1. 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

2. 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

3. 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

4. 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)

5. 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)

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

7. Radiographic Abnormalities Vary
Radiographic Abnormalities Vary

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

9. 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

10. 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

11. 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

12. 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

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

14. 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:
2Machetti and Viscoli. Antimicrob Agents Chemother (9)
3Wheat et al. ICAAC 06

15. 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

16. 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)

17. 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):

18. 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”?

19. Pre-emptive Therapy ? -7 7 14 21 28 35 42 49 56 -14 0.1 1 107 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).

20. 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:

21. 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

22. 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 , Washington, DC.

23. 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): (2002) 2Storek et al. Blood; 97(11) (2001)

24. 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

25. 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

26. 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)