Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
Nagasaki University Hospitalestablished in September 20, 1861
2011/9/9 1
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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~
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
Koichi IZUMIKAWA, M.D., Ph.D.
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
??
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki 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 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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
14
World First RCT in CPA treatment
Kohno, Izumikawa et al: J Infect, 2010
2011/9/9
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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*
Kohno, Izumikawa et al: J Infect, 2010
2011/9/9
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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*
Kohno, Izumikawa et al: J Infect, 2010
2011/9/9
New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZ
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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(%)
Kohno, Izumikawa et al: J Infect, 2010
2011/9/9
New Evidence of CPA treatment Frequency of side effect MCFG v.s. VRCZVisual disturbance & hepatic dysfunction
Department of Molecular Microbiology and ImmunologyNagasaki 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 1×108/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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Another route of antifungal administration
nebulized L-AMB & MCFG IPA murine model
GMS stain×400
Day3pathology
Control MCFG (i.p.) L-AMB L-AMB + MCFG (i.p.)
Takazono, Izumikawa: AAC 53: 3508-3510, 2009
2011/9/9 19
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
Another route of antifungal administration
nebulized L-AMB & MCFG IPA murine model
Takazono, Izumikawa: AAC 53: 3508-3510, 2009
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
23
ErgosterolErgosterol
Toxic sterolToxic sterol
ErgosterolErgosterol
P450P45014DM14DM
Erg3Erg3
Erg3Erg3
AzolesAzoles
Toxic sterolToxic sterol
ErgosterolErgosterol
P450P45014DM14DM
Erg3Erg3
+ Triazole+ Triazoleー Triazoleー Triazole
Mechanism of actionazole antifungals
Cowen LE. Eukaryotic Cell 2008 7:747-764
2011/9/9
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
24
Toxic sterolToxic sterol
ErgosterolErgosterol
P450 14DMP450 14DM Erg 3Erg 3
Erg 3Erg 3
TriazolesTriazoles
MutationMutation
Alternate sterolAlternate sterol
ErgosterolErgosterol
P450 14DMP450 14DM Erg 3Erg 3
TraizolesTraizoles TriazolesTriazoles
ErgosterolErgosterol
P450 14DMP450 14DM Erg 3Erg 3
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Membrane-anchoring regionMembrane-anchoring region
Multi-azole resistance
TR TR L98H
G54 TR+L98H M220
U.K. 12 6 9
Netherland 0 94 3
(%)(%)
2011/9/9
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Drug susceptibility of Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)
frequency of non-WT isolates
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
>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
Drug susceptibility of Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)
frequency of non-WT isolates and cross resistance
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
30
AAC. 2010; 54: 2425-30
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
Membrane-anchoring regionMembrane-anchoring region
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
Drug susceptibility of Aspergillus fumigatusNagasaki University Hospital Clinical Isolates (196 strains)
Comparison Cyp51A mutation with Netherland
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
Azole-resistant A. fumigatus isolated case59 years old, Male
CC: fever, cough, sputumPH: surgical for pneumothorax
2011/9/9 32
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
23/04/10 37
Advertisement
The 86th Japanese Society of Infectious DiseasesAnnual meeting 2012, Nagasaki, JAPAN
The 86th Japanese Society of Infectious DiseasesAnnual meeting 2012, Nagasaki, JAPAN
Department of Molecular Microbiology and ImmunologyNagasaki University Graduate School of Biomedical Sciences
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