The Abbott Plex-ID and RIPL
Tim Brooks, Carrie Turner & Jackie Duggan
Rare & Imported Pathogens Laboratory, PHE Porton Down
Overview
Our lab and why we want the Plex-ID
The instrument & how it works
Performance in trials
Topics for the Tropics
Future steps
Summary
Systemic anthrax
RIPL service & needs
Acute 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 88 990
Manned 24/7
Working hours -> IFS SpR
Out 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
Imported
Service
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 assays
Array cards for panels of PCR’s
Plex ID
Will extend range and fill gaps
Intelligent algorithms for patterns
The Plex-ID
Developed 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 process
Sample
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 answers
Primer 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
Additional wells/plates can
be used to give increased
resolution depending on
assay
Primers 1, 2 and 3 results
combined = p 99.5%
B. pseudomallei
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 on
Anything 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
Burkholderia mallei,
&
S.
Burkholderia pseudomallei
aureus
Staphylococcus hominis,
Staphylococcus aureus,
Streptococcus sp.
EQA
P12/1185
(RG3-01)
B. pseudomallei
Burkholderia mallei,
&
S.
Burkholderia pseudomallei
aureus
Staphylococcus hominis,
Staphylococcus aureus,
Streptococcus sp.
EQA
P12/1186
(RG3-02)
B. anthracis
(pX01 -) & P.
aeruginosa
Bacillus anthracis, Ba-pX02
Pseudomonas aeruginosa
B. melitensis &
S. hominis
Brucella ovis, Brucella abortus,
Brucella suis, Brucella
Francisella tularensis
melitensis, Burkholderia
thailandensis, Vibrio cholerae
EQA
P12/1187
(RG3-03)
Note additional detections over intended result reveal contents
of, and issues in, originators lab!
How does it perform -2
Intended result Front end report
EQA
P12/1190
(RG3-06)
EQA
P12/1190
(RG3-06)
EQA
P12/1191
(RG3-07)
B. abortus &
Y.
enterocolitica
B. abortus &
Y.
enterocolitica
B.
thailandensis
&
E.coli
Brucella ovis, Brucella
abortus, Brucella suis,
Brucella melitensis
Brucella ovis, Brucella
abortus, Brucella suis,
Brucella melitensis
Summary report
Shewanella sediminis†
Shewanella sediminis
Burkholderia thailandensis Escherichia coli
† This organism was best hit; Y enterocolitica
came up as alternative; 1 base difference!
Behind the data -1
Result
EQA
P12/1185 Burkholderia mallei/ Burkholderia pseudomallei
Staphylococcus hominis/ Staphylococcus
aureus/ Streptococcus sp.
EQA
P12/1191 Burkholderia thailandensis
Escherichia coli K-12
*Relevant Primer pairs in this assay
well
*Score
Quality
Level
score
2 of 2
1.00 3089
1 of 1
1.00
2 of 2
1.00 1344
1 of 1
1.00
635
63
Behind the data- detail
Sample P12C001185 Burkholderia data
Quantity
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
1214.2531
Confide
nce
Row Type
1PrimerNumber
1PrimerName
1Observed
1GenomeCounts
1Matched
1Matched
1Matched
1Matched
1Matched
1Matched
Match
Burkholderia mallei NCTC10247
Burkholderia pseudomallei 1710b
Burkholderia pseudomallei 1106a
Burkholderia mallei NCTC10229
Burkholderia mallei ATCC23344
Burkholderia mallei SAVP1
Unintended results:
Food sample intended to demonstrate Brucella
contamination
Score
Quality
Level
1 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
Organism
Coxiella burnetii
Pseudomonas syringae/
Pseudomonas fluorescens/
Pseudomonas putida
1 of 2
Future steps
Evaluate 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.
Summary
Versatile & 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!)