How to select and justify the right rapid microbiological methods

BY DR TIM SANDLE | 7 May 2024

 

As rapid microbiology methods continue to develop, many exciting technologies are starting to emerge. This article presents areas for consideration when looking to select a method and design a business case.

 

 

 

Drivers and barriers

 

Interest in rapid methods has a number of drivers. These include:

 

  • FDA Process Analytical Technology (PAT)
  • Regulatory encouragement (such as EU GMP Annex 1)
  • The outcome of Quality by Design (QbD) assessments
  • Business needs - for example, to help accelerate batch progression
  • Scientific advancement and a desire to account for more microorganisms than conventional methods can account for - including the ‘viable but non-culturable’ attribute

 

On the other hand, barriers to implementation can include:

 

  • Scientific or technological hurdles
  • Regulatory concerns
  • Economics
  • Validation costs and time

 

If the right technology is available for the right application, these barriers are not insurmountable.

 

 

 

Advantages

 

While rapid methods differ in their scope, application and reliability, there are several advantages. For each rapid microbiological method, one or more of the following will apply:

 

  • Faster - reduced time-to-result
  • More accurate - increased sensitivity, accuracy, precision and reproducibility
  • Fewer microbial cells required to produce a result
  • Enhanced detection of stressed organisms
  • Semi-automated or automated
  • Improved throughput
  • Less subjectivity

 

Furthermore, we can consider:

 

  • The significant reduction of testing time to release products more rapidly
  • Lower inventories - raw material, in-process material and finished product
  • The prevention of back orders
  • The reduction of repeat testing, deviations, OOS investigations and product rejection

 

Arguably, Rapid Microbiological Methods (RMMs) also enable a proactive approach to be taken in instances of microbial contamination, especially in relation to out-of-specification results. Here, RMMs enable quicker responses to out-of-trend situations through providing real-time or near real-time results. This allows for corrective actions to be taken earlier.

 

 

 

Typology of rapid methods

 

In terms of available types, a general typology of RMMs would place them in one of the following groups:

 

  • Measurements can be qualitative - determining if microorganisms are either present or absent in a test sample
  • Measurements can be quantitative - determining precisely the number of microorganisms present in a test sample
  • Technologies designed to specifically identify the microorganism.
  • Technologies to detect microbial byproducts (including toxins)

 

We can get a bit more specific with the following groupings:

 

 

Growth-based

 

Growth-based technologies rely on the measurement of biochemical or physiological parameters that reflect the growth of microorganisms. These types of systems require the organisms in a sample to proliferate, either in a solid or liquid medium, in order to be detected and/or quantified.

 

The methods continue to use conventional liquid or agar media. In summary, they include:

 

  • Impedance microbiology - measurable electrical threshold during microbial growth
  • The detection of carbon dioxide (CO2)
  • The utilisation of biochemical and carbohydrate substrates
  • The use of digital imaging and auto-fluorescence for the rapid detection and counting of micro-colonies
  • Fluorescent staining and enumeration of micro-colonies by laser excitation
  • Selective media for the rapid detection of specific microorganisms

 

 

Viability-based

 

Viability-based technologies use viability stains and/or cellular markers to detect and quantify microorganisms without the need for cellular growth.

 

These methods include:

 

  • Demonstration of direct labelling of individual cells with viability stains or fluorescent markers with no requirement for cellular growth
  • Flow cytometry - individual particles are counted as they pass through a laser beam
  • Solid-phase cytometry - staining and laser excitation method

 

 

Artefact-based

 

Artefact-based technologies rely on the analysis of cellular components or the use of probes that are specific for the microbial target sites of interest. Cell component analysis is where the expression of specific cell components offers an indirect measure of microbial presence (e.g., genotypic methods). These methods generally involve the detection and analysis of specific portions of the microbial cell, including ATP, endotoxin, proteins and surface macromolecules. The methods include:

 

  • ATP bioluminescence - the generation of light by a biological process
  • Endotoxin testing (Lumulus Amebocyte Lysate (LAL))
  • Fatty acid analysis - methods that utilise fatty acid profiles to provide a fingerprint for microorganism identification
  • Matrix-Assisted Laser Desorption Ionisation - Time of Flight (MALDI-TOF) mass spectrometry (microbial identification)

 

 

Nucleic acid-based

 

Nucleic acid-based technologies rely on Polymerase Chain Reaction (PCR) DNA amplification, 16S rRNA typing, gene sequencing and other novel applications.

 

These methods include:

 

  • Riboprinting: 16S sequence of rRNA is highly conserved at the genus and species level
  • PCR methods for targeting specific microorganisms - millions of copies of the target DNA in a short period of time
  • Gene sequencing - specific dye labelling

 

 

Optical spectroscopy

 

Optical spectroscopy methods utilise light scattering and other optical techniques to detect, enumerate and identify microorganisms (e.g., ‘real time’ airborne particle counters). These methods include:

 

  • Real-time and continuous detection, sizing and enumeration of airborne microorganisms and total particles. These methods are applied to the monitoring of cleanrooms.

 

 

Micro-electrical-mechanical systems

 

Micro-Electrical-Mechanical Systems (MEMS) utilise microarrays, biosensors and nanotechnology to provide miniaturised technology platforms. These methods include:

 

  • Microarrays (DNA chips), evolved from Southern Blot technology, to measure gene expression (e.g. mycoplasma detection)

 

 

 

Selecting the right method

 

The process of selecting the appropriate rapid method through to its implementation involves several stages, including:

 

  • Initial feasibility assessment
  • Determining the costs for the equipment itself
  • Thinking about the costs of an individual test
  • Development of user requirements
  • Establishing testing protocols and SOPs
  • Staff training
  • The execution of the validation programme
  • Documentation
  • Regulatory submission
  • Technology transfer
  • Site implementation

 

 

When setting out to specify a Rapid Microbiological Method (and as part of the user requirement assessment), the following ten attributes can be useful for helping with the decision-making process:

 

  • Accuracy for the intended purpose
  • Speed in productivity
  • Cost
  • Acceptability by the scientific community
  • Acceptance by regulatory agencies
  • Simplicity of operation, including training requirements and reagents
  • Reputation of the vendor
  • Technical services provided by the vendor
  • Utility requirements
  • Space requirements

 

 

With accuracy, issues to consider include:

 

  • If the rapid method will lead to a reduction in human error
  • If there is a reduction in subjectivity
  • Whether the alternative method will detect more accurately in comparison to a conventional method
  • Whether there is a need for the rapid method to detect what a cultural method cannot

 

 

 

Other considerations include:

 

  • If there is a need for the electronic capture of data
  • Whether the method needs to be automated
  • If there is a need for connecting apparatus or linking the method to a Laboratory Information Management System (LIMS)

 

 

It can also be useful to consider the following reflective questions:

 

  • Is anyone using the method?
  • Does anyone plan to use the method?
  • What are the advantages?
  • What are the disadvantages?
  • What are the obstacles to implementation?
  • What is needed for validation?
  • How long will the process take?

 

 

 

Validation

 

When choosing a rapid microbiological method, consideration should be given to how it is going to be validated. Any methods that are being adopted need to yield results equivalent to or better than the method currently used which already gives an acceptable level of assurance.  In addition, the new method and the method currently used should be run in parallel for a designated time period as a condition of approval.

 

The following validation strategy can be helpful:

 

  • Define the characteristics of the current test that the RMM is to replace
  • Determine the relevant measures that establish equivalence of the RMM to the current method. This may require statistical analysis
  • Demonstrate the equivalence of the RMM to the current method in the absence of the product sample
  • Demonstrate the equivalence of the RMM to the established method in the presence of the test sample

Summary

 

This article has outlined some of the key considerations to be made when deciding whether to adopt a rapid method and the subsequent selection between the different types of rapid methods that are available. The aim was to offer general advice to those tasked with making the selection and undertaking the work required to qualify the method so that it is available for the laboratory or process area to use.

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