Clinicomycological Profile of the Dermatophytes And
CLINICOMYCOLOGICAL PROFILE OF THE DERMATOPHYTES AND
ITS ANTIFUNGAL SUSCEPTIBILITY PATTERN IN PATIENTS
ATTENDING DERMATOLOGY OUT PATIENT DEPARTMENT IN
A TERTIARY CARE HOSPITAL
Dissertation submitted to THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY
in partial fulfillment of the regulations for the award of the degree of
M.D. (MICROBIOLOGY) BRANCH – IV
CHENGALPATTU MEDICAL COLLEGE, THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI – TAMILNADU APRIL 2017 CERTIFICATE
This is to certify that this dissertation titled “CLINICOMYCOLOGICAL
PROFILE OF THE DERMATOPHYTES AND ITS ANTIFUNGAL
SUSCEPTIBILITY PATTERN IN PATIENTS ATTENDING
DERMATOLOGY OUT PATIENT DEPARTMENT IN A TERTIARY CARE
HOSPITAL.’’ is a bonafide record of work done by DR.VISHWA PRIYA.K., during the period of her Post graduate study from 2014 to 2017 under guidance and supervision in the Department of Microbiology, Chengalpattu Medical College and
Hospital, Chengalpattu – 603 001 in partial fulfillment of the requirement for M.D.
MICROBIOLOGY degree Examination of The Tamil Nadu Dr. M.G.R. Medical
University to be held in April 2017.
Dean Dr.A.VIJAYALAKSHMI, M.D.,M.B.A., Chengalpattu Medical College Professor and HOD, & Hospital Department of Microbiology, Chengalpattu – 603 001. Chengalpattu Medical College, Chengalpattu – 603 001. DECLARATION
I declare that the dissertation entitled “CLINICOMYCOLOGICAL
PROFILE OF THE DERMATOPHYTES AND ITS ANTIFUNGAL
SUSCEPTIBILITY PATTERN IN PATIENTS ATTENDING
DERMATOLOGY OUT PATIENT DEPARTMENT IN A TERTIARY CARE
HOSPITAL.” submitted by me for the degree of M.D. is the record work carried out by me during the period of March 2015 to Febraury 2016 under the guidance of Professor Dr.A.VIJAYALAKSHMI, M.D., M.B.A., Department of
Microbiology, Chengalpattu Medical College, Chengalpattu. This dissertation is submitted to the Tamilnadu Dr.M.G.R. Medical University, Chennai, in partial fulfillment of the University regulations for the award of degree of M.D.,
Microbiology (Branch IV) examinations to be held in April 2017.
Place: Chengalpattu Signature of the Candidate
Date: (DR.VISHWA PRIYA.K)
Signature of the guide Prof. Dr.A.VIJAYALAKSHMI, M.D., M.B.A., Professor & HOD, Department of Microbiology, Chengalpattu Medical College, Chengalpattu – 603 001 ACKNOWLEDGEMENT
I humbly submit this work to the Almighty who has given the health, ability and enthusiasm to pass through all the steps in the compilation and proclamation of my dissertation.
I wish to express my sincere thanks to our Dean,
Dr.N.GUNASEKARAN. M.D.,D.T.C.D., Chengalpattu Medical College,
Chengalpattu for permitting me to use institution resources for my study.
First, I would like to express my sincere gratitude to my beloved Head of the Department, my Prof. Dr.A.Vijayalakshmi M.D., M.B.A., Professor& HOD, for her guidance, encouragement, supervision, timely suggestions and support during my carrier.
I extend my whole hearted gratitude to my
Prof. Dr. V. Dillirani, M.D., D.G.O., Department of Microbiology for her constant support, invaluable suggestions and erudite guidance in my study and support in my carrier.
I also convey my sincere thanks to Dr.M.Subha, M.D., D.G.O.,
Associate professor, for her valuable support during the period of my study.
I owe very special thanks to our Assistant professors Dr.J.Rajeshwari M.D.,
Dr.Rathinam Suresh M.D., for their valuable guidance and constant support in my study. I also express my sincere thanks to our former Assistant professors, Dr. A. V. Sowmya M.D., D.C.P., Dr. J. Padmakumari M.D.
Dr.Nithya.C M.D., DR.R.Rajeswari M.D., for their support and encouragement in my study.
My thanks to all the technical and the non technical staffs of Department of Microbiology, for their help at different stages of my study.
I would like to thank the Institutional Ethics Committee for approving my study.
I thank my parents Dr.P.Kumar & Mrs.Rajamani Kumar who have been solid pillars of everlasting support and encouragement and for their heartfelt blessings.
I affectionately thank my husband Dr.L.Babu for his constant love, support and encouragement without which this work would have not been possible.
Finally, I thank my colleague Dr.Ananthakrishnan.P., my senior and junior postgraduates and all staffs of Department of Microbiology, Chengalpattu
Medical College and for those who had enrolled in my study and gave their maximum co-operation and consent for the success.
Last but not the least I am very grateful to all the patients without whom this study would not have been completed.
TABLE OF CONTENTS
S.No TITLE PAGE.NO
1. INTRODUCTION 1
2. AIMS AND OBJECTIVE 7
3. REVIEW OF LITERATURE 8
4. MATERIALS AND METHODS 57
5. RESULTS 74
6. DISCUSSION 101
7. SUMMARY 108
8. CONCLUSION 109 114 9. BIBLIOGRAPHY (i) ABBREVIATIONS 10. (ii) ANNEXURES (iii) PATIENT PROFORMA (iv) FLOW CHART (v) MASTER CHART (vi) KEY TO MASTER CHART (vii) ETHICAL COMMITTEE CERTIFICATE LIST OF TABLES
TABLE-1 Distribution of age groups among the study population TABLE-2 Frequency distribution of gender TABLE-3 Risk factors TABLE-4 Distribution of clinical specimens among study population TABLE-5 Distribution of clinical diagnosis among study population TABLE-6 Evaluation of KOH mount TABLE-7 Evaluation of culture TABLE-8 Correlation between direct microscopy (KOH examination) and culture
TABLE-9 Correlation between direct microscopy (KOH examination) and culture in clinical cases
TABLE-10 Percentage of dermatophytes isolated in the study population TABLE-11 Distribution of cultures in collected samples TABLE-12 Distribution of the genus of the isolates among the dermatophytosis group TABLE-13 Distribution of fungal isolates among the dermatophytosis group TABLE-14 Dermatophyte species isolated from the skin sample
TABLE-15 Dermatophyte species isolated from the nail sample TABLE-16 Dermatophyte species isolated from the hair sample
TABLE-17 Minimal inhibitory concentration(MIC) of the drug griseofulvin
TABLE-18 Antifungal susceptibility pattern of the drug griseofulvin to the isolated dermatophytes TABLE-19 Minimal inhibitory concentration(MIC) of the drug ketoconazole TABLE-20 Antifungal susceptibility pattern of the drug ketoconazole to the isolated dermatophytes LIST OF TABLES
TABLE-21 Minimal inhibitory concentration(MIC) of the drug fluconazole
TABLE-22 Antifungal susceptibility pattern of the drug fluconazole to the isolated dermatophytes TABLE-23 Minimal inhibitory concentration(MIC) of the drug itraconazole TABLE-24 Antifungal susceptibility pattern of the drug itraconazole to the isolated dermatophytes TABLE-25 Minimal inhibitory concentration(MIC) of the drug terbinafine TABLE-26 Antifungal susceptibility pattern of the drug terbinafine to the isolated dermatophytes LIST OF FIGURES
Figure -1 Gender distribution of age groups among the study population Figure -2 Gender distribution Figure -3 Risk factors Figure -4 Distribution of clinical specimens among study population Figure -5 Distribution of clinical diagnosis among study population Figure -6 Evaluation of KOH mount Figure -7 Evaluation of culture Figure -8 Correlation between direct microscopy (KOH examination) and culture Figure -9 Correlation between direct microscopy (KOH examination) and culture in clinical cases
Figure -10 Percentage of dermatophytes isolated in the study population Figure -11 Distribution of cultures in collected samples Figure -12 Distribution of the genus of the isolates among the dermatophytosis group Figure -13 Distribution of fungal isolates among the dermatophytosis group
Figure -14 Dermatophyte species isolated from the skin sample Figure -15 Dermatophyte species isolated from the nail sample Figure -16 Dermatophyte species isolated from the hair sample Figure -17 Minimal inhibitory concentration(MIC) of the drug griseofulvin Figure -18 Antifungal susceptibility pattern of the drug griseofulvin to the isolated dermatophytes Figure -19 Minimal inhibitory concentration(MIC) of the drug ketoconazole Figure -20 Antifungal susceptibility pattern of the drug ketoconazole to the isolated dermatophytes LIST OF FIGURES
Figure -21 Minimal inhibitory concentration(MIC) of the drug fluconazole
Figure -22 Antifungal susceptibility pattern of the drug fluconazole to the isolated dermatophytes Figure -23 Minimal inhibitory concentration(MIC) of the drug itraconazole Figure -24 Antifungal susceptibility pattern of the drug itraconazole to the isolated dermatophytes Figure -25 Minimal inhibitory concentration(MIC) of the drug terbinafine Figure-26 Antifungal susceptibility pattern of the drug terbinafine to the isolated dermatophytes LIST OF IMAGES
Image -1 TINEA CORPORIS Image -2 TINEA UNGUIUM Image -3 KOH WET MOUNT OF DERMATOPHYTOSIS Image -4 TRICHOPHYTON RUBRUM – MACROSCOPY & MICROSCOPY Image -5 TRICHOPHYTON MENTAGROPHYTES- MACROSCOPY & Image -6 TRICHOPHYTON TONSURANS – MACROSCOPY & Image -7 MICROSPORUM CANIS- MACROSCOPY & MICROSCOPY Image -8 MICROSPORUM GYPSEUM - MACROSCOPY & MICROSCOPY Image -9 EPIDERMOPHYTON FLOCCOSUM-
Image -10 HAIR PERFORATION TEST – POSTIVE AND NEGATIVE Image -11 UREASE TEST Image -12 NON DERMATOPHYTIC MOULDS
Image -13 CANDIDA SPECIES- MACROSCOPY & MICROSCOPY Image -14 ANTIFUNGAL DRUGS FRO MIC
Image -15 RPMI 1640 BROTH
Image -16 MICROBROTH DILUTION METHOD – AFST MIC FOR GRISEOFULVIN Image -17 MICROBROTH DILUTION METHOD – AFST MIC FOR KETOCONAZOLE Image -18 MICROBROTH DILUTION METHOD – AFST MIC FOR FLUCONAZOLE Image -19 MICROBROTH DILUTION METHOD – AFST MIC FOR ITRACONAZOLE Image -20 MICROBROTH DILUTION METHOD – AFST MIC FOR TERBINAFINE CLINICOMYCOLOGICAL PROFILE OF THE DERMATOPHYTES AND
ITS ANTIFUNGAL SUSCEPTIBILITY PATTERN IN PATIENTS
ATTENDING DERMATOLOGY OUT PATIENT DEPARTMENT IN A
TERTIARY CARE HOSPITAL.
INTRODUCTION
Dermatophytes are the most common fungal infections worldwide, which consists of a group of morphologically and physiologically related filamentous fungi with the capacity to penetrate keratinized tissues of humans and other animals and produce dermatophytosis (1).These fungi produce proteases that digest and use keratin as a source of nitrogen and permits infection, colonization and invasion of the stratum corneum of the skin, nail and hair shaft(2). These fungi cannot penetrate the deeper tissues of healthy immunocompetent individuals. So, the infection is generally restricted to the dead cornified layers(3,4).
Depending upon the anatomical site and the etiological agent, dermatophytes are classified into different varieties. The terms like ‘tinea’ and ‘ ring worm’ infection are synonyms of word dermatophytosis .The second part of the name of the dermatophytosis identifies the part of the body infected, i.e.in case of Tinea corporis, “corporis ” refers to the body(5, 6). The diseases produced by non dermatophytic fungi infecting skin are called as dermatomycoses, whereas the diseases produced by non dermatophytic fungi infecting hair and nail are called as piedra and onychomycosis respectively (7).
1 World Health Organization estimates that the global prevalence of superficial fungal infections has been found to be 20-25%(8,9). There are several reports on intercontinental variability on the worldwide incidence because of the change in climatic conditions and it is more prevalent in tropical and subtropical countries like India where the heat and humidity is high for most parts of the year, which helps in acquisition and maintenance of the mycotic diseases(1,10,31). Studies on dermatophytosis in India have received increased attention in recent years because one fifth of the world’s population suffers from superficial mycosis(12,11,
12). Primarily it depends on the habits and living condition of the people as it is transmitted through fomites. Sex, race and occupation have a little differential influence upon the frequency of dermatophytosis, however changes in trends are noticed(13,14).
The risk factors include overcrowding, poverty, poor personal hygiene, poor peripheral circulation, immunosuppressive therapy, cancer chemotherapy and immunocompromised conditions(15,16). Mode of transmission is by contact with the infected person / pet animals, fomites or auto inoculation from another body site. The superficial fungal infections of man are the diseases in which the integument and its appendages, the hair and the nail are involved. The non lining cornified layers are generally infected and due to the presence of infectious agent and its metabolic products, variety of changes occurs in the host. Several clinical types with distinct pathology are produced by a single species(17).
2 Based on clinical, morphological and microscopic characteristics, dermatophytes are classified into three genera; (1) Trichophyton, (2)
Microsporum, and (3) Epidermophyton (18).They are differentiated based on macroscopic and microscopic features. Based on their ecological characteristics, dermatophytes are classified into (a) anthropophilic, (b) zoophilic and (c) geophilic.
(a) Anthropophilic fungus:-
- Exclusively affect humans, tend to be chronic with little inflammation
(b) Zoophilic fungus:-
- Exclusively affect domestic and wild animals
(c) Geophilic fungus:-
- Frequently isolated from soil
- Associated with acute inflammation.
-Rural people working in the fields, gardens with manure and decaying organic matter in the soil are all in the risk of getting these fungal infections(19).
Based on the anatomical site and the causative agent involved, dermatophytes are classified into several distinct clinical varieties.
The major role in the pathogenesis of dermatophytes is played by the host response. Most of the clinical manifestations are due to the immune response of the host to the causative fungal agent. The type and severity of the disease are
3 mainly related to the immune status of the host as well as to the strain and species of the fungal agent causing the infection(4). Reactions to a dermatophytes infection may range from mild to severe as consequences of
(i) the emcee’s reaction to the metabolic products of the fungus which diffuse through the Malphigian layer of the epidermis to cause erythema, vesicles and even pustule formation along with pruritis(4,115,30),
(ii) Virulence of the infecting strain or species,
(iii) Anatomical location of the infection and
(iv) Local environmental factors.
Dermatophytosis produces a dermal inflammatory response with intense itching and also of cosmetic problems. The characteristic skin lesion is red and circular scaly patch with sharply demarcated margin(20, 21). The hyphae become old and break into arthrospores, which are shed off in due course of time which is held responsible for the central clearing of the lesion. The estimated life time risk for a person to acquire infection of dermatophytes is 10% -20%. Dermatophytosis is highly contagious and represents a major public health problem. The incidence of dermatophytosis has increased over recent years, particularly in geriatric and pediatric population (22) and in immunocompromised patients (23).
Modified Sabouraud’s Dextrose Agar (SDA) with cycloheximide, chloramphenicol and gentamycin is used for the culture of the dermatophytes(12,24,10). There are many antifungal agents that are available to treat
4 dermatophytes. But not all species of dermatophytes have the same susceptibility pattern and relative or absolute resistance may occur (9). The choice of proper treatment is determined by the site and extent of the infection, the species involved the efficacy, safety profile, and the kinetics of the available drugs.
A breakthrough experimental work done by Gentles in guinea pig in 1958 was the discovery of drug griseofulvin revolutionized the therapeutic approach to this disease. Azoles and its allied groups of antifungal drugs discovered in 1980 had significant impact in the treatment of dermatophytosis(4). In recent decades, the increasing use of antibiotics, immunosuppressive drugs, and prosthetic implanted devices, transplantations of organs, and the appearance of Acquired
Immune Deficiency Syndrome (AIDS) have resulted in dramatic increase in the susceptibility of the patients to the severe fungal infections and a subsequent increase in the development of systemic antifungal agents(25,2).
As fungal infections became a significant public health problem, the antifungal drug resistance also began to emerge. There is a continuous development of newer antifungal agents with broader spectrum activity and also with different target of activity. But in immunocompromised patients, the prognosis is very poor. Therefore it is very essential to diagnose and to start the treatment as soon as possible in order to reduce the emergence of resistant strains and morbidity(26). Now many effective drugs are available for the treatment of fungal infections. Standardization of in vitro antifungal susceptibility testing
5 provides a consistent and reproducible data that may predict clinical response when used in adjunct with individual patients’ risk factors.
Antifungal susceptibility test helps the clinician to select a specific antifungal agent. An ideal antifungal agent should have a broad spectrum activity, with no drug resistance and it should be effective in vivo. The development of
National Committee for Clinical Laboratory Standards (NCCLS) reference method M38A in the1990’s has improved the reproducibility of in vitro antifungal susceptibility test data and facilitated the establishment of interpretive break points for the triazoles (posaconazole,ravuconazole and voriconazole), fluconazoles and itraconazoles(23,27).
The present study is therefore undertaken to know the prevalence of dermatophytic infections and its antifungal susceptibility pattern in our institution by analyzing the patients attending the dermatology OPD in a tertiary care hospital during the period of March 2015 to Febraury 2016.
6 AIMS AND OBJECTIVES
• To study the prevalence of superficial mycoses in patients attending
dermatology outpatient department.
• To isolate and speciate the dermatophytic agents from clinical specimens
like skin, hair and nail obtained from patients attending dermatology OPD.
• To study the clinical and mycological profile of superficial mycoses.
• To identify the commonest prevalent genus and species of dermatophytes.
• To correlate different species of dermatophytes and its clinical
manifestation.
• To study the antifungal susceptibility pattern of the isolated fungi.
• To determine the MIC values of different drugs for the isolates.
7 REVIEW OF LITERATURE
HISTORY
Mycology is the study of fungi came into existence before bacteriology, in
1677 when Hooks studied the yellow spots on the leaves of Demask rose with the help of a magnifying lens and sighted the filamentous organism. Subsequent studies were carried out by Malphigi (1686) ; Mitchelli (1729) and Linnaeus
(1752)(28,29).
Historically, the microbial nature of decaying disease of silkworms
(Bombyx mori) was first elucidated by Agostino Bassi (1835-36).He established clearly that a mold called Beauveria bassiana was the etiological agent of this disease(30,1). These fungal etiologies were described as moulds by Professor
Johann L Schonlein in 1839 and plants were considered to the source of infection. In 1873, hyphal elements in the scalp scrapings were first observed by
Robert Remak. The etiological agent was described by Robert Remak and it was named as Achovion scholeinleini. This discovery was credited by him to his mentor Professor Johann L Schonlein(30,1).
David Gruby, the Parisian physician was the Real founder of Medical
Mycology. Based on his discoveries (1841 -1844) , he published a paper in which he described the isolation of fungus of favus on potato slices and production of the diseases by inoculating onto normal skin .Thus he was the first to establish the role of microorganism in the causation of favus. In addition he described the dermatophyte, Microsporum audouinii from tinea capitis and recognised the
8 endothrix form of Trichophyton(1). Malmsten defined the genus Trichophyton and
Trichophyton tonsurans. In 1847, Charles Robin defined Trichophyton mentagrophytes, who was the first to discuss the topical therapy for dermatophytes infections.
Domenico Majocchi (1849 -1929) first described Majochi’s granuloma, a variant of Tinea corporis, which is an uncommon dermatophytic infection of dermal and subcutaneous tissue .This disorder was named as ‘granuloma tricofitico’ in 1883. In 1892 Raymond Sabouraud, a French dermatologist established the ringworm fungi plurality and integrated the mycological and clinical aspects of ringworm. Based on the advances made by his contemporaries in medicine and veterinary science as well as on his personal observations, in1910
Sabouraud wrote and published his monumental Les-Teignus. He classified dermatophytes into four genus Achorion, Epidermophyton, Microsporum and
Trichophyton based on the clinical aspects of their diseases , culture and microscopic characters(33).
In the 1920, Johns Hopkins and Benham began their scientific study in medical mycology and then Rhoda Williams Benham was called as the ‘Founder of modern medical mycology’. The laboratory at Columbia University was one of the first to study clinical mycology systematically. In 1925 Robert W.Wood, a
Baltimore physicist invented Wood’s lamp for detection of fungal infection of hair(1).
9 Sabouraud’s taxonomic scheme was modified by Chester Emmons in
1934(1). He established current classification of dermatophytes on the basis of spore morphology and other structures and discarded the genus Achorion and redefined the three anamorphic genus based on the colonial morphology and other relatively formed microscopic structures.
(i) Epidermophyton,
(ii) Microsporum and
(iii) Trichophyton
Weitzman et al restudied the teleomorph state of all dermatophytes with sexual phase. The discovery of teleomorphs of Trichophyton by Ajello, Dawsan and Gentles in1959 using hair bait technique of Vanbreusegham led to discovery of teleomorphs of many dermatophytes and related keratinophilic fungi(31). Griffin and Stockdale in 1960s independently obtained teleomorphs of Microsporum gypseum complex, there by proved Nannizzia’s original observation of the sexual stage(1).
In 1958 Griseofulvin was discovered by Gentles after his work on guinea pigs. In 1969, Taplin and co-workers developed Dermatophyte Test Medium
(DTM) to isolate and differentiate dermatophyte from other fungal and bacterial contaminant in cutaneous lesions(35).
Blank and co-workers established the drug dosages and treatment schedules which were widely accepted as the recommended therapy for all forms of dermatophytoses. Many topical preparation have also been introduced of which
10 tolnaftate has become popular. In 1980 discovery of azole derivatives and allied group of antifungal drugs had significant impact on the management of dermatophytosis as topical agents(1). Treatment failures and relapses occur with all the presently available antifungal drugs. Hence there is a need for better therapeutic agents.
MYCOLOGY
Most of the fungal infection of the skin, nail and hair are caused by dermatophytes, Candida and Pityriasis versicolor. Other less frequent infections of skin and hair include tinea nigra and piedra. In addition there are number of non dermatophyte moulds that can cause nail infections (onychomycosis)(36).
TAXONOMY OF DERMATOPHYTES
Kingdom : Eumycota
Phylum : Ascomycota
Class : Euascomy
Order : Onygenales
Family : Arthrodermataceae
Genus : Trichophyton 24 species
Microsporum 16 species
Epidermophyton 2 species
11 The Trichophyton species usually infects skin, hair and nails. Microsporum species infect skin, hair and not the nails and Epidermophyton species infect skin as well as nails but not the hair(1).(Sherne et al 1993)
ECOLOGY
Depending on their ecological characteristics, dermatophytes are classified into 3 groups as,
(i) Geophilic species - soil
(ii) Zoophilic species – animals
(iii) Anthropophilic species – humans
(i) GEOPHILIC SPECIES :-
The soil contains many keratinophilic fungi closely related to the
dermatophytes genera and are secondarily transmitted by animals to
humans(31,36). These include Microsporum fulvum, Microsporum gypseum,
Trichophyton ajelloi, Trichophyton terrestre. Most of these organisms are
rarely isolated from human infections.
(ii) ZOOPHILIC SPECIES :-
Zoophilic species evolved from geophilic species and can cause human
infections(31,36). Infection of the zoophilic dermatophytes like Microsporum
canis can involve the domestic animals as the principal carriers. (DeVroey
1985, McGinnis 1985, Kwong-Chung 1992).
12 Trichophyton verrucosum and Trichophyton mentagrophytes var mentagrophytes are most commonly acquired from cattle. In Tinea corporis,
Tinea capitis and Tinea barbae, fomites play a major role in the transmission of Trichophyton mentagrophytes var quinckeanum (Georg 1960).
Trichophyton mentagrophytes var mentagrophytes (granular variety) is carried by rodents and it can be transmitted to laboratory workers. (Georg 1960,
Sewell 1995)(31).
Infections acquired from zoophilic species are inflammatory hence resolves spontaneously that infection caused by anthropophilic species which are non inflammatory. (Rippon 1988, Kwong-Chung 1992). Zoophilic agents are
Microsporum gallinae, Trichophyton equinum and Microsporum nannum.
(iii) ANTHROPOPHILIC SPECIES :-
Anthropophilic species are the most common cause of human infection and has evolved from zoophilic species(31,36). Since the arthroconidia and chlamydoconidia shed in the environment along with the desquamated epithelium, these species are highly contagious. (Mc Pherson 1957, Kwong-
Chung 1992 )
Fomites also play an important role and infection can also be acquired through aerosolisation of arthroconidia into the air. (eg) Microsporum audouinii – ectothrix Tinea capitis and Trichophyton tonsurans – endothrix Tinea capitis in children (Houchins and Pugliase 1991) and in adults Tinea corporis , Tinea manuum , Tinea unguium (Summerbell , Weitzman 1995).
13 Trichophyton tonsurans can cause nosocomial infection through
aerosolisation. Trichophyton rubrum, Trichophyton mentagrophytes and
Epidermophyton floccosum can cause Tinea pedis, Tinea cruris and Tinea
unguium but varies with the geographical locations. Other anthropophilic
dermatophytes species like Trichophyton violaceum, Trichophyton schonlenii
can also be transmitted through fomites( Kwong-Chung 1992).
Trichophyton concentricum can be vertically transmitted from the mother to
the neonate after birth either directly (Rippon 1988) or via fomites(DeVroey
1985). Tinea pedis is acquired by direct exposure to the fungal spores seen
commonly in shower heads, swimming pool and locker rooms (Gentles 1957).
Epidemiological evidence has analysed by Gentles et al (1957) strongly
documented a threefold increase in the incidence of Tinea pedis cases among
coal miners using the communal shower(31).
EPIDEMIOLOGY
Generally dermatophytes have universal distribution. But some species are endemic to certain parts of the world. It affects 20%-25% of the world’s population and the incidence continues to rise(52,38). Disruption of this epidemiological pattern occurs due to increasing migration of World population.
Hence the importance should be given in identifying the etiology, distribution, pathology for the treatment purposes.
Dermatophytes survive at 25ÛC-28ÛC. Hence in tropical countries, the hot and humid warm condition on human skin supports its growth. In Indians, closure
14 of the infected sites appears to increase the susceptibility to chronic infections by increasing the hydration and the emission of CO2 which enhances the growth of dermatophytes (king et al 1978).
In North India anthropophilic species are the most common pathogen causing tinea capitis. In a study on 153 consecutive patients with tinea capitis,
90% of the patients were aged less than 15 years and the causative strains were
Trichophyton violaceum (38%), Microsporum audouinii (34%), Trichophyton schoenlenii (10%) and Trichophyton tonsurans (10%)(9).
In South India, Trichophyton violaceum is the common species causing tinea capitis. A study in 1978, proved the prevalence of tinea capitis among boys aged 10-17 years due to the unhygienic mass scalp shaving rituals(9). The commonest causative agents of tinea pedis are Trichophyton rubrum,
Trichophyton mentagrophytes and Epidermophyton floccosum.
Immunosuppressive states especially diabetes mellitus and old age, are predisposed to chronic infections(39,40). An inherited pattern of infection with
Trichophyton concentricum is also noted(31). (Serjeantson an Lawrence 1977)
Trichophyton soundanense, Trichophyton gourvili, Trichophyton yaoundei are geographically restricted to Central Africa and West Africa. Trichophyton concentricum, the etiological agent of tinea imbricate is confined to South
America(30). Microsporum ferrugineum is predominantly seen in Japan and its surrounding area. The most common cause of Tinea capitis in children in India,
Nepal, Canada, and Europe is Trichophyton tonsurans, but Trichophyton rubrum
15 continues to be predominant in Tamil Nadu followed by Trichophyton mentagrophytes(41–43).
AGE DISTRIBUTION
Scalp infections are common in children. It can occur rarely after puberty but this infection causes scarring alopecia in adults. The reason for the preponderance of the disease in children is mainly due to the presence of the medium chain fatty acids in sebum since it inhibits the growth of dermatophytes in post pubertal individuals. In contrast to tinea capitis, tinea pedis is commonly seen in adolescents or young adults. Foot infections occur in young children occasionally, but with concomitant skin infection(44). The occurrence of onychomycosis with Tinea pedis is seen more among diabetic patients(45).
Many of the 40 species are distributed worldwide but the remaining strains are confined to specific regions. Hence the study on their ecology and epidemiology are very essential to control the infections.
IMMUNOLOGY(31)
Initially dermatophytes colonize the stratum corneum. The infection and the inflammatory reaction depend on the host immunity and the causative species.
Infection of anthropophilic species elicits less inflammatory response when compared to zoophilic and geophilic species.
16 HOST RESPONSE
x Innate immunity (Non specific)
x Acquired immunity(Specific)
INNATE IMMUNITY
The epidermis and nails synthesize a natural peptide which has antimycotic activities. Dermatophyte antigens acts like chemokines for the leukocytes in inflammatory lesions and these neutrophils kill the pathogen via the oxidative pathway. It activates the alternate complement pathway also(44).
ACQUIRED IMMUNITY
a) HUMORAL MEDIATED IMMUNITY
The humoral immune response does not arrive to help in the elimination of
infection and the highest levels of antibodies are frequently present in
patients with chronic dermatophytosis. Non specific antibodies are
generated which cross react with other dermatophytes and saprophytic
fungi.
‘Immunoglobulin E’ suppresses the cell mediated immunity through its
histamine secretion and humoral mediated immunity is capricious during
dermatophytosis. There is no standard dermatophyte antigen available to
test its sensitivity and specificity (Matsumoto et al 1996).
17 b) CELL MEDIATED IMMUNITY
Cell mediated immunity is by type IV delayed hypersensitivity reaction
arbitrated by cellular immune system which is importunate in eliminating
the infection from stratum corneum, and thus results in both mycological
and clinical cure (Dahl 1993)(51).Trichophytin skin test is positive in normal
population due to their earlier exposure to dermatophytosis or by cross
reactivity to other organisms (Grossman et al 1975)(1).
Defective T-cell mediated immune response causes chronic infection with
Trichophyton rubrum and Trichophyton concentricum. Persistent
dermatophytic infection elicits T helper 2 immune response(44). The
dermatophyte species vary among themselves in eliciting immune response
like Trichophyton rubrum causes chronic or relapsing infection while
Trichophyton verrucosum causes long term resistance to re-infection(44).
PATHOGENESIS
Dermatophytes colonize and accustom to grow in living keratinised layer of stratum corneum , nail bed, nail plate and around hair shafts. Thereby they establish equilibrium with the host and yield only less irritation to the specific host. Dermatophytes slowly flourish specialised methods of reproduction and freely disseminates from host to host, by the formation of arthroconidia(17,36). The arthrospores are the vegetative cells with thick cell wall. They relocate the infecting agents from the original specific hosts.
18 The acquirement of infection depends upon the skin surface factors such as local CO2 tension, moisture and unsaturated transferrin. For penetration it requires, zinc containing metalloproteinase(36). Human genetics also hit a role in the pathogenicity. Autosomal dominant trait is detected in some families with onychomycosis and autosomal recessive trait is detected in tinea imbricata(48).
Various host mediated factors can also curb dermatophytosis, like progesterone (Heoprich et al 1994) hence there is an increased frequency of some dermatophytosis in men. Unsaturated fatty acid in sebum also can hinder the growth of dermatophytes. Thus the generation of sebum in adult scalp defends against tinea capitis(48).
Moreover these fungal infections of hair harvest numerous saprophytic conidia in cultures. On the other hand the anthropophilic species, Trichophyton mentagrophytes var interdigitale fetches only little inflammatory reaction and induces chronic infection and also develop only few saprophytic conidia in cultures(17). On glabrous skin, dermatophytes generate the classical ring worm pattern with centrifugal spreading. Most of the dermatophytosis resolute apparently but may endures as carriers(1).
19 VIRULENCE FACTOR
x Dermatophytes yield a number of keratinoytic proteinases which have
been acknowledged as an important virulence factors. They function
most excellent at acidic pH(49).
x The molecular structure of keratin alters from species to species; hence
various keratinases have been developed with relative specificity(17).
x The cell wall of dermatophytes generates the mannan, another factor
which amplifies the pathogenicity. It acts by annihilating the cell
mediated immunity(48).
x Dermatophytes form catalase enzyme.
x Dermatophytes also secrete enzymes like chitinase or proteinases to
acquire nutrition from the epidermal structure.
CHRONIC DERMATOPHYTIC INFECTION (31,51)
Some patients are liable for chronic or recurrent infection due to the following reasons
x Skin affords an environment favorable for fungal growth and persistence.
x Recurrence owing to under treatment or short term therapy.
x Resistance to antifungal agents.
x Immunosuppression is another source of increased vulnerability to fungal
infection.
x Atopic or those who lack Cell Mediated Immunity to definite
dermatophytes.
20 CLINICAL FEATURES
The clinical feature is the aggregate of direct tissue damage and host immunity. In macerated skin as seen in tinea pedis and tinea cruris, infection is exaggerated(48).
Clinical signs differ with the host and the species of the mold. The lesion is confined in a circular pattern with features of erythema, scaly and pruritis. It diffuses outwards with healing at the centre.
TINEA CAPITIS
Tinea capitis is the generally known dermatophyte infection of the scalp in children. Anthropophilic species produces endemic infections and zoophilic sporadic(44). It can be provoked by sharing of contaminated comb and clothes(49).
The causative agent of tinea capitis is Trichophyton species particularly
Trichophyton tonsurans.
Anthropophilic species are the predominant causative agents in India.
Trichophyton violaceum and Microsporum audouinii are the formative agents in
North India and Trichophyton violaceum in South India(51). Scaling may be occur on the scalp with minimal inflammation but marked with Microsporum canis.
Hairs infected with these fungal agents fluoresce green when exposed to Wood’s lamp(36).
21 Tinea capitis is clinically classified as (9)
(i) Non inflammatory – Microsporum audouinii, Microsporum
ferrugineum
(ii) Inflammatory – Microsporum canis, Microsporum gypseum
(iii) Black dot type – Trichophyton violaceum, Trichophyton tonsurans
Three pattern of invasion exists
(a) Endothrix pattern occurs from infection with anthropophilic fungi in the
genus
Trichophyton and is individualized by non fluorescent arthroconidia within
the hair shaft. The clinical presentation ranges from scaling to ‘black dots’
with patchy alopecia to kerion formation. Trichophyton tonsurans and
Trichophyton violaceum are important creators of endothrix
formation(52,53).
(b) Ectothrix pattern results when arthroconidia are assembled from
fragmented hyphae outside the hair shaft. Cuticular destruction ensues.
Ectothrix infection may be fluorescent (Microsporum) or nonfluorescent
(Microsporum and Trichophyton) as concluded by wood’s lamp
examination.
(c) Favus is the most serious form of dermatophytic hair infection, caused by
Trichophyton schoenleinii. Hyphae and air spaces are noticed within the
hair shaft, and a bluish white fluorescence under wood lamp examination.
Favus manifests as thick yellow crusts made of hyphae and skin debris
(scutula). Scarring alopecia may occur in chronic cases.
22 TINEA BARBAE
Dermatophytosis restricted to post pubertal males and affects the bearded areas of face and neck. Causative agent is Trichophyton mentagrophytes var mentagrophytes and Trichophyton verrucosum. Other agents are Trichophyton schonleinii, Trichophyton violaceum and Trichophyton megninii. Common causative agent of infection was contaminated razors in barber shops. Because zoophilic organisms are the common agent and due to the enormous amount of terminal hair follicles in the diseased areas the clinical presentation tends to be localised, severe with intense inflammation and multiple follicular pustules.
Abscess, sinus tracts, secondary bacterial infection and even kerion like lesion may occur. Some infections are less severe with circular, erythematous, scaly lesions(36).
TINEA FACIEI
The causative species are Trichophyton rubrum and Trichophyton mentagrophyes var mentagrophytes, Trichophyton tonsurans and Microsporum canis. The typical annular lesions are erythematous pruritic, but scaling is often absent. The lesions are often exacerbated by sun exposure(44).
TINEA CORPORIS
Tinea corporis is the most common dermatophytic infection of the skin of the trunk and extremities excluding the hair, nail, palms, soles and groin. Any dermatophyte can possibly cause Tinea corporis, but Trichophyton rubrum is the
23 most common fungal pathogen worldwide followed by Trichophyton mentagrophytes. The mode and spread of infection is from human to human, animal to human or soil to human.
Domestic animals are the important agents in transmission of organisms causing tinea corporis specifically the zoophilic types. Important risk factors in procuring tinea corporis is having personal history or close contact with tinea capitis or Tinea pedis and are generally seen as nodule on the lower part of the legs with the overlying skin appearing red, dry and scaly(44). Other predisposing causes include Immunosuppression, occupational or recreational exposure contact(military camps, sickrooms, athletic clubs, outdoor occupations, wrestling) with contaminated clothes and furniture. The incubation period is 1 to 3 weeks.
Lesions may be arcuate, annular, circinate, oval in shape, scaly plaque with a raised erythematous border and central clearing (lessened or absent in corticosteroid use –tinea incognito). In their most florid form, the lesions can become indurated and pustular which is most common with zoophilic species.
Lesions may also appear vesicular, granulomatous or verrucous in appearance.
Associated symptoms include pruritis and burning sensation in the skin. Clinical variants of tinea corporis include tinea profunda, Majochi’s granuloma and tinea imbricata. Tinea profunda results from excessive inflammatory response to a dermatophyte(analogous to a kerion on the scalp). Majochi’s granuloma caused by Trichophyton rubrum is defined by perifollicular pustules or granulomas.
24 Trichophyton imbricata caused by Trichophyton concentricum is a chronic infection manifesting as concentric annular rings(54).
TINEA IMBRICATA
Tinea imbricata is a variant of tinea corporis caused by Trichophyton concentricum (46). This is a chronic infection described by the development of homogenous sheets or the concentric rings of scaling that can extend to cover large parts of the affected person(36).
TINEA CRURIS
Tinea cruris is the dermatophytic infection of the inguinal region precisely inner aspect of upper thigh and crural folds. Perineal regions are more frequently affected in men since scrotum encourages a moist and warm environment. Other causative factors are obesity and excessive perspiration. Three most common predominant pathogens are the anthropophilic species, Epidermophyton floccosum,Trichophyton rubrum and Trichophyton mentagrophytes(47).
Patient frequently complaints of intense pruritis in the intertrigenous fold between the scrotum and thigh(50). Followed by sharply demarcated lesion with a raised, erythematous, scaly, advancing border which radiate from the groin down the inner border of the thigh. Border may contain pustules or vesicles.
TINEA PEDIS
Tinea pedis is the dermatophyte infection of the soles and interdigital web spaces. It is the most frequent infection around the world affecting both the sexes.
25 The absence of sebaceous glands and the moist environment caused by occlusive shoes are the important causes in development of tinea pedis. Most believe that, it is acquired by walking bare foot. No specific susceptibility has been determined to explain why some people are more likely to acquire than others despite the same level of exposure to common pathogens – Trichophyton rubrum,
Trichophyton mentagrophytes var interdigitale, Epidermophyton floccosum and
Trichophyton tonsurans.
The most common clinical presentation is interdigital or web space infection which generally involves between the fourth and fifth toes. Another common feature associated with Trichophyton rubrum infection is the hyperkeratosis of the sole, which is presented as dry, white scaly lesions. This form of the diseases often tends to be chronic and resistant to treatment.
A third form of tinea pedis, associated with Trichophyton mentagrophytes var interdigitale, is the vesicular lesion involving the soles. This acute lesion commonly resolves spontaneously. Sometimes exacerbation tends to occur under hot and humid conditions. This condition is frequently associated with hyperhidrosis(36).
There are four types of tinea pedis
- Moccasin- extensive involvement of the foot
- Interdigital
- Inflammatory
- Ulcerative
26 TINEA MANUUM
Tinea manuum is the infection of the palm and the inter digital spaces thought to be related to the absence of sebaceous glands in the palm. Causative organisms of tinea manuum are Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes usually associated with Moccasin type of tinea pedis. The infection is usually unilateral. Lesions appearing on the dorsal side of the palm show similar appearance to tinea corporis, with central clearing and distinct border. This appears as a diffuse scaling hyperkeratosis, with accentuation of fissuring in the palmar creases.
TINEA UNGUIUM (ONYCHOMYCOSIS)
Onychomycosis consists of all fungal infections infecting the nail apparatus, i,e, nail matrix, cuticle, nail plate, mesenchymal tissue and nail folds(55). In spite of improved personal hygiene and better living environment, onychomycosis continues to spread and persist as a chronic infection. The prevalence rate of onychomycosis is determined by age, predisposing factor, climate, social class, occupation, living environment and frequency of travel.
The most common pathogens are Trichophyton rubrum, Trichophyton mentagrophytes and Epidermophyton floccosum (less commonly Microsporum species). A single nail may be involved but more frequently multiple nails on one or both hands or feet affected. The responsible pathogen initially invades the nail bed in the region of hyponychium leading to hyperkeratosis of the nail bed. With further advancement of infection there is yellowing and thickening of the distal
27 nail plate as well as onycholysis which are the ideal environment for further proximal invasion and growth of the dermatophyte. Eventually the entire nail bed and plate may become affected (total dystrophic pattern). Severe complication such as cellulitis may arise from onychomycosis specially in patients who are diabetic or immunocompromised(53,56,57).
The various clinical forms of Tinea unguium based on the point of fungal entry into the nail unit are
x Distal and Lateral Subungual Onychomycosis (DLSO)
x White Superficial Onychomycosis(WSO)
x Proximal Subungual Onychomycosis (PSO)
x Total Dystrophic Onychomycosis (TDO)
DISTAL AND LATERAL SUBUNGUAL ONYCHOMYCOSIS (DLSO)
This is the most common type with invasion via the hyponychium. This generally begins as a discolouration and thickening of the distal and lateral border of the nail. Finally it results in demolition of the entire nail plate and separation of the nail from the nail bed.
WHITE SUPERFICIAL ONYCHOMYCOSIS (WSO)
White superficial onychomycosis is common in toe nails with direct invasion into the superficial nail plate and causes white crumbling lesions involving only the nail surface. This condition is most commonly caused by
Trichophyton mentagrophytes var interdigitale.
28 PROXIMAL SUBUNGUAL ONYCHOMYCOSIS (PSO)
Proximal subungual onychomycosis is a rare presentation involving the finger nails with direct invasion under the proximal nail fold
(immunocompromised host). The nail presents as whitish yellow with periungual inflammation.
TOTAL DYSTROPHIC ONYCHOMYCOSIS (TDO)
In total dystrophic onychomycosis, there is complete destruction of nail plate. Onychomycosis should be monitored and treated earlier, as it can disorganize the skin integrity. In untreated cases, these can act as a reservoir and can further facilitate secondary bacterial infections.
The treatment of onychomycosis has been endeavoured throughout ages but only the last two decades has safe and effective systemic treatments. Oral griseofulvin and ketoconazole was the drug of choice for the treatment of onychomycosis. It has been superseded by newer systemic compounds that have better cure and lower relapse rate.
TINEA GLADIATORUM
Tinea gladiatorum is the emerging infections in arms, trunk, head and neck of the wrestlers. The commonest isolate is Trichophyton tonsurans(58).
29 LABORATORY DIAGNOSIS
The diagnosis of dermatophytes is based on commixture of clinical observation supplemented by laboratory investigation(1).
COLLECTION OF SPECIMEN
The specimens of infected skin, hair and nail are collected in a dry sterile container, after decontaminating the affected area(5).
The laboratory diagnosis depends on(1)
x Demonstration of the causative pathogen in tissue by microscopy
x Isolation of fungus in culture
x Serological tests.
DIRECT MICROSCOPY
This is more rapid, simple, reliable method of determining the etiology of an infection when the test is positive(5,1). Moreover it is also helpful in determining whether the organism isolated later in culture is a contaminant or a pathogen and also to select further specific culture media and tests(48).The specimens like skin scales, nail clippings, and hair stubs are commonly collected and examined by suspending a portion of the sample in a clearing agent, KOH
10% used for skin and hair, but 20% for nails.
In KOH wet mount, the fungus is seen as branching hyaline mycelia, which usually show arthrospores production. The demonstration of fungus in nails may be difficult and may be possible only after incubating nail clippings in KOH for
30 overnight. For all clinical specimens, fungal hyphae must be differentiated from other artefacts(1).
MODIFICATION OF KOH MOUNT(31)
x KOH with dyes or blue black ink
x Addition of DMSO (36%) to KOH (20%). Others being dimethyl
acetamide and dimethyl formamide.
x Glycerine (5-10%) to 10-25% KOH
x 10% sodium disulphide solution
OTHER SPECIAL STAINS
x Calcoflour white stain
x Periodic acid shiff stain (PAS)
x Gomori’s methenamine silver stain (GMS)
x Immunofluorescence stain
FUNGAL CULTURE
The collected specimen should be inoculated on fungal culture media irrespective of the findings in direct KOH wet mount microscopic examination(1).
Culture of dermatophytes require media containing antibiotics, because specimen from cutaneous sites almost always contain the normal bacterial flora of the skin, nail and hair in addition to saprophytic fungi from the environment.
31 MEDIA
x Emmons modified Sabouraud’s Dextrose Agar with antibacterial agents
such as chloramphenicol and or gentamycin and cycloheximide to inhibit
the growth of saprophytic fungi.
x Sabouraud’s Dextrose Agar with chloramphenicol should always be
included for culture.
The inoculated cultures are incubated at 25ÛC, 30ÛC and 37ÛC. The growth
is relatively slow and takes 10 days to three weeks. Trichophyton
verrucosum and some strains of Trichophyton tonsurans grow only at
37ÛC.
At the onset of sporulation and pigment production growth is examined by
LactoPhenol Cotton Blue mount. The cultures are examined three times
weekly for four weeks and appropriately sub cultured onto Sabouraud’s
Dextrose Agar.
DERMATOPHYTES TEST MEDIUM (REBELL & TAPLIN 1974)
Dermatophyte Test Medium is used for presumptive identification of dermatophytes(5). All samples of dermatomycosis can also be inoculated onto the
Dermatophyte Test Medium and incubated at 25ÛC. This primary selective medium is helpful in isolating pathogenic species from cutaneous specimen. Thus
Dermatophyte Test Medium can be used to isolate and distinguish dermatophytes from the saprophytic fungus.
32 PRINCIPLE
Dermatophytes utilize the proteins present in the medium and turns the medium red colour by raising the pH (alkaline metabolites) indicating their presence, while most other fungi and bacteria utilizes the carbohydrate in the medium and hence no change of colour or pH occurs.
PROCEDURE
The collected samples should be inoculated onto the agar as soon as possible with a sterile forceps and in inoculated at 22-25Û C for upto 14 days. The culture should be examined daily for change in colour of the medium and evidence of fungal growth for 7-10 days and discarded later.
INTERPRETATION
x Red colour media with white cotton like growth - Presumptive
identification of dermatophyte.
x Yellow colour media with no growth – no dermatophyte in the sample
x No colour change in the medium with white or off white creamy growth-
candida
DISADVANTAGE(31pg236)
It is only a screening media and not a specific media for dermatophytes since non pathogenic species can also produce colour change on prolonged incubation. Pigment production cannot be demonstrated.
33 OTHER MEDIA(5)
x DIM – dermatophyte identification medium
x Sabouraud’s Dextrose Agar with yeast extract
x PDA- Potato Dextrose Agar- to enhance sporulation and pigments are better
x PFA- Potato Flake Agar- to enhance sporulation and pigments are better
x CMA – Corn Meal Agar
x Trichophyton agars(1-7)
x Inhibitory mould agar
x Littman oxgall agar with antibiotics (summerbell et al 1989)
x Casamino acid / erythritol/ albumin
x BCP / casein yeast extract
COLONY MORPHOLOGY(5)
Colonies with one pigment on the obverse surface of the colony and another pigment (other than black) on the reverse are likely to be dermatophytes.
The dermatophyte isolate can be differentiated from contaminant by their condensed growth around the inoculums and by the colour of colony(1).
IDENTIFICATION OF DERMATOPHYTES(5)
The genus Trichophyton, Microsporum and Epidermophyton are the principle etiological agents of Dermatophytosis. Their morphology and cultural characters are as follows
34 Trichophyton species [Malmsten1845]
x This species usually infect skin, hair and nail.
x The genus Trichophyton is characterized by the development of both
smooth walled macroconidia and microconidia.
x Macroscopically the colonies of Trichophyton species are powdery, velvety
and waxy with characteristic pigmentation.
x Microscopically microconidia are abundant, spherical, pyriform to clavate
or of irregular shape which are arranged singly or in clusters along the
hyaline septate hyphae range from 2 to 3 by 2 to 4 µm in size.
x Macroconidia are few or absent in many species.
x Macroconidia are mostly borne directly on the hyphae laterally or on short
pedicels, and are relatively sparse, generally elongated with blunt ends,
smooth, thin or thick walled, clavate to fusiform, spindle or pencil shaped
with 4-6 transverse septa, have distinctive shapes in different species which
helps in species identification and range from 4 to 8 by 8 to 50 µm in size.
x The different species are
(i) Trichophyton mentagrophyes var. nodulare,
(ii) Trichophyton mentagrophytes var. erinacei,
(iii) Trichophyton mentagrophytes var. interdigitale,
(iv) Trichophyton mentagrophytes var. mentagrophytes,
(iv) Trichophyton mentagrophytes var. quinckeanum,
(v) Trichophyton rubrum,
(vi) Trichophyton rubrum downy strain,
35 (vii) Trichophyton rubrum granular strain,
(ix) Trichophyton schoenleinii,
(x) Trichophyton tonsurans,
(xi) Trichophyton verrucosum,
(xii) Trichophyton violaceum,
(xiii) Trichophyton soudanense,
(xiv) Trichophyton terrestre,
(xv) Trichophyton ajelloi,
(xvi) Trichophyton concentricum,
(xvii) Trichophyton megninii,
(xviii) Trichophyton simii,
(xix) Trichophyton equinum.
Trichophyton mentagrophytes
Macroscopy:
Trichophyton mentagrophytes-type-I colony (zoophilic) are generally flat, granular, white to cream in colour, with powdery to granular surface. Reverse pigmentation is usually a yellow to tan or reddish brown colour.
Trichophyton mentagrophytes-type-II colony (anthropophilic) are flat and downy with surface pigment cream to light yellow with white feathery fringes that may become pink with yellow to yellowish orange reverse pigmentation.
36 Microscopy
Type-I: Macroconidia are abundant which are clavate to cigar shape, thin smooth walled with 3-6 cells. Microconidia is also abundant which are globose and unicellular ( en thryses) or in clusters (en grappe).
Type-II: Macroconidia are sparse or absent. Microconidia also are sparse which are clavate or pyriform. Racket hyphae, favic chandeliers and noduar bodies are frequently seen. Both types show branching conidiophores at right angles, arthroconidia chlamydoconidia and spiral hyphae.
Wood’s lamp examination
Ectothrix hair do not fluoresce under wood’s lamp.
Hair perforation test
Positive with 14 days
Urease test
Positive
Clinical significance:
This species is common in human and animals. It is both anthropophilic and zoophilic. It has been associated with tinea manuum, tinea corporis, tinea unguium, tinea pedis, tinea capitis, tinea cruris and tinea barbae.
Trichophyton mentagrophytes var. mentagrophytes is the zoophilic form of
Trichophyton mentagrophytes with a global distribution and a wide range of animal hosts including mice, guinea pigs, kangaroos, cats, horses, sheep and rabbits. It causes acute but severe inflammatory skin or scalp lesions in humans, particularly in rural workers but are readily curable. Kerion of the scalp and beard
37 may occur. Invaded hairs show an ectothrix infection but do not fluoresce under
Wood's ultra-violet light.
T.mentagrophytes var. interdigitale causes more chronic but less inflammatory reaction.
Trichophyton rubrum
Trichophyton rubrum is the commonest species isolated from human (104).
It is an anthropophilic dermatophyte. It may be of two types.
Macroscopy
Type-I (downy type)
White downy to fluffy colony on Emmons modified Sabouraud’s dextrose agar after 10-14 days of incubation at 25ÛC – 30ÛC. The reverse is yellow to blood red.
Type-II (granular type)
This type shows rugose fold with granular texture due to the production of macroconidia. Surface pigment becomes tan, yellow or tinged with red and texture is granular. The reverse pigment may be colourless, tan or yellow to brown but eventually a deep wine red colour appears.
Microscopy
Type-I
Scanty moderate numbers of slender, clavate or tear drop microconidia which are arranged in bird on fence arrangement (34). Macroconidia are sparse or absent. These are narrow, cylindrical with blunt distal ends and thin smooth parallel walls showing 3-8 septations.
38 Type-II
Microconidia are moderate to abundant number of clavate or tear drop forms arranged singly or occasionally in clusters. Macroconidia are also moderate to abundant in numbers, thin walled and of cigar shaped borne directly on the hyphae with broad bases of attachments. Chlamydoconidia, nodular bodies, pectinate hyphae and racquet hyphae are also seen. The macroconidia may or may not have terminal appendages.
Wood’s lamp examination
Ectothrix hair do not fluoresce under wood’s lamp.
Hair perforation test
Negative
Urease test
Negative
Clinical significance
The downy strain has become the most widely distributed dermatophyte of man. It frequently causes chronic infections of skin, nails and rarely scalp.
The granular strain is a frequent cause of tinea corporis.
Trichophyton tonsurans(59)
Macroscopy
After 7–14 days incubation at 25ÛC – 30ÛC on modified Sabouraud's dextrose agar, colonies show considerable variation in texture and colour. They may be suede-like to powdery, flat with a raised centre or heaped, often with radial grooves. The colour may vary from pale-buff to yellow, the so called
39 sulfureum form which resembles Epidermophyton floccosum, to dark-brown. The reverse pigmentation varies from yellow-brown to reddish-brown to deep mahogany colour.
Microscopy
Hyphae are hyaline, septate relatively broad, irregular, much branched with terminal swelling. Barrel shaped arthroconidia are seen in chains. Numerous characteristic ballooning of microconidia varying in size and shape from long clavate to broad pyriform, are borne at right angles to the hyphae, which often remain unstained by lacto phenol cotton blue.
Very occasional smooth, thin walled, short and blunt irregularly clavate or cylindrical shaped 3-8 celled macroconidia may be present on some cultures.
Numerous swollen giant forms of microconidia, intercalary chlamydoconidia, racquet hyphae and spiral hyphae are also produced in older cultures.
Wood’s lamp examination
Do not fluoresce under wood’s lamp.
Hair perforation test
Positive within 14 days
Urease test
Positive at 5 days
Clinical significance:
Trichophyton tonsurans is an anthropophilic fungus with a worldwide distribution which causes inflammatory or chronic non-inflammatory finely scaling lesions of skin, nails and scalp. It produces black dot tinea capitis in
40 children which involves scattered hairs with little inflammation or itching. A fine red scaly lesions of skin can occur. It occasionally causes tinea unguium and tinea pedis. Invaded hairs show an endothrix infection and do not fluoresce under
Wood's ultra-violet light.
Trichophyton verruccosum(59)
Macroscopy:
On modified Sabouraud's dextrose agar supplemented with thiamine and inositol, at 25ÛC – 30ÛC, after 3-4 weeks of incubation, colonies are white to cream coloured, slow growing, small, button-or-disk shaped, with a suede-like to velvety surface, a raised centre, and flat periphery with some deeply submerged growth in the medium. Reverse pigment may vary from non pigmented to yellow colour.
Microscopy
Microscopically distorted broad, irregular hyaline septate hyphae with many terminal and intercalary chlamydospores produced at 37ÛC.
Chlamydospores are often in chains. The tips of some hyphae are broad and club- shaped, and occasionally divided, giving the so-called favic chandeliers("antler" effect).
When grown on thiamine-enriched media, occasional strains produce clavate to pyriform microconidia borne singly along their corresponding hyphae.
Macroconidia are only rarely produced, but when present have a characteristic rat tails or string bean shape.
Growth at 37ÛC: unlike other dermatophytes growth is enhanced at 37ÛC
41 Wood’s lamp examination
Do not fluoresce under wood’s lamp.
Clinical significance
Trichophyton verrucosum is a zoophilic fungus causing highly inflammatory dermatophytoses in cattle and humans which are worldwide in distribution. Infections in humans result from direct contact with cattle or infected fomites and are usually highly inflammatory involving the scalp, beard or exposed areas of the body (ie. nails, skin). Suppurative tinea corporis, kerions, tinea barbae and tinea manuum and ectothrix type of tinea capitis which fluoresce under wood’s lamp are also common. Geographic distribution is worldwide.
Trichophyton schonleinii
Macroscopy
After 2-3 weeks incubation on modified Sabouraud's dextrose agar at
25ÛC-30ÛC, colonies are slow growing, waxy or suede-like with a deeply folded or heaped honey-comb-like thallus and some sub-surface growth. The thallus is cream coloured to yellow to orange brown. Cultures are difficult to maintain in their typical convoluted form, and rapidly become flat and downy. No reverse pigmentation is present.
Microscopy
Microscopically, branched septate hyaline sterile hyphae are seen. No macroconidia and microconidia are seen in routine cultures. However numerous chlamydoconidia may be present in older cultures. However, characteristic antler
"nail head" hyphae also known as "favic chandeliers" may be observed. A few
42 distorted clavate microconidia may be formed by some isolates when grown on polished rice grains.
Wood’s lamp examination
Pale greenish yellow fluorescence under wood’s lamp.
Clinical significance
Trichophyton schoenleinii is an anthropophilic fungus causing favus which is a chronic, scarring form of tinea capitis characterized by saucer-shaped crusted lesions with mouse like odour and scutula formation around the infected hair follicle. It results in permanent hair loss, if not treated promptly. Invaded hairs remain intact and fluorescence pale greenish yellow under Wood's ultraviolet light. Favus is common in Europe, Asia and Africa.
Trichophyton violaceum(17)
Macroscopy
After 14-21 days, at 25ÛC – 30ÛC on modified Sabouraud's dextrose agar, colonies are very slow growing, glabrous or waxy, heaped and folded and a deep violet in colour. Cultures often become pleomorphic, forming white sectors and occasional non-pigmented strains may occur. The reverse pigment of culture is lavender to purple.
Microscopy
Microscopically, sterile distorted, twisted, much branched, relatively broad, tortuous, septate hyaline hyphae, chains of intercalary and terminal chlamydioconidia, swollen hyphal cells containing cytoplasmic granules and favic chandeliers are seen. Young hyphae usually stain well in lactophenol cotton blue,
43 whereas older hyphae stain poorly and show small central fat globules and granules. No conidia are usually seen, although occasional pyriform microconidia have been observed on enriched media. Numerous chlamydoconidia are usually present, especially in older cultures.
Wood’s lamp examination
Do not fluorescence under wood’s lamp.
Clinical significance
Trichophyton violaceum is an anthropophilic fungus causing long standing non-inflammatory finely scaling lesions of skin, nails, beard and scalp, producing the so called "black dot" tinea capitis. It has a geographic distribution all over the world. Invaded hairs show an endothrix infection and do not fluoresce under
Wood's ultra-violet light. Permanent alopecia and kerions can occur.
Microsporum species [Gruby 1843]
Microsporum species infect skin and hair but not nails. The colonies are cottony with white to brown pigmentation. It forms both macroconidia and microconidia on short conidiophores. Macroconidia are hyaline, multiseptate (1-
15), variable in form, fusiform, spindle-shaped to obovate, ranging from 7 to 20 by 30 to 160 µm in size, with thin or thick echinulate to verrucose cell walls.
Their shape, size and cell wall features are important characteristics for species identification. Microconidia are hyaline, single-celled, pyriform to clavate, smooth walled, 2.5 to 3.5 by 4 to 7 µm in size and are not diagnostic for any one species. Seventeen species of Microsporum have been described, however only the more common species are included in these descriptions.
44 (i) Microsporum canis,
(ii) Microsorum canis var. distortum,
(iii) Microsporum gypseum,
(iv) Microsporum nanum,
(v) Microsporum persicolor.,
(vi) Microsporum audouinii
(vii) Microsporum cookei,
(viii) Microsporum equinum,
(ix) Microsporum ferrugineum,
(x) Microsporum fulvum ,
(xi) Microsporum gallinae.
Clinical significance
The genus Microsporum contains a number of important species that are the principle causative agents of animal and human dermatophytoses [tinea and ringworm].
Microsporum gypseum
Macroscopy
After 5-6 days of incubation at 25ÛC, on Sabouraud's dextrose agar, colonies are usually flat, spreading, suede-like to granular, with a deep cream to tawny-buff to pale cinnamon coloured red surface. Many cultures develop a central white downy umbo (dome) or a fluffy white tuft of mycelium and some also have a narrow white peripheral boarder. A yellow-brown pigment, often with
45 a central darker brown spot, is usually produced on the reverse, however a reddish-brown reverse pigment may be present in some strains.
Microscopy
Microscopically, hyaline, septate, branched hyphae with abundant, symmetrical, ellipsoidal, thin-walled, verrucose, echinulate 4-6 celled macroconidia. The terminal or distal ends of most macroconidia are slightly rounded, while the proximal ends (point of attachment to hyphae) are truncate.
Numerous clavate shaped microconidia are also present, but these are not diagnostic.
Wood’s lamp examination
Dull yellow fluorescence under wood’s lamp.
Clinical significance
Microsporum gypseum is a geophilic fungus with a geographical distribution which may cause tinea capitis and tinea corporis infections in animals and humans, particularly children and rural workers during warm humid weather.
Usually produces a solitary raised boggy inflammatory skin lesion and ectothrix type of hair infection. Invaded hairs fluoresce dull yellow green under Wood's ultra-violet light.
Microsporum canis
Macroscopy
After 4-6 days of incubation at 25ÛC, on Sabouraud's dextrose agar, colonies are flat, spreading, white to cream-coloured, with a dense cottony surface which may show some radial grooves. Colonies usually have a bright golden
46 yellow to brownish yellow reverse pigment, but non-pigmented strains may also occur.
Microscopy
Microscopically, hyaline, septate, branched, hyphae with abundant spindle- shaped, verrucose , echinulate 6-15 celled, thick walled asymmetrically beaked apex macroconidia, and often have a terminal knob. A few pyriform to clavate microconidia are also present. Macroconidia and/or microconidia are often not produced on primary isolation media and it is recommended that sub-cultures be made onto boiled polished rice grains to stimulate sporulation.
Pectinate bodies, nodular bodies, racquet hyphae and chlamydoconidia are present.
Wood’s lamp examination
Bright greenish yellow fluorescence under wood’s lamp.
Hair perforation test
Positive at 14 days
Clinical significance
Microsporum canis is a zoophilic dermatophyte of world-wide distribution which is a frequent cause of ringworm in humans, especially children because certain fungicidal fatty acids are absent in them and frequent contact with cats and dogs. Tinea corporis and acute inflammatory ectothrix pattern of grey patch tinea capitis occurs. Cats and dogs are the main sources of infection. Invaded hairs show an ectothrix infection and fluoresce a bright greenish-yellow under Wood's ultra-violet light.
47 Microsporum nanum
Macroscopy
After 7 days, at 25ÛC on modified Sabouraud’s dextrose agar, colonies are initially flat, cream to buff in colour with a suede-like to powdery surface texture.
Young colonies have a brownish-orange pigment which deepens into a dark reddish-brown with age. The reverse pigmentation is brownish red in colour.
Microscopy
Microscopically, the hyaline, septate, branched, hyphae with numerous small ovoid to pyriform macroconidia with 1-3 cells, but mostly 2 cells, with relatively thin, ellipsoidal, finely echinulate (rough) walls, and broad truncate bases. Many macroconidia are borne on conidiophores (stalks) which do not stain readily. Occasional sessile smooth walled clavate to pyriform microconidia are present, which distinguishes M. nanum from some species of Chrysosporium.
Wood’s lamp examination
Do not fluorescence under wood’s lamp.
Clinical significance
Microsporum nanum is a geophilic and zoophilic fungus frequently causing chronic non-inflammatory lesions in pigs and a rare cause of tinea corporis and tinea capitis in humans. Also present in soil of pig-yards. Infections in man usually contacted directly from pig or fomites. Invaded hairs may show a sparse ectothrix or endothrix infection but do not fluoresce under Wood's ultraviolet light. The geographical distribution is world-wide.
48 Epidermophyton species[Sabouraud1907]
It affects mainly the skin and nail but not the hair. The genus of epidermophyton has only one species. The colonies are slow growing, powdery and khaki coloured which do not produce microconidia. Smooth, thin walled, 1-9 celled pear shaped macroconidia are arranged in clusters.
Epidermophyton floccosum
Macroscopy
After 6-12 days of incubation in modified Sabouraud's dextrose agar at
25ÛC-30ÛC, colonies are usually slow growing, white and downy with a suede-like surface, raised and folded in the centre, with a flat periphery and submerged fringe of growth. Older cultures may develop white pleomorphic tufts of mycelium. The reverse pigment is mustard yellow or khaki with yellow fringe. It is sensitive to cold temperature, so the specimens should not be refrigerated.
Microscopy
Microscopic morphology shows thin, hyaline, septate, branched hyphae, smooth thin-walled 5 celled pear or club shaped macroconidia, beaver’s tail appearance which are often produced in clusters growing directly from the hyphae. Numerous chlamydoconidia, few spiral hyphae, racquet hyphae and nodular bodies are formed in older cultures. No microconidia are formed.
Clinical significance
Epidermophyton floccosum, an anthropophilic dermatophyte with a worldwide distribution causes tinea pedis, tinea cruris, tinea corporis and onychomycosis It has no specific growth requirements and it is not known to
49 invade hair in vivo. Epidermophyton floccosum infections may become epidemic among personnel using common shower or gym facilities, e.g. athletic teams, troops, ship crews and inmates of institutions.
MICROSCOPIC METHODS
TEASE MOUNTS (LACTO PHENOL COTTON BLUE)
Cultures are examined microscopically by examining a little portion of the aerial mycelium. The material is placed on a slide in a drop of Lacto Phenol
Cotton Blue. The matted mycelium is gently teased apart with a pair of sterile teasing needle. A coverslip is placed and the morphology observed under light microscope.
RIDDLE’S AGAR BLOCK METHOD (37)
In a petridish with V shaped glass rod, a glass slide, coverslip were placed and sterilised in hot air oven. 1x2 cm Sabourards Dextrose Agar block is placed on the slide and the corners are inoculated with the fungal colony. The whole set is incubated at Room Temperature after adding few drops of distilled water in the bottom of the petridish to maintain moisture.
CELLOPHANE OR SCOTCH TAPE PREPARATION(31pg91)
A two inch piece of cellophane tape is taken and the sticky side is carefully placed on the surface of colony. The speculating colony stick on to it and then the tape is gently placed with the sticky side down on a glass slide with a drop of
Lacto Phenol Cotton Blue dye. All preparations are examined for the presence of hyphae, microconidia, macroconidia, their size, shape and arrangement and other
50 non reproductive vegetative hyphae (spiral hyphae, racquet hyphae, nodular hyphae).
PHYSIOLOGICAL PARAMETERS
Used to identify and speciate the isolates which may show identical morphology especially Trichophyton species. Physiological tests are carried out(31pg237).
They includes,
Nutritional requirement(31)
Trichophyton agar (1-7) is used to distinguish between the Trichophyton species.
Temperature
Most dermatophytes grow at 25-30ÛC.Trichophyton verrucosum grows only at 37ÛC.
Pigment production
Most of the Trichophyton rubrum produces cherry red colour under the colony but to differentiate slow pigment producing variants of Trichophyton rubrum from Trichophyton mentagrophytes, Potato Dextrose Agar and Corn Meal
Agar are used to induce pigment production.
UREASE PRODUCTION(60)
Trichophyton mentagrophytes produces urease and Trichophyton rubrum does not. Hence to differentiate it Christensen’s urease medium is used(Philpot1967,Clayton & midgley1989).Other urease producing Trichophytons
51 are Trichophyton megninii , Trichophyton raubitschekii.(variant of Trichophyton rubrum) and urease negative species is Trichophyton erinacei(31).
INVITRO HAIR PERFORATION TEST (31,61)
Trichophyton mentagrophytes and Microsporum canis can perforate normal human hair (<5years of age) producing wedge shaped tunnels or holes.
Trichophyton rubrum and Microsporum equinum do not perforate and thus can be differentiated.
RICE GRAIN TEST (1)
Except for Microsporum audouinii, all other Microsporum species grow rapidly on sterile rice grains
HAIR BAIT TECHNIQUE
This is performed to isolate geophilic dermatophytes from soil like
Microsporum gypseum.
SEROLOGY (1)
The skin test with trichophytin, which is a crude extract causes delayed type hypersensitivity reaction in most adults (72). The carbohydrate portion is related to an immediate response whereas the portion of peptide moiety to immunity. Absence of these reaction leads to chronic dermatophytoses.
Immunodiffussion test are performed for the diagnosis of dermatophytoses.
ANIMAL INOCULATION(1)
Animal pathogenicity testing is done on laboratory animals like guinea pig, rabbit and mice. Animals can be infected with geophilic dermatophytes and zoophilic dermatophytes. The area to be inoculated with conidia and hyphae is
52 shaved and scarified. The isolates are inoculated and growth is observed in 7 days.
The lesion generally resolves by 3-4weeks in most of the cases.
MOLECULAR TECHNIQUES
To assess the relavance and taxonomic classification of the dermatophytes,
Mitochondrial DNA analysis of dermatophytes using BG II; HaeIII and PCR are being done
ANTIFUNGAL SUSCPTIBILITY TESTING
Antifungal susceptibility testing has obtained much attention with the advent of newer antifungal drugs. But this test is not advanced as with bacteriology. It must afford a reliable measure of the relative activity and also associate with in vivo activity(61). Anti fungal Susceptibility Testing is done to determine the minimum inhibitory concentration by which the in vivo effectiveness of antifungal drugs can be evaluated. Moreover the development of drug resistance can also be assessed. Anti fungal Susceptibility Testing depends on the following parameters(32),
x pH of the medium
x Inoculum size of the isolate
x Medium
x Time and Temperature of incubation
x Invitro and invivo correlation
53 METHODS
x Broth dilution(CLSI M38A)-Macrodilution and Microdilution
x Agar dilution
x Disk diffusion
x E-test
TREATMENT
x Antifungal antibiotics
x Synthetic antifungal drugs
x Miscellaneous drugs.
ANTIFUNGAL ANTIBIOTICS
The drug Griseofulvin is a narrow spectrum antibiotic produced by
Penicillium griseofulvum and Khuskia oryzae. It interferes with polymerised microtubule and spindle formation thereby it inhibits mitosis and is fungistatic agent.
SYNTHETIC ANTIFUNGAL DRUGS
x Thiocarbamate: Topical agents-Tolnaftate Allyl amine and benzylamine:
Selective inhibition of squalene epoxidase which is needed for fungal
ergosterol synthesis. Hence it is fungicidal.
x Azoles: It inhibits cytochrome P450 dependent C14 demethylation in
ergosterol synthesis results in accumulation of abnormal sterols and
ultimately fungal death.
x Imidazole
x Triazoles:
54 MISCELLANEOUS DRUG
x Ciclopiroxolamine: It is Pyridine analogues which inhibits fungal cell wall
synthesis and also inhibit metal dependent enzyme.Used as 1% cream.
x Whitfield’s ointment: It is a combination of benzoic acid (fungistatic) and
salicylic acid (keratolytic) in the ratio of 2:1.It is used for treatment of tinea
pedis.
x Castellani paint- consists of 1.5% carbol fuschin.
x Undecylenic acid: Used as soap and foam for tinea pedis.
x Haloprogin: Used as 1% cream
x Triacetin: Used as 25% cream with cetylpyridinium and chloroxylenol.
PREVENTION(31)
Prevention and control depends on the site of lesion, causative species and its source. For scalp infections, all the close contacts of patient are screened by wood’s lamp examination for fluorescent hair (Microsporum species) including pet animals. In case of non-fluorescent tinea capitis (Trichophyton tonsurans,
Trichophyton violaceum), the scalp is carefully examined for the presence of scaly lesions and spotty alopecia. The suspected lesion should be cultured regularly.
Tinea corporis and tinea cruris can be transmitted with contaminated clothing, towel and bedding hence washed and disinfected regularly.
Avoidance of use of tight fitting inner wear, non absorbent clothes and prolonged exposure to wet cloth and weight reduction can avoid the occurrence of tinea cruris(48). To avoid the occurrence of tinea pedis and onychomycosis,
55 protective footwear can be worn when using public facilities. To avoid recurrence, measures should be taken to reduce foot moisture, like drying foot after baths and applying antifungal powder. For zoophilic infections, the source of infection must be traced and treated. Summerbell and Weitzman (1995) detailed the preventive measures like good sanitation and use of fungicidal sprays.
VACCINE(31)
A live vaccine (LTF130) against Trichophyton verrucosum was introduced for cattles in the former Soviet Union (Segal 1989)
56 MATERIALS AND METHODS
ETHICAL CONSIDERATION
The study was conducted after obtaining approval from the institutional ethical committee of Chengalpattu Medical College, Chengalpattu on 7.1.2016.
Permission to conduct this study was received from the Department of
Dermatology, Chengalpattu Government Hospital, Chengalpattu. Informed consent was obtained from the patients before their enrolment in the study.
STUDY DESIGN
Prospective cohort study
STUDY SETTING
The present study was carried in the Department of microbiology in
Chengalpattu Medical College, Chengalpattu in collaboration with the
Department of Dermatology, Chengalpattu Government Hospital, Chengalpattu.
STUDY PERIOD
One year from March 2015 to Febraury 2016.
SAMPLE SIZE
100 samples
SAMPLE SPECIFICATIONS
The patients who attended the Dermatology Out Patient Department at
Chengalpattu Government Hospital, Chengalpattu with clinical features
57 suggestive of dermatophytosis were selected. Based on the clinical features, respective samples like skin scrapings, hair and nail were collected.
INCLUSION CRITERIA
Patients of all age group irrespective of sex, with clinical features suggestive of dermatophytosis attending Dermatology Out Patient Department.
EXCLUSION CRITERIA
• Patients who are on antifungal therapy
• Defaulters
• Patients who are immunosupressed
- uncontrolled Diabetes mellitus
- Acquired immunodeficiency syndrome
- Antenatal cases
- Malignancy
- Patients on chronic immunosuppressive therapies like steroids, long
term antibiotics, anti-cancerous drug
QUESTIONAIRE RELATED WITH HISTORY
x All relevant details like age, sex, duration of complaint, distribution of
lesion and history of previous similar complaints and treatment history.
x All details about general health and treatment history for Diabetes,
Tuberculosis, Neoplasms, HIV, surgeries(62–64).
x Detailed history of exposure to animals, known cases, pets at home or any
other suspected sources.
58 SPECIMEN COLLECTION(1)
Samples were collected from all patients who have history of skin, scalp or nail lesions clinically diagnosed as superficial fungal infection. detailed history of exposure to animals, pets at home, known cases of dermatophytosis or any other suspected sources were also collected(65,66).
x Skin scrapping
x Nail clipping
x Plucked hair.
SKIN SCRAPPINGS
The affected area of the skin is thoroughly cleaned with 70% alcohol to remove the surface contaminants. Allow the alcohol to dry by evaporation. Using sterilized blunt scalpel, the active edge of the lesion was scrapped. The blunt scalpel was dipped in spirit and passed over flame till the spirit was burnt off.
Allow the scalpel to cool before use. The skin scrapings were collected from the margins of lesion without injuring the skin. This was accomplished by scrapping with the blade kept flat on the skin surface and not in an angular fashion.
HAIR
The similar procedure was followed as for skin scrapings; in addition, using a pair of flame sterilized forceps, a few affected hairs from the scalp was also epilated and collected. As fungus is usually found in the basal portion of the hair, care was taken to collect that area(67).
59 NAIL
After the affected nail was meticulously cleaned with 70% alcohol, the nail was clipped or scrapped deeply enough to obtain recently invaded nail tissue. Nail clippings taken from the discoloured, dystrophic or brittle parts of the nails including the full thickness(66). Scrapings were also collected from the white spots.
TRANSPORT OF SAMPLES(59)
The samples were collected and transported to laboratory in sterilized black paper sachets so that the scales will be seen easily.
PROCESSING OF SPECIMEN(1,5,60,68)
DIRECT MICROSCOPIC EXAMINATION
Direct microscopic examination was performed for all the samples.
PROCEDURE(69)
x A large drop of 10% KOH solution (skin/hair) or 20% KOH solution(nail)
was placed with a pasteur pipette on a clean grease free glass slide.
(Annexure)
x A small amount of the sample was mounted in KOH solution on the glass
slide.
x A clean cover slip was laid gently over the mixture without the formation
of any air bubbles.
x The slide was kept at room temperature for 5-20 minutes (skin/hair
samples) or overnight. (Nail sample).
x Gentle warming over the low flame hastens the keratin digestion (33).
60 x The specimen was observed under low power and high power objectives of
light microscope.
OBSERVATION
The types of hyphae, septation, thickness, branching, arthroconidia and other features were observed.
CULTURE (1,5,60,68)
All collected samples were cultured irrespective of the direct microscopic observation.
MEDIA
The samples were inoculated in duplicate sets into slopes containing,
a) Sabourauds Dextrose Agar with chloramphenicol or gentamicin and
b) Sabourauds Dextrose Agar with chloramphenicol or gentamicin and
cycloheximide. (Annexure)
c) One set of Dermatophyte Test Medium containing chloramphenicol or
gentamicin and cycloheximide (Annexure) was also inoculated and
incubated at 25ÛC.
One set of Sabourauds Dextrose Agar was incubated at 37ÛC and second
set at 25ÛC.
IDENTIFICATION OF FUNGAL ISOLATES (69,70,71,)
Any visible growth on either of the slants was examined for,
1) Colony morphology (yeast/Mould) (5)
a. Topography
b. Rate of growth.
61 c. Surface pigmentation
d. Pigmentation on reverse
e. Presence of diffusible pigment
f. Texture
All the Sabourauds Dextrose Agar tubes were examined every day for a week following incubation and twice weekly thereafter for 4-6weeks(1).
Dermatophyte Test Medium tubes are examined for growth and change in colour of the medium daily for 7-10 days(1,60). Tubes showing no growth after the described period were discarded.
LACTOPHENOL COTTON BLUE (LPCB) (ANNEXURE)(31)
Lacto Phenol Cotton Blue mount was prepared from fungal colonies resembling mould and examined for hyphal morphology, conidia morphology
(microconidia and macroconidia) arrangements of conidia and its pigmentation.
Lactic acid helps in preserving the fungal structure; phenol acts like a disinfectant and cotton blue imparts colour to the structures.
PROCEDURE (1,60)
x A drop of Lacto Phenol Cotton Blue was placed on a dry clean grease free
glass slide.
x Using a straight mycological loop a small portion of the colony to be
examined was placed on the stain.
x With two sterile dissecting needles the mycelial mass was slowly well
teased apart.
62 x A cover slip was placed and examined under low power and high power
objectives.
INTERPRETATION
Morphology of the fungal isolates was observed and the causative fungus identified.
MICRO SLIDE CULTURE (1,5)
The slide culture technique was performed to study the typical morphology of the fungal isolate, like arrangement of conidia, without disturbing the relationship between reproductive structures like conidia, conidiophores and hyphae or the sporulation characteristics of the organism.
PROCEDURE
x A sterile microscope slide was placed on a bent glass rod in a petridish
with cover slip, filter paper and the dish was wrapped and sterilized by
autoclaving at 121Û C for 15mins.
x With a sterile scalpel Sabourauds Dextrose Agar or Potato Dextrose Agar,
a block of 1square centimeter and 2-3 mm deep was cut out of a plate and
was transferred onto the center of the sterilised glass slide. With a heavy
nichrome wire bend loop, the four sides of the agar block were inoculated
with the fungal isolate under identification and the block was covered with
the sterilized cover slip.
x 1-1.5ml of sterile distilled water was pipetted on to the filter paper kept
inside the petri dish so as to avoid drying of the agar block.
63 x The petridish was incubated at room temperature and observed periodically
for growth. Once sporulation had well developed, the cover slip was
carefully removed using a sterile forceps from the agar block, flamed
quickly to fix the fungus with the spores.
x Heat fixed cover slip was placed on a drop of LPCB on a sterile second
glass slide.
x The agar block was gently removed from the original slide, few drops of
Lacto Phenol Cotton Blue were added and new cover slip was placed over
the preparation. Both the slides were then observed under microscope.
When examination is likely to be delayed, the edges of the Lacto Phenol
Cotton Blue mount were sealed with nail polish to avoid drying.
BIOCHEMICAL REACTION
UREA HYDROLYSIS ( 5, 60)
Modified Christensen’s medium (Annexure) for urea hydrolysis was done to differentiate Trichophyton mentagrophytes from Trichophyton rubrum.
PROCEDURE
The slopes were inoculated with a little amount of the fungal culture and were incubated at room temperature along with a control tube. If there is no colour change within 7 days of inoculation at 25ÛC-30ÛC, the test is declared negative(29).
64 INTERPRETATION
x Urease positive within 4 days (deep pink) - Trichophyton mentagrophytes
x Urease negative - Trichophyton rubrum
IN VITRO HAIR PERFORATION TEST (5,61)
Hair perforation test was performed to differentiate between Trichophyton mentagrophytes and Trichophyton rubrum as well as Microsporum canis and
Microsporum equinum. Growth of the fungus in artificial culture media along with the hair was observed.
PROCEDURE
x One cm of sterilized prepubertal or infant hair was placed in a petridish and
autoclaved at 121Û C for 10mins.
x To it 25ml of sterile distilled water, 2-3drops of filter sterilized 10% yeast
extract (Annexure) were added.
x The plate was inoculated with a small fragment of the test fungi.
x The plate was incubated at room temperature (25ÛC) for 4weeks.
x Periodically each hair strands were removed and mounted on a slide
containing Lacto Phenol Cotton Blue and examined under low/high power
objectives of light microscope.
INTERPRETATION
x Positive (wedge shaped perforation) – Trichophyton mentagrophytes and
Microsporum canis.
x Negative – Trichophyton rubrum and Microsporum equinum
65 ANTIFUNGAL SUSCEPTIBILITY TESTING FOR DERMATOPHYTES(60)
MICROBROTH DILUTION METHOD
REQUIREMENTS
Sterile test tubes for drug dilution / inoculum preparation, sterile disposable
Microtitre plates, sterile Gloves / sterile Micro pipette / sterile tips / sterile disposable face masks
MEDIUM
RPMI 1640 contains glutamine, without bicarbonate in MOPS (3N-
Morpholino propane sulphonic acid), buffer sterilized by membrane filtration.
ANTI FUNGAL STOCK SOLUTION
5ml of stock solution prepared for each drug.
Micro-broth dilution method: CLSI M-38 A 2nd edition was followed. (60,69,73,74)
x Reference strain : Candida parapsilosis ATCC® 22019-
Dermatophytes(75,76,77)
KORTING, H.C., et al (1995) stated that no standard reference method for determination of Minimum Inhibitory Concentration to dermatophyte is available; hence broth microdilution method (CLSI M38A) gives good correlation with clinical outcome it is followed.
x Test strain: All isolated dermatophytes.
ANTIFUNGAL AGENTS(60)
Source: Antifungal standards or reference powder having assay potency in ȝg/ml or IU/mg.
66 Weighing antifungal powders: Can be derived by using either of the formulae.
Volume (ml) X Conc. (ȝg/ml)
Weight (mg) of drug = ------
Assay potency (ȝg/mg)
Weight (mg) X Assay potency (ȝg/mg)
Volume of Diluents (ml) = ------
Conc. (ȝg/ml)
PREPARATION OF STOCK SOLUTION
x Antifungal stock solutions were prepared at a concentration of 1280ȝg/ml
for water soluble drug and 1600ȝg/ml for water insoluble drug.
x Water soluble drug like Fluconazole was dissolved in distilled water and
two dilutions were prepared as given in Chart 1.
x Water insoluble drugs, Itraconazole, Ketoconazole and terbinafine were
dissolved in DMSO and diluted with RPMI 1640. Dilutions were prepared
following Chart-2.
67 CHART-1 :SCHEME FOR PREPARING DILUTIONS OF WATER SOLUBLE DRUGS
Drug(µg/ml) Stock 5120 2 4 8 16 32 64 128 256 512 Remark
TUBE TUBE 1 2 3 4 5 6 7 8 9 10
SOUR From stock 1.0 T-1 T-1 T-3 T-3 T-3 T-6 T-6 T-6 T-9 Step 1 CE 1.0 1.0 1.0 0.5 0.5 1.0 0.5 0.5 1.0 Row 1 DRUG RPMI 1640 7.0 1.0 3.0 1.0 1.5 3.5 1.0 1.5 3.5 1.0
INTERMEDITE 640 320 160 80 40 20 10 5 2.5 1.25 DRUG CONC.(µg/ml) Add drug from Row1 1+4 1+4 1+4 1+4 1+4 1+4 1+4 1+4 1+4 1+4 Step 2 (ml)+RPMI(ml) Row 5x(1:4) Final 128 64 32 16 8 4 2 1 0.5 0.25 2X concentration From Row 2 add drug to plate(ml) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Step 3 1:1 Inoculum (ml) to plate 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Step 4
Final drug 64 32 16 8 4 2 1 0.5 0.25 0.125 conc: In each well T= Tube
68 CHART 2 :SCHEME FOR PREPARING DILUTIONS OF WATER INSOLUBLE DRUGS Drug(µg/ml) Stock 2 4 8 16 32 64 128 256 512 Remark TUBE TUBE1 2 3 4 5 6 7 8 9 10 SOURCE From T-1 T-1 T-1 T-4 T-4 T-4 T-7 T-7 T-7 STEP 1 DRUG(ml) stock 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ROW 1 1.0 Solvent DMSO(ml) - 0.5 1.5 3.5 0.5 1.5 3.5 0.5 1.5 3.5 INTERMEDIATE DRUG CONC. 1600 800 400 200 100 50 25 12.5 6.25 3.13 Add drug from 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 0.1+4.9 STEP 2 T1Row1(ml)+ ROW2 RPMI(ml) (1:50)
Final 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 2X concentration From Row 2 add 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 STEP 3 drug to plate(ml) 1:1 Inoculum (ml) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 STEP 4 T.Vol0.2ml Final drug 16 8 4 2 1 0.5 0.25 0.125 0.0625 0.0313 conc: In each well T-Tube
69 NUMBER OF CONCENTRATIONS TESTED(78,79,80)
x Ketoconazole 0.0313 to 16ȝg/ml
x Itraconazole 0.0313 to 16ȝg/ml
x Griseofulvin 0.0313 to 16ȝg/ml
x Fluconazole 0.0125 to 64ȝg/ml
x Terbinafine 0.0313 to 16ȝg/ml
MEDIUM: commercially available Rosewell Park memorial institute (RPMI)
1640(Annexure) with glutamine without bicarbonate.
BUFFER: MOPS (3-N-Morpholino prophanesulfonic acid)
FOR WATER SOLUBLE DRUGS:
Two fold dilutions of a water soluble antifungal agent is used, they may be prepared volumetrically in broth. (eg. Ketoconazole)
FOR WATER INSOLUBLE DRUGS: Diluent DMSO
To prepare for a broth microdilution test series containing a water insoluble drug that dissolves in DMSO, for which the highest desired test concentration is 16ȝg/ml. First weigh 4.8 mg (assuming 100% potency) of antifungal powder and dissolve in 3.0 ml of DMSO. This will provide a stock solution at 1600ȝg/ml. Next prepare further dilutions of this stock solution in
DMSO. The solutions in DMSO will be further diluted 1:50 in the test medium and then diluted two fold when inoculated(69).
DRUG DILUTION
To prepare 5ml volumes of antifungal agent first pipette 4.9 ml volumes of
RPMI 1640 medium into each of 10 sterile test tubes. Use a sterile pipette to add
0.1 ml of DMSO to 4.9 ml of medium which is used as control medium. Then 0.1 70 ml of lowest (3.13microgram/ml) drug concentration in DMSO and then 0.1 ml of the 6.25 ȝg /ml concentration and continue in sequence with increasing the concentration series, each time adding 0.1 ml volumes to 4.9 ml RPMI medium.
These volumes were adjusted according to the total number of test required.
Because there will be 1:2 dilution of the drug when combined with the inoculum, the working antifungal solutions are 2 fold more concentrated than the final concentration (NCCLS).
INOCULUM PREPARATION(81)
Cultures grown on Sabouraud Dextrose Agar at 25°C were used preferably
7-15 days old. Mature colonies were covered with 10ml of sterile saline (0.85 %).
Growth scraped using sterile Pasteur pipette. Heavy particles allowed to settle for about 15-20 minutes at room temperature. Supernatant was mixed with a vortex for 15 seconds. Turbidity of supernatant was adjusted spectrophotometrically to
530nm with 65 -70% absorbance. Each suspension was diluted 1: 50 in RPMI
1640.
INOCULATING RPMI -1640 MEDIUM
Each well will be inoculated on the day of test with 0.1 ml of 2x inoculum suspension. This step will dilute the drug concentration, inoculum densities and solvent used for the final desired test concentration. The growth control wells contains 0.1ml of the corresponding diluted inoculum suspension and 0.1ml of the drug diluents without anti fungal agents.
TEST PROCEDURE(82,27,22)
x Test was carried out using sterile microtitre plates.
x Aliquots of 100ȝl of drug dilutions inoculated in 1-10 microtitre wells. 71 x Concentration of Fluconazole 0.125 - 64 ȝg/ml (Concentration of others
0.039-16ȝg/ml).
x Added 100ȝl of inoculum into each well from 1 to 12.
x Growth control - tube 12 with inoculum and without antifungal drugs.
INCUBATION
All microdilution trays were incubated at 28ºC without agitation.
INTERPRETATION
MIC for azoles was determined by noting 80% inhibition of growth while for terbinafine, 100% growth inhibition.
DRUGS MIC range ȝl/ml Sensitive Resistant Terbinafine 0.0313 -16 0.25 0.25 Ketoconazole 0.0313-16 2 2 Itraconazole 0.0313-16 0.5 0.5 Fluconazole 0.0125-64 1 1 Griseofulvin 0.0313-16 2 2
DRUG END POINT FOR MIC
Itraconazole (100%)-(80) Score “0”
Fluconazole, Ketoconazole Score “2” or less
READING RESULTS(83)
The Minimum Inhibitory Concentration was taken as the lowest concentration of antifungal agent that substantially inhibits the growth of organism as detected visually. For the conventional microdilution procedure, the growth in each MIC well is compared with that of the growth control with the aid
72 of reading mirror. Each micro titer well was then given a numerical score as follows(69)
4 - No reduction in growth
3 - Slight reduction in growth or approximately 80% of growth control (drug free Medium) 2 – Prominent reduction in growth or approximately 50% of growth control
1 – Slight growth or approximately 25% of growth control
0 – Optically clear or absence of growth (NCCLS M-38A)
MIC results recorded in mg/ml.
73 RESULTS
The above study was conducted in a tertiary care hospital with 100 samples of dermatophytosis patients including skin scraping, hair and nail. The KOH mount and fungal culture of the samples were evaluated and the detailed analysis of the results is depicted as below:
Table 1: Distribution of age groups among the study population (n=100)
AGE NUMBER (n) PERCENTAGE (%) <10 6 6 11 to 20 25 25 21 to 30 15 15 31 to 40 24 24 41 to 50 14 14 51 to 60 14 14 61 to 70 2 2 p Value one sample z test = 0.1197
The study population consisted of patients in the age of 4-68 years. Out of 100 patients, maximum cases reported were in the age group of 11-20 years (25%) and the least were in the age group of 61 -70 years (2%). The p value by one sample z test is 0.1197. Figure 1: Distribution of age groups among the study population (n=100)
DISTRIBUTION OF AGE GROUP AMONG THE STUDY POPULATION(n=100) 30 25 24 25 20 15 14 14 15 10 6 5 2 0 <10 11 to 20 21 to 3031 to 40 41 to 50 51 to 60 61 to 70 Number affected Number Age group PERCENTAGE
74 Table 2: GENDER DISTRIBUTION (n=100)
GENDER FREQUENCY PERCENTAGE%
MALE 56 56
FEMALE 44 44
The study population consisted of 56 (56%) males and 44 (44%) females.
The Male: Female ratio was 1.27: 1(p value = 0.3953 one sample test for proportions)
Figure 2: GENDER DISTRIBUTION (n=100)
GENDER DISTRIBUTION (n=100)
44%
56%
MALE FEMALE
75 Table 3: RISK FACTORS (n=100)
RISK FACTORS NO OF PERCENTAGE CASES(n) (%) AGRICULTURER 37 37 HOSTELERS 23 23 CLOSE FAMILY CONTACTS 18 18 ANIMAL CONTACTS 14 14 HYPERHIDROSIS 8 8 TOTAL 100 100
Most of the cases were engaged in occupation related to agriculture 37 (37%) followed by hostelers 23 (23%).
Figure 3: RISK FACTORS (n=100)
RISK FACTORS (n=100)
8% AGRICULTURER
14% HOSTELERS 37% CLOSE FAMILY CONTACTS 18% ANIMAL CONTACTS HYPERHIDROSIS 23%
76 Table 4: DISTRIBUTION OF CLINICAL SPECIMENS AMONG STUDY
POPULATION (n=100)
SPECIMEN SKIN NAIL HAIR TOTAL
NO.OF SAMPLE 74 15 11 100
COLLECTED (n)
Out of 100 specimens collected, 74 were skin scrapings, 15 were nail clippings and 11 were hair samples.
Figure 4: DISTRIBUTION OF CLINICAL SPECIMENS AMONG STUDY
POPULATION (n=100)
DISTRIBUTION OF CLINICAL SPECIMENS AMONG THE STUDY POPULATION ( n=100)
11%
15% SKIN NAIL HAIR
74%
77 Table 5: DISTRIBUTION OF CLINICAL DIAGNOSIS AMONG STUDY
POPULATION (n=100)
SPECIMEN Clinical diagnosis Number Percentage (n) (%) SKIN(n=74 ) Tinea corporis 46 62.2 Tinea cruris 16 21.6 Tinea manuum 6 8.1 Tinea pedis 5 6.8 Tinea faciei 1 1.3 HAIR(n=11) Tinea capitis 9 81.8 Tinea barbae 2 18.2 NAIL (n=15) Tinea unguium 15 100
From skin sample (n=74), Tinea corporis was the predominant dermatomycotic lesion accounted for 46(62.6%) cases followed by tinea cruris 16 (21.6%) cases in this study . Tinea faciei was seen in 1 (1.3%) case. From hair (n=11), 9(81.8%) were from Tinea capitis, 2 (18.2%) were from Tinea barbae.
Figure 5: DISTRIBUTION OF CLINICAL DIAGNOSIS AMONG STUDY
POPULATION (n=100)
Distribution of clinical diagnosis among the study 100 100 population (n=100) 81.8 80 62.2 60 46 40 21.6 16 18.2 15 20 8.1 6.8 9 6 5 1 1.3 2 0
Nail (n=15) Skin (n= 74) Hair (n=11) Number Percentage
78 Table 6: EVALUATION OF KOH MOUNT (n=100)
SAMPLE KOH KOH TOTAL(n) POSITIVE(n) NEGATIVE (n) SKIN 24 50 74 NAIL 8 7 15 HAIR 3 8 11 TOTAL 35 65 100
The direct microscopic examination (KOH) was positive in 35 (35%) samples and was negative in 65(65%) samples.(p value =0.2697)
Figure 6: EVALUATION OF KOH MOUNT (n=100)
SAMPLE Vs KOH 120 100 100
80 74 65 60 50 KOH + 40 35 KOH - 24 15 TOTAL NUMBER OF ISOLATES OF NUMBER 20 8 8 11 7 3 0 SKIN NAIL HAIR TOTAL SAMPLES
79 Table 7: EVALUATION OF CULTURE (n=100)
SAMPLE CULTURE CULTURE TOTAL(n) POSITIVES NEGATIVES SKIN 25 49 74 NAIL 12 3 15 HAIR 3 8 11 TOTAL 40 60 100
The total number of samples which showed growth in cultures was 40 (40%) and the total number of samples which showed no growth in cultures was 60 (60%).
(p value = 0.0029 by Fischer’s Exact Test which is significant)
Figure 7: EVALUATION OF CULTURE (n=100)
SAMPLE Vs CULTURE 120 100 100 80 74 60 60 49 40 CULTURE + 40 25 CULTURE - 20 12 15 11 TOTAL 3 3 8 0 NUMBERISOLATES OF SKIN NAIL HAIR TOTAL SAMPLES
80 Table 8: CORRELATION BETWEEN DIRECT MICROSCOPY (KOH
EXAMINATION) AND CULTURE (n=100)
10% KOH 10% KOH TOTAL(n) POSITIVES NEGATIVES CULTURE 30 10 40 POSITIVES CULTURE 5 55 60 NEGATIVES TOTAL(n) 35 65 100
Out of 100 samples, 35 (35%) isolates were KOH positive and 65 (65%) isolates were KOH negative. 40 (40%) isolates were culture positive and 60 (60%) isolates were culture negative for dermatophytes. p value by Fischer’s Exact Test
= < 0.0001which is very significant. So statistically KOH positive samples were grown in culture and also showed significant growth.
Figure 8: CORRELATION BETWEEN DIRECT MICROSCOPY (KOH
EXAMINATION) AND CULTURE (n=100)
CORRELATION BETWEEN KOH EXAMINATION AND CULTURE OF THE ISOLATES (n=100)
10% KOH POSITIVE 5% CULTURE 30% POSITIVE KOH NEGATIVE CULTURE NEGATIVE KOH POSITIVE 55% CULTURE NEGATIVE
81 Table 9: CORRELATION BETWEEN DIRECT MICROSCOPY (KOH
EXAMINATION) AND CULTURE IN CLINICAL CASES (n-100)
KOH KOH KOH KOH TOTAL Lesion Positive Negative Positive Negative Culture Culture Culture Culture (n) Positive Positive Negative Negative Tinea corporis 11 3 2 30 46 Tinea cruris 7 1 1 7 16 Tinea capitis 2 1 0 6 9 Tinea manuum 1 0 0 5 6 Tinea pedis 1 1 0 3 5 Tinea barbae 0 0 1 1 2 Tinea unguium 8 4 0 3 15 Tinea faciei 0 0 1 0 1 TOTAL 30 10 5 55 100
A total of 30 (30%) samples were positive on direct examination and culture and
55 (55%) samples were negative by both the techniques. Samples positive by direct examination and negative on culture was 5(5%). Further 10(10%) samples isolated on culture were negative on direct examination.
Figure 9: CORRELATION BETWEEN DIRECT MICROSCOPY (KOH
EXAMINATION) AND CULTURE IN CLINICAL CASES (n-100)
Correlation between direct microscopy (KOH) and 120 culture (n=100) 100 100 KOH + CULTURE + KOH - CULTURE + KOH + CULTURE - KOH - CULTURE - 80 55 60 46 40 30 30 16 15 20 11 10 7 7 6 9 5 6 5 8 5 3 2 1 1 2 1 0 1 0 0 1 1 0 3 0 0 1 1 2 4 0 3 0 0 1 0 1 0 Tinea Tinea Tinea Tinea Tinea Tinea Tinea Tinea TOTAL corporis cruris capitis manuum pedis barbae unguim facei
82 Table 10: PERCENTAGE OF DERMATOPHYTES ISOLATED IN THE
STUDY POPULATION (n=100)
NO OF TOTAL NO DERMATOPHYTES OTHERS NO GROWTH OF CASES(n) ISOLATED
100 40 32 28
Out of 100 Samples collected, Dermatophytes isolated were 40 in number.
Non-dermatophytic moulds and candida species were 32 in number.
Figure 10: PERCENTAGE OF DERMATOPHYTES ISOLATED IN THE
STUDY POPULATION (n=100)
PERCENTAGE OF DERMATOPHYTES ISOLATED IN THE STUDY POPOULATION (n=100)
28% 40%
32%
NO OF DERMATOPHYTES ISOLATED OTHERS
83 Table 11: DISTRIBUTION OF CULTURES IN COLLECTED SAMPLES
(n=40)
SAMPLES NUMBERS(n) PERCENTAGE (%)
SKIN 25 25
NAIL 12 12
HAIR 3 3
TOTAL 40 40
Out of 40 culture isolated, 25 (25%) were isolated from the skin sample, 12
(12%) were from nail sample and 3 (3%) were from the hair sample.
Figure 11: DISTRIBUTION OF CULTURES IN COLLECTED SAMPLES
(n=40)
DISTRIBUTION OF CULTURES IN COLLECTED SAMPLES
8%
SKIN 30% NAIL HAIR 62%
84 Table 12: DISTRIBUTION OF THE GENUS OF THE ISOLATES AMONG
THE DERMATOPHYTOSIS GROUP (N=40)
Genus of the No. of Percentage isolates isolates(n) (%) Trichophyton 37 92.5
Microsporum 2 5
Epidermophyton 1 2.5
Total 40 100
Out of 40(100%) cultures isolated, the Genus Trichophyton were 37 (92.5%) cases, 2 (5%) cases were the genus Microsporum and one (2.5%) case was the
Genus Epidermophyton.
Figure 12: DISTRIBUTION OF THE GENUS OF THE ISOLATES
AMONG THE DERMATOPHYTOSIS GROUP (N=40)
Distribution of the genus of the isolates among the dermatophytosis group (n=40) 120 100 100 92.5 80 60 37 40 40 20 2 5 1 2.5 0
No. of isolates Percentage
85 Table 13: DISTRIBUTION OF FUNGAL ISOLATES AMONG THE
DERMATOPHYTOSIS GROUP (n=40)
No. of Percentage Genus Fungal isolate isolates (n) (%) Trichophyton(n=37) Trichophyton rubrum 18 45 T.mentagrophytes 12 30 Trichophyton 7 17.5 tonsurans Epidermophyton Epidermophyton 1 2.5 (n=1) floccosum Microsporum (n=2) Microsporum 1 2.5 gypseum Microsporum canis 1 2.5 Total 40 100 Out of 40 isolates of dermatophytes, 37 (92.5%) isolates belonged to the Trichophyton species of which Trichophyton rubrum 18(45%) was the predominant isolate, followed by Trichophyton mentagrophytes 12 (30%), and Trichophyton tonsurans 7 (17.5%). One isolate belonged to Epidermophyton floccosum 1 (2.5%). 2 (5%) isolates belongs to Microsporum species of which Microsporum gypseum was 1 (2.5%) and another 1(2.5%) isolate was Microsporum canis.
Figure 13: DISTRIBUTION OF FUNGAL ISOLATES AMONG THE
DERMATOPHYTOSIS GROUP (n=40)
DISTRIBUTION OF FUNGAL ISOLATES AMONG DERMATOPHYTOSIS GROUP (n=40) 120 100 100 80 45 60 30 40 40 18 12 17.5 20 7 1 2.5 1 2.5 1 2.5 0
NO. OF ISOLATES OF NO. No. of isolates Percentage FUNGAL ISOLATES 86 Table 14: DERMATOPHYTE SPECIES ISOLATED FROM THE SKIN
SAMPLE (n=25)
FUNGAL
ISOLATES (n) PEDIS TINEA TINEA TINEA TINEA TINEA FACIEI TOTAL CRURIS AGE (%) MANUUM PERCENT CORPORIS T.rubrum 5 4 1 1 - 11 44 T.menta 5 2 - - - 7 28 T.tonsurans 2 1 - 1 - 4 16 E.floccosum - 1 - - - 1 4 M.gypseum 1 - - - - 1 4 M.canis 1 - - - - 1 4 TOTAL(n) 14 8 1 2 - 25 100
Figure 14: DERMATOPHYTE SPECIES ISOLATED FROM THE SKIN
SAMPLE (n=25)
DERMETOPHYTE SPECIES ISOLATED FROM THE NAIL SAMPLE (n=15) 120 100 100 80 60 44 ISOLATES 40 28 25 16 20 11 7 NO OF 4 1 4 1 4 1 4 0
TOTAL(n) PERCENTAGE(%) FUNGAL ISOLATES
87 In Tinea corporis, Trichophyton rubrum 5/14 (35.7%) and Trichophyton mentagrophytes 5/14 (35.7%), were the predominant isolates, followed by
Trichophyton tonsurans 2/14 (14.3%), Microsporum canis 1/14 (7.2%) and
Microsporum gypseum 1/14 (7.2%).
In Tinea cruris, Trichophyton rubrum 4/8 (50%) was the predominant isolates, followed by Trichophyton mentagrophytes 2/8 (25%), Trichophyton tonsurans 1/8 (2.5%) and Epidermophyton floccosum 1/8 (12.5%).
One case 1/1 (100%) of Trichophyton rubrum was isolated from Tinea manuum.
Out of 2 dermatophytes isolated from Tinea pedis, 1 /2(50%) was from
Trichophyton rubrum and1 / 2(50%) was from Trichophyton tonsurans.
Out of 3 dermatophytes isolated from Tinea pedis, 2 were Trichophyton tonsurans and 1 was Trichophyton rubrum. Out of 2 dermatophytes isolated, one each of Trichophyton mentagrophytes and Trichophyton verrucosum were isolated.
88 Table 15: DERMATOPHYTE SPECIES ISOLATED FROM THE NAIL
SAMPLE (n=12)
FUNGAL NO. OF ISOLATES PERCENTAGE ISOLATES (n) (%) T.rubrum 7 58.3
T.mentagrophytes 4 33.3
T.tonsurans 1 8.3
TOTAL 12 100
Out of the 12 isolated dermatophytes in Tinea unguium, Trichophyton rubrum was the Predominant isolate of 7/12 (58.3%) in number, followed by
Trichophyton mentagrophytes 4/ 12 (33.3%) in number and there was 1/12
(8.3%) isolate of Trichophyton tonsurans.
Figure 15: DERMATOPHYTE SPECIES ISOLATED FROM THE NAIL
SAMPLE (n=12)
DERMETOPHYTE SPECIES ISOLATED FROM THE NAIL SAMPLE (n=15) 120 100 100 80 58.3 60 40 33.3 20 8.3 12 7 4 1 0
NO. OF ISOLATES PERCENTAGE(%)
89 Table 16: DERMATOPHYTE SPECIES ISOLATED FROM THE HAIR
SAMPLE (n=3)
FUNGAL Tinea Tinea Total Percentage ISOLATES barbae capitis (n) (%) T.mentagrophytes - 1 1 33.3
T.rubrum - 2 2 66.7
TOTAL - 3 3 100
Out of 3 dermatophytes isolated of Tinea capitis, 2/3 (66.7%) were Trichophyton tonsurans and 1/3(33.3%) was Trichophyton mentagrophytes.
Figure 16: DERMATOPHYTE SPECIES ISOLATED FROM HAIR
SAMPLE (n=3)
DERMATOPHYTE SPECIES ISOLATED FROM THE HAIR SAMPLE (n=3)
120 100 100
80 66.7 60 T.mentagrophytes 40 33.3 T.rubrum 20 TOTAL 1 2 3 1 2 3 0 Tinea capitis Total Percentage(%)
90 TABLE 17: MINIMAL INHIBITORY CONCENTRATION OF THE
DRUG GRISEOFULVIN
MIC MIC SPECIES 0.03 0.06 0.012 0.25 0.5 1 2 4 8 16 50 90 T.rubrum(n=18) - 2 3 8 5 - - - - 0.12 0.25 T.mentagrophytes - 2 4 6 ------0.12 0.25 (n=12) T.tonsurans(n=7) - 1 4 2 ------0.12 0.25 E.floccosum(n=1) - - - 1 ------0.25 0.25 M.gypseum(n=1) - 1 ------0.06 0.06 M.canis (n=1) - 1 ------0.06 0.06
MIC 50 and MIC 90 of Griseofulvin for the species isolated in this study are Trichophyton rubrum was 0.12 and 0.25 µg/ml respectively. Trichophyton mentagrophytes was 0.12 and 0.25 µg/ml respectively. Trichophyton tonsurans was 0.12 and 0.25 µg/ml respectively. Epidermophyton floccosum was 0.25 and 0.25 µg/ml respectively. Microsporum gypseum was 0.06 and 0.06 µg/ml respectively. Microsporum canis was 0.06 and 0.06 µg/ml respectively
Figure 17: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
GRISEOFULVIN
MIC OF THE DRUG GRISEOFULVIN
0.3 0.25 0.25 0.25 0.25 0.25 0.25 0.2 0.15 0.12 0.12 0.12 0.1 0.06 0.06 0.06 0.06 0.05 0
MIC 50 DRUG CONCENTRATION IN (µg/ml) DRUG CONCENTRATION
FUNGAL ISOLATES MIC 90
91 Table 18: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
GRISEOFULVIN TO THE ISOLATED DERMATOPHYTES (n=40)
FUNGAL NO.OF SENSITIVE RESISTANCE MIC µg/ml ISOLATES ISOLATES MIC 0.25 MIC 0.25 Candida Parapsilosis 1 1(100%) - 0.125 ATCC 22019 T.rubrum 18 18(100%) - 0.12-0.25 T.mentagrophytes 12 12(100%) - 0.12-0.25 T.tonsurans 7 7(100%) - 0.12-0.25 E.floccosum 1 1(100%) - 0.25 M.gypseum 1 1(100%) - 0.06 M.canis 1 1(100%) - 0.06
All isolates were sensitive to the drug griseofulvin.
Figure 18: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
GRISEOFULVIN TO THE ISOLATED DERMATOPHYTES (n=40)
ANTIFUNGAL SUSCEPTIBILITY PATTERN OF GRISEOFULVIN TO THE ISOLATED DERMATOPHYTES (n=40)
120% 100% 100% 100% 100% 100% 100% 100% 100% 80% 60% 40% 20% 0%
SENSITIVE MIC 0.25 RESISTANCE MIC 0.25
92 TABLE 19: MINIMAL INHIBITORY CONCENTRATION OF THE
DRUG KETOCONAZOLE
MIC MIC SPECIES 0.03 0.06 0.012 0.25 0.5 1 2 4 8 16 50 90 T.rubrum(n=18) - 1 4 7 5 - - 1 - - 0.12 0.5 T.mentagrophytes - 3 4 5 ------0.12 0.25 (n=12) T.tonsurans(n=7) - 2 2 3 ------0.06 0.25 E.floccosum(n=1) - 1 ------0.06 0.06 M.gypseum(n=1) - - 1 ------0.12 0.12 M.canis(n=1) - 1 ------0.06 0.06
MIC 50 and MIC 90 of Ketoconazole for the species isolated in this study are
Trichophyton rubrum was 0.12 and 0.5 µg/ml respectively. Trichophyton mentagrophytes was 0.12 and 0.25 µg/ml respectively. Trichophyton tonsurans was 0.06 and 0.25 µg/ml respectively. Epidermophyton floccosum was 0.06 and
0.06 µg/ml respectively. Microsporum gypseum was 0.12 and 0.12 µg/ml respectively. Microsporum canis was 0.06 and 0.06 µg/ml respectively.
Figure 19: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
KETOCONAZOLE
MIC OF THE DRUG KETOCONAZOLE
0.6 0.5 0.5 0.4 0.3 0.25 0.25 0.12 0.12 0.12 0.12
(µg/ml) 0.2 0.1 0.06 0.06 0.06 0.06 0.06 0
DRUG CONCENTRATION IN DRUG CONCENTRATION MIC 50 MIC 90
FUNGAL ISOLATES
93 Table 20: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
KETOCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
NO.OF SENSITIVE RESISTANCE MIC FUNGAL ISOLATES ISOLATES MIC 0.25 MIC 0.25 µg/ml Candida parapsilosis 1 1(100%) - 0.25 ATCC 22019 T.rubrum 18 17(94.5%) 1*(5.5%) 0.12-0.5 T.mentagrophytes 12 12(100%) - 0.12-0.25 T.tonsurans 7 7(100%) - 0.06-0.25 E.floccosum 1 1(100%) - 0.06 M.gypseum 1 1(100%) - 0.12 M.canis 1 1(100%) - 0.06
All isolates were sensitive to ketoconazole except for one strain of Trichophyton rubrum.
*MIC= 4 µg /ml
Figure 20: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
KETOCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
ANTIFUNGAL SUSCEPTIBILITY PATTERN OF KETOCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40) 120% 100% 95% 100% 100% 100% 100% 100% 100% 80% 60% 40% 20% 0 5% 0 0 0 0 0 0%
SENSITIVE MIC 2 RESISTANCE MIC 2
94 Table 21: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
FLUCONAZOLE
FUNGAL 0.03 0.06 0.012 0.25 0.5 1 2 4 8 16 MIC MIC ISOLATES 50 90 T.rubrum(n=18) - - - - - 9 6 3 - - 1 4 T.mentagrophytes ------7 3 2 - 2 8 (n=12) T.tonsurans(n=7) ------4 3 - - 2 4 E.floccosum(n=1) ------1 - - 4 4 M.gypseum(n=1) ------1 - - 4 4 M.canis(n=1) ------1 - - - 2 2
MIC 50 and MIC 90 of Fluconazole for the species isolated in this study are Trichophyton rubrum was 1 and 4 µg/ml respectively. Trichophyton mentagrophytes was 2 and 8 µg/ml respectively. Trichophyton tonsurans was 2 and 4 µg/ml respectively. Epidermophyton floccosum was 4 and 4 µg/ml respectively. Microsporum gypseum was 4 and 4 µg/ml respectively. Microsporum canis was 2 and 2 µg/ml respectively.
Figure 21: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
FLUCONAZOLE
MIC OF THE DRUG FLUCONAZOLE
9 8 8 7 6 5 4 4 4 4 4 4 4 3 2 2 2 2 2 1 1 0 DRUG CONCENTRATION IN µg/ml IN DRUG CONCENTRATION
MIC 50
FUNGAL ISOLATES MIC 90
95 Table 22: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG FLUCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40) FUNGAL NO. OF SENSITIVE RESISTANCE MIC ISOLATES ISOLATES MIC 1 MIC 1 µg/ml Candida 1 1(100%) - 4 parapsilosis ATCC 22019 T.rubrum 18 18(100%) - 1 to 4 T.mentagrophytes 12 12(100%) - 2 to 8 T.tonsurans 7 7(100%) - 2 to 4 E.floccosum 1 1(100%) - 4 M.gypseum 1 1(100%) - 4 M.canis 1 1(100%) - 2
All isolates were sensitive to the drug fluconazole
Figure 22: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
FLUCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
ANTIFUNGAL SUSCEPTIBILITY PATTERN OF FLUCONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
120% 100% 100% 100% 100% 100% 100% 100% 100% 80% 60% 40% 20% 0%
SENSITIVE MIC 64 RESISTANCE MIC 64
96 Table 23: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
ITRACONAZOLE
FUNGAL MIC MIC 0.0075 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 ISOLATES 50 90 T.rubrum - - - 3 5 9 - - - 1 0.12 0.25 (n=18) T.menta - - 2 6 4 - - - - - 0.03 0.12 (n=12) T.tonsurans - - - 2 3 - - - - - 0.06 0.25 (n=7) E.floccosum - - 1 - - 2 - - - - 0.03 0.03 (n=1) M.gypseum - - - 1 ------0.06 0.06 (n=1) M.canis - - 1 ------0.03 0.03 (n=1)
MIC 50 and MIC 90 of Itraconazole for the species isolated in this study are Trichophyton rubrum was 0.12 and 0.25 µg/ml respectively. Trichophyton mentagrophytes was 0.03 and 0.12 µg/ml respectively. Trichophyton tonsurans was 0.06 and 0.25 µg/ml respectively. Epidermophyton floccosum was 0.03 and 0.03 µg/ml respectively. Microsporum gypseum was 0.06 and 0.06 µg/ml respectively. Microsporum canis was 0.03 and 0.03 µg/ml respectively.
Figure 23: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
ITRACONAZOLE
MIC OF THE DRUG ITRACONAZOLE 0.3 0.25 0.25 0.25 0.2 0.15 0.12 0.12 0.06 0.06 0.06 0.1 0.03 0.03 0.03 0.03 µg/ml 0.05 0.03 0
DRUG CONCENTARTION IN DRUG CONCENTARTION MIC 50 MIC 90 FUNGAL ISOLATES
97 Table 24: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
ITRACONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
FUNGAL NO.OF SENSITIVE RESISTANCE MIC ISOLATES ISOLATES MIC 0.25 MIC 0.25 µg/ml Candida parapsilosis 1 1(100%) - 0.125 ATCC 22019 T.rubrum 18 17(95 %) 1(5 %) 0.12-0.25 T.menta 12 12(100%) - 0.03-0.12 T.tonsurans 7 7(100%) - 0.06- 0.250 E.floccosum 1 1(100%) - 0.03 M.gypseum 1 1(100%) - 0.06 M.canis 1 1(100%) - 0.03
All isolates were sensitive to the drug itraconazole except one isolate of
Trichophyton rubrum.
Figure 24: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
ITRACONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40)
ANTIFUNGAL SUSCEPTIBILITY PATTERN OF ITRACONAZOLE TO THE ISOLATED DERMATOPHYTES (n=40) 120% 100% 95% 100% 100% 100% 100% 100% 100% 80% 60% 40% 20% 0 5% 0 0 0 0 0 0%
SENSITIVE MIC 0.25 RESISTANCE MIC 0.25
98 Table 25: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG
TERBINAFINE
FUNGAL MIC MIC 0.0075 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 ISOLATES 50 90 T.rubrum(n=18) - 2 6 8 - - - - - 2 0.03 0.06
T.mentagrophytes - 3 4 5 ------0.015 0.06 (n=12) T.tonsurans(n=7) - 1 2 4 ------0.015 0.06
E.floccosum(n=1) - - - 1 ------0.06 0.06
M.gypseum(n=1) - - 1 ------0.03 0.03
M.canis(n=1) - - 1 ------0.03 0.03
MIC 50 and MIC 90 of Terbinafine for the species isolated in this study are Trichophyton rubrum was 0.03 and 0.06 µg/ml respectively. Trichophyton mentagrophytes was 0.015 and 0.06 µg/ml respectively. Trichophyton tonsurans was 0.015 and 0.06 µg/ml respectively. Epidermophyton floccosum was 0.06 and 0.06 µg/ml respectively. Microsporum gypseum was 0.03 and 0.03 µg/ml respectively. Microsporum canis was 0.03 and 0.03 µg/ml respectively.
Figure 25: MINIMAL INHIBITORY CONCENTRATION OF THE DRUG TERBINAFINE
MIC OF THE DRUG TERBINAFINE 0.07 0.06 0.06 0.06 0.06 0.06 0.06 0.05 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.015 0.02 0.015 0.01 µg/ml 0
DRUG CONCENTRATION IN DRUG CONCENTRATION MIC 50 MIC 90 FUNGAL ISOLATES
99 Table 26: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
TERBINAFINE TO THE ISOLATED DERMATOPHYTES (n=40)
FUNGAL NO.OF SENSITIVE RESISTANCE MIC µg/ml ISOLATES ISOLATES MIC 0.25 MIC 0.25 Candida parapsilosis ATCC 22019 1 1(100%) - 0.125
T.rubrum 18 16(88.89 %) 2(11.11 %) 0.03-0.06 T.mentagrophytes 12 12(100%) - 0.015-0.06 T.tonsurans 7 7(100%) - 0.0150.06 E.floccosum 1 1(100%) - 0.06 M.gypseum 1 1(100%) - 0.03 M.canis 1 1(100%) - 0.03
All the isolates were sensitive to the drug terbinafine except two isolates of
Trichophyton rubrum.
Figure 26: ANTIFUNGAL SUSCEPTIBILITY PATTERN OF THE DRUG
TERBINAFINE TO THE ISOLATED DERMATOPHYTES (n=40)
ANTIFUNGAL SUSCEPTIBILITY PATTERN OF TERBINAFINE TO THE ISOLATED DERMATOPHYTES (n=40)
120% 100% 100% 100% 100% 100% 100% 100% 89% 80% 60% 40% 11% 20% 0 0 0 0 0 0 0%
SENSITIVE MIC 0.25 RESISTANCE MIC 0.25
100 DISCUSSION
A total of 100 patients who attended dermatology OPDs and was diagnosed as a case of superficial mycoses were studied during one year period from March 2015 to Febraury 2016.
The age group of the patients were from 4 to 68 years, the mean age being
32.5 years [Table-1] same as that studied by Madhavi et al (2011) in mean age of
28.5 ± 6.32. The common age groups affected were between 11-20 years (25%).
Similar results were shown in the studies of Bindu et al (2002) (66)with 23.3% and Pradeep et al (2012) with 17.7%(21,84,85). Snehu et al (2014) also showed the pupils of age range 13 – 15 years had the highest prevalence rate.
There were 56 males(56%) and 44 females(44%) [Table-2]. The higher incidence in young males could be due to greater physical activity, increased sweating because of environmental conditions such as hot and humid weather and the frequent interaction with different people of the society. Heoprich et al (1994) has attributed the male preponderance to hormonal factor and P Veer et al (2007) to their more outdoor activities hence prone to trauma(15). The male to female ratio is 1.3:1. Males outnumbered females due to prevailing social stigma, illiteracy, poor personal hygiene and environmental conditions. Madhavi et al showed the same male to female ratio of 1.3:1(84). Similar studies were also showed by M.situm et al (1998) who found in 59% male and 41% females(86).
Kennedy et al (2007) who found in 53% and 47% respectively and stated that most studies in and around Chennai showed a male dominance(87,14).
101 In the present study[Table-3], 37 patients were engaged in occupation related to agriculture, 23 patients were hostelers, 18 pateints had a close family contacts, 14 cases had a history of animal contacts and 8 patient were presented with hyperhidrosis.The highest prevalence in agriculture workers coupled with the fact that host susceptibility may be enhanced by moisture, warmth, specific skin chemistry, composition of sebum and perspiration, age, heavy exposure to hot and humid climates, crowded living conditions and genetic predisposition(21).
They are the rural people working in the fields, gardens with manure and decaying organic matter in the soil will add to the further risk of getting these fungal infections(19).
Out of 100 clinical samples, 74 were skin scrapings, 15 were from nail clippings and 11 were from hair specimens [Table-4 and Table -11]. More isolates were from skin scrapings and least from nail and hair samples. P Kannan et al, Rajeswari K.Gangulappa et al were showed similar results(25,88).
Among the skin lesions, tinea corporis was the predominant which occurred in 46 (62.2%) patients followed by tinea cruris in 16 (21.6%), tinea manuum in 6(8.1%) and tinea pedis in 5 (6.8%) [Table-5] which was in accordance with Bindu et al study(66). Verenkar MP et al (1991) has stated that the high incidence of tinea corporis and tinea cruris was probably due to its symptomatic (pruritic) nature which leads the patient to seek medical advice(89). It is observed that most of the patients were involved in exhausting physical work like profuse sweating. Furthermore, they wear tight synthetic clothes resulting in
102 conditions like increased dampness and warmth of the body facilitating the skin surface suitable for the growth of dermatophytes.
A low incidence of tinea pedis 5(6.8%) was also coincides with the study of Venkatesan et al 4(5.6%)(90). The predominance of Tinea pedis in western countries could be because of the regular use of shoes and shocks, resulting in conditions like dampness and warmth of the body thereby facilitating the skin surface for the growth of dermatophytes(90).
Among the hair samples 11 (11%), Tinea capitis was detected in 9(9%) cases in the present study. The incidence of Tinea capitis 9 (9%) in this study was comparable with Suman singh et al study(10). Tinea capitis is less common in
India than in other countries, this may be attributable to the use of hair oils used by Indians and have been shown to have an inhibitory effect on dermatophytosis(91,92).
Out of 100 clinically diagnosed cases, direct microscopic examination
(KOH) showed positivity in 35 samples (35%) [Table-6] which was only slightly varied to JC Mohanty et al 43% (1999)(43). On KOH wet mount examination positivity rates ranging from 23.8% to as high as 91.2% have been reported in various studies(93). Selection criteria of cases and skill involvement in sampling techniques perhaps accounts for the differences. Thus all KOH negative samples should be cultured(94). Non visualization of hyphae on direct microscopy could possibly due to a severe inflammatory reaction which obscures them. This finding elucidates the importance of culture to establish diagnoses(94).
103 The causal agents were isolated by culture in 25 skin lesions, 12 nail samples and 3 hair samples [ Table-7]. Thus the total culture positivity was 40
(40%) which only slightly varied with Surenderan et al 39%, Sumit Kumar et al(2014) 42.4%, Suman singh et al(2003) 44.62%, Pankajalakshmi et al,
Chennai(1981) 44% and Sharma et al (1987) 45%(3,10,61,95,96). It was compared to rates varying from 7% to 49% in other studies of Gupta RN et al(1959) and
Bhaskaran CS et al (1977). The differences in these rates among different studies may be due to the factors involved in the collection, transport, inoculation of specimen, culture condition severity, type and stage of the disease and the effect of antifungal agents
Out of 100 samples collected [Table-8], 35(35%) was KOH positive and
65(65%) was KOH negative. 40(40%) isolates were culture positive and 60(60%) were culture negative for dermatophytes .In this study 10 of the culture positive samples showed no fungal filaments on direct KOH mount because of the fungus in an inactive sporulating phase difficult to be seen by microscopy but able to grow in appropriate medium. Out of the culture negative cases 5 showed fungal elements on KOH mount but failed to grow in culture. Surenderan et al study showed similar results. This could be due to non viability of the fungi prior to inoculation and inappropriate use of antimycotic treatment and self medication before sampling(3).
Samples positive by both the techniques was 30 (30%) and negative by both the techniques was 55 (55%) [Table-9]. Samples positive by direct examination and negative on culture was 5(5%). Further 10 (10%) sample
104 isolated on culture were negative on direct examination which was similar to
Sowmya Nasimuddin et al (2014).
Out of 100 samples, dermatophyte was the major pathogen isolated which accounted for 40(40%) out of culture positive cases followed by candida species and non dermatophytic molds 32(32%) [Table-10]. The result was similar to
Sowmya Nasimuddin et al (2014) 43%, Surenderan et al 39%(3). All three genera of dermatophytes such as Trichophyton 37(92.5%), and Microsporum 2(5%) and
Epidermophyton 1(2.5%) have been isolated as the causative agent in this study
[Table-12]. Totally six species of dermatophytes were isolated. Clarissa et al and
Surenderan et al showed the similar results(3,97).
Out of 40 isolates of dermatophytes, 37(92.5%) isolates belonged to the
Trichophyton species of which Trichophyton rubrum was the predominant isolate
18 (45%)[Table-13]. This coincides with most of the studies like Chitralekha S et al (2013) 43.7%, Gupta sarika et al (2014) 48% and Madhavi S et al (2011)
51%(84,98).Trichophyton mentagrophytes was the second common isolate. This is in conformity with studies of Urmil Mohan et al 28.2%, Nagakatti PS et al 29%
(99)and Madhavi S et al( 2011) 31%(84)..
Trichophyton rubrum was found to be the commonest etiological agent 18
(45%) isolated from tinea corporis, tinea pedis, tinea mannum, tinea faciei, tinea cruris, tinea capitis, and tinea unguium, followed by Trichophyton mentagrophytes 12(30%), Trichophyton tonsurans 7(17.5%), Epidermophyton floccosum 1(2.5%), Microsporum gypseum1(2.5%) and Microsporum canis
1(2.5%) [Table-14-16].As accordance with Suman singh et al,Doddamani PV et
105 al (2012)(10,100).Trichophyton rubrum was the common isolate from tinea corporis. Trichophyton rubrum was the predominant dermatophyte isolated from
Tinea cruris but Suman singh et al showed Epidermophyton floccosum as the chief isolate.
In accordance with P Veer et al, Trichophyton rubrum was the common isolate from tinea unguium(15).
In the present study the MIC range [Table-17-26], MIC 50 and MIC 90 for the drugs griseofulvin, ketoconazole, fluconazole, itraconazoles and terbinafine were determined. This study correlates with the studies conducted by Fernandez
Torres et al, C.J.Jessup et al(22,27,78).
Even though Norries et al and C.J.Jessup et al studies established the inoculums size, optimum condition, optimum medium for conidial formation , incubation time duration and end point determination but standard reference method for antifungal susceptibility testing of dermatophytic infection is lacking(22,27,101). For this study the microdilution method was chosen because of its conveniency, reproducibility and greater ease of performance.
Among dermatophytes, all isolates were sensitive to griseofulvin, fluconazole, terbinafine, itraconazole and ketoconazole except three strains. Out of the resistant strains, one (Trichophyton rubrum) was resistant to terbinafine and ketoconazole, another (Trichophyton rubrum) to terbinafine and the another one (Trichophyton rubrum) to itraconazole. Colin.S.osborne et al (2005) has found that some strains of Trichophyton rubrum isolated showed intrinsic
106 resistance to terbinafine which on prolonged exposure to the drug can raise the
MIC values. Similar findings were shown by Pranab K.Mukherjee et al
(2003,102,103).
Cervelatti EP et al (2006) and Fachin AL et al(2006) has detailed the involvement of ABC transporter gene in the development of resistance to azoles in Trichophyton rubrum.
107 SUMMARY
1. Out of the 100 patient with dermatophytosis studied,
2. The commonest age group affected were 11-20 (25%) followed by 31-
40(24%).
3. Male to female ratio was 1.27:1.
4. It was more common in farmers (37%) followed by hostellers (23%).
5. The commonest skin lesion was tinea corporis 46(62.2%) followed by
tinea cruris 16(21.6%).
6. The direct microscopy (KOH) was positive in 35(35%) and culture in
40(40%) of clinically diagnosed cases.
7. Isolation rate in culture media namely SDA and DTM were the same but
earliest in DTM (5-7days).
8. Samples were skin, hair and nail. The commonest isolates were
dermatophytes 40(40%) followed by candida and nondermatophyte 32
(32%). Among the total dermatophytes isolated, Trichophyton rubrum
18(45%) was the commonest pathogen.
9. Antifungal susceptibility testing was performed by microbroth dilution
method for griseofulvin, ketoconazole, fluconazoles,itraconazle and
terbinafine.
10.Among the total dermatophytes isolated, three isolates showed resistance
to antifungal agents (Terbinafine,ketoconazole and itraconazole). One was
to itraconazoles alone, other to terbinafine alone and the third one was to
both ketoconazole and terbinafine.
108 CONCLUSION
Dermatophytosis accounts the common cutaneous manifestation and especially onychomycosis which can act as a chronic reservoir. The chronicity and tissue disruption can lead to secondary bacterial infection.
Clinical suspicion, early laboratory examination to confirm diagnosis and appropriate treatment is very crucial in this group of patients. Direct microscopic examination and culture identification plays the major part in the management.
Though Sabouraud dextrose agar with antimicrobials which needs incubation for
4-8weeks,is the commonly used media for isolation, Dermatophyte test medium which give presumptive identification within a week can be used instead in these group of patients.
Apart from dermatophytes, which is the commonest isolates in superficial mycoses, other agents like Candida and non dermatophyte moulds which were considered as contaminants or coloniser is also emerging as a pathogen especially in these group of patients. Hence repeated isolation will prove its pathogenicity.
Though early complete course of treatment gives mycological cure, resistant strains are also occurring, which can act as a chronic reservoir of infection. Hence routine antifungal susceptibility testing has to be done routinely for timely interventions.
109 LIST OF ABBREVIATIONS
AFST : Antifungal susceptibility testing
ATCC : American type culture collection
CLSI : Clinical and laboratory standard institute
C.p : Candida parapsilosis
DMSO : Dimethyl sulphoxide
DLSO : Distal and lateral subungual onychomycosis
DTM : Dermatophyte test medium
KOH : Potassium hydroxide02
LPCB : Lactophenol cotton blue
MIC : Minimum inhibitory concentration
NDM : Non dermatophyte mould
PDA : Potato dextrose agar
PSO : Proximal Subungual Onychomycosis
RPMI : Rose Parkwell memorial institute.
SDA : Sabouraud’s dextrose agar
TDO : Total Dystrophic Onychomycosis
T.ment : Trichophyton mentagrophytes
E.floc : Epidermophyton flocossum
WSO : White Superficial Onychomycosis
110 ANNEXURE
SABOURAUDS DEXTROSE AGAR WITH ANTIBIOTICS
Ingredients:
Peptone-10gm
Dextrose-40gm
Agar-20gm
Cycloheximide-500mg
Chloramphenicol-50mg
Gentamicin-20mg
Distilled water-1000ml
All the above mentioned ingredients are autoclaved and adjust pH at 5.6.
Dissolve cycloheximide in 10ml acetone and similarly dissolve chloramphenicol/gentamicin in 10ml of 95% alcohol and added to the boiling medium. Dispense in tubes, allowed to cool in slanted position. Store at 4Û C. It has shelf life of 30 days in test tube and 14 days in petridishes.
DERMATOPHYTE TEST MEDIUM
Phyton-10gm
Dextrose-10gm
Phenol red solution-40ml119
8N HCl-6ml
Actidione-500mg
Gentamicin-100mg
Agar-20gm
111 Distilled water-1000ml
Final pH-5.5+/-0.4
The phenol red solution is 0.5 gm in 15ml of 1N NaOH made upto 100ml with distilled water. Adjust pH to 5.5.
LACTOPHENOL COTTON BLUE MOUNT
Ingredients:
Melted Phenol-20ml
Lactic acid-20ml
Glycerol-40ml
Cotton blue-0.05gm
Distilled water-20ml
Mix all ingredients properly and dissolve 0.05 gm of Cotton blue stain in distilled water before mixing with remaining reagents. The phenol acts as disinfectant. Lactic acid preserves morphology of fungi and glycerol is a hygroscopic agent which prevents drying. The cotton blue stains outer wall of fungus.
POTASSIUM HYDROXIDE MOUNT
Ingredients:
Potassium hydroxide-10gm
Glycerol-10ml
Distilled water-80ml
To solution of 10% KOH, 10ml glycerol is added to prevent drying.
Mix these ingredients properly and store the solution at room temperature.
112 NOTE: For 20% KOH ,20gm Potassium hydroxide is used.
10% YEAST EXTRACT
Yeast extract -10 gm
Distilled water-100ml
Mix the yeast extract and distilled water in a flask and swirl to dissolve. Filter sterilize the solution and store it in sterile flask in a refrigerator until it is used.
123
MODIFIED CHRISTENSEN’S MEDIUM FOR UREA HYDROLYSIS
Peptone-1 gm
Sodium chloride-5 gm
Disodium phosphate-1.2 gm
Monopotassium phosphate-0.8 gm
Phenol red -0.012 gm
Dextrose-1.5 gm
Agar -15 gm
Distilled water - 1000 ml.
Urea, 20%, solution, sterile -100ml
After dissolution of the above ingredients by heat, 5 ml of phenol red solution (0.2% in 50 % alcohol) was added after which autoclaving was done at
115Û C for 15 mins. On cooling to 500 C, 100 ml of urea (20% aqueous solution, sterilized by filtration) was added. The medium was poured into slopes with the butt 1” deep and the slant 1.5” long.
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127 PROFORMA
NAME: AGE/SEX: OP/IP:
OCCUPATION: ADDRESS:
PRESENT COMPLAINTS:
SITE OF LESION:
PRIMARY/RELAPSE:
PET ANIMALS:
CLOSE FAMILY CONTACT :
H/O EXCESSIVE SWEATING:
TREATMENT HISTORY:
PAST HISTORY:
SAMPLE:SKIN/NAIL/HAIR
LAB INVESTIGATION:
KOH MOUNT:
CULTURE:
SDA:
DTM:
GRAM STAIN:
128 LPCB MOUNT:
SLIDE CULTURE:
UREA HYDROLISIS TEST:
IN VITRO HAIR PERFORATION TEST:
INTERPRETATIONS:
129 PATIENT CONSENT FORM
STUDY DETAIL: “CLINICOMYCOLOGICAL PROFILE OF THE DERMATOPHYTOSIS & THEIR ANTIFUNGAL SUSCEPTIBILITY PATTERN IN PATIENTS ATTENDING DERMATOLOGY OUTPATIENT DEPARTMENT IN A TERTIARY CARE HOSPITAL”
STUDY CENTER: CHENGALPATTU MEDICAL COLLEGE & HOSPITAL, CHENGALPATTU
PATIENT NAME: PATIENT AGE: IDENTIFICATION NUMBER: SEX:
I confirm that I have understood the purpose of procedure for the above study. I have the opportunity to ask the question and all my questions and doubts have been answered to my satisfaction. I understand that my participation in the study is voluntary and that I am free to withdraw at anytime without giving any reasons, without my legal rights being affected. I understand that investigator, regulatory authorities and the ethics committee will not need my permission to look at my health records both in respect to the current study and any further research that may be conducted in relation to it, even if withdraw from the study, I understand that my identity will not be revealed in any information released to third parties or published, unless as required under the law. I agree not to restrict the use of any data or results that arise from the study. I agree to take part in the above study and to comply with the instructions given during the study and faithfully cooperative with the study team and to immediately inform the study staff if I suffer from any deterioration in my health or wellbeing or any unexpected or unusual symptoms. I hereby give consent to participate in this study. I also accept to provide my infected skin scrapings / nail clippings / hair for microbiological investigations. I hereby give permission to undergo complete clinical examination and diagnostic test.
Signature/Thumb impression : Place:
Date: Patient name and address:
Study investigator’s name: Place: Date: Signature of the investigator: 130 131 FLOW CHART
Skin / Hair/ Nail
KOH Culture
SDA with SDA with DTM Antibacterial Antimicrobial Agent Agent
Dermatophyte Candida Non x LPCB x GTT Dermatophyte x Urea Hydrolysis x Chrom Agar Mould x Invitro hair x Sugar perforation Test x LPCB Fermantation Test
Antifungal Susceptibility Testing
132 LIST OF IMAGES
Image 1: TINEA CORPORIS
Image 2: TINEA UNGUIUM
133 Image 3 i) KOH WET MOUNT OF DERMATOPHYTOSIS
(ii) KOH WET MOUNT OF PITYRIASIS VERSICOLOR(MEAT AND
SPAGHETTI BALL APPEARANCE)
134 Image 4: Trichophyton rubrum – Macroscopy & Microscopy
Image : Trichophyton rubrum (Bird on fence appearance)-LPCB
135 Image 5:Trichophyton mentagrophytes- Macroscopy & Microscopy
Image : Trichophyton mentagrophytes Image : spiral hyphae
136 Image 6: Trichophyton tonsurans – Macroscopy and Microscopy
Image 7: Microsporum canis- Macroscopy & Microscopy
137 Image 8: Microsporum gypseum –Macroscopy & Microscopy
Image 9: Epidermophyton floccosum – Macroscopy & Microscopy
138 Image 10 :Hair Perforation test – Postive and Negative
Image 11 : Urease test positive
139 Image 12 : Non dermatophytic moulds
Cunninghamella Fusarium species
Aspergillus flavus Aspergillus fumigatus
140 Nigrospora Penicillium
Image 13: candida species – Macroscopy and Microscopy
141 Image 14: ANTIFUNGAL DRUGS FOR MIC
Image 15: RPMI 1640 BROTH
142 Image 16: MICROBROTH DILUTION METHOD – AFST MIC FOR
GRISEOFULVIN
Image 17: MICROBROTH DILUTION METHOD – AFST MIC FOR
KETOCONAZOLE
143 Image 18: MICROBROTH DILUTION METHOD – AFST MIC FOR
FLUCONAZOLE
Image 19: MICROBROTH DILUTION METHOD – AFST MIC FOR
ITRACONAZOLE
144 Image 20: MICROBROTH DILUTION METHOD – AFST MIC FOR
TERBINAFINE
145 MASTER CHART S. No. Age Sex Risk Diagnosis Sample KOH Culture LPCB Urease Hair Perf GRISEO TERBI FLUC ITRA KETO 1 65 M Agri T.unguim Nail + + T.rubrum S S S S S 2 38 F Agri T.corporis Skin - - - 3 4 M Cfc T.corporis Skin + + T.mentagrophytes P P S S S S S 4 5 F Cfc T.corporis Skin - - - 5 40 F Agri T.corporis Skin - - - 6 13 M Host T.corporis Skin + + T.mentagrophytes P P S S S S S 7 32 F Ac T.corporis Skin + - - 8 15 M Host T.facei Skin + - - 9 16 M Host T.corporis Skin - - - 10 18 M Host T.corporis Skin - - - 11 50 M Agri T.cruris Skin + - - 12 44 M Cfc T.corporis Skin - - - 13 55 F Agri T.corporis Skin + + T.tonsurans S S S S S 14 21 M Host T.corporis Skin + + T.mentagrophytes P P S S S S S 15 15 M Host T.corporis Skin - - - 16 48 M Agri T.corporis Skin - - - 17 37 F Agri T.cruris Skin - - - 18 14 M Host T.corporis Skin + + M.gypseum S S S S S 19 68 F Agri T.unguim Nail - - - 20 14 M Host T.unguim Nail + + T.mentagrophytes P P S S S S S 21 24 M Host T.corporis Skin - - - 22 18 M Host T.corporis Skin - - - 23 45 F Agri T.cruris Skin + + T.rubrum S R S S S 24 19 M Host T.capitis Hair - - - 25 40 F Agri T.cruris Skin - - -
146 26 40 M Agri T.corporis Skin + + T.rubrum S S S S S 37 47 M Sweat T.corporis Skin + + T.rubrum S S S S S 28 51 M Agri T.unguim Nail + + T.rubrum S S S S S 29 55 F Cfc T.cruris Skin + + T.rubrum S R S S S 30 33 M Agri T.corporis Skin + - - 31 45 F Agri T.unguim Nail + + T.rubrum S S S S S 32 56 M Agri T.corporis Skin - - - 33 7 MAc T.cruris Skin + + E.floccosum S S S S S 34 21 M Host T.corporis Skin - - - 35 41 M Agri T.unguim Nail + + T.mentagrophytes P P S S S S S 36 30 M Ac T.cruris Skin + + T.mentagrophytes P P S S S S S 37 9 M Host T.capitis Hair + + T.mentagrophytes P P S S S S S 38 13 M Host T.capitis Hair - + T.tonsurans S S S S S 39 21 F Host T.unguiim Nail - + T.mentagrophytes P P S S S S S 40 35 F Sweat T.cruris Skin + + T.rubrum S S S S S 41 35 F Sweat T.cruris Skin - - - 42 18 M Host T.corporis Skin - - - 43 47 F Agri T.corporis Skin - + T.rubrum S S S R S 44 30 F Cfc T.corporis Skin - - - 45 36 M Cfc T.unguim Nail - + T.rubrum S S S S S 46 14 F Host T.unguim Nail + + T.mentagrophytes P P S S S S S 47 24 F Ac T.pedis Skin + + T.rubrum S S S S S 48 14 M Host T.corporis Skin - - - 49 21 F Cfc T.corporis Skin - - - 50 38 F Agri T.corporis Skin - - - 51 54 M Ac T.cruris Skin + + T.tonsurans S S S S S 52 23 F Cfc T.unguim Nail + + T.rubrum S S S S S
147 53 45 M Ac T.corporis Skin - - - 54 16 M Host T.capitis Hair - - - 55 38 M Agri T.barbae Hair - - - 56 20 M Host T.pedis Skin - + T.tonsurans S S S S S 57 25 M Cfc T.unguim Nail + + T.rubrum S S S S S 58 19 M Host T.corporis Skin - - - 59 23 F Ac T.corporis Skin - - - 60 55 F Agri T.corporis Skin + + T.tonsurans S S S S S 61 36 M Agri T.corporis Skin - + T.rubrum S S S S S 62 13 M Host T.corporis Skin - + T.mentagrophytes P P S S S S S 63 17 M Host T.corporis Skin - - - 64 37 F Agri T.manuum Skin + + T.rubrum S S S S S 65 37 F Agri T.cruris Skin - + T.rubrum S S S S S 66 45 F Sweat T.manuum Skin - - - 67 45 F Agri T.corporis Skin - - - 68 55 M Agri T.unguim Nail - + T.tonsurans S S S S S 69 30 F Sweat T.capitis Hair + - - 70 19 F Ac T.cruris Skin + + T.mentagrophytes P P S S S S S 71 3 M Cfc T.capitis Hair + + T.tonsurans S S S S S 72 45 F Cfc T.corporis Skin - - - 73 45 F Cfc T.corporis Skin + + T.rubrum S S S S S 74 45 F Cfc T.corporis Skin - - - 75 55 M Agri T.unguim Nail - - - 76 55 M Agri T.unguim Nail - - - 77 55 M Agri T.unguim Nail - + T.rubrum S S S S R 78 33 F Sweat T.manuum Skin + - - 79 33 F Sweat T.corporis Skin + + T.mentagrophytes P P S S S S S
148 80 33 F Sweat T.cruris Skin - - - 81 18 M Ac T.manuum Skin - - - 82 18 M Ac T.pedis Skin - - - 83 18 M Ac T.corporis Skin + + M.canis S S S S S 84 18 M Ac T.cruris Skin - - - 85 40 F Agri T.corporis Skin - - - 86 40 F Agri T.capitis Hair - - - 87 40 F Agri T.manuum Skin - - - 88 40 F Agri T.pedis Skin - - - 89 40 F Agri T.cruris Skin - - - 90 57 M Agri T.corporis Skin - - - 91 57 M Agri T.cruris Skin - - - 92 57 M Agri T.capitis Hair - - - 93 57 M Agri T.barbae Hair + - - 94 23 F Cfc T.corporis Skin - - - 95 18 M Cfc T.corporis Skin - - - 96 24 M Ac T.pedis Skin - - - 97 40 F Cfc T.corporis Skin - - - 98 25 F Ac T.manuum Skin - - - 99 6 F Cfc T.capitis Hair - - - 100 17 M Cfc T.corporis Skin - - -
149 KEY TO MASTER CHART
M - Male
F - Female
KOH - Potassium Hydroxide
DTM - Dermatophyte Test Medium
MIC - Minimum Inhibitory Concentration
MIC – Keto - MIC – Ketoconazole
MIC – Itra - MIC – Itraconazole
MIC – Griseo - MIC - Griseofulvin
MIC- Fluco - MIC – Fluconazole
MIC- Terbi - MIC – Terbinafine
Agri - Agriculture
Cfc - Close family contacts
Ac - Animal Contacts
Host - Hostelers
Sweat - Excessive sweating
T.corporis - Tinea corporis
T.pedis - Tinea pedis
T.unguim - Tinea unguim
T.versicolor - Tinea versicolor
T.capitis - Tinea capitis
T.barbae - Tinea barbae
150 T.cruris - Tinea cruris
T.mannum - Tinea mannum
T.fasciea - Tinea fasciea
NG - No Growth
T.rubrum - Trichophyton rubrum
T.mentagrophytes - Trichophyton mentagrophytes
T.tonsurans - Trichophyton tonsurans
M.canis - Microsporum canis
M.gypseum - Microsporum gypseum
E.flocossum - Epidermophyton flocossum
P - Positive
S - Sensitive
R - Resistant
151 152