According to Craig et al. [12], the mean serum IgG antibody levels to T. forsythia were higher in a periodontitis group when compared to a healthy group in a sample of the United States (US) Hispanic population (A difference of 2.4 EU [enzyme-linked immunosorbent assay unit] was found). Thus, a difference of 2.4 EU between groups was considered to be relevant. The sample size calculation determined that ≥21 patients per group would provide 80% power and a significance level of 0.05 (two-tailed) for detecting a true difference of 2.4 EU between groups, assuming 2.75 EU as the common standard deviation.

One hundred eight subjects (79 females and 29 males), aged 33 to 82 years (with ≥18 residual teeth) who visited the dental clinics of the Universidad de Antioquia in Medellín, Colombia were invited to participate in this study between January 2009 and December 2011. Informed and written consent was obtained from each participant. The study design was approved by the Ethics Committee on Human Research of the School of Dentistry of the University of Antioquia (ID 02-2008) according to the Declaration of Helsinki on experimentation involving human subjects. Patients with a diagnosis of chronic periodontitis (the diagnostic criteria are described below), ≥18 residual teeth and ≥31 years were considered candidates for the study. Individuals with no evidence of mild, moderate, or severe periodontitis were used as a control group. Of the 108 subjects included, 28 patients belonged to the control group.

Exclusion criteria included diagnosed diabetes and autoimmune diseases. Pregnant women, intake of systemic antimicrobials with the previous six months, nonsteroidal analgesics or anti-inflammatory drugs, and earlier periodontal therapy also served as exclusion criteria.

A medical history and clinical and radiographic examinations were performed for each patient. The diagnosis of chronic periodontitis was made on the basis of principles outlined by Eke et al. [13]; patients were categorized as having moderate periodontitis by ≥2 interproximal sites with clinical attachment level (CAL) ≥4 mm, or by ≥2 interproximal sites with probing depth (PD)≥5 mm (not at the same tooth). Severe periodontitis was defined as ≥2 interproximal sites with CAL ≥6 mm and ≥1 interproximal site with PD≥5 mm (not at the same tooth).

A trained and calibrated clinician performed all clinical examinations. The intraexaminer reproducibility was calculated before and during the study. The intraclass correlation coefficients for the mean PD and CAL were 0.91 and 0.92, respectively; the intraevaluator kappa index was in the range 0.85-0.96. The presence or absence of bleeding on probing (BOP) and plaque were registered. PD and CAL were measured at all proximal, buccal, and lingual surfaces to the nearest millimeter by a calibrated standard probe (UNC-15, Hu-Friedy, Chicago, IL, USA).

Microbial sampling on periodontitis patients was completed on pockets >5 mm. The deepest six pockets were designated for sampling. After removing supragingival plaque with a curette and isolating the area with cotton pellets, paper points (Maillefer, Ballaigues, Switzerland) were inserted into each periodontal pocket for 20 seconds. One paper point from each site was introduced into an empty 1.5-mL microfuge tube for polymerase chain reaction (PCR) analysis.

The presence of T. forsythia was detected by PCR using primers designed to target the relevant 16S rRNA gene sequences, following the approach of Ashimoto et al. [14]. Briefly, PCR mixtures (50 mL) were prepared with 5 mL of bacterial DNA (GoTaq Flexi DNA Polymerase, Promega, Madison, WI, USA), 0.5mM species-specific primers, 10 mL of 5 PCR buffer (Deoxynucleotide Triphosphates, Promega), 1.25 U of Taq DNA polymerase (Deoxynucleotide Triphosphates, Promega), 0.2 mM dNTP mix (Deoxynucleotide Triphosphates, Promega), and 1.5 mM MgCl2 (Promega). Gene-specific amplification was performed in a thermal cycler (MyCycler Termal Cycler, Bio-Rad Laboratories, Hercules, CA, USA) with the following thermal profiles: T. forsythia, initial denaturation step at 95℃ for 2 minutes, followed by 36 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 1 minute and extension at 72℃ for 1 minute, and a final extension at 72℃ for 2 minutes. The PCR products were electrophoresed on 1% agarose gels and stained with 0.5-mg/mL ethidium bromide, and the presence of target bands for each bacterium was confirmed.

Peripheral blood was collected from each patient to determine serum antibodies IgG1 and IgG2 against T. forsythia. Enzyme-linked immunosorbent assay (ELISA) was performed as follows: A 96-well plate (Immulux HB, Dynex Technologies, Chantilly, VA, USA) was covered with 50 µL in a 10-µg/mL concentration of sonicate of T. forsythia. (ATCC43037) of carbonate buffer (pH 9.6) incubated overnight. The plates were blocked with 150 µL of solution of phosphate buffer (PBS; pH 7,2) with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, Mo, USA), for 1 hour and 1% milk and avidin (Avidin/Biotin Blocking Kit, Vector Laboratories Inc., Burlingame, CA, USA). The sera were diluted 1/100 in PBS-Tween-BSA (1%)-biotin solution (Avidin/Biotin Blocking Kit) and were incubated at 37℃ for hour. Anti-IgG1 dilution by 1:5,000 (mouse antihuman IgG1, Invitrogen, Grand Island, NY, USA) or anti-IgG2 dilution 1:10,000 (mouse antihuman IgG2, Sigma-Aldrich) in PBS-Tween-BSA (1%) solution, were incubated for 1 hour at 37℃. Streptavidin peroxidase (Invitrogen) in a 1/1,500 dilution was incubated for 1 hour at room temperature. Between each incubation period, three washes were carried out with 250-µL PBS-tween (0.05%) solution and 2 minutes of stirring (Multiwasher-MW2001, SUMA, Immunoassay Center, Havana, Cuba). Finally, 50-µL phosphate/citrate buffer solution (0.5 M, pH 5) was added with o-phenylenediamine (Opd-Pierce), and a concentration of 1 mg/mL was added and activated with H₂O₂ for 5 minutes at room temperature. The reaction was stopped with a solution of sulphuric acid (2.5 M). Absorbance values were read at 490 nm (Lector Elisa stat fax 2100, Awareness Technology Inc., Palm City, FL, USA). The concentration of IgG1 and IgG2 in the samples for T. forsythia was calculated by linear regression analysis with a known concentration curve (5 µg/mL and 0.152 µg/mL, dilution 1:2) of human immunoglobulin IgG1 o IgG2 (Sigma-Aldrich). Every sample was measured in triplicate.

Antibody titers were calculated as follows: An arbitrary serum confirmed to have a moderate antibody level judged by western immunoblotting was used as a standard and taken as one EU and then serially diluted on every plate to generate a standard curve. As defined by Bishop et al. [15], each patient's serum was compared to the standard curve, and relative titer values were calculated appropriately.

All microbiological and immunological laboratory processes were completed blindly, without awareness of the clinical status of the study patients or of the periodontal sites tested.

Differences in continuous and categorical variables were examined by independent t-test (data were distributed normally) and chi-square test, respectively. IgG1/IgG2 antibody titers against whole-cell T. forsythia in the periodontitis group were related to mean levels of CAL, PD, presence of plaque, and BOP using an independent t-test. Linear regression analysis was applied stepwise in order to analyze whether the IgG1 antibody against whole-cell T. forsythia is a protective factor for CAL; the model was adjusted for age (years), gender (male/female), smoking status (smoker/nonsmoker) and socioeconomic status (amount of monthly personal income≥750 USD; yes/no). Multicollinearity was not found during model fitting. Values of P<0.05 were considered statistically significant. All analyses were performed using PASW Statistics ver. 18.0 (SPSS Inc., Chicago, IL, USA).