Invasive Pulmonary Aspergillosis (Bench to bedside diagnostics)

Question 1

Aspergillus fumigateurs is the

Answer 1

most important opportunistic mould pathogen in immune-compromised humans

Question 2

where are aspergillum fumigatus infections most common

Answer 2

haematological malignancy and allogenic bone marrow transplant patients

Question 3

chronic pulmonary aspergilosis

Answer 3

3 million cases worldwide in patients with underlying lung diseases including asthma

Question 4

allergic bronchopulmonary aspergillosis

Answer 4

4 million cases worldwide in patients with asthma and cystic fibrosis

Question 5

everyone breathes in

Answer 5

spores- can get to lower part of respiratory tract

Question 6

alveolar macrophages use which receptors to recognise fungal PAMP

Answer 6

PPR- Dectin-1

Question 7

neutropenic bone marrow patients have

Answer 7

no immunity- zero WBC count

Question 8

absence of macrophage and neutrophils in the lungs

Answer 8

dangerous

Question 9

aspergilloma

Answer 9

a fungal ball- is a clump of mold which exists in a body cavity such as a paranasal sinus or an organ such as the lung. By definition, it is caused by fungi of the genus Aspergillus.

Question 10

who is aspergilloma common in

Answer 10

those who have had TB and have scar tissue

Question 11

symptoms of aspergilloma

Answer 11

weeping, snuggle to catch breath, cough up

Question 12

CT scan of aspergilloma will show

Answer 12

mass within tissue

Question 13

important to be able to differentiate between

Answer 13

normal breathed in spores and those that growing hyphae

Question 14

process of pulmonary aspergillosis

Answer 14

1) inactive conidia are inhaled
2) conidia lodge in lower respiratory tract
3) condida swell
(block by macrophages
4) condida germinate into hyphae
(block by neutrophils)
5) hyphae invade tissue
(block by neutrophils)
6) hyphae invade blood vessels an disseminate

Question 15

gold standard for IPA

Answer 15

no ‘gold standard’

Question 16

imperative that diagnosis is

Answer 16

made without delay- prognosis worsens signify in the absence of recognition and effective treatment

Question 17

CT scan and invasive pulmonary aspergillosis

Answer 17

cant see- hard to diagnpose

Question 18

when someone comes to the doctor with IPA

Answer 18

• their fever will be going up and down
• fist thing doctor will do is give antibiotic
• fever won’t resolve
• then suspect fungal infection
• window gets small for treatment

Question 19

treatment for IPA

Answer 19

horrendous side effects:
-hallucinations
-fever
due to fungal being very closely related to humans

Question 20

serological detection of aspergllus fumigatus

Answer 20

-elevated tigers of antibodies against A.fumigatus surface component

Question 21

elevated tigers in sera due to

Answer 21

presence of an abundant galactomannoprotein in the cell walls of the pathogen

Question 22

presence of an abundant galactomannoprotein in the cell walls of the pathogen

Answer 22

Elevated titers were shown to be due to the presence of an
abundant galactomannoprotein in the cell walls of the pathogen
(Afmp1p)

Question 23

AFMP1 gene cloned and sequenced and

Answer 23

recombinant Afmp1p

protein produced in E. coli

Question 24

what can be used to prove immunogneicity

Answer 24

Western blot

- purified Afmp1p protein antigen

Question 25

ELISA for the diagnosis of A.fumigatus aspergillosis

Answer 25

- recombinant Afmp1p protein used to coat wells of micro titre plates - ELISA performed using sera taken from patients with aspergilloman, from patients with invasive aspergillosis and from patients with infections caused by the pathogenic fun - ELISA is highly specific for A.fumigatus diangosis

Question 26

when does ELISA not work so well

Answer 26

when patients are neutropenic- poor antibody response

Question 27

structures unique to fungi which are recognised by immune system

Answer 27

- mannans and galactans- highly immunogenic | - making monoclonal antibodies against specific cell membrane proteins

Question 28

invasive pulmonary aspergillosis and galactomannans detection

Answer 28

double AB sandwich ELISA

Question 29

Invasive pulmonary aspergillosis and | galactomannan detection

Answer 29

Traditionally immunological tests for IPA have been centred around the detection of the circulating fungal cell-wall carbohydrate galactomannan (GM) - using EB-A2 to detect

Question 30

reason for false positives within Invasive pulmonary aspergillosis and galactomannan detection

Answer 30

- does cross react with other fund e.g. Fusarium - cant tell if aspergillosis or invasive fusarium - baby milk contains galactomanna- moves into the gut and into the bloodstream - FM in penicillin

Question 31

Penicillin is very closely related to aspergillum

Answer 31

false positives- very dangerous

Question 32

cross reactivity of EB-A2 with G from other fungi

Answer 32

e.g. Fusarium

Question 33

Cross-reactivity of mAb with anti-cancer drug

Answer 33

cyclophosphamide

Question 34

'Pan-fungal detection

Answer 34

when we do not know if a patient is infected with a virus, bacteria or fungi- don't want to waste time treating for viral or bacterial - will pick up all fungi which affect humans

Question 35

Pan-fungal detection picks up

Answer 35

(1-3) B-D glucans from fungal cell wall

Question 36

(1-3) B-D glucans

Answer 36

Glucose polymers Linked to proteins, lipids, mannan, chitin Most fungi, some bacteria, most higher plants, many lower plants Up to 60% dry weight of fungal cells wall

Question 37

(1-3) B-D glucans from the basis of

Answer 37

fungi tell tests-- high rate of false positives

Question 38

(1-3) B-D glucans does not detect

Answer 38

mucromycetes and cryptococcus that lack (1-3) B-D gluons in their cell walls

Question 39

Surrogate (non-GM) antigens for UPA detection

Answer 39

Alternative ‘circulating antigens’ are required as surrogate markers for rapid diagnosis of IPA Most appropriate targets are extracellular, constitutively-expressed antigens Should be able to discriminate between active growth and quiescence

Question 40

which antibody is used

Answer 40

Mouse mAB JF5 | - IgG3 immunoglobulin

Question 41

IgG3 immunoglobulin recognised

Answer 41

an extracellular constitutive, glycoprotein antigen | - antigen is secrets during active growth and not produced by dead spores

Question 42

mAbJF5- IgG3

Answer 42

displays superior specificity to rat mAb EB-A2

Question 43

which is the best marker for J5F (which is only produced when aspergillus fumigates is growing

Answer 43

Immunogold EM