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Featured Articles
Improving the diagnosis of peri-prosthetic joint infections
Prosthetic joint infections are a devastating complication of arthroplasty. Despite this, current culture techniques lack sensitivity and not standardized. We examined the inoculation of peri-prosthetic tissue specimens into blood culture bottles, demonstrating improved sensitivity compared to conventional methods. These findings influence patient care, allowing accurate diagnosis of infection.
by Dr T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, Prof. A. C. Cheng, Dr J. N. Mandrekar and Prof. R. Patel
Background
It is estimated that over 140 000 patients in the USA underwent revision arthroplasty in 2015 [1]. At the time of revision, it is recommended that biopsies of the tissues surrounding the prosthesis be obtained to diagnose or exclude prosthetic joint infection (PJI), a potential cause of arthroplasty failure [2]. PJIs are associated with significant patient morbidity and substantial healthcare costs [1, 3, 4]. In the setting of an ageing population and increasing demand for arthroplasty, the negative impact of PJIs will continue to compound [1, 4]. Despite the recognized impact of PJIs, diagnosis remains challenging owing to two major issues: (i) the lack of a ‘gold standard’ definition for infection and (ii) current imperfect diagnostic techniques [4].
PJI diagnosis and culture conditions
The isolation of an identical microorganism (or microorganisms) from two or more aseptically obtained peri-prosthetic specimens confirms the diagnosis of PJI [3]. In addition to confirmation of infection, microbiological cultures enable antimicrobial susceptibility testing to ensure institution of appropriate, targeted antimicrobial therapy. There are limited studies examining the optimal media for culturing these peri-prosthetic tissue biopsies [4]. Conventional microbiological techniques frequently include culture on various types of aerobic and anaerobic agar plates, and inoculation into broths such as thioglycollate broth [4]. These conventional techniques, alongside various durations of incubation, are not standardized and may have a low sensitivity, as low as 39% in some published studies, particularly in the setting chronic infection [5–7]. Recent research has focused on strategies that optimize the diagnosis of PJI, including the inoculation of peri-prosthetic tissue biopsies into blood culture bottles (Fig. 1).
Assessment of culture conditions
Previous research
Three previous studies examined inoculation of peri-prosthetic tissue specimens into blood culture bottles for the diagnosis of PJI. Baker and colleagues examined inoculation of synovial fluid aspirates and tissue specimens into blood culture bottles using the Sentinel system (Difco Laboratories) automated blood culture system. There were limitations of this study’s design and methodology, including lack of a control group and lack of comparison with conventional techniques [8]. In the second study, by Hughes and colleagues, four tissue culture techniques were examined – culture on blood and chocolate agars and in Robertson’s cooked meat broth, fastidious anaerobic broth and aerobic and anaerobic blood culture bottles. The aerobic and anaerobic blood culture bottles were incubated on the BACTEC 960 platform for 5 days (BD Diagnostic Systems). The authors defined PJI on the basis of histopathological findings, with 23 subjects meeting this criterion for infection from the 141 subjects included in the cohort [9]. Blood culture bottles were more sensitive compared to direct tissue culture and fastidious anaerobic broth (87% [95% confidence intervals {CI}, 72–100%] versus 39% [95% CI, 18–61%]; P = 0.007 and 57% [95% CI, 35–78%]; P = 0.016, respectively). There was no difference in sensitivity between culture in blood culture bottles and cooked meat broth (83% [95%CI, 66–99%]; P = 0.74) [5]. These previous studies used paired-design methodologies which may lead to the miscalculation of the true sensitivity of tests [10]. In a follow up study, Minassian and colleagues examined the optimal duration of incubation for peri-prosthetic tissue samples in blood culture bottles. The majority of blood culture bottles flagged positive within 3 days of incubation. In contrast, Propionibacterium species was isolated after a median of five days incubation (range 3–13 days). Terminal sub-culture of 1000 blood culture bottles at the end of the 14-day incubation period did not isolate clinically significant microorganisms. The authors concluded that prolonged culture for 14 days was not indicated, suggesting an optimal duration of 4 days incubation based on receiver operator characteristics (ROC) analysis [11].
Current study objectives and design
We undertook a larger, prospective cohort study at the Mayo Clinic, Rochester, MN, USA over a 9-month period (August 2013 – April 2014). The study included 369 consecutive subjects undergoing revision arthroplasty, of which 71 subjects (19%) underwent upper limb revision arthroplasty (49 shoulder joint and 22 elbow joint revisions). Overall, 117 study subjects met Infectious Diseases Society of America (IDSA) criteria for PJI. Applying the definitions proposed by Tsukayama and colleagues, the majority of subjects had late chronic infections (82%), 7% had early post-operative infections and 11% had hematogenous infections [7]. The majority (49%) of infections were caused by staphylococci, including Staphylococcus aureus which was isolated in 24% of culture positive cases. In contrast, Propionibacterium acnes was isolated in 41% of shoulder arthroplasty infections.
The study compared the sensitivity and specificity of inoculation of peri-prosthetic tissue specimens into blood culture bottles with standard agar and thioglycollate broth culture. The performance of the different media was analysed using Nemar’s test of paired proportion. In addition, Bayesian latent class modelling was undertaken; this statistical method overcomes potential limitations of traditional analysis as it assumes that no gold standard exists and that the true disease prevalence is unknown, both germane to PJI [10].
Current study results
Inoculation of tissues into blood culture improved sensitivity by 47% compared to conventional agar and broth cultures applying Bayesian latent class modelling (92.1% [95% credible interval, 84.9–97.0%] versus 62.6% [95% credible interval, 51.7–72.5%], respectively); this magnitude of difference was similar when IDSA criteria were applied (60.7% [95% CI, 51.2–69.6%] versus 44.4% [95% CI, 35.3–53.9%], respectively; P = 0.003). The specificity of culture in blood culture bottles was similar to conventional media, if two or more cultures were required to be positive. In 13 subjects (11%) of the 117 subjects meeting the IDSA criteria for PJI, inoculation of peri-prosthetic tissue samples into blood culture bottles detected microorganisms not found using other culture media. However, inoculation of peri-prosthetic tissue specimens into blood culture bottles failed to identify the pathogen that was detected by conventional methods in five PJI subjects (4%).
Microorganisms were detected earlier using blood culture bottles compared to conventional media; aerobic and anaerobic blood culture bottles flagged positive at a median of 21 and 23 hours (interquartile range [IQR] 14–45 and 16–47 hours, respectively) compared to 41 hours (IQR 21–63) for aerobic agar, 62 hours (IQR 43–144) for anaerobic agar and 65 hours (IQR 43–92) for thioglycollate broth.
Discussion
Culture using blood culture bottles has several advantages: it provides a semi-automated method, with positive results automatically flagging by the instrument, potentially yielding faster time-to-positivity than conventional methods; no technologist intervention is required for negative results except for removal of the bottles at the end of the incubation period; furthermore, the technology is available and used in most microbiology laboratories. Use of blood culture bottles overcomes a current limitation of total laboratory automation, the inability to handle anaerobic cultures.
The timely detection of microorganisms in blood culture bottles compared to conventional methods also has a number of clinical advantages particularly as detection of microorganisms in blood culture bottles may be combined with direct species identification using rapid diagnostics, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or rapid nucleic acid amplification tests. Given the speed to detection of microorganisms in blood culture bottles, it is theoretically possible that the causative pathogen could be identified with resistance characterization within as few as 24 hours of revision surgery. This would facilitate optimization of antimicrobial therapy and minimize unnecessary antimicrobial use [12].
Conclusion
This study informs clinical care for patients undergoing revision surgery, demonstrating improved sensitivity for diagnosis of PJI with inoculation of peri-prosthetic tissue specimens into blood culture bottles compared to conventional culture techniques. The use of automated blood culture systems also yields faster results with the potential for pathogen identification within the first day of surgery.
References
1. Kurtz SM, Ong KL, Lau E, Bozic KJ. Impact of the economic downturn on total joint replacement demand in the United States: updated projections to 2021. J Bone Joint Surg Am. 2014; 96: 624-30.
2. International Consensus Meeting on Periprosthetic Joint Infection. Musculoskeletal Infection Society; 2013 (http://www.msis-na.org/wp-content/themes/msis-temp/pdf/ism-periprosthetic-joint-information.pdf).
3. Osmon D, Berbari E, Berendt A, Lew D, Zimmerli W, Steckelberg J, Rao N, Hanssen A, Wilson W, Infectious Diseases Society of America. Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2013; 56:e1–e25.
4. Tande AJ, Patel R. Prosthetic joint infection. Clin Microbiol Rev. 2014; 27: 302–345.
5. Hughes H, Newnham R, Athanasou N, Atkins B, Bejon P, Bowler I. Microbiological diagnosis of prosthetic joint infections: a prospective evaluation of four bacterial culture media in the routine laboratory. Clin Microbiol Infect. 2011; 17: 1528–1530.
6. Font-Vizcarra L, Garcia S, Martinez-Pastor JC, Sierra JM, Soriano A. Blood culture flasks for culturing synovial fluid in prosthetic joint infections. Clin Orthop Relat Res. 2010; 468: 2238–2243.
7. Tsukayama DT, Estrada R, Gustilo RB. Infection after total hip arthroplasty. A study of the treatment of one hundred and six infections. J Bone Joint Surg Am. 1996; 78: 512–523.
8. Baker S, Fraise AP. Use of Sentinel blood culture system for analysis of specimens from potentially infected prosthetic joints. J Clin Pathol. 1994; 47: 475–476.
9. Hughes HC, Newnham R, Athanasou N, Atkins BL, Bejon P, Bowler IC. Microbiological diagnosis of prosthetic joint infections: a prospective evaluation of four bacterial culture media in the routine laboratory. Clin Microbiol Infect. 2011; 17: 1528–1530.
10. Collins J, Huynh M. Estimation of diagnostic test accuracy without full verification: a review of latent class methods. Stat Med. 2014; 33: 4141–4169.
11. Minassian A, Newnham R, Kalimeris E, Bejon P, Atkins B, Bowler I. Use of an automated blood culture system (BD BACTEC) for diagnosis of prosthetic joint infections: easy and fast. BMC Infect Dis.2014; 14: 233.
12. Tamma PD, Tan K, Nussenblatt VR, Turnbull AE, Carroll KC, Cosgrove SE. Can matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) enhance antimicrobial stewardship efforts in the acute care setting? Infect Control Hosp Epidemiol. 2013; 34: 990–995.
The authors
Trisha N. Peel1,2 MBBS, FRACP, PHD, Brenda L. Dylla1 MT (ASCP), John G. Hughes1 MT (ASCP), David T. Lynch1 MT (ASCP), Kerryl E. Greenwood-Quaintance1 MS, Allen C. Cheng3,4 MSSB, FRACP, MPH, PhD, MBIOSTATs, Jayawant N. Mandrekar5 PhD, Robin Patel*1,6 MD
1Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
2Department of Surgery, St Vincent’s Hospital Melbourne, University of Melbourne, Melbourne, Australia
3Department of Infectious Diseases, Alfred Hospital, Melbourne, Australia
4Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia
5Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA
6Division of Infectious Diseases, Department of Medicine Mayo Clinic, Rochester, MN, USA
*Corresponding author
E-mail: Patel.robin@mayo.edu
Mass spectrometry detection of bacterial toxins and antibiotic resistance
Mass spectrometry (MS) and proteomics are gaining popularity in bacterial research and applications; notably, bacterial toxin detection and antibiotic resistance. Currently, several MS platforms exist (Fig. 1) and are described below.
by Angela Sloan, Dr Keding Cheng
Mass pattern and spectra comparison using MALDI-TOF-MS
The United States Food and Drug Administration (US FDA)-approved MALDI Biotyper or VITEK MS are both matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS)-based instruments which perform by ionizing molecules extracted from whole cell culture without specific protease treatment. The bacterial culture is normally treated with a strong solvent such as 1% trifluoroacetic acid (TFA) in 50% acetonitrile (ACN) [1] or 70% formic acid (FA) followed by 50% ACN [2] and centrifuged. Extracted molecules are mixed with chemicals (matrices) such as cyano-4-hydroxy-cinnamic acid (CHCA) and loaded onto a MALDI plate for MS detection. Sample spots are shot by laser energy and mass spectra, represented by mass to change ratios (m/z), are then obtained [1–3]. MS spectra are compared against different MS patterns/fingerprints harboured in the spectra library, and the microorganism is identified as the best match. Alternately, the bacterial culture can be smeared directly onto a MALDI plate and covered by the matrix of choice [3]. Each matching spectra result is a potential identification (ID) and given a confidence score. Generally, an ID showing a score equal to or greater than 2 is considered a correct identification [3]. A score below 1.7 is considered unreliable, from 1.7 to 1.9 indicates probable genus identification, from 2.0 to 2.29 indicates confident genus identification, and from 2.3 to 3.0 indicates highly confident species identification [4]. Culture conditions may affect spectra quality and ID [5], with pure cultures producing the most stable and consistent results [6–7]. MALDI-TOF-MS has been used in to test antibiotic resistance by growing cells in media containing normal and isotope-labelled lysine residues with and without antibiotics. The mass shift of many MS peaks, observed using bioinformatics software, demonstrated cell growth in the presence of the antibiotics, hence signifying antibiotic resistance [8].
Mass fingerprinting for molecules of interest using MALDI-TOF-MS or LC-MS
Peptide mass fingerprinting (PMF) is another form of mass fingerprinting of peptides, often obtained after protease treatment. Experimental mass spectra are compared to theoretical mass spectra using protease digestion patterns, which subsequently allows the protein/molecule of interest to be identified [9]. Proteins are typically prepared using an enrichment process such as gel electrophoresis or chromatography, and enzymatically digested either within the gel or in solution. During in-gel digestion, trypsin penetrates dried gel pieces and digests the protein within. The resulting tryptic peptides are then easily extracted from the gel, vacuum-dried, and analysed by MS. When loaded onto a MALDI plate, samples will be covered with a matrix such as CHCA, for downstream MS analysis [10]. Bacterial extracts for routine MALDI-TOF-MS testing can also be digested, fractionated through chromatography, and loaded onto a MALDI plate to reduce sample complexity on each MALDI spot. Further fragmentation of the selected masses can be performed to obtain peptide sequences and subspecies level identification [11].
MALDI-TOF-MS is now able to detect carbapenemase or β-lactamase activities, indicators of antibiotic hydrolysis, by incubating bacteria from positive blood culture with the antibiotic. If the substrate is hydrolysed and shows a mass shift in MS detection, and antibiotic resistance can be inferred [12–13]. Liquid chromatography-mass spectrometry (LC-MS) has also been used for antibiotic susceptibility testing and metabolite profiling. For example, ampicillin (m/z 350 Da) can be hydrolysed into ampicillin-penicilloic acid (m/z 368 Da) and penilloic acid (m/z 324 Da) in ampicillin-resistant cells, resulting in mass changes easily detected by LC-MS [14].
Kalb et al. and Wang et al. used an Endopep-MS (a MALDI-TOF-MS-based method), to analyse the botulinum toxin [15–16], a highly substrate specific endopeptidase. The Endopep-MS method is based on the masses of cleavage products, which will be unique according to which toxin is present. The authors focused on the light chain of the toxin, providing it with a substrate in vitro that mimics the substrate in vivo. The presence/absence of the toxins was detected with 100% accuracy, and the analysis was not disturbed by the complex sample matrices analysed, such as meat and milk.
Targeted LC-MS/MS for protein identification and quantitation
Targeted protein identification and quantitation can be performed by multiple reaction monitoring (MRM) through a quadrupole MS system [17]. Ions (charged peptides) of interest can be selected from one quadrupole field and fragmented in the next. Peptide sequences are obtained and fragmented ions used for quantitation using stable isotope-labelled standard peptides as identification and quantitation references. Ion selection and fragmentation (MRM transition) is very fast and hundreds of targeted proteins can be identified and quantified in a 1-hour LC-MS/MS run. Either protein standards or peptide standards can be synthesized and used [18–19]. The beauty of using protein standards is that they can be spiked into the test sample, and quantitation of the protein performed accurately since the standards go through the sample preparation process [18]. MRM is highly specific, sensitive, accurate and reproducible. Rees et al. used the unique affinity of organisms for their host, such as bacteriophages for certain bacteria, and analysed phage amplification products through sequence-based LC-MS/MRM to deduce antibiotic resistance properties of host cell [20]. MRM-based quantitation can also be multiplexed to quantitate many molecules of interest per sample run, rendering the process desirable for clinical applications [19]. A recent publication by Charretier et al. showed that with Staphylococcus aureus as a model, bacteria identification, antibiotic resistance, virulence and type profiling could all be obtained using one MRM platform [21].
Moura et al. used two methods of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect the presence of Clostridium difficile toxins in cell culture filtrate: ultra-performance liquid chromatography-tandem mass spec (UPLC-MS/MS) and data independent UPLC-MS/MS [22]. Notably, the label-free data independent method could perform protein identification and quantitation in one MS experiment using alternating high and low energy. UPLC-MS/MS confirmed that digestion with trypsin was efficient and robust with sufficient amino acid coverage to identify the two main C. difficile toxins, TcdA and TcdB. They also showed that the most efficient combination of enzymes for differentiation of the two toxins was trypsin and GluC, providing amino acid coverage of 91% for TcdA and 95% for TcdB. The data independent method quantified toxins at low levels and identified them separately. This is a novel development, as conventional methods typically quantify the total amount of toxin present. The method detected TcdA at 5 ng (1.6 μg/ml) and TcdB at 1.25 ng (0.43 μg/ml). Cell culture filtrate appears to be a novel approach for detection of C. difficile toxins, creating potential for future study.
Staphylococci are Gram-positive bacteria that cause pus-forming infections, food poisoning, and are the major cause of wound infections, nosocomial acquired pneumonia and septicemia [23–26]. Currently different results were obtained for Staphylococci species identification by MS approaches [23–25, 27]. Even so, evidence showed that the emergence of multiple Staphylococcus strains resistant to prescribed antibiotics could be detected by targeted LC-MS/MS by incubating bacteriophages with the bacteria [20]. In addition, Hennekinne et al. used isotope-labelled protein standards spiked into food extracts to identify and quantitate enterotoxins during a S. aureus food-poisoning outbreak using the LC-MS/MS platform. Bacterial toxins were enriched by the immune-affinity method and run on SDS-PAGE. In-gel digestion was performed and the toxin peptides were identified and quantitated based on quantitation standards [26].
Advantages of using MS platforms for bacterial toxin and antibiotic resistance tests
One of the most widely noted benefits of MALDI-TOF-MS is the ability of this platform to produce incredibly fast results, in some cases just a few minutes after loading the sample. MALDI-TOF-MS regularly uses fewer reagents and fewer steps, and requires less information on the organism than methods such as PCR or biochemical testing. Overall analysis is also cheaper since it occupies fewer working hours than traditional methods (the cost per MALDI-TOF-MS sample is reported to be approximately $0.50 to $1.00 [28]. Such rapid analyses would be of great benefit to epidemiological studies and clinical diagnoses where time is of the essence and faster identification will ultimately benefit patients [29].
Conclusion and perspective
An appealing aspect of MS is that any molecule that can be ionized should be detectable by MS with high sensitivity and resolving power. Current studies have confirmed that MS can analyse any cultivatable organism and its related metabolites without much prior knowledge, and it has been shown to be useful in a variety of applications, from fast hospital diagnosis and identification of typical bacteria, to organisms that are difficult to culture. With the gaining popularity of MS instrumentation, growing detectability, and user-friendly hardware and software, MS will certainly play a more substantial role in solving critical problems such as antibiotic resistance and the identification of toxins.
References
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11. Gekenidis MT, Studer P, Wuthrich S, Brunisholz R, Drissner D. Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies. Appl Environ Microbiol. 2014; 80: 4234–4241.
12. Jung JS, Popp C, Sparbier K, Lange C, Kostrzewa M, Schubert S. Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid detection of beta-lactam resistance in Enterobacteriaceae derived from blood cultures. J Clin Microbiol. 2014; 52: 924–930.
13. Carvalhaes CG, Cayô R, Visconde MF, Barone T, Frigatto EA, Okamoto D, Assis DM, Juliano L, Machado AM, Gales AC. Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision. J Antimicrob Chemother. 2014; 69: 2132–2136.
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16. Wang D, Krilich J, Baudys J, Barr JR, Kalb SR. Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates. Bioorg Med Chem. 2015; 23: 3667–3673.
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The authors
Angela Sloan*1 MSc, Keding Cheng1,2 MD
1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada
2Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 0J9, Canada
*Corresponding author
E-mail: angela.sloan@phac-aspc.gc.ca