Application of molecular techniques in Virology Suzan D Pas medical - - PowerPoint PPT Presentation

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Application of molecular techniques in Virology Suzan D Pas medical - - PowerPoint PPT Presentation

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Application of molecular techniques in Virology Suzan D Pas medical molecular microbiologist 1 Viroscience lab, Erasmus MC, Rotterdam, the Netherlands Molecular techniques in virus diagnostics Qualitative and quantitative (RT-)PCRs

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Application of molecular techniques in Virology

Suzan D Pas – medical molecular microbiologist

1Viroscience lab, Erasmus MC, Rotterdam, the Netherlands

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Molecular techniques in virus diagnostics

  • Qualitative and quantitative (RT-)PCRs
  • Qualitative positive/negative answers for clinical decisions
  • RSV for quarantine
  • Respiratory viral pathogens for stopping antibiotics
  • Etc..
  • Quantitative assays
  • Associations with progression to disease
  • Follow-up antiviral treatment
  • Determine genotypic traits that influence clinical decision making
  • Antiviral resistance after therapy failure in HIV/HBV/CMV/HSV
  • Determine sub-/genotype that may negatively influence response

to therapy (HBV/HCV/HPV)

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Course of Hepatitis E virus infection in AlloHSCT recipient

Versluis et al, Blood2013

ALT HEV RNA

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Benefits of molecular diagnostics in clinical virology

  • It saves patient lives
  • It saves nursery costs
  • Compared to virus culture
  • Faster
  • Less hands on time (cheaper)
  • More informative (quantitative)
  • In general robust (not depending on quality of difficult to control

materials like living cells)

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Molecular diagnostic workflow @ Erasmus MC

3. Cito / semi manual line low-medium throughput flexibel, manual pipetting exotic viral diseases

  • 4. Point of impact assays
  • 2. Semi-automated workflow

high throughput, high QC herpes panel (+JC, BK parvo) gastro-enteritis panel, respiratory virus panel

  • 1. CE/IVD automated docked CAP/CTM

high throughput, high QC CatA blood borne viruses : HIV, HBV and HCV qPCR

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Point of impact vs point of care tests

laboratory Point of impact Point of care

X

For virology:

  • resp: FLuA/FluB/RSV
  • HIV at anonymous testing site
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Cepheid geneXprt Aries - Luminex Filmarray - Biomerieux

Simplexa Focus diagnostics

Alere i Genmark Dx Limitations:

  • sensitivity
  • Less variety of tests
  • Expensive!

POC / POI Molecular diagnostics

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Number of targets with in-house real-time (RT-)PCR

Other targets: Mumps; Measles; Rubella; Dengue; YFV; HDV; Hantaviruses; JEV; Lassa; Ebola; Marburg; WNV; Rabies; LCMV; CHV; Pox

1995 2015 2000 2005 2010

HIV-2; HBV; HGV; Phocine Distemper Virus 2010 HEV 2005 Norovirus GI&GII 2002 Enterovirus/ Parecho 2014 Sapovirus Astrovirus Rotavirus resistance markers Influenza (275/pan275/119/ 292/152/198/294) Herpes viruses HSV-1/2 EBV; CMV; VZV; HHV-6/-7/-8; JCV/BKV; Herpes B; Phocine Herpes virus Respiratory viruses Influenza A/B; RSV A/B; HMPV; PIV1-4; Rhino; HCoV-OC43; -229E;

  • NL63; -HKU; -SARS

Boca; Adeno; MPn 2012 HCoV- MERS

In total >90 targets

Zika virus

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Production MDx workgroup Viroscience

Year

1996 1998 2000 2002 2004 2006 2008 2010 2012

# (RT-)PCR

1000 2000 3000 4000 5000 6000 7000 CMV EBV Entero HBV HCV Flu A HIV

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LIMS Middleware software

Automated molecular diagnostic work-flow

Purification RNA/DNA Amplification Detection Report Secondairy Sample handling

PCR

Setup

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Issues to consider in quantification

  • Sampling: distribution (in space and time) of the virus of

interest

  • Respiratory viruses, Varicella Zoster in blisters
  • Quality control
  • Efficiency of nucleic acid extraction and sample type
  • Plasma versus urine, CSF, Faeces etc.
  • Chemistry of downstream detection
  • Variation in the primers/probe region targeted
  • Mismatch tolerance of the enzyme platform
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Issues to consider in quantification

  • Sampling: distribution (in space and time) of the virus of

interest

  • Respiratory viruses, Varicella Zoster in blisters
  • Quality control
  • Efficiency of nucleic acid extraction and sample type
  • Plasma versus urine, CSF, Faeces etc.
  • Chemistry of downstream detection
  • Variation in the primers/probe region targeted
  • Mismatch tolerance of the enzyme platform
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Internal/External controls QC plots (Levey-Jennings charts – Westgard rules)

More info: https://www.westgard.com/lesson12.htm

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1 robustness 2 accuracy 3 Specificity 4A precision – repeatability 4A precision - intermediate precision 5 linearity / efficiency 6 linear range 7 Lower limit of detection (LLOD) 8 Lower limit of quantification (LLOQ) 9 selectivity 10 stability 11 carry-over

QC parameters of molecular diagnostic assays

According to ISO15189:2012 guidelines

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Issues to consider in quantification

  • Sampling: distribution (in space and time) of the virus of interest
  • Respiratory viruses, Varicella Zoster in blisters
  • Quality control
  • Efficiency of nucleic acid extraction and sample type
  • Plasma versus urine, CSF, Faeces etc.
  • Chemistry of downstream detection
  • Variation in the primers/probe region targeted
  • Mismatch tolerance of the enzyme platform
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Efficiency of nucleic acid extraction Universal Internal Viral Control

Phocine Herpes Virus 1 (PhHV)  Herpesvirus  DNA control Phocine Distemper Virus (PDV)  Morbillivirus  RNA control

Sample + known concentration

  • f internal control
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Comparison different clinical samples

Ct values internal control PhHV-1 MagnaPure LC

38 36 34 32 30 28 26 24 Ct values on ABI7700 Plasma Serum CSF Faeces Urine Swabs

* *

38 36 34 32 30 28 26 24 Ct values on ABI7700 Plasma Serum CSF Faeces Urine Swabs 38 36 34 32 30 28 26 24 Ct values on ABI7700 Plasma Serum CSF Faeces Urine Swabs

* *

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Issues to consider in quantification

  • Sampling: distribution (in space and time) of the virus of

interest

  • Respiratory viruses, Varicella Zoster in blisters
  • Quality control
  • Efficiency of nucleic acid extraction and sample type
  • Plasma versus urine, CSF, Faeces etc.
  • Chemistry of downstream detection
  • Variation in the primers/probe region targeted
  • Mismatch tolerance of the enzyme platform
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UL54

Van Doornum et al. JCM 2003

Variation in the primers/probe region targeted - CMV

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Dual target real time PCR

UL54 UL75

Genome CMV 5’-

  • 3’

If there is a mutation in either of the primer/probe sites  the other PCR will ‘take over‘

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Case I – no mutations in UL54 primer/probe site

0,50 1,50 2,50 3,50 4,50 5,50 0,00 1,00 2,00 3,00 4,00 5,00 6,00 08-09-2012 13-09-2012 18-09-2012 23-09-2012 28-09-2012 03-10-2012 08-10-2012 13-10-2012 18-10-2012 23-10-2012 28-10-2012 log (c/ml) LOD UL54 (c/ml) log(IU/ml) LOD UL54+UL75 (IU/ml)

log Viral load (c/ml) log viral load (IU/ml)

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Case II – Merlin strain

0,50 1,50 2,50 3,50 4,50 5,50 0,00 1,00 2,00 3,00 4,00 5,00 6,00 26-02-2011 06-06-2011 14-09-2011 23-12-2011 01-04-2012 10-07-2012 18-10-2012 log (c/ml) LOD UL54 (c/ml) log(IU/ml) LOD UL54+UL75 (IU/ml)

log Viral load (c/ml) log viral load (IU/ml)

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Influence of mastermix composition on primer bindingsite mismatch tolerance

Stadhouders et al., Journal of Mol. Diag. 2010

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rev-primer

2 4 6 8 10 12 14 C-A C-T C-C G-A G-T G-G A-A A-C A-G A-A A-C A-G A-C A-A A-G T-T T-C T-G A-A A-C A-G G-G G-A G-T NT1 NT2 NT3 NT5

Increase in Ct-value

FVMM (Taq) GOLD (Taq) HawkZo5 (rTth) EZ (rTth)

 rTth based RT-PCR: low mismatch tolerance  FVMM (MMLV-Taq based) : high mismatch tolerence

Single primer-template mismatch behavior

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fwd-primer

2 4 6 8 10 12 14 T-T T-G T-C A-C A-G A-A T-T T-G T-C G-T G-G G-A G-T G-A G-G T-T T-G T-C C-T C-A C-C C-T C-C C-A NT1 NT2 NT3 NT5

increase in Ct value

FVMM (Taq) GOLD (Taq) HawkZo5 (rTth) EZ (rTth)

Single primer-template mismatch behavior

Forward primer: high tolerance for Hawk Zo5 high tolerance for FVMM, except for A-A and A-G mismatch at 1st nucleotide !!

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Alignment EMC HeV primers-probe – 5’UTR

forward reverse probe A-A mismatch!!  FVMM discrimination Rev primer Hawk Zo5 (rTth) discrimination

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Use of mismatch tolerance in assay design

* HRV-8 and HRV-9 had a Ct-delay respectively 10 and 17 cycles, fluorescence <0.2 ** HeV-echo7 had a Ct-delay > 20 cycles, fluorescence <0.35 ¹ Lu et al., J.clin. Microbiol. 2008 46:533-539

2 Doornum et al., J. Med. Virology 2007 79:1868-1876 3 Nijhuis et al., J. clin. Microbiol. 2002 40:3666-3670 4 Voermans et al, in preparation

assay

HRV-strains (N=87) HeV-strains (N=54)

EZ (old) HawkZo5 FVMM 2-step Gold (old) HawkZo5 FVMM

HRV

Old EMC HRV set 75 nd 83 nd nd 2 New EMC HRV-set4 nd 87 87 nd 2 1** Published HRV set¹ nd 87 87 nd 46 47

HeV

  • ld EMC

HeV set² 6 nd 86 54 nd 54 New EMC HeV set4 nd 2* nd 54 54 Published HeV set3 nd 42 nd 54 54

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GENOTYPING / DRUG RESISTANCE ANALYSIS

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10 20 30 40 50

A

n=90 % 28% 47% 44% 25%

B

n=23

C

n=39

D

n=103

Flink et al, Am J Gastroenterol. 2006 Feb;101(2):297-303

10 20 30 40 50

A

n=90 % 28% 47% 44% 25% Sanger sequencing: genotyping to predict response

B

n=23

C

n=39

D

n=103

Response by Genotype

HBeAg loss end of follow-up

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Variant detection techniques in virology for drug resistance screening

Fung, Antivir Ther 2004; Locarnini, Antivir Ther 2004

Time HBV Replication

Course of infection Start antiviral therapy

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Genotyping / drug resistance detection Sanger sequencing OR population sequencing

C T A T A T G G A T G A T G T G G

Pas et al. JCV2002

Detection limit: down to 25% of mutant in a population Complete sequence information No detection of double infections

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Genotyping / drug resistance detection Inno Lipa - Reverse hybridization technique

Detection limit: down to 10% of mutant in a population Can detect double infections No complete sequence information

Pas et al., JCM 2008

antiviral therapy

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Detection of oseltamivir resistant influenza A/H1N1 H274Y by real-time discrimination PCR using LNA probes

NTC + control + control NTC + control + control Erhard van der Vries et al., ESCV 2009

Wild-type cluster Mutant cluster

Quantitative real-time techniques: 1-10% LNA/MGB probes (short high affinity probes) Molecular beacons Scorpions

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NGS technique – variance analysis

  • High sensitivity (0.5%) -> what is the clinical relevance?
  • Labor intensive
  • High costs
  • Only cost effective

with high sample-throughput

  • Complicated data analysis,

though automated pipelines are available

  • Longer turn around time

Pas et al, in prep.

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Variant detection techniques in virology

Fung, Antivir Ther 2004; Locarnini, Antivir Ther 2004

Time HBV Replication

Lipa 10% Sanger sequencing 25-50% Allele specific PCR 1-5%

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Take home messages

  • Molecular diagnostics important technique in clinical virology

* virus genome detection (qualitative) * viral load quantification * drug resistance detection * genotyping

  • Pitfalls are a.o. sampling time/place, target diversity, enzyme

mismatch acceptance

  • Solutions are a.o. diversity of probe systems, QC
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s.pas@erasmusmc.nl