A narrative review of nine commercial point of care influenza tests: an overview of methods, benefits, and drawbacks to rapid influenza diagnostic testing.

CONTEXT: Rapid influenza diagnostic tests (RIDTs) are becoming increasingly accurate, available, and reliable as the first line of testing when suspecting influenza infections, although the global burden of influenza infections remains high. Rapid diagnosis of influenza infections has been shown to reduce improper or delayed treatment and to increase access to diagnostic measures in public health, primary care, and hospital-based settings. OBJECTIVES: As the use of RIDTs continues to expand in all healthcare settings, there is a multitude of molecular techniques being employed by these various testing platforms. With this in mind, we compare the sensitivity, specificity, and time to diagnosis for nine highly utilized commercial RIDTs. METHODS: Nine commercially available RIDTs were identified from the US Centers for Disease Control and Prevention (CDC) website, which were also referenced on PubMed by name within the title or abstract of peer-reviewed publications examining the sensitivity and specificity of each test against a minimum of three influenza A virus (IAV) strains as well as seasonal influenza B virus (IBV). Data from the peer-reviewed publications and manufacturers' websites were combined to discuss the sensitivity, specify, and time to diagnosis associated with each RIDT. RESULTS: The sensitivity and specificity across the examined RIDTs were greater than 85.0% for both IAV and IBV across all platforms, with the reverse transcriptase-polymerase chain reaction (RT-PCR) assays maintaining sensitivity and specificity greater than 95.0% for all viruses tested. However, the RT-PCR platforms were the longest in time to diagnosis when compared to the other molecular methods utilized in the examined RIDTs. CONCLUSIONS: Herein, we discussed the benefits and limitations of nine commercially available RIDTs and the molecular techniques upon which they are based, showing the relative accuracy and speed of each test for IAV and IBV detection as reported by the peer-reviewed literature and commercial manufacturers.

A Novel PCR-Based Methodology for Viral Detection Utilizing Mechanical Homogenization.

The impact of viral diseases on human health is becoming increasingly prevalent globally with the burden of disease being shared between resource-rich and poor areas. As seen in the global pandemic caused by SARS-CoV-2, there is a need to establish viral detection techniques applicable to resource-limited areas that provide sensitive and specific testing with a logistically conscious mindset. Herein, we describe a direct-to-PCR technology utilizing mechanical homogenization prior to viral PCR detection, which allows the user to bypass traditional RNA extraction techniques for accurate detection of human coronavirus. This methodology was validated in vitro, utilizing human coronavirus 229E (HCoV-229E), and then clinically, utilizing patient samples to test for SARS-CoV-2 infection. In this manuscript, we describe in detail the protocol utilized to determine the limit of detection for this methodology with in vitro testing of HCoV-229E.

A Rare Variant of Guillain-Barre Syndrome Following Ad26.COV2.S Vaccination.

Efforts to combat the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) range from adequate diagnostic testing and contract tracing to vaccination for the prevention of coronavirus disease 2019 (COVID-19). In the United States alone, three vaccinations have been authorized for emergency use (EUA) or approved to prevent COVID-19. The Ad26.COV2.S vaccine by Johnson and Johnson (New Brunswick, New Jersey) is the only adenovirus-based vaccine and deemed relatively effective and safe by the US Food and Drug Administration (FDA) following its clinical trial. Since its introduction, the US FDA has placed a warning on the vaccine adverse event reporting system (VAERS) after more than 100 cases of Guillain-Barre Syndrome (GBS) were reported. Herein, we outline the hospital course of a generally healthy 49-year-old female who experienced an axonal form of GBS nine days after receiving the Ad26.COV2.S vaccine.

Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: A model for reducing resources needed for accurate testing.

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.

A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E.

BACKGROUND: Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps. METHODS: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 × 106 to 1.2 × 101 copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 µL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. RESULTS: HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 103 viral copies/mL with 96.30% sensitivity. CONCLUSION: We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.

Identification of Non-Nucleoside Inhibitors of the Respiratory Syncytial Virus Polymerase Complex.

Respiratory syncytial virus (RSV) represents a threat to infants, the elderly, and the immunocompromised. RSV entry blockers are in clinical trials, but escape mutations challenge their potential. In search of RSV inhibitors, we have integrated a signature resistance mutation into a recombinant RSV virus and applied the strain to high-throughput screening. Counterscreening of candidates returned 14 confirmed hits with activities in the nano- to low-micromolar range. All blocked RSV polymerase activity in minigenome assays. Compound 1a (GRP-74915) was selected for development based on activity (EC50 = 0.21 µM, selectivity index (SI) 40) and scaffold. Resynthesis confirmed the potency of the compound, which suppressed viral RNA synthesis in infected cells. However, metabolic testing revealed a short half-life in the presence of mouse hepatocyte fractions. Metabolite tracking and chemical elaboration combined with 3D-quantitative structure-activity relationship modeling yielded analogues (i.e., 8n: EC50 = 0.06 µM, SI 500) that establish a platform for the development of a therapeutic candidate.

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening.

UNLABELLED: Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE: This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.