EVALUATION OF LONG PULSED-ND: YAG LASER IN THE TREATMENT OF 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

ببسسمم هللاهللا االلررححممنن االلررححييمم

إقسأ باظن زبك إقسأ باظن زبك االلررًً خخللقق11خخللقق

ااإلإلًًععااىى ههيي

ععللقق22 إإققسسأأ ووززببكك ااألألككسسمم33االلررًً ععللنن ببااللققللنن44ععللنن ااإلإلًًععااىى ههاا للنن ييععللنن55

صصددقق هللاهللا االلععظظييمم ظظووززةة االلععللقق ااآلآلييااثث ))55--11(( AAcckknnoowwlleeddggmmeenntt

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. EEmmaann MMoohhaammeedd 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 ...... 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 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): macroconidia ...... 38 Figure (6): a) Macromorphology of Trichophyton mentagrophytes colonies. b) Macromorphology of colonies front and back view...... 39 Figure (7): microcondia ...... 40 Figure (8): Microsporum colonies in culture ...... 41 Figure (9): Macroconidia of floccosum ...... 42 Figure (10): Colonies of ...... 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 ...... 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): 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 AIDS : Acquired immunedeficiency diseases ALA : aminolevulinic acid Apaf-1 : Apoptotic protease activating factor 1 ATP : Adenosine-5'-triphosphate BCP : Bromcresolpurple C. albicans : C. glabrata : C. krusei :

CO2 : Carbon dioxide C. parapsilosis : Candida parapsilosis C. tropicalis : CHS1 : Chitin synthase 1 gene CMI : Cell-mediated immunity DLSO : Distal and lateral subungual onychomycosis DMSO : Di-methyl sulfoxide DNA : Deoxyribonucleic acid DTH : Delayed-type hypersensitivity DTM : test medium DUBs : Deubiquitinating enzymes EGF : Epidermal growth factor EO : Endonyx onychomycosis Er:YAG : Erbium-doped yttrium aluminium garnet HIV : Human immunodeficiency virus HPV : Human papilloma virus HRQoL : Health related quality of life HSBP1 : Heat shock factor binding protein 1 HSF : Heat shock factor HSP : Heat shock protein HZ : Hertz IL-1 : Interleukin-1 ITS1 : Internal transcribed spacer 1 KOH : Potassium hydroxide KONCPA : Potassium hydroxide treated nail clipping with periodic acid-schiff Laser : Light Amplification by Stimulated Emission of Radiation LPCB : Lacto-phenol cotton blue M. audouinii : M. canis : M. cookie : Microsporum cookie M. ferrugineum : Microsporum ferrugineum M. gypseum : Microsporum gypseum M. nanum : Nacl : Sodium chloride NaOH : Sodium hydroxide Nd: YAG laser : Neodymium-doped yttrium aluminium garnet NDMs : Non-dermatophyes moulds PAS stain : Periodic acid Schiff stain PCR : Polymerase chain reaction PCR-RFLP : Polymerase chain reaction-restriction fragment length polymorphism assay PDA : Potato dextrose agar PDGF : Platelet derived growth factor PDT : Photodynamic therapy Pps : Pulse per second PSO : Proximal subungual onychomycosis QOL : Quality of life REP-PCR : Repetitive Extragenic Palindromic Sequence Polymerase Chain Reaction RNA : Ribonucleic acid ROS : Reactive oxygen species RSM : Rapid sporulating medium SDA : Sabouraud dextrose agar medium spp : Species SWO : Superficial white onychomycosis T. equinum : Trichophyton equinum T. erinacei : Trichophyton erinacei T. megninii : Trichophyton megninii T. rubrum : Trichophyton rubrum T. soudanense : Trichophyton soudanense T. tonsurans : T. violaceum : Trichophyton violaceum T.mentagrophytes : Trichophyton mentagrophytes TDO : Total dystrophic onychomycosis TNF : Tumour necrosis factor TRT : Thermal relaxation time YAG : Yttrium Aluminium Garnet

Introduction

16

INTRODUCTION

nychomycosis is a common persistent fungal infection of O 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, of the Candida species (Evans, 1998).

Onychomycosis is classified clinically as distal and lateral subungual onychomycosis (DLSO), superficial white onychomycosis (SWO), proximal subungual onychomycosis

17

(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 , and 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 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).

18

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).

19

Aim of the Work

20

AIM OF THE WORK

he aim of this work is to explore and evaluate the usage of T long pulsed-Nd:YAG laser 1064nm in treatment of onychomycosis.

21

Review of Literature

22

Onychomycosis

23 Review of Literature Onychomycosis 

Chapter (1) ONYCHOMYCOSIS Definition

nychomycosis is a common persistent fungal infection of O 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.

24 Review of Literature Onychomycosis 

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,

25 Review of Literature Onychomycosis 

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).

26 Review of Literature Onychomycosis 

- 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

27 Review of Literature Onychomycosis 

mechanically and chemically resistant sheet of compacted keratinized cells. Nail hardness is mainly due to the disulfide bonds found in the 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).

28 Review of Literature Onychomycosis 

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

29 Review of Literature Onychomycosis  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 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).

30 Review of Literature Onychomycosis 

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).

31 Review of Literature Onychomycosis 

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

32 Review of Literature Onychomycosis  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).

33 Review of Literature Onychomycosis 

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, , Alternaria, , Penicillium, Scopulariopsis, finally Dermatophytes, which includes species of Microsporum canis and Trichophyton (Abdel-Hafez et al., 1995).

34 Review of Literature Onychomycosis 

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).

35 Review of Literature Onychomycosis 

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).

36 Review of Literature Onychomycosis 

- 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).

37 Review of Literature Onychomycosis  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 that grows in a variety of colours can be seen (Taha, 2011).

38 Review of Literature Onychomycosis 

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

39 Review of Literature Onychomycosis  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

40 Review of Literature Onychomycosis 

(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).

41 Review of Literature Onychomycosis  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).

42 Review of Literature Onychomycosis 

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.

43 Review of Literature Onychomycosis 

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

44 Review of Literature Onychomycosis 

(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, , and 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).

45 Review of Literature Onychomycosis 

II. :

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

46 Review of Literature Onychomycosis  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).

47 Review of Literature Onychomycosis 

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 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

48 Review of Literature Onychomycosis  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

49 Review of Literature Onychomycosis 

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

50 Review of Literature Onychomycosis  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).

51 Review of Literature Onychomycosis 

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).

52 Review of Literature Onychomycosis 

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

53 Review of Literature Onychomycosis  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),

54 Review of Literature Onychomycosis  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

55 Review of Literature Onychomycosis  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).

56 Review of Literature Onychomycosis 

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

57 Review of Literature Onychomycosis  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 Figure (17): b) Nail invasion in PSO (Habif, 2010). 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 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

58 Review of Literature Onychomycosis 

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.

59 Review of Literature Onychomycosis 

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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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).

61 Review of Literature Onychomycosis  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).

62 Review of Literature Onychomycosis  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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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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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

65 Review of Literature Onychomycosis  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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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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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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. 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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. 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

70 Review of Literature Onychomycosis  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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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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Table (1): Diagnosis of onychomycosis caused by dermatophytes (Scher et al., 2007).

Confirmatory Laboratory Clinical examination specimen analysis i. Primary criteria for diagnosis: Positive microscopic evidence White/yellow or orange/brown patches or streaks ii. Secondary criteria for diagnosis: Positive culture of dermatophyte - Onycholysis - Subungual hyperkeratosis/debris - Nail-plate thickening

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

73 Review of Literature Onychomycosis  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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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

75 Review of Literature Onychomycosis  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

76 Review of Literature Onychomycosis  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)

77 Review of Literature Onychomycosis  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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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

79 Review of Literature Onychomycosis  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 - peptone agar with cycloheximide , known with many trade names like mycosel or dermasel, potato flake agar, potato glucose and devised Casamino Acids-erythritol- albumin

80 Review of Literature Onychomycosis  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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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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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

84 Review of Literature Onychomycosis  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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 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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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

87 Review of Literature Onychomycosis  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).

88 Review of Literature Onychomycosis  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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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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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

91 Review of Literature Onychomycosis  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,

92 Review of Literature Onychomycosis  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

93 Review of Literature Onychomycosis  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

94 Review of Literature Onychomycosis  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). 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 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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Table (2): Classification of approved antifungal drugs (Taha, 2011). Target for Group Compound its action 1- Polyenes - Nystatin Ergosterol - Amphotericin B in plasma membrane

2- Azoles - Ketoconazole Cytochrome a) Diazoles - Fluconazole P450 b) TriaZoles - Itraconazole 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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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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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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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%

99 Review of Literature Onychomycosis  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

100 Review of Literature Onychomycosis  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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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

103 Review of Literature Onychomycosis  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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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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Laser and Infections

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Chapter (2) LASER AND INFECTIONS Introduction

he ability of lasers to sterilize surfaces is well known. T 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

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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).

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- 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

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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

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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

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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).

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 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

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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

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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).

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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

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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,

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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)

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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

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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).

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Patient and

Methods

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PATIENTS, MATERIAL AND METHODS Patients

his study includes twenty (20) patients suffering from T 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.

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 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.

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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

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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.

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- 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).

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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.

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. 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.

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 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.

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 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).

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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

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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.

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Results

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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.

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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.

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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.

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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.

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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.

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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

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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

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

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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).

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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).

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Table (11): Clinical presentations of onychomycosis in the total number of fingernails and toenails.

Clinical presentations

Toenail Fingernail Type of onychomycosis 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.

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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).

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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 %

C.krusei 4 20.00

C.albicans 10 50.00

Yeast C.parapsilosis 1 5.00

C.tropicalis 2 10.00

Trichosporon 1 5.00

Dermatophytes T.mentagrophyte 1 5.00

Non - dermatophyte Aspergellus flavus 1 5.00 mould

Total 20 100.00

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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).

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

Figure (40): Trichosporon species showing arthrospores, pseudohyphae and blastospores.

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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.

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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.

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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).

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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 Mycologically Mycologically Total Chronic negative positive paronychia 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 X2 10.128 Chi-Square P- 0.001* value

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.

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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 Mycologically Mycologically Immersion of nails Total in water for long negative positive time N % N % Total % Yes (water for long 5 25.00 5 25.00 10 50.00 time) No (water for long 10 50.00 0 0.00 10 50.00 time) Total patients 15 75.00 5 25.00 20 100.00 X2 8.630 Chi-Square 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.

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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).

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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 Mycologically Mycologically Total Number and types negative positive of nails involved N % N % Total % Multiple 4 20.00 4 20.00 8 40.00 fingernails 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 X2 1.900 Chi-Square P- 0.593 value

Figure (46): Comparison between the mycologically negative and the mycologically positive patients as regards number of nail affection at first follow up results.

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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.

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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

Mycologically Mycologically Total negative positive Duration of onychomycosis 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

X2 4.721 Chi-Square P- 0.094 value 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).

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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

Mycologically Mycologically Total negative positive Clinical presentations 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

X2 5.676 Chi-Square P-value 0.129

Figure (48): Clinical presentations of onychomycosis in mycologically positive and mycologically negative patients at the first follow up.

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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.

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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

Mycologicall Mycologicall y y Total negative positive Fungal Species Member Tota N % N % % l

C.krusei 3 15.00 1 5.00 4 20.00

C.albicans 7 35.00 3 15.00 10 50.00

Yeast 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 T.mentagrophyte 1 5.00 0 0.00 1 5.00 s

Aspergellus 1 5.00 0 0.00 1 5.00 flavus

100.0 Total 15 75.00 5 25.00 20 0

X2 5.777 Chi-Square P-value 0.449

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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.

First follow up

Mycologically Mycologically negative in 1st positive in 1st Total Second follow up follow up follow up N % N % Total % Mycologically negative in 2nd 15 75.00 3 15.00 18 90.00 follow up Mycologically positive in 2nd 0 0.00 2 10.00 2 10.00 follow up Total patients 15 75.00 5 25.00 20 100.00 X2 6.273 Chi-Square P- 0.012* value

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).

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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.

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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).

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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).

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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.

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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

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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.

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Case number (2):

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

Figure (54b): Left middle finger 3 months after treatment.

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Case number (3):

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

Figure (55b): Right big toe 3 months after treatment.

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Case number (4)

Figure (56a): Right index before treatment.

Figure (56b): Right index 6 months after treatment.

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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.

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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.

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Discussion

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DISCUSSION

nychomycosis is a chronic fungal infection of fingernails O 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,

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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,

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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

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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

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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

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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

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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

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(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

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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).

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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

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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.

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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

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(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).

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Conclusions & Recommendations

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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.

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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.

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Summary

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SUMMARY

nychomycosis is a common persistent infection of the nail O 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.

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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%,

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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.

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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.

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Arabic Summary

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الولخص العسبي

إن الفطر الظفري ىو العدوى الفطرية المستمرة الشائعة لمصفوفة الظفر، وصفيحو الظفر وسرير الظفر. وتسببو فطريات الجمد المسماة ديرماتوفايتس والى حد أقل الفطريات الغير جمدية )القوالب الجمدية( والخمائر من أنواع المبيضات. يصنف الفطر الظفرى سريريا الى ظفري تحت الظفر القاصي والجانبي، فطر األظافرالبيضاء السطحية، فطر األظافر تحت الظفر الداني، وظفرى مصاب بخمائر المبيضات المسماه بالكانديدا والظفري المصاب بتشوىات كمية. إن الفطر الظفرى ىو مشكمة شائعة تؤثر عمى كثير من الناس في جميع أنحاء العالم وكثير من الفئات العمرية، فيو يمثل نصف أمراض األظافر في البالغين كما يمثل تيديدا لصحة المسنين وانتشاره يزيد مع التقدم في السن. إن معدل إنتشار الفطر الظفرى يتزايد وذلك بسبب إزدياد أعداد المصابين بأمراض مزمنة مثل داء السكري والذين يعانون من األمراض التي تؤدى لقمع وتقميل دور نظام المناعة وزيادة متوسط األعمار. إن الفطر الظفرى ليس مجرد مشكمة تجميمية بسيطة تسبب اإلحراج ولكن أيضا يشكل عائقا يعوق المريض من ممارسة حياة طبيعية . ويمكن أن يؤدي إلى اإلكتئاب والعزلة االجتماعية أو منع المريض من القيام بأعمال معينة مثل الطابعين أو الخياطين. ويتم تأكيد تشخيص الفطر الظفرى عادة بالفحص اإلكمينيكي بجانب تقنيات التشخيص العادية مثل الفحص المجيرى المباشر ومزرعة الفطريات، التي تعتبر المعيار الذىبي لمتشخيص. إن 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 العالج لفترات زمنية طويمة، وقد تكون العالجات باىظة الثمن وليا العديد من اآلثار الجانبية

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وخاصة عمى الجياز اليضمي والكبد و ليا العديد من التداخالت الدوائية التي يمكن أن تضر المريض وتقمل من اإللتزام بالعالج. إن مرض الفطر الظفري يشكل صعوبة بالغو فى عالجو فى بعض األحيان، وليذا السبب تم تصميم العديد من وسائل و نظم لعالج ىذا المرض فى محاولو لمسيطره عميو وذلك مثل، العالج المسكن لتخفيف وطأة المرض، وأيضا العالج الموضعي وكذلك األدوية الجيازية. من أجل التغمب عمى عيوب ىذه الوسائل ونظم العالج ظيرت مؤخرا طريقة جديده لمعالج غير مدمرة وال تجتاح األظافر وىى إستخدام جياز الميزر. يوجد عدة أنواع من الميزر تستخدم لمعالج بما فى ذلك جياز الميزر أن دى: ياج بالطول الموجي 1064 نانومتر وجياز الدايود ليزر )أشعة الميزر الصمام الثنائي(، وليزر الفيمتو ثانية وأيضا عائمو الياج ليزر قدمت لعالج فطر األظافر ليزر جديد نابض ذو 0.65 ممي ثانية . لقد شممت دراستنا 20 مريضا ثبت بالكشف السريرى و الفحص المعممى لمفطريات انيم يعانون من مرض الفطر الظفرى فى أصابع األيدى أو األقدام وتوقفوا عن أخذ أى عالج أخر لمدة 6 أشير عمى األقل. اليدف من ىذا العمل كان استكشاف وتقييم استخدام الميزر أند:ياج بالطول الموجي 1064 نانومتر، تم إستخدام تدفق في نطاق 50 جول / سم ²، و حجم البقعة قطرىا 3 ممم، و مده النبضو 30 مممي ثانيو وتردد 1ىرتز. تم تطبيق شعاع ليزر لصفيحة الظفر بأكممو من خالل نقل تدريجي لمشعاع بشكل لولبي بدءا بأطراف محيط الظفر وانتياءا في المركز. بعد تعريض صفيحة الظفر بأكمميا لميزر، تم اتخاذ وقفة لمده دقيقتان ثم تكرار العالج مرتين أخريين بنفس الطريقة. العالج تكون من مجموع أربع جمسات مع فاصل اسبوع واحد بين كل جمسة.

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إن عينات الفطريات المعزولو أثناء الدراسو تشمل اإلصابو بفطريات الخمائر تنقسم إلى )الكانديدا( بنسبة 85% )األكثر إنتشارا كان الكانديدا البيضاء(C.albicans) بنسبة 50% ثم كانديدا كروزياى (C.Krusei)بنسبة 20% ثم الكانديدا المداريو (C. tropicalis) بنسبة 10% و أخيرا الكانديدا بارابسيموسس (C. parapsilosis) بنسبة 5%( و فطر الترايكوسبورن (Trichosporon) بنسبة 5% ثم يمييا اإلصابو بالفطريات الغير جمدية )القوالب الجمدية( بنسبة Aspergellus flavus) %5) فطر األسبرجيالس فالفس بينما اإلصابو بالفطريات الجمديو مثمت أيضا 5% متمثمو فى فطر الترايكوفيتون منتاجروفايت (T.mentagrophytes). كان النوع اإلكمينيكى السائد لمفطر الظفرى فى المرضى الخاضعين لمدراسو ىو ظفري تحت الظفر القاصي والجانبي بنسبة 85% ثم يميو فطر األظافر تحت الظفر الداني بنسبة 10% ثم الظفري المصاب بتشوىات كمية بنسبة .%5 فيما يتعمق بمعدالت الشفاء فى الدراسو فى جمسة المتابعو األولى بعد اإلنتياء من جمسات العالج بمدة شير أثبتت النتائج عدم وجود فطريات معمميا عن طريق مزرعة الفطريات والفحص المباشر تحت الميكروسكوب بنسبة 75%، و يعزى ذلك إلى أن كمية الطاقو الموجوده فى الميزر يمكنيا إيقاف نشاط %99-80 من الكائنات العضويو الموجوده فى الظفر ولكن ال يقتل المستعمرات الفطريو كميا عمى الفور بل يتسبب فى إعاقتيم عن التكاثر أو الحياه وفقا أللية الموت الخموى المبرمج لكل خمية. عمى جانب أخر فى جمسة المتابعو الثانيو بعد ثالثة أشير إرتفعت نتائج العالج إلى 90% نسبة شفاء وتم توثيقيا بعدم وجود فطريات معمميا عن طريق مزرعة الفطريات والفحص المباشر تحت الميكروسكوب.

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تم التعرف عمى الفطريات المسببو لنسبة الفشل فى العالج ) 10%( فى العينات المزروعو فى مزرعة الفطريات و ىما فطريات الخمائر )ترايكوسبورن و الكانديدا البيضاء( وكانت األنواع اإلكمينيكيو لمفطر الظفرى عمى التوالى ظفري تحت الظفر القاصي والجانبي، الظفري المصاب بتشوىات كمية. بموجب ىذه النتائج نستنتج أن العالج بجياز الميزر أند:ياج بالطول الموجي 1064 نانومتر يعتبر إلى حد بعيد وسيمة عالج جيدة حيث أنو أمن وفترة العالج قصيرة ونتائجو جيدة خاصة فى الفطريات المقاومو لمعالجات األخرى مثل فطر األسبرجيالس فالفس و ليس لو أعراض جانبية خطيرة.

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تقييم إن دى : ياج ليزر فى عالج فطر األظافر رسالة توطئة للحصول على درجة الماجستير فى األمراض الجلدية والتناسلية و الذكورة مقدم من الطبيبة/ايمان محمد أكمل أحمد بكالسيوض الطب و الجساحت – جاهعت عيي شوط

تحت اشراف األستاذة الدكتورة/ هدى أحمد منيب اظتاذ األهساض الجلديت و التٌاظليت كليت الطب- جاهعت عيي شوط

األستاذ الدكتور/ محمد طه محمود أظتاذ علن الويكسوباث كليت الطب البيطسى- جاهعت الصقاشيق

الدكتورة/ مارى فكرى متى هدزض األهساض الجلديت و التٌاظليت كليت الطب- جاهعت عيي شوط كلية الطب- جامعة عين شمس 2013

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