External Quality Assessment of
laboratories Performing SARS-CoV-2
Diagnostics for the Dutch Population,
February 2021

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Colophon
© RIVM 2021
Parts of this publication may be reproduced, provided
acknowledgement is given to the: National Institute for Public
Health and the Environment, and the authors, title and year of
publication are cited.
John Sluimer1,*
Gabriel Goderski1,*
Sharon van den Brink1
Lisa Wijsman1
Chantal Reusken1
Marion Koopmans2
Richard Molenkamp2
Adam Meijer1
* Equal contribution
1. National Institute For Public Health and The Environment
(RIVM), Centre for Infectious Diseases Research, Diagnostics and
laboratory Surveillance, Antonie van Leeuwenhoeklaan 9, 3721 MA
Bilthoven, The Netherlands.
2. Erasmus Medical Centre Erasmus MC), Department Viroscience,
Dr. Molewaterplein 40, 3015 GD Rotterdam
AM coordinated the project. AM, GG and JS conceptualized the
study, developed and produced the panels and reporting system.
SvdB and LW performed reference PCR analysis. JS performed the
analysis of the reported data and wrote the initial version of the
report. AM critically reviewed the data analysis and report and JS
together with AM wrote the final version.
Corresponding author
Adam Meijer adam.meijer@rivm.nl
This investigation was performed by order, and for the account, of
the Ministry of Health, Welfare and Sport, project number
V/190002/01/PR.

This is a publication of the:
National Institute for Public Health
and the Environment
P.O. Box 1 | 3720 BA Bilthoven
The Netherlands

www.rivm.nl/en

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Contents
Colophon ................................................................................................................................................. 2
1. Introduction ........................................................................................................................................ 6
2 Materials and methods ........................................................................................................................ 6
2.1 Approach ....................................................................................................................................... 6
2.2 Contents of LEQA2 panel .............................................................................................................. 6
2.3 Scoring the workflows................................................................................................................... 9
3. Results ............................................................................................................................................... 10
3.1 Aggregated overview .................................................................................................................. 10
3.1 Used volumes, equipment, kits and reagents............................................................................. 11
3.2 Target genes used for RT‐PCR or other NAAT............................................................................. 17
3.3 Performance of the workflows ................................................................................................... 19
3.4 Comparing results LEQA2 to LEQA1 ............................................................................................ 31
4. Discussion and conclusion ................................................................................................................ 36
5. References ........................................................................................................................................ 38
6. Supplemental material ...................................................................................................................... 39
6.1 Results obtained per target gene per sample............................................................................. 39
6.2 Participating laboratories............................................................................................................ 49

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Summary
Background
Since January 2020 many workflows for molecular diagnostics of SARS‐CoV‐2 were implemented and
checked for performance using specificity and sensitivity panels distributed by the National Institute
for Public Health and the Environment (RIVM). Although panels have been largely similar in load
components for checking SARS‐CoV‐2 Nucleic Acid Amplification Tests (NAAT) performance, they were
not exactly similar in constituents. Because of this heterogeneity in the past, the fact that patchy
quality checks were implemented only when workflows change or laboratories were added to the
network, and because it is considered important by the COVID‐19 WHO reference laboratories at RIVM
and Erasmus Medical Centre (Erasmus MC) and the Dutch Ministry of Health, Welfare and Sport
(‘Dienst Testen’) that the performance of the network of COVID‐19 molecular diagnostic labs is
checked as a whole regularly, a National External Quality Assessment (EQA) (Landelijk EQA; LEQA)
program has been developed. This program consists of three rounds of LEQA. This report includes the
second round of the LEQA program.
Objective
The goal of this LEQA round is to inventorize the quality of the Dutch SARS‐CoV‐2 diagnostics field,
using a panel that consists of 10 simulated clinical specimens, containing heat inactivated SARS‐CoV‐
2, including one variant of concern (VOC) B.1.1.7; 20B/501Y.V1, or other respiratory viruses or genetic
material. Each of the laboratories was asked to conduct molecular detection of SARS‐CoV‐2 according
to their workflows normally used for SARS‐CoV‐2 diagnostics.
Materials and Methods
In January 2021 the LEQA panel was produced and pre‐tested at the RIVM. After obtaining the correct
results per sample, all laboratories performing SARS‐CoV‐2 diagnostics in the Dutch network were
contacted and notified of the distribution of the panel in the first week of February 2021. A number
of workflows, especially the molecular point of care (mPOCT) ones, use expensive cartridges or
pouches of which laboratories only receive a limited number every week. Therefore laboratories with
limited resources that wanted to test their workflows were asked to indicate so. Then they were sent
an email to limit testing for these workflows to samples 3, 5, 8 and 9. Laboratories were asked to
report their results via an online form. Workflows were given a score of 8 for 100% correct results for
the 8 core samples and reduced by 1 point per sample for an incorrect result and 0.5 points for a result
“Indeterminate”, “Equivocal” or “Inconclusive” for a core SARS‐CoV‐2 positive sample. When a
workflow tested the reduced panel (containing samples 3, 5, 8 and 9), 2 points per sample for an
incorrect result and 1 point for a result “Indeterminate”, “Equivocal” or “Inconclusive” for a sample
were subtracted from the maximum score.
Results
Out of 180 workflows reported, 148 scored a 100% correct score for all 8 core specimens (8 points)
and thus met all criteria set for reliable SARS‐CoV‐2 diagnostics, 18 scored between 7‐7.5, making it
likely that only minor adjustments need to be made to meet all criteria and 16 workflows scored a 6.5
or lower, indicating that a lot of improvements still need to be made for these workflows to be reliable
for SARS‐CoV‐2 diagnostics in clinical diagnostic settings and surveillance. For the SARS‐CoV‐2 negative
core samples one false positive result was reported and two workflows gave indeterminate, equivocal
or inconclusive results for non‐SARS‐CoV‐2 containing samples. For the SARS‐CoV‐2‐containing core
samples, false negative results (n=23) and false indeterminate, equivocal or inconclusive results (n=12)
were reported. Some workflows reported a negative result for SARS‐CoV‐2 presence in SARS‐CoV‐2‐
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containing samples due to cut‐off values used in the assay; up to 1.5% of each SARS‐CoV‐2 positive
sample (range: 0% – 1.5%) was reported as SARS‐CoV‐2 negative despite one (or more) of the target
genes against SARS‐CoV‐2 in the assay giving a Ct value. The VOC was detected by 176/177 (99.4%)
workflows. Despite the wide variety of kits, equipment and enzymes that are used in the implemented
workflows, the influence on the quality of molecular diagnostics for SARS‐CoV‐2 was limited.
Compared to the results obtained during the first round of quality control in this program (LEQA1), no
difference in overall quality of the SARS‐CoV‐2 diagnostic laboratory network was detected.
Conclusions
Overall the workflows used for SARS‐CoV‐2 diagnostics perform very well and laboratories using them
provide a reliable network. A small number of workflows should be further optimized to achieve full
potential. The Dutch SARS‐CoV‐2 diagnostics laboratory network performs on a very high level with
the vast majority of workflows detecting the core SARS‐CoV‐2 containing specimens correctly,
including VOC B.1.1.7; 20B/501Y.V1 that has become the major strain (up to 82%) in circulation by
week 9/2021 in The Netherlands. The wide variety of kits, equipment and enzymes used in the Dutch
SARS‐CoV‐2 diagnostic field do not affect adversely the quality of diagnostics. Instead, it allows for
great flexibility during times of shortages in supplies and likely improves the capacity to detect possible
future variants of SARS‐CoV‐2.

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1. Introduction
Since January 2020 many workflows for molecular diagnostics of COVID‐19 were implemented and
checked for performance using specificity and sensitivity panels distributed by the National Institute
for Public Health and the Environment (RIVM). Although panels have been largely similar in load
components for checking SARS‐CoV‐2 Nucleic Acid Amplification Tests (NAAT) performance, they
rapidly varied from SARS‐CoV‐1 RNA initially, to SARS‐CoV‐2 RNA, followed by SARS‐CoV‐2 whole heat
inactivated virus particles, depending on when materials became available. Because of this
heterogeneity in the past, the fact that patchy quality checks were implemented only when workflows
changed or laboratories were added to the network, and because it is considered important by the
COVID‐19 WHO reference laboratories at RIVM and Erasmus Medical Centre (Erasmus MC) and the
Dutch Ministry of Health, Welfare and Sport (‘Dienst Testen’) that we check the performance of the
network of COVID‐19 molecular diagnostic labs as a whole regularly, a National External Quality
Assessment (EQA) (Landelijk EQA; LEQA) program has been developed. This program consists of three
rounds of EQA. In the first week of February 2021 the second round of EQA panels was distributed to
all laboratories performing SARS‐CoV‐2 diagnostics on clinical samples derived from Dutch patients.
This panel consisted of 10 simulated clinical specimens that contained either heat inactivated SARS‐
CoV‐2, including one variant of concern (VOC) B.1.1.7; 20B/501Y.V1 that has become the major strain
(up to 82%) in circulation by week 9/2021 in The Netherlands [1], or other respiratory viruses or
genetic material. Each of the laboratories was asked to conduct molecular detection of SARS‐CoV‐2
on this panel according to their workflows normally used for SARS‐CoV‐2 diagnostics. All data had to
be reported back to the RIVM using an online reporting form.

2 Materials and methods
2.1 Approach
Except for the VOC containing sample the LEQA panel was produced at the RIVM in October 2020 and
pretested at the RIVM and Erasmus MC. Both centers obtained similar results. In January 2021
aliquoted samples for LEQA2 were thawed and analyzed again at RIVM. This confirmed that the
samples were of unchanged quality. The sample containing the VOC was prepared and pretested at
RIVM in January 2021. All 85 laboratories excluding the RIVM performing SARS‐CoV‐2 diagnostics in
the Dutch network by January 2021 were contacted and notified of the distribution of the panel in the
first week of February. All laboratories were asked to report their findings using an online form using
Formdesk software (Wassenaar, The Netherlands) to allow for a more streamlined method of data
collection. Laboratories had until the 21st of February to report their obtained results. After the 21st of
February laboratories that had not reported their results yet were given one week grace time to report
their results, after which the submission was closed on the 28th of February. A number of workflows,
especially the molecular point of care (mPOCT) ones, use expensive cartridges or pouches of which
laboratories only receive a limited number every week. Therefore laboratories with limited resources
that wanted to test their workflows were asked to indicate so. Then they were sent an email to limit
testing for these workflows to samples 3, 5, 8 and 9 (Table 1), covering three different concentrations
of SARS‐CoV‐2 of which one was the VOC and a hCoV‐NL63 containing sample.

2.2 Contents of LEQA2 panel
The LEQA2 panel consisted of 10 simulated clinical specimens (1ml) containing either whole infectious
human respiratory seasonal viruses, genetic material of relevant viruses or heat‐inactivated SARS‐CoV‐
2 viruses. SARS‐CoV‐2 was isolated from clinical specimens on VERO E6 cells and heat‐inactivated by
heat treatment at 60 °C for two hours. The number of detectable copies of SARS‐CoV‐2 positive strand
RNA in the stocks of SARS‐CoV‐2 was back‐calculated from determination of the copy number after
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extraction of RNA by digital SARS‐CoV‐2 E‐gene and RdRP‐gene PCR. For hCoV‐
19/Netherlands/NoordBrabant_10003/2020 at 1.28*10^8 and 1.73*10^8 copies of E‐gene and RdRP‐
gene positive strand RNA/ml, respectively. And for hCoV‐19/Netherlands/NH‐RIVM‐20432/2020 at
3.39*10^9 and 2.32*10^9 copies of E‐gene and RdRP‐gene positive strand RNA/ml, respectively
Because the viruses were not purified from the supernatant, the whole virus preparation contains in
addition to genomic RNA, intermediate replication negative strand genomic RNA and subgenomic E‐
gene RNA that contribute to detection in routine one‐step RT‐qPCR for SARS‐CoV‐2 RNA. Virus
dilutions were made in MEM with Hanks’ salts. HEp2 cells were added to the dilution at a
concentration of 10.000 cells per ml panel sample to simulate a clinical sample. The 10 samples
included in the panel contained the following viruses: SARS‐CoV‐2 (hCoV‐
19/Netherlands/NoordBrabant_10003/2020)
in
various
concentrations,
SARS‐CoV‐2
B.1.1.7/20B/501Y.V1 VOC‐202012/01 (UK) variant (hCoV‐19/Netherlands/NH‐RIVM‐20432/2020),
hCoV‐NL63 (kindly provided by Lia van der Hoek, Amsterdam University Medical Hospital), hCoV‐229E
(ATCC), hCoV‐OC43 (ATCC), SARS‐CoV‐1 (RNA) (kindly provided by Bart Haagmans, Erasmus MC) and
a sample without any virus. Except for the A(H3N2) containing sample in LEQA1 [2] that was replaced
by the VOC SARS‐CoV‐2 in LEQA2, all other samples in LEQA1 and 2 had the same content, but in
different order. In Table 1 all samples are listed together with the expected target specific Ct values
obtained at RIVM with routinely used diagnostic RT‐qPCRs for the respective pathogens and the
expected conclusion for SARS‐CoV‐2 detection in the samples. The digital copies of RdRP‐gene and E‐
gene are also listed in Table 1 for the SARS‐CoV‐2 containing samples.

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Table 1: Composition of LEQA2 together with the target specific expected Ct values1 based on the in‐house assay(s) of the RIVM.

Virus2

E‐gene
RdRP‐gene
Conclusion SARS‐
Number of
Number of
Target specific
(Sarbeco) Ct
(SARS‐CoV‐2) Ct CoV‐2
copies E gene
copies RdRP
Ct4
target/ml
gene target/ml
specimen,
specimen,
determined
determined
with dPCR3
with dPCR3
5
LEQA2_CoV20‐1
SARS‐CoV‐1
‐
‐
NA
28.57 (4/4)
Neg
Negative5
LEQA2_CoV20‐2
SARS‐CoV‐2 (d1)
1.28*10^4
1.73*10^4
NA
28.52 (4/4)
28.37 (4/4)
POSITIVE
ELQA2_CoV20‐3
hCoV‐NL63
‐
‐
28.10 (4/4)
Neg
Neg
Negative
LEQA2_CoV20‐4
hCoV‐229E
‐
‐
17.22 (4/4)
Neg
Neg
Negative
LEQA2_CoV20‐56
SARS‐CoV‐2 (d4)
1.28*10^1
1.73*10^1
NA
36.95 (2/4)
35.59 (2/4)
Weakly POSITIVE
LEQA2_CoV20‐6
SARS‐CoV‐2 (d3)
1.28*10^2
1.73*10^2
NA
34.80 (4/4)
34.88 (4/4)
POSITIVE
LEQA2_CoV20‐7
hCoV‐OC43
‐
‐
27.77 (4/4)
Neg
Neg
Negative
LEQA2_CoV20‐8
SARS‐CoV‐2 (d3)
1.28*10^2
1.73*10^2
NA
34.68 (4/4)
34.74 (4/4)
POSITIVE
LEQA2_CoV20‐97
SARS‐CoV‐2 VOC
3.39*10^5
2.32*10^5
NA
26.86 (4/4)
27.33 (4/4)
POSITIVE
LEQA2_CoV20‐10
No virus
‐
‐
Neg
Neg
Neg
Negative
1
The expected Ct values shown in this table are based on RT‐qPCR tests performed on the panel samples using the routinely used RIVM in‐house assays. The in‐house real‐
time RT‐qPCRs have been performed using the following reagents and volumes: ThermoFisher TaqMan® Fast Virus 1‐Step Master Mix after extraction of 200 μl sample on
Roche MagNA Pure 96 instrument with Roche MagNA Pure 96 DNA and Viral NA Small Volume Kit, elution in 50 μl and 5 μl extract per RT‐qPCR reaction on Roche LightCycler
480 mark I or II. Extractions and subsequent RT‐qPCRs were performed in 4‐fold; after the average Ct value between brackets () the number of times found positive is shown.
SARS‐CoV‐2 E‐gene Sarbeco specific primers and probes are those published by Corman et al.; the RdRP primers and probes are modified from those published by Corman
et al. to become SARS‐CoV‐2 specific and similar in LOD95 compared to the E‐gene Rt‐qPCR.
2
d1, d3 and d4 indicate that d3 is a 1:100 dilution of d1 and d4 is a 1:10 dilution of d3; SARS‐CoV‐2 is heat inactivated. SARS‐CoV‐1 is RNA stabilized with yeast tRNA.
3
dPCR has been performed on + strand genomic RNA for RdRP‐gene and E‐gene; for E‐gene, subgenomic messengers present are also detected. The one‐step E‐gene and
RdRP‐gene diagnostic RT‐qPCR also detects ‐ strand replicative form genomic RNA and the one‐step E gene RT‐qPCR in addition also detects subgenomic messengers, which
probably increases the actual number of target templates for the diagnostic RT‐qPCR in the sample after extraction.
4
For hCoV‐NL63 and hCoV‐229E N‐gene and hCoV‐OC43 M‐gene; n/a = not applicable.
5
Educational specimen. Laboratories using only the Corman E‐gene Sarbeco specific RT‐PCR will epidemiologically rightly label this specimen as SARS‐CoV‐2 positive. The
combination of low Ct with Sarbeco specific PCR and absence of positive signal with another SARS‐CoV‐2 target would prompt further research. One of the two targets
positive with SARS‐CoV‐2 usually only occurs with very low viral load.
6
Educational specimen: repeats of this specimen may have the E‐gene and/or RdRP‐gene negative; only 54.7% of reported workflows having tested this specimen reported
this specimen positive for SARS‐CoV‐2
7
SARS‐CoV‐2 variant B.1.1.7; 20B/501Y.V1 VOC‐202012/01 (UK); TaqPath assay: S‐dropout; TibMolbiol or other N501Y assay: 501Y variant positive. This is a core specimen.
Panel coding

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2.3 Scoring the workflows
The performance of each reported workflow was evaluated after which they were scored on a scale
from 0 to 8, with 8 being the best grade. This scoring system was implemented based on the detection
of the core samples present in the panel. All samples except LEQA2_CoV20‐1 and LEQA2_CoV20‐5
(containing SARS‐CoV‐1 RNA and an educational load of SARS‐CoV‐2, respectively) were deemed core
samples (samples with clinically relevant amounts of virus or no virus). The laboratories were given
the option to evaluate samples with the following scores: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. Each workflow started with 8 points for all correct results for each of the
8 core samples. For each wrongly determined core sample (being positive for a sample containing no
SARS‐CoV‐2 or vice versa) 1 point was deducted (out of 8). When a core sample was scored with an
“Indeterminate”, “Equivocal” or “Inconclusive” result, 0.5 point was deducted from the final mark of
the workflow.
For some workflows (e.g. molecular point‐of‐care test (mPOCT) workflows) an option was given to test
only a smaller subset of samples specimens in order to be able to make a limited statement about the
sensitivity of detection of SARS‐CoV‐2 with the mPOCT used. These workflows only had to test
LEQA2_CoV20‐3, LEQA2_CoV20‐5, LEQA2_CoV20‐8 and LEQA2_CoV20‐9. When notifying the Dutch
SARS‐CoV‐2 diagnostics network, the laboratories were informed about the option to apply for a
reduced (mPOCT) LEQA panel. Especially for these workflows the score “Not tested” was added as an
option. The workflows testing the reduced panel were also graded according to a scale from 0 – 8
points. For each wrongly determined core sample (being positive for a sample containing no SARS‐
CoV‐2 or vice versa) 2 points were deducted (out of 8). When a sample was scored with an
“Indeterminate”, “Equivocal” or “Inconclusive”, 1 point was deducted from the final mark of the
workflow.
When the entire panel was supposed to be tested using a workflow and a sample was given a score of
“Not tested”, 1 point was deducted from the final score for that workflow. This might have occurred
when a laboratory used a second or more workflows for confirmation of some of the results in the
first workflow used.
A workflow scoring 8 out of 8 passed all criteria set for SARS‐CoV‐2 diagnostics in terms of sensitivity
and specificity deemed necessary for SARS‐CoV‐2 diagnostics in accordance with the set requirements
for new workflows and laboratories. [3] Workflows scoring 7.5 or 7 out of 8 might still be valuable for
SARS‐CoV‐2 diagnostics, but need adjustments in order to perform as desired. Adjustments depend
on the type of result, e.g. an “Indeterminate”, “Equivocal” or “Inconclusive” result for low viral load
LEQA2_CoV20‐6 or LEQA2_CoV20‐8 samples is less severe than detection of SARS‐CoV‐2 targets in
specimens which were SARS‐CoV‐2 negative (false positive). Any workflows scoring below 7 out of 8
points needs serious adjustments in order to be fit for SARS‐CoV‐2 diagnostics.

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3. Results
3.1 Aggregated overview
Eighty‐five laboratories were contacted with the announcement of panel distribution for this second
EQA round. Seventy‐nine (92.9%) of these laboratories reported their findings for 180 workflows. The
workflow conclusions reported for each panel sample are summarized in Table 2 (‘Not tested’ result
excluded). The panel scores obtained per laboratory and by number of workflows used are
summarized in Table 3.
Table 2. Aggregated overview of workflow conclusions by LEQA panel sample.
Panel sample

Content

LEQA2_CoV20‐1

SARS‐CoV‐1

№ of workflows SARS‐CoV‐2 detection workflow conclusion
with test result № Positive
№ Indeterminate, № Negative
reported
Equivocal or
1
(n=180)
Inconclusive
161
70 (43.5%)
11 (6.8%)
80 (49.7%)

LEQA2_CoV20‐2
ELQA2_CoV20‐3

SARS‐CoV‐2 (d1)
hCoV‐NL63

160
176

160 (100%)
0

0
1 (0.6%)

0
175 (99.4%)

LEQA2_CoV20‐4
LEQA2_CoV20‐5

hCoV‐229E
SARS‐CoV‐2 (d4)

160
179

0
98 (54.7%)

0
12 (6.7%)

160 (100%)
69 (38.5%)

LEQA2_CoV20‐6

SARS‐CoV‐2 (d3)

162

147 (90.7%)

5 (3.1%)

10 (6.2%)

LEQA2_CoV20‐7
LEQA2_CoV20‐8

hCoV‐OC43
SARS‐CoV‐2 (d3)

160
176

0
157 (89.2%)

0
7 (4.0%)

160 (100%)
12 (6.8%)

LEQA2_CoV20‐9 SARS‐CoV‐2 VOC 177
LEQA2_CoV20‐10 No virus
160

176 (99.4%)
1 (0.6%)

0
1 (0.6%)

1 (0.6%)
158 (98.8%)

Errors

Not applicable,
educational sample
None
False indeterminate,
equivocal or
inconclusive (n=1)
None
Not applicable,
educational sample
False indeterminate,
equivocal or
inconclusive (n=5), false
negative (n=10)
None
False indeterminate,
equivocal or
inconclusive (n=7), false
negative (n=12)
False negative (n=1)
False positive (n=1),
false indeterminate,
equivocal or
inconclusive (n=1)

1 15 of the 180 workflows tested were mPOCT testing samples 3, 5, 8 and 9 only. For 5 of 180 workflows laboratories made

another limited selection of panel samples to test. Therefore the number of workflows with test result per sample is less
than 180.

Table 3. Aggregated overview of scores for core specimens obtained by laboratories using various numbers of workflows.

№ of workflows
per lab
9
7
6
5
4
3
2
1

№ of labs
1
2
1
4
8
10
16
37

№ of workflows per lab with indicated score (No of labs)
Score 8
Score 7 or 7.5
Score < 7
6 (n=1)
0
3 (n=1)
5‐6 (n=2)
1 (n=2)
1 (n=1)
5 (n=1)
1 (n=1)
0
1‐5 (n=4)
1‐4 (n=2)
1 (n=1)
3‐4 (n=8)
1 (n=2)
1 (n=1)
1‐3 (n=10)
1 (n=3)
1 (n=2)
2 (n=10)
0
0
1 (n=26)
1 (n=5)
1 (n=6)

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Despite not all workflows obtained fully correct results with the core specimens (Table 2), nearly all
laboratories (73/79; 92.4%) used at least one workflow with which a score 7‐8 was obtained (Table 3).
All laboratories using two or more workflows had at least one workflow with which a score of 8 for
fully correct results was obtained. Of the laboratories that used one workflow only 26/37 (70.3%) used
a workflow with which a score of 8 was obtained. In the subsequent chapters a more detailed insight
in the background of the results and the results themselves are presented.

3.1 Used volumes, equipment, kits and reagents
Because the sensitivity of a workflow is partly defined by the sample equivalent input volume in the
RT‐qPCR/other NAAT, a subset of questions revolved around the volumes used for testing of clinical
samples for each specific workflow: volume specimen in nucleic acid extraction; elution volume;
volume RNA/total NA in RT‐qPCR reaction or other NAAT; end volume of RT‐qPCR reaction or other
NAAT. Figure 1 shows each of the volumes used for RNA isolation and RT‐qPCR or other NAAT for all
workflows for which results were reported. For those workflows for which extraction input, elution
and RT‐qPCR/other NAAT input volumes were reported the sample equivalent input volume in RT‐
qPCR/other NAAT reaction was calculated and plotted (Figure 1).

Page 11 of 50

Figure 1: Volumes used in the RT‐PCRs or other NAATs described for the workflows reported. Most
commonly used volumes: 200 µl input volume; 50 µl elution volume; 10 µl elution input volume in
reaction; 20 µl end volume of reaction; 20 µl specimen input equivalent.

Other factors that may determine the performance of the workflows are the used kits, equipment
and/or separate enzymes used for extraction and amplification implemented in SARS‐CoV‐2
diagnostics for the Dutch population. Therefore for each workflow these details were inventoried.
Figure 2 shows the kits used for RNA/total NA isolation, Figure 3 shows the RNA isolation equipment,
Figure 4 shows the kits used for the RT‐PCR or other NAAT reaction, Figure 5 shows the separate
enzymes used for the in‐house RT‐PCR or other NAAT reaction and Figure 6 shows the equipment used
for the RT‐PCR or other NAAT reaction. In several occasions the kit used for extraction and for RT‐qPCR
or other NAAT has the same name because these are all‐in‐one kits.

Page 12 of 50

Figure 2: The RNA isolation kits used by workflows testing for SARS‐CoV‐2 together with the number of workflows per kit (n=180)

Page 13 of 50

Figure 3: The RNA isolation equipment used by workflows testing for SARS‐CoV‐2 together with the number of workflows per machine (n=180)

Page 14 of 50

Figure 4: The RT‐qPCR or other NAAT kits used by workflows testing for SARS‐CoV‐2 together with the number of workflows per kit (n=130). Not
all workflows use ‘ready to use’ kits for their RT‐qPCR or other NAAT, so the total N is not equal to the amount of workflows tested. For each kit
the used target genes are listed. Workflows using separate enzymes and primers and probe are listed in Figure 5.

Page 15 of 50

Figure 5: The enzymes used for performing RT‐PCR or other NAAT by workflows testing for SARS‐CoV‐2 together with the number of workflows
per enzyme (n=50). Not all workflows use separate enzymes for their RT‐PCR or other NAAT, so the total N is not equal to the amount of workflows
tested. In total 41/50 of the above mentioned workflows use 1 target gene to test for SARS‐CoV‐2 presence (E‐gene Sarbeco specific (n=33); N2‐
gene (n=1); N‐gene (n=2); ORF1a/b (n=2); RdRP‐gene (n=3)). 9/50 workflows use 2 target genes to test for SARS‐CoV‐2 presence (E‐gene Sarbeco
specific + N1‐gene (n=4); E‐gene Sarbeco specific + N‐gene (n=1); E‐gene Sarbeco specific + RdRP‐gene (n=2); E‐gene SARS‐CoV‐2 specific + N‐
gene (n=1); E‐gene SARS‐CoV‐2 specific + RdRP‐gene (n=1)). Workflows using complete ‘ready to use’ kits for SARS‐CoV‐2 detection are listed in
Figure 4.

Figure 6: The RT‐PCR or other NAAT equipment used by workflows testing for SARS‐CoV‐2 together with the number of workflows per machine
(n=180)

Page 16 of 50

3.2 Target genes used for RT‐PCR or other NAAT
As the sensitivity of a workflow may also depend on the used gene or genes, for all workflows the
target genes used were inventoried. Some workflows used up to 4 target genes. From the 180
workflows a total of 87 workflows used 1 target gene, 78 workflows used 2 target genes, 11 workflows
used 3 target genes and 4 workflows used 4 target genes. In Figure 7, the target genes used in the
order reported for each workflow are shown. In Figure 8 the combinations of target genes used in the
workflows is displayed. Combinations of genes used by number of workflows are listed in Table 4.
Some workflows using more than one gene do not generate separate result for each independent
gene but rather a composite conclusion.

Figure 7: Target genes used in the workflows as reported in the questionnaire (n=180). Color coding genes is shown in the
legend. Black color indicates workflows that do not contain a 2nd, 3rd or 4th gene target.

Page 17 of 50

Figure 8: Combinations of target genes used in the workflows as reported in the questionnaire (n=180). Color coding genes is shown in the legend.

Page 18 of 50

Table 4. Overview of number and type of target genes used per reported workflow.

№ target genes in
workflow

1

2

3

4

Target gene(s)
E‐gene Sarbeco specific
E‐gene SARS‐CoV‐2 specific
N2‐gene
N‐gene
Nsp2‐gene
ORF1a/b
RdRP‐gene
E‐gene Sarbeco specific; N1‐gene
E‐gene Sarbeco specific; N2‐gene
E‐gene Sarbeco specific; N‐gene
E‐gene Sarbeco specific; ORF1a/b
E‐gene Sarbeco specific; RdRP‐gene
E‐gene Sarbeco specific; S‐gene
E‐gene SARS‐CoV‐2 specific; N‐gene
E‐gene SARS‐CoV‐2 specific; RdRP‐gene
N1‐gene; N2‐gene
N‐gene; ORF1a/b
N‐gene; RdRP‐gene
ORF8; RdRP‐gene
M‐gene; S‐gene
E‐gene Sarbeco specific; N‐gene; RdRP‐gene
E‐gene Sarbeco specific; N‐gene; S‐gene
E‐gene SARS‐CoV‐2 specific; N‐gene; RdRP‐gene
E‐gene SARS‐CoV‐2 specific; N‐gene; RdRP‐gene
ORF1a/b; N‐gene; S‐gene
E‐gene Sarbeco specific; N‐gene; RdRP‐gene; S‐gene

№ workflows
39
18
1
7
1
16
5
1
4
30
5
3
1
1
2
7
13
6
1
4
3
1
2
1
4
4

3.3 Performance of the workflows
All laboratories participating in testing of the LEQA were asked to score each of the 10 samples
contained in the panel for the presence of SARS‐CoV‐2 in any of these six categories: Positive,
Negative, Indeterminate, Equivocal, Inconclusive or Not tested. As said before, for some workflows
only a smaller subset of samples needed to be tested. These workflows had to test only LEQA2_CoV20‐
3, LEQA2_CoV20‐5, LEQA2_CoV20‐8 and LEQA2_CoV20‐9. Figure 9 – Figure 18 show the obtained
results for LEQA2_CoV20‐1 up to LEQA2_CoV20‐10 for all target genes tested, in the order how the
genes have been reported and are displayed in Figure 7 and a summary plot for the total workflow
results. Some workflows using more than one gene do not generate separate result for each
independent gene but rather show a composite conclusion. Due to this, some results obtained of
multiple target genes are combined into and shown as one target gene in Figure 9 – Figure 18. The
sample containing B.1.1.7/20B/501Y.V1 VOC‐202012/01 (UK) variant SARS‐CoV‐2 was detected by
176/177 (99.4%) workflows, indicating primary diagnostic tests perform well detecting this variant
(Table 2, Figure 17). A re‐test of the panel was performed on the one workflow for which the
B.1.1.7/20B/501Y.V1 VOC‐202012/01 (UK) variant SARS‐CoV‐2 containing specimen (specimen
Page 19 of 50

number 9) was reported negative for SARS‐CoV‐2. In this second test, the workflow managed to detect
the SARS‐CoV‐2 VOC. During the initial test, this workflow found LEQA2 specimen number 10,
containing no virus, positive for SARS‐CoV‐2 presence, whereas it remained negative during the
second test. It is likely that specimen number 9 and 10 were switched during the first round of testing
the LEQA2 panel. When including the results obtained during the re‐test of this workflow, 100% of the
workflows included in this quality round could detect B.1.1.7/20B/501Y.V1 VOC‐202012/01 (UK)
variant SARS‐CoV‐2. No results on performance of variant specific assays were obtained.

Figure 9: Results obtained for LEQA2_CoV20‐1 containing SARS‐CoV‐1 combined from all workflows. All laboratories participating in testing of the
LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. This sample was not deemed a core sample in the panel. Overall, of 161 workflows that reported
an overall conclusion 80/161 (49.7%) did correctly not detect SARS‐CoV‐2 in this sample. 11/161 (6.8%) workflows reported an indeterminate,
equivocal or inconclusive result and 70/161 (43.5%) reported a positive result, highly likely because in the current epidemiological situation no other
Sarbeco virus than SARS‐CoV‐2 would be expected.

Page 20 of 50

Figure 10: Results obtained for LEQA2_CoV20‐2 containing SARS‐CoV‐2 (with 1.28*10^4 copies E target/ml sample (determined with dPCR) and
1.73*10^4 copies RdRP target/ml sample (determined with dPCR)) combined from all workflows. All laboratories participating in testing of the LEQA
were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. Overall, all 160 workflows that reported an overall conclusion did correctly detect SARS‐CoV‐2 in
this sample.

Figure 11: Results obtained for LEQA2_CoV20‐3 containing hCoV‐NL63 combined from all workflows. All laboratories participating in testing of the
LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. This sample was not deemed a core sample in the panel. Overall, of all 176 workflows that reported
an overall conclusion 175/176 (99.4%) identified this sample as SARS‐CoV‐2 negative and 1/176 (0.4%) with an indeterminate, equivocal or
inconclusive result.

Page 21 of 50

Figure 12: Results obtained for LEQA2_CoV20‐4 containing hCoV‐229E combined from all workflows. All laboratories participating in testing of the
LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. Overall, of all 160 workflows that reported an overall conclusion 160/160 (100%) workflows
correctly did not detect SARS‐CoV‐2 in this sample.

Figure 13: Results obtained for LEQA2_CoV20‐5 containing SARS‐CoV‐2 (with 1.28*10^1 copies E target/ml sample (determined with dPCR) and
1.73*10^1 copies RdRP target/ml sample (determined with dPCR)) combined from all workflows. All laboratories participating in testing of the LEQA
were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. This sample was not deemed a core sample in the panel. Overall, of all 179 workflows that reported
an overall conclusion 98/179 (54.7%) correctly identified this sample as SARS‐CoV‐2 positive and 12/179 (6.7%) workflows gave an indeterminate,
equivocal or inconclusive overall conclusion. 69/179 (38.5%) workflows reported incorrectly a negative result.

Page 22 of 50

Figure 14: Results obtained for LEQA2_CoV20‐6 containing SARS‐CoV‐2 (with 1.28*10^2 copies E target/ml sample (determined with dPCR) and
1.73*10^2 copies RdRP target/ml sample (determined with dPCR)) combined from all workflows. All laboratories participating in testing of the LEQA
were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. Overall, of all 162 workflows that reported an overall conclusion 147/162 (90.7%) correctly
identified this sample as SARS‐CoV‐2 positive and 5/162 (3.1%) workflows gave an indeterminate, equivocal or inconclusive overall conclusion.
10/162 (6.2%) workflows reported incorrectly a negative result.

Figure 15: Results obtained for LEQA2_CoV20‐7 containing hCoV‐OC43 combined from all workflows. All laboratories participating in testing of the
LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. Overall, of all 160 workflows that reported an overall conclusion 160/160 (100%) workflows
correctly did not detect SARS‐CoV‐2 in this sample.

Page 23 of 50

Figure 16: Results obtained for LEQA2_CoV20‐8 containing SARS‐CoV‐2 (with 1.28*10^2 copies E target/ml sample (determined with dPCR) and
1.73*10^2 copies RdRP target/ml sample (determined with dPCR)) combined from all workflows. All laboratories participating in testing of the LEQA
were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative, Indeterminate, Equivocal,
Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were reported) the percentage of
results reported per scoring category are shown. Overall, of all 176 workflows that reported an overall conclusion 157/176 (89.2%) correctly
identified this sample as SARS‐CoV‐2 positive and 7/162 (4.0%) workflows gave an indeterminate, equivocal or inconclusive overall conclusion.
12/176 (6.8%) workflows reported incorrectly a negative result.

Figure 17: Results obtained for LEQA2_CoV20‐09 containing SARS‐CoV‐2 (SARS‐CoV‐2 variant B.1.1.7; 20B/501Y.V1) (with 3.39*10^5 copies E
target/ml sample (determined with dPCR) and 2.32*10^5 copies RdRP target/ml sample (determined with dPCR)) combined from all workflows. All
laboratories participating in testing of the LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories:
Positive, Negative, Indeterminate, Equivocal, Inconclusive or Not tested. For each of the target genes used (which were processed in the order in
which they were reported) the percentage of results reported per scoring category are shown. Overall, of all 177 workflows that reported an overall
conclusion 176/177 (99.4%) correctly identified this sample as SARS‐CoV‐2 positive and 1/177 (0.6%) workflows reported incorrectly a negative
result.

Page 24 of 50

Figure 18: Results obtained for LEQA2_CoV20‐10 containing no virus (negative control) combined from all workflows. All laboratories participating
in testing of the LEQA were asked to score this sample for the presence of SARS‐CoV‐2 in any of these six categories: Positive, Negative,
Indeterminate, Equivocal, Inconclusive or Not tested. For each of the target genes used (which were processed in the order in which they were
reported) the percentage of results reported per scoring category are shown. Overall, of 160 workflows that reported an overall conclusion 176/177
(99.4%) identified this sample as SARS‐CoV‐2 negative and 1/176 (0.4%) with an indeterminate, equivocal or inconclusive result. 1/177 (0.6%)
workflows reported incorrectly a positive result.

As described before, all workflows were graded
using a point system from 0 (being the lowest
grade) up to 8 (highest grade). 148 workflows were
given an ‘8’, five workflows scored a ‘7.5’, 13
workflows scored a ‘7’, one workflow scored a ‘6.5’,
eight workflows scored a “6”, one workflow scored
a ‘5’, one workflow scored a ’3’ and three workflows
scored a ‘1’. It should be noted that all workflows
scoring less than 6 points did not test the full panel
or all samples of the reduced panel, thus these
workflows lost a lot of points due to samples being
scored ‘Not tested’. Figure 19 shows all grades Figure 19: All grades obtained by the reported workflows
out of the maximum of 8 points (n=180).
given to the reported workflows.
An overview containing the results obtained per target gene per panel sample for workflows reporting
Ct values is shown in Figure 20. In this figure for each of the target genes used (shown in the order in
which they were reported) the Ct values are shown for each of the tested samples. Also shown are
the number of tested samples using each of the target genes, the percentage of results showing a Ct
< 50, the number of reported negative results and the percentage of reported negative results (likely
due to implemented cut‐off values) with Ct < 50 .
The obtained scores per workflow are also coupled to the extraction kit or method used, the PCR or
other NAAT test performed and the number of target genes used in order to assess the effect of
different techniques on the performance of workflows. An overview of these factors on the grade is
shown in Figure 21. Unfortunately the specimen input equivalent volume in the PCR could only be
Page 25 of 50

calculated for 90/180 workflows and therefore this factor is not included in Figure 21. For the 90
workflows for which it was calculated the specimen equivalent volume was median 20 µl (range 2 µl
– 400 µl). For the 148 workflows for which specimen input volume in extraction was reported the
median volume was 250 µl (range 50 µl – 1400 µl). The median reaction volume reported for 95
workflows was 20 µl (range 4 µl – 30 µl).
In total one laboratory reported data for 9 workflows, two laboratories reported data for 7 workflows,
one laboratory reported data for 6 workflows, four laboratories reported data for 5 workflows, eight
laboratories reported data for 4 workflows, ten laboratories reported data for 3 workflows, sixteen
laboratories reported data for 2 workflows and thirty‐seven laboratories reported data for 1 workflow.
There are six laboratories which only have scores of < 7 for all reported workflows. The obtained scores
per workflow are sorted (anonymously) per laboratory and shown in Figure 22. Of the 79 laboratories,
68 laboratories reported at least one workflow with fully correct results. When excluding all mPOCT
assays from the analysis, one laboratory reported data for 7 workflows, one laboratory reported data
for 6 workflows, one laboratory reported data for 5 workflows, two laboratories reported data for 4
workflows, nine laboratories reported data for 3 workflows, eighteen laboratories reported data for 2
workflows, forty‐three laboratories reported data for 1 workflow and four laboratories did not report
any other workflows than mPOCT assays. Four laboratories only have scores of < 7 for all reported
workflows. This is shown in Figure 23. It should be noted that all workflows (also including mPOCT
assays) scoring less than 6 points did not test the full panel or all samples of the reduced panel, thus
these workflows lost a lot of points due to samples being scored “Not tested”. Therefore for these
laboratories and workflows a true performance estimate cannot be given.

Page 26 of 50

Figure 20: Results obtained per target gene per panel sample for workflows reporting Ct values. The numbers on the X‐axis indicate which target gene (in order in which they were reported) is used for the detection of each
sample. Underneath these numbers the contents of the sample are shown. All negative values for which no Ct value was given by the reporters have been given an artificial Ct value of 50. Not all negative results have a Ct
value of 50. Some results with Ct < 50 are deemed negative by laboratories, likely due to Ct cutoff values used in the interpretation of an obtained result. Above the graph the number of tests (N) and the percentage of Ct
values below 50 is shown per sample per target gene. Above the graph the number (N) and the percentage of tests with a negative results reported with a Ct value below 50 is shown as well per sample per target gene.
Samples deemed negative with a Ct value below 50 are indicated with a purple diamond inside the graph.

Page 27 of 50

Figure 21: A flow diagram showing all workflows reported to have tested the LEQA panel with extraction method, PCR test, the number of target
genes used and the final score achieved by each workflow. In the alluvial plot PCR tests using 1 target gene are depicted in blue, PCR tests using 2
target genes are shown in red, PCR tests using 3 target genes are shown in green and PCR tests using 4 target genes are shown in purple. For the
target gene combinations used per kit, see Figure 5. Color of trails per workflow are based on the grade obtained for LEQA2 All workflows receiving
grades below 6 are grouped in <6.

Page 28 of 50

Figure 22: Grades obtained per workflow per laboratory (anonymized). For each of the laboratories the amount of reported workflows is
shown on the X‐axis together with their accompanying grades. In total 79 laboratories sent in data of their workflows. There are six
laboratories which only have scores of < 7 for all reported workflows. It should be noted that all workflows scoring less than 6 points did not
test the full panel or all samples of the reduced panel, thus these workflows lost a lot of points due to samples being scored “Not tested”. The
same numbering is maintained as in Figure 23, where all workflows excluding mPOCT assays are listed.

Page 29 of 50

Figure 23: Grades obtained per workflow per laboratory excluding all mPOCT assays (anonymized). For each of the laboratories the amount
of reported workflows is shown on the X‐axis together with their accompanying grades. In total 79 laboratories sent in data of their workflows.
There are four laboratories which only have scores of < 7 for all reported workflows. Four laboratories do not report any other workflows than
mPOCT assays. It should be noted that all workflows scoring less than 6 points did not test the full panel or all samples of the reduced panel,
thus these workflows lost a lot of points due to samples being scored “Not tested”. The same numbering is maintained as in Figure 22, where
all workflows including mPOCT assays are listed.

Page 30 of 50

3.4 Comparing results LEQA2 to LEQA1
Whereas 65 laboratories reported data for 164 workflows for LEQA1, 79 laboratories reported data
for 180 workflows for LEQA2. This is an increase of 21.5% of reporting laboratories and an increase of
9.8% of reported workflows. The grades obtained by the workflows for both LEQA1 and LEQA2 are
listed in Table 5. It should be noted that 7/8 core samples were exactly the same in both panels. The
single different sample was LEQA1 influenza virus A(H3N2) replaced by LEQA2 B.1.1.7/20B/501Y.V1
VOC‐202012/01 (UK) variant SARS‐CoV‐2. Because the concentration of the variant virus in the sample
in LEQA2 was high the number of workflows detecting this variant was expected to be 100%, similar
to expected 100% of workflows not detecting A(H3N2) in panel LEQA1. Therefore, grades can directly
be compared between LEQA1 and LEQA2.

LEQA1
LEQA2

8

7.5

Grade obtained
7
6.5

132
(80.5%)
148
(82.2%)

7
(4.3%)
5
(2.7%)

11
(6.7%)
13
(7.2%)

0
1
(0.1%)

6

<6

8
(4.9%)
8
(4.4%)

6
(3.7%)
5
(2.7%)

Total workflows
tested
164
180

Table 5. Overview of grades obtained by the workflows having tested LEQA1 and LEQA2. All workflows receiving grades below
6 are grouped in <6

The shift in grades obtained by the workflows tested between LEQA1 and LEQA2 are shown in Table
6. Not all workflows were tested for both LEQA1 and LEQA2. These workflows are labelled ‘Not
Tested’ or ‘NT’ abbreviated. This data is also visualized in Figure 24.
Table 6. Overview of grades obtained by workflows that tested LEQA1 and how they scored testing LEQA2. Green cells
indicate workflows performing better in LEQA2 than in LEQA1. Red cells indicate workflows performing worse in LEQA2
than in LEQA1. White cells indicate workflows performing equally in LEQA 2 and LEQA1. All workflows receiving grades
below 6 are grouped in <6

Grade
obtained
for
LEQA2

<6
6
6.5
7
7.5
8
NT2
Subtotal

1
2

<6
1

6

Grade obtained for LEQA 1
6.5
7
7.5

8
4

1
4

3
5

6

8

2

1
2

4
5

3
1

5
2
71
50

11

7

132

NT1
4
4
4
3
66

Subtotal
5
8
1
13
5
148
180

0

164

Short for ‘Not Tested’. These workflows were not used to test LEQA1, but were used to test LEQA2
Short for ‘Not Tested’. These workflows were not used to test LEQA2, but were used to test LEQA1

Page 31 of 50

Figure 24: Overview of grades obtained by workflows that tested LEQA1 and how they scored testing LEQA2. ‘NT’ stands ‘Not Tested’.
These workflows were not used to test LEQA1, but have tested LEQA2 or vice versa. Color of trails per workflow are based on the
grade obtained for LEQA2. All workflows receiving grades below 6 are grouped in <6

In total 65 workflows were not used to test LEQA2 but did test LEQA1. Twenty‐seven of these
workflows were mPOCT assays (41.5%), 22 workflows used ‘ready to use’ kits for their RT‐qPCR
(33.8%) and 16 workflows were in‐house assays (24.%). Of the 81 newly implemented workflows in
LEQA2 (which were not tested in LEQA1), 31 were mPOCT assays (38.3%), 30 workflows used ‘ready
to use’ kits for their RT‐qPCR (37.0%), 2 workflows were Loop‐mediated isothermal amplification
(LAMP) assays (2.5%) and 18 workflows were in‐house assays (22.2%).
In total 78/162 (48.2%) of the workflows tested in LEQA1 could detect the educational load of SARS‐
CoV‐2 (with 12.8 and 17.3 copies E gene and RdRP gene target/ml sample, respectively; dilution d4,
see Table 1) whereas 98/179 (54.7%) of the workflows tested in LEQA2 could detect SARS‐CoV‐2 in
this sample. For LEQA1 both LEQA1_CoV20‐5 and LEQA1_CoV20‐7 contained SARS‐CoV‐2 at a
concentration of 128 and 173 copies E gene and RdRP gene target/ml specimen, respectively (dilution
d3, see Table 1). LEQA1_CoV20‐5 was measured positive for SARS‐CoV‐2 by a total of 131/148 (88.5%)
of the workflows. For LEQA1_CoV20‐7 139/158 (88.0%) of the workflows tested these samples
positive for SARS‐CoV‐2. In LEQA2 identical samples with the same SARS‐CoV‐2 load but with different
labelling were put in the panel: LEQA2_CoV20‐6 and LEQA_CoV20‐8 respectively. LEQA2_CoV20‐6 was
scored positive for SARS‐CoV‐2 by 147/162 (90.7%) workflows, an increase with 2.7% compared to
LEQA1_CoV20‐5. LEQA2_CoV20‐8 was scored positive by 157/176 (89.2%) workflows, an increase with
1.2% compared to LEQA1_CoV20‐7. Another set of specimens with the same SARS‐CoV‐2 loads in both
LEQA panels were LEQA1_CoV20‐1 and LEQA2_CoV20‐2. These samples contained 1.28×104 copies E
gene and RdRP gene target/ml specimen, respectively (dilution d1, see Table 1). LEQA1_CoV20‐1 was
scored positive for SARS‐CoV‐2 by 161/162 (99.4%) workflows, whereas LEQA2_CoV20‐2 was scored
Page 32 of 50

positive by 160/160 (100%) workflows, an increase by 0.6%. So, at the highest SARS‐CoV‐2
concentration (d1), the amount of positive results increased from 99.4% to 100%. The percentage of
positive results for both specimens with the intermediate SARS‐CoV‐2 load (d3) increased from
88.0%/88.5% to 89.2%/90.7%. Lastly the specimens with the lowest SARS‐CoV‐2 load (d4) of both
panels show an increase in positive results from 48.2% to 54.7%. Taken together, for each of the
samples with different SARS‐CoV‐2 concentrations, the workflows included in LEQA2 showed an
increase in the percent of positive results compared to those included in LEQA1. This is an indication
that more‐sensitive workflows have been implemented since LEQA1 or that existing workflows have
been made more sensitive.
In total, 99 workflows used to test LEQA1 were also used to test LEQA2 (Table 6). A detailed description
of a comparison of the performance with these workflows is given below.
In total, 74/99 workflows did not show a change in grade between LEQA2 and LEQA1. One workflow
obtained a grade below 6/8, two workflows obtained a grade of 7/8 and 71 workflows scored a grade
of 8/8 for both LEQA rounds.
Of all workflows scoring a lower grade for LEQA2 than for LEQA1 (n=14/99), 11 (78.6%) previously
scored a grade of 8/8 and 3 (21.4%) scored a grade of 7.5/8 for LEQA1. For LEQA2 the obtained grades
were 6/8 by four (28.6%) workflows; 6.5/8 by one (7.1%) workflow; 7/8 by seven (50%) workflows;
7.5/8 by two (14.3%) workflows. For LEQA2 6 (42.9%) of the workflows reported one false negative
result for one of the lowest concentration SARS‐CoV‐2 containing core specimens (dilution d3, see
Table 1); 2 (14.3%) of the workflows reported a false negative result for both lowest concentration
SARS‐CoV‐2 containing core specimens (dilution d3, see Table 1); 1 (7.1%) of the workflows reported
a false indeterminate/inconclusive/equivocal result for one of lowest concentration SARS‐CoV‐2
containing core specimens (dilution d3, see Table 1); 2 (14.3%) of the workflows reported a false
indeterminate/inconclusive/equivocal result for both lowest concentration SARS‐CoV‐2 containing
core specimens (dilution d3, see Table 1); 1 (7.1%) of the workflows reported one false
indeterminate/inconclusive/equivocal and one false negative result for the lowest concentration
SARS‐CoV‐2 containing core specimens (dilution d3, see Table 1); 1 (7.1%) of the workflows reported
a false negative result for SARS‐CoV‐2 variant B.1.1.7; 20B/501Y.V1 VOC‐202012/01 (UK) and a false
positive result for no virus control; 1 (7.1%) of the workflows reported a false
indeterminate/inconclusive/equivocal result for the no virus control. Most of the incorrect results
have been obtained for the SARS‐CoV‐2 dilution d3 of the core samples (10‐fold higher concentration
than the educational load sample), suggesting that the concerned workflows lost some sensitivity
between both LEQA rounds. This data is summarized in Table 7.
Of all workflows scoring a higher grade for LEQA2 than for LEQA1 (n=11/99), 3 (27.3%) workflows
previously scored a 7.5/8; 4 (36.4%) workflows scored a 7/8; 3 (27.3%) workflows scored a 6/8; 1
workflow (9.1%) scored a 4/8. When testing LEQA2, all these workflows scored an 8/8. So, all core
samples were determined correctly using all the concerned workflows. For LEQA1 3 (27.3%) workflows
reported a false indeterminate/inconclusive/equivocal result for the no virus control; 2 (18.2%) of the
workflows reported one false negative result for the lowest concentration SARS‐CoV‐2 containing core
specimens (dilution d3, see Table 1); 2 (18.2%) workflows reported of the workflows reported a false
indeterminate/inconclusive/equivocal result for both lowest concentration SARS‐CoV‐2 containing
core specimens (dilution d3, see Table 1); 2 (18.2%) workflows reported false negative results for both
lowest concentration SARS‐CoV‐2 containing core specimens (dilution d3, see Table 1); 1 (9.1%)
workflow only tested part of the LEQA1 panel and did not test the reduced mPOCT panel. Most of the
improvements in LEQA2 compared to LEQA1 have been obtained for the SARS‐CoV‐2 dilution d3 of
the core samples (10‐fold higher concentration than the educational load sample), suggesting that the
Page 33 of 50

concerned workflows gained some sensitivity between both LEQA rounds. These results are
summarized in Table 8.

Table 7. Overview of the reasons workflows obtained lower grades for LEQA2 than for LEQA1. For LEQA1 the previously
obtained grades are also shown and how they were obtained.

LEQA1

Higher
Lower

LEQA2

1x false
indeterminate/inconclusive/equivocal for no
virus control
(grade 7.5/8)
1x false
indeterminate/inconclusive/equivocal for
SARS‐CoV‐2 dilution d3
(grade 7.5/8)
1x false negative for SARS‐CoV‐2 dilution d3
(grade 7/8)
2x false
indeterminate/inconclusive/equivocal for
SARS‐CoV‐2 dilution d3
(grade 7.5/8)
1x false negative for SARS‐CoV‐2 dilution d3
AND
1x false
indeterminate/inconclusive/equivocal for
SARS‐CoV‐2 dilution d3
(grade 6.5/8)
1x false negative for SARS‐CoV‐2 dilution d3
(mPOCT panel; grade 6/8)
2x false negative for SARS‐CoV‐2 dilution d3
(grade 6/8)
1x false negative for SARS‐CoV‐2 variant
B.1.1.7; 20B/501Y.V1 VOC‐202012/01 (UK)
AND
1x false positive for no virus control
(grade 6/8)2

1x false
indeterminate/inconclusive/equivocal
for SARS‐CoV‐2 dilution d3
(grade 7.5/8)

No errors (grade
8/8)

na1

n=1

na

n=1

na

n=5

n=2

na

n=1

na

na

n=1

na

n=23

na

n=1

1

na = not applicable.
These samples were likely switched during testing. Upon retesting by the involved laboratory after closure of
the reporting period and receiving the expected results, this workflow correctly determined all core samples of
LEQA2.
3
One of these workflows did find the educational SARS‐CoV‐2 containing specimen (dilution d4; see Table 1)
positive for SARS‐CoV‐2. This might be an indication that the workflow does have a high sensitivity concerning
SARS‐CoV‐2 detection.
2

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Table 8. Overview of the reasons workflows obtained higher grades for LEQA2 than for LEQA1. For LEQA1 the previously
obtained grades are also shown and how they were obtained.

Higher
Lower

LEQA1

LEQA2
No errors (grade 8/8)

1x false indeterminate/inconclusive/equivocal
for SARS‐CoV‐2 dilution d3
(grade 7.5/8)

n=3

1x false negative for SARS‐CoV‐2 dilution d3
(grade 7/8)

n=2

2x false indeterminate/inconclusive/equivocal
for SARS‐CoV‐2 dilution d3
(grade 7/8)

n=2

2x false negative for SARS‐CoV‐2 dilution d3
(grade 6/8)

n=3

1x Testing only part of LEQA panel, but not
mPOCT panel
(grade 4/8)

n=1

On a laboratory level, when testing LEQA1, two laboratories had workflows with grades below 7/8
only. One of these laboratories (laboratory 61 in Figures 22 and 23) has since improved their workflow
and no longer scores a grade below 7 with the implemented workflow. The other laboratory
(laboratory 58 in Figures 22 and 23) unfortunately still scores a grade below 7/8. When testing LEQA2,
unfortunately two new labs have received grades of below 7/8 only, whereas they did not in LEQA1
(laboratories 45 and 71 in Figures 22 and 23). The workflow of laboratory 71 (in Figure 22 and 23)
scored a grade below 7/8 due to missing two core specimen containing SARS‐CoV‐2. This same
workflow however was able to detect SARS‐CoV‐2 in the educational specimen (dilution d4, see Table
1). This might indicate that this workflow does have a high sensitivity for SARS‐CoV‐2 detection. The
reason why the 10‐fold higher concentration has been missed remains unclear.
Also three laboratories who did not participate in LEQA1 have received grades below 7/8 for LEQA2
(laboratories 76, 78 and 79 in Figures 22 and 23). Two of these latter laboratories did not test the full
LEQA2 panel nor the reduced mPOCT panel, leading to a lower grade. And therefore the performance
of these laboratories can in fact not be evaluated using these results.

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4. Discussion and conclusion
Out of 180 workflows reported, 148 scored a 100% correct score for all 8 core specimens (8 points)
and thus met all criteria set for reliable SARS‐CoV‐2 diagnostics, 18 scored between 7‐7.5, making it
likely that only minor adjustments need to be made to meet all criteria and 16 workflows scored a 6.5
or lower, indicating that a lot of improvements still need to be made for these workflows to be reliable
for SARS‐CoV‐2 diagnostics in clinical diagnostic settings and surveillance. All workflows scoring grades
below 6 (n=6) seemed to have obtained a low score due to testing only a small subset of the core
samples while not testing the reduced panel of 4 core samples that has been recommended for
mPOCT testing. Due to this, a lot of samples received a score of “Not tested” and thus decreased the
overall grade of the test. When the workflows on which only the full panel or reduced mPOCT panel
were tested are taken into account (n=174), none of the workflows submitted received a grade below
6.
When considering all workflows each laboratory has access to and have reported LEQA2 results for,
six laboratories have workflows which do not have a grade of 7 or above. Although it is not necessary
to use a workflow scoring a 7 or 7.5 out of 8 for SARS‐CoV‐2 diagnostics, due to slight reduced
sensitivity to detect SARS‐CoV‐2, during the current period with a prevalence of COVID‐19 around 10%
or higher, it is likely that only minor adaptions of the concerned workflows are needed to perform
within the desired criteria detecting all core samples correctly.
Only four laboratories have access to solely mPOCT based assays, limiting their maximal daily
throughput of clinical specimens.
Throughout the reported workflows lots of different target genes and combinations of them are
reported, but the E‐gene as target gene as either a Sarbeco specific or SARS‐CoV‐2 specific target is
most prevalent (115 out of 180 workflows). There was no significant difference in performance
between workflows using different single target genes or combinations of target genes.
False indeterminate/inconclusive/equivocal results have been obtained for samples containing hCoV‐
NL63 (1/176; 0.6%) and no virus control (1/160; 0.6%). Otherwise no such results have been reported
for non SARS‐CoV‐2 containing specimen. This shows that the implemented workflows show a high
specificity to SARS‐CoV‐2 and the RT‐PCRs or other NAATs rarely cross‐react to other human
coronaviruses or other non SARS‐CoV‐2 containing samples. One false positive result for
LEQA2_CoV20‐10 that does not contain virus has been reported by the laboratories. This was likely
due to accidental swapping of specimens LEQA2_CoV20‐9 and ‐10 by the technician. Additionally,
many workflows reported the SARS‐CoV‐1 containing sample (LEQA2_CoV20‐1) positive for
SARS‐CoV‐2 due to using Sarbeco‐specific RT‐PCR. Considering the fact that SARS‐CoV‐1 is not
circulating, this does not pose a problem for the accuracy of SARS‐CoV‐2 diagnostics performed in
clinical and surveillance settings. However, it is striking that for a quite some workflows SARS‐CoV‐2
specific E‐gene primers/probe are reported whilst it still detects SARS‐CoV‐1 (21.74%; Supplemental
Figure 1). It appears that for these workflows most likely the specificity of E‐gene primers/probe is
wrongly labelled as SARS‐CoV‐2 specific rather than Sarbeco specific. Or have a strong cross‐reaction
to SARS‐CoV‐1, which is less likely.
Whereas very little false positive results have been reported for the core samples included in the
panel, a number of workflows reported false negative results for some of the core samples containing
SARS‐CoV‐2. Apparently sensitivity is a bigger issue than specificity for the workflows used for SARS‐
CoV‐2 diagnostics.
Despite some workflows generated a Ct value using SARS‐CoV‐2 RT‐PCR, the sample was deemed
SARS‐CoV‐2 negative, possibly related to a used cut off value or other criterium, e.g. shape of the
amplification curve. Up to 1.5% of each SARS‐CoV‐2 positive sample (range: 0% – 1.5%) was reported
as SARS‐CoV‐2 negative despite one (or more) of the target genes against SARS‐CoV‐2 in the assay
Page 36 of 50

giving a Ct value. For all LEQA samples tested with a workflow providing Ct values as an output, a
broad range of Ct values for the individual SARS‐CoV‐2 containing samples has been reported. The
biggest range of reported Ct values was found for LEQA_CoV20‐9. The average Ct value was 26.89 (SD
2.40; data not shown) where the lowest Ct value reported was 16.29 and the highest value was 41.60
(making a 25.31 difference in Ct value), indicating a wide spread of Ct values reported for the same
sample, generated by the large variety of workflows used. Despite this wide range of Ct values for the
same sample by different workflows, this did not affect the sensitivity of the workflows significantly.
This finding indicates clearly that comparing Ct values between workflows and laboratories should not
be done without calibration using a standard.
A wide array of varying in‐house and kit‐based SARS‐CoV‐2 workflows have been reported, that has
been changed and even broadened compared to LEQA1 [2]. Compared to the 2009 influenza
pandemic, the Dutch clinical diagnostic field for respiratory diagnostics shows a divergent pattern in
use of kits, reagents and equipment. [4] A more divergent use of kits, reagents and equipment can be
quite useful in a laboratory network as a shortage of any of these can be compensated by switching
to different equipment or when certain workflows are less capable of detecting new strains of SARS‐
CoV‐2. This is highly relevant with the rising level of infections with new variants of SARS‐CoV‐2.
Therefore it is encouraging that in the current LEQA2 variant of concern B.1.1.7; 20B/501Y.V1 that has
become the major strain (up to 82%) in circulation by week 9/2021 in The Netherlands [1] was
detected by 176/177 (99.4%) workflows that tested this sample.
Comparing this second round of quality assessment with the first round, 14 additional laboratories
reported their findings. The total number of reported workflows increased with 15 compared to the
earlier assessment. From the workflows initially scoring a grade <6 for LEQA1, 4/6 (66.7%) were not
retested with LEQA2. Similarly, from 5/8 (62.5%) workflows initially scoring a 6, 5/11 (45.5%)
workflows initially scoring a 7, 1/7 (14.3%) workflows initially scoring a 7.5 and 50/132 (37.9%)
workflows initially scoring an 8 were not retested with LEQA2. The relative amount of LEQA1
workflows not re‐tested in LEQA2 is bigger for the poorer scoring workflows (<7) compared to the
better scoring workflows (7‐8) in LEQA1. This data seems to indicate that laboratories use these quality
assessments as a way to improve their performance in SARS‐CoV‐2 diagnostics. The absolute amount
of workflows obtaining a score of <6 only decreased by 1 (from 6 to 5) from LEQA1 to LEQA2. This is
due to an influx of four new workflows compared to LEQA1 with which not the full LEQA2 panel nor
the reduced mPOCT panel was tested, leading to a lower grade. Two of these new workflows belong
to laboratories that did not test the LEQA1 panel. Nevertheless, there seems to be a trend showing a
disuse of lesser performing workflows. However, this data might be biased due to not all workflows
being used by a laboratory have been tested and reported in this second round by all laboratories.
Overall we can conclude that the workflows used for SARS‐CoV‐2 diagnostics for the Dutch population
perform very well. There are some workflows which need some work in order for them to perform as
desired, but all in all the Dutch SARS‐CoV‐2 diagnostics laboratory network appears to perform on a
very high level.

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5. References
1. RIVM. Varianten van het coronavirus SARS‐CoV‐2. 2021 [cited 2021 31‐3‐2021]; Available from:
https://www.rivm.nl/coronavirus‐covid‐19/virus/varianten.
2. J. Sluimer et al., “External Quality Assessment of laboratories Performing SARS‐CoV‐2 Diagnostics
for the Dutch Population, November 2020”, RIVM report. Available from:
https://www.rivm.nl/sites/default/files/2021‐
02/EQA%2520of%2520Laboratories%2520Performing%2520SARS‐CoV‐
2%2520Diagnostics%2520for%2520the%2520Dutch%2520Population%2520November‐2020.pdf
3. RIVM. Aanvullende informatie diagnostiek COVID‐19. 2021 [cited 2021 26‐3‐2021]; 24: Available
from: https://lci.rivm.nl/covid‐19/bijlage/aanvullend.
4. A. Meijer et al., “Preparing the outbreak assistance laboratory network in the Netherlands for the
detection of the influenza virus A(H1N1) variant”, J. Clin. Virol., vol. 45, no. 3, pp. 179–184, 2009.

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6. Supplemental material
6.1 Results obtained per target gene per sample
Here all results (any of these six categories: Positive, Negative, Indeterminate, Equivocal, Inconclusive
or Not tested) obtained per target gene are shown in percentages per panel sample number. Some
workflows using more than one gene do not generate separate result for each independent gene but
rather a composite conclusion. In Supplemental Figure 1‐10 these are shown together as one target
gene.

Supplemental Figure 1: The percentages of the various scores obtained for LEQA2_CoV20‐1 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

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Supplemental Figure 2: The percentages of the various scores obtained for LEQA2_CoV20‐2 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

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Supplemental Figure 3: The percentages of the various scores obtained for LEQA2_CoV20‐3 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

Page 41 of 50

Supplemental Figure 4: The percentages of the various scores obtained for LEQA2_CoV20‐4 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample. This sample was not deemed a core sample from the LEQA panel.

Page 42 of 50

Supplemental Figure 5: The percentages of the various scores obtained for LEQA2_CoV20‐5 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

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Supplemental Figure 6: The percentages of the various scores obtained for LEQA2_CoV20‐6 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

Page 44 of 50

Supplemental Figure 7: The percentages of the various scores obtained for LEQA2_CoV20‐7 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample.

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Supplemental Figure 8: The percentages of the various scores obtained for LEQA2_CoV20‐8 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample

Page 46 of 50

Supplemental Figure 9: The percentages of the various scores obtained for LEQA2_CoV20‐9 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample.

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Supplemental Figure 10: The percentages of the various scores obtained for LEQA2_CoV20‐10 using the various target genes implemented in the workflows
reported. The percentages shown are combined from target genes reported as either target gene 1, 2, 3 or 4 in the various workflows. The results are depicted
in relation to SARS‐CoV‐2 presence in the sample. This sample was not deemed a core sample from the LEQA panel.

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6.2 Participating laboratories
All participating laboratories are listed below. We would like to thank colleagues from these
laboratories for their participation in this round of LEQA for the Dutch SARS‐CoV‐2 diagnostics
laboratory network.
Laboratory
Admiraal de Ruyter Ziekenhuis
Alrijne Zorggroep
ArminLabs GmbH
Atalmedial
Brightlabs B.V.
Canisius Wilhelmina Ziekenhuis
Catharina Ziekenhuis
Certe
Deventer Ziekenhuis
Diagnostiek voor U
Diakonessenhuis Utrecht
Elisabeth Tweesteden Ziekenhuis
Erasmus Medisch Centrum
Eurofins Biomnis France
Eurofins Genomics Europe Applied Genomics GmbH
Eurofins NMDL‐HVL
Eurofins NMDL‐LCPL
Fenelab Consortium ‐ Mérieux Nutrisciences
Fenelab Consortium ‐ NofaLab B.V.
Fenelab Consortium ‐ Normec Biobeheer
Fenelab Consortium ‐ Nutreco Nederland B.V. ‐ MasterLab
Fenelab Consortium ‐ NutriControl
Fenelab Consortium ‐ Nutrilab B.V.
Fenelab Consortium ‐ SGS Nederland B.V.
Fenelab Consortium ‐ Triskelion
Franciscus Gasthuis & Vlietland
Gelre Ziekenhuizen Apeldoorn
GGD Amsterdam Streeklaboratorium
Groene Hart Ziekenhuis
Haaglanden Medisch Centrum
Hagaziekenhuis
IJssellandziekenhuis
Ikazia ziekenhuis
inBiome
Isala
Izore
Jeroen Bosch ziekenhuis
LabMicTA
Labor Dr Wisplinghoff
Laurentius ziekenhuis
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Laboratory
Leiden University Medical Center
Maasstad ziekenhuis
MeanderMC
Medizinisches Versorgungszentrum Dr. Stein + Kollegen
Microbe & Lab
Microvida
Mozand B.V.
Noordwest Ziekenhuisgroep Alkmaar
Novogenia GmbH
OLVG Lab B.V.
Pro Health Medical
RadboudUMC
Reinier Haga MDC
Rijnstate
RLM Dordrecht‐Gorinchem
Royal GD
Saltro, locatie Hudsondreef
Saltro, locatie Mississippidreef
Sanquin, NSS
St. Antonius ziekenhuis
Star‐shl
Stichting PAMM
Streeklab Haarlem
SYNLAB Heppignies
Synlab MVZ Weiden
SYNLAB Jena Oncoscreen
SYNLAB Laboratoire Collard
SYNLAB MVZ Trier
Tergooi ‐ CBSL
TLR International Laboratories
TNO
Universitair Medisch Centrum Groningen
Universitair Medisch Centrum Utrecht
VieCuri MC
Wageningen Bioveterinary Research
Ziekenhuis Gelderse Vallei
Ziekenhuis Rivierenland Tiel
Ziekenhuis StJansdal
Zuyderland Medisch Centrum

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