1. 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

2. Overview 2 12/16/2013 1.Operational characteristics 2.Performance 3.Cost

3. 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 2013 3 12/16/2013

4. Over Generalizations Lead to …. 412/16/2013

5. 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

6. An Incomplete History 1 st LAMP malaria publication (Poon) PCR (Mullins) DNA probes for malaria diagnostics (Franzen) 1 st 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) 1 st LAMP (Notomi) 1 st quantitative PCR for malaria (Hermsen) 1 st malaria PCR multiplex (Kho) Boom Extraction Chemistry Gametocyte quantification (Schneider) 12/16/20136

7. 1. Operational Characteristics Types of PCR Single-step Nested Multiplexed Quantitative Design variables Extraction Choice of target Infrastructure capacity 12/16/20137

8. 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 12/16/20138

9. 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 12/16/20139

10. 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) 12/16/201310

11. 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) 12/16/201311

12. 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 12/16/201312

13. 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 12/16/201313

14. Extraction Landscape PATH unpublished 12/16/201314

15. > 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 12/16/201315

16. Target Implications References 1. Snounou 1996 2. Steenkeste 2009 3. Snounou 1993 4. Farrugia 2011 5. Baltzell 2013 6. Polley 2010 7. Polley 2013 8. Demas 2011 9. Patel 2013 10. Murphy 2012 PATH unpublished 12/16/201316

17. 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 12/16/201317

18. Portability Trends toward level 1 facility: Battery power Reduced mass Ease of use Reduced cost Trade-off: reduced performance Palm PCRt Open PCR B io Fire Tetracore T-CORt 8 POCKIT 16 samples; USB; $599 1 lb requirements gel 2 color real time 10 lbs barcode reader Almassian 2013 Isothermal, end point 12/16/201318

19. 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 12/16/201319

20. Isothermal Methods Overview Niemz 2011 12/16/201320

21. 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 12/16/201321

22. 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 12/16/201322

23. 2. Performance Review of reviews Accuracy Time to results 12/16/201323

24. Review of Point-of-Care Nucleic Acid Amplification Test Platforms Niemz 2011 12/16/201324

25. Review of Malaria Nucleic Acid Amplification Test Vasoo 2013 12/16/201325

26. Review of Malaria Nucleic Acid Amplification Test (continued) Vasoo 2013 12/16/201326

27. Review of Point-of-Care Malaria Diagnostics Platforms Cordray 2012 12/16/201327

28. Cordray 2012 Review of Malaria Nucleic Acid Amplification Test Platforms 12/16/201328

29. Berry 2008 Review of Malaria PCR Methods 12/16/201329

30. Accuracy Analytical – Limit of detection Clinical Robustness 12/16/201330

31. Limit of Detection Cordray 2012 and Vasoo 2013 Depends on: Sample prep/extraction efficiency Amount of blood Sample format and storage 12/16/201331

32. 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 12/16/201332

33. 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??? 12/16/201333

34. Robustness: Quality of Evidence Appropriate use data: lacking! Repeatability: lacking! Inter-operator repeatability Inter-lab repeatability Controlled lab results mirror clinical results 12/16/201334

35. Time to Results Total vs hands-on time: Implications for: Work flow Throughput Cordray 2012 and Hwang 2011 12/16/201335

36. 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 12/16/201336 3. Cost

37. Summary - The Future…. 1 st LAMP malaria publication (Poon) PCR (Mullins) DNA probes for malaria diagnostics (Franzen) 1 st 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) 1 st LAMP (Notomi) 1 st quantitative PCR for malaria (Hermsen) 1 st malaria PCR multiplex (Kho) Boom Extraction Chemistry Gametocyte quantification (Schneider) ? More options Improvements Standardization QOE - Utility 12/16/201337

38. Thank you Photo: © 2012 Diana Mrazikova/Networks/ Senegal, Courtesy of Photoshare For more information | Paul LaBarre, Project Director Kathy Tietje, Project Manager info@path.org www.path.org

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