Melissa J. Karau

C-Reactive Protein, Erythrocyte Sedimentation Rate and Orthopedic Implant Infection

Background C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been shown to be useful for diagnosis of prosthetic hip and knee infection. Little information is available on CRP and ESR in patients undergoing revision or resection of shoulder arthroplasties or spine implants. Methods/Results We analyzed preoperative CRP and ESR in 636 subjects who underwent knee (n = 297), hip (n = 221) or shoulder (n = 64) arthroplasty, or spine implant (n = 54) removal. A standardized definition of orthopedic implant-associated infection was applied. Receiver operating curve analysis was used to determine ideal cutoff values for differentiating infected from non-infected cases. ESR was significantly different in subjects with aseptic failure infection of knee (median 11 and 53.5 mm/h, respectively, p = <0.0001) and hip (median 11 and 30 mm/h, respectively, p = <0.0001) arthroplasties and spine implants (median 10 and 48.5 mm/h, respectively, p = 0.0033), but not shoulder arthroplasties (median 10 and 9 mm/h, respectively, p = 0.9883). Optimized ESR cutoffs for knee, hip and shoulder arthroplasties and spine implants were 19, 13, 26, and 45 mm/h, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 89 and 74% for knee, 82 and 60% for hip, and 32 and 93% for shoulder arthroplasties, and 57 and 90% for spine implants. CRP was significantly different in subjects with aseptic failure and infection of knee (median 4 and 51 mg/l, respectively, p<0.0001), hip (median 3 and 18 mg/l, respectively, p<0.0001), and shoulder (median 3 and 10 mg/l, respectively, p = 0.01) arthroplasties, and spine implants (median 3 and 20 mg/l, respectively, p = 0.0011). Optimized CRP cutoffs for knee, hip, and shoulder arthroplasties, and spine implants were 14.5, 10.3, 7, and 4.6 mg/l, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 79 and 88% for knee, 74 and 79% for hip, and 63 and 73% for shoulder arthroplasties, and 79 and 68% for spine implants. Conclusion CRP and ESR have poor sensitivity for the diagnosis of shoulder implant infection. A CRP of 4.6 mg/l had a sensitivity of 79 and a specificity of 68% to detect infection of spine implants.

Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using Sonication

ABSTRACT Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis.

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

ABSTRACT We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.

A Biofilm Approach to Detect Bacteria on Removed Spinal Implants

Study Design. This is a prospective study comparing the diagnosis of spinal implant infection by conventional peri-implant tissue culture with a technique which uses a combination of vortexing and bath sonication to dislodge bacteria growing as a biofilm on the surface of retrieved spinal implants. Objective. We hypothesized that the biofilm-sampling technique would be more sensitive than peri-implant tissue culture. Summary of Background Data. Culture of peri-implant tissue is inaccurate for the diagnosis of orthopedic device-related infection; cultures taken from the implant may be more sensitive. We have developed a technique which uses vortexing-bath sonication to sample bacterial biofilms on the surface of retrieved hip and knee implants, and shown that it is more sensitive than peri-prosthetic tissue culture for the microbiologic diagnosis of prosthetic knee, hip, and shoulder infection. Methods. We compared peri-implant tissue culture to the vortexing-bath sonication technique which samples bacterial biofilm on the surface of retrieved spinal implants, for the diagnosis of spinal implant infection. In addition, we compared detection of Staphylococcus and Propionibacterium acnes by rapid cycle real-time polymerase chain reaction with culture of sonicate fluid. Results. A total of 112 subjects were studied; 22 had spinal implant infection. The sensitivities of peri-implant tissue and sonicate fluid culture were 73% and 91% (P = 0.046), and the specificities were 93% and 97%, respectively. P. acnes and coagulase-negative staphylococci were the most frequent microorganisms detected among subjects with spinal implant infection, with P. acnes detected in 56 and 45%, and coagulase-negative staphylococci detected in 31 and 40% of peri-implant tissue and sonicate fluid cultures, respectively. Compared with the culture of sonicate fluid, polymerase chain reaction was 100 and 67% sensitive for the detection of culture-positive Staphylococcus and P. acnes spinal implant infection, respectively. Conclusion. Implant sonication followed by culture is more sensitive than peri-implant tissue culture for the microbiologic diagnosis of spinal implant infection.

Implant sonication for the diagnosis of prosthetic elbow infection

BACKGROUND Periprosthetic infection is a potentially devastating complication of elbow arthroplasty, associated with formation of microbial biofilm on the implant surface. The definitive microbiologic diagnosis of periprosthetic infection after elbow arthroplasty may be difficult to establish. Our study aim was to compare the diagnostic accuracy of conventional periprosthetic tissue culture and culture of fluid derived from vortexing and bath sonication of the explanted hardware (a biofilm-sampling strategy). MATERIALS AND METHODS Patients undergoing revision elbow arthroplasty at our institution between July 2007 and July 2010, from each of whom 2 or more periprosthetic tissue cultures and 1 implant sonicate culture were obtained, were studied. A standardized definition of orthopedic implant-associated infection was applied. RESULTS We identified 27 subjects with aseptic failure and 9 with prosthetic elbow infection. Rheumatoid arthritis was the most common underlying disorder. The Coonrad-Morrey prosthesis was the most common type of implant used. The sensitivities of implant sonicate and periprosthetic tissue culture were 89% and 55%, respectively (P = .18), and the specificities were 100% and 93%, respectively (P = .16). Coagulase-negative staphylococci (n = 7) and Staphylococcus aureus (n = 2) were isolated in cases of infection. CONCLUSION Culture of the implant by sonication is at least as sensitive as periprosthetic tissue culture to detect prosthetic elbow infection.

Antimicrobial susceptibility and biofilm formation of Staphylococcus epidermidis small colony variants associated with prosthetic joint infection

We determined the frequency of isolation of non-aureus staphylococcal small colony variants (SCVs) from 31 patients with staphylococcal prosthetic joint infection (PJI) and described the antimicrobial susceptibility, auxotrophy, and biofilm-forming capacity of these SCVs. Eleven non-aureus SCVs were recovered, all of which were Staphylococcus epidermidis, and none of which was auxotrophic for hemin, menadione, or thymidine. Aminoglycoside resistance was detected in 5. Two were proficient, and 7 were poor, biofilm formers. With passage on antimicrobial free media, we observed a fluctuating phenotype in 3 isolates. We also noted a difference in antimicrobial susceptibility of different morphology isolates recovered from the same joints despite similar pulsed-field gel electrophoresis patterns. Our findings suggest S. epidermidis SCVs are common in PJI, and while they have a similar appearance to S. aureus SCVs, they do not necessarily share such characteristics as aminoglycoside resistance; auxotrophy for hemin, menadione, or thymidine; or enhanced biofilm formation. We also underscore the importance of antimicrobial susceptibility testing of all morphologies of isolates recovered from PJI.

Antibacterial and Biocompatible Titanium-Copper Oxide Coating May Be a Potential Strategy to Reduce Periprosthetic Infection: An In Vitro Study

BackgroundPeriprosthetic infections are devastating for patients and more efficacious preventive strategies are needed. Surface-modified implants using antibacterial coatings represent an option to cope with this problem; however, manufacturing limitations and cytotoxicity have curbed clinical translation. Among metals with antibacterial properties, copper has shown superior in vitro antibacterial performance while maintaining an acceptable cytotoxicity profile. A thin film containing copper could prevent early biofilm formation to limit periprosthetic infections. This pilot study presents the in vitro antibacterial effect, cytotoxicity, and copper ion elution pattern of a thin film of titanium-copper oxide (TiCuO).Questions/purposes(1) Do titanium alloy (Ti6Al4V) discs coated with a thin film of TiCuO reduce Staphylococcus epidermidis biofilm and planktonic cell density compared with uncoated discs? (2) Do Ti6Al4V discs coated with a thin film of TiCuO affect normal human osteoblast viability compared with untreated cells? (3) Is copper ion concentration generated by coated discs lower than previously published copper ion concentrations that cause 50% toxicity in similar human cell lines in vitro (TC50)?MethodsNinety Ti6Al4V discs (12.5 mm diameter; 1.25 mm thick) were used in this study. Seventy-two Ti6Al4V discs were coated with a thin film of either titanium oxide (TiO) or TiCuO containing 20%, 40%, or 80% copper using high-power impulse magnetron sputtering (HiPIMS). Eighteen Ti6Al4V discs remained uncoated for control purposes. We tested antibacterial properties of S epidermidis grown on discs in wells containing growth medium. After 24 hours, planktonic bacteria as well as biofilms removed by sonication were quantitatively cultured. Annexin/Pi staining was used to quantify in vitro normal human osteoblast cell viability at 24 hours and Day 7, respectively. Copper elution was measured at Days 1, 2, 3, 7, 14, and 28 using an inductively coupled plasma mass spectrometer to analyze aliquots of culture medium. Copper ion concentration achieved at 24 hours was compared with previously published TC50 for gingival fibroblast, a phenotypically similar cell line with available data regarding copper ion exposure.ResultsDiscs coated with TiCuO 80% copper showed greater biofilm and planktonic cell density reduction when compared with other tested compositions (analysis of variance [ANOVA]; p < 0.001). Discs coated with TiCuO 80% copper showed mean biofilm and planktonic cell density of 4.0 log10 (SD = 0.4) and 5.7 log10 (SD = 0.2). Discs coated with TiCuO 80% showed a mean difference in biofilm and planktonic cell density of 2.5 log10 (95% confidence interval [CI], 1.9–3.1 log10; p < 0.001) and 1.2 (95% CI, 0.6–1.8; p < 0.001), respectively, when compared with uncoated discs. Normal human osteoblast viability did not differ among all groups at 24 hours (ANOVA; p = 0.2) and Day 7 (ANOVA; p = 0.7). Discs coated with TiCuO 80% copper showed a mean difference (95% CI) in relative cell viability (%) at 24 hours and Day 7 of 31.1 (95% CI, −19.4 to 81.7; p = 0.4) and −5.0 (95% CI, −7.8 to 17.9; p = 0.9), respectively, when compared with untreated cells. For all TiCuO-coated discs, copper ion elution peaked at 24 hours and slowly decreased in a curvilinear fashion to nearly undetectable levels by Day 28. Discs coated with TiCuO 80% copper showed mean copper ion concentration at 24 hours of 269.4 µmol/L (SD = 25.2 µmol/L) and this concentration was lower than previously published TC50 for similar human cell lines at 24 hours (344 µmol/L, SEM = 44 µmol/L).ConclusionsThis pilot study demonstrates a proof of concept that a thin-film implant coating with TiCuO can provide a potent local antibacterial environment while remaining relatively nontoxic to a human osteoblast cell line. Further research in an animal model will be necessary to establish efficacy and safety of this technique and whether it might be useful in the design of implants.Clinical RelevanceA thin film coating with TiCuO demonstrates high antibacterial activity and low cellular cytotoxicity to human osteoblasts in vitro. Taken together, these properties represent a potential strategy for preventing periprosthetic infection if further work in animal models can confirm these results in vivo.

The impact of tacrolimus on the immunopathogenesis of staphylococcal enterotoxin-induced systemic inflammatory response syndrome and pneumonia

Staphylococcal superantigens (SAg) are a family of potent exotoxins produced by Staphylococcus aureus. They play an important role in the pathogenesis of staphylococcal shock and pneumonia by causing a robust activation of the immune system and eliciting a strong surge in systemic cytokine and chemokine levels. Given the biological functions of SAg, we evaluated the efficacy of tacrolimus, a potent immunosuppressive agent, in the prophylaxis and therapy of staphylococcal TSS and pneumonia using human leukocyte antigen (HLA)-DR3 transgenic mice. Tacrolimus significantly inhibited staphylococcal SAg induced T cell activation in vitro. In vivo, tacrolimus significantly suppressed the SAg-induced elevation in serum cytokine and chemokine levels when given prophylactically, when administered immediately or even 2 h following systemic SAg challenge. Paradoxically, neither the prophylactic nor post-exposure treatment with tacrolimus protected mice from lethal SAg-induced TSS. A closer examination revealed that tacrolimus failed to suppress SAg-induced T cell proliferation and systemic pathology, including gut dysfunction. Tacrolimus also failed to protect from lethal pneumonia induced by a SAg-producing S. aureus strain. Thus, our study showed that even though T cell activation by SAg plays a major role in the immunopathogenesis of TSS and pneumonia, tacrolimus alone has no beneficial effect.

Linezolid Is Superior to Vancomycin in Experimental Pneumonia Caused by Superantigen-Producing Staphylococcus aureus in HLA Class II Transgenic Mice

ABSTRACT Superantigens (SAg), the potent activators of the immune system, are important determinants of Staphylococcus aureus virulence and pathogenicity. Superior response to SAg in human leukocyte antigen (HLA)-DR3 transgenic mice rendered them more susceptible than C57BL/6 mice to pneumonia caused by SAg-producing strains of S. aureus. Linezolid, a bacterial protein synthesis inhibitor, was superior to vancomycin in inhibiting SAg production by S. aureus in vitro and conferred greater protection from pneumonia caused by SAg-producing staphylococci.

Antibiofilm Activity of Low-Amperage Continuous and Intermittent Direct Electrical Current

ABSTRACT Bacterial biofilms are difficult to treat using available antimicrobial agents, so new antibiofilm strategies are needed. We previously showed that 20, 200, and 2,000 μA of electrical current reduced bacterial biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. Here, we tested continuous direct current at lower amperages, intermittent direct current, and combinations of surface materials (Teflon or titanium) and electrode compositions (stainless steel, graphite, titanium, or platinum) against S. aureus, S. epidermidis, and P. aeruginosa biofilms. In addition, we tested 200 or 2,000 μA for 1 and 4 days against biofilms of 33 strains representing 13 species of microorganisms. The logarithmic reduction factor was used to measure treatment effects. Using continuous current delivery, the lowest active amperage was 2 μA for 1, 4, or 7 days against P. aeruginosa and 5 μA for 7 days against S. epidermidis and S. aureus biofilms. Delivery of 200 μA for 4 h a day over 4 days reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on Teflon or titanium discs. A reduction of P. aeruginosa, S. aureus, and S. epidermidis biofilms was measured for 23 of 24 combinations of surface materials and electrode compositions tested. Four days of direct current delivery reduced biofilms of 25 of 33 strains studied. In conclusion, low-amperage current or 4 h a day of intermittent current delivered using a variety of electrode compositions reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on a variety of surface materials. The electricidal effect was observed against a majority of bacterial species studied.