ISO 18744:2016/DAM 1:2016(en)

Date : 2025-01-27

ISO TC 34/SC 9/WG 6

Project leaders: Rachel Chalmers, Marco Lalle, Tricia Vail

Secretariat: AFNOR

Microbiology of the food chain — Detection of Cryptosporidium and Giardia on fresh leafy green vegetables and berry fruits

AMENDMENT 1: Method validation studies and performance characteristics

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Microbiology of the food chain — Detection of Cryptosporidium and Giardia on fresh leafy green vegetables and berry fruits
AMENDMENT 1: Method validation studies and performance characteristics

Title

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and enumeration

Explanation note: WG6 agreed to remove the term enumeration in the title and text since, due to the nature of the validation, the method cannot be considered quantitative but qualitative and the results should be reported only as presence/absence of the parasite in the matrix. Any enumeration in the method refers to the number of pre-labelled and enumerated non-viable Cryptosporidium oocysts and Giardia cysts that may be used as internal controls. The use of internal controls is recommended not mandatory.

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Microbiology of the food chain — Detection of Cryptosporidium and Giardia on fresh leafy green vegetables and berry fruits

Contents

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Annex D (informative) Method validation studies and performance characteristics

Introduction

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and for their enumeration

1   Scope

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and enumeration

8.1

e)

NOTE 1

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Centrifugation at 2 500 x g can result in a very compact pellet. A lower speed of 1 100 x g for 10 min with no braking has been reported to give at least equivalent recoveries of Cryptosporidium oocysts and Giardia cysts.

i)

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Centrifuge the eluate at 2 500 x g for 10 min with no braking.

NOTE 2

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Centrifugation at 2 500 x g can result in a very compact pellet. A lower speed of 1 100 x g for 10 min with no braking has been reported to give at least equivalent recoveries of Cryptosporidium oocysts and Giardia cysts.

8.2

g)

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Centrifuge the eluate at 2 500 x g for 10 min with no braking.

NOTE 2

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Centrifugation at 2 500 x g can result in a very compact pellet. A lower speed of 1 100 x g for 10 min with no braking has been reported to give at least equivalent recoveries of Cryptosporidium oocysts and Giardia cysts.

k)

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Centrifuge the eluate at 2 500 x g for 10 min with no braking.

NOTE 3

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Centrifugation at 2 500 x g can result in a very compact pellet. A lower speed of 1 100 x g for 10 min with no braking has been reported to give at least equivalent recoveries of Cryptosporidium oocysts and Giardia cysts.

8.5.2.2

NOTE

Third sentence

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Some Cryptosporidium species have diameters up to 8 μm (e.g., C. andersoni and C. muris, which could be associated with infections in immunocompromised persons in rare cases).

10.1

First line

Replace the text with the following: The result should be given as the presence or absence of Cryptosporidium oocysts and/or Giardia cysts detected per weight of fresh produce sample examined.

10.2

i)

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The presence or absence of Cryptosporidium oocysts and Giardia cysts, and any relevant details regarding their identification and verification (e.g. size, DAPI-staining, morphological details);

After Annex C

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1. 
   (informative)
   
   Method validation studies and performance characteristics
   1. Background

The performance data for detection of Cryptosporidium on fresh leafy green vegetables and berry fruits ⁽⁸⁾ and for detection of Giardia on fresh leafy green vegetables (unpublished data, used with permission) were calculated from the interlaboratory study organized by the Central Science Laboratory (CSL), Sand Hutton, York, UK ⁽⁸⁾. The study was organized in 2001. The collaborators were all in the UK. The samples tested in the study were Webb’s lettuce and raspberries. The samples were tested at three different levels of contamination (low, medium and high), plus non-contaminated samples. Artificial contamination was performed by the organising laboratory. The study was supported by the then UK Food Standards Agency.

The test method used in the CSL interlaboratory trial was described by Cook et al., ⁽⁷⁾. This method formed the basis of ISO 18744:2016.

The values of the performance characteristics derived from the interlaboratory studies of Cryptosporidium on fresh leafy green vegetables and berry fruits are shown in Tables D 1 and D 2 respectively. The values of the performance characteristics derived from the interlaboratory studies of Giardia on fresh leafy green vegetables are shown in Table D 3. Where data obtained by collaborators were not included in the calculations this was for technical reasons (e.g. deviations to the protocol).

LOD₅₀, its 95 % confidence interval, the standard deviation of laboratory effects, and intraclass correlation coefficient (ICC) for each interlaboratory study were estimated using the methods described by Wang and Ihrie (2021).^[26] In particular, for the interlaboratory studies for Cryptosporidium on lettuce and Cryptosporidium on raspberries LOD₅₀ and its 95 % confidence interval were estimated based on a fixed effect complementary log-log model. For the interlaboratory study for Giardia on lettuce all parameters were estimated based on a random intercept complementary log-log model, that is generally recommended for an interlaboratory study in ISO 16140-2: 2016 (ISO 2016 Annex F). However, a fitting of this model failed to converge using the data from the interlaboratory studies. The standard deviation of laboratory effects and ICC were estimated based on a linear mixed effects model.

NOTE ICC measures the proportion of total variance in outcomes attributable to between-laboratory variation. Standard deviation of laboratory effects measures the variability in the outcomes among laboratories. Both ICC and standard deviation of laboratory effects measure reproducibility of a detection method among laboratories.

• 1. Performance characteristics of the method for Cryptosporidium oocysts on lettuce and raspberries

Laboratory to laboratory variation in the results was low (i.e., reproducibility was high) as indicated by the small values of ICCs and the standard deviations of laboratory effects.

Table D.1 — Results of data analysis obtained for Cryptosporidium on Webb’s lettuce

Table D.2 — Results of data analysis obtained for Cryptosporidium on raspberries

NOTE LOD₅₀ and its 95 % confidence interval were estimated based on the mean spike concentrations. LOD₅₀ estimates may be biased due to uncertainty in spike concentration. Furthermore, Figure D1 shows that there was a lack of data around the 50 % positive rate. This lack of data may also lead to an inaccurate estimate of LOD₅₀.

Figure D1 — Percent positive recovery across all laboratories at each spike level.

• 1. Performance characteristics of the method for Giardia oocysts on lettuce

Laboratory to laboratory variation in the results was high (i.e., reproducibility was low) as indicated by the large values of ICC and the standard deviation of laboratory effects, thus preventing a robust LOD calculation.

Table D.3 — Results of data analysis obtained for Giardia on Webb’s lettuce

NOTE LOD₅₀ and its 95 % confidence interval were estimated based on the mean spike concentrations. LOD₅₀ estimates may be biased due to uncertainty in spike concentration. Furthermore, Figure D2 shows that there was high variability in the results from laboratory to laboratory. This low reproducibility of data may also lead to an inaccurate estimate of LOD₅₀.

Figure D2 — Percent positive recovery of Giardia on lettuce across all laboratories at each spike level.

Bibliography

Add the following after [25]

[26]   WANG, S. S., & IHRIE J. On the estimation of POD and LOD of qualitative microbiological assays from a multi-laboratory validation study. J. AOAC Intl. 2021, 105 (2) pp. 641-647