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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.
Table (15): Comparison between the mycologically
negative and the mycologically positive
patients as regards exposure to humidity
as a predisposing factor of onychomycosis
at first follow up results. ......................................... 153
Table (16): Comparison between the mycologically
negative and the mycologically positive
patients as regards number of nail affection
at first follow up results. ......................................... 155
Table (17): Comparison of the Duration of
onychomycosis between the mycologically
negative and the mycologically positive
patients at first follow up results. ........................ 157
Table (18): Clinical presentations of onychomycosis in
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…)
Fig. No. Title Page No.
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
List of Figures (Cont…)
Fig. No. Title Page No.
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
negative and the mycologically positive
patients as regards chronic paronychia
as a predisposing factor of
onychomycosis at first follow up results. .......... 152
Figure (45): Comparison between the mycologically
negative and the mycologically positive
patients as regards exposure to humidity
as a predisposing factor of
onychomycosis at first follow up results. .......... 153
List of Figures (Cont…)
Fig. No. Title Page No.
Figure (46): Comparison between the mycologically
negative and the mycologically positive
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
negative and the mycologically positive
patients at first follow up results. ...................... 156
Figure (48): Clinical presentations of onychomycosis
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
Figure (51): Clinical presentations of onychomycosis
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
List of Figures (Cont…)
Fig. No. Title Page No.
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
Review of Literature Onychomycosis
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,
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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
Review of Literature 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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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.
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
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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
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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
Review of Literature Onychomycosis
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
Review of Literature Onychomycosis
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).
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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).
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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
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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),
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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
Review of Literature Onychomycosis
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).
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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
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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
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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.
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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).
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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).
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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).
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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).
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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).
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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
Review of Literature Onychomycosis
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).
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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.,
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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).
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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).
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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
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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).
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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).
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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
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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).
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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
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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
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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)
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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).
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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
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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
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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).
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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
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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).
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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
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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)
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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).
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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
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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).
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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).
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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).
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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
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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,
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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
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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
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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).
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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).
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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.
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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
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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%
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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
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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
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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).
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- 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
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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).
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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).
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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.
Table (15): Comparison between the mycologically negative
and the mycologically positive patients as regards
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*
Figure (45): Comparison between the mycologically negative and the
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
Table (16): Comparison between the mycologically negative
and the mycologically positive patients as regards
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
Figure (46): Comparison between the mycologically negative and the
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
Table (18): Clinical presentations of onychomycosis in
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
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Arabic Summary
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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
48
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