Aspergillosis – New Data and Challenges

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Aspergillosis – new data and challenges

David W. Denning National Aspergillosis Centre University Hospital of South Manchester The University of Manchester Agenda

How many patients are there with serious fungal infection? SAFS Diagnostics – progress and gaps Voriconazole TDM 600 different fungi are human pathogens

Resistance and Virulence changes

Globalization

More species extinction due to fungi than bacteria or viruses

Chytridiomycosis in amphibian spp

The size of the problem

Over 300 million people affected by serious Fungal Infection worldwide

www.fungalresearchtrust.org/HowCommonareFungalDiseases5.pdf

Estimated Life-Threatening Mortality Rates Disease Location Infections / Year at that (% in infected Most common species Locationa populations)a

Opportunistic Systemic Mycoses Aspergillosis worldwide >200,000 30 - 95% Aspergillus fumigatusThe size of the problem Candidiasis worldwide >400,000 46 - 75% Candida albicans Cryptococcosis worldwide >1,000,000 20 - 70% Cryptococcus neoformans Mucormycosis worldwide >10,000 30 90% Rhizopus oryzae – Pneumocystis worldwide >400,000 20 - 80% Pneumocystis jirovecii Endemic Dimorphic Mycoses Blastomycosis Midwestern and ~3,000 <2% - 68% Blastomyces dermatitidis Atlantic U.S. Coccidioidomycosis Southwestern U.S. ~25,000 <1% - 70% Coccidioides immitis Histoplasmosis Midwestern U.S. ~25,000 28 50% Histoplasma capsulatum – Paracoccidioidomycosis Brazil ~4,000 5 27% Paracoccidioides brasiliensis – Penicilliosis SouthEast Asia >8,000 2 75% Penicillium marneffei – The intersecon of serious fungal diseases with TB, AIDS, cancer, asthma and COPD

Crypto PCP Histo

AIDS Cancer COPD CPA IC TB IA Asthma

ABPA SAFS Fungal keratitis The size of the problem Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deicit / Cancer Cryptococcal 1,000’s 1,000,000 1,000’s meningitis Pneumocystis >200,000 >100,000 pneumonia Invasive >100,000 >50,000 >50,000 aspergillosis Chronic pulmonary 3,000,000 aspergillosis Fungal eye 1,000,000 infection Fungal hair 200 million infection The size of the problem Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deicit / Cancer Cryptococcal 1,000’s 1,000,000 1,000’s meningitis Pneumocystis >200,000 >100,000 pneumonia Invasive >100,000 >50,000 >50,000 aspergillosis Chronic pulmonary 3,000,000 aspergillosis Fungal eye 1,000,000 infection Fungal hair 200 million infection The size of the problem Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deicit / Cancer Cryptococcal 1,000’s 1,000,000 1,000’s meningitis Pneumocystis >200,000 >100,000 pneumonia Invasive >100,000 >50,000 >50,000 aspergillosis Chronic pulmonary 3,000,000 aspergillosis Fungal eye 1,000,000 infection Fungal hair 200 million infection Topic 5: The tremendous burden of cryptococcal meningitis in sub-Saharan Africa in persons with HIV/AIDS

Incidence 3,2%, 720,000 cases (144,000-1,3 million) 90d fatality: 90%

Comparison of deaths in sub-Saharan Park BJ et al. Africa due to HIV-related cryptococosis, AIDS 2009 and common infectious diseases excluding HIV, as estimated by WHO 1,170,000 patients (5 year period prevalence) 375,000 annual incident cases ~15% annual mortality The size of the problem Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deficit / Cancer Candida infections Oral thrush 9,500,000 100,000’s millions Oesophageal 2,000,000 candidasis Candida >75 million vaginitis 4x/yr Candidaemia 100,000 200,000 Candida 75,000 peritonitis Allergic lung disease ABPA 4,800,000 SAFS >3,500,000 The size of the problem Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deficit / Cancer Candida infections Oral thrush 9,500,000 100,000’s millions Oesophageal 2,000,000 candidasis Candida >75 million vaginitis 4x/yr Candidaemia 100,000 200,000 Candida 75,000 peritonitis Allergic lung disease ABPA 4,800,000 SAFS >3,500,000 The size of the problem ~24 million patients affected each year

Annual incident and prevalent fungal infections

1,000,000 Candida 12,500,000 Cryptococcus Pneumocystis 11,000,000 Aspergillus 400,000 1,100,000 Fungal keratitis Some fungal infections

Disseminated Penicillium Coccidioidomycosis Aspergillus brain marneffei infection in from Mexico abscess from AIDS from Thailand Pakistan

Oral candidiasis in Allergic fungal AIDS from France sinusitis from India

Chromoblastomycosis from PNG Disseminated Onychomycosis histoplasmosis in AIDS from UK Some fungal infections

Disseminated Penicillium Coccidioidomycosis Aspergillus brain marneffei infection in from Mexico abscess from AIDS from Thailand Pakistan

Oral candidiasis in Allergic fungal AIDS from France sinusitis from India

Chromoblastomycosis from PNG Disseminated Onychomycosis histoplasmosis in AIDS from UK The severity of the problem Deaths per year • Cryptococcal meningitis – 10% death rate in the USA, >80% in Africa. 600,000 deaths. • Invasive aspergillosis – 50% mortality treated, 100% if not. >100,000 deaths • Chronic pulmonary aspergillosis – 15% annual mortality, 450,000 deaths. • Pneumocystis pneumonia - ~15% mortality in AIDS, ~50% non-AIDS, >80,000 deaths. • Candida bloodstream infection - ~40% mortality, 120,000 deaths • SAFS – increased risk of asthmatic death (estimated to be 100,000 annually worldwide) Reality check with TB

TB (2008) Fungal Infection Incident cases 9-10 million >14 million

Prevalent cases 10-13 million ~285 million

HIV related deaths ~550,000 ~650,000

Non-HIV related ~1,500,000 >700,000 deaths Chronic fungal infections Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deicit / Cancer Cryptococcal 1,000’s 1,000,000 1,000’s meningitis Pneumocystis >200,000 >100,000 pneumonia Invasive >100,000 >50,000 >50,000 aspergillosis Chronic pulmonary 3,000,000 aspergillosis Fungal eye 1,000,000 infection Fungal hair 200 million infection Recurrent and chronic fungal infections

Global burden of serious fungal infection (estimates by underlying disease) Fungal Infection None HIV/AIDS Respiratory Immune Critical care deicit / Cancer Candida infections Oral thrush 9,500,000 100,000’s millions Oesophageal 2,000,000 candidasis Candida >75 million vaginitis 4x/yr Candida bloodstream 100,000 200,000 infection Allergic lung disease ABPA 4,000,000 SAFS >3,500,000 The severity of the problem Ill health and morbidity • Oral and oesophageal thrush – unpleasant, reduced food intake and weight loss. • Candida vaginitis – anxiety and impaired sex life • ABPA and SAFS – breathlessness with severe asthma, reducing work capability • Chronic pulmonary aspergillosis – progressive breathlessness and weight loss • Fungal eye infection – unilateral blindness • Fungal hair infection – psychological problems and contagious Asthma and Aspergillus 79 adult asthmatics and 14 controls

Patients sensitised to A. fumigatus compared with non- sensitised asthmatics had: lower lung function (% pred. FEV1 68% vs 88% p < 0.05), more bronchiectasis (68% versus 35% p < 0.05) and more sputum neutrophils (80.9% vs 49.5% p < 0.01).

Fairs et al, Am J Respir Crit Care Med 2010; July 16 Severe asthma and aspergillosis in ICU

57 of 357 (16%) admitted ICU with acute asthma Compared with 755 outpatients with asthma

Aspergillus skin prick test used to screen for aspergillus hypersensitivity, if positive IgE etc for ABPA checked Aspergillus positive ABPA Asthma in ICU 29/57 (51%) 22/57 (39%) Outpatient asthma 90/755 (39%) 155/755 (21%) P value 0.01 0.001

Agarwal et al, Mycoses 2009 Jan 24th Severe asthma with fungal sensitisation (SAFS)

Criteria for diagnosis • Severe asthma (BTS step 4 or 5) AND • RAST (IgE) positive for any fungus OR • Skin prick test positive for any fungus AND • Exclude ABPA (ie total IgE <1,000 iu/mL)

Denning et al, Eur Resp J 2006; 27;27:615 Comparison of ABPA and SAFS serology

ABPA results normal range date 1 date 2 Patient 1

SAFS results

2 Skin prick testing – example of SAFS result

Cladosporium +ve

O’Driscoll, unpublished Fungal sensitisation in severe asthma – skin prick test or RAST for diagnosis?

100% N= 121 patients screened

>23% 50% discordant results }

43 34 10 13 SPT + RAST SPT positive SPT negative SPT negative both positive RAST negative RAST positive RAST negative

O’Driscoll et al, Clin Exp Allergy. In press Fungal sensitisation in severe asthma – number sensitised to one or more fungi

N = 40 13 sensitised to only Aspergillus 8 to Candida 3 to Trichophyton 3 to Penicillium 1 to Alternaria N = 20 1 to Cladosporium

29 11 11 12 7 7 3 1 2 3 4 5 6 7

Sensitisation to one or more fungi

O’Driscoll et al, Clin Exp Allergy. In press Distinguishing different forms of aspergillosis Disease group CCPA ABPA + CCPA ABPA SAFS SAFS n 116 16 98 52 52 2739 2300 370 Median serum 99.8 (26.4-350) (1100-7500) (1100-4550) (140-750) IgE level (IQR) (n=107) (n=16) (n=97) (n=52) Aspergillus 93.6% (103/110) 81.3% (13/16) 65.4% (53/81) 35.9% (14/39) specific IgG Positive fungal 25% (29/116) 25.0% (4/16) 23.5% (23/98) 21.2% (11/52) culture Positive Positive SPT specific IgE Mixed mould N/T N/T 88.9% (8/9) 90.9% (20/30) 100% (2/2) A. fumigatus 37.7% (40/106) 93.8% (15/16) 96.9% (94/97) 78.8% (41/52) 90.9% (20/30) Alternaria 10.0% (1/10) 100% (10/10) 77.5% (55/71) 32.5% (13/40) 47.4% (9/19) alternata C. albicans 33.3% (3/9) 90.0% (9/10) 81.4% (57/70) 37.5% (15/25) 52.6% (10/19) Cladosporium 20.0% (2/10) 80.0% (8/10) 70.4% (50/71) 24.4% (10/41) 35.5% (6/17) herbarum Penicillium 27.3% (3/11) 100% (10/10) 85.3% (58/68) 30.0% (12/40) 43.8% (7/16) chrysogenum Trichophyton 33.3% (2/6) 100% (3/3) 65.2% (30/46) 25.0% (9/36) 23.1% (3/13) mentagrophyte Unpublished Distinguishing different forms of aspergillosis Disease group CCPA ABPA + CCPA ABPA SAFS SAFS n 116 16 98 52 52 2739 2300 370 Median serum 99.8 (26.4-350) (1100-7500) (1100-4550) (140-750) IgE level (IQR) (n=107) (n=16) (n=97) (n=52) Aspergillus 93.6% (103/110) 81.3% (13/16) 65.4% (53/81) 35.9% (14/39) specific IgG Positive fungal 25% (29/116) 25.0% (4/16) 23.5% (23/98) 21.2% (11/52) culture Positive Positive SPT specific IgE Mixed mould N/T N/T 88.9% (8/9) 90.9% (20/30) 100% (2/2) A. fumigatus 37.7% (40/106) 93.8% (15/16) 96.9% (94/97) 78.8% (41/52) 90.9% (20/30) Alternaria 10.0% (1/10) 100% (10/10) 77.5% (55/71) 32.5% (13/40) 47.4% (9/19) alternata C. albicans 33.3% (3/9) 90.0% (9/10) 81.4% (57/70) 37.5% (15/25) 52.6% (10/19) Cladosporium 20.0% (2/10) 80.0% (8/10) 70.4% (50/71) 24.4% (10/41) 35.5% (6/17) herbarum Penicillium 27.3% (3/11) 100% (10/10) 85.3% (58/68) 30.0% (12/40) 43.8% (7/16) chrysogenum Trichophyton 33.3% (2/6) 100% (3/3) 65.2% (30/46) 25.0% (9/36) 23.1% (3/13) mentagrophyte Unpublished Randomised studies of antifungals and ABPA and/or asthma

Disease Antifungal, Benefit? Author, year duration ABPA Natamycin inh, 52 No Currie, 1990 wks ABPA Itraconazole, 32 Yes Stevens, 2000 wks ABPA Itraconazole, 16 Yes Wark, 2003 wks “Trichophyton” asthma Fluconazole, 20 wks Yes Ward, 1999

SAFS Itraconazole, 32 Yes Denning, 2009 wks Therapy of allergic aspergillosis

Knutsen et al. J All Clin Immunol 2012;129:280 Proof of concept RCT of itraconazole Rx in Severe Asthma with Fungal Sensitisation – quality of life improvement

P= 0.014

Denning et al, Am J Resp Crit Care Med 2009; 179:11 Second and third line antifungal therapy for ABPA and/or asthma

• 26 patients, ABPA (n = 21) or SAFS (n = 5). • All patients had failed itraconazole (n=14) or developed adverse events (AEs) (n=12) • 34 courses of therapy, 25 with voriconazole and 9 with posaconazole.

•

Chishimba et al, J Asthma 2012;49:423 VORICONAZOLEVORICONAZOLE AND AND POSACONAZOLE POSACONAZOLE FOR FOR ABPA ABPA AND AND SAFS 427

ResponseResponse to to Therapy Therapy doi/suppl/[doinumber]).doi/suppl/[doinumber]). While While modest falls were seen in some patients at 3 and 6 months, it was only at 9 and 12 ClinicalClinical response response at at 3, 3, 6, 6, and and 12 12 months months of of voriconazole voriconazole or or some patients at 3 and 6 months, it was only at 9 and 12 months that sustained and statistically significant total IgE posaconazoleposaconazole treatment treatment is is summarized summarized in in Table Table 2. 2. Overall Overall months that sustained and statistically significant total IgE falls were seen. At 12 months, the median IgE decreased clinicalclinical improvement improvement to to voriconazole voriconazole treatment treatment was was falls were seen. At 12 months, the median IgE decreased by 27.3% from baseline (median IgE 895, range 64–7200) observedobserved in in 17 17 (68%; (68%; ABPA ABPA 13,13, SAFS SAFS 4)4) of of 25 25 patients patients by 27.3% from baseline (median IgE 895, range 64–7200) ¼ ¼ to a median IgE of 475 kU/L (range 51–3100). at3months,15(75%)of20at6months,and12(70.6%)of17at3months,15(75%)of20at6months,and12(70.6%)of17¼ ¼ to a median IgE of 475 kU/L (range 51–3100). RAST to Aspergillus fell, but was only significant at 12 atat 12 12 months, months, compared compared with with 7 7 of of 9 9 (78%) (78%) at at 3, 3, 6, 6, and and 12 12 RAST to Aspergillus fell, but was only significant at 12 months and beyond from 23.2 kUa/L (range 0.6–294) at monthsmonths for for posaconazole posaconazole (Table (Table 2). 2). On On the the basis basis of of clinical clinical months and beyond from 23.2 kUa/L (range 0.6–294) at baseline to 17.7 kUa/L at 12 months (range 0.6–57.4; parameters,treatmentfailuretovoriconazolewasobservedinparameters,treatmentfailuretovoriconazolewasobservedin baseline to 17.7 kUa/L at 12 months (range 0.6–57.4; p .0056; Supplementary Table E2, Figure 2). We also 11 of of 20 20 (5%) (5%) patients patients at at 3 3 months, months, none none of of 15 15 (0%) (0%) at at 6 6 p .0056; Supplementary Table E2, Figure 2). We also observed¼¼ marked heterogeneity in IgE responses with a months,months, and and 2 2 of of 15 15 (15%) (15%) at at 12 12 months. months. No No treatment treatment failure failure observed marked heterogeneity in IgE responses with a few dramatic falls, most varying erratically through the waswas observed observed with with posaconazole. posaconazole. few dramatic falls, most varying erratically through the course of the therapy and some rising. There was no con- ThereThere was was a a marked marked reduction reduction in in OCS OCS and and short-acting short-acting course of the therapy and some rising. There was no con- beta-2beta-2 agonist agonist (SABA) (SABA) use, use, health-care health-care utilization utilization due due to to sistentsistent response response between between IgE IgE and clinical impact and so the asthma,asthma, and and improvement improvement in in overall overall health health status, status, as as sub- sub- decisiondecision to to continue continue or or discontinue discontinue therapy was based on jectivelyjectively perceived perceived by by individual individual patients patients in in domains domains such such clinicalclinical parameters parameters (i.e., (i.e., response and adverse effects). asas physical physical well-being, well-being, functioning, functioning, energy, energy, increased increased ET, ET, TotalTotal IgE IgE antibody antibody trend trend in ABPA by individual patient andand reduction reduction in in patients patients’’overalloverall symptoms symptoms (see (see Table Table 3). 3). atat baseline baseline and and on on therapy therapy at 3, 6, 9, and 12 months is ThereThere was was a a direct direct correlation correlation between between OCS OCS use use and and other other shownshown in in Supplement Supplement Figure Figure E1. None of our patients clinicalclinical parameters parameters of of clinical clinical response. response. Although Although the the receivedreceived omalizumab omalizumab (Xolair). (Xolair). dosagedosage of of ICS ICS was was reportedly reportedly modified modified during during antifungal antifungal Lung Function therapytherapy in in some some patients, patients, it it was was difficult difficult to to quantify quantify retro- retro- Lung Function spectively,spectively, and and overall overall was was probably probably not not markedly markedly reduced. reduced. Overall,Overall, lung lung function function improved improved throughout the treatment ThereThere were were 10 10 patients patients who who had had had had frequent frequent hospital hospital period.period. Of Of the the eight eight patients patients with spirometry values at both admissionsadmissions at at the the start start of of voriconazole voriconazole therapy therapy and and no no baselinebaseline and and 3 3 months, months, there was a nonsignificant admissionsadmissions were were observed observed in in 9 9 of of them them (90%) (90%) at at 3 3 months. months. improvementimprovement of of FEV1 FEV1 5.5% 5.5% from median baseline value ThisThis benefit benefit was was maintained maintained at at 6 6 and and 12 12 months months of of therapy. therapy. ofof 1.64 1.64 ( (pp .25).25) and and FVC FVC improved by 16.9% from a ¼ VoriconazoleVoriconazole therapy therapy reduced reduced the the frequency frequency of of recurrent recurrent mediummedium value value¼ of of 2.67 2.67 ( (pp .11). There were no positive or ¼ chestchest infections infections or or acute acute exacerbations exacerbations in in 17 17 of of 24 24 (70%), (70%), 9 9 negativenegative statistically statistically significant significant¼ changes or trends wit- ofof 19 19 (47%), (47%), and and 9 9 of of 17 17 (52.9%) (52.9%) patients patients at at 3, 3, 6, 6, and and 12 12 nessednessed beyond beyond 3 3 months. months. months,months, respectively respectively (Table (Table 3). 3). Posaconazole Posaconazole therapy therapy For personal use only. For personal use only. reducedreduced the the frequency frequency of of chest chest infections infections or or acute acute exacer- exacer- RadiologicalRadiological Response Response bationsbations in in 7 7 of of 9 9 (78%) (78%) patients patients at at 3 3 months months and and throughout throughout OfOf the the voriconazole-treated voriconazole-treated patients who had baseline and thethe 12-month 12-month period period (Table (Table 3). 3). follow-upfollow-up radiology radiology ( (nn 11), 5 of 10 (50%), 4 of 11 (36.4%),(36.4%), and and 4 4 of of 7 7 (57.1%) (57.1%)¼¼ patients showed improvement ImmunologicalImmunological Response Response inin radiological radiological abnormalities abnormalities including bronchiectasis PatientPatient median median total total IgE IgE values values (kU/mL) (kU/mL) over over 12 12 months months (severity(severity and/or and/or distribution, distribution, n 3), fibrosis (n 2), con- withwith ABPA ABPA only only are are summarized summarizedImpact in in Figure Figure of 1 1voriconazole and and solidationsolidation ( (nn 4),and4), mucus mucus plugging¼ (n 5), lobar¼ collapse SupplementarySupplementary Table Table E1 E1 (http://informahealthcare.com/ (http://informahealthcare.com/ ((nn 3),3), pulmonary pulmonary¼¼ nodules nodules (n 7),¼ and/or cavitations posaconazole on ABPA¼¼ and SAFS - ¼

TTABLEABLE2.2.——OverallOverall clinical clinicalretrospective response response to to therapy therapy at at different different times times on on treatment treatment by by disease disease groups. J Asthma Downloaded from informahealthcare.com by The University of Manchester on 08/23/12 J Asthma Downloaded from informahealthcare.com by The University of Manchester on 08/23/12 ClinicalClinical outcome outcome of of courses courses of therapy (%)

33 months months 6 6 months months 12 months

ABPAABPA VoriconazoleVoriconazole Improved Improved 13/20 13/20 (65) (65) 11/15 11/15 (73) (73) 9/13 (69) StableStable 2/20 2/20 (10) (10) 2/15 2/15 (13) (13) 2/13 (15) FailureFailure 1/20 1/20 (5) (5) 0/15 0/15 2/13 (15) DiscontinuedDiscontinued (AEs) (AEs) 4/20 4/20 (20) (20) 2/15 2/15 (13) (13) 0/13 PosaconazolePosaconazole Improved Improved 7/9 7/9 (78) (78) 7/9 7/9 (78) (78) 7/9 (78) StableStable 2/9 2/9 (22) (22) 2/9 2/9 (22) (22) 0/9 FailureFailure 0/9 0/9 0/9 0/9 2/9 (22) DiscontinuedDiscontinued (AEs) (AEs) 0/9 0/9 0/9 0/9 0/9 SAFSSAFS VoriconazoleVoriconazole Improved Improved 4/5 4/5 (80) (80) 4/5 4/5 (80) (80) 3/4 (75) StableStable 1/5 1/5 (20) (20) 1/5 1/5 (20) (20) 1/5 (20) FailureFailure 0/5 0/5 0/5 0/5 0/5 DiscontinuedDiscontinued (AEs) (AEs) 0/5 0/5 0/5 0/5 0/5

Notes:Notes: AEs, AEs, adverse adverse events; events; ABPA, ABPA, allergic allergic bronchopulmonary bronchopulmonary aspergillosis; aspergillosis; SAFS, SAFS, severe severe asthma asthma with with fungal fungal sensitization. sensitization. () indicates %.

Chishimba L J Asthma 2012;49:423 Second and third line antifungal therapy for ABPA and/or asthma

• 18/24 (75%) discontinued oral corticosteroids, 12 of them within 3 months of starting antifungal therapy. • 6/23 (26%) patients on voriconazole had AEs requiring discontinuation before 6 months compared to none on posaconazole (p=0.15). • 4 relapsed (57%), 1 at 3 months and 3 at 12 months after discontinuation.

Chishimba et al, J Asthma 2012;49:423 Itraconazole inhaled steroid interaction

• Itraconazole reduces the metabolism of inhaled steroids • Documented for beclomethasone, fluticasone • Ciclosenide probably not • No interaction with prednisolone, dexamethasone, hydrocortisone • Reduces metabolism of methylprednisolone • [Voriconazole reduces prednisolone metabolism, but probably no interaction with inhaled steroid]

Fungal Infection Impact

No studies assessing:

Disability Adjusted Life Years (DALY) Quality Adjusted Life Years (QALY) Quality-adjusted life expectancy (QALE) Population health-related quality of life (HRQOL) Diagnostic improvements in fungal diagnosis in last 20 years

• Aspergillus antigen testing • Susceptibility testing of Candida and Aspergillus • Chromagar • CT scanning of the chest • PCR for Pneumocystis, Aspergillus, Candida and Trichophyton • Molecular identification of fungi and discovery of numerous cryptic species • Direct identification from blood culture or agar plates • Rapid dip-stick test for cryptococcal meningitis Direct detection of resistance mutations in clinical specimens, without positive cultures

Laboratory result ABPA CPA Normals

Culture positive for A. fumigatus 0/19 7/42 (16.7%) 0/11 qPCR positive for Aspergillus spp 15/19 30/42 4/11 (78.9%) (71.4%) (36.4%) A. fumigatus CYP51A mutation detected directly from qPCR positive sample 6/8 (75%) 12/24 (50%) NT

Denning, Clin Infect Dis 2011;52:1123 Evaluation of processing methods for Aspergillus – sputa and bronchoscopy samples

Literature review Journal of Microbiological Methods 85 (2011) 75–81

Contents lists available at ScienceDirect Journal of2 Microbiologicalpapers Methods journal homepage: www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 85 (2011) 75–81 Homogenisation of cystic ﬁbrosis sputum by sonication — An essential step for Aspergillus PCR Contents lists available at ScienceDirect Caroline G. Baxter a,b,c,⁎, Andrew M. Jones b,c, Kevin Webb b,c, David W. Denning a,c

a The National Aspergillosis Centre, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK b Manchester Adult Cystic Fibrosis Unit, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK c The UniversityJournal of Manchester and the Manchester of Academic Microbiological Health Science Centre, Oxford Road, Manchester, M13 9PL, Methods UK

Medical Mycology May 2012, 50, 433–438 article info abstract

Article history:journal homepage:The www.elsevier.com/locate/jmicmeth importance of Aspergillus as a lung pathogen in cystic fibrosis (CF) is becoming increasingly recognised. Received 10 November 2010 However, fungal culture of CF sputum is unreliable and there is no consensus for identifying phenotypes Received in revised form 14 January 2011 beyond ABPA that may benefit from antifungal therapy. There are no published studies using real-time PCR to Accepted 21 January 2011 Routine processing detectproceduresAspergillus in CF sputum. for The majorisolating barrier to sensitive ! lamentous detection of Aspergillus using PCR is sputum Available online 28 January 2011 homogenisation. This study aimed to optimise sputum homogenisation utilising sonication to improve fungi from respiratoryAspergillus sputum samples may Keywords: DNA extraction. Sonication amplitude and duration that enabled sputum homogenisation but fi Aspergillus ensured preservation of DNA integrity were rst determined. 160 sputum samples were collected from CF Cysticunderestimatefibrosis fungalpatients. prevalence 49 of the sputum samples were split, one half was used for standard culture and the other half was Sputum homogenised with NALC–NaOH before undergoing DNA extraction. The subsequent 111 samples were Homogenisation of cysticSonication fibrosis sputumhomogenised with dithiothreitol by sonication plus sonication prior to culture and— DNA extraction.An Real-time essential PCR targeting step for CATHERINE H. PASHLEY , ABBIE FAIRS , a JOSEPH portion ofP. theMORLEY 18S rDNA , SHREEYA of Aspergillus TAILORwas performed , JOSHUA on AGBETILE all DNA extractions. , In the 49 samples with no sonication 8 (16%) were culture positive but only 4 of these were PCR positive. However, PCR was positive in Aspergillus MONA BAFADHEL , CHRISTOPHER E. BRIGHTLING11 culture negative & ANDREWsamples. PCR J. after WARDLAW sonication showed a significant improvement in sensitivity: 33 (30%) PCR Institute for Lung Health, Department of Infectionwere culture, Immunity andPCR and positive, In! ammation 48 (43%), University were culture of Leicester negative,, butLeicester PCR positive, UK (pb0.0001) and 30 (27%) were culture and PCR negative. The combination of dithiothreitol and sonication to homogenise sputum a,b,c, b,c increases PCR yield, with PCR beingb, substantiallyc more sensitive than culture. a,c Caroline G. Baxter ⁎, Andrew M. Jones Colonization, Kevin of the airways Webb by ! lamentous, fungi David can occur W. in asthma, Denning© 2011 chronic Elsevier obstructive B.V. All rights reserved. pulmonary disease (COPD) and cystic ! brosis. A recent study found IgE sensitization to a The National Aspergillosis Centre, University Hospital of South Manchester, Aspergillus Southmoor fumigatus to Road, be associated Manchester, with reduced M23 9LT,lung UKfunction. Signi! cantly higher b Manchester Adult Cystic Fibrosis Unit, University1. Introduction Hospital of Southrates Manchester, of A. fumigatus Southmoor were detected Road, forin Manchester, sputum diagnosis from and M23asthmatics treatment 9LT, are sensitized UK unclear. Allergicto this fungus hypersensitivity is compared to non-sensitized asthmatics.the The most rate common of positive presentation cultures in was the far immunocompetent higher than host. c The University of Manchester and the Manchester Academic Health Science Centre, Oxford Road, Manchester, M13 9PL, UK Cystic fibrosis (CF) is a commonequivalent genetic condition historical which samples primarily analysedAlthough by the there local is clinical growing laboratory evidence forfollowing the use proto- of antifungals in affects water and ion transport acrosscols recommended epithelial surfaces. by Thethe majorUK HealthABPA Protection (Nepomuceno Agency et al.,(HPA). 1999; This Skov study et al., compares 2002; Stevens et al., cause of morbidity and mortality is pulmonary disease. Pulmonary 2000), there have been no studies to evaluate if there is benefit from secretions are thick and respiratorythe cilia HPA have procedure impaired with movement. our sputumantifungal processing treatment method, for patients whereby colonised sputum with plugs or are sensitised to Recurrent bacterial infections causeseparated progressive from damage saliva to and the aliquots lungs. ofAspergillus approximately. Colonisation 150 mg is linkedare inoculated to risk of hospitalisationdirectly onto and lower article info Although bacterialabstract infections are responsiblepotato dextrose for the agar. majority A total of this of 55 sputumlung function samples (Amin from et 41 al., patients 2010) but with causality COPD is were in question and pulmonary damage there is an increasinganalyzed, recognition comparing of fungal the role recovery of prospective of ! ve dilutions data is required. of sputa Sensitisation on two media. has beenIsolation linked to lower fi fungal infections. The most commonof A. fumigatus fungus identiin cultureed inwas the signi! lungcantly function higher inusing CF (theKraemer research et al., approach 2006) and compared non-CF asthmatic Article history: secretions of CF patientsThe is importanceAspergillus fumigatus of (AspergillusBakare et al., 2003as). a lungpatients pathogen (Fairs et al., in 2010 cystic). In orderfibrosis to establish (CF) ifisAspergillus becoming increasingly recognised. Aspergilllus is a ubiquitous fungusto the that HPA causes standard a number method of different for mycologicalcolonisation investigations and sensitisation ( P Ͻhave 0.001). a pathogenic There was role inalso lung function Received 10 November 2010 clinical presentationsHowever, in CF. Thesea include fungalsigni! allergiccant culture difference bronchopulmonary of in CFthe recovery sputumdecline, rate is andof unreliable A. to fumigatus monitor antifungal ( andP Ͻ 0.05) there treatment, between is accurate no media. consensus methods to for identifying phenotypes Received in revised form 14 January 2011aspergillosis (ABPA), allergic sensitisation,This highlights aspergilloma the need and invasiveforfi a standardizeddetect Aspergillus approachin to CF fungal respiratory detection secretions which are is needed. more beyond ABPA that may bene t from antifungal therapy. There are nofi published studies using real-time PCR to Accepted 21 January 2011 aspergillosis (IA) (Stevens et al.,sensitive 2003). Aspergillus than the bronchitismethod recommended in CF CF by sputum the HPA. is often extremely viscous and dif cult to liquefy. The has been describeddetect more recentlyAspergillus (Shoseyov etin al., CF 2006 sputum.) but criteria Theoptimal major method barrier for culture to sensitive is unknown detection and varies as of differentAspergillus using PCR is sputum Available online 28 January 2011 organisms thrive in different growth conditions (Borman et al., 2010). homogenisation. Keywords This Aspergillus study fumigatus aimed , yeast to , optimise culture methods sputum , respiratory homogenisation samples, fungal utilising sonication to improve growth media Techniques such as selection of purulent material, bedside gram stain and culture, selective media, homogenisation and quantitative ⁎ Corresponding authorAspergillus at: 2nd Floor EducationDNA & extraction. Research Centre, University Sonication amplitude and duration that enabled sputum homogenisation but Keywords: cultures have been attempted to optimise culture yields. Organisms Hospital of South Manchester, Southmoor Road, Manchester M23 9LT, UK. Tel.: +44 fifi fi Aspergillus 161 291 5811; fax: +44ensured 161 291 5806. preservation of DNA integritygrowing were in bio rstlms aredetermined. also dif cult to 160 grow sputum (Randall, 2008 samples). were collected from CF fi fi E-mail address: [email protected]. 49 of the(C.G. sputumBaxter). samplesMolecular were split, methods one of identi halfcation was usedhave shown for standardthat culture only culture and the other half was Cystic brosis have a deleterious effect without ful! lling all criteria neces- 0167-7012/$ Introduction– see front matter © 2011 Elsevier B.V. All rights reserved. Sputum doi:10.1016/j.mimet.2011.01.024homogenised with NALC–NaOHsary before for a diagnosis undergoing of ABPA. DNAOne of extraction. the strongest risk The subsequent 111 samples were Sonication Airway diseaseshomogenised such as asthma, chronic with obstructive dithiothreitol pul- factors plus of sonication ! lamentous fungi prior in toCF cultureis decreased and lung DNA func- extraction. Real-time PCR targeting monary disease (COPD) and cystic ! brosis (CF) are com- tion, even after exclusion of patients diagnosed with ABPA. mon, importanta portion causes of of disease the 18S and rDNA ill health. of AspergillusHowever, it iswas unclear performed whether fungal on colonization all DNA contrib- extractions. In the 49 samples with no Colonization of thesonication airways by ! 8lamentous (16%) fungi were can culture occur positiveutes to lower but lung only function 4 ofor theseis a marker were of more PCR severe positive. However, PCR was positive in in all three disease groups, although the clinical relevance 11 culture negative samples. PCRlung after disease sonication and aggressive showed therapy. Incidence a signi fiofcant recovery improvement in sensitivity: 33 (30%) is unclear. Allergic bronchopulmonary aspergillosis (ABPA) of at least one fungal species from an individual is around is well recognizedwere as a severe culture complication and PCR of airway positive, col- 4840%, (43%) with prevalence were culture rates having negative, signi! cantly but increased PCR positive (pb0.0001) and 30 (27%) onization associatedwere with culture a " orid hypersensitivity and PCR negative.reaction in The the last combination decade [3]. In contrast of dithiothreitol to CF [3,4], there has and been sonication to homogenise sputum to Aspergillus fumigatus reported in up to 8% of asthmatics increases PCR yield, with PCR beingno comprehensive substantially studies more looking sensitiveat fungal colonization than culture. in [1] and 13% of CF patients [2]. Fungal colonization may asthma or COPD. Those that exist mostly focus on patients suspected of having ABPA and primarily report© only 2011 Elsevier B.V. All rights reserved. Received 22 March 2011; Received in ! nal form 7 July 2011 ; Accepted A. fumigatus . 16 August 2011 Sputum samples are frequently used to study airway Correspondence: Catherine Pashley, Aerobiology Unit, Department of in" ammation in respiratory diseases and to perform Infection, Immunity and In" ammation, University of Leicester, Leices- ter, LE1 9HN, UK. Tel: ϩ 44 116 2522936; Fax: ϩ 44 116 2525030; E-mail: microbiological investigations of respiratory infections. In [email protected] comparison to bronchoalveolar lavage (BAL), sputum 1. Introduction for diagnosis and treatment are unclear. Allergic hypersensitivity is © 2012 ISHAM DOI: 10.3109/13693786.2011.615762 the most common presentation in the immunocompetent host. Cystic fibrosis (CF) is a common genetic condition which primarily Although there is growing evidence for the use of antifungals in affects water and ion transport across epithelial surfaces. The major ABPA (Nepomuceno et al., 1999; Skov et al., 2002; Stevens et al., cause of morbidity and mortality is pulmonary disease. Pulmonary 2000), there have been no studies to evaluate if there is benefit from secretions are thick and respiratory cilia have impaired movement. antifungal treatment for patients colonised with or sensitised to Recurrent bacterial infections cause progressive damage to the lungs. Aspergillus. Colonisation is linked to risk of hospitalisation and lower Although bacterial infections are responsible for the majority of this lung function (Amin et al., 2010) but causality is in question and pulmonary damage there is an increasing recognition of the role of prospective data is required. Sensitisation has been linked to lower fungal infections. The most common fungus identified in the lung function in CF (Kraemer et al., 2006) and non-CF asthmatic secretions of CF patients is Aspergillus fumigatus (Bakare et al., 2003). patients (Fairs et al., 2010). In order to establish if Aspergillus Aspergilllus is a ubiquitous fungus that causes a number of different colonisation and sensitisation have a pathogenic role in lung function clinical presentations in CF. These include allergic bronchopulmonary decline, and to monitor antifungal treatment, accurate methods to aspergillosis (ABPA), allergic sensitisation, aspergilloma and invasive detect Aspergillus in CF respiratory secretions are needed. aspergillosis (IA) (Stevens et al., 2003). Aspergillus bronchitis in CF CF sputum is often extremely viscous and difficult to liquefy. The has been described more recently (Shoseyov et al., 2006) but criteria optimal method for culture is unknown and varies as different organisms thrive in different growth conditions (Borman et al., 2010). Techniques such as selection of purulent material, bedside gram stain and culture, selective media, homogenisation and quantitative ⁎ Corresponding author at: 2nd Floor Education & Research Centre, University cultures have been attempted to optimise culture yields. Organisms Hospital of South Manchester, Southmoor Road, Manchester M23 9LT, UK. Tel.: +44 fi fi 161 291 5811; fax: +44 161 291 5806. growing in bio lms are also dif cult to grow (Randall, 2008). E-mail address: [email protected] (C.G. Baxter). Molecular methods of identification have shown that culture only

0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.01.024 Voriconazole TDM 110 patients randomised; 14% poor metabolizers [2C19A], mean age 55 years, 77% hematologic disease

Primary outcome was a reduction in adverse events