Observational Study of the Association Between Periodontal Disease and Cardiovascular Disease
Condition: Periodontal Disease; Coronary Artery Disease
MeSH terms: Periodontal Diseases; Cardiovascular Diseases; Coronary Artery Disease
Study type: Observational

Summary: 
      The prevalence of cardiovascular disease is rising; new methods must be created to assess the
      cardiovascular status of patients. If cardiovascular disease can be predicted, it may
      facilitate prevention. An association between periodontal disease and cardiovascular has been
      established, but a definitive mechanism is not understood. A good first step in finding that
      mechanism is to look at the correlation between periodontal disease and cardiovascular
      disease, both of which have an inflammatory component. This study observes the level of
      cardiovascular disease in patients and correlates it with the presence and degree of
      periodontal pathogens.
    

Detailed description: 
      A possible association between periodontal disease and cardiovascular disease was first
      hypothesized in the 1980s. Epidemiological studies performed over the last 20 years have
      shown a strong correlation between periodontal disease and cardiovascular disease. However,
      the literature is somewhat divided, as some studies have shown moderate correlation while
      others have shown independent (insignificant) correlations between the two. This leaves a gap
      in the investigators knowledge that must be brought together through specific studies. There
      have been reports made to suggest that 52% of atherosclerotic plaques studied have been found
      to contain the same pathogens that cause periodontitis. This is the reason that these are the
      bacteria that will be focused on during the course of this study.

      Periodontal disease, known as either gingivitis or periodontitis, is caused by the presence
      of bacteria in the gingival pockets in the mouth which over time can cause systemic
      inflammation through endotoxin production. Some examples of the bacteria that cause
      periodontal disease include Porphyromonas gingivalis and Aggregatibacter
      actinomycetemcomitans. These are gram-negative, anaerobic bacteria that are able to thrive in
      the sub-gingival space after they are able to successfully become established. These two
      bacteria have been known to invade oral epithelial cells, creating the possibility that they
      could invade epithelial cells elsewhere such as in coronary vessels. These bacteria will be
      cultured from the patient and the relative amounts of them as determined by culture
      techniques will be used to assess the prevalence of periodontal disease in the patient. P.
      gingivalis is of special importance because it is known to be found in patients with active
      periodontal disease and not commonly found in patients without periodontal disease. It can be
      identified by the black pigment produced upon culture. Tryptic
      Soy-Serum-Bacitracin-Vancomycin (TSBV) Agar is a selective medium that is able to isolate A.
      actinomycetemcomitans. The identity of A. actinomycetemcomitans can be confirmed based on the
      growth of "star-like" colonies on the TSBV Agar that are catalase positive. The relative
      amount of these bacteria as determined by culture will be used to correlate with the level of
      coronary artery disease based on the groups that each subject is assigned to. This is how the
      basis for an association between the two conditions can be determined.

      Systemic inflammation occurs as a result of the release of pro-inflammatory cytokines in
      response to an infection. Some of the cytokines released, include high sensitivity C-reactive
      protein (hs-CRP), interleukin-1 (IL-1), and interleukin-6 (IL-6). Hs-CRP is a protein that is
      active during the acute phase response to inflammation. Its level in the blood has been shown
      to be markedly raised in chronic inflammation and has consistently been found to be elevated
      in patients with acute coronary syndromes. IL-1 is a pro-inflammatory cytokine that is also
      released as a result of systemic inflammation. Specifically, it has been found to be secreted
      in response to lipopolysaccharide (LPS) from gram-negative bacteria and has been found to be
      elevated in patients with increased periodontal disease. IL-6, like IL-1, is a
      pro-inflammatory cytokine that is part of the acute phase response to infection. It has been
      strongly correlated with increasing levels of coronary artery disease and has even been
      demonstrated as a predictive marker of mortality in Coronary Artery Disease (CAD). In
      addition to its role in CAD, IL-6 has also been found to be elevated in periodontal disease,
      creating the possibility for the basis of the link between these two diseases.

      The aim of this study is to achieve a better understanding of the relationship between
      periodontal disease and coronary artery disease by correlating the severity of coronary
      artery disease with the presence and amount of the periodontal pathogens P. gingivalis and A.
      actinomycetemcomitans. The level of systemic inflammation caused by these pathogens and and
      coronary artery disease will also be assessed. This will determine if coronary artery disease
      can be associated with one of these specific pathogens more than the other and what levels of
      systemic inflammation can be associated with both of these conditions together.

      Samples will be obtained from periodontal tissue, cultured, and the amount of the specified
      pathogens present will be correlated with the type and extent of the patient's cardiovascular
      disease. Blood tests will also be taken to assess the level of systemic inflammation
      occurring as a result of periodontal disease and cardiovascular disease.

      Subjects will be assigned to one of 3 groups based on their cardiovascular status:

        -  Group 1 will include patients with no known history of CAD.

        -  Group 2 will include patients with a known history of CAD without previous Myocardial
           Infarction (MI).

        -  Group 3 will include patients with a known history of CAD and previous MI.

      Charts for patients to be seen in the Cardiology clinic will be pre-screened by a member of
      the study team to determine if the patient is eligible for the study. Patients will be
      consented in a private setting and a cardiovascular disease history pertinent to the study
      will be taken for each patient who consents to participating in the study. Clean floss that
      has been will be used to collect a sample of gingival fluid from between the lower teeth of
      the patient. No measures have been taken to sterilize the floss, so appropriate aseptic
      techniques including the use of sterile gloves to prevent cross contamination. Three samples
      will be collected: in the midline of the teeth, behind the right or left lateral incisor, and
      in front of the right or left first molar. The decision to sample the right or left of the
      patient's mouth will be randomized (coin flip) unless the patient is missing teeth on one
      side and the sample must be taken from the side that will yield a proper sample. At least 2
      out of the 3 samples must be obtained. These samples will be placed in 3 labeled (by study
      identification number and site of sample) sealed vials of thioglycollate broth and cultured
      for 48 hours. A sample will then be removed from the bottom of thioglycollate medium using a
      pipette (to extract anaerobic bacteria) and cultured on specific media for the presence of P.
      gingivalis, and A. actinomycetemcomitans using anaerobic precautions in the University
      Medical Center (UMC) microbiology laboratory. P. gingivalis will be cultured on a specialized
      Columbia agar supplemented with sheep's blood, bacitracin, colistin, and nalidixic acid to
      select for the isolation of P. gingivalis. A. actinomycetemcomitans can be isolated using a
      specialized tryptic soy-serum-bacitracin-vancomycin (TSBV) agar, a specialized agar that
      inhibits gram-positive and gram-negative aerobes and anaerobes. Each sample will be streaked
      onto an agar plate that has been labeled with the study ID and tooth location. The bacteria
      will be cultured using the quad-streak method and cultured for 24-48 hours and the pathogenic
      bacteria will be identified. P. gingivalis will be identified based on the growth of black
      pigmented colonies on the selective agar and A. actinomycetemcomitans will be identified
      based on the growth of star-like colonies on the TSBV agar. The level of periodontal disease
      in the patient will be assessed by the amount of growth on the plates, with a number assigned
      to each quadrant that the bacteria grow in. if they only grow in the first quadrant, they
      will be given a number designation of 1, and growth in the second through fourth quadrants
      will be designated by 2 through 4 respectively. Using this method, the level of the bacteria
      can be quantified, with 4 being the designation for highest relative amount of bacteria. The
      level of each pathogen found in the patients' mouths will be correlated with the severity of
      coronary artery disease in order to determine if there is a significant correlation. It will
      also be determined if one of the pathogens can be correlated with the severity.

      In addition to the gingival sampling, a small portion (3 mL or 0.6 tsp) of the patient's
      blood will be used to test for hs-CRP, IL-6, and IL-1 as systemic markers of inflammation.
      The sample used will be taken during the initial clinic visit when the patient has given
      consent and the gingival sample is taken. The sample will be drawn by a nurse in order to
      obtain the necessary tests. 1 mL (0.2 tsp) of serum will be drawn for each test (0.6 tsp
      total) and sent to the UMC pathology lab for analysis. These levels will be compared to the
      normal values of IL-1 (0-5 pg/mL) and IL-6 (5-15 pg/mL) while hs-CRP will be correlated based
      on increasing levels. This will allow determination of the level of systemic infection caused
      by periodontitis (based on IL-1 level), coronary artery disease (based on hs-CRP level), or
      both (based on IL-6 level). Individual results will be recorded for statistical analysis.
    

Observational model: Cohort
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        All patients from ages 18-89 years who are being seen in the Cardiology clinic at TTUHSC
        who meet inclusion and exclusion criteria, and who will consent to participating in the
        study. There will be 20 to 30 patients in each group.