The Abbott Plex-ID and RIPL

Tim Brooks, Carrie Turner & Jackie DugganRare & Imported Pathogens Laboratory, PHE Porton Down

OverviewOur lab and why we want the Plex-ID

The instrument & how it works

Performance in trials

Topics for the Tropics

Future steps

SummarySystemic anthrax

RIPL service & needsAcute diagnostic services for unusual and imported fevers

24/7 helpline for clinicians

Same day telephone results for critical diagnoses

24/7 service with 2-6 hour turn around for emergencies e.g. VHF

Main hub for National Imported Fever Service

PCR: real time and some block assays

Serology (mostly automated)

Culture

Primary pneumonia from Leptospirosis, Malaysia

The breadth of the problem

In time of need…

Single phone number 0844 77 88 990Manned 24/7Working hours -> IFS SpROut of hours -> on-call Consultant rota• HPA/NHS - RIPL; Liverpool TIDU; HTD/UCLH; HPA

•Weekly IFS teleconference• For discussion of interesting cases, get help with differentials

•Bi-monthly IFS steering committee•Governance & service development

Imported Fever Service Imported Fever Service

Differential Diagnosis Algorithms

Divided into 10 world regions

Divided into 8 broad symptom categories:

Undifferentiated fever

Fever with haemorrhage

Fever with rash

Fever with skin/soft tissue involvement

Fever with respiratory symptoms

Fever with GI symptoms

Fever with jaundice or hepato/splenomegaly

Fever with neurological symptoms

Next generation assaysArray cards for panels of PCR’s

Plex IDWill extend range and fill gaps

Intelligent algorithms for patterns

The Plex-IDDeveloped by IBIS for detection of designated Biothreat agents

IBIS acquired by Abbott and device rebranded as Plex-ID

Additional panels developed for wide range of pathogens

Theoretical capability:

All bacteria

All viruses

All fungi } Known & Unknown

Dengue haemorrhagic fever

Plex-ID Features• Broad range PCR combined with Electrospray-Ionisation Time of Flight

Mass Spectrometry (ESI TOF MS)

• Broad identification of microbes (bacteria, viruses, fungi, protozoa)

• Can detect complex mixtures of microbes

• No culture required

• Expected and unexpected pathogens in a single sample

• High resolution genotyping, strain identification and drug resistance testing

Cowpox, UK

Overview of processSample Nucleic acid extraction

Broad range PCR with multiple primers (8 or 16 well assays)

Determination of the amplicon weights by MS

Calculation of the base compositions (A:G:T:C)

Automated bioinformatics analysis

Triangulating the answersPrimer pair 1

Product = 95% p of B. pseudomallei

Primer pair 2

Product = 90% p of B. pseudomallei

Primer pair 3

Product = 90% p of B. pseudomallei

Primers 1 + 2 results =p 98% B pseudomallei

Primers 1, 2 and 3 results combined = p 99.5%

B. pseudomalleiAdditional wells/plates can be used to give increased resolution depending on assay

Assay panels • Bacterial, antibiotic resistance, candida (6024 species, 10205 strains

detected)

• Broad bacterial identification (5885 species, 10561 strains detected)

• Broad viral (human) (173 species, 2611 strains detected)

• Broad fungal (2108 species, 3044 strains detected)

• Food borne (89 species, 2573 strains detected)

• Respiratory virus (116 species, 13080 strains detected)

• Vector borne (324 species, 1020 strains detected)

• Biothreat (177 species, 3997 strains detected)

• Flu (2 species, 14980 strains detected)

• MDR TB (25 species, 127 strains, drug resistance)

Courtesy Abbott

What you can use it onAnything you can extract!

RIPL sample evaluation• 3 assay panels

– Biothreat (anthrax, plague, tularaemia, Rickettsia, Q fever, alphaviruses, filoviruses)

– Vector-borne panel (Borrelia, Rickettsia, malaria, Anaplasma, Babesia)– Custom designed Tropical fever panel (Alphaviruses, flaviviruses,

bunyaviruses, hantaviruses, malaria etc)• In development subject to contract

Rattus norvegicus, reservoir of Seoul virus in current UK outbreak

How does it perform -1

Sample Expected result Front end report Summary report

EQA P12/1185 (RG3-01)

B. pseudomallei & S. aureus

Burkholderia mallei, Burkholderia pseudomallei

Staphylococcus hominis, Staphylococcus aureus, Streptococcus sp.

EQA P12/1185 (RG3-01)

B. pseudomallei & S. aureus

Burkholderia mallei, Burkholderia pseudomallei

Staphylococcus hominis, Staphylococcus aureus, Streptococcus sp.

EQA P12/1186 (RG3-02)

B. anthracis (pX01 -) & P. aeruginosa

Bacillus anthracis, Ba-pX02 Pseudomonas aeruginosa

EQA P12/1187 (RG3-03)

B. melitensis & S. hominis

Brucella ovis, Brucella abortus, Brucella suis, Brucella melitensis, Burkholderia thailandensis, Vibrio cholerae

Francisella tularensis

Note additional detections over intended result reveal contents of, and issues in, originators lab!

How does it perform -2 Intended result Front end report Summary report

EQA P12/1190 (RG3-06)

B. abortus & Y. enterocolitica

Brucella ovis, Brucella abortus, Brucella suis, Brucella melitensis

Shewanella sediminis†

EQA P12/1190 (RG3-06)

B. abortus & Y. enterocolitica

Brucella ovis, Brucella abortus, Brucella suis, Brucella melitensis

Shewanella sediminis

EQA P12/1191 (RG3-07)

B. thailandensis & E.coli

Burkholderia thailandensis Escherichia coli

† This organism was best hit; Y enterocolitica came up as alternative; 1 base difference!

Behind the data -1

Result *Score Quality score Level

EQA P12/1185 Burkholderia mallei/ Burkholderia pseudomallei 2 of 2 1.00 3089

Staphylococcus hominis/ Staphylococcus aureus/ Streptococcus sp. 1 of 1 1.00 635

EQA P12/1191 Burkholderia thailandensis 2 of 2 1.00 1344

Escherichia coli K-12 1 of 1 1.00 63

*Relevant Primer pairs in this assay well

Behind the data- detail

Sample P12C001185 Burkholderia data

QuantityConfidence Row Type Match

1214.2531 1PrimerNumber  1214.2531 1PrimerName  1214.2531 1Observed  1214.2531 1GenomeCounts  1214.2531 1Matched Burkholderia mallei NCTC102471214.2531 1Matched Burkholderia pseudomallei 1710b1214.2531 1Matched Burkholderia pseudomallei 1106a1214.2531 1Matched Burkholderia mallei NCTC102291214.2531 1Matched Burkholderia mallei ATCC233441214.2531 1Matched Burkholderia mallei SAVP1

Unintended results:Food sample intended to demonstrate Brucella

contamination

OrganismScore Quality Level

Coxiella burnetii1 of 1 & 1 of 2 0.98 166

Brucella ovis/ Brucella abortus/ Brucella suis/ Brucella melitensis

2 of 2 1.00 1226

bacterium EF 1 of 1

0.86 167 Pseudomonas syringae/ Pseudomonas fluorescens/ Pseudomonas putida

1 of 2

Future stepsEvaluate Plex in comparison with extant tests for whole range of samples

Work up tropical fever panel

Generate data to support future validation for marketing

Determine the relative frequency of different pathogens

Evaluate large sample extraction e.g. for typhoid

Long term:

If commercially available, use Plex as 2nd line diagnostic system across full range of capability.

SummaryVersatile & capable system

Single instrument has massive coverage

Needs multiple plates to achieve full potential

Currently relatively slow & expensive

Ideal for lower throughput assays and unusual problems

Can be used as basic typing tool

With thanks to:

ABBOTT staff, especially Ranga Sampath

RIPL staff

At risk of glanders? (not a case this

time!)