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Evidence Review Group on Malaria Diagnostics in Low-Transmission Settings, World Health Organization, Geneva December 16, 2013
•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
Over Generalizations Lead to ….
412/16/2013
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)
12/16/2013 6
1. Operational Characteristics
Types of PCR•Single-step•Nested•Multiplexed•Quantitative
Design variables•Extraction•Choice of target•Infrastructure capacity
12/16/2013 7
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
12/16/2013 8
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
12/16/2013 9
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
12/16/2013 12
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
• 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)
• 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
12/16/2013 17
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
12/16/2013 18
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
12/16/2013 19
Isothermal Methods Overview
Niemz 2011 12/16/2013 20
Loop-Mediated Isothermal Amplification
• Results in 30 minutes w/ tube scanner (Surabattula 2013)
• RealAmp using intercalating dyes for Pv (Patel 2013)
For more information | Paul LaBarre, Project DirectorKathy Tietje, Project [email protected] www.path.org
12/16/2013 39
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