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Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 1 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
CURRENT VERSION AMENDMENTS Each Policy and Procedure has an individual record of amendments. The amendments
for the current version are listed below.
Amendment
Number/ Date
Version no.
Discarded
Version
no. Issued
Section Amendment
11/11.11.19 4
4.1 Contact
details
Remove [email protected]
Services
offered
Remove Listeria serotyping
WGS Amended reference to wgs starting dates
Antibiotic
resistance
Updated
Specimen
rejection
Repeat isolates from same site rejected if within
3 months of previous isolate
New
section
Bioinformatics software used
Change Control No: MIC032/19
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 2 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
National Salmonella, Shigella & Listeria Reference
Laboratory
Users Guide
Contact details
Head of Department: Prof. M.Cormican (091) 544146
Laboratory Tel: (091) 544628
Fax: (091) 542238
e-mail: [email protected]
Address
National Salmonella, Shigella & Listeria Reference Laboratory
Department of Medical Microbiology
University Hospital Galway
Galway
Role of NSSLRL and relationships with other Agencies
Fig.1 NSSLRL Organisational Chart
Primary clinical laboratories identify Salmonella, Shigella and Listeria to species
level and determine the isolates susceptibility to those antimicrobial agents
Clinical Labs
NSSLRL
Public Health
HPSC ECDC
CDC
FSAI
Veterinary Labs Food Labs
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 3 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
immediately relevant to patient treatment. This provides immediate information to the
clinician for treating an individual patient.
Isolates are referred to the NSSLRL for confirmation and for subtyping. The NSSLRL
confirms the result of the clinical laboratory.
The NSSLRL also adds a national public health dimension to the work of the clinical
laboratories by recognition and confirmation of links between individual cases of
infection, even where outbreaks are widely dispersed. This information is primarily
to guide public health intervention to recognise and control transmission of infection.
Trends of Salmonella infections in Ireland can also be more closely followed. Typing
of isolates from food and animal sources help in tracing the sources of infection.
The NSSLRL works in tandem with numerous agencies in protecting public health.
The laboratory which identifies the pathogen and any clinician involved in the care of
the patient is obliged to notify the case to Public Health (PH). The 9 PH departments
in the country regularly review surveillance data locally to determine if incidence is
increased or if trends are occurring in age groups, areas and concerns are discussed
with the NSSLRL. PH contacts the case to try and determine the source of infection
and give advice to prevent spread of infection to others. The above actions may be
carried out by medical staff from PH with the assistance of Environmental Health
officers. Stool specimens may subsequently be submitted to clinical laboratories to
determine if contacts of the cases are also infected (outbreak) and/ or food (and
occasionally water) samples may be sent to a food and water laboratory to try to
determine a source of infection. If a relevant pathogen is isolated in those laboratories
the isolates will be sent to the NSSLRL for comparison with the isolate from the first
case or outbreak. As part of the investigation a questionnaire is administered to
investigate possible risk-factors (contact with animals, occupation, etc), travel history
and recent food history. When more than one case is detected cross-comparison of
questionnaires may allow a putative source to be identified through statistical
analysis.
The Health Protection Surveillance Centre (HPSC) looks at national trends. The two
tier surveillance ensures local trends are picked up by PH and that national trends can
be detected by HPSC. When major outbreaks occur an outbreak control team (OCT)
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 4 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
may be set up at the discretion of the Director of Public Health (regional level) or by
the Health Protection Surveillance Centre (National Level). Staff of NSSLRL are
consulted regarding the need for an OCT and the consultant microbiologist from
NSSLRL (and sometimes other staff of NSSLRL) are involved as members of the
OCT to guide the interpretation of the microbiology results. Other agencies involved
include HPSC, PH and Food Safety Authority of Ireland (FSAI).
The NSSLRL and HPSC liase with the European Centre for Disease Control (ECDC)
to help identify and control international dimensions to outbreaks. This involves
sharing data, countries issuing outbreak alerts and monthly outbreak summaries.
Services offered
The NSSLRL only performs analysis on isolates received on nutrient agar bijoux. The
NSSLRL does not test primary samples, e.g. faeces, blood or food, therefore results
are qualitative and not quantitative.
The NSSLRL types isolates from food and animal sources as well as those of human
origin. The primary role of the NSSLRL is to protect human health. Clients should be
aware that if animal or food isolates are similar to those from sporadic or outbreak-
associated human isolates this information will be shared with relevant bodies, e.g.
Food Safety Authority of Ireland (FSAI), Health Protection Surveillance Centre
(HPSC) and European Centre for Disease Control (ECDC) when it is necessary to do
so to protect public health. In general users will be informed in advance of sharing
the source laboratory information with these agencies. These bodies may then require
further information from the client laboratories.
If users wish to use data from the NSSLRL in publications they must contact the
laboratory director at [email protected] or in his absence one of the scientific
staff.
Salmonella - Analysis of Whole Genome Sequences (non-accredited)
Shigella - Analysis of Whole Genome Sequences (non-accredited)
Listeria - Analysis of Whole Genome Sequences (non-accredited)
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 5 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
Ninety-five percent of samples will be reported within 28 days and specimens that are
identified by telephone call as urgent will be prioritized.
Whole Genome Sequencing
The speed and accuracy of sequencing has increased and the cost has decreased
dramatically in recent years. It is now possible to use sequencing of bacterial
pathogens in outbreak investigations. Analysis of whole genome sequences (WGS) is
rapidly replacing conventional phenotypic. Serotyping and antimicrobial
susceptibility testing was discontinued on Salmonella and Shigella isolates received in
the NSSLRL after the 01/10/18 as analysis of WGS replaced these tests. Listeria
serotyping was discontinued on Listeria monocytogenes isolates from beginning of
2019.
Our primary interest when analysing sequences is to determine relatedness between
bacterial isolates in a timely manner.
The most widely used methods are;
1) Whole genome multi locus sequence typing (wgMLST) or a variation such as
core genome (cg)MLST.
2) Single nucleotide polymorphisms (SNP) analysis.
wgMLST
MLST was first used in 1998 to type Neisseria meningitidis and has since been used
to type a huge number of pathogens. As initially developed MLST involved extracting
DNA from bacterial isolates, performing separate PCR reactions on a number of
internal fragments (450-500 base pairs) of housekeeping genes, purifying the PCR
products and sequencing the products using Sanger sequencing. The resultant
sequences are then analysed to determine the alleles at each locus. Each time a novel
allele is detected it is assigned a new number. The numbering system is sequential so
the distance between numbers does not correlate with degree of relatedness.
Differences in allele sequences can arise from point mutations, insertions or deletions
(Indels), recombination events or a combination of the above. A unique combination
of alleles at each locus, an “allelic profile” specifies the sequence type (ST). The
MLST allele sequences and allelic profiles are stored in various curated databases
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 6 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
worldwide and these are collected by the PubMLST site and made easily accessible
[http://pubmlst.org].
Fig. 2 Diagrammatic representation of Multi Locus Sequence Typing (MLST) of
S.Enteritidis.
Fig. 3 Variations in allele sequences arising by single point mutations (strain 2, locus
A &B), insertions and/or deletions or indels (strain 3 locus A &B) and inversions
(strain 4, locus A).
Most Salmonella serotypes are “monophyletic” this means that they consist of
variants of a common ancestral sequence type, e.g. S.Enteritidis = ST11
(5,2,3,7,6,6,11 ), S.Typhimurium = ST19 (10,7,12,9,5,9,2), S.4,[5],12:i:- = ST34
(10,19,12,9,5,9,2), while other serotypes, e.g. S.Newport, have multiple lineages
(polyphyletic) and consist of numerous, often unrelated, sequence types, e.g. ST118
(16,42,39, 2,43,45,36) and ST166 (5,14,6,12,5,14,58).
‘Locus’ (gene) Strain 1 Strain 2 Strain3 Strain 4 A ACTAGAGGGAA ACTAGAGGCAA ACT-GAGGGTAA ACGGGAGATAA
allele 1 allele 2 allele 3 allele 4
B TAGCCAGGGTC TAGCAAGGGTC TAGC---GGTC TAGGCAGGGTC
allele 1 allele 2 allele 3 allele 1
C, D, E, etc…. alleles 5,2,8… alleles 1,4,7… alleles 1,3,9… alleles 6,2,9…
5
2
3
7
6
6
11
ST11
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 7 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
With the advent of WGS an isolates seven gene allele profile and sequence type can
be deduced from the genome sequence without having to do the separate PCRs. Also
instead of analysing just 7 housekeeping genes the number of genes examined can be
greatly increased to look at entire core genomes (genes present in the genomes of the
vast majority of that pathogen) cgMLST or whole genomes wgMLST incorporating
both core and accessory genomes. This obviously greatly increases the discriminatory
power of MLST from the conventional 7 gene MLST schemes.
The wgMLST schemes need constant curation for QC and assigning new allele
numbers. A major advantage is that results are easily comparable when laboratories
use the same schemes.
SNP analysis
wgMLST only takes account of coding sequences. SNP analysis takes account of
mutations throughout the genome. Short reads from isolates would always be
compared against closely related reference genomes, e.g. S.Enteritidis against a
S.Enteritidis reference, S.sonnei against a S.sonnei reference, etc. This method is
harder to standardise as results depend on the reference strain used.
Fig. 4 Dendrogram of hqSNP analysis of Shigella sonnei using BioNumerics
software.
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 8 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
Antbiotic resistance
Fig. 5 Analysis of resistance genes of Shigella isolates in BioNumerics.
Reports on isolates of Salmonella, Shigella and Listeria monocytogenes will include
sequence type (ST), predicted serotype (based on ST) and relevant resistance genes,
i.e. genes that encode resistances to the antibiotics currently tested for in the
NSSLRL, i.e. A (ampicillin), C (chloramphenicol), Su (sulphamethaxazole), T
(tetracycline), Tm (trimethoprim), Na (nalidixic acid), Cp (ciprofloxacin), Caz
(ceftazadime), Gm (gentamicin), Ctx (cefotaxime), Azt (azithromycin), Tig
(tigecycline), Mer (meropenem), Col (Colistin).
In most cases Shigella isolates that are resistant to azithromycin have both erm(B) and
mphA genes while Salmonella with either gene tend to be resistant.
Most high-level ciprofloxacin resistance in Salmonella and Shigella is accounted for
by gyrA and parC mutations. BioNumerics can detect these mutations in Salmonella
but external software, ResFinder, is used to look for these mutations in Shigella
isolates. If for clinical reasons a laboratory wishes us to check ciprofloxacin MICs
please contact us and we can perform broth microdilution on the relevant isolate.
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 9 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
Fig. 6 Minimum spanning tree (MST) of Salmonella Newport core genome MLST
from isolates from humans in the NSSLRL from 2015-18 coloured by designated
cluster.
S.Newport (6,8:e,h:2) is a polyphyletic serotype, i.e. isolates with this antigenic
structure have originated at different times so all did not evolve from a single
ancestor. The numbers on the branches indicates the number of allele differences
between isolates. Epidemiologically related isolates should have the same or closely
related cg and/or wgMLST profiles. Cluster C/17 isolates were fully susceptible to all
antimicrobials tested. Many of the cases were young adults and had a history of travel
to a tourist destination island. In contrast the isolates in cluster D/17 had resistance to
quinolones and were mainly associated with an older cohort in the East of the country.
[The code C/17 is NSSLRL designation for 3rd
such cluster identified in the year
2017]
1
13
60
67
24
28
13
2346
897
1493
107
2409
7
326
354
C/17
D/17
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 10 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
WGS data is continuously analysed for similarities between isolates and if any
evidence of outbreaks or linked cases are detected then the relevant public health
authorities will be informed.
Bioinformatics software used
Bioinformatics analysis in the NSSLRL is performed primarily with BioNumerics
software (Applied Maths) although other programs are used occasionally.
BioNumerics software 7.6.3
E.coli functional genotyping plug-in version1.2
Salmonella functional genotyping plug-in version 0.1
Listeria functional genotyping plug-in version 1.0
SeqSero
CGE software http://www.genomicepidemiology.org/
KmerFinder
ResFinder
PubMLST https://pubmlst.org/
Databases
Species identification by ribosomal MLST
EnteroBase http://enterobase.warwick.ac.uk/
Opening times
Mon – Fri 9:00am- 5:00pm (lunch from 1:00 – 2:00pm)
Out of hours work
This is performed at the discretion of the Consultant Microbiologist or the laboratory
scientific staff on urgent samples, e.g. during an outbreak investigation.
Laboratory Contact Out of Hours: (091) 544411
Specimen Rejection Policy
Specimens sent to the NSSLRL will be rejected if:
- isolates are sent on agar plates, plastic universals or large bijoux
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 11 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
- specimens contain a mixed bacterial culture
- specimen slope is broken
- specimen form and/or slope are unlabeled, mismatched or incomplete
- transportation of samples to the NSSLRL is not followed correctly, i.e.
slopes not enclosed in a crushproof container
external packaging not labeled correctly
- if for any other reason the specimen is not in a safe condition for processing
and/or can not be clearly identified.
- Duplicate isolates from patients from the same site (e.g. faeces) received within
three months of the initial isolate, even if they are from different hospitals. When
this occurs the sequencing results of the initial test will be included in the report
along with a note stating “An isolate from this patient was recently received from
another hospital. WGS was not performed on this isolate. The sequencing results
in this report are from the previous isolate”
- If a repeat isolate from the same site is received from the same hospital this will
be rejected with a note stating “An isolate from this patient was recently
sequenced. Please see report .....”.
- If isolates are received from multiple sites from the same patient WGS will be
performed on all isolates.
- Laboratories should send only one nutrient agar slope per patient site. If a
laboratory sends two slopes only one will be processed. The second slope will be
held and processed only if there is a problem with the first slope, e.g. no growth
or growth of a non-Salmonella/Shigella/Listeria isolate.
Minimum requirements for completion of NSSLRL request forms
NSSLRL request forms must be completed in legible handwriting or typing and all
details must be entered.
- Human isolates
The patient name and referring lab number and/or date of birth must be on
both the form and side of slope.
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 12 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
If there is reason to suspect the isolate could be a Salmonella Typhi or
Salmonella Paratyphi, e.g. patient from an endemic country, this must be
noted on the form.
If the isolate is suspected to be part of an outbreak this must be noted on the
form.
- Non-human isolates
The referring lab number and isolate source must be on both the form and side
of slope
Isolate source must be clearly stated, e.g. pork, chicken, beef, otherwise a
report will not be issued.
Transportation of samples
Slopes must be packaged and transported according to ADR (Carriage of Dangerous
Goods by Road) regulations.
Grow isolate overnight on a small nutrient agar slope and remove any fluid
accumulating at the bottom of the slope using a sterile Pasteur pipette.
Place slope(s) into an inner crushproof hard plastic container and place this
into a cardboard box or envelope.
Label with an emergency contact number for the sender and an Infectious
substance label.
Label box with UN3373 and add sticker with “BIOLOGICAL SUBSTANCE,
CATEGORY B”.
This can be then sent to the Reference laboratory via a courier company, e.g.
Hays DX (01-8421088), Capital Freight (01-8852064), Biomnis (1800-
252967) or Nightline couriers (091-795100), MedLab Pathology (01-2933690)
A number of slopes may be sent in each crush-proof container but each slope
must be individually wrapped in an adsorbent material to prevent breakage
during transit.
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 13 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
Retention times
Additional examinations may be requested during specimen storage time by
telephoning the NSSLRL. Agar slopes (apart from CL3 isolates) are kept for a
minimum of 6 weeks while isolates are stored at -25oC for 5 years.
Analytical failures
In the event of a specimen being unsuitable for processing or where there is an
analytical failure, the laboratory will be informed by phone or in writing.
Discussion of reports
Please contact Prof. M.Cormican at [email protected], Dr. Deirbhile Keady at
[email protected], Dr.Una NiRiain at [email protected], Dr. Eithne McCarthy
at [email protected] , Dr. Teck Wee Boo at [email protected] and Dr.
Dimitar Nashev at [email protected].
Confidentiality Policy
It is the responsibility of all staff, as defined in their contract of employment, to
ensure that all information which they have access to as part of their work is treated in
the strictest confidence and protected from unauthorised access. All staff are asked to
sign a confidentiality agreement during their laboratory induction programme.
Service Level Agreement
The request form for the NSSLRL serves as the formal ‘Service Level Agreement’
between the National Salmonella, Shigella & Listeria Reference Laboratory
[NSSLRL], Diagnostics Directorate, Galway University Hospital (GUH) and the
Service user.
Complaints
Consumer Affairs and the National Advocacy Unit, Quality and Patient Safety
Directorate have responsibility for developing and implementing best practice models
of customer care within the HSE and promotes service user involvement across the
organisation through the concept of “Your Service Your Say”.
Dept of Medical Microbiology, Division of Clinical Microbiology, Galway University Hospitals
NSSLRL Users Guide Version: 4.1 Ref: NSRLLP001
Prepared by: Niall De Lappe Issue Date: 11.11.2019 Page 14 of 14
Q-Pulse Document Control Authorised by: Martin Cormican
Note: If users have a complaint or feel that any part of the service is
unsatisfactory or could be improved on in any way they should contact the
laboratory.