Toronto; November 23, How do we know that diagnostic assays do what we think they do? Dr. Markus Hess - altona Diagnostics GmbH

Size: px

Start display at page:

Download "Toronto; November 23, How do we know that diagnostic assays do what we think they do? Dr. Markus Hess - altona Diagnostics GmbH"

Noah Griffith
5 years ago
Views:

1 Toronto; November 23, 2015 How do we know that diagnostic assays do what we think they do? Dr. Markus Hess - altona Diagnostics GmbH

2 How do we know that diagnostic assays do what we think they do?

3 How do we know that diagnostic assays do what we think they do? User Expectations

4 How do we know that diagnostic assays do what we think they do? User Expectations Assays Performance Characteristics

6 Symptoms Patient History

7 Symptoms Patient History

8 Symptoms Clinical Chemistry Molecular Diagnostics Serology Patient History

9 Symptoms Clinical Chemistry Molecular Diagnostics Diagnosis Serology Patient History

10 Therapy

11 Molecular Diagnosics Ø Real-Time PCR Ø Isothermal Amplification Ø Lateral flow Ø Pyrosequencing Ø NGS Ø POC Ø

12 Molecular Diagnosics Ø Real-Time PCR Ø Isothermal Amplification Ø Lateral flow Ø Pyrosequencing Ø NGS Ø POC Ø Ø Screening Assay Ø Detection Assay Ø Monitoring Assay Ø Quantification Assay Ø Multiplex Assays Ø

13 Molecular Diagnosics Ø Real-Time PCR Ø Isothermal Amplification Ø Lateral flow Ø Pyrosequencing Ø NGS Ø POC Ø Ø Screening Assay Ø Detection Assay Ø Monitoring Assay Ø Quantification Assay Ø Multiplex Assays Ø Ø Sample Material Ø NA Extraction Systems Ø Cyclers

cause a severe hemorrhagic fever with a relatively high mortality rate of 20-90% Many different

14 Filoviruses (-) ss RNA viruses All known Ebola- and Marburgvirus species are endemic in Africa except RESTV which is endemic in South-East Asia After transmission to humans, filoviruses can cause a severe hemorrhagic fever with a relatively high mortality rate of 20-90% Many different mammalian species are susceptible to filovirus infections; in particular chimpanzees and gorillas

15 Filoviruses Symptoms are rather unspecific at the beginning of the disease including general malaise, fever and pain in different body parts Disease is therefore often mistaken for Malaria, Typhoid fever or other febrile diseases common in Sub-Saharan Africa Infectious virus titer and RNA concentration during acute disease are usually high Laboratory diagnostics is preferably done using RT-PCR from plasma, serum or whole blood samples Many patients (especially with fatal outcome) do not develop detectable antibody titers during the course of the disease at all

16 Filoviruses Ebolavirus and Marburgvirus are genera within the family Filoviridae Genus Marburgvirus contains a single species termed Marburg marburgvirus (MARV) Genus Ebolavirus contains five species: Bundibugyo ebolavirus (BEBOV) Reston ebolavirus (RESTV) Sudan ebolavirus (SEBOV) Tai Forest ebolavirus (TAFV) Zaire ebolavirus (ZEBOV)

17 Outbreak 2014/15 WHO Report September WHO Report September

18 RealStar Kits RealStar Filovirus Screen RT-PCR Kit 1.0 pan-filovirus assay typing of genus (EBOV and MARV) IVD-CE; WHO RealStar Filovirus Type RT-PCR Kit 1.0 second-line assay typing of species RUO RealStar Ebolavirus RT-PCR Kit U.S. Ebolavirus (genus) based on Filovirus Screen EUA RealStar Zaire Ebolavirus RT-PCR Kit 1.0 Zaire ebolavirus specific based on Filovirus Screen RUO

19 How do we know that diagnostic assays do what we think they do?

20 How do we know that diagnostic assays do what we think they do? What is the intended use of a certain diagnostic assay?

21 How do we know that diagnostic assays do what we think they do? What is the intended use of a certain diagnostic assay? What are the performance characteristics of a certain diagnostic assay?

22 How do we know that diagnostic assays do what we think they do? What is the intended use of a certain diagnostic assay? What are the performance characteristics of a certain diagnostic assay? Quality Control

23 Quality Assurance Assay Development and Performance Characteristics Design Input Development Verification Validation Production Quality Control Post Market Surveillance

24 Design Input Qualitative detection and differentiation of Ebolavirus and Marburgvirus specific RNA (pan filovirus assay) Real-Time RT-PCR Internal Control Defined real-time PCR instruments

25 Design Input The RealStar Filovirus Screen RT-PCR Kit 1.0 was developed and validated to be used with the following real-time PCR instruments: Mx 3005P QPCR System (Stratagene) Versant kpcr Molecular System AD (Siemens) ABI Prism 7500 SDS and 7500 Fast SDS (Applied Biosystems) LightCycler 480 Instrument II (Roche) Rotor-Gene 3000/6000 (Corbett Research) Rotor-Gene Q 5/6 plex Platform (QIAGEN) CFX96 system real-time system (BIORAD)

26 Development Target region Primer/probe design Buffer optimization Evaluation (sensitivity, specificity, stability, robustness) Evaluation with virus material Implementation of internal control Definition of positive control and QC controls

a positivity rate of 95% determined by analysis of dilution series of MARV Popp,

27 Verification Analytical Sensitivity defined as the concentration (copies per µl of the eluate) of Ebolaor Marburgvirus specific RNA molecules that can be detected with a positivity rate of 95% determined by analysis of dilution series of MARV Popp, SEBOV Gulu and ZEBOV Gabon 2003 specific in vitro transcripts (IVT) of known concentration

28 Analytical Sensitivity LightCycler 480 Instrument II (Roche) ZEBOV Gabon 2003 IVT 95% LoD = 1.4 copies/µl Confidence interval = 0.7 to 5.3

29 Analytical Sensitivity LightCycler 480 Instrument II (Roche) ZEBOV Gabon 2003 IVT SmartCycler II (Cepheid) ZEBOV Gabon 2003 IVT 95% LoD = 1.4 copies/µl Confidence interval = 0.7 to % LoD = 40 copies/µl Confidence interval = 20 to 250

30 Verification Analytical Specificity in situ analysis Reactivity Cross-reactivity Negative specimen

31 Verification Analytical Specificity Species Strain Zaire ebolavirus Gabon 2003 in situ analysis Reactivity Cross-reactivity Zaire ebolavirus Mayinga Zaire ebolavirus 2014/ Gueckedou-C05 Sudan ebolavirus Tai Forest ebolavirus Reston ebolavirus Gulu N/A N/A Negative specimen Bundibugyo ebolavirus Marburg marburgvirus Marburg marburgvirus Marburg marburgvirus N/A Musoke Popp Leiden

32 Verification Analytical Specificity Species Strains Species Strain Japanese encephaliitis virus Lassa virus Nig08-A37; CSF; Lib /121; AV Saint Louis encephalitis virus Junin virus XJ in situ analysis Reactivity West Nile virus NY99; NY99D; Uganda Machupo virus Carvallo Yellow Fever virus Sabia virus SPH Murray Valley encephalitis virus Guanarito virus Zika virus Vesicular stomatitis virus Indiana Cross-reactivity Tick borne encephalitis virus Rift Valley fever virus MP 12 Negative specimen Usutu virus Hantaan virus Dengue virus 1 to 4 Rotavirus Wa HAV RSV A; B HCV Plasmodium malariae N/A HEV E. coli N/A CCHFV Afg

33 Verification Analytical Specificity in situ analysis Reactivity Cross-reactivity Negative specimen

34 Verification Interfering Substances Bilirubin (30mg/dl) Hemoglobin (2g/dl) Triglyceride (1g/dl) Human Serum Albumin (6g/dl) gdna (10µg/dl)

35 Verification Precision Intra-assay variability (variability within one experiment) Inter-assay variability (variability between different experiments) Inter-lot variability (variability between different production lots) Total variance

36 Validation Cell culture supernatants, serially diluted, RNA extracted and tested in replicates Zaire ebolavirus Mayinga uv RealStar Filovirus Screen 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3 Panning L-Gen Screen 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 Gibb EBOV GP 2/2 2/2 2/2 2/2 0/2 0/2 0/2 0/2 0/2 Gibb MARV GP n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Marburgvirus Leiden uv RealStar Filovirus Screen 3/3 3/3 3/3 3/3 3/3 3/3 0/3 1/3 0/3 Panning L-Gen Screen 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 0/3 Gibb EBOV GP n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Gibb MARV GP 2/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 Zaire ebolavirus 2014/Gueckedou-C05 uv RealStar Filovirus Screen 3/3 3/3 3/3 3/3 3/3 1/3 0/3 0/3 0/3 Panning L-Gen Screen 3/3 3/3 3/3 3/3 3/3 0/3 0/3 0/3 0/3 Gibb EBOV GP 2/2 2/2 2/2 2/2 0/2 0/2 0/2 0/2 0/2 Gibb MARV GP n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Sudan Ebola virus Gulu uv RealStar Filovirus Screen 3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Panning L-Gen Screen 3/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Gibb EBOV GP 2/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 Gibb MARV GP n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Panning L Gene Panning M, Laue T, Olschlager S, et al. Diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all European biosafety level 4 laboratories. J Infect Dis 2007; 196 Suppl 2:S Gibb EBOV GP Gibb TR, Norwood DA, Jr., Woollen N, Henchal EA. Development and evaluation of a fluorogenic 5' nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan. J Clin Microbiol 2001; 39: Gibb MARV GP Gibb TR, Norwood DA Jr., Woollen N, Henchal EA Development and evaluation of a fluorogenic 5 - nuclease assay to identify Marburg virus. Mol Cell Probes 2001; 15:

37 Clinical Performance Evaluation Mock Clinical Study LoD of the RealStar Ebolavirus RT-PCR Kit 1.0 for Zaire ebolavirus 2014/Gueckedou-C05 is 1 PFU/ml Viral RNA at different concentrations prepared in AE buffer spiked into 45 samples composed of AVL buffer and independent human EDTA plasma specimen

38 Clinical Performance Evaluation Mock Clinical Study Positive Specimen 2.25xLoD n=15 Positive Specimen 3xLoD n=15 Positive Specimen 200xLoD n=15 Negative Specimen n=100 Positive Results Negative Results Total n= Positive Agreement 45/45 100% Negative Agreement 100/ %

39 Production Quality Control: Reagents altona Diagnostics Buffer altona Diagnostics Oligonucleotides Enzymes

40 Production Quality Control: Reagents altona Diagnostics Buffer altona Diagnostics Oligonucleotides Enzymes

41 Production Quality Control: Reagents altona Diagnostics Buffer altona Diagnostics Oligonucleotides Enzymes

42 Quality Control Quality Control: Kits Sensitivity Specificity Reactivity Reproducibility

43 Post Market Surveillance KitControl: search for new sequences Proficiancy panel testing Feedback from customers and network

44 Customer How do we know that diagnostic tests do what we think they do? Read the manual Contact technical support in house validation Proficiancy panel testing

Future MOFINA (Mobile Filovirus Nucleic Acid Test) Innovative Medicines Initiative 2 Ebola and other Filoviral Haemorrhagic Fevers Programme (IMI2 Ebola+) Public Health England United Kingdom altona

45 Future MOFINA (Mobile Filovirus Nucleic Acid Test) Innovative Medicines Initiative 2 Ebola and other Filoviral Haemorrhagic Fevers Programme (IMI2 Ebola+) Public Health England United Kingdom altona Diagnostics GmbH Germany Alere Technologies GmbH Germany Bernhard Nocht Institute for Tropical Medicine Germany Istituto Nazionale Malatie Infettive L. Spallanzani Italy FIND Foundation of Innovative New Diagnostics Switzerland

46 Acknowledgment QUANDHIP Consortium Dr. Antonino Di Caro Dr. Stephan Günther Dr. Markus Eickmann Bernhard Nocht Institute for Tropical Medicine Dr. Stephan Günter Dr. Toni Rieger

WHO Emergency Use Assessment and Listing for Zika IVDs PUBLIC REPORT. Product: RealStar Zika Virus RT-PCR Kit 1.0 EUAL Number: EAZ

EAZ 0003-002-00 WHO EUAL ZIKV Public Report WHO Emergency Use Assessment and Listing for Zika IVDs PUBLIC REPORT Product: RealStar Zika Virus RT-PCR Kit 1.0 EUAL Number: EAZ 0003-002-00 Emergency Use Assessment