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«EVALUATION OF ORAL NEUTROPHIL LEVELS AS A QUANTITATIVE MEASURE OF PERIODONTAL INFLAMMATORY LOAD IN PATIENTS WITH SPECIAL NEEDS By Anita Moosani BSc, ...»

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Acquisition of Oral Swab Data is Feasible The oral swab technique for procurement of oral PMNs and assessment of inflammatory load is non-invasive and safe. When compared to the periodontal probe, the oral swab does not enter the gingival sulci, avoids the risks associated with being bitten due to application on the buccal surfaces only, and provides minimal trauma to the dental hard and soft tissues if bitten, the latter being common when examining or treating patients with special needs. This study has effectively demonstrated that it was possible to acquire the oral swabs on uncooperative patients with special needs while awake in the ambulatory dental clinic. As would be expected, patient-related factors led to some noted difficulties in oral swab performance (e.g. patient movement or resistance), yet this is consistent with the challenges faced while performing an oral examination or toothbrushing in this group of patients.

Despite the limitations due to cooperation, it was exciting to find that it was indeed possible to obtain measurable oral PMN counts from all of the swabs performed at the recall appointment.

Correlation of the VAS with Traditional Periodontal Measures and PMN Counts Though not a focus of this study, but 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. As noted earlier, the use of the VAS in this manner is not noted in the literature, but is often the only periodontal-type measure that can be obtained in the limited time available for examination of the uncooperative patient in the dental clinic. The VAS for gingival inflammation provides a subjective yet quantitative measure of inflammation, as in the case of the conventional periodontal indices, and in this study the VAS was noted to positively correlate with the multiple indices that were recorded. Therefore, it could be argued that measurement of the VAS could replace the multiple other indices when performing recall examinations, thereby providing the same level of information regarding the patients‟ gingival condition. However, it should also be noted that the VAS did not correlate well to PMN counts, which importantly provides an objective and quantitative measure of inflammation. Again, this could speak to the value of the PMN assay which provides reliable and reproducible information about disease activity, and demonstrates that the PMN assay is reflecting information (i.e. disease activity and extent of gingival inflammation or total inflammatory load), that is different from the information obtained via conventional periodontal parameters.

POTENTIAL LIMITATIONS

Patients with Down Syndrome Periodontal disease is significantly prevalent in patients with Down syndrome for multiple reasons, including neutrophil dysfunction (Morgan, 2007). As noted in the exclusion criteria for this study, patients with altered neutrophil levels were not eligible for participation in this study. However, patients with Down syndrome were included (4 patients in total), to allow for an appropriate sampling of the special needs population at the Mount Sinai Hospital Dental Clinic. Interestingly, though it would be expected that the inclusion of patients with Down syndrome could confound or obscure the results for prevalence of disease, it was observed that the one and only patient that was found to be healthy in this study was a patient with Down syndrome. Two patients had gingivitis, and one patient had severe periodontal disease. Thus, the inclusion of patients with Down syndrome contributes important information about disease distribution and these results were therefore combined with the other patient data.

Patients Taking Medications Various medications have been noted in the literature to significantly affect gingival disease, specifically anticonvulsant Dilantin, calcium channel blockers, and immunosuppressants (Armitage, 1999; Mariotti, 1999; Kinane & Marshall, 2001). As noted in the exclusion criteria for this study, patients taking these medications were not eligible for participation in this study. Albeit rare, it has been noted that other medications may impact gingival disease (Anderson, Rapley, & Williams, 1997), and MPO levels (Frimat et al., 1997). However, patients taking medications other than those specifically noted in the exclusion criteria were included to allow for a true representation of the patient population at Toronto‟s Mount Sinai Hospital Dental Program for Persons with Disabilities, and specifically the uncooperative patients with special needs cohort.

Other Sources of Peroxidases The oral cavity is a source of many peroxidases, with contributions from salivary glands and cellular components (Castagnola et al., 2011). The colourimetric reaction used in this study is a reaction that involves peroxidase and is not specific to myeloperoxidases from PMN granules. Therefore, it may be argued that this could have led to an over-estimation of PMN counts. However, the PMN is the most abundant source of MPOs (Klebanoff, 2005), and thus has been cited as an indicator for PMN activity, quantity, and tissue inflammation (Cao & Smith, 1989). Also, any contribution of the oral biofilm to the PMN counts may be considered to be consistent for the pre- and post-treatment swabs, as the high plaque index noted at GA did not show a significant change at follow-up. In addition, healthy control samples of saliva using the oral rinse technique have shown that PMN levels are barely detectable (M. Glogauer, personal communication, July 7, 2010), indicating that the levels of salivary peroxidase are negligible and do not impact the PMN assay. In spite of this, efforts were made to limit the contribution of salivary peroxidases, via suctioning of the oral cavity and retraction of lips during oral swab acquisition.





Loss to Follow-Up Of the 49 patients assessed at the GA appointment, a significant proportion (39%) was lost to follow-up. As previously noted, half of the patients that were lost to follow-up did not attend their regularly scheduled appointment, and 38% of patients in this study overall also did not attend their initially scheduled appointment. Missed and/or infrequent appointments are common in this population for a number of reasons. Patients may not be able to attend due to distances involved in travelling to the clinic, which can often be greater than one hour for one way of travel due to difficulties in accessing dental care in the local community. Limitations may also include vehicle availability, reliance on public transportation, weather or traffic delays, illness, and poor cooperation (i.e. not sitting in the car or not leaving the vehicle on arrival to the clinic). In many cases, caregivers are simply overwhelmed by appointments and so it is understandable that patients may not be able to attend regularly scheduled recall appointments. The loss to follow-up and consequent reduction in sample size for the recall data set may account for the lack of statistically significant results when comparing conventional clinical parameters of gingival inflammation with PMN counts obtained at recall. It is important to note that these patients have subsequently been seen for on-going care, but not at the ideal recall interval or within the study period.

Plaque Barrier The magnitudes of correlations in this study are lower than expected and were likely limited due to the poor oral hygiene and high plaque index measures noted in all of the patients before and after treatment. A significant plaque barrier was noted during GA and recall appointments and may have prevented absorption of cells onto the oral swab – leading to underestimation of the inflammatory load. Though the biofilm would have also contained pathogens and PMNs within it, the plaque aggregate that was collected on the swab was discluded from the samples that were measured using the FLUOstar Optima microplate reader. Pre-measurement wipe of excess plaque and debris was considered prior to the study to allow for better access to the gingival sulcus during data collection, but could cause bleeding in the presence of gingival inflammation – leading to exaggerated results of cell counts and therefore was not done. Despite this limitation, it was possible to obtain a measurable level of PMNs from the oral swabs at the GA and recall appointments, and it was possible to ascertain a difference between the swabs when PMN counts were compared preand post-treatment.

Diagnostic Assessment Ideally, this study would have validated the PMN assay using diagnostic assessments, including calculations of sensitivity, specificity, positive and negative predictive values, and evaluation of a receiver operator characteristic (ROC) curve. However, these calculations require a similar proportion of patients in the healthy and diseased categories to allow for appropriate assessment. As discussed earlier, in this study only one patient was found to be healthy. Therefore, the results in this study do not lend themselves to diagnostic calculations, and so were not performed. Future studies with a greater sample size and variant population gingival health status would allow for diagnostic tests for the PMN assay.

Lack of Control Group This study design did not include a control group for comparison of PMN counts, and could limit the interpretation of results. Though a healthy population was measured in this study via oral PMN quantification of the residents participating in the calibration exercises, this is not appropriate for comparison with the study population due to differing sample sizes and population types, and so was not done. Though it is important to note that a review of the PMN counts obtained from the resident group illustrates an appreciable difference in PMN counts when compared to the special needs population, it would be interesting to compare healthy and cooperative special needs groups with the uncooperative special needs population for appropriate comparison in future studies.

Multi-Examiner Approach The periodontal and oral swab assessments were performed by 11 calibrated residents, including graduate paediatric dental and hospital dental residents, over the course of the study. This could be interpreted as a limitation due to the potential source of variation which could obscure positive findings. On the other hand, the fact that significant findings were found in this study despite the multi-examiner approach emphasizes the robustness of results obtained by the PMN assay. Alternatively, this can be viewed as a positive feature of the study protocol, since the PMN assay using oral swabs is meant to be used for multiple practitioners with minimal training and calibration.

One Piece of the Puzzle Recognizing the complex nature of periodontal disease pathogenesis illustrates the importance of analyzing the results of a test that looks at one component of disease (PMN presence or activity), within the backdrop of the entire clinical picture to allow for individualizing test results to the patient. Though the information obtained from the PMN assay regarding inflammatory load are encouraging, they should be evaluated in the context of the overall gingival health status as assessed by the currently standard periodontal examination. A greater number of PMNs could indicate more severe inflammation, or could be due to the interaction of PMNs with subgingival plaque microbes (Smith, Hinrichs, & Melnyk, 1986). Increased MPO levels can indicate PMN activity and the degree of gingival inflammation, but do not specifically indicate periodontitis (Cao & Smith, 1989). It is difficult to interpret the significance of a single study that evaluates levels of any particular marker of inflammation. Nevertheless, promising results from smaller studies need to be confirmed in large, randomized controlled trials. The potential advantage of salivary analyses in aiding the diagnoses of systemic disease indicates that further studies are justified in this field (Kaufman & Lamster, 2002). The goal of being able to differentiate sites that are vulnerable to progress to periodontal disease is still elusive. In this study, identification of gingival inflammation has been shown to be possible using MPO levels to quantify PMNs.

However, discriminating between sites that will progress to periodontal disease may not be possible until destruction has occurred. Thus, risk assessment, decisions about treatment intervention, and monitoring the results of the treatment may be the best solution for now (Fine & Mandel, 1986). Therefore, the PMN assay will serve as a valuable tool in providing quantitative information that will aid in the diagnosis of gingival health status in uncooperative patients with special needs.

CLINICAL SIGNIFICANCE

Besides the advantage of providing an objective and quantitative measure of the gingival status in a safe, non-invasive, and atraumatic fashion, in the special needs population the PMN assay could reveal total inflammatory load, guide treatment decisions and recall intervals, provide screening to prioritize treatment times for GA based on extent of disease, and allow for monitoring of responses after treatment is delivered. Perhaps most importantly, because the PMN assay correlated to the subjective impression of the dental examiner, the PMN assay may be more appropriately used by medical professionals or nurse practitioners to recognize disease and allow for appropriate referral to the dental team.

Appropriate diagnosis would guide the clinician to specific treatment needs and direct individualized treatment plans, leading to a state of improved or optimal oral health in patients with special needs. The ultimate goal is that by providing treatment that leads to improved oral health, we will ultimately reduce patients‟ risks for other general health problems including diabetes, cardiovascular diseases, and pulmonary infections.

In the general population, the PMN assay could allow for site-specific measurement of inflammation, monitoring of treatment responses, and reveal total inflammatory load. It may also serve as a simple monitoring tool for other at-risk populations for oral disease such as the medically compromised or patients with long hospital admissions. Of course, further studies are required to firmly establish the value of the PMN assay in the aforementioned functions.

FUTURE DIRECTIONS



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