Invasive Pulmonary Aspergillosis (Bench to bedside diagnostics) Flashcards
Aspergillus fumigateurs is the
most important opportunistic mould pathogen in immune-compromised humans
where are aspergillum fumigatus infections most common
haematological malignancy and allogenic bone marrow transplant patients
chronic pulmonary aspergilosis
3 million cases worldwide in patients with underlying lung diseases including asthma
allergic bronchopulmonary aspergillosis
4 million cases worldwide in patients with asthma and cystic fibrosis
everyone breathes in
spores- can get to lower part of respiratory tract
alveolar macrophages use which receptors to recognise fungal PAMP
PPR- Dectin-1
neutropenic bone marrow patients have
no immunity- zero WBC count
absence of macrophage and neutrophils in the lungs
dangerous
aspergilloma
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.
who is aspergilloma common in
those who have had TB and have scar tissue
symptoms of aspergilloma
weeping, snuggle to catch breath, cough up
CT scan of aspergilloma will show
mass within tissue
important to be able to differentiate between
normal breathed in spores and those that growing hyphae
process of pulmonary aspergillosis
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
gold standard for IPA
no ‘gold standard’
imperative that diagnosis is
made without delay- prognosis worsens signify in the absence of recognition and effective treatment
CT scan and invasive pulmonary aspergillosis
cant see- hard to diagnpose
when someone comes to the doctor with IPA
- 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
treatment for IPA
horrendous side effects:
-hallucinations
-fever
due to fungal being very closely related to humans
serological detection of aspergllus fumigatus
-elevated tigers of antibodies against A.fumigatus surface component
elevated tigers in sera due to
presence of an abundant galactomannoprotein in the cell walls of the pathogen
presence of an abundant galactomannoprotein in the cell walls of the pathogen
Elevated titers were shown to be due to the presence of an
abundant galactomannoprotein in the cell walls of the pathogen
(Afmp1p)
AFMP1 gene cloned and sequenced and
recombinant Afmp1p
protein produced in E. coli
what can be used to prove immunogneicity
Western blot
- purified Afmp1p protein antigen
ELISA for the diagnosis of A.fumigatus aspergillosis
- 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
when does ELISA not work so well
when patients are neutropenic- poor antibody response
structures unique to fungi which are recognised by immune system
- mannans and galactans- highly immunogenic
- making monoclonal antibodies against specific cell membrane proteins
invasive pulmonary aspergillosis and galactomannans detection
double AB sandwich ELISA
Invasive pulmonary aspergillosis and
galactomannan detection
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
reason for false positives within Invasive pulmonary aspergillosis and
galactomannan detection
- 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
Penicillin is very closely related to aspergillum
false positives- very dangerous
cross reactivity of EB-A2 with G from other fungi
e.g. Fusarium
Cross-reactivity of mAb with anti-cancer drug
cyclophosphamide
‘Pan-fungal detection
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
Pan-fungal detection picks up
(1-3) B-D glucans from fungal cell wall
(1-3) B-D glucans
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
(1-3) B-D glucans from the basis of
fungi tell tests– high rate of false positives
(1-3) B-D glucans does not detect
mucromycetes and cryptococcus that lack (1-3) B-D gluons in their cell walls
Surrogate (non-GM) antigens for UPA detection
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
which antibody is used
Mouse mAB JF5
- IgG3 immunoglobulin
IgG3 immunoglobulin recognised
an extracellular constitutive, glycoprotein antigen
- antigen is secrets during active growth and not produced by dead spores
mAbJF5- IgG3
displays superior specificity to rat mAb EB-A2
which is the best marker for J5F (which is only produced when aspergillus fumigates is growing
Immunogold EM
