A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

O objetivo deste estudo foi descrever um método de "nested" PCR baseado na fração 16S do rDNA para investigar a ocorrência de Campylobacter gracilis em infecções orais. Amostras foram coletadas de dez casos de canais radiculares infectados, dez casos de abscesso perirradicular agudo e oito casos de periodontite do adulto. O DNA extraído das amostras foi inicialmente amplificado usando "primers" universais para o gene do 16S rDNA. Uma segunda etapa de amplificação empregou os produtos de PCR gerados na primeira reação para detectar C. gracilis usando "primers" desenhados a partir de uma região específica para essa espécie localizada no gene do 16S rDNA. O método usado neste estudo apresentou um limite de detecção de 10 células de C. gracilis e ausência de reatividade cruzada com outras espécies bacterianas orais. C. gracilis foi detectado nos três tipos de infecções orais investigadas - 4/10 canais radiculares infectados; 2/10 casos de abscesso perirradicular agudo; e 1/8 espécimes subgengivais de casos de periodontite do adulto. O método proposto neste estudo foi altamente sensível e específico na detecção direta de C. gracilis em amostras clínicas de infecções endodônticas, abscessos e placa subgengival. Nossos achados confirmam que C. gracilis pode ser um membro da microbiota associada com infecções orais distintas e seu papel específico em tais doenças requer posterior elucidação.The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification

Detecção de Treponema denticola em casos de abscesso perirradicular agudo

Nosso objetivo foi detectar Treponema denticola em casos de abscesso perirradicular agudo. O DNA extraído das amostras de pus foi examinado pelo método da "Polymerase Chain Reaction" direcionada para o gene do RNAr (fração 16S). A amplificação usando o "primer" da espécie Treponema denticola permitiu detectá-la em 5 dos 6 casos de abscessos examinados. Apenas uma banda de tamanho esperado foi observada para as amostras positivas para esta bactéria, o que foi confirmado pela comparação com o DNA de referência do Treponema denticola (controle positivo). Até o momento, este é o primeiro relato da presença desta espiroqueta, considerada um importante patógeno periodontal em infecções endodônticas. Os resultados sugerem que Treponema denticola também pode ser um importante patógeno endodôntico.The purpose of this study was to report the detection of Treponema denticola in five out of six cases of acute periradicular abscesses. The 16S rRNA gene directed Polymerase Chain Reaction was the method utilized. This is probably the first report hitherto of the occurrence of this spirochete in acute periradicular abscesses

Desenvolvimento de lesões perirradiculares em ratos normais e diabéticos

Evidence suggests that diabetic patients are more significantly affected by problems of endodontic origin. This study sought to radiographically and histologically examine the development of periradicular inflammation in control and in diabetic rats after induction of pulpal infection. The pulps of the mandibular first molars of normal and streptozotocin-induced diabetic rats were exposed and left in contact with their oral cavities for 21 and 40 days. Afterwards, the animals were sacrificed, the mandibles were surgically removed, fixed in formalin and then radiographed in a standardized position. The radiographic images of the periradicular lesions were scanned and computerized images were evaluated for the total area of the lesions using a specific software. Representative specimens were also prepared for histopathological analysis. Radiographic analysis revealed that diabetic rats presented significantly larger periradicular lesions when compared with control rats, regardless of the experimental period (pEvidências indicam que pacientes diabéticos são mais significativamente afetados por problemas de origem endodôntica. Este estudo avaliou radiográfica e histologicamente o desenvolvimento de inflamação perirradicular em ratos controle e diabéticos depois da indução de infecção pulpar. As polpas dos primeiros molares inferiores de ratos normais ou diabéticos foram expostas e deixadas em contato com a cavidade oral por 21 e 40 dias. Em seguida, os animais foram sacrificados, as mandíbulas removidas cirurgicamente, fixadas em formalina e depois radiografadas em posição padronizada. As imagens radiográficas das lesões perirradiculares foram escaneadas e as imagens computadorizadas avaliadas quanto à área total das lesões usando software específico. Espécimes representativos foram preparados também para análise histológica. A análise radiográfica revelou que os ratos diabéticos apresentaram lesões periradiculares significativamente maiores quando comparados com os ratos normais, independentemente do período experimental (

PRILE 2021 guidelines for reporting laboratory studies in Endodontology: A consensus-based development

Reproducible, skilfully conducted and unbiased laboratory studies provide new knowledge, which can inform clinical research and eventually translate into better patient care. To help researchers improve the quality and reproducibility of their research prior to a publication peer-review, this paper describes the process that was followed during the development of the Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines and which used a well-documented consensus-based methodology. A steering committee was created with eight individuals (PM, RO, OP, IR, JS, EP, JJ and SP), plus the project leaders (PD, VN). The steering committee prepared an initial checklist by combining and adapting items from the modified Consolidated Statement of Reporting Trials checklist for reporting in vitro studies of dental materials and the Clinical and Laboratory Images in Publications principles as well as adding several new items. The steering committee then formed a PRILE Delphi Group (PDG) and PRILE Online Meeting Group (POMG) to provide expert advice and feedback on the initial draft checklist and flowchart. The members of the PDG participated in an online Delphi process to achieve consensus on the items within the PRILE 2021 checklist and the accompanying flowchart for clarity and suitability. The PRILE checklist and flowchart developed by the online Delphi process were discussed further by the POMG. This online meeting was conducted on 12 February 2021 via the Zoom platform. Following this meeting, the steering committee developed a final version of the PRILE 2021 guidelines and flowchart, which was piloted by several authors when writing up a laboratory study for publication. Authors are encouraged to use the PRILE 2021 guidelines and flowchart to improve the clarity, completeness and quality of reports describing laboratory studies in Endodontology. The PRILE 2021 checklist and flowchart are freely available and downloadable from the Preferred Reporting Items for study Designs in Endodontology website (http://pride-endodonticguidelines.org/prile/)

Unprepared root canal surface areas: causes, clinical implications, and therapeutic strategies

Abstract: Chemomechanical preparation is intended to clean, disinfect, and shape the root canal. This step is of utmost importance during treatment of infected teeth with apical periodontitis, because treatment outcome depends on how effectively the clinician eliminates bacteria, their products, and necrotic tissue that would serve as substrate for bacterial regrowth. Nonetheless, curvatures and complex internal anatomical variations of the root canal system can pose a high degree of difficulty in reaching these goals. In infected teeth, bacteria may persist not only in difficult-to-reach areas such as isthmuses, ramifications, dentinal tubules, and recesses from C-shaped or oval/flattened canals, but also in areas of the main canal wall that remain untouched by instruments. If bacteria withstand chemomechanical procedures, there is an augmented risk for post-treatment apical periodontitis. This article discloses the reasons why some areas remain unprepared by instruments and discusses strategies to circumvent this issue and enhance infection control during endodontic treatment/retreatment of teeth with apical periodontitis

Development of periradicular lesions in normal and diabetic rats

Evidence suggests that diabetic patients are more significantly affected by problems of endodontic origin. This study sought to radiographically and histologically examine the development of periradicular inflammation in control and in diabetic rats after induction of pulpal infection. The pulps of the mandibular first molars of normal and streptozotocin-induced diabetic rats were exposed and left in contact with their oral cavities for 21 and 40 days. Afterwards, the animals were sacrificed, the mandibles were surgically removed, fixed in formalin and then radiographed in a standardized position. The radiographic images of the periradicular lesions were scanned and computerized images were evaluated for the total area of the lesions using a specific software. Representative specimens were also prepared for histopathological analysis. Radiographic analysis revealed that diabetic rats presented significantly larger periradicular lesions when compared with control rats, regardless of the experimental period (p<0.05). Histopathological examination of representative specimens revealed larger periradicular lesions and more severe inflammatory exudate in the group of diabetic rats when compared with the control group. Data from the present study indicated that diabetic rats can be more prone to develop large periradicular lesions, possibly due to reduction in the defense ability against microbial pathogens