De acordo com as condições experimentais avaliadas, os resultados obtidos permitiram concluir que:
A PDT mediada pelo PDZ em associação com a luz LED promoveu redução significativa dos valores de UFC/mL de C. albicans resistente a fluconazol presentes nas lesões bucais dos camundongos com candidose oral induzida, resultando em redução equivalente a 1,91 e 1,96 log10, para os grupos P+L+h e P+L+s, respectivamente;
A aplicação de PDZ ou luz LED isoladamente não reduziram os valores de UFC/mL de C. albicans;
A análise histológica revelou que a PDT não promoveu efeitos adversos no tecido do dorso lingual dos camundongos;
A PDT não alterou a capacidade de C. albicans em aderir e formar biofilme em superfície abiótica e não inibiu o seu crescimento em forma filamentar;
REFERÊNCIAS
1. Abaci O. Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individuals denture wearers and genotypic distribution of Candida albicans strains. Curr Microbiol. 2011; 62(4): 1308-14.
2. Andes D, Forrest A, Lepack A, Nett J, Marchillo K, Lincoln L. Impact of antimicrobial dosing regimen on evolution of drug resistance in vivo: fluconazole and Candida albicans. Antimicrob Agents Chemother. 2006; 50(7): 2374-83.
3. Banting DW, Greenhorn PA, McMinn JG. Effectiveness of a topical antifungal regimen for the treatment of oral candidiasis in older, chronically ill, institutionalized, adults. J Can Dent Assoc. 1995; 61(3): 199-200.
4. Banting DW, Hill SA. Microwave disinfection of dentures for the treatment of oral candidiasis. Spec Care Dentist. 2001; 21(1): 4-8.
5. Barros LM, Boriollo MF, Alves AC, Klein MI, Gonçalves RB, Hölfling JF. Genetic diversity and exoenzymes activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brasilian periodontal patients. Arch Oral Biol. 2008; 53(12): 1172-8.
6. Bertoloni G, Reddi E, Gatta M, Burlini C, Jori G. Factors influencing the haematoporphyrin-sensitized photoinactivation of Candida albicans. J Gen Microbiol. 1989; 135(4): 957-66.
7. Bliss JM, Bigelow CE, Foster TH, Haidaris CG. Susceptibility of Candida species to photodynamic effects of Photofrin. Antimicrob Agents Chemother. 2004; 48(6): 2000-6.
8. Bonnett R, Martínez G. Photobleaching of sensitisers used in photodynamic therapy. Tetrahedron. 2001; 57: 9513-47.
9. Budtz-Jorgensen E, Holmstrup P, Krogh P. Fluconazole in the treatment of Candida- associated denture stomatitis. Antimicrob Agents Chemother. 1988; 32(12): 1859-63. 10. Buytaert E, Dewaele M, Agostinis P. Molecular effectors of multiple cell death
pathways initiated by photodynamic therapy. Biochim Biophys Acta. 2007;1776(1): 86-107.
11. Calderone RA, Fonzi WA. Virulence factors of Candida albicans.Trends Microbiol. 2001; 9(7): 327-35.
12. Carmello JC, Pavarina AC, Dovigo LN, Bagnato VS, Mima EGO, Giampaolo ET, et al. Efetividade da terapia fotodinâmica mediado pelo Photodithazine® na inativação de espécies de Candida [resumo PNf082]. Braz Oral Res. 2011; 25(1): 364.
13. Carmello JC. Efetividade da terapia fotodinâmica mediada pelo fotossensibilizador Photodithazine® na inativação de Candida albicans in vivo [dissertação de mestrado]. Araraquara: Faculdade de Odontologia da UNESP; 2011.
14. Chabrier-Rosello Y, Foster TH, Perez-Nazario N, Mitra S, Haidaris CG. Sensitivity of Candida albicans germ tubes and biofilms to photofrin-mediated phototoxicity. Antimicrob Agents Chemother. 2005; 49(10): 4288-95.
15. Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol. 2001; 183(18): 5385-94.
16. Chorilli M, Zague V, Scarpa MV, Leonardi GR. Influência da viscosidade do veículo na liberação in vitro da cafeína. Revista Eletrônica de Farmácia. 2007; 4(1): 52-60. 17. Coletti TMSFA. Efeito fotodinâmico da Curcumina em micelas cetrimida em cepas
de Candida resistentes e suscetíveis a fluconazol [dissertação de mestrado]. Araraquara: Faculdade de Ciências Farmacêuticas da UNESP; 2013.
18. Colussi VC, Nicola EMD, Nicola JH. Fototerapia, fotoquimioterapia e alguns fotossensibilizadores. Rev Assoc Med Bras. 1996; 42(4): 229-36.
19. Correa JC. Fotodegradacao do Photodithazine® e citotoxicidade dos fotoprodutos formados apos irradiacao com laser [dissertação de mestrado]. São Carlos: Instituto de Química da USP; 2006.
20. Demidova TN, Hamblin MR. Effect of cell-photosensitizer binding and cell density on microbial photoinactivation. Antimicrob Agents Chemother. 2005; 49(6): 2329- 35.
21. Donnelly RF, McCarron PA, Tunney MM. Antifungal photodynamic therapy. Microbiol Res. 2008; 163(1): 1-12.
22. Dovigo LN, Pavarina AC, Carmello JC, Machado AL, Brunetti IL, Bagnato VS. Susceptibility of clinical isolates of Candida to photodynamic effects of curcumin. Lasers Surg Med. 2011; 43(9):927-34.
23. Dovigo LN, Pavarina AC, Mima EG, Giampaolo ET, Vergani CE, Bagnato VS. Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata. Mycoses. 2011; 54(2): 123-30.
24. Farah CS, Ashman RB, Challacombe SJ. Oral candidosis. Clin Dermatol. 2000; 18(5): 553-62.
25. Ferreira J, Menezes PFC, Kurachi C, Sibata C, Allison RR, Bagnato VS. Photostability of different chlorine photossensitizers. Laser Phys Lett. 2008; 5(2): 156-61.
26. Fontana CR, Lerman MA, Patel N, Grecco C, de Souza Costa CA, Amiji MM et al. Safety assessment of oral photodynamic therapy in rats. Lasers Med Sci. 2013; 28(2): 479-86.
27. Iacopino AM, Wathen WF. Oral candidal infection and denture stomatitis: a comprehensive review. J Am Dent Assoc. 1992; 123(1): 46-51.
28. Ito T. Photodynamic action of hematoporphyrin on yeast cells--a kinetic approach. Photochem Photobiol. 1981; 34(4): 521-4.
29. Ito T. Toluidine blue: the mode of photodynamic action in yeast cells. Photochem Photobiol. 1977; 25(1): 47-53.
30. Jackson Z, Meghji S, MacRobert A, Henderson B, Wilson M. Killing of the yeast and hyphal forms of Candida albicans using a light-activated antimicrobial agent. Lasers Med Sci. 1999; 14(2): 150-7.
31. Jeganathan S, Lin CC. Denture stomatitis--a review of the aetiology, diagnosis and management. Aust Dent J. 1992; 37(2): 107-14.
32. Ji QX, Zhao QS, Deng J, Lü R. A novel injectable chlorhexidine thermosensitive hydrogel for periodontal application: preparation, antibacterial activity and toxicity evaluation. J Mater Sci Mater Med. 2010; 21(8): 2435-42.
33. Jorge AOC, Totti MAG, Almeida OP, Scully C. Oral candidiasis established in the sialoadenectomised rat. J Oral Pathol Med. 1993; 22(2): 54-6.
34. Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, et al. Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg Med. 2006; 38(5): 468-81.
35. Junqueira JC, Martins Jda S, Faria RL, Colombo CE, Jorge AO. Photodynamic therapy for the treatment of buccal candidiasis in rats. Lasers Med Sci. 2009; 24(6): 877-84.
36. Junqueira JC, Vilela SF, Rossoni RD, Barbosa JO, Costa AC, Rasteiro VM et al. Oral colonization by yeasts in HIV-positive patients in Brazil. Rev Inst Med Trop Sao Paulo. 2012; 54(1): 17-24.
37. Juzeniene A. Chlorin e6-based photosensitizers for photodynamic therapy and photodiagnosis. Photodiagnosis Photodyn Ther. 2009; 6(2): 94-6.
38. Kato IT, Prates RA, Sabino CP, Fuchs BB, Tegos GP, Mylonakis E et al.Antimicrobial Photodynamic Inactivation inhibits Candida albicans virulence factors and reduces in ivo pathogenicity. Antimicrob Agents Chemother. 2013; 57(1): 445-51.
39. Kömerik N, Nakanishi H, MacRobert AJ, Henderson B, Speight P, Wilson M. In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model. Antimicrob Agents Chemother. 2003; 47(3): 932-40.
40. Konopka K, Goslinski T. Photodynamic therapy in dentistry. J Dent Res. 2007; 86(8): 694-707. Erratum in: J Dent Res. 2007; 86(11): 1126.
41. Konsberg R, Axell T. Treatment of Candida-infected denture stomatitis with a miconazole lacquer. Oral Surg Oral Med Oral Pathol. 1994; 78(3): 306-11.
42. Kulak Y, Arikan A, Delibalta N. Comparison of three different treatment methods for generalized denture stomatitis. J Prosthet Dent. 1994; 72(3): 283-8.
43. Kumar R, Shukla PK. Amphotericin B resistance leads to enhanced proteinase and phospholipase activity and reduced germ tube formation in Candida albicans. Fung Biol. 2010; 114(2-3): 189-97.
44. Kvello Strenstrom AG, Moan J, Brunborg G, Eklund T. Photodynamic inactivation of yeast cells sensitized by hematoporphyrin. Photochem Photobiol. 1980; 32(3): 349- 52.
45. Lambrechts SAG, Aalders MCG, Van Marle J. Mechanistic study of the photodynamic inactivation of Candida albicans by cationic porphyrin. Antimicrob Agents Chemother. 2005; 49(5): 2026-34.
46. Mang TS, Mikulski L, Hall RE. Photodynamic inactivation of normal and antifungal resistant Candida species. Photodiagnosis Photodyn Ther.2010; 7(2):98-105.
47. Mantareva V, Kussovski V, Angelov I, Wöhrle D, Dimitrov R, Popova E et al. Non- aggregated Ga(III)-phthalocyanines in the photodynamic inactivation of planktonic and biofilm cultures of pathogenic microorganisms. Photochem Photobiol Sci. 2011; 10(1): 91-102.
48. Martin MV. Antifungal agents. In: Samaranayake LP, Macfarlane TW. Oral candidosis. London: Wright; 1990. p. 238-51.
49. Martins J da S, Junqueira JC, Faria RL, Santiago NF, Rossoni RD, Colombo CE, et al. Antimicrobial photodynamic therapy in rat experimental candidiasis: evaluation of pathogenicity factors of Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011; 111(1): 71-7.
50. Mathaba LT, Davies G, Warmington JR. The genotypic relationship of Candida albicans strains isolated from the oral cavity of patients with denture stomatitis. Med Microbiol. 1995; 42(5): 372-9.
51. Melo AS, Colombo AL, Arthington-Skaggs BA. Paradoxical growth effect of caspofungin observed on biofilms and planktonic cells of five different Candida species. Antimicrob Agents Chemother. 2007; 51(9): 3081-8.
52. Mima EG, Pavarina AC, Dovigo LN, Vergani CE, Costa CA, Kurachi C, et al. Susceptibility of Candida albicans to photodynamic therapy in a murine model of oral candidosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010; 109(3): 392-401.
53. Mima EG, Vergani CE, Machado AL, Massucato EM, Colombo AL, Bagnato VS, et al. Comparison of photodynamic therapy versus conventional antifungal therapy for the treatment of denture stomatitis: a randomized clinical trial. Clin Microbiol Infect. 2012; 18(10): E380-8.
54. Munin E, Giroldo LM, Alves LP, Costa MS. Study of tube formation by Candida albicans after photodynamic antimicrobial chemotherapy (PACT). J Photochem Photobiol B.: Biology. 2007; 88(1): 16-20.
55. Negri M, Henriques M, Svidzinski TI, Paula CR, Oliveira R. Correlation between Etest, disk diffusion, and microdilution methods for antifungal susceptibility testing of Candida species from infection and colonization. J Clin Lab Anal. 2009; 23(5): 324-30.
56. Neppelenbroek KH, Pavarina AC, Palomari Spolidorio DM, Sgavioli Massucato EM, Spolidorio LC, Vergani CE. Effectiveness of microwave disinfection of complete dentures on the treatment of Candida-related denture stomatitis. J Oral Rehabil. 2008; 35(11): 836-46.
57. Niimi M, Firth NA, Cannon RD. Antifungal drug resistance of oral fungi. Odontology. 2010; 98(1): 15-25.
58. Nikawa H, Hamada T, Yamamoto T. Denture plaque-past and recent concerns. J Dent. 1998; 26(4): 299-304.
59. Paardekooper M, De Bruijne AW, Van Steveninck J, Van den Broek PJ. Intracellular damage in yeast cells caused by photodynamic treatment with toluidine blue. Photochem Photobiol. 1995; 6(1): 84-9.
60. Parvinen T, Kokko J; Yli-Urpo A. Miconazole lacquer compared with gel in treatment of denture stomatitis. Scand J Dent Res. 1994; 102(6): 361-6.
61. Paulino TP, Ribeiro KF, Thedei Jr G, Tedesco AC, Ciancaglini P. Use of hand held photopolymerizer to photoinactivate Streptococcus mutans. Arch Oral Biol. 2005; 50(3): 353-9.
62. Pereira CA, Romeiro RL, Costa AC, Machado AK, Junqueira JC, Jorge AO. Susceptibility of Candida albicans, Staphylococcus aureus, and Streptococcus mutans biofilms to photodynamic inactivation: an in vitro study. Lasers Med Sci. 2011; 3(3): 341-8.
63. Perezous LF, Flaitz CM, Goldschmidt ME, Engelmeier RL. Colonization of Candida species in denture wearers with emphasis on HIV infection: a literature review. J Prosthet Dent. 2005; 93(3): 288-93.
64. Pinto E, Ribeiro IC, Ferreira NJ, Fortes CE, Fonseca PA, Figueiral MH. Correlation between enzyme production, germ tube formation and susceptibility to fluconazole in Candida species isolated from patients with denture-related stomatitis and control individuals. J Oral Pathol Med. 2008; 37(10): 587-92.
65. Price MF, Wilkinson ID, Gentry LO. Plate methods for detection of phospholipase activity in C. albicans. Sabouraudia. 1982; 20(1): 7-14.
66. Ramage G, Rajendran R, Sherry L, Williams C. Fungal biofilm resistance. Int J Microbiol. 2012; 2012: 528521.
67. Rüchel R. Proteinases of pathogenic fungi. Mycoses. 1999; 42 Suppl1: 48-52.
68. Sardi JC, Duque C, Höfling JF, Gonçalves RB. Genetic and phenotypic evaluation of Candida albicans strains isolated from subgingival biofilm of diabetic patients with chronic periodontitis.Med Mycol. 2012; 50(5):467-75.
69. Scully C, el-Kabir M, Samaranayake LP. Candida and oral candidosis: a review. Crit Rev Oral Biol Med. 1994; 5(2): 125-57.
70. Silva WJ, Seneviratne J, Parahitiyawa N, Rosa EA, Samaranayake LP, Del Bel Cury AA. Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Dent J. 2008; 19(4): 364-9.
71. Skoglund A, Sunzel B, Lerner UH. Comparison of three test methods used for diagnosis of candidiasis. Scand J Dent Res. 1994; 102(5): 295-8.
72. Soll DR. Candida commensalism and virulence: the evolution of phenotypic plasticity. Acta Trop. 2002; 81(2): 101-10.
73. Strackhovskaya MG, Belenikina NS, Ivanova EV, Chemeris YK, Stranadko EF. The photodynamic inactivation of the yeast Candida guilliermondii in the presence of Photodithazine®. Microbiology. 2002; 71(3): 298-301.
74. Struillou X, Boutigny H, Badran Z, Fellah BH, Gauthier O, Sourice S, et al. Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate. J Mater Sci Mater Med. 2011; 22(7): 1707-17. 75. Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamaguchi H, et al. A novel
murine model of oral candidiasis with local symptons characteristic of oral thrush. Microbiol Immunol. 2003; 47(5): 321-6.
76. Tegos GP, Anbe M, Yang C, Demidova TN, Satti M, Mroz P, et al. Protease-stable polycationic photosensitizer conjugates between polyethyleneimine and chlorin (e6) for broad-spectrum antimicrobial photoinactivation. Antimicrob Agents Chemother 2006; 50(4): 1402-10.
77. Teichert MC, Jones JW, 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(2): 155-60. 78. Thompson DS, Carlisle PL, Kadosh D. Coevolution of morphology and virulence in
Candida species. Eukaryot Cell. 2011; 10(9): 1173-82.
79. Totti MA, dos Santos EB, de Almeida OP, Koga-Ito CY, Jorge AO. Oral candidosis by Candida albicans in normal and xerostomic mice. Braz Oral Res. 2004; 18(3): 202-7.
80. Trindade FZ, Pavarina AC, Ribeiro AP, Bagnato VS, Vergani CE, Costa CA. Toxicity of photodynamic therapy with LED associated to Photogem®: an in vivo study. Lasers Med Sci. 2012; 27(2): 403-11.
81. Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother. 1998; 42(1): 13-28.
82. White TC, Marr KA, Bowden RA. Clinical, cellular and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev. 1998; 11(2): 382-402. 83. Wilson J. The aetiology, diagnosis and management of denture stomatitis. Br Dent J.
1998; 185(8): 380-4.
84. Wilson M, Mia N. Sensitisation of Candida albicans to killing by low-power laser light. J Oral Pathol Med. 1993; 22(8): 354-7.
APÊNDICES
Apêndice 1
Preparo da Concentração de 100mg/L de PDZ para o volume final de 250μL (5 animais com aplicação de 50μL em cada): 5000mg/L--- 100% 100mg/ L --- x X = 2% 250μL ---100% y --- 2% y = 5μL de PDZ
Apêndice 2
Cálculo do tempo de iluminação:
Dose de luz (J/cm2) = Potência (W/cm2) x Tempo de iluminação (seg)
Dose de luz = 37,5 J/cm2
Potência do aparelho = 44,5 mW = 0,0445 W
37,5 J/cm2 = 0,0445 W/cm2 x T T = 842,7 s = 14 min
Apêndice 3
Tabela A - Valores originais obtidos da coleta de C. albicans resistente a fluconazol, das línguas dos camundongos, após 48 horas da semeadura em placas de Petri.
Número de colônias viáveis
Grupos Amostra Duplicata 1 Duplicata 2 Média Diluição UFC/mL Log10(UFC/mL)
1 44 51 47,5 102 1,90E+05 5,28 2 41 50 45,5 102 1,82E+05 5,26 P-L- 3 71 74 72,5 102 2,90E+05 5,46 4 54 56 55 102 2,20E+05 5,34 5 42 55 48,5 102 1,94E+05 5,29 1 66 70 68 102 2,72E+05 5,43 2 52 65 58,5 102 2,34E+05 5,37 P-L+ 3 74 79 76,5 102 3,06E+05 5,49 4 50 51 50,5 102 2,02E+05 5,31 5 29 31 30 102 1,20E+05 5,08 1 53 53 53 102 2,12E+05 5,33 2 42 49 45,5 102 1,82E+05 5,26 P+L-h 3 51 54 52,5 102 2,10E+05 5,32 4 50 51 50,5 102 2,02E+05 5,31 5 68 77 72,5 102 2,90E+05 5,46 1 36 40 38 102 1,52E+05 5,18 2 67 78 72,5 102 2,90E+05 5,46 P+L-s 3 72 68 70 102 2,80E+05 5,45 4 52 65 58,5 102 2,34E+05 5,37 5 40 46 43 102 1,72E+05 5,24 1 90 93 91,5 10° 3,66E+03 3,56 2 111 120 115,5 10° 4,62E+03 3,66 P+L+h 3 99 102 100,5 10° 4,02E+03 3,60 4 68 72 70 10° 2,80E+03 3,45 5 16 18 17 10° 6,80E+02 2,83 1 62 64 63 10° 2,52E+03 3,40 2 80 85 82,5 10° 3,30E+03 3,52 P+L+s 3 48 48 48 10° 1,92E+03 3,28 4 30 35 32,5 10° 1,30E+03 3,11 5 80 84 82 10° 3,28E+03 3,52
Apêndice 4
Receita - Meio Ágar Proteinase
Meio Albumina 1 L Albumina bovina 2,0g Riboflavina 0,2g Ácido Nicotínico 0,4g Hidrocloreto de tiamina 0,4g Água destilada 1L
Esterilizar o meio albumina por filtração em membrana Milipore (0,22 µm). Reservar.
Meio Ágar 1 L Dextrose 20,0g Fosfato de Potássio 1,0g Sulfato de magnésio 0,5g Ágar 15,0g Água destilada 1 L
Auclavar o meio Ágar a 120 oC por 15 minutos. Após resfriamento a 50 °C, adicionar o meio albumina.
Apêndice 5
Receita - Meio Ágar Fosfolipase
Meio Ágar Base 1 L Peptona Glicose 10,0g 30,0g Cloreto de Sódio 57,3g Cloreto de Cálcio 0,55g Ágar 20,0g Água destilada 1L
Auclavar o meio Ágar a 120 oC por 15 minutos. Após resfriamento a 50 °C, adicionar a emulsão de ovo a 50% (Egg Yolk).
ANEXO
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Araraquara, 28 de março de 2013. Fernanda Alves