Aspergillus Fumigatus

Aspergillus Fumigatus


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Microbiology Education Series - Mycology No.2

 

"Aspergillus fumigatus"

 

This web text will specifically focus on Aspergillus fumigatus but reference may be made to other Aspergillus species and possibly other fungi.

1

Background / History

  • First discovered - Fresenius first described Aspergillus fumigatus in 1863. A. fumigatus is a common saprotropic fungus naturally occuring in the environment.
  • Name origin - In Roman Catholic Churches priests used an instrument called an "aspergillum" to sprinkle holy water on members of their congreagtion during mass. The aspergillum looked similar to the conidial head of the Aspergillus genus hence the name of this group of fungi was created. There is also reference to a classic psalm which uses the word "asperges", meaning to sprinke.
  • Spore concentration - in air can reach up to 600 spores / Metre cubed and up to 10,000,000 spores / Metre cubed have been recorded in late Autumn, particularly in farmyard barns, used for storing hay.
  • Fermentation research on Aspergillus fumigatus has shown Spirotryprostatin B to be an anti-mitotic drug with anti cancer properties.
  • A. fumigatus spores can be found in the respiratory tract of healthy individuals although they are quickly removed by a healthy immune system.
  • There are over 900 species of Aspergillus, of which A. fumigatus, A. flavus, and A. niger are responsible for approximately 95% of human infections. Of these infections, A. fumigatus is by far the most prevalent. However, other species of Aspergillus have been documented as pathogenic to man although they will not be alluded to in this text.
  • Distinguishing features - A. fumigatus may be identified by its ability to grow well at temperatures around 45 degrees Centigrade and its pathogenicity may be attributable to the fact that it can grow extremely well at body temperature. It can survive temperatures in excess of 70 Degrees Centigrade.
  • "Farmers Lung" - A. fumigatus is responsible for this disease affecting the lungs. Farmers Lung is similar to "Maltsters Lung" which is caused by Aspergillus clavatus as it grows on both hay and barley, barley being used in the brewing industry.

2

Classification

  • Kingdom
    • Members are, eukaryotic, heterotrophic, non vascular.
    • Lack chloroplasts, do not possess cell walls of chitin.
    • Store Food as glycogen. 
  • Phylum -  Ascomycota or sac fungi
    • possess sexual / asexual reproduction.
    • Form ascospores - spores born inside.
    • Posess asci - elongated bunches of ascospores.
    • Posess asocarp - cup like dikaryon structures attached to the asci.
    • Possess conidia - externally born asexual spores.
  • Class - Ascomycetes
    • Sexual spores called an asci inside an ascus in all members.
  • Order - Eurotiales, (blue / green moulds).
    • Scattered asci positioned on the surface of the closed asocarp.
  • Family - Tricocomaceae
    • Tufts of threads that release ascospores after breakdown of the asci.
  • Genus - Aspergillus
    • Highly aerobic environments, ubiquitous in nature.
    • The majority are plant / animal symbionts.
    • Many food contaminants.
  • Species - A. fumigatus
    • Most common pathogenic species.
    • Sexual reproduction identified in 2008
    • Causes Aspergillosis in immunocompromised individuals.

See table below for latest summarised classification.

Classification

 

Kingdom

Fungi

Phylum

Ascomycota

Subphylum

 

Class

Eurotiomycetes

Order

Eurotiales

Family

Trichocomaceae

Genus

Aspergillus

Species

A. fumigatus

 

Fig. 1. Shows a Phylogenetic Tree Depicting the lineage of A. fumigatus.

This phylogenetic tree shows some species in the Eurotiales order and was compiled using molecular data, (the SSU rDNA gene). Aspergillus fumigatus has been circled in red and is closely related to Aspergillus niger, which is used to make citric acid. It is also close to Penicillium chrysogenum, used to make the antibiotic Penicillin. This highlights the fine line between fungal pathogenicity causing disease to the other end of the spectrum where Fungi closely related to A. fumigatus can be extremely beneficial to mankind if harnassed in the correct way.

3

Morphology

  • Colony appearance - Aspergillus colonies grown on agar plates can manifest themselves in a variety of colours including; black, yellow, green, red white and brown including many shades of each of these colours. A. fumigatus is more commonly light green / white.
  • GrowthA. fumigatus normally grows much faster than many of the fungi in the the same genus.
  • Temperature - A. fumigatus can survive temperatures up to and beyond 70 Degrees Centigrade, especially in compost heaps. It grows well at temperatures up to 50 Degrees Centigrade and survives well in the body.

Fig. 2. Shows the Sporing Head of A. fumigatus

Fig. 3. Shows the sporing head of a Penicillium species to highlight how easy it might be to confuse it with Aspergillus fumigatus.

Fig. 4. Shows an A. fumigatus colony growing on an agar plate

Fig. 5. Electron micrograph of Aspergillus fumigatus

  • Mycelium
      • Well developed
      • Branching hyaline - present
      • Multinucleate cells
      • Vigorous growth of mycelium
      • Abundant grey / green conidia, (2-3um) 
      • Long erect conidiophores
  • Genome - A. fumigatus has a stable haploid genome around 30Mb in size sequenced by the Sanger Institute plus The Institute for Genomic Research, (TIGR) and published in the journal, Nature, December 2005 along with two other species of Aspergillus.
  • Morphological criteria for identification of Aspergillus species;
    • Growth Condition
      • Temperature, light, medium, age and source
    • Colony characteristics
      • Growth rate
      • Growth pattern
        • texture  colour.
        • Basal mycelium.
        • Surface mycelium.
        • Number / arrangement of heads, cleistothecia and sclerotia.
    • Conidial stage
      • Heads
        • How formed
        • Form
        • Colour
        • overall size
      • VesicleShape
        • Shape
        • Size
        • Colour
        • Fertile region
      • Philalides
        • Development
        • Arrangement - uniseriate / biseriate
        • Primaries - colour / dimension
        • Secondaries - colour / dimension
      • Condiophore
        • Length
        • Diameter
        • Wall details
      • Conidia
        • Dimension
        • Wall detail
        • Colour
      • Hulle Cells and other elements
        • Shape
        • Dimension
      • Sclerotia / sclerotium type structures
        • Form / structure
        • Colour
        • Dimension
    • Ascoporic stage
      • Cleistothecia 
        • Origin
        • Form / Structure
        • Colour
        • Dimension
        • Quantity
      • Asci
        • Shape
        • dimensions
      • Ascospores
        • Colour
        • Pattern
        • Dimension

Adapted from Raper & Thom: The genus Aspergillus, Baltimore 1965, William & Wilkins.

 

4

Replication/ Life Cycle

  • Reproduction - Until 2008 it was widely understood that A. fumigatus reproduced asexually as meiosis or mating had never been visualised in this species of fungus. However, in 2008 it was discovered that A. fumigatus has a sexual reproductive cycle.

Fig. 6. Generalised Sexual Life Cycle of Ascomycota

Haploid hyphae connect which is termed, "Plasmogamy". Fused plasma membranes are described as, in a dikaryon stage, (n+n). Karyogamy is where the two nuclei fuse whilst the cells membrance forms into an ascus which is an elongated oval cell. The diplod ascus then divides meiotically forming four ascospores which are released from the cup form of the fungi or the dikaryon ascocarp into the environment. Surviving ascospores grow to form haploid mycelia and the process is repeated. A. fumigatus was not thought to reproduce sexually until 2008 although it is still widely beleived that asexual reporduction is favoured under most conditions and in most habitats.

 

5

Clinical Disease

  • In general, clinical disease falls into three categories;
  • 1. Allergic Aspergillosis
      • 10-20% of asthmatics react to A. fumigatus and asthmatics with eosinophillia are subject to a more severe form.
      • Characterised by fleeting lung shadows and lung consolidation on chest X-rays.
      • Fungal growth produces fungal mycelium plugs which may block off the bronchal lumen thus reducing the available surface area for gaseous exchange in the lungs.
      • Exposure to high spore counts can cause allergic alveolitis.
      • Symptoms that can be evident within eight hours of exposure are;
        • Breathlessness
        • Fever
        • malaise
        • discomfort
      • Repeated attacks can cause severe lung damage similar to "Maltsters Lung" caused by Aspergillus clavatus.
  • 2. Aspergilloma
      • A. fumigatus can colonise cavities in the lung that may have been created by Tuberculosis
      • A compact ball of mycelium is formed with a dense firbrous wall.
      • patients are either asymtomatic or have a moderate cough with sputum production and Haemoptysis may occur.
      • Where blood vessels are invaded fatal complications can occur.
      • Surgery may be necessary to remove the fungus ball.
  • 3. Invasive Aspergilliosis
      • Occurs in severely immunocompromised patients with neutropenia as a predisposing factor.
      • This disease is responsible for significant mortality in bone marrow transplants.
      • The lungs are the sole site of infection for around 70% of patients.
      • There can be widespread destructive growth in the lungs.
      • A. fumigatus can invade blood vessels causing thrombosis.
      • Poor prognosis.
  • Endocarditis
      • Infection of heart tissue can occur in immunocompromised heart surgery patients.
      • Rare.
      • Poor prognosis.
  • Paranasal Granuloma  
      • Invades paranasal sinuses.
      • may spread through the bone to eye and brain regions.
      • Seen in warm, dry climates, for example, Sudan and Northern India.
  • Allergic Bronchopulmonary aspergillosis, (ABPA)
      • ABPA is characterised by an allergy to the spores of Aspergillus and is common in asthmatics. Up to 5% of adult asthmatics may contract ABPA sometime during their life.
      • Cystic fibrosis patients are commonly affected by ABPA through adolescense and into adulthood with similar symptoms to asthma with intermittent episodes of being unwell plus coughing and wheezing. Brown coloured plugs of mucus can be coughed up by infected persons. Diagnosis can take the form of X-Ray, skin, sputum and blood tests.
      • ABPA can lead to permanent lung damage including fibrosis if not treated.
      • Treatment - The steroid prednisolone is used to treat ABPA sufferers during attacks. Itraconazole may also be used so that the level of steroid administered by aerosol can be reduced. Unfortunately prednisolone can give rise to serious side effects, for example, osteoporosis plus weight gain over a long period of time. Benefits to patients are difficult to measure.  

 

6

Pathogenicity

  • A. fumigatus can penetrate the cellular bilayer from around 14-16 hours after inoculation according to researchers.
  • Galactomannan levels - can be specifically linked to A. fumigatus fungal invasion.
  • Invasive Aspergilliosis - the mortality rate for this disease is around 50%.
  • Gliotoxin - A. fumigatus produces this toxin which slows capillary beating. 
  • Elastases and Proteases - A. fumigatus releases these enzymes which can break down elastin and lung proteins.  

7

Immunology

  • IgE Production - The human body increases IgE production in response to being invaded by A. fumigatus.
  • The surface of the A. fumigatus conidia binds to crude fibrinogen in the lung tissue. A. fumigatus then releases elastases and proteases breaking down elastin and lung proteins.
  • Colonisation of respiratory mucosa causes sensitisation of lung tissue which causes Allergic bronchopulmonary aspergillosis, (ABPA).
  • Both Neutrophils and Monocytes prevent a full blown invasive infection in helathy individuals.
  • Bronchoalveolar macrophages are a first line of defence by readily engulfing A. fumigatus conidia. Those conidia which evade capture and begin to germinate into hyphae are targeted by granulocytes which adhere to the hyphae and degranulate them, killing them in the process.
  • Phagocytosis - This process plays a significant role in a healthy persons immune system when invaded by A. fumigatus.
  • Humoral Response in Allergic Aspergillosis 
      • Patients with hypersensitivity pneumonitis or extrinsic allergic alveolitis develop IgG preceptin antibodies.
      • IgG antibody increase induces pulmonary immune complex deposition which in turn increases inflamation.
  • Humoral Response in Aspergilloma
      • Strong IgG response for 99% of Aspergilloma patients.
      • Response does not provide protection.
      • Degree of immunosuppresion very important.
  • Humoral Response in invasive Aspergillosis
      • Patients are usually immunosupressed so mount no significant immune response.
  • Cellular Response in Allergic Aspergillosis
      • Cell mediated response leading to bronchocentric granulamatosis.
      • Lymphocyte increase.
  • Cellualr Response in Aspergilloma
      • Very little cell mediated response.
      • Inflammatory response.
  • Cellular Response in Invasive Aspergillosis
      • Non oxidative and oxidative fungicidal activity involves release of granular myeloperoxidase and myeloperoxidase independant oxidants onto the surface of the A. fumigatus.
  • Neutrophil released antifungal agents include;
      • Lactoferrin
      • Lysosome
      • Cathepsin G
      • Azarocidin
      • Various defencins
  • T-Lymphocytes are of little importance in defnce against A. fumigatus.
  • Neutrohils are far more important in defence against A. fumigatus so AIDS sufferers with low CD4 T-cell Counts are not as susceptible to infection as neutropenic patients. 

8

Epidemiology

  • Common spores - As already mentioned in the background section, A. fumigatus spores are found in abundance in air, up to 600 spores / metre cubed and up to 10,000,000 spores / metre cubed in August in farmyard barns containing mould hay or barley.
  • A. fumigatus can be found on rotting vegetable matter, for example on overipe tomatoes in greenhouses, etc. This fungus is found universally on woodland and farms where workers are placed at risk with the increased spore count during late summer.
  • Contaminant - A. fumigatus frequently contaminates laboratory media and clinical specimens.
  • Risk factors - persons suffering the conditions below are at greater risk of infection with A. fumigatus;
      • Aspergillus - associated allergies are common.
      • Pulmonary abormalities, for example, Emphysema, smoking and Tuberculosis.
      • Immunodefficiency, for example, HIV1/2, Leukaemia, bone marrow transplant.
      • Immunosuppresive therpay.
  • Resistance - It is estimated that 2.1% of A. fumigatus strains are resistant to itraconazole.
  • Invasive Apergillosis - Invasive Aspergillosis has a prognosis of around 29 days after diagnosis so early treatment is critical.

Mortality rate linked to treatment

Treatment started after diagnosis   Mortality Rate

  Less than 10 days   40%

 More than 11 days   90%

9

Laboratory Diagnosis

  • A. fumigatus may prove difficult to detect as there is a large variation among strains within the species as well as there being a very large number of Aspergillus species. It is therefore difficult to develop serological tests to Aspergillus species. It has to be understood that many characteristics from this Genus overlap.  
  • Allergic Aspergilliosis
    • Clinical
      • Fleeting shadows and lung consolidation on chest X-ray.
      • Fungal mycelia contained in coughed up sputum.
      • Breathlessness, malaise, fever, wheeze and discomfort.
    • Laboratory diagnosis
      • X-ray, (see above).
      • A. fumigatus identified in sputum.
      • Microscopy
        • Vesiculate conidiophores with numerous small conidia with columnar spore mass.
        • Conidiophore - short stalks, absent metulae, crowned phialides pointing upwards on upper two thirds of vesicle.
        • Conidia - rounded shape, 2.5um to 3.5um and slightly roughened.
  • Aspergilloma
    • Clinical
      • May be asymptomatic
      • Symptomatic
    • Laboratory diagnosis
      • Direct Microscopy
        • Abundant fungus in sputum for allergic aspergilliosis.
        • Fungus usually difficult to find for aspergilloma.
        • Invasive aspergilliosis microscopy difficult.
        • Best viewed after staining with Periodic Acid Schiff, (PAS) or methanamine - silver.
      • Culture
        • Grows readily at 25 to 45 Degrees Centigrade. The colony diameter can reach 50mm in just one week with a flat, spreading topography and powdery to felt like texture. It generally forms a blue green colony with a white margin and is usually cream on the reverse. 
        • Use Sabaraud agar without cyclohexamide. can also use Malt extract agar and Czapek Dox agar and glucose peptone agar.
        • Blood cultures.
      • Skin tests
        • Detection of A. fumigatus antigen
        • All patients give an immediate Type I reaction
        • 70% of patients with pulmonary eosinophilia give a delayed Type III reaction.
      • PCR Performed on BAL, (antigen detection)
        • High specificity.
        • negative predictive accuracy.
      • PCR Tissue
        • fast, (15 mins).
        • no long enzymatic reactions.
        • single step extraction.
        • no harmful phenol / chloroform materials.
        • highly specific.
      • Serological tests, (antibody detection)
        • Immunodiffusion.
        • CIE.
        • ELISA - Sensitivity is < 80%.

 

10

Recent Developments

  • Voriconazole plus caspofungin combination therpay - salvage therapy, (may be reduced by TB drugs).
  • Antifungal to combat resistant A. fumigatus;
      • Voriconazole - best antifungal used as monotherapy.
      • Terbinafine
      • Ravuconazole
      • Micafungin
      • Posaconazole
  • There is now more support for combination therapy for strains resistant to drugs which is prompting further research.  

11

Prophylaxis / Control

  • New
      • aerosolised amphotericin B for bone marrow transplants.
      • I.V amphotericin B for bone marrow transplants.
      • Oral itraconazole plus nasal amphotericin B.
  • Prevention
      • Remove all potted plants from hospitals.
      • Install High efficiency Particulate Filters, (HEPA) filters.
      • Foodstuffs, for example, pepper, nuts, spices and cereals can contain higher than average A. fumigatus spores. Such foods should not be given to immunocompromised patients.
      • Building works can increase infection incidence, (areas should be carefully sealed off and and dusty environments should be reduced).

12

Treatment

  • Allergic Aspergilliosis
      • Prednisolone, 30mg / day.
      • possible alternative treatment of itraconazole.
      • inhaled corticosteroids to alleviate allergic symptoms.
      • Anti-fungal drugs not known to help.
  • Aspergilloma
      • surgical removal of developed mycelial ball.
      • Mainly lobectomy performed but occasionally segmental of wedge resection can be sufficient.
      • Percutaneous injection of Amphotericin B if surgery is contraindicated.
      • 10-20mg in 10-20ml of distilled water, 2-3 times / week.
      • Itraconazole does not appear effective.
  • Infection of paranasal sinuses
      • Surgical removal if appropriate.
      • drainage and airation of infected site.
      • itraconazole prevents relapse, 200mg / day for up to 6 weeks.
      • Neutropenic patients - Amphotericin B, 1mg/kg / day.
  • Endocarditis
      • Surgical removal if appropriate.
      • Amphotericin B, 1mg/kg / day.
  • Acute Invasive Aspergillosis
      • Within 96 hours of onset of infection, treatment must be administered for any chance of success.
      • Amphotericin B i.v, 250mg/kg  / day. (given intravenously), may cause kidney damage / renal impairment.
      • Itraconazole or variconazole if amphotericin B is ineffective.
      • Caspofungin if drugs above are ineffective. (can only be administered intravenously).

 

13

Acknowledgements

  • Sincere thanks to Dr Petrou from the Mycology Reference Laboratory in Hammersmith Hospital, London. For Peer reviewing web text and supplying images.  
  • Dr Richard Hastings - Biocote Ltd. For help and support with the web design. 
  • Biological Sciences Department. University of Surrey for the image of the Penicillium Species.
  • Wikipedia for Aspergillus fumigatus culture and microscopy images.

14

References

  • Campbell, C., K, et al, (1996), Identification of Pathogenic Fungi. Frome, Somerset, Butler and Tanner, Chapter 7, pp116-123. 
  • Colier, L., Balows, A., Sussman, M., (1998). Topley & Wilson's Microbiology and Microbial Infections. Volume 4, Medical Mycology, Ninth Edition, New York, Oxford University Press inc. Chapter 16, pp281-312.

 

15

Websites

  • Wikipedia. (2009). Aspergillus fumigatus. [online], available at:http://en.wikipedia.org/wiki/Aspergillus_fumigatus, [accessed], 7th March 2009.
  • Aspergillus. (2009). Aspergillus. [online], available at: http://www.aspergillus.org.uk/languages/english.php, [accessed], 8th March 2009.
  • Aspergillus fumigatus. (2008). Aspergillus fumigatus. [online], available at: http://bioweb.uwlax.edu/bio203/s2008/miller_melo/Classification.htm, [accessed], 8th March 2009.

16

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17

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