Clinical AND ADVANCE pathology

TOPIC: DIAGNOSIS FOR INFECTIONS

LABORATORY DIAGNOSIS FOR FUNGAL INFECTIONS 5.01

Tinea corporis- E.
floccosum/tricho
1. WHAT ARE THE BEST SPECIMEN FOR phyton,
MYCOLOGIC DIAGNOSIS? GIVE REPRESENTATIVE microsposrum
CONDITIONS. Tinea imbricate-
- Skin scrapings: candida, microsporum, tricophyton
trichophyton, epidermophyton, blastomyces, concentricum
dermatidis Tinea barbae,
- Nail clippings: aspergillus, epidermophyton, tinea cruris, tinea
trichophyton manuum
- Hair: microsporum, trichophyton (interdigital areas
- Scraping from mucous membrane – throat: and palmar
candida albicans, Geotrichum candidum, lungs: surfaces), tinea
candida albicans, aspergillus, rhizopus, pedis, tinea
penicillium, histoplasma capsilatum, unguium,
blastomyces dermatitidis, coccidiodes immitis
- Crust scraping
- Aspirated pus
Subcutan Involves Scrapin
- Tissue biopsy
eous dermis, g from
- Blood: Candida spp., blastomyces dermatidis, H.
mycoses subcutan mucous
capsulatum, C. neoformans
eous membr
- CSF: cyrptococcus neoformans, candida spp.,
tissue, ane,
histoplasma, coccisdioides immitis
muscles aspirate
- Urine: candida spp.
and fascia d pus,
- Genital tract: candida albicans
tissue
Fungal involeve specime fungi biopsy
infection ment n
Systemic Primarily Blood,
Superficia Outermos Skin Malassezia furfur mycoses lungs and CSF,
l mycoses t layers of scraping (Pityriasis spreading tissue
skin and s, hair versicolor), to other biopsy
hair Exophiala organs
wernekii (Tinea
Opportun Fungi of Mucous
nigra),
istic no secretio
Trichosporon
mycoses significan ns, CSF,
beigelii (white
ce or low
piedra), Piedraia
virulence
hortae (balck
infect
piedra)
humans
Cutaneou Extending Skin Dermatophytes: w/
s mycoses deeper scraping microsporum comprom
into the , nail (tinea capitis), ised
epidermis clipping Trichophyton immune
and its s, hair violaceum, system
integume epidermophyton
nts (dermatophytosis
/ ringworm) 2. WHAT ARE THE METHODS USED UN DIRECT
Candida albicans- EXAMINATION OF FUNGI? EXPLAIN EACH
candidiasis METHOD.

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 Patho 2 LABORATORY DIAGNOSIS FOR
FUNGAL INFECTIONS

- 10-30% of KOH- dissolved non fungal materials - Bird seed/ caffeic acid agar- selective and
in skin, hair and nail samples, specimen is placed differential, Cryptococcus neoformans (black to
on a slide, drop of KOH is added and coverslip is brown colonies due to the activity of the phenol
placed left for 20 min in the incubator at 37C to oxidase), may contain chloramphenicol
degest keratin, then examined microscopically - Cornmeal Agar with Tween 80- used to
- Calcofluor white stain- fluorochrome stains differentiate candida species, blastoconida-
chitin, not absorbed by human tissue, UV light, budding yeast, chlamydoconidia- C. albicans,
fungi appear white to blue green, KOH used to Pseudohyphae, Blastoconidia germinate,
clear debris constricted at the septate, arthroconidia- begin
- Histological stains (H&E, PAS, grams stain, as true hyphae but break apart at cross walls
Methenamine Silver stain, Giemsa Stain)
- India ink- reveals capsules surrounding C.
neoformans in CSF, low sensitivity, replaced by 4. HOW ARE THE FOLLOWING DONE IN THE
direct antigen detection DIAGNOSIS OF FUNGAL INFECTIONS?
- Wet mount- used to view fungal elements such A. SELECTION OF MEDIA
as hyphae, conidia and budding yeast. It has - Type of media for fungal culture in important. It
limited use and is most commonly applicable for is recommended that this culture media need to
vaginal secretions to diagnose vaginosis have special substance that will help support in
- Gram stain can be used to view budding yeast the growth of the organism and inhibit the
- Lactophenol cotton blue wet mount- used to growth of other fungi for identification or inhibit
stain and preserve fungal elements in culture bacterial decontamination
isolates, phenol- kill any live oragnisms, lactic - The media with or without cyclohexamide:
acid- preserves fungal structures, cotton blue- inhibits the growth of rapidly growing
stains chitin in fungal cell walls contaminating molds
- Saline wet mount- used to view hyphae, conidia, - The media with or without antimicrobial agent
budding yeast, mostly used forvaginal secretions such as chloramphenicol, gentamicin and
to diagnose vaginitis ciprofloxacin are commonly used.
Culture media description
3. WHAT ARE THE COMMONLY USED CULTURE Brain heart infusion Non selective, permits
MEDIA IN FUNGAL ISOLATION? growth of all clinically
- (SDA) sabouraud dextrose agar- general relevant fungi, primary
purpose, nutritionally poor, mildly selective for recovery of saprophytic
fungi, acidic pH 5.6 to inhibit bacterial growth, and dimorphic fungi
- SABHI- Sabouraud brain heart infusion- non- Czapek’s agar Used in subculture of
selective for all fungi, contains dextrose, aspergillus spp. For
peptone and brain heart infusion, can be made differential diagnosis
selective for dimorphic fungi with addition of Inhibitory Mold Primary recovery of
cyclohexamide, chloramphenicol and Agar dimorphic pathogenic
gentamicin fungi. However
- BHIB- brain heart infusion agar with blood- used saprophytic and
to grow most fungi from sterile sites, contains dermatophytes will not
brain and heart infusion and sheep blood, can be be recovered
made selective for dimorphic fungi with addition Mycosel/mycobiotic Used to recovery
of cyclohexmaide, chloramphenicol and agar (SDA with dermatophytes
gentamicin cycloheximide +
- Potato dextrose agar- used to enhance conidia chloramphenicol)
development, enhances pigment development Niger seed agar Used to identify C.
of Trichophyton rubrum neoformans

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 Patho 2 LABORATORY DIAGNOSIS FOR
FUNGAL INFECTIONS

Potato dextrose Rich medium for 6.

agar growing wide range of A. SEROLOGIC IDENTIFICATION
fungi
Sabourad’s heart Primary recovery of - Serological methods utilizes the reaction and
infusion agar saprophytic and properties of serum, it is a non-cultured based
dimorphic fungi- technique in identifying fungal infection
fastidious strains - Antigen and antibodies are easier to detect via
Sabourad’s dextrose Sufficient for the the blood , CSF, urine and Brochoalveolar lavage
agar recovery of samples
dermatophytes from - Generally, Sensitivity 80%, Specificity 80% this
cutaneous samples and may vary depending on the test.
yeasts from vaginal - Immunoblot analysis of the serological response
culture is a useful tool for the identification of
Potato flake agar Recovery of saprophytic immunogenic fungal components that elicit a
and dimorphic fungi, specific antibody response in invasive disease.
particulary fastifious This method, and others, have been successfully
and slow growing applied to the study of the immune response to
strains several fungi, including Candida, Aspergillus and
Rhizopus.
B. INOCULATION AND INCUBATION Serological techniques for diagnosis of fungal
 Each sample is cultured in two set of culture infection | Request PDF. Available from:
media and is incubated at two https://www.researchgate.net/publication/206
59157_Serological_techniques_for_diagnosis_o
 different temperatures at 30oC (Room
f_fungal_infection [accessed Jan 18 2018].
temperature) and at 35oC
 All fungal cultures are incubated for a
minimum of 30 days before discarding - This techiniques includes the detection of
 as negative. specific host immune responses to fungal
 The choice between the use of culture tubes antigens using immunologic reagents.
or plate is optional. For tube, the
 media is poured in thick slants to prevent - Amplification and detection of specific fungal
nucleic acid sequences
dehydration during prolonged
 incubation period. After the medium is - Detection of and quantification of specific fungal
inoculated, do not screw down the cap metabolite products
 too tightly because fungi require breathing.
- Targets of serological test: antigen. Antibody.
Metabolites
C. BLOOD CULTURES
- Blood is with draw from both arms right and left
- Decision of fungal serologic test is based on the
1 ml each side and will be place in BHI in sterile
clinical presentation, exposure history and risk
technique and be incubated. If it should turbidity
factor of infection
in the sample, it will be cultured with the
appropriate media to isolate the organism.
Examples:
Culture media are highly sensitive and specific.
Once it shows growth in this media plates, direct 1. agglutination
examination/ microscopy is done for evaluation. - principle: antigen antibody interaction
- will from agglutinates, positive reaction
5. EXPLAIN THE FOLLOWING TECHNIQUES IN - advantage: long shelf life 4-6 mos, rapid
RELATION TO FUNGAL IDENTIFICATION:

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 Patho 2
- disadvantage: false positive,
exoantigens with sodium
metaperioidate / pretrating serum with
2 B mercaptoethanol
2. immunodiffusion,
Principle: patient samples are placed in wells in
an agar plate surrounded by a larger well
containing purified antigen, Ab and Ag will
diffuse out of their respective wells and a
complex will form a visible precipitation band in
between the wells (positive test)
Advantage: 80-90%$ sensitivity, >90% specific
Disadvantage: costly, difficult to standardize,
long TAT
3. complement fixation test,
- principle: ability of the Ag-Ab complex to
fix complement, antibody + fungal
antien = complex  inactivates
exogenouly added complement
- results:
o specific ab + = complement
fixed, RBC pellet
o specific ab - = complement lyse
RBCs
4. ezyme linked immunosorbent assay (ELISA),
- …
5. lateral flow assay,
- principle: analyte + Ab conjugated to
gold nanoparticles = test line band pos,
immune chromatography
6. counter immune electrophoresis,
7. radio immunosorbent assay
B. MOLECULAR INDENTIFICATION
- Polymerase chain reaction based
techniques and other molecular
methods have been proven, in several
cases, to be fast, cheap and reliable
- May provide faster and more sensitive
diagnostics
- Can detect fungi which grew slowly such
as dermatophytes
Transcribed by: John Velasco #makingEAC-SMGreatAgain
LABORATORY DIAGNOSIS FOR
FUNGAL INFECTIONS
- Can detect fungi which cannot be
cultured such us aspergillus in the blood
C. MORPHOLOGIC IDENTIFICATION
- Morphology may be distinct to the fungi
- Fungal structure:
a. Hyphae- long brancing filaments
that come tohether to form
mycelium
- It could be septated – cellular separation
or cross walls from 3-6 um
- Pseudohyphae- chain cells formed from
budding that resemble a true hyphae.
b. Vegetative hyphae- food absorbtion
and are portion which extends
below the agar
c. Aerial hyphae- extends above the
agar function is to support
reproductive structures – conidia
d. Conidia formation of imperfect
fungi: asexual reproduction
- Important in fungal identification
- Microconidia: single celled, small
- Macroconidia: multicellular, large
- Athroconidia: fragmentation of hyphae
- Blastoconidia: conidia in budding
- Chlamydoconidia: terminal cells in the
hyphae that enlarge and have thick walls
- Poroconidia: conidia pushed through a
small pore in the parent cell
- Phialoconidia: tube shape
- Annelloconida: vase shape , saw toothe
appearance
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 Patho 2 LABORATORY DIAGNOSIS FOR
FUNGAL INFECTIONS

D. SUSCEPTIBILITY

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 Clinical AND ADVANCE pathology
TOPIC: DIAGNOSIS FOR INFECTIONS
LABORATORY DIAGNOSIS FOR FUNGAL INFECTIONS 5.01

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 Patho 2 LABORATORY DIAGNOSIS FOR
FUNGAL INFECTIONS

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