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 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

http://www.nhsggc.org.uk/smrl




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Controlled document 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).




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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




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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




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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

[email protected]

Deputy Director: Prof. Andrew Smith 0141 201 8536

[email protected]

Section Manager: Mr Alistair Brown 0141 201 8658

[email protected]

Clinical Scientist: Dr Diane Lindsay 0141 201 8657

[email protected]

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.

mailto:[email protected]








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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.




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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




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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




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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




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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.




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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




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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




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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.




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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




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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






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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.




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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.




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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




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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.




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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.




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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




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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.

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