Implementation and applications for use of MALDI-TOF for ... · use of MALDI-TOF for clinical and food testing . Dominick Centurioni. Research Scientist. ... Rowland LP. N Engl J

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)

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

June 4, 2018 11

Ricin/Abrin Toxin via MALDI-TOF/MS

1 ng Ricin toxin in infant formula

118 Da

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

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

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