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TINEA CAPITIS AMONG SCHOOL CHILDREN
IN OKELELE COMMUNITY, KWARA STATE
BY
DR. EKUNDAYO, HALIMAT AYODELE
M.B.; B.S. (ILORIN)
A DISSERTATION SUBMITTED TO THE NATIONAL
POSTGRADUATE MEDICAL COLLEGE OF NIGERIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF FELLOWSHIP OF
THE COLLEGE IN PATHOLOGY
NOVEMBER, 2013.
2
DECLARATION
I, DR. EKUNDAYO, HALIMAT AYODELE hereby declare that this
dissertation is original unless otherwise acknowledged. This dissertation
has not been presented in part or whole to any college for fellowship nor
has it been submitted elsewhere for publication.
Signature ------------------------------------------ Date--------------------
DR. EKUNDAYO, HALIMAT AYODELE
3
CERTIFICATION
We hereby certify that Dr. Ekundayo, Halimat Ayodele of the Department
of Medical Microbiology and Parasitology, University of Ilorin Teaching
Hospital, Ilorin, carried out the research work under our supervision. We
also supervised the writing of the dissertation.
Signature--------------------------------------- Date------------------------
Prof. Nwabuisi, Charles (M.Sc., FMC Path)
Professor of Microbiology and Head of Department
Department of Microbiology and Parasitology
University of Ilorin Teaching Hospital
Ilorin.
Signature----------------------------------------- Date------------------------
Dr. Fadeyi, Abayomi (B.Sc., MB; BS, FMC Path)
Consultant and Infectious Disease Physician
Department of Microbiology and Parasitology
University of Ilorin Teaching Hospital
Ilorin.
4
DEDICATION
This work is dedicated to God, for HIS mercies and blessings and to my
late husband, Ekundayo Mosuidi Mudashiru, for his love for education.
May his soul rest in perfect peace. Adieu my love.
5
ACKNOWLEDGEMENTS
I am sincerely grateful to my teacher and supervisor, Professor Nwabuisi,
Charles for his mentorship throughout my residency training. Your support
and advice have been invaluable. To my second supervisor, Dr. Fadeyi,
Abayomi I thank you for your unflinching support and for lending a
shoulder to lean on at my time of need. Many thanks to Dr. Akanbi, Ajibola
also a consultant in the Department of Microbiology, for his advice and
encouragement.
I appreciate the assistance of the following members of staff in the
department- Mr. Abiodun Alarape, Mr. Abdulraheeem Jimoh, Mr. Raheem
Razak, and Mr. Mauruf Ilufoye. Special thanks to my field assistants Mr.
Abu-saeed Buhari, who was very supportive throughout the period of this
study, Mr. Lukman Oyekunle as well as Mr. Stephen Olalere.
I am grateful to Dr. Nyamngee, Amanse a lecturer in the Department of
Microbiology, University of Ilorin, Dr. Ameh, Oluwatoyin a consultant
nephrologist in the Department of Internal Medicine, University of Ilorin
Teaching Hospital(UITH) and Dr. Uthman, Mubashir, a consultant public
health physician in the Department of Public Health, UITH for their
immense assistance in statistical analysis. I appreciate Dr. Saka,
Mohammed also a consultant in the Department of Public Health and his
6
wonderful wife, Dr. Saka, Oluwatoyin a consultant paediatrician in the
Department of Paediatrics both of UITH, for their useful suggestions and
advice on this work. To my colleagues in the department Dr. Tinuke
Suleiman, Dr. Adejoke Ikugbaigbe, Dr. Ugochukwu Odoemelam and Dr.
Femi Lawani, I appreciate you all. Many thanks to all members of staff and
pupils of the primary schools in Okelele community for making this work a
reality, by their co-operation.
I appreciate the concern of my wonderful mum, Mrs. Aminat Adebayo.
You are a mother indeed. To my children the ‘Ayos’ (Temidayo, ’Sayo,
Ayobami, Ayomide and Ayodeji) I thank you for giving me the joy that your
names depict and for making me proud by your excellent academic
performances while I struggle with my career. To my late husband, your
love will forever fill my heart. No one can be like you. Above all I praise
God for seeing me through my sojourn in the residency training.
7
TABLE OF CONTENTS
CONTENT PAGE
DECLARATION………………………………………………………… ii
CERTIFICATION……………………………………………………….. iii
DEDICATION…………………………………………………………… iv
ACKNOWLEDGEMENTS…………………………………………..… v
TABLE OF CONTENTS………………………………………………. vii
LIST OF ABBREVIATIONS…………………………………………… x
LIST OF TABLES……………………………………………………… xi
LIST OF FIGURES…………………………………………………….. xii
LIST OF PLATES……………………………………………………… xiii
SUMMARY……………………………………………………………… xiv
CHAPTER ONE……………………………………………………….. 1
INTRODUCTION, JUSTIFICATION & OBJECTIVES……………… 1
CHAPTER TWO………………………………………………………. 6
8
LITERATURE REVIEW………………………………………………… 6
CHAPTER THREE…………………………………………………….. 50
MATERIALS AND METHODS……………………………………….. 50
CHAPTER FOUR……………………………………………………… 72
RESULTS………………………………………………………………. 72
CHAPTER FIVE………………………………………………………. 80
DISCUSSION…………………………………………………………. 80
CONCLUSION………………………………………………………… 87
RECOMMENDATION………………………………………………… 87
LIMITATION …………………………………………………………… 88
REFERENCES……………………………………………………….. 89
APPENDIX I (STUDY PROFORMA)……………………………….. 104
APPENDIX II (INFORMATION SHEET)…………………………… 109
APPENDIX III (INFORMED CONSENT)…………………………… 113
APPENDIX IV (TABLE OF ANTHROPOMETRIC INDICATORS).. 114
9
APPENDIX V (OYEDEJI’S SOCIO-ECONOMIC CLASSIFICATION
SCHEME)....................................................................................... 115
APPENDIX VI (MATERIALS AND REAGENTS)………………...... 116
APPENDIX VII (CLSI CRITERIA)…………………………………… 117
10
LIST OF ABBREVIATIONS
ALT --ALANINE TRANSAMINASES
AST --ASPARTATE TRANSAMINASES
ATCC --AMERICAN TYPE CULTURE COLLECTION
BUN --BLOOD UREA NITROGEN
CLSI --CLINICAL AND LABORATORY STANDARDS INSTITUTE
DID --DOUBLE IMMUNODIFFUSION
DMSO--DIMETHYLSULPHOXIDE
FBC -- FULL BLOOD COUNT
FDA --FOOD AND DRUG ADMINISTRATION
FRN --FEDERAL REPUBLIC OF NIGERIA
KOH --POTASSIUM HYDROXIDE
LA --LATEX AGGLUTINATION
LGA --LOCAL GOVERNMENT AREA
LFT --LIVER FUNTION TESTS
NCCLS-NATIONAL COMMITTEE FOR CLINICAL LABORATORY
STANDARDS
PCR --POLYMERASE CHAIN REACTION
P/E --PHYSICAL EXAMINATION
RFT --RENAL FUNCTION TESTS
UV --ULTRA VIOLET LIGHT
WHO --WORLD HEALTH ORGANIZATION
11
LIST OF TABLES
TABLE PAGE
TABLE 1—Socio-demographic characteristics of the participants.. 75
TABLE 2—Age and sex prevalence of Tinea capitis……………… 76
TABLE 3—Susceptibility pattern of the fungal agents of
Tinea capitis……………………………………………... 78
TABLE 4—Risk factors associated with Tinea capitis…………….. 79
12
LIST OF FIGURES
FIGURE PAGE
FIGURE 1—Aetiological agents of Tinea capitis………….. 77
13
LIST OF PLATES
PLATES PAGE
PLATE 1—Types of clinically suggestive scalp ringworm lesions… 118
PLATE 2—Micrograph of KOH preparation showing chains of
arthroconidia, ectothrix and endothrix hair invasion…... 119
PLATE 3—Isolates on Mycosel agar plates………………………… 120
PLATE 4—Lactophenol cotton blue preparation of fungal isolates. 121
PLATE 5—Hair perforation test by T. mentagrophytes……………. 122
14
SUMMARY
Tinea capitis impacts negatively on the health of children, consequently
affecting their education. Its prevalence is unknown in many African
communities. Tinea capitis is faced with therapeutic challenges as
resistance to all classes of antifungal agents continues to emerge. The
objective of this study was to determine the prevalence of tinea capitis
among primary school children in Okelele community in Kwara State,
highlighting the aetiological agents of the disease and their susceptibility
pattern, as well as the predisposing factors to tinea capitis among the
school children.
Three hundred and one pupils aged 5-14years with scalp lesions clinically
suggestive of tinea capitis attending the seven primary schools in Okelele
community were recruited, using a multistage sampling technique. Their
socio-demographic characteristics along with relevant clinical information
were obtained through a proforma designed for the study. Hair and/or
scalp scrapings and/or swabs were collected as specimens. The
specimens were examined microscopically and then cultured on Mycosel
agar plates. The susceptibility pattern of the isolates was determined by
the disc diffusion method using griseofulvin (25µg), terbinafine (30µg),
15
itraconazole (8µg), fluconazole (25µg), ketoconazole (10µg), miconazole
(10µg) and clotrimazole (10µg) discs.
Two hundred and twenty eight of the children, with a mean age of 9.8
years±2.4, had mycologically proven tinea capitis, giving a prevalence of
75.7%. Tinea capitis was more common among males (76.8%) than in
females (69.0%). The identified aetiological agents of tinea capitis in the
study population were Trichophyton rubrum 156(68.4%), Microsporum
ferrugineum 51(22.4%), Trichophyton mentagrophytes 17(7.4%) and
Trichophyton verrucosum 4(1.8%). Following susceptibility testing T.
rubrum showed 78.8% sensitivity to clotrimazole and miconazole. M.
ferrugineum and T. mentagrophytes were 100.0% sensitive to
clotrimazole, miconazole and terbinafine. In addition, T. mentagrophytes
showed 100.0% intermediate susceptibility to fluconazole. T. verrucosum
was 100.0% resistant to all the antifungal drugs tested. Large family size
was found to be significantly associated with the occurrence of tinea
capitis.
The prevalence of tinea capitis among the school pupils with clinically
suggestive disease is high. There is the need for health education, in the
schools and the community on the modes of transmission, with emphasis
on the identified associated factors, and preventive measures. Terbinafine
16
is recommended for empiric treatment of tinea capitis while clotrimazole or
miconazole is suggested for use as adjunct therapy among the school
children in Okelele community. Further susceptibility studies are desired
on T. rubrum and T. verrucosum to identify the effective drugs for the
treatment of tinea capitis due to these agents.
CHAPTER ONE
INTRODUCTION
Tinea capitis is a fungal infection of the scalp hair follicles and surrounding
skin caused by dermatophytes. It is also known as scalp ringworm or
dermatophytosis and is predominantly seen among prepubescent
children.1It is the commonest cause of acquired hair loss in children.2
Tinea capitis is highly contagious and frequently spreads among family
members and classmates.3 Tinea capitis should be considered in the
diagnosis of any child over the age of three months with a scaly scalp until
ruled out by negative mycology.4
Tinea capitis is widely distributed throughout the world, but more prevalent
in hot humid tropical climates than in cold dry regions.3 It may occur
sporadically or in epidemics. It is endemic in many developing countries
17
making it a significant infectious dermatological disease.2 The prevalence
of tinea capitis remains low in developed countries.5 In developing
countries however, tinea capitis is an important public health problem. In
Africa, it has been reported to affect 10-30% of school age children.6 In
Ivory Coast (West Africa), the prevalence of tinea capitis in a cross-
sectional study performed among primary school children was 11.3%.7
The disease is one of the most prevalent dermato-mycoses in Nigeria and
represents a major public health problem.8 Reports from field surveys and
dermatologic clinics indicate that tinea capitis is endemic in Nigeria.2, 9
Tinea capitis can occur at any age but it is more common in children,
particularly between the ages of 4 and 14 years.10 Males are more
commonly affected with a frequency of about two to five times higher than
in females.11
The causative agents of tinea capitis vary from one geographical region to
another, but are largely due to Trichophyton and Microsporum species.
The predominant dermatophyte causing tinea capitis in a given
geographical region can also change over time, 1 hence there is a need
for constant surveillance to determine the epidemiological trends of the
disease.
18
Tinea capitis is of public health importance, yet it is not a notifiable
disease and as a result, the actual prevalence are unknown in many
endemic areas.8,10 In Nigeria, very few studies have focused on the
prevalence and aetiological agents of tinea capitis in schools in different
parts of the country.8,12 Adefemi et al13 in a study on the prevalence of
dermatophytoses among primary school children in Oke-Oyi, a rural
community in Ilorin, reported tinea capitis as the commonest
dermatophytosis, where it was found to account for 76.1% of cases. In
another study on the prevalence of tinea capitis among primary school
children, carried out in the North central states of Nigeria, Ayanbimpe et
al11 reported 31.2% prevalence for tinea capitis. However, the
susceptibility patterns of the isolates obtained from both studies were
lacking. Antifungal susceptibility testing continues to be an area of intense
interest. With the steady introduction of new antifungal drugs, both pre-
existing and new classes, the interest in and the need for clinically
relevant susceptibility testing has increased.14
In Ilorin, the capital city of Kwara State, there is paucity of reports on tinea
capitis. Furthermore, the variations and changing pattern of the
aetiological agents of tinea capitis among other factors necessitate this
study. The aim of this work therefore, is to study tinea capitis among
school children in Okelele community, Ilorin, Kwara State.
19
1.1 JUSTIFICATION FOR THE STUDY
Despite advances in preventing and treating tinea capitis, it remains a
worldwide public health problem, especially among primary school
children in economically underdeveloped countries. In Africa, tinea capitis
has been reported to affect 10-30% of school age children.6 In West Africa
sub region, a prevalence of 11.3% was reported for tinea capitis in a
cross-sectional study performed in Ivory Coast.7 In Nigeria, tinea capitis is
a major public health problem being endemic and also one of the most
prevalent dermato-mycoses.8 Acquired hair loss or alopecia is a common
complication which is usually temporary, but may become permanent.
This can lead to disfiguring and social stigma.
Tinea capitis can be unsightly. Scratching due to irritation of the affected
scalp could affect child’s education by distracting attention during lessons
and imposing psychological trauma.15 Moreover, like all contagious
diseases, tinea capitis can spread from one person to another. On
account of these, victims may experience varying degrees of mental
stress and anxiety.15
Being an endemic disease also, tinea capitis can assume an epidemic
proportion if environmental and human factors that favour transmission
are enhanced. Surveillance is therefore important in the control and
20
prevention.15 With the introduction of new drugs, the interest in and the
need for clinically relevant antifungal susceptibility testing has increased.14
This work, which intends to study tinea capitis among school children in
Okelele community, will serve not only as a control measure but also
establish the antifungals for use in the locality.
1.2 OBJECTIVE OF THE STUDY
To study tinea capitis among school children in Okelele community in
Kwara state.
1.2 SPECIFIC OBJECTIVES
1. To investigate the prevalence of tinea capitis among school
children with clinical evidence of the infection in Okelele community
in Kwara state.
2. To determine the fungal agents causing tinea capitis among school
children in Okelele community in Kwara state.
3. To document the antifungal susceptibility pattern of the causative
agents.
4. To identify the factors predisposing school children in Okelele
community in Kwara state to tinea capitis .
21
CHAPTER TWO
LITERATURE REVIEW
2.1 INTRODUCTION TO TINEA CAPITIS
Dermatophytosis, also known as ringworm or tinea, which affects the
scalp, glabrous skin and nails is caused by a closely related group of fungi
known as dermatophytes.16 For many people, the name ”ringworm” still
evokes the spectre of social stigma, with visions of dirt, slums and shaven
scalps that were evidence of the vast epidemics in European cities
hundred years ago.15 Dermatophytosis can be unsightly or disfiguring,
impede mobility and performance, cause disability or possibly pain, and
when contagious, spread from one part of the body to another or from
person to person. Consequently, victims may experience varying degrees
of mental stress and anxiety.15
22
Dermatophytoses constitute an important public health problem among
children worldwide including Nigeria. These diseases remain endemic in
Nigeria, largely because of lack of information on its prevalence as well as
Government approved protocols for control.6 The factors affecting the
distribution and transmission of dermatophytoses in different parts of the
world are largely dependent on the source of the infection, 17 which can be
animal, soil, or human. The epidemiology of the fungal species differs
according to age, geographical distribution, other demographic factors,
environmental conditions and sampling methods of the patients
investigated. In addition, species tend to change over time within a
locality.18 Infections of the scalp, hair and general body surface are most
frequent during childhood. Although fungal disease of the hands, feet or
nails is certainly seen before puberty, it is more common after puberty.19
Dermatophytes, the aetiological agents of dermatophytoses are a unique
group of fungi that infect keratinous tissue of lower animals and humans.
They are characterized by their ability to invade the superficial layers of
the epidermis, particularly the stratum corneum and high keratin
containing appendages: the hair, and nails of the living host.20
The dermatophytes are classified into three anamorphic genera
epidermophyton, microsporum and trichophyton. On the basis of primary
23
habitat association, they may be grouped as anthropophilic
dermatophytes, which primarily infects humans and are transmitted from
human to human, for example Trichophyton rubrum;zoophilic
dermatophytes which primarily infects animals and can be transmitted to
humans, for example Trichophyton verrucosum and geophilic organisms,
which live in the soil as saprophytes and can infect both lower animals and
humans, for example Microsporum gypseum.21 Familiarity with the ecology
of dermatophytes is helpful both in tracing the source of infection and in
preventing re-infection.22 Anthropophilic species cause the greatest
number of human infections, produce mild and chronic infections and may
be difficult to eradicate. On the other hand, zoophilic and geophilic species
produce more acute inflammatory infections that tend to resolve quickly
because they are less adapted to the human host.21
Infection takes place by deposition of viable arthrospores or hyphae on the
skin surface of susceptible persons. The source of infection is usually an
active lesion on a human or on animal, formites and soil may also serve
as other sources of infection.22 The usual incubation period of
dermatophytoses is 1-3 weeks.
Dermatophyte infections are classified into different types depending on
the site infected on the human body. These include tinea capitis (ringworm
24
infection of the scalp), tinea corporis (ringworm infection of the skin), tinea
pedis, otherwise known as athlete’s foot (ringworm infection of the foot),
tinea unguim otherwise known as onychomycosis (ringworm infection of
the nails of the feet or hands), tinea cruris otherwise known as Jock itch
(ringworm infection of the groin), tinea manus (ringworm infection of the
hands or fingers) and tinea barbae (ringworm infection of the bearded
region). Among the diseases caused by dermatophytes, tinea capitis is the
commonest fungal infection.23
Tinea capitis, also known as scalp ringworm or dermatophytosis is a
cutaneous fungal infection of the scalp. The disease is primarily caused by
dermatophytes in the Trichophyton and Microsporum genera that invade
the hair shaft. Scalp ringworm can occur in isolation or concurrently with
tinea corporis and / or tinea unguim infections.24
Tinea capitis is usually classified by the pattern of hair invasion into three
types. Ectothrix invasion in which the fungal branches or hyphae and
spores or arthroconidia cover the outside of the hair shaft. The infected
hairs break off a few millimeters above the scalp forming circular areas of
partial alopecia. The cuticle of the hair is destroyed and infected hairs
fluoresce bright greenish yellow or violet colour under Wood’s ultraviolet
(UV) light.25 Fine scaling is characteristic and inflammation varies from
25
minimal with anthropophilic species such as Microsporum audouinii to
moderate or severe with zoophilic agents like Microsporum canis or
geophilic dermatophytes for example Microsporum gypseum.
Microsporum audouinii produces a grey patch with hair tinged a dull grey
colour.
In endothrix pattern, fungal arthroconidia invade the hair shaft. Infected
hairs become very fragile, breaking off at the level of the follicular orifice
and producing a black dot in patients with black hair. Hair loss can be
minimal with little inflammation. The cuticle of the hair remains intact and
infected hairs do not fluoresce under Wood’s UV light. All endothrix
producing agents are anthropophilic for example Trichophyton tonsurans
and Trichophyton violaceum. The third pattern called favus or tinea favosa
is usually caused by Trichophyton schoenleinii. Mycelia invade the hair
medulla but then regress and leave tunnels containing air within the hair
shaft producing a honey comb appearance. Favus is characterized by the
formation of an inflammatory crust or scutulum that is composed of
neutrophils and serous exudates around individual hair shaft. With time,
this amalgamates over the surface of the scalp so that the hair appears to
be matted together with a thick crust that is said to have a mousy odour.
26
In some ectothrix infections, a pustular form of dermatophytosis develops
called kerion. This is commonly associated with zoophilic species. In most
kerions, the pustules are not a sign of secondary bacterial infections,
although this may occur under adherent crust. Kerion is less common in
endothrix infections. More inflammatory presentations including kerion
tend to be seen in children who mount a vigorous cellular immune
response to fungal antigens such as trichophytin.26 Besides enhancing the
inflammation of tinea capitis, a vigorous immune response to the fungus
can result in manifestations distant from the site of infection. This is called
dermatophytid or id reactions, and results from interaction between the
host and fungal antigens.27 Dermatophytid reactions can develop with any
type of tinea, but in children, they are especially likely to be associated
with a kerion or other forms of inflammatory tinea capitis. The trunk and
the proximal extremities are the most frequently involved in id-reactions,
but may also be seen on the face especially the forehead and behind the
ears. The id- reactions may present clinically as symmetric dermatitis or a
papular rash resembling pityriasis rosea. In rare cases, systemic signs
such as fever or leucocytosis are associated. A dermatophytid reaction is
most likely to be encountered as therapy is initiated and should not be
interpreted as a drug reaction.
2.2 AETIOLOGY OF TINEA CAPITIS
27
The genera Microsporum and Trichophyton belong to the phylum
Ascomycota (Ascomycetes) which are perfect fungi. The ascomycetes
reproduce sexually by the production of spores (ascospores) which are
produced in a sac like structure called ascus. Asexual reproduction is via
conidia production known as arthroconidia (arthrospores). Both genera
infect superficial keratinized tissue. Trichophyton infects the skin, hair and
nails while Microsporum infects the skin and hair only. They are classified
morphologically as moulds.21During growth they produce hyphae and as
the hyphae grow, they intertwine to form a loose network of mycelium.
These dermatophytes grow best at an incubation temperature of between
25-300C (room temperature). A few of the dermatophytes causing tinea
capitis grow at between 35-370C for example Trichophyton verrucossum
grows best at 370C.28Thesedifferences in incubation temperature is used
in their identification.21
2.3 EPIDEMIOLOGY OF TINEA CAPITIS
Tinea capitis is widely distributed throughout the world, but more prevalent
in hot humid tropical climates than in cold dry regions.3Its epidemiology
varies within different geographical areas throughout the world. This
variation reflects people’s habits, standard of hygiene, climatic conditions
and levels of education.15It may occur sporadically or in epidemics. It is
28
endemic in many developing countries making it a significant infectious
dermatological disease.2
The prevalence of tinea capitis remains low in developed countries and
there is usually a history of indirect or direct contact with immigrant
population originating from endemic areas for tinea capitis.5 Tinea capitis
is an important public health problem in Africa, where it has been reported
to affect 10-30% of school age children.6 A cross-sectional study
performed among primary school children in Ivory Coast (West Africa),
reported a prevalence of 11.3% for tinea capitis .7 The disease is one of
the most prevalent dermato-mycoses in Nigeria and represents a major
public health problem.8 Reports from field surveys and dermatological
clinics indicate that tinea capitis is endemic in Nigeria.2,9
Tinea capitis can occur at any age but it is more common in children,
particularly between the ages of 4 and 14 years10 and less frequently seen
in adults.1 This is attributed to the low level of fungistatic, short and
medium chain fatty acids in the scalp of these children,3 frequent contact
at school and at play with infected persons and exposure to domestic
animals and pets.8,10 Males are more commonly affected with a
frequency of about two to five times higher than in females.11 This may be
29
due to shortness of the hair in males which facilitates easy reach of the
fungal spores to the scalp.
The fungi that cause tinea capitis infections thrive in warm, moist areas.
Ringworm is most common in children who attend day care and
kindergarten. Inanimate objects like pencils, door handles, chairs and
tables can transmit these fungi. The risk of scalp ringworm is increased
when there are minor scalp injuries and poor hygiene. In overcrowded
conditions, excessive sweating and sharing of personal care items such
as combs in a large family all increase the risk for tinea capitis infection.
Immunosuppression predisposes to chronic and extensive tinea capitis
infection, 22 and in Nigeria, malnutrition is a predisposing factor. 29
2.4 PATHOGENESIS OF TINEA CAPITIS
The dermatophytes secrete extracellular enzymes that aid in the
colonization of keratinous tissue. These extracellular enzymes that are
associated with virulence include keratinase, elastase and lipase.30
Transfer of infecting organisms from soil, animals or humans is
accomplished by means of arthrospores which are vegetative cells with
thickened cell walls formed by dermatophyte hyphae in-vitro and in-vivo.
These structures are shed by the primary host along with shed skin scales
30
or hair. It has been shown that dermatophyte arthrospores can survive for
considerable periods outside the host, in some cases for more than 15
months.28 There is little understanding of the process of transfer of the
arthrospores, but invasion of the skin appears to follow adherence of
fungal cells to keratinocytes in-vitro, a process that is maximal after about
2 or 3 hours of contact. Keratinocytes from different sites do not appear to
differ in their binding capacity for arthrospores. Subsequent germination
will then lead to invasion.28
Tinea capitis infection begins with hyphal invasion of the skin of the scalp,
with subsequent spreading downwards to the keratinized wall of the hair
follicle.21 Infection of the hair takes place just above the hair root. The
hyphae grow downward on the non- living portion of the hair at the same
rate as the hair grows upward. The infection produces dull gray, circular
patches of alopecia, scaling and itching. 21 As the hair grows out of the
follicle, the hyphae of Microsporum species produce chain of spores that
form a sheath around the hair shaft (ecthothrix). In contrast, Trichophyton
species produce spores within the hair shaft (endothrix).
2.5 CLINICAL FEATURES OF TINEA CAPITIS
Tinea capitis may present as either a non-inflammatory or inflammatory
condition.31 Symptoms may be mild with non-inflammatory presentation
31
such as dandruff-like scaling of the scalp, slight pruritus and minimal hair
loss which will return as fungal infection clears. With the inflammatory form
of the disease (kerions), the infected area may be boggy, oozing, and
have pustular masses. Kerions may sometimes be incorrectly diagnosed
as bacterial abscesses leading to inappropriate and ineffective treatment.
Kerion and other inflammatory forms of tinea capitis may result in
permanent scarring.4 The clinical trial of scalp scaling, alopecia and
lymphadenopathy (occipital and/ or posterior cervical) is typically
associated with tinea capitis. A differential diagnosis of tinea capitis should
include other common scalp disorders that may present with scaling and
head and neck lymphadenopathy, such as atopic dermatitis, psoriasis,
seborrhoeic dermatitis.
It is important to note that not all individuals with tinea capitis will manifest
symptoms. This asymptomatic carrier state is estimated to be 5% in the
general population of United States. However, in urban school age
population, the percentage increases to nearly 15%.32
2.6 DIAGNOSIS OF TINEA CAPITIS
This can either be clinical or laboratory. The clinical criteria for establishing
a diagnosis are the triad of scaling, itching and alopecia. This clinical
diagnosis is unreliable, as the infection may mimick other common scalp
32
lesions. Microscopic examination and / or fungal culture should be used
to confirm the clinical diagnosis of tinea capitis because of the extended
nature of most treatment regimen.33
The laboratory diagnosis of tinea capitis infection involves taking
appropriate specimens, specimen collection and transportation, Wood’s
lamp examination, microscopy, culture, the identification of the
dermatophyte isolates, other available diagnostic methods aside culture,
and antifungal susceptibility testing.
Specimens
These are hair and scalp scrapings.
Methods of specimen collection and transportation
Hairs are collected from areas of scaling or alopecia by the following
methods:-
Plucking them with forceps.34 Vigorous plucking may simply break the
hair, leaving the infected proximal portion behind in the scalp, and
resulting in a negative culture. This is because fungal hyphae initially
proliferate in the hair’s proximal portion; the distal hair shaft is less likely to
yield a positive culture.
33
Short hairs that have broken off near the scalp are collected from the
periphery of the infected area by gently rubbing the scalp with a dull
surface such as a moistened gauze pad or the edge of a tongue
depressor.19
Scalp scrapings are obtained from the erythematous advancing edge of
the lesion, 25 by the following methods:-
The use of the edge of a microscope slide or the blunt edge of a scalpel
blade.34 This method may be traumatic.
Scales and hairs of tinea capitis lesions can be obtained with a cotton
swab.35 This is a reliable, inexpensive and less traumatic method.
Swabbing the suspected fungal lesion with a cotton applicator involves
rubbing and rotating a moistened swab over the affected area. Swab
specimens are generally inadequate for the recovery of moulds.25
The use of a cytobrush is also a reliable, inexpensive and less traumatic
means.36
Scrapings and hairs are collected into a dry envelope, petridish or other
suitable containers for transport to the mycology laboratory for microscopy
and inoculation into the appropriate medium.25 Ringworm specimens are
best transported in paper packages (rather than screw cap containers) to
34
reduce humidity and the multiplication of bacteria. Spores of ringworm
fungi resist drying and remain viable for several months when stored in
paper.37
Wood’s lamp examination
Wood’s lamp examination is a screening procedure for patients with scalp
infections.28 It involves the use of filtered ultraviolet (UV) light in the range
of 300-400nm to view infected hairs in tinea capitis infection.37
Microsporum species produce a chain of spores that form a sheath around
the hair shaft (ectothrix). These spores impart a greenish-yellow to silvery
fluorescence when the hairs are examined under Wood’s light (365nm).21
However, Trichophyton species infections do not fluoresce, apart from
favus in which the hairs appear yellowish.
Fluorescent hairs are infected. Apart from its use as a screening
procedure, Wood’s light examination may be helpful as a method of
selecting hairs for microscopy and culture.28 However, Wood’s lamp
inspection of the scalp for fluorescence is not a reliable means of
discovering fungal infection as very few cases of tinea capitis are
presently caused by dermatophytes that fluoresce.19
Microscopy
35
Direct microscopic examination of clinical specimens can often provide the
first clue to the aetiology of disease in patients with fungal infections. This
is the most rapid method currently available.25 It may provide an
immediate presumptive diagnosis for the physicians.
Methods for direct microscopic examination include potassium hydroxide
(KOH) preparation, Calcofluor white (CW) stain, CW-KOH preparation,
and histology-biopsy specimen.
KOH preparation:- Traditionally, this is the recommended method for the
direct microscopic examination of specimens.38 The principle is based on
the use of 10% or 20% KOH to dissolve the keratin present in skin or hairs
thereby clearing out any background scales or cell membranes to allow
visualization of hyphal elements.25 As a caution, KOH reagent is corrosive
and should be handled with care.
The KOH preparation is made by suspending specimen in a few drops of
10% or 20% KOH on a grease-free microscope slide. This is then covered
with a coverslip. The slide is then placed in a petri-dish or any other
container with a lid, together with a damp piece of filter paper or cotton
wool to prevent the preparation from drying out. This is then left for five-
ten minutes if the specimen is hair or twenty to thirty minutes for crusts
and skin scales. Thereafter, the preparation is examined microscopically
36
using the 10x and 40x objectives with condenser iris diaphragm closed
sufficiently to give good contrast. The clearing process can be hastened
by gently heating the preparation over a spirit lamp or pilot flame of a
Bunsen burner, taking care to prevent drying or splatter of the corrosive
KOH solution. Excessive heat will cause crystallization of the KOH
rendering the material useless.19
Modifications of the KOH preparation
KOH with dark coloured blue or black ink:- Dark coloured blue or black ink
if added to the preparation will enhance contrast between hyphae and the
surrounding material.19 However; the ink is not specific for fungi. It also
stains cells and other components in the skin.
Dimethylsulphoxide (DMSO)–KOH reagent:- This reagent can be
prepared through the addition of 40% DMSO to 19% KOH in distilled
water. The use of this improved KOH reagent obviates the need for
heating andenables specimens to be examined immediately or only after a
few minutes.19, 37
Characteristic features of dermatophytes seen in direct
microscopical examination of clinical specimen
37
SKIN—hyaline septate hyphae are commonly seen, chains of
arthroconidia may be present. These are indicative of dermatophyte
infection.34
HAIR—arthroconidia on periphery of hair shaft producing a sheath are
indicative of ectothrix infection; arthroconidia formed within the hair shaft
are indicative of endothrix infection; long channels within the hair shaft are
indicative of favic infection.34
Limitations of the KOH preparation
Caution should be exercised regarding potential misdiagnoses with KOH
preparations:-
A negative KOH preparation should not be taken as an absolute evidence
of a lack of infection. Positive findings can be overlooked, and not all hairs
or scales in an involved area contain hyphae or spores.38
Epidermal cell outlines, elastic fibres, and artefacts such as intracellular
cholesterol and strands of cotton or vegetable fibres may appear like
ringworm fungal hyphae. Ringworm fungal hyphae can be differentiated
from these structures by their branching uniform width, and cross walls
(septa) which can be seen when using 40x objectives.38
38
Calcofluor White (CW) stain:- This is believed to be superior to KOH
preparation.39 Slides preparation by this method may be observed using
fluorescent or bright field microscopy as used for the KOH preparation.
The fluorescent microscopy is optimal in that fungal cells will fluoresce.34
The principle is based on the use of calcofluor white, an industrial
brightener, which binds non-specifically to chitin and other elements in
fungal cell wall. Calcofluor white fluorescence occurs maximally at a
wavelength of 440nm. Under these conditions fungal elements will
fluoresce blue-white.34The presence of KOH in the solution dissolves
human cellular elements and debris, which allows for easier visualization
of fungal elements.
CW-KOH preparation:-The CW-KOH preparation is made with a drop of
CW reagent and a drop of 10% KOH in the centre of a grease-free
microscope slide. A portion of the clinical specimen is then added to the
CW-KOH solution and a cover slip is applied. This is then allowed to stand
for five minutes to allow particles to dissociate. A gentle pressure on the
cover slip will enhance dissociation. Fungal elements are then examined
using a fluorescent microscope with a 500-520nm barrier filter. Slides are
scanned at10x objective for fluorescent fungal elements.34The presence
and nature of fungal elements is discerned using 40 x objectives.
39
Histology:- Biopsy of an infected area stained with periodic acid-
Schiff(PAS) reveals fungal elements in and about the hair and hair follicle
and also in the stratum corneum, accompanied by a cellular infiltrate
consisting of neutrophils, lymphocytes, and histiocytes, sometimes with
neutrophils penetrating the stratum corneum.19
Culture
Selection of culture media:- A battery of culture media for the recovery
of fungi from clinical specimens need not be elaborate, although the
recovery rate may be somewhat enhanced by using a variety of isolation
media.25 Two general types of culture media are essential to ensure the
primary recovery of all clinically significant fungi from clinical specimens:-
Non-selective media:- These will permit the growth of virtually all
clinically relevant fungal species. For dermatophytes from cutaneous
samples, Sabouraud’s Dextrose Agar (SDA) is sufficient as a primary
isolation medium.25 Sabouraud’s Agar is a standard mycological medium
that allows for a reliable examination of colonial morphology of
dermatophytes causing tinea capitis. It consists of glucose (40g), peptone
(10g) and agar (20g) dissolved in 1000ml of water.19 Potato Dextrose Agar
40
(PDA) containing potato flakes and dextrose agar is another non-selective
medium that can be used for the isolation of dermatophytes. Sabouraud’s
Dextrose Agar (2%) is most useful as a medium for the subculture of fungi
recovered from an enriched medium such as Sabouraud’s heart infusion
(SABHI) agar to enhance typical sporulation and provide the more
characteristic colonial morphology.25
Selective media:- SDA can be made selective by the addition of
antibiotics to the medium. The use of either MycoselR or MycobioticR agar,
which essentially is SDA with cycloheximide and chloramphenicol added,
is considered for the primary recovery of dermatophytes. Cycloheximide
(0.5mg/ml) and chloramphenicol (16µg/ml) are added to the medium to
inhibit the growth of saprophytic fungi and contaminating bacteria
respectively.34 Better results have been achieved using a combination of
5µg/ml of gentamicin and 16µg/ml of chloramphenicol as antibacterial
agents. Ciprofloxacin at a concentration of 5µg/ml may be used.
Dermatophyte Test Medium (DTM):- DTM is another selective medium. It
is extremely useful because of its selectivity and ability to change to a
deep red colour with the growth of dermatophytes. It is a phytone dextrose
agar to which is added chlortetracycline, cycloheximide and gentamicin
along with buffered phenol red.40 Metabolites released by the growth of a
41
dermatophyte turn DTM from natural tan of agar to a deep red colour by
causing alkalinization of the medium and a change in the colour of the
phenol red.19 It is important to note that this colour change should be
observed within about fourteen days and that the medium should be
incubated at room temperature without being tightly capped, to allow for
free gas exchange. Colonies normally begin to grow out in approximately
one week at room temperature, but cultures should be incubated for about
three weeks to be certain no growth is occurring. If cultures are incubated
over a period longer than three weeks other organisms such as Candida
species may eventually produce a red colour. In addition colour change
alone should not be interpreted as indicative of dermatophyte growth.
Some examination of colonial morphology is essential.19
Agar plates or screw-capped agar tubes are satisfactory for the recovery
of fungi, but plates are preferred because they provide better aeration of
cultures, a large surface area for better isolation and greater ease of
handling by technologists making microscopic preparations for
examination.34 However, agar plates are more prone to contamination and
tend to dehydrate during the extended incubation period required for
fungal recovery. Dehydration can be avoided by placing the plates in a
sealed, moisturized polyester bag, sealing the edge of the petri-dish with
oxygen permeable tape or placing an open pan of water on the bottom of
42
shelf, if an incubator is used only for fungal cultures. The water provides
the moisture needed for optimal recovery.25
For low volume laboratories, staffed with personnel who may not be as
familiar with the handling of fungi, or where incubator and storage space
may be at a premium, the use of tubes is recommended. Larger culture
tubes (150 x 25mm) with tightly fitted screw cap lids are recommended.25
Compared with agar plates, screw-capped culture tubes are more easily
stored, more easily handled, and have a lower dehydration rate and are
less hazardous.34 The disadvantages of agar tubes include relatively poor
isolation of colonies, a reduced surface area for culturing, and a tendency
to promote anaerobiosis. It is important to note that after inoculation, tubes
should be placed in a horizontal position for at least one to two hours to
allow the specimen to absorb on to the agar surface and avoid settling at
the bottom of the tube. Cotton plugged agar tubes are unsatisfactory for
fungal cultures.30
Inoculation and incubation:- A pair of culture media is inoculated per
specimen. It is currently recommended that all fungal cultures be
incubated at a controlled temperature of 300C.25 Cultures should be
examined at least three times weekly during incubation.34 All fungal
cultures should be incubated for a minimum of thirty (30) days before
43
discarding as negative.25 Isolates on culture should be examined both
microscopically and macroscopically.
Identification of dermatophyte isolates
Dermatophytes are identified on the basis of their growth rate, colonial
morphological features, microscopic morphological features, hair
perforation test and biochemical tests.
Growth rate:-The growth rate may be of limited value in identification,
because the growth rate of certain fungi is variable, depending on the
amount of viable organisms present in a clinical sample. The
determination of growth rate can be most helpful when examining a mould
culture, but it must be used in combination with other features before a
definitive diagnosis is made.34
Colonial morphology:-The colonial morphological features may be of
limited value in identifying the moulds because of natural variation among
isolates and colonies grown on different culture media. Colonial
morphology is an unreliable criterion, 34 and should be used only to
supplement the microscopic morphologic features. Incubation conditions
and culture media must be considered.
44
Microscopy:-The microscopic morphological features of the moulds are
stable and exhibit minimal variation.34It is the most definitive means for
identification. The definitive identification is based on the characteristic
shape, method of reproduction and arrangement of the spores. However,
the size of the hyphae also provides useful information. Small hyphae,
approximately 2 µm in diameter, may suggest the presence of a
dermatophyte.
The tease mount, the transparent tape preparation and the microslide
culture technique are the three commonly used methods for the
microscopic examination of filamentous moulds.25The procedure most
commonly used by most laboratories is the transparent tape preparation.
This is because it can be prepared easily and quickly and often is
sufficient to make the identification for most of the fungi. However, the
tease mount is preferred by some laboratories, and a microslide culture
may be used when greater detail of the morphologic features is required
for identification.25
Procedure for tease mount:-
A pair of dissecting needles or pointed applicator sticks is used to dig out
a small portion of the colony to be examined, including portions of the
subsurface agar. The colony fragment is then placed on a drop of
45
lactophenol cotton blue on a glass microscope slide. The dissecting
needle is used to tease the colony apart and a cover slip is applied. A
gentle pressure is applied on the surface of the cover slip, with eraser end
of a pencil, to disperse the mount. This is then examined microscopically
under 10x objective and then 40x objective or 100x objectives if
suspicious fungal structures are seen.34
It is worthy of note that teasing the colony often disrupts the delicate
fruiting structures of the filamentous moulds, making it difficult in some
instances to observe the characteristic spore arrangements necessary for
a definitive identification. In such cases, a transparency tape mount or a
microslide culture may be required.34
Procedure for transparency tape preparation:-
The sticky side of unfrosted, clear cellophane tape is pressed firmly to the
surface of the colony to pick up a portion of the aerial mycelium. One end
of the tape is then stuck to the surface of the slide adjacent to the drop of
lactophenol cotton blue placed on a glass microscope slide. The tape is
stretched over the stain, by gently lowering it so that the mycelium
becomes permeated with the stain. The tape is then pulled taut and the
opposite end of it is stuck to the glass slide, avoiding as much as possible
46
the trapping of air bubbles. The preparation is then observed
microscopically under 10x and 40x objectives.34
It is important to note that the transparency tape preparation should
always be performed under a safety hood and for maximum safety gloves
should be worn. The transparency tape method is often helpful because
the spore arrangements of the more delicate filamentous moulds are
better preserved. It is inexpensive, rapid, and simple to perform and with a
few exceptions, allows one to make an accurate identification.34
Hair perforation test:-The hair perforation test, also known as in vitro hair
perforation test, is a laboratory test used to distinguish isolates of
dermatophytes particularly Trichophyton mentagrophytes and
Trichophyton rubrum.41 The test is performed by placing the
dermatophyte isolate in petridish containing sterile water, yeast extract
and hair. Human hair sample that has not been bleached, permed or
exposed to spray, regardless of the colour, age or sex of the donor can be
used for the test.42
Biochemical test::-
Key biochemical tests may be needed to differentiate closely related
genera or species within a given group.25The biochemical test available for
47
differentiating the species of dermatophytes is the urease test. The test
organism is cultured in a medium which contains urea and the indicator
phenol red. When the strain is urease producing, the enzyme will break
down urea by hydrolyzing it to give ammonia and carbon dioxide (CO2).
With the release of ammonia, the medium becomes alkaline as shown by
a change in the colour of the medium from tan or mauve to pink-red.34
The colonial morphology, growth rate and microscopic identification
of common Microsporum and Trichophyton species encounterd in
clinical laboratory25:-
The more commonly encountered agents of tinea capitis in clinical
laboratories include Microsporum audouinii, Microsporum canis,
Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton
rubrum, Trichophyton tonsurans, Trichophyton schoenleinii, and
Trichophyton violaceum.25
The genus Microsporum is characterized by the production of many
macroconidia, and a few or no microconidia. The macroconidia are
multicelled, thick-walled, and have a thick, echinulate, or verrucose cell
wall. The species identification is based on differences in morphology of
the macroconidia. The microconidia are small, hyaline, and tear drop or
elliptical in shape and attach directly to the sides of the hyphae.25
48
The genus Trichophyton is characterized by the production of many
microconidia, and a few or no macroconidia. The macroconidia when
formed in contrast to those of the Microsporum species, are thin walled
and smooth. The size and arrangement of the microconidia are important
in making species identification.
The production of pigment, urease activity, hair perforation/penetrating
capabilities, and differential growth patterns on culture media with or
without thiamine and niacin (Trichophyton Differential Agars) are also
helpful in making species identification. For example Trichophyton rubrum
does not perforate hair in vitro or produce urease while Trichophyton
mentagrophytes perforates hair and produces urease within 2-3 days after
inoculation on to Christensen’s urea agar.34
Preliminary observations suggestive of dermatophytes:-
Colonies that grow within 3-5 days with distinct margins.
Colony surface that is cottony to granular in consistency and the presence
of surface pigmentation with colours ranging from grey white to bluff in
colour.
Hyphae that are narrow, septate and hyaline.
49
The production of microconidia and macroconidia, the size, shape, and
arrangement of which provide for genus and species identification.
Characteristics of dermatophyte causing tinea capitis commonly
recovered in the clinical laboratory34
DERMATOPHYTE COLONIAL MORPHOLOGY GROWTH
RATE
MICROSCOPIC IDENTIFICATION
Microsporum audouinii Downy white to salmon-pink colony;reverse
tan to salmon-pink
2 weeks Terminal chlamydoconidia,macroconidia has bizarre
shape; microconidia rare or absent.
Microsporum canis Membranous with feathery periphery;
centre white to buff over orange-yellow;
lemon-orange apron and reverse
1 week Thick-walled, spindle shaped, multiseptate
roughwalled macroconidia, some with curved tip,
miroconidia; rarely seen.
Microsporum gypseum Cinnamon-coloured; powdery colony;
reverse light tan
1 week Thick walled, rough, elliptical, multiseptate
macroconidia;microconidia few or absent.
Trichophyton rubrum Colour vary from white downy to pink
granular and fluffy varieties; reverse buff to
reddish brown
2 weeks Microconidia usually tear drop most commonly borne
along sides of the hyphae; macroconidia usually
absent but when present are smooth, thin- walled and
pencil shaped.
Trichophyton mentagrophytes White granular and fluffy occasionally light
yellow periphery in young cultures;reverse
buff to reddish brown
7-10days Round to globular microconidia most commonly born
in grape-like clusters or laterally along the hyphae;
Macroconidia are thin walled smooth, club shaped
and multiseptate, numerous or rare depending on the
strain.
Trichophyton tonsurans White tan to yellow or rust, suedelike to
powdery wrinkled with heaped or sunken
centre, reverse yellow to tan to rust red
7-14days Microconidia are tear drop or club- shaped with flat
bottoms, vary in size but usually larger than other
dermatophytes; macroconidia rare, balloon forms
50
found when they are present.
Trichophyton schoenleinii Irregularly heaped smooth white to cream
coloured with radiating grooves, reverse
white
2-3weeks Hyphae usually sterile, many antler-type hyphae
seen.
Trichophyton violaceum Port wine to deep violet, may be heaped or
flat with waxy glabrous surface; pigment
may be lost on subculture
2-3weeks Branched tortuous hyphae, chlamydoconidia
commonly aligned in chains
Trichophyton verrucosum Glabourous to velvety white, rare strains
produce yellow-brown colour
2-3weeks Microconidia rare, large and tear drop when seen
macroconidia extremely rare.
Adapted from Bailey and Scott’s Diagnostic Microbiology
Diagnosis by non-culture techniques
Serology:- Serological studies are required in some instances to establish
a definitive clinical diagnosis, assess the status of a previously diagnosed
mycosis, or determine the efficacy of therapy.25 Most common serological
tests include Latex Agglutination (LA), Double Immunodiffusion (DID),
Complement Fixation (CF), and Enzyme Immunoassays (EIA)
LA is best for detection of IgM
DID & CF usually detect IgG
EIA detects both IgG and IgM
Antigen-protein:- Antigen- protein based assays are not useful for the
detection or identification of dermatophytes.34 Fungi are poor antigens and
therefore sensitivity is low.
51
Nucleic acid probes:- Nucleic acid probe assays are being used with
increasing frequency to provide an early diagnosis. Nucleic acid
sequencing offers promise of becoming the standard method for fungal
identification in reference laboratories.43 Due to the technical expertise
required and the cost of instrumentation and supplies, this technology is
available mainly in developed countries.
Polymerase chain reaction (PCR):- The conventional laboratory
procedures for the identification of dermatophytes are either slow or lack
specificity. The nucleic acid amplification technology provides a rapid and
precise identification of dermatophytes. Studies by Liu et al,44 have shown
that when one of the four random primers (OPAA 11, OPD 18, OPAA 17,
OPU 15) was used in arbitrarily primed polymerase chain reaction (AP-
PCR), up to 20 of the 25 dermatophyte species or subspecies under
investigation could be distinguished on the basis of characteristics band
patterns detected on agarose gel electrophoresis.
The drawback of this technology include contamination which can lead to
the generation of spurious products, lack of ready availability of primers
and high running cost which can lead to delay in the processing of
specimens. However, PCR is now widely used in clinical mycology
laboratories.45
52
Antifungal susceptibility testing
Various methods are available for performing antifungal susceptibility
testing.46 The Clinical Laboratory Standard Institute(CLSI) formerly known
as the National Committee for Clinical Laboratory Standards(NCCLS),
provides documents and sets standards for antifungal susceptibility
testing:-
NCCLS broth based methods for yeasts:- The M27-A method for yeasts
incorporates both macro- and micro- dilution testing methods that allows
for the determination of MIC. This method specifies inoculum size and
preparation, test medium, incubation time and temperature as well as end
point readings for various antifungals. It provides quality control limits at
24 and 48 hours for various classes of antifungal drugs such as
amphotericin B, flucytosine, the azoles and the echinocandins
(caspofungin).47 This method has the greatest advantage of interlaboratory
reproduceability.
NCCLS methods for moulds:- Direct adaptation of the M27-A method to
moulds such as Aspergillus species, Fusarium species, Pseudallescheria
boydii and a host of others were shown to generate reproducible results.48
This method published as NCCLS M38-P,49 was developed using isolates
of Aspergillus species, Fusarium species, Rhizopus species,
53
Pseudallescheria boydii and sporothrix schenckii. Due to differences in
size and light scattering properties of many spores that can be generated
by these fungi, the M38 method specifies different optical density ranges
for each genus. Careful preparation of the inoculum is important, since a
concentration outside the specified range will result in an elevated MIC to
most antifungal drugs.50
Epsilometer test (E-test):- This is a proprietary, commercially available
method for antimicrobial susceptibility testing. MICs are determined from
the point of intersection of a growth inhibition zone with a calibrated strip
impregnated with a gradient of antimicrobial concentration and placed on
an agar plate uniformly inoculated with the microbial isolate under test.
This methodology has been adapted to a number of antifungal drugs.51
Both non-uniform growth of the fungal inoculum and the frequent presence
of a feathered or trailing growth edge can make end-point determination
difficult. However, with experience and standardized techniques, the
correlation between this method and the reference method has been
acceptable for most Candida species and the azole antifungal agents.51
Other agar based testing methods:- Disc based susceptibility testing is
convenient and economical. Adaptations with good correlation with the
reference broth method have been shown for some azole antifungal
54
(fluconazole).52 Flooding the surface of the plate with methylene blue
(0.5µg/ml) appear to improve edge definition and facilitates reading.53
Flow cytometry and use of viability dyes:- Flow cytometry has been
long recognized as a possible tool for antifungal susceptibility testing and
has been developed for yeasts in studies that focused principally on
Candida species.54,55 The potential for correlation of flow cytometry-based
technique with reference methods has been explored in recent works.56,57
One of these studies suggested that flow cytometry might be especially
useful for detection of amphotericin B resistance.55
Testing for dermatophytes:- Dermatophytes grow slowly, and therefore
agar based methods have been employed.58 The disc diffusion
susceptibility method is simple and it is advocated for use in routine
clinical testing. This method is not only reproducible and accurate but it is
economical and easy to perform.59
Laboratory safety in the handling of dermatophytes:-
There are risks associated with the handling of fungal specimens.
Therefore, it is important to protect the laboratory from contamination and
workers from becoming infected. The dermatophytes are moulds and
belong to risk group three organisms. All mould cultures and clinical
55
specimens must be handled in a class II biological safety cabinet, with no
exceptions.
Use of an electric incinerator or a gas flame is suitable for the
decontamination of wire used for transfer of cultures. Cultures containing
pathogenic organisms should be sealed with tape to prevent laboratory
contamination and should be autoclaved as soon as definitive
identification is made
2.7 IMMUNITY TO TINEA CAPITIS
Both specific and non-specific immune responses are involved. The non-
specific immune response is provided by the presence of inhibitory fatty
acids in sebum. These fatty acids are of low level in children compared to
adults, hence children are more susceptible to the infection than adults.3
Other skin surface factors thought to be important in determining the
outcomeof infection include local carbon dioxide tension and the presence
of surface moisture. Sweat and serum also contain inhibitory substances,
such as transferrin, which in its unsaturated state is inhibitory to the
growth of dermatophytes.60
The specific immune response is via cell mediated immune response
which has two parts:-
56
The main efferent limb of the immunologic resistance is the T-lymphocyte.
The induced helper T-cells are of particular importance. Studies have
shown that the development of the delayed type hypersensitivity in
children with naturally acquired scalp ringworm caused by Trichophyton
tonsurans correlates with recovery and that experimentally infected
humans develop both the delayed type skin reactions to trychophytin and
T-lymphocyte blastogenic responses at recovery.61
The afferent limb of the immune response is provided by epidermal
langerhans cells which have been shown to act as antigen presenting
cells in mixed cultures with human lymphocytes. The mechanisms by
which T-lymphocytes affect recovery are less well understood.
Phagocytes, mainly neutrophils and to a lesser extent macrophages can
kill dermatophytes both intracellularly and extracellularly, mainly via
oxidative pathways.60Dermatophyte antigens have been shown to be
chemotactic to human leucocytes and may activate the alternate pathway
of complement system. However, except in inflammatory ringworm,
neutrophils are not commonly seen as part of the inflammatory infiltrate in
dermatophytosis.
2.8 TREATMENT OF TINEA CAPITIS
57
To treat tinea capitis, systemic anti-fungal rather than topical treatment is
required. Topical anti-fungal treatments are not able to adequately
penetrate the hair shaft to eliminate the infection.32, 33 Griseofulvin
(GrivulvinR) and Terbinafine (Lamisil granulesR) are the two medications
approved by the United States Food and Drug Administration (FDA) for
the treatment of tinea capitis.62 Newer anti-fungal agents are available and
these have been shown to be safe, effective, and have shorter duration of
treatment, but are not yet approved by FDA for the treatment of tinea
capitis.62 The azole anti-fungal agents such as fluconazole and
itraconazole are currently being prescribed off-label for tinea capitis. Off-
label use refers to the use of legally available medications being
prescribed for a function outside of the medication’s approved label.63
Griseofulvin:- The use of griseofulvin is currently the standard treatment
for tinea capitis infections in the paediatrics population. It is an oral anti-
fungal antibiotic produced by a species of penicillium. Its mechanism of
action consists of binding to microtubular proteins within the fungus and
disrupting mitotic spindle function resulting in inhibition of hyphal growth. It
has a narrow spectrum of activity being effective only against a small
number of dermatophyte species.62
58
Following oral administration, it is poorly absorbed and concentrated in
keratinized tissue. Absorption of griseofulvin is increased by “microsizing“
or reducing the particle size of drug. Absorption is even more rapid with
the “ultramicrosized“ form of the drug that allows for a dosage reduction of
33 to 50%. The microsized formulation of the medication should be
administered at a dose of 20 to 25mg/kg/day, while the ultramicrosized
preparation should be at 10-15mg/kg/day. The normal duration of
treatment is six to eight weeks (up to sixteen weeks) depending on clinical
and mycological cure. It has poor water solubility and should be taken with
food or milk to enhance absorption. The microsized preparation is
available in liquid form.62
Studies researching into the efficacy of griseofulvin noted that after six to
eight weeks of treatment, griseofulvin was nearly 68% effective when
treating the Trichophyton species and 88% effective when treating the
Microsporum species.62 ,64 This concern over the decrease in sensitivity of
griseofulvin is supported by changes in the initial response and cure rates,
and increases in recommendations for dosages and duration of treatment
with griseofulvin.62,65 Mycological cure rates for griseofulvin have been
shown to range from 71 to 92%, 66 depending on the infective organism,
the dose of the medication and duration of therapy. The most common
side effect of the drug is headache which usually resolves without
59
discontinuation of the drug. Less commonly observed side effects are
gastrointestinal disturbances, drowsiness and hepatotoxicity.21Increased
sensitivity to the sun may occur. Patients should be advised to use
sunscreen and avoid tanning booths. Griseofulvin may affect the efficacy
of oral contraceptives. Adolescent girls taking griseofulvin should be
instructed to use a different or an additional form of contraception while
taking griseofulvin. Blood Urea Nitrogen (BUN), creatinine (RFT) and liver
function tests (LFT) should be monitored after eight weeks of oral therapy
with griseofulvin.67
Terbinafine:- Terbinafine is currently FDA - approved to treat tinea capitis
in children four years of age and older. It is an allylamine drug available as
oral medication.21
It acts by blocking ergosterol synthesis, through the inhibition of the
enzyme squalene epoxidase. It is a fungistatic drug and has a narrow
spectrum of activity being effective mainly against dermatophyte
infections. The dose of terbinafine is 125mg/day (25kg weight),
187.5mg/day (25-35kg weight), and 250mg/day (greater than 35kg
weight). The maximum is 250mg/day. Children should be treated once
daily for six weeks. The oral granules should be taken with non-acidic
60
foods, such as pudding or mashed potatoes, but should not be taken with
apple sauce or other fruit- based foods.
The most common side effects reported when using terbinafine are
headache, nasopharyngitis, rash and gastrointestinal symptoms. There is
also the risk of hepatotoxicity. For this reason terbinafine should not be
used in patients with a pre-existing liver condition. Pre–treatment liver
serum transaminases (ALT & AST) and a potassium level should be
obtained. If treatment is to continue beyond six weeks, liver transaminases
and a potassium level should be obtained alongside full blood count (FBC)
with differential to monitor the absolute neutrophil count.
A meta-analysis of randomized clinical trials comparing terbinafine to
griseofulvin for the treatment of tinea capitis was conducted, and results
favoured terbinafine after twelve weeks of treatment.67 More recent
randomized controlled trials comparing griseofulvin to terbinafine oral
granules supported the results of the earlier meta-analysis in patients
infected with tinea from Trichophyton species. Rates of mycological and
clinical cure were significantly higher for terbinafine than for griseofulvin
for patients with Trichophyton tonsurans. For patients with Microsporum
canis, mycological and clinical cure rates were significantly higher for
61
patients treated with griseofulvin than those treated with terbinafine oral
granules.68
Topical agents:- Scalp dermatophytoses are best treated with oral drugs.
Topical treatments for scalp infections are generally ineffective.69
Adjunct therapies:- Transmission of tinea capitis between individuals
may be reduced when anti-fungal shampoos are used in conjunction with
oral therapy. Selenium sulphide shampoos have been shown to have
success in eliminating the shedding of viable tinea capitis spores.70 Both
the 1% and 2.5% preparations of the shampoo are available. These
preparations, when used twice weekly, have been found to be superior to
a non-medicated shampoo in terms of the time required to eliminate the
shedding of viable spores.71
There is no difference between the two preparations of selenium sulphide
in terms of time required to produce negative culture. The 1% selenium
sulphide shampoo which is commercially available is as equally effective,
but less expensive alternative adjunct, to the oral treatment of tinea
capitis.
ketoconazole shampoo 2% was also found to reduce the number of viable
spores in tinea capitis infections caused by Trichophyton tonsurans.72 The
62
shampoo alone produced a 3% clinical cure rate after eight weeks of
therapy. Complete culture cure was obtained in 33% of cases using
ketoconazole shampoo 2% as a treatment for tinea capitis, and these
children remained culture negative for at least one year after treatment.72
2.9 PREVENTION AND CONTROL OF TINEA CAPITIS
It is best to prevent tinea capitis. The fungus is very easily transmitted.
The majority of people acquire the fungus long before they have any
symptoms.33 Maintaining a descent personal hygiene through regular hand
washing and scalp washing with the use of shampoo are important.
Sharing of personal care items in schools or any type of social gathering
should be avoided. Families with children having tinea capitis should avoid
sharing items such as combs, towels, bathing soap, barbing clippers etc,
because the spores of the infecting organisms may be carried by these
items.33 Children should also avoid contact with sick pets. Sick pets should
be taken to the veterinary clinic once they are sick. If a child or pet is
suspected of having an infection, selenium sulphide shampoo should be
applied regularly. 71
At schools and homes children’s scalp should be examined every now and
then so that the infection can be detected early and prompt and
appropriate treatment instituted to prevent transmission of infection to
63
playmates at school and siblings at home. Health Education on how tinea
capitis infection can be contacted and transmitted to others as well as
treatment and prevention should form part of schools’ Education Health
Programmes .Management of complications, particularly alopecia which is
usually associated with inflammatory forms of the disease, is of utmost
importance to avoid social stigma. In most cases the alopecia is
temporary, occasionally it can become permanent. Parents’ worries
should be alleviated.
Research work to keep update about changing aetiological agents and
their susceptibility patterns, as well as screening for carriers and treatment
of carriers to reduce or stop transmission can serve as control measures.
NUTRITIONAL ASSESSMENT USING ANTHROPOMETRIC
MEASUREMENTS/ INDICES
Anthropometric assessment remains the most widely used method for
evaluating the nutritional status of children.73It is the objective
measurement of body muscle and fats. Anthropometric measurement is a
non-invasive, inexpensive method that is readily adapted for field
nutritional surveys.73 Anthropometric measurements include the weight,
height/length (measured in children under two years), skin fold thickness,
occipito-frontal circumference and mid-arm circumference.
64
Anthropometric indices are combinations of measurements. Indices
utilized in nutritional assessment include Weight for Height, Height for
Age, Weight for Age and Body Mass Index. The anthropometric definitions
of malnutrition73include:-
Stunting: stunted growth refers to low height-for-age, when a child is short
for his/her age but not necessarily thin. It is also known as chronic
malnutrition and carries long term developmental risks.
Underweight: underweight refers to low weight-for-age, when a child can
either be short or thin for his/her age. This reflects a combination of
chronic and acute malnutrition.
Wasting: wasting refers to low weight-for-height where a child is thin for
his/her height but not necessarily short. It is also known as acute
malnutrition.
65
CHAPTER THREE
MATERIALS AND METHODS
3.1 STUDY DESIGN
This was a cross sectional descriptive study.
3.2 STUDY AREA
This study was carried out among primary school children in Okelele
community in Ilorin East Local Government Area (LGA) of Kwara state.
Ilorin East LGA is located on latitude 805 N and longitude 40 E 74. The
annual rainfall in the state ranges from 1000-1500mm, while maximum
average temperature ranges between 30 and 35oC.74The state is situated
in the transitional zone between the Northern and Southern parts of
66
Nigeria. The Local Government shares boundaries with Ilorin south, Ilorin
west, Moro and Ifelodun LGAs and has three districts namely Iponrin,
Gambari and Are. Okelele falls under Gambari district. The headquarters
of the Local Government is Oke-oyi, about 16 kilometers from Ilorin
Township.
The people of Okelele community `are mainly of low socioeconomic class
as justified by the type of occupation they practice, which are mainly local
cloth weaving, farming, mat making, dyeing and embroidery. They also
rear domestic animals and pets which live in proximity with them and their
children.
There are seven primary schools within the community.75 The names of
the schools and the population of each school as obtained from the data
compiled by the Ilorin East Local Government Education Authority as at
October, 2011were as follows:- Akerebiata primary school (236);Army
Children School (A.C.S) Sobi (259); Mogaji Are primary School
(320);Dada primary school A (601); Dada primary school B (611); Okelele
primary school B (740); Okelele primary school A (771); Therefore, the
total population of the school children in the community as at study period
was 3,538.
3.3 STUDY POPULATION
67
The study population consisted of children aged 5-14 years attending
primary schools in Okelele community, Ilorin East LGA of Kwara state.
3.4 SAMPLE SIZE DETERMINATION
The minimum sample size was determined as follows:-
Using the Fisher’s formula 76 for population greater than 10 000;
n = z2pq
d2
Where, n = desired sample size (when population is greater than 10, 000)
z = the standard normal deviate, usually set at 1.96 (at 95%
confidence level)
p = the prevalence of tinea capitis. Prevalence of 31.2%
11reported from a previous study was used.
q = 1.0 – p = 0.69
d = degree of accuracy desired set at 0.05
Thus, n = (1.96)2 x 0.31 x 0.69
(0.05)2
n = 329
68
Sample sizewhen population is less than 10,000 was calculated using the
following formula;
nf = n÷ 1+(n)/N76
Where, nf= the desired sample size when population is less than 10,000
n= the desired sample size when the population is more than
10,000
N= the estimate of the population.
When, n = 329
N = 3,538
Therefore,
nf = 329÷1+(329/3,538)
nf = 329÷1.09
nf= 301
3.5 ETHICAL CONSIDERATION
Ethical clearance was obtained from the Ethics and Research Committee
of the University of Ilorin Teaching Hospital. A written informed consent
was obtained from parents or guardian of the children and assent of each
69
child was obtained before recruitment. Permission was also obtained from
Ilorin East Local Government Education Authority before embarking on
field work, while approval and cooperation of the head teachers and class
teachers of the selected schools were sought.
3.6 SELECTION OF SUBJECTS
The subjects for this study were selected from the seven primary schools
in Okelele community using a multi-stage sampling technique:-
First stage:- Proportional allocation was used to select the number of
pupils from each school and from each class(six classes in each school).
For the schools, the number of pupils selected per school (n) was
obtained by dividing the population of pupils in each school (n1) by the
total population of pupils in the area (N), multiplied by the sample size (S).
For example, for Akerebiata primary school;
n = n1 / N multiplied by S76
When, n1= 236
N =3,538
70
S =301
Then, n= the number of pupils selected was 236/3,538 x 301 = 20.0
Therefore, the proportion of pupils taken from each school was as
follows:- Akerebiata school (20); A.C.S. Sobi (22); Magaji Are (27); Dada
A (51); Dada B (52); Okelele B (63) and Okelele A (66).
Similarly, the number of pupils selected from each class was obtained by
dividing the number of pupils in each class (n1) by the population of pupils
in the school (N) multiplied by the number selected from each school (n).
For example the total number of pupils in class one (n1) in Akerebiata is
24, the population of the school (N) is 236, the number of pupils selected
from the school (n) was 20. Therefore the number of pupils selected from
class one is 24/236 x 20 = 2.
Second stage:- From among children aged 5-14years, purposive
sampling technique was used to select pupils with scalp lesions
suggestive of tinea capitis.
Third stage:– Simple random sampling by balloting was then used to
select the subjects until the minimum sample size was achieved.
Inclusion criteria
71
1. School pupils aged 5-14years in Okelele.
2. Children with clinical evidence of tinea capitis who have given
assent to participate and whose parents/ guardians have given their
consent.
Exclusion criteria
1. School pupils < 5 or > 14 years.
2. Non primary school pupils at Okelele.
3. Failure to get consent and child’s assent.
4. Antifungal use within 14 days to recruitment.
Clinical examination
Tinea capitis was identified clinically by the presence of scaly scalp,
alopecia (hair loss) or pus filled sores.33 (Plate 1) Pupils who had these
clinical lesions and fell within the age range were recruited into the study.
These pupils were examined for the presence of fever and
cervical/occipital lymphadenopathy. Anthropometric measurements
(weight and height) were also taken. The weight was measured to the
nearest 0.1kg using Hanson’s bathroom scale and was recorded in
72
kilogrammes while the standing height was measured to an accuracy of
0.1cm using Seca’s standiometer and was recorded in meters.
Anthropometric indices:- Weight for Age and Body Mass Index (BMI)
derived from the weight and height measurements were used to assess
the nutritional status of the pupils, by making reference to a standard table
(Appendix IV).The children were divided into two age groups, the 5-9 and
10-14 year age groups. For the 5-9year age group, the Weight for Age,
calculated using observed weight (kg) / expected weight for age (kg)
multiplied by one hundred formula73was used for their nutritional
assessment. The BMI calculated using the weight (kg) / (height in m) 2
formula,77 was used to determine the nutritional status of the 10-14 year
age group (adolescent).78.
Data collection
A study proforma (Appendix I) was used to obtain information on;
1. Socio-demographic characteristics
2. Clinical profile
3. Anthropometric measurements
4. Laboratory report
73
Socio-Economic Index Score
Socio-economic index scores were awarded to the occupations and
educational attainments of the participants’ parents or caregivers using the
socio-economic classification scheme by Oyedeji (Appendix V). The mean
of four scores (two for the father and two for the mother) approximated to
the nearest whole number was the social class assigned to the child as
proposed by Oyedeji.79 For example, if the mother was a junior, school
teacher (score=3) and father a senior teacher (score=2) and the
educational attainment of the mother was primary six (score=4), and the
father was a school certificate holder (score=2),the socio-economic index
score for this child was; 3+2+4+2/4=2.75 to the nearest whole number,
gave 3.Socio economic classes I and II are the high socio-economic class,
class III is the middle class while socio economic classes IV and V are the
low socio-economic class.
Three research assistants were recruited and trained. They were
responsible for taking weight and height measurements. They also took
part in filling of the study proforma, as well as labeling and transportation
of the specimens. The assistants were supervised by the investigator.
Subjects’ selection, specimen collection and processing were performed
solely by the investigator.
74
3.7 SAMPLE COLLECTION AND LABORATORY PROCEDURES
The affected area of the scalp sampled was cleaned with methylated spirit
swab to remove surface bacterial contaminants.80 Infected hairs were
collected from the margin of the lesion with a pair of small scissors by
cutting the hair shaft from the proximal end. Scissors was disinfected in
sodium hypochlorite (household JIK) and then 70% alcohol before and
after use on each subject. Scalp scrapings/crusts were collected from the
active, erythematous, advancing edge of the lesion using the side of a
clean microscope glass slide.34Onemicroscope slide per person was used.
For purulent/exudative lesions, sterile cotton swab stick was used for
sample collection, 35 and specimen was obtained in duplicate.
Hairs and scalp scrapings were collected on pieces of clean, dry brown
paper. The papers were folded to enclose the specimens and paper clips
were used to close them to form an envelope.37 Swab sticks were put back
into their sterile containers and sealed. Each specimen in an envelope or
on swab stick was properly labeled to indicate the code number for the
subject, the type of specimen and date of collection. All specimens were
transported within four hours of collection, 80 to the Mycology Laboratory,
Department of Microbiology U.I.T.H. Ilorin, and processed immediately.
75
Each specimen was divided into two parts – one part was used for direct
microscopy and the other was used for culture.81Similarly, one of the swab
stick specimens was used for microscopy while the other was used for
culture. All specimens irrespective of microscopy results (whether positive
or negative) were cultured, using a pair of MycoselR agar plates81per
specimen. A subculture was made onto plain Sabouraud’s Dextrose Agar
(SDA), from the growth on the primary plate for identification and
susceptibility tests.
3.7.1 Direct microscopic examination of specimens
Potassium hydroxide (KOH) preparation
Principle:- The principle is based on the use of 10-20% KOH to dissolve
the keratin present in skin or hairs thereby clearing out any background
scales or cell membranes to allow visualization of hyphal elements.25 The
clearing time is 5-10 minutes if specimen is hair or 20-30 minutes for
crusts and scrapings. The process of clearing can be hastened by gently
heating the preparation over a spirit lamp or pilot flame of a Bunsen
burner.
Method:-A wet mount of each specimen (hair, scalp scrapings, crust or
pus on swab stick) was prepared using a sterile pair of forceps to pick
76
hair/scrapings or crust on to few drops of 20% KOH solution or gently
rubbing a swab stick in the drops of KOH placed on a clean, grease-free
microscope slide. A 22 by 22mm cover slip was applied. The slide was
warmed over a low Bunsen burner flame for a few seconds to digest the
keratin and free the fungal elements. Thereafter the slide was left to stand
for five minutes. Each treated slide was examined under low (10x) and
high (40x) objectives for the presence of spores and their distribution
pattern in hairs (ectothrix, endothrix or favic type), and hyphae in scalp
scrapings, crusts or pus. The size and distribution of the spores on the
hair provided information about the species of dermatophyte.80
Interpretation of results:-
Scalp skin—Septate hyphae and/or chains of arthroconidia are indicative
of dermatophyte infection .20 (Plate 2)
Hair--Arthroconidia on periphery of hair shaft producing a sheath is
indicative of ectothrix (Plate 2); arthroconidia formed within the hair shaft
is indicative of endothrix infection (Plate 2); long channels within the hair
shaft is indicative of favic infection.20
3.7.2 Culture
77
Principle:- MycoselR or MycobioticR agaris used for the primary recovery
of dermatophytes. It is essentially SDA with cycloheximide and
chloramphenicol added. The cycloheximide will inhibit the growth of
saprophytic fungi, while chloramphenicol will inhibit the growth of
contaminating bacteria and therefore, allow for the recovery of pure
isolate.
Method:- Each specimen was inoculated into a pair of MycoselR agar
plates by placing some of the hair stubs and/or scrapings centrally on the
surface of the medium by means of a sterile straight wire or forceps.
Where specimen was obtained on sterile swab stick, the swab stick was
used to inoculate the surface of the medium directly by gentle rubbing. For
each specimen, one plate was incubated at room temperature (25-300C)
and the other at between 35- 370C. The culture plates were examined
every other day for evidence of growth. Cultures were not considered
negative for growth until after four weeks of incubation. After the growth of
a dermatophyte is established, a subculture was made onto SDA for
further identification and susceptibility testing.82
Interpretation of results:-
Colonies appearing cottony, wooly, powdery or fluffy are suggestive of the
growth of a dermatophyte.
78
3.7.3 Characterization and identification of isolates
Isolates on culture were identified based on their growth rate, colonial
morphology, microscopic examination of lactophenol cotton blue wet
mount, hair perforation test, urease test and colour change observed on
Dermatophyte Test Medium (DTM).
Growth rate:-The time taken for growth to appear on culture plates was
noted.
Colonial morphology:- Growth on plates were examined
macroscopically. Characteristic features such as mycelia texture, folding
of the colonies and radial grooves were noted. Changes in the colouration
of the medium due to the production of alkalinizing agents such as
ammonium, by the dermatophytes, as well as the reverse appearance of
the colonies were also noted. The cultural features observed were
compared with those contained in a practical mycology text.80 (Plate 3)
Microscopy:-Lactophenol cotton blue mount of colonies on SDA were
examined microscopically. The tease mount method was used. The
procedure was performed under a safety hood with hand gloves on.25
A tease needle was used to dig out a small portion of the colony to be
examined including portions of the subsurface agar. The colony fragment
79
was then placed on a drop of lactophenol cotton blue on a grease free
microscope slide. A 22 by 22mm cover slip was applied. Gentle pressure
was then applied on the surface of the cover slip with the eraser end of a
pencil to disperse the mount. The preparation was observed
microscopically under 10x and 40x objectives. Characteristic features of
conidia such as the shapes, sizes as well as the septate pattern of the
conidia and hyphae were taken into consideration in identification. The
microscopic features observed were compared with that contained in a
practical mycology text.80 (Plate 4)
Hair perforation test for dermatophyte:-The hair perforation test, also
known as in vitro hair perforation test, is a laboratory test used to
distinguish isolates of dermatophytes such as Trichophyton
mentagrophytes and its variants.41
Method:- Pre-pubertal hair obtained from volunteer children with no
clinical evidence of tinea capitis infection, were cut into short pieces(1cm).
The cut pieces were placed in glass petri-dish and autoclaved at 1200C for
20 minutes. The sterilized hair pieces were placed in water vial and yeast
extract was added. This was inoculated with small fragments of the test
fungus. The vial was then incubated at room temperature. Individual hairs
80
were removed at intervals, of up to four weeks and examined
microscopically in lactophenol cotton blue.
Controls:-Locally identified T. mentagrophtye (positive control) and T.
rubrum (negative control) strains were similarly inoculated, incubated and
examined microscopically in lactophenol cotton blue.
Interpretation of results
Marked area of pitting / marked erosion is indicative of T. mentagrophytes
infection (Plate 5); absence of pitting / erosion is indicative of T. rubrum
infection.
Urease test
Principle:-The test organism is examined for urease production by
inoculating it on to a medium which contains urea. The indicator in the
urea medium is phenol red. When the strain is urease producing, the
enzyme will break down urea by hydrolyzing it to give ammonia and CO2.
With the release of ammonia, the medium becomes alkaline as shown by
a change in colour of the medium from tan or mauve to pink-red.
81
Method:-A sterile straight wire was used to inoculate the test organism on
to a urea agar slope contained in a bijou bottle. With the lid partially closed
to allow for aeration, the bottle was incubated at between 35-370C
overnight and thereafter observed for colour change.
Controls:-Locally identified strains of T. mentagrophtes (positive control)
and T. rubrum (negative control) were similarly inoculated, incubated and
examined for colour change.
Interpretation of result:-
Pink colouration of the urea medium------------------------------------------
Positive urease test
No change in the colour of the urea medium-------------------------------
Negative urease test
Colour change on Dermatophyte Test Medium (DTM)
Principle:- DTM is a selective medium for dermatophytes. It is a phytone
dextrose agar to which is added chlortetracycline, cycloheximide and
gentamicin along with buffered phenol red.40 Metabolites released by the
growth of a dermatophyte will turn DTM from natural tan of agar to a deep
82
red colour by causing alkalinization of the medium and a change in the
colour of the phenol red.
Method:- A sterile straight wire was used to inoculate the test organism on
to a DTM agar slope contained in a sterile sample bottle. With the lid
partially closed to allow for aeration, the bottle was incubated at room
temperature and observed on daily basis for a colour change over a
period of fourteen days.
Interpretation of result:-
Deep red colour of the medium-------------------------------------Positive DTM
test
No change in the colour of the medium---------------------------Negative DTM
test
3.7.4 Antifungal susceptibility testing
Agar–Based Disc Diffusion Assay for Susceptibility Testing of
Dermatophytes was used.83
Principle of disc diffusion susceptibility test37: - Discs containing
known concentrations of antimicrobial agent are placed on a plate of
83
sensitivity testing agar uniformly inoculated with the test organism. The
antimicrobial diffuses from the disc into the medium. Strains sensitive to
the antimicrobial are inhibited at a distance from the disc, whereas
resistance strains have smaller zones of inhibition or grow up to the edge
of the disc.
Method:-
Preparation of standardized inoculum84
Isolates stored on SDA slants at room temperature were sub-cultured onto
chloramphenicol supplemented SDA slants in bijou bottles. With the lids
partially closed to allow for aeration, the bottles were incubated at room
temperature for five to seven days until luxuriant growths were obtained.
On to each young culture of fungal isolate, 3mls of normal saline was
dispensed. With the aid of a sterile glass rod, the dermatophyte isolates
were gently scrapped to make a suspension. The organisms’ suspensions
(mixture) were filtered using whatmann filter paper No.1 to remove
mycelia and hyphae. The filtrate which contained the spores was then
84
standardized using 0.5 McFarland’s standard by adjusting the turbidity of
the broth cultures spectrophotometrically at a wavelength of 520nm to a
transmittance of 70-72% to give an inoculum size of between 2 x106 to 4 x
106 CFU/ml.
Preparation of Griseofulvin, Terbinafine and Itraconazole antifungal
discs83, 85
Tablets of Griseofulvin 500mg (Maxheal), Terbinafine 250mg (Novartis)
and Itraconazole 100mg (Hanmi) were purchased locally. Each tablet was
dissolved in sterile distilled water. Different concentrations were made to
the final working concentration of 1mg/ml or 1000µg/1000µl. For example
for griseofulvin, the 500mg tablet was initially dissolved in 10ml of sterile
distilled water to give a drug concentration of 50mg/ml. From the 50mg/ml
solution, 1ml was taken and added to 9ml of sterile distilled water to give a
5mg/ml drug concentration. Finally from this drug concentration 2ml was
taken and added to 8ml of sterile distilled water to give the working
concentration of 1mg/ml or 1000µg/1000µl. Sterile whatmann filter paper
No.4 was cut into 6mm diameter discs using a sterile paper punch. These
discs were then arranged in a sterile glass Petri-dish using sterile forceps.
With the aid of an adjustable micropipette, the desired concentrations of
the antifungal drugs were impregnated into the discs. For example for
85
25µg griseofulvin disc, the micropipette was adjusted to 25µl to pick this
concentration from the working solution of 1mg/ml and dropped onto the
discs in the glass Petri-dish. The discs were dried in an oven at 450C for 5-
10 minutes. After cooling, the prepared discs were placed in sterile screw
capped tubes and stored at between 2-80C until ready for use.
The commercially available antifungal discs included Fluconazole 25µg by
Oxoid, Ketoconazole 10µg, Miconazole 10µg and Clotrimazole 10µg all
from Mast Diagnostica.
Disc diffusion method83
SDA plates were streaked evenly with sterile swab sticks dipped into the
standardized inoculum suspensions of the isolates. The lids of the Petri-
dishes were left ajar in a laminar flow cabinet to allow excess surface
moisture to be absorbed into the agar. The antifungal discs were then
applied to the surface of the inoculated agar plates. The inoculum of a well
identified local strain of T. mentagrophyte was similarly prepared and
inoculated as control. Plates were inverted and incubated at room
temperature for two to five days. The plates were examined daily for
growth. After growth the zones of inhibition for the test and control
organisms were measured using a ruler calibrated in millimeters. These
measurements were compared with the CLSI standard (Appendix VII).
86
3.8 DATA ANALYSIS
Statistical analysis was performed using the Statistical Package for Social
Sciences (SPSS) version 16 software (SPSS Inc. Chicago, Illinois).
Descriptive analyses of variables were used to summarize data. The
frequencies and mean±sd were generated for categorical and continuous
variables respectively. Quantitative and qualitative demographic
characteristics were summarized. Results were presented in tabular
forms. Comparisons were made using standard statistical method in which
categorical data were compared by Pearson chi square. To determine the
predictor(s) of the presence of tinea capitis, binary logistic regression
analysis was done. Odds ratio were determined with respective
confidence interval. A p-value< 0.05 was taken as statistically significant.
Conclusions and recommendations were based on scientific evidence
from the results.
87
CHAPTER FOUR
RESULTS
Out of the 301 school children with clinically suggestive disease studied,
228 had mycologically proven tinea capitis, giving a prevalence of 75.7%
(95% Confidence Interval = 73.0 -78.5).
As presented in table 1, majority, 66(21.9%), of the participants came from
the most populous school (Okelele A primary school) while the least,
20(6.6%) of the participants were from Akerebiata primary school. The
largest number, 67(22.3%) of the participants were in primary three while
the least, 41(13.6%) were in primary five.
88
Two hundred and fifty nine (86.0%) of these children were males while
42(14.0%) were females, giving a male to female ratio of 6.2:1.0.The
mean age of the pupils was 9.8years±2.4.Nineteen (6.3%) of the pupils
belonged to high socio-economic class (SEC), while 282(93.7%) were in
the low SEC.
Table 2 presents the prevalence of tinea capitis according to age and sex.
There was no significant difference in the prevalence of tinea capitis
between the two age groups (p=0.270) and across gender (p=0.275).
Figure 1 is a pie chart representing the aetiological agents of tinea capitis
among the school children. Trichophyton rubrum was the predominant
isolate 156(68.9%), followed by Microsporom ferrigineum which accounted
for 51(22.4%) of the isolates. Trichophyton mentagrophytes accounted for
17(7.4%) while Trichophyton verrucosum accounted for 4(1.8%) of the
total isolates.
As presented in table 3, 78.8% of the T. rubrum isolates were sensitive to
clotrimazole and miconazole while 21.2% were resistant to both drugs. T.
rubrum was 100.0% resistant to griseofulvin, tebinafne, itrconazole,
fluconazole and ketoconazole. M. ferrugineum and T. mentagrophytes
were 100.0% sensitive to clotrimazole, miconazole and terbinafine. In
addition, T. mentagrophytes showed 100.0% intermediate susceptibility to
89
fluconazole. T. verrucosum was 100.0% resistant to all the antifungal
drugs tested.
Table 4, shows that the odds of tinea capitis occurring in children with
large family size was two times higher compared to those with small family
size (OR=2.025, 95% CI=1.185-3.461, p=0.010).
More of the undernourished children 127(55.7%) had tinea capitis
compared to the well-nourished children 101(44.3%), but there was no
statistically significant association between nutritional status and tinea
capitis (OR=0.863, 95%CI=0.509-1.464, p=0.586).
Two-thirds (66.7%) of children having family members with similar scalp
lesion were found to have tinea capitis while one-third (33.3%) of those
with no family history of similar scalp lesion had tinea capitis. However, no
statistically significant association was found between similar scalp lesion
in family members and tinea capitis (OR=0.717, 95%CI=0.417-1.232,
p=0.228).
Over ninety two percent of the pupils with tinea capitis belonged to low
SEC, while 17(7.5%) of them from high SEC were found to have tinea
capitis. However, these observed differences were not statistically
significant (OR=0.350, 95% CI=0.079-1.551, P=0167).
90
Table 1: Socio-Demographic Characteristics of the Participants
Variable Frequency
(N=301) Percentage (%)
Primary Schools Akerebiata 20 6.6 A.C.S. Sobi 22 7.3 Mogaji Are 27 9.1 Dada A 51 16.9 Dada B 52 17.3 Okelele B 63 20.9 Okelele A 66 21.9 Primary School Classes 1 50 16.6 2 44 14.6 3 67 22.3 4 54 17.9 5 41 13.6 6 45 15.0 Sex Male 259 86.0
91
Female 42 14.0 Age group (years) 5-9 132 43.9 10-14 169 56.1 Mean age + Sd 9.8 + 2.4 Socio-Economic Class High 19 6.3 Low 282 93.7
Table 2: Age and Sex Prevalence of Tinea capitis
Age/Sex Number with Scalp lesions N=301
Number Positive (%) 𝑥2 p-value
Age groups (years)
5-9 132 98(74.2)
10-14 169 130(77.0) 11.089 0.270
Gender
Male 259 199(76.8)
Female 42 29(69.0) 1.193 0.275
92
Figure 1: Fungal agents of Tinea capitis
15668%
5122%
178%
42%
Trchophyton rubrum
Microsporum ferrugineum
Trichophyrton mentagrophytes
Trichophyton verrucosum
93
94
95
CHAPTER 5
DISCUSSION
The high prevalence (75.7%) of tinea capitis as found in this study
demonstrates that the infection is common among the school children
studied. Several factors such as low standard of living, poor sanitary and
hygienic conditions that are characteristics of rural communities especially
in developing countries might have accounted for this. The predominance
of anthropophilic dermatophytes as reported later in this study could be
responsible for perpetuating tinea capitis among the school children and
may also explain the high prevalence obtained. It has been reported1 that
fungal particles from anthropophilic dermatophytes are viable for months
and are also found among asymptomatic carriers, that is, individuals who
do not show signs and symptoms of tinea capitis but from whom
dermatophytes can be isolated when samples of their hair and/or skin are
cultured. This prevalence was higher than the 31.2% reported for tinea
capitis by Ayanbimpe et al.11This difference could be due to lack of
surveillance and control measures that is also typical of developing
countries resulting in increase in the incidence of tinea capitis over time.
Low standard of living, overcrowding and poor hygiene are factors which
increase susceptibility to tinea capitis. In a cross-sectional study
96
performed among primary school children in Ivory Coast (Western Africa),
Menan et al7 reported a prevalence of 11.3% for tinea capitis. In
Mozambique, Sidat et al88 reported a prevalence of 9.6% for tinea capitis,
while in Africa tinea capitis was reported6 to affect 10-30% of school age
children. These prevalences are markedly lower compared to the
prevalence reported in present study. These prevalences represented
prevalences of clinically suggestive tinea capitis as opposed to prevalence
of mycologically proven tinea capitis among children with clinically
suggestive disease as reported in this study and in the study by
Ayambimpe et al11. The variations in prevalence could also be due to the
type of sampling methods used and could be a reflection of people’s
habits, climatic conditions, standards of hygiene and levels of education all
of which can influence predisposition to tinea capitis.16 Adefemi et al13 in a
prevalence study among school children in Oke-oyi reported tinea capitis
as the predominant dermatophytosis where it was found to account for
76.1% of cases. Though this percentage is comparable to the one
obtained in the present study, it represented the proportion of children with
tinea capitis.
As found in this study there was no significant association between age
and tinea capitis. Likewise, there was no significant association between
gender and tinea capitis, even though tinea capitis was found to be more
97
common among males (76.8%) than in females (69.0%). These probably
indicate similar exposure to infection irrespective of age and sex. Previous
studies1,4,7,11 also reported tinea capitis to be commoner in males than in
females. The age groups studied have been reported11 to be the most
affected as they constitute the most active group, and they are often left
alone to cater for themselves in terms of bathing and cleaning being often
ignorant about the prevention and control of fungal infections.
The predominant causative agent of tinea capitis among the children was
T. rubrum(68.4%). Others were M. ferrugineum(22.4%),T.
mentagrophytes(7.4%) and T. verrucosum(1.8%). Out of these
dermatophyte species T. rubrum and M. ferrugineum are anthropophilic,
T. verrucosum is a zoophilic dermatophyte while T. mentagrophytes has
both anthropophilic and zoophilic varieties. This finding shows a
predominance of anthropophilic dermatophytes. The ecology of these
dermatophytes indicates transfer of infection from person to person as
well as from animals to humans. The affected children might be sharing
personal items such as combs, towels, hats and beddings which could
serve as vehicles for the transfer of infective agents between them. Also,
close interaction at home and at school with infected family members and
playmates might have exposed them to infection with anthropophilic
agents. The children in Okelele community live in proximity to domestic
98
animals and pets reared by their parents and this could have predisposed
them to infection with zoophilic dermatophytes. As against this study,
Ayanbimpe et al11 found T. soudanese as the predominant causative
agent of tinea capitis among other isolates. Also unlike this study, T.
mentagrophtes was reported as the major isolate by Adefemi et al13. In
faraway Turkey, Altindis et al89 reported T. violaceum as the predominant
isolate while in Ivory Coast, Menan et al7 reported T. soudanese as the
commonest etiological agent of tinea capitis. In Mozambique, M. audouinii
was reported88 as the major isolate. These findings indicate variations
over time, in the aetiological agents of tinea capitis within the same
geographical area and from one geographical area to the other as earlier
reported.3,10
When these isolates were subjected to susceptibility testing, T.
mentagrophytes was the most susceptible to the antifungal drugs used as
it was 100.0% sensitive to clotrimazole, miconazole, terbinafine and it also
showed 100.0% intermediate susceptibility to fluconazole. This was
followed by M. ferrugineum which was 100.0% sensitive to clotrimazole,
miconazole and terbinafine. Over 78% of T. rubrum isolates were
sensitive to clotrimazole and miconazole; the remaining 21.2% were
resistant to both drugs, while T. verrucosum was 100.0% resistant to all
the antifungal drugs tested. This pattern of susceptibility suggests
99
clotrimazole and miconazole for treatment, possibly as adjuncts as both
were active against all susceptible isolates.
All the four fungal agents reported in this study, were found to be resistant
to griseofulvin. Griseofulvin is the drug of choice for tinea capitis and has
been in use for several decades. Repeated use could have partly,
accounted for this resistance. In addition non-compliance due to
prolonged usage required for griseofulvin therapy could be another factor
contributing to the resistance. This observation was previously
documented by Al-Refai90 thus suggesting possible reasons for treatment
failures that maybe experienced with the use of the drug. Mukherjee et
al91also documented primary resistance of T. rubrum to terbinafine and
this may suggest why all the T. rubrum isolates in this study could have
intrinsic resistance to terbinafine as this drug is not commonly used for the
treatment of tinea capitis in our environment. The few T. verrucosum
isolates could be resistant strains resulting from mutation which may result
to alteration in binding site with reduced affinity for binding by drugs or
reduction in the import of drug into the cell resulting in loss of activity.92
It was also observed that none of the isolates was sensitive to
itraconazole and ketoconazole. The relative safety and ease of delivery of
the azoles has led to the wide spread use of these drugs both for
100
prophylactic and therapeutic purposes. Repeated exposure to these drugs
could result in secondary resistance.92 The intermediate susceptibility
observed with fluconazole could mean that higher drug concentration
would be needed to achieve therapeutic effect. Some studies85,93,94
however, have documented fluconazole to have less activity against
dermatophytes. These susceptibility results indicate therapeutic
challenges with tinea capitis, hence further susceptibility studies and
research for new drugs are desirable.
As it is with any contagious disease, tinea capitis has associated risk
factors. This study found an association between family size and tinea
capitis. Children from large families were twice as likely to have tinea
capitis compared to those with small families. Large family size with
limited income for family upkeep encourages the sharing of personal items
such as combs, towels, hats and beddings. These items could act as
vehicles facilitating the transfer of the fungal agents of tinea capitis from
one infected child to the other and this can lead to the spread of the
disease. With inadequate living space, large family size could lead to
overcrowding which may encourage close interaction between infected
and non-infected family members also leading to transmission of infection,
although this study did not find an association between overcrowding and
tinea capitis. These findings stress the importance of good personal
101
hygiene and improved standard of living as measures necessary in the
prevention of tinea capitis among the school children.
Out of 228 children with tinea capitis, more than three-quarter (79.8%)
were exposed to domestic animals and pets, but there was no significant
association between exposure to domestic animals/ pets and tinea capitis.
These children probably have less contact with these animals and pets as
they may be involved minimally in their care.
The occurrence of tinea capitis, as found in this study, was not
significantly associated with Socio-Economic Class. This possibly
indicates interaction in the community and possibly at school between
children from high Socio-Economic Class and children of low Socio-
Economic Class who have tinea capitis. Tinea capitis was present in the
undernourished and well-nourished categories in comparable proportions,
(55.7% of the undernourished and 44.3% of the well-nourished) indicating
no significant association between nutritional status and tinea capitis.
Nutrition can possibly influence the immune status and so affect the
occurrence of disease. However, these children could have had similar
exposure to the infective agents of tinea capitis regardless of their
nutritional status as the level of fungistatic fatty acids present in the scalp
that protects against disease is generally low in children.3 These findings
102
are against reports from previous studies3, 10, 11 where these risk factors
were found to be associated with tinea capitis.
5.1 CONCLUSION
The prevalence of tinea capitis among the school children in Okelele
community is high.
Antthropophilic and zoophilic dermatophytes were found to be responsible
for tinea capitis.
Clotrimazole and miconazole demonstrated activities against majority of
the dermatophyte isolates. T. rubrum and T.verrucosum demonstrated
resistance to the three classes of drugs available for the treatment of tinea
capitis which are griseofulvin, terbinafine and the azoles.
Large family size was identified as the predisposing factor for tinea capitis.
5.2 RECOMMENDATIONS
1. Health education in the schools on the mode of transmission, with
emphasis on the identified risk factors and ways of preventing tinea
capitis is recommended and should form part of the school health
programme in all the primary schools in Okelele community.
2. To the teachers at schools and parents at homes children’s scalp
should be examined frequently so that the infection can be detected
103
early and prompt and appropriate treatment instituted to prevent
transmission of infection to playmates at school and siblings at home.
3. Terbinafine is recommended for the empiric treatment of tinea capitis
while clotrimazole or miconazole is suggested for use as adjunct
therapy in the locality.
4. Further susceptibility studies on T. rubrum and T. Verrucosum are
needed to identify the effective drugs for the treatment of tinea capitis
due to these agents.
5. Multicentre research work in Kwara state and Nigeria to define current
aetiological agents of tinea capitis and their susceptibility pattern is
necessary.
5.3 LIMITATION OF THE STUDY
Some antifungal discs could not be obtained commercially and had to
be prepared locally. This might have affected the result of the
susceptibility test.
104
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APPENDIX I
STUDY PROFORMA
TINEA CAPITIS INFECTION AMONG SCHOOL CHILDREN IN
OKELELE COMMUNITY, KWARA STATE.
SCHOOL’S NAME / IDENTITY---------------------------------------------------------
A. DEMOGRAPHIC DATA
1. Code no-------------------------------------------------------
2. Serial no---------------------------------------------------
3. Age---------------------------------------------------------
4. Sex a) Male b) Female
5. Class ------------------------------------------------------
6. No in class-------------------------------------------------
7. School’s population
B. SOCIO-ECONOMIC PROFILE
8. Mother’s/ Guardian’s Highest Educational level
a) Post tertiary b) Tertiary c) Secondary
d) Primary e) None
9. Father’s/ Guardian’s Highest Educational Level
a) Post tertiary b) Tertiary c)
Secondary
d) Primary e) None
10. Mother’s Occupation: -------------------------------------------
11. Father’s Occupation: --------------------------------------------
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12. Parent’s marital status
a) Married b) Single c) Divorced
d) Separated e) Widowed
13. Family type a) Monogamy b) Polygamy
14. How many siblings do you have?
a) 1-2 b) 3-4 c) ≥5
15. What type of apartment do you live in?
a) Rented b) Private
16. Do you share the building with other families? YES /NO
17. How many rooms are there in the house? ---------------------------
18. How many people sleep per room? ------------------------------------
19. Father’s/ Mother’s average income/ month---------------------------
20. What is the family’s domestic source of water supply?
a) Well b) Spring c) Tap
d) Bore hole e) others (specify)
C. CLINICAL PROFILE
21. Type of scalp lesion
a) Itching scalp YES/NO
b) Bald areas due to hair loss YES/NO
c) Pus filled sores YES/NO
d) Small black dots YES/NO
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22. How long have you had this scalp lesion?
a) <3MTHS b) 3<6MTHS
c) 6<9MTHS d) >9MTH
23. Any prior history of scalp injury before this lesion? YES/NO
24. How frequently do you take your bath?
a) Everyday b) Every other day
c) Twice a week d) Once a week
25. What type of soap do you use for bathing?
a) Toilet soap b) Medicated soap c) Washing soap
26. Do you sweat excessively? YES/NO
27. Where do you barb your hair?
a) Saloon b) Local barber c) Home (Parents)
28. What type of barbing instrument do you use?
a) Clippers b) Knife c) Blade d) Scissors
29. Is there history of similar scalp lesion in any other member of the
family?
a) Father b) Mother
c) Brother d) Sister
30. Do you share personal care items at home, other than clippers?
a) Combs YES/NO b) Towels YES /NO
c) Hats YES/NO d) Beddings YES/ NO
e) Others (specify)
31. (i) Do you keep pets at home? YES/NO
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(ii) If yes, which ones
a) Cat b) Dog c) Others (specify)
32. (i) Are you on any treatment for this scalp lesion? YES/NO
(ii) If yes, what is the nature of the treatment?
a) Topical (cream/ointment applied) b)Oral(Tablets) c) Injections
(iii) If yes, source
a) Traditional b) Orthodox
(iv) For how long have you been on the treatment?
a) < 2weeks b) > 2weeks
(v) Has there been healing with recurrence? YES/NO
D. PHYSICAL EXAMINATION
Weight :----------------------------------( kg)
Height :-----------------------------------( m)
Temperature: ---------------------------( 0C )
Lymphadenopathy
Occipital------------------ Cervical---------------
Weight for age---------------------%
Height for age----------------------%
Weight for height------------------%
BMI=Weight ------------------kg/m2
(Height) 2
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E. LABORATORY REPORT
Fungal isolate
Susceptibility pattern:- Griseofulvin (25µg) -------------------
Terbinafine (30µg) -------------------
Itraconazole (8µg) -------------------
Fluconazole (25µg) --------------------
Ketoconazole (10µg) -------------------
Miconazole (10µg) -------------------
Clotrimazole (10µg) -------------------
KEY:- S-Sensitive; R-Resistant; I-Intermediate Susceptibility.
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APPENDIX II
INFORMATION SHEET
INTRODUCTION
My name is Dr. Ekundayo, Halimat Ayodele of the Department of Medical
Microbiology and Parasitology, University of Ilorin Teaching Hospital,
Ilorin. My work is designed to study tinea capitis among school children in
Okelele community, Kwara state, Nigeria. Before you decide whether or
not to partake in the study, what is expected of you, the risks and the
benefits will be explained to you.
PURPOSE OF THE STUDY
This study will determine the prevalence of tinea capitis, the predisposing
factors and the types of fungal agents causing tinea capitis, as well as the
antifungal susceptibility patterns of the fungal isolates of tinea capitis
among children attending primary school in Okelele community, Ilorin,
Kwara State.
STUDY PROCEDURES
In the course of this study, a study proforma will be used to obtain
information from each child recruited for the exercise, in the language best
understood by the respondent. Each child will also be examined in detail
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including weight and height measurements. Hair strands and scalp
scrapings from lesions will be collected into a brown paper envelope using
a pair of small scissors and the side of a glass microscope slide
respectively. In the case of exuding scalp lesions a sterile cotton swab
stick will be used to collect specimen. All specimens will be taken for
laboratory investigation.
WHAT IS EXPECTED OF YOU IF YOU AGREE TO PARTICIPATE
You will be expected to append your signature or thumbprint on the
consent form to agree to your child’s or ward’s participation in the study
and your child’s assent to participate will also be obtained.
YOUR PARTICIPATION IS VOLUNTARY
Your child’s participation is voluntary and you may withdraw him or her at
any time from the study without any repercussion.
BENEFIT OF THE STUDY TO PARTICIPANTS
The participating children will benefit by knowing whether their scalp
lesions are due to fungal infection or not. Health Education will also be
given on tinea capitis to pupils of the various schools in the community,
and those found to have tinea capitis following the result of laboratory test
will be given free treatment.
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RISKS AND/OR DISCOMFORTS
I do not see any form of risks or discomforts that these study procedures
may cause your child/ward. If however, there are potential risks that you
envisage that may make it important for younot to allow your child or ward
partake in the study, please share with the researcher. I will be happy to
ensure your safety in all possible ways.
FINANCIAL INVOLVEMENT
There is no financial cost for participating in this study.
CONFIDENTIALITY
The information obtained will be treated in absolute confidence. No part or
whole of such information shall be divulged to anyone except the
investigator. I owe it a duty to keep your child’s records absolutely secret.
SPONSORSHIP
The study will be sponsored in its entirety by the researcher (self-
sponsorship)
CONFLICT OF INTEREST
In the course of the study, the researcher will attempt to avoid all actions
that may be considered a conflict of interest.
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PROBLEMS OR QUESTIONS
If you ever have any questions about this study, or if you have a research-
related harm, you should contact the researcher (name and address as
given above). My phone number is 08038295976 and my e-mail address
I will greatly appreciate your permission to allow your child or ward
participates in the study.
Thank you.
128
APPENDIX III
INFORMED CONSENT
(To be explained to the enrollee in the language they understand best)
Mr/Mrs/Chief/Alhaji/Alhaja/Dr.:----------------------------------------------
Whose address is: -------------------------------------------------------------
Hereby give consent for my child/ ward to participate in the research titled:
Tinea capitis infection among school children in Okelele community,
Kwara state.
The research has been explained to me and I am aware that the test to be
carried out will not harm my child/ward and that the information on my
child/ward will be kept in strict confidence.
I am also aware that I have the right to withdraw my child/ward at any
point if I so wish.
All other terms of this consent have been explained to me in a language
that I understand.
Sign/thumbprint------------------------------- Sign---------------------
Child’s Parent/Guardian Date---------------------
Date-------------------------------- Interviewer
Time------------------------------- Witness
------------------------------------
Sign Date
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APPENDIX IV
Table of Anthropometric Indicators for Nutritional Status73
ANTHROPOMETRIC
INDICATOR
NORMAL MILD TO MODERATE SEVERE
CHILDREN
a. Weight for age > or = 80% 60 – 80 %
( -3 to -2z )
< 60 %
( < 3z )
b. Height for age > or = 90% 80 – 90 % < 80 %
c. weight for height > or = 80 % 70 – 79 %
( -3 to – 2z)
< 70 %
( < 3z )
ADOLESCENTS
BMI
> or =17 16 – 17 < 16
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APPENDIX V
Socio-Economic Classification Scheme by Oyedeji
CLASS OCCUPATIONAL STATUS EDUCATIONAL STATUS
I
Senior public servant,
Professionals, Managers,
Large scale traders, Business
men and Contractors
University graduates or its
equivalent
II Intermediate grade public
servants and Senior school
teachers
School certificate holders,
O/L GCE who also had
teaching or other professional
training
III Junior school teachers, drivers,
artisans
School certificate or grade II
certificate holders or its
equivalent
IV Petty traders, labourers,
messengers
Modern three and primary six
certificate holders
V Unemployed, full-time house
wives, students and
subsistence farmers
Those who could just read
and write or are illiterates
131
APPENDIX VI
MATERIALS AND REAGENTS
MICROSCOPE SLIDES
COVER SLIPS
20% KOH
COMPOUND LIGHT MICROSCOPE
BUNSEN BURNER
MYCOSEL AGAR PLATES
SABOURAUD DEXTROSE AGAR
FORCEPS
INCUBATOR
PRE-PUBERTAL HAIR
STERILE DISTILLED WATER
YEAST EXTRACT
UREA AGAR SLOPE
STRAIGHT WIRE
TEASE NEEDLE
DERMATOPHYTE TEST MEDIUM
0.5 MCFARLAND’S TURBIDITY STANDARD
SPECTROPHOTOMETER
STERILE SWAB STICKS
132
APPENDIX VII
CLSI Criteria for Susceptibility and Resistance of Antifungal Discs85
Antifungal drugs/ Potency Zone diameter in mm
Sensitive Intermediate Resistance
Clotrimazole 10 μg ≥20 12-19 ≤11
Fluconazole 25 μg ≥22 15-21 ≤14
Griseofulvin 25 μg ≥10 - No zone
Ketoconazole 15 μg ≥30 23-29 ≤22
Miconazole 10 μg ≥20 12-19 ≤11
Terbinafine 30 μg ≥20 12-19 ≤11
Itraconazole 8µg >23 14-22 ≤13
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