MALDI-TOF: Rapid Pathogen Identification

Question 1

Mass Spectrometry

Answer 1

Analysis of molecules to determine molecule weight.

Question 2

MALDI-TOF

Answer 2

Matrix Associated Laser Desorption Ionization-Time of Flight mass spectrometry
Enabling ID of peptide and proteins of bacteria.
2013; FDA approves first mass spectrometer system in the US for ID of bacteria, yeasts.
New system ID 193 different yeasts and bacteria known to cause illness.
Candida, fungi, cryptococcus.
-Vitek MS
-Bruker Biotyper; 280 different micro organisms.

Question 3

m/z:

Answer 3

Mass to charge ratio (what is really measured in mass spectrometry).

Question 4

Strengths of MALDI-TOFF

Answer 4

Sensitive: A few small colonies
Results in minutes after colonies appear.
Cost: ~0.20$ for consumables/sample
Automated
No gram stain required, no special handling.
Works for GP, GN, yeast, and fungi.
Culture is what slows things down; maybe we don’t have to have a visual colony?

Question 5

Steps in MADLI-TOF

Answer 5

Sample=whole bacteria, whole cells put on the slide.
1. Ionization; 30000 volts, use laser pulse, blast colony with laser; ionizes; want charged particles; comes out are charged proteins and peptides; basically injected into flight tube.
2. Acceleration; time of flight; flight tube under high vacuum; depends on size of molecule on how fast it can travel and hit detector.
Give measurable quantity of kinetic energy.
All given same push
depends on charge and mass of protein, we can correlate time it takes to get across flight tube to hit detector WITH the molecular weight or mass and charge ratio.
3. Detection
Goal: Examine proteins and large peptides that are characteristic of pathogenic organisms.

Question 6

Physics

Answer 6

Kinetic Energy=1/2mv^2
E=acceleration energy
m/z-2E/V^2
TOF=time of flight
m/z is proportional to the TOF^2
Big and small protein; the small one will get through the flight tube faster.

Question 7

TOF: The Great Race

Answer 7

A drag race between vehicles of different sizes, but all having identical engines:
“Start line”=orthogonal accelerator; “finish line”=TOF detector.
Just as all vehicles have the same engine (horsepower), all ions are pushed up the flight tube with the same kinetic energy.
Since m=2E/v^2, the smaller vehicle/ions will reach the finish line/detector before the larger ones.
Smaller peptide gets to the detector first; bigger protein takes longer to get there.
Acceleration happens on the nanosecond level (20-30 times/second).

Question 8

Direct Analysis of Bacterial Colonies

Answer 8

1. Touch colony from agar plate with a loop or toothpick.
2. MALDI plate (different positions)
3. Apply HCCA Matrix (HCCA= alpha-cyano-4-hydroxycinnamic acid); important, allows mass spec proteins and peptides; matrix absorbed energy from laser and transmits to the proteins and peptides; without this, it blows everything apart and you can’t see anything; effectively transfers energy to proteins and peptides.
   Typically has an absorption range over the frequency of the laser.
   MALDI plate slides inside; there is an automated program and you press go.
   Takes 2-3 seconds to generate a spectrum; pattern matching algorithm; outcome is a chart that has green, yellow and red.

Question 9

Outcome Bacterial Fingerprint in a few minutes

Answer 9

Minutes meaning the entire process, not just time in the machine.
Fingerprints are typically obtained by analyzing each spot for 2 seconds!
Automated matching to a database for identification.
Proteins and peptides characteristic of the bacterium.
30 organisms every minute.

Question 10

Database match for microbial fingerprint

Answer 10

Green=got a good match
Target position=position on plate where you put bacterium
Test Organism= standards and other test organisms (patient number).
Score Value:
Green=species level match; high confidence ID.
Yellow=still okay, but low confident ID.
Red=problems with matching; no ID possible.
Gives an idea of what the organism is and what the confidence of the match is.

Question 11

Each bacterium has its own unique profile

Answer 11

Bottom: m/z ratio.
See peaks at different m/x ratios between GN and GP organisms.
Developed database where take clinical isolates, analyzed, spectra, database, the spectra are compared to the clinical isolates.
Methicillin resistant and sensitive S. aureus
See different profiles depending on resistance.
Therapy much different depending on organism.
Spectra look different between resistant or sensitive based on software; tells what AB to use or susceptibility testing.
Not every type of resistance can be ID at this point, but some can.

Question 12

Direct Identification

Answer 12

Yeast grow slower than most bacteria.
Culture takes the longest part of this process.
Filtration and concentrate pathogens directly form urine samples.
Sample of urine, concentrate, de salt, centrifuge to yield pellet take pellet and apply to MALDI plate and do ID; 2-3 hours.
Specific types of infections (UTI)
Depending on what infection is, it isn to necessary to go through steps of culturing plate.

Question 13

Clinical Impact of MALDI-TOF

Answer 13

Want to get appropriate AB to patient in short time period.
MADLI-TOF provided 1.45 days earlier on average (P<0.001).
Albicans not that much different; some bacteria significant differences.
2012-back when starting to get this working.
Good Improvement.
Clinical costs:
189,969 (old school molecule biology methods) vs 87,555 (MALDI-TOF)
Do require some maintenance for the instrument.
2013; didn’t have to grow fully, can grow to see under microscope, then move it in contrast to having full plate of colony to do studies.
+ ID by MALDI and susceptibility after and then AB stewardship.
Not all susceptibilities will be apparent with MALDI.
Then communicate and adjust therapy.
ID; 36 to 11 hours starting with blood culture draw.
MALDI sped things up nicely.
Length of stay and cost outcomes in survivors
2 days less stay
1 day less in ICU, difference in cost
45 vs 26 thousand in total hospital cost stay.

Question 14

Sepsityper

Answer 14

ID and rapid susceptibility testing.
Direct analysis of sample from blood culture bottle.
At risk patients have sepsis.
High risk, high mortality rates.
Faster can ID, faster prescribe AB to physician the better.
Positive blood culture bottle:
-Harvest 1 mL blood culture liquid in an Eppendorf tube; 1 min.
-Add 200 mL lysis buffer and mix; 10 +/- 5 sec.
-Centrifuge (2 min, 13.000 rpm) discaard supernatant; 2 min
-Add 1mL washing buffer and mix; 1.5 min
-Centrifuge (1 min., 13.000 rpm) discard supernatant; 1 min
-Proceed with sample preparation (move sample onto plate, add matrix)
Comes directly from blood culture bottle; direct early analysis.
Using this approach can get ID within 4 hours (some within hour of positive blood culture, have positive ID and can start optimizing therapy).
See trying to grow organism going away.