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Eyes for the invisible: microfluidic paper-based analytics (µPads) and

3rd - generation sequencing in clinical microbiology

John WA Rossen, PhD, MMM

Principle Investigator - Genomics for Infection Prevention

Head Molecular Unit

Department of Medical Microbiology, University of Groningen, University Medical

Center Groningen, Groningen, The Netherlands

Disclosure of speaker’s interests

(Potential) conflict of interest None

Potentially relevant company relationships in

connection with eventNone

Sponsorship or research funding

Fee or other (financial) payment

Shareholder

Other relationship

· Interreg IVa-funded projects EurSafety

Heath-net (III-1-02=73) and SafeGuard

(III-2-03=025)

· None

· None

· None

Disclosure slide for speaker at further training events

Eyes for the invisible

A Dutch tradition?

Antoni van Leeuwenhoek (1632-1723)

Martinus Beijerinck (1851 - 1931)

Filtration experiments Virus

Questions the patients have regarding infections/infectious diseases

1. Did you prevent colonization and infection today?

2. Do I have an infection/ID and which one?

3. What is the optimal therapy?

Molecular Diagnostics – a powerful tool

• Detection (Real-time PCR/NASBA/LAMP)

– Unculturable micro-organisms

– Viral/bacterial load

– Therapeutic monitoring

• Typing (PCR, DNA arrays, Sequencing, AFLP)

– Surveillance of infectious diseases

– Outbreak investigation (epi-typing)

– Pathogenesis and course of infection (patho-typing) • drug resistance (genes) and virulence genes

Does NGS fits it all?

Wet-lab technicians – practical work

Analysis performed by

• E-lab Technicians (Sigrid/Erwin)

• Master Kai Zhou teaches

Post-docs and PhDs

Amplicon-based approach

• Use of the Hospital Acquired Infections (HAI) BioDetection approach for:

– rapid identification of negative clinical samples

– detection of microbes and resistance/virulence genes

10

HAI BioDetection Kit (BioInnovation Solutions SA)

• amplicon-based NGS

• 298 probes (150-190 bp fragments sequenced)

= 144 PCRs

0 hour

Clinical sample

2 hours

DNA isolation

4.5 hours

Work flow

Sample prep (24)

10 hours

Template prep (12)

13.1 hours

Sequencing (12)

≈14 hours

Results (12)

Whole (full) genome sequencing

• Resolve resistance mechanisms

• Find virulence genes

• Outbreak investigation

Klebsiella pneumoniae KPC-outbreak

• KPC-KP ST258

• Hospital and nursing home (July – Dec 2013)

• 6 positive patients

• Extensive environmental contamination

Weterings et al., in revision

WGS and typing by a gene-by gene approach

Using Ridom’s Seqsphere

WGS

Resistance genes:

• Blakpc-2

• BlaSHV-12

ST 258 in Europe

(Curated) databases for early-warning required

Outbreak in a reha-center

• Between May and September 2012

• CTX-M-15 producing K. pneumoniae

• first occurred in a university hospital and later spread to a nearby rehabilitation center

• sequence type (ST) known to be an epidemic clone

• May 2013 - similar CTX-M-15 producing ST15 K. pneumoniae isolated from a patient admitted to the university hospital

Patient Referral Network in the Netherlands

Donker et al. Math Biol 2012

Prof H. Grundmann, UMCG

University Hospitals

Regional Centers

Local Hospital

Phylogenetic analysis of K. pneumoniae isolates

Zhou et al., submitted

Combining data

• An epidemiological link was found between the 2012 and 2013 isolates

• Supported by whole-genome sequencing (WGS) only a few single-nucleotide polymorphisms (SNPs) were detected

• WGS analysis of environmental isolates indicates a possible role for the environment in dissemination of outbreak clones.

The putative transmission chains of the 2012 outbreak

Outbreaker (Jombart et al., 2014) Zhou et al., submitted

Outbreak clone specific Dx-test

Using BRIG to identify

variable regions

Designing primers to amplify

Unique marker

Screen patient isolates

The results of multiplex PCR specific to the outbreak clone

Tailor-made diagnostics(Unique gene analysis - UGA)

Classical

Screening DiagnosticOutbreak Typing Action

E. coli ESBL VRE KPN

Next Gen

NGS UGA

“Precision microbiology”

Conclusions

• WGS allows typing and molecular characterization of the outbreak clone (AMR, virulence)

• The outbreak-specific multiplex PCR facilitated rapid patient screening procedures

• The study emphasizes the necessity of regional collaborations for efficient infection control measures and indicates the potential of WGS for optimized outbreak management in hospital settings

A certain microbe found = Infection?

Diagnostics today:

ID Species -> Clinical orientationfound in blood/wound etc.Resistance -> Therapy advice

Sub-/Genotype -> Infection prevention/Public Health

Pygocentrus nattereri (Piranha) Strepotoccus pyogenes (A-streptococcus)

?

Infectious Disease Staging/Therapy follow-upby diagnostic gene expression profiling

Whole transcriptome sequencing

R

N

AMetagenome sequencing

-> Collaborate with Pathology, Oncology, Genetics

Pharmacomicrobiomics

High-throughput sequencing for everyone ?

Phase 1: more is better

Phase 2: smaller is better

Phase 3: single-molecule

Phase 4: nanopores

“democratization” of next-generation sequencing

The MinION device is adaptable for DNA sequencing,

protein sensing and other nanopore sensing techniques

$ 1000

Costs of Sequencing and costs of data analysis

Sboner et al. (2011). Genome Biol. 12: 125 [PubMed].

Real Cost of Sequencing

Sboner et al. (2011). Genome Biol. 12: 125 [PubMed].

www.worldmapper.org

Mortality due to Infectious diseases

Microfluidics smaller sample volume - increased speed and multiplexing possibilities

"The origins and the future of microfluidics" - G.M. Whitesides, Nature, 2006. DOI:10.1038/nature05058

Microfluid-based point-of-care testing

Relevance of POCT

Clinic Relevance POCT Influence

Sepsis, Meningitis, Pneumonia

High – specially in (N)ICUs Direct impact on clinical outcome

Tuberculosis High for identifying drug-resistant strains

Direct impact on clinical outcome

Respiratory and GI-viruses Prevention of outbreaks and ABS

Direct impact on disease/patient management

Antimicrobial resistance Monitoring emerging resistance – outbreak prevention

Direct impact on disease/patient management

STDs Low

POCT in resource-limited settings

• No costly laboratory based systems

• No need for well-trained technicians

• No need for good sample transport networks

• Self-contained quality control

• Fast – same day result

• Linked to a site where clinical decision making is available at the same patient visit

New technological trends• Smart Phone Diagnostics

• μPADs (micro-paper based analytical devices)

Partially from Martinez et al., Anal. Chem. (2010)

Strategy for performing inexpensive bioassays in remote locations

Martinez et al., Anal. Chem. (2008)

Personalized Diagnostics using Nanocapsules

Courtesy : Mesa+ Nanolab, University of Twente, Netherlands

EHEC O157 please go to

your GP

From (molecular) microbiologist to nanobiologist ?

WGS

DNA Chips

Lab-on-a-chip

Doctor-on-a-chip

Nanotechnology

μPADs

Method centered diagnostics

CostTime

Quality

€-hour-systematic100 h * 3€ = 1h * 300€

Cost

Prevention

Quality

Time

Therapy

Diagnostic

Patient-centered diagnostics

MMB mission

Protect patients against infections by

• Fast and precise Dx

• Advising in optimal treatment

• Obtain and share knowledge

in the patient’s interest

Application of on-demand in-house developed and validated

diagnostics: personalized theragnostics

“Voici mon secret. Il est très simple: on ne voit bien qu'avec le cœur.

L'essentiel est invisible pour les yeux.”

Le Petit Prince (1943) by Antoine de Saint Exupéry

Translation: “Here is my secret. It is very simple: It is only with the heart

that one can see rightly; what is essential is invisible to the eye.”

ESCMID Postgraduate Technical Workshop

Capacity-building Workshop: rapid NGS for characterization and typing of resistant gram negative bacilli

October 7th – 9th, 2015, UMCG Groningen, The Netherlands