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Novel materials and methods for the
detection, traceable monitoring and
evaluation of antimicrobial resistance
Digital PCR as a reference measurement procedure for HIV quantification
Jim Huggett
Principal Scientist (NML) & Senior Lecture (University of Surrey)
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Un
cert
ain
ty
Metr
olo
gic
al
traceab
ilit
y
Reference method2ndary reference
material
1ary reference material
Copies (IU/ml)
Test
Routine sample
Result
Materials Measurement
Procedures
Reference
laboratory
Diagnostic
manufacturer
Testing
laboratory
Molecular measurement
dPCR
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Anal Bioanal Chem. 2014 Oct;406(26):6471-83
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The HIV Life Cycle
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HIV-1
• Retrovirus (single stranded RNA genome)
• Quantified clinically:
Guide treatment
Monitor resistance
• Measured internationally as copies(IU)/ml blood, plasma, serum
<50->100,000 copies/ml
RT-qPCR
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Materials for full analytical workflow
Quantity: Viral
DNA/RNA per cell
Quantity:
Genomic copies
Quantity:
Gene copies
Quantity:
Whole Virus
Quantity:
RNA gene copies
Quantity:
RNA genomes
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Comparison of Reverse Transcriptase choice (LTR/GAG assay)
0
0,5
1
1,5
2
2,5
Re
lative
diffe
ren
ce
s c
om
pa
red
to
O
ne
Ste
p
IVT fragment
Viral Genomes
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Comparison of Reverse Transcriptase choice (LTR/GAG assay)
0
0,5
1
1,5
2
2,5
Re
lative
diffe
ren
ce
s c
om
pa
red
to
O
ne
Ste
p
IVT fragment
Viral Genomes
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HXB2 RNA Fragment
Nanodrop
Qubit
Bioanaly
ser
FIREScrip
t
Maxim
a
SuperScrip
t III
Oneste
p1010
1011
1012
HIV LTR assay
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Synthetic gene fragment ‘UB49’
env
5’ LTR gag
pol
vif
vpr
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HXB2 RNA Fragment
Nanodrop
Qubit
Bioanaly
ser
FIREScrip
t
Maxim
a
SuperScrip
t III
Oneste
p1010
1011
1012
HIV LTR assay
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Nanodrop
Qubit
Bioanaly
ser
FIREScrip
t
Maxim
a
SuperScrip
t III
Oneste
p1010
1011
1012
HIV pol assay
HXB2 RNA Fragment
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Quantity: Viral DNA/RNA per cell
Quantity: Genomic copies
Quantity: Gene copies
Quantity: Whole virus
Quantity: RNA gene copies
Quantity: RNA genomes
Materials for full analytical workflow
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17
HIV-1 RNA Extraction Results
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HIV-1 (RNA) Ringtrial 2019
HIV-1 (RNA) Virus Genome Detection Program (360)
Additional HIV-1 (RNA) Training Program (382)
PTB and LGC participated using dPCR
Other 140 laboratories used qPCR
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Acceptance range ±log(0.6) for HIV Rili-BAEK
HIV-1 (RNA) Ringtrial 2019
10
100
1000
10000
100000
1000000
1 2 3 4 5 6 7 8
HIV
-1 c
op
ies/
ml
EQA material
Consensus
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Acceptance range ±log(0.6) for HIV Rili-BAEK
HIV-1 (RNA) Ringtrial 2019
10
100
1000
10000
100000
1000000
1 2 3 4 5 6 7 8
HIV
-1 c
op
ies/
ml
EQA material
Consensus
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10
100
1000
10000
100000
1000000
1 2 3 4 5 6 7 8
HIV
-1 c
op
ies/
ml
EQA material
Consensus
Acceptance range ±log(0.6) for HIV Rili-BAEK
HIV-1 (RNA) Ringtrial 2019
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Acceptance range ±log(0.6) for HIV Rili-BAEK
HIV-1 (RNA) Ringtrial 2019
10
100
1000
10000
100000
1000000
1 2 3 4 5 6 7 8
HIV
-1 c
op
ies/
ml
EQA material
Consensus
PTB
NML
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10
100
1000
10000
100000
1000000
1 2 3 4 5 6 7 8
HIV
-1 c
op
ies/
ml
EQA material
Consensus
PTB
NML
Acceptance range ±log(0.6) for HIV Rili-BAEK
HIV-1 (RNA) Ringtrial 2019
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Conclusion
• dPCR shows good potential as a reference measurement
procedure for HIV-1 RNA quantification
• Further work is required to explore impact of
• Instrument
• Assay (PCR & RT)
• Sequence complexity
• Continue and expand assessment of pre analytical steps
• SI traceable orthogonal comparisons
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P199 –HIV-1 RNA
• Stakeholder relevance
– Viruses (e. g. HIV, Hep C) pose a global problem with severe health effects
– HIV-1 viral load monitoring is directly relevant to clinical management of patient treatment
• Proposed study plan (Duration: Sept 2018 –March 2020)
– Three materials:1. Low concentration genome fragment
2. High concentration genome fragment for orthogonal SI traceable verification
3. Low concentration whole genome material
• Coordinator: LGC with NIBSC contribution
• 13 NMIs participating. Results presented April 2020
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Future
Explore the role of dPCR in ensuring
traceability in laboratory medicine
ISO17511Xpert MTB/RIF
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NML• Alison Devonshire• Denise O’Sullivan• Alexandra Whale• Simon Cowen• Alison Woolford• Helen Parkes• Carole Foy
NIST• Peter Vallone• Megan Cleveland NIB
• Mojca Milavec • Jana Zel
PTB• Andreas Kummrow• Samreen Falak• Annabell Plauth
Acknowledgements
GBD• Heinz Zeichhardt, • Hans-Peter Grunert• Martin Kammel
NIBSC• Neil Almond• Clare Morris• Mei Mei Ho
UCL (Hospitals)• Eleni Nastouli• Kathryn Harris• Tim Mchugh