Medical microbiology laboratories in the
Netherlands can detect animal type A influenza
viruses well. External Quality Assessment 2023

RIVM report 2023-0392
S. Zoomer et al.

Medical microbiology laboratories in the
Netherlands can detect animal type A influenza
viruses well. External Quality Assessment 2023
RIVM report 2023-0392

RIVM report 2023-0392

Colophon

© RIVM 2023
Parts of this publication may be reproduced, provided acknowledgement
is given to the National Institute for Public Health and the Environment,
and the title and year of publication are cited.
RIVM attaches a great deal of importance to the accessibility of its
products. However, it is at present not yet possible to provide this
document in a completely accessible form. If a part is not accessible, it
is mentioned as such. Also see www.rivm.nl/en/accessibility
DOI 10.21945/RIVM-2023-0392
S. Zoomer (author), RIVM
G. Goderski (author), RIVM
S. van den Brink (author), RIVM
J. Sluimer (author), RIVM
R. Fouchier (author), Erasmus MC
O. Vuong (author), Erasmus MC
E. van der Vries (author), Royal GD
M. Houben (author), Royal GD
A. Meijer (author), RIVM
Contact:
Adam Meijer
IDS, Emerging en Endemische Virussen
adam.meijer@rivm.nl
This investigation was commissioned by and performed on behalf of the
Ministry of Health, Welfare and Sport, within the framework of the
’Kamerbrief Reactie DB-Z advies Vogelgriep’ of June 1st 2022, which
aims to organise a quality control program for all diagnostic laboratories
for the detection of potentially zoonotic bird flu viruses.

Published by:
National Institute for Public Health
and the Environment, RIVM
P.O. Box 1 | 3720 BA Bilthoven
The Netherlands
www.rivm.nl/en
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Synopsis

Medical microbiology laboratories in the Netherlands can detect
animal type A influenza viruses well. External Quality
Assessment 2023
Type A and B influenza viruses can cause severe disease in humans. The
type A influenza virus has many subtypes, which also infect birds and
swine. In the Netherlands, what are known as ‘medical microbiology
laboratories’ test whether people are infected with an influenza virus.
This provides insight into the amount of people infected and whether
they were infected by type A or B influenza virus.
The H5 subtype of type A caused a worldwide outbreak of bird flu in
2022, the largest since 2003. The H5 subtype also infected a growing
number of mammals, raising the concern about a growing possibility
that this virus subtype can infect humans (zoonosis). The same concern
exists about swine influenza viruses. The chance of zoonosis might
increase further when avian and swine influenza viruses mix with each
other. A zoonosis like that could potentially lead to a pandemic. That is
why an external quality assessment (EQA) has been carried out to
investigate whether the laboratories can detect bird and swine influenza
viruses. Fifty laboratories in the Netherlands, Aruba, Bonaire and
Curaçao participated.
All laboratories have at least one routine test with which they were able
to identify animal influenza viruses in the EQA as a type A influenza
virus. A number of these tests identified the subtype of the human
viruses, but most did not identify the subtype of the avian and swine
viruses. Using additional tests, four laboratories identified the H5
subtype of avian influenza viruses. A number of laboratories reported
that they are able to perform non-routine tests to identify the subtype.
It is important that medical doctors are aware of the possibility of
humans getting infected by an animal influenza virus. If they suspect
such an infection in a patient with influenza-like symptoms, further
investigation is needed. That is also the case if a laboratory detects a
type A influenza virus in a patient that is not of a human subtype. This
investigation is conducted by the National Influenza Centre (RIVM and
Erasmus MC), which can discriminate between all human and animal
influenza viruses.
Keywords: swine flu, avian flu, H5N1, medical microbiology laboratory,
National Influenza Centre, molecular diagnostic test, subtyping,
influenza

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Publiekssamenvatting

Medisch microbiologische laboratoria in Nederland kunnen
dierlijke type A griepvirussen goed aantonen. Kwaliteitscontrole
2023
Typen A en B griepvirussen kunnen ernstige ziekte bij mensen
veroorzaken. Het type A griepvirus heeft veel subtypen en komt ook bij
vogels en varkens voor. In Nederland testen de zogeheten ‘medisch
microbiologische laboratoria’ of mensen besmet zijn met een griepvirus.
Dat geeft inzicht in hoeveel mensen besmet zijn met een griepvirus en
of het virus van type A of B is.
Het H5 subtype van type A veroorzaakte wereldwijd een vogelgriep
uitbraak in 2022, de grootste sinds 2003. Ook raken steeds meer
zoogdieren door subtype H5 besmet. De zorg bestaat dat hierdoor de
kans groter wordt dat dit virus ook mensen kan gaan besmetten
(zoönose). Dat geldt ook voor varkensgriepvirussen. De kans dat
mensen besmet raken wordt nog groter als vogel- en
varkensgriepvirussen zich met elkaar vermengen. Zo’n zoönose zou een
nieuwe pandemie kunnen veroorzaken. Daarom is met een
kwaliteitscontrole onderzocht of de laboratoria vogel- en
varkensgriepvirussen kunnen opsporen. Vijftig laboratoria in Nederland,
Aruba, Bonaire en Curaçao hebben deelgenomen.
Alle laboratoria hebben minimaal één routinetest waarmee ze van de
dierlijke griepvirussen in de kwaliteitscontrole konden aantonen dat het
om een type A griepvirus gaat. Een aantal van deze testen herkende het
subtype van menselijke virussen, maar de meeste testen herkenden het
subtype van de vogel en varkens virussen niet. Met extra testen
herkenden vier laboratoria het H5 subtype van vogelgriepvirussen. Een
aantal laboratoria gaf aan speciale, niet-routine testen in te kunnen
zetten om het subtype te herkennen.
Het is belangrijk dat artsen zich bewust zijn van een mogelijke
besmetting bij mensen met een dierlijk griepvirus. Als artsen zo’n
besmetting vermoeden bij een patiënt met griepachtige klachten is
verder onderzoek nodig. Dat is ook het geval als een laboratorium bij
een patiënt een type A griepvirus aantoont dat geen menselijk subtype
is. Dat onderzoek gebeurt bij het Nationaal Influenza Centrum (het
RIVM en Erasmus MC), dat alle menselijke en dierlijke griepvirussen kan
onderscheiden.
Kernwoorden: varkensgriep, vogelgriep, H5N1, medisch microbiologisch
laboratorium, Nationaal Influenza Centrum, moleculair diagnostische
test, subtypering, griep

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Content

Summary — 9
1

Introduction — 11

2
2.1
2.2
2.3

Materials and methods — 13
Approach — 13
Panel preparation and contents — 13
Dataset and analysis — 13

3
3.1
3.2
3.3
3.4
3.5

Results — 17
Aggregated overview — 17
Cq values per target gene — 17
Subtyping — 18
Response from manufacturers and laboratories — 19
Follow-up suspect animal influenza case — 21

4

Discussion and conclusion — 27

5

Acknowledgements — 31

6

References — 33

7
7.1
7.2
7.3
7.4
7.5

Supplementary material — 37
Equipment and kits used — 37
Performance per workflow — 41
In house RIVM results of commercial kits — 43
Correlation Cq value and number of copies/specimen — 43
Participating laboratories — 46

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Summary

Background
During the global outbreak of highly pathogenic H5N1 (clade 2.3.4.4b)
type A influenza virus in over one hundred million wild birds and poultry
in 2022 and 2023, an expanding range of mammals has been found
infected. A total of eight infected people has also been reported, most
after direct contact with affected poultry. This amplified the existing
concern about the enzootic circulation of the H5N1 influenza virus and
transmission to humans. Therefore, the need for correct detection and
identification of animal influenza A viruses increased. It is important that
the influenza molecular diagnostic tests routinely used in medical
microbiology laboratories are able to detect animal influenza viruses.
After a positive generic test for influenza A virus and reported animal
exposure in the clinical history of the patient, rapid subtyping at the
National Influenza Centre (NIC) can be requested to identify a patient
with animal influenza virus infection, in line with the International Health
Regulations of the World Health Organization (WHO). Most commercial
Nucleic Acid Amplification Tests (NAATs) for generic detection of
influenza A virus have been validated by the manufacturer for animal
influenza A virus detection. However, in many cases this validation is
not frequently updated for recently circulating or emerging viruses.
Objective
As requested by the Ministry of Health, Welfare and Sport, the primary
aim of the current External Quality Assessment (EQA) was to investigate
whether the influenza molecular diagnostic tests routinely used in the
medical microbiology laboratories in the Netherlands are capable of
detecting avian influenza viruses. Due to concerns about the
transmission of swine influenza viruses to humans and the possible
mixing of swine influenza viruses and avian influenza viruses, the NIC
has extended this aim to include (potentially) zoonotic swine influenza
viruses. Apart from detecting animal influenza A viruses, the secondary
aim of the EQA was to investigate if the laboratories were able to
identify the subtype of the detected influenza A viruses, and if so, if they
could correctly identify at least the H-subtype.
Material and Methods
To assess the performance of the molecular diagnostic tests, the RIVM
produced a panel with various inactivated influenza A viruses; human
A(H1N1)pdm09 and A(H3N2), swine A(H1N1)v, A(H1N2)v and A(H3N2)
and avian A(H5N1), A(H5N6) and A(H7N2). In the second week of
March 2023 this panel was distributed to the 50 medical microbiology
laboratories that signed up for this EQA. The laboratories were asked to
run their routine diagnostic molecular tests for influenza virus detection
on the panel specimens and to report their outcomes using Microsoft
Forms. Every reported test was seen as a separate workflow and labs
could report multiple workflows. The resulting data was collected in an
Excel file and further analysis was performed using Excel and Graphpad
Prism.
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Results
A total of 120 workflows were reported by 50 different laboratories.
Nearly all tests detected the influenza A viruses, including the animal
influenza viruses. The exceptions were three commercial kits, in which
there seemed to be an issue with the sensitivity for one or two animal
viruses, especially the A(H1N1)v zoonotic swine influenza virus, thus
generating a negative influenza A virus detection result. This A(H1N1)v
containing specimen also produced the highest median Cq value across
all viruses in the panel, indicating the relatively low viral load, but was
still detectable by most workflows. However, these three commercial
kits were capable of detecting two other animal influenza virus
containing specimens with a similarly low or even lower viral load. Only
4/50 laboratories (8%) have a test operational to specifically identify
avian influenza H5. A higher number of laboratories (20/50; 40%), with
24 workflows, have tests operational that can H-subtype human H3
and/or H1pdm09 viruses. With the exception of two, all the workflows
with subtyping capacity identified human H1pdm09, and all workflows
identified human H3 influenza virus correctly. According to their
Instructions for Use, three commercial assays included H-subtyping of
swine H1 and H3 viruses. However, only 2/3 workflows using the same
singular assay identified the swine A(H3N2) virus as ‘Influenza A H3’
while the other two assays did not identify the subtype of the swine
A(H3N2) virus. None of the three assays identified the A(H1N1)v
(12/12) and A(H1N2)v (5/5) zoonotic swine viruses as ‘Influenza A H1’.
Conclusions
Overall, the detection rate of animal influenza A viruses with the
molecular diagnostic tests current routinely used in the Netherlands is
high. However, further subtyping is necessary in order to know what
kind of animal influenza virus is detected. When molecular tests that are
capable of subtyping do not identify a human H1(pdm09) or H3 subtype,
this is a first sign that there could be an animal influenza virus involved.
In that case referral for further investigation at the NIC is required. But,
referral to the NIC should also take place in case the patient was
recently exposed to infected animals and if the subtyping assay leads to
the classification H1(former seasonal), H1(pdm09) or H3. The attending
physician needs to be aware of potential exposure to animals and should
ask specific questions about it, in order to enhance the identification of a
possible case of animal influenza virus infection. Nevertheless, with the
current molecular diagnostic tests routinely used by medical
microbiology laboratories in the Netherlands, the presence of animal
influenza A virus can be reliably detected.

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1

Introduction

Current situation regarding H5N1 influenza virus
Since the largest global outbreak of highly pathogenic H5N1 (clade
2.3.4.4b) type A influenza virus in over a 100 million wild birds and
poultry in 2022 and 2023, an expanding range of mammals has been
found to be infected with this H5N1 virus, sometimes with substantial
mortality (1, 2, 3). This follows on previous reports of a Spanish farm
with transmission of H5N1 in minks in October 2022 (4, 5). Since 2021,
27 wild mammals - especially foxes - in the Netherlands have also been
found to be infected with this specific H5N1 virus. Infection was often
detected because of strange behaviour due to neurological and/or
respiratory disease (6, 7).
Worldwide, eight people with H5N1 clade 2.3.4.4b influenza virus
infection have been reported so far, mostly following direct contact with
infected poultry. Three people were hospitalised and one case was fatal
(8, 9, 10). Historical data of infections with other H5N1 influenza virus
clades since 1997 indicate an overall case-fatality rate of over 50% (11).
However, it is important to realise that this case-fatality rate is mainly
based on the count of hospitalised patients. The actual number of infected
individuals is unknown, and therefore the infection-fatality rate is very
likely lower.
Notification obligation
In The Netherlands, animal influenza virus infection in humans – by avian
or swine influenza virus – is a notifiable group B1 disease (12). This
means that in case of a probable infection with animal influenza virus a
report must be made to the municipal health service (GGD) and the RIVM
National Coordination Centre for Communicable Disease Control (RIVMLCI). Once the infection with animal influenza virus is confirmed, source
finding and contact tracing are started immediately, as part of public
health measures.
Indications for diagnostics
In The Netherlands, targeted diagnostics for H5N1 are currently
performed by the National Influenza Centre (collaboration of RIVM,
Bilthoven and Erasmus MC, Rotterdam). According to the guideline and
scenarios, diagnostics are performed for patients who develop respiratory
complaints or conjunctivitis after contact with infected birds, including the
culling of H5N1-infected poultry farms and other professional encounters
with H5N1-infected birds or mammals (12). Prophylactic oseltamivir and
vaccination with a human flu vaccine are considered for professionals who
encounter avian influenza but have no complaints. When a probable case
is detected in regular health care, according to the guideline it is decided
in consultation with RIVM-LCI whether additional diagnostics and
measures are necessary. As this guideline is about the infection of
humans with animal influenza viruses, the same applies when a case of
infection with swine influenza virus is suspected or detected in regular
healthcare.
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Expert consultation avian influenza
In 2022 a Consultation of Experts on Zoonoses was held, after which the
Minister of Health, Welfare and Sport assigned RIVM several tasks. These
included a check of the molecular diagnostic tests used for diagnosing
human influenza cases on their capability to detect influenza viruses of
animal origin, both avian and swine (13). Swine influenza viruses included
in response to the recent failure of a commercial molecular diagnostic test
in a human infection with swine influenza virus in 2020, and the finding of
another case of swine influenza virus infection in the national influenza
virus surveillance in 2022 (14, 15, 16). In 2023, an additional case of
swine A(H1N1)v influenza virus was detected through communityparticipatory surveillance of acute respiratory infections (17).
Commercial Nucleic Acid Amplification Tests (NAAT)
Because there is not always a suspicion of animal influenza virus in a
patient infected with an animal influenza virus, it is important that the
molecular diagnostic tests in use can at least detect animal influenza
viruses, so that subtyping at the NIC can be requested after a positive
generic test for influenza A virus and reported animal exposure in the
clinical history of the patient. Commercial NAATs for generic detection of
influenza A virus may be validated for animal influenza A virus detection
by the manufacturer. This will be stated in the Instructions for Use (IfU).
However, often this validation is not regularly updated for recently
circulating viruses. It is complicated to check whether the used primers
and probes are in principle suitable to detect recent animal and human
influenza A viruses, since detailed information on used primers and
probes is not included in the IfU and in general the manufacturers of
these kits do not share this information. Subtyping of the hemagglutinin
genome segment (H-subtyping) is usually not built into commercial
assays for influenza virus detection, as there is generally no clinical
implication. The most common built-in subtyping for human H1pdm09
and H3 influenza A virus subtypes is relatively rare in commercial NAATs,
and the avian H5 influenza A virus subtype is only sporadically included in
a commercial NAAT. As a result, using these tests it is hardly possible to
identify an H5N1 case or any case of animal influenza virus infection in
general, when detecting an influenza A virus without a suspicion of
exposure to animals that are potentially infected with influenza virus in
the clinical history of the patient.
External Quality Assessment
To address this lack of information on used primers and probes in
commercial diagnostic tests, the NIC and the Animal Health Service
(Royal GD, Deventer) have prepared an external quality assessment
(EQA) ring trial on behalf of the Ministry of Health, Welfare and Sport. In
this EQA, generic detection of type A influenza viruses (of human and
animal origin) using commercial NAATs and Laboratory Developed Tests
(LDT) currently routinely used in medical microbiology laboratories in the
Netherlands is being tested. This EQA also includes an assessment for
correct H-subtyping, when included in the routinely used commercial kit
for influenza virus detection or when available as separate additional LDT.
This EQA should provide an answer to the question if the medical
microbiology laboratories in the Netherlands can detect animal influenza
viruses, in particular the currently circulating H5N1 virus.
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2

Materials and methods

2.1

Approach
All medical microbiology laboratories in The Netherlands were invited to
participate in the animal influenza EQA through a direct email from the
RIVM and a message sent to the members of the Dutch Society for
Medical Microbiology (NVMM) in March 2023. Fifty laboratories signed up
to participate, including one laboratory from Aruba, one from Bonaire
and one from Curaçao.
The animal influenza EQA panel was produced and pre-tested at the
RIVM, using LDTs and three commercial NAATs routinely used in
surveillance and emergency diagnostics at RIVM. In the second week of
March 2023, the panels were distributed to all diagnostic laboratories
that signed up for the EQA. Laboratories were given till the first week of
May 2023 to report their results. Laboratories using molecular point of
care tests (mPOCT) were given the option to test a limited panel to
reduce the costs. The limited panel consisted of specimens 1-5 (see
Table 3.1).

2.2

Panel preparation and contents
Human and animal viruses were selected to cover recent challenges in
human influenza A(H1N1)pdm09 and A(H3N2) detection and avian and
swine influenza viruses recently infecting humans or potentially capable
of infecting humans (Table 2.1). Viruses were grown on a mixture of
MDCK-I and MSCK-SIAT cells, at biosafety level 3 when necessary.
Panel specimens were prepared in Minimal Essential Medium (MEM) with
Hank’s salts and 10,000 HEp2 cells/ml to simulate a clinical specimen.
Before preparing the specimens, the viruses were inactivated by heating
them for 2.5 hour at 65ºC. Table 2.1 shows the panel composition and
the expected results. Extracts for RT-qPCR were obtained by extracting
200 µl of specimen with Roche MagNA Pure 96 DNA and Viral NA Small
Volume Kit on Roche MagNA Pure 96 and elution in 50 µl Tris-EDTA (TE)
buffer. The Cq values shown were determined by in-house RT-qPCR
testing on 5 µl of extract of the panel specimens with Applied
Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix on Roche
LightCycler® 480 Instrument II, which are currently used at RIVM for
generic influenza virus type A detection and indicated subtyping.
Because primers and probes of the various RT-qPCR assays have a
perfect or less than perfect match with the specific viruses, the Cq
values cannot be used directly as a proxy to compare the concentration
of viruses in the specimens. Therefore, using digital RT-qPCR with the
RIVM in-house influenza A matrix gene primers and probes, the number
of RNA copies was determined for each panel specimen (Table 2.1).
In the Supplementary material section, additional RIVM results are
provided for the three commercial kits that were also tested as a
reference for the evaluation of results from participants (Supplementary
Table 7.1).

2.3

Dataset and analysis
All participating laboratories were asked to run a molecular detection for
influenza A virus for the 10 panel specimens that were sent to them and
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to determine at least the H-subtype, if this test was available. Using
Microsoft Forms, they reported their data to the RIVM. They had to
submit a new form for every workflow. The laboratories were asked to
provide information on the workflow that was used. Among other things
they were asked which kits they used for RNA extraction and
amplification, if they used an in-house method, which equipment they
used, and if they were able to do influenza A virus subtyping and if so,
for which subtypes. For every workflow the laboratories had to indicate
which target gene(s) were used.
After that, results were asked for every panel specimen. For every
target gene a positive, negative or inconclusive result (pre-set reporting
options) had to be reported, as well as the Cq/RFU values, when
available. Furthermore, a final conclusions on the presence or absence
of influenza A virus had to be reported and - when applicable - which
subtype was identified.
In some workflows a limited panel could be tested, using only the first
five specimens of the panel to lower the costs of testing, in particular for
expensive mPOCT assays. Therefore, not every panel specimen has the
same number of workflows it was tested in.
Finally, the question was asked what the laboratory would do if a
detected influenza A virus were from a patient suspected of animal
influenza virus infection (free text answer).
The reports in Microsoft Forms were downloaded into an Excel file and
further analysis was done using Excel version 2202 and Graphpad Prism
version 9.5.1 (733). Before analysis, the data was cleansed. Cleansing
included harmonization of commercial kit names and a review of kit IfUs
for used target genes and precise result interpretation. If required, a
laboratory was contacted to clarify entries or to provide kit information.
One laboratory reported four different tests in one form. These were
separated into four workflows.
For every workflow a score was calculated by giving 1 point for every
correct final result for each of the 10 panel specimens, making 10 points
the highest score that could be obtained. An ‘Inconclusive’ final result
was given 0.5 point. If a limited panel was tested, the total score of the
five specimens was multiplied by 2. No scores were given for subtyping
results. The score results for each workflow can be found in
Supplementary Figure 7.5.
When commercial kits generated deviant results, the manufacturer was
contacted to inform them and to provide them with an opportunity to
give a comment for publication. Laboratories with deviant LDT results
were also contacted to give a comment for publication.

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Table 2.1 Panel contents and expected results animal influenza EQA 2023

Cq value by host specificity, type/subtype and target gene
InfA MP
dPCR
Generic
Human
Avian
genome
N1
InfA
N2
N2
H1
H5 H7 set
copies per µl
H3
pdm09
N1
MP
292R
119E
pdm09
setB
HA-1
specimen
275H

Panel coding

Virus1

Host

A subtype2

Passage
history3

EQA.1_AI23-01

Avian

H7N2

E2

639

29.06

Human

H3N2

MDCK-MIX2

1350

27.22

EQA.1_AI23-03

A/Mallard_Duck/Netherlands/2/20194
A/Netherlands/11715/2022 MP
A12T+C136T5,6
No virus

na

na

na

0

-

-

-

EQA.1_AI23-04

A/Netherlands/10370-1b/2020

Swine/Human

H1N1v

MDCK-MIX2

137

29.87

-

EQA.1_AI23-05

Avian

H5N1 d1

MDCK1

335

26.54

2220

EQA.1_AI23-07

A/Black-headed_Gull/Netherlands/6/2022
A/Netherlands/11772/2022 MP
C124A+G141A5,6
A/Swine/Netherlands/34973/19998

EQA.1_AI23-08

EQA.1_AI23-02

EQA.1_AI23-06

5,7
4

Human

H1N1pdm09 MDCK-MIX2

-

-

-

-

-

-

30.06

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

28.67

-

27.66

26.32

-

-

-

26.96

28.86

-

-

-

27.80 30.62 31.43

Swine

H3N2

MDCK-MIX5

145

27.84

-

30.189

-

-

-

-

A/Domestic Duck/Netherlands/EMC-2/2018

Avian

H5N6

MDCK1

2150

25.64

-

-

-

-

-

24.78

-

EQA.1_AI23-09

A/Netherlands/11748/2022

Swine/Human

H1N2v

MDCK-MIX2

444

26.57

-

-

-

-

-

-

-

EQA.1_AI23-10

A/Black-headed_Gull/Netherlands/6/2022

Avian

H5N1 d2

MDCK1

20

30.18

-

-

-

-

-

32.63

-

4

5,7
4

na means not applicable; ‘-‘ means tested with a negative result; grey cells mean not tested.

1. Sequences of the viruses are available from GISAID (gisaid.org) under accession numbers EPI_ISL_14751138, EPI_ISL_717720, EPI_ISL_13429294, EPI_ISL_15544293,
EPI_ISL_305416, EPI_ISL_15348505 and EPI_ISL_13429294 respectively, except for A/Mallard_Duck/Netherlands/2/2019 and A/Swine/Netherlands/34973/1999 for which sequences are
available on request.
2. H5N1 d2 is a 1:10 dilution of H5N1 d1 but copies/µl were individually determined.
3. E = Egg; MDCK = Madin-Darby Canine Kidney cells; MDCK-MIX = 1:1 mixture of MDCK-SIAT and MDCK-I cells; Number after cell line is the number of passages on the cell line.
4. From the Erasmus MC collection, courtesy of Ron Fouchier and Oanh Vuong. The H5 viruses both have clade 2.3.4.4b HA.
5. From the RIVM collection.
6. Indicated are recently emerging nucleotide mutations in the MP genome segment of these viruses that have led to underreporting in various assays (18, 19)*. The positions of the
mutations are relative to the first ATG of M1/M2 genes in the MP genome segment.
7. Originating from recent human infections with swine influenza virus in the Netherlands.
8. From the collection of Royal GD, Deventer, courtesy of Erhard van der Vries and Manon Houben.
9. The genome segment for the neuraminidase of the swine A(H3N2) virus cross-reacts with low fluorescence with primers and probe of the human 292R N2 SNP and subtyping RT-qPCR.
* Another source was personal communication from UKHSA WHO National Influenza Centre, London UK

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3

Results

3.1

Aggregated overview
For this EQA 50 laboratories reported a total of 117 workflows
(participating laboratories are summarised in Chapter 7.5. One
laboratory submitted four workflows in one form; this dataset was split
into four separate workflows. This created 120 workflows - two of which
were for subtyping only - that were analysed. Table 3.1 provides a
summary of the results per panel specimen. Most workflows (109/118;
92%) found the animal and human influenza virus containing specimens
correctly positive for influenza virus type A. However, panel specimen 4,
with an A(H1N1)v human/swine influenza virus, had the most detection
errors, given that 7/118 (6%) reported workflows were not able to
detect this virus. Overall, 9/118 (8%) reported workflows had errors.
Six reported a negative result for one or two animal influenza virus
containing specimens: 1/3 Abbott ID NOW INFLUENZA A & B 2, 2/3
Roche ePLEX Respiratory Pathogen Panel 2, and 3/5 Seegene Allplex RV
Essential or Master Assay. For BioMerieux BIOFIRE Respiratory panel 2.1
plus 2/7 returned an inconclusive result for two or three animal
influenza virus containing specimens. One of 21 workflows using an LDT
reported the human A(H1N1)pdm09 containing virus specimen negative.
However, all 118 workflows reported avian influenza virus A(H5N1) and
A(H5N6) containing specimens influenza A virus positive. Furthermore,
every workflow reported a correct ‘influenza A virus negative’ final
conclusion for the negative (no virus) control panel specimen.
Figure 3.1 shows a visual representation of the reported final conclusion
of each workflow. It shows that nearly all reported workflows (92%)
were able to detect all influenza viruses that were provided in the panel
as an influenza A virus. The exception was panel specimen 4, containing
A(H1N1)v influenza virus, for which multiple workflows reported a
‘Negative’ final conclusion for influenza A virus presence. Although the
number of copies in specimen 4 was in the lower range, these workflows
did detect influenza A virus in the specimens that contained a similar or
even lower number of copies (specimens 7 and 10) (Table 2.1).
More information about the performance of workflows used by each
laboratory can be found in Supplementary Figure 7.5, which also
includes a score per reported workflow.

3.2

Cq values per target gene
Out of the 120 reported workflows 98 (82%) used an in-house LDT or
commercial detection kit that reports Cq values (more information on
kits and equipment used can be found in Supplementary Figures 7.1 7.4), indicating the presence of a virus by detection of a certain target
gene. Figure 3.2 depicts the Cq values for every workflow that reported
these values per target gene. The far most common target gene used is
the Matrix gene, which is used by a wide variety of kits and all LDTs.
Many reported workflows were for results from Cepheid GeneXpert kits
(n=42) that included influenza virus A/B detection. These kits use two
channels, Flu A1 and Flu A2, for influenza virus type A detection. Flu A1
contains primers and probes against human derived influenza A matrix-,
PA- and PB2-genes and Flu A2 contains avian derived primers and
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RIVM report 2023-0392

probes against the matrix-gene, but is not limited to detect avian
influenza type A viruses only (personal communication, Cepheid).
Between the two channels there is great overlap, so when either or both
channels produce a PCR curve, a positive result will be given for
detection of influenza A virus. For all 42 Cepheid workflows the Flu A1
channel Cq values were reported and for 36/42 the Flu A2 channel Cq
values. Two workflows reported result for the Seegene Allplex RV
Essential Assay kit, which uses a Flu A channel (according to IfU) which
also has the M-gene as target (personal communication, Seegene).
Because of outlier results this kit is depicted separately in Figure 3.2.
Figure 3.2 shows that the Cq values for panel specimen 3 - the negative
control - were all negative. The matrix gene shows a larger spread of Cq
values per specimen than the Cepheid targets, but the matrix gene Cq
values are derived from a large variety of different commercial kits and
LDTs. However, even a closed system like the Cepheid GeneXpert
generates a cloud of Cq values with the same specimen, although
narrower in spread. All workflows show a similar shift in Cq values
between specimens, roughly correlating with the copy number per µl
specimen. Supplemental Figure 7.6 shows the correlation between the
Cq values and the number of copies per specimen, where panel
specimen 4 (H1N1v) had the highest overall Cq value for all target
genes (median=33.48, mean=37.14; copy number 137/µl) and panel
specimen 8 (H5N6) the lowest (median=26.11, mean=27.43; copy
number 2.150/µl).
Furthermore, the Seegene Allplex RV Essential Assay kit (Flu A target
gene, actual being the M-gene; personal communication, Seegene)
shows some outliers. Higher end Cq values were measured for all panel
specimens with this kit in comparison to the results of most other tests
(Figure 3.2), while panel specimen 4 (H1N1v) was not detected at all by
this kit. Moreover, panel specimen 1 (H7N2) was also not detected by
one of the two Seegene Allplex RV Essential Assay workflows.
Considered together, these observations indicate that this workflow
likely has a sensitivity issue with the detection of certain influenza A
viruses.
3.3

Subtyping
Of the 120 workflows, 24 (20%) had a varying capability for subtyping
reported by 20/50 (40%) laboratories, and they could indeed subtype
the viruses in part of the specimens that were provided on the panel
(Table 3.2).
Specimen 2, which is a human A(H3N2) influenza virus strain, was
correctly subtyped by 22 workflows that were capable of subtyping H3
(Table 3.2). All six reported commercial kits that include subtyping of
H1pdm09 subtyped the human A(H1N1)pdm09 virus correctly. With the
exception of one kit none of the commercial kits were able to subtype
the animal hemagglutinin subtypes avian H5 or H7 and swine H1 or H3.
The latter is correct, as these subtypes are not included in the design of
most kits. By design the BIOFIRE kit from BioMerieux should be capable
to identify the swine H1 and H3 subtypes. Indeed, 2/3 workflows that
used this kit on the A(H3N2) swine influenza virus containing specimen
reported it as subtype H3. However, none of seven workflows and none
of three workflows using this kit on the A(H1N1)v and A(H1N2)v
zoonotic swine influenza viruses included in the panel could identify the
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RIVM report 2023-0392

H-subtype of these viruses. Furthermore, the IfU of two commercial kits,
Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel and Roche ePlex
Respiratory Pathogen Panel 2, indicate the capability to subtype swine
H1 and H3 viruses. However, none of the workflows using these kits
identified the subtypes of swine viruses (Table 3.2). Although the eight
in-house LDTs for subtyping subtyped the human H3 containing
specimen correctly, two of them were not able to subtype the human
H1pdm09 containing specimen, which raised some concerns. All four
LDT workflows for H5 subtyping reported by four different laboratories,
subtyped the three avian H5 containing panel specimens correctly. Only
one of two LDT workflows that included subtyping of avian H7 correctly
identified the H7 subtype for the A(H7N2) containing specimen.
3.4

Response from manufacturers and laboratories
The manufacturers of four commercial kits (Abbott, BioMerieux, Seegene
and Roche) and five laboratories with deviant results were individually
approached for comments on the findings. Eight of them responded.
The one laboratory that had a negative result for panel specimens
containing A(H1N1)v and A(H1N1)pdm09 with their LDT for generic
influenza A virus detection provided the following explanation:
Unfortunately, using our LDT two out of nine virus containing specimens
(both H1N1) were not detected. Theoretically the mentioned primerprobe sets should be able to detect all mentioned influenza strains. We
have contacted the laboratory from which we have obtained the primer
and probe sequences of our currently used LDT. They have informed us
that they are aware of the problem, and are currently working on
optimalisation of said primers and probes. Fortunately, we also have two
commercial PCRs running which were able to detect all virus containing
specimens (with good Cq values) in the panel, so for the time being we
will only use these commercial PCRs for the detection of influenza virus
until the issues with our LDT have been resolved.
Three laboratories submitted a workflow that was capable of subtyping
H1, H3 and H5, but did not report a subtype for the A(H1N1)pdm09
containing specimen. These panel specimens were found positive in
generic influenza A detection, but no subtype was identified. One lab
reported that for their H1pdm09 subtyping PCR the forward primer
contained three mismatches and the probe they used had one mismatch
with the panel virus, explaining why the subtype was not recognized in
their PCR. From the H1 primers and probe information that a second
laboratory submitted with their results it became clear that they had
specificity for the former seasonal H1 subtype and not for the H1pdm09
subtype, and therefore could not have identified the H1pdm09 subtype.
This laboratory confirmed that they indeed included subtyping for former
seasonal H1 and not H1pdm09 by omission. The third laboratory
answered that the reported results of the Pathofinder RespiFinder®
2Smart kit are based on results up to genus level and not species level
and that although this kit does have a test to identify the
A(H1N1)pdm09 species, it would be reported only as ‘Influenza A
positive’ to a requester of the test.
The laboratory that failed to subtype the H7 virus in the panel with their
H7 subtyping test provided the following explanation: Our laboratory
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RIVM report 2023-0392

participated in the External Quality Assessment panel for animal
influenza viruses organized by RIVM. All influenza virus containing panel
specimens were identified correctly for influenza A virus. In subsequent
virus typing by PCR one panel member containing H7N2 virus could not
be subtyped. In this case all subtyping assays remained negative (while
this panel member was positive for influenza A virus in the detection
PCR). Our subtyping assay should be able to correctly subtype H1
(former seasonal), H1pdm09, H3, H5, H7 and H10 viruses. Analysis of
genome sequences revealed a minor mismatch in one of the primers
that could lead to a slightly lower sensitivity of the subtyping PCR for
this H7N2 panel member. In practice, when analysing specimens from
clinical cases, such a result (positive result for influenza A detection
PCR, but negative result in all virus subtyping PCRs) will be followed by
whole genome sequencing. It is therefore unlikely that we would miss
an H7N2 infection. Nevertheless, we continuously evaluate, develop and
improve our PCR assays with the latest information and will use this
result as input to update our assays.
BioMerieux provided the following comment on the observed subtyping
of swine H3 and lack of subtyping swine H1 with the BIOFIRE
Respiratory Panel 2.1 (plus) kit: The results of the animal influenza EQA
panel testing are consistent with expected performance and reporting by
the BIOFIRE Respiratory Panel 2.1 (plus) according to the product
design and description provided in the instruction for use. The intended
use of the device is first and foremost to identify the presence of
potential infectious agents in a patient specimen, so that the test results
can be used in conjunction with clinical history of the patient (including
human and animal contact and/or travel) and the epidemiological status
of the region. Results aid in the diagnosis, management, and treatment
of the symptomatic patient. Additional targeted testing may be required
to monitor or screen for zoonotic transmission events and/or emerging
variants within a region or patient population and as part of public
health missions for pandemic preparedness.
Roche provided the following comment on the observed lack of detection
of the A(H1N1)v swine influenza virus by some users of the
ePlex® Respiratory Pathogen Panel 2 kit: Using the sequence
information provided (GISAID, EPI_ISL_717720, A/Netherlands/103701b/2020), a bioinformatics assessment was done against the primers
and probes used in the RP2 Panel assays for influenza. The ePlex RP2
Panel includes a unique assay for the influenza A matrix gene and
separate, independent assays for the H1, H1pdm09 and H3 subtypes.
This redundancy helps mitigate the risk of a missed result due to (a)
new mutation(s). Analysis of the ePlex RP2 influenza A matrix gene
assay found 1 mismatch in the forward primer and also 3 mismatches in
the capture probe region. This data suggests that amplification will
perform as expected, but detection of this specific Eurasian swine-like
influenza A strain may be variable due to these mismatches. Additional
analysis of the RP2 influenza A H1 assay also has multiple mismatches,
suggesting that this strain may not be detected by the RP2 Panel. The
ePlex RP and RP2 Panels are designed to be inclusive of as many
circulating and recently circulating strains of influenza A in humans.
However, as new variants emerge, there may be mutations that are not
detected due to mutations in the regions targeted by the ePlex assays.
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RIVM report 2023-0392

GenMark and Roche conduct routine influenza surveillance of circulating
strains to monitor for inclusivity of new strains in circulation to ensure
the test is performing as expected. Furthermore, the risk of transmission
of a zoonotic virus into humans and from-human-to-human is closely
monitored, to be prepared in case immediate actions are required.
Seegene provided the following comment on the failure to detect
specimens 1 (H7N2) and 4 (H1N1v) with the generic influenza A virus
target component of several workflows using the Seegene Allplex RV
Essential and Master Assays: The IfU show that the assays are validated
to detect several animal influenza A virus subtypes, but that emerging
animal viruses infecting humans might be missed. Seegene is working
on updating the respiratory assays in which these observations will be
addressed in the future.
3.5

Follow-up suspect animal influenza case
The question what a laboratory would do if a positive influenza A case
proved to be a suspect animal influenza case was answered by 47/50
laboratories; two considered it not applicable and one did not answer
the question. Most laboratories (40/47; 85%) answered that they would
follow-up with subtyping/sequencing of the detected influenza A virus,
either by themselves (5/40) or by the National Influenza Centre
(35/40). One out of 40 answered that they would not follow-up, 3/40
that they would first inform RIVM-LCI and the Municipal Health Service
before following up with a subtyping action and 3/40 said they would
discuss appropriate action with their own staff first. Of the 40
laboratories that would follow-up with subtyping, seven indicated that
they would also inform RIVM-LCI and the Municipal Health Service. Two
out of these 40 laboratories said they would also inform the staff of
Infection Prevention.

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RIVM report 2023-0392

Table 3.1 Summary of reported outcomes per panel specimen

Panel specimen

Content

EQA.1_AI23-01

A/Mallard_Duck/Netherlands/2/20191 H7N2

EQA.1_AI23-02

A/Netherlands/11715/2022 MP
A12T+C136T1

H3N2

118

118

0

0

None

EQA.1_AI23-03

No virus1

None

118

0

118

0

None

EQA.1_AI23-04

A/Netherlands/10370-1b/20201

H1N1v

118

109

7

2

False negative results (n=7)
Inconclusive results (n=2)

H5N1 d1

118

118

0

0

None

H1N1pdm09 82

81

1

0

False negative results (n=1)

EQA.1_AI23-05
EQA.1_AI23-06

A/Blackheaded_Gull/Netherlands/6/20221
A/Netherlands/11772/2022 MP
C124A+G141A

A subtype

Number of Conclusion workflow detection influenza virus
workflows
with
No.
No.
No.
Errors
reported
Positive Negative Inconclusive
test results
False negative results (n=1)
118
115
1
2
Inconclusive results (n=2)

EQA.1_AI23-07

A/Swine/Netherlands/34973/1999

H3N2

82

82

0

0

None

EQA.1_AI23-08

A/Domestic Duck/Netherlands/EMC2/2018

H5N6

82

82

0

0

None

EQA.1_AI23-09

A/Netherlands/11748/2022

H1N2v

82

80

1

1

EQA.1_AI23-10

A/Blackheaded_Gull/Netherlands/6/2022

False negative results (n=1)
Inconclusive results (n=1)

H5N1 d2

82

82

0

0

None

1. Panel specimens EQA.1_AI23-01 to EQA.1_AI23-05 were part of the limited panel

Page 22 of 47

RIVM report 2023-0392

A green line represents a correct result, a red line an incorrect result and an orange line an inconclusive result. Panel specimen EQA.1_AI23-06 till
EQA.1_AI23-10 were not part of the limited panel, and therefore these boxes remain white. This is also indicated by separating the boxes containing
the workflows with a limited or a full panel. At the far right of the figure the panel specimen and corresponding virus are depicted (host: Av = avian; Hu
= human; Sw = swine; v = variant, indicating a human infected with swine virus; H5N1 Av d2 is a 1:10 dilution of H5N1 Av d1).

Figure 3.1 Heatmap of reported final conclusion for each workflow.

Page 23 of 47

RIVM report 2023-0392

For every panel specimen the Cq values per workflow are shown. Each dot represents a separate workflow, and each color represents a target gene. At
the top of the Y-axis the negative results are shown. The target gene for LDTs and most commercial tests is the M-gene. For the Cepheid GeneXpert
tests (personal communication with manufacturer) the target genes for Flu A1 are the acidic polymerase (PA), basic polymerase (P2B) and matrix (M)
genes for broad coverage, and for Flu A2 primers and probes designed against avian-derived matrix gene sequences, but not limited to detect avian
influenza viruses only. For the Seegene RV Essential Assay kit the FluA target is actually the M-gene (personal communication with manufacturer), but
results are shown separately because of deviant results. Only workflows that generate Cq values are shown in this figure (n=98). With the virus names
the host is shown: Av = avian; Hu = human; Sw = swine; v = variant, indicating a human infected with swine virus; H5N1 Av d2 is a 1:10 dilution of
H5N1 Av d1.

Figure 3.2 Cq values per target gene.

Page 24 of 47

RIVM report 2023-0392

Table 3.2 Overview of panel specimen results with workflows capable of subtyping.

Amplification kit

BioMerieux Diagnostics.
BIOFIRE® Respiratory
panel 2.1 plus

In-house. LDT

Qiagen. QIAstat-Dx®
Respiratory SARS-CoV-2
Panel
Roche.
ePlex® Respiratory
Pathogen Panel 2
Seegene. Allplex™
Respiratory Panel 1A
Siemens. FTD™
Respiratory pathogens 21
Pathofinder.
RespiFinder® 2Smart

Number of
workflows that
used this kit
(n=24)

Subtyping integrated in
workflow/additional tests2

7

H1(former seasonal), H1pdm09,
H3 3

8

H1pdm09, H3
H1pdm09, H3
H1(former seasonal), H1pdm09,
H3, H5, H7
H1pdm09, H3
H1pdm09, H3
H1pdm09, H3, H5
H1, H3, H5
H1(former seasonal), H1pdm09,
H3, H5, H7, H10

Panel
specimen1
EQA.1_AI23- 01
02
04
05
06
07
08
Subtype
H7N2 H3N2 H1N1v H5N1 (d1) H1N1pdm09 H3N2 swine H5N6
Limited Panel
No
A(H3)
A(H1)pdm09
A(H3)
✖
✖
✖
✖
Yes
A(H3)
✖
✖
✖
No
A(H3)
A(H1)pdm09
✖
✖
✖
✖
✖
Yes
A(H3)
✖
✖
✖
Yes
A(H3)
✖
✖
✖
No
A(H3)
A(H1)pdm09
A(H3)
✖
✖
✖
✖
Yes
A(H3)
✖
✖
✖
No
A(H3)
A(H1)pdm09
✖
✖
✖
✖
✖
No
A(H3)
A(H1)pdm09
✖
✖
✖
✖
✖
No

A(H7)

A(H3)

No
No
No
No

✖
✖
✖
✖

A(H3)
A(H3)
A(H3)
A(H3)

No
Yes

2

H1(former seasonal), H1pdm09,
H3 3

3

H1(former seasonal), H1pdm09,
H3 3

2

H1(former seasonal), H1pdm09,
H3

No
No
Yes
No
No

1

H1pdm09

1

H1pdm09

✖

A(H3)

✖

A(H3)

✖
✖
✖
✖
✖

A(H3)
A(H3)
A(H3)
A(H3)
A(H3)

✖

A(H3)

No

✖

✖

No

✖

✖

Yes

✖

✖

A(H5)

A(H1)pdm09

✖

✖

-

✖
✖
✖
✖
✖

-

-

-

-

-

-

-

-

-

A(H1)pdm09
A(H1)pdm09
A(H1)pdm09
A(H1)pdm09

✖
✖
✖
✖

✖
✖
✖
✖

✖
✖
✖
✖

✖
✖
✖
✖

✖

✖

✖

✖
✖
✖
✖
✖
✖
✖

✖
✖
✖
✖
✖

A(H5)

A(H5)

✖

✖
✖
✖
✖
✖

✖

A(H1)pdm09
A(H1)pdm09
✖4
✖5

✖

H5N1 (d2)

A(H5)

✖
✖
A(H5)
A(H5)

✖

H1N2v

A(H5)

A(H1)pdm09

✖

10

✖

A(H5)

✖
✖
✖
✖

09

✖
✖
✖
✖

✖

A(H1)pdm09

✖

✖

✖6

✖

✖
✖
A(H5)
A(H5)

✖

✖
✖
✖
✖

✖

1. Results are shown for each individual reported workflow using the indicated amplification kit. The subtype is the subtype of the panel specimen. If the test did detect a subtype, then the outcome of the test is depicted in the table. A
cross (X) means the kit did not detect a subtype, which in 14 cases is considered a deviation from the intended specificity of the test (red cross). A dash (-) means that the panel specimen was excluded from testing (limited panel).
Specimen EQA.1_AI23-03 is excluded since it is the negative control.
2. For each individual reported workflow for ‘In-house. LTD’, the subtyping capability is indicated.
3. Might become positive with swine H1 and H3 subtypes according to Instructions for Use and personal communication with the manufacturers.
4. The laboratory explained that primers and probe were not recently reviewed and updated and therefore likely could not subtype the virus.
5. The laboratory provided primers and probe information showing the specificity of the H1 subtyping was for former seasonal H1 and not for H1pdm09.
6. The laboratory explained that it does not report H1pdm09 subtyping results to the requester for a test, and therefore the result in this EQA was not reported either. However, at RIVM we showed that this commercial assay was capable
of subtyping H1pdm09 correctly (Supplementary Table 7.1).

Page 25 of 47

✖
✖
A(H5)
A(H5)

✖

RIVM report 2023-0392

Page 26 of 47

RIVM report 2023-0392

4

Discussion and conclusion

For this EQA, a panel with human and animal type A influenza viruses
was distributed to medical microbiology laboratories in the Netherlands,
to determine whether the molecular influenza diagnostic tests they
routinely use are able to detect animal influenza viruses. The results
show that overall the detection of animal influenza viruses is good. The
vast majority of used tests found all panel specimens containing human,
avian or swine influenza type A viruses positive for an influenza virus
type A. Five commercial tests did not detect one or two animal influenza
viruses, and one LDT did not detect the human A(H1N1)pdm09 virus. All
reported workflows detected the avian A(H5) viruses. A decent number
of laboratories have commercial tests or LDTs that correctly identified
human H1pdm09 and H3 subtypes, only a few have tests that correctly
identified avian H5 and only one has a test that correctly identified avian
H7. With the exception of the BioMerieux BIOFIRE assay that identified
swine H3 with some of the workflows using this assay, none of the tests
could identify the subtype of the swine influenza viruses in the panel.
Most tests use the Matrix gene as a target for generic type A influenza
virus detection, and this appeared to work very well. The Cepheid
GeneXpert commercial tests use multiple targets - the Matrix, PA and
PB2 genes - in one channel, and another Matrix gene-based
amplification in a second channel. All the workflows using the Cepheid
GeneXpert tests produced correct results for the influenza A virus
containing specimens of the panel, indicating that using multiple target
genes can be an effective way for generic detection of type A influenza
viruses. Similarly, the BioMerieux BIOFIRE assay, which uses two pan
influenza A amplifications, found all influenza A virus containing
specimens positive. Including multiple targets in a test or testing
strategy potentially prevents failing detection of a virus if this virus has
mutations in primers and/or probe sites on one of the target genes.
In particular panel specimen 4, an A(H1N1)v zoonotic swine virus, was
difficult to detect for four commercial tests: the Seegene Allplex RV
Essential and Master assays, the Abbott ID NOW Influenza A&B 2 and
the Roche ePlex Respiratory Pathogen Panel 2. Most of the other
molecular tests did detect this virus, but the Cq values were quite high
compared to those of the other panel specimen. It is important to
mention that the expected Cq values, measured at the RIVM, were also
high (Table 2.1), reflecting the relatively low viral load in this specimen.
The Cq values reported for this specimen were also the highest
compared to the other panel specimens, indicating that sensitivity for
this particular virus might be an issue. However, two other animal
influenza virus containing specimens with similar or lower target copy
numbers were properly detected using these tests. This could be the
result of mismatched primers or probes used in these tests. As the
sequences of these primers and probes are not released by the
manufacturers, the manufacturers have been provided with the virus
sequences and were asked to comment on the obtained results. Testing
these commercial kits again with a dilution range of this particular virus
and some other viruses could provide more clarity about the true limit of
detection. Nevertheless, the responses from the manufacturers indicate
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RIVM report 2023-0392

that they are aware that emerging viruses can potentially be missed. As
a result they follow developments closely to be able to adapt their
assays, if deemed necessary.
Several results for some animal influenza viruses were reported as
‘Inconclusive’. These were all from workflows that used the BioMerieux
BIOFIRE Respiratory Panel 2.1 plus molecular test. This is probably
caused by correct use of the IfU, providing an ‘Equivocal’ result for the
indicated virus. This was then translated into ‘Inconclusive’ in the
Microsoft Forms report by design. However, other workflows using the
same test reported these viruses as ‘Influenza A virus Positive’,
suggesting the laboratories involved may have had additional arguments
to consider the ‘Equivocal’ results as actually ‘influenza A virus Positive’.
Similar to the Cepheid GeneXpert tests, the BioMerieux BIOFIRE assay
also uses two channels for influenza A virus detection. If only one
channel yields a positive result and subtyping is negative, the final
conclusion of this test for influenza A virus detection will be ‘Equivocal’.
This could actually be an advantage, because it could be a first
indication that the tested specimen contains an animal influenza virus.
BioMerieux BIOFIRE also mentions this in the IfU, indicating that nonhuman influenza A viruses will generally produce an ‘Equivocal’ or
‘Influenza A (no subtype detected)’ result.
The previously indicated issue with the impaired detection of human
A(H1N1)pdm09 and A(H3N2) viruses with specific mutations in the
matrix gene by several commercial assays (17, 18) was not detected
using any of the commercial assays with the viruses containing these
mutations that were included in the panel. This is reassuring for the
quality of the tests used for the detection of recent human
A(H1N1)pdm09 and A(H3N2) influenza viruses.
Not many molecular tests were able to detect the subtype of detected
influenza A viruses in the panel specimens. Only 24 out of 120 (20%)
workflows were able to perform a subtype identification of detected
influenza A viruses. Therefore, most tests can detect the presence of an
influenza A virus, but cannot indicate whether it is a human or animal
influenza virus subtype. This stresses the importance of sending
influenza A virus positive patient specimens to the National Influenza
Centre (NIC) for further subtyping and characterization when contact
with infected animals is suspected or mentioned in the clinical history of
the patient by a GP or an attending physician. This is of particular
importance for the monitoring and surveillance of animal influenza
viruses that have proved to be able to infect humans. For this purpose,
RIVM-LCI (12) has published guidelines and has recently raised
awareness of the subject by communication through the Dutch Society
for Medical Microbiology (20) and by the publication of a paper targeted
at GPs (21).
All tests that were able to do a defined subtype detection produced
correct results, except for two A(H1)pdm09 subtyping LDTs and one
workflow using a commercial assay that were negative for the specimen
containing A(H1N1)pdm09 virus, and one H7 subtyping LDT that was
negative with the specimen containing A(H7N2) virus. The inability to
subtype the A(H1N1)pdm09 virus was caused by a lack of updated
Page 28 of 47

RIVM report 2023-0392

primers/probe, using H1(former seasonal) instead of H1pdm09 specific
primers/probe, and the policy of a laboratory using a commercial assay
to not report the subtype to requesters. The failure to subtype the H7
virus was explained by mismatched primer. Although by design the
BioMerieux BIOFIRE Respiratory Panel 2.1 plus should be capable of
subtyping swine H1 and H3, similar to the Qiagen QIAstat-Dx®
Respiratory SARS-CoV-2 Panel and Roche ePlex® Respiratory Pathogen
Panel 2, only some of the workflows using the BIOFIRE workflow
provided the H3 subtype for the swine H3 virus and none identified the
subtype of swine H1 viruses. This may be indicative of the difficulty
manufacturers have to keep up with the large variety of Eurasian and
North American lineages of swine viruses in the design of primers and
probes to identify swine H1 and H3 subtypes. It also implies continuous
thorough validation of commercial assays is needed to comply with FDA
and CE marking for In Vitro Diagnostic use. Although the BioMerieux
BIOFIRE kit identified the H3 subtype of the A(H3N2) swine influenza
virus, laboratories can interpret this as a normal, human seasonal H3
influenza virus, since there is no further indication that it could be an
animal influenza virus. This carries the risk of missing a patient infected
with animal influenza virus, if no further questions are asked about
animal exposure in the clinical history of the patient.
Except for the three commercial assays mentioned, other commercial
molecular tests that were tested in this EQA are only capable of
identifying H1(former seasonal), H1(pdm09) and H3, because avian
hemagglutinin variants like H5 and H7 and swine hemagglutinin variants
are not included in the design of these tests. Only a couple of LDTs that
were reported in this EQA proved capable of identifying avian H5 and
H7. Unfortunately, identification of avian or swine influenza virus
variants with the commercial kits routinely used in The Netherlands is
generally not possible, and when there is no suspicion of animal
influenza virus infection it becomes difficult to detect and identify such
cases. Therefore, as explained earlier above, awareness in GPs and
attending physicians in hospitals and further questioning about possible
contact with animals in the clinical history of the patient are imperative.
In conclusion, the current molecular diagnostic tests routinely used in
the Netherlands to detect influenza A viruses perform well for animal
influenza virus detection. However, in order to know what kind of animal
influenza virus is detected further subtyping is necessary. When
molecular tests that are normally capable of subtyping human seasonal
influenza virus subtypes cannot identify an H1(pdm09) or H3, this is a
first sign that it could be an animal influenza virus subtype. These
specimens need to be sent to the NIC for further investigation. In
addition, to improve the diagnostic strategy to identify a patient infected
with an animal influenza virus, GPs and attending physicians in hospitals
need to be aware of possible exposure of a patient to animals and must
ask specific questions about possible contact with animals.

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Page 30 of 47

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5

Acknowledgements

We thank all colleagues in all participating laboratories for reporting
their very valuable testing data of the panel specimens. In particular we
also thank our colleagues and the manufacturers of commercial kits for
their adequate responses to our questions concerning the clarification of
obtained results.

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6

References

1.

European Food Safety Authority, European Centre for Disease
Prevention and Control, European Union Reference Laboratory for
Avian Influenza, Adlhoch C, Fusaro A, Gonzales JL, et al. Avian
influenza overview December 2022 – March 2023. EFSA Journal.
2023;21(3):e07917.
2. European Food Safety Authority, European Centre for Disease
Prevention and Control, European Union Reference Laboratory for
Avian Influenza, Adlhoch C, Fusaro A, Gonzales JL, et al. Avian
influenza overview September – December 2022. EFSA Journal.
2023;21(1):e07786.
3. European Food Safety Authority, European Centre for Disease
Prevention and Control, European Union Reference Laboratory for
Avian Influenza, Adlhoch C, Fusaro A, Gonzales JL, et al. Avian
influenza overview March – April 2023. EFSA Journal.
2023;21(6):e08039.
4. Tammiranta N, Isomursu M, Fusaro A, Nylund M, Nokireki T,
Giussani E, et al. Highly pathogenic avian influenza A (H5N1) virus
infections in wild carnivores connected to mass mortalities of
pheasants in Finland. Infection, Genetics and Evolution.
2023;111:105423.
5. Aguero M, Monne I, Sanchez A, Zecchin B, Fusaro A, Ruano MJ, et
al. Highly pathogenic avian influenza A(H5N1) virus infection in
farmed minks, Spain, October 2022. Euro Surveill. 2023;28(3).
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E, et al. Highly Pathogenic Avian Influenza A(H5N1) Virus in Wild
Red Foxes, the Netherlands, 2021. Emerg Infect Dis.
2021;27(11):2960-2.
7. Bordes L, Vreman S, Heutink R, Roose M, Venema S, PritzVerschuren SBE, et al. Highly Pathogenic Avian Influenza H5N1
Virus Infections in Wild Red Foxes (Vulpes vulpes) Show
Neurotropism and Adaptive Virus Mutations. Microbiol Spectr.
2023;11(1):e0286722.
8. World Health Organization. Influenza at the human-animal interface
summary and assessment, 3 March 2023 2023. Available from:
https://www.who.int/publications/m/item/influenza-at-the-humananimal-interface-summary-and-assessment-3-march-2023.
Accessed: 3 August 2023
9. World Health Organization. Assessment of risk associated with
recent influenza A(H5N1) clade 2.3.4.4b viruses 2022. Available
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Accessed: 3 August 2023
10. World Health Organization. Ongoing avian influenza outbreaks in
animals pose risk to humans 2023. Available from:
https://www.who.int/news/item/12-07-2023-ongoing-avianinfluenza-outbreaks-in-animals-pose-risk-to-humans. Accessed: 3
August 2023

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11. World Health Organization. Cumulative number of confirmed human
cases for avian influenza A(H5N1) reported to WHO, 2003-2023, 24
April 2023 2023. Available from:
https://www.who.int/publications/m/item/cumulative-number-ofconfirmed-human-cases-for-avian-influenza-a(h5n1)-reported-towho-2003-2023-24-april-2023. Accessed: 3 August 2023
12. National Institute for Public Health and the Environment LCI.
Influenza van dierlijke oorsprong. Available from:
https://lci.rivm.nl/richtlijnen/influenza-van-dierlijke-oorsprong.
Accessed: 3 August 2023
13. Ministerie van Volksgezondheid Welzijn en Sport. Kamerbrief over
DB-Z advies vogelgriep 2023. Available from:
https://www.rijksoverheid.nl/documenten/kamerstukken/2023/05/0
8/kamerbrief-over-db-z-advies-vogelgriep. Accessed: 3 August
2023
14. Fouchier R, Hooiveld M, Meijer A, Eggink D, Jenniskens L, Raven S,
et al. Influenzaseizoen 2021/2022 in Nederland Griep is terug na de
COVID­19 pandemie. Ned Tijdschr Med Microbiol. 2023;31(1):9.
15. World Health Organization. Antigenic and genetic characteristics of
zoonotic influenza A viruses and development of candidate vaccine
viruses for pandemic preparedness 2021. Available from:
https://cdn.who.int/media/docs/default-source/influenza/whoinfluenza-recommendations/vcm-northern-hemisphererecommendation-20212022/202103_zoonotic_vaccinevirusupdate.pdf?sfvrsn=97ae1340_1
3. Accessed: 8 August 2023
16. World Health Organization. Genetic and antigenic characteristics of
zoonotic influenza A viruses and development of candidate vaccine
viruses for pandemic preparedness 2023. Available from:
https://cdn.who.int/media/docs/default-source/influenza/whoinfluenza-recommendations/vcm-northern-hemisphererecommendation-20232024/20230224_zoonotic_recommendations.pdf?sfvrsn=38c739fa_
4. Accessed: 8 August 2023
17. World Health Organization. Disease Outbreak News; Influenza
A(H1N1) variant virus - the Netherlands. 2023. Available from:
https://www.who.int/emergencies/disease-outbreaknews/item/2023-DON486 Accessed: 13 Septemer 2023
18. Jørgensen RL, Lerche CJ, Pedersen MS, Kirkby NS, Botnen AB,
Trebbien R, et al. Emergence of circulating influenza A H3N2 viruses
with genetic drift in the matrix gene: be alert of false-negative test
results. Apmis. 2022;130(10):612-7.
19. Association of Public Health Laboratories. APHL Influenza News
Available from:
https://www.magnetmail.net/actions/email_web_version.cfm?ep=b
UmBbNAxVmYRYhz4GHY5JfQfcSWYHJbzrcKJpSCWOqWp8ImcrwBN3D6Ritehg_6
GNtxSqfO_ArJ3TgbKvJUndNZSV_edSq6iAwyBeRDH6jRYICDKvW6ey
0CdOGPo2Of. Accessed: 2 April 2023

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20. NVMM. Aviaire influenza (H5N1): verhoogde alertheid na
vermoedelijke transmissie tussen wilde zoogdieren 2023. Available
from: https://www.nvmm.nl/vereniging/nieuws/aviaire-influenzah5n1-verhoogde-alertheid-na-vermoedelijke-transmissie-tussenwilde-zoogdieren/. Accessed: 8 August 2023
21. Bemelmans-Kremer L, Hackert V. Zoönosen: Een dreiging van nu en
alle tijden. Huisarts Wet. 2023;66(6):20-3.

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7

Supplementary material

7.1

Equipment and kits used
All participating labs were asked to provide information on which kit and
equipment was used to generate the reported results. In Supplemental
Figures 7.1 – 7.4 this information is summarised.
7

Seegene. STARMag 96x4 Universal Cartridge Kit

1

Roche. MP24 Total NA kit
Roche. MagNA Pure 96 DNA and Viral NA Large Volume Kit

3

Roche. ePlex® Respiratory Pathogen Panel 2

3
7

Roche. DNA and Viral NA Small Volume kit

9

Roche. Cobas® Liat SARS-COV-2/FluA/FluB
Roche. Cobas® Liat FluA/FluB

1

Roche MP96 DNA en RNA Large Volume

1
3

Qiagen. QIAsymphony DSP Virus/Pathogen Mini Kit
Qiagen. QIAstat-Dx® Respiratory SARS-CoV-2 Panel

2

Qiagen, NeuMoDx Flu A-B/RSV/SARS-CoV-2 Vantage Assay

2

Promega Maxwell. Viral Total Nucleic Acid purification kit

1

Molgen. PurePrep Pathogens

1

Magcore. MagCore® Viral Nucleic Acid Extraction Kit

1

Life Technologies. MagMax pathogen RNA/DNA kit

1
5

Hologic.® Panther Fusion™ SARS-CoV-2/Flu A/B/RSV

2

Hologic.® Panther Fusion™ Flu A/B/RSV

5

Hologic.® Panther Fusion® Open Access™ RNA/DNA Enzyme…

1

ELITe InGenius® SP 200

2

Cepheid. Xpert® Xpress Flu/RSV

40

Cepheid. Xpert® Xpress CoV-2/Flu/RSV plus

3

BioMerieux. NUCLISENS® easyMAG®

1

BioMerieux. eMAG Specific A

7

BioMerieux Diagnostics. Respiratory panel 2.1 plus

1

BD. BD Respiratory Viral Panel
BD. BD MAX™ ExK™ TNA-3

3

Abbott. ID NOW™ INFLUENZA A & B 2

3

Abbott. Alinity SamplePrep kit 2

1

Abbott. 4plex AlinityM

1
0

5

10

15

20

25

30

Number of workflows
Figure 7.1 The RNA isolation kits used for the reported workflows.

Page 37 of 47

35

40

45

RIVM report 2023-0392

1
2
3

ThermoFischer Scientific. KingFisher Flex Purification System
Seegene. STARlet AIOS
Seegene. STARlet

11

Roche. Magna Pure 96

1

Roche. MagNA Pure 24

10

Roche. Cobas® Liat

3
2
2
1
1
1

Qiagen. QIAsymphony
Qiagen. QIAstat-Dx
Qiagen. NeuMoDx 96
Promega. Maxwell® RSC Instrument
Molgen. PurePrep 96
Magcore. MagCore®

12

Hologic. Panther® Fusion

2
3
1

Hamilton. Genomic STARlet
GenMark ePlex® System
ELITe InGenius®

41

Cepheid. GeneXpert®

1

BioMerieux. EMAG®
BioMerieux Diagnostics. easyMAG®

3

BioMerieux Diagnostics. Biofire FilmArray

4
3
3

BD. BD MAX™
Abbott. ID NOW™
Abbott. Alinity m System

0

7

10

20

30

Number of workflows
Figure 7.2 The RNA isolation equipment used for the reported workflows.

Page 38 of 47

40

RIVM report 2023-0392

3

VIASURE Flu A. Flu B & RSV Real Time PCR Detection Kit

1

Siemens. FTD™ Respiratory pathogens 21
Seegene. Allplex™ RV Master Assay

3

Seegene. Allplex™ RV Essential Assay

2

Seegene. Allplex™ Respiratory Panel 1A

2

SARS-CoV-2 PLUS ELITe MGB® Kit

1

Roche. ePlex® Respiratory Pathogen Panel 2 *

3

Roche. Cobas® Liat SARS-CoV-2/FluA/FluB *
Roche. Cobas® Liat FluA/FluB *

9
1

Qiagen. QIAstat-Dx® Respiratory SARS-CoV-2 Panel

2

Qiagen. NeuMoDx Flu A-B/RSV/SARS-CoV-2 Vantage Assay

2

Pathofinder. RespiFinder® 2Smart

1
21

In-house. LDT

7

Hologic.® Panther Fusion™ SARS-CoV-2/Flu A/B/RSV

5

Hologic.® Panther Fusion™ Flu A/B/RSV
Cepheid. Xpert® Xpress SARS-CoV-2/Flu/RSV *

1

Cepheid. Xpert® Xpress Flu/RSV *

1
40

Cepheid. Xpert® Xpress CoV-2/Flu/RSV plus
BioMerieux Diagnostics. Respiratory panel 2.1 plus *

7
1

BD. BD Respiratory Viral Panel
Abbott. ID NOW™ INFLUENZA A & B 2 *

3

Abbott. Alinity m Resp-4-Plex assay

2
0

10

20

30

Number of workflows
*Molecular point of care tests (mPOCT)

Figure 7.3 The RNA amplification kits used for the reported workflows.

Page 39 of 47

40

50

RIVM report 2023-0392

2

Seegene. STARlet AIOS

7

Roche. Lightcycler 480 II

1

Roche. Lightcycler 480

10

Roche. Cobas® Liat
QIAstat-Dx

2

Qiagen. Rotor-Gene Q

2

Qiagen. NeuMoDx96

2
12

Hologic. Panther® Fusion

3

GenMark ePlex® System

1

ELITe InGenius®

42

Cepheid. GeneXpert®
Bio-Rad. CFX96™ Real-time PCR System

7

BioMerieux Diagnostics. Biofire FilmArray

7
4

BD. BD MAX™

2

Applied Biosystems™. QuantStudio™ 5 Real-Time PCR System

8

Applied Biosystems™. 7500 Real-Time PCR

1

ABI quantstudio 5 én ABI 7500 fast

3

Abbott. ID NOW™

2

Abbott. Alinity m System
0

10

20

30

Number of workflows
Figure 7.4 The equipment used for RNA amplification for the reported workflows.

Page 40 of 47

40

50

RIVM report 2023-0392

7.2

Performance per workflow
In Supplementary Figure 7.5 the number of workflows reported per
laboratory is shown, including the score obtained for each workflow for
influenza A virus detection; subtyping results were not considered for
the scoring. Each laboratory was given an ID number to maintain
anonymity.
Only one lab scored an 8 overall for the one workflow which was
reported. This was a limited panel, and the kit used was the BIOFIRE
Respiratory panel 2.1 plus kit from BioMerieux, for which inconclusive
results were reported. However, strictly speaking this is actually correct
according to the IfU for an ‘Equivocal’ result, as is also shown in
Supplementary Table 7.1 for the RIVM reference results for this kit. For
this workflow, two of seven laboratories reported a final inconclusive
result for the same panel specimens (specimens 1, 4 and 9). As
mentioned in the Discussion section, this is not necessarily a bad thing,
since it can provide a first indication for the detection of an animal
influenza virus. Of the other five laboratories using this workflow, two
reported ‘inconclusive/positive’ for the ‘target result/final result’ and
three reported ‘positive/positive’. This is not exactly according to the
interpretation as dictated by the IfU. However, the laboratories reporting
these ‘target result/final result’ combinations may have used information
from additional testing to generate the ‘positive’ final result, as is also
described in the IfU when an ‘Equivocal’ result is obtained.
Seven other workflows generated a negative result, especially for
specimen 4 A(H1N1)v (7/7) and specimens 1 A(H7N2) (1/7), 6
A(H1N1)pdm09 (1/7) and 9 A(H1N2)v (1/7), resulting in a score lower
than 10. Of these seven workflows, one used an LDT, one the Abbott ID
NOW INFLUENZA A & B 2, two the Roche ePLEX Respiratory Pathogen
Panel 2 and three the Seegene Allplex RV Essential or Master Assays.
See chapter 3.4 for a response from the manufacturers and laboratories
concerning these deviant results.
Nevertheless, most (42/50; 84%) laboratories reported only workflows
scoring a 10, indicating that the performance of the tests used to detect
animal influenza A virus is good.

Page 41 of 47

RIVM report 2023-0392

Every lab was assigned a number to maintain anonymity.

Figure 7.5 Number of workflows submitted per lab and the scores obtained by
these workflows.

Page 42 of 47

RIVM report 2023-0392

7.3

In house RIVM results of commercial kits
Before distributing the panel containing the different viruses, it was
tested at RIVM using in-house LTD as ‘gold standard’ and additionally
with three commercial kits as a reference: the BioMerieux BIOFIRE®
Repiratory Panel 2.1 plus, the Cepheid GeneXpert and the Pathofinder
Respifinder 2Smart assays. The results are shown in supplementary
Table 7.1.
The results of the laboratories using the BioFire kit were very similar to
the results obtained in-house at RIVM. When inconclusive results were
reported (the reporting module option for inconclusive or equivocal
results), they were in line with the Equivocal Influenza A readouts we
obtained.
For Cepheid GeneXpert workflows the highest Cq values were reported
for panel specimen 4, the H1N1v zoonotic swine virus, which is
consistent with the results we obtained.
The Pathofinder RespiFinder® 2Smart was only reported once in the
EQA. This workflow was able to detect influenza A virus. However, it is
also able to do a H1(pdm09) subtype detection, but this was not
reported in the work (this is standard practice of the laboratory
involved). As our results show, this test detects the H1(pdm09) subtype
in panel specimen 6 correctly indeed, besides a successful generic
influenza A virus detection for all specimens.

7.4

Correlation Cq value and number of copies/specimen
For the panel, the number of virus genome copies per specimen was
determined using digital PCR (dPCR). In Supplementary Figure 7.6 the
correlation between the number of copies and the Cq values of all
reported workflows is depicted. A linear trendline is shown and indicates
a correlation between the number of copies per specimen and the Cq
value, despite a considerable bandwidth in Cq values among workflows
per specimen. This indicates that the higher the Cq value is, the lower
the number of copies in the specimen. However, an absolute correlation
between Cq value and number of copies does not exist and is workflow
dependent.
Panel specimen 4 with H1N1v virus had the higher number of deviant
results. This specimen contained 137 copies/µl, which did not make it
the specimen with the lowest number of copies, as this was panel
specimen 10 (H5N1 d2, 20 copies/µl). A specimen with a similar number
of copies was panel specimen 7 (H3N2 Swine, 145 copies/µl). However,
these two specimens were found to have similar or lower Cq values by
the workflows that failed to detect A(H1N1)v. This indicates that the
number of copies is unlikely to be the source of the deviant results for
panel specimen 4.

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RIVM report 2023-0392

Table 7.1 Results of three commercial kits evaluated with the EQA panel at RIVM.

Specimen code
EQA.1_AI23-01
EQA.1_AI23-02
EQA.1_AI23-03
EQA.1_AI23-04
EQA.1_AI23-05
EQA.1_AI23-06
EQA.1_AI23-07
EQA.1_AI23-08
EQA.1_AI23-09
EQA.1_AI23-10

Virus
H7N2
H3N2
n/a
H1N1v
H5N1 d1
H1N1pdm09
H3N2 swine
H5N6
H1N2v
H5N1 d2

BIOFIRE1 qualitative readout

Detected
None
Influenza A H3
None
None
Influenza A (no subtype detected)
Influenza A H1-2009
Influenza A H3
Influenza A (no subtype detected)
None
Influenza A (no subtype detected)

1. BioMerieux Diagnostics. BIOFIRE® Respiratory panel 2.1 plus
2. Cepheid. GeneXpert Xpress CoV-2/FLU/RSV plus
3. Pathofinder. RespiFinder® 2Smart

Page 44 of 47

Equivocal
Influenza A
None
None
Influenza A
None
None
None
None
Influenza A
None

GeneXpert2 Cq
value
FLU A1 FLU A2
29.5
30.8
26.4
29.0
No Cq
No Cq
31.9
32.9
24.5
26.5
26.3
27.4
28.7
29.9
23.1
26.3
27.7
27.6
29.0
30.8

Respifinder3 qualitative
readout
Influenza A H1N1pdm09
Positive
Negative
Positive
Negative
Negative
Negative
Positive
Negative
Positive
Negative
Positive
Positive
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative

RIVM report 2023-0392

For every specimen the Cq values are shown as colored dots. On the x-axis the copies/µl specimen are depicted. With a nonlinear fit a trendline was
created. With the virus names in the legend the host is shown: Av = avian; Hu = human; Sw = swine; v = variant, indicating human infected with
swine virus; H5N1 Av d2 is a 1:10 dilution of H5N1 Av d1.

Figure 7.6 Correlation between Cq values and number of copies per specimen.

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7.5

Participating laboratories
List of participating laboratories.
Name laboratory, city/island
Afd Medische Microbiologie. Radboudumc. Nijmegen
Atalmedial. Medisch Microbiologisch Laboratorium. sectie Moleculaire Biologie.
Amsterdam
Canisius Wilhelmina ziekenhuis. Medische microbiologie en infectieziekten. Nijmegen
Catharina Ziekenhuis Eindhoven. Algemeen klinisch laboratorium. Eindhoven
CBSL. Tergooi MC. Hilversum
Certe. MMB Friesland en Noordoostpolder. Leeuwarden
Certe. Divisie MMB locatie Groningen/Drenthe. Groningen
Eurofins Medische Microbiologie. Leiden
Eurofins PAMM. Veldhoven
Fundashon Mariadal. Bonaire
Groene Hart Ziekenhuis. Gouda
Klinisch Microbiologisch Laboratorium Afd. Medische Microbiologie LUMC. Leiden
LABHOH/FSLMA. Aruba
Labmicta. Hengelo
Laboratorium voor Medische Diagnostiek. Ziekenhuis Rivierenland. Tiel
LMMI. Isala klinieken. Zwolle
Maasstad Laboratorium Maasstad Ziekenhuis. Rotterdam
MeanderMC. Amersfoort
Medical Laboratory Services. Curaçao
Medisch Microbiologisch en Immunologisch Laboratorium. Rijnstate Ziekenhuis. Arnhem
Medisch Microbiologisch Laboratorium. IJssellandziekenhuis. Capelle aan den IJssel
Medisch Microbiologisch Laboratorium. Ikazia Ziekenhuis. Rotterdam
Medisch Microbiologisch Laboratorium. OLVG Lab. Amsterdam
Medische Microbiologie & Hygiëne en Infectiepreventie. Jeroen Bosch Ziekenhuis.
‘s-Hertogenbosch
Medische Microbiologie & Infectiepreventie. UMC Groningen. Groningen
Medische Microbiologie en Immunologie. St. Antonius Ziekenhuis. Nieuwegein
Medische Microbiologie en Infectiepreventie. Eurofins Gelre. Apeldoorn
Medische Microbiologie. Noordwest Ziekenhuisgroep. Alkmaar
Medische Microbiologie. UMCU. Utrecht
Medische Microbiologie. Viecuri Medisch Centrum. Venlo
Medische Microbiologie. Ziekenhuis Gelderse Vallei; organisatie: Dicoon. Ede
Medische Microbiologie. Deventer Ziekenhuis. Deventer
Medische Microbiologie. Franciscus Gasthuis en Vlietland. Rotterdam
Medische Microbiologie. Haaglanden Medisch Centrum. Den Haag
Medische Microbiologie. Hagaziekenhuis. Den Haag
Medische Microbiologie, infectieziekten en infectiepreventie (MMI). Maastricht UMC+.
Maastricht
Medische microbiologie. Laurentius ziekenhuis. Roermond
Medische Microbiologie. Ziekenhuis St Jansdal. Harderwijk
Medische Microbiologie. Zuyderland Medisch Centrum. Heerlen
Microvida. Laboratorium voor Medische Microbiologie en Immunologie. Tilburg
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RIVM report 2023-0392

MMI Adrz. Goes/Vlissingen
MMI Diakonessenhuis Utrecht. Utrecht
Moleculaire technieken. afdeling medische microbiologie en infectiepreventie.
AmsterdamUMC. Amsterdam
Mozand B.V. Eindhoven
Regionaal Laboratorium Medische Microbiologie Dordrecht-Gorinchem.
Dordrecht/Gorinchem
Reinier Haga MDC. Delft/Zoetermeer
Saltro. Unilabs. Utrecht
Star-shl. Etten-Leur/Rotterdam
Streeklab Haarlem. Haarlem
Unit Klinische Virologie. Viroscience. Erasmus MC. Rotterdam

Page 47 of 47

Published by:
National Institute for Public Health
and the Enviroment, RIVM
P.O. Box 1 | 3720 BA Bilthoven
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September 2023

Committed to
health and sustainability