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A Tale of Two Tests

The coming of age of multiplexed nucleic acid

amplification tests in the Microbiology lab

Thomas Novicki PhD DABMM

Clinical Microbiologist

Table of Contents (2)

Review nucleic acid amplification tests (NAAT)

Describe the advantages and pitfalls of NAATs

Review the clinical presentations and causes of acute respiratory infection and acute gastroenteritis

Describe the current FDA-cleared highly multiplexed NAATs for these two syndromes, and what these assays have to offer over traditional diagnostic studies

Disclaimer (3)

I served as the site principle investigator for the Phase III clinical trial of the Prodesse ProGastro SSCS (Hologic Gen-Probe, Inc.) NAAT for bacterial gastrointestinal pathogens

THE TECHNOLOGY

NAATs, History (5)

The first NAAT, the polymerase chain reaction (PCR), was invented in the early 1980’s by Dr. Kary Mullis et al. It relies on rounds of thermal cycling and a thermostable DNA polymerase (e.g. Taq polymerase) to amplify a single piece of DNA into millions of copies, thus allowing for its detection by a variety of methods: Agarose gel

Fluorescent DNA probes

Melting temperature of the amplified product

Some Variations of the Original PCR (6)

Multiplex PCR Amplifies >1 target in a single reaction

RT-PCR RNA is amplified by adding a Reverse Transcriptase enzyme to first convert the RNA into a complimentary DNA (cDNA) molecule that is then amplified by PCR

Real Time PCR Product detection occurs during amp cycling in a closed system instead of afterwards (reducing contamination)

Quantitative PCR Uses real time PCR to determine the input amount of nucleic acid

Non-PCR based amplification methods

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NAATs-Advantages and Pitfalls (7)

Advantages

Able to detect minute quantities of microbes (theoretically 1 cell/viral particle)

Can detect ‘unculturable’ microbes

Rapidly produces results, generally within a few hours, vs. days to weeks for culture

May be relatively inexpensive after the initial equipment outlay (but patent protection and commercial markup of reagents drives up cost

NAATs-Advantages and Pitfalls (8)

Pitfalls

Contamination (‘amplicon’ or environmental) can cause false positive results Real-time NAATs are closed systems

Linear workflow in the lab

Amplification chemistry mods (e.g. UNG)

Inhibitors endogenous to the sample causing false negative results Better extraction protocols Internal controls

NAATs-Advantages and Pitfalls (9)

Pitfalls, continued

NAAT techniques often foreign to lab staff Undergraduate, med tech schools now teach it

Primer/probe sets are designed to be highly specific to avoid false positive results, but naturally occurring genetic variants may not be detected S. aureus mecALGA251 : OXA-R / mecA PCR Negative*

(*L Garcia-Alvarez, MTG Holden, H Lindsay et al. 2011. Lancet Inf. Dis. 11:595)

THE DISEASES

Acute Respiratory Infection (ARI) (11)

ARI is a collection of syndromes affecting the upper or lower respiratory tract airways

The ear and mastoid bones as sites contiguous to the upper airways are often secondarily infected

May be caused by exogenous inhaled microbes (primarily viruses) or endogenous flora (bacteria)

ARI Impact (12)

Collectively, the most common type of human infection. Worldwide:

Estimated >94 million Disability-adjusted life years (DALYs) annually

Estimated 3.9 million attributable deaths annually

WHO http://www.who.int/vaccine_research/diseases/ari/en/index.html (accessed 11/11/13)

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ARI Syndromes (13)

Upper Airway ARI Lower Airway ARI

The common cold1 Bronchitis2

Pharyngitis2 Bronchiolitis1

Laryngitis2 Pneumonia3

Laryngotracheobronchitis (Croup)1 Acute exacerbations of chronic lower

airway disease3 Otitis media3

Sinusitis4

Epiglottitis4

Etiology 1Predominantly or universally viral. 2Primarily viral; plus ‘atypical’ bacteria. 3Viral and bacterial. 4Predominantely or universally bacterial.

ARI-Etiologic Agents That Begin in the Upper Airways (14)

Viruses ‘Atypical’ Bacteria

Adenovirus Bordetella pertussis

Bocavirus (?) Chlamydophilia pneumoniae

Coronavirus Mycoplasma pneumoniae

Enterovirus

Human Metapneumovirus

Influenza A

Influenza B

Parainfluenza 1-4

Respiratory Syncytial Virus

Rhinovirus

ARI-Etiologic Agents: What & Where (15)

Cold Pharyngitis Laryngitis Bronchitis Bronchiolitis

Adenovirus X X X X X

Bocavirus +/- +/- +/- +/- +/-

Coronavirus X X X X X

Enterovirus X

Human Metapneumovirus X X X X

Influenza A X X X X X

Influenza B X X X X X

Parainfluenza X X X X X

Respiratory Syncytial Virus X X X

Rhinovirus X X X X X

Bordetella pertussis X

Chlamydophilia pneumoniae X X

Mycoplasma pneumoniae X X

Notes 1. Infections with more than 1 agent is not uncommon.

2. Not an exhaustive list of etiologic agents 3. for a given syndrome, some agents are more common than others

ARI-To Summarize… (16)

For many ARIs, the syndromes overlap

The etiologic agents may be viral, bacterial, or a combination of both

Treatment will vary by agent (i.e. antibacterial, antiviral, or no treatment)

If the patient is being admitted to hospital, knowing the etiologic agent allows for cohorting

Antibacterial agents (i.e. ‘antibiotics’) while often worthless for ARI are nevertheless frequently prescribed (and we all know what that’s leading to… pan-resistant bacteria that cannot be treated)

Acute Gastroenteritis (AG) (17)

Defined: inflammation of the gastrointestinal tract leading to some combination of diarrhea, vomiting and abdominal pain. Fever and other systemic symptoms are also common (agent-dependent)

Globally, AG causes

Estimated 2,000 million cases annually

Estimated 1.8 million deaths annually

WHO http://www.who.int/mediacentre/factsheets/fs330/en/index.html (Accessed 11/11/13)

AG-Symptomology (18)

Unlike ARI, AG presents differently based upon the etiologic agent: Presence and proportion of the 3 main symptoms

(diarrhea, vomiting, cramps)

Duration of illness

Travel and behavior history

Presence/absence of systemic symptoms

Type of diarrhea: secretory (watery), dysenteric (bloody), inflammatory (stool WBC+)

This allows the clinician to choose the appropriate diagnostic studies

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AG-The Etiologic Agents (19)

Bacteria

Aeromonas Campylobacter Clostridium difficile

E. coli O157, other Shiga toxigenic serotypes

Other entero-pathogenic E. coli

Entero-pathogenic Klebsiella pneumoniae

Plesiomonas Salmonella Shigella

Vibrio cholerae Yersinia enterocolitica Other bacteria

Parasites

Cryptosporidium Cyclospora Entamoeba histolytica

Giardia Isospora Other intestinal parasites

Viruses

Adenovirus Norovirus Rotavirus

Other viruses LAB ASSAYS

Current Lab Assays (21)

ARI

Bacterial culture

Immunofluoresence assay

Influenza/RSV NAAT

Rapid antigen tests

Viral culture

AG

Bacterial culture

Ova & Parasite exam

Parasite/virus antigen IA

Shiga toxin EIA

Norvirus NAAT

Current Lab Assays–Some Limitations (22)

Test Limitation

Bacterial culture Longer TAT, requires viable cells, labor intensive

Immunofluoresence assay Requires fluorescent microscope, trained techs, good collection technique

Influenza/RSV NAAT Doesn’t detect other respiratory viruses

Norvirus NAAT Not readily available

Ova & Parasite exam Labor-intensive, toxic chemicals, insensitive for common AG agents

Rapid antigen tests Lacks sensitivity

Viral culture Longer TAT, requires cell culture lab

NAATs in the Diagnostic Lab (23)

NAATs are not new to infectious disease diagnostics Often have been lab-developed (i.e. not FDA-

cleared/approved) with inconsistent inter-lab performance, but many commercial 1-3 plex NAATs now available

NAATs, do not produce a viable isolate for AST (antimicrobial susceptibility tests), a downside for bacterial NAATs

NAATs have thus had more applicability to viral diagnostics In fact, virus culture has been eliminated at many academic

centers in favor of NAATs

Highly Multiplexed NAATs (hmNAAT) (24)

hmNAAT (>3 targets in a single reaction, my definition) have recently caught the attention of commercial vendors

hmNAATs are of most value when an isolate of the agent is not needed for further studies AST

Typing (a function of the public health lab, we’ll return to this…)

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hmNAATs-What’s on the Market? (25)

ARI AG

FilmArray Respiratory Panel (BioFire Diagnostics, Inc.)

ProGastro SSCS (Hologic Gen-Probe, Inc.)

xTAG Respiratory Viral Panel (Luminex Molecular Diagnostics, Inc.)

xTAG Gastrointestinal Pathogen Panel (Luminex Molecular Diagnostics, Inc.)

eSensor Respiratory Viral Panel (GenMark Diagnostics, Inc.)

Others are in development

hmNAATs vs Conventional Tests (26)

hmNAATs…

Advantages Disadvantages

Offers the opportunity to produce results in a clinically relevant timeframe

Slower than rapid antigen tests

More sensitive, and equally specific, than other methods

Per-test costs higher than other methods, often by a large margin

Less labor-intensive than culture (and sometimes conventional NAATs too)

Requires specialized equipment

May offer high-throughput testing (platform-dependent)

Requires at least some molecular lab-type practices to avoid contamination (platform-dependent)

Susceptible to other NAAT limitations (e.g. genetic drift leading to false negative results)

Clinical utility not yet proven

xTAG RVP (27)

Targets

Adenovirus Influenza B

Human Metapneumovirus Parainfluenza1

Influenza A Respiratory Syncytial Virus

Rhinovirus

Features

Two versions, v1 and FAST

FAST is, well, faster (6hr vs 8+hr)

But FAST is also less sensitive

Off-line extraction, amplification and hybridization steps make full molecular lab capability a must

Throughput: 96 per run

Open system: contamination a risk

Detection is by fluorescence sorting

Off-line PCR equipment, and Luminex 100/200 instrument may be used for other assays

While v1 is the first of this group, its been passed by in some ways

May make sense if your lab uses the Luminex 100/200 detection system for other assays

1 xTAG only

FilmArray RP (28)

Targets Viruses Bacteria

Adenovirus Bordetella pertussis Coronavirus Chlamydophilia pneumoniae

Enterovirus/Rhinovirus Mycoplasma pneumoniae Human Metapneumovirus

Influenza A Influenza B

Parainfluenza Respiratory Syncytial Virus

Features

Small footprint, relatively low capital equipment costs

Uniquely, includes 3 atypical bacteria

Self-contained: low chance of lab contamination; ease of use

Detection is by melting curve analysis

Low throughput: ~1/hr

Reported to have reduced sensitivity for Adenovirus; Co. recently modified assay to address the shortcoming

More assays available and in development for this platform

Reasonable as a single respiratory virus NAAT in a smaller setting, or in a larger one as an adjunct to higher throughput influenza/RSV NAATs

eSensor RVP (29)

Targets

Adenovirus Influenza B

Human Metapneumovirus Parainfluenza

Influenza A Respiratory Syncytial Virus

Rhinovirus/Enterovirus

Features

Off-line extraction, amplification require full molecular lab capability

Throughput: 24samples/ instrument/6-7hr

Detection by electrochemical detection

Open system: contamination a risk

GenMark has other assays for this instrument

An improvement over the xTAG RVP, this assay makes sense for high volume molecular labs

Respiratory hmNAATs-Performance (30)

Sensitivity (%)

Specificity (%)

xTAG RVP v1 93 100

xTAG RVP FAST 84 100

eSensor RVP 98 99

FilmArray RP1 85 100

Comparisons of all respiratory hmNAATs are rare. In general however, all of these perform equal to or better than conventional methods

Questions remain

What population(s) to test?

Use as a 2o test with influenza/RSV NAAT, or as the single test platform

Should we be testing for the ‘other’ respiratory pathogens at all?

Mostly less virulent

No proven antiretroviral treatment

Clinical outcomes studies are lacking

Our experience: docs like to know in seriously ill (i.e. hospitalized) patients

1 Pre-FDA cleared version: lacked 3 bacterial targets; lacked upgraded Adenovirus detection released with v1.7 EB Popowitch, SS O’Neill, and M Miller. 2013 J. Clin. Microbiol. 51:1528

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AG hmNAATs (31)

Targets ProGastro SSCS xTAG GPP

Bacteria

Campylobacter X X

Clostridium difficile X

E. coli O157, and other Shiga-toxin+ E. coli X X

Enterotoxigenic E. coli X

Salmonella X X

Shigella X X

Parasites

Giardia X

Cryptosporidium X

Viruses

Norovirus X

Rotavirus X

xTAG GPP (32) Features

96 samples/6-7hrs

Off-line extraction, amplification and hybridization steps make full molecular lab capability a must

Open system: contamination a risk

Off-line PCR equipment, and Luminex 100/200 instrument may be used for other assays (e.g. xTAG RVP, others)

Challenges

FDA considers these results presumptive, and requires confirmatory testing. SSCS does not have this limitation.

Detecting bacteria, parasites and fungi in a single assay is a new paradigm, requiring buy-in from medical staff, compliance and fees committees, insurance providers, and patients.

ProGastro SSCS (33) Features

Medium throughput a good fit for many labs, and is scalable by adding additional extraction capacity and SmartCycler units.

Makes a good replacement test for fecal culture + Shiga toxin EIA: easier to sell all-around

Detection is by real time PCR, so is a closed system and contamination is limited. However, a dedicated molecular lab is still required

Results are not considered presumptive

Gastrointestinal hmNAATs-Performance (34)

Targets ProGastro SSCS1 xTAG GPP2

Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%)

Campylobacter 96 94 90 99

Clostridium difficile -- -- 91 100

Shiga toxins 1,2 (i.e. E. coli O157 and other STEC3) 100 100 -- --

Enterotoxigenic E. coli/Shiga toxins 1,2 -- -- 94 100

E. coli O157:H7 -- -- 100 100

Salmonella 100 100 92 100

Shigella 100 100 93 100

Giardia -- -- 95 99

Cryptosporidium -- -- 100 100

Norovirus -- -- 96 100

Rotavirus -- -- 100 100

1BW Buchan, WJ Olson, M Pezewski et al. 2013. J. Clin. Microbiol. ahead of print doi 10.1128/JCM.02056-13 2JF Navidad, DJ Griswold, MS Gradus et al. 2013. J. Clin. Microbiol. 51:3018 3STEC, Shiga toxin producing E. coli

Challenges (35)

There are many unanswered questions…

Are these capital-intensive, supply-expensive assays really better? Do they lead to better patient outcomes?

Will they lead to less antimicrobial (mis)use?

Do they save money in the lab?

Do they save money for the institution?

What level of multiplexing is right?

How do we bill for them? CPT codes are lacking, and the AMA always seems behind the curve

Insurers are still skeptical, and need to be convinced

More studies are needed!

Conclusions (36)

hmNAATs are now a viable alternative to conventional methods in the clinical microbiology lab (although they don’t always fit well there), and more are on the horizon

They offer superior performance with a shorter turn-around time compared to conventional methods

The microbiology lab is one of the last bastions of manual testing in clinical laboratory medicine: those walls are beginning to come down however

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THE END. THANK YOU. QUESTIONS?