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Evidence Review Group on Malaria Diagnostics in Low-Transmission Settings, World Health Organization, Geneva December 16, 2013
Photo: © 2012 Diana Mrazikova/Networks/Senegal, Courtesy of Photoshare
Overview of Nucleic Acid-Based Diagnostic Techniques
Introduction
Advantages
•High sensitivity •Earlier detection of infections•Quantification •Species differentiation•Strain identification to distinguish new and recrudescent infections•Potential for high throughput
Disadvantages
•Can be time consuming•Expensive•Extensive training required•Mixed infections require more elaborate assay designs•Requires cross contamination provisions •Standardization is complicated
Attributes of molecular assays
Murphy 2013312/16/2013
Drivers of Molecular Diagnostics Innovation
• Malaria surveillance programs• Epidemiology research• Blood bank screening• Travel medicine• Hunt for sub-patent asymptomatics• Vaccine and drug studies• Passive case detection• Genotyping for origin and resistance• Competition for intellectual property – financial gains
Everyone wants something just a little better – or different
512/16/2013
An Incomplete History
1st LAMP malaria
publication (Poon)
PCR (Mullins)
DNA probes for malaria
diagnostics (Franzen)
1st polymerase chain reaction (PCR)
publication in Science magazine
PCR; Chelex boiling; Dried blood spot on
filter paper (Kain)
Nested PCR sequence-specific amplification
differentiation (Snounou)
(Mullins)
WOW! Year-round asymptomatics
(Roper)
1st LAMP (Notomi)
1st quantitative PCR for malaria
(Hermsen)
1st malaria PCR multiplex
(Kho)Boom
Extraction Chemistry Gametocyte
quantification (Schneider)
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1. Operational Characteristics
Types of PCR•Single-step•Nested•Multiplexed•Quantitative
Design variables•Extraction•Choice of target•Infrastructure capacity
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Single-Step PCR
• Fewer steps and time compared to nested• Less risk of contamination• Electrophoresis gel readout• Range of limit of detection from 0.002-30 p/µl
(Alemayehu 2013)• Trade-off – less sensitive than nested (Singh 1999)
Low frills – no bells and whistles
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Nested PCR
• Increased specificity – two sets of primers
• Two reactions with sample transfer between them
• First assay to detect presence of fewer than 10 parasites from the 4 human malaria species (Snounou 1993)
• Time and cost• Opening tubes risks contamination
Increased specificity with trade-offs
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Multiplexed PCR
• Simultaneous, multiplex PCR to detect malarial species present (Padley 2003)
• Cost and time savings• Primer competition – decreased
sensitivity compared to monoplex; 0.2- 5 p/µL (Alemayehu 2013)
• Overcome decrease in sensitivity with novel targets and probes (Taylor 2010)
Convenience at a cost
(Demas 2011)12/16/2013 10
Quantitative PCR (qPCR)
• Visualization• Precision• Simultaneous detection• Quantification of target DNA• Increased capital and reaction costs
…also known as Real Time PCR (but never RT-PCR)
(Elsayed 2006 and Alemayehu 2013)12/16/2013 11
Standardization?
• PCR machine used can influence the results (Mens 2010)
• PCR assay parameters: • Specific polymerase• Specific temperatures/time/cycles – important to note that a 1°C change in temperature can have
a huge impact on the results – yet calibrated thermocouples can only hold +/1 0.5°C• Hot start• 2 temperatures vs 3 temperatures • Intercolating dyes vs fluorescent probes• Batch-to-batch variations in enzymes, mastermix; cold chain requirements, etc• Other thermocycler machine design considerations (Almassian 2013)
• Peltier vs exotherm, thin resistive film vs continuous flow heat• Bonnet heating vs oil (LaBarre et al unpublished)
• Ramp time between cycles (LaBarre et al unpublished)• Machine maintenance/calibration: Who?/How often?
• Choice of target gene and primers• Standardization should start at the point of sampling• MIQE standard for qPCR reporting (Bustin 2009)
• Standard DNA for comparison of techniques – WHO NAT STD DNA (Padley 2008)
• Repeatable results require standardized tactics supported by standardized tools and process
Many variables to consider
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Extraction
• Highest efficiency from chaotropic and silica binding (Boom 1990)
• BUT, high cost, steps, time, centrifuge requirements• Boil and spin – possible if inhibitors are not a problem (e.g.,
isothermal methods)• Chelex-100• Immiscible fluids• Many syringe-based alternatives
Concentrate sample, lyse cells, isolate nucleic acid, eliminate inhibitors
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• > 65 Primer sets; > 5 targets (Murphy 2013)
• 18S target is the most common• Moderate copy number• Well conserved
• BUT, it is not the perfect target• Sequence variation – Po and Pm – poor annealing/false negatives
(Erdman 2008)
• Actually 4 small subunits rRNA genes expressed during Plasmodium lifecycle (Murphy 2012)
• Alternatives:• Cytochrome b gene (Farrugia 2011)
• Mitochondrial genes (Polley 2010)
• Var (Farrugia 2011)
• Stevor (Berry 2008)
Target selection has enormous impact
Targets and Primers
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Target Implications
References
1. Snounou 19962. Steenkeste 2009 3. Snounou 1993 4. Farrugia 2011 5. Baltzell 2013 6. Polley 2010 7. Polley 2013 8. Demas 2011 9. Patel 201310. Murphy 2012
PATH unpublished12/16/2013 16
Detection of Total Nucleic Acid (DNA and RNA)
• Amplification of total nucleic acid (18S rRNA genes) significantly increases the analytical sensitivity of the assay... roughly a log improvement (Kamau 2011)
• Detection of total nucleic acid (cyt b) via simultaneous qPCR and reverse transcriptase qPCR resulted in a 3-log reduction in the LOD as compared to DNA only (Waitumbi 2011)
• Cost: Add $1.00 for reverse transcriptase step (personal communication, Kamau 2013)
• Trade-off – RNA is inherently more friable, and, therefore, (despite nice results in Jones 2012 with filter spots) more difficult and potentially more expensive to transport samples
Reverse transcriptase
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PortabilityTrends toward level 1 facility:
•Battery power•Reduced mass•Ease of use•Reduced cost•Trade-off: reduced performance
16 samples;
USB; $599
1 lbrequirements
gel
2 colorreal time
10 lbs barcode reader
Almassian 2013
Isothermal, end point
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Nucleic Acid Amplification at Level Zero?
• NALFIA or molecular RDTs: using hybridization of labeled amplicon (Mens 2011)
• Exothermic heat (Singleton 2013)
• Magnetically-driven sample in/results out disposable lab
Enabling technologies focus on instrument-free, minimal complexity
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Loop-Mediated Isothermal Amplification
• Results in 30 minutes w/ tube scanner (Surabattula 2013)
• RealAmp using intercalating dyes for Pv (Patel 2013)
• N=272 in Uganda (Hopkins 2013)
• 705 travelers (Polley 2013)
• Mitochondrial targets increase sensitivity (Polley 2010)
2013 updates
Ghani 2012
Polley et al 2010
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Quantitative Nucleic Acid Sequence-Based Amplification
Reverse transcriptase built into the assay
• Gametocyte quantification: Pfs25 mRNA (Schneider 2004)
• Reduced sample prep compared with PCR due to less inhibition (Schneider 2004)
• High sensitivity due to abundance of rRNA (Schneider 2005)
• Detect all four Plasmodium species causing human disease targeting 18S rRNA gene (Mirangi 2009)
Niemz 2011
Schneider 2003
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Limit of Detection
Cordray 2012 and Vasoo 2013
Depends on:
•Sample prep/extraction efficiency•Amount of blood•Sample format and storage
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Method LOD (p/µl) Microscopy 50 Nested PCR 6 RDT > 100 Lab-based PCR < 5 qPCR 0.1-10 Pf 0.7 Pv 4 Po 1.5 Total nucleic acid qPCR Pan 0.002 Pf 1.2 4 primer qPCR Pf 0.02 Pv 0.02 Po 0.004 Pm 0.006 LAMP 0.2-5 NASBA 0.01-0.1 NALFIA 0.3-3
Clinical Accuracy
Average density affects• Sensitivity• Specificity
a————
a + c
d————
b + d
a————
a + b
d————
c + d
Prevalence affects• PPV• NPV
Population dependent
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Sample Compartments
Buppan 2010 and Nwakanma 2009
Less invasive = less sensitive
• Typical mosquito proboscis is 1.5 to 2.0 mm in length with an inner fascicle diameter of 20 m• Designed for intradermal sampling
What about intradermal sampling???
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Robustness: Quality of EvidenceAppropriate use data: lacking!Repeatability: lacking!
• Inter-operator repeatability
• Inter-lab repeatability• Controlled lab results
mirror clinical results
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Time to ResultsTotal vs hands-on time: Implications for:
• Work flow• Throughput
Cordray 2012 and Hwang 201112/16/2013 35
Total time (h) Hands-on time (h) Microscopy 0.8 0.8
qPCR 2.5 1 Multiplex 4.5 2
Nested 10 3
Method Time (h) Microscopy 0.3/slide
RDT 0.3 Lab-based PCR 1
qPCR 1 PCR-ELISA 6
LAMP 0.5-2 NASBA 1 NALFIA 1-1.5
Cost Estimates
Cordray 2012, Canier 2013, Hsiang 2012, and Erdman 2008
How will PCR costs change when patents expire? •Taqman polymerase•Taqman primers
* Includes extraction, Canier 2013
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Method Per sample (USD) Equipment Microscopy $0.20-$0.26 $200-$700
RDT $0.45-$1.50 Lab-based PCR $1.50-$4.00 $500-$5,000
qPCR $2.75*-$5.00 > $20,000 PCR LDA $0.30 NA
LAMP $4.00-$5.00 $500-$5,000 NASBA $5.00-$20.00
3. Cost
Summary - The Future….
1st LAMP malaria
publication (Poon)
PCR (Mullins)
DNA probes for malaria
diagnostics (Franzen)
1st PCR publication in Science magazine
PCR; Chelex boiling; Dried blood spot on
filter paper (Kain)
Nested PCR sequence-specific amplification
differentiation (Snounou)
(Mullins)
WOW! Year-round asymptomatics
(Roper)
1st LAMP (Notomi)
1st quantitative PCR for malaria
(Hermsen)
1st malaria PCR multiplex
(Kho)Boom
Extraction Chemistry Gametocyte
quantification (Schneider) ?
More optionsImprovements
StandardizationQOE - Utility
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Thank youPhoto: © 2012 Diana Mrazikova/Networks/ Senegal, Courtesy of Photoshare
For more information | Paul LaBarre, Project DirectorKathy Tietje, Project Managerinfo@path.org www.path.org
12/16/2013 39
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