Human subjects' protection
The details of the methods section have been published previously.24 This study was approved by the Ethics (institutional review) Board of Kuopio University Hospital and the University of Kuopio in 1995. Written informed consent was obtained from all participants. The longitudinal portion of the study was approved by the Boston University Institutional Review Board in 2005. This project adhered to the guidelines set forth by the Declaration of Helsinki and the Belmont Accord to assure the protection of human research subjects.
Study population
The description of the study population and the endpoint have been published previously24 but are briefly reiterated here. The Kuopio Oral Health and Heart (KOHH) study was started in 1995-1996 to explore the association between oral health and coronary artery disease (CAD). For the longitudinal part of the study, the mortality data (median follow-up of 18.8 years) were added to the baseline data to create a prospective follow-up study assessing oral infection impacts on CVD mortality. At baseline, 256 consecutive patients attending the Kuopio University Hospital coronary angiography unit and with a confirmed diagnosis of CAD were recruited to participate in the KOHH study. Also, 250 age- and sex-matched controls were recruited from the general surgery or otorhinolaryngology departments at the same hospital. The controls were determined by 'not having heart disease' based on their medical history and the pre-admission tests. The controls resided in the same geographic area where the cases arose. The same exclusion and inclusion criteria were applied to the control subjects. For further details regarding this cohort, please refer to the previous publications.24,25
Endpoint determination
The CVD mortality data were obtained from the Finnish Death Registry in every year from 2009-2015. The current study used the mortality report of 2015. Using the World Health Organisation's International Classification of Diseases-10 codes, I00 through I99 were considered CVD mortality due to atherosclerotic heart disease and stroke. The reliability of these data was very high, with 99% after comparing the 2009 and 2011 records in a random sample of 100 records.
Predictor assessment
At the initiation of this study (1995-1996), a single examiner (MS) conducted dental examinations. For the current study, the edentulous subjects who cannot floss were excluded. The exposure, that is, OHS, was assessed by questionnaire. Toothbrushing was assessed in four categories: 1) brush once or less frequently a week; 2) brush several times a week; 3) brush once a day; and 4) brush more than once daily. We created a dichotomy of brushing by combining the lower two and upper two groups. Similarly, a dichotomy of flossing was created from the four categories by collapsing the first two and the last two categories: 1) never; 2) once a week; 3) several times per week; and 4) daily.
Although large amounts of plaque accumulation on teeth surfaces are a risk factor for periodontitis,26 host factors, such as genetics, and lifestyle factors, such as smoking, obesity, diabetes and OHS, also contribute to periodontitis development.27 To assess how mouthwash changes oral microbe proportions, we collected plaque samples from the worst-affected periodontal sites and analysed by rapid multiplex rt-PCR tests using species-specific 16S rRNA gene primers.28 The periodontal pathogens assessed were Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Tannerella forsythia.28 Similarly, gram-positive microbes were tested from the same plaque samples. The samples were cultured and Streptococcus mutans and Lactobacilli spp. were identified using the analytical profile index kits (Biomerieux, Espoo, Finland)29 at the Kuopio University Hospital laboratory.
Mouthwash usage was assessed by questionnaire. If the patient used mouthwash daily or several times a week, it was considered exposed, and never used or used less frequently than several times weekly were considered as controls. We do not know which patients used what brand, but the brand names of the mouthwashes include chlorhexidine, Listerine (essential oils), products containing 0.05% cetylpyridinium chloride and Meridol (amine fluoride).
Confounding factors
Age in years and smoking in three categories (never, past and current smokers) were assessed. Total cholesterol, triglyceride and high-density lipoprotein cholesterol (HDL) were measured by the automated enzymatic technique. We assessed dyslipidemia by total/HDL cholesterol ratio which was proven the best predictor of future atherosclerosis.30 Diabetes was ascertained by medical record review. Subjects were considered to have diabetes if documented diagnoses were in the medical records or if they were being treated for diabetes. To avoid confounding by affluence and high socioeconomic status, we adjusted educational levels, income and private insurance status.
Inflammatory markers
C-reactive protein (CRP) was measured by immunoturbidimetry utilising the Hitachi 717 analyser (Boehringer Mannheim, Mannheim, Germany). All blood samples were collected after fasting if required and analysed immediately in the hospital laboratory.
Salivary lysozyme (SLZ) is a proteolytic enzyme in the oral innate immune system expressed by neutrophil leucocytes and macrophages in response to exposure to bacteria.31 SLZ is capable of cleaving peptidoglycan of the bacterial cell wall, resulting in bactericidal action and activates the nucleotide-binding oligomerisation domain-like receptors. Thus, SLZ is a marker for oral innate immune activation, which can rupture both gram-positive and gram-negative bacterial cell walls. SLZ was quantified utilising Micrococcus lysodeikticus (Sigma Chemical Co., St. Louis, MO, USA), human milk lysozyme (Sigma Chemical Co.) and bovine serum albumin (Sigma Chemical Co.) as standards according to the methods described previously.32
Dental plaque scores were created, assigning: 0 = if no visible plaque was present; 1 = if plaque covered gingival 1/3 of the tooth surface; 2 = if plaque covered gingival 2/3; and 3 = if plaque covered the whole surface evaluated. Then, mean dental plaque indices were calculated by summing all plaque indices and dividing by the sum of the surfaces evaluated. Mean gingival bleeding indices were created similarly by summing all surfaces with gingival bleeding and dividing by the sum of the surfaces evaluated.