e-mail [email protected] Laboratory Diagnostics ... · always a drop ahead. Introduction . Stephan Ölschläger altona Diagnostics GmbH, Hamburg, Germany (since April 2013)

altona Diagnostics GmbH Mörkenstr. 12 22767 Hamburg Germany phone +49 40 548 06 76 - 0 fax +49 40 548 06 76 - 10 e-mail [email protected]

February 25th 2015

Laboratory Diagnostics of Filovirus Infections

always a drop ahead.

Introduction

Stephan Ölschläger altona Diagnostics GmbH, Hamburg, Germany (since April 2013) Emerging and tropical diseases (MERS-CoV, Filovirus, RVFV, Dengue

Type, Zika virus, …) At altona we currently only work with real-time PCR technology!

always a drop ahead.

What is your role in your institution?

Who is responsible for laboratory diagnostics? Who is responsible for treatment? Who will advice public health measures?

always a drop ahead.

How does the ideal diagnostic tool look like? Sensitivity high Specificity 100% Easy-to-use Fast < 30 min. Cheap Internal control Quantitative No cool chain required No power supply needed

RELIABLE!

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Basic Considerations on the Workflow

1) Biosafety Protect yourself and others!

2) Reliability

Use the correct method! Prevent contamination and wrong results!

3) Efficiency

Deliver results quickly!

Importance

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Equipment and Methods

Nucleic acid extraction: Manual extraction

More “hands-on” time The reference methods are usually very good! Flexible up to a certain extend (input and output volumes, sample

material) Validate in combination with PCR assay!

Automation

Good and bad systems out there! Closed systems

Is it possible to include internal controls? Expensive Laboratory footprint Needs to be validated for every assay!

always a drop ahead.

Sample Inactivation

Trizol LS Agent AVL

Buffer : Sample 4 : 1

3:1 Buffer : sample

AT LEAST!

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Equipment and Methods

First-pick real-time Cycler:

Mx 3005P™ QPCR System (Stratagene) VERSANT™ kPCR Molecular System AD (Siemens)

ABI Prism® 7500 SDS and 7500 Fast SDS (Lifetechnologies)

LightCycler® 480 Instrument II (Roche)

Rotor-Gene™ 3000/6000 (Corbett) Rotor-Gene™ Q 5/6 plex Platform (QIAGEN)

CFX96™/Dx Real-Time System (BioRad) CFX96 Deep Well™ Real-Time System (BioRad)

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Equipment and Methods

2nd choice real-time Cycler:

PikoReal Real-Time PCR System StepOne™

SmartCycler® II

LightCycler® 2.0 Instrument

Inter-module variation - Glass capillaries

- Discontinued

Optical systems / multiplexing

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Organisation of the Laboratories

Kitchen Office Office Office

Sample Prep

PCR Set-up II

PCR Set-up I Amplification

Negative Room

Oligo production

Oligo production

Storage

Entrance Color code: Red: infectious AND/OR highly positive Green: no DNA/RNA Orange: low positive

Example – altona Diagnostics Access

restricted

Prevention of contamination!

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Diagnostic Scenarios

Preparedness for imported cases (initial testing) Unknown agent, broad differential diagnostics (several VHF, Malaria,

Leptospira spp., sepsis, ?) Well equipped Laboratory Few samples Not routinely performed

Old reagents (?) Reliability do you trust unfrequently used methods?

Constant training required workshops (ENIVD), participation in EQAs

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Diagnostic Scenarios

Epidemic (follow-up testing) Known etiology Specialized assays Differential diagnostics less important Bad Infrastructure High throughput Logistics

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Diagnostic Scenarios

Screening Contact screening Screening of healthy individuals (e.g. point of entry: airports, boarders)

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Diagnostic Scenarios

Preparedness Outbreak

Aetiology Unknown known

Differential diagnosis

Broad Less important

Infrastructure Good Bad/difficult

Situation Sudden Timed

Routine Method used unfrequently – reliable?

Method used more often – confident

Work load Low Throughput High Throughput (?)

Training Continuous, low level

Regularly, maneuvers

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In-house or Commercial Kit Assays?

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Reliability

• Performance Evaluation

• Stability

• Controls

• Quality Control

• Post Market Surveillance

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Reliability

in house Solutions

?

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Reliability

• Performance Evaluation

• Stability

• Controls

• Quality Control

• Post Market Surveillance

Analytical Sensitivity

Analytical Specificity

Cross-Reactivity

Interfering Substances

Total Failture Rate / Robustness

Accuracy of Measurement

Linearity

Equivalence Diagnostic Evaluation Studies

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Reliability

• Performance Evaluation

• Stability

• Controls

• Quality Control

• Post Market Surveillance

in use

Room temperature

+4°C

Freeze & Thaw

Transport

Long term / shelf life

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Reliability

• Performance Evaluation

• Stability

• Controls

• Quality Control

• Post Market Surveillance

Positive Controls

Quantification Standards

Negative Controls

High Positive Controls

Low Positive Controls

Sensitivity Controls

Reference Standards

Calibration Standards

Working Standards

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Reliability

• Performance Evaluation

• Stability

• Controls

• Quality Control

• Post Market Surveillance

Reagents

Kits

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Reliability

Quality Control - Specificity

Pos. Control / QS

NTC Neg. Matrix Eluate

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Reliability

Quality Control – Standard Curve

Pos. Control / QS

NTC Neg. Matrix Eluate

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Reliability

Quality Control - Slope

Pos. Control / QS

NTC Neg. Matrix Eluate

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Reliability

Quality Control – Ct-values

Pos. Control / QS

NTC Neg. Matrix Eluate

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Reliability

Quality Control - Fluorescence

Pos. Control / QS

NTC Neg. Matrix Eluate

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Reliability

Quality Control - Sensitivity

Pos. Control / QS

NTC Neg. Matrix Eluate

Sensitivity Control

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Reliability

Quality Control - Trueness

Pos. Control / QS

NTC Neg. Matrix Eluate

Sensitivity Control High Pos. Control

Low Pos. Control

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Reliability

Quality Control – Internal Control

Pos. Control / QS

NTC Neg. Matrix Eluate

Sensitivity Control High Pos. Control

Low Pos. Control

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Reliability

Quality Control – Reagents / Enzyme

Enzyme lot A: pos. Control - sensitivity control - NTC Enzyme lot B: pos. Control - sensitivity control - NTC

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Diagnostic Markers in Filovirus Infections

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Filovirus Infections – Diagnostic Markers

Marburg virus patients

BEBOV

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When does testing make sense and how do you interpret the results?

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Diagnostic Technologies

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Isothermal amplification

High and low temperature methods: NASBA, RPA, LAMP, SDA, … Advantages

No cycler needed Fast Cheap

Disadvantages

Unspecific detection and unreliable (LAMP) If used for real-time detection, more sophisticated instrumentation/software

needed. Multiplexing? Internal Control? More complicated assay design – higher conservation of sequences

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RPA for Ebola and Marburg Detection

Sensitivity similar to real-time RT-PCR Target gene for RPA: NP Parallel assays for EBOV, SUDV, MARV 1 and MARV 2 “Lab in a suitcase”

http://www.dpz.eu/de/startseite/einzelansicht/news/labor-im-koffer-zu-ebola-entwickelt.html

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Polymerase Chain Reaction (PCR)

Agarose gel PCR Risk of contamination Integration of Internal control difficult No multiplexing No quantitation …

Real-Time PCR Instrument requirements Less prone to contamination Multiplexing Quantitation …

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Real-Time RT-PCR

Observation of reaction kinetics allows quantification.

Gel detection will show different band intensity

Cycles

96 replicates

real-time PCR-detection

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Assays with FDA Emergency Use Authorization

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Roche and TibMolbiol

LightMix® Ebola Zaire rRT-PCR Test Zaire ebolavirus specific Internal control FDA Emergency Use Authorization Inactivated virus used Discussion: PCR indeterminate or negative?

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Roche and TibMolbiol

LightMix® Ebola Zaire rRT-PCR Test Zaire ebolavirus specific Internal control FDA Emergency Use Authorization Inactivated virus used Discussion: PCR indeterminate or negative?

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BioFire

Specific for Zaire ebolavirus Two versions both with FDA EUA FilmArray NGDS BT-E Assay FilmArray Biothreat-E test “The EUA for the FilmArray Biothreat-E test enables hospitals with laboratories that fit these characteristics to conduct a PCR test for Ebola in-house, without having to send the sample to an outside lab” Analytical sensitivity: 10e+4 PFU/ml (inactivated virus) Live virus is at least 10 x better:

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Department of Defense and CDC Assays

DOD: Zaire ebolavirus specific CDC: Zaire ebolavirus specific

100 µl sample in 90 µl eluate ~ 650 TCID50/ml

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Department of Defense and CDC Assays

DOD: Zaire ebolavirus specific CDC: Zaire ebolavirus specific

100 µl sample in 90 µl eluate ~ 650 TCID50/ml

External inhibition controls

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altona Diagnostics

RealStar Ebolavirus RT-PCR Kit 1.0 (derivative of the Filovirus Screen kit) internal control

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EUA Sensitivity Data

Assay PFU/ml (TCID50/ml) Geq/ml

DOD 1,000 ?

CDC NP (6,500)

CDC VP40 (650)

Biofire 1 10,000* 436,000

Biofire 2 600,000* ?

LightMix 4,781* ?

RealStar 1 3,500

* Inactivated gamma irradiated virus

Forget these data and wait for international standard…

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Other PCR Kits

Fast-Track Zaire specific (CE-IVD) No access to validation files

Genesig / Primerdesign Zaire specific (RUO) No validation data available

Bioneer AccuPower® EBOV Real-Time RT-PCR Kit (RUO) Zaire specific ~46 copies/rxn (LoD 95%)

altona Diagnostics GmbH RealStar Filovirus Screen RT-PCR Kit (CE-IVD, WHO prequal.) pan-filovirus sensitivity species dependent (~10-70 copies/rxn)

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Other PCR Kits

Lipsdiag Zaire and Sudan Kits (RUO) No validation data

Liferiver Zaire specific (CE-IVD) LOD 1,000 copies/ml (“depending on extraction”)

Sacace Ebola Zaire (RUO?) No validation data

…

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Rapid Tests

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Why Rapid Tests

Models show diminished infection rates

Bell et al. Nature Reviews Microbiology 4, S7–S20 (September 2006) | doi:10.1038/nrmicro1525

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Rapid Tests

Find and WHO defined a “wish list” for rapid simple tests Clinical sensitivity >95% Clinical specificity >99% Corgenix ReBOV is prequalified by WHO What will you do with a result like this?

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Rapid lateral Flow Systems

?

?

?

? ? More?

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Cepheid gene Xpert

Expensive Equipment Powersupply

Similar to BioFire

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Future Diagnostics

Several systems (pouches, cartridges, chips) are currently under development…