Laboratory Diagnosis of
Fungal Infections
MBI 531
Introduction
Toconfirm clinical suspicion to establish
fungal cause of disease.
To help in -
Choosing a therapeutic agent
Monitoring the course of disease
Confirming mycological cure
Sites & Types of Specimens
Specimencollection depends on the
corresponding disease.
Veryimportant to proceed for a final
diagnosis.
(a) Superficial Mycosis
Clean the part with 70% alcohol
Collect the material in a sterile paper or
a sterile petridish to -
Allow drying of the specimen
Reduce bacterial contamination
Maintain viability
(a) Superficial Mycosis
Dermatophytic lesion – spreads outward in
a concentric fashion with healing in the center
– scrape outwards from the edge of the lesion
with a scalpel blade or use Cellophane tape
Scalp lesion – scraping with a blunt
scalpel, including hair stubs, scales &
contents of plugged follicles.
(a) Superficial Mycosis
Scalp lesion – Woodlamp’s examination of
infected hair – fluorescence
Hairbrush sampling technique
Onychomycosis – stop antifungals one
week
prior to collection
Mucosal infections – mucosal scrapings
(b) Subcutaneous Mycosis
Scrapings or crusts from the superficial
parts of lesions
Pus aspirates
Biopsy
(c) Systemic Mycosis
Pus CSF
Biopsy Blood
Feces Scrapings or swabs
Urine from the edge of
Sputum
lesions.
Collection & Transport of
specimen
Proper collection of specimen and in
adequate quantity.
Early transport to the lab to avoid
overgrowth
of contaminant
Respiratory specimens
Sputum – early morning sample, after mouth wash,
flakes to be used for culturing
Bronchoscopy – if non productive cough
Bronchial brushings or lung biopsy – to rule out
invasion or colonisation
Collection & Transport of
specimen
Blood
In biphasic Brain Heart Infusion agar
Inoculated in 2 bottles – for dimorphic fungi
Cerebrospinal fluid
Should be immediately processed else
stored at RT or at 30°C in an
incubator
Centrifuge & use sediment for culture
Collection & Transport of
specimen
Skin, Hair & Nail
Taken for dermatophytic infections
Hair – plucked with forceps
Tissue, BM & Body fluids
Tissues – grind or mince before culturing
Body fluids – centrifuge & use sediment for culture
Urine – centrifuge & use sediment for culture
Laboratory Diagnosis
Direct examination
Fungal culture
Serological tests
Skin tests
PCR & other molecular methods
Direct Examination
Very decisive in the diagnosis of fungal
infections
Wet mounts
Slide & tube KOH mounts – 10 to 20% KOH –
digests protein debris, dissolves keratin. DMSO can
be added to KOH to hasten clearing in skin
scrapings & nail clippings
Calcofluor white – fluorescent stain – excellent
morphology of pathogenic fungi
India ink – capsulated fungi
CFW – yeast form of
Blastomyces
KOH - Aspergillus India ink -
Cryptococcus
Direct Examination
Gram stain – fungi are
Gram +ve
Histopathology - yeast cells, hyphae, pseudohyphae,
arthrospores, chlamydospores, and spherules.
Routine stain – Hematoxylin & Eosin (HE) - very useful
to visualize the host's response
1. Superficial infection – acute, subacute or chronic
dermatitis with folliculitis
2. Subcutaneous & systemic infections – granulomatous
reaction with fibrosis or pyogenic inflammation
Direct Examination
Histopathology
Special stains – PAS (Per Iodic
acid), GMS (Grocott Gomori
Methanamine Silver), Mayer’s
mucicarmine, Gridley’s stain
GMS is more advantageous since
it stains old and nonviable fungal
elements more efficiently than the
others
Mucin stains, like Mayer's
mucicarmine stain the
mucopolysaccharide capsule
of Cryptococcus neoformans
Direct Examination
Fluorescent- antibody staining
To detect fungal Ag in clinical specimen such
as pus, blood, CSF, tissue sections
Adv – can detect fungus even when few
organisms are present
Fungal Culture
Sabouraud Dextrose Agar (SDA)
Contains 2% dextrose, antibiotics (gentamicin,
chloramphenicol) and cycloheximide
Selective media
Corn meal agar (CMA) – sporulation, chlamydospore
formation
Bird seed agar – cryptococcus, forms brown
colonies
Brain Heart Infusion (BHI) agar – dimorphic & other
fastidious fungi
Corn Meal Agar
Bird Seed Agar
Fungal Culture
Temperature requirement
Majority of fungi – 37°C
Superficial mycosis – 30°C
Dimorphic fungi – 25°C & 37°C
Incubation time
At least 4 weeks
Usually positive cultures are obtained in 7-10
days
Candida & Aspergillus - 24 to 72 hrs
Fungal Culture
Specimens should be cultured on agar
slants:
Safe
Require less space
More resistant to drying during prolonged
incubation
Blood cultures should be inoculated in to
biphasic blood culture bottles
Interpretation of Fungal
Culture
Isolation of an established pathogen like
H. capsulatum or C. neoformans – evidence of
infection
Isolation of commensal or opportunistic fungi
like Candida or Aspergillus – consider following
points:
1. Isolation of same strain in all culture tubes
2. Repeated isolation of same strain in multiple
specimens
3. Isolation of same strain from different sites
4. Immune status
5. Serological evidence
Identification of fungal
cultures
Colony morphology – colour, texture,
pigment
Identification of fungal
cultures
Fungal morphology
under microscope –
using Lactophenol Cotton
Blue (LPCB) stain
Composition of LPCB
Lactic acid - preserves fungal
structure
Phenol – kills any live organism
Glycerol – prevents drying
Cotton blue – imparts
blue color to structures
Identification of fungal
cultures
Special culture techniques – Slide culture
to see sporing structures & spore
arrangement, CHROM agar for candida sps.
Biochemicals – ability to assimilate carbon
& nitrogen, sugar fermentation
C.tropicalis
C.krusei
C.albicans
CHROM Agar
Serology & Immunology
Detection of Ag or Ab in serum or body fluids
Ab detection:
Diagnosis of systemic & subcutaneous mycoses
Assess prognosis of the disease
Assess response to treatment
Ag detection:
Early stages of infection
In patients with impaired immunity
Delayed hypersensitivity tests – with Ags like
candidin, histoplasmin, etc.
Serological tests used in Medical
Mycology
Agglutination
Whole cell agglutination
Latex particle agglutination
Passive haemagglutination
Immunodiffusion – most widely used
Counter immunoelectrophoresis (CIEP)
Indirect fluorescent Ab detection
ELISA, RIA
Other Methods
PCR – Polymerase Chain Reaction
RFLP - Restriction fragment length
polymorphism
Protein electrophoresis
Nucleic acid probes
Serotyping
Karyotyping
