Clinical and Microbiological Effects of Subgingival and Gingival Marginal Irrigation With Chlorhexidine Gluconate David L. Jolkovsky, * Marc Y. Waki,* Michael G. Newman, * Joan Otomo-Corgel,* Miles Madison,* Thomas F. Flemmig, * Sushma Nachnani, * and Hessam Nowzari*
Recent interest in the local delivery of antimicrobial and anti-inflammatory agents has stimulated interest in the efficacy of various treatment regimens. Chlorhexidine gluconate (CHX) delivered daily by home-applied marginal irrigation as a 0.04% solution in combination with a single professional irrigation of 0.12% CHX was tested over a 3month period. Sixty periodontal maintenance patients each having at least 2 pockets > 4 mm probing depth, and bleeding on probing were assigned to either Group 1: one professional subgingival 0.12% CHX (Peridex) irrigation (Perio Pik) followed by adjunctive daily home marginal 0.04% CHX irrigation (Pik Pocket); Group 2: one professional subgingival 0.12% CHX irrigation followed by adjunctive daily home marginal water irrigation; Group 3: one professional subgingival water irrigation followed by adjunctive daily home marginal water irrigation; or Group 4: control. At baseline and 3 month visits, subgingival plaque samples were taken from 2 sites per patient. Cultural microbiological analysis was performed using non-selective and selective media. Plaque Index, Gingival Index, pocket probing depths, and gingival recession were assessed. Scaling and root planing (supportive periodontal treatment) was provided for each patient followed by subgingival irrigation as outlined above. At 3 months the Gingival Index and pocket probing depths were both significantly reduced (P < .05) in all irrigation groups compared to baseline. There were no significant changes in clinical parameters in the control group from baseline to 3 months. In Group 1 the GI was significantly reduced (P < .05) compared to Group 4 at 3 months. W. recta and black-pigmented Bacteroides sp were significantly reduced in Group 1 at 3 months compared to baseline. The findings suggest that it is possible to achieve beneficial clinical and microbiological effects from adjunctive professional 0.12% CHX and home 0.04% CHX subgingival irrigations in periodontal maintenance patients receiving supportive periodontal treatment. / Periodontal 1990; 61:663-669.
Key Words: Chlorhexidine/therapeutic
use; dental
prophylaxis;
Supportive periodontal treatment (maintenance therapy) is an important but often difficult task for both the patient and the periodontist. The difficulty in the complete removal of plaque and calculus from tooth surfaces has been well documented. The effectiveness of brushing to remove plaque appears limited to the supragingival area and in some cases as little as 0.9 mm into the gingival sulcus.1 Even with open flap scaling and root planing, deposits can be left on root
surfaces.2 When accretions
are
left
on
the root
surface,
"UCLA School of Dentistry, Section of Periodontics, Los Angeles, CA. TVA Medical Center, West Los Angeles, CA. *University of California, San Francisco, School of Dentistry, Division of Periodontology, San Francisco, CA.
oral
hygiene/methods.
the bacteria in these accretions can multiply and there is a potential need for selected retreatment.3 Clearly, adjunctive therapeutic modalities which improve the success of traditional treatment would be welcome. Research and clinical use of chemotherapeutic agents delivered by oral irrigation to enhance plaque and bacterial control have increased. Adjunctive oral irrigation with and without chemotherapeutic agents is effective in reducing gingivitis.4-6 Unfortunately, traditional supragingival oral irrigation devices do not appear to be able to access the apical extent of periodontal pockets where root accretions may remain even after the active phase of periodontal therapy has been completed.7"9 Two modifications of the tra-
EFFECTS OF GINGIVAL IRRIGATION WITH CHLORHEXIDINE
664
PreTx
Initial Tx
Home Irr
1 week4- 3 months Clinical indices Plaque sampling Maintenance visit (Gl, PI, PD, Professional and CEJ) Patient selection subgingival
2. Patients who had serious systemic diseases. 3. Patients who had taken systemic antibiotics within 2 months prior to the study. 4. Patients who had clinical/radiographic evidence of
3 Month Tx
*
irrigation Dispense home irrigators and
Plaque sampling Clnical indices
(Gl, PI, PD,
and CEJ) Maintenance visit
medications
1: Experimental Protocol. Pre Tx Pretreatment exam; Initial Initial treatment visit; Home Irr Tx 3-month home irrigation phase; 3-month Tx 3-month treatment visit; CI Gingival Index; PI Plaque Index; PD probing depth; CEJ CEJ index.
Figure
=
=
=
=
=
=
=
=
ditional Water Pik§ have been developed to deliver fluids deeper into periodontal pockets. One of these modifications has been shown to deliver fluids to the base of periodontal pockets.10 Neither of these modifications has been clinically evaluated in periodontal maintenance patients. The purpose of this investigation was to evaluate the clinical and microbiological benefits of an irrigation regimen which could be integrated into the routine periodontal maintenance visit and the patient's home care regimen. METHODS AND MATERIALS Clinical Methods This study was a single-blind (examiner) investigation involving 60 patients for 3 months. Twelve females and 48 males with an age range of 22 to 75 years (mean age 56.0 years) were selected for this study. Patients were seen at the University of California, Los Angeles, School of Dentistry and at the Veteran's Administration Medical Center, West Los Angeles. The study consisted of two or three patient visits: a pretreatment exam for patient selection and less than 7 days later, an initial treatment visit, and a 3month treatment visit. Seventeen patients had separate pretreatment exams and initial treatment visits and 43 patients had their pretreatment exam combined with their initial treatment visit. A home irrigation phase lasted for 3 months between the initial treatment visit and the 3-month treatment visit (Fig. 1). Patient selection was made at the pretreatment exam and each patient was currently in supportive periodontal treatment. Additionally, each patient had at least two pockets with probing depths > 4 mm which exhibited signs of gingivitis to include bleeding on probing (persistent periodontal disease). Each selected patient was assigned a sequential code number from 1 to 60. A randomly generated computer list was used to assign the patient to one of the four treatment groups (15 patients each). Subjects were excluded if any of the following findings were noted: 1. Patients who were pregnant or are likely to become pregnant during the course of the study.
5Teledyne-Water Pik,
Fort
Collins, CO.
J Periodontol November 1990
pulpitis, periapical radiolucencies, impacted teeth, or retained root fragments. 5. Patients receiving medications such as anti-inflammatory, cardiac, epilepsy, or other medications which could affect periodontal health. 6. Patients having a history of prosthetic joint replace-
ments, rheumatic heart disease, bacterial endocarditis, heart murmur, artificial heart
valves, or mitral valve prolapse. The sequence of events for the pretreatment exam was as follows: 1. Medical and dental histories were reviewed. 2. Informed consent was obtained (research protocol was approved by the UCLA Human Subject Protection Committee and the VAMC, West Los Angeles). 3. Extraoral and intraoral hard and soft tissue examinations were performed. 4. Plaque Index11 was assessed on all teeth. 5. Pocket probing depths of all teeth were measured with an electronic pressure-sensitive probe11 set to 25 gr and a color-coded probe tip having 3 mm increments.11 6. Gingival Index was assessed on all teeth." 7. The distance from the cemento-enamel junction to the gingival margin was determined on all teeth. The initial treatment visit followed 1 week after the pretreatment visit when these visits were separate. The sequence of events for the initial treatment visit were as follows: 1. One subgingival plaque sample was taken from the same two teeth on each patient at each visit. Experimental sites had to have > 4 mm probing depths with BOP. All plaque samples were taken from proximal surfaces of the teeth. 2. Full mouth scaling and root planing was performed. 3. Subgingival irrigation was begun with the two experimental sites and ended with full mouth irrigation around all remaining teeth. All sites with probing depths > 4 mm had 3.5 ml (20 seconds of irrigation) of the appropriate irrigant delivered subgingivally using the Perio Pik8 at a 1 inch (0.55 mm power setting of 2 out of 10. A 24 g 25 mm) Max-I-Probe# was placed to the apical extent of the pockets. 0.12% Chlorhexidine gluconate (Peridex**) was the irrigant for Groups 1 and 2; sterile water was the irrigant for Group 3. Time for full mouth irrigation was approximately 2 to 7 minutes per patient. Group 4 had no
irrigation (Table 1). 4. The home marginal irrigation devices were dispensed to patients in Groups 1, 2, and 3 with written and verbal instructions for use. The home delivery system consisted "Electronic Periodontal Probe Model 250, Vine Valley sex, NY. 'No. 12, Hu-Friedy Mfg., Chicago, IL. *MPL, Inc. Chicago, IL. "The Procter & Gamble Co., Cincinnati, OH.
Research, Middle-
Volume 61 Number 11
JOLKOVSKY, WAKI, NEWMAN, OTOMO-CORGEL, MADISON, FLEMMIG, NACHNANI,
Table 1: Treatment
Groups Professional
Group
Subgingival Irrigation
1 2 3 4
0.12% CHX 0.12% CHX Sterile water None
Marginal Irrigation
Home
0.04% CHX
Tap water Tap water None
commercially available oral irrigator (Water Pik, Model WP-20§) and a soft rubber marginal irrigation tip (Pik Pocket8) that was designed to be placed between the tooth and the marginal gingiva to deliver fluid into the periodontal pocket. The diameter of the opening in the rubber tip was 0.021". Patients in Group 1 irrigated with 180 ml 0.04% Chlorhexidine gluconate once daily for 3 months (diluted Peridex), Groups 2 and 3 with 180 ml tap water once daily for 3 months, and Group 4 did not irrigate. All groups continued routine oral hygiene procedures used prior to entrance into the study. All home irrigation was performed at the lowest power setting of the oral irrigator. 5. Minnesota Daily Use Cards12 were used to assess compliance. They were addressed and stamped by the investigator and were furnished to patients in Groups 1, 2, and 3. These cards were mailed monthly to the investigators by the patient. of
a
6. The 3-month treatment visit was scheduled. Clinical parameters were assessed at 6 sites on all teeth: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual. When patients had their pretreatment exam and initial therapy visit combined, plaque samples were taken prior to the clinical periodontal examination using the last recall probing depths as a guide to choose the plaque sampling site. If the selected site did not bleed after taking the sample or on probing, it was discarded and another site chosen. At the 3-month treatment visit, subgingival plaque samples were taken from the same 2 sites on each patient that were sampled at the initial treatment visit. Clinical indices were taken as in the initial treatment visit. The patients then received supportive periodontal therapy.
Microbiology
Subgingival plaque sampling was performed with a curette13 after the tooth had been isolated and supragingival plaque had been removed with a sterile gauze. Plaque samples were immediately placed in 8 ml of cold reduced transfer solution originally described by van Palenstein-Helderman and Winkler14 and modified to inactivate Chlorhexidine gluconate.15-16 These vials were immediately flushed with nitrogen, refrigerated, and processed within 24 hours.15 All samples were dispersed in an ultrasonic bath++ for 15 seconds and then vortexed for 10 seconds. All vials with plaque samples were 20 fold serially diluted with 1/4 strength "Ultrasonic T-14,
Kearny,
NJ.
NOWZARI
665
Ringer's solution and plated using 0.1 ml aliquots. Anaerobic incubation was performed in an anaerobic glove box (80% N2, 10% C02, 10%H2). Each sample was plated on three types of media as described below. Trypticase Soy Agar supplemented with 5% sheep blood** (TSA-SB) was used as a non-selective medium. TSA-SB plates were plated with dilutions 1:20, 1:400, and 1:8000 and then incubated anaerobically for 5 to 7 days. At this time all colonies were counted and characterized by morphology, Gram stained,17 and aerotolerance tested. All surface translocating bacteria (STB) colonies and pitted colonies were streaked for isolation on TSA-SB plates. Biochemical tests including nitrate,18 oxidase,19 indole,17"18 and catalase17 were performed. Antibiotic disc susceptibility tests with vancomycin, kanamycin, and Clindamycin were performed to identify key pathogens. Trypticase Soy Agar supplemented with 5% rabbit blood** (TSA-RB) was used as a differential medium for anaerobic growth of black-pigmented Bacteroides species. TSA-RB plates were plated with dilutions 1:400 and 1:8000 and then incubated anaerobically for 5 to 7 days and counted. Black-pigmented colonies had fluorescence,19"20
indole,17-18 4-methylumbelliferyl-beta-D-galactopyranoside (MUG),19 and alpha-D-glucosidase tests performed for presumptive identification. Trypticase Soy Vancomycin Bacitracin (TSVB)21 medium was used as a selective media for the growth of Actinobacillus actinomycetemcomitans in a C02 environment. These were plated from the vial and dilution 1:20 and then incubated in jars with C02 gas packs (Gas Pack Anaerobic System**). After incubation for 4 days, all colonies were counted and characterized. Presumed Actinobacillus actinomycetemcomitans colonies had MUG, alpha-D-glucosidase, catalase tests, and Gram staining performed. From these tests, black-pigmented Bacteroides (including B. gingivalis and B. intermedias), Actinobacillus actinomycetemcomitans, Wolinella recta, and Eikenella corrodens were identified and counted.17"19
Statistical Analysis Except for the microbiology, the experimental unit was the patient. Clinical and microbiological results were analyzed to determine intergroup differences at the 3-month treatment visit and also intragroup changes between the pretreatment examination and the 3-month treatment visit using ANOVA and paired f-tests, respectively. For microbiological analysis, logarithmic transformation (log1()) of colony forming units (CFUs) was performed. Intergroup comparisons using pairwise i-tests were performed with Bonferroni adjustment to insure the overall Type 1 error (alpha) < 0.05. Thus, individual values between 0.01 and 0.05 were interpreted as "marginally significant," suggesting possible microbiologically significant differences. ttBBL Microbiology Systems, Cockeysville, MD.
666
Table 2:
Gingival
Index
Group
Pre Exam
1
1.33 1.29 1.48 1.32
2 3 4
Table 4:
3 Month Visit 0.89 (.22) 1.05 (.25) 1.25 (.42) 1.23 (.32)
(.27) (.22) (.26) (.22)
Change -0.44 (.21)* -0.24 (.19)* -0.23 (.26)* -0.09 (.22)
%
Change -33.1 -18.6 -15.5 -6.8
'Statistically significant reduction at < .05 from baseline; bracket inGroup 1 improved significantly better over 3 months than Group .05). Numbers in parenthesis are standard deviations.
dicates 4 (P
Recent interest in the local delivery of antimicrobial and anti-inflammatory agents has stimulated interest in the efficacy of various treatment regimens. Chlorhexidine gluconate (CHX) delivered daily by home-applied marginal irrigation as a 0.04% solution in combination with a single professional irrigation of 0.12% CHX was tested over a 3month period. Sixty periodontal maintenance patients each having at least 2 pockets > 4 mm probing depth, and bleeding on probing were assigned to either Group 1: one professional subgingival 0.12% CHX (Peridex) irrigation (Perio Pik) followed by adjunctive daily home marginal 0.04% CHX irrigation (Pik Pocket); Group 2: one professional subgingival 0.12% CHX irrigation followed by adjunctive daily home marginal water irrigation; Group 3: one professional subgingival water irrigation followed by adjunctive daily home marginal water irrigation; or Group 4: control. At baseline and 3 month visits, subgingival plaque samples were taken from 2 sites per patient. Cultural microbiological analysis was performed using non-selective and selective media. Plaque Index, Gingival Index, pocket probing depths, and gingival recession were assessed. Scaling and root planing (supportive periodontal treatment) was provided for each patient followed by subgingival irrigation as outlined above. At 3 months the Gingival Index and pocket probing depths were both significantly reduced (P < .05) in all irrigation groups compared to baseline. There were no significant changes in clinical parameters in the control group from baseline to 3 months. In Group 1 the GI was significantly reduced (P < .05) compared to Group 4 at 3 months. W. recta and black-pigmented Bacteroides sp were significantly reduced in Group 1 at 3 months compared to baseline. The findings suggest that it is possible to achieve beneficial clinical and microbiological effects from adjunctive professional 0.12% CHX and home 0.04% CHX subgingival irrigations in periodontal maintenance patients receiving supportive periodontal treatment. / Periodontal 1990; 61:663-669.
Key Words: Chlorhexidine/therapeutic
use; dental
prophylaxis;
Supportive periodontal treatment (maintenance therapy) is an important but often difficult task for both the patient and the periodontist. The difficulty in the complete removal of plaque and calculus from tooth surfaces has been well documented. The effectiveness of brushing to remove plaque appears limited to the supragingival area and in some cases as little as 0.9 mm into the gingival sulcus.1 Even with open flap scaling and root planing, deposits can be left on root
surfaces.2 When accretions
are
left
on
the root
surface,
"UCLA School of Dentistry, Section of Periodontics, Los Angeles, CA. TVA Medical Center, West Los Angeles, CA. *University of California, San Francisco, School of Dentistry, Division of Periodontology, San Francisco, CA.
oral
hygiene/methods.
the bacteria in these accretions can multiply and there is a potential need for selected retreatment.3 Clearly, adjunctive therapeutic modalities which improve the success of traditional treatment would be welcome. Research and clinical use of chemotherapeutic agents delivered by oral irrigation to enhance plaque and bacterial control have increased. Adjunctive oral irrigation with and without chemotherapeutic agents is effective in reducing gingivitis.4-6 Unfortunately, traditional supragingival oral irrigation devices do not appear to be able to access the apical extent of periodontal pockets where root accretions may remain even after the active phase of periodontal therapy has been completed.7"9 Two modifications of the tra-
EFFECTS OF GINGIVAL IRRIGATION WITH CHLORHEXIDINE
664
PreTx
Initial Tx
Home Irr
1 week4- 3 months Clinical indices Plaque sampling Maintenance visit (Gl, PI, PD, Professional and CEJ) Patient selection subgingival
2. Patients who had serious systemic diseases. 3. Patients who had taken systemic antibiotics within 2 months prior to the study. 4. Patients who had clinical/radiographic evidence of
3 Month Tx
*
irrigation Dispense home irrigators and
Plaque sampling Clnical indices
(Gl, PI, PD,
and CEJ) Maintenance visit
medications
1: Experimental Protocol. Pre Tx Pretreatment exam; Initial Initial treatment visit; Home Irr Tx 3-month home irrigation phase; 3-month Tx 3-month treatment visit; CI Gingival Index; PI Plaque Index; PD probing depth; CEJ CEJ index.
Figure
=
=
=
=
=
=
=
=
ditional Water Pik§ have been developed to deliver fluids deeper into periodontal pockets. One of these modifications has been shown to deliver fluids to the base of periodontal pockets.10 Neither of these modifications has been clinically evaluated in periodontal maintenance patients. The purpose of this investigation was to evaluate the clinical and microbiological benefits of an irrigation regimen which could be integrated into the routine periodontal maintenance visit and the patient's home care regimen. METHODS AND MATERIALS Clinical Methods This study was a single-blind (examiner) investigation involving 60 patients for 3 months. Twelve females and 48 males with an age range of 22 to 75 years (mean age 56.0 years) were selected for this study. Patients were seen at the University of California, Los Angeles, School of Dentistry and at the Veteran's Administration Medical Center, West Los Angeles. The study consisted of two or three patient visits: a pretreatment exam for patient selection and less than 7 days later, an initial treatment visit, and a 3month treatment visit. Seventeen patients had separate pretreatment exams and initial treatment visits and 43 patients had their pretreatment exam combined with their initial treatment visit. A home irrigation phase lasted for 3 months between the initial treatment visit and the 3-month treatment visit (Fig. 1). Patient selection was made at the pretreatment exam and each patient was currently in supportive periodontal treatment. Additionally, each patient had at least two pockets with probing depths > 4 mm which exhibited signs of gingivitis to include bleeding on probing (persistent periodontal disease). Each selected patient was assigned a sequential code number from 1 to 60. A randomly generated computer list was used to assign the patient to one of the four treatment groups (15 patients each). Subjects were excluded if any of the following findings were noted: 1. Patients who were pregnant or are likely to become pregnant during the course of the study.
5Teledyne-Water Pik,
Fort
Collins, CO.
J Periodontol November 1990
pulpitis, periapical radiolucencies, impacted teeth, or retained root fragments. 5. Patients receiving medications such as anti-inflammatory, cardiac, epilepsy, or other medications which could affect periodontal health. 6. Patients having a history of prosthetic joint replace-
ments, rheumatic heart disease, bacterial endocarditis, heart murmur, artificial heart
valves, or mitral valve prolapse. The sequence of events for the pretreatment exam was as follows: 1. Medical and dental histories were reviewed. 2. Informed consent was obtained (research protocol was approved by the UCLA Human Subject Protection Committee and the VAMC, West Los Angeles). 3. Extraoral and intraoral hard and soft tissue examinations were performed. 4. Plaque Index11 was assessed on all teeth. 5. Pocket probing depths of all teeth were measured with an electronic pressure-sensitive probe11 set to 25 gr and a color-coded probe tip having 3 mm increments.11 6. Gingival Index was assessed on all teeth." 7. The distance from the cemento-enamel junction to the gingival margin was determined on all teeth. The initial treatment visit followed 1 week after the pretreatment visit when these visits were separate. The sequence of events for the initial treatment visit were as follows: 1. One subgingival plaque sample was taken from the same two teeth on each patient at each visit. Experimental sites had to have > 4 mm probing depths with BOP. All plaque samples were taken from proximal surfaces of the teeth. 2. Full mouth scaling and root planing was performed. 3. Subgingival irrigation was begun with the two experimental sites and ended with full mouth irrigation around all remaining teeth. All sites with probing depths > 4 mm had 3.5 ml (20 seconds of irrigation) of the appropriate irrigant delivered subgingivally using the Perio Pik8 at a 1 inch (0.55 mm power setting of 2 out of 10. A 24 g 25 mm) Max-I-Probe# was placed to the apical extent of the pockets. 0.12% Chlorhexidine gluconate (Peridex**) was the irrigant for Groups 1 and 2; sterile water was the irrigant for Group 3. Time for full mouth irrigation was approximately 2 to 7 minutes per patient. Group 4 had no
irrigation (Table 1). 4. The home marginal irrigation devices were dispensed to patients in Groups 1, 2, and 3 with written and verbal instructions for use. The home delivery system consisted "Electronic Periodontal Probe Model 250, Vine Valley sex, NY. 'No. 12, Hu-Friedy Mfg., Chicago, IL. *MPL, Inc. Chicago, IL. "The Procter & Gamble Co., Cincinnati, OH.
Research, Middle-
Volume 61 Number 11
JOLKOVSKY, WAKI, NEWMAN, OTOMO-CORGEL, MADISON, FLEMMIG, NACHNANI,
Table 1: Treatment
Groups Professional
Group
Subgingival Irrigation
1 2 3 4
0.12% CHX 0.12% CHX Sterile water None
Marginal Irrigation
Home
0.04% CHX
Tap water Tap water None
commercially available oral irrigator (Water Pik, Model WP-20§) and a soft rubber marginal irrigation tip (Pik Pocket8) that was designed to be placed between the tooth and the marginal gingiva to deliver fluid into the periodontal pocket. The diameter of the opening in the rubber tip was 0.021". Patients in Group 1 irrigated with 180 ml 0.04% Chlorhexidine gluconate once daily for 3 months (diluted Peridex), Groups 2 and 3 with 180 ml tap water once daily for 3 months, and Group 4 did not irrigate. All groups continued routine oral hygiene procedures used prior to entrance into the study. All home irrigation was performed at the lowest power setting of the oral irrigator. 5. Minnesota Daily Use Cards12 were used to assess compliance. They were addressed and stamped by the investigator and were furnished to patients in Groups 1, 2, and 3. These cards were mailed monthly to the investigators by the patient. of
a
6. The 3-month treatment visit was scheduled. Clinical parameters were assessed at 6 sites on all teeth: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual. When patients had their pretreatment exam and initial therapy visit combined, plaque samples were taken prior to the clinical periodontal examination using the last recall probing depths as a guide to choose the plaque sampling site. If the selected site did not bleed after taking the sample or on probing, it was discarded and another site chosen. At the 3-month treatment visit, subgingival plaque samples were taken from the same 2 sites on each patient that were sampled at the initial treatment visit. Clinical indices were taken as in the initial treatment visit. The patients then received supportive periodontal therapy.
Microbiology
Subgingival plaque sampling was performed with a curette13 after the tooth had been isolated and supragingival plaque had been removed with a sterile gauze. Plaque samples were immediately placed in 8 ml of cold reduced transfer solution originally described by van Palenstein-Helderman and Winkler14 and modified to inactivate Chlorhexidine gluconate.15-16 These vials were immediately flushed with nitrogen, refrigerated, and processed within 24 hours.15 All samples were dispersed in an ultrasonic bath++ for 15 seconds and then vortexed for 10 seconds. All vials with plaque samples were 20 fold serially diluted with 1/4 strength "Ultrasonic T-14,
Kearny,
NJ.
NOWZARI
665
Ringer's solution and plated using 0.1 ml aliquots. Anaerobic incubation was performed in an anaerobic glove box (80% N2, 10% C02, 10%H2). Each sample was plated on three types of media as described below. Trypticase Soy Agar supplemented with 5% sheep blood** (TSA-SB) was used as a non-selective medium. TSA-SB plates were plated with dilutions 1:20, 1:400, and 1:8000 and then incubated anaerobically for 5 to 7 days. At this time all colonies were counted and characterized by morphology, Gram stained,17 and aerotolerance tested. All surface translocating bacteria (STB) colonies and pitted colonies were streaked for isolation on TSA-SB plates. Biochemical tests including nitrate,18 oxidase,19 indole,17"18 and catalase17 were performed. Antibiotic disc susceptibility tests with vancomycin, kanamycin, and Clindamycin were performed to identify key pathogens. Trypticase Soy Agar supplemented with 5% rabbit blood** (TSA-RB) was used as a differential medium for anaerobic growth of black-pigmented Bacteroides species. TSA-RB plates were plated with dilutions 1:400 and 1:8000 and then incubated anaerobically for 5 to 7 days and counted. Black-pigmented colonies had fluorescence,19"20
indole,17-18 4-methylumbelliferyl-beta-D-galactopyranoside (MUG),19 and alpha-D-glucosidase tests performed for presumptive identification. Trypticase Soy Vancomycin Bacitracin (TSVB)21 medium was used as a selective media for the growth of Actinobacillus actinomycetemcomitans in a C02 environment. These were plated from the vial and dilution 1:20 and then incubated in jars with C02 gas packs (Gas Pack Anaerobic System**). After incubation for 4 days, all colonies were counted and characterized. Presumed Actinobacillus actinomycetemcomitans colonies had MUG, alpha-D-glucosidase, catalase tests, and Gram staining performed. From these tests, black-pigmented Bacteroides (including B. gingivalis and B. intermedias), Actinobacillus actinomycetemcomitans, Wolinella recta, and Eikenella corrodens were identified and counted.17"19
Statistical Analysis Except for the microbiology, the experimental unit was the patient. Clinical and microbiological results were analyzed to determine intergroup differences at the 3-month treatment visit and also intragroup changes between the pretreatment examination and the 3-month treatment visit using ANOVA and paired f-tests, respectively. For microbiological analysis, logarithmic transformation (log1()) of colony forming units (CFUs) was performed. Intergroup comparisons using pairwise i-tests were performed with Bonferroni adjustment to insure the overall Type 1 error (alpha) < 0.05. Thus, individual values between 0.01 and 0.05 were interpreted as "marginally significant," suggesting possible microbiologically significant differences. ttBBL Microbiology Systems, Cockeysville, MD.
666
Table 2:
Gingival
Index
Group
Pre Exam
1
1.33 1.29 1.48 1.32
2 3 4
Table 4:
3 Month Visit 0.89 (.22) 1.05 (.25) 1.25 (.42) 1.23 (.32)
(.27) (.22) (.26) (.22)
Change -0.44 (.21)* -0.24 (.19)* -0.23 (.26)* -0.09 (.22)
%
Change -33.1 -18.6 -15.5 -6.8
'Statistically significant reduction at < .05 from baseline; bracket inGroup 1 improved significantly better over 3 months than Group .05). Numbers in parenthesis are standard deviations.
dicates 4 (P
