Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03668015 |
| Other study ID # |
CRP.3.MAR14.7 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
March 5, 2015 |
| Est. completion date |
June 4, 2015 |
Study information
| Verified date |
September 2021 |
| Source |
The University of The West Indies |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Chewing gum containing xylitol may help prevent caries by reducing levels of mutans
streptococci (MS) and lactobacilli in saliva and plaque. Very little is known about other
species which are possibly beneficial to oral health. In this study, the investigators
employed high-throughput sequencing of the 16S ribosomal ribonucleic acid (rRNA) gene to
profile microbial communities of saliva and plaque following short-term consumption of
xylitol and sorbitol containing chewing gum. Participants (n = 30) underwent a washout period
and were randomly assigned to one of two groups. Each group chewed either xylitol or sorbitol
gum for 3 weeks, before undergoing a second four-week washout period after which they
switched to the alternate gum for three weeks.
Description:
Subjects: Study protocols were approved by the Ethics Committees of the UWI, St. Augustine.
Thirty healthy volunteers from The University of the West Indies (UWI), St. Augustine,
Trinidad were enrolled in the study. To be eligible, subjects must have had at least 20
teeth, provided written informed consent and been willing to comply with study procedures.
Subjects with systemic, infectious or inflammatory diseases or taking medicines, antibiotics
or fluoride in the last month, habitual consumers of xylitol / sorbitol-containing products
and mouth rinses, with abnormal salivary flow (<1ml / min), pregnant, on contraceptive pills,
or with abnormal dietary habits were excluded. Consent obtained at the initial visit was
verified at the second visit, prior to sample collection. The subjects were examined in the
dental chair after thorough medical and dental histories were recorded. The clinical
examination involved examination of the soft tissues and then dental hard tissue charting for
presence of decayed, missing or filled teeth. No radiographs were used. The presence of
untreated dental caries or periodontitis were not used as exclusion criteria. The decayed,
missing, and filled teeth for each individual at the initial visit was documented for
calculation of the index of decayed-missing-filled permanent teeth (DMFT) score.
Chewing gums: Xylitol gum (Epic Spearmint; 1.5g/pellet) designated Gum X contains 70% xylitol
in addition to gum base, natural flavors, soy lecithin, gum arabic, titanium dioxide, carnuba
wax. Gum S (Eclipse Spearmint), was similar except that xylitol was replaced by 63% sorbitol
and 2% maltitol and aspartame was included. Gums were packed in colour-coded containers.
Codes were kept confidential from the participants and researchers who interacted with them
until study completion.
Study design: This prospective cross-over, double-blind, randomised study lasted 14 weeks
(March - June, 2015). Throughout, subjects were instructed not to use mouthwashes or xylitol
products, to consume a normal diet, continue their usual tooth brushing and to report use of
antimicrobial medications. Subjects reporting the latter were dropped.
Subjects were randomly allocated to two groups, A and B (see Figure 1). Both groups entered a
4-week "washout period" during which no gum was chewed, followed by a 3-week treatment period
(treatment period 1) during which Group A used Gum X and Group B used Gum S (2 gum pieces, 3
times daily after meals for 6 minutes). Both groups then underwent another 4-week washout
period before entering treatment period 2 during which Group A used Gum S and Group B used
Gum X for 3 weeks.
Sample collection: Saliva and plaque were collected from participants immediately before and
after each treatment period (Figure 1). Subjects were instructed not to brush their teeth or
use any other oral hygiene procedures at least 24 hours before sample collection, and not to
eat or drink at least 1 hour before. For saliva collection, subjects chewed sterile paraffin
wax and whole saliva produced was collected for 5 minutes in sterile tubes. The subject was
asked to drool into the labelled 50ml collection tube (Falcon sterile, conical polypropylene
tube with flat-top screw cap). This process was repeated multiple times in order to collect
larger volumes of saliva (2-5 ml). The saliva was transferred via sterile pipettes into
labelled sterile centrifuge tubes which were stored at -70˚C until use.
Supragingival plaque was collected with a curette from 2 molars (#16 and #36), 2 premolars
(#24 and #44), and 2 incisors (#21 and #41); Plaque collection involved using a Gracey
curette to remove all of the supragingival plaque from the buccal surface of the selected
index tooth with as many strokes as necessary. The curette tip was immersed in the sterile
DNase-free Tris-EDTA (TE) buffer in the centrifuge tube for 4-5 seconds. The face of the
curette was wiped on the inside edge of the collection tube and then wiped off with sterile
gauze so as to not let the buffer on the tip go into patient's mouth. The site was then
immediately sampled again using the same procedure. The lid on the tube was replaced and then
the tube shaken for 4-5 seconds in an attempt to maximize dispersion of the specimen in the
fluid. The tube was immediately placed on ice in a Ziploc bag and stored at -70˚C until use.
Samples were labelled by group (A/B), treatment period (1/2), gum used (X/S in the case of
those samples collected at the end of a given treatment period) and type (saliva (s) or
plaque (p)). Samples (n=232) were then shipped on dry ice to the J. Craig Venter Institute
(JCVI) USA, La Jolla campus for DNA extraction and sequencing.
DNA extraction: Samples were thawed at 4˚C and vortexed thoroughly prior to DNA extraction
from 500ul of saliva or plaque suspension using bead beating Lysing Matrix B tubes (MPBio
Inc.), then lysozyme digest, phenol/chloroform isoamyl alcohol extraction and ethanol
precipitation. Precipitated DNA was resuspended in 1x TE buffer.
Library Preparation and Sequencing: DNA from each sample was quantified using a Nanodrop
spectrophotometer (Thermo Fisher Scientific, Inc, Waltham, MA). Amplicons were generated
using adaptor and barcode ligated polymerase chain reaction (PCR) primers targeting the V4
region of the 16S rDNA gene (16S) and purified using Qiaquick PCR purification kits (Qiagen,
Inc) following manufacturer's instructions. Purified amplicons were quantified using SybrGold
(Thermo Fisher Scientific, Inc, Waltham, MA), normalized to ensure equimolar quantities of
each sample, and pooled in preparation for Illumina MiSEQ sequencing. The 16S library pool
was sequenced using the Illumina MiSEQ dual index 2x250bp V2 chemistry kit according to
manufacturer's specifications.
16S RNA sequence data processing: Sequences for each sample were binned according to
corresponding dual indices and exported as individual .fastq files using CASAVA v1.8.2
(Illumina Inc. La Jolla, CA). Sequences were processed to ensure that only quality sequences
were retained, as stringent settings were kept to ensure no barcode mismatches were permitted
during demultiplexing. Processed sequences were applied to the Infernal pipeline [35] for
additional quality control (QC) checks. Bacterial sequences were taxonomically assigned based
on the Genomic-based 16S ribosomal RNA Database (GRD; http://metasystems.riken.jp/grd/).
Statistical analyses. Distribution by age, sex and DMFT score for subjects in groups A and B
were compared using independent-samples T- test, Pearson's Chi-Squared test and Wilcoxon rank
sum test respectively, with a cut-off of p <0.05. To avoid possible sequence errors,
operational taxonomic unit (OTU) count tables were filtered such that OTUs present in fewer
than 0.1% in all samples were discarded. OTU tables were then transformed to relative
abundances before community analyses were performed using the R statistical computing
language (Cite R). Kruskal-Wallis test was used to assess statistical significance in
microbial community composition across treatments. Wilcoxon test was used for pairwise
comparison.
