1. Session 2: Laboratory Investigations Good Laboratory Practice
Facilitator notes:
Control of Multidrug Resistant Micro-Organisms in Health Care Settings
Session 2: Laboratory Investigations Good Laboratory Practice
Revision: April 2018

2. Factors influencing laboratory investigation
Clinical suspicion
Quality and type of sample
Speed of transfer to laboratory
Information provided with request
Laboratory methods of detection
Sensitivity testing methods
Available resources
There will always be variation between Laboratories

3. Clinical requirements from laboratory
Rapid diagnosis with clear guidance for treatment
Organism identification to species level and sensitivity results
Infection Control requirements
Early identification of outbreaks/incidents
Subtyping of organisms sufficient to confirm true cross infection/outbreak
Surveillance requirements Public Health
Consistent methods of collection, analysis and reporting of results
A small error, e.g. over-diagnosis by 5% does not matter as long as it is consistent

4. Quality standards of laboratory
Laboratory should quality assure activities. Users should be aware of standards.
Internal quality assurance
Use control organisms or samples to ensure ability to detect relevant organisms
External quality assurance
e.g. HPA NEQAS scheme to ensure detection of relevant pathogens in samples with relevant clinical history
Laboratory Accreditation
Laboratory systems reviewed by external assessor to ensure meeting minimum standards acceptable e.g. Clinical Pathology Accreditation UK
Compliance with Health and Safety
Appropriate containment level; usually 2 and 3

5. Laboratory Methods for organism detection
Bacterial culture
Serology for antigens / antibodies - not considered further
Molecular techniques - Polymerase chain reaction (PCR)
Mass Spectrometry - MALDI-TOF

6. Bacterial Culture Traditional method Dependent on quality of sample
How well collected
Use of transport medium
How fast received by laboratory
Organisms on dry swabs can be lost rapidly
Transport medium can improve detection by stabilising sample and preventing overgrowth of contaminants

7. IMPORTANT TO BE AWARE OF LIMITATIONS OF EACH SAMPLE TYPE
Bacterial Culture
Results dependent on site of sample
Sputum
Likely to have upper respiratory contaminants; may miss ‘true’ pathogen
Patient did not receive appropriate treatment; report a contaminant as significant
Blood/Sterile sites
more likely to detect a true pathogen
Urine
may get perineal contamination
IMPORTANT TO BE AWARE OF LIMITATIONS OF EACH SAMPLE TYPE

8. Detection of pathogens from bacterial culture:
Requires sufficient weight of organisms in sample to detect
not a very sensitive method
Requires selection of appropriate media to detect likely pathogens
May require selective media that suppress growth of unwanted commensals
E.g. GC medium for Neisseria gonorrhoea
indicator media e.g. Sorbitol MacKonkey for E Coli 0157
Must know clinical history to direct culture methods appropriately

9. Bacterial culture: Advantages Materials costs low but labour intensive
Have culture for further investigations
Limitations
Insensitive
Will miss pathogens if antibiotics already received
May report wrong organism
Results take hours

10. Automated and semi-automated culture methods
Detection of bacterial growth e.g.
Blood culture system - BACTEC /phoenix
Can get gram film results at 8 hours
Does not identify organism, but narrows the likely organisms
Requires culture for full identification and sensitivity (though new developments in this area)
Urine system - ALFRED 60
Can perform culture, residual antimicrobial testing and susceptibility testing
Results may be available in 4 hours

11. Molecular Testing Methods
Polymerase chain reaction

12. Molecular Testing Methods
Polymerase Chain Reaction (PCR) can detect key DNA / RNA sequences
Must have necessary primers to detect suspected pathogens
Will detect presence of organism after antibiotics and at subclinical levels
Result may be available in one hour
Can be automated
Introduces option of near patient testing, e.g. screening for MRSA, C. difficile, e.g. GeneXpert (Cepheid)
Have to culture all positive samples to confirm result and perform sensitivity testing
Automated systems: Require expensive capital equipment but can batch process specimens, less labour intensive

13. New Technologies - Example
MALDI – TOF ( Matrix Assisted, Laser Desorption Ionisation, Time of Flight)
A form of Mass Spectrometry.
Can identify organisms once isolated in culture from pattern of printout.
Systems in development to identify organisms during growth phase e.g. linked to BACTEC

14. Typing and subtyping of organisms
Required to identify linked cases / clusters / outbreaks (same or closely associated strains)
Usually undertaken by Specialist / Reference laboratories
Must be discriminatory
Too insensitive - cannot differentiate between strains
Too sensitive - cannot detect linked strains (clusters)
Must be relatively stable over time
Ribotyping – C difficile
Phage typing – Salmonella typhimurium
Full gene sequencing is too costly for ‘routine’ detection and surveillance

15. Sensitivity Testing Requires pure culture Aim:
To detect phenotypic sensitivity profile (not all resistance genes are expressed)
To detect inducible resistance

16. Methods
Disc testing
Measure zone size - labour intensive, can be very accurate but prone to operator error
Minimum Inhibitory Concentration
E.g. E-test - can determine very specifically the concentration of antibiotic in vitro that inhibits the growth of an organism,
Breakpoint testing
Gives range between which MIC lies
Agar methods
Automated methods

17. Reporting of Results Who is the report aimed at? Clinician
Infection Control
Public Health
National Surveillance

18. Clinician
Need to get report to appropriate ward, where the clinician will act on it
Phone urgent results, e.g. CSF film at any time
Phone other results when clinician is available to act
What is the value of a result at 2.00am, if no-one will use it then?
Electronic reporting - need to flag positive results
Paper reports
Need to get to clinician at appropriate time of day to act - not always easy –
Lab transport systems often dependent on other hospital priorities

19. Infection Control
Need to get information to appropriate infection control personnel
Phone urgent results to on-call Infection Control team
Discussion of appropriate infection control interventions
Paper reports - audit trail should be in place

20. Public Health Frequency of reporting Immediate:
confirmed meningococcal meningitis
Within 24 hours
suspected outbreak e.g. Salmonella
Weekly
standard reports from which PH compile trends

21. National Surveillance
Usually report retrospectively, may compile through local public health or reference laboratory or directly from diagnostic laboratory

22. Summary of Good Laboratory Practice
Quality of a laboratory report depends on:
Quality of sample
Information submitted
Systems must be in place to quality assure laboratory activity
Bacterial culture is the mainstay, but new automated systems are improving identification and turnaround
Users must be familiar with the advantages and limitations of various methods

23. Plenary feedback
Opportunity for group activity and discussion around laboratory investigative methods in each member state to be fed back in a plenary session.

24. Group exercise Group activities:
Participants should be grouped, as far as possible, in own member state groups
Discussion should centre around laboratory investigations in their member state in preparation for plenary feedback

25. Facilitator notes:
Acknowledgements The creation of this training material was commissioned in 2011 by ECDC to Health Protection of Scotland, National Services Scotland, University of Chester and University of Dundee with the direct involvement of Dr Anne Eastaway, C Wiuff, A Seaton, J Reilly, Miss Michelle Rivett, P Davey, A Bryan, D Robertson Adapted / modified by: S. Rosales Klintz, Karolinska Institutet, Stockholm, Sweden, 2015, ECDC MDRO course Diamantis Plachouras, ECDC, 2015, ECDC MDRO course Oliver Kacelnik, Norwegian Institute of Public Health, 2016, ECDC MDRO course The revision and update of this training material was commissioned in 2017 by ECDC to Transmissible (Netherlands) with the direct involvement of Rita Szabo, Remco Schrijver and Arnold Bosman

26. ---- This last slide or page must always be included with this work ----
Author: Dr Anne Eastaway, C Wiuff, A Seaton, J Reilly, Miss Michelle Rivett, P Davey, A Bryan, D Robertson (2011)
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ATTENTION AUTHORS & ADAPTERS: All external sources should be mentioned on individual pages, diagrams, tables, photos within the work. 
Adapted / Modified by:
S. Rosales Klintz, Karolinska Institutet, Stockholm, Sweden, 2015, ECDC MDRO course
Diamantis Plachouras, ECDC, 2015, ECDC MDRO course
Oliver Kacelnik, Norwegian Institute of Public Health, 2016, ECDC MDRO course
Rita Szabo, Remco Schrijver in collaboration with Transmissible (Netherlands), 2018
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