Draft - Canadian requirements for determining the equivalence of food microbiological methods of analysis

Date published: November 4, 2023

Table of Contents

• Purpose
• Demonstration of equivalency
• Requirements for qualitative and quantitative methods of analysis
  • Requirements for selectivity studies
    • Inclusivity study
    • Exclusivity study
  • Relevant food
  • Relevant strains
  • Relevant stress
  • Relevant equilibration
• Additional requirements specific to qualitative methods of analysis
  • Inoculation levels
  • Co-inoculation
  • Number of samples
  • Performance parameters
• Additional requirements specific to quantitative methods of analysis
  • Performance parameter
• Requirements for colony identification methods
  • Requirements for selectivity studies
    • Inclusivity
    • Exclusivity
  • Performance parameters

Purpose

This document defines the performance parameters and sets out other requirements that shall be met to determine that a method is equivalent to a microbiological reference method, for the purposes of Division 30 of the Food and Drug Regulations.

This document does not replace existing method validation guidelines from Health Canada for microbiological methods of analysis.

This document is published and maintained by Health Canada and is incorporated by reference into Division 30 of the Food and Drug Regulations.

Demonstration of equivalency

To determine if an alternative method is equivalent to a reference method set out in column 2 of the Table of Microbiological Reference Methods for Food, that method shall be validated using procedures that will provide sufficient evidence to evaluate the performance of the method against its respective microbiological reference method using performance parameters described in this document.

Requirements for qualitative and quantitative methods of analysis

Requirements for selectivity studies

Inclusivity study

The ability of the method to detect a wide variety of strains representing the target microorganism (inclusivity) shall be demonstrated by:

• Testing at least 50 pure strains of the target microorganism, except for Salmonella methods for which testing at least 100 pure cultures of different serotypes of Salmonella is required.
• Selecting strains that cover the range of diversity of the target microorganism, such as biochemical characteristics, serotypes, phage types and geographical distribution.

Exclusivity study

The ability of the method to resist interference by cross-reactivity with non-target microorganisms likely to be found in the tested food (exclusivity) shall be demonstrated by:

• Testing at least 30 pure strains of non-target microorganisms, including:
• strains that are suspected to cause interference;
• strains naturally occurring in food tested; and
• strains closely related to the target microorganism.

Selectivity studies (inclusivity and exclusivity) can be omitted for methods such as aerobic plate counts and total plate counts, where the level of natural microorganism of a food commodity is measured.

Relevant food

The ability of the method to detect target microorganisms in foods typical of the samples to be tested, including foods that may present a challenge to the performance of the method, shall be demonstrated. A sufficient number of foods shall be tested, to have a proper representation of the diversity of that food.

Relevant strains

The ability of the method to detect target microorganisms shall be demonstrated using strains likely to be detected in the foods tested.

Relevant stress

The ability of the method to detect target microorganisms in a stressed state shall be demonstrated only when the manufacturing processes or conditions of the food are likely to cause injury to the target microorganism.

Relevant equilibration

The ability of the method to detect target microorganisms shall be demonstrated after the food (inoculated with the target microorganism in a stressed or unstressed state, as appropriate) has been stored at the normal storage temperature for that food, for a relevant period, as follows:

• Frozen food: at least 2 weeks at -18°C or less
• Refrigerated food: at least 48h at 2–8°C
• Food stored at room temperature: at least 2 weeks at 20–25°C

Additional requirements specific to qualitative methods of analysis

A qualitative method of analysis is a method whose result is either that the target microorganism is detected or not detected either directly or indirectly in a unit of mass or volume.

Inoculation levels

The ability of the method to detect target microorganisms shall be demonstrated using two inoculation levels. The low level of inoculation shall result in a fractional recovery of positive samples, which is as close as possible to 50% (acceptable range: 25–75%) and confirmed positive by the microbiological reference method. The high level of inoculation shall result in all or nearly all samples testing positive.

Co-inoculation

When detection of the target microorganism may be impeded by the presence of non-target microorganisms, the ability of the method to detect target microorganisms in the presence of non-target microorganisms shall be demonstrated.

Number of samples

The ability of the method to detect target microorganisms shall be demonstrated using a sufficient number of contaminated samples for each food. The number of samples shall provide high probability of detecting a difference in performance between the alternative method and the reference method.

Performance parameters

The method shall meet or exceed the following performance parameters when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:

• Sensitivity ≥ 98%
• Specificity ≥ 90.4%
• False negative rate < 2.0%
• False positive rate ≤ 9.6%
• Efficacy ≥ 94%
• Level of detection (LOD) of the method must be comparable to or exceed the lower limit of detection of the reference method.

Additional requirements specific to quantitative methods of analysis

A quantitative method of analysis is a method where the result is the quantity of the target microorganism in a unit of mass or volume, measured either directly, or indirectly, for example through colour absorbance or impedance.

Performance parameter

The method shall meet the following performance parameter when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:

• Strict equivalence between the alternative method and reference method covering the range of target microorganism concentrations set out in columns 5 and 6 of the Table of Microbiological Criteria for Food shall be demonstrated based on recognized statistical approaches.

Requirements for colony identification methods

A colony identification method is a method used to confirm the identity of target microorganisms that have been isolated as pure colonies on agar media.

Requirements for selectivity studies

Inclusivity

The ability of the method to detect a wide variety of strains representing the target microorganism (inclusivity) shall be demonstrated by:

• Testing at least 100 pure strains of the target microorganism, except for Salmonella methods for which testing at least 150 pure cultures of different serotypes of Salmonella is required.
• Selecting strains that cover the range of diversity of the target microorganism, such as biochemical characteristics, serotypes, phage types and geographical distribution.

Exclusivity

The ability of the method to resist interference by cross-reactivity with non-target microorganisms likely to be found in the tested food (exclusivity) shall be demonstrated by:

• testing at least 50 pure strains of non-target microorganisms, including:
• strains that are suspected to cause interference;
• strains naturally occurring in food tested;
• strains closely related to the target microorganism.

Performance parameters

The method shall meet or exceed the following performance parameters when compared to the corresponding microbiological reference method set out in column 2 of the Table of Microbiological Reference Methods for Food:

• False negative rate ≤ 1%
• False positive rate ≤ 2.0%