Infection following treatment of a fracture is a burden for patients, treating physicians, and health care economies.1 The “gold-standard” for diagnosis of an implant-associated musculoskeletal infection is often considered to be the isolation and identification of a pathogen from an appropriately obtained culture.2,3 This review will examine recent changes in, or the development of, clinical criteria, sonication, biomarkers, detection of host immune response, histopathology, and molecular methods for detecting bacterial presence to aide in the diagnosis of infection in patients that have undergone fixation of fractures. There are a host of terms, including “surgical site infection,” that have been used to describe this clinical entity, but in this manuscript we will refer to this infection after fracture fixation as a “fracture-related infection” (FRI).4
FRI is obvious when it is obvious. This is echoed by often-used systems for infection surveillance/diagnosis. For example, the second International Consensus criteria (Fig. 1) for defining periprosthetic joint infection (PJI) has as one of the major criteria the presence of a sinus tract communicating with the joint.5 The Centers for Disease Control and Prevention criteria for deep organ space infection references “purulent drainage” and “deep incision that spontaneously dehisces, or is deliberately opened or aspirated.”6 However, clinicians recognize that the diagnosis of infection is not always obvious, and this explains why there are minor criteria associated with these systems that incorporate imaging tests, laboratory values, and cultures.
In the setting of Orthopedic trauma, development of infection after fracture surgery has not been rigorously defined. A recent study looked at 100 randomized controlled trials in fracture surgery, noting only 2% used a validated definition in the methodology of ascertainment of infection, and only 30% provided a description of what constituted an infection for that study.7 The need for a standardized definition of FRI, similar to what has been done for joint arthroplasty, was recently attempted to be addressed through an international expert group generating a consensus definition (Fig. 2).4 The authors of the definition recognize the need for validation studies of this newly proposed definition. As validation commences with the expected modifications, an accepted definition for FRI that does not necessarily rely on culture results is a welcome method to aide in the diagnosis of implant-associated infection after fracture surgery.
BIOFILMS: A CONTRIBUTOR TO THE DIFFICULTY IN DIAGNOSING FRI
Even in the setting of “obvious” FRI, there is reported 7% to 9% of “culture-negative” infections (false negatives).8–10 At least part of the discordance between clinical infection and microbiological “proof” of infection is related to the shortcomings of microbiological culture as the conventional diagnostic tool used in hospitals. It has been increasingly recognized that infections resulting from bacterial biofilms are not readily diagnosed because of limitations of the culture method and physiological adaptations of bacteria during the biofilm mode of growth. The culture-based methods used today were developed in the late 19th century11 and have largely been replaced throughout microbiology, except in medical microbiology.12
Biofilms are a sessile community of microorganisms attached to biotic or abiotic surfaces and each other that are encapsulated in an extracellular polymeric matrix of polysaccharide, nucleic acids, and proteins.13 Biofilm microbes exhibited different gene transcription and growth rates compared planktonic counterparts.14 Within the biofilm, bacteria and/or fungi are exposed to gradients of nutrients, oxygen, metabolic wastes, and bacterial signaling compounds; microbe adapt to these local microenvironments exhibiting differential gene expression and protein production throughout the community.15,16 Unlike planktonic bacteria, bacteria in the biofilm mode of growth are recalcitrant to clearance by the host immune response and antimicrobial therapies (Fig. 3).17 Biofilm bacteria exhibit 100 to 1000 times more tolerance to antibiotics than planktonic bacteria.18 Mechanisms contributing to antibiotic tolerance include reduced antibiotic penetration because of the biofilm matrix or polysaccharides, antibiotic-induced expression of efflux pumps in the biofilm phenotype, inhibited oxidative response, and the presence of metabolically down-regulated or inactive cells.19,20 Metabolically dormant bacteria within the biofilm are viable but nonculturable (VBNC) bacteria.
Efforts to improve the sensitivity of microbial culture have used sonication of removed hardware and culture of the sonication fluid.21 Despite improved detection of bacterial pathogens following sonication, the membranes of gram-negative bacteria lack rigidity and are susceptible to ultrasound disruption. Optimal recovery of viable bacteria requires strict adherence to sonication conditions, temperature, and time.22,23 This simple and low-cost technique increases the percent of positive cultures, but false-negative results may persist because of improper culture conditions (incorrect culture media or incubation period) or antibiotic therapies before revision surgery.24,25 The VBNC state is 1 phenotype contributing to false-negative results. VBNC bacteria fail to grow on bacterial culture media under the routine conditions used for a particular species, but bacterial viability is verified by reverse transcriptase polymerase chain reaction(PCR) to identify bacterial messenger RNA.26,27 Further, sonication may increase the false-positive rate by identifying bacteria that have colonized the implant, but are not responsible for clinical infection. Specifically dealing with sonication in FRI, a recent systematic review concluded that there is no strong evidence to favor sonication at this time.3 This is largely because of heterogeneity of the studies reviewed, low power, and lack of standardized processes and reference criteria by which to evaluate the effectiveness of sonication as a diagnostic modality compared with conventional tissue culture. A similar conclusion was reached in a primary paper where the author’s found no benefit of sonication over routine tissue culture in patients with obvious clinical infection or no culture in clinically “aseptic” nonunions.28
False-positive results from culture are also a possibility. Contamination, or the introduction of microorganisms to sterile locations,29 would reflect the true false-positive rate; the culture is positive, but it is not from bacteria obtained from the patient (eg, environmental contamination from air, surface contact, laboratory personnel, etc.). There is also the possibility of bacteria being obtained from the patient, but not being reflective of a clinical disease entity. This is exemplified by the human microbiome, or collection of all the microorganisms living in association with the human body, and codified by the question “What is the definition of implant ‘colonization’ versus implant-related infection?” addressed at the Proceedings of the Second International Consensus Meeting on Musculoskeletal Infection. The voted on answer as it applied to joint replacement surgery was “Colonization is the presence of microbiota in a joint, with growth and multiplication of the organism, but without interaction between the host’s immune response, thus avoiding any clinical expression. Infection is the invasion of a joint by disease-causing organisms that result in an interplay with the host’s immune response causing a clinical expression and disease state.”30 Interestingly, published data suggest closed fractures could be colonized.31,32
Clinical infection is a phenotypical expression of the host’s response to pathogenic bacteria. This represents a true positive and is the final possible positive culture result. Measuring the body’s response to an infection through various methods captures a large proportion of the literature on non–culture-based methods to aide in the diagnosis of implant-associated infection after fracture surgery.
HOST RESPONSE TO INFECTION
The FDA BEST Resource on biomarkers defines a biomarker as “a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions.”33 Seven classes of biomarkers are recognized, one of which is a diagnostic biomarker defined as “a biomarker used to detect or confirm the presence of a disease or condition of interest or to identify individuals with a subtype of the disease.” The ideal diagnostic biomarker has high sensitivity and specificity compared with a “gold-standard” reference of the disease process and has been validated against the intent to diagnose population using reproducible processes. Unfortunately, there is no truly applicable biomarker for FRI to date.
Among the suggestive criteria for FRI diagnosis is elevated levels of the conventional inflammatory serologic markers: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and white blood cell count (WBC).4 The screening tests for these markers are low-risk, routinely accessible, and highly sensitive, but other underlying conditions or injuries that generate an inflammatory response can diminish test specificity for infection. Another consideration is the postoperative elevation of ESR for 6 weeks and CRP for 2 weeks reported for PJI.34 Several studies of CRP kinetics following fracture surgery report a similar drop to normal CRP levels at 2 weeks postsurgery.35–37 Diagnostic performance of these tests for PJI diagnosis has substantial clinical evidence, but the diagnostic sensitivity and specificity has not been established in large-scale studies of FRI patients.38,39 Studies of PJI patients report sensitivity and specificity for ESR at 91% and 72% and for CRP at 94% and 74%.40,41 In studies examining infectious complications of long bone fractures, sensitivity and specificity for CRP was 60% and 86% in nonunion patients, whereas 82% and 18% in posttraumatic osteomyelitis patients.42,43 Two studies of patients with nonunions comparing positive intraoperative culture to WBC, ESR, and CRP have shown that accuracy of these serum measures increases as the number of them that are positive increases.43,44 Both studies showed 100% predicted probability of infection when 3 of the tests were positive. Wang et al43 also included serum interleukin-6 (IL-6) measurements. Though IL-6 has been associated with systemic inflammation following polytrauma, there is limited literature relating IL-6 to FRI.43,45,46 In a recent meta-analysis regarding serum inflammatory markers in diagnosing FRI compared with intraoperative cultures as the reference test, ESR pooled sensitivity and specificity were 45.1% and 79.3%, respectively, and WBC pooled sensitivity and specificity were 51.7% and 67.1%, respectively.47 The authors concluded that elevated WBC, ESR, and CRP are insufficient as confirmatory or rule out criteria to diagnose late FRI. With respect to early deep infection after fracture surgery diagnosed by “microbiological tests,” the time course of CRP preoperatively, on postoperative day 2, and after the fourth postoperative day may be useful.35 Serum procalcitonin has been investigated for diagnosis of non–implant-associated septic arthritis and acute osteomyelitis.48
Orthopedic infections are also diagnosed by screening for proteins or biomarkers locally produced in response to host detection of the microbial pathogen. Biomarkers commonly used in PJI diagnosis include the antimicrobial peptide α defensin and leukocyte esterase (LE) produced by neutrophil granulocytes in the sampling site. Synovial fluid is analyzed for these biomarkers in cases of suspected PJI; therefore, specimen availability in suspected FRI may limit the biomarkers’ utility in diagnosis. The synovial fluid α defensin assay has >95% sensitivity and specificity for diagnosing PJI.49–52 Of note, elevated α defensin levels in plasma have been observed during sepsis, but systemic levels may be elevated in patients with other conditions, for example, HIV infection and cancer.53–55 Overall, specimens from the infection site generate the best direct correlation between α defensin levels and infection, for example, elevated α defensin reported in the gastric juice of Heliobacter pylori patients or in the bronchoalveolar lavage fluid of tuberculosis patients.56,57 In comparison, the LE test has sensitivity and specificity of 81% and 100% in diagnosing PJI using synovial fluid samples.58 This rapid diagnostic method takes 2 to 3 minutes using a LE strip test, which was developed to screen urine and diagnose urinary tract infections.58,59 Glucose reagent and LE strips have also been used in combination to diagnose native septic arthritis. Compared with defined criteria for septic arthritis, Omar et al60 showed ≥90% sensitivity, specificity, positive predictive value (PPV), and negative predictive value. In this setting, glucose concentrations are decreased because of bacterial metabolism.
Other biomarkers have also been investigated for diagnosing PJI. In 1 study, 5 proteins from a panel of 46 biomarkers were tested in synovial fluid samples and compared with standard diagnostic criteria for PJI yielding receiver operating characteristic curves with and area under the curve >0.9.61 These proteins were IL-6, IL-8, α2-macroglobulin, vascular endothelial growth factor, and CRP. To the authors’ knowledge, there are no high-quality studies regarding the use of local biomarkers to diagnose FRI, posing an opportunity for ample future research. Furthermore, the opportunity to measure multiple biomarkers at multiple time points lends itself to dynamic network analysis. This has been used successfully to predict nosocomial infection in patients that have sustained blunt trauma.62
Host-specific Immune System Response to Bacterial Presence
In the setting of clinical infection in an immune-competent host, there will be an interaction between the host’s immune system and pathogen. This is also true in the setting of biofilms. As a specific example, Staphylococcus aureus chronic osteomyelitis biofilms generate specific proteins that result in antibody production by the host.63 It has been shown that at least one of these antibodies may be used to diagnose PJI.64 Although there are no published studies related to FRI, an unpublished study65 showed a 0.81 area under the receiver operating characteristic curve for presence of the Staphylococcus antigen SACOL0688 (a putative ATP-binding cassette transporter) compared with positive S. aureus culture obtained at the time of surgery for infection. This was a prospective study of 185 patients surgically treated for open fractures, or fractures of the tibial plateau, tibial pilon, or foot. Examples of using antibodies as biomarkers for diagnosis are ubiquitous in medicine (eg, viral hepatitides) and represent an exciting opportunity for point of care screening and potential for early diagnosis. In addition to validation studies, future work will need to elucidate parameters regarding whether a positive test represents a history of exposure versus active disease.
NON–CULTURE-BASED IDENTIFICATION OF BACTERIAL PRESENCE AND SPECIATION
Histopathology is another method used to diagnose FRI. Nearly 2 decades ago Simpson and colleagues showed that histologic assessment of nonunion tissue has an accuracy of 91% compared with clinical and microbiological diagnoses. Further, in 1 quarter of cases where the diagnosis could not be established by clinical and microbiological methods, histology was able to make a diagnosis of infection.66 In a series of 95 nonunions, a negative gram stain was 100% specific.44 A positive gram stain is a confirmatory criteria in the recent consensus definition of FRI,4 but Gram stain has been shown to have only 25% sensitivity.44 Finally, a recent paper compared quantitative histology with the consensus definition on FRI in a cohort of 156 nonunions.67 The authors showed that >5 neutrophils per high power field had 100% PPV for infected nonunion, whereas no neutrophils per high power field had a 98% PPV of no infection (aseptic nonunion). This criteria has been added to the confirmatory list of the consensus definition for FRI in fractures >4 weeks old.68
Nucleic Acid-based Tests
Although the host response to infection may be harnessed to diagnose infection, the response does not identify the bacteria that is present, with the exception of the possibility of detecting specific antibodies. Pathogen identification has historically been the role of culture for speciation and antibiotic susceptibility testing. However, there is a growing body of literature on the use of nucleic acid testing for infectious disease.69 This technology can both detect the presence and identify bacteria by extraction of DNA or RNA from the sample being tested.12 Bacteria contain DNA sequences not found in the human genome [eg, 16S ribosomal RNA (rRNA) subunit]. Further, specifics of the genome can be used to distinguish the species of bacteria.70 Advances in molecular biology techniques have the potential to overcome limitations of culture for diagnosis.
Although microbiological culture remains the “gold-standard” for diagnosis of orthopedic infections, molecular diagnostic techniques offer higher sensitivity with the ability to target difficult to culture or low abundance etiologic agents. It may be particularly useful in settings of “culture-negative” infections.70,71 Biopsy or aspirate specimens for obtaining DNA from pathogenic microorganisms by PCR is one alternative. Multiplex PCR analysis uses multiple species-specific primers to identify the pathogenic bacteria or antibiotic resistance genes, and this method has been evaluated in patients with infections after fracture fixation.72 Limitations of this standard method include restricted identification because of the species-specific primer sets used in the amplification reactions. In contrast, 16S rRNA gene sequencing identifies more bacterial species by using universal primers to amplify the hypervariable region of the 16S rRNA gene, which is sequenced and queried against rRNA gene sequence database.73 Despite improved bacterial identification with this approach, false-positive results from detection of bacteria in very low numbers remain a concern. The clinical relevance of bacteria that exist in very low numbers remains unknown. In addition, genomic material from dead bacteria remains an issue. Extraction of bacterial rRNA from the specimen and generation of complementary DNA by reverse transcriptase PCR overcomes this issue, thereby targeting metabolically active bacteria and reducing the number of false positives.74,75 The drawback of the 16S typing technique is that identification of the etiologic agent requires DNA sequencing, which increases the technical difficulty and requires DNA sequencers in the clinical laboratory. 16S sequencing has detected bacterial presence at the time of surgery in closed fractures31 and has been used to characterize the microbiome colonizing open fractures.76 In the closed fracture study,31 there was no follow-up to infection. In the open fracture study of 30 patients,76 3 patients ultimately had cultures taken, and the cultures did match 16S data.
IBIS is another broad-range PCR method targeting 16S and 18S rRNA for the identification of bacteria and fungi, respectively. Antimicrobial resistance markers to determine antibiotic sensitivity can also be tested. Instead of sequencing the amplicons, this technique utilizes electrospray ionization mass spectrometry to determine the mass of individual amplicons within a complex mixture. Nucleotide composition is derived from the mass and then compared with a database of nucleotide compositions for known microorganisms. IBIS technology offers high sensitivity with the ability to detect any microorganism in a specimen at >1% of the total microbial population and has a searchable database of >3000 microorganisms.77 IBIS identification of etiologic agents is a rapid process with results in <6 hours, but not likely to result during standard duration surgical procedures for infection. One obstacle to the widespread clinical use of the IBIS T5000 biosensor is the associated high cost from the mass spectrometry unit. Few medical institutions have invested in this technology for their clinical laboratory. IBIS has shown utility in identifying bacteria in synovial fluid and tissue specimens from patients with aseptic loosening and tissue biopsies from long bone nonunions.78–81 With respect to PJI, in one study 5/7 (71%) of patients undergoing index total knee arthroplasty were positive using IBIS, but only 1 of these patients progressed to revision surgery for infection, and the bacteria that was identified at revision surgery were different than that at index surgery.82 Another study compared culture positivity with positive results from molecular methods for a cohort of patients undergoing long bone fracture nonunion surgery. Detection of bacteria with IBIS was positive at a rate 3.7 times that of culture; 24% positive cultures and 88% positive specimens on the basis of molecular diagnosis.83 This finding has led some to question whether the results of intraoperative cultures obtained during nonunion surgery should be trusted, particularly ones that are negative, as culture may not be efficient at picking up “occult” or “subclinical” infection. Diagnosing infection at the time of nonunion operation is important, as not doing may increase the risk of failed treatment.84
Visualization of the etiologic agent using microscopy imaging has also shown efficacy in the reclassification of culture-negative biopsies. One method to detect metabolically active bacteria is fluorescent in situ hybridization (FISH) that uses fluorescently labeled oligonucleotide probes to target bacterial 16S rRNA sequence. 16S rRNA is an efficient target because of high copy number, stability, and domain composition with both conserved and variable regions.85 FISH analysis can be performed with universal probes to target many eubacteria or specific probes to target a specific genus or species in the specimen.86 This has been recently demonstrated by Lippmann et al,87 who showed 95% sensitivity with FISH compared with both clinical and histopathologic classifications. Further, using species-specific probes, they identified all cases of Pseudomonas aeruginosa and S. aureus that were positive by microbiological culture. FISH can differentiate between sample contaminations and implant infection by visualizing the adhered microbes. Confocal microscopy of FISH-labeled implants has demonstrated bacteria on a prosthetic joint in aseptic loosening case after culture-negative result.78 This method has also confirmed infections and identified bacteria in previously culture-negative cases of nonunions.81,83 Though able to detect and identify the bacteria present,12,88 an issue with molecular diagnostic and microscopy methods is the inability to diagnosis the infection before biopsy and/or surgical intervention.
Though nucleic acid testing seems to be more sensitive than culture for detecting bacteria, it may depend on how the samples are obtained. One study has shown that taking 3 cultures from the implant-tissue interface had a significantly higher area under the receiver operating characteristic curve for diagnosing infection compared with PCR swab of the implant surface.89 Although there has been 1 large clinical study using molecular diagnostics in the setting of culture-negative infection,70 the study population was heterogenous (only 47% of the patients were FRIs, which included spine procedures). The remainder of studies have had relatively low numbers of patients, making them underpowered to define the role of these methods in diagnosing FRI. Next-generation sequencing is another method for detecting bacterial nucleic acids in specimens. The authors are not aware of any study on FRI that has used next-generation sequencing. In the setting of PJI, next-generation sequencing may perform better than IBIS in terms of false positives compared with the reference Musculoskeletal Infection Society criteria.90 With respect to FRI, all nucleic acid-based tests are translational, and further study is necessary to elucidate their role in clinical medicine.
Postoperatively a fracture patient’s course exists on a continuum of fracture healing and the possibility of developing clinically relevant infection (Fig. 4). The interplay of the biological processes of fracture healing and host response to infection are complex.91 Although we have some clinical names for the different states a patient may exist in, and these clinical states have relatively “tried and true” treatment strategies, a key element is accurate and timely diagnosis of clinical infection when necessary. It is imperative to remember that clinical infection depends not only on detection of bacteria, but also requires a host response. This will help avoid over treatment of false-positive cases because of contamination and colonization. Despite clinical criteria, scoring systems,92 biomarkers, and molecular diagnostics, the “gold-standard” for microbiological diagnosis of infection at present is still considered to be isolation/identification of a pathogen from an appropriately acquired tissue biopsy. For FRIs, the tissue sample is commonly obtained during surgery for infection at the time of debridement, though it would be clearly beneficial to have tests to detect early infection, with the goal being different treatment paradigms to avoid and/or limit the magnitude of surgical intervention. Data show that molecular diagnostic tests routinely identify bacteria with a higher sensitivity than traditional culture methods.31,82,83
Specific diagnostic gaps stemming from reliance on microbiological culture include the speed of diagnosis, quantitative capability, and false-negative results. These may be overcome in the future by leveraging unique aspects of molecular methods to allow point of care testing (either in clinic or in the operating room), quantitative evaluation of bacterial presence, and not depending on whether a pathogen is able to be cultured with traditional methods. However, there is minimal data at this time showing the clinical relevance of this improved “sensitivity” given the lack of validation against the clinical phenotype of FRI. On the basis of the success seen in PJI research, there is no doubt that these emerging technologies will improve the diagnosis of implant-associated infection after fracture surgery. Continued study is warranted, with a focus on well-designed clinical trials with control groups, described and reproducible processes, and comparison with defined/standardized clinically identifiable phenotypes.
The authors thank Krista Brown for editing and formatting of manuscript.
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