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The Abbott Plex-ID and RIPL Tim Brooks, Carrie Turner & Jackie Duggan Rare & Imported Pathogens Laboratory, PHE Porton Down.

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Presentation on theme: "The Abbott Plex-ID and RIPL Tim Brooks, Carrie Turner & Jackie Duggan Rare & Imported Pathogens Laboratory, PHE Porton Down."— Presentation transcript:

1 The Abbott Plex-ID and RIPL Tim Brooks, Carrie Turner & Jackie Duggan Rare & Imported Pathogens Laboratory, PHE Porton Down

2 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

3 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

4 The breadth of the problem

5 In time of need… Single phone number 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 Service

6 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

7 Next generation assays Array cards for panels of PCR’s Plex ID Will extend range and fill gaps Intelligent algorithms for patterns

8 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

9 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

10 Overview of process SampleNucleic 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

11 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 results =p 98% B pseudomallei Primers 1, 2 and 3 results combined = p 99.5% B. pseudomallei Additional wells/plates can be used to give increased resolution depending on assay

12 Assay panels Bacterial, antibiotic resistance, candida (6024 species, strains detected) Broad bacterial identification (5885 species, 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, strains detected) Vector borne (324 species, 1020 strains detected) Biothreat (177 species, 3997 strains detected) Flu (2 species, strains detected) MDR TB (25 species, 127 strains, drug resistance) Courtesy Abbott

13 What you can use it on Anything you can extract!

14 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

15 How does it perform -1 SampleExpected resultFront end reportSummary 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-pX02Pseudomonas 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!

16 How does it perform -2 Intended resultFront end reportSummary 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 thailandensisEscherichia coli † This organism was best hit; Y enterocolitica came up as alternative; 1 base difference!

17 Behind the data -1 Result *Score Quality score Level EQA P12/1185Burkholderia mallei/ Burkholderia pseudomallei 2 of Staphylococcus hominis/ Staphylococcus aureus/ Streptococcus sp. 1 of EQA P12/1191 Burkholderia thailandensis 2 of Escherichia coli K-12 1 of *Relevant Primer pairs in this assay well

18 Behind the data- detail Sample P12C Burkholderia data Quantity Confide nceRow TypeMatch PrimerNumber PrimerName Observed GenomeCounts MatchedBurkholderia mallei NCTC MatchedBurkholderia pseudomallei 1710b MatchedBurkholderia pseudomallei 1106a MatchedBurkholderia mallei NCTC MatchedBurkholderia mallei ATCC MatchedBurkholderia mallei SAVP1

19 Unintended results: Food sample intended to demonstrate Brucella contamination Organism ScoreQualityLevel Coxiella burnetii 1 of 1 & 1 of Brucella ovis/ Brucella abortus/ Brucella suis/ Brucella melitensis 2 of bacterium EF 1 of Pseudomonas syringae/ Pseudomonas fluorescens/ Pseudomonas putida 1 of 2

20 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.

21 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!)


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