current applications testing specimens from antibiotic pre ... · professor of pathology at the ......

Current Applications of Real-time PCR technology in Diagnostic Bacteriology Dr. Udo Reischl Institute of Medical Microbiology and Hygiene Un i ve r s i t y Ho s p i t a l o f Re gen s b ur g Regensburg, Germany

Reasonable Indications for requesting PCR-based tests

When the highly desired same-day-results can not be obtained by conventional diagnostic procedures (i.e., microscopy, serology, antigen detection)

and/or: testing specimens from antibiotic pre-treated patients

when molecular detect ion of known resistance genes or pathogenicity factors contribute to the reliability of diagnostic reports in the case of unclear phenotypic results

for rapid epidemiological strain typing (i.e., MLST)

Reischl, U., Indikationen für die molekulare Diagnostik - Bakterien, Pilze, Eukaryonten. In:Leitfaden Molekulare Diagnostik (Thiemann, Cullen, Klein, Edts.), Wiley-VCH, Weinheim, 2006, pp.175-183.

Antibody- based assays ( e . g . , E L I S A , W e s t e r n B l o t )

Principles of Molecular Diagnostics

Specific detection of nucleic acids

(e.g., hybridization)

In vitro amplification of nucleic acids (e.g., PCR, NASBA)

DNA RNA

Pathogen

D N A RNA

Pr o t e i n

Automated DNA sequencing

AG CT

Culture, microscopy,biochem. differentiation,susceptibility testing Agglutination tests

Computer-assistedcomparison with known

microbial DNA sequences(GenBank, SmartGeneTM)

1989

Phenol / Chloroform / EtOH

1993Affinitäts-Säulchen

1999

2005AutomatisierteDNA/ RNA Extraktion

C l o s e d Cartridge 2009

Pyro-sequencing

MALDI-TOF SBH

In vitro Amplification DNA-SequencingSample preparation

Technological Evolution

Added value

Speed

U. Reischl/RIMMH/03.2007

of traditional block cycler PCR:

Melting curve analysis Option for Nested-PCR Quantitative results Multiplexing (?)

Real-time PCR in Diagnostic Microbiology

by using Real-time PCR instead

Evolution of the Real-Time PCR Platforms or how technology meets the various demands of molecular diagnostic laboratories... one uniform thermocycle protocol fits

to the majority of our PCR assays

H. pylori EHECL. p,eumophila B. pertussis C. p,eumo,iae C. albica,s

InnovativeConcept Commo, Protocol

U. Reischl /RIMMH/05.2004

The remarkably creativegenius behind

real-time PCR technology:

Real-time PCR

Rapid Cycle Real-time PCR

Current Spectrum of Real-Time PCR Platforms

Carl T. Wittwer Professor of Pathology at the

University of Utah Medical School and Director of Flow Cytometry and the Advanced Technology Group at ARUP, Salt Lake City.

HybridizationProbes

AACC Hall of Fame

Melting Curve Analysis

High Resolution Melting Analysis


TaqMan:

HSV-1,2

Virus

Adeno

HHV-6

Parvo

EBV

CMV

HBV

HCV

HDV

HEV

VZV

HIV

BKV

JCV

Nocardia spp. Pseudomonas aeruginosaStreptococcus pneumoniaeStrep. pyogenes (A-Strep)

Haemophilus influenzae Legionella pneumophila Moraxella catharrhalis Mycobact. tuberculosis Mycoplasma pneumoniae

Bordetella pertussis Bordetella parapertussis Chlamydia pneumoniae Chlamydia psittaci Corynebact. diphtheriae

Our PCR-Assay Portfolio

EHEC, ETEC, EPEC, EIEC Salmonella enterica H e l i c o b a c t e r p y l o r i S. aureus Enterotoxine Yersinia enterocolitica Campylobacter spp. Clostridium difficile Toxin Chlamydia trachomatis Strep. agalactiae (B-Strep)

Neisseria gonorrhoeae Borrelia burgdorferi Neisseria meningitidis Tropheryma whipplei

Treoponema pallidum

Bacteria

Coxiella burnetii (Q-Fieber)

Clarithroymcin Resistance

Vancomycin Resist. (VRE)

Bacteria spp. (16S rDNA)

Listeria monocytogenes

Rifampicin Resistance

Methicillin Resistance

Staph. aureus / mecA

Isoniazid Resistance

Bartonella henselae Brucella melitensis

Bacillus anthracis

Leptospira spp.

Yersinia pestis Trichophyton verrucosum

Blastomyces dermatitidis

Histoplasma capsulatum

Paracoccidioides brasil.

Aspergillus fumigatus

Fungi spp. (28S rDNA)

Coccidioides immitis

Microsporum canis

Cryptococcus spp.

Candida albicans

Fungi

s t a t u s March 2009U. Reischl /RIMMH/03.2009

Cryptosporidium spp.

Pneumocystis carinii

Acanthamoeba spp.

Toxoplasma gondiiPlasmodium spp.

Protozoeae

Entamoeba spp.

Leishmania spp.

Highly specific

Rapid (<1 h)

Closed system

Real-time PCR Hybridization Probes

Evaluation of Diagnostic Protocols in Clinical Bacteriology

FRET: fluorescence resonance energy transfer

Pre-a,alyticalworkup

ClinicalSpecimens(Respiratory andnon-respirartory)

NAA - Diagnostic Workflow

Enzymatic(Lysozyme, Prot.K)

Physical (Heat, Sonication)

Sample preparatio,

Chemical(Alkaline Lysis)

SDA (Becton Dickinson)

TMA (bioMerieux, Hain)

DNA / RNA amplificatio,

PCR(Roche)

Real-time PCR (fluorescent probes)

ELISA (sequence specific)

Line-Blot (sequence specific)

Agarose-Gel(size specific)

Amplico, detectio,

... we ususally encounter a broad spectrum of clincal specimens:

� Tissue (solid or soft tissue biopsies) � Swabs (nasal swabs, wound swabs, rectal swabs) � Blood (whole blood, blood culture, serum)

� Bone marrow aspirate � Respiratory specimens (sputum, BAL, tracheal aspirate)

� Cerebrospinal fluid (CSF)

� Gastric juice aspirate � Stool � Urine � Others ...

Sample Preparation Issues

U. Reischl /RIMMH/09.00

PCR-Dipstick-Test

Block Cycler PCR

in-house ELOSA

Real-time PCR

S: Sample preparation; AMP: Amplification; DP: Detection; INT: Interpretation of results

PCR Workflow Assessment-Amplification / Detection -

S

S A M P D P

S

S

1 2 3 4 5 6 7 8 hours

same day results next day results

AMP DP

AMP

AMP

DP

time-to-result

DP

automated p h y s i c a l manipulation

Pathogen- specific qualitative or quantitative

Real-time PCR assays

Clinical specimens

Economic Sample Preparation


total

DNAa n d / o r

RNA

Prot. K Incubation

M anu al or

Automated

DNA / RNAPreparation

5 C c e s o f 5 Zyklen vo boil & freeze b o i & f r e e z e

- Efficient- Simple - Rapid

- Non-

infectious

Reischl, U., Pulz, M , Ehret, W and Wolf, H (1994) PCR-based detection of mycobacteria insputum samples using a simpleand reliable DNA extractionprotocol BioTechniques 17,

844-846

- 196°C

Rational design of primers und real-time PCR hybridization probes:

Reischl, U., N. Lehn, G.N. Sanden, and M. Löf felholz (2001) Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii. J. Clin. Microbiol. 39, 1963-1966.

RepetitiveInsertion-

sequenceIS481


- Bordetella pertussis -

Benefits of "Same Day Results" in Diagnostic Microbiologyor how rapid testing and rapid results can impact patient management...

Mikrobiology vs. PCR:

Throat swab

Nasopharyngealswab

Bordetella pertussis - Diagnostic Workflow

DNA- Preparation

DiagnosticCulture

( gold standard)

SerologySpecific IgG, IgM, IgA

antibodies (> day 14 of disease)

Microscopy( G r a m s t a i n )


Prerequisites: -Calcium alginate swab -Catarrhal stage (< day 14)

-Modified Bordet-Gengou medium - Charcoal-horseblood agar (Regan-Lowe)

- BCYE agar

day 0

Gram +/ - � rods / cocci

low sensitivity and specificity

Serology � often

inconclusive sensitivity < 50%

PCR highly sensitive

and very specific

Sensitiviy less than 50%

when positive

day 2-4 day 4-10

Biochemical testse.g., urease / oxidase

& susceptibility testing

Speciesidentification

Antibiogram


u r e as e

ox i d as e

Culture result

B. parapertussisPCR B. pertussis PCR

- Bordetella pertussis / parapertussis - Comparative study on diagnostic culture vs. PCR-based detection of

B. pertussis & B. parapertussis DNA:

I N S T A N D - R e i > W v K _ 0 8 / 0 4 - 0 4



No. of patients (n = 208)

Positive Negative Positive NegativePositive )20010 0

20 0 Negative b) 2 4 1 6 4 8

190

a) Includes 2 patients whose samples were both positive by PCR and culture for B. pertussis and B. parapertussis. b) Includes 2 patients whose samples were both positive by PCR and negative by culture for B. pertussis and B. parapertussis.

Microscopy (Gram stain)

Blood agar +ABDiscs

day 0

Mannitol Salt agar

Oxacillin Mannitol Bouillon

MRSA PCR

Staphylococcus PCR

mecA PCR

ß-Globin PCR

Coagulase/MRSAAgglut.-test

MRSA - Diagnostic Workup


Catalase Prod.

Oxacillin Screen plate

S. aureus

MH-Agar +ABDiscs

day 1

neg. 3 h

MRSA / MSSA

Tubed coagulase

Reading of AB Discs& Oxa screening plate

Antibiogram

day 2

Decisive winning marginwith respect to timelyinitiation of selective

and efficient isolation measures by

the availability of "same day results"

from clinical specimens

MRSA Direct detection of

Assay principles and diagnostic target genes.

Current spectrum of PCR assaysfor direct detection of MRSA

U Reischl /R IMMH/02 99

S. aureusCoNS mecA

Please note: Analytical sensitivity is mainly determined by sample lysis.

Separate Detection of:

PCR-based Direct Testing for MRSA (Benefit of same-day-results)

Flexible assay design: nuc, pSA422, coa, 16S rDNA, ITS, etc., can be used as molecular species markers...

PCR results 'not interpretable' when S. aureus, CoNS and a mecA gene are present simultaneously.

Occasionally false-negative results in the presence of 'rare' coagulase-negative Staphylococci. Several PCR reactions have to be performed.

'Relaxed' patent situation with target genes.

Please note: Analytical sensitivity is mainly determined by sample lysis.U. Reischl/RIMMH/09.2008

Assay principles and diagnostic target genes.


SCCmec S. aureus genome

2002 We introduced PCR-based MRSA screening for patients at risk.



SCCmec types


at least 6'different'SCCmectypes...


SCCmec-based PCR assays

Single PCR reaction with yes or no result for MRSA.

Growing number of heterogeneous SCCmec-types.

S. aureus isolates may harbor a SCCmec cassette with deleted or mutated mecA gene (> false-positive results).

'Narrow' patent situation with SCCmec target gene.


2007


GeneXpert MRSA Lab in a

cartridge

POCT

"Lab in a Cartridge"

GeneXpertMRSA

Lab in a cartridge

POCT

POCT in Medical Microbiology


GeneXpertMRSA

Lab in a cartridge

POCT

This concept seems to work

in cinical practice !!

POCT in Medical Microbiology

GeneXpertMRSA

GeneXpert MRSA

BD

i,,-house

HAIN

QC-Report April 2008

Reischl / Linde/ Wolf

I N S T A N D

R e i 0 9 . 2 0 0 9

GeneXpert MRSA

cMRSAPVL-

positive S. aureusIsolates

Ring Trials Bacterial Genome Detection status 09.2008 Reischl / Linde / Straube / Maaß/ Jacobs/ Wolf

Bi-annual QC since April 2003: BACTERIAL GENOME DETECTION

PCR-/NAT C. trachoeatis& N. go,,o,rhoeae RV 530, March 2004

BACTERIAL GENOME DETECTION PCR-/NAT Cfllae3dia trachoeatis

RV 531, April 2003

BACTERIAL GENOME DETECTION PCR-/NAT Bo,detella pertussis

RV 533, April 2003

BACTERIAL GENOME DETECTION PCR-/NAT Heli4oba40er pØori

RV 535, April 2003

BACTERIAL GENOME DETECTION PCR-/NAT EHEC / STEC

RV 537, April 2003

BACTERIAL GENOME DETECTION PCR-/NAT 2o11elia burgdo,feri

RV 535, April 2003

Regensburg

BACTERIAL GENOME DETECTIONPCR-/NAT M39oplasea p,,eueo,,iae

RV 541, April 2008

BACTERIAL GENOME DETECTION PCR-/NAT Legio,,ella p,,eu..opJ,ila

RV 536, March 2004 BACTERIAL GENOME DETECTION

PCR-/NAT Sal0o,,ella e,,terica RV 537, March 2004

BACTERIAL GENOME DETECTION PCR-/NAT MRSA / cMRSA

RV 539, March 2006

BACTERIAL GENOME DETECTION PCR-/NAT Cfllae3dia p,,eu0o,,iae

RV 540, March 2006

Ne* in 2008:

BACTERIAL GENOME DETECTION PCR-/NAT Lis0eria spp. RV 538, March 2004

I N S T A N D - R e i 0 9 . 2 0 0 8

External Q u a l i t y -C o n t r o l PCR / NAT

133.-€ /set


2 3 4

1

Institut für Medizinische Mikrobiologie und HygieneUniversi tät Regensburg, FJS-Al lee 11, 93053 Regensburg

http://***.udo-reischl.de

I N S T A N D e. V.Gesellschaft zur Förderung der qualitätssicherung

in medizinischen Laboratorien e.V.ht tp: / / *** . instand-ev.de cMRSA

High Troughput Real-time PCR Testing For screening purposes.

LC480


100 µl

384-well96-well

20 µl5 µl

Detection of the 25 most important pathogens found in blood stream infections Identification of 20 groups relevant for decision making Covering approx. 90 % of the nosocomial blood stream infections

DNA from the following bacterial and fungal species can be detected and indentifiedby the Roche LightCycler® SeptiFast® test within a timeframe of 6 h:

Species Coverage of the Roche SeptiFast5 Test



cMRS A-U R 12 ( lu kFS Ass ay)

High throughput screening of S. aureus strains for the lukFS gene.

LightCylcer 480 PCR

M a g N A P u r e L C

time-to-result < 2 hfor

up to 384 samples !!

- lukFS real-time PCR assay (LC 480) -


Template DNA of S. aureus

strains

C8

H9

C9posit iv e c o n t r o l (CA- MRSA)

90 PVL-negative S. aureus strains

N e g . c o n t r o l

Reference: Reischl et al.,

CM ID (2007) 26:131-135

lukFS (PVL) in-house

LightCycler assay(Regensburg)

Tm~ 59°C ( � l u k F S )

Tm-analysis [F2]


cultureds t r a i n s

Multiplex and multi-channel concept:

Gram-positives Gram-negatives

Concept of the Roche SeptiFast5 Test


LightCycler® 2.0 instrument (CE-IVD):


Rapid and sensitive detection of bacterial and fungal sepsis from EDTA blood samples.

Roche

SEPTI Fast5

Im SeptiFast Test konnte zwar ein ähnlicher Prozentsatz grampositiver Erreger wie inder Blutkultur nachgewiesen werdenjedoch ist der Anteil Koagulase-negativer Staphylokokken (CoNS) geringer und es findet sich mehr Enterococcus faecium.

Desweiteren zeigt sich die bessere Identifikation von Pilzen.

Ergebnisse der Evaluierung:

Evaluierung diagnostischer PCR-Protokollein der Klinischen Bakteriologie


current applications testing specimens from antibiotic pre ... · professor of pathology at the ......

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