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Bugs, Drugs & Silver Bullets

Lindsay Kalan, PhD


PCN West
May 28, 2015

Faculty/Presenter
Disclosure

Faculty: Dr. Lindsay Kalan


Relationships with commercial interests:
o Other: Employee of Exciton Technologies Inc. (Manufacturer of
Exsalt Wound Dressings)

This program has received in-kind support from


Exciton Technologies in the form of clinical information tools
and literature.

Potential for conflict(s) of interest:


o Dr. Lindsay Kalan has received salary from Exciton whose
product(s) will being discussed in this program.
o Exciton Technologies Inc develops and benefits from the sale
of a product that will be discussed in this program: Exsalt SD7
and T7 Wound Dressings with Ag Oxysalts.

There is a potential for bias in slides 40-44. This has been mitigated
by providing full references, referring to scientific names and
providing materials/methods and raw data for experimental results.

Disclosure of
Commercial Support

This program has received in-kind support from


Exciton Technologies in the form of clinical information tools
and literature.

Potential for conflict(s) of interest:


o Dr. Lindsay Kalan has received salary from Exciton whose
product(s) will being discussed in this program.
o Exciton Technologies Inc develops and benefits from the sale
of a product that will be discussed in this program: Exsalt SD7
and T7 Wound Dressings with Ag Oxysalts.

Mitigating Potential
Bias

There is a potential for bias in slides 40-44. This has been


mitigated by providing full references, referring to scientific names
and providing materials/methods and raw data for experimental
results.

Learning Objectives
1. Antibiotic Resistance and Wound Infection
2. Biofilms 101
1. Biofilms in chronic wounds

3. The use of silver as a topical antimicrobial


4. Applying antibiotic stewardship and new research
knowledge to clinical methodology and practice
5

The Cost of a Wound

$ 3.9 Billion CND/year more than care for stroke


30-50% of all health care involves wounds
In the US >$25 Billion USD/year1
o 30% of DPN cost go towards wounds ($10B/yr)
o Additional $35-45B/yr spent on SSI2

Dont include indirect and intangible costs


associated with quality of life and society as a
whole
Sen et al. 2009. Human Skin Wounds: A Major Snowballing Threat to Public Health and the Economy
Scott, R.D. 2009. The Direct Medical Cost of Hospital Acquired Infection in US Hospitals and the Benefits of Prevention

1
2

Antibiotic Resistance
In 2013 - 23 million antibiotic Rx were written in
Canada
o 45% respiratory, 16% upper respiratory, 14% UTI, 10% ear, 20% other
o Many of these infections may not have required antibiotic therapy (eg.
virus)

The CDC estimates >$20B in excess health care


costs
o >$35B in other societal costs and >8 million additional hospital days

http//www.sciencephoto.commedia11428enlarge
http://phil.cdc.gov/phil/home.asp

Staphylococcus aureus
Enterococcus faecium
VRE

Enterobacter spp.
CRE

MRSA/VRSA

No ESKAPE

Pseudomonas aeruginosa
MDRPA

Klebsiella pneumoniae
CRE

Acinetobacter baumannii
MDRAB

Plus: Totally drug resistant TB, C. difficile, N.


gonorrhoeae and more

Why is this happening?

Q. Where do antibiotics
come from?
Alexander Flemings Nobel Laureate Speech

A. Bacteria

Environmental microbes make antibiotics


and have self resistance which can be
transferred to pathogens

http://microbewiki.kenyon.edu/index.php/Streptomyces

The Antibiotic Resistome


Clinical Pathogens
Environmental
Organisms
Producers
Precursor
Genes
Resistance Genes

Antibiotic Resistance is Natural

Bacteria in these caves have been isolated for an estimated 4 million years
They are on average resistant to 3-4 clinically used antibiotics

Glycopeptide Antibiotics
Front-line treatment for serious Gram-positive
infection (MRSA)
Two used clinically vancomycin and
teicoplanin
Three second-generation televancin (2009),
oritovancin, dalbavancin (2014)
All have the same primary mechanism of
action
Resistance exists for all

How Old is it?


Probe DNA extracted from permafrost cores for
resistance genes
6 permafrost soil cores from eastern Beringia
(Yukon)
25.3ky (stadial)
80-90ky (interstadial)
740ky (interglacial)

Resistance mechanism consistent


with contemporary (VRE) clinical
pathogens discovered
DCosta et al. 2011. Nature

Resistance is Widespread and Diverse


A. balhimycina
S.
toyocaensis

murF

ABh
ASt

ABh

Producers

VRE vanA
R

VRE vanB
R

VVE
H

APa

Clinical
Pathogens

P. apiarius PA-B2B
R

S. coelicolor
S

ASc

VVE strains have recently been isolated from patients


across Canada. They have high level resistance that
evolves after vancomycin administration and include
wound isolates

Environmental

Why is this important?

Number of
Antimicrobial
NDA = 1

Stewardship and alternate infection control strategies are of equal


importance to new drug discovery

We have a lot left to learn


On June 26, 2000 the first draft of the human
genome was sequenced the project started in
1990
2001
Canadian microbiologist Dr. Julien Davies quickly
pointed out: Our existence is dependent on
bacterial speciesliving in and on us!
Microbiologist Dr. Joshua Lederberg coins the term
microbiome

We have 10x more bacterial cells than


human cells in our bodies that contribute to
our health and homeostasis

17

Considerations
Antibiotics and subsequent resistance are natural
phenomena
Microbes can adapt quickly
Antibiotics impact the healthy microbiome and
select for resistance in human pathogens
Alternate strategies are desperately needed

So Why am I here?
What does this have to do with wound care?

19

The Microbial Influence on Healing


Increasing clinical problems

Contamination
Haemostasis

Colonization
Inflammation

Vigilance required

Critical
ColonizationRemodeling
Infection
Proliferation

Intervention required

Wounds become chronic and stuck in an inflamed state


Note: Localized infection may or may not be accompanied by the classical
signs and symptoms of inflammation

20

Principles of best practice: Wound Infection in clinical practice. An International Consensus. London: MEP Ltd, 2008.
Available from www.mepltd.co.uk

21

Biofilm Formation in a Wound


Contamination

Infection

Colonization

Critical Colonization
Harrison et al. 2008. Nature Rev Micro
Phillips et al. 2010. Wounds International

What are biofilms?


And are they really that important?

Microbial Biofilms are everywhere

General Characteristics of a Biofilm


Surrounded
by
a
slime

EPS:
an
electrostatically
charged
mix
of
eDNA,
polysaccharides, and protein
Structurally heterogeneous and fluid
(structure can change to form ridges,
layers, or microcolonies - depending on
environment and genetics)
Adherence to a surface (biotic or abiotic)

Unique biochemistry: signaling, and metabolism,

**Note: requirement for adherence is a topic of contention

How does the biofilm lifestyle help the microorganism?


Protection from shear forces
Entrapment of particles in slime leading to the formation of
microscopic niches metabolic heterogeneity
Tolerance to UV radiation
Protection from grazing by protozoans and from cells of the
mammalian immune system
Enhanced horizontal transfer of genetic material
Protection from a wide range of antimicrobials (such as antibiotics,
detergents, biocides and heavy metals)
More energy efficient, protective environment, with reduced
susceptibility to antimicrobials, invaders, and chemicals

Biofilm Physiology
Aerobic
Neutral pH
Readily available
nutrients
High antimicrobial
penetration

A.
B.
C.
D.

Anaerobic,
Acidic pH
Scarce nutrients/by-products
Low antimicrobial penetration

Different metabolism (metabolic heterogeneity) and chemical species


Population preferences
More dormant populations in the interior
Results in more resilient infection (dead cells and EPS can provide
additional protection from antimicrobials)

Mixed vs Single Species Biofilm


A

In a multispecies biofilm

Co-metabolism and cooperation

High rate of gene transfer

A may not be pathogenic, but is a


good film former.

B is pathogenic and now has


protection in As EPS.

C may promote aggregation of A

(examples will be given for each)

The CDC and FDA state >60% of human infections involve


biofilms
In these cases the biofilm provides for a constant inoculation of the
infectious organism. The result is cycles of infection.

VLU Polymicrobial Biofilm?

Microbial composition in wound swabs

43 yr old male with a


recurring chronic leg
wound (5-8 yrs).

Liver disease, PVD

Prior to study was dressed


with nanocrystalline Ag

Bacterial Genera: >35


At onset: >80% S. aureus
W4: Reduced to 23% relative
abundance in tissue swabs

T=0

D1

T=0 D1

W1 W2 W3 W4

W2

Family Comamonadaceae
Curvibacter sp.
Family Comamonadaceae
Family Burkholderiales
Sphingomonas sp.
Family Rhodospirillaceae
Family Bradyrhizobiaceae
Family Caulobacteraceae
Peptoniphilus sp.
Finegoldia sp.
Streptococcus sp.
S. aureus
Paenibacillus sp.
Corynebacterium sp.

W3

29

Biofilms in Wounds

Wound biofilms are often not


detected.

They are intrinsically antibiotic


resistant but can be caused by
multi-drug resistant organisms

They may be caused by previously


unknown pathogens or skin
commensals (more research is
needed)

Depending on the wound systemic


antibiotics may not even reach the
site of infection and have collateral
damage to beneficial microbes

The biofilm acts as a continual


source of re-inoculation

30

Duplantier and van Hoek. 2013. Front. Immunol. Doi:10.3389/fimmu.2013.00

Where do we go from here? focus on


wound care
Start testing antimicrobials against biofilm populations
Try other antimicrobials: metals are showing promise at eradicating
biofilms and persister cells:
Metals

Metals have multiple targets in


the cell (non-specific biocides)

Biofilm tolerance is time


dependent

VS

Antibiotics

Antibiotics are directed toward a handful


of targets, cells may not grow, but they
dont die

Persister cells are not as susceptible

Multidrug resistance easily developed

Antimicrobial Silver

Phoenicians
Water purification

1000 BCE

Silver sutures and foil


used in surgery

Golden Era of Antibiotic


Resurgence of silver usage
Discovery

400 BCE 1500-1895 1900-20 1940-70

Hippocrates healing
and anti-disease

Widespread use of silver


in medicine

1980-now

Emergence of antibiotic resistance

32

Silver has many targets


Ag+

Gram (-)

Ag+

Gram (+)

Ag+
Ag+

SH

OUT
IN

Morones-Ramirez et al. 2013. Sci. Trans. 33


Med

Basic Redox Chemistry


Reducing Agent

Oxidizing Agent

e-

Reduced

Blue loses electrons,


becoming oxidized

Oxidized

Oxidized

e-

Red gains electrons,


becoming reduced

Reduced

ee-

Silver Science: Redox


Potential
Potential to take
electrons
Reaction
Ag3+

+ e Ag2+

Ag2+

+ e Ag+

Ag2O + 2H+ + 2e 2Ag0 + 2H2O


Ag+

+ e Ag0

Ag2SO4 + 2 e- 2Ag + SO42-

Reduction
Potential (V)

+ 1.80
+ 1.98
+ 1.17

+ 0.80
+ 0.64

Ag2O

+ H2O + 2e 2Ag0 + 2OH-

+ 0.34

AgCl

+ e Ag0 + Cl

+ 0.22

Reduction potential
(+) potential (V)
= need for electrons
oxidation
All silver species are
different

Reactivity

35

Ag Found Commonly in Dressings


Ag2O

AgCl

Very insoluble (1.9mg/L) and


stable ie. does not react with
water

Ag+(aq) + Cl-(aq) AgCl(s)

Light exposure causes


degradation to metallic silver
(no activity) and chlorine.
Mostly used in photography

Ag2O formed by surface


oxidation of Ag metal
Releases Ag+

Ag2O(s) + H2O(aq) Ag(OH)2-(aq)

OH- causes pH spike = pain


High [Ag] needed for efficacy

O2

O2

O2
Ag2O

Ag

Ag

Ag7NO11

Ag+, Ag2+, Ag3+

37

Silver Wound Dressings how to


choose?
Oxidation State

Silver in Dressing
(mg Ag/100 cm2)

Silver Release
(4h in SWF)

Silver Oxysalts

Ag3+, Ag2+, Ag1+

40

107

Metallic Silver,
Silver (I) Oxide

Ag1+, Ag0

160

28

Metallic Silver

Ag1+, Ag0

440

Silver Na H
Zr Phosphate

Ag1+

210

Silver Sulfate

Ag1+

120

18

Silver Chloride

Ag1+

12

28

Silver Type

Adapted from the respective product documentation/marketing materials


Spina, Carla J., Lischuk, David, Motta, Glenda, Silver Dressings 101: Silver Science

38

Silver Availability
4 hr silver release into RO H2O

400
200

ppm Ag+/dressing
ppm Ag+/mg Ag(s)

500
400
300
200
100
0

24

48

72

96 120 144 168 192

Time (hrs)

40

MRSA 4 hr Log Reduction

30
20
10
0

Log 10 value [cfu/mL]

Silver release (ppm)

600

Silver release (ppm)

Cumulative Ag+ Release from Ag7NO11


600

10
8

2
LOD
1

Efficacy of Silver Dressings


106 cfu/mL
72 hrs., 250 rpm, 37C Rinse gauze Treatment (4 hrs)
3X with
sterile saline
Simulated Wound Fluid

+/- Nutrient Agar

200 M

200 M

10 M

10 M

20 M

10 M

10 M

1 M

SEM P. aeruginosa biofilm on gauze


fibers DOI: 10.1111/wrr.12232
Adapted from Seth et al. 2015. Wound Repair and Regeneration

SEM - S. aureus biofilm on


gauze fibers

40

Not all Silver Bullets are the Same


4 hr exposure on
complex media

4 hr exposure in saline

log cfu/gauze

AgCl

8
6
4
2
0

P.aeruginosa (blavim-2)

**** ****

****
****
*

****

10

log cfu/gauze

10

Ag Oxysalts Wound Dressing


HF Ag/BC/EDTA
HF Ag
Negative

8
6
4
2
0

****
***

***

In Vitro In Vivo
Efficacy
Porcine Burn Biofilm Model
Log 10 value [cfu/mL]

10

5
LOD
0

Burn Wound Inoculated


with P. aeruginosa
Ask Questions!
What was the test method?
What was being measured?
What kind of environment was the
dressing tested in?
What is the type and quantity of silver?
Are there other components that reduce
bioburden or keep the wound bed
healthy?
What is the construction of the dressing?

Clinical Case Study #1


T=0

W2

W1

W4

72 yr old male with a recurring DFU

Type II diabetic, renal failure, overweight,


immunocomprimised

Insulin, steroids, anticoagulants, oral


hypoglycemics

Prior to study was dressed with


nanocrystalline Ag

Dressing was changed 2x weekly at


home

Microbial composition in wound


swabs
Roseateles
sp.

Bacterial Genera : >50


At onset: High abundance of
Staphyloccocus sp. and
Paenibacillus sp. W3:
Staphylococcus sp. reduced to
<2% abundance.
W4: Wound healed

T=
D4

W
1

W2

W3

Curvibacter sp.
Comamonadaceae
family
Burkholderiales order
Sphingobium sp.
Caulobacteraceae
family
Peptoniphilus sp.
Helcococcus sp.
Finegoldia sp.
Anaerococcus sp.
S. aureus
Staphylococcus sp.
Macrococcus sp.
Paenibacillus sp.

43

Clinical Case Study #2


T=0

W1

W3

W2

W4

Wound size over time

Microbial composition in wound swabs


H. parainfluenzae
Caulobacteraceae sp.
Allobaculums sp.
Peptoniphilus sp.
Helococcus sp.
Finegoldia sp
Anaerococcus sp
Streptococcus sp
S. aureus
Staphylococcus sp.
T=
0

D1

W1

W2

W3

W4

66 yr old male with a recurring


DFU (several yrs)

Type II diabetic , diabetic


neuropathy, metabolic disorder,
poor perfusion, overweight

Osteomyelitis and awaiting tendon


surgery

44

Silver Wound Dressings


Silver dressing on the market today have different types of silver.
Most products offer Ag0 or Ag1+
New products offer Ag2+ & Ag3+
Silver Dressings are Used To:
Prevent - where there is a high risk of infection in:
Contaminated wounds
Partial- or full-thickness burns
Large and/or deep wounds
Reduced healing because of patient-related factors
Restore Wound Balance - where there is increased bioburden:
local infection
local infection that is associated with a widespread (systemic)
infection
45

Additional Considerations
Infected wounds may require additional
interventions such as systemic antibiotics (oral or
intravenous), dressing selection to support
moisture balance and debridement of necrotic
tissue.
The wound management team, including the
patient, must have clearly identified endpoints
(goals or outcomes) with rationale for all
decisions related to wound care.

46

Summary
Systemic antibiotics might not be effective even in
the absence of a resistant microbe Antibiotic
stewardship is critical
Many if not all chronic wounds have a multi-species
biofilm established with intrinsic antibiotic resistant
The patient may have co-morbidities significantly
impacting healing
We still have a lot to learn about the influence of
microbes on healing pathways
The wound team is multi-disciplinary
Innovative treatment strategies start with you
47

Instrumental Policy & Clinical


Practice Documentation

Wound Infection in Clinical Practice An International


Consensus (2008)

http://www.woundsinternational.com/clinical-guidelines/wound-i
nfection-in-clinical-practice-an-international-consensus

International Consensus - Appropriate Use of Silver


Dressings in Wounds (2012)

http://www.woundsinternational.com/clinical-guidelines/intern
ational-consensus-appropriate-use-of-silver-dressings-in-woun
ds

A Closer Look at Silver A Clinical Information Tool (CIT) to


help you to help you choose the right silver wound dressing
for your patient (2013)

https://www.scribd.com/doc/261794917/Clinical-Information48
Tool-a-Closer-Look-at-Silver

Thank-You
The Canadian Wound Care Community Collaborators
Michele Suitor
Dr. Andrew Myles
Jane Ratay
Dr. Raymond Turner
Marlene Varga

Dr. Joe Lemire

Dr. David Keast

Dr. Ben Willing

Heather Orsted (and eQuadra


Solutions team)

Dr. Mi Zhou
Westview Health Center Wound Clinic
Alberta Provincial Microbiology Lab

Exciton Technologies Inc.


Steven Miller
Deanna Pepin
Dr. Imran Ul-Haq
Karen Cuvelier
Melanie Ussyk
Rod Precht
49

Questions and
Discussion

50

Supplementary

51

What about Biofilms?


Ag can exist with different oxidation states and aqueous availability
Ag2+/3+ has antimicrobial and antibiofilm activity at lower
concentrations than Ag1+ in vitro
Minimum Biocidal Concentration
4 hr exposure to Ag

2000
1500

****

****

****

E.coli
1250
P.aeruginosa
S.aureus

850

500

80

**
60
40

50
40

*
*

E.coli
P.aeruginosa
S.aureus

30
20

20

10

Planktonic populations of I) E.coli (JM109), II) P.


aeruginosa (PAO1), and III) S.aureus (ATCC
25923)

*
*

450

Conc. of Metal [M]

Conc. of Metal [M]

1000

Minimum Biofilm Eradication Concentration


Established Biofilms - 24 hr Treatment

24h established biofilms grown for 24h in SWF


and then treated with varying concentrations of
52
Ag compounds for 24h. n 4 SD. v

Clinical Performance
50 patients, mixed etiology wounds
o 25 men and 25 women

Median/Avg. Age = 67 years


o range 33-95 years

Median wound duration 3.5 months


o Range, 2 days to 24 months

Wound treatment with exsalt SD7 weekly,


minimum 4 weeks, with BWAT wound scores,
analogue pain scale, and cost analysis.

Spina, C.J., Lischuk, D.L, CE Mark Technical File: Scientific and Clinical Report. Exciton Technologies Inc.,
December 2011, In Support of European Regulatory Submission

53

Multi-Center Clinical
Evaluation
patients
entered (4 week) study with
chronic (non-healing) wounds
93% of patients perceived a reduction in pain
(with no pain reported upon dressing application)

Day 0

98% of wounds had a decrease in wound exudate


25% of wounds healed within the 4 week evaluation
48% cost savings observed over standard of care

Conclusion:
Higher oxidation state silver (in Exsalt wound dressing)
is effective for chronic and critically colonized wounds.

Day 6
Day 16
Data on file - GMA Multi-Site
Study

54

Mixed Etiology Case Series


20 Patient case series*
10 Chronic Leg Ulcers
10 Chronic Foot Ulcers

Positive outcomes
Size in 16 out of 20 stalled chronic wounds
Decrease in cumulative pain scores

Patricia
Patricia Coutts, Grace
Grace Modelski,
Modelski, Laurie Goodman, Judy Ryan, R. Gary Sibbald
Sibbald M.D.
M.D.
** 20
20 out
out of
of 30
30 total
total patients
patients analyzed
analyzed to
to date
date

Chronic Wound Case


Series
6 Patient case series*
Mixed etiology
Localized infection or at risk of infection
Exsalt SD7 applied twice weekly for four weeks

Positive outcomes
Reducing wound size: 52.3 cm2 to 34.1 cm2
Visual improvement in bacterial burden
Decrease in exudate
Symptom

# Improved

Pain

Oedema

Stalled Healing

Malodor

Bakeer
Bakeer M,
M, Vair
Vair A,
A, Keast
Keast D,
D, Evaluation
Evaluation of
of Silver-impregnated
Silver-impregnated Dressings
Dressings in
in a Clinical
Clinical Setting:
Setting: Observations
Observations on
on Efficacy
Efficacy and
and
Practicality,
Practicality, CAWC 2012

Microbial Ecology of
Wounds
INCLUSION AND EXCLUSION
CRITERIA
Inclusion

Exclusion
Systemic antibiotics in
previous 2 weeks

Age >17 yrs


Chronic nonhealing wound >
6 wk
Requires at least
weekly care

stview Health Center, Stony Plain, AB

Debride wound collect


Swab surface of wound
T=0

Day 1

V1 V2
V1

Swab

Known skin sensitivity


Participation in another
study

Extract total DNAAmplify 16S rDNA geneNGS 454 platform

Week1
V3

Debridement

Week2
V4

Week3
V5

Dressing Change

Week4
V6

57

Diabetic Foot Ulcer


February 12, 2014 - Started on
Exsalt
Feb. 20, 2014

November, 2013: Started on Acticoat Flex


December, 2013: Switched to promogram
January, 2014: Restarted on Acticoat Flex because
wound got worse
Mid Jan, 2014: Promogram added to Flex

Bacterial Genera : >10


High proportion of streptococcus on all visits.
From tissue swabs 92% relevant abundance from
T=0 (before exsalt application) to 41% relative
abundance on week 4 of exsalt treatment.

March 13, 2014 Beginning to


Epithelialize

exsalt cut
to size,
foam
secondary
dressing
Note: Debridement samples have an inverse
correlation to streptococcus (assuming dead
slough). DNA analysis does not indicate
58
live/dead cells.

Infected Surgical
Wound

Exsalt SD7 fan-folded and packed into


wound.
Week One: the wound decreased 70% in
size.
Week Three: switched to T7 for packing
Secondary dressing changed according to
level of exudate

59

Right Flank

Fragile skin, wound caused


by cell phone rubbing
against leg

Day 10: Wound healed

Prior to study treated with Hydrofera Blue


Treated with exsalt T7 and secured with film (eg.
Tegaderm)
Healed before week 2 visit.

Non-Healing VLU
December 4, 2013: Started on
Aquacel Ag + Mepilex
December 12, 2013: NU-GEL
+ Alldress
December 19, 2013: Biopsy
taken, NU-GEL, calcium
alginate, Alldress
January, 2014: Mepilex Ag and
coban wrap

Day 0, pre- exsalt application

Day 1 Jan 28, 2014

Bacterial Genera: >25


Before treatment: Mostly
betaproteobacteria
(Comamonadaceae)
W4: Mainly Allobaculum and
Caulobacter (associated with
skin).

Week 2

Week 4

Wound fully healed on next follow up visit

61

Foot Wound - PVD

Day 0, pre- exsalt application

Dressing treated with Inadine


(iodine antimicrobial) prior to
exsalt study)

Day 2

Week 2 - Healed

Patient comments: Wound


less painful and happy with
improvement
62

Recurring Cellulitis

Patient had an infection to the lower leg, after treatment


with exsalt a notable improvement to periwound skin was
observed (breakdown was occurring).
63

Incisional Hernia skin


graft
Week 2

Day 0

Pre-study: Hypergranulation, dressed


with Biatain Ag
Week 3

Purulent discharge ceased by week 2


Week 4

Exsalt packed into undermining and dressed center of wound64

Stasis Ulcer failed


graft
Day 0

Week 1

Pre-study: On Acticoat flex

Wound measurements
improving

Week 2

Week 4

Increase of granulation

Continued improvement,
wound eventually closed

65

Fistula infection 1
month post surgery
Day 0

Week 4

Day 0 - 1 month post surgery. Wound epithelializing by


week 2, healed by week 4.

66

Recurrent VLU
Week 1
Day 0

Week 2

Week 3

Week 4

67

Questions?

68

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