Microbial Profiling in Pockets Related to Chronic Periodontitis Patients Using 16s RNA Metagenomics Sequencing

Ajman University

Status: Completed

Conditions: Periodontal Diseases

Treatments: Genetic: 16s RNA Metagenomics sequencing

Study type: Interventional

Funder types: Other

Identifiers

NCT04425343

F-H-17-11-03

Details and patient eligibility

About

The study was developed in compliance with the Helsinki Declaration on medical research involving the Ethics Committee at Ajman University (2017-A-DN-04). Informed consent from all the participants was obtained before participation in the study. The participants were systemically healthy with no history of antibiotics for the past three months. Patients recruited for the study were diagnosed as Stage II Generalized periodontitis according to the classification from the 2017 world workshop on the "classification of periodontal and peri-implant disease and conditions". Periodontal status indicating the severity of interdental clinical attachment loss of 3-4mm, radiographic bone loss between 15%-33% and with no tooth loss were included. The complexity of periodontitis with a maximum probing depth of ≤ 5mm with horizontal bone loss and having an extent and distribution with >30% teeth involved were included in the study. A total of 80 plaque samples were collected with the clinical characteristics of the patient comprising of age between 25-39 years, 36 females and 44 males. The subgingival plaque was collected using a sterile curette from the buccal aspect of maxillary molars and lingual aspect of mandibular incisors, in a vial containing 200µl of Buffer CL.

Full description

The collected plaque samples were then freshly prepared for the DNA isolation using ABIOpure TM Total DNA (version 2.0) (Cat No: M501DP100) according to the manufacturer's instruction. All the samples were assessed for DNA quantification using spectrophotometry and further quantified using the fluorometric method. This was performed using DeNovix DS-11 FX (DeNovix). Further to the DNA quantification, assessment of DNA integrity was resolved on a 0.8% agarose gel with ethidium bromide. For NGS library preparation, all the samples underwent PCR amplification of the 16S rRNA gene in the isolated bacterial DNA. The primers used had targeted in the V3-V4 region of the 16S rRNA gene. The full length primer sequences, using standard IUPAC nucleotide nomenclature, to follow the protocol targeting this region are:

16SAmpliconPCRForwardPrimer=5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 16SAmpliconPCRReversePrimer=5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC Gel electrophoresis method was used for the size selection and the bioinformatics pipeline used for processing microbiome 16S sequence data was QIIME

Enrollment: 80 patients

Sex: All

Ages: 18 to 60 years old

Volunteers: Accepts Healthy Volunteers

Inclusion criteria

• Patients recruited for the study were diagnosed as Stage II Generalized periodontitis
• Medically fit

Exclusion criteria

• Patients having gingivitis or stage III periodontitis
• Medically unfit

Trial design

Primary purpose: Health Services Research

Allocation: Randomized

Interventional model: Single Group Assignment

Masking: Single Blind

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80 participants in 2 patient groups

Periodontitis, Adult

Experimental group

Description:

Plaque samples were taken from subgingival pocket and send to the lab for metagenomic analysis

Treatment:

Genetic: 16s RNA Metagenomics sequencing

Metgenomic analysis

Experimental group

Description:

Analysis for whole bacterial count

Treatment:

Genetic: 16s RNA Metagenomics sequencing