Antimicrobial disk susceptibility testing

Quality in The Clinical Microbiology Laboratory

Dr.S.Hekmat

Reference Health Laboratory

Total laboratory Quality Program

quality management(QM)

Continuous quality Improvement (CQI) or

Performance improvement (PI)

Quality Control (QC)

Quality Assurance (QA)

Test Cycle

Preanalytical

(sampling , labelling , transport , storage , specimen clinical information

↓Analytical

(macroscopic evaluation , microscopy , culture , identification , interpretation , antibiogram) .. ,

↓Post analytical

(Final report to physician)

Quality management ( QM )

QM is:

A process of looking of what is done

How it is done

Identifying opportunities for improvement

Making the appropriate changes

Assessing the impact

QC and QA are integral and essential components of QM.

CQI and PI

CQI and PI went a step further by seeking to improve patients care by placing the emphasis on not making mistake on first place; CQI and PI advocate continuous training to guard against having to correct

deficiencies

QUALITY ASSURANCE PROGRAMME QA

• A quality assurance (QA) programme is concerned with sampling, specifications and testing as well as with organization, documentation and release procedures that ensure that the necessary and relevant steps have been taken to ensure satisfactory quality.

• The bacteriology and immunology laboratory is responsible for providing accurate and relevant information that is of use for clinical diagnosis of a patient or in support of a public health activity. The accuracy and clinical value of the laboratory analysis of the clinical specimen and microbial isolates are dependent upon a QA programme that

•      assesses the quality of the specimen,

•      documents the validity of the test method,

•      monitors the performance of test procedures, reagents, media, instruments and personnel,

•      and reviews test results for errors and clinical relevance

An effective QA programme is dependent on a continuous process of assessment and

improvement. It has two broad components:

• Quality assurance = Internal quality control + External quality assessment

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QA is the sum of activities to ensure that test results are accurate , reproducible and timely

However

The speed , the cost , and the usefulness or clinical relevance of the test is important.

 

It must be comprehensive,

concentrate on the most critical steps ,

provide continuous monitoring of test procedures ,

able to detect and correct errors as they occur.

Internal quality control

• Internal quality control (IQC) is the set of procedures undertaken by the staff of a laboratory for continuously and concurrently assessing laboratory work and the emergent results, to decide whether they are reliable enough to be released. These results may be required for a wide variety of purposes; e.g. support of clinical decision making or epidemiological surveillance or research. Internal quality control procedures have an immediate effect on the laboratory’s activities and should actually control – as opposed to merely examining – the

laboratory’s output.

Internal Quality Control IQC

Practical

Realistic

Economical

QC program

The laboratory director is primarily responsible for QC and QA programs. However all laboratory personnel must actively participate in both program

Sources of error that effect the reliability and reproducibility

Sampling , transport,storage, processing :Specimens

Environmental factors: Inadequate space working :ventilation, temperature, excessive noise, unsafe :work conditions..,

Personnel:Education, training , experience, condition of employment

Laboratory materialsQuality or reagents, stains, chemicals, culture media, ,

Test method ; some methods are more reliable

Substandard or poorly maintained instruments:Equipments

Examination and reading :Hurried Reading of results , failure to examine

sufficient number of microscopic fields,…Reporting

Procedures

•Begins with proper laboratory operations based on SOPS for:

Collection of specimen

Registeration of specimen

Processing of specimen.

Care of equipment

Preparation and storage of culture media , stains , reagents and antisera

Antibiotic susceptibility tests

Stock cultures

Safety precautions

Personal hygiene

Handling and disposal of infected material

 

ParameterGuidelines

Specimen collection and transportation

Provide instructions for collection and transport

Establish criteria for acceptable specimens

Establish rejection criteria for unacceptable specimens

PersonnelUse sufficient qualified personnel depending upon volume and complexities of work

Provide continuous technical education

Provide written performance standards

Evaluate annually

QC recordsRecord all QC results on prescribed forms

Report all out-of-control observations to supervisor

Note corrective actions on QC form

Review QC records monthly

Patient reportsReport only to authorized personnel

Notify test requester of important values immediately

Provide normal ranges where appropriate

Correct errors in patient’s reports in timely fashion

Retain records for at least two years

Information should be filled

Patient name

Hospital or laboratory number

Ordering physician

Whether the patient receiving antimicrobial therapy

Suspect agent or syndrome

Written collection instruction

Test selection criteria

Patients selection criteria

Timing of specimen collection (e.g ,.

Before antimicrobial are administration)

Optimal specimen collection site

Approved specimen collection method

Specimen transport medium

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Specimen transport time and temperature

Specimen holding instructions if it cannot be transported immediately (e.g. hold at 4C for 24 hours)

Availability of test (on –site or sent to reference laboratory )

Hours test performed (daily or batch)

Turn-around time

Result reporting procedures

Criteria for unacceptable specimens

Unlabeled or mislabeled specimensUse of improper transport medium

Excessive transport timeImproper temperature during transport or storageImproper collection site for test requestSpecimen leakage out of transport container

Personnel

It is laboratory director's responsibility to employ sufficient qualified personnel for the volume and complexity of the work performed.

Document competency and training twice a year

Continuing education program should be provided

All documentation should maintained in personnel file

External quality assessment

• External quality assessment (EQA), which was earlier known as proficiency testing, refers to a system of objectively assessing the laboratory performance by an outside agency. The assessment is retrospective and periodic in clinical microbiology unlike industrial microbiology such as production of vaccines and immunobiologicals which, as per the laws of the land, require testing by an agency independent of the manufacturer before their release for use.

•The main objective of external quality assessment is to establish inter-laboratory comparability. This will influence the reliability of future testing. In contrast, the main objective of internal quality control is to ensure day-to-day consistency. Hence, both internal quality control and external quality assessment are complementary in ensuring the reliability of procedures, their results and finally the quality of the product

quality control of equipment

Biological safety cabinets

Autoclave

Oven

Incubator

Refrigerator

Water- bath

Microscope

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Maintenance records should be retained in the laboratory for the life of instrument. Specific guidelines regarding periodicity of testing for autoclaves, biological safety cabinets ,centrifuges ,incubators, microscope, refrigerators ,freezers, water bathes , heat blocks and other microbiology laboratory can be

found in reference books

Culture media

Ordering and storage of dehydrated media

Order quantities for 6 months or at most 1 year.

The overall quantity should be packed in containers that will be used up in 1-2 months.

Write date of receipt on each container

Store in a dark , cool, well ventilated place

When a container is opened , write the date of opening on it

Discard all dehydrated media that are either caked or darkened

•Storage of prepared media

•Distilled or demineralized water should be used for preparation of media

•Protect against sunlight •Protect against heat. Media containing blood, antibiotics ,

… should be stored in refrigerator

•The shelf-life of prepared media when stored in cool , dark place depend on the type of container used.

•Tybes with cotton – wool plygs 3 weks

•Tubes with loose caps 2 weeks

• containers with screw – caps 3 months

• Petri dishes , sealed in plastic bags 4 weeks

Commercially Prepared Culture Media

The NCCLS subcommittee on media quality control collected data over several` years regarding the incidence of QC failure of commonly used microbiology media Based on its finding the subcomm ittee published a list of media that did not require retesting in the user's laboratory if purchased from a manufactures who follow

NCCLS guidelines

pH testing

Prepared media should not be checked routinely.

If it is prepared in house from basic ingrediente it should be allowed to cool before the pH tested.

Solid media with a surface electrode or after macetration in distilled water.

If pH differs 0.2 units from the specification, adjust with acid or alkali or prepare a new batch.

Sterility Check

A representative sample of each media which blood or their components are added after autoclaving should be test for sterility; 3-5% of any batch is tested.Sterility is routinely checked by incubating the medium for 48 hours at the 35 C temperature . More than 2 colonies per plate if seen , discard

the whole batch. .

Performance testing

When medium dose need to quality controlled because it was prepared “in house (in the laboratory) or because it is complex, several basic

rules must be followed:

All media must be tested before use

Each medium must be tested with organisms expected to give positive reaction as well as with organism expected either not to grow or produce a negative reaction

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Performance tests for different commonly used media arePerformed by different microorganisms ( stock culture )

Blood agar :Performance test *( hemolysis and growth )

s.Pyogeness.PneumoniaeMedium Negative Bacitracin disc positive

s. pyogenes (zone) E.faecalis blood agar

شلشق ذمخخیperformed by differrent microorganisms ( stock cultures )

User-Prepared and Noncommercially prepared Media

QC forms for user-prepared media should contain:

The amount of prepared

The source of each ingredient

The lot number

The preparation date

The expiration date (Usually 1 month for agar plate and 6 month for tube media)

Sterlization methods:

All user prepared colures media also should checked for:

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Proper color

Depth

Smoothness

Clarity

Hemolysis

Excessive bubbles

Contamination

Stain and Reagents

Containers of stains and reagents should be labeled as to contents, concentration ,storage requirements, date prepared (or received) date placed in service ,expiration date, source (commercial manufacture or user prepared) and lot number .All stains and reagents should be stored according manufacture's recommendations and tested with positive and negative controls before

use .

Antisera

The lot number, date received, condition received ,and expiration date must be recorded for all shipments of antisera.In addition ,the antisera should be dated when opened .New lots must be tested concurrently with previous lots, and testing must include positive and negative controls .

Use of Stock Cultures

To operate a quality control program, stock culture must be maintained by all laboratories .They are available from many sources.

Commercial sources

Clinical specimens

Proficiency testing

Reference laboratories

Official Culture Collection (ATCC)

Preservation

Long –term

Lyophilization

Storage at -70C TSB with 15 % Glycerol

CTA for Neisseria and streptococci

Cooked-meat medium for anaerobes

Short-term

TSA slants

Store in refrigerator

Transfer every 2 weeks.

Stock strains for quality control suggusted by CLSI

Escherichia coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

Staphylococcus aureus ATCC 25923

Streptococcus pneumoniae ATCC 49619

Enterococcus faecalis ATCC 29212

Haemophilus influenzae ATCC 49247

ATCC 70063 Pneumoniae Klebsiella

Antimicrobial Susceptibility Testing( AST )

METHODS OF AST

Diffusion Dilution Diffusion&

Kirby – Bauer method MIC Dilution

Test method ٍٍٍٍٍٍٍٍٍE 1) Broth dilution

2 )Agar dilution

Reagents for the Disk Diffusion Test

1-Selection of Antimicrobial Disks (source of infection and the isolated pathogen )

2 -Mueller- Hinton Agar medium-prepared from commercially dehydrated powder

-4mm depth ( 25-30 ml for plates with100mm diameter )

-Store in 2-8 C within 7days

0.5 McFarland ) from isolated colony) 3-StandardTurbidity

Mueller Hinton Agar Medium

Preparation of Mueller Hinton Agar

Moisture

PH

Effects of Thymidine or Thymine

Effects of Variation in Divalent Cations

Testing Strains That Fail to Grow Satisfactorily

Turbidity Standard

0.5 McFarland standard

(0.5 ml of 0.048 mol/ml BaCl2 added to

99.5 ml of 0.18 mol/L H2SO4(1% v/v)

density in 625 nm = 0.08 - 0.1

Keep in 4-6 ml aliquots in screw cap

tubes in dark , room temperature

Replace or verify density month

04/19/23 AST 42

04/19/23 AST 43

Common sources of error

Inhibitory substance thymine and thymidine:Enterococcus faecalis ATCC 29212 or 33186 tested by SXT disc . An inhibition

.zone ≥ 20 mm and free of colony.

Control zones too small may be due to: Discs with low potency for bad storage or expired discs .

Too dense inoculum .

Too thick culture media.

small zones for tetracyclines and beta- lactams indicate pH is too high.

zones for aminoglycosides which are too small indicate pH is too low

Salmonella and Shigella

:Fecal isolates

Ampicilin , a quinolone , SXT

Extraintestinal isolates of Salmonella;

In addition chloramphenicol and a third generation cephalosporin should be tested and reported.

Vibrio cholerae

MIC is the best method :

Broth or agar dilution method

Antibiotic agents:

Ampicillin

Tetracycline

SXT

Chloramphenicol

Antibiotic susceptibility of Neisseria gonorrhoeae

Medium : GC agarInoculum ; Direct colony suspensionIncubation ; 35 C , 5% CO2 , 20-24h

Disk diffusion tests with ampicillin , penicillin , rifampin are unreliable for Nisseria meningitidis , MIC is necessary.

Positive β - lactamase test predicts resistance to ampicillin , penicillin ,amoxicillin.

Gonococci with 10-unit penicillin disk zone ≤ 19 mm are likely to beβ - lactamase producing strains.

β - lactamase test remains preferable to other susceptibility methods for rapid , accurate penicillin resistance

Patient Report

The laboratory should established a system for supervisory of all laboratory reports. This review should involve checking the specimens workup to verify that the correct conclusion were drawn and no clerical errors were made in reporting results. Reports should be given only given only

authorized by law to receive them .

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Clinician should be notified about ‘’panic values’’ immediately. Panic values are potential-threatening results, for example positive Gram stain for CSF or a positive blood culture. All patients records should be

maintained for least 2 years .