Mta dvs chxgel


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Mta dvs chxgel

  1. 1. CONSORT Randomized Clinical TrialAntibacterial Efficacy of MTAD Final Rinse and Two PercentChlorhexidine Gel Medication in Teeth with ApicalPeriodontitis: A Randomized Double-blinded Clinical TrialGevik Malkhassian, DDS, MSc,* Aldo J. Manzur, DDS, MSc,* Milos Legner, PhD,†Edward D. Fillery, PhD,† Sheela Manek, BSc,† Bettina R. Basrani, DDS, PhD,*and Shimon Friedman, DMD*AbstractIntroduction: Clinical assessment of the efficacy ofnovel root canal disinfection protocols is an importantfocus in endodontic research. This randomized double- T he causative role of bacteria in the pathogenesis of apical periodontitis (1) under- lines the goal of elimination of bacteria as a critical step in root canal therapy. Bacte- rial elimination is attempted by mechanical instrumentation, irrigation withblinded study assessed the antibacterial efficacy of antibacterial agents, and medication with intracanal dressing. The commonly used anti-a final rinse with BioPure MTAD (MTAD) and intracanal bacterial regimen includes the use of sodium hypochlorite (NaOCl) for irrigation andmedication with 2% chlorhexidine gel (CHX) in teeth calcium hydroxide for medication. In spite of the good efficacy of this regimen, bacteriawith apical periodontitis. Methods: Canals in 30 teeth might still survive in small numbers (2), which might adversely affect the outcome of(single-rooted and multi-rooted) were prepared by using treatment (3). Therefore, alternatives and complements to the common regimen1.3% NaOCl, rinsed with MTAD or saline in random continue to be investigated, with the aim to improve bacterial elimination duringsequence, medicated with CHX for 7 days, irrigated root canal treatment (2).with 1.3% NaOCl, and filled. Bacteriologic root canal Instrumentation of the canal walls forms a smear layer that might be an obstacle tosamples were obtained by aspiration before (1A) and effective disinfection of the root canal system (4). The smear layer can be removed withafter (1B) canal preparation, after the final rinse (1C), a chelating agent such as ethylenediaminetetraacetic acid and citric acid (4). Recently,after CHX was flushed (2A), and after final irrigation a novel compound has been developed with combined chelating and antibacterial(2B). Bacteria were enumerated by epifluorescence- properties (5). MTAD (BioPure MTAD; Dentsply Tulsa Dental Specialties, Tulsa, OK)microscopy (EFM) by using 2 staining methods and by is a mixture of doxycycline, citric acid, and Tween 80 (5). It is applied as a 5-minutecolony-forming-unit (CFU) counts after 14 days of incu- final rinse after canal instrumentation and irrigation with 1.3% NaOCl (6). Althoughbation. Results: Bacterial counts (EFM) in 1B were preliminary in vitro studies have suggested effective elimination of root canal bacteriagreater than 95% decreased from 1A. Low bacterial by MTAD (7–10), subsequent studies did not support those results (11–15). At thedensities in 1B, 1C, 2A, and 2B did not differ significantly time this study was initiated, in vivo studies on the antibacterial efficacy of MTADfrom each other. EFM counts were consistently higher were lacking.than CFU counts. Conclusions: The final rinse with Intracanal medication, most commonly calcium hydroxide, is applied primarily toMTAD and medication with CHX did not reduce bacterial prevent proliferation of bacteria that survive canal instrumentation and irrigation, ifcounts beyond levels achieved by canal preparation with treatment cannot be completed at the same session (2). Several in vivo studies haveNaOCl. (J Endod 2009;35:1483–1490) suggested that calcium hydroxide can effectively curtail residual root canal bacteria (16, 17), whereas others have noted only a modest effect or even a moderate bacterialKey Words regrowth (18–20).Chlorhexidine gel, MTAD One alternative to calcium hydroxide is chlorhexidine gluconate (CHX) (21). CHX is a wide-spectrum antimicrobial with a potential to impart substantive antimicrobial activity, referred to as substantivity (21). In high concentrations CHX damages and permeates the bacterial cell wall and then causes precipitation of the cytoplasm that From the *Department of Endodontics and †Department of prevents repair of the cell membrane, leading to cell death. Both 2% CHX solutionOral Microbiology, Faculty of Dentistry, University of Toronto, and gel have been used for intracanal medication in vivo (19, 22, 23). Paquette etToronto, Ontario, Canada. Address requests for reprints to Dr Gevik Malkhassian, al (22) reported a moderate increase in bacterial counts in canals medicated withEndodontics, Faculty of Dentistry, 124 Edward St, Toronto, On- CHX solution for 7–14 days. Manzur et al (19) reported no change in bacterial countstario M5G 1G6, Canada. E-mail address: gevik.malkhassian@ in canals medicated for 7 days with CHX gel, calcium hydroxide, or a mixture of hydroxide and CHX.0099-2399/$0 - see front matter The in vivo antibacterial efficacy of endodontic treatment regimens has been as- Copyright ª 2009 American Association of Endodontists.doi:10.1016/j.joen.2009.08.003 sessed by enumeration of bacteria recovered from root canal samples before and after the use of test regimens. Samples have usually been acquired by means of paper points and recovered bacteria cultivated on growth media. Although this method detects viable cultivable bacteria, it cannot detect many noncultivable bacterial species (24). There- fore, more sensitive methods of microbial detection have been introduced to endodontic research, including direct enumeration of bacteria by using epifluores- cence microscopy (22, 25). Epifluorescence microscopy is more sensitive than cultureJOE — Volume 35, Number 11, November 2009 Clinical Study of MTAD and CHX Gel 1483
  2. 2. CONSORT Randomized Clinical TrialTABLE 1. Inclusion and Exclusion Criteria Considered for Enrollment of Subjects in the Study Consideration Included Excluded Patient ASA I or ASA II; 18 y of age and older; consented to ASA III and higher; pregnant female; nursing participate; able to comply with protocol; good oral mother; cognitively impaired; known allergy hygiene to CHX, tetracycline (doxycycline), citric acid, or Tween 80 Tooth Adequate coronal structure; probing depth of #4 mm; Previous endodontic treatment; aberrant anatomy; radiographic evidence of apical periodontitis; negative maxillary molar; radiographic evidence of response to cold test internal or external root resorptionASA, American Society of Anesthesiologists Physical Status classification quantifying bacteria, and it is also capable of distinguishing live and 30 teeth, 12 (40%) presented with spontaneous pain, 7 (23.3%)dead bacteria in the sample (23, 26). with a history of swelling, 5 (16.6%) with a sinus tract, and 20 The aim of this in vivo epifluorescence microscopy study was to (66.6%) with percussion sensitivity.assess the efficacy of a final rinse with MTAD, followed by root canal Subjects were randomly assigned to 2 equal groups, experimentalmedication with 2% CHX gel, in curtailing residual bacteria after canal (MTAD) or control (sterile saline solution). To satisfy the requirementsinstrumentation and irrigation. of a double-blinded trial, the saline control was colored to mimic MTAD. The randomization, by drawing from a pool of lots, and prepa- Material and Methods ration of the solutions were carried out in advance by a laboratory tech-Study Cohort and Groups nician (S.M.), who delivered the test solutions to the treatment provider. Before recruitment of the study cohort, the University of Toronto In this manner, both the treatment provider and patients were blindedHealth Sciences Research Ethics Board approved the protocol of this to the group allocation.randomized, controlled, double-blinded clinical trial. Eligible partici-pants were recruited from November 2005 until December 2006 Sample Size Calculationfrom among patients who attended the Graduate Endodontics Clinic In the absence of data on the clinical efficacy of MTAD, theat the Faculty of Dentistry, University of Toronto. Subjects were selected sample size calculation was based on the data from our previous studyin accordance with specific inclusion and exclusion criteria (Table 1). (23). In that study the mean difference in bacterial densities beforeThirty subjects consented to participate in the study (Fig. 1), including and after medication with 2% CHX liquid was 8.4 Æ 8.6 (natural loga-15 men and 15 women with ages ranging from 25–78 years (mean, rithm-transformed DAPI counts). With SamplePower 2 software (SPSS51.9 years). Each subject had 1 tooth that required endodontic treat- Inc, Chicago, IL), the sample size was established as 30 teeth dividedment, for a total of 15 maxillary (6 anterior and 9 premolars) and into 2 equal groups, for an analysis at 94% power and with an alpha15 mandibular (4 anterior, 7 premolars, and 4 molars) teeth. Of the of 0.05. Assessed for eligibility (n = 34) Excluded (n = 4) Enrollment Not meeting inclusion criteria (n = 2) Refused to participate (n = 2) Randomly allocated to intervention MTAD group (n = 15) Saline group (n = 15) Allocation All received allocated intervention All received allocated intervention Lost to follow-up (n = 0) Follow-Up Lost to follow-up (n = 0) Analyzed (n = 15) Analyzed (n = 15) Excluded from analysis Analysis Excluded from analysis (n = 1; 1B sample from CFU) (n = 1; 1A sample from CFU)Figure 1. The Consort flowchart.1484 Malkhassian et al. JOE — Volume 35, Number 11, November 2009
  3. 3. CONSORT Randomized Clinical TrialTABLE 2. Dilutions of original samples (1.0 mL RTF and approximately 0.3 mL sample), aliquots taken and RTF added to make the final subsamples constant at 1mL for each EFM technique and 0.5 mL for CFU Enumeration method DAPI/DHET and BacLight CFU Sample Original aliquot (mL) RTF added (mL) Original aliquot (mL) RTF added (mL) 1A 200 800 250 250 1B, 1C, 2A, 2B 400 600 250 250 1AC, 2AC 300 700 250 250CFU, colony-forming unit; DAPI, 40 ,6-diamidino-2-phenylindole; DHET, dihydroethidium; EFM, epifluorescence microscopy; RTF, reduced transport fluid.Preclinical Laboratory Procedures instruments (Dentsply Maillefer) used in the manufacturer-recommen- For the collection of root canal bacterial samples, labeled micro- ded sequence to the WL. The apical diameter was gauged with hand filestubes (1.5 mL) were prepared each with 1.0 mL of reduced transport and enlarged 1 size beyond the first gauging file. After each instrument,fluid (RTF) mixed with dithiothreitol (DTT; Sigma-Aldrich, St Louis, the canal was irrigated with 1.5 mL of 1.3% NaOCl with a 30-gauge nee-MO) (22). Empty micro-tubes were also prepared for measuring the dle, for a total minimum of 10.5 mL of NaOCl. The second root canalvolume of the treated canals. Each micro-tube was then weighed 3 times sample (1B) was obtained, and the RCV was measured as described ¨with a precision balance (Sartorius, Gottingen, Germany), and the above. The final rinse with the test (MTAD) or control (colored saline)average weight was calculated (22). solution was delivered in identical 5-mL syringes with a 30-gauge needle inserted within 2 mm of WL. Initially, 1 mL of solution was slowly in-Clinical Procedures, Sampling, and Canal Volume jected into the canal by using an up and down motion, followed byMeasurement manual agitation with a size 15 K-type file. After 5 minutes, the canal was rinsed with the remaining 4 mL of solution. In the absence of The basic clinical and experimental procedures, including infec- a known inactivation protocol for MTAD (8, 9, 11), an effort wastion control, inactivation of antibacterial agents, sampling procedures, made to thoroughly rinse it out with 3 mL of sterile saline. Similarly,and root canal volume (RCV) measurements, were performed as the colored saline solution was only rinsed out, having shown no anti-described by Paquette et al (22); they are briefly outlined below. bacterial effect in the pilot test. The third root canal sample was then Subjects were treated in 2 sessions 7 days apart. At the start of each obtained (1C), and the canal was dried with paper points. Two percenttreatment session teeth were isolated, pumiced, and surface-decontami- CHX gel (20% aqueous solution of CHX digluconate, methylcellulosenated with 30% hydrogen peroxide and 5% iodine tincture. The antibac- powder, and sterile distilled water; University of Toronto) was injectedterial agents were inactivated with 5% sodium thiosulfate (J. T. Baker, into the canal from a preloaded syringe and distributed with a lentuloPhillipsburg, N.J.) (27), and the tooth surface was rinsed with sterile instrument (Dentsply Maillefer). A sterile sponge pellet was placed insaline. A sterility control sample (1SC) was then obtained as follows. the chamber, and the access was sealed with 2 layers of resin-modifiedA sterile cotton pellet was used to rub the tooth surface and then placed glass ionomer cement (Photac Fil Quick Aplical; 3 M ESPE, St Paul,in a micro-tube filled with RTF. Drills and root canal instruments were MN).all heat-sterilized. Sterile saline was used for cooling during drilling. In the second treatment session, the sterility control sample (2SC) In the first treatment session, caries was removed, and the prelim- was obtained, the temporary seal was removed short of penetrating theinary access was prepared. The access cavity was decontaminated, and chamber, the access control sample was obtained (2AC), and a newthe disinfectants were inactivated and rinsed similarly to the surface drill was used to complete removal of the temporary seal from thedecontamination described above. The access control sample (1AC) chamber. The canal was flushed with 3 mL sterile saline and the CHXwas obtained as follows. RTF was injected into the access cavity by using gel inactivated with 3 mL of 0.3% L-a-lecithin (Sigma Aldrich) anda 27-gauge needle (Monoject; Sherwood Medical, St Louis, MO) and 3% Tween 80 (28). The canal was flushed again with 3 mL of sterilethen aspirated into a syringe containing 0.3 mL of RTF. The syringe saline and dried, and the fourth sample (2A) was obtained. The canalcontents were promptly emptied into a labeled micro-tube with 1.0 was irrigated with 1.3% NaOCl, prepared with the last hand file used or 1mL of RTF/DTT and transferred to the microbiology laboratory for pro- size larger, and dried, and the fifth and final sample (2B) was obtained.cessing within 2 hours. A new drill was used to extend the access cavity At the conclusion of treatment, the canal was dried, filled with verticallyinto the pulp chamber and canal orifice. With only saline for irrigation, compacted warm gutta-percha by using System B (Sybron Dentalan ISO size 10 or 15 stainless steel K-type file (Flexofile; Dentsply Mail- Specialties, Orange, CA) and Thermaseal sealer (Dentsply Tulsa Dentallefer, Ballaigues, Switzerland) was negotiated apically, and the working Specialties), backfilled with injected warm gutta-percha (Obtura II;length (WL) was established with an electronic apex locator (Root ZX II; Obtura Spartan, East Earth City, MO), and the access was restoredJ. Morita, Irvine, CA). The first root canal sample (1A) was acquired with Photac Fil.similarly to the access cavity sample, and the canal was dried with paperpoints. To express the bacterial concentration per RCV, the canal wasfilled with sterile water dispensed from a micro-pipette. The weight of Processing of Samplesthe dispensed water was established and converted to volume, assuming All the samples were transferred to the microbiology laboratory fora specific gravity of 1.0. In teeth with more than 1 canal, the sampling processing within 2 hours.and volume measurement procedures were performed in all the canals Samples were processed for vital microscopy by using direct epi-at the same time. fluorescence microscopy (EFM) counts based on 2 vital staining tech- The canals were negotiated with ISO size 15 and 20 stainless steel niques. To enable comparisons with other studies, samples were alsoK-type files to the WL, flared coronally with Gates-Glidden drills number processed for culture to obtain colony-forming unit (CFU) counts. To2 and 3 (Dentsply Maillefer), and shaped with engine-driven ProTaper this end, each sample was divided into 3 subsamples. The final volumeJOE — Volume 35, Number 11, November 2009 Clinical Study of MTAD and CHX Gel 1485
  4. 4. CONSORT Randomized Clinical Trial used for processing was constant at 1 mL for each EFM technique and TABLE 3. Mean and Standard Deviation Values of Bacterial Densities (counts per mL of root canal volume) in Root Canal Samples Obtained at Different Treatment Steps from Teeth Medicated with MTAD or Saline 5.47 Â 106 Æ 5.44 Â 106 1.09 Â 105 Æ 1.34 Â 105 2.51 Â 104 Æ 1.89 Â 104 2.39 Â 105 Æ 7.14 Â 105 4.40 Â 104 Æ 6.48 Â 104 2.52 Â 106 Æ 2.96 Â 106 1.44 Â 105 Æ 2.61 Â 105 6.16 Â 104 Æ 1.04 Â 105 6.29 Â 104 Æ 1.11 Â 105 5.95 Â 104 Æ 6.48 Â 104 0.5 mL for CFU (Table 2). The EFM subsamples were processed with 2 vital staining Total methods. For each sample, 1 subsample was stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes Inc, Eugene, OR). The second subsample was stained with dihydroethidium (DHET) (Molecular Probes) and DAPI (40 ,6-diamidino-2-phenylindole; Sigma-Aldrich) (23). For the culture subsamples and the sterility control samples, 2.97 Â 106 Æ 3.30 Â 106 1.13 Â 104 Æ 1.43 Â 104 4.46 Â 103 Æ 3.93 Â 103 8.18 Â 104 Æ 2.41 Â 105 5.28 Â 103 Æ 4.21 Â 103 1.11 Â 106 Æ 1.10 Â 106 6.53 Â 104 Æ 2.19 Â 105 6.30 Â 103 Æ 1.08 Â 104 2.72 Â 104 Æ 7.81 Â 104 6.18 Â 103 Æ 9.93 Â 103 aliquots of 52 mL per sample were inoculated on a blood agar plate BacLight (Difco, Becton Dickinson, Sparks, MD) by using a Spiral System Plater Dead (Spiral System, Cincinnati, OH). All plates were incubated at 37 C for 14 days. Root canal cultures were kept under strict anaerobic condi- tions in sealed jars with a gas mixture of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen; access cavity cultures were incubated sepa- rately for both aerobic and anaerobic bacteria, and the sterility control 2.50 Â 106 Æ 3.11 Â 106 9.76 Â 104 Æ 1.29 Â 105 2.07 Â 104 Æ 1.68 Â 104 1.57 Â 105 Æ 3.56 Â 104 3.88 Â 104 Æ 5.33 Â 104 1.41 Â 106 Æ 2.60 Â 106 7.90 Â 104 Æ 1.46 Â 105 5.53 Â 104 Æ 9.34 Â 104 3.56 Â 104 Æ 4.33 Â 104 5.33 Â 104 Æ 5.74 Â 104 cultures were incubated aerobically. A count template was used to establish CFU counts at the end of the incubation period. Live Bacterial Enumeration Bacterial density (counts per mL of RCV) EFM images were captured by using a Leica DMIRE2 (Leica Micro- systems, Wetzlar, Germany) epifluorescence microscope, equipped with a high sensitivity, black and white charge-coupled device camera CFU, colony-forming unit; DAPI, 40 ,6-diamidino-2-phenylindole; DHET, dihydroethidium; MTAD, mixture of tetracycline isomer, acid, and detergent; RCV, root canal volume. (Hamamatsu, Hamamatsu City, Japan), and Modular Imaging Software 5.90 Â 106 Æ 4.23 Â 106 7.93 Â 104 Æ 8.39 Â 104 6.66 Â 104 Æ 5.24 Â 104 2.45 Â 105 Æ 4.65 Â 105 2.32 Â 105 Æ 6.96 Â 105 2.96 Â 106 Æ 2.18 Â 106 1.40 Â 105 Æ 3.24 Â 105 1.39 Â 105 Æ 3.31 Â 105 1.50 Â 105 Æ 2.13 Â 105 6.09 Â 104 Æ 7.82 Â 104 (Openlab 4.0.2; Improvision, Lexington, MA). With the BacLight stain- ing, live bacteria fluoresced green and dead bacteria fluoresced red. Total The signals for green and red were captured in 2 separate channels (L5 and TX2 cubes; Leica Microsystems). With the DHET/DAPI staining, live bacteria fluoresced red, whereas all bacteria fluoresced blue. The red and blue signals were captured in 2 separate channels (A4 and TX2 cubes; Leica Microsystems). The dead bacteria count was calculated as the difference between the live and total counts. 4.40 Â 106 Æ 3.93 Â 106 3.85 Â 104 Æ 5.60 Â 104 5.70 Â 104 Æ 4.95 Â 104 2.27 Â 105 Æ 4.56 Â 105 2.13 Â 105 Æ 7.00 Â 105 1.95 Â 106 Æ 1.75 Â 106 1.03 Â 105 Æ 2.75 Â 105 1.22 Â 105 Æ 3.25 Â 105 1.32 Â 105 Æ 2.03 Â 105 4.79 Â 104 Æ 6.47 Â 104 After manual focusing, the microscope was programmed to DAPI/DHET capture one image and to switch automatically to the appropriate filter Dead cube set to capture the second image. The captured paired images were saved in TIF (Tag Image File) format on a personal computer. On average, 130 digital images were saved for each 1A sample and approx- imately 250 images for each subsequent sample from the same tooth. An image analysis package (Optimas 6.5; Media Cybernetics, Silver Spring, MD) was used to enumerate bacteria with both EFM methods. 1.51 Â 106 Æ 1.98 Â 106 4.08 Â 104 Æ 7.75 Â 104 9.58 Â 103 Æ 8.75 Â 103 1.79 Â 104 Æ 2.76 Â 104 1.86 Â 104 Æ 2.29 Â 104 1.01 Â 106 Æ 1.21 Â 106 3.66 Â 104 Æ 6.67 Â 104 1.73 Â 104 Æ 2.71 Â 104 1.72 Â 104 Æ 2.70 Â 104 1.30 Â 104 Æ 2.29 Â 104 Automated counts of bacterial cells in the microscopic images were made with a macro developed specifically for this purpose. Images Live were converted to 32-bit gray scale, background noise was reduced (3 Â 3 median filter), edges were enhanced with a filter (8-point compass), and an attempt was made to separate individual bacterial cells from clumped or overlapping bacteria with a water-shed algorithm. After processing with edge detection, cells were automatically counted. 3.52 Â 105 Æ 5.83 Â 105 6.04 Æ 1.13 Â 101 5.41 Â 104 Æ 1.04 Â 105 6.66 Æ 1.01 Â 101 Counts were enumerated by CFU and 2 vital microscopy methods. To verify the proper performance of the macro, bacteria from samples CFU 14 days collected in a pilot project were counted visually, and the results were 9.37 Â 10À1 Æ 1.74 3.71 Æ 4.69 1.59 Æ 2.32 2.30 Æ 3.45 2.11 Æ 2.50 7.82 Â 10À1 Æ 1.59 compared with those obtained with the automated procedure. Culture plates were examined for absence or presence of bacterial growth. When growth occurred, CFU counts were established with the aid of a count template. The bacterial densities recorded by both EFM and CFU were expressed as the number of bacteria per mL RCV (22). Sample Analysis 1A 2A 1A 2A 1B 1C 2B 1B 1C 2B SAS 9.1 (SAS Institute Inc, Cary, NC) was used for data management and analysis. The main outcome variables were the mean EFM bacterial Group densities transformed by natural logarithms. The main experimental MTAD Saline variables were group (MTAD versus saline) and sample (1C, 2A, 2B). As a result of the dependent nature of the multiple treatment steps,1486 Malkhassian et al. JOE — Volume 35, Number 11, November 2009
  5. 5. CONSORT Randomized Clinical TrialTABLE 4. Means and Standard Deviations of the Bacterial Counts in the Access Cavity Samples at 2 Treatment Sessions, Obtained with Different EnumerationMethods, and the Proportion of Bacteria-positive Cultures in the Samples Count per access cavity Enumeration method 1AC 2AC CFU 14 days Aerobic 7.11 Â 101 Æ 2.92 Â 102 8.89 Æ 2.17 Â 101 Anaerobic 3.30 Â 101 Æ 6.23 Â 101 2.41 Â 101 Æ 3.81 Â 101 DAPI/DHET Live 2.62 Â 105 Æ 5.77 Â 105 1.08 Â 105 Æ 1.49 Â 105 Dead 1.75 Â 106 Æ 2.81 Â 106 2.24 Â 106 Æ 5.96 Â 106 Total 2.01 Â 106 Æ 2.76 Â 106 2.35 Â 106 Æ 6.06 Â 106 Baclight Live 1.15 Â 106 Æ 3.41 Â 106 3.12 Â 105 Æ 2.39 Â 105 Dead 1.21 Â 105 Æ 3.58 Â 105 7.83 Â 104 Æ 9.17 Â 104 Total 1.27 Â 106 Æ 3.75 Â 106 3.90 Â 105 Æ 2.69 Â 105CFU, colony-forming unit; DAPI, 40 ,6-diamidino-2-phenylindole; DHET, dihydroethidium.repeated-measures analysis of covariance was used to detect significant Sterility Control and Access Cavity Samplesdifferences in the bacterial densities at the different samples. In this anal- Sterility control samples 1SC and 2SC yielded bacterial growth inysis, bacterial densities in samples 1A and 1B were considered baseline 20% and 37% of the samples, respectively. Bacterial growth occurredvalues and used as covariates after 2-sample t-tests were used to in 30% of 1AC and 17% of 2AC samples after aerobic incubation andcompare bacterial densities between the test (MTAD) and control 43% and 40%, respectively, after anaerobic incubation. The bacterial(saline) groups in samples 1A and 1B. In addition, logistic regression counts in the access cavity samples are presented in Table 4. Theanalysis was used to detect significant differences in the proportion of CFU counts were consistently low, and the EFM counts were at least 5positive cultures between sample 1B and the subsequent samples. Statis- orders of magnitude higher. Both the CFU and EFM live counts appearedtical tests were 2-tailed and interpreted at the 5% significance level. A to decrease from 1AC to 2AC samples. The counts of dead bacteria ap-Bonferroni correction was used to account for multiple testing. peared higher in the DAPI/DHET-stained samples than in the BacLight- stained samples. The access cavity dataset was not analyzed statistically. ResultsFinal Data Set Effect of Root Canal Preparation The bacterial densities (counts per mL RCV) obtained with the 3 The vital bacterial counts (both CFU and EFM values) decreasedenumeration methods are presented in Table 3. The CFU counts from substantially from 1A to 1B samples (Table 3). In both samples 1Asubject 18 (control group) were excluded because no bacterial growth and 1B, the counts did not differ significantly between the MTAD andoccurred in sample 1A, and the CFU count of sample 1B from subject 5 control groups.(MTAD group) was excluded because of contamination of the cultureplate. Abundant CFUs were recorded in 1A samples, in sharp contrast Significant Effects, MTAD, CHX, and Final Preparationto the few CFUs recorded in all subsequent samples. Because 1B, 1C, Table 5 summarizes the analyses of covariance for the EFM results.2A, and 2B samples frequently had no bacterial growth (below detec- The bacterial counts in 1A samples showed no effect on counts at subse-tion threshold), they were analyzed only for presence or absence of quent samples and were dropped from the analyses. With 2 exceptionsgrowth on culture plates. (BacLight-dead and DAPI/DHET-live), the bacterial counts in the 1B EFM detected live bacteria in all samples except the DHET/DAPI- samples explained the bacterial counts at the subsequent samples;stained 2A sample from subject 14 (MTAD group). Dead bacteria thus, higher counts at 1B samples corresponded to higher subsequentwere also detected in all samples, except the BacLight-stained 2B counts. Group (MTAD or saline) had no significant effect on the counts.sample from subject 12 (MTAD group). The mean live bacterial counts Samples (1A, 2A, 2B) had a significant difference only for BacLight-obtained with both EFM methods were consistently higher than the cor- dead and DAPI/DHET-total, showing higher counts in 2A than in 1Cresponding CFU counts. The discrepancy was particularly evident in 1B and 2B. Group and sample had a confounded effect for BacLight-liveand subsequent samples. and BacLight-total. In particular, the MTAD counts were lower thanTABLE 5. P Values for Repeated-measures Analysis of Covariance for Vital Microscopy (EFM) Methods Effects Covariate Vital microscopy method Group Sample Group*Sample 1B BacLight Live .6703 .5768 .0407* .0147* Dead .1305 .0215* — .0711 Total .8134 .1660 .0327* .0369* DAPI/DHET Live .8849 .5413 — — Dead .2336 .2430 — .0269* Total .3271 .0452* — .0043*†DAPI, 40 ,6-diamidino-2-phenylindole; DHET, dihydroethidium.Group = MTAD, saline; sample = 1C, 2A, 2B; empty cell = model rerun without this nonsignificant interaction or covariate.Samples 1A and 1B were used as covariates in the analysis. All 1A results were nonsignificant and dropped from the model.*Statistically significant at alpha = 0.05.† Only remaining significant value after Bonferroni correction.JOE — Volume 35, Number 11, November 2009 Clinical Study of MTAD and CHX Gel 1487