1 Detection of Microbes in Orthopaedic Infections

Michael Henry, Andy O. Miller, and Barry D. Brause

A very broad range of microorganisms cause orthopaedic infections. Modern diagnosis depends on traditional culture techniques, which remain in common use, and on molecular testing, which is advancing rapidly as a field. Advances in culture-based techniques include modifications in specimen collection, incubation, and identification. Identification of pathogens through detection and analysis of microbial nucleic acids, without culturing the organism, is the focus of molecular microbiologic diagnostics. A variety of polymerase chain reaction (PCR) tests can identify single or multiple pathogens in a single PCR reaction. 16S PCR uses conserved DNA sequences to identify a very broad array of pathogens. Newer techniques (next-generation sequencing) avoid the limitations of PCR and can detect an even broader, theoretically unlimited range of pathogens by sequencing all of the nucleic acids in entire samples. The place for these technologies in orthopaedics is evolving. While anecdotal reports and some studies show molecular diagnostics’ advantages over culture, traditional cultures still remain the most accessible, affordable, and reliable in most clinical scenarios. However, further improvements are likely to alter the landscape of microbial diagnosis of orthopaedic infections.

Osteomyelitis – prosthetic joint infection – bacteria – microbiology – biofilm – PCR – next-generation sequencing

1 Detection of Microbes in Orthopaedic Infections

Practical Tips

When obtaining cultures, one should take specimens of deep tissue and fluid prior to antibiotic administration; swabs and samples of draining sinuses or postoperative wounds have low culture yield.
It is ideal to obtain three to five cultures at a time using separate surgical instruments.
Samples should be transported in blood-culture bottles and enriched media to the lab in under 2 hours, and these cultures should be grown on both solid and liquid media culture. Gram stains are not recommended.
The optimal incubation period for anaerobic cultures is 14 days to increase culture yield.
Molecular techniques that improve organism identification include polymerase chain reaction (PCR) to identify single or multiple pathogens or 16S conserved DNA sequences, or next-generation sequencing to detect an even broader range of pathogens.

1.1 Introduction

There is a broad range of microorganisms that cause orthopaedic infections. Many microbiologic diagnostic techniques are available to identify these pathogens. Pathogen identification has traditionally been performed with standardized laboratory culture and biochemical analytic techniques, many of which have been in use for over a century. The increasing sophistication and availability of molecular microbiologic techniques have the potential to transform the way organisms are identified. They hold promise in augmenting the sensitivity of traditional techniques, shortening the time required to identify an organism, and broadening the spectrum of pathogens to include those that have been difficult to isolate in culture. Molecular technology remains less widely available, more expensive, and sometimes more difficult to interpret. In addition, many of these tests are laboratory-derived single-center assays, and lack of standardization can lead to varying accuracy between the performing laboratories.

Traditional culture-based techniques remain the backbone of orthopaedic infection diagnosis. Much scholarship has gone into improving and streamlining these well-established methods. Active areas of study to maximize the sensitivity of these tests without sacrificing specificity have included: specimen acquisition, specimen number, biofilm culture methods, incubation techniques, improvements in culture media, and duration of incubation.

1.2 Culture-Based Microbiology

Orthopaedic infections can develop in native bone or synovium, or can involve orthopaedic hardware, tissue grafts, or other foreign bodies. The most commonly encountered orthopaedic infections are osteomyelitis and septic arthritis. As with infections at other sites in the body, the specific organisms one expects to encounter in each patient is dictated by many host factors. Being able to anticipate which organisms to expect allows the clinician to better provide an optimal approach to the microbiology workup and to understand the limitations of each technique. The overwhelming majority of orthopaedic infections develop via hematogenous spread, via extension to bone from a contiguous site or via direct inoculation in the setting of trauma or surgery. The range of potential pathogens varies greatly as a result of a number of host and environmental factors. Differences in age, immune status, as well as an array of comorbidities, such as diabetes, peripheral vascular disease, and hemoglobinopathies, can all inform which organisms are more likely to be encountered. The most salient variable dictating which organisms will be the cause of infection is the presence or absence of orthopaedic hardware or other foreign material. The presence of orthopaedic hardware creates an area of focal immunodeficiency, as immune effectors such as leukocytes and antibody are often unable to function in close proximity to foreign surfaces. In addition, orthopaedic hardware, which often has large surface areas, permits the development of chronic bacterial biofilms. This allows many generally nonpathogenic organisms to cause infection.

Recent guidelines published by the Infectious Diseases Society of America (IDSA) and American Society for Microbiology (ASM) outline the optimal approach to obtaining and processing tissue specimens for culture, including bone and joint tissue. ¹ Regardless of the type of infection, the use of swabs to obtain specimens is strongly discouraged in almost all situations. ² ^, ³ ^, ⁴ Swabs hold an extremely small volume of specimen and are prone to picking up extraneous organisms. The winding fibers that make up the bulb also entrap organisms, preventing efficient release when the swab is used to inoculate liquid or solid media. ⁵ This further reduces an already limited yield. Draining sinus tracts or postoperative wounds is an inviting target for swab cultures, but repeated demonstrations have shown the inaccuracy of superficial cultures for delineating the pathogens in deep infection. ⁶ ^, ⁷ ^, ⁸ Instead, cultures of deep tissue and fluids from the site of the infection are the most valuable specimens to submit for culture to more readily establish the microbiological diagnosis.

The IDSA/ASM guidelines also recommend that specimens be acquired prior to the administration of antibiotic. Once a specimen is collected it should be kept at room temperature and transported to the lab in under 2 hours. Extended transport time decreases the population of viable organisms, which can delay or prevent their recovery in the microbiology lab. ⁹ Once the specimen arrives in the lab, there are no widely accepted standards for the microbiologic workup for orthopaedic infections. ¹⁰ In general, the basic protocols for culturing bone and prosthetic hardware once the specimen arrives in the microbiology lab are modeled on the techniques and protocols that have been refined over decades to process blood cultures. Direct examination can be performed, typically a Gram stain. If the pathogen is present in sufficient quantity, Gram staining can provide immediate visual detection of a wide array, but not all, organisms that typically cause orthopaedic infection. However, Gram staining rarely yields a pathogen in nonpurulent orthopaedic infections, and many institutions no longer recommend its routine use in this setting. Clinical specimens are then processed and inoculated onto solid agar media and into liquid media (broth), followed by incubation for aerobic and anaerobic bacteria (and also mycobacteria and fungi if desired). Often several different media are employed, enriched with nutrients or otherwise modified to identify a specific type or range of microorganisms. When microbial growth is noted in the initial cultures, it undergoes further testing to identify the organism and its antimicrobial susceptibility profile. This may be done through manual or automated methods, via the analysis of a wide variety of the characteristics of the organism including growth characteristics, morphology, and biochemical and metabolic characteristics. Antimicrobial sensitivity is performed with disk diffusion or dilution methods. Much of this analysis is now automated.

In addition to being plated onto solid media, liquid media culture is typically performed as well. These cultures frequently include thioglycolate or similar solutions and are designed to support anaerobic bacterial growth. Liquid media is also able to support the recovery of smaller quantities of inoculated microorganism and may be more sensitive than solid media. The use of more sensitive media comes at the expense of an increased rate of isolating contaminants. Detected growth in liquid media is plated onto solid media (sub-cultured) before further analysis of the isolate can be completed.

Because longer incubation duration increases the isolation rate of nonspecific contaminants, the standard incubation time for blood cultures is 5 days; the incubation period for bacteria in tissue cultures and body fluid is variable from lab to lab but is usually between 2 and 5 days. ¹¹ Some microbiology labs, both academic and commercial, incubate tissue (including bone and synovial fluid) culture for only 48 to 72 hours. As discussed below, the optimal incubation duration can greatly extend beyond 5 days, depending on the organism and clinical scenario.

1.2.1 Limitations of Culture-Based Microbiology

The majority of the bacteria that routinely cause orthopaedic infection can be grown in culture using standard media. However, recovering these bacteria in the setting of an orthopaedic infection can be a frustrating experience even in an experienced microbiology lab. For example, the cultures from 10 to 50% of orthopaedic prosthesis infections, with results varying on the population under study, fail to recover any organisms. ¹² The concordance between preoperative aspirate cultures and intraoperative tissue culture in chronic prosthetic joint infection (PJI) has been reported at 60%. ¹³ Several characteristics inherent to orthopaedic infections reduce the efficacy of traditional culture techniques. The most important factor may be the presence of biofilm.

1.2.2 Detection of Microbes in Biofilm

Traditional culture techniques are optimized for recovering bacteria in their active growth phase (planktonic growth). However, many orthopaedic infections, particularly those that are chronic or associated with hardware, persist due to the presence of a biofilm. The formation of biofilm is induced by specific conditions hostile to planktonic growth, and marked by significant changes in gene expression, allowing the microorganisms to attach to solid, preferentially inert, surfaces or dead tissue, forming microcolonies. ¹⁴ As the biofilm matures, bacteria secrete a complex mixture of polysaccharides, DNA, and protein, ¹⁵ allowing the microcolonies to aggregate, to become enmeshed in a complex extracellular matrix, and to develop into complex and functionally heterogenous communities. This increases the ability of the colony to survive regardless of the type of metabolic stress encountered. The extracellular matrix (aka slime, glycocalyx) resists the effects of antibodies, oxidative stress, host immune cells, and many chemical and enzymatic detergents, ¹⁶ and provides a structural framework within which bacteria can remain mechanically sheltered. While most culture-based techniques are optimized for bacteria in planktonic growth phase, most organisms within a biofilm are in stationary growth phase. The dramatic differences in phenotype greatly hinder the sensitivity of traditional culture methods.

1.2.3 Infections with Atypical Organisms

Traditional culture techniques can also fail in the setting of a wide array of less common causes of infection that are difficult or impossible to identify in this manner. Many of these bacteria, such as Cutibacterium acnes, Brucella spp., and nutritional variant streptococci are more indolent, requiring a prolonged incubation period and/or have specific nutritional requirements not met by standard enriched culture media. ¹⁷ ^, ¹⁸ C. acnes can require up to 14 days of anaerobic incubation to be detected; Brucella spp. can require up to 4 weeks. ¹⁹ Other causes of orthopaedic infections such as Neisseria gonorrhoeae require specialized handling and specific environmental conditions to enhance growth in culture. ²⁰ Lyme arthritis is caused by Borrelia burgdorferi, which cannot be cultured in routine clinical labs. ²¹ Mycobacteria and fungi are also uncommon, but important, causes of orthopaedic infection. ¹⁷ Some fungi, such as Candida species, grow readily in standard bacterial culture media. Otherwise, almost all these organisms require specifically tailored culture media to support their growth; the duration of incubation for these organisms is often many weeks. To diagnose many fungal or mycobacterial infections, or to detect the wide range of bacteria that grow poorly with traditional culture methods, a high index of suspicion is required. In order to grow these organisms in culture, appropriate tests need to be specifically requested when submitting tissue or body fluid for culture.

To improve the yield of orthopaedic fluid and tissue culture, many adaptations and variations of the standard microbiologic approach have been evaluated for their ability to maximize the sensitivity of the cultures while avoiding a loss of specificity. Targets of study include improvements in the methodology of specimen collection and variations on the laboratory testing parameters, including the tissue preparation, duration of incubations, as well as the use of enhanced and/or more selective media. Over the past decade there have been dramatic advances in molecular diagnostic techniques, including PCR sequencing, and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, allowing for the identification of many organisms that have proven difficult to isolate and identify by traditional culture-based techniques. These advances in culture technique are an active area of study in both septic arthritis and osteoarthritis, but because of the high burden of biofilm-driven infections, the majority of investigation has centered on orthopaedic hardware infection.

1.2.4 Orthopaedic Hardware Infections

The presence of hardware, such as prosthetic joint or fracture-fixation hardware, can complicate any attempt to establish a microbiologic diagnosis. From a technical standpoint, the common causes of hardware-related infection, staphylococcal and streptococcal species, enteric gram-negative bacilli, and Enterococcus, can be easily recovered in the microbiology lab. However, the rate of culture-negative workups can be substantial, exceeding 20 to 25% in some series. ²² ^, ²³ Methods to maximize the sensitivity of culture-based diagnostics of orthopaedic hardware infection without sacrificing specificity have been an active area of research for the past several decades. While some researchers have focused on spinal and fracture-fixation hardware infections, PJIs have been the main focus of inquiry.

Hardware Infections: Number of Cultures

Sending multiple cultures from the site of infected orthopaedic hardware, especially when there is concern for a low virulence or fastidious organism, improves the likelihood of a successful microbiologic diagnosis. Obtaining multiple specimens increases the overall yield of the cultures and can also aid in differentiating whether a cultured organism is a pathogen or a contaminant. Bacteria making up the normal skin flora can be asymptomatically introduced to the site of orthopaedic hardware at the time of surgery, only to present with infection months to years later. Often these subacute and chronic orthopaedic hardware infections can present without systemic or even local signs of infection or inflammation or may only come to attention after they have led to mechanical loosening, fracture nonunion, or other forms of hardware failure. Determining whether an isolated organism is the cause of infection or is merely a contaminant based on a single specimen can be very difficult.

Several early studies underscored the importance of obtaining multiple cultures in patients with PJIs. A 1981 prospective study of 63 infected and 30 uninfected patients found that collection of five cultures allowed for the investigators to differentiate infection from contamination, concluding the growth in “one or two of five biopsy samples was a strong indication of contamination, while growth in all five biopsies strongly correlated with the presence of an infection.” ²⁴ A much larger prospective trial published in 1998 found, through mathematical modeling, that the optimal number of culture specimens to send is five to six and that finding the same organism in at least three of the specimens strongly correlates with the presence of infection. ²⁵ This observation was corroborated in a 2016 prospective, multicenter study enrolling 264 patients with suspected infection, using patients as their own controls. Using a random-sampling method, repeated 1,000 times per case, the authors found that obtaining four separate specimens and inoculating them on three distinct culture media to be equivalent to obtaining five specimens. This reduction in specimens required was a result of leveraging newer methodologies, including specimen preparation, choice of culture media, and duration of incubation, underscoring the many factors that can impact on the yield of orthopaedic intraoperative cultures. ²⁶ Peel et al, in 2016, reported that the greatest accuracy of diagnosis was observed when four tissue cultures were performed. However, when directly inoculating tissue specimens into blood culture bottles, the optimal number of tissue specimens required decreased, without sacrificing sensitivity, to three specimens. ²⁷

Hardware Infection: Sample Acquisition

Separate surgical instruments should ideally be used to collect intraoperative tissue specimens. Swab cultures, whether taken intraoperatively or preoperatively (such as from a draining sinus), have been repeatedly shown to be less sensitive and less specific than deep tissue culture; as a result, the use of swab cultures in the orthopaedic setting is strongly discouraged. ² ^, ³ Cultures should be taken prior to any extensive debridement, suctioning, or electrocautery. ² ^, ⁴ Which type of tissue has the highest diagnostic value is unclear. Expert guidelines recommend the surgeon take tissue cultures from the “most suspicious” areas ²⁸ and target “visibly inflamed or abnormal tissue” ² and in the setting of infected fracture fixation hardware, tissue from the “site of perceived infection,” including “necrotic bone, site of pseudarthrosis or nonunion or the surrounding deep tissue bed,” ⁴ whether it be synovial tissue, periprosthetic tissue including the bone–implant interface and periprosthesic membrane, or the orthopaedic implant component. In spite of the extensive literature evaluating the microbiologic workup of orthopaedic hardware infection, very little of it has focused on comparing sites of tissue acquisition. ²⁹ The periprosthetic membrane/bone–implant interface has been touted by some to have a higher rate of culture positivity as compared to neosynovium by some authors, ³⁰ ^, ³¹ while others have found no difference. ²⁹ Bone cultures were found to be of low diagnostic yield in one study. ³²

Hardware Infection: Use of Blood Culture Bottles and Enriched Media

The direct inoculation of both synovial fluid and intraoperative tissue into blood culture bottles has been shown to improve the sensitivity of the cultures without a rise in false positive results. ³³ ^, ³⁴ The observation that blood culture bottles for synovial fluid could improve the yield and isolate more fastidious organisms was first made in the 1980s, although hypothesized much earlier. ³⁵ This technique was later adopted for prosthetic joint synovial fluid cultures. Small retrospective studies in the 2000s reported increased sensitivity, ³³ reporting significant improvements with the recovery of anaerobes when compared to traditional cultures, as well as faster recovery of microorganisms. ³⁶ These findings were confirmed in a prospective study using automated blood culture systems. ³⁷ Recent work by Peel et al found the use of a semi-automated method of tissue culture using blood culture bottles improved the sensitivity for tissue cultures without an increase in false positives, as well as shortening the time to positivity. ³⁴ Why blood culture bottles outperform traditional culture techniques is not entirely understood, although several mechanisms have been proposed. ³³ ^, ³⁸ The large volume of media in blood culture broth dilutes the host inflammatory cells that are present within the synovial fluid inoculated into the bottle; the presence of these inflammatory cells can inhibit of bacterial growth. In addition, the use of blood culture bottles allows for a larger volume of synovial fluid to be cultured in a single culture, as compared to the volume of synovial fluid that can be plated on to solid media. Also, lytic agents present in blood culture bottles allow phagocytized organisms to be released from white blood cells. From a practical standpoint, the use of blood culture bottles also allows the lab to use automatic culture systems, which reduces contact with the environment and diminishes exposure to aerobic conditions. A recent workflow analysis at a referral center for revision arthroplasty reported that the use of blood culture bottles for tissue culture reduced cost and labor time when compared to conventional methods. ³⁹ The routine use of blood culture bottles for synovial fluid and intraoperative tissue culture is strongly advocated. ² There has been little formal study of the role blood culture bottles for infected fracture-related hardware and spinal instrumentation.

Other than the use of blood culture bottles, exceptionally little research has been done comparing the effect of different culture media. The only prospective evaluation of culture media (including blood culture bottles) involved 178 patients and found the sensitivities of blood culture bottles (87%) and two other enriched media, cooked meat broth (83%), and fastidious anaerobic broth (57%) to be superior to traditional direct plating method (39%). ³⁷

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