Abstract

Hyaluronic acid fillers are considered to be the most safe fillers but rare side effects can
appear and must be known. A severe reaction is represented by the development of biofilm.
Herein, we report a case of a 57 year-old female patient who returned in our clinic 48 hours after
a injection of 1,2 ml of hyaluronic acid filler in both nasolabial folds. The patient presented
erythema, edema and pain on the left zygomatic arch, the left malar region and the left side of the
nose. The blood tests showed an acute inflammation of bacterial etiology. Antibiotic and steroid
therapy was performed without success and the process extended to the right zygomatic arch and
the right malar region. The decision of changing the antibiotic and administrating also
hyaluronidase was effective. The diagnostic of biofilm is difficult especially because the most
accurate methods , such as fluorescent in situ hybridization, are not available. Further studies are
necessary to understand how biofilms can be prevented, promptly diagnosed and combated.

Keywords
Skin aging, hyaluronic acid, facial rejuvenation, hypersensitivity reactions, edema,
erythema, modulse, vascular occlusion, foreign ody granuloma, biofilm, hyaluronidase injection.

Introduction
Skin aging is a complex process induced by multiple factors: extrinsic (environmental
factors such as air pollution, prolonged sun exposure, nicotine, miscellaneous lifestyle, poor
nutrition) and intrinsic (the genetic background), resulting in morphological alterations mainly of
the dermis, in sun-exposed areas, and of the epidermis in sun-protected areas. [1, 2]
The major components involved in tissue repair are glycosaminoglycans and particularly
hyaluronic acid (HA) , a high molecular weight molecule with the capacity to bind and retain
water molecules. HA is found naturally in every cell of the human body and represents a major
component of the synovial fluid, articular cartilage, ligaments and tendons and skin (50% of the
bodies HA in found here). [3]
Since 2002 -the approval of FDA-, the use of HA became one of the most popular
procedures for facial rejuvenation (lip augmentation, scars treatment, glabellar, marionette, neck,
nasolabial wrinkles). HA has an intrinsic solubility translated to a rapidly clearance when
injected therefore modifications to its mechanical properties – such as gel firmness and half-life
of the HA-product- were necessary. This process varies from a manufacturer to another and this
is the reason why there are differences in longevity and in viscosity of the different products [4,
5] .
Even though HA is the safest filler agent in cosmetic indications today due to its
biocompatibility and biodegradability, rare side effects may appear and must be known. These
complications can be divide into early (hours to days post procedure) and delayed onset
complications (weeks to years post procedure). [6]
The most common early complications are caused by local technical problems during the
injection or by the introduction of a foreign material with a secondary acute inflammatory
response and include pain, edema, erythema and echymosis- mild side effects that usually last 3-
5 days and can be managed by application of ice. Other early complications include
hypersensitivity reactions attributed to the impurities from the bacterial fermentation process or
to the hyaluronic associated protein component [7] ; bacterial infections caused by the skin
surface breakage or viral infections ( reactivation of herpes simplex infection especially when the
procedure is performed in the lips area ) can be managed by prescribing antibiotics respectively
antivirals. If the filler is injected superficially in the skin, small blue papules or nodules can
appear – the Tyndall effect-[8]. As a result of a poor technique, too much material can
accumulate in an area causing a non-inflammatory, well-defined and highly mobile nodule. This
type of nodule will resolve with hyaluronidase treatment [9].
One early complication but also the most concerning and potentially irreversible one is
vascular occlusion resulting in skin necrosis or blindness or cerebral ischemic events. In most
cases it is caused by the intravascular injection into an artery causing an embolism or by
compression. The injection sites most commonly believed to have a greater risk for this
concerning side effect are the glabella and the nasolabial fold and the risk factors include the
deep injection of HA at or near the site of named vessel, a large amount of filler injected or the
presence of deep tissue scars. The gold standard treatment consists in hyaluronidase injection.
[10, 11, 12]
The delayed complications of HA injection include foreign body granulomas,
depigmentation, scarring and biofilms. Foreign body granulomas are a chronic inflammatory
reaction that can occur after the injection of any dermal filler. In HA fillers, small amounts of
proteins contaminants can be present. The foreign body is trapped by the inflammatory reaction
preventing the adsorption of injected material (secondary to protein adsorption, macrophage
adhesion, macrophage fusion and crosstalk) [13] and appears as a cystic granuloma with a firm
consistency. The treatment consists in hyaluronidase injection, intralesional corticosteroids or
surgery. Also, intralesional 5-fluorouracil and laser were used successfully [6, 13].
A biofilm represents a group of microorganisms in which cells stick, using an
extracellular matrix composed of extracellular polymeric substances, to each other and often to a
surface. It represents a key factor of bacteria resistance. [14]
One of the most important features of microbial biofilms is their high antibiotic
resistance. The biofilm matrix has also the role of barrier that delays the diffusion of antibiotics.
The time required for an antibiotic to penetrate the matrix is longer then the duration of antibiotic
treatment. Also, a subpopulation of persister cells is formed. Their particularity is that this cells
have a very slow growth and are highly tolerant to antibiotic treatment, being the only surviving
cells after the treatment, creating a reservoir of cells.[15] Another protective mechanisms of
biofilms are represented by nutrient limitations and the adaptive stress responses – peripheral
cells consume the nutrients while the diffusion through the biofilm is reduced inducing a
repression of division and growth leading to a higher amino acid synthesis and the survival of
bacteria. [16]
Within the human body, first biofilms discovered were on dental plaques – many species
of bacteria such as Streptococcus mutans and fungi such as Candida albicans-. Biofilms are
involved in a wide variety of microbial infections (60-80%), from urinary tract infections,
catheter infections, sinusitis, gingivitis to endocarditis, infections of heart valves, different types
of prosthesis and osteonecrosis and osteomyelitis. [17]
Biofilm is a very rare but dangerous complication of HA-based filler injections.
Concerning the formation of biofilms after this procedure, several studies demonstrated the
presence of bacteria in dormant state followed by activation (awakening) after a disturbance of
its local environment. This disturbance can be represented by trauma, injection, manipulation etc.
[19, 20]. This is the reason why patients with chronic dental problems, chronic sinusitis or other
infections have a greater risk to develop this side effect [21]. Also, injecting a HA-filler induces
a normal body reaction, in which lymphocytes, macrophages, plasma cells are involved.
Normally this reaction has no clinical significance. During the injection of HA-filler, bacteria
can be introduced causing the formation of biofilm, especially if the injection is deeply placed.
Pre- and post- procedure aseptic precautions are essential and this procedure should not be
considered a “semi-sterile procedure” [22].
The diagnosis of biofilm is difficult. Clinically, the patient presents with redness,
swelling, pain, local heat and, in some cases, pus or fistula. The microbiological exam will reveal
a sterile abscesses. The most accurate method of diagnosis is FISH ( fluorescence in situ
hybridization) [23].
Case Report
A 57 year-old Caucasian woman, former economist, presented for facial rejuvenation.
The medical history revealed no relevant chronic diseases, no treatment with any medicine and
no past history of using dermatological fillers. The patient denies smoking or drinking alcohol
and states prolonged sun-exposure. On physical examination, the patient presented forehead
transverse furrows, fine wrinkles and lines on the lower lid, the lateral aspect of the orbital
region, deep nasolabial folds, deep corner of the mouth lines and marionette lines ( Fig.
1,2,3,4,5) .
Fig 1. Forehead transverse furrows, deep nasolabial folds,deep corner of the mouth lines

Fig 2,3. Deep forehead transverse furrows

Fig 4,5. Deep nasolabial folds, deep corner of the mouth lines and marionette lines.

After discussing the treatment options with the patient, she signed the informed consent and
after disinfection according to surgical standards, the treatment prescribed was 40 UI of
Abobotulinumtoxin A for the upper region and 1 ml of hyaluronic acid filler for volume of the
lips and 1,2 ml of hyaluronic acid filler for the nasolabial folds.
The patient tolerated the procedure well but, she returned to the our clinic two days after with
pain, redness, swelling on the left zygomatic arch, the left malar region and the left side of the
nose
(Fig 6,7).
 Fig. 6,7 Redness and swelling on the left zygomatic arch, the left malar region and the left side
of the nose, 48 hours post intervention.

The therapy prescribed was antibiotic therapy ( Azithromycin 1g per day , Metronidazole 500
mg per day and Amoxiciline clavulanate 3g per day )for three days along with Prednison (40 mg
per day in the first day, 30 mg per day in the second day and 20 mg per day in the third day) but
the treatment failed to improve the patient’s condition. The process extended to the right
zygomatic arch (Fig 8,9).
Fig. 8,9 Redness and selling on both zygomatic arches and malar region ( 5 days post
intervention)

Laboratory blood tests showed an acute inflammation of bacterial etiology ( Tabel 1).

TEST RESULT CLINICAL REFERENCE

White blood cells 11,38 4-10 109/L
Neutrophils 8,17 2-8 109 /L
Lymphocytes 2,2 1-4 109 /L
Eosinophils 0,2 0,0-0,5 109 /L
CRP 13,13 0-5 mg/L
Tabel 1

The antibiotic therapy was continued for three days along with nonsteroidal anti-inflammatories (
Piroxicam 800 mg per day). The result of the treatment was still unsatisfactory so, the decision
made was to change the antibiotics prescribed to Ciprofloxacine 1g per day along with
antihistamines and the administration of Hyaluronidase 1500UI with significant improvement.

Discussion
Hyaluronic acid-based fillers are well tolerated, severe reactions are rare. Biofilm is a dangerous
complication not only because the correct is difficult but especially because one of the biofilms
feature is their high resistance to antibiotics. There are different theories about how the use of
fillers can increase the risk of biofilms. One of these considers the improper disinfection of the
skin along with the presence of potential pathogens in a patient with decreased immunity to be
the cause. Another theory is that the bacteria are present in a dormant state and the trauma or the
injection can activate (awaken) them [19,20,22]. Also, there is the possibility for the filler to
carry the risk so, the production methods must be very carefully studied.
In the reported case, we couldn’t identity the most possible cause for the biofilm development.
The diagnostic was made based on history, clinical aspects and the available tests results
showing an acute inflammation of bacterial etiology. The limitation of the management of this
case consists in the unavailability of more diagnostic tests such as fluorescent in situ
hybridization which represents the most accurate method of diagnosis when facing a probable
biofilm-related infection. In the reported case, the treatment of the patient was in accordance
with the guidelines (antibiotics, corticotherapy, antihistamines ) but the success of the therapeutic
plan as mainly the consequence of using hyaluronidase to remove the hyaluronic acid, conform
to the theory ,, no foreign substance no biofilm’’.

Disclosure
The authors report no conflict of interest in this work.

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