Kerryl E. Greenwood-Quaintance

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.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

ABSTRACT Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster. IMPORTANCE Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of 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.

Clinical Characteristics and Outcomes of Prosthetic Joint Infection Caused by Small Colony Variant Staphylococci

ABSTRACT Small colony variants (SCVs) are naturally occurring subpopulations of bacteria. The clinical characteristics and treatment outcomes of patients with prosthetic joint infection (PJI) caused by staphylococcal SCVs are unknown. This study was a retrospective series of 113 patients with staphylococcal PJI, with prospective testing of archived sonicate fluid samples. SCVs were defined using two-investigator review. Treatment failure was defined as (i) subsequent revision surgery for any reason, (ii) PJI after the index surgery, (iii) prosthesis nonreimplantation due to ongoing infection, or (iv) amputation of the affected limb. There were 38 subjects (34%) with SCVs and 75 (66%) with only normal-phenotype (NP) bacteria. Subjects with SCVs were more likely to have been on chronic antimicrobials prior to surgery (P = 0.048), have had prior surgery for PJI (P = 0.03), have had a longer duration of symptoms (P = 0.0003), and have had a longer time since joint implantation (P = 0.007), compared to those with only NP bacteria. Over a median follow-up of 30.6 months, 9 subjects (24%) with SCVs and 23 (32%) with only NP bacteria experienced treatment failure (P = 0.51). Subjects infected with Staphylococcus aureus were more likely to fail than were those infected with Staphylococcus epidermidis (hazard ratio [HR], 4.03; 95% confidence interval [CI], 1.80 to 9.04). While frequently identified in subjects with PJI and associated with several potential predisposing factors, SCVs were not associated with excess treatment failure compared to NP infections in this study, where they were primarily managed with two-stage arthroplasty exchange. IMPORTANCE Bacteria with the small colony variant (SCV) phenotype are described in small case series as causing persistent or relapsing infection, but there are insufficient data to suggest that they should be managed differently than infection with normal-phenotype bacteria. In an effort to investigate the clinical importance of this phenotype, we determined whether SCVs were present in biofilms dislodged from the surfaces of arthroplasties of patients with staphylococcal prosthetic joint infection and assessed the clinical outcomes associated with detection of SCVs. We found that prosthetic joint infection caused by SCV staphylococci was associated with a longer duration of symptoms and more prior treatment for infection but not with an increased rate of treatment failure, compared to infection caused by normal-phenotype staphylococci. Bacteria with the small colony variant (SCV) phenotype are described in small case series as causing persistent or relapsing infection, but there are insufficient data to suggest that they should be managed differently than infection with normal-phenotype bacteria. In an effort to investigate the clinical importance of this phenotype, we determined whether SCVs were present in biofilms dislodged from the surfaces of arthroplasties of patients with staphylococcal prosthetic joint infection and assessed the clinical outcomes associated with detection of SCVs. We found that prosthetic joint infection caused by SCV staphylococci was associated with a longer duration of symptoms and more prior treatment for infection but not with an increased rate of treatment failure, compared to infection caused by normal-phenotype staphylococci.

Low sensitivity of periprosthetic tissue PCR for prosthetic knee infection diagnosis

We previously described a rapid PCR panel targeting bacteria associated with prosthetic joint infection (PJI) for use on biofilms dislodged from explanted hip and knee arthroplasties. Herein, we tested the PCR panel on periprosthetic tissues from 95 subjects undergoing resection knee arthroplasty, all of whom had had tissue cultures performed. A subset also had synovial fluid culture (n=89) and/or device vortexing and sonication with culture (n=58), and a subset of these PCR (n=36) of resulting sonicate fluid, performed. Of the 64 subjects with PJI, 10 and 44 had positive tissue PCR and cultures, respectively, one of whom had a positive tissue PCR with negative tissue cultures. The overall sensitivity of tissue PCR (16%) was much lower than that of tissue (69%), synovial fluid (72%) and sonicate fluid (77%) culture as well as sonicate fluid PCR (78%) (P < 0.00001, P < 0.00001, P < 0.00001, and P=0.0003, respectively). Periprosthetic tissue PCR, as performed herein, has poor sensitivity for PJI diagnosis.

Diagnosis of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry

ABSTRACT We compared PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) to culture using sonicate fluid from 431 subjects with explanted knee (n = 270) or hip (n = 161) prostheses. Of these, 152 and 279 subjects had prosthetic joint infection (PJI) and aseptic failure, respectively. The sensitivities for detecting PJI were 77.6% for PCR-ESI/MS and 69.7% for culture (P = 0.0105). The specificities were 93.5 and 99.3%, respectively (P = 0.0002).

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.

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing.

Thymidine Auxotrophic Staphylococcus aureus Small-Colony Variant Endocarditis and Left Ventricular Assist Device Infection

ABSTRACT We describe a thymidine-dependent small-colony variant of Staphylococcus aureus associated with left ventricular assist device infection and prosthetic valve and pacemaker endocarditis.