Novel molecular diagnostics in AMR · Guided pathway flow in nitrocellulose and between two glass/plastic layers. 11/22/2018 3 Carbon nanoparticles in rapid diagnostics

11/22/2018

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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

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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

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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

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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

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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

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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

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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

+

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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

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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

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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

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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

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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)

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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


Some software possibilities:

User interface

Spots are recognised

Multi-spot intensity increase

Slopes of the curves correlate with

concentrations of analytes


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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

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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

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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

[email protected]

• Cindy Dierikx (RIVM)• Hans Dijk, Ben Nitsche,

David Lukow, Holger Eickhoff (Scienion)

• Jeroen Veen, Hugo Arends (HAN University of Applied Sciences)

Documents

Novel molecular diagnostics in AMR · Guided pathway flow in nitrocellulose and between two glass/plastic layers. 11/22/2018 3 Carbon nanoparticles in rapid diagnostics