Evaluating clinical periodontal measures as surrogates for bacterial exposure: The Oral Infections and Vascular Disease Epidemiology Study (INVEST)

Ryan T Demmer 0 Panos N Papapanou 2 David R Jacobs Jr 1 5 Mose Desvarieux 0 3 4 0 Department of Epidemiology, Mailman School of Public Health, Columbia University , New York, NY , USA 1 Division of Epidemiology and Community Health, School of Public Health, University of Minnesota , Minneapolis, MN , USA 2 Division of Periodontics, Section of Oral and Diagnostic Sciences, College of Dental Medicine, Columbia University , New York, NY , USA 3 Ecole des hautes etudes en sante publique , Paris et Rennes , France 4 INSERM, UMR-S 707, Paris, F- 75012; Universite Pierre et Marie Curie-Paris6, UMR S 707, Paris , F-75012 , France 5 Department of Nutrition, University of Oslo , Oslo , Norway Background: Epidemiologic studies of periodontal infection as a risk factor for cardiovascular disease often use clinical periodontal measures as a surrogate for the underlying bacterial exposure of interest. There are currently no methodological studies evaluating which clinical periodontal measures best reflect the levels of subgingival bacterial colonization in population-based settings. We investigated the characteristics of clinical periodontal definitions that were most representative of exposure to bacterial species that are believed to be either markers, or themselves etiologic, of periodontal disease. Methods: 706 men and women aged 55 years, residing in northern Manhattan were enrolled. Using DNA-DNA checkerboard hybridization in subgingival biofilms, standardized values for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were averaged within mouth and summed to define bacterial burden. Correlations of bacterial burden with clinical periodontal constructs defined by the severity and extent of attachment loss (AL), pocket depth (PD) and bleeding on probing (BOP) were assessed. Results: Clinical periodontal constructs demonstrating the highest correlations with bacterial burden were: i) percent of sites with BOP (r = 0.62); ii) percent of sites with PD 3 mm (r = 0.61); and iii) number of sites with BOP (r = 0.59). Increasing PD or AL severity thresholds consistently attenuated correlations, i.e., the correlation of bacterial burden with the percent of sites with PD 8 mm was only r = 0.16. Conclusions: Clinical exposure definitions of periodontal disease should incorporate relatively shallow pockets to best reflect whole mouth exposure to bacterial burden. - Background There are several models for studying infectious origins of cardiovascular disease(CVD) [1]. Many models rely on serum antibody titer to define infectious exposure. This has the advantage of capturing historical infectious exposure, but the possible disadvantage of not requiring the presence of active infection at the time of measurement. Another method of assessing infectious bacterial exposure is to directly measure bacterial colonization levels. In this regard, one infection model that may be useful is periodontal disease, because active bacterial infections with inflammatory consequences are often present for years and are easily accessible in the subgingiva[2-5]. However, microbiological assessment is resource intensive and is rarely done in epidemiologic studies investigating periodontal-CVD associations. Consequently, studies exploring associations between periodontal infection and CVD frequently use one of a wide variety of whole mouth summaries of clinical periodontal measures (i.e. pocket depth, attachment loss and bleeding on probing) as a surrogate measure of underlying microbiology. Methodological research is needed to clarify which clinical periodontal constructs act as surrogates for such bacterial infection. In doing so, hypothesis testing in future studies can be more specifically focused on infectious exposure as opposed to clinical periodontal characteristics which can be the result of non-infectious exposures such as smoking[6-8], diabetes[9], or endodontic lesions[10]. In this paper we investigated how well whole mouth clinical periodontal constructs reflect exposure to bacteria that are associated with risk of periodontal disease. Methods The Oral Infections and Vascular Disease Epidemiology Study (INVEST) is a randomly sampled prospective population-based cohort study investigating whether oral infections increase the risk of carotid atherosclerosis and stroke[11]. Briefly, 1056 subjects were randomly selected from Northern Manhattan, an area between 145th Street and 218th Street, bordered westward by the Hudson River, and separated eastward from the Bronx by the Harlem River. Hispanics, Blacks, and Whites live together in this area and have similar access to medical care. The selection process was derived from the Northern Manhattan Study (NOMAS) in which patients are also enrolled[12]. Of 841 dentate participants enrolled at baseline, both clinical periodontal exams and subgingival plaque samples were available for 706 subjects, who entered these analyses. The Institutional Review Board at Columbia University approved the study, and all subjects provided informed consent. Dental history and oral examination Subjects were interviewed and examined by trained research assistants and calibrated dental examiners [4,11]. Teeth were counted and localized. Plaque samples in the two most posterior teeth (mesiolingual in the upper jaw and mesiobuccal in the lower jaw) were collected before probing. Assessment of periodontal status for all teeth present included presence/absence of dental plaque, probing depth in millimeters, and location of the gingival margin in relation to the cementoenamel junction at six locations for each tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual and distolingual) using a UNC-15 manual probe with ruler markings at each millimeter from 1 to 12 (HuFriedy, Chicago, IL). Attachment loss was calculated as the sum of pocket depth and gingival margin. Subgingival plaque collection and laboratory processing A maximum of eight subgingival plaque samples (mean 7; median 8) were collected from each subject by a single scaling stroke from the base of the periodontal pocket using a sterile Gracey curette. Samples were collected from the two most posterior teeth in each quadrant (mesiopalatal sites in the maxilla and mesiobuccal sites in the mandible). The collected plaque mass from each site was transferred into an individual Eppendorf tube containing 200 l of sterile T-E buffer (10 mM Tris HCl, 1.0 mm EDTA, pH 7.6). The tubes were immediately transferred into the laboratory and the plaque pellet was re-suspended, vigorously vortexed, and 200 l of a 0.5 M NaOH solution were added. The samples were kept at +4C until immobilization onto nylon membranes, within a few days from sample collection. A total of 4,922 bacterial plaque samples (collected from n = 706 participants) are included presently. Four bacterial species (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerell (...truncated)