rapid automated multiplex pcr diagnostics for blood pathogens
TRANSCRIPT
Presented at the 112th General Meeting of the American Society for Microbiology, June 2012Minor revisions have been made to this poster since its presentation at ASM (June 2012).
CONTACT INFORMATIONAndrew Hemmert, Ph.D.Idaho Technology, [email protected]
I. Ota1, E. Barker1, A. Hemmert1, B. Baarda1, O. Cham1, C. Heyrend1, B. Barrett1, N. Garrone1, S. Thatcher1, R. Crisp1, P. Lephart5, S. Silbert6, C. Kubasek6, A. Blaschke3, J. Daly2, H. Salimnia4, R. Widen6, & K. Ririe1
1Idaho Technology, Inc. (ITI), 2Primary Children’s Medical Center (PCMC), 3University of Utah (UU), 4Detroit Medical Center University Laboratories (DMCUL), 5Wayne State University School of Medicine (WSUSM), 6Tampa General Hospital Clinical Laboratory (TGH)
Rapid Automated Multiplex PCR Diagnostics for Blood Pathogens On-Site Testing in Clinical Microbiology Laboratories
CONCLUSION• The FilmArrayBCID exhibited high sensitivity and specificity for all organisms and
antibiotic resistance genes when compared to conventional culture methods.
• LowprevalenceorganismswillbeevaluatedatITItoconfirmsensitivityandspecificity.
• FilmArray BCID is currently being evaluated for 510k FDA clearance.
UPDATED ABSTRACTRapid identification of the numerous organisms causing sepsis, and identification ofantimicrobial resistance genes could improve patient outcomes. The ITI FilmArray®, whichstreamlinespathogenidentificationandprovidesresultsin~1hwouldfacilitatethisprocess. The FilmArray comprises a unique lab-in-a-pouch and countertop instrument which automatically extracts nucleic acids, performs nested multiplex PCR and data analysis. We describe results from beta testing of the development version of the FilmArray BloodCulture Identification(BCID)system,whichaimstodetect~90%ofpathogensisolated from positive aerobic blood cultures (PABCs) and select antibiotic resistance genes.
PABCs from children and adults were tested using IRB approved protocols in FilmArray BCID pouches at three sites, PCMC, DMCUL and TGH, and compared to conventional blood culture and susceptibility testing. An aliquot of PABC (250 µL) was simultaneously tested in a BCID pouch with nested multiplexed PCR assays for gram positive and negative bacteria, antibiotic resistance genes, and fungi.
Atotalof403bloodculturesweretestedbyFilmArrayBCID.Conventionalcultureidentified467 pathogens; the FilmArray contained assays to detect 430 of them. Of these 430 pathogens,386(90%)wereidentifiedbytheFilmArray.Systemsensitivity(truepositives/thesumoftruepositivesandfalsenegatives),was95%orgreaterformostorganismsincluding Staphylococcus aureus, Streptococcus, Enterococcus, Enterobacteriaceae, Pseudomonas aeruginosa and Candida species. For antibiotic resistance genes greater than95%sensitivitywasobtained.Systemspecificity(truenegatives/thesumoftruenegativesandfalsepositives)wasequallygoodwith95%orgreaterspecificityfornearlyall tests.
UPDATEDuring a second round of beta testing (data shown in center), 130 PABCs were tested usingtheFilmArrayBCIDpanel(DMCUL).Conventionalcultureidentified137pathogens;theFilmArraycontainedassaystodetect128.Ofthese128,122(95.3%)wereidentifiedcorrectly by the FilmArray.
The FilmArray BCID system demonstrated accurate identification of pathogens andantibiotic resistancegenes inPABCsamples.Rapid identificationofa large rangeofpathogens in blood culture could improve the medical management of sepsis.
INTRODUCTIONRapid identification of the numerous organisms causing sepsis and identification ofantimicrobial resistance genes could improve patient outcomes. The ITI FilmArray, which streamlinespathogenidentification,providingresultsin~1hr,wouldfacilitatethisprocess.We describe results from beta testing of the development version of the FilmArray BCID systemwhichaimstodetect~90%ofpathogensisolatedfromPABCsamplesandselectantibiotic resistance genes.
FilmArray BCID Panel
Gram Positive Bacteria Staphylococcus species• Staphylococcus aureus• Coagulase negative
StaphylococciEnterococcus speciesListeria monocytogenesStreptococcus species• Streptococcus agalactiae
• Streptococcus pneumoniae
• Streptococcus pyogenes• Viridans Streptococci
Gram Negative BacteriaAcinetobacter baumanniiEnterobacteriaceae family:• Citrobacter
• Enterobacter• Enterobacter cloacae
• Escherichia• Escherichia coli
• Klebsiella• Klebsiella oxytoca• Klebsiella pneumoniae
• Salmonella• Shigella
• Serratia• Proteus HaemophilusinfluenzaeNeisseria meningitidisPseudomonas aeruginosa
Antibiotic resistance genesmecA geneKPC gene
vanA genevanB gene
FungiCandida albicansCandida glabrataCandida kruseiCandida parapsilosisCandida tropicalis
Clinical Beta Testing Results
Gram Positive Bacteria
TP FN Sensitivity TN FP Specificity
Enterococcus 50 1 98.0% 474 8 98.3%S. aureus 76 2 97.4% 454 1 99.8%Staphylococcus* 61 3 95.3% 62 4 93.9%S. agalactiae 6 1 85.7% 526 0 100.0%S. pneumoniae 6 0 100.0% 526 1 99.8%S. pyogenes 7 0 100.0% 526 0 100.0%Streptococcus 58 4 93.5% 462 9 98.1%
Gram Negative Bacteria
TP FN Sensitivity TN FP Specificity
A. baumannii 8 0 100.0% 523 2 99.6%E. cloacae* 6 1 85.7% 123 0 100.0%Enteric 114 1 99.1% 412 6 98.6%E. coli 61 1 98.4% 463 8 98.3%H.influenzae 1 0 100.0% 530 2 99.6%K. oxytoca 2 0 100.0% 527 4 99.2%K. pneumoniae* 8 0 100.0% 122 0 100.0%
L. monocytogenes* 0 0 0.00% 533 0 100.0%
N. meningitidis 1 0 100.0% 532 0 100.0%
Proteus* 4 0 100.0% 125 1 99.2%
P. aeruginosa 17 1 94.4% 506 9 98.3%
Serratia 4 0 100.0% 528 1 99.8%
Fungi
TP FN Sensitivity TN FP Specificity
C. albicans 14 0 100.0% 517 2 99.6%C. glabrata 8 0 100.0% 525 0 100.0%C. krusei 3 0 100.0% 530 0 100.0%C. parapsilosis 10 0 100.0% 522 1 99.8%C. tropicalis 4 0 100.0% 528 1 99.8%
Antibiotic Resistance Genes
TP FN Sensitivity TN FP Specificity
KPC 2 0 100.0% 531 0 100.0%mecA* 46 1 97.9% 73 10 88.0%vanA/B 14 0 100.0% 519 0 100.0%*Duetosignificantassay/protocolchanges,onlythesecondroundofbetadataisshown.
ACKNOWLEDGEMENTS• Development of the FilmArray BCID panel is supported by grant number U01 AI082184 from the
NIAID, NIH.• Special thanks to all those who provided samples for preliminary testing:
• ARUP, Salt Lake City, UT• Dr. Judy Daly, Primary Children’s Medical Center• Dr. Hossein Salimnia, Detroit Medical Center University Laboratories • Dr. Raymond H. Widen, Tampa General Hospital Clinical Laboratory
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Figure 1
A. Fitment with freeze-dried reagentsB. Plungers- deliver reagents to blistersC. Sample lysis and bead collectionD. Wash stationE. Magnetic bead collection blisterF. Elution StationG. Multiplex Outer PCR blisterH. Dilution blisterI. Inner Nested PCR array
ITIhasdevelopedalab-in-a-pouchsystemcalled“FilmArray”.Itisamedium-scalefluidmanipu-lationsystemperformed inaself-contained,disposable, thin-filmplasticpouch.TheFilmArrayplatformprocessesasinglesample,fromnucleicacidpurificationtoresult,inafullyautomatedfashion. These system characteristics are ideal for the multiplex testing of pathogens in standard diagnostic sample matrices.
The FilmArray Test SystemA FilmArray test is initiated by injecting re-hydration solution and a patient sample into the FilmArray pouch and placing it in the FilmArray instrument. The user enters the sample and pouch type (using a barcode reader) into the software and initiatesarun.Resultsareprovidedin~1hour.
Thefilmportionofthepouchhasstationsfor:
1. Cell lysis (Blister C)2. Magnetic-bead based nucleic acid purification(D&E)3. First-stagemultiplexPCR(F&G)4. Array of 102, second-stage nested
PCRs (I)
C D
E
F
G
I
Figure 2A
Figure 2C
Melt curve analysis data for a positive blood culture sample:DMCULidentifiedthissampletobepositivefor K. pneumoniae which contained KPC. FilmArray correctly identified the organism and the antibioticresistance gene it contained.
Blue = KPC assayPink = K. pneumoniae assayPurple = Enteric assay
Melt curve analysis data for a Pseudomonas aeruginosa PABC sample from PCMC.
Figure 2B
Melt curve analysis data for a Staphylococcus aureus (mecA+) and Group A Streptococcus co-infection from TGH.
Red = mecA assayGreen = S. aureus assayPink = S. pyogenes assay Grey = Streptococcus assay
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METHODSPABC samples from children and adults from three different sites (PCMC, DMCUL and TGH) were tested using IRB approved protocols in a FilmArray BCID pouch. FilmArray results were compared to conventional blood culture and susceptibility testing. One 250 µl aliquot from each PABC was simultaneously tested in a pouch with nested multiplexed PCR assays for gram positive and gram negative bacteria, fungi and antibiotic resistance genes.
PCRprimersaredriedintothewellsofthearrayandeachprimersetamplifiesauniqueproductofthefirst-stagemultiplexPCR.ThesecondstagePCRproductisdetectedinreal-timeusingafluorescent-double-strandedDNAbindingdye,LCGreen® Plus.