wm, westmead hospital, sydney, australia wieland meyer phd molecular mycology laboratory westmead...

WM, Westmead Hospital, Sydney, Australia

Wieland Meyer PhD

Molecular Mycology LaboratoryWestmead Hospital/The University of Sydney

Molecular Techniques for the Identification of

Pathogenic Fungi


Issues in Mycology Significant increase opportunistic fungal infections due to the

increase in immunocompromised hosts

Cosmopolitan environmental fungi have emerged as causes of potentially life-threatening diseases

Many emerging pathogenic fungi are inherently resistant to antifungal drugs

A number of pathogenic fungi are non-viable in tissue samples Traditional identification techniques lack sensitivity & specificity, are

slow, labor-intensive and require skilled personnel

Clinical and economic consequences


Conventional Fungal Identification Techniques

Identification Method Concerns

Culture ID - only in 26% positive if < 1cfu/ml blood

Serological Tests - limited value (e.g. Iatron Crypto Kit)

- suboptimal sensitivity & specificity

Germ-tube Formation - useful screening test only for C. albicans

(does not differentiate C. dubliniensis)

Carbohydrate Assimilation - limited species in the databases

(e.g. Vitek & API strips) - can misidentify certain pathogenic species

Morphological Characters - subjective measure,

- high degree of skills required


Fungal Identification is Currently Based on the Analysis of:

Morphological Characters Culture and Microscopy

Physiological/Biochemical Characters e.g. Vitek and API

These techniques are often time-consuming, labour intensive and difficult to interpret.


Results Obtained with Vitek-YBC and API

Vitek-YBC API ID32C

Total number of isolates tested 81 80

# of isolates included in the database 55 69

# of isolates correctly identified 48 (87.3%) 50 (76.8%)

# of isolates incorrectly identified 7 (12.7%) 16 (23.2%)


Culture-ID Blood cultures positive in only 20-58% of Invasive

Candidaisis Only in 26% positive if < 1cfu/ml blood, and 10% of Aspergillosis

Serological Tests e.g.:Iatron Cryptococcus Kit

95% accurate with Cryptococcus neoformans

Serotype specific antisera, problems AD strains

Iatron Candida Check Kit

95% accurate with Candida specific antisera

In general suboptimal sensitivity and specificity


Ideal ID/Diagnostic Test

- Sensitive and specific

- High positive predictive value

- High negative predictive value

- Useful for monitoring

- Simple, rapid and inexpensive


Why ?Confirmation of medical diagnosis, choice of therapy, follow-up and prevention are critical to a successful infectious disease management.

- Facilitate earlier diagnosis

- Initiating earlier intervention with aggressive

antifungal treatment to improve patient outcome

- Reduce empiric use of antifungal agents

How ?- Detection of fungal genomic sequences


Why Molecular Methods?

Phenotypic Characters are unstable and can change with environmental changes

Identification methods based on Genotypic Characteristics would be

advantageous and potentially more accurate, reproducible, simple and rapid


Proposed Applications of DNA Protocols

Organism detection in blood

Organism detection in body fluids

Organism detection in tissue

Identification of the fungal agent

Quantification of the fungal load

Monitoring of antifungal treatment

Molecular ID


DNA Technology

DNA probes– Southern Hybridization– In-situ Hybridization– Microarray– Macroarray– Reversed line blot

PCR primers– SSCP– Genotyping– Panfungal PCR– Multiplex PCR– Nested PCR– Real Time PCR– Sequencing– PCR Fingerprinting– AFLP– PCR-RFLP

Karyotyping


Samples used: Blood:Should be collected in tubes containing EDTA (1mg/ml)

!! To not use heparin as anticoagulant !!

Biopsy Tissue:

!! Fresh tissue is always better !!

Paraffin embedded tissue is not always amenable to PCR amplification because of DNA modification due to cross-linking induced by the

fixative or fixation time!

In addition time-dependent physical degradation of DNA in paraffin-embedded tissue limits the length of the DNA fragment amplified.


Targets for PCR, PCR-RFLP, Sequencing& In-situ Hybridization:

Universal fungal primers for:- Multi-copy Genes

rDNA gene cluster 18S, ITS1/2, 5.8S, 28S, 5S, IGS

- Single-copy Genes Actin, Alkaline Protease (ALP), Chitin Synthase,GP43, Lanosterol - - demethylase (LIA1), URA5, Secreted Aspartic Protease (SAP), Beta glucan synthetase (FKS), Histone, etc.

Genus- or species-specific primers 18S, ITS1/2, 28S rDNA, Mitochondrial DNA, Histone

e.g. Candida, Cryptococcus, Aspergillus


Tandem Repeat of the Ribosomal Gene Cluster

Vilgalis Lab, Duke University, NC, USA


1 = Cryptococcus neoformans

2 = Cryptococcus albidus

3 = Candida albicans

Mitchell et al. JCM 1994 32(1) 253-255

Species-Specific Amplification


PCR Amplification of Specific Genes

Clinical specimen Direct PCR of a single colony

form a primary isolation plate, tissue sample or clean culture

Agarose gel electrophoresis ID via comparison with the

data base

Species Level

ITS1, 5.8S, ITS2 region

Nicolas Latouche


Commercial Fungal Molecular Identification Kits

Fungal ID Kits for:

- Blastomyces dermatitidis

- Coccidioides immitis

- Histoplasma capsulatum

- Single-stranded DNA probe targeted to the ribosomal RNA

- Selection reagent differentiates between non-hybridized and hybridized probe

- Labelled DNA:RNA hybrids measured in a GEN-PROBE luminometer


Protocol of the Kits


Histone H3/H4 gene pair is arranged differently between yeast and humans

H3 H4

H3 H4

yeast

Human

MicroBioGenPrimers designed to amplify yeast H3-H4 region should not amplify human sequences

• Four members of histone gene family: H2A, H2B, H3, and H4• The genes are organized into two pairs of genes separated by divergent promoter regions• Histone genes are very highly conserved between species

Highly conserved coding region

Unique species-specific sequence

Highly conserved coding region

Product of amplification using Histone loci


S. cerevisiae cellsS. cerevisiae ID

Z. rouxii ID

K. m

arxianus ID

MicroBioGen

Nested PCR Histone locus

•use degenerate consensus primers to amplify unknown

•use species specific primers to do nested amplification

•use SYBR green to detect PCR product


PCR-RFLP(Restriction Fragment Length Polymorphism Analysis)

Clinical specimen Direct PCR of a single colony

form a primary isolation plate, tissue sample, or clean culture

Agarose gel electrophoresis Digestion with restriction

enzymes Agarose gel electrophoresis ID via comparison with the data

base

Species Level ITS1, 5.8S, ITS2 region

Nicolas Latouche


RFLP MAPSCandida albicans/dubliniensis

“Atlas of Clinical Fungi” 2nd Edition 2000by: GS de Hoog, J Guarro, J Gené & MJ Figueras

Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands

BioloMICS at: www.cbs.knaw.nl

by: V. Robert

Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands and BioAware, Belgium

Atlas of Clinical Fungi de Hoog et al 2000

RFLP maps available from:


Real Time PCR

ABI 7700 System (TaqMan) Reporter Dye and Quencher Probe Detection

Quantitative DNA and Species Detection e.g. Candida sp. detection Guiver et al. J. Clin. Pathol. 2001 54:362-366

Roche LightCycler SYBR Green Detection

Quantitative DNA Detectione.g. Pneumocystis carinii detection Kaiser et al. (2001) J. Microbiol. Meth. 45:113-118

Hybridization Probes (Donor Fluor and Acceptor Fluor) Detection

Quantitative DNA and Species Detection

e.g. C. albicans and A. fumigatus detection Loeffler et al. (2000) JCM 38:586-590

Diagnostics


LightCycler Candida Kit MGRADE

All necessary reagents for amplification and detection of Candida albicans

Targets the ITS region Wide range of biological specimens including swabs,

sputum, urine as well as blood cultures and isolated colonies

Detect Candida albicans in less than 3 hours using the PCR workflow system

Runs under a common thermal profile with other Microbiology specific kits eg. Enterococcus, Pseudomonas and Staphylococcus kits

Diagnostics


PCR-FingerprintingRAPD (Random Amplified Polymorphic DNA)

AFLP (Amplified Fragment Length Polymorphism)

Clinical specimen Pure culture DNA extraction PCR amplification with Mini- or Microsatellite specific primers Agarose gel electrophoresis ID via comparison with the data bank

Species and Strain LevelHomology

Primer Intra-species Inter-speciesM13 75-95% 5-25%

(GACA)4 74-94% 6-26%

(Data obtained from 80 Candida species and 150 strains)

Multilocus approaches:

Meyer et al. (1997) Electrophoresis, 18: 1548-1559

Primer: M13


PCR Fingerprinting Database Development

DNA extraction, PCR fingerprinting and gel running conditions standardized

Reference profiles - 70 anamorph-teleomorph pairs

- type cultures and clinical strains (>300 individual strains)

Pattern analysis via GelcomparII and

Integrated database/web access via

Collaboration is welcome: [email protected]

Heide-Marie Daniel/Krystyna Marszewska/Vincent Robert


Fungal ID via Sequencing Clinical specimen Direct PCR of a single colony from a primary isolation plate,

tissue sample or clean culture DNA extraction PCR amplification Sequencing ID via comparison with the EMBL or GenBank data bases

Species and Strain Level


rDNA gene cluster

18S rDNA or SSU 5.8S rDNA 28S rDNA or LSU 1800 bp 159 bp 3396 bp

IGS ITS 1 ITS 2 IGS 361 bp 231 bp

D1 D2 D3 D4/5 D6/7a/7b D8 D9/10 D11/12 V1/2 V3/4 V5 V7 V8 V9

SR1R SR6R/ITS1

5.8S/ITS2

ITS3 LR1/ITS4

LROR LR12LR16

ITS1

ITS2

LSU

5.8S

SSU

Sequence variability

Discriminatory power

ITS1

ITS2

5.8S

LSU

SSU

Heide-Marie Daniel/Wieland Meyer


Sequence Data Bases EMBL at: www.ebi.ac.uk

GenBank at: www.psc.edu/general/software/packages/genbank/genbank.html

BioloMICS at: www.cbs.knaw.nl

International European rRNA database (Candida)at: www.rrna.uia.ac.be.Isu

Sequence variation used for fungal ID

Sequence Analysis: LSU rDNA PLB1 URA5 Intra-species variation: 0 - 0.5% 0 - 0.8% 0 - 0.3% Inter-species variation: 0 - 17.5% 0.8 - 16% 0.8 - 15%

(Data obtained from sequences of 82 Candida species)

Heide-Marie Daniel/Nicolas Latouche/Stuart Jackson


Not all

fungal species

are

sequenced!!!

Candida species 18S ITS1 ITS2 28S

Candida castelii Y

Candida catenulata Y Y

Candida ciferri Y

Candida famata var.famata Y Y Y Y

Candida famata var. flareri Y Y Y Y

Candida (Torulopsis) glabrata Y Y Y Y

Candida guilliermondiiCandida humicola Y Y

Candida inconspicua Y

Candida intemedia Y

Candida kefyr Y

Candida krusei Y Y Y Y

Candida lambica Y Y

Candida lipolytica Y Y

Candida lusitaniae Y Y Y Y

Candida nitrativoransCandida norvegenisis Y Y

Candida norvegica Y

Candida parapsilosis Y Y Y Y

Candida (Torulopsis) pintolopesii Y Y Y Y

Candida pseudotropicalisCandida pulcherrima Y Y

Candida rugosa (var rugosa)Candida saitoana Y Y

Candida sake Y Y

Candida sphaericaCandida utilisCandida validaCandida viswanathii Y Y Y

Candida zeylanoides Y Y Y

Candida lusitaniae Y Y

e.g. GenBank 1.10.2002


MicroSeq® Workflow

or Sample from colony or pure culture

MicroSeq PCR module (1 reaction) < 2 hours

MicroSeq Cycle sequencing module (2 reactions) < 2 hours

MicroSeq analysis software and rDNA database < 30 minutes

Final identification report

PrepMan Ultra DNA isolation < 30 minutes


The Future

DNA Extraction Robot Real Time PCR Sequencing

Extraction Detection Identification


DNA Array Technology Small glass or silicon matrices on which potentially

thousands of short oligonucleotides can be immobilized

e.g. - species-specific DNA probes

- antifungal resistance genes

- virulence genes

Macroarray Microarray

C. glabrataC. parapsilopsisC. lipolyticaC. kruseiC. norvegensisC. lusitaniaeC. multigemmisC. tropicalisC. utilis

C. g

.

C. p

.

C. l

.

C. k

.

C. n

.

C. l

i.

C. m

.

C. t

.

C. u

.


PCR Methods for Fungal IDMethod Culture Pattern Reproducibility Identification Turn- Cost

Requirements Stability Level around Efficiency Time

PCR-Amplification single colony very good very good species 1 days good

of Specific Genes tissue sample

PCR-RFLP single colony very good very good species 2 days good

Real Time PCR single colony very good very good species 1-2 hours good

PCR-Fingerprinting pure culture very good very good species/strain 2 days good

(RAPD/AFLP) (RAPD: good-poor)

Sequencing single colony/ best best species/strain/ 2 days expensive

tissue sample single mutation

Reference Labs only! Average Medical Mycology Lab


PCR Assay Sensitivity

Multi-copy Genes:e.g. 18S rRNA 1-5 CFU

ITS 10 CFU/ml blood

Single-copy Genes:e.g. Actin 1-10 CFU

1.4- lanosterol demethylase 10-100 CFU/ml bloodHeat-shock protein 90 10-100 CFU


Role of Molecular Diagnosis for Candidiasis

- Blood / Serum

+ve PCR good

Consecutive +ve PCR very good

- ve PCR cannot justify ceasing therapy


Precautions for clinical PCR

Standard approaches for the three major phases of clinical PCR:

- Sample preparation- Target and probe selection- PCR and post-PCR analysis- Positive and negative controls

PCR contamination!!!


Pitfalls of DNA based Identification

PCR reaction; contamination risk

e.g. Qiagen DNA extraction columns contaminated with fungal DNA

Detection of false positives or false negatives

Disease causing agent or colonization


Sources of Contamination

Pre-PCR Contamination

No routine sample collection methods have been established for PCR-diagnosis

Contaminating DNA can originate from:

Any persons skin, hair, door handles, surfaces in the laboratory

Clinical equipment (maybe sterile but not DNA free)

Reagents Use only PCR grade!!

Taq polymerase (can contain procaryotic or eucaryotic DNA)

PCR components (e.g: gelatine, BSA)

PCR products from previous PCR reactions (e.g. if diagnostic PCR is repeatedly performed)


Floor Plan for a Clinical PCR Laboratory


Many in-house DNA based fungal ID techniques exist:

Problem all of them lack standardization

There is a lack of commercial interest to develop DNA based fungal ID systems, because of: - the limited market

- limited antifungal spectrum available

- high development costs

Commercially available DNA based ID kits exist only for:

- Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis

(AccuProbe Kits, Gen-Probe, USA)

- Candida albicans (Roche)

- Universal Fungal ID via Sequencing MicroSeq (Applied Biosystems)

In summary, consistently reliable, universally applicable and standardized methods for fungal ID are still to be established.


Acknowledgements

Molecular Mycology Laboratory Microbiogen/Macquarie University Krystyna Marszewska Dr. Phillip Bell

Mathew Huynh

Sarah Kidd CBS, The Netherlands

Dr. Nicolas G. Latouche Dr. Vincent Robert

Dr. Heide-Marie Daniel David Yarrow

Dr. Catriona L. Halliday


Molecular Mycology Reference Laboratory

Samples Should be Directed to:Westmead Hospital

ICPMRDarcy Road

Westmead, NSW 2145

Marked: Molecular Mycology Laboratory

Contact Persons: Dr. Wieland Meyer Ph.: 61-2-98456895Fax: 61-2-98915317E-mail: [email protected]

More Info soon at: www.usyd.edu.au/~cidm

wm, westmead hospital, sydney, australia wieland meyer phd molecular mycology laboratory westmead...

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