Mycology -introduction and lab diagnosis with QC

1. INSTITUTE OF PUBLIC HEALTH, DHAKA
Department of Laboratory Medicine
BSc in Health Technology (Laboratory)- L4
MYCOLOGY
Lecture –Introduction & Lab Diagnosis
By
Sk. MIZANUR RAHMAN
Assistant Bacteriologist,
PhD Candidate
MS in BGE; MS in Microbiology; MPH in Epidemiology

2. The Fungi
Learning outcomes- Students should be able to:
• Establish familiarity with the scientific terminology
peculiar to mycology
• Describe the dimorphic nature of the pathogenic fungi
used in making a clinical diagnosis.
• Emphasize the eukaryotic nature of the fungi.
• To explore the nature of the pathogenesis of fungal
infections.
• To gain familiarity with the classification of medically-
important fungi.
• To develop an understanding of the nature and mode of
action of anti-fungal agents

3. Recommended Textbooks
Theory
• Medical Mycology and Human Mycoses ES Beneke and AL Rogers Star
Publishing Company 1996 Belmont CA
Laboratory
• Identifying Filamentous Fungi: A Clinical Laboratory Handbook St-
Germain G and Summerbell R Rogers Star Publishing Company 1996
Belmont CA
Additional Reading
• - Introductory Mycology Alexopoulos CJ Mims CW Blackwell M Fourth
Edition John Wiley &Sons Inc 1996 New York NY
• - Medical Mycology KJ Kwon-Chung and JE Bennett Lea &Febiger 1992
Philadelphia PA
• - Medical Microbiology A Laboratory Manual: Section II WG Wu Third
Edition Star Publishing Company 1995 Belmont CA

4. Medical Mycology Iceberg

8. What is a Fungus ?
• Eukaryotic – a true nucleus
• Do not contain chlorophyll
• Have cell walls
• Produce filamentous structures
• Produce spores

10. Species of Fungi
• 100,000 – 200,000 species
• About 300 pathogenic for man

11. Fungal Morphology and Structure
Eukaryotic organisms, distinguished by a rigid cell
wall composed of chitin and glucan, and a cell
membrane in which ergosterol is substituted for
cholesterol as the major sterol component.
Fungal taxonomy relies heavily on morphology and
mode of spore production
Fungi may be unicellular or multicellular.
The simplest grouping based on morphology divides
fungi into either yeast or mold forms.

12. Features of Fungi and its value in our
life:
The fungi are a ubiquitous and diverse organisms, that degrade organic
matter.
Fungi have heterotrophic life; they could survive in nature as:
Saprophytic: live on dead or decaying matter
Symbiotic: live together and have mutual advantage
Commensal: one benefits and other neither benefits nor harmed.
Parasitic: live on or within a host, they get benefit and harm the other.
Fungi mainly infect immunocompromised or hospitalized patients with
serious underlying diseases.
The incidence of specific invasive mycoses continues to increase with
time
The list of opportunistic fungal pathogens likewise increases each year “It
seems there are no non-pathogenic fungi anymore ! “
This increase in fungal infections can be attributed to the ever-growing
number of immunocompromised patients.

13. Characteristics of fungi
A. eukaryotic, non- vascular organisms
B. reproduce by means of spores (conidia), usually wind-
disseminated
C. both sexual (meiotic) and asexual (mitotic) spores may be
produced, depending on the species and conditions
D. typically not motile, although a few (e.g. Chytrids) have a motile
phase.
E. like plants, may have a stable haploid & diploid states
F. vegetative body may be unicellular (yeasts) or multicellular
moulds composed of microscopic threads called hyphae.
G. cell walls composed of mostly of chitin and glucan.

14. More Characteristics of Fungi
H. fungi are heterotrophic ( “other feeding,” must feed on
preformed organic material), not autotrophic ( “self feeding,”
make their own food by photosynthesis).
- Unlike animals (also heterotrophic), which ingest then digest,
fungi digest then ingest.
-Fungi produce exoenzymes to accomplish this
I. Most fungi store their food as glycogen (like animals). Plants
store food as starch.
K. Fungal cell membranes have a unique sterol, ergosterol, which
replaces cholesterol found in mammalian cell membranes
L. Tubule protein—production of a different type in microtubules
formed during nuclear division.

15. Structure
•The body of fungi is termed thallus (non-
reproductive)
•The thalli of yeast are small, globular and are single
celled
•The thalli of mold are composed of long, branched
tubular filaments called hyphae.

16. Structure
• The thallus of a mold is composed of hyphae
intertwined to form a tangled mass called
mycelium.

17. Mycotic Diseases
(Four Types)
1. Hypersensitivity
– Allergy
2. Mycotoxicosis
– Production of toxin
3. Mycetismus (mushroom poisoning)
– Pre-formed toxin
4. Infection

18. The Clinician Must Distinguish Between:
• COLONIZATION
• FUNGEMIA
• INFECTION

19. EYE
SKIN
UROGENITAL TRACT
ANUS
MOUTH
RESPIRATORY
TRACT
Portal of Entry
•SKIN
•HAIR
•NAILS
•RESPIRATORY TRACT
•GASTROINTESTINAL
TRACT
•URINARY TRACT

20. EYE
SKIN
UROGENITAL TRACT
ANUS
MOUTH
RESPIRATORY
TRACT
Colonization
Multiplication
of an organism
at a given site
without harm
to the host

21. EYE
SKIN
UROGENITAL TRACT
ANUS
MOUTH
RESPIRATORY
TRACT
Infection
Invasion and
multiplication
of organisms
in body tissue
resulting in
local cellular
injury.

22. Classification of fungi
They are classified by several methods:
1- Morphological classification
2- Systematic classification
3- Clinical classification

23. Fungal Morphology
Yeast
Hyphae (threads)
making up a mycelium
Mould
Encapsulated yeast
Cryptococcus neoformans

24. • Unicellular fungi
• Fission yeasts divide symmetrically
• Budding yeasts divide asymmetrically
Saccharomyces and Candida
Yeasts

25. Yeast Reproduction
• FISSION
• “even” reproduction, nucleus divides forming
two identical cells, like bacteria
• BUDDING
• “uneven” reproduction, parent cell’s nucleus
divides and migrates to form a bud and then
breaks away

27. Molds
• Multicellular, tubular structures (hyphae)
• Hyphae can be septate (regular crosswalls) or
nonseptate (coenocytic) depending on the
species (grow by apical extension)
– Vegetative hyphae grow on or in media (absorb
nutrients); form seen in tissue, few distinguishing
features
– Aerial hyphae contain structures for production of
spores (asexual propagules); usually only seen in
culture

28. • The fungal thallus consists of hyphae; a
mass of hyphae is a mycelium.
Molds

29. Moulds are multicellular organisms consisting of threadlike tubular
structures called Hyphae that elongate by apical extension.
Hyphae are either:
Coenocytic: hollow and multinucleate
Septate: divided by partitions or cross-walls
Hyphae form together to produce a mat-like structure
called a Mycelium.
Vegetative hyphae, grow on or under surface of culture medium,
Aerial Hyphae: project above surface of medium
Aerial H. produce Conidia (asexual reproductive elements)
Conidia can easily airborne and disseminate the fungus.
Many medical fungi are termed dimorphic because they exist in yeast
and mould forms.

30. Septate hyphae Non-Septate hyphae Myceliu
m

33. Dimorphic Fungi
• Growth as a mold or as a yeast
• Most pathogenic fungi are dimorphic fungi
• At 37o C yeast-like
• At 25o C mold-like
• Can also occur with changes in CO2
• Fungi grow differently in tissue vs
nature/culture; often dictated by temp

34. • Some fungi are
dimorphic
depending on
environmental
conditions
• These organisms
produce both
yeast-like and
mold-like thalli
• Many are
pathogenic
• Candida albicans
Dimorphism

35. Dimorphism
Many pathogenic fungi are dimorphic,
forming moulds at ambient temperatures but
yeasts at body temperature.

36. Clinical Classification of
Mycoses
•Cutaneous
•Subcutaneous
•Systemic
•Opportunistic

37. Cutaneous Mycoses
Skin, hair and nails
Rarely invade deeper tissue
Dermatophytes

38. Subcutaneous Mycoses
• Confined to subcutaneous tissue and
rarely spread systemically. The
causative agents are soil organisms
introduced into the extremities by
trauma

39. Systemic Mycoses
• Involve skin and deep viscera
• May become widely disseminated
• Predilection for specific organs

40. Opportunistic Fungi
Ubiquitous saprophytes and occasional
pathogens that invade the tissues of
those patients who have:
• Predisposing diseases:
Diabetes, cancer, leukemia, etc.
• Predisposing conditions:
Agammaglobulinemia, steroid or antibiotic
therapy.

41. • Systemic mycoses Deep within body
• Subcutaneous mycoses Beneath the skin
• Cutaneous mycoses Affect hair, skin, nails
• Superficial mycoses Localized, e.g., hair
shafts
• Opportunistic mycoses Caused by normal
microbiota or
fungi
Fungal Diseases (mycoses)

42. Laboratory Diagnosis

44. Detection and recovery of fungi from
clinical specimens
 Dermatophytosis and Agents of Superficial Mycosis
 Specimen and direct microscopic examination
 Skin, nail scraping and hair shaft
 Placed in one or two drops of 10-20% KOH
 A cover slip is placed on top and the preparation
 is heated gently
 Nails may require a strong alkali solution (25%KOH) and
long clearing time
 N. B. combination of KOH plus Dimethyl sulfaoxide
(DMSO) may be used for nail specimens

45. Fungal Culture, Non-Dermal Sites (FCUL): Fungal culture only; no smear
Fungal Culture and Smear, Non-Dermal (FCULSM): Fungal culture and smear
Fungus CSF Culture/CAD (FUNCSF): For use with CSF only; fungal culture and
cryptococcal antigen detection
Cryptococcus Ag Detection (CAD): Cryptococcal antigen on serum only; does not
include culture
Fungal Blood Culture (HISTCL): Histoplasma blood or bone marrow culture
Fungus Screen (FUNGSC): Fungal screen only; for use when looking for yeast only
Fungal Culture and Smear: Hair, Skin, Nail (FHSNSM): Fungal culture plus smear
on hair, skin, nail
Fungal Culture: Hair, Skin, Nails (ACFSC): Culture only for dermatophytes (hair,
skin, nail); does not include smear
Mycology order codes

46. Human Mycosis-1
• Dermatophytosis /Superficial Mycoses/ Cutaneous
Mycoses/ Ringworm / Tinea ..
• Involve superficial keratinize.. Dead tissues.. skin, hair,
Nails.. Caused by Dermatophytes: Trichophyton -
Microsporium -, Epidermophyton species
• Worldwide distribution.. Spores, Hyphae fragments..
Common in nature, skin human, animals.
• Tinea versicolor / Pityriasis versicolor, Yeast
• Clinical Features: Erythematic Skin lesions..Rare
inflammation.. Allergic reaction.. Common under stress
conditions.. Fever, Unknown Factors.

47. Human Mycosis-2
• Skin spots commonly affect the back, underarm, upper arm,
chest, lower legs, and neck. Occasionally it can also be present
on the face.
• The yeasts can often be seen under the microscope within the
lesions with typically round yeasts & filaments. Light to Dark
patches on skin. Difficult to culture.
• Hair: Tinea capitis, Hairshaft /hair follicles. Scalp, Endo-
Exothrix, Common in Children.. Rare Adults.. Infection
Outbreaks .
• Nail: Tinea unguium &Tinea pedis.. Feet fingers, Feet
interspace, moist skin lesions, Common in Adults, develop
Chronic
• Causative agents: Dermatophytes.. Trichophyton -
Microsporium -, Epidermophyton species.

48. Important considerations:
 Proper collection
 Rapid delivery to the laboratory
 Prompt and correct processing
 Inoculation into proper and appropriate medium
 Incubation at a suitable temperature
Collection, handling and processing of clinical
mycology specimens

49. Transport of Specimen:
 Antibiotics may be incorporated in body fluid
specimens to prevent proliferation of bacteria:
 50, 000 units of Penicillin
 100,000 units of Streptomycin
 0.2 mg of Chloramphenicol
Collection, handling and processing of clinical
mycology specimens

50. Transport of Specimen:
 Storage temperature of specimen for fungal culture:
 Blood & CSF: 30 – 37 OC
 Dermatological: 15 – 30 OC
 Others: 4 OC
Collection, handling and processing of clinical
mycology specimens

51. SPUTUM
first early morning sample
 Deep cough specimen; may be induced by:
o Aqueous aerosol
o Bronchial tap
 Volume: 5 – 10 ml
Collection, handling and processing of clinical
mycology specimens

52.  BLOOD and BONE MARROW
 Transport medium: at 1:10 proportion
 TSB-Tryptic soy broth or TSA-Tryptic soy Agar
(biphasic agar or broth)
 BHI(Brain heart infusion) transport medium
 Thioglycollate broth
 Volume: 10 ml
Collection, handling and processing of clinical
mycology specimens

53.  CEREBROSPINAL:
 Transport immediately. Do not refrigerate.
 For suspected Cryptococcus, Coccidioides
infections, containers must be leak proof and
lab manipulations should be done under a hood
Collection, handling and processing of clinical
mycology specimens

54. DERMATOLOGICAL SPECIMENS
SKIN LESIONS
 Sterilized area with
70% alcohol or sterile
water
 Collect at the the active
border
Collection - usually by physician/nursing staff /Lab scientist/MT
Collection, handling and processing of clinical
mycology specimens

55. • Skin - cleaned with 70% alcohol to remove dirt, oil and surface
saprophytes
Collection, handling and processing of clinical
mycology specimens

56. • Nails - cleaned same as for skin. Usually clipped; need to be
finely minced before inoculating to media
Collection, handling and processing of clinical
mycology specimens

57. NAILS
Clean with 70% alcohol If:
Dorsal plate:
scrape the deeper portion
Nail plate:
scrape beneath the nail plate
Whole nail or clippings
Collection, handling and processing of clinical
mycology specimens

58. HAIR
Collect from:
 Areas of scaling
 Alopecia
 Hair that fluoresce under
Wood’s lamp
Collection, handling and processing of clinical
mycology specimens

59. • Hair - obtained from edge of infected area of scalp,. Use a
Wood's lamp (fluorescence) to help locate infected hair. Hair
can be obtained by plucking, brushing, or with a sticky tape.
Collection, handling and processing of clinical
mycology specimens

60. • Body fluids - normal sterile collection procedures
Collection, handling and processing of clinical
mycology specimens

61.  EXUDATES & PUS
 Undrained or unruptured abscess
 Aspirate using sterile syringe, recap
needle and transport to lab
immediately
 Failed aspiration, do skin biopsy
Collection, handling and processing of clinical
mycology specimens

62.  URINE
 First early morning
 Transport and perform test ASAP (as
soon as possible) within 2 hours
 If not possible, refrigerate specimen.
Collection, handling and processing of clinical
mycology specimens

63.  VAGINAL SECRETIONS
 Sterile swabs
 Put in transport medium or
primary isolation broth
immediately (ex: TSB-Tryptic
soy broth)
Collection, handling and processing of clinical
mycology specimens

64.  TISSUES & Biopsy specimens:
 Collect aseptically at the center and edge of the
lesion
 Place in between sterile gauze wet with sterile
NSS (Normal Saline Solution) or transport medium.
Collection, handling and processing of clinical
mycology specimens
Respiratory Mycoses
Disseminated fungal infection

66. Diagnosis
Common Tests

67. 1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and histopathology
6. Culture
7. DNA probes
Diagnosis

68. 1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and histopathology
6. Culture
7. DNA probes
Diagnosis

69. 1.Direct Microscopic examination
a) Wet preparation
- uses KOH or NaOH as clearing agent
b) Calcofluor white stain
- shows fungal elements in exudats & small skin scales under fluorescent
microscope
c) Nigrosin or India Ink
d) Wright stain or Giemsa stain

71. Direct Microscopic Observation
• 10 % KOH
• Gentle Heat

74. KOH Wet Mount

75. Budding Yeast/Germ Tubes

76. Capsulated Yeast / Cryptococcus
neoformans (India ink test)

77. Candida Trush

79. Diagnosis
1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probes

80. Skin Testing
(DERMAL HYPERSENSTIVITY)
Use is limited to :
– Determine cellular defense mechanisms
– Epidemiologic studies

82. Diagnosis
1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probes

83. FUNGI ARE POOR ANTIGENS

84. Fungal Serology Antibodies
• Latex Agglutination IgM
• Immunodiffusion IgG
• EIA IgG & IgM
• Complement Fixation IgG

85. for detection of antigen or antibody
-complement-fixation, Agglutination, Precipitin test
-useful only for systemic & opportunistic mycoses
• complement-fixation is used in suspected cases of
coccidiodomycoses, blastomycoses, histoplasmosis
Immunologic

87. Most serological tests for fungi measure
antibody. Newer tests to measure antigen
are now being developed
ANTIGEN DETECTION PRESENTLY AVAILABLE
 Cryptococcosis
 Histoplasmosis
 Aspergillosis

88. Diagnosis
1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probes

89. DIRECT FLUORESCENT ANTIBODY
1. HISTOLOGIC SECTIONS
2. CULTURE
• Viable organisms
• Non-viable organisms
CAN BE APPLIED TO

91. Diagnosis
1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probe

92. • Periodic Acid Shift
• Gomori Methenemine Silver Stain
• Calcofluor white
• Fluorescent Antibody Stain
- for rapid diagnosis of fungal; cell wall
Histologic stains

93. Inflamatory Reaction
• Normal host
–Pyogenic
–Granulomatous
• Immunodeficient host
–Necrosis

94. Polymorphic Nuclear Leukocytes

95. Giant Cell

96. GMS(Gomori Methenamine Silver Stain)

97. Diagnosis
1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probes

98. • Slow growers
• Medium: Sabouraud Dextrose Agar, Potato Dextros Agar, Blood
agar Corn Meal Agar
IDENTIFICATION OF FUNGUS
a. Macroscopic examination
- study the mycotic colony, mycelium & the pigment produced
b.Microscopic examination
- uses a drop of Lactophenol Cotton Blue (LPCB)
-observe the size, shape, septation & color of spores
Culture

99. •IDENTIFICATION OF YEAST CULTURES
- is based on morphologic characteristics & biochemical tests
•IDENTIFICATION OF FILAMENTOUS FUNGAL CULTURES
- uses an immunologic method called exoantigen test
* antigen extracted are immunodiffused against known antisera

100. Isolation Media
SABOURAUD DEXTROSE AGAR
(pH ~ 5.6)
•Plain
•With antibiotics
•With cycloheximide

101. Incubation Temperature
• 370 C - Body temperature
• 250 C - Room temperature

102. 1. Wet Mount
2. Skin test
3. Serology
4. Fluorescent antibody
5. Biopsy and
histopathology
6. Culture
7. DNA probes
Diagnosis

103. DNA Probes
• Rapid (1-2 Hours)
• Species specific
• Expensive
DNA probe test
-identify colonies growing in culture at an earlier stage of
growth
-available for coccidioides, histoplasmas, blastomyces,
Cryptococcus