Surveillance of human papillomavirus through salivary diagnostics - A roadmap to early detection of oropharyngeal cancer in men.

Human papillomavirus (HPV) is the most common sexually transmitted disease. Certain strains have the potential to cause malignancy in multiple anatomical sites if not cleared by the immune system. In most infected people, HPV is cleared within two years. However, HPV may persist in susceptible individuals with certain risk factors, eventually leading to malignancy. New evidence suggests that over 75% of all oropharyngeal cancers (OPC) are directly attributable to HPV. It is estimated that prophylactic HPV vaccination alone may take at least 25 years to have a significant impact on reducing the incidence of OPC. The temporal link between detection of oral HPV, persistence of the infection and the subsequent development of OPC have been well established. Moreover, men have threefold higher risk than women for acquiring HPV-OPC. This comprehensive review focuses on OPC development in men, highlighting the risk factors associated with malignant transformation of HPV-OPC. Current evidence is insufficient to determine whether early identification of at-risk demographics, screening, and prompt diagnosis result in improved outcomes. Hitherto, the effectiveness of an oral HPV screening program in this regard has not been investigated. Nevertheless, the potential to emulate the success of the cervical screening program remains a very real possibility.

Associations between salivary cytokines and periodontal and microbiological parameters in orthodontic patients.

ABSTRACT: Orthodontic treatment can lead to microbial-induced gingival inflammation and aseptic periodontal inflammations. The aim of this study was to investigate the relationship between salivary pro-inflammatory cytokines levels with gingival health status and oral microbe loads among patients undergoing orthodontic treatment.The present investigation was a cross-sectional study among a sample of 111 consecutive orthodontic patients (mean age 18.4 ±â€Š4.4 years). Clinical examinations were conducted to assess the gingival health status employing the Modified Gingival Index, Gingival Bleeding Index, and Plaque Index. Salivary microbiological assessments of total aerobic and anaerobic bacteria count, streptococci count, and lactobacilli count were undertaken. Saliva immunological assessments included Interleukin-1Beta (IL-1ß) and macrophage migration inhibitory factor (MIF) ELISA assays.The mean ±â€Šstandard deviation of salivary IL-1ß was 83.52 ±â€Š85.62 pg/ml and MIF was 4.12 ±â€Š0.96 ng/ml. Moderate positive correlations were found between salivary IL-1ß levels and total aerobic and anaerobic bacteria count, streptococci count, and lactobacilli count (r = 0.380-0.446, P < .001), and weak positive correlations between salivary MIF levels and total salivary aerobic and anaerobic bacteria counts (r = 0.249-0.306, P < .01) were observed. A positive correlation was found between salivary IL-1ß levels and Bleeding Index (r = 0.216, P < .05).The level of salivary IL-1ß positively correlates with oral bacterial load among orthodontic patients; the relationship between inflammatory cytokines and oral microflora deserved further study.

Exopolysaccharide matrix of developed Candida albicans biofilms after exposure to antifungal agents.

This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.

Bioactivity and architecture of Candida albicans biofilms developed on poly(methyl methacrylate) resin surface.

The aim of this study was to evaluate the bioactivity and architecture of Candida albicans biofilms developed on the surface of poly(methyl methacrylate) (PMMA) resin. To do this, surface roughness (SR) and surface free energy of PMMA specimens were measured. Next, the biofilms of two different C. albicans strains (ATCC 90028 and SC5314) were allowed to develop on the PMMA surface and evaluated at 24, 48, and 72 h after adhesion. The bioactivity of the biofilms was measured by the XTT reduction assay. Biofilm topography was evaluated by scanning electron microscopy. Confocal microscopy was used to evaluate the architectural properties of bio-volume, average thickness, biofilm roughness, surface area/volume ratio and the proportion of live/dead cells in the different biofilm development stages. SR and SFE had no influence on biofilm development. Each strain exhibited a different biofilm activity (P < 0.001). Confocal images showed different architectures for the different biofilm development stages. We conclude that the main differences detected in biofilm bioactivity and architecture were related to the characteristics of each C. albicans strain and to biofilm development time.

Improvement of XTT assay performance for studies involving Candida albicans biofilms.

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

Exopolysaccharide matrix of developed candida albicans biofilms after exposure to antifungal agents

This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.

Este estudo avaliou o efeito da exposição de fluconazol ou nistatina a biofilmes de Candida albicans desenvolvidos, considerando a matriz de polissacarídeos extracelulares. Inicialmente uma cepa referência de C. albicans (ATCC 90028) foi submetida ao teste de concentração inibitória mínima (CIM) utilizando-se o fluconazol ou nistatina como agentes antifúngicos. Após, espécimes foram confeccionados em resina acrílica de polimetilmetacrilato (PMMA) de acordo com as recomendações do fabricante e tiveram sua rugosidade de superfície padronizada. Após, biofilmes de C. albicans foram desenvolvidos na superfície dos espécimes durante 48 h. Em seguida, os biofilmes foram expostos a fluconazol ou nistatina nas concentrações de CIM, 10 x CIM ou 100 x CIM, por 24 h. A atividade metabólica dos biofilmes foi avaliada pelo teste de XTT. A produção de polissacarídeos extracelulares solúveis e insolúveis, bem como dos polissacarídeos intracelulares foi avaliada pelo método fenol-sulfúrico. A arquitetura dos biofilmes e proporção de células vivas e mortas foi investigada utilizando-se microscopia confocal a laser. Os resultados foram analisados por ANOVA seguido do teste de Tukey, utilizando-se o nível de significância de 5%. A presença do fluconazol ou nistatina em concentrações maiores que CIM resultaram em uma redução significativa da atividade metabólica (p<0,001). Nas concentrações de CIM e 10 x CIM, biofilmes expostos ao fluconazol apresentaram quantidades significativas de polissacarídeos intracelulares (p<0,05), enquanto não houve alterações na quantidade de polissacarídeos extracelulares (p>0,05). A presença de nistatina também não alterou a matriz de polissacarídeos extracelulares em todas as concentrações investigadas (p>0,05). A arquitetura dos biofilmes não foi afetada por ambos os agentes antifúngicos, em qualquer concentração testada (p>0,05). A nistatina apresentou maior proporção de células mortas (p<0,05). Conclui-se que tanto para o fluconazol quanto para a nistatina, concentrações maiores que CIM reduziram a atividade metabólica dos biofilmes de C. albicans; no entanto, tais concentrações não alteraram a matriz de polissacarídeos extracelulares nem a arquitetura dos biofilmes.

Improvement of XTT assay performance for studies involving Candida albicans biofilms

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay has been used to study Candida biofilm formation. However, considering that the XTT reduction assay is dependent on cell activity, its use for evaluating mature biofilms may lead to inaccuracies since biofilm bottom cell layers tend to be relatively quiescent at later stages of biofilm formation. The aim of this study was to improve XTT reduction assay by adding glucose supplements to the standard XTT formulation. Candida albicans ATCC 90028 was used to form 24-, 48- and 72-h biofilms. The oxidative activity at 90, 180 and 270 min of incubation was evaluated. The control consisted of standard XTT formulation without glucose supplements, and was modified by the addition of 50, 100 and 200 mM of glucose. The XTT assay with 200 mM glucose showed more accurate and consistent readings correlating with biofilm development at 24, 48 and 72 h. Biofilm growth yield after 180 min incubation, when evaluated with the 200 mM glucose supplemented XTT, produced the most consistent readings on repetitive testing. It may be concluded that glucose supplementation of XTT could minimize variation and produce more accurate data for the XTT assay.

O teste de redução do 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) tem sido utilizado para mensurar o desenvolvimento de biofilmes de Candida. Contudo, a reação de XTT é dependente da atividade celular e o seu uso para biofilmes maduros pode ser questionado, considerando que diferentes camadas celulares têm atividade metabólica diferenciadas. O objetivo deste estudo foi avaliar se a adição de glicose à formula de XTT diminuiria a variabilidade na mensuração da atividade metabólica. Biofilmes de Candida albicans ATCC 90028 com tempos de crescimento de 24, 48 e 72 h foram utilizados. Para avaliar o melhor tempo de incubação do XTT, este foi mantido a temperatura de 37 °C, em tempos de 90 180 e 270 min. A fórmula padrão do teste XTT (controle) foi modificada com a adição de 50, 100 e 200 mM de glicose para os grupos experimentais. Os melhores resultados para a incubação foi observado com tempo de 180 min e para a suplementação de glicose à concentração de 200 mM (p<0.001). Concluiu-se que a incubação de 180 min utilizando a suplementação de 200 mM de glicose apresenta resultados de atividade metabólica celular com a menor variação para o estudo de biofilmes de Candida albicans.