Easy-Plex Molecular Diagnostics
Dmitry Kuevda, PhDAusDiagnostics Pty Ltd, Australia
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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SPIKE verifies PCR reaction
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Human Reference Gene for Adequacy of Sampling
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