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Issued by the Standards Unit, Microbiology Services Division, HPABacteriology --- Identification | ID 20 | Issue no: 2.1 | Issue date: 21.10 .11 | Page: 1 of 12

UK Standards for Microbiology Investigations Identification of Shigella s pecies

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Identification ofShigella species

Bacteriology --- Identification | ID 20 | Issue no: 2.1 | Issue date: 21.10 .11 | Page: 2 of 12UK Standards for Microbiology Investigations | Issued by the Standards Unit, Health Protection Agency

Acknowledgments

UK Standards for Microbiology Investigations (SMIs) are developed under the auspices of theHealth Protection Agency (HPA) working in partnership with the National Health Service (NHS),Public Health Wales and with the professional organisations whose logos are displayed belowand listed on the website http://www.hpa.org.uk/SMI/Partnerships. SMIs are developed,reviewed and revised by various working groups which are overseen by a steering committee(see http://www.hpa.org.uk/SMI/WorkingGroups).The contributions of many individuals in clinical, specialist and reference laboratories who haveprovided information and comments during the development of this document areacknowledged. We are grateful to the Medical Editors for editing the medical content.

For further information please contact us at:Standards UnitMicrobiology Services Division

Health Protection Agency61 Colindale AvenueLondon NW9 5EQE-mail: [email protected]

Website: http://www.hpa.org.uk/SMI UK Standards for Microbiology Investigations are produced in association with:

The Royal College of PathologistsPathology: the science behind the cure
http://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMI/WorkingGroupshttp://www.hpa.org.uk/SMI/WorkingGroupshttp://www.hpa.org.uk/SMI/WorkingGroupsmailto:[email protected]:[email protected]:[email protected]://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMImailto:[email protected]://www.hpa.org.uk/SMI/WorkingGroupshttp://www.hpa.org.uk/SMI/Partnerships

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UK Standards for Microbiology Investigations# : Status

Users of SMIsThree groups of users have been identified for whom SMIs are especially relevant:

SMIs are primarily intended as a general resource for practising professionals in the fieldoperating in the field of laboratory medicine in the UK. Specialist advice should beobtained where necessary.

SMIs provide clinicians with information about the standard of laboratory services theyshould expect for the investigation of infection in their patients and the documentsprovide information that aids the electronic ordering of appropriate tests from hospitalwards.

SMIs also provide commissioners of healthcare services with the standard ofmicrobiology investigations they should be seeking as part of the clinical and publichealth care package for their population.

Background to SMIsSMIs comprise a collection of recommended algorithms and procedures covering all stages ofthe investigative process in microbiology from the pre-analytical (clinical syndrome) stage tothe analytical (laboratory testing) and post analytical (result interpretation and reporting)stages.Syndromic algorithms are supported by more detailed documents containing advice on theinvestigation of specific diseases and infections. Guidance notes cover the clinical background,differential diagnosis, and appropriate investigation of particular clinical conditions. Qualityguidance notes describe essential laboratory methodologies which underpin quality, forexample assay validation, quality assurance, and understanding uncertainty of measurement.Standardisation of the diagnostic process through the application of SMIs helps to assure theequivalence of investigation strategies in different laboratories across the UK and is essentialfor public health interventions, surveillance, and research and development activities. SMIsalign advice on testing strategies with the UK diagnostic and public health agendas.

Involvement of Professional OrganisationsThe development of SMIs is undertaken within the HPA in partnership with the NHS, PublicHealth Wales and with professional organisations.The list of participating organisations may be found athttp://www.hpa.org.uk/SMI/Partnerships. Inclusion of an organisations logo in an SMI impliessupport for the objectives and process of preparing SMIs. Representatives of professionalorganisations are members of the steering committee and working groups which developSMIs, although the views of participants are not necessarily those of the entire organisationthey represent.SMIs are developed, reviewed and updated through a wide consultation process. The resultingdocuments reflect the majority view of contributors. SMIs are freely available to view athttp://www.hpa.org.uk/SMI as controlled documents in Adobe PDF format.

# UK Standards for Microbiology Investigations were formerly known as National Standard Methods.Microbiology is used as a generic term to include the two GMC-recognised specialties of Medical Microbiology (which includes Bacteriology,Mycology and Parasitology) and Medical Virology.
http://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMI/Partnershipshttp://www.hpa.org.uk/SMI/Partnerships

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Quality AssuranceThe process for the development of SMIs is certified to ISO 9001:2008.NHS Evidence has accredited the process used by the HPA to produce SMIs. Accreditation isvalid for three years from July 2011. The accreditation is applicable to all guidance producedsince October 2009 using the processes described in the HPAs Standard Operating Procedure

SW3026 (2009) version 6.SMIs represent a good standard of practice to which all clinical and public health microbiologylaboratories in the UK are expected to work. SMIs are well referenced and represent neitherminimum standards of practice nor the highest level of complex laboratory investigationpossible. In using SMIs, laboratories should take account of local requirements and undertakeadditional investigations where appropriate. SMIs help laboratories to meet accreditationrequirements by promoting high quality practices which are auditable. SMIs also provide areference point for method development. SMIs should be used in conjunction with other SMIs.UK microbiology laboratories that do not use SMIs should be able to demonstrate at leastequivalence in their testing methodologies.

The performance of SMIs depends on well trained staff and the quality of reagents andequipment used. Laboratories should ensure that all commercial and in-house tests have beenvalidated and shown to be fit for purpose. Laboratories should participate in external qualityassessment schemes and undertake relevant internal quality control procedures.

Whilst every care has been taken in the preparation of SMIs, the HPA, its successororganisation(s) and any supporting organisation, shall, to the greatest extent possible underany applicable law, exclude liability for all losses, costs, claims, damages or expenses arising outof or connected with the use of an SMI or any information contained therein. If alterations aremade to an SMI, it must be made clear where and by whom such changes have been made.SMIs are the copyright of the HPA which should be acknowledged where appropriate.

Microbial taxonomy is up to date at the time of full review.

Equality and Information GovernanceAn Equality Impact Assessment on SMIs is available at http://www.hpa.org.uk/SMI. The HPA is a Caldicott compliant organisation. It seeks to take every possible precaution toprevent unauthorised disclosure of patient details and to ensure that patient-related recordsare kept under secure conditions.

Suggested citation for this document:Health Protection Agency. (2011). Identification of Shigellas pecies. UK Standards forMicrobiology Investigations. ID 20 Issue 2.1. http://www.hpa.org.uk/SMI/pdf.
http://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMI/pdfhttp://www.hpa.org.uk/SMI/pdfhttp://www.hpa.org.uk/SMI/pdfhttp://www.hpa.org.uk/SMI/pdfhttp://www.hpa.org.uk/SMIhttp://www.hpa.org.uk/SMI

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ContentsACKNOWLEDGMENTS ............................................................................................................ 2

UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: STATUS ............ ............ ......... ............ ..... 3

AMENDMENT TABLE ............................................................................................................... 6

SCOPE OF DOCUMENT ........................................................................................................... 7

INTRODUCTION ..................................................................................................................... 7

TECHNICAL INFORMATION/LIMITATIONS ................................................................................... 7

1 SAFETY CONSIDERATIONS ............................................................................................ 8

2 TARGET ORGANISMS ................................................................................................... 8

3 IDENTIFICATION ......................................................................................................... 8 4 IDENTIFICATION OFSHIGELLAFLOWCHART .................................................................. 10

5 REPORTING .............................................................................................................. 11

6 REFERRALS ............................................................................................................... 11

REFERENCES ........................................................................................................................ 12

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Amendment Table

Each SMI method has an individual record of amendments. The current amendments are listedon this page. The amendment history is available from [email protected]. New or revised documents should be controlled within the laboratory in accordance with thelocal quality management system.

Amendment No/Date. 3/ 21.10 .11

Issue no. discarded. 2

Insert Issue no. 2.1

Section(s) involved. Amendment.

Whole document. Document presented in a new format.

References. Some references updated.

Amendment No/Date. 2/09.11.07

Issue no. discarded. 1.1

Insert Issue no. 2

Section(s) involved. Amendment.

Front page. Northern Ireland logo added.

Flowchart. Title changed and flowchart put into Visio format.Contents of flowchart updated.

6 Referrals. Links to reference laboratory user manuals inserted.

References. References reviewed and updated.

All. PDF links inserted to cross-reference SMI documents.
mailto:[email protected]:[email protected]:[email protected]:[email protected]

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Scope of DocumentThis SMI describes the identification ofShigella species with particular reference to isolationfrom faeces.

This SMI should be used in conjunction with other SMIs.

Introduction

TaxonomyThe genus Shigella belongs to the family Enterobacteriaceae and consists of four species;Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei. Each of the species,with the exception of S. sonnei , is subdivided by serotype.

CharacteristicsShigella species are small Gram-negative rods. They produce pink colonies on XLD medium

and colourless colonies on DCA.Shigella species are facultative anaerobes, are non-motile,oxidase-negative, urease-negative, do not decarboxylate lysine and all except S. dysenteriae type 1 are catalase-positive1. The species may be differentiated by biochemical tests andserology of their lipopolysaccharides2. The majority ofShigella species, except S. flexneri 6, and S. boydii 13 and 14, ferment sugars without gas production. S. boydii, S. flexneri and S. sonnei ,with a few exceptions, ferment mannitol; S. dysenteriae does not. S. sonnei, and S. dysenteriae type 1 are the only species that are ONPG-positive. S. boydii 13 are Ornithine positive, andsome may be ONPG positive.Shigella species are highly infective. The infective dose is particularly low withS. dysenteriae,which may require as few as 10-100 organisms to cause infection 2.

Principles of IdentificationIsolates from primary culture are identified by colonial appearance, biochemical tests andserology (agglutination with specific antisera).Plesiomonas shigelloides cross reacts with S.sonnei antisera. If confirmation of identification is required, isolates should be sent to theReference Laboratory. All identification tests should ideally be performed from non-selectiveagar.

Technical Information/Limitations

N/A

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1 Safety Considerations 3-13 Most Shigella species are in Hazard Group 2. An important exception is Shigella dysenteriaetype 1. All work onShigella dysenteriae type 1 must be performed under Containment level 3conditions.

Shigella dysenteriae type 1 causes severe and sometimes fatal disease.Laboratory acquired infections have been reported. Low numbers of Shigella species arerequired for an infective dose 2.Refer to current guidance on the safe handling of all Hazard Group 2 organisms documentedin this SMI.

Laboratory procedures that give rise to infectious aerosols must be conducted in amicrobiological safety cabinet.The above guidance should be supplemented with local COSHH and risk assessments.Compliance with postal and transport regulations is essential.

2 Target Organisms

Genus ShigellaAll species cause human infectionsShigella dysenteriae (15 serotypes)Shigella boydii (20 serotypes)Shigella flexneri (6 serotypes which can be sub-divided into sub-serotypes)Shigella sonnei (1 serotype, 2 variants - rough and smooth)

3 Identification

3.1 Microscopic AppearanceN/A

3.2 Primary Isolation MediaXLD agar incubated in air at 35-37C for 18---24 hrDCA incubated in air at 35-37C for 18-24 hr

3.3 Colonial AppearanceShigella species on XLD agar produce 1-2 mm diameter red colonies (no black centre).Colonies on DCA are colourless (S. sonnei may form pale pink colonies because of late lactosefermentation).

3.4 Test Procedures

3.4.1 AgglutinationAgglutination with shigella antiserum (not all the serotypes are contained in polyvalentantisera).

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3.4.2 Biochemical testsUrease (TP 36 -Urease Test)Shigella species do not produce ureaseOxidase (optional) (TP 26 -Oxidase Test)

Shigella species are oxidase-negativeCommercial identification kitIn house identification kit

3.5 Further IdentificationN/A

3.6 Storage and ReferralIf required, save the pure isolate on a nutrient agar slope for referral to the ReferenceLaboratory.
http://www.hpa.org.uk/SMI/pdf/Testprocedureshttp://www.hpa.org.uk/SMI/pdf/Testprocedureshttp://www.hpa.org.uk/SMI/pdf/Testprocedureshttp://www.hpa.org.uk/SMI/pdf/Testprocedureshttp://www.hpa.org.uk/SMI/pdf/Testprocedureshttp://www.hpa.org.uk/SMI/pdf/Testprocedures

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4 Identification of Shigella species Flowchart

Clinical specimenPrimary isolation plate

DCA -colourless coloniesXLD agar -red colonies

CLED purity plate

Oxidase testAn oxidase test will distinguish between the organismsS. sonnei (oxidase neg) & P. shigelloides (oxidase pos)

Positive Negative

Discard

Urease(37 C for up to 4 hr in air)

Negative Positive

Positive Negative

Pure culture

Discard

Biochemical tests

General agglutination s

Pure cultureSpecific agglutinations

S. sonnei

Negative Positive

S. flexneri Polyvalent(1-6,x,y)

Negative Positive

S. boydii PolyvalentNot all serotypes are contained

in polyvalent antisera(1-6, 7-11, 12-15)

Negative Positive

S. dysenteriae Polyvalent

NegativePositive

Discard

Further identification if clinicallyindicated. Commercial identification

kits or other biochemical identificationor send to the Reference Laboratory.If required save the pure isolate on

an agar slope.

Mannitolnegative

Mannitolpositive

The flow chart is for guidance only

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5 Reporting

5.1 Presumptive IdentificationIf appropriate growth characteristics, colonial appearance, urease and serology results aredemonstrated.

5.2 Confirmation of Identification

Further biochemical tests and/or molecular methods and/or reference laboratory report.

5.3 Medical MicrobiologistInform the medical microbiologist of presumptive or confirmed Shigella dysenteriae O1isolates, according to local protocols.The medical microbiologist should also be informed of a presumptive or confirmed Shigella species if the request card bears relevant information eg:

enterocolitis, dysentery (especially if complicated by haemolytic-uraemic syndrome). neurological dysfunction or confusional states. history of recent foreign travel or laboratory work. food poisoning. investigations of outbreak situations.

Follow local protocols for reporting to clinician.

5.4 CCDCRefer to local Memorandum of Understanding.

5.5 Health Protection Agency 14 Refer to current guidelines on CDSC and COSURV reporting.

5.6 Infection Control TeamInform the infection control team of presumptive or confirmed isolates of Shigella species.

6 Referrals

6.1 Reference LaboratoryFor information on the tests offered, turn around times, transport procedure and the otherrequirements of the reference laboratory refer to: http://www.hpa.org.uk/cfi/lep/default.htmLaboratory of Enteric PathogensMicrobiology Services DivisionHealth Protection Agency61 Colindale AvenueLondonNW9 5HT

Contact main Microbiology Services Divisions switchboard: Tel. +44 (0) 20 8200 6173
http://www.hpa.org.uk/cfi/lep/default.htmhttp://www.hpa.org.uk/cfi/lep/default.htmhttp://www.hpa.org.uk/cfi/lep/default.htm

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References1. Rowe B, Gross RJ. Genus II Shigella. In: Krieg NR, Holt JG, editors. Bergey's Manual of Systematic

Bacteriology.Vol 1. Baltimore: Williams and Wilkins; 1984. p. 423-7.

2. Emmerson AM, Gillespie SH. Shigella . In: Emmerson AM, Hawkey PM, Gillespie SH, editors. Principles andPractice of Clinical Bacteriology. Chichester: John Wiley & Sons; 1997. p. 389-98.

3. Advisory Committee on Dangerous Pathogens. The Approved List of Biological Agents. Her Majesty'sStationery Office. Norwich. 2004. p. 1-21

4. Infections at work: Controlling the risks. Her Majesty's Stationery Office; 2003.

5. Advisory Committee on Dangerous Pathogens. Biological agents: Managing the risks in laboratories andhealthcare premises. HSE. 2005.

6. Control of Substances Hazardous to Health Regulations. The control of susbstances hazardous to healthregulations 2002. 5th ed. HSE Books; 2002.

7. Health and Safety Executive. Five Steps to Risk Assessment: A Step by Step Guide to a Safer and HealthierWorkplace. HSE Books. 2002.

8. Health and Safety Executive. A Guide to Risk Assessment Requirements: Common Provisions in Healthand Safety Law. HSE Books. 2002.

9. British Standards Institution (BSI). BS EN12469 - Biotechnology - performance criteria for microbiologicalsafety cabinets. 2000.

10. British Standards Institution (BSI). BS 5726 - Microbiological safety cabinets. Part 2: Recommendations forinformation to be exchanged between purchaser, vendor and installer and recommendations forinstallation. 1992.

11. British Standards Institution (BSI). BS 5726 - Microbiological safety cabinets. Part 4: Recommendations forselection, use and maintenance. 1992.

12. Health Services Advisory Committee. Safe Working and the Prevention of Infection in Clinical Laboratories

and Similar Facilities. HSE Books. 2003.13. Department for transport. Transport of Infectious Substances, 2011 Revision 5.

http://www.dft.gov.uk/426155/425453/800_300/infectioussubstances.pdf.

14. Health Protection Agency. Laboratory Reporting to the Health Protection Agency: Guide for DiagnosticLaboratories. 2010.
http://www.dft.gov.uk/426155/425453/800_300/infectioussubstances.pdfhttp://www.dft.gov.uk/426155/425453/800_300/infectioussubstances.pdf

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