UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara FERNANDA ALVES EFICÁCIA DAS TERAPIAS FOTODINÂMICA E SONODINÂMICA COMO MÉTODOS PARA INATIVAÇÃO DE ESPÉCIES DE CANDIDA. ESTUDO IN VITRO E CLÍNICO. Araraquara 2017 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara FERNANDA ALVES EFICÁCIA DAS TERAPIAS FOTODINÂMICA E SONODINÂMICA COMO MÉTODOS PARA INATIVAÇÃO DE ESPÉCIES DE CANDIDA. ESTUDO IN VITRO E CLÍNICO. Tese apresentada ao programa de Pós- Graduação em Reabilitação Oral Área de Prótese, da Faculdade de Odontologia de Araraquara, da Universidade Estadual Paulista para título de Doutor em Reabilitação Oral. Orientadora: Profª Drª Ana Cláudia Pavarina Araraquara 2017 Alves, Fernanda Eficácia das terapias fotodinâmica e sonodinâmica como métodos para inativação de espécies de Candida. Estudo in vitro e clínico / Fernanda Alves.-- Araraquara: [s.n.], 2017 106 f. ; 30 cm. Tese (Doutorado em Prótese) – Universidade Estadual Paulista, Faculdade de Odontologia Orientadora: Profa. Dra. Ana Cláudia Pavarina 1. Fotoquimioterapia 2. Terapia por Ultrassom 3. Candida 4. Resistência a medicamentos 5. Estomatite sob prótese I. Título FERNANDA ALVES EFICÁCIA DAS TERAPIAS FOTODINÂMICA E SONODINÂMICA COMO MÉTODOS PARA INATIVAÇÃO DE ESPÉCIES DE CANDIDA. ESTUDO IN VITRO E CLÍNICO. Comissão julgadora Tese para obtenção do grau de Doutor Presidente e orientador Profª Drª Ana Cláudia Pavarina 2º Examinador Profª Drª Fernanda de Freitas Anibal 3º Examinador Profº Drº Gilberto Ubida Leite Braga 4º Examinador Profª Drª Ana Carolina Pero Vizoto 5º Examinador Profº Drº Carlos Alberto de Souza Costa Araraquara, 02 de Agosto de 2017. DADOS CURRICULARES FERNANDA ALVES NASCIMENTO: 28/10/1987 – Araraquara – São Paulo FILIAÇÃO: Selma Salum Alves José Vitor Roncada Alves 2006/2010 Curso de Graduação pela Faculdade de Odontologia de Araraquara – UNESP. 2007/2010 Estágio de Iniciação Científica na Disciplina de Prótese Parcial Removível. 2011/2013 Curso de Pós-Graduação em Reabilitação Oral, Área de concentração em Prótese, nível de Mestrado, pela Faculdade de Odontologia de Araraquara - UNESP. 2012/2016 Estágio de docência nas Disciplinas de Prótese Parcial Removível I e II do Departamento de Materiais Odontológicos e Prótese da Faculdade de Odontologia de Araraquara – UNESP. 2015/2015 Estágio de docência nas Disciplinas de Prótese Total II do Departamento de Materiais Odontológicos e Prótese da Faculdade de Odontologia de Araraquara – UNESP. 2016/2016 Doutorado Sanduíche na Faculdade de Farmácia da Ulster University – Reino Unido. 2013/2017 Curso de Pós-Graduação em Reabilitação Oral, Área de concentração em Prótese, nível de Doutorado, pela Faculdade de Odontologia de Araraquara - UNESP. Agradecimentos Agradeço a todos aqueles que contribuíram de forma direta ou indireta na realização deste trabalho. Em especial agradeço... A Deus, por nunca me desamparar e sempre ser tão bom para mim, mesmo com tantas imperfeições. Aos meus pais, Selma e Vitor, por todo investimento, ensino de vida, apoio, dedicação e amor por mim. Agradeço também ao meu irmão, Renan, pela amizade e companheirismo. Ao meu marido, Danilo, por ser meu companheiro, melhor amigo, acreditar no meu potencial, sempre me incentivar positivamente, me apoiar e, principalmente, por me amar incondicionalmente. À minha orientadora Profª Drª Ana Cláudia Pavarina por todo ensinamento em todos estes anos de convivência. Há dez anos que sou orientada por você e o ensinamento transmitido é imensurável. Com você aprendi a escrever artigos, a amar a pesquisa e ensino, a realizar pedidos de bolsa e auxílio à pesquisa, a orientar alunos de iniciação científica, a escrever projetos de pesquisa, a ter postura profissional, dentre outras coisas. Sem dúvida te levarei eternamente como um exemplo de Professora, Orientadora e Pesquisadora. Ao Profº Drº Ewerton Garcia de Oliveira Mima por ser um pilar em todas as etapas da minha vida profissional: iniciação científica, mestrado, doutorado sanduíche e doutorado. Com você aprendi desde pipetar soluções a escrever artigos. Também te levarei eternamente como um exemplo de Professor, Orientador e Pesquisador. Às minhas amigas de pós-graduação e de vida Juliana Cabrini Carmello e Gabriela Caroline Alonso. Obrigada pelo tempo dedicado a este trabalho, vocês foram peças fundamentais para que esta pesquisa fosse possível. Além disso, agradeço pela amizade e carinho durante todos estes anos de convivência. Ao Profº Drº Vanderlei Salvador Bagnato pela confecção dos aparelhos de luz LED e fornecimento do Photodithazine. Aos 65 pacientes que participaram do estudo clínico. Vocês contribuíram para a minha vida profissional e, mais ainda, para a comunidade científica em pesquisa com Terapia Fotodinâmica. Aos Professores da Disciplina de Prótese Parcial Removível: Drª Janaína Habib Jorge, Drª Ana Lúcia Machado, Drº Carlos Eduardo Vergani e Eunice Giampaolo. Aos Professores da Disciplina de Prótese Total: Drª Ana Carolina Pero Vizoto, Drº João Neudenir Arioli Júnior, Drº Francisco de Assis Mollo Júnior, Drº Sérgio Sualdini Nogueira e Drº Marco Antônio Compagnoni pelos ensinamentos, por indicar pacientes para a pesquisa clínica e por abrir as portas da clínica da Graduação para o acompanhamento dos pacientes. À Profª Drª Marlise Inez Klein Furlan por todo o ensinamento em Microbiologia e Biologia Molecular e pela prontidão em ajudar na realização desta pesquisa. Você contribuiu imensamente para o meu crescimento profissional. Aos meus queridos amigos de pós-graduação Beatriz Helena Dias Panariello, Kássia Dias, Jefferson Trigo, Elkin Florez, Bruna Pimentel, Camila Cristina de Foggi, Carmélia Lobo, Guilherme Rocha, Jéssica Bernegossi, Lucas Portella, Vinícius Sakima, José Francisco Rocha e Midian Castillo. À secretária da Clínica de Pesquisa do Departamento de Materiais Odontológicos e Prótese, Tânia, por me ajudar com a triagem e agendamento de pacientes. À técnica do microscópio confocal, Drª Paula Aboud Barbugli. Às bolsistas técnicas do Laboratório de Microbiologia Aplicada: Geisiane Helena Gomes Bueno, Érica de Carvalho, Sarah de Annunzio, Bruna Michelli Novelli, Luana Sales e Lígia Sabino. À Faculdade de Odontologia de Araraquara, por ter fornecido toda estrutura para realização do curso de pós-graduação, contribuindo para minha formação profissional. Tenho orgulho de fazer parte desta Faculdade. A todos os Professores do Curso de Pós-graduação em Reabilitação Oral. Aos funcionários do Departamento de Materiais Odontológicos e Prótese, aos funcionários da Sessão de Pós-graduação e da Biblioteca da Faculdade de Odontologia de Araraquara. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES pela bolsa de Doutorado concedida para a realização deste trabalho. À Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP pelas bolsas de Doutorado no país (Processo 2014/01262-1) e Bolsa Estágio de Pesquisa no Exterior (BEPE) concedidas para a realização deste trabalho. I would like to thank Professor John Francis Callan from Ulster University – Northern Ireland (Coleraine Campus) for the opportunity of being part of your group. I am proud to say that I made part of "John's group", even for 6 months. I admire your work, your experience, your responsability with research and your students. It was a great experience for my carreer and my personal life. Likewise, I also would like to thank David Costley, Dean Nicholas, Christopher O’Kane, Adam Johnston, Noorjahan Aibani, Sheng Jie, Sukanta Kamila, Heather Nesbitt, Keiran Logan, Jordan Atchison, Atul Nayak, Scarlett, Diego and Nino for being my friends and receiving me at Ulster University. All of you made this experience unforgettable! The technicians Barry Hyland, Chris Millar, Svetlana Tretsiakova-McNally and Bernadette Doherty from Ulster University. The Professors Dr. Bridgeen Callan, Dr. Anthony McHale, Dr. Kathryn Burnett, and Dr. Webba da Silva from Ulster University. Finally, I would like to thank my dear friends Marie Kristine, Ellen, Vincent, Adrian, Virginia and Siobhan for being my family when I was in Coleraine. I cannot explain how important you were for me and I will be eternally grateful. Muito obrigada! Alves F. Eficácia das Terapias Fotodinâmica e Sonodinâmica como métodos para inativação de espécies de Candida. Estudo in vitro e clínico [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017. Resumo Este trabalho avaliou a eficácia das Terapias Fotodinâmica Antimicrobiana (aPDT) e Sonodinâmica (SDT) na inativação de espécies de Candida. O estudo 1 avaliou a efetividade da aPDT na inativação de biofilmes de Candida albicans susceptível e resistente ao fluconazol, bem como seus efeitos sobre os fatores de virulência das cepas. Para isso, biofilmes destas cepas foram tratados com aPDT mediada pelo Photodithazine e luz LED. Após a aPDT, as células foram recuperadas e os fatores de virulência avaliados. A capacidade de adesão foi analisada pelos testes de XTT (atividade metabólica) e UFC/mL (viabilidade celular). A capacidade de formar biofilme foi avaliada pelos testes de XTT, UFC/mL e biomassa total. A síntese de exoenzimas foi avaliada por testes fluorimétricos. Os dados foram analisados por ANOVA a 2 ou 3 critérios (p≤0,05). Verificou-se que a aPDT reduziu a viabilidade das cepas, entretanto não alterou os fatores de virulência. No estudo 2, foi avaliada a efetividade da aPDT no tratamento de pacientes com estomatite protética em comparação com a Nistatina (NIS). Os pacientes do grupo aPDT (n=30) foram submetidos a 6 sessões de aPDT (3 vezes/semana, 15 dias) e os pacientes do grupo NIS (n=35) utilizaram o antifúngico 4 vezes/dia, durante 15 dias. Coletas microbiológicas das próteses e palatos foram realizadas e cultivadas em ágar sangue e chromagar (UFC/mL). Fotografias dos palatos foram tomadas para avaliação clínica da lesão. Os dados foram analisados pelo Modelo Linear de Medidas Repetida (p≤0,05). Foi observado que a aPDT foi mais efetiva em reduzir a microbiota total do que a NIS. A aPDT foi tão eficaz quanto a NIS em reduzir Candida spp. do palato. A aPDT e a NIS apresentaram percentual de melhora clínica da lesão similar. O estudo 3 avaliou a eficácia da aPDT, SDT e a combinação de ambos os tratamentos na inativação de C. albicans. Suspensões foram tratadas com aPDT e SDT e a efetividade foi determinada por UFC/mL. Os biofilmes foram tratados com aPDT, SDT e a combinação de ambos os tratamentos (aPDT+SDT). Os tratamentos foram avaliados por UFC/mL, biomassa total e microscopia. Os dados foram analisados por ANOVA (p≤0,05). Embora a aPDT e SDT tenham erradicado suspensões, estes tratamentos tiveram pouco efeito sobre os biofilmes. A associação da aPDT+SDT reduziu a viabilidade celular e a biomassa total. Palavras-chave: Fotoquimioterapia. Terapia por Ultrassom. Candida. Resistência a medicamentos. Estomatite sob prótese. Alves F. Eficacy of Antimicrobial Photodynamic and Sonodynamic Therapies for the inactivation of Candida species. In vitro and clinical study. [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017. Abstract This work evaluated the efficacy of Antimicrobial Photodynamic (aPDT) and Sonodynamics (SDT) Therapies in the inactivation of Candida species. The study 1 evaluated the efficacy of aPDT in the inactivation of susceptible and fluconazole resistant Candida albicans biofilms, as well as its effect on the virulence factors of the strains. For this, biofilms were treated with aPDT mediated by Photodithazine and LED light. After aPDT, cells were recovered and the virulence factors were evaluated. The adhesion capacity was assessed by XTT assay (metabolic activity) and CFU/mL (cell viability). The ability to form biofilm was evaluated by XTT, CFU/mL and total biomass. The synthesis of exoenzymes was evaluated by fluorimetric tests. Data were analyzed by ANOVA-2 or 3 way (p≤0.05). It was found that aPDT reduced the viability of the strains, however aPDT did not alter the virulence factors. The study 2, evaluated the effectiveness of aPDT in the treatment of patients with denture stomatitis in comparison with Nystatin (NYS). Patients of the aPDT group (n=30) underwent 6 sessions of aPDT (3 times/week, 15 days). Patients of the NYS group (n=35) rinsed the antifungal, 4 times/day, for 15 days. Microbiological collections of dentures and palates were performed and cultured on blood agar and chromagar (CFU/mL). Photographs of the palates were taken for clinical evaluation. Data were analyzed by the Linear Model of Repeated Measures (p≤0.05). It was observed that aPDT was more effective in reducing the total microbiota than NYS. The aPDT was as effective as NYS to reduce Candida spp. of the palate. aPDT and NYS showed similar clinical improvement percentage. The study 3 evaluated the efficacy of aPDT, SDT and the combination of both treatments in the inactivation C. albicans. Planktonic cultures were treated with aPDT and SDT. Cell survival was determined by CFU/mL. Biofilms were treated with aPDT, SDT and the combination of both treatments (aPDT+SDT). Efficacy in treating biofilms was evaluated by CFU/mL, total biomass and microscopy. Data were analyzed by ANOVA (p≤0.05). Although aPDT and SDT completely eradicated planktonic cultures, these treatments had no effect on biofilms. However, the combined treatment (aPDT+SDT) reduced the cell viability and total biomass. Key words: Photochemotherapy. Ultrasonic Therapy. Candida. Drug Resistance. Denture Stomatitis. SUMÁRIO 1 INTRODUÇÃO ............................................................................................................... 12 2 PROPOSIÇÃO ................................................................................................................ 17 3 PUBLICAÇÕES .............................................................................................................. 18 3.1 Publicação 1 ...................................................................................................................... 18 3.2 Publicação 2 ...................................................................................................................... 38 3.3 Publicação 3 ...................................................................................................................... 58 4 DISCUSSÃO .................................................................................................................... 84 5 CONCLUSÃO ................................................................................................................. 89 REFERÊNCIAS .............................................................................................................. 90 APÊNDICE A .................................................................................................................. 95 APÊNDICE B................................................................................................................... 99 ANEXO A ....................................................................................................................... 103 ANEXO B ....................................................................................................................... 104 12 1 INTRODUÇÃO Muitas espécies de Candida são isoladas da cavidade oral dos seres humanos em relação de comensalismo (Soll 53 , 2002). Porém, sob determinadas situações, como por exemplo quando o indivíduo possui imunossupressão relacionada à síndrome da imunodeficiência adquirida (AIDS), faz uso de medicamentos imunossupressores, antibióticos de amplo espectro, terapias antineoplásicas ou quando o indivíduo é diabético, estes fungos são responsáveis pelo desenvolvimento de infecções, tais como a candidose oral (Iacopino, Wathen 27 , 1992). A Candida albicans é o principal agente etiológico desta infecção (Nikawa et al. 40 , 1998), no entanto, outras espécies do mesmo gênero também têm sido isoladas e são frequentemente associadas ao desenvolvimento de infecções, tais como a Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei e Candida guilliermondi (Samaranayake, Samaranayake 49 , 2001). A expressão de fatores de virulência pela C. albicans auxilia na colonização do hospedeiro (Ferreira et al. 17 , 2010). A produção de enzimas, como as proteinases e fosfolipases, hidrolisa importantes constituintes da membrana citoplasmática da célula hospedeira, como os fosfolipídios e proteínas. Esse processo resulta na ruptura da membrana celular e consequente invasão do microrganismo (Hube, Nagllik 26 , 2001; Niewerth, Korting 39 , 2001). A habilidade de formar biofilmes tem sido intimamente associada à capacidade de causar infecções, e pode ser considerada um importante fator de virulência (Ramage et al. 45 , 2012). Os biofilmes são comunidades biológicas com um elevado grau de organização e complexidade. Estas comunidades encontram-se envoltas por matriz extracelular produzida pelas próprias células. Os biofilmes podem desenvolver-se em qualquer superfície que esteja em contato com fluidos do corpo, seja biótica ou abiótica. A associação dos organismos em biofilmes constitui uma forma de proteção ao seu desenvolvimento, favorece relações simbióticas e permite a sobrevivência em ambientes hostis (Ramage et al. 45 , 2012). A utilização de próteses dentais removíveis tem sido associada à manifestação de uma forma de candidose que afeta normalmente o palato de aproximadamente 65% dos pacientes usuários de próteses (Chandra et al. 9 , 2001), conhecida como estomatite protética. Como as espécies de Candida são capazes de aderir à resina acrílica, a superfície interna das próteses torna-se um reservatório de microrganismos, e passa a ser colonizada por um biofilme complexo e consistente (Salermo et al. 48 , 2011), com capacidade de armazenar e proteger os microrganismos que, por sua vez, podem colonizar outras regiões da cavidade bucal (Monroy et al. 35 , 2005). Outros fatores locais associados à prótese, como trauma local, xerostomia, uso 13 contínuo da prótese e alteração do pH da saliva, também são frequentemente relacionados ao desenvolvimento da estomatite protética (Figueiral et al. 19 , 2007). Essa infecção caracteriza-se pela presença de múltiplos pontos hiperêmicos na mucosa subjacente às próteses removíveis dos pacientes, e, em casos mais avançados, também podem ser observadas áreas eritematosas difusas e hiperplasia papilar do palato (Telles et al. 54 , 2017). Apesar de muitas vezes ser assintomática, a infecção pode causar prurido, ardência, dor, sabor desagradável, desconforto, hemorragia na mucosa bucal, alteração no paladar, halitose, xerostomia e lesões associadas, como queilite angular e glossite rombóide mediana (Hoshi et al. 25 , 2011; Scully 51 , 2013). Os tratamentos direcionados à estomatite protética são variados, podendo ser terapia antifúngica tópica, medicação antifúngica sistêmica e procedimentos de higienização e desinfecção das próteses (Banting, Hill 3 , 2001; Goldman et al. 22 , 2004). O grau de infecção, condições de saúde geral e bucal, idade e gênero do paciente e idade da prótese são aspectos que devem ser avaliados na escolha do tratamento (Yarborough et al. 57 , 2016). Em qualquer situação clínica, é necessário orientar devidamente os pacientes quanto à higienização das próteses e à sua remoção durante o período noturno (Hoshi et al. 25 , 2011). Além disso, é indicado que as próteses com mais de cinco anos de uso ou em condições inadequadas sejam substituídas (Hoshi et al. 25 , 2011). Para o tratamento da estomatite protética a prescrição de agentes antifúngicos tópicos, como a Nistatina tem sido indicada (Pappas et al. 41 , 2009). Para pacientes imunocomprometidos ou em risco de candidemia, a terapia antifúngica sistêmica, como por exemplo os azoles, tem sido usada (Akpan, Morgan 2 , 2002). No entanto, o aumento na utilização dos medicamentos azoles, combinado com vários casos de recorrência da infecção, tem tornado estes fungos resistentes aos tratamentos antifúngicos convencionais (Goldman et al. 22 , 2004). A resistência aos medicamentos antifúngicos é definida como a falha de um tratamento antifúngico, que resulta na persistência ou progressão da infecção (White et al. 56 , 1998). Diante das dificuldades relacionadas a resistência fúngica, uma modalidade terapêutica alternativa vem sendo sugerida como promissora para inativação microbiana (Donnelly et al. 13 , 2008). A Terapia Fotodinâmica Antimicrobiana (do inglês, Antimicrobial Photodynamic Therapy, aPDT) baseia-se na administração de um composto químico, um fotossensibilizador (Fs), que submetido a irradiação com luz visível, na presença de oxigênio, produz danos celulares específicos que inativam os microrganismos. Na área médica, esta terapia também tem sido amplamente estudada para o tratamento de câncer bucal, pulmão e pele (Agostinis et al. 1 , 2011). Dois diferentes mecanismos oxidativos parecem ocorrer após a 14 fotoativação do fotossensibilizador. No primeiro, ocorre uma reação fotoquímica, na qual o fotossensibilizador interage com uma biomolécula produzindo radicais livres, já no segundo mecanismo, há produção do oxigênio singlete ( 1 O2). Estas moléculas que são produzidas (radicais livres e oxigênio singlete) são responsáveis pela inativação celular (Donnelly et al. 13 , 2008). Atualmente, FSs de segunda geração vem sendo amplamente empregados na aPDT. Dentre estes compostos estão as clorinas, porfirinas hidrofílicas reduzidas que apresentam forte banda de absorção na região vermelha do espectro fotomagnético. O Photodithazine ® (PDZ) é uma clorina e6 preparada na Rússia, obtida a partir da cianobactéria Spirulina platensis. Esse FS vem sendo aplicado atualmente com sucesso na PDT contra o câncer, devido ao seu alto rendimento quântico de formação de oxigênio singlete (Ferreira et al. 18 , 2008). A efetividade da aPDT mediada pelo PDZ na inativação in vitro de espécies de Candida têm sido relatada (Dovigo et al. 14 , 2013; Quishida et al. 44 , 2013). Dovigo et al. 14 (2013), observaram que a aPDT associada ao PDZ e luz LED promoveu redução no número de colônias e no metabolismo celular dos biofilmes de C. albicans, C. glabrata e C. tropicalis em aproximadamente 62,1%, 76,9% e 76% respectivamente (Dovigo et al. 14 , 2013). Quando um biofilme misto formado por C. albicans, C. glabrata, e Streptococcus mutans foi submetido a aPDT mediada pelo PDZ foi observada uma redução significativa na viabilidade das três espécies avaliadas, além disso, foi observada uma redução significativa na atividade metabólica dos biofilmes (Quishida et al. 44 , 2015). Apesar de o tratamento da estomatite protética por meio da aPDT mediada pelo PDZ ainda não ter sido avaliado, estudos in vivo mostraram a eficácia da aPDT no tratamento de candidose oral induzida em camundongos. Carmello et al. 8 (2015) observaram que a aplicação de 100 mg/L de PDZ e 37,5 J/cm² de luz LED foi capaz de reduzir 4,36 log10 na viabilidade de C. albicans presentes na língua de camundongos com candidose oral. Em outro estudo, cinco aplicações sucessivas de aPDT mediada pelo PDZ foram tão efetivas quanto a Nistatina para o tratamento de candidose oral experimental em camundongos (Carmello et al. 7 , 2016). Ambos os estudos concluíram que a aPDT mediada pelo PDZ pode ser aplicada de forma segura, uma vez que não foi verificada qualquer alteração microscópica nos tecidos dos animais. O rosa-bengala (RB) (sal dissódico 4,5,6,7-tetracloro-2,4,5,7-tetra-iodo-fluoresceínas), é um corante de xanteno hidrofóbico aniônico caracterizado por absorção em comprimentos de onda de luz na faixa de 450 a 600 nm. O RB tem várias aplicações na medicina que incluem o diagnóstico de dano ocular, a detecção de lesões pré-cancerosas orais e é usado 15 como agente revelador de placa dentária (Fischer et al. 20 , 2004; Feenstra, Tseng 16 , 1992). Devido a sua capacidade de produzir espécies reativas de oxigênio ao ser irradiado, este corante também tem sido investigado como FS para mediar a aPDT. Freire et al. 21 (2014) tratou células planctônicas de C. albicans ATCC 18804 (10 6 ) com aPDT mediada pelo RB nas concentrações de 0,78 a 400 μM durante 5 min, e luz LED verde (532 nm, 42,63 J/cm²). Os autores observaram completa erradicação de C. albicans quando incubadas com 12,5 μM. Quando este FS foi avaliado na concentração de 40 μM na inativação suspensão de isolados clínicos de C. albicans, a maior redução encontrada foi de 1,97 log10 (Costa et al. 10 , 2012). Em outro estudo, quando a aPDT foi mediada pelo RB a 200 μmol/L por 20 min, foram observadas reduções de 4 log10 e 6 log10 de C. albicans nas densidades celulares de 10 7 e 10 6 células/mL, respectivamente, quando este FS foi iluminado nas doses de 40 e 80 J/cm² (Demidova, Hamblin 12 , 2005). Desta forma, dependendo da cepa, concentração do FS, fonte e dose de luz avaliados, observam-se diferentes resultados da efetividade da aPDT mediada pelo RB. Recentemente, a Terapia com Sonodinâmica (SDT) surgiu como uma alternativa à aPDT para o tratamento de tumores (McHale et al. 32 , 2016) e para a inativação de micro- organismos (Harris et al. 23 , 2014). Enquanto a aPDT utiliza luz para ativar o FS, a SDT utiliza o ultrassom (US). A vantagem da utilização do US é que esse se propaga mais profundamente no tecido. Embora o mecanismo da aPDT seja bem conhecido, o mecanismo exato para a geração de oxigênio singlete na SDT ainda é controverso (Hiraoka et al. 24 , 2006). Sabe-se que na SDT a cavitação inercial pelo ultrassom, um processo caracterizado pela formação, oscilação e colapso de bolhas gasosas no meio irradiado com o ultrassom, é a reação central para a produção de oxigênio singlete. Quando o fenômeno de cavitação é dominado por forças de inércia, as bolhas colapsam violentamente, acarretando na elevação da temperatura local e na produção de sonoluminiscência. Sugere-se que, na SDT, a geração de oxigênio singlete seja resultado da foto-ativação indireta da droga sensibilizante via sonoluminescência (McHale et al. 32 , 2016). Uma vez excitado, o sensibilizador produz oxigênio singlete exatamente da mesma maneira que na aPDT. O uso da SDT como tratamento antimicrobiano é uma abordagem relativamente nova, por isso as publicações relacionadas a este assunto são escassas. Alguns estudos nesta área relataram inativação de bactérias por meio do US mediado por diferentes sensibilizadores, incluindo rose-bengal (RB) (Nakonechny et al. 37 , 2013), curcumina (Wang et al. 55 , 2014) e porfirina (Zhuang et al. 58 , 2014). Nakonechny et al. 37 (2013), observaram que a SDT, utilizando-se o US na frequência de 28 kHz, mediada pelo RB foi efetiva na erradicação das 16 suspensões de Staphylococcus aureus e Escherichia coli. Zhuang et al. 58 (2014) demonstraram que a incubação de 50 μg/L com um éster monometílico de hematoporfirina seguida de irradiação ultrassônica (1 MHz, 6 W/cm², 30 min) promoveram redução de ~ 2 log10 de suspensões de S. aureus (Zhuang et al. 58 , 2014). Em outro estudo, culturas planctônicas de S. aureus e Pseudomonas aeruginosa tratadas com SDT utilizando um peptídeo antimicrobiano conjugado ao RB resultou em reduções de 5 e 7 log10 na viabilidade celular, respectivamente (Costley et al. 11 , 2015). Quando uma suspensão de S. aureus resistente a meticilina (MRSA) foi tratada usando SDT com curcumina, a viabilidade bacteriana foi reduzida em 5 log10 (Wang et al. 55 , 2014). Dessa forma, a SDT tem demonstrado ser uma abordagem promissora para a inativação de bactérias planctônicas, no entanto, ainda não há relatos que descrevam a eficácia da SDT para a inativação de suspensões ou biofilmes de C. albicans. Por isso, o objetivo deste trabalho foi avaliar a efetividade da aPDT medida pelo PDZ, em inativar biofilmes de C. albicans susceptíveis e resistentes ao fluconazol (Publicação 1). Também foram avaliados os efeitos desta terapia sobre os fatores de virulência de Candida (adesão, capacidade de formação de biofilme, produção de fosfolipase e proteinase). Em outra etapa (Publicação 2), foi verificada a efetividade da aPDT, mediada pelo PDZ, no tratamento de pacientes com estomatite protética em comparação com o antifúngico tópico Nistatina. Adicionalmente, foi avaliada a capacidade das Terapias Sonodiâmica, aPDT e a combinação de ambos os tratamentos, mediados pelo PDZ ou RB, em inativar suspensões e biofilmes de C. albicans (Publicação 3). 17 2 PROPOSIÇÃO Publicação 1: Avaliar a eficácia da aPDT medida pelo PDZ e luz LED na inativação de biofilmes de C. albicans susceptíveis e resistentes ao fluconazol. Também foram avaliados os efeitos desta terapia sobre os seguintes fatores de virulência de Candida: adesão e capacidade de formação de biofilme, e produção de exoenzimas (fosfolipase e proteinase). Hipótese: A aPDT é capaz de inativar biofilmes de C. albicans susceptíveis e resistentes ao fluconazol, além de reduzir os fatores de virulência destes micro-organismos Publicação 2: Avaliar a efetividade da aPDT, mediada pelo PDZ e luz LED, no tratamento de pacientes com estomatite protética em comparação com o antifúngico tópico Nistatina. Hipótese: A aPDT pode ser empregada como tratamento alternativo de pacientes com estomatite protética. Publicação 3: Avaliar a eficácia da aPDT, SDT e a combinação de ambos os tratamentos na inativação de suspensões e biofilmes de C. albicans. Os tratamentos foram mediados pelo PDZ ou ou RB associados a luz LED ou ao US. Hipótese: Tanto a aPDT quanto a SDT são efetivas em inativar biofilmes de C. albicans. 18 3 PUBLICAÇÕES 3.1 Publicação 1 Virulence factors of fluconazole-susceptible and –resistant Candida albicans after antimicrobial photodynamic therapy. Fernanda Alves, MSc 1 Ewerton Garcia de Oliveira Mima, PhD 1 Renata Caroline Polato Passador, DDS 1 Vanderlei Salvador Bagnato, PhD 2 Janaína Habib Jorge, PhD 1 Ana Cláudia Pavarina, PhD 1† 1 Department of Dental Materials and Prosthodontics, Araraquara Dental School, UNESP – Univ. Estadual Paulista, Rua Humaitá, 1680, 14801-903, Araraquara, SP, Brazil. 2 Biophotonics Lab, Group of Optics, Physics Institute of São Carlos, University of São Paulo - USP, Av. Trabalhador São-carlense, 400, 13566-590, São Carlos, SP, Brazil. † Corresponding Author: Dr Ana Claudia Pavarina e-mail: pavarina@foar.unesp.br Phone: 55 16 33016544 Fax: 55 16 33016406 Adress: Rua Humaitá, 1680, CEP 14801-903, Araraquara, SP, Brazil. *Artigo publicado em (Autorização de reprodução pela revista - ANEXO B): Alves F, de Oliveira Mima EG, Passador RCP, Bagnato VS, Jorge JH, Pavarina AC. Virulence factors of fluconazole-susceptible and fluconazole-resistant Candida albicans after antimicrobial photodynamic therapy. Lasers Med Sci. 2017;32(4):815-826. mailto:pavarina@foar.unesp.br 19 Virulence factors of fluconazole-susceptible and –resistant Candida albicans after antimicrobial photodynamic therapy. Abstract This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on virulence factors of fluconazol-susceptible (CaS) and –resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (10 7 ), and after 48h of biofilm formation, these strains were incubated with PDZ (100 mg/L) for 20 minutes and exposed to LED light (660nm, 37.5J/cm 2 ). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, the metabolic activity tests (XTT assay) and cell viability (CFU/mL) were applied. To evaluate the biofilm forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p ≤ 0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and -resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability, and enzymatic production. Keywords: Candida albicans, Photodynamic inactivation, Drug Resistance, Virulence Factors 20 Introduction Candida albicans is commonly found in human beings in a relationship of comensalism, however when there is imbalance in the host immune system, this fungus may invade tissues and cause superficial infections such as oropharyngeal candidosis (OPC) [1]. The pathogenesis of OPC may be related to the host conditions and virulence of the microorganism [2]. Diabetic patients, users of immunosuppressive medications and broad spectrum antibiotics; individuals submitted to atineoplasic therapies and AIDS patients present a high incidence of infection by Candida [3]. Moreover, C. albicans presents virulence factors that aid the colonization and invasion of host tissues [4]. Organization into biofilms may be considered an important virulence factor [5], since it is intimately associated with the capacity to cause infections, protect development of the microorganism in hostile environments, and prevent the penetration of antifungal drugs [5]. Furthermore, the production of enzymes, such as proteinases and phospholipases play an important role in the pathogenicity of these fungi, since these enzymes are able to hydrolyze important phospholipids and proteins of the cytoplasmic membrane of the host cell, resulting in rupture of this organelle [6,7]. Topical and systemic antifungal agents (polyenes, and azoles, such as Amphotericin B, and itraconazol) have been used in the treatment of OPC, however, these medications may promote the development of hepatoxicity and antifungal resistance [8]. Prophylactic treatment with fluconazol, recommended for HIV- positive patients, has been shown to cause substitution of susceptible C.albicans strains by those resistant to fluconazole [9]. Moreover, the fungistatic activity of azoles has also been associated with failure of antifungal treatment in immunocompromised patients. Another aspect related to antifungal resistance and recurrence of infection is the capacity of Candida spp to form biofilms on surfaces [10,11]. Due to the antifungal resistance and difficulties associated with the use of conventional medications, antimicrobial photodynamic therapy (aPDT) has been suggested for inactivating of Candida spp., and for the treatment of superficial fungal infections [12-14]. Photodithazine® (PDZ), a second generation photosensitizer (FS), derived from chlorine that has been successfully applied with aPDT against cancer [15]. In vitro studies have indicated that aPDT mediated by PDZ was effective in inactivating bacteria and fungi. Dovigo et al. [16] observed high rates of reduction in metabolic activity of biofilms formed from clinical isolates of C. albicans, Candida tropicalis and C. glabrata after exposure to aPDT. Significant reduction in metabolic activity and cell viability has also been observed in multi-specie biofilms [17]. Studies have demonstrated that the species of Candida present susceptibility to aPDT [12], however, there are still some aspects that need to be clarified. Due to non-specific oxidant agents, the organisms resistant to conventional antifungal agents may be inactivated by aPDT. However, it has been verified that strains fluconazole-resistant of C. albicans and Candida glabrata exhibit reduced sensitivity to aPDT compared to fluconazole- sensitive reference strains (ATCC), suggesting that the mechanisms of resistance of microorganisms to traditional antifungal medications may reduce the efficacy of aPDT [13]. In addition, the response of strains to aPDT has not been homogeneous among the resistant strains of the same species [13,18]. Since the most promising advantage of aPDT would be to treat infections resistant to antifungal agents, the susceptibility of fluconazole-resistant C.albicans needs to be better understood. Moreover, both the action of aPDT mediated by PDZ against fluconazole-resistant C. albicans and the effect of aPDT on the virulence factors of fluconazole-susceptible and -resistant C. albicans are unknown. 21 Therefore, the aim of this study was to evaluate the expression of virulence factors of fluconazole- susceptible and -resistant C. albicans after aPDT mediated by PDZ associated with LED light. The virulence factors evaluated were: capacity of adhesion and biofilm formation, and the capacity for specific degradative enzyme synthesis (protease and phospholipase). Materials and Methods: Photosensitizer and Light Source Photodithazine® (PDZ- VETA- GRAND Co., Moscow, Russia), a chlorin e6 derivative, was used as PS. PDZ was diluted in physiological solution (0.85 % NaCl) in a concentration of 100 mg/L. The FS was excited by a light emitting diode (LED) in the red region of the spectrum (660 nm). This device is composed of red LEDs (LXHL-PR09, Luxeon® III Emitter, Lumileds Lighting, San Jose, California, USA) uniformly distributed, with a constant power output of 71 mW/cm 2 . The dose of light evaluated was 37.5 J/cm 2 . Obtaining the biofilms and performing Antimicrobial Photodynamic Therapy The standard strains of C. albicans ATCC 90028 (fluconazole-susceptible; CaS) and C. albicans ATCC 96901 (fluconazole-resistant; CaR) were used to form biofilm. The strains were defrosted and reactivated in Agar Sabouraud Dextrose (SDA, Acumedia Manufactures Inc., Baltimore, MD) culture medium containing 5 µg/mL of gentamycin and incubated (37 o C / 48 h). After this period, the cell cultures were inoculated in 5 mL of RPMI - 1640 (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37º C for 16 h, at 75 rpm in an orbital agitator (AP 56, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil). After incubation, the cells were submitted to the steps for biofilm formation, described by Dovigo et al. [16]. In summary, the cells were washed and resuspended in phosphate-buffered saline (PBS). Cell concentration was adjusted to 10 7 cells/mL in a spectrophotometer (540 nm) and 100 μL of this suspension was transferred to a 96-well plate. The plate was incubated at 37º C for 1.5 hours in an incubator shaker at 75 rpm (adhesion stage). After this period, RPMI – 1640 was added, and the plate was incubated at 37º C at 75 rpm for 48 h (biofilm maturation). After biofilm formation, the culture medium was removed and the biofilm was washed twice with PBS. To perform the aPDT, 100 µL of PDZ was added to each biofilm. After this, the plates were incubated in the dark for 20 min (pre-irradiation time), and then illuminated with LED light for 9 min (37.5 J/cm²) (Groups P+L+CaS and P+L+CaR). Additional samples were treated with PDZ (P+L-CaS or P+L-CaR) only, or with LED light only (P-L+CaS and P-L+CaR). The control group received no treatment with PDZ or with LED light (P-L- CaS and P-L-CaR). After application of the treatments, tests were performed to evaluate the effectiveness of aPDT and its effect on the virulence factors. All the tests were performed in triplicate on three different occasions. It is important to point out that for each test and occasion performed, individual plates were used. Therefore, the evaluations were made directly on the cells coming from the biofilms that were submitted to the treatments described, according to the flowsheet (Figure 1). mailto:cirelli@foar.unesp.br 22 Fig 1 Flowchart of the methods used for evaluation of the treatments and the virulence factors after the treatments Evaluation immediately after treatments To evaluate the efficacy of aPDT against the biofilms, the following methods were used: quantification of colonies (CFU/mL), evaluation of cell metabolism (XTT assay) and total biomass. For quantifying the colonies (CFU/mL), on conclusion of treatments, the biofilms were detached from the wells with the aid of a sterile swab (Johnson) and aliquots of 25 μL of the serial dilution were plated on SDA medium. After incubation at 37 °C for 48 h, colony forming units per milliliter (CFU/mL) values were determined and transformed into log10 [16]. Biofilm cell metabolism was evaluated by means of the yellow tetrazolium salt XTT reduction assay. For this purpose, after application of the treatments, 200 μL of XTT solution (containing 158 μL of PBS with the addition of 200 mM glucose, 40 μL of XTT({2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H tetrazolium hydroxide}), and 2 μL of menadione) were added to each well. The plates were incubated at 37 °C in the dark for 3 h and colorimetrically measured in a microplate reader (Thermo Plate / TP Reader) at 492 nm [19]. The total biomass of biofilm was quantified by means of crystal violet (CV) staining. After being submitted to treatments, the biofilms were washed with PBS and then fixed with 200 μL of methanol for 15 min. The methanol was removed and the plates were kept at 37 °C for 20 minutes to dry. Subsequently, 200 µL of CV (1 % v/v) was added and maintained for 5 minutes. The wells were washed with ultrapure water, and after this, 33% acetic acid was added to remove the dye. The result of this chemical reaction was measured, using the microplate reader at 570 nm [19]. Evaluation of virulence factors Additional biofilms that received the previously described treatments were evaluated with regard to the following virulence factors: capacity for adhesion to abiotic surfaces; biofilm forming capacity, and capacity for specific degradative enzyme synthesis (proteinase and phospholipase). 23 To evaluate the capacity for adhesion to abiotic surface, the treated biofilms were detached from the bottom of the wells; the cells were transferred to a new 96-well plate, and submitted to the steps described previously to adhesion of the cells as reported by Dovigo et al. [16]. After the adhesion stage, the adhered cells were evaluated according to the following previously described methods: quantification of colonies (CFU/mL) and cell metabolism (XTT assay). The values obtained after adhesion were statistically compared with those obtained from biofilms immediately after aPDT. To evaluate the biofilm forming capacity on an abiotic surface, the biofilms submitted to the treatments were detached from the plate and the microorganisms were submitted to the same procedures as those described previously for the development of the biofilms [16]. After biofilm formation, quantification of colonies (CFU/mL), evaluation of cell metabolism (XTT) and total biomass were performed. The values obtained after biofilm formation were statistically compared with those obtained from biofilms immediately after aPDT. The capacity for phospholipase and proteinase synthesis was evaluated by means of a fluorimetric test. To measure the phospholipase production after aPDT the biofilms were resuspended in 200 µL of lysis buffer and sonicated for 20 s. The biofilms were removed from the bottom of the culture plate wells with the aid of a pointer, transferred to eppendorf tubes and centrifuged (10000 rpm/5 min). The Amplex® Red Phosphatidyl choline-Specific Phospholipase C Assay Kit (cod#A-12218) was used, in accordance with the manufacturer’s instructions. The assays were performed in black 96-well plates in a total volume of 200 µL per well (100 µL of the centrifugation product of the biofilms and 100 µL of the working solution). Fluorescence was read in the fluorescence microplate reader (Fluoroskan Ascent Microplate™, USA) at 544 nm excitation and 590 nm emission. The fluorescence values (nm) were recorded and used for comparisons with the fluorescence values of the positive controls provided by the manufacturer (purified PL-C from Bacillus cereus and hydrogen peroxide) [20]. To evaluate proteinase enzyme production, after performing the proposed treatments, 200 µL of RPMI medium were added in each orifice of the samples. After 24h of incubation at 37 °C under agitation (75 rpm), the biofilms were removed from inside the wells with the aid of a pointer, transferred to eppendorf tubes and centrifuged at 10000 rpm for 5 min. To evaluate Proteinase secretion, the EnzChek® Protease Assay Kit (cod#E- 6638) was used, in accordance with the manufacturer’s instructions. The assays were performed in black 96-well plates in a total volume of 200 µL per well (100 µL of the centrifugation product of the biofilms and 100 µL of the working solution). Fluorescence readout was obtained in Fluoroskan at 485 nm excitation and 538 emission. The fluorescence values were used in linear equations derived from the standard curves obtained from trypsin solutions, and the enzymatic activity was expressed in ng/mL [20]. Statistical Analysis Homogeneity of variance and normality of the data were verified by the Levene and Shapiro-Wilk tests respectively. The results of biofilm adhesion and formation were analyzed statistically by means of three-way analysis of variance (three-way ANOVA): time (immediately after treatments and after biofilm adhesion or formation subsequently to aPDT), strain (susceptible and resistant) and treatment groups (P-L-, P+L-, P-L+, P+L+). For multiple comparisons, the post-hoc Tukey test was used for the homoscedastic data and the Games Howell test for the heteroscedastic data. On the other hand, when the assumptiom of normality was not found, non-parametric analysis was used (ANOVA on Rank) and the Tukey post-hoc test. The proteinase and 24 phospholipase data were analyzed by two-way ANOVA and the Tukey post-hoc test for homoscedastic data and the Games Howell test for the heteroscedastic data. The level of significance adopted was 5% (α=0.05). It was also performed Pearson Correlation between enzymatic production (proteinase and phospholipase) and the Log10(CFU/mL) values (α=0.01). These analyses were performed using the software SPSS (IBM ® SPSS ® Statistics, version 20, Chicago, IL, USA). Results Capacity for adhesion to the abiotic surface Regarding the quantification of colonies (CFU/mL), the biofilms presented immediately after aPDT a reduction in the viability of 19.09% and 18.7%, equivalent of 1.20 log10 and 1.14 log10 compared with the control groups (P-L-CaS or P-L-CaR) for CaS and CaR, respectively. In general, it was observed that all the cells submitted to the treatments presented reduced capacity for adhesion. Comparing the groups at the times evaluated (immediately after treatments and after adhesion subsequently to aPDT), 68.9% of the cells of P-L- CaS group adhered to the polystyrene plate, while 63% of the cells of P+L+CaS group presented capacity for adherence. For CaR, 74.2% of the cells of P-L-CaS group adhered to the polystyrene plate, and 73.9% of the cells of P+L+CaS group presented capacity of adherence. Three-way analysis of variance revealed that there was no interaction among the 3 factors evaluated: strain, time and treatment groups (p = 0.503). However, significant interaction was demonstrated between the factors “strain” and “time” (p < 0.001), for this reason, the Tukey post-hoc test was used, since data showed normal distribution (Shapiro-Wilk test, p≥ 0.05) and were homoscedastic (Levene test, p < 0.001). The Tukey post-hoc test demonstrated a significant difference between the time intervals evaluated, and similarity between the strains studied. Therefore, the factors “group” and “strain” were not significant for the capacity for adhesion, since only the factor “time” showed significant influence on the capacity for adhesion of the cells (Figure 2). Fig 2 Mean values and standard deviations of log10(CFU/mL) of the CaS and CaR, in the different time intervals of evaluation (I = immediately after treatments, A = after adhesion), independent of the treatment groups (n=36). Equal letters denote statistical similarity between the factors evaluated (p>0.05) 25 The analysis of the XTT assay showed that the biofilms submitted to aPDT demonstrated a reduction of 79.5 and 50.4% in metabolic activity for CaS and CaR, respectively, in comparison with the control groups (P-L- CaS or P-L-CaR) (Figure 3). Subsequently, after aPDT, the capacity for adhesion of the viable cells was also evaluated by the XTT assay. When comparing the groups at the times evaluated (immediately after aPDT and after adhesion subsequently to aPDT) it was observed that adhesion of the cells reduced to 15.5% for the P-L- CaS group, while for P+L+CaS group the reduction was equivalent to 15.8%. For CaR strain, 19% of the cells of P-L-CaR group were metabolically active, and for P+L+CaR group the metabolic activity reduced to 13%. Three-way analysis of variance revealed significant interaction among the three factors evaluated (p < 0.001). Therefore, the multiple comparisons test of Games Howell was applied, since data showed a normal distribution (Shapiro Wilk test, p >= 0.64) but was heteroscedastic (Levene test, p < 0.001). The Games Howell test demonstrated that the adhesion capacity for all groups of the CaS strain was significant different (p<0.05) to that observed for the groups of the CaR strain. The time was also a significant factor (p<0.05), since a reduction in the metabolic activity of the adhered cells was observed for all the groups evaluated (controls and aPDT). Moreover, the groups treated with aPDT showed significant difference (p=0.0) compared with the other groups (P-L-, P+L- and P-L+), which do not show significant differences among them (p=0.0). Fig 3 Mean values and standard deviations of the metabolic activity of the experimental groups for the two C. albicans strains evaluated immediately after application of the treatments and after the cell adhesion (n=9) Capacity for biofilm formation on abiotic surface The quantification of colonies demonstrated a reduction of 16.18% and 13.5% of the biofilms submitted to aPDT compared with the control group (P-L-CaS or P-L-CaR), equivalent to 1.01 log10 and 0.81 log10, for CaS and CaR, respectively. When comparing the groups at the times evaluated (immediately after aPDT and after biofilm formation) 100% of the cells from P-L-CaS and P+L+CaS groups were able to form biofilm. For CaR 96.2% and 94.3% of the cells from P-L-CaS and P+L+CaS groups, respectively, formed biofilm on the polystyrene plate. Three-way analysis of variance revealed that there was no significant interaction (p = 0.466) 26 among the 3 factors evaluated: strain, time and treatment groups. However, a significant interaction (p<0.001) was found between group versus strain (Figure 4) and time versus strain (Figure 5). The Games Howell post-hoc test was used to evaluate the interaction between group x strain, since data showed normal distribution (Shapiro- Wilk test, p >= 0.074) and was heteroscedastic (Levene test, p = 0.017). The Games Howell test showed that aPDT groups (CaS and CaR) was significant different (p < 0.001) from the other groups (P-L-, P-L+ and P+L-), which showed no significant difference (p > 0.487) among them (Figure 4). Furthermore, the Tukey post-hoc test used to analyze the interaction between strain x time (homoscedastic data, Levene test, p = 0.136), demonstrated for CaR a significant (p <= 0.007) reduced ability to form biofilm (Figure 5). Fig 4 Mean values and standard deviations of log10(CFU/mL) of the CaS and CaR strains from different groups (P-L-, P-L+, P+L-, P+L+), independent of the time evaluated (n=18). Equal letters denote statistical similarity between the factors evaluated (p>0.05) 27 Fig 5 Mean values and standard deviations of log10(CFU/mL) of the CaS and CaR at different time intervals of evaluation (I = immediately after treatments, A = after biofilm formation), independent of the treatment group (n=36). Equal letters denote statistical similarity between the factors evaluated (p>0.05) The result of the XTT assay demonstrated that aPDT promoted a reduction of 78.23 and 39.1% in the metabolic activity of CaS and CaR strains, respectively (Figure 6). After aPDT the biofilm formation ability of the viable cells was also evaluated by the XTT assay. In general, it was observed that only the CaR strain submitted to the treatments presented a reduction in the metabolic activity. When comparing the groups at the times evaluated (immediately after aPDT and after biofilm formation subsequently to aPDT) it was observed that 56% of the cells from the P-L-CaS group was able to grow and form biofilm, while for P+L+CaS group, this value was equivalent to 250%, indicating that after aPDT the CaS presented an elevated ability for multiplication and biofilm formation. For CaR, 61.7% and 67.2% of the cells from P-L-CaR and P+L+CaR groups grew as biofilm (Figure 6). Since the assumption of normality was not found (Shapiro-Wilk test, p<=0.021) a non-parametric analysis was used (ANOVA on Rank). The non-parametric test revealed a significant interaction (p<0.001) among the three criteria evaluated (time, strain and groups), and the Tukey post-hoc test demonstrated that immediately after application of the treatments there were significant differences (p <= 0.001) between the groups treated with aPDT (for both CaS and CaR) and the other groups, which were similar among them (p >= 0.127). However, the biofilm formation ability was statistically similar (p >= 0.069) among all the groups, demonstrating that aPDT was not able to reduce the ability of the two strains (CaS and CaR) to grow as biofilm. It was also verified a significant difference (p = 0.001) between CaR and CaS strains immediately after aPDT, but after biofilm formation both strains were similar (p >= 0.069) between them, independent of the group. 28 Fig 6 Mean values and standard deviations of the XTT assay of the experimental groups for the two strains of C. albicans evaluated immediately after application of the treatments and after biofilm formation (n=9). Equal letters denote statistical similarity between the factors evaluated (p>0.05) Finally, the crystal violet assay demonstrated that immediately after aPDT, the CaS strain demonstrated no reduction in total biomass compared with the other groups; while the resistant strain (P+L+CaR) showed a reduction of 23.1% in the total biomass compared with the control (P-L-CaR) (Figure 7). After application of the treatments, the total biomass of the viable cells grown as biofilm was also quantified by crystal violet stain. It was observed that the total biomass values after treatments of all groups were lower than the initial values, showing that even the biofilms of the control group presented lower total biomass values (Figure 7). When comparing the groups at the times evaluated (immediately after aPDT and after biofilm formation), reductions of 32.8% and 32.1% in the total biomass were observed for P-L-CaS and P+L+CaS groups, respectively. For CaR strain, a reduction of 49.5% in the total biomass was observed for P-L-CaR group, while for P+L+CaR group the biomass reduction was equivalent to 32.5%. The three-way analysis of variance revealed a significant interaction (p < 0.001) among the 3 criteria evaluated: strain, time and treatment groups. The Games-Howell post-hoc test was applied since data showed normal distribution (Shapiro-Wilk, p >= 0.070), but not homogeneity of variance (Levene test, p < 0.001). The Games-Howell test demonstrated for CaS strain no significant difference (p=1.000) between aPDT (P+L+CaS group) and the other groups immediately after application of the treatments. After biofilm formation subsequently to the treatments, the total biomass of all groups remained similar among them (p >= 0.984). The total biomass of the CaS biofilm was significant lower (p<0.001) than the initial value, demonstrating that even the biofilms of the control group showed a lower total biomass values. For CaR strain, immediately after aPDT, a significant reduction (p<=0.028) in the total biomass of the biofilm submitted to aPDT (P+L+CaR) was observed compared with the control and P+L- groups. However, after biofilm formation subsequently to the treatments, the biomasses of all groups were similar among them (p>=0.995), showing that aPDT was not able to reduce the biofilm formation ability. It was also verified that, after receiving some type of treatment, the biomass value of the CaR biofilm was lower than the initial value (p<0.001). Lastly, a significant difference (p<0.001) was also observed between CaS and CaR strains, both immediately after application of the treatments and after biofilm formation. 29 Fig 7 Mean values and standard deviations of total biomass of the experimental groups for both C. albicans strains evaluated immediately after application of the treatments and after biofilm formation (n=9). Equal letters denote statistical similarity between the factors evaluated (p>0.05) Proteinase Production The fluorescence values obtained in the readouts were normalized in accordance with the standard curve and the control reagents (tris-Hcl buffer and RPMI), and the value of proteinase production was obtained in the scale of ng/mL (Figure 8). The two-way analysis of variance was applied, once there were 2 factors involved: strain and treatment. This test demonstrated a significant interaction (p=0.0) between the two factors. For this reason, the Games-Howell post hoc test was used (normal distribution, Shapiro-Wilk test, p >= 0.061 and heterogeneity of variance, Levene test, p < 0.001) and demonstrated that for CaS strain only the P+L-CaS and P- L+CaS groups were similar between them (p=0.995) and the others were significantly different (p < 0.001). For CaR strain, no significant difference was observed between P+L-CaR and P-L+CaR groups (p=0.965), P-L+CaR and P+L+CaR (p=0.066), while the others groups showed significant different (p <= 0.001) among them. It was also observed a significant difference (p=0.00) in proteinase production between the strains evaluated, with the resistant strain producing significantly less proteinase than the susceptible strain (p<0.001). In addition, the Pearson Correlation demonstrated a significant (p=0.007) and positive correlation between the enzyme production and the values of Log10(CFU/mL) (r = 0.315), which means that the production of this enzyme was influenced by the quantity of viable cells. 30 Fig 8 Mean values and standard deviations of Proteinase production (ng/mL) of the experimental groups for both C. albicans strains evaluated. Equal letters denote statistical similarity (p> 0.05) Phospholipase Production The results of proteinase production were also analyzed by two-way analysis of variance, once there were 2 factors involved: strain and treatment. The two-way analysis of variance showed a significant interaction between the factors group and strain (p=0.0). The Tukey post-hoc test was applied (normal distribution, Shapiro- Wilk test, p >= 0.156 and homogeneity of variance, Leve test, p = 0.470) and demonstrated that for the CaS all groups differed among them and between the groups of the CaR (p<0.001). For CaR strain, a significant difference was observed only between P+L+CaR and P-L-CaR groups (p=0.002); P+L+CaR and P-L+CaR, in addition to all the groups of the CaS (Figure 9). It was also observed that there was significant difference (p=0.026) in phospholipase production between the strains evaluated, with the resistant strain producing significantly less of this enzyme than the susceptible strain (p<0.001). As the same way as for the Proteinase, it was also observed a significant (p=0.004) and positive correlation between the enzyme production and the values of Log10(CFU/mL) (r = 0.338), which means that the production of this enzyme was influenced by the quantity of viable cells. 31 Fig 9 Mean values and standard deviations of Phospholipase production (ng/mL) of the experimental groups for both C. albicans strains evaluated. Equal letters indicate statistical similarity (p> 0.05) Discussion C. albicans expresses diverse virulence factors that contribute to its pathogenicity, such as, polymorphism, the capacity for adhesion and biofilm formation; and degradative enzyme secretion [21]. The free radicals and singlet oxygen produced during aPDT may interact with diverse cell structures of the microorganisms (proteins, lipid membranes and nucleic acids) resulting in cell death by apoptosis or necrosis [22]. Furthermore, there are reports that ROS generated during aPDT application may change the virulence profile of fungi [23,24]. Therefore, the aim of this study was to evaluate the expression of virulence factors of fluconazole-susceptible and resistant C. albicans after the aPDT mediated by PDZ in association with LED light. In the present study, it was observed that immediately after application of the treatments, the biofilms submitted to aPDT demonstrated a reduction of 79.5 and 50.4% in metabolic activity, for CaS and CaR, respectively. This finding is in agreement with those found in the literature in studies that evaluated the photoactivation of strains of Candida by using PDZ as FS, however, it is important to point out that these studies did not evaluate fluconazole-resistant strains. Dovigo et al. [16] verified that aPDT mediated by PDZ associated with LED light reduced the metabolic activity of clinical isolated biofilms of C. albicans, C. tropicalis and C. glabrata by 62,1%; 76,0% and 76.9%, respectively. In the study conducted by Quishida et al. [17], the multispecie biofilm of C. albicans, C. glabrata and S mutans incubated with PDZ (100, 150, 175 or 200 mg/L) and irradiated with LED light (37.5 Jcm²) presented 36% of reduction in metabolic activity when compared with the control group. With regard to the colony quantification test (CFU/mL) in the present study, it was observed that immediately after aPDT, compared with the control group (P-L-CaS or P-L-CaR), the biofilms submitted to aPDT presented a reduction equivalent to 1.20 and 1.14 log10, for CaS and CaR, respectively. These results are promising, since the pattern of inactivation observed for the susceptible and resistant strains were similar. Studies have demonstrated that aPDT promoted the inactivation of suspensions of fluconazole-resistant C. albicans; however different FSs were used. Paz-Cristobal et al. [25] evaluated the effectiveness of aPDT, 32 mediated by hypericin or dimethyl methylene blue, for the inactivation of suspensions of clinical isolates of CaS and CaR and observed complete inactivation of the suspensions. In the study conducted by Chien et al. [26], chitosan was used to enhance the efficiency of aPDT (mediated by toluidine blue) against clinical isolates of CaS and CaR and they demonstrated that 30 minutes of incubation with chitosan combined with aPDT promoted photoinactivation of the strains in suspension. Mang et al. [27], demonstrated that suspensions of clinical isolates of antifungal resistant Candida spp. presented equivalent susceptibility to aPDT mediated by Photofrin as reference strain. In the study of Dovigo et al. [13], suspensions and biofilms of resistant and standard strains of C. albicans and C. glabrata were treated with Photogem, followed by irradiation with LED light. It was observed that the effectiveness of aPDT was dependent on the species and the resistant strains were less susceptible to the effects of aPDT. The authors also observed that the strains organized as biofilms were less susceptible to aPDT. In general, these studies demonstrated that aPDT has the capacity for inactivating C. albicans in suspension; however, complete inactivation of this yeast organized as biofilm continues to be an important challenge. The capacity for adhesion of both strains was evaluated after the application of the aPDT treatments, by means of the XTT and CFU/mL tests. The adhesion of the microorganisms to biotic or abiotic surfaces is the first step of colonization and subsequent infection and, therefore adhesion is considered an important virulence factor [28]. According to the pertinent literature, this is the first study that evaluated the capacity of adhesion of fluconazole-susceptible and -resistant C. albicans to an abiotic surface after the application of aPDT. The results of the XTT assay demonstrated that aPDT reduced the capacity of adhesion of cells and there was significant difference between CaS and CaR (only 15.8 and 13% of the CaS and CaR, respectively, were capable to adhere to the polystyrene plates). However, the tendency of reduction observed in XTT was not observed in the CFU/mL counts, since it was not possible to verify significant difference in the capacity for adhesion between the cells treated with aPDT in comparison with the other groups. These findings corroborate the results obtained by Soares et al., who evaluated the effect of aPDT mediated by toluidine blue on the capacity of adhesion of Candida species to bucco-epithelial cells (BEC) [23]. These authors observed that the greater the effectiveness of aPDT against the Candida species, the greater was the reduction in adhesion of the yeast to the bucco- epithelial cells. Two isolates of fluconazole-sensitive C. albicans presented elevated reduction in the number of viable cells (mean of 5.20 log10), and the adhesion of these strains to the BECs was also significantly inhibited (mean reduction of 61.5%). On the other hand, C. albicans isolate that presented the lowest reduction in cell viability (mean of 2.15 log10) demonstrated lower inhibition for adhesion to the BECs (mean reduction of 34.5%). Furthermore, one application of aPDT reduced 3.54 and 1.95 log10 of clinical isolates of fluconazole resistant C. albicans and C. tropicalis, respectively, and adhesion to the BECs was reduced by around 61% and 66%, respectively. These authors suggested that the change in the capacity of adhesion might be the effect of aPDT on the cytoplasmic membrane of the Candida spp. cells [23]. Another virulence factor evaluated was the capacity of biofilm formation of C. albicans after aPDT. According to the results obtained in the colony quantification tests (CFU/mL), aPDT was capable to reduce the capacity of both strains of C. albicans to form biofilm (16.1 and 13.5% for P+L+CaS and P+L+CaR groups, respectively). Moreover, CaR strain presented lower biofilm formation ability in comparison with the CaS strain. This result is in agreement with the study conducted by Rosseti et al., who also observed that aPDT, mediated by toluidine blue, reduced the capacity of C. albicans to form biofilm [29]. Furthermore, the authors demonstrated 33 that aPDT promoted an increase in ROS production and cell permeability. According to the authors, ROS production and increase in the cell permeability caused by aPDT were responsible for inhibiting the cell proliferation and formation of biofilms. Therefore, it could be suggested that cells that survive to aPDT are more susceptible to future treatments. Another important factor that must be observed is the result obtained in the total biomass test, by means of crystal violet staining. Immediately after application of the treatments, aPDT reduced the total biomass of CaR by 23.1% in comparison with the control, and there was no reduction in the biomass of CaS. After application of the treatments, the surviving cells were submitted to the procedures of biofilm formation and total biomass was measured again and no difference was verified between the strains that received treatments with aPDT and their respective control groups. However, it is important to point out that the total biomass value of the CaS was higher in all the periods evaluated in comparison to the values of the CaR. These findings do not corroborate those found in the literature. Vavala et al. [30] characterized biofilms (24, 48, and 72 hours) of clinical isolates of susceptible and resistant C. albicans and verified that the resistant strains presented 50% higher total biomass values than the susceptible strain. The differences between the results obtained in the these studies may be attributed to C albicans strain evaluated, because in the present study reference strains (ATCC) were used, while Vavala et al. [30] evaluated clinical isolates. The results of the present study also demonstrated that aPDT changed the capacity of both strains evaluated for secreting Proteinase in comparison with the control group (P-L-). Moreover, CaR presented lower production of this enzyme in comparison with the CaS. As regards Phospholipase, it was also observed that the groups treated with aPDT differed statistically from the control groups and the CaR presented lower ability to secrete this enzyme. However, it was observed a positive correlation between enzyme production and the values of Log10(CFU/mL) (r = 0.315 for proteinase, and r = 0.338 for phospholipase), which means that the production of the enzymes was influenced by the quantity of viable cells. It is important to emphasize that, although the Correlation Coefficient of Proteinase and Phospholipase were positive and significant, the values obtained show a very weak correlation (r=0.3), for this reason, these results should be read carefully. These results partially corroborate the data obtained by Martins et al. [31] who evaluated the effects of aPDT on the capacity of C. albicans for producing phospholipase and proteinase in a murine model of oral candidosis, when methylene blue was used in association with laser light. These authors observed the inhibitory effects of aPDT on the capacity of C. albicans to secrete proteinase; however, this treatment was not capable to reduce the secretion of phospholipase. The in vitro study conducted by Freire et al. [32] evaluated the effects of aPDT mediated by methylene blue and laser light on the expression of genes of C. albicans biofilms responsible for the enzymes proteinase (SAP5) and phospholipase (PLB2), by qPCR technique. The authors observed that aPDT was capable to reduce 60% of the expression of SAP5 gene and 50% of the expression of PLB2 gene in the samples; however, there was no significant difference when compared with the group of non-treated biofilms [32]. In addition to these enzymes produced by Candida, the activity of the lipase enzyme was also evaluated by Freire et al. [32] after the application of aPDT mediated by methylene blue. The cited authors observed that aPDT reduced by 50% the expression of the gene responsible for codifying this enzyme (LIP9) in C. albicans. However, it's important to evaluate if the reduction in the enzyme production is not associated with the reduction of cell viability after aPDT. Therefore, in the present investigation a correlation between cell viability and enzyme production was performed and showed a positive correlation. 34 The production of proteinase and phospholipase enzymes by CaS was greater in comparison with that of the CaR. Some studies have shown an increase in the production of proteinase by CaR after the use of sub- inhibitory concentrations of amphotericin B. Kumar et al. [33] verified higher proteinase enzyme production in strains resistant to amphotericin B compared with susceptible strains. Similarly, studies have verified that phospholipase secretion by strains resistant to medications is higher [34,35]. Our results do not corroborate with those of these studies, once all of these studies used clinical isolates strains of Candida spp., whereas in the present study a standard strain (ATCC) of fluconazole-resistant C. albicans was used. We can hypostasize that the resistance of the Candida strain evaluated in this study is not related to the virulence factors (proteinase and phospholipase), however it could be associated with efflux pumps and other resistance mechanisms (ergosterol synthesis). Apart from these virulence factors assessed in the present study, the effects of aPDT on the expression of other pathogenicity factors of C. albicans have been evaluated. The transition of C. albicans into hyphae is responsible for the invasion of this fungus into the epithelial tissue, favoring penetration and growth of this microorganism among epithelial cells. The germinative tube is the first stage of the morphological transition from blastopore to hypha [36]. Munin et al. [24], observed that aPDT mediated by methylene blue and laser light reduced the formation of C. albicans hyphae. Whereas, Kato et al. [37], observed that aPDT mediated by methylene blue inhibited the formation of germinative tubes; however, the authors considered this effect transitory, since this alteration was not observed in the daughter cells. Other studies have also evaluated the effects of aPDT on virulence factors of bacteria [38-40]. aPDT mediated by toluidine blue reduced the activity of lipopolysaccharides (LPS) and proteases of the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa [38], as well as the activity of proteolytic enzymes of suspensions of Porphyromonas gingivalis [39]. Similarly, aPDT mediated by methylene blue was capable of inhibiting the activity of enzymes V8 protease, α- haemolysin, phingomyelinase of the Staphylococcus aureus [40]. Conclusion Based on the results obtained in the present study, the authors could conclude that aPDT reduced the viability of both strains, fluconazole-susceptible and -resistant C. albicans. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability, and enzymatic production. Acknowledgements: We thank the Physics Institute of São Carlos (University of São Paulo, São Carlos, SP, Brazil) for designing the LED device used in the present investigation. Funding information: This work was supported by São Paulo Research Foundation (FAPESP – grants number 2012/21201-1 and 2012/02942-0) and Centro de Pesquisa em Óptica e Fotônica (CEPOF – grant number 2013/ 07276-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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Lasers Med Sci 15:24-30. 40. Tubby S, Wilson M, Nair SP (2009) Inactivation of staphylococcal virulence factors using a light- activated antimicrobial agent. BMC Microbiol 9:211. 10.1186/1471-2180-9-211 38 3.2 Publicação 2 A Randomized clinical trial evaluating Photodithazine-mediated Antimicrobial Photodynamic Therapy as a treatment for denture stomatitis. Fernanda Alves a , MSc. Gabriela Caroline Alonso a , DDs. Juliana Cabrini Carmello a , Ph.D. Ewerton Garcia de Oliveira Mima a , Ph.D. Vanderlei Salvador Bagnato b , Ph.D. Ana Cláudia Pavarina †a , Ph.D. a Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, Rua Humaitá, 1680, 14801-903, Araraquara, SP, Brazil. b Biophotonics Lab, Group of Optics, Physics Institute of São Carlos, University of São Paulo - USP, Av. Trabalhador São-carlense, 400, 13566-590, São Carlos, SP, Brazil. † Corresponding Author: Dr. Ana Cláudia Pavarina e-mail: pavarina@foar.unesp.br Phone: +55 16 33016544 Fax: +55 16 33016406 Address: Humaitá Street, 1680, ZIPCODE 14801-903, Araraquara, SP, Brazil. *Artigo a ser submetido na revista Medical Mycology. 39 Abstract This randomized clinical trial assessed the ability of antimicrobial Photodynamic Therapy (aPDT) mediated by Photodithazine (PDZ) to treat patients with denture stomatitis (DS). Sixty-five patients with DS were selected and randomly assigned to the two study groups: aPDT group (n=30) and Nystatin group (NYS, n=35). Patients of the aPDT group were submitted to 6 aPDT sessions, 3 times a week for 15 days, which consisted in the topical application of PDZ 200 mg/L for 20 min on the palate and upper denture of patients and, then, irradiated with red LED light (660 nm – 50 J/cm²). Patients of the NYS group received Nystatin medication (oral suspension). For the antifungal application, patients rinsed 1 dropper of the suspension for 1 minute before being discarded, 4 times a day, for 15 days. Microbiological collections of dentures and palates were performed and cultured on blood agar and chromagar Candida. The values of colony forming units per milliliter (CFU/mL) were determined. Standardized photographs of the palates were taken at the baseline (initial), at the end (final), and on days 15, 30 and 45 after the completion of treatments. Data was analysed by Repeated Measure Linear Model test and Bonferroni (p≤0.05). aPDT was more effective to reduce the total microbiota present in the palate and denture than NYS. Moreover, aPDT was as effective as NYS to reduce Candida spp. and to improve the oral lesions. However, the recurrence of DS was observed in both groups. In conclusion, PDZ-mediated aPDT is a promising treatment for DS. Key words: antimicrobial photochemotherapy; candida; oral candidiasis 40 Introduction Longer life expectancy has led to an increase in the number of elderly population worldwide. Consequently, the number of people requiring removable dentures is increasing proportionally 1 . The lack of appropriate manual dexterity and the rising number of immunocompromised patients (patients under cancer treatment, human immunodeficiency virus-infected patients) increase the susceptibility of the elderly people to opportunistic oral mucosal infections 2 . Besides that, the tissue surface of the acrylic resin denture acts as a reservoir which protects microorganisms and facilitates the development of a complex community of mixed microorganisms, called biofilm 3 . Denture stomatitis is the most common denture-related infection of the oral cavity, present in up to 65% denture wearers and the fungus Candida albicans is the main etiological agent. C. albicans is an opportunistic fungal pathogen found in the oral flora of healthy patients and may cause superficial or serious invasive infections. Other Candida species have also been isolated from patients with DS, such as Candida glabrata, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida kefyr 4 . Although most of the DS cases are asymptomatic, various symptoms such as burning, painful sensation, change of taste and swallowing difficult may be reported 6 . Based on clinical criteria, DS can be classified as type I, II and III, according to Newton 7 . The DS type I is characterized by localized simple inflammation or pin-point hyperemia. Type II, the most common type, is presented as diffuse erythema and edema of palatal mucosal areas covered by dentures. Type III is described as granular surface or inflammatory papillary hyperplasia in central palate 7 . The available treatments for Candida-associated DS currently are topical and systemic. For mild cases of DS, the prescription of topical antifungal agents, such as Nystatin, have been indicated 8 . For immunocompromised patients or at risk for disseminated candidiasis, systemic antifungal therapy, e.g. azoles, have been used 9 . Besides that, denture hygiene should be evaluated and reinforced, dentures should be checked for proper fitting and the age of the denture should be considered when treating DS 8 . Nevertheless, the increased use of azoles, combined with several cases of treatment failures has led to antifungal drug resistance 10 . Clinical resistance to antifungal medications is defined as failure of an antifungal treatment, which results in persistence or progression of the infection 11 . A promising approach to treat DS is Antimicrobial Photodynamic Therapy (aPDT). The basis of aPDT requires microbial exposure to a photosensitizer (PS), followed by visible light energy that is able to excite the PS resulting in the production of singlet oxygen and 41 other reactive oxygen species that react with intracellular molecules and, consequently, produce cell death 12 . The photosensitizer Photodithazine (PDZ) is a chlorine e6 derivative extracted from Spirulina platensis, which has absorption bands in the red region of the spectrum (660 nm), which facilitates the penetration into tissues 13 . The treatment of DS through PDZ-mediated aPDT have not been evaluated yet. However, previous studies showed the effectiveness of this treatment in the management of induced oral candidosis in mice. Carmello et al. 14 observed that the application of 100 mg/L of PDZ and 37.5 J/cm 2 of LED light was able to reduce 4.36 log10 in the viability of C. albicans present on the tongue of mice with oral candidosis. In another study, five applications of PDZ-mediated PDT were as effective as Nystatin for the treatment of experimental oral candidosis in mice 15 . Both studies concluded that aPDT mediated by PDZ may be safely applied, once any alteration on the tongue tissue was verified. For this reason, the present randomized clinical trial study evaluated the effectiveness of aPDT mediated by PDZ for the treatment of patients with denture stomatitis. Materials and Methods Ethics Committee Acceptance This randomized clinical trial was approved by the Ethics Committee of the School of Dentistry, Araraquara, São Paulo State University (Permit number – CAAE: 23558614.8.0000.5416 - ANEXO A), and was performed in compliance with the Declaration of Helsinki. All individuals were informed about the aim of the present investigation, about all procedures that would be performed and about the benefits and risks. Then, all selected participants signed voluntarily the informed consent form (Ficha clínica: APÊNDICE A, Termo de consentimento: APÊNDICE B). Criteria and randomization Edentulous denture wearers who frequent the School of Dentistry – Araraquara or lived in the São Francisco Nursing Home - Araraquara were clinically evaluated for DS diagnostic. The DS was classified based on the Newton’s criteria (type I, II, III) 7 . Medical and additional information about each individual were collected during the enrolment. The following predisposing factors for Candida colonization were considered as exclusion criteria: patients who received antibiotics, antifungal or steroids in the past 3 months prior to the beginning of the research, women in the reproductive phase, patients who had worn the 42 same denture in the past 10 years, diabetics, anemics, immunocompromised and under cancer treatment. Some risk factors were considered and they were equally distributed into the two groups of study: degree of inflammation (DS type I, II or III), demographic characteristics (age and gender), denture (age, support, retention, stability and fitting), denture hygiene habits, nocturnal wearing of the denture, xerostomia, smoking habit and the use of medication. Thus, the study groups were similar and homogeneous at the baseline. Sixty-five patients were selected and randomly assigned to the two study groups. Photosensitizer and light sources The chlorin e6 derivative, Photodithazine ® (PDZ), produced in Russia by VETA- GRAND Co., was used as PS. The PDZ was formulated in Natrosol hydrogel (Pharmacy Santa Paula, Araraquara, SP, Brazil) at the concentration of 200 mg/L. The hydrogel formulation was chosen with the aim to facilitate the clinical application of the PS. The absorption bands of PDZ is shown in Figure 1. Two light emitting diode (LED) devices in the red region of the spectrum (peak at 660 nm) were designed by the Instituto de Física de São Carlos (Physics Institute, University of São Paulo, São Carlos, Brazil). The light apparatus used to irradiate the denture was a biotable composed of 24 LEDs with a light intensity of 50 mW/cm² (Figure 2A). The light device used to illuminate the patient’s palate was composed of 10 LEDs, with a light intensity of 240 mW/cm², in a circular shape (Figure 2B). Both LED devices had an air cooler to avoid heating. Besides that, the LED apparatus of the palate had a Peltier in order to dissipate the heat created by the LED light. The amount of energy applied to irradiate the patient’s denture and palate, in other words, the light dose (J/cm²) was determined by the output power of the LED apparatus (I) and the illumination time. The light dose of 50 J/cm² was selected to irradiate both palate and denture. The following formula was used to calculate the time of illumination: Light dose (J/cm²) = I (W/cm²) x t (sec.). For this reason, taking in account the light dose and the output power of each LED device, the palate was illuminated for 4 minutes and the denture was irradiated for 17 minutes. Treatment of DS Prior to the beginning of the treatments, every patient received instructions about oral hygiene (brush their dentures with soft toothbrush and toothpaste after every meal and before 43 going to sleep). All volunteers were also instructed to remove their denture before going to sleep and to immerse it in filtered water overnight. All these recommendations were given at the baseline and at the follow-up period (days 15, 30 and 45) time intervals. The individuals selected were allocated in one of the two groups of treatment: Nystatin (NYS, n=35) or aPDT (n=30). Patients belonging to the NYS group received the topical Nystatin-based antifungal medication (oral suspension). For the antifungal application, patients were instructed to remove their dentures from the oral cavity, then rinse the volume of 1 dropper (100,000 UI/mL) of the suspension for 1 minute before being discarded. The application of the medication was carried out 4 times a day, for a period of 15 days 16 . Patients belonging to the aPDT group had his/her maxillary denture and palate individually submitted to aPDT. The denture was incubated with the PDZ gel at 200 mg/L in the dark for 20 minutes 14,15 . After this period, the denture was positioned inside the LED biotable and illuminated for 17 minutes (50 J/cm 2 ). The palate was also incubated with the PDZ gel for 20 min 14,15 . After that, the palate was irradiated with the other LED device. The LED apparatus was handled by the operator and positioned inside the patient’s mouth (Figure 2). The palate was illuminated for 4 minutes (50 J/cm 2 ). aPDT was performed 3 times per week, for 15 days (6 sessions) in each patient 16 . Evaluation of the treatments To document the clinical progression of the oral lesion before and after the treatments, standardized photographs of the palates were taken at the baseline (initial), at the end (final) and at the follow-up time intervals: 15, 30 and 45 days after the end of the treatments. All the photographs were taken with the same digital camera (NIKON D7000, Tokyo, Japan), by the same operator, under the same conditions (place, light, position). After that, all photographs were analyzed by two individuals blinded to the treatment performed, in order to classify the oral lesion in type I, II or III in all time intervals. The efficacy of the treatments was also verified microbiologically at the baseline (initial), at the end (final) and at the follow-up time intervals: 15, 30 and 45 days after the end of the treatments. For each patient, oral swabs were rubbed onto the palatal mucosa and on the tissue surface of the denture for 1 minute to recover the microorganisms. Each swab was placed into a falcon tube containing 5 mL of 0.9% sterile saline solution and vortexed for 1 minute to suspend the microorganisms from the swab. To evaluate Candida spp. survival and to presumptively identify Candida species by color, serial 10-fold dilutions from 10 0 to 10 1 were plated in duplicate on Chromagar Candida (Probac do Brasil Produtos Bacteriológicos, 44 São Pa