supervised by prof. dr. hoda ahmed monieb

EVALUATION OF LONG PULSED-ND: YAG LASER IN THE

TREATMENT OF ONYCHOMYCOSIS

Thesis

Submitted for Partial Fulfillment of Master Degree in

Dermatology, Venereology and Andrology

By Eman Mohamed Akmal Ahmad

(M.B.B.CH) Faculty of Medicine – Ain Shams University

Supervised By

Prof. Dr. Hoda Ahmed Monieb Professor of Dermatology and Venereology

Faculty of Medicine Ain-Shams University

Prof. Dr. Mohammed Taha Mahmoud

Professor of Microbiology Faculty of Veterinary Medicine

Zagazig University

Dr. Mary Fikry Matta Lecturer of Dermatology and Venereology

Faculty of Medicine Ain-Shams University

Faculty of Medicine - Ain-Shams University

2013

بسم الله الرحمن الرحيمبسم الله الرحمن الرحيمبسم الله الرحمن الرحيم

زبكزبكزبك باظنباظنباظن إقسأإقسأإقسأ

111خلقخلقخلق الرًالرًالرً خلقخلقخلق

هيهيهي الإًعاىالإًعاىالإًعاى

222علقعلقعلق إقسأإقسأإقسأ

وزبكوزبكوزبك

333الأكسمالأكسمالأكسم الرًالرًالرً

444بالقلنبالقلنبالقلن علنعلنعلن علنعلنعلن

لنلنلن هاهاها الإًعاىالإًعاىالإًعاى

555يعلنيعلنيعلن

صدق الله العظيم صدق الله العظيم صدق الله العظيم ظوزة العلقظوزة العلقظوزة العلق

(((555---111 ) ) )الآياثالآياثالآياث

AAAccckkknnnooowwwllleeedddgggmmmeeennnttt

First of all, thanks to Allah the most merciful for giving me the strength to complete this work.

I would like to express my gratefulness and respect to Prof.

Dr. Hoda Ahmed Monieb, Professor of Dermatology, Venereology and Andrology, Ain Shams University for her moral and sincere scientific support and for kind observation and valuable advice that were essential for this work to be achieved.

I would like to express my thanks and gratitude to Prof.

Dr. Mohammed Taha Mahmoud, Professor of Microbiology, Faculty of Veterinary medicine-Zagazeg University for his priceless help and his kind supervision.

I would like to express my gratitude and respect to Dr.

Mary Fekry Matta, Lecturer of Dermatology, Venereology and Andrology, Ain Shams University for her valuable time, remarkable efforts, help and guidance.

I would like to express my gratefulness and respect to

Assist Prof.Dr Ahmed Fathy Albedewey, Assit Professor of Dermatology, Venereology and Andrology, National Center for Radiation Research and Technology for his observation and valuable advice that were essential for this work to be achieved.

I would like to express my gratitude and respect to Dr.

Noha Fawzy Ibrahim , Lecturer of Dermatology, Venereology and Andrology, National Center for Radiation Research and Technology for her valuable time, remarkable efforts, help and guidance.

Finally I wish to express my deepest gratitude and appreciation to all my Family for their patience, moral support and encouragement.

EEEmmmaaannn MMMooohhhaaammmeeeddd

List of Contents Title Page No.

Introduction ......................................... Error! Bookmark not defined.

Aim of the Work .................................................................................... 21

Review of Literature

Onychomycosis .......................................................................... 24

o Definition ....................................................................... 5

o Nail Apparatus .............................................................. 5

o Epidemiology of onychomycosis .................................. 13

o Etiology ........................................................................ 16

o Pathophysiology and clinical presentation ................ 33

o Complications .............................................................. 44

o Differential diagnosis .................................................. 46

o Diagnosis of onychomycosis ........................................ 53

o Management................................................................ 74

Laser and Infections .............................................................. 107

o Introduction ................................................................. 87

o Bactericidal effect of lasers ......................................... 87

o Fungicidal effects of lasers ......................................... 90

Patients and Methods ....................................................................... 122

Results .................................................................................................. 134

Discussion ............................................................................................ 174

Conclusion ............................................................................................ 188

Recommendations .............................................................................. 189

Summary .............................................................................................. 191

References ................................................................................................ 1

Arabic Summary

List of Tables

Table No. Title Page No.

Table (1): Diagnosis of onychomycosis caused by

dermatophytes .............................................................73

Table (2): Classification of approved antifungal drugs .........96

Table (3): Sex distribution of patients in the study. ........... 134

Table (4): Age distribution of patients in the study. ........... 135

Table (5): Residence distribution of patients in the study. ...... 136

Table (6): Occupations of patients in the study. .................. 137

Table (7): Predisposing factors of onychomycosis in

patients of the study. ............................................... 139

Table (8): Number of diseased nails in patients of the

study. ........................................................................... 140

Table (9): Duration of onychomycosis (years). ...................... 142

Table (10): Patient’s complaints of the disease

(onychomycosis). ....................................................... 143

Table (11): Clinical presentations of onychomycosis in

the total number of fingernails and

toenails. ...................................................................... 144

Table (12): Identification of fungi into genera and

species of the 20 fungal isolates obtained

from cases of onychomycosis. ................................. 146

Table (13): Mycological response to treatment at the

first follow up ............................................................ 150

Table (14): Comparison between the mycologically

negative and the mycologically positive

patients as regards chronic paronychia as a

predisposing factor of onychomycosis at

first follow up results. ............................................. 152

List of Tables (Cont…)

Table No. Title Page No.



patients as regards exposure to humidity

as a predisposing factor of onychomycosis

at first follow up results. ......................................... 153



patients as regards number of nail affection

at first follow up results. ......................................... 155

Table (17): Comparison of the Duration of

onychomycosis between the mycologically


patients at first follow up results. ........................ 157


mycologically positive and mycologically

negative patients at the first follow up. .............. 158

Table (19): Comparison of the fungal species isolate

between the mycologically negative and the

mycologically positive patients at first

follow up results. ...................................................... 160

Table (20): Relation between first and second follow

up. ................................................................................ 161

Table (21): Mycological response to treatment at the

second follow up. ....................................................... 162

Table (22): Side effects noticed during the treatment. ......... 166

List of Figures

Fig. No. Title Page No.

Figure (1): Schematic drawing of nail anatomy

sagittal section .......................................................... 25

Figure (2): The proximal matrix forms the

superficial part of the nail plate and the

distal matrix makes the under-surface

part of the nail plate ................................................ 26

Figure (3): A diagram showing front view of nail

anatomy ...................................................................... 28

Figure (4): Schematic diagram of spores of the three

common genera of dermatophytes: ....................... 37

Figure (5): Trichophyton macroconidia .................................... 38

Figure (6): a) Macromorphology of Trichophyton

mentagrophytes colonies.

b) Macromorphology of Trichophyton

rubrum colonies front and back view. ................. 39

Figure (7): Microsporum microcondia ...................................... 40

Figure (8): Microsporum colonies in culture ........................... 41

Figure (9): Macroconidia of Epidermophyton

floccosum .................................................................... 42

Figure (10): Colonies of Epidermophyton floccosum ............. 43

Figure (11): C.Albicans under microscope ................................. 47

Figure (12): C.albicans on saburaud’s dextrose agar .............. 48

Figure (13): Microscopic morphology of Scopulariopsis

showing chains of single-celled globose to

pyriform, usually truncate, with a

rounded distal portion conidia ................................ 49

Figure (14): Scytalidium species in culture produce

woolly colonies and both surface and

reverse colony colour range from white

to brown pigmented hyphae ................................... 50

Figure (15): Clinical picture &nail invasion in DLSO ............ 55

Figure (16): Clinical picture & nail invasion in SWO ............. 57

List of Figures (Cont…)


Figure (17): a) Clinical picture of PSO.

b) Nail invasion in PSO. ........................................ 58

Figure (18): Clinical picture &nail invasion in

endonyx onychomycosis .......................................... 59

Figure (19): Candida onychomycosis with chronic

paronychia.................................................................. 60

Figure (20): Chronic mucocutaneous candidiasis .................... 61

Figure (21): Total dystrophic onychomycosis............................ 63

Figure (22): Nail Psoriasis showing onycholysis and

oil drop ........................................................................ 67

Figure (23): Lichen planus showing onychorrhexis

and angel wing deformity ....................................... 68

Figure (24): Contact dermatitis of the nail showing

onycholysis and nail dystrophy ............................. 69

Figure (25): Pitting in organized transverse rows

giving the nail a "hammered brass"

appearance ................................................................. 70

Figure (26): The Heat Shock Response (Physical or

chemical stress induces production of

unfolded or misfolded proteins. ........................... 118

Figure (27): Schematic representation of apoptosis

(ROS generated by mitochondria are

essential mediators of apoptosis. ........................ 120

Figure (28): Long pulsed Nd:YAG laser 1064 nm

(Candela, Wayland, MA, USA). .......................... 126

Figure (29): Laser irradiation a spiral pattern

starting at the nail periphery and

finishing in the nail centre ................................... 132

Figure (30): Sex distribution of patients in the study. ......... 134

Figure (31): Age distribution of patients in the study. ......... 135

Figure (32): Residence distribution of patients in the

study. ......................................................................... 136

Figure (33): Occupations of patients in the study. ................ 137



Figure (34): Predisposing factors of onychomycosis in

patients of the study. ............................................ 139

Figure (35): Number of diseased nails in patients of

the study. .................................................................. 141

Figure (36): Duration of onychomycosis (years). .................... 142

Figure (37): Clinical presentations of onychomycosis

in the total number of fingernails and

toenails. .................................................................... 144

Figure (38): Identification of fungi into genera and

species of the 20 fungal isolates obtained

from cases of onychomycosis. ............................... 147

Figure (39): Candia albicans, C.tropicalis & C.krusei

on chromogen agar. ................................................ 148

Figure (40): Trichosporon species showing

arthrospores, pseudohyphae and

blastospores. ............................................................ 148

Figure (41): a) T.mentagrophytes culture on SDA.

b) Microscopy of T.mentagrophytes

showing macroconidia and microconidia. ......... 149

Figure (42): Aspergellus flavus culture on SDA. ................... 149

Figure (43): Mycological response to treatment at the

first follow up. ......................................................... 150

Figure (44): Comparison between the mycologically


patients as regards chronic paronychia

as a predisposing factor of

onychomycosis at first follow up results. .......... 152



patients as regards exposure to humidity

as a predisposing factor of

onychomycosis at first follow up results. .......... 153





patients as regards number of nail

affection at first follow up results....................... 155

Figure (47): Bar chart Comparing the Duration of

onychomycosis in the mycologically


patients at first follow up results. ...................... 156


in mycologically positive and

mycologically negative patients at the

first follow up. ......................................................... 158

Figure (49): Comparison of the fungal species isolate

between the mycologically negative and

the mycologically positive patients at

first follow up results. ........................................... 159

Figure (50): Mycological response to treatment at the

second follow up. ..................................................... 162


in mycologically positive and

mycologically negative patients at the

second follow up. ..................................................... 164

Figure (52): Comparison of the fungal species isolate

between the mycologically negative and

the mycologically positive patients at

second follow up results. ....................................... 165

Figure (53): a) Left big toe before treatment.

b) Left big toe 3 months after treatment. ......... 167

Figure (54): a) Left middle finger before treatment.

b) Left middle finger 3 months after .................. 168

Figure (55): a) Right big toe before treatment.

b) Right big toe 3 months after treatment .......... 169



Figure (56): a) Right index before treatment.

b) Right index 6 months after treatment .......... 170

Figure (57): a) All fingers of left hand except little

finger before treatment.

b) All fingers of left hand except little

finger 3 months after treatment. ........................ 171

Figure (58a): a) All fingers of right hand before

treatment.

b) All fingers of right hand after

treatment ................................................................. 172

List of Abbreviations

Abb. Full term

Acquired immunedeficiency diseases : AIDS

aminolevulinic acid : ALA

Apoptotic protease activating factor 1 : Apaf-1

Adenosine-5'-triphosphate : ATP

Bromcresolpurple : BCP

Candida albicans : C. albicans

Candida glabrata : C. glabrata

Candida krusei : C. krusei

Carbon dioxide : CO2

Candida parapsilosis : C. parapsilosis

Candida tropicalis : C. tropicalis

Chitin synthase 1 gene : CHS1

Cell-mediated immunity : CMI

Distal and lateral subungual onychomycosis

: DLSO

Di-methyl sulfoxide : DMSO

Deoxyribonucleic acid : DNA

Delayed-type hypersensitivity : DTH

Dermatophyte test medium : DTM

Deubiquitinating enzymes : DUBs

Epidermal growth factor : EGF

Endonyx onychomycosis : EO

Erbium-doped yttrium aluminium garnet : Er:YAG

Human immunodeficiency virus : HIV

Human papilloma virus : HPV

Health related quality of life : HRQoL

Heat shock factor binding protein 1 : HSBP1

Heat shock factor : HSF

Heat shock protein : HSP

Hertz : HZ

Interleukin-1 : IL-1

Internal transcribed spacer 1 : ITS1

Potassium hydroxide : KOH

Potassium hydroxide treated nail clipping with periodic acid-schiff

: KONCPA

Light Amplification by Stimulated Emission of Radiation

: Laser

Lacto-phenol cotton blue : LPCB

Microsporum audouinii : M. audouinii

Microsporum canis : M. canis

Microsporum cookie : M. cookie

Microsporum ferrugineum : M. ferrugineum

Microsporum gypseum : M. gypseum

Microsporum nanum : M. nanum

Sodium chloride : Nacl

Sodium hydroxide : NaOH

Neodymium-doped yttrium aluminium garnet

: Nd: YAG laser

Non-dermatophyes moulds : NDMs

Periodic acid Schiff stain : PAS stain

Polymerase chain reaction : PCR

Polymerase chain reaction-restriction fragment length polymorphism assay

: PCR-RFLP

Potato dextrose agar : PDA

Platelet derived growth factor : PDGF

Photodynamic therapy : PDT

Pulse per second : Pps

Proximal subungual onychomycosis : PSO

Quality of life : QOL

Repetitive Extragenic Palindromic Sequence Polymerase Chain Reaction

: REP-PCR

Ribonucleic acid : RNA

Reactive oxygen species : ROS

Rapid sporulating medium : RSM

Sabouraud dextrose agar medium : SDA

Species : spp

Superficial white onychomycosis : SWO

Trichophyton equinum : T. equinum

Trichophyton erinacei : T. erinacei

Trichophyton megninii : T. megninii

Trichophyton rubrum : T. rubrum

Trichophyton soudanense : T. soudanense

Trichophyton tonsurans : T. tonsurans

Trichophyton violaceum : T. violaceum

Trichophyton mentagrophytes : T.mentagrophytes

Total dystrophic onychomycosis : TDO

Tumour necrosis factor : TNF

Thermal relaxation time : TRT

Yttrium Aluminium Garnet : YAG

16

Introduction

17

INTRODUCTION

nychomycosis is a common persistent fungal infection of

the nail bed, matrix or plate. It is the most common nail

disorder in adults, accounting for one third of all fungal skin

infections and up to 50 percent of all nail diseases worldwide

including psoriasis, atopic dermatitis, nail trauma, contact

irritants, and lichen planus (Schlefman, 1999 and Ghannoum,

2000).

Toenails are affected more often than fingernails and the

incidence is greater in older adults (Evans, 1998). Individuals

who are especially susceptible include those with chronic

diseases such as diabetes, circulatory problems, smokers,

patients with psoriasis and those with diseases that suppress the

immune system (e.g. HIV-positive patients, extremes of age,

patients on long term corticosteroids therapy). Other risk

factors include a family history, previous trauma to the nails,

warm climate, and occlusive or tight footwear (Gupta et al.,

2004).

The causative agents of onychomycosis include

dermatophytes, to a lesser extent non dermatophyte moulds and

rarely, yeasts of the Candida species (Evans, 1998).

Onychomycosis is classified clinically as distal and

lateral subungual onychomycosis (DLSO), superficial white

onychomycosis (SWO), proximal subungual onychomycosis

O

18

(PSO), candidal onychomycosis and total dystrophic

onychomycosis (Summerbell, 1989).

Onychomycosis is very difficult and sometimes

impossible to treat, and therapy is often long-term with high

relapse rates 50 – 85 %. Several management and treatment

regimens were designed to control and cure this disease such as

palliative care, topical as well as systemic drugs (Gupta, 2003).

Among the orally delivered systemic drugs terbinafine,

itraconazole and fluconazole are most frequently used, but with

several unpleasant side effects as headache, gastrointestinal

symptoms, liver enzyme abnormalities (Scher, 1999).

Nail debridement chemically or surgically is another

treatment option, but it is considered by many to be primitive

compared with topical or systemic treatment (Donald et al.,

2002). The choice of therapy is influenced by the presentation

and severity of the disease, other medications that the patient is

taking, physician and patient preference, and cost (Gupta et al.,

2003).

A novel non-invasive approach for treatment of

onychomycosis is the application of laser energy to the nail

plate targeting the fungal cells themselves. By using a laser

with a specific wavelength of laser light energy, the fungus

could be directly targeted in the nail and heated to the point it is

killed, but without burning the surrounding tissue and with

leaving the skin and nail intact (Kozarev and Vizintin, 2010).

19

The Nd-Yag (neodymium-doped yttrium aluminium

garnet; Nd: Y3Al5O12) laser is such a device with a

wavelength 1064nm that will pass through the nail plate and

into the nail bed resulting in superheating of the fungal

material. Exposure of fungi to high temperatures inhibits their

growth as well as causing cell damage and death (Hashimoto

and Blumenthal, 1978).

Several other types of lasers are introduced for the

treatment of onychomycosis including diode laser (Landsman

et al., 2010) femtosecond infrared titanium sapphire laser

(Manevitch et al., 2010). A novel 0.65-millisecond pulsed

Nd:YAG is also introduced for the treatment of onychomycosis

(Mozena and Haverstock, 2010).

20

Aim of the

Work

21

AIM OF THE WORK

he aim of this work is to explore and evaluate the usage of

long pulsed-Nd:YAG laser 1064nm in treatment of

onychomycosis.

T

22

Review of

Literature

23

Onychomycosis

Review of Literature Onychomycosis

24

Chapter (1)

ONYCHOMYCOSIS

Definition

nychomycosis is a common persistent fungal infection of

the fingernail or /& toenail (Jesudanam et al., 2002). It is

a general term used for any fungal infection of the nail that can

be caused by dermatophytes, yeasts or other non-dermatophyes

moulds (NDMs) (Jennings et al., 2002). This contrasts with the

term tinea unguium that is specifically used for infection of the

nails caused strictly by dermatophytes (Zaias, 1990). It may

involve any component of the nail unit, including the nail

matrix, nail bed, or nail plate (Zaias, 1990).

Nail Apparatus

Nails are keratinous horny plates that form protective

coverings on the dorsal surface superior to the distal or ungual

phalanges of the fingers and toes (Daniel, 2004).

It is important to know the structural and functional

organization of the nail unit and the process of nail growth to

understand pathogenesis of fungal infection of the nail unit

(Haneke, 2006).

A. Structure of the Nail unit:

The term “nail unit” is used to describe the nail and its

surrounding structural components.

O


25

Anatomic structures of the nail (Figures 1 & 3) include,

from proximal to distal; the nail matrix (nail root) and the

lunula, the proximal nail fold, eponychium, the cuticle the

lateral nail folds (perionychium), the nail plate, the nail bed, the

onychodermal band and the hyponychium (Rich, 2005 and

Ximena and Gregor, 2006).

Figure (1): Schematic drawing of nail anatomy sagittal section

(Wolff, 2009).

1. The matrix (Nail root): it is the germinative epithelium

that gives rise to majority of nail plate. It consists of

proximal (dorsal) and distal (intermediate) portions. The

proximal portion of the matrix lies beneath the nail folds and

the distal curved edge. It can usually be seen through the

nail plate as the lunula (half moon) which is whitish,


26

crescent-shaped and contains nerves, lymph, blood vessels

(Elewski, 1998). The proximal matrix forms the superficial

(dorsal) part of the nail plate and the distal matrix makes the

under-surface (ventral) part of the nail plate (Figure 2) (De-

Berker et al., 2007). Its proliferation activity is higher in its

proximal portion than distally so that more nail substance is

formed proximally and the nail plate achieves a natural

convex curvature from proximal to distal (Rich, 2005). It

has lateral matrix horns that reach further proximally than

the central part of the matrix (Haneke, 2006).

Figure (2): The proximal matrix forms the superficial part of the nail

plate and the distal matrix makes the under-surface part of the nail plate

(Jiaravuthisan et al., 2007).

2. The nail folds:

- The proximal nail fold is the cutaneous fold covering the

proximal end of the nail (Haneke, 2006). It is continuous

with the cuticle, which is the horny end product that is

shed from the underside of the proximal nail fold

(Elewski, 1998 and Rich, 2005).


27

- The lateral nail folds (the paronychium) are the

cutaneous folds on the lateral sides of the nail, where it

meets the skin of the finger (Zook, 2003). Its function is

to surround, support and protect the nail (Rich, 2005).

3. The Eponychium: it is the small band of epithelium that

binds the nail to the underlying skin. It is located proximally

on the dorsal surface of the nail extending from the base of

the nail. Precisely, it is located at the end of the proximal

nail fold above the cuticle (Elewski, 1998).

4. The nail bed: it is the vascular bed beneath the nail plate

extending from the lunula to the hyponychium. It is

composed of two layers, the deeper dermis, which is the

living tissue fixed to the bone containing capillaries and

glands; and the superficial epidermis, which is the layer just

beneath the nail plate that moves forward with it. The

epidermis is attached to the dermis by tiny longitudinal

"grooves" known as the matrix crests (De-Berker et al.,

2007), forcing the nail plate to grow forwards (Perrin,

2008). The nail bed is sometimes called the sterile matrix

and probably contributes some cells to the under-surface of

the nail plate, allowing the nail to grow continuously while

adhering to the nail bed (Rich, 2005).

5. The nail plate: it is the smooth translucent structure that is

the end product of the keratinocyte differentiation in the nail

matrix (De-Berker et al., 2007). It is formed of a strong

flexible material made of several layers of dead, flattened

http://en.wikipedia.org/wiki/Eponychium

http://en.wikipedia.org/wiki/Epithelium

http://en.wikipedia.org/wiki/Dermis

http://en.wikipedia.org/wiki/Epidermis_%28skin%29

http://en.wikipedia.org/wiki/Nail_plate


28

mechanically and chemically resistant sheet of compacted

keratinized cells. Nail hardness is mainly due to the

disulfide bonds found in the keratin in the nail plate. It also

contains 0.1% calcium, which contributes a little to the

hardness of the nail plate (Rich, 2005).

6. The Hyponychium: is the epithelium located beneath the

nail plate at the junction between the free edge and the skin

of the fingertip (Elewski, 1998). It forms a seal that protects

the nail bed and obliterates the distal groove called

onychodermal band located just under the free edge, in that

portion of the nail where the nail bed ends (Perrin, 2008).

7. The onychodermal band: it is an ill-defined transverse

band of a deeper pink, approximately 1 to 1.5 mm in width

that marks the transition of the nail bed to the hyponychium.

Its integrity is important for the health of the nail bed.

(Haneke, 2006), as it represents the first barrier to

penetration of materials beneath the nail plate (De-Berker et

al., 2007).

Figure (3): A diagram showing front view of nail anatomy (Rich, 2005).

http://en.wikipedia.org/wiki/Epithelium


29

B. Types of cells in the nail apparatus:

1. Nail matrix:

The nail matrix is made of epidermis and dermis. It is

composed of squamous epithelium but has no granular layer. It

has long rete ridges characteristically descending at a slightly

oblique angle, their tips pointing distally. Laterally, the matrix

rete ridges are less marked, whereas those of the nail bed and

nail folds become prominent. Distally, there is often a step

reduction in the epithelial thickness at the transition of the

matrix with the nail bed which represents the edge of the

lunula. Germinal cells in the matrix become larger and paler

and eventually the nucleus disintegrates. There is progression

with flattening, elongation and further pallor. There are some

melanocytes and dendritic cells found in the epibasal layers and

most prominent in the distal matrix. There is only a thin layer

of dermis dividing the matrix from the terminal phalanx. This

has a rich vascular supply and an elastin and collagen

infrastructure giving attachment to periosteum (De-Berker et

al., 2007).

2. Nail folds:

The lateral and proximal nail folds are similar in

structure to the adjacent skin but are normally devoid of

demographic marking and sebaceous glands. Keratinization

within the nail folds proceeds via keratohyaline formation in

the granular layer (De-Berker et al., 2007). The dorsal part of


30

the proximal nail fold is formed of epidermis and dermis that is

the continuation of the skin of the dorsal digit with sweat gland

but no hair follicles and sebaceous glands. The skin of the

ventral surface of proximal nail fold is thin, does not show rete

ridges- dermal papillae pattern or epidermal appendage (Rich,

2005).

3. Nail Bed:

It is made of deep epidermal layers -as it has no granular

layer- and dermis. There is no subcutaneous tissue in the nail

bed, so immediately beneath the nail bed lies the periostium of

the distal phalanx (Rich, 2005).

The epidermis of the nail bed is thin but becomes thicker

at the nail folds where it develops rete ridges. The dermis is

sparse, with firm collagenous adherence to the underlying

periosteum and no sebaceous or follicular appendages. Sweat

ducts can be seen at the distal margin (De-Berker et al., 2007).

4. Nail plate:

It is composed of multilayered, stacked sheet of

compacted keratinized epithelial cells that are intimately fused

and translucent. These cells derived from anucleate

onychocytes that arise from the germinal matrix epithelium

where the proximal matrix gives rise to the superficial (dorsal)

part of the nail plate and the distal matrix makes the under-

surface (ventral) part of the nail plate (De-Berker et al., 2007).


31

The cells of the proximal part of the nail plate are not

completely anucleated (parakeratotic cells in horny layer) so

they are whitish in colour (Ximena and Gregor, 2006).The nail

plate appears pink because it transmits the coloration of blood

vessels of the nail bed beneath (Daniel, 2004).

5. The hyponychium:

It has a granular layer unlike the matrix and nail bed

(Rich, 2005). Cells of the hyponychium are larger than cells of

the nail plate proper (Daniel, 2004).

C. Nail growth:

Nail growth record can show the history of recent health

and physiological imbalances, and can be used as an important

diagnostic tool (Elewski, 1998). Nail growth occurs by the

addition of keratinizing cells from the nail matrix onto the nail

plate over the nail bed (Daniel, 2004).

As new nail plate cells are incubated, they emerge from

the matrix round and white to push older nail plate cells

forward towards the free margin and in this way yet older cells

become compressed, flattened and translucent making the pink

colour of the capillaries in the nail bed below visible (Haneke,

2006). Fingernails grow continuously, at a rate of

approximately 0.1 mm/day or 3 mm a month. Toenails grow at

about one-half to one-third the rate of fingernails meaning that

toenail growth rate is 1mm/month (Rich, 2005).


32

Epidemiology of onychomycosis

Onychomycosis affects approximately 5% of the

population worldwide and it is increasing, because of many

factors such as diabetes, immunosuppression and increasing

age (Murray and Dawber, 2002 and Iorizzo et al., 2007). It

accounts for one third of all fungal skin infections (Migdley et

al., 1994) and up to 50% of all nail diseases world wide

(including psoriasis, atopic dermatitis, nail trauma, contact

irritants, and lichen planus) (Drake et al., 1996).

- Sex:

It affects males more commonly than females. However,

candidal infections are more common in women than in men,

affecting the toenails 4-7 times more than that of the fingernails

(Elewski, 1998).

- Age:

It has been estimated that 15% to 20% of individuals

between the ages of 40-60 years may suffer from this problem

(Jesudanam et al., 2002).

Children have infection rates 30 times lower than adults.

The reason for this decrease may be due to, reduced exposure

to fungus because of less time spent in environments containing

pathogens, faster nail growth, smaller nail surface for invasion

and lower prevalence of tinea pedis. Onychomycosis


33

prevalence in children younger than 18 years ranges from 0%

in USA up to 2.6% in Guatamala (Gupta et al., 1997c).

- Residence & its effect on onychomycosis

Dermatophytes are the fungi most commonly responsible

for onychomycosis in the temperate western countries (Chi et

al., 2005). Non dermatophtes moulds (NDMs) are more

frequent in tropical and subtropical areas with a hot and humid

climate (Haneke, 1991 and Chi et al., 2005).

- Incidence and prevalence:

According to small studies, prevalence of

onychomycosis varies from 4% to 18% depending upon the

age, geographic distribution and population studied (Erbagci,

2005).

However, the incidence of onychomycosis has been

reported to be 2% to 13% in North America (Raujo et al.,

2003), while a multicenter survey in Canada showed the

prevalence of onychomycosis at 6.5% (Summerbell, 1997).

In addition, studies in the United Kingdom, Spain, and

Finland found prevalence rates of onychomycosis to be 3% to

8% (Iorizzo et al., 2007).

The „Achilles‟ project which is the largest survey of

onychomycosis in 20 European countries, revealed an

incidence rate of 29% (Burzykowski et al., 2003).

http://www.uptodate.com/contents/onychomycosis/abstract/2




34

Unlike the Western countries where it is a frequent cause

of nail disorder, the prevalence of onychomycosis in South East

Asia is very low in comparison. A large-scale survey at late

1990s confirmed a prevalence of 3.8 % in Tropical countries &

up to 18% in the subtropical countries (Bramono, 2001).

In developing countries, higher priorities directed to

socioeconomic concerns and health issues for other diseases,

have resulted in low awareness of onychomycosis by

physicians and the general public alike (Kaur et al., 2008a) so

not much data is available regarding the prevalence of

onychomycosis there (Seebacher et al., 2008).

But a few literatures about prevalence of onychomycosis

in some developing countries reveal that it affects 2.4% to 3.5%

in some cities of Iran caused mainly by dermatophytoses

(Falahati et al., 2003 and Chadeganipour et al., 1997), and in

some cities of Algeria prevalence was estimated to be 4.6%

(Djeridane et al., 2006).

In EGYPT, literatures stated that onychomycosis is

infrequent among their patients affecting approximately 6% of

a sample of 50 patients in a study by El-Said (El-Said, 2002

and Abdel-Hafez et al., 1995). It was mainly caused by NDMs

including Cochliobolus Lunatus, Aspergillus, Alternaria,

Fusarium, Penicillium, Scopulariopsis, finally Dermatophytes,

which includes species of Microsporum canis and Trichophyton

(Abdel-Hafez et al., 1995).


35

Some large epidemiological studies showed the

prevalence of onychomycosis in the diabetic population to be as

high as 35%, compared to estimates ranging from 2% to 13% in

the general population (Kemna and Elewski, 1996).

Another study showed that psoriatic patients had 56%

higher odds of developing onychomycosis compared with non-

psoriatic patients (Gupta et al., 1997a).

Etiology

A. Causative Organisms

Onychomycosis is a pattern of superficial mycoses

caused by three main classes of fungi: dermatophytes, yeasts,

and NDMs (Evans, 1998).

The number of patients affected by onychomycosis is

continually growing and every year the number of

microorganisms recognized as capable of parasitizing the nail

plate directly is growing all over the world (Greer, 1995). With

the continued increase in the prevalence of onychomycosis, it is

important to keep in mind that all isolated filamentous or

pseudohyphal organisms should be evaluated as potential

pathogens when diagnosing fungal infections (Bassiri-Jahromi

and Khaksar, 2010).

The causative fungi as Dermatophytes, yeasts or NDMs

differ according to geographic location (Midgley et al., 1994).

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term=%20Bassiri-Jahromi%2BS%5bauth%5d


36

Dermatophytes are by far the most common cause of

Onychomycosis. However, NDMs are becoming more common

worldwide. In addition, onychomycosis due to Candida is

prevalent but not that common (Tosti et al., 2003).

However, in a large-scale North American study of

fungal isolates from the nails to detect the frequency of

onychomycosis, it was found that NDMs and yeasts represented

20 % each (Ghannoum et al., 2000).

I. DERMATOPHYTES:

Dermatophytes are a group of filamentous fungi

composed of three genera that are morphologically and

physiologically related, some of them can cause well-defined

infections called dermatophytoses (tineas or ringworm)

(Weitzman and Summerbell, 1995).

They possess two important properties: they are

keratinophilic and keratinolytic. This means that they have the

ability to digest and utilize keratin in vitro in their saprophytic

state and some members can invade tissues in vivo and provoke

tineas (Hay, 1992).

Dermatophytes can be classified by many ways

according to their morphological, ecological or genetic

characters (Taha, 2011).


37

- Morphological Classification:

Morphology in the parasitic growth phase is different

from the morphology exhibited in culture or in vitro

(Simpanya, 2000).

Dermatophytes can reproduce asexually & produce

micro and macroconidia. Based on that, particularly on

differences in conidial morphology, dermatophyte species can

be classified into three genera which are Epidermophyton,

Microsporum, and Trichophyton (Figure 4) (Summerbell,

1997).

Figure (4): Schematic diagram of spores of the three common genera of

dermatophytes: (a) It represents the macroconidia and microconidia of

Trichophyton genus. (b) It represents the macroconidia of

Epidermophyton genus. (c) It represents the macroconidia and

microconidia of Microsporum genus (Deacon, 2005).


38

a) Trichophyton genus

Under the microscope: The macroconidia are large

smooth, thin walled septate (0-10 septa) and cigar- shaped

(Figure 5). Microconidia are spherical, pyriform to clavate or of

irregular shape, and range from 2 to 4 μm in size.

Figure (5): Trichophyton macroconidia (Khurana et al., 2011).

In culture the Macromorphology of the colonies (Figure

6a) differ according to the species itself. In Trichophyton

mentagrophytes the front surface is flat, white to cream in

colour (Ellis and Watson, 1996) and pale yellowish brown in

colour from reverse. Trichophyton rubrum shows cottony white

colonies with reddish periphery and red ring or red colour from

reverse (Figure 6b) (Taha, 2011). Trichophyton rubrum can

show a granular velvety type with creamy surface and dark

brownish red colour from reverse. Also loose aerial mycelium

that grows in a variety of colours can be seen (Taha, 2011).


39

Figure (6a): Macromorphology of Trichophyton mentagrophytes colonies

(Ellis, 2007).

Figure (6b): Macromorphology of Trichophyton rubrum colonies front

and back view (Taha, 2011).

Trichophyton genus includes 30 species the commonest

of them are Trichophyton rubrum, Trichophyton

mentagrophytes (Kaur et al., 2008b). Other members present in

this genus are Trichophyton tonsurans, Trichophyton


40

violaceum, Trichophyton kanei, Trichophyton raubitscheckii

and others (Elewski, 1998).

Some species such as T. tonsurans produce numerous

microconidia and rarely produce macroconidia (Ellis and

Watson, 1996).

b) Microsporum genus

Under the microscopy (Figure 7) the macroconidia are

rough thin or thick walled, spindle shapes, multicellular having

an average of 2-12 cells according to species. The essential

distinguishing feature of this genus is the echinulations on the

macroconidial cell wall (Simpanya, 2000) while microconidia

are pyriform and nearly about 2-3μm (Taha, 2011).

Figure (7): Microsporum microcondia (Salvo et al., 2004).

In culture, the colonies are arranged singly along the

hyphae. They are usually flat & white to yellow in colour


41

(Figure 8) (Ellis and Watson, 1996). Microsporum canis shows

cottony white colonies with yellowish periphery and yellowish

brown colour from reverse (Taha, 2011).

Figure (8): Microsporum colonies in culture (Ellis, 2007).

It includes 18 species that can infect skin and hair, but

rarely nails examples are Microsporum canis, Microsporum

gypseum, Microsporum audounii, Microsporum nanum,

Microsporum persicolour, Microsporum ferrugineum and

others (Aly, 1994).

It has been shown that the ontogeny of the holothallic

conidia of Microsporum and Trichophyton is essentially the

same. Their only difference is the macroconidial cell-wall

thickness and presence of echinulations in Microsporum

species which are absent in Trichophyton species (Simpanya,

2000).


42

c) Epidermophyton genus

The only important pathogenic member is

Epidermophyton floccosum.

Under the microscopy (Figure 9) the macroconidia are

known to be thin-walled, with bifurcated hyphae which are

multiple smooth club-shaped and cluster in bunches, while no

microcondidia are produced (Ellis and Watson, 1996).

Figure (9): Macroconidia of Epidermophyton floccosum

(Salvo et al., 2004).

Colonies in culture (Figure 10) are slowly growing,

greenish-yellow coloured with flat furrows at the periphery and

raised folded centre, ranging from whitish yellow to brown

colour from reverse surface. They can infect skin and nails and

rarely hair (Taha, 2011).


43

Figure (10): Colonies of Epidermophyton floccosum

(Salvo et al., 2004).

Trichophyton is known to be the most frequent

dermatophyte genus affecting nails, mainly T. rubrum

accounting for 70% of cases followed by T. mentagrophytes

accounting for 20% of all cases (Elewski, 1998) and to some

extend T. tonsurans. Other dermatophytes less commonly

invade the nail.

- Ecological Classification:

The dermatophytes are classified to three ecological

groups epidemiologically according to their habitat Geophiles,

Zoophiles and Anthropophiles. The differences in host

specificity have been attributed to the differences in keratin of

the hosts (Taha, 2011).

a) Anthropophylic:

It is usually restricted to human only, transmitted from

man to man by close contact or through contaminated objects.


44

The most important members are Epidermophyton fluccosum,

T.rubrum, and T.mentagrophytes (Taha, 2011).

b) Zoophilic:

It usually inhabits animals then is transmitted to man by

close contact with animals (cats, dogs, and cows) or with their

contaminated products. The most important members include

T. verrucosum, T. erinacei, M. canis and T. equinum (Taha,

2011).

c) Geophylic:

It is usually found in soil and transmitted to man by

direct exposure such as M. cookei, M. gypseum, M. nanum and

others (Hainer, 2003).

- Phylogenetic classification:

Due to difficulties in some dermatophyte species

identification by morphological criteria only, modern molecular

biological investigation such as analysis of serological antigens,

comparison of DNA base compositions , fatty acid composition

and enzyme isoelectric focusing are done to help in

differentiation of morphologically and physiologically similar

species (Simpanya, 2000).

These new analytic investigations gave rise to a

completely new classification where members are put in

complexes depending on their close phylogenetic characters


45

(Taha, 2011); using internal transcribed spacer 1 (ITS1) region

ribosomal DNA sequences that demonstrated the mutual

phylogenetic relationships of dermatophytes of the genera

Trichophyton, Microsporum, and Epidermophyton.

Trichophyton genus and Microsporum genus form a cluster in

the phylogenetic tree with Epidermophyton floccosum as an

outgroup (Makimura et al., 1999). Stating some of the

examples of these complexes in the new classification:

a) Trichophyton mentagrophyte complex

It contains most varieties of Trichophyton

mentagrophytes (Makimura et al., 1999).

b) Trichophyton rubrum complex

It contains the most common agents of dermatomycoses

primarily causing tinea pedis, onychomycosis, tinea corporis,

and tinea capitis like T. rubrum, T. megninii and others

(Gräser et al., 2000).

c) The Arthroderma otae Complex

The most common species worldwide in this complex is

M. canis. In addition, there are M. audouinii and M.

ferrugineum (Gräser et al., 2000).


46

II. YEAST:

Yeasts are species with a unicellular cell that reproduce

by budding. They are part of the normal flora on the skin and

mucous membrane (Fisher and Cook, 1998).

They are classified into two groups related

phylogenetically, which are ascomycetes & basisdiomycetes.

The class of our concern is ascomycetes as it contains one of

the most medically important yeasts, which is candida genus

that comprises about 170 species the most important of them is

Candida albicans (Taha, 2011). In addition, Blastoschizomyces

genus contains only one species Blastoschizomyces capitatus, a

soil saprophyte that was reported to rarely cause

onychomycosis (D’Antonio, 1999).

C. albicans predominates in most yeast-caused

onychomycosis cases (Pontes et al., 2002 and Seebacher et al.,

2007). However, C.parapsilosis, C. krusei, C. glabrata, and C.

tropicalis have been reported too but less frequently

(Faergemann, 1996).

C. parapsilosis has low pathogenicity mostly occurring in

toes & not usually associated with chronic paronychia

(Haneke, 1991).

Candida species is opportunistic yeast that needs an

altered immune response as a predisposing factor to be able to


47

penetrate the nail but it was shown that it might have a role in

keratinolysis of the nails (Zaias et al., 1996).

However, Yeasts are neither keratinolytic nor capable of

colonizing healthy nails, but they may possibly have some

proteolytic activity that can destroy the integrity of keratin.

This is why yeasts need predisposing factors like trauma or

alteration in immune response to invade the non healthy nail

stratum corneum and cause onychomycosis (Haneke, 1991).

The most important member is candida albicans that is

known under the microscopy to produce true hyphae,

pseudohyphae and clusters of blastoconidia & chlamydoconidia

(Figure 11) (Taha, 2011).

Figure (11): C.Albicans under microscope (Salvo et al., 2004).

Moreover, it is known in culture by creamy colour, pasty

& smooth colonies on Sabouraud's dextrose agar (Figure12)

(Taha, 2011).


48

Figure (12): C.albicans on saburaud‟s dextrose agar (Salvo et al., 2004).

Other methods for further identification of candida

species include:

- Culture on other media such as potato dextrose agar that

produces whitish, buff colonies.

- Culture on corn meal agar or rice agar then microscopic

examination for demonstration of chlamydospores and

pseudohyphae.

- Culture on Chromogen Candida agar where colonies‟ colour

strongly identifies the canida species (green for C. albicans,

blue for C. tropicalis, pink for C. krusei and creamy white

for C. parapsilosis) (Taha, 2011).

III. NON –DERMATOPHYTIC MOULDS:

Non dermatophtic moulds are filamentous fungi common

in soil and decaying plant debris (Tosti et al., 2000) but a


49

number of species (spp.) were described as etiological agents of

onychomycosis such as, Aspergillus spp., Fusarium spp,

Scopulariopsis brevicaulis, Scytalidium dimidiatum,

Onychocola canodiensis, S. hyalinum and Acremonium spp.

(Summerbell et al., 2005).

Acremonium, Fusarium, and Scopulariopsis species

(Figure 13) are the most common isolates among the NDMs

followed by Scytalidium species and Aspergillus species

(Ghannoum et al, 2000).

Figure (13): Microscopic morphology of Scopulariopsis showing chains

of single-celled globose to pyriform, usually truncate, with a rounded

distal portion conidia (Ellis, 2007).

Scopulariopsis nail infection causes brown

discolouration of the nail and a crumbly texture commonly

causing nail dystrophy, while Scytalidium infection of nail is

often characterized by dark discolouration and onycholysis


50

without significant thickening (Midgley et al., 1997).

Scytalidium species (Figure 14) is the only mould proved to be

capable of primary nail infection (Hay and Moore, 1998).

Figure (14): Scytalidium species in culture produce woolly colonies and

both surface and reverse colony colour range from white to brown

pigmented hyphae (Xavier et al., 2010).

IV. Mixed infections:

Mixed infections caused by Dermatophytes and non-

Dermatophytes are present although they need more

investigations to determine them accurately (Summerbell, 1997

and Elewski, 1998).

B. Predisposing Factors

Several risk factors contribute to the rising incidence of

onychomycosis including, cohabitation with family members

suffering from onychomycosis, increasing age of the

population, prior or repeated trauma, nail biting, smoking, poor

hygiene (Daniel, 1996). Other factors include, warm climate,

occlusive footwear, contamination of communal bathing places

by infected users because disinfecting the floors of such


51

facilities is very difficult as fungal elements are protected in

small pieces of keratin (Detandt and Nolard, 1995).

Patients with associated chronic diseases have an

increased risk of developing onychomycosis e.g. diabetes,

circulatory problems, tinea pedis and cancer chemotherapy

(Sigurgeirsson and Steingrímsson, 2004) hemiplegia

(Siragusa et al., 2001), chronic venous insufficiency (Del-mar

et al., 2001), poor peripheral circulation and peripheral

neuropathy (Elewski, 1996).

Immune suppression plays an important role in

onychomycosis. In individuals suffering from HIV,

onychomycosis is one of the important dermatologic signs of

the disease progression (Conant, 1994). In addition,

immunosuppression due to other reasons such as drug intake

like corticosteroids or other immunosuppressants makes

patients more susceptible for this fungal disease (Joish and

Armstrong, 2001).

Some occupations that require wetness and humidity also

play a role in developing onychomycosis such as nurses,

miners, housewives and cooks (Roberts et al., 1990).

In addition, some dermatological diseases are suggested

to be associated with onychomycosis for example, pemphigus

vulgaris patients show prevalence rate around 25% to 47% for

onychomycosis (Schlesinger et al., 2002).


52

An increased prevalence of onychomycosis among the

patients with psoriasis was found as dystrophic nails in

psoriasis patients are more predisposed to fungal infections

(Zisova et al., 2011).

Pathophysiology and clinical presentation

Dermatophytes, NDMs or Yeasts invade the nail

depending on their virulence factors, which include cell wall

consistency, enzymes produced or arthroconidia. Fungi can

infect the nail by adherence then invasion aided by overcoming

host immune defence (Hung and Finlay, 1997).

Three elements of the body defence system protect it

against deep penetration of fungal pathogens, namely the cell-

mediated immunity (CMI), the serum inhibitory factor (Dahl,

1993) and the myeloperoxidase-hydrogen peroxide-chloride

system (MacCarthy and Dahl, 1989).

Cell-mediated immunity is triggered when fungal antigen

activates lymphocytes and macrophages to produce

inflammation intensely and destroy the skin epidermal barrier.

This process is called delayed-type hypersensitivity (DTH)

reaction (Dahl, 1993). However, some dermatophytes can

produce certain glycoprotein called Mannans that diminish the

immune response and facilitate fungal infection. In addition,

this may contribute to the chronicity of the disease (Peres et al.,

2010).


53

The serum inhibitory factor is unsaturated transferrin that

enters the extra-vascular space and inhibits fungal growth by

robbing the organism of iron (Dahl, 1993).

Some fungi especially T. rubrum could activate

complement by the alternative pathway during invasion, which

activates neutrophils and cause adhesion of neutrophils to

hyphae leading to interferance with hyphal growth.

The myeloperoxidase - hydrogen peroxide-chloride

system may be involved in the actual destruction of the fungal

pathogens (MacCarthy and Dahl, 1989).

Any alteration of immunity affecting neutrophil

chemotaxis and phagocytosis, or impaired CMI, will result in

increased susceptibility to both Candida and dermatophytic

infections (Blanco and Garcia, 2008).

Fungi may invade the nails in four different ways,

leading to four separate types of onychomycosis with specific

clinical features, prognosis and response to treatment. The type

of nail invasion depends on the fungus responsible and the host

susceptibility (Tosti et al., 2003).

The main types are distal and lateral subungual

onychomycosis (DLSO), superficial white onychomycosis

(SWO), proximal subungual onychomycosis (PSO), endonyx

onychomycosis (EO) and candidal onychomycosis. Total

dystrophic onychomycosis (TDO) refers to the most advanced


54

form of any subtype of them. Patients may have a combination

of these subtypes (Baran et al., 1998).

1. Distal and lateral subungual onychomycosis

DLSO is the most common type accounting for the

majority of cases and is almost always due to dermatophyte

infection most commonly by T. rubrum (Faergemann and

Baran, 2003) rarely yeasts & moulds (Faergemann, 1996).

It spreads from epithelium of nail bed & via the

hyponychium, often at the lateral edges initially, and spreads

proximally along the nail bed resulting in an inflammatory

response causing subungual hyperkeratosis and onycholysis

(Figure 15).

Clinically the nail shows focal parakeratosis followed by

subungual hyperkeratosis and yellow to white colour

onycholysis (Kaur et al., 2008a). Yellow streaks / onycholytic

areas in the central portion of the nail plate are commonly

observed (Figure 15) (Elewski, 1998). The edge of the affected

nail is usually uneven and often one or more streaks of

dystrophic discoloured nail extend towards the distal border

(Tasic et al., 2001). When complicated by infection with

pigmented moulds or bacteria, nails may appear dark green to

black (Hay et al., 2001).

DLSO may be confined to one side of the nail or spread

sideways to involve the whole of the nail bed as in (Figure 15),


55

and progress until it reaches the proximal nail fold causing

paronychia (Elewski, 1998). Eventually the nail plate becomes

friable and may break up, often due to trauma, although nail

destruction may be related to invasion of the plate by

dermatophytes that have keratolytic properties (Roberts et al.,

2003).

Figure (15): Clinical picture &nail invasion in DLSO (Tosti et al., 2003).

2. Superficial white onychomycosis (SWO)

It is much less common than DLSO (Gupta and

Summerbell, 1999). It is confined to toenails and manifested as

a small, white, speckled or powdery patch on the surface of the

nail plate (Figure 16). Discolouration is white rather than

creamy and the surface of the nail plate is noticeably flaky

(Figure 16) (Roberts et al., 2003). It can extend and involve the

whole surface and at this point the nail becomes rough, soft and

crumbly (Gupta, 2001). In addition, onycholysis is not a


56

common feature of SWO and intercurrent foot infection is not

as frequent as in DLSO (Roberts et al., 2003).

As many species are involved, many classifications

appeared. Classic SWO in healthy people is always due to

dermatophyte infection most commonly caused by

T.mentagrophytes var interdigitale while NDMs cause deep

white superficial onychomycosis (Piraccini and Tosti , 2004

and Zias et al., 1996), and Candia can be involved specially C.

albicans (Faergemann, 1996).

T. rubrum affects the surface of the nail plate directly

rather than the nail bed (Figure 16). Later infection may extend

to the cornified layer of the nail bed and hyponychia. It may

also be responsible for SWO especially in children with

occlusion of the nail plate by an over- riding toe.

In the later example “occlusion of the nail plate by an

over- riding toe”, there may be DLSO-SWO and PSO caused

by T.rubrum or T. interdigitale on the nail of the same patient

(Meisel and Quadripur, 1992 and Baran et al., 1998).


57

Figure (16): Clinical picture & nail invasion in SWO (Tosti et al., 2003).

3. Proximal subungual onychomycosis (PSO)

It is the least common type (Faergemann, 1996) mostly

due to T.rubrum infection. It occurs in immunosuppressed

patients‟ e.g. Diabetes, AIDS and in peripheral vascular

disease (Hay et al., 2001).

It presents as subungual hyperkeratosis, proximal

onycholysis and leukonychia in the proximal nail plate that

moves distally with nail growth (Figure 17a) (Elewki, 1998). In

PSO caused by NDMs, periungual inflammation is usually

present (Tosti et al., 2000) and the leukonychia is typically

associated with marked paronychia (Fisher and Cook, 1998) as

a secondary nail involvement by C. albicans (Baran et al.,

1998). On the other hand, the fungus penetrates the nail matrix

through the proximal nail fold & the cuticle then colonizes at

the deep portion of proximal nail plate causing its

inflammation(Figure 17b) (Roberts et al., 2003). Inflammation


58

of the proximal nail fold followed by nail dystrophy is the

cause of complaint most of the times (Roberts et al., 1990).

Figure (17): a) clinical picture of

PSO (Habif, 2010).

Figure (17): b) Nail invasion in

PSO (Tosti et al., 2003).

4. Endonyx Onychomycosis:

This type is a variant of distal lateral subungual

onychomycosis (Tosti et al., 1999) caused mainly by T.

soudanense and T. violaceum which normally produce

endothrix scalp infections (Baran et al., 1998). This form of

infection involves invasion of the superficial surface of the nail

via the skin as well as deeper penetration of the nail plate

(Figure 18) without resulting nail bed hyperkeratosis,

onycholysis or nail bed inflammatory changes.

It presents as a milky white discolouration of the nail

plate, but no evidence of subungual hyperkeratosis or

onycholysis is present (Gupta, 2001) and is characterized by


59

specific lamellar splitting of the plate & white discolouration

(Figure 18) (Tosti et al., 1999).

Figure (18): Clinical picture &nail invasion in endonyx onychomycosis

(Tosti et al., 2003).

5. Candidal onychomycosis

Infection of the nail apparatus with Candida yeasts may

present in one of four ways, chronic paronychia with secondary

nail dystrophy, distal nail infection, chronic mucocutaneous

candidiasis, and secondary candidiasis (Roberts et al., 2003).

These forms occur more commonly in women than in men and

caused by Candida albicans, which can be observed commonly

in immunocompromised patients (Tasic et al., 2001).

i. Chronic paronychia with secondary nail dystrophy

It mainly affects the proximal and lateral folds presenting

with inflammatory signs, Beaus‟ lines, and cuticular

detachment.


60

It only occurs in patients with wet occupations. Swelling

of the proximal nail fold is secondary to chronic immersion in

water, and the cuticle becomes detached from the nail plate thus

losing its watertight properties. Microorganisms, both yeasts

and bacteria, enter the subcuticular space causing further

swelling of the proximal nail fold and further cuticular

detachment, i.e. a vicious circle. Infection and inflammation in

the area of the nail matrix eventually lead to a proximal nail

dystrophy (Figure 19) (Roberts et al., 2003).

Figure (19): Candida onychomycosis with chronic paronychia

(Singal and Khanna, 2011).


61

ii. Chronic mucocutaneous candidiasis

It has multifactorial aetiology leading to diminished cell-

mediated immunity. Clinical signs vary with the severity of

immunosuppression but in more severe cases gross thickening

of the nails occurs, affecting all digits with involvement of

mucous membranes (figure 20) (Cohen et al., 1992) oftenly

taking on a bulbous or drumstick appearance called (candida

granuloma) ending by irregular, convex, rough, dystrophic nail

plate (Kaur et al., 2008a and Roberts et al., 2003).

Figure (20): Chronic mucocutaneous candidiasis (Tosti et al., 2003).

iii. Distal nail infection

It is uncommon and occurs in patients of Raynaud‟s

phenomenon or suffering of some other form of vascular

insufficiency. It is characterized by onycholysis though it is

unclear whether the underlying vascular problem gives rise to

onycholysis as the initial event or whether yeast infection

causes the onycholysis first (Gary, 2007).


62

iv. Secondary candidiasis

It occurs in other diseases of the nail apparatus, most

notably psoriasis (Shirwaikar et al., 2008).

Despite the frequent isolation of Candida from the

proximal nail fold or the subungual space of patients with

chronic paronychia or onycholysis, candida is only a secondary

colonizer not an invader (Shirwaikar et al., 2008) but In

chronic mucocutaneous candidiasis, the yeast infects the nail

plate and eventually the proximal and lateral nail folds (Gary,

2007).

6. Total dystrophic onychomycosis (TDO)

Any of the above varieties of onychomycosis may

eventually progress to total nail dystrophy where there is

complete dystrophy of the nail plate (Elewski, 1998). It

presents as a thickened, opaque, and yellow-brown nail (figure

21) (Baran et al., 2006).

This can be classified into two main forms:

i) Secondary total dystrophic onychomycosis:

This results from complete progression of any of the

different types of destructive nail dystrophy (Roberts et al.,

1990).


63

ii) Primary total dystrophic onychomycosis:

This type occurs in chronic mucocutenous candidiasis,

where all the tissues of the nail apparatus may be involved

simultaneously, including nail folds (Baran et al, 1998).

Figure (21): Total dystrophic onychomycosis

(Singal and Khanna, 2011).

It is established that the fungus in TDO penetrates from

the nail plate into the skin epidermis, connective tissue, bone

and into the bone marrow of the phalanx (Parkhomenko et al.,

2001).


64

Complications

Onychomycosis is not life threatening, but it is a

common chronic disease with a substantial negative effect and

significant reduction in the quality of life (QOL) of the majority

of patients (Eros and Karoli, 2002).

QOL is impaired in all domains, including physical,

psychological, mental and social (Drake et al., 1999).

Particular problems include thickening which may produce

serious physical and occupational limitations due to loss of part

or all of a nail bed. Functions such as manipulating small

objects and repetitive finger use (e.g. typing) may be severely

compromised when fingernails are affected. Also prolonged

standing, walking can be impaired by severe toenail disease

(Bending, 2002). Psychosocial and emotional effects can lead

to lowered self-confidence, depression and social isolation that

result from disfigurements of nails (Elewski, 1995).

Another main problem is that infected nails serve as a

chronic reservoir of infection, which can give rise to repeated

mycotic infections of the skin (Jesudanam et al., 2002). In

patients with complicating factors, deformed nails can lead to

surrounding tissue damage and once again promote secondary

bacterial infection (Shirwaikar et al., 2008) that makes infected

toenails risk factors for the development of bacterial cellulitis

of the lower legs (Roujeau et al., 2004). On very rare

occasions, it could even disseminate via blood, resulting in

sepsis and patient death (Arrese et al., 1996).


65

Differential diagnosis

Many dermatological diseases can present with nail

affection and destruction so confirming the diagnosis of

onychomycosis needs certain criteria including both laboratory

and clinical features (Roberts et al., 2003).

First step in the clinical approach to the patient is done

by obtaining proper history from the patients, beside the

clinical appearance of the nail.

A careful history may reveal many environmental and

occupational risk factors and help differentiating fungal from

non-fungal aetiologies of nail dystrophies (Elewski, 1998).

As onychomycosis is usually asymptomatic, patients

usually present to the clinic with cosmetic complaint without

any physical complaints. As the disease progresses,

onychomycosis may interfere with standing, walking, and

exercising. Patients may report paresthesia, pain, discomfort,

and loss of dexterity (Milles, 1998). They also may report

psychological troubles like loss of self-esteem and lack of

social interaction (Lubeck, 1998).

Second step, is performing a meticulous dermatological

examination to differentiate True onychomycosis from

common nail changes that are unrelated to onychomycosis but

resemple it specially some clinical types like white superficial

onychomycosis & proximal subungual onychomycosis. These


66

nail changes can be onycholysis, nail plate thickening,

longitudinal or transverse ridging, pits, onychoschizia, dryness

of the nail plate and Surface leukonychia (Scher et al., 2007).

Also by clinical examination, subtypes of onychomycosis can

be identified on basis of their usual presenting clinical features.

Many dermatological diseases can present with nail

affection and may mimic onychomycosis so that they are

clinically indistinguishable from it so care should be taken to

correctly identify their other signs and symptoms (Elewski and

Hay, 1996).

These diseases include psoriasis, lichen planus, bacterial

infections, contact dermatitis, traumatic onychodystrophies,

pachyonychia congenita, nail bed tumors, yellow-nail

syndrome, and idiopathic onycholysis (Brodell and Elewski,

1997).

Psoriasis

The most common of these diseases is psoriasis.

Distinguishing between onychomycosis and psoriasis can be

difficult, since subungual hyperkeratosis, onycholysis, splinter

hemorrhages, and diffuse crumbling are clinical signs of both

conditions. The finding of a positive fungal culture does not

rule out psoriasis because dermatophytes or other fungi can

occasionally colonize psoriatic nails especially when the nail

plate is grossly deformed (Stander et al., 2001).


67

Three clinical symptoms of psoriasis can guide the

differentiation between it and onychomycosis, which include,

the presence of fine pitting on nail surface, the small salmon-

coloured oil-drop sign of onycholysis that is present in psoriasis

but absent in onychomycosis (Figure 22), and the frequent

involvement of nails in both hands in cases of psoriasis.

In addition, evidence of psoriasis at other sites such as

elbows and/or knees is also helpful in differentiating between

the two conditions. Sometimes a nail biopsy may be required to

obtain a definitive diagnosis (Salomon et al, 2003).

Figure (22): Nail Psoriasis showing onycholysis and oil drop

(Jiaravuthisan et al., 2007).

Lichen planus

Lichen planus is an inflammatory skin disease that may

involve the nails in 10% of affected patients (Cohen et al.,


68

1992). It may involve the nails on both hands and both feet

(twenty nail dystrophy) (Elewski, 1998).

The most common manifestations are onychorrhexis i.e.

exaggerated longitudinal ridging, and angel wing deformity

i.e. the central portion of the nail is raised, and the lateral

portion is depressed (Figure 23) (Albert et al., 1998). A key

clinical finding that differentiates lichen planus from

onychomycosis is the presence of Wickham's striae i.e irregular

white streaks, in typical lesions of the skin or mucous

membranes. Pterygium i.e scar of the cuticle may also be seen

in patients with lichen planus but not in those with

onychomycosis (Elewski and Hay, 1996).

Figure (23): Lichen planus showing onychorrhexis and angel wing

deformity (Holzberg, 2006).


69

Contact dermatitis

Contact dermatitis of the skin overlying the nail matrix

may produce hyperkeratosis and other nail changes that might

mimic onychomycosis (Figure 24) (Elewski and Hay, 1996).

Contact dermatitis is usually occupation-related, and a

differential diagnosis can easily be made by obtaining a careful

clinical history and performing a patch test (Elewski and Hay,

1996).

Figure (24): Contact dermatitis of the nail showing onycholysis and nail

dystrophy (Oppel and Korting, 2003).


70

Alopecia Areata

Nail changes caused by alopecia areata are pitting,

thinning of the nail plate (figure 25) and sometimes red-lunula

(Brodell and Elweski, 1997).

Figure (25): Pitting in organized transverse rows giving the nail a

"hammered brass" appearance (Kane et al., 2002).

Others

Repeated trauma to the nails can be responsible for

onycholysis that resembles onychomycosis. In traumatic

onychodystrophies, microorganisms that produce pigmentation

of the onycholytic area can colonize the onycholytic space. A


71

differential diagnosis can be made by clipping the onycholytic

nail.

In traumatic onychodystrophy, it reveals a normal nail

bed unless the trauma is chronic, compared to the

hyperkeratotic nail bed in onychomycosis (Elweski, 1998).

Nail bed tumours should also be considered in the

differential diagnosis of onychomycosis and can be ruled out by

obtaining a radiograph. Melanomas of the nail can be

differentiated from onychomycosis by biopsy (Elweski and

Hay, 1996).

A rare nail disease that sometimes needs differentiation

from onychomycosis is yellow-nail syndrome. This condition is

described as a triad of yellow nails, primary lymphedema and

chronic obstructive pulmonary disease (Samman and White,

1964). Clinical manifestations of this yellow nail in the

syndrome include absence of cuticles, yellow pigmentation, an

excessive curve in the nail, and cessation of nail growth

(Elweski and Hay, 1996).

In Darier‟s disease, there is an involvement of the nail

resulting in onychorrhexis, with splitting and fragility, red and

white longitudinal streaks and subungual hyperkeratosis with

overlying V-shaped notches at the free edge differentiated by

presence of the lesion in other sites as behind ears (Allevato,

2010).


72

In addition, nail products containing formaldehyde may

cause onycholysis and yellowish discolouration in all exposed

nails (Elweski, 1998).

Other causes may include habit Tic, pachyonychia

congenita, Idiopathic onycholysis and bacterial infections

(Brodell and Elweski, 1997).

Moreover, some drugs can cause nail dystrophy. The

changes range from mild pigmentation to nail shedding and

matrix scarring. Drugs involved include antimicrobial specially

tetracycline, antineoplastic, antimalarials, penicillamine as well

as other drugs (Daniel and Scher, 1984). For example,

dyschromia and dystrophy of the nail plate occurred in one

report after treatment with the antineoplastic drug docetaxel

(Correia et al., 1999).

Diagnosis of onychomycosis

The use of appropriate confirmatory diagnostic

techniques is essential as to start the specific correct therapy.

Although the cost of diagnostic tests may be high but the cost is

always relatively higher in case of inappropriate and

unnecessary treatment (Roberts et al., 2003).

As explained by table-1, diagnosis of onychomycosis is

commonly confirmed by clinical examination side by side with

regular diagnostic techniques such as direct microscopy and

fungal culture, which are considered the golden standards of

diagnosis (Gupta and Ricci, 2006).


73

Table (1): Diagnosis of onychomycosis caused by

dermatophytes (Scher et al., 2007).

Clinical examination Confirmatory Laboratory

specimen analysis

i. Primary criteria for diagnosis:

White/yellow or orange/brown

patches or streaks

Positive microscopic evidence

ii. Secondary criteria for diagnosis:

- Onycholysis

- Subungual hyperkeratosis/debris

- Nail-plate thickening

Positive culture of dermatophyte

In order to achieve an accurate diagnosis of

onychomycosis; proper collection of the specimen, suitable

transport to the laboratory, correct interpretation of the findings

on direct microscopic examination, use of appropriate culture

media and correct identification of the causative organisms is

needed (Khafagy et al., 1998 and Haneke and Roseeuw,

1999).

Valuable methods of diagnosis but less frequently

performed include PCR, Nucleic acid hybridization and

amplification methods, immunohistochemistry and dual-flow

cytometry (Pierard et al., 1996 and Lemont, 1997).

1- Specimen Collection:

Proper specimen collection is essential to accurate

diagnosis (Elewski et al., 1995) which is provided by collection


74

of an adequate amount of suitable clinical material from the

active site of infection (Ellis, 1999).

While collecting the specimen first, remove nail polish,

creams & ointments if present from the nail to be sampled.

o Wipe the nail with 70% alcohol on gauze.

o Collect debris from under the nail and place it in a clean

envelope or plastic tube.

o Scrape the outer surface of the nail and discard the

scraping.

o Collect scrapings from the deeper diseased areas of the

nail (Miller and Michael, 1999).

Because the sites of invasion and localization of the

infection differ in the different clinical types of onychomycosis,

different approaches are done depending on the primitive

clinical diagnosis:

- Distal subungual onychomycosis:

In this type, dermatophytes invade the nail bed rather

than the nail plate, so the specimen must be obtained from the

nail bed, where the concentration of viable fungi is greatest

(Elewski, 1995).

The nail should be clipped short with nail clippers, and

the specimen should be taken from the nail bed as proximal to

the cuticle as possible (Elewski et al., 1995).


75

Material should also be obtained from the underside of

the nail plate, from the advancing infected edge most proximal

to the cuticle. This is the area most likely to contain viable

hyphae and least likely to contain contaminants (Shemer,

2009).

- Proximal subungual onychomycosis:

Because the fungus invades under the cuticle before

settling in the proximal nail bed while the overlying nail plate

remains intact, the healthy nail plate should be gently pared

away with scalpel blade. A sharp curette can then be used to

remove material from the infected proximal nail bed as close to

the lunula as possible (Elewski et al., 1995 and Elewski, 1995).

- White superficial onychomycosis:

Since the infection affects the nail plate surface, a

scalpel blade or sharp curette can be used to scrape the white

spots on the nail while the outer most surface is discarded. The

white debris directly underneath is then collected (Midgley et

al., 1997).

- Candidal onychomycosis:

Material is needed from the proximal and lateral nail

edges. If Candida onycholysis is suspected, the lifted nail bed

should be scraped. If insufficient debris is present in the nail


76

bed, scrapings can be taken from the under-surface of the nail

(Roberts et al., 1990).

- Total dystrophic onychomycosis:

Specimen can be collected from any abnormal area of

the nail plate or bed (Elewski et al., 1995).

Finally, the last step is that all collected samples must be

examined and analyzed as soon as possible (Taha, 2011).

2- Specimen Analysis:

i) Direct Microscopy

Direct examination by microscopy is a rapid, easy,

simple and inexpensive technique (Weinberg et al., 2003). It is

used as an efficient screening test to identify presence or

absence of fungi in the nail specimen (Elewski, 1998) simply

by visualizing the fungal structure (hyphae & spores), not to

identify the specific etiological pathogen itself (Weitzman and

Summerbell, 1995), though it might help in differentiation

between yeast cells, dermatophyte hyphae, and non-

dermatophyte moulds (Clayton, 1992).

Although, it plays an important role in diagnosis of

onychomycosis direct Microscopy faces many limitations, as it

is often time-consuming because nail debris is thick and coarse.

Moreover, hyphae are usually sparsely present (Elewski, 1995).

In addition, it was reported that it shows false negative results


77

at a rate of approximately 5% to 15% (Weitzman and

Summerbell, 1995). That is caused by many reasons the most

important of them includes; inappropriate material obtained for

the study with inactive spores or low amount of hyphae ,

incorrect KOH solution used , an inadequate time hydrolyzing

the material (either not long enough or a very long time)

(Daniel, 1991). It may also give false positive results,

especially if saprophytic fungal spores are present, mosaic

fungus (incompletely dissolved hyphae) (Markus et al., 2001),

debris that is interpreted as fungal elements, Pus may contain

artefacts that may superficially resemble hyphae and budding

forms of fungi. Occasional fibres like wool or cotton fibres may

produce artefacts as well (Sparkes et al., 1993). Experienced

interpretation may be needed to distinguish hyphal elements

from various artefacts (Hassab-El-Naby et al., 2011)

Before visualizing the specimen by microscope, it should

be left in Potassium hydroxide preparation (KOH) which is the

most common formula used to soften and clear the nail

specimen (Pierard et al., 1996).

Nail is put in a solution of 10%-30% KOH or NaOH

mixed with 5% glycerol, heated for 1 hr at 51 °C to 54 °C in

order to emulsify lipids (Weitzman and Summerbell, 1995)

and clear the cells of the stratum corneum. Then the material

obtained is placed on a glass slide, left for 15 to 20 minutes

before examining the sample by direct microscopy (Roberts et

al., 2003) under low (10x) and high power (40x objective lens)


78

microscope for visualization of fungal elements such as

refractile septate hyphae, pseudo-hyphae, budding cells or

spores (Zaias et al., 1996).

Sensitivity of KOH can be increased by using an

alternative formulation, which consists of 20% potassium

hydroxide (KOH) preparation in 36% di-methyl sulfoxide

(DMSO). This preparation does not require heating or

prolonged incubation (Elewski, 1998). Added to that, it gives a

more rapid maceration and clearing than KOH alone (Ellis,

1999).

The detection of spores and fungal hyphae can be

enhanced by adding stains to the KOH such as Parker blue /

black ink (1 part KOH to 1 part ink), Polychrome Multiple

Stain, or fluorochromes (Eleweski, 1996).

Fluorochromation for fluorescence dyes (e.g. with

Blankophor, Calcofluor, or Fungiqual) is particularly helpful,

especially where hyphae are sparse, because it selectively

highlights the chitin of fungal walls (Pierard et al., 1996).

Calcofluor white and Blankophor P are non-specific

fluorochrome stains that detect fungi by binding with

polysaccharides in their chitin cell wall, and found to be

significantly more sensitive than the KOH wet mount in

observing fungal pathogens (Chander et al., 1993).


79

Congo red also is a non-specific flurochrome but it stains

cellulose and chitin to reveal fungal hyphea (Slifkin and

Cumbie, 1988).

The specimen can also be counter-stained with chitin-

specific stain such as, Chlorazol black E which is helpful in

identification of fungi when the number of fungal elements is

low. It acts by staining the carbohydrate rich wall of the hyphae

a blue-black colour accentuating it. This stain is especially

useful because it does not stain likely contaminants such as

cotton or elastic fibres, which can help prevent false-positive

identifications (Elewski and Charif, 1997 and Elewski, 1996).

Another method to enhance demonstration of fungal

hyphae in nail clippings is (Potassium hydroxide treated nail

clipping with periodic acid-schiff) (KONCPA) which is done

by centrifuging the clippings treated by KOH & staining the

sediment with PAS (Taha, 2011). This stain depends on

chemical identification of fungal cell wall, staining certain

polysaccharides, especially glycogen and mucoproteins

(Elewski, 1996).

Direct microscopic examination is done by using

different techniques like bright- field illumination, phase-

contrast or dark ground illumination (Pierard et al., 1996).

They greatly facilitate the observation of hyphae, which are

seen as fine filaments that traverse the tissue unimpeded by the


80

host cell structure (Milne, 2001) and also increase resolution in

some specimens (Pierard et al., 1996).

It is important to observe the hyphae and arthrospores

closely to determine if they are typical of dermatophyte fungi

or have features of non-dermatophyte moulds or yeasts

(Eleweski, 1998).

ii) Culture

A fungal culture is an important diagnostic technique that

must be used as it is the only way to identify the species of the

pathogen itself (Elewski, 1996).

Cultures should be obtained from crushed nail scrapings

or clippings to enhance fungal growth in media (Gupta and

Ricii, 2006).

Isolation of fungi depends mainly on the specific type of

media because non-dermatophytic moulds may be resistant to

the conventional therapy used for the more common

dermatophytes (Tosti et al., 2000). Therefore, two types of

growth media should be used, one primary media, selective to

dermatophytes only added to it cycloheximide to inhibit growth

of NDMs & yeasts. Some examples of selective media are

(dermatophyte test medium [DTM], Sabouraud glucose-

peptone agar with cycloheximide , known with many trade

names like mycosel or dermasel, potato flake agar, potato

glucose and devised Casamino Acids-erythritol- albumin


81

medium). The other secondary media are without

cycloheximide such as (Sabouraud glucose agar, Littman oxgall

medium, or inhibitory mould agar) to isolate other yeasts and

NDMs (Elewski, 1995).

As nails are non-sterile specimens, antibiotics like

chloramphenicol or gentamicin should be added to these

medias as an additional precaution to inhibit bacterial growth

and eliminate bacterial contamination (Kaur et al., 2008a).

Each media is important in identifying a specific

pathogen, Some examples include:

I. For identification of dermatophytes:

1. Sabouraud dextrose agar medium (SDA): It is

satisfactory for the isolation of the majority of fungal

pathogens of the nail (dermatophytes, NDMs and

yeast) (Weitzman and Summerbell, 1995).

2. Sabouraud glucose-peptone agar with cycloheximide

(Emmos modification): This medium provides the

basic nutrition for fungal growth (Milne, 2001), but

adding cycloheximide make it specific to

dermatophytes only (Fisher and Cook, 1998).

3. Dermatophyte test medium (DTM): considered for

screening purposes only as it gives false negative

reactions. It is used mainly for dermatophytes but other

NDMs can react to it too (Pariser and Opper, 2002).


82

The dermatophyte colony turns the media red by its

alkalinity (Elewski, 1996) though some non-pathogenic

fungi (e.g. Trichophyton terrestre) induce the red

colour too (Weitzman and Summerbell, 1995) while

some saprophytes give rise to green colour (Lemont,

1997).

4. Littman oxgall agar: is used for the selective isolation

and cultivation of fungi, especially Dermatophytes

(Ronald, 2000). It can reduce the colony diameters of

fast-growing contaminants, so allowing growth of

slower-growing etiologic agents (Summerbell et al.,

1989).

5. Devised Casamino Acids-erythritol- albumin medium:

a highly selective medium to isolate dermatophytes

from lesions heavily contaminated by bacteria or by the

cycloheximide-tolerant C.Albicans as egg albumin

inhibits those specific yeasts. That makes it useful in

diabetics & immune compromised (Fischer and Kane,

1974).

6. Bromcresolpurple (BCP)-casein-yeast extract agar:

This grows all dermatophytes but is designed for the

rapid recognition of microcolonies of T.verrucosum

(Kane and Smitka, 1978).

7. Urea agar or broth: used to differentiate urease negative

species. It is mainly used to differentiate T. rubrum


83

from T. mentographytes and some candida species

(Koneman and Roberts, 1991).

8. Rapid sporulating medium (RSM) of dermatophytes:

These Media enhance microscopic structure appearance

so it can enhance microconidia & macroconidia production.

a. Sabaroud dextrose agar plus 3 - 5% Nacl: used for

T.mentagrophytes and M. persicolour (Kane and

Fischer, 1973).

b. Potato dextrose agar (PDA): It is for primary isolation

and speeding the identification of T. rubrum (Weitzman

and Summerbell, 1995).

c. A series of vitamin and amino acid test agars are

available and used to confirm the identity of several

species through the distinctive responses to the growth

substances in the media e.g. the Trichophyton agars and

elucidated on autoclaved polished rice grains. M.

Audouinii grows poorly on it and secretes a brownish

pigment while M.canis grows well and secretes a yellow

pigment (Rippon, 1988).

II. For yeast identification:

1. Sugar assimilation agar used for identification of yeast

by determining carbohydrate assimilation (Koneman and

Roberts, 1991).


84

2. Yeast fermentation broth: it is used for identification of

yeast by determining carbohydrate fermentation

(Koneman and Roberts, 1991).

3. Rapid sporulating medium (RSM) of yeast:

Corn meal agar: It stimulates fungal conidial production

(Aly, 1994 and Milne, 2001). Yeast is identified by the

presence of hyphae, blastoconidia, chlamydoconidia, or

arthroconidia (Aghamirian and Ghiasian, 2010). After primary

culture these isolates are seen under microscopy (Taha, 2011).

Culture is incubated at room temperature (25°C to 30°C)

for four weeks and should be examined regularly. With this

period of incubation, a sufficient volume of medium should be

used and the humidity in the incubator should be maintained to

prevent drying of the agar. In addition, a good oxygen supply is

mandatory for fungal growth (Midgley et al., 1994).

Fungal isolates in the primer culture are identified based

on their growth rate, media of growth, macroscopic and

microscopic examinations of fungal colonies specific character

like pigmentation, or texture and morphologic characteristics

such as microconidia, macroconidia, spirals, pectinate branches

and pedicels are important. Other methods of identification

include biochemical reactions (Midgley and moore, 1996),

physical characters and sometimes a differential media is


85

needed (Taha, 2011). Successive specimens can be used

specially to identify non-dermatophytes (Kaur et al., 2008a).

If growth occurs on both types of media, the infective

agent is probably a dermatophyte, while growth occurring only

on the cycloheximide-free medium indicates that the infective

agent may be a non-dermatophyte such as Scopulariopsis

brevicaulis, Scytalidium dimidiatum, or Scytalidium hyalinum

(Elewski, 1998).

Yeasts and NDMs tend to grow more rapidly than

dermatophytes, taking days instead of weeks (Gupta and Ricii,

2006).

It is difficult to evaluate if non-dermatophyte fungi

cultured from the nail specimen are laboratory contaminants or

pathogens. Summerbell suggested a categorization for non-

filamentous non-dermatophytes identified in nail tissue:

Normal mammalian surface commensal organism

Transient saprobic colonizer (colonizer of accessible

surface molecules but non-invasive)

Persistent secondary colonizer (colonizer of material

infected by a dermatophyte but incapable of remaining

after the dermatophyte is eliminated)

Successional invader (species that can cause infection

after gaining entry into a nail via the disruption caused

by a primary pathogen)


86

Primary invader (is able to infect and cause

onychomycosis in a previously uncolonized nail).

Such an analysis has the value of identifying non-

dermatophytic infections that are truly invasive and

subsequently guiding their treatment (Summerbell, 1997).

Commonly encountered problems:

Unfortunately, culturing was reported to have a false-

negative rate of about 30% (Hay, 2005). This may arise when

the nail sample contains only dead or nonviable mycotic

organisms, which can occur when sample is taken from the

distal end of the nail plate & nail bed distal to fungal growth.

To avoid this problem sample should be taken more

proximally, at the junction between the diseased and normal-

appearing nail (Midgley et al., 1994).

Other causes like insufficient incubation temperature,

the nature and size of the inoculums, not collecting an adequate

sample or not crushing it thoroughly can result in false negative

results (Mehregan and Gee, 1999 and Guillot et al., 2001).

A negative mycological result does not rule out

onychomycosis, because direct microscopy may be negative in

up to 10% of cases and culture in up to 30% of cases (Tosti et

al., 2010).


87

There are other analytic techniques for diagnosis of

onychomycosis, which include histologic analysis,

immunohistochemistry, flow cytometry, in vivo confocal

microscopy, scanning electron microscopy, and polymerase

chain reaction (PCR) (Gupta and Ricci, 2006).

iii) Histopathological examination

This method usually is not mainly required in

onychomycosis diagnosis and can be limited to other causes of

nail dystrophy (Roberts et al., 2003) as the histology could aid

in their diagnosis making it easily differentiated from

onychomycosis specially in psoriasis (Weinberg et al .,2003).

Histologic examination depends on obtaining nail plate

and nail bed specimens. Many techniques can be used such as

punch and scalpel biopsy but these are not favoured because of

the potential risk of permanent nail dystrophy (Suarez et al.,

1991).

Nail clippings can give results as good as those obtained

from biopsy, but in a faster, more economical and painless

manner (Borkowski et al., 2001), so it is suggested as a good

alternative to either incisional or punch biopsy (Pierard et al.,

1996 and Machler et al., 1998).

Typically, the most distal portion of the nail plate is

clipped, fixed in 10% formalin and treated with 4% phenol for

softening then specimens are processed and embedded in


88

paraffin blocks and about 3-µm thin slices are taken and

mounted on glass slides (Pierard et al., 1994).

Then it is submitted for histopathologic evaluation using

a specific stain like periodic acid–Schiff stain (Machler et al.,

1998), Haematoxylin and Eosin, Toluidine blue, Grocott,

Blancophor or MacManus stains (Saurez et al., 1991).

Periodic acid–Schiff stain demonstrates the presence of

certain polysaccharides, specifically glycogen and

mucoproteins, which are present in the walls of the fungal

hyphae (Lemont, 1997). It also assesses the topographic

distribution, density, and nature of fungi present in the

subungual hyperkeratosis and in the nail plate itself (Arrese et

al., 1993).

A positive result is determined when the fungal hyphae

appear as bright red dots (Lemont, 1997).

It is considered the method of choice for the evaluation

of onychomycosis, as it is the most sensitive and superior to

other methods in its negative predictive value (Weinberg et al.,

2003). It can detect dermatophytes missed by microscopic

examination of KOH mounts with a sensitivity of 85% and

missed by nail cultures with a sensitivity of 94% and it is even

more sensitive when combined with Sabouraud‟s culture

(Lawry et al., 2000).


89

iv) Diagnostic PCR

Polymerase chain reaction allows for direct identification

of the nucleic acids of the fungal species and thus bypasses the

need for cultures that are traditional in morphological studies

(Seifert, 2009).

It involves the amplification of a specific target DNA

using a heat-stable enzyme, Taq polymerase, and a set of DNA

primers (short DNA oligonucleotides), leading to an

exponential amplification of this specific DNA. As a result,

minute quantities of DNA become detectable, in addition,

further analysis and testing are made possible (Arca et al.,

2004).

Some of the important types of PCR include:

1- Repetitive Extragenic Palindromic Sequence Polymerase

Chain Reaction (REP-PCR):

In this technique, primers are designed to complete the

interspersed repetitive sequences in the genome. As a result,

different-sized DNA fragments are produced consisting of

unique DNA sequences between these REP elements and used

as strain-specific identification method (Versalovic et al.,

1991).


90

2- Real-time quantitative PCR:

It is a commonly used quantitation method (Orlando et

al., 1998). In this technique, accumulation of the PCR product

is monitored and the amount of initial target DNA is quantified

using calibration curves from the log-linear phase of

amplification (Tsuboi et al., 2002).

3- LightCycler PCR:

It is an application of real-time PCR which has proved to

be a rapid and sensitive method for the detection and

quantitation of DNA and thus the infectious agent. In this

method, the level of measured fluorescence is proportional to

the amount of DNA synthesized in the closed PCR reaction,

leading to quantitation of this reaction and a reduction in the

risk of contamination (Tsuboi et al., 2002).

4- Reverse Transcriptase-PCR:

This technique has the ability to follow gene expression

and thus the viability of the organism by converting the

messenger RNA into a type of DNA termed complementary

DNA that can then be amplified using PCR and the gene

activity can be studied and even quantified using real-time PCR

(Ginzinger, 2002).


91

5- Nested PCR:

It is targeting the chitin synthase 1 gene (CHS1) gene

and targeting internal transcribed spacer gene 1 (ITS1) which

are dermatophyte-specific primers proved to be more sensitive

than the traditional methods (Nagao et al., 2005 and Garg et

al., 2007).

6- Polymerase chain reaction-restriction fragment length

polymorphism assay (PCR-RFLP):

It is more specific and sensitive as NDMs can be

identified with certainty as the infectious agents of

onychomycosis, and discriminated from dermatophytes as well

as from transient contaminants. It can also identify the

infectious agent when direct nail mycological examination

shows fungal elements, but negative results are obtained from

fungal culture (Tosti et al., 2000).

PCR may prove to be a rapid, sensitive, and specific test,

but the available techniques like PCR-restriction fragment

length polymorphism analysis and single-round PCR are either

too complex or not sensitive enough (Arca et al., 2004 and

Arrese et al., 1999).

v) Immunohistochemistry

It is useful if there were many fungi in the nail specimen

as in mixed infections because it enables in situ demonstration


92

of each specific fungal species, which is helpful in excluding

the possibility of the presence of contaminants that are just

attached to the nail surface (Pierard et al., 1996).

It is done by exposing the nail sample to antibodies

specific to certain fungi like monoclonal anti-Trichophyton spp

(dermatophytes), anti-Candida spp (yeasts) and anti-

Aspergillaceae spp (moulds) antibodies. If the fungi

corresponding to that specific antibody applied are present, they

are labelled and appear by direct immunofluorescence,

immunoperoxidase, or avidin-biotin complex methods. When

combined with image analysis, it allows quantification of the

fungal load in the nail plate (Pierard et al., 1996).

vi) Flow Cytometry

It is a diagnostic technique for fungal infections based on

the ability to identify molecular variances among fungal

species. Dissociated nail samples are filtered to collect single

fungal cells that are analyzed with a flow cell sorter based on

DNA and protein detection mainly and also, cell size and cell

granularity. Collection and analysis of data is used to identify

each fungal species (Pierard, 1996).

vii) Scanning electron microscopy

Scanning electron microscopy generates three-

dimensional images of high resolution, high magnification, and

large focal depth. Cross-sectioned nail specimens are fixed,


93

dehydrated and dried to be viewed allowing a detailed imaging

of the spatial orientation, penetration, and integration of fungal

elements in the nail plate (Scherer and Scherer, 2004).

viii) In vivo confocal microscopy

It is a rapid non-invasive technique of optical sectioning

through intact tissue by using a source of reflected light for

example YAG laser (Boston et al., 2000) to penetrate nail on

various depth. Fungi can be seen as a network of branching

hyphae (Hongcharu et al., 2000). This technique has the

advantage of increasing the volume of tissue available for

examination as nails are imaged in the native state as a living

tissue at high resolution and contrast without fixing, sectioning

or staining (Hongcharu et al., 2000).

Management

The increased prevalence of dermatophytic infections

and its associated morbidity make them an important public

health problem (Ellis, 1999 and Aly, 1994).

There are several options for therapy of onychomycosis

but treatment is influenced by the presentation and severity of

the disease; severity of nail involvement and the number of

affected nails. Decision of therapy also depends on other

medications that the patient is already taking and previous

therapies for onychomycosis that have already been attempted

(and their effects). Patient‟s age, compliance, general medical


94

condition, cost and patient preference have to be put in mind

(Gupta et al., 2003).

Treatment modalities of onychomycosis include topical

or systemic antifungal therapy, which can be either alone or

combined. Surgical interference and nail debridement

chemically or surgically is another treatment option, but it is

considered primitive compared with topical or systemic

treatment (Donald et al., 2002). In addition, patients should be

instructed in the proper general care of their nails (Eleweski

and Hay, 1996).

The primary aim of treatment is to eradicate the

organism as demonstrated by microscopy and culture. This is

defined as the primary end-point. Clinical improvement and

clinical cure are secondary end-points based on a strict scoring

system of clinical abnormalities in the nail apparatus (Roberts

et al., 2003). Also, important goals are to reduce morbidity and

to prevent complications (Zaias and Rebell, 2004).

1. Antifungal drugs

Topical treatment:

Although they have insufficient nail plate penetration

and low success rate of 20% (Zuber and Baddam, 2001), they

may be useful in SWO, and possibly very early DLSO (Roberts

et al., 2003) also in preventing the relapse of chronic tinea

pedis that often accompanies onychomycosis. Combining it


95

with newer oral antifungal agents may result in a more rapid

cure (Eleweski, 1998). Topical treatment has a primary role as

prophylactic, in treatment in mild infection and if systemic

antifungals are contraindicated for any reason (Kaur et al.,

2008b).

There are several topical antifungal preparations, in

which the active antifungal agents are imidazoles, allylamine or

apolyene, or a preparation that contains a chemical with

antifungal, antiseptic and sometimes keratolytic agents

(Roberts et al., 2003).

Systemic treatment:

Oral antifungal agents are one of the most effective

agents available to treat onychomycosis even more successful

than topical treatment (Roberts et al., 2003). Griseofulvin was

used as a classic first line treatment option but it was widely

replaced by newer Triazole and allylamine antifungal drugs

such as itraconazole, and terbinafine, as these agents offer

shorter treatment courses, higher cure rates and fewer relapses

and adverse effects (Rezabek and Friedman, 1992).

In addition, fluconazole and ketoconazole are also

prescribed for onychomycosis (Gupta and Tu, 2006). The

efficacy of the newer antifungal agents lies in their ability to

penetrate the nail plate within days of starting therapy (Cohen

et al., 2003).


96

Table (2): Classification of approved antifungal drugs (Taha,

2011).

Group Compound Target for

its action

1- Polyenes - Nystatin

- Amphotericin B

Ergosterol

in plasma

membrane

2- Azoles

a) Diazoles

b) TriaZoles

- Ketoconazole

- Fluconazole

- Itraconazole

Cytochrome

P450

Lanosterol

14á-

Demethylase

3- Allyamines Terbinafine squalene

4- Others Griseofulvin Nucleic

division

a) Griseofulvin

Griseofulvin was the first oral antifungal drug approved

by the US Food and Drug Administration for the treatment of

onychomycosis. It is fungistatic in nature and active against

dermatophytes, including Trichophyton spp, Microsporum spp,

and Epidermophyton floccosum. It is not effective against

Candida spp and the non-dermatophyte moulds.

Griseofulvin inhibits fungal reproduction by preventing

the formation of the fungal intracellular microtubules,

disrupting the mitotic spindle, and preventing the division of

fungal cells (Debruyne and Coquerel, 2001) which will inhibit

DNA synthesis and RNA binding (Rosen, 1997).


97

Its effectiveness is not satisfactory, as it requires long

course of treatment that may take more than one year especially

in toenails provided with low compliance, low cure rates and

high relapse rates due to its narrow spectrum against

dermatophytes only (Scher, 1999).

Allergic reactions to griseofulvin occur in 5%-7% of

cases (Zurcher and Krebs, 1992). Other common side effects

include headache and nausea, vomiting, diarrhoea,

photosensitivity, urticaria, fatigue, fever, and menstrual

irregularities up to liver function abnormalities and rarely,

granulocytopenia (Elewski and Hay, 1996).

Drug interactions also occur like reduced efficacy of

birth control pills and inhibition of warfarin's effect (Gupta et

al., 1994).

b) Azoles group antifungals:

As mentioned in table-2 these are medications used to

treat onychomycosis include itraconazole, ketoconazole, and

fluconazole. They are fungistatic against a broad spectrum of

pathogens, including dermatophytes, NDMs and candida

species.

They act primarily by selectively inhibiting fungal

cytochrome P450 (14 α-sterol demethylase), which is an

enzyme responsible for converting lanosterol to 14 α -

demethyllanosterol in the ergosterol biosynthesis pathway.


98

Ergosterol is essential to fungus because it is a major

membrane sterol; impairing the synthesis of ergosterol

ultimately interfering with the function and permeability of the

cell membrane of the fungal organism (Gupta and Tu, 2006).

i. Ketoconazole

It was one of the first broad-spectrum oral antifungals

developed with mycologic cure rate of 15% to 30% for toenails

and 50% to 70% for fingernails (Zurcher and Krebs, 1992).

The side effects may include nausea, vomiting,

abdominal pain, diarrhea, pruritus, headache, abnormalities in

liver function in < 10% of patients (Clissold, 1990) in addition

to significant drug interactions including potentiation of

warfarin's effect, decreased absorption of rifampicin, increase

in levels of cyclosporine and an interaction with terfenadine

that leads to cardiac dysrhythmia (Gupta et al., 1994).

Dosage was prescribed as 200 mg/d for 4-6 months for

fingernails and 200 mg/d for 10-18 months for toenails

(Elewski and Hay, 1996).

ii. Itraconazole

Of all antifungal drugs, it has the broadest spectrum and

due to its lipophilic tendencies, its concentration in nail remains

high for months after stoppage of the drug (Rosen, 1997). For

that cause and for its affinity to keratinized tissues, mycologic


99

cure rates of Itraconazole range from 45% to 70 % and clinical

cure rates range from 35% to 80% (Elewski, 1998).

Headache, rash and gastrointestinal upset are usual

adverse effects in about 7% of treated patients (Gupta et al.,

1998). Because it is metabolized by the hepatic cytochrome

P450 system, significant drug interactions can occur. Notably,

it interacts with quinidines, atorvastatin and benzodiazepines.

Though hepatic toxicity is rare, monitoring liver enzymes is

recommended every four to six weeks (Katz and Gupta, 1997).

Dosage is 200 mg once daily taken continuously for

twelve weeks to treat toenail infections and for six weeks to

treat fingernail infections (Gupta et al., 1998).

Pulse treatment consists of 200 mg taken twice daily for

one week per month, with the treatment repeated for two to

three months (De-Doncker et al., 1997).

iii. Fluconazole

IT is one of the new agents of Azole antifungals

characterized by decreased lipophilicity, protein binding,

resistance to metabolism and higher potency and specificity

(Gupta et al., 1997b).

Adverse effects, including nausea, headache, pruritus and

liver enzyme abnormalities, are reported in approximately 5%


100

of treated patients (Scher, 1999). It also has important drug

interactions (Katz and Gupta, 1997).

Attention has focused on 150 mg once weekly until nail

is normal or acceptably improved (treatment often requires 6 to

9 months) associated with a cure rate of 72% to 89% and nearly

total mycologic eradication (Scher et al., 1998).

C. Terbinafine

As mentioned in table-2 Terbinafine is an allylamine

antifungal agent that is active against dermatophytes, which are

responsible for the majority of onychomycosis cases. This

agent is notably less effective against NDMs and yeasts

(Rodgers and Bassler, 2001).

It acts by inhibition of squalene epoxidase synthesis so

inhibiting ergosterol formation that makes it fungicidal (Scher,

1999).

Dosage of terbinafine is 250 mg per day given

continuously for twelve weeks to treat toenail infections and for

six weeks to treat fingernail infections. Studies have shown that

the regimen for toenails results in a mycologic cure rate of 71%

to 82% and a clinical cure rate of 60 to 70 percent (Goodfield et

al., 1992 and Hofmann et al., 1995).

Adverse effects are rare and range from headache, rash

and gastrointestinal upset (Roberts, 1997) to serious drug


101

interactions though minimal such as cholestatic hepatitis, blood

dyscrasias and Stevens-Johnson syndrome (Del Rosso and

Gupta, 1997).

It is considered safer, with better efficacy and more

favourable results (more than 50% to 80% cure rate) however,

relapse is common (De Cuyper and Hindryckx, 1999).

2. Mechanical procedures in treatment

Mechanical procedures include surgical or chemical nail

avulsion that may be useful in patients with severe onycholysis,

extensive nail thickening or longitudinal streaks or spikes in the

nail (Baran and Hay, 1985).

i. Chemical nail Debridement

The objective of debridement is to reduce the thickness

of the nail plate so that any associated pain is reduced and the

probability of success of oral/topical therapies is enhanced. The

thickened, ragged, misshaped toenail or fingernail can be

buffed (shaping nail by emery board and filing the nail edges

and nail plate to reduce nail ridges and cause thinning of nail

plate) (Rich and kwak, 2010) ) and smoothed down to prevent

further trauma caused by clothing or footwear (Markinson et

al., 1997).

Chemical nail avulsion is done by many ways such as

application of 40% to 50% urea ointment to the nail, and the


102

entire distal end of the digit is kept bandaged for 1 week. The

urea ointment enables the diseased nail to be removed, leaving

the healthy tissue intact (Gupta and Tu, 2006). Also phenol can

be used for chemical matricectomy. In this procedure, 88%

phenol is applied to the nail matrix. It may be applied in three

cycles of 30 seconds each. A tourniquet has to be applied

before using phenol to ensure a bloodless field, as blood is

known to inactivate phenol. Finally, phenol is neutralised with

isopropyl alcohol and an appropriate dressing is done (Bostanci

et al., 2001). An alternative chemical agent is using 10%

sodium hydroxide, either applied for 2 min or 1 min combined

with curettage. It has been claimed to cause less tissue damage

than phenol (Ozdemir et al., 2004).

ii. Surgical nail avulsion

Although surgery can be a useful therapeutic adjuvant in

the treatment of onychomycosis, surgical avulsion is painful

and disfiguring. General indications for surgical treatment

include pachyonychia associated with pain, contraindications to

oral antifungal drugs administration, the presence of

onychomycosis due to drug-resistant non-dermatophytic fungi

or candida species (Elewski and Hay, 2006).

Surgical nail plate avulsion is an ambulatory procedure

done under local anaesthesia (Elewski and Hay, 2006).


103

- Simple nail trimming at the most proximal part of the

diseased nail plate is useful for managing onycholysis

(Elewski and Hay, 2006).

- Partial avulsions: it is advisable to remove only the

diseased part of the nail plate.

- Total nail avulsion.

- Distal transversal nail plate avulsion.

- Hemi avulsion.

These other three types of nail avulsion have to be

discouraged as the distal nail bed may shrink or dislocate

dorsally. In addition, the relief of the contour pressure may

allow expansion of the distal soft tissue of the re-growing nail

plate to embed itself (Kaur et al., 2008a).

In conclusion, a systemic treatment is always required in

proximal subungual onychomycosis (PSO) and in distal lateral

subungual onychomycosis (DLSO) involving the lunula region.

While, white superficial onychomycosis (WSO) and distal

lateral subungual onychomycosis (DLSO) limited to the distal

nail can be treated with a topical agent (Manevitch et al.,

2010).

Most yeast infections can be treated topically,

particularly those associated with paronychia. Antiseptics can

be applied to the proximal part of the nail and allowed to wash

beneath the cuticle, thus sterilizing the subcuticular space. An


104

imidazole lotion alternating with an antibacterial lotion is

usually effective. Itraconazole is the most effective agent for

the treatment of candidal onychomycosis where the nail plate is

invaded by the organism (Gupta and Shear, 1999).

NDMs‟ response to systemic antifungal agents is

variable. Although terbinafine is probably the drug of choice,

some consider nail avulsion followed by an oral agent during

the period of re-growth probably the best method of restoring

the nail to normal (Gupta and Gregurek-Novak, 2001).

3. General hygienic measures in treatment

Patients with onychomycosis often need to break old

habits and learn new, healthier habits to achieve an optimal

therapeutic response and prevent re-infection.

1) Appropriate nail care which is:

a- Keeping nails short.

b- Clip toenails straight across to prevent ingrown

toenails.

c- File and buff hypertrophic nails.

d- Avoid trauma and irritants.

e- Use cotton gloves for dry manual work and vinyl

gloves for wet work.

f- Change instruments between care of normal and

infected nails (Elewski and Hay, 1996).


105

2) Wearing loose shoes, choosing breathable footwear,

wearing 100% cotton socks and changing them often.

3) Stopping a range of potentially risky behaviours (going

barefoot in public places, wearing other people‟s shoes, and

exposing feet or hands to damp environments).

4) Protecting feet in shared bathing areas.

5) Keeping feet dry throughout the day.

6) Recognizing and treating tinea pedis.

7) Maintaining and improving chronic health conditions (e.g.,

controlling diabetes, quitting smoking) (Elewski, 1998 and

Rodgers and Bassler, 2001).

4. Laser treatment

Discussed in chapter (2).


106

Laser and

Infections

107

Chapter (2)

LASER AND INFECTIONS

Introduction

he ability of lasers to sterilize surfaces is well known.

Adrian and Groos showed that the CO2 laser could

sterilize a scalpel blade that had been contaminated with spores

(Adrian and Gross, 1979). The CO2 laser and Argon laser have

also been shown to be capable of sterilizing endodontic reamers

(Hooks et al., 1980 and Powell and Whisenant, 1991).

Laser energy had been shown to have the potential to

eliminate some protozoal, bacterial and fungal infections. Saks

and Roth had reported the eliminating effects of Ruby laser on

various protozoa (Saks and Roth, 1963).

A. Bactericidal effect of lasers:

Thermal and photo-disruptive effects were considered

the principal reasons for the laser to eliminate the bacteria by

inducing cellular wall damage that affect mainly gram positive

bacteria and also by causing denaturation of internal cellular

protein (Ando et al., 1996).

- Anti bacterial effect of ruby laser

Klein and colleagues using low power Ruby laser by

fluence greater than 250 J/cm had found that it inhibited growth

of Pseudomonas aeroginosa but did not have any effect on

T

108

Staphylococcus aureus (Klein et al., 1965). However, when

McGuff and Bell used Ruby, Nd:YAG and Helium-Neon

lasers on the same microorganisms, they found no inhibitory

effect at all (McGuff and Bell, 1966).

- Antibacterial effect of diode laser

A diode laser (light emitting diode 670 nm) emitting a

constant beam of coherent, continuous monochromatic light

with a power of 30 mw and Pulsing of 146Hz was found to

have a bactericidal effect on Streptococcus mutans when

irradiated above 10 seconds exposure. A diluted bacterial

suspension contained 1 ml volume was exposed to the laser

light at different times (5, 10, 15, 20, and 25 seconds). Results

showed that the numbers of colonies were decreased with

increasing length of irradiation exposed to bacterial suspension

(Ahmed et al., 2011).

Furthermore, Moritz and colleagues reported that the

diode laser has a bactericidal effect on specific bacteria, such as

Actinobacillus actinomycetemcomitans, Prevotella intermedia,

and Porphyromonas gingivalis (Moritz et al., 1998).

It was also found that a 980-nm diode laser can eliminate

bacteria (enterococcus faecalis) that have immigrated deep into

the dentin (Gutknecht, 2004).

109

- Antibacterial effect of Er:YAG laser

Er:YAG laser has a high bactericidal potential on

common implant surfaces. Irradiation at pulse energies of 60

and 120 mJ (low energy densities) and a frequency of 10 pps

was performed on Streptococcus sanguinis and showed

decrease in number of colonies (Kreisler et al., 2002).

- Antibacterial effect of Nd:YAG laser

The Nd:YAG laser was found to have a bactericidal

effect and was capable of killing both pigmented and non-

pigmented bacteria at different energy levels. Long pulsed

Nd:YAG laser is effective in destroying heat resistant bacteria

(Rooney et al., 1994). It also, has bactericidal effect on α-

hemolytic streptococcus, Bacteroides fragilis, Neisseria,

Streptococcus salivarius, Staphylococcus aureus (Meral et al.,

2003). Also Pseudomonas aeruginosa and Escherichia coli

exhibited decreased viability when 0.5 ml bacterial suspension

was exposed to Nd:YAG laser light energy with density greater

than 1,667 J/cm during a 10 seconds exposure (Schultz et al.,

1986).

This antibacterial effect level is totally depending on

environmental factors like microorganism count, type and

pigmentation of tissue (Meral et al., 2003). When an Nd: YAG

laser is used at high power settings and for a long time, the rise

110

in temperature has deleterious effects on periodontal tissues

(Pick, 1993).

B. Fungicidal effects of lasers:

The use of lasers to treat nail fungus is not new.

Researchers have been trying to use lasers to treat

onychomycosis since the 1980s. The approach at that time was

to use the laser to create holes or channels in the nail. This was

meant to give topical onychomycosis treatments better access

to the fungus. However, it did not meet with much success

(Rothermel and Apfelberg, 1987).

As lasers were evolving, better approaches were adopted.

Several types of lasers are available for this modality of

treatment for example diode laser (dual-wavelength 870- and

930-nm near-infrared diode laser) (Landsman et al., 2010),

femtosecond infrared titanium sapphire laser (Manevitch et al.,

2010), also, the treatment of onychomycosis can be achieved

with many novel variants like novel 0.65-millisecond pulsed

Nd: YAG (Mozena and Haverstock, 2010).

Laser treatment of onychomycosis infections uses the

principle of selective photothermolysis. The Theory of

selective photothermolysis is concerned with how to target a

specific chromophore in the skin with minimal unwanted

thermal injury to the surrounding tissue. And each

chromophore has its own unique absorption coefficient curve

111

i.e. absorptive power of photon energy in different

wavelengths. A specific wavelength of photon energy is

emitted by the laser to the target tissue trying to achieve

optimal absorption of energy by the target but minimizing

absorption of the photon energy by other non-target tissues

According to that, thermal destruction of a specific target will

occur if sufficient energy is delivered at a wavelength absorbed

by the target within a time period less than or equal to its

thermal relaxation time (TRT) (the time it takes for the target to

cool to the half of its baseline temperature and transfer the heat

to the surrounding structures) (Anderson and Parrish, 1983).

Most of these lasers depend on delivering a high

concentration of laser energy in a small area with the ability to

penetrate deeper into the nail plate. The differences in laser

energy absorption and thermal conductivity between the fungal

infection and the surrounding tissue will cause eradication of all

residual fungal elements by targeting and killing the fungus in

the nail and leaving the skin and nail intact. The absorption of

light energy by the fungi results in the conversion of the energy

into heat or mechanical energy (Altshuler et al., 2001). As

fungi are heat sensitive above 55°C, so absorption of laser

energy results in sustained photothermal heating of the

mycelium causing its fungicidal effects. However, heating

dermal tissue to temperatures above 40°C results in pain and

necrosis; therefore, the laser energy format must either be

pulsed to allow the dissipation of heat by the tissue or delivered

112

at a moderate energetic level to prevent tissue damage

(Hashimoto and Blumenthal, 1978 and Bergman and

Casadevall, 2010).

Photodynamic therapy using an excimer-dye laser

Photodynamic therapy (PDT) uses visible spectrum light

to activate a topically applied photosensitizing agent, which

generates reactive oxygen species that initiate apoptosis in

fungi (Harris and Pierpoint, 2011). Studies have demonstrated

that fungi can be effectively photosensitized after topical

delivery of 5-aminolevulinic acid (ALA) and killed at dose

rates much lower than those that kill keratinocytes (Smijs et al.,

2008). Studies were done on T.rubrum and NDMs (Fusarium

oxysporum and Aspergillus terreus). These studies indicate that

the nail plate should be pre-treated with urea ointment to soften

the nail plate prior to application of the photosensitizer. They

used 16-20% of ALA or its methyl ester and excimer-dye laser

as the source of irradiation. The treatment site was irradiated

with pulsed laser light at a wavelength of 630 nm at 100 J/cm2

(Watanabe et al., 2008). Another study used broadband red

light with same wavelength of 630 nm and irradiation dose of

37 J/cm2 (Piraccini et al., 2008). Another study used 635 nm

emiting diode lamp at 37 J/cm2; all showed 100% mycological

clearance (Gilaberte et al., 2011).

113

Near-infrared diode laser

The use of near infrared diode laser with wavelength of

870 and 930 nm light would kill the microbial pathogens

(Neuman et al., 1999) in vitro studies showed that these

wavelengths (870 nm and 930 nm) can photo inactivate T.

rubrum and Candida albicans, and have a minimal negative

effect on cultured fibroblasts (Bornstein et al., 2009). Using the

photo lethal characteristics in each of both wavelengths at

lower energies and the consequently lower temperatures

generated; there is no harm to tissues being exposed. Important

also is that the wavelengths used are far from the ultraviolet

range and, therefore, pose no potential for mutagenic effect

(Matsumu and Ananthaswamy, 2004). Its mechanism of

action depends on the interaction of the wavelengths with

plasma and mitochondrial membranes and the generation of

endogenous radical oxygen species causing photodamage and

photoinactivation of fungi and bacteria (Bornstein et al., 2009).

Femtosecond infrared titanium sapphire laser

This laser emits red and near-infrared light in the range

from 650 to 1100 nm. Its laser energy is capable to penetrate to

the deeper levels of the nail plate without collateral damage.

Near infrared diode laser was used on T. rubrum cultures in

vitro using the parameters of 200 fs pulses at a frequency of 76

MHz. It has the advantage of nonlinear interactions with

biological media (which means the matter responds in a

114

nonlinear manner to the incident radiation fields, endows the

media a characterization to change the wavelength. It is only

observed at very high intensity (electric field) of incoming

light). This quality, combined with its deeply penetrating

nature, allows elimination of deeply seeded nail dermatophytes

without associated collateral damage (Manevitch et al., 2010).

Nd:YAG Laser

This laser produced significant inhibitory effects on

fungi like T.rubrum and Candida albicans (Meral et al., 2003).

- Q-switched Nd:YAG laser:

The Q-switched Nd:YAG laser with 532-nm and 1064-

nm inhibitory effect is likely due to pigment-related

photothermolysis rather than inhibition due to simple

nonspecific thermal damage. The 532-nm setting is well

absorbed by red pigment in xanthomegnin in T. Rubrum The

1064-nm setting is beyond the absorption spectrum of

xanthomegnin, but its effectiveness is postulated to be due to

another absorbing chromophore, such as melanin dark pigments

which are an essential constituent of fungal cell wall having a

broad absorption spectrum (Vural et al., 2008).

Q-switched lasers have a pulse duration in the

nanosecond range and they emit the highest peak power per

pulse of all the Nd:YAG lasers. Another effective way of

inhibition of fungi by Q-switched Nd:YAG laser includes its

115

short pulse width (i.e., nanoseconds). These short pulses will

induce microcavitation and acoustic shock waves that could

result in significant inhibition of the fungal colonies. The short

pulse times (much shorter than the thermal relaxation time) will

also induce thermal shocks in the target chromophore via rapid

heating and cooling. These extreme thermal cycles and shock

waves do not occur when the laser light is delivered in relative

long pulse times (Suthamjariya et al., 2004).

- Short pulse Nd:YAG 1064 nm laser:

Flashlamp pumped short pulse Nd:YAG 1064 nm lasers

are used to inactivate many fungal species. The use of a shorter

pulse allows for greater safety and convenience, reducing damage

to underlying tissue and eliminating the use of cooling during the

procedure (Hochman, 2011). One study used short pulse Nd:Yag

1064 nm laser with pulse length of 0.3 ms, energy fluence of 13

J/cm2 and a frequency of 6 Hz. Follow-up mycological cultures

was negative in 95% of patients (Waibel, 2011). Another study

introduced ultra short pulsed novel 0.65-millisecond Nd:YAG

laser for the treatment of onychomycosis. It is used by applying a

2 mm spot size, with fluence 223 J/cm 2 in the absence of any

cooling device (Mozena and Haverstock, 2010).

- Long pulsed Nd:YAG 1064 nm laser:

Long pulsed Nd: YAG laser was reported to have a high

fungicidal effect on Candida albicans suspension (Meral et al.,

2003).

116

Long pulsed Nd:YAG laser pulse duration is in the

millisecond range. It employs a near infrared wavelength of

1064 nm; this wavelength causes local hyperthermia,

destruction of pathogenic microorganisms, and stimulation of

the reparative process. And it is well known to cause cellular

photo damage in the absence of exogenous dyes or other

photosensitive drugs and chemicals (Kozarev and Vizintin,

2010).

One of the mechanisms of action of infrared laser is that

it penetrates through the nail plate and produces heat deep

(temperature of 45°C±5) within the dermis and nail tissue

without burning the surrounding tissue but heating fungal cells

to the point of structural and functional impairment in their

infectious activity which can deactivate the fungi (Kozarev and

Vizintin, 2010).

Laser beam deactivates the unwanted organisms by

damaging its cytoskeleton morphology through denaturing or

partially denaturing one or more of the molecules within the

pathogens. The beam accomplishes this by initiating a photo

biological or photochemical reaction that attacks the pathogen

cell or by inducing an immune response that attacks the

organism (Dayan et al., 2003).

The effects of laser on onychomycotic nails are

multifactorial but a major role by laser beams done in this

process is local hyperthermia that causes Heat-shock stress that

117

causes alteration of microbial cellular metabolism such as DNA

replication & protein synthesis and generation of radical

oxygen species (ROS). Heat shock response is a natural way for

cells to protect against environmental stress. (Figure 25) the

extracellular stress induces a heat shock response which

involves induction of heat shock proteins (HSP) under genetic

control which can be classified into three categories according

to their molecular size: (I) high-molecular size, with molecular

weight between 69 and 120 kDa, (II) medium-molecular size,

with molecular weight between 39 and 68 kDa, and (III) low-

molecular size, with molecular weight below 38 kDa (Chen

and Chen, 2004). In yeast, increase in production of the

transmembrane yeast protein Wscl-Hcs77 acts as a heat shock

sensor. It is thought to sense cell wall stress and membrane

fluidity (Philip and Levin, 2001). They cause inactivation and

aggregation of various cellular proteins which become further

insoluble (Dubois et al., 1991) this includes drastic repression

of normal transcription and translation pathways and activation

of a family of heat shock genes which is mediated by the

interaction of heat shock factor (HSF) transcription factors with

heat shock elements in the heat shock protein gene promoter

regions (Voellmy, 1994) which leads to severe protein

denaturation leading to apoptosis of the fungal cell. If the stress

is over the thermotolerance of the cell, denatured proteins

disrupt cellular homeostasis and increase ROS level which are

highly toxic to microbial cells through their detrimental effects

on important biological macromolecules including DNA,

118

proteins and cellular membrane. ROS causes modification of

protein by formation of carbonyl groups caused by oxidation of

specific amino acids residues (Davies and Goldberg, 1987).

Severe protein denaturation leads to apoptosis of the fungal cell

(Kozarev and Vizintin, 2010). Apoptosis is a regulated cell

death that results from an organized process ending by

fragmentation of the genome (Lin et al., 1999).

Figure (26): The Heat Shock Response (Physical or chemical stress

induces production of unfolded or misfolded proteins. Heat shock factor

(HSF) monomers in the cytoplasm form trimers, then become

phosphorylated and translocate into the nucleus where it bind to heat

shock protein (HSP) gene promoter regions, leading to induction of HSP

gene transcription. Hsp70 gene transcription is down-regulated by

interaction of Hsp70 or HSBP1 with the HSF trimers) (Pockley, 2003).

Also the thermal injury may initiate a death signal, target

certain heat labile proteins, or cause direct or indirect DNA

damage leading to apoptosis, which is the result of a

combination of the thermal destruction (directly or indirectly)

119

of apoptosis protecting molecules with a concurrent production

of killing molecules that induce cell death (Cuende et al.,

1993).

Membrane lipid ceramide is the simple sphingolipid

produced mainly from sphingomyelin by sphingomyelinases or

by a de novo pathway. Membrane lipid ceramide has been

proposed as a signaling molecule that converts extracellular

stresses into intracellular signals. In response to heat shock,

ceramide levels increased which has been proposed as a second

messenger of heat shock (Elia and Santoro, 1994).

It was found that ceramide could induce cell death

without heat shock response including blockage of protein

synthesis and Hsp synthesis (Kim and Lee, 2002).

Reactive oxygen species (ROS) is located in the

endoplasmic reticulum; it is a cytochrome P450 complexes,

which generate superoxides to metabolize toxic hydrophobic

compounds (Shenkman, 1993). It has been suggested as a

second messenger generated by growth factors and cytokines,

including platelet derived growth factor (PDGF), epidermal

growth factor (EGF), angiopoietin-1, tumour necrosis factor

(TNF), and interleukin-1 (IL-1) in non-phagocytic cells (figure

26). ROS deactivates the deubiquitinating enzymes (DUBs)-

which are a large group of proteases that regulate ubiquitin-

dependent metabolic pathways-, which have redox sensitive

120

cysteines in their active sites, resulting in the accumulation of

misfolded proteins (Kim et al., 2007).

Denatured proteins disrupt cellular redox homeostasis

and increase ROS level that induces protein misfolding. When

mis-folded proteins are produced, proteolytic machinery is

turned on to remove them which in turn induce Hsp expression

and cell death (Deng and Podack, 1993).

Another mechanism of action of ROS is inactivating

specific phosphatases and prolongs the phosphorylation status

of signaling proteins (Kim et al., 2007).

Figure (27): Schematic representation of apoptosis (ROS generated by

mitochondria are essential mediators of apoptosis. Together with

cytochrome C, Apaf-1, and ATP, released from mitochondria, ROS

activate proteolytic enzymes, termed caspases, which promote

deoxyribonuclease, and thereby destroy targeted cells (Salganik, 2001).

121

Patient and Methods

122

PATIENTS, MATERIAL AND METHODS

Patients

his study includes twenty (20) patients suffering from

onychomycosis randomly selected from Dermatology

Outpatient Clinic of Ain-Shams university hospitals.

- Inclusion criteria:

Age group from 18 to 60 years (the age of adult‟s

consent and the prevelance of onychomycosis increases

with age).

Toenail or fingernail fungal infection.

Clinical types of onychomycosis can be: Total dystrophic

onychomycosis, proximal subungual onychomycosis,

Distal and lateral subungual onychomycosis, Superficial

white onychomycosis.

- Exclusion criteria:

Age less than 18 years and more than 60 years (age less

than 18 years can‟t give informed concent).

Usage of topical or systemic anti-fungal therapy in the

preceding 6 months.

Use of drugs inducing photosensitivity e.g. tetracyclins,

systemic retinoids.

T

123

Permanent or semi-permanent discoloration of the nail

plate (e.g. topical therapeutics, cosmetics or professional

dye exposure) as they can cause various physiological

changes to the nail plate in addition to discoloration.

Subungual hematoma, nevoid subungual formation,

bacterial nail infections, and concomitant nail disorders

such as psoriasis of nail plate, lichen planus and atopic

dermatitis.

Any generalized skin disease.

Immunodeficiency e.g. HIV infected patients, patients

with organ transplantation and currently on

immunosuppressive drugs for long periods or patients

under chemotherapy or radiotherapy.

Pregnancy.

- Our patients were subjected to:

1. Written informed consent.

2. Detailed history.

3. Thorough Clinical and dermatological examination to

exclude skin diseases with associated nail disorders.

4. Photographing of affected nails before treatment, during

the follow up and after 3 months.

5. Mycological examination by direct examination of nail

scrapings in 20% KOH and mycological cultures.

124

6. Four sessions of Long pulsed Nd:YAG 1064 laser

treatment.

7. Follow up visits after one month of the final laser session

and three month after.

Materials

- Chemicals used:

a. KOH:

20% KOH solution was done by dissolving 20 gm KOH

in 100 ml distilled water, and then slowly mixed until KOH is

completely dissolved and kept at 2-8 C.

b. Culture media:

Sabouraud’s dextrose agar medium (SDA) with

chloramphenicol:

Composition (SDA +C):

Dextrose 40 gm

Peptone 10 gm

Agar 20 gm

Distilled water (D.W) 1 Liter

Chloramphenicol 100 mg dissolved in 3 ml alchol

Alcohol 3 cc

125

The ingredients were mixed in 1 liter of distilled water

and dissolved by heat. The pH was adjusted to (5.6). It was

autoclaved at 115ºC for 15 minutes and poured into 10 ml tubes

as slopes. They were kept at 2-8 C. The addition of

chloramphenicol is to inhibit the growth of bacteria. This

medium was used for yeast and non dermatophytic moulds

isolation.

Sabouraud’s dextrose agar with cyclohexamide and

chloramphenicol (SDA +C+C):

It was the same as the previous medium with the addition

of Dermasil supplement (Oxoid) company contains

cyclohexamide and chloramphenicol dissolved in 3 ml alcohol

was used for 500 ml of the medium. It was used for isolation of

dermatophytes and Candida albicans.

Chromgenic Candida Agar (Oxoid):

Glucose 20 gm

Peptone 10 gm

Agar 15 gm

Chromgenic mix 2 gm

Distilled water (D.W) 1 Liter

The ingredients were boiled then poured in tubes as

slopes.

126

- Other material:

Waxy carver/ Nail clipper

Plastic petridishs

Glass slides

Sterile glass cover

Bacteriological loop

Glass tubes

- Devices used

Long pulsed Nd:YAG laser 1064 nm(Candela, Wayland,

MA, USA).

Figure (28): Long pulsed Nd:YAG

laser 1064 nm(Candela, Wayland, MA,

USA).

127

Methods:

1. Written informed consent:

Detailed information about the procedure and other

alternative treatments was explained to the patient.

2. Detailed history including:

Personal history: name, age, sex, occupation, Special

habits of medical importance, marital status and

residence.

The most annoying complain of the patient.

Present history: Number of nails affected, colour and

dystrophic changes, onset, course & duration of nail

lesion, swelling and pain around nails (Paronychia).

Probable predisposing factors as: use of water, exposure

to trauma, previous medication, other associated diseases

as diabetes mellitus, peripheral vascular disease, hepatic

diseases and the presence of other fungal infections.

3. General examination to exclude skin diseases with

associated nail disorders and clinical examination of the

diseased nail:

Cutaneous diseases with nail disorders (lichen planus,

psoriasis).

The number and site of infected nails.

Nail surface, consistency and configuration.

128

Clinical signs of onychomycosis which include

onycholysis, subungual hyperkeratosis, dystrophy,

paronychia and discoloration.

4. photographing documentation :

Photographs were taken using the same camera settings,

lighting, and nail position by the digital camera (Yashica-

China) before the treatment and at each follow up after 1 month

of the final laser session and 3 month after.

5. Mycological examination:

Nail specimen collection:

The nail specimens were either:

- Nail clippings.

- Nail scrapings. (In cases where the nail bed was not

found raised, nail scraping was taken from the surface of

the nail plate).

- Subungual curettage.

The nail of the patient was first disinfected by 70%

alcohol using a piece of sterile gauze to reduce contamination,

then the specimen after that was taken in a sterile plastic

petridish. It was taken in consideration to obtain as much

specimen as possible to be enough for both direct microscopy

and culture, also to increase the chance of possible positive

results.

129

Direct Microscopic Examination:

Direct microscopic examination by KOH was performed.

In case of nail clips they were fragmented first by scalpel

blade in to small particles. Fine particles of specimen were

placed in the center of a clean slide with 20% KOH and

covered with a cover slide, gently heated then examined after

half an hour under the low (x10) and high power (x40) lenses

of the light microscope.

Evidence was considered to be positive if fungal

elements such as yeast cells, branched and septated, thread- like

structures (hyphae or pseudohyphae), and / or beaded,

spherical, occasionally double contoured structures (spores)

were seen.

Culture:

Specimens were inoculated in four to five sites in tubes

containing media (SDA+C and SDA+C+C) then cultures were

incubated at 25°C under aerobic conditions and observed for

growth for a period of two weeks.

The results were usually interpreted after two weeks, but

whenever no growth in the tubes was observed, the period of

observation was extended up to one month. The tubes were

examined for up to 4 weeks before being discarded as negative.

130

Identification of fungal isolates by:

- Macroscopic examination:

The colonies were identified by rate of growth whether

slow or rapid and by number of colonies, shape, size, colour

and surface whether rough, smooth, fissured, corrugated or

waxy. And sometimes a differential media is needed such as

Chromogenic Candida Agar to identify yeast colonies by colour

into Canida species as the following:

Green colonies: Candida albicans

Blue colonies: Candida tropicalis

Pink colonies: Candida krusei

Creamy white colonies: Candida parapasillosis

Pink colonies with white border: Candida glabrata

- Microscopic examination:

It was done by taking small portion of the colony by

dissecting needles and place it in a drop of lacto-phenol cotton

blue (LPCB) on a clean microscopic slide.

In cases of dermatophytes: the sample was examined

under the microscope for size, shape and arrangements of

macroconidia, microconidia and modification of hyphae after

(Taha, 2011).

131

For non- dermatophytic moulds: the isolates were

examined for hyaline and coloured hyphae or conidiogenous

hyphae (phialides).

Identification of Candida by: The isolates were examined

for presence of pseudohyphae and by shape as well as culture

on Chromgenic Candida Agar to identify Yeast into species as

above.

6. Long pulsed Nd:YAG 1064 nm laser treatment

Laser therapy was performed using long pulsed Nd:YAG

laser 1064 nm(Candela, Wayland, MA, USA) using the

following parameters:

Fluence: 50 J/ cm²

Pulse duration: 30 ms

Spot size: 3 mm

Frequency: 1 Hz

Cooling cryogen spray was turned off completely.

Hyperkeratotic nail bed material was removed manually by

subungual curettage with waxy carver or by chemical agent

such as urea cream (Carbamide cream contain 10gm urea) used

once daily in between laser sessions.

Each nail plate was fully covered with a transcutaneous

laser irradiation in a spiral pattern starting at the nail periphery

132

and finishing in the nail centre. In one session three passes of

laser beams across each nail plate with two minutes pauses

between passes were applied. The total therapy consists of four

sessions with a one week interval between each session.

Figure (29): Laser irradiation a spiral pattern starting at the nail periphery

and finishing in the nail centre (Kozarev and Vizintin, 2010).

If the culture results after 3 months follow up are still

positive, another 4 sessions of laser treatment are done.

7. Follow up visits

All subjects of the study were evaluated after 1 and 3

months for clinical observation and photographic

documentation of clinical signs of onychomycosis, observation

of any possible adverse reactions (acute paronychia, pain) and

mycological examination at 1 and 3 months after laser

treatment to evaluate the effectiveness of the treatment.

133

Results

134

RESULTS

Demographic data of patients in our study:

Our study included 20 patients with Onychomycosis

affecting fingernails and/or toenails that were treated with 4

weekly sessions of long pulsed Nd:YAG laser.

a) Age and Sex:

Patients included in the study were 18 (90%) females and 2

males (10%) (Table 3), (Figure 30). Patients ranged from 18 to 54

with mean age of 35.2(±10.26) years (Table 4), (Figure 31).

Table (3): Sex distribution of patients in the study.

Sex

N %

Female 18 90.00

Male 2 10.00

Total 20 100.00

Figure (30): Sex distribution of patients in the study.

135

Table (4): Age distribution of patients in the study.

Age

N %

(Young) <40y. 13 65.00

(middle age) >40y. 7 35.00

Total 20 100.00

Figure (31): Age distribution of patients in the study.

136

b) Residence:

Six (30%) patients lived in rural areas while 14(70%)

patients lived in urban areas (Table 5), (Figure 32).

Table (5): Residence distribution of patients in the study.

Residence

N %

Rural 6 30.00

Urban 14 70.00

Total 20 100.00

Figure (32): Residence distribution of patients in the study.

137

c) Occupation:

Ten of patients (50%) were housewives; 4 patients (20%)

reported working by their hands (in a dry clean, bakery,

restaurant or as a carpenter) and 6 patients (30%) worked as

students or employees such as a banker, teacher or a doctor

(Table 6), (Figure 33).

Table (6): Occupations of patients in the study.

Occupation

N %

House wife 10 50.00

Handy worker 4 20.00

Others 6 30.00

Total 20 100.00

Figure (33): Occupations of patients in the study.

138

d) Associated conditions (predisposing factors to

onychomycosis):

Ten (50%) of the patient‟s had occupations that required

to put their hands in water for long times (housewives and

working in a dry clean) while 6 (30%) of the patients reported

using irritating chemicals and detergents in their work

(carpenter, worker in restaurant and some housewives).

Presence of coexisting fungal infection in other parts of

the body was discovered during examination in 3 (15%) who

were found to have associated tinea pedis.

Nine of our patients (45%) were found to have

onychomycosis associated with chronic paronychia. Other

predisposing factors such as family history of fungal nail

infection were detected in 4 patients (20%) and patients who

received intermittent short courses of topical and/or systemic

antifungal treatment over the long course of their disease were

5 (25%) (Table 7), (Figure 34) but all were off treatment for at

least 6 months before being included in this study.

139

Figure (34): Predisposing factors of onychomycosis in patients

of the study.

Table (7): Predisposing factors of onychomycosis in patients

of the study.

Predisposing factors

N %

Immersion of fingers in water for long times 10 50.00

Associated Tinea pedis 3 15.00

Associated chronic paronychia 8 40.00

Long history of intermittent short courses of treatment 5 25.00

Positive family history 4 20.00

Using chemicals & detergents in washing or work 6 30.00

Total 20 100.00

140

e) Number of nails affected:

Both finger &toenails were clinically examined and

investigated for the presence of fungal nail infection in all cases

with nail changes.

Our results revealed finger nails involved in 13 cases

(65%) while Toe nails were involved in 7 cases (35%).

Eight patients with multiple fingernails affection

represented (40%) of the study while 5 patients (25%) had

single fingernail affection, also 5 (25%) of patients had single

toenail affection while only 2 (10%) of patients had multiple

toenails affected (Table 8), (Figure 35).

Table (8): Number of diseased nails in patients of the study.

Number of affected nails

N %

Multiple finger nails 8 40.00

Single fingernail 5 25.00

Multiple toe nails 2 10.00

Single toenail 5 25.00

Total 20 100.00

141

Figure (35): Number of diseased nails in patients of the study.

142

f) Duration of the disease:

The duration of the disease among patients ranged from

1 month to 10 years (Table 9), (Figure 36) with mean duration

2.98 (±3.41) years.

Table (9): Duration of onychomycosis (years).

duration

N %

<1Y. 5 25.00

1-5Y 9 45.00

>5Y 6 30.00

Total 20 100.00

Figure (36): Duration of onychomycosis (years).

143

g) Complaint:

90% of our patients (18/20) complained of cosmetic

disfigurement, 60% complained of pain (12/20) due to

onycholysis and/or associated paronychia, 40% (8/20)

complained of itching, 2 patients (10%) reported having bad

odor and only 2 patients (10%) complained of slow nail growth

(Table 10).

Table (10): Patient‟s complaints of the disease

(onychomycosis).

Complaints

N %

cosmetic disfigurement 18 90.00

pain 12 60.00

itching 8 40.00

bad odor 2 10.00

slow nail growth 2 10.00

Total 20 100.00

h) Clinical types of onychomycosis:

As regards the different clinical presentations, the most

prevalent was distal-lateral subungual onychomycosis (85%)

followed by total dystrophic onychomycosis accounting for

(10%) and the least common was proximal subungual

onychomycosis representing only one (5%) patient (Table 11),

(Figure 37).

144

Table (11): Clinical presentations of onychomycosis in the

total number of fingernails and toenails.

Clinical presentations

Type of

onychomycosis

Toenail Fingernail

N % N % Total %

DLSO 7 35.00 10 50.00 17 85.00

PSO ---- -- 2 10.00 2 10.00

TDO --- -- 1 5.00 1 5.00

Total 7 35.00 13 65.00 20 100.00

Figure (37): Clinical presentations of onychomycosis in the total number

of fingernails and toenails.

145

Mycological data:

Subungual specimens and nail clipping samples were

cultured on different Medias such as Sabouraud dextrose agar

(SDA), Sabouraud dextrose agar with cyclohexamide and

chloramphenicol (SDA +C+C) and Chromogenic Candida agar

for further identification of fungal genera, rate of growth, shape

of colony, texture and pigmentation.

Fungal isolates in the present study were grouped into

Yeast 90% (18/20), non dermatophyte mould infection in 5%

(1/20) while dermatophyte infection was detected in 5% (1/20)

only (Table 12), (Figure 38).

146

Table (12): Identification of fungi into genera and species of

the 20 fungal isolates obtained from cases of

onychomycosis.

Fungi classification

Fungal group Genera & Species N %

Yeast

C.krusei 4 20.00

C.albicans 10 50.00

C.parapsilosis 1 5.00

C.tropicalis 2 10.00

Trichosporon 1 5.00

Dermatophytes T.mentagrophyte 1 5.00

Non -

dermatophyte

mould

Aspergellus flavus 1 5.00

Total 20 100.00

147

Figure (38): Identification of fungi into genera and species of the 20

fungal isolates obtained from cases of onychomycosis.

Identification of yeast isolates revealed candida species

in (85%) which were identified into c.albicans (50%), c.krusei

(20%), c.tropicalis (10%) and c.parapsilosis (5%) (Figure 39),

and Trichosporon species (5%) (Figure 40).

148

Figure (39): Candia albicans, C.tropicalis & C.krusei on chromogen agar.

Figure (40): Trichosporon species showing arthrospores, pseudohyphae

and blastospores.

149

Only one dermatophyte isolate was identified as T.

Mentagrophyte (5%) (Figure 41a & b).

a b Figure (41): a) T.mentagrophytes culture on SDA. b) Microscopy of

T.mentagrophytes showing macroconidia and microconidia.

On the other hand only one non dermatophyte mould

isolate was obtained and identified as Aspergellus flavus

(Figure 42).

Figure (42): Aspergellus flavus culture on SDA.

150

Results obtained at the first follow up (one

month after treatment):

Mycological results obtained one month after

treatment

The results revealed mycological clearance (by culture

and KOH) in 15 out of 20 (75%) patients at one month follow-

up while no response to treatment was detected in 5 patients

(25%) (Table 13), (Figure 43).

Table (13): Mycological response to treatment at the first

follow up

1st follow up one month after treatment

N %

Mycologically Negative patients 15 75.00

Mycologically Positive patients 5 25.00

Total 20 100.00

Figure (43): Mycological response to treatment at the first follow up.

151

Clinical response one month after treatment at first

follow up

There was no clinical response after one month of

treatment as the growth rate of the new nail plate emerging

from the matrix is 3 mm a month for the healthy fingernails and

1mm a month for toenails.

Clinical and mycological data of the 5 patients that

were still mycologically positive one month after

treatment in first follow up:

i. Demographic Characteristics:

All 5 patients were females. The ages of patients ranged

from 28 to 48 with mean age of 36.2 (±7.50). Two of the 5

patients (40%) lived in rural areas while 3 (60%) patients lived

in urban areas. Four of the 5 patients (80%) were housewives

and only one patient (20%) worked in a bakery.

ii. Associated conditions (predisposing factors of

onychomycosis):

All of the 5 mycologically positive patients had

onychomycosis associated with chronic paronychia (P-value =

00.1) which is highly significant (Table 14), (Figure 44).

152

Table (14): Comparison between the mycologically negative

and the mycologically positive patients as regards

chronic paronychia as a predisposing factor of

onychomycosis at first follow up results.

Predisposing factor chronic paronychia in all patients of the study

Chronic

paronychia

Mycologically

negative

Mycologically

positive Total

N % N % Total %

Yes (present) 4 20.00 5 25.00 9 45.00

No (absent) 11 55.00 0 0.00 11 55.00

Total patients 15 75.00 5 25.00 20 100.00

Chi-Square

X2 10.128

P-

value 0.001*

Figure (44): Comparison between the mycologically negative and the

mycologically positive patients as regards chronic paronychia as a

predisposing factor of onychomycosis at first follow up results.

153

All of the 5 patients reported immersion of their hands in

water for long times (P-value = 0.003) (Table 15), (Figure 45)

which is highly significant.



exposure to humidity as a predisposing factor of

onychomycosis at first follow up results.

Predisposing factor prolonged water use in the 20 patients

Immersion of nails

in water for long

time

Mycologically

negative

Mycologically

positive Total

N % N % Total %

Yes (water for long

time) 5 25.00 5 25.00 10 50.00

No (water for long

time) 10 50.00 0 0.00 10 50.00

Total patients 15 75.00 5 25.00 20 100.00

Chi-Square X

2 8.630

P-value 0.003*


mycologically positive patients as regards exposure to humidity as a

predisposing factor of onychomycosis at first follow up results.

154

Other predisposing factors found in these 5 patients were:

Only 2 of the 5 patients (40%) reported using irritating

chemicals and detergents. Family history of fungal nail

infection was found in only one patient (20%) and also one

patient (20%) received intermitted short courses of systemic

and topical antifungal treatment.

iii. Number of nails affected:

Our results revealed that fingernails are involved in all the

mycologically positive patients after treatment and there was no

toenails involvement. Four of the 5 patients (80%) had multiple

fingernails affection and only one patient (20%) had single

fingernail affection (Table 16), (Figure 56).

155



number of nail affection at first follow up results.

Number of nails affected in patients at first follow up

Number and types

of nails involved

Mycologically

negative

Mycologically

positive Total

N % N % Total %

Multiple

fingernails 4 20.00 4 20.00 8 40.00

Single fingernail 4 20.00 1 5.00 5 25.00

Multiple toenails 2 10.00 0.00 0.00 2 10.00

Single toenail 5 25.00 0.00 0.00 5 25.00

Total 15 75.00 5 25.00 20 100.00

Chi-Square

X2 1.900

P-

value 0.593


mycologically positive patients as regards number of nail affection at first

follow up results.

156

iv. Duration of the disease:

The duration of the disease among those 5 patients

ranged from 1 year to 10 years (Table 17), (Figure 47) with

mean duration 3.4 (±3.78) years with no significant difference

between the 5 mycologically positive and the rest of the

mycologically negative patients (P value = 0.094).

Figure (47): Bar chart Comparing the Duration of onychomycosis in the

mycologically negative and the mycologically positive patients at first

follow up results.

157

Table (17): Comparison of the Duration of onychomycosis

between the mycologically negative and the

mycologically positive patients at first follow up

results.

Duration of the disease in all patients at the first follow up

Duration of

onychomycosis

Mycologically

negative

Mycologically

positive Total

N % N % Total %

<1 year 5 25.00 0.00 0.00 5 25.00

1-5 years 5 25.00 4 20.00 9 45.00

>5 years 5 25.00 1 5.00 6 30.00

Total 15 75.00 5 25.00 20 100.00

Chi-Square

X2 4.721

P-

value 0.094

v. Clinical types of onychomycosis:

As for the clinical presentations, the most prevalent in 3

out of 5 (60%) of the mycologically positive patients was

distal-lateral subungual onychomycosis which represents (15%)

of the patients in our study. This is followed by total dystrophic

onychomycosis in one patient (5%) of the total patients in our

study and also proximal subungual onychomycosis in one

patient (5%) of the total patients in our study (Table 18),

(Figure 48).

158


mycologically positive and mycologically negative

patients at the first follow up.

Clinical presentations in all patients at the first follow up

Clinical

presentations

Mycologically

negative

Mycologically

positive Total

N % N % Total %

DLSO 14 70.00 3 15.00 17 85.00

TDO 0 0.00 1 5.00 1 5.00

PSO 1 5.00 1 5.00 2 10.00

Total patients 15 75.00 5 25.00 20 100.00

Chi-Square

X2 5.676

P-value 0.129

Figure (48): Clinical presentations of onychomycosis in mycologically

positive and mycologically negative patients at the first follow up.

159

vi. Identified fungal isolates

Identification of the isolates in the cultured specimens

showed that the commonest isolated fungal species were

candida infection in 80% (4 out of 5 mycologically positive

patients in first follow up) representing 20% of patients in the

study, followed by Trichosporon in one patient only

representing 5% of cases in our study (Table 19), (Figure 49).

The most prevalent candida species isolated from the

mycologically positive patients in first follow up was candida

albicans in 3 patients followed by candida krusei in one patient.

Figure (49): Comparison of the fungal species isolate between the

mycologically negative and the mycologically positive patients at first

follow up results.

160

Table (19): Comparison of the fungal species isolate between

the mycologically negative and the mycologically

positive patients at first follow up results.

Fungal isolates in first follow up

Fungal Species Member

Mycologicall

y

negative

Mycologicall

y

positive

Total

N % N % Tota

l %

Yeast

C.krusei 3 15.00 1 5.00 4 20.00

C.albicans 7 35.00 3 15.00 10 50.00

C.parapsilosis 1 5.00 0 0.00 1 5.00

C.tropicalis 2 10.00 0 0.00 2 10.00

Trichosporon 0 0.00 1 5.00 1 5.00

Dermatophyte

s T.mentagrophyte 1 5.00 0 0.00 1 5.00

Aspergellus

flavus 1 5.00 0 0.00 1 5.00

Total 15 75.00 5 25.00 20 100.0

0

Chi-Square

X2 5.777

P-value 0.449

161

The results of treatment at the 2nd follow up

(after 3 months):

The results revealed complete response to treatment

proven by mycological clearance (by culture and KOH) in 3 out

the 5 patients who were mycologically positive in the first

follow up after one month of treatment (P-value = 0.012) which

is highly significant (Table 20).

Table (20): Relation between first and second follow up.

Second follow up

First follow up

Mycologically

negative in 1st

follow up

Mycologically

positive in 1st

follow up

Total

N % N % Total %

Mycologically

negative in 2nd

follow up

15 75.00 3 15.00 18 90.00

Mycologically

positive in 2nd

follow up

0 0.00 2 10.00 2 10.00

Total patients 15 75.00 5 25.00 20 100.00

Chi-Square

X2 6.273

P-

value 0.012*

This increases the response to long pulsed Nd:YAG laser

treatment to 18 patients (90%) showing full clearance

mycologically at the second follow up after 3 months and

leaving only two patients (10%) mycologically and clinical

positive after 3 month (Table 21), (Figure 50).

162

Table (21): Mycological response to treatment at the second

follow up.

2nd follow up 3 months after treatment

N %

Mycologically Negative patients 18 90.00

Mycologically Positive patients 2 10.00

Total 20 100.00

Figure (50): Mycological response to treatment at the second follow up.

163

Clinical and mycological data of the two patients who were

resistant to treatment:

They were females, housewives, aged 38 and 48 years

with mean age of 33(±7.00), lived in urban areas and they had

multiple fingernails affection.

They had common predisposing factors such as

immersion of hands in water for long times and chronic

paronychia. One patient reported other predisposing factors like

using irritating chemicals and detergents and receiving

intermittent short courses of systemic or topical antifungal

treatment.

One patient had onychomycosis for 1 year and the other

had it for 10 years with mean duration 5.5(± 6.36) years.

Clinical presentations of onychomycosis were distal-

lateral subungual onychomycosis and total dystrophic

onychomycosis (Figure 51).

164

Figure (51): Clinical presentations of onychomycosis in mycologically

positive and mycologically negative patients at the second follow up.

Identification of the fungal isolates in the cultured

specimens showed that both patients had yeast infection, one

isolated species was candida albicans and the other isolated

species was Trichosporon (Figure 52).

165

Figure (52): Comparison of the fungal species isolate between the

mycologically negative and the mycologically positive patients at second

follow up results.

One of these two patients refused to repeat the 4 weekly

sessions of long pulsed Nd:YAG laser and continued on

medical treatment and the other patient (38 years old female,

house wife with total dystrophic onychomycosis of all fingers

of both hands of 10 years duration with isolated fungi candida

albicans) repeated the 4 sessions of long pulsed Nd:YAG laser

and reached a mycological clearance by culture and KOH.

Side effects of the treatment:

All patients complained of mild to moderate heat

sensation causing pain during the laser procedure and few hours

after, but it didn‟t require anesthesia though two patients took

over the counter analgesic at the same day of the procedure.

166

Slight yellowish-brownish temporary discoloration of affected

nails occurred in 3 patients. Other complications noticed were

acute paronychia developed in 2 patients 3 weeks after the end

of the 4 weekly sessions, decreased rate of growth of toenails in

2 patients after the procedure (Table 22).

Table (22): Side effects noticed during the treatment.

Side effects

N %

Pain (heat sensation) 20 100.00

Discoloration of the treated nails 3 15.00

Acute paronychia 2 10.00

Slow growth rate of nails 2 10.00

Total 20 100.00

167

Examples of results of onychomycosis treated

with long pulsed Nd:YAG laser 1,064 nm

A. Patients who responded clinically and

mycologically to treatment: Case number (1):

Figure (53a): Left big toe before treatment.

Figure (53b): Left big toe 3 months after treatment.

168

Case number (2):

Figure (54a): Left middle finger before treatment.

Figure (54b): Left middle finger 3 months after treatment.

169

Case number (3):

Figure (55a): Right big toe before treatment.

Figure (55b): Right big toe 3 months after treatment.

170

Case number (4)

Figure (56a): Right index before treatment.

Figure (56b): Right index 6 months after treatment.

171

B. A patient who didn’t show clinical response to treatment

though mycologically negative results:

Case number (5):

Figure (57a): All fingers of left hand except little finger before

treatment.

Figure (57b): All fingers of left hand except little finger 3 months

after treatment.

172

C. A patient who failed treatment:

Case number (6):

Figure (58a): All fingers of right hand before treatment.

Figure (58b): All fingers of right hand after treatment.

173

Discussion

174

DISCUSSION

nychomycosis is a chronic fungal infection of fingernails

and/or toenails leading to gradual destruction of the nail

plate (Hay, 2005 and Seebacher et al., 2007). It is caused by

many species, dermatophytes or yeasts or other non-

dermatophyes moulds (NDMs) (Jennings et al., 2002).

It is classified according to its clinical presentation into

distal and lateral subungual onychomycosis (DLSO),

superficial white onychomycosis (SWO), proximal subungual

onychomycosis (PSO), candidal onychomycosis, endonyx

onychomycosis and total dystrophic onychomycosis (TDO)

(Mahoney et al., 2003).

Children have infection rates 30 times lower than adults.

The reason for this decrease may be due to, reduced exposure

to fungus because of less time spent in environments containing

pathogens, faster nail growth, smaller nail surface for invasion

and lower prevalence of tinea pedis (Gupta et al., 1997c).

Onychomycosis is commonly confirmed by clinical

examination beside the direct microscopy and fungal culture

(Gupta and Ricci, 2006).

Onychomycosis is a frequent nail disease that can cause

various complications for example, physical and occupational

limitations due to loss of part or all of a nail bed, prolonged

standing, walking can be impaired in toenail onychomycosis,

O

175

psychosocial and emotional effects, infected nails serve as a

chronic reservoir of fungal infection, can promote to secondary

bacterial infection and significantly impair health-related

quality of life (HRQoL). Therefore; onychomycosis must be

considered a significant medical problem (Szepietowski and

Reich, 2009).

Many ways of treatment modalities are used that include

topical or systemic antifungal therapy, also surgical

interference and nail debridement (Rodgers and Bassler, 2001).

The ability of lasers to sterilize surfaces is well known

(Adrian and Gross, 1979). Also, laser beams are known to

eliminate bacteria by inducing cellular wall damage and

causing denaturation of its internal cellular protein (Ando et al.,

1996). Further more, the use of lasers to treat fungal nail

infection is not new. Researchers have been trying to use lasers

to treat onychomycosis since the 1980s. The approach at that

time was to use the laser to create holes or channels in the nail

(Rothermel and Apfelberg, 1987).

A non-invasive approach for treatment of onychomycosis

by laser is using a specific wavelength to kill the fungal cells

themselves (Kozarev and Vizintin, 2010). Lasers introduced for

the treatment of onychomycosis including diode laser

(Landsman et al., 2010), femtosecond infrared titanium

sapphire laser (Manevitch et al., 2010), short pulsed 0.65-

millisecond pulsed Nd:YAG laser (Mozena and Haverstock,

176

2010) and Long pulsed 1064 Nd:YAG laser (Kozarev and

Vizintin, 2010).

Our study included 20 patients with fingernail and/or

toenail onychomycosis treated by 4 weekly sessions of long

pulsed 1064 Nd:YAG laser.

Although it is commonly reported that onychomycosis

prevalence increases with age ranging between 40-60 years old

(Jayatilake et al., 2009) with highest prevalence above 55 years

old (Heikkala and Stubbs, 1995 and Hay, 1993) other authors

reported a high prevalence in the age group of 20-40 years

(Grover, 2003) which matches with our observation as the ages

of our patients ranged from 18 to 54 with mean age of

35.2(±10.26) years. The high prevalence in adults can be

attributed to the more consciousness about discoloration and

disfigurement of their nails.

On evaluating onychomycosis in our study we found it to

be more common in females (90%) than males (10%) in a ratio

of (9:1). A similar female predominance was reported in a

study in Indonesia that included 113 patients with female: male

ratio (2:1) (Bramono and Budimulja, 2005). This female

predominance was further reported in a study in South Asia

included (China, South Korea and Taiwan) (Haneke and

Roseeuw, 1999) and in a study on 255 patient in Tunis with a

female percentage of (63.5%) (Anane et al., 2001). This can be

attributed to the fact that females do household wet work as

177

laundry and house cleaning. On the other hand these results

differ from some other studies that showed onychomycosis

more common in males than females (Scher, 1994 and Ahmad

et al., 2010). The latter study explained this by the higher risk

of traumas and by the more common usage of occlusive foot-

wear in males (Ahmad et al., 2010).

In the present study onychomycosis was more common

in patients living in urban areas (70%) compared to those living

in rural areas (30%), this was in accordance to a study that was

carried out on 60 patients in India documenting that (85%) of

their patients lived in urban areas, and (15%) lived in rural

areas (Kaur et al., 2008b). This can be related to the fact of

presence of a higher socio-economic class in the urban

community and also that medical services are better and more

available in urban areas. Our results were in contrast to another

study showed that 94.12% of their patients lived in rural areas

(Jesudanam et al., 2002).

There were many patients in our study predisposed in

their work to minor traumas, injuries and wet work. Their role

as predisposing factors in onychomycosis is well established

(Gupta et al., 2004).

Housewives formed the largest group (50%) in our study

and this could be due to the involvement of housewives in

domestic activities that include wet work like cleaning and

laundry. They are also more concerned about cosmetic

178

appearance of their nails. Similar results were reported by other

studies, in a study on 448 patients with nail abnormalities at

Visakhapatnam, India during a one year period from 1998 to

1999 reported that 33.3% of their patients were housewives

(Jesudanam et al., 2002). Other cross-sectional study carried

out on 182 patients from 2006 to 2007 at Kathmandu, Nepal

reported that housewives represent 28.5% of their patients

(Neupane et al., 2009).

On evaluating risk factors of onychomycosis in our

study, 50% of the patients had occupations that required them

to put their hands in water for long time. This highlights the

fact that warm and moist environment promotes the growth of

fungi and predisposes to onychomycosis. These results are

consistent with other studies (Ahmad et al., 2010).

Using irritating chemicals and detergents is another risk

factor reported by 30% of our patients. It is documented that

chronic exposure to moisture, trauma and chemicals including

smoke, detergents and soap breach local immunity at the nail

complex and can predisposes to onychomycosis (Jayatilake et

al., 2009).

One of the most important predisposing factors of

onychomycosis is simultaneous superficial fungal infection at

other sites such as tinea pedis and chronic paronychia.

Concomitant fungal infections affected 60% of patients in our

study that is similar to other studies like Ching and colleagues

179

also Wang and Ching which recorded 62.4% and 62.6% in their

studies respectively (Ching et al., 2005 and Wang and Ching

2005)

In our study, onychomycosis was significantly higher in

patients with associated chronic paronychia (45%) which might

be attributed to the predominance of females in our study that

were mostly housewives with chronic exposure to wet

environment causing swelling of the proximal nail fold causing

cuticle detachment so that both yeasts and bacteria cause

proximal nail fold inflammation (Roberts et al., 2003). These

results were higher compared to other studies that recorded

chronic paronychia of 14.3% in a study on 182 patients in

Nepal (Neupane et al., 2009).

Furthermore (15%) of patients in our study were found

to have associated tinea pedis. Many studies indicated that

toenail onychomycosis and tinea pedis tend to coexist and that

tinea pedis can also predispose to subclinical onychomycosis of

the toe nails (Szepietowski et al., 2006 and Walling, 2009).

In our study fingernails were more common being

involved in (65%) of patients while Toenails were involved in

(35%) of patients. These results are similar to the results of a

study in Indonesia (Bramono and Budimulja, 2005), and is an

agreement with other studies in India, Jammu where fingernails

were involved in 56.5% of patients, toenails involved in 17% of

patients (Ahmad et al., 2010). Most of the studies from India

180

show that fingernails onychomycosis is more common than

toenails onychomycosis (Grover, 2003 and Kaur et al., 2008b).

In contrast to Western studies

that show toenails

onychomycosis is more common than fingernails

onychomycosis (Haneke, 1991 and Perea et al., 2000).

The low incidence of toenails onychomycosis in our

country may be attributed to open footwear and less concern for

appearance of feet and toe nails in our people.

The duration of onychomycosis among our patients

ranged from 1 month to 10 years. Twenty-five percent (25%) of

patients suffered from onychomycosis for less than one year

and 45% of them had it for less than 5 years. This may be due

to the fact that if the nail changes remain for a longer time

patients get used to living with it and may consider it as a part

of normal physiology.

The predominant clinical type of onychomycosis in our

study was distal-lateral subungual onychomycosis (85%)

followed by proximal subungual onychomycosis (10%) and the

least common was total dystrophic onychomycosis (5%). This

was confirmed in a Brazilian study that showed Distal and

lateral subungual onychomycosis (62.5%) being the most

prevalent followed by (20.8%) with Proximal subungual

infection and (12.5%) with Total dystrophic onychomycosis

(Matos and Mariano, 2010 and Raujo et al., 2003). Our results

are in accordance with results recorded by other studies

181

(Sujatha et al., 2000 and Veer et al., 2007) but lower than that

recorded by Jesudanam and colleagues where Distal and

lateral subungual onychomycosis was not the most common

clinical type (38.5%) (Jesudanam et al., 2002).

Identification of the fungal isolates showed that the

commonest isolated fungal groups were yeast infection in 90%

mainly candida species (the most common was candida

albicans 50%, candida krusei 20%, candida tropicalis 10% and

candida parapsilosis 5%) and Trichosporon species 5%.

followed by non dermatophyte mould (NDMs) infection in 5%

(Aspergellus flavus 5%) and dermatophyte infection

(T.mentagrophyte) was also detected in 5%. In contrast to most

studies that showed the most common fungal isolate to be

dermatophyte mainly T. rubrum followed by yeast mainly

candida albicans (Matos and Mariano, 2010 and Kaur et al.,

2008b and Vinod et al., 2000).

Our results might be in agreement with results of a study

by Godoy and colleagues (2009) though the huge difference in

percentages representing each species but this study showed

that candida species were the most frequent agents in fingernail

onychomycosis (38.3%) mainly C. albicans 18.3% and C.

parapsilosis 13.8% which were the most frequent species. The

dermatophytes did have a major role in fungal infection of

toenails onychomycosis (31.6%).The authors suggest that the

higher proportion could be the result of more traumas and the

greater use of occlusive footwear in male and tight fitting shoes

182

with high heels in females. T.rubrum in 33.2% followed by T.

mentagrophytes in 6.3% were the most frequent species in

toenail onychomycosis. The non-dermatophytic fungi were

etiologic agent in 7.4% of cases (Godoy et al., 2009).

Long pulse 1064 nm Nd:YAG laser (Candela, Wayland,

MA, USA) was used as a treatment for our patients with

parameters: fluence: 50 J/ cm², pulse duration: 30 ms, spot

size: 3 mm and frequency: 1 Hz.

Cooling cryogen spray was turned off completely. The

laser beam was applied to the entire nail plate three times by

moving the beam in a spiral pattern taking 2 minute pause twice

after each nail irradiaded completely. Patients were evaluated

after 1 and 3 months clinically, mycologicaly and by

photographic documentation.

In the first follow up after one month the results revealed

mycological clearance (by culture and KOH) in (75%) patients

while (25%) of patients were still mycologically positive (by

culture and KOH). This can be attributed to the fact that the

amount of laser energy that can deactivate 80-99% of the

organisms present in an affected nail (deactivating dose) does

not instantly kill the fungal colonies but results in their

disability to replicate or survive according to the apoptotic

mechanism (Kozarev and Vizintin, 2010).

183

These results are in agreement with another study in

Thailand on 25 nails of 14 patients used long pulsed Nd:YAG

1064 nm for 4 sessions at one week interval and the parameters

were set with fluence ranged from 35-45 J/ cm², pulse duration

ranged from 30-35 ms, spot size 4 mm and frequency 1 Hz.

They reported a response rate of 48% one month after treatment

at the one month follow up (Wanitphakdeedecha, 2011).

In the second follow up 3 months after treatment 90% of

the patients showed mycological clearance (by culture and

KOH) (P-value = 0.012) which is highly significant and only

10% remained mycologically positive.

Interestingly, these results are in accordance to similar

study used long pulsed Nd:YAG 1064nm the parameters were

set with fluence ranged from 35-45 J/ cm², pulse duration 35

ms, spot size 4 mm and frequency 1 Hz. That study showed

that 3 months after treatment at the follow up, 83% of their

patients cleared of all fungal infections while (17%) of patients

were still with present infection (Kozarev and Vizintin, 2010).

Also another study used long pulsed Nd:YAG 1064nm used

parameters with fluence 40 J/ cm², pulse duration 25 ms, spot

size 4 mm and frequency 1 Hz, showed that 3 months after

treatment in the follow up, 93% of their patients were cleared

of all nail fungal infections (Kozarev, 2009).

A large scale study on 162 patients with 413 affected

nails used long pulsed Nd:YAG 1064nm the parameters were

184

set with fluence ranged from 35-45 J/ cm², pulse duration 35

ms, spot size 4 mm and frequency 1 Hz, showed mycological

clearance of 99.98% (160 patients) at the follow up 6 months

after treatment (Kozarev, 2012).

Several other types of lasers are introduced for the

treatment of onychomycosis including diode laser (Landsman

et al., 2010) and novel 0.65-millisecond pulsed Nd:YAG laser

(Mozena and Haverstock, 2010).

The trial by diode laser consisted of 4 sessions at 1, 14,

42 and 120 days. In each session two exposures directed at the

toenail: a 4-min exposure applying both wavelengths (870 and

930 nm) simultaneously and a second exposure with 930 nm

alone for 2 min combined with topical antifungal starting from

the second session. Results showed (63%) improvement of the

affected nails which are close but lower than results proved by

our procedure (Landsman et al., 2010).

Short pulsed (0.65 millisecond) Nd:YAG laser was also

used as a treatment of onychomycosis. It is used by applying a

2-mm spot, with fluence 223 J/cm 2 in the absence of any

cooling device. Each infected nail was treated in a criss-cross

pattern with two alternating passes of laser pulses to cover the

full nail, one pass applied vertically and the second horizontally

over the nail surface. Treatment takes two to three sessions with

each session spaced at least 3 weeks apart.

185

An antifungal cream is provided to each patient after

treatment, to be applied to the nails daily as a preventive

measure against re-infection.

Results of this modality of treatment at follow up showed

87.5% mycological clearance which shows similar results to

our procedure (Mozena and Haverstock, 2010).

In our study, patients that didn‟t respond to treatment

were females; housewives aged 38 and 48, living in urban areas

and having multiple fingernails affection and having common

predisposing factors such as immersion of hands in water for

long times and onychomycosis associated with chronic

paronychia, but these data were statistically not significant in

our study.

As for the clinical presentations, one patient suffered

from distal-lateral subungual onychomycosis and the other

suffered from total dystrophic onychomycosis which is similar

to a study that showed treatment failure in a patient with total

dystrophic onychomycosis (Kozarev, 2009).

Fungal isolates in the cultured specimens were yeast

infection (candida albicans and Trichosporon). In contrast

another study that showed the resistant patient was suffering

from dermatophyte infection (T.rubrum) (Kozarev, 2009).

As for side effects and complaints from the treatment in

our study, the most common that occurred in all patients

186

(100%) was mild to moderate heat sensation causing pain

during the laser procedure and few hours after. Also in 15% of

patients a slight yellowish-brownish temporarily discoloration

of affected nails occurred. Two patients (10%) developed acute

paronychia 3 weeks later after the end of the 4 weekly sessions.

Also, 2 patients (10%) complained of decreased rate of growth

of their toenails after the procedure but relation between these

complications and our procedure is still unknown.

Side effects reported in other studies included mild to

moderate pain that decrease every session also yellowish

discoloration was reported as common side effects to the

procedure (Kozarev and Vizintin, 2010).

187

Conclusions & Recommendations

188

CONCLUSION

a) Long pulsed Nd:YAG laser 1064 nm is a safe and effective

option for treatment of onychomycosis. We recommend its

use in the management of any type of onychomycosis.

b) If the patient after 3 months follow up is still mycologically

positive another 4 sessions of laser treatment are advised.

c) Although long pulsed Nd:YAG laser 1064 nm is safe, with

minimal time consumption, good outcome but it is highly

expensive as it needs specialized laser centers and needs

standardization of treatment parameters.

189

RECOMMENDATIONS

a. Further prospective studies on large population are

recommended.

b. Longer follow up time of the patients is needed.

c. Correlating the effect of treatment on wider spectrum of

fungal species.

d. Correlating the effect of treatment by grading

onychomycosis to mild, moderate and sever.

e. Comparing the effectiveness of long pulsed Nd:YAG laser

1064 nm with other types of lasers as treatment of

onychomycosis.

f. Comparing other treatment parameters with the ones used in

an attempt for standardization of treatment parameters.

190

Summary

191

SUMMARY

nychomycosis is a common persistent infection of the nail

unit by fungi either dermatophytes, yeasts or non

dermatophyte moulds (NDMs).

It is classified clinically as distal and lateral subungual

onychomycosis (DLSO), superficial white onychomycosis

(SWO), proximal subungual onychomycosis (PSO), candidal

onychomycosis, endonyx onychomycosis and total dystrophic

onychomycosis (TDO).

Onychomycosis is not just a simple cosmetic problem

but a handicap as it hinders the patient from practicing normal

life and prevent the patient from doing certain jobs like typists

or tailors.

It is commonly confirmed by clinical examination side

by side with regular diagnostic techniques such as direct

microscopy and fungal culture, which are considered the golden

standards of diagnosis.

Onychomycosis is a challenging problem to both the

patient and the physician. The patient suffers from a disease

that reduces his quality of life while the physician struggle with

long time, expensive treatments with many side effects and

drug interactions that can harm the patient.

O

192

It is very difficult to treat onychomycosis, for this reason

several treatment regimens were designed to cure this disease

such as palliative care, topical as well as systemic drugs. In

order to overcome the drawbacks of such regimens a non-

invasive approach for treating onychomycosis by laser beams

was introduced.

Several types of lasers can be used for the treatment of

onychomycosis including long pulsed-Nd:YAG laser 1064nm,

diode laser, femtosecond infrared titanium sapphire laser and

0.65-millisecond pulsed Nd:YAG laser.

We performed our study on 20 patients with finger

and/or toenail onychomycosis. Our aim was to explore and

evaluate the usage of long pulsed-Nd:YAG laser 1064nm as a

treatment of onychomycosis with parameters of; fluence 50 J/

cm², pulse duration 30 ms, spot size 3 mm, frequency 1 Hz and

the cooling cryogen spray was turned off.

Each nail plate was fully covered with a transcutaneous

laser irradiation in a spiral pattern starting at the nail periphery

and finishing in the nail centre. In one session three passes of

laser beam across each nail plate with two minutes pause

between passes were applied. The total therapy consists of four

sessions with a one week interval between each session.

The fungal isolates in the study were 90% yeast

infection, mainly candida species (candida albicans 50%,

193

candida krusei 20%, candida tropicalis 10% and candida

parapsilosis 5%) and Trichosporon species in 5% of patients.

Followed by non dermatophyte mould infection (Aspergellus

flavus) detected in 5% of patients and dermatophyte infection

(T.mentagrophyte) detected in 5% of patients.

The predominant clinical type of onychomycosis in the

study was distal-lateral subungual onychomycosis (85%)

followed by proximal subungual onychomycosis (10%) and the

least common was total dystrophic onychomycosis (5%).

As regards cure rates of our procedure, the results

revealed mycological clearance (by culture and KOH) in (75%)

of patients at the one month follow-up. This is due to the fact

that the amount of laser energy that can deactivate 80-99% of

the organisms present in an affected nail does not instantly kill

the fungal colonies but results in their disability to replicate or

survive according to the apoptotic mechanism. By the second

follow up after 3 month the results reached 90% cure rate

documented by mycological and clinical clearance using KOH

and culture.

Identification of the fungal isolates in the cultured

specimens showed that the 10% failure rate was yeast infection

(candida albicans and Trichosporon) and the clinical

presentations were distal-lateral subungual onychomycosis and

total dystrophic onychomycosis.

194

We hereby conclude that long pulsed-Nd:YAG laser

1064nm is by far a good treatment of onychomycosis as it is

safe, with minimal time consumption, helpful in fungal species

resistant to medical treatment such as Aspergellus flavus, has

good outcome and doesn‟t cause any serious side effects.

195

References

1

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Arabic Summary

45

الولخص العسبي

إن الفطر الظفري ىو العدوى الفطرية المستمرة الشائعة لمصفوفة الظفر، لى . وصفيحو الظفر وسرير الظفر وتسببو فطريات الجمد المسماة ديرماتوفايتس وا .والخمائر من أنواع المبيضات (القوالب الجمدية)حد أقل الفطريات الغير جمدية

يصنف الفطر الظفرى سريريا الى ظفري تحت الظفر القاصي والجانبي، فطر الأظافرالبيضاء السطحية، فطر الأظافر تحت الظفر الداني، وظفرى مصاب

.بخمائر المبيضات المسماه بالكانديدا والظفري المصاب بتشوىات كميةإن الفطر الظفرى ىو مشكمة شائعة تؤثر عمى كثير من الناس في جميع

أنحاء العالم وكثير من الفئات العمرية، فيو يمثل نصف أمراض الأظافر في نتشاره يزيد مع التقدم في السن .البالغين كما يمثل تيديدا لصحة المسنين وا

إن معدل إنتشار الفطر الظفرى يتزايد وذلك بسبب إزدياد أعداد المصابين بأمراض مزمنة مثل داء السكري والذين يعانون من الأمراض التي تؤدى لقمع

.وتقميل دور نظام المناعة وزيادة متوسط الأعمار

إن الفطر الظفرى ليس مجرد مشكمة تجميمية بسيطة تسبب الإحراج ولكن ويمكن أن يؤدي إلى .أيضا يشكل عائقا يعوق المريض من ممارسة حياة طبيعية

الإكتئاب والعزلة الاجتماعية أو منع المريض من القيام بأعمال معينة مثل الطابعين .أو الخياطين

ويتم تأكيد تشخيص الفطر الظفرى عادة بالفحص الإكمينيكي بجانب تقنيات التشخيص العادية مثل الفحص المجيرى المباشر ومزرعة الفطريات، التي تعتبر

.المعيار الذىبي لمتشخيصإن الفطر الظفرى مشكمة صعبة لممريض والطبيب عمى حد سواء فالمريض

يعاني من مرض يقمل من جودة حياتو، ويتطمب منو أن يغير عادات حياتو أو وظيفتو كما يتطمب الصراع مع المرض من الطبيب أن يستمر العلاج لفترات زمنية طويمة، وقد تكون العلاجات باىظة الثمن وليا العديد من الآثار الجانبية

46

وخاصة عمى الجياز اليضمي والكبد و ليا العديد من التداخلات الدوائية التي . يمكن أن تضر المريض وتقمل من الإلتزام بالعلاج

إن مرض الفطر الظفري يشكل صعوبة بالغو فى علاجو فى بعض الأحيان، وليذا السبب تم تصميم العديد من وسائل و نظم لعلاج ىذا المرض فى محاولو لمسيطره عميو وذلك مثل، العلاج المسكن لتخفيف وطأة المرض، وأيضا

من أجل التغمب عمى عيوب ىذه . العلاج الموضعي وكذلك الأدوية الجيازيةالوسائل ونظم العلاج ظيرت مؤخرا طريقة جديده لمعلاج غير مدمرة ولا تجتاح

. الأظافر وىى إستخدام جياز الميزرتستخدم لمعلاج بما فى ذلك جياز الميزر أن يوجد عدة أنواع من الميزر

أشعة الميزر الصمام )الدايود ليزر نانومتر وجياز 1064ياج بالطول الموجي : دى، وليزر الفيمتو ثانية وأيضا عائمو الياج ليزر قدمت لعلاج فطر الأظافر (الثنائي

. ممي ثانية 0.65ليزر جديد نابض ذو بالكشف السريرى و الفحص المعممى مريضا ثبت20لقد شممت دراستنا

لمفطريات انيم يعانون من مرض الفطر الظفرى فى أصابع الأيدى أو الأقدام اليدف من ىذا العمل . أشير عمى الأقل6وتوقفوا عن أخذ أى علاج أخر لمدة

نانومتر، تم 1064ياج بالطول الموجي :كان استكشاف وتقييم استخدام الميزر أندمده ممم، و3، و حجم البقعة قطرىا ²سم / جول 50 في نطاق تدفقإستخدام تم تطبيق شعاع ليزر لصفيحة الظفر . ىرتز1 مممي ثانيو وتردد 30النبضو

بأكممو من خلال نقل تدريجي لمشعاع بشكل لولبي بدءا بأطراف محيط الظفر . وانتياءا في المركز

بعد تعريض صفيحة الظفر بأكمميا لميزر، تم اتخاذ وقفة لمده دقيقتان ثم العلاج تكون من مجموع أربع جمسات . تكرار العلاج مرتين أخريين بنفس الطريقة

. مع فاصل اسبوع واحد بين كل جمسة

47

إن عينات الفطريات المعزولو أثناء الدراسو تشمل الإصابو بفطريات الأكثر إنتشارا كان الكانديدا )% 85بنسبة (الكانديدا)الخمائر تنقسم إلى

بنسبة (C.Krusei)ثم كانديدا كروزياى% 50 بنسبة (C.albicans)البيضاءو أخيرا الكانديدا % 10 بنسبة (C. tropicalis) ثم الكانديدا المداريو % 20

و فطر الترايكوسبورن (%5 بنسبة (C. parapsilosis)بارابسيموسس (Trichosporon) القوالب )ثم يمييا الإصابو بالفطريات الغير جمدية % 5 بنسبة

فطر الأسبرجيلاس فلافس بينما (Aspergellus flavus)% 5بنسبة (الجمديةمتمثمو فى فطر الترايكوفيتون % 5الإصابو بالفطريات الجمديو مثمت أيضا

. (T.mentagrophytes)منتاجروفايت كان النوع الإكمينيكى السائد لمفطر الظفرى فى المرضى الخاضعين

ثم يميو فطر % 85لمدراسو ىو ظفري تحت الظفر القاصي والجانبي بنسبة ثم الظفري المصاب بتشوىات كمية بنسبة % 10الأظافر تحت الظفر الداني بنسبة

5%. فيما يتعمق بمعدلات الشفاء فى الدراسو فى جمسة المتابعو الأولى بعد

الإنتياء من جمسات العلاج بمدة شير أثبتت النتائج عدم وجود فطريات معمميا عن و ، %75طريق مزرعة الفطريات والفحص المباشر تحت الميكروسكوب بنسبة

% 99-80يعزى ذلك إلى أن كمية الطاقو الموجوده فى الميزر يمكنيا إيقاف نشاط من الكائنات العضويو الموجوده فى الظفر ولكن لا يقتل المستعمرات الفطريو كميا عمى الفور بل يتسبب فى إعاقتيم عن التكاثر أو الحياه وفقا لألية الموت الخموى

. المبرمج لكل خميةعمى جانب أخر فى جمسة المتابعو الثانيو بعد ثلاثة أشير إرتفعت نتائج

نسبة شفاء وتم توثيقيا بعدم وجود فطريات معمميا عن طريق % 90العلاج إلى . مزرعة الفطريات والفحص المباشر تحت الميكروسكوب

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فى (%10)تم التعرف عمى الفطريات المسببو لنسبة الفشل فى العلاج ترايكوسبورن و )العينات المزروعو فى مزرعة الفطريات و ىما فطريات الخمائر

وكانت الأنواع الإكمينيكيو لمفطر الظفرى عمى التوالى ظفري (الكانديدا البيضاء .تحت الظفر القاصي والجانبي، الظفري المصاب بتشوىات كميةياج بالطول :بموجب ىذه النتائج نستنتج أن العلاج بجياز الميزر أند

نانومتر يعتبر إلى حد بعيد وسيمة علاج جيدة حيث أنو أمن وفترة 1064الموجي العلاج قصيرة ونتائجو جيدة خاصة فى الفطريات المقاومو لمعلاجات الأخرى مثل

. فطر الأسبرجيلاس فلافس و ليس لو أعراض جانبية خطيرة

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ياج ليزر فى علاج فطر الأظافر: تقييم إن دى

رسالة

توطئة للحصول على درجة الماجستير

فى الأمراض الجلدية والتناسلية و الذكورة

مقدم من

ايمان محمد أكمل أحمد /الطبيبةجاهعت عيي شوط – بكالسيوض الطب و الجساحت

تحت اشراف

هدى أحمد منيب / الأستاذة الدكتورةاظتاذ الأهساض الجلديت و التٌاظليت

جاهعت عيي شوط - كليت الطب

محمد طه محمود / الأستاذ الدكتورأظتاذ علن الويكسوباث

جاهعت الصقاشيق - كليت الطب البيطسى

مارى فكرى متى / الدكتورةهدزض الأهساض الجلديت و التٌاظليت

جاهعت عيي شوط - كليت الطب

جامعة عين شمس - كلية الطب2013

supervised by prof. dr. hoda ahmed monieb

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