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Medical Microbiology and Immunology (2022) 211:105–117
https://doi.org/10.1007/s00430-022-00730-z
ORIGINAL ARTICLE
Impaired detection ofomicron bySARS‑CoV‑2 rapid antigen tests
AndreasOsterman1 · IrinaBadell1· ElifBasara1· MarcelStern1 · FabianKriesel1· MarwaEletreby1·
GamzeNazÖztan1· MelanieHuber1· HannaAutenrieth1· RicardaKnabe1· PatriciaM.Späth1·
MaximilianMuenchho1,2,3 · AlexanderGraf4 · StefanKrebs4 · HelmutBlum4 · JürgenDurner5,6·
LudwigCzibere5 · ChristopherDächert1,2 · LarsKaderali7 · Hanna‑MariBaldauf1 · OliverT.Keppler1,2,3
Received: 9 February 2022 / Accepted: 12 February 2022 / Published online: 20 February 2022
© The Author(s) 2022
Abstract
Since autumn 2020, rapid antigen tests (RATs) have been implemented in several countries as an important pillar of the
national testing strategy to rapidly screen for infections on site during the SARS-CoV-2 pandemic. The current surge in
infection rates around the globe is driven by the variant of concern (VoC) omicron (B.1.1.529). Here, we evaluated the
performance of nine SARS-CoV-2 RATs in a single-centre laboratory study. We examined a total of 115 SARS-CoV-2 PCR-
negative and 166 SARS-CoV-2 PCR-positive respiratory swab samples (101 omicron, 65 delta (B.1.617.2)) collected from
October 2021 until January 2022 as well as cell culture-expanded clinical isolates of both VoCs. In an assessment of the
analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 1.77 × 106 to 7.03 × 107 RNA cop-
ies subjected to the RAT for omicron compared to 1.32 × 105 to 2.05 × 106 for delta. To score positive in these point-of-care
tests, up to 10-fold (LoD50) or 101-fold (LoD95) higher virus loads were required for omicron- compared to delta-containing
samples. The rates of true positive test results for omicron samples in the highest virus load category (Ct values < 25) ranged
between 31.4 and 77.8%, while they dropped to 0–8.3% for samples with intermediate Ct values (25–30). Of note, testing of
expanded virus stocks suggested a comparable RAT sensitivity of both VoCs, questioning the predictive value of this type
of invitro-studies for clinical performance. Given their importance for national test strategies in the current omicron wave,
awareness must be increased for the reduced detection rate of omicron infections by RATs and a short list of suitable RATs
that fulfill the minimal requirements of performance should be rapidly disclosed.
Keywords SARS-CoV-2 rapid antigen test· Nucleocapsid protein· Diagnostic test· Sensitivity· Specificity· VoC· Lateral
flow
Edited by: Matthias J. Reddehase.
* Lars Kaderali
lars.kaderali@uni-greifswald.de
* Hanna-Mari Baldauf
baldauf@mvp.lmu.de
* Oliver T. Keppler
keppler@mvp.lmu.de
1 Max Von Pettenkofer Institute andGene Center, Virology,
National Reference Center forRetroviruses, LMU München,
Feodor-Lynen-Str. 23, 81377Munich, Germany
2 German Center forInfection Research (DZIF), Partner Site,
Munich, Germany
3 COVID-19 Registry oftheLMU Munich (CORKUM),
University Hospital, LMU Munich, Munich, Germany
4 Laboratory forFunctional Genome Analysis, Gene Center,
LMU München, Munich, Germany
5 Labor Becker MVZ GbR, Munich, Germany
6 Department ofConservative Dentistry andPeriodontology,
University Hospital, LMU München, Goethestr. 70,
80336Munich, Germany
7 Institute ofBioinformatics, University Medicine Greifswald,
Felix-Hausdorff-Str. 8, 17475Greifswald, Germany
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106 Medical Microbiology and Immunology (2022) 211:105–117
1 3
Introduction
During the SARS-CoV-2 pandemic, new variants of the
virus continue to emerge. The variant of concern (VoC)
omicron was first detected in November 2021 in Southern
Africa. Omicron’s ability for immune escape [1–5] and
high transmissibility in the population [6–10] have resulted
in omicron’s rapid spread around the globe [11, 12] and
have fueled the pandemic with numbers of new infections
reaching all-time highs despite increasing COVID-19 vac-
cination rates. In early February 2022, numbers of mostly
unvaccinated, at-risk COVID-19 patients with omicron
that require hospitalization or even intensive care treat-
ment are high in some countries, including Israel and the
United States. Omicron is characterized by a large number
of mutations in the spike protein [13] and currently has
two prominent sub-lineages based on Pangolin nomencla-
ture, namely BA.1 and BA.2.
In many countries, rapid antigen tests (RATs) for the
detection of SARS-CoV-2 continue to be a central com-
ponent of national testing strategies offering quick, inex-
pensive and laboratory-independent, point-of-care diagnos-
tics. However, the evaluation by independent laboratories
[14–25] and a Cochrane meta-analysis [26] have indicated
a highly variable performance of RATs resulting in an ongo-
ing controversy over these tests’ utility for the detection of
acute SARS-CoV-2-infected individuals in different settings
relevant for clinical diagnosis and containment strategies.
Some of these RATs have been approved for use by lay-
person and are frequently used as a tool for public health
interventions. In light of the somewhat limited capacities of
laboratory-based RT-qPCR for the quantitative detection of
SARS-CoV-2 RNA, test results from RATs alone are cur-
rently being suggested by national health authorities both to
reliably diagnose COVID-19 or to document an “uninfected”
or “non-infectious” status for ending quarantine or isolation
of persons without symptoms, respectively.
The nucleocapsid protein is most commonly detected
by SARS-CoV-2 antigen tests [13]. Since the majority
of RATs was developed prior to the emergence of VoCs,
the latter carrying different mutational patterns in the
nucleocapsid protein [19] (Suppl. Table1), it is of utmost
importance to perform VoC-specific RAT evaluations. In
omicron, besides more than 30 non-synonymous muta-
tions in the spike protein, the nucleocapsid protein of the
BA.1 sub-lineage carries four mutations compared to the
reference sequence of the Wuhan-hu-1 virus, i.e. P13L,
DEL31/33, R203K and G204R, and the additional muta-
tion S413R is present in BA.2 (Suppl. Table1). Three of
these mutations (P13L, DEL31/33 and S413R) are unique
to VoC omicron compared to alpha, beta, gamma or delta,
rendering predictions of the performance of RATs difficult.
Notably, first reports on the clinical and analytical per-
formance of RATs for omicron are partly contradictory: a
high-risk occupational case cohort of 30 individuals with
daily testing during an omicron outbreak in December 2021
showed a median of two days of apparently false-negative
RAT results with four recorded transmission events during
the period preceding the positive PCR result [27]. A preprint
study reports on 296 persons seeking COVID-19 testing at a
walk-up San Francisco community site in January 2022, who
tested positive by RT-qPCR. In these individuals, simultane-
ous nasal rapid antigen testing with BinaxNOW™ detected
95.2% of high viral load-omicron cases and 65.2% of all
PCR-positive cases [28]. A comparative study on isolates
classified as delta or omicron, that had been expanded in
tissue culture, found no VoC-specific differences in the ana-
lytical sensitivity of ten RATs, concluding the effectiveness
of these antigen lateral flow tests for omicron [29]. Other
investigators have suggested that an analytical and com-
parative validation with cultured VoCs might be a proxy
for clinical accuracy, but also stated that this cannot replace
clinical evaluations [14].
The aim of the current study was to compare nine dif-
ferent SARS-CoV-2 RATs that all detect the nucleocapsid
protein of SARS-CoV-2, for their analytical performance
using both clinical respiratory material and cultured viruses.
We evaluated respiratory specimen, identified as either delta
or omicron that had been collected from patients seen at dif-
ferent hospitals, nursing homes, outpatient clinics, COVID-
19 testing centers or seen by primary care physicians in the
Greater Munich area during the fourth and fifth pandemic
wave in Germany in the period October 2021 until January
2022. In addition, dilution series of isolates of delta and
omicron that had been expanded in cell culture in a biosafety
level 3 laboratory, were assessed.
Materials andmethods
Respiratory swabs
In the period October 30, 2021 to January 17, 2022 (delta)
and November 26, 2021 to January 19, 2022 (omicron),
health care professionals collected respiratory swabs (naso-
pharyngeal or nasal) from individuals who were seen on
clinical units or in the emergency room of the LMU Klini-
kum, the second-largest University Hospital in Germany, and
three teaching hospitals of the LMU Munich (Helios Amper
Hospital Dachau, Helios Hospital München West and Helios
Hospital München Perlach). Furthermore, SARS-CoV-2 in
respiratory swabs identified as omicron by Labor Becker
MVZ GbR in Munich, Germany, a diagnostic laboratory
that receives samples from regional hospitals, COVID-19
testing centers, nursing homes and primary care physicians'
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107Medical Microbiology and Immunology (2022) 211:105–117
1 3
offices, completed the study panel. All samples from this
period were randomly included in the study depending on
the availability of sufficient sample volume. No informa-
tion on the persons’ vaccination status, previous infections,
symptoms or clinical course were available. For this study,
flocked swabs were collected in different liquid transport
media and analyzed by RT-qPCR for SARS-CoV-2 RNA.
Dry swabs were resuspended in sterile 0.9% NaCl. Patient
samples in liquid transport medium with denaturing poten-
tial were excluded from the study. All samples with a Cp/
Ct value < 40 by RT-qPCR, which was quantified less than
24h after sample collection and under accredited diagnostic
laboratory conditions, were scored “SARS-CoV-2-positive”.
Original respiratory swabs and transport media were stored
at 2–8°C for up to 1week. If longer storage periods were
required, samples were frozen at − 20°C and thawed once
before antigen testing was performed. 166 SARS-CoV-
2-PCR-positive and 115 PCR-negative respiratory samples
were analyzed in this study.
SARS‑CoV‑2 antigen tests
The methodological procedure for the evaluation of the
RATs in this study follows the publication on the com-
parative sensitivity evaluation for CE-marked rapid diag-
nostic tests for SARS-CoV-2 antigen as "corresponding to
the current state-of-the-art" of the Paul-Ehrlich-Institute
[21]. For testing a particular specimen, a 50µl aliquot
was added and completely absorbed using the sampling
devices (swab) supplied with the respective kit. This cor-
responds to a spike-in after step 1 in Fig.1. Swabs were
then eluted in the test-specific buffer, following the respec-
tive manufacturer's instructions. After application of the
specimen/buffer solution to the test cassette, visual read-
ing of the control and target lines was performed after
a 15min-incubation period. The appearance of the RDT
control line was mandatory for a valid test result. Invalid
tests were documented and repeated if possible. Any vis-
ible colored band in the test area was considered a positive
result regardless of the intensity of the band. Scoring the
RAT was performed under constant lighting conditions
and by a trained person who was blinded to the preceding
RT-qPCR result of the sample. This procedure deviates in
part from manufacturer specifications regarding the use of
liquid transport medium rather than direct swabs and the
fact that the sample was not immediately inserted into the
assay. These study design-related adaptations in the pro-
tocol are unlikely to affect the comparative assessment of
the RATs for VoCs delta and omicron. For comparability,
this study reports the number of SARS-CoV-2 RNA copies
subjected to each test (determined by absolute quantifica-
tion of RT-qPCR—see below).
123
Extraction bufferSwab Positive test result
TC
represents > 1 Mio. RNA equivalents
Fig. 1 Schematic overview of the nasal swab sampling and testing
procedure. Step 1: viral particles are collected with the provided col-
lecting device (swab) from the anterior part of the nasal cavity. In
general, the test sensitivity is influenced by the amount of nucleocap-
sid antigen collected with the swab from nasal mucosa and secretions.
Step 2: antigen-containing sample material is eluted from the swab
into an extraction buffer. The sensitivity of the assay can be affected
by the efficiency of the physical elution of the antigen from the col-
lection device. Step 3: extracted nucleocapsid antigen is applied onto
the rapid antigen test cassette. In this step, manufacturer-specific rec-
ommendations for the volume of antigen-containing buffer that are
applied to the test cassette (known as “input ratio”) exist. The amount
of “RNA copies subjected to assay” investigated in this study refers to
an input equivalent at step 1
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108 Medical Microbiology and Immunology (2022) 211:105–117
1 3
Eight of the RATs examined in this study are recom-
mended by the manufacturers for self-testing and were listed
as a CE-labeled antigen test by the Bundesinstitut für Arz-
neimittel und Medizinprodukte (BfArM) in Germany at the
time of purchase, fulfilling the minimum criteria required for
these test devices in Germany according to the information
provided by the manufacturers. Of note, these eight assays
investigated were among the first rapid SARS-CoV-2 antigen
tests approved by BfarM for lay use in March 2021.
In detail, the products and manufacturers are: for test
1: FUJIFILM COVID-19 Ag Test (Fujifilm Cooperation),
research-use-only kits were provided for this study by the
manufacturer. Except for the labels, these are identical to the
final product, which meets the above criteria and was intro-
duced after approval. Test 2: Novel Coronavirus 2019-nCoV
Antigen Test (Colloidal gold) (Beijing Hotgen Biotech Co.,
Ltd.); test 3: NanoRepro SARS-CoV-2 Antigen Schnelltest
(Viromed) (NanoRepro AG); test 4: CLINITEST Rapid
COVID-19 Antigen Test (Healgen Scientific LLC); test 5:
Lyher Novel Coronavirus (COVID-19) Antigen Test Kit
(Colloidal Gold) (Hangzhou Laihe Biotech Co., Ltd.); test
6: COVID-19 Ag BSS self-test (Biosynex Swiss SA); test 7:
rapid SARS-CoV-2 Antigen Test Card (Xiamen Boson Bio-
tech Co., Ltd.); test 8: rapid SARS-CoV-2 Antigen Test Card
(MP Biomedicals Germany GmbH) and test 9: Medicovid-
AG SARS-CoV-2 Antigen Rapid Test Card-nasal (Xiamen
Boson Biotech Co., Ltd.). Tests 7 and 9 appear to be the
same antigen cassette product from one manufacturer (Ref
No.: 1N40C5 and 1N40C5-4), but they may differ in other
kit components (swab and buffer) in addition to package
size. Eight of these nine RATs have been evaluated by the
Paul-Ehrlich-Institute [21] and the European Commission
[30]. An overview of the RATs used in the current study and
their characteristics is provided in Suppl. Table2.
Quantitative viral load determination
In the accredited routine diagnostics laboratory of the
Max von Pettenkofer Institute, the following RT-qPCR
assays were used and quantified as described previously
[19] using the respective formulas: the nucleocapsid
(N1) reaction (Center for Disease Control (CDC) proto-
col [31] (
x=e
(
y
−48.597)
−1.461 ) on a LightCycler 480 system, the
Roche Cobas SARS-CoV-2 E-Gen reaction on a Cobas
6800 system (
x=e
(
y
−44.576)
−1.401 ) or the Xpert Xpress SARS-
CoV-2 (
x=e
(
y
−50.859)
−
1.887 ), Xpert Xpress SARS-CoV-2/Flu/
RSV (
x=e
(
y
−45.904)
−1.5 ) and Xpert Xpress CoV-2/Flu/RSVplus
(
x=e
(
y
−46.747)
−
1.501 ) run on a GeneXpert System, with x equals Geq
per ml and y equals the Ct/Cq value. For nucleic acid extrac-
tion, the QIAsymphony DSP Virus/Pathogen Kit was used
with the QIAsymphony instrument from QIAGEN GmbH.
In general, the calculations for quantification do not take
into account variability between separate RT-qPCR runs.
However, since this variability applies to all study groups,
they do not affect the interpretation of the results in this
study.
SARS‑CoV‑2 variant‑specific PCR andwhole‑genome
sequencing
Protocols for SARS-CoV-2 variant-specific PCR have been
reported [32]. In brief, nucleic acids from patients’ respira-
tory samples were extracted and eluates used for melting
curve analyses using commercially available PCR kits from
two vendors. For whole genome sequencing, amplicon pools
covering the SARS-CoV-2 genome were prepared accord-
ing to the ARTIC network nCoV-2019 sequencing protocol
v2 and analyzed utilizing the Artic bioinformatics protocol
[33]. The consensus sequences and associated sample meta-
data were uploaded to the GISAID repository.
Expansion ofSARS‑CoV‑2 frompatient material
SARS-CoV-2 was expanded as reported [5]. In brief, for
generation of high titer SARS-CoV-2 virus stocks, Vero-E6
cells were challenged with respiratory specimens containing
either delta or omicron. Virus stocks were harvested three
days post infection when approximately 50% of cells were
detached displaying a strong cytopathic effect. Supernatants
were cleared and stored at −80°C. Expanded stocks were
analyzed by whole genome sequencing (B.1.617.2: GISAID
3233464; B.1.1.529: GISAID 7808190) and RNA copies per
mL were determined as the mean from three independent
biological experiments with technical unicates or duplicates.
Statistical analyses
Statistical analysis was performed in R version 4.1.2. Bino-
mial confidence intervals for sensitivities and specificities
were computed using the Wilson score interval. To further
analyze analytical sensitivities, we used logistic regression,
with viral loads and RNA copy numbers subjected to the test
as independent and test outcomes as the dependent variable,
yielding detection probabilities for each viral load level.
Results
Evaluation ofRAT specificity
To evaluate the specificity of all RATs in a comparable
experimental setting (Fig.1), test collection devices (swabs)
were individually incubated with 50µl each of the individual
115 PCR-negative nasal/nasopharyngeal swabs and applied
to the individual assay’s extraction buffer. Under these
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109Medical Microbiology and Immunology (2022) 211:105–117
1 3
conditions, the specificity of all nine RATs was 100% (CI
96.3–100%) (Suppl. Table3).
Characterization ofrespiratory specimens
fromCOVID‑19 patients containing VoCs delta
oromicron
Next, we quantified the viral load in PCR-positive naso-
pharyngeal swabs, of which 65 were classified as delta
(B.1.617.2) and 101 as omicron (B.1.1.529), respectively, by
variant-specific PCR and next-generation sequencing [32].
The omicron sequences in all respiratory swabs were clas-
sified as belonging to the BA.1 sublineage. The viral loads
of delta specimen ranged from 1.74 × 104 to 6.72 × 108 Geq/
ml (median 6.67 × 106 Geq/ml, interquartile range 6.61 × 105
– 5.14 × 107 Geq/ml, median absolute deviation 9.73 × 106
Geq/ml) (Fig.2a, b, e), the viral loads of omicron speci-
men ranged from 9.3 × 102 to 2.26 × 109 Geq/ml (median
6.97 × 106 Geq/ml, interquartile range 9.45 × 105–5.14 × 107
Geq/ml, median absolute deviation 1.02 × 107 Geq/ml)
OmicronDelta
9
7
5
3
log10 viral load (Geq/ml)
6
10
2
0
10
20
30
2610
48
0
10
20
2610
48
log10 viral load (Geq/ml)
Number of samples
040 10020 60 80
log10 viral load (Geq/ml)
Number of samples
04020 60
Frequency
log10 viral load (Geq/ml)
Frequency
log10 viral load (Geq/ml)
e
ab
cd
n.s.
Delta
Omicron
6
10
2
Fig. 2 SARS-CoV-2 viral load distribution of respiratory samples
included in the study containing either delta or omicron. a, c Shown
is the log10 viral load (Geq/ml) distribution of all 65 delta (a) and
all 101 omicron (c) sorted by ascending magnitude from left to right.
Each dot indicates one patient and the sample ID is indicated. b, d
Histogram of the viral load distribution in specimen containing delta
(b) or omicron (d) categorized into the indicated ranges of log10 viral
load. Each bar depicts the number of samples in the respective viral
load range. e The horizontal line in the box plots shows the median of
the samples shown in a and c, bound between upper and lower quar-
tiles, and whiskers between minimum and maximum are indicated.
n.s. = not significant by Wilcoxon rank sum test with continuity cor-
rection and by two-sample Kolmogorov–Smirnov test
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110 Medical Microbiology and Immunology (2022) 211:105–117
1 3
(Fig.2c, d, e). Importantly, the median and distribution of
viral loads were comparable and statistically indistinguish-
able between both groups of VoC-containing respiratory
samples (Fig.2e), allowing a direct comparison of the ana-
lytical sensitivity of RATs for the detection of these two
most recently emerged VoCs.
Comparative analysis ofanalytical RAT sensitivity
forVoCs delta andomicron inclinical specimens
We used this panel of SARS-CoV-2 RT-qPCR-positive res-
piratory specimen to determine the analytical sensitivity
of the nine RATs for the detection of delta and omicron
in a direct comparison. The overall sensitivities for delta-
containing specimen ranged between 34.92 and 58.46%
(Table1), corresponding to 22 of 63 and 38 of 65 samples,
respectively, that were tested true positive. For omicron-
containing specimens, the overall sensitivities ranged from
22.22 to 57.43% (Table2), corresponding to 22 of 99 and
58 of 101 SARS-CoV-2 PCR-positive samples, respectively,
that were correctly scored.
We determined the 50% (dotted line in pink vertical area)
and 95% (dotted line in yellow vertical area) limits of detec-
tion (LoD) based on a logistic regression model as recently
reported [16] (Fig.3a, b). For test 1, the RNA copy numbers
for LoD50 and LoD95 were 1.19 × 106 and 7.03 × 107 for delta-
containing specimen, respectively. The detection of omicron-
containing samples was in contrast impaired: the LoD50 and
LoD95 values corresponded to 1.11 × 107 and 7.12 × 109 RNA
copies, respectively, thus requiring 9- to 101-fold higher RNA
copy numbers than those for delta-containing specimens to
reach these detection thresholds. For test 2, the LoD50 and
LoD95 values for delta-containing samples corresponded to
1.54 × 105 and 3.86 × 106 RNA copy numbers, respectively.
Those for omicron-containing specimens were 9- to 17-fold
higher with 1.38 × 106 and 6.64 × 107 RNA copy numbers,
respectively. The performance of test 3 was comparable to test
2 with LoD50 and LoD95 values for delta-containing sam-
ples of 2.05 × 105 and 5.04 × 106 RNA copy numbers, respec-
tively, and for omicron-containing samples of 2.12 × 106 and
1.35 × 108 RNA copy numbers, respectively, i.e. ranging 10-
to 27-fold higher for the latter. The SARS-CoV-2 RNA copy
numbers corresponding to LoD50 and LoD95 for test 4 for
delta- versus omicron-containing specimens were 8- to ten-
fold higher for the latter with 1.77 × 105 and 4.52 × 106 versus
1.33 × 106 and 4.37 × 107, respectively. For test 5, the LoD50
and LoD95 values for delta-containing samples corresponded
to 1.34 × 105 and 3.08 × 106 RNA copies, respectively. These
LoD values were 3- and 5-times higher for omicron-containing
samples with 4.54 × 105 and 1.43 × 107 RNA copies, respec-
tively. The LoD50 and LoD95 values for test 6 were 1.82 × 105
and 3.02 × 106 for delta- and 6.93 × 105 and 2.08 × 107 for
omicron-containing specimen, respectively, corresponding to
a 4- to sevenfold difference. For test 7, the RNA copies with
50% and 95% detection rates determined for delta-containing
specimens were 3.31 × 105 and 1.77 × 106, respectively. Detec-
tion of omicron was similar to test 1 and strongly impaired
as LoD values were 8- to 99-fold higher with 2.62 × 106 and
1.75 × 108 RNA copies, respectively. The relative performance
of test 8 was among the best of the RATs tested in this study:
the LoD50 and LoD95 values for delta and omicron differed at
maximum by a factor of 2: values for delta-containing samples
corresponded to 1.32 × 105 (LoD50) and 3.86 × 106 (LoD95),
and for omicron samples 3.24 × 105 and 3.94 × 106 RNA cop-
ies, respectively. Test 9 performed similarly well in our analy-
sis: the LoD50 and LoD95 for delta samples were 1.82 × 105
and 3.02 × 106 RNA copies and for omicron samples 1.94 × 105
and 3.41 × 106, respectively.
Table 1 Determination of assay sensitivity for nine SARS-CoV-2
rapid antigen tests in SARS-CoV-2 PCR-positive respiratory swabs
classified as delta
Binomial confidence intervals were computed using the Wilson score
interval
Assay Sensitivity (%) 95% CI (%) True
positive/
total
Test 1 34.92 24.33–47.25 22/63
Test 2 56.25 44.09–67.71 36/64
Test 3 53.85 41.85–65.41 35/65
Test 4 55.38 43.34–66.83 36/65
Test 5 58.46 46.34–69.64 38/65
Test 6 55.38 43.34–66.83 36/65
Test 7 49.21 37.27–61.24 31/63
Test 8 58.46 46.34–69.64 38/65
Test 9 55.38 43.34–66.83 36/65
Table 2 Determination of assay sensitivity for nine SARS-CoV-2
rapid antigen tests in SARS-CoV-2 PCR-positive respiratory swabs
classified as omicron
Binomial confidence intervals were computed using the Wilson score
interval
Assay Sensitivity (%) 95% CI (%) True
positive/
total
Test 1 22.22 15.16–31.36 22/99
Test 2 35.64 26.99–45.35 36/101
Test 3 31.68 23.42–41.29 32/101
Test 4 35.64 26.99–45.35 36/101
Test 5 47.52 38.06–57.18 48/101
Test 6 43.00 33.73–52.78 43/100
Test 7 29.70 21.67–39.23 30/101
Test 8 51.49 41.86–61.00 52/101
Test 9 57.43 47.69–66.62 58/101
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111Medical Microbiology and Immunology (2022) 211:105–117
1 3
In summary, a reduction in the analytical sensitivity for
the detection of omicron- compared to delta-containing res-
piratory specimens, determined by the LoD50 and LoD95,
was seen for eight of the nine RATs with factors of up to
10.3-fold and up to 101-fold more virus genome equiva-
lents, respectively, required to score positive, reaching sta-
tistical significance for tests 2, 3, 4 and 7. This suggests
a general impairment for a virus load-stratified detection
of an omicron infection compared to a delta infection by
SARS-CoV-2 antigen testing with a RAT-specific degree
of sensitivity reduction.
Analytical RAT sensitivity incell culture‑expanded
virus stocks
Based on an initial report of comparable sensitivity for
invitro-expanded virus stocks [29], we assessed in paral-
lel cell-culture expanded delta and omicron stocks. The
expanded delta isolate (GISAID 3233464) carries the D63G,
R203M and D377Y mutations in the nucleocapsid protein
as well as the commonly reported G215C mutation [34].
The expanded omicron isolate (GISAID 7808190; BA.1 sub-
lineage) carries the expected P13L, del31/33, R203K and
G204R mutations. Neither culture medium alone nor the
virus-inactivating detergent Triton X-100 had an impact on
the performance of the RATs (not shown).
As shown in Suppl. Figure1, rapid antigen tests 1, 5, 8
and 9 were able to detect delta and omicron virus stocks
down to 2.5 × 106 RNA copies, whereas the other five tests
were less sensitive requiring up to 8-times more SARS-
CoV-2 RNA to score positive. Interestingly, there was a
trend for a slightly better detection of cell-culture expanded
omicron compared to delta, in particular for tests 2, 4, 6 and
7. In summary, this analysis confirms first studies by other
laboratories [14, 35] indicating a comparable detection of
a
b
0
50
100
0
50
100
0
50
100
0
50
100
Test 6
Test 5Test 1Test 2
Test 7
Test 3
Test 8
Test 4
Test 9
Probability (%)Probability (%) Probability (%)Probability (%)
Delta
Omicron
Omicron
Delta
8624
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
10 8624 10 8624 10 8624 10
log
10
RNA copies
subjected to test
8624 108624
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
log
10
RNA copies
subjected to test
10 8624 10 8624 10 8624 10
Fig. 3 Limit of detection analyses of respiratory samples positive for
either delta (top panels) or omicron (bottom panels) by RT-qPCR for
nine SARS-CoV-2 RATs. a test #1–5, b test #6–9. The log10 RNA
copies subjected to the test of quantified samples on the x axis were
plotted against a positive (+ 1) or negative (0) test outcome on the y
axis. For readability of the figure, slight normal jitter was added to
the y values. Blue (delta) and red (omicron) curves, respectively,
show logistic regressions of the viral load on the test outcome; verti-
cal dashed lines indicate log10 RNA copies subjected to the test at
which 50% (LoD50) and 95% (LoD95), respectively, of the samples
are expected positive based on the regression results. Significant
differences for LoD50: test 2, 3, 4 and 7. Significant difference for
LoD95: test 7
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112 Medical Microbiology and Immunology (2022) 211:105–117
1 3
invitro-expanded omicron and delta virus stocks by rapid
antigen testing. However, this stands in clear contrast to the
above evaluation of clinical respiratory specimens, which
show an impaired sensitivity for the detection of an omicron
infection in COVID-19 patients with comparable viral loads
in respiratory specimens.
Comparative, Ct value‑stratified evaluation
ofanalytical RAT sensitivity
Next, we sought to directly compare the analytical sensi-
tivities of the nine RATs for the detection of “non-delta and
non-omicron” SARS-CoV-2, evaluated and published by the
Paul-Ehrlich-Institute, to VoCs delta or omicron assessed in
our study in respiratory specimen in predefined corridors of
SARS-CoV-2 RNA copy numbers. To this end, we harmo-
nized the corresponding Ct/Cp values between our study
results and those reported by Puyskens etal. and Scheiblauer
etal. [21, 36]. This allowed us to plot and compare the per-
centages of test samples that scored positive in one of the
three categories of Ct/Cp values, i.e. < 25, 25–30 and > 30.
The following observations were made (Table3): (1) for
the seven RATs, for which all three variant data sets were
available, the overall sensitivity of the “non-delta and non-
omicron” SARS-CoV-2 scored highest in four RATs, closely
followed by three RATs for delta. (2) In eight out of nine
RATs, the overall sensitivity was higher for delta- compared
to omicron-containing respiratory samples. (3) In the highest
viral load category with Ct values < 25, omicron detected the
lowest percentage of cases in eight out of nine RATs with
test 9 posing the exception. (4) In the intermediate viral load
corridor reflected by Ct values 25–30, none of the respira-
tory specimen scored positive in six of the RATs, and with
low percentages of 4.2–8.3% for tests 5, 8 and 9. (5) Samples
with Ct values > 30 were generally not detected with a single
exception for the “non-delta and non-omicron” specimen
group (Table3). In summary, these results indicate that the
analytical sensitivity of RATs for omicron-containing respir-
atory samples cannot be deduced from previous evaluations
of other SARS-CoV-2 variants. For the majority of RATs
evaluated in this study, an impaired detection of the emer-
gent VoC omicron compared to previous variants, including
delta, was noted.
Discussion
In the current study, we evaluated the performance of nine
commercially available SARS-CoV-2 raid antigen tests that
were launched on the European market during the corona-
virus pandemic for the detection of the viral nucleocapsid
protein. We find that for the majority of RATs evaluated,
detection of omicron is impaired compared to delta, requir-
ing up to 10.3-fold and up to 101-fold more SARS-CoV-2
RNA of omicron in respiratory specimens to score positive
in LoD50 or LoD95 analyses, respectively.
We examined two groups of specimens: the first group
encompassed respiratory samples collected from patients in
the period October 2021 until January 2022. A total of 115
SARS-CoV-2 PCR-negative and 166 SARS-CoV-2 PCR-
positive specimen (65 delta, 101 omicron) were tested. The
second group consisted of cell culture-expanded clinical
isolates of these VoCs. The latter was important to assess
the predictive value of sensitivity evaluations of RATs using
SARS-CoV-2 variants expanded in tissue culture as a poten-
tially rapid proxy for their diagnostic performance.
Limitations of the current study include the lack of infor-
mation on the vaccination status, previous infections, symp-
toms or stage of COVID-19 of individuals from whom the
respiratory swabs were taken. We focused on the analytical
sensitivity in relation to the viral loads in respiratory swabs
to allow a direct comparison of specimens containing omi-
cron and delta (the current study) and “non-omicron/non-
delta” SARS-CoV-2 (studies by the Paul-Ehrlich-Institute
[21, 36]) in the assessment of RAT performance.
A potential confounder in the methodological procedure
for specificity assessment is the rather small volume of
biological material from the original swab that is actually
transferred to the RAT extraction buffer. Here, swab kits
containing 1ml virus transport medium were used, result-
ing up front in a 1:20 dilution of the input sample. This may
have potentially reduced biological components that may
trigger a false-positive result. Therefore, the RATs’ specific-
ity of 100% observed in the current study is only informative
for the chosen experimental setting to evaluate the analytical
sensitivity, but is unlikely to reflect the specificity of the
investigated RATs in a point-of-care setting.
The reasons underlying the discrepancy of the RAT per-
formance comparing delta and omicron between respiratory
samples, on the one hand, and expanded virus stocks, on the
other hand, are currently unclear. As proposed in a cryo-
electron tomography study [37], nucleocapsid assembly
of SARS-CoV-2 is driven by the intracellular interaction
between the nucleocapsid protein and the genomic viral
RNA. In a clinical setting, our data suggest that the relative
ratio of nucleocapsid protein to SARS-CoV-2 RNA may be
higher, on average, in the extracellular space and on the res-
piratory mucosa of COVID-19 patients infected with delta
compared to those infected with omicron.
Several scenarios can be envisioned that may under-
lie this difference: (1) a variant-specific degree of virus-
induced cell death or cytolysis triggered by the host’s
immune response may contribute to a VoC-specific level
of nucleocapsid protein found on the respiratory mucosa.
(2) A higher severity of disease has been associated with
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113Medical Microbiology and Immunology (2022) 211:105–117
1 3
increased transcriptional levels of nucleocapsid RNA
[38], providing a potential explanation for an enhanced
nucleocapsid per viral RNA ratio in COVID-19 as a
consequence of a delta infection compared to an omi-
cron infection. (3) Tissue culture-expanded virus stocks
are harvested at a time point of considerable lysis of the
Table 3 Comparative evaluation
of the analytical sensitivity of
nine SARS-CoV-2 rapid antigen
tests stratified for Ct/Cp value
ranges based on studies by the
Paul-Ehrlich-Institute (non-
delta/non-omicron*) and the
current study for respiratory
samples containing VoCs delta
and omicron
* Data published by Puyskens etal. (collected between March and September 2020 (Panel 1V1; [36]) and
Scheiblauer etal. (additionally collected between October 2020 and January 2021 (Panel 1V2); [21]) and
“SARS-CoV-2 antigen rapid diagnostic tests passing the sensitivity criteria” of the Paul-Ehrlich-Institute
(date of access: 31.1.22)
1 The Ct values of test specimens in the study performed by the Paul-Ehrlich-Institute and our study were
harmonized to allow a direct comparison of data sets. Harmonization was achieved based on the publica-
tion by Scheiblauer etal. and the formula derived to convert virus loads into Ct values:
y = − 1.455lnx + 41.154 with x equals Geq per ml and y equals the Ct/Cq value. n.a. not available;– these
test results were no longer included in the latest data set released by the Paul-Ehrlich-Institute on January
31, 2022
NCt < 25
(%)1Ct 25–30
(%)1Ct > 30
(%)1Overall
sensitivity
(%)
Test 1
Non-Delta/non-Omicron* n.a 85.0 10.0 0 38.0
Delta 63 50.0 5.6 0 34.9
Omicron 99 31.4 0 0 22.2
Test 2
Non-Delta/non-Omicron* n.a 100 47.8 0 56.0
Delta 64 74.4 22.2 0 56.2
Omicron 101 50.0 0 0 35.6
Test 3
Non-Delta/non-Omicron* n.a 94.1 – – –
Delta 65 72.7 16.7 0 53.9
Omicron 101 44.4 0 0 31.7
Test 4
Non-Delta/non-Omicron* n.a 100 87 0 76.0
Delta 65 72.7 22.2 0 55.4
Omicron 101 50.0 0 0 35.6
Test 5
Non-Delta/non-Omicron* n.a 94.4 17.4 0 42.0
Delta 65 77.3 22.2 0 58.5
Omicron 101 63.9 8.3 0 47.5
Test 6
Non-Delta/non-Omicron* n.a 100 78.3 11.1 74.0
Delta 65 75.0 16.7 0 55.4
Omicron 100 60.6 0 0 43.0
Test 7
Non-Delta/non-Omicron* n.a 100 43.5 0 54.0
Delta 63 72.1 0 0 49.2
Omicron 101 41.7 0 0 29.7
Test 8
Non-Delta/non-Omicron* n.a 100 43.5 0 54.0
Delta 65 75.0 27.8 0 58.5
Omicron 101 70.8 4.2 0 51.5
Test 9
Non-Delta/non-Omicron* n.a – – – –
Delta 65 75.0 16.7 0 55.4
Omicron 101 77.8 8.3 0 57.4
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114 Medical Microbiology and Immunology (2022) 211:105–117
1 3
virus-producing Vero-E6 cells, potentially leveling out
VoC-specific differences in the ratio of released nucle-
ocapsid protein relative to viral RNA compared to a more
physiological setting. (4) Moreover, one can speculate that
SARS-CoV-2-specific antibody responses in COVID-19
patients, either through vaccination or previous infections,
could differentially impact on the VoC-specific positivity
rate of RATs.
It is widely communicated that individuals with an omi-
cron infection with high viral loads are reliably detected
by RATs that are available on the German market, and that
these tests’ quality is sufficient to have a positive impact
on national pandemic management [39, 40]. These general
claims stand in contrast to the following aspects: (1) among
the approximately 580 RATs [41] currently available on the
German market not even half has been evaluated by either
the Paul-Ehrlich-Institute or independent diagnostic labo-
ratories for any SARS-CoV-2 variant, let alone omicron.
The Paul-Ehrlich-Institute stated that first results of their
omicron validations will be made publically available at the
end of February 2022; (2) general claims that reactivity in a
RAT reliably identifies the group of “truly” infectious indi-
viduals or individuals with super-spreader potential under
normal human interaction conditions are not substantiated
by published scientific literature. Examples of an apparent
super-spreader, long before the emergence of VoCs with
enhanced transmissibility, with Ct values of ≥ 27 [42] or cul-
tivation of SARS-CoV-2 from specimen with Ct values ≥ 35
[43, 44] have been reported. Similarly, experimental studies
have estimated that around 1000 virus particles may be suf-
ficient for infection of a new host [45, 46], while viral loads
required for a positive score in SARS-CoV-2 RATs range
more than 1.000-fold higher. (3) The determination of the
analytical sensitivity alone is not sufficient to draw conclu-
sions on the clinical performance. Preliminary reports indi-
cate that COVID-19 patients with omicron may shed even
less virus than individuals infected with previous VoCs [7].
This could further worsen the clinical performance in light
of the lower analytical sensitivity of the majority of RATs
examined in this study. Since omicron is considered more
contagious than previous VoCs based on epidemiological
findings [47, 48], these COVID-19 patients, with or without
symptoms, would be assumed to potentially be infectious
despite a lower viral load in respiratory swabs. Furthermore,
clinical studies are just beginning to assess the level of virus
shedding and infectivity in the context of omicron infections
and breakthrough infections in vaccinated individuals [7–9,
27].
Modeling studies suggest that the limited sensitivity of
RATs to identify infected and infectious individuals may
be compensated for by multiple repeated testing. This is
based, in part, on claims that a “recalibrated absolute sen-
sitivity” of RATs is around 80% [49], which is, however,
not supported by the majority of independent laboratory-
based or field studies [26]. On the other hand, especially in
the initial phase of symptomatic disease, a time window of
a few hours may be sufficient to repeat a rapid antigen test-
ing with low sensitivity in suspected disease to enhance
the likelihood of detecting an infection. A recent study
suggests that the viral load during this period can rapidly
and markedly increase for RATs to score positive [50].
Interestingly, epidemiological studies by the Robert
Koch-Institute suggested that 67% of individuals with a
negative RAT result believe that they are non-infectious
for at least 48h, potentially affecting their behavior regard-
ing preventive hygiene measures. In part, this perception
results from overly optimistic communications on the reli-
ability of this class of tests.
Our current study suggests that the emergence of future
VoCs of this pandemic β-coronavirus with an altered muta-
tional pattern in the nucleocapsid protein should result in
an immediate reassessment of the performance of RATs.
Since the rapidly spreading BA.2 sub-lineage of omicron
carries an additional S413R mutation in the nucleocapsid
protein, it is difficult to predict the RAT performance for
this VoC. The minimum requirements stated by interna-
tional organizations and the Paul-Ehrlich-Institute for the
performance of SARS-CoV-2 rapid antigen tests include
an overall diagnostic sensitivity of > 80% and a specific-
ity of > 97% [51, 52]. Independently validated RATs that
do not meet these criteria, should be taken off the market
immediately.
In light of first international reports [14, 29] and the
results from our present study, current communications by
the Paul-Ehrlich Institute [51] and the Bundesministerium
für Gesundheit [40] on the apparent reliability of RATs for
the detection of an omicron infection seem premature. More-
over, critical pre-analytical factors, including the timing of
the swab relative to the onset of symptoms, the vaccination
status, the swabbing practices and test procedures, in par-
ticular when not performed by trained health care profes-
sionals in COVID-19 testing centers, may also have a great
impact on the “real-world” diagnostic sensitivity of RATs.
The latter factors should underlie a constant optimization
and a regular and stringent assessment by health authorities.
Vaccination campaigns have achieved a substantial cover-
age of the adult population in first-world countries, from the
perspective of health care systems markedly improving the
starting point for the control of future VoCs that may arise
from the original evolutionary trait of SARS-CoV-2, from
which VoCs alpha, beta, gamma and delta arose. Alternative
testing concepts including sample pooling for PCR or iso-
thermal RT-LAMP nucleic acid-based detection of SARS-
CoV-2 should be optimized to reduce the potential burden
of infections and COVID-19 in the context of large social
events with conditions favoring SARS-CoV-2 transmission.
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115Medical Microbiology and Immunology (2022) 211:105–117
1 3
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00430- 022- 00730-z.
Acknowledgements We are grateful to members of the diagnostic
laboratory of virology at the Max von Pettenkofer Institute for support.
Author contributions AO and OTK designed the study. IB, EB, FK,
GNÖ, ME, RK, MH, PS, MS, HA, AG, SK, performed experiments.
MC and JD provided and characterized clinical samples. AO, MM, HB,
CD, LK, H-MB, OTK analyzed data. LK performed statistical analysis.
AO, MS, CD, LK, H-MB, and OTK wrote the manuscript. All authors
discussed the results and commented on the final manuscript.
Funding Open Access funding enabled and organized by Projekt
DEAL. This work was supported by the Free State of Bavaria (research
initiatives Bay-VOC (M.M., A.G., S.K., H.B., O.T.K.) and FOR-
COVID (M.M., O.T.K.)).
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics approval Ethical approval was not required because all samples
used were residual samples. Samples were identified in the laboratory
and assigned a number without reference to patient or clinical details.
Results from testing using the study assays did not impact on clinical
care.
Data and materials availability Not applicable.
Code availability Not applicable.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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