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

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  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)

Course of Hepatitis E virus infection in AlloHSCT recipient HEV RNA ALT Versluis et al, Blood2013

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)

Molecular diagnostic workflow @ Erasmus MC 1. CE/IVD automated docked CAP/CTM high throughput, high QC CatA blood borne viruses : HIV, HBV and HCV qPCR 2. Semi-automated workflow high throughput, high QC herpes panel (+JC, BK parvo) gastro-enteritis panel, respiratory virus panel 3. Cito / semi manual line low-medium throughput flexibel, manual pipetting exotic viral diseases 4. Point of impact assays

Point of impact vs point of care tests Point of care X laboratory Point of impact For virology: - resp: FLuA/FluB/RSV - HIV at anonymous testing site

POC / POI Molecular diagnostics Aries - Luminex Cepheid geneXprt Genmark Dx Filmarray - Alere i Biomerieux Limitations: Simplexa - sensitivity Focus - Less variety of tests diagnostics - Expensive!

Number of targets with in-house real-time (RT-)PCR 1995 2000 2005 2015 2010 Herpes viruses Respiratory viruses resistance markers HSV-1/2 Influenza A/B; Influenza EBV; RSV A/B; HMPV; (275/pan275/119/ HIV-2; CMV; PIV1-4; Rhino; 292/152/198/294) HBV; VZV; HCoV-OC43; -229E; HGV; HHV-6/-7/-8; -NL63; -HKU; -SARS Phocine Distemper Virus 2010 HEV JCV/BKV; Boca; Adeno; MPn Herpes B; 2012 HCoV- Phocine Herpes virus MERS 2014 2002 Sapovirus 2005 Enterovirus/ Astrovirus Norovirus Parecho Rotavirus GI&GII In total >90 targets Zika virus Other targets: Mumps; Measles; Rubella; Dengue; YFV; HDV; Hantaviruses; JEV; Lassa; Ebola; Marburg; WNV; Rabies; LCMV; CHV; Pox

Production MDx workgroup Viroscience 7000 CMV EBV 6000 Entero HBV HCV 5000 Flu A HIV 4000 # (RT-)PCR 3000 2000 1000 0 1996 1998 2000 2002 2004 2006 2008 2010 2012 Year

Automated molecular diagnostic work-flow Secondairy Purification Amplification PCR Report Sample RNA/DNA Detection Setup handling Middleware software LIMS

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

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

Internal/External controls QC plots (Levey-Jennings charts – Westgard rules) More info: https://www.westgard.com/lesson12.htm

QC parameters of molecular diagnostic assays 1 robustness 2 accuracy 3 Specificity precision – repeatability 4A 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 According to ISO15189:2012 guidelines

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

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 of internal control

Comparison different clinical samples Ct values internal control PhHV-1 MagnaPure LC 38 38 38 36 36 36 * * 34 34 34 Ct values on ABI7700 Ct values on ABI7700 Ct values on ABI7700 * * 32 32 32 30 30 30 28 28 28 26 26 26 24 24 24 Plasma Plasma Plasma Serum Serum Serum CSF CSF CSF Faeces Faeces Faeces Urine Urine Urine Swabs Swabs Swabs

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

Variation in the primers/probe region targeted - CMV UL54 Van Doornum et al. JCM 2003

Dual target real time PCR Genome CMV 5’ - - 3’ UL54 UL75 If there is a mutation in either of the primer/probe sites  the other PCR will ‘take over‘

Case I – no mutations in UL54 primer/probe site 6,00 log (c/ml) 5,50 5,00 LOD UL54 (c/ml) log viral load (IU/ml) log(IU/ml) log Viral load (c/ml) 4,50 LOD UL54+UL75 (IU/ml) 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,00 0,50 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

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

Influence of mastermix composition on primer bindingsite mismatch tolerance Stadhouders et al., Journal of Mol. Diag. 2010

Single primer-template mismatch behavior FVMM (Taq) rev-primer GOLD (Taq) HawkZo5 (rTth) EZ (rTth) 14 12 10 Increase in Ct-value 8 6 4 2 0 C-T C-C G-G A-C A-G A-C A-G A-C A-G T-T T-C T-G A-C A-G G-G C-A G-A G-T A-A A-A A-A A-A G-A G-T NT1 NT2 NT3 NT5  rTth based RT-PCR: low mismatch tolerance  FVMM (MMLV-Taq based) : high mismatch tolerence

Single primer-template mismatch behavior FVMM (Taq) fwd-primer GOLD (Taq) HawkZo5 (rTth) EZ (rTth) 14 12 10 8 increase in Ct value 6 4 2 0 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 Forward primer: high tolerance for Hawk Zo5 high tolerance for FVMM, except for A-A and A-G mismatch at 1 st nucleotide !!

Alignment EMC HeV primers-probe – 5’UTR probe forward reverse A-A mismatch!!  FVMM Rev primer  Hawk Zo5 discrimination (rTth) discrimination

Use of mismatch tolerance in assay design HRV-strains (N=87) HeV-strains (N=54) assay EZ (old) HawkZo5 FVMM 2-step Gold (old) HawkZo5 FVMM Old EMC 75 nd 83 nd nd 2 HRV set New EMC HRV nd 87 87 nd 2 1** HRV-set 4 Published nd 87 87 nd 46 47 HRV set¹ old EMC 6 nd 86 54 nd 54 HeV set² New EMC HeV nd 0 2* nd 54 54 HeV set 4 Published nd 0 42 nd 54 54 HeV set 3 * 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

GENOTYPING / DRUG RESISTANCE ANALYSIS

Sanger sequencing: genotyping to predict response Response by Genotype HBeAg loss end of follow-up 47% 47% 50 50 44% 44% % % 40 40 28% 28% 30 30 25% 25% 20 20 10 10 0 0 B C D A A B C D n=90 n=23 n=23 n=39 n=39 n=103 n=103 n=90 Flink et al, Am J Gastroenterol. 2006 Feb;101(2):297-303

Variant detection techniques in virology for drug resistance screening Start antiviral therapy Course of infection HBV Replication Time Fung, Antivir Ther 2004; Locarnini, Antivir Ther 2004

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 Detection limit: down to 25% of mutant in a population Complete sequence information No detection of double infections Pas et al. JCV2002