(3) Collection, Handling, Processing of Clinical Mycology Specimens

Question 1

Enumerate the Techniques to obtain culture material for slide preparation

Answer 1

1. Tease Mount Method
2. Cellophane Tape Method
3. Slide Culture/ Microculture/ Riddel’s Method

Question 2

Techniques to obtain culture material for slide preparation

• One of the most common technique done in the laboratory.
• A dissecting needle, commonly a bent wire, is used to pull apart a fungal colony which is placed on a slide.
• Usually done when placing an LPCB stain on a culture sample.

Answer 2

Tease Mount Method

Go over the procedure

Question 3

Techniques to obtain culture material for slide preparation

Cellophane tape is used to transfer aerial hyphae from the colony to a microscope slide for examination

Answer 3

Cellophane Tape Method

Go over the procedure

Question 4

Techniques to obtain culture material for slide preparation

Allows for in-situ analysis of fungi with as little disruption on the structures as possible

Answer 4

Slide Culture/ Microculture/ Riddel’s Method

go over the procedure

Question 5

Enumrate the Biochemical Tests

Answer 5

1. Urease Hydrolysis Test
2. Germ Tube Production
3. Carbohydrate Assimilation Test
4. Red Colonial Pigmentation
5. Woods Lamp Examination

Question 5

Biochemincal Tests

Tests an organism’s ability to produce exoenzyme, called urease, which hydrolyzes urea to ammonia and carbon dioxide.

Answer 5

Urease Hydrolysis Test

Question 6

Biochemincal Tests

what is the positive reaction for Urease Hydrolysis Test

Answer 6

PINK

Urease (+) organisms generate enough ammonia to turn the phenol red indicator in the media into PINK.

Question 7

Biochemincal Tests

what are the Urease positive fungies

Answer 7

Cryptococcus neoformans and
Trichophyton mentagrophytes

Question 8

Biochemincal Tests

hyphae-like extensions of young yeast cells showing parallel non-septate sides

Answer 8

Germ Tube Production

Question 9

Biochemincal Tests

what are teh fungi positive for Germ tube

Answer 9

Candida albicans and Candida dubliniensis

go over the procedure

Question 10

Biochemincal Tests

• Determine the ability of a yeast to use a carbohydrate as its sole carbon source.
• Uses a yeast nitrogen base broth

Answer 10

Carbohydrate Assimilation Test

Question 11

Biochemincal Tests

A (+) result for Carbohydrate ASsimilation test indicated by the growth of yeast means

Answer 11

the fungi can use carbohydrate as its carbon source

Question 12

Biochemincal Tests

• Use the potato dextrose
  agar to enhance the pigment production in some dermatophytes

red pigment is then observed in the sub-surface of the agar surrounding the
fungi

Answer 12

Red Colonial Pigmentation

Question 13

Biochemical Tests

What fungi are red pigment producers

Answer 13

Trichophyton rubrum
and Rhodotorula species

Question 14

Biochemical Test

Diagnostic test in which the skin or hair is examined while exposed to the black light emitted by a wood lamp

Answer 14

Woods Lamp Examination

Question 15

Biochemical Test

Infected skin or hair (+) for wood lamps examination, dermatophytes will fluoresce what color?

Answer 15

bright green to yellow-green

The test works for both zoonotic and human infections.

Question 16

Important in cases of some fungal infections where the fungal polysaccharide or proteins are shed out in body fluids

Answer 16

Serology-Antigen Detection Test

Question 17

Serology- Antigen used

Polysaccharide capsular antigen of Cryptococcus neoformans

Answer 17

Glucuronoxylomannan

Question 18

Serology- Antigen used

Mannan Antigens

Answer 18

For Candida species

Question 19

Serology- Antigen used

Galactomannan Antigens

Answer 19

For Aspergillus species

Question 20

Match the antigen

1. Galactomannan
2. Glucuronoxylomannan
3. Mannan

a. Polysaccharide capsular antigen
of Cryptococcus neoformans
b. Aspergillus species
c. Candida species

Answer 20

1. b
2. a
3. c

BACcla

Question 21

Provides information that aids the physician in selecting the appropriate anti-fungal medicine totreat a specific fungal infection.

Answer 21

Antifungal Susceptibility Testing (AST)

Question 22

AST results for fungi are easily influenced by methodological factors such as?

Answer 22

• Isolate growth phase and inoculum size
• Incubation time and temperature
• Type of media used
• Method of reading

Question 23

Enumerate the Type of AST

Answer 23

1. Brothe Dilution
2. Disk Diffusion Method

Question 24

# Types of AST * Different concentrations of **one anti-microbial agent (AMA)** against one fungal isolate.

Answer 24

Broth Dilution

Question 25

# Types of AST Determines the minimum inhibitory concentration (MIC)—the lowest concentration of AMA that can inhibit the growth of fungi

Answer 25

Broth Dilution

Question 26

# Type of AST * AMA are impregnated onto paper disks. * Several AMA with standardized concentrations against one isolate

Answer 26

Disk Diffusion Method

Question 27

These standards were formed due to the wide factors that can affect the results of antifungal susceptibility test

Answer 27

CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI) STANDARDS FOR AST

Question 28

# AST what document pertains to reference method for broth dilution antifungal susceptibility testing of **YEASTS**

Answer 28

Document M27-A4

Question 29

# AST Reference method for broth dilution antifungal susceptibility testing of **FILAMENTOUS FUNGI**

Answer 29

Document M38-A3

Question 30

# AST DOCUMENT M38-A3 is of Microdilution method for molds that causes invasive and cutaneous infections including what spp?

Answer 30

**Aspergillus species**, **Fusarium species**, and **Zygomycetes**

Question 31

# AST Method for antifungal **DISK DIFFUSION** susceptibility testing of **yeasts**

Answer 31

Document M44-A2

Question 32

DOCUMENT M44-A2 are intended only for testing this spp and not for other genera

Answer 32

Candida species

Question 33

# identify what AST document Final Inoculum: **0.5-2.5x 103 CFU/mL** Test Medium:Roswell Park Memorial Institute **(RPMI)** 1640 with 0.2% glucose Growth Temperature: **35 ‘C** Incubation Time: **24 to 48 hours** Endpoint: **50% inhibition** Reading of Endpoint: Visually

Answer 33

DOCUMENT M27-A4

Question 34

# identify what AST document Final Inoculum: **0.4-5 x 104 CFU/mL** Test Medium: Roswell Park Memorial Institute (RPMI) 1640 with 0.2% glucose Growth Temperature:** 35 ‘C** Incubation Time: **48 hours** Endpoint: **100% inhibition** Reading of Endpoint: Visually

Answer 34

DOCUMENT M38-A3

Question 35

Identify the Antifungal agents

Answer 35

1. Polyene Macrolide Antifungals 2. Azole Antifungal Drugs 3. Polyene Macrolide Antifungals

Question 36

# Antifungal agents This group of antifungal agents **BINDS TO THE ERGOSTEROL** of the fungal cell membrane causing the membrane to weaken and results to fungal cell death

Answer 36

Polyene Macrolide Antifungals

Question 37

# Antifungal agents what are the Polyene Macrolide Antifungals

Answer 37

- Amphotericin B - Nystatin - Griseofulvin - 5-Fluorocytosine or Flucytosine

Question 38

# Antifungal agents This group of antifungal agents INHIBITS THE FUNGAL **CYTOCHROME P450 ENZYMES** o These enzymes are needed for the synthesis of ergosterol in the cell membrane of fungi

Answer 38

Azole Antifungal Drugs

Question 39

# Antifungal agents what are the Azole Antifungal Drugs

Answer 39

* Clotrimazole and Miconazole * Fluconazole * Ketoconazole * Itraconazole * Voriconazole * Posaconazole * Anidulafungin * Micafungin

Question 40

# Antifungal agents This group of antifungal agents **INHIBITS BETA-(1,3)-D-GLUCAN SYNTHASE** which is an important enzyme for the synthesis of the components of fungal cell wall.

Answer 40

Polyene Macrolide Antifungals

Question 41

# Antifungal agents What are the polyene macrolide antifungals used

Answer 41

* Caspofungin * Selenium Sulfide * Potassium Iodide

Question 42

# Antifungal agents INHIBITS **SQUALENE EPOXIDASE** which is needed for the synthesis of ergosterol in the fungal cell membrane

Answer 42

Terbinafine or Lamisil

Question 43

ti

Answer 43

te