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The Science of Speed:How Fast Biological Indicators have Changed Quality Control in Reprocessing
Janet PrustDirector – Global Standards
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Devil’s CascadeRainy Lake
Northwestern Ontario
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Hexagenia Mayfly
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Common Loon
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Tullibee Cisco
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Freshwater Mussels - Ontario
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What do these four species have in common?
Bioindicators (indicator species) • Species or a group of species whose function, population or status can reveal
the qualitative status of the environment in which it lives.
What environment are they bioindicators for? • Clean water. Clean water indicators are sensitive to pollution and are
a good indicator of water quality changes.
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Viability is reflective of it’s environment
Common Biological Indicator
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Learning Objectives
1. Review how biological indicators work
2. Understand how rapid readout biological indicators work
3. Discuss the biological indicators role in sterilization quality control.
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Director – Global Standards
3M Health Care Employee
Medical Solutions Division
Janet Prust - Disclosure
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Biological Indicator Basics
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Biological Indicator – Definition:
A test system containing viable microorganisms providing a defined resistance to a specified sterilization process.
ISO/TS 11139: 2018. Sterilization of health care products — Vocabulary. International Organization for Standardization.
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Types of Biological Indicators
Spore stripsInoculated carriers Liquid BIs
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Self-contained Biological Indicators
Cap
Filter
Growth Media
with pH Indicator
Spore Strip Sleeve
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The Microbiology of Biological Indicators
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Bacterial Spores
Some types of bacteria can form spores to protect themselves from a hostile environment, such as lack of nutrients
• e.g. some Bacillus sp., Clostridium sp.
• Like nuts or seeds - no biological activity - dormant
Very tough and hard to kill – challenges the sterilization process
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Bacterial Spores
Geobacillus stearothermophilus Bacillus atropheus
Too small to see . . . about 1-2 micrometers (.000001 meters)
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Growth Media
• Mixture of sugars and other nutrients
• Formula optimized for the biological indicator microorganisms
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Bacterial Spores – Activation and Germination
Google Images
A spore “sprouts”A spore “sprouts”Like a seed sprouts . . .
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Bacterial cell
Spore
Biochemical and physical transformation steps
Bacterial Spores – Activation and Germination
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Bacterial Growth
Bacterial cell
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Biological Indicator Standards
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ISO 11138 Series
11138 – 2: Ethylene oxide
11138 – 3: Moist heat (steam)
11138 – 4: Dry heat
11138 - 5: Low temperature steam formaldehyde
CAN/CSA-ISO 11138-1:17 Scope
This document specifies general requirements for production, labelling, test methods and performance characteristics of biological indicators, including inoculated carriers and suspensions, and their components, to be used in the validation and routine monitoring of sterilization processes.
This document specifies basic and common requirements that are applicable to all parts of ISO 11138. Requirements for biological indicators for particular specified processes are provided in the relevant parts of ISO 11138. If no specific subsequent part is provided, this document applies.
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Biological Indicator Performance Requirements
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Key BI Performance Parameters
Population (number of test organisms)• Number of organisms that must be killed to get a negative BI• The more organisms, the longer it takes to inactive the BI• Measured by distributing the spores on plates and counting• Expressed in exponents (e.g. 106 = 1,000,000)
D-value (resistance)• A measure of how resistant the organisms are to the process
(how hard to kill)• Measured as the time to reduce the population by 90%• Expressed in time units (usually minutes)
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Biological Indicator Incubation Time
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Biological Indicator IncubationProcess for providing the spores in the biological indicator with the perfect conditions to support the growth of the organisms
- Nutrients (food)- Optimal temperature- Optimal pH
The incubation conditions are designed to be able to recover a very small number of organisms that remain alive in the biological indicator after exposure to the sterilization process, if there is a sterilization process failure.
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Biological Indicator Incubation
These biological processes happen during incubation . . .
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Biological indicator incubation time is the time required to make a final determination that the BI is negative• If the biological indicator turns positive at any point, the test is
complete and appropriate action must be taken.
Biological Indicator Incubation Time
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Biological Indicator Incubation Time
“An incubation period is commonly recognized to be 7 days for established sterilization processes . . .”
ISO 11138 (2017) Sterilization of health care products – Biological indicators –Part 1: General requirements
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Reduced Incubation Time (RIT)
Reduced Incubation Time is any established biological indicator incubation time that is less than 7 days• For example: 48 hours, 24 hours, 3 hours, 1 hour or 24 minutes
• The biological indicator manufacturer compares (validates) the BI response at the RIT to the BI response after 7 days, against established criteria.
How is this validation accomplished?
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RIT Test Methods
Pharmacopeias- No methodology defined in USP, EP
ISO Standards- No specific methodology defined in ISO 11138 series standards- No specific methodology defined in ISO 14161- Standard in Draft – ISO CD 16342
U.S. Food and Drug Administration (FDA)- Method defined in FDA 510k Guidance document
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Fractional cycles and MPN
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Fractional cycles and MPN
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U.S. FDA RIT VALIDATION
http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm071261.htm
Reduced incubation time validation is part of the legal requirements for biological
indicators used in hospitals in the United States.
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http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm071261.htm#a2
U.S. FDA –RIT Methodology
30+’s → MPN = 0.357• Range 0-3 spores/BI
80+’s → MPN = 1.609• Range 0-7 spores/BI
AAMI WG 4 Presentation to U.S. FDA; May 26, 2016
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Positive Biological Indicators• Any spores that survive the sterilization cycle (a sterilization cycle failure)
are exposed to the growth media, then germinate (change from spores to cells), and start to grow:
• As they germinate and grow, they create many chemical reactions, that can be detected as a “signal”, that the biological processes are underway.
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Positive Biological Indicator “signals”
TurbidityBacterial cells scatter light
Media Color ChangepH shift caused by metabolic by-products
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3M Rapid Readout Technology
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3M Rapid Readout Technology
• Introduced in 1991
• Now used to monitor 3 different sterilization technologies (steam, ethylene oxide, hydrogen peroxide)
• Electronic detection of a fluorescent signal produced by the action of an enzyme system active in the germination and outgrowth phases of the test organism- Geobacillus stearothermophilus (steam, hydrogen peroxide): alpha-
glucosidase- Bacillus atropheus (ethylene oxide): beta-glucosidase
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Glucosidase Enzyme Systems
Bacterial glucosidase enzymes play an important role in uptake and utilization of nutrients
“The enzyme activity observed by sterilization assurance assays appears likely to come from heat stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth.”
Setlow P, Korza G, Setlow B. Analysis of alpha-glucosidase enzyme activity used in a rapid test for steam sterilization assurance. J Appl Microbiol 2016; 120: 1326-1335.
Vihinen M, Mäntsälä P (1989) Microbial amylolytic enzymes. CritRev Biochem Mol Biol 24:329–418
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3M™ Attest™ Rapid Technology
Enzyme acts on a non-fluorescent substrate in the media, resulting in a fluorescent end product, which is
detected by the Auto-reader (“signal”)
Non-fluorescent substrate (MUG)
Fluorescent end-product (4-MU)
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Rapid Attest™ Technology
Enzyme acts on a non-fluorescent substrate in the media, resulting in a fluorescent end product, which is
detected by the Auto-reader (“signal”)
Non-fluorescent substrate (MUG)
Fluorescent end-product (4-MU)
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Positive Biological Indicator “Signals”
TurbidityBacterial cells scatter light
Media Color ChangepH shift caused by metabolic by-products
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Positive Biological Indicator “Signals”
TurbidityBacterial cells scatter light
Media Color ChangepH shift caused by metabolic by-products
FluorescenceFluorescent response generated by metabolic enzyme action
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Optimized for Signal Production and Detection
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Optimized for Signal Production and Detection
Media optimized to produce fluorescent signal
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Optimized for Digital Production and Detection
Physical design to concentrate the signal
Advanced optics and software to detect the signal
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Rapid Readout Biological Indicators Use Worldwide
Malaysia JapanChinaTaiwanAustraliaNew ZealandIndiaThailandIndonesiaKoreaSingaporePhilippinesVietnam
United StatesCanadaMexicoBrazilColombiaChilePeruArgentinaUruguayCaribbean Region
SpainItalyGreeceBelgiumFranceNetherlandsSaudi ArabiaTurkeyPolandSouth AfricaUKIrelandRussiaIsrael
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Use of Biological Indicators in Sterilization Quality Control
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Sterilization Process Monitoring
Goal of the sterilization process?• To kill all microorganisms!
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Biological Indicators
Chemical Indicators
Physical Monitors
Sterilization Process Monitoring Tools
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Physical Monitors
•Information from sensors in the sterilizer chamber walls or drain (e.g. temperature, pressure)
•Can confirm that the correct cycle was selected
•Provides a record of the cycle • Sensor placement
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Chemical Indicators
Test system that reveals change in one or more predefined process variables based on a chemical or physical change resulting from exposure to a processISO/TS 11139:2005 Sterilization of health care products — Vocabulary
Bowie-Dick Tests
Pack indicatorsProcess indicators
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Biological indicatorsBiological indicators are recognized by most authorities as being closest to the ideal monitors of the sterilization process because they measure the sterilization process directly by using the most resistant microorganisms (i.e., Bacillus spores), and not by merely testing the physical and chemical conditions necessary for sterilization. Since the Bacillus spores used in biological indicators are more resistant and present in greater numbers than are the common microbial contaminants found on patient-care equipment, the demonstration that the biological indicator has been inactivated strongly implies that other potential pathogens in the load have been killed.
CDC Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008
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Biological indicators
Biological indicators are recognized by most authorities as being closest to the ideal monitors of the sterilization process because they measure the sterilization process directly by using the most resistant microorganisms (i.e., Bacillus spores), and not by merely testing the physical and chemical conditions necessary for sterilization. Since the Bacillus spores used in biological indicators are more resistant and present in greater numbers than are the common microbial contaminants found on patient-care equipment, the demonstration that the biological indicator has been inactivated strongly implies that other potential pathogens in the load have been killed.
CDC Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008
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Biological indicators
Biological indicators are recognized by most authorities as being closest to the ideal monitors of the sterilization process because they measure the sterilization process directly by using the most resistant microorganisms (i.e., Bacillus spores), and not by merely testing the physical and chemical conditions necessary for sterilization. Since the Bacillus spores used in biological indicators are more resistant and present in greater numbers than are the common microbial contaminants found on patient-care equipment, the demonstration that the biological indicator has been inactivated strongly implies that other potential pathogens in the load have been killed.
CDC Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008
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Process Challenge Devicesprocess challenge device (PCD): Item designed to constitute a defined resistance to a sterilization process and used to assess performance of the process.
ISO/TS 11139: 2005. Sterilization of health care products — Vocabulary. International Organization for Standardization.
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Use of PCDs
• Biological indicators should be inside of the load, but this is not feasible
• PCDs are easily retrieved without disturbing any of the load items
• PCDs make it more difficult for the sterilant to reach the BI, like the BI was located inside of the load
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Frequency of use – biological indicators
Steam sterilization
Test CAN/CSA-Z314.-18 U.S.: ANSI/AAMI ST 79 (2017) Comprehensive guide to steam sterilization and sterility assurance in health care facilities
Routine Efficacy Monitoring BI PCD. Each day the sterilizer is used for each type of cycle
BI PCD. Weekly, preferably daily; Each cycle type used.
Load release – non-implant Physical parameters. BI PCD with CI provides increased sterility assurance
Optional. PCD with BI, Type 5, Type 6.
Load release - implants Every load. PCD with BI + Type 5 or 6 CI. Quarantine until BI result is available.
Every load. PCD with BI + Type 5 or 6 CI. Quarantine until BI result is available.
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Considerations for monitoring every load with a BI/PCD:• Uniform standard of care for all patients
• Highest level of quality control
• Reduce the possibility of monitoring mistakes (implant vs. non-implant)
• Fast readout BIs facilitate frequent monitoring
Note: 53% of US Tier A hospitals monitor every load with BI PCD.
Source: 3M survey 2017, 2018.
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Considerations for monitoring every load with a BI/PCD:• Uniform standard of care for all patients
• Highest level of quality control
• Reduce the possibility of monitoring mistakes (implant vs. non-implant)
• Fast readout BIs facilitate frequent monitoring
7 days 2 days 3 hours 1 hour Minutes!
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Steam sterilization quality issues . . .
• Most steam sterilization failures are related to problems with the steam quality, rather than time or temperature (e.g. air, non-condensable gases, wet steam, dry steam)
• Physical monitors and most chemical indicators cannot detect these problems
• Poor steam quality reduces the killing power of the process. Biological indicators directly measure the effectiveness of the process, so they do detect steam quality problems.
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SUMMARY1. Biological indicators are the direct measure of lethality of
the sterilization process, because they measure the effect of the process on spores.
2. Biological indicators can detect cycle quality problems.
3. Biological indicators incubation times have been reduced by detecting biological signals faster.
4. Fast incubation times make BIs easier to use, and enable better quality control by increased monitoring frequency.
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Thank you for your time and attention.
Contact information:
Janet Prust3M Medical Solutions DivisionSt. Paul, MN USA651 736 1546