Public Health Ontario National Conference on Laboratory ......Microbiology Lab Today? •Bruker MALDI Biotyper –Microflex LT, FlexControl software –Bruker microorganism database

11th National Conference on Laboratory Aspects of Tuberculosis, April 23rd 2019

Frances Jamieson, MD FRCPC

Medical Lead, TB and Mycobacteriology Laboratory, Public Health Ontario

PublicHealthOntario.ca

Declaration of Conflict of Interest

• I have no conflicts to declare


Outline and objectives

1. Overview of TB and Mycobacteriology laboratory, Public Health Ontario and mycobacteria epidemiology in Ontario

2. General review of MALDI-ToF technology and application in PHO laboratories

3. Evaluation: MALDI-ToF extraction method development, and MALDI-ToFvalidation

4. Evolution: MALDI-ToF implementation and experience, interesting observations and lessons learned


TB and Mycobacteriology Labs in Ontario

Timmins

Ottawa

TORONTO

London

Public Health Ontario Lab Network

PHL-Toronto and 3 Regional PHLs

Ottawa Hospital

Dynacare (community lab)


TB @PHOL: By the Numbers…

• Largest TB and Mycobacteriology laboratory in North America (by volume) - avg. 65,000 specimens processed annually (56,000 in Toronto)

• Provide >95% of diagnostic testing and 100% of reference testing for Ontario

• Over 200 AFB smears are read daily (one primary reader and one secondary) – Toronto lab

• Over 200 culture positive specimens and referred cultures identified weekly – Toronto lab

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Nu

mb

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Year

Total positive

NTM

TB

Proportion of MtbC and NTM Isolated-PHL Toronto


14.7%

63.0%

5.4%

10.7%

5.7%

0.5%

% Mycobacteria spp. 2017 (n=11,919)

Mtb

MAC (M. avium and M.intracellulare)**

M. xenopi

Common Rapid growers(non-pigmented)*

Other NTM

M. kansasii

*M. fortuitum, M. chelonae, M. abscessus group, M. peregrinum, M. mucogenicum, M. senegalense

**M. avium 90.7% and M. intracellulare 8.5% of MAC isolates

8

Incubate

up to 7

wks

Smear Culture

TB DST;

report in

8-10 days

New smear

positive, smear

negative on

request

Daily

TB/MAC NAAT

Smear read and

reported in 24

hours TB or NTM ID:

MPT64Ag lat flow (TB)

MALDI/line-probe or

sequencing and

report ID

Laboratory Testing Algorithm - PHOL

MALDI-ToF: a type of Mass Spectrometry

• Matrix Assisted

• Laser Desorption/ Ionizing

• Time of Flight

• Mass Spectrometry

Your sample is co-crystallized with “matrix” keeps proteins intact, absorbs light

A pulse of laser light forces molecules into gas phase (desorption) and ionizes them

Ions “fly“ down a high-vacuum flight tube then strike the detector and the ToF is measured

Figure courtesy J. Kus, PHO

What is available for the Clinical Microbiology Lab Today?

• Bruker MALDI Biotyper– Microflex LT, FlexControl software– Bruker microorganism database– Class I Medical Device – Health

Canada; database RUO– CE Mark

• bioMeriéux VITEK MS– Shimadzu hardware with

microbial database acquired from AnagnosTec

– RUO database –SARAMIS– IVD database – MS-ID - Class I

Medical Device – Health Canada– CE Mark

Figure courtesy J. Kus, PHO


Implementation of MALDI-ToF at PHOL

11

• First MALDI instrument - 2013:• Anaerobes – June 2013

• Phased in implementation of Gram-positive and Gram-negative organisms (e.g. enterococci, staphylococci, HACEK) from 2013 - 2016

• Enterobacteriaceae – May 2017

• Second MALDI instrument - 2016:• Purchased as back-up for first MALDI instrument and for additional

capacity

• Mycobacteria spp. - March 2017 (M. chimaera module Dec 2017)

• Yeast – 2016

• Nocardia/aerobic actinomycetes – 2017

• Additional organisms currently under evaluation (e.g. N. gonorrhoeae, moulds)


Mycobacteriology MALDI Evaluation:Extraction Validation

12

• To determine optimal method for isolate identification in isolates from liquid media (MGIT 960)

• Method must provide an acceptable score > 1.8 in > 70% of isolates tested

• Evaluated 5 extraction methods: Bruker, Ottawa Hospital (courtesy of Dr. M. Desjardins), 3 modifications of Brukermethod

• Each method assessed using Type strains: 2 M. avium, 3 M. fortuitum, and 1 M. gordonae

• Final method (best scores obtained in all 6 isolates):• Modification of Bruker method utilizing an extra wash step and bead

beating


MALDI-ToF for Mycobacteria spp Identification Evaluation:Summary of Protocol

13

• Test a representative sample size for each species based on the most common species encountered (min 5 – 10 common species; 1 – 3 rare, and non-mycobacteria)

• Isolates selected from fresh cultures, subcultures from frozen archived isolates (identified by Hain LPA; HPLC; 16S, hsp65, ITS sequencing), and Mycobacterium spp Type strains

• “Mixed cultures (2 organisms)” were run to assess MALDI performance

• Reproducibility testing within runs, between runs, day-to-day, and between users

• Sequencing was reference “gold standard” for discordant results arbitration

• Agreement to species > 90% (reportable scores > 1.79)

• Evaluation run from October 2016 – January 2017

• Post-implementation performance tracked over 6 months

14

Organism 2014 2015 Organism 2014 2015

M. abscessus 296 341 M. longobardum 0 2

M. agri 0 0 M. mageritense 8 3

M. arupense 1 5 M. malmoense 2 1

M. asiaticum 0 1 M. marinum 10 10

M. avium 4256 5900 M. mucogenicum 103 152

M. bohemicum 2 1 M. nebraskense 0 1

M. bovis 0 1 M. neoaurum 7 7

M. bovis BCG 17 8 M. nonchromogenicum 0 0

M. branderi 0 1 M. novocastrense 0 1

M. celatum 1 1 M. obuense 1 5

M. chelonae 106 91 M. palustre 0 0

M. cosmeticum 2 0 M. paraterrae 0 1

M. elephantis 0 3 M. peregrinum 22 26

M. flavescens 0 1 M. phlei 2 0

M. fortuitum 257 336 M. scrofulaceum 13 5

M. gilvum 3 0 M. senegalense 21 23

M. goodii 4 1 M. shimoidei 13 4

M. gordonae 747 838 M. simiae 20 14

M. heckeshornense 1 0 M. smegmatis 2 0

M. interjectum 3 5 M. szulgai 2 10

M. intermedium 0 0 M. triplex 0 1

M. intracellulare 525 552 M. triviale 1 0

M. iranicum 1 1 M. vaccae 1 0

M. kansasii 95 70 M. vulneris 0 0

M. kyorinense 0 0 M. wolinskyi 1 1

M. kubicae 0 2 M. xenopi 842 793

M. kumamotonense 8 11 M. tuberculosis 1556 1491

M. lentiflavum 77 140 TOTAL 9029 10861


Type strain (ATCC, CIP)

n = 40

Line probe assay (Hain)

Solid media n = 193

n = 318

Sequencing (16S, hsp65 )

n = 71

HPLC

n = 14

n = 649

Type strain (ATCC, CIP)

n = 27

Line probe assay (Hain)

Liquid media n = 222

n = 331

Sequencing (16S, hsp65 )

n = 24

HPLC

n = 58

Isolates selected for MALDI validation

Number of clinical isolates by identification method

and Type strains

LJ

MGIT


Precision Verification:

16

Mixed Cultures Evaluation:• 31 cultures with 2 organisms tested

• 5/31 (16.3%) had reportable scores and accurately identified both organism species

• 24/31 (51.6%) only 1 of the organisms present was accurately identified with a reportable score

DetailsNumber of isolates

includedResults

Reproducibility10 previously tested isolates were repeated,

different days10 100% concordance

Within Run10 previously tested isolates were repeated on

the same run10 100% concordance

Between Run10 previously tested isolates were repeated on 2

different runs10 100% concordance

Between Users10 previously tested isolates were repeated on 2

different runs, different users10 100% concordance


Accuracy Verification:

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Performance(Bruker MBT Compass

V4.1)

Total testedN=649

%Agreement

SolidN=318

%Agreement

LiquidN=331

% Agreement

Correct identification to species -all

(score > 1.79)580/645 90.2 289/315 91.7 291/330 88.2

Correct species identification

580/582 99.7 288/289 99.7 290/291 99.7

Score < 1.79 63 25 38

Incorrect species identification (score > 1.79)

2 1 1

Species not in library 2 1 1

Excluded (discrepancies not resolved)

4 3 1


SpeciesMALDI (>1.79)

Validation%

MALDI (>1.79) Post -implementation

%

M. abscessus 37/40 92.5 194/212 91.5

M. avium 108/118 91.5 2090/2645 79.0

M. chelonae 16/20 80.0 44/57 77.2

M. fortuitum 30/31 96.8 157/172 91.3

M. gordonae 30/34 88.2 277/353 78.5

M. chimaera-intracellulare 67/71 94.4 * *

M. kansasii 14/14 100 26/30 86.7

M. lentiflavum 18/19 94.7 44/53 83.0

M. marinum 10/12 83.3 6/6 100.0

M. mucogenicum-phocaicum group 19/21 90.5 57/61 93.4

M. peregrinum 3/4 75.0 3/4 75.0

M. senegalense 13/13 100 11/11 100.0

M. simiae 8/8 100 8/9 88.9

M. xenopi 28/33 84.8 108/161 67.1

M. tuberculosis complex19/21 90.5 13/16 81.3


M. chimaera-intracellulare Subtyping Module* Validation

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Performance(*Bruker MBT Compass V5.0)

Total testedN=29

%Agreement

Correct identification to species/group -all (score > 1.79)

27/29 93.1

Correct species identification 26/26 100

Score < 1.79 0

Incorrect species identification (score > 1.79)

0

Failed to distinguish within M. chimaera-M. intracelluare group

4 (2 M. chimaera and 2 M. intracellulare)

• 29 isolates tested: 12 M. chimaera and 12 M. intracellulare (confirmed by sequencing)3 M. chimaera/intracellulare group (ID by MALDI) –no sequence consensus ID2 M. marseillense (confirmed by sequencing)

• Implemented December, 2017

Centrifuge @ 15000 rpm for 10 min

then re-suspend in 300 µl H2O

Laboratory Workflow for MALDI-TOF MS ID of Mycobacteria spp. from Culture (MGIT or LJ)

MGIT –

vacuum

growth in 1.2

ml

LJ – suspend ≥ 1

large colony in

300 µl H2O

Centrifuge @ 15000

rpm for 10 min, then

remove all liquid

Re-suspend in 500 µl H2O

then vortex* @ 3000rpm

for 1 min

Ensure no

clumps

remain!

Vortex* @

3000 rpm

for 1 min

Heat

inactivation 20

min @ 95 C

Add 900 µl EtOH then

vortex* @3000rpm

for 1 min

Centrifuge @

15000 rpm for

3 min

Decant

supernatant

Centrifuge @

15000 rpm

for 3 min

Remove residual

liquid with micro-

pipette

CL3

CL2

Place open tubes in pass-through

(will dry during this time)

Add 0.1 mm beads

= pellet size

Add 40 µl ACN and

40 µl formic acid

Load tubes in chilled

BeadRuptor adaptor

Bead beat at 3.1 m/sec

for 120 seconds

Centrifuge @ 14000

rpm for 5 min

Pipette 1 µl in

duplicate to target

Add matrix

with Galaxy

Run target on

MALDI instrument!

*Disruptor Genie


MGIT or

LJ positive

AFB on

smear?Contaminated / repeat smear

Patient history and

smear consistent

with referable

organism?

Mixed? (Smear or

previous mixed culture)

Refer to previous

culture ID

Cording

present?

MPT-64

Positive?

Report

MALDI-

TOF

Good ID?

HAIN CM/AS Suspect

Mixed?

SequencingGood ID?

Report

Yes

Yes

Yes

Yes

Report

No

Yes

No No

Yes

Yes

Yes

No*

No

Yes

Positive Culture Identification Algorithm

Assess status of

other media

No

No

No

No

*do not sequence if suspect mixed

or no “genus” band observed


MALDI-ToF: Cost Analysis: March 1st 2018-Feb 28th 2019

22

• Materials and labour included (MALDI, Hain GenoType Mycobacterium CM/AS, 16S sequencing)

• MALDI – 1 FTE, Hain CM/AS – 2 FTE

• 6501 isolates from culture

• 5516 identified by MALDI alone (84.8%)

• MALDI - $12.27 CAD/test

• Hain CM/AS - $48.12 CAD/test

• 16S sequencing - $125.00 CAD/test

• Savings differential: $35.85/test (MALDI over Hain)

• 1 FTE savings absorbed into other components of the ID algorithm, and number of isolates requiring sequencing decreased by 85%


Observations, Lessons Learned and Pearls:“Mycobacterium malmoense” ID from LJ

23


Observations, Lessons Learned and Pearls:M. avium and intermediate score (1.6 – 1.79)

24


Observations, Lessons Learned and Pearls:MALDI “Mixed” Messages

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• MALDI isn’t optimal for identifying “mixed” cultures, but can be useful if careful attention is paid to the “top ten” list:

• A top score (green) isn’t necessarily the correct ID - when the rest of the list is reviewed, a closely related 2nd species may appear several times below (e.g. M. fortuitum and M. senegalense) with the second isolate confirmed as the ID on repeat or by other ID method

• 6 or more appearances in the top 10 list (not good scores) of an organism indicates it is in the culture, and will often be identified with a good score on repeat


PHOL TB and Mycobacteriology Laboratory 2019

Thank-you!!

Public Health Ontario National Conference on Laboratory ......Microbiology Lab Today? •Bruker MALDI Biotyper –Microflex LT, FlexControl software –Bruker microorganism database

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