Nd:YAGNd:YAG LASER LASER TREATMENT OF TREATMENT OF

ONYCHOMYCOSIS ONYCHOMYCOSIS

JasminaJasmina KozarevKozarevDermamedicaDermamedica Dr. Dr. KozarevKozarev

Private Dermatology Clinic, SerbiaPrivate Dermatology Clinic, Serbia

Introduction: Introduction: OnychomycosisOnychomycosis

OnychomycosisOnychomycosis

is a is a fungal infection of the fungal infection of the

toenails and fingernails toenails and fingernails that results in that results in

thickening, thickening, discoloration, discoloration,

splitting of the nails,splitting of the nails,as well as as well as liftinglifting

of the of the

nails from the nail bed.nails from the nail bed.

NAIL FUNGUS NAIL FUNGUS (ONYCHOMYCOSIS)(ONYCHOMYCOSIS)

It has It has increased by 18.5%increased by 18.5% during the past several decadesduring the past several decades

OneOne--third of people withthird of people with diabetesdiabetes develop nail fungus develop nail fungus

Antibiotics & corticosteroids increase risk of fungal Antibiotics & corticosteroids increase risk of fungal infections infections

Artificial nails and overzealous manicuring contribute to nail Artificial nails and overzealous manicuring contribute to nail fungus developmentfungus development

48% of the population48% of the population may be affected by age 70 may be affected by age 70

RingwornRingworn

A characteristic feature A characteristic feature of of dermatophytedermatophyte

infections is an infections is an inflammatory pattern at inflammatory pattern at

the edge of the skin the edge of the skin lesion, noted by lesion, noted by

redness and scalingredness and scalingor occasionally, or occasionally,

blister formationblister formation

Treatment optionsTreatment options

Multiple therapies, topical and oral Multiple therapies, topical and oral methodsmethods,,

including surgicalincluding surgical

tratmentstratments, ,

chemicalchemical

ablationablation,,

and new laser tratments.and new laser tratments.

OOralral

therapies result in better outcomes. therapies result in better outcomes.

However, none of these treatment options However, none of these treatment options provides lasting high cure rates. provides lasting high cure rates.

Laser treatments are new modalities Laser treatments are new modalities including waveleth of 1064 nm, 930 nm and including waveleth of 1064 nm, 930 nm and 870 nm870 nm, PDT, , PDT, femtosecondfemtosecond

800nm laser…800nm laser…

Laser systems, wavelengths, and Laser systems, wavelengths, and fluencesfluences used during the initial phase of the studyused during the initial phase of the study

Laser system Wavelength (nm) Fluence (J/cm2)

Intense pulsed light (Lumenis) 695 to 1,000 38, 45, 57Intense pulsed light (Lumenis,) 755 to 1,000 38, 45, 57Pulsed dye laser (Candela) 585 8, 11, 14Q-switched Nd:YAG laser (Surgical Laser Technology) 532 8, 10Q-switched Nd:YAG laser (Surgical Laser Technology) 1,064 6, 8,10, 12Erbium YAG laser (Sciton) 2,940 25KTP laser (Laserscope) 532 2, 4, 6, 8

Vural et al. T. rubrum isolates and colony growth inhibition in vitro.

Growth of fungal colonies receiving different Growth of fungal colonies receiving different laser treatmentslaser treatments* * ((VuralVural

E at all. E at all. Laser Med Sci 2008 Laser Med Sci 2008

2323:349:349--353353))

Treatment with 1064nm laser at 4 and 8 J/cm2 significantly reducTreatment with 1064nm laser at 4 and 8 J/cm2 significantly reduced ed fungal growth rate compared to the average growth of all laser fungal growth rate compared to the average growth of all laser treatmedtreatmed colonies.colonies.

Aim of Study:Aim of Study:

To investigate (in vitro and in vivo) topicalTo investigate (in vitro and in vivo) topicalnear infrared (1064 nm wavelenth ) photonear infrared (1064 nm wavelenth ) photo--

inactivationinactivation

of T. Rubrum, of T. Rubrum, T.Mentagrophytes, Aspergilus niger, T.Mentagrophytes, Aspergilus niger,

Candida sp., and molds Candida sp., and molds as a major fungi that cause onychomycosisas a major fungi that cause onychomycosis

and and

superficial fungal skin infectionsuperficial fungal skin infection

Study Design: Study Design: Inclusion study criteria:Inclusion study criteria:

TToenailoenail

fungus, finger nail fungal infection in all fungus, finger nail fungal infection in all four clinical types of fungal nail infection four clinical types of fungal nail infection

total dystrophic form, total dystrophic form,

distal distal subungualsubungual

onychomycosisonychomycosis, ,

proximal proximal subungualsubungual

onychomycosisonychomycosis,,

endonyxendonyx

onychomycosisonychomycosis

Superficial skin fungal infection (Superficial skin fungal infection (tineatinea cutis cutis glabraeglabrae) ) with nail involvement.with nail involvement.Ages between 18Ages between 18--45 years old. 45 years old. SSignedigned

informed consent before laser procedure. informed consent before laser procedure.

Treatment of sTreatment of systemicystemic

antifungal therapy or oral antifungal therapy or oral

antifungalsantifungals

6 months before laser procedure6 months before laser procedure..

Treatment with local Treatment with local antifungalsantifungals..

UUsage of local antifungal therapy such as sage of local antifungal therapy such as CastellaniCastellani

sol.sol., which are changing nails pigmentation., which are changing nails pigmentation.

UUsage of nail sage of nail coloringcoloring

dyes or dyes or photosensibilisatorsphotosensibilisators

which are changing nails pigmentation. which are changing nails pigmentation.

SubungualSubungual

exostosisexostosis..

Exclusion study criteria:Exclusion study criteria:

Exclusion study criteria: cont.Exclusion study criteria: cont.

PPregnancyregnancy

EExistancexistance

of of subungualsubungual

hematoma or nevoid hematoma or nevoid

subungualsubungual

formationformation, , bacterial nail infection bacterial nail infection which are changing nail pigmentationwhich are changing nail pigmentation..

EExistancexistance

of concomitant nail disorders such asof concomitant nail disorders such as

psoriasis of nail plate, lichen psoriasis of nail plate, lichen planusplanus

and atopic and atopic dermatitis.dermatitis.

Special attention was made about factor Special attention was made about factor which cause additional nail pigmentationwhich cause additional nail pigmentation

NNail polishes which may contain magnesiumail polishes which may contain magnesiumor ironor iron

PProfessionalrofessional

exposure to dyes and asphalt exposure to dyes and asphalt

Attention was made about other drugs:Attention was made about other drugs:

Vasodilators which are increasing the blood flow Vasodilators which are increasing the blood flow through the nail region (and thus enable the through the nail region (and thus enable the quicker cooling)quicker cooling)

WarfarinWarfarin

therapy according to INR datatherapy according to INR data

Special attention was made about factor which Special attention was made about factor which cause or increase skin photosensitivity and cause or increase skin photosensitivity and

additional changes in skin pigmentationadditional changes in skin pigmentation

SSyystestemicmic

therapy with photosensitive effecttherapy with photosensitive effect: :

RetinoidesRetinoides, c, ceephphalosporinalosporinss, griseofulvin, , griseofulvin, nalidiksinalidiksic acidc acid,,

tetraciklilntetraciklilnss, hinolon, hinolonss, NSAIM, , NSAIM,

antimalaricantimalaricss, antimitotic, antimitoticss, diuretic, diureticss

(furosemid, (furosemid, triamteren), trankvilitriamteren), trankvilissererss, ben, benssodiaodiassepinepinss, , antiaritmicantiaritmicss,sulfonamid,sulfonamidss......

LoLoccalal

chemical chemical photosensitisersphotosensitisers::

AArsen, rsen,

busulfan, mebusulfan, mecchloretaminhloretaminee, 5 , 5 ffluorouracil...luorouracil...

Methode:Methode: Fungal nail infectionFungal nail infection

72 patients, 194 affected nails72 patients, 194 affected nails

162 patients, 413 infected nails162 patients, 413 infected nails

Follow up was performed at 3,6 and 12 months, with mycological check ups at 3 and 6 months.

2009.2009.

20112011..

112 patients2 patients

withwith

tineatinea

2009.2009.36 36 patientspatients

withwith

tineatinea

2011.2011.

Number of affected Number of affected patientspatients

LocalisationLocalisation

of of tineatinea

2 42 4 faceface44 1212 extremitiesextremities2 42 4 bodybody4 164 16 footfoot

Follow up was performed at 2,4, 8 and 12 weeks.2009. 2011.

Fungal superficial skin infection

Methode:Methode:

Sample Sample collection process performed thorough collection process performed thorough cleansing of the nail area with alcohol to cleansing of the nail area with alcohol to remove contaminants. remove contaminants.

TThe abnormal nail was clipped proximally and he abnormal nail was clipped proximally and the nail bed and underside of the nail plate the nail bed and underside of the nail plate were scraped with a 1were scraped with a 1--2 mm serrated curette2 mm serrated curette..

The sampled material was dThe sampled material was deevided into two vided into two portions: one for direct microscopy and the portions: one for direct microscopy and the remainder for cultureremainder for culture..

Direct microscopy: 10% KOH with 40% DMSO Direct microscopy: 10% KOH with 40% DMSO

solution was used for direct microscopic examinationsolution was used for direct microscopic examination

Candida spCandida sp

Candida sp.

Trichphyton rubrum

Trichophyton mentagrophytes Molds

Diagnostic Tests:Diagnostic Tests:

Fungal CulturesFungal Cultures

DTM (DTM (DermatophyteDermatophyte Test Medium)Test Medium)

Yellow to red is (+). Yellow to red is (+).

Nickerson’s MediaNickerson’s Media

YeastYeast

Black growth is (+)Black growth is (+)

Sabouraud’sSabouraud’s MediaMedia

MoldsMolds

Pre laser treatment nail preparationPre laser treatment nail preparation

Reduction of hyperkeratosisReduction of hyperkeratosis

Er:YAGEr:YAG

ablationablation

Chemical keratin melting (urea)Chemical keratin melting (urea)

DermabraderDermabrader

or or sanpapersanpaper

nail plate nail plate

abrasionabrasion

LP Nd:YAG laser settingsLP Nd:YAG laser settings

Long pulse 1064 nm wavelenth laser (Fotona Dualis Long pulse 1064 nm wavelenth laser (Fotona Dualis SPII platform)SPII platform)

FFluence in the range of about 35 to 40 J/cm2 luence in the range of about 35 to 40 J/cm2

SSpotpot

size of 4 mmsize of 4 mm

PPulsewidt 25ulsewidt 25--35 ms35 ms

Pulse rate 1 HzPulse rate 1 Hz

The fluence can be selected based on the thickness of The fluence can be selected based on the thickness of the nail to be treated, a thicker nail can require a the nail to be treated, a thicker nail can require a higher fluence. higher fluence.

Laser beam is applied to substantially the entire nail Laser beam is applied to substantially the entire nail plateplate..

FFluence 30 J/cm2 luence 30 J/cm2 SSpotpot

size 6 mmsize 6 mm

PPulsewidt 25 msulsewidt 25 ms

Application of laser beamApplication of laser beam

Laser beam is appliedLaser beam is applied

by moving by moving the beam in spiral pattern. the beam in spiral pattern.

After substantially all of the entire After substantially all of the entire nail plate was irradiated, the nail plate was irradiated, the treatment is stopped for a minute treatment is stopped for a minute and repeated by going over the and repeated by going over the nail two more additional times. nail two more additional times.

Each treatment consisted of Each treatment consisted of moving the laser beam over the moving the laser beam over the nail for three cycles, once per nail for three cycles, once per week, for 4 total treatments. week, for 4 total treatments. (2)(2)

No topical medicine was applied No topical medicine was applied to the nail.to the nail.

Results: Results: Clinical types of fungal nail infection in treated Clinical types of fungal nail infection in treated groupgroup

Type of Type of onychomycosisonychomycosis

Number of Number of patients patients

(%)(%)Total dystrophicTotal dystrophic 6 ( 8.3%)6 ( 8.3%)Distal Distal subungualsubungual 38 38

(52.8%)(52.8%)Proximal Proximal subungualsubungual 22 22

(30.5%)(30.5%)EndonyxEndonyx 6 (8.3%)6 (8.3%)

2009. 2011.2009. 2011.

Number of Number of patients patients (%)(%)

13 13 ( ( 88..0202%)%)

86 86 (5(533%)%)

3434((2121%)%)

2929 ((1717..99%)%)

ResultesResultes

of fungal nail isolates in of fungal nail isolates in a primary a primary isolation medium isolation medium SabouraudSabouraud

PPeptoneeptone--GGlucoselucose

AAgargar

Type of fungal Type of fungal isolates isolates

Number of patients Number of patients

Candida sp.Candida sp. 1010 (13.9%)(13.9%)

T. T. rubrumrubrum 3737 (51.4%)(51.4%)

T. T. mentagrophytesmentagrophytes 2222 (30.5%)(30.5%)

AspergilusAspergilus nigerniger 33 (4.16%)(4.16%)

Number of patientsNumber of patients

94 (58%)94 (58%)

40 (24.7%)40 (24.7%)

224 (14.8%)4 (14.8%)

4 (2.5%)4 (2.5%)

2009.2009.

2011.2011.

Petri dish with T. Petri dish with T. mentagrophytesmentagrophytes

before and 3 before and 3 days after LP days after LP NdYAGNdYAG

laser irradiation. laser irradiation.

Note the inhibition of growth in treatedNote the inhibition of growth in treated

ccoloniesolonies

At high radiant energy each pulse creates an At high radiant energy each pulse creates an obvious crater.obvious crater.

At At lower radiant energieslower radiant energies blanching appears in the blanching appears in the

center of the beam on the center of the beam on the target surface and a small target surface and a small plume of ablated material plume of ablated material is emitted from the target is emitted from the target surface.surface.

Treatment efficacy (according to microscopy Treatment efficacy (according to microscopy findings) at four control points: 3, 6, 9 and 12 findings) at four control points: 3, 6, 9 and 12

months (2011., No of patients:162)months (2011., No of patients:162)

80,00%

82,00%

84,00%

86,00%

88,00%

90,00%

92,00%

94,00%

96,00%

98,00%

100,00%

3M 6M 9M 12M

Duration of followDuration of follow--up intervals of interviewed up intervals of interviewed patients (2011, No of patients:162)patients (2011, No of patients:162)

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

12-18 M 18-24 M 24-30 M > 30 M

Subjective evaluation of resultsSubjective evaluation of results

All interviewed patients reported to have no All interviewed patients reported to have no problems with nails after the treatment. 93,5% problems with nails after the treatment. 93,5% of them reported to have fully clear nail plates, of them reported to have fully clear nail plates, while 6,5% of responders were not sure if their while 6,5% of responders were not sure if their nail plates are fully clear. nail plates are fully clear.

93.5%

6.5%

Our idea from the start of the study: Single shot generated aprox. 45-50°C, after 7 shots temp.

increased to aprox. 75-80°C

(Without cooling with Cryo 6)

Cooling down

Measurement of nail temperature after Nd:YAG laser Measurement of nail temperature after Nd:YAG laser scanningscanning

Fluence 40 J/cm2Fluence 40 J/cm2, P, Pulseulsewidthwidth

25msec25msec, , Spot size 4mm, Frequency Spot size 4mm, Frequency 1Hz.1Hz.

°C

35

40

45

50

55

60

0 10 20 30 40 50 1:00 1:10

The nail is fully covered in approximatelly 15 sec, reaching cca 50°C and the cooling down fase below 40°C is usually longer than 1 minute

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

30,4°C

39,5°C

32

34

36

38

Toenail Nd:YAG laser irradiationToenail Nd:YAG laser irradiation

1. 2. 3. 4.

5. 6. 7. 8. 9.

10. 11.

Cooling down

Starting point

The nail is fully covered in approximatelly 15 sec, reaching cca 50°C and the cooling down fase below 40°C is usually longer than 1 minute

Candida spCandida sp

before after 3 months after 9 months

Aspergilus nigerAspergilus niger

before after 6 months

Trichophyton rubrumTrichophyton rubrum

before after 6 months after 12 months

Trichophyton mentagrophytesTrichophyton mentagrophytes

before after 12 months

Trichohyton rubrumTrichohyton rubrum

Before after 9 months

Trichophyton rubrumTrichophyton rubrum

1.0-1.8 mm/monthbefore after 12 months

Candida sp, Fusarium sp.Candida sp, Fusarium sp.

Before after 3 months after 12 months

Trichophyton rubrumTrichophyton rubrum

Before after 6 months

Aspergilus nigerAspergilus niger

before after 12 months

Trichophyton rubrumTrichophyton rubrum

Before after 12 months

DisscusionDisscusion::Volumetric heat production Volumetric heat production Nd:YAGNd:YAG

We still have a lot of questionsWe still have a lot of questions

1.1. How many treatments do we really need?How many treatments do we really need?2.2. Mechanism of action?Mechanism of action?3.3. Is it LLLT or not?Is it LLLT or not?4.4. Is it non ablative laser Is it non ablative laser photodestructionphotodestruction??5.5. Is it specific or non specific antifungal Is it specific or non specific antifungal

therapy?therapy?

LLLT

35 J/cm2

Nd:YAGNd:YAG laser LLLTlaser LLLT

5 J/cm2 16 J/cm2

Positive structural and cellular response.An increase in cellular migration, viability, proliferation and cytokine expression

Ihibitory effects on cellular migration, a decrease in cellular viability, proliferation and cytokine expression, An increase of cellular and genetic damage.

Ref: Houreld and Abrahamse 2006

HOST

Time between treatmentsTime between treatments

There is no hard and fast rule concerning There is no hard and fast rule concerning treatment intervals, but this study treatment intervals, but this study demostrateddemostrated that appropriate time between that appropriate time between treamtentstreamtents was was more effective than single more effective than single irrardiationirrardiation

Adaptive response?Adaptive response?

The first exposure prepares cells for subsequent irradiation

HOST

FungusFungus

>300 °C>300 °C Vaporization, burningVaporization, burning

>150 °C>150 °C Carbonisation Carbonisation

9090--100 °C100 °C DehydrationDehydration

80 °C80 °C Damage of membraneDamage of membrane

6060--65 °C65 °C Denaturisation of proteinDenaturisation of proteinee

4040--45 °C45 °C Formation of edemaFormation of edema

3737--60 °C60 °C Heating, and Heating, and pretransitionpretransition in the unfolding of in the unfolding of ribonucleaseribonuclease S, due to partial unfolding of the S protein/S S, due to partial unfolding of the S protein/S peptide complex.peptide complex.The dissociation occurs at higher temperature. The dissociation occurs at higher temperature. Mechanism of sequential Mechanism of sequential denaturationdenaturation means that at least one means that at least one partial unfolding step comes before the main conformational partial unfolding step comes before the main conformational transition, which is instead a concerted, final transition, which is instead a concerted, final unfolding/dissociation step. unfolding/dissociation step.

Trichophyton Trichophyton mentagrophytesmentagrophytes

Laser Laser ––

Nail tissue interactionNail tissue interaction

Due to the light absorbing substance present in fungus, laser irradiation results in excessive heat production which leads to the destruction of the pathogen and therefore to the termination of the infection condition.

This device employ 1064 nm infrared wavelength that is well known to cause cellular photodamage in the absence of exogenous dyes or other photosensitive drugs and chemicals.

Pigment related Pigment related photothermolysisphotothermolysis

Lack of growth inhibition in our

study

might be a

supporting

evidence regarding pigment-related photothermolysis

of T.rubrum

rather than inhibition

due to nonspecific thermal

damage.

Although the wavelength of Nd:YAG

laser at 1,064

nm is beyond the absorption spectrum of xanthomegnin, we have observed inhibitory effects

on

the colonies treated with this wavelength.

This might be

due to another chromophore

absorbing

at 1,064 nm, such

as

melanin,

as it is known that Trichophyton

species contain

melanin in their cell

walls.

Heat shock responsHeat shock respons

Heat shock response is nature’s device to protect cellsHeat shock response is nature’s device to protect cells against environmental and physiological against environmental and physiological stresstress.s.

External stresses including heat shockExternal stresses including heat shock induce the induce the generation of ROS and generation of ROS and denaturationdenaturation of cellular proteins.of cellular proteins. Activations of signaling pathways in response to a stress Activations of signaling pathways in response to a stress vary depending on the strength of stress resulting in the vary depending on the strength of stress resulting in the generation of various amounts of ROS and denatured generation of various amounts of ROS and denatured proteins.proteins.

Strong stress which is overflowing the rescuing capacity Strong stress which is overflowing the rescuing capacity of cells, induce cell death. of cells, induce cell death.

Membrane lipid Membrane lipid ceramideceramide has been proposed as a has been proposed as a signaling moleculesignaling molecule that converts extracellular stresses into that converts extracellular stresses into intracellular signals. In response to heatintracellular signals. In response to heat shock, shock, ceramideceramide levels increased in normal HLlevels increased in normal HL--60 cells60 cells..

HSP 70 in T. rubrumHSP 70 in T. rubrum

HSP70HSP70

in in TrichophytonTrichophyton

rubrumrubrum

is already detected is already detected

and carefully and carefully characterisedcharacterised..

Reactive oxygen species (ROS)Reactive oxygen species (ROS)

has recently beenhas recently been

suggested as a second messenger generated by suggested as a second messenger generated by growthgrowth

factors and cytokines, including PDGF, factors and cytokines, including PDGF,

EGF, angiopoietinEGF, angiopoietin--1,1,

TNFαTNFα, and IL, and IL--1 in 1 in nonphagocyticnonphagocytic

cells. cells.

ApoptosisApoptosis

Denatured proteins disrupt cellular Denatured proteins disrupt cellular redoxredox homeostasis homeostasis and increase ROS levels and and increase ROS levels and ROS inducesROS induces protein protein misfoldingmisfolding.. When When misfoldedmisfolded proteins are produced, proteins are produced, proteolyticproteolytic machinery is turned on to remove themmachinery is turned on to remove them..

Most severe protein Most severe protein denaturationdenaturation leads apoptosis of leads apoptosis of fungal cellfungal cell -- a programmed cell death or sometimes cell a programmed cell death or sometimes cell suicide which plays asuicide which plays an in i mportantmportant role in a wide variety role in a wide variety of normal and pathological processesof normal and pathological processes..

ConclusionConclusion

1064 nm l1064 nm laseraser irradiation with the capability of irradiation with the capability of deliveringdelivering destructive high energy pulses to specific destructive high energy pulses to specific targets withtargets with minimized surrounding tissue damage minimized surrounding tissue damage would seem to bewould seem to be well suited for the task of eradicating well suited for the task of eradicating nail fungnail fungus and us and tineatinea infection.infection.

LLPP Nd:YAGNd:YAG photophoto--inactivate fungal pathogens to a inactivate fungal pathogens to a depth below the nail tissue surface leaving the depth below the nail tissue surface leaving the surrounding tissue intact, using safe energy densities in surrounding tissue intact, using safe energy densities in vitro and in vivo at physiologic temperatures.vitro and in vivo at physiologic temperatures.