Current Applications testing specimens from antibiotic pre ... · Professor of Pathology at the ......
Transcript of 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
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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
U. Reischl/RIMMH/03.2007
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
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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
Evaluation of Diagnostic Protocols in Clinical Bacteriology
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
Evaluation of Diagnostic Protocols in Clinical Bacteriology
- 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 )
Evaluation of Diagnostic Protocols in Clinical Bacteriology
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
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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
Evaluation of Diagnostic Protocols in Clinical Bacteriology
U. Reischl/RIMMH/08.2004
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
Evaluation of Diagnostic Protocols in Clinical Bacteriology
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.
Current spectrum of PCR assaysfor direct detection of MRSA
SCCmec S. aureus genome
2002 We introduced PCR-based MRSA screening for patients at risk.
Current spectrum of PCR assaysfor direct detection of MRSA
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SCCmec types
PCR-based Direct Testing for MRSA (Benefit of same-day-results)
at least 6'different'SCCmectypes...
Current spectrum of PCR assaysfor direct detection of MRSA
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.
PCR-based Direct Testing for MRSA (Benefit of same-day-results)
2007
Current spectrum of PCR assaysfor direct detection of MRSA
GeneXpert MRSA Lab in a
cartridge
POCT
"Lab in a Cartridge"
GeneXpertMRSA
Lab in a cartridge
POCT
POCT in Medical Microbiology
U. Reischl/RIMMH/04.2007
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
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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
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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
Evaluation of Diagnostic Protocols in Clinical Bacteriology
U. Reischl/RIMMH/03.2007
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) -
Evaluation of Diagnostic Protocols in Clinical Bacteriology
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]
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cultureds t r a i n s
Multiplex and multi-channel concept:
Gram-positives Gram-negatives
Concept of the Roche SeptiFast5 Test
Evaluation of Diagnostic Protocols in Clinical Bacteriology
LightCycler® 2.0 instrument (CE-IVD):
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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
U. Reischl/RIMMH/10.2007