Laboratory Diagnosis of Fungal Infections: Specimen Collection and Transport
L
a
b
o
r
a
t
o
r
y
D
i
a
g
n
o
s
i
s
o
f
F
u
n
g
a
l
I
n
f
e
c
t
i
o
n
s
MBI 531
I
n
t
r
o
d
u
c
t
i
o
n
To confirm clinical suspicion to
establish
fungal cause
of
disease.
To
help
in
-
Choosing a
therapeutic
agent
Monitoring the
course of
disease
Confirming mycological
cure
S
i
t
e
s
&
T
y
p
e
s
o
f
S
p
e
c
i
m
e
n
s
Specimen
collection
depends
on
the
corresponding
disease.
Very
important
to
proceed for
a
final
diagnosis.
(
a
)
S
u
p
e
r
f
i
c
i
a
l
M
y
c
o
s
i
s
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
)
S
u
p
e
r
f
i
c
i
a
l
M
y
c
o
s
i
s
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
)
S
u
p
e
r
f
i
c
i
a
l
M
y
c
o
s
i
s
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
)
S
u
b
c
u
t
a
n
e
o
u
s
M
y
c
o
s
i
s
Scrapings or crusts from the
superficial
parts
of
lesions
Pus
aspirates
Biopsy
(
c
)
S
y
s
t
e
m
i
c
M
y
c
o
s
i
s
Pus
Biopsy
Feces
Urine
Spu
t
um
CSF
Blood
Scrapings or
swabs
from
the
edge of
lesions.
C
o
l
l
e
c
t
i
o
n
&
T
r
a
n
s
p
o
r
t
o
f
s
p
e
c
i
m
e
n
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
C
o
l
l
e
c
t
i
o
n
&
T
r
a
n
s
p
o
r
t
o
f
s
p
e
c
i
m
e
n
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
C
o
l
l
e
c
t
i
o
n
&
T
r
a
n
s
p
o
r
t
o
f
s
p
e
c
i
m
e
n
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
L
a
b
o
r
a
t
o
r
y
D
i
a
g
n
o
s
i
s
Direct
examination
Fungal
culture
Serological
tests
Skin
tests
PCR & other
molecular
methods
D
i
r
e
c
t
E
x
a
m
i
n
a
t
i
o
n
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
C
F
W
–
y
e
a
s
t
f
o
r
m
o
f
B
l
a
s
t
o
m
y
c
e
s
K
O
H
-
A
s
p
e
r
g
i
l
l
u
s
I
n
d
i
a
i
n
k
-
C
r
y
p
t
o
c
o
c
c
u
s
D
i
r
e
c
t
E
x
a
m
i
n
a
t
i
o
n
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
D
i
r
e
c
t
E
x
a
m
i
n
a
t
i
o
n
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
D
i
r
e
c
t
E
x
a
m
i
n
a
t
i
o
n
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
F
u
n
g
a
l
C
u
l
t
u
r
e
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
C
o
r
n
M
e
a
l
A
g
a
r
B
i
r
d
S
e
e
d
A
g
a
r
F
u
n
g
a
l
C
u
l
t
u
r
e
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
F
u
n
g
a
l
C
u
l
t
u
r
e
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
I
n
t
e
r
p
r
e
t
a
t
i
o
n
o
f
F
u
n
g
a
l
C
u
l
t
u
r
e
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
I
d
e
n
t
i
f
i
c
a
t
i
o
n
o
f
f
u
n
g
a
l
c
u
l
t
u
r
e
s
Colony morphology
–
colour, texture,
pigment
I
d
e
n
t
i
f
i
c
a
t
i
o
n
o
f
f
u
n
g
a
l
c
u
l
t
u
r
e
s
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
I
d
e
n
t
i
f
i
c
a
t
i
o
n
o
f
f
u
n
g
a
l
c
u
l
t
u
r
e
s
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
.
t
r
o
p
i
c
a
l
i
s
C
.
k
r
u
s
e
i
C
.
a
l
b
i
c
a
n
s
C
H
R
O
M
A
g
a
r
S
e
r
o
l
o
g
y
&
I
m
m
u
n
o
l
o
g
y
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.
S
e
r
o
l
o
g
i
c
a
l
t
e
s
t
s
u
s
e
d
i
n
M
e
d
i
c
a
l
M
y
c
o
l
o
g
y
Agglutination
Whole
cell
agglutination
Latex
particle
agglutination
Passive
haemagglutination
Immunodiffusion
– most widely
used
Counter immunoelectrophoresis
(CIEP)
Indirect fluorescent
Ab
detection
ELISA,
RIA
O
t
h
e
r
M
e
t
h
o
d
s
PCR – Polymerase
Chain
Reaction
RFLP - Restriction
fragment
length
polymorphism
Protein
electrophoresis
Nucleic acid
probes
Serotyping
Karyotyping
In the laboratory diagnosis of fungal infections, proper collection and transportation of specimens are crucial for accurate diagnosis and treatment. Different sites and types of specimens require specific collection techniques to avoid contamination and ensure viability. From superficial to systemic mycoses, appropriate sampling methods are essential to confirm clinical suspicion, choose the right therapeutic agent, and monitor disease progression. Early transport of specimens to the lab is vital to prevent contaminant overgrowth and obtain reliable results.
Download Presentation
Please find below an Image/Link to download the presentation.
The content on the website is provided AS IS for your information and personal use only. It may not be sold, licensed, or shared on other websites without obtaining consent from the author. Download presentation by click this link. If you encounter any issues during the download, it is possible that the publisher has removed the file from their server.
E N D
Presentation Transcript
Laboratory Diagnosis of Fungal Infections MBI 531
Introduction To confirm 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 Specimen collection depends on the corresponding disease. Very important 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 CSF Blood Scrapings or swabs from the edge of lesions. Pus Biopsy Feces Urine Sputum
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 usefulto visualize the host's response Superficial infection acute, subacute or chronicdermatitis with folliculitis Subcutaneous & systemic infections granulomatousreaction with fibrosis or pyogenic inflammation 1. 2.
Direct Examination Histopathology Special stains PAS (Per Iodic acid), GMS (Grocott Gomori Methanamine Silver), Mayer s mucicarmine, Gridley s stain GMS is more advantageous sinceit stains old and nonviable fungal elements more efficiently than the others Mucin stains, like Mayer's mucicarmine stain the mucopolysaccharide capsule of Cryptococcusneoformans
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 MealAgar Bird SeedAgar
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 Repeated isolation of same strain in multiple specimens Isolation of same strain from different sites Immune status Serological evidence 2. 3. 4. 5.
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 - preservesfungal structure Phenol kills any live organism Glycerol preventsdrying 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 CHROMAgar
Serology & Immunology Detection of Ag or Ab in serum or body fluids Ab detection: Diagnosis of systemic & subcutaneousmycoses 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
