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
Specimens: Sampling , transport,storage, processing
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
:
Equipments Substandard or poorly maintained instruments
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 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 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
Bacitracin disc
positive
Negative Medium
s. pyogenes (zone)
E.faecalis
blood agar
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
Klebsiella Pneumoniae ATCC 70063
Antimicrobial Susceptibility Testing
( AST )
METHODS OF AST
Diffusion
Kirby – Bauer method
Dilution
MIC
2) Agar dilution
Diffusion &
Dilution
Test method
Eٍٍٍ
1) Broth 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
3-StandardTurbidity )0.5 McFarland ) from isolated colony
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
3/14/2016
AST
42
3/14/2016
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 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 β - lactamase test predicts resistance to ampicillin , penicillin ,amoxicillin.
Gonococci with 10-unit penicillin disk zone ≤ 19 mm are likely to be
β - 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.