Diagnosis of Invasive Fungal

Infections

Tri Wibawa
Dept of Microbiology
FM UGM
A. fumigatus infects by
inhalation of conidia, which
germinate to hyphae in the
lung tissue.
A. fumigatus is
angioinvasive
 Nodular skin lession

Pulmonary cryptococcosis Cryptococcomas in MRI

imaging

Bird seed agar plate

showing the typical
brown
colour effect seen with
C. neoformans.
secondary oral pseudomembraneous
candidiasis infection in HIV/AIDS patient
Invasive Fungal Infection (IFI)
Importance in immunocompromised
patients
◦ Malignancy  high dose of chemotherapy
◦ Allogenic stem cells transplantation
◦ Pre term neonates
◦ Intensive care patients
◦ HIV/AIDS
Sypthoms in not specific
 Current Problems of IFI
Overall survival for immunocompromised
patients with invasive fungal infections
remains too low
Large variations according to underlying
disease.
Early diagnosis and subsequent early
initiation of therapy improves outcome
Diagnosing invasive fungal infections can be
difficult.
 Diagnosis of Invasive Fungal Infection
Convensional Methods
◦ Microscopics
◦ Culture
◦ Histopathology
New Diagnosis Tools
◦ Galactomannan
◦ I,3 - beta - D – glucan
◦ PCR
◦ Other biomarkers
Direct Wet Mount
a sputum sample
from a patient with
allergic
bronchopulmonary
aspergillosis.

cutaneous
aspergillosis
Aspergillus fumigatus conidiophores & free conidia
(LPCB)
Aspergillus fumigatus
Intracellular yeasts
(arrows) of H. capsulatum in a liver
biopsy specimen (hematoxylin and eosin stain).
Aspergillus fumigatus
Conventional methods
Current ‘conventional methods’ are very
limited for the diagnosis of invasive fungal
infections.
◦ Blood cultures have a low sensitivity for the
diagnosis of candidaemia (~50%)
◦ cultures other than blood are non-specific and
can take too long to became positive.
◦ Time consuming
◦ diagnoses of Candida pneumonia that are based
solely on microbiological data are often incorrect
For the diagnosis of invasive aspergillosis,
cultures of the respiratory tract secretions lack
sensitivity.
◦ Aspergillus is grown from sputum in only 8% to 34%,
◦ from broncho-alveolar lavage (BAL) in 45% to 62%
Blood, cerebrospinal fluid (CSF) and bone
marrow specimens rarely yield Aspergillus spp
Immunohistological staining using polyclonal
fluorescent antibody reagents may help  it
need biopsy
Galactomannan
Galactomannan is a cell wall
polysaccharide released by Aspergillus spp.
during fungal growth in tissue
A commercially available sandwich ELISA
Circulating galactomannan may be detected
at a median of five to eight days before
clinical manifestation of aspergillosis
may therefore be used to monitor the
response to treatment
High rates of false-positive results among
paediatric patients and neonates (~83%)
◦ Related with the diet in the guts
◦ cross-reactivity with Bifidobacterium bifidum.
the galactomannan test is a highly specific
diagnostic tool (94% to 99%), even though
sensitivity has ranged from 50% to 93% in
patients with haematologic malignancy
False positive in patient receiving synthetic
/fungal origin antibiotics
1,3-beta-D-glucan
isa cell wall component of yeast and filamentous
fungi, which is detectable in the blood during
most invasive fungal infections
Sensitivity 67% -100%; Specificity 84% -100%
Detect : invasive aspergillosis and candidosis,
also infection by Fusarium, Trichosporon,
Saccharomyces and Acremonium
Not detect: oral candidosis and cryptococcosis
PCR
PCR is at lest 19 times more sensitive than
culture for A. fumigatus
PCR-based molecular diagnostic tests for
aspergillosis are not commercially available
Remain largely unstandardized
Sensitivity of 79% to 100%
Specificity of 81% to 93%
More sensitive then cluture for candidemia
Similar specificity with ELISA  97%
PCR
Three fundamental steps:
◦ The selection of a specific target region of
DNA/RNA to identify the fungus;
◦ Extraction of total community DNA/RNA
from the environmental sample;
◦ A method to identify the presence of the target
DNA/RNA region in the sample
 PCR: quantitative PCR, Real time PCR
 Hybridization: Probing, micro array
Selection of target DNA
Primer design 
◦ Sequence data base
◦ Cloned and sequence
Forfungal diagnostics  ribosomal genes
Nuclear ribosomal DNA (rDNA) consist of three
genes
◦ The large subunit gene (25S)
◦ The small subunit gene (18S)
◦ The 5.8S gene,
 Separated by internal transcribed spacer (ITS)
regions, in a unit repeated many times
internal transcribed spacer (ITS) regions
Importance to fungal diagnostics, it
contained
◦ Highly concerve  universal primer
◦ Highly variable  species identification

Other then ITS:

◦ β-tubulin gene
◦ mating type genes
NUCLEIC ACID HYBRIDIZATION PROBES
(Wengenack & Binnicker, 2009)
Limitation of real time PCR for fungal
diagnosis
Highly sensitive  colonizing or causing a
subclinical infection may be detected

Fungal PCR assays may be prone to false-

positive results because of contamination with
environmental fungi

Growth in culture is still required if antifungal

susceptibility testing is to be performed
Markers for Invasive Candidosis
Mannan
◦ Correlation between detectable mannanemia
and tissue invasive in patients with
candidaemia
◦ rapidly cleared from the blood and occurs in
low levels
◦ Expensive
D-arabinitol
◦ Metabolite of certain species of Candida that
accumulates in the urine of patients with
invasive candidosis
◦ sensitivity for D-arabinitol is only ~50%,
◦ does not detect C. krusei and C. glabrata
Enolase
◦ Most promising
◦ Spesificity 54% to 75%
◦ Highly specific for Candida spp.,
◦ is not present in superficial Candida
colonisation
FUTURE DIRECTIONS IN MOLECULAR
FUNGAL DIAGNOSTICS
Sensitive
Specific
rapiddetection
However, many fungi still require culture-
and morphology based identification
Promising:
◦ Luminex
◦ Micro array