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Online Journal - URL: http://bioline.bdt.org.br/bf Effects of a Chlorhexidine Varnish on the Mutans Streptococci and on Dental Caries W.A. BRETZ^1,^4, E. V. do VALLE^2, R. ALMEIDA^2, C. DJAHJAH^2, Y-M. CHEN^3, M.A. SCHORK^3.
1. University of Texas, Houston, TX, USA
Author for correspondence:
Date received: 15th July 1997
Code Number:BF97002
Sizes of Files:
Text: 25.3K
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ABSTRACT The purpose of this study was to determine the effects of a chlorhexidine varnish-sealant on the mutans streptococci and on caries outcome variables, in 113 female school-children 10 to 15 years-old, in Rio de Janeiro, Brazil. Subjects were randomly allocated to control (C) and treatment (T) groups, n=55 and n=58, respectively. At baseline the number of decayed, restored, and white spot lesions on teeth surfaces was determined by a calibrated examiner, and the salivary levels of the mutans streptococci were assessed with a dip-slide test. Both groups had their existing carious lesions restored and they were given a prophylaxis. Subjects who belonged to the T group had a 10% chlorhexidine varnish-sealant applied to their entire dentition once or twice (with a 1 week interval) if they had =/< 2.5 x 10^5 CFU of mutans streptococci/dip-slide, respectively. The same procedures performed at baseline were performed after 3 months for each group. After 6 months all subjects received caries examination and salivary analysis for the mutans streptococci. Results showed significantly lower levels of the mutans streptococci in the T group when compared to the C group after 6 months (p<0.0001). No significant differences were observed between T and C groups from baseline to 6-month examinations in caries scores. Treatment group was the only predictor (p<0.0001-final model) of the salivary levels of the mutans streptococci after 6 months. It is concluded that the use of a chlorhexidine varnish-sealant in the T group when compared to the C group had a significant effect in reducing the levels of the mutans streptococci after 6 months which could not be translated into a significant effect on caries outcome variables when T and C groups were compared. Therefore, we suggest that protocols with multiple applications of chlorhexidine varnishes for a finite period of time may be necessary in longitudinal studies of the effects of chlorhexidine varnishes on the mutans streptococci and on caries outcome variables. Keywords: chlorhexidine varnishes mutans streptococci dental caries INTRODUCTION The mutans streptococci are cariogenic organisms which have been strongly associated with human dental decay. The mutans streptococci are prominent members of dental plaque biofilms present on tooth surfaces of patients experiencing dental decay (Loesche, 1986). There is evidence in the literature suggesting that the levels of the mutans streptococci present on dental plaque biofilms may be reflective of their levels in saliva (Loesche, 1986; Bowden, 1997). Suppression or elimination of detectable mutans streptococci from the oral environment for prolonged periods of time would be desirable, especially if this suppression/elimination could result in clinical benefits for dental patients by preventing or reducing dental decay development. Studies on the clinical applications of antimicrobial treatment, a two-stage chlorhexidine varnish-sealant, used to prevent the development on tooth surfaces of dental plaque biofilms containing high proportions of the mutans streptococci, have demonstrated that this varnish can be effective in suppressing/eliminating detectable mutans streptococci in adults and in children wearing orthodontic appliances for prolonged periods of time (Sandham et al., 1988; Sandham et al., 1991; Sandham et al., 1992). The protocols designed for these studies varied in terms of: 1) the concentration of the antimicrobial agent present in the varnish-sealant; 2) delivery of a dental prophylaxis prior to varnish application; 3) coverage of the dentition by the varnish-sealant; and 4) the study length. Despite variations in the protocols, the frequency of application of the varnish-sealant in these studies was consistent and always included four weekly applications. We have implemented a simpler protocol to assess the efficacy of the chlorhexidine varnish-sealant on various oral health parameters in children and adolescents. Some of the results of these clinical trials have been described elsewhere (Bretz et al., 1995; Valente et al., 1996). The present study describes the effects of the chlorhexidine varnish-sealant on the mutans streptococci (ms) salivary levels and on dental caries. MATERIALS & METHODS One-hundred and thirteen female school-children, aged 10 to 15 years, residing in an orphanage in Rio de Janeiro, Brazil, participated in this study. Subjects were randomly allocated to control (C) and treatment (T) groups, n=55 and n=58, respectively. The average age for participants in C and T groups was 12.0+/-1.3 and 12.2+/-1.3 years. FIGURE Study Protocol
Baseline 3 Months 6 Months
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Caries Examination
C^a + + +
T^b + + +
Saliva Samples
C + + +
T + + +
Prophylaxis
C + +
T + +
Varnish Application
C
T + +
a Control group
b Test group
------------------------------------------------------------------
The study protocol (Figure) included caries examination at baseline, where the number of decayed, restored, and white spot lesions on teeth surfaces was determined by a calibrated examiner, and the salivary levels of the mutans streptococci were assessed semi-quantitatively with a dip-slide test (CARITEST^TM). The CARITEST is similar to the commercially available CARISCREEN^TM. Test scores ranged from 0 (non-detected) to 6 (>10^6 colony-forming units/dip-slide). C and T groups were balanced on all caries and microbiological parameters at baseline (Table 1).
TABLE 1 Multiple Regression Analysis of Caries Scores at 6 Months (Final Model)
--------------------------------------------------------------------------
Number of Number of Number of Number of
Predictor Sound Surfaces Decayed Surfaces Restored White
At 6 Months At 6 Months At 6 Months Spot Surfaces
Variables At 6 Months
---------------------------------------------------------------------------
Number of
Sound Surfaces p<0.0001
At Baseline
Number of
Decayed Surfaces p<0.0001
at Baseline
Number of
Restored Surfaces p<0.0001
at Baseline
Number of
White Spot p<0.0001
at Baseline
Age NS NS NS NS
Group
Control/Test NS NS NS NS
Caries at Baseline
not Restored NS NS p<0.0001 p=0.0016
Yes/No
NS- not significant
---------------------------------------------------------------------------
Both groups were to have their existing carious lesions restored, and they were given a prophylaxis (supragingival plaque removal with a rubber cup) in order to eliminate any dental plaque biofilm present on tooth surfaces. Subjects who belonged to the T group had a 10% chlorhexidine varnish-sealant (CHLORZOIN^TM) applied to their entire dentition once or twice (with a 1 week interval) if they had =/< 2.5 x 10^5 CFU of mutans streptococci/dip-slide, respectively. The same procedures performed at baseline were performed after 3 months for each group (Figure). After 6 months, subjects in C and T groups received a final caries examination and salivary analysis for the mutans streptococci. Unfortunately, some of the subjects who belonged to C and T groups still had retentive sites and open carious lesions at the 3-month visit which should have been restored prior to the application of the chlorhexidine varnish-sealant at baseline. Therefore, statistical analysis was performed in all subjects from C and T groups, and then on subjects who had their restorative needs met at baseline in C (n=42) and T (n=47) groups. Subject attrition was minimal during the course of the study because subjects resided in the orphanage. Statistical analyses using t-tests were completed to determine if there were differences between treatment regimens (control and test) for each variable separately at baseline, and 3 and 6 months thereafter. Such analyses were completed for sound teeth, decayed teeth, restored teeth, white spot lesions and salivary levels of the mutans streptococci, as well as age at baseline. Next, t-tests were employed to assess if changes in these variables from baseline to 6 months were different between C and T groups. Finally, multiple regression analysis to determine the effects of predictors including age, treatment group and corresponding baseline values on 3 and 6 months follow-up values for sound, decayed and restored teeth, white spot lesions and salivary levels of mutans streptococci, were carried out. RESULTS The occurrence of dental decay parameters after 6 months in this study, i.e., the number of sound, decayed and restored surfaces, and the number of white spot lesions, was significantly predicted by their respective baseline values for analyses based on all subjects (Table 1). Age and treatment group were not predictors of dental decay parameters after 6 months. The presence of carious lesions in some subjects of C and T groups at baseline, which should have been restored, was a significant predictor of the number of restored surfaces and of the number of white spot lesions after 6 months in all subjects. Based on that, analysis of dental decay experience between baseline and 6 months was performed only on subjects who had their restorative needs met at baseline (Table 2). TABLE 2 Caries Scores of Control and Test Groups at Baseline and After 6 Months
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% of Sound % of Decayed % of Restored % of White
Surfaces Surfaces Surfaces Spot Lesions
-------------- -------------- -------------- --------------
Group BL^a 6 mo^b BL 6 mo BL 6 mo BL 6 mo
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(42)^c (40) (42) (40) (42) (40) (42) (40)
CONTROL 104.8^d 114.3 0.04 0.12 2.3 2.6 9.1 2.7
(14.9) (12.4) (0.3) (0.4) (2.8) (3.3) (6.3) (3.6)
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(47) (43) (47) (43) (47) (43) (47) (43)
TEST 106.8 116.3 0 0.12 2.8 3.2 9.7 3.0
(14.3) (11.8) (0) (0.4) (3.5) (3.8) (5.5) (2.3)
--------------------------------------------------------------------------
a BL = Baseline b 6 mo = 6 months c ( ) = number of subjects d mean +/- (SD) Table 2 shows analysis of dental decay parameters at baseline and after 6 months. None of the parameters were significantly different in T and C groups between baseline and 6 months examinations, with the exception of the number of sound surfaces which were significantly increased at the 6 months examination in both C and T groups. It is possible that the number of reversals of white spot lesions after 6 months may have contributed to an increase in the number of sound surfaces after 6 months. No significant differences on dental decay parameters were found between T and C groups at the 6-month examination. We next evaluated the effects of the chlorhexidine varnish-sealant applications on the mutans streptococci after 3 and 6 months using multiple regression analysis. This analysis revealed that randomized group (control vs treatment) was a significant predictor of the salivary levels of the mutans streptococci after 6 months. Age was also a significant predictor of the salivary levels of the mutans streptococci after 3 months. TABLE 3 Multiple Regression Analysis of Mutans Streptococci (MS) Salivary Levels at 3 and 6 Months (Final Model) --------------------------------------------------------------------------- Predictor Salivary Levels of Salivary Levels of Variables MS At 3 Months MS At 6 Months --------------------------------------------------------------------------- Salivary Levels of NS NS MS at Baseline Age p<0.0001 NS Group Control/Test NS p<0.0001 Caries at Baseline NS NS not Restored Yes/No NS = not significant --------------------------------------------------------------------------- TABLE 4 Mutans Streptococci (MS) Salivary Levels** in Test and Control Subjects at Baseline, and After 3 and 6 Months
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Group Baseline 3 Months 6 Months
---------------------------------------------------------------------------
N* 42 40 40
CONTROL MS 2.8+/-1.3 2.8+/-1.9 4.1+/-1.3
NS# NS p<0.0001
TREATED MS 2.5+/-1.3 2.7+/-1.5 2.9+/-1.5
N 47 46 43
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* N = number of subjects, # NS = not significant ** mutans streptococci salivary levels are expressed in mean +/- standard deviation test scores ranging from 0 to 6 ( see Materials & Methods for corresponding levels).
Table 4 depicts the salivary levels of the mutans streptococci in T and C groups at baseline, 3 and 6-month examinations. There were no significant differences between T and C groups after 3 months on the salivary levels of the mutans streptococci. After 6 months the C group exhibited significantly higher levels of the mutans streptococci when compared to the T group. DISCUSSION Since the advent of chlorhexidine varnishes (Balanyk and Sandham, 1985; Friedman et al., 1985; Schaeken et al., 1989; Huizinga et al., 1990) investigators have been interested in determining the effects of these varnishes on selected oral health parameters. Despite variations in the protocols of chlorhexidine varnish treatments, studies have demonstrated that these varnishes can be effective in suppressing significantly the salivary mutans streptococci or the mutans streptococci present in tooth dental plaque biofilms for periods ranging from 4 weeks (Sandham et al., 1988; Sandham et al., 1991; Sandham et al., 1992; Schaeken and De Haan, 1989; Twetman et al., 1995; Pienihakkinen et al., 1995) up to 3 to 4 months (Petersson et al., 1991; Hildebrandt et al., 1992; Le and Schaeken, 1993; Pienihakkinen et al., 1995; Twetman and Petersson, 1997). A few studies have reported a significant suppression of the mutans streptococci present in saliva or in tooth dental plaque biofilms for periods ranging from 7 to 9 months (Schaeken et al., 1991; Sandham et al., 1992). The variations in the protocols of studies on chlorhexidine varnishes included the study length, age of subjects, chlorhexidine varnish concentration, and the frequency of application of the chlorhexidine varnishes. Nevertheless, these studies have unequivocally shown that chlorhexidine varnishes can be effective in suppressing and/or eliminating the mutans streptococci from saliva, and from occlusal and interdental dental plaque biofilms for prolonged periods of time. With the exception of three studies to our knowledge (Huizinga et al., 1990; Schaeken et al., 1991; Bratthall et al., 1995) none of the studies on chlorhexidine varnishes looked at the effects of these varnishes on caries outcome variables. The clinical utility of chlorhexidine varnishes can only be meaningful if a reduction of the cariogenic flora from dental plaque biofilms, i.e., the mutans streptococci, is paralleled by a reduction in the development of dental caries. We have implemented this study in order to determine the effects of a chlorhexidine varnish-sealant on the salivary levels of the mutans streptococci and on dental caries. Our protocol included one or two applications (see Material & Methods) of the chlorhexidine varnish-sealant in the T group at baseline and after 3 months, while after 6 months the overall effects were determined (Figure). The results of this study have shown that our protocol did not have an effect on caries outcome variables (Tables 1 & 2) after 6 months when T and C groups were compared. The applications of the chlorhexidine varnish-sealant were able at best to maintain the levels of the mutans streptococci stabilized in the T group throughout the study period. However, a significant difference in the levels of the mutans streptococci was observed between T and C groups after 6 months (Table 4). Huizinga et al., (1990) and Schaeken et al., (1991) demonstrated a significant effect on root remineralization and on root caries arrestment after 2 weeks and 9 months, respectively, with a 1% chlorhexidine-thymol varnish (Huizinga et al., 1990) and with a 40% chlorhexidine varnish (Schaeken et al., 1991). Bratthall et al., (1995) showed a significant reduction in fissure caries development of teeth treated with a 1% chlorhexidine-thymol varnish when compared to non-treated fissures after two years. We acknowledge the fact that the length of our study may have not been adequate in order to demonstrate a significant effect of chlorhexidine varnish-sealant treated subjects on caries outcome variables. The frequency of application of chlorhexidine varnishes seems to be critical for the long-term suppression of the mutans streptococci (Sandham et al., 1988 Sandham et al., 1991). These investigators have used a protocol with 4 applications one week apart for a month, demonstrating long-term suppression of the mutans streptococci in study participants. In fact, a recent study (Twetman and Petersson, 1997) implemented a protocol on the effects of chlorhexidine varnishes in an intensive mode (3 applications within 7 to 10 days) and in a monthly mode (3-month period) on the mutans streptococci levels. After 3 months, a significant reduction in the levels of the mutans streptococci was demonstrated in the intensively treated subjects as opposed to that observed in monthly treated subjects. Our protocol (1 or 2 applications at baseline and after 3 months) was marginally effective in significantly suppressing the levels of the mutans streptococci in subjects treated with a chlorhexidine varnish-sealant. Collectively, our results and the results of other studies suggest that protocols with multiple applications of chlorhexidine varnishes for a finite period of time may be necessary in longitudinal studies of the effects of chlorhexidine varnishes on the mutans streptococci and on caries outcome variables. REFERENCES Balanyk, T.E., Sandham, H.J.,1985, Development of sustained-release antimicrobial varnishes effective against Streptococcus mutans in vitro. J Dent Res 64:1356-1360. Bowden, G.H., 1997, Does assessment of microbial composition of plaque/saliva allow for the diagnosis of disease activity of individuals? Comm Dent Oral Epid 25:76-81. Bratthall D., Serinirach, R., Rapisuwon, S. et al., 1995, A study into the prevention of dental caries using an antimicrobial varnish. Int Dent J 45:245-254. Bretz, W.A.; Djahjah, C.A.; Almeida, R.S.; Villar do Valle, E.; Fonseca, C.; Valente, I.; Seabra, G.; Chiesa, C.,1995, Effects of a chlorhexidine varnish on caries lesions. Oral Health 85:29-30. Friedman, M., Harari, D., Raz, H., Golomb, G., Brayer L., 1985, Plaque inhibition by sustained release of chlorhexidine from removable appliances. J Dent Res 64:1319-1321. Hildebrandt, G.H.; Pape, H.R.; Syed, S.A.; Gregory, W.A.: Friedman, M., 1992, Effect of slow-release chlorhexidine mouthguards on the levels of selected salivary bacteria. Caries Res 26:268-274. Huizinga, E. D.; Ruben, J; Arends, J., 1990, Effect of an antimicrobial- containing varnish on root demineralisation in situ. Caries Res 24;130-132. Le, Y.L. and Schaeken, M.J.M., 1993, Effect of single and repeated application of chlorhexidine varnish on mutans streptococci in plaque from fissures of premolar and molar teeth. Caries Res 27:303-306. Loesche, W. J., 1986, Role of Streptococcus mutans in human dental decay. Microbiol Rev 50:353-380. Petersson L.G., Maki, Y., Twetman, T., Edwardsson, S., 1991, Mutans streptococci in saliva and interdental spaces after topical applications of an antibacterial varnish in schoolchildren. Oral Microbiol Immunol 6:284-287. Pienihakkinen, K., Soderling, E., Ostela, I., Leskela, I., Tenovuo, J., 1995, Comparison of the efficacy of 40% chlorhexidine varnish and 1% chlorhexidine-fluoride gel in decreasing the level of salivary mutans streptococci. Caries Res 29:62-67. Sandham, H.J., Brown, J., Phillips, H.I., Chan, K.H., 1998, A preliminary report of long term elimination of detectable mutans streptococci in man. J Dent Res 67:9-14. Sandham, H.J., Brown, J., Chan, K.H., Phillips, H.I., Burgess, R.C., Stokl, A.J., 1991, Clinical trial in adults of an antimicrobial varnish for reducing mutans streptococci. J Dent Res 70:1401-1408. Sandham, H.J., Nadeau, L., Phillips, H.I., 1992, The effect of chlorhexidine varnish treatment on salivary mutans streptococcal levels in child orthodontic patients. J Dent Res 71:32-35. Schaeken, M.J.M., De Haan, P.,1989, Effects of sustained-release chlorhexidine acetate on the human dental plaque flora. J Dent Res 68:119-123. Schaeken, M.J.M., van der Hoeven, J.S., Hendriks, J.C.M., 1989, Effects of varnishes containing chlorhexidine on the human dental plaque flora. J Dent Res 68:1786-1789. Schaeken, M.J.M., Schouten, M.J., van der Kieboom, C.W.A., van der Hoeven, J.S., 1991, Influence of contact time and concentration of chlorhexidine varnish on mutans streptococci in interproximal dental plaque. Caries Res 25:292-295. Schaeken, M.J.M., Keltjens, H.M., Van der Hoeven, J.S.,1991, Effects of fluoride and chlorhexidine on the microflora of dental root surfaces and progression of root surface caries. J Dent Res 70:150-153. Twetman, S.; Hallgren, A.; Petersson, L.G., 1995, Effect of a antibacterial varnish on mutans streptococci in plaque from enamel adjacent to orthodontic appliances. Caries Res 29:188-191. Twetman, S. and Petersson, L. G., 1997, Effect of different chlorhexidine varnish regimens on mutans streptococci levels in interdental plaque and saliva. Caries Res 31:189-193. Valente, I.; Seabra, G.; Chiesa, C.; Almeida, R.; Djahjah, C.A.; Fonseca, C.; Villar do Valle, E.; Bretz, W.A.,1996, Effects of a chlorhexidine varnish on the gingival status of adolescents. J Can Dent Assoc 62:46-48. Copyright remains with the author. Published by Bioline Publications.
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