2011/9/9 1 Nagasaki University Hospital established in September 20, 1861 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 2 Fungus diseases in literature 150 years ago Fungal diseases 1 st Case report Candida Aspergillus Mucor Actinomyces Coccidioides 1839, Langenbeck 1847, Sluyter 1855, Kurchenneuster 1857, Lebert 1892, Posadas, Wernicks 1894, Busse Cryptococcus Okudaira M, Jpn J Pathology： 74, 61 -91, 1985 Describe of microorganism 1839, Langenbeck 1729，Micheli 1880, Lindt, Paltauf 1891, Wolff & Isarael 1900, Ophuls & Moffitt 1895, Sanfelics Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 3 IUMS 2011, Sapporo, JAPAN Respiratory Mycoses Pulmonary Aspergillosis: Pathogenesis and Treatment Chronic pulmonary aspergillosis ~new treatment evidence and emergence of azoleresistant Aspergillus fumigatus in Japan~ Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Koichi IZUMIKAWA, M. D. , Ph. D. Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN 54 years old, male, SON 4 82 years old, male, father slowly progressive inflammatory pulmonary syndrome due to Aspergillus spp. Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN investigation of circumstances 5 over 50 years old wooden house humid and old Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Systemic Mycosis in Japan from Autopsy Data MCZ 1980 5 -FC 1979 FLCZ 1989 ITCZ 1993 F-FLCZ 2004 VRCZ 2005 MCFG 2002 L-AMB 2006 AMPH-B 1962 (%) 5 6 Total number of Mycosis 4 Candidiasis Frequency (%) ? Aspergillosis Cryptococcosis Mucor 3 2 1 0 1960 1970 1980 Year Kume et al. Med Mycol J 52: 117 -127, 2011 1990 2007 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 7 Proposed classification and pathogenesis of chronic pulmonary aspergillosis Preexisting pulmonary defect, with cavity Aspergillus exposure Colonization of pulmonary cavity Generalized immunocompromised state (e. g. diabetes, AIDS, alcoholism) Subtle generalized or pulmonary defense defect Immune dysregulation Nodular or consolidation, w/ or w/o cavitations: Multiple cavities w/ surrounding inflamation ±aspergilloma: subacute invasive pulmonary aspergillosis (subacute IPA) chronic cavitary pulmonary aspergillosis (CCPA) or or chronic necrotizing pulmonary aspergillosis (CNPA) complex aspergilloma normal/weak fibrosis response continuing cavity formations and local inflamation Denning et al: Clin Infect Dis 37 Suppl 3: S 265 -80, 2003 No local generalized defect Resolution of infection or asymptomatic, stable single aspergilloma: simple aspergilloma strong fibrosis response Extensive pluero/pulmonary fibrosis: chronic fibrosing pulmonary aspergillosis (CFPA) Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 8 Clinical features of CPA patients Japan (n=198) UK (n=126) Nam et al. Int J Infect Smith et al. ERJ, 2010 Dis, 2010 Reference Avg. age Korea (n=43) 69. 5 60. 0 59 (alive) male 145 (73. 2) 34 (79. 1) 75 (59. 5) female 53 (26. 8) 9 (20. 9) 51 (40. 5) 17. 6 17. 5 - 92 (46. 5) 40 (93. 0) 21 (16. 7) NTM 20 (15. 9) COPD 34 (17. 2) 6 (14. 0) 42 (33. 3) Diabetes 25 (12. 6) 5 (11. 6) Sex BMI Underlying diseases Old Tbc. (%) Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Reported TB cases New and relapse cases (per 100 000 population) 9 60 50 France Germany 40 Italy Japan 30 Netherlands Portugal Spain 20 Sweden United Kingdom of Great Britain and Northern Ireland United States of America 10 0 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 WHO, Communicable Diseases Report Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
![2011/9/9 10 CPA case [case]65 Y, Male [CC] hemosputum, cough [PH] n. p. [PI]](https://present5.com/presentation/6e795f172457f284378dba9fc6bce5b9/image-10.jpg "2011/9/9 10 CPA case [case]65 Y, Male [CC] hemosputum, cough [PH] n. p. [PI]")
2011/9/9 10 CPA case [case]65 Y, Male [CC] hemosputum, cough [PH] n. p. [PI] 1998：right upper lobectomy (Tbc) 2005~：cough, hemosputum 2006~: hemosputum increased chest CT：fungus ball like shadows in right lower lung. Platelia EIA: positive, Aspergillus Ab: positive β-D-gulucan 35. 0 pg/ml admission for further treatment [PE] Height 161 cm, Weight 44. 3 kg, BMI 17. 1, Body temp. 36. 8℃, pulse 68/min, regular rhythm Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 11 CPA case ITCZ oral solution treatment 2/16 2/22 3/19 BIPM 0. 6 g/day MCFG 150 mg/day ITCZ 400 mg/day 10/11 BIPM 0. 6 g/day VRCZ 200 mg/day ITCZ 200 mg/day BALF: A. fumigatus 37 ℃ 10/4 6 months BALF: sputum: A. terreus hemosputum WBC(/μl) 8700 6700 A. fumigatus A. niger A. versicolor A. terreus 5700 5300 6500 8000 7500 0. 19 0. 20 0. 93 1. 46 3. 24 0. 72 CRP(mg/dl) 1. 65 5. 70 β-D-glucan(pg/ml) 39. 5 117. 8 90. 9 68. 2 47. 4 25. 9 28. 9 Aspergillus antigen (EIA) 0. 65 0. 976 0. 985 1. 025 0. 441 0. 461 0. 591 0. 517 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Drug CPA ITCZ Evidence of CPA treatment 12 Response rate (%) Route Design Year Author Reference CNPA+Aspergilloma 60 -66 oral CS 1998 De Buele Mycoses ITCZ CNPA+Aspergilloma 71 -93 oral CS 1990 Dupont J Am Acad Dermatol ITCZ Aspergilloma 30 oral CS 1991 Campbell Thorax ITCZ CNPA 67 oral CS 1996 Caras Mayo Clin Proc ITCZ CNPA 67 oral CS 1997 Saraceno Chest ITCZ Aspergilloma 63 oral CS 1997 Tsubura Kekkaku ITCZ CPA 71 oral CS 2003 Denning Clin Infect Dis ITCZ CNPA 58 oral CS 2009 Nam Int J Infect Dis POSA CPA 46 -61 oral CS 2010 Felton Clin Infect Dis MCFG CNPA+Aspergilloma 55 -67 IV CS 2004 Kohno Scand J Infect Dis MCFG CPA 78 IV CS 2007 Izumikawa Med Mycol MCFG CNPA+Aspergilloma 58 IV CS 2009 Yasuda Nihon Kokyuki Gakkai Zasshi VRCZ CCPA 64 oral CS 2006 Jain J Infect VRCZ CNPA+CCPA 50 -80 oral CS 2006 Sambatakou Am J Med VRCZ CNPA+CCPA 70 oral CS 2007 Camuset Chest Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
CPA treatment IDSA GL 2011/9/9 13 Treatment Primary CNPA (Subacute IPA) VRCZ CCPA ITCZ or VRCZ Alternative Aspergilloma none or SURGERY L-AMB ITCZ MCFG posaconazole ABLC caspofungin Monthly treatment and orally administrative azoles are recommended Innateimmune defects demonstrated Longterm therapy IFN-γ The role of medical therapy in treatment of aspergilloma is uncertain NO RCT existed !! P. O. first and I. V. is optional Clin Infect Dis 2008; 46: 327 -360 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 14 World First RCT in CPA treatment Kohno, Izumikawa et al: J Infect, 2010 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
New Evidence of CPA treatment Overall efficacy MCFG v. s. VRCZ 2011/9/9 （％） 15 P＝ 0. 543* 100 60. 0％ （30/50） efficacy 53. 2％ （25/47） 50 0 MCFG Kohno, Izumikawa et al: J Infect, 2010 VRCZ Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 New Evidence of CPA treatment Frequency of side effect MCFG v. s. VRCZ 16 P＝ 0. 0004* （％） 61. 1％ （33/54） 70 60 frequency 50 40 30 26. 4％ （14/53） 20 10 0 Kohno, Izumikawa et al: J Infect, 2010 MCFG VRCZ Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
New Evidence of CPA treatment Frequency of side effect MCFG v. s. VRCZ Visual disturbance & hepatic dysfunction 2011/9/9 （％） 70 P＜ 0. 0001* （％） 70 50 29. 6％ （16/54） 40 30 20 10 0 30 26. 4％ （14/53） 40 30 Visual events Kohno, Izumikawa et al: J Infect, 2010 20 10 VRCZ 35. 2％ （19/54） 50 20 0. 0％ （0/53） MCFG 40 P=0. 025* 60 frequency 50 （％） 70 50. 0％ （27/54） 60 frequency 60 P=0. 012* 17 10 0 0 15. 1％ （8/53） MCFG VRCZ Adverse effects except visual related events MCFG VRCZ Hepatic events Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 18 Another route of antifungal administration nebulized L-AMB & MCFG IPA murine model Day-2, 0：Cyclophosphamide 200 mg/kg i. p. +Cortisone. Acetate 250 mg/kg s. c. Day 0： MF-13 conidia 1× 108/ml: 50μl intratracheal inoculation Day 1～ 5： L-AMB 1. 2 mg/ml： 8 ml nebulize once/day MCFG 1 mg/kg/day intraperitoneal Group 1: n. L-AMB + MCFG Group 2: n. L-AMB Group 3: MCFG Group 4: Control Day-2 immunosupression ICR, ♀, 8 weeks Day 0 Day 1～ 5 Inoculum(i. t. ) L-AMB inhalation and/ or MCFG Takazono, Izumikawa: AAC 53: 3508 -3510, 2009 i. p. Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Another route of antifungal administration 19 survival rate nebulized L-AMB & MCFG IPA murine model 1 n L-AMB+MCFG . 8 N L-AMB . 6 MCFG . 4 control . 2 0 0 2 Control 4 6 8 10 MCFG (i. p. ) 12 14 16 L-AMB Time (DAYS) L-AMB + MCFG (i. p. ) Day 3 pathology GMS stain× 400 Takazono, Izumikawa: AAC 53: 3508 -3510, 2009 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Another route of antifungal administration 20 nebulized L-AMB & MCFG IPA murine model L-AMB inhalation conc. AMPH-B concentration Lung (mg/kg) control serum (μg/ml) 0. 1 <0. 02 L-AMB 1. 2 mg/ml 35. 5± 4. 2 0. 02 L-AMB 2. 6 mg/ml 73. 2± 15. 8 0. 06 L-AMB 4. 0 mg/ml 94. 2± 20. 2 0. 06 Takazono, Izumikawa: AAC 53: 3508 -3510, 2009 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 number 250 21 ITCZ resistant A. fumigatus Nijmegen, Netherland 6 % 5 200 4 150 3 100 2 50 0 1 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 PLo. S Med. 2008 November; 5(11): e 219 0 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Azole resistant A. fumigatus Manchester, U. K. 100 Number of patient cases 22 20 90 % 80 70 Multi-azole 5 60 50 10 0 VRCZ 7 ITCZ Susceptible 5 7 0 ITCZ & POSA 0 0 30 20 17 5 3 40 14 09 20 08 20 07 20 06 20 05 20 04 20 03 20 02 20 01 20 00 20 99 19 98 19 19 97 0 Year Bueid et al. JAC 65: 2116 -2118, 2010 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
Mechanism of action azole antifungals 2011/9/9 ー Ｔｒｉａｚｏｌｅ 23 ＋ Ｔｒｉａｚｏｌｅ Ｅｒｇｏｓｔｅｒｏｌ Ｔｏｘｉｃ ｓｔｅｒｏｌ Ａｚｏｌｅｓ Ｅｒｇｏｓｔｅｒｏｌ Ｐ４５０ Ｅｒｇ３ １４ＤＭ Ｔｏｘｉｃ ｓｔｅｒｏｌ Ｅｒｇ３ Cowen LE. Eukaryotic Cell 2008 7: 747 -764 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
Mechanism of resistance azole antifungals 2011/9/9 Alternation, mutation and over expression of P 45014 DM Alternation of synthesis Mutation of Erg 3 24 Overexpression of Efflux pumps Alternate sterol Triazoles Traizoles Triazoles P 450 14 DM Erg 3 Ergosterol P 450 14 DM Erg 3 Cowen LE. Eukaryotic Cell 2008 7: 747 -764 P 450 14 DM Mutation Erg 3 Toxic sterol Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 25 Mechanism of azole resistant A. fumigatus Cyp 51 A mutation hot spot, Netherland & U. K. G 54 M 220 U. K. 12 6 9 Netherland Multi-azole resistance TR+L 98 H 0 94 3 (%) TR L 98 H TR M 220 F-helix Promoter G 448 G-helix Cyp 51 A Multi-azole resistance G 54 G 138 ITCZ resistance POSA resistance Membrane-anchoring region Verweij PE, et al. Lancet Infect Dis 9: 789 -795, 2009 Membrane-anchoring region Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Drug susceptibility of Aspergillus fumigatus 26 Nagasaki University Hospital Clinical Isolates (196 strains) Strains： Clinical isolated A. fumigatus between 1994 and 2010 Method of identification： microscopic morphology ability to grow at 48˚C molecular confirmation (sequence of ITS and D 1/D 2) Drug susceptibility test： CLSI M 38 A-2 Tested antifungals： FLCZ, ITCZ, VRCZ, POSA, MCFG, AMPH-B Molecular epidemiology STR/microsatellite analysis Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 27 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) frequency of non-WT isolates Itraconazole ≧ 2 μg/ml (14/196) 7. 1 % Voriconazole ≧ 2 μg/ml (8/196) 4. 1% Posaconazole ≧ 1 μg/ml (5/196) 2. 6 % Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 28 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) frequency of non-WT isolates and cross resistance POSA VRCZ ITCZ Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 29 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Comparison of positive rate of Cyp 51 A mutation to Europe Nagasaki, JAPAN U. K. Netherland Positive rate of Cyp 51 A mutation 87. 5% 57% 88% Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
Drug susceptibility of Aspergillus fumigatus 2011/9/9 30 Nagasaki University Hospital Clinical Isolates (196 strains) Comparison Cyp 51 A mutation with Netherland G 54 M 220 none Nagasaki 75. 0 0 0 11. 1 Netherland Multi-azole resistance TR+ L 98 H 1. 5 80. 1 3. 8 12. 3 (%) TR L 98 H TR M 220 F-helix Promoter G 448 G-helix Cyp 51 A Multi-azole resistance G 54 G 138 ITCZ resistance POSA resistance Membrane-anchoring region AAC. 2010; 54: 2425 -30 Membrane-anchoring region Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Azole-resistant Aspergillus fumigatus 31 Nagasaki University Hospital Clinical Isolates (196 strains) Correlation of ITCZ exposure amount and drug susceptibility Cumulative ITCZ exposure （ mg） 180000 150000 120000 90000 r = 0. 5916 p <0. 0001 60000 30000 0. 125 0. 5 1 4 2 ITCZ MIC（μg/ml） 8 >8 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Azole-resistant A. fumigatus isolated case 59 years old, Male 32 CC: fever, cough, sputum PH: surgical for pneumothorax Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Azole-resistant A. fumigatus isolated case 59 years old, Male 1 2 3 2001 4 5 βD glucan 1999 2000 6 8 9 10 11 ITCZ 200 mg 53. 5 15. 7 Aspergillus Ag 2. 9 Sputum culture MCFG 150 mg Serum ITCZ conc. 1. 148 μg/ml 2. 1 0. 6 5. 1 7 12 24. 0 6. 9 5. 3 2. 5 1 st isolated ITCZ low-sensitive A. fumigatus ITCZ 400 mg 2002 33 6. 6 53. 4 9. 1 ITCZ 200 mg 132. 2 87. 0 4. 9 2. 9 1. 5 VRCZ 300 mg 2003 0. 8 0. 6 ITCZ 200 mg 7. 2 0. 1 MCFG 300 mg Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
Azole resistant A. fumigatus isolated case 2011/9/9 MCFG 150 mg 8/16 2000/08/15 59 years old, Male ITCZ 400 mg 12/14 2000/12/14 6/4 2002/02/27 2002/05/22 34 ITCZ 200 mg 1/23 5/16 2001/10/22 2001/05/17 VRCZ 300 mg 2002/08/27 2002/01/23 8/25 2003/04/14 2011/08/29 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Azole-resistant A. fumigatus isolated case 35 59 years old, Male, summary of isolated strains and cases Cumulative Cumulati ITCZ ve ITCZ exposed term (days) dose (mg) strain Isolated date STR cluster type MF-368 2000/8/16 3 189 MF-367 2000/8/16 3 MF-370 2000/9/7 MIC (μg/ml) Cyp 51 A mutation ITCZ POSA VRCZ 37, 800 0. 5 0. 06 0. 5 I 266 N 189 37, 800 0. 5 0. 06 0. 25 I 266 N 3 189 37, 800 0. 25 0. 06 0. 25 I 266 N MF-439 2001/10/19 3 507 144, 850 2 0. 5 0. 25 G 54 E I 266 N MF-452 2002/4/3 3 589 161, 650 >8 0. 5 MF-454 2002/4/17 3 589 161, 650 2 0. 5 0. 125 G 54 E I 266 N MF-460 2002/5/8 3 589 161, 650 4 2 0. 25 G 54 E I 266 N MF-468 2002/5/22 3 589 161, 650 4 0. 5 0. 25 G 54 E I 266 N MF-469 2002/5/29 3 589 161, 650 8 1 0. 25 G 54 W I 266 N I 266 N Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 SUMMARY CPA treatment and drug resistance 36 Treatment →New evidence become available by first RCT →Development of newer treatment is required Azole-resistance →It is few in Japan →Cyp 51 A mutation is common in drug resistant strains →Resistant may be acquired by exposure of azole Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2018/3/16 Advertisement 37 The 86 th Japanese Society of Infectious Diseases Annual meeting 2012, Nagasaki, JAPAN Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
2011/9/9 Nagasaki University Shigeru Kohno Takayoshi Tashiro Katsunori Yanagihara Yoshihiro Yamamoto Hiroshi Kakeya Taiga Miyazaki Yoshifumi Imamura Shigeki Nakamura Takahiro Takazono Masato Tashiro Katsuji Hirano 38 Acknowledgement National Institutes of Infectious Diseases Yoshitsugu Miyazaki Hideaki Ohno Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences
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