Role of Fungi in Disease

And
Laboratory Diagnosis of
Fungal Diseases
KELOMPOK 5 :
1. Arsuciah Lisfika Putri
2. Aqila Fadila Haya
3. Elsa
4. N. Rizka Nerisandi
5. Purnama Eka Sari
Role of Fungi in Disease
Mycotic diseases in humans develop as pathogenic
processes in one or more organ systems. Because the
development of a fungal infection depends on factors that
often outweigh the virulence potential of the infecting
organism
• as the immune status of the host
• The opportunity for interaction between host and fungus
(e.g.,Is the fungus endogenous to the patient or
exogenous?),
• the potential infectious dose (e.g., in the case of an
endemic dimorphic fungus)
• in determining the possibility of a fungal infection, the
significance of the microbiologic data (e.g., culture
results), and the necessity to treat and with what agent.
Summary of Fungi Associated with Human Disease
System Affected Pathogens
Upper Respiratory Infections
Oropharyngeal Candida spp., Cryptococcus
neoformans, Histoplasma capsulatum,
Blastomyces dermatitidis,
Paracoccidioides brasiliensis,
Penicillium marneffei, Geotrichum
candidum
Esophageal Candida spp.
Ear Infections Aspergillus niger, Candida spp.
External otitis
Eye Infections Candida spp., Aspergillus spp.,
Endophthalmitis Blastomyces dermatitidis, Coccidioides
immitis/posadasii, Fusarium
spp., Histoplasma capsulatum,
Cryptococcus neoformans
Other Infections Candida spp., all others very rare
Prosthetic joint
Hematogenous dissemination Candida spp., Histoplasma capsulatum,
Blastomyces dermatitidis, Coccidioides
immitis/posadasii,
Cryptococcus neoformans/gattii,
Paracoccidioides brasiliensis, Sporothrix
schenckii, Aspergillus
spp., Fusarium spp., Trichosporon spp.,
Malassezia spp., Blastoschizomyces
capitatus, Penicillium
marneffei, others (e.g., Rhodotorula,
Acremonium, Saccharomyces spp. in
neutropenic or
transplant patients)
Intraabdominal Infections Candida spp., Rhodotorula spp.,
Peritonitis Trichosporon spp., Aspergillus spp. (rare)

Skin and Soft-Tissue Infections

Subcutaneous Dematiaceous molds, Fusarium spp.,
Acremonium spp., Scedosporium
apiospermum, Sporothrix
schenckii, Basidiobolus sp., Conidiobolus
Laboratory Methods for Diagnosing Fungal Disease
Conventional Microbiologic Methods
• Direct microscopy (Gram, Giemsa, and calcofluor
• white stains)
• Culture
• Identification
• Susceptibility testing
Histopathologic Methods
• Routine stains (H&E)
• Special stains (GMS, PAS, Mucicarmine)
• Direct immunofluorescence
• In situ hybridization
Immunologic Methods
• Antibody
• Antigen
Molecular Methods
• Direct detection (nucleic acid amplification)
• Identification
• Strain typing
Biochemical Methods
• Metabolites
• Cell wall components
• Enzyme
Laboratory Diagnosis of
Fungal Diseases
• CLINICAL RECOGNITION OF FUNGAL INFECTIONS
Thus diagnosis of fungal infections depends
on three basic laboratory approaches
1. microbiologic,
2. immunologic, and
3. Histopathologic
These approaches may be supplemented by molecular and
biochemical methods of organism detection and identification.
Use of the newer methods for detection of fungal antigens and
nucleic acids offers great promise for rapid diagnosis of fungal
infections.
CONVENTIONAL LABORATORY DIAGNOSIS

• Specimen Collection and Processing

As with all types of infectious processes, the laboratory
diagnosis of fungal infection is directly dependent on the
proper collection of appropriate clinical material and
prompt delivery of the specimens to the clinical laboratory.
Selection of specimens for culture and microscopic
examination is based not only on information obtained
from clinical examination and radiographic studies but also
on consideration of the most likely fungal pathogen that
may cause a specific type of infection.
Infection Site and Specimen Options Collection Diagnostic
Infecting Methods Procedure
Organism
Blood
Candida, Whole blood Venipuncture Culture, broth,
Cryptococcus (sterile) culture,
neoformans, lysiscentrifugation
Histoplasma
capsulatum,
Fusarium,
Aspergillus
terreus,
Penicillium
marneffei,
Trichosporon
Serum Venipuncture Antigen (Candida,
(sterile) Cryptococcus,
and Histoplasma),
nucleic acid
amplification
• Stains and Direct Microscopic Examination
Direct microscopic examination of tissue sections and clinical
specimens is generally considered to be among the most rapid
and cost-effective means of diagnosing fungal infections.
Microscopic detection of fungi in tissue serves to guide the
laboratory in selecting the most appropriate means to culture
the specimen and also is helpful in determining the significance
of culture results.
used most often in the clinical mycology laboratory include the
fluorescent reagent calcofluor white or staining of smears and
touch preparations with either Gram or Giemsa stains.
Calcofluor white stains the cell walls of fungi, causing the fungi
to fluoresce for easier and faster detection
Gram stain of Cryptococcus neoformans. Variable-sized,
encapsulated, budding yeasts showing a stippled pattern
resulting from uneven retention of crystal violet stain.
Gram stain of Aspergillus. T his specimen did not retain the
crystal violet stain and appears gram-negative.
Giemsa stain showing intracellular yeast forms of Histoplasma
capsulatum.
Culture
The most sensitive means of diagnosing a fungal infection
is usually considered to be isolation of the fungus in
culture. Culture is also necessary, in most instances, to
identify the etiologic agents.
Identifying Characteristics of Various Fungi
Determination of the identity of the specific etiologic agent
of mycotic disease may have a direct bearing on prognosis
and therapeutic considerations. Microscopic examination
provides further delineation and often is all that is required
for identification of many fungi.
Culture
Fungus Microscopic Macroscopic Microscopic Additional Tests
Morphologic (Morphologic (Morphologic for identification
Features in Features in Features in
Clinical Culture) Culture)
Specimens
Penicillium Oval Colonies Septate Demonstration of
marneffei intracellular produce hyphae with thermal
yeast cells diffusible metulae, dimorphism
with septum red pigment at phialides with
25° C chains of
conidia in a
“paint brush”
distribution
(25° C). Yeast
cells divide
by fission (37°
C)
IMMUNOLOGIC, MOLECULAR, AND BIOCHEMICAL
MARKERS FOR DIRECT DETECTION OF INVASIVE FUNGAL
INFECTIONS
Detection of fungal cell wall and cytoplasmic antigens and
metabolites in serum or other body fluids represents the most
direct means of providing a serologic diagnosis of invasive fungal
infection. Example :
• Organism : Candida
• Cell Wall or Capsule : Mannans, LA, RIA, EIA, 1,3-β-glucans,
Limulus test, Chitin, Spectrophotometry
• Components Cytoplasmic Antigens Metabolites :Enolase, EIA,
Immunoblot, Antienolase antibody, EIA, 47-kD breakdown
product of HSP-90, Enzyme-linked dot, Immunobinding assay
• Genomic DNA : D-Arabinitol, Rapid enzymatic ClC/FID, Mass
spectroscopy/GLC
• Sequences* : Actin, Chitin synthase, P450, ITS, Ribosomal RNA
genes