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Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Nagasaki University Hospital established.

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Presentation on theme: "Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Nagasaki University Hospital established."— Presentation transcript:

1 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Nagasaki University Hospital established in September 20, /9/91

2 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Fungus diseases in literature 150 years ago Fungal diseases1 st Case reportDescribe of microorganism Candida1839, Langenbeck Aspergillus1847, Sluyter 1729 Micheli Mucor1855, Kurchenneuster1880, Lindt, Paltauf Actinomyces1857, Lebert1891, Wolff & Isarael Coccidioides1892, Posadas, Wernicks 1900, Ophuls & Moffitt Cryptococcus1894, Busse1895, Sanfelics Okudaira M, Jpn J Pathology 74, 61-91, /9/92

3 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/93 IUMS 2011,Sapporo, JAPAN Respiratory Mycoses Pulmonary Aspergillosis: Pathogenesis and Treatment Chronic pulmonary aspergillosis ~new treatment evidence and emergence of azole- resistant Aspergillus fumigatus in Japan~ Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Koichi IZUMIKAWA, M.D., Ph.D.

4 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 82 years old, male, father54 years old, male, SON Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN slowly progressive inflammatory pulmonary syndrome due to Aspergillus spp. 2011/9/94

5 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN investigation of circumstances over 50 years old wooden house humid and old 2011/9/95

6 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 6 Kume et al. Med Mycol J 52: , (%) Frequency (%) Year Total number of Mycosis Candidiasis Aspergillosis Cryptococcosis Mucor AMPH-B FC 1979 MCZ 1980 FLCZ 1989 ITCZ L-AMB 2006 VRCZ 2005 F-FLCZ 2004 MCFG 2002 Systemic Mycosis in Japan from Autopsy Data 2011/9/9 ?

7 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/97 Proposed classification and pathogenesis of chronic pulmonary aspergillosis Denning et al: Clin Infect Dis 37 Suppl 3: S265-80, 2003 Colonization of pulmonary cavity Nodular or consolidation, w/ or w/o cavitations: subacute invasive pulmonary aspergillosis (subacute IPA) or chronic necrotizing pulmonary aspergillosis (CNPA) Multiple cavities w/ surrounding inflamation ±aspergilloma: chronic cavitary pulmonary aspergillosis (CCPA) or complex aspergilloma Resolution of infection or asymptomatic, stable single aspergilloma: simple aspergilloma normal/weak fibrosis response continuing cavity formations and local inflamation strong fibrosis response Extensive pluero/pulmonary fibrosis: chronic fibrosing pulmonary aspergillosis (CFPA) Generalized immuno- compromised state (e.g. diabetes, AIDS, alcoholism) Subtle generalized or pulmonary defense defect Immune dysregulation No local generalized defect Aspergillus exposure Preexisting pulmonary defect, with cavity

8 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 8 Clinical features of CPA patients Japan (n=198)Korea (n=43)UK (n=126) Reference Nam et al. Int J Infect Dis, 2010 Smith et al. ERJ, 2010 Avg. age (alive) Sex male 145 (73.2)34 (79.1)75 (59.5) female 53 (26.8)9 (20.9)51 (40.5) BMI Underlying diseases Old Tbc. 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) (%) 2011/9/9

9 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/99 Reported TB cases New and relapse cases (per population) France Germany Italy Japan Netherlands Portugal Spain Sweden United Kingdom of Great Britain and Northern Ireland United States of America WHO, Communicable Diseases Report

10 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/910 [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 35.0pg/ml admission for further treatment [PE] Height 161cm, Weight 44.3kg, BMI 17.1, Body temp. 36.8, pulse 68/min, regular rhythm CPA case

11 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/911 WBC (/μl) CRP(mg/dl) β-D-glucan(pg/ml) / /11 MCFG 150mg/day sputum:A. terreus 3/19 VRCZ 200mg/day ITCZ 400mg/day BIPM 0.6g/day 10/4 BALF:A. fumigatus A. niger A. versicolor A. terreus 2/22 ITCZ 200mg/day BIPM 0.6g/day BALF: A. fumigatus Aspergillus antigen (EIA) months hemosputum CPA case ITCZ oral solution treatment

12 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 12 Evidence of CPA treatment DrugCPAResponse rate (%)RouteDesignYearAuthorReference ITCZCNPA+Aspergilloma60-66oralCS1998De BueleMycoses ITCZCNPA+Aspergilloma71-93oralCS1990DupontJ Am Acad Dermatol ITCZAspergilloma30oralCS1991CampbellThorax ITCZCNPA67oralCS1996CarasMayo Clin Proc ITCZCNPA67oralCS1997SaracenoChest ITCZAspergilloma63oralCS1997TsuburaKekkaku ITCZCPA71oralCS2003DenningClin Infect Dis ITCZCNPA58oralCS2009NamInt J Infect Dis POSACPA46-61oralCS2010FeltonClin Infect Dis MCFGCNPA+Aspergilloma55-67IVCS2004KohnoScand J Infect Dis MCFGCPA78IVCS2007IzumikawaMed Mycol MCFGCNPA+Aspergilloma58IVCS2009YasudaNihon Kokyuki Gakkai Zasshi VRCZCCPA64oralCS2006JainJ Infect VRCZCNPA+CCPA50-80oralCS2006SambatakouAm J Med VRCZCNPA+CCPA70oralCS2007CamusetChest 2011/9/9

13 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/913 Clin Infect Dis 2008; 46: CPA treatment IDSA GL Treatment PrimaryAlternative CNPA (Subacute IPA) VRCZ L-AMB ITCZ MCFG posaconazole ABLC caspofungin Monthly treatment and orally administrative azoles are recommended CCPA ITCZ or VRCZ Innateimmune defects demonstrated Longterm therapy IFN-γ Aspergillomanone or SURGERY The role of medical therapy in treatment of aspergilloma is uncertain NO RCT existed !! P.O. first and I.V. is optional

14 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 14 World First RCT in CPA treatment Kohno, Izumikawa et al: J Infect, /9/9

15 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 15 New Evidence of CPA treatment Overall efficacy MCFG v.s. VRCZ / / MCFGVRCZ efficacy P * Kohno, Izumikawa et al: J Infect, /9/9

16 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences / /54 MCFGVRCZ frequency P * Kohno, Izumikawa et al: J Infect, /9/9 New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ

17 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences / /54 P=0.012 * / /54 P=0.025 * Visual events Hepatic events Adverse effects except visual related events MCFGVRCZ P * 0.0 0/ /54 MCFGVRCZ frequency MCFGVRCZ frequency Kohno, Izumikawa et al: J Infect, /9/9 New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ Visual disturbance & hepatic dysfunction

18 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Another route of antifungal administration nebulized L-AMB & MCFG IPA murine model Day-2,0 Cyclophosphamide200mg/kg i.p.+CortisoneAcetate250mg/kg s.c. Day0 MF-13 conidia 1×10 8 /ml:50μl intratracheal inoculation Day1 5 L-AMB 1.2mg/ml 8ml nebulize once/day MCFG 1mg/kg/day intraperitoneal Day-2Day0 immunosupression Inoculum(i.t.) Day1 5 L-AMB inhalation ICR,,8weeks Group1: nL-AMB + MCFG Group2: nL-AMB Group3: MCFG Group4: Control and/ or MCFG i.p. Takazono, Izumikawa: AAC 53: , /9/918

19 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences survival rate Time (DAYS) control MCFG N L-AMB n L-AMB+MCFG Another route of antifungal administration nebulized L-AMB & MCFG IPA murine model GMS stain×400 Day3 pathology ControlMCFG (i.p.)L-AMBL-AMB + MCFG (i.p.) Takazono, Izumikawa: AAC 53: , /9/919

20 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Another route of antifungal administration nebulized L-AMB & MCFG IPA murine model Takazono, Izumikawa: AAC 53: , 2009 L-AMB inhalation conc. Lung (mg/kg)serum (μg/ml) control0.1<0.02 L-AMB 1.2mg/ml35.5± L-AMB 2.6mg/ml73.2± L-AMB 4.0mg/ml94.2± AMPH-B concentration 2011/9/920

21 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 21 ITCZ resistant A. fumigatus Nijmegen, Netherland PLoS Med November; 5(11): e219 number % 2011/9/9

22 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 22 Azole resistant A. fumigatus Manchester, U.K. Bueid et al. JAC 65: , Multi-azole ITCZ & POSA VRCZ ITCZ Susceptible Year Number of patient cases % 2011/9/9

23 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 23 Mechanism of action azole antifungals Cowen LE. Eukaryotic Cell : /9/9

24 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 24 Toxic sterol Ergosterol P450 14DM Erg 3 Triazoles Mutation Alternate sterol Ergosterol P450 14DM Erg 3 Traizoles Triazoles Ergosterol P450 14DM Erg 3 Alternation, mutation and over expression of P450 14DM Alternation of synthesis Mutation of Erg 3 Overexpression of Efflux pumps Cowen LE. Eukaryotic Cell : /9/9 Mechanism of resistance azole antifungals

25 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 25 Mechanism of azole resistant A. fumigatus Cyp51A mutation hot spot, Netherland & U.K. Verweij PE, et al. Lancet Infect Dis 9: , 2009 Cyp51A M220G448 F-helixG-helix ITCZ resistance POSA resistance Membrane-anchoring region G138 Multi-azole resistance G54 Promoter Membrane-anchoring region Multi-azole resistance TR L98H G54TR+L98HM220 U.K.1269 Netherland0943 (%) 2011/9/9

26 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 26 Drug susceptibility of Aspergillus fumigatus 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 D1/D2) Drug susceptibility test CLSI M38 A-2 Tested antifungals FLCZ, ITCZ, VRCZ, POSA, MCFG, AMPH-B Molecular epidemiology STR/microsatellite analysis 2011/9/9

27 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences % 2.6 % 4.1% Itraconazole 2 μg/ml (14/196) Posaconazole 1 μg/ml (5/196) Voriconazole 2 μg/ml (8/196) 2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) frequency of non-WT isolates

28 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences ITCZ VRCZ POSA 2011/9/928 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) frequency of non-WT isolates and cross resistance

29 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 29 Positive rate of Cyp51A mutation Nagasaki, JAPAN87.5% U.K.57% Netherland88% 2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Comparison of positive rate of Cyp51A mutation to Europe

30 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 30 AAC. 2010; 54: Cyp51A M220G448 F-helixG-helix ITCZ resistance POSA resistance Membrane-anchoring region G138 Multi-azole resistance G54 Promoter Membrane-anchoring region Multi-azole resistance TR L98H G54 TR+ L98H M220none Nagasaki Netherland (%) 2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Comparison Cyp51A mutation with Netherland

31 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences ITCZ MIC μg/ml ITCZ MIC μg/ml Cumulative ITCZ exposure mg Cumulative ITCZ exposure mg > /9/931 Azole-resistant Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Correlation of ITCZ exposure amount and drug susceptibility r = p <0.0001

32 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Azole-resistant A. fumigatus isolated case 59 years old, Male CC: fever, cough, sputum PH: surgical for pneumothorax β-D glucan Aspergillus Ag Aspergillus Ab Sputum culture (bacteria) Sputum culture (Tbc) Sputum culture pg/ml C.O.I. A. fumigatus 2011/9/932

33 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences ITCZ 200mg ITCZ 400mgITCZ 200mg VRCZ 300mg ITCZ 200mg MCFG 150mg MCFG 300mg βD glucan Aspergillus Ag Sputum culture 1 st isolated ITCZ low-sensitive A. fumigatus Serum ITCZ conc μg/ml Azole-resistant A. fumigatus isolated case 59 years old, Male 2011/9/933

34 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2000/08/152000/12/142001/05/ /10/ /01/23 MCFG 150mg 8/1612/14 ITCZ 400mg ITCZ 200mg 5/16 1/ /02/ /05/ /08/ /04/14 6/4 8/25 VRCZ 300mg Azole resistant A. fumigatus isolated case 59 years old, Male 2011/9/ /08/29

35 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences strain Isolated date STR cluster type Cumulative ITCZ exposed term (days) Cumulati ve ITCZ exposed dose (mg) MIC (μg/ml) Cyp51A mutation ITCZPOSAVRCZ MF /8/ , I266N MF /8/ , I266N MF /9/ , I266N MF /10/ , G54EI266N MF /4/ ,650 >80.5 I266N MF /4/ , G54EI266N MF /5/ , G54EI266N MF /5/ , G54EI266N MF /5/ , G54WI266N Azole-resistant A. fumigatus isolated case 59 years old, Male, summary of isolated strains and cases 2011/9/935

36 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/936 SUMMARY CPA treatment and drug resistance Treatment New evidence become available by first RCT Development of newer treatment is required Azole-resistance It is few in Japan Cyp51A mutation is common in drug resistant strains Resistant may be acquired by exposure of azole

37 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2014/3/3137 Advertisement The 86 th Japanese Society of Infectious Diseases Annual meeting 2012, Nagasaki, JAPAN

38 Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences 2011/9/938 Acknowledgement National Institutes of Infectious Diseases Yoshitsugu Miyazaki Hideaki Ohno Nagasaki University Shigeru Kohno Takayoshi Tashiro Katsunori Yanagihara Yoshihiro Yamamoto Hiroshi Kakeya Taiga Miyazaki Yoshifumi Imamura Shigeki Nakamura Takahiro Takazono Masato Tashiro Katsuji Hirano


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