Improved Diagnosis of Orthopedic Implant-Associated Infection by Inoculation of Sonication Fluid into Blood Culture Bottles

Sonication improved the diagnosis of orthopedic implant-associated infections (OIAI). We investigated the diagnostic performance of sonication fluid inoculated into blood culture bottles in comparison with that of intraoperative tissue and sonication fluid cultures. Consecutive patients with removed orthopedic hardware were prospectively included and classified as having OIAI or aseptic failure (AF) according to standardized criteria. The diagnostic procedure included the collection of five intraoperative tissue cultures and sonication of the removed device, followed by conventional culture of the sonication fluid. Cultures were incubated for 7 days (aerobic) or 14 days (anaerobic). In addition, 10 ml of sonication fluid was inoculated into each aerobic and anaerobic BacT/Alert FAN blood culture bottle and incubated in the automated blood culture system for 5 days. Of 75 included patients, 39 had OIAI and 36 AF. The sensitivity of sonication fluid inoculated into blood culture bottles (100%) was higher than that of conventional sonication fluid (87%; P = 0.05) or intraoperative tissue cultures (59%; P < 0.01). Previous antibiotic therapy reduced the culture sensitivity of conventional sonication fluid to 77% and that of intraoperative tissue to 55%, while it remained 100% for blood culture-inoculated sonication fluid. The time to positivity was shorter in blood culture-inoculated sonication fluid, with detection of 72% of microorganisms after 1 day of incubation, than for intraoperative tissue and conventional sonication fluid cultures, with detection of 18% and 28% of microorganisms, respectively. In conclusion, compared to conventional sonication fluid and intraoperative tissue cultures, sonication fluid inoculated into blood culture bottles improved the diagnosis of OIAI and considerably reduced the time to culture positivity.     

Diagnosis of spacer-associated infection using quantitative cultures from sonicated antibiotics-loaded spacers: implications for the clinical outcome

Recent studies showed that a positive microbiological result from sonication of the PMMA spacer was associated with poor outcome of patients, but no quantitative analysis has yet been performed. For this purpose, a prospective analysis of 50 spacers (46 patients) was performed. All spacers were processed according to a previously described protocol, including centrifugation and quantitative culture. Clinical data and outcome were also analysed. A statistical relationship between the results of the cultures and the outcome of the patient was assessed. Sixteen patients were diagnosed with spacer-associated infection. Thirteen out of 50 spacers gave a positive culture. Nine of 13 presented with growth of an organism not isolated in the first-stage cultures, and in 7 out of 13 the organisms count was high (>10,000 CFU/ml). We have detected a significant statistical relationship between poor outcome and positive cultures, high colony counts, isolation of different organisms, positive periprosthetic cultures and spacer-associated infection. The detection in a sonicated, antibiotic-loaded PMMA spacer of organisms other than those isolated in the first surgical samples or high colony counts of any organisms is diagnostic with regard to spacer-associated infection.     

Improved Diagnosis of Infection Associated with Osteosynthesis by Use of Sonication of Fracture Fixation Implants

Previous studies have shown that sonication fluid cultures from removed orthopedic devices improved the microbiological diagnosis of orthopedic implant-associated infections; however, few of these investigations have applied sonication to the removed fracture fixation devices to evaluate its utility for the diagnosis of osteosynthesis-associated infection (OAI). We compared sonication fluid to conventional tissue cultures from 180 subjects with different sizes of plates and screws (n = 156), spinal implants (n = 26), and intramedullary nails (n = 3), of whom 125 and 55 subjects had OAI and noninfected osteosynthesis (NIO), respectively. The sensitivity for detecting OAI was 90.4% for sonication fluid culture and 56.8% for periprosthetic tissue cultures (P < 0.05), and the specificities were 90.9% and 96.4%, respectively. Sonication fluid culture detected more pathogens than peri-implant tissue culture (113 versus 71; P < 0.001), while polymicrobial infections were diagnosed by sonication fluid cultures and tissue cultures in 20.8% and 8% (P < 0.001), respectively. Microbiological diagnosis was achieved exclusively by sonication fluid cultures for 47 (90.4%) subjects, and among them, 18 (38.3%) had previously received antibiotics, whereas in five (9.6%) infected subjects, tissue culture was positive and the sonication fluid culture was negative. Among 39 (31.2%) OAI cases receiving antibiotics, the identification of the organisms occurred in 38.5% and 82.1% of the tissue and sonication fluid cultures, respectively (P < 0.049). We demonstrated that sonication fluid culture from removed osteosyntheses has the potential for improving the microbiological diagnosis of OAI.     

Sonication of Explanted Cardiac Implants Improves Microbial Detection in Cardiac Device Infections

The sonication technique has been shown to be a promising tool for microbiological diagnosis of device-related infections. We evaluated the usefulness of the sonication method for pathogen detection in 80 explanted cardiac components collected from 40 patients, and the results were compared with those of conventional cultures. Forty subjects undergoing cardiac device removal were studied: 20 had cardiac device infection, and 20 subjects underwent elective generator replacement or revision in the absence of infection. Sonication of explanted devices was more sensitive than traditional culture for microbial detection (67% and 50%, respectively; P = 0.0005). The bacterial count detected in sonication fluid culture was significantly higher than that detected in traditional culture in both infected (P = 0.019) and uninfected (P = 0.029) devices. In the infected patients, sonication fluid culture yielded a significantly higher rate of pathogen detection in explanted electrodes than traditional culture (65% versus 45%; P = 0.02), while no differences were found in the generators. Ten strains were detected only through sonication fluid culture: 6 Staphylococcus epidermidis strains, 1 Staphylococcus hominis strain, 2 Corynebacterium striatum strains, and 1 Brevundimonas sp. Neither the type nor the duration of antimicrobial therapy before device removal had an effect on the diagnostic performance of sonication fluid culture (P = 0.75 and P = 0.56, respectively). In the patients without infection, sonication fluid culture was positive in 8 cases (40%), whereas conventional culture was positive in only 4 (20%). In summary, the sonication technique improves the microbiological diagnosis of explanted cardiac devices.     

Comparison of Heat Inactivation and Cell Disruption Protocols for Identification of Mycobacteria from Solid Culture Media by Use of Vitek Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Two novel protocols for inactivation and extraction were developed and used to identify 107 Mycobacterium clinical isolates, including Mycobacterium tuberculosis complex, from solid cultures using Vitek matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The protocol using heat inactivation with sonication and cell disruption with glass beads resulted in 82.2% and 88.8% species and genus level identifications, respectively.     

Sonication of Explanted Prosthesis Combined with Incubation in BD Bactec Bottles for Pathogen-Based Diagnosis of Prosthetic Joint Infection

Prosthetic joint infection (PJI) is a rare but refractory complication of arthroplasty. Accurate identification of pathogens is a key step for successful treatment of PJI, which remains a challenge for clinicians and laboratory workers. We designed a combined culture method with sonication of implants and incubation in a BD Bactec system to improve the effectiveness of pathogen diagnosis in PJI. The aims of this study were to investigate the diagnostic accuracy of sonicate fluid cultures in the BD Bactec system and to compare the results with those of synovial fluid cultures in the BD Bactec system. The prosthetic components removed were sonicated in Ringer''s solution, and then sonicate fluid was incubated in Bactec bottles for 5 days. Synovial fluid was incubated in Bactec bottles for 5 days as a control. Synovial fluid cultures with Bactec bottles and sonicate fluid cultures with Bactec bottles showed sensitivities of 64% and 88%, respectively (P = 0.009), with specificities of 98% and 87% (P = 0.032), respectively. Sonicate fluid cultures with Bactec bottles were more sensitive than synovial fluid cultures with Bactec bottles regardless of whether antimicrobial agents were used within 14 days before surgery (81% versus 52%; P = 0.031) or not (93% versus 72%; P = 0.031). Sonication of explanted prostheses followed by incubation of the resulting sonicate fluid in Bactec bottles detected many more pathogens than did synovial fluid cultures with Bactec bottles. This method is also effective in cases with antibiotic treatment before surgery.     

Evaluation of a Microarray-Based Assay for Rapid Identification of Gram-Positive Organisms and Resistance Markers in Positive Blood Cultures

Rapid identification of pathogens directly from positive blood cultures can play a major role in reducing patient mortality rates. We evaluated the performance of the Verigene Gram-Positive Blood Culture (BC-GP) assay (Nanosphere Inc., Northbrook, IL) for detection of commonly isolated Gram-positive organisms as well as associated resistance markers from positive blood cultures. Positive blood cultures (VersaTREK; Trek Diagnostic Systems, Independence, OH) from 203 patients with Gram-positive organism infections were analyzed using the BC-GP assay within 12 h for the detection of 12 different organisms, including staphylococci, streptococci, and enterococci, as well as for the presence of 3 resistance markers (mecA, vanA, and vanB). Results were compared to those of routine laboratory methods for identification and susceptibility testing. For identification of organisms and detection of resistance markers in 178 monomicrobial positive blood cultures, the BC-GP assay showed 94% and 97% concordance, respectively, with routine methods. After 25 polymicrobial cultures were included, the results showed 92% and 96% agreement for identification and resistance markers, respectively, for a total of 203 positive cultures. In 6/25 polymicrobial cultures, at least 1 isolate was not detected. Concordance levels for detection of major pathogens such Staphylococcus aureus (n = 45) and enterococci (n = 19) were 98% and 95%, respectively. Agreement levels for detection of resistance markers such as mecA and vanA/B were 92% and 100%, respectively. The BC-GP assay is capable of providing rapid identification of Gram-positive cocci as well as detection of resistance markers directly from positive blood cultures at least 24 to 48 h earlier than conventional methods.     

The application of sonication in diagnosis of periprosthetic joint infection

Periprosthetic joint infection (PJI) is a catastrophic complication after total joint arthroplasty. It has always been difficult to diagnose PJI, which is characterised by existence of biofilm around the implants. The application of sonication has proven advantageous for pathogen detection. This meta-analysis of clinical trials was performed to evaluate the diagnostic value of sonication and to compare it with traditional bacterial culture. We assessed 16 studies that evaluated sonication fluid cultures (SFC) for the diagnosis of PJI. It was shown that sonication may be of great value in PJI diagnosis, with a pooled sensitivity of 0.79 (95 % confidence interval [CI]?=?0.76–0.81), specificity of 0.95 (CI?=?0.94–0.96), DOR of 71.20 (CI?=?31.08–163.10), PLR of 15.25 (CI?=?6.44–36.15), and NLR of 0.23 (CI?=?0.18–0.30). The AUC value of the SROC was 0.90. The results of this meta-analysis showed that culture of fluid after sonication was of great value for PJI diagnosis. Sonication was more sensitive than traditional tissue culture with lower specificity, especially for patients previously taking antibiotics.     

Comparison of Perirectal versus Rectal Swabs for Detection of Asymptomatic Carriers of Toxigenic Clostridium difficile

For long-term care and spinal cord injury patients, the sensitivity, specificity, and positive and negative predictive values of perirectal versus rectal cultures for detection of asymptomatic carriers of Clostridium difficile were 95%, 100%, 100%, and 97%, respectively. Perirectal cultures provide an accurate method to detect asymptomatic carriers of C. difficile.     

Concentration of Sonication Fluid through Centrifugation Is Superior to Membrane Filtration for Microbial Diagnosis of Orthopedic Implant-Associated Infection

Microbial identification of orthopedic implant-associated infections using sonication fluid (SF) submitted to a concentration step by membrane filtration (SMF) was compared with the standard centrifugation (SC) method. Among 33 retrieved infected implants, sonication identified microorganisms in 26 (78.8%). The sensitivity of SC was higher than that of SMF (78.8% versus 30.3%; P < 0.001).     

How Should Long-Term Tunneled Central Venous Catheters Be Managed in Microbiology Laboratories in Order To Provide an Accurate Diagnosis of Colonization?

Guidelines recommend the roll-plate technique for short-term central venous catheter (CVC) tip cultures. However, the issue of whether the roll-plate technique is better than the sonication method for long-term CVCs remains unresolved. In addition, no data are available for predicting the value of direct Gram staining in anticipating catheter colonization or catheter-related bloodstream infection (CRBSI) in these long-term CVCs. Our objectives were to compare the roll-plate technique and the sonication method and to define the validity values of Gram staining for the prediction of colonization and CRBSI in patients with long-term tunneled CVCs. During the study period, all tunneled CVCs removed at our institution were prospectively and routinely sent to the microbiology laboratory for Gram staining (first) and tip culture (the Maki technique and sonication, in a random order). We received 149 tunneled CVCs, 39 (26.2%) of which were colonized and 11 (7.4%) of which were associated with CRBSI. Overall, the roll-plate method detected 94.9% of the colonized catheters, whereas sonication detected only 43.6% (P < 0.001). The validity values of Gram staining for the detection of colonization and CRBSI were as follows: a sensitivity of 35.9% to 60.0%, a specificity of 100% to 94.2%, a positive predictive value of 100% to 42.9%, and a negative predictive value of 81.5% to 97.0%. The roll-plate technique proved to be better than sonication for the detection of bacteria in long-term tunneled CVCs. Gram staining of the tips of tunneled CVCs can anticipate a positive culture and rule out CRBSI. In our opinion, direct Gram staining should be incorporated into routine microbiological assessments of long-term catheter tips.     

Evaluation of the Verigene Gram-Positive Blood Culture Nucleic Acid Test for Rapid Detection of Bacteria and Resistance Determinants

Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.     

Diagnosis of triple-lumen catheter infection: comparison of roll plate, sonication, and flushing methodologies.

In a recent clinical trial, 248 triple-lumen catheters were removed from patients in an intensive care unit, and their tip and subcutaneous segments were cultured by both the sonication and roll plate methods; for 191 of these catheters, flush cultures of all three catheter lumens were also performed. Previously published quantitative endpoints were used to define significant catheter colonization. By using a composite index as a definition of colonization (any of the seven types of cultures meeting quantitative criteria), sonication of the subcutaneous segment was the most sensitive at detecting colonization (58%), followed by sonication of the catheter tip (53%). Sonication of both the subcutaneous and tip segments was 20% more sensitive than sonication of an adjacent catheter segment by the roll plate method (P < 0.05). The greater sensitivity of the sonication method could be attributed to its greater ability than the roll plate method to detect catheter lumen colonization (82 versus 57%, respectively; P = 0.01). A greater number of positive catheter segment cultures were found for colonized catheters from patients with associated bacteremia than for colonized catheters from patients without bacteremia (57 versus 37%; P = 0.004), making any culture method more likely to identify them. For catheters with significant colonization of only one site, the localization was as follows: 36.7% subcutaneous segment, 36.7% catheter lumen, and 26.6% tip segment. These findings suggest that the current practice of culturing a single segment of a central vascular catheter is inadequate and needs to be reexamined. They further suggest that initial colonization of the catheter lumen and tip segments may be more important than previously thought and may require a change in thinking of strategies designed to prevent catheter infection.     

Cultures of Needleless Connectors Are Useful for Ruling Out Central Venous Catheter Colonization

Semiquantitative cultures of skin surrounding intravascular catheter entry sites and catheter hubs have high negative predictive values for catheter tip colonization. However, culturing samples from the inner side of the hub requires the catheter to be manipulated, thus increasing the risk of migration of microorganisms into the bloodstream. Today, hubs are closed using needleless connectors (NCs). Cultures of NCs could predict catheter colonization. Our objective was to compare the yield of NC sonicate cultures for prediction of catheter colonization with that of hub cultures. For 6 months, we prospectively collected all short-term central lines and systems removed from patients admitted to the cardiac surgery postoperative care unit, irrespective of the reason for withdrawal. Hub cultures were obtained immediately before withdrawal and were cultured using a semiquantitative method. Catheter tips were cultured using the roll-plate technique and sonication, and NCs were cultured using a semiquantitative technique after sonication. We considered NCs to be colonized when ≥1 culture was positive. We collected a total of 75 central systems. The catheter colonization rate was 10.7%. The rates for hub and NC colonization were 6.7% and 12.0%, respectively. The validity values for hubs and NCs for prediction of catheter colonization were as follows: sensitivity, 25.0% and 87.5%; specificity, 95.5% and 97.0%; positive predictive value, 40.0% and 77.8%; negative predictive value, 91.4% and 98.5%; validity index, 88.0% and 96.0%, respectively. Cultures of closed NCs can be used to rule out catheter tip colonization and are superior to hub cultures in ruling out short-term central venous catheter colonization.     

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10³ CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.     

Direct Testing of Blood Cultures for Detection of Streptococcal Antigens

A direct, rapid, and simple method for the detection of streptococcal antigens of Lancefield groups A, B, C, D, and G from blood cultures was developed by using a coagglutination test. Fifty-five clinical specimens and 117 simulated blood cultures containing gram-positive cocci were tested. Out of 6,261 clinical blood cultures screened, 55 cultures from 53 patients were positive, with organisms resembling streptococci, by Gram stain. Of these cultures, 78% (43 of 55) were pure cultures of streptococci, and 22% (12 of 55) were mixed with at least one other organism. Of the 43 pure cultures only, correct reactions were obtained (grouping correctly or giving no cross-reactions, or both) with 86% (37 of 43) of the isolates, 12% (5 of 43) exhibited cross-reactions, and 2% (1 of 43) gave false-negative reactions. All of the cross-reacting isolates were Streptococcus pneumoniae, which reacted with the group C reagent, and the false-negative reaction occurred with a Streptococcus bovis isolate. However, by using a direct modified bile solubility test, the correct identification of the S. pneumoniae isolates was obtained. Therefore, by using the modified bile solubility test in conjunction with the direct grouping method, 98% (42 of 43) of the isolates in pure culture could be identified accurately and rapidly after the detection of a positive Gram stain. Correct grouping reactions were obtained with 83% (10 of 12) of the mixed blood cultures, and false-negative results occurred with 17% (2 of 12) of them. Both cultures contained an enterococcus and a gram-negative rod. Of the 117 simulated blood cultures, there was only one incorrect grouping reaction; this occurred with an S. bovis isolate that cross-reacted with the group C reagent. The direct grouping reaction was positive when blood cultures contained a minimum of 1 × 10⁸ to 8 × 10⁸ colony-forming units per ml. In general, this procedure provided information on the identification of the organism 24 h earlier than by conventional identification methods.     

Comparison of sonicated and nonsonicated specimens for the isolation of Chlamydia trachomatis

Cultures of sonicated and nonsonicated chlamydial specimens were compared using a Heat Systems sonicator with a cup-horn attachment for sonication. Chlamydial inclusions were detected in 71 of 780 cultures tested; 44 (62%) were positive from both, while 12 (16.9%) were positive in nonsonicated only, and 15 (21.1%) in sonicated specimens only. The mean number of inclusions from sonicated specimens was more than double that from nonsonicated specimens. The cultures of sonicated specimens could be screened more quickly because of statistically significant higher inclusion counts, less toxicity, and the absence of overlying epithelial cells.     

Rapid Time to Results and High Sensitivity of the CarbaNP Test on Early Cultures

For rapid results, we adapted the CarbaNP test by using 5-hour-old cultures; the sensitivity and specificity for detection of carbapenemase production were 100% for non-OXA-producing organisms. The sensitivity from early cultures was 94% for detection of carbapenemase production in Enterobacteriaceae strains. Utilizing younger cultures allows the test to be incorporated into a single day''s workflow, facilitating timely patient care and antimicrobial stewardship.     

Evaluation of the use of sonication of retrieved implants for the diagnosis of prosthetic joint infection in a routine setting

In order to evaluate the usefulness of sonication of retrieved implants for the diagnosis of prosthetic joint infection (PJI) in a large group of patients in a routine setting, we designed a 3-year retrospective study. Patients were classified into two groups: those meeting the clinical criteria of PJI and those that did not (control group). Two hundred patients and 276 samples were included. The types of infection were early (n?=?44), delayed (n?=?53), positive intraoperative cultures (n?=?13) and late-acute (n?=?8). The culture sensitivities of sonicate fluid, periprosthetic tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid were 69.5, 52.8, 54.8 and 60.2%, respectively. The specificities were 97.6, 90.3, 93.0 and 89.9%, respectively. Sonicate fluid culture of implants was more sensitive than peri-implant tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid for all infection types, though it was especially useful in delayed infection: 91.3% vs. 60.0% (p?=?0.0015), 63.2% (p?=?0.0005) and 66.7% (p?=?0.0001), respectively. When sonicate fluid culture of implants was performed in addition to conventional cultures, the sensitivity increased significantly in total (from 60.2 to 77.1%) and delayed PJI (from 45.1 to 71.7%). On the other hand, for early PJI, sonicate fluid culture of prosthesis was not superior to conventional diagnostic methods.     

Performance of the Verigene Gram-Negative Blood Culture Assay for Rapid Detection of Bacteria and Resistance Determinants

Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated.