Observational Study of the Association Between Periodontal Disease and Cardiovascular Disease

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.

Inclusion Criteria:

- Patients with known CAD with and without previous MI, OR

- Patients with no known CAD

- Ages 18-89 years

Exclusion Criteria:

- Patients with a known history of rheumatoid arthritis.

- Patients with a known history of lupus.

- Patients currently under the care of a rheumatologist.

- Patients who have had a MI in the two weeks prior to entering the study.

- Patients with a previous history of coronary artery bypass graft.

- Patients who have taken antibiotics in the two weeks prior to entering the study.

- Patients who are unable to provide at least 2 out of the 3 gingival samples.

- Current smokers.