Directed therapy for fungal infections - latest advances Rosemary Barnes Focus on aspergillosis

Total UK antifungal expenditure c £112 million Rising by 9% pa Problem Antifungal expenditure is completely out of proportion with the scale of the problem Incidence of IFD in ICU (candida) <0.6% Aspergillus infection in haematological malignancy (0.5-12%) Aspergillus in SOT <5% Harrison D et al Fungal Infection Risk Evaluation (FIRE) Study. Health Technol Assess 2013; 17(3). Pagano L et al. Haematologica 2006; 91: 1068-1075 Pagano L et al. Clin Infect Dis 2007; 45: 1161-1170

Reasons Infection associated with significant morbidity and mortality Signs and symptoms of systemic infection are nonspecific Conventional diagnostic techniques are suboptimal Delays in treatment associated with poorer outcome

Choices choices choices Febrile despite antibiotics Empirical therapy Diagnosis Change antibiotics

Decision tree Fever yes no

Decision tree yes no Fever yes No diagnosis no yes no

Decision tree Fever No diagnosis Anxiety yes no yes yes no yes no no

Decision tree Fever No diagnosis Anxiety Out of hours yes no yes yes

Decision tree Fever Empirical therapy No diagnosis Anxiety yes no yes yes no no yes yes no yes no yes Fever yes no no Empirical therapy No diagnosis Anxiety Out of hours no yes yes yes no no yes no no yes yes no no yes no No therapy

Aim of a directed strategy include all patients likely to have invasive fungal infection and treat them with the safest and most effective drug exclude all patients unlikely to have invasive fungal disease and adopt a WAIT-and-SEE policy

Maertens et al. (2012) Haematologica 97(3): 325-327.

Consensus criteria Designed for use in clinical trials Aimed to provide definitions for proven, probable and possible fungal infection that could facilitate clinical research Designed for use in clinical trials Highly selective population Not representative of real life clinical practice Focus on specific radiological signs Focus on defining DISEASE needs to shift towards INFECTION Needs a diagnostic approach biomarkers De Pauw et al CID 2008, 46

The biomarkers Antigen tests Molecular Galactomannan (aspergillus) Beta D glucan (pan-fungal-ish) Lateral flow device Molecular Aspergillus specific Panfungal Commercial (…….)

Galactomannan - in serum useful test in surveillance: high NPV Performance in haematological malignancy better than in SOT neutropenic > corticosteroid treated group Adults >children Influenced by pre-test probability (ie sensitivity increases with prevalence) EORTC/MSG criteria heavily dependent on test being performed Recommended by ECIL

Galactomannan – meta-analyses 30 studies > 7000 patients Prevalence 7.7% sensitivity78% (61% to 89%) specificity 81% (72% to 88%). cut-off 0.5: 100 patients: 2 patients with IA, will be missed, 17 patients will be treated unnecessarily cut-off 1.5 OD: 3 IA patients will be missed 5 patients will be treated unnecessarily results were very heterogeneous. Insufficient data to look at clinical utility http://www.thecochranelibrary.com

BAL 0.5 approved by FDA On the basis of clinical validity PPV of GM BAL is 100% at an OD index cutoff of ≥3 only 76% at ≥ 0.5 (but NPV is high) Pre test probability PUO GM pos CT BAL Maertens et al CID 2009

Beta D Glucan 4 different commercial tests Heterogenous data : retrospective vs. prospective; Different cut offs “panfungal” – except cryptococcus and mucoracous moulds Sensitivity, specificity variable but NPV high High false-positives: up to 30% - bacteraemia, antibiotics, pre-/analytical contaminations complex analytical procedures Analytical validity established Utility data limited Included in EORTC/MSG criteria

Beta D Glucan-meta-analysis 16 studies in 2979 patients Included case-controlled studies Included critical care, HM and solid organ cancer patients Cut off 10-1000 pg/ml Sensitivity 76.8% (67.1%–84.3%) specificity 85.3% (79.6%–89.7%) “area under ROC curve 0.89” “good diagnostic accuracy” Karageorgopoulos et al Clin Infect Dis 2011;52(6):750

PCR The UK Fungal PCR consensus group 2004 technically validated candida PCR Made recommendations for aspergillus PCR 2006 European Aspergillus PCR Initiative set up 86 participants in 69 centres in 24 countries defined a standard for PCR for Aspergillus Whole blood Serum plasma optimal methodology to evaluate the performance and impact QCMD available White et al J Molec Diagn 2006; 8: 376 White et al J Clin Micro 2010: 48 1231 www.eapcri.eu/

PCR Single negative PCR to exclude disease 2 consecutive PCRs to diagnose IA Sensitivity 88% Specificity 75% DOR22 Mengoli et al Lancet Infectious Diseases. 2009; 9: 89-96

“Directed/pre-emptive” therapy References Country Clinical HRCT GM Micro-biological PCR Non-comparative studies Maertens 2005 Belgium X Girmenia 2010 Italy (X) Aguilar-Guisado 2010 Spain Barnes 2009 UK Dignan 2009 Randomised, comparative studies Cordonnier 2009 France Hebart 2009 Germany Observational studies Pagano 2012

Maertens et al. Clin Infect Dis 2005; 41: 1242 Galactomannan EIA Open study 136 episodes of neutropenia Patients receiving flucon prophylaxis daily EIA GM + early CT scanning in neutropenic febrile episodes Antifungal given if 2 consecutive EIA GM results +ve (index ≥ 0.5) and confirmed by BAL or CT Maertens et al. Clin Infect Dis 2005; 41: 1242

Maertens et al 35% of episodes met criteria for empirical antifungal but only7.7% treated on basis of pre-emptive therapy Duration of fever not affected 22 cases of IFD only one missed 3 breakthrough infections 2 candidaemias 1 mucorales No excess mortality or fungal related death No impact on overall antifungal usage despite deceased empirical use

Cordonnieret al CID 2009 48:1043 293 patients randomised empirical or pre-emptive therapy empirical arm received antifungals if they had persistent/recurrent fever after 4 days pre-emptive patients given antifungal only if they showed clinical and radiological signs of pneumonia/sinusitis positive GM index ≥ 1.5 Aspergillus colonization Septic shock CNS signs/periorbital inflammation Diarrhoea/mucositis ≥ grade 3 fever > 14 days

Cordonnieret al Survival was not significantly “Non inferiority” demonstrated pre-emptive patients had more IFI 9.1% vs 2.7% pre-emptive patients received significantly less antifungals no significant cost savings were achieved Used ampho B deoxycholate first -line

Empirical vs. pre-emptive antifungal therapy IFI in Pre-emptive IFI in Empirical Cordonnier et al, Clin Infect Dis, 2009; 48: 1042-1051

Pagano et al Haematologica 2011; 96:1363 Observational: Empiric versus “pre-emptive” Data collection 397 HM patients 190 empiric ; 207”pre-emptive” More IFD in pre-emptive arm Increased mortality and antifungal use in “pre-emptive arm” Fever driven, no screening, diagnostic work up not standardized some GM usage, no PCR Pre-emptive group largely diagnosed on basis of HRCT

PCR Nested PCR to guide antifungal therapy 42 patients with cancer, neutropenia AmB required in only 2 patients randomised study of a PCR directed versus an empirical antifungal more than 400 SCT patients Safe Improved survival at 30 days (not 100) No reduction in antifungal drug use. Lin et al. Clin Infect Dis. 2001;33:1621-1627 Hebart et al. Blood 2004;104: 59A.

In Cardiff 549 high-risk haematology patients entering neutropenic pathway 2005-2010 audited and followed up for a minumum of 12 months Twice weekly antigen and PCR testing (or GvHD) Itraconazole prophylaxis or AmBisome 7mg/kg/weekly Empiric antifungals not used unless Clinical/mycological evidence of disease Itraconazole levels were subtherapeutic or unmeasured First 125 patients analysed for safety and proof of concept Data collected on compliance, incidence of IFD and efficacy of prophylaxis Of the 549 patients, 238 were stem cell transplant patients while 310 were high/intermediate risk haematology patients Suffering mainly from leukaemias and lymphomas. Not really empiric therapy as there is some evidence of disease Barnes et al Journal of Clinical Pathology 2009

Incidence of IFD (2005-2011) Invasive aspergillosis 9.6% 6 histologically proven (2 postmortem) 4 pulmonary (2 with dissemination) 2 invasive sinusitis 47 probable (23 possible IA) Invasive Candidal infection 2% 12 proven 4 C. albicans, 3C. glabrata, 2C. tropicalis, 1C. parapsilosis, 1C. guilliermondii, 1 mixedC. albicans + C. glabrata 1 probable 2 non-aspergillus moulds 1 Mucoraceous mould, 1 Scedosporium prolificans Incidence of proven/probable IFD 12.3% Incidence within our study group

IA disease status of subjects By EORTC/MSG diagnostic criteria Proven – 6 Probable – 47 Possible – 23 NEF – 473 248 had clinical signs including haemoptysis, pleuritic chest pain, abnormal CXRs, persistent fever 225 truly had NEF Having both EIA and PCR positive shows the tests are not likely to be false negatives 248 of NEF showed some signs suggestive of IA EIA positive n=36 PCR positive n=136 EIA and PCR positive n=75 Aspergillus isolated n=5

Diagnostic accuracy Explore analytical validity. Clinical validity, clinical utility Sensitivity specificity PPV, NPV, LR, DORs Use ROC analysis to explore different thresholds for defining “cases” EORTC/MSG EORTC – GM EIA EORTC + PCR Dual biomarker positivity Multiple positives versus single

Provena, probableb vs No IFDc EIA Single 96.2 (87.3-99.0) Population Assay Positivity Threshold Sensitivity (%) Specificity (%) PPV (%) NPV (%) LR+ LR- DOR Provena, probableb vs No IFDc EIA Single 96.2 (87.3-99.0) 76.7 (72.7-80.3) 31.7 (25.0-39.2) 99.5 (98.0-99.9) 4.14 0.05 84.15 Multiple 62.3 (48.8-74.1) 91.8 (88.9-93.9) 45.8 (34.8-57.3) 95.6 (93.3-97.1) 7.55 0.41 18.36 Provena, probableb, possibled vs No IFDc 67.1 (55.9-76.6) 93.6 (93.7-97.6) 2.89 0.43 6.73 43.4 (32.9-54.6) 91.0 (88.1-93.2) 5.27 0.62 8.54 Provena, probable incorporating PCRe vs No IFDc 72.9 (61.5-81.9) 95.0 (92.4-96.8) 3.13 0.35 8.86 47.1 (35.9-58.7) 92.1 (89.4-94.3) 5.72 0.58 9.93 PCR 92.5 (82.1-97.0) 55.6 (51.1-60.0) 18.9 (14.6-24.1) 98.5 (96.2-99.4) 2.08 0.14 15.34 73.6 (60.4-83.6) 79.9 (76.1-83.3) 29.1 (22.1-37.3) 96.4 (94.1-97.9) 3.66 0.33 11.08 86.8 (77.5-92.7) 23.9 (19.3-29.3) 96.3 (93.4-98.0) 1.96 0.24 8.27 65.8 (54.6-75.5) 34.5 (27.2-42.5) 93.6 (90.7-95.6) 3.28 7.65 94.3 (86.2-97.8) 2.12 0.10 20.66 71.4 (60.0-80.7) 95.0 (92.4-96.7) 3.56 0.36 9.95 PCR or ELISA 98.1 (93.4-100) 47.8 (42.0-53.5) 17.4 (11.9-22.9) 99.6 (98.5-100) 1.88 0.04 47.6 79.2 (65.3-93.2) 76.7 (71.9-81.6) 27.6 (0.19-36.7) 97.1(94.9-99.2) 3.41 0.27 12.6 90.8 (82.5-99.1) 21.8 (16.0-27.6) 97.0 (94.2-99.8) 1.74 0.19 9.0 69.7 (56.6-82.9) 32.5 (23.341.7) 94.0 (91.0-97.1) 3.00 0.39 7.6 98.6 (92.3-100) 21.8 (0.17-0.28) 99.6 (96.1-100) 1.89 0.03 63.13 90.0 (80.8-95.1) 36.4 (27.6-46.2) 98.1 (95.5-99.3) 3.87 0.13 29.7 Both PCR and ELISA 90.6 (79.8-95.9) 84.4 (80.8-87.4) 39.3 (31.1-48.2) 98.8 (97.1-99.5) 5.79 0.11 51.76 Multiple (1 assay) 71.7 (56.2-87.2) 87.7 (84.0-91.5) 39.6 (27.1-52.1) 96.5 (94.3-98.7) 5.85 0.32 18.13 Multiple (both assays) 56.6 (43.3-69.1) 94.9 (92.6-96.6) 55.6 (42.4-68.0) 95.1 (92.8-96.7) 11.16 0.46 24.40 63.2 (51.9-73.1) 93.4 (90.7-95.4) 4.04 0.44 9.24 50.0 (35.7-91.5) 91.6 (88.4-94.90 4.08 0.57 7.16 39.5 (29.3-50.7) 90.7 (87.8-93.0) 7.78 0.64 12.20 68.6 (57.0-78.2) 94.8 (92.2-96.5) 4.38 0.37 11.76 54.3 (39.4-69.2) 92.8 (89.8-95.9) 4.43 0.52 8.5 42.9 (31.9-54.5) 91.8 (89.1-93.9) 8.45 0.60 14.03

Statistical parameters Sensitivity Specificity PPV NPV LR+ LR- DOR Proven, probable vs No IFD PCR or GM EIA Single 98.1 47.8 17.4 99.6 1.88 0.04 47.6 multiple 79.2 76.7 27.6 97.1 3.41 0.27 12.6 PCR and GM EIA single 90.6 84.4 39.3 98.8 5.79 0.11 51.76 56.6 94.9 55.6 95.1 11.16 0.46 24.4 By testing biomarkers twice weekly it is possible to use the data obtained to get the best diagnostic statistics (see those highlighted) Comparing proven and probable results against those with no evidence of fungal disease using both biomarkers either independently or together allows ruling out disease (NPV) as well as giving strong indications to those with disease (PPV) In essence, Multiple positives show a good accuracy for diagnosing Invasive fungal disease (PPV) whilst single positives could indicate that more diagnostic procedures are necessary (NPV) NPV high throughout all permutations of the data showing it is possible to confidently rule out disease using these tests. PPV low as low prevalence of disease and EORTC criteria downgraded from infection to disease in 2002 The DOR of over 50 shows the test to have good diagnostic utility. By EORTC/MSG criteria

Proven/probable disease versus no IFD Diagnostic odds ratio Ascertainment bias This graph shows all permutations of biomarker results, incorporating them into the diagnostic criteria to see those which gave us the best accuracy. Can see that point here from the table previously. However, if we incorporate PCR into the diagnostic criteria we were able to increase the utility of these tests further by using a singly positive EIA or PCR give an even better diagnostic utility. The large cone using a singly positive EIA is an ascertainment bias as EIA is used in the original EORTC criteria.

Positive likelihood ratio 15.8 11.2 10.5

Negative likelihood ratio 0.03 0.05

ROC plot PCR + EIA curve (AUC): 0.910 (95% CI: 0.872-0.948) Proven/prob/poss Proven/prob curve (AUC): 0.910 (95% CI: 0.872-0.948)

Performance of PCR Utility in proven/probable n=53 First marker positive PCR in 23 EIA in 15 PCR and EIA simultaneously positive in 7 radiological features in 8 In 85% biomarkers preceded specific radiological signs (range 1-118d) So looking at the performance of PCR within the proven and probable group shows it was often the 1st marker of disease. Out of 53

Diagnostic accuracy Use antifungals more cost effectively Screening by PCR AND GM EIA can enable a diagnosis of IA to be excluded Positive PCR +GM EIA or multiple positive PCRs or EIAs can be used to accurately diagnosis IA specificity 84.4%; sensitivity >90% DOR>50 Biomarkers are earliest markers in 85% of cases Use antifungals more cost effectively Can be excluded because of the high negative predictive value. If doing multiple biomarker testing you can select the best diagnostic utility by choosing the best combinations to get the best statistical evidence.

Antifungal expenditure What all this means is that antifungals can be used more selectively and so more cost effectively. The graph shows antifungal expenditure fell in first 6 months and remained on a downward trend with minor fluctuations since, which contrasts with other sites of similar size where expenditure has increased Similar units typically spending £1-2 mill pa

Use of biomarkers Regular screening throughout period or risk Screening during fever only Diagnostic testing during refractory fever only Confirmation when specific radiological signs are present None - empiric therapy

Strategy Used Influenced by Risk of IFD Prophylaxis used Prevalence affects utility of diagnostic tests ECIL recommend screening if IFD 5-10% Prophylaxis used Mould active reduces utility of diagnostic tests Availability of Diagnostic tests Protective environments/HEPA filtered air

Incidence of IFD after posaconazole therapy Pagano et al Haematological 2012; 97:963

Effect of antifungal therapy Group Days post infection 1 2 3 4 5 Test qPCR GM PCR Infected controls 2/3 0/3 3/3 1/3 Amphotericin B Caspofungin Posaconazole Uninfected controls McCulloch et al J Clin Path 2012; 65:83 Marr K A et al. Clin Infect Dis. 2005;40:1762-1769

Example High risk patient Prevalence8- ≥10% No Mould active prophylaxis – Screening regime Mould active prophylaxis used – Diagnostic regime Twice weekly screening of blood samples: Galactomannan, And Aspergillus PCR HRCT and BAL when infection suspected Diagnostic testing during refractory fever with Beta D glucan (serum) and Aspergillus PCR (BAL and blood or serum), galactomannan (BAL and serum) Targeted antifungal therapy for clinically diagnosed infection only with biomarker confirmation Single Positive biomarker Continue screening process >1 biomarker positive* triggers diagnostic workup to include relevant radiology and BAL if indicated No consistent clinical signs or symptoms indicates need for possible pre-emptive therapy Any consistent clinical signs or symptoms indicates need for antifungal therapy *For example: PCR and GM, or Multiple GM

at risk exposure infection disease Pre-emptive Prophylaxis Targetted

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