[2S] Exercise 6: Laboratory Diagnosis of Common Fungal Diseases

Question 1

constitute a major part of the ecosystem and are also known potential pathogen in human and animals

Answer 1

Fungi

Question 2

are specialized fungi that produce the enzyme keratinases that degrade keratin for their nutrition

Answer 2

Keratophilic fungi

Question 2

Soil rich in _______ materials favours the growth of these fungi

Answer 2

keratinous

Question 3

specific method for isolation of keratophilic fungi in soil

Answer 3

Hair Baiting Technique

Question 3

PRINCIPLE: Temperature, pH, soil humidity, texture, as well as depth of soil are the climactic factors to be considered for cultivation of keratophilic fungi. Human hair is a keratin-rich substrate often used as bait to trap and isolate keratophilic fungi

Answer 3

Hair Baiting Technique

Question 4

source of nutrition

Answer 4

Keratin

Question 5

• Substrate
• Rich in keratine

Answer 5

human hair

Question 5

Mainly composed of Hyphomycetes

Answer 5

Keratinophilic fungi

Question 6

is a keratin-rich substrate often used as bait to trap and isolate keratophilic fungi

Answer 6

human hair

Question 7

Media used for Hair Baiting

Answer 7

Saboraud Dextrose agar (with cycloheximide, penicillin and streptomycin)

Question 8

HAIR BAIT PROCEDURE

T/F: The collected hairs should be washed thoroughly with water then air dry at room temp

Answer 8

T

Question 9

HAIR BAIT PROCEDURE

T/F: Cleanse the collected hair baits with 70% alcohol. Air dry

Answer 9

T

Question 10

HAIR BAIT PROCEDURE

Sterilized the cleaned hair by autoclaving at ____ for __ min

Answer 10

15 lbs./sq inch; 20 min

Question 11

HAIR BAIT PROCEDURE

Sterile petri dishes are then half-filled with soil and moistened with _____ of sterile distilled water

Answer 11

15 to 30 mL

Question 12

HAIR BAIT PROCEDURE

Cover the plates and incubate the preparation at _______ in a ______ or cupboard for about 1-2 months. Soil may be moistened when necessary

Answer 12

room temperature (20—25°C); dark place

Question 13

HAIR BAIT

Examine the plates periodically for the development of ______ on the hair filaments. This can be done by taking a small amount of colony on hair bait, mount on a clean slide with saline or LPCB and examine the preparation under the microscope

Answer 13

mycelium

Question 14

HAIR BAIT

After fungal colonies have grown around the hair, subculture the colonies in a ____________ medium containing cycloheximide.

Answer 14

Sabouraud Dextrose agar

Question 15

HAIR BAIT

The keratinophilic fungi isolated and identified in this technique, particularly if it is a _____________ or its variant, may be used as test fungi for the Hair Perforation test

Answer 15

Trichophyton spp.

Question 16

The ability to perforate hair and utilize its keratin content through digestion by its keratinase enzymes is evident in the majority of the dermatophytes

Answer 16

Hair Perforation Technique

Question 17

An in vitro hair perforation test used to differentiate
keratophilic dermatophytes, particularly the Trichophyton spp. and its variant

Answer 17

Hair Perforation Technique

Question 17

HAIR PERFORATION

Positive control strains

Answer 17

Trichophyton mentagrophytes

Question 18

The ability to perforate hair and utilize its keratin content through digestion by its ______ enzymes is evident in the majority of the dermatophytes

Answer 18

keratinase

Question 19

PRINCIPLE: Petri dish containing sterile water and yeast extract is inoculated with test fungi and human hair samples

Answer 19

Hair Perforation Technique

Question 20

PRINCIPLE: After 7 days of incubation at room temperature, hair is examined microscopically and observed for deep, narrow wedge-shaped tears or slashes indicative of presence of hair perforations

Answer 20

Hair Perforation Technique

Question 21

HAIR PERFORATION Negative control strains

Answer 21

Trichophyton rubrum

Question 22

HAIR PERFORATION PROCEDURE Collect about 10-20 pieces of hair samples and cut it into short pieces about ___ in length

Answer 22

1 cm

Question 23

HAIR PERFORATION PROCEDURE Transfer the sterile short pieces of hair into a screw capped tube containing ___ of sterile water

Answer 23

5 mL

Question 23

HAIR PERFORATION PROCEDURE Incubate the tubes at _______

Answer 23

room temperature

Question 24

HAIR PERFORATION PROCEDURE Sterilize the cut hair samples by autoclaving at ___C for ___ minutes and store in a sterile container

Answer 24

121 °C for 10

Question 25

HAIR PERFORATION PROCEDURE After _ days of incubation, take an aliquot amount of the hair sample and place in a clean glass slide with lactophenol cotton blue (LPCB) stain.

Answer 25

7

Question 25

HAIR PERFORATION T/F: Steps 6 and 7 may be repeated at intervals of 7 days until the 4th week.

Answer 25

T

Question 26

HAIR PERFORATION PROCEDURE After 7 days of incubation, take an aliquot amount of the hair sample and place in a clean glass slide with _______ stain.

Answer 26

lactophenol cotton blue (LPCB)

Question 26

HAIR PERFORATION can be used as positive control and should be processed and tested in the same manner as that of the specimen or test sample

Answer 26

Prepubertal hair or child’s scalp hair

Question 27

INTERPRETATION OF RESULTS Hair inoculated with isolates of _______ _______ produce marked localized areas of pitting and marked erosion or deep, narrow wedge-shaped scars or slashes indicative of presence of hair perforations

Answer 27

POSITIVE: Trichophyton mentagrophytes

Question 28

INTERPRETATION OF RESULTS Absence of deep, narrow wedge-shaped perforations on surface of hair sample inoculated with _______ _____

Answer 28

NEGATIVE: Trichophyton rubrum

Question 29

RAPID TEST FOR DIAGNOSIS OF FUNGAL DISEASES * non-culture-based diagnostic tool * Presumptive

Answer 29

Detection of patient antibodies (serologic tests)

Question 30

RAPID TEST FOR DIAGNOSIS OF FUNGAL DISEASES Standard method for diagnosing cryptococcosis, and to some extent, aspergillosis, and candidiasis

Answer 30

Antigen detection test

Question 30

T/F: Establishing the presence of antibodies to fungi in serum/plasma and spinal fluid or the direct detection of the fungal antigens themselves, can be vital in the diagnosis of bacterial disease

Answer 30

F; mycotic disease

Question 31

Caused by opportunistic fungi C. neoformans and C. gattii

Answer 31

Cryptococcosis

Question 31

RAPID TEST FOR DIAGNOSIS OF FUNGAL DISEASES Routine serologic assay identifying many fungal diseases including coccidioidomycosis, histoplasmosis, aspergillosis

Answer 31

Antibody detection

Question 32

RAPID TEST FOR DIAGNOSIS OF FUNGAL DISEASES 3 Important serologic techniques for the rapid diagnosis of fungi

Answer 32

* Immunodiffusion (ID) * Complement Fixation (CF) * Enzyme immunoassay (EIA)

Question 33

Cryptococcosis MOT

Answer 33

inhalation of dust from pigeon droppings or bird feces contaminated with fungi

Question 33

A latex agglutination test wherein a carrier particle such as latex coated with antibodies specific to Cryptococcus capsular antigen

Answer 33

Cryptococcal antigen test

Question 34

T/F: At risk of Cryptococcosis are individuals with lowered or impared cell-mediated immune function causing meningoencephalitis, pneumonia, septicemia

Answer 34

T

Question 35

Cryptococcosis cause (3)?

Answer 35

Meningoencephalitis, pneumonia, septicemia

Question 36

Cryptococcal antigen test is mixed with what specimen?

Answer 36

CSF / Serum / Plasma

Question 37

Reagent used for Cryptococcal antigen test

Answer 37

Pronase reagent

Question 37

Positive result for Cryptococcal antigen test

Answer 37

formation of visible clumps or aggregates

Question 38

To detect Cryptococcal capsular polysaccharide antigen could be taken as the early diagnostic method of cryptococcal meningitis

Answer 38

Latex agglutination test

Question 39

CAT PREPARATIONS: CSF Centrifuge specimen at _____ g for __ mins

Answer 39

1000 x; 15 mins

Question 40

CAT PREPARATIONS: CSF Using a serologic pipette, aspirate ____ of CSF and place it in a sterile glass tube

Answer 40

0.5 or 1 mL

Question 41

CAT PREPARATIONS: CSF Inactivate the CSF sample in a water bath set at __C for _ minutes. Allow to cool for ___ mins before testing

Answer 41

56C; 5 mins; 3-4 mins

Question 42

CAT PREPARATIONS: CSF Prepare 1:100 dilution of CSF sample by mixing ___ of the heat inactivated specimen with ____ of sterile normal saline

Answer 42

25 uL; 2475 uL

Question 43

CAT PREPARATIONS: CSF Prepare _____ dilution of CSF sample by mixing 25 uL of the heat inactivated specimen with 2475 uL of sterile normal saline

Answer 43

1:100

Question 44

CAT PREPARATIONS: SERUM In a clean glass tube, place ____ of serum and add ___ of pronase reagent.

Answer 44

300 uL; 50 uL

Question 45

CAT PREPARATIONS: SERUM Centrifuge ______ at 1000x g for 15 mins.

Answer 45

clotted blood

Question 46

CAT PREPARATIONS: SERUM Mix the specimen and reactant by using a ______.

Answer 46

vortex mixer

Question 47

CAT PREPARATIONS: SERUM Inactivate the mixture by heating it in a water bath set at __C for ___ minutes.

Answer 47

56C, 30 mins

Question 48

CAT PREPARATIONS: SERUM Prepare 1:100 dilution of CSF sample by mixing ___ of the heat inactivated specimen with ____ of sterile normal saline

Answer 48

25 uL; 2475 uL

Question 49

CAT TEST PROCEDURE T/F: 1. Label the test/reaction areas of the disposable card as follows: one for positive control, one for negative control, one for the undiluted heat inactivated specimen, and one for 1:100 dilution of clinical specimen (e.g., CSF, serum/Plasma)

Answer 49

T

Question 50

CAT TEST PROCEDURE 2. On separate areas on the disposable card slide, place __ uL of heat inactivated specimen, __ uL of the 1:100 diluted specimen, and __ uL of positive and negative controls respectively. Spread each sample on the labeled reaction area.

Answer 50

25

Question 51

CAT TEST PROCEDURE Add __ uL of latex reagent in each of the four labeled wells.

Answer 51

25

Question 52

CAT TEST PROCEDURE Mix well the specimen-latex reagent mixture using a disposable _____, making sure that the mixture covers the entire reaction area.

Answer 52

stirrer

Question 53

CAT TEST PROCEDURE Continue mixing by rotating the reaction card at ___ rpm for __ minutes using a mechanical rotator

Answer 53

100 rpm, 15 mins

Question 54

CAT TEST PROCEDURE T/F: All positive test results should be doubly titrated and maximum titer showing agglutination reported

Answer 54

T

Question 55

CAT INTERPRETATION OF RESULT Denotes presence of clumping or granular aggregates, indicative of the presence of cryptococcal antigen

Answer 55

Positive

Question 56

CAT INTERPRETATION OF RESULT Presence of homogenous suspension

Answer 56

Negative

Question 57

Principle of Rapid Cryptococcal Antigen Test

Answer 57

Immunochromatographic assay

Question 58

will bind to the gold-conjugated antibodies

Answer 58

CrAg

Question 59

gold-conjugated antibody-CrAg complex moves up the test strip through ______ ______

Answer 59

capillary action

Question 59

gold-conjugated antibody-CrAg complex is captured/immobilized with ________ ________ against CrAg, forming a red band

Answer 59

monoclonal antibodies

Question 60

Specimen used for RCAT

Answer 60

* Cerebrospinal fluid (CSF) * Serum/Plasma * Urine

Question 60

RCAT PROCEDURE 1. On a clean kahn test tube or ____ tube, add 1 drop of specimen diluent

Answer 60

Eppendorf

Question 61

RCAT PROCEDURE 2. Using a disposable dropper pipette, place ___ (1 drop) of the patient specimen to the tube.

Answer 61

40 uL

Question 62

RCAT PROCEDURE 3. Insert the ______ strip into the tube

Answer 62

lateral flow assay (LFA)

Question 63

RCAT PROCEDURE 4. Immerse the strip into the specimen for ____

Answer 63

10 minutes

Question 64

RCAT INTERPRETATION OF RESULT Absence of red line or a line only at the test region

Answer 64

Invalid

Question 65

RCAT INTERPRETATION OF RESULT Presence of two red lines-one at the control region and the other at the test region

Answer 65

Positive

Answer 66

Invalid

Question 66

RCAT INTERPRETATION OF RESULT Only one red line at the control region

Answer 66

Negative