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ePaper created 2015-11-17, 15:11:47 | version 1.38.0
Volume 1 | Issue 1/2015 35Journal of Oral Science & Rehabilitation Epste i n–B arr vi ru s and pe r i i mplanti ti s by a combination of EBV and Gram-negative anaerobic rods.8 The aim of the present study is to compare the presence of EBV in periimplanti- tis-affectedandhealthyperiimplantsites. Materials&methods From January 2013 to December 2014, 50 con- secutivesubjectswithimplantsaffectedbyperi- implantitis and 50 subjects with healthy im- plants attending for a routine check-up or spon- taneous visits during the study period in three private clinics (Rome and Genoa, Italy, and Bel- grade,Serbia)wereenrolledinthisclinicalstudy. The inclusion criteria were systemically healthynonsmokersubjectstreatedwithatleast oneimplantthathadbeenfunctioningforatleast one year. Patients were periodontally healthy and had nottaken anysystemic antibiotics, anti- inflammatory drugs or oral antimicrobial agents withintheprecedingsixmonths. Periimplantitis is commonly defined as re- ported by the Estepona consensus meeting: an infectionwith suppuration associatedwith clini- callysignificantprogressingcrestalbonelossaf- ter the adaptive phase.1 However, in the present study, to find a clinically feasible threshold, ac- cording to Renvert et al., periimplantitis was de- fined when an implant presented radiographic presenceofbonelossof> 3 mmafterimplantin- tegration,with a pocket probing depth of≥ 4 mm, bleedingonprobingand/orsuppuration.9 This human case–control study was con- ducted in accordance with the Declaration of Helsinki and all subjects provided written in- formedconsentpriortotheirentryintothestudy. ItconformedwiththeStrengtheningtheReport- ing of Observational Studies in Epidemiology guidelines.10 All clinical examinations were performed by the same operators (LC, PP and MR) and sub- gingival plaque samples were collected with the GUIDOR Perio-Implant Diagnostic Kit (Sunstar Iberia, Barcelona, Spain). The sampling kit is in- tended for the collection and transport of sam- ples containing periodontal and periimplant pathogens. Briefly, prior to subgingival plaque sampling, each tooth was isolated with cotton rolls. Absorbent paper points were inserted into the periodontal pockets. After 15s, these paper pointswereremovedandplacedintoa2 mltube. Thetubescontainingthesampleweresenttothe Institut Clinident laboratory (France) in the pro- vided mailing envelopes. The internal connec- tions and external surfaces ofthe implants were evaluated. Q u anti tati ve re al- ti me po lyme rase chai n re acti o n assays f o r the Epste i n–B arr vi ru s Quantitative real-time polymerase chain reac- tion (PCR) assays were performed to detect the presence orabsence ofand quantifyEBVDNAin the paper points. First, total DNA was isolated using the QIAxtractor DNA Plasticware and QI- Axtractor DX Reagents (Qiagen, Hilden, Ger- many) according to the manufacturer’s guide- lines. Then, real-time PCR was carried out for EBV using the Epstein-Barr virus quantitative Real Time PCR kit (Diagenode, Liège, Belgium) and the Rotor-Gene Q thermal cycling system (Qiagen,Hilden,Germany). Briefly, quantitative real-time PCR assays were performed in a volume of 25 μl, composed of 12.5 μl of MasterMix Optima Multiplex 2X DNA,2.5 μlofEBVprimersanddouble-dyeprobe (FAM, emission 520 nm), 2.5 μl of internal con- trol DNA, 2.5 μl of internal control primers and double-dye probe (Yellow Dye, emission 548 nm),and5 μlofDNAextractorEBV-positive control or EBV-negative control or DNA Stan- dard (for quantitative standard curve; all prod- ucts by Diagenode, Liège, Belgium). Five EBV DNA dilutions were used for the standard curve (from 200 copies to 2,000,000 copies of EBV amplicon/PCRreaction). Assays were carried out on the Rotor-Gene Q thermal cycling system with the following program: 50 °C for 2 min, 95 °C for 10 min, fol- lowed by 45 cycles of 15 s at 95 °C, and 60 s at 60 °C. Fluorescence signals (FAM, emission 520 nm; Yellow Dye, emission 548 nm) were measured every cycle at the end of the exten- sion step. The resulting data were analyzed us- ing Rotor-Gene Q Series Software (Qiagen, Hilden, Germany). Stati sti cal me tho ds Normality of variables was assessed by graphi- cal methods (mean of histograms) and con- firmed by the Shapiro–Wilk normality test and the Levene test of homogeneity of variance. All characteristics were summarized using mean (standard deviation) median (range) or frequen- cies (percentages).