department of molecular microbiology and immunology nagasaki university graduate school of...

Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences

Nagasaki University Hospitalestablished in September 20, 1861

2011/9/9 1


Fungus diseases in literature150 years ago

Fungal diseases 1st Case report Describe of microorganism

Candida 1839, Langenbeck 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

Okudaira M, Jpn J Pathology ： 74, 61-91, 1985

2011/9/9 2


2011/9/9 3

IUMS 2011,Sapporo, JAPANIUMS 2011,Sapporo, JAPAN

Respiratory MycosesPulmonary Aspergillosis: Pathogenesis and Treatment

Chronic pulmonary aspergillosis ~new treatment evidence and emergence of azole-

resistant Aspergillus fumigatus in Japan~


Koichi IZUMIKAWA, M.D., Ph.D.


82 years old, male, father82 years old, male, father54 years old, male, SON54 years old, male, SON

Chronic forms of Pulmonary Aspergillosisfamily case, Sasebo, JAPAN

slowly progressive inflammatory pulmonary syndrome due to Aspergillus spp.

2011/9/9 4


Chronic forms of Pulmonary Aspergillosisfamily case, Sasebo, JAPAN

investigation of circumstances

over 50 years old wooden househumid and oldover 50 years old wooden househumid and old

2011/9/9 5


6

Kume et al. Med Mycol J 52: 117-127, 2011

0

1

2

3

4

5(%)

Frequency

(%)

1960 1970 1980 1990 2000Year

Total number of Mycosis

Candidiasis

Aspergillosis

Cryptococcosis

Mucor

AMPH-B 1962

5-FC 1979MCZ 1980

FLCZ 1989ITCZ 1993

6

2007

L-AMB 2006

VRCZ 2005F-FLCZ 2004

MCFG 2002

Systemic Mycosis in Japan from Autopsy Data2011/9/9

??


2011/9/9 7

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) orchronic necrotizing pulmonary aspergillosis (CNPA)

Multiple cavities w/ surrounding inflamation ±aspergilloma:chronic cavitary pulmonary aspergillosis (CCPA) orcomplex 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 exposurePreexisting pulmonary defect, with cavity


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 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) 5 (11.6)

(%)(%)

2011/9/9


2011/9/9 9

Reported TB casesNew and relapse cases (per 100 000 population)

0

10

20

30

40

50

60

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

France

Germany

Italy

Japan

Netherlands

Portugal

Spain

Sweden

United Kingdom of GreatBritain and Northern IrelandUnited States of America

WHO, Communicable Diseases Report


2011/9/9 10

[case]65 Y, Male

[CC] hemosputum, cough

[PH] n.p.

[PI] 1998 ： right upper lobectomy (Tbc)2005~ ： cough, hemosputum2006~: hemosputum increasedchest CT ： fungus ball like shadows in right lower lung.Platelia EIA: positive, Aspergillus Ab: positiveβ-D-gulucan 35.0pg/mladmission for further treatment

[PE] Height 161cm, Weight 44.3kg, BMI 17.1, Body temp. 36.8 , pulse 68/min, regular rhythm ℃

CPA case


2011/9/9 11

WBC(/μl)

CRP(mg/dl)

β-D-glucan(pg/ml)

8700 6700 80005700 5300 6500 7500 7500

117.8 47.439.5 90.9 25.9 28.968.2

2/16

37℃

10/11

MCFG 150mg/day

sputum:A. terreus

3/19　

VRCZ 200mg/day

ITCZ 400mg/day

BIPM 0.6g/day

10/4

BALF: A. fumigatusA. nigerA. versicolorA. terreus

2/22

ITCZ 200mg/day

BIPM 0.6g/day

BALF: A. fumigatus

Aspergillus antigen (EIA)

1.65 1.465.70 0.20 3.240.19 0.93 0.72

0.65 0.976 0.985 0.591 0.5171.025 0.441 0.461

6 months

hemosputum

CPA caseITCZ oral solution treatment


1212Evidence of CPA treatment

Drug CPAResponse rate

(%)Route Design Year Author Reference

ITCZCNPA+Aspergilloma

60-66 oral CS 1998 De Buele Mycoses

ITCZCNPA+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

MCFGCNPA+Aspergilloma

55-67 IV CS 2004 Kohno Scand J Infect Dis

MCFG CPA 78 IV CS 2007 Izumikawa Med Mycol

MCFGCNPA+Aspergilloma

58 IV CS 2009 YasudaNihon Kokyuki Gakkai Zasshi

VRCZ CCPA 64 oral CS 2006 Jain J Infect

VRCZ CNPA+CCPA 50-80 oral CS 2006Sambatakou

Am J Med

VRCZ CNPA+CCPA 70 oral CS 2007 Camuset Chest

2011/9/9


2011/9/9 13

Clin Infect Dis 2008; 46:327-360

CPA treatmentIDSA GL

Treatment

Primary Alternative

CNPA

(Subacute IPA)

VRCZL-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

Aspergilloma none

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

World First RCT in CPA treatment

Kohno, Izumikawa et al: J Infect, 2010

2011/9/9


1515

New Evidence of CPA treatmentOverall efficacy MCFG v.s. VRCZ

60.0 ％（ 30/5

0 ）

53.2 ％（ 25/4

7 ）

0

50

100

MCFG VRCZ

efficacy

（％） P ＝ 0.543*


2011/9/9


1616

26.4 ％（ 14/5

3 ）

61.1 ％（ 33/5

4 ）

MCFG VRCZ

frequency

0

10

20

30

40

50

60

70

（％）

P ＝ 0.0004*


2011/9/9

New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ


1717

26.4 ％（ 14/5

3 ）

50.0 ％（ 27/5

4 ）

P=0.012*

15.1 ％（ 8/53 ）

35.2 ％（ 19/5

4 ）

P=0.025*

Visualevents

Hepatic events

Adverse effects except visual related events

MCFG VRCZ

P ＜0.0001*

0.0 ％（ 0/53 ）

29.6 ％（ 16/5

4 ）

MCFG VRCZ

frequency

0

10

20

30

40

50

60

70（％）

MCFG VRCZ

frequency

frequency

0

10

20

30

40

50

60

70（％）

0

10

20

30

40

50

60

70（％）


2011/9/9

New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZVisual disturbance & hepatic dysfunction


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

Day-2 Day0

immunosupression Inoculum(i.t.)

Day1 ～ 5

L-AMB inhalationICR,♀,8weeks

Group1: nL-AMB + MCFG

Group2: nL-AMB

Group3: MCFG

Group4: Control

and/ or MCFG i.p.

Takazono, Izumikawa: AAC 53: 3508-3510, 2009

2011/9/9 18


0

.2

.4

.6

.8

1

surv

ival

rat

e

0 2 4 6 8 10 12 14 16 Time (DAYS)

control

MCFG

N L-AMB

n L-AMB+MCFG


nebulized L-AMB & MCFG IPA murine model

GMS stain×400

Day3pathology

Control MCFG (i.p.) L-AMB L-AMB + MCFG (i.p.)


2011/9/9 19



nebulized L-AMB & MCFG IPA murine model


L-AMB inhalation conc.

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

L-AMB 4.0mg/ml 94.2±20.2 0.06

AMPH-B concentration

2011/9/9 20


21

ITCZ resistant A. fumigatusNijmegen, Netherland

0

50

100

150

200

250

1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 20070

1

2

3

4

5

6

PLoS Med. 2008 November; 5(11): e219 PLoS Med. 2008 November; 5(11): e219

numbernumber%%

2011/9/9


22

Azole resistant A. fumigatusManchester, U.K.

Bueid et al. JAC 65: 2116-2118, 2010Bueid et al. JAC 65: 2116-2118, 2010

0

10

20

30

40

50

60

70

80

90

100

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

Multi-azole

ITCZ & POSA

VRCZ

ITCZ

Susceptible

YearYear

Num

ber

of p

atie

nt c

ases

Num

ber

of p

atie

nt c

ases

00 0077

33

55

00 00

55

55

77

17171414

2020 %%

2011/9/9


23

ＥｒｇｏｓｔｅｒｏｌＥｒｇｏｓｔｅｒｏｌ

ＴｏｘｉｃｓｔｅｒｏｌＴｏｘｉｃｓｔｅｒｏｌ


Ｐ４５０Ｐ４５０１４ＤＭ１４ＤＭ

Ｅｒｇ３Ｅｒｇ３


ＡｚｏｌｅｓＡｚｏｌｅｓ

ＴｏｘｉｃｓｔｅｒｏｌＴｏｘｉｃｓｔｅｒｏｌ


Ｐ４５０Ｐ４５０１４ＤＭ１４ＤＭ


＋Ｔｒｉａｚｏｌｅ＋ＴｒｉａｚｏｌｅーＴｒｉａｚｏｌｅーＴｒｉａｚｏｌｅ

Mechanism of actionazole antifungals

Cowen LE. Eukaryotic Cell 2008 7:747-764

2011/9/9


24

Toxic sterolToxic sterol

ErgosterolErgosterol

P450 14DMP450 14DM Erg 3Erg 3

Erg 3Erg 3

TriazolesTriazoles

MutationMutation

Alternate sterolAlternate sterol



TraizolesTraizoles TriazolesTriazoles



Alternation, mutation and over expression of

P45014DM

Alternation, mutation and over expression of

P45014DM

Alternation of synthesisMutation of Erg 3

Alternation of synthesisMutation of Erg 3

Overexpression ofEfflux pumps

Overexpression ofEfflux pumps

Cowen LE. Eukaryotic Cell 2008 7:747-764

2011/9/9

Mechanism of resistanceazole antifungals


25

Mechanism of azole resistant A. fumigatusCyp51A mutation hot spot, Netherland & U.K.

Verweij PE, et al. Lancet Infect Dis 9: 789-795, 2009

Cyp51ACyp51A

M220 G448

F-helixF-helix G-helixG-helix

ITCZ resistancePOSA resistance

Membrane-anchoring regionMembrane-anchoring region

G138

Multi-azole resistance Multi-azole resistance

G54PromoterPromoter


Multi-azole resistance

TR TR L98H

G54 TR+L98H M220

U.K. 12 6 9

Netherland 0 94 3

(%)(%)

2011/9/9


26

Drug susceptibility of Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)

Strains ：Clinical isolated A. fumigatus between 1994 and 2010

Method of identification ：microscopic morphologyability 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 epidemiologySTR/microsatellite analysis

Strains ：Clinical isolated A. fumigatus between 1994 and 2010

Method of identification ：microscopic morphologyability 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 epidemiologySTR/microsatellite analysis

2011/9/9


27

7.1 %7.1 %

2.6 %2.6 %

4.1%4.1%

Itraconazole Itraconazole ≧≧2 μg/ml (14/196)2 μg/ml (14/196)

Posaconazole Posaconazole ≧≧1 μg/ml (5/196)1 μg/ml (5/196)

Voriconazole Voriconazole ≧≧2 μg/ml (8/196)2 μg/ml (8/196)

2011/9/9


frequency of non-WT isolates


>80

0.58

01

40

24

00.5

40

0.54

00.5

20

>162

02

20

0.52

00.5

20

0.52

00.5

22

02

00

00

20

20

02

00

20

02

00

20

02

00

20

ITCZ

VRCZ

POSA

2011/9/9 28


frequency of non-WT isolates and cross resistance


29

Positive rate of Cyp51A mutation

Nagasaki, JAPAN 87.5%

U.K. 57%

Netherland 88%

2011/9/9

Drug susceptibility of Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)Comparison of positive rate of Cyp51A mutation to Europe


30

AAC. 2010; 54: 2425-30

Cyp51ACyp51A

M220 G448

F-helixF-helix G-helixG-helix

ITCZ resistancePOSA resistance


G138

Multi-azole resistance Multi-azole resistance

G54PromoterPromoter


Multi-azole resistance

TR TR L98H

G54TR+

L98HM220 none

Nagasaki 75.0 0 0 11.1

Netherland 1.5 80.1 3.8 12.3

(%)(%)

2011/9/9


Comparison Cyp51A mutation with Netherland


ITCZ MICITCZ MIC （（ μg/mμg/mll ））

Cum

ulat

ive

ITC

Z e

xpos

ure

Cum

ulat

ive

ITC

Z e

xpos

ure

（（m

gm

g））

0.1250.125 0.250.25 0.50.5 11 22 44 88 >8>8

3000030000

6000060000

9000090000

120000120000

150000150000

180000180000

2011/9/9 31

Azole-resistant Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)

Correlation of ITCZ exposure amount and drug susceptibility

r = 0.5916p <0.0001


Azole-resistant A. fumigatus isolated case59 years old, Male

CC: fever, cough, sputumPH: surgical for pneumothorax

2011/9/9 32


ITCZ 200mg

ITCZ 400mg ITCZ 200mg

VRCZ 300mg

ITCZ 200mg

MCFG 150mg

MCFG 300mg

1 2 3 4 5 6 7 8 9 10 11 12

1999

2000

2001

2002

2003

2.9 5.3 2.5

2.1 0.6

5.1 6.6 9.1 4.9 2.9 1.5

0.8 0.6 0.1

24.0

53.5 15.7 6.9

53.4 132.2 87.0

7.2

βD glucan

Aspergillus AgSputum culture

1st isolated ITCZ low-sensitive A. fumigatus

Serum ITCZ conc. 1.148 μg/ml

Azole-resistant A. fumigatus isolated case59 years old, Male

2011/9/9 33


2000/08/152000/08/15 2000/12/142000/12/14 2001/05/172001/05/17 2001/10/222001/10/22 2002/01/232002/01/23

MCFG150mg

8/16 12/14

ITCZ400mg

ITCZ200mg

5/16 1/23

2002/02/272002/02/27 2002/05/222002/05/22 2002/08/272002/08/27 2003/04/142003/04/14

6/4 8/25

VRCZ300mg

Azole resistant A. fumigatus isolated case59 years old, Male

2011/9/9 34

2011/08/292011/08/29


strainIsolated

date

STR cluster type

Cumulative ITCZ

exposed term (days)

Cumulative ITCZ exposed

dose (mg)

MIC (μg/ml)

Cyp51AmutationITCZ POSA VRCZ

MF-368 2000/8/16 3 189 37,800 0.5 0.06 0.5 I266N

MF-367 2000/8/16 3 189 37,800 0.5 0.06 0.25 I266N

MF-370 2000/9/7 3 189 37,800 0.25 0.06 0.25 I266N

MF-439 2001/10/19 3 507 144,850 2 0.5 0.25 G54E I266N

MF-452 2002/4/3 3 589 161,650 >8 0.5 0.5

I266N

MF-454 2002/4/17 3 589 161,650 2 0.5 0.125 G54E I266N

MF-460 2002/5/8 3 589 161,650 4 2 0.25 G54E I266N

MF-468 2002/5/22 3 589 161,650 4 0.5 0.25 G54E I266N

MF-469 2002/5/29 3 589 161,650 8 1 0.25 G54W I266N

Azole-resistant A. fumigatus isolated case59 years old, Male, summary of isolated strains and cases

2011/9/9 35


2011/9/9 36

SUMMARYCPA 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


23/04/10 37

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2011/9/9 38

Acknowledgement

National Institutes of Infectious DiseasesYoshitsugu MiyazakiHideaki Ohno

Nagasaki UniversityShigeru KohnoTakayoshi TashiroKatsunori YanagiharaYoshihiro YamamotoHiroshi KakeyaTaiga MiyazakiYoshifumi ImamuraShigeki NakamuraTakahiro TakazonoMasato TashiroKatsuji Hirano

department of molecular microbiology and immunology nagasaki university graduate school of...

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