Assessing the progression of chronic periodontitis using subgingival pathogen levels: a 24-month prospective multicenter cohort study

This study was performed as a clinical research project for diagnosis of periodontitis, as sponsored by the Japanese Society of Periodontology. Subjects were recruited in cooperation with 17 facilities (16 university hospitals and one clinic) in Japan, and were registered between February 2009 and February 2012. An additional file shows this in more detail [see Additional file 1]. The study enrolled a total of 163 follow-up patients, that is, individuals with chronic periodontitis who had completed active treatments such as initial therapy or periodontal surgery. Each diagnosis was based on the Guidelines of the American Academy of Periodontology [19]. All individuals were of age ≥ 30 years, systemically healthy, possessed at least 20 teeth, and had not taken systemic antibiotics, anti-inflammatory drugs, or immunosuppressive drugs within 3 months prior to enrollment.

The follow-up patients were seen trimonthly over a 24-month period. At each visit, the patients received treatment only to remove supragingival plaque and calculus, if detected. To identify disease progression, a full-mouth periodontal examination, except for the third molars, was performed every 6 months. The following clinical parameters were recorded: plaque index (PlI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL). PlI was recorded at 4 sites per tooth (mesial, buccal, distal, and lingual). PPD, CAL, and BOP were recorded at 6 sites per tooth (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and distolingual). Patients with at least one site of CAL loss of ≥3 mm at a given site over the 24-month study period were considered to be exhibiting periodontitis progression [6, 7, 20, 21]. Intra- and inter-examiner calibration sessions were conducted at the beginning and middle of the study period.

At baseline, samples of subgingival plaque from the deepest pockets, saliva, and blood were collected from each patient. Samplings were carried out according to the following order; blood samples, saliva samples, and subgingival plaque samples. Whole saliva was collected from each subject by having the individual chew on a gum base, containing neither fragrance nor flavored ingredients, for 5 min. The subgingival plaque was collected as follows: after removal of the supragingival plaque, samples of subgingival plaque from the deepest pockets (except for those at the third molars) were obtained by consecutive insertion (at a given site) of two sterile number-40 paper points (Zipperer Absorbent Paper Points, VDW GmbH, Munich, Germany) into the periodontal pocket for 10 s per point [22]. The plaque and saliva samples were sent immediately to a medical laboratory (BML Corporation, Tokyo, Japan) for bacterial analysis [6, 17, 23]. Blood sampling was performed using a commercial kit (DEMECAL Blood Test Set; Sunstar Inc, Osaka, Japan). In brief, device-treated serum was obtained from a 50-μL sample of whole capillary blood collected from the middle fingertip. The samples of sera were sent immediately to a commercial laboratory (Leisure Inc., Tokyo, Japan) for immune analysis [7, 24‐27].

Quantitative analysis of the total bacterial count and periodontopathic bacterial counts, including P. gingivalis, P. intermedia, and A. actinomycetemcomitans, was performed using a modification of the Invader PLUS assay [7, 22, 28, 29]. Briefly, bacterial DNA was extracted from samples of the subgingival plaque from the deepest pockets by suspending each plaque sample in 1 mL of phosphate-buffered saline, pH 7.4 and processing using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Similarly, bacterial DNA was extracted from the 100-μl whole saliva samples using the MagNA Pure LC Total Nucleic Acid Isolation Kit. The individual sequences of each bacterial species were obtained from a public database (National Center for Biotechnology Information, Bethesda, MD). Primers for each species were designed based on a region of the 16S rRNA gene. A pair of universal primers and a universal probe were used to determine the total number of bacteria. Primary probes and Invader oligos were designed using the Invader technology creator (HOLOGIC, Madison, WI) and were based on sequences in the amplified regions [7, 28‐30].

Template DNA was added to a 15-μL reaction mixture containing primers for each species, 50 μM dNTPs, 700 nM primary probe, 70 nM Invader oligo, 2.5 U polymerase chain reaction (PCR) enzyme (EagleTaq DNA polymerase, Roche, Basel, Switzerland), and the Invader core reagent kit (Cleavase XI Invader core reagent kit, HOLOGIC, Madison, WI) containing a fluorescence resonance energy transfer (FRET) mix and an enzyme/MgCl₂ solution. The reaction mixture was preheated at 95 °C for 20 min, and a 2-step PCR reaction (95 °C for 1 s and 63 °C for 1 min) was performed for 35 cycles using an ABI PRISM 7900 thermocycler (Applied Biosystems, Foster City, CA). Fluorescence values of carboxyfluorescein (FAM; wavelength/bandwidth: excitation, 485/20 nm; emission, 530/25 nm) were measured at the end of the incubation/extension step at 63 °C for each cycle.

The limit of detection for this method was determined for each species with dilutions of bacterial DNA. Standard curves were constructed based on a crossing point determined by the fit point method.

Next, the proportions of the 3 pathogens within the total bacterial counts were determined [10, 31]. Bacterial ratios (%) and bacterial counts (log₁₀) for each species also were used in various comparison and diagnostic analyses.

Serum IgG antibody titers against periodontal pathogens were determined by Leisure Inc. using enzyme-linked immunosorbent assay (ELISA) [32]. The method and selection of bacterial antigens were based on previous reports [7, 24‐27]. Briefly, sonicated preparations of P. gingivalis FDC381, P. intermedia ATCC25611, and A. actinomycetemcomitans ATCC29523 were used as representative bacterial antigens. Each antigen was diluted to 10 μg/mL with 0.1 M carbonate buffer (pH 9.6). An aliquot of this diluted solution (100 μL) then was added to each well in a flat-bottomed microtiter plate (Greiner Co. Ltd., Frickenhausen, Germany) and the plate was stored overnight at 4 °C. Each well with immobilized antigen was washed three times with PBS containing 0.05% Tween-20 (PBST). Subsequently, a diluted serum sample (3100-fold dilution with PBST) was added to each well. After incubation at 37 °C for 2 h, each well was washed three times with PBST and bound/free (B/F) separation was carried out. Next, a 100-μl aliquot of 1:5000 diluted alkaline phosphatase-conjugated goat antihuman IgG antibody (Jackson Immuno Research Laboratories, Inc., Baltimore, MD) was added to each well. After incubation at 37 °C for 2 h, each well was washed three times with PBST and B/F separation was carried out. Thereafter, 50 μl of p-nitrophenyl phosphate (Wako Pure Chemical Industries, Ltd., Osaka, Japan) adjusted to 1 mg/ml with 10% diethanolamine buffer (pH 9.8) was added to each well as substrate. The plate then was incubated at room temperature for 10–20 min. The enzymatic reaction was terminated by adding 50 μl of 3 M NaOH, and optical density (measurement at 405 nm; reference at 490 nm) was measured in a Micro ELISA Auto Reader (Bio-Rad Laboratories, Hercules, CA).

The standard titration curves were formulated using serial dilutions of pooled control serum collected from participants without periodontitis. The absorbance of each sample after reaction was defined as an ELISA unit (EU), such that 100 EU corresponded to 1:3200 dilution of the control sample. The antibody titers were expressed as standardized values calculated as follows: (EU for study serum samples—EU for control samples)/2 standard deviations (SD), using SDs as determined based on ELISA of the control samples [25].

Descriptive analysis of the collected data was performed; the results are presented as medians. The Mann Whitney U test was used to compare clinical parameters, periodontal bacterial amounts, and IgG titers with or without progression of chronic periodontitis (CP). Only correlation with sex was performed using the Fisher’s exact test. To determine an optimal test for distinguishing follow-up patients with periodontal progression or stability, we constructed receiver operating characteristic (ROC) curves and set the cutoff point based on the minimum difference between sensitivity and specificity [6, 25]. On the basis of the calculated cutoff point, we then made appropriate cross-tabulations. Diagnostic values were calculated by the Fisher’s exact test to identify statistically significant differences. To check the correlation of periodontal bacteria with disease progression, logistic regression analysis was carried out to eliminate possible confounding variables. This study was originally designed to evaluate the diagnostic salivary levels of P. gingivalis in patients with or without the progression of CP. Calculation of the sample size was described in our previous report [7]. For the present study, post-hoc power analysis calculation using the cross table for patients with or without progression of CP against positive or negative for P. gingivalis suggested that groups would have to consist of at least 41 subjects each. A similar analysis revealed that groups of at least 26 subjects each would be required to detect differences in P. gingivalis counts (log₁₀) in subgingival plaque from the deepest pockets. All analyses were performed by independent statisticians (Y.N., E.K., and N.H.) using IBM SPSS Statistics ver. 19.0 software (IBM Japan, Tokyo, Japan) and S-plus (ver. 6.0, NTT DATA, Tokyo, Japan).