Laboratory diagnosis of fungal

infection
Practical of Microbiology
Definitive diagnosis of invasive fungal infection is
usually based on
recovery and identification of a specific etiological
agent from clinical specimens by
(i)Microscopic demonstration of fungi with distinctive
morphological features (e.g., encapsulated
Cryptococcus neoformans cells in cryptococcosis).
(ii)Culture media
(iii)Detection of specific host antibody responses.
(iv) Detection of fungal genome.
EXAMINATION OF
SPECIMENS
Specimens should be examined macroscopically
and microscopically.
Microscopic detection of a fungus in clinical
specimens can alert the physician to the etiology of
the disease and alert the laboratory staff to select
the appropriate media and inoculation techniques
that will enhance the recovery of the fungus.
MACROSCOPIC
EXAMINATION:
Before inoculating a specimen to the appropriate
isolation media, the specimen is examined
macroscopically for caseous, purulent or bloody
areas, and necrotic material. Specimens from cases
of mycetoma are examined with the dissecting
microscope for the presence of granules before
proceeding.
MICROSCOPIC
EXAMINATION:
When there is not a sufficient quantity of a
specimen for both a culture and direct exam, the
culture takes priority over the smear because it is
more sensitive than microscopic examination.
Observing a fungus in a clinical specimen is,
however, often valuable in establishing its
significance and in providing early information that
may be crucial for determining appropriate
therapy for the patient.
Methods of Microscopic Examination:
1. Potassium Hydroxide Procedure
 Principle: KOH may be used to examine hair, nails, skin scrapings,
fluids, exudates, or biopsies.
 In unstained preparations (KOH without ink or specimen with no
reagent), the fungal structures may be enhanced by using a phase-
contrast microscope.
 Specimens placed in a drop of 15% KOH will dissolve at a greater
rate than fungi because fungi have chitinous cell walls. The
clearing effect throughout the clinical specimen can be accelerated
by gently heating the KOH preparation.
Solution
Microscope filter system: An epifluorescent
microscope equipped with a mercury vapor lamp
and either an ultraviolet (UV) or blue-violet (BV)
excitation filters .
Fungi stain bright green or blue-white depending
upon the filters used
India Ink
 India ink can be added to specimens such as spinal
fluids or exudates to provide a dark background
that will highlight hyaline yeast cells and capsular
material (halo effect).
Hence, it should be used to examine specimens
suspected of containing Cryptococcus neoformans.
Wright stain, or Giemsa stain
Buffy Coat for Histoplasma capsulatum -
Hematological smears may be used for examining
specimens for Histoplasma which usually occurs as
an intracellular yeast.
Gram Stain
Gram stain is usually a poor stain to use when
examining a specimen for a fungus.
Gram stain may be used when examining smears of
Candida, Malassezia, and Sporothrix but should not
be relied upon to demonstrate the yeast of the other
dimorphic fungi.
A gram stain will demonstrate the filaments of
Nocardia and Actinomyces.
CULTURE MEDIA
All inoculated media should be read every 2 days
following incubation and twice weekly thereafter.
If the specimen is from a contaminated site, it is
important to include media that contain
inhibitory substances such as chloramphenicol,
gentamicin, or cycloheximide.
Chloramphenicol or gentamicin will inhibit most
bacterial contaminants, cycloheximide inhibits
most saprobic moulds
ISOLATION MEDIA
Sabouraud Dextrose Agar (SDA)
1. SDA agar
(Emmons' modification contains 2% glucose and is
slightly acidic (pH 6.5). It is the standard medium
for recovery and maintenance of a wide variety of
fungi commonly isolated in the clinical laboratory.
The original SAB formulation specifies 4% glucose.
Emmons' modification with less glucose is
preferred as an isolation medium because some
isolates, notably Blastomyces dermatitidis may not be
recovered using the original Sabouraud
formulation.
2. SAB + Chloramphenicol + Cycloheximide
are commercially prepared media containing SAB
agar, 1% glucose, chloramphenicol, and
cycloheximide. These media are used for the
selective recovery of dimorphic fungi and
dermatophytes.
 Serological diagnosis of fungal infections
Methods have been developed for the detection of both
circulating antibodies and antigens, but the
usefulness of antibody detection may be limited when
the patients under investigation are immunosuppressed
and/or heavily colonized but uninfected.
limitations for serological diagnosis of fungal
infection include
(i) cross-reactions among different species,
(ii) presence of antibodies to common environmental or
commensal fungi,
(iii) lack of standardization of antigens and methods for
detecting and quantifying antibodies.
DETECTION OF SPECIFIC
NUCLEIC ACIDS
Nucleic acid hybridization and amplification methods
are fundamental to molecular diagnosis. These
methods have the potential to provide both high
detection rates and identification of specific fungal
pathogens.As the latter becomes increasingly
important with the widespread use of antifungal
therapy and the problem of antifungal resistance.
Target Selection
Targets that have been used in molecular diagnostic
tests for fungal infections include single and
multicopy nuclear and mitochondrial genes and
RNA.
In general, molecular diagnostic methods targeting
multicopy genes have better detection thresholds than
those targeting single-copy genes.
Among multicopy genes, mitochondrial DNA has
been used in the PCR-based detection of C. albicans
and Aspergillus species.
However, the variability of mitochondrial DNA among
different strains may be a limiting factor.
Other genes that have been used in molecular
diagnosis of fungal infections are:
Actin
Chitin synthase
P450
18S rRNA