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Issued by the Standards Unit, Microbiology Services Division, HPA

Bacteriology | B 2 | Issue no: 5.2| Issue date: 29.03.12 | Page: 1 of 16

UK Standards for Microbiology Investigations

Investigation ofE

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wabs andC

analicularP

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Investigation of Eye Swabs and Canalicular Pus

Bacteriology | B 2 | Issue no: 5.2 | Issue date: 29.03.12 | Page: 2 of 16

UK Standards for Microbiology Investigations | Issued by the Standards Unit, Health Protection Agency

AcknowledgmentsUK 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 below

and listed on the websitehttp://www.hpa.org.uk/SMI/Partnerships.SMIs are developed,reviewed and revised by various working groups which are overseen by a steering committee(seehttp://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 DivisionHealth Protection Agency61 Colindale AvenueLondon NW9 5EQ

E-mail:[email protected]

Website:http://www.hpa.org.uk/SMI

UK Standards for Microbiology Investigations are produced in association with:

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UK Standards for Microbiology Investigations: StatusUsers 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, for

example 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 at

http://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/SMIas 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.

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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 produced

since October 2009 using the processes described in the HPAs Standard Operating ProcedureSW3026 (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 athttp://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 DocumentHealth Protection Agency. (2012). Investigation of Eye Swabs and Canalicular Pus. UKStandards for Microbiology Investigations. B 2 Issue 5.2 http://www.hpa.org.uk/SMI/pdf.

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ContentsACKNOWLEDGMENTS ............................................................................................................ 2UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: STATUS ............ ............ ......... ............ ..... 3AMENDMENT TABLE ............................................................................................................... 6SCOPE OF DOCUMENT ........................................................................................................... 7INTRODUCTION ..................................................................................................................... 7TECHNICAL INFORMATION/LIMITATIONS ................................................................................... 91 SPECIMEN COLLECTION, TRANSPORT AND STORAGE ....................................................... 92 SPECIMEN PROCESSING ............................................................................................. 103 REPORTING PROCEDURE ............................................................................................ 144 NOTIFICATION TO THE HPA ........................................................................................ 14REFERENCES ........................................................................................................................ 15

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Amendment TableEach SMI method has an individual record of amendments. The current amendments are listedon this page. The amendment history is available [email protected].

New or revised documents should be controlled within the laboratory in accordance with thelocal quality management system.

Amendment No/Date. 8/29.03.12

Issue no. discarded. 5.1

Insert Issue no. 5.2

Section s) involved/ Page no. Amendment.Whole document. Document transferred to an updated template.

Amendment No/Date. 7/27.03.12

Issue no. discarded. 5

Insert Issue no. 5.1

Section s) involved/ Page no. Amendment.

Whole document.

Document presented in a new format.

The term CE marked leak proof container replacessterile leak proof container (where appropriate) and isreferenced to specific text in the EU in vitro DiagnosticMedical Devices Directive (98/79/EC Annex 1 B 2.1) andto Directive itself EC1,2.

Edited for clarity.

Reorganisation of [some] text.

Minor textual changes.

Sections on specimen collection,transport, storage and processing.

Reorganised. Previous numbering changed.

References. Some references updated.

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Scope of DocumentType of SpecimensEye swabs.

Canalicular pus.

ScopeThis UK Standards for Microbiology Investigation (SMI) describe the processing andbacteriological investigation of specimens from the eyes with the exception of those fromkeratitis, endophthalmitis, hypopyon and post surgical infections for these refer toB 52 -Investigation of intraocular fluids and corneal scrapings.New molecular techniques are nowavailable to diagnose chlamydia infections from eye swabs. These are not covered in this SMI.

This SMI should be used in conjunction with other SMIs.

IntroductionInfections of the eye can be caused by a variety of microorganisms. Swabs from eyes may becontaminated with skin microflora, but any organism may be considered for furtherinvestigation if clinically indicated.

Exogenous organisms may be introduced to the eye via hands, fomites (eg contact lenses),traumatic injury involving a foreign body, following surgery, or simply by spread from adjacentsites3,4.

InfectionsCommon mild eye infections include conjunctivitis (inflammation of the conjunctiva) andblepharitis (inflammation of the eyelid). Conjunctivitis may occur in association with infectionof the eyelid (blepharoconjunctivitis) or of the cornea (keratoconjunctivitis). Less common andmore severe infections include keratitis (inflammation of the cornea) and endophthalmitis(infection inside the eye itself). Haematogenous spread from a focus elsewhere in the bodycan also occur5. Other periocular infections include dacryoadenitis (inflammation of thelacrimal gland), dacryocystitis (inflammation of the lacrimal sac), canaliculitis (infection of thelacrimal puncta and canaliculi), and preseptal and orbital cellulitis6. Invasive specimens may berequired for optimal investigation of severe eye infections, and these are dealt with inB 52 -Investigation of intraocular fluids and corneal scrapings.Separate swabs in appropriatetransport media are needed for the diagnosis of viral and chlamydial infections.

Eye infections occurring in the first 4 weeks of life caused by ChlamydiatrachomatisorNeisseria gonorrhoeaeare notifiable as ophthalmia neonatorum.

Eye swabs may be received from patients with any of these conditions, but may need handlingdifferently according to the type and severity of infection.

BlepharitisBlepharitisis associated with7,8:

Staphylococcus aureus. Staphylococcus epidermidis. Corynebacteriumspecies.

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Propionibacterium acnes.However, these organisms may be isolated from the eyelids of normal healthy individuals,necessitating careful interpretation of such cultures.

ConjunctivitisConjunctivitis may be acute or chronic. The conjunctiva is the most commonly infected oculartissue, and infectious conjunctivitis is one of the most common causes of red or sticky eyes.Common bacterial causes include:

S. aureus. Streptococcus pneumoniae. Haemophilus influenzae.

Less common causes include Lancefield group A, C and G streptococci, Neisseria cinerea9,10

.

P. acnes, Moraxellaspecies and other Gram negative rods, and anaerobes such as Eubacterium

species and Peptostreptococcusspecies11,12

. Moraxella catarrhaliscauses acute conjunctivitisand Moraxella lacunatacauses a chronic infection

12. However, many of these organisms mayalso be isolated from the surrounding areas (skin), and so the interpretation of the significanceof their presence is difficult.

Conjunctivitis caused by Neisseriaspecies is uncommon in developed countries. The mostimportant ocular pathogen in this genus is Neisseria gonorrhoeae. In adults it is associatedwith concomitant genital infection. In neonates it is an important cause of ophthalmianeonatorum, which may cause blindness if left untreated. Neisseria meningitidishas also beenimplicated in hyperacute conjunctivitis. Treatment of this is important to reduce the risk ofdissemination, and rifampicin prophylaxis may be indicated on household contacts and thepatient to eliminate throat carriage.

Conjunctivitis in neonates is caused by the pathogens commonly found in adult cases11,13.Additional organisms include12:

N. gonorrhoeae. Haemophilus parainfluenzae. Lancefield group B streptococci and enterococci. Enterobacteriaceae eg Klebsiella pneumoniaeand Proteus mirabilis. Pseudomonas aeruginosa.

Chlamydial and viral conjunctivitisChlamydial and viral conjunctivitis also occur. Inclusion conjunctivitis and trachoma are causedby various serotypes of Chlamydia trachomatis. Trachoma is associated with serotypes A-C.This occurs in rural under-developed areas, whereas inclusion conjunctivitis is associated withtypes D-K, and is a feature of developed urban communities14. These serotypes are associatedwith sexual transmission. The most common causes of viral conjunctivitis are adenoviruses.canthamoeba species

Acanthamoebaspecies can cause severe keratitis, usually in contact lens wearers or afterocular trauma. These protozoa may be isolated from corneal scrapings, as well as fromcontact lenses and storage cases (B 52 - Investigation of intraocular fluids and cornealscrapingsandB 31 - Investigation of specimens other than blood for parasites ).

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Orbital cellulitisOrbital cellulitis is the infection of orbital tissue. It can result from trauma, surgery, or anextension of paranasal sinus infections. It is a serious infection and may cause blindness, septicthrombosis of the cavernous sinus or intracranial infections. The most common pathogens inadults are S. aureus, streptococci and anaerobes. In children H. influenzaestill remains

prevalent, but the capsulated (type b) strain is rarely seen. Streptococci, staphylococci,peptostreptococci and P. aeruginosamay cause necrosis15. Eye swabs are of limited value inthe investigation of orbital and preseptal cellulitis. Ideally aspirates from the affected tissuesshould be obtained and treated according to the procedures outlined inB 26 - Investigation offluids from normally sterile sites.Blood cultures are also useful in diagnosis (refer toB 37 -Investigation of blood cultures (for organisms other than Mycobacterium species)).

CanaliculitisCanaliculitis is a rare condition. Infections are usually chronic and caused by anaerobicactinomycetes such as Actinomyces israeliior by Propionibacterium propionicus16,17. Swabs ofsamples of the canalicular pus are preferable to eye swabs for diagnosis.

For further information about serious eye infections, including examination for Acanthamoebaspecies, refer toB 52 - Investigation of intraocular fluids and corneal scrapings.

Technical Information/LimitationsSuperficial swabs, although not ideal, may be all that is available. Deep-seated samples ifavailable should be sought.

Specimen containers1,2SMIs use the term CE marked leak proof container to describe containers bearing the CE

marking used for the collection and transport of clinical specimens. The requirements forspecimen containers are given in the EU in vitro Diagnostic Medical Devices Directive(98/79/EC Annex 1 B 2.1) which states: The design must allow easy handling and, wherenecessary, reduce as far as possible contamination of, and leakage from, the device during useand, in the case of specimen receptacles, the risk of contamination of the specimen. Themanufacturing processes must be appropriate for these purposes.

1 Specimen Collection, Transport and Storage1,21.1 Safety considerations18-28Use aseptic technique.Collect specimens in appropriate CE marked leak proof containers and transport specimens insealed plastic bags.

Collect swabs into Amies transport medium with charcoal and transport in sealed plasticbags29.

Compliance with postal and transport regulations is essential.

1.2 Achieving optimal conditions1.2.1 Time between specimen collection and processingCollect specimens before antimicrobial therapy where possible.

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Prepare a thin smear from the swab or pus on a clean microscope slide for Gram staining.

2.5 Culture and investigation2.5.1 Pre-treatmentN/A

2.5.2 Specimen processingInoculate each agar plate with swab or pus (Q 5 - Inoculation of culture media (formerly B54)).

For inoculation methods performed at the patients side, refer to local protocols.

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2.5.3 Culture media, conditions and organisms for all specimens:Clinical details/conditions

Standardmedia Incubation Culturesread Target organism s)TempC Atmos TimeBlepharitis

Conjunctivitis

Sticky eye

If no clinical detailsavailable, treat as asticky eye

Chocolate agar 35-37 5-10%CO2

40-48 hr daily H. influenzae

Lancefield groupA,B,C and Gstreptococci

Moraxellaspecies

N. gonorrhoeae

N. meningitidis

P. aeruginosa

S. aureus

S. pneumoniaeOther organisms(see section 2.6.1)

Blood agar 35-37 5-10%CO2

40-48 hr daily

For these situations, add the following:

Clinical details/conditions

Supplementarymedia Incubation Culturesread Target organism s)TempC Atmos TimeGUM clinic sticky eye

Neonates

GC selectiveagar

35-37 5-10%CO2

40-48 hr 40 hr N. gonorrhoeae

Immunocompromised

Chronic blepharitis

Sabouraud agar 28-30 air 40-48 hr* 40 hr Fungi

Canaliculitis

Orbital cellulitis

Dacryocystitis

Dacryoadenitis

Keratitis

Endophthalmitis

Hypopyon

Post surgery

Post trauma

Fastidiousanaerobe agar

35-37 anaerobic 40-48 hr* 40 hr Anaerobes

10 d 40 hr,at 7 dand 10 d

Actinomycetes

Sabouraud agar 28-30 air 40-48 hr* 40 hr Fungi

If Gram negative rodsseen in Gram film

CLED agar 35-37 air 16-24 hr 16 hr Enterobacteriaceae

Other organisms for consideration - Chlamydia species and viruses*incubation may be extended to 5 days; in such cases plates should be read at 40 h and then left in theincubator/cabinet until day 5.

extend incubation time to 10 days if clinically suspected or Gram-positive branching rods present in Gram stain.

Refer toB 52 - Investigation of intraocular fluids and corneal scrapings.

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2.6 IdentificationRefer to individual SMIs for organism identification.

2.6.1 Minimum level of identification in the laboratoryActinomycetes "actinomycetes" level

Anaerobes "anaerobes" level

ID 14 -Identification of non-sporing, non-branchinganaerobes

ID 8 - Identification of Clostridium species

ID 25 - Identification of anaerobic Gram negative rods

Coagulase-negative staphylococci "coagulase-negative" level

Diphtheroids "diphtheroid" level

Enterobacteriaceae "coliforms" level

Enterococci species level

Fungi genus level

Haemophilus influenzae species level

Lancefield groups A, B, C and G streptococci Lancefield group level

Moraxellaspecies species level

Neisseria meningitidis species level

P. aeruginosa species level

Pseudomonads "pseudomonads" level

S. aureus species level

S. pneumoniae species level

-haemolytic streptococci "-haemolytic" level

Yeasts "yeasts" level

Organisms may be further identified if clinically or epidemiologically indicated.

2.7 Antimicrobial susceptibility testingRefer to British Society for Antimicrobial Chemotherapy (BSAC) guidelines. Prudent use ofantimicrobials according to local and national protocols is recommended.

2.8 Referral to reference laboratoriesFor information on the tests offered, turn around times, transport procedure and the otherrequirements of the reference laboratoryclick here for user manuals and request forms.

Organisms with unusual or unexpected resistance and whenever there is a laboratory orclinical problem, or anomaly that requires elucidation should be sent to the appropriatereference laboratory.

2.9 Referral for outbreak investigationsN/A

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3 Reporting Procedure3.1 MicroscopyReport on WBCs and organisms detected.

3.2 CultureReport:

Clinically significant organisms isolated orother growth, eg No significant growth orabsence of growth

3.2.1 Culture reporting timeClinically urgent results: to be telephoned or sent electronically.

Written report: 16---72 hr stating, if appropriate, that a further report will be issued.3.3 Antimicrobial susceptibility testingReport susceptibilities as clinically indicated.

4 Notification to the HPA30,31The Health Protection (Notification) regulations 2010 require diagnostic laboratories to notifythe HPA when they identify the causative agents that are listed in Schedule 2 of theRegulations. Notifications must be provided in writing, on paper or electronically, within sevendays. Urgent cases should be notified orally and as soon as possible, recommended within 24

hours. These should be followed up by written notification within seven days.

For the purposes of the Notification Regulations, the recipient of laboratory notifications is thelocal HPA office. If a case has already been notified by a registered medical practitioner, thediagnostic laboratory is still required to notify the case if they identify any evidence of aninfection caused by a notifiable causative agent.

Notification under the Health Protection (Notification) Regulations 2010 does not replacevoluntary reporting to the HPA. The vast majority of NHS laboratories voluntarily report a widerange of laboratory diagnoses of causative agents to the HPA and many HPA offices haveagreements with local laboratories for urgent reporting of some infections. This shouldcontinue.

Note: The Health Protection Legislation Guidance (2010) includes reporting of HIV & STIs,HCAIs and CJD under Notification Duties of Registered Medical Practitioners: it is not notedunder Notification Duties of Diagnostic Laboratories.

Other arrangements exist in Scotland32and Wales33.

Note: Isolation of N. meningitidisshould be reported to the CCDC.

Note: Ophthalmia neonatorum is a notifiable disease.

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References1. European Parliament. UK Standards for Microbiology Investigations (SMIs) use the term "CE marked leak proof

container" to describe containers bearing the CE marking used for the collection and transport of clinicalspecimens. The requirements for specimen containers are given in the EU in vitro Diagnostic Medical Devices

Directive (98/79/EC Annex 1 B 2.1) which states: "The design must allow easy handling and, where necessary,reduce as far as possible contamination of, and leakage from, the device during use and, in the case ofspecimen receptacles, the risk of contamination of the specimen. The manufacturing processes must beappropriate for these purposes".

2. Official Journal of the European Communities. Directive 98/79/EC of the European Parliament and of theCouncil of 27 October 1998 on in vitro diagnostic medical devices. Official Journal of the EuropeanCommunities 1998;1-37.

3. Das T, Choudhury K, Sharma S, Jalali S, Nuthethi R. Clinical profile and outcome in Bacillus endophthalmitis.Ophthalmology 2001;108:1819-25.

4. Barequet IS, Jabbur NS, Barron Y, Osterhout GJ, O'Brien TP. Perioperative microbiologic profile of theconjunctiva in photorefractive keratectomy. J Refract Surg 2001;17:55-62.

5. Matsuo K, Nakatuka K, Yano Y, Fujishima W, Kashima K. Group B streptococcal metastatic endophthalmitis inan elderly man without predisposing illness. Jpn J Ophthalmol 1998;42:304-7.

6. Brook I, Frazier EH. Aerobic and anaerobic microbiology of dacryocystitis. Am J Ophthalmol 1998;125:552-4.

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