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

Overview

212/16/2013

1. Operational characteristics2. Performance3. Cost

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

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

Concentrate sample, lyse cells, isolate nucleic acid, eliminate inhibitors

12/16/2013 13

Extraction Landscape

PATH unpublished12/16/2013 14

• > 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

12/16/2013 15

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

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)

• N=272 in Uganda (Hopkins 2013)

• 705 travelers (Polley 2013)

• Mitochondrial targets increase sensitivity (Polley 2010)

2013 updates

Ghani 2012

Polley et al 2010

12/16/2013 21

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

12/16/2013 22

2. Performance

Review of reviews

Accuracy

Time to results

12/16/2013 23

Review of Point-of-Care Nucleic Acid Amplification Test Platforms

Niemz 2011 12/16/2013 24

Review of Malaria Nucleic Acid Amplification Test

Vasoo 201312/16/2013 25

Review of Malaria Nucleic Acid Amplification Test (continued)

Vasoo 201312/16/2013 26

Review of Point-of-Care Malaria Diagnostics Platforms

Cordray 2012 12/16/2013 27

Cordray 2012

Review of Malaria Nucleic Acid Amplification Test Platforms

12/16/2013 28

Berry 2008

Review of Malaria PCR Methods

12/16/2013 29

Accuracy

Analytical – Limit of detection

Clinical

Robustness

12/16/2013 30

Limit of Detection

Cordray 2012 and Vasoo 2013

Depends on:

•Sample prep/extraction efficiency•Amount of blood•Sample format and storage

12/16/2013 31

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

12/16/2013 32

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???

12/16/2013 33

Robustness: Quality of EvidenceAppropriate use data: lacking!Repeatability: lacking!

• Inter-operator repeatability

• Inter-lab repeatability• Controlled lab results

mirror clinical results

12/16/2013 34

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

12/16/2013 36

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

12/16/2013 37

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

1. Abdul-Ghani R, Al-Mekhlafi AM, Karanis P. Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta Tropica. 2012; 122(3):233–240.

2. Alemayehu S, Feghali KC, Cowden J, et al. Comparative evaluation of published real-time PCR assays for the detection of malaria following MIQE guidelines. Malaria Journal. 2013; 12(277):doi: 10.1186/1475-2875-12-277.

3. Almassian DR, Cockrell LM, Nelson WM. Portable nucleic acid thermocyclers. Chemical Society Reviews. 2013; 42(22):8769–8798. 4. Baltzell KA, Shakely D, Hsiang M, et al. Prevalence of PCR detectable malaria infection among febrile patients with a negative Plasmodium falciparum

specific rapid diagnostic test in Zanzibar. The American Journal of Tropical Medicine and Hygiene. 2013; 88(2):289–291. 5. Berry A, Benoit-Vical F, Fabre R, et al. PCR-based methods to the diagnosis of imported malaria. Parasite. 2008; 15(3):484–488. 6. Boom R, Sol CJ, Salimans MM, et al. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology. 1990; 28(3):495–503.7. Buppan P, Putaporntip C, Pattanawong U, et al. Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine

samples from symptomatic malaria patients in a low endemic area. Malaria Journal. 2010; 9(72):doi: 10.1186/1475-2875-9-72. 8. Bustin SA, Benes V, Garson JA, et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical

Chemistry. 2009; 55(4):611–622. 9. Canier L, Khim N, Kim S, et al. An innovative tool for moving malaria PCR detection of parasite reservoir into the field. Malaria Journal. 2013;

12(405):doi: 10.1186/1475-2875-12-405. 10. Cordray MS, Richards-Kortum RR. Review: Emerging Nucleic Acid Based Tests for Point-of-Care Detection of Malaria. The American Journal of Tropical

Medicine and Hygiene. 2012; 87(2):223–230. 11. Demas A, Oberstaller J, DeBarry J, et al. Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S

rRNA. Journal of Clinical Microbiology. 2011; 49(7):2411–2418. 12. Elsayed S, Plewes K, Church D, et al. Use of molecular beacon probes for real-time PCR detection of Plasmodium falciparum and other plasmodium

species in peripheral blood specimens. Journal of Clinical Microbiology. 2006; 44(2):622–624. 13. Erdman LK, Kain KC. Molecular diagnostic and surveillance tools for global malaria control. Travel Medicine and Infectious Disease. 2008; 6(1-2):82–99. 14. Farrugia C, Cabaret O, Botterel F, et al. Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers. Journal of

Clinical Microbiology. 2011; 49(6):2191–2195. 15. Hopkins H, Gonzalez IJ, Polley SD, et al. Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-

mediated isothermal amplification kit in a remote clinic in Uganda. The Journal of Infectious Diseases. 2013; 208(4):645–652. 16. Hwang SY, Kim SH, Lee GY, et al. A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low

parasitized individuals. Acta Tropica. 2011; 120(1-2):40–45. 17. Jones S, Sutherland C, Hermsen C, et al. Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes. Malaria

Journal. 2012; 11(1):266. 18. Kamau E, Tolbert LS, Kortepeter L, et al. Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for

detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes. Journal of Clinical Microbiology. 2011; 49(8):2946–2953.

19. Kamau E, Alemayehu S, Feghali KC, et al. Multiplex qPCR for detection and absolute quantification of malaria. PLoS One. 2013; 8(8):e71539. 20. Murphy SC, Prentice JL, Williamson K, et al. Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum

infections in malaria human challenge trials. The American Journal of Tropical Medicine and Hygiene. 2012; 86(3):383–394.

References

12/16/2013 40

21. Murphy SC, Shott JP, Parikh S, et al. Malaria Diagnostics in Clinical Trials. The American Journal of Tropical Medicine and Hygiene. 2013; 89(5):824–839. 22. Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for infectious diseases. Trends in Biotechnology. 2011; 29(5):240–250. 23. Nwakanma DC, Gomez-Escobar N, Walther M, et al. Quantitative Detection of Plasmodium falciparum DNA in Saliva, Blood, and Urine. The Journal of

Infectious Diseases. 2009; 199(11):1567–1574. 24. Padley D, Moody AH, Chiodini PL, et al. Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present. Annals

of Tropical Medicine and Parasitology. 2003; 97(2):131–137. 25. Padley DJ, Heath AB, Sutherland C, et al. Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA

for nucleic acid amplification technique (NAT)-based assays. Malaria Journal. 2008; 7(139):doi: 10.1186/1475-2875-7-139.26. Patel JC, Oberstaller J, Xayavong M, et al. Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of

Plasmodium vivax. PLoS One. 2013; 8(1):e54986. 27. Polley SD, Mori Y, Watson J, et al. Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification.

Journal of Clinical Microbiology. 2010; 48(8):2866–2871. 28. Polley SD, Gonzalez IJ, Mohamed D, et al. Clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria. The Journal of

Infectious Diseases. 2013; 208(4):637-644. 29. Schneider P, Schoone G, Schallig H, et al. Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative

nucleic acid sequence-based amplification. Molecular and Biochemical Parasitology. 2004; 137(1):35–41. 30. Schneider P, Wolters L, Schoone G, et al. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification

of Plasmodium falciparum. Journal of Clinical Microbiology. 2005; 43:402–405. 31. Singh B, Bobogare A, Cox-Singh J, et al. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic

studies. The American Journal of Tropical Medicine and Hygiene. 1999; 60(4):687–692. 32. Singleton J, Zentner C, Buser J, et al. Instrument-free exothermic heating with phase change temperature control for paper microfluidic devices. SPIE

Proceedings. 2013; 8615:86150R–86150R. 33. Snounou G, Viriyakosol S, Jarra W, et al. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction

and detection of a high prevalence of mixed infections. Molecular and Biochemical Parasitology. 1993; 58(2):283–292. 34. Snounou G. Detection and identification of the four malaria parasite species infecting humans by PCR amplification. Methods in Molecular Biology.

1996; 50:263–291. 35. Steenkeste N, Incardona S, Chy S, et al. Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.

Malaria Journal. 2009; 8(86):doi: 10.1186/1475-2875-8-86. 36. Surabattula R, Vejandla MP, Mallepaddi PC, et al. Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal

amplification (LAMP). Experimental Parasitology. 2013; 134(3):333–340. 37. Taylor SM, Juliano JJ, Trottman PA, et al. High-throughput pooling and real-time PCR-based strategy for malaria detection. Journal of Clinical

Microbiology. 2010; 48(2):512–519. 38. Waitumbi JN, Gerlach J, Afonina I, et al. Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid

amplification in a malaria-endemic region during the peak malaria transmission season. Tropical Medicine and International Health. 2011; 16(7):786–793.

39. Vasoo S, Pritt BS. Molecular diagnostics and parasitic disease. Clinics in Laboratory Medicine. 2013; 33(3):461–503.

References