Gaining detailed insights into the role of host immune responses in viral clearance is critical for understanding COVID-19 pathogenesis and future treatment strategies. Although studies analyzing humoral immune responses against SARS-CoV-2 were available rather early during the pandemic, cellular immunity came into focus of investigations just recently. For the present work, we have adapted a protocol designed for the detection of rare neoantigen-specific memory T cells in cancer patients for studying cellular immune responses against SARS-CoV-2. Both CD4+ and CD8+ T cells were detected after 6 d of in vitro expansion using overlapping peptide libraries representing the whole viral protein. The assay readout was an intracellular cytokine staining and flow cytometric analysis detecting four functional markers simultaneously (CD154, TNF, IL-2, and IFN-γ). We were able to detect SARS-CoV-2-specific T cells in 10 of 10 COVID-19 patients with mild symptoms. All patients had reactive T cells against at least 1 of 12 analyzed viral Ags, and all patients had Spike-specific T cells. Although some Ags were detected by CD4+ and CD8+ T cells, VME1 was mainly recognized by CD4+ T cells. Strikingly, we were not able to detect SARS-CoV-2-specific T cells in 18 unexposed healthy individuals. When we stimulated the same samples overnight, we measured significant numbers of cytokine-producing cells even in unexposed individuals. Our comparison showed that the stimulation conditions can profoundly impact the activation readout in unexposed individuals. We are presenting a highly specific diagnostic tool for the detection of SARS-CoV-2-reactive T cells.Flagellin is an immunodominant Ag in Crohn disease, with many patients showing anti-flagellin Abs. To study the clonality of flagellin-reactive CD4 cells in Crohn patients, we used a common CD154-based enrichment method following short-term Ag exposure to identify Ag-reactive CD4 cells. CD154 expression and cytokine production following Ag exposure compared with negative control responses (no Ag exposure) revealed that only a small fraction of CD154-enriched cells could be defined by Ag-reactive cytokine responses. This was especially true for low-frequency flagellin-reactive CD4 cells compared with polyclonal stimulation or Candida albicans Ag exposure. Moreover, we found that culture conditions used for the assay contributed to background CD40L (CD154) expression in the CD154-enriched CD4 cells. Using a cut-off rule based on flow cytometry results of the negative control CD154-enriched CD4 cells, we could reliably find the fraction of Ag-reactive cells in the CD154-enriched population. Ag-reactive CD4 cytokine production was restricted to CD4 cells with an effector memory phenotype and the highest levels of induced CD154 expression. This has important implications for identifying Ag-specific T cells of interest for single cell cloning, phenotyping, and transcriptomics.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.During the ongoing coronavirus disease 2019 (COVID-19) outbreak, robust detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key element for clinical management and to interrupt transmission chains. selleck products We organized an external quality assessment (EQA) of molecular detection of SARS-CoV-2 for European expert laboratories. An EQA panel composed of 12 samples, containing either SARS-CoV-2 at different concentrations to evaluate sensitivity or other respiratory viruses to evaluate specificity of SARS-CoV-2 testing, was distributed to 68 laboratories in 35 countries. Specificity samples included seasonal human coronaviruses hCoV-229E, hCoV-NL63, and hCoV-OC43, as well as Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and human influenza viruses A and B. Sensitivity results differed among laboratories, particularly for low-concentration SARS-CoV-2 samples. Results indicated that performance was mostly independent of the selection of specific extraction or PCR methods.We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (Tm s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific Tm s or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA.