FIELD OF THE INVENTION

The present invention relates to the diagnosis and reduction of dental caries which involves monitoring and reducing Streptococcus mutans levels in the oral cavity.

BACKGROUND OF THE INVENTION

U.S. Patent No. 4,496,322 issued to Sandham and Balanyk discloses a varnish which may be applied to teeth which contains a dentally acceptable antimicrobial agent, such as chlorhexidene acetate, a benzoin gum, and an orally acceptable solvent. The composition, once applied to the teeth, is allowed to dry thereon and gives a transparent, translucent or tooth coloured film which is effectively invisible, but provides sustained release of the antimicrobial agent to an infection site over a period of a few days. To further improve the effect of the applied composition, the above inventors in their U.S. Patent No. 4,883,534, describe the use of a sealing composition applied over the varnish. The sealing composition is preferably solvated polyurethane which upon evaporation of solvent is cured.

Sandham and Balanyk in their initial proposal relative to the use of the one or two stage antimicrobial coating postulated the necessity of one or more treatment per patient in order to achieve the therapeutic endpoint which was the absence of Streptococcus mutans in the oral cavity. Furthermore, every patient was tested by a laboratory plate count reader which clearly presented cost and compliance problems in the patient population and by dentists (family practitioners).

BST1TUTE SMFKT

In U.S. Patent No. 5,178,870 issued January 12, 1993 to Schaeken et al., there is described a single stage antimicrobial composition which uses chlorhexidene as the antimicrobial agent for the purpose of eliminating Streptococcus mutans in one treatment. The compositions in this instance comprise a physiological acceptable varnish base and an antimicrobial agent, chlorhexidene or a salt thereof, the latter in an amount of more than 30% by weight based on the weight of the total composition, with this amount being apparently sufficient for elimination of Streptococcus mutans in one treatment. The amount of antimicrobial agent proposed in this formulation is substantially higher than that proposed by Sandham and Balanyk. The Schaeken patent proposes a single treatment regardless of the patient profile or the Streptococcus mutans levels found in the patient.

In Canadian Patent No. 1,235,986 which issued May 3, 1988 to Jordan and Marmel, there is disclosed a test kit and method for the determination of Streptococcus mutans in the oral cavity of a dental patient. The test is a semi-quantitative determination which is suitable for use by dentists and dental professionals in a non-laboratory environment. The test kit is sold commercially under the trade-mark CARIESCREEN SM by APO Diagnostics Inc. of Markham, Ontario, Canada. The test is an in vitro semi-quantitative dip-slide culture test for the detection of Streptococcus mutans in the oral cavity by use of a selective culture medium. The test uses a selective medium which, when used in conjunction with the diluent plus a dissolved bacitracin tablet, inhibits the growth of most salivary bacteria except for Streptococcus mutans. A carbon dioxide generating tablet is used to provide a carbon dioxide environment which enhances the growth of Streptococcus mutans.

The CARIESCREEN SM test kit has been on the market for some time, but very little use has been made of it, in the clinical setting.

SUBSTITUTE SHEET

SUMMARY OF THE INVENTION

It has now been found that the use of a two stage antimicrobial coating proposed by Sandham and Balanyk, together with a Streptococcus mutans monitoring test, such as that provided commercially under the trade-mark CARIESCREEN SM may be used most effectively in combination to not only monitor and reduce the levels of Streptococcus mutans bacteria, but also to determine the number and timing of two stage antimicrobial coating treatments necessary for a particular patient, and also to identify patients with high levels of Streptococcus mutans who may have dentician problems which normally would not receive early diagnosis and attention. The proposed method for the diagnosis and reduction of dental caries in patients would substantially reduce the cost of the antimicrobial coating for Streptococcus mutans bacteria control, since it would only be applied in instances where it was required, and further its use in the required instances would be in accordance with the levels of Streptococcus mutans found in monitoring tests. In addition, the method provides an early warning indicator of dental problems which may otherwise go unnoticed. Thus the method allows for treatment cost reduction and the problems of patient compliance are substantially eliminated.

Thus, in one aspect of the present invention there is provided a method for the diagnosis and reduction of dental caries in patients comprising testing the level of Streptococcus mutans in an oral cavity of a patient to determine the level of Streptococcus mutans in the oral cavity and establish what treatment, if any, the patient requires in accordance with the following guidelines: a. for a patient with normal level of Streptococcus mutans, the patient is not subjected to any treatment, but is asked to return for routine checkups and testing of Streptococcus mutans levels;

SUBSTITUTE SHEΞ

b. for a patient with above normal levels of Streptococcus mutans, the patient is given one application of a two stage antimicrobial coating to reduce the level of Streptococcus mutans in the oral cavity, which comprises as a first stage, an antimicrobial varnish comprising at least one antimicrobial agent, a resin binder and an orally acceptable liquid vehicle for painting upon the teeth, and as a second stage, a dentally acceptable, biodegradable polymeric sealant in an orally acceptable liquid vehicle which is applied over the varnish when the varnish has dried to a translucent, transparent or tooth coloured film and asking the patient to return after three months for a further Streptococcus mutans test; c. for a patient with substantially greater than normal or persistently above normal levels of Streptococcus mutans, giving the patient from about two to about four once a week applications of the above described two stage antimicrobial coating; and d. for a patient whose Streptococcus mutans levels remain unaffected by the two stage application set out in paragraph c, subjecting the patient to a careful re-examination of the dentician for previously undetected decay, recurrent root decay or leaking restorations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The monitoring of the levels of Streptococcus mutans in dental patients has proved to be an indispensable component when treating patients with the product sold under the trade-mark CHLORZOIN for a number of reasons.

SUBSTITUTE SHEET

Firstly, routine testing using the CARIESCREEN SM kit or another suitable testing means such as STRIP MUTANS® manufactured by Ivoclar- Vivadent, during new patient examinations and recall examinations allows a dentist to identify those patients who have high Streptococcus mutans counts. Clearly, this sets up a target group for patients who would benefit from treatment with CHLORZOIN two stage antimicrobial coating. In determining what therapy is required from the testing for Streptococcus mutans levels, one has to view the patient as a whole and thus be familiar with the dental and medical history of the patient, the oral hygiene habits of the patient, what medications the patient may be taking, and determine what orthodontic brackets and/or appliances are in place, since these provide natural sites for colonization of Streptococcus mutans.

Generally what constitutes normal, higher than normal and substantially higher than normal levels of Streptococcus mutans bacteria is dependant on all of the previously mentioned factors. However, it is generally understood that levels of Streptococcus mutans ranging from 0 or nothing detected to 250,000 colonies per ml constitute a low level, for which no treatment would be indicated.

A person who would have slightly above normal levels and who would benefit from at least one treatment would have Streptococcus mutans levels ranging from 250,000 to 500,000 colonies per ml. Substantially higher than normal levels would range from upwards of 500,000 colonies per ml and such a person would necessarily require multiple treatments over the space of corresponding weeks, often about four treatments over about four weeks, with one treatment per week being applied.

The use of the Streptococcus mutans testing clearly helps to demonstrate the efficacy of the CHLORZOIN antimicrobial two stage coating. Such testing clearly indicates how many CHLORZOIN coating applications are required, and whether in fact they are working.

SUBSTITUTE SHEET

It is quite apparent that the only way to determine whether the multiple applications of the CHLORZOIN two stage coating is effective is to retest using for example the CARIESCREEN SM test. In this regard, the CARIESCREEN SM testing readily identifies patients who are refractory to the CHLORZOIN two stage coating. When the Streptococcus mutans count drops only slightly or remains the same after both single or multiple CHLORZOIN two stage coating applications, this alerts the dentist and careful re-examination of the dentician for previously undetected decay, recurrent root decay or leaking restorations should be undertaken. Finally, the use of the CARIESCREEN SM testing subsequent to the

CHLORZOIN coating treatments allows a dentist to determine when retreatment with CHLORZOIN coating is required on an individual basis. The means for testing the level of Streptococcus mutans in the kit and method of the present invention may be selected from any suitable test available, examples of which include that commercially available under the trade-mark CARIESCREEN SM, which is described in detail in Canadian Patent No. 1,235,986 and which may be described generally in the following manner.

The CARIESCREEN SM test kit generally comprises the following components:

1. a sealed container for an antiseptic solid substrate, which substrate has at least one surface coated with a selected solid medium to reduce the growth of Streptococcus mutans and a predetermined amount of saccharide compound; 2. a second sealed container for a buffered saline solution into which a test sample is deposited for determining Streptococcus mutans level; 3. a solid bacitracin composition in an amount and concentration, which in combination with the quantity of saccharide is not sufficient to preclude growth of Streptococcus mutans, but is sufficient to

6

SUBSTITUTE SHEET

prevent growth of substantial amounts of any interfering microorganism;

4. a carbon dioxide generating composition for providing a carbon dioxide atmosphere for incubation of the solid substrate; 5. means are included to examine the surface of the incubated solid substrate for the colony density of Streptococcus mutans;

6. means are provided to prepare the colony density of the tested Streptococcus mutans with a comparison standard in order to determine in a semi-quantitative manner colony density of the Streptococcus mutans of the test sample; and

7. an incubator container for incubation of the solid substrate surface after contact by the buffered saline solution containing the bacitracin composition and the test sample. The carbon dioxide generating composition is placed in this incubation container and after the solid substrate has been allowed to remain in the incubator for a period of time, it is then examined and compared to a standard to determine the colony density of the Streptococcus mutans in the test sample. Other types of Streptococcus mutans bacteria test kits are available commercially and may be used in this method. The two stage antimicrobial coating composition comprises at least one antimicrobial agent which is effective against Streptococcus mutans and which preferably may be selected from chlorhexidene, salts of chlorhexidene and erythromycin. Chlorhexidene acetate is a preferred salt, but other orally and biologically acceptable salts of chlorhexidene may be used such as hydrochloride or gluconate. Chlorhexidene acetate is preferred because of its solubility in water and in the solvent ethanol.

The resin binder may be selected from natural and synthetic resins which are physiologically acceptable, namely substances acceptable for application onto skin or mucosa and which do not cause negative taste perception. The antimicrobial agent must be soluble in the resin or varnish

SUBSTITUTE SHEET

base. Preferred resins include benzoin gum, namely sandarac benzoin, in particular Sumatra or siam benzoin or fractions or recombined fractions thereof. Generally, the resin is dissolved in a solvent, such as ethanol or water. Typically, the solvent must be physiologically acceptable and ethanol is the solvent of choice. Other alternatives would be apparent to the person skilled in the art.

The relative proportions of binder, antimicrobial agent and solvent in the composition may vary widely. The lower solvent limit is fixed only by the maximum solubility of the other ingredients in the coating. The ratio of antimicrobial agent to solvent can be anywhere from 0.001% w/v up to a saturated solution thereof e.g. 20%. The ratio of antimicrobial agent to binder is suitably in the range of 10:1 to 1:10 by weight preferably 5:1 to 1:5 by weight, and most preferably 2:1 to 1:2 by weight. Precise preferred ratios depend to some extent on the rate of release of the antimicrobial agent from the film, once applied to the teeth. It is preferred to subject the site of infection initially to relatively large amounts of antimicrobial agents to reduce the chance of formation of antimicrobial resistance in the infection. In such instances, the amount of antimicrobial agent may range from about 10% to about 30%, preferably about 10% by weight. It is possible to include other ingredients in the antimicrobial coating.

No ingredient likely to have the effect of discolouring the dental site to which the coating is applied should be incorporated. In addition, any ingredient selected should not impart an unacceptable taste or texture, rendering it unpleasant to the user. Examples of suitable additives include fluoride, such as sodium fluoride, stannous fluoride, amine fluoride and sodium monoflourophosphate. Fluoride is known to strengthen the hard tooth tissues against decalcification. It also has an additional effect on the suppression of Streptococcus mutans. The fluoride may be present in an amount ranging from about 0.1 to 1% by weight with respect to the weight of the complete coating.

SUBSTITUTE SHEET

The second stage of the coating, namely the polymeric sealant may be selected from a variety of materials known in the art to be physiologically acceptable for such application. Generally, the preferred sealants are selected from varnishes such as polyurethane, acrylic, cellulose acetate, nitrocellulose and solid polyamide based, which are soluble in and used in suitable physiologically compatible solvents such as acetone or methylenechloride, with acetone being preferred. Another preferred sealant comprises polyethyl methacrylate in ethyl acetate. The polyethyl methacrylate may be selected from those commercially available under the trade-mark EIVACITE 2042, available from Dupont Canada Inc. and from ESSCHEM Co. of Essington, Pennsylvania, U.S.A. Alternatively, CHEMFIL II Varnish, available from De Trey Division Dentsply Limited of Surrey, England, may be used. Generally the methyl methacrylate will be present in from 9.0 to 11.0% w/w in ethyl acetate. An example of a commercially available product is that sold under the trade-mark FLUOR-PROTECTOR. This formulation includes fluoride and is manufactured by Ivoclar-Vivadent. Other commercially acceptable materials are sold under the trade-marks ADHESIT and PLUS-PROTECTOR. The latter formulation is similar to the FLUOR-PROTECTOR composition, without the fluoride component. In some instances, these formulations use ethanol or FREON as solvents.

As is described in U.S. Patent No. 4,883,534, the application of the polymeric sealant is made after the antimicrobial varnish layer has been applied and allowed to dry. The sealant is applied in liquid form, usually in solution with a suitable solvent. It may be applied by painting over the entire area which is already covered by the antimicrobial layer. The form of the sealant allows it to infiltrate crevices and other tooth surfaces whereby the already applied antimicrobial agent is sealed in position so that it maintains communication with the target site and hinders egress into the oral cavity. The sealant once applied is allowed to cure by evaporation of

SUBSTITUTE SHEET

solvent to form a hard transparent or translucent film over the first coating layer. Usually the sealing layer is dried rapidly and instantaneously. Drying may be assisted by use of an air syringe of the type usually employed by a dentist. The formulations suitable for the antimicrobial coating layer are fully described in U.S. Patent No. 4,883,534. Typically, the product available commercially which falls within the scope of this patent is that sold under the trade-mark CHLORZOIN.

EXAMPLES

The invention is further described in the following, specific, nonlimited examples. All parts and percentages are by weight, unless otherwise indicated.

EXAMPLE 1

The use of CARIESCREEN SM test for Streptococcus mutans was combined with the application of CHLORZOIN antimicrobial two stage coating in patients in a dental centre in Ottawa, Ontario, Canada. At this specific dental centre, 56 patients were tested with CARIESCREEN SM test for Streptococcus mutans counts and 68% were found to have Streptococcus mutans levels of 250,000 colonies per ml of saliva or higher. It was determined that these patients would benefit best from the two stage antimicrobial coating sold under the trade-mark CHLORZOIN. In this clinical experience, 6 out of 17 or 35% of patients had Streptococcus mutans counts in excess of 250,000 colonies per ml. As a result, each of these patients was given one CHLORZOIN antimicrobial two stage coating application, and the levels noted subsequently were found three months after application.

10

SUBSTITUTE SHEET

Seven patients in this study had received multiple CHLORZOIN coating applications. Only four of these patients were retested with CARIESCREEN SM test. Multiple applications of the CHLORZOIN antimicrobial two stage coating resulted in two of these patients having Streptococcus mutans counts significantly below 250,000 per ml, while the other two remained high with very little change.

In seven patients who showed little response to the CHLORZOIN antimicrobial coating application at this dental centre, all of these were thoroughly re-examined, and as a result, six of these patients were found to have previously undetected decay.

The CARIESCREEN SM test allows the dentist to determine when retreatment with the CHLORZOIN antimicrobial two stage coating is required on an individual basis. The objective is to keep the Streptococcus mutans counts below 250,000 colonies per ml in all patients and this is achievable by monitoring with the CARIESCREEN SM test on a three month basis.

Following is a history of patients studied who were refractory to the CHLORZOIN two stage antimicrobial treatment.

In these examples, individual subjects are described, who proved to be refractory to the CHLORZOIN antimicrobial two stage treatment. The study of these patients is ongoing, but clearly, these annecdotal descriptions indicate that the combination of the testing for Streptococcus mutans levels along with CHLORZOIN antimicrobial two stage coating provides a diagnostic tool which permits the discovery of hard to diagnose dental problems.

SUBJECT NO. 1

When this patient first visited the centre the patient was tested for Streptococcus mutans level using CARIESCREEN SM test kit. The

11

SUBSTITUTE SHEET

Streptococcus mutans level found was 250,000 colonies per ml of saliva. A single two stage coating of CHLORZOIN antimicrobial treatment was given to the patient, and in three months, upon recall, a further test for Streptococcus mutans level indicated it to be a 500,000 colonies per ml of saliva. A return visit one month later involved treating the patient for root surface restorations. Subsequently, in the two and three months following, the patient was the recipient of four treatments of CHLORZOIN antimicrobial two stage coating, and after another month, the patient was once again tested with CARIESCREEN SM for Streptococcus mutans and the level was found to be 100,000 colonies per ml of saliva.

SUBJECT NO. 2

When this patient first visited the centre, a CARIESCREEN test for Streptococcus mutans level indicated 500,000 colonies per ml of saliva. At that time, one treatment of CHLORZOIN antimicrobial two stage coating was given to the patient. Three months later, the patient was recalled and a further testing for Streptococcus mutans level using CARIESCREEN SM was undertaken and this revealed a count of 200,000 colonies per ml of saliva. Three months afterwards, this patient was treated for two root surface restorations. One month later, further testing with CARIESCREEN SM for Streptococcus mutans revealed a count of 250,000 colonies per ml of saliva. The patient was required to return to be reexamined for more decay.

SUBJECT NO. 3

When this patient first visited the centre where a Streptococcus mutans level of 650,000 colonies per ml of saliva was found in a CARIESCREEN SM test. The patient then received one treatment of

12

SUBSTITUTE SHEET

CHLORZOIN antimicrobial two stage coating. Four months later, the patient returned and a count of 500,000 colonies per ml of saliva of Streptococcus mutans was found upon the use of CARIESCREEN SM test. Three months thereafter, the patient received three fillings, at the same time, the patient received three treatments, for a three week period of

CHLORZOIN antimicrobial two stage coating. The patient was required to return in two months for follow up testing of Streptococcus mutans level.

SUBJECT NO. 4

This patient was tested for Streptococcus mutans using CARIESCREEN SM and the level was found to be 400,000 colonies per ml of saliva. Three and four months later, this patient was treated with three applications of the CHLORZOIN two stage antimicrobial coating. Two months afterwards, the patient was retested with CARIESCREEN SM test for Streptococcus mutans level and this was found to be 500,000 colonies per ml of saliva. Further examination revealed active decay at 37 mo root surface.

SUBJECT NO. 5

This patient was tested with CARIESCREEN SM test for S. mutans level which was found to be 500,000 colonies per ml of saliva. At this time, this patient was given one treatment of CHLORZOIN antimicrobial two stage coating. Subsequently three months later, this patient was retested with CARIESCREEN SM for Streptococcus mutans level and this was found to have remained at 500,000 colonies per ml of saliva. Five months later, the patient was the recipient of one filling for root decay. One month thereafter, the patient was the recipient of three CHLORZOIN antimicrobial two stage coating applications.

13

SUBSTITUTE SHEET

SUBJECT NO. 6

This patient was found upon testing with CARIESCREEN SM test, to have an Streptococcus mutans level of 500,000 colonies per ml of saliva. The patient was the recipient of one CHLORZOIN antimicrobial two stage coating application. Three months later, further testing with CARIESCREEN SM test revealed an Streptococcus mutans level of 500,000 colonies per ml of saliva. A further three months thereafter, this patient was the recipient of three CHLORZOIN antimicrobial two stage coating application. One month thereafter, this patient was retested with

CARIESCREEN SM test and found to have a Streptococcus mutans count of 100,000 colonies per ml of saliva. No decay was found and it is apparent that multiple treatments can decrease the level of Streptococcus mutans in an oral cavity.

SUBJECT NO. 7

This patient was tested with CARIESCREEN SM for Streptococcus mutans level. The count was found to be 1,000,000 colonies per ml of saliva. The patient was then subjected to very careful examination and found to be suffering from deep root decay. Two extractions and two fillings were required. One month later, the patient was the recipient of four separate antimicrobial two stage applications.

EXAMPLE 2

Seven subjects with moderate or high counts of mutans streptococci, as measured by CARIESCREEN, participated in the study. Five of them were treated once with CHLORZOIN utilizing CHEMFIL as a Stage II, and two were treated with CHLORZOIN utilizing polyurethane-acetone, as a

14

SUBSTITUTE SHEET

control. At intervals of between 7 and 28 days after his/her CHLORZOIN treatment, each subject was re-assessed for hi/her mutans streptococcal count using CARIESCREEN.

The results (Table I) demonstrated that CHLORZOIN was equally effective in reducing mutans streptococcal counts when CHEMFIL was used as a Stage II as when polyurethane-acetone was used. Both Stage II formulations had an effectiveness similar to those observed in our past studies when we utilized a Stage II formulation comprised or polyurethane- methylene chloride. CHEMFIL, which contains polyethylmethacrylate-ethyl acetate, is as effective as a Stage II for CHLORZOIN treatment as is polyurethane- acetone. The effectiveness CHLORZOIN treatment with both is similar to our observation in a previous study utilizing a Stage II consisting of polyurethane-methylene chloride, in which a single treatment reduced the salivary mutans streptococcal counts in 27 children from a mean of 550,000 CFU/ml before treatment to a mean of 23,000 CFU/ml one week after treatment {Sandham, et al, J Dent Res 71:32-35, 1992).

15

SUBSTITUTE SHEET

TABLE I

COMPARISON OF THE EFFECTS OF CHLORZOIN

WITH CHEMFIL OR POLYURETHANE AS STAGE II

ON COUNTS OF MUTANS STREPTOCOCCI

(colony-forming units/ml saliva)

Type of Stage Subject* Mutans streptococcus counts Days post II Rx

Before After

NI 250K, >1,000K 250K 11

CHEMFIL AP 1000K, 500K, 500K 50K 7

MA 500K, >1000K. 250K 50K 8

HR 1000K >10K 28

GF 250K 0 14

POLY-U BE 500K <50K 21

MAR 1000K 0 8

11 subjects had a moderate or high initial count of mutans streptococci.

16

SUBSTITUTE SHEET