An insight into the National
Microbiology Reference
Laboratories
John E Coia
1
Overview
• What is a Microbiology Reference Laboratory?
• How do we decide we need a Reference Laboratory?
• Microbial Typing
– What do we mean by typing?
– Why do we want to type?
– How do we type?
• What Microbiology Reference services are there in
Scotland?
• Do we make a difference?
2
What is a Microbiology Reference
Laboratory?
3
Types of Microbiology laboratory
• Primary Diagnostic Laboratory
• Specialist Diagnostic Laboratory
• Reference Laboratory
4
Primary Diagnostic Laboratory
• “Local” laboratory
– Increasingly rationalised especially elsewhere in the UK
• Receives samples directly from patients
– E.g. blood cultures, wound swabs, urines, sputa etc.
• Front line service for immediate patient management
• Still predominantly based on conventional culture
techniques and antimicrobial sensitivity testing
• Automated technologies and molecular diagnostics
starting to become established (already very well
established in virology)
5
Specialist Diagnostic
Laboratory
• Usually centralised/regionalised
• Performs low volume and/or high costs
tests
– Not practicable to provide locally
– Not cost effective to provide locally
• Receives samples directly from patients
and/or indirectly via primary diagnostic
laboratories
6
Reference Laboratory
• Single national centre
• Specialised expertise in particular
pathogen/pathogens
• Receives organisms isolated in primary
diagnostic labs
• May in addition have a specialist diagnostic
role, so may receive patient samples
• Why reference?
– Provides a reference point and resource
– Samples are “referred”
7
Organisation of reference
laboratories in Scotland
• Funded by NHS Scotland via National Services Scotland
(NSS)
• Health Protection Scotland & National Services Division
(HPS/NSD) have a statutory role in commissioning on
behalf of Scottish Government
• Further oversight, monitoring the reference work,
assessing public health impact and reviewing the
reference laboratory function is undertaken by HPS/NSD
with advice from a multidisciplinary group of clinicians
and managers (Reference Laboratories’ Working
Group)
8
How do we decide we need a
Reference Laboratory?
9
Criteria for establishing a
Microbiology Reference Service
• A reference function is called for when there
is a significant recognised need in Scotland
on the grounds of health protection for
secondary investigations related to a
microbiological agent e.g.
–
–
–
–
–
subtyping
antimicrobial resistance
virulence markers
cluster analysis
vaccine failure
10
Additional functions of the
Reference Service
• confirmation of rare or difficult diagnoses
• help in management of individual, unusual
cases or outbreaks
• advise on policy-making
• research and development related to the
public health aspect of the subject
• evaluation of new technology
• education and training for the wider service
• identify emerging public health threats
11
Criteria for Entry as a
Reference Laboratory 1
1. The laboratory must provide a reference service for
which there is a recognised need in Scotland
2. The laboratory must possess established expertise in
the relevant area (clinical, scientific and technical)
3. The laboratory must form part of an existing
laboratory with CPA or equivalent accreditation
4. The laboratory must demonstrate satisfactory
participation in recognised relevant EQA schemes
5. The laboratory must have the support of the HB/
Operating Division in which it is hosted and the
capacity to provide a Scotland-wide service
12
Criteria for Entry as a
Reference Laboratory 2
6.
The laboratory should be co-located with routine
diagnostic services to maintain appropriate skills
and possess the ability to appraise new techniques
7. The laboratory must have an established record of
good communication with HPS and similar referral
centres in the UK
8. The laboratory must be able to demonstrate good
communication links with users of the Service
9. The Head of the laboratory should be a medically
qualified Consultant or Consultant Clinical Scientist
10. Clinical advice should at all times be made
available from a medically qualified person
13
Outputs Expected from a
Reference Laboratory
• The laboratory should undertake reference tests and provide
advice in line with set targets.
• The laboratory must produce an annual report
–
–
–
–
–
–
–
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Aims and objectives of the Reference Service
Service activity (i.e. numbers and types of samples tested)
Range of tests undertaken
Range of tests likely to be undertaken in the coming year
Archive usage
QA performances
Research and Development
Publications in peer-reviewed (and other) journals and any other
relevant quality outputs.
– Presentations
– Staffing [present and projected]
– Financial Summary [present and projected]
• Service contracts renewed every 3 years as appropriate.
14
Key relationships
Veterinary
Labs
CsPHM
NHS
Labs
Clinicians
Other
Labs
HPS
Other UK
Agencies
International
Agencies
Ref
Labs
Other
Reference
Labs
Funded Reference Activity
(commissioned by HPS/NSD)
Research
Initiatives
Universities
Activity Supported
From Other Sources
15
What do we mean by typing?
16
•
•
Two different individuals
Genetically similar enough to be
members of the same species
–
•
Genetically different enough to be
different individuals
–
–
•
Homo sapiens
Genotype is the underlying genetic
make up of the organism encoded
in the nucleic acid (DNA)
Phenotype is the sum of the
observable characteristics of the
organism and is the product of
expression of the underlying
genes and their interaction with
the environment
We perceive these two individuals
to be different on the basis of the
phenotypic expression of their
underlying genotype
17
•
•
Some phenotypic traits
are more useful than
others in reliably
differentiating
individuals of the same
species e.g.
fingerprints are more
useful than hair colour
or eye colour
Rather than using
phenotypic
characteristics to
discriminate individuals
we can use genotypic
methods which use
direct DNA-based
analysis differences in
the underlying genetic
code of the individual
e.g. forensic DNA
fingerprinting or
profiling
18
Microbial Typing
• It is also possible to differentiate
between different individuals or “strains”
of microbes
• The process of differentiating strains
based on their phenotypic and
genotypic differences is known as
'typing'
19
Criteria for evaluating
typing systems
1. Typeability: Is the ability to obtain a result for each strain
analysed
2. Reproducibility: Is the ability of the technique to yield the
same result when the same strain is tested repeatedly
3. Discriminatory Power: Is the ability to differentiate among
unrelated strains. Ideally, each unrelated isolate is detected as
unique
4. Ease of performance: To be widely useful, a typing method
should be applicable to a broad range of microorganisms as
well as inexpensive and technically easy
5. Ease of interpretation: The typing tests must produce
reproducible and unambiguous results that can be interpreted
easily
6. Portability: The technique should be capable of
standardisation between laboratories, and data should be
easily comparable between laboratories
20
Why do we want to type?
21
Some reasons for typing
• Is this an outbreak?
• Is this vehicle (food/water/etc) the source of an
outbreak?
• Is this strain more pathogenic, virulent or
antibiotic resistant than other strains within a
species?
• Are changing numbers associated with
changing strains?
• How do these isolates compare with those from
elsewhere?
• Was this strain covered by the current vaccine?
22
Other points to note about
typing
• How much variability do you want?
– Sometimes it is helpful to be able to “lump”
as well as “split” e.g. are isolates part of a
group with a recent common ancestor
• How common are particular strains in
the background population?
23
How do we type?
24
Phenotyping
•
•
•
•
Biotyping
Phage typing
Serotyping
Antibiogram typing
25
Genotyping
•
•
•
The discrimination of bacterial strains based on
their genetic content
Has progressively replaced the majority of
phenotypic methods
Three main types of approach
1.
2.
3.
•
•
DNA-banding pattern based
DNA-hybridisation based
DNA-sequence based
DNA-banding patterns still widely used, especially
in outbreak settings, but are rapidly being replaced
by DNA-sequence based approaches
DNA-hybridisation based (microarrays) are less
commonly used for reference work
26
Phenotyping versus genotyping
Typeability
low to high
high
Reproducibility
low to moderate
medium to high
Discriminatory power
low to moderate
high to very high
Ease of performance
Highly variable, often labour intensive,
variable costs, reagents are often highly
organism specific
Variable but many modern
methods lend themselves to
automation. Costs variable
but falling. Applicable to
broad range of organisms.
Ease of interpretation
Highly variable, and may require
considerable expertise, skills not easily
transferrable
May require considerable
expertise, but lends itself to
automated methods
Portability
Highly variable, may be very difficult to
standardise
Older gel-based methods
difficult to standardise.
Modern methods highly
transportable
27
Pulsed field gel
electrophoresis (PFGE)
• Very well established
band-based method
• Applicable to wide
range of organisms
• Highly discriminatory
• Difficult to standardise
and compare large
numbers of organisms
Extract genomic DNA
Digest with rare-cutting restriction endonuclease
Separate fragments by electrophoresis on an agarose gel
28
Multi-locus VNTR analysis
(MLVA)
• Modern band-based
technique
• Bands can be
accurately measured by
capillary electrophoresis
rather than gels
• Highly discriminatory
• Wide applicability
• Highly portable data
Amplification of highly polymorphic VNTR loci using PCR
Separate fragments by electrophoresis on an
agarose gel or by capillary electrophoresis
Calculate the number of repeats at each locus
Generate identity “code” e.g. 4321156
= 4 repeats at locus 1, 3 repeats at locus 2, etc.
29
Multi-locus sequence typing
(MLST)
• Sequence-based
technique
• Sequence a small
number of
housekeeping
• Less discriminatory,
but very stable
• Wide applicability
• Highly portable data
Extraction of genomic DNA
Amplification of 7 housekeeping genes
Compare sequences in a database
Generate code based upon the sequence type at each locus
30
Whole-genome sequencing
• Sequence-based technique
• Rapid sequencing and
assembly of whole genome
sequence of the organism
• Ultimate level of
discrimination
• Wide applicability
• Highly portable data
• Requires complex
bioinformatics to analyse
• Will replace other typing
techniques as costs fall
– 2005 $500,000 per genome
– 2013 $100 per genome
Extraction of genomic DNA
Rapid sequencing using “next generation” techniques
(hours versus months by conventional methods)
Compare sequences in a database
Discriminate strains on whole sequence or by looking at
differences in key sites (SNPs).
Possibility to perform “super MLST”
31
Whole genome sequencing
and the Scottish reference labs
• Activities to date
– Collaborative studies that have taken advantage of extensive
well-curated collections maintained by reference services
• Planned activities in short term
– More focussed collaborations that will seek to develop
potential specific public health benefits of NGS
• Likely impact longer term
– Replacement of existing methodologies
– “Disruptive” technology
• Management of transition
– Within the reference laboratories
– Within the wider public health context
32
What Microbiology Reference
services are there in Scotland?
33
http://www.hps.scot.nhs.uk/reflab/index.aspx#
34
Do we make a difference?
35
C.difficile Reference Service
• Services
–
–
–
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Confirmation of identity
Antimicrobial sensitivity testing (E-test)
PCR ribotyping
MLVA typing
• Designed to: – Support mandatory national surveillance
•
Characterise the epidemiology of C. difficile in Scotland
•
Identify new emerging strains
– Support outbreak investigation
– Support AMR surveillance
• Isolates from severe cases, suspected outbreaks &
suspected 027
• Representative surveillance program
36
36
Local impact
37
Is this an ongoing outbreak?
• Contacted by IPCT from HB A
• Increased numbers of CDI
• Ongoing issue despite stringent
IPC precautions
• Possible epidemiological links,
but complicated picture
• PCR ribotyping demonstrated a
number of different ribotypes
• Recommended looking more
closely at prescribing
• Number of issues identified
• Contacted by IPCT from HB B
• Increased numbers of CDI
• Possible epidemiological links,
but complicated picture
• PCR ribotyping demonstrated
most isolates of the same
unusual PCR ribotype
• Evidence of spread
• IPC measures had been
increased but issue around a
difficult to clean environment
38
Importance of subtyping
• Cluster of C.difficile in
one HB in Scotland
• All typed as PCR
ribotype 027
• High resolution
subtyping by MLVA
revealed 3 different
groupings
• MLVA was essential to
understanding the
epidemiology of the
“outbreak”
39
National impact
40
PCR ribotypes in Scotland
Severe & Outbreak Cases
300
250
200
Others
027
150
001
106
100
50
0
07- 08- 08- 08- 08- 09- 09- 09- 09- 10- 10- 10- 10- 11- 11- 11- 11- 12- 12Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2
41
All (n=560)
Others (n=148)
020 (n=28)
078 (n=86)
014 (n=28)
023 (n=28)
002 (n=56)
005 (n=43)
015 (n=38)
027 (n=40)
001 (n=39)
106 (n=26)
Antibiotic sensitivities
12 months until June 2012
100
90
80
70
60
50
40
30
Levofloxacin
20
Cefotaxime
10
Clindamicin
0
42
•078 seen by SSSCDRL since 2007
•Modest year on year increases
•Sudden increase in Q1 & Q2 2012
•Seen in 12/14 HBs
•Outbreaks in 2 HBs
43
Wiuff C et al., The epidemiology of Clostridium difficile in Scotland, J Infect (2011), doi:10.1016/
j.jinf.2011.01.015
44
44
45
Internationally
46
WGS and SNP analysis of C.difficile ribotype 027 was used to
elucidate the spread of this pathogen from North America to Europe,
and throughout the UK
47
Activities of other Reference
Services in Glasgow 1
• SMRSARL
– Outbreak support
– Surveillance e.g. SABs
– Virulence factors e.g. PVL producing
organisms
– EARS-net
– Monitoring other resistance trends e.g.
Mupirocin
48
Activities of other Reference
Services in Glasgow 2
• SHLMPRL
– Outbreak support
– Circulating pneumococcal and
meningococcal types
– Antimicrobial resistance e.g. penicillinresistant pneumococci
– Legionella testing and typing e.g. support
for recent local investigations in Edinburgh
49
Activities of other Reference
Services in Glasgow 3
• SPDRL
– Large specialist diagnostic remit
• Malaria, schistosomiasis etc.
– Antimicrobial resistance in malaria
parasites
– Cryptosporidium subtyping (outbreak
support)
50
Summary
• Microbiology reference laboratories play a key public
health role in supporting and augmenting the
activities of front line diagnostic laboratories,
clinicians, IPCTs and AMTs
• This work underpins key activities of HPS and
several national surveillance programmes including
those for CDI and SABs
• Modern molecular subtyping helps to inform and
support local, national and international surveillance,
target interventions and assess their impact
• Surveillance of circulating strains, their associated
virulence factors and antimicrobial susceptibilities
helps to detect and monitor emerging public health
threats
• Specialist diagnostic testing provides confirmation of
rare or difficult diagnoses
51
Acknowledgements
• All staff of the Reference laboratories
• HPS/NSD
• Staff in NHS Boards
52