Determining Time

to Results

Validating an Automated Rapid Method:

with Andrew Sage Ph. D

• Background: Compendial method incubation and implications of use of RMMs

• Experimental Design Strategies for setting time to results (TTR)

• Case Study 2: EM air testing optimizing incubation profile, and TTR determination

• Summary

Overview

Background

Compendial Method Incubation Parameters and Time to Results

• Two temperatures of 22.5°C and/or 32.5°C or both serially

• Water testing: 32.5°C, 3 D (USP) > 5 days (EP)

• Higher temperature, longer incubation allow growth of oligotrophic, possibly stressed water species

• Bioburden: Single temperature (22.5°C or 32.5°C): 3-5 D (USP, EP)

• Lower or higher temperature allows mold/yeast or bacteria to be more effectively recovered

• Environmental Monitoring (air & surfaces, personnel): serial incubation –

• 22.5°C (3-5 D), 32.5°C (2-3 D) (WHO, EMA)

• Low to high or high to low temperature shift will allow mold/yeast and bacteria both to be detected

• Low to high allows temperature sensitive molds to develop – but they may then over grow bacteria

• High to low allows bacteria to develop and be counted before mold can develop with the lower temperature incubation

• Alternate incubation regimes used:

• Example: Single temperature for EM of 28°C

• Presume that this temperature will support growth of mold and bacteria equally

Implications of Use of RMMs

• Use of RMMs are meant to reduce Time-to-Result of testing

• Results to be as good or better than current test method (i.e. accuracy, precision, LoD etc.)

• Important that RMMs provide time savings that are worthwhile for ROI for user

• Different technologies are available – each comes with pluses and minuses (read the fine print)

• Real time detection: Answer is immediate, but there is no colony to count or isolate to ID

• Complicates comparison to compendial method, tracking of facility bioburden

• Growth based: Longer TTR, but there is a colony to identify for tracking

• Both must be shown to proide accurate results vs. the compendial methods

Pitfalls of a Wrong TTR Selection

• Time to Result is selected to balance need for accuracy with need for a timely answer

• Compendial time to results are recommended to essentially balance these needs

• One can often incubate longer, at another temperature and recover more or different colonies

• With RMM, must also balance between a faster answer and accuracy

• Wrong TTR can have serious results:

• Too short: possible missed contamination and possible tainted product

• Too long: lose any advantage to use the RMM

• For growth-basedRMMs, TTR needs to be determined and then confirmed by equivalence testing

• Non-growth based (e.g. epiflourescence not applicable)

Performing TTR Determination

• Requirements for determining growth-based TTR

• Need a regular, if not continuous analysis of growth

• Need an assay method that is non-destructive

• To simplify the process, need a system that:

• Performs automated, non-destructive analysis of colony numbers

• Analysis to be at measured intervals during incubation

• This discussion uses the Growth Direct™ System for the case study presented (process is applicable to any such system)

• Growth-based detection

• Automated sample handling

• Non-destructive detection technology

The Growth Direct™ Technology Platform

TTR Process Simplified by Automation

• Use of automated, non-destructive system simplifies TTR determination

• Automation allows analysis to be performed at measured times, on schedule at all hours without interruption

• Difficult to perform without automation

• Need really dedicated lab personnel

• A non-destructive assay allows repeated analysis of the same sample

• Final count at end of incubation as defined by standard method

• Impossible to perform with destructive tests

Experimental Design Strategies

for Setting TTR

Flow Chart for TTR Determination

Define protocol method

Define test organisms and/or

samples

Run Experiments Analyze Data

Equivalence TestingSet presumptive

TTR

Complete

Flow Chart for TTR Determination

• Compare detection of time course of standard incubation vs. final count

• Set test samples:

• In-house isolates associated with process or environment

• Samples from the process environment

• If needed, prepare stressed populations that will reflect environment of sample source

• Product matrix (e.g. pH osmiarity

• Clean room (e.g. disinfectant, bleach treatment)

• For single incubation run RMM on same media, temperature as reference method

• Run for the full time of compendial or standard method

• First time point at which RMM is >70% detection for all the test organisms over the course of the reference incubation can be selected as TTR

• May add time to ensure >70%, or other considerations

• Can focus on specific frame based on vendor claims (for example ~50% of standard method)

• For serial incubation (case study)

• Run RMM at same temperature profile and media

• Minimum time of compendial or standard method of each temperature

• Use vendor claims (e.g. ~50% of standard method)

• First time point where RMM is >70% of reference at the end of the serial incubation can be defined as TTR

Case Study: Determining Best Incubation Profile and

TTR for Air Testing

Goal: Determine optimal incubation profile and TTR for an active air testing application

Questions to answer during this study:

• What is the best incubation profile for optimal detection of microbial contaminants?

• With the selected profile, determine the Time-to-Result (70% of total count)

• Does the selected TTR provide results that are comparable to the standard method

Background

• Environmental monitoring (air, surfaces, personnel) can be performed using single or serial incubation

• Single incubation: 22.5°C or 32.5°C

• 22.5°C presumed good for molds, 32.5°C for bacteria

• To recover both – run 22.5°C and 32.5°C serialy

• Questions to answer during this study:

• Is single incubation sufficient?

• If serial, do 32.5°C first or 22.5°C first

• How long an incubation for each temperature?

• What TTR to select for the chosen incubation profile?

Samples for Testing

• For incubation profile and TTR determination

• Isolated from EM air testing in dirty environments

• Range of fast and slow-growing strains

• Mold and bacterial species

• No real world samples

• Equivalence testing: Growth Direct™ vs. standard method in air testing in facility

• Real world samples compared over time

Procedure

• Based on ~50% TTR claim for time to result, all strains should be detected within 4 days on system –run 96th

• 1st: What is optimal incubation profile?

• Is serial really the best method?

• Test detection of colonies at different incubation parameters:

• 22°C, 96 hours

• 22°C, 96 hours, 48 hours followed by 32°C, 96 hours 48 hours

• 32°C, 96 hours, 48 hours followed by 22°C, 96 hours 48 hours

• 32°C, 96 hours

• 2nd: What is TTR at optimal incubation profile

• Compare detection vs. final count of test organisms

• 3rd: Confirm TTR is appropriate in side-by-side

EM Isolate Detection Curves at 4 Profiles: Bacteria

32°C provides faster detection for bacteria

EM Isolate Detection Curves at 4 Points: Molds

32°C prevents recovery of some mold species. Need 22°C first for mold recovery

TTR Determination at Incubation Profile

22 C, 48 hr; 32 C, 48 hr

Selected incubation profile provides about 50% shorter TTR

TTR 70%, ~60 hr

Equivalence Testing: Co-Trending of Air Samples

Equivalence testing shows co-trending of RMM result and control standard method

Considering Alternative Strategies

• Additional option: one can optimize TTR for a RMM using an alternate testing strategy

• With new method, can look to improve testing

• Example: air testing TTR

• 22°C and 32°C alone are not effective

• Move to 28°C for single incubation?

• Advantages of single temperature

• For RMM, higher throughput

• Possible reduced TTR

• Lower resource uses

Alternative Incubation (28 C) TTR

Possible obtain same TTR and reduce resource use

TTR 70%, ~56 hr

70% detection

Using Real World Samples

• Use of real world samples in problematical

• Great to use if possible

• Provide microbes in appropriate physiological state (stressed)

• Sampling a good

• Most samples have limited or no contamination

• Clean rooms for EM, final product for bioburden/sterility – Difficult to use

• In-house isolated obtained during normal testing can be treated to mimic stress of environment

• Mimic stress state that exists for real world samples – Examples:

• Surfaces – treat with antimicrobial compounds used in clean room

• Water testing – expose in-house isolates to same water to induce nutrient, osmotic stress

• Final product – expose test organisms to product matrix or active antimicrobial ingredients

• Provide an approximation of the response of organisms from the test environment

Summary

• As with any technology, growth-based RMMs must be validated. A part of the process can involve selecting and confirming a Time-to-Result for the RMM

• Automated, non-destructive platforms provide the means to most effectively carry out experimentation to set and validate a Time-to-Result

• The basic strategy for setting TTR is to determine at which point comparable recovery of microbial test samples is obtained vs. the normal reference method

• Test samples can be in-house or real world samples

• Isolates treated to approximate the stress state of the sample matrix can also be used

• Selected TTRs should be confirmed by side-by-side equivalency testing

• TTR selection may be influenced by other factors such as personnel availability

• Setting TTR was illustrated by determining the optimal incubation profile, and time-to-result for environmental air sample testing on the Growth Direct™ System using in-house EM isolates

• The effectiveness of an alternate incubation strategy was also shown by determination of the TTR using a subset of the same test organisms

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