Management of Orthopaedic Infections (eBook)

1 Detection of Microbes in Orthopaedic Infections

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

Abstract

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.

Keywords: Osteomyelitis, prosthetic joint infection, bacteria, microbiology, biofilm, PCR, next-generation sequencing

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...