Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV Test.

With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Invader assay for RNA quantitation.

The Invader assay is a homogeneous, isothermal, signal amplification system for the quantitative detection of nucleic acids. The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the hybridization of two oligonucleotides to the target sequence. The combination of sequence-specific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms directly from genomic DNA as well as highly homologous mRNAs in closely related gene families. Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence.

Invader technology for DNA and RNA analysis: principles and applications.

Concomitant advances made by the Human Genome Project and in the development of nucleic acid screening technologies are driving the expansion of pharmacogenomic research and molecular diagnostics. However, most current technologies are restrictive due to their complexity and/or cost, limiting the potential of personalized medicine. The invader assay, which can be used for genotyping as well as for gene expression monitoring without the need for intervening target amplification steps, presents an immediate solution that is accurate, simple to use, scaleable and cost-effective.

Multiplexed gene expression analysis using the invader RNA assay with MALDI-TOF mass spectrometry detection.

A mass spectrometric approach for measuring gene expression levels has been developed. This technique utilizes a signal amplification system and analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Signal amplification from the targeted RNA employs a recently developed invasive cleavage assay that does not require prior PCR amplification. The assay uses a set of target-specific probes (oligonucleotides), which hybridize to the RNA being measured to create an overlap structure with a single-stranded flap. This flap is enzymatically cleaved and accumulates linearly in a target-specific manner. The products of the reaction, short DNA oligomers, are well suited for quantitative detection by MALDI-TOF mass spectrometry. Multiplexing is achieved by designing the assays so that reaction products for different mRNA targets have discrete masses that can be resolved in a single mass spectrum. Simultaneous analysis of human cytokine in vitro transcripts IL-1beta, TNF-alpha, and IL-6, with GAPDH as a reference standard, was used as a model system to demonstrate this novel method of gene expression analysis.