molecular diagnosis of fungal pathogens

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


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.


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



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



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



PCR

Microarrays

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PCRReal-time PCRBroad-range PCR


Deep sequencing

Example of Microarray

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Leaw et al, J Clin Microbiol. 2007, 45:2220



PCR

Molecular Diagnosis Methods

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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



PCR

Deep Sequencing

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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

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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


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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


Molecular diagnosis methods

The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate


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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

molecular diagnosis of fungal pathogens

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