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cellular basis of immunity > 16 - bench-bedside diagnostics (aspergillus) > Flashcards

16 - bench-bedside diagnostics (aspergillus) Flashcards

1
Q

features of aspergillus

A

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

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2
Q

typical patient of aspergillosis

A

immunocompromised

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

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3
Q

most acute/serious version of aspergillosis

A

invasive pulmonary aspergillosis (IPA)

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4
Q

less prevalent diseases caused by aspergillus

A

chronic pulmonary aspergillosis

allergic bronchopulmonary aspergillosis

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5
Q

immune recognition of aspergillus

A

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

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6
Q

immune response once aspergillus is recognised

A

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

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7
Q

why is aspergillus detrimental in immunocompromised

A 
no WBC (neutropenic) due to treatment or cancer
therefore no immunity
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8
Q

aspergilloma

A

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

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9
Q

role of macrophages in immunit against aspergillus

A

block germination of spores into hyphae

recruit neutrophils

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10
Q

role of neutrophils in aspergillus immunity

A

block hyphae invasion of tissues and blood vessels to prevent dissemination

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11
Q

diagnosis for invasive pulmonary aspergillosis (IPA)

A

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

window of opportunity small - early diagnosis important

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12
Q

role of radiology department in IPA diagnosis and next steps

A

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

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13
Q

serum sample removed from patient in diagnosis of IPA

A

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

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14
Q

serological detection of aspergillus fumigatus

A

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

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15
Q

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

A

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

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16
Q

method of using serum

A

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

17
Q

use of ELISA to diagnose A. fumigatus aspergillosis

A

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

18
Q

benefits of ELISA in diagnosis

A

allows large scale screening of patients

19
Q

major component of fungalcell wall

A

chitin

20
Q

antibodies created against beta glucans

A

not that effective

21
Q

benefits of mannans and galactans

A

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

22
Q

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

A

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

use DAS-ELISA

23
Q

lots of false positives in galactomannan detection

A

due to cross-reactivity with other fungi

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

24
Q

importance of pan-fungal tests

A

can pick up all the fungi that can infect humans

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

25
Q

method of pan-fungal tests

A

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

26
Q

limitations of fungitell test

A

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

27
Q

Surrogate (non-GM) antigens for IPA detection

A

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

28
Q

most appropriate target for diagnostic detection

A

extracellular, constitutively-expressed antigens

should be able to discriminate between active
growth and quiescence(dormancy of spores)

29
Q

IgG3 mouse antibody

A

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

30
Q

JF5

A

humanised monoclonal antibody

used in detection of invasive pulmonary aspergillosis

31
Q

gold particles in gold-EM

A

represent where antigen is bound to antibody

32
Q

lateral flow assay

JF5 MAb

A

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

33
Q

negative test in LFA

A

no immobilised antibody - antigen - antibody complex

no gold particle precipitation

34
Q

Aspergillus-LFD and Human Serum

A

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

35
Q

bronchoalveolar lavage (BAL)

A

fluid in bronchi

used for diagnostic test

36
Q

method of BAL test

A

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

37
Q

benefits of BAL test

A

good for early detection

38
Q

limitations fo BAL test

A

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

cellular basis of immunity (21 decks)

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