The Effectiveness of Brushing and Flossing Sequence on Control of Plaque and Gingival Inflammation-A Randomized Controlled Clinical Trial in Klinik Pergigian, MMMC, Melaka
Phase: NA
Conditions: Gingival Inflammation, Plaque, Gingival Bleeding, Plaque Control
Sponsor: Melaka Manipal Medical College

 
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COVER LETTER                 
04/09/2019 
 
To whomsoever it may concern, 
 
This is to certify that this is the bonafide title of our study- “The Effectiveness of Brushing 
and Flossing Sequence on Control of Plaque and Gingival Inflammation- A Randomized 
Controlled Clinical Trial in Klinik Pergigian, MMMC, Melaka.” with a NCT number: 
NCT03989427.  
We hereby assure that this was a bonafide study approved by Institutional Ethics and 
Research Committee of Melaka-Manipal Medical College (MMMC/FOD/AR/B6/E C-2019 
(21). To the best of our knowledge all the information provided in this study is true and is 
being reported is being reported in compliance with the declaration of Helsinki for clinical 
trials.  
                      NAME OF RESEARCHERS 
                    Prof. Dr. Rajesh Hosadurga 
                    Prof. Dr. Htoo Htoo Kyaw Soe 
                    Sacha Miriam Augustus 
                      Deevatharshini a/p Jayabalan  
                      Jayahneiswary a/p Ganesan 
 
 
 
 
 
 
 
 
 
 

 
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Table of Contents 
OPERATIONAL DEFINITIONS 
 
LIST OF ABBREVIATIONS 
CHAPTER 1: INTRODUCTION 
1.1 Research Background 
1.2 Problem Statements 
  
 
 
1.3 Research Justification 
1.4 Research Objectives 
1.4.1 General Objective 
1.4.2 Specific Objective 
1.5 Research Questions 
1.6 Research Hypothesis (Null hypothesis) 
 
CHAPTER 2: LITERATURE REVIEW 
 
CHAPTER 3: THEORETICAL FRAMEWORK/CONCEPTUAL FRAMEWORK 
 
CHAPTER 4: METHODOLOGY 
 
4.1 Study Design 
 
     4.1.1 Type of study 
 
     4.1.2 Sampling method 
 
     4.1.3 Randomization if any  
 
4.2 Study Period 
 
     4.2.1 Reference Population 
 
     4.2.2 Source Population 
 
     4.2.3 Sampling Frame 
 
 
 
- Inclusion Criteria 
 
 
 
- Exclusion Criteria 
 
4.3 Sample Size Calculation 

 
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4.4 Variables and Research Tools 
4.5 Statistical Analysis 
 
4.6 Data Collection and Flow Chart 
 
4.7 Ethical Consideration 
 
CHAPTER 5: RESULTS 
 5.1 Descriptive Statistic/Sociodemographic Profiles 
  5.2 Table/ Graphs 
 5.3 Inferential Statistic answering the objectives 
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Operational Definitions 
 
1. Rustogi Modified Navy Plaque Index(RMNPI): 
 
This  index divides buccal and lingual surfaces into nine areas(A to I) that  are scored for the 
presence (score=1) or absence (score=0) of plaque. 
Whole mouth=Areas A,B,C,D,E,F,G,H and I; Marginal areas A,B and C; Interdental D and F 
 
 
 
 
Presence of plaque: Score 1 
 Absence of plaque: Score 0 
 
Buccal surface- 9 areas (A to I) 
Lingual surface- 9 areas (A to I) 
Interdental - D and F 
 
RMNPI Score= (𝑆𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 "′1′"  𝑚𝑎𝑟𝑘𝑠 )/(𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑖𝑡𝑒𝑠 𝑠𝑐𝑜𝑟𝑒) 
 
 
2. BPI-Bleeding point index (bleeding point index, BPI; Lenox et al, 1973) 
Bleeding on probing is recorded  and  provides  evaluation  of  gingival  inflammation  
around  each  tooth  in patient’s mouth 
 
 Evaluates level of oral hygiene performance  
 A periodontal  probe is inserted 1 mm into the sulcus at the buccal, lingual, 

 
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mesial and distal surfaces 
 After 20–30s scores will be recorded 
 0 – No bleeding 
 1 – Bleeding present 
Percentage of BPI = (𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑙𝑒𝑒𝑑𝑖𝑛𝑔 𝑠𝑢𝑟𝑓𝑎𝑐𝑒)/(𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑛𝑡𝑎𝑙 
𝑠𝑢𝑟𝑓𝑎𝑐𝑒𝑠) × 100 
The percentage of the number of bleeding surfaces will be calculated by:  
 
 
 
 
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑙𝑒𝑒𝑑𝑖𝑛𝑔 𝑠𝑢𝑟𝑓𝑎𝑐𝑒
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑛𝑡𝑎𝑙 𝑠𝑢𝑟𝑓𝑎𝑐𝑒𝑠 × 100 
 
 
Gingivitis- Inflamed gum tissue around their teeth, caused by bacteria found in dental plaque.2 
main types of gingivitis generalized and localized. Generalized has marginal gingivitis and diffuse 
gingivitis. Localized has marginal gingivitis, diffuse gingivitis and papillary gingivitis. 
 
Dental plaque-Resilient clear to yellow-greyish substances.Primarily composed of bacteria in a 
matrix of  salivary glycoproteins and extracellular polysaccharides .Considered to be a 
biofilm.Impossible to remove by rinsing or using sprays. 
 
 
 
List of abbreviation 
 
FB-Flossing and brushing 
BF-Brushing and flossing 
BPI-Bleeding Point Index 
RMNPI-Rustogi modified navy plaque index 
DJ- Deevatharshini Jayabalan 
SA- Sacha Augustus 
JG- Jayahneiswary Ganesan 
DHE-Dental Health Education 
MMMC- Melaka-Manipal Medical College. 

 
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Chapter 1: Introduction 
 Dental plaque removal is critical for preventing and treating periodontal disease. Toothbrush             
and toothpaste are the most reliable means of plaque control. Cleaning should be through and 
performed at regular intervals. Toothbrush has limited ability to reach interdental areas. 
Interdental areas are prone for plaque accumulation and gingival inflammation. Regular 
interdental cleaning results in less gingivitis. Most effective tool has been reported to be dental 
floss. 
1.1 Research Background 
Periodontal disease is the disease of the supporting dental tissues and the most common 
form is plaque-induced gingivitis.1 
 
The removal of dental plaque is the most crucial action for preventing and  treating 
periodontal disease.2 Mechanical methods for plaque removal along with oral 
hygiene instructions are regarded as the first step in periodontal treatment. 
Toothbrush along with toothpaste are still the most reliable means of plaque control, 
provided cleaning is thorough and performed at regular intervals.2 
 
Dentifrice is used as an adjunct to tooth brushing. The indication of use of dentifrices is 
mainly based on the presence of fluoride, antimicrobial agents that aim at further 
reducing the plaque formation or removal of previously established plaque. They have 
been used as plaque removal aids, especially because of their abrasive agents. In 
addition to fluoride and presence of antimicrobial agents, the actual adjunct role of 
dentifrices to mechanical removal of dental plaque is contradictory.2 
 
Toothbrush has a limited ability to reach interdental areas. These areas are prone for 
plaque accumulation and gingival inflammation. Regular cleaning can reduce 
inflammation. Various products have been designed for accessing these sites such as 
dental flosses, interdental brushes and wooden dental sticks.2 The most effective tool 
has been reported to be dental floss. It is effective in type 1 embrasure form. According 
to studies, the sequence of using toothbrush and dental floss may influence the 
removal of dental plaque and consequently reducing the bleeding on probing. 
Bleeding is one of the objective signs of periodontal disease. Therefore, it is possible 

 
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that the sequence of using a toothbrush and dental floss may influence the removal 
of dental plaque and consequently the BPI.1 
1.1. Problem Statements 
Regular oral hygiene using toothbrush and toothpaste are the most reliable for dental 
plaque control. But the cleaning  should  be  thorough  and  performed at  regular 
intervals. The  reason why  the plaque control  is  most  often  inadequate is due to 
limited ability of the toothbrush to reach interdental areas which are more prone for 
plaque accumulation and inflammation.1 
 
According  to  a  systematic  review, flossing  in  addition   to  tooth brushing  is   better  to  
reduce gingivitis compared to only tooth brushing. It is possible that while using the 
tooth brush and dental floss,  the sequence of using toothbrush and dental floss may 
influence removal of dental plaque and gingival bleeding. Recent study by Mazhari et al 
in a plaque regrowth model has shown that flossing first is better than brushing first and 
then flossing. This has given rise to the conflict whether brushing first and then flossing 
is better or flossing first and then brushing is better. (reference) 
 
1.2. Research Justification 
Mechanical  plaque  removal has been  directly related  to  prevention  and  treatment  
of periodontal disease.  Interdental  areas are  the  sites  where   plaque  and  
inflammation  are  most  pronounced. Dental  floss  is an effective tool to access 
these sites. Bleeding on probing is a sign of local inflammatory  response  related  to  
destructive  periodontitis. Tooth brushing and flossing significantly reduces 
gingivitis.1  It  is  possible that the  sequence of using  toothbrush and dental   floss  may  
influence the removal of dental plaque and consequently the gingival bleeding. A study  
done using plaque regrowth model has reported that flossing first and then brushing is more effective. But, plaque 
regrowth model does not necessarily replicate the clinical situation. Cross -over clinical trials of at least  two weeks 
duration in each phase not lasting less than two weeks with a wash out period of seven days (7 days) does provide 
more credible evidence. (put torkzaban article as reference). There are very  few  studies addressing  this 
issue. No  studies have been  done among Malaysian population. Hence, this is be the first  
study  assessing the effectiveness of  sequence of flossing and brushing on gingival 
inflammation among dental students of MMMC.. 

 
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1.3. Research Objectives 
1.3.1 
General Objective 
To study the effectiveness of brushing and flossing sequence on plaque control and 
gingival inflammation 
 
1.3.2 
Specific Objectives 
a) To evaluate the effectiveness of brushing first and flossing later sequence on 
plaque control 
b) To evaluate the effectiveness of brushing first and flossing later sequence on 
gingival bleeding 
c) To evaluate the effectiveness of Flossing first and brushing later sequence on 
plaque control 
d) To evaluate the effectiveness of Flossing first and brushing later sequence on 
gingival bleeding 
e) To compare the effectiveness of Brushing first and flossing later versus Flossing 
first brushing later 
 
1.4. Research Questions 
a) What is the effectiveness of brushing first and flossing later sequence on plaque 
control? 
b) What is the effectiveness of brushing first and flossing later sequence on gingival 
bleeding? 
c) What is the effectiveness of flossing first and brushing later sequence on plaque 
control? 
d) What is the effectiveness of flossing first and brushing later sequence on gingival 
bleeding? 
e) Which is better brushing first and flossing later or flossing first and brushing later? 
 
 

 
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1.5. Research Hypothesis (Null hypothesis) 
The  sequence  of  brushing  and  flossing  does not influence plaque control and 
gingival inflammation.

 
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Chapter 2: Literature Review 
 
 
 
 
 
 
 

 
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Chapter 3: Theoretical Framework/Conceptual Framework 
 
 
 
 
 
 

 
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Chapter 4: Methodology 
 
4.1 Study Design 
4.1.1 Type of study 
        Single blind, cross-over randomized clinical trial 
 
4.1.2 Sampling method 
          Non probability sampling and recruiting volunteers  
 
 
4.1.3 Randomization 
         Computer generated randomization sequence (AB/BA) 
 
4.2 Study Period 
 
   4.2.1 Reference Population 
           Dental students studying in MMMC, Melaka 
 
   4.2.2 Source Population 
              Phase II (Year 4 & 5) dental students studying in MMMC, Melaka 
 
   4.2.3 Sampling Frame 
 
            -Inclusion Criteria: Healthy students of either gender. 
-Exclusion Criteria: Probing depth and attachment loss ≥ 3mm,  use    
of antibiotics in the past 3 months, smoking,  active orthodontic 
treatment, orthodontic retainers. 
4.3 Sample Size Calculation 
Since there is no previous study available with mean and SD, A pilot study was 
conducted on 30 participants.  
 
 
                    4.4 Variables and Research Tools 
Independent Variable: Sequence of brushing and flossing 

 
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Dependent Variable: Rustogi  Modified  Navy  Plaque  Index (RMNPI) scores  and  bleeding point 
index (BPI) Scores 
 
Tools: Head cap, Face mask, Gloves, Mouth mirror, Oral prophylaxis paste, Oral prophylaxis cup/brush, 
Micromotor, Disclosing agent, William’s probe. 
 
Inter examiner reliability 
Prior  to  the  start  of  the  study, the  examiner  was  trained  by  an  experienced  periodontist 
until reaching  informal agreement on the diagnostic criteria. After that, calibration with 10 
participants was performed. Two assessments   of the scoring criteria of the index   was 
performed with 1-hour interval. When the strength of the kappa coefficient was 0.78, the 
data was recorded. 
 
Experimental procedure 
Clinical Trial was registered in ClinicalTrials.gov PRS on July 4th 2019 (ID: NCT03989427). 
Following selection of participants after applying inclusion and exclusion criteria, written informed 
consent was obtained. Participants received  instructions about oral hygiene from DJ. They were 
subjected to a thorough dental prophylaxis for complete removal of dental plaque from JG. 
Then, dental  plaque was  stained  with disclosing  agent  followed  by  a  mouth  rinsing with 
water for 1 min. All erupted teeth, except for third molars, were examined by a blind examiner 
(SA) at  6 sites per tooth (mesio-buccal, mid-buccal, distobuccal, mesio-lingual,  mid-lingual, and 
distolingual surfaces) for RMNPI score and BPI score. 
 
 To  ensure  standardization,  the participants were briefed on the type of toothbrush, toothpaste 
and dental floss to be used in the study. The amount of dentifrice used was  equivalent to half-length 
of the toothbrush’s head. Additionally, the Bass  method  of  brushing  and  Spool method of 
flossing  was  taught  to  the participants during the baseline appointment. . The methods of 
toothbrushing and flossing were explained to the participants by a step-by-step video. The 
participants were instructed to brush for two minutes which corresponds to 1 min for each hemiarch, 
30s for buccal surfaces and 30 s for lingual/palatine surfaces.  
 

 
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 Cross-over design was used.  The participants were studied in two phases  with  a 1-week wash-
out  interval. In  the  first  phase of  the  study,  15 participants  were  asked to  floss  first  and  brush  
later  for  two  weeks(method FB) and the remaining 15 participants were asked to brush first 
and floss later for two weeks(method BF) . In  the  second phase, the  participants were  asked  to  
do  the  opposite, i.e. floss  after  brushing,  for  another  two  weeks (method BF) and vice versa. In  
the wash-out period, the  participants  were asked  to  use  their  toothbrush  and  floss  according to 
their  usual routine. Dental prophylaxis was  repeated  at  the beginning  of  the  second  phase  in  
order  to  create the same  baseline  conditions  as  at  the  start  of  the  first  phase  and  study  indices 
were measured again. For  measuring RMNPI score,  disclosing tablets were given to the participants 
to chew and rinse. The index score was calculated according to the guidelines.  
 
Bleeding  point  index  were  determined  at  the  buccal,  lingual  and proximal  surfaces  using  the  
BPI. Periodontal  probe was inserted  1  mm  into  the  sulcus  at  the  distal aspect  of   the tooth  and  
moving  it  across the  length of  the  sulcus  to  the  mesial  on  the   facial   and  lingual surfaces. 
Bleeding  surfaces  after 20–30 s  was recorded. The  percentage  of  the number  of  bleeding surfaces 
was calculated. 
 
ADA    specification  of  toothbrush: Soft -bristled, No. of   bristle  are 80-85  per  tufts, No.of   rows  
are  2- 4  rows  per  brush,  Length: 25.4-31.8mm. Width: 7.9-9.5mm. Modified  bass   technique 
Dental floss: A thin waxed, fluoridated dental floss used to clean between  the  teeth3, spool method  
 
Dentrifice: toothpaste containing 1450 ppm fluoride. Brushed for three minutes with their assigned 
toothbrush with 1.5 g of toothpaste3 
 
 
 
 
 
 
 
 
 
 

 
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4.5StatisticalAnalysis  
 
We used Microsoft Excel for data entry and SPSS version 18 was used for data analysis. We calculated 
change score (Post – Pre) for outcome variables such as BPI scores and RMNPI scores. Descriptive 
statistics such as mean and standard deviation were calculated the outcome variables.  
4.6 Data Collection and Flow Chart  
 
 

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                    4.7 Ethical Consideration 
a. 
Ethical approval was obtained  from Institutional  Ethics and Research Committee of Melaka-Manipal 
Medical College(MMMC/FOD/AR/B6/E C-2019 (21) 
b. 
Written  consent  form  was  given  to  the  participants  before  conducting  the    study    and 
informed consent  taken. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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 References 
 
1. 
Torkzaban P, Arabi SR, Sabounchi SS, Roshanaei G. The Efficacy of Brushing and 
Flossing Sequence on  Control of Plaque and Gingival Inflammation. Oral Health Prev Dent 
13. 2015;3:267-273 
2. 
Berchier CE, Slot DE, Haps S, Van der Weijden GA. The Efficacy of Dental Floss in 
Addition to a Toothbrush on Plaque and Parameters of Gingival Inflammation: A Systematic 
Review. Int J Dent Hygiene 2008;6:265–279. 
3. 
Caves ES, Woo RC, Jones AA, Newbold DA, Manwell MA, Kornman KS. Relationship 
of Bleeding on Probing and Gingival Index Bleeding as Clinical Parameters of Gingival 
Inflammation. J Clin Periodontol 1993;20:139–143. 
4. 
Crocombe LA, Brennan DS, Slade GD, Loc DO. Is Self Interdental Cleaning Associated 
with Dental Plaque Levels, Dental Calculus, Gingivitis and Periodontal Disease. J Periodont 
Res 2012;47:188–197. 5. Mazhari F, Boskabady M, Moeintaghavi A, Habibi A. The Effect of 
Toothbrushing and Flossing Sequence on Interdental Plaque Reduction and Fluoride 
Retention: A Randomized Controlled Clinical Trial. J Periodontol.  2018;89:824– 832. 
6. 
Bagramian RA, Garcia-Godoy F, Volpe AR. The Global Increase in Dental Caries. A 
Pending Public Health Crisis. American Journal of Dentistry 2009;22(1):3-8. 
7. 
Lenox JA, Kopczyk RA. A Clinical System for Scoring a Patient’s Oral Hygiene