Digital PCR in HIV Reservoir quantification Ward De Spiegelaere

Transcription

1 Digital PCR in HIV Reservoir quantification Ward De Spiegelaere HIV Cure Research Center Dept. Internal Medicine Ghent University Belgium

2 Outline 1) What is digital PCR 2) Comparison of digital PCR with qpcr 3) Applications of digital PCR in Virology 4) Future perspectives

3 What is digital PCR Sven Sachsalber

4 Quantification (Using the Poisson distribution) Unknown: concentration of target molecules (λ) Known: The ratio of positive partitions (P) λ = - ln(1- pˆ)

5 What is digital PCR

6 dpcr is not a novel technology! Limiting dilution of samples for single molecule PCR Β-globulin

7 Single molecule PCR for HIV quantification env HAT 3 (359 bp) pbh10+r3 clone (317 bp) gag

8 Why did people stop using digital PCR? 100 µl reactions! Assay costs! Automatization? Operating costs and troubles:

9 New technologies make dpcr possible Arrays: Multiple partitions on a plate

10 New technologies make dpcr possible picodroplets: Droplets in oil

11 A typical dpcr read-out

12 Outline 1) What is digital PCR 2) Comparison of digital PCR with qpcr 3) Applications of digital PCR in Virology 4) Future perspectives

13 Theoretical benefits of digital PCR Direct quantification of DNA molecules No standard curve No variability due to lower efficiency Higher accuracy Higher precision Higher sensitivity Higher reproducibility

14 dpcr is more precise compared to qpcr Precision: ddpcr is superior when compared to qpcr CV 4 to 20-fold lower in ddpcr compared to qpcr Strain et al., Plos One (2013) 8: e55943

15 dpcr is more precise compared to qpcr Comparison of ddpcr versus seminested qpcr for HIV DNA quantification Coeficient of Variation (CV) between triplicates n replicates CV ddpcr CV qpcr Fold difference Standard curve % 43% 3 Patient samples % 29% 3.5 Bosman et al., Scientific Reports :13811

16 Accuracy: Bias compared to qpcr Comparison of HIV DNA between ddpcr and seminested qpcr No significant bias compared to qpcr on HIV DNA measurement Bland Altman plot Bosman et al., Scientific Reports :13811

17 dpcr Performance of current technology - Better accuracy and precision - Less refractory to inhibition - Better reproducibility - More sensitive?

18 dpcr and inhibition Inhibition dpcr outperforms qpcr comparing inhibitory substances (SDS, EDTA and Heparin) Dingle et al., Clin Chem (2013)59:1670-2

19 dpcr and inhibition: Total DNA content HIV template in background of HIV negative DNA gdna (ng/µl ddpcr mix) gdna (ng/µl ddpcr mix) gdna (ng/µl ddpcr mix) gdna (ng/µl ddpcr mix) 33 ng/µl DNA = 660ng/20µl reaction = cell equivalents per reaction Malatinkova et al., Journal of Clinical Microbiology (2015) 53: (supplemental data)

20 dpcr and inhibition: priming mismatches Mismatches in the probe region Match Mismatch 1 Mismatch 2 Mix Lower fluorescence would harm qpcr but less harm to ddpcr Trypsteen et al., Anal Bioanal Chem (2015) 407: (suppl data)

21 dpcr Performance of current technology - Better accuracy and precision - Less refractory to inhibition - Better reproducibility - More sensitive?

22 reproducibility Replicate experiments on 3 consecutive days Factor of 7 more reproducible compared to qpcr Hindson et al., Nature Methods (2013) 10:1003-5

23 Copies/million PBMCs 2.5yrs dpcr is highly reproducible Day years n=8 DNA isolation HIV ddpcr (triplicate) RPP30 ddpcr (duplicate) DNA isolation HIV ddpcr (triplicate) RPP30 ddpcr (duplicate) Total HIV-1 DNA copies/million PBMCs day 0 Median CV 6% Unpublished data

24 dpcr Performance of current technology - Better accuracy and precision - Less refractory to inhibition - Better reproducibility - More sensitive?

25 Sensitivity Some reports indicate higher sensitivity of ddpcr At the limit of detection, more samples are positive with ddpcr But false positives in ddpcr Kiselinova et al., Plos One :e85999; Bosman et al., Scientific Reports :13811

26 False positives in ddpcr NTCs No forward primer No forward primer Artefacts? Kiselinova et al., Plos One (2014) 9: e85999

27 dpcr Performance - Better accuracy and precision - Less refractory to inhibition - Better reproducibility - More sensitive? x When to use dpcr or qpcr depends on the question: Template present? go for standar PCR or nested How much? go for digital PCR

28 Outline 1) What is digital PCR 2) Comparison of digital PCR with qpcr 3) Applications of digital PCR in Virology 4) Future perspectives

29 Applications of digital PCR in virology - HIV reservoir analysis in Cure research - Difficult samples - Mutation detection - Relative quantification

30 HIV reservoirs Healthy subject HIV infection cart cart stop T cells HIV reservoir HIV

31 HIV reservoir in early ART p=0.015* No ART (n=10) Late ART (n=32) Early ART (n=25) LTNP (n=17) Malatinkova, E., Spiegelaere, W.D., Bonczkowski, P., Kiselinova, M., Vervisch, K., Trypsteen, W., Johnson, M., Verhofstede, C., Looze, D., Murray, C., Loes, S.K., Vandekerckhove, L., Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs. elife 4, e09115.

32 Applications of digital PCR in virology - HIV reservoir analysis in Cure research - Difficult samples - Mutation detection - Relative quantification

33 Difficult samples Low sample volumes - Direct quantification after lysis Pavsic et al., Digital PCR for direct quantification of viruses without DNA extraction. Analytical and Bioanalytical Chemistry (2016) Vol 408:67-75

34 Direct quantification of RNA viruses in wastewater Detection of Rotavirus in wastewater No RNA isolation step Racki et al., One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses. Analytical and Bioanalytical Chemistry (2014) 406:

35 Applications of digital PCR in virology - HIV reservoir analysis in Cure research - Difficult samples - Mutation detection - Relative quantification

36 Oseltamivir Resistant mutants in influenza A H1N1 Higher sensitivity of dpcr to detect resistant mutations Alexandra S. Whale et al. J. Clin. Microbiol. 2016;54:

37 Applications of digital PCR in virology - HIV reservoir analysis in Cure research - Difficult samples - Mutation detection - Relative quantification

38 Human Herpes Virus 6 Discriminating chromosomal integrated (ci)hhv-6 from genuine viral replication. Sedlak R.H. et al. Identification of Chromosomally Integrated Human Herpesvirus 6 by Droplet Digital PCR. Clinical Chemistry (2013) 60:

39 Relative quantification Merkel cell polyomavirus in cancer IHC positive IHC negative Merkel Cell carcinoma Basal cell carcinoma Actinic keratosis Ota et al., Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel Cell Polyomavirus infection in skin tumor

40 Perspectives of dpcr in virology Several applications possible: Low level quantification Mutation/resistance detection Relative quantification Current drawbacks: False positives New technologies? Need for objective data analysis Has received little analysis, but new technique, new issues.

41 What is positive, what is negative??... False neg??... False pos? Vynck et al., The Future of Digital Polymerase Chain Reactin in Virology. Molecular Diagnosis & Therapy (2016) 20:

42 Future perspectives: devices and software Automatic threshold setting (extreme value theory) Open source software: ddpcrquant: Trypsteen et al., Anal Bioanal Chem (2015) 407: (suppl data)

43 General Conclusion Current state of the ART Specific applications Do we need the sensitivity? Future perspectives May partially/totally replace real time PCR Need for hight throughput and low cost Ease of analysis, ease of interlaboratory standardization

44 Acknowledgements Ghent University: HIV Cure Research Center Prof. Linos Vandekerckhove Ward De Spiegelaere Magdalena Sips Eva Malatinkova Karen Vervisch Laura De Clercq Maja Kiselinova Pawel Bonczkowski Sam Kint Wim Trypsteen Marie-Angélique De Scheerder Sofie Rutsaert Clarissa Van Hecke Basiel Cole Dorien Vanderroost Jon Huyghe All participating participating patients! Collaborators: University College London Sabine Kinloch-de Loes University of Amsterdam Prof. Ben Berkhout Alexander Pasternak University of Utrecht Prof. Monique Nijhuis Prof. Annemarie Wensing Kobus Bosman Petra Vanham Ghent University Prof. Olivier Thas Matthijs Vynck Prof. Bruno Verhasselt Prof. Chris Verhofstede The AIDS Reference Center Prof Dirk Vogelaers Prof Chris Verhostede Financial Support: