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 laboratorys activities and
should actually control as opposed to merely examining the
laboratorys 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 materials
, ,Quality 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

Parameter

Guidelines

Specimen collection and Provide instructions for collection and transport

transportation
Establish criteria for acceptable specimens
Establish rejection criteria for unacceptable specimens

Personnel

Use sufficient qualified personnel depending upon volume and complexities of work
Provide continuous technical education
Provide written performance standards
Evaluate annually

QC records

Record 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 reports

Report only to authorized personnel

Notify test requester of important values immediately
Provide normal ranges where appropriate
Correct errors in patients 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 specimens
Use of improper transport medium
Excessive transport time
Improper temperature during transport or storage
Improper collection site for test request
Specimen 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 are
Performed by different microorganisms ( stock culture )
Blood agar
Performance test *( hemolysis and growth ) :
s.Pyogenes
s.Pneumoniae
Medium Negative
Bacitracin disc
positive
s. pyogenes (zone)

E.faecalis

blood agar

performed 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
Kirby Bauer method
Test method E

Dilution
MIC

Diffusion
Dilution
1) Broth dilution

Agar dilution( 2

Reagents for the Disk Diffusion Test

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

Mueller- Hinton Agar medium- 2

prepared from commercially dehydrated powder 4mm depth ( 25-30 ml for plates with100mm diameter ) Store in 2-8 C within 7daysMcFarland ) from isolated colony)

StandardTurbidity

3- 0.5

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
McFarland standard 0.5
ml of 0.048 mol/ml BaCl2 added to 0.5)
ml of 0.18 mol/L H2SO4(1% v/v) 99.5
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

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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 agar
Inoculum ; Direct colony suspension
Incubation ; 35 C , 5% CO2 , 20-24h
Disk diffusion tests with ampicillin , penicillin , rifampin are unreliable for Nisseria
.meningitidis , MIC is necessary
.Positive - lactamase test predicts resistance to ampicillin , penicillin ,amoxicillin
Gonococci with 10-unit penicillin disk zone 19 mm are likely to be
. - lactamase producing strains
- 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