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Journal of Oral Science & Rehabilitation 58 Volume 2 | Issue 4/2016 S u r g i c a l t r e a t m e n t o f p e r i i m p l a n t i t i s M i c r o b i o l o g i c a l a n a l y s i s Sampling for microbiological analysis was per- formed by a single researcher. Samples were obtained from three types of sites in each pa- tient: (a) the periimplant sulcus of each implant; (b) the gingival sulcus of the neighboring teeth; and (c)the connection’s inside andthe abutment surface of each implant. Samples were obtained before the cleaning procedures and 12 months after treatment. Sampling was performed using the GUIDOR Perio-Implant Diagnostic Kit (Sunstar Iberia, Barcelona, Spain; provided by Institut Clinident, Aix-en-Provence, France), consisting offive ste- rile absorbent paper tips and an empty sterile 2mLEppendorftube.Priortosubgingivalplaque sampling, supragingival plaque was eliminated from the implants and teeth using a curette or cotton roll, without penetrating the gingival or periimplant sulcus. Cotton rolls were used for relative isolation and the sampling sites were dried with a pistol. The paper tips were inserted into the gingival or periimplant sulcus for 30 s. One drop of RNA- and DNA-free water (WaterMolecularBiologyReagent,codeW4502, Sigma-Aldrich, St. Louis, Mo., U.S.) was placed insidethe implant connection andthe papertips were inserted for 30 s. Quantitative real-time polymerase chain reaction (PCR) assays were carried out for total bacterial count and for ten pathogens at Institut Clinident: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerellaforsythia(Tf), Trepone- madenticola(Td),Prevotellaintermedia (Pi),Pep- tostreptococcus micros (Pm), Fusobacterium nucleatum (Fn), Campylobacterrectus (Cr), Eike- nella corrodens (Ec) and Candida albicans (Ca). Quantitative real-time PCR assays was per- formed in a volume of 10 μL composed of 1× QuantiFast SYBR Green PCR (Qiagen, Berlin, Germany), 2 μLof DNAextract and 1 μM of each primer. The species-specific PCR primers (Me- tabion, Martinsried, Germany) used inthis study were provided by Institut Clinident. The bacte- rial primers used were derived from previously publishedribosomal16SribosomalRNAsequen- ces15, 16 and were adapted to the real-time PCR conditions. The Ca primers used in this study were derived from 18S/28S ribosomal RNA se- quences. Assays were carried out on the Rotor-Gene Q thermal cycling system (Qiagen) with the fol- lowing program: 95 °C for 5 min, followed by 40 cycles of10 s at 95 °C, 10 s at 60 °C, and 35 s at72°C.Afinalmeltingcurveanalysis(70–95°C in 1 °C steps for 5 s increments) was performed. Fluorescence signals were measured every cy- cle at the end of the extension step and conti- nuously during the melting curve analysis. The resulting data were analyzed using Rotor-Gene Q Series Software (Qiagen). Serial dilutions of bacterial standard DNA, provided byInstitut Clinident,were used in each reactionasexternalstandardsforabsolutequan- tification of the targeted bacterial pathogens. StandardbacterialstrainsusedforstandardDNA production were obtained from the DSMZ (Braunschweig, Germany), the Collection of In- stitutPasteur(Paris,France)andfromtheBCCM/ LMG Bacteria Collection (Ghent, Belgium): Aa (DSM No. 8324), Pg (DSM No. 20709), Tf (CIP No. 105220), Td (DSM No. 14222), Pi (DSM No. 20706), Pm (DSM No. 20468), Fn (DSM No. 20482), Cr (LMG No. 18530) and Ec (DSM No. 8340). A Ca standard DNA (DSM No. 6659) was also used as external standard for identifi- cation and semiquantification. S t a t i s t i c a l a n a l y s i s The statistical analysis was performed using a commercially available software program (IBM SPSS Statistics forWindows,Version 20.0, IBM Corp.,Armonk,N.Y.,U.S.).Meanvaluesandstan- darddeviationswerecalculatedforeachvariable usingthe implant asthe statisticalunit.The data rows were examined with the Kolmogorov– Smirnov test and proven to be normally distrib- uted. Forthe statistical evaluation ofthe chang- Figs. 3a–c Figs. 3a–c Radiographic sequence of the site at the time of periimplantitis detection and at the time of surgery. a b c