Nagasaki University Hospital established in September 20, 1861

Nagasaki University Hospital established in September 20, 1861
2011/9/9 1 Nagasaki University Hospital established in September 20, 1861 This is where I work and our medical school is oldest in Japan and it was established 150 years ago.

Fungus diseases in literature
2011/9/9 2 Fungus diseases in literature 150 years ago Fungal diseases 1st Case report Describe of microorganism Candida 1839, Langenbeck Aspergillus 1847, Sluyter 1729，Micheli Mucor 1855, Kurchenneuster 1880, Lindt, Paltauf Actinomyces 1857, Lebert 1891, Wolff & Isarael Coccidioides 1892, Posadas, Wernicks 1900, Ophuls & Moffitt Cryptococcus 1894, Busse 1895, Sanfelics When you look through the fungus diseases in the literature, about 150 years ago, first case report article of aspergillosis was published by Slutyer. Okudaira M, Jpn J Pathology： 74, 61-91, 1985

Chronic pulmonary aspergillosis
2011/9/9 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~ Good morning, ladies and gentleman. It is my honor to have a talk in this seminar and would like to show my appreciation to organizers of IUMS and Prof. Niki, chair person, in this session. My topic of this symposium today is Chronic pulmonary aspergillosis. And I would like to introduce two major topics including our world first RCT study and azole resistance in our hospital. Department of Molecular Microbiology and Immunology Nagasaki University Graduate School of Biomedical Sciences Koichi IZUMIKAWA, M.D., Ph.D.

Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN
2011/9/9 4 Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN 54 years old, male, SON 82 years old, male, father slowly progressive inflammatory pulmonary syndrome due to Aspergillus spp.

Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN
2011/9/9 5 Chronic forms of Pulmonary Aspergillosis family case, Sasebo, JAPAN investigation of circumstances over 50 years old wooden house humid and old

Systemic Mycosis in Japan from Autopsy Data
2011/9/9 6 FLCZ 1989 F-FLCZ 2004 MCZ 1980 ITCZ 1993 VRCZ 2005 5-FC 1979 MCFG 2002 AMPH-B 1962 L-AMB 2006 (%) 1 2 3 4 5 Total number of Mycosis Candidiasis Aspergillosis Cryptococcosis Mucor ? Frequency (%) This is the juts published data indicating the relative frequency of systemic mycosis in Japan. This data acquired from whole record of autopsy cases in Japan. As you can see here, recently total number of mycosis is little bit decreasing after I guess that it would be influenced by the newer antifungal drugs become available in Japan. The point is that aspergillosis is overwhelming candidiasis. 1960 1970 1980 1990 2000 2007 Year Kume et al. Med Mycol J 52: , 2011 6

2011/9/9 Proposed classification and pathogenesis of chronic pulmonary aspergillosis Preexisting pulmonary defect, with cavity Aspergillus exposure Colonization of pulmonary cavity Generalized immuno-compromised state (e.g. diabetes, AIDS, alcoholism) Subtle generalized or pulmonary defense defect Immune dysregulation No local generalized defect 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 Dr. Denning is proposing this classification of chronic pulmonary aspergillosis. These classifications are based on the mainly radiological findings, etiology and symptoms. normal/weak fibrosis response strong fibrosis response continuing cavity formations and local inflamation Extensive pluero/pulmonary fibrosis: chronic fibrosing pulmonary aspergillosis (CFPA) Denning et al: Clin Infect Dis 37 Suppl 3: S265-80, 2003

Clinical features of CPA patients
2011/9/9 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 69.5 60.0 59 (alive) Sex 　male 145 (73.2) 34 (79.1) 75 (59.5) 　female 53 (26.8) 9 (20.9) 51 (40.5) BMI 17.6 17.5 - 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) (%) 8

Reported TB cases New and relapse cases (per 100 000 population)
2011/9/9 Reported TB cases New and relapse cases (per population) 60 50 France Germany 40 Italy Japan 30 Netherlands Portugal 20 Spain The other reason is that we still have lots of tuberculosis patinets and patients with tuberculosis sequelae. As I showed before cavitary lesion by tbis a one of the important risk factors of CPA in Japan. This slide shows that number of reported tbc cases is still in high compared to other countries like U.S. or U.K. Sweden United Kingdom of Great 10 Britain and Northern Ireland United States of America 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 WHO, Communicable Diseases Report

CPA case [case]65 Y, Male [CC] hemosputum, cough [PH] n.p. [PI]
2011/9/9 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 35.0pg/ml admission for further treatment [PE] Height 161cm, Weight 44.3kg, BMI 17.1, Body temp. 36.8℃, pulse 68/min, regular rhythm First, I want to introduce one of our CPA case. The patient is 65 years old with tuberculosis treatment history including right upper lobectomy in past history. He complained hemosputum, and cough. The chest xray and laboratory findings showed fungus ball like shadows in right lower lung. And serum tests showed positive of antigen and antibody and β-D-gulucan also elevated.

CPA case ITCZ oral solution treatment
2011/9/9 CPA case ITCZ oral solution treatment 2/16 2/22 3/19　 6 months 10/4 10/11 BIPM 0.6g/day BIPM 0.6g/day MCFG 150mg/day VRCZ 200mg/day ITCZ 400mg/day ITCZ 200mg/day BALF: A. fumigatus A. niger A. versicolor A. terreus BALF: A. fumigatus sputum:A. terreus 37℃ hemosputum WBC(/μl) 8700 6700 8000 5700 5300 6500 7500 This is the clinical course of the patinet. He admitted in our hospital and received BIPM for 6days and no improvement was seen. We performed the BF and nothing but A. fumigatus was isolated, no bacteria was isolated. We then started MCFG i.v. for one month followed by ITCZ oral solution for six months in outpatinet clinic. However, it relapsed and these aspergillus were isolated. We then used VRCZ and he recovered. As you can see here, there is no daut that CPA requires longer period of treatment and even longer treatment is not enough. So we should think how to deal with this disease. CRP(mg/dl) 1.65 1.46 5.70 0.20 3.24 0.19 0.93 0.72 β-D-glucan(pg/ml) 117.8 47.4 39.5 90.9 25.9 28.9 68.2 Aspergillus antigen (EIA) 0.65 0.976 0.985 0.591 0.517 1.025 0.441 0.461

Evidence of CPA treatment
2011/9/9 Evidence of CPA treatment 12 Drug CPA Response rate (%) Route Design Year Author Reference ITCZ CNPA+Aspergilloma 60-66 oral CS 1998 De Buele Mycoses 71-93 1990 Dupont J Am Acad Dermatol Aspergilloma 30 1991 Campbell Thorax CNPA 67 1996 Caras Mayo Clin Proc 1997 Saraceno Chest 63 Tsubura Kekkaku 71 2003 Denning Clin Infect Dis 58 2009 Nam Int J Infect Dis POSA 46-61 2010 Felton MCFG 55-67 IV 2004 Kohno Scand J Infect Dis 78 2007 Izumikawa Med Mycol Yasuda Nihon Kokyuki Gakkai Zasshi VRCZ CCPA 64 2006 Jain J Infect CNPA+CCPA 50-80 Sambatakou Am J Med 70 Camuset This Islide indicates the summary of CPA treatment evidence. As you can see here, reports are well seen in iTCZ followed by few POSA, VRCZ and MCFG. 12

P.O. first and I.V. is optional
2011/9/9 CPA treatment IDSA GL Treatment Primary Alternative CNPA (Subacute IPA) VRCZ L-AMB ITCZ MCFG posaconazole ABLC caspofungin Monthly treatment and orally administrative azoles are recommended CCPA or Innateimmune defects demonstrated Longterm therapy IFN-γ Aspergilloma none SURGERY The role of medical therapy in treatment of aspergilloma is uncertain This table shows the recommendation of treatment for CPA, we know that there is no RCT as a evidence. And IDSA recommends VRCZ or ITCZ oral treatment and IV is optional. NO RCT existed !! P.O. first and I.V. is optional Clin Infect Dis 2008; 46:

World First RCT in CPA treatment
2011/9/9 World First RCT in CPA treatment This is the world first RCT in CPA comparing intravenous MCFG and VRCZ. Kohno, Izumikawa et al: J Infect, 2010

New Evidence of CPA treatment Overall efficacy MCFG v.s. VRCZ
2011/9/9 New Evidence of CPA treatment Overall efficacy MCFG v.s. VRCZ 15 （％） P＝0.543* 100 60.0％（30/50） efficacy 53.2％（25/47） 50 Overall efficacy was 60 % in MCFG and 53% in VRCZ and there was no statistical difference. However, we should notice that it is still about 50-60% efficacy even using average three weeks long administration of these expensive antifungals. MCFG VRCZ Kohno, Izumikawa et al: J Infect, 2010 15

New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ
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 26.4％（14/53） 30 20 10 MCFG VRCZ Kohno, Izumikawa et al: J Infect, 2010 16

New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ
2011/9/9 New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ Visual disturbance & hepatic dysfunction 17 P=0.012* （％） P＜0.0001* （％）（％） P=0.025* 10 20 30 40 50 60 70 10 20 30 40 50 60 70 10 20 30 40 50 60 70 50.0％（27/54） frequency frequency frequency 35.2％（19/54） 29.6％（16/54） 26.4％（14/53） 15.1％（8/53） 0.0％（0/53） Various visual events including photophobia, xanthopsia, abnormal vision, defective color vision, vision blurred, and visual disturbance occurred only in the VRCZ group and all visual events were transient and resolved without intervention. All of the adverse effects other than visual events recovered during the treatment or after the end of treatment, except in one case with alkaline phosphatase elevation, which was mild. Table 5 shows the treatment-related adverse events and reasons for discontinuation from the study in patients who received at least one dose of study drug. Significantly fewer adverse effects were observed in the MCFG group than in the VRCZ group. Hepatic events were the most frequent adverse events in both the MCFG and VRCZ groups (8 cases in the MCFG group and 15 cases in the VRCZ group). There were four cases in which VRCZ was discontinued due to severe hepatic events (at least one of the values of glutamic oxaloacetic transamidase, glutamic pyruvic transamidase, alkaline phosphatase, or gamma-glutamyl transpeptidase exceeded 2.5 times the value of the upper normal limit). Various visual events including were transient and resolved without intervention. All of which was mild MCFG VRCZ MCFG VRCZ MCFG VRCZ Visual events Adverse effects except visual related events Hepatic events Kohno, Izumikawa et al: J Infect, 2010 17

Another route of antifungal administration
2011/9/9 18 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 1×108/ml:50μl intratracheal inoculation Day1～5：　L-AMB　1.2mg/ml：8ml nebulize once/day MCFG 1mg/kg/day intraperitoneal Group1: nL-AMB + MCFG Group2: nL-AMB Group3: MCFG Group4: Control Another option is that we may try is another administration of antifungals. Since CPA occurred in the patients with existing lung diseases, which means that systemic antifungal administration might not reach to the site of infection enough. This is the Invasive Pulmonary Aspergillosis murine model treated with inhalation of ambisome with or without MCFG as combination treatment. Day-2 Day0 immunosupression Inoculum(i.t.) Day1～5 L-AMB inhalation ICR,♀,8weeks and/ or MCFG i.p. Takazono, Izumikawa: AAC 53: , 2009 18

survival rate Another route of antifungal administration Time (DAYS)
nebulized L-AMB & MCFG IPA murine model 2011/9/9 19 1 n L-AMB+MCFG .8 survival rate N L-AMB .6 MCFG .4 control .2 Although single administration of inhaled AMB and intraperitonelal MCFG is not showing statistically different in survival, however, when they combined the survival rate dramatically increased. We still do not know what is going to happen in CPA model. 2 4 6 8 10 12 14 16 Time (DAYS) Control MCFG (i.p.) L-AMB L-AMB + MCFG (i.p.) Day3 pathology GMS stain×400 Takazono, Izumikawa: AAC 53: , 2009 19

Another route of antifungal administration
nebulized L-AMB & MCFG IPA murine model 2011/9/9 20 L-AMB inhalation conc. AMPH-B concentration Lung (mg/kg) serum (μg/ml) control 0.1 <0.02 L-AMB 1.2mg/ml 35.5±4.2 0.02 L-AMB 2.6mg/ml 73.2±15.8 0.06 Although single administration of inhaled AMB and intraperitonelal MCFG is not showing statistically different in survival, however, when they combined the survival rate dramatically increased. We still do not know what is going to happen in CPA model. L-AMB 4.0mg/ml 94.2±20.2 0.06 Takazono, Izumikawa: AAC 53: , 2009 20

ITCZ resistant A. fumigatus
2011/9/9 ITCZ resistant A. fumigatus Nijmegen, Netherland number 50 100 150 200 250 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 1 2 3 4 5 6 % Recently some reports from europe has reported the emergence of azole-resistant Aspergillus fumigatus. This is data from netherland and it is strikingly increasing. PLoS Med November; 5(11): e219 21

Azole resistant A. fumigatus
2011/9/9 Azole resistant A. fumigatus Manchester, U.K. 100 20 % 90 80 70 Multi-azole ITCZ & POSA VRCZ ITCZ Susceptible 5 14 Number of patient cases 60 17 50 5 7 3 40 30 5 This is the data from U.K. and Dr. Denning will explain this later. As you can see here , it is also increasing in U.K. 20 7 10 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 Year Bueid et al. JAC 65: , 2010 22

Cowen LE. Eukaryotic Cell 2008 7:747-764
Mechanism of action azole antifungals 2011/9/9 ーＴｒｉａｚｏｌｅ＋ＴｒｉａｚｏｌｅＥｒｇｏｓｔｅｒｏｌＴｏｘｉｃｓｔｅｒｏｌＴｏｘｉｃｓｔｅｒｏｌＥｒｇｏｓｔｅｒｏｌＰ４５０１４ＤＭＥｒｇ３Ａｚｏｌｅｓ This slide indicates the mechanism of action of azole to fungus. Azole inhibit the 14 alpha dimethylase and fungus is not able to produce ergosterol. ＥｒｇｏｓｔｅｒｏｌＰ４５０１４ＤＭＥｒｇ３ Cowen LE. Eukaryotic Cell :

Mechanism of resistance azole antifungals
2011/9/9 Mechanism of resistance azole antifungals Alternation, mutation and over expression of P45014DM Alternation of synthesis Mutation of Erg 3 Overexpression of Efflux pumps Toxic sterol Ergosterol P450 14DM Erg 3 Triazoles Mutation Alternate sterol Triazoles Ergosterol P450 14DM Erg 3 Ergosterol P450 14DM Erg 3 Traizoles This is the resistant mechanism. There are three major resistant mechaanism #1 aletrnation or mutation of targeted enzyme, 14 alpha dimethylase, #2 alternation of synthesis pathway due to mutation of Erg3, #3 Over expression of efflux pumps. Cowen LE. Eukaryotic Cell :

2011/9/9 Mechanism of azole resistant A. fumigatus Cyp51A mutation hot spot, Netherland & U.K. G54 TR+L98H M220 U.K. 12 6 9 Netherland 94 3 Multi-azole resistance (%) TR L98H M220 G448 F-helix G-helix Cyp51A This slide indicates hot mutation of Cyp51a coding 14alpha dimethylase. As you can see here, profiles of mutation differs in U.K. and Netherland and it is interesting. I assume that Resistant in Netehrland might be acquired from environmanet since 94% of resistant starins possess same mutation of Cyp51A. ITCZ resistance POSA resistance Membrane-anchoring region G138 Multi-azole resistance G54 Promoter Membrane-anchoring region Verweij PE, et al. Lancet Infect Dis 9: , 2009

Drug susceptibility of Aspergillus fumigatus
2011/9/9 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 Now I would like to show you our data from Nagasaki, Japan. We have total of 196 A. fumigatus strains in our hand and performed drug susceptibility test by CLSI methods. We also investigated strains by short tandem repeat molecular analysis to reveal their epidemiological analysis.

7.1 % 4.1% 2.6 % Drug susceptibility of Aspergillus fumigatus
2011/9/9 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% We investigated the triazole, amphotericin B, and micafungin susceptibilities of 196 A. fumigatus clinical isolates obtained in Nagasaki, Japan. The percentages of non-wild-type (non-WT) isolates for itraconazole, posaconazole, and voriconazole were 7.1%, 2.6%, and 4.1% respectively. G54 mutation in cyp51A was detected in 64.2% (9/14 isolates) and 100% (5/5 isolates) of itraconazole and posaconazole non-WT isolates, respectively. The proportions of azole non-WT isolates in Japan were similar to those reported in other countries. Wild-type (WT) isolates of A. fumigatus (MIC ≤ ECV) were distinguished from non-WT isolates (MIC > ECV) which may exhibit acquired low susceptible mechanisms. ECVs used in this study were as follows: itraconazole, 1 μg/ml; posaconazole, 0.5 μg/ml; voriconazole, 1 μg/ml as previously suggested (9, 25). Posaconazole ≧1 μg/ml (5/196) 2.6 %

2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) frequency of non-WT isolates and cross resistance POSA VRCZ ITCZ This slide indicates the cross drug resistance in azole antifungals. Red indicates resistance and yellow indicates intermediate and blue shows susceptible. As you can see there is a trend that POSA and ITCZ may have relative cross resistant trend. But not for VRCZ. It may be resulted from the differnce of structure of VRCZ and POSA and ITCZ which are resembles to each other.

2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Comparison of positive rate of Cyp51A mutation to Europe Positive rate of Cyp51A mutation Nagasaki, JAPAN 87.5% U.K. 57% Netherland 88% Mutation frequency in Nagasaki strains indicated almost 90% of all of resistant strains. I266Nの変異も含めてしまうと、88.9％（8株/9株）になります。 G54に限定すると、66.7％（6/9）になります。 8株はITCZ MIC=>4あるいはPOSA MIC=>1を示すものです。

2011/9/9 Drug susceptibility of Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Comparison Cyp51A mutation with Netherland G54 TR+ L98H M220 none Nagasaki 75.0 11.1 Netherland 1.5 80.1 3.8 12.3 Multi-azole resistance (%) TR L98H M220 G448 F-helix G-helix Cyp51A And 75% indictaed aminoacid alternationat the 54 , from glycin to something else. Of all 22 non-WT isolates in our study of Japanese isolates, cyp51A gene mutations were detected as follows: G54W, two isolates; G54R, one isolate; I266N, two isolates; G54E + I266N, four isolates, G54W + I266N, three isolates. No TR/L98H-bearing isolates were detected. The I266N mutation, which has (to our knowledge) not been reported previously, also was seen in other azole-susceptible isolates; therefore, it might not be directly related to azole resistance. W: トリプトファン、 R: アルギニン　I:イソロイシン、N：アスパラギン ITCZ resistance POSA resistance Membrane-anchoring region G138 Multi-azole resistance G54 Promoter Membrane-anchoring region AAC. 2010; 54:

Azole-resistant Aspergillus fumigatus
2011/9/9 Azole-resistant Aspergillus fumigatus Nagasaki University Hospital Clinical Isolates (196 strains) Correlation of ITCZ exposure amount and drug susceptibility ITCZ MIC（μg/ml） Cumulative ITCZ exposure （mg） 0.125 0.25 0.5 1 2 4 8 >8 30000 60000 90000 120000 150000 180000 r = p <0.0001 This slide indicates that cumulative ITCZ exposure before the strains isolation. Strains with higher MIC is related to total exposure amount of ITCZ.

Azole-resistant A. fumigatus isolated case
2011/9/9 Azole-resistant A. fumigatus isolated case 59 years old, Male 32 β-D glucan Aspergillus Ag Aspergillus Ab Sputum culture (bacteria) Sputum culture (Tbc) Sputum culture 53.5 2.9 （+）（-） pg/ml C.O.I. A. fumigatus CC: fever, cough, sputum PH: surgical for pneumothorax I would like to present the case with azole-resistant A, fumigate isolated. The patients was 59 years old and complained ever, cough and sputum. He had history of surgical treatment for pneumonothorax. These serological and mycological data were positive for diganosis of CPA. As you can see here, there are dead space due to surgical treatment and fungus ball. There is also pericavity infiitrates with niveau lije shadow. 左上肺野に壁肥厚を伴う空洞あり左中肺野縦隔側にair-fluid levelを伴う空洞あり全体的に肺野の透過性は亢進右肺尖部胸膜肥厚あり【症例】 59歳　男性【主訴】発熱、咳嗽、喀痰【既往歴】 1950年　肋膜炎との診断でパラアミノサリチル酸 (PAS)で半年治療。 1970年　両側気胸を発症。以後気胸を繰り返す。 1977年　難治性気胸に対し、左上葉＋舌区切除術、右肺部分縫縮術施行。【生活歴】喫煙：40本/日　30歳～46歳　B.I.：640 飲酒：焼酎3合/日【現病歴】 1996年　湿性咳嗽が出現、以後増悪。 1998年 37℃台の発熱、血痰が出現。近医で精査。胸部CT、アスペルギルス抗体陽性（抗原：陰性）の結果より、肺アスペルギルス症と診断、 ITCZ cap 200mg/日を189日間内服継続し終了、症状は軽減。 2000年治療終了半年後より、全身倦怠感が増悪、38℃台の発熱、湿性咳嗽が出現。別の近医入院。抗菌薬の投与を受けるも改善なく、当科紹介となった。

59 years old, Male 2011/9/9 33 1 2 3 4 5 6 7 8 9 10 11 12 βD glucan 24.0 1999 ITCZ 200mg 53.5 15.7 6.9 2000 Aspergillus Ag 2.9 5.3 2.5 Serum ITCZ conc μg/ml Sputum culture MCFG 150mg 1st isolated ITCZ low-sensitive A. fumigatus 2001 2.1 0.6 ITCZ 400mg ITCZ 200mg This is the clinical course of this case, this symbol inidicates azole senssitive fumigatus and this idicates resistanat strains, As you noticed here, before isolation of resistant strains, the patinet had been administrated long term ITCZ. After voriconazole treatment here, no fumigtus has been detected from this patient. 慢性アスペルギルス症の多分に漏れず、この方も非常に長い経過を示していますので、簡略化して示します。一番上の1999年が、先ほど当科紹介される1年前に初めてアスペルギルス症と診断され、イトラコナゾールによる治療を受けた年です。それから当科紹介された2000年から2005年までの経過を示しています。示されているデータは、使用された抗真菌薬とその期間及び量を色つきのバーで示しています。並列で示したデータは、茶色字のβDグルカン、青字のアスペルギルス抗原、赤の記号の喀痰培養結果です。喀痰培養の結果は、A. fumigatusが分離された場合プラスの形で示し、陰性だった場合はマイナスの形で示しています。さらに、アゾールの感受性が低下した株が分離された場合は、赤で塗りつぶしたプラスの形で示しています。 1999年に初めて肺アスペルギルス症の診断を受けた時は、治療前のβDグルカンが24程度を示していたようです。この黄色バーで示すように、約半年間イトラコナゾール200mgによる治療を受けたのち、症状が軽減したとのことで経過を見られています。しかし、2000年8月ごろに再度症状増悪し、喀痰培養陽性、βDグルカン上昇を認めています。ここでミカファンギンの治験が入りまして、4か月ほどミカファンギンで治療され症状が改善しております。その後維持療法としてイトラコナゾールカプセル高用量の800mgで半年ほど継続され、そこからさらにイトラコナゾールカプセル200mgに減量され2002年の初めまで継続されました。 1年以上、イトラコナゾールカプセルによる治療を行ったことになります。なお、その血中濃度は400mg投与中は1ガンマを超えていたようです。200mg投与中の血中濃度は測定されておりませんでした。イトラコナゾールの累積投与期間が1年を超えてから、喀痰から分離されるA. fumigatusのアゾール感受性が低下し始めました。維持療法としてのイトラコナゾールの投与を終了し、半年後より再度症状の増悪、喀痰から低感受性株が分離される頻度の増加、βDグルカン及びアスペルギルス抗原の上昇を認めています。当時は分離されたA. fumigatusの感受性はわからなかったのですが、偶然、ボリコナゾールの治験に入りまして、ボリコナゾールによる治療が行われました。 βDグルカンは一時的に上昇しているように見えますが、症状は軽減し、ボリコナゾール治療後は喀痰から一度もA. fumigatusが分離されておりません。アスペルギルス抗原及びβDグルカンも以後低下していきました。以後、2003年、2004年とミカファンギンやイトラコナゾールが投与されていますが、細菌感染とアスペルギルス症増悪の鑑別が困難なまま抗菌薬も同時に投与されているパターンがほとんどですので、イトラコナゾールの治療効果があったのか、なかったのか、判断は困難です。 2005年以降は、2011年の現在に至るまで特に増悪なく経過しています。 53.4 132.2 87.0 2002 5.1 6.6 9.1 4.9 2.9 1.5 VRCZ 300mg 7.2 2003 0.8 0.6 0.1 ITCZ 200mg MCFG 300mg

Azole resistant A. fumigatus isolated case
59 years old, Male 2011/9/9 34 MCFG 150mg ITCZ 400mg ITCZ 200mg 8/16 12/14 5/16 1/23 2000/08/15 2000/12/14 2001/05/17 2001/10/22 2002/01/23 VRCZ 300mg 6/4 8/25 Thhese are the radiological findings of this patient, As you can see, it worsened after resistant srain isolation. Although it does not look like become better after VRCZ treatment, no sumigatus has been islated after 2003. Fungus ballを伴った空洞の時間的経過を示します。同時に抗真菌薬の治療経過及び喀痰培養の経過を示します。赤で塗りつぶしたものは低感受性株です。初めの写真が当科初診時のものです。壁が肥厚した空洞と、その右下に膿汁の貯留したブラを認めます。この膿汁を貯留したブラは、CTガイド化にドレナージを行い、以後改善しております。ミカファンギン、イトラコナゾール400mgの治療が経過した頃、当初あった空洞は縮小していくように見え、また2001年5月には全周性に壁が剥がれ落ちているように見えます。イトラコナゾール200mgの治療が継続されている間、その剥がれ落ちたものは徐々に分厚くなっていくように見え、空洞内にアスペルギローマがあり、その中にさらに空洞があるような珍しい形になっていきました。なお、この途中より分離されるA. fumigatusのアゾール感受性が低下していっております。よって、この間のイトラコナゾールの治療効果があったのか否かが重要なところですが、イトラコナゾール200mg投与中も壁から剥がれたアスペルギルス菌体と疑われる部分が分厚く成長していっておりますので、この間の治療効果はなかったのではないかと推測します。ただ、カプセル200mg投与中のイトラコナゾールの血中濃度は測定されていませんし、このように空洞内で物理的に壁から離れた状態で、菌体にどこまで血中から薬剤が移行するかも疑問がありますので、分離されたA. fumigatusの薬剤感受性のみと治療効果の関係について論じることは困難と思われます。その後、低感受性株が立て続けに分離され、ボリコナゾールの治験が入る中で、空洞内のFungus ballと思われるものは、その内部が徐々に充満していきました。ボリコナゾールの治療効果は、臨床症状やラボデータの改善及び喀痰から培養されなくなったことから、治療効果ありと思われますが、レントゲン上ははっきりとした変化は言い切れません。 2002/02/27 2002/05/22 2002/08/27 2003/04/14 2011/08/29

59 years old, Male, summary of isolated strains and cases 2011/9/9 35 strain Isolated date STR cluster type Cumulative ITCZ exposed term (days) Cumulative ITCZ exposed dose (mg) MIC (μg/ml) Cyp51A mutation ITCZ POSA VRCZ MF-368 2000/8/16 3 189 37,800 0.5 0.06 I266N MF-367 0.25 MF-370 2000/9/7 MF-439 2001/10/19 507 144,850 2 G54E MF-452 2002/4/3 589 161,650 >8 MF-454 2002/4/17 0.125 MF-460 2002/5/8 4 MF-468 2002/5/22 MF-469 2002/5/29 8 1 G54W This is the summary of strains isolated in this case. STR cluster inidicated all sensitive and resistant starins during two years were genetically identical. And Mutation of Cyp51A indicated alternation og glycin of positin 54 to tryptophan ( W) or glutamine acid( E) Finally this case inidciated the possibity that drug resitant has been acquired in the body of patient due to longer exposure of low dose of azoles. Resembel case report was published by Dr. Denning before. 全経過中に分離されたA. fumigatusの薬剤感受性をレトロスペクティブに確認しております。それに合わせ、それぞれ株が分離されるまでに投与されたイトラコナゾールの累積使用期間及び累積量のデータも合わせて示しております。また、A. fumigatusのアゾール耐性機序の一つとして知られている、アゾール標的酵素のcyp51Aの変異も調べています。この表で示すように、分離された株は後半になるに従い徐々にイトラコナゾール及びポサコナゾールへの感受性が低下していきました。それはイトラコナゾールの累積使用期間が500日を超えてからでした。また、その感受性低下の原因として、アゾール標的酵素cyp51AのG54変異を確認できました。それ以外に存在したI266Nという変異は未報告のものですが、初めから存在するので、おそらく感受性には関与しない変異と思われます。

CPA treatment and drug resistance
2011/9/9 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 Here is summarized slide.

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Acknowledgement Nagasaki University Shigeru Kohno Takayoshi Tashiro
2011/9/9 Acknowledgement Nagasaki University Shigeru Kohno Takayoshi Tashiro Katsunori Yanagihara Yoshihiro Yamamoto Hiroshi Kakeya Taiga Miyazaki Yoshifumi Imamura Shigeki Nakamura Takahiro Takazono Masato Tashiro Katsuji Hirano National Institutes of Infectious Diseases Yoshitsugu Miyazaki Hideaki Ohno In last, I would like to appreciate to all members of our department and Dr. Miyazaki in Japanese NIID. Thank you.

Nagasaki University Hospital established in September 20, 1861

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