Early Diagnosis and Pre-emptive Therapy of Fungal Pneumonia in High Risk Patients: Current Thinking Kieren Marr MD Fred Hutchinson Cancer Research Center University of Washington Seattle, WA

58 year old F with Hodgkin’s lymphoma received autologous BMT after conditioning with “BuMELT”. 2 days after BMT noted to have what appeared to be a cellulitis in R foot, CNS bacteremia. Treated with imipenem and vancomycin. 3 days afterwards, developed hypotension requiring 3 pressors, acute renal failure, severe metabolic acidosis, DIC. Small myocardial infarct likely by enzymes; TTE showed good EF. Exam revealed early gangrenous lesion on R 2nd toe. CXR- ‘bibasilar atelectasis’. Received imipenem, ciprofloxacin, vancomycin, caspofungin. Hemodialysis as tolerated. 3 days later, better. Off pressors. Exam: foot worse (all toes). Pelvic ‘ischemia’ (no signs of soft tissue infection). Blood cultures all without growth. A case

CT abd and pelvis: fluid. Lungs: BL consolidations What do you want to do: BAL. Continue current antifungal therapy BAL. Change to caspofungin to voriconazole BAL. Change to lipid based amphotericin B formulation Change to voriconazole empirically. Obtain serum GM EIA None of the above

Early Diagnosis and Pre-emptive Therapy Antifungal therapy administered late is rarely successful Dependent on immune system of the host and extent of disease What is late?? With culture documentation of disease High disease burden when radiographic abnormalities become apparent Sensitivity of culture is very poor Organisms are difficult to cultivate in the lab Establishing culture-defined diagnoses are difficult Review of 391 cases of IFI in patients with hematological malignancies, 20011 Diagnosis made pre-mortem 79% BAL culture sensitivity 66% 1Pagano et al. Haematologica 86 (2001)

How to Diagnose Early CXR screening lacks sensitivity Patients at risk require screening based on more sensitive parameters Early CT scans with signs / symptoms of disease Serial CT scans in patients at risk Day 0: halo Day 4: size, halo Day 7: air crescent Caillot et al, J Clin Oncol 2001: 19(1)

Lee et al. Brit J Radiol 78 (2005) Halo Signs Lee et al. Brit J Radiol 78 (2005)

Radiographic Abnormalities Vary     Radiographic Abnormalities Vary

Post-engraftment IA Kojima et al. BBMT 11(7): 506-11 (2005)

1Horger et al. Eur J Radiol 55 (2005) Sequential CT characterized in 45 patients with IPA1 No radiographic finding predicted outcome PET scans may be useful for early diagnosis 2,3 Radiography 1Horger et al. Eur J Radiol 55 (2005) 2Hot et al. ICAAC 2006 M1307 3Li et al. ICAAC 2006 M1684

BAL for diagnosis Nonspecific findings warrant evaluation for microbial etiology Different therapies; frequent co-pathogens Problem: BAL culture is not sensitive Sensitivity 50-65% of cases of documented IA Ways to make facilitate diagnosis? Culture under different conditions Adjunctive tests Serum based assays Galactomannan EIA, qPCR, glucan Adjunctive assays on BAL fluid Galactomannan EIA, qPCR

Diagnostic Tests for Aspergillosis Natural history of infection not well understood When do people become infected? How do you analyze sensitivity and specificity with multiple test results in one patient? Per-patient analysis Per-test analysis Imperfect gold standard tests False or true positive? Diagnostic Tests for Aspergillosis Marr and Leisenring. Clin Infect Dis 2005:41: S381

GM Evolution of Testing Methods dsELISA: Bio Rad Platelia EIA Measures GM using rat EBA-2 monoclonal antibody as acceptor and detector 0.5-1 ng/mL galactomannan Results: OD index GM Evolution of Testing Methods Mennenk-Kersten et al Lancet Infect Dis 2004 4 349

Aspergillosis Galactomannan EIA Frequent false-positives in children Marr and Leisenring Clin Infect Dis 2005; 41:S381

Issues Antifungal administration decreases sensitivity of the assay1 Variability in the literature Challenges current preventative paradigms False positive tests occur b lactam antibiotics containing GM (or cross-reactive antigen) GI tract translocation of GM (or cross-reactive antigen) Other infections: Histoplasmosis3 1Marr et al. Clin Infect Dis 2005; 40: 1762-9 2Machetti and Viscoli. Antimicrob Agents Chemother 2005 49(9) 3Wheat et al. ICAAC 06

Diagnostic tests relying on identification of (1-3)--D-Glucan Activates Limulus amebocyte lysate Factor G initiates cascade. Output measured by Turbidity after gel clot: WB003 (Wako Pure Chem. Indust.)1 Chromogenic substrate: Fungitec G test (Seikagaku) and Fungitell, (Assoc. Cape Cod)2 Endotoxin (1-3)-b-D-glucan Factor C Activated Fact. C Activated Fact G Factor G Factor B Activated Fact. B Proclotting Enzyme Clotting Enzyme Coagulogen Coagulin (gel) 1 Chromogenic method 2

Ostrosky-Zeichner et al. Clin Infect Dis 2005; 41:654 Performance not calculated from large numbers of patients with fungal pneumonia Smaller studies: sensitivity in setting of IA – 80% Recent observations 555 assays, 320 patients 74 positive tests 49 patients proven / probable IFI Sensitivity 71% Positive in several IFIs PCP b D glucan Ostrosky-Zeichner et al. Clin Infect Dis 2005; 41:654 Koo et al. ICAAC 2006 (M-1600) Marty et al. ICAAC 2006 (M-1606)

Utility of Galactomannan Detection in BAL Samples # pt 160 Sens (%) Spec PPV NPV Serum 47 93 73 82 BAL 85 100 88 Becker et al. Br J Haematol 2003; 121: 448 Musher et al. J Clin Microbiol 2004: 42(12): 5517-22

Early Diagnostics Current standard of relying on culture based detection of filamentous fungi is not adequate Need to incorporate adjunctive diagnostic tests in patients who have signs of disease (radiographic abnormalities) Can these tests be used “pre- emptively”?

Pre-emptive Therapy ? -7 7 14 21 28 35 42 49 56 -14 0.1 1 10 7 14 21 28 35 42 49 56 -14 0.1 1 10 Granulocytes Day Prophylaxis Empirical Pre-emptive approach Risk based approach Biomarker approach 63 Among other potential preventative strategies being explored is the use of circulating antigen to apply mould-active drugs (similar to CMV).

Screening for Early Diagnosis PCR assays and immunoassays (GM EIA) have been studied Particularly strong negative predictive values Can diagnostic assays be used to spare empiric therapy in patients who are receiving prophylaxis? Nested PCR to guide antifungal therapy1 42 patients with cancer, neutropenia AmB required in only 2 patients 1Lin et al. Clin Infect Dis. 2001;33:1621-1627.

Maertens et al. Clin Infect Dis 2005; 41: 1242 Galactomannan EIA Followed 136 episodes to neutropenia to see if GM EIA can be used to avoid empirical therapy Patients receiving fluconazole prophylactically 3 breakthrough infections 2 candidemias 1 Zygomycetes Maertens et al. Clin Infect Dis 2005; 41: 1242

Risk-based approach: Posaconazole in SCT Recipients with GVHD Randomized, double-blind Posa: 200 mg po tid (301 Pts) Flu: 400 mg po qd (299 Pts) Drug: GVHD-- to 112 days (16 wks) Outcomes measured after 16 weeks Decreased probability of IFI; IA Many patients who developed IA had a positive GM EIA at randomization Can this be used to guide therapy? Ullmann AJ, et al. Presented at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. Dec. 16-19, 2005. Washington, DC.

Targeted therapy Consider observation that everyone exposed to this organism, yet only 10% develop disease Are there “biologic risks” that can be measured and more predictive than clinical variables? Epidemiologic studies: role of cellular immunity in conferring risks for late IA1 CD4+ T cells with Th1-type cytokine: protective Immune-reconstitution studies confirmed importance of cellular immunity2 Developed functional assays to measure Aspergillus-specific PBMC responses Upcoming study to measure Aspergillus-specific immune reconstitution in allogeneic HSCT patients 1Marr et al. Blood; 100(13):4358-66 (2002) 2Storek et al. Blood; 97(11) 3380-89 (2001)

Our patient Serum GM EIA- negative BAL performed No growth on culture Galactomannan index 0.8 / 1.4 qPCR (light cycler assay) detected fungal DNA, but not Aspergillus What would you do? Continue caspofungin Change caspofungin to voriconazole Change caspofungin to Ambisome Add voriconazole Add Ambisome Our patient

Rhizopus microsporus var. rhizopodiformis Progressive pulmonary infection; therapy withdrawn Autopsy Large fungal infarcts in both upper and lower lung lobes bilaterally Erythema and necrosis of vagina, urethra, lower ¾ of bladder mucosa and uterine cervix: invasive mould Gangrenous foot with vascular involvement of mould Splenic infarcts Culture of lungs, pelvic swab Outcome Rhizopus microsporus var. rhizopodiformis

Conclusions Early therapy is an important goal Microbe-specific given toxicities, differential activities of drugs, and changing epidemiology Current culture-based standards are not adequate Multiple adjunctive tests being developed Need to learn how to apply them Clinical study is tricky given inadequate ‘gold standard’ (culture)