Scottish Bacterial Sexually Transmitted Infections Reference Laboratory

Scottish Bacterial Sexually
Transmitted Infections
Reference Laboratory
(SBSTIRL)
User Manual
K Eastick March 2012
Review date: March 2014
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Purpose
The Scottish Bacterial Sexually Transmitted Infections Reference Laboratory
(SBSTIRL) is commissioned by Health Protection Scotland (HPS) to provide a
national reference service for the management of bacterial sexually
transmitted infections throughout Scotland.
Scope of service
The following services are provided for referred isolates of presumptive Neisseria
gonorrhoeae:
• Identification of ‘problem’ isolates
• Antibiotic resistance surveillance
• Molecular typing of gonococci by Neisseria gonorrhoeae multi-antigen
sequence typing (NG-MAST) and, where appropriate, by other methods
• Characterisation of medico-legal isolates
• Identification of N. gonorrhoeae by nucleic acid amplification test (NAAT) for
non-viable isolates.
The following tests are performed on clinical specimens:
• Supplemental NAAT for N. gonorrhoeae (GC NAAT)
• Real-time PCR for T. pallidum on exudates from ulcers that are suspected
to be syphilitic
• Real-time PCR for Lymphogranuloma venereum (LGV) on Chlamydiapositive samples from cases of suspected LGV.
The SBSTIRL also provides these general services:
• Clinical and microbiological advice
• Assessment of new methodologies
• Participation in national/international surveillance and quality assurance
projects
• Provision of six-monthly reports to users
• Education, training, audit and collaborative research
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Contact details
Name and Designation
Director
Dr Kirstine Eastick
Deputy Director and Medical
Microbiology Adviser
Dr A Paddy Gibb
Telephone numbers and Email
address
0131 242 6032
[email protected]
[email protected]
0131 242 6070
[email protected]
Specialist Biomedical Scientists
Mr John Anderson
Mr Brian Malloy
Ms Alison Fleming
0131 242 6078
Administrator
Mrs Janis Shade
0131 242 6081
[email protected]
Departmental confidential fax
0131 242 6008
The service is managed overall by NHS Lothian. If we are unable to deal with
an issue and you
wish to discuss a matter with NHS Lothian Management then contact:
Mr Mike Gray
Tel: 0131 242 6076
[email protected]
Laboratory Service Manager, NHS Lothian
Royal Mail Postal Address
Scottish Bacterial Sexually Transmitted Infections Reference Laboratory
(SBSTIRL)
Microbiology Department
Edinburgh Royal Infirmary
51 Little France Crescent
Edinburgh
EH16 4SA
Hays DX Address
DX 6231202
Edinburgh 96EH
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Hours of work
Core laboratory hours are 9am – 5pm Monday to Friday. There is no service
on Public Holidays or weekends.
Submission of samples
Please complete the relevant SBSTIRL request form (see end of document).
Printed request forms can be provided by SBSTIRL upon request. There are
four separate request forms for submission of N. gonorrhoeae cultures,
specimens for supplemental N. gonorrhoeae NAAT, T. pallidum and LGV real
time PCRs.
All specimens must be labelled with the following information:
Unique Identifier (Surname & Forname/CHI number/NaSH number)
Date of birth
Sender’s Laboratory Number
All specimens must be accompanied by the appropriate request form
with the following information:
Patient’s unique Identifier (Surname & Forname/CHI number/NaSH number)
Patient’s date of birth and gender
Specimen type/site
Sender’s Laboratory Number
Consultant to whom a report should be addressed
Location of Sender
Any relevant clinical information
The specimen types that are appropriate for each reference test are outlined
in the table below
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Table of Specimen types appropriate for each reference test
Test
N. gonorrhoeae
culture,
identification,
sensitivities and
typing
Specimen type
‘Chocolate agar’ slope of the culture after a
minimum of 6 hours incubation at 35-37ºC, 5%
CO2.
or
Overnight growth of organism transferred to an
Amies charcoal agar transport swab
Supplemental GC
Any positive or equivocal GC NAAT specimen
NAAT
in any media as recommended by the NAAT
manufacturer
N. gonorrhoeae
Any NAAT specimen which is GC positive by
typing directly from a two different NAATs or two different targets
clinical specimen
within the same NAAT
T. pallidum PCR
Any swab from ano-genital or oral
ulcers/lesions that are suspected to be
syphilitic, in a liquid transport medium or dry.
Lymphogranuloma
venereum (LGV)
PCR
Any NAAT specimen that is chlamydia positive
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Comment
Please ensure this arrives Monday-Friday.
Viability in transit cannot be guaranteed beyond 2
days.
Please note that there is no delivery service on
Public Holidays.
Please indicate on supplemental GC NAAT
request form which test has already been
performed and the result.
A supplemental GC NAAT is not required but the
specimen is very useful for sequence typing (and
prediction of antibiotic susceptibility) if no cultured
isolate is available.
Use the supplemental GC NAAT request form.
Use a T. pallidum PCR request form. Tests are
charged to the requesting laboratory at £25 per
specimen.
Use an LGV PCR request form.
Suitable specimens are those from symptomatic
patients, contacts of LGV and chlamydia-positive
rectal swabs from HIV-positive patients.
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Tests performed
Test
Methodology
Frequency
Turnaround
time
N.gonorrhoeae
culture
identification
Oxidase test
Gram stain
Rapid Carbohydrate Utilisation Test (including βlactamase test); API NH test when necessary
Nitrocefin test
Phadebact
Minimum inhibitory concentration for 7 antibiotics
Daily
4 days
Twice weekly
7 days
Weekly
11 days
Daily
3 days
Monthly
12 weeks
Weekly
Weekly
7 days
7 days
N. gonorrhoeae
antibiotic
susceptibility
Typing
NG-MAST (Neisseria gonorrhoeae multi-antigen
sequence typing) on all episodes of infection
Opa typing (when required)
Supplemental GC Assays targeting PorA (in-house realtime PCR) and 16s
NAAT
RNA (Aptima GC, Genprobe)
N. gonorrhoeae
NG-MAST using nested PCR to achieve sensitivity
typing directly on
clinical
specimens
T. pallidum PCR
Real time PCR detecting PolA gene
Real time PCR detecting pmpH gene
LGV PCR
Turnaround time excludes weekends and public holidays
Further description of these methodologies is given below:
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Methods used at SBSTIRL
N. gonorrhoeae
1.
Identification
The identity of all referred cultures of N. gonorrhoeae is confirmed using the
following tests:
Oxidase test: Positively identifies organisms that constitutively produce
cytochrome oxidase
Gram stain: Gram negative diplococci are identified for further testing
Rapid Carbohydrate Utilisation Test (RCUT): Utilisation of glucose and not
maltose, sucrose, lactose or fructose distinguishes N. gonorrhoeae from other
oxidase-positive Gram-negative diplococci. A test for β-lactamase is included
in this assay, which identifies penicillinase producing Neisseria gonorrhoeae
(PPNG).
Phadebact GC monoclonal test: A species-specific co-agglutination reaction
between monoclonal antibody reagents with the major porin protein positively
identifies N. gonorrhoeae and subdivides strains into two serogroups WI or
WII/III types.
API NH test: A panel of biochemical tests; this is performed only if RCUT or
Phadebact results are inconclusive.
2.
Antibiotic sensitivity
All positively identified N. gonorrhoeae isolates are tested by the agar plate
dilution method for their sensitivity to the following seven antibiotics:
Azithromycin (0.015-2mg/L)
Penicillin (0.015-2mg/L)
Cefixime (0.015-0.12mg/L)
Spectinomycin (16-128mg/L)
Ceftriaxone (0.015- 0.12mg/L)
Tetracycline (0.25-4mg/L)
Ciprofloxacin (0.004-1mg/L)
E-tests are used for further analysis of isolates with MICs that exceed the
concentration range covered by the agar plate dilution and for those that give
an MIC at a resistance category threshold.
3.
Sequence typing
Molecular typing is performed using the Neisseria gonorrhoeae multiantigen
sequence typing (NG-MAST) method (Martin et al. JID 2004:189;1497-1505).
Sequence typing is performed on all episodes of gonorrhoea. A patient with
multiple sites of infection has typing performed on genital, oral and rectal
specimens. NG-MAST is also performed on clinical specimens that are N.
gonorrhoeae positive by NAAT if no cultured isolate from the patient is
available (where sufficient DNA is present).
NG-MAST is a highly discriminatory typing scheme that differentiates between
isolates on the basis of the sequence variation observed in two genes coding
for the por protein and transferrin binding protein b. Each sequence type (ST)
is unique and differs from any other by at least one nucleotide. Clusters of
isolates sharing the same ST can be observed over time and both phenotypic
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and demographic data support the notion that the clusters are valid,
containing related isolates.
4.
Supplemental NAAT testing for N. gonorrhoeae
A second test that targets a different specific gene is required to ensure a true
positive GC result. We provide alternative assays to ensure that what ever
the first GC NAAT used at your laboratory, there will be a suitable second test
available for supplemental testing. Assays currently in use are an an in-house
PCR which targets the PorA pseudogene and the Aptima GC test (GenProbe), which targets N. gonorrhoeae 16s RNA.
5.
Additional tests
NAAT for non-viable cultures
Material from any referred isolate that fails to grow is tested for the presence
of N. gonorrhoeae DNA. Currently the Gen-probe Aptima GC test is used.
Opa typing (O’Rourke et al Mol Microbiol 1995:17;865-75).
This is a restriction fragment length polymorphism (RFLP) method that may
be used to supplement the discrimination achieved by NG-MAST typing on
selected groups of isolates where necessary.
Tests for other Sexually Transmitted bacterial
infections
T. pallidum PCR
A real-time PCR to detect a region of the DNA polymerase A gene is
performed (Koek et al Clin Micro Infect 2006 12:1233-6) and uses an ABI
7500 platform. This test is not funded under our Service Level Agreement with
Health Protection Scotland, and requesters will be charged £25 per specimen
tested.
LGV PCR
The presence of C. trachomatis DNA is first confirmed by a nucleic acid
amplification test.
A real-time PCR to detect the polymorphic membrane protein H gene is
performed (Morré et al. Emerge Infect Dis 2005:11;1311-2.) and uses an ABI
7500 platform.
Referred tests
Occasionally, specimens and isolates may be referred to the Sexually
Transmitted Bacteria Reference Laboratory at the Health Protection Agency’s
Centre for Infections, Colindale, for further specialist testing unavailable in
Scotland. All such referrals should be made through SBSTIRL in order to be
funded by HPS.
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Additional tests
Gonococcal isolates and medicolegal specimens are stored indefinitely, and
DNA extracts from specimens for NAAT are stored for at least one year. For
retrospective testing or typing within these time-frames, please contact the
laboratory.
Reporting of Results
Written reports are issued and sent to the requesting consultant via mail.
For gonococcal cultures, a preliminary report of identification is issued
followed by a further report of antimicrobial susceptibility and sequence type.
NAAT reports are issued as soon as possible.
For NAAT-positive samples an updated report containing sequence typing
data will be issued at a later date where there are no culture isolates for an
episode of infection.
Positive T. pallidum PCR and LGV PCR results are telephoned directly to the
requesting consultants.
The laboratory is currently introducing a facility for the transmission of results
by email.
Accreditation details
SBSTIRL, as part of the Department of Laboratory Medicine, Microbiology
Laboratory, has been accredited by CPA (CPA Reference Number 2496) and
found to be in compliance with “Standards for the Medical Laboratory”
incorporating ISO 15189:2003.
External audit schemes
National External Quality Assurance (NEQAS)
SBSTIRL, as part of the Department of Laboratory Medicine, Microbiology
Laboratory participates in NEQAS.
Quality Control for Molecular Diagnostics (QCMD).
SBSTIRL participates in the QCMD scheme for N. gonorrhoeae NAATs, which
produces one distribution of 10 samples annually.
Quality assurance exchange with the Health Protection Agency (HPA)
SBSTIRL exchanges the following specimens with the Sexually Transmitted
Bacteria Reference Laboratory (STBRL) at the HPA:
Specimen
20 gonococcal isolates
Test
Antimicrobial susceptibility
5 gonococcal DNA lysates
10 ulcer specimens
10 swab specimens
NG-MAST
T.pallidum PCR
LGV PCR
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Frequency
Twice each
year
Annually
Annually
Annually
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SBSTIRL activities and dissemination of
data
The SBSTIRL works closely with colleagues in microbiological clinical and
epidemiological disciplines to provide timely and relevant data; where possible
this is both collected and disseminated in electronic formats. Staff members
make every effort to attend national and international meetings within the
bacterial STI field to present work and maintain awareness of the most up to
date findings. The reference laboratory activities are summarized in the
organogram below:
Gonococcal
Antibiotic
Surveillance in Scotland
Data published in
Participation in
HPS Weekly Report
national &
annual report (GASS)
Six-monthly reports to
users by electronic format
international STI
working groups
Collection of
Antimicrobial Resistance
testing of all isolates
Quarterly data
epidemiological data
submission to HPS
(via STISS)
Quality assurance by established EQA
or exchange programmes
Dissemination of Data
Scottish
Bacterial STIs
Reference Laboratory
Audit of
gonococcal
Presentations at
conferences & peer
reviewed publications
antibiotic
resistance levels in Scotland
Sequence typing
of all cases of
gonorrhoea
Consultation with BASHH
Scottish branch
Recommendation for change
Supplemental
NAAT testing for
gonorrhoea
Real time PCR for
T. pallidum from
ulcer material.
Real time
PCR for
LGV
in treatment guidelines
Surveillance report of
Identification of problem isolates
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Weekly summary of typing
data sent directly to GUM
clinics
positive cases to HPS
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Scottish Bacterial Sexually Transmitted Infections
Reference Laboratory (SBSTIRL)
Telephone 0131 242 6032 Fax:0131 242 6008
Hays DX: DX 6231202, Edinburgh 96EH
To:
From:
SBSTIRL
Microbiology Department
Edinburgh Royal Infirmary
51 Little France Crescent
Edinburgh EH16 4SA
REQUEST
Date Taken:
Date Posted:
Case No/Patient:
CHI No:
Category of patient:
DOB:
/
Sex:
M
Specimen/
Site
GUM
GP
Hospital
/
F
Beta-Lactamase
producer
Sender’s Lab
Reference No
For SBSTIRL
use
Urethra
Cervix
Rectum
Throat
Others
(Please state)
_______________________________________________________________
Comment:
Please supply details of clinician whom we can contact for epidemiological information
Name:
Address:
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Supplemental Neisseria gonorrhoeae NAAT Request
Scottish Bacterial Sexually Transmitted Infections Reference Laboratory
(SBSTIRL)
Microbiology Department, Edinburgh Royal Infirmary
51 Little France Crescent, Edinburgh EH16 4SA
Hays DX: DX 6231202, Edinburgh 96EH
FROM:
Microbiology Laboratory
Consultant Name
Address
Tel:
______________________________________________________________
REQUEST
Date taken:
Date posted:
Case No/Patient:
Category of patient:
GUM
DOB
GP
Hospital
Sex:
M
F
______________________________________________________________
SPECIMEN: The referred specimen should be N. gonorrhoeae positive or
equivocal by your own NAAT. Please send :
a)
your sample transferred to an aptima urine or swab collection tube or
b)
the remainder of the clinical specimen
Your Laboratory Number:…………………………………………………………….
Specimen type
The GC NAAT you used
Abbott real time CT/NG
Aptima Combo 2
BD Probetec
Roche Cobas 4800
Other (specify target)
Cervical
Vulvo-vaginal
Urine
Urethral
Rectal
Throat
Other (specify)
Your GC NAAT result
Positive
Other (specify)
Positive
Other (specify)
Positive
Other (specify)
Positive
Other (specify)
Positive
Other (specify)
Was the NAAT result reproducible? Yes
No
Not done
Sequence typing only required as GC NAAT already confirmed ?
___________________________________________________________________
For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab)
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Treponema pallidum PCR Test Request
Scottish Bacterial Sexually Transmitted Infections Reference Laboratory
Microbiology Department, Edinburgh Royal Infirmary
51 Little France Crescent, Edinburgh EH16 4SA
Hays DX: DX 6231202, Edinburgh 96EH
FROM:
Microbiology Laboratory
Consultant Name
Referring Clinic (e.g. GUM)
Consultant Name
Address
Address
Tel:
Tel:
______________________________________________________________
REQUEST
Date taken:
Date posted:
Case No/Patient:
DOB:
Sex
M
F
______________________________________________________________
SPECIMEN:
A swab of exudate from the depth of the lesion in virus transport
medium
Site of suspect primary lesion:
Genital
Anal
Oral
Other (specify)
__________________________________________________________________________
ADDITIONAL INFORMATION:
Known contact of patient with syphilis
Yes
No
Signs/Symptoms
Yes
No
If yes, please give details……………………………………………………………….
Epidemiological treatment
Yes
No
If yes, please give treatment and date….………………………………………………..
…………………………………………………………………………………………..
Dark ground microscopy
Positive
Negative
Not done
For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab)
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Lymphogranuloma Venereum (LGV) Test Request
Scottish Bacterial Sexually Transmitted Infections Reference Laboratory
Microbiology Department, Edinburgh Royal Infirmary
51 Little France Crescent, Edinburgh EH16 4SA
Hays DX: DX 6231202, Edinburgh 96EH
FROM:
Microbiology Laboratory
Consultant Name
Referring Clinic (e.g. GUM)
Consultant Name
Address
Address
Tel:
Tel:
______________________________________________________________
REQUEST
Date taken:
Date posted:
Case No/Patient:
DOB
Sex:
M
F
______________________________________________________________
SPECIMEN: The referred specimen should be chlamydia positive
Your Laboratory Number:
Specimen site
(specifiy)
Chlamydia test used
SDA
Urethral
Rectal
Taqman/Cobas
Aptima Combo
Abbott real time CT/NG
Other
Other (specify)
__________________________________________________________________________
REASON FOR REQUEST:
Symptomatic patient
Proctitis
Inguinal Lymphadenopathy
Other (specify)
LGV contact
Chlamydia positive rectal swab from HIV positive patient
For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab)
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