Microbiological and Clinical evaluation of Neem gel and Chlorhexidine gel on dental plaque and gingivitis in 20-30 years old adults: A Randomized Parallel-Armed, Double-blinded Controlled Trial

INTRODUCTION It has been well established that oral health is an integral part of general health and well-being. Oral health and general health comorbidity is well established. Throughout the world, dental diseases contribute as major public health problem. Worldwide, dental caries affects 60%–90% children and approximately 100% adults.[1] Periodontal disease in its simplest form is chronic gingivitis,[2] and, regardless of age, sex, or race, affects more than 90% of the population.[3] The etiology for dental caries and periodontal diseases is the supragingival and subgingival plaque.[4] Dental plaque is a biofilm, which is formed by the interaction between microbial deposits and salivary coating immersed in an extracellular polysaccharide matrix.[5] The most common bacteria responsible for plaque formation are Streptococcus mutans and Lactobacilli.[6] The most crucial factor in terms of preventive and curative treatment of dental diseases is therefore the removal of bacterial biofilm. The most feasible and cost-effective method for gingivitis control is use of mechanical agents.[7] However, this mechanical plaque control has failed as effective control measure as proven by worldwide prevalence of gingivitis. It is difficult to achieve an “ideal” mechanical plaque control and this resulted in exploring chemical antimicrobial agents inhibiting biofilm formation.[8] Although numerous chemical agents for plaque control have been tried over the years, all have shown some side effects. Thus, patients are more inclined toward herbal preparations, which are effective and do not cause any side effects. Hence, the search for unconventional product continues. The neem tree has been deeply rooted in Indian culture.[9] The neem leaves are cheap and easily available as well as widely accepted socially in the Indian society. It has been tested for its antibacterial activity,[10111213] including its therapeutic use for gingivitis and periodontitis.[38,1415] In this study, attempts were made to prolong the contact time of therapeutic dosage of agent to allow sufficient time period of contact[16] and improve compliance by the virtue of sustained release drug-delivery system of bioadhesive nature without any side effects. Thus, there is need to study this indigenous agent in depth to establish its efficacy in gel form for the prevention of oral diseases. On extensive literature review, we were unable to find adequate studies conducted to assess effect of gel form of chemical plaque control agents. Hence, this study was conducted with an aim to evaluate and compare the effect of neem gel and chlorhexidine (CHX) gel on plaque, gingivitis, and the bacterial count of S. mutans and Lactobacilli during a period of 90 days use in a city of western Maharashtra, India. MATERIALS AND METHODS Preparation of gel formulations The gels were formulated with the help of pharmacology department of BioGreen Healthcare, Mulund, Mumbai, India––a commercial manufacturer of ayurvedic and herbal medicines. The aqueous liquid extract of Azadirachtaindica was prepared by boiling 20g of neem leaves in 1000 mL of water, which was reduced to 100g of neem extract. It was observed that the neem extracts showed a minimum inhibitory concentration at 25 µg/mL for S. mutans and Lactobacilli by in vitro testing. Hence, this concentration was used for the preparation of the neem gel. 20g of carbopol 934P was added in 1000 mL of water and soaked for 24h to prepare the base gel. To this base gel 2.5% neem extract and 0.2% CHX powder was added along with 0.06% peppermint oil and preservatives to prepare the respective gels. The gels were tested and found stable for 1 year. The gels had similar color and were packed in opaque collapsible tubes, which were labelled as gel A and gel B by pharmacologist for allocation concealment. Sample size calculation It was carried out according to the expected minimum reduction in dental plaque scores in control group, as observed in a previous study[16] along with consideration of 10% attrition, which was derived to be 30 in each group. Recruitment, randomization, and clinical examination Ethical approval from institutional ethical committee and informed consent from study participants in local language were obtained prior to start of this randomized parallel arm double-blind clinical trial. All the examinations and recording of the parameters were carried out by a single trained and calibrated examiner. After obtaining approval from school authorities to conduct study among school teachers, participants were evaluated for inclusion and exclusion criteria. Those with similar socioeconomic status, oral hygiene methods, having fair plaque score, and moderate gingivitis assessed by Plaque Index[17] and Gingival Index,[18] respectively, and no loss of attachment were included in study. Those with overhanging restorations, antibiotic therapy in last 3 months before commencing the study, using any dentifrice/mouthwash having anti-inflammatory action, presence of grossly decayed teeth, and any systemic disorders that require the use of medications with side effects known to affect oral health were excluded from the study. A total of 60 participants were randomly allocated by lottery method into two groups of 30 each such that both groups had equal gender distribution. The two study groups were as follows: Group A––2.5% neem gel (n = 30, men = women = 15 each) Group B––0.2% CHX gel (n = 30, men = women = 15 each) The preparatory phase for the study started from October 2014, which included preparation of test formulations, stability testing, obtaining permissions from schools, and screening for selection of study participants. The actual study period of clinical trial extended from July 14, 2015 to October 23, 2015. Type 3 clinical examination (classification by the American Dental Association 1970)[19] was followed throughout the study during intraoral examinations. The study participants were availed with their respective gels and instructions were given of its use as, twice daily––once in morning after consuming breakfast and at night just before sleeping. Approximately 1g of gel should be dispensed on index finger and applied thoroughly in the oral cavity. This was shown with the help of video. They were instructed that after gel application they were not supposed to eat or drink for 30 min. They were provided with 20g of gel tube every 10th day. They were asked to maintain their normal dietary habits throughout the study. No instructions were given concerning brushing technique and duration. Compliance evaluation checklist was given in which participants had to tick mark in corresponding boxes after every application of gel. Data were collected using predesigned recording form and by clinical examinations at baseline, 30th day and 90th day. The recording form had two parts: In first part, the general information regarding the participant’s demographic profile, past and present medical history, dental history, and oral hygiene practices, which was collected through an interview. Second part included the clinical examination of the oral cavity and assessment of plaque and gingival scores at baseline, 30th day and 90th day using: Löe:[17] Plaque Index Löe and Silness:[18] Gingival Index Total colony counts of S. mutans and Lactobacilli from dental plaque samples were entered into recording form after obtaining results from the microbiologist. The CONSORT (Consolidated Standards of Reporting Trials) 2010 guidelines were followed throughout the study as shown in Figure 1.Figure 1: Flow chart of study design (Consolidated Standards of Reporting Trials [CONSORT] 2010 guidelines)Plaque sample collection Instructions were given to the participants that they should not eat or drink at least 1h before the plaque sample collection. To control the circadian variations, samples were collected between 8:00 AM and 11:00 AM. All patients were asked to wash their mouth with normal saline prior to dental plaque sampling. The study participants were seated on a chair to collect plaque from the cervical thirds of the buccal surfaces and from the pit and fissure areas using explorer. The collected samples were transferred into appropriately coded 2 mL sterile Eppendorf vials containing 1 mL normal saline and carried in a vaccine carrier with freezing mixture to the laboratory. Microbial analysis of plaque The plaque sample was homogenized and vortexed manually for 2 min and stirred by stirrer. 100 µL of this mixture was diluted till a dilution of 1:1000 was obtained and used for microbial analysis. Streptococcus mutans was cultured on Mitis salivarius-bacitracin (MSB) agar and Lactobacilli on De Man, rogosa and sharpe (MRS) agar being selective media for culture of these organisms. Using 5 µL of the 1:1000 dilution, sample was streaked on MSB and MRS agar under strict aseptic conditions by the aid of an inoculation loop. The MSB agar and MRS agar plates were incubated for 48h at 37°C, anaerobically using candle jar. After 48h of incubation period, tiny, raised, rough, and adherent colonies of S. mutans appeared. For Lactobacilli colonies were large and white. Electronic colony counter was used for counting identified colonies. After the completion of 90 days period of use of gels, the study participants were educated regarding the oral health, oral hygiene maintenance, and proper method of tooth brushing with the aid of health education models and charts. Emphasizing on routine regular dental checkup, the participants who needed immediate care were directed to the required departments in Dental College and Hospital. Statistical analysis was performed with the Statistical Package for the Social Sciences software program, version 21.0 for Windows (IBM, Armonk, New York). Data were presented as mean and standard deviation (SD). For all tests, P < 0.05 was considered for statistical significance. Unpaired t test compared the difference between Groups A and B

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• Gingival Crevicular Fluid (GCF): A Diagnostic Tool for the Detection of Periodontal Health and Diseases · 2021

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