author by automated molecular testing of infectious

Automated molecular testing of infectious diseases

Prof. Dr. Alexander H. Dalpke

Dept. for Infectious DiseasesMedical Microbiology and HygieneUniversity Hospital Heidelberg

© ESCMID eLibrary by a

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Heidelberg University Hospital

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


manual

Full automation„walk away“

partial/modularautomation


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

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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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http://en.wikipedia.org/wiki/File:Computer-aj_aj_ashton_01.svg


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

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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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