16 - bench-bedside diagnostics (aspergillus)

Question 1

features of aspergillus

Answer 1

fungi mould pathogen
opportunistic
causes invasive disease (aspergillosis)
reaches terminal air spaces of lungs –> travels in airborne spores

Question 2

typical patient of aspergillosis

Answer 2

immunocompromised

e.g. suffering from haematological malignancies or allogenic bone marrow transplant patients

Question 3

most acute/serious version of aspergillosis

Answer 3

invasive pulmonary aspergillosis (IPA)

Question 4

less prevalent diseases caused by aspergillus

Answer 4

chronic pulmonary aspergillosis

allergic bronchopulmonary aspergillosis

Question 5

immune recognition of aspergillus

Answer 5

dectin-1 receptors on alveolar macrophages in lungs
recognition of beta-glucan on cell wall
activation of complement

Question 6

immune response once aspergillus is recognised

Answer 6

activation of complement system
phagocytosis and classic killing of pathogen within phagolysosome
chemokines/cytokines activated and recruit neutrophils

Question 7

why is aspergillus detrimental in immunocompromised

Answer 7

no WBC (neutropenic) due to treatment or cancer
therefore no immunity

Question 8

aspergilloma

Answer 8

fungal ball colonises within lungs
typically seen in chronic infection
scars develop in lung tissue and spores of aspergillus get stuck in the scar

Question 9

role of macrophages in immunit against aspergillus

Answer 9

block germination of spores into hyphae

recruit neutrophils

Question 10

role of neutrophils in aspergillus immunity

Answer 10

block hyphae invasion of tissues and blood vessels to prevent dissemination

Question 11

diagnosis for invasive pulmonary aspergillosis (IPA)

Answer 11

no ‘gold standard’ test
relies of cumulation of variety of data

window of opportunity small - early diagnosis important

Question 12

role of radiology department in IPA diagnosis and next steps

Answer 12

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

Question 13

serum sample removed from patient in diagnosis of IPA

Answer 13

needle stuck into remove sample from abnormality seen in CT scan
grown in agar to see what is present

Question 14

serological detection of aspergillus fumigatus

Answer 14

serum shows elevated levels of antibodies against A. fumigatus surface components

Question 15

elevated levels of antibpdies against what surface component seen in serum of aspergillus patietns

Answer 15

abundant galactomannoprotein in the cell walls of the pathogen
(Afmp1p)

Question 16

method of using serum

Answer 16

look for antigen-sepcific antibodies in the serum and multiply using E.Coli
using ELISA and western blotting

Question 17

use of ELISA to diagnose A. fumigatus aspergillosis

Answer 17

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

Question 18

benefits of ELISA in diagnosis

Answer 18

allows large scale screening of patients

Question 19

major component of fungalcell wall

Answer 19

chitin

Question 20

antibodies created against beta glucans

Answer 20

not that effective

Question 21

benefits of mannans and galactans

Answer 21

highly immunogenic
species specific
(carbohydrates organised on fungi in unique manner)
allows production of specific monoclonal antibodies against them

Question 22

Traditionally immunological tests for IPA have been centred around the detection of….

Answer 22

…the circulating fungal cell-wall carbohydrate galactomannan (GM)

use DAS-ELISA

Question 23

lots of false positives in galactomannan detection

Answer 23

due to cross-reactivity with other fungi

and detection of galactomannan in food and other drugs e.g. penicillin

Question 24

importance of pan-fungal tests

Answer 24

can pick up all the fungi that can infect humans

we dont know whether an infection may be caused by bacteria/virus/fungi

Question 25

method of pan-fungal tests

Answer 25

looks for beta-1,3-glucans they are found in MOST fungi 'fungitell' test

Question 26

limitations of fungitell test

Answer 26

some bacteria also contain the beta-1,3-glucan component giving false positives some fungi lack the component in their wall therefore not detected

Question 27

Surrogate (non-GM) antigens for IPA detection

Answer 27

Alternative ‘circulating antigens’ are required as surrogate markers for rapid diagnosis of IPA

Question 28

most appropriate target for diagnostic detection

Answer 28

extracellular, constitutively-expressed antigens should be able to discriminate between active growth and quiescence(dormancy of spores)

Question 29

IgG3 mouse antibody

Answer 29

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

Question 30

JF5

Answer 30

humanised monoclonal antibody | used in detection of invasive pulmonary aspergillosis

Question 31

gold particles in gold-EM

Answer 31

represent where antigen is bound to antibody

Question 32

lateral flow assay | JF5 MAb

Answer 32

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

Question 33

negative test in LFA

Answer 33

no immobilised antibody - antigen - antibody complex | no gold particle precipitation

Question 34

Aspergillus-LFD and Human Serum

Answer 34

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

Question 35

bronchoalveolar lavage (BAL)

Answer 35

fluid in bronchi | used for diagnostic test

Question 36

method of BAL test

Answer 36

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

Question 37

benefits of BAL test

Answer 37

good for early detection

Question 38

limitations fo BAL test

Answer 38

patient feels like theyre drowning with fluid forced into lungs some patients go on to suffer from pneumona (have to be treated in ICU)