Microbiota Around Periodontal Teeth and Implants Affected by Peri-implant Disease.
Condition: Peri-Implantitis; Periodontal Diseases
MeSH terms: Periodontal Diseases; Peri-Implantitis
Study type: Observational [Patient Registry]

Summary: 
      The purpose of the study was to evaluate, from a microbiological point of view, microbiota
      around teeth and implants in the same patient affected by periodontal disease and
      peri-implant disease, all compared with a healthy tooth. From the identification of
      particular bacterial species in the examined sites, it is intended to be traced back to the
      identification of a clear etiopathogenic process, which may shed light on the similarities
      and differences between the two diseases. In recognition of these mechanisms, the
      investigators think to develop new therapeutic strategies for the future.
    

Detailed description: 
      Harvested via sterile paper points, for endodontic use, the sulcular fluid present around a
      dental element affected by periodontitis, and, in the same patient, the fluid present inside
      a peri-implant sulcus of a implant affected periimplantitis. If more implants are considered
      affected by perimplantitis, partecipant will choose the worst as sample site. The same
      principle will be followed in the case of the tooth affected by periodontal disease. The
      samples must be underwent to bacterial genomic meta-analysis for the precise identification
      of bacterial. For a better comparation of the flora, even a healthy tooth from the same
      patient will be choose like test site and a gingival crevicular fluide will be harvested on
      it.

      The investigators think that the perfect knowledge of the pathogen species is the starter
      point from a correct therapeutic strategy. But is really important take in mind the
      individual characteristcs of oral microflora.

      The data to be noted at the outset are:

      Signs and symptoms of periodontal disease, with precise framing within the Armitage 1999
      Classification, and peri-implant disease. For the aim of the study definition of
      perimplantitis concern an implant with a 4 mm minimal probing depth with bleeding on probing
      and or suppuration (BOP and Supp) and radiographic bone loss (BL).

      Evidence of BL recorded in order of definition of 8th European Consensus Conference of
      Periodontology Working group 4: BL > 2 mm from the expected bone level if a preliminary rx
      was absent; Rx 2-3 times the SD of the measurement record (1-1,5 mm) in case a preliminary rx
      was present.

        1. Detections diagnostic radiographic and pre-intervention performed with parallel
           technique (CBCT optional).

        2. Photographs pre, intra and post op (if present)

        3. Follow up to 6 months / 1 year (optional) The study provides a GCF harvested around
           periodontal and peri-implant sulcus in patients recruited for microbiological
           investigation. The analyzes of the samples are performed "blind". The Probe used is a
           UNC15 and only one calibrated clinician perform the probing. The pathogen microflora
           will be compaired with a healthy tooth microflora harvested around a healthy tooth in
           the same patient.

      Pretreatment of the sample sites in order to avoid contamination of the samples by the
      supragingival bacterial plaque will proceed as follows:

      - 2 days prior to sampling, the patient undergoes professional cleaning cups and brushes with
      the sole purpose of eliminating subgingival plaque. also we will be provided appropriate oral
      hygiene instruction with brushing twice / day and use of the other instruments where
      required.

      Sampling procedure: The harvested will be carried out through sterile paper point the extent
      to tip 30 and ISO taper 4%. The sampling technique will be that shown in the figure and
      marked with the letter c (intracrevicular deep method) until minimum resistance is felt. Each
      paper point will remain in place for 5 sec, then it will be readily stored in a special
      sterile container, stored in a refrigerated and delivered within 60 minutes Institute
      environment that will carry out the examination. Any harvested that show blood contamination
      will be excluded from the exam.

      Microbiological Protocol

        1. Sample collection and DNA isolation For the metagenomic analysis, samples of paper strip
           / paper point by patients included in the study, they will be eluted overnight in PBS
           (phosphate buffered saline) at 4 ° C. Subsequently, the samples will be centrifuged at
           10,000 rpm for 15 min at 4 ° C. After removal of the paper point, the supernatant will
           be used for the extraction of DNA using the QIAamp DNA Mini kit . The DNA will be
           quantified with Qubit® 2.0 Fluorometer (Thermo Fisher) using QUBIT Kit dsDNA HS ASSAY
           KIT (Life Technologies).

        2. Analysis of 16S rRNA - Metagenomics The metagenomic analyzes will be conducted on MiSeq
           platform Illumina. The gene libraries will be sequenced through the MiSeq v3 reagent
           kits. The 16S rRNA hypervariable regions will be amplified using a DNA concentration of
           5 ng / l with the primers V5-V7.

      The libraries will be purified by magnetic beads (AgencourtAmpure XP, Beckman), and the
      concentration and distribution of the fragments of the libraries will be evaluated on DNA
      Chip 1000 in the microanalyzer 2100 (Agilent Technologies). The analysis of the sequences
      (amplicon data sets) and the determination of OTU (OperationalTaxonomicUnits) will be
      performed by QIIME pipeline using as 16S rRNA gene database Green genes .

      Inclusion criteria:

        -  Assets in service for at least one year without any technical GBR (therefore excluded
           from the study also post-extraction)

        -  entered Works on native bone

        -  Fill out a medical history form and a periodontal board dedicated to the event, as well
           as a card for the clinician which builds on the work published in J ClinPeriodontol
           2012; 39 (Suppl 12): 224-244

        -  Reporting in the type of system board (optional surgical technique)

      Exclusion criteria:

        -  Patients in subintrant systemic conditions that contraindicate the insertion of implants

        -  Patients in the last three months, after surgery have taken antibiotics or follow any
           therapy for the resolution of acute events. The same may qualify for the study later.

        -  Women who are pregnant or nursing or undergoing hormone therapies
    

Intervention name(s): Harvested
Intervention type(s): Other
Intervention description(s): Metagenomic exam of Gingival crevicular fluid harvested by adsorbent paper point around periodontal teeth and implant affected by peri-implantitis
Observational model: Other
Time perspective: Other
Sampling method: Probability Sample
Study population: 
        People who received at least one implant loaded for at least one year and affected by
        peri-implantitis and contemporary present a periodontal tooth and a healthy tooth in own
        mouth
      
Biospecimen description: 
      gingival crevicular fluid around teeth affected by periodontal disease and implant affected
      by peri-implant disease. samples obtained by inserting paper cone in periodontal and
      peri-implant sulcus.
    
Keywords: Peri-implant pocket microbiota; Periodontal pocket microbiota