Quantitative And Qualitative Analysis Of Microorganisms In Root-filled Teeth With Persistent Infection: Monitoring Of The Endodontic Retreatment.

The aim of this study was to investigate in vivo microorganisms detected in root-filled teeth with post-treatment apical periodontitis and quantify colony-forming units (CFU) during endodontic retreatment. Fifteen root-filled teeth had their previous gutta-percha removed and were randomly instrumented before being divided into three groups and medicated with either [Ca(OH)2 + 2% CHX gel], [Ca(OH)2 + 0.9% NaCl] or 2% CHX gel. Samples were taken after removal of gutta-percha (S1), after chemomechanical preparation using 2% CHX gel (S2), and after inter-appointment dressing (S3) for 7 or 14 days later. Cultivable bacteria recovered from infected root canals at the three stages were counted and identified by means of culture and PCR assay (16S rDNA). Quantitative data were statistically analyzed by using Mann-Whitney test in which pairs of groups were compared (P < 0.05). CFU counts decreased significantly from S1 to S2 (P < 0.05). No significant difference was found between S2 and S3 (P = 0.3093) for all three experimental groups. Chemomechanical preparation and intra-canal dressing promoted significant median reductions of 99.61% and 99.57%, respectively, in the number of bacteria compared to S1 samples. A total of 110 cultivable isolates were recovered by culture technique from 32 different species and 7 different genera. Out of the 13 target species-specific primer of bacteria analyzed, 11 were detected during endodontic retreatment. The great majority of taxa found in post-treatment samples were Gram-positive bacteria, although Gram-negative bacteria were found by molecular methods. Moreover, our results showed that gutta-percha removal and chemomechanical preparation are effective for root canal disinfection, whereas additional intra-canal dressing did not improve disinfection.7302-

Identificação de patógenos endodônticos por PCR e quantificação de DNA em amostras extraídas de dentes com periodontite apical pós-tratamento

Objetivo: O objetivo deste estudo clínico foi quantificar a concentração de DNA e detectar algumas espécies bacterianas de amostras de dentes tratados endodonticamente com periodontite apical após a remoção da guta-percha (S1), após o preparo químico-mecânico na primeira sessão (S2), 5 dias após o preenchimento do canal com solução fisiológica estéril (S3), após reinstrumentação na segunda sessão (S4), e 14 dias após a inserção da medicação intracanal na terceira sessão (S5). Métodos: Quinze dentes tratados endodonticamente foram selecionados. A remoção da guta-percha foi realizada por meio da técnica coroa-ápice. Utilizaram-se limas manuais associadas à clorexidina gel a 2% durante o preparo químico-mecânico. A medicação intracanal selecionada foi à base de hidróxido de cálcio. DNA foi isolado das amostras e foram investigadas 14 espécies bacterianas (primer espécie-específi co16S rDNA). A concentração de DNA foi quantifi cada utilizando o espectrofotômetro NanoDropTM 2000. Resultados: Em todos os casos foram detectadas bactérias, como revelado por meio do primer universal. DNA foi isolado de todas as amostras, com uma concentração média de 4,24 ± 2,9 ng/µL (S1), 3,39 ± 1,54 ng/ µL (S2), 4,0 ± 1,94 ng/µL (S3), 2,66 ± 0,98 ng/µL (S4) e 3,97 ± 2,32 ng/µL (S5). Parvimonas micra e Enterococcus faecalis (S1), P. micra (S2), Porphyromonas endodontalis e E. faecalis (S3), E. faecalis e Prevotella nigrescens (S4/S5) foram as espécies mais frequentemente detectadas. A concentração de DNA diminuiu entre S3 e S4 (p = 0,0256), ao passo que um aumento foi observado entre S4 e S5. Conclusão: Uma ampla variedade de espécies bacterianas foi detectada em canais radiculares de dentes tratados endodonticamente com periodontite apical. Além disso, o uso da medicação intracanal não potencializou a redução da concentração de DNA bacteriano.Aim: The aim of this clinical study was to quantify the concentration of DNA and to detect selected bacterial species from samples of infected root-fi lled teeth with post-treatment apical periodontitis after removal of gutta-percha (S1), after chemo-mechanical preparation at the fi rst appointment (S2), 5 days after the canal was fi lled with sterile physiological solution (S3), after reinstrumentation at the second appointment (S4), and 14 days after an intracanal dressing was placed at the third appointment (S5). Methods: Fifteen root-fi lled teeth were selected. Removal of gutta-percha was performed using the crown-down technique. Chemo-mechanical preparation was performed with hand fi les associated with 2% chlorhexidine gel. An intracanal dressing based on Ca(OH)2 was used. DNA was extracted from the samples and 14 endodontic 16S rDNA species-specifi c primers were tested. The concentration of DNA was quantifi ed using a NanoDropTM 2000 spectrophotometer. Results: Bacteria were present in all cases at all sampling times, as revealed by a universal primer. DNA was isolated from all samples, with an average concentration of 4.24 ± 2.9 ng/µL (S1), 3.39 ± 1.54 ng/µL (S2), 4.0 ± 1.94 ng/µL (S3), 2.66 ± 0.98 ng/µL (S4) and 3.97 ± 2.32 ng/µL (S5). Parvimonas micra and Enterococcus faecalis (S1), P. micra (S2), Porphyromonas endodontalis and E. faecalis (S3), E. faecalis and Prevotella nigrescens (S4/S5) were the species most frequently deteced. DNA concentration reductions were detected between S3 and S4 (p = 0.0256), whereas an increase was found between S4 and S5. Conclusion: A wide variety of bacterial species was detected in root-fi lled teeth with post-treatment apical periodontitis. Moreover, the use of an intracanal dressing was unable to further reduce the concentration of bacterial DNA

Effectiveness in the removal of bioceramic sealers versus conventional sealers: systematic review and meta-analysis

This study aimed to carry out a systematic review to evaluate the effectiveness in removing bioceramic sealers compared to conventional sealers commonly used, evaluated through the percentage of remaining material. The electronic search was carried out in the following databases: MEDLINE (PubMed), Embase, Web of Science, Scopus, Cochrane Library, and in gray literature, at the Brazilian Digital Library of Theses and Dissertations (BDTD). &nbsp;Two independent researchers conducted the survey to identify the studies, without restrictions on year and language of publication, using the PICO strategy until the month of August 2020. A total of 80 titles were retrieved in the initial search, however, only 9 studies were included for the qualitative synthesis and 5 studies were included in the quantitative synthesis. The descriptive results indicated that the average time taken to remove the filling material was longer for bioceramics by approximately 67% of the studies that evaluated this condition, and with regard to the establishment of patency, no difference was detected between the sealers. It could be observed that bioceramic sealers presented a lower amount of remaining material than conventional sealers (p = 0.01) both in the overall analysis and in the analysis of subgroups. The removal of conventional sealer proved to be inferior to bioceramic sealers, with greater amounts of material remaining in the root canals after endodontic retreatment. The use of bioceramic sealers has gained space in the endodontic practice. However, it is not yet known whether these sealers affect the removal of root canal fillings during retreatments

Comparison of fusobacterium nucleatum and porphyromonas gingivalis lipopolysaccharides clinically isolated from root canal infection in the induction of pro-inflammatory cytokines secretion

The aim of this study was to compare the biological activity of lipopolysaccharides (LPS) purified from Fusobacterium nucleatum and Porphyromonas gingivalis strains, both isolated from primary endodontic infection (PEI) in the levels of IL-1β and TNF-α released by macrophage cells. Moreover, LPS was purified from F. nucleatum and P. gingivalis American Type Collection (ATCC) and its biological activity was compared to respectively clinical isolates strains. F. nucleatum and P. gingivalis strains clinically isolated from PEI had their identification confirmed by sequencing the 16S rRNA gene. LPS from F. nucleatum and P. gingivalis and their respective ATCC strains were extracted by using Tri-reagent method. Macrophages (Raw 264.7) were stimulated with LPS at 100 ng/mL for 4, 8 and 12 h. Secretion of IL-1 β and TNF-α was also determined. Paired t-test, repeated measures ANOVA and one-way ANOVA were employed. All LPS induced significant production of IL-1β and TNF-α, with the former being secreted at higher levels than the latter in all time-points. F. nucleatum induced a higher expression of both cytokines compared to P. gingivalis (p<0.05). No differences were observed between clinical and ATCC strains, as both presented the same potential to induce pro-inflammatory response. It was concluded that F. nucleatum and P. gingivalis LPS presented different patterns of activation against macrophages as seen by the IL-1β and TNF-α production, which may contribute to the immunopathogenesis of apical periodontitis. Moreover, clinical and ATCC strains grown under the same in vitro environment conditions presented similar biological activity272202207CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302575/2009-0; 150557/2011-6; 308162/2014-510/19136-1; 10/17877- 4; 11/50051-5; 11/50510-0; 11/09047-4O objetivo deste estudo foi comparar a atividade biológica de lipopolissacarídeos (LPS) purificados a partir de linhagens de Fusobacterium nucleatum e Porphyromonas gingivalis, ambas isoladas de infecções endodônticas primárias (IEP) nos níveis de IL-1β e TNF-α produzidos por macrófagos. Adicionalmente, LPS foi purificado de F. nucleatum e P. gingivalis "American Type Collection" (ATCC) e sua atividade comparada às respectivas linhagens clinicamente isoladas. Linhagens de F. nucleatum e P. gingivalis isoladas clinicamente de IEP tiveram sua identificação confirmada por sequenciamento do gene 16S rRNA. LPS de F. nucleatum e P. gingivalis e das respectivas linhagens foram extraídos com o uso do método "Tri-reagent". Macrófagos (Raw 264.7) foram estimulados com LPS a 100 ng/mL por 4, 8 e 12 h. A secreção de IL-1β e de TNF-α foi determinada. Foram usados os testes t-pareado, ANOVA de medidas repetidas e ANOVA de um fator. Todos os LPS induziram a produção significante de IL-1β e TNF-α, sendo o primeiro secretado em mais altas concentrações que o último em todos os tempos avaliados. F. nucleatum induziu uma maior expressão de ambas as citocinas comparativamente ao P. gingivalis (p<0,05). Não foram observadas diferenças entre as linhagens clínica e ATCC, uma vez que ambas apresentaram o mesmo potencial de indução da resposta pró-inflamatória. Conclui-se que F. nucleatum e P. gingivalis possuem diferentes padrões de ativação dos macrófagos, como visto pela produção de IL-1β e TNF-α, o que pode contribuir para a imunopatogênese da periodontite apical. Ainda, linhagens clínica e ATCC mantidas no mesmo ambiente in vitro apresentaram ativação biológica semelhant

Clínica ampliada em Odontologia: experiência precursora avaliada pelos usuários

Objetivou-se analisar a atenção em saúde bucal prestada pela Clínica Ampliada (CA) da Universidade Estadual de Maringá, bem como avaliar satisfação com atendimento odontológico e autonomia do paciente sob percepção de usuários. Trata-se de estudo observacional, transversal, descritivo, retrospectivo, com usuários que frequentaram o serviço odontológico da CA. Foi aplicado questionário estruturado sobre variáveis socioeconômicas, humanização, autonomia, acesso, encaminhamento e satisfação sobre atenção odontológica da CA. Os dados descritivos foram analisados no Programa EpiInfo. O principal motivo de procura da CA foi dor (37,8%), sendo escolhida principalmente pela gratuidade do atendimento (43,4%). Todos pacientes citaram que os dentistas os escutaram com atenção, tiveram um bom relacionamento e confiança no seu trabalho. A maioria dos pacientes mudou seu comportamento sobre hábitos saudáveis em relação à sua saúde, aumentando autonomia e sua responsabilização com manutenção da saúde bucal. A maioria dos pacientes avaliou satisfatoriamente a CA em relação aos recursos humanos, estrutura e limpeza. Todos avaliaram o atendimento odontológico como muito bom/bom, sendo que acharam que o dentista fez bom trabalho e recomendariam a CA. Conclui-se que esta experiência pioneira está sendo positiva, qualificando a atenção odontológica em relação à humanização, aumentando autonomia do paciente e satisfação do usuário com serviço prestado

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress