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Calibration Analyses and Reliability Testing Of the 14 residents scheduled to be in the operating room during the study period, 11 residents completed the calibration exercise, consisting of 3 hospital dental residents and 8 paediatric dental graduate residents. Seven of the eleven residents were scheduled on clinical rotation when participating patients were assessed under GA, and four of the eleven residents were available for recall assessments. Table 1 illustrates the intra-class correlation coefficients (ICC) for the 7 trained evaluators, who demonstrated excellent or substantial inter-rater reliability in their measurements when comparing sessions 1 and 2. Table 2 shows the ICC for the 7 evaluators, to assess for inter-calibration reliability. In this case, the participant number represents the person that was measured, and the ICC score represents the agreement between examiners when assessing that participant. Again, evaluators demonstrated excellent or substantial inter-rater reliability in their measurements of the 6 participants. It is also interesting to note that all groups improved in their measurements at the second session, except in the case of measuring participant 3. However, even then the ICC reveals substantial inter-calibration reliability, and none of the evaluators required retraining.

Oral Neutrophil Quantification at Calibration Sessions Oral PMNs were quantified from nine residents during the calibration sessions. These residents ranged in age from 24 to 34 years, and consisted of 3 males and 6 females. Medical histories for all residents were non-contributory, and gingival health status was measured via probing depths classified according to the description above (see the Acquisition of Periodontal Examination Data section). Five residents were healthy, while 4 had gingivitis.

A summary of resident characteristics is illustrated in Table 3.

Patient Recruitment During the study period, 101 patients had dental treatment under GA at the Mount Sinai Hospital, and the patient flow and reasons for exclusion from this study are represented in Table 4. Fifty-eight patients (57%) consented to participate in this study. Nineteen patients were excluded from the study after chart review, and therefore were not sent letters of invitation to participate in the study. Most of these were due to the lack of evaluator availability in the operating room, despite efforts to maximize available evaluators during the calibration exercises. Nine patients were excluded if the anaesthetic procedure was difficult, or if the operating room had scheduling delays. Some patients were not assessed if an evaluator could not attend as scheduled, or if the case was cancelled entirely. Patient recruitment was a challenge, due to the patient population involved. Most patients were unable to provide consent for themselves, and instead consent was provided by parents, caregivers, group home managers, or substitute decision makers, who usually did not reside or attend appointments with the patient. Thus, consent had to be obtained before the GA appointment. Eight decision-makers did not receive the consent form in time prior to the scheduled GA appointment. Eighteen decision-makers opted not to participate and most did not provide a reason. It is suspected that the paragraph contained within the consent form In case you are harmed in this study”, caused some decision-makers to decline participation in this investigation. Though there were no foreseeable risks or harms as a result of the study technique or protocol, this paragraph had to be included in the informed consent form at the behest of the Hospital Research Ethics Board. In any case, since caregivers were not required to provide reasons for non-participation, it can only be speculated at this time as to why some did not participate.

Demographics The mean age of patients that were assessed under GA was 31.3 ± 11.3 years, consisting of 31 (63%) males and 18 (37%) females. Participant age, Frankl behaviour ratings, sex, and total number of teeth at the time of GA and recall appointments can be found in Table 5, and are consistent for the two groups. All patients assessed under GA had some extent of developmental delay as shown in Figure 1. Thirty-four patients were rated as Definitely Negative at their last clinic appointment, and 15 were rated as Negative. None were in the positive range, as seen in Figure 2. In contrast, at the recall assessments seven patients were noted to be Positive, which may be due to the differences in responses to treatment being attempted before GA appointments (e.g. procedure requiring local anaesthesia compared to toothbrushing at recall). Differences could also be due to patient-specific factors, where the patient could be having a “good day” or “bad day”, translating into differences in Frankl behaviour rating scores. Twenty-five patients (51%) were non-verbal. Many patients also had concurrent medical conditions, including seizure disorder (n = 21), autism (n = 13), and cerebral palsy (n = 13), as the most common conditions. A complete list of medical conditions noted in the participating patients‟ medical histories is shown in Table 6. Table 7 illustrates the complete list of medications that were noted in the patients‟ medical histories.

The data showed that 80% of patients were taking some type of daily medication.

Prevalence of Gingival Inflammation in Patients with Special Needs According to the classification of gingival health status as used in this study, only one patient in the uncooperative patients with special needs cohort was found to be healthy. About 45% of the patients had gingivitis, while an equal proportion of patients were assessed as having mild (22.5%) or moderate (22.5%) periodontitis. A small proportion of patients (8%) had severe disease. The distribution of patients‟ gingival health status is represented in Figure 3.

Loss to Follow-up Of the 49 patients assessed at the GA appointment, 19 (39%) were lost to follow up. A summary of reasons for loss to follow-up and number of patients involved is available in Table 8. Four patients did not return because they are on an annual GA schedule, due to very poor cooperation. Three patients did not attend recall appointments at the Mount Sinai Hospital Dental Clinic and opted to continue regular follow-ups at their community dentist.

One patient was made edentulous during the GA appointment, and one patient attended the ambulatory clinic at an un-scheduled appointment time and thus, the recall examination was performed by an un-trained evaluator and so study-specific data was not collected for analysis. One patient passed away prior to their scheduled recall appointment, and of course could not be seen. Three patients were scheduled outside of the study period, and six patients “re-scheduled” outside of the study period, where “re-scheduled” may refer to cases where the patient did not attend a confirmed appointment (i.e. “no show”), or re-scheduled in advance of the confirmed appointment. Interestingly, of the 30 patients that did attend recall, the same number of patients “re-scheduled” within the study period to allow for assessment.

In other words, a total of 18 (38%) patients did not attend the originally scheduled recall appointment. Figure 4 represents the time interval at which patients presented for recall, and illustrates that all but one patient attended the recall appointment within or after 4 months.

Patients who did not return for follow-up were only included in the GA appointment data set.

Recall Patient Exclusions for Neutrophil Counts As previously noted, thirty (61%) patients returned for recall examination. Of these 30 patients, three patients were excluded from data analyses involving PMN counts, for reasons described below. Hence, when examining the recall patient neutrophil data, the sample size used was 27 rather than 30.

For one patient, oral swab data were not collected by the trained evaluators in light of the clinical circumstances as follows. This patient had severe developmental delay, was nonverbal, and had concurrent medical conditions noted in the health history. The patient constantly wore a helmet with full face shield due to a history of self-biting as well as of biting others. The helmet was maintained in place with three separate locks. During the recall appointment, the patient was restrained by three people, including care workers and dental staff while the helmet was un-locked and removed and dental assessment and toothbrushing were performed by the trained evaluator. Though the trained evaluator commented that oral swabs could have been performed despite the poor cooperation, it was decided not to proceed in order to maximize the amount of time available for the dentist while the helmet was off. Interestingly, the toothbrush that was used on both the maxillary and mandibular arches was swabbed by two swabs, and measurable PMN counts were obtained. However, these PMN counts were not included in the study as the appropriate swab technique or study protocol had not been followed.

For two patients, oral swabs were performed at recall but PMN counts were not obtained due to failure of the computer associated with the FLUOStar OPTIMA microplate reader. Both patients had been assessed on the same day, and the one-time computer failure resulted in loss of data for both patients. Because the ABTS colour reaction is time-sensitive, the patient samples could not be salvaged once the computer error was noted.

Correlation of Periodontal and Neutrophil Variables for Assessments under GA The correlation between clinically obtained periodontal measurements and PMN variables for assessments under GA, using the Spearman Correlation Coefficient (rs) are shown in Table 9. Total PMN counts obtained from the maxillary and mandibular dental arches are listed both separately and combined. Most of the relationships do not show statistically significant results because the number of teeth that were swabbed was not considered in these calculations. However, as shown in Table 10, multiple statistically significant results were actually demonstrated when the number of teeth that were measured using swabs was controlled for in the calculations. This has basic face-validity since PMNs are collected from specific sites along the gingival sulcus. Therefore, the surface area covered and the PMNs absorbed onto the swab must depend upon the number of teeth present in the measured area.

Hence, this illustrates the importance of controlling for number of teeth present when interpreting the oral inflammatory load of patients based on the numbers of collected PMNs.

It is also noteworthy that consistent relationships for most periodontal variables were shown with PMN counts obtained from the swabs, whether obtained from the maxilla or mandible.

Also, the number of PMNs obtained from the maxilla and mandible correlated significantly with the total maxillary and mandibular PMN values (rs = 0.902 and 0.956 respectively, p 0.01), indicating that it might be possible in the future to take samples only from one arch, thus simplifying the test method even more for this difficult to manage population.

The periodontal parameters are based on the mean score of the whole mouth rather than just the maxilla or mandible separately, therefore the total PMNs per tooth of the combined swabs would reveal the true relationships between the variables and will be evaluated further.

Spearman‟s correlation allowed for comparisons between periodontal parameters and oral PMN counts and demonstrated positive correlations as seen in Table 10. For measures using the periodontal probe, positive correlations were noted for mean probing depths (rs = 0.286, p 0.05), number of probing depth sites ≥ 5 mm (rs = 0.350, p 0.05), and gingival inflammation category (rs = 0.294, p 0.05), indicating that the PMN assay may be helpful to identify cases of inflammation where patients fall into the conventional categories of periodontitis. There was also a positive correlation between the calculus index and PMN counts (rs = 0.385, p 0.01), indicating the presence of a higher inflammatory load. It is interesting that a positive correlation between PMN counts and tooth mobility was found (rs = 0.467, p 0.01). This is important because of all periodontal parameters used for assessment of prognosis, mobility is one of the most significant ones since excessively mobile teeth are usually removed regardless of what other measures might show. Therefore, measurement of PMNs using the PMN assay may be helpful in measuring past and clinically relevant disease activity in the population of patients with special needs. Lastly, the modified gingival index also showed a positive relationship (rs = 0.285, p 0.05), indicating that the PMN assay positively correlated to the subjective impression of the dental examiner when evaluating inflammation based on overall gingival condition.

There were no statistically significant differences found for the following variables: number of probing depth sites ≥ 4 mm, plaque index, bleeding on probing, and VAS for gingival inflammation. This may be as a result of these periodontal variables having consistently higher scores, which may overestimate the presence of gingival disease when compared to levels of PMN cells, which were likely under-estimated as will be described later.

Correlation of Periodontal and Neutrophil Variables for Assessments at Recall The correlation between clinical periodontal measurements and PMN counts made at recall examination is represented in Table 11. There were no statistically significant relationships apparent in this assessment. This could be due to the decreased number of PMNs, which may not allow for findings of association. However, the significant relationship indicated before between the individual swabs and the total inflammatory load while controlling for number of teeth is maintained in this data set (rs = 0.954 and 0.947 respectively, p 0.01), signaling that performing one swab per person could be sufficient for periodontal assessment.

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