caribbean regional microbiology standard operating ... · title: culture of sputum for bacterial...

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

Sputum Culture for Bacterial Pathogens other than Mycobacteria – SOP No: CRM-SOP 13

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

Sputum Culture

�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


Title: Culture of Sputum for bacterial SOP No: CRM-SOP: 13

pathogens other than mycobacteria Version: 1

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

Methods Drafting Group Review Date: 1st September �008

Acknowledgements 3

Introduction 5

Amendment Procedure 6

Process Flow Chart 7

1. Title 8

�. Purpose 8

3. Introduction 8

4. Scope 9

5. Staff Competency Requirements 9 6. Safety Instructions 9

7. Pre-Examination Procedures 10 7.1 Sample Type 10

7.� Sample Collection 10

7.3 Sample Transport & Storage 10

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

9. Examination Procedures 12

9.1 Quality Control 12

9.� Microscopy 12

9.3 Culture 13 9.4 Identification 13

9.5 Susceptibility Testing 15

9.6 Sample Referral 16

10. Post-Examination Procedures 17 10.1 Interpretation of Results 17 10.� Reporting 17

10.3 Sample Retention, Storage & Disposal 18

11. Limitations and Pitfalls of the Procedure 18

1�. References 18

Sputum Culture

3STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN





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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




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

Controlled Document Reference CRM-SOP 14

Controlled Document Title Standard Operating Procedure for Sputum Culture for Bacterial Pathogens other than Mycobacteria

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

Sputum Culture



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

SPUTUM CULTURE




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1. Title

Sputum Culture.

2. Purpose

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

3. Introduction

Lower respiratory tract samples are primarily submitted for the detection of the causative agents of pneumonia. The most commonsampletypeisexpectoratedorinducedsputumalthoughthisisnottheoptimalsampleasculturefrequentlyreflectsupperrespiratorytractfloraandnormalcarriage.Bronchial lavage,washes,brushingsandtranstrachealaspiratesarethesamples of choice for the diagnosis of pneumonia. They may be received but require additional handling procedures.

The most common causative agents of bacterial pneumonia in non-hospitalized patients are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.

In hospitalized patients Staphylococcus aureus, Klebsiella pneumoniae, Acinetebacter sp. Escherichia coli, and Pseudomonas aeruginosa are more common and are frequently multi-resistant as a result of prolonged antibiotic use.

Legionellae should also be considered as a cause of pneumonia.

Pneumocystis carinii is a major cause of pneumonia in immunocompromised patients.

Cysticfibrosis(CF).

Sputum samples frompatientswith cystic fibrosis should target the culture of organisms specifically associatedwith thedisease. These include, most commonly, S. pneumoniae (mainly isolated in early stage of disease), H. influenzae, S. aureus, and P. aeruginosa. In the later stages of disease B. cepacia complex and Stenotrophomonas maltophilia may also be isolated and are of considerable concern in both the treatment of patients and spread of infection to others. Organisms from CF patients do not always appear as typical colonies on primary isolation. Strains of P. aeruginosa may appear as mucoid or non-mucoidcolonies,withorwithouttheproductionofpigment.Allcolonialvariantsshouldbeidentifiedandhavesusceptibilitytests performed. Aspergillus sp. and tuberculosis may also be associated with infection in this group of patients.

As P. aeruginosa is isolated from themajority of samples the presenceof other significant organismsmaybemaskedorinhibited.Theuseofspecificselectivemediaisstronglyrecommended.

Recoveryandidentificationoforganismsdependonseveralfactors:• the quality of the sample.• theavoidanceofcontaminationbyupperrespiratorytractflora.• the use of suitable microscopic and culture methods that will also allow for the growth of fastidious organisms.• whether or not antimicrobial therapy has been initiated. • the use of appropriate selective media for some organisms.




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3. Introduction continued

Care should be taken when interpreting the results of Gram-stained smears of sputum from individuals who are on antibiotics. Organisms that are visible on the smear may be non-viable.

4. Scope

This procedure provides detailed instructions for processing sputum samples for culture and sensitivity, as offered by regional laboratories. It may be adopted or adapted by any laboratory as needed, provided that such adaptations use 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 � containment; require all personal protective equipment (PPE).

All procedures, especially primary handling of samplesamplesamples should be performed under a Biosafety cabinet (BSC) level II.

Level 3 containment if possible; require all personal protective equipment (PPE).

Gloves and masks must be worn.

All microbiological samples for processing should be considered a potential source of transmissible infections so universal safety precautions should be observed.

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

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

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

Samplesandcultureplatesshouldbeautoclavedbeforefinallydiscarding.




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Sputum Culture



7. Pre-Examination Procedures

7.1 Sample Type Expectorated or induced sputum.

7.2 Sample Collection

Sample should be collected before antimicrobial therapy is initiated where possible.

Collect a minimum of 1mL expectorated or induced sputum into a sterile, screw-capped, leakproof container (early morning samples are generally more suitable). Samples are best collected as soon as the patient wakes and before any mouth-wash is used.

Adequate safety precautions must be taken to prevent the spread of infectious organisms, and to avoid contaminating the outside of the container.

Wipe the outside of the container with disinfectant on receipt in the laboratory.

Ensure that:• the sample is clearly labeled with the patient’s name, number, date and time of collection, and type of

sample.• the sample is placed in a sealed plastic bag.• the sample is transported to the laboratory along with the requisition form as soon as possible, preferably

within two hours of collection.

If pulmonary tuberculosis is suspected, three separate samples should be sent for Acid Alcohol Fast Bacilli (see CRM-SOP 1 ‘Microscopy of sputum to detect Acid Alcohol Fast Bacilli (AAFB)’.

7.3 Sample Transport & Storage

Samples should be transported as soon as possible to the laboratory. If a delay in processing is unavoidable, the sample must be refrigerated at �-8ºC for up to 48 hours.

Delays of over 48 hours are undesirable.

Any delay in processing beyond �-3 hours may compromise the isolation of more fastidious organisms such as Haemophilus influenzae and Streptococcus pneumoniae, and may result in the overgrowth with enterobacteriaceae.

7.4 Rejection Criteria

• unlabelled or incorrectly labeled sample. • sample sent in unsterile or inappropriate container..• leaking/ contaminated samples.• sample not accompanied by properly completed requisition form.




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7. Pre-Examination Procedures continued

• unrefrigerated samples > � hours old.• sample > 48 hours old.• sample is of poor quality as determined by the Gram Stain.• sputum samples for mycobacteria and legionella culture, from immuno-suppressed and neutropenic patients,

andfromthosethataredifficulttoobtainsuchaswithintubatedpatients,shouldnotberejectedbasedonthe quality of the sample.

• rejected samples should be stored in the refrigerator at 4°C for up to 48 hours before appropriately discarding according to safe, acceptable standards.

7.5 Relevant Clinical Information

• date of onset of symptoms.• antimicrobial therapy if any.• fever, abnormal chest X-ray results, chronic, productive cough.• travel to known MDR areas.• previous anti-tuberculosis therapy.• contact with known positive TB case.

8. Table of Media, Reagents, Materials and Equipment




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Equipment/Material Media/Reagents

Biosafety CabinetWire loopBacti-cinerator/BunsenflameO� Incubator 35-37ºCCO� incubator 35-37ºCorcandle Jar Light MicroscopeImmersion oilLens cleanerLens PaperSterile Pasteur PipettesPaper Towels/Tissue PaperStaining RackAntibiotic susceptibility DiscsGrease Pencil/ MarkerSterile glass tubesMicroscope slidesCover slips

5% sheep blood agarChocolate agarMac Conkey agarAPI/Biochemicals/VitekMueller Hinton (MH) agarMH-Blood AgarGram Stain reagents Distilled waterSterile saline (0.85%)70% alcoholFreshly prepared 10% bleach solution

Sputum Culture

1�STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN


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.

Ensure all required QC is performed.

Perform appropriate Quality Control (QC) according to the SOP for all media, biochemicals and antibiotics discs used inindividualstaining,cultureandidentificationtechniques.

Appropriate American Type Culture Collection (ATCC) Control strains should be used wherever possible.

Results of all QC testing should be recorded on the appropriate QC forms available in the department.

Results of all tests are invalid if the QC test results are not as expected.

Ensure that reagents, stains and media are not used beyond the expiry date.

Check all reagents and stains before use to ensure that they are free from contamination, debris or deposits.

All media used should be examined visually just before use to ensure that there is no contamination or deterioration appearing as lysis, discolouration, drying, shrinkage, or cracking of the media.

9.2 Microscopy

a. Label a new clean slide with the patient’s information using a pencil.b. Preparation of the smear and culture should be undertaken in the Biosafety cabinet.c. Prepare a thin smear from the most purulent material in the sputum, by transferring a portion of the sample

to the centre of the slide using a sterile loop, swab or stick.d. AllowtoairdryinaBiosafetycabinetlevelII,thenheat-fixorfixinmethanol.e. Heatfixbypassingtheslideuppermost,threetimesthroughtheflameofabunsenburner.(Donotoverheat-

it should be possible to rest the back of the slide on the hand comfortably). Allow slide to cool before staining.

f. Formethanol fixation, floodsmearwithmethanoland leave forabout twominutesoruntil themethanolevaporates.

g. Stain the dry smear using Grams Stain. Refer to CRM-SOP 7: Gram Stain.h. Allow the stained smear to dry before examining under low power (x10 objective) for the presence of white

blood cells (WBC’s) and squamous epithelial cells (SECs). Examine under oil immersion (100 x objectives) for the presence of organisms.

i. Recordfindings.j. Reject sample if excessive oropharyngeal contamination is demonstrated ie large numbers of SECs and few

WBCs.




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9. Examination Procedures continued

9.3 Culture

a. Describe and report the appearance of the sample ie whether salivary, muco-salivary, purulent, muco purulent, mucoid, blood stained.

b. Using a sterile swab or loop, select a purulent portion of the sample. c. Inoculate media and streak for the isolation of single colonies using a sterile loop. d. Incubate plates as indicated below:

9.4 Identification

9.4.1 Target Organisms

Organism Minimum Level of Identification

• Haemophilus influenzae• Streptococcus pneumoniae• Staphylococcus aureus• Pseudomonas aeruginosa• Moraxella catarrhalis• Klebsiella pneumoniae• Burkholderia cepacia complex• Stenotophomonas maltophilia• Enterobacteriaceae• Fungi• Pasteurella sp.• Pseudomonads• Yeasts• Legionella




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Media Incubation

5% Sheep blood agarChocolate agar (+ bacitracin disc if CF sample)

MacConkey agar

B. cepacia selective agar(if CF sample)

Mannitol Salt agar (if CF sample)

CO�,35-37˚Cfor24-48hoursCO�,35-37˚Cfor24-48hours

O�,35-37˚Cfor24-48hours

O�,35-37˚Cfor24-48hoursthenatroom temperature further 3 days

O�,35-37˚Cfor24-48hours

• species level• species level• species level• mucoid or non-mucoid species level• species level• species level• species level• species level• “coliforms” level• genus level• species level• “pseudomonads” level• “yeasts” level• refer to Reference laboratory

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9.4.2 Organisms Associated with Community Acquired Pneumonia

• Streptococcus pneumoniae• Haemophilus influenzae• Moraxella catarrhalis• Klebsiella pneumoniae• Legionella pneumophila

9.4.3 Organisms Associated with Hospital Acquired Pneumonia

• Pseudomonas aeruginosa• Acinetobacter spp• Klebsiella spp• Enterobacter spp• MRSA

Many of the above organisms may be antibiotic resistant due to prolonged antibiotic use.

9.4.4 Organisms Associated with Cystic Fibrosis

• S. aureus• H. influenzae• S. pneumoniae• P. aeruginosa - mucoid and non-mucoid strains• B. cepacia complex• S. maltophilia• Aspergiluus fumigatus

Sputum Culture




9.4.5 Plate Reading Procedure

Examine plates for growth and perform the following tests based on the colonial morphology, haemolysis and Gram reaction of the colonies isolated.

9.5 Susceptibility Testing

Perform antimicrobial susceptibility testing (AST) as per CLSI guidelines on heavy growth or pure growth of the following:

• H. influenzae,• S. pneumoniae• B. catarrhalis

Thefollowingorganisms,ifpresent,mayrepresentsuperficialcolonizationonly(exceptincysticfibrosis).Susceptibilitytesting, reporting and treatment may not be indicated. Discussion with the medial microbiologist and clinician to assessclinicalsignificanceisrecommendedbeforetestingandreportingsusceptibilityresults:

Alpha-haemolytic streptococci

Beta-haemolytic streptococci

Haemophilus spp.

Oxidase positive, Gram-negative diplococci

Staphylococci

Enterobacteriacea, Pseudomonas sp. (each colonial type)B. cepacia complexS. maltophilia

PerformtheoptochintesttoconfirmorexcludeS. pneumoniae

Susceptibility,PerformLancefieldgroupingorbacitracinsusceptibilitytoconfirmorexcludeGroup A BHS

PerformXandVfactorstestingtoconfirmorexclude H. influenzae

ConfirmB-lactamasepositiveGram Stain – Perform rapid sugar fermentation, CTAsugarsorcommercialkitstoconfirmor exclude B. catarrhalis. Also test for beta-lactamase production

Performcoagulaseorlatextesttoconfirmorexclude S. aureus

Identify using normal method ie API, MICRO ID, In House Biochemicals, VITEK or Microscan




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• S. aureus (reportandtestifcysticfibrosis).• P. aeruginosa(reportandtesteachcolonialtypeifcysticfibrosis).• B. cepacia complex(reportandtestifcysticfibrosis).• Acinetobacter sp.• Stenotrophomonas sp.

Report susceptibility test results as clinically indicated.

9.6 Sample Referral

Samples requesting Legionella pneumophila or Pneumocystis cariniispecifically.

Anyfungiisolatedthatrequiresidentificationandsusceptibilitytesting.

Isolates associated with outbreaks and where clinically indicated.

Any organism isolated with unusual or unexpected resistance pattern, or if there is a laboratory or clinical problem that requires elucidation.

Always notify Reference Laboratory before shipping samples for further testing, especially if an urgent sample is included in the shipment.

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

When samples require overseas shipping, there should be strict adherence to IATA regulations.




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10. Post-Examination Procedures

10.1 Interpretation of Results 10.1.1 Interpretation of Gram Stain:

10.1.2 Interpretation of Cultures:

a. After �4 hours incubation, examine the plates. If using selective agar refer to manufacturer’s instructions on colonial morphology.

b. Ifrequired,performappropriateteststodeterminetheorganism(s)present.Anyplateswithsignificantgrowthofapotentialpathogenmustbequantified,identified,andhavesusceptibilitytestingperformedifclinicallyindicated.

c. Re-incubateplatesshowingonlynormalfloraforanother24hoursandre-readthefollowingday.d. After 48 hours incubation note any changes to the culture result and performthe appropriate testing required

to identify the organism(s). Perform susceptibility testing if indicated.

10.2 Reporting 10.2.1 Gram Stained smear:

• Reportwithquantificationthepresenceofpuscells,organisms,andepithelialcells.• Report fungal hyphae if present.• If the sample is unsuitable for culture according to the table above in 10.1.1, report the Gram result and add

the comment: “Gram smear indicates excessive oropharyngeal contamination, therefore the sputum sample is not suitable for culture. Please resubmit”.

• Urgent microscopy results should be promptly telephoned and /or sent electronically as soon as they become available.

• A written report should be issued within 18-7� hours of receipt of a sample, and if necessary, it should state thatafinalreportistofollow.

Group No. Cells per x10 magnification field

Leucocytes Squamous Epithelial Cells (WBC’s) (SEC’s)

6543�1

>�510 - �5

<�5>�5

10-�5<10

<�510 - �5

<10>�5>�5>�5

Groups 1, �, and 3: evidence of oral contamination – need not be cultured unless special request.

Groups 4, 5, and 6 (WBC’s present): good, purulent sample with little or no oral contamination - culture and examine under oil immersion (x100 objective) for organisms.




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10. Post-Examination Procedures continued

10.2.2 Culture:

• Quantifyandreportallclinicallysignificantpathogensandreportwithappropriateantimicrobialsusceptibilityresults.

• Ifonlynormaloralflorapresent,reportas“NoSignificantGrowth”.• If no growth, report as “No growth.” or report the absence of growth for a particular targeted organism eg. in

CF patients.

10.3 Sample Retention, Storage & Disposal

Sample should be held for a minimum of 3 days after results are sent out.

Samplesshouldbeautoclavedbeforefinaldiscardingaccordingtothecurrentstandardsfordisposalofhazardouswaste.

11. Limitations and Pitfalls of the Procedure

Poorqualitysamplesmustnotbeculturedunlessbyspecificrequest. Ifcultured,acommentregardingthepoorquality of the sample should be included in the report.

Procedure does not allow for isolation of other agents such as Legionella sp. These samples must be referred to the Reference Laboratory.

Failuretouseselectivemediaforspecifictargetorganismsonsamplesfromcysticfibrosispatientsmayresultinsomeorganisms being overlooked or overgrown with Pseudomonas sp.

12. References

1) Microbiology QA Practices & Procedures: Sputum / Lower Respiratory Culture, Oct. �004.�) Mario Clarke Martin and Parimi Prabhakar, Caribbean Procedure Guidelines for Diagnostic Microbiology. 3) Health Protection Agency (�005). Investigation of Bronchoalvelolar Lavage, Sputum and Associated Samples. National Standard Method. CRM-SOP 57. Issue 1.




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