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3/9/15
1
Behind the Scenes of a MALDI Biotyper Intergration in a Clinical
Laboratory
Indre Budvytiene, M.S., CLS (ASCP)
Objectives
• Describe principles of Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI TOF) Mass Spectrometry.
• Review MALDI TOF validation and integration into the clinical laboratory.
• Summarize our experience of MALDI TOF MS usage for the routine laboratory testing.
Microbiology Laboratory
• Serving: ▫ Stanford Hospital : 613 licensed
beds ▫ Lucile Packard Children's
Hospital: 311 licensed beds
• Staffed 24/7 00 specimens per
• Test ~ 180,000 specimens per year
Before MALDI TOF
Isolation of single colony (24h)
Gram Stain
Sample plating
Incubation (24h)
Growth characteristics
Before MALDI TOF
• Biochemical characteristics
Catalase test Indole test Fermentation of sugars and H2S production
Before MALDI TOF • Commercial identification systems (4-24h) ▫ Microscan, Vitek ▫ API , Rapid ANA
API identification system
Rapid ANA identification system
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Problems with phenotypic methods
Growth after 48h Ambiguous Gram stain result
Problems with phonotypical methods • Biochemical readings are subject of variation
and dependent on individual interpretation and expertise.
Molecular identification Methods
• Real-time PCR ▫ Fast and accurate. ▫ Limited to specific
targets. ▫ Requires additional
skills for data interpretation.
Molecular Identification Methods
• DNA Sequencing (10h) ▫ Very accurate, specific ▫ Time consuming ▫ Requires advanced
experience for test performance and data interpretation. ▫ Expensive
Why MALDI TOF MS? • Increased accuracy • Broad applicability
Broad Applicability
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Why MALDI TOF MS? • Increased accuracy • Broad applicability • Low influence on culture conditions and incubation
time.
• Spectrum peak intensity is higher from the BAP media • No score value difference observed with isolate from different
culture media. PA, BAP plate
PA, CHOC plate
PA, MAC plate
Culture media effect
Cultivation time effect • No score value difference observed with cultures incubated 8h, 24h and 48h.
• Recommendation : 16 hours minimum incubation time ( to avoid spotting agar or mixed colonies)
K.pne 48h growth
K.pne 24h growth
K.pne 8 h growth
Why MALDI TOF MS? • Increased accuracy • Broad applicability • Low influence on culture conditions incubation
time. • Faster turn-around time • Targeted antibiotic therapy • Decreased price in reagent cost • Independent of personal expertise and individual
interpretation • Robust and simple to operate
How does Maldi-TOF work?
α-cyano-4-hydroxycinnamic acid
Courtesy of Mayo Clinic
MALDI TOF MS
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Data interpretation • Identification is performed by computerized comparison of the
acquired spectra to a database of reference spectra composed of previously well-characterized isolates
,
≥2.0 = species identification
1.7-1.999 = genus identification
<1.7 = no identification
Direct Transfer Smear biological material directly on target spot.
Formic Acid (FA)
Overlay with matrix.
Dry
Overlay with FA. Dry
Overlay with matrix. Dry
Formic Acid/ Ethanol
H2O, organism
Ethanol, 100% Ethanol
Formic acid, Acetonitrile
Supernatant to test
Sample Preparation
Validation design
Finding a space for MALDI Biotyper
Selecting validation team
Selecting validation organism groups Defining performance
specifications
Defining result acceptance criteria
Defining test performance limits
Collecting data
Performance specifications
• Run controls for 20 days.
Precision (repeatability)
• Perform RTC runs for frequent and infrequent isolates that were previously identified with phenotypic, biochemical or molecular methods (gold standard).
• Resolve all discrepant results using molecular methods (PCR and DNA sequencing).
Accuracy, Sensitivity, Specificity
• Validation acceptable if identifications by MALDI TOF MS agrees with the gold standard for 95% of all organisms.
Reference interval
• Evaluate test performance using different media and incubation time.
Reproducibility
Selecting validation organism groups
▫ Anaerobic bacteria ▫ Aerobic bacteria ▫ Yeast ▫ Mold ▫ Aerobic actinomycetes and AFB
• Frequently isolated organisms: 10 isolates per species • Infrequently isolated organisms: ≥3 isolates per species • To investigate species identification capability using lower
scores.
49.85%
20.70%
9.16%
4.73%
3.29% 2.04%
2.48% 2.06% 1.90% 1.62% 0.72%
0.66% 0.46%
0.20% 0.14%
Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
Proteus spp.
Enterobacter cloacae
Klebsiella oxytoca
Citrobacter spp.
Stenotrophomonas maltophilia
Enterobacter aerogenes
Serratia marcescens
Morganella morganii
Achromobacter xylosoxidans
Acinetobacter baumannii
Salmonella spp.
Burkholderia cepacia
Gram-negative rods, 2012
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Validation design
Finding a space for MALDI Biotyper
Selecting validation team
Selecting validation organism groups Defining performance
specifications
Defining result acceptance criteria
Defining test performance limits
Collecting data
Organism Groups tested
Anaerobes , 160
Gram-neg rods, 387
Gram-pos cocci, 280
Gram-pos rods, 64
Aerobes, 731
Total: 891
Group N Species level iden8fica8on No Iden8fica8on/
Misiden8fica8on ≥1.7 ≥1.8 >2.0
Enteric Gram-‐neg rods
178
1 0.6%
8 4.5%
168 94.3%
1/0 0.6%
Non-‐fermenters 155 5 3.2%
14 9.0%
128 82.6%
1/7 5.2%
Other Gram-‐neg rods 54
1 1.8%
9 16.7%
42 77.8%
2/0 3.7%
Gram-‐posi8ve cocci 280
11 3.9%
44 15.8%
223 79.6%
2/0 0.7%
Gram-‐posi8ve rods 64 4
6.2% 14
21.9% 43
67.2% 3/0 4.7%
Anaerobes 160 3
1.9% 6
3.8% 130 81.2%
17/4 13.1%
Validation summary Bacterial
group MALDI DNA SEQ
Enterobacteriaciae 1/0
no peaks
Pantoea dispersa
Non-‐fermenters 1/7
Pseudomonas citronellolis (2) Pseudomonas libanensis Acinetobacter guillouiae Acinetobacter parvus Acinetobacter tjernbergiae Cryseobactyerium gleum No peaks
Pseudomonas delhiensis Pseudomonas extremorientalis Acinetobacter bereziniae Acinetobacter calcoaeAcus Acinetobacter beijerinckii Cryseobacterium joostei Acinetobacter sp
Other gram-‐neg rods 2/0
No reliable idenGficaGon
Asias species
Gram-‐pos cocci 2/0
No reliable idenGficaGon (2) Streptococcus lactarius Kytococcus schroeteri
Gram-‐pos rods 2/0
No reliable idenGficaGon (3)
Corynebacterium aurimucosum Corynebacterium mycetoides Arthrobacter cumminsii
No identification/ Misidentification
Bacterial group
MALDI DNA SEQ
Anaerobes No reliable identification (17)
Bacteroides vulgatus Fusobacterium canifelinum AcAnomyces oris Peptoniphilus harei
Prevotella veroralis (n=1) Cetobacterium somerae (n=1) Porphyromonas somerae (n=3) Porphyromonas uenonis(n=1) Alloprevotella rava (n=1) Eubacterium tenue (n=1) Oscillibacter sp. (n=1) AcAnomyces georgiae (n=1) Robinsonella peroiensis (n=1) Varibaculum cambriense (n=1) AcAnomyces cardiffensis(n=1) Peptostrptococcus stomaAs (n=1) Anaerococcus octavious (n=1) Murdochiella asaccharolyAca (n=1) Peptoniphilus lacrimalis (n=1) Bacteroides dorei Fusobacterium nucleatum AcAnomyces neuii Peptoniphilus indolicus
Species Complex Assignment • Acinetobacter species ▫ Acinetobacter baumannii ▫ Acinetobacter lwoffi ▫ Acinetobacter species
• Pseudomonas species group assignment ▫ Pseudomonas fluorescens group (27) ▫ Pseudomonas putida group (8) ▫ Pseudomonas aeruginosa group (14) ▫ Pseudomonas stutzeri group (3)
• Enterobacter cloacae complex ▫ Enterobacter cloacae ▫ Enterobacter asburiae ▫ Enterobacter hormaechei ▫ Enterobacter kobei ▫ Enterobacter ludwigii ▫ Enterobacter nimipressuralis
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Species Complex Assignment
• S.gallolyticus, subspecies pasteurianus and S.gallolyticus, subspecies gallolyticus can be distinguished to sub-species level if best match score is used.
MALDI TOF Limitations
E. coli vs Shigella • Very closely related, cannot be differentiated. • For sterile body sites: use PCR to r/o Shigella. • Use different method for stool cultures.
Streptococcus pneumoniae vs Streptococcus mitis group • Very closely related, cannot be differentiated. • Use different method: bile solubility and optochin disk.
Bordetella pertussis vs Bordetella bronchioseptica
• Very closely related, cannot be differentiated. • Rarely cultured.
Bacillus cereus vs Bacillus anthracis • Bacillus cereus cross-reacts with Bacillus anthracis
Overcoming limitations
Organism Bioterrorism agent that cannot not be ruled out
Alterna8ve iden8fica8on methods
Bacillus cereus
Bacillus anthracis
1.Do moGlity test and check for beta-‐hemolysis 2. Send to county (If non-‐moGle and non–hemolyGc)
Bacillus mycoides
Yersinia pseudotuberculosis
Yersinia pesAs Send to county
Burkholderia thailandensis
Burkholderia pseudomallei / Burkholderia mallei
Give to molecular bench for DNA sequencing
Vibrio albensis Vibrio cholerae Send to county
Expanding Library • A Blood culture set turned positive for Gram-negative coccobacilli, that grew on
Blood and Chocolate agar after 48h. • Colony was spotted on target and subjected to MALDI TOF analysis, but results
gave no reliable identification:
Real time classification result Spectra comparison
Score: 1.306 (Staphylococcus lutrae)
Expanding Library • Colonies were subjected to DNA sequencing of 16s rRNA gene: • DNA sequencing ID: Psychrobacter sanguinis (>99%)
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7
Expanding Library • Proteins were extracted using Formic Acid/ Ethanol tube extraction method and
generated spectrum added to Stanford library.
Expanding Library • Seven months later…. • Another patients blood culture turned positive with Gram-negative coccobacilli. • 1 mL of blood was directly extracted for protein analysis. • MALDI TOF: Psychrobacter sanguinis (2.048 score)
Psychrobacter species • Psychrobacter sp. are
psychrotolerant and halotolerant environmental microorganisms.
• They have been observed in marine environments such as deep sea or sea ice and in a wide range of food products, including seafood.
• It is normal respiratory flora for seals and sea lions
• It is rare opportunistic human pathogen.
Courtesy of Marine Mammal Center , Sausalito
Unfamiliar Species Names … • Lysinibacillus macroides • Herbaspirillum huttiense • Cupriavidus gilardii • Brachybacterium rhamnosum • Actinomyces funkei • Kerstensia gyiorum • Propionimicrobium lymphophilum • Theilavia terrestris • Staphylococcus pettenkoferi
Lysis buffer
Wash buffer
.
Positive Blood Culture Workup
• Species Identification in ~30 min
Software matters
Total Extracted % ID
Overall 341 290 85%
GNR 172 159 92% GPC 112 96 86% GPR 27 19 70% Yeast 23 9 39%
Regular protocol software
Blood Culture protocol software
Total Extracted % ID
Overall 174 160 92%
GNR 90 85 94% GPC 59 54 92% GPR 19 15 79% Yeast 6 6 100%
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Regular vs Blood Culture Protocol
Regular protocol Campylobacter jejuni Score: 1.632
Blood culture Protocol
Campylobacter jejuni
Score: 2.054
Benefits of Rapid Positive Blood Culture Identification
• Species identification results are available at least 30h earlier.
• Improved antibiotic stewardship: MALDI TOF results are emailed to pharmacist 24/7. Pharmacist recommends de-escalation or adjustment of therapy based on the rapid ID.
• Reduced hospitalization time, reduced hospitalization cost .
• Helps to resolve ambiguous Gram-stain results.
Benefits of Rapid Positive Blood Culture Identification
Blood culture turned positive with beaded Gram-positive rods at 6am. Gram-stain result was changed to Mycobacterium mucogenicum at 6:50 am.
Benefits of Rapid Positive Blood Culture Identification
• Positive blood culture bottle from pediatric patient was sent for bacterial identification together with subcultures blood and chocolate agar plates that did not have any growth.
• Protein extraction was performed directly from bottle and MALDI TOF analysis gave identification of Streptobacillus moniliformis.
Streptobacillus moniliformis • Infectious agent of “rat bite
fever”.
• Extremely fastidious Gram-negative rods that need microaerophilic conditions to grow.
• Rat is the dominant natural reservoir of S. moniliformis, which is a member of the commensal flora of the rat's upper respiratory tract.
• Pediatric patient got infected while sharing food from the same plate with his pet rat.
Courtesy of CDC
Yeast Validation
Isolates tested (80)
• Candida spp. (55) • Cryptococcus spp.
(20) • Other yeast (5)
Sample preparation
• Formic Acid • Formic acid/
Ethanol tube
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Yeast Validation Total
Species level identification No Iden8fica8on/ Misiden8fica8on
(≥1.7) (≥1.8) (>2.0)
80 3
3.7%
10 12.6%
64 80%
3 (3.7%) MALDI: No reliable idenGficaGon SEQ: Cryptococcus laurenAi (n=1) MALDI : No reliable idenGficaGon SEQ: Cryptococcus neoformans (n=2)
• ExtracGon: FA and FA/ Etahnol • Score for species and genus level idenGficaGon 1.700-‐3.000 • Can idenGfy Cryptococcus gaSi. • New technology, new names:
• Magnusiomyces capitatus • Cyberlindnera fabianii • Sporidiobolus salmonicolor
Mold Validation
Vortex for 10 min
Centrifuge at 13,000 rpm for 2 min
Vortex for 5 min Vortex for
5 min
ETH, 70% Beads
FA, 70%
ACT, 100%
• Libraries for spectrum match :
Bruker and NIH
• Tested : 118 isolates • Score for species level ID: 1.700 – 3.000. • Species identification in ~30 min
Spot 1 µL on target Overlay with Matrix
Protein Extraction
Mold Validation
Aspergillus species, 29
Dermatophytes, 15
Fusarium species, 8 Dematiaceous
Mold , 32
Mucorales, 14
Other mold, 20
Total: 118 Mold Validation Mold groups Sensi8vity
Genus(≥1.7) Sensi8vity Species(≥1.7)
No iden8fica8on/ Misiden8fica8on
Aspergillus sp. 100% (29/29)
96.6% (28/29)
A. sydowii idenGfied as A. versicolor
Dema8aceous Moulds 89% (24/27)
100% (4/4)
Bipolaris sp. idenGfied as Curvularia sp. (3)
Dermatophytes 100% (15/15)
90% 9/10
T. verrucosum idenGfied as T. mentagrophytes
Fusarium 100% (8/8)
100% (2/2)
Mucorales 100% (14/14)
100% (4/4)
Pseudallescheria boydi complex/Scedosporium prolificans
100% (5/5)
100% (5/5)
Other Hyaline Molds 100% (20/20)
100% (10/10)
Other challenges
Name May cross-‐iden8fy with Aspergillus flavipes Candida albicans Aspergillus nidulans Emericella quadrilineata Aspergillus oryzae Aspergillus flavus Aspergillus sydowii Aspergillus versicolor Bipolaris hawaiiensis Curvularia lunata Curvularia lunata Curvularia pallescens Emericella quadrilineata Aspergillus nidulans Fusarium oxysporum Fusarium proliferatum and Fusarium verAcillioides Fusarium proliferatum Fusarium oxysporum Fusarium verAcillioides Fusarium oxysporum and Fusarium moniliforme Mucor circinelloides Stenotrophomonas maltophilia Trichoderma longibrachiatum Trichoderma viridae
Trichophyton verrucosum Trichophyton mentagrophytes
Species group assignment
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Benefits of Mold identification by MALDI TOF Skin tissue turned positive for mold. No sporulation, only sterile hyphae observed during 4 weeks of incubation.
Lacto phenol cotton blue stain , 500x
Benefits of Mold identification by MALDI TOF
• Part of colony was extracted and subjected to MALDI TOF MS. • MALDI TOF MS: Trichophyton rubrum (1.840 score)
Benefits of Mold identification by MALDI TOF
• 68 y/o man with h/o rheumatoid arthritis maintained on prednisone and methotrexate presents with 3 months on increasing pain and swelling in the right forearm and wrist.
• Ulnar styloid aspirated: 1 mL of turbid yellow fluid.
• Gram stain: Rare number of PMNs, no organisms seen.
• Fluid culture: 3 waxy colonies appeared on blood agar plate after 48h. Preliminary report: Yeast .
Gram stain, 500x
Benefits of Mold identification by MALDI TOF
Courtesy of Niaz Banaei
• Colony was spotted on MALDI target for species identification. • MALDI TOF: Sporothrix schenckii (score: 2.044 )
Benefits of Mold identification by MALDI TOF Expanding Mold Library
Courtesy of Laleh Ghafghaichi
• BAL culture turned positive for very fastidious MOLD after 3 weeks of incubation. • No identification specific characteristics seen with Lacto phenol cotton blue stain.
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Expanding Mold Library • MALDI TOF MS: no reliable identification. • Score: 1.56 (Coccidioides immitis)
Expanding Mold Library • DNA sequencing of ITS2 and D2 regions gave Coccidioides immitis/
Coccidioides posadasii. • Spectrum added to Stanford library.
No Test is Perfect • No susceptibility information is provided.
• Technology is not useful for direct testing of clinical specimens.
• Technology is not reliable for identification of mixed cultures.
• MALDI TOF MS does not work on refrigerated cultures.
• No spectrum in library = No ID.
• Sometimes there are no peaks generated upon all extraction methods.
• Need to have a backup in case of maintenance or malfunction.
Successful result depends on good target spotting
(garbage in = garbage out)
Small colony poorly smears on target . Agar introduced with the colony.
Test repeated next day with more grown colony and better spotting.
MALDI TOF MS workflow adjustments
• PCR, DNA sequencing, one Vitek, Microscan - STAY • Biochemicals, API panels, Remel rapid panels one Vitek- GO
Which one goes, which one stays?
• One CLS per shift
Who performs the test
• Daily at 9am, 12pm, 3pm, once per PM shift, once per midnight shift.
How many times per day is test performed?
• Interpretation- CLS performing MALDI TOF MS • Result reporting- CLS performing bench level identification
Who interprets and reports results?
MALDI TOF MS workflow adjustments
• New bacterial, fungal, yeast names. • No/few CLSI breakpoints.
Microorganism Identification
• Safety steps need to be defined. When to accept ID? Agree with Gram stain? To which ID level report ?
Automation of IDs
• MALDI for sterile sites. • Use rapid IDs for urines, latex IDs, indole, colony
morphology.
Rapid Vs. MALDI IDs
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Microbiologists concern: “Loosing the excitement of recognizing the bacterial face”
Thanks to MALDI TOF MS validation team for excellent job!
• Dr. Niaz Banaei • Amy Cheng • Cheryl D’souza • Farnaz Foroughi • Laleh Ghafghaichi • Nathan Taylor • Cheryl Tau • Jennifer Kuschel
“The rapid identification of organisms, especially those in blood cultures, is extremely valuable for the quick treatment of critical infections” . Amy K. “MALDI TOF has probably saved many lives since we began using it. Our physicians are able to provide the most effective treatment much more rapidly since they know the precise identification of what they are treating”. Diane “A revolutionary tool in identification of microorganisms : fast, easy, convenient, accurate”. Manju “MALDI made our lives easier in the lab and help patients to get treatment much faster “. Abbas “Astonishingly fast and accurate results in what seems like children play with toothpicks”. Vasu “It is an awesome machine : no more waiting days to be able to identify organisms”. Veronica “Accurate, simple, fast results. Revolutionary change in workflow”. Cheryl T “The most efficient and convenient way to get an organism identification. It has saved us time and money from doing extra work that is unnecessary. The precision of this instrument has helped us in resulting patient reports with confidence”. Shirley