Novel Quantitative and Qualitative Screening Assays for Blood Borne Pathogens based on Multi-Dye Technology

John Saldanha 27th May, 2010, IPFA/PEI Workshop, Zagreb, Croatia picture placeholder

Overview

•  Multi-dye technology

•  Roche cobas s 201 system and cobas® TaqScreen DPX Test

•  Roche cobas s 201 system and cobas® TaqScreen MPX Test, v2.0

•  Conclusions

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Multi-dye Technology

Multi-dye PCR

Multiple Target Detection & Identification •  Real-time multiple target detection and identification

–  Target identification by target-specific primers –  Multiple reporter fluorescent dyes –  Multiple detection channels to detect specific fluorescent dyes –  Software to interpret multiple signals in a single test

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Multi-dye Tests

Tests currently in development •  cobas® TaqScreen DPX Test* –  Multiplex, multi-dye test for simultaneous detection and identification of B19 (quantitative) and HAV (qualitative) –  Tests for B19 and HAV on the same cobas s 201 system to be used by both plasma fractionators and blood banks

•  cobas® TaqScreen MPX Test, v2.0 –  Multiplex, multi-dye test for simultaneous detection and identification of HBV, HCV and HIV (HIV-1 groups O & M and HIV-2) –  Eliminates need for secondary testing to identify viral target

*CE Mark Pending

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cobas® TaqScreen DPX Test

Test currently in development; CE Mark pending

cobas® TaqScreen DPX Test

Overview •  Dual target test: quantitative parvovirus B19 result + qualitative HAV detection –  Can be installed to only provide B19 result •  In-process test for plasma for further manufacture •  Test recovered plasma for B19 titer as required by plasma fractionators •  Runs on cobas s 201 system •  Pools of 1, 96 and 480 pooling algorithms •  User-defined B19 titre to initiate pool deconstruction –  Definable for initial and resolution pool sizes

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Overview •  Quantitation Standard (QS) is directly traceable to the WHO B19 International Standard •  QS + Internal Control (IC) for HAV target co-extracted and co-amplified with each sample •  Generic nucleic acid preparation •  Amplification/detection of B19 DNA, HAV RNA, QS and IC in single, closed tube •  Fluorescently labeled probes detected in separate channels to discriminate B19, HAV, QS and IC •  AmpErase (UNG) reduces potential contamination by previously amplified material (amplicon) Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

CT Plot Ct plot of cobas® TaqScreen DPX test

•  B19: 1.00E +08 IU/mL •  HAV: 3.18 IU/mL

Test currently in development; CE Mark pending

cobas® TaqScreen DPX Test

Overview Test kit

External Control kit

•  Ready-to-use reagents

•  Negative Control

•  96 tests (two sets of 48 T cassettes)

•  Dual Low Positive Control

•  After initial use, reagents stable –  for 30 days at 2-8°C

–  B19 Low Titre –  HAV Reactive

•  B19 High Positive Control –  up to 6 instrument runs and maximum of 48 cumulative hours on instrument

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Analytical Sensitivity •  95% LOD by PROBIT Analysis

Virus

LOD (IU/mL)

95% Lower Confidence Limit

95% Upper Confidence Limit

HAV

1.06

0.94

1.24

Parvovirus B19

11.48

10.56

12.91

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Linearity of parvovirus B19 Quantitation

® •  B19 genotype 1 sample calibrated B19 Linearity of cobas TaqScreen DPX Test against WHO Standard (99/800)

•  2 lots of reagents used •  Linear range 75 - 3.0 x 1E8 IU/mL •  Genotypes 2 and 3 also demonstrated linear quantitation

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

B19 and HAV Genotype Inclusivity

•  HAV –  Clinical specimens: 10 genotype 1, 1 genotype 1B –  Transcripts: 2 genotype 1B, 1 genotype IIA,, 1 genotype IIB, 3 genotype IIIA, and 2 genotype IIIB

•  B19 –  Clinical specimens: genotypes 1A, 3A and 3B –  2 plasmids: genotype 2

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

B19 Precision Standard Deviation of B19 Titres, log10 IU/mL (n = 20) B19 Panel Members (IU/mL)

Reagent Lot 1

Reagent Lot 2

Lots 1 and 2 Combined

106

0.073

0.103

0.088

105

0.051

0.083

0.070

104

0.071

0.071

0.070

103

0.218

0.112

0.176

Over all (103 - 106)

0.123

0.094

0.110

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Co-infection

•  B19 1.00E + 08 IU/mL HAV 3.18 IU/mL

•  B19 3.75E + 02 IU/mL HAV 1.00E + 07 IU/mL

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Test Performance

Study

Results

Lot Interchangeability

Equivalent performance with tested combinations of reagent, control and wash reagent kits

IC/QS Performance

Batch failure rate 0.75 % IC failure rate 0.44 %

Reproducibility

HAV reactive rate overlapped each other when compared day to day, lot to lot, and instrument to instrument

Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Correlation to Established Device – Qiagen artus •  Testing done at DRK Hagen •  cobas® TaqScreen DPX Test: B19 accuracy equivalent to Artus RT-PCR B19 Test –  Deming Regression and Bland Altman bias plot –  Mean bias of -0.35 log10 in range 1E + 03 IU/mL to 1E + 06 IU/mL (acceptable criterion not exceeding ± 0.5 log10) •  cobas® TaqScreen DPX Test: B19 equivalent sensitivity to Artus RT-PCR B19 Test (214 clinical samples) •  cobas® TaqScreen DPX Test: HAV more sensitive than Artus RT-PCR HAV Test at low concentration levels of 5, 10 and 50 IU/mL but equivalent at higher concentration levels Test currently in development; CE Mark pending

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cobas® TaqScreen DPX Test

Correlation to Established Device – Qiagen artus

Test currently in development; CE Mark pending

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cobas® TaqScreen MPX Test, v2.0

Test currently in development

cobas® TaqScreen MPX Test, v2.0

Preliminary 95% LOD TaqScreen MPX, v2.0

TaqScreen MPX (v1) PI

95% Probit

LCL/UCL

95% Probit

LCL/UCL

HIV-1 M*

48.3

33.0 – 88.6

49.0

42.4 – 58.1

HBV*

2.4

2.0 – 3.1

3.8

3.3 – 4.4

HCV*

7.1

5.9 – 9.2

11.0

7.0 – 21.7

HIV-1 O**

21.3

13.9 – 47.0

89

56 - 217

HIV-2**

65.1

54.7 – 80.4

59.3

51.9 – 69.7

* Calibrated against WHO International Standards, IU/mL ** Calibrated against FDA Standards, copies/mL Test currently in development

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cobas® TaqScreen MPX Test, v2.0

Genotype/Subtype Inclusivity - Summary •  HBV: A – H

•  HCV: 1 -6 (including sub-genotypes)

•  HIV-1, group M: A – J + group N + recombinants AE, AG, BG (or G)

•  HIV-1, group O

•  HIV-2 Test currently in development

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cobas® TaqScreen MPX Test, v2.0

Co-infected Sample

Test currently in development

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cobas® TaqScreen MPX Test, v2.0

Co-infected Sample

Test currently in development

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cobas® TaqScreen MPX Test, v2.0

Co-infected Sample

Test currently in development

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Summary

Advantage of Real-time PCR

•  Target-multiplexing reduces number of tests to run •  Multi-dye capacity enables real-time viral target detection & discrimination •  Quantitative as well as qualitative tests •  Multi-dye assays readily automated on current instrumentation •  Allows for rapid development of assays for emerging pathogens (dengue, chikungunya, ) if need is demonstrated

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cobas® TaqScreen DPX Test

Test Performance Summary Study Analytical Sensitivity

Results B19: HAV:

11.5 IU/mL 1.06 IU/mL

Linearity – dynamic range B19

75 - 3E + 08 IU/mL

HAV genotype Inclusivity

Genotype I, II and III showed equivalent LOD

Verification of Genotype Linearity for B19

Genotypes 2 and 3 showed Linear performance

B19 genotype 95% LOD

1A: 2: 3A: 3B:

13.57 IU/mL 6.07 IU/mL 3.94 IU/mL 5.31 IU/mL

Cut Off/‌Specificity

B19: HAV:

96.3% 99.6%

Correlation/Established Device Quantitation difference: 0.35 log IU/mL Test currently in development; CE Mark pending

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Acknowledgements

•  DRK Blutspendedienst West •  Dr. V. Schottstedt •  Dr. L. Pichl

•  RMS, Pleasanton •  Dr. Y. Ohhashi •  K.Lu •  Dr. K. Malhotra •  N. Dyer •  C. Harkleroad

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