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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
2. Disclosure of speaker’s interests
(Potential) conflict of interest None
Potentially relevant company relationships in
connection with event
None
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
4. Antoni van Leeuwenhoek (1632-1723)
Martinus Beijerinck (1851 - 1931)
Filtration experiments Virus
5. 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?
6. 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
9. Analysis performed by
• E-lab Technicians (Sigrid/Erwin)
• Master Kai Zhou teaches
Post-docs and PhDs
10. 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
11. HAI BioDetection Kit (BioInnovation Solutions SA)
• amplicon-based NGS
• 298 probes (150-190 bp fragments sequenced)
= 144 PCRs
14. Klebsiella pneumoniae KPC-outbreak
• KPC-KP ST258
• Hospital and nursing home (July – Dec 2013)
• 6 positive patients
• Extensive environmental contamination
15. 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
16. ST 258 in Europe
(Curated) databases for early-warning required
17. 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
18. Patient Referral Network in the Netherlands
Donker et al. Math Biol 2012
Prof H. Grundmann, UMCG
University Hospitals
Regional Centers
Local Hospital
20. 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.
21. The putative transmission chains of
the 2012 outbreak
Outbreaker (Jombart et al., 2014)Zhou et al., submitted
22. Outbreak clone specific Dx-test
Using BRIG to identify
variable regions
Designing primers to amplify
Unique marker
Screen patient isolates
23. The results of multiplex PCR specific to
the outbreak clone
24. Tailor-made diagnostics
(Unique gene analysis - UGA)
Classical
Screening DiagnosticOutbreak Typing Action
E. coli ESBL VRE KPN
Next Gen
NGS UGA
“Precision microbiology”
25. 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
26. A certain microbe found = Infection?
Diagnostics today:
ID Species -> Clinical orientation
found in
blood/wound etc.
Resistance -> Therapy advice
Sub-/Genotype -> Infection prevention/Public Health
Pygocentrus nattereri (Piranha) Strepotoccus pyogenes (A-streptococcus)
?
27. Infectious Disease Staging/Therapy follow-up
by diagnostic gene expression profiling
Whole transcriptome sequencing
R
N
A
Metagenome sequencing
-> Collaborate with Pathology, Oncology, Genetics
Pharmacomicrobiomics
28. 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
29. The MinION device is adaptable for DNA sequencing,
protein sensing and other nanopore sensing techniques
$ 1000
30. Costs of Sequencing and costs of data analysis
Sboner et al. (2011). Genome Biol. 12: 125 [PubMed].
31. Real Cost of Sequencing
Sboner et al. (2011). Genome Biol. 12: 125 [PubMed].
33. 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
35. 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
36. 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
37. New technological trends
• Smart Phone Diagnostics
• μPADs (micro-paper based analytical devices)
Partially from Martinez et al., Anal. Chem. (2010)
38. Strategy for performing inexpensive
bioassays in remote locations
Martinez et al., Anal. Chem. (2008)
39. Personalized Diagnostics using Nanocapsules
Courtesy : Mesa+ Nanolab, University of Twente, Netherlands
EHEC O157
please go to
your GP
43. “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.”
44. 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
Notas do Editor
A maximum likelihood tree was constructed based on the alignments of a 4.4Mb genome, defined as the core genome in this study. The tree was rooted on the isolate KP-21F. The outbreak clones are shown as a red triangle. The non-outbreak isolates of this study are indicated as a blue dot, and the others, retrieved from GenBank, are indicated as black dots. The numbers represent the percentage of bootstrap support (> 90). The inset shows the close-up phylogenetic tree of isolates from each outbreak. The outbreak isolates of patients are shown as red (2012) and yellow (2013) dots, and the environment isolates are shown as green dots. The number of SNPs is indicated on the branches.
The transmission route was predicted with patient trace data combined with genetic data. Nodes with numbers represent patients, and arrows indicated a possible transmission event from one patient to another. The blue arrows represent a transmission even between patients predicted by both epidemiological data and genetic data, and the red arrows indicate the equally parsimonious transmission link which cannot be resolved by neither epidemiological data nor genetic data