External quality assessment (EQA) schemes to support European surveillance of legionnaires’ disease 2019-2020 : EU/EEA countries.
Date: 2020-12-31
Institutions: European Centre for Disease Prevention and Control
Subjects: European Economic Area, European Union, disease prevention, health control, infectious disease, public health, scientific cooperation, traveller

TECHNICAL REPORT

External quality assessment (EQA) 
schemes to support European sur-
veillance of Legionnaires’ disease 
2019-2020 - EU/EEA countries

www.ecdc.europa.eu

ECDC TECHNICAL REPORT 

External quality assessment (EQA) 
schemes to support European surveillance 
of Legionnaires’ disease 20192020 

EU/EEA countries 

 
 
This report was commissioned by the European Centre for Disease Prevention and Control (ECDC Framework 
contract ECDC/2019/024 – Legionella EQA, specific contract CPN/2019/OCS/10918, coordinated by Lara Payne 
Hallstrom and produced by the Food and Environmental Proficiency Testing Unit and United Kingdom National 
External Quality Assessment Service on behalf of Public Health England. 

Authors 
Nita Patel – Head of Food and Environmental Proficiency Testing Unit  
Jennifer Henderson – UK NEQAS Scheme Organiser for Bacteriology and Mycology 
Shila Seaton – UK NEQAS Scheme Manager for Bacteriology and Mycology. 

This report was sent for consultation to:  
Dr Sandra Lai – Unit Head of the Food, Water and Environmental Microbiology Laboratory, Colindale 
Dr Vicki Chalker - Head of the Respiratory and Vaccine Preventable Bacteria Reference Unit, Colindale 
Dr Baharak Afshar - Head of Respiratory and Systemic Bacteria Section, Colindale.  

Acknowledgements 
We would like to thank all staff within the EQA services (FEPTU and UK NEQAS) that helped to support the delivery 
of this survey, staff in the reference laboratory that provided the characterised strains and did the quality control 
testing, all laboratories that participated in the survey and all national focal points for Legionnaires’ disease in the 
relevant countries. 

Suggested citation: European Centre for Disease Prevention and Control. External quality assessment (EQA) 
schemes to support European surveillance of Legionnaires’ disease 2019-2020  EU/EEA countries. Stockholm: 
ECDC; 2020. 

Stockholm, December 2020 

ISBN 978-92-9498-526-2 
doi: 10.2900/436611 
Catalogue number TQ-04-20-737-EN-N 

© European Centre for Disease Prevention and Control, 2020 

Reproduction is authorised, provided the source is acknowledged 

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TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease, 2019-2020  EU/EEA countries 

Contents 
Abbreviations ............................................................................................................................................... iv 
Executive summary ........................................................................................................................................ 1 
1. Introduction .............................................................................................................................................. 3 
Background .............................................................................................................................................. 3 
EQA exercise 2019/2020 ........................................................................................................................... 3 
2. Study design and methods .......................................................................................................................... 5 
Organisation of EQA .................................................................................................................................. 5 
EQA exercise scenario and sample design ................................................................................................... 6 
3. Results ...................................................................................................................................................... 9 
Intended results for 2019/2020 exercise ..................................................................................................... 9 
4. Discussion ............................................................................................................................................... 17 
General .................................................................................................................................................. 17 
Limitations of this EQA exercise ............................................................................................................... 20 
EQA benefits .......................................................................................................................................... 21 
Participant feedback on this EQA .............................................................................................................. 22 
5. Conclusions ............................................................................................................................................. 23 
6. Recommendations .................................................................................................................................... 24 
References .................................................................................................................................................. 25 
Annex 1 ...................................................................................................................................................... 26 

Figures 
Figure 1. Turnaround time for reporting results - clinical laboratories ................................................................ 16 
Figure 2. Turnaround times for reporting results - environmental laboratories ................................................... 16 

Tables 
Table 1. Countries within the EU/EEA that participated in the clinical and environmental EQA ............................... 6 
Table 2. Clinical specimens 5706–5715 provided in the distribution (4 November 2019) ....................................... 9 
Table 3. Environmental samples 5716–5725 provided in the distribution (4 November 2019) ................................ 9 
Table 4. Legionella pneumophila (5706) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 10 
Table 5. Legionella pneumophila (5707) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 10 
Table 6. Negative for Legionella (5708) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 10 
Table 7. Legionella pneumophila (5709) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 11 
Table 8. Legionella pneumophila (5710) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 11 
Table 9. Legionella pneumophila (5711) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 11 
Table 10. Legionella pneumophila (5712) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 11 
Table 11. L. pneumophila antigen not detected (5713) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories ................................................................................... 12 
Table 12. Legionella longbeachae. (5714) - intended results reported by the PHE reference laboratory and overall 
concordance with participating laboratories .................................................................................................... 12 
Table 13. L. pneumophila antigen not detected (5715) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories ................................................................................... 12 
Table 14. Serogroup and sequence type concordance for simulated sputum specimens containing Legionella spp. 12 
Table 15. Urinary antigen result concordance ................................................................................................. 12 
Table 16. Examinations done on cultured samples .......................................................................................... 13 
Table 17. Molecular methods ........................................................................................................................ 14 
Table 18. Data on enumeration results .......................................................................................................... 14 

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EQA schemes to support surveillance of Legionnaires’ disease in EU/EEA countries 20192020  

TECHNICAL REPORT 

Abbreviations 

BCYE 
BAL 
CFU 
COVID-19 
DFA 
ECDC 
EQA 
ELDSNet 
EU 
EEA 
ELISA 
FEPTU 
GVPC 
GU/L 
ID 
LUA 
MALDI-TOF  
PCR 
PFGE 
PHE 
MLST 
MVLA 
RVPBRU  
Sg 
SBT 
SNP 
ST 
TALD 
UK 
UK NEQAS 
WGS 

Buffered charcoal yeast extract  
Broncho-alveolar lavage 
Colony forming units 
Disease caused by coronavirus SARS-CoV-2 
Direct fluorescent antibody 
European Centre for Disease Prevention and Control 
External Quality Assessment 
European Legionnaires’ Disease Surveillance Network 
European Union 
European Economic Area 
Enzyme-Linked Immunosorbent Assay 
Food and Environmental Proficiency Testing Unit (Public Health England) 
Glycine vancomycin polymyxin B cycloheximide 
Genome units per litre 
Identification 
Legionella pneumophila urinary antigen 
Matrix Assisted Laser Desorption Ionisation Time-of-Flight 
Polymerase chain reaction 
Pulsed Field Gel Electrophoresis 
Public Health England 
Multilocus Sequence Typing 
Multiple-Locus Variable-Number Tandem-Repeat Analysis 
Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England  
Serogroup 
Sequence Base Typing 
Single Nucleotide Polymorphism 
Sequence Type 
Travel-associated Legionnaires’ disease 
United Kingdom 
United Kingdom National External Quality Assessment Service 
Whole genome sequencing 

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TECHNICAL REPORT 

EQA scheme to support European surveillance of Legionnaires’ disease - EU/EEA countries 20192020 

Executive summary 

In 2019, ECDC implemented the start of an EQA scheme for the European Legionnaires’ Disease Surveillance 
network, for the detection, isolation, identification and enumeration of Legionella spp. This was organised under a 
framework contract with the Food and Environmental Proficiency Testing Unit (FEPTU) of Public Health England 
(PHE) and the United Kingdom National External Quality Assessment Service (UK NEQAS).  

This is the first EQA exercise for laboratories participating in surveillance from the ELDSNet network that has been 
organised by ECDC since 2015 and the EQA format and arrangements have changed. The current EQA scheme 
uses an outbreak scenario with a package of clinical and environmental samples for the participating laboratories to 
process, depending on their technical capacity and protocols. 

The purpose of the 20192020 EQA exercise was to determine the accuracy of Legionella testing and results 
reported by individual laboratories in order to enable comparison of results between laboratories and within 
countries across Europe. This report presents an analysis of participants' results for the 2019 EQA exercise for the 
EU/EEA countries.  

For each round, up to two nominated laboratories per EU/EEA country were allowed to participate (to cover clinical 
and/or environmental testing). 

Only one round was completed during 20192020 due to the impact of COVID-19.  

In summary, there was one delivery of EQA samples which was sent on 4 November 2019. This EQA distribution 
was sent to a maximum of two laboratories per country for a total of 28 EU/EEA countries invited to take part via 
their national focal point for Legionnaires’ disease. Laboratories were selected based on their involvement in the 
management of public health incidents associated with Legionella in their country. 

The distribution comprised a total of 20 simulated samples; 10 representing clinical material and 10 representing 
environmental samples. Strains of Legionella was provided by the Respiratory and Vaccine Preventable Bacteria 
Reference Unit (RVPBRU) and these strains were fully characterised using conventional methods and an analytical 
profile index system. 

Laboratories only needed to report whether the sample/specimen contained a Legionella spp., and then provide 
identification, enumeration, serogroup and sequence type. 

Individualised reports were generated for each laboratory that included the results for their individual examinations 
and the overall results submitted by all laboratories for this exercise. 

This report provides an analysis of the EQA performance of laboratories in the 28 EU/EEA countries in relation to 
the detection/isolation, identification, enumeration and quantification of Legionella spp. and the further 
characterisation of L. pneumophila, by serogrouping and sequence-based typing, from both clinical and 
environmental samples, where applicable. The report is split into two parts - clinical and environmental analysis - 
as the aim of this first new EQA exercise was to assess the baseline testing. 

A separate survey was also organised on methods/kit information and frequency of testing performed for each 
method/kit by the laboratories. 

For this EQA the scenario was a simulation of an outbreak associated with a spa facility. The selected outbreak 
strain, Legionella pneumophila, was serogroup (Sg) 1, sequence type (ST) 47. 

Laboratories were given the opportunity to examine samples they would routinely test in their laboratory. For the 
clinical element, 27 laboratories examined the sputum samples and 24 examined the urine samples. For the 
environmental element, 26 of the laboratories examined the water samples and 24 examined the swab samples. 
Where the results reported were not in accordance with the intended exercise, laboratories were advised by 
contractors to investigate in order to determine the cause. 

A total of 27 clinical laboratories from countries within the EU/EEA were sent the clinical distribution (Austria, 
Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, 
Ireland, Italy, Latvia, Lithuania, Netherlands, Norway, Poland, Portugal, Romania, Slovak Republic, Slovenia, Spain, 
Sweden and the United Kingdom). All laboratories returned results for this EQA distribution.  

For the clinical laboratories, all 27 identified the sample, while 26 identified the serogroup and 14 identified the 
sequence type.  

For the identification of Legionella pneumophila in simulated sputum samples, there was excellent concordance 
with the intended results, with 100% of participants reporting the correct result (specimens 5706, 5710 and 5712). 
Overall determination of Sg and ST was excellent, with 100% of the participating laboratories achieving the 
intended result. One specimen (5714) contained Legionella longbeachae, for which only 17 out of 27 participants 
reported the correct identification. A further 6/27 reported either Legionella species, or that the specimen was not 
Legionella pneumophila, both of which were considered correct responses. Overall, 27 participants reported the 
identification, 26 for Sg and 14 for ST. One specimen (5708) contained commensals only, with no Legionella spp. 

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EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

present. In total, 92.6% of participants reported the correct negative result, however two reported the presence of 
Legionella. All laboratories reported isolation and identification using culture-based methods. A total of 22/27 then 
went on to use molecular methods to detect Legionella and 7/27 performed whole genome sequencing.  

The performance for urinary antigen testing was very good, with an overall mean concordance of 95.9% of 
participating laboratories returning a correct result. However, one country reported an incorrect positive LUA result 
for specimens 5709 and 5710.  

Overall, performance in the identification, serogroup, sequence type and urinary antigen test detection was very 
good.  

A total of 27 environmental laboratories were sent this EQA. The laboratories represented 27 EU/EEA countries 
(Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, 
Hungary, Ireland, Italy, Latvia, Lithuania, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovak Republic, 
Slovenia, Sweden and the United Kingdom). All 27 laboratories returned a result.  

For Legionella isolation, the overall performance was 96%, 27 of the laboratories examined the water samples, 
with 24 examining the swab samples. Six of the ten simulated samples contained a Legionella spp.. The overall 
performance for correctly identifying the Legionella spp. when a sample contained this organism was 99%. Seven 
of the ten samples were simulated water samples which allowed laboratories to report an enumeration result. The 
overall performance for the enumeration results correctly reported within the intended range was 86%. The overall 
performance for reporting a correct serogroup was very good with 98%. For sequence type the overall 
performance of reporting a correct sequence type was 93% for five of the samples that contained a L. 
pneumophila.  

For the environmental laboratories, 27 laboratories reported a result for isolation and identification, with 25 
reporting a serogroup, 24 providing an enumeration count and 14 offering a serotype. With regard to molecular 
methods, eight laboratories analysed the samples for L. pneumophila and five looked at those for Legionella spp.. 
The overall isolation performance for culture was 95%, with 27 laboratories reporting a result. The overall 
performance for molecular methods was very good, with eight laboratories reporting a result for kits detecting L. 
pneumophila. The routine application of molecular methods for water and environmental samples is still being 
developed in laboratories due to the fact there are currently no guidelines for interpretation of molecular results 
(GU/L). Therefore, culture remains the preferred method.  

The overall performance of the clinical laboratories was very good for the three sets of samples and clearly 
demonstrated that the fourth paired sample was not associated with the ‘outbreak’. 

Laboratories have demonstrated that they can undertake testing to an acceptable level of at least 80% 
concordance with intended results and this is relevant for both clinical and environmental laboratories. This data 
provides a limited assurance of EU/EEA laboratories’ ability to undertake effective public health investigations for 
Legionella pneumophila. Further EQA rounds will provide more data on performance and the robustness of testing. 

In conclusion, the performance of laboratories in the EU/EEA countries was very good for culture based/detection 
methods, which were used by both the clinical (96.2%) and environmental laboratories (95%). 

In replies to an ECDC questionnaire, laboratories indicated that this EQA exercise was very useful and overall there 
was very positive feedback on the new ECDC EQA Legionella scheme. 

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TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

1. Introduction 

Background 

Legionnaires’ disease is a severe and sometimes fatal form of infection caused by the Gram-negative bacteria, 
Legionella spp. These bacteria are found in freshwater and soil worldwide and can contaminate man-made water 
systems. There are at least 60 species of Legionella and over 20 have been associated with human disease. 
Legionella pneumophila is the most common species isolated both from the environment and from infections. 
Based on surface antigens, this species can be divided into at least 16 serogroups, of which L. pneumophila 
serogroup 1 is the most common cause of outbreaks. The strains of serogroup 1 most commonly associated with 
disease share a common epitope, as shown by monoclonal subtyping. It is important to routinely be able to 
differentiate between L. pneumophila and other Legionella spp. and to be able to distinguish serogroup 1 from the 
other serogroups of L. pneumophila. 

Humans are infected through the inhalation of contaminated aerosols containing Legionella bacteria. Legionnaires’ 
disease is classically described as a severe pneumonia that may be accompanied by systemic symptoms and may 
lead to a fatal outcome. Cases of Legionnaires’ disease are mainly reported in older people (>50 years), especially 
males. Other known risk factors for Legionnaires’ disease are smoking, chronic obstructive pulmonary disease, 
diabetes, immune system compromise and receipt of transplant or chemotherapy. In Europe, most cases 
(approximately 70%) are community-acquired and sporadic. About 20% of the cases are travel-related and 
identification of the source of infection often requires international collaboration. 

Legionnaires’ disease is a statutorily notifiable disease in all EU/EEA countries, but is thought to be under-reported 
for two reasons:  

 

 

it is underdiagnosed by clinicians who may not test patients for Legionnaires’ disease before empirically 
prescribing antibiotics likely to cover Legionella spp.; 
some health professionals may fail to notify cases to health authorities. Furthermore, under-ascertainment 
and differences in laboratory practice may also partly explain the variations in notification rates observed 
among EU/EEA countries. 

Legionnaires’ disease surveillance has been carried out at European level since 1987, firstly through a dedicated 
surveillance network funded by the European Commission and then, since April 2010, through ELDSNet (European 
Legionnaires’ Disease Surveillance Network) coordinated by ECDC. ECDC also coordinates the collation of annual 
surveillance data on Legionnaires’ disease in the EU/EEA with Member States. The resulting surveillance data is 
available through the European Surveillance Atlas on ECDC’s website. A second ELDSNet surveillance system 
focuses on Travel Associated Legionnaires’ Disease (TALD) cases.  

The aim of ELDSNet is to detect and communicate on clusters and outbreaks of TALD. The network supports the 
Member States and other countries involved in sharing information and collaborating on response actions to 
provide better protection from TALD, both domestically and abroad.  

TALD surveillance objectives [1] are: 

 

 
 
 

 

to rapidly detect cases and clusters of TALD reported in the EU/EEA and affecting European residents, both 
in their own countries or abroad; 
to disseminate information on TALD and respond in a coordinated fashion; 
to promote awareness of TALD to support primary preventive action and collaborative investigations; 
to assist in detecting and understanding the extent of common-source outbreaks of Legionnaires’ disease 
worldwide by promptly notifying reported travel-related cases and clusters; 
to reduce the incidence of TALD among EU residents by increasing awareness of active control and 
prevention measures at accommodation sites. 

A laboratory’s role during Legionnaires’ disease outbreaks includes identifying and characterising the pathogen via 
clinical and/or environmental samples to support epidemiological investigation, patient treatment/management and 
source control. Legionnaires’ disease cases and environmental findings are reported to the above European 
surveillance programmes, with cases reported according to agreed case definitions [2]. 

EQA exercise 20192020 

In 2019, ECDC organised an EQA exercise for the detection/isolation, identification, enumeration and quantification 
of Legionella spp and the further characterisation of L. pneumophila through serogrouping and sequence-based 
typing from both clinical and environmental samples, where applicable. The EQA was organised in collaboration 
with the Food and Environmental Proficiency Testing Unit (FEPTU) of Public Health England (PHE) and the United 
Kingdom National External Quality Assessment Service (UK NEQAS).  

This is the first EQA exercise organised by ECDC since 2015 and the EQA format and arrangements have changed. 
The purpose of this EQA exercise was to determine the accuracy of Legionella testing and results reported by 

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EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

individual laboratories, to allow comparison of results between laboratories and within countries across Europe. 
This report presents an analysis of participants' results for the 2019 EQA exercise in the EU/EEA countries. The 
results provided ECDC with information on the laboratories’ capabilities of accurately performing Legionella testing. 
This helped to provide confidence in data submitted for surveillance; identify where further support is needed for 
individual laboratories or countries and allow laboratories to understand their own capabilities, if testing demand 
were to increase due to an outbreak.  

The overall objectives of the 20192020 EQA were:  

 

 
 

to provide a baseline understanding of the level of testing undertaken in laboratories in response to routine 
outbreak scenarios, for both clinical and environmental samples; 
to determine where there were any general performance issues; 
to provide individual technical support to laboratories as a follow up to the exercise, if requested by the 
countries. 

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TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

2. Study design and methods 

Organisation of EQA 

This EQA was organised by FEPTU and UK NEQAS for Microbiology in collaboration RVPBRU, PHE and ECDC as part 
of an ECDC Framework contract (ECDC/2019/024). The EQA exercise was for laboratories nominated through 
ECDC National Focal Points for Legionnaires’ disease within ELDSNet and up to two nominated laboratories per 
EU/EEA country (to cover clinical and/or environmental samples) could participate per round. Two rounds were 
foreseen for 20192020, however due to the COVID-19 situation, only one could be implemented. 

The laboratories chosen were those involved in the management of public health incidents in their country and/or 
undertaking expert reference testing for specialised examinations. A unique laboratory identification was created 
and user name and passwords generated for each one. This allowed the laboratory to return results and view 
individualised reports through a secure web portal and it meant that the results were anonymised results for ECDC. 

Both FEPTU and UK NEQAS are UKAS accredited EQA providers under ISO/IEC 17043:2010 (Conformity 
assessment- General requirements for proficiency testing) and all these principles and practices was applied to the 
ECDC EQA scheme.  

 

 

 

 

 

 

 

The EQA distribution was sent on 4 November 2019 to a maximum of two laboratories per country in a total 
of 28 EU/EEA countries. 
ECDC invited ECDC National Focal Points for Legionnaires’ disease to propose up to two laboratories per 
country to take part in the EQA exercise, one that undertakes clinical examination of specimens and one 
that examines environmental samples. One laboratory could also be nominated to participate in both clinical 
and environmental, if they usually processed both types of sample. Participating laboratories needed to be 
contributing to national surveillance data or environmental findings that are shared through ELDSNet 
surveillance activities. 
The EQA organiser sent a letter of invitation to the nominated laboratories informing them of the EQA 
arrangements and the objectives of the exercise. The letter also provided an opportunity for the 
laboratories to confirm their interest in participating and that their details in the system were correct. 
The distribution comprised a total of 20 simulated samples; 10 representing clinical material and 10 
representing environmental samples. Sample/specimen design and format was agreed in advance with 
ECDC and PHE Legionella experts.  
PHE undertook testing of the samples in accordance with published methods, to replicate where possible 
testing methods that would be used by the participants. Detection, identification, enumeration, confirmation 
and further characterisation tests (serogrouping (Sg) and sequence-based typing (SBT)) were also 
undertaken. 
PHE also ran a separate survey on methods/kit information and frequency of testing performed for each 
method/kit by the laboratories. 
The distribution exercise simulated an outbreak associated with a spa facility. The outbreak Legionella 
pneumophila strain chosen was serogroup (Sg) 1, sequence type (ST) 47. 

A total of 27 environmental and 27 clinical laboratories from 28 EU/EEA countries took part in the EQA, table 1. 
Each laboratory was provided with a unique laboratory identification. Of those taking part, 20/34 participating 
countries tested both the clinical specimens and environmental samples. 

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Table 1. Countries within the EU/EEA that participated in the clinical and environmental EQA  

Country 

Clinical EQA samples 

Austria 
Belgium 
Bulgaria 
Croatia 
Cyprus 
Czech Republic 
Denmark 
Estonia 
Finland 
France 
Germany 
Greece 
Hungary 
Ireland 
Italy 
Latvia 
Lithuania 
Malta 
Netherlands 
Norway 
Poland 
Portugal 
Romania 
Slovak Republic 
Slovenia 
Spain 
Sweden 
United Kingdom 

Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
No 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 

Environmental EQA 
samples 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
No 
Yes 
Yes 

Number of participating 
laboratories per country 
1 
1 
1 
1 
2 
1 
1 
1 
2 
2 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
2 
1 
2 
1 
1 
1 
2 

An EQA protocol was drawn up and sent with the samples which were dispatched in approved United Nations 
containers. This protocol included information on the sample/specimen details, instructions on how to process 
samples/specimens, information on the results to be provided, a copy of a method questionnaire (information to be 
returned electronically) and safety information. All information was also provided electronically to all participants 
and NFPs for Legionnaires’ disease in ELDSNet and was available on the UK NEQAS web portal.  

A dedicated web page was available on the UK NEQAS website for participants to enter and submit their results. 
Participants could access instructions for using the secure web portal and download the protocol describing the 
process for specimen examination via the web page. Detailed instructions were included on how to access the 
secure website via a unique user ID and password provided for each participant. The deadline for final submission 
of results was stated on the paperwork detailing the sample/specimen information. For convenience, there was 
also a copy of the web reply form available for participants to download so that they could manually record test 
results before submitting them online. For this first exercise participants were allowed six weeks (42 days) from the 
date of dispatch of both clinical and environmental samples to examine the EQA specimens/samples and return all 
their results. The length of time allowed for this exercise was due to the length of time required to isolate the 
Legionella spp. on culture media (minimum 10 days) and undertake the relevant confirmatory testing, which 
includes the time a reference laboratory may take to provide a result for specialist tests, such as ST. 

Six weeks after the date of dispatch (4 November 2019), the web platform was closed for results submission and 
the intended results were published on the secure website on 20 December 2019 where they could be accessed by 
participating laboratories. Participants were notified by email that the intended results were available for viewing. 
Individualised reports were made available ten weeks after the closing date on 6 March 2020.  

From 223 April 2020, ECDC conducted a short online survey to obtain feedback on the EQA exercise and enable 
the laboratories to suggest improvement for the next distribution. A summary of this feedback can be found in the 
discussion section 4. 

Certificates of participation were sent electronically to the laboratories on 21 April 2020. A hard copy of the 
certificate was available on request.  

EQA exercise scenario and sample design 

The strains selected for the November 2019 exercise were chosen in consultation with PHE Legionella experts in 
clinical and environmental microbiology. Sample/specimen design was also developed in collaboration with the PHE 
UK NEQAS and ECDC experts and approved by ECDC.  

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TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Five outbreak environmental samples were supplied to represent spa pool water, a swab of the biofilm from the spa 
pool pipeline and water from three cooling towers in the same vicinity as the spa. In addition, five routine 
monitoring samples were supplied: water and swabs from hot and cold water systems and water from a spa pool.  

Clinical samples were taken from six patients with suspected Legionella symptoms (sputum and or urine samples.) 

The strain of Legionella pneumophila serogroup 1, ST47 used for this EQA exercise is considered a leading cause of 
Legionnaires’ disease in north-western Europe, however it is rarely isolated from the environment. The Legionella 
strains were provided, tested and fully characterised (before and after sample/specimen preparation) by the 
Respiratory and Vaccine Preventable Bacteria Reference Unit (RVPBRU).  

Legionella pneumophila serogroup 3, ST2630 was also included, as this is a unique ST with a single documented 
isolation from a community-acquired clinical case in the UK. Legionnaires’ disease attributed to L. pneumophila 
serogroup 3 is less common than detections of serogroup 1 infection. Many commercially available urinary antigen 
kits for Legionella pneumophila are not designed and validated for the detection of non-serogroup 1 type L. 
pneumophila antigens. 

Strains of Legionella were provided by RVPBRU as fully characterised isolates; commensal/background flora was 
taken from a bank of organisms held by the EQA organisers and these strains were fully characterised using 
conventional methods and an analytical profile index system. All isolates were clinical isolates from patients with 
pneumonia. In 2018, 80% of respiratory samples from patients with Legionella were positive for Legionella 
pneumophila serogroup 1, of which ST47 is one of the more commonly isolated strains from patients in the UK. 
Non-serogroup 1 infections are detected at a much lower frequency. L bozemanii is rarely isolated from patients in 
the UK. 

Samples/specimens were prepared and quality-controlled by the EQA organisers and the panels was dispatched as 
distribution 4680 (clinical) and 4681 (environmental).  

All packages with samples were dispatched at ambient temperature, in accordance with the latest International Air 
Transport Association (IATA) regulations, using an approved airfreight company. 

Additional data was collected and analysed through a questionnaire on methodology and the annual number of 
tests done using this method, materials and EQA participation. This information was captured electronically and 
findings are shown in the annex of this report. 

Provision of individual feedback to the participating laboratories on their results was provided in an environmental 
EQA report. This included recommendations (where necessary), actions to take and method performance (if 
applicable.) The PHE Legionella expert for clinical and water microbiology provided comments on these reports 
based on performance and strains used.  

The individualised laboratory reports detailed a laboratory’s reported results for each examination requested and 
the intended results for each sample (including the simulated microbiological contents). This included the 
identification of the Legionella species, serogroup, serotype, enumeration results, where applicable. The report also 
provided an overall performance for each examination based on all the laboratories reported results.  

Clinical 

A total of 27 sets of specimens were distributed to 27 participating countries. Ten clinical samples were prepared in 
each set (five simulated sputum samples and five liquid urine samples.) An overview of samples is provided in 
Table 2. 

Participants were asked to provide an organism identification, serogroup and sequence type (simulated sputum 
samples) and LUA result (urine specimens). Simulated sputum samples were prepared in a lyophilised format. The 
freeze-dried sample matrix was composed of inositol serum broth with variable concentrations of the pathogen 
Legionella pneumophila or other species. To simulate the specimen close to an authentic clinical material, the freeze-
dried vials contained a strain of the pathogen and included commensal flora commonly isolated from lower respiratory 
tract infections. The serogroups and species of Legionella to be used were approved by the commissioned experts at 
PHE. Participants’ results were analysed and considered ‘concordant’ if the reported categorisation agreed with the 
PHE reference laboratory (RVPBRU) interpretation. In addition, participants were asked to complete a questionnaire 
survey to provide further information on methods used, both in general and for this EQA exercise. Additional data was 
collected and analysed via a questionnaire on methodology and the annual number of tests done for this method, 
materials and EQA participation. 

The yield of the pathogen after reconstitution of the lyophilised vials, ranged between 102104 colony-forming units per 
mL. The yield of the commensal flora following lyophilisation, ranged between 102103 colony-forming units per mL. 

Two simulated specimens with no pathogens were also included in the set of 10 specimens. 

7 

 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Instructions provided to participants included: 

 

 

 

how to reconstitute lyophilised specimens with 1mL of nutrient broth (the pellet had to be fully dissolved in 
the liquid media to attain a homogenous suspension); 
how to inoculate the appropriate media with the appropriate incubation conditions to isolate any potential 
pathogens; 
information on reporting results (absence or presence of Legionella pneumophila or other species.) 

The simulated sputum samples were examined using the national documents SMI ID18 Identification of Legionella 
species and SMI B 57 Investigation of bronchoalveolar lavage, sputum and associated specimens. This is in 
accordance with the requirements for clinical laboratories accredited to ISO 15189:2012 (Medical laboratories - 
Requirements for quality and competence). 

Environmental 

Ten environmental samples were prepared as LENTICULE discs. This method of preparing samples has been 
extensively validated and proven to preserve organisms over long periods of time. Samples were tested in the 
FEPTU laboratory according to the international method ISO 11731:2017 (Water quality - Enumeration of 
Legionella) for water, sludge and swab samples. This is in accordance with water laboratories being accredited to 
ISO/IEC 17025:2010 (General requirements for the competence of testing and calibration laboratories).  

The simulated sample designs included a selection of the following to make the 10 samples required: water taken 
from various sites such as cooling towers, hot and cold water systems, spa pools and swab samples.  

The samples positive for Legionella spp. contained bacteria at varying levels from <102 – 105 colony-forming 
units/L.  

Background organisms were included that were relevant to the sample type in order to simulate a real sample, but 
also to challenge the laboratories’ processing techniques, such as acid/heat treatment, and to confirm the 
performance of the selective agar used. Participants were not asked to report on the background flora included.  

Samples were authorised for inclusion in a distribution if: 

 
 
 

they were homogeneous; 
they passed quality control testing prior to the distribution date; 
the sample contents matched those obtained from RVPBRU for identification, serogroup and sequence type. 

Samples were quality-controlled as they would have been by the participant. This step involved rehydration and 
culturing onto Glycine Vancomycin Polymyxin Cycloheximide (GVPC) as neat, following heat and acid treatment. 
Agar plates were incubated for up to 10 days aerobically at 37oC and read on Day 3, 6 and 10. Any suspected 
Legionella spp. was ascertained by means of confirmatory testing.  

Background flora selected for inclusion in the samples were those that would compete with the Legionella spp. in 
the sample. During processing for the isolation of Legionella, heat and acid treatment is employed to kill competing 
organisms - if this is done correctly.  

Homogeneity and stability results were analysed using local robust statistics to ensure suitability for use and that 
defined criteria were met. 

Results for environmental samples were analysed according to ISO 13528:2015 (Statistical methods for use in 
proficiency testing by inter-laboratory comparison). For enumeration values the participants’ median was used as 
the assigned value and the intended range calculated using robust statistics (5 and 95% percentiles).  

Data was displayed graphically. Detected/not detected serogroup and sequence type results were analysed against 
the intended results which were based on RVPBRU confirmation. For molecular examination, the samples were 
examined according to the procedures in ISO/TS 12869:2019 - Water quality - Detection and quantification of 
Legionella spp. and/or Legionella pneumophila by concentration and genic amplification using a quantitative 
polymerase chain reaction (qPCR).  

8 

 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

3. Results 

The methods questionnaire sent to participants to gather details on processes and methods was analysed as part of 
this EQA exercise and findings can be found in Annex 1. Key results are integrated into the separate sections below. 

Intended results for the 20192020 exercise 
Sample contents for the specimens included in the clinical and environmental distributions are described in Tables 2 
and 3, including the serogroup and sequence base type when Legionella pneumophila was present.  

The intended results to be returned by participating laboratories for each specimen are listed in Tables 415 for 
clinical samples and Tables 1618 for environmental samples.  

Table 2. Clinical specimens 5706–5715 provided in the distribution (4 November 2019) 

Sample contents 

Sg 

SBT 

Details 

Specimen 
number 

Patient 

Sample 

5706 

5707 

5708 

5709 

5710 

5711 

5712 

5713 

5714 

5715 

1 

2 

3 

4 

5 

6 

1 

2 

1 

2 

1 

2 

1 

2 

5 

6 

Sample 
type 

Sputum 

Legionella pneumophila 
Streptococcus oralis 
Streptococcus salivarius 

Urine 

Legionella pneumophila 

Sputum 

Neisseria sicca 
Streptococcus mitis 

1 

1 

47 

47 

Patient lives in location of 
cooling tower, has visited spa 

Patient lives in location of 
cooling tower, has visited spa 

Urine 

Legionella pneumophila 

1 

47 

Sputum 

Legionella pneumophila 
Streptococcus oralis 
Streptococcus salivarius 
Moraxella catarrhalis 

Urine 

Legionella pneumophila 

Sputum 

Legionella pneumophila 
Streptococcus mitis 
Streptococcus salivarius 

Urine 

Legionella pneumophila 

Sputum 

Legionella longbeachae 
Streptococcus oralis 
Streptococcus salivarius 

Urine 

No organisms  

1 

1 

3 

3 

Patient lives in location of 
cooling tower, has visited spa 

47 

47 

2630  Asthmatic elderly male, lives 

at home local area, rarely 
goes out, except to the shops 
and local public inn 

2630 

Community acquired 
pneumonia, keen gardener, 
elderly 
Patient on ECMO, severe 
pneumonia 

Table 3. Environmental samples 5716–5725 provided in the distribution (4 November 2019) 

Sample type 

Sample contents 

Sg 

SBT 

Comments 

Specimen 
number 

5716 

5717 

Spa water from site 1 

Biofilm swab from spa water 
pipeline (site 1) 

5718 

Cooling tower water (site 2) 

5719 

Cooling tower water (site 3) 

5720 

Cooling tower water (site 4) 

5721 

Hot and cold water system 

5722 

Biofilm swab from hot and cold 
water system 

5723 

Hot and cold water system 

5724 

5725 

Spa water 

Biofilm swab from hot and cold 
water system 

Legionella pneumophila 
Microbacterium luteolum  
Legionella pneumophila 
Staphylococcus saprophyticus  
Klebsiella pneumoniae 
Staphylococcus haemolyticus 
Enterococcus faecium  
Legionella pneumophila 
Enterococcus faecalis  
Klebsiella pneumoniae 
Staphylococcus haemolyticus  
Enterococcus faecium  

Legionella pneumophila 
Brevundimonas vesicularis 
Aerococcus viridans  
Pseudomonas putida  
Staphylococcus epidermidis  
Legionella bozemanii 
Acinetobacter junii 
Pseudomonas stutzeri  
Legionella pneumophila 
Citrobacter braakii  
Roseomonas aestuarii 
Pseudomonas aeruginosa  

1 

1 

47 

47 

1 

47 

1 

48 

6 

2923 

Samples taken as 
part of one outbreak 
investigation 

Samples taken as 
part of routine 
quality monitoring of 
water 

9 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Clinical 

A total of 27 of participating laboratories from 27 countries reported results for the simulated sputum samples, 
compared to 24 for urine samples. 

From the methods questionnaire, it was determined that a total of 22/27 participants had reported they were 
clinical diagnostics laboratories, as well as reference laboratories. One laboratory noted that it was only a clinical 
diagnostics laboratory. The remaining 4/27 were reference laboratories.  

A total of 17/27 laboratories participated in a national EQA scheme. However, this was only a mandatory exercise 
for seven of them. The most commonly tested sample types reported by the 27 laboratories for routine testing 
were sputum, broncho-alveolar lavage and urine. BCYE (7) and BCYE/BMPA (7) were the most frequently used 
media for the isolation of Legionella spp., together with MALDI-TOF (16) and serology (19) as the confirmatory 
tests. (See results in Annex 1). 

Participants were only requested to report information on Legionella spp. and not on the background flora included 
to simulate a specimen. 

Specimen 5706: This specimen contained Legionella pneumophila serogroup 1: ST47. An excellent concordance 
with intended results was achieved for all participating laboratories. 

Table 4. Legionella pneumophila (5706) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 

Serogroup 1 

Sequence type 47 

27/27 

25/25 

14/14 

100 

100 

100 

Specimen 5707: This specimen was positive for a Legionella pneumophila urinary antigen. An excellent 
concordance with intended results was achieved for all 24 participating laboratories returning a result.  

Table 5. Legionella pneumophila (5707) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 
antigen detected 

24/24 

100 

Specimen 5708: This specimen contained Neisseria sicca and Streptococcus mitis only, no Legionella spp. was 
present. A good concordance with intended results was achieved. Incorrect results reported included the presence 
of Legionella spp. (n=1) and Legionella pneumophila (n=1).  

Table 6. Negative for Legionella (5708) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Negative 

25/27 

92.6 

10 

 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Specimen 5709: The specimen was positive for L. pneumophila urinary antigen. A good concordance with 
intended results was achieved. Two laboratories incorrectly reported results as ‘antigen not detected’ and a further 
three stated that this specimen was ‘not examined’. 

Table 7. Legionella pneumophila (5709) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Legionella pneumophila 
serogroup 1 antigen 
detected 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

22/24 

91.7 

Specimen 5710: The specimen contained Legionella pneumophila serogroup 1: ST47. An excellent concordance 
with intended results was achieved for all participating laboratories. 

Table 8. Legionella pneumophila (5710) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 

Serogroup 1 

Sequence Type 47 

27/27 

26/26 

14/14 

100 

100 

100 

Specimen 5711: The specimen was positive for L. pneumophila urinary antigen. Two laboratories incorrectly 
reported the results as ‘antigen not detected’. A good concordance with intended results was achieved for the 
majority of participating laboratories.  

Table 9. Legionella pneumophila (5711) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories  

Intended interpretation 

Legionella pneumophila 
antigen detected 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

22/24 

91.7 

Specimen 5712: The specimen contained Legionella pneumophila serogroup 3: ST2630. An excellent 
concordance with intended results was achieved for all participating laboratories.  

Table 10. Legionella pneumophila (5712) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation 

Number of laboratories 
returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 

Serogroup 3 

Sequence Type 2630 

26/26 

26/26 

14/14 

100 

100 

100 

11 

 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Specimen 5713: The specimen was negative for L. pneumophila urinary antigen. An excellent concordance with 
intended results was achieved for all participating laboratories.  

Table 11. L. pneumophila antigen not detected (5713) - intended results reported by the PHE 
reference laboratory and overall concordance with participating laboratories 

Intended interpretation  Number of laboratories 

returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 
antigen not detected 

24/24 

100 

Specimen 5714: The specimen contained Legionella longbeachae. Three laboratories reported the result 
incorrectly as Legionella pneumophila and one laboratory did not test the sample. A good concordance with 
intended results was achieved for the majority of participating laboratories within the EU/EEA.  

Table 12. Legionella longbeachae. (5714) - intended results reported by the PHE reference laboratory 
and overall concordance with participating laboratories 

Intended interpretation  Number of laboratories 

returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella longbeachae 
Legionella species, not L. 
pneumophila 

17/27 

6/27 

88.5 

Specimen 5715: The specimen was negative for L. pneumophila urinary antigen. A very good concordance with 
intended results was achieved for the majority of participating laboratories.  

Table 13. L. pneumophila antigen not detected (5715) - intended results reported by the PHE 
reference laboratory and overall concordance with participating laboratories 

Intended interpretation  Number of laboratories 

returning correct 
results/total number 
reporting a result 

Overall concordance 
(%) 

Legionella pneumophila 
antigen not detected 

23/24 

95.8 

Summary of results by specimen type 

Overall determination of Sg and ST was excellent, with 100% of participating laboratories achieving the expected result.  

Table 14. Serogroup and sequence type concordance for simulated sputum specimens containing 
Legionella spp. 

Specimen number 

Serogroup (Sg) 

Sequence type (ST) 

5706 

5710 

5712 

100 (n=25) 

100 (n=26) 

100 (n=26) 

100 (n=14) 

100 (n=14) 

100 (n=14) 

The performance for urinary antigen testing was very good, with an overall mean concordance of 95.9% of 
participating laboratories returning a correct result.  

Table 15. Urinary antigen result concordance  

Specimen number  LUA result L. pneumophila antigen 

Overall concordance(%) 

5707 
5709 
5711 
5713 
5715 

12 

Detected 
Detected 
Detected 
Not detected 
Not detected 

100 
91.7 
91.7 
100 
95.8 

TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Environmental samples 

Ten simulated environmental samples were sent to 27 laboratories in 27 EU/EEA countries. All 27 laboratories 
returned a result within the six-week timeframe for this EQA exercise. 

Sample numbers: 5716–5720 were samples taken as part of one outbreak investigation. 

Sample numbers: 5721–5725 were samples were taken as part of routine monitoring. 

Sample numbers: 5717, 5722 and 5725 were swab samples. 

Sample numbers: 5716, 5718, 5719, 5720, 5721, 5723 and 5724 were water samples. 

Performance of the laboratories on these samples were split into culture-based methods (Table 16) and molecular 
methods (Table 17). Culture-based method analysis included results reported for isolation, identification, 
enumeration, serogroup and sequence type results. An overall performance assessment column as a percentage 
has been captured for culture-based method results by sample number and by each examination (including 
isolation, identification, enumeration, serogroup and serotype). Overall performance by sample was calculated 
using the mean value across each of the five examinations. For molecular methods and culture results the overall 
performance is shown by examination only.  

Table 18 shows in more detail the enumeration results reported by the laboratories.  

Only eight datasets were returned (25 laboratories examined the sample for isolation) with culture-based methods 
and molecular methods providing molecular results and quantification results. Therefore these were not statistically 
analysed as the data generated would not be robust. 

Table 16. Examinations done on cultured samples 

Contents 

Isolation 

Identification 

Enumeration 

Serogroup 

Sequence 
type 

Overall % 
performance by 
sample 

Sample 
number 

5716 

5717 

5718 

5719 

5720 

5721 

5722 

5723 

5724 

5725 

N 

% 

N 

% 

N 

% 

N 

% 

N 

% 

26/27 

96 

26/26 

100 

23/24 

96 

25/25 

100 

12/13 

92 

L. pneumophila sg 1, 
ST47 

L. pneumophila sg 1, 
ST47 

24/24 

100 

24/24 

100 

No Legionella 

25/25 

100 

- 

- 

- 

- 

- 

- 

23/23 

100 

13/14 

92 

- 

- 

- 

- 

L. pneumophila sg 1, 
ST47 

24/25 

96 

24/24 

100 

20/22 

91 

23/23 

100 

10/12 

83 

No Legionella 

25/25 

100 

- 

- 

- 

L. pneumophila sg 1, 
ST48 

23/27 

85 

23/23 

100 

19/24 

No Legionella 

24/24 

100 

- 

Legionella bozemanii 

24/27 

88 

23/24 

- 

96 

- 

19/22 

L. pneumophila sg 6, 
ST2923 

24/27 

88 

24/24 

100 

18/23 

No Legionella 

24/24 

100 

- 

Overall performance by 
examination 

95 

- 

- 

99 

- 

79 

- 

86 

78 

- 

86 

- 

- 

- 

- 

22/22 

100 

11/11 

100 

- 

- 

- 

- 

- 

- 

- 

- 

9/10 

90 

8/8 

100 

- 

- 

98 

- 

- 

93 

97 

98 

100 

94 

100 

93 

100 

90 

91 

100 

13 

 
 
 
 
 
 
 
 
 
 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Table 17. Molecular methods  

Sample 
number 

Identification 

Intended results 
for Legionella 
pneumophila 

Molecular 
results 
Legionella 
pneumophila 
(%) 

5716 

5717 

Legionella 
pneumophila 

Legionella 
pneumophila 

Detected 

8/8 (100) 

Detected 

7/7 (100) 

5718 

No Legionella 

Not detected 

7/7 (100) 

Quantification 
Legionella 
pneumophila 

2 (154000, 
2600000) 

Intended 
results for 
Legionella 
spp. 

Molecular 
results 
Legionella 
spp. (%) 

Quantification 
Legionella 
spp. 

Detected 

3/4 (75) 

2 (139000, 
2600000) 

- 

- 

Detected 

2/3 (67) 

Not detected 

3/4 (75) 

- 

- 

5719 

Legionella 
pneumophila 

Detected 

8/8 (100) 

1 (177000) 

Detected 

3/5 (60) 

1 (172000) 

5720 

No Legionella 

Not detected 

6/6 (100) 

- 

Not detected 

3/5 (60) 

5721 

Legionella 
pneumophila 

Detected 

8/8 (100) 

2 (9849, 310000) 

Detected 

4/5 (80) 

1 (<608) 

2 (7852, 
480000) 

5722 

No Legionella 

Not detected 

5/6 (83) 

- 

Not detected 

2/3 (67) 

- 

5723 

5724 

Legionella 
bozemanii 

Legionella 
pneumophila 

Not detected 

7/7 (100) 

1 (<840) 

Detected 

5/5 (100) 

Detected 

6/7 (86) 

2 (6728, 330000) 

Detected 

4/4 (100) 

5725 

No Legionella 

Not detected 

5/6 (83) 

- 

Not detected 

2/3 (67) 

Overall performance 

95 

75 

2 (16300, 
850000) 

2 (6986, 
260000) 

1 (<608) 

Table 18. Data on enumeration results 

Identification 

Participants 
median (cfu/L) 

Intended range (cfu/L) 

Number of 
results 

Number of outlying 
counts 

Sample 
number 

5716 

5717 

5718 

5719 

5720 

Legionella pneumophila 

3.1x104 

3.3x103 – 6.9x104 

Legionella pneumophila 

No Legionella 

- 

- 

- 

- 

Legionella pneumophila 

2.2x104 

1.1x103 – 8.7x104 

No Legionella 

- 

- 

5721 

Legionella pneumophila 

1.1x103 

98 – 3.9x103 

5722 

5723 

No Legionella 

- 

- 

Legionella bozemanii 

5.5x103 

87 – 2.8x104 

5724 

Legionella pneumophila 

1.3x103 

1.1x102 – 9.8x103 

5725 

No Legionella 

- 

- 

24 

- 

- 

22 

7 

24 

- 

22 

23 

- 

1 high 

- 

- 

2 high 

- 

3 (1 low, 2 high) and 2 < 
values 

- 

3 (1 low, 2 high) 

3 (1 low, 2 high) and 2 < 
values 

2 < values 

Sample 5716: This sample was water from spa (site 1) which contained a Legionella pneumophila serogroup 1 
sequence type ST47 at levels of approximately 105 colony-forming units per litre. The background flora was 
Microbacterium luteolum.  

Performance was very good with 26/27 (96%) of participants reporting the correct isolation result, 26/26 (100%) 
for identification, 23/24 (96%) of the laboratories reporting a count within the intended range, 25/25 (100%) 
reporting the correct serogroup and 12/13 (92%) of the laboratories reporting the correct sequence type. The 
overall performance for examinations by culture was 97%. 

Eight laboratories examined the sample using a molecular kit which only detects L. pneumophila and all eight 
reported the correct result. In addition, four of these laboratories also examined the sample using a molecular kit 
that detects Legionella spp., with 3/4 reporting a correct result. 

Sample 5717: This sample was a swab from the biofilm of a spa water pipeline (site 1) which contained a 
Legionella pneumophila serogroup 1 sequence type ST47 at levels of approximately 105 colony-forming units per 
litre. The background flora was Staphylococcus saprophyticus.  

14 

 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Performance was very good, with 24/24 (100%) of participants reporting the correct isolation result, 24/24 (100%) 
for identification, 23/23 (100%) reporting the correct serogroup and 13/14 (92%) of the laboratories reporting the 
correct sequence type. The overall performance for examinations by culture was 98%. 

Seven laboratories examined the sample using a molecular kit which only detects L. pneumophila and all seven 
reported the correct result. In addition, three of these laboratories also examined the sample using a molecular kit 
that detects Legionella spp., with 2/3 reporting a correct result. 

Sample 5718: This sample was water taken from a cooling tower (site 2) which contained no Legionella spp.. 
Background flora were Klebsiella pneumoniae, Staphylococcus haemolyticus and Enterococcus faecium. 

Performance was excellent with 25/25 (100%) of the laboratories reporting the correct isolation result. In addition, 
8/8 (100%) laboratories reported a low censored value which is considered to be a correct result, although this 
data is not shown in Table 16. The overall performance for examinations by culture was 100%. 

Seven laboratories examined the sample using a molecular kit which only detects L. pneumophila and all seven 
reported the correct result. In addition, four of these laboratories also examined the sample using a molecular kit 
that detects Legionella spp., with 3/4 reporting a correct result. 

Sample 5719: This sample was water taken from a cooling tower (site 3) which contained a Legionella 
pneumophila serogroup 1 sequence type ST47 at levels of approximately 104 colony-forming units per litre. 
Background flora was Enterococcus faecium.  

Performance was very good, with 24/25 (96%) of participants reporting the correct isolation result, 24/24 (100%) 
for identification, 20/22 (91%) of the laboratories reporting a count within the intended range, 23/23 (100%) 
reporting the correct serogroup and 10/12 (83%) of the laboratories reporting the correct sequence type. The 
overall performance for examinations by culture was 94%. 

Eight laboratories examined the sample using a molecular kit which only detects L. pneumophila and all eight 
reported the correct result. In addition, five of these laboratories examined the sample using a molecular kit that 
detects Legionella spp., with 3/5 reporting a correct result. 

Sample 5720: This sample was water taken from a cooling tower (site 4) which contained no Legionella spp.. 
Background flora were Klebsiella pneumoniae, Staphylococcus haemolyticus and Enterococcus faecium. 

Performance was excellent, with 25/25 (100%) of the laboratories reporting the correct isolation result. In addition, 
7/7 (100%) laboratories reported a low censored value which is considered to be a correct result, although this 
data is not shown in Table 16. The overall performance for examinations by culture was 100%. 

Six laboratories examined the sample using a molecular kit which only detects L. pneumophila and all six reported 
the correct result. In addition, five of these laboratories examined the sample using a molecular kit that detects 
Legionella spp., with 3/5 reporting a correct result. 

Sample 5721: This sample was a water taken from a hot and cold water system which contained a Legionella 
pneumophila serogroup 1 sequence type ST48 at levels of approximately 102 colony-forming units per litre. 
Background flora were Brevundimonas vesicularis and Aerococcus viridans.  

Performance was good, with 23/27 (85%) of participants reporting the correct isolation result, 23/23 (100%) for 
identification, 19/24 (79%) of the laboratories reporting a count within the intended range, 22/22 (100%) 
reporting the correct serogroup and 11/11 (100%) of the laboratories reporting the correct sequence type. The 
overall performance for examinations by culture was 93%. 

Eight laboratories examined the sample using a molecular kit which only detects L. pneumophila and all eight 
reported the correct result. In addition, five of these laboratories examined the sample using a molecular kit that 
detects Legionella spp., with 4/5 reporting a correct result. 

Sample 5722: This sample was a swab of a biofilm from a hot and cold water system which contained no 
Legionella spp.. Background flora were Pseudomonas putida and Staphylococcus epidermidis.  

Performance was excellent, with 24/24 (100%) of the laboratories reporting the correct result.  

Six laboratories examined the sample using a molecular kit which only detects L. pneumophila and 5/6 reported 
the correct negative result for Legionella. In addition, three of these laboratories also examined the sample using a 
molecular kit that only detects Legionella spp., with 2/3 reporting the correct negative result for Legionella. 

Sample 5723: This sample was water taken from a hot and cold water system which contained Legionella 
bozemanii at levels of approximately 103 colony-forming units per litre. Background flora were Acinetobacter junii 
and Pseudomonas stutzeri.  

Performance was good with 24/27 (88%) of participants reporting the correct isolation result, 23/24 (96%) for 
identification and 19/22 (86%) of the laboratories reporting a count within the intended range. The overall 
performance for examinations by culture was 90%. 

15 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Seven laboratories examined the sample using a molecular kit which only detects L. pneumophila and all seven 
reported the correct result. In addition, five of these laboratories examined the sample using a molecular kit that 
detects Legionella spp. and all five reported a correct result. 

Sample 5724: This sample was water from a spa which contained a Legionella pneumophila serogroup 6 
sequence type ST2923 at levels of approximately 103 colony-forming units per litre. Background flora was 
Citrobacter braakii.  

Performance was good, with 24/27 (88%) of participants reporting the correct isolation result, 24/24 (100%) for 
identification, 18/23 (78%) of the laboratories reporting a count within the intended range, 9/10 (90%) reporting 
the correct serogroup and 8/8 (100%) of the laboratories reporting the correct sequence type. The overall 
performance for examinations by culture was 91%. 

Seven laboratories examined the sample using a molecular kit which only detects L. pneumophila and 6/7 reported 
the correct result. In addition, four of these laboratories also examined the sample using a molecular kit that 
detects Legionella spp. and all reported a correct result. 

Sample 5725: This sample was a swab of the biofilm from a hot and cold water system which contained no 
Legionella spp. Background flora were Roseomonas aestuarii and Pseudomonas aeruginosa. 

Performance was excellent, with 24/24 (100%) of the laboratories reporting the correct isolation result. 

Six laboratories examined the sample using a molecular kit only which detects L. pneumophila and 5/6 reported 
the correct result. In addition, three of these laboratories examined the sample using a molecular kit that detects 
Legionella spp., with 2/3 reporting a correct result. 

Culture methods are considered to be the gold standard and are still the method of choice for many laboratories.  

Figures 1 and 2 below illustrate the turnaround time for reporting results via the on-line secure system to PHE to 
complete the exercise. 

Figure 1. Turnaround time for reporting results - clinical laboratories 

>40

36-40

31-35

26-30

20-25

s
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10

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4

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6

8

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12

Number of participants

Figure 2. Turnaround times for reporting results - environmental laboratories 

6

6

4

3

15

0

2

4

6

8

10

12

14

16

No. of participants

s
y
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36-40

31-35

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16 

 
 
 
 
 
 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

4. Discussion 

General 

Environmental and clinical laboratories play a vital role in protecting the public’s health, by helping to ensure public 
health incidents are effectively detected and managed through the provision of quality results for samples 
analysed. Laboratories may also be required to report detected cases of Legionnaires’ disease to their national 
surveillance systems if this is a requirement in their country.  

External quality assessment provides laboratories with an independent external assessment of their performance. 
Regular participation in proficiency testing schemes is an important part of laboratory quality procedures and helps 
to ensure that the results of their tests are accurate. It also ensures high quality for the surveillance data reported. 

Overall, the performance of laboratories participating in the 20192020 EQA was very good. There were no 
significant issues arising for species identification, serogroup, enumeration, or sequence type. 

The outbreak strain chosen to simulate samples and specimens was L. pneumophila Sg 1, ST47. Two clinical 
specimens (5712 and 5713) contained Legionella pneumophila serogroup 3, ST2630. This is a unique ST, with a 
single documented isolation from a community-acquired clinical case in the UK. Legionnaires’ disease attributed to 
L. pneumophila serogroup 3 is less common than serogroup 1 infection and many of the commercially-available 
urinary antigen kits are not designed or validated for the detection of L. pneumophila non-serogroup 1 antigens.  

Comparing clinical and environmental isolates using serological and molecular techniques can help identify the 
source of Legionnaires’ disease during potential outbreak investigations. Legionella is frequently found in the 
environment, and examination of clinical isolates can help interpret the findings of an environmental investigation. 

There were no issues encountered with the preparation of the simulated specimens/samples. Homogeneity, 
stability and viability were consistent throughout all the stages of production and distribution. To maintain these 
parameters, proven technology for preserving organisms/levels of organisms were used, such as lyophilised or 
LENTICULE® discs. This preservation technique, used to produce simulated EQA samples/specimens, meant that 
the stability of the organisms would probably be guaranteed during transit to the 28 EU/EEA countries. This was 
important, given that transit time would probably be longer than that for local or national distribution of samples to 
designated laboratories.  

From the results reported in this EQA it was ascertained that the majority of laboratories for clinical samples 
identified the pathogen and serogroup, but a significant number did not report the ST (Tables 1, 5 and 7). For 
environmental samples, the ISO 11731:2017 requires that suspect colonies are identified to at least L. 
pneumophila. Laboratories reported L. pneumophila correctly and went further, reporting a serogroup. For the 
clinical findings, 14 laboratories only examined the sample for ST. 

Clinical discussion 

The clinical aspect of this EQA was a qualitative exercise designed to assess simulated sputum and urine 
specimens. The panel of sputum samples were used to ascertain the absence or presence of L. pneumophila and 
when, following isolation of the respiratory pathogen, full identification to species level were requested, with 
accompanying Sg and ST. Examination to detect the urinary antigen for Legionella pneumophila was requested in 
the simulated urine samples. 

Based on published guidance by PHE in the UK, the three most commonly-described sample types analysed were 
urine and lower respiratory fluids, including sputum and broncho-alveolar lavage (BAL) [4]. Using this information, 
simulated sputum and urine specimens were designed for distribution as part of the EQA exercise. A survey of 
methods (Annex 1) was sent out simultaneously with the EQA panel and this confirmed the most common 
specimen types examined routinely by participating laboratories to be sputum 100% (27/27), urine 92.6% (25/27) 
and BAL 100% 27/27. 

Three paired (sputum/urine) simulated specimens (5707, 5708; 5709, 5710; 5711, 5712) with relevant 
accompanying clinical details were sent for evaluation. These specimens were designed to mimic an outbreak. 

Patient 1 
Both the simulated sputum (specimen 5706) and the urine sample (specimen 5707) contained Legionella 
pneumophila Sg1 ST47, one of the most prevalent circulating serogroups for Legionella pneumophila.  

Of the 27 of laboratories reporting an identification, 100% achieved the correct result confirming the presence of 
Legionella pneumophila. A total of 25/27 laboratories reported the correct serogroup but, interestingly, two 
laboratories did not report a Sg, one of which was a public health laboratory. Failure to report a serogroup is a 
concern and may need follow up to understand why the Sg was not reported for the two laboratories in question. 

Only 51.9% (14/27) of the participants examined for sequence type. Of those reporting an ST, all 14/14 
laboratories achieved the correct result. Only 14/27 reporting a ST result may be a reflection of the level of 

17 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

services provided by the participating laboratories. Participants were both clinical diagnostic and regional reference 
laboratories (RRL), and one would expect all RRL to report the ST. The low number of laboratories reporting a ST 
may be due to the lack of materials available (e.g. sequence base typing) at their facilities. It is currently under 
consideration as to whether this information should be requested in subsequent EQA exercises, by asking 
participants to enter the method used to determine the ST in the online reply form. 

All 24 laboratories that tested for urinary antigen (specimen 5707) achieved the correct positive result, and can 
evidently meet the desired quality of service in laboratory reporting for clinical cases of Legionnaires’ disease. 
However, three laboratories did not examine this specimen type. 

Patient 2 
This reflected a possible scenario in patient screening/or samples taken from a patient presenting with 
Legionnaires’ disease symptoms, but not infected with Legionella spp.. Patient 2 sputum specimen 5708 was 
negative for the presence of the pathogen and 92.6% (25/27) of laboratories correctly reported the absence of the 
organism. Of the two laboratories reporting an incorrect result, one general hospital laboratory reported Legionella 
spp. and a public health laboratory reported L. pneumophila. 

In contrast, the urine for patient 2 (specimen 5709) was a simulated urine containing a urinary Legionella antigen 
and 91.7% (22/24) of laboratories reported the correct result. One laboratory that reported a negative result stated 
that this sample was referred to another hospital for testing. The second was a national reference laboratory for 
Legionella testing. Three laboratories did not examine these specimens. As the corresponding sputum sample 
tested negative for this patient, it could be that local protocols state that no further testing needs to be performed.  

To conclude, the overall intended results for this patient could represent the possibility of an inadequate sputum 
specimen having been received, and only the positive urine result having been taken into consideration for 
interpretation of Legionnaires’ disease. In this case, a request for a repeat sputum or more sensitive specimen 
(e.g. BAL) would be appropriate. Further investigations into the two laboratories that achieved an incorrect result 
and the three laboratories that failed to examine the urine sample are warranted.  

Patient 3 
With the third set of samples, the sputum specimen (5710) contained a high yield of L. pneumophila Sg1, ST47 
and a very high level of urinary antigen (specimen 5711) for L. pneumophila Sg1, ST47. A 100% concordance was 
achieved with 27/27 of laboratories reporting correctly to species level. A further 26/27 reported the correct 
serogroup but only 14 of the 27 reported a ST47. Interestingly, 13 laboratories did not report sequence type and 
this may warrant further investigation, to find out why they did not report for this test. Of the urinary antigen 
results, 91.7 % (22/24) of laboratories reported the correct result, detecting the Legionella antigen, with only two 
failing to detect it.  

As regards concordance with the intended results, performance was very good to excellent for all three sets of 
simulated patient samples. This demonstrates the competence level of participating laboratories in isolating and 
identifying the causative agent and detecting the presence of the circulating antigen.  

The methods survey questionnaire was used to obtain information on the various methods applied to identify the 
pathogen and the most commonly reported were conventional testing (culture) and MALDI-TOF. The most 
frequently reported confirmatory tests included serology and MALDI-TOF, with some laboratories also reporting use 
of fluorescent microscopy. A small number also reported using Direct Fluorescent Antibody (DFA) staining.  

Patient 4 
One set of simulated samples (5712 and 5713) contained L. pneumophila Sg 3, ST2630 and this ST was included 
as a patient suspected of contact with Legionella, with indicative accompanying clinical details (asthmatic elderly 
male, lives at home local area, rarely goes out, except to the shops and local pub). This set of patient samples is 
not associated with the simulated outbreak, based on the reported Sg and ST and an absence of the antigen for L. 
pneumophila Sg1 being detected in the urine.  

A 100% concordance was achieved with 26/26 reporting the correct species, 26/26 reporting the correct Sg and all 
14/14 reporting the correct ST.  

There are a plethora of testing kits available for use in clinical diagnostic laboratories which are designed and 
validated for the detection of the surface antigen for Sg 1 in urine (serogroup 1 being the predominant circulating 
antigen). Results for specimen 5713 concluded that the kits used by the participants do not detect the Sg3 
circulating antigen and this was confirmed by all 24/24 laboratories reporting the absence of Legionella antigen. 

As regards concordance with the intended results, performance was very good to excellent for all four sets of 
samples, demonstrating the competence level of participating laboratories in isolating and identifying the causative 
agent and detecting the circulating Legionella antigen.  

18 

 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Patient 5 
Legionella longbeachae is the second most commonly reported causative agent of Legionnaires’ disease. L. 
longbeachae was distributed in a simulated sputum specimen (5714) as an educational objective. This proved a 
little challenging, with only 88.5% of participants reporting the correct result. Overall, 17 laboratories reported the 
presence of L. longbeachae and six reported the absence of L. pneumophila. This illustrates the different testing 
methods adopted by the reporting laboratories. More details on the methodologies used will be obtained and 
collated in the next exercise. 

Patient 6 
EQAs often include a negative sample in the assessment. It is just as important for the participating laboratories to 
be able to provide a true negative result as it is to determine a positive one. Specimen 5715 was a simulated urine 
specimen, containing no antigen. Participant performance was excellent with 23/24 (95.8%) of laboratories 
reporting the absence of a circulating L. pneumophila antigen. 

The source of infection can be identified by comparing clinical and environmental L. pneumophila isolates using 
various typing methods. A variety of rapid identification and sensitivity methods have been developed for isolates 
from clinical samples. These include molecular techniques such as Real-time Polymerase Chain Reaction (PCR), 
Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Multiple-Locus Variable-Number 
Tandem-Repeat Analysis (MVLA), Single Nucleotide Polymorphism (SNP) assays, Whole Genome Sequencing 
(WGS) and Matrix Assisted Laser Desorption Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry. Although 
these applications enable subtyping of unrelated strains, the accuracy, precision and reproducibility are not 
comparable. Within this EQA exercise all participants (n=27) reported culturing the simulated sputum specimens. 
Other methods used included Gram stain (7), UV microscopy (9), serology (19), MALDI-TOF (16), semi-automated 
methods (1), ELISA (2), direct fluorescent staining (6), monoclonal antibody typing (14), mip sequencing (10) and 
WGS (7). 

Most failures with EQA specimens can be a result of inadequacies in the other components of the quality system, 
including methodologies used. 

Environmental discussion 

The environmental aspect of this EQA was a qualitative and quantitative exercise designed to assess simulated 
environmental water and swabs. The environmental samples were used to ascertain the presence or absence of 
Legionella spp. and, upon isolation/detection of the organism, a full identification to species level. There was also 
an option to report enumeration/quantification with accompanying Sg and ST, if applicable and as requested.  

Legionella spp. are found in cooling towers, hot and cold water systems, air conditioners, spa equipment, 
fountains, humidifiers and showers, misting devices, decorative fountains and water features, dentistry tools and 
thermostatic mixing valves (TMVs). The main mode of transmission is through inhalation of airborne droplets 
contaminated with Legionella spp..  

The detection of Legionella by culture is the gold standard method for detecting Legionella colonies on buffered 
charcoal yeast extract (BCYE)/glycine vancomycin polymyxin B cycloheximide (GVPC) agar plates. This is a labour-
intensive approach which takes ten days to complete. Recovery of Legionella bacteria by culture can be challenging 
as Legionella colonies on BCYE agar media can be overgrown or inhibited by competing microbial flora, masking 
the presence of Legionella colonies. Therefore, acid and heat treatment of samples is the key to reducing the 
amount of background flora [3]. 

Polymerase chain reaction (PCR) method, is a molecular technique that only takes a few hours to complete and can 
be a useful method to screen environmental and water samples. The disadvantage of this method is that dirt and 
debris can have an impact on the test outcome. Molecular testing is not widely used to test water and 
environmental samples for Legionella and only a few commercial laboratories offer this service routinely. Moreover, 
the detection of DNA from dead Legionella cells has limited public health significance. In 2015, the ISO/TS 12869 
standard method was published for the detection and quantification of Legionella and/or L. pneumophila by 
concentration and genic amplification using real-time polymerase chain reaction (qPCR) in order to standardise this 
methodology. 

A methods survey questionnaire (Annex 1), sent out simultaneously with the EQA panel, confirmed that the most 
common sample types routinely examined by participating laboratories are water from hot/cold water systems 
100% (27/27), cooling towers waters 93% (25/27), water from spas 100% (27/27) and swabs from biofilms 89% 
(24/27).  

The overall performance of the laboratories in the EU/EEA countries was very high. A total of 19 laboratories 
examining water samples for Legionella bacteria indicated that they followed ISO 11731:2017 (Water quality - 
Enumeration of Legionella). All 24 laboratories that returned information responded that they filtered the water 
sample, 19 cultured the sample untreated, 22 carried out an acid treatment and 20 also used a heat treatment.  

 

For isolation: the overall performance for isolation of Legionella was 96%, with 2327 of the laboratories 
reporting a result. Three laboratories did not examine the swab samples. However, performance was lower 
with sample 5721 (23/27 – 85%) when compared to the other samples containing a Legionella spp.. The 

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EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

low level of L. pneumophila in the sample at 9.8x102 colony-forming units per litre may have contributed to 
this finding. This level of organism content may have been around the lower end of the detection limit for 
methods used by the laboratories, which is why it is important that they know their detection limits and the 
potential impact this may have on results. The most common isolation media used was GVPC and/or BCYE. 
There was variation among laboratories in the use of other culture media and acid and/or heat treatment.  
For identification: six of the ten samples contained a Legionella spp.. The overall performance for correctly 
identifying the Legionella spp. was 99%, with 2326 of the laboratories reporting a result. Sample 5723 
contained a Legionella bozemanii, one laboratory incorrectly reported the Legionella as Legionella 
pneumophila.  
For enumeration: seven of the ten samples were simulated water samples. Of these, five contained a 
Legionella spp.. The overall performance for counts being reported correctly as a censored value or within 
the intended range was 90%. Performance was lower with three samples, 5721 (79%), 5723 (86%) and 
5724 (78%), due to the bacterial load being at the lower end of the detection limit for methods used (102-
103). For swab samples, the number of enumeration results reported was low.  
For serogroup: the overall performance for serogroup confirmation was very good, with 98% of results 
being reported correctly. One laboratory reported an incorrect serogroup 3 for sample 5724 when the strain 
was a serogroup 6. According to the data from the questionnaire, a majority of the laboratories used the 
‘Oxoid Dryspot’ Legionella latex test. 
For sequence type: the overall performance in reporting a correct sequence type was 93% for five of the 
samples that contained L. pneumophila (5716, 5717, 5719, 5721 and 5724). Between 814 laboratories 
reported the ST results. This may have been due to lack of expertise or linked to the method implemented 
in the laboratory. Samples 5716 and 5717 contained a L. pneumophila sg1 ST47. One laboratory reported 
the ST as ‘other’ and did not provide further details. Sample 5719 also contained a L. pneumophila sg1 
ST47; two laboratories reported the ST as ‘other’ and did not provide further detail. Further analysis showed 
that one laboratory reported an incorrect ST for all samples 5716, 5717 and 5719. The most common 
method used for ST analysis was Sanger sequencing and/or Whole Genome Sequencing (WGS).  
For molecular methods: 58 laboratories analysed the samples for L. pneumophila using molecular methods 
and culture methods concurrently. The overall performance with detection/absence of L. pneumophila for 
the 10 samples was 95%. One laboratory reported an incorrect result for samples 5722, 5724 and 5725: 
samples 5722 and 5725 did not contain L. pneumophila. For molecular detection/absence of Legionella spp. 
the overall performance was 75%. A total of 25 laboratories examined the samples using a molecular 
method for Legionella spp.. One laboratory reported an incorrect result for samples 5718, 5720 and 5722; 
this was the same laboratory mentioned above that incorrectly reported the presence of L. pneumophila. 
One laboratory reported correctly that L. pneumophila was ‘detected’ for some samples but ‘not detected’ 
for Legionella spp.. An analysis of the kits from the method questionnaire did not indicate that one specific 
molecular test was commonly being used.  
For quantification: one or two laboratories reported a quantification result in terms of genomic copies per 
litre. Due to the low number of data sets returned, it was impossible to analyse the values. However, where 
a censored value was reported for samples containing no Legionella spp. this can be assumed to be correct.  
The overall performance between the results reported by the clinical laboratories and those reported by 
environmental laboratories was very good for the three sets of samples and clearly demonstrated that the 
fourth paired sample was not associated with the ‘outbreak’. 

 

 

 

 

 

 

 

The detection and acceptable level of Legionella spp. is also an important factor in determining the effectiveness of 
control measures in an artificial water system. Other types of Legionella spp. besides L. pneumophila have also 
been implicated in causing infection, particularly in nosocomial cases. However, the EQA organisers are aware that 
national guidance documents may only refer to L. pneumophila and not necessarily include the requirement to test 
other species of Legionella. 

Limitations of this EQA exercise 

This EQA was only able to evaluate the analytical and post analytical stages of the total testing process. The pre-
analytical stage of the process was not evaluated. The pre-analytical stages would include the demographics of the 
patient sample, correct sample type, volume of sample, correct tests requested and suitable container, all of which 
were pre-determined for this EQA panel. 

The EQA scheme was only available to a maximum of two selected laboratories per EU/EEA country, therefore the 
breadth of the cohort was limited to those who received a panel and returned results. 

A period of six weeks was given for laboratories to return results. This period of time was allocated to allow 
sufficient time for the panel to arrive at the laboratories via air freight. The time allowed for the return of results 
was not meant to reflect the expected turnaround times for clinical or environmental laboratories when 
investigating and returning results. Nevertheless, the number of days taken to report results from the receipt date 
by the laboratory was recorded (Figures 1 and 2). Interestingly, the mode for returning results was determined to 
be 35 days for clinical and 40 days for environmental samples. The turnaround time to report results indicates that 
the participating laboratories may not have treated the EQA specimens as they would routine samples (having 
several staff analyse the results prior to reporting online rather than just one member of staff processing and 

20 

TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

reporting). For a service provider turnaround times of 35 and 40 days respectively would be unacceptable. 
However, one limitation to the system used to report results is that it does not allow for the capture of preliminary 
results, as some laboratories do. There is a need to understand whether the six-week period given to report results 
truly reflects the way in which laboratories work with genuine samples/specimens. 

The clinical samples sent in lyophilised format did not represent the matrix of an authentic liquid purulent sputum 
which would normally be received by a diagnostic laboratory. However, the specimens distributed for detection of 
urinary antigens were authentic clinical liquid urine, spiked with species antigen and provided in plastic tubes to 
resemble a true sample. 

For the environmental water samples, once the LENTICULE discs were rehydrated this would constitute one litre of 
water which would not be representative of the chemical constituents normally be found in real samples. For swabs 
the laboratory was instructed to rehydrate the sample and then absorb the material onto a swab before suspending 
the swab into a diluent. This was the most practical way to simulate a swab sample, however this is not 
representative of how a swab sample would be received in a laboratory for analysis.  

For environmental samples, the molecular quantification results could not be assessed due to the low number of 
data set returned. 

An EQA is of limited value without at least some of the other components of a quality system, such as adequate 
documentation, training of staff and internal quality control (IQC). 

EQA benefits 

The importance of an EQA is to ascertain and assess the level of competency of the participating laboratories in 
delivering a service to examine clinical specimens and water samples for presence and detection of Legionella spp.. 

The benefits of participating in this EQA are: 

 
 
 
 
 
 
 
 
 

to provide laboratories with an insight into their performance; 
to help improve local standards; 
to reveal unsuspected areas of difficulty; 
to provide an educational stimulus for improvement; 
to check the efficacy of internal quality control procedures; 
to demonstrate a commitment to quality to colleagues and customers; 
to provide a method performance evaluation  
to provide independent evidence of performance for accreditation bodies; 
to enable the participants to monitor, evaluate and improve their own performance and training needs, 
since dealing with discrepant EQA results will improve testing performance which, in turn, would directly 
improve the management of public health incidents and clinical service. 

A comprehensive quality assurance system will cover such areas as provision and control of standard operating 
procedures, education and training, planned maintenance and calibration of equipment and the monitoring of 
response times. Many laboratories are formally accredited in order to acknowledge compliance with defined 
objectives and quality standards such as those detailed in ISO 17025:2017 or ISO 15189:2012. 

Results of consistently good quality can be expected only when all the components of a quality system are in place. 

21 

 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Participant feedback on this EQA 

A short feedback evaluation survey was sent to all participating laboratories by ECDC after the first exercise, with 
the online survey open from 213 April 2020 and then extended to 23 April 2020. 

A total of five questions were asked. 

Question 1.   Regarding any of your analytical test results that did not conform with the intended results, can you 

specify which corrective action(s), if any, was/were taken (e.g. review and adjust SOPs, verify 
reagents)?  

Question 2.  Are the results of this EQA exercise to be used as documentation for accreditation and/or licensing 

purposes for the method(s) used in your laboratory?  

Question 3.  Overall, is this EQA exercise important for your laboratory to assure its diagnostic capability?  
Question 4.  Were you satisfied with the EQA report of results specific to your laboratory?  
Question 5.  Do you see a benefit in participating in this EQA scheme compared to other (commercial) EQAs for 

Legionella? Please describe why or why not.  

Feedback was provided by 18 of the 34 EU/EEA participating laboratories, representing a response rate of 51%. 

Among the respondents, 9/18 had participated in both clinical and environmental distributions, 4/18 in the clinical 
distribution and five in the environmental distribution only.  

Four of the EU/EEA respondents indicated that corrective actions were taken based on the EQA distribution results. 
The types of corrective action were that environmental samples were now sent to a specific environmental 
laboratory; sample preparation and handling procedures had been extended for different matrices; procedures had 
been adopted for environmental samples to check for overgrowth (in the event of high background flora) and 
antigen kits or reagents had been replaced with different types. 

In all, 17/18 participating laboratories indicated they would use the results of the EQA exercise as evidence for 
accreditation and/or licencing purposes for the methods used. 

A total of 5/18 laboratories indicated the exercise was important but not essential, 13/18 responded that it was 
very important. The reasons given for its high importance were: 

 
 

 

use of results as documentation for accreditation; 
lack of other available EQA schemes for the laboratory to participate in (e.g. complex diagnostics for 
Legionella; no other available EQA covering culture/clinical samples; no urine antigen EQA available); 
a means of ensuring the high-quality testing protocols (e.g. good laboratory practice; review of sensitivity 
and specificity of tests using the lab methods). 

Suggested areas of consideration for the next EQA round were:  

 
 
 

a better assessment of quantitative counts for environmental samples; 
inclusion of all data, such as sequence types, allele profile, Dresden typing;  
making the environmental report clearer so that it was easier to see where the method results came from. 

Responses from EU/EEA laboratories on the performance of this EQA compared with other providers included: 

 
 
 

 

the importance of including an outbreak situation in an environmental scheme; 
the fact that this scheme covered all methods at the same time; 
the fact that the scheme was not limited to one restricted matrix assay, having different ranges of 
microorganisms contained in the sample (also negative samples) which tests the robustness of laboratory 
work over a short time period; 
the fact that it simulated a real outbreak investigation situation where all the tests required had been 
evaluated simultaneously which was a good exercise to prepare laboratories for possible outbreaks and the 
matching of sources. 

Overall, there was a very positive feedback on this new ECDC EQA Legionella scheme. 

Although the comments and feedback represent only half of the laboratories that participated in this distribution, 
they indicated that offering this EQA scheme to the wider ELDSNet network of EU/EEA countries is beneficial for 
the surveillance and response to Legionnaires’ disease in Europe. Moreover, the format of this EQA scheme as an 
outbreak simulation comprising environmental samples and clinical specimens was considered an added value 
when compared to other EQA services available in Europe. 

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TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

5. Conclusions 

The performance of laboratories from the 28 EU/EEA countries in this exercise was very good for culture-
based/detection methods used by both the clinical (96.2%) and environmental laboratories (95%). 

Both clinical and environmental laboratories demonstrated that they could undertake testing to an acceptable level 
of at least 80% correctness. Although the data provide some assurance of the laboratories’ ability to undertake 
effective public health investigations for Legionella pneumophila, more EQA data is required to determine the 
actual robustness. If laboratories report accurate data this also ensures that the information provided to 
surveillance systems is accurate. 

Laboratories were provided with the opportunity to examine samples that they would routinely test. For clinical 
samples, 27 laboratories examined the sputum samples and 24 examined the urine. For environmental samples, 27 
of the laboratories examined the water samples and 24 examined the swab samples. Where results reported were 
not in accordance with those intended, laboratories were advised by contractors to investigate in order to 
determine the root cause.  

For the clinical laboratories, all 27 reported an identification, 26 for serogroup and 14 for sequence type. All 
laboratories reported isolation and identification using culture-based methods. Isolation in culture remains the gold 
standard for the diagnosis of infection caused by Legionella spp., due to the low sensitivity and specificity 
associated with serotyping. MALDI-TOF MS is then frequently used to identify isolates to species level. 
Differentiation and typing of strains can be achieved using a range of molecular techniques, including SBT and RT-
PCR methods. A total of 22/27 participants went on to perform molecular methods for the detection of Legionella. 

For the environmental laboratories, 27 laboratories reported a result for isolation and identification, 25 reported a 
serogroup, 24 reported an enumeration count and 14 a serotype. For molecular methods, eight laboratories 
analysed the samples for L. pneumophila and five for Legionella spp. The overall isolation performance for culture 
was 95%, with 27 reporting a result. The overall performance for molecular methods was 95%, with eight 
laboratories reporting a result for kits detecting L. pneumophila. It is known that laboratories are still developing 
the routine application of molecular methods for water and environmental samples due to the fact there are 
currently no guidelines for the interpretation of molecular results (GU/L). Therefore, culture remains the preferred 
method.  

The results of a survey carried out by ECDC indicated that laboratories found this EQA exercise to be very useful. 
Overall, the feedback on this new ECDC EQA Legionella scheme was very positive. 

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TECHNICAL REPORT 

6. Recommendations 

This exercise will continue to provide a baseline understanding of the level of testing undertaken in the 
laboratories, determine any performance issues and, where possible, provide support to laboratories/countries who 
have identified limitations in addressing improvement of their testing capabilities or capacity building.  

Main recommendations for future EQA exercises 

Sample/specimen design 

 

 

 

 

To continue providing this EQA exercise and include different L. pneumophila serogroups, sequence types 
(STs) and Legionella species. This will allow a better understanding of a laboratory’s ability to undertake 
testing to the level required for successful management of public health incidents.  
To identify through further EQA exercises if there are issues when less commonly encountered species, SG 
or ST are included. A single EQA distribution cannot confirm this. 
For environmental samples to include levels of Legionella spp. that are at the lower end of the detection 
limit for culture. In addition, to confirm if molecular assessment is of value since, with low numbers of 
laboratories currently using this method of examination, performance cannot be assessed. 
To continue to include more than one species of Legionella spp. within the simulated samples/specimens 
set. This will help educate and improve knowledge and experience with organisms which are otherwise not 
frequently encountered.  

Methods  

 

 

 

To gather more information on the methods used to report results on the samples/specimens – this will be 
required when returning results. 
To link the method information more closely to the results reported in order to identify tests that 
laboratories do routinely but did not report a result for or instances where the organisers did not allow for 
these examination results to be reported. 
To include the option for laboratories to examine clinical specimens using molecular methods, as the 
evidence from the methods questionnaire suggests that laboratories do undertake this type of testing.  

Scoring 

 

 
 

To determine if performance assessment should be scored and design an appropriate scoring system to 
make it easier to identify those laboratories that experience significant, ongoing difficulties with their 
examinations.  
The allocation of scores is provided as a visual management tool to help assess performance.  
Ongoing performance assessment for a laboratory can only be done if the same laboratory takes part in all 
the EQA distributions. 

Future objectives 

To improve awareness of the different Legionella spp. that may be isolated from clinical specimens and 
environmental samples. 
To improve awareness of the confirmatory tests done and their limitations when confirming Legionella spp. 
Isolates in samples. 
To improve awareness of the importance of following standardised methods when managing public health 
incidents. 
To encourage regular participation in the EQA by the same laboratories in the countries as it is an important 
element of their quality procedures and helps to ensure that the results of their tests are accurate. 
Laboratories should participate regularly throughout the year in order to review performance on an ongoing 
basis. Ongoing performance assessment is designed to identify genuine problems. 
To determine if performance assessment should be scored and design the appropriate scoring system to 
make it easier to identify those laboratories that experience significant, ongoing difficulties with their 
examinations. The allocation of scores is provided as a visual management tool to help assess performance.  
To determine if participating in EQA exercises can improve understanding of the link between clinical and 
environmental laboratories within countries when dealing with outbreaks to make the management of public 
health incidents more effective.  
To explore participants’ feedback from the evaluation survey in greater depth to improve the exercise (e.g. 
improving information in the individual EQA reports.)  
To update the IT platform so that results on methods used to can be provided. 
To communicate the results of this EQA at future ELDSNET and Legionella conference meetings to increase 
awareness of the importance of EQAs for the quality of Legionella detection in laboratories.  

 

 

 

 

 

 

 

 
 

24 

 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

References 

1.  

2. 

3. 

4. 

European Legionnaires’ Disease Surveillance Network (ELDSNet); 2017. Available at: 
https://www.ecdc.europa.eu/sites/default/files/documents/ELDSNET_2017-revised_guidelines_2017-
web.pdf 

Publications Office 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 decisions (text with EEA relevance). Available at: 
https://op.europa.eu/en/publication-detail/-/publication/57ed0506-80e4-11e8-ac6a-
01aa75ed71a1/language-en  

Control of Biohazards, Legionella detection and sampling methods. [cited 1 December 2020.] Available at: 
https://www.legionella.com/legionella-detection-and-sampling-methods 

National Infection Service, Reference Laboratories Colindale, Bacteriology Reference Dept., User manual, 
Public Health England Publications: Gateway Number: GW-604, Version 11. October 2019. 

25 

 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Annex 1. Findings from methods survey questionnaire 

As part of the EQA exercise, a questionnaire was sent out on the methods used to analyse the samples/specimens. 
The data presented below is for all EU/EEA and non-EU/EEA countries.  
Methods survey findings for clinical specimens 

Distribution 4680, which closed on 16 December 2019, consisted of 10 simulated clinical samples. A questionnaire 
sent out together with the EQA was completed by all 27 of the participating laboratories in the 28 EU/EEA countries. 

General information 
A total of 22/27 participants reported that, in addition to being reference laboratories, they were clinical diagnostic 
laboratories. One laboratory reported that they were only a clinical diagnostic laboratory. The remaining 4/27 were 
reference laboratories only.  

The majority of laboratories (63% - 17/27) participated in a national EQA scheme. Of these, the national scheme 
was mandatory for only 41.2% (7/17).  

Figure A3. Sample types analysed for Legionella by participating laboratories in the EU/EEA 

Tracheal fluid

Respiratory smear

Pleural fluid

Blood/serum

Tissue/ biopsy

Sputum

Broncho-alveolar lavage

Urine

l

s
e
p
m
a
s

f
o

e
p
y
T

0

5

10

15

20

25

30

Number of laboratories

Figure A4. Different types of media used for the detection of Legionella 

GVPC

BMPA, BCYE & GVPC

BMPA

BCYE, MWY & BMPA

BCYE & GVPC

BCYE & MWY

BCYE & BMPA

BCYE

d
e
s
u
s
d
o
h
t
e
m
e
r
u
t
l
u
c

f
o
s
n
o
i
t
a
n
b
m
o
C

i

0

1

2

3

4

5

6

7

8

Number of laboratories

Codes: 
BCYE: Buffered charcoal yeast extract agar  
GVPC: Glycine vancomycin polymyxin B cycloheximide 
MWY: Modified Wadowsky Yee 
BMPA: Legionella BMPα selective media with activated charcoal, yeast extract and ACES- buffer 

Comments: 
All laboratories noted culture prior to confirmatory testing. The majority of laboratories (n=19) used commercial media, while the 
others (n=8) used in-house media. The manufacturers included Oxoid, Biomerieux, BioMaxima, Liofilchem, Beckton Dickinson, 
Thermo Fisher scientific, PVL and E&O Laboratories.  

26 

 
  
 
 
 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Table A19. Incubation period of culture plates (in days) 

Incubation period (days) 

Number of laboratories 

3 
5 
7 
>=10 

1 
1 
9 
17 

Figure A5. Confirmatory tests used for detection of Legionella 

s
t
n
a
p
i
c
i
t
r
a
p
f
o
r
e
b
m
u
N

20
18
16
14
12
10
8
6
4
2
0

Method

Figure A6. Distribution of reported use of PCR molecular methods by EQA clinical participating 
laboratories 

Real Time PCR

Conventional PCR

PCR not used

Comments: 
A total of 22/27 laboratories performed molecular testing, the majority of these (n=20) used Real Time PCR. The two other labs 
used conventional PCR. The manufacturers listed vary, there is no predominant kit or supplier used.  

27 

 
 
 
 
 
 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Figure A7. Typing and species identification methods, including phenotypic and genotypic 

monoclonal antibody typing

WGS

mip sequencing

d
e
s
u
s
d
o
h
t
e
M

0

5

10

15

20

25

30

Number of laboratories

yes

no

didn’t answer

Comments: 
Only five labs outsourced molecular testing 

All of the laboratories performing WGS (n=7) were reference laboratories. There appears to have been more 
laboratories performing sequence-based typing than actually reported here (n=14 reporting results for sequence 
type.) 

Methods survey findings for environmental samples 

A questionnaire was sent to all participants who participated in distribution 4681. The objective was to gather 
information on the method/processes used for this EQA exercise. 

In all, 24/27 (88%) of the participating laboratories provided information on their methods/processes. No replies 
were provided for Italy, Lithuania or Hungary. Of the responders, the total numbers will not always correspond to 
24 as some participants did not provide information on all the questions and some questions allowed for more than 
one option to be selected. 

The method data shown is for information only. It does not evaluate or associate the data with a failure in the EQA 
or method/process used and it does not attempt to compare performance of the various molecular kits/processes.  

All data is presented for all EU/EEA laboratories that participated in the EQA November 2019 exercise.  

General information 

Figure A8 below shows the approximate number of water and environmental samples examined for Legionella spp. 
in a year (n=19). 

Figure A8. Number of samples examined per year for Legionella 

>1000

500-1000

100-500

10-100

3

0

1

2

5

5

6

3

4

5

6

7

Number of laboratories

s
e
l
p
m
a
s

f
o
r
e
b
m
u
N

28 

 
 
 
 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Figure A9. Type of samples analysed by laboratories for Legionella (n=27) 

Wastewater

 Swabs from biofilms

Spa water

Industrial water

Hot and cold water systems

Cooling towers

Bathing waters

s
e
l
p
m
a
s

f
o
e
p
y
T

0

5

10

15

20

25

Number of laboratories

Figure A10 shows whether laboratories participated in their national EQA schemes programme and if these 
schemes are voluntary or mandatory (n=24). 

Figure A10. Participation in national EQA schemes 

No

3

2

Yes

0

10

5

9

15

10

20

Number of laboratories

Voluntary

Mandatory

Not applicable

Information on water examination 

Figure A11 shows the published methods used to examine environmental and water samples (n=26)  

Figure A11. Published methods used to examine swabs and water 

s
d
o
h
t
e
M

Other

1

2

ISO 11731:2017 and in house

1

ISO 11731:2017

14

4

ISO 11731-2:2004

1

In house

1

1

AFNOR NF T90-431:2017

1

Swab and water

Water

Swab

0

5

10

15

20

Number of laboratories

Figure A12 shows the method and volume used to examine the water samples in this exercise (n=24). 

29 

 
 
 
 
 
 
 
 
 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Figure A12. Method and volume used to examine water samples 

Filtration, neat and vortexing

1

Filtration and neat

2

5

3

2

Filtration, centrifugation and neat

Filtration and centrifugation

1

1

d
o
h
t
e
M

Filtration

2

7

0

2

4

6

8

10

12

14

Number of laboratories

100mL

500mL

1000mL

Other

Figure A13 provides details of how the sample was processed (n=23).  

Figure A13. Information on sample processing 

18

16

14

12

10

8

6

4

2

0

s
e
i
r
o
t
a
r
o
b
a
l

f
o
r
e
b
m
u
N

17

2

2

1

1

Acid

Heat and acid

Untreated and
acid

Untreated and
heat

Untreated, heat
and acid

Treatment type

For the laboratories that undertook acid treatment as part of the examination, the information in Figure A14 
provides details on the stage of the process at which the acid was applied (n=21). 

Figure A14. Stage at which acid was applied 

On concentrated sample

Directly on membrane

4

5

e
g
a
t
S

After filtration and bacterial released from
membrane and directly on membrane
After filtration and bacterial released from
membrane

After centrifugation, on concentrated sample and
directly on membrane

After centrifugation and after filtration and bacterial
released from membrane

1

1

1

9

0

1

2

3

4

5

6

7

8

9

10

Number of laboratories

Figure A15 shows the different types of media used (n=24). 

30 

 
 
 
 
 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Figure A15. Different types of media used 

20
18
16
14
12
10
8
6
4
2
0

s
e
i
r
o
t
a
r
o
b
a
l

f
o
r
e
b
m
u
N

12

14

18

BCYE

BCYE with antibiotics

GVPC

Media used

1

MWY

Codes: 
BCYE: Buffered charcoal yeast extract agar without L-cysteine 
BCYE with antibiotics: Buffered charcoal yeast extract agar with selective supplements 
GVPC: Glycine vancomycin polymyxin B cycloheximide 
MWY: Modified Wadowsky Yee. 

Figure A16 shows the combinations of media used and the source of this media (n=24). 

Figure A16. All media used and source of media 

a
i
d
e
M

GVPC

BCYE, GVPC

BCYE, BCYE with antibiotics and MYW

BCYE, BCYE with antibiotics  and GVPC

BCYE with antibiotics and GVPC

BCYE with antibiotics

0

2

4

6

8

Number of laboratories

Commercial

In-house

Both in-house and commercial

Other

Codes: 

BCYE: Buffered charcoal yeast extract agar without L-cysteine 
BCYE with antibiotics: Buffered charcoal yeast extract agar with selective supplements 
GVPC: Glycine vancomycin polymyxin B cycloheximide 
MWY: Modified Wadowsky Yee. 

31 

 
 
 
 
 
 
EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

TECHNICAL REPORT 

Table A20 provides information on the confirmation tests done on presumptive colonies of Legionella (n=24).  

Table A20. Confirmation tests undertaken on presumptive colonies of Legionella 

Confirmation tests 

MALDI-TOF 

Subculture in parallel on BCYE with and without antibiotics, and MALDI-TOF MS 

Subculture on BCYE and BCYE with cysteine 

Subculture on BCYE and PCR 

Subculture on BCYE with cysteine 

Subculture on BCYE with cysteine and MALDI-TOF 

Subculture on BCYE with cysteine and PCR 

Subculture on BCYE with cysteine, on TSASB and MALDI-TOF 

Subculture on BCYE, BCYE with cysteine and PCR 

Subculture on BCYE, BCYE without cysteine and R2A 

Subculture onto GVPC and BCYE 

Codes: 
BCYE: Buffered charcoal yeast extract agar without L-cysteine 
BCYE with antibiotics: Buffered charcoal yeast extract agar with selective supplements 
GVPC: Glycine vancomycin polymyxin B cycloheximide 
MWY: Modified Wadowsky Yee.  
TSASB: Trypticase soy agar with blood 

Number of laboratories 

1 

1 

6 

1 

7 

1 

1 

1 

2 

1 

1 

Figure A17 details the kits used to carry out the serogroup testing, with some laboratories using a combination of 
kits (n=27). 

Figure A17. Kit used for serogroup testing 

Prolex™ Legionella pneumophila Latex

Oxoid: Dry spot Legionella latex test

e
m
a
n
t
i
K

Monoclonal antibody MAb 3/1 of the Dresden Panel

Microgen® Legionella Latex Agglutination Kit

MAST® ASSURE Legionella Agglutinating Antisera

In-house monoclonal antibody direct fluorescence

Denka Seiken™ Agglutinating Sera Legionella 

Bio-Rad Legionella pneumophila and Species Latex Kit

1

1

1

2

2

3

3

14

0

2

4

6

8

10

12

14

16

Number of laboratories

Information on molecular testing 

Figure A18 shows the method used to determine the sequence base type (n=13).  

Figure A18. Method used to determine the SBT 

SBT using WGS

SBT using Sanger sequencing and WGS

SBT using Sanger sequencing

In-House

d
o
h
t
e
M

32 

0

1

2

3

4

5

6

7

Number of laboratories

 
 
 
 
 
TECHNICAL REPORT 

EQA schemes to support European surveillance of Legionnaires’ disease 2019-2020 - EU/EEA countries 

Table A21 below shows how samples are processed for molecular examination (n=6).  

Table A21. Sample processing for molecular examination 

Process  

Number of laboratories 

Filtration of 100 mL and filtration of 10 mL on 2 different membrane 

Filtration of 1L of water sample 

Filtration of 500 mL 

Real time PCR was used on isolated Legionella strains from samples  

The table below shows the DNA extraction kits used. 

Table A22. List of DNA extraction kits used 

1 

1 

2 

2 

DNA extraction kit  

Number of laboratories 

2 

2 

1 

2 

2 

1 

Bio-Rad Aquadien™ Bacterial DNA Extraction and Purification 

Invitrogen™ i-prep  

NucliSens easyMAG  

Other 

Roche Diagnostics MagNA Pure LC DNA Isolation Kit III 

Sacace™ Legionella pneumophila RealTM  

The table below shows the volume of extracted DNA for use in assays. 

Table A23. Volume of extracted DNA used in assays 

Volume (mL) 

Number of laboratories 

2.5 

5 

10 

Other 

1 

5 

2 

1 

The table shows the commercial assay that was used. 

Table A24. Commercial assays used 

Commercial assay 

Number of laboratories 

AmpliSens® Legionella pneumophila-FRT  
Bio-Rad iQ-Check® Quanti Legionella spp. and L. pneumophila Real-Time PCR Quantification 
Kit 
Bio-Rad iQ-Check® Screen Legionella spp. and L. pneumophila Kit 

In-house  

Other 

Sacace™ Legionella pneumophila RealTM  

The table below shows the amplification platforms used. 

Table A25. Amplification platforms used 

1 

1 

1 

1 

2 

1 

Amplification platform  

Number of laboratories 

Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System 

Bio-Rad CFX96 Touch™ Deep Well RT-PCR Detection System 

Other 

Roche LightCycler ® 2.0 

RotorGene 

1 

2 

1 

1 

1 

33 

 
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