SIST EN ISO 18744:2016/oprA1:2025

Microbiology of the food chain - Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits - Amendment 1: Method validation studies and performance characteristics (ISO 18744:2016/DAmd 1:2025)

Mikrobiologie der Lebensmittelkette - Nachweis und Zählung von Cryptosporidium und Giardia in frischem grünem Blattgemüse und Beeren -Änderung 1 (ISO 18744:2016/DAmd 1:2025)

Microbiologie de la chaîne alimentaire - Recherche et dénombrement de Cryptosporidium et Giardia dans les légumes verts frais à feuilles et les fruits à baies - Amendement 1: Études de validation des méthodes et caractéristiques de performance (ISO 18744:2016/DAmd1:2025)

Mikrobiologija v prehranski verigi - Ugotavljanje prisotnosti in števila "Cryptosporidium" in "Giardia" v sveži zeleni listnati zelenjavi in jagodičju - Dopolnilo A1: Študije validacije metode in značilnosti delovanja (ISO 18744:2016/DAmd 1:2025)

General Information

• Status: Not Published
• Public Enquiry End Date: 30-May-2025
• ICS: 07.100.30 - Food microbiology
• ICS: 67.080.01 - Fruits, vegetables and derived products in general
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 03-Apr-2025
• Due Date: 21-Aug-2025
• Completion Date: 02-Jun-2025
• Ref Project: EN ISO 18744:2016/prA1:2025 - Microbiology of the food chain - Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits - Amendment 1: Method validation studies and performance characteristics (ISO 18744:2016/DAmd 1:2025)

Relations

• Amends: SIST EN ISO 18744:2016 - Microbiology of the food chain - Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits (ISO 18744:2016)
• Effective Date: 31-Jul-2024

Overview

SIST EN ISO 18744:2016/oprA1:2025 is an important amendment to the original ISO 18744:2016 standard, focusing on the microbiological safety of the food chain. Specifically, it covers the detection of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits, addressing method validation studies and performance characteristics. Developed under the European Committee for Standardization (CEN) and ISO Technical Committee 34/Subcommittee 9 (Food products–Microbiology), this amendment refines procedures to ensure reliable identification of these protozoan parasites in food products.

This standard update removes enumeration as a quantitative measure and instead focuses on qualitative detection: the presence or absence of Cryptosporidium oocysts and Giardia cysts per sample weight. The amendment incorporates enhanced protocol details, including centrifugation parameters and notes on method validation from interlaboratory studies demonstrating reproducibility and sensitivity.

Key Topics

• Detection of Cryptosporidium and Giardia: The method targets fresh leafy greens and berries, food groups at risk of protozoan contamination.
• Method Validation Studies: Presents data from international interlaboratory trials, analyzing reproducibility (ICC values), limit of detection (LOD), and laboratory effects.
• Performance Characteristics: Provides sensitivity, specificity, and confidence intervals for detection efficacy at various contamination levels (low, medium, high).
• Centrifugation Protocols: Refines centrifugation speeds (e.g., 2,500 x g or alternatively 1,100 x g with no braking) optimizing recovery of oocysts and cysts without overcompacting pellets.
• Qualitative Reporting: Emphasizes reporting results as presence or absence, rather than counting organisms, reflecting the qualitative nature of current validation.
• Inclusion of Internal Controls: Use of non-viable, pre-labelled oocysts and cysts is recommended for quality control but not mandatory.

Applications

• Food Safety Testing Laboratories: Provides standardized method validation and performance metrics to adopt in routine screening of leafy greens and berries for Cryptosporidium and Giardia contamination.
• Regulatory Compliance: Supports national and international food safety regulations requiring reliable detection of protozoan contamination in fresh produce.
• Food Industry Quality Control: Enables producers and distributors to implement validated detection techniques to safeguard against parasitic contamination risks.
• Public Health Monitoring: Facilitates surveillance of foodborne parasites critical to reducing outbreaks linked to fresh produce consumption.
• Research and Development: Offers a validated reference framework for developing new or improved detection methods targeting protozoan pathogens in the food chain.

Related Standards

• ISO 16140-2:2016 – Method validation for microbiological methods, referenced for statistical modeling and interlaboratory study design.
• ISO 18744:2016 – The base standard on which this amendment builds by refining detection methods and validation data.
• Other ISO TC 34 Food Microbiology Standards – Complementary standards covering a range of microbiological safety testing and validation methodologies for food products.

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Keywords

Cryptosporidium detection, Giardia detection, microbiology food chain, fresh produce testing, method validation, performance characteristics, food safety standards, leafy green vegetables microbiology, berry fruits contamination, ISO 18744 amendment, protozoan parasites food, foodborne pathogen detection, interlaboratory study, limit of detection (LOD), reproducibility in microbiological assays.

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2025
Mikrobiologija v prehranski verigi - Ugotavljanje prisotnosti in števila
"Cryptosporidium" in "Giardia" v sveži zeleni listni zelenjavi in jagodičevju -
Dopolnilo A1: Študija validacije metode in značilnosti delovanja (ISO
18744:2016/DAmd 1:2025)
Microbiology of the food chain - Detection and enumeration of Cryptosporidium and
Giardia in fresh leafy green vegetables and berry fruits - Amendment 1: Method
validation studies and performance characteristics (ISO 18744:2016/DAmd 1:2025)
Mikrobiologie der Lebensmittelkette - Nachweis und Zählung von Cryptosporidium und
Giardia in frischem grünem Blattgemüse und Beeren -Änderung 1 (ISO
18744:2016/DAmd 1:2025)
Microbiologie de la chaîne alimentaire - Recherche et dénombrement de
Cryptosporidium et Giardia dans les légumes verts frais à feuilles et les fruits à baies -
Amendement 1: Études de validation des méthodes et caractéristiques de performance
(ISO 18744:2016/DAmd1:2025)
Ta slovenski standard je istoveten z: EN ISO 18744:2016/prA1:2025
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.080.01 Sadje, zelenjava in njuni Fruits, vegetables and
proizvodi na splošno derived products in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
Amendment
ISO 18744:2016/
DAM 1
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Detection and enumeration of
Cryptosporidium and Giardia in
Voting begins on:
2025-03-25
fresh leafy green vegetables and
Voting terminates on:
berry fruits
2025-06-17
AMENDMENT 1: Method validation
studies and performance
characteristics
Microbiologie de la chaîne alimentaire — Recherche et
dénombrement de Cryptosporidium et Giardia dans les légumes
verts frais à feuilles et les fruits à baies
AMENDEMENT 1: Études de validation des méthodes et
caractéristiques de performance
ICS: 07.100.30
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ISO 18744:2016/DAM 1:2025(en)
ISO 18744:2016/DAM 1:2025(en)
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ii
ISO 18744:2016/DAM 1:2025(en)
Foreword
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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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iii
ISO 18744:2016/DAM 1:2025(en)
Annex D (informative) Method validation studies and performance characteristics

Introduction
Line 13
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and for their enumeration
1  Scope
Line 1
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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.

iv
ISO 18744:2016/DAM 1:2025(en)
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
...