key: cord-1043656-u158dd5k
authors: Sluimer, John; Goderski, Gabriel; van den Brink, Sharon; Broeders, Maaike; Rahamat-Langendoen, Janette; Then, Euníce; Wijsman, Lisa; Wolters, Femke; van de Bovenkamp, Jeroen; Melchers, Willem JG; Meijer, Adam
title: Multi-center evaluation of Cepheid Xpert® Xpress SARS-CoV-2/Flu/RSV molecular point-of-care test
date: 2021-09-24
journal: Journal of clinical virology plus
DOI: 10.1016/j.jcvp.2021.100042
sha: 258bd5a9d7ab4348e20ccd602abc0348ee2a16b5
doc_id: 1043656
cord_uid: u158dd5k

Background SARS-CoV-2 is taking a huge toll on society while influenza and RSV detection are also becoming more important. These viruses pose a high burden on health care. Rapid and accurate diagnostics for these pathogens are important for swift triage in the hospital. Fast molecular point of care test (mPOCT) assays for these pathogens can prove an alternative. Here a multi-center evaluation of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is reported. Study design The Xpert® Xpress SARS-CoV-2/Flu/RSV assay was compared to three reference assays at three Dutch medical microbiology laboratories. An external quality assessment panel consisting of 16 specimens containing SARS-CoV-2, influenza viruses, RSV or human seasonal coronaviruses, or a combination thereof were used. Clinical specimens containing SARS-CoV-2 (n=57), influenza viruses (n=21) or RSV (n=12), at a wide range of relevant concentrations were used. One laboratory also tested zoonotic avian and swine influenza viruses, and eight relevant SARS-CoV-2 variants. Results The Xpert® Xpress SARS-CoV-2/Flu/RSV assay showed equal performance compared to the reference assays. All SARS-CoV-2 variants of interest and variants of concern were accurately detected. Human seasonal coronaviruses were not detected. All four circulating seasonal influenza virus subtypes/lineages and both RSV types were accurately detected as well as a set of recent zoonotic avian and swine influenza viruses. The clinical specimens showed 98.2% concordance using this assay. Conclusion The Xpert® Xpress SARS-CoV-2/Flu/RSV assay is a good alternative for accurate detection for SARS-CoV-2, influenza type A virus, influenza type B virus and RSV types A and B detection in a short timeframe.

Key words: SARS-CoV-2, COVID-19, Influenza virus, RSV, Xpert Xpress, Molecular point-of-care test

In December 2019 the first patient suffering from SARS-CoV-2 was reported in Wuhan, China and on the 11 th of

March 2020 SARS-CoV-2 was declared a pandemic. [1, 2] Since then over 182 million confirmed cases and over 3.9 million deaths worldwide have been attributed to the virus which continues to circulate and cause morbidity and mortality across the globe. [3] The symptoms induced by a SARS-CoV-2 infection can vary a great amount from causing an asymptomatic infection to severe respiratory failure. [4] Traditionally, specimens derived from patients with suspected SARS-CoV-2 infection are tested using RT-PCR, however the process of testing can be time consuming.

To provide a quick platform for SARS-CoV-2 diagnostics, Cepheid developed the Xpert® Xpress SARS-CoV-2 point-of-care test in April 2020 which has a hands on time limited to 2−3 min and a run-time of 45−50 min. [5] Although the convenience of this test has been shown, one of the downsides of this assay is that it can only test for the presence of SARS-CoV-2. In the more temperate regions of the Northern Hemisphere, influenza virus A/B and RSV A/B infection levels normally peak each year from December -March. Infections with SARS-CoV-2 and RSV can show similar symptoms [6, 7] . In order to allow diagnostic laboratories to be able to test patient derived specimens on a multitude of respiratory diseases, Cepheid has developed the Xpert® Xpress SARS-CoV-2/Flu/RSV cartridges which allows for testing of patient material for the presence of SARS-CoV-2, Influenza A/B and RSV A/B virus using a single cartridge. The aim of this study is to evaluate the performance of the Xpert® Xpress SARS-CoV-2/Flu/RSV test and determine if it is a useful addition to the respiratory viral diagnostic repertoire.

The external quality assessment (EQA) panel used for this evaluation was prepared at the Dutch National Table 1 ). The panel comprised a total of 16 specimens containing either one or a combination of two viruses mentioned above. SARS-CoV-2 was also included as dilution series to determine sensitivity. All specimens were made in MEM with Hanks' salts and to each specimen 10,000 HEp2 cells (ATCC® CCL-23) were added per milliliter to simulate a human specimen in virus transport medium. Details of the EQA panel with expected positivity indicated by Ct values are listed in Table 1 .

As an extra evaluation of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay laboratory 3 also tested specimens Table 2 provides an overview of the specifications of the selected clinical specimens.

The EQA panel was shipped frozen on dry ice ensuring the same amount of freeze-thaw cycles for all locations.

Both panel specimens and clinical specimens were processed according to the routine diagnostic protocol as performed in each lab (see Table 3 and Table 4 ). Laboratory 1 did not perform RSV typing. Laboratory 2 did not perform influenza virus type A subtyping, no influenza virus type B lineage determination nor RSV typing. Panel specimens and clinical specimens for the Xpert® Xpress SARS-CoV-2/Flu/RSV assay were processed according to manufacturer's instruction and only required 300 μl specimen input. The Xpert® Xpress SARS-CoV-2/Flu/RSV assay contains primers and probes targeting the E-gene and N2-gene of SARS-CoV-2, the matrix protein, PB2

and PA genes of influenza virus type A, the matrix protein and non-structural protein genes of influenza virus type B and nucleocapsid genes of RSV-A and RSV-B. The assay provides results for SARS-CoV-2, influenza virus by type (type A two channels) and RSV without typing.

Laboratory 1 did not test the EQA panel with the routine workflow (the Xpert® Xpress SARS-CoV-2 assay). This was deemed unnecessary as their routine SARS-CoV-2 diagnostics assay was already validated by Wolters et al. Table 5 . Taking into account false negatives for the 17.3 and 86.5 copies SARS-CoV-2 RdRP-gene/ml specimens and no false negatives with 10-fold higher concentration specimens, the SARS-CoV-2 LOD for the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is estimated roughly between 86.5 and 173 copies RdRP-gen/ml specimen. All specimens containing avian and swine influenza viruses tested positive for influenza virus type A using the Xpert® Xpress SARS-CoV-2/Flu/RSV assay.

The SARS-CoV-1 containing specimen as well as all SARS-CoV-2 variants were reported positive for SARS-CoV-2 by the Xpert® Xpress SARS-CoV-2/Flu/RSV assay. All SARS-CoV-2 variants tested by laboratory 3 were accurately detected in a high and low concentration at 1.14E06-6.59E06 and 1.14E04-6.59E04 dPCR RdRP-gene copies/ml, respectively.

At all three laboratories the clinical specimens containing influenza virus or RSV, tested negative for SARS-CoV-2. All specimens tested positive for influenza and RSV with the Xpert® Xpress SARS-CoV-2/Flu/RSV assay, except for one specimen from laboratory 3 that tested negative for RSV. This specimen contained a low concentration of RSV-A confirmed by re-testing. All high titer SARS-CoV-2 containing clinical specimens were confirmed SARS-CoV-2 positive upon testing with the Xpert® Xpress SARS-CoV-2/Flu/RSV assay. Of the low titer clinical SARS-CoV-2 specimens laboratory 1 showed an N-gene only positive result for two specimens using their regular diagnostic workflow. The E-gene result was negative for these specimens. One of these two specimens also was negative upon retesting with the Xpert® Xpress SARS-CoV-2/Flu/RSV assay, confirming low viral content. From the low load SARS-CoV-2 positive specimens, three specimens selected by laboratory 3

were not reconfirmed using the routine assay. In two of these three specimens SARS-CoV-2 could still be detected using the Xpert® Xpress SARS-CoV-2/Flu/RSV assay. All other selected clinical specimens tested positive for SARS-CoV-2 by both the routine SARS-CoV-2 diagnostic workflows and the Xpert® Xpress SARS-CoV-2/Flu/RSV assay at all locations. This data is summarized in Table 6 .

In this study we have shown that Cepheid's Xpert® Xpress SARS-CoV-2/Flu/RSV assay is a highly sensitive SARS-CoV-2, influenza A/B and RSV-A/-B specific mPOCT. The performance of this assay using a variety of EQA and clinical specimens of high and low viral load was highly similar to that of the SARS-CoV-2, influenza A/B and RSV-A/-B assays routinely performed by the participating laboratories. In another multicenter European validation this same conclusion was reached with 95% concordance of all tested clinical specimens. [8] An advantage of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is the runtime of only 45−50 minutes as well as the option for random access. Decreased runtime of diagnostics can decrease length of stay on an emergency ward, expedite intake of antiviral drugs and lessen the number of tests ordered. The latter is especially true for multiplex assays as they test for multiple pathogens per run. [9, 10] As an extra control several SARS-CoV-2 variants, namely Alpha, Beta, Gamma, Epsilon, Zeta and three other variants (pangolineage B.1.177, B.1.258.21 and C.2.1) were tested using the assay. All variants were accurately detected in a high and low concentration. The capacity of the assay to detect (novel) variants of SARS-CoV-2 makes it a robust assay. The Cepheid's Xpert® Xpress SARS-CoV-2/Flu/RSV assay uses two target genes for both SARS-CoV-2 and influenza detection. An advantage of this is that it decreases the chance of false negative test results when testing (novel) genetic variants of those pathogens. For example mutations in SARS-CoV-2 variant B.1.1.7/20B/501Y.V1 can cause an S-gene dropout in certain assays. [11] If an assay uses only one target gene for detection of a pathogen, there is a higher chance of false-negative results when confronted with novel variants than in assays with multiple target genes. There is a limited chance that a novel variant avoids detection of two or more target genes at the same time.

In addition, recent zoonotic avian and swine influenza viruses were accurately detected by the assay. Swine and avian influenza variants are known to infect the human population. [12] The ability of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay to detect these strains increases its usability in potential outbreaks. Currently there is an RSV epidemic in the Netherlands which does not fit in the regular yearly (winter) cycle in which the disease normally is present. [13] This finding is similar to the delayed increase in RSV infections found in France, Australia, Argentina, Chili and South-Africa. [14] [15] [16] [17] [18] [19] [20] As SARS-CoV-2, (zoonotic) influenza and RSV diseases show similar symptoms it is very convenient to triage patients quickly into the correct hospital ward. [4, 6, 7] This decreases the chance on cross-contamination between different patient groups.

Considering the high throughput of suspected SARS-CoV-2 /influenza/RSV patients in certain hospital wards, it is highly practical to have a multiplex assay capable of testing for both diseases in the same run. The LOD of Cepheid's Xpert® Xpress SARS-CoV-2/Flu/RSV assay is estimated roughly between 86.5-173 digital copies of positive strand genomic SARS-CoV-2 RNA/ml. This is similar to the LOD of 131 copies/ml reported by the manufacturer.

[21] The only false positive result with the Gene Xpert® SARS-CoV-2/Flu/RSV assay when testing any of the specimens was obtained with SARS-CoV-1. It is known that the E-gene component of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is specific for the SARS-like betacoronavirus group to which SARS-CoV-1 belongs. Considering the fact that SARS-CoV-1 currently does not circulate in the human population, there is no reason to question whether a clinical specimen might be SARS-CoV-1 positive when the assay reports it as SARS-CoV-2 positive. All in all the Gene Xpert® SARS-CoV-2/Flu/RSV assay is a reliable method for rapid detection of SARS-CoV-2, influenza type A virus, influenza type B virus and RSV types A and B.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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