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

The genus Aspergillus - importance to humanity 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 on the negative side:

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

Spores inhaled Germination Mass of hyphae (plateau phase) Hyphal elongation and branching Aspergillus Life-cycle

A. nidulans – may be amphotericin B resistant The genus Aspergillus – ~800 species, ~50 have caused disease (able to grow at 37C) Common in the environment A. niger A. terreus – resistant to AmB A. flavus -sometimes amphotericin B resistant A. fumigatus low frequency of azole resistance Aspergillus fumigatus conidial head

CLASSIFICATION OF ASPERGILLOSIS Persistence without disease - colonisation of the airways or nose/sinuses Airways/nasal exposure to airborne Aspergillus 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 allergic aspergillosis

Global opportunistic infections Disease Most common species Location Estimated Life- Threatening Infections / Year Mortality Rates (% in infected populations) Opportunistic Systemic Mycoses Invasive aspergillosis Aspergillus spp. worldwide>200, % Invasive candidiasis Candida spp. worldwide>400, % Cryptococcosis Cryptococcus neoformans worldwide>1,000, % Mucormycosis Rhizopus oryzae worldwide>10, – 90% Pneumocystis Pneumocystis jirovecii worldwide>400, % Brown et al, Sci Transl Med 2012;4:165rv13

Interaction of Aspergillus with the host A unique microbial-host interaction Immune dysfunction Frequency of aspergillosis Immune hyperactivity Frequency of aspergillosis Acute IA Subacute IA Tracheobronchitis Aspergilloma Chronic cavitary Chronic fibrosing ABPA Allergic sinusitis. Normal immune function

Interaction of Aspergillus with the host A unique microbial-host interaction Immune dysfunction Frequency of aspergillosis Immune hyperactivity Frequency of aspergillosis Acute IA Subacute IA Aspergillus keratitis Aspergillus bronchitis Chronic pulmonary Otitis externa Onychomycosis ABPA Severe asthma with fungal sensitisation Allergic sinusitis. Lung/tissue damage

Changing incidence of fatal invasive mycoses in non-HIV patients in USA Rate per 100,000 population Candidiasis Aspergillosis McNeil et al, Clin Infect Dis 2001;33:641

Aspergillosis burden in Europe ECDC report published February 2013 Type of aspergillosis Predominant risk groups Risk population size (000’s) Aspergillosis rate Annual aspergillosis burden (000’s) ABPA Asthma35,4742.5%887 (248 – 1,242) Cystic fibrosis2915%4.3 SAFS Severe asthmaa3,54733% 1,170 (886 – 1,774) Chronic pulmonary aspergillosis COPD, TB, sarcoidosis, ABPA, Pneumothorax >13, % 240 Invasive aspergillosis Myeloid leukaemia, Other haematological HSCT % COPD hospital admissions 3,6001.2%34 Solid organ transplantation %0.25 Medical ICU1,100 ( all ICU)2%22 Total aspergillosis annual burden All--2, ,250 2,061,300

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

Invasive pulmonary aspergillosis Normal lung IPA IPA occurs in ~7% of acute leukaemia patients, 10-15% allogeneic BMT patients

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

Recent examples of the frequency of invasive aspergillosis Underlying conditionIncidenceReference/year Acute myeloid leukaemia8%Cornet, 2002 Acute lymphatic leukaemia6.3%Cornet, 2002 Allogeneic HSCT11-15% Grow, 2002; Marr, 2002 Lung transplantation % Minari, 2002; Singh,2003 Heart-lung transplantation11%Duchini, 2002 Small bowel tranplantation11%Duchini, 2002 AIDS2.9%Libanore, 2002

Gillies & Campbell, 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?

How does Aspergillus fumigatus cause thrombosis (clotting of vessels) and also bleeding? Filler et al, Blood 2004;103:2134; Paris et al, Infect Immun 1997;65:1510. Interaction of conidia and endothelial cell projections Internalisation of conidia (and hyphae) by endothelial cells with injury apparent at 4 hours

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

Early diagnosis of invasive aspergillosis is important Treatment started 11d Mortality 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 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: ; and others

Use of serology, PCR, and GM to establish patient groups 130 patients not on azoles Total IgE, specific IgE aspergillus, specific IgG aspergillus, sputum galactomannan, sputum PCR Cluster analysis – software Mplus version 6.1 Baxter, J Allerg Clin Immunol 2013;132:560

Outcome from invasive aspergillosis – amphotericin B therapy Lin et al, Clin Infect Dis 2001;32:358

Risk groups and frequencies of invasive aspergillosis – different test performances Herbrecht, Ann NY Acad Sci 2012;1271:23

Sub-acute invasive aspergillosis in AIDS

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

Aspergillus otitis externa Almost always A. niger Acute otitis affects 1 in 250 persons annually Chronic otitis affects 3-5% of the population ( million) ~10% are fungal in origin

Aspergillus onychomycosis Almost always toenails 0.5-3% of all cases of onychomycosis 10’s to 100’s millions with onychomycosis worldwide ? 1 million cases

Aspergillus keratitis Usually A. flavus or A. fumigatus ~50% of keratitis fungal, and ~50% of these are due to Aspergillus million affected worldwide, especially in India, Nepal, Sri Lanka, Myanmar. Usually farmers and labourers, occasionally contact lens wearers

Aspergillus bronchitis Complicates cystic fibrosis and bronchiectasis Recurrent infection and/or mucus plugging Superficial invasion of the bronchial wall UK CF patients, 5000 adults. Aspergilus bronchitis affects ~1,500 Non-CF patients unclear

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 Denning, Clin Microbiol Infect 2001;7(Suppl 2): Right upper lobe. Patient has diabetes and pulmonary mycobacterium avium- shows small cavitary lesion PT MS Right upper lobe showing circular shadow partly filled by a mass. PT MS 1997 Same lobe shows expansion of the shadow, still partially filled with a mass. Pt MS 1998 Right lobe shows huge cavity containing some debris, with +ve aspergillus precipitins.Pt MS 2000

CLASSIFICATION OF ASPERGILLOSIS Persistence without disease - colonisation of the airways or nose/sinuses Airways/nasal exposure to airborne Aspergillus 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 Langley, ATS 2004 Types of aspergillosis of the airways Colonisation (no disease – could be at risk) Obstructing Aspergillus tracheobronchitis /Mucus impaction (non-invasive) Aspergillus bronchitis/tracheobronchitis (superficially invasive only) Ulcerative Aspergillus tracheobroncitis (locally invasive) (lung transplants – at anastomosis) Pseudomembranous Aspergillus tracheobronchitis (Extensive disease, locally invasive, associated with IPA and may disseminate)

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

Aspergillus bronchitis Young et al. Medicine (Balt) 1970;49:147

Aspergillus tracheobronchitis Review of 58 patients in literature for normal and immuno compromised patients - risk factors % None (ie normal)25 Heart / Lung transplant18 Solid tumour15 BMT13 Leukaemia13 HIV/AIDS 8 Other 8 Kemper et al, Clin Infect Dis 1993; 17: 344

Spectrum of pulmonary aspergillosis Hope. Med Mycol 2005:43 (Suppl 1) S207

Different patterns of CPA Radiological response varies by subtype of CPA

Aspergilloma Patient RT December 2002 Fungus ball

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

Chronic pulmonary aspergillosis - serology All 18 patients had positive Aspergillus precipitins ( ) 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 September 1992 Relapse in normal lung Patient RW December 1991 Pre surgical resection

Chronic cavitary pulmonary aspergillosis Patient RW July

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 Patient JP July 2001 Denning DW et al, Clin Infect Dis 2003; 37:S265

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

Prognosis CPA + subacute IA Korea ( ) CPA Japan (2001-9) 75% mortality 80% mortality Nam Int J Infect Dis 2010;14:e479; Ohba et al, Resp Med 2012; 106:724

Mannose Binding Lectin (MBL)- a key part of the innate immune system Crosdale et al J Infect Dis 2001;184:653

Mannose Binding Protein 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 Mutations 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= % 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 Persistence without disease - colonisation of the airways or nose/sinuses Airways/nasal exposure to airborne Aspergillus 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 Rickett et al. Arch Intern Med 1983; 143: 1553; Patterson, Chest 2000;118:7 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

ABPA versus SAFS Denning et al, Med Mycol 2013:51:361

How common is ABPA in asthma? 13/500 (2.6%) 10/1390 (0.72%) 9/255 (3.5%) 6/264 (2.3%) + 5/200 (2.5%) China Donnelly, Irish J Med Sci 1991;160:288; Eaton, Chest 2000;118:66; Al-Mobeireek, Resp Med 2001;98:341

ABPA Before bronchoscopy After bronchoscopy

ABPA mucous plugging

ABPA - CT showing central bronchiectasis

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:

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 &

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 Fungal-associated asthma ABPA Treatment of ABPA and pilot data Severe asthma linked with fungal sensitisation Frequency of fungal sensitisation 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:

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 Langley, ATS 2004 AllergenNo asthma n= 111 Mild asthma FEV 1 >75% <90% n= 67 Moderate asthma FEV 1 >60% <75% n= 42 Severe asthma FEV 1 >60% n= 42 House dust mite 61%71%45%77% Cats*49%51%38%35% Moulds # 17%19%36%31% * P = 0.05 # p = 0.01