EWMA FOCUS ON ANTIMICROBIALS AND …...T he European Wound Management As-sociation (EWMA) has a...

Volume 13Number 1April 2013

Published byEuropeanWound ManagementAssociation

EWMA FOCUS ON ANTIMICROBIALS AND DEBRIDEMENT

Ann-Mari Fagerdahl

EWMA Council

The EWMA JournalISSN number: 1609-2759

Volume 13, No 1, April, 2013

The Journal of the EuropeanWound Management Association

Published twice a year

Editorial BoardSue Bale, UK, Editor

Jan Apelqvist, SwedenGeorgina Gethin, Ireland

Martin Koschnick, GermanyMarco Romanelli, Italy

Rytis Rimdeika, LithuaniaJosé Verdú Soriano, Spain

Rita Gaspar Videira, Portugal Salla Seppänen, Finland

EWMA web sitewww.ewma.org

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Copies printed: 13.000

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in hard copies to members as part of their EWMA membership.

EWMA also shares the vision of an “open access” philosophy,

which means that the journal is freely available online.

Individual subscription per issue: 7.50€Libraries and institutions per issue: 25€

The next issue will be published in October 2013. Prospective material for

publication must be with the EWMA Secretariat as soon as possible

and no later than August 1st 2013.

The contents of articles and letters inEWMA Journal do not necessarily reflect

the opinions of the Editors or the European Wound Management Association.

All scientific articles are peer reviewed by EWMA Scientific Review Panel.

Copyright of published materialand illustrations is the property of

the European Wound ManagementAssociation. However, provided prior

written consent for their reproduction, including parallel publishing

(e.g. via repository), obtained from EWMA via the Editorial Board of the Journal,

and proper acknowledgement, such permission will normally

be readily granted. Requests to reproduce material should state

where material is to be published, and, if it is abstracted, summarised,

or abbreviated, then the proposed new text should be sent to the

EWMA Journal Editor for final approval.

All issues of EWMA Journal are CINAHL listed.

CO-OPERATING ORGANISATIONS’ BOARD

Esther Armans Moreno, AEEVHChristian Thyse, AFISCeP.beTommaso Bianchi, AISLeCRoberto Cassino, AIUCAníbal Justiniano, APTFeridasGilbert Hämmerle, AWAJan Vandeputte, BEFEWOVladislav Hristov, BWAEls Jonckheere, CNCLenka Veverková, CSLRIvana Vranjkovic, CWAArne Buss, DGfWBo Jørgensen, DSFSHeidi Castrén, FWCSPedro Pacheco, GAIFJ. Javier Soldevilla, GNEAUPP

Christian Münter, ICWAleksandra Kuspelo, LBAASusan Knight, LUFLoreta Pilipaityte, LWMACorinne Ward, MASCHunyadi János, MSKTSuzana Nikolovska, MWMAAnne Wilson, NATVNSKristin Bergersen, NIFSLouk van Doorn, NOVWArkadiusz Jawień, PWMASeverin Läuchli, SAfW (DE)Hubert Vuagnat, SAfW (FR)Goran D. Lazovic, SAWMAMária Hok, SEBINKOF. Xavier Santos Heredero, SEHER

Sylvie Meaume, SFFPCSusanne Dufva, SSISJozefa Košková, SSOORLeonid Rubanov, STW (Belarus)Guðbjörg Pálsdóttir, SUMSCedomir Vucetic, SWHS SerbiaMagnus Löndahl, SWHS SwedenAlison Hopkins, TVSJasmina Begić-Rahić, URuBiHZoya Ishkova, UWTOBarbara E. den Boogert-Ruimschotel, V&VNJulie Jordan O’Brien, WMAISkender Zatriqi, WMAKNada Kecelj Leskovec, WMASMustafa Deveci, WMAT

Paulo Jorge Pereira Alves, PortugalCaroline Amery, UKJan Apelqvist, SwedenSue Bale, UKMichelle Briggs, UKStephen Britland, UKMark Collier, UKRose Cooper, UKJavorka Delic, SerbiaCorrado Maria Durante, ItalyBulent Erdogan, TurkeyAnn-Mari Fagerdahl, SwedenMadeleine Flanagan, UK Milada Franců, Czech RepublicPeter Franks, UKFrancisco P. García-Fernández, Spain

Magdalena Annersten Gershater, SwedenGeorgina Gethin, IrelandLuc Gryson, BelgiumEskild W. Henneberg, DenmarkAlison Hopkins, UKGabriela Hösl, AustriaDubravko Huljev, CroatiaGerrolt Jukema, NetherlandsNada Kecelj, SloveniaKlaus Kirketerp-Møller, DenmarkZoltán Kökény, HungaryMartin Koschnick, Germany Severin Läuchli, SchwitzerlandMaarten J. Lubbers, NetherlandsSylvie Meaume, France Zena Moore, UK

EWMA JOURNAL SCIENTIFIC REVIEW PANEL

Magdalena Anner-sten Gershater

Jan ApelqvistPresident

Zena MooreImmediate Past President

Mark CollierBarbara E. den Boogert-Ruimschotel

Paulo Alves Javorka Delic

Salla SeppänenPresident Elect

Robert StrohalRytis RimdeikaElia Ricci José Verdú SorianoSebastian Probst

Gerrolt JukemaScientific Recorder

Corrado M. DuranteTreasurer

Dubravko HuljevSecretary

Martin KoschnickNada Kecelj-LeskovecGeorgina Gethin

Christian Münter, GermanyAndrea Nelson, UKPedro L. Pancorbo-Hidalgo, Spain Hugo Partsch, AustriaPatricia Price, UKSebastian Probst, SchwitzerlandElia Ricci, ItalyRytis Rimdeika, LithuaniaZbigniew Rybak, PolandSalla Seppänen, FinlandJosé Verdú Soriano, Spain Robert Strohal, AustriaRichard White, UKCarolyn Wyndham-White, SwitzerlandGerald Zöch, Austria

Sue BaleEWMA Journal Editor

2

Science, Practice and Education

Organisations

Cochrane Reviews

EWMA

4 Editorial

7 The Biofilm-forming capacity of staphylococcus aureus from chronic wounds can be useful for determining Wound-Bed Preparation methodsYuliya Yarets, L. Rubanov, I. Novikova, N. Shevchenko

15 In vitro efficacy of various topical antimicrobial agents against multidrug-resistant bacteriaMarianna Hajská, L. Slobodníková, H. Hupková, J. Koller

20 The mTOR inhibitors and the skin wound healingFarida Benhadou, V. del Marmol

24 A Review of Evidence for Negative Pressure Wound Therapy (NPWT) use Post Spinal SurgeryRoss A. Atkinson, K. J. Ousey, S. Lui, J. B. Williamson

27 A randomized study on the effectiveness of a new pressure-relieving mattress overlay for the prevention of pressure ulcers in elderly patients at riskElia Ricci, C. Roberto, A. Ippolito, A. Bianco, M. T. Scalise

35 Motorcycle ride position, venous return, and symptoms of chronic venous insufficiencyEllie Lindsay, P. Vowden, K. Vowden, J. Megson

38 Archagathus – History’s first wound expertElia Ricci

40 Abstracts of Recent Cochrane ReviewsSally Bell-Syer

46 EWMA Journal Previous Issues and Other Journals

49 EWMA Antimicrobial DocumentFinn Gottrup

52 Finn Gottrup – Honorary lecturer of the EWMA Conference 2013Jan Apelqvist

55 EWMA Document: Résumé: Debridement – An updated overview and clarification of the principle role of debridementRobert Strohal, Dissemond, J., O’Brien, J., Piaggesi, A., Rimdeika R., Young, T., Apelqvist, J

63 EWMA policy paper: Patient Safety & Pressure UlcersZena Moore

66 E-Health Symposium at EWMA 2013, CopenhagenClaus Duedal Pedersen, Jan Apelqvist

68 The Diabetic Foot Symposium: A Nordic PerspectiveKarel Bakker, Jan Apelqvist

74 Conference Calendar

75 Report from the 4th Pisa International Diabetic Foot Course Alberto Piaggesi

76 Report from the 1st Charcot Foot CourseMaximillian Spraul

77 Russian Wound Conference In NorgorodAlexey Baindurashvili, Vladimir Kenis

78 WAWLC Symposium at EWMA 2013David Keast

80 AAWC NewsRobert J. Snyder

82 AEEVH: Spanish Association of Vascular Nursing and WoundsEsther Armans

84 AWMA: Australian Wound Management Association in 2012Bill McGuiness

86 Review of the 5th Symposium on Chronic Wounds with International Participation: Atypical Wounds Nastja Kucišec-Tepeš

88 SWHS: Report from First Congress for treatment of chronic woundsJavorka Delic, Simon Pandjatijan

90 EWMA Cooperating Organisations

The April 2013 edition of the EWMA Journal Electronic Supplement consist of all the accepted abstracts for

the EWMA 2013 Conference in Copenhagen. The Electronic Supplement is divided into

Oral presentations and Poster presentations and it is possible to download individual abstracts

as well as the entire supplement (including all the abstracts) at:

http://ewma.org/english/publications/ewma-journal/electronic-supplement.html

ELECTRONIC SUPPLEMENT

3

Join us for our symposia on the 15th May 2013 which will be held at EWMA 2013 in Copenhagen

* At smith&nephew our products promote wellbeing which helps to reduce the human and economic cost of wounds.

© Smith & Nephew January 201340823

Wound Management Smith & Nephew, Medical Ltd, 101 Hessle Road, Hull, HU3 2BN, UK T +44 (0)1482 225181 - F +44 (0)1482 328326 - www.smith-nephew.com/wound

The European Wound Management As-sociation (EWMA) has a track record of presenting in-depth investigations of selected wound management topics. During the past year, EWMA has focused on debridement and the use of antimicrobials in infected wounds. The EWMA document “Debridement – An up-dated overview and clarification of the principal role of debridement” was published in the Journal of Wound Care last January. EWMA now looks forward to presenting the ”EWMA Antimicrobial Document” at EWMA 2013 in Copenhagen from May 15 to 17. Further information about these two documents is available on pages 49 and 55 of this issue of the EWMA Journal.

EWMA has grown in significance in recent years due to its participation in a wide and growing va-riety of activities. The EWMA documents play a central role by incorporating the objectives of our association in practice, bringing specific topics into focus to advocate for policy change at the national or European level, and improving the work and education of health care professionals. The docu-ments are interdisciplinary where EWMA recruit and involve specialists and co-workers from various disciplines to be able to present and analyse each topic from a broad perspective.

The EWMA documents aim to:n Present in-depth investigations of specific

treatment strategies and methodologies.n Provide an overview of the best available

evidence and/or how this evidence may be achieved.

n Highlight a particular topic that has not yet been sufficiently described in the existing literature.

n Bring specific topics into focus to advo-cate for policy change at the national and European levels.

n Ensure that the documents are edited by editors appointed by the EWMA Council.

n Include authors that represent the views and expertise of both EWMA as well as other stakeholders.

n Present information from a clinical and sci-entific viewpoint and with a focus on benefits to the patient.

During 2013, EWMA will produce two new docu-ments. The first document will focus on wound care in home care settings across Europe; a work-ing group has been established and is meeting to assemble this document. The second document will outline multidisciplinary approaches with a specific focus on organisational and political aspects of wound care.

We welcome participants who plan to attend the EWMA 2013 conference in Copenhagen to also at-tend the sessions that present the documents on de-bridement and the use of antimicrobials in wound care.

Sue Bale, EWMA Journal Editor and Jan Apelqvist, EWMA President

Editorial

Presenting In-depth Wound Care Topics

EWMA Journal 2013 vol 13 no 1 5

BSN medical GmbH · Quickbornstraße 24 · 20253 Hamburg · www.bsnmedical.com

85%healing success*

Teaming up for more success.

Venous Leg Ulcer Therapy

Cutimed® Sorbact®

Cutimed® Siltec

JOBST ® CompriforeJOBST ® UlcerCARE TM

A European case study of BSN medical showed that the combination of Advanced Wound Care and Compression Therapy signifi cantly improved 85 % of

venous leg ulcers and completely healed 53 % of all cases within 12 weeks.*

With its products Cutimed® Sorbact®, Cutimed® Siltec , JOBST® Comprifore and JOBST® UlcerCARE TM, BSN medical enables effective management of venous

leg ulcers while offering ease of use for doctors and patients – the perfect combination for optimal wound healing and high patient compliance.

*Currently in preparation: A European Approach for Successful Venous Leg Ulcer Healing (VERUM).

Advanc

ed Woun

d Care

Compres

sion The

rapy

123321_AZ_VenousLegUlcerTherapy_A4.indd 1 15.03.13 11:34

Yuliya Yarets1, MD, PhD

Leonid Rubanov2, MD

Iryna Novikova1, MD, PhD, Professor

Natalia Shevchenko3, MSc

1Clinical Laboratory Diagnostic Department, Gomel State Medical Uni-versity (Gomel, Belarus)

2Regional Centre for Thermal Injury, Wounds, Wounds Infection, and Reconstructive Surgery and President of Gomel Wound Management Society (Gomel, Belarus)

3Bacteriologist, Clinical-Diagnostic Laboratory Department, Republican Centre of Radiation Medi-cine and Human Ecology (Gomel, Belarus)

Correspondence: [email protected]

Conflict of interest: none

ABSTRACTIntroduction: Staphylococcus aureus is frequent-ly found in chronic wounds. Bacterial biofilms within chronic wounds impact on the surgical closure of the wounds with skin grafting. Meth-ods of wound bed preparation (WBP) influence the microorganisms within the biofilm. We used in vitro monitoring to analyse the possible influ-ence of WBP methods on the in vivo formation of biofilms.Aim: To conduct a comparative analysis of the impact of WBP treatment methods on the capac-ity of S. aureus isolated from chronic wounds to form biofilms in vitro.Methods: We modeled S. aureus biofilm forma-tion in 96-well plates. We assayed the capacity of bacteria isolated from chronic wounds to form biofilms at the time of patient admission and af-ter treatment with either ultrasound debridement (UD) and topical negative pressure (TNP; Main Group) or standard dressings (Control Group). We also compared biofilm formation by bacteria from patients with different grafting outcomes.Results: The treatment of chronic wounds with UD and TNP reduced the capacity of S. aureus to synthesise a major biofilm substance. S. aureus iso-lates from patients with favourable skin-grafting results had a lower capacity to form biofilms in vitro compared with isolates from patients with poor skin-grafting results. The use of UD and TNP for surgical closure reduced the length of the skin-graft healing process compared with the use of standard bandages. Conclusion: The isolation from chronic wounds of significant titers of S. aureus strains with a high capacity to form biofilms within 2 to 6 hours of incubation in vitro highlights the advantage of us-ing hardware methods (UD and TNP) for WBP for surgical closure.

INTRODUCTIONAll chronic wounds harbor a diverse microflora that contributes directly and indirectly to their non-healing phenotype[1,2]. Exposed and devital-ised tissue in chronic wounds provides a favour-able environment for colonisation by a wide va-riety of microorganisms[3,4]. Frequently, chronic wounds harbor aerobic gram-positive cocci (Staphylococcus aureus is reported to be present in frequencies varying from 43% to 88% of the ulcers)[5,6]. Biofilms are a significant factor that differentiates chronic wound infections from acute wound infections[3]. The key characteristics of biofilm-forming bacteria are their resistance to host defenses and their tolerance of antimicrobi-als[7,8]. Bacterial biofilms are a complex microen-vironment consisting of single or mixed species that are attached to one another or to surfaces and encased within extracellular polymeric substances (EPSs)[9]. The EPS generally consists of polysac-charides, and the bacteria within the EPS induce chronic inflammation that delays healing[10,11]. The polysaccharide components of S. aureus cell walls facilitate adherence to extracellular matrix components such as fibronectin or collagen in wounded tissues[1,2]. Different methods of treat-ing pathogenic biofilms in chronic wounds can improve patient outcomes[3,7,11].

Sibbald et al. (2000) and Falanga (2000) first de-scribed the concept of wound bed preparation (WBP)[12,13]. The approach stresses that the suc-cessful diagnosis and treatment of chronic wounds require holistic care and a team approach[14,15]. The three main objectives of care in the WBP model are debridement, bacterial balance, and exudate management. The achievement of the key objectives should lead to a well-stabilised wound supported by a sufficiently vascularised wound bed.

The Biofilm-forming capacity of staphylococcus aureus from chronic wounds can be useful for determining Wound-Bed Preparation methods


BSN medical GmbH · Quickbornstraße 24 · 20253 Hamburg · www.bsnmedical.com

85%healing success*

Teaming up for more success.

Venous Leg Ulcer Therapy

Cutimed® Sorbact®

Cutimed® Siltec

JOBST ® CompriforeJOBST ® UlcerCARE TM

A European case study of BSN medical showed that the combination of Advanced Wound Care and Compression Therapy signifi cantly improved 85 % of

venous leg ulcers and completely healed 53 % of all cases within 12 weeks.*

With its products Cutimed® Sorbact®, Cutimed® Siltec , JOBST® Comprifore and JOBST® UlcerCARE TM, BSN medical enables effective management of venous

leg ulcers while offering ease of use for doctors and patients – the perfect combination for optimal wound healing and high patient compliance.

*Currently in preparation: A European Approach for Successful Venous Leg Ulcer Healing (VERUM).

Advanc

ed Woun

d Care

Compres

sion The

rapy

123321_AZ_VenousLegUlcerTherapy_A4.indd 1 15.03.13 11:34

EWMA 2012

ienna


Debridement promotes healing by removing non-viable tissue and biofilms that shield bacterial colonies[16]. Ul-trasonic assisted debridement (UD) is a relatively painless method of removing non-viable tissue. UD allows a rapid transition to secondary procedures, and its effectiveness in biofilm disruption is supported by in vitro studies and a decreased bioburden in patients[17,18]. Topical negative pressure (TNP) is reported by clinical and experimental studies to decrease bacterial colonisation. The seal created by the foam and drape used to apply TNP reduces the risk of bacterial colonisation; and TNP causes improved blood perfusion that may increase resistance to infections[19]. The complex use of UD and TNP methods in WBP prior to skin transplant may impact on biofilm formation fol-lowing the transplant and thus reduce the frequency of post-surgery complications.

Our study aimed to conduct a comparative analysis of the in vitro biofilm-forming capacity of S. aureus isolated from chronic wounds before and after wound treatment with various WBP treatment methods (standard bandages or UD/TNP) prior to skin transplant.

MATERIALSWe enrolled 55 patients (20 females and 35 males, aged 25 to 70 years) who sought treatment for chronic wounds at the Gomel Regional Centre for Thermal Injury, Wound, Wounds Infection, and Reconstructive Surgery (Gomel City Clinical Hospital No1, Belarus) during 2011. The chronic wounds consisted of venous leg ulcers (n=16), pressure ulcers (n=6), traumatic ulcers (n=10), and in-flammatory ulcers (n=13). In each case, the duration of the ulcers was more than 4 weeks prior to the patient’s enrollment in the study. Because UD and TNP are al-ready used in the Gomel Regional Centre to treat chronic wounds, approval from the local ethical committee was not necessary. All patients gave written informed consent before being admitted to the study. In all cases, prior treat-ments, including domiciliary outpatient treatment with local remedial therapy and empirical antibiotic therapy, were unsuccessful. At the time of admission to the Gomel Regional Centre, each patient had wounds with clinical signs of inflammation.

METHODSWe divided the patients into two groups, depending on the treatment methods. The pre-procedural wound-treat-ment protocol for the Control Group (n=30) included antiseptic bandages, chlorhexidine, povidone iodine, and polyethylene glycol-based ointments (e.g., Levomecol). We performed WBP 7-10 days after starting the pre-proce-

dural treatment. We changed the patients’ bandages once each day. After the pre-procedural treatment and WBP, the patients received skin grafts.

The pre-procedural wound-treatment protocol for the Test Group (n=25) included UD and TNP. We applied UD at 25 kH using a «Sonoca-185» (Soring Inc., Germany) apparatus. Each patient underwent two UD treatments. We conducted the first UD treatment on the 2nd or 3rd day after beginning the pre-procedural wound-treatment protocol. We performed TNP each day for 5 to 7 days us-ing the Visma-Planar (Belarus) negative pressure-therapy system. The optimal target pressure was 75-125 mmHg; the pressure was dependent on the patient’s tolerance. We changed the wound dressings for the patients in the Test Group every 48 hours. After receiving TNP, the patients in the Test Group underwent a second UD procedure and concomitant skin grafting.

We performed wound closure for the patients in both groups using a split-thickness (0.4 mm) skin graft. None of the patients had intra-procedural complications. We changed the wound dressings for the patients in both groups once per day during the post-procedural period.

OUTCOME ASSESSMENTWe collected data characterising the clinical efficacies of the pre-procedural treatments via detailed visual assess-ment of the regeneration process. Our data included the amount of wound effluent, the condition of the tissue surrounding the wound, the degree of granulation, the wound size and surface condition, and the appearance of edge epithelialisation.

To assess the effectiveness of the skin grafts, we consid-ered the recommended clinical signs of wound readiness: absence of inflammation, expressed exudation, purulent discharge, wound adhesiveness, mature red or bright pink granulations, and the presence of edged epitheli-alisation[12]. We assessed the process of graft healing by considering not only the terms and signs of the fixation, but also the complete graft healing, including the colour and degree of fixation of the graft and the degree of exuda-tion after surgical wound closure. We defined the grafting procedure as successful when graft fixation occurred on the 3rd or 4th day after surgery and complete graft healing occurred within 7 to 9 days after surgery. We determined the presence of graft instability based on paleness and graft failure. We defined graft failure as rejection or dissolution of the graft in the immediate post-procedural period (day 8, ±3 days, on average). If a graft failed, then the patient required future repetition of the procedure.


BACTERIAL STRAINSWe isolated S. aureus from chronic wounds. Previous work by our group demonstrated that Staphylococcus species pre-dominate in cultures isolated from chronic wounds [20].

BIOFILM ASSAYWe modeled biofilm formation in sterile 96-well polys-terene microtiter plates[21]. We used Congo red and crystal violet stains to visualise both the matrix and the bacterial cells [22, 23]. We used 95% ethanol to extract the stain connected to the biofilm. We measured the optical den-sity (OD) of each sample at a wavelength of 540 nm (for crystal violet/ethanol solution) and 490 nm (for Congo red/ethanol solution) using a Sirio microplate reader (Seac Radium Group, Italy).

We isolated S. aureus from the patients’ wounds before and after WBP and compared the biofilm formation capacities of the isolates from patients with different skin-grafting outcomes. We performed all of our experiments in the Clinical-Diagnostic Laboratory of the Republican Centre of Radiation Medicine and Human Ecology and at the Clinical Laboratory Diagnostic Department of Gomel State Medical University (Gomel, Belarus). We presented the results as medians with lower and upper quartiles (M [25-75%]). We used a non-parametric Mann-Whitney U-test to compare the two study groups the Wilcoxon test to compare dependent samples (variables). We considered results to be statistically significant when p105 c.f.u.). We isolated 20 strains from patients in the Control Group and 15 strains from patients in the Test Group. We con-sidered bacterial contamination to be significant at a titer


Fig. 1. Complete graft healing terms of the patients with different WBP treatment methods.


of 103-105 c.f.u. At the first bacteriological examination, all of the isolates had titers >105 c.f.u. After treatment with UD and TNP, the titers decreased up to 103-105c.f.u. In all of the patients in the Control Croup, the titers remained high, more than 105 c.f.u., prior to surgery. During the post-procedural period, we identified 14 strains of S. aureus with titers of 103-105 c.f.u. in the patients in the Control Group. During the same period, we identified nine such strains in the patients in the Test Group.

Figure 2 shows the in vitro biofilm-forming capacity of the S. aureus strains isolated during the first bacteriological ex-amination (Fig. 2). We noted an increase in the OD of the crystal violet-stained eluate from 0,171 (0,057; 0,308) units at 2 hours to 0,568 (0,281; 0,611) units by 4 hours (p=0.015). This change reflects the active reproduction of S. aureus. Dur-ing the remaining incubation time (from 4 to 48 hours), the biomass of the S. aureus isolates in the model biofilms was stable. An increase in the OD of Congo red-stained eluate (p=0.03) indicated a change in the dynamics of the synthesis of the exopolysaccharide (Fig. 2). The absorption parameters did not change during the incubation period from 6 to 24 hours, and we observed simultaneous decreases in EPS accumulation and the OD of Congo red staining (p=0.015).

We observed changes in the dynamics of biofilm formation by the S. aureus strains isolated from the wounds of the Test Group patients after WBP but prior to surgery. After 4 and 6 hours of incubation, the OD values of the Congo red-stained eluates from the samples obtained after WBP were lower than those obtained prior to treatment (p=0.003). This finding indicates a decrease in biofilm polysaccharide production after complex WBP. The in vitro biomass production of the post-WBP isolates from the Test Group decreased 18 hours after

Fig. 2. Biofilm formation by S. aureus strains isolated from patient chronic wounds.

Fig. 3. Biofilm formation by S. aureus strains isolated from chronic wounds during the first examination and after WBP with UD and TNP.


Science, Practice and EducationScience, Practice and Education

the reduction in polysaccharide production (p=0.013; Fig. 3). The biofilm formation at 18 to 24 hours among the strains isolated immediately prior to surgery did not differ from that among the strains isolated at the time of hospital admission. The exopolysaccharide synthesis by the strains isolated after WBP, however, decreased. In addition, the Congo red OD values were lower after 48 hours of incubation than they were initially (p=0.038; Fig. 3).

The dynamics of biofilm formation among the S. aureus strains isolated from the patients in the Control Group following WBP did not differ from those among the strains isolated from the same patients at the time of admission (Fig. 4).

We examined the differences in the dynamics of the graft-healing process and the frequency of graft loss during the post-procedural period (Figs. 5 and 6) by measuring biofilm formation among the strains isolated from loss grafts (Control Group) and at the moment of skin-graft healing (Test Group). The Congo red-absorption parameters of the isolates from the wounds that experienced graft loss were greater than those of the isolates obtained after complete graft healing (p=0.037, p=0.004). This result indicates that graft loss is accompanied by a higher capacity for S. aureus to form biofilms. The S. aureus isolates from the wounds that experienced graft loss demonstrated a lower capacity for biomass formation and an increased capacity for exopolysaccharide accumulation at the beginning of the incubation period (2-4 hours). This finding was confirmed by lower crystal violet-extinction parameters for isolates from the wounds that experienced graft loss (Fig. 6). We observed higher biomass (p=0.01) after 6 hours of incubation, although the OD values for crystal violet stain-ing did not differ between the groups during the rest of the incubation time.

Fig. 4. Biofilm formation dynamics in vitro by S. aureus strains isolat-ed from patient wounds after standard treatment.

Fig. 5. Differences in the capacity for exopolysaccharide synthesis of the biofilm from patients depending on the results of wound closure outcome. Results are shown for patients with favourable skin grafting outcome from the Test Group and for patients with graft failure from the Control Group.


DISCUSSIONExperimental studies describe biofilm formation by different bacteria, including Staphylococcus species[23,24]. Bacteria attach to one another within several minutes and begin to develop the initial EPS within 2 to 4 hours. After beginning to form the EPS, the bacteria become increasingly tolerant to biocides within 6 to 12 hours. Within 24 hours, the bacteria develop mature biofilm colonies that are extremely resistant to biocides and shed planktonic bacteria. Therefore, the application of appropriate treatment at the early stages of biofilm formation is critical for biofilm management in chronic wounds.

UD treatment involves the removal of contaminated tissue and disrupts the formation of the biofilm in the wound by destroying the EPS[17,18]. The use of TNP, which involves the removal of wound exudates and the reduction of bacteria during WBP, prevents bacterial attachment[19]. The complex use of UD and TNP facilitates the formation of granulation tissue and further helps to promote the wound-healing proc-ess[13,14,15]. Our experience is that the combination of UD and TNP is an excellent method for WBP prior to skin grafting, in contrast to other standard treatment methods.

The results of our in vitro studies of biofilm formation by S. aureus strains isolated from chronic wounds support the hypothesis that the complex use of UD and TNP prior to skin grafting has a significant, positive effect on treatment out-comes. Treatment with UD and TNP resulted in a decreased capacity for S. aureus to form biofilms in vitro. The reduced capacity to from biofilms was a predictor of skin-grafting out-come; and the result was confirmed by the differences in OD values for Congo red staining measured at 2 to 18 hours in the Test Group and the Control Group. We detected higher crystal violet-absorbance parameters during the early stages of incubation (2-4 hours) in the patients with favourable skin-grafting outcomes compared with those in the patients with graft loss. The isolates from patients with favourable outcomes exhibited increased proliferation and decreased biofilm-form-ing capacity (Fig. 6). The absence of a thick layer of ESP did not prevent the influence of local factors for wound protection, and thus, S.aureus contamination in wounds treated with UD and TNP did not cause infection or prevent healing. There-fore, we propose that the presence of S. aureus strains with a low biofilm-forming capacity will not influence the process of graft fixation. On the other hand, the presence of S. aureus strains that quickly form biofilms will affect the contact char-acteristics of the wound bed and impede the process of graft fixation, resulting in graft rejection. Hence, the influence of S. aureus biofilm formation should be taken into consideration when choosing the methods for WBP.

The application of standard treatment methods did not alter the capacity of S. aureus to form biofilms. As a result, the

Fig. 6. The formation of the cellular biomass of S. aureus isolated from wounds of patients with different skin graft healing outcomes. Results are shown for patients from the Test Group with favourable skin grafting outcomes and for patients from the Control Group with graft failure.


standard treatment methods did not achieve more favour-able results in graft healing; in the Control Group, 20% of the patients experienced graft failure. Also, patients in the Control Group that had successful graft healing during the post-procedural period exhibited unstable graft signs; as a result, healing took longer in the Control Group than in the Test Group. Therefore, the conventional treatment of chronic wounds appears to be of little use in improving the outcome of skin grafts, and this notion is supported by other studies[24,25].

The complex use of UD and TNP did not influence the key stages of biofilm formation in vitro; these methods, however, directly influenced the pathophysiological mech-anisms of chronic wounds. UD removes non-viable tissues and reduces bacterial load[17,18]. TNP promotes healing via the removal of soluble healing inhibitors from the wound and thus increases tissue perfusion, inactivates capillary au-toregulation, allows the proliferating cells to rest between cycles of cell division and produce new cellular compo-nents, stimulates angiogenesis and epithelialisation, and draws the wound edges closer together[19].

Overall, our isolation of etiologically significant S. aureus strains from chronic wounds and our determination of their capacity to form biofilms with 2 to 6 hours of incuba-tion support the complex use of UD and TNP for WBP before surgical closure.

Implications for Clinical PracticeStudies of the capacity of S. aureus isolates to form a biofilms in vitro can influence the choice of methods for WBP prior to skin grafting. If S. aureus strains with an active capacity to form biofilms in vitro within 2 hours of incubation are isolated from a chronic wound during an initial bacteriological examination, then the patient is a good candidate for UD and TNP, because the bacterial strains in their wounds are likely to influence the healing of the graft. The influence of the thick layer of the main biofilm substance on the process of graft fixation should also be taken into account. Because S. aureus strains with a low capacity for biofilm formation, evidenced by EPS accumulation after 6 hours of incubation in vitro, do not influence graft fixation and healing, conservative methods of standard wound dressing will be effective for WBP in wounds harbouring those strains.

Further researchIn the future, we will repeat these studies with increased numbers of patients in the experimental group and con-firm the results in a practical setting. We are also studying the influence of different methods of wound treatment on biofilm formation by other etiologically important strains of bacteria, such as Enterobacteriaceae, Enterococcus faecalis, and non-fermenting gram-negative rod bacteria.

CONCLUSIONThe biofilm-formation capacity of bacteria from chronic wounds can be used for choosing the best treatment meth-od for WBP prior to skin grafting. m

References

1. Stephens P. Anaerobic cocci populating the deep tissues of chronic wounds impair cellular wound healing responses in vitro. Brit J Dermatol 2003:148:1-11.

2. Wall I, [et al]. Potential role of anaerobic cocci in impaired human wound healing. Wound Repair Regen 2002:10:346-53.

3. James G.A, [et al]. Biofilms in chronic wounds. Wound Rep Regen 2008:16:37-44.

4. Bowler P.G, [et al]. Wound microbiology and associated approaches to wound management. Clin Microbiol Rev 2001:141:247-269.

5. Brook I., Frazier, E. H. Aerobic and anaerobic microbiology of chronic venous ulcers. International Journal of Dermatology 1998:37:426-8.

6. Hansson C, [et al]. The microbial flora in venous leg ulcers without clinical signs of infection. Acta Dermato-Venereologica 1995:75:24-30.

7. Bjarnsholt T, [et al]. Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen 2008:16:2-10.

8. Potera C. Forging a link between biofilm and disease. Science 1999:283:1837-1839.

9. Davis D.G, [et al]. The involvement of cell-to cell signals in the development of a bacterial biofilm. Science 1998:280:295-298.

10. Mack D, [et al]. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear b-1,6-linked glucosaminoglycan: purification and structural analysis. J Bacteriol 1996:178:175-183.

11. Davis S.C, [et al]. Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen 2008:16:23-29.

12. Falanga V. Classifications for wound-bed preparation and stimulation of chronic wounds. Wound Repair Regeneration 2000:8:347-352.

13. Sibbald R.G, [et al]. Preparing the wound bed: debridement, bacterial balance, and moisture balance Ostomy/Wound Management 2000:11(46):14-35.

14. Sibbald R.G, [et al]. Best practice recommendations for preparing the wound bed: Update 2006. Wound Care Canada 2006:1(4): 15-29.

15. Schultz G.S, [et al]. Wound bed preparation: a systematic approach to wound management. Wound Repair and Regeneration 2003:1(11):1-28.

16. Allan N, [et al]. The impact of VERSAJET hydrosurgi-cal debridement on wounds containing bacteria biofilms. Wound Rep Reg 2010:18:A 88.

17. Ensing G.T, [et.al] The combination of ultrasound with antibiotics released from bone cement decreases the viability of planktonic and biofilm bacteria: an in vitro study with clinical strains. J Antimicrob Chemother 2006:58:6:1287-1290.


18. Breuing K.H, [et al]. Early experience using low-frequency ultrasonic in chronic wounds. Annals of plastic surgery 2005:2(55):183-187.

19. European Wound Management Association (EWMA). Position Document: Topical negative pressure in wound management. London: MEP Ltd, 2007. 17 P.

20. Yarets Y.I, Shevchenco N.I, Rubanov L.N. Monitoring of strains and antibiotic sensitivity in Gomel regional centre of thermal injury, wound, wounds infection and reconstructive surgery. Infections in Surgery 2011:3:8-11.

21. Judith H, [et al]. Growing and Analyzing Static Biofilms. In: Current Protocols in Microbiology. John Wiley & Sons, Inc; 2005. Supplement 22. Units 1B.1.1-1B.1.17.

22. Christensen G.D, [et al]. Adherence of slime-produc-ing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982:37:318-326.

23. Deighton M.A, [et al]. Methods for studying biofilms produced by Staphylococcus epidermidis. Methods Enzymol 2001:336:177-195.

24. Bester E, [et al]. Metabolic differentiation in biofilms as indicated by carbon dioxide production rates. Appl Environ Microbiol 2010:76(4):215-233.

25. Rhoads

26. D.D, [et al]. Evidence of biofilms in wounds and potential ramifications. In: Gilbert P, [et al], editors. Biofilms: Coming of Age. Manchester, UK: The Biofilm Club, Manchester University; 2007. p. 131-143.


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ABSTRACTAim: To evaluate the in vitro efficacy of nine topical antimicrobial agents frequently used in our burn centre against five multidrug-resistant bacterial strains isolated from burn wounds of our patients. Material and methods: A simple and re-producible in vitro model was used to evaluate the effects of the following topi-cal antimicrobials: 1% silver sulfadiazine, neomycin with bacitracin, 1% acetic acid, 0,2% nitrofurazone, 1% acetic acid with 1% silver sulfadiazine, Acticoat®, Aquacel®Ag, Atrauman®Ag, and Ialugen®Plus. Survival of five bacteria planktonic forms (Pseu-domonas aeruginosa, methicillin-resistant Staphylococcus aureus, Staphylococcus haemo-lyticus, Enterococcus faecalis, and Escherichia coli) was evaluated 24-hours after applica-tion. Results: The highest efficacies against all strains were found with 1% silver sul-fadiazine, 1% silver sulfadiazine + 1% acetic acid, Acticoat®, Aquacel®Ag and Ialugen®Plus. The combination of neomy-cin with bacitracin in ointment form and Atrauman®Ag were absolutely ineffective.Conclusions: Topical antimicrobial agents play an important role in the treatment of burns, but they should be used accord-ing to the efficacy against bacterial strains in wounds. Thanks to our results we can modify the spectrum of topical antimicrobi-als used in our burn centre. In the future, we would like to examine the efficacy of the topical agents using an in vitro biofilm model.

INTRODUCTIONImprovements in burn wound care have led to longer survival and potentially an extended hospital stay. The most likely cause of death is an infectious complication after surviving the initial burn insult and resuscitation period[1]. It is now estimated that approximately 75% of the mortality following thermal injuries is directly re-lated to infection[2,3,4]. Burn patients are at high risk for infections caused by nosocomially-acquired multidrug-resistant organisms because of generalised humoral and cellular immunodeficiency, gastrointestinal transloca-tion of microorganisms, prolonged hospital stay, and invasive diagnostic and therapeutic procedures[2,5]. The standard of care at specialised burn centres worldwide is early excision of necrotic tissues and grafting, which results in decreased mortality. However, the benefit of topical antimicrobial agents is that they can be used at all levels of care. Another benefit of topical antimi-crobials is that they can be applied directly to the site of colonisation or infection and can be used for both prophylaxis and treatment of burn wound infections[6]. According to 43% of burn specialists worldwide, the antimicrobial activity in a burn dressing is considered to be essential[7]. Bacteria with resistance to multiple systemic antimicrobials are increasing in prevalence, which raises the concern for a subsequent increase in resistance to topical antimicrobial agents[6,8,9]. In the last decades, one of the most commonly applied topi-cal agents for partial-thickness burns has been silver sulfadiazine[10,11]. Despite the possible but uncom-mon side effects attributed to silver sulfadiazine[9,12], this agent is also a reference standard therapy in many studies.

The most frequently used topical agent in our burn centre is a combination of 1% silver sulfadiazine cream and 1% acetic acid solution. In addition to our own clinical experience, several studies have affirmed the efficacy of this solution[13-17]. According to the Euro-pean Wound Management Association position docu-ment [Management of wound infection (2006)], the selection of antimicrobial agents to reduce or eradicate micro-organisms must be influenced by the specificity and efficacy of the agent. However, the decision for

Marianna Hajská1 M.D., PhD Student

Lívia Slobodníková2

PhD., Clinical Micro-biologist

Helena Hupková2 Assoc. Prof. – Head of the Department

Ján Koller1 Assoc. Prof. – Head of the Department

1 Teaching Department of Burns and Reconstructive Surgery, Medical Faculty of Comenius University and University Hospital, Bratislava, Slovakia

2 Institute of Microbiology, Medical Faculty of Comenius University and University Hospital, Bratislava, Slovakia

Corresponding author: mariannahajska@ gmail.com


In vitro efficacy of various topical antimicrobial agents against multidrug-resistant bacteria


selection of a particular topical antimicrobial agent in our burn centre has so far been mostly empiric. The common standard is to take a swab from the burn wound at admis-sion and at least once per week, after which the sample is sent to the microbiological laboratory for evaluation of wound flora and susceptibility testing. The results inform us of bacterial susceptibility to most of the systemic anti-biotics, but not to topical ones. From the high variety of topical antimicrobials only the minimal inhibition con-centration of nitrofurazone and bacitracin are routinely tested in our laboratory so far.

AIMThe aim of this study was to evaluate and compare the effi-cacy of nine topical antimicrobials and investigate whether the multidrug-resistant bacteria isolated from burns of our hospitalised patients are still susceptible against the most frequently applied agents in our burn centre.

MATERIAL AND METHODSMultidrug-resistant bacteria isolated in the year 2011 from burn wounds of patients hospitalised in the Brati-slava Burn Centre were used to create a bacterial collec-tion stored in the research laboratory of the Institute of Microbiology. We tested the susceptibility of five bacte-rial strains [Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus haemolyticus, Enterococcus faecalis, and Escherichia coli] in planktonic form against nine topical antimicrobials (Table 1) using the modified quantitative method originally introduced by Hammond A. et al.[18]. In comparison with the original method, solutions and impregnated dressings were also tested in addition to ointments.

We placed three sterile 6-mm cellulose disks on a petri dish with Luria Bertani agar (LB-agar), and 10 µl bacte-rial suspension containing 10²-10³ colony forming units

(CFU) was dropped on each disk (Figure 1). The surface of the dish was covered with a sterile gauze square (5 × 5 cm) saturated with topical antimicrobials, or with one of the commercially produced impregnated dressings. From each cream/ointment, 1 g was uniformly spread on the gauze. From solutions, 2-ml aliquots were dropped to saturate the gauze. A sterile gauze square without topical antimicrobials was used as a control.

A sterile small glass petri dish was placed on the gauze to maintain direct contact between the agent and the in-oculated disks (Figure 2). The dishes were incubated at 37°C for 24 hours, after which the gauze squares were removed and each disk transferred into a sterile tube con-taining 1 ml phosphate buffered saline (PBS). The tubes were vigorously vortexed three times for 2 minutes to de-tach bacteria from the disks. Suspended cells were serially diluted four times (a 10-fold dilution was used) in PBS and 10-µl aliquots of each dilution were inoculated on LB agar plates. These plates were incubated at 37°C for 16 hours and the numbers of CFU were counted (Figure 3). The final result recorded represented ³ 108 CFU/disk if the bacterial growth was so massive that the number of CFU obtained was uncountable even in the highest dilution.

RESULTS Using five multidrug-resistant bacterial strains in plank-tonic form our results demonstrated strong bactericidal action of five topical antimicrobial agents: 1% silver sul-fadiazine, 1% silver sulfadiazine with 1% acetic acid, Ac-ticoat®, Aquacel®Ag, and Ialugen®Plus. In all of these samples tested no bacterial growth was detected 24 hours after their application. The same bactericidal effect against MRSA, S. haemolyticus, and P. aeruginosa was found with the application of 1% acetic acid. On the other hand, this agent had only a bacteriostatic effect against E. coli and E. faecalis. Nitrofurazone in a 0,2 % concentration

Table 1: List of the tested topical antimicrobial agents

Solutions 1% Acetic Acid Prepared by hospital pharmacy

0,2% Nitrofurazone Prepared by hospital pharmacy

Creams /Ointments

1% Silver Sulfadiazine Dermazin®1% crm, Sandoz Pharmaceuticals

Neomycin with Bacitracin Framykoin® ung, Zentiva

Combination 1% Silver Sulfadiazine with 1% Acetic Acid

Dermazin®1% crm, Sandoz Pharmaceuticals with 1% Acetic acid prepared by hospital pharmacy

Impregnated dressings Acticoat® Smith and Nephew

Consists of three layers: an absorbent rayon/polyester inner core sand-wiched between outer layers of a nanocrystalline silver-coated low-adher-ent polyethylene net

Aquacel®Ag Convatec

Non-woven pad or ribbon dressing composed of sodium carboxymethylcel-lulose and 1.2% ionic silver

Atrauman®Ag Hartmann

Polyamide fibres coated with elemental silver; the impregnation contains Caprylic/Capric/Stearic Triglyceride;Bis-diglyceryl-polyacyladipate-2 and Macrogol 2000

Ialugen®Plus IBSA

1% silver sulfadiazine, 0,2% natrii hyaluronas


was highly effective as well, with the exception against P. aeruginosa where no effect was observed. On the other hand, neomycin with bacitracin and Atrauman®Ag were found ineffective against all of the bacteria tested, as we observed the same bacterial growth using these agents as in the control samples with sterile gauze only. The results are shown in Figure 4.

DISCUSSIONBacterial resistance belongs to the most severe problems in modern medicine. Some authors suggest that resistance against topical antimicrobials increases similar to resistance seen against systemic antibiotics[19]. Animal and clinical studies are performed to determine the efficacy of topi-cal antimicrobials in vitro. While studies on patients are focused on comparisons between new agents and standard therapy[12,20,21], in vitro models are designed to test more agents[1,19,22,23]. To show the efficacy of topical antimi-

Figure 1: Three sterile 6-mm cellulose disks with bacterial suspension placed on the sur-face of an LB-agar plate.

Figure 2: Sterile gauze square placed on top of disks and covered by a small sterile glass petri dish.

Figure 3: Bacterial growth in 10-μl aliquots of the serial dilutions of bacteria detached from the testing disk after 24 hours of incubation with the tested agent.

Figure 4: Bacteria counts (CFU/disk) after 24 hours of action of the tested topical antimicrobials (bacterial count 108 CFU per disk corresponds to ³108 CFU per disk) (1% AA: 1% acetic acid; 1% SS: 1% silver sulfadiazine; neo+bac: combination of neomycin and bacitracin)

crobials against bacterial flora from the wounds of patients it is necessary to establish a standard in vitro model. This is important to test and compare the efficacy of different forms (solutions, ointments, etc.) of agents. We proved that a modification of the method introduced by Ham-mond A. et al. (2011) provides this possibility. Our in vitro method for testing and comparing antimicrobial efficacy is simple and reproducible, and the quantitative analysis of the surviving microbial inoculum is fully suf-ficient to demonstrate the efficacy of the tested agents.The majority of results obtained in our study are in con-cordance with results of other already published studies. For instance, we demonstrated resistance of P. aeruginosa to 0,2% nitrofurazone, which, on the other hand, had a bactericidal effect on the rest of the bacterial strains tested. These findings are similar to those of Conly et al.[24]. We also found high efficacy of 1% silver sulfadi-azine, despite it having been the most frequently used topical antimicrobial in our burn centre for many years and that evidence for bacterial resistance to silver has been reported[25-27]. Similarly high efficacy of this agent was demonstrated by Koo et al.[28]. In addition to our results, the findings of Ryssel et al.[13-16] have shown a bacterio-



static and even a bactericidal effect of acetic acid on various bacterial strains. The clinical efficacy of dressings combin-ing hyaluronic acid and silver sulfadiazine (Ialugen®Plus) was previously shown in a clinical study[29] and has been realised in our burn centre. Now its high antibacterial ef-ficacy was demonstrated in the in vitro model presented in this study. Acticoat® and Aquacel®Ag exhibited similarly excellent antimicrobial activity in our study too, which is in concordance with results published by other groups.[30-34]. We demonstrated excellent efficacy of five topical antimicrobials in preventing wound infection; however, all of our tests were performed in vitro on planktonic bacteria only, and further research on bacterial biofilms and in the clinical setting will be necessary.

CONCLUSIONSWe demonstrated that the topical antimicrobial agents most frequently used in our burn centre (1% silver sul-fadiazine and 1% acetic acid) have high efficacy against

multidrug-resistant bacteria isolated from burn wounds of our patients.

Implication for clinical practiceThe use of topical antimicrobials is very important not only according to usual practice but also according to the establishment of their efficacy in vitro. Even if we were not able to test all varieties of bacteria from wounds of our patients, our having tested the efficacy of the most regularly used agents against resistant strains of bacteria can help burn specialists to prevent and treat burn wound infection more effectively.

Further researchWe are currently performing tests on bacteria in a biofilm form of growth to find out the efficacy of the tested agents in burns treated several hours/days after injury. This test-ing will also be expanded to a larger number of clinical bacterial strains. m


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http://www.ncbi.nlm.nih.gov/pubmed/20542641http://www.ncbi.nlm.nih.gov/pubmed/20542641http://www.ncbi.nlm.nih.gov/pubmed/20542641http://www.ncbi.nlm.nih.gov/pubmed?term=%22Guggenheim M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Thurnheer T%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Gm�r R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Giovanoli P%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Guggenheim B%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Daryabeigi R%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=fundermol burn dressinghttp://www.ncbi.nlm.nih.gov/pubmed/21130579http://www.ncbi.nlm.nih.gov/pubmed/21130579http://www.ncbi.nlm.nih.gov/pubmed/21130579http://www.ncbi.nlm.nih.gov/pubmed?term=%22Neely AN%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Gardner J%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Durkee P%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Warden GD%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Greenhalgh DG%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Gallagher JJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Herndon DN%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Tompkins RG%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Kagan RJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/19060725http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gupta SS%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Singh O%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Bhagel PS%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Moses S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shukla S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Mathur RK%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/22279383http://www.ncbi.nlm.nih.gov/pubmed/21991222http://www.ncbi.nlm.nih.gov/pubmed/21991222http://www.ncbi.nlm.nih.gov/pubmed/21991222http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ge SD%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hu ZL%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Cheng YL%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Fang ZY%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Tu SZ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Xu BR%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Jiang XT%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/3664316http://www.ncbi.nlm.nih.gov/pubmed/2108696http://www.ncbi.nlm.nih.gov/pubmed/2108696http://www.ncbi.nlm.nih.gov/pubmed?term=%22Conly JM%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Byrne S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22McLeod J%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hoban S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Robertson G%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ronald AR%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/3937563http://www.ncbi.nlm.nih.gov/pubmed?term=%22Koo DS%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhen S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Zhen ZD%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shi XW%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Xiang SJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/2757770http://www.ncbi.nlm.nih.gov/pubmed/15700744http://www.ncbi.nlm.nih.gov/pubmed?term=Ulk�r E%5BAuthor%5D&cauthor=true&cauthor_uid=16011879http://www.ncbi.nlm.nih.gov/pubmed?term=Oncul O%5BAuthor%5D&cauthor=true&cauthor_uid=16011879http://www.ncbi.nlm.nih.gov/pubmed?term=Karagoz H%5BAuthor%5D&cauthor=true&cauthor_uid=16011879http://www.ncbi.nlm.nih.gov/pubmed?term=Yeniz 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AA%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Haneef MN%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Haneef MN%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Nasir NA%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Halim AS%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Singh KK%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Dorai AA%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Haneef MN%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Haneef MN%5BAuthor%5D&cauthor=true&cauthor_uid=20576085http://www.ncbi.nlm.nih.gov/pubmed/20576085http://www.ncbi.nlm.nih.gov/pubmed?term=Mabrouk A%5BAuthor%5D&cauthor=true&cauthor_uid=22100189http://www.ncbi.nlm.nih.gov/pubmed?term=Boughdadi NS%5BAuthor%5D&cauthor=true&cauthor_uid=22100189http://www.ncbi.nlm.nih.gov/pubmed?term=Helal HA%5BAuthor%5D&cauthor=true&cauthor_uid=22100189http://www.ncbi.nlm.nih.gov/pubmed?term=Zaki BM%5BAuthor%5D&cauthor=true&cauthor_uid=22100189http://www.ncbi.nlm.nih.gov/pubmed?term=Maher A%5BAuthor%5D&cauthor=true&cauthor_uid=22100189http://www.ncbi.nlm.nih.gov/pubmed/22100189

DM Systems Heelift Anatomy_FullPage_EWMA_April2013.indd 1 2/14/13 11:51 AM

Farida Benhadou, MD

Veronique del Marmol, MD, PhD

Dermatology depart-ment, ULB Erasme Hos-pital, Brussels, Belgium

Correspondence:Farida.Benhadou@

ulb.ac.be


The mTOR inhibitors and the skin wound healing

EWMA 2012

ienna


ABSTRACTBackground: The wound healing process is di-vided into four phases: haemostasis/coagulation, inflammation, proliferation, and wound remodel-ling. The wound closure needs a perfect control of these phases thus wound management is always a challenge. Numerous factors, like medications, can markedly affect most aspects of the wound healing process.Aim: We want to focus our attention on the mam-malian target of rapamycin (mTOR). Deregu-lation of the mTOR signalling pathway occurs frequently in human malignancies. The pharma-cological inhibition of mTOR with rapamycin and its analogs is used in organ transplantation to prevent rejection and to slow skin carcinogenesis in organ recipients.Methods: This article reviews the recent literature on the mTOR inhibitors and the potential cel-lular and/or molecular mechanisms involved in the wound healing process.Results and conclusions: The mTOR inhibi-tors can affect all steps of the healing process by decreasing the inflammatory cell number, ang-iogenesis, and myofibroblast proliferation. The frequent association, among organ recipients treated by mTOR inhibitors, with other immu-nosuppressive therapies and comorbidities exac-erbate the risk of wound healing complications. The complexity of the mTOR pathway is not completely understood but its role in the wound healing process is crucial. The indication for the mTOR therapy has to be discussed carefully for each patient.

INTRODUCTIONThe mTOR inhibitors are new immunosuppres-sive agents mainly used in transplant medicine and in oncology.

The first agent developed was the rapamycin or rapamune and was initially isolated in the soil of Easter Island and was used as a fungicide(1).

The discovery of the immunosuppressant properties of rapamycin led to the understanding of the mammalian target of rapamycin (mTOR) pathway.

The mTOR pathway is a complex network playing a key role in the synthesis of cellular pro-teins important for angiogenesis, metabolism and cell proliferation(2).

Many clinical trials have shown the effective-ness of rapamycin in decreasing the incidence of malignancy and skin cancers in solid organ trans-plant recipients(3,4).

The development of rapamycin and its analogs is a promising therapeutic option but important adverse events have been described such as hy-perlipidemia, anaemia, leukopenia, and throm-bocytopenia(5). Their cutaneous side effects and especially their effect on the wound healing proc-ess will be discussed in this article.

MECHANISMS OF ACTIONThe mTOR is an important kinase necessary for physiological cellular activities acting by forming two complexes; the mTOR complex 1 and the mTOR complex 2. The result of the activation of complex 1 is the promotion of cell proliferation, the angiogenesis process and protein synthesis. The mTOR complex 2 activity is essential for the transformation and vitality of a number of can-cer cell types, but in many normal cells, mTOR complex 2 activity is less essential.

The rapamycin can inhibit the activation of the mTOR complex 1 by binding to an intracel-lular receptor FKBP12, but how this interaction antagonises the mTOR complex 1 is not well understood.

In that way, rapamycin is able to cause an im-munosuppression by inhibiting the signal trans-duction pathway required for the progression of cytokine-stimulated T-cells from G1 into S phase. In addition, many enzymes along the signalling pathway that are inhibited by the rapamycin play



a role in the development and progression of different cancers and metabolic disorders such as diabetes or athero-sclerosis.

The mTOR pathway is upregulated in many other conditions such as in polycystic kidney disease and neu-rofibromatosis.

The mTOR pathway is a complex network with a variety of positive and negative regulators but all these mechanisms are not completely explained(6,7,8).

SIDE EFFECTSDespite their attractive pharmacological properties, side effects are associated with mTOR use in 20-40%(9) of patients. Some side effects are easily manageable, whereas others lead to discontinuation of the drug. The anaemia, thrombocytopenia, neutropenia, proteinuria, lymphedema and hyperlipidemia are the most reported dose dependent side effects(10,11).

Cutaneous side effects have also been reported such as wound healing impairment. Such skin side effects have also been reported with the use of anti-vascular endothelial growth factor (VEGF) therapy and could therefore be a limiting factor for their use(12).

CUTANEOUS SIDE EFFECTSPruritic follicular papulo-pustular eruption represents a typical side effect occurring early after the initiation of the mTOR inhibitors therapy. This effect is mostly temporary and usually improves within a few weeks(13).

Pruritus and xerosis are frequently reported by the pa-tients receiving mTOR inhibitors therapy(13).

Cases of angioedema have been reported but all the patients were simultaneously treated with angiotensin-converting enzyme inhibitors. The resolution of the an-gioedema was observed after the withdrawal of the angi-otensin-converting enzyme inhibitors(14).

The development of buccal ulcerations and stomati-tis are a common and potentially dose limiting toxicity associated with the use of mTOR inhibitors in cancer treatment(13).

The interactions of the mTOR inhibitors on the heal-ing process are important cutaneous side effects. Wound infections, incisional hernias and wound dehiscence have also been reported(15).

WOUND HEALING IMPAIRMENTA normal healing process can be divided in four steps: haemostasis and coagulation, inflammation, proliferation and remodelling. A perfect control on each step is neces-sary for a correct healing(16).

The mTOR inhibitors can directly interfere with each step of the healing process but more specifically on the inflammation and proliferation stages. They are able to inhibit the angiogenesis process by decreasing the level of the VEGF, they can also decrease the activity of the intraepithelial gd T cells and cause an inhibition of the smooth muscle cells, fibroblast proliferation and matrix deposition. This would decrease the formation of scar tis-sue and compromise blood flow to the defect.

The crucial role played by the mTOR in the healing process has yet to be examined in detail(17).

The mTOR inhibitors can also indirectly interfere with the repair process. Their immunosuppressant properties increase the risk of infection. The anaemia, hypoproteine-mia and lymphedema secondary to the mTOR inhibitors therapy are also risk factors for healing complication and delayed healing(18,19).

MANAGEMENT The management of wound healing complications in patients treated by mTOR therapy is difficult. The clini-cians have to be aware of the potential healing impairment caused by mTOR therapy.

We have reviewed the recent literature concerning mTOR therapy and its impact on the wound healing process. There is no standardised guideline concerning the management of healing complications. We have found some recommendations; with a level three of evidence based medicine, proposed for the use of the mTOR inhibi-tors in the transplantation medicine(20).

We have summarised the important points:n Before starting the mTOR inhibitors therapy, it is

recommended that the risk factors for wound heal-ing complications are checked. Factors like the use of concomitant immunosuppressive therapy, obesity, and smoking are modifiable risk factors. The pa-tient’s age, sex and ethnic origin are non–modifiable risk factors. Any modifiable risk factor should be ad-dressed. If a non-modifiable risk factor is identified, a risk–benefit analysis should be performed and, if appropriate, an alternative treatment to mTOR in-hibitors should be considered.

n Some clinicians are narrowing or even discontinu-ing the use of steroids in the early post-transplant period. It is also recommended to avoid the use of the mTOR inhibitors during the first week post-transplantation.

n The impaired healing is a dose-dependent side effect. A cumulative rapamycin dose of more than 35 mg during the first four days post-transplantation is a risk factor for impaired healing. It is highly recom-


References:

1 Sharp ZD, Strong Rather role of mTOR signaling in controlling mammalian life span: what a fungicide teaches us about longevity. J Gerontol A Biol Sci Med Sci 2010: 65:580–589.

2 Karar J, Maity A.J PI3K/AKT/mTOR pathway in angiogenesis. Front Mol Neurosc 2011:4:51-64.

3 Tee A, Blenis J. mTOR, translational control and human disease. Seminars in Cell & Developmental Biology 2005:16:29–37.

4 Hofbauer G et al. Swiss clinical practice guidelines for skin cancer in organ transplant recipients. Swiss med Wkly2009 :139(29_30) :407- 415.

5 Brewer J et al. The effects of sirolimus on wound healing in dermatologic surgery. Dermatol Surg 2008:34:216-223.

6 Hara K et al. Raptor, a binding partner of target of rapamycin, mediates TOR action. Cell 2002:110:177-189.

7 Kim D et al. G betaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 2003:11:895-904.

8 Suryawan A et al. Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway J Anim Sci Biotechnol.2012 : 3(3).

9 Rostaing L et al. mTOR inhibitor/proliferation signal inhibitors: entering or leaving the field. J nephrol .2010:23 (02) : 133-142.

10 Kim M et al. Marked erythrocyte microcytosis under primary immunosuppression with sirolimus. Transpl Int.2006:19 :12-18.

11 Kasiske B et al. Mammalian target of rapamycin inhibitor dyslipidemia in kidney transplant recipients. Am J Transplant.2008:8 : 1384-1392.

12 Kamba T et al. Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer. 2007 :96(12): 1788–1795.

13 Mahe E et al. Cutaneous adverse events in renal transplant recipients receiving sirolimus-based therapy. Transplantation 2005:79 :476-482.

14 Zuckermann A et al. Clinical experience with Certican in maintenance heart transplant patients at the medical University of Vienna. J. Heart Lung Transpl 2005:24 : 206-209.

15 Brewer J et al. The effects of sirolimus on wound healing in dermatologic surgery. Dermatol Surg 2008:34:216-223.

16 Di Pietro L A, Guo. Factors affecting wound healing. J Dent Res. 2010 ;89(3):219-29

17 Squarize C et al. Accelerated Wound Healing by mTOR Activation in Genetically Defined Mouse Models. Plos One 2010:5 (5) :10643.

18 Mills R et al. Defects in skin T cell function contribute to delayed wound repair in rapamycin-treated mice .J Immunol 2008 :181(6): 3974-3983.

19 Schäffer M et al. Sirolimus impairs wound healing. Langenbecks Arch Surg 2007: 392:297-303.

20 Campistol JM et al. European Society for Organ Transplantation 2009: 22(7) : 681-687.


mended to avoid the loading doses and to initiate and maintain rapamycin at a dose of 2–4 mg/day, with an increase in dose above this level only if target levels are not reached by day seven. Recommended target levels are between 5 and 10 ng/ml, depending on concomitant immunosuppressant therapy.

n It is also important to know that some medications can increase the blood level of the rapamycin like the inhibitors of CYP3A4 (verapamil, ketoconazole, erythromycin…) and their concomitant use with the mTOR inhibitors therapy can be a risk factor for impaired healing.

n Concerning the risk for surgery, it is important to identify the type of surgery. For major surgery, the mTOR inhibitors have to be stopped five to 10 days before the surgery and restarted one to three months after the surgery.

n Regarding non-surgical wounds such as traumatic wounds and vascular ulcers, the recommended at-titude is classical wound care management. The risk factors for a wound healing complication have to be checked and corrected if possible. The diagno-sis and treatment of infections are important steps for helping the correct healing. The withdrawal or the decreasing of the dose of the mTOR inhibitors therapy has to be considered only for complicated wounds where there is an inability to correct the risk factors(20).

CONCLUSIONS:The development of the mTOR inhibitors is an interesting therapeutic option thanks to their different immunosup-pressive properties. They are mainly used in transplant medicine and oncology. The involvement of the mTOR pathway in many disorders such as neurofibromatosis and polycystic kidney disease has enlarged their indications.

One of the major benefits of rapamycin is that it is an immunosuppressant that inhibits carcinogenesis, whereas other immunosuppressants are thought to increase car-cinogenesis like the calcineurin inhibitors. The effect of the mTOR inhibitors on carcinoma is likely inhibition of angiogenesis and an associated decrease in vascular en-dothelial growth factor.

Unfortunately the use of the mTOR inhibitors is as-sociated with many side-effects. Some of these are manage-able, whereas others could be limiting factors for mTOR use.

This article gives you a general overview of the effects of mTOR inhibitor therapy. Our article sought to point out their important impact on the skin healing process.

The management of this side effect is based on a number of recommendations with a level three of evidence based medicine. Control of the risk factors for wound complication is a crucial step before starting such therapy. The complexity of the mTOR pathway is not completely understood but its role in the wound healing process is crucial. The indication for the mTOR therapy has to be fully discussed and the risk-benefits balance must be con-sidered carefully for each patient.

It would also be interesting in the future to develop guidelines regarding their use in the oncology field. m

HHH

The Coloplast logo is a registered trademark of Coloplast A/S. © [YYYY-MM.] All rights reserved Coloplast A/S, 3050 Humlebæk, Denmark.

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2013

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CPWSC_New_BS_Prelaunch_Teaser_Ad_A4_EWNA.indd 1 20/02/13 10.19

Ross A. Atkinson1

PhD, Spinal Research Coordinator

Karen J. Ousey2

PhD, RGN, Reader,

Steve Lui2 MSc, Senior Lecturer,

John Bradley Williamson3

MBChB, FRCS, Consultant in

Spinal Surgery

1Greater Manchester Neurosciences Centre;

and Honorary Research Associate – The University

of Manchester, Manchester Academic

Health Science Centre, Salford Royal NHS Foundation Trust,

C235 Clinical Sciences Building, Stott Lane,

Salford, M6 8HD.

2School of Human and Health Sciences,

Centre for Health and Social Care Research,

University of Huddersfield, Queensgate, Huddersfield, West Yorkshire, HD1 3DH.

3Salford Royal NHS Foundation Trust,

Stott Lane, Salford, M6 8HD

Correspondence:[email protected]


ExTENDED ABSTRACT

Aims To systematically search, critically appraise and summarise randomised controlled trials (RCTs) and non-RCTs assessing the effectiveness of nega-tive pressure wound therapy (NPWT) in patients with a surgical spinal wound.

MethodsA systematic review based on search strate-gies recommended by the Cochrane Back and Wounds Review Groups was undertaken using the Cochrane Library, MEDLINE, EMBASE and CINAHL databases. Any publications between 1950 and 2011 were included. Funding to under-take this systematic review was received from the University of Huddersfield Collaborative Venture Fund and KCI Medical.

ResultsNine retrospective studies(1-9) and five case stud-ies(10-14) of patients with spinal wound complica-tion were included in this systematic review. No RCTs were found. Only one study described more than 50 patients(4). Generally, a pressure of -100 to -125 mmHg was used in adult patients(1,8,12).

Duration of NPWT in situ ranged from three to 186 days(2,5,6,8,13). Wound healing was assessed every two to three days and generally complet-ed between seven days and 16 months(1-5,9-14). NPWT is contraindicated in the presence of active cerebrospinal fluid leak(1), metastatic or neoplastic disease in the wound(9,10), in patients with an al-lergy to the NPWT dressing(9), and in those with a bleeding diathesis(1).

DiscussionWe identified no RCTs discussing the use of NPWT in the management of surgical spinal wounds, and limited low quality evidence dem-onstrating that NPWT can be used effectively in this type of patient. In an RCT in obese patients undergoing total knee arthroplasty, no difference in the time taken to achieve a dry wound with NPWT as compared with a sterile gauze has been reported(15). Importantly, that study was termi-nated early due to the presence of skin blisters associated with the NPWT dressing; an adverse effect which has not been reported in the spine lit-erature. Furthermore, Dorafshar and colleagues(16) concluded that NPWT did not provide superior

A Review of Evidence for Negative Pressure Wound Therapy (NPWT) use Post Spinal Surgery


References

1. Jones GA, Butler J, Lieberman I, Schlenk R. Negative-pressure wound therapy in the treatment of complex postoperative spinal wound infections: complications and lessons learned using vacuum-assisted closure. J Neurosurg Spine 2007: 6(5):407-411.

2. Labler L, Keel M, Trentz O, Heinzelmann M. Wound conditioning by vacuum assisted closure (V.A.C.) in postoperative infections after dorsal spine surgery. Eur Spine J 2006: 15(9):388-396.

3. Mehbod AA, Ogilvie JW, Pinto MR, Schwender JD, Transfeldt EE, Wood KB et al. Postoperative deep wound infections in adults after spinal fusion: management with vacuum-assisted wound closure. J Spinal Disord Tech 2005: 18(1):14-17.

4. Ploumis A, Mehbod AA, Dressel TD, Dykes DC, Transfeldt EE, Lonstein JE. Therapy of spinal wound infections using vacuum-assisted wound closure: risk factors leading to resistance to treatment. J Spinal Disord Tech 2008: 21(5):320-323.

5. Antony S, Terrazas S. A retrospective study: clinical experience using vacuum-assisted closure in the treatment of wounds. J Natl Med Assoc 2004: 96(8):1073-1077.

6. Canavese F, Gupta S, Krajbich JI, Emara KM. Vacuum-assisted closure for deep infection after spinal instrumentation for scoliosis. J Bone Joint Surg Br 2008: 90(3):377-381.

7. Canavese F, Gupta S, Emara KM, Krajbich JI. Use of the vacuum assisted closure in instrumented spinal deformities for children with neuromuscular scoliosis who developed post-operative deep spinal infection. Developmental Medicine and Child Neurology 2009: 51(5):50.

8. Horn PL, Ruth B, Kean JR. Use of wound V.A.C. therapy in pediatric patients with infected spinal

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