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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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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