CARIBBEAN REGIONAL MICROBIOLOGY STANDARD ...Laboratory detection and reporting of bacteria with Extended Spectrum ß-lactamases (ESBL). 2. Purpose To ensure the correct, validated

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

ESBL Detection – SOP No: CRM-SOP 5

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

ESBL Detection

�STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEANA 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 9

5. Staff Competency Requirements 9 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 10

9. Examination Procedures 11

9.1 Quality Control 11

9.� Microscopy 11

9.3 Culture 11 9.4 Identification 11

9.5 Susceptibility Testing 12

9.6 Sample Referral 12

10. Post-Examination Procedures 12 10.1 Interpretation of Results 12 10.� Reporting 13

10.3 Sample Retention, Storage & Disposal 13

11. Limitations and Pitfalls of the Procedure 13

1�. References 13


Title: ESBL Detection SOP No: CRM-SOP: 5

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

Methods Drafting Group Review Date: 1st September �008

ESBL Detection

3STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEANA CARIFORUM Project Funded by the European Union and Implemented by CAREC



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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 5

Controlled Document Title Standard Operating Procedure for ESBL Detection

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

ESBL Detection


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

ESBL



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ESBL Detection


1. Title

Laboratory detection and reporting of bacteria with Extended Spectrum ß-lactamases (ESBL).

2. Purpose

To ensure the correct, validated procedure is followed for detecting ESBL’s, and provide accurate, reliable, reproducible results having clinical utility.

3. Introduction

ESBL are acquired, class A plasmid-mediated enzymes which that hydrolyse and confer resistance to oxyimino – ß-lactams such as the ‘�nd and 3rd generation’ cephalosporins, e.g. cefuroxime, cefotaxime, ceftazidime and ceftriaxone are the most important.

Many TEM related have been described but there are also other non-TEM related enzymes which are capable of conferring resistance to other anitibiotics such as cefoxitin and some carbapenems. ESBLs occur mostly in Enterobacteriaceae (e.g. E. coli, Klebsiella species and Enterobacter species) and rarely in nonfermenters (e.g. P. aeruginosa).

ESBLsareclinicallyimportantbecausetheydestroycephalosporins–antibioticswhichareusedasfirstlineagentsincriticallyill patients, including those with intra-abdominal infections, pneumonias and bacteraemias.

Some ESBL producers achieve outbreak status, spreading among patients and locales, perhaps owing to particular pathogenicity traits.

Many ESBL producers are multi-resistant to non-ß-Lactam antibiotics such as quinolones, aminoglycosides, and trimethoprim, thus, limiting treatment options.

ESBL-mediated resistance is not always obvious in vitro to all cephalosporins.

The basic strategy for detecting ESBL producers is to use an indicator cephalosporin to screen for likely producers, then to seek cephalosporin/ clavulanate synergy, which distinguishes ESBL producers from, for example, AmpC or K1 enzymes producers.

In all strains with ESBL, the zone diameter for one or more of the indicator cephalosporins is expanded by the clavulanate. Another distinguishing mark is that all ESBL producers are in vitro sensitive to Cefoxitine; others are resistant.

The ideal indicator cephalosporin is one to which all ESBLs confer resistance, even when their production is scanty. Choice is predicated by the following general traits:

TEM & SHV ESBLs – obvious resistance to ceftazidime, variable to cefotaxime.CTX-M ESBLs – obvious resistance to cefotaxime: variable to ceftazidime.All ESBLs – obvious resistance to cefpodoxime.Cefuroxime, cephalexin and cephradine (see below) are unreliable indicators.

It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance.



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ESBL Detection


4. Scope

This procedure provides detailed instructions for the detection of ESBL’s, 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 II containment; require all personal protective equipment (PPE).

Any procedure that is likely to produce aerosols should be performed in a biosafety cabinet.

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

All Microbiological samples for processing should be considered a potential risk of causing transmissible infections and, therefore, universal safety precautions should be observed.

All work surfaces should be disinfected 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.

7. Pre-Examination Procedures

7.1 Sample Type

Isolates suspected of being ESBL producing organisms.

7.2 Sample Collection

N/A–sampleswillbeprocessedaccordingtotheirspecificSOP,evenifsentforscreeningforESBL.



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7. Pre-Examination Procedures continued

7.3 Sample Transport & Storage

If isolates are referred for testing a completed request form should accompany the specimen.

Adherence to international transport regulations is essential.

7.4 Rejection Criteria

Documentation of any discrepancies found on receipt must be made by laboratory personnel and a record kept in the laboratory for future reference.

N/A

Referredisolatesthatarenotaccompaniedbyacompletelyandcorrectlyfilledinrequisitionform.

Isolates that are received in leaking containers or in inappropriate containers.

Isolates sent in transport medium showing signs of obvious contamination.

Samples that have not been properly stored before being received by the Lab.

Samples that are too old- prolonged delay before receipt in the Lab.

7.5 Relevant Clinical Information

N/A.

8. Table of Media, Reagents, Materials and Equipment



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Media Reagents Materials Equipment

MH-agar medium

Mac Conkey agarmedium

ESBL-positiveE.coli strains

ESBL-negativeE.coli strains

E-test ESBL strips

CTX discs 30μg

CAZ discs 30μg

Cefpodoxime discs 10μg

Sterile physiologicalsaline

Sterile Loops

Grease Pencilor Marker

35-37°C O�incubator

Bunsen Burner or bacti-cinerator

ESBL Detection


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.

Quality control should be performed on all media, stains, and reagents used in the examination of specimens.

Ensure that reagents, stains and media are not used beyond the expiration dates.Check all reagents and stains before use to ensure that they are free from contamination, debris or deposits.

Examine all plates just prior to use to ensure that there is no contamination or signs of deterioration such as drying, cracking, shrinkage or discoloration of media.

Document the results of all QC tests on the appropriate forms available in the department for that purpose.

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

Refer to SOPs for media, Gram stain and relevant biochemical tests for further details.

QC of the cefpodoxime, CTX and/or CAZ discs used in primary screening should be in accordance with standard BSAC or CLSI recommendations, as appropriate.

The NCTC recommends as ESBL- positive control, E.coli strains: NCTC 13353, NCTC 13351, NCTC 1335�.

The CLSI and AB Biodisk (E-test) recommend K. pneumoniae ATCC 700603 as an ESBL-positive control.

E.coli NCTC 10418 or ATCC �59�� should also be used as a negative control.

9.2 Microscopy

N/A.

9.3 Culture

AllspecimensarealsoplatedontoMacConkeyagarspecificallyfortheisolationofEnterobacteriaceae.Refer to CRM-SOP ��: Inoculation of Culture Media.

9.4 Identification

Identificationshouldbeperformedbyautomatedsystemsorconventionalbiomedicaltests. It isessential thattheorganismisidentifiedtospeciesleveltoenablecorrectinterpretationoftestresults.



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

9. Examination Procedures continued

9.5 Susceptibility Testing

The indicator drugs should be included in primary susceptibility testing done using a standardized method.

Enterobacteriaceae isolates resistant to any indicator cephalosporin in the screening tests outlined above should besubjectedtoconfirmatorytests.ConfirmationofESBLproductiondependsondemonstratingsynergybetweenclavulanate and those indicator cephalosporin(s) to which the isolate was initially found resistant.

9.6 Sample Referral

N/A

10. Post-Examination Procedures

10.1 Interpretation of Results

The BSAC zone diameter breakpoints for cefpodoxime have recently been revised to <=19 (R) and >=�0 (S). It is acknowledged that isolates with zone diameters of �1-�5 mm may deserve further investigation, although they are unlikely to produce an ESBL.

If CLSI methodology is followed, attention should be paid to the Standards’ low breakpoints for ESBL detection, not only their (much higher) therapeutic breakpoints.

10.1.1 Combination Disc Methods

(Oxoid or Becton Dickinson ‘Combination Discs’ and Mast ‘MAST DD’).

These compare the zones of cephalosporin discs to those of the same cephalosporin plus clavulanate. According to the supplier, either the difference in zone diameters, (Oxoid) or the ratio of diameters, is compared (Mast and BD) with zone diameter increases of >5 mm�1 or >50%�� in the presence of the clavulanate implying ESBL production. These tests are cheap and do not require critical disc spacing.



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Antibiotic & disc content Zone breakpoints (mm) MIC (mg/L)

R, < S, > R, < S, >

Cefotaxime, 30 ug �9 30 1 1

Ceftazidime, 30 ug E. coli & Kleb �1 ��

Ceftazidime, 30 ug, other species �7 �8 � �

Cefpodoxime, 10 ug �5* �6* 1 1

ESBL Detection


10. Post-Examination Procedures continued

10.1.2 Etest ESBL strips

(AB Biodisk, Solna, Sweden; Bio-Stat, Stockport, UK). These have a cephalosporin gradient at one end and a cephalosporin + clavulanate gradient at the other. These compare the zones of cephalosporin discs to those of the same cephalosporin plus clavulanate

Users should follow the manufacturer’s instructions, including for a heavier inoculum than in BSAC disc tests. ESBL production is inferred if the MIC ratio for cephalosporin alone: cephalosporin + clavulanate MIC is >8.

These are accurate and precise, but more expensive than combination discs. 10.2 Reporting Report ESBL producers resistant to ALL penicillins, cephalosporins (except cefoxitin) and to aztreonam, irrespective of routine susceptibility results.

Note: Carbapenems (e.g. imipenem and meropenem) are active against ESBL producers, as long as these do not have additional resistance mechanisms.

10.3 Sample Retention, Storage & Disposal

ESBL positive strains isolated from suspected outbreaks may be stored on nutrient agar slope at room temperature pendingreferraltoaReferenceLaboratoryforconfirmationandtyping.

11. Limitations and Pitfalls of the Procedure

Enterobactericeae with inducible Amp C chromosomalal enzymes (e.g. Enterobacter species, C. Freundii, M. morganii, Providencia species and Serratia species) should be tested with an Amp C stable cephalosporin (i.e. cefepime or cefpirome) in the clavulanate synergy test.

The Amp C enzymes may be induced by clavulanate (which inhibits them poorly) and may then attack the cephalosporin, masking synergy arising from inhibition of the ESBL.

ESBL methods were not developed for P. aeruginosa, Acinetobacter species and S. maltophilia and should not be used for them.

10 – �0% of K. oxytoca isolates, susceptible to CAZ, with high level resistance to TZP and CXM due to hyperproduction of their class A “K1” chromosomal ß-lactamase, may be confused with ESBL producers.

12. References

Health Protection Agency (�006). Laboratory detection and reporting of bacteria with extended spectrum ß-lactamases. National Standard Method QSOP 51 Issue �.

CLSI M�-Ag vol.�6 pg. 1g



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

Methods Drafting Group Review Date: 1st July �008

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CARIBBEAN REGIONAL MICROBIOLOGY STANDARD ...Laboratory detection and reporting of bacteria with Extended Spectrum ß-lactamases (ESBL). 2. Purpose To ensure the correct, validated