EFFICACY AND SAFETY OF LULICONAZOLE (1%) CREAM VERSUS

CLOTRIMAZOLE (1%) CREAM IN TINEA INFECTIONS OF SKIN

A COMPARATIVE STUDY.

DISSERTATION SUBMITTED TO

THE TAMILNADU DR.M.G.R MEDICAL UNIVERSITY

IN PARTIAL FULFILLMENT FOR THE AWARD OF THE DEGREE OF

DOCTOR OF MEDICINE

IN

PHARMACOLOGY

DEPARTMENT OF PHARMACOLOGY

TIRUNELVELI MEDICAL COLLEGE

TIRUNELVELI – 11 APRIL 2017 BONAFIDE CERTIFICATE

This is to certify that the dissertation entitled

“EFFICACY AND SAFETY OF LULICONAZOLE (1%) CREAM VERSUS

CLOTRIMAZOLE (1%) CREAM IN TINEA INFECTIONS OF SKIN

- A COMPARATIVE STUDY” submitted by DR.LAKSHMI PRABHA M to the Tamilnadu Dr. M.G.R. Medical University, Chennai, in partial fulfillment of the requirement for the award of the Degree of Doctor of

Medicine in Pharmacology during the academic period 2014 – 2017 is a bonafide research work carried out by her under direct supervision & guidance.

PROFESSOR AND H.O.D. DEAN

Department of Pharmacology, Tirunelveli Medical College,

Tirunelveli Medical College, Tirunelveli.

Tirunelveli. CERTIFICATE

This is to certify that the dissertation entitled

“EFFICACY AND SAFETY OF LULICONAZOLE (1%) CREAM VERSUS

CLOTRIMAZOLE (1%) CREAM IN TINEA INFECTIONS OF SKIN

- A COMPARATIVE STUDY” submitted by DR.LAKSHMI PRABHA M is an original work done by her in the Department of Pharmacology,

Tirunelveli Medical College, Tirunelveli for the award of the Degree of

Doctor of Medicine in Pharmacology during the academic period of

2014-2017.

Place : Tirunelveli GUIDE

Date : Department of Pharmacology

Tirunelveli Medical College

Tirunelveli. DECLARATION

I solemnly declare that the dissertation titled “EFFICACY AND

SAFETY OF LULICONAZOLE (1%) CREAM VERSUS CLOTRIMAZOLE

(1%) CREAM IN TINEA INFECTIONS OF SKIN-A COMPARATIVE

STUDY” is done by me in the Department of Pharmacology, Tirunelveli

Medical College, Tirunelveli.

The dissertation is submitted to The Tamilnadu Dr.M.G.R.Medical

University in partial fulfillment for the award of the degree of Doctor of

Medicine in Pharmacology.

Place: Tirunelveli DR. LAKSHMI PRABHA. M,

Date: Postgraduate student,

M.D Pharmacology,

Department of Pharmacology,

Tirunelveli Medical College,

Tirunelveli-627011.

ACKNOWLEDGEMENT

First of all, I am grateful to the Almighty for the good health and wellbeing that were necessary to complete this research work.

I am greatly indebted to Dr. K. Sithy Athiya Munavarah MD.,

Dean, Tirunelveli Medical College and Dr. S.M. Kannan MD., Vice

Principal, Tirunelveli Medical College, Tirunelveli for their valuable support and generous permission for doing this research work.

I wish to thank with due respect and deep gratitude to Dr. J.Ezhil

Ramya M.D., Associate Professor and Head of the Department of

Pharmacology for her inspiration, guidance and kind help rendered in completing this dissertation.

I would like to express my deepest thanks to my guide

Dr.B.Meenakshi M.D., Associate Professor for giving kind advices, aspiring guidance, motivation and encouragement throughout the research.

I sincerely thank Dr.P. Nirmala Devi , M.D, Professor and HOD,

Department of Dermatology for her support. With deep sense of gratitude, I thank the faculties of the department of dermatology and microbiology for sharing their implicit knowledge, support and encouragement during the course of this research work. I am immensely grateful to Dr. A. Dulcie Celia M.D., Assistant

Professor, Dr.A.Geetha Rani M.D., Assistant Professor, & Dr. M. Shanthi

M.D., Assistant Professor for their support and advice provided during the research work.

I would like to thank the post graduates of Department of

Pharmacology, for their friendly advice and sharing their illuminating views related to this study.

I also express my sincere thanks to my family members for their moral support and encouragement to carry out this research successful as a postgraduate. CONTENTS

S.No. Topics Page No.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 4

3. AIM OF THE STUDY 60

4. METHODOLOGY 61

5. RESULTS 69

6. DISCUSSION 93

7. CONCLUSION 98

8. APPENDIX

I. Informed Consent Form

II. Study Proforma

III. Master Chart

9. BIBILIOGRAPHY INTRODUCTION

Dermatophytosis is a common superficial fungal infection of the stratum corneum, hair and nails which contain keratin (1, 2). The fungi causing dermatophytosis belongs to the genera Trichophyton, Microsporum and

Epidermophyton (3, 4). The specific causative agents of tinea corporis and tinea cruris most commonly are Trichophyton mentagrophytes, Trichophyton rubrum and Microsporum canis (5). The prevalence of dermatophytosis is around

20-25% worldwide, and its incidence continues to rise (6).

Age, sex, host factors, immunological factors, environmental conditions and variety of other epidemiological factors contribute to the development of dermatophyte infections. Direct microscopic examination with potassium hydroxide (KOH) mount and culture of dermatophytes in suitable media are the two most common investigations that can be done to establish the diagnosis of dermatophytosis. Pottasium hydroxide mount aids in the detection of fungal hyphal elements while the culture aids in the identification of species of dermatophytes (7, 8).

The pathogenesis of dermatophytosis is unique because the fungi do not invade the living tissues. Dermatophytes can form colonies only in the keratin containing tissues of the host but the mere presence of dermatophytes or its products elicits an allergic and an inflammatory response in the host .The host response developed vary in type and severity according to the nature of the

1 particular species of dermatophyte causing the infection. The dermatophytes remain as the only fungi to be dependent on human or animal hosts for their survival and dissemination (9).

Topical therapy is sufficient for the treatment of tinea infections however systemic agents are needed when the area of involvement is large or when there is secondary infection and also in immuno-compromised individuals(5). At present, topical azoles and allylamines remain as the treatment of dermatophytosis. The main disadvantage with those agents is long duration of therapy, which results in poor compliance and a high rate of relapse.

Many newer agents are introduced with advantages like once-daily application, short duration of therapy and hence lower relapse rates (10).The traditional imidazole antifungal agent Clotrimazole is effective in the treatment of dermatophytosis with cure rate of 60-100% on applying twice daily for

4 weeks (11). The vast majority of antifungals are applied twice daily, although the newer ones introduced are applied only once daily. At present the research is focused towards shortening the frequency of application of antifungals and the duration of therapy in order to increase the patient compliance, to increase the cure rates and to decrease the relapse rates.

In the past few years, many new extended-spectrum topical antifungals have been introduced into the market. Luliconazole is one among those drugs which offers a good efficacy and tolerability with a short duration of treatment (12).

2 Luliconazole is topical imidazole antifungal agent which inhibits the ergosterol biosynthesis more effectively. It requires only once daily application because the reservoir property in the stratum corneum is greater for luliconazole (13).

The present study was done to compare the efficacy and safety of topical luliconazole versus topical clotrimazole in tinea infections of skin.

3 REVIEW OF LITERATURE

HISTORY:

Dermatophytes were the earliest fungi discovered to cause infection in human beings and this discovery marked the beginning of medical mycology(14).Dermatophytes were found to cause superficial skin infections.Greek Physicians were aware of ring worm infections. The earliest records of dermatophytosis were in 16th century by the Dutch explorers (14).

Robert Remak (1835 )was the first person to identify the fungi isolated from the lesions by observing via the microscope as rods and cones(15).But the mycotic nature of these was described by Schonlein in 1839.The clinical features of dermatophyte infection was first described by David Gruby and he also demonstrated that dermatophytosis is contagious in nature(16).

Raymond Sabouraud, a french dermatologist had classified the dermatophytes into four genera namely Achorion, Epidermophyton, Microsporum and

Trichophyton. He published his work ‘Les Teignes’ in 1910.He was the person who described about the methods to culture dermatophytes and also about the therapy for such infection. Then Emmons eliminated the genus Achorion and modified the taxonomy to lay out the current classification of dermatophytes which includes only three genera (15).

4 NOMENCLATURE:

The word 'Tinea' or 'dermatophytosis' refers to infections caused by dermatophytes. The word “Tinea” is derived from Latin and it means worm or

‘clothes moth’(15).The tinea infections are commonly referred as ringworm lesions due to the distinct ring shaped lesions(17).The tinea infections are usually named in accordance with the anatomic sites involved(18).

CLASSIFICATION:

Ecological classfication:

Ecological classification of dermatophytosis is based on the host preference of these pathogenic fungi. The variation in keratin composition leads to the difference in their host preference (19).

The three types of dermatophytes according to their host preference are as follows:

 Anthrophiles:

The primary host of these fungi is human beings. These fungi might also

affect animals. The primary mode of transmission is from human to

human. Some examples for anthrophiles are Trichophyton rubrum,

Trichophyton concentric, Trichophyton tonsurans, Epidermophyton

floccosum, Microsporum gypseum and Microsporum audouinii (20).

5  Zoophiles:

The primary host of these fungi is animals but can also affect human

beings. The mode of acquisition of infection in human beings is from

infected pet animals. The important examples of this class are

Trichophyton rubrum from rodents and Microsporum canis from cats.

 Geophiles:

These pathogens are mainly present in soil and they affect human beings

very rarely. Those organisms are mainly saprophytic and obtain their

nutrition from keratineceous materials. The important examples of this

group are Trichophyton ajelloi, Epidermophyton stockdaleae and

Microsporum gypseum (20).

Mycological classification:

The dermatophytes are classified into three genera mycologically by

Emmons(21).They are known as Epidermophyton, Microsporum and

Trichophyton. The classification is based on the characters of conidia such as their number, structure, shape and formation(22).

 Epidermophyton:

The prototype of this class is Epidermophyton floccosum. Only two

known species belong to this group and Epidermatophyton floccosum is

the pathogenic organism among them. The morphological features of

this group are presence of abundant, smooth walled, septate and clavate

shaped macroconidia and absence of microconidia(23).

6  Trichophyton:

The prototype is Trichophyton tonsurans. The morphological features

include the presence of abundant number of microconidia than

macroconidia. Microconidia are globose or pyriform shaped born in

hyphae either singly or in clusters. Macroconidia are pencil shaped,

smooth walled and septate in nature.

 Microsporum:

The classical agent of this group is Microsporum audouinii.

Macroconidia are fusiform, septate, rough walled and verrucose by

nature. Microconidia are present either singly or in racemes(23).

EPIDEMIOLOGY:

The dermatophytes vary in their distribution and the distribution is influenced by the geographical area, environment, social, cultural and economic conditions(24). So differences in epidemiological data exist among the various parts of the world. The most common dermatophyte species distributed widely in the world are Trichophyton rubrum and Trichophyton mentagrophytes .The distribution of other species of dermatophytes is confined to particular geographical area or region or country.

S.no Geographical Region Common Dermatophytes

1. Africa T. violaceum,T. soudanense

2. North America T. rubrum and T. tonsurans

3. South America T. rubrum and M. canis

7 4. Australia T. rubrum and T. mentagrophytes var.

interdigitale

5. China T. rubrum and T. mentagrophytes

6. Northern Europe T.rubrum

7. Southern Europe M. canis , E. floccosum

8. India T. rubrum, T. mentagrophytes, T. violaceum ,

M. audouinii

9. Middle east countries T. violaceum, T. tonsurans , M. canis

The wide range of differences in the global distribution may be also due to variations in individual hygiene or may be due to variations in the therapeutic facilities available in each region(25).A small amount of variation in distribution can be attributed to the differences in methodology of diagnosis.

Data regarding epidemiology are not available for some regions as they are unknown or underreported. It is not mandatory to report all the dermatophyte infections in all parts of the world. Rules and regulations differ for each country. Hence the data available may not represent the original prevalence of the disease and the original prevalence values must be much greater than what is reported. The exact prevalence is not known for dermatophytosis and the available data is based on only small isolated study samples(26).

8 FACTORS AFFECTING DISTRIBUTION OF DERMATOPHYTES:

 Age and sex:

There is variation in the incidence of dermatophyte infection

among different age groups and sex and this is due to numerous

factors like difference in clothing, varying immunity with age,

differences in rate of exposure to the infectious agent and also

due to the variation in sebum production according to age, sex

and hormonal factors(7).The incidence of Tinea capitis is high in

children.

 Race and ethnicity:

Prevalence of Tinea capitis is more in people of certain race and

ethnicity. Genetic factor is also suggested to have an etiological

role in the form of autosomal recessive inheritance.

 Occupation:

Tinea infections are found to be more prevalent in athletes and

persons working under sun for a very long period of time.

 Climate:

It is more prevalent in tropical and sub tropical countries because

hot and humid climate favors the occurrence of dermatophytes.

The presence of moisture favors the germination of the

arthrospores which are responsible for the infection.

9  Temperature and microenvironment:

Dermatophytes fail to grow when the temperature exceeds 37

degree celcius. This property retains the infecting organism in the

epidermis. Increase in carbon dioxide tension will help in

adherence and penetration of organisms.

 Immunodeficient states:

Dermatophytosis is more common among the diabetic patients,

immuno compromised patients with HIV infection, malnutrition

and patients taking steroids or other immunosuppressants due to

lack of resistance to infection and deficient cellular immunity(27).

 Skin disease:

Persons suffering from ichthyosis and collagen vascular disease

are more likely to develop chronic dermatophytosis(28).

TRANSMISSION OF DERMATOPHYTOSIS:

The most distinguishable nature of dermatophytosis is its communicability. The mode of transmission of fungi may be either via direct or indirect contact with affected animals or human beings. The spread of infection is via the arthrospores produced by dermatophytes. The spores of dermatophytes may survive for upto 12 to 20 months outside the host cell(28). The risk of acquiring infection increases with exposure to fungi. Fomites play a vital role in transmission of fungus but transmission also depends largely on host factors such as injury to skin, presence of moisture or wearing occlusive and tight clothes. The major modes of transmission from man to man are via combs,

10 towels, bedding, toilet seats, pillow covers and exchange of clothing from infected person(15).

PATHOGENESIS:

The dermatophytes cause infection by the process of colonization of skin, hairs and nails. The colonies thus produced by the dermatophytes are well equipped of spreading the infection through production of conidia and spores(20). The most important defense mechanism of the host against these infectious agents is the renewal process of stratum corneum which is done by keratinocytes. This leads to shedding of epithelium to the possible removal of the fungus (29).The starting point of the primary infection is by a small breach in the continuity of the skin. This breach is brought about by secretion of an enzyme keratinase that breaks down keratin(30,31,32). The keratinase enzyme is a primary virulent factor in the pathogenesis of dermatophytosis(33). The pathogenesis is dependent on the involvement of various virulence factors secreted by the dermatophytes or the invading organisms. The three important processes involved in the pathogenesis of superficial dermatophytosis infection are as follows

 Adherence

 Invasion

 Development of host response

Incubation Period:

The incubation period is around 1 to 2 weeks in human beings(34).

11 Adherence:

The first and foremost step in the pathogenesis is the adherence of the fungi to keratinized cells in the stratum corneum of human beings. This occurs either by direct contact with the fungal elements from infected animals or humans or it may occur by indirect contact through the contaminated fomites. The infective fungal elements are found to be the arthroconidia or the arthrospores which adhere to surface of the skin and germinate further. The number of spores adhering to surface of the host is time dependent.

The factors mediating adherence:

o Galactose and mannose on skin surface functioning as adhesins.

o Proteases (eg:Subtilisin) secreted by the dermatophytes.

Invasion:

The next step after adherence is the invasion. Following adherence the spores germinate and then penetration occurs. Penetration involves the degradation of keratin. The degradation of keratin depends on virulence factors secreted by the dermatophytes. The virulence factors may be enzymatic or non enzymatic in nature.

The virulence factors involved in invasion are as follows:

 Virulence enzymes:

They are secreted by the dermatophytes and include the protease, lipase

and cellulase. Their function is to extract the nutrients needed for the

survival from the host. They can also elicit inflammatory response by

acting as antigens. Among the enzymatic virulence factors proteases

12 have extensively studied. Proteases act by reducing the disulfide bridges

of the keratin which is responsible for the integrity of keratin and thus

exhibit keratinolytic action eliciting an immune response.

 Non enzymatic virulence factors:

The two important virulence factors that are non enzymes are the

xanthomegnin and melanin or melanin like compounds(35).

DEVELOPMENT OF HOST RESPONSE:

After invasion the host develops an innate immune response to the infection.

The cell wall components such as chitin, glycopeptides or the keratinases secreted or the carbohydrates may act as the antigenic stimulus resulting in development of immune response. The response developed to each antigen is different(29). The keratinases secreted from dermatophytes can influence the immune defenses(36). The infection can result in either type 1 or type 4 hypersensitivity reactions. Acute dermatophytosis is due to delayed type or type 4 hypersensitivity reaction whereas the reason for chronic dermatophytosis is immediate or type 1 reaction(37).The main response is Th1 mediated and it results in production of inflammatory cytokines to stop the spread of infection(38). But in chronically infected persons there may be immunosuppression instead of stimulation of immune response.

13 A figure to illustrate the pathogenesis of dermatophytosis infection :

Arthrospores/conidia

Adherence to host(Keratin of skin, hair and nails)

Spore germination

Invasion (entry into Stratum corneum)

Secretion of Keratinases

Host Immune response

Centripetal invasion(results in classical ring shaped lesions)

CLINICAL SYMPTOMS:

The presenting clinical symptoms and signs vary depending on the site of infection, species involved and also upon the immune status of the individual.

Several species can cause a similar single clinical manifestation. A single species can also result in many clinical manifestations depending on the anatomic location(27).

14 The varying clinical pictures of dermatophytosis are discussed below:

Tinea capitis:

Tinea capitis is the dermatophyte infection of the scalp. It is often marked by the development of erythematous papule around the hair follicle which later develops into centrifugal patch involving the entire hair shaft and hair follicles in the infected region. This patch is also seen with raised borders studded with vesicles all around. The hair in the affected area of scalp becomes thin and lustreless. Clinical diagnosis of tinea capitis is made based on the presence of scaly, erythematous patches of alopecia which is itchy in nature or the presence of boggy kerion like lesion(27).

Tinea barbae:

It is the superficial fungal infection involving the beard area of the face and the neck. It is otherwise known as Barber's itch or Tinea sycosis(39).Tinea barbae is very rare to occur(40).The hair follicles in the infected region may be involved and the affected hair can be easily epilated. The clinical symptoms may vary from itching in superficial infections to tenderness or pain in case of deeper structures involvement. The pustules may extend and affect the underlying deep sinuses in the area giving rise to tenderness. The variance clinical presentation depends solely on the etiological agent(27). Zoophilic dermatophytes cause an inflammatory lesion where as anthrophilic dermatophytes cause mild non inflammatory lesions(40).

15 Tinea faciei:

Tinea faciei is the superficial fungal infection of the face. It occurs commonly in female and children. Tinea faciei presents as erythematous hives with minute patches and elevated borders. They are itchy in nature and commonly caused by T.tonsurans(41).

Tinea favosa:

Tinea favosa is the chronic dermatophyte infection of the scalp characterized by yellow crusts and scutulae. On removing the crusts there is erythematous base. The lesion often begins as a small reddish yellow puncta which spreads later. The characteristic mousy odor is specific to this lesion.

Tinea corporis:

Tinea corporis is the dermatophyte infection of the glabrous skin of the trunk.

Lesions vary in size, shape and clinical symptoms. An erythematous or hyper pigmented ring shaped lesion with papules in the border and central clearance is the most common presentation. The lesion may present as a simple scaly patch to deep granulomatous patch. The most common presenting complaint is the itching of the lesion. Sometimes secondary infection occurs due to super invasion by bacteria characterized by pustules.

16 Tinea imbricata:

It is the chronic dermatophyte infection of the skin caused by one particular species of dermatophyte. The etiological agent is the Trichophyton concentricum. T.concentricum does not invade the hair and nails. The classical presentation is the concentrically arranged annular ring shaped patches with non healing centres. The central area is raised and separated from the periphery by well developed fissures. The lesion is characterized by intense itching.

Tinea cruris:

The other name for Tinea cruris is jock itch. Tinea cruris is the dermatophyte infection of the groin and the perineal region. The lesion in the groin is erythematous with sharply demarcated raised borders studded with papulo vesicles. Secondary infection may occur due to scratching. Extensive scratching may also lead to maceration. The infected person has severe itching.

The lesions are scaly in nature. Lichenification and hyper pigmentation are the characteristic features of chronic dermatophytosis in the groin(27).

Tinea pedis:

Tinea pedis involves the foot region. The lesion typically begins in web spaces and then extends to the plantar and dorsal surface of the digits. There may be a foul smelling odor. Hyperkeratosis is a characteristic feature of tinea pedis. The clinical presentation of tinea pedis vary from scaly patches or vesicles to hyperkeratotic plaques or fissures.

17 Tinea mannum:

It is the fungal infection of the skin of the hand. The most distinct nature of tinea manuum is the diffuse hyperkeratosis of the palms. It may affect one or both the hands. The typical feature is the prominence of the creases in the palms. The infection may involve the dorsum of the hand(27).

Tinea unguium:

It is the dermatophyte infection of the nail. It is characterized by discoloration and hypertrophy of the nails and nail bed. The nails become dull, lustreless and brittle(27).

DIAGNOSTIC METHODS FOR DERMATOPHYTOSIS:

The history and the clinical presentation provide the basis for the diagnosis of dermatophytes infections. The clinical diagnosis is confirmed by mycological assessment. When a patient presents with a characteristic clinical lesion which appears ring shaped spreading centrifugally associated with erythema and presence of well defined scaly red borders with asymmetrical distribution it is very likely to be a dermatophyte infection. But the diagnosis is uncertain when there is secondary infection or in case of atypical presentation or in already treated cases. It is highly advisable to perform fungal examination before initiating therapy to identify the infecting dermatophyte as well to assess response to therapy. The conventional methods which remain as the main procedures in lab to diagnose dermatophyte infection are the direct microscopic examination and culture of the dermatophytes.

18 Specimen collection:

 Hair

In patients suspected to have dermatophytosis, the specimen of hair should be collected carefully keeping the roots of hairs intact. Removing hair without root or cutting hair is not a suitable method for collection of sample. The hairs which are affected are easy to recognize as they appear dull, lustreless, broken and can be easily epilated with the help of fine scissors. This may aid in collecting the correct sample for examination. It is very suitable method for diagnosing dermatophyte infection of scalp and beard. For the diagnosis of kerions in which the etiological agent is Trichophyton verrucosum, examination of multiple hairs is essential to find out the infected hair because here a severe form of disease is caused by a small amount of dermatophytes.

Scraping of the scalp would yield the suitable material in certain cases where the hairs are broken and appear very short. Here the affected hair roots will present as small stumps in the scales collected. Vellus hair examination is suitable for diagnosing dermatophytosis treated with steroids or for finding out tinea incognito. Trichophyton rubrum can affect the shaft of the hair or it may invade the follicle of the hair. The samples collected by brush provide suitable material for the culture of dermatophytes in case of infection of the scalp.

But direct microscopy cannot be performed on the hair samples collected. For the collection of brush samples, a clean and sterile scalp massager made of plastic or a toothbrush which is disposable can be used. Brush the hair firmly

19 with the massager or the brush for a minimum of ten times and then is firmly pressed in the culture medium for dermatophytes kept in a Petri dish. It is a simple, cost effective and sensitive technique which can be used easily for screening a large sample of children. Large scale screening of children to diagnose Tinea capitis is effectively done using Wood's light microscopy and brush sample culture. Cultures obtained from affected children will produce fungal colonies from multiple inoculation points whereas the cultures from the contacts of the affected children will produce only a small number of colonies. When the sampling of hair is painful or difficult to obtain a swab can be used to gently wipe over the lesion of the scalp and this method is excellent to collect enough number of conidia to yield a positive culture(7).

 Skin:

Solid type disposable scalpel blades should be kept vertically to the surface of the skin to collect the scrapings. Or else blunt scalpels which are banana shaped are sterilized by heat can be used to collect the scrapings. If the patient has applied any medication or powder cleansing the area of the lesion is essential.

The scraping should always be obtained from the active border if it has a definitive edge and the scales should be obtained. When there is very little scaling or there is no active edge scraping can be just generalized without confining to the edges. Often it is very hard to get enough or sufficient scraping material for fungal culture in tinea infections. To rectify this issue transport swabs can be used which provide a good alternative or back up support(7).

20 If there is presence of blisters or vesicles the roof of the lesions are cut with fine scissors and the fluid can be sent for direct microscopy and for culture.

The skin scrapings are collected and usually transported to lab in thin sheet of folded paper. This will help the specimen to be free from overgrowth of any bacteria by keeping it dry. Plastic boxes are not suitable for transport, because there is adherence of the skin to the sides of the container which is hard to remove. Glass slides are frequently used as other means of transport but have the disadvantage of being easily broken. Bright colored paper square cards folded and fastened by the paper clip is an ideal transport equipment.

Recording of the name, age and other details of the patient along with the exact anatomic location of the lesion and date of collection of specimen is absolutely essential. The samples can also be collected in specifically designed collection packs available commercially for hair, skin and nails. Dermatophytes remain viable for weeks in the collected specimen samples.

 Nail:

Onchyomycosis is very difficult to diagnose as it is difficult to isolate the causative dermatophyte from the sample obtained from nail for culture than from samples of skin and hair. In case of infections of nail the sample material is mostly obtained from the distal part in spite of the fact that the infection of the nail spreads proximally. The hyphae obtained from the distal end of the nail are often not viable. But it is not possible to obtain samples from the proximal or advancing edge of the infection as it is located deep and will cause pain to the patient. The sample obtained must enclose the entire thickness of the nail

21 because the infections mostly have hyponychium as the starting point. A flat ended dental probe is used for scraping the debris present underneath the nail which is an excellent source of sample for diagnosis of dermatophytosis.

To diagnose the proximal subungual nail infection or white onychomycosis, it is not essential to collect the full thickness nail sample. Instead the superficial scrapings may provide the necessary sample for diagnosis. A sample collected from associated skin lesions is essential in case of onychomycosis as it is hard to isolate the fungal pathogen from the nail sample. And while sending the samples collected to the laboratory it is mandatory to mention the site of collection whether is it a finger nail or a nail from toe because certain group of dermatophytes are commonly prevalent in one site than the other site(7).

Wood's lamp:

It is based on the principle of fluorescence by UV light. The dermatophyte affected hair can be recognized with the help of the specific fluorescence in ultraviolet light that is filtered by Wood's lamp. Wood’s glass is made up of barium silicate which has nine percent nickel oxide. This glass can transmit light rays of wavelength greater than 365nm. Any source of ultraviolet rays of light is suitable for the transmission. The dermatophytes producing the tryptophan metabolites is found to be responsible for the fluorescence obtained from infected hair(42).The fluorescence of the infected hair is not affected by the viability of the dermatophytes. The sample continues to remain fluorescent even if the dermatophyte becomes unviable.

22 The pH of the solution will determine the color emitted by the fluorescent hair(43). The dermatophytes grown in culture or in vitro do not yield fluorescence. Hence, the fluorescence is attributed to some substance that is produced as a result of interaction between the causative fungal pathogen and the hair probably pteridine. There are exceptions. All the dermatophytes which invade the hair do not produce fluorescence. Only dermatophytes belonging to certain genus and family invading the hair can produce fluorescence. The most common example is fungi belonging to genus Microsporum.

Example : Microsporum canis and Microsporum audounii. The hairs invaded by genus Microsporum yield a brilliant fluorescence of green color identified in a dark room. The fluorescence develops only when the shaft of the hair is entirely infected by the pathogen where as in case of early infections the fluorescent part would not appeared out from the follicle and is made out only after epilation.

The species which always produce fluorescence are Microsporum canis var distortum and Microsporum ferrugineum while Microsporum gypseum and

Microsporum nanum induce fluorescence only occasionally. A pale green fluorescence is produced by species of Trichophyton schoenleinii. The infections caused by microsporum species produce short, thin, broken hair shafts and stumps in contrast to the cases of favus, where the fluorescent hairs remain long. Wood's lamp is an absolutely needed equipment to diagnose

Microsporum infections in areas of its prevalence to effectively aid in control of the dermatophyte epidemics in those areas.

23 It can be very easily transported to the schools, colleges and other places for rapid detection of dermatophytes among the contacts. The Wood's lamp is not free from disadvantages in spite of these benefits. Occurrence of errors in diagnosing is the commonest problem encountered. Errors might appear due to various causes.

The common mistakes committed in diagnosing through Wood's lamp are as follows:

 Petrolatum present in ointments can produce blue fluorescence.

 Darkness of the room may be inadequate.

 White coat worn by the investigator may reflect light.

 All dermatophytes do not emit fluorescence.

The results of the Wood's light examination are specific only when the procedure is performed correctly and interpreted exactly. Wood's lamp is also helpful in diagnosing subclinical state of infection and in assessing the rate of response to the treatment provided or the cure rate.

 Other uses of Wood's lamp:

1. To diagnose Pityriasis versicolor where yellow fluorescence is produced.

2. It is also useful to diagnose Malassezia species in severe dandruff or cradle cap occurring in infants where a yellow fluorescence is emitted(7).

Direct microscopy:

Examination of the collected specimen under direct microscope is the most effective way of diagnosing a fungal infection. It is a screening procedure to identify the causative agent of dermatophytosis. Direct microscopy can identify

24 the genus of the etiological fungi. The method chosen for microscopy depends on the type of specimen and the resources available in the laboratory. The usual specimens collected for examination of dermatophytes include skin scrapings, hair, or clippings or scrapings of the nail.

The various procedures under direct microscopy include using Potassium hydroxide (10-20% solution), calcofluor white or Periodic acid -Schiff stain is used. The sensitivity of the direct microscopy in finding out fungal infection can be increased by the use of fluorescent microscope or fluorescent dyes(15).

 KOH mount:

KOH mount is the most useful and least expensive diagnostic method in the field of mycology. Potassium hydroxide is the most commonly used preparation in direct microscopy to visualize the dermatophytes. A drop of

10 -20% KOH is put on a slide and to this drop a small amount of the collected specimen is added. After this step a cover slip is placed over the preparation and heated gently. The potassium hydroxide will digest the proteinaceous material , disrupts the cellular sheets of keratin making the dermatophyte visible. The dermatophyte is visible as a hyaline, septate, branched or unbranched hyphae with arthroconidia. In specimens of hair, the conidia may appear on the outside referred to as ectothrix invasion or on the inside referred to as endothrix invasion or may co occur with bubbles and tunnels known as favic invasion(15).

25  Modifications of Potassium hydroxide mount:

Several modifications are done in basic 10% KOH preparation to aid in rapid detection of fungal elements. They are discussed below

1. Addition of Parker superchrome blue black ink to KOH and this helps in selective staining of the dermatophytes.

2. Addition of 36% DMSO (Dimethyl sulfoxide) to KOH preparation to visualize the fungal elements without heating.

3. Addition of 5-10% glycerin in nail preparations of KOH to avoid and prevent dehydration and crystallization of KOH(23).

The fungal hyphal elements should be differentiated from the mosaic fungus.

The artifacts like fibers, cotton wool that may resemble the hyphae are referred as the mosaic fungus. The cholesterol artifacts are the ones which are difficult to distinguish but can be identified by regular outline, abrupt changes in width and the absence of internal organelles(15).

 Calcofluor white preparation:

It is a whitening agent commonly used in paper and textile industry. It avidly binds to fungal cell wall elements such as chitin and cellulose and fluoresces on excitation either by UV rays or visible rays. The advantage is that the dermatophyte detection is easy with low technical expertise and hence saves time. But this technique requires a fluorescent microscope with a good UV ray source and proper filter. Hence it is too expensive which is a greater

26 disadvantage. Background elements may also fluoresce but the fungus appear brighter and easily recognizable(15).

 Periodic Acid Schiff Stain(PAS):

Fungal elements on combining with the PAS stain gives a red violet fuschin color.

 Advantages of direct microscopy:

1. Easy, simple, cost effective and readily available technique

2. The fungal spores or hyphae can be visualized clearly

3. The arrangement of spores, their location whether endothrix or ectothrix can be made out easily.

 Disadvantages:

1. Direct microscopy can just detect the presence of dermatophyte spores but cannot give any information regarding the viability of the spores.

2. There is no possibility to rule out false negative results.

3. Any topical antifungal used has to be stopped one week prior to examination. Or else the results may be falsely negative.

Culture methods:

Culture of dermatophytes is essential only if the diagnosis is not certain or if the lesion is not responding to treatment .Simple cases with clear-cut clinical

27 features do not require culture. Culture is usually done to identify the species of dermatophyte(34).

 Growth media for dermatophytes :

The commonly used culture media for the growth of dermatophytic fungi is

Sabouraud's dextrose agar medium(SDA). It has cyclohexamide and chloramphenicol added as supplement for the isolation of dermatophytes. The other media used for the isolation of dermatophytes include Potato dextrose agar medium, Corn meal agar medium and the dermatophyte test medium.

. Sabouraud's dextrose agar medium:

This is the most standard medium used for the growth of dermatophytic fungi.

The purpose of addition of chloramphenicol (0.05%) as a supplement is for inhibition of the growth of bacteria while the purpose of cyclohexamide is for the inhibition of growth of saprophytic fungi like candida and aspergillus.

. Dermatophyte test medium :

It is a selective medium for isolation of dermatophytes and it has phenol red as a pH indicator. When there is growth of dermatophytes in the medium the phenol red changes the color of the medium from yellow to red. The color change is mainly due to the production of alkaline metabolic substances by the dermatophytes. Hence DTM is used as screening medium for the dermatophyte isolation and not as a diagnostic medium. Apart from phenol red DTM has many supplements like gentamycin, tetracycline and also cyclohexamide solely

28 for the purpose of inhibition of the growth of most bacteria and saprophytic fungi.

. Corn meal agar medium:

It is mainly used for the formation of spores by suppressing the vegetative growth of the fungi. Addition of dextrose along with this medium is used for the differentiation of T.rubrum from T. mentagrophytes based on the differential pigmentation formed.

. Potato dextrose agar medium:

It is a medium which can be used for the culture of dermatophytic fungi on slides. It causes sporulation of dermatophytic fungi.

 Morphology:

The morphological features of different species of dermatophytes differ on the

Sabouraud's dextrose agar medium .The uniqueness of each dermatophyte species can be established by their differential growth characteristics on SDA as well as on some other tests described below.

. Rate of growth:

Usually most of the dermatophytes grow in seven to ten days but the duration of growth is still longer for species like T.verrucosum and T.tonsurans.

29 . Colony characters:

The color of the colonies formed the texture of the surface of colonies, their topography and the pigmentation produced helps to differentiate different species of dermatophytes.

 Nutritional requirements:

Nicotinic acid is required for culture of T. equinum, histidine is

essential for the growth of T. megninii, while thiamine is needed for the

colonies of T. violaceum and T. tonsurans. Thus nutritional

requirements aid in identification of dermatophyte species.

 Temperature :

The optimum temperature for the growth of dermatophytes is from

25°C to 35°C but some exceptional species can survive in extremes of

temperature. For example, the commonest dermatophyte T. rubrum

grows on albumin agar at the optimum temperature mentioned above

within seven days and produces red pigment while T.mentagrophytes

cannot do so.

 Tests for differentiation:

. Urease Test :

Differentiation between the species of T. rubrum and T. mentagrophyte

can be made out easily with the help of urease test. The agents used in

this test are Christensen's urea agar or broth. The test yields positive

result for T. mentagrophyte as it a urease producing strain whereas it is

negative for T. rubrum.

30 . Hair perforation Test:

The hair perforation test is basically used in differentiating the atypical

strains of T.mentagrophyte from T. rubrum. It is also used to distinguish

M. equinum and M. canis. Hair perforation is positive for species of T.

mentagrophyte and also M. canis and is negative for strains of T.

rubrum as well as for M. equinum .

. Rice Grain Test

The rice grain test can easily distinguish the features of M. audouinii

from other species of Microsporum. The rice grain is polished and then

sterilized with the help of hot air oven before inoculating it with the

test fungi .Then it is incubated at 28°C for seven to ten days. All

microsporum species yield sufficient growth except M. audouinii which

grows very poorly.

A sample should be cultured for three weeks at the least, though most of the positive cultures can be made out after 1-2 weeks(44). Negative culture does not rule out dermatophytosis(34). The level of sensitivity for culture is very low.

Culture may be repeated again after a negative culture if clinically fungal infection is suspicious prior to initiation of treatment(44).

Causes for negative culture(45):

 The initial clinical diagnosis is wrong

 Sampling errors occurring due to improper collection technique

 Sampling errors occurring due to collection of inadequate specimen

31  Due to overgrowth of the contaminant saprophytic fungi

 When the patient has taken anti-fungal drugs before the collection of the

sample

 While there is a delay for the specimen collected to reach the laboratory

 Mistakes of lab procedures

 Slow growing dermatophytes or organism

ANTI - FUNGAL SUSCEPTIBILITY TESTING:

 Microdilution method:

Broth micro dilution assay is used for antifungal susceptibility testing of dermatophytes. It is a modified version of the standard method of Clinical and

Laboratory Standards Institute. A standard inoculum is created by the count of the number of microconidia. The cultures are then grown in SDA slants for a period of seven days at 35°C. 85% normal saline is then added to the SDA agar slant. Then the suspension is slowly transferred to a centrifuge tube. The suspension thus made is subjected to count on a hemocytometer and then diluted to the desired concentration. Then the micro dilution plates are set and incubated at 35°C according to the reference method. Finally the result is read out after four days. MIC or minimum inhibitory concentration is made out. It is the concentration in which 80% of the growth of the fungus is inhibited (46).

 Determination of MFC:

MFC is determined by removing the assay wells with no growth and streaking them on the SDA medium plates. Then the plates are to be incubated for 7 days

32 again at 30°C .MFC is being defined as the lowest concentration of the drug at which there is no visible growth of fungus.

Histopathology:

Histology is very useful in the diagnosis of a variant of Tinea called Majocchi’s granuloma ,a condition in which the potassium hydroxide mount does not show the presence of fungal hyphal elements. The fungal hyphae can be easily made out by H & E staining of stratum corneum. Some other special stains which are useful for visualizing hyphal elements are PAS and Gomori methanamine silver(46).

MOLECULAR DIAGNOSIS:

 Polymerase chain reaction:

The PCR tests results in the rapid and early diagnosis of dermatophyte infection as well as aids in the determination of drug resistance(47).They are of 2 types as follows

 Uniplex PCR :

It is done for direct detection of dermatophytes in clinical sample. The

PCR ELISA assay is 80.1% sensitive and 80.6% specific when compared with culture methods which yield low sensitivity.

 Multiplex PCR:

This is the most common method available commercially available. It can result in simultaneous detection of twenty one dermatomycotic pathogens

And subsequent detection of DNA sequence with help of agarose gel electrophoresis(46).

33 NEWER MOLECULAR METHODS:

 Matrix assisted laser desorption by mass spectrometry:

This test is based on the principle of detection of biochemical characters, degradation products resulting from proteolysis which occurs as a result of fungal infections or other noninfectious diseases. This is shown by proteolytic degradation of native proteins. The pattern of peptides in infected samples is identified and compared with the known peptide sequences obtained from other skin disorders which are stored in a database. It is a time saving procedure because it enables simultaneous identification of around sixty four dermatophyte species and the results are available within 24 hours(48).

 Reflectance confocal microscopy:

It is an in vivo imaging technique of the epidermis and also the superficial dermis with resolution at cellular level which is useful for detection cutaneous fungal infections and parasitic infestations(49).The branches of fungal hyphal elements are detected over a red circular scaly patch. The advantage of this test is that it is noninvasive with a sensitivity of 100% and also it is a safe method for diagnosing the cutaneous dermatophytic infections clinically should be supplemented with a mycologic confirmation. It is a method which is easy to use and detect involvement of vellus hairs and hence helps in the decision of treatment option.

Fungal culture and antifungal susceptibility testing are expensive methods of diagnosis and more specialized and most centers does not have the methods readily available.

34 Molecular methods like PCR and others like reflectance confocal microscopy are primarily used for research at present(46).

TREATMENT OF DERMATOPHYTOSIS:

General principles:

. Mycological confirmation is necessary before prescription of any

therapy in case of dermatophytosis.

. The spontaneous healing is very uncommon in tinea infections. So

initiation of therapy is very important for the cure.

. Several antifungals are approved for use in dermatophytosis. But for

effective therapy selection of most appropriate drug for dermatophytosis

is essential.

. To make sure the efficient working of topical antifungals work, these

drugs should be able to the horny layer of the skin.

. Topical agents must also be non-irritant as well as tolerable on applying

in the skin.

. Skin lesions on trunk require therapy for 2 to 3 weeks.

. Cases of tinea pedis and capitis should require therapy for 4 to 6 weeks

. Tinea infection of nails known as onychomycosis are to be treated with

oral antifungal agents for 3-6 months as it does not respond to the

topical agents.

35 The treatment of dermatophytosis should be based on various factors in selecting an antifungal or treatment schedule as follows:

 Patient factors:

Individual preference

Risk /benefit ratio

 Drug factors:

Pharmacokinetics

Efficacy

Duration of treatment

Cost of the drug

Levels attained in the horny layer of the skin

 Disease factors:

Percentage of recurrence in long term

Species of dermatophytes causing tinea

ORAL ANTIFUNGALS IN DERMATOPHYTOSIS TREATMENT:

Class Drug

1.Imidazoles Ketoconazole

2.Triazoles Fluconazole,Itraconazole,Voriconazole

3.Allylamines Terbinafine

4.Heterocyclic Benzofuran Griseofulvin

36 Indications for use of systemic antifungal agents in

dermatophytosis

 Extensive tinea corporis.

 Simultaneous occurrence of tinea in more than one part( E.g. Tinea

corporis and Tinea cruris, Tinea pedis and Tinea Cruris)

 Extensive Tinea pedis with involvement of the entire

 Sole of the feet or the heel or dorsum of the foot

 Recurrent tinea infections

 Tinea unguium

 Tinea capitis

INDIVIDUAL DRUGS:

 Griseofulvin:

It is the standard oral anti fungal drug used in the treatment of dermatophytosis at present except the case of Tinea unguium. Its antifungal property was observed by Gentles in 1960s(50). The source of this griseofulvin is Penicillium sp., and it successfully eradicates the dermatophyte infections. It is commonly used in both children and adults. The safety profile of griseofulvin is excellent. It has exclusive action against dermatophytes and acts by interfering with DNA synthesis.

Griseofulvin has poor gastrointestinal tract absorption. The rate of absorption is influenced by size of the drug particle, amount of fat in the diet and also on the rate of dissolution of different preparations. Formulations which are micronized or ultra micronized are absorbed better(51).Bioavailability

37 of griseofulvin ranges from 25% to 70%.The highest concentration of the drug is obtained in outer layer of stratum corneum. Unbound drug is carried by ECF in sweat and is excreted via transdermal fluid loss(52).On reaching the stratum corneum, there is reversible protein binding of the drug and lipid solubility results in concentration of griseofulvin in the horny layer of the skin. When the administration of the drug is discontinued the drug concentrated is cleared rapidly. The elimination of griseofulvin occurs as metabolites both in the urine and faeces and hence it requires a daily dosing. As griseofulvin is cleared rapidly from the site of infection once the patient stops taking the drug, the drug must be continuously taken till the occurrence an apparent clinical cure (52)

The usual dosage is 10 mg/kg/day in tinea corporis / tinea cruris, but higher dose of 20 to 30 mg/kg/day is commonly required in cases of tinea capitis. The occurrence of is uncommon clinically.

Adverse effects are also not common when griseofulvin is taken with meals. The most common side effect of griseofulvin is headache but it disappears usually when the drug is stopped. The other side effects which occur rarely involve the skin, nervous, genitourinary, the gastrointestinal and the musculoskeletal systems.

Azole derivatives:

Azoles are classified into imidazoles (have 2 nitrogen atoms in the azole ring) and triazoles (have 3 nitrogen atoms).Azole derivatives are fungistatic but exerts fungicidal effects in higher concentrations. All azoles have similar mechanism of action and the action is executed by inhibition the demethylation

38 of carbon-14 of sterol which is a component of fungal cell wall. So there occurs the inhibition of synthesis of normal ergosterol which leads to arrest growth and replication of fungi. Triazoles like fluconazole, itraconazole, voriconazole,posaconazole and ravuconazole are used in systemic treatment of fungal infections.

 Ketaconazole:

Ketoconazole was introduced in 1980 as an antifungal agent. It is an azole derivative. When the drug was introduced into the market it was considered as an important advance in the therapy of mycosis but now it is not so. It is mainly because of the fact that ketaconazole therapy is significantly associated with the incidence of idiosyncratic reaction which resulted in hepatic toxicity(53).The high incidence of 1in 3000 patients is the cause for concern .

 Itraconazole:

Itraconazole is a triazole derivative. It is poorly water soluble. The bioavailability of the drug is better when it is given with a fatty meal(54). The oral absorption of itraconazole is dose dependent. Itraconazole is highly protein bound (>99.8%).The binding is mainly with albumin. The drug is highly lipophilic .So it achieves higher concentrations in fat/ omentum/skin

/nails and vaginal/cervical tissues(54).

The accumulation of itraconazole occurs slowly in skin.

But the concentration achieved in skin persists for one month even after the discontinuation of the drug. The concentration obtained in skin is 3 to 10 times higher than the concentration in plasma(55).

39 The terminal elimination half life of itraconazole is 20 to

60 hours and hence it takes around 2 weeks of continuous therapy to achieve steady state concentration. Around 65% of itraconazole is excreted in faeces and remaining 35% is excreted in urine after metabolism. No dose adjustment is needed in hepatic and renal dysfunction.

Itraconazole appears to be a safe drug(54), as the frequency of adverse effects of the drug depends solely on the length of therapy. The incidence of side effects is approximately around 7% to 12%(54). The most common side effects are nausea, vomiting and headache. The occurrence of liver function abnormalities is less common involving less than 1% of patients.

The drug is available both in liquid and capsule formulations. The liquid solution has cyclodextrin and this is the cause of concern in its use. This concern is due to the fact that cyclodextrin is carcinogenic and animal studies have proved the occurrence of pancreatic adenocarcinomas in rats when used at doses used in human beings. The advantages of cyclodextrin containing formulation is that it increases the absorption of itraconazole to a greater extent, can be taken while fasting and also there is no significant drug interaction with H2 blockers and proton pump blockers(56). Above all it can also be used in therapy of mucosal candidiasis topically .

The disadvantage of the liquid formulation is that it is unpalatable and unpleasant to taste. Currently research work is on process in

40 several countries to convert the liquid formulation in parenteral form and its use on deep mycosis.

 Fluconazole:

Fluconazole is again a triazole derivative like itraconazole. It is easily soluble in water. The absorption is also very good orally(52). It has high bioavailability and it is not affected by concurrent food intake, antacid administration or pH. The protein binding of the drug is very minimal; hence this property precludes the drug-drug interactions.

Fluconazole has a wide distribution in the body tissues as well as in cerebrospinal fluid. The half-life of fluconazole is long and ranges from 22-30 hours in adults. Hence it takes 6 - 10 days to attain the steady state concentration after the initiation of therapy. The route of excretion of the drug is via kidneys in urine and the drug is excreted unchanged as it does not undergo any hepatic metabolism.

Elimination from the skin occurs more slowly .And so the clinical cure could be obtained even after the therapy is withdrawn. Dose adjustment is needed in patients with renal dysfunction. The incidence of side effects is approximately 16%(52).The most common adverse effects are nausea, vomiting and elevations in level of liver function tests.

 Voriconazole:

Voriconazole is a triazole derivative. It is a third generation azole recently approved by Father broad spectrum of activity of voriconazole renders it active against yeasts (including species such as Candida

41 glabrata, C. krusei and Candida lusitaniae that are resistant to fluconazole usually).Infections caused by the fungi like Aspergillus , Cryptococcus neoformans and other emerging organisms including species of Fusarium.,

Acremonium., Scedosporium ., Trichosporon and S. apiospermum also respond well to therapy with voriconazole(57,58).

Pharmacokinetics show that the bioavailability of voriconazole is up to 90% after oral administration. Absorption is less when taken with food(57).The drug also is widely distributed throughout the body. It is

65% plasma protein bound in humans. Around 78% to 88% of the drug is metabolized and then excreted in urine while only less than 5% of the drug is not metabolized remains unchanged. From a dermatological point of view it is interesting to note that voriconazole is active in vitro against dermatophytes and Malassezia species.

Voriconazole is active against dermatophytes.

Currently, voriconazole is available both in oral formulation as well as in an intravenous formulation with cyclodextrin. Drug interactions are seen when

Voriconazole is administered to patients on cyclosporine and warfarin. Enzyme inducers such as rifampicin, rifabutin and phenytoin when administered concurrently with voriconazole will reduce the efficacy of the drug(57,58).

 Terbinafine :

It is a fungicidal drug. It belongs to the class allylamines.

Terbinafine is absorbed well from the gastrointestinal tract on oral administration providing a bioavailability of around 70-80%.It reaches peak

42 plasma concentration rapidly ,within 2 hours approximately(59). The drug avidly binds to plasma proteins while about 8% of the given binds to blood cells(52).Higher concentration is obtained in skin and skin structures. Then terbinafine is metabolized and 80% is excreted in urine.

Dose of the drug should be reduced in patients suffering from severe hepatic dysfunction or renal dysfunction(52,59). The concentration in stratum corneum is retained at high level of 0.1 μg/ml till the end of 8 weeks even after the withdrawal of therapy. Hence it is enough to use a short course of therapy when terbinafine is used.

There is an around 10% side effect on using terbinafine(52).The adverse effects are common in the initial weeks of treatment but disappears with continuation of therapy. Terbinafine has least drug - drug interactions when compared with other oral agents.

 Novel systemic antifungals:

Systemic antifungals are rarely used in the treatment of tinea cruris or corporis.

Only a few novel systemic antifungals are approved in the last few decades.

And among them, most are reserved only for use in severe, life threatening invasive fungal infections. There are only a few evidences for their use in superficial fungal infections. Posoconazole is a recently approved systemic antifungal which is effective in cases of extensive dermatophytosis and onchyomycosis with CARD9 mutation(60).

43 Oral antifungals for tinea corporis/tinea cruris(46):

DRUG NAME DOSAGE DURATION

Itraconazole 200 mg/day 2 weeks

Fluconazole 150 to 300 mg/week 3 to 4 weeks

Terbinafine 250 mg/day 2 to 3 weeks

Griseofulvin(micronized) 500 mg/day 2 to 4 weeks

Griseofulvin(ultramicrosize) 300 to 375 mg/day 2 to 4 weeks

TOPICAL ANTIFUNGAL AGENTS USED IN TREATING

DERMATOPHYTOSIS

1.Allylamines:

 Naftifine

 Terbinafine

2.Imidazole derivatives:

 Bifonazole, butoconazole, clotrimazole, eberconazole,

 Econazole, fenticonazole, flutrimazole, isoconazole, ketoconazole,

 Luliconazole, miconazole, oxiconazole, sertaconazole, sulconazole,

 Terconazole, tioconazole.

44 3.Benzylamine derivatives:

 Butenafine

4.Morpholine derivatives:

 Amorolfine

5.Miscellaneous compounds:

 Tolnaftate

 Whitfield´s ointment

 Undecilenic acid.

 Ciclopirox olamine

 Haloprogin(61)

Indications of topical therapy in dermatophyte infections:

 Localized and limited lesions which are not widespread.

 When patient is on other drugs which interacts with systemic antifungal

agents.

 Patients who are not compliant to the systemic therapy regimen.

 Used as an adjunct along with oral antifungal treatment.

 As a prophylaxis after oral therapy to prevent recurrence.

 When there is absolute contraindications to systemic therapy.

 Pregnancy.

 Lactation.

 To minimize the duration of systemic therapy.

45 TOPICAL ANTIFUNGAL AGENTS:

 Allylamines

This inhibits synthesis of ergosterol by the fungi which is a vital component of the fungal cell membranes via inhibition of the enzyme squalene epoxidase.

So it also results in accumulation of squalene to toxic concentration inside the fungi that is fungicidal. The drugs in this class include naftifine and terbinafine. On topical application there is rapid response in dermatophytosis infection due to the fungicidal nature of allylamines. In addition to this naftifine also has anti-inflammatory activity where as Terbinafine has additional activity against pityriasis versicolor(62) .

 Butenafine :

It is a bencylamine derivative. It blocks the enzyme squalene epoxidase. It is fungicidal. It is active against both Candida and dermatophytes. It is available as various formulations to be used topically.

 Imidazoles:

This group contains a large number of drugs which includes bifonazole, clotrimazole, sertaconazole, oxiconazole and other agents. These drugs inhibit the enzyme 14 alpha demethylase and thus inhibit fungal ergosterol synthesis.

Imidazoles are fungistatic. In addition to activity against dermatophytes these drugs are active against Pityrosporum yeast as well as Candida. They are available in a wide range of formulations like creams, powders, suspensions, nail lacquer and also as sprays. Combined formulations are also available that contain imidazoles and corticosteroids but their role of is controversial. They

46 are only occasionally useful for accelerating the resolution of symptoms when the affected skin is inflamed and pruritic. On the other side it may cause delay in response to the antifungal and can also mask the underlying persistent infection.

 Morpholines:

Amorolfine is the drug of this class. It inhibits synthesis of ergosterol in two stages. It is fungicidal. This drug is commonly available as a 5% amorolfine nail lacquer and is useful for the therapy of onchyomycosis. The unique advantage is its usefulness against dermatophytes as well as against

Scytalidium and Scopulariopsis species that cause onychomycosis. The nail lacquer is water-resistant and hence it can be used as a once weekly or twice weekly application. But twice-weekly regimen is more effective (63). Cure rates are around 50% at the end of six months. It can act along with other systemic antifungal agents like azoles and allylamines synergistically and this action can improve the cure rates .

 Polyenes:

Nystatin belongs to polyene group of antifungals. It is named after New York

State health laboratory because the drug was discovered there. It binds to ergosterol and damages fungal cell membrane .It is fungistatic at low concentrations but fungicidal at higher concentration .It is not very effective against dermatophytes but has good activity against Candida species. Nystatin is available as cream, lozenges, ointment, oral suspension and pessaries.

47  Ciclopirox olamine:

It is a hydroxypyridone compound. It has a different mode of action. It does not inhibit fungal sterol synthesis directly. Instead it binds to Fe3+ with high affinity, which is an essential component for the function of vital enzymes of the fungal cell, including the cytochromes. So all the metabolic pathways linked to those vital enzymes are disturbed including the electron transport chain in mitochondria which renders energy. It has excellent activity against a wide spectrum of dermatophytes, yeasts as well as moulds like Scytalidium species and Scopulariopsis species. Ciclopirox is commercially available as creams, lotions or else as shampoos, powders and as well as a lacquer for nail(64).

 Tolnaftate:

It is a thiocarbamate derivative . It acts by inhibiting squalene epoxidase. It is an over the counter topical preparation used in the treatment of dermatophyte infections. It is found less effective than other antifungal agents.

 Undecylinic acid:

It is a monounsaturated fatty acid used as an antifungal agent topically in therapy of dermatophyte infection. It has fungistatic, antiseptic and also antiviral properties. It is available in various formulations but found to be less effective than other newer antifungal agents.

 Whitfield’s ointment

Whitfield’s ointment is a combination of 3% salicylic acid and 6% benzoic acid. It is effective for treating the superficial dermatophyte infections. Its

48 chief advantage is its low cost but it is not suitable cosmetically to all and also has irritant property. It has additional antiseptic property due to the presence of benzoyl peroxide(65).

NEWER TOPICAL ANTIFUNGALS:

 Luliconazole is a novel azole antifungal agent. It is fungicidal against

Trichophyton species. Its efficacy is comparable with terbinafine. It is

available as 1% cream formulation. It is effective in dermatophytosis on

applying once daily for 1-2 weeks. It is approved recently by FDA(66).

 Econazole nitrate in foam preparation is effective in treatment of tinea

pedis(67).

RECENT ADVANCES:

 Recently, the use of special carrier systems in which the parent drug is

attached to the carriers. The carriers may be of various types such as

micelle, nanoparticles, micro emulsions, liposomes, ethosomes,

niosomes and transfersomes. The use of the carriers enhances the

penetration of the drug and increases the bioavailability of the drug and

helps in obtaining improved therapeutic response(68)

 At present, the lipid based amphotericin B gel drug formulation has

promising pharmacologic properties which are found to be fruitful in the

therapy of several mucocutaneous fungal infections and

dermatophytosis with nil adverse effects(69).

 Also Amphotericin B when incorporated in micro emulsion results in

100% increase in skin retention. This formulation increases the in vitro

49 antifungal activity of the drug against the species of T. rubrum(70).But

the important concern is that the use of topical amphotericin can lead to

occurrence of its resistance. So its use is limited to invasive fungal

infections alone.

 When the drug griseofulvin is used in Micro emulsion formulations it is

found to show better cure rates in case of dermatophytosis(71).

 And then there is a novel formulation of the drug terbinafine which is

called as terbinafine film forming solution. It forms a thin layer of film

on the skin on topical application. Its fungicidal effect persists for

13 days after single application(72).

NEW POTENTIAL THERAPIES:

 There are some natural remedies effective in dermatophyte infections.

One among them is the plant extract from some Chinese herbs.

 Macrocarpal C is an active plant ingredient extracted from the leaves of

the plant called Eucalyptus globulus Labill. This extract was found to

have activity against species of both T. mentagrophytes and

T. rubrum(73).

 Demicidin is another plant extract with antifungal property. It is a

peptide that provides a novel therapeutic target for dermatophytosis(74).

50 TREATMENT REGIMEN FOR DERMATOPHYTOSIS:

 TOPICAL ANTIFUNGALS FOR TINEA CORPORIS/TINEA

CRURIS:

Drug name Formulation Frequency of Duration of therapy application

1% clotrimazole Cream, lotion Twice a day 4 to 6 weeks

1% Econazole Cream Once or twice a 4 to 6 weeks day 1% Miconazole Cream, lotion Twice a day 4 to 6 weeks

2% Oxiconazole Cream, lotion Twice a day 4 weeks

2% Sertaconazole Cream Twice a day 4 weeks

1% Luliconazole Cream, lotion Once a day 2 weeks

1% Eberconazole Cream Once a day 2 to 4 weeks

Terbinafine Cream, Twice a day 2 weeks Powder 1% Naftifine Cream Once or Twice a 2 weeks beyond day resolution

1% Butenafine Cream Once or Twice a 2 to 4 weeks day

0.25% Amolorfine Cream Twice a day 4 weeks

0.1% Lipid based Twice a day 2 weeks Amphotericin B gel

51 UNMET NEEDS IN TREATMENT OF DERMATOPHYTOSIS:

Complete clearance of fungal elements requires adequate treatment with antifungal agents. The main problem with prolonged treatment with antifungal agents is the compliance. Noncompliance to therapy is common when there is reduction in clinical signs and symptoms. It leads to survival of dermatophytes in partially treated lesions which leads to recurrence. So the enormous challenge is to develop an antifungal agent which has fungicidal property and has highest rate of mycological cure with shortest duration of usage(66).

PROPERTIES OF AN IDEAL TOPICAL ANTIFUNGAL AGENT FOR

TINEA INFECTIONS:

 Broad spectrum of activity

 High efficacy

 Fungicidal property

 Convenient dosing schedules

 Easy penetration in stratum corneum with reservoir effect

 High mycological and clinical cure rates

 Low cost and low incidence of adverse events

 Lack of resistance

The present topical therapies have many drawbacks like low efficacy, noncompliance and higher recurrence.

52 CLOTRIMAZOLE:

Clotrimazole is a broad spectrum antifungal agent that is commonly used to treat Candida infections as well as other fungal infections. The antifungal property of clotrimazole was demonstrated in late 1960s.It shows activity against Trichomonas infection that is resistant to metronidazole and effective against some gram positive bacteria(75) .

Chemistry:

Its molecular formula is C22H17ClN2. The structure of clotrimazole is unique and is given below. Chemically clotrimazole is (1[(2-chlorophenyl)

- diphenylmethyl]1H-imidazole). It has four aromatic rings that is bonded to a tetrahedral carbon atom.One of the aromatic ring is an imidazole ring.It mediates electron transfer in biological systems. The remaining aromatic rings contain a triphenylmethyl system which aids in formation and stabilization of radical intermediates. Clotrimazole is an achiral molecule and exhibit enantiotropism.

53 Therapeutic class:

It is a member of azole group of antifungal agent. It belongs to imidazole subclass in azole group of antifungals.

Mechanism of action:

It interferes with ergosterol synthesis an essential component of fungal cell wall. The specific action of all azoles including clotrimazole is the inhibition of cytochrome P450 dependent 14 alpha-lanosterol demethylation, a step that occurs in ergosterol synthesis(76). As a result there is depletion of ergosterol as it is replaced with other abnormal 14-alpha-methylsterol that affects the normal cell membrane permeability and fluidity. So there occurs leakage of cell contents. Clotrimazole is an anti mycotic that exerts fungistatic but becomes fungicidal at high concentrations.

Uses:

Clotrimazole is considered to be the drug of choice for the topical therapy of tinea pedis , tinea cruris(jock's itch)and tinea corporis(77).

It is used to treat vulvovaginal candidiasis and or pharyngeal candidiasis topically.

Adverse effects:

Topical clotrimazole cream and ointment are reasonably safe. They do not cause serious side effects. But, there are some case reports of allergic contact dermatitis occurring with clotrimazole cream. However it is due to the vehicle or the excipient and not attributable to the active ingredient.

54 Disadvantages:

Variable cure rate-60 to 100%.

Longer duration of therapy - minimum of 4 weeks.

Rising incidence of clotrimazole resistance.

CLINICAL STUDIES OF TOPICAL CLOTRIMAZOLE IN TINEA

CORPORIS/ TINEA CRURIS:

1. In a clinical study done by Clayton et al, the antifungal activity of topical clotrimazole was in par with the use of nystatin or whitfield ointment in treating ringworm infections of the skin(78).

2. A clinical study done by Van Dersarl et al, for comparing the efficacy of 1% clotrimazole solution with 1% haloprogin solution in tinea infections demonstrated that topical clotrimazole was significantly more efficacious than topical haloprogin both clinically as well as mycologically . And further it also showed that significantly more number of patients treated with topical clotrimazole had lesser relapse rate(79).

3. In a phase III study done by Del Palacio et al, in 2001, a newer topical antifungal eberconazole was compared with topical clotrimazole , applied twice daily for four weeks in dermatophytosis. The analysis at the end of the study showed that there were no significant differences in response to eberconazole when compared to clotrimazole(80).

4. In a study done by Singal et al , the results showed that topical butenafine had exhibited high clinical cure rates when compared with topical clotrimazole at the end of one week , as well as those patients had exhibited high

55 mycological cure rate also (61.7% vs. 17.6%). But, this difference was insignificant by the end of 4 weeks and 8 weeks of therapy(81).

5. In a recent clinical study done by Banerjee et al, in 2011, topical amorolfine was found to be equally safe and effective as topical clotrimazole in treating tinea corporis(82)

LULICONAZOLE:

Chemistry

Luliconazole is an imidazole anti-fungal agent which was first synthesized by

Nihon Nohyak & Co in Japan. The unique feature of luliconazole is the incorporation of the imidazole moiety into the ketene dithioacetate ring. It is an optically similar compound to lanoconazole variation being the addition of

2,4-dichlorophenyl group to the ketene dithioacetal structure. The structural formula of luliconazole is (E)-[4(2,4-dichlorophenyl)-1,3dithiolan-2 ylidene]

1imidazolylacetonitrile. R-enantiomer is the active form of luliconazole and is more potent than lanoconazole.

1% Luliconazole cream was approved in Japan in the year 2005 for treating tinea infections, followed by FDA approval in November 2013 for treating tinea pedis, tinea corporis and tinea cruris. It is used as once-daily application for a period of 1 week in cases of tinea corporis and cruris while 2 weeks therapy is indicated in tinea pedis.

56 Chemical structure of luliconazole :

Preclinical studies

The minimum inhibitory concentration of luliconazole against Trichophyton is

2–4 times lower when compared to that of lanoconazole. And it is the lowest

MIC among the topical antifungals used(83) .

Safety

Different strengths of topical luliconazole cream are available. All strengths are generally well tolerated. Mild side effects like pruritis , redness, irritation , pain have been reported in clinical trials using 1% luliconazole and these reactions did not warrant any discontinuation of therapy. Allergic contact dermatitis has been reported with luliconazole 1% cream(84).

57 Advantages over other azole antifungals:

Azole antifungals are generally known to be fungistatic. The mechanism of action of luliconazole is same as that of clotrimazole as mentioned before, but luliconazole exhibits strong fungicidal activity in dermatophytosis against

Trichophyton species. The potent antifungal activity is due to strong in-vitro antifungal activity as well as favorable pharmacokinetic profile in the skin. The reduced frequency of application (once daily) with short duration of therapy

(1 week for tinea corporis/cruris , 2 weeks for interdigital tinea pedis) is an advantage when compared with other topical regimens(85).

CLINICAL STUDIES OF LULICONAZOLE IN TINEA INFECTIONS:

1. In a study done by Jerjani et al, topical sertaconazole showed better symptomatic relief than terbinafine or luliconazole while the mycological cure rate was comparable in all the three groups with no significant differences(86).

2. In a study done by Lakshmi et al, in 2013, two-week therapy with both terbinafine 1% cream and luliconazole 1% cream achieved 100% cure rate.

Luliconazole , a newer topical azole has fungistatic activity when compared to fungicidal effect of terbinafine. But luliconazole has equal efficacy as that of terbinafine in dermatophytosis with better control of pruritus and thus improving the quality of life(87).

58 3. In a study done by Khare et al, in 2016, the results show that cure rate to the topical monotherapy with sertaconazole 2% and luliconazole 1% are similar and higher than that of 1% clotrimazole and 1% terbinafine in patients with tinea corporis or tinea cruris. Topical luliconazole cream 1% had more convenience due to a short course of application(88).

So with this detailed review of literature we have done this study is undertaken to compare the efficacy and safety of 1% luliconazole cream vs.

1% clotrimazole cream in tinea corporis and tinea cruris.

59 AIM OF THE STUDY

To compare the efficacy and safety of luliconazole (1%) cream versus clotrimazole (1%) cream in tinea infections of skin.

60 METHODOLOGY

STUDY DESIGN:

An open labeled, prospective, randomized, controlled, single centre, comparative, parallel group study.

STUDY PERIOD:

From March 2015 to September 2016(a period of 18 months).

STUDY CENTRE:

Dermatology OPD, Department of Dermatology, Tirunelveli medical college.

STUDY POPULATION:

Patients attending Dermatology OPD diagnosed to have tinea corporis / tinea cruris.

SAMPLE SIZE:

100(50 patients in each study group)

This study was done after obtaining approval from the Institutional Ethical

Committee of Tirunelveli Medical College. Written informed consent was obtained in local vernacular language from all the participants patients before enrolling them for the study.

61 INCLUSION CRITERIA:

1. Patients of either sex.

2. Age > 12 years of age.

3. Patients who are diagnosed with tinea corporis or tinea cruris (involving

<20% of the BSA).

EXCLUSION CRITERIA:

1. Immuno compromised individuals (H/o HIV infection, Diabetes

mellitus, steroid intake).

2. Pregnant and lactating females.

3. H/o hypersensitivity reaction to azole antifungals.

4. Patients with other types of tinea infections

(T.capitis,T.pedis,T.mannum).

5. Patients with extensive dermatophytoses (>20% involvement of the body

surface area).

6. Patients having superadded bacterial infection.

7. Patients with contact dermatitis, atopic dermatitis, psoriasis.

8. Patients who received topical antifungal within 1 week before baseline

visit.

9. Patients who received systemic antifungals within 4 weeks before

baseline visit.

62 STUDY PROCEDURE:

Screening and Recruitment:

Patients attending the skin OPD, who were diagnosed clinically with tinea corporis or tinea cruris were screened. Detailed medical and drug intake history were elicited at screening. Random blood sugar estimation was done at screening and patients with RBS > 200 mg/dl were excluded from the study.

Baseline clinical parameters such as erythema, scaling, pruritis and papules were noted during the first visit. And these clinical parameters were graded on a 4 point scale. Patients whose total score were ≥ 5 were only eligible to participate in the study.

Enrollment and Randomization:

Patients who satisfied the study criteria were enrolled in the study.

Demographic data of patients like age and sex were recorded. The patients were randomly allotted to Group A or Group B by using a computer generated random table in the ratio of 1:1.

Treatment Phase:

In the treatment phase, Group 1 received 1% luliconazole cream topically and

Group 2 received 1% clotrimazole cream topically as treatment for tinea corporis or tinea cruris. Total duration of treatment of group 1 was 2 weeks and for group 2 the total duration was 4 weeks. The patients in group 1 were advised to apply the 1% luliconazole cream to the affected area and 1 inch

63 surrounding the affected area in thin layer once daily for 2 weeks. The patients in group 2 were advised to apply 1% clotrimazole cream to the affected area and 1 inch surrounding the affected area twice daily for 4 weeks. All the patients were advised to clean and dry the affected area before applying the medications.

Direct microscopy and fungal culture were done at baseline and at each follow up visit. Patients were given a week’s supply of medication at each visit. The patients were followed up every week for safety and efficacy parameters.

Mycological assessment:

Preparation of KOH mount and examination under microscope:

The infected areas were scraped from the edge of lesions using a scalpel blade of size 15. Scrapings were collected in a black paper or directly on the slide. Two to three drops of 10%Potassium hydroxide (KOH ) was added to the scrapings and the slide was covered by a cover slip(62,89).

Prepared slides were mounted and examined under direct microscopy using low power of magnification at first(10X), next with 40X power and ultimately under high resolution of 100 X to confirm the presence of fungal hyphal elements(62,89).

Procedure for fungal culture:

Specimens (scales) collected from each patient enrolled in the study were inoculated into the culture plates with Sabouraud’s dextrose agar medium and incubated at room temperature. The culture media were examined weekly for

64 the growth of dermatophytes. The culture plates were examined at least for

3 weeks before declaring negative.

Clinical assessment:

At each visit after baseline, both the groups were evaluated for improvement in clinical symptoms and signs (pruritus, erythema, scaling, papules).This improvement was assessed using a 4-point scale by the investigator.

Scoring of symptoms/signs:

 Score 0=absent

 Score 1=mild

 Score 2=moderate

 Score 3=severe

Global assessment score was calculated at each follow up visit by summation of individual scores on all four signs(erythema, scaling, pruritis, papules) in a patient.

Follow up phase:

At the end of treatment phase, there was a ‘Follow-up Phase’ until 4 weeks where the patients were assessed clinically and mycologically for potential relapse. Patients who missed out more than one week of medications were treated as drop outs.

During each visit the patients were assessed for clinical cure, mycological cure and clearance of lesions and improvement from baseline. Photographs of the

65 lesions were taken for evidence of improvement. At each visit, clinical examination of the target lesion and KOH test was done. Safety and tolerability were assessed by monitoring treatment related adverse events at each visit.

Mycological assessment was done at baseline, at the end of treatment phase and at the end of follow up phase.

PRIMARY OUTCOME:

1. Proportion of patients achieving complete clearance.

SECONDARY OUTCOMES:

1. Clinical cure rate and mycological cure rate.

2. Relapse rate.

3. Safety and adverse effect profile of the drug.

OPERATIONAL DEFINITIONS:

 Complete clearance: Mycological cure with complete absence of

clinical signs and symptoms.

 Clinical cure: No signs or symptoms of tinea infection( erythema,

scaling, pruritis, papules) / (Global assessment score ≤ 2)

 Mycological cure: Negative KOH microscopy and culture.

 Relapse: Positive KOH after 2 weeks of completion of treatment.

66 STUDY PARTICIPANT FLOW CHART:

Assessed for eligibility (n =158)

Excluded (n = 54)

1.Not meeting inclusion Enrollment criteria(n = 34) 2.Refused to participate (n = 20)

Randomized (n =104)

Allocated to Allocated to 1%Luliconazole 1% Clotrimazole (n =54) (n = 50) 1.Received allocated 1.Received allocated intervention (n = 50) intervention (n =50) 2.Did not receive 2. Did not receive

Allocation medication(n=4) medication(n=0) (withdrew consent)

Lost to follow up Lost to follow up (n = 2) (n = 8)

Discontinued Discontinued Follow up intervention (n = 0) intervention (n = 1) ( Due to Hypersensitivity)

Analyzed (n = 50) Analyzed (n = 50)

Excluded from analysis Excluded from analysis Analysis (n = 4) (n=0) 4 -Did not receive intervention

67 STATISTICAL ANALYSIS

 The statistical analysis was done with SPSS version 17( Statistical

Package for the Social Sciences)

 Mean and Standard deviation were calculated for continuous data while

the categorical data was expressed in percentages and in absolute

frequencies.

 The baseline demographic characteristics of both the groups were

matched by unpaired student 't' test and Pearson's Chi square test.

 The baseline disease characteristics like type, number and duration of

lesions were compared by using Pearson's Chi square test.

 The efficacy of the individual drugs was analyzed with the help of

Wilcoxon signed rank test.

 Mann Whitney U test was used for comparison between the two groups.

 Mycological cure was analyzed using Fischer's exact test.

 p value <0.05 were considered as significant.

 All the analyses were carried out in the intention to treat population

(ITT) .ITT group contained all the patients who were randomized and

had one baseline visit.

 Missing data were computed with last observation carried forward

method (LOCF).

68 RESULTS

Patients who were newly diagnosed with Tinea corporis / Tinea cruris in the

Dermatology Outpatient Department during the period May 2015 to December

2015 were screened. Totally one hundred and fifty eight patients were screened out of which, 34 patients did not meet the inclusion criteria, 20 patients were not willing to give consent for the study. One hundred and four patients were included in the study and randomly assigned into 2 groups using a computer generated random table. Patients in Group 1 (n=54) were allocated to receive 1% Luliconazole topical cream and Group 2(n=50) to receive

1% Clotrimazole cream.

In luliconazole group, 4 patients withdrew consent after screening and enrollment and hence did not receive any study medication whereas in clotrimazole group all the patients who were randomized received the drug.

In luliconazole group, 2 patients lost to follow up. In clotrimazole group 8 patients lost to follow up and 1 patient discontinued treatment due to hypersensitivity reaction to clotrimazole. Hence a total of 48 patients completed the study in luliconazole group and 41 patients completed the study in clotrimazole group.

69 TABLE -1:

MEAN AGE:

Group Mean S.D t df 'p' value

Luliconazole 29.6 13.900 0.385

0.872 98 (p>0.05) Clotrimazole 31.98 13.386

p value is statistically insignificant

Table-1: Shows the mean age and standard deviation in both luliconazole and clotrimazole group.

From the insignificant p value we can interpret that both groups were comparable or similar in terms of mean age distribution.

70 FIGURE -1

Mean Age Distribution

Luliconazole Clotrimazole 50

40 29.6 31.98 30

20

Mean Age Age Meanin years 10

0 Luliconazole Clotrimazole

Figure -1: Displays the pictorial representation of mean age with standard deviation in both the groups.

The mean age in the luliconazole group (29.6) is similar to the mean age in clotrimazole group(31.98).

71 TABLE-2:

GENDER DISTRIBUTION:

Sex Chi

Group Male Female square df 'p' value

Luliconazole 24 26

Clotrimazole 23 27 0.40 1 0.84

p value> 0.05(insignificant)

Table-2: shows the gender distribution in both the groups.

The insignificant p value indicates that both the groups were similar with due regard to the gender distribution.

72 FIGURE 2

GENDER DISTRIBUTION

27 Female 26

23 Male 24

0 5 10 15 20 25 30 35 40 45 50

Clotrimazole Luliconazole

Figure-2: shows the bar chart representing the gender distribution in luliconazole and clotrimazole group.

The number of males and females in both the groups were similar.

73 TABLE-3 :

BASELINE DISEASE CHARACTERISTICS:

Baseline disease Chi 'p' Luliconazole Clotrimazole df Characters square value Tinea 34 36 Corporis Type Tinea 1.152 1 0.28 16 14 Cruris

=1 36 34 Number 0.048 1 0.82 ≥2 14 16 < 3 days 24 25 ≥ 3 ≤ 7 20 21 Duration days 0.448 2 0.80 >7 days 6 4 KOH mount 50 50 1 1 1 Mycology positive Culture 50 50 1 1 1 positive

p value >0.05 - statistically insignificant

Table-3: compares the baseline disease characters in both groups.

It shows that both groups were similar with respect to type of lesion, number of lesions, duration of the disease and mycological assessment at baseline.

74 TABLE-4:

BASELINE SYMPTOM SCORES:

Luliconazole Clotrimazole Symptoms 'p'value (Mean±S.D) (Mean±S.D)

1.Erythema 2.44 ± 0.61 2.28 ± 0.75 0.354

2.Scaling 2.38 ± 0.56 2.32 ± 0.55 0.568

3.Pruritis 2.54 ± 0.57 2.64 ± 0.48 0.451

4.Papules 2.12± 0.71 2.36 ± 0.63 0.055

5.Total 9.42±1.25 9.52±1.33 0.637 score(GAS)

p value>0.05-statistically insignificant

GAS-Global assessment score.

Table-4: Shows the distribution of baseline clinical parameters in both the groups

The insignificant p value implies that the baseline parameters like erythema, scaling, pruritis and papules were similar in both luliconazole and clotrimazole groups.

75 TABLE-5:

EFFICACY OF LULICONAZOLE:

Mean 95% CI Parameters Duration p value difference Lower Upper

BL_1 WK 1.36 1.17166 1.54834 <0.0001*

Erythema 1WK_2WKS 0.94 0.806453 1.073547 <0.0001*

2WKS_6WKS 0.02 -0.08726 0.127263 0.705

BL_1 WK 1.88 1.652933 2.107067 <0.0001*

Scaling 1WK_2WKS 0.36 0.210717 0.509283 <0.0001*

2WKS_6WKS 0.02 -0.10166 0.141663 0.739

BL_1 WK 1.38 1.21 1.55 <0.0001*

Pruritis 1WK_2WKS 1 0.859359 1.140641 <0.0001*

2WKS_6WKS 0.14 0.025024 0.254976 0.020*

BL_1 WK 1.46 1.31 1.61 <0.0001*

Papules 1WK_2WKS 0.54 0.365753 0.714247 <0.0001*

2WKS_6WKS 0.06 -0.02913 0.149134 0.180

BL_1 WK 5.44 5.076141 5.803859 <0.0001*

GAS 1WK_2WKS 2.88 2.523093 3.236907 <0.0001*

2WKS_6WKS 0.14 -0.21399 0.493985 0.054

*p value<0.05 significant

Table -5: shows the efficacy of luliconazole with respect to duration of treatment.

The clinical parameters such as erythema, scaling, pruritis, papules and global assessment score improved significantly from baseline to 1 week and from

1 week to 2 weeks.

76 FIGURE -3

Efficacy of Luliconazole 3

2.5

2 Erythema 1.5 Scaling 1 Pruritis Papules 0.5

0 Baseline 1 week 2 weeks(EOT) 6 weeks(Follow Up)

Figure-3: shows the improvement in clinical parameters from baseline.

The steep decrease in slope from baseline to first week in all parameters indicated that most of the patients in group achieved significant reduction in signs and symptoms by the end of 1 week of treatment.

77 FIGURE-4

GAS in luliconazole group 10 9 8 7 6 5 4 3 2 1 0 GAS_BL GAS_1WK GAS_2WKS GAS_6WKS

Figure-4: shows trend of reduction in the global assessment score from baseline to the end of follow up period (6 weeks)

A marked reduction in GAS was noted from baseline to 2 weeks (end of treatment period)

78 TABLE -6:

EFFICACY OF CLOTRIMAZOLE:

95% CI Parameters Duration Mean difference 'p' value Lower Upper BL_1 WK 0.12 -0.09205 0.332053 0.253 1WK_2WKS 0.8 0.685167 0.914833 <0.0001* 2WKS_3WKS 0.48 0.315184 0.644816 <0.0001* Erythema 3WKS_4WKS 0.68 0.546083 0.813917 <0.0001* 4WKS_8WKS -0.1 -0.309 0.108999 0.314 BL_1 WK 0.62 0.469289 0.770711 <0.0001* 1WK_2WKS 0.36 0.190063 0.529937 <0.0001* 2WKS_3WKS 0.46 0.305829 0.614171 <0.0001* Scaling 3WKS_4WKS 0.58 0.438308 0.721692 <0.0001* 4WKS_8WKS -0.08 -0.31562 0.155617 0.322 BL_1 WK 0.92 0.698816 1.141184 <0.0001* 1WK_2WKS 0.1 -0.13847 0.338468 0.398 2WKS_3WKS 0.6 0.437602 0.762398 <0.0001* Pruritis 3WKS_4WKS 0.36 0.200056 0.519944 <0.0001* 4WKS_8WKS 0.42 0.18264 0.65736 0.002* BL_1 WK 0.72 0.547368 0.892632 <0.0001* 1WK_2WKS 0.36 0.210717 0.509283 <0.0001* 2WKS_3WKS 0.48 0.315184 0.644816 <0.0001* Papules 3WKS_4WKS 0.48 0.336574 0.623426 <0.0001* 4WKS_8WKS 0.14 -0.05898 0.338979 0.162 BL_1 WK 2.32 1.998058 2.641942 <0.0001* 1WK_2WKS 1.62 1.31139 1.92861 <0.0001* 2WKS_3WKS 2.06 1.857734 2.262266 <0.0001* GAS 3WKS_4WKS 1.94 1.58882 2.29118 <0.0001* 4WKS_8WKS 0.5 -0.2131 1.213097 0.105

*p value<0.05 -statistically significant

Table-6: shows the efficacy of clotrimazole in improving scores of clinical parameters. Significant improvement was noted in erythema, pruritis, scaling, papules and GAS from baseline till the end of treatment period (4 weeks).

79 FIGURE-5

Efficacy of Clotrimazole

3

2.5

2

1.5

1 Erythema

0.5 Scaling Pruritis 0 Papules

Figure 5: Represents the picture of improvement in clinical parameters with respect to duration of therapy.

The reduction in mean scores of all the parameters was gradual with each week of therapy and clinical cure achieved by the end of 4 weeks

80 FIGURE -6

Change in GAS in Clotrimazole group 12

10

8

6

4

2

0 BL 1 WEEK 2 WEEKS 3 WEEKS EOT(4 WEEKS) FOLLOW UP(8 WEEKS)

Figure 6: Pictorial representation of GAS with respect to duration.

Mean GAS was gradually decreasing from baseline with increase in duration of treatment and the change in score was maximum by the end of 4 weeks.

81 TABLE-7 :

COMPARISON OF EFFICACY OF LULICONAZOLE VS

CLOTRIMAZOLE:

Parameters Duration Group Mean S.D P value Luliconazole 1.08 0.53 Erythema at 1 week <0.0001* Clotrimazole 2.16 0.65 ERYTHEMA Luliconazole 0.14 0.40 Erythema at EOT 0.303 Clotrimazole 0.2 0.40 Luliconazole 0.5 0.58 Scaling at 1 week <0.0001* Clotrimazole 1.7 0.46 SCALING Luliconazole 0.14 0.40 Scaling at EOT 0.078 Clotrimazole 0.3 0.54 Luliconazole 1.2 0.45 Pruritis at 1 week <0.0001* Clotrimazole 1.72 0.54 PRURITIS Luliconazole 0.2 0.40 Pruritis at EOT <0.0001* Clotrimazole 0.66 0.48 Luliconazole 0.66 0.66 Papules at 1 week <0.0001* Clotrimazole 1.64 0.48 PAPULES Luliconazole 0.12 0.33 0.016* Papules at EOT Clotrimazole 0.32 0.47 Luliconazole 3.48 1.13 GAS at 1 week <0.0001* Clotrimazole 7.2 1.28 GAS Luliconazole 0.6 0.93 GAS at EOT <0.0001* Clotrimazole 1.58 1.14

*p <0.05-Statistically significant

Table-7: shows the comparison of improvement in clinical parameters between two groups.Luliconazole was more effective reducing mean scores of all the clinical parameters significantly by the end of one week when compared to clotrimazole.

82 FIGURE -7

CLINICAL SYMPTOM IMPROVEMENT(1WEEK) 12

10

8

6 CLOTRIMAZOLE 4 LULICONAZOLE

2

0 ERYTHEMA SCALING PRURITIS PAPULES GAS

Figure7 : shows the pictorial representation of comparison of clinical parameters by the end of 1 week of treatment.

FIGURE-8

CLINICAL SYMPTOM IMPROVEMENT(EOT) LULICONAZOLE VS CLOTRIMAZOLE 2

1.5

1 LULICONAZOLE CLOTRIMAZOLE 0.5

0 ERYTHEMA SCALING PRURITIS PAPULES GAS

Figure 8: shows the comparison of improvement in clinical parameters by the end of treatment period (2 weeks in luliconazole and 4 weeks in clotrimazole group).

83 TABLE 8:

COMPARISONS BETWEEN PROPORTIONS OF PATIENTS

ACHIEVING COMPLETE CLEARANCE:

Complete clearance Chi 'p' Duration Group df Not square value Achieved achieved

Luliconazole 11 39 1 week 10.22 1 0.0014* Clotrimazole 0 50

Luliconazole 49 1 EOT 11.294 1 0.0008* Clotrimazole 36 14

p value<0.05 is statistically significant.

Table 8: shows the number of patients who achieved complete clearance at the end of 1 week and at the end of treatment periods.

A higher proportion of patients achieved complete clearance in luliconazole group compared to clotrimazole group and this difference was found to be statistically significant both at end of 1 week and at the end of treatment period.

84 FIGURE-9

COMPLETE CLEARANCE

EOT

CLOTRIMAZOLE LULICONAZOLE 1 WEEK

0 10 20 30 40 50

Figure 9-shows the number of patients who achieved complete clearance.

In clotrimazole group none achieved complete clearance at the end of 1 week of treatment whereas in luliconazole group 22% of patients achieved complete clearance.

By the end of treatment period 98% in luliconazole group achieved complete clearance whereas it was only 72% in clotrimazole group.

85 TABLE 9:

CLINICAL CURE RATE COMPARISON:

Clinical cure Chi 'p' Duration Group df Not square value Achieved achieved

Luliconazole 11 39 10.22 1 0.0014* 1 week Clotrimazole 0 50

Luliconazole 49 1 3.39 1 0.065 EOT Clotrimazole 43 7

p value<0.05 statistically significant.

Table-9: shows the clinical cure rates in luliconazole and clotrimazole group at

1 week and at EOT.

Significant change in clinical cure rate was seen at 1 week in luliconazole group whereas there was no difference in clinical cure by the end of treatment period between the groups.

86 FIGURE-10

CLINICAL CURE RATE COMPARISON 60

50

40

30 LULICONAZOLE CLOTRIMAZOLE 20

10

0 1 WEEK EOT

Figure 10-Shows the graphical representation comparison of clinical cure rates in both the groups.

Clinical cure achieved at 1 week in clotrimazole group was 0% whereas it was around 22% in luliconazole group.

87 1% LULICONAZOLE GROUP

BASELINE AFTER 1 WEEK AT EOT(2WEEKS) 1% CLOTRIMAZOLE GROUP

BASELINE AFTER 1 WEEK AT EOT(4 WEEKS) TABLE-10 :

MYCOLOGICAL CURE:

Mycological cure Chi Duration Group df 'p' value Not square Achieved achieved

Luliconazole 39 11 At 1 41.37 1 <0.0001* week Clotrimazole 6 44

Luliconazole 49 1 At EOT 6.537 1 0.0106* Clotrimazole 40 10

*p value<0.05 -Statistically significant

Table 10: Shows the mycological cure rate in both the groups at 1 week and at

EOT.

Significant p value indicated that high mycological cure rate was achieved with luliconazole group than clotrimazole group.

88 FIGURE-11

MYCOLOGICAL CURE AT END OF 1 WEEK

CLOTRIMAZOLE NO YES LULICONAZOLE

0 10 20 30 40 50

Figure 11: shows the pictorial representation of mycological cure by the end of

1 week in both the groups.

FIGURE-12

MYCOLOGICAL CURE AT END OF TREATMENT

CLOTRIMAZOLE 80% NO YES LULICONAZOLE 98%

0 10 20 30 40 50

Figure 12: shows the graphical representation of mycological cure by the end of treatment period in both the groups.

89 TABLE-11:

RELAPSE RATE COMPARISON:

Relapse 'p' Group Chi square df value Present Absent

Luliconazole 2 48 0.03 1 0.0312* Clotrimazole 10 40

*p value<0.05 statistically significant.

Table 11- shows the comparison of relapse rate between groups.

In luliconazole group, the lesions reappeared in 2 patients after the end of treatment period whereas in clotrimazole group 10 patients developed relapse and this difference in relapse rate was found to be statistically significant.

90 FIGURE-13

RELAPSE RATE

clotrimazole

luliconazole

0 2 4 6 8 10 12

Figure 13-shows the pictorial representation of relapse rate comparison between two groups.

In clotrimazole group 20% had relapse while only 4% of the patients had relapse in luliconazole group.

91 TABLE-12:

ADVERSE DRUG REACTION COMPARISON:

LULICONAZOLE CLOTRIMAZOLE ADR (N=22)(%) (N=21)(%)

BURNING 17(77.2%) 14(66.7%) SENSATION

RASH 0(0%) 1(4.7%)

NUMBNESS 2(9.1%) 4(19%)

TINGLING 3(13.6%) 2(9.5%)

Table 12- shows the adverse drug reactions that occurred during the study period.

A total of 22 ADRs were noted in luliconazole group and 21 ADRs were noted in clotrimazole group. The most common ADR noted in both the groups was the occurrence of mild burning sensation on topical application of the drugs.

92 DISCUSSION

Superficial fungal infections occur in around 25% of world's population. It is associated with increase in morbidity, health care expenditure and poor quality of life (24). Aerobic fungi like dermatophytes are the most common cause for superficial fungal infections. These dermatophytes digest keratin for their growth. They replicate in the superficial layers of the epidermis. As a result, clinically the body parts rich in keratin such as the hair, skin, and nails are the most affected areas. Prolonged survival of embedded arthroconidia in epidermis results in frequent recurrence or relapse (66).

Oral and topical antifungals can be used in the treatment of dermatophytosis.

All the available antifungals inhibit ergosterol synthesis which is an essential component of fungal cell wall, but the drugs act at different enzymes which make them to vary in their efficacy. Topical imidazole antifungals work by inhibiting 14 alpha lanosterol demethylase enzyme. However, conventional use of older imidazole antifungal such as clotrimazole resulted in various disadvantages like emergence of resistance, non adherence due to longer treatment course and twice daily application altogether resulting in frequent recurrence of tinea infections (89).

Luliconazole is a novel imidazole antifungal having R enantiomer in its structure in addition to one chiral centre. Luliconazole has the highest antifungal activity against Trichophyton species among the currently available

93 topical antifungal drugs and it was also highly effective against Candida species (90.91).

The present study evaluated the efficacy and safety of 1% clotrimazole versus

1% luliconazole in tinea corporis and tinea cruris. The mean age distribution in this study clearly indicates that tinea infections are more common in 2nd and

3rd decade. They contribute to the majority of the working population which leads to increase in outdoor activities, thus more prone for increase in sweating which favors the growth of dermatophytes (92, 93). In this study, 72% of luliconazole group and 74% of clotrimazole group fall under the age group of

12 to 40 years. This may be due to the fact that the patients in the younger age group experience greater impact of the disease on their quality of life. Tinea infections are contagious, spread readily and produce itching which affects the daily activities and sleep. Hence the people in this age group seek treatment earlier.

The incidence of dermatophytes in males and females is found to be equal in contrast to other studies where males were predominantly infected(92,93).This might be due to the fact that females at present day are equally employed as male and work outdoors.

Tinea corporis was the most common dermatophyte infection in this study followed by tinea cruris. In this clinical study, 70 % of the patients presented with tinea corporis while only 30% presented with tinea cruris. This is similar

94 to the results of a study published by Lakshman et al, in 2015(94), in which tinea corporis was the commonest clinical presentation.

The time of presentation to dermatologist is much earlier in both the groups, with 88% and 92% presenting to the OPD within one week of noticing the lesion in luliconazole and clotrimazole group respectively.

Mycological assessment was done before randomization. All the patients who were positive for KOH mount and culture were included in the study.

Trichophyton rubrum (74%) was the most common isolated species in culture of dermatophytes while Trichophyton mentagrophytes was isolated in 26% of the samples which is in correlation with other studies (94) and none of the samples had Epidermatophyton or Microsporum species.

Clinical assessment for symptoms like erythema, pruritis, scaling, papules and global assessment scores with their mean scores revealed that both the groups were similar at baseline. The reduction in mean score of clinical symptoms at the end of 1 week in luliconazole group was greater than clotrimazole group.

This difference was statistically significant. However, by the end of treatment period (i.e. 2 weeks in luliconazole group and 4 weeks in clotrimazole group) the mean reduction in scores was same in both the groups. This clearly showed that both the groups achieved symptomatic relief by the end of treatment period but the duration to achieve was shorter in luliconazole group.

The primary endpoint was complete clearance which was defined as attaining both clinical cures (global assessment score ≤2) as well as mycological cure

95 (negative KOH microscopy). The number of patients who achieved complete clearance of the lesions was much higher in luliconazole group (98%) when compared to clotrimazole group (72%). This was in par with a study done by

Jerajani et al (86), where the complete clearance was about 95%.

Patients who achieved negative KOH microscopy and culture after therapy were taken to be mycologically cured. Around 78% achieved mycological cure at the end of 1 week in luliconazole group while only 12% were mycologically cured in clotrimazole group. About 98% of patients achieved mycological cure in luliconazole group by the end of treatment period (i.e. 2 weeks). But it was only 80% by the end of treatment (i.e. 4 weeks) in clotrimazole group. This result is similar to a study done by Koga et al, where 14 day regimen of once daily 1% Luliconazole achieved 100% mycological cure in the study population (83).

Clinical cure was defined as the attainment of Global assessment score ≤ 2 with no evidence of tinea infection clinically. In the present study around 22% attained clinical cure by the end of 1 week in luliconazole group while none attained clinical cure in clotrimazole group. Clinical cure rate by the end of therapy in both the groups revealed that luliconazole was a better agent in resolving symptoms achieving a clinical cure rate of 98% compared to clotrimazole group, where it was only 86%. This result is similar to the one obtained in a study done by Lakshmi et al , in which the clinical cure and mycological cure was 100% at the end of 2 weeks on treating with 1% luliconazole with no relapse(87).

96 When KOH microscopy was positive for fungal hyphal elements after 2 weeks of completion of therapy, the patient was diagnosed to have a relapse. In this study, the relapse rate was higher with clotrimazole group (20%) while it was only 4% in luliconazole group.

The onset of clinical and mycological cure was earlier in luliconazole group than in clotrimazole group.

A high proportion of patients completed the study in luliconazole group (96%) compared to clotrimazole group (86%) which might be attributed to the simple regimen of once daily application of the luliconazole for 2 weeks. Long duration of therapy led to poor compliance and greater loss to follow up in clotrimazole group.

A total of 43 adverse drug reactions such as burning sensation, itching, rashes, tingling and numbness were reported, out of which 22 were in luliconazole group and 21 in clotrimazole group. All of them were mild except one who developed rashes, itching and hypersensitivity reaction to topical clotrimazole application and the treatment was discontinued. In general both the drugs were well tolerated.

Limitations:

The study was conducted only with small sample size and did not include patients having other forms of dermatophytosis except tinea corporis and tinea cruris.

97 CONCLUSION

In conclusion, topical luliconazole 1% was superior to topical clotrimazole 1% in achieving complete clearance, faster clinical cure and mycological cure.

However the proportion achieving clinical cure was same at the end of treatment period in both the groups, but the duration of treatment period was short (only 2 weeks) in luliconazole group. Relapse rate was lower in luliconazole group. Both the drugs were safe and well tolerated. Thus, the present study proves that 1% topical luliconazole is more efficacious than

1% clotrimazole in dermatophytosis.

98 ABBREVIATIONS

KOH :Potassium hydroxide

HIV :Human immunodeficiency virus

Th1 :T helper cells 1

UV :Ultraviolet

DMSO :Dimethyl sulfoxide

PAS :Periodic acid schiff stain

SDA :Sabouraud's dextrose agar

DTM :Dermatophyte test medium

MIC :Minimum inhibitory concentration

MFC :Minimum fungicidal concentration

H& E :Hematoxylin and eosin stain

PCR :Polymerase chain reaction

DNA :Deoxy ribonucleic acid

ECF :Extracellular fluid

CARD9 :Caspase recruitment domain containing protein 9

FDA :Food and Drug Administration

OPD :Outpatient department

BSA :Body surface area

SPSS : Statistical Package for the Social Sciences ITT :Intention to treat population

LOCF :Last observation carried forward

SD :Standard deviation df :Degree of freedom

GAS :Global assessment score

BL :Baseline

CI :Confidence interval

WK :Week

EOT :End of treatment

ADR :Adverse drug reaction APPENDIX –I

INFORMED CONSENT FORM

Study Title : EFFICACY AND SAFETY OF LULICONAZOLE (1%) CREAM VERSUS CLOTRIMAZOLE

(1%) CREAM IN TINEA INFECTIONS OF SKIN-A COMPARATIVE STUDY.

Study Number ______Subject's Full Name ______Date of Birth/Age______Address ______1. I confirm that I have read and understood the information sheet dated for the above study and have had the opportunity to ask questions. OR I have been explained the nature of the study by the Investigator and had the opportunity to ask questions 2. I understand that my participation in the study is voluntary and that I am free to withdraw at any time, without giving any reason and without my medical care or legal rights being affected. 3. I understand that the sponsor of the clinical trial/project, others working on the Sponsor's behalf,the Ethics Committee and the regulatory authorities will not need my permission to look at my health records both in respect of the current study and any further research that may be conducted in relation to it, even if I withdraw from the trial. However, I understand that my Identity will not be revealed in any information released to third parties or published. 4. I agree not to restrict the use of any data or results that arise from this study provided such a use is only for scientific purpose(s) 5. I agree to take part in the above study Signature (or Thumb impression) of the Subject/Legal Representative: ______Signatory's Name ______Date ______Signature of the Investigator ______Date ______Study Investigator's Name ______Date ______Signature of the Witness ______Date ______Name of the Witness úSôVô°LÞdÏ A±®l× Utßm Jl×Rp T¥Ym UÚjÕY Bn®p TeúLtTRtÏ BnÜ ùNnVlTÓm RûXl×: OÅah fh®nghÇÞ (gl®jhkiu) nehahËfS¡F ÿÈfdn[hš (1%) fË«ò mšyJ ¡sh£iunkn[hš (1%) fË«ò jlÉ gy‹ k‰W« ghJfh¥ò F¿¤J X® x¥òa®î MŒî. TeÏ ùTßTY¬u ùTVo : TeÏ ùTßTY¬u YVÕ : TeÏ ùTßTYo CRû] ϱdLÜm 1 Sôu úUúX ϱl©hÓs[ UÚjÕY Bn®u ®YWeLû[ Sôu T¥jÕ ×¬kÕ ùLôiúPu. GuàûPV NkúRLeLû[ úLhLÜm ARtLô] RÏkR ®[dLeLû[ ùT\Üm YônlT°dLlThÓs[Õ G] A±kÕ ùLôiúPu. 2 Sôu CqYôn®p Ru²fûNVôL Rôu TeúLt¡ú\u. GkR LôWQj§]ôúXô GkR LhPj§Ûm, GkR NhP £dLÛm EhTPôUp Sôu CqYôn®p CÚkÕ ®X¡ ùLôs[Xôm Gußm A±kÕ ùLôiúPu. 3 CkR BnÜ NmTkRUôLúYô. CûRf NôokÕ úUÛm BnÜ úUtùLôsÞm úTôÕm CkR Bn®p TeÏ ùTßm UÚjÕYo GuàûPV UÚjÕY A±dûLûV TôolTRtÏ GuàûPV AàU§ úRûY«pûX G] A±kÕ ùLôs¡ú\u. Sôu Bn®p CÚkÕ ®X¡d ùLôiPôÛm CÕ ùTôÚkÕm G] A±¡ú\u. 4 CkR An®u êXm ¡ûPdÏm RLYûXúVô. Ø¥ûYúVô TVuTÓj§d ùLôs[ UßdL UôhúPu. 5 CkR Bn®p TeÏ ùLôs[ Jl×d ùLôs¡ú\u. G]dÏ ùLôÓdLlThP A±ÜûWL°uT¥ SPkÕ ùLôsYÕPu BnûY úUtùLôsÞm UÚjÕ A¦dÏ EiûUÙPu CÚlúTu G] EߧV°d¡ú\u. Gu EPp SXm Tô§dLlThPôúXô. ApXÕ G§oTôWôR YZdLj§tÏ Uô\ô] úSônϱ ùRuThPôúXô EPú] CûR UÚjÕY A¦«Pm ùR¬®lúTu G] Eߧ A°d¡ú\u. TeúLtTY¬u ûLùVôlTm /……………..…….. CPm ……………….. úR§ ...... LhûP ®Wp úWûL TeúLtTY¬u ùTVo Utßm ®XôNm ……………………………………………...……… BnYô[¬u ûLùVôlTm /……………..…….. CPm ……………….. úR§ ...... BnYô[¬u ùTVo ………………………………………………………………………….. ûUVm…………………Lp®V±Ü CpXôRYtÏ (ûLúWûL ûYjRYoLÞdÏ) CÕ AY£Vm úRûY Nôh£«u ûLùVôlTm /……………..…….. CPm ……………….. úR§ ...... ùTVo Utßm ®XôNm ………………………………………………………… APPENDIX - II

PROFORMA

Name: Age: Sex: Male/Female

OP no: Diagnosis: Tinea corporis/ Tinea cruris

Medical History: H/o Diabetes / HIV

Drug History: H/o steroid / immuno suppressant / systemic antifungal intake

H/o Hypersensitivity to antifungals

Size of lesion: No. of lesions: Duration of lesions:

EFFICACY PARAMETERS:

Characteristics Baseline 1 2 3 4 6 8 week weeks weeks weeks weeks weeks 1.KOH(+/-)

2.Culture(+/-)

3.Erythema

4.Scaling

5.Papules

6.Pruritis

7.GAS OUTCOME:

Cure Mycological cure Clinical cure Complete

clearance

1 week(+/-)

EOT(+/-)

CULTURE REPORT DATE

SPECIES APPENDIX 3 1% LULICONAZOLE GROUP S No Age Sex Type Size No Duration KOH CLINICAL SYMPTOM SCORE 1-M 1-corporis (cm) Of 1-P 2-N ERYTHEMA SCALING PRURITIS PAPULES GAS 2-F 2-cruris Lesions 1-3days B I II VI B I II VI B I II VI B I II VI B I II VI B I II VI 1-1 2-3-7days 2-≥2 3->7days 1 39 1 1 3×2 1 1 1 2 2 2 2 1 0 0 2 1 0 0 3 1 0 0 2 1 0 0 9 4 0 0 2 48 2 1 4×5 2 1 1 2 2 2 3 2 1 0 3 1 0 0 3 1 0 0 2 0 0 0 11 4 1 0 3 48 1 1 8×5 1 1 1 2 2 2 2 1 0 0 2 0 0 0 2 1 0 0 3 2 1 0 9 4 1 0 4 20 2 1 6×4 2 1 1 2 2 2 2 1 0 0 3 1 0 0 3 1 0 0 2 1 0 0 10 4 0 0 5 18 1 1 6×5 1 1 1 2 2 2 2 1 0 0 3 0 0 0 2 1 1 0 2 0 0 0 9 2 1 0 6 40 2 1 2×4 1 2 1 2 2 2 3 1 0 0 3 1 0 0 2 1 0 0 2 1 0 0 10 4 0 0 7 30 2 1 4×5 2 1 1 2 2 2 3 2 0 0 3 1 0 0 2 2 1 0 2 2 0 0 10 7 1 0 8 26 1 2 10×6 2 2 1 2 2 2 3 1 0 0 3 1 0 0 3 2 0 0 2 0 0 0 11 4 0 0 9 12 2 1 10×7 1 1 1 2 2 2 3 1 0 0 3 1 0 0 2 1 0 0 2 0 0 0 10 3 0 0 10 15 2 2 5×4 1 2 1 2 2 2 2 1 0 0 3 0 0 0 3 2 0 0 2 0 0 0 10 3 0 0 11 20 2 1 3×3 1 3 1 1 2 2 3 1 0 0 2 0 0 0 2 1 0 0 2 0 0 0 9 2 0 0 12 14 1 1 2×2 1 1 1 1 2 2 2 1 0 0 2 0 0 0 3 2 1 0 1 0 0 0 8 3 1 0 13 17 1 2 5×6 1 1 1 2 2 2 3 2 1 0 2 0 0 0 2 1 0 0 2 0 0 0 9 3 1 0 14 57 2 1 3×5 2 1 1 2 2 2 2 1 0 0 2 0 0 0 3 1 0 0 3 1 0 0 10 3 0 0 15 20 2 2 4×6 1 2 1 1 2 2 3 1 0 0 2 0 0 0 2 1 0 0 2 0 0 0 9 2 0 0 16 28 2 1 3×3 1 3 1 1 2 2 2 1 0 0 2 0 0 0 3 2 1 0 3 2 0 0 10 5 1 0 17 12 1 1 4×4 1 1 1 2 2 2 3 2 0 0 3 0 0 0 3 1 0 0 2 1 0 0 11 4 0 0 18 12 1 1 3×3 1 2 1 2 2 2 2 1 0 0 3 1 0 0 3 1 0 0 2 1 1 0 9 4 1 0 19 42 1 1 6×5 1 3 1 2 2 2 3 1 0 0 2 0 0 0 3 2 0 0 3 1 0 0 11 4 0 0 20 16 1 1 3×3 1 1 1 2 2 2 3 2 1 0 2 1 0 0 3 1 0 0 2 0 0 0 10 4 1 0 21 45 2 1 4×3 1 1 1 2 2 2 2 1 0 0 2 0 0 0 3 1 0 0 2 0 0 0 9 2 0 0 22 20 1 1 3×3 1 1 1 2 2 2 3 1 0 1 2 0 0 0 3 2 1 0 3 1 0 0 11 4 1 1 23 50 2 1 10×6 1 2 1 2 2 2 3 1 0 0 3 0 0 0 3 1 0 0 2 1 0 0 11 3 0 0 APPENDIX 3 1% LULICONAZOLE GROUP 24 13 1 2 5×8 2 3 1 2 2 2 2 2 0 0 3 0 0 0 3 1 0 0 3 2 0 0 11 5 0 0 25 42 2 1 4×5 1 2 1 2 2 2 2 1 0 0 3 2 1 0 2 1 0 0 2 1 0 0 9 5 1 0 26 36 2 1 4×4 1 2 1 2 2 2 1 0 0 0 3 0 0 0 2 1 0 0 1 0 0 0 6 1 0 0 27 13 1 1 3×4 1 2 1 2 2 1 2 0 0 1 2 1 0 1 3 2 1 1 2 1 1 1 9 4 2 4 28 20 2 2 5×4 2 2 1 1 2 2 2 2 0 0 3 0 0 0 2 1 0 0 1 0 0 0 8 3 0 0 29 22 1 2 3×4 1 1 1 2 2 2 3 1 0 0 3 0 0 0 3 2 0 0 2 1 0 0 11 4 0 0 30 35 1 2 6×5 2 3 1 1 2 2 3 1 0 0 2 0 0 0 2 1 0 0 2 1 0 0 9 3 0 0 31 15 1 1 5×8 1 2 1 2 2 2 1 0 0 0 2 0 1 0 3 1 0 0 3 1 0 0 9 2 1 0 32 14 1 1 4×6 2 1 1 1 2 2 2 1 0 0 1 1 0 0 3 1 0 0 3 2 1 0 9 5 1 0 33 35 1 2 2×4 2 1 1 2 2 1 3 0 0 0 2 0 0 0 3 1 0 0 2 1 0 0 10 2 0 0 34 59 2 1 4×6 1 3 1 2 2 2 2 1 0 0 2 1 0 0 3 1 0 0 2 0 0 0 9 3 0 0 35 40 2 1 3×4 1 2 1 2 2 2 1 0 0 0 3 1 0 0 3 1 0 0 3 1 0 0 10 4 0 5 36 33 2 1 5×4 1 1 1 2 2 1 3 1 0 1 3 1 0 2 2 1 0 1 2 1 0 1 10 4 0 5 37 34 1 2 10×6 2 1 1 1 2 2 2 2 1 0 3 0 0 0 3 2 0 0 1 0 0 0 9 5 1 0 38 31 2 2 4×5 2 1 1 1 2 2 2 1 0 0 3 0 0 0 2 1 1 0 3 1 1 1 10 3 2 1 39 47 2 2 3×5 1 1 1 1 2 2 3 1 0 0 1 1 0 0 3 1 0 0 2 0 0 0 9 3 0 0 40 52 2 2 4×4 1 1 1 2 2 2 2 1 0 0 2 1 0 0 2 1 0 0 2 0 0 0 8 3 0 0 41 15 1 2 2×4 1 2 1 2 2 2 3 1 0 0 2 0 0 0 3 1 0 0 3 1 0 0 11 3 0 0 42 42 2 1 3×3 1 1 1 2 2 2 2 1 0 0 2 0 0 0 2 1 0 0 3 1 0 0 9 3 0 0 43 22 2 2 4×6 2 2 1 2 2 2 3 1 0 0 2 1 0 0 3 1 0 0 3 1 1 0 11 4 1 0 44 17 1 2 3×5 2 2 1 2 2 2 2 1 0 0 2 1 1 0 2 1 1 0 2 1 0 0 8 4 2 0 45 42 2 1 6×5 1 2 1 2 2 2 3 1 0 0 2 0 0 0 2 1 0 0 2 0 0 0 9 2 0 0 46 23 1 1 4×5 1 2 1 2 2 2 3 1 0 0 2 1 0 0 3 2 0 0 2 1 0 0 10 5 0 0 47 27 2 1 2×4 1 2 1 2 2 2 3 1 0 0 2 1 1 0 3 1 1 0 2 0 0 0 10 3 2 0 48 18 1 1 10×7 1 1 1 2 2 2 3 1 0 0 2 0 0 0 2 1 0 0 1 0 0 0 8 2 0 0 49 29 2 1 4x4 1 2 1 1 1 1 2 2 2 2 2 2 2 2 1 1 1 1 1 0 0 0 5 5 5 5 50 55 1 1 5x4 1 2 1 2 2 2 3 1 1 1 3 1 1 1 1 0 0 0 1 0 0 0 8 2 2 2 B-Baseline, I-1 week, II-2 weeks, VI-6 weeks,GAS-Global Assessment Score 1% CLOTRIMAZOLE GROUP KOH CLINICAL SYMPTOM SCORE 1-P 2-N

F) ERYTHEMA SCALING PRURITIS PAPULES GAS - M,2 - Age SNo Type Duration Size(cm) No o Lesions o No Sex(1 I I I I I I II II II II II II B B B B B B III III III III III III IV IV IV IV IV IV VIII VIII VIII VIII VIII VIII

1 35 2 1 2×4 2 1 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 2 38 2 1 4×5 1 2 1 2 2 2 1 1 3 3 2 0 0 2 2 2 1 1 1 2 3 2 2 2 0 2 2 1 1 0 0 1 10 8 6 4 1 7 3 24 1 2 10x6 2 1 1 1 1 1 2 2 3 2 2 1 0 0 2 1 1 1 0 0 3 2 2 1 0 0 2 1 1 1 0 0 10 6 6 4 0 0 4 15 2 2 10×7 2 1 1 1 1 1 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 5 3 1 0 5 19 1 2 5×4 1 3 1 1 1 1 2 2 3 2 2 1 1 0 2 2 1 0 0 0 3 2 2 1 1 0 2 2 1 1 1 0 10 8 6 3 3 0 6 14 1 1 3×3 1 2 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 7 40 2 1 2×2 1 1 1 1 1 1 2 2 2 2 1 1 0 0 3 2 2 1 1 0 3 2 2 0 0 0 3 2 1 0 0 0 11 8 6 2 1 0 8 70 1 1 5×6 1 1 1 2 2 2 2 2 3 2 2 2 1 0 2 1 1 1 1 0 3 1 2 1 0 0 1 1 1 0 0 0 9 6 6 4 2 0 9 25 1 2 3×5 2 1 1 1 1 1 2 2 2 1 1 0 0 0 1 1 1 0 0 0 3 2 1 2 0 0 2 1 1 0 0 0 7 5 4 2 0 0 10 25 1 1 6×5 1 1 1 1 1 1 2 2 1 2 1 0 0 0 2 1 1 0 0 0 3 2 2 1 1 0 3 1 1 1 0 0 9 6 5 2 1 0 11 50 1 1 3×3 1 1 1 1 1 1 2 2 3 2 2 2 1 0 2 1 2 2 2 1 3 2 2 2 1 0 3 2 1 0 0 0 11 7 7 6 4 1 12 35 2 1 4×3 2 2 1 2 2 2 1 1 3 1 0 0 0 2 3 2 1 1 0 2 3 2 2 1 1 2 2 2 1 0 0 1 11 7 4 2 1 7 13 34 2 1 9×5 1 2 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 14 45 2 1 6×5 1 1 1 1 1 1 1 1 3 2 1 0 0 1 2 1 0 0 0 2 3 2 2 1 1 1 3 1 1 1 0 1 11 6 4 2 1 4 15 45 2 1 3×3 1 2 1 1 2 2 2 2 1 2 1 1 0 0 2 2 3 2 1 0 3 2 2 1 1 0 2 1 2 0 0 0 8 7 8 4 2 0 16 15 2 1 4×3 1 1 1 1 1 1 2 2 2 2 1 1 0 1 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 5 3 1 1 17 45 2 1 3×3 1 1 1 1 1 1 2 2 2 2 1 2 1 1 2 1 1 1 0 0 3 2 2 1 0 0 2 1 1 1 0 0 9 6 5 5 1 1 18 40 2 1 10×6 1 2 1 1 1 1 2 2 1 1 0 0 0 0 2 2 1 0 0 0 3 2 2 1 0 0 3 2 1 0 0 0 9 7 4 1 0 0 19 49 1 1 10×7 2 1 1 1 1 1 2 2 2 2 1 1 1 0 1 2 2 1 1 0 3 3 1 1 0 0 2 2 2 1 1 0 8 9 6 4 3 0 20 21 2 2 5×4 2 1 1 1 1 2 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 5 3 1 0 21 26 2 1 3×3 1 1 1 1 1 1 2 2 3 3 2 2 1 0 2 1 1 1 1 0 3 1 2 1 0 0 1 1 1 0 0 0 9 5 6 4 2 0 1% CLOTRIMAZOLE GROUP 22 15 1 1 2×2 1 1 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 23 16 1 1 5×6 1 2 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 24 14 1 2 3×5 1 1 1 1 2 2 2 2 3 2 2 2 1 0 2 1 2 2 2 1 3 2 2 2 1 0 3 2 1 0 0 0 11 7 7 6 4 1 25 44 1 2 4×3 1 2 1 1 1 1 1 1 3 2 1 0 0 1 3 2 1 1 1 1 2 1 0 0 0 2 2 2 1 1 0 1 10 7 3 2 1 5 26 36 2 1 6×5 1 2 1 1 1 1 2 2 2 3 2 2 1 0 2 1 1 1 1 0 3 1 2 1 0 0 1 1 1 0 0 0 7 6 6 4 2 0 27 40 1 1 5×4 1 2 1 2 2 2 1 1 3 2 1 1 0 1 2 2 1 1 0 2 3 2 2 1 0 0 2 1 1 0 0 1 10 7 5 3 0 4 28 14 1 2 10×6 2 1 1 1 1 1 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 6 3 1 0 29 20 1 1 6×5 2 3 1 1 1 1 2 2 1 1 0 0 0 0 2 2 1 0 0 0 3 2 2 1 0 0 3 2 1 0 0 0 9 7 4 1 0 0 30 42 2 2 5×4 1 1 1 1 1 1 1 1 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 31 16 1 1 6×5 1 1 1 1 1 1 2 2 2 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 10 9 7 5 2 0 32 27 2 1 3×3 2 1 1 1 1 2 2 2 1 1 0 0 0 0 2 1 1 0 0 0 2 1 2 1 1 0 2 1 1 1 0 0 7 4 4 2 1 0 33 46 2 1 10×6 1 2 1 1 1 1 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 5 3 1 0 34 28 2 1 5×4 2 2 1 1 1 1 2 2 2 2 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 10 8 7 5 2 0 35 30 1 1 3×3 1 1 1 1 1 1 2 2 1 2 1 0 0 0 2 1 1 0 0 0 3 2 2 1 1 0 3 1 1 1 0 0 9 6 5 2 1 0 36 43 2 1 2×2 1 2 1 1 1 1 2 2 2 2 2 2 1 1 2 2 1 2 1 1 2 1 2 1 1 0 2 1 0 1 0 0 8 6 5 3 6 2 37 28 1 1 5×6 1 2 1 1 1 1 2 2 2 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 10 9 7 5 2 0 38 36 2 1 5×4 1 3 1 1 1 1 2 2 1 1 1 0 0 0 2 1 1 0 0 0 2 3 2 1 0 0 2 1 1 1 0 0 7 6 4 1 0 0 39 18 1 2 2×4 2 2 1 1 2 2 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 1 1 0 9 7 5 4 2 0 40 14 2 1 3×3 1 1 1 1 1 1 1 1 1 2 1 0 0 1 3 2 1 1 1 1 2 1 0 0 0 2 2 2 1 1 0 1 7 6 3 2 3 6 41 46 1 2 4×8 1 1 1 1 1 1 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 4 3 1 0 42 19 1 2 2×6 2 2 1 1 1 1 2 2 1 2 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 9 8 7 5 2 0 43 33 2 1 3x6 1 2 1 1 1 1 2 2 2 3 2 2 1 0 2 1 1 1 1 0 3 1 2 1 0 0 1 1 1 0 0 0 7 6 6 4 2 0 44 42 2 1 5×4 2 3 1 2 2 2 1 1 3 2 1 1 0 1 2 2 1 1 0 2 3 2 2 1 0 0 2 1 1 0 0 1 10 7 5 3 0 4 45 22 1 1 3x8 1 2 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 46 35 1 2 5x3 2 1 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 47 28 1 1 4×3 2 2 1 2 2 2 1 1 3 1 0 0 0 2 3 2 1 1 0 2 3 2 2 1 1 2 2 2 1 0 0 1 11 7 4 2 1 7 48 65 2 2 9×5 1 2 1 1 1 1 2 2 3 3 2 1 0 0 3 2 2 1 0 0 2 2 1 1 1 0 3 2 2 2 1 0 11 9 7 5 2 0 49 43 1 1 4x6 1 1 1 1 1 1 1 1 3 2 1 0 0 1 2 1 0 0 0 2 3 2 2 1 1 1 3 1 1 1 0 1 11 6 4 2 1 4 50 29 2 1 5x8 1 2 1 1 1 1 2 2 2 2 1 1 0 0 2 2 1 1 0 0 3 1 2 1 1 0 2 2 1 0 0 0 9 7 5 3 1 0 B-Baseline, I-1 week,II-2 weeks,III-3 weeks,IV-4 weeks, VIII-8 weeks ,P-positive , N-negative, GAS-global assessment score BIBILIOGRAPHY

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