The promotional effect of bone wax on experimental Staphylococcus aureus osteomyelitis.

The consequences of using surgical bone wax are not well studied. We evaluated the infection-promoting potential of sterile bone wax in a rat model of chronic Staphylococcus aureus osteomyelitis. The addition of bone wax greatly reduced the quantitative bacterial inoculum (log colony-forming units) required to establish chronic osteomyelitis in 50% and 100% of challenged animals. The 50% infection rate was reduced from log 6.9 to 2.6 and the 100% infection rate from 8.2 to 4.4, respectively (p less than 0.015, t test for parallelism). Separate experiments were done 10 to 30 minutes after inoculation with only log 6.4 staphylococci. Tibiae of animals that received bone wax yielded more organisms than those that did not (log 2.76 +/- 0.68 versus 1.72 +/- 0.94, p less than 0.01). At 24 hours quantitative colony counts were not significantly different whether animals received wax or not (log 5.02 +/- 0.42 versus 4.43 +/- 0.65, p greater than 0.09). These studies suggest that the routine surgical use of bone wax should be reassessed.

Multistrain comparison of three antimicrobial prophylaxis regimens in experimental postoperative Pseudomonas endophthalmitis.

We compared subconjunctivally administered ceftazidime and BMY 28142, two third-generation cephalosporins, to a regimen of gentamicin and cefazolin for their ability to prevent experimental Pseudomonas postoperative endophthalmitis in rabbits. After extracapsular lens extraction, an inoculum of Pseudomonas was injected into the vitreous; one of the three antimicrobial regimens was then administered subconjunctivally. All 25 eyes treated with gentamicin and cefazolin become infected (P = 1). Two of 25 eyes treated with BMY 28142 became infected (P less than .001). None of the 25 eyes treated with ceftazidime became infected (P less than .001).

Immune complex glomerulonephritis associated with Klebsiella pneumoniae infection.

The kidneys of three patients who died of pneumonia due to Klebsiella pneumoniae were studied at autopsy by light and immunofluoerescent microscopy. One had no clinical evidence of renal disease; two had only microscopic hematuria and mild proteinuria. Light microscopy revealed focal proliferative glomerulonephritis in all three cases. Also in all three, immunofluorescent microscopy revealed a granular deposition of capsular polysaccharide antigens of Klebsiella pneumoniae in association with immunoglobulins and complement components in the mesangium and along the glomerular basement membrane. Furthermore, the glomerular bound immunoglobulins were eluted and demonstrated to contain antibodies specific to a capsular polysaccharide antigen of Klebsiella pneumoniae isolated from each patient. These findings may illustrate that the capsular polysaccharides of Klebsiella pneumoniae are antigenic, and that the immune complex deposition in the kidney during infection with this agent can be associated with renal morphological changes. Whether or not clinical evidence of nephritis occurs may depend on the characteristics of the infection and the host factors.

A comparison of laterally condensed gutta-percha, thermoplasticized gutta-percha, and mineral trioxide aggregate as root canal filling materials.

The purpose of this study was to evaluate the potential of using mineral trioxide aggregate as a root canal filling material by comparing its apical sealing ability with that of laterally condensed gutta-percha with sealer and high-temperature thermoplasticized gutta-percha with sealer in extracted bovine teeth. Sixty bovine incisors with single canals were prepared in a standard manner using LightSpeed instruments, randomly divided into three groups of 20 teeth, and obturated. The sealing ability of each technique was assessed by immersion in 1% methylene blue dye for 3 days. The teeth were cleared, and the linear extent of dye penetration was measured with a stereomicroscope. Data were analyzed by the Kruskal-Wallis one-way ANOVA followed by Dunn's test. Canals filled with laterally condensed gutta-percha or thermoplasticized gutta-percha showed significantly less apical dye penetration than canals obturated with mineral trioxide aggregate (p < 0.001). There was no significant difference in leakage between the laterally condensed group and the thermoplasticized group. The results suggest that gutta-percha obturation may provide an apical seal that is superior to MTA.

Antimicrobial activity of several calcium hydroxide preparations in root canal dentin.

The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)2) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degrees C in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)2 USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)2 USP (0.1 g/ml H2O); group 3, Pulpdent TempCanal paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)2 can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)2 may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.

Effect of the wicking behavior of multifilament sutures.

The purpose of this study was to compare the wicking propensity of multifilament sutures. Dexon II, Vicryl, and black silk suture (BSS) were dipped in saline or soaked for 48 h, then suspended on a microscope slide. Fluorescein isothiocyanate-dextran (FITC-D) was placed at the suture mid points, and its movement was observed using fluorescence microscopy. The experiment was repeated, replacing the FITC-D with mixture of S. salivarius and saline, incubating the suture specimens in culture medium, and evaluating microbial growth. Dipped sutures showed FITC-D movement in the Dexon II group only. All 48-h soaked sutures demonstrated FITC-D movement with significant (p < 0.005) differences in mean times: BSS 179 +/- 42 s; Vicryl 120 +/- 26 s; and Dexon II 32 +/- 2 s. Dexon II suture demonstrated wicking of S. salivarius, whereas Vicryl and BSS did not (p < 0.05). These results suggest that BSS and Vicryl sutures do not wick as readily as Dexon II does.

Determination of periodontal ligament cell viability in long shelf-life milk.

The purpose of this study was to determine the ability of long shelf-life milk to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability on avulsed teeth. PDL cells were plated onto 24-well culture plates and allowed to attach for 24 h. Minimal Essential Medium was replaced with regular pasteurized milk (refrigerated milk), long shelf-life milk (Parmalat), or Save-A-Tooth. Tap water served as the negative control, and Minimal Essential Medium served as the positive control. The tissue culture plates were incubated at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined using a cell proliferation assay (CellTiter 96 AQ Assay) and absorbance read at 490 nm. ANOVA indicated that all media performed significantly better than tap water at all time periods. At 8 h, PDL cell viability in regular pasteurized milk and long shelf-life milk were significantly greater than in Save-A-Tooth (p < or = 0.001). There was no significant difference between regular pasteurized milk and long shelf-life milk at any time period. These results suggest that long shelf-life milk, which has the advantage of not requiring refrigeration, is as effective a storage medium for avulsed teeth as regular pasteurized milk and more effective than Save-A-Tooth.

Disseminated candidiasis: a comparison of two immunologic techniques in the diagnosis.

Eighty-five subjects were tested for the presence of circulating candidal antigen (CAg) and anti-candidal antibody (CAb) using both an enzyme immunoassay (ELISA) and counterimmunoelectrophoresis (CIE). The 72 studied controls included laboratory volunteers; hospitalized patients without evidence of infection; febrile hospitalized patients without evidence of candidiasis; and patients with superficial candidiasis and candiduria. The control subjects were compared with 13 patients with proven disseminated candidal infection (disease prevalence = 15%). The ELISA CAb test was of greater individual sensitivity (92%) in separating patients with systemic candidiasis from all controls combined than the ELISA CAg, CIE CAg, or CIE CAb test (61%, 15%, 69%, respectively). The CIE CAg test, though specific (100%), was insensitive. Sensitivity, specificity, and predictive values were generally enhanced by employing combinations of tests. Sera from patients with disseminated candidiasis were much more likely to yield a positive result by two or more serologic tests than were control sera (p = less than 0.0004). The sensitivity of combinations ranged from 15% to 92%. The specificity of combinations ranged from 21% to 100%. The predictive value positive of combinations test ranged from 40% to 100%. Predictive value negative of combinations ranged from 69% to 98%. Patients with a variety of superficial and deep candidal infections apparently have detectable circulating CAb and/or CAg. The ELISA CAb test was superior to the other tests in identifying patients with disseminated candidiasis. Combinations of serologic tests may be superior to individual tests in the diagnosis or exclusion of serious disease due to Candida albicans.

The effect of various intracanal oxidizing agents on the push-out strength of various perforation repair materials.

OBJECTIVE: The purpose of this study was to determine the effect of intracanal oxidizing agents on the strength of materials used to repair root perforations. STUDY DESIGN: Standardized perforations in bovine root samples were repaired with mineral trioxide aggregate (MTA), Super-EBA cement (S-EBA), or intermediate restorative material (IRM). After 7 days, 10 samples from each group were tested for push-out strength with an Instron machine (controls). The remaining samples were immersed in NaOCl, sodium perborate mixed with saline (SPB+S), Superoxol (SO), sodium perborate mixed with Superoxol (SPB+SO), or saline for 7 days to investigate the effect of irrigating and walking bleach compounds on the perforation repair materials. Push-out strength values were compared with those of the dry materials to determine whether any loss of integrity had occurred. RESULTS: MTA was statistically significantly less resistant across conditions to displacement than S-EBA or IRM. IRM was consistent across treatment conditions, whereas S-EBA lost strength when exposed to NaOCl, SPB+S, or SPB+SO. Exposure to SPB+S had the greatest effect on all 3 materials. CONCLUSIONS: IRM performed consistently as a perforation repair material despite exposure to oxidizing agents, whereas MTA was less resistant to dislodgement than either IRM or S-EBA and was more affected than IRM by sodium perborate-containing bleaching solutions.

Effect of ibuprofen on gross pathology, bacterial count, and levels of prostaglandin E2 in experimental staphylococcal osteomyelitis.

Infections with Staphylococcus aureus were induced in rat tibiae without sclerosing agents. Animals received ibuprofen or 0.9% NaCl. Both ibuprofen-treated and control animals developed a progressively more-destructive disease over 12 days. Gross tibial pathology was significantly reduced in animals receiving ibuprofen for both six and 12 days postinfection. Radiographic evidence of osteomyelitis was attenuated at 12 days. Geometric mean counts of S. aureus were, however, not significantly changed by ibuprofen treatment. Mean levels of prostaglandin E2 (PGE2) were highest in untreated controls. Ibuprofen treatment of infected animals was associated with a much-reduced mean value of PGE2. Ibuprofen-treated infected tibiae disclosed less PGE2 than did either ibuprofen- or NaCl-treated uninfected tibiae.

Detection of specific IgG antibody in sera from patients infected with Bacteroides fragilis by enzyme-linked immunosorbent assay.

During a period of 13 months, 28 serious infections caused by Bacteroides were seen in 27 patients. Sixteen patients yielded Bacteroides fragilis; sera from 13 (81%) of these 16 had increased levels of IgG specific for B. fragilis lipopolysaccharide (LPS) antigens by enzyme-linked immunosorbent assay (ELISA). Sera from 20 normal controls did not have increased specific IgG. Sera from 22 of 23 patients with bacteremia caused by other gram-negative rods also failed to yield increased levels of specific antibody (P less than 0.0012). Analysis of sera from patients with B. fragilis infections disclosed a significant correlation between the levels of specific IgG to B. fragilis LPS measured by ELISA and the IgG antibody to the infecting B. fragilis by indirect immunofluorescence (r = 0.84, P less than 0.012). Two of the remaining 12 infections caused by Bacteroides not apparently due to B. fragilis organisms were also associated with increased levels of specific IgG to B. fragilis LPS antigens. Specific IgG antibody response may be an important adjunct in diagnosis of common B. fragilis infections and may allow better management of antimicrobial agents.

In vivo glycocalyx expression by Staphylococcus aureus phage type 52/52A/80 in S. aureus osteomyelitis.

Osteomyelitic rat tibiae were examined by scanning electron microscopy for the extracellular glycocalyx of Staphylococcus aureus. S. aureus and fractured tibiae from normal rats were incubated together in vitro and examined similarly. Low magnification of endosteal Haversian portals from tibiae studied in vivo and in vitro disclosed adherent S. aureus exuding glycocalyx that buried the organism in dense, coccoid-studded biofilms. The biofilm became progressively more dense over time in vitro and was exuberant at day 70 in vivo. S. aureus incubated in vitro without tibiae disclosed no glycocalyx. Bone chips studied in vitro disclosed staphylococci more commonly near the endosteal Haversian portals than on the intervening endosteal surfaces (mean +/- SE, 280 +/- 75 vs. 12 +/- 3 per 2,500-micron 2 field; P less than .002 by Student's t test). Organisms within ostia were not counted, although they occluded 10%-40% of the ostium. Staphylococci were adherent to exposed woven material, perhaps collagen.

Treatment of chronic experimental Staphylococcus aureus osteomyelitis with LY146032 and vancomycin.

LY146032 and vancomycin were compared as therapeutic agents in the treatment of chronic Staphylococcus aureus osteomyelitis in the rat. Quantitative cultures disclosed that one of 16, none of 16 and two of 17 tibiae were sterile from the control LY146032, and vancomycin groups, respectively. From positive cultures, geometric mean staphylococcal CFU per gram of bone were as follows: control, 5.13 +/- 1.58; LY146032, 5.36 +/- 0.43 (p = 0.57); and vancomycin, 4.33 +/- 1.73 (p = 0.078). Mean gross pathology was decreased significantly in both treatment groups. LY146032 was no more effective than vancomycin in reducing bacterial counts.

Arachidonic acid facilitates experimental chronic osteomyelitis in rats.

Arachidonic acid was used as a facilitating agent in experimental rat Staphylococcus aureus osteomyelitis and compared with the more commonly used agent, sodium morrhuate. The injection of arachidonic acid or sodium morrhuate and S. aureus into rat tibiae caused increased quantitative bacterial bone counts, gross bone pathology, roentgenographic changes, and weight loss. The doses required to produce these changes appeared to be lower for arachidonic acid.

Detection of specific bacterial antigen in urine of patients infected with Bacteroides fragilis.

An indirect enzyme-linked immunosorbent assay was developed to detect specific Bacteroides fragilis antigen(s) in human urine. Specimens collected within 72 hr of a positive culture were centrifuged, dialyzed, and treated with Tween 20, polyethylene glycol, and bovine serum albumin. Goat hyperimmune gamma-globulin to B. fragilis strain ATCC 23745 was added and incubated, and supernatants were tested for antibody activity to polysaccharide-protein antigen of the same organism. Mean +/- SD results, reported as percentage inhibition of control values and interpreted blindly, were as follows: 29 normal subjects, 9.8% +/- 6.0%; 22 patients with Enterobacteriaceae bacteremia, 6.0% +/- 5.1%; six patients with nonbacteremic infections due to B. fragilis, 22.3% +/- 10.3%; and nine patients with B. fragilis bacteremia, 28.7% +/- 10.2%. Three of six nonbacteremic patients and eight of nine bacteremic patients yielded values greater than 2 SD of control values. None of 22 patients with Enterobacteriaceae bacteremia was falsely positive (specificity, 100%).

Enzyme-linked immunospecific antibody test for detecting antibody to Klebsiella.

The enzyme-linked immunospecific antibody test was performed in standard test tubes and microtiter plates to meausre high-titer antibody against Klebsiella capsular polysaccharide. Initial studies were conducted with rabbit sera; other studies were conducted with the serum of a patient infected with type 9 Klebsiella. Both immunized rabbits and an infected patient disclosed high titers of anticapsular antibody. Control sera from other immunized rabbits and other infected humans failed to show this substantial antibody titer against type 9 Klebsiella. Comparisons between counterimmunoelectrophoresis and indirect immunofluorescence disclosed that the sensitivity of the enzyme-linked immunospecific antibody test for anti-Klebsiella antibody ranged between 400 and 10,000 times that of these tests.

Assessment of lipopolysaccharide and outer membrane of Bacteroides fragilis by an antibody-inhibition enzyme-linked immunosorbent assay in physiologic fluids and infected animals.

LPS antigen of Bacteroides fragilis (CDC strain 5462) was measured in vitro in physiologic buffer and undilute human sera by using an antibody-inhibition ELISA system. Other studies were performed to assess detection of the outer membrane antigen from this organism. LPS was repetitively detected at 20 to 50 ng/ml dry weight, and outer membranes were detected at 200 ng/ml total protein in physiologic buffers and human membranes were detected at 200 ng/ml total protein in physiologic buffers and human sera. LPS of other type strains was also detected. Prior incubation of the reagent antibody with multiple whole Enterobacteriaceae organisms and Pseudomonas aeruginosa did not alter test results. Bacteremic rats were easily separated into those with B. fragilis (N = 15) and Escherichia coli (N = 14) bacteremias. Sera from rats in which subcutaneous abscesses were produced with 18 strains of Enterobacteriaceae inhibited detection antibody significantly less than did sera from 30 rats in which abscesses were produced with 11 strains of b. fragilis (p less than 0.01). Although values from the group of animals challenged with B. fragilis were significantly different from the group challenged with Enterobacteriaceae, the present results lack significant sensitivity and specificity for clinical application.

An antibiotic resistant experimental model of Pseudomonas osteomyelitis.

We report a model of chronic Pseudomonas aeruginosa osteomyelitis in the rat that was reproducible, simple and inexpensive. No promoting agent was required to cause infection. Infected animals yielded consistent pseudomonal colony counts (log): 4.98 +/- 0.32 (SD)/g cortical tibia (n = 16). The 95% confidence interval of the mean was 4.83-5.14. The inoculum required to infect 50% of challenged rats (ID50) was log 4.0; the ID 100 was log 6.4. Ceftazidime (50 mg/kg/8 h, subcutaneously), alone and in combination with tobramycin (40 mg/kg/12 h, subcutaneously), produced no significant change in quantitative bacterial count or gross bone pathology when used to treat established disease.

Model of experimental chronic osteomyelitis in rats.

We describe here a Sprague-Dawley rat model for chronic osteomyelitis. Staphylococcus aureus and sodium morrhuate were implanted by either microdrilling or direct needle injection into the tibiae of rats. Of 107 rats, 87 (81%) developed osteomyelitis when a high-speed drill was used for implantation, and 27 (51%) of 53 rats developed osteomyelitis by direct needle inoculation (chi square = 9.81, P less than 0.01). Demonstrated histopathological changes included the presence of resorption bays filled with osteoclasts. Quantitative microbiological monitoring of tibial count confirmed disease chronicity, yielding stable numbers of CFU (10(6.29 +/- 0.27) ) of S. aureus over 70 days. Infected animals became anemic and lost weight. The erythrocyte sedimentation rates and leukocyte counts were not elevated. Roentgenograms provided the best correlation with the number of organisms in infected tibiae (r2 = 0.80). Rats with infected tibiae were treated with either oxacillin (120 mg/kg per day) or ceftriaxone (50 mg/kg per day). Treatment over 14 or 28 days reduced S. aureus counts in tibiae but did not reliably sterilize infected bones, suggesting that this model was resistant to prolonged antimicrobial therapy.

Comparison of two enzyme-linked immunosorbent assays for antigen quantitation: direct competition and antibody inhibition.

Two enzyme-linked immunosorbent assays for antigen quantitation, direct competition and antibody inhibition, were used to measure rabbit immunoglobulin G in polystyrene microtiter plates and were compared for sensitivity and reproducibility. Both procedures repetitively detected this antigen in the 1- to 100-ng/ml range. Both procedures were predictably reproducible, with direct competition procedures having steeper slopes in ranges tested. Antibody inhibition does not require conjugated antigen and can detect sample antigens if available stock antigens can be passively bound to a solid-phase polystyrene plate.