Plaque disclosing agent as a guide for professional biofilm removal: A randomized controlled clinical trial

The oral cavity is the natural habitat of a heterogeneous population of bacteria.1 Both soft and hard surfaces are the substrate where microorganisms adhere and grow, forming the oral biofilm.1,2 Biofilm quantity and complexity increase with time and affect the environment, leading to the development of caries, gingivitis2,3 and periodontitis,4 according to individual susceptibility and risk factors. Vice versa, the environment and local factors can influence the growth of biofilm, leading to its diversification in distinct areas even of the same tooth.2 The regular disruption of biofilm through professional mechanical plaque removal and home oral hygiene is a critical point in the prevention of caries and periodontal disease.5-8 Professional mechanical plaque removal in cariology involves biofilm and calculus removal from the supra-gingival area while, in periodontology, it extends to the sub-marginal space.8 While manual and ultrasonic instrumentation constitutes the traditional professional mechanical plaque removal procedure, air-polishing with low-abrasiveness Received: 16 August 2019 | Revised: 1 April 2020 | Accepted: 23 April 2020 DOI: 10.1111/idh.12442

powder is of more recent introduction and is regarded as a promising way to manage supra-and sub-gingival biofilm, with advantages in terms of time and comfort. [9][10][11] The clinical results during periodontal maintenance therapy are comparable with the ones obtained via traditional scaling and root planing. 10,12 Regardless of the instruments used and time, complete biofilm removal from hard surfaces is hardly achievable. 13,14 The aim of professional mechanical plaque removal is to keep the bacterial population below the "critical mass," that is where an equilibrium with the host can exist. 15 Being individual tolerance highly variable and non-definable, 16 it is essential to keep oral biofilm level as low as possible.
Oral biofilm is mostly colourless. Disclosing tablets and liquids can allow its visualization for clinical and research purposes. 17 Disclosing is proven to ensure complete cleaning of molar occlusal surfaces before sealants, 18 increase biofilm control on dentures, 19 allow a more efficient debridement of root surfaces during periodontal resective surgery 20 and, in case of agents able to identify acid-producing bacterial populations, assist in caries risk assessment. 21 The ability to see the biofilm can also improve patients education and motivation and guide their self-performed oral hygiene. [22][23][24] To date, no studies are available involving the use of plaque disclosing agents as a guide for the clinician during professional mechanical plaque removal.
In the research field, application of disclosing agents and subsequent photograph software analysis can be used as an advanced plaque quantification tool, 25,26 allowing to overcome classic plaque indices limitations, such as variability between different examiners and centres. 17 Comparisons between planimetric methods and conventional indices show that the former ones are more precise, objective, sensitive and reproducible, and can detect even small changes in plaque area. 17,25,26 The aim of the present study was to evaluate through computer software analysis-also known as planimetric plaque analysis-the efficacy of the use of a plaque disclosing agent as a visual guide for biofilm removal during professional mechanical plaque removal and compare it with the same procedure without any visual aid in terms of post-treatment residual plaque area (RPA).

| Intervention
A total of 32 eligible subjects were randomized in two groups: the test group received a session of professional mechanical plaque removal guided by the application of a plaque disclosing agent as a visual guide for the clinician (named by the authors Guided Biofilm Therapy-GBT), while the Control group received the same professional mechanical plaque removal procedure without any visual aid.
After the placement of a lips and cheeks retractor (OptraGate ® , Ivoclar Vivadent) and the collection of Plaque Index (PI), 27 the patients were allocated to one of the groups (GBT or Control) via randomization list and numbered opaque envelopes. In the GBT group, the plaque disclosing agent (MIRA-2-TON ® 60 mL bottle, HAGER WERKEN) was then applied by the operator with a micro-brush to cover the entire tooth surface and thoroughly rinsed with water

| Image analysis
The clinical photographs were processed by an operator blinded to the group allocations through ImageJ software (National Institutes of Health). The area covered by the disclosing agent (residual plaque area-RPA) was calculated as % of the total teeth area. blue channels. The green-channel elaborations were chosen for the next step, as green is the colour that better highlights the pink-purple tint of the plaque disclosing agent, shown as dark-grey/black.
Though the colour threshold selection function, the range within the 0-255 greyscale corresponding to the disclosing agent was set, and the pixel-based percentage (hereafter indicated percentage of area with residual plaque) of the disclosing-coloured areas was calculated ( Figure 6).

| Statistical analysis
The sample size was computed assuming a two independent group comparison based on t test allowing for different variances (Welch's test). We assumed 5% and 10% residual plaque (% of plaque are over total teeth inspected area), respectively, and a 60% coefficient of variation for both groups. Considering an 80% power and a 5% significance level, we computed a total sample size of N = 32 (16 for each group). To allow for potential deviations from normality assumption for percentages, we also computed sample size using a Wilcoxon-Mann-Whitney simulation based on 2000 Monte Carlo samples from the null distributions (with parameters as specified above) achieving a consistent (software: PASS 13). Patients were randomized using a computer-generated randomization list. The random allocation sequence was generated with uninformative labels (A and B) and using block randomization algorithm (block size = 4).
All data analyses were carried out according to a pre-established analysis plan by a biostatistician blinded to group allocation. The percentage of area with residual plaque was modelled at tooth level using a linear mixed models (LMM) using a random intercept model with Patient as a random component to account for data clustering.
Residual area values were transformed on logit prior to modelling.
Estimated PI at baseline was computed after aggregation within patients, that is PI was computed as the number of sites with plaque within the subject. This was modelled using a GLM with negative

| RE SULTS
Results are reported as estimate and 95% confidence interval.
Proportional variation is expressed as the variation going from Control to GBT expressed as a percentage relative to Control starting value. it was not possible to use the same planimetric analysis method for initial plaque quantification.
The residual plaque area (RPA) measurements for both treatments on the Gingival and the Coronal surfaces are presented in Table 2 and Graphic 1. A statistically and clinically significant difference between treatments is evident in both location, with GBT achieving a lower RPA, with a proportional reduction going from 49.2% (P-value = .018) on the Gingival surface to more than 60% (P-value = .002) on the Coronal surface. Overall, we also observed a higher RPA on the Gingival surface compared with the Coronal one.

| D ISCUSS I ON
The present study represents the first of a series aimed to investigate and validate the concept of Guided Biofilm Therapy (GBT), whose significant novelties are the use of plaque disclosing as a visual guide and the predominant use of an air-polishing device for biofilm removal. The choice of the authors is due to the desire to progress towards a minimally invasive professional mechanical plaque removal concept and is supported by the evidence that supra-and sub-gingival air-polishing is safe and conservative on both soft and hard tissues, more time-efficient and more comfortable for the patient. 10,28-32 Furthermore, it allows reducing the use of ultrasonic/ manual instrumentation to the minimum required to remove hard calculus.
Plaque disclosing through tablets and liquids is a well-known tool to help patients visualize the oral plaque and improve their self-performed hygiene and compliance, both in a professional and home setting. 33,34 It is also proven to ensure complete cleaning of molar occlusal surfaces before fissure sealing, 18 to increase biofilm control on dentures 19 and to allow better debridement of root surfaces during resective periodontal surgery. 20 In the context of professional oral hygiene, one could assume that the plaque disclosing can be beneficial not only for the patient but also for the clinician as a guide for biofilm removal, allowing immediate feedback, especially for those areas difficult to access and for those individuals at high risk of carious or periodontal pathology. To date, no clinical trials are available to prove the assumption; hence, the present study aimed to measure the potential advantage of the use of a plaque disclosing agent as a visual guide for the clinician during professional mechanical plaque removal, compared with the same treatment without any aid.  While some studies report the use of camera-to-head positioning frames, 25 in some others the photogrphs are taken freely but with the same focal distance and settings. 35 In the present study, we decided to use an extra-oral camera and standardized settings.
Even if a frame for camera-to-head positioning was not used, we are confident that through the use of the same settings, the same expert operator and the randomization process, the results are accurate and reproducible.
A limitation of the image elaboration process adopted could be the necessity to manually select and cut the teeth areas of the images, eliminating soft tissues and background, with the risk of not being able to identify the gingival margin and papillae accurately.
Nevertheless, Smith et al 25 show that manual selection does not impair the intra-and inter-operator reliability, which is still excellent.
Most importantly, as in Smith et al, 25 our protocol does not involve manual area tracing of plaque regions, but an automatized colour encoding by the ImageJ software, eliminating human error in the crucial step of plaque and non-plaque areas discrimination.
At baseline (Table 1), both experimental groups show homogeneity of PI. Because of the design of the study, especially the intervention in the control group, it was not possible to use the same planimetric analysis method for initial plaque quantification.
At the end of the professional mechanical plaque removal session, the RPA in the GBT group was significantly lower than in the Control group. An example of results obtained with GBT and Control is shown in Figures 7 and 8, comparing the subjects clinically and via software analysis. When considering the Gingival and Coronal surfaces separately, the GBT group showed, respectively, half and a third of the mean RPA area of the Control group ( Table 2, Graphic 1).
The decision to analyse the Gingival portion of the clinical crown separately comes from the fact that biofilm at and below gingival  Figure 7, with the plaque disclosing agent highlighted in red G R A P H I C 1 Estimated average percentages of areas with residual plaque and corresponding 95% confidence intervals, grouped for treatment and position know for sure this threshold of tolerance 16 ; hence, the necessity to reduce biofilm as much as possible, especially in highly susceptible patients, such as periodontal, paediatric or orthodontic patients. 5,7,14 The major limitation of the present study is the fact that the computer analysis protocol chosen can be confidently applied only to anterior teeth, since a validated method to take standardized photographs of posterior areas still does not exist, and intra-oral cameras cannot provide the same level of resolution as the extra-oral ones.
Images with dissimilar illumination and angulation can impair the reliability of the software colour analysis and area calculation, hindering the results. Plaque accumulation in the posterior areas is of paramount importance when considering the overall bacterial load and patient's adherence to hygiene instructions, and further investigations are needed to shed some light on this aspect. Furthermore, the software analysis is performed on a 2D image, with limited power to give a real measurement of the interproximal plaque, a crucial area to be kept free-of-plaque in susceptible patients. As mentioned above, another limitation comes from the limited sample size and the type of population selected for the present study, which might not represent the clinical reality for most professional mechanical plaque removal sessions. In future research, it would be of major interest to investigate the role of plaque disclosing in more complex and higher-risk patients and, when the technology will allow it, to perform image software analysis of the posterior areas of the dental arches, where the access for professional mechanical plaque removal is limited. It would also be interesting to conduct the same investigation in conjunction with different protocols of professional mechanical plaque removal, such as the traditional ultrasonic debridement and polishing with a rubber cup and prophylaxis pastes.
In conclusion, within the limitations of the present study, the application of a plaque disclosing agent to guide plaque removal (GBT) seems to lead to better plaque removal, especially in areas of more difficult access.

| Scientific rationale for study
To date, no studies are available involving the use of plaque disclosing agents as a guide for the clinician during professional biofilm removal.

| Principal findings
The application of a plaque disclosing agent seems to lead to better plaque removal, especially in areas of more difficult access.

| Practical implications
The regular use of plaque disclosing agents may improve the level of professionally delivered oral hygiene.