J Oral Maxillofac 50:1055-l
Surg
059. 1992
Evaluation of the Substantivity of a Chlorhexidine Oral Rinse in Irradiated Head and Neck Cancer Patients JOSEPH A. TOLJANIC, DDS,* JAMES C. HAGEN, PHD, MPH,t YASUNOMBU TAKAHASHI, DDS, PHD,$ AND ROBERT D. SHAPIRO,
DDS§
A trial was conducted to evaluate the substantivity of chlorhexidine in a population of patients who had undergone primary or adjunctive radiation therapy for tumors of the head and neck. Subjects were instructed to first rinse with 0.12% chlorhexidine as per manufacturer instructions, followed by a releasing rinse provided 1 minute, 1 hour, and 4 hours later. All expectorants were collected, pH adjusted, and introduced to the test microorganism. Zones of bacterial inhibition were then measured and recorded. The results suggest that 0.12% chlorhexidine is retained in the oral cavity for at least 4 hours after an initial rinsing and that the property of substantivity remains active in spite of radiation-induced changes in the oral cavity and salivary glands.
Chlorhexidine gluconate has been extensively studied for almost 20 years as an adjunct to currently accepted oral health care measures. It has been shown to be clinically safe and effective in preventing the development of plaque-associated gingivitis.‘-I4 Its effect is achieved primarily via two mechanisms. First, chlorhexidine is both bactericidally and bacteriostaticly active against organisms responsible for the formation and adherence of plaque to dental structures. This action is most likely the result of the disruption of bacterial cell wall function.‘5-‘7 Second, chlorhexidine exhibits the property of substantivity, a phenomenon whereby a substance binds to a structure it contacts, in this case probably glycoproteins coating the oral mucosa and dentition. Such binding, which is hypoth-
esized as being electrostatic in nature, is reversible and thus permits a steady release of the active agent into the oral cavity. ‘8,‘9-2’This affords a prolongation of effect and provides continuous protection until the next rinse cycle is performed. Oral health care for patients who have undergone therapeutic irradiation for tumors of the head and neck is complicated by radiation-induced regional changes. As a result of these changes, such patients are placed at increased risk for developing significant dental pathology, including gingival and periodontal disease, while concurrently possessing a decreased capacity to provide host defense mechanisms and effect physiological repair. It would appear that this patient population could benefit dramatically from the use of chlorhexidine as a preventive adjunct in the maintenance of oral health. However, it is as yet unclear whether the changes seen in the oral milieu may act to decrease the effective substantivity of chlorhexidine by decreasing salivary output and depriving the patient of important sources of potential protein molecules that may act as binding sites. If chlorhexidine can be shown to retain substantivity in irradiated patients, it could be included in the current postradiation dental protocols now recommended. The purpose of this investigation was to evaluate and quantify the level of substantivity of chlorhexidine in individuals who have received cancericidal doses of radiation for tumors of the head and neck.
* Assistant Professor of Clinical Surgery; Chief, Prosthetics, Section of Oral and Maxillofacial Surgery/Dentistry, University of Chicago. Chicago. t Associate Professor, Department of Periodontics, Microbiology and Biochemistry, Loyola University, School of Dentistry. Chicago. $ Instructor, Department of Removable Prosthodontics, Osaka University, Faculty of Dentistry, Osaka, Japan. 5 Associate Professor of Clinical Surgery. Chief, Section of Oral and Maxillofacial Surgery/Dentistry, University of Chicago, Chicago. Address correspondence and reprint requests to Dr Toljanic: University ofchicago, Zolles Dental Clinic, 584 S Maryland Ave. Chicago. IL 60637. 0 1992 American
Association
of Oral and Maxillofacial
Surgeons
0278-2391/92/5010-0007$3.00/O
1055
1056
CHLORHEXIDINE USE IN IRRADIATED PATIENTS
Materials and Methods PRESTUDY STANDARDIZATIONPROCEDURES AND SUSCEPTIBILITY TESTING
Prestudy screening of selected clinical and standard microbial strains was performed to identify an organism with a reproducibly high level of sensitivity to chlorhexidine. Microorganisms screened for included Streptococcus mitis, Streptococcus salivarius, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Actinomyces israelii, Bacteriodes gingivalis, Peptostreptococcus anaerobius, and Actinobacillus actinomycetemcomitans. Serial twofold dilutions of sterile chlorhexidine were prepared in test tubes containing enriched Todd-Hewitt Broth (enriched with 0.5% yeast extract, 0.05% hemin, and 0.0005% menadione) for use with obligate anaerobes and trypticase soy broth for use with facultative flora. Into each prepared tube, 0.1 mL of an overnight culture of the test microorganism was introduced to a final concentration of 10’ organisms/ml. The tubes were then incubated at 37” for 24 hours either aerobically or anaerobically, as indicated. Results were visually read as either growth or no growth, with the last tube possessing turbidity recorded as the minimum inhibitory concentration (MIC) of chlorhexidine for each microorganism evaluated. In this manner, S epidermidis was found to be the most reproducibly sensitive organism and therefore was used throughout the study. A prestudy standard curve of chlorhexidine concentration versus S epidermidis growth was created using a modification of the procedure described by Ericsson and Sherris22 and applied to chlorhexidine by Emilson.23 This was accomplished by first seeding media plates containing trypticase soy agar with an overnight culture. Into the prepared media of each plate, five evenly spaced wells were created using a k-in-diameter cork bore. A commercially available formula containing 0.12% chlorhexidine gluconate (Peridex, Proctor & Gamble, Cincinnati, OH) as the active ingredient was prepared to a 1: 10 dilution and then adjusted to a pH of 7. In triplicate, 0.32 mL of this solution was introduced into the media plate wells at the following concentrations: 64, 32, 16, 8, 4, 2, 1, and 0.5 pg/mL. Each plate was then incubated for 18 to 24 hours. The diameters of the resultant zones of inhibition surrounding each well were measured by hand to the nearest ‘/iOmm using a millimeter ruler. From these measures, a standard curve was generated (Fig 1). ESTABLISHMENTOFTESTPROTOCOL
A protocol was developed and tested using two nonirradiated, dentate volunteers (the authors) who pos-
‘II 0.5
1 1.5
2 2.5
3
4 Peridex
5
6
7
6
9
10
Conc.(mglmU
FIGURE 1. Standard curve of chlorhexidine concentration versus growth of test microbial organism.
sessed normal salivary flow characteristics, with adequate volume and consistency. Each subject was instructed to vigorously rinse with ‘/2oz of the commercially prepared formula containing 0.12% chlorhexidine gluconate as the active ingredient. After 30 seconds, they were instructed to expectorate (as per manufacturer instructions). This was followed exactly 1 minute later by a second aqueous rinse of 1% acetic acid, which was again expectorated. The purpose of the second rinse was to release any residual bioactive substance retained on oral tissues that would then be collected in the expectorant.24 A solution of 1% aqueous acetic acid has been previously shown to be safe as an oral rinse under selected conditions.” The preceding procedures were then repeated on two separate occasions during which time only the interval between the 0.12% chlorhexidine rinse and the 1% acetic acid rinse was changed: to 1 and 4 hours, respectively. The expectorated 1% acetic acid rinse was collected from each subject for all test periods and the acidity was neutralized to pH 7, as measured using an ion analyzer (Model 601A, Orion Company, Cambridge, MA), by the addition of aqueous NaOH. Media plates containing trypticase soy agar were seeded with the test organism and wells prepared as previously described. Into each well 0.32 mL of the neutralized expectorants were introduced in duplicate using a micropipette. The prepared media plates were then incubated for 18 to 24 hours and the resultant zones of inhibition measured. Each measurement was compared with the standard curve previously developed. TESTPROTOCOL
Six subjects volunteered to participate in the trial. Each was selected from the pool of patients previously
1057
TOLJANIC ET AL
treated with primary or adjunctive radiotherapy at the University of Chicago Hospitals for tumors of varying sites contained within the head and neck region following full study disclosure (Table 1). Each patient was on a schedule of routine follow-up in the Section of Oral and Maxillofacial Surgery/Dentistry. All patients were either partially or fully dentate, with the minimum number of 2 1 teeth present. Ages ranged from 16 to 82 years. The subjects were instructed to rinse vigorously with both the 0.12% chlorhexidine and 1% acetic acid as per the protocol previously described. Resultant expectorants after each of the l-minute, I-hour, and 4hour rinse cycles were collected and placed in -65°C frozen storage for later data analysis. Additionally, a fourth rinse cycle was performed using the 1% acetic acid without a previous rinse of 0.12% chlorhexidine. The collected expectorant served as a within-patient control. Expectorants were neutralized to a pH of 7 by the addition of aqueous NaOH and media plates containing trypticase soy blood agar were seeded with the test organism and wells prepared as previously described. Into each well, 0.32 mL of the neutralized trial expectorants was introduced in duplicate using a micropipette. All prepared media plates were then incubated for 18 to 24 hours and the resultant zones of inhibition measured. Each measurement was compared with the standard curve previously developed. Results
Table 2 lists the zones of inhibition obtained with all rinse cycles for each subject. As the expectorants were plated in duplicate, the values represent the average of two measures. Measurable zones of inhibition were noted with all cycles for five of the six subjects. A zone of inhibition was not evident with the 4-hour rinse cycle expectorant obtained from subject B.K. The data generated were compared with the pretrial standard curve developed as described in the Methods and Materials section. This was done to quantify the
Table 1. Primary Tumor Site and Radiation Dose Delivered Subject
Primary Tumor Site
Dose GY)
B.K.
Left tonsil, soft palate, and base of tongue Nasopharynx Nasopharynx Right lateral tongue border Supraglottic larynx Floor of mouth
60.4 50 64.8 60 66 65
W.H. K.B. W.K. N.S. B.B
Table 2. Microbial Inhibition Measured After Incubation With Test Expectorants
Subject
Time Interval
B.K.
I Minute I Hour
W.H.
K.B.
W.K.
N.S.
B.B.
4 Hour I Minute I Hour 4 Hour I Minute 1 Hour 4 Hour 1 Minute I Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour
Zone of Inhibition (cm) 1.5 1.2 0 1.6 1.35 I.1 1.35 1.3
1 1.5
1 0.9 1.55 1.3 1.3 1.5 1.4 1.05
Zones of inhibition represent average of two measurements.
levels of antibacterial activity for the expectorants at each rinse cycle relative to a commercial product containing 0.12% chlorhexidine at known dilutions. The results of this comparison are shown in Table 3. and indicate the concentration of bioactive substance contained within each collected expectorant. Furthermore, a decrease in this bioactive substance activity was apparent for the expectorants as the rinse cycle time interval increased in all but two trial periods (between 1 minute and 1 hour for subject K.B. and between 1 hour and 4 hours for subject W.K.). When control expectorants were evaluated, no zones of inhibition were generated using the resultant expectorants for any subject. Discussion
The results of this study indicate that when a commercial product containing 0.12% chlorhexidine is used as an oral rinse in a population of patients irradiated for certain tumors of the head and neck, an antibacterial element is retained and then gradually released into the oral cavity over time. This shows that the property of substantivity, as ascribed to chlorhexidine, remains clinically in effect in this patient population and provides prolongation of antibacterial action. Zones of inhibition were noted in all six subjects at the 1-minute and 1-hour trial periods and zones of inhibition were noted in five of six subjects for the 4-hour trial period. From this it may be deduced that the substantivity of chlorhexidine appears to operate despite
1058
CHLORHEXIDINE
Table 3. Chlorhexidine Concentration in the Expectorant at Each Trial Period
Subject
Time Interval
B.K.
I Minute I Hour
W.H.
K.B.
W.K.
N.S.
B.B.
4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute I Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour
Concentration (mg/mL) 5.35 2.17 0 >10.7 10.7 1.08 2.17 2.17
I .08 10.7 0.27 0.54 > 10.7 5.35 2.17 10.7 5.35 1.08
the physiological and histologic tissue changes induced by therapeutic radiation. Over the course of 4 hours, less bioactive agent remains available as indicated by the steady decrease in zone size obtained over time in four of six subjects. This would presumably be due to gradual release of a bioactive substance into the oral cavity. Further review of subjects K.B. and WK. was undertaken to elicit an explanation for the discrepancies in zone size changes over time. During the course of the study, subject W.K. complained of difficulty in vigorously swishing the test rinses for the prescribed period. The subject would tend to stop swishing altogether, if not subjected to constant observation and encouragement. No performance status variations could be determined for subject K.B., who had no difficulty in performing the study tasks. As measurable zones of inhibition were noted at the 4-hour time interval in five of six subjects, it would be expected that chlorhexidine would remain biologically available and provide antibacterial action for some time beyond this period. However, the total time during which bioactivity could be anticipated was not assessed in the present study. Although the study involved a small sample, these results show that the property of substantivity remains active in patients who rinse with a commercial preparation containing chlorhexidine following therapeutic doses of radiation for oral and oropharyngeal neoplasms. This capacity remains intact for at least 4 hours, and quite likely beyond, providing antibacterial action in the oral cavity. This finding may be of major sig-
USE IN IRRADIATED PATIENTS
nificance. Oral health is adversely effected subsequent to irradiation, with the postradiation xerostomia resulting in, among other sequelae, an increase in gingival inflammation that can progress to active periodontal disease. Periodontal disease and the resultant need for tooth extraction have long been recognized as a major etiologic factor in the development of osteoradionecrosis of the jaw bones. Hence, irradiated patients not only face the danger of dramatically decreased dental health, with the attendant discomfort and loss of function, but also the additional risk of developing a serious, potentially untreatable, pathologic bone condition. Although measures that effectively combat some of the posttreatment complications associated with radiation therapy have been developed and instituted over the years, a substantial percentage of patients fail to respond to this dental maintenance. Once dental pathology has progressed to a significant extent, salvage becomes extremely difficult. Use of chlorhexidine as a long-term measure in oral health maintenance could provide important benefits in selected circumstances. First, it could serve to augment the currently accepted regimen of oral care in specific populations of irradiated patients showing active and recalcitrant oral disease in spite of application of the standard regimen. These patients would be identified through the course of regular follow-up dental examinations. Second, chlorhexidine could be added to the standard regimen for those individuals who are unable and/or unwilling to consistently comply with the standard regimen. Such individuals might include those who are ineffective in toothbrushing, who could benefit from the additional protective mechanisms provided by chlorhexidine. Inclusion of long-term use of chlorhexidine into the accepted standard of care, however, may potentially create problems for patients. Although prolonged use has been demonstrated to be a safe and effective measure in the oral health care of nonirradiated patient populations, no such longitudinal studies have been performed in irradiated patients.3~8-‘0,‘2-‘4Soft-tissue response in light of a compromised regional vasculature, alterations in bacterial response and population, and the issue of tooth/restoration staining need to be addressed in this group. Further, patient compliance in the regular use of chlorhexidine over an extended period of time would have to be evaluated. To obtain the answers to these questions will require longitudinal clinical trials in a larger sample of irradiated patients. References 1. American Dental Association, Council on Dental Therapeutics: Council on dental therapeutics accepts Peridex. J Am Dent Assoc 117:516, 1988 2. Banting D. Bosma M, Bollmer B: Clinical effectiveness of a 0.12% chlorhexidine mouthrinse over two years. J Dent Res 68: 1716, 1989
1059
TOLJANIC ET AI
3. Briner W, Buckner R. Rebitski G, et al: Effect of two years of 0.12%chlorhexidine on plaque bacteria. J Dent Res 68:1719, 1989 4. Briner WW, Grossman E, Buckner RY, et al: Assessment of 5. 6. 7.
8.
9.
10.
I i. 12.
13.
14.
susceptibility of plaque bacteria to chlorhexidine after six months oral use. J Periodont Res 1653, 1986 (suppl) Case DE: Safety of hibitane. 1. Laboratory experiments. J Clin Periodontol4:66, I977 Foulkes DM: Some toxicological observations on chlorhexidine. J Periodont Res 8:55, 1973 (suppl) Gjermo P, Eriksen HM: Unchanged plaque inhibiting effect of chlorhexidine in human subjects after two years of continuous use. Arch Oral Biol l9:3 17, 1974 Loe H, Schiott CR. Glavind L. et al: Two years oral use ofchlorhexidine in man. 1. General design and clinical effects. J Periodont Res 11:135, 1976 MacKenzie IC. Nuki K. Loe H, et al: Two years oral use of chlorhexidine in man. V. Effects on stratum corneum of oral mucosa. J Periodont Res 11:165. 1976 Nuki K. Schlenker R. Loe H, et al: Two years oral use of chlorhexidine in man. VI. Effect on oxidative enzymes in oral epithelia. J Periodont Res I I: 172, 1976 Rushton A: Safety of hibitane. II. Human experience. J Clin Periodontol 4~73. 1977 Schiott CR, Briner WW. Loe H: Two year oral use of chlorhexidine in man. II. The effect on salivary bacterial flora. J Periodont Res 11:145, 1976 Schiott CR, Briner WW, Kirkland JJ, et al: Two years oral use of chlorhexidine in man. III. Changes in sensitivity of the salivary flora. J Periodont Res I 1:153, 1976 Schiott CR. Loe H, Briner WW: Two year oral use of chlorhexidine in man. IV. Effect on various medical parameters. J Periodont Res 11:158. I976
15. Hennessey TD: Antibacterial properties of hibitane. J Clin Peri-
odontol4:36, 1977 16. Hugo WB, Longworth AR: The effect of chlorhexidine on the electrophoretic mobility. cytoplasmic constituents, dehydrogenase activity and cell walls of escherichia coli and staphylococcus aureus. J Pharm Pharmacol I8:569. I966 17. Walker CB: Microbiological effects of mouth rinses containing antimicrobials. J Clin Periodontol I5:499. 1988 18. Gjermo P: Chlorhexidine and related compounds. J Dent Res 68:1602. 1989 19. Bonesvoll P: Retention and plaque-inhibiting effect in man of chlorhexidine after multiple mouth rinses and retention and release of chlorhexidine aFtertoothbrushing with chlorhexidine gel. Arch Oral Biol 23:295. 1978 20. Bonesvoll P, Lokken P. Rolla G. et al: Retention of chlorhexidine in the human oral cavity after mouth rinses. Arch Oral Biol I9:209. 1974 21. Rolla G. Loe H. Schiott CR: The affinity of chlorhexidine for hydroxyapatite and salivary mucins. J Periodont Res 5:90. 1970 22. Ericsson HM, Shenis JC: Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbial Stand [B] 217:9. 1971 (suppl) 23. Emilson CG: Susceptibility of various microorganisms to Chlorhexidine. Stand J Dent Res 85:255. 1977 24. Gjermo P. Bonesvoll P, Rolla G: Relationship between plaqueinhibiting effect and retention of chlorhexidine in the human oral cavity. Arch Oral Biol 19:103 I, 1974 25. Mashberg A: Reevahiation of toluidine blue application as a diagnostic adjunct in the detection of asymptomatic oral squamous carcinoma. Cancer 46:758. 1980
Surg
059. 1992
Evaluation of the Substantivity of a Chlorhexidine Oral Rinse in Irradiated Head and Neck Cancer Patients JOSEPH A. TOLJANIC, DDS,* JAMES C. HAGEN, PHD, MPH,t YASUNOMBU TAKAHASHI, DDS, PHD,$ AND ROBERT D. SHAPIRO,
DDS§
A trial was conducted to evaluate the substantivity of chlorhexidine in a population of patients who had undergone primary or adjunctive radiation therapy for tumors of the head and neck. Subjects were instructed to first rinse with 0.12% chlorhexidine as per manufacturer instructions, followed by a releasing rinse provided 1 minute, 1 hour, and 4 hours later. All expectorants were collected, pH adjusted, and introduced to the test microorganism. Zones of bacterial inhibition were then measured and recorded. The results suggest that 0.12% chlorhexidine is retained in the oral cavity for at least 4 hours after an initial rinsing and that the property of substantivity remains active in spite of radiation-induced changes in the oral cavity and salivary glands.
Chlorhexidine gluconate has been extensively studied for almost 20 years as an adjunct to currently accepted oral health care measures. It has been shown to be clinically safe and effective in preventing the development of plaque-associated gingivitis.‘-I4 Its effect is achieved primarily via two mechanisms. First, chlorhexidine is both bactericidally and bacteriostaticly active against organisms responsible for the formation and adherence of plaque to dental structures. This action is most likely the result of the disruption of bacterial cell wall function.‘5-‘7 Second, chlorhexidine exhibits the property of substantivity, a phenomenon whereby a substance binds to a structure it contacts, in this case probably glycoproteins coating the oral mucosa and dentition. Such binding, which is hypoth-
esized as being electrostatic in nature, is reversible and thus permits a steady release of the active agent into the oral cavity. ‘8,‘9-2’This affords a prolongation of effect and provides continuous protection until the next rinse cycle is performed. Oral health care for patients who have undergone therapeutic irradiation for tumors of the head and neck is complicated by radiation-induced regional changes. As a result of these changes, such patients are placed at increased risk for developing significant dental pathology, including gingival and periodontal disease, while concurrently possessing a decreased capacity to provide host defense mechanisms and effect physiological repair. It would appear that this patient population could benefit dramatically from the use of chlorhexidine as a preventive adjunct in the maintenance of oral health. However, it is as yet unclear whether the changes seen in the oral milieu may act to decrease the effective substantivity of chlorhexidine by decreasing salivary output and depriving the patient of important sources of potential protein molecules that may act as binding sites. If chlorhexidine can be shown to retain substantivity in irradiated patients, it could be included in the current postradiation dental protocols now recommended. The purpose of this investigation was to evaluate and quantify the level of substantivity of chlorhexidine in individuals who have received cancericidal doses of radiation for tumors of the head and neck.
* Assistant Professor of Clinical Surgery; Chief, Prosthetics, Section of Oral and Maxillofacial Surgery/Dentistry, University of Chicago. Chicago. t Associate Professor, Department of Periodontics, Microbiology and Biochemistry, Loyola University, School of Dentistry. Chicago. $ Instructor, Department of Removable Prosthodontics, Osaka University, Faculty of Dentistry, Osaka, Japan. 5 Associate Professor of Clinical Surgery. Chief, Section of Oral and Maxillofacial Surgery/Dentistry, University of Chicago, Chicago. Address correspondence and reprint requests to Dr Toljanic: University ofchicago, Zolles Dental Clinic, 584 S Maryland Ave. Chicago. IL 60637. 0 1992 American
Association
of Oral and Maxillofacial
Surgeons
0278-2391/92/5010-0007$3.00/O
1055
1056
CHLORHEXIDINE USE IN IRRADIATED PATIENTS
Materials and Methods PRESTUDY STANDARDIZATIONPROCEDURES AND SUSCEPTIBILITY TESTING
Prestudy screening of selected clinical and standard microbial strains was performed to identify an organism with a reproducibly high level of sensitivity to chlorhexidine. Microorganisms screened for included Streptococcus mitis, Streptococcus salivarius, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Actinomyces israelii, Bacteriodes gingivalis, Peptostreptococcus anaerobius, and Actinobacillus actinomycetemcomitans. Serial twofold dilutions of sterile chlorhexidine were prepared in test tubes containing enriched Todd-Hewitt Broth (enriched with 0.5% yeast extract, 0.05% hemin, and 0.0005% menadione) for use with obligate anaerobes and trypticase soy broth for use with facultative flora. Into each prepared tube, 0.1 mL of an overnight culture of the test microorganism was introduced to a final concentration of 10’ organisms/ml. The tubes were then incubated at 37” for 24 hours either aerobically or anaerobically, as indicated. Results were visually read as either growth or no growth, with the last tube possessing turbidity recorded as the minimum inhibitory concentration (MIC) of chlorhexidine for each microorganism evaluated. In this manner, S epidermidis was found to be the most reproducibly sensitive organism and therefore was used throughout the study. A prestudy standard curve of chlorhexidine concentration versus S epidermidis growth was created using a modification of the procedure described by Ericsson and Sherris22 and applied to chlorhexidine by Emilson.23 This was accomplished by first seeding media plates containing trypticase soy agar with an overnight culture. Into the prepared media of each plate, five evenly spaced wells were created using a k-in-diameter cork bore. A commercially available formula containing 0.12% chlorhexidine gluconate (Peridex, Proctor & Gamble, Cincinnati, OH) as the active ingredient was prepared to a 1: 10 dilution and then adjusted to a pH of 7. In triplicate, 0.32 mL of this solution was introduced into the media plate wells at the following concentrations: 64, 32, 16, 8, 4, 2, 1, and 0.5 pg/mL. Each plate was then incubated for 18 to 24 hours. The diameters of the resultant zones of inhibition surrounding each well were measured by hand to the nearest ‘/iOmm using a millimeter ruler. From these measures, a standard curve was generated (Fig 1). ESTABLISHMENTOFTESTPROTOCOL
A protocol was developed and tested using two nonirradiated, dentate volunteers (the authors) who pos-
‘II 0.5
1 1.5
2 2.5
3
4 Peridex
5
6
7
6
9
10
Conc.(mglmU
FIGURE 1. Standard curve of chlorhexidine concentration versus growth of test microbial organism.
sessed normal salivary flow characteristics, with adequate volume and consistency. Each subject was instructed to vigorously rinse with ‘/2oz of the commercially prepared formula containing 0.12% chlorhexidine gluconate as the active ingredient. After 30 seconds, they were instructed to expectorate (as per manufacturer instructions). This was followed exactly 1 minute later by a second aqueous rinse of 1% acetic acid, which was again expectorated. The purpose of the second rinse was to release any residual bioactive substance retained on oral tissues that would then be collected in the expectorant.24 A solution of 1% aqueous acetic acid has been previously shown to be safe as an oral rinse under selected conditions.” The preceding procedures were then repeated on two separate occasions during which time only the interval between the 0.12% chlorhexidine rinse and the 1% acetic acid rinse was changed: to 1 and 4 hours, respectively. The expectorated 1% acetic acid rinse was collected from each subject for all test periods and the acidity was neutralized to pH 7, as measured using an ion analyzer (Model 601A, Orion Company, Cambridge, MA), by the addition of aqueous NaOH. Media plates containing trypticase soy agar were seeded with the test organism and wells prepared as previously described. Into each well 0.32 mL of the neutralized expectorants were introduced in duplicate using a micropipette. The prepared media plates were then incubated for 18 to 24 hours and the resultant zones of inhibition measured. Each measurement was compared with the standard curve previously developed. TESTPROTOCOL
Six subjects volunteered to participate in the trial. Each was selected from the pool of patients previously
1057
TOLJANIC ET AL
treated with primary or adjunctive radiotherapy at the University of Chicago Hospitals for tumors of varying sites contained within the head and neck region following full study disclosure (Table 1). Each patient was on a schedule of routine follow-up in the Section of Oral and Maxillofacial Surgery/Dentistry. All patients were either partially or fully dentate, with the minimum number of 2 1 teeth present. Ages ranged from 16 to 82 years. The subjects were instructed to rinse vigorously with both the 0.12% chlorhexidine and 1% acetic acid as per the protocol previously described. Resultant expectorants after each of the l-minute, I-hour, and 4hour rinse cycles were collected and placed in -65°C frozen storage for later data analysis. Additionally, a fourth rinse cycle was performed using the 1% acetic acid without a previous rinse of 0.12% chlorhexidine. The collected expectorant served as a within-patient control. Expectorants were neutralized to a pH of 7 by the addition of aqueous NaOH and media plates containing trypticase soy blood agar were seeded with the test organism and wells prepared as previously described. Into each well, 0.32 mL of the neutralized trial expectorants was introduced in duplicate using a micropipette. All prepared media plates were then incubated for 18 to 24 hours and the resultant zones of inhibition measured. Each measurement was compared with the standard curve previously developed. Results
Table 2 lists the zones of inhibition obtained with all rinse cycles for each subject. As the expectorants were plated in duplicate, the values represent the average of two measures. Measurable zones of inhibition were noted with all cycles for five of the six subjects. A zone of inhibition was not evident with the 4-hour rinse cycle expectorant obtained from subject B.K. The data generated were compared with the pretrial standard curve developed as described in the Methods and Materials section. This was done to quantify the
Table 1. Primary Tumor Site and Radiation Dose Delivered Subject
Primary Tumor Site
Dose GY)
B.K.
Left tonsil, soft palate, and base of tongue Nasopharynx Nasopharynx Right lateral tongue border Supraglottic larynx Floor of mouth
60.4 50 64.8 60 66 65
W.H. K.B. W.K. N.S. B.B
Table 2. Microbial Inhibition Measured After Incubation With Test Expectorants
Subject
Time Interval
B.K.
I Minute I Hour
W.H.
K.B.
W.K.
N.S.
B.B.
4 Hour I Minute I Hour 4 Hour I Minute 1 Hour 4 Hour 1 Minute I Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour
Zone of Inhibition (cm) 1.5 1.2 0 1.6 1.35 I.1 1.35 1.3
1 1.5
1 0.9 1.55 1.3 1.3 1.5 1.4 1.05
Zones of inhibition represent average of two measurements.
levels of antibacterial activity for the expectorants at each rinse cycle relative to a commercial product containing 0.12% chlorhexidine at known dilutions. The results of this comparison are shown in Table 3. and indicate the concentration of bioactive substance contained within each collected expectorant. Furthermore, a decrease in this bioactive substance activity was apparent for the expectorants as the rinse cycle time interval increased in all but two trial periods (between 1 minute and 1 hour for subject K.B. and between 1 hour and 4 hours for subject W.K.). When control expectorants were evaluated, no zones of inhibition were generated using the resultant expectorants for any subject. Discussion
The results of this study indicate that when a commercial product containing 0.12% chlorhexidine is used as an oral rinse in a population of patients irradiated for certain tumors of the head and neck, an antibacterial element is retained and then gradually released into the oral cavity over time. This shows that the property of substantivity, as ascribed to chlorhexidine, remains clinically in effect in this patient population and provides prolongation of antibacterial action. Zones of inhibition were noted in all six subjects at the 1-minute and 1-hour trial periods and zones of inhibition were noted in five of six subjects for the 4-hour trial period. From this it may be deduced that the substantivity of chlorhexidine appears to operate despite
1058
CHLORHEXIDINE
Table 3. Chlorhexidine Concentration in the Expectorant at Each Trial Period
Subject
Time Interval
B.K.
I Minute I Hour
W.H.
K.B.
W.K.
N.S.
B.B.
4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute I Hour 4 Hour 1 Minute 1 Hour 4 Hour 1 Minute 1 Hour 4 Hour
Concentration (mg/mL) 5.35 2.17 0 >10.7 10.7 1.08 2.17 2.17
I .08 10.7 0.27 0.54 > 10.7 5.35 2.17 10.7 5.35 1.08
the physiological and histologic tissue changes induced by therapeutic radiation. Over the course of 4 hours, less bioactive agent remains available as indicated by the steady decrease in zone size obtained over time in four of six subjects. This would presumably be due to gradual release of a bioactive substance into the oral cavity. Further review of subjects K.B. and WK. was undertaken to elicit an explanation for the discrepancies in zone size changes over time. During the course of the study, subject W.K. complained of difficulty in vigorously swishing the test rinses for the prescribed period. The subject would tend to stop swishing altogether, if not subjected to constant observation and encouragement. No performance status variations could be determined for subject K.B., who had no difficulty in performing the study tasks. As measurable zones of inhibition were noted at the 4-hour time interval in five of six subjects, it would be expected that chlorhexidine would remain biologically available and provide antibacterial action for some time beyond this period. However, the total time during which bioactivity could be anticipated was not assessed in the present study. Although the study involved a small sample, these results show that the property of substantivity remains active in patients who rinse with a commercial preparation containing chlorhexidine following therapeutic doses of radiation for oral and oropharyngeal neoplasms. This capacity remains intact for at least 4 hours, and quite likely beyond, providing antibacterial action in the oral cavity. This finding may be of major sig-
USE IN IRRADIATED PATIENTS
nificance. Oral health is adversely effected subsequent to irradiation, with the postradiation xerostomia resulting in, among other sequelae, an increase in gingival inflammation that can progress to active periodontal disease. Periodontal disease and the resultant need for tooth extraction have long been recognized as a major etiologic factor in the development of osteoradionecrosis of the jaw bones. Hence, irradiated patients not only face the danger of dramatically decreased dental health, with the attendant discomfort and loss of function, but also the additional risk of developing a serious, potentially untreatable, pathologic bone condition. Although measures that effectively combat some of the posttreatment complications associated with radiation therapy have been developed and instituted over the years, a substantial percentage of patients fail to respond to this dental maintenance. Once dental pathology has progressed to a significant extent, salvage becomes extremely difficult. Use of chlorhexidine as a long-term measure in oral health maintenance could provide important benefits in selected circumstances. First, it could serve to augment the currently accepted regimen of oral care in specific populations of irradiated patients showing active and recalcitrant oral disease in spite of application of the standard regimen. These patients would be identified through the course of regular follow-up dental examinations. Second, chlorhexidine could be added to the standard regimen for those individuals who are unable and/or unwilling to consistently comply with the standard regimen. Such individuals might include those who are ineffective in toothbrushing, who could benefit from the additional protective mechanisms provided by chlorhexidine. Inclusion of long-term use of chlorhexidine into the accepted standard of care, however, may potentially create problems for patients. Although prolonged use has been demonstrated to be a safe and effective measure in the oral health care of nonirradiated patient populations, no such longitudinal studies have been performed in irradiated patients.3~8-‘0,‘2-‘4Soft-tissue response in light of a compromised regional vasculature, alterations in bacterial response and population, and the issue of tooth/restoration staining need to be addressed in this group. Further, patient compliance in the regular use of chlorhexidine over an extended period of time would have to be evaluated. To obtain the answers to these questions will require longitudinal clinical trials in a larger sample of irradiated patients. References 1. American Dental Association, Council on Dental Therapeutics: Council on dental therapeutics accepts Peridex. J Am Dent Assoc 117:516, 1988 2. Banting D. Bosma M, Bollmer B: Clinical effectiveness of a 0.12% chlorhexidine mouthrinse over two years. J Dent Res 68: 1716, 1989
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TOLJANIC ET AI
3. Briner W, Buckner R. Rebitski G, et al: Effect of two years of 0.12%chlorhexidine on plaque bacteria. J Dent Res 68:1719, 1989 4. Briner WW, Grossman E, Buckner RY, et al: Assessment of 5. 6. 7.
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I i. 12.
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susceptibility of plaque bacteria to chlorhexidine after six months oral use. J Periodont Res 1653, 1986 (suppl) Case DE: Safety of hibitane. 1. Laboratory experiments. J Clin Periodontol4:66, I977 Foulkes DM: Some toxicological observations on chlorhexidine. J Periodont Res 8:55, 1973 (suppl) Gjermo P, Eriksen HM: Unchanged plaque inhibiting effect of chlorhexidine in human subjects after two years of continuous use. Arch Oral Biol l9:3 17, 1974 Loe H, Schiott CR. Glavind L. et al: Two years oral use ofchlorhexidine in man. 1. General design and clinical effects. J Periodont Res 11:135, 1976 MacKenzie IC. Nuki K. Loe H, et al: Two years oral use of chlorhexidine in man. V. Effects on stratum corneum of oral mucosa. J Periodont Res 11:165. 1976 Nuki K. Schlenker R. Loe H, et al: Two years oral use of chlorhexidine in man. VI. Effect on oxidative enzymes in oral epithelia. J Periodont Res I I: 172, 1976 Rushton A: Safety of hibitane. II. Human experience. J Clin Periodontol 4~73. 1977 Schiott CR, Briner WW. Loe H: Two year oral use of chlorhexidine in man. II. The effect on salivary bacterial flora. J Periodont Res 11:145, 1976 Schiott CR, Briner WW, Kirkland JJ, et al: Two years oral use of chlorhexidine in man. III. Changes in sensitivity of the salivary flora. J Periodont Res I 1:153, 1976 Schiott CR. Loe H, Briner WW: Two year oral use of chlorhexidine in man. IV. Effect on various medical parameters. J Periodont Res 11:158. I976
15. Hennessey TD: Antibacterial properties of hibitane. J Clin Peri-
odontol4:36, 1977 16. Hugo WB, Longworth AR: The effect of chlorhexidine on the electrophoretic mobility. cytoplasmic constituents, dehydrogenase activity and cell walls of escherichia coli and staphylococcus aureus. J Pharm Pharmacol I8:569. I966 17. Walker CB: Microbiological effects of mouth rinses containing antimicrobials. J Clin Periodontol I5:499. 1988 18. Gjermo P: Chlorhexidine and related compounds. J Dent Res 68:1602. 1989 19. Bonesvoll P: Retention and plaque-inhibiting effect in man of chlorhexidine after multiple mouth rinses and retention and release of chlorhexidine aFtertoothbrushing with chlorhexidine gel. Arch Oral Biol 23:295. 1978 20. Bonesvoll P, Lokken P. Rolla G. et al: Retention of chlorhexidine in the human oral cavity after mouth rinses. Arch Oral Biol I9:209. 1974 21. Rolla G. Loe H. Schiott CR: The affinity of chlorhexidine for hydroxyapatite and salivary mucins. J Periodont Res 5:90. 1970 22. Ericsson HM, Shenis JC: Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol Microbial Stand [B] 217:9. 1971 (suppl) 23. Emilson CG: Susceptibility of various microorganisms to Chlorhexidine. Stand J Dent Res 85:255. 1977 24. Gjermo P. Bonesvoll P, Rolla G: Relationship between plaqueinhibiting effect and retention of chlorhexidine in the human oral cavity. Arch Oral Biol 19:103 I, 1974 25. Mashberg A: Reevahiation of toluidine blue application as a diagnostic adjunct in the detection of asymptomatic oral squamous carcinoma. Cancer 46:758. 1980
