1. Antimicrobial disk susceptibility testing

2. Quality in The Clinical Microbiology Laboratory
Dr.S.Hekmat
Reference Health Laboratory

3. Total laboratory Quality Program
quality management(QM)
Continuous quality Improvement (CQI) or
Performance improvement (PI)
Quality Control (QC)
Quality Assurance (QA)

4. ( Final report to physician )
Test Cycle
Preanalytical
(sampling , labelling , transport , storage , specimen clinical information ↓
Analytical
( macroscopic evaluation , microscopy , culture , identification , interpretation , antibiogram , .. )
Post analytical
( Final report to physician )

5. 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 .

6. 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

7. 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

8. Continue….
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.

9. 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.

10. Internal Quality Control IQC
Practical
Realistic
Economical

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

12. 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

13. 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

14. Parameter
Guidelines
Specimen collection and transportation
Provide instructions for collection and transport
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

15. Information should be filled
Patient name
Hospital or laboratory number
Ordering physician
Whether the patient receiving antimicrobial therapy
Suspect agent or syndrome

16. 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

17. Continue… 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

18. 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

19. 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

20. 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

21. quality control of equipment
Biological safety cabinets Autoclave Oven Incubator Refrigerator Water- bath Microscope

22. Continue..
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

23. 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

24. 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

25. 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

26. 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 units from the specification, adjust with acid or alkali or prepare a new batch.

27. 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..

28. 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

29. Continue.. 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
ذمخخی شلشق
performed by differrent microorganisms ( stock cultures )

30. 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 :

31. Continue. Proper color Depth Smoothness Clarity Hemolysis
Excessive bubbles
Contamination

32. 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.

33. 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.

34. 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)

35. 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.

36. Stock strains for quality control suggusted by CLSI
Escherichia coli ATCC Pseudomonas aeruginosa ATCC Staphylococcus aureus ATCC Streptococcus pneumoniae ATCC Enterococcus faecalis ATCC Haemophilus influenzae ATCC ATCC Pneumoniae Klebsiella

37. Antimicrobial Susceptibility Testing ( AST )
METHODS OF AST
Diffusion Dilution Diffusion &
Kirby – Bauer method MIC Dilution
Test method ٍٍٍE ) Broth dilution
2) Agar dilution

38. 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 ( ml for plates with100mm diameter )
-Store in 2-8 C within 7days
0.5 McFarland ) from isolated colony) 3-StandardTurbidity

39. 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

40. Turbidity Standard 0.5 McFarland standard
( 0.5 ml of mol/ml BaCl2 added to
99.5 ml of mol/L H2SO4(1% v/v)
density in 625 nm =
Keep in 4-6 ml aliquots in screw cap
tubes in dark , room temperature
Replace or verify density month

42. 4/17/2017
AST

43. 4/17/2017
AST

44. Common sources of error
Inhibitory substance thymine and thymidine:
Enterococcus faecalis ATCC or 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

45. 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.

46. Vibrio cholerae MIC is the best method : Broth or agar dilution method
Antibiotic agents :
Ampicillin
Tetracycline
SXT
Chloramphenicol

47. 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

48. 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.

49. Continue..
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.