[OS 217 - IDS] LEC 04 Diagnostic Mycology

8/15/2019 [OS 217 - IDS] LEC 04 Diagnostic Mycology

1/6

ENDENCIA, EUSEBIO, FACTOR, FERNANDEZ UPCM 2016: XVI, Walang Kapantay! 1of 6

OS 217: SYSTEMIC DISEASES Infectious Diseases ModuleLEC 04: Diagnostic Mycology

Exam 1| Alice Alma S. Bungay, MD | 19 June 2013

OUTLINE

I. Diagnostic Mycology

II. Specimens

A. Skin Scraping SpecimenB. Hair Specimen

C. Nail Specimen

D. Subcutaneous Tissue

III. Microscopic Examination

IV. Fungal Culture

V. Biochemical TestsVI. Laboratory Precautions

VII. Appendix

DIAGNOSTIC MYCOLOGY

Ancient Diagnostic Evidence

A petrified sporangium of Coccidioides immitis, the cause of

Valley fever, was unearthed in the lungs of a 600-1000 year old

American Indian skeleton.

Specimen Collection

The specimen is the beginning. All diagnostic information from

the laboratory depends on the knowledge by which specimens

are chosen and thhe care with which they are collected and

transported.

General guidelines:

Sterile collection method and devices

Sufficient quantity

Accurate and complete label

Actual infection site to avoid normal flora

Prompt delivery to lab :avoid overgrowth of fungal or

bacterial contaminants

Physician has suspected diagnosis for special proceduresor specimen treatment

Collect the right specimen:

- Superficial : skin and hair

- Cutaneous : skin, hair, and nails

- Subcutaneous: biopsy, granules

- Systemic: abscess, blood, CSF, sputum

Diagnostic Laboratory Tests \

Specimens

○ Biopsy materialsor exudate from granulomatous and

ulcerative lesions, skin, nails and hair

Microscopic Exam

○

Samples can be examined directly using 10% KOH or withuse of calcofluor white (an optical brightener)

Culture

○ Using slide culture or Riddell technique

○ Inoculate onto Saboraud’s dextrose agar (SDA) or

mycobiotic agar and incubated at 27-35 degrees C. Use

Lactophenol cotton blue (LPCB)

Serology

○

For yeast samples using latex agglutination tests

SPECIMENS

Skin Scraping Specimen

For skin specimen, first clean lesion & periphery with 70%

alcohol. Scrape (with scalpel)

o Scalpel should be sterile and cooled

o Use sterile scalpel or edge of microscope slide, scrape

perpendicular to the skin

o If with ring, scrape outer edge. The center of lesion

heals first, so the laboratory results are negative using

this sample.

o Scrape area with active infection. Scrape around the

active edge where the fungus is actively growing.

o If the lesion is inflamed or with fissures, clean it with

sterile distilled water.

o Collect skin scrapings in paper envelope or petri dish,

or place between 2 slides.

o

Store at room temperature.

Adhesive tape

o If patients are young children and are scared of the

scalpel, use can use scotch tape to collect specimen

for microscopy

o Press on surface of lesion

o Used for rapid mounting of sporulating fungi (keeps

the

reproductive structures intact)

Athelete’s foot : wash first with water, do not use cotton swabs

Collect moist exudate for candida


2/6

OS 217 - IDS LEC 04: Diagnostic Mycology


Figure 1. Tinea versicolor sticky tape strip showing a typical cluster of round

budding cells and mycelial elements of Malassezia furfur. Methylene blue

stain is used.

Direct Examination

For fungi that exhibit characteristic structures in clinical

specimens that can be seen microscopically using a brightfield

or phase contrast microscope.

Prepare a wet mount, a specimen plus sterile water or NSS, or

specimen alone, like exudates.

KOH

o

10% to 30%, depending upon the type of specimen,

for skin use 10%, for nails use a stronger

concentration, 30%.

o Makes the fungal cell wall, which is resistant to alkali,

visible.

o Use Parker Superquink blue-black ink. It will stain the

fungal structures to appear bluish green.

o Do gentle warming. The preparation is passed 2 or 3

times over an alcohol lamp or bunsen burner, to

hasten the reaction.

o If the result is negative, especially in nail scrapings,

you can leave the preparation overnight on the lab

table, to give time for digestion to occur.

Fungal Detection- Direct Microscopy

Three stains are used to identify fungi

o KOH

o Calcofluor white

o Methenamine silver

Hair Specimen

To get hair specimen, you need to use the ff:

o Scissors

o

Tweezerso Paper/envelope

Figure 2. Example of an infected hair specimen. In some cases, more than

one hair shaft have concretions.

Figure 3. 3-D presentation of a hair shaft penetrated by fungi that are

categorized as keratin lovers.

1. Ectothrix Hair Invasion

Formation of arthroconidia on the outside of hair shaft

Cuticle of hair is destroyed

Will fluoresce under a wood’s UV lamp, unlike endothrix

Endothrix- refers to dermatophyte infections that invade the hair shaft and

internalize into the hair cell

Ectothrix – dermatophyte infections that remain confined to the hair surface

2. Use of Wood’s Lamp


3/6



Wood’s UV light

Infected hairs (ectothrix infections) will fluoresce bright green or

yellow green

Figure 4. Cutaneous Mycosis: Tinea Capitis(a) Ringworm of the head caused

by Microsporum audouinii (b) Close-up utilizing a Wood’s lamp

Nail Specimen

Clean with 70% alcohol

Scrape off outer surface and discard!

Scrape deeper portion

Collect whole nail or clippings

Collect all the debris that you would see around the nail

Use paper or envelope

Figure 5. Cutaneous Mycosis: Tinea Unguium. Ringworms of the nails caused

by Trichophyton rubrum. It is very hard to treat nail fungal infections

Figure 6.

Ringworm of the

extremities. (a)

Trichophyton infection spreading

over the foot in a

“moccasin”

pattern. The

chronicity of the

tinea pedis is attributed to the lack of fatty-acid-forming glands in the feet.

(b) Ringworm of the nails. Invasion of the nail bed causes some degree of

thickening, accumulation of debris, cracking, and discoloration; nails can be

separated from underlying structures as shown.

Subcutaneous Tissue: Lesions, Abscesses

Biopsy/Needle Aspiration

Clean surface with 70% alcohol

Add tincture of iodine Add sterile water

Aspirate with sterile needle and syringe

Do the incision

Remove the aspirate with sterile Pasteur pipette

Place in sterile test tube

MICROSCOPIC EXAMINATION

Figure 7. Microscopic morphology of yeasts. (a) Scanning electron

micrograph of brewer’s or baker’s yeast Saccharomyces cerevisiae (b)

Formation and release of yeast buds (c) Formation of pseudohypha (a chain

of budding yeast)

Saline Wet Mount

Visible: budding yeast, hyphae, pseudohyphae

Specimen used: skin scrapings, nail hair

Major Disadvantage: lack of contrast, thus difficult identification

of fungal elements

Figure 8. Saline wet mount

Figure 9. (L) Microscopic appearance of molds. (R) Microscopic appearance

of yeast

India Ink Preparation

Used to identify capsules of Cryptococcus neoformans Stains the background but gives a halo appearance on organism

Disadvantages

o WBCs and artifacts can be mistaken as capsules

o

Capsule negative in AIDS

Replaced by direct antigen testing for crypto capsular proteins

Figure 10. India Ink Preparation

Figure 11. Microscopic examination of infected CSF using india ink.

Cryptococcus is monomorphic (yeast form). It’s capsule is impenetrable by

the ink, therefore they appear white against a black background .

Encapsulation is a useful diagnostic sign for crytococcosis, although the

capsule is fragile and may not show up in some preparations.

These are included to show the difference is microscopic appearance of

other organisms.

Figure 12. (L) Microappearance of Aspergillus. A large, branched septate

mycelium is the most common finding in tissues, but the characteristic

conidial heads are also present in some specimens. These molds are not

dimorphic and exist only in hyphal stage. (R) The morpholog of

Paracoccidiodes. A Gridley stain from a skin lesion reveals the central round

mother cell with a series of narrow-necked buds that lock like spokes of a

wheel.


4/6



Lactophenol Cotton Blue (LPCB)

Components

○ Lactic acid – clearing agent

○ Glycerol – prevents drying

○ Phenol – disinfectant

○ Aniline blue - dye

Gives the morphology of the conidiogenous cells and the conidia

that they give rise to in order to identify a filamentous fungus

(hyaline fungal structures)

Permanently seal slides with Permount/clear nail polish

Used in Tease preparation (wet mount)/slide cultures – slow

portion of actively gorwing fungal culture

Gram Stain

\Used for yeast form

Useful for:

○ Candida albicans

○

Malassexia furfur

○ Sporothrix schenkckii (appear as cigar-shaped bodies)

Also used for Nocardia and Actinomyces

Methenamine-Silver Nitrate Stain For screening of clinical specimens

Stain shows good contrast and staining of fungal elements

Fungi appear black against a pale background

For viable and non-vialbe fungi stain

Modified type: Gomori – used for fungi in histological specimens

(A) (B)Figure 13. (A) Pneumocystis carinii in the lung tissue of an AIDS patient (B)

Silver stain used in tissue sections to visualize fungi

Periodic Acid-Schiff (PAS)

Oxidizes hydroxyl in CHO of cell walls to form aldehydes which

react with fuschin dye, forming pink-purple complex

Counterstained with fast green for contrast

FUNGAL CULTURE

Fungal Detection Culture

Identification of yeasts and filamentous fungi depends on:○ YEAST – Candida and Cryptococcus, sugar fermentation and

assimilation biochemistry (e.g. Urea test + for Cryptococcus)

○ FILAMENTOUS FUNGI – Macroscopic (colony morphology

and pigmentation) and microscopic (hyphae, conidia, and

sexual structures) morphology

Fungi are cultured on Agar media such as SDA that either mimic

the environment (low pH and room temperature), or mimic the

host (pH 7, added blood and nutrients, incubation at 35oC

In vitro, culture at room temperature with low pH and minimal

nutrients support the growth of environmental or mycelial

phase

Incubation at body temperature and supplemented with blood

and amino acids supports the yeast phase of the fungi

Figure 14. Cellular and cultural characteristics of Histoplasmacapsulatum.

(A) A colony at 25oC produces a fuzzy mycelium (B) A yeast colony at 37

oC is

dense and waxy

Figure 15. Lab diagnosis culture – flat, spreading suede-like to granular

cinnamon growth with yellow brown pigment on reverse of colony

Culture Media

Non-Selective

○ Saboraud’s dextrose agar (SAB/SDA)

Selective

○ SDA with chloramphenicol and cyclohexamide (Mycosel or

Mycobiotic agar)

○ Dermatophyte test medium – for dermatophyte fungi

(e.gEpidermophyton, Microsporum, Trichophyton spp.)▪ Phenol Red – indicator; red in alkaline medium

○ Potato dextrose agar – for fungi that attack living or

decaying dead plant matter; sporulating medium for fungi

○

Chromogenic agar

○

Guizotiaabsynica cysteine agar – for Cryptococcus

Incubation is at room temperature for at least 2 weeks

BIOCHEMICAL TESTS

Metabolism & Biochemical Identification

few methods of serology & biochemical identification used in

mycology

o only exception: yeasts

many use fermentation pathways – this, plus the microorganism’s

morphology, is used to identify yeasts

o commercial ID systems using array of biochemical substrates

allow for rapid identification of yeasts

TABLE X: Biochemical Tests for Fungi Identification

Tests Fungi Description

Urea Utilization T. mentagrophytes,

T. rubrum

-

Amino Acid

Utilization

T. tonsurans Thiamine

In vitro hair

perforation

T. mentagrophytes,

T. rubrum

Unshampooed hair,

horse hair

preferable, animal

hoof

Multiple test system Yeasts Uses substrate array

for rapid

identification


5/6



Figure 16: A hair sample with T. mentagrophytes that tested positive for

hair perforation (left), and a sample that tested positive for urease

production (rightmost test tube, right)

Serology

there are few antigen detection methods used in mycology

1. Latex Agglutination Method

used to detect yeast formation of Cryptococcus neoformans

antibodies prepared against a large polysaccharide capsule are

tagged to latex beads

fluids such as CSF, blood and urine are mixed with latex bead

solution

presence of capsule in the body fluid results in visible

agglutination

2. Immunodiffusion Testing

used to detect coccidioidomycosis

small wells punched into agar & patient’s sera placed into

numbered wells, with central well containing fungal antigen (ex.

coccidioidin)

Figure 17: Agar setup of immunodiffusion testing

lines of precipitation between outer wells and central well

indicate reaction between antibodies in serum and antigen

diffusing from the central wello ex. in Figure 17 (above), reactions for sera 1, 5, 6 show positive

evidence for infection; no infection in sera 2, 3, 4 due to

absence of antibodies and infection

Identification

gross color and texture

microscopic characteristics

confirm/compare with written descriptions, drawings,

photographs

microscopic examination of growth

o beginning of growth: conidia and spores for identification

Figure 18: Circinella (note sporangia, sporangiospores, nonseptate hyphae)

Figure 19: Penicillium (note brush arrangement of phialospores)

Figure 20: Sporothrix schenckii (daisy-like microconidia; this organism is

dimorphic – yeast form in the human host, mycelial form in environments

outside human body)

Figure 21: verrucous lesions (cauliflower-like)

Figure 22: lesions with sclerotic bodies (large dark brown/copper-coin-like

structures with or without transverse septa)

Figure 23: Eumycotic mycetoma


6/6



Figure 24: Coccidioides immitis mature spherules (contains endospores)

Figure 25: dimorphic nature of Blastomyces dermatitidis: hyphal filaments(left ) that are lollipop-shaped; tissue phase from a sputum sample ( right )

LABORATORY PRECAUTIONS

conidia, spores remain dormant in air

inhalation common route of infection

all work must be performed in a biological cabinet

must observe Biosafety Level III in the laboratory

never smell fungal culture

APPENDIX: LABORATORY PROCEDURES FOR SPECIFIC SPECIMEN TYPES

END OF TRANSCRIPTION

Bea: LUV!!!! Thanks for coming to our first team building session! Hope you all had fun!! Lets make the most of this year and enjoooyyy!!

Lets all watch Monsters University when it comes out!

Trish: Ma’am said everything we need to know is in her ppt . To my awesome block, B4TTC! Hi phierless!

Documents

[OS 217 - IDS] LEC 04 Diagnostic Mycology