MALDI TOF Mass Spectrometry A New Era in Diagnostic

MALDI TOF Mass Spectrometry A New Era in Diagnostic

Microbiology

Dr Barry Wong, Scientific Officer (Medical) Microbiology Laboratory, United Christian Hospital

19 May 2015

1

2021

1995

1973

United Christian Hospital

3

Traditional Microbiology

How to identify the pathogen??

Colony characteristics Direct microscopy

Biochemical tests

Antigen detection

protein bacterial cell

DNA

genome proteome

DNA fingerprint

3000 5000 7000 9000 11000 13000

Mass ( m/ z)

2117. 3

0

10

20

30

40

50

60

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80

90

100

% Intensity

V o y a g e r S p e c # 1 = > A d v B C ( 3 2 , 0 . 5 , 1 . 0 ) = > N F 0 . 7 [ B P = 4 4 2 3 . 9 , 2 1 1 7 ]

44

24

.2

94

76

.2

48

61

.0

88

23

.5

58

97

.8

62

71

.2

80

54

.3

66

29

.0

74

24

.9

12

27

1.4

47

38

.6

69

84

.6

83

83

.8

67

86

.7

65

33

.3

90

75

.2

31

32

.3

61

37

.3

72

09

.8

89

03

.3

86

10

.6

10

11

0.8

47

65

.4

10

97

5.2

40

26

.9

45

35

.6

33

11

.8

10

49

3.9

34

91

.4

96

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.5

mass fingerprint

Species identification

Microbial taxonomy: genomics vs proteomics

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Technology advancement

What is MALDI TOF MS?

MALDI-TOF MS(Matrix Assisted Laser Desorption / Ionization – Time-Of-Flight Mass Spectrometry). Mass Spectrometry (MS) is an analytical technique to determine the elemental composition of a sample. The MS principle consists of ionizing chemical compounds to generate charge molecules and to measure their mass-to-charge ratio. The first attempt of using MS technology started in 1970s and was mainly used for Chemical Pathology. A type of MS which can allow the screening proteins directly from intact bacteria.

5

Technology breakthrough in 1996….

The use of MALDI-TOF for the whole cell fingerprinting and identification of bacteria from cell surface proteins, ribosomal proteins, high abundance

cold-shock protein, and acid resistance proteins 6

Matrix Assisted Laser Desorption / Ionization – Time-Of-Flight

The analyte with matrix was dried on a target plate

Matrix

Analyte (organism)

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Target plate


High energy laser

The laser fired at the matrix dried sport and cause desorption: matrix absorbs the energy from laser and becomes ionized (by addition of a proton)

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The matrix will transfer the proton to the analyte molecule making it becomes a positive charge [M-H]+ ion.

Finally, laser causes the sample and matrix volatilize

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The ions formed are accelerated by a high voltage supply and allow to drift down a flight tube where they are separated according to their mass

The positively charged proteins draft in the flight tube, with the smaller proteins draft faster

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MALDI-TOF is a technology that can develop a protein pattern from an unknown isolate. This unknown pattern is used to match with known library and presents with a score for that isolate to facilitate species identification.

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Target preparation MALDI-TOF

measurement Bacterial identification

Workflow of MALDI TOF

12 96 tests / 1 hour

Meaning of score value

Meaning of consistency category A, B, C Category Description

A Species consistency: The best match was classified as green. Further green matches are of the same species as the first one. First yellow match are at least the same genus as the first one.

B Genus consistency: The best match was classified as green or yellow. Further green or yellow matches are of the same genus as the first one. The conditions of species consistency are not fulfilled.

C No consistency: Neither species nor genus consistency. Check for synonyms of names or microbial mixtures. 13

All green matches belong to the same species

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Class A report – Species consistent result

All the green matches belong to the same genus

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Class B report – Genus consistent result

One green and three yellow matches belong to different genus

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Class C report – Non consistent result

Limitation in data base • E. coli / Shigella

• Streptococcus pneumoniae / S. mitis / S. oralis

• Salmonella species

• Raoultella ornithinolytica / Klebsiella oxytoca

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Need supplementary tests

Evaluation data • From 15 Jan to 15 April 2015

• 431 bacteria and yeasts

• Clinical isolates and ATCC strains

• < 3 days isolates

• Positive blood culture (94):

– Positive bottles in the morning, use BA for MALDT testing in the afternoon (~5-6 hrs incubation)

• Compared with conventional tests, commercial kits and gene sequencing

• Agar: BA, CHO, MH, MHBA, HTM, SAB

Results Number (percentage)

Species Level

382 (88.6%)

Genus Level

45 (10.4%)

Mismatch with conventional tests

4 (1%)

18 99% identified to genus + species level

MALDT TOF result -Organism (no) Species level No. (%) Genus level No. (%) Wrong ID No. (%) Comment/In house result

E. Coli (18) 18 (100%) 0 (0%) 0 (0%) Closely related with Shigella

Enterobacter cloacae (3) 3 (100%) 0 (0%) 0 (0%)

Enterobacter kobei (2) 0 2 (100%) 0 (0%) E. cloacae, E. species

Enterobacter aerogenes (2) 2 (100%) 0 (0%) 0 (0%)

Enterobacter gergoviae (1) 0 (0%) 1 (100%) 0 (0%) E. aerogenes

Citrobacter freundii (7) 7 (100%) 0 (0%) 0 (0%)

Citrobacter koseri (3) 3 (100%) 0 (0%) 0 (0%)

Citrobacter youngae (1) 0 1 (100%) 0 (0%) Citrobacter species

Achromobacter xylosoxidans (1) 1 (100%) 0 (0%) 0 (0%)

Klebsiella pneumoniae (9) 9 (100%) 0 (0%) 0 (0%)

Klebsiella oxytoca (3) 3 (100%) 0 (0%) 0 (0%)

Raoultella ornithinolytica (2) 0 (0%) 0 (0%) 2 (100%) Klebsiella oxytoca

Morganella morganii (6) 6 (100%) 0 (0%) 0 (0%)

Proteus mirabilis (7) 7 (100%) 0 (0%) 0 (0%)

Proteus vulgaris (4) 4 (100%) 0 (0%) 0 (0%)

Providencia alcalifaciens (1) 1 (100%) 0 (0%) 0 (0%)

Providencia rettegeri (2) 2 (100%) 0 (0%) 0 (0%)

Providencia stuartii (1) 1 (100%) 0 (0%) 0 (0%)

Plesiomonas shigelloides (2) 2 (100%) 0 (0%) 0 (0%)

Acinetobacter baumannii (3) 3 (100%) 0 (0%) 0 (0%)

Acinetobacter guillouiae (1) 0 (0%) 1 (100%) 0 (0%) Acinetobacter species

Acinetobacter tandoii (1) 0 (0%) 1 (100%) 0 (0%) Acinetobacter species

Acinetobacter ursingii (1) 0 (0%) 1 (100%) 0 (0%) Acinetobacter species

Aeromonas caivae (2) 2 (100%) 0 (0%) 0 (0%) Aeromonas caivae by API, report as Aeromonas sp

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20


Aeromonas hydrophila (3) 3 (100%) 0 (0%) 0 (0%) Aeromonas hydrophila by API, report as Aeromonas sp

Aeromonas veronii (1) 0 (0%) 1 (100%) 0 (0%) Aeromonas species

Burkholderia cepacia (2) 2 (100%) 0 (0%) 0 (0%)

Burkholderia cenocepacia (3) 3 (100%) 0 (0%) 0 (0%) Burkholderia cepacia

Campylobacter jejuni (2) 2 (100%) 0 (0%) 0 (0%)

Campylobacter coli (2) 2 (100%) 0 (0%) 0 (0%)

Pseudomonas aeruginosa (5) 5 (100%) 0 (0%) 0 (0%)

Salmonella species (11) 0 (0%) 11 (100%) 0 (0%) Confirmed by serology

Serratia marcescens (8) 8 (100%) 0 (0%) 0 (0%)

Stenotrophomonas maltophilia (4) 4 (100%) 0 (0%) 0 (0%)

Vibrio parahaemolyticus (3) 3 (100%) 0 (0%) 0 (0%)

Vibrio vulnificus (3) 3 (100%) 0 (0%) 0 (0%)

Myroides odoratimimus (1) 0 (0%) 1 (100%) 0 (0%) Myroides sp (vitek)

Yersinia enterocolotica (1) 1 (100%) 0 (0%) 0 (0%) RCPA QAP

Elizabethkingia meningoseptica (2) 2 (100%) 0 (0%) 0 (0%)

Chryseobacterium indologenes (1) 0 (0%) 1 (100%) 0 (0%) Chryseobacterium species

Chryseobacterium gleum (1) 0 (0%) 1 (100%) 0 (0%) Chryseobacterium indologenes (Vitek)

Moraxella catarrhalis (3) 3 (100%) 0 (0%) 0 (0%)

Moraxella nonliquefaciens (2) 1 (50%) 1 (50%) 0 (0%) M. nonliquefaciens by sequencing (1) (eye), Moraxella species (1)

Moraxella osloensis (2) 1 (50%) 1 (50%) 0 (0%) Moraxella osloensis (1), Moraxella species (1)

Neisseria mucosa (1) 0 (0%) 1 (100%) 0 (0%) N. sicca (Vitek)

Neisseria gonorrhoeae (6) 6 (100%) 0 (0%) 0 (0%)

Haemophilus influenzae (11) 11 (100%) 0 (0%) 0 (0%) Non-haemolytic

Kingella kingae (1) 1 (100%) 0 (0%) 0 (0%)

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Aerococcus viridans (1) 1 (100%) 0 (0%) 0 (0%)

Aerococcus sanguinicola (1) 1 (100%) 0 (0%) 0 (0%)

Enterococcus avium (3) 3 (100%) 0 (0%) 0 (0%)

Enterococcus faecium (6) 5 (83%) 1 (17%) 0 (0%) Enterococcus species (1) (MSU)

Enterococcus faecalis (17) 17 (100%) 0 (0%) 0 (0%)

Streptococcus agalactiae (10) 10 (100%) 0 (0%) 0 (0%)

Streptococcus constellatus (4) 4 (100%) 0 (0%) 0 (0%)

Streptococcus intermedius (3) 3 (100%) 0 (0%) 0 (0%) Streptococcus intermedius (2), Streptococcus milleri (1)

Streptococcus pneumoniae (14) 14 (100%) 0 (0%) 0 (0%) Closely related with S. mitis / S. oralis

Streptococcus pyogenes (10) 14 (100%) 0 (0%) 0 (0%)

Staphylococcus aureus (12) 12 (100%) 0 (0%) 0 (0%) MRSA (5), MSSA (7)

Staphylococcus epidermidis (11) 4 (36%) 7 (64%) 0 (0%) S. epidermidis (4), CNS (7)

Staphylococcus haemolyticus (3) 2 (67%) 1 (33%) 0 (0%) S. haemolyticus (2), CNS (1)

Staphylococcus lugdunensis (2) 1 (50%) 1 (50%) 0 (0%) S. lugdunensis (1), CNS (1)

Staphylococcus saprophyticus (3) 3 (100%) 0 (0%) 0 (0%)

Candida albicans (11) 11 (100%) 0 (0%) 0 (0%) FAE (4)

Candida glabrata (13) 9 (69%) 4 (31%) 0 (0%) Candida species (4), FAE (5)

Candida parapsilosis (1) 1 (100%) 0 (0%) 0 (0%) FAE

Candida tropicalis (4) 2 (50%) 2 (50%) 0 (0%) Candida species (2), FAE (2)

Cryptococcus neoformans (2) 2 (100%) 0 (0%) 0 (0%) FAE (2)

Bacteroides fragilis (3) 3 (100%) 0 (0%) 0 (0%)

Clostridium difficile (5) 5 (100%) 0 (0%) 0 (0%) Detected by PCR

Clostridium sordellii (2) 2 (100%) 0 (0%) 0 (0%)

Fusobacterium varium (1) 1 (100%) 0 (0%) 0 (0%)

ATCC strain MALDI-TOF results

ATCC 51299 Enterococcus faecalis

20 ATCC stains identified to species level

ATCC 29212 Enterococcus faecalis

ATCC 12386 Streptococcus agalactiae

ATCC 49619 Streptococcus pneumoniae

ATCC 19615 Streptococcus pyogenes

ATCC BA1708 Staphylococcus aureus

ATCC 43300 Staphylococcus aureus



ATCC 12228 Staphylococcus epiderimidis

ATCC 25922 Escherichia coli

ATCC 35218 Escherichia coli

ATCC 49766 Haemophilus influenzae

ATCC 49247 Haemophilus influenzae

ATCC 70063 Klebsiella pneumoniae

ATCC 13883 Klebsiella pneumoniae

ATCC 43069 Neisseria gonorrhoeae

ATCC 12453 Proteus mirabilis

ATCC 27853 Pseudomonas aeruginosa

ATCC 14028 Serratia marcescens

ATCC 13880 Salmonella typhimurium Identified to genus level ATCC 12022 Shigella flexneri

Identified as E. coli ATCC 9290 Shigella sonnei 22

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Positive blood culture bottles


E. Coli (44) 44 (100%) 0 (0%) 0 (0%)

Klebsiella pneumoniae (13) 13 (100%) 0 (0%) 0 (0%)

Proteus mirabilis (5) 5 (100%) 0 (0%) 0 (0%)

Enterobacter aerogenes (2) 2 (100%) 0 (0%) 0 (0%)

Salmonella species (2) 0 (100%) 2 (100%) 0 (0%) Confirmed to group level by API and serology

Serratia marcescens (1) 1 (100%) 0 (0%) 0 (0%)

Bacteroides fragilis (3) 3 (100%) 0 (0%) 0 (0%)

Staphylococcus aureus (9) 9 (100%) 0 (0%) 0 (0%) 8 MSSA, 1 MRSA

Corynebacterium striatum (2) 2 (100%) 0 (0%) 0 (0%)

Clostridium perfringens (1) 1 (100%) 0 (0%) 0 (0%)

Enterococcus avium (1) 1 (100%) 0 (0%) 0 (0%)

Parvimonas micra (1) 1 (100%) 0 (0%) 0 (0%)

Staphylococcus epidermidis (4) 2 (50%) 2 (50%) 0 (0%) S. epidermidis (2), CNS (2)

Staphylococcus hominis (1) 0 (100%) 1 (100%) 0 (0%) CNS

Streptococcus agalactiae (3) 3 (100%) 0 (0%) 0 (0%)

Streptococcus dysgalactiae (1) 1 (100%) 0 (0%) 0 (0%)

Streptococcus mitis (2) 1 (50%) 1 (50%) 0 (0%) S. mitis (1), Viridans strep (1)

Streptococcus oralis (1) 1 (100%) 0 (0%) 0 (0%)

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Special consideration

• E. coli / Shigella – Lactose fermenting – Motility – Serology

• Streptococcus pneumoniae / S. mitis / S. oralis – Optochin susceptibility – Slidex pneumo-kit

• Salmonella species – Serology typing – API 20E for S. typhi, S. paratyphi and blood culture isolates

• Raoultella ornithinolytica / Klebsiella oxytoca – Ornithine decarboxylase test

• Not include in current database: Bacillus anthracis, Brucella species, Burkholderia mallei, Clostridium botulinum, Vibrio cholera

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Implementation date in UCH 15 April 2015

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Challenges

• Staff training • Testing and reporting algorithm • Quality control issues

– BTS, Standard strain, blank control, data transfer

• Equipment and consumable maintenance – Life span of laser, plate

• Re-design workflow • Laboratory accreditation • Integration with other equipment - AST • Laboratory automation • Back up plan

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Positive

• Faster TAT – life threatening diseases – MALDI-TOF + PCR for rapid identification of MRSA in

blood culture – Same date result as for gram stain vs 2-3 days later

• Reduce in consumable cost • Reduce manual work error • Reduce unnecessary workload • Workflow reengineering

– Formation of mega-bench – Extend service hours

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Thank you

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Acknowledgement

Dr Kitty Fung (CONS) Dr Sandy Chau (AC) Mr Ricky Ng (MT) Mr Jason Cheng (AMT) Mr Kelvin Tsui (AMT)

UCH Microbiology Team Members

Documents

MALDI TOF Mass Spectrometry A New Era in Diagnostic