European external quality assessment programme for influenza virus : 2018.
Date: 2019-03-04
Institutions: European Centre for Disease Prevention and Control
Subjects: disease prevention, health control, infectious disease, public health, research body, research report

European external quality 
assessment programme for 
influenza virus

2018

ECDC/WHO REGIONAL OFFICE FOR EUROPE TECHNICAL REPORT 

European external quality assessment 
programme for influenza virus 

2018 

 
 
This report was jointly commissioned by the European Centre for Disease Prevention and Control (ECDC) 
(framework contract ‘Support of surveillance activities of human influenza in Europe 2017-2021- External quality 
assessment (EQA)’ reference ID 7268; first specific contract 1 ECD.7737 implementing Framework Contract 
ECDC/20I7/002), coordinated by Cornelia Adlhoch (ECDC) and the World Health Organization (WHO) Regional 
Office for Europe (Agreement for Performance of Work ‘External Quality Assurance (EQA) for influenza virus culture 
and antiviral susceptibility 2018’ reference WCCPRD6918934 2018/804002), coordinated by Dmitriy Pereyaslov 
(WHO). It was produced by Dr Adam Meijer, National Institute for Public Health (RIVM), the Netherlands, on behalf 
of participants in the European Reference Laboratory Network for Human Influenza (ERLI-Net) in the European 
Union/European Economic Area (EU/EEA) and the network of National Influenza Centres (NICs) in the WHO 
European Region. 
Authors 
Gabriel Goderski, Ton Marzec, Mariam Bagheri, Pieter Overduin, Sharon van den Brink and Lisa Wijsman (RIVM) 
and Ruud van Beek and Prof Dr Guus Rimmelzwaan (Erasmus Medical Centre, Department of Viroscience, 
Rotterdam, the Netherlands) contributed to the design and planning of the exercise and production of the panel at 
the Respiratory Viruses Group, Division of Virology, Department of Respiratory and Enteric Viruses, Centre for 
Infectious Diseases Research, Diagnostics and Laboratory Surveillance, RIVM, Bilthoven, the Netherlands. 

Pre-testing of the panel with specimens labelled in random order was performed at Erasmus Medical Centre, 
Department of Viroscience, Rotterdam, the Netherlands and Centre National de Référence Virus des Infections 
Respiratoire, Laboratoire de Virologie, Institut des Agents Infectieux, Lyon, France. 

Panel distribution and data services were provided by Quality Control for Molecular Diagnostics (QCMD), Glasgow, 
UK. 

Natalja Kuznetsova (NIC-Tallinn, Estonia) took care of Russian documentation translations and Russian-speaking 
laboratory support. 

Colin Steel and Caterina Di Lorenzo, QCMD, and Dr Adam Meijer, RIVM, worked jointly to perform an initial analysis 
on the submitted data. Further analysis and report preparation were done by Dr Meijer. 

Suggested citation: European Centre for Disease Prevention and Control and WHO Regional Office for Europe. 
European external quality assessment programme for influenza virus – 2018. Stockholm and Copenhagen: ECDC 
and WHO; 2019. 

Stockholm, February 2019 

ISBN 978-92-9498-305-3 
DOI 10.2900/689354 
Catalogue number TQ-04-19-132-EN-N 

© European Centre for Disease Prevention and Control, 2019 

Cover picture: © Centers for Disease Control and Prevention/Frederick Murphy 

Reproduction is authorised, provided the source is acknowledged. 

For any use or reproduction of photos or other material that is not under the EU copyright, permission must be 
sought directly from the copyright holders. 

ii 

 
 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Contents 
Abbreviations ................................................................................................................................................ v 
Executive summary ........................................................................................................................................ 1 
1 Introduction ............................................................................................................................................... 3 
Objectives ................................................................................................................................................ 4 
2 Study design ............................................................................................................................................... 5 
2.1 Organisation ....................................................................................................................................... 5 
2.2 Panel composition, preparation and validation ....................................................................................... 5 
2.3 Participation ....................................................................................................................................... 6 
2.4 Testing ............................................................................................................................................... 6 
2.5 Data reporting .................................................................................................................................... 7 
2.6 Data analysis ...................................................................................................................................... 8 
3 Results ....................................................................................................................................................... 9 
3.1 Participating laboratories...................................................................................................................... 9 
3.2 Molecular detection ............................................................................................................................. 9 
3.3 Virus isolation and antigenic and genetic characterisation ..................................................................... 12 
3.4 Antiviral susceptibility ........................................................................................................................ 18 
3.5 Accreditation requirements ................................................................................................................. 25 
4 Discussion ................................................................................................................................................ 28 
5 Conclusions .............................................................................................................................................. 30 
6 Recommendations ..................................................................................................................................... 31 
Molecular detection, typing, type A H- and N-subtyping and type B lineage determination ............................. 31 
Virus isolation ......................................................................................................................................... 31 
Strain characterisation ............................................................................................................................. 31 
Antigenic characterisation ........................................................................................................................ 31 
Genetic characterisation .......................................................................................................................... 32 
Antiviral susceptibility testing ................................................................................................................... 32 
Genetic antiviral susceptibility testing ....................................................................................................... 32 
Phenotypic antiviral susceptibility testing ................................................................................................... 32 
Accreditation .......................................................................................................................................... 33 
References .................................................................................................................................................. 34 
Annexes: extra tables and figures .................................................................................................................. 37 

Figures 

Figure 1. Calendar days between starting molecular testing and return of results for molecular detection, typing 
and subtyping/lineage determination ............................................................................................................... 9 
Figure 2. Overview of cumulative performance scores for molecular detection, typing (A/B) and type A H-
subtyping. ................................................................................................................................................... 11 
Figure 3. Overview of cumulative performance scores for molecular detection, typing (A/B), type A H-and N-
subtyping and type B lineage determination ................................................................................................... 11 
Figure 4. Calendar days used for virus isolation and antigenic and genetic characterisation ................................ 13 
Figure 5 Overview of cumulative performance scores for virus isolation ............................................................ 14 
Figure 6. Overview summarising reported antigenic characterisation categories by specimen code ...................... 16 
Figure 7. Overview of cumulative performance scores for genetic characterisation ............................................. 17 
Figure 8. Calendar days used for genetic and phenotypic antiviral susceptibility testing ...................................... 18 
Figure 9. Overview of cumulative performance scores for genetic antiviral susceptibility determination, amino acid 
substitution analysis and interpretation of this analysis .................................................................................... 20 
Figure 10. Overview of reported IC50 values by specimen code ........................................................................ 23 
Figure 11. Overview of calculated IC50 fold-change values for EISN_AV18-01 and 02 specimens ......................... 24 
Figure 12. Overview of cumulative performance scores for phenotypic antiviral susceptibility determination ......... 25 
Annex Figure 1. Antigenic cartography maps created based on HI-assay data for A(H1N1)pdm09 (A), B/Victoria (C) 
and B/Yamagata influenza viruses (D) and based on virus neutralisation data for A(H3N2) influenza virus (B) ...... 37 
Annex Figure 2A. Phylogenetic tree of full HA of A(H1N1)pdm09 influenza viruses with common amino acid 
changes for viruses at indicated branch ......................................................................................................... 38 
Annex Figure 2B. Phylogenetic tree of full HA of A(H3N2) influenza viruses with common amino acid changes for 
viruses at indicated branch ........................................................................................................................... 39 
Annex Figure 2C. Phylogenetic tree of full HA of B/Victoria influenza viruses with common amino acid changes for 
viruses at indicated branch ........................................................................................................................... 40 

iii 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 2D. Phylogenetic tree of full HA of B/Yamagata influenza viruses with common amino acid changes 
for viruses at indicated branch ...................................................................................................................... 41 
Annex Figure 3. Neuraminidase inhibition curves for determination of IC50 for oseltamivir of B/Victoria NA- E105K 
mutant (A) and A(H1N1)pdm09 N1-H275Y(mixH/Y) mutant (B) using native virus and inactivated by Triton X-100 
or formalin .................................................................................................................................................. 42 
Annex Figure 4. Molecular methodologies used by 55 laboratories in detection of influenza virus types A and B (A), 
type A H-subtyping (B), type A N-subtyping (C) and type B lineage determination (D) ....................................... 48 
Annex Figure 5. Methodologies used by 44 laboratories in virus isolation; type of cells or eggs for A(H1N1)pdm09 
(A), A(H3N2) (B) and type B viruses (C); assay type used for confirmation of virus growth (D), type of red blood 
cells used in haemagglutination assay (E) ...................................................................................................... 51 
Annex Figure 6. Source and species of sera used for antigenic characterisation in HI assay and virus neutralisation  56 
Annex Figure 7. Methods used for genetic antiviral susceptibility determination ................................................. 64 
Annex Figure 8. Overview of reported IC50 values by method and participant ID of laboratories participating in the 
phenotypic antiviral susceptibility determination challenge ............................................................................... 65 
Annex Figure 9. Overview of calculated IC50 fold-change values by method and participant ID of EISN_AV18-01 
and 02 specimens ........................................................................................................................................ 70 

Tables 
Table 1. Panel composition, 2018 EEQIAP ........................................................................................................ 5 
Table 2. Expected results panel, EEQIAP 2018 molecular detection, virus isolation and antigenic and genetic 
characterisation ............................................................................................................................................. 7 
Table 3. Expected results panel, EEQIAP 2018 antiviral susceptibility .................................................................. 8 
Table 4. Breakdown of number of participants by challenge type........................................................................ 9 
Table 5. Overview of molecular detection, typing, type A H- and N-subtype and type B lineage determination 
results by specimen code .............................................................................................................................. 10 
Table 6. Overview of molecular detection, typing, type A H- and N-subtype and type B lineage determination 
results by challenge type .............................................................................................................................. 12 
Table 7. Overview of virus isolation results by specimen code .......................................................................... 13 
Table 8. Overview summarising reported genetic characterisation categories by specimen code .......................... 18 
Table 9. Overview summarising the reported identified amino acid substitutions associated with reduced antiviral 
susceptibility by specimen code ..................................................................................................................... 19 
Table 10. Overview summarising reported interpretation of amino acid substitution identification associated with 
reduced antiviral susceptibility by specimen code ............................................................................................ 19 
Table 11. Overview of phenotypic antiviral susceptibility testing results by specimen code .................................. 21 
Table 12. Methodologies used by laboratories to determine and evaluate IC50 values ......................................... 22 
Table 13. Summary of survey on laboratory accreditation and participation in external quality assessment 
programmes ................................................................................................................................................ 25 
Annex Table 1. List of participants ................................................................................................................. 43 
Annex Table 2. Overview in which challenge type each participating laboratory participated ............................... 45 
Annex Table 3. Overview of molecular detection and typing and type A H-subtype, type A N-subtype and type B 
lineage determination results by participant with performance score and used methodology ............................... 46 
Annex Table 4. Overview of virus isolation results with performance score and used methodology ...................... 49 
Annex Table 5. Overview of virus antigenic characterisation results with used methodology ............................... 52 
Annex Table 6. Overview of genetic characterisation results with performance score and used methodology ........ 57 
Annex Table 7. Overview of genetic antiviral susceptibility testing results with performance score and used 
methodology ............................................................................................................................................... 61 
Annex Table 8. Overview of phenotypic antiviral susceptibility testing results with performance score and used 
methodology (assay type only)...................................................................................................................... 71 

iv 

 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Abbreviations 

AANI 
AAHRI 
AARI 
AV18 
CDC 
CPA 
CPE 
Ct 
EEIQAP 
EISN 
EISNINF_MD18 

EISNINF_VI18 

EISNINF_VS18 

EISS 
ELISA 
EQA 
EQAP 
ErasmusMC 
ERLI-Net 
GISAID 
GISRS 
HA 
HI 
HRI 
IC50 
INF18 

No amino acid substitution previously associated with (highly) reduced inhibition 
Amino acid substitution previously associated with highly reduced inhibition 
Amino acid substitution previously associated with reduced inhibition 
Influenza antiviral susceptibility determination specific specimens of the EEIQAP 2018 panel 
Centers for Disease Control and Prevention 
Clinical Pathology Accreditation 
Cytopathic effect 
Cycle threshold 
European External Influenza Quality Assessment Programme 
European Influenza Surveillance Network 
Programme in EEIQAP 2018 comprising molecular detection, typing, type A H- and N-
subtyping and type B lineage determination 
Programme in EEIQAP 2018 comprising virus isolation and antigenic and genetic 
characterisation 
Programme in EEIQAP 2018 comprising antiviral (neuraminidase inhibitors) susceptibility 
determination 
European Influenza Surveillance Scheme 
Enzyme linked immunosorbent assay 
External quality assessment 
External quality assessment project 
Erasmus Medical Centre 
European Reference Laboratory Network for Human Influenza 
Global Initiative on Sharing All Influenza Data 
Global Influenza Surveillance and Response System 
Haemagglutinin 
Haemagglutination inhibition 
Highly reduced inhibition 
50% inhibitory concentration 
Influenza virus detection, typing, subtyping and lineage determination, virus isolation and 
genetic and antigenic characterisation and antiviral susceptibility determination specimens of 
EEIQAP 2018 panel 
Interquartile range 
Information Technology EQA Management System 
Madin-Darby Canine Kidney 
20-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid 
Neuraminidase 
Naval Medical Research Unit 
Nucleic acid amplification technologies 
Next-generation sequencing 
Normal inhibition 
National influenza centre 
No interpretation possible due to partial NA segment information 
Nanomolar 
Not performed 
Polymerase chain reaction 
Public Health England 
Quality Control for Molecular Diagnostics 
Red blood cells 
Reduced inhibition 
National Institute for Public Health and the Environment 
Reverse transcription polymerase chain reaction 
Human alpha-2,6-sialyltransferase 
Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek 
Single nucleotide polymorphism 
Standard operating procedure 
50% tissue culture infectious dose 
The European Surveillance System 
Virus neutralisation 
Virus transport medium 
WHO Collaborating Centre for Reference and Research on Influenza 

IQR 
ITEMS 
MDCK 
MUNANA 
NA 
NAMRU 
NAT 
NGS 
NI 
NIC 
NIP 
nM 
NP 
PCR 
PHE 
QCMD 
RBC 
RI 
RIVM 
RT-PCR 
SIAT 
SKML 
SNP 
SOP 
TCID50 
TESSy 
VN 
VTM 
WHO CC 
WHO CC London  WHO CC at the Francis Crick Institute Worldwide Influenza Centre 

v 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Executive summary 

Influenza epidemics occur every winter with high impact on disease burden, hospitalisations and excess mortality 
in countries in the WHO European Region. To understand the characteristics of circulating influenza viruses during 
seasonal epidemics, virological influenza surveillance is performed and detected viruses are further characterised at 
national influenza centres (NICs) that are part of the wider network of the WHO Global Influenza Surveillance and 
Response System (GISRS). External quality assessments (EQAs) are important instruments in assessing the quality 
of the generated data that are reported nationally and internationally through The European Surveillance System 
(TESSy) and presented in Flu News Europe, the joint ECDC-WHO Regional Office for Europe weekly influenza 
update (http://www.flunewseurope.org). 

From March through July 2018, a European external influenza quality assessment programme (EEIQAP) exercise 
was held for NICs and other national influenza reference laboratories in the WHO European Region. The exercise 
covered influenza virus molecular detection, isolation, strain characterisation and antiviral susceptibility testing. It 
was the fifth detection, isolation and strain characterisation panel and the fourth antiviral susceptibility testing 
panel that have all been organised with the support of the European Union (EU) and ECDC. EEIQAP 2018 was 
organised by the contractor RIVM for the European Reference Laboratory Network for Human Influenza (ERLI-Net; 
previously called Community Network of Reference Laboratories for Human Influenza in Europe; CNRL) with the 
support of ECDC. Participation in EEIQAP 2018 by laboratories in countries outside the EU/European Economic Area 
(EU/EEA) region was supported by the WHO Regional Office for Europe. For the first time, results are jointly 
presented for the WHO European Region as a whole. 

The objectives of the exercise were to collect information on the capacity and capability of the network regarding 
rapid molecular influenza virus detection, A/B typing, type A H- and N-subtyping and B lineage determination by 
reverse transcription polymerase chain reaction (RT-PCR) influenza virus isolation and strain characterisation using 
antigenic and/or genetic techniques and antiviral susceptibility testing using genetic and/or phenotypic techniques, 
within a defined reporting timeframe. 

This exercise aimed to provide participants with an independent assessment of their own laboratory’s performance 
and a comparison with other reference laboratories for influenza in the region. Additionally, performance in EEIQAP 
2018 provided a validity check of data reported to TESSy. All 63 reference laboratories for influenza in the region 
were actively approached to participate and 56 laboratories in 46 of the 50 countries with at least one reference 
laboratory for influenza participated in at least one of the three components of EEIQAP 2018. 

The panel consisted of seven simulated clinical specimens containing variable amounts of live virus (one 
A(H1N1)pdm09 clade 6B.1, two A(H3N2) clade 3C.2a1, two B/Victoria clade 1A, two B/Yamagata clade 3), one 
negative simulated clinical specimen and two inactivated specimens, one containing A(H1N1)pdm09 N1-275YH mix 
showing highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and one containing B/Victoria 
NA-E195K showing reduced inhibition by oseltamivir and highly reduced inhibition by zanamivir. 

One of the 56 laboratories performed virus detection by virus isolation only. As with previous panels, the 
performance of 55 laboratories for rapid molecular detection, A/B typing and type A H-subtyping was of high 
accuracy, with 52 (95%) of these laboratories reporting correct results. Of the 21 laboratories that reported on the 
type A N-subtype, all reported correct results. Except for one laboratory that reported all results in the incorrect 
random order, 45/46 (98%) laboratories that reported on the type B lineage returned correct results. One 
laboratory reported a false positive result detecting type B influenza virus in the negative specimen and another a 
false negative result for an A(H3N2) influenza virus containing specimen. These results highlight the wide capability 
to perform molecular diagnostics across the region and the high quality of data generated by the national reference 
laboratories reported to TESSy. 

Of the 44 laboratories participating in the virus isolation component, 27 (61%) reported correct results. This low 
percentage was mainly caused by the failure of 17/44 (39%) laboratories to isolate type B influenza viruses from 
one or more of the four type B influenza virus containing specimens. In 2013, when the panel had also included 
two type B viruses of each lineage (but different from the 2018 panel), only 4/32 (13%) laboratories failed at 
influenza type B virus isolation. The percentage of laboratories capable of isolating the virus from the four type B 
influenza virus containing specimens in the EEIQAP 2018 ranged from 30/44 (68%) for the specimen with the 
lowest isolation rate through 40/44 (91%) for the specimen with the highest isolation rate. This low performance 
was possibly linked to a low sensitivity of the applied methods as the specimens with the lowest amount of virus 
posed greater difficulties. All participants except one succeeded in isolating A(H3N2) viruses. Additionally, three 
laboratories could not isolate the virus from the specimen with A(H1N1)pdm09 virus. The decreased number of 
isolated viruses limited the number of viruses that could be antigenically characterised and tested phenotypically 
for antiviral susceptibility. 

Antigenic strain characterisation reported by 32 laboratories was fairly concordant across laboratories for the four 
type B viruses, ranging from 13/23 (57%) for the specimen with lowest concordance to 20/25 (80%) for the 

1 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

specimen with highest concordance and the A(H1N1)pdm09 virus (23/30, 77%). The reporting of the antigenic 
group for A(H3N2) viruses caused some uncertainties as to which group the viruses belonged. Similar numbers of 
participants reported the A(H3N2) panel viruses as ‘old’ A/Hong Kong/4801/2014-like vaccine strain (TESSy 
reporting category for 2016/17 and 2017/18 seasons) or the ‘new’ A/Singapore/INFIMH-16-0019/2016-like vaccine 
strain (TESSy reporting category for 2017/18 season). The result for A(H3N2) viruses highly likely reflects the 
diversity in reagents used for haemagglutination inhibition (HI) assays (source of sera, source species of red blood 
cells (RBCs), use of oseltamivir to inhibit neuraminidase-associated haemagglutination that both viruses showed) 
as only two laboratories used virus neutralisation. 

Genetic strain characterisation through sequencing of the haemagglutinin (HA) genome segment was more 
straightforward. Seventy-three percent (19/26) of reporting laboratories provided correct results. Five laboratories 
assigned A(H3N2) viruses to older clade 3C.2a1 or even clade 3C.2a representative strains, indicating some 
difficulty in interpreting phylogenetic and amino acid substitution data. Similarly, four laboratories incorrectly 
allocated B/Victoria viruses to the new deletion subgroups characterised by a 2-amino acid deletion at 162-163 or a 
3-amino acid deletion at 162-164 in the HA as the test viruses did not have these deletions. One laboratory 
allocated a B/Yamagata virus to a B/Victoria category. 

The results for antigenic and genetic characterisation indicate that weekly analysis and interpretation of these data 
during the season requires some caution. However, accession numbers for HA sequences are also reported to 
TESSy. They offer an opportunity for additional analysis and validation of the genetic category data reported. 

The results for detection of amino acid substitutions associated with (highly) reduced inhibition (HRI or RI) by the 
neuraminidase inhibitors oseltamivir and zanamivir and the results for IC50 determination of wild-type viruses and 
viruses with (H)RI amino acid substitutions were broadly encouraging. All 28 laboratories participating in the 
antiviral susceptibility component of EEIQAP 2018 correctly identified the A(H1N1)pdm09 N1-H275Y amino acid 
substitution, although only 17/28 (61%) identified the N1-275YH mixed nature of the specimen. Of the 25 
laboratories testing the B/Victoria NA-E105K specimen, 23 (92%) identified the amino acid substitution. One 
laboratory erroneously reported the R292K HRI substitution as both wild-type A(H3N2) viruses and two 
laboratories erroneously reported the H275Y HRI substitution as wild-type A(H1N1)pdm09 virus. A major challenge 
was the correct interpretation of the amino acid composition analysis data in the context of the level of testing, 
e.g., single nucleotide polymorphism (SNP) test versus full-length neuraminidase (NA) genome segment 
sequencing. A definitive conclusion can only be drawn when detecting a substitution associated with (highly) 
reduced inhibition or sequencing full-length NA. Of the 28 laboratories, 16 (57%) reported one or more 
interpretation errors. These data need to be interpreted with some caution. Accession numbers for NA sequences 
are also reported to TESSy and offer an opportunity for additional analysis and validation of the reported amino acid 
composition data. 

Phenotypic antiviral susceptibility results from the 23 laboratories that participated in this component of the EEIQAP 
2018 were highly accurate for all specimens except one, including a (highly) reduced inhibited phenotype for the 
A(H1N1)pdm09 N1-275YH mixture-containing specimen. The exception was the B/Victoria NA-E105K-containing 
specimen tested for oseltamivir susceptibility, for which seven laboratories reported a too low IC50 and hence a 
‘normal inhibited’ instead of ‘reduced inhibited’ phenotype. This result is partly due to the use of chemiluminescent 
neuraminidase inhibition assays; the four laboratories that used this type of assay reported this artefact. Three 
laboratories incorrectly identified one wild-type B/Victoria that had been isolated in the laboratory with a ’reduced 
inhibited’ phenotype. However, one laboratory reported that this was caused by outgrowth of a minority variant 
with NA-T106P amino acid substitution present in the simulated clinical specimen. Virus isolation-induced reduced 
inhibition is a phenomenon laboratories should be aware of. Only one IC50 data interpretation error was made 
classifying the A(H1N1)pdm09 N1-275YH mix as ‘reduced inhibited’ instead of ‘normal inhibited’ by zanamivir. 

The majority of laboratories (43/55, 78%) are ISO 15189-accredited or are preparing to apply for it and about 90% 
are required to participate in EQAs for detection and A/B typing, type A subtyping and B lineage determination to 
obtain and maintain this accreditation. However, only 44%-67% of laboratories require EQA participation for the 
other tests challenged in EEIQAP 2018. Adding all these tests to the scope of ISO 15189 accreditation could 
provide an additional instrument for systematically addressing issues identified by EQA. 

The participating laboratories were provided the expected results with some background information as soon as 
the last laboratory had reported its results. After the analysis of the results, the final draft version of the current 
report was provided to the individual laboratories together with the participant code in order to assess their own 
results in the context of those achieved by other laboratories. 

In conclusion, the overall performance of the participating laboratories was good. Certain laboratories are 
encouraged to enhance their testing performance by evaluating the sensitivity and specificity of the assays in place 
and apply necessary updates accordingly. Other issues (e.g. incorrect translation into TESSy categories) will be 
addressed jointly with ECDC and the WHO Regional Office for Europe through training or adapting validation and 
analysis of data captured in TESSy (e.g. making better use of reported HA and NA sequences and 
organising/providing training, especially focusing on sequence analysis and interpretation and reporting). 

2 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

1 Introduction 

Influenza viruses cause a highly contagious acute respiratory disease that can spread rapidly, causing important 
morbidity and mortality in Europe. Influenza viruses evolve rapidly from season to season through point mutations, 
leading to genetic drift that sometimes results in antigenic drift. The segmented nature of the influenza genome 
also makes genomic reassortment an important mechanism for producing genetic diversity, which may lead to 
antigenic shift if new H- and/or N-subtypes are introduced to which humans are vulnerable. This process is 
particularly important in influenza A virus because of its potential to generate new pandemic strains [7–8]. 

Early detection and characterisation of circulating influenza viruses is of great importance for timely risk 
assessment, treatment recommendations and vaccine formulation. The laboratory network responsible for virologic 
surveillance of influenza in the WHO European Region is part of the WHO Global Influenza Surveillance and 
Response System (GISRS) [1]. The network consists of national influenza laboratories in 50 countries in the region, 
a WHO Collaborating Centre for Reference and Research on Influenza at the Francis Crick Institute Worldwide 
Influenza Centre, London, United Kingdom (WHO CC London), a WHO Essential Regulatory Laboratory (ERL) at the 
National Institute for Biological Standards and Control, Potters Bar, United Kingdom [2] and three WHO H5 
reference laboratories in France, Russia and the United Kingdom [3]. National influenza laboratories in 43 countries 
in the WHO European Region are recognised by WHO as national influenza centres (NICs) [4] and laboratories in 
30 countries of the EU/EEA participate in the European Reference Laboratory Network for Human Influenza (ERLI-
Net) coordinated by ECDC [5]. The European Influenza Surveillance Network (EISN), which includes ERLI-Net, is a 
dedicated network for the epidemiological and virological surveillance of influenza in the EU/EEA. 

The introduction of nucleic acid amplification technologies (NAT) has led to the replacement of less sensitive rapid 
antigen detection assays by molecular methods that can rapidly detect influenza viruses with high sensitivity and 
specificity. These new techniques allow for simultaneous identification of the type of virus (A or B), the H- and N-
subtype of influenza A viruses (H1N1, H3N2, H7N9) and the genetic lineage of influenza B viruses (B/Victoria/2/87-
like: Victoria and B/Yamagata/16/88-like: Yamagata). As a result, these tests assume great practical relevance in 
diagnosing individual patients and surveillance. 

Through phylogenetic and amino acid substitution analysis, it is possible to genetically characterise the 
haemagglutinin genome segment of influenza viruses and categorise circulating viruses in genetic (sub)clades or 
(sub)groups. This provides data on the evolution and possible emergence of variants that may escape (vaccine-
induced) immunity, i.e. the match of vaccine strains with circulating strains, and on known markers for increased 
virulence. Similarly, genetic characterisation of the neuraminidase genome segment provides useful information on 
known markers for (highly) reduced inhibition by neuraminidase inhibitors (oseltamivir and zanamivir). However, 
the ability to accurately determine the antigenic susceptibility profile of an influenza virus still requires the ability to 
isolate the virus in cell culture or embryonated eggs and carry out serological tests (haemagglutination inhibition 
[HI] or virus neutralisation [VN] assays). Likewise, virus isolates are phenotypically tested (neuraminidase enzyme 
activity inhibition assays) to measure their level of susceptibility to neuraminidase inhibitors. 

It is essential to assess such technologies through effective quality control to ensure the reliability and 
comparability of results reported to physicians and disease surveillance systems at the national and European levels 
[9]. An integral part of quality control is performed through external quality assessments (EQAs), which provide a 
means of independent and objective laboratory performance evaluation. Starting with the predecessors of EISN, 
the influenza laboratory network in Europe has performed EQA studies on all aspects of laboratory influenza 
surveillance as described above. The first was performed in 2000, with antiviral susceptibility testing added in 2010 
[10–12]. The European influenza EQA programme was the first of its kind to include virus isolation, strain 
characterisation and antiviral susceptibility determination. Molecular EQA is covered bythe WHO external quality 
assessment programme (EQAP) for NICs since 2007 [13]. In the past, the European Influenza Surveillance Scheme 
(EISS) also participated in other international programmes that included human and zoonotic avian influenza virus 
subtypes [14]. Antiviral susceptibility determination has been added on an optional basis to the WHO EQAP since 
2013, but only targeting A(H1N1)pdm09 NA-H275Y [13]. There is no international EQA programme available for 
virus isolation and antigenic and genetic strain identification, although the first virus isolation EQA was reported 
from the WHO South East Asia and Western Pacific Regions in 2017 [15]. However, that EQA did not include strain 
identification through antigenic or genetic characterisation. Hence, the European External Influenza Quality 
Assessment Programme (EEIQAP) still fills a gap integrating all aspects of laboratory influenza surveillance in one 
EQA panel: molecular detection, typing, type A H- and N-subtyping and type B lineage determination, virus 
isolation, antigenic and genetic strain characterisation and antiviral susceptibility determination. These comprise all 
aspects of routine influenza surveillance data published in Flu News Europe, the joint ECDC-WHO Regional Office 
for Europe weekly influenza online update (http://www.flunewseurope.org). 

In 2018, a framework contract with ECDC was put in place for EEIQAP covering the period from 2017–2021 to 
ensure the reliability and comparability of results reported to TESSy and identify needs for improvement in 
laboratory influenza surveillance and diagnostic capability. An Agreement for Performance of Work with the WHO 

3 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Regional Office for Europe ensured the participation of NICs in countries outside the EU/EEA in EEIQAP 2018. For 
the first time, results are jointly presented for the WHO European Region as a whole. 

Objectives 
The goal of ECDC-supported EQAs is to appraise the proficiency of public health microbiology laboratories in using 
microbiological test methods that underpin capabilities in the following areas [9]: 

• 

• 
• 
• 

diagnostic confirmation of disease for reporting to TESSy in accordance with EU case definitions for 
52 notifiable diseases and antimicrobial resistance 
outbreak detection, investigation and response 
control of communicable diseases (e.g., tuberculosis isolation and treatment); and 
preparedness (e.g., avian influenza viruses). 

The operational public health objectives of ECDC-supported EQAs are [9]: 

• 
• 

assessment of the quality and comparability of surveillance data reported by EU/EEA Member States; and 
support of threat detection capabilities for emerging diseases, epidemic diseases and drug resistance. 

These objectives are consistent with the laboratory and public health objectives of EQAs outlined in the WHO 
laboratory quality management system handbook [17]. 

Translated into operational procedures in the laboratory, the main purposes of EQAs, as also intended for the 
current EEIQAP 2018, include: 

• 
• 
• 
• 
• 

• 

assessment of general performance standards 
assessment of the effects of analytical procedures (method, principles and techniques) 
evaluation of individual laboratory performance 
identification and justification of problem areas 
provision of continuing (self-) education (testing against specimens of known status) and comparison with 
other laboratories; and 
identification of training needs. 

This report presents the results of EEIQAP 2018 for influenza reference laboratories in the WHO European Region, 
designed and prepared by the contractor and funded by ECDC and the WHO Regional Office for Europe. 

The major objective of EEIQAP 2018 was to assess the performance of individual influenza reference laboratories in 
the following areas: 

• 

• 

• 

rapid detection by reverse transcription polymerase chain reaction (RT-PCR) or other NAT, including typing, 
type A H- and N-subtyping and type B lineage determination, within a defined reporting timeframe of seven 
calendar days 
virus isolation, including follow-up strain characterisation by haemagglutination inhibition (HI) assay or virus 
neutralisation (VN) and/or sequencing within a defined reporting time frame of 36 calendar days; and 
determination of susceptibility to neuraminidase inhibitors oseltamivir and zanamivir by genotypic and/or 
phenotypic methods within a defined reporting time frame of 36 calendar days. 

Although objective 1 is covered by WHO EQAP and other available EQA schemes and objective 3 is partly covered 
by WHO EQAP, objective 2 is not covered by any widely available EQA scheme and none integrate all three 
objectives in one comprehensive panel. 

A secondary objective was to collect information on the (desired) accreditation status of laboratories and how EQA 
fits into this accreditation. 

4 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

2 Study design 

2.1 Organisation 
The EEIQAP panel was designed by staff from the contractor, National Institute for Public Health and the 
Environment (RIVM), and the final composition was agreed upon with ECDC. The panel was prepared and tested 
by the Respiratory Viruses Group of the Department Emerging and Endemic Viruses, Division of Virology, Centre for 
Infectious Diseases Research, Diagnostics and Laboratory Surveillance, RIVM, Bilthoven, the Netherlands. Further 
pretesting was performed by subcontractors Erasmus Medical Centre (ErasmusMC), Department of Viroscience, 
Rotterdam, the Netherlands and Centre National de Référence Virus des Infections Respiratoire, Laboratoire de 
Virologie, Institut des Agents Infectieux, Lyon, France. The panel contents, frozen on dry ice, were distributed to 
participants from March to July 2018 by specialist courier organised by subcontractor QCMD. Participants submitted 
results to a web-based database, Information Technology EQA Management System (ITEMS), that is hosted by 
QCMD and was adapted to the needs of EEIQAP 2018. 

2.2 Panel composition, preparation and validation 
The EEIQAP panel consisted of eight simulated clinical specimens containing live influenza viruses of subtypes that 
currently circulate or have recently circulated in humans, including human influenza viruses A(H1N1)pdm09 and 
A(H3N2) and both genetic lineages of influenza B viruses (Table 1). One negative specimen with no virus 
completed the live virus part of the panel. In addition, two specimens of two vials each specifically designed for 
antiviral susceptibility testing were included in the panel that contained inactivated virus with amino acid 
substitutions causing HRI or RI by the neuraminidase inhibitors oseltamivir and/or zanamivir (Table 1). 

Table 1. Panel composition, 2018 EEQIAP 

Specimen 
code EISN_ 

Matrix1 

Strain; antiviral amino acid substitution; GISAID 
isolate accession number2 

Type and 
subtype/lineage 

Ct 
value3 

TCID50/
ml4 

INF18-01 
INF18-02 
INF18-03 
INF18-04 
INF18-05 
INF18-06 
INF18-07 
INF18-08 

AV18-01 

AV18-01 

AV18-02 

AV18-02 

A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 
A549 cells in VTM 

VTM; Triton X-100-
inactivated 

VTM; heat-
inactivated 

VTM; formalin-
inactivated 
VTM; heat-
inactivated 

A/Netherlands/751/2017; none; EPI_ISL_270970 
B/Netherlands/2424/2017; none; EPI_ISL_255661 
B/Netherlands/076/2014; none; EPI_ISL_166326 
A/Netherlands/502/2017; none; EPI_ISL_247427 
No influenza virus 
B/Netherlands/2423/2017; none; EPI_ISL_255660 
A/Netherlands/757/2017; none; EPI_ISL_270971 
B/Netherlands/3066/2015; none; EPI_ISL_233380 
Mixture of A/Netherlands/3165/2017 NA-275Y 
(EPI_ISL_312679) and A/Netherlands/502/2017 NA-
275H A(H1N1)pdm09 
Mixture of A/Netherlands/3165/2017 NA-275Y 
(EPI_ISL_312679) and A/Netherlands/502/2017 N1-
275H A(H1N1)pdm09 

B/Netherlands/2518/2016; NA-E105K; EPI_ISL_239749 

B/Netherlands/2518/2016; NA-E105K; EPI_ISL_239749 

A(H3N2) 
B/Yamagata 
B/Victoria 
A(H1N1)pdm09 
n/a 
B/Victoria 
A(H3N2) 
B/Yamagata 

A(H1N1)pdm09 
(provided) 

A(H1N1)pdm09 
(provided) 

B/Victoria 
(provided) 
B/Victoria 
(provided) 

IC50 
(nM)5 
Z 
O 

0.10  0.30 
3.0 
24 
22 
3.1 
0.39  0.44 
n/a 
n/a 
3.2 
15 
0.13  0.25 
1.4 
14 

15.92 
18.04 
17.15 
16.82 
n/a 
19.09 
25.79 
16.57 

2795 
2795 
559 
1250 
n/a 
167 
4180 
374 

12.35 

n/a 

135  0.36 

13.15 

n/a 

n/a 

n/a 

15.94 

n/a 

375  338 

20.53 

n/a 

n/a 

n/a 

1VTM: virus transport medium. 
2For all panel members, HA and NA segments are available in GISAID; for 5 full genomes and for 1 full genome except PB2. 
3Matrix-gene based RT-PCR for type A influenza viruses and non-structural gene based RT-PCR for type B influenza viruses. 
450% tissue culture infectious dose (TCID50) determined on Madin-Darby Canine Kidney (MDCK)-MIX cells, MDCK-I and MDCK 
cells stable expressing human alpha-2,6-sialyltransferase (MDCK-SIAT) in 96-well plate, static incubation and end-point 
determination of cytopathic effect (CPE) in each well; this produces a valuable estimate for relative comparison of the amount of 
live virus in the panel members used for virus isolation, but likely produces an underestimate of the true number of live viruses in 
the panel members as using routine rolling tube virus isolation in contractors’ laboratory dilution of these panel members was 
positive in virus isolation well below the 0 TCID50 dilution. n/a: not applicable. 
5IC50: 50% inhibitory concentration; O: oseltamivir; Z: zanamivir; n/a: not applicable. 

All viruses were selected based on known antigenic (Annex Figure 1), genetic (Annex Figure 2) and antiviral 
susceptibility characteristics (Table 1) previously determined at RIVM and ErasmusMC. All viruses were grown in 
monolayers of MDCK-MIX cells, consisting of MDCK-I cells and MDCK cells stable expressing human alpha 2,6-
sialyltransferase MDCK-SIAT, to stocks with a sufficient concentration to prepare the required number of panels for 
distribution to the intended number of laboratories. Initially, the specimens with viruses exhibiting reduced 
inhibition by antivirals were inactivated with 1% Triton X-100 for one hour at room temperature as this procedure 
preserves neuraminidase activity and RNA quality for direct sequencing [18]. However, validation of these panel 
specimens at RIVM showed that Triton X-100 inactivation of the specimen containing the B/Victoria NA-E105K 
mutant caused an atypical inhibition curve with neuraminidase inhibitors different from that obtained with native 

5 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

virus, complicating IC50 determination (Annex Figure 3). Using 0.02% formalin inactivation for 18 hours at 37°C 
[18] of this virus, the inhibition curve was similar to that of the native live virus, although with a slightly higher IC50 
(Annex Figure 3). Therefore, specimen EISN_AV18-02 containing this virus was prepared using formalin 
inactivation. 

A mixture of wild-type and N1-H275Y A(H1N1)pdm09 virus for specimen EISN_AV18-01 was prepared using the 
wild-type specimen EISN_INF18-04 virus to be able to compare the IC50 results of specimen EISN_AV18-01 with 
that of the wild-type specimen. Triton X-100 inactivation shifted the IC50 of the mixture for oseltamivir slightly to a 
higher value, but still with an inhibition curve having a typical slope for a mixture (Annex Figure 3). The live virus 
specimens were diluted to a TCID50 high enough for successful virus isolation in static virus isolation procedures in 
microtitre plates (Table 1) and the inactivated virus specimens were diluted to a neuraminidase enzyme activity 
high enough for direct use in phenotypic antiviral susceptibility testing. The live virus specimens were prepared in a 
virus transport medium (VTM) with a final concentration of 1x105/ml adenocarcinomic human alveolar basal 
epithelial (A549) cells to simulate a real clinical specimen. All panel members were aliquoted and stored frozen at -
80°C until dispatch to QCMD for further distribution. One panel was thawed and pretested at RIVM using in-house 
methods and panels with random numbering of specimens different from the numbering of the final panel were 
sent frozen on dry ice to the two independent laboratories for pretesting. Pretesting by these laboratories also 
included pretesting of the online reporting system ITEMS at QCMD. During pretesting of the inactivated virus 
specimens and follow-up study at RIVM, it became clear that the inactivation procedure using Triton X-100 or 
formalin preserved the neuraminidase activity well, but that with longer storage times at -80°C, one-step full-
length sequencing of the neuraminidase segment became cumbersome. Using heat inactivation for one hour at 
60°C and longer storage time at -80°C worked well for one-step full-length neuraminidase segment sequencing. 
Because heat inactivation inactivates the neuraminidase [18], the specimens for antiviral susceptibility testing were 
inactivated by Triton X-100 or formalin for phenotypic testing and separately by heat for genotypic testing. The 
final panels were shipped frozen on dry ice to the laboratories from March to early July 2018. Expected results for 
all panel members are listed in Tables 2 and 3. For a final check on the viability of live viruses in the panel, one 
panel stored frozen at -80°C similar to the distributed panels was thawed at the end of September 2018 and the 
specimens on MDCK-MIX cells at the RIVM were cultured. All specimens with live virus became positive within 
seven calendar days, confirming viability after long storage time and freeze-thaw cycles similar to that in the 
participating laboratories. 

2.3 Participation 
Participation in EQAs is one of the key tasks of ECDC ERLI-Net laboratories [5] and plays a key role in 
strengthening the WHO GISRS’ diagnostic capacity and preparedness to effectively respond to influenza 
outbreaks [1]. Participation in components of EEIQAP 2018 for which NICs/national influenza reference laboratories 
in the WHO European Region routinely report data to TESSy was strongly recommended. All laboratory contact 
points of ERLI-Net and NICs outside the EU/EEA and the WHO CC in London were notified of the EEIQAP 2018 
exercise in advance on 19 January (EU/EEA by ECDC) and 22 January 2018 (non-EU/EEA by WHO) and asked to 
sign up by 10 February 2018 for participation in any of the four areas: molecular detection, virus isolation, virus 
characterisation and antiviral susceptibility determination. They were actively contacted if no response was 
received. A final list of participants in EEIQAP 2018 can be found in Annex Table 1. 

2.4 Testing 
All participating laboratories were expected to perform the molecular detection, typing, type A H- and N-subtyping 
and type B lineage determination components of EEIQAP 2018 using the tests routinely used in the laboratory, the 
weekly results of which are reported to TESSy. If usual laboratory procedures included virus isolation and strain 
characterisation and/or antiviral susceptibility testing and resulting data were reported to TESSy, it was strongly 
recommended to also complete the corresponding parts of EEIQAP 2018. For any tests used, the laboratories were 
asked to provide detailed information. 

The laboratories were instructed to test for: 

EISNINF_MD18 programme – molecular detection, typing, type A H- and N-subtyping and type B lineage 
determination; specimens EISN_INF18-1 through -8) using RT-PCR or other NAT 
EISNINF_VI18 programme – virus isolation and antigenic and genetic characterisation; specimens 
EISN_INF18-1 through -8. Virus isolation was to be performed in cell culture and/or embryonated eggs. For 
antigenic characterisation, the viruses had to be isolated and propagated first, followed by HI assay or VN. 
For genetic characterisation, the simulated clinical specimen or virus isolate had to be sequenced by Sanger 
or next-generation sequencing (NGS) techniques. 
EISNINF_VS18 programme – antiviral (neuraminidase inhibitors) susceptibility determination; specimens 
EISN_AV18-1 and -2 and specimens EISN_INF18-1 through -8 by available phenotypic (neuraminidase 
enzyme activity inhibition assay) and/or genotypic (SNP RT-PCR, Sanger sequencing or NGS or 

• 

• 

• 

6 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

pyrosequencing) antiviral susceptibility determination methods. EISN_AV18 tubes labelled ‘phenotypic’ 
contained virus inactivated by validated methods that retain neuraminidase activity and could be used 
directly for phenotypic antiviral susceptibility testing. The EISN_AV18 tubes labelled ‘sequencing’ contained 
virus inactivated by heat-preserving RNA integrity and could be used directly for genotypic antiviral 
susceptibility testing, including one-step full segment Sanger or NGS. EISN_INF18-1 through -8 specimens 
first had to be isolated and propagated for phenotypic antiviral susceptibility testing. 

The manual for testing was made available to the participants through QCMD ITEMS in English and Russian. 

2.5 Data reporting 
The deadline for reporting molecular detection and typing, type A H- and N-subtyping and type B lineage 
determination results was within seven calendar days of receipt of the panel. The deadline for reporting virus 
isolation and strain characterisation and antiviral susceptibility testing results was within 36 calendar days of receipt 
of the panel. Data were reported in the web-based QCMD-owned database ITEMS adapted for collating EEIQAP 
2018 data. The documentation for reporting to TESSy for the 2016/17 and 2017/18 seasons and guidance for 
strain-based reporting of antiviral susceptibility and antigenic and genetic characterisation data were used for 
adapting ITEMS [19–21]. The expected results for each programme are displayed in Tables 2–3. 

For the molecular detection programme of EEIQAP 2018, the laboratories were asked to report type (A/B), H- and 
N-subtype for influenza A viruses and lineage for influenza B viruses using drop-down pick lists. We also asked for 
details on the tests used. 

For virus isolation, participants were asked to report whether and by which method the virus was isolated, using 
drop down-pick lists and which methods were used to confirm virus isolation in predefined categories with the 
‘other’ option. 

For strain characterisation, participants were asked to report the result of antigenic and/or genetic characterisation 
using drop-down lists with categories reflecting the TESSy categories for the 2016/17 and 2017/18 seasons. In 
addition, details were asked on the methods used for antigenic and/or genetic characterisation. 

For genotypic antiviral susceptibility testing, participants were asked to report the relevant amino acid positions 
screened as marker positions for a change in susceptibility to oseltamivir and zanamivir with the amino acid found 
in the format as requested for reporting to TESSy. 

Table 2. Expected results panel, EEQIAP 2018 molecular detection, virus isolation and antigenic and 
genetic characterisation 

Specimen code 

Programme 
EISNINF_MD18 
Type and 
subtype/lineage 

Programme EISNINF_VI18 

Virus 
isolation 

Type and 
subtype/lineage 

Antigenic category1 

Genetic category2 

EISN_INF18-01 

A(H3N2) 

Positive 

A(H3N2) 

EISN_INF18-02 

B/Yamagata 

Positive 

B/Yamagata 

EISN_INF18-03 

B/Victoria 

Positive 

B/Victoria 

EISN_INF18-04 

A(H1N1)pdm09 

Positive 

A(H1N1)pdm09 

EISN_INF18-05 

n/a 

Negative 

n/a 

EISN_INF18-06 

B/Victoria 

Positive 

B/Victoria 

EISN_INF18-07 

A(H3N2) 

Positive 

A(H3N2) 

EISN_INF18-08 

B/Yamagata 

Positive 

B/Yamagata 

A(H3) A/Singapore/INFIMH-
16-0019/2016 (H3N2)-like 
(has neuraminidase induced 
HA) 
B(Yam) lineage not attributed 
to category (low reactor 
B/Phuket/3073/2013) 

B/Brisbane/60/2008-like 
(B/Victoria/2/87 lineage) 

A(H1)pdm09 
A/California/7/2009 (H1N1)-
like 
n/a 
B(Vic) lineage not attributed 
to category; low reactor 
B/Brisbane/60/2008 
A(H3) A/Hong 
Kong/4801/2014 (H3N2)-like 
(has neuraminidase induced 
HA) 

B/Phuket/3073/2013-like 
(B/Yamagata/16/88-lineage) 

A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-16-
0019/2016 subgroup 
B(Yam)-lineage clade 3 
representative  
B/Phuket/3073/2013 
B(Vic)-lineage clade 1A 
representative 
B/Brisbane/60/2008 
A(H1)pdm09 group 6B.1 
representative 
A/Michigan/45/2015 
n/a 
B(Vic)-lineage clade 1A 
representative 
B/Brisbane/60/2008 
A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-16-
0019/2016 subgroup 
B(Yam)-lineage clade 3 
representative 
B/Phuket/3073/2013 

1Annex Figure 1 
2Annex Figure 2. 

7 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Table 3. Expected results panel, EEQIAP 2018 antiviral susceptibility 

Specimen code 

Phenotypic1 

Programme EISNINF_VS18 
Genotypic (expected result when full NA is sequenced)2 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

EISN_INF18-01 
EISN_INF18-02 
EISN_INF18-03 
EISN_INF18-04 
EISN_INF18-05 
EISN_INF18-06 
EISN_INF18-07 
EISN_INF18-08 

EISN_AV18-01 

NI 
NI 
NI 
NI 
n/a 
NI 
NI 
NI 

HRI 

NI 
NI 
NI 
NI 
n/a 
NI 
NI 
NI 

NI 

EISN_AV18-02 

RI 4 

HRI 4 

AANI 
AANI 
AANI 
AANI 
n/a 
AANI 
AANI 
AANI 

AAHRI N1-H275Y(mixH/Y) 3 

AANI 
AANI 
AANI 
AANI 
n/a 
AANI 
AANI 
AANI 

AANI 

AANI for NA-E105K based on 
published phenotypic data5 
AARI for NA-E105K based on 
phenotypic data EISN_AV18-02 
virus isolate4 

AARI for NA-E105K based on 
published phenotypic data5 
AAHRI for NA-E105K based on 
phenotypic data EISN_AV18-02 
virus isolate4 

Accession number 
NA in GISAID 
EPI1030650 
EPI991483 
EPI541780 
EPI959344 
n/a 
EPI975564 
EPI1030658 
EPI832038 
EPI1244483 
(H275Y)/EPI959344 

EPI868728 

1NI: normal inhibition (fold-change IC50; type A <10; type B <5); RI: reduced inhibition (fold-change IC50; type A ≥10 & ≤100; 
type B ≥5 & ≤50); HRI = highly reduced inhibition (fold-change IC50; type A >100; type B >50); n/a: not applicable. 
2AANI: has no amino acid substitutions previously associated with RI or HRI; AARI: has amino acid substitutions previously 
associated with RI; AAHRI: has amino acid substitution previously associated with HRI; n/a: not applicable. 
3AAHRI also correct when determined using partial NA segment sequencing, pyrosequencing or SNP RT-PCR. 
4Fold-change compared to median IC50 of recent Dutch B/Victoria viruses for oseltamivir determined at 32 (RI) and for zanamivir 
at 115 (HRI) at the contractors’ laboratory. 
5According to WHO reference table: 
http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/NAI_Reduced_Susceptibility_Marker_Table_WHO.pdf. 

Participants were also asked to provide an interpretation of the generated results using drop-down lists with the 
categories used for reporting to TESSy, taking into account the level of testing, e.g. SNP test versus full length 
neuraminidase (NA) genome segment sequencing. A ‘No interpretation for this drug possible from testing 
performed’ category due to incomplete analysis of the neuraminidase segment was added. For phenotypic antiviral 
susceptibility testing, participants were asked to report IC50 values for oseltamivir and zanamivir and provide an 
interpretation of their results using drop-down lists with the categories used for reporting to TESSy. For both 
methodologies, details to put the reported results in the context of the used methodologies were asked. Additional 
data were collected on the accreditation status of laboratories, which EQA programmes they participate in for each 
aspect of laboratory surveillance of influenza and whether these are required for accreditation. 

2.6 Data analysis 
All challenges of EEIQAP 2018 were considered ‘educational’ and therefore no pass/fail criteria were defined. A 
scoring system was used in which a correct result for a specimen scored 0 (for 0 errors). Depending on the level of 
testing, a specimen with an error reported obtained a score of 1, 2 or 3, with the maximum score for an error kept 
equal for different aspects within one challenge (Figure 2 footnote). For each challenge, each laboratory received a 
cumulative performance score by summing up the individual specimen scores. For network performance, the 
percentages of laboratories with a specific cumulative performance score were calculated and plotted in overview 
bar graphs. The detailed scoring system used for each challenge is provided in the footnotes to these graphs and 
the individual laboratory result tables in the annexes. In addition, explanations of the judgements to conclude an 
error are provided for individual specimens in the footnotes of the individual laboratory result tables in the 
annexes. If a laboratory did not perform a particular test because it is not available in that laboratory, it was not 
counted as an error. Accordingly, the individual and network cumulative scoring is not a reflection of the capability 
of the individual laboratory or the network to perform a specific test. To that end, in the overview results tables, 
the number of laboratories that have performed a specific test is shown as a denominator, overall or for individual 
panel specimens as applicable. As the same panel was used in the molecular detection and virus isolation 
challenges, laboratories had already determined the type/subtype or lineage of the viruses. Since there was no 
clear proof of confirmation of the type/subtype or lineage of isolated viruses, although reported, these results are 
neither shown nor included in the scoring. 

An ‘Expected results’ document for self-evaluation was shared with each participant through QCMD ITEMS through 
an email notification from the QCMD neutral office after the last participant had submitted its results in July 2018. 
The ‘Expected results’ document was made available in English and Russian. After the analysis of the results, the 
final draft version of the current report was provided to the individual laboratories in November 2018 through 
QCMD ITEMS. This was accompanied by email notification from the QCMD neutral office to each individual 
participant and included the participant code in order to submit their own results in the context of those achieved 
by other laboratories. 

8 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

3 Results 

3.1 Participating laboratories 
A total of 56 laboratories in 46 of the 53 countries in the WHO European Region participated in EEIQAP 2018: 35 in 
28 of the 31 EU/EEA countries and the other 21 in 18 non-EU/EEA countries. An aggregated breakdown of 
participating laboratories by challenge type is shown in Table 4 and participating laboratory in Annex Table 2. Only 
16/56 (29%) laboratories participated in the full programme. 

Table 4. Breakdown of number of participants by challenge type 

Region 

Molecular 
detection 

Virus 
isolation 

Yes  No  Yes  No 

EU/EEA (n=35)  35 
Total (n=56)  55 

0 
6 
29 
1  44  12 

Number of participants for each challenge 

Characterisation 

Antiviral susceptibility testing 

Full 
programme 

Antigenic 
only 
6 
12 

Genetic 
only 
5 
6 

Both  None 

16 
20 

8 
18 

Genetic 
only 
4 
6 

Phenotypic 
only 
0 
1 

Both  None  Yes 

18 
22 

13 
27 

13 
16 

No 

22 
40 

3.2 Molecular detection 
A total of 55 of the 56 laboratories participated in the molecular detection, typing, type A H- and N-subtype and 
type B lineage determination challenges. The median delay between starting testing and reporting of results was 
four calendar days (range 0–34) with 50% (interquartile range) of the reports delayed by 2–6 calendar days, 75% 
of the reports by six calendar days and 89% available within the seven calendar day limit (Figure 1). 

Figure 1. Calendar days between starting molecular testing and return of results for molecular 
detection, typing and subtyping/lineage determination 

Start date of molecular testing taken as proxy for date of panel arrival in the laboratory. Large horizontal bar represents median, 
whiskers represent interquartile range. 

An aggregated breakdown of the results is shown in Table 5 by specimen code, Table 6 by challenge type and 
Annex Table 3 by participating laboratory. The used methodologies are listed in aggregated form in Annex Figure 4 
and by laboratory in Annex Table 3. An overview of the percentage of laboratories by cumulative performance 
score for detection, A/B typing, type A H-subtyping and the full challenge is shown in Figures 2 and 3 and by 
participating laboratory in Annex Table 3. 

9 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Table 5. Overview of molecular detection, typing, type A H- and N-subtype and type B lineage 
determination results by specimen code 

Specimen 
code 
(EISN_) 

INF18-01 
INF18-02 
INF18-03 
INF18-04 
INF18-05 
INF18-06 
INF18-07 
INF18-08 

Expected 
results 

A(H3N2) 
B/Yamagata 
B/Victoria 
A(H1N1)pdm09 
Negative 
B/Victoria 
A(H3N2) 
B/Yamagata 

Ct value 
by 
contractor 
laboratory 
15.9 
18.0 
17.2 
16.8 
- 
19.1 
25.8 
16.6 

Correct type & A H-subtype 

Molecular detection by type, A H- and N-subtyping and B lineage determination (n=55)1 
Correct type and A H- and N-
subtype/B-lineage 
Result 
A(H3N2) 
B/Yamagata 
B/Victoria 
A(H1N1)pdm09 
Negative 
B/Victoria 
A(H3N2) 
B/Yamagata 

Result 
Negative 
A(H1)pdm09 
B/Yamagata 
B/Victoria 
B; A(H3) 
B/Yamagata 
B/Victoria 
A(H3) 

Result 
A+H3 
B 
B 
A+H1pdm09 
Negative 
B 
A+H3 
B 

n  % 
96.4 
53 
98.2 
54 
98.2 
54 
98.2 
54 
96.4 
53 
98.2 
54 
98.2 
54 
98.2 
54 

n  % 
32.7 
18 
81.8 
45 
81.8 
45 
34.5 
19 
96.4 
53 
81.8 
45 
34.5 
19 
81.8 
45 

n  % 
3.6 
2 
1.8 
1 
1.8 
1 
1.8 
1 
3.6 
2 
1.8 
1 
1.8 
1 
1.8 
1 

Incorrect result 2 

Ct: cycle threshold. 
1Methodologies used are shown in Annex Figure 4. 
2One laboratory reported results for all specimens in the incorrect random order and is therefore considered incorrect for all 
specimens. 

Most laboratories (52/55, 95% with a cumulative performance score of 0) correctly identified influenza virus type 
and type A H-subtype in all eight panel specimens (Figure 2). One laboratory reported results for all eight 
specimens in the incorrect random order, one laboratory reported a false negative and another a false positive 
result (Tables 5–6). Only 17/55 (31%) of the participants reported a fully correct result (cumulative performance 
score of 0) when the reported identification of the type A N-subtype and type B lineage were included (Figure 3). 
Many laboratories (34/55, 62%) did not report on type A N-subtyping and 9/55 (16%) did not report on type B 
lineage determination, likely because such assays were not available in the laboratory (Table 6, Annex Figure 4). Of 
the 19 laboratories that included type A N-subtyping, 18 (95%) identified the correct N subtype of the three type A 
influenza viruses included in the panel (Table 6). One laboratory did not detect influenza virus in one of the three 
type A influenza virus containing specimens and therefore did not perform N-subtyping of this specimen. Excluding 
the laboratory reporting all results in the incorrect random order, all 45 laboratories that included type B lineage 
determination correctly identified the lineage of the four influenza virus type B containing specimens (Table 6). 

The majority of laboratories used in-house assays or primers and probes from the Centers for Disease Control and 
Prevention (CDC) for virus detection by type (A/B) (24/55, 44% and 20/55, 36% respectively), type A H-subtyping 
(22/55, 40% and 20/55, 36% respectively), type A N-subtyping (14/19, 74% and 1/19, 5% respectively) and type 
B lineage determination (27/46, 59% and 18/46, 39% respectively; Annex Figure 4). The high performance of 
laboratories using a wide variety of assays for the specific challenge types (Table 6) indicates that none of these 
assays had any issues (Annex Figure 4). 

10 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Figure 2. Overview of cumulative performance scores for molecular detection, typing (A/B) and type 
A H-subtyping. 

Scoring system used: 
Type A viruses – correct type and H-subtype, 0; correct type, 1; all other results, 3. 
Type B viruses – correct type, 0; all other results, 3. 
Negative specimen – negative, 0; all other results, 3. 

Figure 3. Overview of cumulative performance scores for molecular detection, typing (A/B), type A 
H-and N-subtyping and type B lineage determination 

Scoring system used: 
Type A viruses – correct type and H- and N-subtype, 0; correct type and H-subtype, 1; correct type, 2; all other results, 3. 
Type B viruses – correct type and lineage, 0; correct type, 1; all other results, 3. 
Negative specimen – Negative, 0; all other results, 3. 
*: missing N-subtype of type A influenza viruses. 
#: missing N-subtype of type A influenza viruses and lineage of type B influenza viruses. 

11 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Table 6. Overview of molecular detection, typing, type A H- and N-subtype and type B lineage 
determination results by challenge type 

Evaluation 

Performance for all 55 participating laboratories 

Performance for laboratories that used assays appropriate for indicated 
challenge type (column one) 

Challenge type 
(number of 
specimens with 
this challenge) 

Labs 
with 
fully 
correct 
result 

Labs with 
error or 
missing 
data 

Specimens 
tested in 
challenge 
with correct 
result 

Specim
ens 
with 
error or 
missing 
data 

Type of error 
(number of 
specimens) 

Labs 
included in 
evaluation1 

Labs 
with 
fully 
correct 
result 

Labs 
with 
error 

Specimens 
tested in 
challenge 
with correct 
result 

Specimens 
with error 

Type of error 
(number of 
specimens) 

positive/negative 
(8) 

94.5% 

type A/B (7) 

96.4% 

H1pdm09 
subtype (1) 

98.2% 

H3 subtype (2) 

96.4% 

3 

2 

1 

2 

97.7% 

10 

97.9% 

98.2% 

97.3% 

8 

1 

3 

N1pdm09 
subtype (1) 

34.5% 

36 

34.5% 

36 

N2 subtype (2) 

34.5% 

36 

33.6% 

73 

Victoria lineage 
(2) 

Yamagata 
lineage (2) 

81.8% 

10 

81.8% 

81.8% 

10 

81.8% 

20 

20 

false positive (1); 
false negative (1); 
incorrect order (8) 

false negative (1); 
incorrect order (7) 

incorrect order (1) 

false negative (1); 
incorrect order (2) 

N-subtype missing 
(35); 
incorrect order (1) 
N-subtype missing 
(70); 
false negative (1); 
incorrect order (2) 
B-lineage missing 
(18); 
incorrect order (2) 
B-lineage missing 
(18); 
incorrect order (2) 

54 

96.3% 

2 

99.5% 

54 

54 

54 

98.1% 

100% 

98.1% 

19 2 

100% 

1 

0 

1 

0 

99.7% 

100% 

99.1% 

100% 

19 2 

98.1% 

1 

97.4% 

45 3 

100% 

45 3 

100% 

0 

0 

100% 

100% 

2 

1 

0 

1 

0 

1 

0 

0 

1Laboratory excluded that reported results of all specimens in the incorrect random order. 
2Laboratories excluded that did not report on type A N-subtyping. 
3Laboratories excluded that did not report on type B lineage determination. 

false positive 
(1); 
false negative 
(1) 

false negative 
(1) 

false negative 
(1) 

false negative 
(1) 

3.3 Virus isolation and antigenic and genetic 
characterisation 
A total of 44 of the 56 laboratories participated in the virus isolation and antigenic and genetic characterisation 
challenge. All 44 participated in the virus isolation, 20 participated in both the antigenic and genetic 
characterisation, 12 in antigenic characterisation only and six in genetic characterisation only. Six did not perform 
characterisations. 

With a median of one day (IQR 0–5 days) after start of molecular testing, virus isolation was started (Figure 4). 
This indicates that virus isolation was started very shortly after confirming the presence of the virus and 
determining type and type A H-subtype in the specimens, likely to select the appropriate cell type for virus 
isolation. Virus isolation was completed after a median of 15 days (IQR 10–23 days; Figure 4). Results of this 
challenge were available 30 days (median; IQR 26–35 days) after starting molecular testing and for 81% of 
participating laboratories, within in time limit of 36 calendar days (Figure 4). The difference in days between start 
of virus isolation and reporting characterisations (12 days) indicates the number of days needed for antigenic 
characterisation. 

12 

  
  
  
  
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Figure 4. Calendar days used for virus isolation and antigenic and genetic characterisation 

Start date of molecular testing taken as proxy for panel arrival date in laboratory. Large horizontal bars represent median and 
whiskers interquartile range. 

3.3.1 Virus isolation 

A total of 44 of the 56 laboratories participated in the virus isolation challenge (aggregated breakdown of results in 
Table 7; by participating laboratory and cumulative performance score in Annex Table 4). The methodologies used 
are listed in aggregated form in Annex Figure 5 and by laboratory in Annex Table 4. An overview of percentage of 
laboratories by cumulative performance score is shown in Figure 5. 

Table 7. Overview of virus isolation results by specimen code 

0.13 

Virus 

TCID50/ml1; 
Ct value 

EISN_INF18-01 
EISN_INF18-02 
EISN_INF18-03 
EISN_INF18-04 
EISN_INF18-05 
EISN_INF18-06 
EISN_INF18-07 
EISN_INF18-08 

A(H3N2) 
B/Yamagata 
B/Victoria 

2795; 15.9 
2795; 18.0 
559; 17.2 
A(H1N1)pdm09  1250; 16.8 

Negative 
B/Victoria 
A(H3N2) 
B/Yamagata 

-; - 
167; 19.1 
4180; 25.8 
374; 16.6 

Expected 
results 

Viral isolation results (n=44) 

Correct results 

Result 

n  % 
Positive/Not attempted2  43  97.7 
Positive 
40  90.9 
Positive 
Positive 
37  84.1 
Positive 
Positive 
Positive 
40  90.9 
Positive 
Negative  Negative/Not attempted2  44  100.0 
30  68.2 
Positive 
Positive 
43  97.7 
Positive 
Positive 
35  79.5 
Positive 
Positive 

Incorrect results 
Result 
Negative 
Negative 
Negative 
Negative 
- 
Negative 
Negative 
Negative 

n  % 
2.3 
1 
4 
9.1 
7  15.9 
9.1 
4 
0 
0 
14  31.8 
1 
2.3 
9  20.5 

1TCID50 determined on MDCK-MIX cells (mixed monolayer of MDCK-I and MDCK cells stable expressing human 2,6-sialtransferase 
[MDCK-SIAT]) in 96-well plate, static incubation and end-point determination of cytopathic effect in each well. This produces a 
valuable estimate for relative comparison of the amount of live virus in the panel members used for virus isolation, but likely 
produces an underestimate of the true number of live viruses in the panel members. 
2Not attempted: laboratory did not attempt virus isolation because molecular testing was negative (Annex Tables 3–4) and the 
laboratories’ algorithms likely include only virus-positive specimens in virus isolation. ‘Not attempted’ results are therefore 
considered correct. 

13 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Figure 5 Overview of cumulative performance scores for virus isolation 

Scoring system used: 
Virus-containing specimens – positive and not attempted (because of negative result in molecular detection), 0; negative and all 
other results, 1. 
Specimen without virus: negative and not attempted (because of negative result in molecular detection), 0; all other results, 1. 

Just over 60% of the laboratories (27/44; 61%) had fully correct results (cumulative performance score of 0) 
reporting all influenza virus containing specimens with the correct isolated virus and the negative specimen as 
negative (Figure 5). Of all 44 laboratories, one failed to isolate the virus from both A(H3N2) containing specimens, 
four failed to isolate the virus from the A(H1N1)pdm09 containing specimen and 16 failed to isolate the virus from 
one or more of the four influenza B virus-containing specimens. Laboratories performed less well on the B/Victoria 
and B/Yamagata containing specimens with lower viral load compared to those with higher viral load (Table 7). No 
false positives were reported for virus isolation. 

All laboratories used MDCK cells for virus isolation, although the type or types of MDCK cell lines used varied 
largely (Annex Figures 5A-C). MDCK-SIAT cells alone or in combination with other MDCK types were used more 
often for isolation of A(H3N2) influenza viruses (24/44, 55%) than for isolation of A(H1N1)pdm09 (13/44, 30%) 
and type B influenza viruses (15/44, 34%). The frequent failure to isolate type B influenza viruses was not clearly 
linked to the use of specific types of MDCK cells. However, 5/10 (50%) laboratories using MDCK-SIAT cells alone 
had one or more errors; four of the five failed to isolate B/Victoria virus from EISN_INF18-6. Of the five 
laboratories using another MDCK cell type in addition to MDCK-SIAT, three (60%) failed to isolate type B influenza 
viruses from at least one specimen and none of the three could isolate B/Yamagata from 
EISN_INF18-8. Among 29 laboratories not using MDCK-SIAT alone or in combination with other MDCK cell types, 
eight (28%) reported one or more failures to isolate type B influenza viruses and none of the eight could isolate 
B/Victoria virus from EISN_INF18-6. 

The majority of laboratories used cytopathic effect (CPE; 35/44, 80%), mainly combined with haemagglutination 
(28/44, 64%) for detection and confirmation of virus growth (Annex Figure 5D). In addition to haemagglutination 
(37/44, 84%), RT-PCR (20/44; 46%) and neuraminidase activity assay (9/44, 21%) were most often used to detect 
and/or confirm growth of the virus. For haemagglutination, a wide variety of RBCs was used (Annex Figure 5E). 
Several laboratories used a combination of RBCs (Annex Table 4) or a specific type of RBC dependent on the 
influenza virus type and type A H-subtype (details not shown). 

3.3.2 Antigenic characterisation 

The evaluation of the reported antigenic characterisation results compared with the expected results was 
challenging. The returned antigenic characterisation category as preset in the TESSy reporting system largely 
depends on the specificity of the antisera used in HI and VN assays and the characteristics of the used assays, like 
the type of RBCs used in HI assay or the deployment of oseltamivir in the HI assay for viruses with neuraminidase-
related haemagglutination (e.g. many of the current A(H3N2) influenza viruses). Individual specimen radar graphs 
to summarise the antigenic characterisation results were chosen to address these subjectivities and the wide range 
of reported categories per specimen (aggregated breakdown of the reported results in Figure 6; by participating 
laboratory in Annex Table 5). The used methodologies are listed by laboratory in Annex Table 5. Of the 32 
laboratories reporting on antigenic characterisation, 31 (97%) used HI assay, one virus neutralisation and one HI 
assay or virus neutralisation on individual viruses. Of the 31 laboratories that used HI assay, one (3%) used 

14 

 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

oseltamivir with all isolated viruses and 12 (39%) with the isolated A(H3N2) viruses only. In Annex Figure 6, an 
aggregated breakdown of type of sera used in HI assay and virus neutralisation is shown. WHO CC (London or 
Atlanta) or in-house ferret sera were used alone or in combination with other sera by all laboratories except one 
that used in-house chicken hyperimmune sera. 

The one or two categories per specimen reported with the highest counts (Figure 6) can be considered correct. At 
the same time, this reflects the variety of results that can be reported to TESSy for the same virus depending on 
the laboratory that performed the analysis. The A(H1N1)pdm09 virus was mainly categorised as 
A/Michigan/45/2015-like (23/30, 77%; TESSy category for the 2016/17 and 2017/18 seasons) rather than the 
expected A/California/7/2009 (TESSy category for the 2016/17 season). This finding likely reflects the replacement 
of antisera against A/California/7/2009 with antisera against A/Michigan/45/2015, the A(H1N1)pdm09 vaccine 
component introduced in the 2017/2018 northern hemisphere vaccine. However, these two strains do not greatly 
differ antigenically (within fourfold HI titre difference, Annex Figure 1A). The two A(H3N2) viruses were mainly 
reported either as A/Singapore/INFIMH16-0019/2016-like (TESSy category for the 2017/18 season) or A/Hong 
Kong/4801/2014-like (TESSy category for the 2016/17 and 2017/18 seasons), possibly reflecting the difficulty to 
distinguish between these viruses or the limited use of multiple antisera against A(H3N2) viruses in HI assay or VN. 
Both A(H3N2) viruses showed neuraminidase-induced haemagglutination. However, there was no clear difference 
in reported categories between laboratories that used oseltamivir in the HI assay and those that did not (Annex 
Table 5). Both B/Victoria viruses were reported mainly as B/Brisbane/60/2008-like, although one of them 
(EISN_INF18-06) was expected to be a low reactor with antisera against B/Brisbane/60/2008-like vaccine virus 
(‘not attributed to category’ to be reported to TESSy). Similarly, one of the B/Yamagata viruses (EISN_INF18-02) 
was expected to be a low reactor with antisera against B/Phuket/3073/2013-like vaccine virus (‘not attributed to 
category’ to be reported to TESSy), whereas both B/Yamagata viruses were reported mainly as 
B/Phuket/3073/2013-like. 

15 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Figure 6. Overview summarising reported antigenic characterisation categories by specimen code 

Total number of laboratories that obtained a virus isolate for characterisation indicated with each sample code. Expected result 
categories were: EISN_INF18-01: A(H3) A/Singapore/INFIMH-16-0019/2016 (H3N2)-like; EISN_INF18-02: B(Yam) lineage not 
attributed to category; EISN_INF18-03 – B/Brisbane/60/2008-like (B/Victoria/2/87 lineage); EISN_INF18-04: A(H1)pdm09 
A/California/7/2009 (H1N1)-like; EISN_INF18-05: N/A; EISN_INF18-06 – B(Vic) lineage not attributed to category; EISN_INF18-
07 – A(H3) A/Hong Kong/4801/2014 (H3N2)-like; EISN_INF18-08: B/Phuket/3073/2013-like (B/Yamagata/16/88-lineage). Both 
A(H3N2) influenza viruses had neuraminidase-induced haemagglutination. The B(Yam) and B(Vic) lineage viruses with expected 
result ‘not attributed to category’ were low reactors for B/Phuket/3073/2013 and B/Brisbane/60/2008 respectively. 

16 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

3.3.3 Genetic characterisation 

A total of 26 laboratories performed genetic characterisation (aggregated breakdown of reported results in Table 8; 
by participating laboratory and overall performance score in Annex Table 6). An overview of the percentage of 
laboratories by cumulative performance score is shown in Figure 7. The used methodologies are listed by 
laboratory in Annex Table 6. Of the 26 laboratories, 22 performed sequencing on the simulated clinical specimen, 
two on the simulated clinical specimen or the virus isolate, one on both and one on the virus isolate only. Nineteen 
of the laboratories used Sanger sequencing, six NGS and one Sanger sequencing and NGS. 

Figure 7. Overview of cumulative performance scores for genetic characterisation 

Scoring system used: correct: 0; incorrect: 1. Detailed arguments for allocating results as correct or incorrect inAnnex Table 6 
footnote. 

Just over 70% of the 26 laboratories (19/26, 73%) that performed genetic characterisation reported correct results 
for all virus-positive specimens (cumulative performance score of 0; (Figure 7, Table 8). The B/Victoria virus of 
specimen EISN_INF18-06 was assigned by some laboratories to genetic groups within clade 1A with deletion of 
amino acids 162-164 (representative B/Hong Kong/269/2017) or amino acids 162-163 (representative 
B/Norway/2409/2017) in HA1 (Table 8). However, this virus did not have these deletions and should have been 
assigned to the root 1A clade with B/Brisbane/60/2008 representative (Annex Figure 2C). Some laboratories 
assigned the A(H3N2) viruses to the older 3C.2a1 subgroup representative A/Bolzano/7/2016 subgroup or the root 
3C.2a clade representative A/Hong Kong/4801/2014 subgroup (Table 8). However, both A(H3N2) panel viruses 
have the N121K amino acid substitution defining the 3C.2a1 subgroup represented by A/Singapore/INFIMH16-
0019/2016 (Annex Figure 2B). EISN_INF18-01 virus has the N171K amino acid substitution further characterising 
this subgroup. The EISN_INF18-07 virus has a further amino acid substitution at this position, namely K171R, 
which is only discovered when a phylogenetic analysis in the context of the recommended reference viruses is 
performed (Annex Figure 2B). 

17 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Table 8. Overview summarising reported genetic characterisation categories by specimen code 

Specimen code 

EISN_INF18-01 

EISN_INF18-02 

EISN_INF18-03 

EISN_INF18-04 

Expected results 
Genetic category 

A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-
16-0019/2016 
subgroup 

B(Yam)-lineage 
clade  3 
representative  
B/Phuket/3073/2013 

B(Vic)-lineage clade 
1A representative 
B/Brisbane/60/2008 

A(H1)pdm09 group 
6B.1 representative 
A/Michigan/45/2015 

EISN_INF18-05 

N/A 

EISN_INF18-06 

B(Vic)-lineage clade 
1A representative 
B/Brisbane/60/2008 

EISN_INF18-07 

EISN_INF18-08 

A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-
16-0019/2016 
subgroup 
B(Yam)-lineage 
clade  3 
representative  
B/Phuket/3073/2013 

Participants 
with results 
n 

26 

26 

26 

26 

N/A 

26 

26 

25 

Correct 

Incorrect1 

n  % 

Result 

n  % 

Total 

Result 

n  % 

Result 

n  % 

Total 

Genetic characterisation results 

Result 
A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-
16-0019/2016 
subgroup 

B(Yam)-lineage 
clade 3 
representative 
B/Phuket/3073/2013 

24  92.3 

92.3% 

25  96.2 

96.2% 

A(H3) clade 3C.2a1 
representative 
A/Bolzano/7/2016 
subgroup 

B(Vic)-lineage clade 
1A (dell162-164 
subgroup) 
representative 
B/Hong 
Kong/269/2017 

2  7.7 

1  3.8 

B(Vic)-lineage clade 
1A representative 
B/Brisbane/60/2008 

22  84.6 

B/Johannesburg/3964/ 
2012 (Clade 1A, 
B/Brisbane/60/2008) 2 

2  7.7 

92.3% 

B Victoria - clade 1A 
- not branched 

1  3.8 

- 

- 

- 

- 

7.7% 

- 

- 

3.8% 

B(Vic)-lineage 
clade 1B 
representative 
B/Hong 
Kong/514/2009 

1 

3.8 

7.7% 

A(H1)pdm09 group 
6B.1 representative 
A/Michigan/45/2015 

25  96.2 

N/A 

N/A  N/A 

A/Slovenia/2903/2015 
(A(H1)pdm09 group 
6B.1 
A/Michigan/45/2015) 3 
N/A 

1  3.8  100.0% 

- 

- 

- 

- 

- 

- 

- 

N/A  N/A 

N/A 

N/A 

N/A  N/A 

B(Vic)-lineage clade 
1A representative 
B/Brisbane/60/2008 

23  88.5 

88.5% 

22  84.6 

84.6% 

A(H3) clade 3C.2a1 
representative 
A/Singapore/INFIMH-
16-0019/2016 
subgroup 
B(Yam)-lineage 
clade 3 
representative 
B/Phuket/3073/2013 

B(Vic)-lineage clade 
1A (dell162-163 
subgroup) 
representative 
B/Norway/2409/2017 

A(H3) clade 3C.2a 
representative 
A/Hong 
Kong/4801/2014 
subgroup 

2  7.7 

1  3.8 

N/A 
B(Vic)-lineage 
clade 1A 
(dell162-164 
subgroup) 
representative 
B/Hong 
Kong/269/2017 
A(H3) clade 
3C.2a1 
representative 
A/Bolzano/7/2016 
subgroup 

N/A  N/A 

N/A 

1 

3.8 

11.5% 

3  11.5  15.4% 

25  100.0 

100.0% 

- 

- 

- 

- 

- 

- 

- 

1Details on why the reported category is considered ‘incorrect’: in Annex Table 6 footnote. 
2Although B/Johannesburg/3964/2012 is a more precise indication of the allocation in phylogenetic analysis (Annex Figure 2C), 
but still a B(Vic)-lineage clade 1A representative B/Brisbane/60/2008 and therefore scored ‘correct’, it is not a current TESSy 
reporting category. 
3Although A/Slovenia/2903/2015 (Annex Figure 2A) has less in common in phylogenetic analysis with the current TESSy reporting 
category A(H1)pdm09 group 6B.1 representative A/Michigan/45/2015, it is a clade 6B.1 virus and is therefore considered a 
correct result. 

3.4 Antiviral susceptibility 
Twenty-nine laboratories participated in the antiviral susceptibility challenge; 22 performed genetic and phenotypic 
testing, six genetic testing only and one phenotypic testing only. 

Antiviral testing results were reported with a median of 31 days (IQR 27–38 days) after the start of molecular 
testing , with 21/29 (72%) of laboratories reporting within the requested 36 calendar days (Figure 8). 

Figure 8. Calendar days used for genetic and phenotypic antiviral susceptibility testing 

Molecular testing start date taken as proxy for panel arrival date in laboratory. Large horizontal bar represents median and 
whiskers represent interquartile range. 

18 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

3.4.1 Genetic testing 

A total of 28 laboratories performed genetic testing. However, some tested only a subset of specimens depending 
on the type of tests available and deployed (e.g. laboratories with only RT-PCR for N1-H275Y tested only  
specimens containing A(H1N1)pdm09; Annex Table 7). Genetic testing results are shown in two tables: Table 9 
with an aggregated breakdown of the identified amino acid substitutions and Table 10 with an aggregated 
breakdown of the reported interpretations. Results on amino acid substitution identification and interpretation by 
participating laboratory and cumulative performance score are shown in Annex Table 7. The used methodologies 
are listed in aggregated form in Annex Figure 7 and by laboratory in Annex Table 7. An overview of the percentage 
of laboratories by cumulative performance score is shown in Figure 9. 

All 28 laboratories identified the N1-H275Y amino acid substitution in specimen EISN_AV18-01 that contained a 
mixture of mutant N1-275Y and wild-type N1-275H. However, only 17/28 (61%) laboratories reported the 
mutant/wild-type mixed nature of the specimen. The specimen was prepared with SNP RT-PCR Ct values of 24 for 
275H and 22 for 275Y and about 80% 275Y by sequencing. One laboratory reported Ct values of 22 for 275H and 
20 for 275Y and eight laboratories reported a median percentage of 82% (range 70%–97%) for 275Y after 
sequencing, reflecting the intended nature of the mixture. 

The NA-E105K amino acid substitution in the B/Victoria virus in specimen EISN_AV18-02 was reported by 23/25 
(92%) laboratories that tested this specimen. One laboratory reported 150R instead. This was most likely a 
reporting error as the interpretation ‘no amino acid substitutions associated with reduced inhibition’ (AANI) for 
oseltamivir and zanamivir was given. The other laboratory reported ‘no amino acid substitution’ identified. 

Two laboratories reported wild-type A(H1N1)pdm09 virus with N1-H275Y, likely a reporting error as both 
laboratories reported as interpretation either AANI for oseltamivir or ‘no interpretation possible’ (NIP). One of 24 
laboratories reported the detection of N2-R292K amino acid substitution associated with (highly) reduced inhibition 
by neuraminidase inhibitors for both wild-type A(H3N2) viruses, likely a reporting error as the interpretation was 
reported as AANI for oseltamivir and zanamivir. 

Table 9. Overview summarising the reported identified amino acid substitutions associated with 
reduced antiviral susceptibility by specimen code 

Identification of amino acid substitutions3 

Correct 

Specimen 
code 
(EISN_) 
AV18-01 
AV18-02 
INF18-01 
INF18-02 
INF18-03 
INF18-04 
INF18-05 
INF18-06 
INF18-07 
INF18-08 

Number 
tested1 

28 
25 
24 
24 
24 
26 
0 
24 
24 
24 

Expected 
results2 

275YHmix 
E105K 

Result 
257YHmix 
E105K 

n  %  Result 
17  60.7 
257Y 
23  92.0 
No substitutions  No substitutions  23  95.8 
No substitutions  No substitutions  24  100 
No substitutions  No substitutions  24  100 
No substitutions  No substitutions  24  92.3 

No virus 

- 

- 

No substitutions  No substitutions  24  100 
No substitutions  No substitutions  23  95.8 
No substitutions  No substitutions  24  100 

n  % 
11  39.3 

Total 
100% 
92.0% 
95.8% 
100% 
100% 
92.3% 

100% 
95.8% 
100% 

Incorrect 

Result 
- 
None, 150R 
292K 
- 
- 

n  % 
- 
2  8.0 
1  4.2 
- 
- 

275Y, 275YHmix  2  7.7 

- 
- 
292K 
- 

- 
- 
1  4.2 
- 

1A number of laboratories did not perform genotypic antiviral susceptibility testing for all specimens depending on available tests. 
2No substitutions: no amino acid substitutions associated with a reduction in neuraminidase inhibitor susceptibility following full 
NA segment sequencing. 
3Reported result was translated from amino acid profiles reported and judged as correct or incorrect regardless of whether NA 
segment was fully or partially sequenced or whether only SNP detection assay was used; in the interpretation scoring these 
results, the level of testing was taken into account (Table 10; details: in Annex Table 7 footnote). 

Table 10. Overview summarising reported interpretation of amino acid substitution identification 
associated with reduced antiviral susceptibility by specimen code 

Specimen 
code 
(EISN_) 

Number 
tested1 

AV18-01 
AV18-02 
INF18-01 
INF18-02 
INF18-03 
INF18-04 
INF18-05 
INF18-06 
INF18-07 
INF18-08 

28 
25 
24 
24 
24 
26 
0 
24 
24 
24 

Expected 
interpretation 
for 
oseltamivir2 
AAHRI 

Results interpretation 
for oseltamivir3 

Correct 

Incorrect 

Result 
AAHRI 

n  %  Result  N  % 
24 85.7  AARI, NIP  4  14.3 

Expected 
interprettation 
for zanamivir2 

AANI 

Result 
AANI 

Results interpretation 
for zanamivir3 

Correct 

Incorrect 

AANI or AARI  AANI or AARI 22 88.0  AANI, NIP  3  12.0  AARI or AAHRI  AARI or AAHRI  21 84.0 

AANI 
AANI 
AANI 
AANI 
No virus 
AANI 
AANI 
AANI 

AANI 
AANI 
AANI 
AANI 
- 
AANI 
AANI 
AANI 

19 79.2  AANI, NIP  5  20.8 
20 83.3  AANI, NIP  4  16.7 
21 87.5  AANI, NIP  3  12.5 
21 80.8  AANI, NIP  5  19.2 
- 
- 
21 87.5  AANI, NIP  3  12.5 
20 83.3  AANI, NIP  4  16.7 
21 87.5  AANI, NIP  3  12.5 

- 

- 

AANI 
AANI 
AANI 
AANI 
No virus 
AANI 
AANI 
AANI 

AANI 
AANI 
AANI 
AANI 
- 
AANI 
AANI 
AANI 

n  % 
n  % 
Result 
20 71.4 AANI, AARI, NIP  8  28.6 
4  16.0 
AANI, NIP 
17 70.8 AANI, AARI, NIP  7  29.2 
4  16.7 
AANI, NIP 
20 83.3 
3  12.5 
AANI, NIP 
21 87.5 
5  19.2 
AANI, NIP 
21 80.8 
- 
- 
- 
3  12.5 
AANI, NIP 
21 87.5 
4  16.7 
AANI, NIP 
20 83.3 
3  12.5 
AANI, NIP 
21 87.5 

- 

1A number of laboratories did not perform genotypic antiviral susceptibility testing for all specimens depending on available tests. 

19 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

2Expected result when full NA segment has been sequenced or specific amino acid substitution has been identified with any test; 
AANI: no amino acid substitution previously associated with (highly) reduced inhibition; AARI: amino acid substitution previously 
associated with reduced inhibition; AAHRI: amino acid substitution previously associated with highly reduced inhibition; NIP: no 
interpretation possible due to partial NA segment information (SNP RT-PCR, partial or pyrosequencing). 
3Judged as correct or incorrect after comparison with expected result taking into account the level of testing. For viruses without 
amino acid substitution, AANI is correct if full NA segment has been sequenced and no amino acid substitution previously 
associated with reduced antiviral susceptibility has been identified, otherwise NIP should have been reported. If full NA segment 
has been sequenced, NIP is incorrect as the presence or absence of an amino acid substitution previously associated with 
reduced antiviral susceptibility could have been identified (details in Annex Table 7). 

Thirteen laboratories had errors in the interpretation of their amino acid substitution analysis, often with the same 
underlying problem for all specimens (Table 10, Annex Table 7). 

Common errors made were over the interpretation of results from partial sequencing or SNP detection by 
pyrosequencing or RT-PCR only. Five laboratories that used this approach reported wild-type viruses as AANI that 
should have been reported as NIP because the full-length NA was not analysed. On the other hand, two 
laboratories that had applied full-length NA sequencing under-interpreted their results and reported all or a number 
of wild-type viruses as NIP, whereas the more specific interpretation AANI for wild-type viruses should have been 
used. These errors due to not taking the level of genetic testing into account in the interpretation were reported by 
six laboratories for the EISN_AV18-01 and EISN_AV18-02 specimens. Examples are reporting detection of N1-
H275Y with SNP assay or partial sequencing as AANI (over-interpretation, as should be NIP) or with full NA 
segment sequencing as NIP (under-interpretation, as should be AANI) for zanamivir. 

Four laboratories detected N1-H275Y, but reported ‘amino acid substitution associated with reduced inhibition’ 
(AARI; n=2) or NIP (n=2) for oseltamivir instead of what should have been ‘amino acid substitution associated 
with highly reduced inhibition’ (AAHRI). Similarly, two laboratories detected B/Victoria NA-E105K, but reported NIP 
for zanamivir instead of what should have been AARI or AAHRI. 

Figure 9 shows the cumulative performance score for the amino acid substitution analysis and interpretation. Only 
8/28 (29%) laboratories had fully correct results (cumulative performance score of 0; specimens not tested were 
not scored). Given the high percentage of correct results for the amino acid substitution analysis by specimen 
(Table 9, Annex Table 7), this relatively low percentage of laboratories with fully correct results is obviously caused 
by failing to report the 275YH mixed nature for EISN_AV18-01 (11/28; 39%) and interpretation errors (13/28; 
46%; Table 10, Annex Table 7). 

Figure 9. Overview of cumulative performance scores for genetic antiviral susceptibility 
determination, amino acid substitution analysis and interpretation of this analysis 

Scoring system used (only those specimens scored for which a result was reported): 
EISN_AV18-01 substitutions – 275HY mix found, 0; 275Y found, 1; none found, 2; not tested, not scored. 
EISN_AV18-01 interpretation oseltamivir – 275Y and any test and ‘amino acid substitution associated with highly reduced 
inhibition’ (AAHRI), 0; rest, 1; not tested, not scored. 
EISN_AV18-01 interpretation zanamivir – full NA sequenced AND ‘no amino acid substitution associated with (highly) reduced 
inhibition’ (AANI), 0; SNP OR partial sequenced and ‘no interpretation possible’ (NIP), 0; any other, 1; not tested, not scored. 
EISN_AV18-02 substitutions: E105K found, 0; none found, 1; not tested, not scored. 
EISN_AV18-02 interpretation oseltamivir – E105K and any test and AANI or ‘amino acid substitution associated with reduced 
inhibition’ (AARI), 0; rest, 1; not tested, not scored. 

20 

 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

EISN_AV18-02 interpretation zanamivir – E105K and any test and AARI or AAHRI, 0; rest, 1; not tested, not scored. 
EISN_INF18-01 – 08 (except 05) substitution – none found, 0, any other, 1; not tested, not scored. 
EISN_INF18-01 – 08 (except 05) interpretation oseltamivir and zanamivir – full NA sequenced and AANI, 0; SNP or partial 
sequenced and NIP, 0; any other, 1; not tested, not scored. 

3.4.2 Phenotypic testing 

A total of 23 participants performed phenotypic testing. However, not all specimens were tested by all laboratories 
and not all were tested for both oseltamivir and zanamivir (Table 11). Although the virus first had to be isolated for 
specimen EISN_INF01-08, at least one laboratory used one or more of the specimens without preceding virus 
isolation. Figure 10 shows an aggregated overview of the IC50 values reported and Figure 11 an aggregated 
overview of IC50 fold changes of EISN_AV18-01 and 02 calculated using IC50 values reported for wild-type 
EISN_INF18-04 and wild-type EISN_INF18-03,06 respectively (by participating laboratory in Annex Figures 8–9). 
Table 11 shows an aggregated breakdown of the interpretations of the reported IC50 values and by participating 
laboratory with cumulative performance score in Annex Table 8. An overview of the percentage of laboratories by 
cumulative performance score is shown in Figure 12. One laboratory included in Table 11 could not be included in 
Figures 10–11 because this laboratory indicated that it reported IC50 fold change values instead of the requested 
IC50 values. The methodologies used are listed in aggregated form in Table 12 and by laboratory for the used type 
of neuraminidase inhibition assay only in Annex Table 8. 

Table 11. Overview of phenotypic antiviral susceptibility testing results by specimen code 

Oseltamivir phenotypic testing 

Zanamivir phenotypic testing 

Specimen code 
(EISN) 

Number 
tested1 

Expected 
results2 

Results phenotypic testing 
Incorrect 

Correct 

Result  n  %  Result  n  % 

Number 
tested1 

Expected 
results2 

AV18-01 
AV18-02 
INF18-01 
INF18-02 
INF18-03 
INF18-04 
INF18-05 
INF18-06 
INF18-07 
INF18-08 

23 
23 
21 
20 
16 
19 
0 
15 
21 
18 

HRI or RI  HRI or RI  23  100 
HRI or RI  HRI or RI  16  69.6 
21  100 
20  100 
16  100 
19  100 
- 
15  100 
21  100 
18  100 

NI 
NI 
NI 
NI 
No virus 
NI 
NI 
NI 

NI 
NI 
NI 
NI 
- 
NI 
NI 
NI 

- 

- 
NI3 
- 
- 
- 
- 
- 
- 
- 
- 

- 
7  30.4 
- 
- 
- 
- 
- 
- 
- 
- 

22 
22 
20 
19 
15 
18 
0 
14 
20 
17 

Results phenotypic testing 
Incorrect 

Correct 

NI 

NI 

21  95.5 
HRI or RI  HRI or RI  22  100 
20  100 
19  100 
15  100 
18  100 
- 
11  78.6 
20  100 
17  100 

Result  n  %  Result  n  % 
1  4.5 
- 
- 
- 
- 
- 
- 
3  21.4 
- 
- 

NI 
NI 
NI 
NI 
No virus 
NI 
NI 
NI 

RI4 
- 
- 
- 
- 
- 
- 
RI5 
- 
- 

NI 
NI 
NI 
NI 
- 
NI 
NI 
NI 

- 

1A number of laboratories that participated in the antiviral susceptibility challenge did not perform any phenotypic testing; some 
laboratories tested only a limited number of specimens and not always for both oseltamivir and zanamivir; not testing is mainly 
due to not having isolated a virus. 
2Type A viruses: normal inhibition (NI)=IC50 fold-change < 10; reduced inhibition (RI)=IC50 fold-change ≥ 10 – ≤ 100; highly 
reduced inhibition (HRI)=IC50 fold change > 100. Type B viruses: NI=IC50 fold-change < 5; RI = IC50 fold-change ≥ 5 – ≤ 50; 
HRI = IC50 fold change > 50. 
3In five cases for which it could be confirmed, IC50 fold change compared to EISN_INF18-3,6 was <5 and NI interpretation of own 
result is correct, but it was incorrect compared to expected result; in one case, the IC50 fold change compared to EISN_INF18-3,6 
was >5; in one case, no EISN_INF18-3,6 data was available for confirmation of the IC50 fold change. 
4IC50 fold change compared to EISN_INF18-4 <10. 
5Likely due to culture selected NA-T106P amino acid substitution confirmed to be present as minority variant in simulated clinical 
specimen and in one of three isolates. For two laboratories, the fold change relative to wild-type EISN_INF18-3 could be 
calculated >5 and < 50, indicating interpretation was correct, but incorrect compared to expected result. 

The overall performance of the laboratories in phenotypic testing was good; 12/23 (52%) laboratories had all 
specimens correct and the other 11/23 (48%) reported an incorrect result only for one specimen and one antiviral 
(Figure 12, Annex Table 8). A variety of techniques and approaches for IC50 measurement and calculations were 
used (Table 12), although the majority of laboratories used an in-house MUNANA substrate-based assay with pre-
titration of NA activity and an ‘HPA Excel template’ for IC50 calculation, reflecting training activities performed in the 
past at Public Health England. 

The EISN_AV18-01 specimen with a mixture of A(H1N1)pdm09 N1-275YH was correctly determined as HRI (n=18) 
or RI (n=5) for oseltamivir by all 23 laboratories (Table 11, Figures 10– 11). However, when comparing reported 
IC50 values with those available for wild-type EISN_INF18-04, one laboratory reporting HRI had an RI fold change 
(79-fold) and two laboratories reporting RI had an HRI fold change (139-fold and 691-fold; Figure 11). 

21 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Table 12. Methodologies used by laboratories to determine and evaluate IC50 values 

Method 
Assay type1 
MUNANA in-house 
NA-Fluor Kit 
NA-STAR Kit 
NA XTD Kit 
Drug range (nM) tested 
Median 
Mean 
Minimum 
Maximum 
Pre-titration NA activity 
Yes 
No 
Measurements 
Duplicate 
Single 
Control viruses2 
In-house 
ISIRV 
CDC 
ISIRV; in-house 
CDC; WHO London 
Reference strain 
Vaccine reference strains 
3 
Software to calculate IC50
HPA Excel template 
GraphPad Prism 
Excel template 
SigmaPlot 
MyAssay (http://www.myassays.com) 
Evaluation of IC50 against4 
wild-type virus 
median previous 
mean current 
mean previous; wild-type virus 
mean previous 
median current 
median current; mean current 
median previous; mean previous 
median previous; median current 
median previous; median current; wild-type virus 
wild-type virus; known resistant virus 

Number of laboratories 

Highest concentration 
4 000 
16 759 
1 000 
200 000 

Lowest concentration 
0.010 
0.028 
0 
0.32 

16 
3 
3 
1 

21 
2 

21 
2 

8 
8 
2 
2 
1 
1 
1 

14 
6 
1 
1 
1 

7 
5 
2 
2 
1 
1 
1 
1 
1 
1 
1 

1MUNANA: 20-(4-methylumbelliveryl)-a-D-N-acetylneuraminic acid substrate producing a fluorescent product after neuraminidase 
cleavage; NA: neuraminidase 
2ISIRV: International Society for Influenza and other Respiratory Virus Diseases antiviral working group ( ISIRV-AVG stopped 
providing reference viruses in 2018); CDC: Centers for Disease Control and Prevention, International Reagent Resource; WHO 
London: WHO Collaborating Centre, London, UK. 
3HPA Excel template: Excel template file provided by Health Protection Agency, currently Public Health England. 
4previous: previous season; current: current season. 

Among the laboratory-specific fold changes that could be calculated for 19 laboratories reporting IC50 values for 
both EISN_AV18-01 and EISN_INF18-04, 17 resulted in an HRI fold change (15 laboratories reported HRI and 2 
RI) and two in an RI fold change (one laboratory reported HRI and one RI; Figure 11). The differences in 
interpretation likely depend on what is used as a reference for the calculation of the fold changes (Table 12). For 
zanamivir and specimen EISN_AV18-01, one laboratory erroneously reported RI, although the laboratory-specific 
fold change compared to EISN_INF18-04 was <10, indicating an NI phenotype for zanamivir (Figure 11). Overall, 
22/23 (96%) laboratories had a correct result for this specimen (Figure 12). 

22 

 
 
 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Figure 10. Overview of reported IC50 values by specimen code 

Red dots indicate specimens with incorrect result: IC50 level not as expected and/or interpretation not as expected (details by 
laboratory in Annex Table 8, Annex Figure 8). Large horizontal bars represent median and whiskers represent interquartile range. 

23 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Figure 11. Overview of calculated IC50 fold-change values for EISN_AV18-01 and 02 specimens 

Laboratory-specific fold change values calculated for laboratories that also reported IC50 values for wild-type viruses of same 
subtype or lineage included in EISN_INF18 specimens, EISN_INF18-04 and mean EISN_INF18-03, 06 (3 RI outliers excluded) 
respectively. Large horizontal bars represent median and whiskers represent interquartile range. 
IC50 fold change categories definitions for type A viruses: NI – IC50 fold-change < 10; RI – IC50 fold change ≥ 10 – ≤ 100; HRI – 
IC50 fold change > 100, fir type B viruses: NI – IC50 fold change < 5; RI – IC50 fold change ≥ 5 – ≤ 50; HRI – IC50 fold change > 
50. Red dots: specimens with incorrect result; IC50 level not as expected and/or interpretation not as expected (details by 
laboratory in Annex Table 8, Annex Figure 9). Orange dots: laboratory correctly reported RI compared to expected result, 
although fold-change calculated with mean of EISN_AV18-03,06 indicated fold change below RI threshold. Blue dots: laboratory 
reported RI, while fold change calculated with EISN_INF18-04 indicated HRI. Purple dot: laboratory reported HRI, while fold 
change calculated with EISN_INF18-04 indicated RI. 

All 22 laboratories that tested the EISN_AV18-02 specimen (B/Victoria NA-E105K) reported correct results with HRI 
(n=12) or RI (n=10) for zanamivir (Table 11, Figures 10–11). However, when laboratory-specific IC50 fold changes 
relative to the average of the EISN_INF18-03,06 specimens were calculated, the RI result of one laboratory 
returned an NI fold change (Figure 11). IC50 measurement and interpretation for the EISN_AV18-02 specimen and 
oseltamivir was challenging, as seven laboratories reported an NI interpretation of the IC50 obtained (Table 11). For 
six of them, the laboratory-specific fold change relative to the average of the EISN_INF18-03,06 specimens could 
be calculated and five turned out as NI (Figure 11). Of these five, three were obtained with the NA-STAR or NA 
XTD assay and two with the MUNANA in-house assay. Furthermore, an additional NA-STAR assay and MUNANA in-
house assay result reported as RI returned an NI result after a laboratory-specific fold change calculation (Figure 
11). These results indicate that chemiluminescent assays experience difficulties in generating an RI result for this 
specimen that is mildly RI for oseltamivir when using fluorescent-based MUNANA or NA-Fluor assay types (Figure 
11, Annex Table 8). Overall, 16/23 (70%) laboratories reported a correct result for this specimen (Figure 12). 

Among the seven EISN_INF18 specimens with wild-type viruses tested by 21 laboratories, three laboratories 
reported an incorrect result for the same single specimen (EISN_INF18-06; B/Victoria): RI instead of the expected 
NI for zanamivir (Table 11, Figure 11). However, for two of the three laboratories, it was confirmed by comparing 
the IC50 values with those of the EISN_INF18-3 specimen that the laboratories had correctly reported RI (fold 
changes of 7.7 and 17.6; Figure 10). As reported by one of the laboratories, the most likely explanation is that cell 
culture-induced amino acid substitution NA-T106P caused this shift in IC50 for zanamivir, similar to what is observed 
for the NA-E105K amino substitution in specimen EISN_AV18-02. Deep sequencing of the original simulated clinical 
specimen showed the presence of NA-T106P as a minority variant. Overall, 18/21 (86%) laboratories had a correct 
result for this specimen (Figure 12). 

24 

 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Figure 12. Overview of cumulative performance scores for phenotypic antiviral susceptibility 
determination 

Scoring system used (only those specimens scored for which a result was reported): 
EISN_AV18-01: oseltamivir: HRI or RI, 0; other, 1; zanamivir: NI, 0; other, 1; not tested or no isolate, not scored. 
EISN_AV18-02: oseltamivir: RI or HRI, 0; other, 1; zanamivir: HRI or RI, 0; other, 1; not tested or no isolate, not scored. 
EISN_INF18-01 - 08 (except 05): oseltamivir and zanamivir: NI, 0; other 1; not tested or no isolate, not scored. 

3.5 Accreditation requirements 
Participation in EQA programmes is an important element in laboratory accreditation. Table 13 outlines th 
accreditation status of the laboratories, their participation in other EQA programmes and the requirement for the 
laboratory to participate in such EQAs for accreditation purposes. 

Table 13. Summary of survey on laboratory accreditation and participation in external quality 
assessment programmes 

Question and response1 

Is your laboratory accredited? 
Yes 

ISO 15189 
ISO 17025 
WHO 
ISO 9001 
CPA 
ISO 15189; ISO 17025 
ISO 15189; WHO 
Ministry of Health Slovenia 
National Academy of Medical Science of Ukraine 

Total 
No, in the process of obtaining accreditation 

ISO 15189 
ISO 15189; ISO 17025 

Total 
No, and not in the process of obtaining accreditation 
Question and response 
Which EQA panel(s) do you use for detection, typing, 
subtyping and lineage determination? 

WHO 
WHO; ERLI-NET 
WHO; QCMD 
CDC 
ERLI-NET 
QCMD 
CDC; INSTAND 
EQA Finland; ERLI-NET 
INSTAND 

Further specification and numbers of laboratories 
n 

16 
6 
4 
2 
1 
1 
1 
1 
1 
33 

9 
1 
10 
12 
Further specification and numbers of laboratories 

Detection and typing (n)  Type A subtyping (n) 

26 
4 
4 
2 
2 
2 
1 
1 
1 

26 
5 
4 
2 
2 
2 
1 
1 
1 

Type B lineage 
determination (n) 
22 
5 
2 
3 
3 
2 
1 
1 
1 

25 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Question and response 

Further specification and numbers of laboratories 

WHO; ERLI-NET; INSTAND 
WHO; ERLI-NET; EQUALIS; NEQAS 
WHO; ERLI-NET; VSR, local NIC; EQUALIS; NEQAS 
WHO; INSTAND 
WHO; PHE 
WHO; PNEQA 
WHO; QCMD, local NIC 
WHO; QCMD; NEQAS 
WHO; QCMD; NEQAS; CAP 
WHO; QCMD; SKML 
None 

NA 
Total 
Required for accreditation? (number/number with response) 
Which EQA panel(s) do you use for virus isolation? 

ERLI-NET 
WHO 
ERLI-NET; NEQAS 
CAP 
ERLI-NET; WHO 
INFHT, QCMD 
Local NIC 
SKML 
None 
N/A 
Total 
Required for accreditation? (number/number with response) 
Which EQA panel(s) do you use for virus characterisation? 

ERLI-NET 
ERLI-NET; WHO 
WHO 
ERLI-NET; WHO; local NIC 
CAP 
FluPEP-SEQ, CDC 
INFHT, QCMD 
Local NIC 
PHE 
None 
None (performed at other NIC location) 
N/A 
Total 
Required for accreditation? (number/number with response) 
Question and response 
Which EQA panel(s) do you use for antiviral susceptibility 
testing? 

ERLI-NET; WHO 
WHO 
ERLI-NET 
PHE 
WHO; PHE 
None 
N/A 
Total 
Required for accreditation (number/number with response) 

1 
- 
1 
1 
1 
1 
1 
1 
1 
1 
- 
- 
52 
19/21 

In cells (n) 
18 
4 
2 
1 
1 
1 
1 
1 
7 
3 
39 
7/11 
Antigenic (n) 
14 
1 
4 
- 
1 
- 
1 
1 
- 
9 
1 
2 
34 
4/9 

1 
- 
1 
1 
1 
- 
1 
1 
1 
1 
- 
- 
52 
18/20 

1 
1 
- 
1 
1 

1 
1 
- 
1 
2 
2 
51 
16/18 

In eggs (n) 
4 
1 
- 
- 
- 
- 
1 
- 
18 
7 
31 
2/4 
Genetic (n) 
8 
4 
4 
1 
- 
1 
1 
1 
1 
14 
- 
2 
37 
6/9 

Further specification and numbers of laboratories 

n 

11 
8 
6 
1 
1 
11 
2 
40 
7/11 

1CDC: Centers for Disease Control and Prevention; CPA: Clinical Pathology Accreditation, UK based; EQUALIS: Equalis AB; ERLI-
NET: European Reference Laboratory Network for Human Influenza; FluPEP-SEQ, CDC: Influenza Molecular Diagnostic 
Performance Evaluation Panel, CDC; INFHT: influenza virus hemagglutinin typing panel QCMD; INSTAND: Gesellschaft zur 
Förderung der Qualitätssicherung in medizinischen Laboratorien e.V.; N/A: not applicable; NEQAS: UK NEQAS; NIC: national 
influenza centre; PHE: Public Health England; PNEQA: unknown EQA programme abbreviation; QCMD: Quality Control for 
Molecular Diagnostics; SKML: Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek; VSR: unknown EQA programme 
abbreviation; WHO: World Health Organization; although not always explicitly indicated, assumed to refer to EQAP. 

Data on accreditation were available for 55 laboratories. Of the 55 laboratories, 33 (60%) were accredited, of 
which 18 (55%) by ISO 15189 (medical laboratories). A further 10/55 (18%) laboratories were in the process of 
obtaining ISO 15189 accreditation. The laboratories reported to have participated in a wide variety of EQA panels 
(Table 13). Although not reported by all laboratories in the survey, all have used the current EEIQAP 2018 and 
many EU/EEA laboratories (31 in 2008, 38 in 2015 for molecular rapid testing) also participated in previous 
ERLI-Net-provided panels for external quality assessment [22]. A high percentage reported the use of the WHO 

26 

 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

EQAP panel for detection and typing (43/52, 83%), subtyping influenza A viruses (43/52, 83%), lineage 
determination of type B viruses (36/51, 71%) and antiviral susceptibility testing (20/40, 50%). Participation in 
WHO EQAP panels for detection and typing and type A H-subtyping is a requirement for WHO-recognised NICs. 
Type A N-subtyping, type B lineage determination and antiviral susceptibility testing are so far optional in that 
project. The ERLI-Net panel is the most often used panel for virus isolation (21/29, 72%), antigenic 
characterisation (15/34, 44%) and genetic characterisation (13/37, 35%). A relatively high number of laboratories 
reported not using EQA panels for virus isolation, antigenic and genetic characterisation and antiviral susceptibility 
testing. This corresponded with a low percentage of laboratories reporting EQA for these techniques as a 
requirement for accreditation. However, EEIQAP 2018 shows a high percentage participation in molecular 
detection, virus isolation and antigenic and/or genetic characterisation and a lower percentage participation in 
antiviral susceptibility testing (Table 4). 

27 

 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

4 Discussion 

There was a high participation in EEIQAP 2018 among the 53 WHO European Region Member State countries. 
Forty-six of the 50 countries with at least 1 NIC/national influenza reference laboratory participated. Of the 63 
laboratories in the WHO European Region actively approached to sign up, 56 laboratories participated. As such, 
EEIQAP 2018 provides a good overview of the capabilities and performance of laboratories in the WHO European 
Region Member States and WHO GISRS and ECDC EISN ERLI-Net influenza reference laboratories for different 
aspects of influenza surveillance in the region. 

Panel distribution started in March 2018 and the last panel was delivered in early July 2018 due to delayed 
distribution to non-EU/EEA countries and the required paperwork for passing local customs. The return result was 
relatively good, with 49/55 (89%) laboratories reporting within the expected time limit of seven calendar days for 
molecular detection and 35/43 (81%) and 21/29 (72%) laboratories reporting within the expected time limit of 36 
calendar days for virus isolation with strain characterisation and antiviral susceptibility testing respectively. The time 
required for sequencing was not separately recorded. Nevertheless, many laboratories used genetic strain 
characterisation (26/56, 46%) and sequencing for antiviral susceptibility marker identification (25/56, 45%). This 
indicated that these laboratories have the capability for a relatively rapid (within one week) genetic strain 
characterisation and antiviral susceptibility marker analysis using sequencing directly on clinical specimens when an 
emerging situation requires it. Virus isolation is the limiting factor for antigenic characterisation and phenotypic 
testing for antiviral susceptibility, taking a median of 15 days until completion. 

Fifty-five laboratories performed very well in applying molecular testing to influenza virus detection, A/B typing and 
type A H-subtyping of the current seasonal circulating A(H1N1)pdm09, A(H3N2), B/Victoria and B/Yamagata 
influenza viruses. Overall, 52/55 (95%) laboratories reported correct results for all specimens, reconfirming the 
correct and reliable reporting of surveillance detection data by the network laboratories to TESSy. The percentage 
of laboratories with correct results was higher than that reported by 38 ERLI-Net laboratories in EU/EEA countries 
in the 2015 EQA study (90%) and 46 NICs in the WHO European Region in the 2017 WHO EQAP study (89%), 
which also included avian type A subtypes [22,23]. EEIQAP 2018 challenged participating laboratories to determine 
the Nsubtype and B-lineage for the first time. Fewer laboratories performed N-subtyping of type A influenza viruses 
(21/55, 38%) than lineage determination of type B viruses (46/55, 84%), both included in the dataset for reporting 
to TESSy. Nevertheless, all laboratories that reported the N-subtype and/or B-lineage did so correctly when the 
incorrect results of one laboratory due to incorrect random order reporting were excluded. Both type A N-subtyping 
and type B-lineage determination are important capabilities for influenza reference laboratories. N-subtyping is 
important for early detection of H and N reassortants. In 2001, the emergence of H1N2 reassortant virus was 
detected late because seasonal viruses were not widely N-subtyped [24]. This emergence led to inclusion of the N-
subtype in the EISS database (predecessor of TESSy for influenza) to be able to determine its spread [25]. In 
2018, one H1N2 reassortant virus was rapidly identified in routine surveillance because N-subtyping was 
included [26]. B-lineage is important to know for the (re)emergence and distribution of lineages [25], the lineage 
match with the strain included in trivalent vaccine and the differential impact of both lineages, e.g. lineage-specific 
vaccine effectiveness [27]. 

After the proportion of laboratories with correct results increased from 21/30 (70%) in the 2010 ERLI-Net EQA to 
26/32 (81%) in the 2015 ERLI-Net EQA [22], it dropped to 27/44 (61%) in EEIQAP 2018. This is explained by the 
high number of isolation failures for influenza virus type B, especially for specimens with the lowest concentration 
of virus and was also observed in the first virus isolation EQA in the Asia-Pacific region [15]. Type B isolation 
failures in EEIQAP 2018 were randomly distributed among laboratories. However, if a laboratory failed to isolate 
B/Victoria from the EISN_INF-06 specimen, the laboratory often also failed to isolate type B viruses from one or 
more of the other type B specimens. There was no obvious correlation with the wide variety of MDCK cells used 
and known to be suitable for influenza virus isolation [28]. However, of laboratories using MDCK-SIAT cells alone, 
50% faced influenza type B virus isolation failures compared to 28% of those that did not use MDCK-SIAT cells 
alone or in combination with other MDCK cell types. MDCK-SIAT has been developed specifically to support cell-
based assays for measuring neuraminidase inhibitor susceptibility of human influenza viruses by increased 
expression of the human variant of the influenza virus receptor containing sialic acid alpha(2,6) linked to galactose 
[29]. These MDCK-SIAT cells were shown to support the growth of A(H1N1), A(H3N2) and type B influenza viruses 
better than native MDCK cells [30]. Therefore, the observed high number of failures to isolate influenza type B 
viruses cannot be solely explained by the use of MDCK-SIAT cells. Similar to the observations for the Asia–Pacific 
region [15], there is clearly an issue with the sensitivity of influenza type B virus isolation procedures. As virus 
isolation is required for antigenic characterisation and phenotypic antiviral susceptibility testing, failure to isolate 
the virus had an immediate effect on the number of EEIQAP 2018 specimens that could be included in these 
analyses. In a broader perspective, failure to isolate the influenza virus dependent on the type of virus or viral load 
in the clinical specimen reduces the number of characterisations that can be reported to TESSy and the selection of 
viruses with specific characteristics to be forwarded to WHO CC London. 

28 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Comparable antigenic characterisation of influenza viruses across laboratories remains a challenging task, even 
among WHO CCs. Assays are difficult to standardise and changes in the viruses may prevent them from 
agglutinating RBCs or cause neuraminidase-induced haemagglutination [31,32]. This is also reflected in the 
struggle of NICs to generate accurate antigenic characterisation data as shown in previous EISN EQAs [22] and the 
current EEIQAP 2018. Low reactors of the B/Victoria and B/Yamagata viruses included in EEIQAP 2018 were not 
readily identified in HI assay with ferret sera against B/Brisbane/60/2008 and B/Phuket/3073/2013 respectively. 
Both A(H3N2) viruses were attributed almost equally to the A/Singapore/INFIMH-16-0019/2016 and the older 
A/Hong Kong/4801/2014 reference TESSy reporting categories. Despite the high variability of the assays used, 
there was good concordance between laboratories in the reported antigenic characterisation categories for all 
virus-containing panel specimens. However, the exercise also demonstrates that antigenic characterisation data 
reported to TESSy should be interpreted with caution. 

Genetic characterisation of the haemagglutinin segment of the panel viruses after sequencing was more 
straightforward, with 19/26 (73%) laboratories reporting correct results for all panel specimens, slightly less than in 
2015 (15/20, 75%) [22]. However, four laboratories did not genetically characterise all panel specimens in 2015. 
The main error made with A(H3N2) viruses in EEIQAP 2018 was allocating them to an older category of clade 
3C.2a viruses, probably a result of not fully taking into account the phylogenetic position and amino acid 
substitution characteristics for clade 3C.2a1 viruses represented by A/Singapore/INFIMH-16-0019/2016, namely 
N121K and N171K [20]. A specific challenge with EISN_INF18-07 was to interpret the K171R amino acid 
substitution correctly as a further evolution of the clade-characterising N171K amino acid substitution. A particular 
error made with both B/Victoria viruses was allocating them to the subclades of clade 1A that harbour the deletion 
in HA at positions 162-163 or 162-164, although the viruses included in the panel did not have these amino acid 
deletions. Similar errors in data reported to TESSy might lead to flawed estimates when analysing the emergence 
and spread of these deletion variants and laboratories should carry out appropriate analysis to avoid such errors. 
This could be complemented by timely automated analysis of available sequence data to validate the categories 
reported to TESSy, especially if they concern emerging variants with an impact on vaccine match like deletion variants. 

Reporting of amino acid substitution as required for TESSy reporting [21] was cumbersome, as the relevant amino 
acid positions were often not reported accurately. Nevertheless, determination of the A(H1N1)pdm09 N1-275Y/H 
mixed virus and B/Victoria NA-E105K virus was very good, with  all 28 (100%) laboratories detecting 275Y and 
23/25 (92%) detecting NA-E105K. This is consistent with the high accuracy in detecting N1-275Y in the 2017 WHO 
EQAP and 2015 ERLI-Net EQA [23,33], a sporadic amino acid substitution in influenza B/Victoria, NA-I221L in the 
2015 ERLI-Net EQA [33,34] and NA-E105K in the current EEIQAP 2018 [35]. Identification of the A(H1N1)pdm09 
N1-275Y/H mix, in which N1-275H was present at approximately 20%, was achieved by 17/28 (61%) laboratories, 
a decline from 17/23 (74%) laboratories in the 2015 ERLI-Net EQA [33]. This observation is possibly explained by 
the percentage of 20% N1-275H in the mixture being close to the detection limit for Sanger sequencing or the 
inclusion of a higher number of laboratories with less extensive training on this aspect of antiviral susceptibility 
monitoring in EEIQAP 2018. Still, the capability to accurately detect a mixture is important as specimens with a 
(highly) reduced inhibited virus quasispecies are a common scenario for laboratories that receive clinical specimens 
as part of a surveillance programme or treatment evaluation. Several laboratories reported amino acid substitutions 
associated with (highly) reduced inhibition for wild-type specimens, likely a reporting error as in all cases, the 
interpretation reported was different throughout. The major problem in genetic antiviral susceptibility testing was 
similar to that in the 2015 ERLI-Net EQA [33] related to the interpretation of the amino acid substitution analysis 
relative to the level of testing performed, e.g. SNP RT-PCR versus full-length NA sequencing. Frequently, data were 
either over- or under-interpreted. These errors were often made by the same laboratories for all or a large number 
of specimens. In the past, webinars specifically addressed this issue and they are still available on the EISN 
extranet. Phenotypic testing for antiviral susceptibility and interpretation of IC50 values were highly accurate for all 
specimens except the B/Victoria NA-E105K specimen and oseltamivir. Although the provided interpretations ‘normal 
inhibited’ for this specimen for the determined IC50s for oseltamivir by seven laboratories was correct, the 
determined IC50s were too low, resulting in fold changes compared to wild-type B/Victoria viruses included in the 
panel just below the ‘reduced inhibited’ threshold. All four laboratories that used chemiluminescent-based assays 
had this issue, compared to three of 16 laboratories that used fluorescent-based assays. Chemiluminescent assays 
were also associated with reduced sensitivity to generate a ‘reduced inhibited’ result with an A(H1N1)pdm09 N1-
275Y/H mixture in the 2015 ERLI-Net EQA [33]. In EEIQAP 2018, this issue was not observed, likely because of the 
relatively high percentage of approximately 80% of 275Y in the specimen. A particular issue previously described in 
surveillance of antiviral susceptibility of influenza viruses is virus isolation-induced amino acid substitution in the 
neuraminidase resulting in (highly) reduced inhibition [36]. This can only be addressed by sequencing the 
neuraminidase gene segment of both the clinical specimen and virus isolate and must be done especially if an 
unknown amino acid substitution is found in a virus isolate with a (highly) reduced inhibited phenotype. 

A relatively high number of laboratories in the network have obtained ISO 15189 accreditation for medical 
laboratories or is working towards obtaining this standard. This is a good development, although many laboratories 
reported that EQAs on several aspects of laboratory influenza surveillance challenged in EEIQAP 2018 and other 
EQAs are not required for accreditation. As ISO 15189-accredited tests require recorded corrective action when 
failing in EQAs, adding tests to the scope of the accreditation could be helpful in addressing errors and further 
improving the performance of laboratory testing and surveillance of the network as a whole. 

29 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

5 Conclusions 

Overall, the performance of the network in molecular detection, simultaneous typing and type A H-subtyping of 
seasonal influenza viruses can be rated as very good. Laboratories determining the N-subtype of type A influenza 
viruses and lineage of type B viruses demonstrated excellent performance. However, an increase in the number of 
laboratories with these latter capabilities would be desirable. Surveillance data derived from molecular testing can 
be considered accurate. 

EEIQAP 2018 has clearly identified an issue in the performance of the network in virus isolation, especially for type 
B influenza viruses. This is possibly linked to a lack of sensitivity of the procedures used and probably at least 
partly due to inappropriate use of only MDCK-SIAT cells. Not being able to isolate the virus has immediate 
repercussions on the number of isolates available for antigenic characterisation and phenotypic antiviral 
susceptibility testing in  laboratories and reporting the results to TESSy. It also limits the provision of viruses to 
WHO CC London for further analysis in an even more international context aimed to inform the WHO vaccine 
composition recommendation meetings. 

Although there is good concordance among laboratories in the reported antigenic characterisation categories for 
the EEIQAP panel specimens, the results also indicate that subtle antigenic differences between viruses are not 
picked up accurately and results reported to TESSy in the predefined categories should therefore be interpreted 
with caution. 

Genetic characterisation of the HA of seasonal influenza viruses by the network is highly accurate. However, certain 
laboratories had difficulties with allocating certain viruses to the correct predefined categories. Therefore, genetic 
categories reported to TESSy should also be analysed and interpreted with caution. 

Genetic and phenotypic testing of antiviral susceptibility by the network is of high accuracy. However, amino acid 
substitution data and their interpretation in the TESSy format and categories are inaccurate for a relatively high 
number of laboratories. This complicates the analysis of antiviral susceptibility data reported to TESSy. 

A high number of laboratories have obtained ISO 15189 accreditation or are in the process of applying for it, 
suggesting that the performance of tests for laboratory surveillance of influenza should be of high standard if 
included in the scope. For molecular detection and typing, A-subtyping and B-lineage determination, this holds as 
shown in EEIQAP 2018. However, for other tests challenged in EEIQAP 2018, many laboratories indicated that 
EQAs were not required for ISO 15189 accreditation. 

30 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

6 Recommendations 

Molecular detection, typing, type A H- and N-subtyping and 
type B lineage determination 
One participant only taking part in the molecular detection component of the panel reported the correct number of 
viruses by type, type A H-subtype, type B-lineage and a negative specimen, but in the incorrect random order. This 
indicates an issue in the reporting of data rather than a testing protocol issue. A review of standard operating 
procedures (SOPs) for the reporting of test data should resolve this issue. 

One participant reported a false positive in the molecular detection component of the panel. This indicates an issue 
with specimen contamination or switching and therefore a specimen handling error rather than a testing protocol 
issue. A review of SOPs for specimen reception and sample handling should resolve this issue. 

Another participant reported a false negative for an A(H3N2)-containing specimen and therefore did not perform 
further analyses on this specimen. This is likely an error in the generic influenza virus type A detection assay. 
Repeating this specimen and a review of used primers and probes should resolve this issue. 

Laboratories currently not performing type A N-subtyping and/or type B-lineage determination should consider 
adding this capability to their assay repertoire. 

Seasonal reviews of primers and probes as to whether they are still fit for purpose for currently circulating viruses 
should be part of the routine of preparing for the upcoming season. For in-house tests, this is the responsibility of 
the laboratory. For commercial or non-commercial assays obtained from another laboratory, this is the responsibility 
of the provider if sequences are not released. 

Virus isolation 
A high number of laboratories failed to isolate influenza type B viruses. Furthermore, some laboratories failed to 
isolate the A(H1N1)pdm09 virus and one failed to isolate all except one B/Yamagata virus despite multiple 
attempts. A review of all relevant SOPs is recommended as these errors are usually due to reagent, cell or 
sensitivity issues. Laboratories using MDCK-SIAT cells alone may want to consider using an additional MDCK cell 
type. This lack of sensitivity must be further addressed in discussion with the laboratories concerned. 

Continued support of and training for virus isolation is important to maintain this capability within Europe. This is 
particularly relevant for countries with decreasing capacity, especially with the increased use of direct sequence 
analysis from clinical specimens putting antigenic characterisation and phenotypic antiviral susceptibility testing 
capabilities of laboratories under pressure. 

Strain characterisation 
The number of laboratories performing antigenic and/or genetic strain characterisation is considerable (38/56; 
68%). However, given that 27 of these laboratories are based in the EU/EEA, it suggests somewhat unequal 
distribution across the WHO European Region. Increasing this capability in a number of additional laboratories 
would improve timely surveillance of circulating variants across the whole region. 

Antigenic characterisation 
Antigenic characterisation of influenza viruses is considered of good quality based on concordance in the reported 
antigenic TESSy categories. However, it appeared difficult to allocate A(H3N2) viruses to one category, possibly 
reflecting differences in used ferret sera and other assay characteristics, such as species source of RBCs andthe 
use of oseltamivir in HI assay to inhibit neuraminidase-induced haemagglutination of current A(H3N2) viruses. 
Standardisation of assays and the reagents used and testing with an increased number of ferret sera covering 
strains seen in multiple seasons may be an approach to increasing accuracy. However, distribution of high volumes 
and numbers of ferret sera to all network laboratories is likely impossible due to production limitations and limited 
comparability of multiple batches of ferret sera raised against the same strain. In addition, for accurate reporting in 
TESSy, a mechanism should be in place for timely updating of the antigenic categories in response to the 
emergence of new antigenic variants. This should be accompanied by making the appropriate ferret reference sera 
and influenza reference strains for antigenic characterisation available. 

31 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Genetic characterisation 
A couple of laboratories had difficulty allocating certain viruses to the correct TESSy categories. Errors made were 
possibly due to incorrect interpretation of obtained results after phylogenetic and amino acid substitution analysis. 
For laboratories with errors, training in these types of analysis and appropriate use of online tools like FluSurver 
[37] is recommended. 

One laboratory correctly identified a B/Yamagata-containing specimen, but incorrectly reported it as a B/Victoria 
lineage B/Hong Kong/269/2017 virus with deletion 162-164 in its HA. This could be the result of sample switching 
or a reporting error. Review of the original sequencing results against the reported category should resolve this 
issue. In the SOP for specimen processing and reporting, correlation of results across all analyses done on a 
specimen and validation of reported results by a second person should avoid this type of error. 

Reporting of correct data to TESSy is the responsibility of the submitting laboratory. However, as laboratories are 
also asked to report Global Initiative on Sharing All Influenza Data (GISAID) accession numbers for HA sequences 
to TESSy, interpretation of genetic strain characterisation data reported to TESSy is backed up by the possibility to 
perform additional sequence analysis to validate suspicious entries, e.g. when preparing Flu News Europe. 

Reporting of appropriate genetic categories to TESSy requires updating these categories to reflect emerging clades 
during the season. In the absence of such updates, laboratories that wish to correctly report the outcomes of 
phylogenetic analysis are forced to allocate viruses to an inappropriate category or report it in a free text comment, 
which complicates weekly analysis for Flu News Europe. A mechanism to update genetic categories more 
frequently would be desirable. 

Antiviral susceptibility testing 
Relatively few (29/56; 52%) laboratories are performing some level of antiviral susceptibility testing and they are 
unequally distributed across the region (22 in the EU/EEA). Increasing this capability in a number of additional 
laboratories would benefit the timely estimation of antiviral susceptibility across the Region and reporting to TESSy. 

Genetic antiviral susceptibility testing 
Two laboratories reported the detection of N1-H275Y in the wild-type A(H1N1)pdm09 virus specimens and one 
laboratory (ID 200) reported R292K in both wild-type A(H3N2) specimens. These laboratories did not report the 
HRI interpretation for the amino acid substitutions. Errors may have been made in the reporting of the amino acid 
positions screened and results obtained. Several laboratories reported the amino acid substitutions obtained from 
FluSurver [37] by comparison with the nearest vaccine strain instead of using the amino acid positions associated 
with reduced antiviral susceptibility that can be found one layer deeper in FluSurver [37] or in the list provided by 
the WHO working group on surveillance of influenza antiviral susceptibility [38]. In addition, a considerable number 
of laboratories made over- or under-interpretation errors based on the level of testing (e.g. SNP RT-PCR versus full-
length NA segment sequencing). These observations suggest that laboratories are not fully familiar with data 
interpretation and reporting to TESSy. 

Similar to genetic strain characterisation, reporting correct data is the responsibility of the submitting laboratory. 
However, laboratories are also asked to report GISAID accession numbers for NA sequences to TESSy. Therefore, 
interpretation of genetic antiviral susceptibility data reported to TESSy is backed up by the possibility of performing 
additional sequence analysis for validation of suspicious results. 

A number of laboratories did not report the mixed nature of the A(H1N1)pdm09 N1-275YH mixed specimen. 
Careful review of SNP detection or sequencing results should identify whether the wild-type was not detected or 
overlooked after detecting 275Y. Alternatively, these laboratories may not have been familiar with how to report the 
mixed nature of this specimen. 

Laboratories would benefit from training on interpretation and reporting of antiviral susceptibility data. Recordings 
of previous webinars and instruction documents are available on the EISN extranet. 

Phenotypic antiviral susceptibility testing 
A number of laboratories incorrectly identified the B/Victoria NA-E105K containing virus with an IC50 value 
considered ‘normal inhibited’. This was not a testing error in itself, but mainly the result of using a 
chemiluminescent assay that is known to be less sensitive in detecting mixtures of wild-type and (highly) reduced 
inhibited virus, the A(H3N2) NA-E119V variant and other variants like the B/Victoria NA-E105K variant with mildly 
reduced inhibition. Laboratories using a chemiluminescent assay should consider switching to in-house MUNANA or 
commercial fluorescent-based neuraminidase inhibition assay for routine use. 

32 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Accreditation 
ISO 15189 recommends external EQA for self-evaluation and addressing test issues in a systematic way. 
Laboratories could probably make better use of the methodology to address issues as provided by interpretations 
of the ISO 15189 accreditation. Laboratories would also benefit from inclusion of tests used in routine laboratory 
surveillance of influenza in the scope of ISO 15189 accreditation. 

33 

 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

References 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

World Health Organization (WHO). Global Influenza Surveillance and Response System (GISRS) [Internet]. 
Geneva: WHO; 2019. Available from: http://www.who.int/influenza/gisrs_laboratory. 

World Health Organization (WHO). WHO Collaborating Centres for influenza and their terms of reference 
[Internet]. Geneva: WHO; 2018. Available from: 
http://www.who.int/influenza/gisrs_laboratory/collaborating_centres/list. 

World Health Organization (WHO). WHO reference laboratories for diagnosis of influenza A/H5 infection 
[Internet]. Geneva: WHO; 2017. Available from: 
http://www.who.int/influenza/gisrs_laboratory/h5_reflabs/list. 

World Health Organization (WHO). National Influenza Centres – European Region of WHO [Internet]. 
Geneva: WHO; 2018. Available from: 
http://www.who.int/influenza/gisrs_laboratory/national_influenza_centres/list/en/index3.html. 

European Centre for Disease Prevention and Control. European Reference Laboratory Network for Human 
Influenza (ERLI-Net) [Internet]. Stockholm: ECDC; 2017. Available from: 
http://ecdc.europa.eu/en/healthtopics/influenza/laboratory_network/Pages/laboratory_network.aspx. 

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

Wu A, Su C, Wang D, Peng Y, Liu M, Hua S, et al. Sequential Reassortments Underlie Diverse Influenza 
H7N9 Genotypes in China. Cell Host Microbe. 2013 Oct 16;14(4):446-52. 

Westgeest KB, Russell CA, Lin X, Spronken MI, Bestebroer TM, Bahl J, et al. Genomewide Analysis of 
Reassortment and Evolution of Human Influenza A(H3N2) Viruses Circulating between 1968 and 2011. J 
Virol. 2014 Mar;88(5):2844-57. 

European Centre for Disease Prevention and Control. Strategy for the external quality assessment of public 
health microbiology laboratories. Stockholm: ECDC; 2018. Available from: 
http://ecdc.europa.eu/publications-data/strategy-external-quality-assessment-public-health-microbiology-
laboratories. 

10. 

Valette M, Aymard M. Quality control assessment of influenza and RSV testing in Europe: 2000-01 season. 
Euro Surveill. 2002 Nov;7:161-5. Available from: 
http://www.eurosurveillance.org/content/10.2807/esm.07.11.00375-en. 

11.  Meijer A, Valette M, Manuguerra JC, Pérez-Breña P, Paget J, Brown C, et al. Implementation of the 

community network of reference laboratories for human influenza in Europe. J Clin Virol. 2005 Oct;34(2):87-
96. 

12. 

Thompson CI, Lackenby A, Daniels RS, McCauley JW, Pereyaslov D, Broberg EK, et al. Evaluation of 
influenza virus antiviral susceptibility testing in Europe: results from the first external quality assessment 
exercise. J Clin Virol. 2013 Mar;56(3):212-8. 

13.  World Health Organization. WHO External Quality Assessment Project for the detection of influenza viruses 

by PCR. Geneva: WHO; 2012 Available from: 
http://www.who.int/influenza/gisrs_laboratory/external_quality_assessment_project. 

14.  Mackay WG, van Loon AM, Niedrig M, Meijer A, Lina B, Niesters HG. Molecular detection and typing of 
influenza viruses: are we ready for an influenza pandemic? J Clin Virol. 2008 Jun;42(2):194-7. 

15. 

Reading PC, Leung VK, Buettner I, Gillespie L, Deng YM, Shaw R, et al. The first external quality assessment 
of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016. J Clin Virol. 
2017 Dec;97:54-58. 

16. 

Snacken R, Brown C. New developments of influenza surveillance in Europe. Euro Surveill. 
2015 Jan 29;20(4). Available from: http://www.eurosurveillance.org/content/10.2807/ese.20.04.21020-en. 

17.  World Health Organization. Laboratory quality management system: handbook. Geneva: WHO; 2011. 

Available from: http://www.who.int/ihr/publications/lqms. 

Jonges M, Liu WM, van der Vries E, Jacobi R, Pronk I, Boog C, et al. Influenza Virus Inactivation for Studies 
of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling. J Clin Microbiol. 2010 
Mar;48(3):928-40. 

18. 

34 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

19. 

20. 

21. 

22. 

European Centre for Disease Prevention and Control. Influenza virus characterisation guidelines for the 
northern hemisphere influenza season 2016-2017. Stockholm: ECDC; October 2016. 

European Centre for Disease Prevention and Control. Influenza virus characterisation guidelines for the 
northern hemisphere influenza season 2017-2018. Stockholm: ECDC; October 2017. 

European Centre for Disease Prevention and Control. Reporting and interpretation of antiviral susceptibility 
data. Stockholm: ECDC; November 2014. 

European Centre for Disease Prevention and Control. External quality assessment scheme for detection, 
isolation and characterisation of influenza viruses for the European Reference Laboratory Network for 
Human Influenza – 2015. Stockholm: ECDC; 2016. Available from: http://ecdc.europa.eu/publications-
data/external-quality-assessment-scheme-detection-isolation-and-characterisation. 

23.  World Health Organization. Detection of influenza viruses by reverse transcription polymerase chain 

reaction: WHO external quality assessment programme summary analysis, 2017. Wkly Epidemiol Rec. 2018 
Jan 12;93(2):9-16. 

24. 

25. 

Gregory V, Bennett M, Orkhan MH, Al Hajjar S, Varsano N, Mendelson E, et al.. Emergence of Influenza A 
H1N2 Reassortant Viruses in the Human Population during 2001. Virology. 2002 Aug 15;300(1):1-7. 

Paget WJ, Meerhoff TJ, Goddard NL; EISS. Mild to moderate influenza activity in Europe and the detection 
of novel A(H1N2) and B viruses during the winter of 2001-02. Euro Surveill. 2002 Nov;7(11):147-57. 
Available from: http://www.eurosurveillance.org/content/10.2807/esm.07.11.00373-en. 

26.  Meijer A, Swaan CM, Voerknecht M, Jusic E, van den Brink S, Wijsman LA, et al. Case of seasonal 

reassortant A(H1N2) influenza virus infection, the Netherlands, March 2018. Euro Surveill. 2018 Apr;23(15). 
Available from: http://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2018.23.15.18-00160. 

27. 

Kissling E, Valenciano M, Pozo F, Vilcu AM, Reuss A, Rizzo C, et al. 2015/16 I-MOVE/I-MOVE+ multicentre 
case-control study in Europe: Moderate vaccine effectiveness estimates against influenza A(H1N1)pdm09 
and low estimates against lineage-mismatched influenza B among children. Influenza Other Respir Viruses. 
2018 Jul;12(4):423-437. 

28. 

Dukes JD, Whitley P, Chalmers AD. The MDCK variety pack: choosing the right strain. BMC Cell Biol. 
2011 Oct 7;12:43. 

29.  Matrosovich M, Matrosovich T, Carr J, Roberts NA, Klenk HD. Overexpression of the α-2,6-Sialyltransferase 

in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors. J Virol. 
2003 Aug;77(15):8418-25. 

30. 

31. 

32. 

33. 

34. 

35. 

36. 

Hatakeyama S, Sakai-Tagawa Y, Kiso M, Goto H, Kawakami C, Mitamura K, et al. Enhanced Expression of an 
α2,6-Linked Sialic Acid on MDCK Cells Improves Isolation of Human Influenza Viruses and Evaluation of 
Their Sensitivity to a Neuraminidase Inhibitor. J Clin Microbiol. 2005 Aug;43(8):4139-46. 

Ampofo WK, Azziz-Baumgartner E, Bashir U, Cox NJ, Fasce R, Giovanni M, et al. Strengthening the influenza 
vaccine virus selection and development process: Report of the 3rd WHO Informal Consultation for 
Improving Influenza Vaccine Virus Selection held at WHO headquarters, Geneva, Switzerland, 
1-3 April 2014. Vaccine. 2015 Aug 26;33(36):4368-82. 

Hampson A, Barr I, Cox N, Donis RO, Siddhivinayak H, Jernigan D, et al. Improving the selection and 
development of influenza vaccine viruses - Report of a WHO informal consultation on improving influenza 
vaccine virus selection, Hong Kong SAR, China, 18-20 November 2015. Vaccine. 2017 Feb 22;35(8):1104-
1109. 

European Centre for Disease Prevention and Control. External quality assessment scheme for influenza 
antiviral susceptibility for the European Reference Laboratory Network for Human Influenza – 2015. 
Stockholm: ECDC; 2016. Available from: http://ecdc.europa.eu/publications-data/external-quality-
assessment-scheme-influenza-antiviral-susceptibility-european. 

Escuret V, Collins PJ, Casalegno JS, Vachieri SG, Cattle N, Ferraris O, et al. A Novel I221L Substitution in 
Neuraminidase Confers High-Level Resistance to Oseltamivir in Influenza B Viruses. J Infect Dis. 2014 Oct 
15;210(8):1260-9. 

Fujisaki S, Takashita E, Yokoyama M, Taniwaki T, Xu H, Kishida N, et al. A single E105K mutation far from 
the active site of influenza B virus neuraminidase contributes to reduced susceptibility to multiple 
neuraminidase-inhibitor drugs. Biochem Biophys Res Commun. 2012 Dec 7;429(1-2):51-6. 

Lackenby A, Besselaar TG, Daniels RS, Fry A, Gregory V, Gubareva LV, et al. Global update on the 
susceptibility of human influenza viruses to neuraminidase inhibitors and status of novel antivirals, 2016-
2017. Antiviral Res. 2018 Sep;157:38-46. 

35 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

37. 

A*STAR Bioinformatics Institute. FluSurver Influenza surveillance – Prepared for the next wave. Singapore: 
A*STAR; 2019. Available from: http://flusurver.bii.a-star.edu.sg. 

38.  World Health Organization. Summary of neuraminidase amino acid substitutions associated with reduced 

inhibition by neuraminidase inhibitors. Geneva: WHO; 2018. Available from: 
http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/NAI_Reduced_Susceptibility_Marker_T
able_WHO.pdf. 

36 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annexes: extra tables and figures 

Annex Figure 1. Antigenic cartography maps created based on HI-assay data for A(H1N1)pdm09 (A), 
B/Victoria (C) and B/Yamagata influenza viruses (D) and based on virus neutralisation data for 
A(H3N2) influenza virus (B) 

generic HI ac_file_acmacs H1N1 ^3.8400 >none [9:7]

A 

A/SINGAPORE/GP1908/15_IVR
A/Michigan/45/2015-like strain

generic HI ac_file_acmacs H3N2 ^82.3533 >none [9:8]

B 

VIRUS NEUTRALISATION

A/NL/2413/16

A/NL/502/17

A/NL/2917/15

A/NL/148/15

A/NL/148/15

A/NL/502/17

A/NL/602/09

A/SINGAPORE/GP1908/15_IVR180

A/NL/2917/15

A/CALIFORNIA/004/09

A/CALIFORNIA/007/09_X-181

A/CALIFORNIA/007/09_X-181

A/NL/3792/16

A/NL/047/17

A/NL/602/09

A/CALIFORNIA/007/09

A/NL/1810/15

A/NL/499/17

NL/2413/16

NL/499/17

NL/1810/15

A/HK/4801/14

HK/4801/14

A/HK/4801/14 X-263B

A/NL/757/17

NL/2249/13

A/NL/751/17

X-263B

NL/757/17

A/NL/2249/13

A/Switzerland/9715293/2013 NIB-88

NIB-88

C 

generic HI ac_file_acmacs BVic ^92.2342 >none [9:9]

D 

generic HI ac_file_acmacs BYam ^17.0169 >none [10:6]

B/MALAYSIA/2506/04

B/MALYSIA/2506/04

B/BRISBANE/060/08_FS09033

B/NL/2241/13

B/NL/1588/17

B/NL/2334/17

B/NL/2424/2017

B/NL/2648/17

B/BRISBANE/060/08

B/NL/076/14

B/BRISBANE/060/08_FS16050

B/NL/2241/13

B/NL/076/14

B/NL/2914/15

B/NL/441/12

B/NL/385/09

B/NL/2423/17

B/NL/2914/15

B/NL/441/12

B/NL/385/09

B/NL/3680/16

B/NL/2423/17

B/NL/3066/15

B/NL/2424/17
B/NL/257/2014

B/PHUKET/3073/2013

B/NL/1551/2015

B/NL/1551/15

B/NL/3066/2015

B/PHUKET/3073/2013

B/FLORIDA/4/2006

B/NL/257/14

B/FLORIDA/4/2006

B/NL/830/17

Generated at the Dutch National Influenza Centre, ErasmusMC, Rotterdam, the Netherlands. Vaccine viruses indicated with red 
dot, viruses included in the panel indicated with blue dot and other recent viruses indicated with green dot. Spacing between grid 
lines is one unit of antigenic distance, corresponding to a twofold dilution of antiserum in the HI or virus neutralisation assay. 

37 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 2A. Phylogenetic tree of full HA of A(H1N1)pdm09 influenza viruses with common 
amino acid changes for viruses at indicated branch 

S183P, E235D, N260D, V520A

A/Netherlands/10010/2018_EPI1143294

A/Netherlands/3523/2017_EPI1143321

S164T

A/Netherlands/3546/2017_EPI1143272

A/Paris/1289/2017_EPI1043315

S74R, I295V

A/Hong_Kong/2214/2017_EPI1019935

A/Hong_Kong/2245/2017_EPI1019951

A/Norway/2146/2017_EPI1036454

A/Netherlands/050/2016_EPI731589

A/Dakar/09/2017_EPI1054433

I324V

A/Hong_Kong/2111/2017_EPI1019907

A/Thuringen/130/2017_EPI1054449

A/Slovenia/2903/2015_EPI687827

A/Bayern/81/2017_EPI995824

A/Torino/93/2017_EPI1019973

6B.1

6B

I166V, S183P, R205K, G482E

A/Netherlands/3165/2017_EPI1244482

S84N, S162N, I216T

A/Netherlands/502/2017_EPI907344

A/Michigan/45/2015_EPI849370_VACCINE_SH_2017

A215G

A/Hong_Kong/2199/2017_EPI1019925

A/OSAKA/170/2017_EPI1044569

A/Netherlands/1037/2015_EPI590367

K163Q, A256T

A/Ghana/FS-16-0249/2016_EPI770123

K283E, E499K

A/Israel/Q-504/2015_EPI697729

V152T, V173I, E491G, D501E

A/Jilin-Ningjiang/SWL1297/2017_EPI1030093

6B.2

D97N

S185T

S451N

A/South_Africa/3626/2013_EPI466626

A/Dakar/04/2014_EPI539474

6C

A/Hong_Kong/5659/2012_EPI390473

A/St_Petersburg/27/2011_EPI319527

A/St_Petersburg/100/2011_EPI320141

A/Czech_Republic/32/2011_EPI319447

A/Norway/120/2013_EPI416411

E374K

S203T

I295V

A/Christchurch/16/2010_EPI278607

A/Astrakhan/1/2011_EPI319590

A/Hong_Kong/3934/2011_EPI326206

A/Hong_Kong/2212/2010_EPI279895

A/Bayern/69/2009_EPI253705

A/California/07/2009_EPI176620_VACCINE_2009-2018

0.003

A/Dakar/20/2012_EPI417122

Reference virus set recommended by ECDC for analysis of viruses to be reported to TESSy supplemented with relevant viruses 
from the Netherlands have been used to infer the phylogenetic tree. Vaccine viruses indicated in bold red font and other 
reference viruses for reporting TESSy categories indicated in bold red italic font. Viruses included in panel indicated in bold blue 
font. 

38 

 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Figure 2B. Phylogenetic tree of full HA of A(H3N2) influenza viruses with common amino acid 
changes for viruses at indicated branch 

K135N

T135K

A/Hong_Kong/3163/2017_EPI1073834

A/England/80220578/2018_3C_2a1b+135N_EPI1152695

3C.2a1b
+ 135N

E62G, R142G

A/Hong_Kong/2286/2017_EPI1073820

A/Victoria/653/2017_3C_2a1b+135K_EPI1132322

3C.2a1b
+ 135K

A/Netherlands/741/2017_EPI957638

A/Netherlands/3385/2016_EPI873299

A/Netherlands/495/2017_EPI885210

K92R, H311Q

I58V

A/Hong_Kong/3156/2017_EPI1043977

A/Victoria/10/2017_EPI1031656

A/Netherlands/1992/2017_EPI922192

A/Netherlands/2335/2017_EPI957655

A/Oman/2585/2016_EPI769531

Q75H, R142G, K171R, I192N, A530V

A/Netherlands/757/2017_EPI1030659

A/Netherlands/1085/2017_EPI957646

N121K

T135K

A/Qatar/10-VI-17-0001178/2017_EPI960116

A/Greece/4/2017_EPI967219

A/Hong_Kong/3090/2017_EPI1060852

N96S

A/Netherlands/3458/2016_EPI878333

3C.2a1a

G484E

G479E

A/Netherlands/3344/2016_EPI873259

A/Idaho/37/2016_3C_2a1a_EPI858103

T160K, L194P

A/Norway/3806/2016_EPI922445

3C.2a1

N171K, I406V

I140M

A/Netherlands/2947/2017_EPI972307

A/Netherlands/751/2017_EPI907333

3C.2a

A/Singapore/Infimh-16-0019/2016_EPI1047604_VACCINE_2018-19

A/Netherlands/2007/2017_EPI922203

A/Bolzano/7/2016_EPI773595

A/Antananarivo/2398/2017_EPI1050811

A/Netherlands/3535/2017_EPI1143254

A/Netherlands/3521/2017_EPI1143313

T131K, R142K, R261Q

A/Lithuania/6165/2017_EPI1043042

A/Hong_Kong/3160/2017_EPI1073828

A/Brisbane/321/2016_3C_2a2_EPI919282

3C.2a2

A/Hong_Kong/3115/2017_EPI1060858

A/Netherlands/1415/2015_EPI590370

N122D, S262N

A/Antsirabe/2961/2017_EPI1042914

A/Netherlands/3782/2016_EPI878325

A/Analavory/2821/2017_EPI1042912

A/Norway/2603/2017_EPI1043054

N121K, S144K

A/Singapore/TT1374/2016_3C_2a3_EPI874032

I58V, S219Y

A/Hong_Kong/3105/2017_EPI1043909

3C.2a3

A/Hong_Kong/3125/2017_EPI1043945

L3I, N144S, F159Y, K160T, N225D, Q311H, D489N

A/Cote_D_Ivoire/544/2016_EPI781596

A/Hong_Kong/4801/2014_EPI539576_VACCINE_2015-16_2017-18

N31S, D53N, R142G, S144R, N171K, I192T, Q197H

A138S, F159S, N225D

K326R

T128A, R142G

A/Switzerland/9715293/2013_EPI530687_VACCINE_2015-16
A/Stockholm/6/2014_EPI530677

A/Hong_Kong/3042/2017_EPI1036313

A/Saudi_Arabia/252773/2017_EPI1050879

3C.2a4

A/Auckland/500/2017_3C_2a4_EPI1126195

A/Iceland/69/2017_EPI1015889

A/Florida/48/2017_EPI1058341

E62K, K83R, N122D, L157S,
Q197H, R261Q, V347K, D489N

A/Netherlands/865/2016_EPI731539

A/Netherlands/1726/2015_EPI590373

3C.3b

A/Samara/73/2013_EPI460558

A/Texas/50/2012_EPI391247_VACCINE_2014-15

0.002

Reference virus set recommended by ECDC for analysis of viruses to be reported to TESSy supplemented with relevant viruses 
from the Netherlands have been used to infer the phylogenetic tree. Vaccine viruses indicated in bold red font and other 
reference viruses for reporting TESSy categories indicated in bold red italic font. Viruses included in the panel indicated in bold 
blue font. 

39 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 2C. Phylogenetic tree of full HA of B/Victoria influenza viruses with common amino acid 
changes for viruses at indicated branch 

B/Netherlands/10414/2018_EPI1211066

D129G

B/Netherlands/10367/2018_EPI1208808

I180V, R498K

B/Norway/2409/2017_EPI1015818

Δ162-163

B/Colorado/06/2017_EPI1056637_VACCINE_2018-2019

B/Maryland/15/2016_EPI924546

B/Dakar/08/2017_EPI1046132

B/Analavory/2827/2017_EPI1053518

B/Kazakhstan/7440/2017_EPI1053560

B/Ireland/3154/2016_EPI702299

B/Netherlands/3094/2017_EPI975868

B/Netherlands/1581/2017_EPI922169

I180T, K209N

B/Hong_Kong/269/2017_EPI1053546

B/Hong_Kong/286/2017_EPI1053548

Δ162-164

B/Ukraine/7760/2017_EPI1046166

B/Netherlands/2874/2016_EPI770519

B/Georgia/523/2017_EPI1036494

B/Arizona/34/2017_EPI1033871

B/Dnipro/241/2017_EPI1023610

B/Zaporizza/162/2017_EPI1046168

B/Hong_Kong/245/2017_EPI1022738

B/Netherlands/1229/2016_EPI770493

B/Nordrhein-Westfalen/1/2016_EPI702285

B/Netherlands/619/2016_EPI844484

B/Netherlands/2523/2016_EPI762122

B/Netherlands/1840/2016_EPI770503

B/St_Petersburg/297/2017_EPI1040389

B/Moldova/422.18/2017_EPI1022801

B/Kyiv/197/2017_EPI1023630

B/Bremen/4/2017_EPI995898

1A

I117V, N129D, V146I

B/Georgia/566/2017_EPI1036470

B/Netherlands/2423/2017_EPI957662

B/Netherlands/363/2016_EPI844475

B/Bratislava/275/2017_EPI1022716

K209N

B/Bilthoven/4311400416/2014_EPI533366

B/Netherlands/076/2014_EPI541781

B/Johannesburg/3964/2012_EPI406272

B/Bilthoven/4311300491/2013_EPI533281

A169E

B/Bilthoven/4311300591/2013_EPI533285

B/Formosa/V2367/2012_EPI406250

B/South Australia/81/2012_EPI468076

B/Malta/MV636714/2011_EPI346732

N75K, N165K, S172P, I199T

B/Brisbane/60/2008_EPI172555_VACCINE_2009-2018

L58P

B/Hong_Kong/514/2009_EPI243627

B/Guangxi-Changzhou/31/2015_EPI586422

1B

B/Heilongjiang-Aihui/1234/2016_EPI768754

B/Malaysia/2506/2004_EPI175755_VACCINE_2007

0.002

Reference virus set recommended by ECDC for analysis of viruses to be reported to TESSy supplemented with relevant viruses 
from the Netherlands have been used to infer the phylogenetic tree. Vaccine viruses indicated in bold red font and other 
reference viruses for reporting TESSy categories indicated in bold red italic font. Viruses included in the panel indicated in bold 
blue font. 

40 

 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Figure 2D. Phylogenetic tree of full HA of B/Yamagata influenza viruses with common amino 
acid changes for viruses at indicated branch 

B/Netherlands/10141/2018_EPI1188196
B/Netherlands/3537/2017_EPI1143258

B/England/42/2017_EPI1053534

B/Netherlands/10545/2018_EPI1228383
B/Netherlands/10091/2018_EPI1188172
B/Netherlands/10293/2018_EPI1192965

B/Netherlands/10202/2018_EPI1188238

B/Netherlands/10016/2018_EPI1159918

B/Hong_Kong/253/2017_EPI1053542

B/Netherlands/10006/2018_EPI1143286

B/Arizona/32/2017_EPI1025598

B/Netherlands/10506/2018_EPI1211088

B/Netherlands/10055/2018_EPI1188152

B/Saarland/3/2017_EPI1046146

B/South_Africa/R07204/2017_EPI1055586

B/Ukraine/7758/2017_EPI1046162

B/Hong_Kong/256/2017_EPI1022754
B/Hong_Kong/264/2017_EPI1022766

B/Netherlands/3383/2016_EPI873283
B/Netherlands/3517/2017_EPI1143216

B/KANAGAWA/61/2017_EPI1054598

B/Norway/1460/2017_EPI1022805

B/Wellington/19/2017_EPI1044819

3

K253R

B/Slovenia/1666/2017_EPI1010256

B/Papeete/1291/2017_EPI1043331
B/Christchurch/505/2017_EPI1044839

B/Hong_Kong/260/2017_EPI1022760

B/Netherlands/2304/2016_EPI817061

B/Netherlands/2334/2017_EPI957654
B/Netherlands/1588/2017_EPI922170

B/Netherlands/2424/2017_EPI957663

B/Netherlands/3066/2015_EPI831913

B/Taiwan/68/2017_EPI1054202

B/Norway/2155/2017_EPI1023648

B/Bucuresti/210437/2017_EPI1053526
B/Norway/2162/2017_EPI1023650

M251V

D229N

B/Netherlands/1498/2015_EPI590313

B/Rheinland-Pfalz/3/2017_EPI1046144

B/Netherlands/365/2016_EPI844477

B/Netherlands/100/2015_EPI590295

L172Q

B/Netherlands/537/2014_EPI590294

B/Netherlands/252/2015_EPI590296

N116K, K298E, E312K

N202S

B/Netherlands/1031/2015_EPI590307

B/Hong_Kong/3417/2014_EPI536447

S150I, N165Y, G229D

B/Phuket/3073/2013_EPI540675_VACCINE_2015-16_quadri_2016-18

B/Wisconsin/01/2010_EPI271545_VACCINE_2012-13

B/Stockholm/12/2011_EPI346827
B/Massachusetts/02/2012_EPI438406_VACCINE_2013-15

R48K, P108A

T181A

B/Estonia/55669/2011_EPI319345
T234R

B/Poprad/320/2016_EPI773377

2

B/Rwanda/637/2016_EPI825739

B/BRISBANE/3/2007_EPI370742_VACCINE_2008-09

B/Florida/4/2006_EPI133760_VACCINE_2008-09

0.002

Reference virus set recommended by ECDC for analysis of viruses to be reported to TESSy supplemented with relevant viruses 
from the Netherlands have been used to infer the phylogenetic tree. Vaccine viruses indicated in bold red font for reporting TESSy 
categories. Viruses included in the panel indicated in bold blue font. 

41 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 3. Neuraminidase inhibition curves for determination of IC50 for oseltamivir of 
B/Victoria NA- E105K mutant (A) and A(H1N1)pdm09 N1-H275Y(mixH/Y) mutant (B) using native 
virus and inactivated by Triton X-100 or formalin 

A 

B 

42 

 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 1. List of participants 

Country 

Albania 

Armenia 

Austria 
Azerbaijan 

Belarus 

Belgium 

Bosnia and Herzegovina 

City 

Tirana 

Yerevan 

Vienna 
Baku 

Minsk 

Brussels 

Sarajevo 

Bosnia and Herzegovina, Republic of Srpska 

Banja Luka 

Bulgaria 

Cyprus 

Czech Republic 

Denmark 
Estonia 

Finland 

Sofia 

Nicosia 

Prague 

Copenhagen 
Tallinn 

Helsinki 

Former Yugoslav Republic of Macedonia 

Skopje 

France 

France 

Georgia 
Germany 

Greece 

Greece 

Hungary 

Iceland 

Ireland 

Israel 

Italy 

Kazakhstan 

Kyrgyzstan 

Latvia 

Lithuania 
Luxembourg 

Malta 

Moldova 

Montenegro 

The Netherlands 

The Netherlands 

Norway 

Lyon 

Paris 

Tbilisi 
Berlin 

Athens 

Thessaloniki 

Budapest 

Reykjavik 

Dublin 

Ramat Gan 

Rome 

Almaty 

Bishkek 

Riga 

Vilnius 
Luxembourg 

Msida 

Chisinau 

Podgorica 

Bilthoven 

Rotterdam 

Oslo 

Organisation 
Albanian National Institute of Public Health National 
Virology Laboratory, National Influenza laboratory 
National Centre for Disease Control and Prevention 
(NCDCP) 
Center of Virology, Medical University Vienna 
Republican Anti-Plague Station – virological laboratory 
Republik Research-Practical Centre of Epidemiology & 
Microbiology 
National Influenza Centre, Scientific Institute of Public 
Health 
OU Clinical Microbiology, Clinical Center University of 
Sarajevo 
Public Health Institute of the Republic of Srpska 
Department of Virology, National Laboratory ‘Influenza 
and ARD’ 
Microbiology Department, Nicosia General Hospital 
National Institute of Public Health, Centre for Epidemiology 
and Microbiology, Reference Laboratory for Influenza 
Statens Serum Institut 
Central Laboratory of Communicable Diseases 
National Institute for Health and Welfare (THL), Expert 
Microbiology Unit 
Institute of Public Health of Republic of Macedonia, 
Laboratory for Virology and Molecular Diagnostics 
CNR Virus des infections respiratoires, Laboratoire de 
Virologie, Centre de Biologie & Pathologie Nord – IAI 
Institut Pasteur, Centre National de Reference Virus des 
infections respiratoires (dont la grippe) 
National Center for Disease Control & Public Health 
Robert Koch-Institut, NRZ Influenza 
Hellenic Pasteur Institute, National Influenza Reference 
Laboratory for Southern Greece 
National Influenza Centre for Northern Greece, 
Microbiology Department, Medical School, Aristotle 
University of Thessaloniki 
National Public Health Institute, Directorate of Clinical and 
Public Health Microbiology , Department Respiratory 
Viruses 
Virus laboratories, Landspital –- National University 
Hospital 
UCD National Virus Reference Laboratory, University 
College Dublin 
Central Virology Laboratory, Sheba Medical Centre 
National Influenza Centre, Department of Infectious 
Diseases, Istituto Superiore di Sanità 
National Reference Laboratory for Control of Viral 
Infections, Research-Practical Center of Sanitary-
Epidemiological Expertise and Monitoring 
Department of the State Sanitary and Epidemiological 
Surveillance, Ministry of Health, Centre of Molecular-
Genetic and Microbiological Investigations 
Riga East University Hospital, Latvian Centre of Infectious 
Diseases, National Microbiology Reference Laboratory 
National Public Health Surveillance Laboratory 
Laboratoire National de Santé, Service Virologie-Serologie 
Molecular Diagnostics, Pathology Department, Mater Dei 
Hospital 
National Center for Public Health, Laboratory of Viral 
Infections 
Institute of Public Health of Montenegro 
Department Emerging and Endemic Viruses, Division 
Virology, Centre for Infectious Disease Research, 
Diagnostics and laboratory Surveillance, National Institute 
for Public Health and the Environment (RIVM) 
Erasmus Medical Centre, Department of Viroscience 
Norwegian Institute of Public Health, Department of 
Influenza 

43 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

City 

Lisboa 

Bucharest 

Moscow 

Novosibirsk 

Saint Petersburg 

Belgrade 

Bratislava 

Ljubjana 

Barcelona 

Madrid 

Valladolid 

Solna 

Geneva 

Ankara 

Kyiv 

London 

Glasgow 

Cardiff 

Tashkent 

Organisation 
Instituto Nacional de Saúde Dr. Ricardo Jorge, Laboratório 
Nacional de Referência Vírus Gripe 
Institutul Național de Cercetare-Dezvoltare Medico-Militară 
(INCDMM) ‘Cantacuzino’, Laboratory for Viral Respiratory 
Infections, National Influenza Center Romania 
Influenza Etiology and Epidemiology Laboratory, D.I. 
Ivanovsky Institute of Virology FSBI ‘N.F. Gamaleya 
NRCEM’, Ministry of Health of the Russian Federation 
Federal Budgetary Research Institution - State Research 
Center of Virology and Biotechnology VECTOR, 
Rospotrebnadzor (FBRI SRC VB VECTOR, 
Rospotrebnadzor), Department of Zoonotic Infections and 
Influenza 
Research Institute of Influenza, WHO-recognised National 
Influenza Centre of the Russian Federation 
Institute of Virology, Vaccine and Sera ‘Torlak’, Respiratory 
Department 
National Influenza Center, Public Health Authority of the 
Slovak Republic 
Laboratory for Public Health Virology, National Laboratory 
for Health, Environment and Food 
Laboratory of Microbiology, Hospital Clinic 
Instituto de Salud Carlos III, Influenza and Respiratory 
Virus Laboratory, National Center for Influenza Madrid 
Centro Nacional de Gripe de Valladolid, Servicio de 
Microbiología e Inmunología, Hospital Clínico Universitario 
de Valladolid 
Public Health Agency of Sweden, Unit for Laboratory 
Surveillance of Viral Pathogens and Vaccine Preventable 
Diseases 
National Center of Influenza, University of Geneva 
Hospitals, Laboratory of Virology 
Public Health Institution of Turkey, Virology Reference and 
Research Laboratory, National Influenza Centre 
L.V. Gromashevsky Institute of Epedemiology and 
Infectious Diseases, National Academy of Medical Science 
of Ukraine 
Public Health England, Respiratory Virus Unit, Virus 
Reference Department, National Infection Service, 
Colindale 
West of Scotland Specialist Virology Centre 
Wales Specialist Virology Centre, Public Health Wales 
Microbiology Cardiff, University Hospital of Wales 
Republican Centre of State Sanitary Epidemiological 
Surveillance of the Ministry of Health of the Republic of 
Uzbekistan 

Country 

Portugal 

Romania 

Russia 

Russia 

Russia 

Serbia 

Slovakia 

Slovenia 

Spain 

Spain 

Spain 

Sweden 

Switzerland 

Turkey 

Ukraine 

United Kingdom, England 

United Kingdom, Scotland 

United Kingdom, Wales 

Uzbekistan 

44 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 2. Overview in which challenge type each participating laboratory participated 

Participant 
ID1 

50 
95 
112 
117 
200 
1159 
1299 
1643 
2125 
2126 
2253 
2258 
2270 
2271 
2272 
2274 
2276 
2277 
2278 
2820 
3442 
10007 
10014 
10023 
10078 
10080 
10104 
10115 
10144 
10205 
10461 
10462 
10464 
10465 
10466 
1600 
1991 
2295 
2812 
2813 
2814 
2815 
2817 
2826 
3558 
4344 
10053 
10142 
10206 
10248 
10261 
10492 
10493 
10494 
10498 
10507 

Molecular 
detection 
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 
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 
Yes 
Yes 
Yes 
Yes 
Yes 

Virus 
isolation 
Yes 
Yes 
No 
No 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
No 
No 
Yes 
No 
No 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
No 
No 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
No 
Yes 
No 
Yes 
Yes 
No 
No 
Yes 
Yes 

Challenge type 

Characterisation 
(antigenic, genetic) 
Antigenic only 
Both 
None 
None 
Both 
Both 
Both 
Genetic only 
Both 
Both 
Antigenic only 
None 
None 
Both 
None 
None 
Both 
Antigenic only 
Both 
Antigenic only 
Both 
Both 
Both 
Genetic only 
None 
Genetic only 
Genetic only 
Antigenic only 
Both 
None 
Antigenic only 
Genetic only 
Both 
Both 
Both 
Antigenic only 
None 
None 
None 
None 
Antigenic only 
None 
Both 
Antigenic only 
Both 
Both 
Both 
None 
Antigenic only 
None 
Antigenic only 
None 
None 
None 
Genetic only 
Antigenic only 

Antiviral susceptibility testing 
(genetic, phenotypic) 
None 
Both 
None 
None 
Both 
Both 
Both 
Both 
Both 
Both 
None 
None 
None 
Both 
None 
None 
Both 
None 
None 
None 
Both 
Genetic only 
Genetic only 
Both 
None 
Both 
Both 
Both 
Both 
Genetic only 
Genetic only 
None 
Both 
Both 
Both 
None 
None 
None 
None 
None 
None 
None 
Both 
Genetic only 
Both 
Both 
Genetic only 
None 
None 
None 
Phenotypic only 
None 
None 
None 
Both 
None 

1Cell with orange shading: laboratory located in EU/EEA member country.

Full 
programme 

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

45 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Table 3. Overview of molecular detection and typing and type A H-subtype, type A N-subtype and type B lineage determination results by participant 
with performance score and used methodology 

01 

02 

03 

04 

05 

06 

07 

08 

A(H3N2) 

B/Yam 

B/Vic  A(H1N1)pdm09  Negative  B/Vic  A (H3N2)  B/Yam 

A(H3) 
A(H3N2) 

B/Yam 
B/Yam 

B/Vic 
B/Vic 

A(H1pdm09) 
A(H1N1)pdm09 

Negative 
Negative 

B/Vic 
B/Vic 

A(H3) 
A(H3N2) 

B/Yam 
B/Yam 

A(H3) 

B 

B 

A(H1N1)pdm09 

Negative 

B 

A(H3) 

B 

A(H3) 
A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 
A(H3N2) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 

A(H1pdm09) 
A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1N1)pdm09 
A(H1pdm09) 

Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 

A(H3) 
A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 
A(H3N2) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 

A(H3N2) 

B/Yam 

B/Vic 

A(H1N1)pdm09 

Negative 

B/Vic 

A(H3N2) 

B/Yam 

A(H3N2) 
A(H3) 

B/Yam 
B/Yam 

B/Vic 
B/Vic 

A(H1N1)pdm09 
A(H1pdm09) 

Negative 
Negative 

B/Vic 
B/Vic 

A(H3N2) 
A(H3) 

B/Yam 
B/Yam 

A(H3) 

A(H3) 

A(H3) 

A(H3) 
A(H3) 
A(H3) 
A(H3) 
A(H3) 
A(H3) 
A(H3) 
A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 

B 

B 

A(H1pdm09) 

Negative 

B 

A(H3) 

B 

B/Yam 

B/Vic 

A(H1pdm09) 

Negative 

B/Vic 

A(H3) 

B/Yam 

B 

B 

A(H1pdm09) 

Negative 

B 

A(H3) 

B 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 
B 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 
B 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 

A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 

Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 
B 
B/Vic 
B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 

A(H3) 
A(H3) 
A(H3) 3 
A(H3) 
A(H3) 
A(H3) 
A(H3) 
A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 
B 
B/Yam 
B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 

A(H3) 

B 

B 

A(H1pdm09) 

B 

B 

A(H3) 

B 

A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 
B/Yam 
B/Yam 

B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 
B/Vic 
B/Vic 

A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1N1)pdm09 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 

Negative 
Negative 
Negative 
Negative 
Negative 
Negative 
Negative 

B/Vic 
B/Vic 
B/Vic 
B 
B/Vic 
B/Vic 
B/Vic 

A(H3N2) 
A(H3N2) 
A(H3N2) 
A(H3) 
A(H3) 
A(H3) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B 
B/Yam 
B/Yam 
B/Yam 

Type 
and A H-
subtype 
score 4 
0 
0 

0 

0 
0 
0 
0 
0 
0 
0 
0 
0 

0 

0 
0 

0 

0 

0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

3 

0 
0 
0 
0 
0 
0 
0 

Specimen 
(EISN_INF18) 
Expected result 
Participant ID1 
50 
95 

112 

117 
200 
1159 
1299 
1643 
2125 
2126 
2253 
2258 

2270 

2271 
2272 

2274 

2276 

2277 

2278 
2820 
3442 
10007 
10014 
10023 
10078 
10080 
10104 
10115 
10144 
10205 
10461 

10462 

10464 
10465 
10466 
1600 
1991 
2295 
2812 

46 

Overall 
score 5 

Assay type 

Type A/B 

A H-subtype 

A N-subtype 

B-lineage 

In-house 

In-house 

3 
0 

6 

3 
0 
0 
0 
3 
3 
3 
0 
3 

0 

0 
3 

7 

3 

7 

3 
3 
3 
3 
7 
3 
7 
0 
0 
0 
3 
7 
3 

10 

0 
0 
0 
7 
3 
3 
3 

CDC 
In-house6 
In-house, Luminex 
NxTag RPP kit 
In-house 
In-house 
In-house 
In-house 
In-house 
CDC 
In-house 
CDC 
CDC 
Sacace Influenza A/B 
kit 
In-house 
CDC 
Fast Track Diagnostics 
Respiratory Screen 33 
In-house 
EliGene Influenza 
A/B/Pandemic LC kit 
In-house 
In-house 
In-house 
In-house 
Luminex NxTag RPP 
In-house 
CDC 
In-house 
In-house 
In-house, CDC 
CDC 
In-house 
Luminex NxTag RPP 
Seegene Allplex 
Respiratory Panel 1 
CDC 
CDC 
In-house 
CDC 
CDC 
CDC 
CDC 

CDC 
In-house 
In-house, Luminex 
NxTag RPP kit 
In-house 
In-house 
In-house 
In-house 
In-house 
CDC 
In-house 
CDC 
CDC 

CDC 
CDC 
Fast Track Diagnostics 
A-Subtyping 
In-house 
EliGene Influenza 
A/B/Pandemic LC kit 
In-house 
In-house 
Prodesse ProFast+ kit 
In-house 
Luminex NxTag RPP 
In-house 
CDC 
In-house 
In-house 
In-house, CDC 
In-house, CDC 
In-house 
CDC 
Seegene Allplex 
Respiratory Panel 1 
CDC 
In-house, CDC 
In-house 
CDC 
CDC 
CDC 
CDC 

Not performed 
In-house 

CDC 
In-house 

Not performed 

Not performed 

Not performed 
In-house 
In-house 
In-house 
Not performed 
sequencing 
Not performed 
CDC 
Not performed 

In-house 
Not performed 

In-house 
In-house 
In-house 
In-house 
In-house 
CDC 
In-house 
CDC 
CDC 

In-house 

CDC 
CDC 

Not performed 

Not performed 

Not performed 

In-house 

Not performed 

Not performed 

Not performed 
Not performed 
Not performed 
sequencing 
Not performed 
Not performed 
Not performed 
In-house 
In-house 
In-house 
Not performed 
Not performed 
Not performed 

In-house 
In-house 
In-house 
In-house 
Not performed 
In-house 
Not performed 
In-house 
In-house 
In-house 
In-house 
Not performed 
CDC 

Not performed 

Not performed 

Sequencing 
In-house 
In-house 
Not performed 
Not performed 
Not performed 
Not performed 

CDC 
In-house 
In-house 
Not performed 
CDC 
CDC 
CDC 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Specimen 
(EISN_INF18) 
Expected result 
Participant ID1 
28132 
2815 

2817 

2826 
3558 

01 

02 

03 

04 

05 

06 

07 

08 

A(H3N2) 

B/Yam 

B/Vic  A(H1N1)pdm09  Negative  B/Vic  A (H3N2)  B/Yam 

Negative  A(H1pdm09)  B/Yam 
B/Vic 
B/Yam 

A(H3) 

B/Vic 
A(H1pdm09) 

A(H3) 
Negative 

B/Yam 
B/Vic 

B/Vic 
A(H3) 

A(H3) 
B/Yam 

A(H3N2) 

B/Yam 

B/Vic 

A(H1N1)pdm09 

Negative 

B/Vic 

A(H3N2) 

B/Yam 

Negative 
A(H3N2) 

B/Yam 
B/Yam 

B/Vic 
B/Vic 

A(H1N1)pdm09 
A(H1N1)pdm09 

Negative 
Negative 

B/Vic 
B/Vic 

A(H3N2) 
A(H3N2) 

B/Yam 
B/Yam 

4344 

A(H3N2) 

B/Yam 

B/Vic 

A(H1pdm09) 

Negative 

B/Vic 

A(H3N2) 

B/Yam 

10053 
10142 
10206 
10248 

A(H3) 
A(H3) 
A(H3) 
A(H3) 

B 
B/Yam 
B/Yam 
B/Yam 

B 
B/Vic 
B/Vic 
B/Vic 

A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 
A(H1pdm09) 

Negative 
Negative 
Negative 
Negative 

B 
B/Vic 
B/Vic 
B/Vic 

A(H3) 
A(H3) 
A(H3) 
A(H3) 

B 
B/Yam 
B/Yam 
B/Yam 

10261 

A(H3N2) 

B/Yam 

B/Vic 

A(H1N1)pdm09 

Negative 

B/Vic 

A(H3N2) 

B/Yam 

10492 
10493 
10494 
10498 
10507 

A(H3) 
A(H3) 
A(H3N2) 
A(H3) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 

A(H1pdm09) 
A(H1pdm09) 
A(H1N1)pdm09 
A(H1pdm09) 
A(H1pdm09) 

Negative 
Negative 
Negative 
Negative 
Negative 

B/Vic 
B/Vic 
B/Vic 
B/Vic 
B/Vic 

A(H3) 
A(H3) 
A(H3N2) 
A(H3) 
A(H3) 

B/Yam 
B/Yam 
B/Yam 
B/Yam 
B/Yam 

Type 
and A H-
subtype 
score 4 
24 
0 

0 

3 
0 

0 

0 
0 
0 
0 

0 

0 
0 
0 
0 
0 

Overall 
score 5 

Assay type 

Type A/B 

A H-subtype 

A N-subtype 

B-lineage 

24 
3 

In-house 
In-house 

0 

3 
0 

1 

7 
3 
3 
3 

0 

3 
3 
0 
3 
3 

AmpliSens Influenza 
A/B-FL 

In-house 
In-house 

AmpliSens Influenza 
A/B-FL 

CDC 
CDC 
CDC 
CDC 

AmpliSens Influenza 
A/B-FL 

CDC 7 
In-house 
In-house 
CDC 
CDC 

In-house 
In-house 
AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) (H1 swine) 
In-house 
In-house, CDC 
AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) (H1 swine), 
CDC 
CDC 
CDC 
CDC 
CDC 
AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) (H1 swine) 
CDC 7 
In-house 
In-house 
CDC 
CDC 

Not performed 
Not performed 
AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) (H1 swine) 5 
In-house 
In-house 

AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) 

Not performed 
Not performed 
Not performed 
Not performed 
AmpliSens Influenza 
virus A-FL (H1N1, 
H3N2) (H1 swine) 8 
Not performed 
Not performed 
In-house 
Not performed 
Not performed 

In-house 
In-house 

Sequencing 

In-house 
In-house 

CDC 

Not performed 
CDC 
CDC 
CDC 

In-house 

CDC 7 
In-house 
In-house 
CDC 
CDC 

1Cell with orange shading = laboratory located in EU/EEA member country. 
2Laboratory returned correct number of virus detections of specific type and subtype/lineage, but in incorrect random order and therefore all results are considered incorrect; hence scores of 24 and 24 
respectively in the scoring columns. 
3Laboratory returned ‘Negative’ for N-subtype. 
4Scoring for detection with type and type A H-subtyping only: 
•  A viruses – correct type and H-subtype (green or yellow shading), 0; correct type without H-subtype (shading not applicable), 1; all other results (red shading), 3 
•  B viruses – correct type (green or yellow shading), 0; all other results (red shading), 3; and 
•  Negative specimen – negative (green shading), 0; all other results (red shading), 3. 
5Scoring for detection with type, type A H- and N-subtyping and type B lineage determination: 
A viruses: correct type and H- and N-subtype (green shading), 0; correct type and H-subtype without N-subtype (yellow shading), 1; correct type without H- and N-subtype (shading not applicable), 2; 
all other results (red shading), 3. 
B viruses: correct type and lineage (green shading), 0; correct type without lineage (yellow shading), 1; all other results (red shading), 3. 
Negative specimen: Negative (green shading), 0; all other results (red shading), 3. 
6In-house: in own laboratory developed or implemented or modified from published or personally obtained from elsewhere primers and probes or if not further specified; use of CDC primers and 
probes or kit indicated separately. 
7Laboratory actually reported WHO; this presumably refers to CDC primers and probes used widely in eastern part of region. 
8Kit insert of H1 swine test shows that assay is unsuitable for N-subtyping. 

47 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 4. Molecular methodologies used by 55 laboratories in detection of influenza virus 
types A and B (A), type A H-subtyping (B), type A N-subtyping (C) and type B lineage determination 
(D) 

A 

C 

B 

D 

In-house: in own laboratory developed or implemented or modified from primers and probes published or personally obtained 
from elsewhere or if methodology not further specified; use of CDC primers and probes or kit indicated separately as CDC. 
*One laboratory actually reported WHO; this presumably refers to CDC primers and probes used widely in region. 
#Kit insert of (H1N1, H3N2) test states that assay should not be used for H1pdm09-subtyping and the (H1 swine) test used 
instead. 
$Kit inserts of (H1N1, H3N2) and (H1 swine) tests state that assays are unsuitable for N1pdm09-subtyping. 
&Two of three laboratories reporting using sequencing for N-subtyping did not report N-subtype in molecular diagnostics. 
Numbers in bars indicate number of laboratories.

48 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 4. Overview of virus isolation results with performance score and used methodology 

Specimen 
(EISN_INF18) 
Subtype/lineage 
Expected result 
Participant ID1 
50 
95 

200 

1159 
1299 

1643 

2125 

2126 

2253 

2271 

2276 

2277 

2278 

2820 
3442 

10007 

10014 

100232 
10078 

10080 

10104 

10115 

10144 

10205 

10461 

10462 

01 

02 

03 

04 

05 

06 

07 

08 

Confirmation method virus isolation (n specimens)7 

A(H3N2) 

B/Yam 

B/Vic 

A(H1N1)pdm09 

Negative 

B/Vic 

A(H3N2)  B/Yam 

Positive 

Positive  Positive 

Positive 

Negative 

Positive  Positive  Positive 

Positive 
Positive 

Positive 
Positive 

Positive 
Positive 

Positive 
Positive 

Negative 
Negative 

Positive 
Negative  Positive 
Negative  Positive  Negative 

Positive 

Positive 

Positive 

Positive* 

Not attempted 

Positive 

Positive  Negative* 

Positive 
Positive 

Positive  Negative 
Positive 
Positive 

Positive 
Positive 

Negative 
Negative 

Negative  Positive 
Positive 
Positive 

Positive 
Positive 

Positive 

Negative  Positive* 

Negative 

Negative 

Negative  Positive  Negative 

Positive 

Positive 

Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Positive 

Positive*  Positive* 

Positive 

Negative 

Positive 

Positive  Positive* 

Positive 

Positive 

Positive 

Positive* 

Positive 

Positive 

Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Not attempted 

Positive  Positive*  Positive 

Positive* 

Positive 

Positive 

Positive 

Not attempted 

Positive  Positive*  Positive 

Positive 

Positive 

Positive 

Positive 

Negative* 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Positive 
Positive 

Positive 
Positive 
Positive  Negative 

Positive 
Negative 

Negative 
Negative 

Positive 
Positive 
Positive 
Negative  Positive  Negative 

Positive* 

Positive 

Positive 

Positive 

Not attempted 

Positive  Positive*  Positive 

Positive* 

Positive 

Positive 

Positive 
Positive 

Negative  Negative 
Positive 
Positive 

Positive 

Positive 

Positive 

Positive 

Positive 
Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Negative 
Negative 

Negative  Positive  Negative 
Positive 
Positive 
Positive 

Negative 

Positive 

Positive 

Positive 

Positive* 

Positive 

Positive 

Positive* 

Not attempted 

Positive  Positive*  Positive 

Positive 

Positive 

Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Positive* 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive* 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Negative 

Negative  Positive*  Positive 

Negative 

Positive 

Positive 

Positive 

Negative* 

Positive  Positive*  Positive 

Negative 

Positive 

Positive 

Positive 

Overall 
score5 

Isolation method 
(n specimens)7 

HA-assay 

NA activity 

RBC species  MUNANA  NA-STAR 

RT-PCR 

IF  CPE  Other 

1 
2 

1 

2 
0 

4 

0 

0 

0 

0 

0 

0 

0 

0 
4 

0 

0 

4 
0 

0 

0 

0 

1 

0 

0 

0 

MDCK-I (8) 
MDCK-SIAT (8) 
MDCK-SIAT (6) 
MDCK (2*) 
MDCK-SIAT (8) 
MDCK (8) 
MDCK-SIAT (7) 
Embryonated egg (1*) 

MDCK (8) 

MDCK.1 (5) 
MDCK-SIAT (3*)6 
MDCK-II (8) 
MDCK-SIAT (2*) 
MDCK London line (5) 
MDCK-SIAT (2*) 
MDCK (5) 
MDCK-I (7) 
MDCK-II (1*) 
MDCK (8) 
MDCK-SIAT1 (8) 
MDCK-SIAT (8) 
MDCK-SIAT (8) 
MDCK-SIAT (2*) 
MDCK (5) 
MDCK-SIAT (1*) 
MDCK (7) 
MDCK (8) 
MDCK (8) 
MDCK-SIAT and 
MDCK-I mixed cells (8) 
MDCK-SIAT (3*) 
MDCK.1 (4) 

MDCK-I (8) 

MDCK-SIAT (2*) 
MDCK (6) 
MDCK-SIAT (8) 
MDCK-SIAT (3*) 
MDCK.1 (5) 
MDCK.1 (8) 

Human (8) 
Turkey (8) 

Guinea pig (1) 

HA not used 
Guinea pig (8) 
Turkey (8) 
Guinea pig (8) 
Turkey (1) 
Guinea pig (8) 
Turkey (5) 
Guinea pig (2) 
Human (8) 

Guinea pig (7) 

Turkey (7) 
Guinea pig (7) 

Human (8) 

Human (8) 
Chicken (2) 
Guinea pig (8) 
Guinea pig (8) 
Turkey (5) 
Guinea pig (2) 

Guinea pig (8) 

HA not used 
HA not used 

HA not used 

Guinea pig (5) 

Guinea pig (4) 
Chicken (6) 
Turkey (5) 
Human (1) 
HA not used 
Human (3) 
Chicken (6) 
Human (8) 

0 
0 

0 

0 
0 

6 

0 

0 

0 

0 

7 

0 

0 

0 
0 

0 

0 

0 
0 

8 

0 

7 

7 

0 

0 

0 

0 
7 

0 

0 
0 

0 

7 

0 

0 

0 

0 

0 

0 

0 
0 

0 

0 

0 
0 

0 

0 

0 

0 

0 

0 

0 

0 
0 

1 

8 
0 

1 

0 

0 

8 

0 

0 

0 

8 

0 
0 

0 

0 

3 
4 

7 

0 

0 

8 

8 

8 

0 

0 
0 

1 

0 
0 

7 

0 

0 

0 

0 

0 

0 

8 

0 
0 

0 

0 

0 
8 

0 

0 

0 

0 

0 

8 

0 

8 
8 

7 

8 
8 

7 

8 

8 

7 

0 

7 

0 

8 

8 
0 

0 

0 

8 
8 

8 

7 

8 

8 

8 

8 

8 

0 
0 

0 

0 
0 

0 

0 

0 

0 

0 

0 

0 

0 

0 
0 

0 

0 

0 
0 

0 

7 8 

8 9 

0 

0 

0 

0 

49 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Specimen 
(EISN_INF18) 
Subtype/lineage 
Expected result 
Participant ID1 

01 

02 

03 

04 

05 

06 

07 

08 

Confirmation method virus isolation (n specimens)7 

A(H3N2) 

B/Yam 

B/Vic 

A(H1N1)pdm09 

Negative 

B/Vic 

A(H3N2)  B/Yam 

Positive 

Positive  Positive 

Positive 

Negative 

Positive  Positive  Positive 

Overall 
score5 

Isolation method 
(n specimens)7 

HA-assay 

NA activity 

RBC species  MUNANA  NA-STAR 

RT-PCR 

IF  CPE  Other 

10464 

10465 

10466 

1600 
1991 
2295 
2814 
2815 

2817 

2826 
3558 

4344 

Positive* 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 

Positive 
Positive 
Positive 
Positive 
Positive 

Positive 
Positive 
Positive 
Positive 
Positive 

Positive 
Positive 
Positive 
Positive 
Positive 

Positive 

Positive 

Positive 

Positive 
Positive 
Positive 
Positive 
Positive 

Negative 

Positive  Positive*  Positive 

Negative 

Positive 

Positive 

Positive 

Negative 

Positive 

Positive 

Positive 

Negative 
Negative 
Negative 
Negative 
Negative 

Positive 
Positive 
Positive 
Negative  Positive 
Positive 
Negative  Positive  Negative 
Positive 
Positive 
Positive 
Positive 
Positive 
Positive 

Positive 

Positive  Negative 

Positive 

Negative 

Negative  Positive 

Positive 

Not attempted 4  Positive 
Positive 

Positive 

Positive 
Positive 

Positive 
Positive 

Not attempted 
Not attempted 

Positive 
Positive 

Positive 
Positive 

Positive 
Positive 

Positive 

Positive  Negative 

Positive 

Not attempted  Negative  Positive  Negative 

10053 

Positive* 

Positive#  Positive 

Positive# 

Not attempted 

Positive  Positive*  Positive 

10206 

10261 

10492 
10498 

105073 

Positive 

Positive 

Positive 

Positive 

Not attempted  Negative  Positive 

Positive 

Positive* 

Positive  Negative 

Positive 

Negative 

Positive  Positive*  Positive 

Positive 
Positive 

Negative  Positive 
Negative  Positive 

Negative 
Positive 

Negative 
Not attempted 

Negative  Positive  Negative 
Positive 
Positive 
Positive 

Negative 

Positive  Negative 

Negative 

Negative 

Negative  Negative  Negative 

0 

0 

0 

0 
1 
2 
0 
0 

2 

0 
0 

3 

0 

1 

1 

4 
1 

6 

MDCK-SIAT (2*) 
MDCK (6) 
MDCK.2 (8) 

MDCK-SIAT (8) 

MDCK  (8) 
MDCK London line (8) 
MDCK NAMRU line (8) 
MDCK-II (8) 
MDCK-I (8) 

MDCK (8) 

MDCK (6) 
MDCK-SIAT (7) 

MDCK (7) 

MDCK-SIAT (2*) 
MDCK-I (2#) 
MDCK-II (3) 

MDCK-SIAT (7) 

MDCK-SIAT (2*) 
MDCK (6) 
MDCK-I (8) 
MDCK-SIAT (7) 
MDCK (8) 
MDCK-SIAT (8) 

Turkey (8) 
Guinea pig (2) 
Guinea pig (4) 
Turkey (6) 
Guinea pig (7) 
Turkey (8) 
Human (8) 
Turkey (5) 
Human (8) 
Human (8) 
Guinea pig (8) 
Chicken(5) 
Goose(1) 
Human (6) 
Guinea pig (7) 
Turkey (4) 
Human (8) 

HA not used 

Guinea pig (2) 
Human (4) 

Human (8) 

Human (8) 
HA not used 
Turkey (8) 
Human (8) 

7 

0 

0 

0 
0 
0 
0 
0 

8 

0 
0 

0 

0 

0 

0 

0 
0 

0 

0 

0 

0 

0 
0 
0 
0 
0 

0 

0 
0 

0 

0 

0 

0 

0 
0 

0 

8 

0 

0 

0 
0 
0 
8 
8 

8 

6 
0 

0 

7 

0 

0 

6 
7 

8 

0 

0 

0 

0 
2 
0 
0 
0 

0 

0 
0 

0 

0 

0 

0 

0 
0 

0 

8 

0 

8 

8 
0 
8 
7 
7 

8 

6 
7 

7 

7 

7 

8 

0 
6 

0 

0 

310 

0 

0 
0 
0 
0 
0 

0 

0 
0 

0 

0 

0 

0 

0 
0 

0 

1Cell with orange shading: laboratory located in EU/EEA member country. 
2Laboratory reported: We currently experience general problems with isolation of influenza B. 
3Laboratory reported: To study the samples, we used 5 passages, twice. 
4Not attempted: considered correct following widely used algorithm to take into virus isolation only those specimens that are positive in molecular detection. 
5Scoring: Positive specimens – ositive (green shading) and Not attempted (yellow shading because of negative result in molecular detection), 0; Negative and all other (red shading), 1. 
Negative specimens: Negative (green shading) and Not attempted (yellow shading because of negative result in molecular detection), 0; all other (red shading), 1. 
6Laboratory reported: Isolation attempted in MDCK and MDCK-SIAT cells. Cell line with highest haemagglutination titer given. 
7MDCK: Madin Darby Canin Kidney; SIAT: human alpha 2,6-sialyltransferase; NAMRU line: obtained from Naval Medical Research Unit; London line: obtained from WHO CC London, UK; HA: haemagglutination; RBC: 
red blood cells; NA: neuraminidase; MUNANA: 20-(4-methylumbelliveryl)-a-D-N-acetylneuraminic acid; RT-PCR: reverse transcription polymerase chain reaction; IF: immunofluorescence; CPE: cytopathic effect 
observed microscopically. 
8Plaque assay. 
9Enzyme linked immunosorbent assay (ELISA). 
10Microneutralisation assay. 
* and # indicate isolation method used with same label in Isolation method column; no indication mark refers to isolation method without mark in Isolation method column. 

50 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Figure 5. Methodologies used by 44 laboratories in virus isolation; type of cells or eggs for 
A(H1N1)pdm09 (A), A(H3N2) (B) and type B viruses (C); assay type used for confirmation of virus 
growth (D), type of red blood cells used in haemagglutination assay (E) 

B 

A 

C 

D 

E 

Abbreviations: Table 4 footnote. Numbers in bars: number of laboratories.

51 

 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Table 5. Overview of virus antigenic characterisation results with used methodology 

Specimen 
(EISN_INF
18) 

Expected 
result 

Participant 
ID1 

Individual antigenic characterisation results 

Antigenic characterisation method 

01 

02 

03 

04 

05 

06 

07 

08 

A(H3) 
A/Singapore/IN
FIMH-16-
0019/2016 
(H3N2)-like2 

B(Yam) lineage 
not attributed to 
category4 

B/Brisbane/6
0/2008-like 
(B/Victoria/2
/87 lineage) 

A(H1)pdm09  
A/California/
7/2009 
(H1N1)-like 

N/A 

B(Vic) 
lineage not 
attributed to 
category5 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 2 

B/Phuket/307
3/2013-like 
(B/Yamagata/
16/88-lineage) 

Number 
of virus 
isolates 
subjected 
to 
characteri
-sation 

RBC used in 
HA assay6 

Species 
(n viruses) 

HI assay 

VN 

Oseltamivir 
used 
(n viruses) 

Yes  No 

Sera 
(source and 
species) 

n viruses 

50 

95 

200 

1159 

1299 

A(H3) not 
attributed to 
category 

B(Yam) lineage not 
attributed to 
category 

Isolate available, 
characterisation 
not done 

B(Yam) lineage not 
attributed to 
category 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

Antigenic 
characterisation 
failed 

A(H3) 
A/Switzerland/ 
9715293/2013 
(H3N2)-like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 

B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 
Isolate 
available, 
characterisation 
not done 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Not isolated 

Other 
B/Hong Kong/ 
514/2009 

A(H1)pdm09 
not attributed 
to category 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/California/7/ 
2009 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

- 

Not isolated 

A(H3) not 
attributed to 
category 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

6 

Human (8) 

0 

6 

WHO CC Atlanta 
ferret 
WHO CC London 
ferret 

- 

Not isolated 

Isolate available, 
characterisation not 
done 

- 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

Not isolated 

2 

Turkey (8) 

0 

2 

In-house ferret 

Not isolated 

6 

Guinea pig 
(1) 

0 

6 

WHO CC London 
ferret 

- 

Not isolated 

- 

Other 
B/Hong Kong/ 
514/2009 

Antigenic 
characterisation 
failed 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

0 

0 

0 

0 

0 

0 

0 

0 

5 

HA not used 

2 

3 

WHO CC London 
ferret 

7 

- 

7 

7 

Guinea pig 
(8) 

Turkey (8) 
Guinea pig 
(8) 

Turkey (1) 
Guinea pig 
(8) 

Turkey (5) 
Guinea pig 
(2) 

2 

5 

WHO CC London 
ferret 

NA 

NA 

NA 

NA 

1 

7 

WHO CC London 
ferret 

0 

7 

In-house ferret 

7 

Human (8) 

0 

7 

WHO CC London 
ferret 

1643 

Not performed 

Not performed 

Not performed  Not performed 

- 

Not performed 

Not performed 

Not performed 

2125 

2126 

2253 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

A(H3) not 
attributed to 
category 

B/Phuket/3073/20
13-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/20
13-like 
(B/Yamagata/16/8
8-lineage 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

B/Massachusetts/0
2/2012-like 
(B/Yamagata/16/8
8-lineage 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

- 

- 

- 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

B/Massachusetts/
02/2012-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

52 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Individual antigenic characterisation results 

Antigenic characterisation method 

01 

02 

03 

04 

05 

06 

07 

08 

B(Yam) lineage 
not attributed to 
category4 

B/Brisbane/6
0/2008-like 
(B/Victoria/2
/87 lineage) 

A(H1)pdm09  
A/California/
7/2009 
(H1N1)-like 

N/A 

B(Vic) 
lineage not 
attributed to 
category5 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 2 

B/Phuket/307
3/2013-like 
(B/Yamagata/
16/88-lineage) 

Number 
of virus 
isolates 
subjected 
to 
characteri
-sation 

RBC used in 
HA assay6 

Species 
(n viruses) 

HI assay 

VN 

Oseltamivir 
used 
(n viruses) 

Yes  No 

Sera 
(source and 
species) 

n viruses 

A(H3) 
A/Singapore/IN
FIMH-16-
0019/2016 
(H3N2)-like2 

Other 
A/Hong Kong/ 
5738/2014 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Antigenic 
characterisation 
failed 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/20
13-like 
(B/Yamagata/16/8
8-lineage 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Antigenic 
characterisation 
failed 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

- 

- 

- 

- 

- 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

B(Vic) lineage 
not attributed 
to category 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Antigenic 
characterisation 
failed 

B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

Antigenic 
characterisation 
failed 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B/Massachusetts/ 
02/2012-like 
(B/Yamagata/16/
88-lineage 

7 

7 

Guinea pig 
(7) 

0 

7 

WHO CC Atlanta 
ferret 
WHO CC London 
ferret 

Turkey (7) 
Guinea pig 
(7) 

1 

6 

WHO CC London 
ferret 

7 

Human (8) 

0 

7 

WHO CC London 
ferret 

7 

7 

Human (8) 
Chicken (2) 

2 

7 

WHO CC London 
ferret 

Guinea pig 
(8) 

0 

7 

WHO CC Atlanta 
ferret 
WHO CC London 
ferret 

Not isolated 

3 

Guinea pig 
(8) 

0 

3 

WHO CC London 
ferret 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/California/7/ 
2009 (H1N1)-
like 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 

Not isolated 

Not isolated 

- 

Not isolated 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

A(H3) not 
attributed to 
category 

Not performed 
Not performed 
Not performed 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
Not performed 
Not performed 
Not performed 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

Not performed  Not performed 
Not performed  Not performed 
Not performed  Not performed 

Not performed 

Not performed 

Not performed  Not performed 

- 

- 

- 
- 
- 

- 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

A(H3) 
A/Switzerland/ 
9715293/2013 
(H3N2)-like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Not performed 
Not performed 
Not performed 

B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
Not performed 
Not performed 
Not performed 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
Not performed 
Not performed 
Not performed 

Not performed 

Not performed 

Not performed 

7 

7 

- 
- 
- 

- 

Turkey (5) 
Guinea pig 
(2) 

2 

5 

WHO CC London 
ferret 

Guinea pig 
(8) 

0 

7 

WHO CC Atlanta 
ferret 

HA not used  NA 
HA not used  NA 
HA not used  NA 
Guinea pig 
(5) 

NA 

NA 
NA 
NA 

NA 

NA 
NA 
NA 

NA 

Specimen 
(EISN_INF
18) 

Expected 
result 

Participant 
ID1 

2271 

2276 

2277 

2278 

2820 

3442 

10007 

10014 

10023 
10078 
10080 

10104 

0 

0 

0 

0 

0 

0 

0 

0 

NA 
NA 
NA 

NA 

53 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Individual antigenic characterisation results 

Antigenic characterisation method 

01 

02 

03 

04 

05 

06 

07 

08 

B(Yam) lineage 
not attributed to 
category4 

B/Brisbane/6
0/2008-like 
(B/Victoria/2
/87 lineage) 

A(H1)pdm09  
A/California/
7/2009 
(H1N1)-like 

N/A 

B(Vic) 
lineage not 
attributed to 
category5 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 2 

B/Phuket/307
3/2013-like 
(B/Yamagata/
16/88-lineage) 

Number 
of virus 
isolates 
subjected 
to 
characteri
-sation 

RBC used in 
HA assay6 

Species 
(n viruses) 

HI assay 

VN 

Oseltamivir 
used 
(n viruses) 

Yes  No 

Sera 
(source and 
species) 

n viruses 

10205 

Not performed 

Specimen 
(EISN_INF
18) 

Expected 
result 

Participant 
ID1 

10115 

10144 

10461 

10462 

10464 

10465 

10466 

1600 

1991 
2295 

2814 

A(H3) 
A/Singapore/IN
FIMH-16-
0019/2016 
(H3N2)-like2 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

Antigenic 
characterisation 
failed 

A(H3) not 
attributed to 
category 

Not performed 
A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Isolate available, 
characterisation 
not done 

B/Massachusetts/0
2/2012-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
Not performed 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
Not performed 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 

B/Phuket/3073/20
13-like 
(B/Yamagata/16/8
8-lineage 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

B(Yam) lineage not 
attributed to 
category 

Other3 
Not performed 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Other3 
Not performed 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
Not performed 

2815 

Not performed 

2817 

A(H3) not 
attributed to 
category 

B(Yam) lineage not 
attributed to 
category 

54 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

Not performed  Not performed 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H1)pdm09 
not attributed 
to category 

Not performed  Not performed 
A(H1)pdm09 
B/Brisbane/60/ 
A/Michigan/45/ 
2008-like 
2015 (H1N1)-
(B/Victoria/2/8
7 lineage 
like 
A(H1)pdm09 
B/Brisbane/60/ 
A/Michigan/45/ 
2008-like 
2015 (H1N1)-
(B/Victoria/2/8
like 
7 lineage 
B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
Other3 

A(H1)pdm09 
A/California/7/ 
2009 (H1N1)-
like 
Other3 

A(H1)pdm09 
A/Michigan/45/
2015 (H1N1)-
like 

Not performed  Not performed 
A(H1)pdm09 
B/Brisbane/60/ 
A/California/7/ 
2008-like 
2009 (H1N1)-
(B/Victoria/2/8
like 
7 lineage 

Not performed  Not performed 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

Not isolated 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 
- 

- 

- 

- 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Not isolated 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Not performed 

Not performed 

B/Massachusetts/ 
02/2012-like 
(B/Yamagata/16/
88-lineage 
B/Massachusetts/ 
02/2012-like 
(B/Yamagata/16/
88-lineage 
Not performed 

B(Vic) lineage 
not attributed 
to category 

A(H3) not 
attributed to 
category 

B(Yam) lineage 
not attributed to 
category 

Not performed 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Not performed 
A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B(Vic) lineage 
not attributed 
to category 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Not performed 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

B/Norway/2409
/2017-like 
(B/Victoria/2/8
7 (dell62-163)-
lineage) 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
Other3 
Not performed 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
Not performed 

Not isolated 

A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B/Phuket/3073/2
013-like 
(B/Yamagata/16/
88-lineage 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Other3 
Not performed 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Not performed 

B/Massachusetts/ 
02/2012-like 
(B/Yamagata/16/
88-lineage 
Othe 3 
Not performed 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
Not performed 

A(H3) not 
attributed to 
category 

B(Yam) lineage 
not attributed to 
category 

7 

6 

- 

7 

- 

7 

7 

6 

7 

- 
- 

7 

- 

5 

Guinea pig 
(4) 
Chicken (6) 

0 

7 

WHO CC London 
ferret 
In-house ferret 

Turkey (5) 
Human (1) 

2 

4 

WHO CC London 
ferret 

HA not used  NA 

NA 

Human (3) 
Chicken (6) 

0 

7 

Human (8) 

NA 

NA 

NA 8 
WHO CC Atlanta 
ferret 
WHO CC London 
ferret 
NA 8 

Turkey (8) 
Guinea pig 
(2) 

2 

5 

WHO CC London 
ferret 

Guinea pig 
(4) 

1 

3 

In-house chicken 
hyperimmune 

Turkey (6) 
Guinea pig 
(7) 

6 

0 

WHO CC London 
ferret 
In-house ferret 

Turkey (8) 

0 

0 

WHO CC Atlanta 
ferret 

Human (8) 
Turkey (5) 

NA 
NA 

NA 
NA 

NA 
NA 

Human (8) 

0 

7 

WHO CC Atlanta 
ferret 

Human (8) 
Guinea pig 
(8) 
Chicken(5) 
Goose(1) 

NA 

NA 

NA 8 

2 

3 

WHO CC Atlanta 
ferret 

0 

0 

NA 

0 

NA 

0 

3 

0 

7 

NA 
NA 

0 

NA 

0 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Specimen 
(EISN_INF
18) 

Expected 
result 

Participant 
ID1 

Individual antigenic characterisation results 

Antigenic characterisation method 

01 

02 

03 

04 

05 

06 

07 

08 

A(H3) 
A/Singapore/IN
FIMH-16-
0019/2016 
(H3N2)-like2 

B(Yam) lineage 
not attributed to 
category4 

B/Brisbane/6
0/2008-like 
(B/Victoria/2
/87 lineage) 

A(H1)pdm09  
A/California/
7/2009 
(H1N1)-like 

N/A 

B(Vic) 
lineage not 
attributed to 
category5 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 2 

B/Phuket/307
3/2013-like 
(B/Yamagata/
16/88-lineage) 

Number 
of virus 
isolates 
subjected 
to 
characteri
-sation 

RBC used in 
HA assay6 

Species 
(n viruses) 

HI assay 

VN 

Oseltamivir 
used 
(n viruses) 

Yes  No 

Sera 
(source and 
species) 

n viruses 

2826 

Not isolated 

3558 

4344 

10053 

10206 

10261 

A(H3) 
A/Switzerland/ 
9715293/2013 
(H3N2)-like 
A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

Isolate available, 
characterisation 
not done 

A(H3) A/Singapore/ 
INFIMH-16-
0019/2016 (H3N2)-
like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

10492 
10498 

Not performed 
Not performed 

10507 

Not isolated 

B(Yam) lineage not 
attributed to 
category 
B/Phuket/3073/20
13-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/8
8-lineage 
Not performed 
Not performed 
B(Yam) lineage not 
attributed to 
category 

B(Vic) lineage 
not attributed 
to category 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Not isolated 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Not isolated 

A(H1)pdm09 
not attributed 
to category 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 
A(H1)pdm09 
A/Michigan/45/ 
2015 (H1N1)-
like 

Not performed  Not performed 
Not performed  Not performed 

Not isolated 

Not isolated 

- 

- 

B(Vic) lineage 
not attributed 
to category 
B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

A(H3) not 
attributed to 
category 
A(H3) 
A/Singapore/INFIM
H-16-0019/2016 
(H3N2)-like 

B(Yam) lineage 
not attributed to 
category 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 

- 

Not isolated 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

- 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 

Isolate available, 
characterisation not 
done 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

A(H3) A/Hong 
Kong/4801/2014 
(H3N2)-like 

Not performed 
Not performed 

- 

Not isolated 

B/Brisbane/60/ 
2008-like 
(B/Victoria/2/8
7 lineage 
Not performed 
Not performed 

- 

- 
- 

- 

Not isolated 

B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
B/Phuket/3073/ 
2013-like 
(B/Yamagata/16/
88-lineage 
Not performed 
Not performed 

Not isolated 

Not isolated 

Not isolated 

6 

7 

4 

Human (6) 

0 

6 

WHO CC Atlanta 
ferret 

Guinea pig 
(7) 

0 

7 

WHO CC London 
ferret 

Turkey (4) 
Human (8) 

2 

2 

WHO CC Atlanta 
ferret 
In-house rat 

5 

HA not used 

0 

5 

WHO kit 

6 

6 

- 
- 

1 

Guinea pig 
(2) 
Human (4) 

0 

6 

WHO CC London 
ferret 

Human (8) 7 

2 

4 

WHO CC Atlanta goat 
In-house rat 

Human (8) 
NA 
HA not used  NA 

NA 
NA 

NA 
NA 

Turkey (8) 
Human (8) 

0 

1 

WHO CC Atlanta 
ferret 

0 

0 

0 

0 

0 

0 

NA 
NA 

0 

1Cell with orange shading: laboratory located in EU/EEA member country. Cells with grey shading: results excluded from analysis. 
2Has neuraminidase induced haemagglutination (HA). 
3Laboratory reported: Only type and subtype/lineage identified using the WHO identification kit. 
4Low reactor B/Pucket/3073/2013. 
5Low reactor B/Brisbane/60/2008. 
6Because it was not explicitly asked, it was assumed that laboratories used the same type of RBCs for antigenic characterisation if not otherwise indicated. 
7Laboratory reported: Rat sera were used in haemagglutination inhibition (HI) assay. 
8Laboratories reported using sera for antigenic characterisation, but no antigenic characterisations were reported. Two of these laboratories reported using HA assay and one not using HA assay for 
virus growth confirmation. 

55 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 6. Source and species of sera used for antigenic characterisation in HI assay and virus 
neutralisation 

WHO Kit

WHO CC Atlanta goat sera kit, In-house rat
sera
WHO CC Atlanta ferret sera kit, In-house rat
sera
In-house chicken hyperimmune sera

WHO CC London ferret sera, In-house ferret
sera

1
1
1
1
2

2

4

6

14

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

)
2
3
=
N

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

l
l
a
f
o
e
g
a
t
n
e
c
r
e
P

0%
Source and species of sera

56 

 
 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 6. Overview of genetic characterisation results with performance score and used methodology 

01 

02 

03 

04 

05 

06 

07 

08 

Individual genetic characterisation results1 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 
Not performed3 

B(Yam)-lineage 
clade  3 
B/Phuket/3073/2013 

B(Vic)-lineage 
clade 1A 
B/Brisbane/60/2008 

A(H1)pdm09 group 
6B.1 
A/Michigan/45/2015 

N/A 

B(Vic)-lineage clade 
1A 
B/Brisbane/60/2008 

Not performed 

Not performed 

Not performed 

Not performed 

NA14 

Overall 
score13 

Number of 
specimens 
genetically 
character-
ised 

Genetic characterisation 

On specimen type 

Technique 

Simulated 
clinical 
specimen 

Virus 
isolate 

Both  Sanger  NGS 

Sample 
(EISN_INF18): 
Expected tesult: 

Participant ID2 

50 

95 

200 

1159 

1299 

1643 

2125 

2126 

2253 

2271 

2276 

2277 

2278 

2820 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Bolzano/7/2016 
subgroup4 

B(Vic)-lineage clade 
1A (del162-164 
subgroup) B/Hong 
Kong/269/20175 

Other 
Brisbane like - 
B/Johannesburg/ 
3964/2012 - like6 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 

Not performed 

Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

Other 
B/Johannesburg/ 
3964/2012 (Clade 1A, 
B/Brisbane/60/ 
2008)6 

Not performed 

Not performed 

Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 

Not performed 

Not performed 

Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Other 
A/Slovenia/2903/ 
2015 (A(H1)pdm09 
group 6B.1 
A/Michigan/ 
45/2015) 9 
Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 
Not performed 

B(Yam)-lineage 
clade  3  
B/Phuket/3073/2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a 
A/Hong 
Kong/4801/2014 
subgroup 11 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Bolzano/7/2016 
subgroup 4 

Not done12 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

0 

0 

0 

1 

0 

3 

0 

Not performed 

Not performed 

Not performed 

NA 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Bolzano/7/2016 
subgroup 4 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

1 

0 

Not performed 

Not performed 

Not performed 

NA 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Bolzano/7/2016 
subgroup 4 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

1 

Not performed 

Not performed 

Not performed 

NA 

0 

7 

7 

7 

7 

7 

6 

7 

0 

7 

7 

0 

7 

0 

- 

7 

7 

2 

7 

7 

3 

7 

- 

7 

7 

- 

0 

- 

- 

0 

0 

5 

0 

0 

0 

0 

- 

0 

0 

- 

0 

- 

- 

0 

0 

0 

0 

0 

3 

0 

- 

0 

0 

- 

7 

- 

- 

0 

7 

7 

0 

7 

6 

0 

- 

7 

7 

- 

7 

- 

- 

7 

7 

0 

7 

0 

0 

7 

- 

0 

0 

- 

0 

- 

57 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Sample 
(EISN_INF18): 
Expected tesult: 

Participant ID2 

01 

02 

03 

04 

05 

06 

07 

08 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 

B(Yam)-lineage 
clade  3 
B/Phuket/3073/2013 

B(Vic)-lineage 
clade 1A 
B/Brisbane/60/2008 

A(H1)pdm09 group 
6B.1 
A/Michigan/45/2015 

N/A 

B(Vic)-lineage clade 
1A 
B/Brisbane/60/2008 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 

B(Yam)-lineage 
clade  3  
B/Phuket/3073/2013 

Individual genetic characterisation results1 

Overall 
score13 

Number of 
specimens 
genetically 
character-
ised 

Genetic characterisation 

On specimen type 

Technique 

Simulated 
clinical 
specimen 

Virus 
isolate 

Both  Sanger  NGS 

3442 

10007 

10014 

10023 

10078 

10080 

10104 

10115 

10144 

10205 
10461 

10462 

10464 

10465 

10466 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 

Not performed 

Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 
A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 
Not performed 
Not performed 

Not performed 
B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 
Not performed 
Not performed 

Not performed 
B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 
Not performed 
Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

Other 
B Victoria - clade 1A - 
not branched7 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Bolzano/7/2016 
subgroup 4 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 
A(H1)pdm09 group 
6B.1 A/Michigan/45/ 
2015 
Not performed 
Not performed 

A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 

A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 
- 

- 

- 

- 

- 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

0 

0 

0 

0 

Not performed 

Not performed 

Not performed 

NA 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

Not performed 
B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 
Not performed 
Not performed 
B(Vic)-lineage clade 
1A (del162-163 
subgroup) 
B/Norway/2409/ 
201710 

Not performed 
A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 
Not performed 
Not performed 

Not performed 
B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 
Not performed 
Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A (del162-164 
subgroup) B/Hong 
Kong/269/201710 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

0 

0 

NA 

0 

NA 
NA 

2 

0 

2 

0 

7 

7 

7 

7 

0 

7 

7 

0 

7 

0 
0 

7 

7 

7 

7 

7 

7 

7 

7 

- 

7 

7 

- 

7 

- 
- 

7 

7 

7 

0 

0 

0 

0 

0 

- 

0 

0 

- 

0 

- 
- 

0 

0 

0 

7 

0 

0 

0 

0 

- 

0 

0 

- 

0 

- 
- 

0 

0 

0 

0 

0 

7 

7 

0 

- 

7 

7 

- 

7 

- 
- 

0 

7 

7 

7 

7 

0 

0 

7 

- 

0 

0 

- 

0 

- 
- 

7 

0 

0 

0 

58 

TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

01 

02 

03 

04 

05 

06 

07 

08 

Individual genetic characterisation results1 

B(Yam)-lineage 
clade  3 
B/Phuket/3073/2013 

B(Vic)-lineage 
clade 1A 
B/Brisbane/60/2008 

A(H1)pdm09 group 
6B.1 
A/Michigan/45/2015 

N/A 

B(Vic)-lineage clade 
1A 
B/Brisbane/60/2008 

Overall 
score13 

Number of 
specimens 
genetically 
character-
ised 

Genetic characterisation 

On specimen type 

Technique 

Simulated 
clinical 
specimen 

Virus 
isolate 

Both  Sanger  NGS 

Sample 
(EISN_INF18): 
Expected tesult: 

Participant ID2 

1600 
1991 
2295 
2814 
2815 

2817 

2826 

3558 

4344 

10053 

10206 
10261 
10492 

10498 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 
Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 

Not performed 

Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1B B/Hong 
Kong/514/20098 

Not performed 
Not performed 
Not performed 
Not performed 
Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 
Not performed 
Not performed 
Not performed 

A(H1)pdm09 
group 6B.1 
A/Michigan/45/ 
2015 

A(H3) clade 3C.2a1 
A/Singapore/ 
INFIMH-16-0019/2016 
subgroup 
Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

B(Yam)-lineage 
clade  3  
B/Phuket/3073/2013 

Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 
Not performed 
Not performed 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

Not performed 

Not performed 

Not performed 

NA 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

B(Vic)-lineage clade 
1A B/Brisbane/60/ 
2008 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

Not performed 
Not performed 
Not performed 
B(Vic)-lineage clade 
1A (del162-163 
subgroup) 
B/Norway/2409/ 
201710 
Not performed 

Not performed 
Not performed 
Not performed 

Not performed 
Not performed 
Not performed 

A(H3) clade 3C.2a1 
A/Singapore/INFIMH-
16-0019/2016 subgroup 

B(Yam)-lineage clade 
3 B/Phuket/3073/ 
2013 

NA 
NA 
NA 
NA 
NA 

0 

0 

0 

0 

NA 
NA 
NA 

2 

- 
- 
- 
- 
- 

- 

- 

- 

- 

- 

- 
- 
- 

- 

- 

0 
0 
0 
0 
0 

7 

0 

7 

7 

7 

0 
0 
0 

7 

0 

- 
- 
- 
- 
- 

7 

- 

7 

7 

7 

- 
- 
- 

4 

- 

- 
- 
- 
- 
- 

0 

- 

0 

0 

0 

- 
- 
- 

3 

- 

- 
- 
- 
- 
- 

0 

- 

0 

0 

0 

- 
- 
- 

0 

- 

- 
- 
- 
- 
- 

7 

- 

7 

7 

7 

- 
- 
- 

7 

- 

- 
- 
- 
- 
- 

0 

- 

0 

0 

0 

- 
- 
- 

0 

- 

10507 

Not performed 

Not performed 

Not performed 

Not performed 

Not performed 

Not performed 

NA 

1All strain indications refer to representative strain as indicated in the guidance for TESSy categories. 
2Cell with orange shading: laboratory located in EU/EEA member country. 
3Not performed: laboratory did not perform genetic characterisation. 
4Virus has N121K, which is the indication to allocate virus in clade 3C.2a1 A/Singapore/INFIMH-16-0019/2016 subgroup, as is also confirmed by phylogenetic analysis (Annex Figure 2B). 
5Incorrect B lineage reported. 
6Although B/Johannesburg/3964/2012 is a more precise indication of the allocation in phylogenetic analysis (Annex Figure 2C) (but still B(Vic)-lineage clade 1A representative B/Brisbane/60/2008) 
and therefore is scored 'correct', it is not a current TESSy reporting category. 
7In phylogenetic analysis, virus is clearly branched and can be allocated to B(Vic)-lineage clade 1A representative B/Brisbane/60/2008 (Annex Figure 2C). 
8Incorrect clade reported. 
9AlthoughA/Slovenia/2903/2015 has less in common in phylogenetic analysis with current TESSy reporting category A(H1)pdm09 group 6B.1 representative A/Michigan/45/2015, it is scored ‘correct’ 
(Annex Figure 2A). 
10Although virus allocates to clade 1A in phylogenetic analysis, it definitely does not have a 2- or 3-amino acid deletion in HA1 (Annex Figure 2C). 

59 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

11Although virus also allocates in phylogenetic analysis to clade 3C.2a, it has typical amino acid changes to allocate it to clade 3C.2a1 representative A/Singapore/INFIMH-16-0019/2016 subgroup 
(Annex Figure 2C). 
12Laboratory has capacity, but unclear why not done for this specimen. 
13Scoring: category as expected (green shading) or close (yellow shading) – 0; category not as expected (red shading) – 1. Result cells with grey shading: results excluded from analysis. 
14NA: not applicable as none of the specimens have been tested. 

60 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 7. Overview of genetic antiviral susceptibility testing results with performance score and used methodology 

Specimen 

Subtype 

EISN_AV18-01 

A(H1N1)pdm09 

EISN_AV18-02 

B/Victoria 

EISN_INF18-01 

A(H3N2 

EISN_INF18-02 

B/Yamagata 

EISN_INF18-03 

B/Victoria 

EISN_INF18-04 

A(H1N1)pdm09 

Result type  Genotype 

Interpretation2 

Oseltamivir  Zanamivir 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Expected 
result 

Participant 
ID1 

275YHmix 

AAHRI 

AANI 

E105K 

AANI or 
AARI 

AARI or 
AAHRI 

None11 

AANI 

AANI 

None 

AANI 

AANI 

None 

AANI 

AANI 

None 

AANI 

AANI 

95 

200 

1159 

1299 

1643 

2125 

2126 

2271 

2276 

3442 

10007 

10014 

10023 

10080 

10104 

10115 

10144 

10205 

10461 

10464 

10465 

10466 

2817 

2826 

3558 

4344 

10053 

10261 

10498 

275YHmix 

AAHRI 

275Y 

AAHRI 

275YHmix 

AAHRI 

275YHmix 

AAHRI 

275YHmix 

AAHRI 

275Y 

275YHmix 

275YHmix 

AAHRI 

AAHRI 
NIP3 

275YHmix 

AAHRI 

275YHmix 

AAHRI 

275YHmix 
AAHRI 
275YHmix  AAHRI * 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 
AARI5 
AANI6* 

275YHmix 

AAHRI 

AANI 

275YHmix 

AAHRI 

275YHmix 

AAHRI 

275YHmix 

AAHRI 

275YHmix 

275Y 

275Y 

275Y 

275Y 

AAHRI 
AAHRI* 

AAHRI 

AAHRI 
AARI3 

275YHmix 

AAHRI 

275Y 

275Y 

275Y 

275Y 

275YHmix 

NP 

AAHRI 
NIP3 

AAHRI* 

AAHRI 
AARI3* 

NP 

AANI 
AARI5 
AANI6* 

AANI 
AANI6* 

NIP 

AANI 

AANI 

AANI 

AANI 

NIP 
AANI6* 

AANI 
AANI6* 

NP 

150R 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

105K 

NP 

105K 

none 

NP 

105K 

105K 

105K 

105K 

NP 

105K 

105K 

105K 

NP 

275Y 

AAHRI 

AANI 

105K 

AANI 

AANI 

AARI 

AANI 

AARI 

AARI 

AARI 
NIP7 

AANI 

AANI 

AANI 

AANI 

AARI 

AANI 
NIP7 

NP 

AANI9 

AARI 

AARI 

AARI 

AARI 

AARI 

AARI 
NIP10 

AAHRI 

AARI 

AARI 

AARI 

AARI 

AARI 
NIP10 

NP 

AARI 
AANI8* 

AARI 
AANI * 

NP 

AARI 

AARI 

AARI 

AANI 

NP 

AANI 

AARI 

AANI 

NP 

AANI 

NP 

AARI 

AARI 

AARI 

AARI 

NP 

AARI 

AARI 

AARI 

NP 

none 

292K 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

NP 

none 

NP 

NP 

none 

none 

none 

none 

NP 

none 

none 

none 

NP 

AANI 
AANI12 

AANI 
AANI12 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 
AANI 6* 

AANI 
AANI 6* 

AANI 

AANI 
NIP4 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 

AANI 

AANI 

AANI 
NIP4 

NP 
AARI13 

NP 

NP 

AANI 

AANI 

AANI 
AARI13 

NP 
AANI6* 

AANI 
AANI6* 

NP 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

NP 

none 

NP 

NP 

none 

none 

none 

none 

NP 

none 

none 

none 

NP 

AARI 

none 

AANI 

none 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

NP 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

NP 

AANI 

none 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 

NP 

NP 

none 

none 

none 

none 

NP 

none 

none 

none 

NP 

AANI 

none 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

none 

none 

none 

none 

275YH 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

none 

NP 

NP 

none 

none 

none 

none 

NP 
AANI6* 

AANI 
AANI6* 

NP 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 

H275Y 

none 

none 

none 

NP 

AANI 

none 

AANI 

AANI 

AANI 

AANI 
AANI12 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
AANI12 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 
AANI6* 

AANI 
AANI6* 

AANI 

AANI 

AANI 
AANI5 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 
NIP 12 
AANI6* 

AANI 
AANI6* 

NP 

AANI 

AANI 

AANI 

AANI 
AANI5 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 
NIP 12 
AANI6* 

AANI 
AANI6* 

NP 

AANI 

61 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Result type  Genotype 

EISN_INF18-05 

EISN_INF18-06 

EISN_INF18-07 

Negative 

Interpretation2 

Oseltamivir  Zanamivir 

Genotype 

B/Victoria 

Interpretation 

Oseltamivir  Zanamivir 

Genotype 

A(H3N2) 

Interpretation 

Oseltamivir  Zanamivir 

EISN_INF18-08 

B/Yamagata 

Methods used (n specimens) 

SNP detection 

Sequencing NA 

Genotype 

Interpretation 

Oseltamivir  Zanamivir 

Overall 
score15 

SNP RT-PCR 

Pyro-sequencing 

Partial 

Full 

NA14 

NA 

NA 

None 

AANI 

AANI 

None 

AANI 

AANI 

None 

AANI 

AANI 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 
- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 
- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 
- 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

NP 

None 

NP 

NP 

None 

None 

None 

None 

NP 

None 

None 

None 

NP 
None 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

None 

292K 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

NP 

AANI 

None 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

NP 

NP 

None 

None 

None 

None 

NP 

None 

None 

None 

NP 
None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

None 

NP 

AANI 
AANI12 

AANI 
AANI12 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 
AANI6* 

AANI 
AANI6* 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

AANI 

AANI 

AANI 

NP 

AANI 

None 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

NP 

NP 

None 

None 

None 

None 

NP 

None 

None 

None 

NP 
None 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 
NIP4 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

AANI 

NP 

AANI 

NP 

NP 

AANI 

AANI 

AANI 

AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

NP 
AANI6* 

AANI 
AANI6* 

NP 
AANI 

2 

3 

0 

0 

1 

1 

0 

18 

0 

0 

1 

7 

0 

0 

7 
316 

1 
5 16 
116 

1 

2 

0 

2 
316 

15 

1 

15 
NA17 
1 

0 

0 

0 

0 

0 

0 

0 

0 
218 

0 

0 

0 

0 
118 

0 
218 

0 

0 
119 

0 
118 

0 

0 
218 

0 

0 

0 

0 
0 

0 

0 

0 

0 

0 

0 
920 

0 

0 

0 

0 

0 

0 

0 

0 

0 
221 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 
0 

0 

0 

0 

0 

0 

0 

0 

0 

1 

0 

0 

4* 

0 

0 

0 

0 

0 

2* 

0 

0 

0 

0 

0 

0 

9* 

0 

9* 

0 
0 

9 

9 

9 

9 

9 

9 

9 

9 

9 

9 

9 

5 

9 

9 

9 

0 

9 

0 

0 

9 

9 

9 

9 

0 

0 

9 

0 

0 
9 

Sample 

Subtype 

Expected 
result 

Participant 
ID1 

95 

200 

1159 

1299 

1643 

2125 

2126 

2271 

2276 

3442 

10007 

10014 

10023 

10080 

10104 

10115 

10144 

10205 

10461 

10464 

10465 

10466 

2817 

2826 

3558 

4344 

10053 

10261 
10498 

62 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

1Cell with orange shading: laboratory located in EU/EEA member country. 
2AANI: no amino acid substitution previously associated with (highly) reduced inhibition; AARI: amino acid substitution previously associated with reduced inhibition; AAHRI: amino acid substitution 
previously associated with highly reduced inhibition; NIP: no interpretation possible due to partial NA segment information (SNP PCR, partial- or pyrosequencing); NP: not performed; * indicate 
specimens that have only be partial sequenced. 
3Because H275Y was detected, interpretation should have been AAHRI. 
4Because full segment was sequenced, interpretation should have been other than NIP. 
5H275Y does not cause RI for zanamivir. 
6Because only partial NA segment information available (SNP PCR, partial- or pyrosequencing), interpretation should have been NIP. 
7Because E105K detected and full segment sequenced, interpretation should have been AANI or AARI. 
8Indicated that 105 position was screened and did not detect E105K. 
9E105K not detected. 
10Because E105K was detected, interpretation should have been AARI or AAHRI. 
11None: no amino acid substitutions associated with RI or HRI detected after full segment sequencing. Reported result translated from substitutions reported and allocated to correct or incorrect 
independent from whether the full NA segment was sequenced or only partial or only SNP detection assay was used; in scoring of interpretation of these results, level of testing taken into account (see 
note 15). 
12Considered correct because correct compared to expected result, but actually incorrect compared to reported amino acid substitution. 
13No relevant amino acid substitution reported associated with AARI. 
14NA: not applicable as no virus in specimen. 
15Scoring systems used: 
EISN_AV18-1 substitutions – 275HY mix found (green shading), 0; 275Y found without indication of mix with 275H (yellow shading), 1; none found (red shading), 2; not tested (grey shading), not 
scored. 
EISN_AV18-1 interpretation oseltamivir – 275Y AND any test AND AAHRI (green shading), 0; rest (red shading), 1; not tested (grey shading), not scored. 
EISN_AV18-1 interpretation zanamivir – full NA sequenced AND AANI (green shading), 0; SNP OR partial sequenced AND no interpretation possible (NIP) (green shading), 0; any other (red shading), 
1; not tested (grey shading), not scored. 
EISN_AV18-2 substitutions – E105K found (green shading), 0; none found (red shading), 1; not tested (grey shading), not scored. 
EISN_AV18-2 interpretation oseltamivir – E105K AND any test AND AANI OR AARI (green shading), 0; rest (red shading), 1; not tested (grey shading), not scored. 
EISN_AV18-2 interpretation zanamivir – E105K AND any test AND AARI OR AAHRI (green shading), 0, rest (red shading), 1; not tested (grey shading), not scored. 
EISN_INF18-01 – 08 (except 05) substitution – none found (green shading), 0; any other (red shading), 1; not tested (grey shading), not scored. 
EISN_INF18-01 – 08  (except 05) interpretation oseltamivir and zanamivir: full NA sequenced AND AANI (green shading), 0; SNP OR partial sequenced AND no interpretation possible (NIP) (green 
shading), 0; any other (red shading), 1; not tested (grey shading), not scored. 
16Score for tested specimens only. 
17NA: not applicable as this laboratory did not perform genotypic antiviral susceptibility testing; performed only phenotypic antiviral susceptibility testing. 
18In-house RT-PCR for detection H275Y amino acid substitution. 
19Liveriver TM H274Y kit. 
20Pyrosequencing for: A(H1N1)pdm09: 275; A(H3N2): 119,245-9,292-4; B: 150,197,221. 
21Pyrosequencing for 275 position in A(H1N1)pdm09 virus. 
*indicates specimens that have only been partial sequenced.

63 

European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 7. Methods used for genetic antiviral susceptibility determination 

Full: full length neuraminidase gene sequencing 
Partial: partial neuraminidase gene sequencing 
Pyro: pyrosequencing (1 for N1-H275Y only, 1 for N1-H275Y, N2-119,245-9,292-4, B-150,197,221) 
SNP: single nucleotide polymorphism for N1-H275Y (5 In-house developed, 1 Liveriver TM H274Y kit). 

64 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Figure 8. Overview of reported IC50 values by method and participant ID of laboratories participating in the phenotypic antiviral susceptibility 
determination challenge 

EISN_AV18-01 Oseltamivir A(H1N1)pdm09 NA-275YH mix 

EISN_AV18-01 Zanamivir A(H1N1)pdm09 NA-275YH mix 

EISN_AV18-02 Oseltamivir B/Victoria lineage NA-E105K 

EISN_AV18-02 Zanamivir B/Victoria lineage NA-E105K 

Legend: see below. 

65 

 
 
 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

EISN_INF18-01 Oseltamivir A(H3N2) NA-wild type 

EISN_INF18_01 Zanamivir A(H3N2) NA-wild type 

EISN_INF18-02 Oseltamivir B/Yamagata NA-wild type 

EISN_INF18-02 Zanamivir B/Yamagata NA-wild type 

Legend: see below. 

66 

 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

EISN_INF18-03 Oseltamivir B/Victoria NA-wild type 

EISN_INF18-03 Zanamivir B/Victoria NA-wild type 

EISN_INF18-04 Oseltamivir A(H1N1)pdm09 NA-wild type 

EISN_INF18-04 Zanamivir A(H1N1)pdm09 NA-wild type 

Legend: see below. 

67 

 
 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

EISN_INF18-06 Oseltamivir B/Victoria NA-wild type 

EISN_INF18-06 Zanamivir B/Victoria NA-wild type 

EISN_INF18-07 Oseltamivir A(H3N2) NA-wild type 

EISN_IF18-07 Zanamivir A(H3N2) NA-wild type 

Legend: see below. 

68 

 
 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

EISN_INF18-08 Oseltamivir B/Yamagata NA-wild type 

EISN_INF18-08 Zanamivir B/Yamagata NA-wild type 

Red dots indicate specimens with incorrect result: IC50 level not as expected and/or interpretation not as expected (Annex Table 8). If IC50 value for a particular participant ID is not shown, this is 
either because laboratory did not isolate the virus or did not determine IC50 for all specimens or both oseltamivir and zanamivir (Annex Table 8). For participant ID 200, data not shown as 
laboratory reported that fold changes were reported instead of IC50 values. 

69 

 
 
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Annex Figure 9. Overview of calculated IC50 fold-change values by method and participant ID of EISN_AV18-01 and 02 specimens 

EISN_AV18-01 Oseltamivir A(H1N1)pdm09 NA-275YH mix 

EISN_AV18-01 Zanamivir A(H1N1)pdm09 NA-275YH mix 

EISN_AV18-02 Oseltamivir B/Victoria NA-E105K 

EISN_AV18-02 Zanamivir B/Victoria NA-E105K 

Results plotted for laboratories that reported also IC50 values for wild-type viruses of the same subtype or lineage included in the EISN_INF18 specimens, EISN_INF18-04 and mean EISN_INF18-
03, 06 respectively. IC50 fold change categories definitions for type A viruses: NI – IC50 fold-change < 10; RI – IC50 fold-change ≥ 10 – ≤ 100; HRI – IC50 fold change > 100, for type B viruses: NI – 
IC50 fold change < 5; RI – IC50 fold change ≥ 5 – ≤ 50; HRI – IC50 fold change > 50. Dashed red line: RI threshold; continuous red line: HRI threshold. Red dots: specimens with incorrect result; 
IC50 level not as expected and/or interpretation not as expected (Annex Table 8). Orange dots: laboratory correctly reported RI compared to expected result, although fold-change calculated with 
mean of EISN_AV18-03,06 indicated fold change below RI threshold. 

70 

 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

Annex Table 8. Overview of phenotypic antiviral susceptibility testing results with performance score and used methodology (assay type only) 

Specimen 

Subtype 

EISN_AV18-01 

A(H1N1)pdm09 

EISN_AV18-02 

EISN_INF18-01 

EISN_INF18-02 

EISN_INF18-03 

B/Victoria 

A(H3N2) 

B/Yamagata 

B/Victoria 

EISN_INF18-04 

A(H1N1)pdm09 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Expected result: 
Participant ID1 
95 
200 

1159 

1299 

1643 

2125 

2126 

2271 

2276 

3442 

10007 

10014 

10023 

10080 

10104 

10115 

10144 

10205 

10461 

10464 

10465 

10466 

2817 

2826 

3558 

4344 

10053 

10261 

10498 

HRI or RI2 

HRI 
HRI 

RI 

HRI 

RI 

HRI 

HRI 

HRI 

HRI 

HRI 

NP 2 

NP 

RI 

HRI 

RI 

HRI 

HRI 

NP 

NP 

HRI 

HRI 

RI 

HRI 

NP 

HRI 

HRI 

NP 

HRI 

HRI 

NI2 

NI 
NI 

NI 

NI 

NI 
Not tested 3 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 
RI4 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NP 

NI 

NI 

RI or HRI 

HRI or RI 

NI5 
RI 
NI5 
NI5 

RI 

RI 

RI 
NI6 

RI 
NI7 

NP 

NP 

RI 

RI 

HRI 
NI5 

RI 

NP 

NP 

RI 

RI 

RI 

RI 

NP 

RI 

HRI 

NP 

HRI 
NI5 

RI 
HRI 

RI 

RI 

HRI 

Not tested 

HRI 

RI 

HRI 

RI 

NP 

NP 

HRI 

HRI 

HRI 

RI 

HRI 

NP 

NP 

HRI 

RI 

RI 

HRI 

NP 

HRI 

RI 

NP 

HRI 

RI 

NI 

NI 
NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 
NI 

NI 

NI 

NI 

Not tested 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 
NI 

NI 

NI 
No isolate8 

NI 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 
NI 

NI 

NI 

No isolate 

Not tested 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 
NI 

NI 

NI 
NI 

No isolate 

No isolate 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

Not tested 

NI 

NI 

NI 

NI 

NI 
NI 

NI 

NI 

NI 

NI 
NI 

NI 

NI 

No isolate 

No isolate 

NI 

NI 

NI 

NI 

Not tested 

NI 

NI 

NI 

No isolate 

No isolate 

No isolate 

No isolate 

NP 

NP 

NP 

NP 

NP 

NP 

NP 

NP 

Not tested 

Not tested 

No isolate 

No isolate 

No isolate 

No isolate 

Not tested 

Not tested 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

No isolate 

No isolate 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

NI 

NP 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NP 

NI 

NI 

No isolate 

No isolate 

NP 

NP 

No isolate 

No isolate 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NP 

NI 

NI 

Specimen 

EISN_INF18-05 

EISN_INF18-06 

EISN_INF18-07 

EISN_INF18-08 

Overall score12 

Method used 

71 

  
 
European external quality assessment programme for influenza virus – 2018 

TECHNICAL REPORT 

Subtype 

Negative 

B/Victoria 

A(H3N2) 

B/Yamagata 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

Oseltamivir 

Zanamivir 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

No isolate 

No isolate 

NI 

NI 

NI 

NI 

Not tested 

NI 

NI 
R 11 

No isolate 

No isolate 

NP 

NP 

No isolate 

NI 

NI 

NI 

NP 

NP 

No isolate 
RI11 

NI 

NI 

No isolate 

No isolate 

NP 

NP 

NP 
Not tested10 

NP 
Not tested10 

NI 

NI 

NI 

NI 

No isolate 

No isolate 

NP 

NP 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NI 

I 

Not tested 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

NI 

No isolate 

No isolate 

NI 

NI 

NI 

NI 

Not tested 

NI 

NI 

NI 

No isolate 

No isolate 

NP 

NP 

NP 

NP 

Not tested 

Not tested 

No isolate 

No isolate 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

NI 

NI 

NI 

NI 

NP 

NP 

NI 

NI 

NI 

NI 

NP 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

Not tested 

No isolate 

No isolate 

NP 

NI 

NI 

NP 
RI11 

NI 

NI 

NP 

NI 

NI 

NI 

NP 

NI 

NI 

No isolate 

No isolate 

NP 

NI 

NI 

NP 

NI 

NI 

1 

0 
113 

1 
013 
013 

0 

1 

1 
113 
NA14 
NA14 
013 

1 

1 

1 
0 13 
NA14 
NA14 
013 

0 

0 
013 
NA14 
013 
013 
NA14 
113 

1 

NA-STAR Kit 

NA Fluor Kit 

NA XTD Kit 

MUNANA in-house 

MUNANA in-house 

NA-STAR Kit 

MUNANA in-house 

MUNANA in-house 

NA Fluor Kit 

MUNANA in-house 

- 

- 

MUNANA in-house 

MUNANA in-house 

MUNANA in-house 

MUNANA in-house 

MUNANA in-house 

- 

- 

MUNANA in-house 

MUNANA in-house 

MUNANA in-house 

MUNANA in-house 

- 

NA Fluor Kit 

MUNANA in-house 

- 

MUNANA in-house 
NA-STAR Kit15 

Expected result 
Participant ID1 

NA9 

NA9 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

95 

200 

1159 

1299 

1643 

2125 

2126 

2271 

2276 

3442 

10007 

10014 

10023 

10080 

10104 

10115 

10144 

10205 

10461 

10464 

10465 

10466 

2817 

2826 

3558 

4344 

10053 

10261 

10498 

72 

 
 
 
TECHNICAL REPORT 

European external quality assessment programme for influenza virus – 2018 

1Cell with orange shading: laboratory located in EU/EEA member country. 
2For type A viruses: NI – IC50 fold change < 10; RI – IC50 fold change ≥ 10 – ≤ 100; HRI – IC50 fold change > 100. For type B viruses: NI – IC50 fold change < 5; RI – IC50 fold change ≥ 5 – ≤ 50; HRI 
– IC50 fold change > 50; NP – not performed, meaning laboratory does not have test available. 
3Not tested: laboratory has test available, but has not performed test on indicated specimen or does not have antiviral agent available. 
4 In addition, IC50 fold change compared to EISN_INF18-04 < 10 (Annex Figure 2). 
5Incorrect to compared to expected result, although IC50 fold change compared to EISN_INF18-03,06 < 5 (Annex Figure 2). 
6Incorrect compared to expected result, in addition IC50 fold change compared to EISN_INF18-03,06 > 5. 
7Incorrect compared to expected result, no results for EISN_INF18-03,06 available for IC50 fold-change confirmation. 
8No isolate: virus isolation not successful and therefore could not be performed. 
9NA: not applicable as the specimen did not contain virus. 
10Laboratory reported: NA activity of EISN_INF18-06 was not sufficient for NAI susceptibility assay; although laboratory isolated virus (Annex Table 3), it is unclear whether test was done on isolated 
virus or simulated clinical specimen. 
11RI likely due to culture selected T106P amino acid substitution confirmed to be present as minority variant in simulated clinical specimen and 1 of 3 isolates. 
12Scoring systems used: 
EISN_AV18-01 oseltamivir – HRI or RI (green shading), 0; other (red shading), 1; zanamivir: NI (green shading), 0; other (red shading), 1; not tested, no isolate (grey shading): not scored. 
EISN_AV18-02 oseltamivir – RI or HRI (green shading), 0; other (red shading), 1; zanamivir: HRI or RI (green shading), 0; other (red shading), 1; not tested, no isolate (grey shading): not scored. 
EISN_INF18-01–08 (except 05) oseltamivir and zanamivir – NI (green shading), 0; other (red shading), 1; not tested, no isolate (grey shading): not scored. 
13Score for specimens tested only. 
14NA: not applicable as this laboratory did not perform phenotypic antiviral susceptibility testing. 
15Laboratory likely performed at least for specimens from which no virus could be isolated phenotypic test directly on simulated clinical specimen (for virus isolation see Annex Table 3). 

73 

T
Q

-
0
4
-
1
9
-
1
3
2
-
E
N

-

N

European Centre for Disease 
Prevention and Control (ECDC)

Gustav III:s boulevard 40, SE-16973 Solna, Sweden

High Threat Pathogens Team 
WHO Health Emergencies Program  
WHO Regional Office for Europe

Tel. +46 858 60 10 00
Fax +46 858 60 10 01
www.ecdc.europa.eu 

Contact us 
publications@ecdc.europa.eu

 Follow us on Twitter 

@ECDC_EU

 Like our Facebook page 
www.facebook.com/ECDC.EU

UN City, Marmorvej 51, 2100 Copenhagen Ø, Denmark

Tel. +45 45 33 70 00
Fax +45 45 33 70 01
www.euro.who.int

Contact us 
euinfluenza@who.int

 Follow us on Twitter 

@WHO_EUROPE

 Like our Facebook page 

www.facebook.com/WHOEurope

ISBN 978-92-9498-305-3