Safety and Accuracy Assessment of MALDI-TOF Mass Spectrometry Platforms for the Detection of Biological Threats
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1 Safety and Accuracy Assessment of MALDI-TOF Mass Spectrometry Platforms for the Detection of Biological Threats James T. Rudrik, Ph.D. Michigan Department of Health and Human Services
2 Preparation Safety Study Determined viability of BT agents using direct, extended direct, and tube extraction Bacillus anthracis Sterne, Brucella abortus Strain 19, Francisella tularensis LVS, Yersinia pestis A1122, Burkholderia thailandensis ATCC 70038, Clostridium spp (botulinum types A, B, E, and perfringens) Some isolates of B. anthracis, C. botulinum, F. tularensis, Y. pestis, and B. abortus survived the direct and extended direct prep methods None survived the tube extraction B. anthracis, B. thuringiensis, B. cereus, B. subtilis survived tube extraction in other studies Use tube extraction AND 0.1 µm filter for hazardous organisms
3 Study Design Determine ability of Bruker and Vitek software libraries to accurately identify BT agents Test IVD, ROU, and Security Libraries independently Panel of 6 strains and 6 near neighbors for each agent B. anthracis, Y. pestis, F. tularensis (5), C. botulinum (4), B. mallei, B. pseudomallei, Brucella spp. Select isolates from AOAC Stakeholder Panel on Agent Detection Assays (SPADA) inclusivity and exclusivity panels Each isolate tested in triplicate Challenges No SPADA panels for C. botulinum or Brucella spp. Isolate availability and shipping No tube extraction procedure for Vitek MS
4 Study Limitations Samples were prepared by four labs, shipped and stored frozen Some participants tested extracts beyond the 45 day time frame established in the study protocol Some participants prepared new targets for testing each software library
5 Bruker Microflex Software libraries: IVD (version 1, n = 210), RUO (version 3, n = 2,500), Security (version 1, n = 12) Sample score of > 2.0 was considered accurate ID to genus and species Published studies validated samples scores between 1.7 and 1.9 for species level identification for some organisms
6 (# of strains B. anthracis (6) B. thuringiensis B. circulans B. cereus B. mycoides B. megaterium B. subtilis Reported Identification IVD Library RUO Library Security Library ID % (>2.0) ID % (>2.0) ID % (>2.0) No reliable ID 1.13 (90) 1.38 (45) 1.1 (40) B. cereus 1.88 (62) 8.3 B. anthracis 2.08 (68) 50 B. pseudomycoides 1.56 No reliable ID 1.1 (12) 1.33 (9) 1.12 (9) B. cereus 2.01 (9) 22.2 B. anthracis 2.06 (9) 38.9 No reliable ID 1.06 (12) 1.33 (5) 1.01 (18) B. circulans 1.87 (13) 16.7 No reliable ID 1.15 (12) 1.35 (9) 1.23 (9) B. cereus 2.08 (9) 44.4 B. anthracis 2.14 (9) 50 No reliable ID 1.15 (15) 1.61 (7) B. mycoides 1.77 (7) B. anthracis 1.77 (18) B (4) weihenstephanensis No reliable ID 1.12 (12) 1.35 (3) 1.06 (18) B. megaterium 1.98 (15) 55.6 No reliable ID 1.04 (15) 1.47 (13) 0.98 (18) B. subtilis 1.68 (5)
7 (# of strains Reported Identification IVD Library RUO Library Security Library ID % (>2.0) ID % (>2.0) ID % (>2.0) Y. pestis (6) Y. ruckeri Y. pseudotuberculosis (3) Y. enterocolitica (2) No reliable ID 1.47 (8) 1.5 (10) 1.46 (14) Y. pseudotuberculosis 2.15 (72) (98) 81.5 Y. pestis 2.16 (94) 82.4 No reliable ID 1.67 (2) 1.68 (2) Y. ruckerii 1.88 (9) Y. pseudotuberculosis 1.75 (8) 1.82 (6) Y. pestis 1.82 (16) Y. enterocolitica 1.72 (2) 1.88 (3) No reliable ID 1.64 Y. pseudotuberculosis 2.04 (38) (53) 86.8 Y. pestis 1.96 (53) 35.8 No reliable ID 1.64 (7) Y. enterocolitica 2.18 (17) (36) 100 Y. pseudotuberculosis 2.14 (7) 29.1 Y. pestis 1.97 (29) 33.3
8 (# of strains F. tularensis (5) F. philomiragia (3) F. novicida (2) H. influenzae Reported Identification IVD Library RUO Library Security Library ID % (>2.0) ID % (>2.0) No reliable ID 1.11 (60) 1.33 (87) 1.34 (46) ID % (>2.0) F. tularensis 1.77 (41) 4.6 No reliable ID 1.14 (39) 1.43 (14) 1.16 (54) F. philomiragia 1.94 (40) 22.2 No reliable ID 1.1 (24) 1.36 (36) 1.45 (29) F. tularensis 1.73 (6) No reliable ID 1.56 (5) 1.04 (18) H. influenzae 2.1 (10) (18) 83.3
9 (# of strains B. mallei (6) B. thailandensis B. cepacia B. cenocepacia B. multivorans S. maltophilia Reported Identification IVD Library RUO Library Security Library ID % (>2.0) ID % (>2.0) ID % (>2.0) No reliable ID 1.27 (75) 1.56 (47) B. thailandensis 1.87 (58) 7.4 B. pseudomallei 2.16 (5) 3.7 B. mallei 2.17 (103) 71.3 B. vietnamiensis 1.33 (3) No reliable ID 1.36 (15) B. thailandensis 2.11 (18) 94.4 B. pseudomallei 1.96 (11) 22.2 B. mallei 1.99 (7) 16.6 No relaible ID 1.34 (18) B. cepacia complex 2.05 (15) 53.3 B. cepacia 2.17 (12) 66.6 B. cenocepacia 2.01 (2) 5.6 B. pyrrocina 2.07 (4) 11.1 No reliable ID 1.27 (18) B. cepacia complex 2.04 (15) 60 B. cenocepacia 2.24 (18) 100 No reliable ID 1.42 (18) B. multivorans 2.18 (15) (18) 77.7 No reliable ID 0.95 (18) S. maltophilia 2.02 (15) (18) 94.4
10 (# of strains B. pseudomallei (6) Reported Identification (# replicates) IVD Library RUO Library Security Library Species level ID % (>2.0) No reliable ID 1.27 (74) 1.54 (28) ID % (>2.0) B. thailandensis 1.84 (79) 5.6 ID % (>2.0) B. 2.1 (100) 78.5 pseudomallei B. mallei 2.06 (7) 6.5
11 (# of strains C. botulinum (4) C. perfringens C. difficile C. septicum C. sordellii C. innocuum C. butyricum Reported Identification IVD Library RUO Library Security Library Species ID % (>2.0) level ID % (>2.0) ID % (>2.0) No reliable ID 1.08 (51) 1.47 (26) 1.29 (51) C. sporogenes 1.86 (46) C. botulinum 1.84 (21) 15.3 No reliable ID 1.0 (3) 1.05 (18) C. perfringens 2.22 (12) (18) 100 No reliable ID 0.97 (3) 1.04 (17) C. difficile 2.14 (12) (17) 100 No reliable ID 1.12 (15) 1.06 (18) C. septicum 2.30 (18) 100 No reliable ID 1.07 (15) 1.09 (18) C. sordellii 2.12 (18) 83.3 No reliable ID 1.18 (15) 1.55 (2) 1.06 (18) C. innocuum 2.20 (16) 83.3 No reliable ID 1.10 (15) 1.36 (3) 0.98 (18) C. butyricum 2.32 (15) 77.8
12 (# of strains Reported Identification IVD Library RUO Library Security Library ID % (>2.0) ID % (>2.0) ID % (>2.0) B. melitensis (2) B. abortus (2) B. suis B. canis B. neotomae B. ovis O. anthropi B. pinnipedialis B. ceti O. ureolytica No reliable ID 1.04 (18) 1.3 (36) 1.6 (2) B. melitensis 2.17 (34) 83.3 No reliable ID 1.03 (23) 1.31 (35) B. melitensis 2.17 (35) 82.9 No reliable ID 1.04 (12) 1.27 (18) 0 B. melitensis 2.17 (17) 94.4 No reliable ID 0.75 (12) 1.21 (18) 1.63 (9) B. melitensis 2.05 (9) 44.4 No reliable ID 1.16 (15) 1.3 (18) B. melitensis 1.96 (18) 22.2 No reliable ID 1.06 (15) 1.32 (18) 1.36 B. melitensis 1.88 (17) 11.1 No reliable ID 1.04 (15) (18) Ochrobactrum spp (13) O. intermedium 1.82 No reliable ID 1.13 (15) 1.43 (15) 1.63 (3) B. melitensis 1.81 (15) Ochrobactrum tritici 1.77 (3) No reliable ID 1.1 (13) 1.34 (18) B. melitensis 1.98 (18) 38.9 No reliable ID 1.27 (2) (18) O. ureolytica 1.88 (13) (17) 22.2
13 Vitek MS Vitek MS (biomerieux Inc., Durham, NC) equipped with the IVD (version 2.0, n = 193) and RUO (version 4.12, n = 1,388) software libraries Identification score of >60% was considered accurate ID to genus and species
14 (# of strains Reported Identification IVD Library RUO Library B. anthracis (6) B. thuringiensis B. circulans B. cereus No Identification 0 (12) 0 (19) B. thuringiensis/b. cereus/b. mycoides 33.3/33.3/33.3 (36) B. cereus group 94.9 (35) No Identification 0 B. mycoides/b. cereus/b. thuringiensis 33.3/33.3/33.3 (9) B. cereus group 81.6 (8) No Identification 0 (7) 0 (7) B. circulans 45 (2) Serratia rubideae/mycobacterium 25/25 smegmatis E. coli No Identification 0 (3) 0 (3) B. mycoides/b. cereus/b. thuringiensis 33.3/33.3/33.3 (6) B. cereus group 95.3 (5) Capnocytophaga ochacea/sputigena 78.4
15 (# of strains Reported Identification IVD Library RUO Library B. mycoides B. megaterium B. subtilis No Identification 0 (3) 0 B. mycoides/b. cereus/b. thuringiensis 33.3/33.3/33.3 (6) B. cereus group 91.5 (3) B. weihenstephanensis 84.7 (5) 55.6 No Identification 0 (2) B. megaterium 94.8 (9) (3) 33.3 B. megaterium/b. coagulans/b.amyloliquefaciens 82.4 (3) B. coagulans/b. megaterium 76.6 No Identification 0 (6) B. amyloliquefaciens/b. subtilis 50/50 (9) B. subtilis 82 (3) 33.3
16 (# of isolates Reported Identification IVD Library RUO Library Y. pestis (6) Y. ruckeri Y. pseudotuberculosis (3) Y. enterocolitica (2) No Identification 0 (2) 0 (23) Y. pseudotuberculosis/y. frederiksenii 51.7/48.2 (7) Y. ruckerii/y. pseudotuberculosis/y. 33.3/33.4/33.2 frederiksenii Y. pseudotuberculosis 99.9 (17) (18) 33.3 Y. pestis Yersinia spp (13) No Identification 0 (5) Y. ruckerii 99.7 (4) 44.4 Yersinia spp (9) No ID 0 Y. pseudotuberculosis 99.6 (20) (20) 95.2 Y. enterocolitica No Identification 0 Y. enterocolitica 99.9 (8) (11) 91.7 Y. pseudotuberculosis 86 Y. pseudotuberculosis/y. enterocolitica 49.1/50.9 (3)
17 (# of strains Reported Identification IVD Library RUO Library F. tularensis (5) No ID 0 (33) 0 (31) F. tularensis 77.5 (14) 24.4 Enterobacter cloacae/e. asburiae 50/50 Streptococcus constellatus Streptococcus pluranimalium 64.5 (3) 4.4 Vibrio mimicus 33.6 Kocuria varians/gemella bergeri 33.3/33.4 Cellulosmicrobium cellulans/s. equi ssp equi 46.2/53.7 F. philomiragia (3) F. novicida (2) H. influenzae Acinetobacter johnsonii 79.8 (2) 4.4 Kytococcus sedentarius No Identification 0 (27) 0 (25) Microsporum canis Enterococcus spp No ID 0 (16) 0 (8) F. tularensis 92.2 (10) 55.6 Erwinia rhapontici Vibrio alginolyticus No Identification 0 (4) 0 (6) H. influenzae 99.9 (4) (3) 33.3 H. influenzae/h. haemolyticus/s. mitis/s. oralis 33/33/33
18 (# of isolates B. mallei (6) B. thailandensis B. cepacia B. cenocepacia Reported Identification IVD Library RUO Library No Identification 0 (51) 0 (31) B. multivorans Burkholderia spp (6) Streptococcus porcinus 95.3 (2) 3.7 Staphylococcus carnosus Escherichia coli Yersinia spp No Identification 0 (9) 0 (5) Burkholderia spp (3) Staphylococcus epidermidis 75 No Identification 0 (4) 0 B. cepacia 99.4 (2) Burkholderia spp. 85 (7) B. cepacia/b. vietnamensis 50/50 No Identification 0 (3) 0 (5) B. cepacia 99.8 (5) 55.6 Burkholderia spp (4) B. cepacia/b. vietnamensis 50.0/49.9
19 (# of isolates Reported Identification IVD Library RUO Library B. pseudomallei (6) B. multivorans S. maltophilia No Identification 0 (54) 0 (47) Burkholderia spp (2) Staphylococcus aureus Streptococcus oralis E. coli Yersinia spp. 75 No Identification 0 (3) 0 (4) B. multivorans 88.8 (6) (3) 33.3 Burkholderia spp (2) No Identification 0 (2) 0 (3) S. maltophilia 99.9 (7) (6) 66.7
20 (# of strains Reported Identification IVD Library RUO Library C. botulinum No Identification 0 (9) 0 (35) (4) Clostridium sporogenes 99.5 (24) 66.7 Mycobacterium bovis/m. tuberculosis/c. sporogenes 33/33/33 (3) Candida krusei C. perfringens C. perfringens 99.9 (9) (9) 100 C. difficile No Identification 0 (3) 0 C. difficile 96.5 (6) (8) 88.9 C. septicum C. sordellii C. innocuum No Identification 0 C. septicum 99.9 (9) (8) 88.9 No Identification 0 (9) C. sordellii 92.6 (8) 88.9 C. sordellii/l. monocytogenes 58.6/41.4 No Identification 0 (8) 0 (7) Citrobacter freundii Candida norvegensis Staphylococcus aureus C. butyricum C. butyricum 99.9 (6) (9) 100
21 (# of isolates Reported Identification IVD Library RUO Library B. melitensis (2) B. abortus (2) B. suis B. canis No ID 0 (16) 0 (7) Brucella spp (11) Enterococcus avium 35.6 Prevotella disiens No ID 0 (15) 0 (9) Brucella spp (9) Listeria seeligeri 50.1 Alloiococcus otitis 51.4 Actinomyces radingae/l. seeligeri 56.4/43.5 No identification 0 (6) Brucella spp (6) No Identification 0 (8) 0 (5) Brucella spp (3) Gordonia rubripertincta/ralstonia 21.4/21.4/21.4 mannitolilytica/ L. seeligeri Candida glabrata
22 (# of isolates Reported Identification IVD Library RUO Library B. neotomae B. ovis O. anthropi B. pinnipedialis B. ceti O. ureolytica No Identification 0 (9) Brucella spp (9) No Identification 0 (9) 0 (3) Brucella spp. 84 (6) No Identification 0 (5) 0 O. anthropi 99.9 (4) (3) 33.3 Ochrobactrum spp (5) No Identification 0 (5) Brucella spp (6) E. asburiae/o. anthropi/e. cloacae/v. 25/25/25/25 parahaemolyticus O. anthropi 99.9 (3) Ochrobactrum spp. 81 (2) No Identification 0 (9) 0 Brucella spp (7) No Identification 0 (3) 0 (4) O. ureolytica 99.9 (5) (3) 33.3 Oligella spp (2) L. seeligeri/o. ureolytica 43.2/56.7
23 Identification Errors Organism Concerns B. anthracis B. cereus B. cereus group Split identifications B. thuringiensis B. anthracis B. cereus B. anthracis Y. pestis Y. pseudotuberculosis Yersinia spp. Split identifications Y. pseudotuberculosis Y. pestis Y. enterocolitica Y. pestis F. philomiragia F. tularensis Organism Concerns B. mallei B. pseudomallei B. pseudomallei B. mallei B. thailandensis B. pseudomallei B. mallei C. botulinum C. sporogenes B. melitensis Brucella spp. B. abortus, suis, canis, ovis, ceti pinnipedialsis B. melitensis Brucella spp.
24 Conclusions Software libraries for BT agents need improvement Addition of BT agents to IVD software Availability of alternative libraries i.e. MicrobeNet Laboratories must be aware of software limitations Partial or incorrect identifications Additional training by manufacturer or BT coordinator MALDI-TOF currently not reliable for ID of BT agents Critical thinking about ID does the result make sense Continue use of rule out and refer protocols
25 Study Participants Michigan Department of Health and Human Services New York Department of Health, Wadsworth Center, Biodefense Laboratory North Carolina State Laboratory of Public Health New York City Department of Health and Mental Hygiene Florida Department of Health, Bureau of Public Health Laboratories Jacksonville Branch Minnesota Department of Health State Hygienic Laboratory at the University of Iowa Texas Department of State Health Services, Laboratory Services Section, Austin Center for Microbial Genetics and Genomics, Northern Arizona University
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