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Comparing Periodontal and Neutrophil Variables Obtained at GA and Recall There was a statistically significant difference in mean modified gingival index, calculus index, mobility, VAS, and total PMNs controlling for number of teeth (p 0.05), when comparing outcomes both before (i.e. at GA) and after treatment (Wilcoxon Signed Ranks Test; Table 12). The difference in PMN counts before and after treatment indicates that the PMN assay may be used to monitor responses to treatment. It also emphasizes that the treatment provided at GA allowed for a significant improvement in the extent of periodontal inflammation as measured by conventional periodontal parameters and oral PMN levels. The mean plaque index did not change significantly when comparing pre- and post-treatment values, and remained consistent over time. This indicates the presence of an undisturbed biofilm or plaque due to continued poor oral hygiene measures.

Factors Influencing Oral Neutrophil Counts An Analysis of Covariance (ANCOVA) was done to assess the factors influencing oral PMN counts at recall, while adjusting for baseline PMN counts and age (Table 13). Notably, there was a significant difference in PMN counts when differentiating for the severity of developmental delay. In fact, patients with severe developmental delay had almost double the number of PMNs at recall when compared to those with mild or moderate developmental delay. An ANCOVA was also performed to examine the influence of the Frankl behaviour rating scale, age, sex, and extent of developmental delay, controlling for baseline levels of PMNs, on the PMNs collected at recall examination. The results (in Table 14) indicate that the extent of developmental delay was the only significant factor to influence the number of PMNs collected at follow-up (F(2) = 3.816, p 0.05). Finally, another ANCOVA was done to examine the influence of Frankl behaviour ratings, age, sex, and extent of developmental delay, on the noted difference in PMNs counts before and after treatment. Again, the severity of developmental delay was the only confounding factor that contributed to changes in this model (see Table 15). This is consistent with the finding that patients with severe developmental delay have poor cooperation (see Figure 5), and so accordingly, they cannot maintain oral hygiene independently, and tend to have poor oral hygiene.

Feasibility of Oral Swab Data Acquisition Despite the lack of cooperativity in this population (see Figure 2), it was possible to use the oral swabs on 100% of the patients that attended recall and were assessed while awake. As noted before, one patient did not have the swabs done by the evaluator, but the evaluator did note that it would have been possible to obtain swabs had they been attempted. Importantly, the skill-set required to obtain the swabs for each dental arch was noted to be the same as would be required to perform an oral exam and/or toothbrushing in this population.

In fact, breakage of the swab was the only test-related complication, and was encountered in 10/98 cases at GA and 4/58 cases at recall examination; or 14 cases in total. In 13/14 cases, swab breakage occurred on the wooden aspect due to operator pressure, and did not cause trauma to the patient or examiner, or interfere with data collection. The swabs used in this study were 6 inches long, and could have been easily broken with finger pressure. This could be remedied by using shorter swabs (i.e. 3 inches), so that the finger pressure would be applied along a shorter length and require greater force to result in breakage. In one case at recall, the patient bit on the swab after data collection was completed, resulting in breakage of the swab head into the patient‟s mouth. The swab head was retrieved in one piece using the evaluator‟s fingers and dental instruments. Again, no trauma was caused to the patient (or evaluator) as a result of this incident.

Patient-related factors that led to difficulties in use of the oral swab were also noted by the evaluators and included patient movement or resistance resulting in decreased visibility of tooth surfaces. Some swabs were acquired after multiple breaks rather than one continuous motion. This may have resulted in the same tooth being swabbed twice (over-estimation of PMNs), or some tooth surfaces not being swabbed at all (under-estimation of PMNs). Lip smacking and tightness of oral musculature were also noted as challenges to oral swab acquisition at recall, resulting in the swab contacting the labial or buccal mucosa. Despite the limitations due to cooperation, it was possible to obtain measurable oral PMN counts from all of the swabs taken at the recall appointments.

Correlation of the VAS with Traditional Periodontal Measures and PMN Counts As a point of interest, the VAS for gingival inflammation as measured at GA and recall was compared to the traditional periodontal variables and the PMN counts obtained by the oral swabs. Table 16 demonstrates a moderately positive correlation of the VAS with measures obtained via periodontal probing. The plaque index, calculus index, modified gingival index, and bleeding on brushing parameters also demonstrated positive correlations with the VAS at GA and recall. With the exception of the calculus index, parameters that were measured at both GA and recall consistently had statistically significant correlations with the VAS.

Notably, mobility and PMN counts did not show statistically significant correlations with the VAS at GA or recall examination.

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High Prevalence of Gingival Inflammation in Patients with Special Needs As expected, the prevalence of gingival inflammation in the uncooperative special needs patients in this cohort was found to be quite high, where a conventional measurement of disease, as well as assessment of PMN levels at one point in time (at GA) demonstrated a high prevalence of disease and high inflammatory load. These results point to the importance of diagnosis of a very prevalent condition in this particular population of patients, where until now, there were no reliable diagnostic tools for identification of periodontitis; at least not until such patients were placed under GA, by which time there might be inadequate time or preparation for treatment, or disease may have progressed to a hopeless state. As described earlier, the failure to identify and treat periodontitis adequately and the persistence of a high inflammatory load can have negative oral and general health consequences.

As far as we know, this is the first study to have measured the prevalence of disease in adult patients with special needs, where a periodontal examination with numerous indices was conducted on patients who were defined as uncooperative, and where the population of interest did not consist of a majority of patients with Down syndrome (see Anders & Davis, 2010, for a recent systematic review of oral health in persons with disabilities). Therefore, the paucity of dental literature in this regard does not allow for comparisons to be made, but indicates that further studies in this area would be invaluable to recognize, manage, and hopefully prevent periodontal diseases in this at-risk population group.

Positive Treatment Outcomes in Uncooperative Patients with Special Needs A profound and unexpected finding was made when comparing the measured clinical parameters of gingival inflammation, including traditional periodontal parameters and PMN levels, before treatment (i.e. at GA) and after treatment (i.e. at recall). A significant improvement in all of the measured clinical parameters (except plaque index), and a lower inflammatory load was revealed – a treatment effect that was discernible despite a longer than ideal recall interval (greater than 4 months in most cases). The positive treatment outcomes that were noted as a result of one session of non-invasive periodontal treatment (scaling and root planing with ultrasonic instruments), are especially encouraging for the clinicians involved in treating this patient population everyday.

In this study, the mean plaque index did not change significantly when comparing pre- and post-treatment values, and remained consistent over time. This indicates the presence of an undisturbed biofilm or plaque due to poor oral hygiene measures. Though these results are consistent with findings from patients with chronic periodontal disease (Cobb, 1996; Drisko, 2001; Cobb, 2002; Suvan, 2005), similar studies have not been done in uncooperative patients with special needs. In the face of poor oral hygiene, the provision of regular dental care is often met with a sense of inevitable deterioration of the oral condition. At best, it is hoped that dental treatment may prolong the certain and eventual loss of teeth in this patient population. However, this study has shown that the provision of dental treatment as provided at the Mount Sinai Hospital Dental Program for Persons with Disabilities is effective, and moreover provides a direct benefit in reducing the oral inflammatory load. The difference in PMN counts before and after treatment also indicates that PMNs respond to treatment and that the PMN assay may be used to monitor treatment needs and responses to therapy, which could not be measured before in the uncooperative special needs population.

Correlation of Periodontal and Neutrophil Variables for Assessments under GA For measures using the periodontal probe, positive correlations were noted for mean probing depths, number of probing depth sites ≥ 5 mm, and gingival inflammation category, indicating that the PMN assay may be helpful to identify cases of inflammation where patients fall into the conventional periodontitis categories. The calculus index correlated positively with PMN counts, indicating the presence of a higher inflammatory load consistent with the presence of calculus where more gram negative bacteria would be expected (Socransky & Haffajee, 1992). It is interesting that mobility had the strongest positive correlation, which is an index that occurs when disease is present and clinical attachment loss has already occurred. Therefore, the PMN assay may be helpful in measuring past disease activity. Lastly, the modified gingival index was also positive, indicating that the PMN assay correlated to the subjective impression of the dental examiner when evaluating overall gingival condition.

As described earlier, the number of probing depth sites ≥ 4 mm, plaque index, bleeding on probing, and VAS for gingival inflammation did not have statistically significant relationships. One explanation for this is that these types of periodontal variables generally produce higher scores, which may overestimate the presence of gingival disease when compared to PMN levels, which were likely under-estimated as will be discussed later.

Nevertheless, with PMN counts it is possible to obtain an understanding of disease activity versus signs of disease. This could also speak to the value of the PMN counts as a measure of gingival inflammation, where the conventional measures of periodontal assessment are lacking as noted previously. This area of the analyses also demonstrated the importance of controlling for the number of teeth present when interpreting the oral inflammatory load of patients based on the number of collected PMNs, and also indicated that measuring PMN levels from one arch may be sufficient to identify the patient‟s gingival health status.

Correlation of Periodontal and Neutrophil Variables for Assessments at Recall An assessment of the correlation between PMN counts and periodontal parameters at recall did not reveal any statistically significant relationships. The lack of correlations could be due to the fact that by the time of recall, which occurred after periodontal therapy (scaling and root planing), there was a marked reduction in PMN counts. Moreover, given a response to treatment, multiple clinical periodontal measures would have been recorded as „0‟. This is consistent with studies showing a reduction in leukocytes after treatment, attributable to a reduction of the bacterial load in the periodontal pocket. Leukocytes may still be present in the pocket after treatment due to residual bacteria in the environment, or due to cellular activities related to the healing process (Boretti, Zappa, Graf, & Case, 1995). Our study is also consistent with another study showing a decrease in clinical indices and MPO levels after periodontal treatment, but without significant associations at follow-up with the clinical parameters (Smith, Hinrichs, & Melnyk, 1986). In this case, it was suggested that the narrow range of values for the clinical indices may not allow for an association to be detected between MPO activity and clinical variables. The lack of association may also occur because MPO levels reflect factors other than those associated with the currently used clinical parameters of periodontal health status, which are descriptors of disease history as opposed to disease activity (Smith, Hinrichs, & Melnyk, 1986), and which have also failed to demonstrate significant relationships with systemic markers of inflammation (Beck & Offenbacher, 2002).

Severity of Developmental Delay Influences Gingival Inflammation Another notable finding in this study was the contribution of developmental delay severity to the results, where the extent of developmental delay had a significant influence on oral PMN counts and extent of gingival inflammation. This is consistent with findings in the literature, where patients with severe developmental delay have poor cooperation, and have poor oral hygiene, due to an inability to maintain oral hygiene independently and/or lack of cooperativity to allow for oral health care needs to be met (Gabre, Martinsson, & Gahnberg, 1999; Lindemann, Zaschel-Grob, Opp, Lewis, & Lewis, 2001). About 5% of patients with special needs will require dental treatment under GA to manage their behaviour appropriately, and to allow for provisions of optimal dental care (Roeters & Burgersdijk, 1985; Milam, 1986). The importance of assessment in this small but considerably underserved proportion of the special needs population is undeniable. This further highlights the importance of diagnosis and timely and appropriate management of disease in a population that is at significant risk for oral disease and its systemic consequences (Teng et al., 2002;

Sigal & Sigal, 2006), and underscores the importance of this study and further studies in this field.

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