1. Fungal infections in patients with hematological malignancies: advances in diagnosis and prevention. Yoshinobu Kanda Division of Hematology, Saitama Medical Center, Jichi Medical University

2. Diagnosis of invasive fungal infection If we don’t do thorough examinations to make a diagnosis of fungal infection, a proven diagnosis can be obtained only postmortem.

3. Eligible patients: 1) Patients who underwent chemotherapy and neutropenia (<500) for at least 10 days was expected. 2) Patients who developed grade II-IV acute GVHD or extensive chronic GVHD after allogeneic stem cell transplantation. 3) Patients who were receiving steroid for at least 3 weeks.

4. Methods: Blood tests: Real-time PCR for aspergillus DNA (PCR; Kami et al. Clin Infect Dis 2001; 33:1504–12) ELISA for aspergillus galactomannan antigen (GM; Platelia) Beta-1,3-D-glucan (BDG; Wako) Frequency: Once a week. Diagnosis ： Proven/probable EORTC (Serum test not included)

5. Receiver operating characteristic (ROC) curve GM*1 GM*2 PCR*1 PCR*2 BDG*1 BDG*2 (Kawazu et al. J Clin Microbiol 2004;42:2733-2741) n=149 Proven IA: 9 Probable IA: 2 Area under the ROC curve was greatest for GM, using two consecutive positive results. The best cuttoff for the GM was 0.6. Sensitivity 100%, Specificity 93% Positive predictive value 55% Negative predictive value 100%

6. Aspergillus galactomannan detection in the diagnosis of invasive aspergillosis in cancer patients (Herbrecht et al. J Clin Oncol 2002;20:1898-1906)

7. Aspergillus galactomannan enzyme immunoassay for diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid (Musher et al. J Clin Microbiol 2004;42:5517–5522) Galactomannan test is a very useful test for the diagnosis of aspergillosis if the cutoff around 0.6 is used.

8. Retrospective study to evaluate the incidence of false-positive GM antigen test after allogeneic stem cell transplantation. Positive galactomannan: two consecutive GM results with O.D.I above 0.6 Incidence: 16% at day 100, 32% at 1-year, 38% at 2-year (Asano-Mori et al. J Antimicrob Chemother 2008;61:411-416) n=157

9. Classification of the positive GM episode by reviewing the clinical information. False-positive results were frequent, especially within 100 days after transplantation and in patients who developed gut GVHD. (Asano-Mori et al. J Antimicrob Chemother 2008;61:411-416) The damage of the gastrointestinal tract mucosa and translocation of dietary GM may be the cause of false-positive results

10. Prognosis of invasive fungal infection

11. Aspergillosis: Fatality rate Underlying disease or conditions (Lin et al. Clin Infect Dis 2001;32:358-366) Solid organ transplantation

12. (Herbrecht et al. N Engl J Med 2002;347:408-415) Patients: proven or probable aspergillosis

13. Approaches to prevent overt invasive fungal infection 1. Prophylaxis 2. Empiric therapy 3. Preemptive/presumptive therapy Approaches to prevent overt invasive fungal infection 1. Prophylaxis 2. Empiric therapy 3. Preemptive/presumptive therapy

14. ↓Start of chemotherapy Prophylaxis ↓For persistent febrile neutropenia (FN) Empiric therapy Preemptive / ↓Serological / Imaging evidence ↓Persistent FN + Serological / Imaging evidence Presumptive therapy ↓Proven or probable diagnosis Targeted treatment Timing to start antifungal agents Candida prophylaxis with FLCZ -> Empiric therapy with anti-molds

15. IDSA guideline 2002 for neutropenic patients with cancer (Clinical Infectious Diseases 2002; 34:730–751) Despite the use of broad antibiotics..based on very old and small randomized trials…

16. Empiric antifungal therapy in febrile granulocytopenic patients Empiric antifungal therapy in febrile granulocytopenic patients 132 patients remaining febrile and granulocytopenic despite broad-spectrum antibiotic therapy for four days ↓ Randomization to receive or not to receive empiric amphotericin-B (0.6 mg/kg/day iv) (EORTC Am J Med 1989:86;668-672)

17. Amph-BNil (n=68)(n=64) Response rate (fever) 69%53% Documented fungal infection 1 6 Mortality (day 30) 1114 Cause of death fungal infection 0 4 bacterial infection 1 2 pulmonary infection 1 0 Empiric antifungal therapy in febrile granulocytopenic patients Empiric antifungal therapy in febrile granulocytopenic patients (EORTC Am J Med 1989:86;668-672)

18. More than half of the patients who received empiric antifungal treatment did not have invasive fungal infection...

19. Strategies to reduce an unnecessary use of antifungal agents…

20. Universal prophylaxis with fluconazole Empiric treatment strategy Fluconazole was empirically switched to anti-mold agents for patients with persistent or recurrent FN for 7 days or longer. Presumptive treatment strategy For patients with persistent or recurrent FN for 7 days or longer, anti-mold agents were started as an early presumptive treatment for aspergillosis only after patients developed positive serum test and/or infiltrates or nodules on X-ray or CT-scan. Chest X-ray, galactomannan test, beta-D-glucan test: Weekly Chest CT scan: bi-weekly or at the discretion of attending physician Presumptive treatment strategy early after transplantation

21. 114 patients who underwent allogeneic transplantation between September 2002 and December 2005. Patients with a previous history of aspergillosis were excluded. All patients received antifungal prophylaxis with fluconazole. Primary endpoint: Early invasive aspergillosis (Until one week after engraftment) Diagnostic criteria: Probable or proven by EORTC/MSG 2002 Presumptive treatment strategy early after transplantation (Oshima et al. J Antimicrob Chemother 2007;60:350-355)

22. Treatment and outcome of patients who were managed in presumptive strategy Presumptive anti-Aspergillus treatment 60 patients (+) (-) 2 patients 1 patients 4 patients 56 patients Development of early IA (Oshima et al. J Antimicrob Chemother 2007;60:350-355) Persistent or recurrent FN for 7 days Beta-D-glucan: 1 X-ray or CT scan: 3 These findings suggested the feasibility of presumptive strategy for invasive aspergillosis in transplantation recipients before engraftment, provided that they were treated in HEPA- filtered laminar air flow rooms. Anti-mold empiric therapy early after transplantation might be unnecessary for patients without a history of aspergillosis. Considering the low incidence of early invasive aspergillosis in this series, most patients in the past might have been unnecessarily exposed to empiric anti-mold agents.

23. Prophylaxis necessary? As we administered FLCZ as prophylaxis in these clinical studies, antifungal agents are frequently used in daily practice.

24. Fluconazole 400 mg/day as prophylaxis in bone marrow transplant recipients SYSTEMIC FUNGUS SUPERFICIAL FUNGUS COLONIZATION SYSTEMIC AMPHO-B DEATH RATE 7%0%77%38%13%18%7%86%55%21% FLUCONAZOLE n = 152 PLACEBO n = 149 (SLAVIN et al. J. Infect. Dis. 1995;171:1545) However, in patients who underwent chemotherapy for hematological malignancies, the effect of fluconazole prophylaxis has been inconsistent.

25. Antifungal prophylaxis with fluconazole: a meta-analysis TrialFluconazole armControl arm (first investigator)RegimenSubjects(BMT)RegimenSubjects(BMT) Goodman JLoral 400 mg or iv 200 mg179(179)placebo177(177) Winston DJoral 400 mg or iv 200 mg123(0)placebo132(0) Chandrasekar PHoral 400 mg or iv 400 mg23(11)placebo23(11) Schaffner Aoral 400 mg or iv 200 mg75(8)placebo76(7) Slavin MAoral 400 mg152(152)placebo148(148) Egger Toral 400 mg or iv 400 mg43(14)oral nystatin 24000000 U46(19) Kern Woral 400 mg36(0)none32(0) Rotstein Coral 400mg141(62)placebo133(58) Akiyama Horal 200 mg71(0)oral AMPH-B 2400 mg59(0) Ellis MEoral 200 mg42(10)clotrimazole troche 20 mg48(13) Meunier Moral 200 mg30(9)oral AMPH-B 1200 mg29(9) Menichetti Foral 150 mg420(420)oral AMPH-B 2000 mg400(0) Ninane Joral 3 mg/kg236(58)oral nystatin 200000 U/kg or oral AMPH-B 100 mg/kg249(64) Groll AHoral 3 mg/kg25(0)oral nystatin 50000 U/kg25(0) Philpott-Howard JNoral 50 mg256(60)oral nystatin 4000000 U or oral AMPH-B 2000 mg255(50) Rozenberg-Arska Moral 50 mg25(0)oral AMPH-B 800 mg25(0) (Kanda et al. Cancer. 2000;89:1611-1625)

26. Endpoint: Proven systemic fungal infection (Kanda et al. Cancer. 2000;89:1611-1625)

27. Endpoint: Proven systemic fungal infection Arranged by the incidence of systemic fungal infection in the control patients Patients who will undergo less intensive chemotherapy do not need fluconazole prophylaxis.

28. (Winston et al. Ann Intern Med 2003;138:705-713) Randomized controlled trial comparing fluconazole vs. itraconazole in allogeneic SCT

29. (Winston et al. Ann Intern Med 2003;138:705-713) Early phase : prophylaxis against Candida Middle phase: prophylaxis against Aspergillus Incidence of invasive fungal infection

30. Voriconazole vs. Itraconazole as anti-mold prophylaxis in patients with GVHD after allogeneic stem cell transplantation. Multicenter randomized controlled trial on going.

31. Which approach should we choose to prevent invasive fungal infection? 1. Prophylaxis 2. Empiric therapy 3. Preemptive/presumptive therapy … depends on the intensity of immunosuppression and environment where the patient is treated.

32. For example … 1. Patients with non-Hodgkin ’ s lymphoma who will undergo CHOP or ESHAP chemotherapy. 2. Patients who will undergo remission induction chemotherapy for acute leukemia. 3. Patients who will undergo allogeneic transplatation in a clean unit and who do not have a history of aspergillosis. 4. Patients who are receiving steroid for chronic GVHD in an outpatient clinic. For example … 1. Patients with non-Hodgkin ’ s lymphoma who will undergo CHOP or ESHAP chemotherapy. 2. Patients who will undergo remission induction chemotherapy for acute leukemia. 3. Patients who will undergo allogeneic transplatation in a clean unit and who do not have a history of aspergillosis. 4. Patients who are receiving steroid for chronic GVHD in an outpatient clinic.

33. Ideal environment for clinical trials of fungal infection. Surrounded by nothing but rice paddy fields: Very very humid!! Everlasting construction!! Worst environment for stem cell transplantation recipients. Currently, we are administering anti-mold agents as prophylaxis for most patients with hematologic malignancies.