16 - bench-bedside diagnostics (aspergillus) Flashcards
features of aspergillus
fungi mould pathogen
opportunistic
causes invasive disease (aspergillosis)
reaches terminal air spaces of lungs –> travels in airborne spores
typical patient of aspergillosis
immunocompromised
e.g. suffering from haematological malignancies or allogenic bone marrow transplant patients
most acute/serious version of aspergillosis
invasive pulmonary aspergillosis (IPA)
less prevalent diseases caused by aspergillus
chronic pulmonary aspergillosis
allergic bronchopulmonary aspergillosis
immune recognition of aspergillus
dectin-1 receptors on alveolar macrophages in lungs
recognition of beta-glucan on cell wall
activation of complement
immune response once aspergillus is recognised
activation of complement system
phagocytosis and classic killing of pathogen within phagolysosome
chemokines/cytokines activated and recruit neutrophils
why is aspergillus detrimental in immunocompromised
no WBC (neutropenic) due to treatment or cancer therefore no immunity
aspergilloma
fungal ball colonises within lungs
typically seen in chronic infection
scars develop in lung tissue and spores of aspergillus get stuck in the scar
role of macrophages in immunit against aspergillus
block germination of spores into hyphae
recruit neutrophils
role of neutrophils in aspergillus immunity
block hyphae invasion of tissues and blood vessels to prevent dissemination
diagnosis for invasive pulmonary aspergillosis (IPA)
no ‘gold standard’ test
relies of cumulation of variety of data
window of opportunity small - early diagnosis important
role of radiology department in IPA diagnosis and next steps
CT chest scan of patient
any abnormalities sent to histopathology and microbiology units and serum sample sent off
needle stuck in to abnormality seen in CT scan
serum sample removed from patient in diagnosis of IPA
needle stuck into remove sample from abnormality seen in CT scan
grown in agar to see what is present
serological detection of aspergillus fumigatus
serum shows elevated levels of antibodies against A. fumigatus surface components
elevated levels of antibpdies against what surface component seen in serum of aspergillus patietns
abundant galactomannoprotein in the cell walls of the pathogen
(Afmp1p)
method of using serum
look for antigen-sepcific antibodies in the serum and multiply using E.Coli
using ELISA and western blotting
use of ELISA to diagnose A. fumigatus aspergillosis
Recombinant Afmp1p protein used to coat wells of microtitre plates
bathe using serum from patients with aspergilloma (IPA patients)
- ELISA highly specific for A. fumigatus diagnosis
benefits of ELISA in diagnosis
allows large scale screening of patients
major component of fungalcell wall
chitin
antibodies created against beta glucans
not that effective
benefits of mannans and galactans
highly immunogenic
species specific
(carbohydrates organised on fungi in unique manner)
allows production of specific monoclonal antibodies against them
Traditionally immunological tests for IPA have been centred around the detection of….
…the circulating fungal cell-wall carbohydrate galactomannan (GM)
use DAS-ELISA
lots of false positives in galactomannan detection
due to cross-reactivity with other fungi
and detection of galactomannan in food and other drugs e.g. penicillin
importance of pan-fungal tests
can pick up all the fungi that can infect humans
we dont know whether an infection may be caused by bacteria/virus/fungi
method of pan-fungal tests
looks for beta-1,3-glucans
they are found in MOST fungi
‘fungitell’ test
limitations of fungitell test
some bacteria also contain the beta-1,3-glucan component giving false positives
some fungi lack the component in their wall therefore not detected
Surrogate (non-GM) antigens for IPA detection
Alternative ‘circulating antigens’ are required as surrogate markers for rapid diagnosis of IPA
most appropriate target for diagnostic detection
extracellular, constitutively-expressed antigens
should be able to discriminate between active
growth and quiescence(dormancy of spores)
IgG3 mouse antibody
recognises an extracellular, constitutive, glycoprotein antigen that is only present in active growth
Much more specific than rat
used to develop lateral flow assay diagnostic test
JF5
humanised monoclonal antibody
used in detection of invasive pulmonary aspergillosis
gold particles in gold-EM
represent where antigen is bound to antibody
lateral flow assay
JF5 MAb
JF5 MAb conjugated to gold particles
Solution enters containing target antigen
Antigen migrates along and picks up gold particles conjugated to JF5 MAbs
Complexes migrate along test line
Same JF5 antibody immoblised on membrane
Pulls antibody bound to glycoprotein complex
Positive test line if antibody sandwich forms
negative test in LFA
no immobilised antibody - antigen - antibody complex
no gold particle precipitation
Aspergillus-LFD and Human Serum
diagnostic test to detect JF5 antigen in human serum
Galactomannan circulate causes antibodies and serum components to bind to it (highly immunogenic)
Sample has to be treated and heated to dissociate complement proteins bound
Low sophistication –> no special technology, just requires heating block
bronchoalveolar lavage (BAL)
fluid in bronchi
used for diagnostic test
method of BAL test
Take patient
Put bronchoscope down into lung
Feed another tube down that contains fluid and force fluid into lung
Pull fluid back out again
(patient feels like they’re drowning)
Might see aspergillus sporing within lung tissue
Send off for testing
If picking up in bloodstrean/serum infected already invasive
benefits of BAL test
good for early detection
limitations fo BAL test
patient feels like theyre drowning with fluid forced into lungs
some patients go on to suffer from pneumona (have to be treated in ICU)
