Diagnostic solutions for mycobacterial animal pathogens · 2020-04-03 · 4 Diagnostic For Animal Productivity and Zoonotic Diseases - Offering advanced diagnostic solutions for farm

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November, 7th 2016 - Prague

Diagnostic solutions for mycobacterial animal pathogens

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• 50,000 employees

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We enable our customers to make the world

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We help customers monitor and

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farm animal diseases.

We help maintain herd health,

which may lead to improved

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We help prevent the spread of

zoonotic disease at the farm and

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Healthier

Cleaner

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Passion. Applied.

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Diagnostic For Animal Productivity and Zoonotic Diseases

- Offering advanced

diagnostic solutions for farm

animal designed to meet your

challenges of today and

tomorrow

- Workflow optimization from

sampling to result

- Sample prep solutions

- ELISA, qPCR kits and

machines

- Next-generation sequencing

(NGS) and genotyping by

sequencing (GBS) solutions

for the livestock and animal

health markets.

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Let’s work today on Mycobacteria

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Speakers

Dr Vet Med Jean Louis Moyen

Director

Laboratoire Départemental

d’Analyse et de Recherche,

Coulounieix-Chamiers, France

Dr Björn Schröder

Global Product Manager

Thermo Fisher Scientific,

Schlieren Zurich, Switzerland

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Mycobacterial infections – Impact on animal

health and innovative solutions of their

pathogens

Dr Björn Schröder

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Mycobacterial infections - Content

• Mycobacterial animal pathogens and diagnostic solutions

− Mycobacterium avium subsp. avium,

− Mycobacterium avium subsp. Paratuberculosis

− Mycobacterium tuberculosis

• Applied Biosystems™VetMAX™ M. tuberculosis Complex PCR

kit

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Mycobacterial infections - Overview

• Mycobacterium is a genus of Actinobacteria given its own

family, the Mycobacteriaceae. The genus includes

pathogens known to cause serious diseases in mammals,

including tuberculosis and leprosy.

• Robert Koch identified and described on March 24th 1882 the

first time the Mycobacterium tuberculosis.

• The distinguishing characteristic of all Mycobacterium species is

that the cell wall is thicker than in many other bacteria, being

hydrophoci, waxy, and rich in myclic acids/mycolates.

http://medicineworld.org/images/blogs/11-

2007/mycobacterium-tuberculosis-299290.jpg

Robert Koch

(1843* – 1910†)

http://de.wikipedia.org/w/index.php?title=Datei:Robert_Koch.jpg&filetimestamp=20071015135418

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Mycobacterial infections - Overview

• Mycobacterium avium subsp. Avium

- M. avium avium belongs to the M. avium complex members

which do not necessarily clinical symptoms in pigs but can cause

zoonotic diseases in man

• Mycobacterium avium subsp. Paratuberculosis

- M. paratuberculosis is the cause of Johne’s disease

• Mycobacterium bovis

- M. bovis belongs to the M. tuberculosis complex (MTBC)

members that are causative agents of human and animal

tuberculosis.

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Mycobacterium avium

subsp. avium

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Mycobacterium avium - Infection with zoonotic potential

• Mycobacterium avium (MA) belongs to the M. avium complex (MAC)

• It is an important pathogen in both animals and humans.

• 28 MAC serotypes have been identified of which to M. avium subsp. avium (MAA) and to M. avium subsp. hominissuis (MAH)

• Zoonotic transmissions from pigs to humans due occur, and can induce a broad range of symptoms.

• Especially immunocompromised people are at higher risk.

• An infection with MA in pigs develops mostly subclinically, only seen as granulomatous lesions in the submaxillary and mesenteric lymph nodes.

• The current diagnosis, based on palpation and the incision of lymph nodes, is not specific and not sensitive

Preferred alternative to pathological examination:

Applied Biosystems™ PrioCHECK™ M. avium Antibody

ELISA kit, porcine

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PrioCHECK M. avium Antibody ELISA kit, porcine

• Risk-based surveillance schemes - only a fraction of pigs are tested

• Helps to save time and resources compared to pathological examination

• Allows efficient implementation of appropriate control measures

Convenient

• No false positive results due to lesions caused by Rhodococcus equi or other infective agents •More sensitive than the routine pathological examination

Preferred alternative

• Fast and robust test, follows a short 4 step protocol

• Allows efficient testing of large numbers of samples

• Uses the same sample in other PrioCHECK tests for swine diseases

• Allows efficient addressing of the herd health status

Efficient

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Mycobacterium avium

subsp.

Paratuberculosis

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MAP - Paratuberculosis

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Unique MAP offering

• Antibody ELISA tests*

• Applied Biosystems™ LSIVet™ Ruminant

Paratuberculosis Advanced Serum ELISA kit

• Applied Biosystems™ PARACHEK 2 Kit

• Real-time PCRs*

• Applied Biosystems™ VetMAX™ MAP Real

Time PCR screening Kit

• Applied Biosystems™LSI VetMAX™

M.paratuberculosis advanced kit

• Applied Biosystems™ LSI VetMAX™ Triplex

M. paratuberculosis kit

*For Veterinary Use Only. For In Vitro Use Only. Regulatory requirements vary by

country; products may not be available in your geographic area.

• Sample extraction (including

instruments)

• Applied Biosystems™ MagMAXTM Total

Nucleic Acid Isolation Kit

• Thermo Scientifc™

KingFisher™ instrument

• Real-time PCR Instruments

• e.g:QuantStudio 5 Real-Time PCR System

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MAP - Samples and testing purposes

Faeces

Milk

•qPCR for screening and

confirmation

•ELISA and qPCR

Serum

•ELISA - Screening

Environmental samples

•qPCR

Milk powder, cheese

•qPCR

Organs

•qPCR

Bacterial colonies /

broth culture

•qPCR

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Mycobacterium bovis

and other members of

the Mycobacteria

Tuberculosis Complex

(MTBC)

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Time after infection

CFT CCT SCT

BOVIGAM Serology

PCR Culture

Products – The bTb Disease

The interferon-gamma field trial: background, principles and progress M. Vordermeier, A. Goodchild, R. Clifton-Hadley, R. de la Rua Veterinary Record (2004) 155, 37-38

World Free of Bovine Tuberculosis World Free of Bovine Tuberculosis

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Bovine Tuberculosis Solution

Lelystad Tuberculin

PPD for Skintest

CCT, CFT or SCT

(PPDB or PPDB+PPDA)

Primary Test (PPD tuberculin for Skin test)

To be

slaughtered

+++

Necropsy

(Lymph nodes)

-

Auxiliary Test (IFN-y Release Assay High prevalence)

(Antibody ELISA very low prevalence)

BOVIGAM ELISA +

Stimulant

Confirmatory Test (PCR or Cell culture)

Lelystad Tuberculin PPD

and/or Peptide Cocktails

and/or Pokeweed

To be

slaughtered

+++ - +

Staying

alive

+ -

+

A generalized TB program decision tree – Serial testing

VetMAX M. tuberculosis

Complex PCR

Herd

Closed

Herd

Open

Testing regime

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Introducing our new VetMAX M.

Tuberculosis Complex

PCR kit (MTBC)

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VetMAX M. tuberculosis Complex

A new tuberculosis PCR – Why ? • The Applied Biosystems™ VetMAX M. tuberculosis Complex PCR kit will be

used as a confirmatory test to determine the TB status of a suspicious

animal.

• The VetMAX M. tuberculosis Complex PCR completes the tuberculosis

solution portfolio for Thermo Fisher Scientific.

• The new VetMAX M. tuberculosis Complex PCR is an improved version of

the current TUB2W100 PCR kit.

Duplex, single well instead of a double well for customer convenience

and cost optimization.

Multiple species approach

Increased robustness against inhibitors

Increased sensitivity by having an excellent specificity

Detects all TB strains belonging to the MTBC including M.bovis,

M.tuberculosis, M.microti, M.caprae, M.pinnipedi, M.africanum, M.canetti

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Workflow from sample to result

Three different workflows have been investigated running the LDMETHOD (UG/µL per spiked

matrix) for Applied Biosystems™ LSI MagVet™ Universal Isolation kit 1 UG/ µL and QIAamp®

DNA Mini Kit 1 UG/ µL

Individual sample :

• Lymph nodes

Sample in

validation :

• Other tissues

• Milk

Automated purification system :

Thermo ScientificTM KingFisherTM mL

or Flex Magnetic Particle instruments

Magnetic bead based kit:

MagVet Universal Isolation kit

PCR instruments :

Applied Biosystems™ 7500, 7500 Fast

and QuantStudioTM 5 Real-Time PCR

System

Real-time PCR kit :


• Single real-time PCR

• Duplex: Tb & internal positive control

• Ready-to-use master mix

~3 hrs

Samples Nucleic acid extraction Amplification

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• Kit Format

Duplex kit instead of two wells kit. Single well approach, no separated

IPC

• Robustness

Less sensitive to inhibitors than original kit. Change of MMx; Change

of probe IPC Ct values between 26-34; Rate of re-test <5%.

• Stability

Less sensitive to inhibitors than original kit. Change of MMx; Change

of probe. IPC Ct values between 26-34; Rate of re-test <5%.

• IPC

Duplex kit instead of two wells kit. Change of MMx; Change of probe

IPC Ct values between 26-34; Rate of re-test <5%.

• Other

Fast mode.

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• Efficiency The PCR efficiency is close to 100% (99.14%).

• Repeatability

PCR repeatability shows a coefficient of variation (CV) comprised

between 0.70 % and 1.55 % (3x same technician)

• Intermediate Precision TUB PCR intermediate precision shows a coefficient of variation (CV)

comprised between 0.74 % and 1.52 % (different operators, days and

thermal cyclers)

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• Analytical Sensitivity

The detection limit it is 16 copies of nucleic acid per PCR.

• Analytical Specificity

Analytical Specificity (PCR exclusivity) was evaluated on 22

Mycobacterium strains not belonging to the complex and on 20

pathogens typically found in the same ecological niches, or

phylogenetically close, or because they have the same clinical

symptoms in the target species. Analytical Specificity has been

assessed - based on the tested sample panel – to be 100%

Campylobacter fetus fetus, Campylobacter fetus venerealis, Coxiella burnetii, Mycobacterium avium hominisuis, Mycobacterium avium

subsp avium, Mycobacterium avium subsp paratuberculosis, Mycobacterium bourgelatii, Mycobacterium chelonae subs chelonae,

Mycobacterium flavescens, Mycobacterium fortuitum fortuitum, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium

intermedium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium

non chromogenicum, Mycobacterium paraffinicum, Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium smegmatis,

Mycobacterium terrae, Mycobacterium vaccae, Mycobacterium xenopi, Mycobacterium xenopi, Mycoplasma bovis, Mycoplasma

hyopneumoniae, Mycoplasma hyosynoviae, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteurella multocida toxinogène,

Salmonella enteritidis, Salmonella typhimurium, Streptococcus agalactiae

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Diagnostic Sensitivity and Specificity

Species H2SO4/NaOH PBS1X Total

Bovine 445 148 593

Badger 82 3 85

Wild boar 2 2 4

Total 529 153 682

Global Results

(Both sample preparation)

TUB2W100 (PCR currently

used in France)

POS NEG

MTBC POS 307 6

NEG 8 361

Kappa correlation coefficient 0.96

Sensitivity (Se)=VP/(VP+FN) 97.5% [95.1 – 98.9%]

Specificity (Sp)=VN/(VN+FP) 98.4% [96.5 – 99.4%]

Field validation study & Field trial study

French samples tested : lymph

nodes and surrounding tissues

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World field test plan

France; Jean Louis Moyen Laboratoire

Départemental D`Analyses Agriculture et

Vétérinaire:

Testing on lymph node and

surrounding tissues

UK; APHA, New Haw

Addlestone, Surrey KT15 3NB, UK


surrounding tissues

Uruguay; DILAVE,

Montevideo


surrounding tissues

Philippines;

INFINNOMED

ENTERPRISE's

Quezon City; Manila

Philippines


milk

New Zealand; Farina Khan Laboratory

Manager (TB Diagnostics) Infectious Diseases,

Animal Science, Agriresearch

Testing on lymph node and surrounding

tissues

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Take home message

• VetMAX M. tuberculosis Complex PCR kit is a reliable

diagnostic tool to confirm suspect cases of TB infected

animals of different species - derived from mycobacteria

belonging to the MTB Complex.

• The duplex single well approach is customer convenient in

terms of number of tests per plate and analysis speed.

• VetMAX M. tuberculosis Complex PCR kit is embedded

into three well defined workflows

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Timelines

• VetMAX M. tuberculosis Complex PCR kit will be available

for purchase at 15.12.2016.

• If country specific batch release procedure applies in you

country the PCR will be available at a country specific time

point.

• Kits for validation purposes are available free of charge

with immediate effect.

• Please contact Dr. Björn Schröder for further information.

© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise

specified.

Validation of the MTC PCR kit Jean-Louis Moyen EAVLD congress 7th November 2016

Diagnostic of Mycobacterium

tuberculosis complex—back over 9 years of PCR use

Field evaluation of a new qPCR kit from

Thermo Fisher Scientific

J-l. Moyen, L. Brugère, H. Gares

Laboratoire départemental d’Analyse et de Recherche de la Dordogne


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PCR use for bTB diagnostic: French diagnostic scheme

Mycobacterium tuberculosis complex

First Real Time PCR with IS6110

Extraction

Se/Sp evaluation

M. microti

New master mix

Decontamination process

Field evaluation of a new PCR kit from Thermo Fisher Scientific: Comparison with first generation test (current kit)

Comparison with normal or fast protocol

Effect of bacteriological decontamination on PCR

Evolution of PCR test since 2006: SCHEDULE


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First RT PCR with IS6110

Extraction

Se/Sp evaluation

M. microti

New mix







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bTB diagnosis in France

Network of authorized laboratories for diagnostic tests

Trained and managed by the NRL (Anses Maisons Alfort)

Challenge for the network: a rapid direct diagnosis

PCR is used in parallel with bacteriology in compliance with European regulation

LDAR24: is agreed for these tests (about 10.000 bTB pcr, 10.000

bacteriology and 20.000 γ-IFN /year)

Collaboration with ANSES on bTB diagnosis (PHD students)


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how we perform first line PCR: the Network

= PCR / bact (17)

= NRL

First-line diagnosis (official) by network of authorised Regional labs:

= 2012 = 2010- 2011 = 2006-2009

Breakdowns:

Appropriate premises (L3)

Staff trained by the NRL

Follow NRL’s SOPs

Participate to NRL’s Ring Trials

Be accredited (ISO 17025) *

Submit strains / DNA / samples to NRL

(identification/genotyping/expertise) AFNOR NF U 47-104 (bact) & NF U 47-600 (PCR)

*


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NRL molecular diagnostic methods allow: • confirming PCR positive as MTBC (99%)

• confirming PCR first-line positive samples as belonging to a concrete

MTBC entity (95%)

• find the spoligopatterns

This molecular diagnostic scheme provides rapid direct reliable results

Field confidence Gain in the new diagnostic scheme

since 2011, quicker management of infection and avoid further transmission provides very quick info for ameliorating investigation of breakdown origin up to 10% outbreak confirmation by molecular results alone Confirmation of presence of other tuberculous non notifiable agents that confound diagnosis

Confirmation and molecular typing by NRL


Current Techniques of diagnosis

Animal

sampling

Bacterial isolation (Gold

standard): 3 months of culture for

diagnosis

Real-time PCR: 2 days Histology: 8 days

http://www.google.fr/imgres?imgurl=http://idata.over-blog.com/1/98/20/51//blaireau.jpg&imgrefurl=http://spendeursauvage.over-blog.com/article-30851077-6.html&usg=__S7RWs96B2HmVatPvs11l5ILK2Cc=&h=741&w=600&sz=59&hl=fr&start=15&um=1&itbs=1&tbnid=DYChtywk1bNUoM:&tbnh=141&tbnw=114&prev=/images?q=tuberculose+bovine&um=1&hl=fr&sa=N&tbs=isch:1

http://www.google.fr/imgres?imgurl=http://www.bienpublic.com/fr/images/get.aspx?iMedia=17285902&imgrefurl=http://www.bienpublic.com/fr/accueil/article/3012457/La-tuberculose-bovine-inquiete-les-chasseurs.html&usg=__Mvl1m45BmMRveYNdXVlaXRVgvRg=&h=373&w=485&sz=39&hl=fr&start=18&um=1&itbs=1&tbnid=Xnt6QTVjdQ5lVM:&tbnh=99&tbnw=129&prev=/images?q=tuberculose+bovine&um=1&hl=fr&sa=N&tbs=isch:1

http://fr.wikipedia.org/wiki/Fichier:TB_Culture.jpg


~ 6 months

8 days

8-10 days

3 months 1 month

CCID HISTOLOGY CULTURE IDENTIFICATION

& TYPING

Loss of qualification

6-8 weeks

SID

Diagnostic

cull

PCR IFN-g

(PPD+EC)

Diagnostic scheme

2/3 days 2/3 days 1 month


39




Extraction

Se/Sp evaluation

M. microti

New mix







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REMINDER

EAVLD 2010: presentation of the results with LSI 2 wells* kit

Target: IS6110

Inclusion list: all members of the MTBC (M. bovis, M. tuberculosis,

M. caprae, M. microti, M. africanum, M. Canetii, M. pinipedii)

Extraction spin columns or magnetic beads (for hightroughput process)

A better sensitivity than official culture (Se: 95% of the detected samples instead of 90%)

*LSI VetMAX Mycobacterium tuberculosis real time PCR kit complex

(TUB2W100)


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Progress from 2010

• About 70.000 PCR tests realized since 6 years on bovine and wildlife lymph nodes and tissues

• NRL has developed optimized molecular tools for identification, confirmation, typing and epidemiology

• A larger study (2008/2012) to assess Se and Sp • Sensitivity of the PCR 87.7% [82.5 - 92.3%] versus bacteriology 78.1% [72.9 - 82.8%]


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Parameter Mean 95% credible interval

Seculture 78.1 [72.9 – 82.8]

SePCR 87.7 [82.5 – 92.3]

Sehistology 93.6 [89.9 – 96.9]

Spculture 99.1 [97.1 – 100]

SpPCR 97.0 [94.3 – 99.0]

Sphistology 83.3 [78.7 – 87.6]

2008-2012

How well does our PCR method work?

Latent classe analysis


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Extraction

Se/Sp evaluation

M. microti

New mix






• Detected as member of MTBC group

• Non notifiable agent

• Zoonotic (immunocompromised people)

• Wild life /domestic animals

• Better detection by PCR


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0

2

4

6

8

10

12

No

mb

re d

e ca

s

Faune sauvage Animaux domestiques

Other applications PCR+ve samples:

widespread distribution of M. microti

• Michelet et al JCM, 2015 • Michelet et al Vet Rec, 2015 • Michelet et al EID, 2016


45




Extraction

Se/Sp evaluation

M. microti

New master mix







46

Progress since 2010

New master mix (in 2 wells current version):

A new master mix was introduced in mid 2014 by Thermo Fisher Scientific to reduce inhibition.

Today inhibition are unusual (less than 2% analysis with more than 2 CT later)

1/10 Dilution of the extracts is rare. Allows more concentrate extracts (the use of a small volume of PBS

is possible)

Evaluation and comparison of sample preparation with PBS or H2SO4/NaOH Sample preparation (dissection, crushing…) is a critical point H2SO4/NaOH was used in the french standard for bacteriology

(NF U 47-104)

Crushed material was used for culture and PCR (reproducibility) PBS allows more concentrate samples and gives a better Se and

higher DNA concentration for typing All decontamination process have the same effect


48




Extraction

Se/Sp evaluation

M. microti

New mix







49

Field evaluation of the new PCR kit

MTBC PCR Single well internal positive control (IPC) + target

Material Bovine & badgers (+ a few wild boars): - Infected lymph nodes (confirmation by

spolygotyping) - Non infected lymph nodes ( already tested with the

kit TUB2W100) - We have extract (DNA) and crushed material

Method

- Crushed material was thawned and extracted again - Extraction with KF 96 and magnetic beads - Extracts were amplified with the three methods:

Current kit (TUB2W100) New kit (MTBC), New kit with fast method (MTBC fast)

- Amplified with the thermocycler, Applied Biosystems™ 7500 and 7500 fast Real-Time PCR System


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First trial - Experimental kit

78 extracts from infected animals:

• 3 Wild Boars

• 19 Badgers

• 56 Cattle

• 60 extracts from PBS and H2SO4/NaOH crushed material

• 18 extracts from H2SO4/NaOH only

214 crushed material from:

• Badgers: 12 (1 infected)

• Bovine: 202 (10 infected)

• All but 9 were tested after H2SO4/NaOH


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First trial - experimental kit

Extracts (magnetic beads): • Target

- No difference between two methods - High difference between crushing methods (H2SO4/ NaOH versus PBS)

• IPC (internal positive control): - Later Ct with new kit and both methods => better inhibition detection - Same IPC Ct values reproducibility (about 3%)

Crushed material (H2SO4/ NaOH) : 11 infected and 203 non infected • Lower Ct with PBS • Later IPC Ct with new methods

H2SO4/NaOH PBS

target IPC target IPC

TUB2W100 31.3 26.6 26.7 26.7

MTBC 31.0 29.0 26.3 28.7

MTBC fast 31.3 28.8 26.8 29.3


52

Second Trial - Commercial kit

166 animal tested :

95 positive (34 badgers & 61 cattle)

71 negative

77 positive were tested with H2SO4/NaOH and PBS preparation

All samples were tested with the three amplification methods


53

Commercial kit results

Negative samples:

• Same results for all methods: no false positive

Positive samples:

• comparison between the 77 samples with all tests

• Very good correlation between results

• More false negative results with H2SO4/NaOH

H2SO4/NaOH PBS

target IPC target IPC

LSI 2w 32.5 26.4 28.6 26.4

MTBC 31.6 27.8 27.8 27.7

MTBC fast 32.0 27.9 28.2 28.4


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bTB target Ct distribution

No difference between different amplification protocols and kits Large difference between different preparation methods


55

IPC Ct distribution

With the new kit (MTBC): • Same value for both protocols • Higher Ct for MTBC and MTBC fast


56

Summary

Datas from all analysis with PBS protocol for both trials (experimental batch and commercial) :

Sensitivity: (88 infected lymph nodes) *

87/88 positive (86/88 with fast method, not significant)

*in diagnosis animals were tested with at least 3 PCR ( 3 lymph nodes pools/animal)

Specificity: 0 false positive / 274 lymph nodes tested


57

Conclusions

For bTB diagnosis with PCR: PBS preparation should be prefered

→ but decontamination is necessary for bacteriology wich is mandatory in EU regulation

New mix (TUB2W100 – 2014) allows a sharp decrease of inhibitions and a

better sensitivity

Field evaluation of the new kit (2016 MTBC and MTBC fast) No difference of sensitivity between three protocols (TUB2W100/

MTBC/MTBC fast) Rare inhibitions (master mix) Higher detection of inhibition thanks to higher IPC Ct values with new

kit (MTBC or MTBC fast) Easy to use kit :

→ Single well → Ready to use reagents → Same kit for fast protocol


Thank you for your attention

Acknowledgement:

ANSES Maisons Alfort (bTB NRL):

Laura Boschiroli, Sylvie Henault

Technicians of LDAR24

Thermo fisher team

http://www.afsset.fr/

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For Veterinary Use Only. For In Vitro Use Only. Regulatory requirements vary by country; products may not be available in your geographic area. © 2016 Thermo Fisher

Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. QiaAMP is a trademark of Qiagen

Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and

reported results. Parties presenting images, text and material represent they have the rights to do so.

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Diagnostic solutions for mycobacterial animal pathogens · 2020-04-03 · 4 Diagnostic For Animal Productivity and Zoonotic Diseases - Offering advanced diagnostic solutions for farm