11/22/2018
1
Novel molecular diagnostics in AMR
Aart van Amerongen1, Rik Slendebroek1, Kees Veldman2, Dik Mevius2,3 and Jan Wichers1
Emerging technologies in AMR14th of November 2018, Bilthoven
1:Wageningen Food & Biobased Research2:Wageningen BioVeterinary Research
3:Faculty of Veterinary Medicine, Utrecht University
Content
Introduction BioSensing & Diagnostics Detection of antibiotic resistance genes Multiplex PCR and lateral flow assay Initial results of test development A new rapid phenotypic method (Dr. Jeroen Veen, HAN)
Wageningen Campus Wageningen Food & Biobased Research
11/22/2018
2
BioSensing & Diagnostics (BSD)
Over 30 years of experience in the development of rapid and sensitive methods, especially:● Nanoparticles-based; (Nucleic Acid) Lateral flow
and microarray assays / microarray-ELISAs
Technology-driven; broad experience in detection targets:● Proteins, microbial cells, chemical components,
carbohydrates, ......● Nucleic acids: specific DNA/RNA amplicons
BioSensing & Diagnostics (BSD)
Multi-analyte assay formats:● Lateral flow devices with several lines
● Lateral flow / Flow through devices with a microarray of spots
● Microarrays in wells of ELISA plates
● New platforms:● Guided pathway flow in nitrocellulose
and between two glass/plastic layers
11/22/2018
3
Carbon nanoparticles in rapid diagnostics
Aero-allergen:● Mouse Urinary Antigen (MUA)
Airborne (filters) and settled dust (wipe) samples were collected from various places in laboratory animal facilities
Blank correctedLOD: 31 pg/mL
Measurement of Occupational Allergen ExposureEU-MOCALEX; EU-QLRT-2001-00432Coordinator: Gert Doekes, IRAS, University of Utrecht
Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens. Marjo Koets, Anne Renström, Eva Zahradnik, Jelena Bogdanovic, Inge M. Wouters and Aart van Amerongen. J. Environ. Monit., 2011, 13, 3475-3480
Carbon nanoparticles in rapid diagnostics
Dose-response curve for IL-6 antigen - prototype line-test in buffer, incl. standard deviation bars
Funded by the Bill & Melinda Gates Foundation
LoD (blank+3*SD): 2.2 pg/mL
Specific lines were scanned (flatbed) and the total sum of the pixel grey values was plotted against the IL-6 concentration
11/22/2018
4
Carbapenemases PCR-NALFIA and NALMIA
Detection of genes coding for carbapenemases that are involved in antibiotic resistance
Antibiotic resistance genes
Two main classes of resistance against antibiotics● ESBLs (Extended-Spectrum Beta-Lactamases);
degradation of β-lactam antibiotics● Target genes: CTX-M (5 sub-groups), SHV (6
different SNPs), TEM (5 different SNPs), CMY-2● Carbapenemases; hydrolysis of cefalosporins,
penicillins, monobactams and carbapenems● Target genes: NDM-1, OXA-48, KPC, VIM
11/22/2018
5
Detection of resistant microorganisms
Human clinical practice:● Conventional phenotypic culturing● Genotypic methods
● qPCR, Next Generation Sequencing, microarrays
● Mass spectrometry (MALDI TOF MS) Veterinary practice:
● Preferably point-of-care / on-site● Methods mentioned above are too expensive, too
time-consuming and/or too facility-demanding● Timely diagnosis would enable veterinarians to start
a dedicated antibiotic treatment on the same day
Detection of resistant microorganisms
Solution: development of a fast and specific procedure by combining multiplex PCR and Nucleic Acid Lateral Flow (Microarray) ImmunoAssay (NALFIA / NALMIA) First targets: detection of bacterial strains that have a
carbapenemase encoding gene● Additional target: MCR-1; degradation of polymyxins
and colistin Final goals:
● Panel of genes encoding carbapenemases and MCR-1● Panels of (sub-groups of) genes encoding ESBLs● Contamination-free device; automated transfer of
PCR material to detection assay; on-site application● Sample in - Result out
11/22/2018
6
Nucleic acid amplification
DNA to protein and DNA amplification
Multiplex PCR
Tagged primers: forward with (antibody-)specific tag 1reverse with biotin (tag 2)
Multiplex PCR procedure (5 primer sets)● Run time optimized to 30 minutes
11/22/2018
7
Multiplex PCR
Mix of tagged primers:
Target F/R Sequence Label (5’)Amplicon
size (bp)
NDM F ATT AGC CGC TGC ATT GAT FAM
R CAT GTC GAG ATA GGA AGT G Biotin
OXA‐48 F CGA CCC ACC AGC CAA TCT TA DNP
R GTT ACA CGT ATC GGA GCG CA Biotin
KPC F CTC GCT GTG CTT GTC ATC CT DIG
R GGC ACG GCA AAT GAC TAT GC Biotin
VIM F TTT GGT CGC ATA TCG CAA CG Alexa‐Fluor‐488
R CCA TTC AGC CAG ATC GGC AT Biotin
MCR‐1 F CGG TCA GTC CGT TTG TTC Texas Red
R CTT GGT CGG TCT GTA GGG Biotin
154
131
102
500
309
Detection principle
Double-tagged amplicons are formed that can be sandwiched between neutravidin on carbon nanoparticles and tag 1 specific antibody on nitrocellulose
+
11/22/2018
8
DNA template material
Bacterial strains:● NDM-1: E.coli ATCC BAA-2469● OXA-48: K. pneumoniae NCTC 13442● KPC: K. pneumoniae ATCC BAA-1705● VIM-1: K. pneumoniae NTCT 13440● MCR-1: CVI nr.101.62 (no reference strain)
Multiplex PCR with 5 sets of primers (4 carbapenemasesand the MCR-1 encoding genes)● Optimized to 30 minutes
Recent results
Optimized 5-plex PCR:
1 2 3 4 5 6 7 8Lane 1: Primer mix with NDM template: 154 bp productLane 2: Primer mix with OXA template: 131 bp productLane 3: Primer mix with KPC template: 102 bp productLane 4: Marker TrackitTM 50 bpLane 5: Primer mix with VIM template: 500 bp productLane 6: Primer mix with MCR-1 template: 309 bp productLane 7: Negative control; primer mix, no template DNALane 8: Marker TrackitTM 50 bp
11/22/2018
9
Recent results
PCR solution first tested with a lateral flow line assay; Nucleic Acid Lateral Flow ImmunoAssay (NALFIA):
Template materialThe positive control (+ ctrl) is a sample
from a multiplex PCR targetted at genes
encoding E.colivirulence factors
Validation study on OXA and MCR-1 strains
Limited validation performed:● 24 E.coli strains negative for OXA/MCR-1● 48 E.coli strains MCR-1 positive● 18 Shewanella strains OXA-48 positive● 6 Shewanella strains# OXA-48 negative
Samples were spiked with KPC hydrolysate as a positive PCR control
#: Shewanella xiamenensis, marine species found in fish farms
11/22/2018
10
Validation study
Result limited validation:● KPC (control) 100% (n=96)● Negatives 100% (n=24+6)● MCR-1 100% (n=48)● OXA 94% (n=18; 17 positive)
OXA: Shewanella xiamenensis strain 25B contained an OXA-181 gene instead of OXA-48
● The OXA-48 primers did not recognize the OXA-181 gene
Lateral flow Microarray ImmunoAssay (LMIA)
Microarray of 5 x 5 spots Printed antibodies: 10 to 40 nanoliter
● Can be much lower upon analysing results by a dedicated (colorimetric / fluorescent / chemiluminescent) reader
● < 1 nanoliter possible Performance similar to line lateral flow
immunoassay Instead of 1 µL antibody as in line lateral flow
assays (€ 0.05):● 40 nL to < 1 nL per antibody● 25 to 1000 times cheaper
11/22/2018
11
Nucleic Acid LMIA
Example: Verotoxigenic Escherichia coli (VTEC / STEC) Microarray layout:
Verotoxigenic E.coli virulence factors:_| hui: α-Cy5
Multiplex | ehxA: α-DNPPCR | eae: α-DIG
| vt2: α-FITC|_ vt1: α-TxRcontrol: IgG-biotin
flow direction
nitrocellulose membrane
absorption pad
NDM OXA KPC VIM MCR-1 neg.CtrlTemplate material
Carbapenemases/MCR-1 specific NALMIA
Initital carbapenemases / MCR-1 specific Nucleic Acid Lateral flow Microarray ImmunoAssay (NALMIA) Layout:
NALMIA results:
MCR‐1
VIM
KPC
OXA‐48
NDM
Pos.Ctlr
11/22/2018
12
Reading spots in lateral flow assays
Real-time video reader for lateral flow microarray immunoassays
Reading spots by real-time video reader
Initially (April 2015): Box with the digital video camera above + LED illumination in a ring format
Then: 3D printed device
Recent lab-version: Small wireless device
Collaboration with HAN University of Applied Sciences: Jeroen Veen (Digital Signal Processing, Sustainable Energy, Healthcare Technology), Hugo Arends (Embedded Vision Design)
11/22/2018
13
Reading spots by real-time video reader
Collaboration with HAN University of Applied Sciences: Jeroen Veen (Digital Signal Processing, Sustainable Energy, Healthcare Technology), Hugo Arends (Embedded Vision Design)
3D printed reader device:● Final goal: Quantification of biomarkers
Detection of a blood biomarker (home test) Dilution range of
antibodies Accelerated view
of a 5 minutes run
Inert red colour is added during printing for quality purposes
Spot colour is based on specific interaction with carbon
nanoparticles-antibody conjugates
Result:
Development of spots can be followed real-time
Reading spots by real-time video reader
Some software possibilities:
User interface
Spots are recognised
Multi-spot intensity increase
Slopes of the curves correlate with
concentrations of analytes
Collaboration with HAN University of Applied Sciences: Jeroen Veen (Digital Signal Processing, Sustainable Energy, Healthcare Technology), Hugo Arends (Embedded Vision Design)
11/22/2018
14
Reading spots by real-time video reader
Wireless video reader is available● Scienion, November 2018
Real-time video reader for
lateral flow microarray
immunoassays
Dimensions: 10 x 8 x 8 cm
Smartphone app that controls the video reader
and returns and/or transfers the results
Prototype readerMEDICA, Düsseldorf, Germany,
13-16 November 2017
Rapid phenotypic antibiotic susceptibility test
Development at HAN University of Applied SciencesDr. Jeroen Veen
11/22/2018
15
Rapid phenotypic AST
Micro-culture incubation
Automated cell-counting
Real-time image processing
Low-cost focal adjustment
Reference set‐up for automated incubation, microscopy
and real‐time image processing.
Micro‐chamber (20 µL)
with grid pattern.
Automatically annotated image showing
grid and microbe detection.
Cell chains are detected and
separated in the image.
Dr. Jeroen Veen
Technology verification
Viability tested 1-2 hours after incubation start(depending on the organism)
Proof of principle shown
Open-source system under development
Reference system: 5 to 10,000 euro
Prognosis for present system: some hundreds of euros
Autocount results for L. lactis grown in M17 medium at 37°C, w/ and w/o ampicillin, inlay shows p‐value.
Results for VRE with MIC concentrations of vancomycin and linezolid.
Dr. Jeroen Veen
11/22/2018
16
Final remarks
A rapid molecular procedure to test for genes coding for carbapemases and MCR-1 is available Procedure qualifies for extension to panels of genes
encoding other antibiotic resistance proteins Prototype of a multiplex-PCR - NALMIA cartridge is ready
● New project "On-site Nucleic Acid Testing" will start per 1-1-2019
● Subsidy from the Dutch Topsector Agri&Food● Initial subject: meat adulteration tested worldwide
Acknowledgements
• Cindy Dierikx (RIVM)• Hans Dijk, Ben Nitsche,
David Lukow, Holger Eickhoff (Scienion)
• Jeroen Veen, Hugo Arends (HAN University of Applied Sciences)