June 4, 2018 Implementation and applications for use of MALDI-TOF for clinical and food testing Dominick Centurioni Research Scientist NYS Biodefense Laboratory
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June 4, 2018
Implementation and applications for use of MALDI-TOF for clinical and food testing
Dominick CenturioniResearch Scientist
NYS Biodefense Laboratory
June 4, 2018 2
OverviewMALDI-TOF/MS backgroundBotulinum neurotoxin detection
• Workflow, validation, method comparison• Food testing
Ricin toxin detection in foodsBacillus species identification
• Applications and validation / verificationConclusions
June 4, 2018 3
Ionization Source• Matrix Assisted Laser Desorption Ionization
Mass Analyzer• Time-of-Flight • Uniform electromagnetic force is applied to
all ions, causing them to accelerate down a flight tube
• Lighter ions travel faster, arrive at detector first
• Have a wide mass range
MALDI-TOF/MS
June 4, 2018 4
June 4, 2018 5
Botulinum Neurotoxins (BoNT)Produced by anaerobic, gram positive, spore-forming organism
• Clostridium botulinumCauses paralytic disease, botulism
• Disrupts communication at neuromuscular junction
• Descending paralysisRoutes of exposure
• Infant: colonization of GI tract• Foodborne: ingestion of BoNTs • Wound: colonization of wound• Inhalation
Rowland LP. N Engl J Med. 2002 Aug 8;347(6):382-3.
June 4, 2018 6
NYS Botulinum Neurotoxin Detection Workflow
June 4, 2018 7
BoNT MALDI Method Overview
June 4, 2018 8
Validation Work & Method ComparisonSensitivity
• LODSpecificity
• Related organisms• Similar symptoms
Reproducibility• Inter/Intra-assay
Accuracy• Blinded panel• 40 per serotype
analyzed in duplicate
Comparison to “Gold Standard” Mouse Bioassay (MBA)
Presenter
Presentation Notes
LOD – clinically relevant, list LODs, define MLD50 Specificity -
June 4, 2018 9
Food TestingProcessing
• Sample volumeTomato sauce (MALDI vs MBA)
• MBA & PCR Negative Serum/Stool
• MALDI Positive Stool• Sauce with meat and peas
• PCR, MALDI, & MBA Pos.
Provides info on active toxin vs detection of toxin gene
http://thefamilydinnerbook.com/wp-content/uploads/tomatosaucejars.jpghttp://adoptamicrobe.blogspot.com
June 4, 2018 10
Ricin ToxinDerived from castor bean plant
• Ricinus communisRibosome inactivating proteins (RIP)
• Potent cytotoxicity to animal cells• No antidote
AB-toxin • Catalytically active A chain:
• Depurinates a universally conserved adenine in α-sarcin/ricin loop (SRL) of large rRNA
• Stops translation, inactivates ribosomes• B chain binds to cells:
• Binds to carbohydrates on cells
http://adoptamicrobe.blogspot.com
Presenter
Presentation Notes
The B chain
June 4, 2018 11
Ricin/Abrin Toxin via MALDI-TOF/MS
1 ng Ricin toxin in infant formula
118 Da
Presenter
Presentation Notes
Two step process 2nd more specific, digest toxin to confirm Ab/pH differ for abrin
June 4, 2018 12
Bacillus Species IdentificationClinical Isolates
• ID isolates from clinical specimens & food samples
• May require further molecular testing• R/O SA’s• Epidemiological Investigation
MALDI provides rapid, inexpensive data• NGS data analysis• Molecular assays
Bacillus species isolated from the NYS medical marijuana program
http://adoptamicrobe.blogspot.com
June 4, 2018 13
Bacillus Species Identification Validation / Verification
In-house comparison between labs• Over 60 smears, 25 extractions
In-house comparison between instruments• Over 65 smears, 20 extractions
Software / library / database updates• Approximately 15 smears, 10 extractions• Verification panel
Presenter
Presentation Notes
Story about software update – new instrument – panel of known organisms and some didn’t ID – looked at it further, saw libraries were different etc
June 4, 2018 14
Conclusions
• Relatively inexpensive systems perform well for many types of testing
• Can provide assay target activity vs. presence of genes• Eliminate use of animals for laboratory testing• Low sample volume needed for testing• Provides quick, inexpensive info for downstream
applications
June 4, 2018 15
AcknowledgementsNYSDOH
• Michael Perry• Christina Egan• Biodefense Laboratory• Bacteriology Laboratory
CDC• Suzanne Kalb• John Barr
FSIS FERN• Cooperative Agreement
FSIS-C-19-2014-2016
Presenter
Presentation Notes
Work with food testing supported by fern CDC for antibodies, method development
June 4, 2018 16
Infectious Diseases: Applying novel diagnostic tools to emerging infections and outbreaks.
Genetics: Identifying life-threatening conditions through newborn screening.
Environmental Health Sciences: Linking human disease with chemical and toxic exposures.
Lab Management: Operations, regulation and quality assurance.
Logistics• 5 semester, 21 month program• No tuition / fees• Stipend (awarded in 2nd year, based on
academic merit & funding availability)
Training• Practical laboratory skills• Method development• Scientific communication• Lab management, operations• Quality control, assurance
Master of Science in Laboratory Sciences Programat the Wadsworth Center, NYS Department of Health
Admissions• BS in science• GRE or MCAT• TOEFL (if required)• US citizen / permanent resident status
wadsworth.org/mls
Presenter
Presentation Notes
microbiology and infectious disease, molecular diagnostics, newborn screening, clinical and environmental chemistry, immunology, laboratory operations and management 8 core courses, 3 special topics, lab rotations, capstone project
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