Today s Topics. General Quality Control Best Practices. Practices Antimicrobial Effectiveness Testing(AET) Best Practices Environmental Isolates

Transcription

1 Laboratory Best Practices in QC Microbiology

2 Today s Topics General Quality Control Best Practices Growth Promotion Testing (GPT) Best Practices Antimicrobial Effectiveness Testing(AET) Best Practices Environmental Isolates Validation of Prepared Microorganisms

3 Eight Best QC Practices 1. Use quality control materials with known values 2. Include QC in the procedure manual 3. Monitor media, reagents, stains, antigens 4. Monitor equipment 5. Train and monitor personnel 6. Perform verification/validation as needed 7. Keep detailed records 8. Continue to evaluate results

4 1. Use QC Materials with Known Values Run controls with known values: values can be qualitative or quantitative Use controls recommended by regulations, standards, or manufacturer of reagents, media, or kits Controls should be stored and maintained correctly

5 2. Include QC in Procedure Manual In addition to the purpose, principle, and procedural steps, the instructions should include quality control procedures

6 3. Monitor Media, Reagents, Stains, and Antigens Store per manufacturer s instructions Test new shipments with controls Test stains and reagents routinely This includes reagents for instrumentation

7 Monitor Media QC checks Check the condition of the plates Record lot #, expiration date Store media per manufacturer s instructions Test for growth and indicative properties with control organisms

8 4. Monitor Equipment Follow manufacturer s recommendations for preventative maintenance Recheck equipment after it is moved Examples of equipment: Automated commercial identification systems Thermometers CO 2 level in CO 2 incubator Microliter pipettes

9 Identification Systems Includes many components Need to ensure that both the reagents and the equipment are working Optics Lasers Detectors t

10 5. Train and Monitor Personnel Offer continuing education program activities for technicians Personnel should have access to reference material Competence can be assessed by: Direct observation Monitoring work records and results Testing unknowns

11 6. Perform Validation or Verification as Needed Non-standard methods Laboratory designed or developed tests Standard methods used outside their intended use

12 7. Keep Detailed Records Record the activities ities as they happen and the data as you collect it Records include: Nonconformities identified Corrective action taken

13 8. Evaluate Results Review results to prevent the release of bad product if there was a testing error Nonconformities should be identified Corrective action should be taken Strive to continuously improve

14 GPT Refresher Best practices for Growth Promotion Testing How is GPT performed? What are frequent challenges encountered in the lab and how do we overcome them?

15 GPT: the Global Standard GPT determines the suitability of medium used in pharmaceutical tests Used by pharmacopeias throughout the world

16 Suitable Media is Needed for: Type of Product Non-sterile Non-sterile Test Microbial Enumeration Tests Tests for Specified Organisms Purpose Counts the total number of bacteria and fungus in a product sample Looks for objectionable organisms such as Salmonella in a product sample Sterile Sterility Tests Checks sterility of a product sample

17 Four Rules for the GPT 1. Test each new batch of medium 2. Inoculate medium with small number of organisms 3. Organisms should be 5 passages or fewer from Reference Culture 4. Use organisms listed in USP

18 There are Variations in the GPT Test The Microbial Enumeration Tests, Tests for Specified Organisms, and Sterility Tests vary by: Microorganisms Medium Test method

19 Microbial Enumeration Test: Agar Once inoculum is prepared p be sure to compare previously approved medium and new medium with same suspension Previously approved medium New medium

20 Acceptance Criteria for Agar Compare number of colonies on previously approved medium to number on new medium The number must be within a factor of 2 Example: previously approved medium = 40 new batch of medium must = 20 to 80 Previously approved medium New medium

21 Challenge: Factor of 2 The pharmacopeia says, For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum The value of the standardized di d inoculum is obtained by counting the number of colonies on the previously approved media Perform side-by-side testing for direct comparison of new and previously approved media

22 Microbial Enumeration Test: Broth 1. Prepare inoculum 2. Inoculate new and previously approved Soybean-Casein Digest Broth 3. Inoculate Soybean- Casein Digest Agar as a control 4. Incubate Soybean-Casein Digest Broth Soybean-Casein Digest Agar

23 Acceptance Criteria for Broth Count colonies on non- selective agar: there should be 100 Compare turbidity in new medium to turbidity in the previously approved medium Turbidity should be Previously New approved broth comparable broth Control plates Control plates Soybean-Casein Digest Agar

24 Tests for Specified Microorganisms: Medium Tested for Three Criteria Growth promoting properties Indicative properties Inhibitory properties

25 Why Side-by-Side Testing? Only one variable: the medium Other variables eliminated, including: Temperature Atmosphere Technique Method Incubation time

26 Variable - Temperature Candida albicans after 48 hours incubation 25 C 20 C

27 Variable - Atmosphere Aspergillus brasiliensis taped and not taped

28 Variable - Atmosphere Bacillus subtilis with much air and with little air With air Without air

29 Variable Incubation Time Bacillus subtilis will form secondary colonies after 24 hours of incubation

30 Variable -Technique Pipetting angle effects results Microliters Sample Sample Sample Sample Pipetting w/angle Pipetting-no angle

31 Variable-Medium Variances in Media Manufacturer GPT was performed on various brands of Soybean-Casein Digest Agar using the same suspension of EZ-Accu Shot, Pseudomonas aeruginosa, 0484A. The test t was performed in duplicate. Tryptic Soy Agar CFU per 0.1 ml Brand A 36 Brand B 45 Brand C 57 Brand D 38 Brand E 21 Brand F 10 Conclusions CFU results can vary between media manufacturers GPT test is important to determine if medium is suitable

32 Challenge: Selective Medium Test non-selective medium in parallel 0.1 ml 25 CFU 0.1 ml 6 CFU

33 Best Practices Mix thoroughly Use dry plates Use correct growth conditions Verify pipettes deliver correct volume Test in duplicate or triplicate Test a negative control

34 Best Practices Use Correct Growth Conditions Penicillium chrysogenum was inoculated to 2 plates of PDA Plate on left incubated at 25 C Plate on right incubated at 35 C

35 Best Practices for Pour Plates Keep melted medium for pour plates at <50 C (44 C to 46 C is best) Give microorganisms sufficient time to grow. It takes longer for colonies to appear on pour plates than on spread plates A back-lit colony counter can make it easier to see small colonies such as Candida albicans

36 Beware of Overheating The Oxoid Manual 8th Edition: Overheating is a common cause of ph drift, darkening, precipitation, poor gel strength and reduced bacteriological performance.

37 Effects of Overheating The same suspension of E. coli was used to inoculate both pour plates The medium on the left was poured at 45 C The medium on the right was poured at 59 C Both plates were incubated 48 hours

38 Take-aways The purpose of the Growth Promotion Test is to ensure the media used to test products can recover small numbers of organisms Testing in parallel eliminates all variables except for the media Use best practices to obtain accurate and consistent results

39 AET Best Practices for Antimicrobial Effectiveness Testing

40 Preservatives and Antimicrobials A preservative manages contamination from manufacturing processes, product use. It preserves product integrity and quality. An antimicrobial is an agent that kills microorganisms i or inhibits their growth

41 The Importance of Preservatives Preservatives are used to control the growth of a range of microorganisms The organisms may be introduced during manufacturing or by the consumer The picture at the right shows eye cream with and without preservatives

42 There Are Two Goals: The first goal is efficacy. The preservative must prevent the growth of microorganisms The second goal is safety. The preservative must be used at a concentration below a level toxic to humans

43 When is the AET Performed? During development of a new product When the formulation of a product has changed When product ingredients have changed

44 Industries Which Use the AET and Why Pharmaceutical aceut ca Personal Care Products (ie: Cosmetics) Household Products Food Nutraceutical (ie: Vitamins) Textiles, plastics, carpets, paints and inks

45 Tests Methods Can Be Found In: USP <51> EP Japanese Pharmacopoeia ISO CFTA (Personal Care Products Council) ASTM

46 The Methods Differ Slightly In: Product sample volume Size of inoculum per ml of product CFU concentration of inoculum CFU concentration of product Strains required

47 USP <51> We will concentrate on USP <51> The USP sets standards for the identity, strength, quality, and purity of medicines and dietary supplements manufactured worldwide The standards are used in more than 140 countries

48 Antimicrobials Added Vary: Non-sterile Sterile Single Dose Multi-Dose

49 Challenge Test In the challenge test the laboratory adds microorganisms to the product Periodically, the laboratory tests the product to see how many organisms are in the it Depending on how effective the preservative is, the number of organisms may increase, decrease, or stay the same over time.

50 The USP Test for Categories 1, 2, 3 Product Product Product 1. Add microorganism to product. The concentration in product should be 10 5 to 10 6 CFU/ml 2. Incubate at 20 C -25 C 3. Sample product at intervals to see how many organisms remain over a period of time: for example; 7, 14, 28 days.

51 USP Acceptance Criteria The USP has set acceptance criteria for each product category over a period of 28 days More details and examples can be found at microbiologics.com

52 Best Laboratory Practices When Performing the AET 1. Qualify media used in test 2. Validate microorganisms can be recovered from the product 3. Determine the initial CFU concentration when testing a product 4. Test additional organisms if they pose a risk

53 Neutralization If recovery of organisms in the product is less than 50% of the recovery for the control, the product may need to be neutralized USP chapter <1227> describes neutralization. Neutralization methods include: Dilution Chemical inactivation (for example, lecithin for parabens) Membrane filtration/washing Any combination

54 Neutralizing Considerations Must inhibit antimicrobial Must not be toxic to organisms Must not combine with active agent to form toxic compound

55 Why Test the Inocula Instead of the Product? The initial count in the product may be too low because the antimicrobial may kill many microorganisms immediately Therefore, labs must calculate the initial CFU concentration in the product based on the number of CFU added to it

56 Test Additional Microorganisms if They Pose a Risk Species other than the five listed in the USP can be objectionable in certain products For example, B. cepacia in alcohol free mouthwash sickened 12 ICU patients at a German hospital Test additional species if: They pose a risk to the product or user If they are difficult to detect using standard methods

57 In-House Isolates Any isolate collected from your facility Why maintain them?

58 Cost of Contamination Equipment damage Production downtime Investigations Physical changes to product for example, gas, odors, changes in flavor Illness Recalls Lawsuits

59 Cost of Contamination Triad Group and H&P Industries filed for Chapter 11 bankruptcy Triad alcohol wipes were recalled after some were found to be contaminated t with Bacillus cereus The companies listed more than $37 million in liabilities against assets of less than $11 million

60 Which Organisms Cause Contamination? The specified organisms listed in the USP (Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli) Any other objectionable/environmental organisms

61 What is an Objectionable Organism? An objectionable organism is one which: May cause infection May harm the consumer by changing the properties of the product Source: Sutton and Jimenez, American Pharmaceutical Review, Jan/Feb 2012

62 Is an Organism Objectionable? Consider: The use of the product: hazard varies with route of administration (eye, nose, etc) The intended recipient (infants, elderly, immunocompromised patient) USP 2013 <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use

63 How are Objectionable Organisms Found? Testing raw materials and finished i products Monitoring the environment

64 Test Methods which use Environmental Isolates as Controls Disinfectant Efficacy Testing Sanitizer Efficacy Studies Media Growth Promotion Testing Test Method Suitability Validation of Rapid Microbiological Methods Identification Control

65 Common Environmental Isolates Pseudomonas/Ralstonia Causes issues due to the formation of biofilms which interfere with cleaning processes Staphylococcus sp. Normal human skin flora Normal human skin flora Bacillus sp. Normal human skin flora Micrococcus luteus Normal human skin flora

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