Cassuto, Daniel M.D.; Sundaram, Hema M.D.
The aesthetic use of soft-tissue fillers has grown significantly over the past several years. Throughout this period, hyaluronic acid has remained the most popular product. The American Society for Aesthetic Plastic Surgery reported in its annual survey that the number of hyaluronic acid filler procedures was second only to the number of botulinum toxin type A injections and that over 1.4 million hyaluronic acid filler procedures were performed in 2012.1 Calcium hydroxylapatite filler is also growing in popularity and is often combined with hyaluronic acid fillers.
All hyaluronic acid and calcium hydroxylapatite fillers in current use in the United States and Europe are nonanimal derived. They are notably safe, efficacious, and well tolerated, and produce predictable results. The incidence of adverse events is rare, but it is to be expected that a significant increase in filler procedures would be accompanied by a corresponding increase in the number of reported filler complications. There is a potential risk of complications with any procedure. However, a number of authors have expressed the concern that complications are more frequent when procedures are performed by physicians and nonphysicians who lack the requisite understanding of facial anatomy and appropriate injection technique.2,3 Most specialists who inject fillers will, during the course of their careers, encounter a patient with a complication.
In this article, we present a problem-oriented approach to the diagnosis and management of nodules arising from the aesthetic use of hyaluronic acids. The salient features of this approach may also be applied to nodules after the injection of calcium hydroxylapatite filler. Several publications already provide excellent insights and guidelines for self-limited injection sequelae such as transient pain, ecchymosis, and mild tissue swelling, and for the more serious complications of tissue ischemia and necrosis.2–6 Our objective is to offer a structured strategy that we hope will be of value when filler nodules are encountered, and also to dispel some misconceptions. Our rationale for this focus is that filler nodules pose a particular challenge, especially when they occur after treatment by another injector, because the precise details and time course of this prior procedure may not be fully known. We consider it essential to differentiate between inflammatory and noninflammatory nodules, and to always consider an infectious cause even when it is not apparent after initial investigation. We do not consider corticosteroids and/or other immunosuppressants such as 5-fluorouracil to be appropriate interventions unless the possibility of infection, including biofilm, has been completely ruled out. Their use in a situation of undiagnosed infection would inevitably result in a rebound effect once their temporary antiinflammatory effect has ceased.
CORTICOSTEROIDS FOR FILLER NODULES: RATIONALE AND RISKS
The tendency to use corticosteroids for treatment of nodules (Fig. 3) or even diffuse swelling and erythema arising from filler injections is understandable when viewed through the prism of history and clinical extrapolation. Historically, a nodule that appears after filler injection has often been described as a granuloma on the basis of clinical assessment, without the histopathologic evaluation that is a prerequisite to make this diagnosis. From a clinical perspective, intralesional corticosteroids are a mainstay of the dermatologist’s therapeutic methods of treating inflammatory, nodular skin conditions such as granuloma annulare. The subliminal association of anecdotally diagnosed “filler granulomas” with actual, steroid-responsive skin granulomas such as granuloma annulare can lead to the notion that a similar therapeutic approach is indicated.
Further confusion can arise if the diagnosis of filler hypersensitivity is made presumptively and used as the rationale for corticosteroid treatment. This can occur when cultures from a nodule are negative or not performed. It can also occur when anecdotal reports pertaining to one specific filler product are extrapolated to other, unrelated products. For example, a previously available but now discontinued avian-derived hyaluronic acid (Hylaform; Genzyme Corp., Ridgefield, N.J.) was considered contraindicated in patients with known sensitivity to avian proteins. However, currently available hyaluronic acid products are produced by bacterial fermentation rather than being animal-derived, with Streptococcus equi serving as the source for products approved by the U.S. Food and Drug Administration—the Restylane/Perlane family (Medicis, a Division of Valeant Pharmaceuticals, Inc., Scottsdale, Ariz.), the Juvéderm family (Allergan, Inc., Irvine, Calif.), Belotero Balance (Merz Pharmaceuticals, Greensboro, N.C.), and Prevelle Silk (Mentor Corp., Santa Barbara, Calif.). Calcium hydroxylapatite filler (Radiesse; Merz) is manufactured by a synthetic, nonbiological process.
As another example, case reports attest to the propensity of another discontinued hyaluronic acid, Hydrelle/Elevess (Anika Therapeutics, Bedford, Mass., Distributed by Coapt Systems, Inc., Palo Alto, Calif.), to cause nodules and abscesses after injection.7 Analysis of a biopsy specimen from a culture-negative nodule from one patient showed a granulomatous reaction with multinucleated foreign body giant cells.8 However, Hydrelle/Elevess differed from currently available products in a number of important respects. During its manufacture, it was cross-linked with p-phenylene bisetheyl carbodimide and therefore contained trace amounts of this chemical instead of the cross-linkers used for current products: 1,4-butanediol diglycidyl ether for Restylane, Juvéderm, and Belotero, or divinyl sulfone for Prevelle Silk. In addition, Hydrelle/Elevess also contained 0.1% sodium metabisulfite, which has been suggested as a cause of hypersensitivity. It has also been speculated that this product’s high total hyaluronic acid concentration of 28 mg/ml resulted in a higher bacterial protein load than the current formulations of Restylane, Perlane, Belotero, and Juvéderm, which range in total hyaluronic acid concentration from 20 to 24 mg/ml. This theory is indirectly corroborated by a retrospective review of 144,000 patients with adverse events from nonanimal-derived hyaluronic acids. The incidence of reported adverse events, including filler nodules that were presumptively diagnosed as hypersensitivity and granulomatous reactions, declined from one in 650 in 1999 to one in 1800 in 2000 after the introduction of a new hyaluronic acid raw material with trace amounts of proteins that were six times lower than in the previously used raw material.9
Although hypersensitivity to traces of streptococcal proteins remaining in the finished products has been cited on a number of occasions as the major cause of reactions to hyaluronic acid fillers,10 we feel that some caveats are warranted. First, patients who suffered reactions to Hydrelle/Elevess have been treated uneventfully with other, currently available hyaluronic acid fillers.7 From an immunologic perspective, this suggests that their reactions were to unique components of Hydrelle/Elevess rather than to streptococcal proteins.
The second caveat is also immunologic. Streptococcal hypersensitivity has indeed been implicated in the pathogenesis of autoimmune inflammatory rheumatic diseases such as Behçet disease. This is a cytokine-mediated, type III hypersensitivity reaction associated with accumulation of antibody-antigen complexes that gives rise not only to local inflammation but also to a severe systemic inflammatory immune response.11 In contrast, there have been no reports of systemic symptoms associated with filler nodules even when local inflammation was pronounced. Two randomized, prospective, sequential studies of 150 subjects in 10 centers and 283 subjects in 17 centers, respectively, showed serum levels of immunoglobulin G and immunoglobulin E antibodies to hyaluronic acid products (Restylane and Perlane) that were normal or unchanged from preinjection baseline at 6 and 24 weeks after injection; negative intradermal skin test results for immunoglobulin E antibodies at 24 weeks; and no histologic evidence of type IV or type I hypersensitivity.12 Of the total subjects, 7.8 percent had serum immunoglobulin G antibodies to the hyaluronic acid products at preinjection baseline, but antibody levels were unchanged over the study period in all except one subject, and treatment outcomes were not impacted in any of these subjects. Only one of the 433 subjects in these studies was judged by investigators to have a systemic adverse experience (flu/pyrexia) that might be related to filler injection. It is certainly possible that prior exposure to Streptococcus (e.g., from cellulitis or erysipelas) might result in antibodies that react in an immunoassay to trace levels of streptococcal proteins in hyaluronic acid filler products. However, there is no evidence from the controlled study data above that antistreptococcal antibodies are implicated in adverse events arising from hyaluronic acid injection, including filler nodules. The importance of an evidence-based approach and the challenge of forming a hypothesis based on anecdote are underscored by conflicting case reports of patients with inflammatory filler nodules that show either normal or elevated serum levels of immunoglobulin G and immunoglobulin E antibodies to hyaluronic acid products.13,14
The third caveat is clinical. In most case reports of granulomatous or cutaneous hypersensitivity reaction to a filler, the diagnosis has been made presumptively on the basis of swelling, erythema, and induration at the implantation site and in its immediate vicinity.7,9,13,15 However, these signs are simply the classic, nonspecific manifestations of inflammation—as immortalized in the medical encyclopedia of Celsus over 2000 years ago with the aphorism, “calor, dolor, rubor, tumor.” Their presence does not prove the existence of granuloma or hypersensitivity, nor does it disprove the presence of infection. In any case, infection itself can result in secondary granulomas or hypersensitivity.
The fourth caveat is that negative cultures from a filler nodule do not exclude the diagnosis of bacterial or fungal infection. The classic presentation of mycobacterial infection as “sterile” nodules or abscesses has been described with breast implants,16 and cutaneous inflammation at the site of hyaluronic acid filler implantation has been recently associated with Mycobacterium chelonae infection.17
INFECTION AS A PRIMARY CAUSE OF FILLER NODULES
A skin procedure or a procedure room can be clean but is never completely sterile. There are several factors that heighten the risk of bacterial contamination during a filler injection entailing the repeated piercing of nonsterile skin in a nonsterile environment. These include the passage of the injection needle through a sebaceous gland containing bacteria; incidental bacteremia at the time of the procedure resulting from tooth brushing, dental flossing and other everyday activities; and physiologic fluctuations in the skin’s inherent immune surveillance. Furthermore, several pathogenic bacteria possess the ability to use hyaluronic acid as a nutrient source: Staphylococcus and Streptococcus species synthesize the enzymes that are necessary to break glucosidic bonds and grow well on hyaluronic acid–supplemented culture media, and the in vitro growth of mycobacteria is enhanced by the presence of hyaluronic acid.18,19
The ease with which contamination can occur is vividly illustrated by the cases of mycobacterial infection after hyaluronic acid injection, in which pulsed-field gel electrophoresis showed that the causative M. chelonae originated in the clinic tap water from which ice was prepared for preprocedural application to the skin.17 As another example, a case-controlled study used bacteriologic investigation, gene sequencing, random amplification of polymorphic DNA, and pulsed-field electrophoresis to trace 15 cases of mycobacterial infection following breast implantation procedures at a single medical center over a 4-month period to mycobacterial contamination of the body of the surgeon, and ultimately to his outdoor whirlpool at home.20
In light of the above, we invoke the old medical school maxim to first think of horses (what is common) rather than zebras (what is rare) when hoof beats are heard, and recommend that infection should be considered the primary cause of persistent filler nodules until proven otherwise. We also make a more general plea for common sense in its most literal interpretation—that we should apply our sense of what is most common when investigating and diagnosing any condition, including filler nodules. When our accrued, experiential knowledge is combined with the available evidence, we find that images of filler nodules that were previously considered caused by hypersensitivity9 now evoke instead the unmistakable specter of infection, including biofilm.
BIOFILM: POSSIBLE ROLE AND DIFFERENTIATION FROM OTHER INFECTIONS
A biofilm is defined as an aggregate of microorganisms, comprising cells adhering to each other on a surface and embedded within a self-secreted extracellular polymeric substance.21–25 Bacterial and candidal biofilms have been implicated in infections with solid implants, including joint prostheses and heart valves, and inserted devices such as catheters and contact lenses.
The study of biofilms composed of Pseudomonas aeruginosa has provided several insights. A notable feature of biofilms is that they do not generate an immune response. The extracellular polymeric substance is thought to protect microbial cells physically from components of the immune system and possibly also from certain antibiotics. A biofilm can exist in a dormant (persister) state, during which the metabolism of its cells shuts down. This has important sequelae: a dormant biofilm is likely to produce negative in vitro cultures and to be particularly resistant to antibiotics such as penicillins because their rate of bacterial killing is proportional to the rate of bacterial growth and they cannot kill nongrowing cells. Although fluoroquinolones, cephalosporins, and aminoglycosides can kill nongrowing cells, they are more effective in killing cells that are dividing rapidly. It is also hypothesized that there may be biofilm-specific expression of genes affecting cell persistence and perhaps even antibiotic resistance. Activation of a dormant biofilm can occur after local environmental disturbance and may manifest as local nodules, abscesses, granulomas, low-grade infections, or systemic infections.26
Evidence for the role of biofilms in filler complications is circumstantial but strong—particularly with permanent or long-lasting filler products.6,27 Biofilms are believed to present as persistent, erythematous, mildly tender nodules that develop weeks to months after filler implantation. Fluorescence in situ hybridization has demonstrated bacteria in seven of eight biopsy specimens taken from culture-negative nodules arising after implantation of polyacrylamide gel filler.28,29
It is important that the recent focus on biofilms does not engender confusion and prompt clinicians to label all cases of persistent filler nodules as biofilms. The hallmarks of a biofilm distinguish it clearly from other subacute or chronic infections. Specifically, biofilm reactions manifest little or no antibody response, are frequently culture-negative, are resistant to antibiotic treatment, and tend to present as low-grade rather than fulminant inflammation. Although the current evidence for biofilm formation after hyaluronic acid or calcium hydroxylapatite implantation is circumstantial, the possibility should always be considered and, as when any type of infection is suspected, this should obviate the use of systemic or intralesional corticosteroids before a diagnosis has been made.
CLASSIFICATION AND DIAGNOSIS OF FILLER NODULES
Although the impulse of patients is often to remove the offending lump or bump as soon as possible, our responsibility as clinicians is to prioritize definitive diagnosis and safe management. The starting point is a problem-oriented classification for filler nodules (Fig. 1).
Nodules of acute onset (within 48 hours after filler implantation) can be defined as noninflammatory or inflammatory, on the basis of visual inspection and palpation. Diffuse noninflammatory swelling or well-defined nodules may be caused by deep hematoma (which may be associated with a red, violaceous, or dark hue to the overlying skin and pain or tenderness) or by overcorrection with filler—which can become more pronounced if the product is a high-concentration hyaluronic acid that binds water in the hours after implantation. A high-viscosity filler extruded through a needle that is too narrow can generate high extrusion force30 and consequent tissue edema and ecchymosis, as can rapid or uncontrolled injection technique.31 The periocular region is felt to be vulnerable to nodules and swelling because of its anatomically unforgiving nature and its propensity for lymphatic outflow obstruction, which can be triggered by overfilling or inappropriate filler placement.32 A bolus of particulate hyaluronic acid that is placed too superficially, especially in a thin-skinned area, may result in a nodule and impart a bluish color to the overlying skin because of the Tyndall effect (i.e., preferential scattering of blue light back to the observer’s eye).33 Nodule formation may also be more common in regions of high mobility such as the lips.
Inflammatory nodules may also be localized or diffuse. As with noninflammatory nodules, this can depend to some extent on the pattern of placement of the filler. Inflammatory nodules may be more diffuse if they arise after microaliquot or superficial “mesofiller” injections; because these techniques entail multiple skin punctures, it is reasonable to consider infection as a likely cause.
Rarely, nodules can be associated with intermittent or continuous paresthesia. This is presumed to be caused by filler placement close to a branch of the trigeminal nerve (e.g., the infraorbital branch in the case of malar augmentation). Paresthesia can occur or be exacerbated if the filler is a hyaluronic acid that swells after implantation because of water absorption and applies more pressure to the nerve. One of the authors (D.C.) has personally observed two such cases following filler injection to the pyriform fossa and below the zygomatic arch.
Nodules or cords of subacute onset (48 hours to 2 weeks after filler implantation) or late onset (beyond 2 weeks after implantation) may be classified based on whether they are asymptomatic or symptomatic. Depending on their depth and location, asymptomatic nodules may be invisible, visible while animating, or always visible (Fig. 2). Symptomatic nodules may present with no inflammation, mild inflammation, or major inflammation. Symptoms of noninflammatory nodules include edema, paresthesia, and/or limitation of movement. Mild inflammation may be occasional or constant and manifests with one or two of Celsus’s cardinal signs of heat (calor), tenderness (dolor), redness (rubor), and swelling or induration (tumor). Major inflammation is characterized by three or all of these signs (Figs. 3 through 5).
MANAGEMENT OF FILLER NODULES
Diagnosis and management of filler nodules are greatly facilitated by ultrasonographic imaging, and we recommend that it should become the standard of care. Imaging performed by an ultrasonographer who is well versed in facial anatomy can reveal the presence of unreported, previously implanted materials that could form a nidus of infection, help to determine the nature of these materials, and indicate their precise location and whether they encroach on vital structures.34 Both authors have been consulted by many patients with filler complications who were unable to recall accurately and completely the products with which they had been injected. One author (D.C.) has found ultrasonographic evaluation to be invaluable in the diagnosis and management of patients who reported receiving hyaluronic acid filler but had actually received permanent or semipermanent products, including silicone oil and the hyaluronic/acrylic hydrogel Dermalive (DermaTech, Paris, France).
Whatever the cause of a nodule, removal of this cause is always the treatment of choice. Hyaluronidase will enzymatically digest unwanted hyaluronic acid filler (Figs. 4 and 6), and it may also be considered for the tissue surrounding calcium hydroxylapatite nodules to aid in their dispersion. When there is no clinical evidence of inflammation and after ultrasound has ruled out the presence of other materials, hyaluronic acid or calcium hydroxylapatite nodules may be dispersed by gentle passage of a small-gauge needle back and forth within them. Hyaluronidase can be injected simultaneously. The injection of lidocaine suspension during needle dispersion, followed by tissue molding, has been reported to be an efficacious and comfortable method of correcting contour irregularities from calcium hydroxylapatite.35
The evidence for infection as a primary cause of filler nodules that have subacute or late onset or persist is compelling enough to justify empiric, broad-spectrum antibiotic therapy. Because this treatment is intended to address microbial contamination of an implanted foreign body (filler material), it should be more aggressive and prolonged than the short courses of antibiotics that are typically administered for minor infections. Lack of improvement after a few days of antibiotic treatment or negative cultures should raise the suspicion of resistant microbes, mycobacteria, or biofilm. Macrolide antibiotics such as azithromycin and clarithromycin possess the added benefit of polymodal immunomodulatory activity that confers antiinflammatory effects without impairing the host’s immune defenses.36 Fluoroquinolones such as moxifloxacin may also be considered, as this genre of antibiotic readily equilibrates across biofilms and is therefore somewhat effective in impeding their growth.26
External application of radiofrequency or infrared energy to the skin surface above filler nodules has been reported to be a useful adjunct.37 Treatment to a level that produces consistent postprocedural erythema is believed to generate sufficient heat within the tissue to promote nodule dispersion, bacterial killing, and perhaps even some penetration or disruption of a biofilm. The edema that is generated may perfuse the nodule and aid in its dispersion.
Although no procedure that involves breaching of the skin surface can ever be sterile, scrupulous cleansing of the skin before, during, and after filler injection is recommended to minimize the risk of avoidable contamination. A good cleansing agent is 4% chlorhexidine gluconate (Hibiclens; Mölnlycke Health Care, Gothenburg, Sweden), although it should not be used near the eyes.38 Patients should be instructed to remove all makeup before the procedure, as it can be a source of microbes.39 Sterile technique should be used for injection tools such as needles and cannulas, and if filler products are mixed with lidocaine or saline. An injection strategy that minimizes the number of times the skin must be pierced—such as the selection of a blunt cannula where appropriate, rather than a sharp needle—may also be of value.
As illustrated above, none of these precautions can eliminate the risk of contamination, which we consider to be the primary cause of filler nodules. It is therefore important that clinicians should not get lulled into a false sense of security by the wearing of sterile gloves, sterile preparation of the skin, and so forth, and fail to recognize the symptoms and signs of infection if they occur.
From an evidence-based perspective, one of the challenges of filler nodules is that data tend to be retrospective, anecdotal, and case-based and thus of suboptimal evidence level. Therefore, we urge the incorporation of experiential knowledge and simple common sense into the process of diagnosis and management. The empiric use of potentially harmful treatments such as corticosteroids or potentially useless ones such as furosemide and other diuretics should be avoided. It is worth keeping in mind that the development of nodules after filler implantation is not an emergency situation akin to a cardiac or anaphylactic event in which action must be taken before diagnosis.
Aesthetic use of hyaluronic acid and calcium hydroxylapatite fillers is efficacious, carries a high rate of patient satisfaction, and is generally safe and well tolerated. Complications are rare and typically related to aspects of the injection procedure, such as suboptimal technique and bacterial contamination rather than to the products themselves. The diagnosis and management of nodules after filler implantation can pose a particular challenge. We have found a problem-oriented approach based on time of onset and clinical features of the nodules to be of value in achieving an accurate diagnosis—which is a prerequisite for effective treatment.
Patients provided written consent for the use of their images.
The authors express their deep appreciation to David J. Howell, Ph.D., a medical communications specialist in San Francisco, Calif., for assistance with formatting of the manuscript and figures and with fact-checking.
2. Glashofer MD, Cohen JLJones DJ. Complications from soft tissue augmentation to the face: A guide to understanding, avoiding, and managing periprocedural issues. Injectable Fillers: Principles and Practice. 2010 Oxford, United Kingdom Wiley-Blackwell:121–139
3. Glashofer MD, Flynn TCCarruthers J, Carruthers A, Dover JS, Alam M. Complications of temporary fillers. Procedures in Cosmetic Dermatology: Soft Tissue Augmentation. 20133rd ed London Elsevier Saunders:179–187
4. Alam M, Gladstone H, Kramer EM, et al.American Society for Dermatologic Surgery. ASDS guidelines of care: Injectable fillers. Dermatol Surg. 2008;34(Suppl 1):S115–S148
5. Sclafani A, Fagien S. Treatment of injectable soft tissue filler complications. Dermatol Surg. 2009;35:1672–1680
6. Narins RS, Coleman WP III, Glogau RG. Recommendations and treatment options for nodules and other filler complications. Dermatol Surg. 2009;35(Suppl 2):1667–1671
7. Van Dyke S, Hays GP, Caglia AE, Caglia M. Severe acute local reactions to a hyaluronic acid-derived dermal filler. J Clin Aesthet Dermatol. 2010;3:32–35
8. Chabra I, Obagi S. Severe site reaction after injection hyaluronic acid-based soft tissue filler. Cosmet Dermatol. 2011;24:14–21
9. Friedman PM, Mafong EA, Kauvar AN, Geronemus RG. Safety data of injectable nonanimal stabilized hyaluronic acid gel for soft tissue augmentation. Dermatol Surg. 2002;28:491–494
10. Grossman KL. Hyaluronic acid gel fillers: Hypersensitivity reactions. Aesthet Surg J. 2005;25:403–405
11. Hügle T, Bircher A, Walker UA. Streptococcal hypersensitivity reloaded: Severe inflammatory syndrome in Behcet’s disease following 23-valent polysaccharide Streptococcus pneumoniae vaccine. Rheumatology (Oxford). 2012;51:761–762
12. Hamilton RG, Strobos J, Adkinson NF Jr. Immunogenicity studies of cosmetically administered nonanimal-stabilized hyaluronic acid particles. Dermatol Surg. 2007;33(Suppl 2):S176–S185
13. Lupton JR, Alster TS. Cutaneous hypersensitivity reaction to injectable hyaluronic acid gel. Dermatol Surg. 2000;26:135–137
14. Micheels P. Human anti-hyaluronic acid antibodies: Is it possible? Dermatol Surg. 2001;27:185–191
15. Arron ST, Neuhaus IM. Persistent delayed-type hypersensitivity reaction to injectable non-animal-stabilized hyaluronic acid. J Cosmet Dermatol. 2007;6:167–171
16. Rimmer J, Hamilton S, Gault D. Recurrent mycobacterial breast abscesses complicating reconstruction. Br J Plast Surg. 2004;57:676–678
17. Rodriguez JM, Xie YL, Winthrop KL, et al. Mycobacterium chelonae facial infections following injection of dermal filler. Aesthet Surg J. 2013;33:265–269
18. Costagliola C, Del Prete A, Winkler NR, et al. The ability of bacteria to use Na-hyaluronate as a nutrient. Acta Ophthalmol Scand. 1996;74:566–568
19. Lee YN, Kim JD, Lew J. Comparison of mycobacterial growth in Dubos medium, hyaluronate supplemented medium and umbilical cord extract based medium. Yonsei Med J. 1977;18:130–135
20. Rahav G, Pitlik S, Amitai Z, et al. An outbreak of Mycobacterium jacuzzii infection following insertion of breast implants. Clin Infect Dis. 2006;43:823–830
21. Lear G, Lewis GD Microbial Biofilms: Current Research and Applications. 2012 Norwich, United Kingdom Caister Academic Press
22. Parsek MR, Singh PK. Bacterial biofilms: An emerging link to disease pathogenesis. Annu Rev Microbiol. 2003;57:677–701
23. Wolcott RD, Ehrlich GD. Biofilms and chronic infections. JAMA. 2008;299:2682–2684
24. Imamura Y, Chandra J, Mukherjee PK, et al. Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrob Agents Chemother. 2008;52:171–182
25. Auler ME, Morreira D, Rodrigues FF, et al. Biofilm formation on intrauterine devices in patients with recurrent vulvovaginal candidiasis. Med Mycol. 2010;48:211–216
26. Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45:999–1007
27. Rohrich RJ, Monheit G, Nguyen AT, Brown SA, Fagien S. Soft-tissue filler complications: The important role of biofilms. Plast Reconstr Surg. 2010;125:1250–1256
28. Bjarnsholt T, Tolker-Nielsen T, Givskov M, Janssen M, Christensen LH. Detection of bacteria by fluorescence in situ hybridization in culture-negative soft tissue filler lesions. Dermatol Surg. 2009;35(Suppl 2):1620–1624
29. Christensen L. Normal and pathologic tissue reactions to soft tissue gel fillers. Dermatol Surg. 2007;33(Suppl 2):S168–S175
30. Sundaram H, Voigts B, Beer K, Meland M. Comparison of the rheological properties of viscosity and elasticity in two categories of soft tissue fillers: Calcium hydroxylapatite and hyaluronic acid. Dermatol Surg. 2010;36(Suppl 3):1859–1865
31. Glogau RG, Kane MA. Effect of injection techniques on the rate of local adverse events in patients implanted with nonanimal hyaluronic acid gel dermal fillers. Dermatol Surg. 2008;34(Suppl 1):S105–S109
32. Sundaram H, Monheit GD, Goldman MP. Clinical experiences with hyaluronic acid fillers: Roundtable discussion. J Drugs Dermatol. 2012;11:15–27
33. Sundaram H, Flynn T, Cassuto D, Lorenc P. New and emerging concepts in soft tissue fillers: Roundtable discussion. J Drugs Dermatol. 2012;11(Suppl):12–25
34. Grippaudo FR, Mattei M. High-frequency sonography of temporary and permanent dermal fillers. Skin Res Technol. 2010;16:265–269
35. Voigts R, DeVore DP, Neuman P, Grazer J. Dispersion of calcium hydroxylapatite accumulations in the skin: Animal studies and clinical practices. Dermatol Surg. 2010;36(Suppl):798–803
36. Kanoh S, Rubin BK. Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clin Microbiol Rev. 2010;23:590–615
37. Cassuto D, Marangoni O, De Santis G, Christensen L. Advanced laser techniques for filler-induced complications. Dermatol Surg. 2009;35(Suppl 2):1689–1695
38. Sundaram H, Carruthers JCarruthers J, Carruthers A. The glabella and central brow. Procedures in Cosmetic Dermatol: Soft Tissue Augmentation. 20133rd ed London Elsevier Saunders:88–99
39. Wilson LA, Julian AJ, Ahearn DG. The survival and growth of microorganisms in mascara during use. Am J Ophthalmol. 1975;79:596–601