Performance of a flow cytometry-based immunoassay for detection of antibodies binding to SARS-CoV-2 spike protein

The performance of a laboratory-developed IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n = 179), followed by IgA-FCI (n = 177), Roche ECLIA (n = 175), IgG-FCI (n = 172) and Diasorin CLIA (n = 154). For sera collected early after the onset of symptoms (within 15 days) IgG/IgA-FCI also returned the highest number of positive results (52/72; 72.2%). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1 (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.

A method for variant agnostic detection of SARS-CoV-2, rapid monitoring of circulating variants, detection of mutations of biological significance, and early detection of emergent variants such as Omicron

The rapid emergence of new SARS-CoV-2 variants raises a number of public health questions including the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to better understand patterns of transmission and possible load on healthcare resources. Next-Generation Sequencing (NGS) is the primary method for detecting and tracing the emergence of new variants, but it is expensive, and it can take weeks before sequence data is available in public repositories. Here, we describe a Polymerase Chain Reaction (PCR)-based genotyping approach that is significantly less expensive, accelerates reporting on SARS-CoV-2 variants, and can be implemented in any testing lab performing PCR. Specific Single Nucleotide Polymorphisms (SNPs) and indels are identified that have high positive percent agreement (PPA) and negative percent agreement (NPA) compared to NGS for the major genotypes that circulated in 2021. Using a 48-marker panel, testing on 1,128 retrospective samples yielded a PPA and NPA in the 96.3 to 100% and 99.2 to 100% range, respectively, for the top 10 most prevalent lineages. The effect on PPA and NPA of reducing the number of panel markers was also explored. In addition, with the emergence of Omicron, we also developed an Omicron genotyping panel that distinguishes the Delta and Omicron variants using four (4) highly specific SNPs. Data from testing demonstrates the capability to use the panel to rapidly track the growing prevalence of the Omicron variant in the United States in December 2021.

Agreement and Reliability Analysis of Machine Learning Scaling and Wireless Monitoring in the Assessment of Acute Proximal Weakness by Experts and Non-Experts: A Feasibility Study

Assessing the symptoms of proximal weakness caused by neurological deficits requires the knowledge and experience of neurologists. Recent advances in machine learning and the Internet of Things have resulted in the development of automated systems that emulate physicians’ assessments. The application of those systems requires not only accuracy in the classification but also reliability regardless of users’ proficiency in the real environment for the clinical point-of-care and the personalized health management. This study provides an agreement and reliability analysis of using a machine learning-based scaling of Medical Research Council (MRC) proximal scores to evaluate proximal weakness by experts and non-experts. The system trains an ensemble learning model using the signals from sensors attached to the limbs of patients in a neurological intensive care unit. For the agreement analysis, we investigated the percent agreement of MRC proximal scores and Bland-Altman plots of kinematic features between the expert- and non-expert scaling. We also analyzed the intra-class correlation coefficients (ICCs) of kinematic features and Krippendorff’s alpha of the observers’ scaling for the reliability analysis. The mean percent agreement between the expert- and the non-expert scaling was 0.542 for manual scaling and 0.708 for autonomous scaling. The ICCs of kinematic features measured using sensors ranged from 0.742 to 0.850, whereas the Krippendorff’s alpha of manual scaling for the three observers was 0.275. The autonomous assessment system can be utilized by the caregivers, paramedics, or other observers during an emergency to evaluate acute stroke patients.

Performance of the BinaxNOW COVID-19 Antigen Rapid Diagnostic Test for the Detection of SARS-CoV-2

Background: Diagnosis of COVID-19 disease primarily relies on nucleic acid amplification tests (NAAT) that amplify and detect viral RNA in specimens. These methods are expensive and time consuming. Antigen-based rapid diagnostic tests can substantially decrease turnaround time.Methods: We analyzed paired anterior nares swabs collected from symptomatic patients and asymptomatic healthcare workers being tested COVID-19. One swab was used for a direct RDT and the results were compared to NAAT.Results: 89 paired specimens were evaluated. The positive percent agreement (PPA) for the antigen RDT was 68.2%, and the negative percent agreement (NPA) was 98.5%. Despite a low PPA, the Κ statistic was 0.733 indicating substantial agreement with the NAAT result. The median cycle number in paired specimens with concordant results was significantly lower than in discordant specimens (21.3 versus 32.3; P=0.003).Conclusions: The RDT showed modest PPA and high NPA when compared to NAAT. The quick TAT and use of an inexpensive test more frequently could be useful in settings in which results from NAAT testing is delayed.

Saliva swabs are the preferred sample for Omicron detection

The Omicron variant is characterised by more than 50 distinct mutations, the majority of which are located in the spike protein. The implications of these mutations for disease transmission, tissue tropism and diagnostic testing are still to be determined. We evaluated the relative performance of saliva and mid-turbinate swabs as RT-PCR samples for the Delta and Omicron variants. The positive percent agreement (PPA) of saliva swabs and mid-turbinate swabs to a composite standard was 71% (95% CI: 53-84%) and 100% (95% CI: 89-100%), respectively, for the Delta variant. However, for the Omicron variant saliva and mid-turbinate swabs had a 100% (95% CI: 90-100%) and 86% (95% CI: 71-94%) PPA, respectively. This finding supports ex-vivo data of altered tissue tropism from other labs for the Omicron variant. Reassessment of the diagnostic testing standard-of-care may be required as the Omicron variant becomes the dominant variant worldwide.

Comparative evaluation of rapid isothermal amplification and antigen assays for screening testing of SARS-CoV-2

Human transmission of SARS-CoV-2 and emergent variants of concern has continued to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test, had a limit of detection of 30,000 copies, and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.

Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer

The detection of actionable mutations in tumor tissue is a prerequisite for treatment customization in patients with metastatic colorectal cancer (mCRC). Analysis of circulating tumor DNA (ctDNA) for the identification of such mutations in patients’ plasma is an attractive alternative to invasive tissue biopsies. Despite having the high analytical sensitivity required for ctDNA analysis, digital polymerase chain reaction (dPCR) technologies can only detect a very limited number of hotspot mutations, whilst a broader mutation panel is currently needed for clinical decision making. Recent advances in next-generation sequencing (NGS) have led to high-sensitivity platforms that allow screening of multiple genes at a single assay. Our goal was to develop a small, cost- and time-effective NGS gene panel that could be easily integrated in the day-to-day clinical routine in the management of patients with mCRC. We designed a targeted panel comprising hotspots in six clinically relevant genes (KRAS, NRAS, MET, BRAF, ERBB2 and EGFR) and validated it in a total of 68 samples from 30 patients at diagnosis, first and second disease progression. Results from our NGS panel were compared against plasma testing with BEAMing dPCR regarding the RAS gene status. The overall percent of agreement was 83.6%, with a positive and negative percent agreement of 74.3% and 96.2%, respectively. Further comparison of plasma NGS with standard tissue testing used in the clinic showed an overall percent agreement of 86.7% for RAS status, with a positive and negative percent agreement of 81.2% and 92.8%, respectively. Thus, our study strongly supports the validity and efficiency of an affordable targeted NGS panel for the detection of clinically relevant mutations in patients with mCRC.

Validity of the Novel Radiological Classification System of the Distal Femur

Objective Aseptic loosening (AL) is among the most important causes of failure after total knee arthroplasty (TKA). However, while there are numerous underlying causes of AL, the morphometry of the distal femur and intramedullary canal has not been sufficiently demonstrated. This study aimed to show the interobserver and intraobserver reliability and validity of the Citak classification, which has been recently defined according to the morphometry of the distal femur and provides a risk factor definition for AL. Materials and Methods A total of 200 patients whose standardized anteroposterior (AP) and lateral images of the knee joint were obtained between October 2019 and April 2020 were retrospectively evaluated in this study. Patients with a history of extra-articular deformity and knee surgery were excluded from the study. For AL, morphologies of the distal femur were identified by two observers using the new radiological classification system of the distal femur. Mean pairwise Cronbach’s alpha coefficient was used to assess the intra- and interobserver agreement of the classification. Results There was excellent interobserver agreement for the 20 cm proximal and 2 cm proximal to the lateral joint line (PLJL) and adductor tubercle (PAD), respectively. The mean Cronbach’s alpha coefficient was 0.96 (range 0.764–0.944) for the PAD and 0.98 (range 0.734–0.929) for the PLJL. There was also an excellent intraobserver agreement, with 93% average pairwise percent agreement for the index group and 95.5% average pairwise percent agreement for the anatomical classification group. Conclusions The level of inter- and intraobserver agreement for the morphology of the distal femur was excellent in the new radiological classification system, which was shown to be beneficial in the planning of revision knee arthroplasty for AL. However, there is a need for further studies in order to make a correlation of the classification with specific intraoperative findings.

Observational Study of Re-Biopsy in EGFR-TKI-Resistant Patients with EGFR Mutation-Positive Advanced NSCLC

Identification of acquired resistant mutation has been essential in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) active mutations. Re-biopsy plays a pivotal role to select the optimal treatment for patients who develop resistance to initial EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This multicenter, observational study was conducted to investigate the details of re-biopsy in Japanese clinical practice.The primary endpoints were the implementation rate of re-biopsy and the concordance rate for the T790M mutation detection between histology and cytology specimens using the Cobas ® EGFR mutation test v2. 194 patients with EGFR-mutant NSCLC were enrolled and 120 patients developed acquired resistance to EGFR-TKIs. The median age was 68 years (range 20-87), and 52.5% of the patients were women. Re-biopsy was performed on 109 patients with the implementation rate of re-biopsy was 90.8%. The success rate of re-biopsy in total/histology/cytology/liquid biopsy population was 78.0%, 94.9%, 83.3% and 43.8%, respectively. The positive percent agreement and the negative percent agreement in the detection of T790M mutations between the histology and cytology specimens was both of 90.9%. Aggressive obtaining histological or cytological tissue samples at re-biopsy may contribute to improvement of the detection rate of T790M mutation. (trial registration number: UMIN000026019)

Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach

The surging COVID19 pandemic has underlined the need for quick, sensitive, and high-throughput SARS-CoV-2 detection assays. Although many different methods to detect SARS-CoV-2 particles in clinical material have been developed, none of these assays are successful in combining all three of the above characteristics into a single, easy-to-use method that is suitable for large-scale use. Here we report the development of a direct RT-PCR SARS-CoV-2 detection method that can reliably detect minute quantities of SARS-CoV-2 gRNA in nasopharyngeal swab samples as well as the presence of human genomic DNA. An extraction-less validation protocol was carried out to determine performance characteristics of the assay in both synthetic SARS-CoV-2 RNA as well as clinical specimens. Feasibility of the assay and analytical sensitivity was first determined by testing a dilution series of synthetic SARS-CoV-2 RNA in two different solvents (water and AMIES VTM), revealing a high degree of linearity and robustness in fluorescence readouts. Following analytical performance using synthetic RNA, the limit of detection was determined at equal to or less than 1 SARS-CoV-2 copy/ul of sample in a commercially available sample panel that contains surrogate clinical samples with varying SARS-CoV-2 viral load. Lastly, we benchmarked our method against a reference qPCR method by testing 87 nasopharyngeal swab samples. The direct endpoint ultra-fast RT-PCR method exhibited a positive percent agreement score of 98.5% and a negative percent agreement score of 100% as compared to the reference method, while RT-PCR cycling was completed in 27 minutes/sample as opposed to 60 minutes/sample in the reference qPCR method. In summary, we describe a rapid direct RT-PCR method to detect SARS-CoV-2 material in clinical specimens which can be completed in significantly less time as compared to conventional RT-PCR methods, making it an attractive option for large-scale SARS-CoV-2 screening applications.