Virus variant-specific clinical performance of SARS coronavirus two rapid antigen tests in point-of-care use, from November 2020 to January 2022.

OBJECTIVES: Antigen rapid diagnostic tests (RDTs) for SARS coronavirus 2 (SARS-CoV-2) are quick, widely available, and inexpensive. Consequently, RDTs have been established as an alternative and additional diagnostic strategy to quantitative reverse transcription polymerase chain reaction (RT-qPCR). However, reliable clinical and large-scale performance data specific to a SARS-CoV-2 virus variant of concern (VOC) are limited, especially for the Omicron VOC. The aim of this study was to compare RDT performance among different VOCs.
METHODS: This single-centre prospective performance assessment compared RDTs from three manufacturers (NADAL, Panbio, MEDsan) with RT-qPCR including deduced standardized viral load from oropharyngeal swabs for detection of SARS-CoV-2 in a clinical point-of-care setting from November 2020 to January 2022.
RESULTS: Among 35 479 RDT/RT-qPCR tandems taken from 26 940 individuals, 164 of the 426 SARS-CoV-2 positive samples tested true positive with an RDT corresponding to an RDT sensitivity of 38.50% (95% CI, 34.00-43.20%), with an overall specificity of 99.67% (95% CI, 99.60-99.72%). RDT sensitivity depended on viral load, with decreasing sensitivity accompanied by descending viral load. VOC-dependent sensitivity assessment showed a sensitivity of 42.86% (95% CI, 32.82-53.52%) for the wild-type SARS-CoV-2, 43.42% (95% CI, 32.86-54.61%) for the Alpha VOC, 37.67% (95% CI, 30.22-45.75%) for the Delta VOC, and 33.67% (95% CI, 25.09-43.49%) for the Omicron VOC. Sensitivity in samples with high viral loads of ≥106 SARS-CoV-2 RNA copies per mL was significantly lower in the Omicron VOC (50.00%; 95% CI, 36.12-63.88%) than in the wild-type SARS-CoV-2 (79.31%; 95% CI, 61.61-90.15%; p 0.015). DISCUSSION: RDT sensitivity for detection of the Omicron VOC is reduced in individuals infected with a high viral load, which curtails the effectiveness of RDTs. This aspect furthert: limits the use of RDTs, although RDTs are still an irreplaceable diagnostic tool for rapid, economic point-of-care and extensive SARS-CoV-2 screening.

Introduction

Due to their independence of diagnostic infrastructure, short analysis time, self-testing option, and affordability, SARS-CoV-2 rapid diagnostic tests (RDT), technically based on lateral flow enzyme-linked immunosorbent assays, have established as an important diagnostic alternative to quantitative reverse transcription polymerase chain reaction (RT-qPCR) as reference standard.[1, 2] With respect to the different SARS-CoV-2 VOC and the variable VOC dominance in the pandemic course, available RDT performance assessments present heterogeneous results regarding a potential limitation of RDT sensitivity due to a particular VOC. Comparable data from real-life, extensive point-of-care usage is still lacking.[3, 4, 5, 6].

This prospective performance evaluation study compares the accuracy of RDT to normalised RT-qPCR in the daily clinical routine, the main focus being on SARS-CoV-2 VOC dependent test performance as well as sensitivity in highly infectious individuals and specificity in broad screening use.

Methods

Study setting

Clinical performance assessment took place in a tertiary care hospital in Bavaria, Germany as a single-center study. Presented data were collected from November 12, 2020 to January 31, 2022 which included the second to the fifth wave of the COVID-19 pandemic in Germany caused by wild-type SARS-CoV-2, the Alpha VOC, the Delta VOC, and the Omicron VOC.[7] The study continues a previous RDT evaluation up to February 2021 including 5,068 samples.[8].

During the data collection period the spread of SARS-CoV-2 in the Federal State of Bavaria was quantified with an average weekly incidence of 191.61 per 100,000 inhabitants that reached unprecedented maximum incidence values in January 2022 due to increasing rise of Omicron VOC infections (Supplementary Fig. S1).[7, 9, 10].

Test enrolment

As part of measures in place to prevent and reduce the intrahospital spread of SARS-CoV-2, RDT testing in tandem with RT-qPCR was implemented in key situations for prevention of nosocomial SARS-CoV-2 transmission chains. As part of the local SARS-CoV-2 screening conception RDT diagnostics were established for patients and accompanying individuals staying overnight with underage and otherwise dependent patients on admission as well as employees in case of arising symptoms potentially caused by COVID-19.

While during high SARS-CoV-2 incidence periods (February 1, 2021 to June 30, 2021 and November 4, 2021 to January 31, 2022) tandem testing was performed on all patients on admission, during low incidence periods (November 12, 2020 to January 31, 2021 and July 1, 2021 to November 3, 2021) RDT were only performed in critical areas such as emergency departments and the delivery room. This reflects in the weekly number of RDT included in the study visualised in Supplementary Fig. S1. In case of more than one documented RDT per day per person, only the first RDT was considered for data analysis. Patients who fulfilled the inclusion criteria on multiple days of the study period were tested and included once per visit. Individuals with a recent SARS-CoV-2 infection and subsequent deisolation were also excluded due to potential persistent RT-qPCR positivity not related to a risk of viral spread.[8].

Antigen rapid diagnostic tests (RDT)

To ensure continuous supply, RDT of three different manufacturers were selected out of 23 products listed by the German Federal Institute for Drugs and Medical Devices in October 2020 for implementation to clinical point-of-care diagnostics.[8, 11].

NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany)

PANBIO™ COVID-19 Ag Rapid Test (Abbott Laboratories, Abbott Park IL, USA)

MEDsan® SARS-Cov-2 Antigen Rapid Test (MEDsan GmbH, Hamburg, Germany)

The selected three RDT were maintained for the complete study period with randomly varying distribution across the hospital departments over the study time for infrastructural and availability reasons.

In case of an invalid RDT result, the RDT was repeated. As only the first RDT per day per person was included, these repeated RDT were excluded from the analysis.

RDT as well as RT-qPCR specimens were gathered in successive paired oropharyngeal swabs by trained medical staff in accordance with manufacturers’ instructions, all used RDT target SARS-CoV-2 nucleoprotein antigen. As RDT were performed at point-of-care, RT-qPCR results were not available to the performers.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

The RT-qPCR tests and analytical devices as well as the viral load determination are described in detail in the Supplementary Methods. To prioritise RDT positive samples, RDT results were available to RT-qPCR conducting staff.

VOC-specific PCR and determination of SARS-CoV-2 VOC prevalence levels

Between February 3, 2021 and January 19, 2022, all new RT-qPCR positive samples underwent spike protein variant specific PCR to differentiate between present spike protein VOC using the VirSNiP SARS-CoV-2 Spike N501Y, del 69/70, E484K, N501Y, L452R, T478K, and 371L 373P 452R kits (TIB molbiol, Berlin, Germany) on a cobas® z 480 analyser (Roche Diagnostics, Rotkreuz, Switzerland). Determination of VOC by multiple PCR has shown a high agreement with VOC determination by whole genome sequencing.[12] In case of combinations not corresponding to a frequent VOC, the described analytical procedure was followed by spike protein sequencing. Due to the high COVID-19 prevalence and Omicron VOC in 95% of all COVID-19 cases in Germany, VOC determination was interrupted as of January 19, 2022. All positive samples before February 3, 2021 were assumed to be wild-type SARS-CoV-2, all samples after termination of virus variant determination to be Omicron VOC. In case no VOC determination was possible due to low viral loads, the VOC was, if possible, derived from the known SARS-CoV-2 infection source. Otherwise, samples were assumed to belong to VOC responsible for more than 90 % of prevalent SARS-CoV-2 cases in that calendar week in Germany: Wild-type SARS-CoV-2 (November 12, 2020 to January 24, 2021), Alpha VOC (April 19 to May 2, 2021 as well as May 17 to May 30, 2021), Delta VOC (July 12 to December 19, 2021), and Omicron VOC (January 17 to January 31, 2022).[10].

Data collection

Documented RDT and RT-qPCR results, as well as demographic data was collected from the local hospital information system (HIS) SAP ERP 6.0 (SAP, Walldorf, Germany).

Ethical approval

The Ethics committee of the University of Wuerzburg considered the study protocol and waived the need to formally apply for ethical clearance due to the study design (File Number 20210813 01).

Statistics

Data analysis was performed with Excel® 2019 (Microsoft, Redmond WA, USA) and GraphPad Prism 9.0.2 (GraphPad Software, San Diego CA, USA). Confidence intervals were calculated with the Wilson/Brown method,[13] statistical significance levels with Fisher’s exact test, Chi-squared test, Mann Whitney U-test as well as Kruskal-Wallis test. The two-tailed significance level α was set to 0.05. The limit of detection (LOD) analysis was performed in R, version 4.2.1. A logistic regression model was fitted to test results in dependence of log10 viral load; 50% and 95% LOD values with 95% confidence intervals were calculated from the regression curve.

Results

Between November 12, 2020 and January 31, 2022, a total of 35,994 RDT with parallel RT-qPCR were conducted, out of which 35,479 RDT with parallel RT-qPCR taken from 26,940 individuals were taken into consideration for performance assessment. 9,137 (25.75%) RDT were carried out with NADAL®, 17,311 (48.79%) with Panbio™, 9,031 (25.45%) with MEDsan® (Fig. 1 ).

Fig. 1

Distribution of enrolled RDT results. 469 cases of multiple RDT performances per day per person as well as 20 cases of recent deisolation after SARS-CoV-2 infection were excluded from data analysis. 26 carried out RDT with invalid test results (missing positive control or interfering lines, 5 NADAL®, 11 Panbio™, 10 MEDsan®) were also not taken in account. RDT: Antigen rapid diagnostic test. RT-qPCR: Quantitative reverse transcription polymerase chain reaction.

Study population

Enrolled study participants were aged 0 to 101 years (median age: 50.5 years, IQR: 30–69 years). 17,906 (50.43%) RDT were conducted on female, 17,597 (49.56%) on male individuals, and one on an individual with allocation to diverse gender. 30,588 (86.15%) RDT were performed on patients, 598 (1.68%) on employees, and 4,319 (12.16%) on accompanying individuals staying overnight with underage and otherwise dependent patients.

Performance of RDT in comparison to RT-qPCR

In 426 specimens among 35,479 enrolled RDT/RT-qPCR tandems SARS-CoV-2 positivity could be confirmed, corresponding to a SARS-CoV-2 prevalence of 1.20%. 164 (0.46%) of all tandem samples were tested positive by RDT (true positive), 262 (0.74%) negative (false negative). Out of the 35,053 RT-qPCR-negative samples, 34,937 (98.47%) tested negative by RDT (true negative), 116 (0.33%) positive (false positive). Three of the 26 as invalid assessed RDT tested RT-qPCR positive.

The overall RDT sensitivity was calculated as 38.50% (95% CI 34.00%–43.20%), the overall specificity as 99.67% (95% CI 99.60%–99.72%). The positive predictive value (PPV) was 58.57% (95% CI 52.57%–64.19%), the negative predictive value (NPV) 99.26% (95% CI 99.16%–99.34%).

The differences between manufacturers in sensitivity (p=0.31, Fig. 2 A and Supplementary Results) and specificity (p=0.37, Chi-squared test, Fig. 2B) were not significant.

Fig. 2

Sensitivity of antigen rapid diagnostic testing compared to quantitative reverse transcription polymerase chain reaction by manufacturer and viral load. Fig. 2A&B: Sensitivity and specificity of antigen rapid diagnostic tests from three manufacturers (nal von minden NADAL®, Abbott Panbio™, MEDsan®) compared to quantitative reverse transcription polymerase chain reaction as reference standard, n = 35,479. Fig. 2C: Viral load of RT-qPCR positive specimen which tested positive and negative by RDT. Fig. 2D: Sensitivity of RDT compared to RT-qPCR in relation to viral load determined from Ct values. Sensitivity is sharply increasing with higher viral loads, n=426. The dotted lines in Fig. 2C and 2D represent the viral load of 106 SARS-CoV-2 RNA copies per ml assumed as infectivity threshold.[14]. n: Number of samples in each group. RDT: Antigen rapid diagnostic test. RT-qPCR: Quantitative reverse transcription polymerase chain reaction. ****: p<0.0001.

Relation of RDT sensitivity to viral load

Median viral loads were significantly higher in RDT positive (median: 8.22x106 SARS-CoV-2 RNA copies per ml) than in RDT negative samples (median: 6.84x104 copies per ml, p<0.0001, Mann-Whitney U test, Fig. 2C). Sensitivity increased with rising viral loads (Fig. 2D).

Relation of RDT performance to molecularly determined SARS-CoV-2 VOC

In 257 (60.32% of all RT-qPCR-positive) samples, VOC were determined by PCR. 6.23% (16) of specimen were assigned as containing wild-type SARS-CoV-2, 27.24% (70) Alpha VOC, 51.36% (132) Delta VOC, and 14.01% (36) Omicron VOC. In two samples (0.78%) Iota VOI was detected, in one case results of variant specific PCRs as well as spike protein sequencing did not conform to any commonly described VOC.

Including all RDT/RT-qPCR tandems with molecularly determined VOC, RDT sensitivity was 31.25% (5/16, 95% CI 14.16%–55.60%) for wild-type SARS-CoV-2, 45.71% (32/70, 95% CI 34.57%–57.30%) for Alpha VOC, 40.91% (54/132, 95% CI 32.89%–49.44%) for Delta VOC, 36.11% (13/36, 95% CI 22.48%–52.42%) for Omicron VOC, and 50.00% (1/2, 95% CI 2.56%–97.44%) for Iota VOI (Fig. 3 A).

Fig. 3

VOC depending on antigen rapid diagnostic test sensitivity. RDT sensitivity compared to RT-qPCR as reference standard by VOC. Fig. 3A included 257 samples with molecularly confirmed VOC, Fig. 3B included 407 samples with either molecularly confirmed VOC or epidemiologically assigned VOC (in case no VOC was determined molecularly, and the VOC of the infection source was known, or a VOC was responsible for more than 90% of all COVID-19 cases in Germany at the time of sampling). Fig. 3C included 218 samples with a viral load < 106 SARS-CoV-2 RNA-copies per ml and an either molecularly or epidemiologically assigned wild-type, Alpha VOC, Delta VOC, or Omicron VOC. Fig. 3D included 184 samples with a viral load ≥ 106 SARS-CoV-2 RNA-copies per ml and an either molecularly or epidemiologically assigned wild-type SARS-CoV-2, Alpha VOC, Delta VOC, or Omicron VOC. n: Number of samples in each group. RDT: Antigen rapid diagnostic test. RT-qPCR: Quantitative reverse transcription polymerase chain reaction. VOC: Variant of concern. *: p<0.05.

Relation of RDT performance to SARS-CoV-2 VOC including epidemiological assignment

148 further samples could be assigned to a presumed SARS-CoV-2 VOC using epidemiological data, resulting in 405 RT-qPCR positive samples with confirmed or presumed VOC: 20.74% (84) specimen to wild-type SARS-CoV-2, 18.02% (73) to Alpha VOC, 36.30% (147) to Delta VOC, 24.20% (98) to Omicron VOC, and 0.49% (2) to Iota VOI. One sample showed an uncommon VOC. 21 remaining specimens without VOC allocation could not be epidemiologically identified due to VOC prevalence levels below 90% and were consequently not considered for VOC dependent performance evaluation.[10].

Based on this assignment, the following VOC dependent sensitivities were observed:[10] 42.86% (36/84, 95% CI 32.82%–53.52%) for wild-type virus, 43.84% (32/73, 95% CI 33.05%–55.24%) for Alpha VOC, 38.10% (56/147, 95% CI 30.64%–46.15%) for Delta VOC, 33.67% (33/98, 95% CI 25.09%–43.49%) for Omicron VOC, and 50.00% (1/2, 95% CI 2.56%–97.44%) for Iota VOI (Fig. 3B).

Median viral load was significantly lower in wild-type samples (7.71x104 SARS-CoV-2 RNA copies per ml) compared to VOI and VOC samples (p<0.001, Mann-Whitney U test). No significant difference was observed in comparison of Alpha VOC (median: 8.28x105 copies per ml), Delta VOC (median: 7.43x105 copies per ml), Iota VOI (6.84x105 copies per ml), and Omicron VOC (5.30x105 copies per ml, p=0.71, Kruskal-Wallis test).

While no significant differences in RDT sensitivity could be detected in samples with a viral load below 106 SARS-CoV-2 RNA copies per ml (Fig. 3C), differences in sensitivity were observed in samples with a viral load above this threshold: Sensitivity was 79.31% (23/29, 95% CI 61.61%–90.15%) in wild type SARS-CoV-2 containing samples while it decreased in other VOC to 69.44% (25/36, 95% CI 53.14%–82.00%, p=0.41) in Alpha VOC, to 61.64% (45/73, 95% CI 50.17%–71.95%, p=0.11) in Delta VOC, and 50.00% (23/46, 95% CI 36.12%–63.88%, p=0.015, Fisher’s exact test) in Omicron VOC (Fig. 3D).

50% LOD increased from 2.75x105 (95% CI: 6.03x104–1.74x106) SARS-CoV-2 RNA copies per ml for wild-type virus over 2.51x106 (95% CI: 6.46x105–1.17x107) copies per ml for Alpha VOC and 5.13x106 (95% CI: 1.86x106–1.82x107) for Delta VOC to 1.32x107 (95% CI: 2.09x106–3.39x108) for Omicron VOC. 95% LOD increased from 7.59x108 (95% CI: 4.17x107–8.71x1011) SARS-CoV-2 RNA copies per ml for wild-type virus over 1.38x109 (95% CI: 1.20x108–1.74x1012) copies per ml for Alpha VOC and 7.41x109 (95% CI: 7.76x108–6.76x101) for Delta VOC to 2.34x1012 (95% CI: 9.33x109–2.00x1018) for Omicron VOC (Fig. 4 ).

Fig. 4

VOC depending LOD: overall and by manufacturer. Fig. 4 included 402 samples either molecularly confirmed or epidemiologically assigned to wild-type virus, Alpha VOC, Delta VOC or Omicron VOC (in case no VOC was determined molecularly, and the VOC of the infection source was known, or a VOC was responsible for more than 90% of all COVID-19 cases in Germany at the time of sampling). 50% and 95% LOD was marked by dashed vertical lines with 95% confidence intervals visualised in grey (50% LOD) and yellow (95%). The regression curve is shown as straight line with 95% confidence interval visualised in blue. The difference in overall 50% LOD was significantly higher for Omicron VOC compared to wild-type virus, while a non-significant increase in 50% and 95% LOD was observed from wild-type virus over Alpha VOC, Delta VOC in the overall data as well as for Abbott Panbio™ (n=156) and MEDsan® (n=197). Due to limited case numbers, no reliable VOC specific LOD calculation could be obtained for nal von minden NADAL® (n=49). n: Number of samples in each group. LOD: Level of detection. VOC: Variant of concern.

Discussion

With an overall sensitivity of 38.5%, the clinical sensitivity matches the range of a laboratory analysis of the utilised products (36.0% to 64.0%).[15] The overall sensitivity is on the lower end of the range reported by other studies explained by the inclusion of asymptomatic individuals, whereas the majority of previously published clinical evaluation studies are limited to symptomatic individuals.[16, 17, 18, 19] Among the subgroup of specimens with a viral load ≥106 SARS-CoV-2 RNA-copies per ml, suggested as viral load threshold for infectivity,[14] RDT sensitivity was statistically significantly impaired in Omicron VOC samples as compared to wild-type SARS-CoV-2. The 50% LOD for Omicron VOC increased by the factor 48 compared to wild-type SARS-CoV-2, the 95% LOD by the factor 1,425. This is in line with the previously published small-cohort or laboratory studies that claim Omicron VOC as curtailing RDT sensitivity as compared to wild-type SARS-CoV-2 and Delta VOC. The present data supports the previous studies and provides a translation of data to real-life and large-scale clinical conditions.[3, 4, 5, 6].

Data from a laboratory evaluation suggests that the decrease in sensitivity may be caused by a lower nucleoprotein to RNA ratio in Omicron VOC infected individuals.[5] A possible explanation for the fact that this difference was not significant in the category of individuals with a viral load ≤106 RNA-copies per ml may be the higher medium viral load in Omicron VOC compared to wild-type SARS-CoV-2 samples which may compensate for the restriction in sensitivity. Another possible bias might be the differences in immunisation status of general public: While nearly no wild-type SARS-CoV-2 and only a few Alpha VOC infections occurred in vaccinated or convalescent individuals enrolled to this study, the majority of the adults was vaccinated twice or threefold during the Delta and Omicron VOC dominated interval.[7, 20] Recently, this potential influence of immunisation status including vaccination status on RDT sensitivity was discussed in a preprint analysis.[21].

The presented results are limited in several aspects. As RDT point-of-care usage was implemented under real-life clinical conditions and differing product availabilities, absolute numbers, and proportions of used RDT products varied between the several clinical departments and in course of the study period. The participating clinical departments differ regarding patient structure and morbidity. Enrolled study participants were only tested using one of the three chosen RDT, which restricts direct comparability between the selected RDT manufacturers. In a large-scale laboratory performance evaluation, the three RDT used belonged to a moderate sensitivity group. RDT with higher sensitivity have since become available, although their performance regarding Omicron VOC is still unclear.[15] In acceptance of these limitations, the study allowed an analysis of real-life data in a large cohort with a broad demographic structure and consequently high reliable transferability to in vivo conditions including VOC dependence. Due to sample collection for RDT and RT-qPCR by a multitude of skilled, professional operators, influence of potential inhomogeneity in sampling, test execution as well as interpretation could not be avoided. Further, the role of preanalytical quality and accurate specimen collection must be highlighted. Compared to RT-qPCR, RDT are more prone to incorrect swabbing. Consequent lower viral loads of the gained samples may in part be responsible for increasing numbers of false negative results. While NADAL® and MEDsan® RDT were recommended for nasopharyngeal as well as oropharyngeal sampling, the Panbio™ RDT was used with oropharyngeal sampling in contrast to manufacturer’s instructions recommending nasopharyngeal specimen collection which might limit the comparability to studies based on nasopharyngeal sampling.

VOC determination was only performed between January 2021 and January 2022. Therefore, a relevant proportion of wild-type SARS-CoV-2 and Omicron VOC samples could only be epidemiologically assigned. Omicron VOC sublineages could not be differentiated. Due to the inability of VOC determination in several low viral load samples, sensitivity in molecularly determined VOC is biased towards higher values. With only two specimens of Iota VOI, no precise sensitivity data could be determined for this VOI. Compared to previously published studies, the study represents a low SARS-CoV-2 prevalence setting of 1.20% but corresponds to a real-life scenario including RDT testing on asymptomatic individuals.[16, 17, 18, 19, 22].

Omicron VOC reduces RDT sensitivity even in individuals with high viral loads, which limits the use of RDT in the group most relevant for SARS-CoV-2 transmission. However, RDT are still an important strategical tool for rapid identification of highly infectious individuals before the availability of RT-qPCR results and for the regular screening of large cohorts. The self-testing option and straightforward feasibility emphasise these aspects. This points out the importance of further large clinical studies of RDT in large populations not only in times of Omicron VOC prevalence, but also for future possible VOC and VOC sublineages.

Conflicts of interests

None of the authors has any conflict of interest.

Funding

This study was funded by the Federal Ministry for Education and Science (BMBF) via a grant provided to the University Hospital of Wuerzburg by the Network University Medicine on COVID-19 (B-FAST, grant-No 01KX2021) as well as by the Free State of Bavaria with COVID-research funds provided to the University of Wuerzburg, Germany. This work was supported by the Bavarian Staten Ministry of Health and Care via Bay-VOC.

Nils Petri is supported by the German Research Foundation (DFG) funded scholarship UNION CVD.

This study was initiated by the investigators. The sponsoring institutions had no role in study design, data collection, analysis, and interpretation of data as well as in writing of the manuscript. All authors had unlimited access to all data. The first and the corresponding author had final responsibility for the decision to submit for publication.

Access to Data

Applications for relevant anonymous data that underlies the results reported in this article could be submitted to the corresponding author immediately following publication and ending five years following article publication to achieve aims in the approved proposal.

Contribution

All authors had unlimited access to all data. Ms Wagenhäuser and Dr Krone take responsibility for the integrity of the data and the accuracy of the data analysis.

Conception and design: Andres, Wurmb, Ernestus, Forster, Weismann, Weißbrich, Dölken, Liese, Kurzai, Vogel, Krone.

RT-qPCR testing as well as a standardised Cquantification: Knies, Hofmann, Weißbrich.

RDT use and documentation instruction in different departments: Rauschenberger, Eisenmann, Flemming, Andres, Topp, Papsdorf, Verma-Führing, Scherzad, Zeller, Böhm, Gesierich, Seitz, Kiderlen, Gawlik, Taurines, Wurmb, Weismann, Krone.

Usersupport: Rauschenberger, Eisenmann, Vogel, Krone.

Collection of clinical data from patient’s files: Wagenhäuser, Rauschenberger, Eisenman, Petri, McDonogh, Krone.

LOD calculation: Kaderali.

Statistical analysis: Wagenhäuser, Reusch, Gabel, Petri, Krone.

Obtainedfunding: Dölken, Kurzai, Vogel.

First draft of the manuscript: Wagenhäuser, Krone.

Reviewing and modifying the manuscript and approving its final version: Knies, Hofmann, Rauschenberger, Eisenmann, Reusch, Gabel, Flemming, Andres, Petri, Topp, Papsdorf, Petri, McDonogh, Verma-Führing, Scherzad, Zeller, Böhm, Gesierich, Seitz, Kiderlen, Gawlik, Taurines, Wurmb, Ernestus, Forster, Weismann, Weißbrich, Dölken, Liese, Kaderali, Kurzai, Vogel.