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An insight into the National Microbiology Reference

Laboratories

John E Coia

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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?

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What is a Microbiology Reference Laboratory?

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Types of Microbiology laboratory

• Primary Diagnostic Laboratory

• Specialist Diagnostic Laboratory

• Reference Laboratory

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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)

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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

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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”

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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)

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How do we decide we need a Reference Laboratory?

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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

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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

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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

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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

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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– 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.

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Key relationships

RefLabs

NHSLabs

CsPHM

HPS

Other UK Agencies

Clinicians

OtherLabs

ResearchInitiatives

Universities

Funded Reference Activity(commissioned by HPS/NSD)

Activity SupportedFrom Other Sources

International Agencies

OtherReference

Labs

VeterinaryLabs

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What do we mean by typing?

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• Two different individuals• Genetically similar enough to be

members of the same species– Homo sapiens

• Genetically different enough to be different individuals

– 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

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• 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

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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'

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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

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Why do we want to type?

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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?

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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?

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How do we type?

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Phenotyping

• Biotyping• Phage typing• Serotyping• Antibiogram typing

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Genotyping

• The discrimination of bacterial strains based on their genetic content

• Has progressively replaced the majority of phenotypic methods

• Three main types of approach1. DNA-banding pattern based2. DNA-hybridisation based3. 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

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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

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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

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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.

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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

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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 atdifferences in key sites (SNPs).

Possibility to perform “super MLST”

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Whole genome sequencingand 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

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What Microbiology Reference services are there in Scotland?

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http://www.hps.scot.nhs.uk/reflab/index.aspx#

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Do we make a difference?

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C.difficile Reference Service

• Services– 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

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Local impact

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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

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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”

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National impact

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PCR ribotypes in ScotlandSevere & Outbreak Cases

0

50

100

150

200

250

300

07-Q4

08-Q1

08-Q2

08-Q3

08-Q4

09-Q1

09-Q2

09-Q3

09-Q4

10-Q1

10-Q2

10-Q3

10-Q4

11-Q1

11-Q2

11-Q3

11-Q4

12-Q1

12-Q2

Others

027

001

106

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Antibiotic sensitivities12 months until June 2012

10

6 (

n=

26

)

00

1 (

n=

39

)

02

7 (

n=

40

)

01

5 (

n=

38

)

00

5 (

n=

43

)

00

2 (

n=

56

)

02

3 (

n=

28

)

01

4 (

n=

28

)

07

8 (

n=

86

)

02

0 (

n=

28

)

Oth

ers

(n

=1

48

)

All

(n=

56

0)

0

10

20

30

40

50

60

70

80

90

100

Levofloxacin

Cefotaxime

Clindamicin

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•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

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Wiuff C et al., The epidemiology of Clostridium difficile in Scotland, J Infect (2011), doi:10.1016/

j.jinf.2011.01.015

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Internationally

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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

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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

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Activities of other Reference Services in Glasgow 2

• SHLMPRL– Outbreak support– Circulating pneumococcal and

meningococcal types– Antimicrobial resistance e.g. penicillin-

resistant pneumococci– Legionella testing and typing e.g. support

for recent local investigations in Edinburgh

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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)

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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

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Acknowledgements

• All staff of the Reference laboratories

• HPS/NSD

• Staff in NHS Boards