Investigação da presença do bacilo Mycobacterium tuberculosis em amostras sugestivas de tuberculose oral / Investigation of the presence of Mycobacterium tuberculosis bacillus in samples suggestive of oral tuberculosis

A Tuberculose (TB) é um considerável problema de saúde pública mundial. Em 2021, de acordo com a Organização Mundial de Saúde (OMS) estimou-se que, no mundo, cerca de 10,6 milhões de pessoas desenvolveram TB e 1,4 milhão morreu devido à doença. Com isso, tornou-se a principal causa de morte por infecção em todo o mundo e uma das dez principais causas de morte em geral. A TB tem o pulmão como o principal sítio de acometimento, sendo denominada de TB Pulmonar (TBP). Porém pode ser diagnosticada em muitos órgãos do corpo de maneira Extrapulmonar (TBEP), sendo o linfonodo o local mais comum. Porém, o envolvimento pleural, neurológico, sinovial, pericárdico, abdominal, geniturinário e oral tem sido descrito, o que mostra a potencial capacidade de disseminação do Mycobacterium tuberculosis (MTB). A detecção do Bacilos Álcool-Ácido Resistentes (BAAR), geralmente ocorre pela observação das características microscópicas da morfologia dos tecidos, presença de granulomas com necrose caseosa, histiócitos epitelióides e células gigantes do tipo Langhans, associada à coloração para BAAR, pela técnica de Ziehl Neelsen (ZN). Ademais, investigação por imuno-histoquímica (IHQ), testes de amplificação de ácido nucleico pela Reação em Cadeia da Polimerase Hemianinhada (nested-PCR) e pelo sistema de detecção automatizado GeneXpert® MTB/RIF são métodos aplicados para o diagnóstico da infecção. Com isso, este estudo teve como objetivo investigar a presença do bacilo Mycobacterium tuberculosis em amostras orais em parafina que continham granulomas com necrose caseosa. Ao todo, como critério de inclusão, foram selecionadas biópsias que apresentaram granulomas com necrose caseosa, sugerindo o diagnóstico de TB. Foram excluídas aquelas que após a revisão das fichas e histológicas, não apresentavam os granulomas exibindo necrose caseosa e aquelas que foram de biópsias intraósseas. O M. tuberculosis foi procurado por meio da coloração de ZN, IHC, nested-PCR e ensaios GeneXpert® MTB/RIF. Foram então selecionadas nove amostras com granulomas com necrose caseosa. Houve predominância de indivíduos do sexo masculino (2,5:1), com idade média de 50 anos (±23,08; 19-89), sendo a língua o local anatômico mais afetado (n=4). O bacilo não foi identificado pela técnica de ZN em nenhuma amostra, e a coloração por IHC mostrou um padrão granular grosseiro, sugerindo M. tuberculosis, em três delas. Nested-PCR e os ensaios GeneXpert® MTB/RIF foram positivos em duas e três das amostras, respectivamente. Conclui-se que testes moleculares e IHC podem ser métodos auxiliares úteis para casos suspeitos de tuberculose.

Tuberculosis (TB) is a significant global public health issue. In 2021, according to the World Health Organization (WHO), it was estimated that approximately 10.6 million people developed TB worldwide, and 1.4 million died from the disease. Consequently, it became the leading cause of death due to infection worldwide and one of the top ten overall causes of death. TB primarily affects the lungs and is referred to as Pulmonary TB (PTB). However, it can be diagnosed in various organs of the body as Extrapulmonary TB (EPTB), with lymph nodes being the most common site of involvement. Moreover, pleural, neurological, synovial, pericardial, abdominal, genitourinary, and oral involvement have been described, demonstrating the potential for Mycobacterium tuberculosis (MTB) dissemination. The detection of Acid-Fast Bacilli (AFB) typically involves the observation of microscopic tissue characteristics, the presence of granulomas with caseous necrosis, epithelioid histiocytes, and Langhans giant cells, along with AFB staining using the Ziehl-Neelsen (ZN) technique. Furthermore, immunohistochemistry (IHC), nucleic acid amplification tests by Nested Polymerase Chain Reaction (nested-PCR), and the automated detection system GeneXpert® MTB/RIF are methods employed for diagnosing the infection. Therefore, the aim of this study was to investigate the presence of Mycobacterium tuberculosis in paraffin-embedded oral samples containing granulomas with caseous necrosis. Inclusion criteria were based on the selection of biopsies displaying granulomas with caseous necrosis, suggesting a diagnosis of TB. Biopsies without these features upon review of records and histological findings, as well as intraosseous biopsies, were excluded. M. tuberculosis was sought using ZN staining, IHC, nested-PCR, and GeneXpert® MTB/RIF assays. Nine samples with granulomas and caseous necrosis were selected. The majority of individuals were male (2.5:1 ratio), with an average age of 50 years (±23.08; range 19-89), and the tongue was the most affected anatomical site (n=4). AFB was not identified by the ZN technique in any of the samples, and IHC staining exhibited a coarse granular pattern, suggestive of M. tuberculosis, in three of them. Nested-PCR and GeneXpert® MTB/RIF assays yielded positive results in two and three of the samples, respectively. In conclusion, molecular tests and IHC can be valuable auxiliary methods for suspected cases of tuberculosis.

Oral mucositis and microbial status in acute lymphoblastic leukemia subjects undergoing high-dose chemotherapy

Aim: To assess oral microbial status in patients with acute lymphoblastic leukemia (ALL) undergoing high-dose chemotherapy and to unravel possible associations between nosocomial pathogens and the establishment of chemotherapy-induced oral mucositis (CIOM). Methods: Oral mucosa, saliva, and peripheral blood samples were collected from 46 ALL subjects one day prior to chemotherapy (D0) and 2 weeks after treatment initiation (D14). Clinical intraoral inspection was performed by a single practitioner, with mucositis classification performed according to the WHO oral toxicity scale. Blood components were quantified by automatic flow cytometry, while oral Staphylococcus aureus and Pseudomonas aeruginosa were detected by Polymerase Chain Reaction with species-specific primers. Associations among bacteria and clinical findings were determined by Fisher's Exact test, longitudinal bacterial changes by paired Macnemar, and correlations among blood parameters and mucositis status or bacteria via Mann-Whitney. Results: S. aureus displayed higher detection rates at D14 (p < 0.05) and was positively associated with mucositis, adoption of a non-solid diet (all p < 0.001), nausea and fever (all p < 0.05). Conversely, P. aeruginosa did not correlate to CIOM clinical parameters. At the systemic standpoint, lower hemoglobin levels associated with CIOM and fever events (all p < 0.01). Conclusion: The study evidences S. aureus as a potential pathogen in ALL-CIOM, reaffirming microbial control as an important preventive measure during high-dose immunosuppressive therapy. The weight of non-white-blood-cell parameters should be validated as novel CIOM biomarkers in prospective research

Doença cardiovascular e periodontite: análise de série de casos avaliando níveis de Proteína C-Reativa / Cardiovascular disease and periodontitis: case series analysis evaluating C-Reative Protein levels

As periodontopatias podem ser dos principais fatores para o agravamento de doenças, como alterações cardiovasculares. Microrganismos e produtos bacterianos encontrados nessas condições provocam intensa produção de mediadores inflamatórios, incluindo a proteína C-reativa (PCR), marcador para cardiopatias. Avaliou-se a relação dos níveis plasmáticos de PCR em pacientes com doenças cardiovasculares (DCV) e periodontite. Uma série de quatro casos acompanhados/ concluídos na referida clínica (idade entre 24 e 61 anos, todos homens). Constatada a condição de periodontite, o periograma do sextante comprometido foi realizado, junto à requisição de exames para dosagem dos níveis plasmáticos de PCR, antes e após sessão de raspagem e alisamento radicular. Em 21 a 30 dias, um periograma reavaliativo foi realizado para análise comparativa das pro fundidades de sondagem e níveis de PCR antes e após a sessão de instrumentação. Houve redução de 13,7% (paciente "A") e até 2 mm na perda de inserção (pacientes "A" e "C") dos sítios avaliados; não houve nenhuma mudança significativa nas novas dosagens dos níveis de PCR. Conclui-se que não foi possível demonstrar uma correlação entre os níveis de PCR em pacientes que apresentam concomitantemente DCV e periodontite, nesta série de casos... (AU)

Periodontal diseases are one of the main factors for aggravation of diseases, such as cardiovascular alterations. Microorganisms and bacterial products found under these conditions provoke intense production of inflammatory mediators, including C-reactive protein (CRP), a marker for cardiovascular disease. It was evaluated the relationship of plasma levels of CRP in patients with Cardiovascular Diseases (CVD) and periodontitis in the dental clinic of the UPE campus Arcoverde. A series of four cases followed at the clinic (24 to 61 years old, all men). Condition of periodontitis confirmed, the periogram of the compromised sextant was performed, together with the requisition of tests for the determination of plasma levels of CRP, before and after scaling and root planing sessions. Around 21-30 days a re-evaluating periogram was performed for comparative analysis of depths of probing and CRP levels before and after the instrumentation. There was a reduction of 13.7% (patient "A") and up to 2 mm in the loss of insertion (patients "A" and "C") of the evaluated sites; there wasn't significant change in the new doses of CRP levels. It's concluded was not possible to demonstrate a correlation between CRP levels in patients with concomitant CVD and periodontitis in this case series... (AU)

Caracterização genotípica de amostras clínicas de Cryptococcus Spp. e avaliação do perfil de virulência in vitro e em modelo de Galleria Mellonella / Genotypic characterization of clinical samples of Cryptococcus spp. and evaluation of the virulence profile in vitro and in a Galleria mellonella model

A criptococose é uma infecção oportunista comum em pacientes HIV positivos, mas também pode afetar pacientes com outras comorbidades imunológicas ou imunocompetentes. Neste trabalho, cepas clínicas de Cryptococcus spp. provenientes de hospitais de São José dos Campos (SP) foram identificadas pela amplificação do gene SRT. A seguir, foi avaliada a sensibilidades das cepas a fluconazol e anfotericina B (AMB) pela técnica de microdiluição conforme EUCAST. Também foi analisada a capacidade de formação de biofilme por cristal violeta (formação da biomassa) e XTT (viabilidade celular). Foram analisadas in vivo a formação da cápsula durante a infecção, a curva de sobrevivência e do índice de saúde em modelo invertebrado de Galleria mellonella. Seis cepas foram identificadas como C. neoformans var. grubbii e uma como C. gattii. Todas as cepas clínicas foram sensíveis a AMB e a concentração inibitória mínima para fluconazol variou entre 2 e 32 µg/mL. Todos os isolados de Cryptococcus spp. foram capazes de produzir biofilme. A sobrevida de G. mellonella infectada variou entre 6 e 7 dias para C. neoformans, e a cepa clínica 6 se mostrou menos virulenta, com 24% das larvas vivas no último dia de experimento. Para cepa de C. gattii, 20,8% das larvas infectadas permaneceram vivas ao final da análise. Em relação a análise do tamanho da cápsula após inoculação in vivo, as cepas clínicas de ambas espécies apresentaram aumento no tamanho, sendo observado o maior percentual de cápsulas ≥ 30µm na cepa 6. O índice de saúde foi capaz de agregar informações ao resultado da curva de sobrevivência de G. mellonella, uma vez que ele mostrou diferenças de saúde das larvas entre os grupos que apresentaram mesmo perfil de sobrevida. A associação do índice de saúde à curva de sobrevivência amplia a visão da vitalidade das larvas durante os dias de experimento e, ainda, auxilia na comparação de resultados entre laboratórios. O conhecimento das características genotípicas e fenotípicas de Cryptococcus spp. em uma determinada região pode ser ferramenta importante para implementação de políticas públicas de saúde eficazes. (AU)

Cryptococcosis is a common opportunistic infection in HIV-positive patients, but it can also affect patients with other immunological comorbidities or immunocompetent. In this work, clinical strains of Cryptococcus spp. from hospitals in São José dos Campos (SP) were identified by amplification of the SRT gene. Next, the sensitivities of the strains to fluconazole and amphotericin B (AMB) were evaluated by the microdilution technique according to EUCAST. Biofilm formation capacity by crystal violet (biomass formation) and XTT (cellular viability) was also analyzed. Capsule formation during infection, survival curve and health index in an invertebrate model of Galleria mellonella were analyzed in vivo. Six strains were identified as C. neoformans var. grubbii and one as C. gattii. All clinical strains were sensitive to AMB and the minimum inhibitory concentration for fluconazole ranged between 2 and 32 µg/mL. All Cryptococcus spp. were able to produce biofilm. The survival of infected G. mellonella ranged between 6 and 7 days for C. neoformans, and the clinical strain 6 was less virulent, with 24% of larvae alive on the last day of the experiment. For the C. gattii strain, 20.8% of the infected larvae remained alive at the end of the analysis. Regarding the analysis of capsule size after in vivo inoculation, the clinical strains of both species showed an increase in size, with the highest percentage of capsules ≥ 30µm being observed in strain 6. The health index was able to add information to the result of the survival curve of G. mellonella, since it showed differences in the health of larvae between the groups that presented the same survival profile. The association of the health index to the survival curve broadens the vision of the larvae's vitality during the days of the experiment and also helps in comparing the results between laboratories. The knowledge of the genotypic and phenotypic characteristics of Cryptococcus spp. in a given region, it can be an important tool for the implementation of effective public health policies (AU)

Presence of Type 1 Collagen Alpha-2 (COL1A2) (rs42524) Gene Polymorphism and Scar Tissue Formation in Different Areas of Head and Neck

Abstract Objective: To determine the effect type I collagen gene polymorphism alpha-2 (COL1A2) (rs42524) on the formation of scar tissue that is localized in the head and neck areas. Material and Methods: Sixty patients with scars in different areas of the head and neck were examined. The patients were divided into four subgroups, according to the types of scarring: G I: 15 patients with normotrophic scars; G ІІ: 15 patients with atrophic scars; G ІІІ: 15 patients with hypertrophic scars; and G IV: 15 patients with keloid scars. The age of patients ranged from 17 to 54 years. The single-nucleotide polymorphic site of the COL1A2 (rs42524) gene was detected by a polymerase chain reaction and subsequent analysis of restriction fragment lengths. Pearson's chi-squared test with Yates's correction and Fischer's exact test were used. Results: There were no significant changes between the control and basic groups (p=0.83) at analyzing the frequencies of G and C alleles. For the G allele, the calculation of odds ratio between the basic and control groups was 0.93 at 95% confidence interval (CI) (0.50-1.75), for the C allele - OR was 1.07 at 95% CI (0.57-2.01). Conclusion: Our studies may indirectly indicate the activation of the skin's protective reaction to physiological scarring and dosed scar formation in different areas of the head and neck.

Effect of enhanced masticatory force on OPG, RANKL and MGF in alveolar bone of ovariectomized rats

Abstract Menopause induces oral bone loss, leading to various oral diseases. Mastication importantly affects bone metabolism in the jawbone. Objective: To analyze the effect of enhanced masticatory force on osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and mechano-growth factor (MGF) in alveolar bone of ovariectomized rats and to study the mechanics mechanism of the alveolar bone of ovariectomized rats response to enhanced masticatory force. Methodology: Thirty Sprague Dawley rats were randomly divided into three groups: sham-operation group (fat around the removed ovary + normal hard diet), model group (ovariectomy + normal hard diet), and experimental group (ovariectomy + high hard diet). It was a 2-month experiment. Enzyme-linked immunosorbent assay (ELISA) detected serum estradiol (E2), osteocalcin (BGP) and alkaline phosphatase (ALP) in rats. Bone histomorphometric indices in the third molar region of maxilla were detected by micro-CT; protein expressions of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Western blot; and gene expression of OPG, RANKL, and MGF in the third molar region of maxilla was detected by Quantitative Real-Time PCR. Results: Comparing with model group, serum E2 in experimental group increased but not significantly, serum BGP and serum ALP in experimental group decreased but not significantly, OPG in experimental group in alveolar bone increased significantly, RANKL in experimental group in alveolar bone decreased significantly, RANKL/OPG ratio in experimental group decreased significantly, MGF in experimental group in alveolar bone increased significantly, bone volume to total volume fraction increased significantly in experimental group, trabecular thickness increased significantly in experimental group, and trabecular separation decreased significantly in experimental group. Conclusion: Enhanced masticatory force affected the expression of OPG, RANKL, and MGF in alveolar bone of ovariectomized rats, improved the quality of jaw bone of ovariectomized rats, and delayed oral bone loss by ovariectomy.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Risk Factors for Early Childhood Caries Based on Identification of Veillonella spp. Using RT-PCR

Abstract Objective: To identify the occurrence of Veillonella spp. in children using real-time PCR (RT-PCR) and to determine its role as a risk factor for ECC in children aged 2-3 years. Material and Methods: A cross-sectional survey was conducted and samples from 87 children aged 2-3 years, who lived in selected villages in the Bandung City area, Indonesia, were collected. Examination for dental caries was performed using standard checks for decay, missing, and filled surfaces (dfms), and saliva samples were taken. Microbiological examination was performed using RT-PCR with primers consisting of one primary set for Veillonella spp. and one universal primary set for 16S rDNA. We performed statistical testing using the Mann Whitney rank-sum test. Results: A total of 87 children were sampled, and an ECC prevalence of 71.3% was found, with a mean dmfs of 7.1 (± 9.1). The proportion of Veillonella spp. in caries-free children was 2.13 ± 2.30, while in children with ECC, it was 3.29 ± 6.83. Conclusion: The proportion of Veillonella spp. in children with ECC was higher than in caries-free children; therefore, Veillonella spp. may be a risk factor for ECC.

Efeito da irrigação ultrassônica e da medicação intracanal com hidróxido de cálcio na quantidade e no metabolismo de bactérias que persistiram após o preparo dos canais radiculares de dentes com periodontite apical / Effect of ultrasonic irrigation and calcium hydroxide intracanal medication on the levels and metabolism of bacteria that persisted after root canal preparation of teeth with apical periodontitis

Estudos moleculares ressaltam as limitações do protocolo endodôntico tradicional em eliminar bactérias dos canais radiculares. Apesar do preparo químico-cirúrgico (PQC) promover uma drástica redução bacteriana, muitos canais continuam infectados após essa etapa do tratamento. Dessa forma, estudos apontam para a necessidade de complementação técnica para potencializar a desinfecção dos canais radiculares após o PQC. Assim, o objetivo deste estudo clínico foi avaliar, por métodos moleculares baseados em DNA e RNA, o efeito dos métodos complementares ao preparo na desinfecção dos canais radiculares. Coletas microbiológicas dos canais de 20 dentes unirradiculares com periodontite apical foram feitas em diferentes etapas do tratamento endodôntico: previamente ao preparo (S1); após o PQC realizado com sistema Reciproc associado à irrigação com NaOCl 2,5% (S2); após a irrigação ultrassônica passiva, denominada PUI (S3); e após a medicação intracanal à base de hidróxido de cálcio (S4). As amostras foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT-PCR) para confecção da fita dupla de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR, com iniciadores universais para a região 16S rRNA do domínio Bacteria. A atividade metabólica das bactérias foi verificada através da relação entre os níveis de rRNA e rDNA determinados pelos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para amostras pareadas (p < 0,05). As amostras S1 dos 20 casos apresentaram altos níveis de rDNA (mediana: 1,25 x 105, intervalo 1,83 x 104 - 9,2 x 106) e rRNA bacteriano (mediana: 5,47 x 105, intervalo 7,8 x 104 - 5,95 x 107). Dezessete canais (85%) apresentaram reações qPCR positivas para rDNA nas amostras pós-preparo (S2). A redução de rDNA após o preparo foi estatisticamente significativa (p = 0,0003), com mediana de 2,5 x 104 (intervalo 2,26 x 103 - 9,52 x 104) cópias de rDNA em S2. Por sua vez, os níveis de rRNA (mediana: 7,84 x 104, intervalo 2,91 x 103 - 1,09 x 106) foram maiores que os níveis de rDNA (p = 0,01), sugerindo que essas bactérias estavam metabolicamente ativas em S2. Após a PUI, o número de amostras S3 com resultados positivos para rDNA caiu para 12, representando uma redução significativa em relação às amostras S2 (p = 0,008). Além disso, a PUI promoveu uma redução significativa dos níveis de rDNA (mediana 2,94 x 103, intervalo 2,70 x 103 - 1,09 x 105) em relação à amostras S2 (p = 0,01). Na análise baseada em rRNA, os níveis em S3 (mediana: 03 x 104, intervalo 1,82 x 103 - 1,39 x 105) não apresentaram diferença significativa em comparação aos níveis de rDNA (p = 0,07), sugerindo que houve uma redução do metabolismo bacteriano após a PUI. Em S4, o número de casos positivos para rDNA bacteriano (n = 13) e os níveis de rDNA (mediana: 3,73 x 104, intervalo 1,98 x 103 - 3,21 x 105) foram ligeiramente maiores quando comparados aos valores das amostras S3, porém sem diferenças significativas. Entretanto, os níveis de rRNA (mediana: 1,08 x 105, intervalo 3,41 x 103 - 1,60 x 106) foram maiores que os de rDNA (p = 0,02) nas amostras S4, sugerindo que as bactérias retomaram sua atividade metabólica apesar do uso da medicação intracanal. Portanto, foi possível concluir que a irrigação ultrassônica passiva contribuiu para a desinfecção dos canais radiculares, promovendo uma redução do número e do metabolismo de bactérias. Por outro lado, as bactérias persistiram ativas nos canais radiculares após o uso do hidróxido de cálcio como medicação intracanal em dentes com periodontite apical.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Abstract Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Expression of BMP II by human osteoblasts cultivated on dense or porous titanium / Expressão da BMP II por osteoblastos humanos cultivados sobre titânio denso ou poroso

Objective: Modifications of titanium have been described as an important tool improving bone repair and boneimplant contact. The aim of this research was to quantified the expression of the morphogenetic bone protein II (BMP II) produced by human cells with osteoblast differentiation, after cultured over dense or porous samples of pure titanium grade II. Material and Methods: The experimental groups were: control group, dense titanium, porosity of 33.79% and porosity of 41,79% (n=36). The samples were produced by powder metallurgy technique. Mesenquimal steam cells isolated from alveolar bone of healthy donors were stimulated to differentiate, assuming an osteoblastic phenotype, by supplemented medium and plated over the samples. After 7 and 14 days, the RNA was collected to perform reverse transcriptase polymerase chain reaction (RT-PCR) in real time. Data was analysed by t-Student and ANOVA tests. The porosity, the pore morphology and interconnection were evaluated by Scanning Electron Microscopy (SEM). Results: Total porosity (obtained after apply dimensions and density formulas) and surface porosity (SEM) presented significant differencesamong the groups. For the group of total porosity of 33.79%, the superficial porosity was 32.5% (± 7.74%) and for the group of 41.79%, the superficial porosity was 37.4% (± 7.95%), significantly lower. The expression of BMP II was similar in all groups. Conclusion: The present study demonstrated that powder metallurgy has a reduced ability to standardize the porosity in the samples and that the porosity does not interfere in the cellular response of BMP II production, an important inducer of osteoblastic differentiation. (AU)

Objetivo: As modificações do titânio são descritas como importantes ferramentas na melhora do reparo ósseo no contato osso implante. O objetivo deste estudo foi quantificar a expressão da proteína óssea morfogênica II (BMP II) por células humanas com diferenciação osteoblastica, quando cultivadas sobre amostras de titânio puro grau II, denso ou poroso. Material e Métodos: Os grupos experimentais foram: controle, titânio denso, titânio de maior porosidade e titânio de menor porosidade, sendo que, as amostras foram confeccionadas pela técnica da metalurgia do pó. As células isoladas de doadores saudáveis foram plaqueadas sobre as amostras. Após 7 e 14 dias, o RNA foi extraído das células. A qualidade e integridade do RNA foram analisadas qualitativamente por eletroforese e quantitativamente por espectrofotômetro. O cDNA foi confeccionado e a foi utilizada técnica de reação em cadeia da polimerase (PCR) em tempo real. Os dados foram utilizados para quantificação relativa, e o gene constitutivo foi a BetaActina. A morfologia e a interligação dos poros foram comprovadas por Microscopia Eletrônica de Varredura (MEV). Resultados: A porosidade superficial (MEV) teve diferença significativa em relação a porosidade obtida analisando-se volume e massa das amostras. Para o grupo 3,79%, a superficial foi de 32,5% (±7,74%) e para o grupo 41,79% a porosidade superficial foi de 37,4% (±7,95%), significativamente menor. A expressão da BMP II foi semelhante em todos os grupos. Conclusão: Concluiu-se a metalurgia do pó tem reduzida capacidade de padronização da porosidade das amostras por ela confeccionas e que a porosidade não interfere na resposta celular de produção da BMP II, importante indutor de diferenciação osteoblastica.(AU)

Effect of titanium tetrafluoride varnish on fibroblasts (NIH/3T3): viability assay, morphological analysis and cell signals for apoptotic pathways / Efeito do verniz de tetrafluoreto de titânio sobre fibroblastos (NIH/3T3): ensaios de viabilidade, morfologia e sinalização celular para apoptose

Current knowledge supports the application of TiF4 varnish to protect against tooth caries and erosion; however, it is indispensable to know its cytotoxic potential and the mechanism involved on it before applying in patients. Therefore, this study aimed to evaluate 1) The cytotoxic effect of titanium tetrafluoride (TiF4) varnish compared with sodium fluoride (NaF) varnish on murine fibroblast (NIH/3T3), varying the fluoride concentration and time of treatment and 2) The percentage of apoptosis and its mechanism (both mitochondrial mediated by the Bcl-2 family- and death receptorpathways) in human gingival fibroblasts (HGF) and murine fibroblasts (NIH/3T3) treated with TiF4 varnish compared to NaF varnish for 6 h. Step 1) NIH/3T3 were exposed to NaF or TiF4 varnishes containing 0.95, 1.95 or 2.45% F, for 6, 12 or 24 h. MTT viability (n=6) and Hoescht/PI stain assays (n=3) as well as the cells morphology (HE, only for 24 h, n=3) and stiffness (AFM, only for 2.45% F, 6 or 12 h) were analyzed. Both varnishes, at 1.90 and 2.45% F, reduced cells viability by similar extent (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared to control, regardless of the type of fluoride. TiF4 and NaF (2.45% F) reduced cell stiffness to a similar extent, but only TiF4 differed from control. Step 2) HGF and NIH/3T3 were exposed to NaF or TiF4 (2.45% F) varnishes for 6 h. Cells were examined by the TUNEL method using fluorescence microscope. The caspases-3, -8 and -9 activities were assessed. The cDNA for cytocrome c, Bax, Bad, Bcl-2, VDAC-1 and Fas-L was amplified by quantitative PCR (qPCR). Bax, Bcl-2 and Fas-L were further detected by western blot. Both fluorides similarly increased the percentage of apoptosis, while they failed in activating caspases-3, -8 and -9 for both types of cells. Bax/Bcl-2 ratio, cytochrome C and VDAC-1 gene expressions were not altered by both fluoride treatments. However, NaF varnish increased the amplification of Fas-L gene for NIH/3T3 and HGF, while TiF4 varnish induced lower Bad/Bcl-2 ratio expression compared to control for NIH/3T3, but not for HGF. No effect of the fluorides was detected in the proteins analysis. TiF4 and NaF have similar cytotoxicity on NIH/3T3, which is dependent on the F concentration and the exposure time. Both fluorides, at the studied conditions, similarly induce a low percentage of apoptosis, with consequent modest activation of Bcl-2 and Fas-L-dependent signaling pathways.(AU)

Conhecimento atual suporta a aplicação de verniz de TiF4 para proteção contra cárie e erosão dentárias; entretanto, é indispensável conhecer o seu potencial citotóxico e o mecanismo envolvido antes de aplicá-lo em pacientes. Portanto, o objetivo deste estudo foi avaliar 1) o efeito citotóxico do verniz de tetrafluoreto de Titânio (TiF4) comparado ao fluoreto de sódio (NaF), em fibroblastos NIH/3T3, variando a concentração de fluoreto e o tempo de tratamento 2) a porcentagem de apoptose e seus mecanismos (ambos mitocondrial mediado pela família Bcl-2 e pelo receptor de morte celular) em fibroblastos gengivais humanos (FGH) e fibroblastos murinos (NIH/3T3) tratados com verniz de TiF4 comparado com verniz de NaF por 6 h. Etapa 1) NIH/3T3 foram expostos a vernizes de NaF e TiF4 contendo 0,95, 1,95 ou 2,45% F, por 6, 12 ou 24 h. Ensaios de viabilidade por MTT (n=6) e Hoechst 33342/iodeto de propídeo (n=3) bem como a morfologia (HE, apenas para 24 h, n = 3) e a rigidez celular (MFA, apenas para 2,45% F, 6 ou 12 h) foram realizados. Ambos os vernizes com 1,90 e 2,45% F reduziram a viabilidade das células de forma semelhante (33-86% em 6 h, 35-93% em 12 h e 87-98% em 24 h) em comparação com o controle, independentemente do tipo de fluoreto. TiF4 e NaF (2,45%) reduziram de forma similar a rigidez celular, mas somente TiF4 diferiu do controle no período de 6 h. Etapa 2) FGH e NIH/3T3 foram tratadas com verniz de NaF ou TiF4 por 6h. As células foram examinadas pelo método de TUNEL, usando microscopia de fluorescência. A atividade das caspases -3, -8 e -9 foram avaliadas. O cDNA para citocromo C, Bax, Bad, Bcl-2, VDAC-1 e Fas-L foi amplificado e quantificado por PCR em tempo real (qPCR). A expressão das proteínas Bax, Bcl-2 e Fas-L foi quantificada por western blot. Ambos os fluoretos aumentaram de forma semelhante a porcentagem de apoptose, enquanto falharam na ativação de caspases-3, -8 e -9 para ambos tipos celulares. A expressão gênica da relação Bax/Bcl-2, do citocromo C e do VDAC-1 não foram alteradas por ambos fluoretos. No entanto, o verniz NaF aumentou a amplificação do gene Fas-L para ambas as células, enquanto que o verniz TiF4 induziu menor expressão da razão Bad/Bcl-2 em comparação com o controle para NIH/3T3, mas não para FGH. Nenhum efeito foi detectado na análise de proteínas. TiF4 e NaF apresentam citotoxicidade similar em NIH/3T3, a qual é dependente da concentração de F e do tempo de exposição. Ambos os fluoretos, nas condições estudadas, induzem uma baixa porcentagem de apoptose, com consequente modesta ativação das vias de sinalização dependentes de Bcl-2 e Fas-L.(AU)

Dental caries and bacterial load in saliva and dental biofilm of type 1 diabetics on continuous subcutaneous insulin infusion

Abstract Objectives Since most of the studies evaluates diabetics on multiple daily injections therapy and continuous subcutaneous insulin infusion may help gain better metabolic control and prevent complications, the objective of this study was to evaluate the prevalence of dental caries, the unstimulated salivary flow rate and the total bacteria load, Streptococcus spp. levels and Lactobacillus spp. levels in saliva and supragingival dental biofilm of type 1 diabetics on insulin pump. Material and Methods Sixty patients with type 1 diabetes on insulin pump and 60 nondiabetic individuals were included. The dental caries evaluation was performed using ICDAS and the oral hygiene was assessed according to Greene and Vermillion Simplified Oral Hygiene Index. Unstimulated saliva and supragingival dental biofilm were collected. Total bacteria, Streptococcus spp. and Lactobacillus spp. was quantified by qPCR. Results Patients with type 1 diabetes had a higher prevalence of dental caries and filled and missing teeth when compared with the control group. These patients were associated with more risk factors for the development of dental caries, namely a lower unstimulated salivary flow rate and a higher bacterial load in saliva and dental biofilm. Conclusion Some risk factors related to dental caries were associated with type 1 diabetics. An early diagnosis combined with the evaluation of the risk profile of the diabetic patient is imperative, allowing the dental caries to be analyzed through a perspective of prevention and the patient to be integrated into an individualized oral health program.

Microbiological and clinical effects of probiotics and antibiotics on nonsurgical treatment of chronic periodontitis: a randomized placebo- controlled trial with 9-month follow-up

ABSTRACT Objective: The aim of this double-blind, placebo-controlled and parallel- arm randomized clinical trial was to evaluate the effects of Lactobacillus rhamnosus SP1-containing probiotic sachet and azithromycin tablets as an adjunct to nonsurgical therapy in clinical parameters and in presence and levels of Tannerella forsythia, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Material and Methods: Forty-seven systemically healthy volunteers with chronic periodontitis were recruited and monitored clinically and microbiologically at baseline for 3, 6 and 9 months after therapy. Subgingival plaque samples were collected from four periodontal sites with clinical attachment level ≥1 mm, probing pocket depth ≥4 mm and bleeding on probing, one site in each quadrant. Samples were cultivated and processed using the PCR technique. Patients received nonsurgical therapy including scaling and root planing (SRP) and were randomly assigned to a probiotic (n=16), antibiotic (n = 16) or placebo (n = 15) group. L. rhamnosus SP1 was taken once a day for 3 months. Azithromycin 500mg was taken once a day for 5 days. Results: All groups showed improvements in clinical and microbiological parameters at all time points evaluated. Probiotic and antibiotic groups showed greater reductions in cultivable microbiota compared with baseline. The placebo group showed greater reduction in number of subjects with P. gingivalis compared with baseline. However, there were no significant differences between groups. Conclusions: The adjunctive use of L. rhamnosus SP1 sachets and azithromycin during initial therapy resulted in similar clinical and microbiological improvements compared with the placebo group.

The effects of IL-10 gene polymorphism on serum, and gingival crevicular fluid levels of IL-6 and IL-10 in chronic periodontitis

Abstract Objective Anti-inflammatory cytokines play a crucial role in periodontitis by inhibiting synthesis of pro-inflammatory cytokines. The purpose of this study was to evaluate the effect of interleukin-10 (-597) gene polymorphism and genotype distributions on chronic periodontitis (CP) development and IL-6 and IL-10 levels in gingival crevicular fluid (GCF) and serum before and after non-surgical periodontal treatment. Material and Methods The study population consisted of 55 severe generalized CP patients as CP group and 50 healthy individuals as control group. Plaque index, gingival index, probing depth and clinical attachment level were recorded and GCF and blood samples were taken at both the baseline and the sixth week after non-surgical periodontal treatment. PCR-RFLP procedure was used for gene analyses and cytokine levels were measured via ELISA. Results IL-10 genotype distribution was significantly different between CP and control groups (p=0.000, OR:7, 95%CI, 2.83-60.25). Clinical measurements significantly improved in the CP group after periodontal treatment (p<0.05). Periodontal treatment significantly decreased GCF IL-6 and IL-10 levels. No significant difference was found in clinical parameters between IL-10 AA and AC+CC genotypes at both the baseline and the sixth week (p>0.05). Sixth week GCF IL-10 levels were significantly lower in patients carrying IL-10 AC+CC genotype compared to the patients carrying IL-10 AA genotype (p<0.05). Serum IL-6 and IL-10 levels were lower in patients carrying the IL-10 AA genotype compared to patients with IL-10 AC+CC genotype, but the difference was not significant (p>0.05). Conclusion IL-10 AA genotype carriers had lower IL-6 and IL-6/10 levels in serum; however, GCF IL-6/10 levels were similar in both genotypes. Within the limitations of our study, a possible association between IL-10(-597) gene polymorphism and CP might be considered.

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients

Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Microbiological, lipid and immunological profiles in children with gingivitis and type 1 diabetes mellitus

Abstract Objective The aim of this study was to compare the prevalence of periodontal pathogens, systemic inflammatory mediators and lipid profiles in type 1 diabetes children (DM) with those observed in children without diabetes (NDM), both with gingivitis. Material and methods Twenty-four DM children and twenty-seven NDM controls were evaluated. The periodontal status, glycemic and lipid profiles were determined for both groups. Subgingival samples of periodontal sites were collected to determine the prevalence of periodontal microorganisms by PCR. Blood samples were collected for IL-1-β, TNF-α and IL-6 analysis using ELISA kits. Results Periodontal conditions of DM and NDM patients were similar, without statistical differences in periodontal indices. When considering patients with gingivitis, all lipid parameters evaluated were highest in the DM group; Capnocytophaga sputigena and Capnocytophaga ochracea were more prevalent in the periodontal sites of DM children. “Red complex” bacteria were detected in few sites of DM and NDM groups. Fusobacterium nucleatum and Campylobacter rectus were frequently found in both groups. Similar levels of IL-1-β, TNF-α and IL-6 were detected in DM and NDM children. Conclusion Clinical and immunological profiles are similar between DM and NDM children. The presence of Capnocytophaga sputigena and Capnocytophaga ochracea were associated with gingivitis in DM children.

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment.