Sonication of Antibiotic-Loaded Cement Spacers
Sonication of Antibiotic-Loaded Cement Spacers
From March 2009 to January 2011 we performed a two-stage exchange procedure in 21 consecutive patients, 10 women and 11 men, affected by PJI (16 total knee prostheses and 5 hip prostheses). Mean patient age was 66.1 years (range, 55–78 years). The diagnosis of suspected prosthetic infection was made on the basis of clinical signs and symptoms (reported pain, hyperthermia, swelling, redness), preoperative microbiological cultures (material from joint aspiration or sinus track), laboratory studies (leukocytosis, increased CRP), plain radiographs, and nuclear medicine findings (99 m-Tc HMPAO labeled leukocyte scan or 18-fluorodeoxyglucose positron emission tomography (FDG-PET) scan). To be included in this study, that was approved by the institutional review board of the Department of Surgery and Orthopaedics of Federico II University, Naples, Italy and is compliant with the Helsinki Declaration, all individuals gave their oral and written informed consent for the publication of individual clinical details. They met at least one of the following criteria to confirm the infection: i) two or more cultures of joint aspirates or cultures of intraoperative specimens yielding the same microorganism; ii) purulence surrounding the prosthesis at the time of explantation; iii) acute inflammation consistent with infection during pathohistological examination; iv) a sinus tract that communicated with the prosthesis. Removal of the prosthesis with debridement of all infected tissue and placement of a temporary molded antibiotic-loaded cement spacer was performed in each case. All these spacers contained gentamicin plus clindamycin, but in three cases an extra 2 g vancomycin was added. At the time of the first surgical stage, five specimens of periprosthetic tissue were intraoperatively collected under sterile conditions and sent for microbiological and histopathological diagnosis. The prosthetic components were packed into sterile containers, covered with four-hundred millilitres of Ringer's solution, and sent for sonication fluid analysis. The resection of prosthetic components was followed by intravenously administered empirical antibiotic treatment for one week until the cultures and susceptibility tests were available. Combination therapy including drugs showing activity against biofilms was used on an outpatient basis thereafter, according to the sensitivity profile of the cultured microorganisms. In 3 patients (14.3%) with negative pre- and intra-operative cultures and positive histology showing acute inflammation consistent with infection, an empirical combination antibiotic treatment including rifampin was given. The second surgical stage was performed after clinical and laboratory findings had normalized. Antibiotic therapy was discontinued at least two weeks before surgery. The time interval between the removal of the infected prosthesis and the reimplantation of the second prosthesis varied, depending on the clinical and laboratory response to antibiotics and the cultured microorganism (Table 1). At the time of the cement spacer removal, multiple specimens of periprosthetic tissue were collected and sent for microbiological and histopathological studies. The cement spacers were packed into sterile containers, covered with four hundred millilitres of Ringer's solution, and sent for sonication. After the hospital discharge, patients were checked clinically, serologically, and radiographically at 2, 4, 12, and 24 weeks after the second surgery. Thereafter, clinical, serologic (CRP, leukocyte count), and radiographic checks were obtained every 6 months.
Tissue specimens were homogenized in 3 ml of brain-heart-infusion broth for 1 min and the homogenate was inoculated in aliquots of 0.5 ml. Aerobic and aerobic sheep-blood agar were incubated at 35°C to 37°C in 5% to 7% carbon dioxide aerobically and anaerobically for 5 days and 7 days, respectively.
The container was vortexed for 30 seconds using a Vortex-Genie (Scientific Industries, Inc, Bohemia, NY, USA) and then subjected to sonication (frequency 35–40 KHz) in an Aquasonic Model 750 T ultrasound bath (VWR Scientific Products, Radnor, PA, USA) for 5 minutes, followed by additional vortexing for 30 seconds. The resulting sonicate fluid was plated in 500 μl aliquots onto aerobic Columbia sheep blood agar plates for 5 days and anaerobic Shaedler sheep blood agar for 7 days. Microorganisms were enumerated and classified by routine microbiological techniques. A total of 200 ml of sonicate fluid was centrifuged at 2600 rpm for 15 minutes and the sediment was gram stained. All bacteria were counted and identified by standard methods (VITEK Biomerieux, Bagno a Ripoli (FI), Italy).
Continuous variables are expressed as the median and interquartile range (IQR). Categorical variables were analyzed with a chi-squared test or Fisher's exact test. P < 0.05 was considered statistically significant. SPSS software (SPSS Inc., Chicago, Illinois, USA) was used for the database and statistics.
Methods
From March 2009 to January 2011 we performed a two-stage exchange procedure in 21 consecutive patients, 10 women and 11 men, affected by PJI (16 total knee prostheses and 5 hip prostheses). Mean patient age was 66.1 years (range, 55–78 years). The diagnosis of suspected prosthetic infection was made on the basis of clinical signs and symptoms (reported pain, hyperthermia, swelling, redness), preoperative microbiological cultures (material from joint aspiration or sinus track), laboratory studies (leukocytosis, increased CRP), plain radiographs, and nuclear medicine findings (99 m-Tc HMPAO labeled leukocyte scan or 18-fluorodeoxyglucose positron emission tomography (FDG-PET) scan). To be included in this study, that was approved by the institutional review board of the Department of Surgery and Orthopaedics of Federico II University, Naples, Italy and is compliant with the Helsinki Declaration, all individuals gave their oral and written informed consent for the publication of individual clinical details. They met at least one of the following criteria to confirm the infection: i) two or more cultures of joint aspirates or cultures of intraoperative specimens yielding the same microorganism; ii) purulence surrounding the prosthesis at the time of explantation; iii) acute inflammation consistent with infection during pathohistological examination; iv) a sinus tract that communicated with the prosthesis. Removal of the prosthesis with debridement of all infected tissue and placement of a temporary molded antibiotic-loaded cement spacer was performed in each case. All these spacers contained gentamicin plus clindamycin, but in three cases an extra 2 g vancomycin was added. At the time of the first surgical stage, five specimens of periprosthetic tissue were intraoperatively collected under sterile conditions and sent for microbiological and histopathological diagnosis. The prosthetic components were packed into sterile containers, covered with four-hundred millilitres of Ringer's solution, and sent for sonication fluid analysis. The resection of prosthetic components was followed by intravenously administered empirical antibiotic treatment for one week until the cultures and susceptibility tests were available. Combination therapy including drugs showing activity against biofilms was used on an outpatient basis thereafter, according to the sensitivity profile of the cultured microorganisms. In 3 patients (14.3%) with negative pre- and intra-operative cultures and positive histology showing acute inflammation consistent with infection, an empirical combination antibiotic treatment including rifampin was given. The second surgical stage was performed after clinical and laboratory findings had normalized. Antibiotic therapy was discontinued at least two weeks before surgery. The time interval between the removal of the infected prosthesis and the reimplantation of the second prosthesis varied, depending on the clinical and laboratory response to antibiotics and the cultured microorganism (Table 1). At the time of the cement spacer removal, multiple specimens of periprosthetic tissue were collected and sent for microbiological and histopathological studies. The cement spacers were packed into sterile containers, covered with four hundred millilitres of Ringer's solution, and sent for sonication. After the hospital discharge, patients were checked clinically, serologically, and radiographically at 2, 4, 12, and 24 weeks after the second surgery. Thereafter, clinical, serologic (CRP, leukocyte count), and radiographic checks were obtained every 6 months.
Culture of Tissue Specimens
Tissue specimens were homogenized in 3 ml of brain-heart-infusion broth for 1 min and the homogenate was inoculated in aliquots of 0.5 ml. Aerobic and aerobic sheep-blood agar were incubated at 35°C to 37°C in 5% to 7% carbon dioxide aerobically and anaerobically for 5 days and 7 days, respectively.
Sonication Protocol
The container was vortexed for 30 seconds using a Vortex-Genie (Scientific Industries, Inc, Bohemia, NY, USA) and then subjected to sonication (frequency 35–40 KHz) in an Aquasonic Model 750 T ultrasound bath (VWR Scientific Products, Radnor, PA, USA) for 5 minutes, followed by additional vortexing for 30 seconds. The resulting sonicate fluid was plated in 500 μl aliquots onto aerobic Columbia sheep blood agar plates for 5 days and anaerobic Shaedler sheep blood agar for 7 days. Microorganisms were enumerated and classified by routine microbiological techniques. A total of 200 ml of sonicate fluid was centrifuged at 2600 rpm for 15 minutes and the sediment was gram stained. All bacteria were counted and identified by standard methods (VITEK Biomerieux, Bagno a Ripoli (FI), Italy).
Statistical Analysis
Continuous variables are expressed as the median and interquartile range (IQR). Categorical variables were analyzed with a chi-squared test or Fisher's exact test. P < 0.05 was considered statistically significant. SPSS software (SPSS Inc., Chicago, Illinois, USA) was used for the database and statistics.
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