Rapid Molecular Diagnostics of Antimicrobial
Resistance
Dr. Vicky Enne
Centre for Clinical Microbiology
Co-ordinator FP7 RiD-RTI
PI Prof. Ali Zumla
Co-I Dr. Vanya Gant
Talk Outline
• Resistance problem & need for rapid diagnostics
• Complexity of resistance
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Potential benefit of rapid molecular diagnostics
Potential problems of rapid molecular diagnostics
Type of technologies
Examples of point of care systems on/near market
Technology in development at UCL – RiD-RTI
Knowledge gaps & future need
The Problem of Antibiotic Resistance
• Antibiotics used heavily in
human & veterinary
medicine
• Use undoubtedly selects
resistance
• Multi-resistant organisms
e.g. MRSA,VRE & C.
difficile cause serious
problems in hospital
setting
• Biggest current & future
threat thought to come
from Gram-negatives
Penicillin-resistant pneumococci vs.
outpatient beta-lactam sales
(From: Livermore. 2005. Lancet Infectious Diseases
5:450-459)
Complexity of Resistance Genetics in Gram-ve rods
• Enormous complexity complicates both treatment
and diagnosis
• Resistance generally transmitted on mobile
genetic elements – plasmids & transposons
• Infection control needs to prevent not only spread of
individual organisms but also genetic elements
• Several different mechanisms for one resistance
phenotype
• e.g. at least 4 known for fluoroquinolones, can all be
present at one
• One bacterium / plasmid can carry a large number
of different resistance genes – >10 commonplace
Many variants of a
single gene,
sometimes with
different phenotypes.
(From: Cornaglia et al. Lancet Infect Dis 2011)
There are approx. 30
distinct acquired betalactamase families,
among these there can
be up to 370 variants of
a single type e.g. TEM,
OXA
(From: Canton et al. CMI 2012)
The Need for Rapid Diagnostics
• Conventional methods > 48h
to identify antibiotic
susceptibility
• Reference laboratories
often required to identify
specific resistance gene –
slows down outbreak
tracing
• Importance recognised in
recent reports & policy
documents (CMO Report,
UK Strategy etc.)
Potential Benefits of Rapid Diagnostics
• Improved treatment outcome for patient
• Improved infection control and outbreak
monitoring achieved by acquiring more precise
information sooner
• Reduction in empirical antimicrobial prescriptions
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Preservation of broad spectrum antimicrobials
Reduction in duration of treatment
Reduction in cost of treatment
Overall reduction antimicrobial consumption
Potential reduction in levels of resistance
Does reduced prescribing reduce resistance?
8 years
3 years
2 years
8 years
7 months
20
0
F inla nd
I c e la nd
S w eden
UK
I s ra e l
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% C h a n g e -4 0
P re s c rib ing R e d uc tio n
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C o u n tr y
From: Enne 2010 JAC 65: 179-182
Problems Associated with Molecular
Diagnostics of Resistance
• Discrepancy between genotype and phenotype
1. The potential presence of unknown resistance
mechanisms leading to treatment failure
2. Genotypic markers of resistance not resulting in
clinically significant resistance. Patient may be denied
therapeutic drug needlessly.
• Rapid molecular methods should be
supplemented with culture-based techniques
Sources of Pitfalls
• Resistance gene not detected by test
• PCR based technologies may not be able to include all
possibilities – constantly changing epidemiology
• New genes/mechanisms of resistance
• Resistance genes not conferring resistance
• Poorly expressed genes
• Non-functional mutated genes
• Silent genes
• Difficulty in predicting exact resistance phenotype
from genotype e.g. interplay of multiple
simultaneous mechanisms e.g. low level efflux +
mutations
Technologies for Rapid Diagnostics
• Point of care – near-patient systems requiring
minimal user expertise
• Kit-based – provides reagents but skilled operator
& specialist equipment still required
• PCR Based
• Real-time/qPCR
• Droplet PCR
• Microarray – usually for 2ndary detection only
• MALDI-TOF MS + PCR
• Whole genome/next generation sequencing
• Transcriptomics
Point of Care Products on/near Market
• Cepheid GeneXpert – Known for TB diagnostics,
sample in answer out
• Curetis Unyvero – sample in/answer out,
pneumonia cartridge
• Nanosphere Verigene – sample in/answer out
blood culture
• Biofire (Biomerieux) FilmArray – blood culture
• Epistem Genedrive – small, low cost. TB
diagnostics.
Cepheid GeneXpert
• 1st point of care system
introduced to market
 MTB/RIF test, WHO endorsed
 Good evaluation data
• Integrated sample prep & PCR;
cartridge based, < 1 hour
• Low number of targets per test
 Strong focus on colonisation/infection control
 SA/MRSA testing from swabs or positive
blood cultures
 Carbapenemase assay from rectal swabs in
pipeline (KPC, VIM, NDM)
Curetis UnyVero
• Point of Care PCR based
instrument. Test time < 4h
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Separate lysis unit
• First cartridge for pneumonia
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Comprehensive detection of bacterial pathogens ,
including those implicated in HAP & atypical
infections
Diverse list of resistance genes – mecA, macrolides,
ESBLs, KPC, OXA-51, integron markers, FQR
mutations
• Validation data not yet available
Nanosphere Verigene
• Point of Care, PCRbased instrument
• Works on positive
blood cultures, < 2.5 h
• Gram positive test
• mecA, vanA, vanB
• FDA cleared, generally good evaluation results but
mis-ID of streptococci
• Gram-negative test
• KPC, NDM, IMP, VIM, CTX-M, OXA
• No evaluation available yet
In development at UCL – RiD-RTI
• EU FP7 funded project to
develop rapid diagnostics for
respiratory tract infections
• Low cost, point of care device
• Integrated sample prep, cartridge based
• PCR & microarray detection
• Answer in < 2 hours
• Ability to scale up
Unique partnership of SMEs, Universities
and Hospitals
Sample Preparation
&
Real Time PCR assays
Clinical evaluation
using patient
samples
Instrument &
Software design
Microarray chip design
3 RTI Diagnostic products
• Community acquired pneumonia
• Bacteria vs. virus
• Eliminates unnecessary empirical prescriptions
• Resistance markers in phase 2 product
• Hospital acquired pneumonia
• S. aureus & enteric pathogens & non-fermenters
• Comprehensive ESBL and carbapenamase
• Opportunistic respiratory tract infections
• Fungi, bacteria and virus
What is Needed in the Future ?
• Still in early stages of commercialisation – many
tests not comprehensively validated yet
• Many systems require regulatory approval - CE-IVD
(Europe) & FDA (US)
• Clinical evaluation of rapid diagnostics to guide
treatment – will potential benefits be realised?
• More comprehensive tests & scaleable devices
• More focus on global problems, current devices heavily
US based
• More tests simultaneously
• Ability of technology to adapt rapidly to evolving
pathogens & emerging resistances
Acknowledgements – RiD-RTI Consortium
• UCL
Ali Zumla (PI)
Ljuban Grgic
Gareth Platt
Stefan Schwenk
Yoann Personne
Mary Lenahan
• UCLH
Vanya Gant
• NUIG, Ireland
Tom Barry
Nina Tuite
Kate Reddington
• Mobidiag, Finland
Juha Kirveskari
• Genewave, France
Francis Domain
Yann Marcy
Claude Weisbuch
www.rid-rti.eu