Novel developments in the prevention, diagnosis, and treatment of periprosthetic joint infections.

Novel Developments in the Prevention, Diagnosis, and Treatment of Periprosthetic Joint Infections Abstract Javad Parvizi, MD, FRCS Pouya Alijanipour, MD Elie F. Barberi, MD Noreen J. Hickok, PhD K. Scott Phillips, PhD Irving M. Shapiro, BDS, PhD Edward M. Schwarz, PhD Michelle H. Stevens, MD Yi Wang, PhD Mark E. Shirtliff, PhD

From the Rothman Institute (Dr. Parvizi and Dr. Alijanipour) and Sidney Kimmel Medical College (Dr. Hickok and Dr. Shapiro), Thomas Jefferson University, Philadelphia, PA, the Department of Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN (Dr. Berbari), the United States Food and Drug Administration, Beltsville, MD (Dr. Phillips), the Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY (Dr. Schwarz), 3M Healthcare, Woodbury, MN (Dr. Stevens), the United States Food and Drug Administration, Silver Spring, MD (Dr. Wang), and the Department of Microbial Pathogenesis, University of Maryland, Baltimore, MD (Dr. Shirtliff). J Am Acad Orthop Surg 2015;23 (suppl 1):S32-S43 http://dx.doi.org/10.5435/ JAAOS-D-14-00455 Copyright 2015 by the American Academy of Orthopaedic Surgeons.

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Periprosthetic joint infection (PJI) is one of the most challenging complications compromising the outcome of an otherwise successful operation. Considerable efforts have been invested in the recent years to address paradigm shifts in our understanding of the complex microbiological phenomena that contribute to the pathophysiology of PJI, such as microbial adherence, biofilm formation, and resistance to antibiotics. This article is an introduction to some of the recent advancements in the prevention, diagnosis, and treatment of PJI. It describes how industry, academic researchers, and government are increasing collaboration to address PJI through development of novel technologies, therapeutic strategies, and regulatory science that specifically target the unique biofilm-associated aspects of its pathogenesis.

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he biofilm theory of bacterial growth is postulated to play a major role in the pathogenesis of periprosthetic joint infection (PJI) and has significant implications for its prevention, diagnosis, and treatment.1 Prosthetic implantation provides a physiologic niche for pathogenic organisms to cause infection. Bacteria of many genera have been recovered from cases of PJI. Although Staphylococcus species cause most cases of PJI, other pathogenic microorganisms also commonly cause infections. These include Propionibacterium acnes; Acinetobacter and Klebsiella species; Pseudomonas aeruginosa; Enterococcus, Streptococcus, Enterobacter, and Mycobacterium species; and various anaerobic bacterial and mycoidal species (specifically Candida species). Each of these pathogenic genera individually represents a minority of infections compared to those represented by Staphylococcus species. Foreign bodies increase the ability of microbes to cause chronic infection more than 100,000-fold.2 Even if medical devices are successfully

implanted without infection, their continued presence predisposes patients to infection years after implantation. Biofilm is especially likely to be a challenge with implants because of their detrimental effects on immune response. Implants are frequently responsible for reduced blood flow and the compromise of local immunity by impairing the activity of natural killer, lymphocytic, and phagocytic cells, as well as by reducing the amount of superoxide, a mediator of bacterial killing, within professional phagocytic blood cells.3 Another means by which implanted medical devices create local immune compromise is through frustrated phagocytosis,3 which occurs when professional phagocytes undergo apoptosis upon encountering a substrate of a size that is beyond their phagocytic capability. The resulting release of reactive oxygen products and lysosomal enzymes may trigger accidental damage to host tissue as well as local vascular insufficiency. This can result in increased opportunities for bacterial colonization and, thus, development of

Journal of the American Academy of Orthopaedic Surgeons

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Javad Parvizi, MD, FRCS, et al

a chronic infection. Also, a portion of the normal phagocytic processes is directed toward the removal of the implant itself (particularly with metals, methyl methacrylate, and polyglycolic acid), thus wasting the energy and resources of the immune system that typically would be used to counter the infection.4-6 For prosthetic articular implants in particular, the risk of implant infection is known to be increased by several factors. First, certain joint arthroplasties are more vulnerable to infection because they are sited proximally to the skin surface, thus having poor soft-tissue coverage (eg, total elbow arthroplasties).7 Second, polymethyl methacrylate (PMMA) bone cement can inhibit complement function and the activity of white blood cells. Also, heat released during PMMA polymerization may kill the adjacent cortical bone, thereby creating a nonvascularized area. This offers the bacteria a beneficial growth environment while being sealed off from the circulating host defenses. Third, some patient populations are at elevated risk because of underlying conditions or systemic diseases, including patients with diabetes mellitus and rheumatoid arthritis.8 Other risk factors include the development of infection at the site of the prosthesis that is not associated with the prosthesis itself, the presence of malignancy, and a history of joint arthroplasty.9 Additionally, patients who are elderly, obese, or malnourished, or who have undergone

prior surgery at the implantation site, are at increased risk of developing PJI. Unfortunately, many of these risk factors are common to PJI patients and may be related to underlying causes of the need for arthroplasty. In summary, prosthetic implants not only present a physiologic niche for pathogenic organisms but also restrict the capability of the host to effectively deal with the infection, which presents a unique challenge when additional risk factors are present. The intrinsic risk of colonization and subsequent infection associated with implants is further exacerbated by their tendency to become coated in host proteins, such as fibrinogen and fibronectin, shortly after implantation.10 In this way, implants can become a surface to which bacteria easily adhere. Following initial adherence and colonization, bacteria are thought to form a complex matrix of extracellular polymeric substance serving as a protective scaffold in which they can survive despite the competence of the host’s immune system or the presence of antimicrobial agents.11-14 Once established, most bacteria (including staphylococcal species) are able to produce a biofilm. Biofilm is defined as a sessile community composed of microbial cells that are attached to a substratum, an interface, or each other and are embedded in a matrix of extracellular polymeric substance.15 Such microbial communities exhibit a radically altered

phenotype with regard to growth, gene expression, and protein production compared to taxonomically identical microorganisms growing planktonically (ie, single cells).16,17 The biofilm mode of growth is one of the most important mechanisms that bacteria use to persist within the host—particularly on indwelling medical devices and other orthopaedic infections.18 Infections associated with biofilms may account for up to 80% of all infections in modern healthcare facilities,19 which translates into 17,000,000 infections, 550,000 deaths, and $90 billion annually in the United States.20 Once an implant is colonized and a chronic infection develops, implant removal is usually the only intervention capable of resolving the infection. Biofilm allows for the bacterial population to evade the effects of antimicrobial therapy and the immune response of the host.21 Biofilms, once established, become a source of release of planktonic bacteria or biofilm fragments. Depending on the environmental conditions, bacteria within the biofilms may also remain in a metabolically quiescent state and therefore may be even more difficult to detect or eliminate.22 On the other hand, some bacteria, such as Staphylococcus aureus, can be internalized within the osteoblasts, where they can be protected from the extracellular host defense mechanisms or antibiotics, similar to the bacteria lodged in the biofilms.23

Dr. Parvizi or an immediate family member serves as a paid consultant to or is an employee of CeramTec, ConvaTec, Corentec, Johnson & Johnson, Medtronic, Smith & Nephew, TissueGene, and Zimmer; has stock or stock options held in Alphaeon, CD Diagnostics, Corentec, Hip Innovation Technology, Joint Purification Systems, and Physician Recommended Nutriceuticals (PRN); has received research or institutional support from 3M, Cempra, CeramTec, DePuy, the National Institutes of Health (National Institute of Child Health and Human Development and National Institute of Arthritis and Musculoskeletal and Skin Diseases), the Orthopaedic Research and Education Foundation, Smith & Nephew, StelKast, Stryker, and Zimmer; and serves as a board member, owner, officer, or committee member of the Eastern Orthopaedic Association and the Muller Foundation. Dr. Hickok or an immediate family member serves as a paid consultant to or is an employee of AstraZeneca/MedImmune and Bristol-Myers Squibb; has stock or stock options held in Pfizer; and has received research or institutional support from Synergy Medical and Zimmer. Dr. Shapiro or an immediate family member has stock or stock options held in Smith & Nephew. Dr. Schwarz or an immediate family member serves as a paid consultant to or is an employee of DePuy, the Musculoskeletal Transplant Foundation, Regeneron, MedImmune, and Telephus Biomedical; has stock or stock options held in LAGeT and Telephus Biomedical; has received research or institutional support from DePuy, Eli Lilly, LAGeT, and Telephus; and has received non-income support (such as equipment or services), commercially derived honoraria, or other non-research–related funding (such as paid travel) from LAGeT, and Telephus. Dr. Stevens or an immediate family member is an employee of and has stock or stock options held in 3M. Dr. Shirtliff has stock or stock options held in DiFUSION. None of the following authors or any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this article: Dr. Alijanipour, Dr. Berbari, Dr. Phillips, and Dr. Wang. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. The findings and conclusions in this article have not been formally disseminated by the U.S. Food and Drug Administration and should not be construed to represent any Agency determination or policy.

April 2015, Vol 23, Supplement 1


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Novel Developments in the Prevention, Diagnosis, and Treatment of Periprosthetic Joint Infections

By adopting this sessile mode of growth, biofilm-embedded microbes enjoy several advantages over their planktonic counterparts. One major benefit is resistance to clearance by antimicrobial agents24 and cellular and humoral host immune effectors.25-27 In fact, biofilmembedded bacteria are up to 1,000 times more tolerant of antibiotics.28 Therefore, once a surface is colonized, the only successful strategy to resolve the infection is surgical removal of the implant or débridement of the devitalized tissue. This antibiotic tolerance is mediated through low metabolic levels and drastically downregulated rates of cell division of the deeply entrenched microbes,29 including nondividing “persister” cells.30,31 Although low metabolic rates explain a great deal of the antimicrobial resistance properties of biofilms, other factors may also play a role. One example may be the capability of biofilms to act as a diffusion barrier, retarding the infiltration of some antimicrobial agents.24 Another advantage to the biofilm manner of growth for bacteria is the potential for dispersion via detachment. Microcolonies may detach under the influence of fluid mechanical shear or through a genetically programmed response that mediates the detachment process.32 Therefore, growth in the biofilm mode allows an enduring bacterial source population that is resilient to challenge by both antimicrobial agents and the host immune response, while simultaneously enabling continuous shedding to encourage bacterial spread. The pathogenic mechanisms described above partly explain why an established PJI is difficult to eliminate and demands an approach that is focused on addressing the unique aspects of biofilm. The dramatically altered phenotypes of biofilm-embedded microbes present both challenges and opportunities for prevention, diagnosis, and treatment of such infections. This manuscript introduces how industry, government, and researchers are working together to take steps toward specifically addressing these unique aspects of PJI by facilitating novel strategies and technologies that are specifically targeted toward the unique biofilmassociated aspects of its pathogenesis. The

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first step is to develop effective ways to diagnose PJI. The unique properties of biofilm may be exploited by researchers to develop diagnostics specific for antigenic markers of the biofilm phenotype. The second step is development of novel strategies to prevent PJI by preventing bacterial adhesion and biofilm formation on prosthetics. This can be accomplished by leveraging the immune response through the use of vaccines and immunization strategies, or by implant modification to prevent biofilm, both of which are discussed here. The third step is to improved treatment modalities for PJI, including technologies capable of disrupting biofilm once it forms, or targeted antibiotics against cells infected with organisms. In the last step, all of these novel efforts can benefit from increased regulatory science research to expand the scientific basis for evaluation of new technologies and performance claims related to biofilmassociated infections.

Diagnosis of PJI: Present Technologies and Future Prospects Rapid and accurate pathogen detection and identification are needed to allow physicians to react and respond appropriately to PJI. Currently, pathogen identification requires microbial culture followed by diagnostic analyses that normally involve additional rounds of replication in culture, purification of specific bacterial products, or biochemical tests. At best, microbe identification can take place in days to weeks, depending on the growth rate of a specific pathogen. In addition, biofilm infections are particularly difficult to culture. Biofilms are able to encapsulate bacteria in a small nidus of infection that can cause inflammation in large areas of tissue because of secreted toxins and inflammatory mediators. Therefore, if the biopsy misses the small (,0.1 mm3) biofilm population among the large volume of involved tissue, then microbial cultures will be negative, which is often the case. There is also significant difficulty in liberating the bacteria from the biofilm to grow in culture media. Thus, the application of culture for identification of the

infecting microbes often results in negative culture that delays appropriate therapy, thereby allowing the biofilm time to form, mature, and become even more resistant to antimicrobial agents. Other modern diagnostic modalities have been developed to add to or replace standard culture and identification in the clinical microbiology laboratory, but these also have their limitations. One modern method that has allowed for more rapid identification of bacteria without the additional purification or biochemical testing steps includes the protein phenotyping platform, such as the MALDI (Matrix Assisted Laser Desorption Ionization) Biotyper (Bruker). This type of identification system has seen a recent rise in popularity, but it continues to suffer from the limitations of its dependence upon culture. The advent of molecular techniques based upon polymerase chain reaction (PCR) and cultureindependent diagnosis have increased the sensitivity in the speciation of pathogenic microbes. However, problems associated with PCR detection occur, including false negatives (due to poor technique) and false positives (due to poor quality control and exogenous contaminating DNA). In many cases, it is also unknown whether a positive PCR result has clinical significance, especially considering that these genetic analyses are often positive from normally sterile areas of the body that lack clinical signs of pathology. Several other diagnostic techniques rely on imaging technologies. Conventional radiography is often used, but radiographic changes to bone are often difficult to interpret and can take at least 2 weeks after the onset of infection to reach a level that can be visualized.33 Sensitivity and specificity are only 70% and 50%, respectively, making this technique unreliable.34 Other imaging methods, including radiography,33 CT, MRI, and radionuclide scans (indium In-111–labeled white blood cells, technetium Tc-99m scintigraphy) are all used for diagnosis of orthopaedic infections.35 However, the wide-ranging sensitivities and specificities and the use of expensive imaging equipment, together with the time and expertise of the operators, limit the use of imaging techniques and often make them adjunctive to more standard diagnostic techniques.36-38




To improve the diagnosis of PJI, disruptive technologies must be developed to overcome current limitations of culture, molecular, protein-based, and imaging techniques.

Detection of Anti-biofilm Antibodies From Patient Biologic Samples via ELISA or Lateral Flow Immunoassay In previous studies, the Shirtliff research group has found microbial gene products with upregulated production in biofilms.39 Therefore, biologic samples (eg, serum, urine, cerebrospinal fluid, synovial fluid, and/or pus) can be obtained from patients with a biofilm-mediated infection, and then the antibodies within those samples can be labeled and applied to immobilized biofilm antigens either in an enzyme-linked immunosorbent assay (ELISA) assay or lateral flow immunoassay (LFI). Using this approach, serum samples were collected from 21 patients with S aureus infection at patient presentation and 5 days later, in addition to samples from 30 noninfected controls. Serum levels of immunoglobulin G (IgG) antibodies were determined for antibodies specific to an S aureus biofilmupregulated protein, MntC (a manganese transporter SACOL0688 and 55 other staphylococcal antigens, using a beadbased flow cytometry technique (xMAP; Luminex). MntC was the only antigen associated with the highest median initial-to-peak antibody fold-increase for IgG (5.1-fold) and IgA (2.1-fold), and it was the only antigen to show a statistically significant increase.40 A subsequent clinical study was designed to evaluate the efficacy of using the host antibody response to MntC in cases of human chronic implant-associated infection. Synovial joint samples were obtained from 30 patients with infected artificial knees from the Rothman Institute (courtesy of Dr. Javad Parvizi) with a symptomatic period .14 days. Synovial samples were harvested at surgery and cultured (aerobic and anaerobic culture) for microbial infection. Because obtaining accurate culture specimens in the cases of chronic prosthetic implant infections is often problematic, a PCRbased DNA method of detection was also

used.41 Although the DNA method has the false-positive and -negative issues, like all PCR-based methods, it was useful for secondary confirmation in cases in which a sample was shown to have a positive host antibody response but was culture negative. The culture and PCR analysis identified that only one of the patient samples contained S aureus. Following culture and PCR-based analysis, the samples were submitted for analysis to the Shirtliff laboratory, in a blinded fashion. ELISAs were performed on the clinical synovial samples to detect the concentration of host antibody to SACOL0688. Only 1 of the 30 patient samples produced a positive assay for S aureus (3 standard deviations above average), and this matched with the only patient who had been identified as infected with S aureus by the culture and PCR-based methods, when those results became unblinded. This patient had a polymicrobial infection that included Stenotrophomonas, Treponema, and Candida species along with S aureus. A second sample had antibody concentrations that approached a predetermined threshold for a positive result, but this patient had a joint infection resulting from S haemolyticus, which contains a close homolog to MntC. An LFI was then used to specifically, rapidly (,10 minutes), and inexpensively (,$10 USD) diagnose biofilm infections by detecting host antibodies against these biofilm-upregulated antigens.42 LFIs are devices intended to detect the presence of a target in a sample without the need for specialized and costly equipment. One example of an LFI is the most widely used diagnostic in medicine, the pregnancy test. LFIs depend upon spontaneous liquid transfer via capillary beds. Samples of biologic fluids are first applied on an absorbent sample pad, followed by application of a running buffer. The sample is subsequently transported into the conjugation pad, where a conjugate of protein A-gold is present. IgG antibodies present in the biologic sample bind to the conjugate and, in turn, become labeled. As the fluid front of the labeled sample continues to laterally flow toward the absorbent pad along the capillary beds, the host

antibodies encounter a test line. This test line contains an in vivo–expressed biofilm antigen unique to each microbial species. If the patient is infected with this same microbial species, the labeled IgG from the clinical sample will specifically adhere to this test line, producing a red stripe, thereby indicating presence of infection. As the fluid front continues to migrate past the control line containing an anti-protein A antibody, the leftover protein A-gold conjugate binds and appears as a second red stripe to show that the assay is functioning properly. Thus, one dark line indicates a negative result, and two dark lines indicate a positive result.

Biofilm Infection Diagnosis and Localization by Labeled Anti-biofilm Antibodies Previous work in the Shirtliff research group used the phenotypic distinctiveness of biofilm-resident S aureus to elucidate the identities of surface proteins that are upregulated in biofilm. Antibodies (ie, IgG) generated against biofilmupregulated surface proteins were conjugated to a quantum dot fluorophore and introduced to mice infected with experimental S aureus implantassociated osteomyelitis; anti-hen egg lysozyme (HEL) IgG, and mice implanted with sterile pins, were used as controls. Imaging revealed that the anti–S aureus biofilm IgG localized rapidly to the nidus of infection, generating a pinpoint fluorescence signal that was approximately 10 orders of magnitude greater than that in uninfected animals or in those that received anti-HEL IgG. Therefore, administration of IgG targeted specifically to biofilm cells, and conjugated to a marker, represents a novel, sensitive, and inexpensive method of detection of S aureus infection in vivo. This technology will eventually be used in a clinical setting for the rapid detection and localization of S aureus biofilm infection in humans. This strategy may also be used as a model for diagnostic development against other microbial species of orthopaedic concern, including Streptococcus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus, and others.



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Photoacoustic Diagnosis and Photothermal Infection Elimination by Antibodydirected Nanoparticles The Smeltzer research group introduced a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of microbes in tissue and blood.43 This method could be applicable for label-free diagnosis and treatment using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of bacteria cells, two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of a biofilm-upregulated surface-associated markers. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of bacterial cells with threshold sensitivity as low as 0.5 cells/mL, in vivo magnetic enrichment of bacterial cells, PT eradication of biofilm populations, and realtime monitoring of therapeutic efficacy by bacterial cell counting. This PA-PT nanotheranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biologic tool to gain insights on dissemination pathways of bacterial cells, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans.

Prevention of PJI Through Vaccination and Immunization In addition to innovative device technologies, another approach to preventing PJI is through vaccination and immunization. Although immunization is broadly considered to be the best intervention for infectious diseases, an effective vaccine against S aureus remains elusive, and several clinical trials have failed.44-47 However, leaders in the field remain optimistic and point out that we are far from an exhaustive effort because it is

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likely that the protective antigen or combination of antigens has yet to be identified. Additionally, most of the antibodies used in these failed clinical trials were not engineered with state-of-the-art immunomodulatory functions to opsonize, phagocytose, and kill the bacteria.48 In addition, the limited success in these studies may have been to the result of not accounting for the temporal variability in antigen expression and the multiple modes of bacterial growth, namely biofilm and planktonic growth forms. Therefore, there has been a resurgence of research focused on immunizing against multiple antigens essential for S aureus colonization, growth, and survival in the host, to directly promoting humoral immunity or by enhancing bacterial susceptibility to antibiotics.49-51 Immunization to prevent PJI has an additional challenge for active vaccination strategies, which is that most of these patients have some immunodeficiency because of aging, autoimmunity, obesity, and/or diabetes.52-54 Thus, preoperative passive immunization with neutralizing antibodies against critical S aureus proteins appears to be the best way forward. To have the greatest chance of success, the passive immunization should contain a dual-acting monoclonal antibody that has direct antimicrobial effects and dominant effector functions to enhance the host response and bacterial clearance. An example of this is the recent work on the glucosaminidase (Gmd) subunit of autolysin (Atl), which several groups have identified as an immunodominant antigen.39,51,55 Functionally, Atl is known to be critical for cell wall biosynthesis and degradation during binary fission.56-58 Atl is also an adhesin,59 a biofilm enzyme,39 and a potential molecular target of vancomycin.60 Clinically, it has been shown that circulating anti-Gmd antibodies are a serum biomarker of protective immunity against S aureus in patients with orthopaedic infections,61 and preclinical studies have shown that both active and passive immunization against Gmd protect animals for S aureus osteomyelitis.48,50 However, given the remarkable heterogeneity of the single-species and polymicrobial infections evident in an approach that concentrates on single

antigens, targeting multiple antigens may be required. The Shirtliff research group previously performed a study that identified genes that were upregulated in the biofilm mode of growth and recognized by the antibody-mediated immune response in a S aureus osteomyelitis infection.39,62 Based upon these data, a vaccine composed of four biofilmupregulated antigens plus antibiotic administration (used to clear planktonic populations) was able to prevent biofilm infection where vaccination or antibiotic therapy alone failed.62 Subsequently, the Shirtliff group expanded the protective efficacy of the S aureus vaccine to include gene products with upregulated production in biofilms as well as those upregulated in the planktonic mode of growth. Recombinant forms of these gene products were combined into a pentavalent vaccine that showed complete protective efficacy from challenge in a S aureus animal model of infection without the requirement for antibiotic therapy. In these studies, S aureus was eliminated from all of the challenged hosts in murine models of indwelling medical device infection and intraperitoneal sepsis.63 Thus, future clinical development of immunization strategies to prevent and treat PJI remains an important area of investigation.

Implant Modification Techniques to Prevent PJI Most authorities would agree that titanium alloys exhibit outstanding osseointegrative properties as well as remarkable biomechanical strength, features that make them almost ideal for implant use. Despite their extraordinary properties titanium implants can fail as a result of PJI. In the presence of serum proteins, even a small number of organisms can attach onto the implant surface and initiate a laconic and persistent proliferative response. The subsequent development of a biofilm inhibits osseointegration and promotes osteolysis; the eventual loss of bone tissue provides space for the advancing biofilm and loss of attachment of the implant. Additionally, the biofilm impedes the acquired immune response and blunts the effects of antibiotics as well




as antibacterial agents. With the goal of preventing biofilm formation, implant surfaces have been developed that are nanotextured and exhibit antibacterial properties while maintaining the alloy’s bioconductive and bioinductive characteristics. The ultimate goal of these developments is to increase the longevity of the implant, lower surgical costs, and mitigate patient pain and suffering.

Nanodiamond Particles An innovative method to prevent implantrelated infections is to design hardware that can resist colonization by bacteria, whether passively or through active release of antimicrobials. Implant-adherent S aureus can survive vancomycin levels at 100 times minimum inhibitory concentration, whereas non-adherent (ie, planktonic bacteria) retain “normal” antibiotic sensitivity.64,65 Thus, the ideal system will eliminate adherent bacteria and eradicate bacteria in the surrounding soft tissue. Application of ultrasound loosens bacterial adherence66-68 and can restore antibiotic sensitivity. It decreases numbers of adherent bacteria by approximately100-fold at 20 minutes; addition of vancomycin further decreases adherent bacteria by an additional 50% to 90%.69-71 Ultrasound thus needs to be supplemented by treatments to eradicate the bacterial contaminants. To achieve this objective, ultrasoundactivated drug delivery systems, such as ultrasound-mediated release of nanodiamond (ND)-adsorbed antibiotics from nanoporous surfaces, have been used. The advantage of this system is that the NDs remain near the surface of the implant to blanket the area adjacent to the surface of the implant, including the implant itself, with high levels of antibiotic. NDs are chosen as a drug carrier because of their large, accessible and tailorable surface, biocompatibility, and lack of cytotoxicity.72-75 NDs are particles nearly 5 nm in size produced by detonation synthesis in large volumes;76 the inert diamond core is terminated by surface functional groups, such as C=O, COOH, and OH. By tailoring these surface functionalities, antibiotics such as tetracycline can be efficiently adsorbed to NDs, releasing large local quantities. Based on animal toxicity studies that

showed little apparent toxicity with concentration in the lung, spleen, kidney, and liver, where they are cleared by macrophages,77 these ND composites seem to be nontoxic. Based on these findings, NDdoxorubicin conjugates are now in preclinical trials for the treatment of drugresistant tumors.78 NDs can be immobilized in nanoporous surfaces for reserve until stimulation by ultrasound. The application of ultrasound will serve to loosen adherent bacteria in the establishing infection while releasing antibiotic-adsorbed NDs from the nanopore reservoir. Once released, the ND surface groups will also dictate ND aggregation, which will occur and ensure antibiotic release in propinquity to the implant. Furthermore, additional aggregation can be tailored to prolong antibiotic-release kinetics. Thus, NDs serve as an antibioticrelease system whereby release is triggered at a specified time by application of ultrasound. This ultrasound acts both to mobilize adherent bacteria and to release antibiotic-NDs from the surface that then eradicate the mobilized bacteria. In conjunction with systemic antibiotic treatments, these targeted systems can help to significantly reduce the burden of implantrelated infections.

Controlled-release Agents Calcium sulfate, PMMA (which can be loaded with antibiotics), or adsorbed reagents that liberate nitric oxide are other examples of implant coating materials with antibacterial activity.79-81 Another approach has been to coat the alloy with polyglycolic acid, polylactic acid or polyp-dioxanone nanoparticles; these substrates can be loaded with antibiotics or antibacterial agents that, as they degrade, release their cargo in a predictable manner.82,83 In addition, because of the high surface charge, calcium phosphate/ hydroxyapatite coatings have been developed that can deliver bioactive molecules to the forming osseous tissue. Cargos include antiseptic agents such as chlorhexidine, antibiotics such as gentamycin and vancomycin, and bone morphogenetic proteins in an attempt to accelerate bone healing.84,85 In vitro studies have shown that all of these agents remain bioactive and/or promote tissue repair. Although all of these agents would probably prevent

infection in the short term, it remains to be learned whether (1) they remain active over long time periods to counter hematogenous infections; (2) residual nidi of material contribute to biofilm formation; (3) they promote formation of resistant bacterial strains; (4) the agents influence the surface properties of the metal; (5) they slow osseointegration; and (6) they are clinically meaningful.

Elution of Antibiotics From Titanium Nanotubes Popat et al86 and Lin et al87 have both explored the notion that titanium nanotubes can be used as a reservoir for antibiotics. They described methods of preparing titanium nanotubes of different diameters sintered onto the bulk metal surface. The nanotubes are sintered at 500C in dry oxygen onto the bulk titanium surface. The vertically oriented nanotube arrays can then be preloaded with antibiotic (ie, gentamycin), which is then released in a controlled manner. These loaded hybrid materials kill adherent organisms, decrease methicillin-resistant Staphylococcus aureus (MRSA) adhesion and colonization, and prevent biofilm formation.87 Although a change in osteoblast phenotype has been reported, this can be overcome by nanotexturing of the titanium surface through acid etching procedures.88 Popat et al86 also reported a further refinement of the technique by which an apatite layer formed in the nanotubes markedly increased antibiotic-uptake and -release kinetics. To date, although all of these studies have been performed in in vitro systems, they have not been used in a preclinical model. There has been no discussion of the long-term effects of the nanotube fabrication system on the bulk titanium phase of the implant and the effect of the empty nanotubes on subsequent biofilm formation and organism resistance.

Silver Modification of the Implant Surface It has long been known that silver is a powerful antibacterial agent. For example, silver-containing anti-caries compounds have been used outside the United States for many years to treat



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bacterial decay in the primary teeth of children.89 Silver-coated materials have been shown to influence bacterial adhesion, and silver-coated prostheses have been fabricated for clinical testing.90,91 The antibacterial effects of silver result from the release of its ions from the implant surface and the subsequent thiol bonding to the active site of many metabolic enzymes. Silver has been used in combination with calcium phosphate/ hydroxyapatite coatings and ceramics.92 Although effective, there is concern that the silver layer may influence the metabolic status of adherent cells as well as the metallurgical properties of the implant in vivo. There is also concern that when the silver release is complete, the implant surface will no longer function as a microbicidal agent. Altrhough rare, there is also the problem of silver resistance and hypersensitivity to silver ions.93

Anchoring Antibiotics to the Implant Surface Controlled release of antibiotics provides a highly effective modality for the treatment of acute infection. However, at later treatment times, when antibiotic levels fall to sub-therapeutic levels, surviving bacteria can slowly re-establish a community, which can serve as a nidus for biofilm formation. More specifically, controlled-release systems generate supra-therapeutic levels of antibiotic for a short time, after which the antibiotic concentration falls below the minimum inhibitory concentration; when this occurs, there is concern that resistant strains can emerge. Very high levels of antibiotic can also interfere with stem cell recruitment and commitment and osteoblast function, and thus block osseointegration.94 There is concern that immune system surveillance may be disturbed. One other practical problem is that the coating itself is fragile and, as such, it can easily be dislodged or removed during or following surgical insertion of the implant. Addressing this problem, investigators have covalently attached antibiotics to titanium alloy to provide extended protection.79 This type of surface provides a long-lived anti-bacterial layer that should be active over the lifetime of the implant. Once tethered,

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the antibiotic provides a constant level of protection, which might discourage colonization. Because the total amount of the agent is small compared to the quantities used for controlled release, it may be less likely to foster resistance.79,95 Methods for preparing these surfaces have been extensively characterized, and systems have been described for bonding agents to titanium through diphosphonates,96,97 plasma amination,98 and photopolymerization.88 Because the surface of the metal implant is hydrophobic and chemically inert, it needs to be passivated, a process achieved by brief acid treatment.65 This type of treatment promotes the formation of a surface layer of hydroxyl groups, which can then be reacted with aminopropyltriethoxysilane to form a terminal primary amine. Further stabilizing the attachment reaction, each hydroxyl groups forms three siloxy bonds, thus generating a self-assembled monolayer on the metal surface. The primary amine group of the covalently tethered silane can be used to couple other organic compounds to the self-assembled monolayer. This procedure has been used to attach vancomycin as well as other antibiotics to titanium or titanium alloy.95 Once formed, these surfaces exhibit antibacterial activity and specificity without development of resistance. A considerable number of studies have shown that the covalently attached vancomycin is firmly attached to the metal surface and provides a permanent antibacterial layer by blocking bacterial cell wall synthesis and crosslinking.99 When implanted into infected femoral medullary canals in rats, it blocks bacterial proliferation and osteolysis.64 In a sheep osteotomy model, a titanium locking end plate treated with vancomycin using the silanization procedure prevented osteomyelitis and enhanced bone healing across the bone interface.100

Novel Antibiotic Development in PJI Antibiotic resistance in microorganisms affecting patients with PJI is on the rise. MRSA accounted for 24% of PJI in one recent study.101 Vancomycin-resistant enterococci and, more recently,

multidrug-resistant gram-negative enterococci, in patients with PJI have also been reported.102,103 The approval by the FDA of novel systemic antibiotic decreased from 16 approved agents between 1983 and 1987 to 2 between 2008 and 2012.104 This is largely the result of the changing commercial landscape of antibiotic development by pharmaceutical companies, which is driven by the unfavorable economics of antibiotics discovery and development. To date, there are few randomized clinical trials that have assessed the safety and efficacy of antibiotics in the management of patients with PJI. Daptomycin, a novel lipopeptide with excellent anti–gram-positive activity, was approved by the FDA in 2003. An openlabel, phase two, randomized clinical trial performed in 49 patients treated with daptomycin at 6 and 8 mg/kg for up to 6 weeks was safe and appeared to be effective in managing staphylococcal PJI (some of whom were methicillin resistant) using a two-stage revision arthroplasty.105 Linezolid, a novel antibiotic of the oxazolidinone class of drugs, is active against most gram-positive organisms, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Few retrospective, single-center case series on the experience of using linezolid in patients with PJI have been published. Linezolid appears to be an acceptable choice in the therapy of patients with drug-resistant, gram-positive PJI.106 Serious and common adverse events have been associated with prolonged use of linezolid, including anemia, thrombocytopenia, and neuropathy. Ceftaroline, a novel and advancedgeneration cephalosporin with activity against methicillin-resistant staphylococci, was approved by the FDA in 2010. A selection of case reports on its use in PJI has been published.107 Other novel antibiotics, such as dalbavancin, oritavancin, and various b-lactamase inhibitor/b-lactamase combinations are in various stages of advanced development and may play a role in the therapy of drug-resistant organisms in patients with PJI. In response to the changing landscape of novel antimicrobial development, and




in recognition of the need for new and creative approaches to address the problem of the dwindling antibiotic pipeline, the Infectious Diseases Society of America launched the 10 · ’20 Initiative in 2010.104 Some progress in the clinical development of novel antibacterial drugs targeting infections caused by drugresistant gram-negative bacilli and gram-positive cocci has occurred since then, albeit at a slow pace. It is important to develop more sustainable infrastructure for the development of novel antibiotics. This may include development of regulatory tests that minimize the burden of clinical trials without compromising the evaluation of safety and effectiveness, such as organ-on-a-chip technologies. In addition, there is a need for our society to develop appropriate economic incentives for pharmaceutical companies. Until we reach such a point, the preservation of our current armamentarium of effective antibiotics will remain contingent on the development and application of strong antibiotic stewardship policies.

Table 1 Impact of Microbiological Paradigm Shift on Medical Device-associated Infection Model Factor

Classic Planktonic Model

Who What

One organism at a time in most cases Free-floating cells without substantial EPS When Immediately after surgery Where Fluids How

Acute

Biofilm Model Often polymicrobial Surface-bound cells encapsulated in EPS Can be short or long-term Tissue, device surfaces, fluids, blood Colonize and wait for right conditions

EPS = extracellular polymeric substances

Table 2 Major Challenge and Opportunity Areas Identified for Further Research Etiology-related Questions Meaningful ways to quantify biofilm in vivo Association between device colonization, biofilm and clinical outcome Reliable clinical diagnostics for device colonization and biofilm Product-related Questions Interaction of medical device materials with bacteria Use of in vitro testing to predict clinical outcomes

The Regulatory Science of PJI Treating orthopaedic device–associated infections is costly, and the annual cost of infected revision total joint arthroplasty to US hospitals (as one of the most common device-associated infections) has been projected to exceed $1.62 billion by 2020.108 The high cost of PJI is a strong incentive for development of new technologies, such as those discussed above, which are intended to reduce prosthetic device–associated infections and are directed against device colonization and biofilm formation. In addition to the drug-eluting coatings, ultrasound, and vaccines discussed above, other interesting strategies in the literature are too numerous to mention but include quorum-sensing inhibitors, enzymes, anti-adhesion coatings, and electric fields. In the field of regulatory science, the idea of device-protective strategies directed against colonization or biofilm formation is a paradigm shift from the current focus on treating/preventing systemic infection, whereby effectiveness is partially assessed in vitro by measuring

lethality against planktonic organisms. The biofilm paradigm is different from the planktonic paradigm in all of the five H’s (ie, Who, What, When, Where and How) (Table 1). The nature of biofilm varies depending on the type of device and anatomic location, and the dynamic life cycle necessitates complex interdisciplinary studies. While many of the characteristics of biofilm-associated communities have now been well characterized in vitro and in situ,109-112 and although there is increasing scientific evidence for the role of biofilm-associated microbes in deviceassociated infections,113-117 challenges remain for translating this paradigm shift to gain clinically meaningful advantage. The 2009 National Action Plan to Prevent Healthcare Associated Infections: Roadmap to Elimination noted that “the mechanisms whereby biofilm organisms initiate a disease process are still poorly understood.”118 The Office of Science and Engineering Laboratories, in the Center for Devices and Radiological Health in the US FDA, is working in

collaboration with stakeholders to identify and encourage research necessary to fill in gaps in our understanding of how biofilm causes medical device–associated infections. Several laboratory research projects related to medical device biofilms are under way, and additional research through collaborative efforts with institutions specializing in this area of medicine is being pursued. To better engage external stakeholders, a public workshop entitled Biofilms, Medical Devices, and Anti-biofilm Technology: Challenges and Opportunities, cosponsored by the Montana State University Center for Biofilm Engineering, was held in February 2014 at the FDA.119 A panel discussion at the workshop identified areas in which further research is needed. Two of the key areas were etiology and product performance (Table 2). Clinical researchers in the field of orthopaedics are encouraged to consider initiating research efforts to help fill these gaps to make progress in the battle against PJI.



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Conclusions The prospect of the increasing burden of PJI and its tremendous impact on patients, health institutions, and society is a significant public health concern. Therefore, we are in desperate need for novel approaches and technologies to counter the burden of PJI, which poses severe morbidity to the patients and leads to their demise. The paradigm shift from a planktonic to biofilm model for microbes is a major challenge with unique aspects that are being specifically addressed through recent detection, prevention, and treatment approaches. The success of these novel technologies in preclinical or early clinical stages suggests that it is timely to consider translating these approaches from the laboratory bench to the clinic. Current collaborative efforts between industry, academics, and government have unique potential to help to fuel innovation, increase transparency and trust, and ultimately lead to improved patient outcomes. Last but not least, greater support and interest is needed for regulatory science research to identify and fill in remaining gaps in scientific knowledge (especially in the clinical realm) needed for regulatory decision making.

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S43

Diagnosis of periprosthetic infection: novel developments.

[Prevention of periprosthetic joint infections : Not evidence-based strategies].

New developments in the epidemiology, diagnosis, treatment and prevention of respiratory tract infections.

Sonication: a valuable technique for diagnosis and treatment of periprosthetic joint infections.

Prevention of Periprosthetic Joint Infection.

Diagnosis of periprosthetic joint infection.

Diagnosis of periprosthetic joint infection.

Periprosthetic joint infections: clinical and bench research.

[Diagnosis of periprosthetic hip infections].

Biofilm and the Role of Antibiotics in the Treatment of Periprosthetic Hip and Knee Joint Infections.

Diagnosis and Management of Periprosthetic Shoulder Infections.

Periprosthetic joint infections: a clinical practice algorithm.

[Periprosthetic infections of the hip joint : Clinical approach].

Prevention of periprosthetic joint infection: what are the effective strategies?

A Review of the Literature on Culture-Negative Periprosthetic Joint Infection: Epidemiology, Diagnosis and Treatment.

[Revision arthroplasty : Histopathological diagnostics in periprosthetic joint infections].

Synovial IL-6 as inflammatory marker in periprosthetic joint infections.

Management of fungal or atypical periprosthetic joint infections.

Management of fungal or atypical periprosthetic joint infections.

Surgical management of periprosthetic joint infections: two-stage exchange.

What role do periodontal pathogens play in osteoarthritis and periprosthetic joint infections of the knee?

Temporomandibular joint replacement periprosthetic joint infections: a review of early diagnostic testing options.

Developments and challenges in the diagnosis and treatment of ADHD.

Familial hypercholesterolemia: developments in diagnosis and treatment.