caribbean regional microbiology standard...

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEANA CARIFORUM Project Funded by the European Union and Implemented by CAREC

CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE

Enteric Specimens for Bacterial Pathogens – SOP No: CRM-SOP 4

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEANA CARIFORUM Project Funded by the European Union and Implemented by CAREC

Enteric Specimens for Bacterial Pathogens

�STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN

A CARIFORUM Project Funded by the European Union and Implemented by CAREC

TA B L E O F C O N T E N T S

Acknowledgements 3

Introduction 5

Amendment Procedure 6 Process Flow Chart 7

1. Title 8

�. Purpose 8

3. Introduction 8

4. Scope 8

5. Staff Competency Requirements 8 6. Safety Instructions 9

7. Pre-Examination Procedures 9 7.1 Sample Type 9

7.� Sample Collection 9

7.3 Sample Transport & Storage 10

7.4 Rejection Critiria 10 7.5 Relevant Clinical Information 10 8. Table of Media, Reagents, Materials & Equipment 11

9. Examination Procedures 11

9.1 Quality Control 11

9.� Microscopy 12

9.3 Culture 12 9.4 Identification 14

9.5 Susceptibility Testing 16

9.6 Sample Referral 16

10. Post-Examination Procedures 16 10.1 Interpretation of Results 16 10.� Reporting 16

10.3 Sample Retention, Storage & Disposal 17

11. Limitations and Pitfalls of the Procedure 17

1�. References 17

13. Appendices


Title: Enteric Specimens for Bacterial SOP No: CRM-SOP: 4

Pathogens Version: 1

Page No: � of ��

Prepared By: Caribbean Regional Standard Effective Date: 1st September �007

Methods Drafting Group Review Date: 1st September �008


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The Caribbean Regional Standard Methods include a variety of standard, validated methods, produced as a single standard operating procedure (SOP) for use in a variety of levels of microbiology laboratory service. It is intended that these methods provide detailed instructions for microbiology services for microbiological investigations, in order to provide accurate, reliable and reproducible results which will have clinical utility. These methods may be adopted by laboratories within the region, or adapted, provided that such adaptations use an evidence-based validation process.

These methods have been developed by the Caribbean Regional Microbiology Standard Methods Drafting Group (CSMDG) in response to a request by the Caribbean Regional Microbiology Council (CRMC), which was set up by the CARIFORUM Project entitled ‘Strengthening of Medical LaboratoriesintheCaribbean’tostrengthenspecificallythemicrobiology services in the Caribbean Region. The Project was initiated in response to findings which indicated thatthere was an unacceptable level of error in laboratories within the region. External quality assessment results revealed that microbiology laboratories were not performing well and feedback from the region via laboratory staff, lab managers and directors was that they felt that guidance in microbiology requirements was required.

The background for this initiative is a worldwide move to implement standards in all areas, which has now extended to include medical laboratories. As tourism is so vital to the region’s economy, the need for accurate diagnosis and treatment is paramount. It was accepted that there is a requirement for validated methods for accreditation purposes and providing validated standard methods will assist in the move towards accreditation.

The methods will be chosen for standardization by the Caribbean Regional Microbiology Council, and this selection will be based on a review of EQA results, most common and/or critical tests. Part of the method standardization process will be an ongoing review and amendment procedure. The CSMDG consists of microbiology laboratory representatives from most of the CARIFORUM countries, all of whom were nominated to the task by the CRMC.

This initiative should enable the region to implement a standardized and constructive method for ensuring that validated methods are available for the region, and that they are updated as required.

Advantages of using regionally validated methods are to improve quality, make better use of resources, reduce costs, enable central procurement & media preparation, facilitate staff training and transfers due to horizontal integration, a reduction in variability of service provision, an improved quality of surveillance data, and the purchase of appropriate equipment. A major advantage is that the availability of regional standard methods would assist microbiology laboratories with documentation for accreditation.

Although the CSMDG has taken every care with the preparation and issue of these standard procedures, and they have been validated regionally, nationally and internationally, the CSMDG, or any other organization, cannot be responsible for the accuracy of any statement or representation made or the consequences arising from the use of or alteration to any information contained in them. These procedures are intended solely as a resource for practicing microbiology professionalsinthefield,operatingintheCaribbeanregion,and specialist advice should be obtained where necessary. If changes are made to the original publication, it must be made clear where changes have been made to the original document. When referring to these SOPs in successive documentation, the CSMDG should be acknowledged.

I N T R O D U C T I O N
















A M E N D M E N T P R O C E D U R E

Controlled Document Reference CRM-SOP 4

Controlled Document Title Standard Operating Procedure for Enteric Specimens for Bacterial Pathogens

Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting Group. Any amendments should be validated and authorized by an agreed process, and referenced.

Each Regional Standard Method has an individual record of amendments. The current amendments are listed on this page.

On issue of revised or new pages, each controlled document should be updated by the copyholder in the laboratory.

Amendment Issue Number Insert Page Section(s) AmendmentNumber / Date Discarded Issue Number involved




P R O C E S S F L O W C H A R T

ENTERIC SPECIMENSFOR BACTERIALPATHOGENS










1. Title

Enteric Specimens for Bacterial Pathogens.

2. Purpose

To ensure the correct, validated procedure is followed for processing stool specimens to provide accurate, reliable, reproducible results having clinical utility.

3. Introduction

Diarrheaisusuallydefinedasthepassageofthreeormorelooseorwaterystoolsina24hourperiod;aloosestoolbeingonethat would adopt the shape of a container. Stools may be loose, watery, bloodstained or mucoid.

Bacteria, viruses and parasites are all capable of causing diarrhea using a variety of mechanisms. For example, illness may result from multiplication of bacteria in the gut e.g. Salmonella sp or Shigella sp infection, or by ingestion of pre-formed toxins producedbybacteriainfoodpriortoingestione.g.neurotoxicshellfishtoxin,Staph aureus, Clostridium perfringens.

Changesinthegutfloraassociatedwithbroadspectrumantibioticsandchemotherapeuticagents,canresultincolonizationbyClostridium difficile.Thisisthemostcommonidentifiablecauseofantibioticassociateddiarrhea(AAD),especiallyinhospitalpatients.

Diarrhoeal outbreaks can occur by ingestion of contaminated food and water, and also from direct contact with animals or person to person contact. Investigation of outbreaks may also include culture of food and/or water which is suspected as being contaminated. Consumption of food containing irritant chemicals such as heavy metals may also cause illness.

Astool culture is normallydone to assist in the isolationand identificationof thecausative agentofpersistentorbloodydiarrhea.

Culture may also be employed to determine asymptomatic carriage of organisms that may cause episodes of diarrhea such as Salmonella species. This is especially important if these carriers are food handlers, since they are likely to transfer these organisms to other individuals, thus resulting in outbreaks.

Stool cultures may also provide useful information to assist in the management of epidemics.

4. Scope

This procedure provides detailed instruction for Microbiology services for the examination of stool specimens for common bacterial pathogens offered by Regional Laboratories. It may be adopted or adapted by any laboratory as needed, provided that such adaptation uses an evidence-based validation process.

5. Staff Competency Requirements

Laboratory personnel, trained and assessed to be competent to perform this procedure.










6. Safety Instructions

Please refer to CRM-SOP �0: Safety In The Microbiology Laboratory. Keep all books, forms and papers away from technical work surfaces.

Level II containment unless a Containment Level 3 (CL3) organism such as Salmonella typhi or Salmonella paratyphi is suspected in which case all work should be carried out in a category II Biosafety Cabinet in a CL3 laboratory. Require all personal protective equipment (PPE)

A Class II biosafety cabinet should be used when performing procedures that produce aerosols.

Hands should be thoroughly washed with soap and water before and after handling all specimens.

Allbodyfluidsandothersamplesforprocessingshouldbeconsideredapotentialriskofcausingtransmissibleinfectionsand,therefore, all universal safety precautions should be observed.

Disinfect all work surfaces with 70% alcohol or a freshly prepared 10% bleach solution prior to and after processing.

All samples and reagents should be properly discarded according to the current standards for disposal of hazardous waste.

Samplesandcultureplatesshouldbeautoclavedbeforefinallydiscarding.

7. Pre-Examination Procedures

7.1 Sample Type

Fresh stool or rectal swab (rectal swabs do not yield reliable culture results and are inappropriate for the examination for parasites).

7.2 Sample Collection

Stool – passed directly into a clean, dry, leak-proof, wide-mouth screw-capped container as provided by the laboratory.

The container should not be contaminated with any traces of disinfectants, soaps or detergents.

Care must be taken not to contaminate the sample.

1-2gms of sample is sufficient for routine culture. Tests for quantifying food poisoning organisms may requireapproximately 10 grams.

Rectal swab – obtain rectal swab and place in Cary-Blair transport media. Do not use expired transport media for samples. Rectal swabs should be collected only when it is not possible to collect a stool sample.

Samples should be collected as soon as possible after the onset of symptoms.










7. Pre-Examination Procedures continued

Legibly label all specimens with the patient’s name, number, date and time of collection and type of sample.

Samples should be transported without delay to the laboratory, along with the appropriately completed requisition form. 7.3 Sample Transport & Storage

Specimenshouldbetransportedtothelaboratoryassoonaspossibleinasealedplasticbag.Storeat2-8˚Cifadelay of more than �4 hours is foreseen before processing.

7.4 Rejection Criteria

Leaking containers.

Inappropriate containers such as matchboxes, plastic bags and foil paper.

Stool contaminated with water, soil, urine, for example.

Rectal swab not in transport media.

Unlabeled or incorrectly labeled specimens.

Specimens unaccompanied by requisition form.

Stool in preservatives.

Expired transport media used for rectal swab.

7.5 Relevant Clinical Information

Symptoms and date of onset.

Travel history.Any relevant occupation eg food handler, healthcare worker, farmer etc.

Suspected cause or organism eg ingestion of contaminated food or water, part of an outbreak or suspected enteric fever.

Details of any antimicrobial therapy.










8. Table of Media, Reagents, Materials and Equipment

9. Examination Procedures

9.1 Quality Control

Please refer to CRM-SOP 18: Media Preparation and Quality Control.

Please refer to CRM-SOP �1: Quality Control of Reagents and Tests.

Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organsims.

Performance and sterility checks should be performed on all media before use.

Equipment Media Reagents

Bunsen burner/ Bacti cineratorLoops / Straight WireIncubator37˚CIncubator42˚CCampylobacter jarsLight MicroscopeDispose jarLevel � BSCPPEBacteriological straight wire/loop (preferably nichrome) or disposablealternativeU well microtitre platesCampylobacter packsApplicator sticksImmersion oilLens CleanerLens paperGlass slidesDistilled waterSalineSterile glass tubesTissue paper or paper towels

XLDTCBSCCDASMAC

Enrichment broth

Gram Stain reagentsOxidase reagentSalmonella antisera or latex E. coli 0157: Shigella antisera: S. sonnei; S. flexneri(poly);S. boydii(poly);S. dysenteriae (poly).

Reagents for API / Enterotube/ In-house BiochemicalsC. difficile toxin detection kitPPA - Phenyl Pyruvic Acid

If applicable:Salmonella culture0.85% saline (Sterile)Somatic antigen suspensionFlagellar antigen suspension1% formol saline








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9. Examination Procedures continued

All media should be checked again visually just before use to determine that there are no signs of deterioration such as discoloration or drying of the media as is evidenced by cracking, or shrinkage of the plates or other signs of contamination. All reagents used in testing must also be checked for performance.Quality control checks can be done using known Appropriate American Type Culture Collection (ATCC) control organisms.

Ensure that reagents, stains and media are not used beyond the expiration dates.Check all reagents and stains just before use to ensure that they are free from contamination, debris or deposits. Results of all QC tests should be recorded on the appropriate forms available in the department for that purpose.

Test results are acceptable for reporting only if the QC results are as expected.

9.1.1 Quality control organisms for tube and slide agglutinations.

Positive control: Homologousorganismsuspensionspecifictothepolyvalentantiserum.Negative control: Organism suspension in saline only. 9.2 Microscopy

Not applicable directly from sample unless part of an outbreak investigation or the presence of parasites is suspected.

Please refer to CRM-SOP 7: Gram StainOrganism Characteristics.

Salmonellae: Gram-negative, non-sporing, motile rods (very few may be non-motile).

Shigellae: Gram-negative, non-sporing, non-capsulate, non-motile rods.

Escherichia coli: Gram-negative, motile rods.

Campylobacter sp: small delicate, spirally curved, motile and Gram-negative. In stained faecal smears (1% basic fuchsin), the organisms may appear as the wings of gulls, while some may form an “S” or comma shape. Vibrio cholerae: Gram-negative motile (‘darting’ motility) curved rods, measuring about 4-5 x 0.5 µm with a single flagellumatoneend.

9.3 Culture

Culture should include media for the isolation of the most commonly encountered enteric pathogens: Salmonella sp, Shilgella sp, Campylobacter sp and Vibrio sp. Other bacterial pathogens such as Yersinia sp. should be sought if clinical details are indicative.




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Inoculate the appropriate media depending on the clinical details eg if no travel history or no indication of contact with cholera it may be unnecessary to include a TCBS.

Inoculate and spread for isolation of individual colonies.

Refer to CRM-SOP ��: Inoculation of Culture Media. Incubate plates aerobically at 35-37°C for �4 hours. (Incubate CCDA for Campylobacter sp under microaerophilic conditions at 4�°C for 48 hours.)

Observe for growth on plates after overnight incubation. Examine the XLD for non-lactose fermenting (NLF) colonies that may be either Salmonella sp or Shigella sp.

Toassistintheidentificationofsuspectorganisms,inoculateKligler’sIronAgar(KIA),LysineIronAgar(LIA)orTripleSugarIronAgar(TSIA)slantstakenfromtheprimaryculturemediumusingasterilestraightwire.Stabthebuttfirstthen streak the slope. Immediately after inoculation of the slope inoculate a CLED or McConkey purity plate.

The urease test can be used to exclude Proteus sp. Inoculate the urea slope and include a CLED or McConkey purity plate as above. Proteus sp. = urease positive.(pink color develops) Salmonella and Shigella sp. = urease negative

Closethetubewithaloosefittingcapandincubateat35-37°Caerobicallyforupto24hours;examineafterovernightincubation for growth. NB. The slopes frequently become positive before �4 hours. Examine the purity plate carefully before accepting any results.











CCDA Charcoal, cefperazone, amphotericin agar for Campylobacter sp.SMAC Cefixime,tellurite,sorbitolMacConkeyagarforE.coli 0157TCBS Thiosulphate, citrate, bile salt agar for Vibrio sp. XLD Xylose, lysine, deoxycholate agar for Shigella sp & Salmonella spCIN Cefsulodin, irgasan (triclosan), novobiocin agar for Yersinia sp. TSIA Triple Sugar Iron AgarLIA Lysine Iron Agar

9.4 Identification

9.4.1 Salmonella & Shigella (Appendix 1)

9.4.2 Vibrio spp (Appendix 2)

9.4.3 Campylobacter spp (Appendix 3)

Pathogen Selective Enrich Broth Direct Plating Media

Salmonella

Shigella

Campylobacter

Vibrio

E.coli 0157

Tetrathionate/SeleniteBroth(incubateat35-37˚Cfor 18-�4 hours thensubculture on to XLD)

GN Broth(incubate4-6hoursat37˚Cthen culture onto XLD)

Alkaline Peptone Water (APW) (incubate for 6-8 hoursat35-37˚Cthensubculture from surface pellicle’ onto TCBS)

N/A

XLD

(incubateat35-37˚Cfor18-24hoursaerobically)

XLD

(incubateat35-37˚Cfor18-24hoursaerobically)

CCDA(incubateat40-42˚Cfor48hours) under microaerophilic conditions.

TCBS(incubateat35-37˚Cfor24hours aerobically)

SMAC(incubateat35-37˚Cfor24hours aerobically)











9.4.4 E. coli 0157 (Appendix 4)

9.4.5 Clostridium difficile toxin (Appendix 5)

9.4.6 Slide Agglutination Tests

a. Make two adjacent suspensions of the test organism in a drop of saline on a slide.

b. If auto-agglutination occurs or the suspension is rough in saline then discard the slide. The test can only be performed with smooth suspensions.

c. Add a drop of antiserum to one suspension only and mix.

d. Examine for agglutination (clumping) of the suspension (with antiserum) and background clearing of the saline.

Positive result: Agglutination of the suspension.

Negative result: Suspension remains turbid with no agglutination observed.

Minimum level of identification:

Salmonella species genus level

Shigella species species level

Ecoli 0157 H7 species level and serotype

Campylobacter species genus level

Vibrio species species level

Clostridium difficile species level

Clostridium perfringens species level

Staph aureus species level

Organismsmaybefurtheridentifiedifthisisclinicallyorepidemiologicallyindicated.











9.5 Susceptibility Testing

Susceptibility testing is not routinely reported for enteric pathogens, except for S. typhi and S. paratyphi. They are performed for epidemiological purposes only.

9.6 Sample Referral

Samples should be referred to reference laboratory if national or local laboratories do not have the facilities / capabilities to perform the required tests.

Organisms isolated should be referred, where necessary, for further identification / confirmation.Salmonella and Shigella samples can be shipped in TSIA tubes.

Organisms with unusual or unexpected resistance, or whenever there is a laboratory or clinical problem that requires elucidation.

Isolates associated with outbreaks and where epidemiologically indicated.

All referrals must be accompanied by appropriately completed laboratory requisition forms.

Always notify reference labs before sending samples, especially when an urgent specimen is included.

Ensure that all IATA regulations are adhered to when shipping samples overseas for further testing.

10. Post-Examination Procedures

10.1 Interpretation of Results

Refer to Appendices.

10.2 Reporting Describe the appearance of the specimen: Report on the colour – whether green, brown, black, yellow, and the consistencyofthestoolshouldbedocumented–whetherformed,semi-formed,unformedorsoft,fluidorrunny.

The presence of blood, mucus or pus should also be noted.

Report the presence or absence of the named organisms that have been looked for / excluded eg:

Negative report: No “pathogen investigated” isolated.

Positive report: “Named Pathogen” isolated.










10. Post-Examination Procedures continued *Note:Reportsshouldindicateallpathogensthathavespecificallybeenlookedfor/excluded.

Report susceptibilities where clinically indicated.

Clinically urgent results should be promptly telephoned or sent electronically as soon as they become available.

A written report should be issued within 16-7� hours of receipt of a sample and if necessary, the report should indicate thatafinalreportistofollow.

10.3 Sample Retention, Storage & Disposal

Samples should be kept refrigerated for a minimum of 3 days after report is sent out.

Samplesshouldbeautoclavedbeforefinaldiscardingaccordingtothecurrentstandardsfordisposalofhazardouswaste.

11. Limitations and Pitfalls of the Procedure

SomenormalentericflorasuchasProteus spp and Citrobacter spp may resemble Salmonella sp on XLD. Non-specificagglutination.

Some antisera only give weak reactions. NB. The presence of Vi antigen in certain Salmonella sp may mask the O reaction.

False positive agglutination may occur if the isolate has lost its ‘O’ antigen and become rough.

C. difficile toxin is very unstable and degrades over time at room temperature. False negative results occur when specimens are not tested promptly or refrigerated until testing can be done.

12. References

Health Protection Agency (�006). Investigation of faeces specimens for bacterial pathogens. National Standard Method CRM-SOP 30 Issue 5. http://www.cdc.gov/ncidod/dhqp/id_cdiffFAQ_general.html








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