Dermatophytes & Lab diagnosis of fungi

1. DR V S VATKAR
Asso Prof
Microbiology Department
D Y Patil Medical College, Kolhapur

2. 
Objectives
Classification of fungi
Infections caused by fungi like
superficial mycoses, deep mycoses,
opportunistic mycoses
Mycotoxicoses
Laboratory diagnosis of fungi

3. Mykos (Greek word): fungus, study of
fungi
Most of the fungi : saprophytes
Human infections : mainly
opportunistic
Widespread use of modern advances in
treatment: antibiotics, steroids &
immunosuppressive agents, leads to
opportunistic fungal infection
Introduction

4. 
Eukaryotes
Rigid cell wall: chitin, mannan & other
polysaccharides
Cytoplasmic membrane contains sterols
True nuclei with nuclear membrane & paired
chromosomes
Divide asexually, sexually or by both
May be unicellular/multicellular
Characteristics of fungi

5. 
CLASSIFICATION
MORPHOLOGICAL CLASSIFICATION
YEAST YEAST-LIKE MOULDS DIMORPHIC FUNGI
Cryptococcus Candida
Aspergillus
Mucor
Penicillium
Rhizopus
Dermatophytes
Histoplasma capsulatum
Cocciidioides immitis
Sporothrix schnkii
Blastomyces dermatidis
Paracoccidiodes brasiliensis
Penicilliosis marneffei

6. 
Morphological Classification
Yeasts:
 Unicellular
 Reproduce by budding
 Macroscopic : pasty colonies
 Microscopic: spherical/oval
forms, filamentous forms:
not seen
 e g Cryptococcus
neofrmance
Yeast like fungi:
 Unicellular
 Reproduce by budding & by
fission
 Macroscopic : pasty colonies
 Microscopic :
spherical/oval forms,
filamentous forms seen
 e g Candida albicans

7. 
Morphological Classification
 Filamentous fungi/Moulds:
 Hyphae: septe (cross walls,
multicellular) or aseptate
(coenocytic)
 Reproduction: asexual: spore
formation (some exhibit sexual
reproduction)
 Macroscopic:
cottony/woolly/velvety/granu
lar, pigmentation on the reverse
 Microscopic: thread like
filamentous hyphae aseptate
or septate.
 Mycelium : vegetative/arial
& shapes like racquet,
nodular, pectinate , spiral,
root-like etc
 e g Aspergillus fumigatus,
Penicillium, Mucor etc

9. 
Thermally Dimorphic fungi:
 Grow as filaments in culture at 22 0 C & in
environment
 yeast forms at 37 0 C
 e g Histoplasma capsulatum
Morphological Classification

11. 
Phycomycets:
 lower fungi,
 Non septate hyphae
 Forms endogenous spores called sporangeospores :
swollen sac like structures called SPORANGIA
 Sexual spores: oospores in some fungi & zygospores
in others
SYSTEMIC CLASSIFICATION
based on their sexual spore formation

12. 
Ascomycets:
 Highre fungi
 Sexual spores (ascospores) within a sac
 Includes both filamentous & yeast forms

13. 
Basidiomycetes:
 Higher fungi
 Forms sexual spores (basidiospores) on the base/
basidium

14. 
Fungi imperfecti:
 Most of the medically important fungi belongs to
this group

17. 
Pathogenic classification
Primary pathogenic: causes infection in healthy
person
 e g diamorphic fungi
Opportunistic pathogenic: causes infection in
immunocompromised patients

18. Superficial mycoses
Cutaneous mycoses
Subcutaneous mycoses
Deep/systemic mycoses
Opportunistic mycoses
Mycotoxicoses: disease produce due to fungal toxins
 Hypersensitivity (allergic reactions): inhalation of fungal
spores
Fungal infections (Mycoses)

19. Microscopy
Culture
Serology
Skin test
Antifungal susceptibility test
Laboratory Diagnosis

20. 
 Collect from affected site : skin scrapings, nail bed
etc
 Disseminated infections: blood
Specimens

21. Potassium hydroxide (KOH) preparation:
 Tissue specimens like skin scrapings : examined as
wet mount (10% KOH)
 Tissue : kept in KOH for 20 min incubator at 37 0 C,
for affected nail samples 40% KOH is used
 Alkali digest cells & other tissue materials, we can
see fungus clearly
Microscopy

22. 
Calcoflour white:
 Sensitive staining technique, good visualization of
fungal morphology
Gram stain: yeast & Yeast like fungus

23. 
India ink preparations: Negative staining: to
visualise the capsule of fungus

24. 
Methenamine silver stain & Periodic –acid-
schiff (PAS) stain: used to demonstrate fungus in
tissue
Lactophenol cotton blue stain: widely used
method to see morphology

25.  Most of the fungi grow in media used to grow
bacteria but at low pH
 Grow better at 25-30 0 C except fungi causing
systemic mycoses(37 0 C). Aspergillus fumigatus :
grow even at 50 0C
Media :
Specific medias
Non- Specific medias
Culture

26. 
SABOURAUD DEXTROSE agar ,
Sabouraud glucose neopeptone agar
(antibiotics added)
Common culture media
pH 5.4, neutral
Czapek-Dox medium, bird seed agar, Niger
seed agar, corn meal agar
Cyclohexamide : incorporated in it to
prevent contamination by moulds
Cultures : incubated at room temperature (22
0 C) for wk & at 37 0C for days

27. 
Growth characteristics
 Rapidity of growth
 Colour & morphology
of the colony on
obverse
 Pigmentation on the
reverse
 Morphology of hypha:
diameter, septa, conidia
etc

28. 
Non specific media :
Brain heart infusion agar
Brain heart infusion broth
Blood agar

29. Teased mount:
 Bit of fungal colony is teased out from culture plate,
 Place a drop of lactophenol cotton blue stain
 Put a cover slip
 View under low power & then focus under high
power
 Proper morphology is difficult to view in teased
mount
Morphological studies can be studied by teased
mount, slide culture & cellophane tape preparation

30.  Proper morphology of fungus is seen .
 Procedure: sterile glass slide , place it on a bent glass
rod in a petri dish, a 1 cm square block of SDA is
placed on it
 Test strain of fungus is inoculated at four sides of
agar block
 Inoculated block is covered with sterile cover slip
 Incubate it at 25 0 C for 48 hrs
 Take a clean glass slide, put a drop of LCB stain over
it, transfer a cover slip on this slide
 Observe under microscope
Slide Culture

31. 
Cellophane tape mount
 Technically easy
Serology:
 Antibody detection: agglutination, Complement
Fixation, Immunodiffusion, CIEP, ELISA
 Antigen detection: Latex agglutination
(Cryptococcus)
Newer rapid diagnostic tests: Nucleic acid
hybridization, PCR

32. 
 Infection of dead layer of skin & its appendages
 No inflammatory response
 Cosmetic effects
 Cutaneous Infections
 Dermatophytes mainly causative agents
 Candida
Superficial Mycoses

33. 
Pityriasis versicolar (Tinea
versicolar)
 Usually asymptomatic
 Does not beyond stratum
corneum layer of skin
 World wide distribution
 Prevalent in torpics
 Seen in young adults
mainly
 Causitive agent :
Malassezia furfur
 C/F: confluent macular
areas of discolouration or
depigmentation of skin of
chest, abdomen, upper
limbs and back.
 Present on normal skin ,
but opportunistic
pathogen
 Diagnosis: skin scrapings
shows yeast like cells,
short branched filaments,
 Grow on SDA covered
with olive oil

34. Depigmentation of the skin

35. 
Tinea nigra
 Infection of stratum
corneum
 Palms
 Black/brownish
macular lesions
 Seen in tropics
 Causative agent:
Exophila werneckii,
Exophila castellanii
 Diagnosis:
 Skin scraping
 Brownish, branched,
septate hyphae and
budding cells
 SDA colonies: grey or
black colonies

36. Infection of hair
Firm , irregular nodules along the hair shaft
Nodules : composed of fungal elements,
cemented together on the hair shaft
Black piedra : caused by Piedraia hortae
White piedra: caused by Trichosporon
beigelii
Piedra

37. BLACK PIEDRA

39. WHITE PIEDRA

41. 
Commonly called ringworm or tinea
infection
Infection of keratinised structures like
hair, nail & skin caused by keratophilic
Dermatophytes
 acute or chronic
Dermatophytes

42. 
 Characteristics of
dermatophytes:
 Hyaline filamentous
fungi, digest keratin by
producing enzyme
keratinase
 Resistant to
cyclohexamide
 Classified into 3 genera
 Microsporum
 Trichophyton
 Epidermophyton
 Microsporum: M
gypseum, M cannis
 Trichophyton: T rubrum,
T mentagrophytes , t
verrucosum
 Epidermophyton: E
floccosum

43.  Twice common in males than females
 Tinea barbae (barber’s itch): involves beared area of
face & neck
 Tinea corporis: smooth or non-hairy skin of the body
 Tinea imbricata: found in tropics, extensive
concentric rings papulosquamous scaly patches
 Tinea capitis: affects scalp
 Tinea cruris: affects groin & perineum
 Tinea pedis: (athlet’s foot)
 Tinea manuum: affects hands
 Tinea unguium: affects nails
Clinical aspects

44. Tinea barbae
Tinea corporis
Tinea cruris
Tinea capitis

45. Tinea manuum
Tinea pedis
Tinea imbricata
Tinea unguium

46. 
Clinical features
Skin lesions: circular,
dry, erythematous, scaly
& itchy
Hair lesions: kerion,
sacrring & alopecia,
 a) Favus: chronic type of
ringworm, dence crusts
(scutula) develop in the
hair follicles leading to
alopecia & scarring
 b) Kerion: severe boggy
lesions with marked
inflammation sometimes
develop on scalp
 Nail lesions:
 deformed, friable,
discolouration
 Accumulation of debris
under the nails

47. 
 Mechanism : unclear
 Fungal products : responsible for local inflammation
 Hypersensitivity to fungal Ags: sterile vesicular
lesions, sometimes seen in sites distant from
ringworm lesions,
 These lesions are called as dermatophytidis
 Diagnosis : by clinical features, use of Wood’s lamp
Pathogenicity

48. Specimen: skin scrapings (from edges of the
lesions), nail & hair clippings(hair plucked from the
scalp)
Microscopy: wet preparation: scrapings from the
lesion is placed in a drop of 10-20% KOH on a slide,
put a cover slip, keep for 10-20 min to digest keratin.
Then observe the slide under the microscope
Laboratory diagnosis

49. 
 Hair: plucked hair examined under UV light (Woods
lamp)
 Ectothrix : arthrospores seen surrounding the hair
shaft
 Endothrix: spores : inside the hair shaft

50.  Specimen : inoculated on SDA containing
Chloramphenicol or SDA with chloramphenicol &
cycloheximide
 Incubated aerobically at 25-30 0 C upto 21 days
 identification by macroscopic & microscopic appearance
of fungal colonies
 Trichophyton: powdery, velvety or waxy with
pigmentation
 Microsporum: cotton like, velvety or powdery with white
to brown pigmenation
 Epidermophyton: powdery greenish or yellow colonies
Culture

52. 
Trichophyton: Microconidia abundant, scanty
Macroconidia: thin, elongated , blunt ends, spiral
hyphae, racquet mycelium, affects hair, nail & skin
Microsporum: scany microconidia,
Macroconidia: spore form, large, arranged singly on
the end of hyphae, affects hair & skin
Epidermophyton: powdery & greenish yellow,
microconidia : absent, Macroconidia: pear shaped,
arranged in clusters, affects skin & nails
Microscopic examination

56. 
Species identification: physiological
tests
Hydrolyses urea
Grow on polished rice grains
Special amino acids & vitamins can be
tested
Antigen detection: hypersensitivity test
(trichophytin)

57. Mild infection : topical imidazole
Severe infections: griseofulvin orally 4-6 wks
Hair : 3-6 mnths treatment
Nails: upto 1 year
Alternative treatment: oral imedazole like
ketoconazole 200 mg b d daily or itraconazole
100 mg od daily, fluconazole 150 mg od daily
Treatment

58. Human : main host for Anthrophilic
dermatophytes e g Tricophyton rubrum,
Microsporum audouinii, Epidermophyton
floccosum
Zoophilic: affects animals e g T.
verrucosum, M cannis in dogs
Geophilic: naturally in soil, less
pathogenic for human e g M gypseum
Epidemiology & Prevention

59. 
Personal cleanliness
 avoid contact with infectious
material
Proper treatment

60. THANK YOU