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Automated molecular testing of infectious diseases
Prof. Dr. Alexander H. Dalpke
Dept. for Infectious DiseasesMedical Microbiology and HygieneUniversity Hospital Heidelberg
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Disclaimer
I have received honoraria as a speaker from:
Becton Dickinson, Sanofi Pasteur
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Agenda
Automation in molecular testing
• General considerations
• 2 examples: Implementation at Dept. of Infectious Diseases, University Hospital Heidelberg
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Problems/Demands in Molecular Bacteriology
Advantages
• Very high sensitivity
• Specificity
• Flexibility (target gene selection)
• speed, turnaround time (TAT)
• Independency from culture
• Qualitative and quantitative
Disadvanteges
• Specific infrastructure
• Training of staff
• Limited automation
• Complexity of the assays
• Prone to contamination
• Targeted/directed diagnostics(mutants, variants?)
• „low“ throughput (?)
• Costs
limited availability of molecular diagnostics (24/7)
increased demand for timely, flexible molecular diagnostics
availability
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Degrees of automation
Extraction Amplification/DetectionMM
Extraction Amplification/DetectionMM
manual
Full automation„walk away“
partial/modularautomation
Extraction Amplification/DetectionMM
continuous/on demand/randomaccess
batching
Amplification/DetectionMM
Amplification/DetectionMM
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Automation: Where we are
Full automation
• platforms for high-throughput assays (HIV, HCV, HBV, CT/GC, HPV)
• POCT (closed systems)
• (Few systems that combine automation and flexibility)
Extraction + amplification: Partial automation
• Extraction robots for various throughput and materials
• Real-time PCR machines for IVD and in house assays
• In-house PCRs require increased manual handling for development, validation and quality management
Flexibility
han
ds-o
n tim
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Points to consider for automated moleculardetection
• Which is your patient population? Clinical need?
• Where to test? (Ward, core lab, molecular lab?) POCT?
• Experience in molecular test? (modular vs full automation)
• Throughput: low/intermed/high
• Multiple usage of one sample?
• Sample types (lysis efficacy, consistency)
• Flexibility vs. single-assay only (modular vs. full automation)
• Costs
• Integration into workflow, random access vs. batching?
• Technical „dependency“: backup strategy, service availability
• Quality controls: extraction, amplification controls? externalcontrols?
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Partial automationUser-specific combination of extraction + PCR automates
• Small sample series
• Multiple PCRs from one sample
• No integration, comparability of results?
Flexibility +++, hands-on-time +/-, costs +/-
+
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Modular automation
Defined combination of devices fromone company
• Mostly IVD assays
• Multiple assays
• Mostly for larger sample series
Flexibility +, hands-on-time ++
Modular Automation for IVD’s &
User Defined Protocols
QiaSymphony (Qiagen) : 3
modules
Cobas 4800 (Roche) : 2/3
modules
M2000 (Abbott)© ESCMID eLibrary b
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Full automation I
Vivalytic, Bosch
Idylla, Biocartis
Closed test systems
• IVD assays
• Multiple assays
• Individual samples
• Walk-away
• Dependency on the seller‘s assay panel, (e.g. multiplex composition)
Flexibility -, hands-on-time +++,
Costs: often high/sample
Cobas Liat, Roche© ESCMID eLibrary by a
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Alere q Aries, Luminex
BD MAX, BD
Cepheid, GeneXPert
GenMarkDx, EplexFilmarray Biofire
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Full automation II
Closed test systems
• IVD assays
• Few assays
• Large sample series
• Walk-away
• Dependency on the seller‘s assaypanel
Flexibility -, hands-on-time +++
Hologic
Roche
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University Hospital Heidelberg: Implementation of automation
in house PCRs
Commercial PCRs
QiaSymphony
manual NA extraction
Pre-treatment
Light Cycler 480
qTowerABI 7900
FlexCycler
BD ProbeTec
Smart CyclerGeneXPert
nested PCRs
post PCR Hybridisation
Sequencing
Nimbus
CFX96
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Philippe Halsman: The Frenchman – a photographic interview with Fernandel, Taschen Verlag, 2005
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Automation I: Strategic decision for a mid-size automated platform
Needs: in house & IVD, small sample size for most assays (<20), different assays, increased availabilty, labor reduction
• Implementation of automated molecular dx
– Commercial assays > Business plan
– In house assay > flexibility
– Easy to use > not restricted to molecular technicians , 24/7
– Closed > Placement outside molecular lab
– Full automation > reduced hands-on time, cost savings
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BD MAXTM: fully automated but flexibel
DNA extractor
pipetting robot
Real-time PCR, 5 colours
2x12 samplesDNA/RNA
extraction,
automated
Microfluidics PCR
2x24(12), single
lane
pipettor
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IVD Assays:MRSA
Is there a benefit for full automation?
Idea: MRSA BD MAX and BD GeneOhm ACP assays are comparable in chemistry but run on two different platforms:
BD MAX vs. Smart Cycler© ESCMID eLibrary by a
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Resulta
Assay TP FP FN TN Sensitivity
[%]b
Specificity
[%]b
PPV
[%]b
NPV
[%]b
BD MAX MRSA 31 6 2 766 93.9
[79.8;99.3]
99.2
[98.3;99.7]
83.8
[68.0;93.8]
99.7
[99.1;100]
BD GeneOhm
MRSA ACPc
30 13 2 755 93.8
[79.2;99.2]
98.3
[97.1;99.1]
69.8
[53.9;82.8]
99.7
[99.1;100] 1
Automation shows superior performance
Fully-automated versus partial-automated PCR
• Slightly better specifity (99.2%)
• good positive predictive value (PPV) in a low prevalence cohort (4.1%)
• very good negative predictive value (NPV)
• Lower failure rate: 1.2% vs. 4.2% UNR
J Clin Microbiol (2012), 50:3365-3367.
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Workflow GeneOhm vs. BD MAXStreamlining the process!
8 Samples
GeneOhm (Smart cycler)
MRSA (BD Max)
Prep Extr./Amplification/Detection Evaluation
hands-on: 15min
total: 1h55min
hands-on: 30min
total: 1h50min
Prep/Extr Amplification/Detection Evaluation
LIS reporting through
unidirectional interface
no molecular expertise necessaryclosed system > no demands for special infrastructure
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IVD Assays:C.diff
How do two fullyautomated PCR systems compare?
Idea: Compare BD MAX and Cepheid GeneXPert system for detection of C. difficile
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• no significant differences between BD MAX and GeneXpert
• Automated PCR vs EIA– increase in sensitivity
– increase in specificity
– (despite of optimized sample transport, pneumatic tube system)
Automated PCR to improve diagnostic quality
PCR as one-step testing for C.difficileBD Max/GeneXPert/Vidas/culture
J Clin Microbiol 2013, 51:1906-1908
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In house assays
Can the BD MAX be used flexibly for in housePCRs?
Idea: Develop and evaluate BD MAX user-developed protocol (UDP) for detection of P. jirovecii and compare against “manual” PCR
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Pneumocystis jirovecii:why automated molecular diagnostic might have advantages
• detection has direct therapeutic consequence rapid diagnostics 24/7
• molecular detection is superior to IFT, yet quantificationnecessary real-time PCR
PCR
negativ positiv gw <1e2
IFT negativ 108 4 6 118
positiv 1 9 4 14
109 13 10
clinical neg. 4/4 clinical pos. 2/6 questionable
http://www.ppdictionary.com/mycology/jiroveci.htm
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Prospective comparative study, N=278
• Discrepancies:– BD MAX neg/Chromo pos (5)
• 3/5 < 3 log10 copies/ml• 1 possible case• 1 case, in repeat analysis of another sample: pos. by BD MAX
– BD MAX pos/Chromo neg (11)• 2.34 – 5.56 log10 copies/ml• 3 cases, 6 possible cases of Pneumocystis infection; 3 patients pos. by
Chromo in diff. samples
BD MAX
pos neg/unr
Chromo(manual)
pos/borderline 35 5
neg 11 227
J Clin Microbiol (2013), 51:2337-2343
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BD MAX evolution: Avoid pipetting
liquid PCR mastermix,
manual pipetting
pre-aliquoting, ready-to-use
commercial PCR enzymecommercial, ready-to-use primer
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Automation II: Stool diagnostics
• Culture: Laborious, different media, different incubationtimes&conditions, time-to-result>48h
• Molecular diagnostics:
– Panel covering the relevant bacteria (incl. C.diff > replacement of different media and single plex C.diff PCR)
– Same-day result
– Batches of 20-50 samples, 1x/d
– Flexible use of the system for other assays
– Cost effectiveness
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Molecular stool diagnostics
• Since november 2017
• Assay: Seegene, AllplexTM GI-Bacteria(I) Assay
• Mo-Fr: Cam/Sal/Shi/Yer detection is followed by culture
• C.diff ‚only‘: BD MAX, weekends
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Modular automation
Extractor+pipettor, flexible
PCR, different assays
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Results automated molecular stool diagnostics
Molecular: Nov 17-June 18, N=5032Vs.Culture: Nov 16-June 17, N=4173
Comparable Germany-wideepidemiology for Ca,Sa,Sh,Yer(2017 vs 2018)
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Superiority to culture
Nov 17-June 18, N=5032
Pos. (N) Confirmed by culture
C. difficile toxin B 422 n.d.
Aeromonas spp. 146 n.d.
Campylobacter spp. 113 63 (56%)
Salmonella spp. 23 14 (61%)
Shigella spp/EIEC 21 3 (14%)
Vibrio spp 7 4 (57%)
Y. enterocolitica 4 4/4 (100%)
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Automated molecular stool diagnostics: Monetary evaluation
• Replacement of culture by multiplex PCR
• Partial replacement of C.diff PCR (included in multiplex panel)
• Secondary culture only upon pos. PCR result
6.11.16-15.7.17 (culture) 6.11.17-15.7.18 (PCR)N € N €
Culture 4173 11,267.10 168 453.60
C.diff singleplex PCR 4141 63,978.45 938 14,492.10
Multiplex stool PCR 0.00 5032 71,303.44
sum 75,245.55 86,249.14increase costs 114.62%*
*cmp to increased no. of requests
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Summary
• Different solutions for automated moleculardiagnostics of infectious diseases are available
– Modular automation
– Full automation
• Technical solutions are meanwhile robust andreliable
• Implementation of automation needs to considerlab-specific demands
– automation/labor savings vs. flexibility
– workflow
– througput
– availabilty
– backup, redundancy
flexibility
auto
matio
n
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Ursus Wehrli: TIDYING UP ARTPublished by Kein & Aber
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Thanks to
Dept. of Infectious Diseases, U Heidelberg
Marjeta Hofko
Paul Schnitzler
Stefan Zimmermann
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References
• Photos of diagnostic systems: website of the respectivecompanies
• Photos of the laboratory: Dept. of Infectious Diseases, Med. Microbiology
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The molecular diagnostics market
• 20072012 increase from $1.7 billion to $5.4 billions for molecular IVD tests worldwide
• 40% growth/y • Trend to develop broad diagnostic platforms (infectious diseases
> oncology, therapy management, pathology)
Assays• Leading assays: HIV, HCV, HBV, CT/GC (& HPV)
Country specific differences:• Molecular tests: GER 40% of all labs (all universities, 30% in
smaller hospitals and 50% in private labs), UK 80%, Netherlands70%, France 25%, Sweden 20%
• GER, France: more in house tests, UK: mostly IVD © ESCMID eLibrary by a
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Full lab automation: Bacteriology Heidelberg (07/2016)
12h/day (+ on call), 7d/week, 365d/year, Technicians: 23FTE, Doctors: 5 microbiologists, 600-1000 new specimens/day (bacteriology only)
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• MRSA IVD (with ESwabs)– J Clin Microbiol 2012, 50:
3365-3367
• C. diff IVD– J Clin Microbiol 2013, 51:
1906-1908
• Pneumocystis jirovecii, UDP– J Clin Microbiol 2013, 51:
2337-2343
• EHEC from culture, UDP– DGHM 2013
• Pertussis (Diagenode), thirdparty assay
• GeneOhm VanR on BD MAX– ASM2014
• VRE BD MAX, UDP– J. Clin. Microbiol. 2016, 54(9): 2321-2329
• Carba assay, UDP– J Clin Microbiol 2014, 52(5):
1701-4
• StaphSR/MRSA XT, IVD– with different swabs
• J Clin Microbiol 2014, 52(12): 4343-6– use with blood cultures
• J. Clin. Microbiol. 2015, 53(11): 3630-2
• cps/S. pneumoniae, UDP
BD MAX in Heidelberg
3 devices (2x outside molecular lab)
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Room situation
PC
R M
aste
rMix
Sample prep (A)
Extraction (B)
PCR completion (C)
(B)
(B) (A,B)
PC
R/
Am
plif
icat
ion
Po
st-P
CR
/
Dee
tect
ion
(A)-
(C),
Au
tom
atsPC
R M
M11 22 34
(C)
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Bacteriology University Heidelberg
• Borrelia burgdorferi
• Chlamydia pneumoniae
• Chlamydia trachomatis
• Legionella pneumophila
• Mycoplasma pneumoniae
• Mycobacterium tuberculosis, NTM
• Neisseria gonorrhoeae
• Pneumocystis jirovecii
• Universal fungal/bacterial PCR + sequencing
• Carbapenemases (Enterobacteria)
• MRSA (screening, confirmation)
• VRE
• EHEC/STEC
• Enterococci: esp/hyl
• Enterotoxins/TSST+ S.aureus
• PVL+ S. aureus
Identification
Resis
tence
Virule
nce
Devices
in house (60%)
•QiaSymphony
•qTower3(Jena Analytic)
•Light Cycler 480 (Roche)
•Smart Cycler
mainly real-time PCR
with hydrolysis probes
commercially (40%)
BD ProbeTec
Smart Cycler
GeneXpert© ESCMID eLibrary by a
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•sample buffer tube, snap in tubes
for extraction and PCR reagents
(colour coded)
•simple handling: squeeze swab in
SBT and vortex
•no molecular expertise necessary
•closed system > no demands for
special infrastructure
BD MAXTM MRSA Assay
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BD MAXTM: Microfluidic-amplification
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