Molecular Diagnosis of Fungal PathogensSeptember 15, 2012
Hsiu-Jung LoInvestigator
National Health Research InstitutesTel ::::03-7246166-35516
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Tel ::::03-7246166-35516E-mail::::[email protected]
Human Pathogens
Over 1000 agents known to infect humans
~ 550 bacterial species~ 300 fungal species
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~ 300 fungal species~ 70 parasitic protozoa~ 200 virus species
There Are Good and Bad Fungi
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Mushroom
Saccharomyces cerevisiaeBeer and Bread
Candida, Aspergillus, Cryptococcus, etc.Local or systemic infections
Yeast Infections Increased Significantly
Rep
orte
d ca
ses
4
Year
Rep
orte
d ca
ses
Taiwan CDC :Taiwan Nosocomial Infection Surveillance (TNIS) inpatients inintensive care unit (ICU)
Current Challenges for Managing Fungal Infections
1. Risk populations increased.
2. Limited choice of antifungal drugs.
3. Emerging drug resistance isolates.
4.Emerging species causing diseases in humans.
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4.Emerging species causing diseases in humans.
5. How to identify those high-risk populations?
6. How to improve diagnosis of fungal infections?
7. How to develop new effective antifungal drugs?
Diseases caused by primary pathogensBlastomycosisCoccidioidomycosisHistoplasmosisParacoccidiomycosisPenicillosis
Diseases caused by opportunistic pathogens
Fungal Infections in Human
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Diseases caused by opportunistic pathogensCandidasisCryptococcosisAspergillosisZygomycosisOther mycosesPhaeohyphomycosisPneumocystosis
Time to Initiation of Fluconazole TherapyImpacts Mortality in Patients with
CandidemiaAMulti-Institutional Study
30
40
50
Mo
rtal
ity
(%) 40
50
30
7
Garey et al Clin Infect Dis 2006; 43:25-31.
0
10
20
30
C ulture day Day 1 Day 2 Day >= 3
Mo
rtal
ity
(%)
Culture day 1 Day 2 Days ≧≧≧≧ 3 Days
30
20
10
0
Diagnosis Methods-ISymptom: Not easy
Fever with antibiotic treatmentDirect microscopy: Rapid and cost-effective
(Gram, Giemsa, and Calcofluor strains) Culture:
False negative results due to conditionTime consuming
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Time consumingHazards from culturing certain species
Histopathologic Methods:Routine stains (Hematoxylin and Eosin)
Special stains (Gomori Methenamine Silver)In situ hybridization
Diagnosis Methods-II
Immunologic Methods : sensitivity and specificityAntibodyAntigen
Biochemical Methods : sensitivity and specificityMetabolitesCell wall components
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Cell wall componentsEnzymes
Molecular Methods : sensitivity and specificityDirect detection (nucleic acid amplification)False positive
What Do We Need?
Broad identification of microbesRapid detection: culture often not requiredMixtures of microbes are detectedHigh-resolution genotyping and strain identificationNovel microbes can be detected
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Novel microbes can be detectedDrug resistance testing without culture
ACCURATE, CHEAP, and RAPIDMolecular Methods
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
Conventional PCR
Polymerase Chain Reaction
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Conventional PCRReal-time PCRBroad-range PCR
Molecular typingMicroarrays MultiplexingPartial or whole genome sequencing
Deep sequencing
Primers for PCR
18S 5.8S 26S 5S 18S
D1/D2
ITS1
ITS4 NL4
NL1
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ITS: internal transcribed spacerIGS: intergenic spacer
IGS1ITS2
ITS1IGS2
NL4
rDNA: High copy per genomeIncrease sensitivity
primers ITS1, ITS2, CA3, and CA4. Lane 1, 50-bp DNA ladder, C. krusei, C.neoformans, C. albicans, species markers formulated from amplicons of the seven
Results of PCR
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amplicons of the seven major yeast species, C. tropicalis, C. parapsilosis, C. guilliermondii, and C. glabrata.
Chang HC et al, J Clin Microbiol. 2001,39:3466
ckr cne cal ctr cpa cgu cgl7spp
Size of fragments fromC. albicans and C. tropicalis are similar.
DNA ladder, cal: C. albicanscgl: C. glabratacpa: C. parapsilosisCtr: C. tropicalis eco: E. coli,
Multiplex PCR
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eco: E. coli,ecl: E. cloacaeBlood: human blood-: negative control
100 cells
Chang HC et al, J Clin Microbiol. 2001,39:3466
calcgl
calcpa
cglctr
7spp caleco
ctrecl
blood -
50-bp DNA ladde C. pelliculosa, C. famata, species markers Rhodotorula rubra Trichosporon beigeliispecies markers
Identification of Minor Yeast Species
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species markersC. lusitaniaeCandida sp.
R. rubra (lane 5) was misidentified as C. parapsilosis.
Chang HC et al, J Clin Microbiol. 2001,39:3466
cpe cfa 7 spp rru tbe 7 spp clu Candida
Intergenic Spacer (IGS) of Saccharomyces
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Ganley A R D et al. PNAS 2005;102:11787
Size of fragments are similar.Sequencing
18S 5.8S 26S 5S 18S
D1/D2
ITS1
ITS4 NL4
NL1
Intergenic Spacer (IGS) of Trichosporon
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IGS1ITS2
ITS1IGS2
Genotypes of Trichosporon asahii
10 nucleic acid
V
III
III
IV
Suqita T et al, J Clin Microbiol. 2002,40:1826
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
Conventional PCR
Real-time Polymerase Chain Reaction
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Conventional PCR Real-time PCR
Decreased false positive resulting from amplicon carryover
Melting curve analysisExpensive instrument3X higher cost then PCR
Real-time Polymerase Chain Reaction
Zygosaccharomyces bailiiSaccharomyces cerevisiae
Zygosaccharomyces rouxii
Rhodotorula glutinis
Candidakrusei
Melting temperature (ITS)
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Zygosaccharomyces rouxii
Casey GD &Sobson ADW Internal J Food Microbiol 2004:3, 327
Real-time Polymerase Chain ReactionZb Zr Sc Ck Rg
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Zb: Zygosaccharomyces bailii;Zr: Zygosaccharomyces rouxii; Sc: Saccharomyces cerevisiae; Ck: Candida krusei; Rg: Rhodotorula glutinis;
Casey GD &Sobson ADW Internal J Food Microbiol 2004:3, 327
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
Conventional PCR
Molecular Typing
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Conventional PCRReal-time PCRBroad-range PCR
Molecular typingMore for epidemiology Less for identification
Repetitive Sequence-based (REP)-PCR
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From Dr. Li at CDC
BSH II Restriction Endonuclease Analysis of Genomic DNA
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From Dr. Li at CDC
Multilocus Sequence Typing (MLST)
Diploid Sequence Type (DST)
Sequence of 6-7
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Sequence of 6-7 genes
From Dr. Li at CDC
Two Closely Related Fluconazole-Resistant Candida tropicalis Clones
Circulating in Taiwan from 1999 to 2006
140
Res
ista
nt
In both 1999 and 2006 surveys, 18 isolates of DST140 were isolated from 10 different hospitals localized in
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98 Res
ista
nt
different hospitals localized in all four geographic regions in Taiwan. 7 isolates of DST98 were also isolated from 4 different hospitals located in both north and south Taiwan.
MLST: Multilocus sequence type From Dr. Li at CDC
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
PCR
Microarrays
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PCRReal-time PCRBroad-range PCR
Molecular typingMicroarrays MultiplexingPartial or whole genome sequencing
Deep sequencing
Example of Microarray
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Leaw et al, J Clin Microbiol. 2007, 45:2220
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
PCR
Molecular Diagnosis Methods
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PCRReal-time PCRBroad-range PCR
Molecular typingMicroarrays MultiplexingPartial or whole genome sequencing
Deep sequencing
PLEX-ID: PCR/ESI-MS1.1.1.1.無需對樣本進行培養無需對樣本進行培養無需對樣本進行培養無需對樣本進行培養
2.2.2.2.可可可可測測測測對樣本中已知或未知的微生物對樣本中已知或未知的微生物對樣本中已知或未知的微生物對樣本中已知或未知的微生物
3.3.3.3.能把樣本中的基因組片段進行擴增能把樣本中的基因組片段進行擴增能把樣本中的基因組片段進行擴增能把樣本中的基因組片段進行擴增,,,,並對並對並對並對擴增產物的堿基組成進行分析擴增產物的堿基組成進行分析擴增產物的堿基組成進行分析擴增產物的堿基組成進行分析,,,,獨有的分析獨有的分析獨有的分析獨有的分析軟體可把檢測到的堿基組成于資料庫中的資軟體可把檢測到的堿基組成于資料庫中的資軟體可把檢測到的堿基組成于資料庫中的資軟體可把檢測到的堿基組成于資料庫中的資訊進行比對訊進行比對訊進行比對訊進行比對,,,,達到識別微生物的目的達到識別微生物的目的達到識別微生物的目的達到識別微生物的目的
4.4.4.4.資料庫中含有資料庫中含有資料庫中含有資料庫中含有75757575萬條微生物基因組擴增片萬條微生物基因組擴增片萬條微生物基因組擴增片萬條微生物基因組擴增片段堿基組成資訊段堿基組成資訊段堿基組成資訊段堿基組成資訊,,,,從而把擴增產物與特定微從而把擴增產物與特定微從而把擴增產物與特定微從而把擴增產物與特定微
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段堿基組成資訊段堿基組成資訊段堿基組成資訊段堿基組成資訊,,,,從而把擴增產物與特定微從而把擴增產物與特定微從而把擴增產物與特定微從而把擴增產物與特定微生物進行關聯生物進行關聯生物進行關聯生物進行關聯。。。。
5.5.5.5.可檢測細菌可檢測細菌可檢測細菌可檢測細菌、、、、病毒病毒病毒病毒、、、、真菌和原生動物真菌和原生動物真菌和原生動物真菌和原生動物,,,,並並並並鑒定微生物的基因型鑒定微生物的基因型鑒定微生物的基因型鑒定微生物的基因型,,,,檢測毒性標記和抗藥檢測毒性標記和抗藥檢測毒性標記和抗藥檢測毒性標記和抗藥性基因性基因性基因性基因
6.6.6.6.能識別單一樣本中的混合微生物能識別單一樣本中的混合微生物能識別單一樣本中的混合微生物能識別單一樣本中的混合微生物
PLEX-ID: PCR/ESI-MS
Microbial DNAsMicrobial DNAsMicrobial DNAsMicrobial DNAs
PCR by 16 sets of primersPCR by 16 sets of primersPCR by 16 sets of primersPCR by 16 sets of primers
MSMSMSMS
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DatabaseDatabaseDatabaseDatabase
IdentificationIdentificationIdentificationIdentification
微生物微生物微生物微生物
Broad Fungal6 Samples/96 Well PlateSet 1 Set 2
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S1 S2 S3 S5S4 S6 S1 S2 S3 S5S4 S6
Luminex 100 Total Systemwww.luminexcorp.com
Specific probes
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利用利用利用利用DNA DNA DNA DNA 之雙股雜交特性之雙股雜交特性之雙股雜交特性之雙股雜交特性,,,,一次偵測一次偵測一次偵測一次偵測>100 >100 >100 >100 個個個個DNADNADNADNA目標目標目標目標。。。。整合了螢光編碼微球整合了螢光編碼微球整合了螢光編碼微球整合了螢光編碼微球、、、、鐳射檢測鐳射檢測鐳射檢測鐳射檢測、、、、影用流體學影用流體學影用流體學影用流體學、、、、最新的高最新的高最新的高最新的高速數位信號和電腦運算法等多項技術速數位信號和電腦運算法等多項技術速數位信號和電腦運算法等多項技術速數位信號和電腦運算法等多項技術,,,,真正實現了真正實現了真正實現了真正實現了““““高通高通高通高通量量量量””””檢測檢測檢測檢測 ((((基因體研究中心育成中心基因體研究中心育成中心基因體研究中心育成中心基因體研究中心育成中心 ))))。。。。
Luminex 100 Total Systemwww.luminexcorp.com
Candida albicans
Candida tropicalis
Candida glabrata
Specimen 1 Specimen 2
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Candida glabrata
Candida parapsilosis
Candida krusei
Cryptococcus neoformans
Trichosporon asahiiC. albicans C. albicans
C. glabrata
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methodsPolymerase chain reaction
PCR
Deep Sequencing
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PCRReal-time PCRBroad-range PCR
Molecular typingMicroarrays MultiplexingPartial or whole genome sequencing
Deep sequencing
Number of times a nucleotide is read
Uni primer I - a
Deep Sequencing
Sample a Sample b Sample c
Microbial DNAs
Uni primer I - b Uni primer I - c
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Uni primer I - aUni primer II-a
Uni primer I - bUni primer II-b
Uni primer I - cUni primer II-c
Sequencing
Database
Identify
DNA Isolation from Lo’s Laboratory
1. ≧≧≧≧ 3 ml of Blood
2. Blood in tube with
104 102103 10 104 102103 10 + -
0.85% NaCl Blood
Cells
36
tube with EDTA
3. Removal of red andwhite blood cells
UNPOUBLISHED
Laboratories have historically relied on phenotypic methods(i.e., culture and biochemical tests)
Molecular diagnosis methods (EXPENSIVE and TECHNOLOGY )
Polymerase chain reaction
Molecular Diagnosis Methods
37
Polymerase chain reaction Molecular typingMicroarrays MultiplexingPartial or whole genome sequencing
Deep sequencing
50 um
Germ Tube Assay of TSARY CollectionDetects Mixed Yeast Cultures
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RPMI 10% serum at 37℃℃℃℃ for 3.5h
C. albicans C. albicansC. glabrata
C. albicansC. tropicalis
C. albicansC. parapsilosis
Bacterium
a b dc50 um
TSARY: Taiwan Surveillance of Antimicrobial Resistance of Yeasts
UNPOUBLISHED
Identification of Yeasts
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Excellent for detecting multiple species.Only for major species
For subjective identification.
Power of CHROMagar Candida
a
c
b
C. albicans C. glabrata
C. albicans C. tropicalis
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d
e
C. albicans C. parapsilosis
C. glabrataC. tropicalis
C. krusei C. tropicalis
Not good to patch cells on the mediumUNPOUBLISHED
Candida glabrata vs. Candida nivariensis
Color on CHROMagar medium
C. glabrata C. nivariensis
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C. nivariensis can ferment trehalose.
UNPOUBLISHED
C. nivariensis
C. glabrata
Cal
C. tropicalis
Cpa Cor
Limitations of CHROMagar Candida
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Cal: C. albicans; Ckr: C. krusei;Cme: C. metapsilosis; Cor: C. orthopsilosis; Cpa: C. parapsilosis
Ckr
Cme
UNPOUBLISHED
Laboratories have historically relied on phenotypic methods (i.e., culture and biochemical tests)
Molecular diagnosis methods
The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate
Molecular Diagnosis Methods
43
immunocompromised depends on rapid and accuratediagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types.
What Do We Need?
Broad identification of microbesRapid detection: culture often not requiredMixtures of microbes are detectedHigh-resolution genotyping and strain identificationNovel microbes can be detected
44
Novel microbes can be detectedDrug resistance testing without culture
ACCURATE, CHEAP, and RAPIDConventional methodsMolecular Methods
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