EP1173555A1 - Method and kit for indicating the level of bad breath - Google Patents

Abstract

The invention provides a method for the rapid assessment of the degree of halitosis comprising the steps of: a) obtaining a sample of fluid and/or tissue from the oral cavity of a subject, b) assessing the amount of β-galactosidase present in said sample, c) determining the degree of halitosis in said subject, by comparing the result obtained in step b) with appropriate reference values.

Description

METHOD AND KIT FOR INDICATING THE LEVEL OF

BAD BREATH

Field of the Invention

The present invention is concerned with a method for gauging the presence

and degree of bad breath. More specifically, a method for the measurement

of bad breath based on the estimation of β-galactosidase activity is

disclosed, together with a diagnostic kit that employs this method.

Background of the Invention

Oral malodor, (halitosis, fetor ex ore) is a common human condition dating

back to ancient times. Bad breath usually originates within the oral cavity itself, due to the production of putrid smelling gases by deposits of

microorganisms, generally under anaerobic conditions.

Bad breath is considered to be caused chiefly by proteolytic activity of gram

negative organisms. When gram positive bacteria from the mouth are

incubated in the presence of amino acids, little or no odor ensues. However

when gram negative bacteria are incubated in the presence of amino acids,

putrid odors abound.

One of the practical problems related to this condition is that

self-measurements of oral malodor are not reliable. This results, on the one hand, in patients who suffer from bad breath and are not aware of it, and on

the other hand, in patients who are concerned about having this problem,

while in fact they do not suffer from it. Because of the difficulties inherent

in self-estimation of oral malodor, simple testing devices are of potential

importance.

Various tests have been proposed for measuring parameters associated with

bad breath and the degree of improvement following therapy. One approach

which is commonly used is to measure the degree of volatile sulfides using

electronic apparatuses (such as the Halimeter, InterScan Corp., Chatsworth

California), or visual means (such as by precipitation of lead acetate).

However, the first technique involves apparatus which is expensive to purchase and the second is time consuming. Another test which measures

proteolysis, the BANA test, requires extensive work-up, including a period

of heating at elevated temperature in a specialized instrument.

In a previous patent, US 5,270,174, a technique for measuring microbial

activity associated with bad breath is described. It includes swishing the

oral cavity with sterile liquid, followed by introduction of the expectorate to

a vessel containing an indicator of oxygen consumption. This test is messy

and the time required for a color change may be long. There are currently no quick and simple procedures which allow an

individual to objectively test their own level of bad breath. In many social

situations such a test could be invaluable to avoid embarrassment or to

forewarn an individual of the need to take some remedial action to treat the

condition. Such a test could also be of value in clinical situations where a

fast objective test of bad breath could assist in evaluating periodontal

disease as well as measure the effect of various treatments.

It is a purpose of this invention to provide a simple and convenient assay for

the detection of oral malodor.

It is a further purpose of this invention to provide an oral malodor assay

that is useful both for home use and for the evaluation of oral diseases and

their response to treatment, by health care professionals.

Other objects and advantages of the invention will become apparent as the

description proceeds.

SUMMARY OF THE INVENTION

It has now been unexpectedly found that an assessment of the presence and

degree of bad breath can be obtained by measuring the level of

β-galactosidase enzymes in samples taken from the oral cavity. These

enzymes are not known to contribute directly to bad breath odors, and therefore it is a matter of some surprise that their detection and

measurement may be used as an indicator of the presence and severity of

halitosis.

The present invention is primarily directed to a method for the rapid

assessment of the degree of halitosis comprising the steps of:

a) obtaining a sample of fluid and/or tissue from the oral cavity of a subject.

b) assessing the amount of β-galactosidase present in said sample

c) determining the degree of halitosis in said subject, by comparing the

result obtained in step b) with appropriate reference values.

The term "appropriate reference values" refers to any quantitative or

qualitative value assigned to the amount of β-galactosidase found in the

sample, be it a set of discrete values or a calibration curve, such as a

graphical plot of β-galactosidase levels for samples taken from a group of

subjects versus the results of another measurement of bad breath, for

example mean whole-mouth odor scores (based on odor -judge scoring on an

arbitrary intensity scale). This term also refers to the use of the

mathematical equation that describes said calibration curve. Finally, this

term is also used, in the case of semi-quantitative β-galactosidase level

results, to refer to comparison of the β-galactosidase score with a pre-determined odor-intensity scale based on such semi-quantitative

enzyme measurements.

While any suitable fluid or tissue (e.g. superficial mucosal cells taken by

scraping the lingual or buccal mucosa, periodontal pocket exudate, and so

on) may be used in order to carry out the method of the invention, saliva is

the preferred sample type.

In another aspect, the invention provides a kit for the rapid assessment of

halitosis comprising:

a) means for obtaining a fluid and/or tissue sample from the oral cavity;

b) a substrate of β-galactosidase that undergoes a change in color or other

discernible property when broken down by said β-galactosidase, adsorbed

onto a solid support medium;

c) a color or intensity chart or instructions for determining the level of

halitosis from the change in color of the solid support medium.

Any suitable β-galactosidase substrate possessing the properties described

hereinabove may be used in the manufacture of the kit of the invention.

However, a preferred substrate is X-gal (5-bromo-4-chloro-3-indolyl-

β-D - galactopyranoside) . In one preferred embodiment of the kit of the invention, the solid support

medium further comprises a gratuitous inducer of β-galactosidase. While

any such gratuitous inducer may be used, in a preferred embodiment of the

kit of the invention, said inducer is IPTG (isopropyl β-D-thiogalactoside).

All the above and other characteristics and advantages of the invention will

be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof.

Detailed Description of Preferred Embodiments

Example 1

Beta-Galactosidase Activity as an Indicator of Bad Breath

Four subjects complaining of bad breath were tested for the odor levels

(based on odor judge scoring on an increasing intensity scale of 0-5, and measurement of volatile sulfides using a sulfide monitor [InterScan Corp.,

Chatsworth, Ca model 1170]). In addition, the level of beta-galactosidase

activity was measured based on a colorimetric assay as follows: a sample of

the back of the tongue was taken with a plastic spoon. The sample was

removed from the spoon by washing twice with 0.2 mL water which were

pooled into a single sample. X-gal (5-bromo 4 chloro 3 indoyl beta D

galactopyranoside and IPTG (isopropyl thiogalactopyranoside) were added

(0.05 mL of a 20 mg/mL solution and 0.05 mL of a 50 mg/mL solution, respectively) and the samples were incubated for one hour at 37 degrees

Celsius in ELISA plates. The relative amount of enzymatic activity was

recorded as OD at 650 nm.

The results of these measurements are presented in Table I.

Table I

Subject no. ELISA OD Judge Score Volatile Sulfides (ppb)

1 0.15 2 (slight) 20

2 0.27 2.5 (slight-moderate) 20

3 0.30 2.5 (slight-moderate) 30

4 0.52 3 (moderate) 50

It is clear that the increasing ELISA reading of beta-galactosidase activity

is in association with the increasing odor judge scores and volatile sulfide

levels.

It was further found that β-galactosidase activity in saliva or other oral

samples can be measured by simply wetting absorbent discs containing

β-galactoside activity detecting agents and incubating them at room

temperature for short periods of time. By comparing the amount of color

produced to color standards, a semi-quantitative estimate of the

β-galactosidase activity in the oral sample can be determined. Example 2

Color test using paper discs

Five mm discs of absorbent paper were cut from sheets of Whatman

chromato graphic paper (Whatman Ltd., Maidstone, England). 100 mg of

X-gal were dissolved in 5 mL of dimethylform amide. A second solution of 100 mg IPTG in 2 mL of water was prepared. The two solutions were

combined. Twenty microliters of the combined solution were applied to each

of the above paper discs. The discs were then dried for 24 hours before use

and then affixed to a plastic backing.

To use the test, subjects collected a small amount of saliva and used it to

thoroughly wet the above treated discs. The discs were allowed to stand at

room temperature for 10 minutes. The color generated was scored using a

standard color scale provided to the subjects. Breath odor scores from each subject were also measured using a sulfide monitor. A significant degree of

correlation was found between the test scores.

Example 3

Use of paper discs to monitor treatment with mouthrinse

Absorbent paper discs impregnated with 20 mg/ml of X-al and 50 mg/ml of

IPTG were used in a self-administered test to evaluate breath odor before

and after the use of a breath freshening mouthrinse with active ingredient compared to a placebo mouthrinse. The subjects saturated the discs with

samples of their saliva immediately before and 1,2 and 3 hours after using

the treatment or placebo mouthrinse. The amount of color developed on the

discs was scored after standing 5 minutes at room temperature. Breath odor

scores were also measured with a sulfide monitor and an expert panel.

The results obtained with the three different measurements confirmed the

breath freshening action of the treatment product compared to the control.

Example 4

Paper disc-based enzymatic assay for the assessment of oral

malodor

The purpose of this study was to test a simple enzymatic color assay for the

detection of oral malodor, to test its correlation with other oral

malodor-related parameters such as odor judge scores and sulfide monitor

measurements. In addition to the color assay, (organoleptic) measurements

were made by two odor judges. Sulfide monitor measurements, microbial

counts, BANA test and an indole test were similarly carried out.

Subjects

The study included 60 healthy young adult volunteers (mean age 23 ± 2

years, 35 females). Subjects who were smokers or took antibiotics within

one month prior to the study were not allowed to participate. The experiment was conducted according to an approved human subjects

protocol and participants signed an informed consent form.

Participants were asked to refrain from eating or drinking for two hours

prior to measurements. Initially, subjects were tested for malodor-related

parameters: odor judge measurements, sulfide monitor levels, color assays

and microbial counts. The subjects were split randomly into three groups: 1)

treatment group: active chewing gum (with Breathanol TM ). 2) placebo

chewing gum (without Breathanol TM). 3) control group (no treatment). The

subjects were given the chewing gum (or no treatment) and were asked to

chew for 15 minutes. The subjects were reexamined after 1.5 and 3 hours following use. At the beginning of the experiment the subjects were asked to

form an opinion on their own breath by scoring it using the same scale as the odor judges (see below).

Measurements parameters:

Color assay (OK2KS)

Paper discs (6mm) were impregnated with enzyme substrates as described

below:

The paper test was prepared by punching out 6 mm diameter discs from

chromatography paper (Whatman paper no. 3). Two solutions were

prepared, one by dissolving 100 mg of 5 - bromo - 4 — chloro — 3 - indolyl -

D galactopyranoside (X - Gal, Sigma) in 2 ml of N,N - Dimethylformamide (Sigma), and the other by dissolving 100 mg of isopropyl β-D-thiogalactoside

(IPTG, Sigma) in 2 ml of double distilled water. 100 μl from each solution

were combined, vortexed and then 20 μl of the mixture was impregnated on

each paper disc. The discs were dried over-night at 37° C.

Saliva (whole, unstimulated) was collected from each subject at the

beginning of the experiment (before treatment) as well as at 1.5 hours and

after 3 hours. A 20 μL drop of each saliva sample was applied to the paper

disc and following 10 min incubation at room temperature, the results were

recorded after 10 minutes as follows: 0 - no color, 1 - faint color, 2 - dark

color.

BANA and Indole production assays

The BANA reagent card (PerioscanTM , Oral - B Laboratories, Redwood

city, CA) and the indole production slide (DrySlideTM INDOLE, Difco

laboratories, Detroit MI) were used according to manufacturer's

instructions. Samples for these assays were taken from the same posterior

tongue dorsum scrapings which were used to determine tongue odor scoring

by the odor judges. Results were recorded as either: strong reaction = 2,

light reaction = 1, or no color change = 0. Sulfi.de monitor

Determination of intraoral headspace volatile sulfur compounds (VSC) was

carried out using a sulfide monitor (model 1170, InterScan ). Subjects were

asked to refrain from talking for 5 minutes prior to measurements . The

monitor was zeroed on ambient air, and the measurements were performed

by inserting a disposable one quarter inch plastic straw approximately 4 cm

into the partially opened oral cavity. Subjects were asked to breathe

through their nose during measurements. Results were recorded as peak

ppb sulfide equivalents.

Organoleptic measurements

Two odor judges scored whole mouth malodor and for tongue malodor. For

judge scoring of whole mouth subjects were instructed to exhale briefly

through the mouth, at a distance of approximately 10 cm from the nose of

the judge. Tongue malodor was scored by using a plastic spoon to scrape and

scoop material from the far back region of the tongue dorsum, and scoring

the malodor from the spoon by both judges, sequentially. Judge scores were

recorded using a semi-integer scale of 0 to 5, as follows: 0, no appreciable

odor; 1, barely noticeable odor ; 2, slight, but clearly noticeable odor; 3,

moderate odor; 4, strong odor; 5, extremely foul odor. Microbial counts

Viable counts from saliva samples were conducted using Diaslides (Savyon

Diagnostics, Ashdod, Israel) containing tryptic soy agar (TSA) and mitis

salivarius agar (MSA). Diaslides were incubated anaerobically for 72 hours

at 370C. Viable counts included total on TSA and MSA as well as counts of

the blue colonies which formed on the MSA.

Statistical analysis

Spearman correlation coefficients were used to determine the level of

association between he various parameters. One way analysis of variance

(ANOVA) was used to compare the results of the color assay (0,1 and 2) in

terms of the other parameters. Stepwise multiple regression analysis was

carried out in order to test the contribution of the color test results and the

sulfide monitor in predicting the odor judges scores.

Results

Spearman correlation coefficients comparing color assay scores for the three

rounds of measurements (time 0, 1.5 and 3 hours) with the other

parameters are presented in Table II. In this table, the appropriate p value

is shown below each r value. Table II

Judge 1 Judge 2 Microbial counts Microbial assay

Log Whole Whole

Monitor mouth Tongue mouth Tongue TSA MSA Blue BANA Indole

Time zero

OK2KS 0.18 0.39 0.50 0.47 0.48 0.29 0.37 0.38 0.21 0.21 p= 0.086 00..000011 O.000K0.000K0.0001 0.013 0.002 0.002 0.055 0.057

1.5 Hours

OK2KS 0.3290 0.32 0.42 0.46 0.33 0.31 0.42 0.39 -0.12 -0.12 p= 0.005 0.00 <0.0001 <0.0001 0.005 0.007 O.0001 0.002 0.185 0.171

3 Hours

OK2KS 0.41 0.32 0.44 0.49 0.60 0.21 0.25 0.17 0.08 0.15 p= 0.001 0.006 O.OOOKO.OOOKO.OOOl 0.053 0.025 0.136 0.263 0.120

Among the various tests, OK2KS scores were most highly associated with

the odor judge scores for whole mouth (p<0.007) and tongue (p<0.005) odor.

Significant correlations were also observed between OK2KS and monitor

measurements for the last two time points (p<0.005) and the microbial

counts for the first two time points (p<0.013). In contrast, no significant

association was found between OK2KS and the BAΝA or Indole production

assays (p>0.055). The initial correlations between OK2KS scores, sulfide monitor levels,

BANA test results and microbial counts (MSA) are compared with odor

judge scores in Table III below. Correlations between odor judge scores and

OK2KS scores were as high or higher than corresponding correlations with

sulfide monitor scores in all cases. Correlations between organoleptic scores

and the BANA test were less significant, as were correlations between

bacterial counts on MSA and odor judge scores. Indole scores were not correlated significantly with odor judge scores (not shown).

Table III

OK2KS Sulfide Monitor BANA Bacterial

Color test (ppb equivalents) Test Counts

Judεe 1

Whole mouth r= 0.39 0.37 0.25 0.27

P= 0.002 0.002 0.048 0.030

Tongue r= 0.50 0.26 0.26 0.20

P <0.001 =0.036 =0.036 =0.118

Judεe 2

Whole mouth

R= 0.47 0.46 0.22 0.16

P <0.001 <0.001 =0.086 =0.196

Tongue r= 0.48 0.38 0.14 0.18

P <0.001 =0.002 =0.282 =0.160

Stepwise multiple regression analysis of odor judges scores for whole mouth

and tongue odor (at time 0), in terms of color assay scores and log monitor

readings are shown in Table IV.

Table IV

Both sulfide monitor readings and OK2KS scores factored significantly into the regression equation for both judges scores for whole mouth and tongue

odors, yielding multiple r values ranging from 0.47 (judge 2, tongue,

p=0.0007) to 0.60 (judge 2, whole mouth, p<0.0001). The results presented hereinabove show that OK2KS was highly

significantly correlated with odor judges scores for whole mouth and tongue

odor, at all three time points during the study. Furthermore, correlations

between OK2KS and organoleptic scores were as significant, or more

significant than corresponding correlations between the sulfide monitor and

organoleptic scores. When multiple regression analysis was carried out to

try to account for odor judge scores in terms of OK2KS and sulfide levels, both parameters entered into the regression equations, yielding multiple r

values of up to 0.6. The results suggest that (I) OK2KS may be used as an

assay which correlates with odor judge scores; and (ii) OK2KS can be used

alongside sulfide monitor testing to improve the correlation with odor judge

scores. The convenience and low anticipated cost of OK2KS may make it

useful in both clinical and home settings.

While specific embodiments of the invention have been described for the

purpose of illustration, it will be understood that the invention may be

carried out in practice by skilled persons with many modifications,

variations and adaptations, without departing from its spirit or exceeding

the scope of the claims.

Claims

1. A method for the rapid assessment of the degree of halitosis

comprising the steps of:

a) obtaining a sample of fluid and/or tissue from the oral cavity of a subject.

b) assessing the amount of β-galactosidase present in said sample

c) determining the degree of halitosis in said subject, by comparing the

result obtained in step b) with appropriate reference values.

2. A method according to claim 1, wherein the fluid sample is saliva.

3. A kit for the rapid assessment of halitosis comprising:

a) means for obtaining a fluid and/or tissue sample from the oral cavity;

b) a substrate of β-galactosidase that undergoes a change in color or other

discernible property when broken down by said β-galactosidase, adsorbed

onto a solid support medium;

c) a color or intensity chart or instructions for determining the level of

halitosis from the change in color and/or change in intensity of the solid

support medium.

4. A kit according to claim 3, wherein the β-galactosidase substrate is

5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (X-gal).

5. A kit according to claim 3, wherein the solid support medium

further comprises a gratuitous inducer of β-galactosidase.

6. A kit according to claim 5, wherein the gratuitous inducer of

β-galactosidase is isopropyl β-D-thiogalactoside (IPTG).