Easy-Plex Molecular Diagnostics - IACLD Portaliacld.ir › DL › co › 14 › workshop ›...

Easy-Plex Molecular Diagnostics

Dmitry Kuevda, PhDAusDiagnostics Pty Ltd, Australia

www.AusDiagnostics.comwww.AusDiagnostics.com

Multiplexed Diagnostics - What we try to deliver?

One Sample

One Method and one Diagnostic System

One Run

Many targets: bacteria, viruses, protozoa, parasites, resistance genes,oncogene etc.

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Multiplexed Diagnostics - RELIABLE Healthcare

With multiplexed diagnostics you see the whole picture, not just a part

Knowledge of the whole picture can change patient’s treatment ormanagement What if the symptoms were not so clear?

What if there is a multiple infection?

What if there is occult or hidden infection?

What if there is an unusual infection, which was not expected?

etc. 3

Virus N

Virus M

Bacteria XBacteria Y

Parasite A

NEGATIVEVirus N

All others ornegative

Single-plexDiagnostics

MultiplexDiagnostics


Multiplexed Diagnostics - Easy-going Healthcare

Main idea is a consolidation of tests

Multiple prescriptions - one kit

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Order 1: HSV1, HSV2, EVOrder 1: HSV1, HSV2, EV

Order 2: EBV, Adeno, EVOrder 2: EBV, Adeno, EV

Order 3: VZV, HSV1, HSV2Order 3: VZV, HSV1, HSV2

One universal testpanel:HSV1HSV2VZVCMVAdenEV

One universal testpanel:HSV1HSV2VZVCMVAdenEV

Straightforward andeasy


Multiplexed Diagnostics - AFFORDABLE Healthcare

Multiplexing mean lower price per one detected target (per pathogen)

Lower labour costs - just one system delivers all results

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MT-PCR Principle

The whole procedureconsists of

two steps of nucleicacid amplificationamplicon analysis

STEP 1

STEP 2

AMPLICONANALYSIS

MT-PCR - no competition between targets

Easy-PlexTM

Multiple TargetKits


Genital tract Infections (STI) - What do we know aboutthem?

There are more than 15 pathogens, causing genital tract disorders (bacteria, viruses, fungi)

4 of them are obligatory pathogenic, but can be asymptomatic! Chlamydia trachoma's

Neisseria gonorrhoeae

Trichomonas vaginalis

Mycoplasma genitalium

these must be detected among asymptomatic people (screening)

More than 5-7% of infections are mixed (e.g. C.trachomatis & M.genitalium)

Others are conditionally pathogenic and are to be detected among patients with symptoms

Treatment is dramatically differ depending on pathogen detected

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Detection of 4 obligatory pathogenic bacteria usingclassical methods

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Chlamydia trachomatis Direct immunofluorescence microscopy(sensitivity 70-80%) or cell culturing

Neisseria gonorrhoeae Gram microscopy (low sensitivity 30-40%among women) or bac. culturing

Trichomonas vaginalis Wet mount microscopy, microscopy

Mycoplasma genitalium No classical method available

ORjust one multiplexed

PCR test


AusDiagnostics Multiplexed panels for genital infections (STI)

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Short panel Long Panel

Chlamydia trachomatis Chlamydia trachomatis Chlamydia LGV

Neisseria gonorrhoeae Neisseria gonorrhoeae Treponema pallidum

Trichomonas vaginalis Trichomonas vaginalis GBS

Mycoplasma genitalium Mycoplasma genitalium Candida albicans & glabrata

Ureaplasma urealiticum Ureaplasma urealiticum HSV 1 & 2

Ureaplasma parvum Ureaplasma parvum Adenovirus

Mycoplasma hominis Mycoplasma hominis Haemophilus dicrei


Genital Infections (STIs) study in Western Australia

In WA study (Nov 2015): 13 out of 71 samples positive for CT/NG M.genitalium 3/71 (4%) positive M.hominis 2/71 (3%) Ureaplasma 28/71 (39%) ie 46% STIs missed by CT/NG assay in patients presenting with STI

Urinogenital EU (8Plex), Catalogue number: 27115# Description Inclusivity

1 Mycoplasma genitalium Includes all isolates2 Mycoplasma hominis Includes all isolates3 Ureaplasma urealyticum Includes serovars 2, 4, 5 and 7-134 Ureaplasma parvum Includes serovars 1, 3, 6 and 145 Trichomonas vaginalis Includes all stains6 Chlamydia trachomatis (momp) Includes all strains7 Neisseria gonorrhoeae (opa gene) Includes all strains

8 Artificial sequence for assay control &Human reference gene for sample adequacy control NA


Human papillomaviruses (HPV) - What do we know aboutthem? There are two groups of HPV: high and low cancerogenic risk

There is no antiviral therapy

80% of asymptomatic infections eliminate spontaneously without any treatment

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High-risk HPV Low-risk HPV

14 genotypes (16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66, 68) More than 10 genotypes, most common are 6 & 11

Detection recommended in screening settingsto determine risk group for cervical cancer.

Genotyping is recommended

Detection in screening settings or in patientswithout symptoms strongly NOT

RECOMMENDED!!!Only symptomatic patient (condylomas) might be

testedHigh risk HPV should be detected separately

from low riskLimited value of test results as the result will not

change the management / treatment


AusDiagnostic Panel for Human papillomaviruses (HPV)

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High-risk HPV panel - only relevant genotypes

14 genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68

Separate detection of each genotype (genotyping)

Calibrated for cervical screening (P.Meijer’s criteria)


Respiratory Pathogen B Panel - example

Respiratory Viruses Influenza A, Influenza B

Respiratory syncytial virus (RSV)

Parainfluenza 1, 2, 3, 4

Rhinovirus (RV)

Metapneumo virus (MPV)

Adenovirus (AdV)

Bacteria Bordetella pertussis & parapertussis

Legionella pneumophila & longbeache

Chlamydia & Clamydophila

Mycoplasma pneumonia

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Prior to 2014• Respiratory viruses

• B.pertussis/parapertussis (Roche Lightcycler)

• Atypical pneumonia (Referred tests)

September 2014• Respiratory Pathogens (16plex)

Savings:• Staff time

• $54K/annum

Clinical benefits

Consolidation of assays – Respiratory Pathogens

Suspected Whooping cough


Mixed infections among respiratory pathogens

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Faecal Pathogens X - 21 pathogen called for each sample!

Viruses

Bacteria

Parasites

The Easy-Plex™ System Outlook

ProcessorProcessor AnalyserAnalyser

Easy-Plex™ Processor

• Performs all liquid handling steps• Takes 24 or 48 samples on board (model

dependent)• Sets up and performs Step 1 of MT-PCR• Prepares ready-to-run Step 2 PCR plate for

Easy-plex™ Analyser• Hands-on time - 5 minutes to start a run

Preparing Run Has Never Been so Easy

11 Place samples

33 Check tips

22 Load reagents

Easy-Plex™ Analyser

• Performs Step 2 Real-time PCR andamplicon analysis steps

• Available in two variants:• for up to 12 samples (96-well model)• for up to 48 samples (384-well model)

• Could be replaced by other existingin a lab real-time PCR Machine

Start Run in a Few ClicksChoose aPanel &Enter

Samples

Choose aPanel &Enter

Samples

Verify theRobotDeck

Components

Verify theRobotDeck

Components

Automatic Results Calling

All targetsdetected in the

sample are shownon a screen

All targetsdetected in the

sample are shownon a screen

Amplificationcurves

Amplificationcurves

AmpliconAnalysis with

automatic calling

AmpliconAnalysis with

automatic calling


SPIKE verifies PCR reaction

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Human Reference Gene for Adequacy of Sampling

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Easy-Plex Molecular Diagnostics - IACLD Portaliacld.ir › DL › co › 14 › workshop ›...

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Transcript of Easy-Plex Molecular Diagnostics - IACLD Portaliacld.ir › DL › co › 14 › workshop ›...