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THE ISLAMIC UNIVERSITY - GAZA
Biological Sciences Master Program
Improving the Diagnosis of Dermatophytes in Gaza
Strip by using Nested PCR
Submitted in Partial Fulfillment for the Degree of Master of Science in
Biological Sciences / Microbiology
By
Eyad Khalil Ayesh
Supervisor
Dr. Tarek Elbashiti
Assoc. Prof. of Biotechnology
Jun, 2013
II
Declaration
I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by another
person nor material which to a substantial extent has been accepted for the award of any
other degree of the university of other institute, except where due acknowledgment has
been made in the text .
Signature Name Date Eyad Eyad Khalil Ayesh
Copy right.
All Right Reserved : No part of this work can be copied , translated or stored in a
retrieval system , without prior permission of the author.
III
Dedication
To my beloved parents and family
to my wife,
to my sons and daughters
IV
Acknowledgements
This work has been carried out at Remal Clinic laboratories in the Ministry of
Health of Gaza, Palestine and Gene Medical Laboratories.
I would like to express my sincere thanks to all the people who directly or indirectly
have contributed to this work. In particular, I would like to thank Dr. Tarek Elbashiti
my great supervisor.
I would like to extend my thanks to all the staff at the Department of Microbiology, in
Remal clinic and staff at Gene Medical Laboratories.
I would also like to thank my friends and colleagues, for all of their support and
guidance and encouraging; good luck to all.
Finally, I want to say that my beloved family especially my brothers and sisters, my
wife, they always stand beside me and give me encouragement all time and for their
never-ending love and support. I am so proud of my family.
Thank you all.
V
Improving the Diagnosis of Dermatophytes in Gaza Strip by using Nested PCR
Abstract
Dermatophytes are a very related to keratinophilic fungi that can invade
keratinized humans and animal tissues such as skin, hair and nails causing
dermatophytosis. They are the important cause of superficial fungal infection.
Conventional methods like potassium hydroxide (KOH) microscopy and fungal culture
lacks the ability to make an early and specific diagnosis. In this study it is taken into
consideration to evaluate nested polymerase chain reaction (NPCR) using primers
targeting dermatophyte specific sequence of chitin synthase 1 (CHS1) gene and
compared with conventional method potassium hydroxide (KOH) microscopy test in
Remal Clinic in Gaza city.
A total of ninety nine patients were clinically suspected with dermatophytosis including
16 skin specimens 16 nail specimens and 67 hair specimens. For each specimens KOH,
PCR and NPCR tests were carried out.
Having compared the output results of NPCR sequencing with the wild-type gene which
is obtained from the National Center for Biotechnology Information (NCBI) gene bank.
The comparison indicates that the product of NPCR is CHS1 gene according to (NCBI)
gene bank. Additionally, it is considered to compare the results of NPCR with KOH for
dermatophytes which gives that 41.4% are positive indication based on KOH and
18.18% is positive indication according to NPCR.
After carrying out the statistical analysis using SPSS for both tests results obtained from
NPCR and KOH, it is found that 30% of the total sample has to be included for
treatment based on KOH test, although this percent of the sample doesn‟t need to
undergo treatment according to NPCR test. It is also shown that 6% of the sample are
excluded for treatment in KOH test, in spite the NPCR indicated that this percent must
be included in the treatment.
The prominent controversy between the test results (KOH and NPCR) was found
particularly in the nails diagnosis.
Key words: Dermatophytes, KOH method, PCR, Nested PCR
VI
Arabic Abstract
في قطاع غزج NPCR انرضاػف انركزر ذحسي ػهيح فحص انفطزياخ انجهذيح تاسرخذاو ذقيح
انهخص
انكيشاحييت في اإلسا انحيا انفطشياث اندهذيت ي فطشياث يشحبطت بادة انكيشاحي حيث إا حاخى األسدت
انفحصاث انخقهيذيت انخاصت بانفطشياث اندهذيت . حيث أ األظافش حسبب انعذ انفطشيت –انشعش –يثم اندهذ
ت بعضا يفخقش إن انذقت األخش يحخاج إن فخشة طيهت نهخشخيص .( انزاسع انفطشيKOHيثم فحص )
حج يقاست CHS1باسخخذاو حسهسم يعي ندي NPCR انخضاعف انخكشس ز انذساست حى حقييى فحص ففي
عيت نا احخانيت عذ فطشيت 99انسخخذيت في عيادة انشيال )غزة ( حيث حى فحص KOHخائح فحص
.عيت ي انشعش 67-عيت ي انبششة 66-عيت أظافش 66يقست كانخاني
انقاست NCBIانشكز انطي نعهياث انخقيت انحييت يع NPCRقذ حج يقاست خائح انخسهسم نفحص
كاج سبت KOHيع خائح NPCRباإلضافت إن رنك حى يقاست خائح ، CHS1 اندي أكذث أ اناحح
.% 68.68ي NPCR% في فحص 46.4ي KOHفي فحص تانعياث انخب
% ي يدع انعياث دخهج في دائشة انعالج باء عه فحص 03ز انخائح بعذ انخحهيم اإلحصائي حشيش إن أ
KOHعه فحص باء نهعالجى ي أ ز انسبت نيسج في حاخت عه انشغNPCR ، حى 6خذ أ يا سبخ %
ظش NPCRعه فحص باءعه انشغى ي حاخخا ان انعالج KOHإخشاخا ي دائشة انعالج في فحص
. أكثش االخخالف في انخائح في عهيت حشخيص األظافش
VII
Table of contents
Item Page
Biological Sciences Master Program.............................................................................. I
Declaration ..................................................................................................................... II
Dedication ...................................................................................................................... III
Acknowledgements ....................................................................................................... IV
Abstract ........................................................................................................................... V
Arabic Abstract ............................................................................................................ VI
Table of contents ......................................................................................................... VII
List of Figures ................................................................................................................. X
List of Tables ................................................................................................................. XI
Abbreviations .............................................................................................................. XII
Chapter One .................................................................................................................... 1
Introduction .................................................................................................................... 1
1.1 Preface .................................................................................................................... 2
1.2 Dermatophytes Species........................................................................................... 2
1.2.1 Epidermophyton Spp. ...................................................................................... 2
1.2.2 Microsporum Spp. ........................................................................................... 3
1.2. 3 Trichophyton Spp. .......................................................................................... 3
1.3 Keratin and keratinolytic ........................................................................................ 3
1.4 The Role of the Immune System ............................................................................ 4
1.5 Pathogenicity .......................................................................................................... 4
1.6 Epidemiology ......................................................................................................... 4
1.7 Dermatophytes in the Worldwide ........................................................................... 5
1.7.1 Mediterranean Countries ................................................................................. 5
1.7.2 West Bank ....................................................................................................... 5
1.7.3 Arab Land Occupied in 1948.......................................................................... 5
1.7.4 Egypt ................................................................................................................ 5
1.7.5 Gaza Strip ........................................................................................................ 6
1.8Aim of the Study . ................................................................................................... 6
1.8.1 General Objective ............................................................................................ 6
1.8.2 Specific Objectives .......................................................................................... 6
1.8.3 Significance: ................................................................................................... 6
Chapter Two ................................................................................................................... 7
VIII
Literature Review ........................................................................................................... 7
2.1 Dermatophytoses .................................................................................................... 8
2.2 Etiology .................................................................................................................. 9
2.3 Transmission ........................................................................................................... 9
2.4 Disinfection ............................................................................................................ 9
2.5 Infections in Humans ............................................................................................ 10
2.6 Prevalence ............................................................................................................. 10
2.7 Diagnostic Tests ................................................................................................... 11
2.8 Identification of Dermatophyte. ........................................................................ 11
2.8.1 Conventional Method .................................................................................... 11
2.8.2 Conventional PCR ........................................................................................ 11
2.8.3 Nested PCR ................................................................................................... 12
2.9 The Role of the Immune System .......................................................................... 12
2.11 Teleomorphs ....................................................................................................... 14
2.12 Preview of previous studies : ............................................................................. 15
Chapter Three ............................................................................................................... 27
Materials and Methods ................................................................................................ 27
3.1 Materials ............................................................................................................... 28
3.1.1 Instrument ...................................................................................................... 28
3.1.2 PCR primers .................................................................................................. 28
3.1.3 Chemical ........................................................................................................ 28
3.2 Methods ................................................................................................................ 29
3.2.1 Study Area ..................................................................................................... 29
3.2.2 Samples .......................................................................................................... 29
3.2.3 Specimens Collection .................................................................................... 29
3.2.4 Specimens Division. ...................................................................................... 29
3.2.5 Specimens Identification ............................................................................... 29
3.2.6 Questionnaire ................................................................................................. 36
3.2.7 Data Analysis ................................................................................................. 36
Chapter Four ................................................................................................................ 37
Results ............................................................................................................................ 37
4.1 Potassium hydroxide (KOH) microscopy. ........................................................... 38
4.2 Molecular Diagnosis ............................................................................................. 39
4.3 Gene Sequencing .................................................................................................. 39
4.4 Statistical analysis ................................................................................................ 41
4.4.1 Study population ............................................................................................ 41
IX
4.4.2 Relative absolute error .................................................................................. 45
4.4.3 Lab diagnosis exclusion and inclusion errors ................................................ 46
Chapter Five .................................................................................................................. 47
Discussion ...................................................................................................................... 47
Chapter six .................................................................................................................... 52
Conclusions & Recommendations ............................................................................... 52
Conclusions ................................................................................................................ 53
Recommendations ...................................................................................................... 53
References .................................................................................................................. 54
Appendix .................................................................................................................... 62
X
List of Figures
Figure Page
Figure 4.1 Microscopic appearance of positive sample (spores) from hair in KOH. ... 40
Figure 4. 2 Results of First and Nested PCR .................................................................. 41
Figure 4.3 The wild-type gene obtained from the NCBI gene bank accession number
GI: AB 003558) .............................................................................................................. 42
Figure 4.4 The DNA sequencing result of CHS1 out put .............................................. 43
Figure 4.5 Sample classification of gender .................................................................... 44
Figure 4.6 classification of sample type ......................................................................... 44
Figure 4.7 The domestic animal kinds in houses ........................................................... 45
Figure 4.8 Age group distribution. ................................................................................. 45
Figure 4.9 The results of KOH classification ................................................................. 44
Figure 4. 63 result of FPCR classification ...................................................................... 46
Figure 4. 66 The result of NPCR classification .............................................................. 44
Figure 4.62 The results of KOH,FPCR,NPCR classification ........................................ 47
XI
List of Tables
Table Page
Table 2.1: Anamorph genera and species of dermatophytes...........................................15
Table 2.2: Teleomorph-Anamorph State of Dermatophytes ........................................... 16
Table 3.1: list of equipment used in this study. .............................................................. 28
Table 3.2: list of PCR primers used in this study. ......................................................... 28
Table 3.3: list of chemical used in this study. ............................................................... 28
Table 3.4: for first PCR reaction mixture ...................................................................... 32
Table 3.5: Temperature cycling program for FPCR ....................................................... 33
Table 3.6: Nested PCR Master Mix For 25 μ l reactions, the amounts given are per
reaction ........................................................................................................................... 33
Table 3.7: Temperature cycling program for NPCR ...................................................... 33
Table 4.1: sample type and lab methods KOH & NPCR . ............................................. 45
Table 4.2: holding animals in the house relative KOH & NPCR. .................................. 45
Table 4.3: lab diagnosis exclusion and inclusion errors. ................................................ 46
XII
Abbreviations
KOH Potassium hydroxide microscopy
PCR polymerase chain reaction NPCR Nestedpolymerase chain reaction
CHS1 Chitin Synthase 1 gene
ITS polymorphisms within the fungal internal transcribed spacer
µl Micro liter
DNA Deoxyribonucleic acid
nt Nucleotides
dNTPs Deoxynucleotide triphosphates
bp Base pair
g Gram
GEL Gelatin liquifaction
FPCR First polymerase chain reaction
NCBI National Center for Biotechnology Information
EDTA Ethylene diamine tetra acetic acid
6
Chapter One
Introduction
2
Chapter 1
INTRODUCTION
1.1 Preface
The dermatophytes are a group of closely related fungi that have the capacity
to invade keratinized tissue (skin, hair, and nails) of humans and other animals to
produce an infection, dermatophytosis, commonly referred to as ringworm. Infection
is generally cutaneous and restricted to the nonliving cornified layers because of the
inability of the fungi to penetrate the deeper tissues or organs of immunocompetent
hosts. Reactions to a dermatophyte infection may range from mild to severe as a
consequence of the host‟s reactions to the metabolic products of the fungus, the
virulence of the infecting strain or species, the anatomic location of the infection, and
local environmental factors (Shinkafi and Manga,2011; Barry and Hainer, 2003; and
Weitzman and Summerbell, 1995).
1.2 Dermatophytes Species
The etiologic agents of the dermatophytoses are classified in three anamorphic
(asexual or imperfect) genera, Epidermophyton, Microsporum and Trichophyton, of
anamorphic class Hyphomycetes of the Deuteromycota (Fungi Imperfecti) (Weitzman
and Summerbell, 1995).
The most common system to classify dermatophytes as follows (Achterman and
White, 2011).
•Geophilic dermatophytes are found mainly in soil, where they are associated with
decomposing hair, feathers, hooves and other keratin sources. They infect both
humans and animals ( Epidermophyton).
•Zoophilic dermatophytes are mainly found in animals but can be transmitted to
humans ( Microsporum) .
•Anthropophilic dermatophytes are mainly found in humans and are very seldom
transmitted to animals ( Trichophyton)
1.2.1 Epidermophyton Spp. The type species is Epidermophyton floccosum. The macroconidia are broadly
clavate with typically smooth, thin to moderately thick walls and one to nine septa,
range in size 20 to 60 by 4 to 13 mm in size. They are usually abundant and borne
0
singly or in clusters. Microconidia are absent. This genus has only two known species
to date, and only E. floccosum is pathogenic (Taleb, 2010 and Borelli, 1965).
1.2.2 Microsporum Spp. The type species is Microsporum audouinii. Macroconidia are characterized
by the presence of rough walls which may be asperulate, echinulate, or verrucose.
Originally, the macroconidia were described by Emmons as spindle shaped or
fusiform, but the discovery of new species extended the range from obovate (egg
shaped) as in Microsporum nanum to cylindrofusiform as in Microsporum
vanbreuseghemii.
The macroconidia may have thin, moderately thin to thick walls and 1 to 15 septa and
range in size from 6 to 160 by 6 to 25 mm. Microconidia are sessile or stalked and
clavate and usually arranged singly along the hyphae or in racemes as in
Microsporum racemosum, a rare pathogen (Taleb, 2010 and Borelli, 1965) .
1.2. 3 Trichophyton Spp. The type species is Trichophyton tonsurans. Macroconidia, when present,
have smooth, usually thin walls and one to 12 septa, are borne singly or in clusters,
and may be elongate and pencil shaped, clavate, fusiform, or cylindrical. They range
in size from 8 to 86 by 4 to 14 mm. Microconidia, usually more abundant than
macroconidia, may be globose, pyriform or clavate, or sessile or stalked, and are
borne singly along the sides of the hyphae or in grape-like clusters (Taleb, 2010 and
Borelli, 1965).
1.3 Keratin and keratinolytic
Keratin is a major component of hair, feathers and wool and is the most
complex of the cytoskeletal intermediate filament proteins of epithelial. The
durability of keratins is a direct consequence of their complex architecture. In
addition to keratin, keratinaceous materials such as skin, hair, nails, hoofs and horns
contain a large proportion of non-keratin proteins. A large number of fungi,
including yeasts, dermatophytes and other moulds, grow on human skin, hair and
nails. The term „keratinolytic‟ is used for fungi exhibiting the enzymatic ability
to attack and utilize keratin. Degradation of keratin by microorganisms is
performed by specific proteases that is, keratinases (Sharma et al., 2011).
4
1.4 The Role of the Immune System
Fungal virulence is the result of interplay between the infecting organism and
the host. During dermatophyte infection, cell-mediated immunity is widely
considered to be responsible for modulating dermatophyte disease and fungal antigens
activate T-suppressor and T-helper cells. Diverences specific to the host are thought to
be important in determining the relative susceptibility of individuals, with factors such
as age, gender, and genetics all like lytoplayarole )Achterman and White, 2012(.
1.5 Pathogenicity
The dermatophyte species within the three genera Epidermophyton,
Microsporum and Trichophyton differ in their pathogenicity in vivo. While all species
invade the stratum corneum of the epidermis and the follicular ostium of hairs,
different species vary widely in their capacity to invade hair and nail. The reasons for
this observed tissue specificity are unknown, but are thought to be related to specific
nutritional requirements or the enzyme production of individual organisms. Role of
proteolytic enzymes in pathogenicity Self synthesised enzymes serve fungi in a
number of ways. They enhance survival in tissues by chemically or physically altering
the immediate environment and they act directly by digesting host proteins, thus
providing a source of nutrition. Therefore the pathogenic potential of a fungal agent
depends on its ability to produce enzymes. In turn variations in enzymatic potential of
a fungus may be responsible for differences in the pathogenic effects of various
strains (Simpanya, 2000).
1.6 Epidemiology
Dermatophytes are among the few fungi causing communicable disease, that
is, diseases acquired from infected animals or birds or from the fomites they have
engendered. All but one of the species known to cause disease primarily affect
mammals, the exception is Microsporum gallinae, is primarily established in
gallinaceous fowl. Apart from those species usually associated with disease,
transitional species exist which appear to be primarily saprobic organisms
occasionally or rarely causing infection. Finally, some Trichophyton,
Epidermophyton, and Microsporum species closely related to the dermatophytes
appear to be exclusively saprobic or nearly so. The members of these three genera
5
have no collective designation. The term dermatophytes should be restricted to
designate infectious organisms and will be referred to below as dermatophytes and
their congeners. Closely biologically related organisms not included in this group
include Chrysosporium species with teleomorphs in the genus Arthroderma (Ajello,
1974).
1.7 Dermatophytes in the Worldwide
Although dermatophytes can be isolated worldwide, many species are only
encountered in geographically restricted areas of the more than 40 species of
dermatophytes previously identified only about 12 are common causes of human
infection (Elewski, 2000).
1.7.1 Mediterranean Countries T. violaceum and M. canis were also reported to be the predominant scalp ringworm
pathogens in many countries of the Mediterranean, including Suadia Arabia
(Venugopal and Venugopal, 1993), Kuwait (Al-Fouzan et al., 1992) and Iran
(Knosravi et al ., 1994).
1.7.2 West Bank The test seventy-five children cases of tinea capitis (1%) were mycologically proven.
The incidence was higher in young children. T. violaceum was the most common
causative agent 82.7% followed by M . canis (16%) and T. schoenleinii (1.3%)
(Ali-Shtayeh et al., 1997).
1.7.3 Arab Land Occupied in 1948 M. canis was first reported in 1975, since then this dermatophyte has spread
throughout the country becoming an important cause of scalp ringworm (Alteras et
al., 1986).
1.7.4 Egypt The most frequently isolated dermatophyte species was T. violaceum which accounted
for most (71.1%) of all the recovered dermatophyte, followed by M. canis (21.09%)
T. rubrum (6.2%) and M. boullardii (0.49) both E. floccosum and T. tonsurans were
only rarely isolated (0.24%) (Zaki et al., 2008).
6
1.7.5 Gaza Strip As mentioned in the only related study, the most common dermatophyte caused tinea
capitis in north Gaza area was M. canis (92.5%) and (7.5%) was T. mentagrophytes
(Taleb, 2010).
1.8Aim of the Study .
1.8.1 General Objective
The aim of this study is to improving the diagnosis of dermatophytes in Gaza Strip by
using Nested PCR.
1.8.2 Specific Objectives The specific objectives of this research could be summarized in the following points,
includes:
1. To carrying out the traditional diagnosis by direct microscopy by using KOH.
2. To evaluating a nested PCR targeting specific gene for dermatophytes.
3. To comparing between the results of the traditional diagnosis and the new method
by nested PCR.
4. Faster following of residual disease during drug treatment.
1.8.3 Significance: In Gaza Strip labs, the laboratory diagnosis of dermatophytosis routinely involves
direct microscopic examination of clinical specimen and some times followed by in
vitro culture techniques. Microscopic identification of fungal elements directly from
clinical specimen is a rapid diagnostic method but it lacks specificity and sensitivity,
with false negative results. In vitro culture is a specific diagnostic test but it is slow
technique, and may take up to 8 weeks to give the results. The advent of molecular
technology has enabled the development of techniques like polymerase chain
reaction, which is a highly sensitive and specific test and can be used for diagnosis of
various microorganisms including fungal pathogens. In this study, it is considered to
improve and evaluated a nested PCR to obtain rapid and good identification of the
dermophytes fungi to help the doctors to post theraputic strategies. The treatment of
dermatophytoses would be most appropriate when the selection of antimicrobial agent
is based on the identity of the causative agent. The results of this study may shed light
on this tragic condition that will be of interest to improve the health conditions of
people living in the Gaza Strip.
7
Chapter Two
Literature Review
8
Chapter 2
Literature Review
2.1 Dermatophytoses
Because dermatophytes require keratin for growth, they are restricted to hair, nails,
and superficial skin. Thus, these fungi do not infect mucosal surfaces.
Dermatophytoses are referred to as “tinea” infections. They are also named for the
body site involved. Some dermatophytes are spread directly from one person to
another (anthropophilic organisms). Others live in and are transmitted to humans from
soil (geophilic organisms), and still others spread to humans from animal hosts
(zoophilic organisms). Transmission of dermatophytes also can occur indirectly from
fomites (e.g., upholstery, hairbrushes, hats). Anthropophilic organisms are responsible
for most fungal skin infections. Transmission can occur by direct contact or from
exposure to desquamated cells. Direct inoculation through breaks in the skin occurs
more often in persons with depressed cell-mediated immunity. Once fungi enter the
skin, they germinate and invade the superficial skin layers. In patients with
dermatophytoses, physical examination may reveal a characteristic pattern of
inflammation, termed an “active” border. The inflammatory response is usually
characterized by a greater degree of redness and scaling at the edge of the lesion or,
occasionally, blister formation. Central clearing of the lesion may be present and dis-
tinguishes dermatophytoses from other papulosquamous eruptions such as psoriasis or
lichen planus, in which the inflammatory response tends to be uniform over the
lesion. The location of the lesions also can help identify the pathogen. A
dermatophytosis can most likely be ruled out if a patient has mucosal involvement
with an adjacent red, scaly skin rash. In this situation, the more probable diagnosis is a
candidal infection such as perlèche (if single or multiple fissures are present in the
corners of the mouth) or vulvovaginitis or balanitis (if lesions are present in the geni-
tal mucosa). Potassium hydroxide (KOH) microscopy aids in visualizing hyphae and
confirming the diagnosis of dermatophyte infection. Other diagnostic modalities
include Wood‟s lamp examination, fungal culture, and skin or nail biopsy (Barry and
Hainer, 2003).
9
2.2 Etiology
Dermatophytosis is caused by fungi in the genera Microsporum, Trichophyton and
Epidermophyton. These organisms, called dermatophytes, are the pathogenic
members of the keratinophilic (keratin digesting) soil fungi. Microsporum and
Trichophyton are human and animal pathogens. Epidermophyton is a human
pathogen. The dermatophytes were all formerly classifed as members of the phylum
Deuteromycota (Fungi imperfecti). Some are now known to reproduce sexually and
have been reclassifed in the phylum Ascomycota, family Arthrodermataceae. Each of
these fungi now has two species names, one for the stage found in vertebrate hosts,
and one for the form that grows in the environment (the perfect state). Formerly, the
perfect states of Microsporum species were placed in the genus Nannizia and the
perfect states of Trichophyton in the genus Arthroderma. Currently, the perfect states
of both Microsporum and Trichophyton belong to the genus Arthroderma. (Weitzman
and Summerbell, 1995 and ALY,1994).
2.3 Transmission
Infection occurs by contact with arthrospores (asexual spores formed in the hyphae
of the parasitic stage) or conidia (sexual or asexual spores formed in the “free living”
environmental stage). Infection usually begins in a growing hair or the stratum
corneum of the skin. Dermatophytes do not generally invade resting hairs, since the
essential nutrients they need for growth are absent or limited. Hyphae spread in the
hairs and keratinized skin, eventually developing infectious arthrospores.
Transmission between hosts usually occurs by direct contact with a symptomatic or
asymptomatic host, or direct or airborne contact with its hairs or skin scales. Infective
spores in hair and dermal scales can remain viable for several months to years in the
environment. Fomites such as brushes and clippers can be important in transmission.
Geophilic dermatophytes, such as M. nanum and M. gypseum, are usually acquired
directly from the soil rather than from another host (Georg, 1960).
2.4 Disinfection
Dermatophyte spores are susceptible to common disinfectants such as benzalkonium
chloride, dilute (1:10) chlorine bleach, or strong detergents. Chlorhexidine is no
longer considered to be a good environmental decontaminant for these fungi. The
mechanical removal of any material containing keratin, such as shed skin and hairs,
63
facilitates disinfection. Vacuuming is considered to be the best method in many cases
(cfsph.web)
2.5 Infections in Humans
The incubation period in humans is 1 to 2 weeks.
Clinical Signs
Dermatophytes generally grow only in keratinized tissues such as hair, nails and the
outer layer of skin; the fungus usually stops spreading where it contacts living cells
or areas of inflammation. Mucus membranes are not affected. The clinical signs may
vary, depending on the region affected. In humans, pruritus is the most common
symptom. The skin lesions are usually characterized by infammation that is most
severe at the edges, with erythema, scaling and occasionally blister formation.
Central clearing is sometimes seen, particularly in tinea corporis; this results in the
formation of a classic “ringworm” lesion. On the scalp and facial hair, there may be
hair loss. Dermatophytes acquired from animals or the soil generally produce more
inflammatory lesions in humans than anthropophilic dermatophytes. In humans,
dermatophytoses are referred to as “tinea” infections, and are named with reference
to the area of the body involved. Infections can spread to other areas; tinea corporis
in children, for example, is often the result of a tinea capitis infection that has spread
to the face (cfsph.web).
2.6 Prevalence
Although dermatophyte infections are known to be common, their prevalence is
unknown as this disease is not not fable and many infections are treated with over-the-
counter drugs. In the United Kingdom, a survey found dermatophytosis to be the
most common zoonosis; its prevalence was 24%. Infections are more common in
children than adults. The geographic distribution of the various dermatophyte
species, as well as their animal hosts, influences the zoonosis found in humans. M.
canis, usually transmitted by cats and dogs, is more common in people living in
urban areas. T. verrucosum is more often found in rural environments. In
Switzerland, one study reported that 14% of those working with cattle had been
infected. Most dermatophyte infections are not serious in healthy persons; however,
opportunistic bacteria can cause cellulitis in skin damaged by inter digital fungal
infections. These infections are a particular concern in diabetics. Dermatophytosis is
more serious in those who are immunosuppressed. These individuals may have
66
atypical and locally aggressive dermatophyte infections, including extensive skin
disease, sub cutaneous abscesses, and disseminated disease (cfsph.web).
2.7 Diagnostic Tests
Some (but not all) strains of M. canis and M. equinum exhibit green fluorescence
when stimulated by certain wave- lengths of UV light. A Wood‟s lamp can be used to
examine the fur for these fungi. Certain topical preparations may mask the
fluorescence, and alcohol can either suppress the fluorescence or cause non-specific
fluorescence. Microscopic examination of skin scrapings or hairs in potassium
hydroxide (KOH) may reveal hyphae or conidia. A potassium hydroxide-calcofuor
white (CFW) mixture can also be used to visualize dermatophyte structures, using a
fluorescence microscope. Definitive diagnosis usually relies on culture. Skin
scrapings or plucked hair samples may be cultured, or the fur may also be brushed
with a disinfected toothbrush to collect hairs. Species found in dogs and cats will
grow in about 4 to 7 days at 25-28؛C, on a variety of commercial media.
Dermatophyte Test Medium (DTM) contains a pH indicator (phenol red) that will
turn the medium red when a dermatophyte is growing; however, bacteria and fungi
other than dermatophytes can also produce a pH change. Therefore, the growth must
be examined further to differentiate the organism. Dermatophytes are traditionally
identified using a “slide culture” to observe the reproductive structures (conidia) and
hyphae. Species can be identified by the colony structure and color, micro conidia,
macro conidia and other microscopic structures (Moriello k. 2004).
2.8 Identification of Dermatophyte.
2.8.1 Conventional Method The dermatophytosis caused by various dermatophyte species cannot be easily
differentiated on the basis of clinical manifestations methods.
For many years, conventional laboratory methods based on the detection of
phenotypic characteristics, such as microscopy and in-vitro culture, have played an
essential role in dermatophyte identification. However, these procedures generally
suffer from the drawbacks of being either slow or non-specific (Liu et al., 2000).
2.8.2 Conventional PCR Recent developments and applications of nucleic acid amplification technology have
provided the opportunity to enhance the quality and speed of dermatophyte diagnosis.
62
This method by use polymerase chain reaction (PCR) for diagnosis after use nested
PCR (Liu et al., 2002).
2.8.3 Nested PCR Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs
(instead of one pair) of PCR primers are used to amplify a fragment.
The first pair of PCR primers amplify a fragment similar to a standard PCR. However,
a second pair of primers called nested primers (as they lie / are nested within the first
fragment) bind inside the first PCR product fragment to allow amplification of a
second PCR product which is shorter than the first one.
The advantage of nested PCR is that if the wrong PCR fragment was amplified, the
probability is quite low that the region would be amplified a second time by the
second set of primers. Thus, Nested PCR is a very specific PCR amplification (PCR
Station. web).
2.9 The Role of the Immune System
Fungal virulence is the result of interplay between the infecting organism and the
host. During dermatophyte infection, cell-mediated immunity is widely considered to
be responsible for modulating dermatophyte disease and fungal antigens activate T-
suppressor and T-helper cells. Differences specific to the host are thought to be
important in determining the relative susceptibility of individuals, with factors such
As age, gender ,and genetic. The most numerous cells in the epidermis are
keratinocytes, indicating that dermatophytes must primarily interact with these cells.
Interestingly, keratinocytes seem to exhibit a differential response following exposure
to different dermatophyte species (Achterman and White, 2011).
60
2.10 Anamorphic
The anamorphic species of the dermatophytes are listed in Table 1 (Weitzman
and Summerbell, 1995).
Table.2.1 Anamorph genera and species of dermatophytes (Weitzman and Summerbell 1995).
Species Date of exploring Epidermophyton 1907
E. floccosum 1930
Microsporum 1843
M. audouinii 1843
M. canis 1902
M. equinum 1904
M. ferrugineum 1921
M. fulvum 1909
M. gallinae 1929
M. gypseum 1928
M. nanum 1956
M. persicolor 1928
M. praecox 1987
M. racemosum 1965
M. vanbreuseghemii, 1962
Trichophyton 1845
T. concentricum 1895
T. equinum 1902
T. gourvilii 1933
T. kanei 1989
T. megninii 1896
T. mentagrophytes 1896
T. raubitschekii, 1981
T. rubrum 1911
T. schoenleinii 1930
T. simii 1965
T. soudanense 1912
T. tonsurans 1845
T. verrucosum 1902
T. violaceum 1902
T. yaoundei 1957
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2.11 Teleomorphs
Some dermatophytes, mostly the zoophilic and geophilic species of
Microsporum and Trichophyton, are also capable of reproducing sexually and
producing ascomata with asci and ascospores. These species are classified in the
teleomorphic genus Arthroderma (Weitzman et al., 1986), family Arthrodermataceae
of the Onygenales (Currah, 1985), phylum Ascomycota. Previously, the teleomorphs
of the sexually reproducing Microsporum and Trichophyton species and related
keratinophilic fungi had been classified in the genera Nannizzia and Arthroderma,
respectively (Ajello, 1977). However, on the basis of a careful evaluation of the
morphological characteristics used to define these two genera (Weitzman et al., 1986),
concluded that the species making up these genera represented a continuum and that
their minor differences did not merit maintaining them in two separate genera.
Nannizzia and Arthroderma are considered to be congeneric, with Arthroderma
having taxonomic priority (Weitzman and Summerbell, 1995).
Table 2.2 Teleomorph-Anamorph State of Dermatophytes (Weitzman and Summerbell, 1995)
Teleomorph (reference) Anamorph
Arthroderma Microsporum, Trichophyton
A. benhamiae T. mentagrophytesa
A. fulvum M. fulvumb
A. grubyi M. vanbreuseghemii
A. gypseum M. gypseumb
A. incurvatum M. gypseumb
A. obtusum M. nanum
A. otae. M. canis var. canis, M. canis var distortum
A. persicolor M. persicolor
A. simii T. simii
A. racemosum M. racemosum
A. vanbreuseghemii T. mentagrophytesa
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2.12 Preview of previous studies :
Chandran et al. Study (2013)They aimed to evaluate a commercially available PCR
kit for the in vitro detection of dermatophytes and specifically Trichophyton rubrum
in nail specimens with suspected onychomycosis, and to compare the detection rates
of PCR with conventional diagnostic methods.Nail specimens were prospectively
collected from patients with clinically suspected onychomycosis. All nail specimens
were positive on direct microscopic examination. PCR and fungal cultures were
administered, and the detection rates of dermatophytes were compared. In all, 107
nail specimens were analysed. The fungal culture was positive in 57 (53%) specimens
(38 dermatophytes and 19 non-dermatophytes). PCR was positive in 77 (72%)
specimens (63 T. rubrum and 14 pan-dermatophyte). A total of 37 specimens (35%)
were positive for both fungal culture and PCR. PCR detected dermatophytes in 39
specimens that were missed by the fungal culture, increasing the diagnosis of
dermatophyte-positive specimens by 37%. Five dermatophyte-culture-positive
specimens were negative for PCR.The study demonstrates that PCR increases the
sensitivity of detection of dermatophytes in nail specimens. Despite its limitations, the
use of PCR can complement direct microscopic examination and fungal cultures to
aid clinicians in the diagnosis of suspected dermatophytic onychomycosis (Chandran
et al,. 2013)
A total of 218 patients presenting in a surgical practice over 3 months with clinical
signs of tinea pedis and/or onychomycosis were involved in the prospective study. All
patients had predisposing factors for tinea pedis and tinea unguium, such as vascular
insufficiency, diabetes mellitus, and leg ulcers. Nail specimens and skin scrapings
were investigated for fungi using Blancophor preparation, and cultured. In addition
to conventional diagnostics, PCR (polymerase chain reaction) for detection of
dermatophyte DNA was employed. This PCR-Elisa assay is based on the use of
specific primers which target the topoisomerase II gene. This allows the highly
specific molecular identification of Trichophyton (T.) rubrum, T. interdigitale, and
Epidermophyton floccosum directly in clinical samples.
23.9 % of patients were culture-positive for dermatophytes (either T. rubrum, or T.
interdigitale). With PCR, dermatophyte DNA either of T. rubrum or T. interdigitale
66
could be detected in nail samples and skin scrapings from at least 29.9 % of all
patients. Epidermophyton floccosum was not found in this study, neither by
cultivation nor by PCR. The diagnostic sensitivity of the PCR-Elisa assay was
calculated as 79.0% %; the diagnostic specificity as 85.5 %.
PCR-Elisa evaluation makes possible a rapid, specific and sensitive diagnosis of
dermatophytosis of the nails and skin within 24 (maximal 48) hours with
identification of the involved species(Winter et al.,2013)
PCR method based on the amplification of the chitin synthase 1 gene was developed.
The study included 119 strains of dermatophytes and non dermatophytic fungi, eight
dermatophytic reference strains and 201 nail specimens from patients with
dermatophytic onyxis.
PCR positivity was based on the production of a specific 432bp fragment. None of the
investigated non dermatophytic strains was positive. Sensitivity of PCR was higher as
compared to mycological examination (90.5% vs. 81.1%). PCR was positive in 31
onyxis cases with positive direct examination but negative or contaminated culture. In
contrast, PCR was negative in 10 cases where both direct examination and culture
were found positive.
PCR is an adequate tool for the diagnosis of dermatophytic onychomycosis. It is much
adapted to cases where culture is negative or contaminated by overgrowing molds,
which makes the identification of the causal agent problematic (Dhib et al., 2012)
Verrier et al .Study (2012)In this study, they describe a PCR-terminal restriction
fragment length polymorphism (TRFLP) assay to directly and routinely identify the
infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after
dissolving nail fragments in an Na 2 S solution. Trichophyton spp., as well as 12 non
dermatophytes could be unambiguously identified by the specific restriction fragment
size of 5-end-labeled amplified 28 S DNA. This assay enables the distinction of
different fungal infectious agents and their identification in mixed infections.
Infectious agents could be identified in 74% (162/219) of cases in which the culture
results were negative. The PCR-TRFLP assay described here is simple and reliable.
67
Furthermore, it has the possibility to be automated and thus routinely applied to the
rapid diagnosis of a large number of clinical specimens in dermatology laboratories.
Kim et al. study (2011) In this study, the possibility of using multiplex PCR was
investigated to speed up and specify the detection of aflatoxigenic Aspergillus species
in meju, a traditional Korean fermented soybean food starter. Two different sets of
three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes
present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed
that only aflatoxigenic Aspergillus species gave three band patterns in both primer
sets. The detection limits were determined as 125 pg/ml for genomic DNA from
aflatoxigenic A. parasiticus KCCM 35078, and 105 spores/g of meju sample for DNA
extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested
for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were
analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates.
Results showed a good correlation between the presence of the aflatoxin biosynthesis
genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This
suggests that this multiplex PCR method may provide an accurate and specific
detection of aflatoxigenic Aspergillus species in fermented soybean foods
Sharma et al.study (2011) In this study,the present investigation was aimed to
evaluate the in vitro biodegradation of keratin by clinical isolates of
dermatophytes and soil fungi. Ten fungal species, out of which, six
(Chrysosporium indicum, Trichophyton mentagrophytes, Scopulariopsis sp.,
Aspergillus terreus, Microsporum gypseum and Fusarium oxysporum) were
isolated from soil and four clinical (Trichophyton rubrum, Trichophyton
verrucosum, Trichophyton tonsurans and Microsporum fulvum) were obtained
from human skin. The isolates were tested for their keratin degradation ability on
human and animal (cow and buffalo) hair baits. The rate of keratin degradation was
expressed as weight loss over three weeks of incubation. Human hair had the highest
rate of keratin degradation (56.66%) by colonization of C. indicum. Whereas M.
gypseum and T. verrucosum were highly degraded (49.34%) to animal hairs. There
was a significant difference (p < 0.05) in keratin substrate degradation rates by the
examined fungi. Human hair served as an excellent source for the biodegradation of
keratin by the isolated test fungi as compared to animal hair. Releasing protein
showed maceration of the keratin substrates by the test fungi. The present study
68
reveals that, the isolated test fungi play a significant impact on biodegradation of
keratin substrates for betterment of environmental hazards.
De Baere et al. study (2010) In this study,a total of 95 isolates, belonging to 33
species of five dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium
(two), Epidermophyton (one), Microsporum (three) and Trichophyton (12), were
studied using internal transcribed spacer 2 (ITS2)-PCR-RFLP analysis (ITS2-RFLP),
consisting of amplification of the ITS2 region, restriction digestion with BstUI
(CG/CG) and restriction fragment length determination by capillary electrophoresis.
ITS2-RFLP analysis proved to be most useful for identification of species of the
genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish
between several Trichophyton species. The identification results are in agreement
with established and recent taxonomical insights into the dermatophytes; for example,
highly related species also had closely related and sometimes difficult-to-discriminate
ITS2-RFLP patterns. In some cases, several ITS2-RFLP groups could be
distinguished within species, again mostly in agreement with the taxonomic
delineations of subspecies and/or genomovars, confirming the relevance of ITS2-
RFLP analysis as an identification technique and as a useful taxonomic approach.
A new concept for multiplex detection and quantification of microbes is here
demonstrated on a range of infectious fungal species. Padlock probe methodology in
conjunction with qPCR and Luminex™ technology was used for simultaneous
detection of ten fungal species in one single experiment. By combining the
multiplexing properties of padlock probes and Luminex™ detection with the well
established quantitative characteristics of qPCR, quantitative microbe detection was
done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation
reaction forms circular DNA when hybridizing to specific target DNA. The region of
the padlock probe that does not participate in target DNA hybridization contains
generic primer sequences for amplification and a tag sequence for Luminex™
detection. This was the fundamental for well performing multiplexing. Circularized
padlock probes were initially amplified by rolling circle amplification (RCA),
followed by a SybrGreen™ real time PCR which allowed an additive quantitative
assessment of target DNA in the sample. Detection and quantification of amplified
padlock probes were then done on color coded Luminex™ microspheres carrying
69
anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent
reannealing of amplimers by covering the flanks of the address sequence, improved
the signal to noise ratio in the detection step considerably. The method correctly
detected fungi in a variety of clinical samples and offered quantitative information on
fungal nucleic acid (Eriksson et al.,2009)
Uchida et al. Study (2009)The present study was performed to assess the utility of
specific polymerase chain reaction (PCR)-based methods for Trichophyton rubrum
and Trichophyton mentagrophytes as diagnostic tools for dermatophytoses. Both
conventional morphological identification and specific PCR methods based on the
nuclear ribosomal internal transcribed spacer (ITS)1 DNA sequence were performed
to identify dermatophyte species from clinical specimens of patients who visited
Kawasaki Social Insurance Hospital between 16 May and 17 August 2005. Specific
PCR methods were also directly applied to clinical specimens, and the results of the
two methods were compared. The clinical samples examined consisted of 126 skin
scale specimens and 80 nail specimens. The positive rates of culture isolation from
clinical specimens were 67% and 33% for skin scale and nail specimens, respectively.
In contrast, PCR analysis yielded a positive rate of 100% for clinical isolates from
both skin scales and nails, and rates of 95% and 99% were obtained by direct
application to clinical specimens. The results of the present study indicated that
specific PCR is highly advantageous as a diagnostic tool for detection and
identification of dermatophytes on direct application to skin scale or nail specimens.
Garg et al. Study (2009)In their study they have evaluated nested PCR targeting the
Chitin Synthase 1 (CHS1) gene (DDBJ accession no.-AB003558) shared by three
genera, i.e., Trichophyton, Epidermophyton, and Microsporum,Of the 105 clinically
suspected cases of skin dermatophytosis, 63.8% (67/105) were positive for fungal
elements by KOH microscopy. Dermatophytes were detected in 82.8% (87/105) of
the specimens by nested PCR, 49.5% (52/105) by first round PCR and isolated by
culture in 23.8%(25/105) cases. Among the dermatophytes isolated on culture
Trichophyton rubrum was the commonest isolate (48%, 12/25), followed by T.
mentagrophyte (40%, 10/ 25), Trichophyton tonsurans (8%, 2/25), and Trichophyton
violaceum (4%, 1/25). Of 80 specimens negative for dermatophyte isolation by
fungal culture, 4 specimens were positive for nondermatophytic molds and 12
23
specimens for Candida albicans. 37 (46.2%) specimens were positive by first round
PCR and 59 (73.7%) by nested PCR. Of the 87 nested PCR positive specimens
candida albicans was cultured from 5 specimens, thus nested PCR detecting cases
with hidden mixed infections. Nested PCR was positive in 73.7% (28/38) of the KOH
microscopy-negative specimens. In addition, all 59 patients on antifungal therapy
were positive by nested PCR. Among 50 clinically suspected cases of hair
dermatophytosis, positivity by nested PCR was highest 86% (n = 43/ 50) followed by
KOH microscopy 58% (n = 29/50), first round PCR 52% (n = 26/50) and fungal
culture 30% (n = 15/50). Nested PCR was positive for 66.6% (n = 14/ 21) and 80% (n
= 28/35) of the KOH microscopy-negative and culture-negative specimens
respectively. Of twenty specimens negative both by KOH microscopy and fungal
culture, nested PCR was positive in 13 (65%) specimens. In addition, all 28 patients
on antifungal therapy were positive by nested PCR .
The DNA sequencing analyses have demonstrated relatively limited polymorphisms
within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp.
they sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes
belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense,
T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and
developed a novel padlock probe and rollingcircle amplification (RCA)-based method
for identification of single nucleotide polymorphisms (SNPs) that could be exploited
to differentiate between Trichophyton spp. Sequencing results demonstrated
intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense
but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-
bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified.
The RCA assay correctly identified five Trichophyton species. Although the use of
two “group-specific” probes targeting both the ITS1 and the ITS2 regions were
required to identify T. soudanense, the other species were identified by single ITS1-
or ITS2-targeted species-specific probes. There was good agreement between ITS
sequencing and the RCA assay. Despite limited genetic variation between
Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed
potential as a simple, reproducible method for the rapid (2-h) identification of
Trichophyton spp(Kong et al.,2008)
26
Garg et al. Study (2007)In this study, nested PCR using novel primers targeting the
pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) was
compared with KOH microscopy, culture isolation, and single-round PCR for
diagnosis of 152 patients with clinically suspected onychomycosis. Results indicate
that nested PCR may be considered the gold standard for the diagnosis of cases of
onychomycosis for which the etiological agents are dermatophytes.
A rapid two-step DNA extraction method and a multiplex PCR for the detection of
dermatophytes in general and Trichophyton rubrum specifically were developed and
evaluated with DNA extracted from pure cultures and from clinically diseased nails.
A total of 118 nail samples received for routine microscopy and culture for
dermatophytes were subsequently tested by the two PCRs separately and in a
multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte
PCR, the T. rubrum-specific PCR sequentially and in a multiplex format correctly
detected all dermatophytes and additionally correctly identified T. rubrum.
Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail
samples with PCR on DNA directly extracted from the nails showed excellent
agreement between PCR and microscopy, but the number of samples with
dermatophyte species identification was increased considerably from 22.9% to 41.5%,
mainly due to the identification of T. rubrum by PCR in microscopy-positive but
culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to
increase not only the speed but also the sensitivity of investigation for nail
dermatophytosis(Dabrowska et al.,2007)
Newer methods such as PCR are being investigated in order to improve the diagnosis
of invasive aspergillosis. One of the major obstacles to using PCR to diagnose
aspergillosis is a reliable, simple method for extraction of the fungal DNA. The
presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of
the DNA by conventional methods employed for bacteria. Numerous fungal DNA
extraction protocols have been described in the literature. However,these methods are
time-consuming, require a high level of skill and may not be suitable for use as a
routine diagnostic technique. Here, a number of extraction methods were compared: a
22
freeze–thaw method, a freeze–boil method, enzyme extraction and a bead-beating
method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was
compared using real-time PCR. It was found that the use of a bead-beating method
followed by extraction with AL buffer (Qiagen) was the most successful extraction
technique, giving the greatest yield of DNA, and was also the least time-consuming
method assessed(Lisa et al.,2006)
Roque et al. Study (2006)This report describes application of PCR fingerprinting to
identify common species of dermatophytes using the microsatellite primers M13,
(GACA) 4 , and (GTG) 5. The initial PCR analysis rendered a specific DNA fragment
for Microsporum audouinii, which was cloned and sequenced. Based on the
sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers
were designed and verified by PCR to establish their reliability in the diagnosis of M.
audouinii. These primers produced a singular PCR band of 431 bp specific only to
strains and isolates of M. audouinii, based on a global test of 182 strains/isolates
belonging to 11 species of dermatophytes. These findings indicate these primers are
reliable for diagnostic purposes, and we recommend their use in laboratory analysis.
Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from
abnormal nails. To investigate whether these fungi are the aetiological agents of
infection or simply transient contaminants, a PCR/sequencing/RFLP assay was
developed for direct and routine identification of the infecting fungi in
onychomycosis. Fungal DNA was readily extracted using a commercial kit after
dissolving nail fragments in a Na 2 S solution. Amplification of part of the 28S rDNA
by PCR was performed with universal primers and the fungal species were identified
by sequencing. The PCR/sequencing results were comparable with microbiological
identification from the same nail sample. In addition to dermatophytes, Fusarium spp.
and other less frequently isolated non-dermatophyte fungi were identified as single
fungal agents in onychomycosis. Moreover, mixed infections were clearly
demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of
infectious agents could be obtained in 2 days, whilst results from fungal cultures take
1–3 weeks. Rapid and reliable molecular identification of the infectious fungus
expedites the choice of appropriate antifungal therapy, thereby improving the cure
rate of onychomycosis (Monod et al., 2006)
20
Multiple codominant genetic markers from single spores of the arbuscular
mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus
geosporum were amplified by nested multiplex PCR using a combination of primers
for simultaneous amplifi cation of five loci in one PCR. Subsequently, each marker
was amplified separately in nested PCR using specific primers. Polymorphic loci
within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were
characterized by sequencing and single strand conformation polymorphisms (SSCP).
Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to
ensure correct identification of the Glomus spp . spores. Single AM fungal spores
were characterized as multilocus genotypes by combining alleles of each amplified
locus. Only one copy of each putative single copy gene could be amplified from each
spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique
genotypes. The amplification of multiple codominant genetic markers from single
spores by the nested multiplex PCR approach provides an important tool for future
studies of AM fungi population genetics and evolution (Stukenbrock and
Rosendahl .,2004)
They analyzed the population structure of the anthropophilic dermatophyte species
Trichophyton violaceum, which mainly causes tinea capitis, and T. rubrum, the most
frequently isolated agent of dermatophytosis worldwide. A microsatellite marker (T1)
was developed by using the enrichment technique for microsatellites. The T1 marker
containing a (GT) 8-10 repeat was proven to specifically amplify both species,
underlining their close kinship. Four polymorphic alleles were detected within asset of
about 130 strains by using polyacrylamide gel electrophoresis with this marker. An
association with geographic origin of the isolates was apparent. Given the close
relatedness of both species, these data suggest an African origin of the entire
T. rubrum complex, followed by the emergence of a new genotype (B) in Asia with
subsequent spread of this genotype over Europe and the United States (Ohst et al.,
2004)
24
Dermatophytoses such as tinea pedis and tinea unguium are very common diseases in
the field of dermatology. The diagnosis of dermatophytoses is usually performed by
direct microscopy and culture. The identification of species is based on morphological
features of giant culture and slide culture. However, in some cases, it is difficult to
identify the species clearly because the culture shows an atypical appearance or is
false negative. Therefore, several molecular biological methods have been developed
for precise identification of a species. The analysis of patterns of random
amplification of polymorphic DNA (RAPD) and restriction fragment length
polymorphisms (RFLP) of mitochondrial DNA is useful for identifying isolates which
are not clearly identifiable by conventional biological techniques. The phylogenetic
analysis of dermatophytes was made by using DNA direct sequencing of nuclear
ribosomal internal transcribed spacer 1 (ITS1). Sequence analysis of chitin synthase 1
(CHS 1) is a rapid tool for species level identification. They attempted the
identification and viability assessment of dermatophytes based on the quantitative
measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT,
725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and
sequenced. ACT intron based primers were dermatophyte species-specific and primer
pairs crossing the intron were dermatophyte genus-specific. The results indicated that
quantification of dermatophyte ACT mRNA correlated with the results of culture and
KOH examination. It is important that the identification of dermatophyte be done by
combining conventional methods with molecular biological methods. In some cases
results of the two methods do not correspond, and is those the fungal species needs to
be re-examined (Kawai et al., 2003).
For PCR-based identification of Aspergillus species, a common primer of the DNA
topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific
primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger,
A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in
PCR amplifications. The method consisted of amplification of the genomic DNA topo
isomerase II gene by a common primer set, followed by a second PCR with a primer
mix consisting of 5 species-specific primer pairs for each Aspergillus species. By
using the common primer pair, a DNA fragment of approximately 1,200 bp was
amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-
25
specific primer pair, unique sizes of PCR products were amplified, all of which
corresponded to a species of Aspergillus even in the presence of DNAs of several
fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100
fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer
mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical
specimens. These results suggested that this nested PCR method is rapid, simple and
available as a tool for identification of pathogenic Aspergillus to a species level
(Kanbe et al.,2002).
Diagnosis of dermatophytosis employing conventional laboratory procedures has been
complicated by the slow growth and varied morphological features shown by
dermatophytes. After analysis of the nucleotide base sequences of a 1.2-kb fragment
amplified from a dermatophyte fungus Trichophyton rubrum by arbitrarily primed
PCR with random primer OPD18, a pair of primers (TR1F and TR1R) was designed
and evaluated for specific identification of T. rubrum. The sensitivity of the primers
TR1F and TR1R was high, as a specific PCR band of 600 bp was detected from as
little as 7pg of T. rubrum DNA (Liu et al.,2002)
Dermatomycoses are very common infections caused mainly by dermatophytes.
Scytalidiosis is a differential mycological diagnosis, especially in tropical and
subtropical areas. Since a culture-based diagnosis takes 2 to 3 weeks, they set up a
PCR-restriction fragment length polymorphism (RFLP) method for rapid
discrimination of these fungi in clinical samples. The hypervariableV4 domain of the
small ribosomal subunit 18S gene was chosen as the target for PCR. The
corresponding sequences from 19 fungal species (9 dermatophytes, 2 Scytalidium
species, 6 other filamentous fungi, and 2yeasts) were obtained from databases or
were determined in the laboratory. Sequences were aligned to design primers for
dermatophyte specific PCR and to identify digestion sites for RFLP analysis. The
reliability of PCR-RFLP for the diagnosis of dermatomycosis was assessed on fungal
cultures and on specimens from patients with suspected dermatomycosis. Two sets of
primers preferentiallyamplified fungal DNA from dermatophytes (DH1L and
DH1R)or from Scytalidium spp. (DH2L and DH1R) relative to DNA from bacteria,
26
yeasts, some other filamentous fungi, and humans. Digestion of PCR products
withEaeIorBamHI discriminated between dermatophytes and Scytalidiumspecies, as
shownwithcultures of 31 different fungal species.Whenclinical samples weretested
byPCR-RFLP, blindly to mycological findings, the results of the two methods agreed
for 74 of 75 samples. Dermatophytes and Scytalidium spp. can thus be readily
discriminated by PCR-RFLP within 24 h. This method can beapplied to clinical
samples and is suited to rapid etiologic diagnosis and treatment selectionfor patients
with dermatomycosis (Dubach et al.,2001)
27
Chapter Three
Materials and Methods
28
Chapter 3
Materials and Methods
3.1 Materials
3.1.1 Instrument
Table 3.1 list of equipment used in this study.
Equipment Manufacture/country light microscope Zeiss/Germany
electrophoresis apparatus Thermo-electron corporation Ec 105
Vacuum (suction unit) Su-770(association with cannic.lnc) Taiwan
Vortex microspin BioSan/England
micro centrifuge (rpm 14000) Sigma/USA
Digital camera Sony (4*200m)cyber-shot/Japan
A thermal cycler HYBAID, Omnigene/ England b a l a n c e 4 d i g i t F e w – m o d e l : f e j - 2 0 0
UV Transilluminator (Dinco & Reunium Industries Ltd.) /USA
electrophoresis chamber (Owl Scientific Plastics, Inc.)/. USA
Dna/rna – uv – cleaner Uvc/t - BioSan/England
3.1.2 PCR primers
Table 3.2 list of PCR primers used in this study.
Primer name Sequence nucleotides [nt]
CHS1 1S F 5'-CAT CGA GTA CAT GTG CTC GC-3' 70 to 89
CHS1 1R R 5'-CTC GAG GTC AAA AGC ACG CC-3' 485 to 504
CHS1JF2 F 5'-GCA AAG AAG CCT GGA AGA AG-3' 111 to 130
CHS1JR2 R 5'-GGA GAC CAT CTG TGA GAG TTG-3' 378 to 398
3.1.3 Chemical Table 3.3 list of chemical used in this study.
Chemicals potassium hydroxide (KOH ). 0.1% Triton X-100
proteinase K solution
GoTaq Green Master Mix, 2X ( Promega,
USA). phenol-chloroform
Tris-EDTA buffer dimethyl sulfoxide. Ca HCo3 HCL
precipitation solution Isopropanol 75% ethanol TE-buffer
agarose ethiudium bromide
29
3.2 Methods
3.2.1 Study Area
The study was performed at Al-Remal Clinics at Ministry of Health (MOH) and
Gene Medical Labs in Gaza Strip.
3.2.2 Samples
A total of 99 sample from patients clinically suspected with dermatophytosis were
included in the study irrespective of their age or gender.
3.2.3 Specimens Collection
For skin dermatophytoses the clinical specimens collected were epidermal scales. The
scales were scrapped from near the advancing edges of the lesions after disinfecting
the lesions with 70% alcohol. When the advancing edges were not evident, scrapings
were collected from areas representing the whole infected area.
For hair sample dermatophytoses basal root portion of hair were collected by
plucking the hair with sterile forceps. In cases with black dot, scalpel was used to
scrape the scales and excavate small portions of the hair roots.
For nail the first step of the sample collection process is thorough cleansing of the nail
area with alcohol to remove contaminants such as bacteria. Because the sites of
invasion and localization of the infection differ in the different types of nychomycosis
. Nail clippings and nail scrapings of the affected part of the nail .
3.2.4 Specimens Division. According to the modified procedures of (Garg et al., 2009) the collected specimens
were divided into two portions. The first portion of the specimens was examined
microscopically using 20% potassium hydroxide (KOH).The second portion was used
for DNA extraction we put in eppendorf tube
3.2.5 Specimens Identification
3.2.5.1 Direct microscopy by KOH
This method aids visualizing hyphae and confirmation of the diagnosis of
dermatophyte infection. The scale from the active border of a lesion was obtained,
03
and several loose hairs from the affected area were pulled out, in the case of nails, sub
ungual debris was obtained. A moist cotton swab was rubbed vigorously over the
active border of a lesion works as well as a scalpel blade and is safer. The scale, hair,
or debris were transferred to a glass slide, and a few drops of 20% KOH were added.
For nail material or hair the slide was gently warmed. The wetmount preparation was
then examined under a microscope (X400) with back and forth rotation of the focus
knobs. This technique aided the visualization of hyphae (branching, rod-shaped
filaments of uniform width with lines of separation [septa]). In tinea capitis, the
hairshaft may be uniformly coated with minute dermatophyte spores (Barry and
Hainer study, 2003).
3.2.5.2 Molecular Techniques
3.2.5.2.1 DNA Extraction
The crushed specimen were cut and put in eppendorf tube and 200 μl buffer (0.02g
Ca HCo3-30 μl HCL - add water to 10 ml) were Added.The following steps were
followed.
1. Add 5 μl of (proteinase K)
2. Incubation for 2-3 hours at 65C
3. Then using MasterPure TM
Genomic DNA Purification Kit for Blood
(Epicentre Technologies Co., USA) according to the following procedure:
a. Add 250 μl precipitation solution (5M Sodium perchlorate (dissolve
70 g of sodium perchlorate in 80 ml d.w make up 100 ml)
b. Mix by vortex for at least 30 sec, then centrifugation at 14,000 Xg
for 7 min.
c. The supernatant was poured into a new eppendorf tube, and 700 μl of
isopropanol were added. The tube was inverted gently 30-40 times to
visualize the DNA strings.
d. The DNA was precipitated by centrifugation at 14,000 Xg for 10 min.
4. DNA was washed twice with 75% ethanol, by adding 200 μl of 70%
ethanol followed by centrifugation at 14,000 Xg for 3 min.
06
5. DNA pellet was air dried, resuspended in 100 μl of TE (10 mM Tris-
HCl [pH 8.0], 1 mM EDTA) buffer, and then incubated overnight at
room temperature (or incubation for 10 min at 37°C).
6. Finally, the DNA was mixed, quantified using agarose gell
electrophoresis for semi quality and DNA quality evaluation .
3.2.5.2.2. Polymerase Chain Reaction (PCR)
The polymerase chain reaction(PCR) is an in vitro technique which allows the
amplification of a specific DNA fragment that lies between two regions of known
DNA sequence (Newton and Graham, 1997). The amplification of DNA is achieved
by using a short single stranded DNA molecules which are complementary to the ends
of a defined sequence of the DNA template (known as primers), that hybridize to
opposite strands and flank the target DNA sequence that is to be amplified. Under
suitable reaction conditions and in the presence of deoxynucleoside triphosphate
(dNTPs), a DNA polymerase extends the primers annealed to a single stranded DNA
template. As a result, a new DNA strands complementary to the template strands are
synthesized (Newton and Graham, 1997; Bartlett and Stirling, 2003). Repetitive
cycles involving template denaturation, primer annealing, and extension of the
annealed primers by Taq DNA polymerase results in exponential accumulation of a
specific DNA fragments. In other words, the number of target DNA copies
approximately doubles every cycle, since the primer extension products synthesized
in a given cycle can serve as a template in the next cycle (Watson et al., 1992).
3.2.5.2.3 First PCR
The sequence of primers used for specific amplification .
PCR was performed using primer pairs CHS1 1S (5'-CAT CGA GTA CAT GTG
CTC GC-3'; nucleotides [nt] 70 to 89) and CHS1 1R (5'-CTC GAG GTC AAA AGC
ACG CC-3'; nt 485 to 504). These primers amplify a 435-bp DNA fragment of the
dermatophyte-specific CHS1 gene sequence of Arthroderma benhaemiae, a
teleomorph of Trichophyton mentagrophytes (DDBJ accession no. AB003558) (Garg
et al., 2009).
02
3.2.5.2.4 Nested PCR
Nested PCR was performed by designing a novel set of primers, JF2 (5'-GCA AAG
AAG CCT GGA AGA AG-3'; nt 111 to 130) and JR2 (5'-GGA GAC CAT CTG TGA
GAG TTG-3'; nt 378 to 398), amplifying a DNA fragment of 288 bp from the internal
sequence of the amplicon obtained from first-round PCR ( Garg et al., 2009).
PCR Mixture First
The PCR mixture (25 μl) for first-round PCR contained 12.5 μ l of green mix 1 μl
each of primers 0.1MG/ML CHS1 1S and CHS1 1R (Operon, Cologne, Germany),
and 3 μ l of DNA template. Deionised water was added subsequently to achieve the
final volume(Table 3.4). The reaction mixture was initially denatured at 94°C for 30
s, followed by 31 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s,
and extension at 72°C for 60 s. This was followed by a final extension step for 5 min
at 72°C in a thermal cycler(Table 3.5 ) (HYBAID, Omnigene). The PCR mixture for
nested PCR consisted of 1 μl primers JF2 and JR2 and 2 μ l diluted product of the
primary cycle as the DNA template; the rest of the constituents were the same as
those described in (Table 3.6). The running conditions of nested PCR were similar to
the first-round PCR except that 35 cycles were used(Table 3.7). double-distilled
water and DNA of positive controls were used as the negative and positive controls,
respectively.
Table 3.4 For first PCR reaction mixture for 25 μ l, the amounts given are per reaction
Reagents Volume ( μl) Go tag polymerase (ready mix) 12.5 μ l Primer forward 1 μl Primer Reverse 1 μl DNA 3 μ l Water 7.5 μ l Total mix 25 μ l We take DNA Template 2 μ l
00
Table 3.5 Temperature cycling program for FPCR
PCR Machine Cycling Parameters
For first PCR Rounds
initially
denatured 94ºC/30 sec.
31 cycles:
94ºC/30sec.
60ºC/30 sec.
72ºC/ 60 sec.
extension step 72ºC/ 5 min
Hold: 4ºC
Table 3.6 Nested PCR Master Mix for 25 μ l reactions, the amounts given are per reaction
Reagents Volume ( μl)
Go tag polymerase (ready mix) 12.5 μ l
Primer forward 1 μl
Primer Reverse 1 μl
DNA Template 2 μ l
water 6.5 μ l
Total mix 25 μ l
Table 3.7 Temperature cycling program for NPCR
PCR Machine Cycling Parameters
For nested PCR Rounds
initially
denatured 94ºC/30 sec.
35 cycles:
94ºC/30sec.
60ºC/30 sec.
72ºC/ 60 sec.
extension step 72ºC/ 5 min
Hold: 4ºC
04
3.2.5.2.5 Agarose Gel Electrophoresis
The amplified PCR product were resolved by electrophoresis on a 2% agarose gel and
stained with ethidium bromide for analysis. The agarose gel (Life Technologies,
Scotland) was prepared in 1X Tris-Acetate EDTA (TAE) buffer (40 mM Tris base, 40
mM acetic acid, 1mM EDTA), then stained with ethidium bromide (final
concentration is 0.5 μg/μl). The gel casting tray containing the gel is placed into the
electrophoresis chamber (Owl Scientific Plastics, Inc.). Stained PCR products and
DNA molecular weight marker were loaded into the agarose gel. Then the gel was run
at 80 volt (constant voltage) for 45-75 min, according to the gel size used. After that,
the ethiudium bromide-stained DNA was detected by ultraviolet radiation using UV
Transilluminator (Dinco & Rheunium Industries Ltd.) amplicon of 288 bp was taken
as positive for dermatophytes. (Fig. 2) and photographed by digital Camera for
documentation.
3.2.5.2.6 DNA Sequencing To be more confirmed, we conducted DNA sequencing in Makassed Islamic
Charitable Hospital.
Direct DNA sequencing for PCR products that contain the known mutations and
polymorphisms in –( AB003558) JF2 (5'-GCA AAG AAG CCT GGA AGA AG-3'; nt
111 to 130) and JR2 (5'-GGA GAC CAT CTG TGA GAG TTG-3'; nt 378 to 398),
amplifying a DNA fragment of 288 bp from the internal sequence of the amplicon
obtained from first-round PCR
The amplification was performed in 100 µl reaction containing 400 ng of genomic
DNA, 0.4 µg of each primer, and 50 µl of GoTaq Green Master Mix, 2X ( Promega,
USA).
Cycling parameters included an initial denaturation step of 4 min at 94C, followed
by 35 cycles of 94C for 30 sec, 61C for 30 sec, 72C for 30 sec, with a final
extension step of 10 min at 72C.
The amplified products were separated on 2% agarose gel and purified using GFX™
PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech) according
to the following procedure:
Purification of DNA from gel band:
05
An empty 1.5 ml eppendorf tube was weighed to the nearest
10mg.
The slice of agarose containing the DNA band was excised
using a sterile razor blade and transferred to the last preweighed tube.
The weight of the slice was determined by weighing the tube
containing the agarose slice and subtracting the weight of the empty
tube.
Proteins were denatured and agarose was dissolved by adding
10 µl of capture buffer for each 10 mg of gel slice, mixing vigorously,
and incubating the tubes at 60 ºC for 15 min.
During the incubation, one GFX column is placed in a
collection tube for each sample to be purified.
After the agarose was completely dissolved, the sample was
centrifuged briefly (to collect at the bottom of the tube), transferred to
the GFX column and incubated at room temperature for 1 min (so as to
capture the DNA to the glass fiber matrix).
The column with its collection tube was then centrifuged at
15,000 Xg for 30 sec and the flow-through was discarded.
Matrix-bound DNA was washed by adding 500 µl of wash
buffer to the column followed by centrifugation at 15,000Xg for 30
sec.
The collection tube was discarded, and the GFX column was
transferred to a new 1.5 ml eppendorf tube.
The purified DNA is eluted from the GFX column by applying
35 µl of autoclaved double-distilled water to the top of the glass fiber
matrix in the GFX column, incubating at room temperature for 1 min,
and centrifugation at 15,000 Xg for 1 min.
Following the purification, DNA segments were separated
using 3130 Genetic Analyzer. Then the sequence of each sample was
compared to the wild-type gene obtained from the NCBI gene bank
accession number GI: AB 003558).
06
3.2.6 Questionnaire
For each sample, the questionnaire was filled by technician . The questionnaire
sample is given in the appendix. The questionnaire was prepared to find out any
relation between the dermatophytes and age, sex,work, domestic animals in the
house.
3.2.7 Data Analysis
The data were analyzed statistically by SPSS analysis (version 13). The results
presented through histograms, tables,. Relative absolute error and exclusion and
inclusion errors.
07
Chapter Four
Results
08
Chapter 4
Results
4.1 Potassium hydroxide (KOH) microscopy.
This method aids visualizing hyphae or spores and confirmation of the diagnosis
of dermatophyte infection. In tinea capitis, the hair shaft may be uniformly coated
with minute dermatophyte spores (Figure 4.1).
Figure 4. 1: Microscopic appearance of positive sample (spores) from hair in KOH.
As shown in Figure 4.9 and table 4.1 the positive result in KOH method were 41 out
of 99 sample which considered as 41.4% . These results distributed as 3 from skin out
of 16 , 9 from nails out of 16 and 29 from scalp out of 67 (Table 4.1)
The result also show that the higest percentage of positive result was from the nails
samples .
09
4.2 Molecular Diagnosis
The amplified PCR product were resolved by electrophoresis on a 2% agarose gels
and stained by ethidium bromide for analysis (Figure 4.2).
4.2.1 First PCR
As shown in Figure 4.10 the positive result in FPCR were 2 out of 99 sample
which considered as 2.02%only .
4.2.2 Nested PCR
As shown in Figure 4.11 and Table 4.1 the positive results in NPCR were 18 out
of 99 sample which considered as 18.18% . These results distributed as 1 from skin
out of 16 , 3 from nails out of 16 and 14 from scalp out of 67 (Table 4.1).
The results also show that the highest percentage of positive results was from the
scalp,which constitute 20% of all suspected scalp samples.
Figure 4. 2 : Results of First and Nested PCR
Figure (4.2) Results of first and nested PCR of clinical specimens of
dermatomycosis. Lane 1, 100 bp DNA ladder (Molecular Marker); Lane 2, 4, 6 First
PCR negative cases; lane 3, 7 nested PCR positive cases; Lane 5, nested PCR
negative case; lane 8, 9 first and nested PCR positive control respectively (288 bp);
Lane 10, 11 first and nested PCR. Negative control; lane12 blank .
4.3 Gene Sequencing
Figure (4.4) show the gene sequencing of our PCR gene product.By
comparing our result with the wild-type gene obtained from the NCBI gene bank
accession number GI: AB 003558). we found that 95% homology with the reference
43
> dbj|AB003558.1| Arthroderma benhamiae gene for chitin synthase 1,
partial cds
Length=615
Score = 339 bits (183), Expect = 1e-97
Identities = 206/217 (95%), Gaps = 1/217 (0%)
Strand=Plus/Minus
Query 4
CGCGGCTTGAGGATAACCTGGGTGCCCTTGACCTCCATGCCTATCTGGGTGGTATATTCG 63
||||||||||||||||||||||| |||||||||||||||||||||||||
||||||||||
Sbjct 326 CGCGGCTTGAGGATAACCTGGGT-
CCCTTGACCTCCATGCCTATCTGGGCGGTATATTCG 268
Query 64
TAGATGTGAGCAGTGACGTCTTTACCGTTAACCTGCTGCTTAGCAATGCCATCTTGGTAA 123
|||||||||||||||| ||||| ||||||||||||||| || |||||||| ||
||||||
Sbjct 267
TAGATGTGAGCAGTGATGTCTTGACCGTTAACCTGCTGTTTGGCAATGCCGTCCTGGTAA 208
Query 124
ACACCTAGACCGGCAAGGACAGCTCTTGTACGTGGGTTTATCTTTGCACGACCGTCTGAG 183
||||| |||||||||||||||||||| ||||||||
||||||||||||||||||||||||
Sbjct 207
ACACCAAGACCGGCAAGGACAGCTCTCGTACGTGGATTTATCTTTGCACGACCGTCTGAG 148
Query 184 ACGATACAAACGACAATCTTCTTCCAGGCTTCTTTGC 220
|||||||||||||||||||||||||||||||||||||
Sbjct 147 ACGATACAAACGACAATCTTCTTCCAGGCTTCTTTGC 111
sequencer GI: AB 003558 .This finding confirms PCR product to be indeed the
product of CHS1 gene and confirms the PCR product specificity of the CHS1 gene
Figure (4.3) .
Figure 4. 3: The wild-type gene obtained from the NCBI gene bank accession number
GI: AB 003558)
46
Figure 4.4: The DNA sequencing result of CHS1 out put
4.4 Statistical analysis
Of the 99 clinically suspected cases of dermatophytosis, 41.4% were diagnosed as
positive for fungal elements by KOH microscopy. Dermatophytes were detected in
18.18% of the specimens by nested PCR.
4.4.1 Study population
As shown in Figure (4.5 ) 46.32% of our clinical samples were from males. The
sources of the clinical sample was distributed as 16.16% from each skin and nails and
67.68% from scalp Figure (4.6).
42
- Figure 4. 5: Sample classification of gender
Figure 4. 6: classification of sample type
As inferred from the Questionnaire (Appendix) about the presence of domestic
animals in the houses of the suspected patient ,cats were the highest percentage which
was 70.4%.
The percentage of positive results among the suspected patients holding domestic
animals at their houses was 33.3%while it was 22% for the suspected patients not
holding domestic animals Table( 4.2).
40
Figure 4. 7: The domestic animal kinds in houses
The age distribution of the cases is ranged from 1 year to higher than 35 years.The
highest age distribution was ranged between 1-10 years which constitute 64.52%
(Figure 4.8)
Figure 4. 8: Age group distribution.
__
44
0
10
20
30
40
50
60
positive KOHnegative KOH
41
58
Figure 4. 9: The results of KOH classification
Figure 4. 10 result of FPCR classification
0
20
40
60
80
100
positive NPCRnegative NPCR
18
81
Figure 4. 11: The result of NPCR classification
0
20
40
60
80
100
positive FPCRnegative FPCR
2
97
Figure 4. 10 result of FPCR classification
45
0102030405060708090
100
positive KOH
negative KOH
positive FPCR
negative FPCR
positive NPCR
negative NPCR
Figure 4. 12: The results of KOH,FPCR,NPCR classification
4.4.2 Relative absolute error
The following table shows a tabulating of the sample according to the sample type
and the Lab methods with the (relative absolute error).
Table 4.1 Sample type and lab methods KOH & NPCR .
%
Error
Total Nested pcr Result Koh Result Sample Type
Negative Positive Negative Positive
12.50 16 15 1 13 3 Skin
37.50 16 13 3 7 9 Nail
22.39 67 53 14 38 29 Scalp
23.23 99 81 18 58 41 Total
The following table shows a tabulating of the sample according to holding animals in
the house and the Lab methods with the (relative absolute error).
Table 4.2 Holding animals in the house relative to KOH & NPCR.
Total Nested PCR Result KOH Result holding animals
Negative Positive Negative Positive
27 22 5 13 14 Yes
63 53 10 39 24 NO
90 75 15 52 38 Total
46
4.4.3 Lab diagnosis exclusion and inclusion errors
Inclusion and Exclusion errors refer to discrepancies between the diagnosis by NPCR
method and the diagnosis by KOH method. Exclusion error represents the percentage
of negative samples by KOH method that is positive according to NPCR method.
Inclusion error represent the percentage of negative samples by NPCR method which
is positive in KOH method.
Table 4.3 Lab diagnosis exclusion and inclusion errors.
Nested PCR Result vs. KOH Result
KOH Result Total
positive negative
Nested
pcr
Result
positive 12 6 18
negative 29 52 81
Total 41 58 99
Exclusion error = 6/99 = 6.06%
Inclusion error = 29/99 =29.29%
47
Chapter Five
Discussion
48
Chapter 5
Discussion
Dermatophytes are among the few fungi causing communicable diseases; previously
most dermatophyte strains had relatively restricted geographical distribution.
However recently, dermatophytosis has become one of the most common human
infectious diseases in the world and is cosmopolitan in distribution. Dermatophytosis
cannot be easily diagnosed on the basis of clinical manifestations as a number of other
conditions mimic the clinical presentation.The differential diagnosis of
dermatophytoses includes seborrhoeic dermatitis, atopic dermatitis, contact dermatitis,
psoriasis, candidal intertrigo, erythrasma, eczema etc (Barry and Hainer, 2003).
Further it is more difficult to diagnose dermatophytosis in immunocompromised
patients, as clinical presentation is often atypical (Odom,1994).
It is essential that good laboratory methods are available for rapid and precise
identification of the dermatophytes involved, in order to apply appropriate treatment
and prevention measures. The conventional methods of fungal detection have their
own drawbacks; for e.g. KOH microscopy has low specificity and fungal culture is
associated with low sensitivity and takes long time. Further dermatophyte isolates
from patients on antifungal treatment generally do not show characteristic
morphology on culture, thus further compromising the results of culture isolation (liu
et al., 1997). The changing profiles of human dermatophytoses among countries have
further necessitated the development of improved diagnostic methods for
identification of dermatophytes (liu et al., 1997). Thus newer fungal diagnostic meth-
ods are need of the hour as identification of the etiological agent is required not only
for accurate diagnosis, but also for post-therapeutic strategies (Shiraki et al., 2004).
The treatment of dermatophytoses would be most appropriate when the selection of
antimicrobial agent is based on the identity of the causative agent. For e.g.
griseofulvin is effective only for dermatophytic infections, with no activity against
Candida spp. and nondermophytic molds. Terbinafine shows fungicidal activity
against dermatophytes with a cure rate of 80 to 95% but shows only fungistatic
activity against Candida albicans. For nondermatophytic molds infections, the role of
49
terbinafine is not well defined and topical amorolfine lacquer may be effective for
select patients (DW et al., 1995).
Recently, molecular biology-based techniques, such as PCR followed by restriction
fragment length polymorphism (RFLP) (Yang et al, 2008), Real time PCR (Arabatzis
et al, 2007) and multiplex PCR assay (Brillowska et al., 2007) have been adapted for
detection of dermatophytes from clinical specimen. These molecular methods have a
good potential to directly detect dermatophytes in clinical specimens; however these
methods are yet to be standardised for routine clinical laboratories. PCR – RFLP is a
complex technique with poor discriminative power to make an easy and specific
diagnosis. Real time PCR – appears to be promising but is not practical enough for a
large number of laboratories that are either small scale or very tightly budgeted.
Very few studies like our study have compared KOH microscopy and culture with
direct PCR of clinical specimens In a case study, Nagao et al.(2012) detected
Trichophyton rubrum by nested PCR targeting internal transcribed spacer gene 1
(ITS1) in a patient with trichophytia profunda acuta, which was negative by both
KOH microscopy and culture (Nagao et al, 2005). This finding is agree with our
results. Yan et al (2007) evaluated arbitrary primed PCR with conventional methods
in 50 tinea corporis and 58 tinea cruris patients and showed that arbitrary primed PCR
is a rapid sensitive and specific detection method for dermatophytes from skin
scrapings. (Yan et al, 2007). Recently Bergman et al,(2008) performed a PCR-reverse
line blot assay on 819 clinical samples (596 nail, 203 skin and 20 hair) and
demonstrated a positive PCR-RLB result in 93.6% of 172 culture-positive and
microscopy-positive samples. (Bergmans et al, 2008). Garg et.al 2007 study strongly
supports our result they involving 152 clinically suspected patients with
onychomycosis it was established that nested PCR might be considered as gold
standard for the diagnosis of onychomycosis, where the etiological agents are
dermatophytes. In Garg et.al 2009 study results also indicate that nested PCR may be
considered as gold standard for the diagnosis of dermatophytosis and can aid the
clinician in initiating prompt and appropriate antifungal therapy. Which also supports
our finding .
In this study, a total of ninety nine patients were clinically suspected with
dermatophytosis including 16 skin specimens 16 nail specimens and 67 hair
53
specimens Table (4.1). For each specimens both of KOH and NPCR test were carried
out.
Having compared the output results of NPCR sequencing with the wild-type gene
which is obtained from the NCBI gene bank Fig. (4.4). The comparison indicates that
the product of NPCR is CHS1 gene according to (NCBI) gene bank Fig.(4.3).
Additionally, it is considered to compare the results of NPCR with KOH for
dermatophytes which gives that 41.4% are positive indication based on KOH and
18.18% is positive indication according to NPCR Fig.(4.12)and Table(4.3). Our
results reflect the accuracy of NPCR method and eliminate the false – positive and
reconsider few of the false-negative as positive samples.
After carrying out the statistical analysis using SPSS for both test results obtained
from NPCR and KOH, it is found that 30% of the total sample have to be included
for treatment based on KOH test, although this percent of the sample doesn‟t need to
undergo treatment according to NPCR test. It is also shown that 6% of the sample are
excluded for treatment in KOH test, in spite the NPCR indicated that this percent shall
be included in the treatment Table(4.3).
Correct diagnosis of dermatophytic onychomycosis and identification of the causal
agent is a major importance as it allows appropriate antifungal treatment to be
promptly instituted. Diagnosis of onychomycosis is currently performed by direct
mycological examination and culture on Sabouraud dextrose agar medium. The
precise identification of the dermatophyte in cause is based on the macroscopic and
microscopic characters of the grown colonies. However, false negative results of
direct examination occur in 5 to 15% of cases, depending on the skill of the observer
and the quality of sampling (Liu et al, 2000, and Robert and Pihet, 2008).
Furthermore, dermatophyte hyphae are very difficult to distinguish from those of non
dermatophytic fungi like molds which often only occur as transient contaminants and
not as the actual etiological agent of the disease (Brillowska et al, 2007, Ebihara et al,
2009, and Robert and Pihet, 2008).
The present study aimed at evaluating a PCR technique based on the amplification of
the CHS1 gene which is one of the most widely used target in the molecular diagnosis
of dermatophytic onyxis in humans (Garg et al, 2007, Hirai et al, 2003, Kano et al,
1998, Neji et al, 2010, and Spiliopoulou et al, 2011).
The prominent controversy between the test results (KOH and NPCR) was found in
the nails diagnosis Table(4.1). This complies with previous studies but differs in the
56
nature of results as the positive results higher in KOH test and it may be related to
one or more of the following reasons :
- Labs equipment shortage .
- No advanced training for workers in the labs .
- Unqualified technicians to carry out perfect tests to discriminate between
pathogenic fungus and normal flora,contament ,bubbles or oil.
- Nails thickness without being treated enough in KOH. .
On the other hand, our results showed that people who get contact with animals(pets)
are most likely to have dermatophytosis more than other people . This finding is in
accordance with most previously reported studies (Emeka, 2011 and Beraldo et al.,
2011).
52
Chapter six
Conclusions & Recommendations
50
Chapter 6
Conclusions & Recommendations
Conclusions
A total of ninety nine patients were clinically suspected with dermatophytosis
including 16 skin specimens 16 nail specimens and 67 hair specimens. For each
specimens both of KOH and NPCR test were carried out.
Having compared the output results of NPCR sequencing with the wild-type gene
which is obtained from the NCBI gene bank. The comparison indicates that the
product of NPCR is CHS1 gene according to (NCBI) gene bank. Additionally, it is
considered to compare the results of NPCR with KOH for dermatophytes which gives
that 41.4% are positive indication based on KOH and 18.18% is positive indication
according to NPCR.
After carrying out the statistical analysis using SPSS for both test results obtained
from NPCR and KOH, it is found that 30% of the total sample has to be included for
treatment based on KOH test, although this percent of the sample doesn‟t need to
undergo treatment according to NPCR test. It is also shown that 6% of the sample are
excluded for treatment in KOH test, in spite the NPCR indicated that this percent shall
be included in the treatment.
The prominent controversy between the test results (KOH and NPCR) was found
particularly in the nails diagnosis.
The study results approved that the NPCR test has to be considered in dermatophytes
test in Gaza medical Laboratories in addition to the KOH test specially in nail test.
Recommendations
1- Sending directives to the Ministry of Health in the Gaza Strip by introducing
screening NPCR part of routine testing for dermatophytes .
2- Conduct training session for lab technicians to develop their skills in the
diagnosis of dermatophytes by KOH test.
3- Community a wareness in taking necessary measures when dealing with
domestic animals.
4- Inviting researchers to take into account the studies on dermatophytes in the
Gaza Strip
54
References
- Achterman RR.,and White TC., (2011) Dermatophyte virulence factors:
identifying and analyzing genes that may contribute to chronic or acute skin
infections. International Research Journal of Microbiology.vol.(2012),pp:1-8.
- Ajello L. (1977) Taxonomy of the dermatophytes: a review of their imperfect
and perfect states, In K. Iwata (ed.), Recent advances in medicaland veterinary
mycology. University of Tokyo Press, Tokyo, pp. 289–297.
- Ajello L. (1974) Natural history of the dermatophytes and related
fungi.Mycopathol. Mycol. Appl. Vol. 53, pp. 93–110.
- Al-Fouzan A., Wanda A. and Kubec K. (1992) Dermatophytosis of children in
Kuwait ;Aprospective survey . Intl.J.Dermatol., vol. 32, pp.798-801.
- Ali-Shtayeh M., Arda H., and Abu-Ghdeib, S., (1997) Epidemiological study of
tinea capitis in school children in the Nablus area (West Bank). Myco., Vol. 41,
pp. 243-248.
- Alteras I., Feuerman, E., David, M., and segal, R., (1986) The increasing role of
Microsporum.canis and the variety of dermatophytic manifestations in Israel.
Mycopath.,vol. 15, pp.105-107.
- Aly R. (1994) Ecology and epidemiology of dermatophyte infections. J. Am.
Acad. Dermatol.,Vol. 31pp. 21–25.
- Anna Brillowska-Dąbrowska, Ditte Marie Saunte, and Maiken Cavling
Arendrup. (2007) Five-Hour Diagnosis of Dermatophyte Nail Infections with
Specific Detection of Trichophyton rubrum. J Clin Microbiol. Vol. 45(4),pp:
1200–1204.
- Arabatzis M., Bruijnesteijn van Coppenraet LE., Kuijper EJ., de Hoog GS.,
Lavrijsen AP., Templeton K., Raaij-Helmer EM van der., Velegraki A., Gräser
Y., Summerbell RC. (2007) Diagnosis of common dermatophyte infections by a
novel multiplex real-time polymerase chain reaction detection/identification
scheme. Br J Dermatol,Vol 157,pp.681-689.
55
- Barry I.and Hainer M. (2003) Dermatophyte Infections. American family
physician, Vol. 67,pp.101-108.
- Beraldo R., Gasparoto A., Martins A., Siqueira D., Dias A. (2011)
Dermatophytes in household cats and dogs Dermatófitos em gatos e cães
domésticos..
- Bergmans AM., Schouls LM., Ent M van der, Klaassen A, Böhm N
Wintermans RG. (2008) Validation of PCR-reverse line blot, a method for rapid
detection and identification of nine dermatophyte species in nail, skin and hair
samples. Clin Microbiol Infect, Vol.14(8),pp.778-88.
- Borelli D. (1965) Microsporum racemosum nova species. Acta. Med. Venez.
Vol.12,pp.148-151.
- Brillowska DA., Saunte DM., Arendrup MC. (2007) Five-Hour Diagnosis of
Dermatophyte Nail Infections with Specific Detection of Trichophyton rubrum.
Journal of Clinical Microbiologyl, Vol 45,pp.1200-1204.
- Chandran NS., Pan JY., Pramono ZA., Tan HH., Seow CS. (2013)
Complementary role of a polymerase chain reaction test in the diagnosis of
onychomycosis. Australas J Dermatol.Vol.54(2),pp:105-108.
- Chandran NS., Pan JY., Pramono ZA., Tan HH.,Seow CS.
(2013)Complementary role of a polymerase chain reaction test in the diagnosis
of onychomycosis. Australas J Dermatol. Vol.54(2),pp.105-108.
- Currah R., (1985) Taxonomy of the Onygenales: Arthrodermataceae,
Gymnoascaceae, Myxotrichaceae, and Onygenaceae. Mycotaxon.,Vol. 24,pp.1-
216.
- De Baere T, Summerbell R, Theelen B, Boekhout T, Vaneechoutte M. (2010)
Evaluation of internal transcribed spacer 2-RFLP analysis for the identification
of dermatophytes. J Med Microbiol,Vol. 59,pp:48-54.
- Dhib I., Fathallah A., Charfeddine IB., Meksi SG., Said MB., Slama F., Zemni
R. (2012) Evaluation of Chitine synthase (CHS1) polymerase chain reaction
assay in diagnosis of dermatophyte onychomycosis. J. Mycol Med
,Vol.22(3),pp.249-255.
56
- DW , Evans EGV., Kibbler CC., Richardson MD., Roberts MM., Rogers ,
Warnock DW., Warren RE. (1995) Fungal nail disease: a guide to good practice
(report of a Working Group of the British Society for Medical Mycology). Br
Med J,Vol. 311,pp.1277-1281.
- Ebihara M., Makimura K., Sato K., Abe S., Tsuboi R. (2009) Molecular
detection of dermatophytes and non dermatophytes in onychomycosis by nested
polymerase chain reaction based on 28s ribosomal RNA gene sequences. Br J
Dermatol, Vol.161,pp. 1038-1044.
- Elewsk B. (2000) tinea capitis :a current perspective. J. Am. Acad. Dermatol.,
Vol.42,pp.1-20.
- Emeka I. Nweze (2011) Dermatophytoses in Domesticated Animals. Rev. Inst.
Med. Trop. Sao Paulo, vol53(2),pp:95-99.
- Eriksson R, Jobs M, Ekstrand C, Ullberg M, Herrmann B, Landegren U, Nilsson
M, Blomberg J. (2009) Multiplex and quantifiable detection of nucleic acid from
pathogenic fungi using padlock probes, generic real time PCR and specific
suspension array readout. J Microbiol Methods,Vol.78(2),pp:195-202.
- Garg J., Tilak R., Singh S., Gulati AK., Garg A., Prakash P. (2007) Evaluation of
Pan Dermatophyte Nested PCR in Diagnosis of Onychomycosis. J Clin
Microbiol ,Vol.45(10)pp.3443-3445.
- GargJ., TilakR., GargA., PrakashP.,Gulati A., and NathG., (2009) - Rapid
detection of dermatophytes from skin and hair. BMC Res. 2,p.60.
- Georg K. (1960) Epidemiology of the dermatophytoses sources of infection,
modes of transmission and epidemicity. Annals of the New York Academy of
Sciences, Vol.89,pp.69-77.
- Griffiths LJ, Anyim M, Doffman SR, Wilks M, Millar MR, Agrawal SG. (2006)
Comparison of DNA extraction methods for Aspergillus fumigatus using real-
time PCR. J Med Microbiol,Vol. 55( 9,),pp:1187-1191.
- Gupta A. and Cooper E. (2008) Update in Antifungal Therapy of
Dermatophytosis. Mycopathologia,Vol. 166, pp. 353–367.
57
- Hirai A., Kano R., Nakamura Y., Watanabe S., Hasgawa A. (2003) Molecular
taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene
sequences. Antonie van Leeuwenhoek ,Vol.83,pp.11-20.
- Kanbe T, Yamaki K, Kikuchi A. (2002) Identification of the pathogenic
Aspergillus species by nested PCR using a mixture of specific primers to DNA
topoisomerase II gene. Microbiol Immunol. Vol.46(12),pp:841-848.
- Kano R., Nakamura Y., Watanabe S., Takahashi H., Tsujimto H., Hasegawa
A.(1998) Molecular analysis of chitin synthase 1 (CHS1) gene sequences of
Trichophyton mentagrophytes complex and T. rubrum. Curr Microbiol
,Vol.37,pp.236-239.
- Kawai M. (2003) Diagnosis of dermatophytoses: conventional methods and
recent molecular biological methods. Nihon Ishinkin Gakkai Zasshi.,Vol.
44(4),pp:261-264.
- Kim DM., Chung SH., Chun HS. (2011) Multiplex PCR assay for the detection
of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented
soybean food starter. Food Microbiol,Vol.28(7),pp:1402-1408.
- Knosravi R., Aghamirian R., and Mahmoudi M. (1994) Dermatophytoses in
Iran. Mycoses,Vol.37,pp: 43-48.
- Kong F, Tong Z, Chen X, Sorrell T, Wang B, Wu Q, Ellis D, Chen S. (2008)
Rapid identification and differentiation of Trichophyton species, based on
sequence polymorphisms of the ribosomal internal transcribed spacer regions, by
rolling-circle amplification. J Clin Microbiol.,Vol.46(4),pp:1192-1199
- Liu D., Pearce L., L i l l e y g ., Coloe S., B a i r d R ., and Pedersen J. (2002)
PCR identifcation of dermatophyte fungi Trichophyton rubrum, T. soudanense
and T. gourvilii. J. Med. Microbiol, Vol. 51,pp. 117-122.
- Liu D, Coloe S, Baird R, Pederson J. (1997) Molecular determination of
dermatophyte fungi using the arbitrarily primed polymerase chain reaction. Br
Dermatol ,Vol.137,pp:351-355.
- Liu D., Coloe S., Baird R., Pederson J. (2000) Application of PCR to the
identification of dermatophyte fungi. J Med Microbiol ,Vol.49,pp. 493-497.
58
- Machouart-Dubach M, Lacroix C, de Chauvin MF, Le Gall I, Giudicelli C,
Lorenzo F, Derouin F. (2001) Rapid discrimination among dermatophytes,
Scytalidium spp., and other fungi with a PCR-restriction fragment length
polymorphism ribotyping method . J Clin Microbiol,Vol.39(2),pp:685-690.
- Monod M., Bontems O., Zaugg C., Léchenne B., Fratti M. and Panizzon R.
(2006) Fast and reliable PCR/sequencing/RFLP assay for identification of fungi
in onychomycoses. J Med Microbiol , vol. 55 ( 9),pp: 1211-1216.
- Moriello k. (2004) Treatment of dermatophytosis in dogs and cats. Veterinary
Dermatology,Vol.15,pp.99-107.
- Mukesh Sharma, Meenakshi Sharma and Vijay Mohan Rao. (2011) In vitro
biodegradation of keratin by dermatophytes and some soil keratinophiles.
African Journal of Biochemistry Research Vol. 5(1), pp: 1-6.
- Nagao K., Takashi S., Takashi O. (2012) Identification of Trichophyton rubrum
by nested PCR analysis from paraffin embedded. (Microbilogy, 2012)N.
- Nagao, K., S. Takashi, O. Takashi, and N. Takeji. (2005) Identification of
Trichophyton rubrum by nested PCR analysis from paraffin embedded specimen
in trichophyton profunda acuta of the glabrous skin. Jpn. J. Med.Mycol,Vol.
46,pp:129–132.
- Neji S., Makni F., Sellami H., Cheihrouhou F., Sellami A., Ayadi A. (2010)
Molecular identification of dermatophytes isolated in Sfax- Tunisia. J Mycol
Med ,Vol.20,pp.85-90.
- Odom RB. (1994) Common superficial fungal infections in immunosuppressed
patients. J Am Acad Dermatol,Vol. 31,pp.56-59.
- Ohst T, de Hoog S, Presber W, Stavrakieva V, Gräser Y. (2004) Origins of
microsatellite diversity in the Trichophyton rubrum-T. violaceum clade
(Dermatophytes). J Clin Microbiol.,Vol.42(10),pp:4444-4448.
- PCR station http://www.pcrstation.com/nested-pcr/accessed at 9/6/2011
- Robert R., Pihet M. (2008) Conventional methods for the diagnosis of
dermatophytosis. Mycopathologia ,Vol.166,pp.295-306.
59
- Roque D., Vieira R., Rato S., and Luz-Martins M. (2006) Specific Primers for
Rapid Detection of Microsporum audouinii by PCR in Clinical Samples. J. Clin.
Microbiolvol.44 ( 12),pp: 4336-4341.
- Sharma A., Chandra S., Sharma M. (2010) Prevalence of keratinophilic
fungi in semi−arid region with particular reference to soil pH. Asian J Exp
Sci, Vol. 24, pp: 59−63.
- Shin JH., Sung JH., Park SJ., Kim JA., Phd MD., Lee JH. (2003) Species
identification and strain differentiation of dermatophyte fungi using polymerase
chain reaction amplification and restriction enzyme analysis. J Am Acad
Dermatol ,Vol48,pp.553–561.
- Shinkafi A., and Manga B., (2011) Isolation of Dermatophytes and Screening of
selected Medicinal Plants used in the treatment of Dermatophytoses.
International Research Journal of Microbiology, Vol. 2(1) pp:. 040-048.
- Shiraki Y., Soda N., Hirose N., Hiruma M. (2004) Screening Examination and
Management of Dermatophytosis by Trichophyton tonsurans in the Judo Club of
a University. Nippon Ishinkin Gakkai Zasshi,Vol 45(1),pp.7-12
- Simpanya M. (2000) Dermayophytes: Their taxonomy, ecology and
pathogenicity. Revista Iberoamericana de Micologya, pp: 1-12.
- Spiliopoulou A., Bartzavali C., Anastassiou ED., Christofidou M.(2011)
Detection of Trichophyton rubrum by multiplex PCR and all other
dermatophytes from nails. Eur Soc Clin Microbiol Infect Dis. Italy . Vol.
46(2),pp.129-132.
- Stukenbrock EH, Rosendahl S. (2005) Development and amplification of
multiple co-dominant genetic markers from single spores of arbuscular
mycorrhizal fungi by nested multiplex PCR. Fungal Genet
Biol.,Vol.42(1),pp:73-80.
- Taleb M. (2010) Microbiological studies in tintea capitis, and its treatment by
species oils in Gaza strip. Unpublished.
- The center for food security and public health
:http://www.cfsph.iastate.eduaccessed at 25/4/2011.
63
- Turin L., Riva F., Galbiati G., Cainelli T. (2000) simple and highly sensitive
double-rounded polymerase chain reaction assay to detect medically relevant
fungi in dermatological specimens. Eur J Clin Invest ,Vol.30,pp.511-518.
- Uchida T, Makimura K, Ishihara K, Goto H, Tajiri Y, Okuma M, Fujisaki R,
Uchida K, Abe S, Iijima M. (2009) Comparative study of direct polymerase
chain reaction, microscopic examination and culture-based morphological
methods for detection and identification of dermatophytes in nail and skin
samples. J Dermatol,Vol.36(4),pp:202-208.
- Urano S., Shirai S., Suzuki Y., Sugaya K., Takigawa M., Mochizuki T.(2003)
A case of Tinea capitis caused by Trichophyton tonsurans. Jpn J Med Mycol
,Vol. 44,pp.25-29.
- Venugopal P., and venugopal T., (1993) Tinea capitis in Saudi Arabia. Int.J.
Dermatol.,Vol.32,pp. 39-40.
- Verrier J, Pronina M, et al. (2012). Identification of infectious agents in
onychomycoses by PCR-terminal restriction fragment length polymorphism. J.
Clin. Microbiol.,Vol.50(3),pp:553-561.
- Weitzman I., McGinnis A., PadhyeA., and AjelloL., (1986) The genus
Arthroderma and its later synonym Nannizzia. Mycotaxon.,Vol. 25,pp.505-518.
- Weitzman I., and Summerbell R., (1995) The dermatophytes.Clin. Microbiol.
Rev,Vol. 8,pp.240–259.
- Winter I, Uhrlaß S, Krüger C, Herrmann J, Bezold G, Winter A, Barth S, Simon
JC, Gräser Y, Nenoff P. (2013) Molecular biological detection of dermatophytes
in clinical samples when onychomycosis or tinea pedis is suspected. A
prospective study comparing conventional dermatomycological diagnostics and
polymerase chain reaction. Hautarzt, Vol. 64(4),pp:283-289.
- Winter I., Uhrla S., Krüger C., Herrmann J., Bezold G., Winter A., Barth S.,
Simon JC., Gräser Y., Nenoff P. (2013) Molecular biological detection of
dermatophytes in clinical samples when onychomycosis or tinea pedis is
suspected: A prospective study comparing conventional dermatomycological
diagnostics and polymerase chain reaction . Hautarzt.,Vol.64(4),pp.283-289.
66
- Yan D., Li L., Chen DY., Zhang YH., Hu CH., Deng ZH. (2007) Detection of
fungi in liquor workers with tinea corporis and tinea cruris using arbitrarily
primed polymerase chain reaction. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing
Za Zhi,Vol 25(3),pp.133-135.
- Yang G., Zhang M., Li W., An L. (2008) Direct Species Identification of
Common Pathogenic Dermatophyte Fungi in Clinical Specimens by Semi-nested
PCR and Restriction Fragment Length Polymorphism.Mycopathologia,Vol
166(4),pp.203-208.
- Zaki S., Ibrahim N., Aoyama K., Shetaia y., Abdel-Ghany, A. and Mikami Y.
(2008) Dermatophyte infections in Cairo, Egypt, Science and Business Media B.
Mycopath.,Vol.167,pp.133-139.
62
Appendix
استبيان خاص بالبحث
رقـى انؼيــح: انراريـخ:
انؼـــــز: انجس: االســى:
رقى انجىال/ انهاذف: انؼـىا:
أخزي ػم يشرزك روضح يذرسح يكا انؼم أو انذراسح:
ال ؼى هم يىجذ حيىااخ في يكا انسك؟
أخزي أغاو حيز كالب تقز دواج قطط أىاػها:
ال عى هم يىجذ أيزاض يزيح؟
ال عى هم يزضد خالل األشهز األخيزج؟
ال عى هم ذأخذ كىرذيزول؟
ال عى هم ذأخذ يضاداخ حيىيح؟
انؼانج:ذشخيص انطثية
انريجح:
KOH:
First PCR:
Nested PCR:
يالحظاخ:
انرىقيغ: