The extraordinary spectrum of diseases caused by Aspergillus David W. Denning Wythenshawe Hospital University of Manchester

The genus Aspergillus - importance to humanity

on the negative side:

cause invasive and allergic disease in humans and other animals:

A. fumigatus

cause plant and food spoilage and produce mycotoxins: A. flavus and A. parasiticus www.aspergillus.man.ac.uk

The genus Aspergillus - importance to humanity

on the positive side:

composting well-established model organism in cell biology and genetics:

A. nidulans

food production: enzymes and organic acids: A. niger East Asian foods: A. oryzae and A. sojae pharmaceuticals: echinocandins: A. nidulans and A. sydowi lovastatin: A. terreus fumagillin: A. fumigatus www.aspergillus.man.ac.uk

Aspergillus Life-cycle

Spores inhaled Germination Mass of hyphae (plateau phase) Hyphal elongation and branching www.aspergillus.man.ac.uk

The genus Aspergillus –

~180 species, 38 have caused disease (able to grow at 37C) Common in the environment

A. nidulans – may be amphotericin B resistant

A. niger Aspergillus fumigatus

A. flavus -sometimes amphotericin B resistant www.aspergillus.man.ac.uk

CLASSIFICATION OF ASPERGILLOSIS Airways/nasal exposure to airborne Aspergillus Persistence without disease - colonisation of the airways or nose/sinuses Invasive aspergillosis • Acute (<1 month course) • Subacute/chronic necrotising (1-3 months) Chronic aspergillosis (>3 months) • Chronic cavitary pulmonary • Aspergilloma of lung • Chronic fibrosing pulmonary • Chronic invasive sinusitis • Maxillary (sinus) aspergilloma Allergic • Allergic bronchopulmonary (ABPA) • Extrinsic allergic (broncho)alveolitis (EAA) • Asthma with fungal sensitisation • Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)

Immunosuppression and infection • Inhalation of aspergillus spores is a common daily occurrence. A healthy immune system would normally remove the spores and no symptoms or infection would occur.

• In individuals whose immune system may be suppressed either because of illness eg AIDS, cancer patients or drugs, spores may germinate and resulting tissue or systemic aspergillus invasion can result.

• Individuals with allergies such as asthma, can also be vulnerable to aspergillus disease.

Interaction of Aspergillus with the host A unique microbial-host interaction

Acute IA ABPA Allergic sinusitis Subacute IA Tracheobronchitis Aspergilloma Chronic cavitary Chronic fibrosing Immune dysfunction Normal immune

.

Immune hyperactivity www.aspergillus.man.ac.uk

Changing incidence of fatal invasive mycoses in non-HIV patients in USA

1981 1986 1991 Candidiasis Aspergillosis 1996 McNeil et al, Clin Infect Dis 2001;33:641

Invasive pulmonary aspergillosis

Normal lung IPA IPA occurs in ~7% of acute leukaemia patients, 10-15% allogeneic BMT patients www.aspergillus.man.ac.uk

Unequivocal ‘Halo sign’ surrounding a nodule Halo sign Herbrecht, Denning et al, NEJM 2002;347:408-15.

Recent examples of the frequency of invasive aspergillosis Underlying condition Acute myeloid leukaemia Acute lymphatic leukaemia Allogeneic HSCT Lung transplantation Heart-lung transplantation Small bowel tranplantation AIDS Incidence 8% 6.3% 11-15% 6.2-12.8% 11% 11% 2.9% Reference/year Cornet, 2002 Cornet, 2002 Grow, 2002; Marr, 2002 Minari, 2002; Singh,2003 Duchini, 2002 Duchini, 2002 Libanore, 2002

Bleeding as an aspect of disseminated invasive aspergillosis

Fumagillin is anti-angiogenic A haemolysin described from

Aspergillus fumigatus

Other factors that contribute to thrombosis or a coagulopathy?

Gillies & Campbell, www.aspergillus.man.ac.uk

How does Aspergillus fumigatus cause thrombosis (clotting of vessels) and also bleeding?

Interaction of conidia and endothelial cell projections Internalisation of conidia (and hyphae) by endothelial cells with injury apparent at 4 hours Filler et al, Blood 2004;103:2134; Paris et al, Infect Immun 1997;65:1510.

Cerebral aspergillosis (abscess) in chronic lymphocytic leukaemia

Dissemination via the blood stream to the brain occurs in ~5% of cases of invasive aspergillosis, and in ~40% of allogeneic bone marrow (HSCT) recipients www.aspergillus.man.ac.uk

Early diagnosis of invasive aspergillosis is important

Treatment started Mortality <10d >11d 40% 90% Von Eiff et al, Respiration 1995;62:241-7.

Sputum Cultures for Fungus

Bacteriological media inferior to fungal media – 32% higher yield on fungal media A four day A. fumigatus culture on malt extract agar (above). Light microscopy pictures are taken at 1000x, stained with lacto-phenol cotton blue.

Aspergillus Antigen Test

• Diagnosis or surveillance? • Only blood, or BAL, CSF etc • Best OD cut-off - 0.7

• False positives in kids / antibiotics • False negative with antifungal prophylaxis • Not as useful for non-hematology • Not useful if pre-existing antibody Herbrecht et al, J Clin Microbiol 2002;20:1898-906; and others

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Outcome from invasive aspergillosis – amphotericin B therapy Survival Functions by Site of Infection Sinusitis (n =17) Multi-site (n =11) Aspe rgilloma (n =10) Pu lmonary (n =83) 30 60 90 CNS o r Dissemin ated (n =35) 120 150 180 210 Days 240 270 300 330 360 Lin et al, Clin Infect Dis 2001;32:358

Sub-acute invasive aspergillosis in AIDS www.aspergillus.man.ac.uk

Sub-acute invasive aspergillosis

• Less immunocompromised patients • Slower progression of disease (> 1 month) • Cavitary or nodular pulmonary disease typical • Vascular invasion less common • Dissemination less common • Antigen testing less useful • Antibody testing may be helpful in diagnosis www.aspergillus.man.ac.uk

Chronic necrotizing aspergillosis (CNPA)

Chronic necrotizing pulmonary aspergillosis (CNPA) is a subacute process usually found in patients with some degree of immunosuppression.

Usually it is associated with underlying lung disease, alcoholism, or chronic corticosteroid therapy. Because it is uncommon, CNPA often remains unrecognized for weeks or months and causes a progressive cavitary pulmonary infiltrate.

Chronic necrotising pulmonary aspergillosis

Right upper lobe showing circular shadow partly filled by a mass. PT MS 1996 Right lobe shows huge cavity containing some debris, with +ve aspergillus precipitins.Pt MS 1999 Right upper lobe. Patient has diabetes and pulmonary mycobacterium avium- shows small cavitary lesion PT MS 1995.

Same lobe shows expansion of the shadow, still partially filled with a mass. Pt MS 1998 Denning, Clin Microbiol Infect 2001;7(Suppl 2):25-31.

CLASSIFICATION OF ASPERGILLOSIS Airways/nasal exposure to airborne Aspergillus Persistence without disease - colonisation of the airways or nose/sinuses Invasive aspergillosis • Acute (<1 month course) • Subacute/chronic necrotising (1-3 months) Chronic aspergillosis (>3 months) • Chronic cavitary pulmonary • Aspergilloma of lung • Chronic fibrosing pulmonary • Chronic invasive sinusitis • Maxillary (sinus) aspergilloma Allergic • Allergic bronchopulmonary (ABPA) • Extrinsic allergic (broncho)alveolitis (EAA) • Asthma with fungal sensitisation • Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)

Aspergillus and airways

Types of aspergillosis of the airways • Colonisation (no disease – could be at risk) • Obstructing Aspergillus tracheobronchitis impaction ( non-invasive ) • Aspergillus bronchitis/tracheobronchitis (superficially invasive only ) • • Ulcerative Aspergillus tracheobroncitis ( /Mucus locally invasive ) (lung transplants – at anastomosis) Pseudomembranous Aspergillus tracheobronchitis (Extensive disease, locally invasive , associated with IPA and may disseminate) Langley, ATS 2004

Aspergillus tracheobronchitis

Autopsy drawing of a ‘normal’ 3 year old who died over 10 days Wheaton, Path Trans 1890; 41:34-37

Aspergillus tracheobronchitis

Review of 58 patients in literature for normal and immuno compromised patients - risk factors

%

None (ie normal) Heart / Lung transplant Solid tumour BMT Leukaemia HIV/AIDS Other 25 18 15 13 13 8 8 Kemper et al, Clin Infect Dis 1993; 17: 344

Aspergilloma Fungus ball Patient RT December 2002

Chronic pulmonary aspergillosis – pre-existing disease All 18 patients had prior pulmonary disease 9 TB, 5 with atypical mycobacteria 13 smokers or ex-smokers All 18 non-immunocompromised 3 excess alcohol Denning DW et al, Clin Infect Dis 2003; 37:S265

Chronic pulmonary aspergillosis presentation Weight loss Cough Shortness of breath Haemoptysis Fatigue / malaise Chest pain Sputum production ++ Fever 16 / 18 (89%) 15 / 18 (83%) 9 / 18 (50%) 9 / 18 (50%) 5 / 18 (28%) 3 / 18 (17%) 3 / 18 (17%) 2 / 18 (11%) Denning DW et al, Clin Infect Dis 2003; 37:S265

Chronic pulmonary aspergillosis serology

All 18 patients had positive Aspergillus precipitins (1+ - 4+) All 18 patients had elevated inflammatory markers, CRP, PV and / or ESR 14 of 18 (78%) had elevated total IgE (>20), 13 >200 and 7 >400 9 of 14 (67%) had Aspergillus specific IgE (RAST) Denning DW et al, Clin Infect Dis 2003; 37:S265

Chronic cavitary pulmonary aspergillosis (CCPA) Patient RW December 1991 Pre surgical resection Patient RW September 1992 Relapse in normal lung www.aspergillus.man.ac.uk

Chronic cavitary pulmonary aspergillosis www.aspergillus.man.ac.uk

Patient RW July 1993

Chronic Cavitary Pulmonary Aspergillosis Patient JA Jan 2001

Chronic Cavitary Pulmonary Aspergillosis Patient JA Feb 2002

Chronic Cavitary Pulmonary Aspergillosis Patient JA April 2003

Chronic Cavitary Pulmonary Aspergillosis Patient JA July 2003

Chronic cavitary pulmonary aspergillosis Patient JP June 1999 Denning DW et al, Clin Infect Dis 2003; 37:S265

Chronic Cavitary Pulmonary Aspergillosis, with aspergilloma Denning DW et al, Clin Infect Dis 2003; 37:S265 Patient JP July 2001

Chronic Fibrosing Pulmonary Aspergillosis Patient JP April 2002 Denning DW et al, Clin Infect Dis 2003; 37:S265

Mannose Binding Lectin (MBL)- a key part of the innate immune system

D i d n c i c c r e I r i n s Crosdale et al J Infect Dis 2001;184:653

Mannose Binding Protein

Mutations 5 mutations described 2 in promoter region (less important) 3 in open reading frame ( M52 , M54 , M57) Codon 54 mutation present in 16% of Caucasian homozygous in 2% Defects associated with bacterial infections in children and hepatitis B carriage Eisen & Minchinton Clin Infect Dis 2003;37:1496

CCPA and human gene defects

• 8 of 11 (72%) had low MBL genotypes p=<0.05

(compared to normal controls) • 8 of 17 (47%) had low MBL genotypes p=0.0002

• 32% and 21.5% frequency of 2 SPA2 mutations, compared with normals (18% and 11%) (p=0.021 and p=0.044) • not related to coeliac disease (<1 in 30) Crosdale et al J Infect Dis 2001;184:653; Vaid et al, unpublished.

CLASSIFICATION OF ASPERGILLOSIS Airways/nasal exposure to airborne Aspergillus Persistence without disease - colonisation of the airways or nose/sinuses Invasive aspergillosis • Acute (<1 month course) • Subacute/chronic necrotising (1-3 months) Chronic aspergillosis (>3 months) • Chronic cavitary pulmonary • Aspergilloma of lung • Chronic fibrosing pulmonary • Chronic invasive sinusitis • Maxillary (sinus) aspergilloma Allergic • Allergic bronchopulmonary (ABPA) • Extrinsic allergic (broncho)alveolitis (EAA) • Asthma with fungal sensitisation • Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)

ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS – Key diagnostic criteria • Asthma • Blood eosinophilia (>1,000 / cu mm) • History of pulmonary infiltrates • Central bronchiectasis ABPA possible ABPA possible ABPA probable ABPA almost certain • Precipitins against A. fumigatus positive • Aspergillus IgE antibody >2x asthma control • Aspergillus IgG antibody >2x asthma control • Total serum IgE concentration, >1000 iu/mL If 3 tests +ve, then ABPA very likely, If all 4 +ve the diagnosis established Rickett et al. Arch Intern Med 1983; 143: 1553; Patterson, Chest 2000;118:7

Before bronchoscopy

ABPA

After bronchoscopy www.aspergillus.man.ac.uk

ABPA mucous plugging

www.aspergillus.man.ac.uk

ABPA - CT showing central bronchiectasis www.aspergillus.man.ac.uk

ABPA and surfactant

5 surfactant proteins in man, SPA1, SPA2, SPB, SPC and SPD – all ‘collectin’ family Mason et al, Am J Physiol 1998;275:L1-13.

ABPA – surfactant defects

2 exonic polymorphisms, and 2 intronic polymorphisms in SP A2 associated with ABPA A1660G = OR of 4.78; or if combined with G1649C = OR 10.4

Also associated with higher peripheral eosinophilia Saxena et al, J Allergy Clin Immunol 2003;111:1001-7.

Eosinophilic fungal rhinosinusitis or allergic fungal sinusitis

Patient with chronic symptoms of nasal obstruction, loss of smell and nasal polyps Ponikau et al, Mayo Clinic Proc 1999;74:877 & WWW.aspergillus.man.ac.uk

Eosinophilic fungal rhinosinusitis

(link with airborne fungi - ?which most important = Myelin basic protein, highly toxic to local epithelium Ponikau et al, Mayo Clinic Proc 1999;74:877

A link between Aspergillus and asthma?

Fungal-associated asthma – evidence

Severe asthma linked with fungal sensitisation Frequency of fungal sensitisation ABPA Fungal-associated asthma Treatment of ABPA and pilot data High spore counts and asthmatic attacks

Spore counts and asthma attacks and admission to hospital

All circumstantial evidence • Thunderstorm asthma – linked to Alternaria • Asthma deaths (Chicago) linked to high ambient spores counts and season (summer autumn) when spore counts highest • Asthma hospital admission linked to high ambient spore counts (Derby, New Orleans, Ottawa • Asthma hospital attendance linked to high spore counts , but not pollen counts (Canada) • Asthma symptoms increased on days of high spore counts (California, Pennsylvania) O'Hollaren, N Engl J Med 1991; 324: 359; Newson, Occup Environ Med 2000; 57: 786-92.

Fungus at home

Environmental data • Mouldy housing associated with worse asthma, with a correlation between asthma severity and degree of dampness in the home and separately with visible mould growth • In Germany bronchial reactivity in children was associated with damp housing • Mouldy and damp school associated with asthma symptoms and emergency room visits • Highest concentration of Aspergillus fumigatus is at home Williamson, Thorax 1997;52:229. Taskinen, Acta Paediatr 1999; 88:1373.

Severe asthma and moulds

Mild asthma – 564 (50%) Moderate asthma – 333 (29%) Severe asthma – 235 (21%) – linked with fungus skin test positivity Zureik et al, Br Med J 2002;325:411

Asthma severity, house dust mites, cats and moulds

Allergen House dust mite Cats* No asthma n= 111 61% Mild asthma FEV 1 >75% <90% n= 67 Moderate asthma FEV 1 >60% <75% n= 42 Severe asthma FEV 1 >60% n= 42 71% 45% 77% 49% 51% 38% 35% Moulds # 17% 19% * P = 0.05

# p = 0.01

36% 31% Langley, ATS 2004