Fourth external quality assessment on species identification and antimicrobial susceptibility testing of Campylobacter 2018.
Date: 2020-10-28
Institutions: European Centre for Disease Prevention and Control
Subjects: analytical chemistry, disease prevention, harmonisation of standards, health control, public health, research body, research report, zoonosis

TECHNICAL REPORT

Fourth external quality assessment on 
species identification and 
antimicrobial susceptibility testing of 
Campylobacter 

2018

www.ecdc.europa.eu

ECDC TECHNICAL REPORT 

Fourth external quality assessment on 
species identification and antimicrobial 
susceptibility testing of Campylobacter, 
2018 

 
 
This report was commissioned by the European Centre for Disease Prevention and Control (ECDC), coordinated by 
Therese Westrell (ECDC Food- and Waterborne Diseases and Zoonoses Programme), and produced by Jeppe Boel, 
Kristoffer Kiil and Malgorzata Ligowska-Marzeta (Unit for Foodborne Infections, Statens Serum Institut, Artillerivej 
5, DK-2300 Copenhagen S, Denmark). 

Acknowledgements: 

We wish to thank Erika Matuschek (EUCAST Development Laboratory, Clinical Microbiology, Central Hospital, 351 
85 Växjö, Sweden) for testing the strains included in this EQA before they were sent to the participating 
laboratories. 

Suggested citation: European Centre for Disease Prevention and Control. Fourth external quality assessment on 
species identification and antimicrobial susceptibility testing of Campylobacter, 2018. Stockholm: ECDC; 2020. 

Stockholm, October 2020 

ISBN 978-92-9498-521-7 
doi: 10.2900/672723 
Catalogue number TQ-01-20-641-EN-N 

© European Centre for Disease Prevention and Control, 2020 

Reproduction is authorised, provided the source is acknowledged 

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TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Contents 

Abbreviations ............................................................................................................................................... iv 
Executive summary ........................................................................................................................................ 1 
1. Introduction .............................................................................................................................................. 2 
1.1 Background ........................................................................................................................................ 2 
1.2 Surveillance of Campylobacter AMR ...................................................................................................... 2 
1.3 Objectives of the EQA4-AST on Campylobacter ...................................................................................... 3 
2. Study design and methods .......................................................................................................................... 4 
2.1 Organisation ....................................................................................................................................... 4 
2.2 Selection of strain panel ....................................................................................................................... 4 
2.3 Preparation and shipment of the strains ................................................................................................ 4 
2.4 Testing and reporting .......................................................................................................................... 4 
2.5 Data analysis ...................................................................................................................................... 5 
3.1 Participation ....................................................................................................................................... 6 
3.2 Applied methods ................................................................................................................................. 6 
3.3 Campylobacter species identification ..................................................................................................... 7 
3.4 Antimicrobial susceptibility testing of Campylobacter .............................................................................. 7 
Disk diffusion ....................................................................................................................................... 7 
Dilution and gradient strip ..................................................................................................................... 7 
3.4.1 Results by antimicrobial and by strain ............................................................................................ 9 
3.4.2 Individual laboratory results ........................................................................................................ 13 
6.1 Laboratories ..................................................................................................................................... 17 
6.2 FWD-Net .......................................................................................................................................... 17 
6.3 The EQA provider .............................................................................................................................. 17 
References .................................................................................................................................................. 18 
Annex 1. List of participants .......................................................................................................................... 19 
Annex 2. EUCAST ECOFFs used for Campylobacter EQA4-AST .......................................................................... 20 

Figures 

Figure 1. EU/EEA countries (light green) and EU candidate/potential candidate countries (dark green) participating 
in Campylobacter EQA4/AST, 2018 .................................................................................................................. 6 
Figure 2. Distribution of DD (mm) differences compared to the expected result for the mandatory antimicrobials for 
Campylobacter by laboratory  ....................................................................................................................... 13 
Figure 3.Distribution of MIC dilution differences compared to the expected results for the mandatory antimicrobials 
for Campylobacter by laboratory ................................................................................................................... 13 

Tables 

Table 1. Species and resistance profiles of the Campylobacter EQA4-AST test strains ........................................... 4 
Table 2. Laboratories participating (represented by an arbitrary number) from the EU/EEA countries in the 
Campylobacter EQA4-AST, participation with mandatory antimicrobials by method and percentage correct results 
for the test strains ........................................................................................................................................ 8 
Table 3. Performance per antimicrobial for DD and MIC for the three mandatory and one optional antimicrobial .. 10 
Table 4. Distribution of Campylobacter DD values (mm) of the participating laboratories .................................... 11 
Table 5. Distribution of MIC values (mg/L) of the participating laboratories ....................................................... 12 

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TECHNICAL REPORT 

Abbreviations 

AMR 

AST 

DD 

ECDC 

ECOFF 

EFSA 

EQA 

ESBL 

EU/EEA 

EUCAST 

FWD 

Antimicrobial Resistance 

Antimicrobial Susceptibility Testing 

Disk diffusion inhibition zone 

European Centre for Disease Prevention and Control 

Epidemiological Cut-Off Value 

The European Food Safety Authority 

External Quality Assessment 

Extended Spectrum Beta-Lactamase 

European Union/European Economic Area 

The European Committee on Antimicrobial Susceptibility Testing 

Food- and Waterborne Diseases and Zoonoses 

FWD-Net 

European Food- and Waterborne Diseases and Zoonoses Network 

IATA 

MIC 

NA 

ND 

NWT 

R 

S 

SSI 

TESSy 

WT 

International Air Transport Association 

Minimum inhibitory concentration 

Not applicable 

Not determined 

Non-wild type 

Resistant 

Susceptible 

Statens Serum Institut 

The European Surveillance System 

Wild type 

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TECHNICAL REPORT  

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Executive summary 

Since 2008, the EU/ EEA countries have been able to report antimicrobial resistance (AMR) data to the European 
Surveillance System (TESSy) as part of the routine surveillance data for salmonellosis and campylobacteriosis. In 
2014, ECDC published an EU protocol for harmonised monitoring of AMR in human Salmonella and Campylobacter 
isolates (updated in 2016). In addition, ECDC launched an external quality assessment (EQA) scheme for antimicrobial 
susceptibility testing (AST) for Salmonella and Campylobacter to support the implementation of the EU protocol in the 
EU Member States and EEA countries and to get an overview of the quality of the AMR data reported to ECDC.  

This report presents the results of the fourth round of the EQA on antimicrobial susceptibility testing (AST) for national 
public health laboratories for Campylobacter (Campylobacter EQA4-AST) within the Food‐ and Waterborne Diseases 
and Zoonoses Network (FWD-Net). The objectives of this EQA4-AST were to determine the accuracy of quantitative 
AST results reported by participants; to identify common laboratory problems related to the guidance in the EU 
protocol, and to assess the overall comparability of routinely collected AST data from national public health reference 
laboratories across Europe. 

The Campylobacter EQA4-AST covered species identification and AST in Campylobacter spp. Twenty-two national 
public health reference laboratories in the EU/EEA participated in the EQA that took place during the period March to 
December 2018. In addition, six EU candidate/potential candidate countries (EU enlargement countries) participated 
in the EQA. This report focuses only on the results and evaluation from the EU/EEA countries. 

Strains for the EQA were selected according to their current relevance to public health in Europe and shipped to the 
participating laboratories. Participation in the EQA involved testing and reporting on three mandatory antimicrobials 
(ciprofloxacin, erythromycin and tetracycline), with an option to report on an additional antimicrobial, gentamicin. Test 
results from all participants were evaluated and individual feedback provided. 

The test results for antimicrobial susceptibility were analysed using two different approaches. The laboratories 
reported their results as disk diffusion (DD) and minimum inhibitory concentration (MIC) values that were compared 
to the value established by the EQA provider, either by calculating mm difference for disk diffusion values or number 
of dilution differences for minimum inhibitory concentration values. Reported quantitative results were further 
interpreted as wild type or non-wild type (WT or NWT) based on the available Epidemiological Cut-Off Values 
(ECOFFs) from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and compared to the 
expected results determined by the EQA provider. Reporting of the species identification (C. jejuni or C. coli) was 
mandatory as this is a requirement for the correct interpretation using ECOFFs. 

Twenty laboratories reported the Campylobacter species and all identifications were correct, except for a single result 
for strain C18.0005 which one laboratory falsely reported as C. coli. Two laboratories did not submit results for species 
identification. All 22 participating national public health reference laboratories reported results for the mandatory 
antimicrobials ciprofloxacin, erythromycin, and tetracycline and thus fulfilled the requirement for participating in the 
EQA. Fourteen laboratories reported additional results for gentamicin. 

Overall, there was a satisfactory correspondence between the expected results established by the EQA provider and 
the results reported by the participating laboratories. For the mandatory antimicrobials, the relative accuracy, (i.e. the 
percentage of DD and MIC results within the accepted range of the expected result) was 70% for DD and 76% for 
MIC results. When the results were interpreted using EUCAST ECOFFs, 89% of the DD results and 96% of MIC results 
were in accordance with the expected results. For disk diffusion, this is not such a good result as in the previous EQA 
round (EQA3-AST), 79% of DD results were within the expected range and all DD results were correct when 
interpreted with ECOFFs.  

The performance of the individual laboratories varied considerably when comparing the reported results with the 
expected values. For the mandatory antimicrobials, the percentage of correct quantitative results ranged from 37% to 
100% for DD results and from 33% to 100% for MIC results. This large range in results indicates that some 
laboratories may need further support to identify problems and correct them in their laboratory procedures. Two 
laboratories in particular were responsible for many of the incorrect quantitative results that also were incorrect when 
interpreted using ECOFFs. 

No common laboratory problems were identified. However, a few laboratories did not entirely comply with the 
guidance in the harmonised EU AST protocol (e.g. using disk loads of antimicrobials that differed from those 
recommended or establishing MIC values using antimicrobial concentration  ranges that did not comply with the 
recommendations set to cover both the ECOFFs and the clinical breakpoints). 

The surveillance system implemented as part of TESSy relies on the capacity of the European Food- and Waterborne 
Diseases and Zoonoses Network (FWD-Net) laboratories to produce comparable AST results. Overall, the results from 
the Campylobacter EQA4-AST indicate that it is feasible to compare AST results from the national public health 
reference laboratories when applying ECOFFs. However, improvements are warranted at a few of the laboratories. 

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Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

1. Introduction 

1.1 Background 

The European Centre for Disease Prevention and Control (ECDC) is a European Union (EU) agency with a mandate 
to operate European infectious disease networks and to identify, assess, and communicate current and emerging 
threats to human health from communicable diseases. As part of its mission, ECDC fosters the development of 
sufficient capacity within the Community for the diagnosis, detection, identification and characterisation of 
infectious agents, which may threaten public health. One of the ways in which ECDC does this is by supporting the 
implementation of quality assurance schemes [1]. 

External quality assessment (EQA) is part of a quality management system. It evaluates the performance of 
laboratories by using material that is specifically prepared and supplied for this purpose. 

ECDC supports a series of EQAs for EU/EEA countries within the disease networks. The aim of the EQAs is to 
identify areas requiring improvement in laboratory diagnostic capacity and further characterisation as relevant for 
the surveillance of the diseases listed in Commission Implementing Decision 2018/945/EU [2], and to ensure the 
reliability and comparability of results from laboratories in all EU/EEA countries. The main objectives of EQA 
schemes include: 

 
 
 
 
 
 

assessment of the general standard of performance (‘state of the art’); 
assessment of the effects of analytical procedures (method principle, instruments, reagents, calibration); 
evaluation of individual laboratory performance; 
identification and justification of problem areas; 
provision of continuing education; 
identification of training activity requirements. 

The Unit for Foodborne Infections at Statens Serum Institut (SSI) was awarded the framework service contract 
‘External quality assessment on antimicrobial susceptibility testing (AST) for national public health laboratories for 
Salmonella and Campylobacter ‘ for the two lots covering Lot1 Salmonella and Lot2 Campylobacter for the period 
2014–2018. The contract covers the organisation of an EQA exercise to test antimicrobial susceptibility and detect 
Extended Spectrum Beta-Lactamase (ESBL)-producers, acquired AmpC and carbapenemase-producers in 
Salmonella and identify species and test antimicrobial susceptibility in Campylobacter. This report presents the 
results of the fourth EQA exercise under the framework contract (Campylobacter EQA4-AST). 

1.2 Surveillance of Campylobacter antimicrobial resistance 

Antimicrobial resistance (AMR) is a serious threat to public health in Europe, leading to mounting healthcare costs, 
treatment failure and deaths. The issue calls for both concerted efforts at Member State level and close 
international cooperation in order to preserve future antimicrobial effectiveness and access to effective treatment 
for bacterial infections. Surveillance of AMR is a fundamental part of an effective response to this threat, and 
surveillance results constitute an essential source of information on the magnitude and trends of resistance. 

Campylobacteriosis, followed by salmonellosis, is the leading cause of zoonotic foodborne diseases in the EU/EEA, 
with approximately 250 000 laboratory-confirmed cases reported in 2017 [3].  

EU surveillance of AMR in foodborne human infections is carried out within the Food‐ and Waterborne Diseases and 
Zoonoses Network (FWD-Net), under the auspices of ECDC. Since 2008, the EU/EEA countries have been able to 
report AMR data to the European Surveillance System (TESSy) as part of the routine surveillance data for 
salmonellosis and campylobacteriosis. The European Food Safety Authority (EFSA) also collects AMR data on 
zoonoses and zoonotic agents in food‐producing animals and food in accordance with Directive 2003/99/EC [4] and 
Implementing Decision 2013/652/EU [5]. Since 2012, both EFSA and ECDC have strived to harmonise the AMR 
monitoring in zoonoses and zoonotic agents within their respective areas but also between the areas in order to 
obtain data that can be compared across the sectors. This work was also requested by the European Commission 
in its Commission Action Plan on AMR and in 2014, ECDC published an EU protocol for harmonised monitoring of 
AMR in human Salmonella and Campylobacter isolates, which was further updated in 2016 (hereafter referred to as 
the harmonised EU AST protocol) [6]. The harmonised EU AST protocol is primarily designed for use by the 
national public health reference laboratories or other nationally recognised public health laboratories to guide the 
susceptibility testing needed for EU surveillance and reporting to ECDC. Since July 2018, in accordance with 
Commission Implementing Decision 2018/945/EU, the EU Member States have been required to test and report 
AST results for a representative subset of Salmonella and Campylobacter isolates according to the methods and 
criteria specified in the harmonised EU AST protocol [2].  

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TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

The EU surveillance objectives for antimicrobial resistance in zoonotic bacteria, specifically Salmonella spp. and 
Campylobacter spp. [6] are:  

 

 

 

 

 

 

to monitor, in human clinical isolates, trends in the occurrence of resistance to antimicrobial agents relevant 
for treatment of human Salmonella and Campylobacter infections, including comparison with food/animal 
isolates; 
to monitor, in human clinical isolates, trends in the occurrence of resistance to other antimicrobial agents of 
public and animal health importance, including comparison with food/animal isolates; 
to monitor, in human clinical isolates, the prevalence of ESBL, plasmid-encoded Ambler class C β-
lactamases (pAmpC) and carbapenemases;  
to use antimicrobial resistance patterns to characterise human clinical isolates (i.e. as an epidemiological 
marker), to support identification of outbreaks and related cases; 
to identify and monitor, in human clinical isolates, genetic determinants of resistance that are important for 
public health (e.g. to aid recognition of epidemic cross-border spread of multi-drug resistant Salmonella 
strains); 
to monitor, in human clinical isolates, trends in resistance to antimicrobial agents where these agents may 
be needed for future therapeutic use. 

1.3 Objectives of the EQA4-AST on Campylobacter 

The aim of the EQA4-AST was to support implementation of the harmonised EU AST protocol for monitoring 
antimicrobial resistance in human Salmonella and Campylobacter isolates and to assess the quality of the AST data 
obtained using minimum inhibitory concentration (MIC) and/or disk diffusion (DD) at national public health 
reference laboratories across Europe.  

The Campylobacter EQA4-AST covered the laboratory procedure when producing AST data including species 
identification, as this is a prerequisite for interpreting quantitative data following the EUCAST ECOFFs. The 
objectives of the Campylobacter EQA4-AST scheme were: 

 
 

 

to determine the relative accuracy of quantitative AST results reported by participating laboratories; 
to identify common laboratory problems related to the testing of individual antimicrobials and the guidance 
in the harmonised EU AST protocol; 
to assess the overall comparability of routinely collected AST results from national public health reference 
laboratories across Europe based on the results of the EQA.  

The term ‘relative accuracy’ used to describe the quantitative result means that the results from the participating 
laboratories are comparable with the expected result determined by the EQA provider. 

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2. Study design and methods 
2.1 Organisation 
The entire EQA process, from planning to final reporting, took place during the period March to December 2018 
and included species determination and AST of eight Campylobacter spp. strains. 

On 30 April 2018, SSI emailed invitations to the 27 laboratories in the FWD-Net that had been nominated as 
contact points for the EQA by the national focal points for food- and waterborne diseases and zoonoses in the 
FWD-Net. Twenty-two national public health reference laboratories in EU/EEA countries accepted the invitation to 
participate. In addition, six laboratories from EU candidate/potential candidate countries (EU enlargement 
countries) participated in the EQA. The list of participants is presented in Figure 1 and Annex 1.  

The EQA test-strains were sent to the laboratories on 12 July 2018. The participants were asked to submit their 
results using a web-based electronic submission form. All laboratories were assigned an arbitrary laboratory 
number by the EQA provider and these numbers are used throughout this report to ensure the anonymity of the 
participating laboratories.  

2.2 Selection of strain panel 
Strains were selected for the EQA4-AST based on the following criteria:  

 
 

that they should represent strains commonly reported in the EU/EEA; 
that they should remain stable during the preliminary testing period at the organising laboratory.  

The EQA-provider tested 16 Campylobacter spp. strains and selected eight (five C. jejuni and three C. coli), with 
different resistance profiles (Table 1).  

In order to be able to determine the accuracy of the reported results, the EQA provider established expected 
results for MIC and DD values for the test strains. The expected values were established in accordance with the 
harmonised EU AST protocol [6]. The DD values were determined using disks from Oxoid and the MIC values were 
determined using the micro-broth-dilution-based MIC system from TREK diagnostic systems© (Thermo Scientific). 
The expected results were verified by EUCAST’s Development Laboratory for Antimicrobial Susceptibility Testing of 
bacteria, c/o Clinical Microbiology, Central Hospital, Växjö, Sweden. 

Table 1. Species and resistance profiles of the Campylobacter EQA4-AST test strains 

Strain 

Species 

Resistance profile1 (NWT) 

EQA_AST.C18.0001  Campylobacter jejuni  

Ciprofloxacin, tetracycline 

EQA_AST.C18.0002  Campylobacter jejuni  

Ciprofloxacin, erythromycin, tetracycline 

EQA_AST.C18.0003  Campylobacter jejuni  

Ciprofloxacin, tetracycline 

EQA_AST.C18.0004  Campylobacter jejuni  

Tetracycline 

EQA_AST.C18.0005  Campylobacter jejuni  

Ciprofloxacin, erythromycin, gentamicin, tetracycline 

EQA_AST.C18.0006  Campylobacter coli 

Ciprofloxacin, tetracycline 

EQA_AST.C18.0007  Campylobacter coli 

Wild type (i.e. no acquired resistance) 

EQA_AST.C18.0008  Campylobacter coli 

Tetracycline  

1 Based on MIC values and according to EUCAST ECOFFs - see Annex 2. 

2.3 Preparation and shipment of the strains 

Cultures of the test stains were grown on blood agar and transferred to Stuart's transport medium using charcoal 
swabs. The parcels with the strains were shipped on 12 July 2018 from SSI and labelled in accordance with the 
International Air Transport Association (IATA) regulations (UN 3373 Biological Substance, Category B). 

2.4 Testing and reporting 
The EQA4-AST included AST of four first-priority antimicrobials listed in the harmonised EU AST protocol [6] and 
species identification. It was a prerequisite for the participating laboratories to test three of the first-priority 
antimicrobials (ciprofloxacin, erythromycin and tetracycline) and there was an additional option to report results for 
the fourth, gentamicin.  

Instructions for AMR testing were given in the invitation letter, in an email following shipment of strains, and in the 
reporting forms. Participants were asked to follow the harmonised EU AST protocol and could submit results from 

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TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

broth dilution and gradient strip methods (MIC results) or disk diffusion (DD). The participants were asked to 
report the test result as a value (mg/L or mm). The harmonised EU AST protocol mainly refers to the methods 
recommended by EUCAST, available on EUCAST’s website [7] and it was expected that the participants would 
follow these instructions. No instructions were given with regard to species identification and it was anticipated that 
the laboratories would use their own standard method. 

When the test strains were dispatched the laboratories received an email with a link to an electronic submission 
form, constructed using Enalyzer software (www.enalyzer.com), for the reporting of the results in a fixed format. 
The deadline for submitting the results was 28 July 2018. This deadline was extended to 3 August 2018 due to 
delays in the freight handling of packages to some of the laboratories. Data reporting included Campylobacter 
species, quantitative DD and/or MIC results, information about the methods used, growth media, brand of disks for 
DD and brand of strips or panels for MIC determination. 

2.5 Data analysis 

The participating laboratories provided test results (i.e. inhibition zones measured as diameter in mm for disk 
diffusion methods and MIC values for broth dilution and gradient strip methods.) It was mandatory to report the 
species identification (C. jejuni or C. coli) as this information is needed for the correct interpretation using EUCAST 
ECOFFs. 

The test results were analysed using different approaches: 

1. 

2. 

3.  

The laboratories reported their results and these values were compared with the expected results 
established by the EQA provider, either by calculating the difference in mm for DD values or the number of 
dilution differences for MIC values. 

DD results (values in mm) generated with disk loads that deviated from the recommended disk loads were 
excluded from the analysis and classified as ‘ND’ (not determined).  

MIC dilution differences between the reported and expected results were calculated taking into account 
several situations:  

 
 
 

 

If the operator of the reported value was >, results were approximated to = the next dilution step. 
If the operator of the reported value was <=, results were approximated to = the same dilution step. 
If the operator of both the reported value and the expected value were > and the participant’s range 
for a given antimicrobial was wider than the EQA provider’s range, the dilution difference was 
designated as ‘0’. 
If the expected result was outside of the range tested by the participant but within the EQA providers’ 
range, it was not possible to calculate the dilution difference.  

MIC values generated by the use of gradient strips for MIC determination were transformed on a base-2 log scale, 
rounded to the nearest two-fold dilution, and then retransformed to enable comparison with the results from 
dilution methods. 

The quantitative results were categorised into three groups. The first group, designated correct, included DD 
results that were within ±4 mm difference of the expected result and MIC results that were within one dilution 
difference. The second group were results outside the accepted area (incorrect) and the third group included MIC 
results that were not within the relevant range for comparison with expected results (ND). 

Reported qualitative results were interpreted based on the available EUCAST ECOFFs. This interpretation (wild type 
(WT) or non-wild type (NWT)) was compared with the expected result, as determined by the EQA provider. These 
qualitative results where categorised into three groups. The first group included results that complied with the 
expected interpretation (correct), the second group included the interpreted results that did not comply with the 
expected results (incorrect), and the third group included results where this comparison was impossible due to the 
lack of EUCAST ECOFFs for the antimicrobial (NA). The applied EUCAST ECOFFs can be found in Annex 1. In the 
event of incorrect or missing Campylobacter species identification, the reported DD and MIC data were analysed 
using the correct species result determined by the EQA provider. 

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TECHNICAL REPORT 

3. Results 

3.1 Participation 

Twenty-two laboratories from EU/EEA countries participated in the Campylobacter EQA4-AST (Figure 1), along with 
six EU candidate/potential candidate countries (EU enlargement countries). Test results from all participants were 
evaluated and individual feedback provided on 12 October 2018. All participants also received a file sent by e-mail 
on 19 October 2018, with the distributions of all reported MIC and DD (mm) values for all test 
strains/antimicrobials included in the EQA4-AST. 

This report focuses solely on the results and evaluation of data from the EU/EEA countries. The 22 participating 
countries susceptibility-tested the test strains for the mandatory antimicrobials, ciprofloxacin, erythromycin, and 
tetracycline and 13 countries also reported results for gentamicin. Due to delays during shipment, one laboratory 
was unable to recover the strains EQA_AST.C18.0001 and EQA_AST.C18.0005. After consultation with the EQA 
provider, it was decided that this laboratory should not provide results for these two test strains. 

Figure 1. EU/EEA countries (light green) and EU candidate/potential candidate countries (dark 
green) participating in Campylobacter EQA4/AST, 2018 

3.2 Applied methods 

A total of 15 laboratories reported DD results. Most laboratories used the EUCAST recommended disk loads. Three 
laboratories used disk loads that did not comply with the recommendations in the harmonised EU AST protocol. 
One laboratory (L021) used a ciprofloxacin load of 10 μg instead of 5 μg, for erythromycin one laboratory (L024) 
used a disk load of 5 μg instead of 15 μg and for gentamicin one laboratory (L020) used a disk load of 30 μg 
instead of the recommended 10 μg.  

Most of the DD results (86%) were produced on Mueller Hinton agar, supplemented with 5% horse blood and L ß-
NAD at a concentration of 20 mg/L, and the remainder were produced on the same media without the NAD 
component. 

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Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Disks from Oxoid were used to produce 49% of the DD results and disks from Bio-Rad- and Becton Dickinson were 
used to generate 24% and 14% of the results, respectively. Disks from i2A diagnostics and Mastdiscs were used to 
make 8% and 6% of the results, respectively.  

For AST DD testing of Campylobacter, EUCAST recommends an incubation temperature of 41±1°C for 24 hours in 
a micro-aerobic environment. Three laboratories did not follow this recommendation and used an incubation 
temperature of 3537°C. 

Eleven laboratories reported MIC results. Forty-one percent of the MIC results were obtained using gradient strips 
and all gradient strip results were obtained with Etest from bioMérieux. The remaining 59% of the MIC results 
were obtained using broth dilution methods; 31% were generated with the TREK sensititre microdilution system 
(also used by the EQA provider), 18% were made using in-house assays and 8% of the broth dilution results were 
reported as having been produced using the brand ‘SVA’. Two laboratories used in-house assays that applied 
concentration ranges that did not comply with the recommendations in the harmonised EU AST protocol. This 
meant that it was impossible to calculate the dilution difference for some of the quantitative MIC results applying 
the principles described in section 2.5. Consequently, some of the results from these two laboratories were 
classified as ND. 

3.3 Campylobacter species identification 

Twenty laboratories reported Campylobacter species of the test strains. All species identifications were correct 
except for one result relating to strain C. jejuni C18.0005 which one laboratory, L032, reported as C. coli. Two 
laboratories, L12 and L19, did not report the species of any of the test strains.  

3.4 Antimicrobial susceptibility testing of Campylobacter 

The laboratories’ DD and MIC results, along with the percentage of correct qualitative and quantitative results for 
the eight test strains, are presented in Table 2. Eleven of 22 laboratories tested all the mandatory antimicrobials 
only using DD, seven only used MIC determinations, and four laboratories reported both DD and MIC results. Six 
laboratories used broth dilution methods and five laboratories used gradient strip for MIC determinations.  

Disk diffusion 

Fifteen laboratories reported 432 DD results for the test strains. Twenty-four of these results were classified as ND 
due to deviating disk loads. Of the remaining 408 DD results, 292 (72%) were classified as correct and within ±4 
mm of the expected value. For the mandatory antimicrobials, 253/360 (70%) of the DD results were classified as 
correct. The correct DD results from the individual laboratories ranged from 38% to 96%. After interpretation of 
the DD results using EUCAST ECOFFs, the overall proportion of correct qualitative DD results was 89% for the 
mandatory antimicrobials and 100% for gentamicin. Two laboratories had less than 90% correct qualitative results, 
L019 (54%) and L040 (83%), for the mandatory antimicrobials and L019 reported 11 of the total 25 incorrect 
qualitative results. 

Dilution and gradient strip 

Eleven laboratories reported 314 MIC results for the test strains. Twenty of these MIC results were classified as ND 
due to deviating test ranges. Of the 294 remaining MIC results, 229 (78%) were classified as correct and within ± 
one dilution rate of the expected value. For the mandatory antimicrobials, 189/237 (80%) were classified as 
correct. The correct MIC results for the individual laboratories ranged from 46% to 100%. After interpretation of 
the MIC results using EUCAST ECOFFs, the overall proportion of correct qualitative MIC results were 96% for the 
mandatory antimicrobials and 97% for gentamicin. All laboratories except one (L012) reported more than 90% 
correctly interpreted qualitative results. L012 reported 71% correct qualitative results and eight of the 12 MIC 
results that were incorrect when evaluated using the EUCAST ECOFFs (six of these were MIC results for 
tetracycline.) The 20 MIC results classified as ND were reported by two laboratories, L007 (12) and L012 (8). 
Seventeen of the ND MIC results were evaluated as correct when interpreted with ECOFFs and it was not possible 
to evaluate the remaining three MIC results as there was no ECOFF available for gentamicin/C coli. 

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Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

TECHNICAL REPORT 

Table 2. Laboratories participating in the Campylobacter EQA4-AST (represented by an arbitrary number) from the EU/EEA countries, participation with mandatory 
antimicrobials by method and percentage of correct results for the test strains (AST.C18.0001-0008)* 

Mandatory 

n
i
c
a
x
o
l
f
o
r
p
C

i

n
i
c
y
m
o
r
h
t
y
r
E

D
I

y
r
o
t
a
r
o
b
a
L

DD 

D
N

e
n

i
l
c
y
c
a
r
t
e
T

t
c
e
r
r
o
C

e
v
i
t
a
t
i
t
n
a
u
q

s
t
l
u
s
e
r

t
c
e
r
r
o
C

e
v
i
t
a
t
i
l
a
u
q

s
t
l
u
s
e
r

n
i
c
i
m
a
t
n
e
G

t
c
e
r
r
o
C

e
v
i
t
a
t
i
t
n
a
u
q

s
t
l
u
s
e
r

t
c
e
r
r
o
C

e
v
i
t
a
t
i
l
a
u
q

s
t
l
u
s
e
r

Optional 

Mandatory 

Optional 

MIC 

n
i
c
a
x
o
l
f
o
r
p
C

i

B 
G 

B 
B 

B 

n
i
c
y
m
o
r
h
t
y
r
E

B 
G 

B 
B 

B 

e
n

i
l
c
y
c
a
r
t
e
T

B 
G 

B 
B 

B 

t
c
e
r
r
o
C

e
v
i
t
a
t
i
t
n
a
u
q

s
t
l
u
s
e
r

92% 
79% 

46% 
96% 

D
N

50% 

t
c
e
r
r
o
C

e
v
i
t
a
t
i
l
a
u
q

s
t
l
u
s
e
r

100% 
96% 

100% 
100% 

29% 

4% 

71% 

n
i
c
i
m
a
t
n
e
G

B 
G 

B 

B 

D
N

t
c
e
r
r
o
C

e
v
i
t
a
t
i
t
n
a
u
q

s
t
l
u
s
e
r

100% 
63% 

t
c
e
r
r
o
C

e
v
i
t
a
t
i
l
a
u
q

s
t
l
u
s
e
r

100% 
80% 

63% 

100% 

0% 

88% 

80% 

G 

G 

75% 

94% 

88% 
96% 
88% 

67% 

58% 
96% 
38% 
38% 
75% 
50% 
67% 
67% 
88% 

96% 
100% 
100% 

96% 

96% 
100% 
92% 
54% 
92% 
94% 
100% 
96% 
100% 

33% 
33% 

88% 

100% 

38% 

100% 

38% 

100% 

0% 
25% 
88% 
88% 

100% 
100% 
100% 

75% 
67% 
70% 

83% 
96% 
89% 

4% 

63% 
63% 
54% 

100% 
100% 
100%    

G 
B 
G 
G 
B 

G 
B 
G 
G 
B 

B 
B 
G 
G 
B 

71% 
92% 
71% 
83% 
100% 

96% 
100% 
100% 
100% 
100% 

G 
B 
G 

B 

75% 
100% 
0% 

100% 

100% 
100% 
100% 

100% 

76% 

5% 

96%    

63% 

11% 

97% 

L003 
L004 
L006 
L007 
L008 
L011 
L012** 
L015 
L016 
L017 
L019** 
L020 
L021 
L024 
L028 
L030 
L032** 
L034 
L037 
L039 
L040 
L043 
Total 

   : Results for all test strains reported. B: Broth microdilution, G: Gradient strip.  

* Results classified as NA are excluded from the total number of results.  

** The quantitative results were evaluated using species identification determined by the EQA provider.  

*** No gentamicin ECOFF has been established for C. coli and disk diffusion. 

8 

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
 
  
 
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
 
  
  
     
  
  
  
  
  
  
  
  
  
 
TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

3.4.1 Results by antimicrobial and by strain 

Table 3 gives an overview of the DD and MIC results by antimicrobial and also presents the MIC results separately 
for gradient strip and broth dilution methods. 

With regard to DD, the lowest scores for correct quantitative results were recorded for gentamicin and 
erythromycin (61% and 64% of the results respectively were correct.) The highest score was for ciprofloxacin with 
80% of results correct. For MIC methods overall, between 70% and 86% of the quantitative results were correct 
for the four antimicrobials tested. When divided by type of MIC method, the lowest scores for gradient strips were 
observed for gentamicin and ciprofloxacin (46% and 55%, respectively), and with broth dilution for tetracycline 
(76%). The highest score for gradient strip results was for tetracycline (95%) and with broth dilution, similar 
results were achieved for ciprofloxacin, erythromycin and gentamicin (8688%).  

The proportion of correct EUCAST ECOFF interpreted qualitative results by antimicrobial ranged from 89% to 
100%, where the lowest proportion of correct results, 89%, was observed for erythromycin DD results and for 
tetracycline broth dilution results.  

The distribution of DD values (mm) and the distribution of MIC values (mg/L) for the participating laboratories are 
presented in Table 4 and 5, respectively. 

Control strain 
EUCAST has defined acceptance criteria relating to the size of the inhibition zone for the control strain, C. jejuni 
ATCC 33560, for ciprofloxacin, erythromycin and tetracycline [8]. For ciprofloxacin and tetracycline, two and three 
of the reported inhibition zones for the control strain, respectively, were too large (Table 4). For erythromycin, one 
DD result was too small and one was too large. For gentamicin, the reported values for the control strain were 
compared with the expected valued established by the EQA provider. Two DD values for gentamicin were outside 
the accepted range, with one value being too low and one being too high. One laboratory, L043, did not report any 
DD results for the control strain for any antimicrobial, L21 did not report DD results for ciprofloxacin, and L024 did 
not report DD results for erythromycin. The incorrect results for the control strains were submitted by six 
laboratories. 

EUCAST has not defined acceptance criteria for MIC values for the control strain C. jejuni ATCC 33560 and 
therefore values established by the Clinical and Laboratory Standard Institute were used as guidance [9]. The 
accepted range included values based on incubation at both 37°C for 48 hours and 42°C for 24 hours. For 
erythromycin and gentamicin, all results for the control strain were within the accepted range (Table 5). For 
ciprofloxacin, one result was one dilution higher that the accepted range and for tetracycline, two values were one 
dilution lower than the accepted range. 

Test strains 
The reported DD values were generally in accordance with the expected values (Table 4). Strain C18.005 deviated 
markedly from the expected results for erythromycin as nine of the 13 results were incorrect and all nine incorrect 
results exhibited varying inhibition zones. In general, most of the incorrect results were due to the zones being too 
large, most noticeably for gentamicin. For three strain/antimicrobial combinations (C18.004/ciprofloxacin, 
C18.006/erythromycin, and C18.007/erythromycin), the accepted zone deviations included values on the ‘wrong 
side’ of the ECOFF and three such results were reported for erythromycin (i.e. quantitatively correct but 
qualitatively incorrect). 

A high number of incorrect MIC values were observed for ciprofloxacin (Table 5). Most of the incorrect MIC values 
were generated using gradient strip methods (Table 3). The same phenomenon was observed for gentamicin where 
most of the incorrect values were also generated using gradient strip methods. The problem with erythromycin for 
strain C18.005 was also seen in the MIC results. For tetracycline, most of the incorrect DD values were due to the 
zones being too small. In addition, L012 performed rather poorly for MIC, with only 29% correct results using an 
in-house broth dilution method (Table 2). 

9 

 
 
Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

Table 3. Performance per antimicrobial for DD and MIC for the three mandatory and one optional antimicrobial 

Antimicrobial 

Number of laboratories 
performing DD 

Numbers of DD results within 
the accepted four mm 
difference of the total tested 

Number of correct results 
when using EUCAST ECOFF 

Ciprofloxacin 

Erythromycin 

Tetracycline 

Gentamicin* 

Total DD 

Ciprofloxacin 

Erythromycin 

Tetracycline 

Gentamicin* 

Total MIC 

Ciprofloxacin 

Erythromycin 

Tetracycline 

Gentamicin* 

Total MIC gradient 
strips 

Ciprofloxacin 

Erythromycin 

Tetracycline 

Gentamicin* 

Total MIC broth 
dilution 

14 

14 

15 

8 

Disk diffusion 

90/112 (80%) 

72/112 (64%) 

91/120 (76%) 

39/64 (61%) 

292/408 (72%) 

105/112 (94%) 

100/112 (89%) 

114/120 (95%) 

40/40 (100%) 

359/384 (93%) 

Number of laboratories 
performing MIC (both 
gradient strips and broth-
dilution) 

Numbers of MIC results within 
the accepted one dilution 
difference of the total tested 

Number of correct results 
when using EUCAST ECOFF 

11 

11 

10 

8 

5 

5 

3 

3 

6 

6 

7 

5 

MIC total 

58/81 (72%) 

72/84 (86%) 

59/72 (82%) 

40/57 (70%) 

85/86 (99%) 

83/86 (97%) 

72/78 (92%) 

38/40 (95%) 

229/294 (78%) 

278/290 (96%) 

MIC gradient strips 

21/38 (55%) 

32/38 (84%) 

21/22 (95%) 

11/24 (46%) 

38/38 (100%) 

35/38 (92%) 

22/22 (100%) 

14/15 (96%) 

85/122 (70%) 

109/113 (96%) 

MIC broth dilution 

37/43 (86%) 

40/46 (87%) 

38/50 (76%) 

29/33 (88%) 

47/48 (98%) 

48/48 (100%) 

50/56 (89%) 

24/25 (98%) 

144/172 (84%) 

169/177 (95%) 

Results classified as NA and ND excluded.  

* EUCAST has not determined a gentamicin ECOFF value for C. coli. 

10 

  
  
  
  
  
 
TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Table 4. Distribution of Campylobacter DD values (mm) among the participating laboratories 
A n t i m i c r o b i a l

2 0

1 0

1 1

2 2

2 6

1 2

1 3

1 4

1 5

1 6

1 7

1 8

1 9

2 1

2 3

2 4

2 5

2 7

6

7

8

9

2 8

n
i
c
a
x
o
l
f
o
r
p
C

i

n
i
c
y
m
o
r
h
t
y
r
E

e
n

i
l
c
y
c
a
r
t
e
T

n
i
c
i
m
a
t
n
e
G

11 
9 
10 
1 
13 
14 
1 

12 

4 
1 

3 
8 
13 
12 
12 

1 

S t r a i n
ATCC 33560 
C18.0001 
C18.0002 
C18.0003 
C18.0004 
C18.0005 
C18.0006 
C18.0007 
C18.0008 
ATCC 33560 
C18.0001 
C18.0002 
C18.0003 
C18.0004 
C18.0005 
C18.0006 
C18.0007 
C18.0008 
ATCC 33560 
C18.0001 
C18.0002 
C18.0003 
C18.0004 
C18.0005 
C18.0006 
C18.0007 
C18.0008 
ATCC 33560 
C18.0001 
C18.0002 
C18.0003 
C18.0004 
C18.0005 
C18.0006 
C18.0007 
C18.0008 

1 
1 

2 
1 

1 

1 

1 

1 

1 
1 
1 

2 

1 

1 

1 

2 
3 

1 

1 

2 

1 
1 

1 

1 

1 
3 
1 

1 
2 

1 
1 

1 

1 

1 

1 

2 

1 

3 

1 

1 

1 

1 

1 

2 9

3 0

3 1

3 2

3 3

3 4

3 5
2 

3 6
1 

3 7
2 

3 8
2 

3 9

4 0
1 

4 1
2 

4 2
1 

4 3
1 

4 4

4 5
1 
1 

4 6

 > 4 6

1 

2 

1 

1 

2 

2 

2 

1 

1 

1 
1 

1 
1 

2 
1 

1 

1 

1 
1 
1 

1 

2 

1 

1 

1 
1 
1 
2 

1 

1 
2 

1 

3 
1 
1 

1 

2 

1 
2 

1 

1 

3 

3 

4 
1 
1 

1 
3 
2 

1 

1 
1 
2 
1 

1 

1 

1 

1 

1 

1 
2 

1 
3 
1 
2 
4 
3 

1 
1 
2 
3 
1 
1 

2 

1 

1 

2 

1 

2 

1 
1 

1 

2 
1 

1 

1 
1 

4 

1 
1 

1 
2 

1 

1 

2 

1 

1 
1 

1 

1 
1 

2 
3 

1 
1 
2 
1 

1 
1 

1 

1 

4 

4 
1 
2 

1 

1 
1 

3 

2 

1 

2 

2 

1 

2 

1 

1 

2 

1 

1 

2 
3 
1 

1 

2 
1 

1 
1 

1 

1 

1 

1 

1 

1 
1 

2 
1 

1 

1 

1 
3 

1 

1 

1 

4 

1 

1 

1 

2 

1 
1 

1 

2 
1 

1 

1 

1 

1 
1 

1 
1 

   : Expected value    : Accepted range 

The red line indicates ECOFF according to EUCAST for the respective antimicrobial, wild type/susceptible to the right of the red line.  
The expected values for the control strain ATCC 33560 were from EUCAST, except for gentamicin, where the expected values were determined by the EQA provider. 

1 
1 
1 

1 

1 

1 
1 
2 

1 

2 
1 
1 

1 

1 

11 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Table 5. Distribution of MIC values (mg/L) among the participating laboratories 

MIC results for the Campylobacter strains tested 

l
a
i
b
o
r
c
i
m

i
t
n
A

n
i
c
a
x
o
l
f
o
r
p
C

i

n
i
c
y
m
o
r
h
t
y
r
E

e
n

i
l
c
y
c
a
r
t
e
T

n
i
c
i
m
a
t
n
e
G

Strain 

ND  0.03  0.06  0.12  0.25  0.5  1  2 

4 

8 

16  32  64  128  256  512 

ATCC 33560 

2 

1 

3 

3 

1 

C18.0001 

C18.0002 

C18.0003 

C18.0004 

C18.0005 

C18.0006 

C18.0007 

C18.0008 

ATCC 33560 

C18.0001 

C18.0002 

C18.0003 

C18.0004 

C18.0005 

C18.0006 

C18.0007 

C18.0008 

ATCC 33560 

C18.0001 

C18.0002 

C18.0003 

C18.0004 

C18.0005 

C18.0006 

C18.0007 

C18.0008 

ATCC 33560 

C18.0001 

C18.0002 

C18.0003 

C18.0004 

C18.0005 

C18.0006 

C18.0007 

C18.0008 

1 

1 

1 

1 

1 

1 

1 

1 

1 

1 

1 

2 

1 

1 

1 

1 

1 

1 

1 

1 

3 

5 

4 

1 

1 

2 

1 

1 

3 

4 

3 

3 

5 

6 

1 

3 

1 

1 

1 

1 

3 

5 

1 

1 

1 

1 

3 

1 

1 

2 

4 

2 

1 

1 

3 

4 

1 

4 

3 

3 

4 

1 

1 

1 

3 

4 

2 

2 

1 

3 

2 

1 

1 

1 

2 

1 

5 

3 

4 

4 

1 

1 

2 

2 

3 

1 

4 

3 

3 

1 

1 

5 

1 

1 

2 

6 

1 

3 

3 

3 

1 

1 

2 

1 

3 

2 

2 

1 

4 

6 

7 

4 

1 

2 

6 

6 

1 

1 

1 

1 

1 

1 

2 

1 

1 

1 

1 

2 

2 

4 

2 

1 

2 

1 

3 

3 

1 

1 

1 

2 

1 

7 

3 

2 

3 

5 

2 

1 

5 

4 

3 

6 

4 

1 

2 

1 

3 

2 

2 

2 

3 

1 

1 

2 

1 

   : Expected value    : Accepted range 

The red line indicates ECOFF according to EUCAST for the respective antimicrobial, wild type/susceptible to the left of the red 
line.  
The expected values for the control strains ATCC 33560, are from the Clinical and Laboratory Standard Institute CLSI [9] and 
include values based on incubation at both 37°C for 48 hours and 42°C for 24 hours. 

12 

 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 
TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

3.4.2 Individual laboratory results 

Disk diffusion 
The proportion of correct DD results for the mandatory antimicrobials reported by individual laboratories varied 
from 38% to 100% (Figure 2). One laboratory (L024) reported 100% correct DD results and two laboratories (L006 
and L016) had more than 90% correct results. The lowest proportion of correct results, 37%, was reported by two 
laboratories (L017 and L019). 

Figure 2. Distribution of DD (mm) differences compared to the expected result for the mandatory 
antimicrobials for Campylobacter by laboratory 

L024
L006
L016
L030
L004
L007
L020
L040
L021
L043
L028
L011
L015
L017
L019

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0-1

2-4

>4

Dilution and gradient strip 
The proportion of correct MIC results for the mandatory antimicrobials reported by individual laboratories ranged 
from 29% to 100% (Figure 3). One laboratory (L040) reported 100% correct MIC results and three laboratories 
(L008, L003 and L034) reported more than 90% correct MIC results. Two laboratories (L007 and L012) reported 
MIC results that were classified as ND due to deviations in the testing ranges, one of them (L007) for half of the 
test results. However, it should be noted that all results from the latter laboratory were categorised correctly using 
ECOFFs (Table 2). 

Figure 3. Distribution of MIC dilution differences compared to the expected results for the mandatory 
antimicrobials for Campylobacter by laboratory 

L040
L008
L003
L034
L039
L004
L021
L032
L037
L007
L012

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0

1

>1

ND

13 

 
 
 
Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

4. Discussion 

Since 2008, the EU Member States and EEA countries have been able to report AMR data to TESSy as part of the 
routine surveillance data for campylobacteriosis. In 2014, ECDC published the harmonised EU AST protocol 
(updated in 2016) with guidance on laboratory procedures and the interpretation of data [6]. The purpose of the 
EQA4-AST on Campylobacter was to evaluate the quality of the AST data generated in the FWD laboratory network 
when following the harmonised EU AST protocol. The data submitted were used to determine the relative accuracy 
of AST data and to assess its overall comparability. An additional aim was to collect information on the methods 
used by each laboratory to produce data on antimicrobial susceptibility. 

Twenty-seven national public health reference laboratories in EU/EEA countries were invited to participate in the 
EQA and 22 countries accepted the invitation, meaning that the number of participating laboratories was similar to 
that for the previous Campylobacter EQAs. All laboratories submitted results for the mandatory antimicrobials and 
fulfilled the requirement for participating in the EQA. 

Overall the logistics of the EQA went well. All laboratories, except one, were able to recover the test strains and the 
submission of results on the Enalyzer platform was efficient. 

Some laboratories did not fully adhere to the recommendations given in the harmonised EU AST protocol but in 
general, the laboratories used disks with the recommended concentration of antimicrobials, and the media, 
incubation conditions and test ranges prescribed by EUCAST. 

When interpreting the AST values for C. jejuni and C. coli  different criteria apply and correct species identification 
is essential for the correct interpretation of AST data in relation to Campylobacter. For the test strains in this EQA 
the reported species was in line with the expected results, although two laboratories did not report any species for 
the test strains. 

Overall, there was a satisfactory agreement between the quantitative results reported by the participants and the 
expected results established by the EQA provider. With a few exceptions, the test strains exhibited DD zones and 
MIC values that were distinct from the ECOFF values and, consequently, the interpreted qualitative results were 
generally better than the quantitative results. 

The results submitted for erythromycin for strain C18.005 deviated from the expected results, both for the DD and 
MIC values. The strain was consistently resistant to erythromycin when tested by the EQA provider. High-level 
macrolide resistance in Campylobacter is commonly caused by a base substitution in the 23S rRNA gene, 
specifically A2075G, and less frequently A2074C/G [10]. DNA sequencing of C18.005 revealed that the strains 
carried the A2075G substitution in two of three copies of the 23S rRNA gene. It has been reported that ribosomal 
substitutions may not always occur in all three copies of the gene, and this can result in a lower level of resistance 
[10]. This could be one explanation for the deviation in the result for erythromycin obtained in relation to strain 
C18.005. Generally the laboratories demonstrated proficiency in erythromycin AST testing for the other test 
strains. 

Most of the quantitative DD results for the wild type strains that differed from the expected results had inhibition 
zones that were too large. Some laboratories reported a number of DD results with very large inhibition zones, and 
a single laboratory, L19, reported 12 DD results that were 60 mm. 

As with the EQA3-AST on Campylobacter [11], the number of correct MIC results for ciprofloxacin were rather low, 
as only 72% of the results were in accordance with the expected value. All incorrect MIC results generated by 
broth dilution were reported by the same laboratory that submitted results for six strains using an in-house 
method. All the other 17 incorrect results except one were gradient strip results, reported with MIC values of >32 
where the expected broth dilution results were 8 or 16. The reason for this difference is unknown, but the 
difference in the MIC results did not affect the qualitative results. 

The harmonised EU AST protocol recommends (micro-) broth dilution as the preferred testing method for 
monitoring purposes. However, validated methods of gradient strip diffusion or disk diffusion according to EUCAST 
protocols are also accepted. In the present EQA, 72% and 70% of the diffusion-based methods (DD and gradient 
disk results respectively) were correct when compared with the expected values, whereas 84% of the reported MIC 
results were correct. This is similar to the findings in the previous Campylobacter EQAs which support the 
recommendation that broth dilution should be the preferred testing method. 

The performance of the individual laboratories varied substantially. For DD, the percentage of correct results for the 
mandatory antimicrobials varied from 37% to 100% by laboratory and for MIC results, the variation was from 33% 
to 100% correct results. This large range in results indicates that some laboratories may need further support to 
identify problems and, if necessary, adjust their laboratory procedures. Two laboratories in particular, one 
submitting MIC results and one submitting DD results, were responsible for many of the incorrect quantitative 
results that also were incorrect when interpreted using ECOFFS. 

14 

TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

When interpreting the results with ECOFFs, the laboratories reported a total of 89% and 96% correct DD and MIC 
results, respectively for the mandatory antimicrobials. For the optional antimicrobial, gentamicin, they reported 
100% and 97% respectively. These results are slightly lower than the results obtained in the EQA3-AST performed 
in 2017. They are also slightly lower than the results in the most recent EU reference laboratory network 
proficiency test for antimicrobial resistance [12], where the qualitative MIC results for the four antimicrobials tested 
in this EQA were all above 98.7%. Overall, the results from the Campylobacter EQA4-AST indicate that it is feasible 
to compare AST results from the national public health reference laboratories when applying ECOFFs. Nevertheless, 
improvements are warranted at a few of the FWD-Net laboratories.  

15 

 
 
Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

5. Conclusions 

A total of 22 national public health reference laboratories from the FWD network participated in the EQA. All 
laboratories, except two, performed Campylobacter species identification and all reported results for the mandatory 
antimicrobials ciprofloxacin, erythromycin, and tetracycline, thus fulfilling the requirement for participating in the 
EQA. In addition, fourteen laboratories reported results for gentamicin.  

There were no issues identified linked to the content of the harmonised EU AST protocol. The problems arose 
among the few laboratories that did not entirely comply with the protocol (e.g. using disk loads of antimicrobials 
that differed from those recommended or establishing MIC values using test ranges for antimicrobials that did not 
comply with the recommendations.) 

Overall, there was a satisfactory correspondence between the expected results established by the EQA provider 
and the results reported by the participating laboratories. For the mandatory antimicrobials the relative accuracy, 
(i.e. the percentage of DD and MIC results that were within the accepted range from the expected result) was 70% 
for disk diffusion and 76% for MIC results. When the results were interpreted with EUCAST ECOFFs, 89% of the 
DD results and 96% of MIC results were in accordance with the expected results. 

When compared with the expected values, the performance of individual laboratories varied substantially. For the 
mandatory antimicrobials, the percentage of correct quantitative results ranged from 37% to 100% for DD results 
and from 33% to 100% for MIC results. This large range in results indicates that some laboratories may need 
further support to identify problems and, if necessary, to adjust their laboratory procedures. Two laboratories in 
particular were responsible for many of the incorrect quantitative results that also were incorrect when interpreted 
using ECOFFs. 

Overall, the results from the Campylobacter EQA4-AST indicate that it is feasible to compare AST results from the 
national public health reference laboratories when applying ECOFFs. Nevertheless, improvements are warranted at 
a few FWD-Net laboratories.  

16 

 
 
TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

6. Recommendations 

6.1 Laboratories 

The laboratories should comply with the recommendations set out in the harmonised EU AST protocol which 
stipulates that the EUCAST guidelines should be followed. These guidelines include specifications for control 
strains, media, incubation temperature, disk loads for DD testing, concentration range for MIC determination, etc. 
The results from this and other EQAs show that the results generated using MIC broth dilution methods are 
generally better than the diffusion-based methods (disk diffusion and gradient strip based methods). Therefore, if 
possible, laboratories should consider implementing broth dilution methods. 

6.2 FWD-Net 

In order to ensure the comparability of AST data reported to TESSy it is important to apply standardised testing 
and interpretation of data in the Member States. ECDC and the EQA provider will continue to provide consultancy 
facilities for the FWD-Net laboratories, however specific support may also be required by individual laboratories. 

6.3 The EQA provider 

In order to assist with troubleshooting, the current reporting scheme will be further developed for a more detailed 
and uniform collection of results, method, manufacturer, growth medium and incubation temperature used by the 
participating laboratories. 

17 

 
 
Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

References 

1.  European Parliament and Council of the European Union. Regulation (EC) no 851/2004 of the European Parliament and of the 
Council of 21 April 2004 establishing a European centre for disease prevention and control. Article 5.3. Strasbourg: European 
Parliament and European Council; 2004. Available from: http://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX:32004R0851 

2.  European Parliament and Council of the European Union. Commission Implementing Decision (EU) 2018/945 of 22 June 2018 
on the communicable diseases and related special health issues to be covered by epidemiological surveillance as well as 
relevant case definitions. Available from: https://eur-lex.europa.eu/legal-
content/EN/TXT/PDF/?uri=CELEX:32018D0945&qid=1578988407694&from=EN 

3.  European Centre for Disease Prevention and Control. Campylobacteriosis. In: ECDC. Annual epidemiological report for 2017. 

Stockholm: ECDC; 2019. Available from: https://www.ecdc.europa.eu/sites/portal/files/documents/AER_for_2017-
campylobacteriosis.pdf 

4.  Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and 

zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC. Available from: 
https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32003L0099&qid=1578988446780&from=EN 

5.  2013/652/EU: Commission Implementing Decision of 12 November 2013 on the monitoring and reporting of antimicrobial 

resistance in zoonotic and commensal bacteria. Available from: https://eur-lex.europa.eu/legal-
content/EN/TXT/PDF/?uri=CELEX:32013D0652&qid=1578988480431&from=EN 

6.  European Centre for Disease Prevention and Control. EU protocol for harmonised monitoring of antimicrobial resistance in 

human Salmonella and Campylobacter isolates – June 2016. Stockholm: ECDC; 2016. Available from: 
https://ecdc.europa.eu/sites/portal/files/media/en/publications/Publications/antimicrobial-resistance-Salmonella-
Campylobacter-harmonised-monitoring.pdf 

7.  The European Committee on Antimicrobial Susceptibility Testing – EUCAST, Recommended AST methods. Available from: 

http://www.eucast.org/ast_of_bacteria/ 

8.  European Committee on Antimicrobial Susceptibility Testing - Routine and extended internal quality control for MIC 

determination and disk diffusion as recommended by EUCAST. Available from: 
http://www.eucast.org/ast_of_bacteria/qc_tables/ 

9.  Clinical and Laboratory Standards Institute (CLSI): M45: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of 

Infrequently Isolated or Fastidious Bacteria, 3rd Edition, 2015, CLSI, Wayne, PA, USA. 

10. Bolinger H, Kathariou S. 2017. The current state of macrolide resistance in Campylobacter spp.: trends and impacts of 

resistance mechanisms. Appl Environ Microbiol 83:e00416-17. Available from: https://aem.asm.org/content/83/12/e00416-17 

11. European Centre for Disease Prevention and Control. Third external quality assessment on species identification and 

antimicrobial susceptibility testing of Campylobacter – 2017. Stockholm: ECDC; 2019. Available from: 
https://www.ecdc.europa.eu/sites/default/files/documents/campylobacter-external-quality-assessment.pdf 

12. The 23rd EURL-AR Proficiency Test Salmonella, Campylobacter and genotypic characterisation 2017. Available from: 
https://www.eurl-ar.eu/CustomerData/Files/Folders/19-reports-eqas-reports/431_salm-camp-gen-report-2017-final-
isbnreg.pdf 

18 

TECHNICAL REPORT 

Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018 

Annex 1. List of participants 

Country 

EU status  Name of laboratory 

Name of institution 

Albania 

Austria 

Belgium 

Croatia 

Cyprus 

Enlargement  Laboratory of Enterobacteriology 

Institute of Public Health 

EU/EEA 

NRL Campylobacter 

Austrian Agency for Health and Food Safety 

EU/EEA 

LHUB-ULB site Porte de Hal - 
Microbiologie 

CHU Saint-Pierre 

EU/EEA 

Department for Clinical Microbiology 

University Hospital for Infectious Diseases 

EU/EEA 

Reference Laboratory for Salmonella and 
other Enteric Pathogens 

Nicosia General Hospital 

Czech Republic 

EU/EEA 

National reference laboratory for 
antibiotics 

National Institute of Public Health 

Denmark 

EU/EEA 

Diagnostic and Typing of Gastrointestinal 
Bacteria 

Statens Serum Institut 

England 

EU/EEA 

Gastrointestinal Bacteria Reference Unit 

National Infection Service 

Estonia 

France 

EU/EEA 

Laboratory of Communicable Diseases 

Health Board 

EU/EEA 

French National Reference Center for 
Campylobacter 

CHU Pellegrin 

Germany 

EU/EEA 

FG11 

Robert Koch Institute 

Iceland 

Ireland 

Italy 

Kosovo 

Latvia 

Lithuania 

EU/EEA 

Dept. of Clinical Microbiology 

Landspítali University Hospital 

EU/EEA 

EU/EEA 

National Salmonella, Shigella and Listeria 
Reference Lab (NSSLRL) 

NSSLRL 

Antimicrobial resistance and special 
pathogens 

Istituto Superiore di Sanità 

Enlargement  Microbiology 

National Institute of Public Health of Kosovo 

EU/EEA 

EU/EEA 

National Microbiology Reference 
Laboratory of Latvia 

Riga East University Hospital 

National Public Health Surveillance 
Laboratory 

National Public Health Surveillance 
Laboratory 

Luxembourg 

EU/EEA 

Bacteriologie-Mycologie-
Antibiorésistance-Hygiéne hospitalière 

Laboratoire National de Santé 

Macedonia 

Enlargement  Laboratory of bacteriology and AMR 

Institute of Public Health of Macedonia 

Malta 

Portugal 

EU/EEA 

Bacteriology Laboratory 

Mater Dei Hospital 

EU/EEA 

NRL for Gastrointestinal Infections 

Republic of Serbia 

Enlargement 

Referee laboratory for Campylobacter 
and Helicobacter 

Republic of Srpska, 
Bosnia and 
Herzegovina 

Enlargement  Department of Microbiology 

Romania 

EU/EEA 

Bacterial Enteric Infections Laboratory 

National Institute of Health  
Dr Ricardo Jorge 

Center for Microbiology 

Public Health Institute, Republic of Srpska, 
Bosnia and Herzegovina 

National Institute of Medico-Military 
Research and Development Cantacuzino 

Slovenija 

EU/EEA 

Oddelek za medicinsko mikrobiologijo 
Nova Gorica 

NLZOH 

Spain 

EU/EEA 

Unidad de Enterobacterias 

Centro Nacional de Microbiología 

The Netherlands 

EU/EEA 

NRL on AMR in animals 

Turkey 

Enlargement 

National Reference Laboratory for Enteric 
Pathogens 

Wageningen Bioveterinary Research 
(WBVR) 

General Directorate of Public Health 

19 

 
 
 
Fourth EQA on species identification and antimicrobial susceptibility testing of Campylobacter, 2018  

TECHNICAL REPORT 

Annex 2. EUCAST ECOFFs used for 
Campylobacter EQA4-AST 

MIC determination (µg/mL) 

Disk Diffusion (mm) 

Antimicrobial 
agent 
Mandatory 

Ciprofloxacin 

Erythromycin (C. 
jejuni) 
Erythromycin (C. 
coli) 
Tetracycline (C. 
jejuni) 
Tetracycline (C. 
coli) 
Optional 

Gentamicin (C. 
jejuni) 
Gentamicin (C. 
coli) 

EUCAST ECOFF 

WT ≤ 

 NWT > 

0.5 

0.5 

4 

8 

1 

2 

2 

2 

4 

8 

1 

2 

2 

2 

EUCAST Clinical 
breakpoint 

EUCAST ECOFF 

EUCAST Clinical 
breakpoint 

S ≤ 

0.5 

4 

8 

2 

2 

R > 

0.5 

4 

8 

2 

2 

WT ≥ 

NWT < 

S ≥ 

R < 

26 

20 

24 

30 

30 

26 

20 

24 

30 

30 

26 

22 

24 

30 

30 

20 

26 

22 

24 

30 

30 

20 

20 

 
  
  
  
  
  
  
  
  
 
 
 
 
 
 
 
 
 
 
 
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Prevention and Control (ECDC)

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