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Evaluator training consisted of a calibration exercise, which consisted of 2 separate evening sessions. In the first session, the residents attended a thirty minute presentation given by the trainer (A.M.), which provided an overview of the research study, reviewed the study protocol, and the definitions and techniques involved in data collection. Each evaluator then demonstrated the oral swab technique intra-orally on another evaluator, while being observed by the trainer to ensure proper technique. The oral swabs (MedPro Sterile Cotton Tipped Applicators 6”, AMG Medical Inc., Montreal, Canada) were then taken to the lab for oral

PMN quantification. Next, each evaluator performed periodontal probing (periodontal probe:

UNC-15, Hu-Friedy Mfg. Co., Inc., Chicago, USA), on six selected teeth of three individuals, and these probing depths were compared to those done by the gold standard on the same evening. For ethical and logistical reasons, it was not possible for evaluators to be calibrated on patients with severe chronic periodontitis. However, one of the three individuals that were measured by the evaluators had probing depths that were greater than or equal to 5 mm. The gold standard (GS) for dental probing in this study was a Periodontist (H.C.T.), with over 25 years of experience in the field.

In the second session, the evaluators attended a fifteen minute presentation given by the same trainer, which reviewed the definitions and techniques involved in data collection. Each evaluator then demonstrated the oral swab technique intra-orally on another evaluator, while being observed by the trainer. The oral swabs were then taken to the lab for oral PMN quantification. Next, each evaluator performed periodontal probing on the same six teeth of the same three individuals from session one. These probing depths were compared to the probing depths that had been performed by the same gold standard on the evening of the second session (inter-rater reliability), and those performed by the evaluator at the first session (intra-rater reliability).

A summary sheet of the study protocol was given to the residents on the second training session to allow for review of definitions prior to their clinical rotations. The same summary sheet was also available in the operating room for reference. Where possible, the trainer also attended the first clinical rotations for all evaluators to ensure similarity of assessment for the periodontal indices.

Patient Selection Patients attending the Mount Sinai Hospital Dental Program for Persons with Disabilities in Toronto, Ontario, Canada, for comprehensive dental care served as potential participants for this study. At the time of initial consultation and subsequent recall examination, each patient has a comprehensive medical and dental history review, as well as an oral examination, which includes an assessment of dental hard and soft tissues, including periodontal health status. Depending on the extent and severity of disease, treatment needs, and level of cooperation assessed according to the Frankl Behaviour Rating Scale (Frankl, Shiere, & Fogels, 1962; see Appendix 1), patients may require dental examination and treatment under GA. The patient is then put on a prioritized waiting list, currently twelve months long, to have comprehensive dental treatment under GA in the operating room. Based on a chart review of patients that are on this waiting list and planned to have dental treatment under GA, a review of the medical and dental history was conducted to identify potential patients using the following inclusion and exclusion criteria.

Inclusion Criteria

1. Patients aged greater than or equal to 18 years.

2. Patients that received Frankl Behaviour Ratings of Negative and Definitely Negative at the most recent clinical examination.

3. Informed consent obtained from patient, parent, legal guardian, and/or designated substitute decision maker.

Exclusion Criteria

1. Patients taking medications associated with gingival hyperplasia (i.e. anticonvulsant Dilantin, calcium channel blockers, immunosuppressants).

2. Patients who revealed gingival inflammation or disease due to causes other than

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3. Patients who had been treated with systemic steroids or immunosuppressants within 30 days of the dental appointment.

4. Patients with diseases related to altered neutrophil function or levels (e.g.

autoimmune or immune deficiency disease, neutropenia).

5. Patients who had an edentulous arch (maxillary or mandibular), or less than 14 clinically visible teeth on the last available odontogram.

Patients were further excluded from this study on the day of the general anaesthetic appointment if the anaesthetic procedure was difficult, in order to minimize the time that the patient was under GA and to maximize patient safety. Patients were also not included if the operating room was running behind schedule, to ensure that data collection would not further delay hospital staff, or result in cancellation of another patient that was scheduled later the same day.

Obtaining Consent Eligible patients or their parents, guardians, and/or substitute decision makers were sent a letter of invitation, research information, pre-stamped envelope, and consent form by mail (see Appendix 2). If the consent form was not returned in the allotted time, a follow-up telephone call was made. If participation was declined, no further contact was made.

Consent was further re-affirmed by the trained evaluator on the day of the appointment. An overview of the study patient flow is available in Appendix 3.

Operating Room Algorithm The GA for the dental patient was provided by the hospital anaesthesiologist. After nasotracheal intubation and standard draping, a Molt mouth prop (Molt Mouth Gag Adult Size, Hu-Friedy Mfg. Co., Inc., Chicago, USA), was used to maintain mouth opening, and the oral

cavity and oropharynx were suctioned. Data collection was then performed as follows:

1. Acquisition of Oral Swab Data A cursory oral examination was followed by the acquisition of the oral swab. The oral swab data was collected first to limit alterations in the gingival crevicular fluid and potential blood transfer to the swab. A sterile oral swab (MedPro Sterile Cotton Tipped Applicators 6”, AMG Medical Inc., Montreal, Canada), was traced in a continuous motion at a 45 degree angle towards the gingival sulcus until the gingiva was noted to blanch, on the buccal surfaces of the maxillary dental arch from first permanent molar to the contralateral first permanent molar. The swab head was also rotated periodically to ensure coverage of the entire swab head. If an edentulous space was encountered then the swab was lifted off and then approximated on the next tooth in sequence. Another sterile oral swab was traced in the same manner on the mandibular arch, resulting in two oral swabs per patient. Each swab was placed in an individual Eppendorf tube (AdvanTech Research Products, Mississauga, Canada), containing 0.5mL of 0.9% sterile saline to create a solution, and transported to an off-site lab for processing as described later.

2. Acquisition of Periodontal Examination Data The periodontal examination consisted of recording conventional parameters on six selected teeth, tooth numbers 16, 21, 24, 36, 41, 44 (Ramfjord, 1959). If the selected tooth was not present, the adjacent and most similar tooth was substituted.

The following clinical parameters were recorded: gingival pocket depths including 6-point probing of selected teeth (periodontal probe: UNC-15, Hu-Friedy Mfg. Co., Inc., Chicago, USA), bleeding on probing, recession, mobility, plaque index, calculus index, modified gingival index, and VAS for gingival inflammation, using a standardized method for data collection. Definitions of the clinical parameters used in this study are noted in Appendix 4. Depending on the parameter measured, the 6 or 36 individual tooth scores for each variable were averaged, resulting in a mean score for each parameter.

The gingival health status of patients was categorized based on probing depth

measurements from 36 tooth points according to the following classification:

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3. Clinical and Radiographic Examination The soft tissues of the oral cavity were examined for pathology, followed by a hard tissue examination of teeth that were present for caries. Radiographic examination then followed based on the clinical findings.

4. Dental Treatment Review of the complete clinical and radiographic examinations was followed by treatment planning and throat pack placement, scaling and root planing with ultrasonic instrumentation, dental prophylaxis, and then restorations and/or extractions as per the individualized dental treatment plan.

5. Oral Neutrophil Quantification Oral swabs were transported to an off-site laboratory (University of Toronto, Fitzgerald Building Room 241, 150 College Street, Toronto, Ontario, Canada).

Swabs were stored at 4ºC to preserve the cells prior to transportation and until processed. Each oral swab had been contained in an individual Eppendorf tube containing 0.5 mL of 0.9% sterile saline, resulting in solution. Oral swab solutions were then placed in the centrifuge (Hettich Rotina 35R, Kare Scientific, Edmonton, Canada), at 2500 rotations per minute for 10 seconds at 21ºC. The swabs were then removed from the tube while squeezing the swab head, resulting in PMN lysis and a MPO-containing solution. Twenty milligrams of ABTS (AO; Sigma Chemical, Burlington, Ontario, Canada), was dissolved in 3.6 milliliters of phosphocitrate buffer to produce a 1X concentrated solution. Hydrogen peroxide (45.6 microliters) was then added to double distilled water (3.952 milliliters) to produce a homogenous solution. For each sample, 50 microliters of ABTS solution followed by 50 microliters of hydrogen peroxide solution were combined to allow for the characteristic blue colour change. Finally, each sample was placed in one or two wells of a 96 well plate, and the absorbance measured using the FLUOstar Optima microplate reader (BMG LabTech, Germany). The absorbance was measured at 420 nanometers light for ten cycles at 180 seconds per cycle. Absorbance measures obtained from each well were averaged, and the PMN concentration was calculated from a standard curve, obtained from blood PMN quantification in a parallel investigation (M. Glogauer, personal communication, July 7, 2010). Using this standard curve, the concentration can be read for any unknown sample given its absorbance reading, using the equation for the slope of the line of best fit, resulting in the PMN counts obtained per swab for each patient. The slope of the line used in this study to calculate oral PMN counts was y = 41025x - 6494.8, where x = absorbance

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After the completion of dental treatment, the throat pack was removed, the patient was extubated and then monitored in the post-anaesthetic care unit, and then dismissed with an appointment for follow-up in the dental clinic.

Follow-up Appointment Every patient was scheduled for a follow-up and recall examination at the Mount Sinai Hospital Dental Clinic after the GA appointment, according to the patient‟s usual dental recall schedule (i.e. at 3 or 6 month intervals). At the recall examination, consent for participation in the study was re-affirmed by the trained evaluator. A review of the medical and dental histories was done, in addition to clinical extra-oral and intra-oral examinations. The oral swab technique described earlier (see the Acquisition of Oral Swab Data section above), was then completed for each dental arch. Swabs were typically performed by using the non-dominant hand to support the patients‟ head and retract the lips, and using the dominant hand to collect the swabs as per the study protocol. Most patients required protective stabilization by dental staff and attending caregivers. Periodontal examination was performed and conventional gingival parameters that were assessed at this appointment were the same as those collected while under GA, except for parameters which required dental probing, namely plaque index, calculus index, modified gingival index, mobility, VAS for gingival inflammation, and bleeding on brushing. Definitions of the clinical parameters used in this study are noted in Appendix 4. Finally, scaling and root planing with ultrasonic instrumentation or hand instruments, and/or dental prophylaxis with rotary instruments or toothbrushing was conducted depending on the level of cooperation.

Statistical Analyses All statistical analyses in this study were calculated by IBM SPSS Statistics 20 software (Chicago, USA).

Calibration Analyses Inter-examiner reliability testing was conducted, to ensure reliability of the periodontal probing measurements when compared to the gold standard. Intra-examiner reliability testing was also carried out to ensure reliability within the individual. Reliability testing was accomplished using the intra-class correlation coefficient (ICC), and ICC scores of 0.61 or greater (i.e. substantial agreement), were considered as an appropriate level of agreement.

An ICC score of 0.60 or less would necessitate re-training of the evaluator.

Patient Analyses Non-parametric analyses were used in this study because all variables demonstrated nonnormal distributions. Spearman‟s Correlation was used to assess the correlation between PMN counts and periodontal variables at GA and recall. The Wilcoxon Signed Ranks Test was used to evaluate the effect of periodontal treatment at GA on the periodontal and PMN variables obtained at recall. An Analysis of Covariance (ANCOVA) was used to evaluate potential influencing factors on PMN levels. Statistical tests were interpreted at the 5% significance level (2-tailed). Finally, descriptive statistics were used to assess the feasibility (ease of data collection) of the oral swab technique on the uncooperative special needs patients at recall.

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