of yeast cell growth The effect of light and dye combinations, the photodynamic antimicrobial effect, was determined by plating 100 l of the yeast suspension onto SDA agar in duplicate and incubat-
Photodynamic inactivation of normal and anti-fungal resistant
Figure 1 (a) Phototoxicity of Photofrin and increasing light dose, on colony forming ability of various ATCC Candida cultures. (b) Phototoxicity of Photofrin and increasing 630 nm light doses on the colony forming ability of ATCC cultures of C. glabrata.
ing for 48 h at 37 ◦ C in a humidified incubator. Colony forming unit (CFU) assays were used to determine CFU macroscopically. Specifically, under conditions of darkness, an aliquot was removed from each vial and diluted with sterile 0.85% NaCl. 0.10 ml of this was plated to SDA and spread with a glass rod spreader. All plates were incubated for 48 h at 37 ◦ C. Only at the time of counting were the yeast growth plates exposed to light. Total colony counts were done on each plate. The total count was averaged for each condition from the triplicate plates. Untreated controls were manipulated in parallel and used for reference and to determine the plating efficiency.
Statistical analysis The effect of each photosensitizing drug and varying light dose will be expressed as colony forming Unit (CFU) survival and analyzed by means of the Mann—Whitney U-test. Values of p less than .05 will be considered statistically insignificant.
Results The effect on the viability of the examined ATCC strains of Candida after exposure to porfimer sodium alone, light alone and the PDT combination of porfimer sodium and light, using escalating light doses is shown in Fig. 1a and b. Exposure of the organisms to increasing doses of laser light at 630 nm following a single dose 24 h incubation porfimer sodium concentration, 25 g/ml, resulted in a light dose dependent
101 decrease in viable colony counts. In the absence of light in suspensions incubated with porfimer sodium, changes in colony counts were not significant and there was little or no evidence of dark toxicity. Exposure to laser illumination in the absence of porfimer sodium did not result in any significant changes or decreases in cell viability as determined by colony counts. All Candida strains tested showed significant sensitivity to light and drug combinations though there were some differences. In the experiments conducted to compare the different strains, C. Krusei demonstrated less sensitivity to the lower light doses applied, than the other strains examined. However, at the highest light dose utilized, 135 J/cm2 , significant cell kill was observed. C. glabrata also demonstrated significant killing effect by the combination of porfimer sodium and light (Fig. 1b). A second series of experiments were conducted to determine the effect of drug incubation time on the overall cell killing efficacy of PDT. It was determined that effective cell kill using the same light dose parameters, could be achieved using a 1 h incubation vs. 24 h, in media containing 25 g/ml porfimer sodium concentration, demonstrating rapid uptake of the drug. All subsequent experiments were carried out using the porfimer sodium concentration of 25 g/ml for a 1 h incubation period prior to light exposure, while maintaining the light dose parameters of 150 mW/cm2 power density and light dose exposures of 45, 90 and 135 J/cm2 . Another series of experiments was conducted to determine if antifungal resistant Candida species differ in their susceptibility, compared to ATCC strains, to the photodynamic killing action of porfimer sodium activation by 630 nm laser light. FL and/or AP resistant strains of C. glabrata, C. albicans, C. krusei, C. guilliermindi, and C. parapsilosis, isolated from HIV positive patients were grown under the same conditions of the ATCC strains and subjected to the identical experimental conditions as previously demonstrated effective in producing significant cell kill. Figs. 2—6 show the light dose-dependent cell killing effect when cultures previously incubated with 25 g/ml porfimer sodium for 1 h, are exposed to the increasing doses of 630 nm light. The comparison of ATCC C. albicans (Fig. 2a) to the FL resistant isolate of C. albicans is shown (Fig. 2b). The lowest light dose demonstrated approximately 92% killing (p = 0.002) which subsequently increased with increasing light dose. All resistant or sensitive strains examined showed significant susceptibility to the killing effect of PDT under the conditions tested as demonstrated in the following isolate strains examined: C. glabrata (Fig. 3a; FL resistant, AP sensitive), C. glabrata (FL and AP resistant Fig. 3b); C. krusei (FL and AP resistant, Fig. 4); C. guilliermindi (AP resistant FL sensitive, Fig. 5) and 2 (FL and AP sensitive), isolates of C. parapsilosis (Fig. 6a and b). The isolate strain of C. krusei demonstrated similar response characteristics to the lower light doses tested as did the ATCC strain (Fig. 4). However, the resistant strain was also susceptible to the killing effects of PDT at the highest light dose tested (p = 0.003).
Discussion In vitro susceptibility of bacterial and fungal species to the photodynamic effects of various photosensitizing dyes and
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Figure 2 (a) Efficacy of Photofrin and 630 nm light on colony forming ability of ATCC culture of C. albicans. Cultures were irradiated with light doses ranging from 45 to 135 J/cm2 using a dose rate of 150 mW/cm2 . Significant reductions are seen (p = 0.002) when compared to unirradiated controls. (b) Efficacy of Photofrin and 630 nm light on colony forming ability of fluconazole resistant isolate of C. albicans. Significant reductions in Colony forming ability seen at all light doses (p = 0.001).
illumination with appropriate wavelengths of light has been receiving increased attention. Many organisms found within the flora of the oral cavity have demonstrated susceptibility to various regimens in vitro and in animal models. Some studies have shown various strains to be relatively resistant to the killing effects of PDT. In the oral cavity oral candidiasis is primarily caused by Candida albicans, a dimorphic organism that typically is present in the oral cavity in a non-pathogenic state in healthy individuals. Often harmless in the normal human cavity, Candida species, especially C. albicans, can lead to mucosal infections [21,22]. Recent evidence suggests that OPC infections with mixed C. albicans and other Candida organisms in HIV patients present more severe symptoms and are more difficult to treat than those infections sue solely to C. albicans [23]. A wide variety of Candida organisms have been shown to cause infection, with C. dubliensis, C. glabrata, C. krusei and C. tropicalis being the most commonly described. C. dublienis is still commonly described as C. albicans. In the
vast majority of clinical cases of OPC, distinction between C. dublienis and C. albicans is not necessary for successful treatment. C. glabrata is the second or third leading cause of Candidiasis comprising 5—8% of Candida isolates and reports of OPC sue solely to C. glabrata have been increasing. OPC with C. krusei is only rare and C. krusei is found mixed with C. albicans and in C. glabrata. C. tropicalis in OPC has been reported [24]. Primary fluconazole resistance has been defined by the Subcommittee for Antifungal Testing of the Clinical Laboratory Standards Institute as resistance in the absence of prior fluconazole exposure. Secondary resistance occurs as a result of treatment with fluconazole. Fluconazole-resistant oropharyngeal candidiasis has involved both C. albicans and non-albicans species. Following the widespread use of fluconazole in the 1990’ the proportion of C. albicans isolates decreased and other Candida species, including C. krusei, C. dubliensis and C. glabrata increased. In a study by Campisi [25] C. albicans was most often recovered in patients with HIV, isolated in 19/26 HIV+ carriers (73.1%). C. glabrata was found in 1
Photodynamic inactivation of normal and anti-fungal resistant
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Figure 3 (a) Phototoxicity of Photofrin and 630 nm light on irradiated cultures of fluconazole resistant and amphotericin B sensitive C. glabrata. Reductions in CFU for all light doses studied demonstrated significance (p = 0.009—0.008) (b) Phototoxicity of Photofrin and 630 nm light on cultures of fluconazole and amphotericin B resistant C. glabrata (p = 0.0001).
Figure 4 Efficacy of Photofrin induced PDT on fluconazole and amphotericin B resistant isolates of C. Krusei. At the lowest light doses colony forming ability was reduced at a significance of (p = 0.011—0.006). Continued irradiation yielded greater cell killing (p = 0.003) when compared to unirradiated controls.
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Figure 5
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Photofrin PDT phototoxicity of amphotericin B resistant and fluconazole sensitive isolates of C. guilliermondi (p = 0.0001).
subject (3.8%) and combinations of Candida species were also found [24]. Worldwide C. parapsilosis is among the most common species causing invasive disease. It has been demonstrated to be the second most frequent bloodstream isolate in candidemia patients and increasing in frequency [26]. Photodynamic drugs have routinely been administered by IV injection. This mode of delivery would not generally meet with acceptance in the treatment of fungal diseases, as systemic administration of a photosensitizing drug, in particular porfimer sodium, results in the primary side effect of cutaneous photosensitivity for a period of approximately 3—4 weeks. Some photosensitizers which have gained regulatory approval demonstrate shorter periods of cutaneous photosensitivity yet still require IV injection and periods of time for selective uptake to occur. While the side effect, cutaneous photosensitivity, is much less serious than those demonstrated with current therapeutic modalities to treat oral candidiasis, IV drug delivery would not, most likely, result in significant uptake of the photosensitizer drug by Candia species in the oral cavity. Further this method of
Figure 6
drug delivery would result in decreased selectivity of drug uptake as healthy tissues would retain some level, albeit lower concentration, than diseased tissue or in this scenario, fungal organisms. Ultimately this results in dosimetric considerations necessary to avoid collateral damage to healthy tissues. This is further illustrated in Fig. 1a and b. Generally, when a patient is given porfimer sodium IV, there is a 48 h period in which uptake and retention, to provide a reasonable therapeutic ratio, occurs. In vitro studies using this drug in mammalian cells have generally employed a 24 h incubation period to ensure adequate uptake and localization. Therefore the short incubation time in which the Canadida actively take up sufficient drug would provide a clear therapeutic ratio between the target and normal tissue which results in a selective therapeutic advantage. A topical mode of drug delivery for oral candidiasis is highly desirable in this patient population. This mode of delivery has the advantage of avoiding systemic toxicity and drug interactions as well as saving systemic agents for invasive disease. At present there are no efficacious topical agents for oral candidiasis in patients at risk for developing
Phototoxicity of photofrin induced PDT on fluconazole and amphotericin B sensitive isolate of C. parapsilosis (p = 0.007).
Photodynamic inactivation of normal and anti-fungal resistant the infection. PDT may be a promising topical treatment for the control of oral Candida growth and by extension oropharyngeal candidiasis. The drug porfimer sodium has shown promise in the treatment of superficial disease but as yet has not been developed as a topical treatment. In people with a weakened immune system due to steroid therapy, cancer chemotherapeutic regimens or radiotherapy, or diseases such as AIDS, candidal infections are a considerable challenge. The infection can occur locally regionally or throughout the body and can be life threatening. The therapy of choice for oral candidiasis is a course of systemic antifungal agents such as the azole antifungal fluconazole and amphotericin B. Generally the azole medications are prescribed as they can be used topically or through oral administrations. Amphotericin B is reserved for more serious systemic fungal infections. Some forms of the drug are used for superficial candidal infections and thrush. Further, in patients who have demonstrated fluconazole and or amphotericin B resistant strains PDT represents a possible alternative to or adjunctive therapy for the treatment of oral candidiasis. We demonstrate in our series of experiments the susceptibility of control strains of Candida obtained from the ATCC and various isolates of Candida, obtained from adult patients with AIDS, which had demonstrated fluconazole and amphotericin B resistance, to the killing effects of Photofrin mediated PDT. The demonstration of the sensitivity of these organisms to the photodynamic toxicity of an agent which is clinically accepted is relevant to the use of this agent in the treatment of patients with oral candidiasis. The ultimate key to successful treatment of Candidaassociated infection is eradication of the Candida biofilm. Photodynamic therapy may provide a means to the successful elimination of denture biofilm if uptake of the photosensitizing dye by the microbes may be achieved with topical administration and their subsequent destruction upon illumination demonstrated in a manner consistent with today’s clinical practice.
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105 [7] Wilson BD, Mang TS, Cooper M, Stoll H. Use of photodynamic therapy for the treatment of extensive basal cell carcinomas. Facial Plast Surg 1989;6:185—9. [8] Wilson BD, Mang TS, Stoll H, Jones C, Cooper M, Dougherty TJ. Photodynamic therapy for the treatment of basal cell carcinoma. Arch Dermatol 1992;128:1597—601. [9] Weishaupt KR, Gomer CJ, Dougherty TJ. Identification of singlet oxygen as the cytotoxic agent in photoinactivation of a murine tumor. Cancer Res 1976;36:2326—9. [10] Dougherty TJ, Marcus SL. Photodynamic therapy: review. Eur J Cancer 1992;28A:1734—42. [11] Chavarria AP, Clark JH. The reaction of pathogenic fungi to ultraviolet light. Am J Hyg 1924;4:639—49. [12] Dickey RF. Investigative studies in fungicidal powers of photodynamic action. Invest Dermatol 1962;39:7—19. [13] Bliss JM, Bigelow CE, Foster TH, Haidaris CG. Susceptibility of Candida species to photodynamic effects of photofrin. Antimicrob Agents Chemother 2004;48:2000—6. [14] Teichert MC, Jones BA, Usacheva MN, Biel MA. Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:155—60. [15] Rosello YC, Foster TH, Perez-Nazario N, Mitra S, Haidaris CG. Sensitivity of Candia albicans germ tubes and biofilms to photofrin-mediated phototoxicity. Antimicrob Agents Chemother 2005;49:4288—95. [16] Chadbourne K, Mikulski L, Mang TS. Photodynamic therapy of oral microbes. In: Abstracts 35th annual Meeting of the AADR. 2006. [17] Thorsrud KJ, Mang TS, Hall RE. In vitro photodynamic treatment of S. Mutans with m-THPC. In: Abstract 90th annual meeting of the AAOMS Seattle Wash. 2008. [18] Powderly WG, Mayer KH, Perfect JR. Diagnosis and treatment of oropharyngela candiddiasis in patients infected with HIV: a critical reassessment. AIDS Res Hum Retroviruses 1999;15:1405—12. [19] Wai So C, Tsang PWK, Lo PC, Seneviratne CJ, Samaranayake LP, Fong WP. Photodynamic inactivation of Candida albicans by BAM-SiPc. Mycoses 2009. March early view online 1—6. [20] Dovigo LN, Pavarina AC, deOliveira Mima EG, Giampaolo ET, Vergani CE, Bagnato VS. Fungicidal effect of photodynamic therapy against fluconazole resistant Candida albicans and Candida glabrata. Mycoses 2009. November early view online 1—8. [21] Barchiesi F, Maracci M, Radi B, et al. Point prevelance, microbiology and fluconazole susceptibility patterns of yeast isolates colonizing the oral cavities of HIV-infected patients in the era of highly active antiretroviral therapy. J Antimicrob Chemother 2002;50:999—1002. [22] Nolte FS, Parkinson T, Falconer DJ, et al. Isolation and characterization of fluconazole and amphotericin B-resistant Candida albicans from blood of two patients with leukemia. Antimicrob Agents Chemother 1997;44:196—9. [23] Nevill BW, Damm DD, Allen CM, Bouquot JE. Oral and maxillofacial pathology. 2nd ed. Philedelphia: W.B. Saunders; 2002. [24] Davies r, Bedi R, Scully C. Oral health care for patients with special needs. Br Med J 2000;21:19—26. [25] Campisi G, Pizzo G, Milici ME, Mancuso S, Margiotta V. Candidal carriage in the oral cavity of human immunodeficiency virus-infected subjects. Oral Surg Oral Med Oral Pathol 2002;93:281—6. [26] Trofa D, Gácser A, Nosanchuk JD. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 2008;21:606—25.
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Photodynamic inactivation of normal and antifungal resistant Candida species T.S. Mang MD, PhD ∗, L. Mikulski, R.E. Hall University at Buffalo, School of Dental Medicine, Department of Oral and Maxillofacial Surgery, Squire 112, 3435 Main St., Buffalo, NY 14214, United States Available online 8 April 2010
KEYWORDS PDT; Candida; Antifungal resistance; AIDS
Summary Background: Susceptibility of bacterial and fungal species to the photodynamic killing effects of various photosensitizing dyes has received increasing attention. In the oral cavity oral candidiasis is primarily caused by Candida albicans. Evidence suggests that Oropharyngeal Candidiasis, found frequently in patients with immunodeficiency, present with mixed Candida organisms and are more difficult to treat than those solely due to C. albicans. In the present study we demonstrate the ability to efficiently kill antifungal resistant isolates of Candida using Photofrin induced PDT. Methods: Candida strains from the ATCC as well as fluconazole and amphotericin B resistant and sensitive isolates from adults with AIDS were grown cultures and grown under standard conditions. Photofrin was added to appropriate cultures as dictated by experimental design. Light was delivered to assigned cultures using a 630 nm laser source at a power density of 150 mW/cm2 , for appropriate time to deliver 45—135 J/cm2 . Colony forming assays were used to determine survival. Results: After illumination cultures treated with Photofrin had significant reduction in colony forming ability at all light doses examined. Isolates from AIDS patients which had demonstrated antifungal resistance showed equivalent sensitivity to photodynamic killing as did control ATCC cultures of the same strain. Conclusion: This study demonstrates Photofrin induced PDT can eliminate Candida species with significant efficiency as revealed by colony forming ability. Further Candida isolates from AIDS patients that had demonstrated fluconazole and amphotericin B resistance were equally susceptible to photodynamic killing. © 2010 Elsevier B.V. All rights reserved.
Introduction/background Cancer patients receiving chemotherapy are at high risk of morbidity and mortality from disseminated fungal infection.
∗
Corresponding author. E-mail address: [email protected] (T.S. Mang).
Patients with lymphoreticular malignancies are especially vulnerable because of suppressed cellular immune function. Oral Candida infections are common opportunistic infections that have emerged as a major side effect in cancer patients receiving chemotherapy or radiation to the head and neck area and have been identified risk factor for Candida dissemination. Patients with hematological and head and neck malignancies are especially vulnerable because of
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Photodynamic inactivation of normal and anti-fungal resistant suppressed leukocyte function in the former and compromised salivary flow in the latter. Oral Candida infections cause inflammation of the oral mucosa, pain and dysglusia. It is a common source of distress in cancer patients and an identified risk factor for more invasive lesions. Yeasts are the only fungi commonly found in the oral cavity of healthy individuals. In a healthy individual the strain most prevalent is C. albicans and is found in about 60—70% of all isolates. In a review by Odds [1], a compilation of 32 papers on yeast isolations from the ‘‘normal’’ oral cavity showed the median carriage frequency was 34.4% for all yeasts and 17% for C. albicans alone. C albicans is followed in frequency by C. glabrata, C. krusei, C. tropicalis and other Candida species. Data obtained for the frequency of yeast in the oral cavity are dependent on isolation techniques and time of sampling. Often harmless in the normal human cavity, Candida species, especially C. albicans, can lead to mucosal infections. Oropharyngeal Candidiasis (OPC) is a frequently found infection in patients with immunodeficiency like HIV, nutritional deficiencies, metabolic disorders such as diabetes, malignancies, xerostomia (secondary to radiation therapy), medication side effects, aging, Sjorgrens syndrome, and denture prostheses. The development of Candidiasis greatly depends on the host defense system and is considered to be an independent predictor of immunodeficiency in patients with acquired immune deficiency syndrome (AIDS). C. albicans has been described as the predominant organism and still remains so. Studies have also described the emergence of yeast other than C. Albicans as the causative pathogens in OPC both as co-infecting organisms with C. albicans and as sole pathogens themselves [2,3]. A wide variety of Candida organisms have been shown to cause infection, with C. dubliensis, C. glabrata, C. krusei and C. tropicalis being the most commonly described [2]. C. glabrata is the second or third leading cause of Candidiasis comprising 5—8% of Candida isolates and reports of OPC sue solely to C. glabrata have been increasing. OPC with C. krusei is only rare and C. krusei is found mixed with C. albicans and in C. glabrata. C. tropicalis in OPC has been reported. Recent evidence suggests that OPC infections with mixed C. albicans and other Candida organisms in HIV patients present more severe symptoms and are more difficult to treat than those infections sue solely to C. albicans. Erthematous Candidiasis (EC) also known as denture stomatitis and denture sore mouth is a disease that involves pathologic changes in denture-bearing oral mucosa. Candida species can be isolated in up to 93% of patients with denture stomatitis and in 46—78% of healthy denture wearers. The mucosal changes can be numerous but symptoms infrequent. Treatment requires removal and disinfection of the prosthesis, use of antimycotic drugs and identification of factors that may have lowered host resistance. In a study by Crockett [4] at least 5 species of Candida were found in EC, with C. albicans being the most prevalent. It was also found that strains of C. albicans associated with EC are similar to those found in healthy subjects wearing full dentures [3]. The therapy of choice for oral candidiasis is a course of systemic antifungal agents such as the azole antifungal fluconazole or amphotericin B. These antifungal therapies are effective, but re-occurrence of candidiasis is common [5]. Maintenance fluconazole therapy is used to combat
99 recurrent infections. The concomitant risk of antifungal resistance, fluconazole interactions with other medical regimens and organ toxicity are potential adverse events. Azole resistant oropharyngeal and esophageal candidiasis is a refractory form of the opportunistic infection occurring particularly in HIV infected patients [6]. Amphotericin B is well known for severe and potentially lethal side effects when administered intravenously. Oral preparations of the drug are less toxic.
Photodynamic therapy Photodynamic therapy, PDT, is a therapeutic modality involving the use of a photosensitizing agent and the local application of visible light. A photosensitizing agent approved for this use is Photofrin (porfimer sodium) (Axcan Pharma, Mt. St. Hilaire, QC, Canada). PDT has been employed in a wide range of cutaneous malignancies with excellent clinical and cosmetic response [7,8]. Fundamentally PDT involves application of a photosensitizing agent which for various reasons accumulates preferentially in malignant tissue and/or clears faster from normal tissues. Subsequently the photosensitizer is activated by the appropriate wavelength of light that results in the cytotoxic photodynamic reaction, involving the production of singlet oxygen [9]. The emergence of porphyrin mixtures in the treatment of cancer was described in a series of studies by Dougherty [10]. These studies investigated the application of Hematoporphyrin derivative (HPD) induced PDT in a broad range of malignancies in pre-clinical and clinical trials. These studies were focused on determining the sites of action, toxicity, drug and light dosimetry and efficacy of HPD-PDT. Through their success, PDT has become an emerging modality for the treatment of cancer today.
PDT for microbial diseases In 1924 Chavarria and Clark [11] produced dermatophytic cutaneous lesions in guinea pigs with Trichophyton acuminatum and treated them with eosin and visible light. Guinea pigs treated with eosin and visible light were cured of cutaneous dermatophyte infections while the control groups showed slight (light alone) or no improvement. In 1962 Dickey [12] demonstrated the photodynamic fungicidal effects of neutral red, methylene blue and other dyes on Candida albicans and Trichophyton mentagrophytes. Studies conducted to determine the susceptibility of pathogenic Candida species to photodynamic effects of photosensitizing agents’ demonstrated unique sensitivity of various strains in vitro which showed organisms damaged in a drug dose and light dose-dependent manner. Subsequent studies conducted in biofilms and in vivo have likewise concluded that photodynamic therapy could potentially be used to treat patients with oral candidiasis [13—15]. Photodynamic inactivation of viruses, bacteria and fungi by photoactive dyes and visible light is a well-documented phenomenon. Analysis of the data demonstrates that S. mutans and Candida cells grown in planktonic cultures are highly susceptible to killing with minimal exposures to PDT. Although dye-light therapy has shown to cure superficial bac-
100 terial and fungal infections the treatment has lost out to modern-day nonstaining antibiotics and antifungals [16,17]. Systemic azole antifungals however, rarely achieve enduring responses and have resulted in growing antifungal drug resistance issues [6]. Several studies have demonstrated the utility of PDT, utilizing a growing number of photosensitizing agents and light activation sources, for the inactivation of Candida species [13—19]. Dovigo et al., [20] have also demonstrated that PDT using a porphyrin based photosensitizer, and activation wavelengths in the visible blue light region, is effective against C. albicans and C. glabrata. That study demonstrated that under the same conditions fluconazole resistant strains of C. albicans and C. glabrata show some resistance to the action of PDT as well. In the present study we have examined the ability of porfimer sodium mediated PDT to treat different species of Candida as well as fluconazole (FL) and amphotericin B (AP) resistant and sensitive isolates of the following; C. albicans, C. glabrata, C. guilliermindi, C. parapsilosis and C. krusei, from a collection of isolates from adults with AIDS.
Materials and methods Yeast strains and growth conditions Candida albicans ATCC (American Type Culture Collection, Rockville, MD, USA) 90028, C. glabrata ATCC 90030, C. parapsilosis 22019, C. krusei 6258, and C. tropicalis 42678 were used for direct comparison of susceptibility to killing by Photodynamic antimicrobial chemotherapy. Cells were transferred from a 16 h SBA plate (Difco Sabouraud Dextrose Agar, Becton Dickinson, Sparks, MD, USA) into RPMI medium 1640 (GIBCO Invitrogen, Grand Island, NY, USA) with the addition of 5% newborn calf serum (Crane Laboratories, Syracuse, NY, USA). Cells were grown with shaking at 37◦ until the log growth phase was reached. At this point the optical density at 600 nm was 0.30—0.40 after 3—4 h of growth. The cells were centrifuged for 2 min at 14,000 rpm, resuspended in O.85% NaCl and adjusted to an O.D. of 0.10 at 600 nm.
Yeast growth and concentration Growth curves of C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis were performed to determine the range of the log phase of growth. It was at this phase that the yeast was used for subsequent experiments. A fixed optical density was used in the vials during Photofrin and light exposure to ensure yeast concentrations were consistent. Various dilutions after the treatment process were used to determine an optimal number of organisms on the plates that could be easily counted. Yeast isolates Strains are from the oral mucosa of adults with Candida infections C. albicans, (1 FL resistant) C. glabrata, (1 FL and AP resistant; 1 FL resistant) C. guilliermondi, (1 AP resistant), C. parapsilosis (1 FL sensitive) and C. krusei, (1 FL and AP resistant). The strains were pulled from a collection of isolates from a previous study on adults with AIDS. Resis-
T.S. Mang et al. tance and sensitivity were determined by standard Etest (AB BIODISK, Solna Sweden) for antifungal susceptibility. Each isolate was grown on SDA (Sabouraud Dextrose Agar) in a humidified incubator at 32 ◦ C. Inoculums were prepared from individual colonies (≥1 mm diameter) on the SDA into 5 ml of sterile 0.85% saline to a density of 0.5 McFarland standards to equalize the yeast cell density. A 1:10 dilution of the same suspension was then made for photodynamic illuminations.
Photosensitizing agent Photofrin® porfimer solution, (Axcan Scandipharm, Inc., Birmingham, AL, USA) stock solution was adjusted to 7.5 mg/ml in 0.85% sterile NaCl and frozen at −50 ◦ C until use. The final media, concentration used for all experiments was 25 g/ml.
Photofrin exposure to yeast cells The adjusted cells were aliquoted into 4.0 ml sterile glass vials (Wheaton, Millville, NJ, USA). Each control and test condition was done in triplicate. The conditions were as follows: For the controls, NLNP (no light exposure, no porfimer sodium), NLP (no light exposure, porfimer sodium), LNP (light exposure, no porfimer sodium), and for the test condition, LP (light exposure, porfimer sodium). Under conditions of darkness, porfimer sodium was added at a concentration of 25 g/ml and sterile saline was added in the same amount to the vials without porfimer sodium. The drug was added to growing yeast cultures at either 24 or 1 h prior to aliquot preparation for illumination. All subsequent procedures were carried out in darkness to avoid ambient light activation of the drug in incubated samples.
Laser light exposure to yeast cells The light source for PDT is generally derived from a laser and delivered via a fiberoptic device designed for that specific application. Light was delivered using a KTP: YAGdye laser combination (Laserscope, CA) tuned to deliver light at 630 nm or a Diomed 630 nm PDT laser (Diomed Inc. Cambridge UK). These systems are equivalent and utilized interchangeably in pre-clinical and clinical studies. The light from the laser was focused on suspensions of yeast cells following incubation with either porfimer sodium using a lens fiberoptic. This fiberoptic focuses light in a homogenous field over diameters of 1 mm to 15 cm. Light was delivered using a power density of 150 mW/cm2 for varying light doses according to the experimental plan. The vials were exposed to laser light directed from the bottom of the vial at a light level of 45, 90, or 135 J/cm2 . All vials not being treated were kept in darkness for periods of time equivalent to light treatment durations.
Plating and enumeration of yeast cell growth The effect of light and dye combinations, the photodynamic antimicrobial effect, was determined by plating 100 l of the yeast suspension onto SDA agar in duplicate and incubat-
Photodynamic inactivation of normal and anti-fungal resistant
Figure 1 (a) Phototoxicity of Photofrin and increasing light dose, on colony forming ability of various ATCC Candida cultures. (b) Phototoxicity of Photofrin and increasing 630 nm light doses on the colony forming ability of ATCC cultures of C. glabrata.
ing for 48 h at 37 ◦ C in a humidified incubator. Colony forming unit (CFU) assays were used to determine CFU macroscopically. Specifically, under conditions of darkness, an aliquot was removed from each vial and diluted with sterile 0.85% NaCl. 0.10 ml of this was plated to SDA and spread with a glass rod spreader. All plates were incubated for 48 h at 37 ◦ C. Only at the time of counting were the yeast growth plates exposed to light. Total colony counts were done on each plate. The total count was averaged for each condition from the triplicate plates. Untreated controls were manipulated in parallel and used for reference and to determine the plating efficiency.
Statistical analysis The effect of each photosensitizing drug and varying light dose will be expressed as colony forming Unit (CFU) survival and analyzed by means of the Mann—Whitney U-test. Values of p less than .05 will be considered statistically insignificant.
Results The effect on the viability of the examined ATCC strains of Candida after exposure to porfimer sodium alone, light alone and the PDT combination of porfimer sodium and light, using escalating light doses is shown in Fig. 1a and b. Exposure of the organisms to increasing doses of laser light at 630 nm following a single dose 24 h incubation porfimer sodium concentration, 25 g/ml, resulted in a light dose dependent
101 decrease in viable colony counts. In the absence of light in suspensions incubated with porfimer sodium, changes in colony counts were not significant and there was little or no evidence of dark toxicity. Exposure to laser illumination in the absence of porfimer sodium did not result in any significant changes or decreases in cell viability as determined by colony counts. All Candida strains tested showed significant sensitivity to light and drug combinations though there were some differences. In the experiments conducted to compare the different strains, C. Krusei demonstrated less sensitivity to the lower light doses applied, than the other strains examined. However, at the highest light dose utilized, 135 J/cm2 , significant cell kill was observed. C. glabrata also demonstrated significant killing effect by the combination of porfimer sodium and light (Fig. 1b). A second series of experiments were conducted to determine the effect of drug incubation time on the overall cell killing efficacy of PDT. It was determined that effective cell kill using the same light dose parameters, could be achieved using a 1 h incubation vs. 24 h, in media containing 25 g/ml porfimer sodium concentration, demonstrating rapid uptake of the drug. All subsequent experiments were carried out using the porfimer sodium concentration of 25 g/ml for a 1 h incubation period prior to light exposure, while maintaining the light dose parameters of 150 mW/cm2 power density and light dose exposures of 45, 90 and 135 J/cm2 . Another series of experiments was conducted to determine if antifungal resistant Candida species differ in their susceptibility, compared to ATCC strains, to the photodynamic killing action of porfimer sodium activation by 630 nm laser light. FL and/or AP resistant strains of C. glabrata, C. albicans, C. krusei, C. guilliermindi, and C. parapsilosis, isolated from HIV positive patients were grown under the same conditions of the ATCC strains and subjected to the identical experimental conditions as previously demonstrated effective in producing significant cell kill. Figs. 2—6 show the light dose-dependent cell killing effect when cultures previously incubated with 25 g/ml porfimer sodium for 1 h, are exposed to the increasing doses of 630 nm light. The comparison of ATCC C. albicans (Fig. 2a) to the FL resistant isolate of C. albicans is shown (Fig. 2b). The lowest light dose demonstrated approximately 92% killing (p = 0.002) which subsequently increased with increasing light dose. All resistant or sensitive strains examined showed significant susceptibility to the killing effect of PDT under the conditions tested as demonstrated in the following isolate strains examined: C. glabrata (Fig. 3a; FL resistant, AP sensitive), C. glabrata (FL and AP resistant Fig. 3b); C. krusei (FL and AP resistant, Fig. 4); C. guilliermindi (AP resistant FL sensitive, Fig. 5) and 2 (FL and AP sensitive), isolates of C. parapsilosis (Fig. 6a and b). The isolate strain of C. krusei demonstrated similar response characteristics to the lower light doses tested as did the ATCC strain (Fig. 4). However, the resistant strain was also susceptible to the killing effects of PDT at the highest light dose tested (p = 0.003).
Discussion In vitro susceptibility of bacterial and fungal species to the photodynamic effects of various photosensitizing dyes and
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Figure 2 (a) Efficacy of Photofrin and 630 nm light on colony forming ability of ATCC culture of C. albicans. Cultures were irradiated with light doses ranging from 45 to 135 J/cm2 using a dose rate of 150 mW/cm2 . Significant reductions are seen (p = 0.002) when compared to unirradiated controls. (b) Efficacy of Photofrin and 630 nm light on colony forming ability of fluconazole resistant isolate of C. albicans. Significant reductions in Colony forming ability seen at all light doses (p = 0.001).
illumination with appropriate wavelengths of light has been receiving increased attention. Many organisms found within the flora of the oral cavity have demonstrated susceptibility to various regimens in vitro and in animal models. Some studies have shown various strains to be relatively resistant to the killing effects of PDT. In the oral cavity oral candidiasis is primarily caused by Candida albicans, a dimorphic organism that typically is present in the oral cavity in a non-pathogenic state in healthy individuals. Often harmless in the normal human cavity, Candida species, especially C. albicans, can lead to mucosal infections [21,22]. Recent evidence suggests that OPC infections with mixed C. albicans and other Candida organisms in HIV patients present more severe symptoms and are more difficult to treat than those infections sue solely to C. albicans [23]. A wide variety of Candida organisms have been shown to cause infection, with C. dubliensis, C. glabrata, C. krusei and C. tropicalis being the most commonly described. C. dublienis is still commonly described as C. albicans. In the
vast majority of clinical cases of OPC, distinction between C. dublienis and C. albicans is not necessary for successful treatment. C. glabrata is the second or third leading cause of Candidiasis comprising 5—8% of Candida isolates and reports of OPC sue solely to C. glabrata have been increasing. OPC with C. krusei is only rare and C. krusei is found mixed with C. albicans and in C. glabrata. C. tropicalis in OPC has been reported [24]. Primary fluconazole resistance has been defined by the Subcommittee for Antifungal Testing of the Clinical Laboratory Standards Institute as resistance in the absence of prior fluconazole exposure. Secondary resistance occurs as a result of treatment with fluconazole. Fluconazole-resistant oropharyngeal candidiasis has involved both C. albicans and non-albicans species. Following the widespread use of fluconazole in the 1990’ the proportion of C. albicans isolates decreased and other Candida species, including C. krusei, C. dubliensis and C. glabrata increased. In a study by Campisi [25] C. albicans was most often recovered in patients with HIV, isolated in 19/26 HIV+ carriers (73.1%). C. glabrata was found in 1
Photodynamic inactivation of normal and anti-fungal resistant
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Figure 3 (a) Phototoxicity of Photofrin and 630 nm light on irradiated cultures of fluconazole resistant and amphotericin B sensitive C. glabrata. Reductions in CFU for all light doses studied demonstrated significance (p = 0.009—0.008) (b) Phototoxicity of Photofrin and 630 nm light on cultures of fluconazole and amphotericin B resistant C. glabrata (p = 0.0001).
Figure 4 Efficacy of Photofrin induced PDT on fluconazole and amphotericin B resistant isolates of C. Krusei. At the lowest light doses colony forming ability was reduced at a significance of (p = 0.011—0.006). Continued irradiation yielded greater cell killing (p = 0.003) when compared to unirradiated controls.
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Figure 5
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Photofrin PDT phototoxicity of amphotericin B resistant and fluconazole sensitive isolates of C. guilliermondi (p = 0.0001).
subject (3.8%) and combinations of Candida species were also found [24]. Worldwide C. parapsilosis is among the most common species causing invasive disease. It has been demonstrated to be the second most frequent bloodstream isolate in candidemia patients and increasing in frequency [26]. Photodynamic drugs have routinely been administered by IV injection. This mode of delivery would not generally meet with acceptance in the treatment of fungal diseases, as systemic administration of a photosensitizing drug, in particular porfimer sodium, results in the primary side effect of cutaneous photosensitivity for a period of approximately 3—4 weeks. Some photosensitizers which have gained regulatory approval demonstrate shorter periods of cutaneous photosensitivity yet still require IV injection and periods of time for selective uptake to occur. While the side effect, cutaneous photosensitivity, is much less serious than those demonstrated with current therapeutic modalities to treat oral candidiasis, IV drug delivery would not, most likely, result in significant uptake of the photosensitizer drug by Candia species in the oral cavity. Further this method of
Figure 6
drug delivery would result in decreased selectivity of drug uptake as healthy tissues would retain some level, albeit lower concentration, than diseased tissue or in this scenario, fungal organisms. Ultimately this results in dosimetric considerations necessary to avoid collateral damage to healthy tissues. This is further illustrated in Fig. 1a and b. Generally, when a patient is given porfimer sodium IV, there is a 48 h period in which uptake and retention, to provide a reasonable therapeutic ratio, occurs. In vitro studies using this drug in mammalian cells have generally employed a 24 h incubation period to ensure adequate uptake and localization. Therefore the short incubation time in which the Canadida actively take up sufficient drug would provide a clear therapeutic ratio between the target and normal tissue which results in a selective therapeutic advantage. A topical mode of drug delivery for oral candidiasis is highly desirable in this patient population. This mode of delivery has the advantage of avoiding systemic toxicity and drug interactions as well as saving systemic agents for invasive disease. At present there are no efficacious topical agents for oral candidiasis in patients at risk for developing
Phototoxicity of photofrin induced PDT on fluconazole and amphotericin B sensitive isolate of C. parapsilosis (p = 0.007).
Photodynamic inactivation of normal and anti-fungal resistant the infection. PDT may be a promising topical treatment for the control of oral Candida growth and by extension oropharyngeal candidiasis. The drug porfimer sodium has shown promise in the treatment of superficial disease but as yet has not been developed as a topical treatment. In people with a weakened immune system due to steroid therapy, cancer chemotherapeutic regimens or radiotherapy, or diseases such as AIDS, candidal infections are a considerable challenge. The infection can occur locally regionally or throughout the body and can be life threatening. The therapy of choice for oral candidiasis is a course of systemic antifungal agents such as the azole antifungal fluconazole and amphotericin B. Generally the azole medications are prescribed as they can be used topically or through oral administrations. Amphotericin B is reserved for more serious systemic fungal infections. Some forms of the drug are used for superficial candidal infections and thrush. Further, in patients who have demonstrated fluconazole and or amphotericin B resistant strains PDT represents a possible alternative to or adjunctive therapy for the treatment of oral candidiasis. We demonstrate in our series of experiments the susceptibility of control strains of Candida obtained from the ATCC and various isolates of Candida, obtained from adult patients with AIDS, which had demonstrated fluconazole and amphotericin B resistance, to the killing effects of Photofrin mediated PDT. The demonstration of the sensitivity of these organisms to the photodynamic toxicity of an agent which is clinically accepted is relevant to the use of this agent in the treatment of patients with oral candidiasis. The ultimate key to successful treatment of Candidaassociated infection is eradication of the Candida biofilm. Photodynamic therapy may provide a means to the successful elimination of denture biofilm if uptake of the photosensitizing dye by the microbes may be achieved with topical administration and their subsequent destruction upon illumination demonstrated in a manner consistent with today’s clinical practice.
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