LABORATORY PROCEDURE SHLMPRL UM 2014 REVIEW...
Transcript of LABORATORY PROCEDURE SHLMPRL UM 2014 REVIEW...
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL User Manual Controlled document Page 1 of 22
Title SHLMPRL User Manual
LABORATORY PROCEDURE SHLMPRL_UM_2014
NUMBER / VERSION 2.1
DATE OF ISSUE 18/08/2014
REVIEW INTERVAL 2 Years
AUTHORISED BY Dr. B. Jones
AUTHORS A. Brown
COPY 1 of 1 Master file in Q-Pulse
LOCATION OF COPY GG&C website www.nhsggc.org.uk/smrl
DOCUMENT REVIEW HISTORY
All review / revision details are available in Q-Pulse
Date Amendment Initials
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
Controlled document Page 2 of 22
Scottish Haemophilus, Legionella,
Meningococcus and Pneumococcus
Reference Laboratory
User Manual
2014
We aim to select our test repertoire for the benefit of our users and their patients. If you
have any suggestions for improving our service please contact us.
CONFIDENTIALITY POLICY
NHSGG&C Standing Financial Instructions and Fraud Policy ensure that users’
confidential information is protected and that this department cannot undertake
activity that would diminish confidence in its impartiality.
Users’ confidential information is also governed by our procedure RL_MP_010
‘Management of data & information’ and by NHSGG&C I.T. Policy.
Activity that would diminish confidence in impartiality or integrity is also
prohibited by the Health & Care Professions Council code of conduct.
Complaints procedure:-
We will:-
1. Take all complaints seriously.
2. Deal with the client in a courteous manner.
3. Try to resolve the issue immediately at a local level.
4. Inform the client about the progress of the complaint.
5. Make corrective action as soon as possible.
6. Investigate root cause analysis to prevent recurrence.
If you have a complaint, contact the laboratory manager (see page 5).
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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MANUAL
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Section One: The Scottish Haemophilus, Legionella, Meningococcus and
Pneumococcus Reference Laboratory
Introduction 4
Laboratory hours 4
Contact details 5
Section Two: Services provided by the SHLMPRL
Submission of samples 6
Specimen acceptance & rejection criteria 6
Transportation of specimens 7
Inappropriate use of services 7
Enhanced surveillance of meningococcal disease 8
Enhanced surveillance of Pneumococcal disease 8
Enhanced surveillance of H. influenza disease 8
Enhanced surveillance of travel associated
Legionnaires’ Disease 9
Neisseria meningitidis referrals 9
Streptococcus pneumoniae referrals 11
Haemophilus influenzae referrals 11
Legionella referrals 12
Invasive Group A Strep (G.A.S) 14
Request turnaround times 15
Reporting of results 16
Other services 17
Section Three: Interpretation of SHLMPRL Results
Interpretation of meningococcal testing 18
Interpretation of pneumococcal testing 20
Interpretation of H. influenzae testing 20
Interpretation of Legionella testing 21
Interpretation of invasive G.A.S testing 22
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
Controlled document Page 4 of 22
Section One:
The Scottish Haemophilus, Legionella, Meningococcus and Pneumococcus
Reference Laboratory.
INTRODUCTION
The laboratory was founded in 2009 as a result of a merger between the Scottish Legionella
Reference Laboratory and the Scottish Meningococcus and Pneumococcus Reference
Laboratory and is part of the Diagnostics Directorate of the Acute Division of NHS Greater
Glasgow & Clyde. It is funded via the commissioning agent Health Protection Scotland
(HPS).
All tests are provided without charge to submitting diagnostic laboratories in Scotland. The
laboratory is able to provide information relating to the diagnosis and monitoring of infections
caused by Haemophilus influenzae, Legionella, meningococci and pneumococci. General
advice and the interpretation of results can be sought through the Director in the first instance,
by telephone or in writing to the above address.
If you have any problems in relation to the SHLMPRL service then minor problems can be
resolved by telephone. If there are any major problems then you should write to the above
address. If you have a complaint please telephone or write to the Director who will
investigate. Any complaint will result in a written acknowledgement within 48 hours.
LABORATORY HOURS:
Monday to Friday: 8:45 am to 5 pm
Saturday morning: Specimen reception only.
Public holidays: Specimen reception only.
Emergency situations: As required after discussion with Director or Medical Director.
Availability of advice: Normally 8:45 am to 5pm, Monday to Friday
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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MANUAL
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CONTACT DETAILS
Scottish Haemophilus, Legionella, Meningococcus and Pneumococcus Reference Laboratory
(SHLMPRL)
Level 5, New Lister Building
10-16 Alexandra Parade
Glasgow Royal Infirmary
GLASGOW
G31 2ER
Telephone:- 0141 201-8659
Fax:- 0141 201-8729
Director: Dr Brian Jones 0141 201 8567
Deputy Director: Prof. Andrew Smith 0141 201 8536
Section Manager: Mr Alistair Brown 0141 201 8658
Clinical Scientist: Dr Diane Lindsay 0141 201 8657
Meningococcal/Pneumococcal Mrs Barbara C Denham 0141 201 8732
Surveillance Co-ordinator: [email protected]
Enquires: Contact Mr A Brown on 0141 201 8658 in the first instance, but in the event of
difficulty any of the contacts on this page.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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Section Two: Services provided by the SHLMPRL
Specimen acceptance & rejection criteria
Please complete a SHLMPRL request form. Sample and request form information must be
compatible. The minimum information that should be provided is as follows:
ESSENTIAL DESIRABLE
Sample Patient’s Full Name *
Date of Birth and/ or
Hospital Unit Number
CHI number, etc.
Date and Time
Destination of Report
Request Form Patients’ Full Name*
Date of Birth and/or
Hospital Unit Number,
CHI number, etc
Name of requesting
microbiologist.
* or Proper Coded Identifier
Clinical Information
Date and Time of sample
Collection
Patients Address
Referring microbiologist’s
Contact Number
main symptoms
diagnosis
date of onset
vaccination history
investigation (s) required
These details are essential for samples processing, interpretation of test results and for
enhanced epidemiological surveillance. On the request form please also indicate where
reports should be sent.
Improperly Labelled specimens/ Request Forms
Sample or request forms received without the minimum essential identification will be referred
back to the requesting laboratory.
Samples and forms that are mismatched
The requesting microbiologist (or appropriate laboratory staff) will be informed that the form
& sample did not match and had been discarded.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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Samples that arrive with no form
If a sample arrives with no form, a blank request form will be filled out with the details taken
from the specimen, booked in and stored for future testing.
Forms that arrive with no sample
The form will be booked in & a report issued stating that no sample was received.
Samples that are inappropriately labelled
Samples that arrive with no details on them may still be processed, however the report will
state “No name on specimen but received in the same bag as request form”.
Under the direction of a senior member of staff further action might be:
Processing the specimen and withholding results
Storage of the specimen
Requesting a fresh specimen and request form.
Damaged/ Leaking Samples
The action taken will often depend on the preciousness of the sample. Some may be difficult
to repeat and it may be necessary to try to save and use what has been received.
Transportation of specimens
All submissions should be sent by first class mail and must comply with UN3373 postal regulations.
Alternatively use the DX courier service: -
DX Number – 6490200, Exchange – Bishopbriggs 90G.
Isolates of N. meningitidis, S. pneumoniae or H. influenzae for characterisation should be
transported to the SHLMPRL on blood/heated agar slopes or transport swabs. Isolates of
Legionella spp. should be sent on BCYE agar plates or slopes. Approximately 2ml of serum,
whole blood, urine or respiratory secretions, not less than 100l of CSF or 10ml of other body
fluid (e.g. pleural fluid, synovial fluid) are required for other investigations.
Inappropriate use of services
The SHLMPRL has undergone significant rationalisation over the past few years in order to
provide a more effective service. This rationalisation includes the rejection of inappropriate
sample requests, such as the following:
specimen duplication
sample not suitable for requested test
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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MANUAL
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request not justified by information supplied
test no longer carried out routinely
A report indicating the reasons for rejection will be issued for every inappropriate request.
Enhanced surveillance
Enhanced surveillance of meningococcal disease (MIDAS)
The laboratory confirmation of meningococcal disease is very important as it provides
valuable information for treatment, short-term management of outbreaks, long-term
epidemiological data and vaccine-related information. MIDAS officially began in November
1999 to fulfil these requirements and centralise the information gained from the various
sources. It is therefore imperative that the correct samples are taken from the patient and that
the correct laboratory tests are requested. Guidelines for the investigation of suspected
meningococcal disease have been published.
Enhanced surveillance of pneumococcal disease (SPIDER)
A number of pneumococcal conjugate vaccines are currently being evaluated, two of which
have been licensed and are available in the UK (PPV23 and PCV7). It is therefore important
to determine certain data on pneumococcal infection so that effective public health decisions
may be made. Such data include the incidence of pneumococcal disease in Scotland, the most
common infecting serotypes, and the occurrence of antibiotic resistance. SPIDER also began
in November 1999 and has improved to the extent that over 95% of pneumococci routinely
reported to the HPS are now also sent to the SHLMPRL for serotyping. We therefore request
your continuing co-operation in sending all pneumococcal isolates from sterile sites for typing
and antibiotic MIC determination. All invasive samples from children five years and under
are routinely faxed to the relevant Health Board and a surveillance form is completed by
return. Details are then included in the SPIDER data set.
Enhanced surveillance of Haemophilus influenzae disease
Conjugate vaccines against H. influenzae type b have been available since 1992. There is
therefore a possibility of a decrease in protection in those immunised early on in the campaign
and the potential for a shift in serotype distribution. It is therefore important to determine the
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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serotype of H. influenzae strains causing invasive disease in Scotland. Such data helps inform
future vaccine policy and we therefore request your continuing co-operation in sending all
H.influenzae isolates from sterile sites for typing. The SHLMPRL will also forward paediatric
isolates to the HPA Haemophilus Reference Unit, Colindale.
Enhanced surveillance of travel associated Legionnaires’ Disease
This website is managed by the co-ordinating centre in London but is in the process of being
transferred to ECDC in Stockholm. The information provided on it has been prepared by
members of EWGLI and the European Surveillance Scheme for Travel Associated
Legionnaires’ Disease (EWGLINET). In the event that hotels or other accommodation sites
are named on this website, it is with the agreement and support of the European countries that
they are following the procedures outlined in the European Guidelines for Control and
Prevention of Travel Associated Legionnaires' Disease. SHLMPRL is a collaborator in
association with HPS to alert EWGLInet of any travel associated cases.
Neisseria meningitidis referrals
Clinical isolates of Neisseria meningitidis
Biochemical confirmation, serogrouping, genogrouping, genotyping and multi-locus sequence
typing (MLST) are performed. Antimicrobial susceptibility (MIC) against penicillin,
rifampicin, cefotaxime, ciprofloxacin, chloramphenicol and sulphamethoxazole are also
tested. Genotyping (porA) and MLST are now performed by DNA sequencing. Genotyping
involves the sequencing of three variable regions (VRs) within the porA gene. The three VRs
can, to some extent, be back-related to the traditional phenotypic method although DNA
sequencing provides greater discrimination than that possible by traditional phenotyping
which utilises monoclonal antibodies that recognise specific serosubtypes. MLST involves the
sequencing of seven housekeeping genes to provide a characteristic allelic profile. Both
genotyping and MLST results are digital and transportable between laboratories worldwide.
The routine introduction and reporting of these methods enables strains of meningococci to be
better characterised for both immediate public health management purposes and longer term
epidemiological surveillance. Serotyping of meningococci is no longer performed because it
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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does not provide discrimination beyond that provided by porA and MLST sequencing. Non-
invasive meningococci should also be sent for confirmation, serogrouping, MIC
determination, genotyping and MLST.
Figure 1 Enhancement of meningococcal characterisation by DNA sequencing
Traditional phenotyping N. meningitidis C, 2a, P1.2,5
New genotyping N. meningitidis C, subtype 5-2, 2-1, 36-2: ST11
serogroup porA VR1 porA VR2 porA VR3 MLST
Detection of meningococcal DNA
A PCR assay which detects N. meningitidis, S. pneumoniae and H. influenzae is performed
using CSF, serum or whole blood (EDTA or other un-clotted sample). Bacterial DNA is
extracted from the CSF or blood sample upon receipt at the SHLMPRL to greatly reduce the
presence of PCR inhibitory factors and concentrate the DNA present in the sample. For
meningococci, the ctrA gene is used for detection. When positive, further PCR assays can be
performed which may provide a serogroup (siaD gene), subtype (porA gene) and MLST
profile. Such information is equal to that gained by culture characterisation. It is therefore
very important that whole blood samples are sent to the SHLMPRL for PCR testing as such
testing can provide diagnostic and additional typing information in the absence of a culture
isolate.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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Streptococcus pneumoniae referrals
Clinical isolates of Streptococcus pneumoniae
Serotyping, MLST and antimicrobial susceptibility (MIC) testing against penicillin,
erythromycin, cefotaxime, ciprofloxacin and moxifloxacin are performed. Non-invasive
pneumococci may also be sent for confirmation or MIC determination although these are not
processed for MLST unless clinically relevant or after discussion with the Director. The
SHLMPRL can, but does not routinely confirm the identity of other streptococci.
Detection of pneumococcal DNA
As mentioned previously, the SHLMPRL provides a PCR assay which detects N.meningitidis,
S. pneumoniae and H. influenzae. This follows the same requirements/parameters as stated
previously. For pneumococci, the lytA gene is used as a genetic target for detection. In
addition the SHLMPRL also has a genotypic assay for determining the common capsular
types by non-culture.
Detection of pneumococcal antigen
The multiplex PCR detects meningococcal, pneumococcal and H. influenzae. Antigen testing
on urine and sputum should be performed by the diagnostic laboratory although the
SHLMPRL is able to confirm positive results from tests performed elsewhere. Such requests
must be made clear on the request form.
Haemophilus influenzae referrals
Clinical isolates of Haemophilus influenzae
SHLMPRL offer identity confirmation, serotyping, genotyping and MLST of invasive isolates
of H. influenzae. Antibiotic susceptibility testing is not performed.
Detection of H. influenzae DNA
As mentioned previously, the SHLMPRL provides a PCR assay which detects N.
meningitidis, S. pneumoniae and H. influenzae. This follows the same
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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requirements/parameters as stated previously. For H. influenzae, the bexA and/or the hel gene
may be used for detection.
Requests for Haemophilus influenzae type b antibodies should be directed to Moira Thomas,
Immunology department, Southern General hospital, Glasgow.
Legionella referrals
Clinical and environmental isolates of Legionella spp.
The laboratory welcomes submission of all suspected Legionella isolates from clinical and
environmental sources for confirmation and surveillance purposes.
Currently, there are over 50 named species of legionellae comprising more than 60
serogroups. The most commonly encountered species, Legionella pneumophila, comprises 16
serogroups.
Confirmation of species, serogroup and monoclonal subtype where appropriate is performed
by latex agglutination and immunofluorescent antibody typing (IFA) using polyclonal and
monoclonal antibodies. Molecular techniques available include gel based AFLP and digital
Sequenced Based Typing (SBT).
Detection of Legionella Urinary Antigen
This test detects the presence of Legionella pneumophila antigen in the acute phase of
Legionnaires’ disease. Urinary antigen is a diagnostic service not routinely provided by
SHLMPRL, but we will undertake confirmation of any positive results from the diagnostic
service. This is a useful test as usually antigen excretion is detectable before sero-conversion
takes place. Specimens should be collected as soon as possible after onset of symptoms.
Excretion of antigen usually continues for at least 2 weeks after onset. It should be noted that
a negative result does not exclude infection with a Legionella sp. other than L pneumophila
serogroup 1. We recommend the sending of a convalescent serum sample. For the diagnosis
of Legionnaires’ disease, the laboratory uses an EIA test to detect a heat stable antigen
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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specific to L. pneumophila Sg1 (Bartels) which gives a result in just over an hour. An
Immunochromatographic test (ICT) is also available for rapid results (15 – 45 minutes).
Detection of Legionella antibodies
The estimation of antibodies directed against all human associated Legionella species is
available. Antibody levels are determined using the indirect immunofluorescent antibody test
(IFAT). The IFA test will pick up disease caused by many species not detected in the urinary
antigen test, therefore it is important to submit serum samples in addition to urine where a
diagnosis of Legionnaires’ disease is suspected. Where possible, paired sera should be
submitted, separated by at least 5 days. If the most likely diagnosis is Legionnaires’ disease,
an acute serum sample should be sent without delay as titres suggestive of infection can often
be seen soon after onset.
Detection of legionellae in Clinical Material
A PCR assay targeting the 16sRNA gene found in all Legionellae is available.
The most commonly referred samples are sputum and bronchoalveolar lavage (BAL) although
the laboratory will accept any appropriate clinical samples for examination.
All samples submitted will be cultured to attempt isolation of Legionella spp.
Environmental Services
While the laboratory is not funded to provide an environmental legionellae isolation service,
this service can be provided by arrangement and a charge will be made (unless in association
with a possible outbreak investigation or from cases with possible links to domestic
dwellings).
The methods employed include both conventional culture methods and a PCR technique for
the detection of Legionellae in water.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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Invasive Group A Strep referrals
The principal purpose of this service is to support public health by enhancing the
epidemiological surveillance and understanding of Group A streptococcal strains, causing
invasive disease, circulating in Scotland. The laboratory will perform Lancefield grouping,
emm (M) typing and in some cases MLST on submitted isolates.
Typing of Group A Streptococci (and sometimes other streptococcal groups) will also be
provided when required to support outbreak investigations in acute and maternity healthcare
settings as agreed with NHS board infection control teams.
Submission criteria for further typing
All group A streptococcal isolates from normally sterile body sites (blood, CSF, joint
aspirates, pericardial/peritoneal/pleural fluids, bones, endometrium, deep tissue or abscess at
operation or post mortem)
Group A streptococcal isolates from a normally non-sterile site in combination with severe
clinical presentation, such as streptococcal toxic shock syndrome (STSS) or necrotising
fasciitis
On identification of a suspected or confirmed GAS outbreak in acute health care or maternity
setting, the reference laboratory should be informed and isolates sent for further typing.
Further guidance during Group A streptococcal suspected/confirmed outbreaks:
An outbreak should be considered if there are two or more cases of suspected GAS infection
related to person or place. These cases will usually be within a month of each other
Include hospital in patients, patients recently discharged (<7 days) and women who gave birth
in any other setting including at home
Clearly labelled isolates should be sent to the lab on an SHLMPRL request form
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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For all isolates submitted to the laboratory, an enhanced surveillance form for iGAS
(available from [email protected] ) must be completed by the sending lab and
submitted to HPS (e-mail to [email protected] )
Request Turnaround Times
Specimen Test Reporting Time1
Clinical isolates
N. meningitidis geno/serogrouping 2 working days 2
antimicrobial susceptibility tests 4 working days
genotyping and MLST 3 10 working days
S. pneumoniae serotyping 2 working days
antimicrobial susceptibility tests 4 working days
MLST 3 10 working days
H. influenzae identification and serotyping 3 working days
MLST 3 10 working days
Legionella sp. identification and serotyping 2 -12 working days
Invasive GpA Strep emm typing 10 working days
Polymerase chain reaction (PCR)
CSF, whole blood PCR for meningococcal, 3 working days
and serum pneumococcal and H. influenzae DNA
Serogrouping/sero-subtyping and MLST 10 working days
on meningococcal PCR positive samples
Respiratory secretions Culture and PCR for Legionella 16sRNA 2 – 7 working days
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
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Serology
Serum Legionella antibodies 1 – 2 working days
Antigen detection
Urine Legionella urinary antigen 1 -2 working days
1 Reporting times assume that the correct sample and pure cultures are received.
2 All N. meningitidis culture serogrouping results are telephoned within one working day.
3 MLST is multi-locus sequence typing.
Reporting of results
All results of diagnostic importance or epidemiological value are telephoned directly to the
Consultant Microbiologist and/or senior BMS staff at the submitting laboratory. Copies of the
results are also e-mailed to the appropriate Consultant in Public Health Medicine. We
routinely inform CPHMs and HPS of results that may have public health significance but
users should be aware that formal notification of meningococcal disease is the responsibility
of the clinician making the clinical diagnosis.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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MANUAL
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Other services
Specimen Referral
The SHLMPRL works closely with other Reference Laboratories on disease trends and the
development of new diagnostic tests.
Epidemiological Data
The SHLMPRL supplies enhanced surveillance data for meningococcal, pneumococcal, H.
influenzae and invasive G.A.S. disease to Health Protection Scotland. Annual published
reports of all Reference Lab organisms are available on the HPS website.
Service development
The SHLMPRL continually endeavours to improve its service. The laboratory assesses new
techniques or tests as they become available and works to offer them as a service to users as
they prove useful. Users are invited to discuss any potential developments in the service with
the Director.
Teaching and research
We can provide research facilities at undergraduate, postgraduate and postdoctoral levels. The
SHLMPRL has national and international collaborations with other reference laboratories and
universities. Invitations for collaborations for research or other purposes are welcome.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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Section Three: Interpretation of SHLMPRL Results
This guidance is provided to assist in the interpretation of SHLMPRL results regarding the
laboratory confirmation of Legionella, meningococcal, pneumococcal, H. influenzae and
invasive G.A.S. disease. Although all assays have been evaluated and validated, a small
number of cases will require individual interpretation. Clinical information should be taken
into account where necessary when interpreting SHLMPRL results.
Interpretation of meningococcal tests
Detection of meningococcal DNA
PCR is a highly sensitive technique that now has many applications. It has played an
important part in the non-culture diagnosis of meningococcal disease for many years. The
SHLMPRL uses a PCR assay which detects meningococcal, pneumococcal and H. influenzae
DNA. Overall, the test has shown a higher sensitivity compared to culture-confirmed cases.
The test is batched but usually run every working day according to demand. It should be
noted that the PCR test may be negative even when blood cultures are positive for
N.meningitidis. Importantly, a negative PCR results does not exclude meningococcal disease.
Genogrouping, genotyping and MLST assays may be performed on samples that are positive
by PCR. These assays require more DNA than the initial detection PCR because the sample is
processed for DNA sequencing after amplification. Therefore, not all samples that are positive
by the initial PCR will yield enough DNA for further testing. However, for those that do, the
information provided is equivalent to that produced when characterising a meningococcal
culture isolate.
Meningococcal isolate typing
Meningococci are serogrouped on the basis of their capsule. There are 13 serogroups of
meningococci and the SHLMPRL is able to characterise eight, namely A, B, C, Y, W
(formerly known as W135), X, Z and E (formerly known as 29E). In addition a number of
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
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MANUAL
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meningococci may not possess a capsule (cnl gene), as a consequence the SHLMPRL has a
PCR assay to detect the absence of a capsule. Serogroup B is responsible for most invasive
meningococcal disease in the UK whilst serogroups Y and W are often associated with
pneumonia, joint infections and non-capsulated are often associated with carriage. However,
it should be noted that serogroup Y accounts for around a third of invasive infection in the
United States and therefore imported infections may not be due to the usual B serogroup.
Individuals who have returned from the sub-Saharan epidemic belt may be infected with
serogroup A or W and serogroup W was implicated during previous Hajj pilgrimages. While
group C disease has been largely eliminated in Men C immunised cohorts, occasional cases
are still seen. Meningococci are further characterised using the outer membrane protein, porA,
and by multi-locus sequence typing (MLST). With regards the porA gene, the SHLMPRL
performs DNA sequencing of three variable regions known as VR1, VR2 and VR3. Such
analysis provides more data than that provided by traditional serological methods. Whereas
traditional serotyping and serosubtyping was limited to a few serotypes and serosubtypes,
DNA sequencing of the three VRs provides an unlimited number of possible subtypes. For
example, a subtype previously designated P1.4 may now be 7-2, 4, 36. For MLST, the seven
alleles that are DNA sequenced provide a sequence type (ST) which is then reported. For
example, a typical allelic profile result would be 7, 5,1,13,36,53,15 which would be reported
as ST213.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
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Interpretation of pneumococcal tests
Pneumococcal isolate typing
Serotyping of pneumococcal isolates is achieved by co-agglutination. The SHLMPRL now
also performs MLST on pneumococci and this is reported as for meningococci as a ST type.
Pneumococcal DNA
S. pneumoniae DNA can now be detected using a PCR assay which detects meningococcal,
pneumococcal and H. influenzae DNA. Overall, the test has shown a higher sensitivity
compared to culture-confirmed cases. Again, it should be noted that the PCR test may be
negative when blood cultures or urinary antigen are positive for S. pneumoniae due to the
different sensitivities of each method. Importantly, a negative PCR result does not exclude
pneumococcal disease.
Interpretation of H. influenzae tests
H. influenzae isolate typing
Serotyping of H. influenzae isolates is achieved by co-agglutination for serotypes a, b, c d, e
and f. This can then be confirmed by a genotyping PCR. MLST is also performed and is
reported as for meningococci and pneumococci as an ST type.
H. influenzae DNA
H. influenzae DNA can now be detected using a PCR assay which detects meningococcal,
pneumococcal and H. influenzae DNA. Overall, the test has shown a higher sensitivity
compared to culture-confirmed cases.
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
Controlled document Page 21 of 22
Interpretation of Legionella tests
IFAT Serology
A four-fold rise in titre to L. pneumophila Sg 1 is classed as a definitive case of LD. A four-
fold rise in titre to other serogroups or species and a single high titre to all Legionellae is
classed as presumptive. An initial antibody peak attributed to IgM is seen 6 to 20 days post
disease onset. The antibody response in general then falls and increases again after 25 days
post onset as a result of an increase in the IgG response.
Urinary antigen
Urinary antigen is normally detected in the acute phase of disease around 5-10 days post
onset. A positive urinary antigen is definitive of a case. The current urinary antigen tests are
sensitive and specific for L. pneumophila Sg 1 and therefore may not detect infection caused
by another serogroup or species. A negative urinary antigen is not definitive and should be
followed up by serology or culture where available. Urinary antigen detection is dependent on
availability, sample timing and antibiotic therapy.
Legionella isolate typing
Culture is definitive for the diagnosis of LD. SHLMPRL can type all Legionella species using
a variety of polyclonal, monoclonal and genotyping techniques. We can further subtype by
Sequence based typing (SBT) to provide a Sequence type of the isolate. AFLP is used to
identify similarities in non L. pneumophila cases.
Legionella DNA
A routine 16s RNA PCR is performed on all respiratory secretions and has been found to be
more sensitive and quicker than culture. It identifies both Legionella species and
L. pneumophila by using a probe hybridization step. If the sample does not yield a culture and
is PCR positive the sample can be directly sequence based typed.
Environmental samples
Water and compost samples can be tested and are reported as cfu/L or cfu/g. If a water sample
contains greater than 1000 cfu/L then action must be taken to reduce and control the growth
Scottish Microbiology Reference Laboratories SHLMPRL_UM_2014
NHS Greater Glasgow and Clyde Authorised by: Dr. B. Jones
Issued: 18/08/14 Author: A. Brown
SHLMPRL USER
MANUAL
Controlled document Page 22 of 22
of the organism in the associated water system. If the samples contain Legionella but in
quantities less than 1000cfu/L, systems should be monitored and insurances sought that the
system is well maintained.
Invasive G.A.S emm typing
M/emm typing of GAS isolates can aid in the classification of sporadic, linked and outbreak
strains. However there are two very common M/emm types (1 and 89) and emm typing results
should be viewed in conjunction with epidemiological and clinical data to determine whether
cases are linked or part of an outbreak. The isolates can be further typed by MLST but
common M types tend to have the same ST therefore the advantage of identifying further
differences by MLST is limited and only performed on a select number of isolates.