Prevalence of SARS-CoV-2 in hemoglobinopathies is modified by age and race.

Prior literature has established a positive association between sickle cell disease and risk of contracting SARS-CoV-2. Data from a cross-sectional study evaluating COVID-19 testing devices (n = 10,567) was used to examine the association between underlying health conditions and SARS-CoV-2 infection in an urban metropolis in the southern United States. Firth's logistic regression was used to fit the model predicting SARS-CoV-2 positivity using vaccine status and different medical conditions commonly associated with COVID-19. Another model using the same method was built using SARS-CoV-2 positivity as the outcome and hemoglobinopathy presence, age (<16 Years vs. ≥16 Years), race/ethnicity and comorbidities, including hemoglobinopathy, as the factors. Our first model showed a significant association between hemoglobinopathy and SARS-CoV-2 infection (OR: 2.28, 95 % CI: (1.17,4.35), P = 0.016). However, in the second model, this association was not maintained (OR: 1.35, 95 % CI: (0.72,2.50), P = 0.344). We conclude that the association between SARS-CoV-2 positivity and presence of hemoglobinopathies like sickle cell disease is confounded by race, age, and comorbidity status. Our results illuminate previous findings by identifying underlying clinical/demographic factors that confound the reported association between hemoglobinopathies and SARS-CoV-2. These findings demonstrate how social determinants of health may influence disease manifestations more than genetics alone.

Investigation of Blood Plasma Viral Nucleocapsid Antigen as a Marker of Active Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Infection.

Background: Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. Methods: We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Results: Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Conclusions: Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.

Pivot, persevere, or perish: how Ellume Health overcame development and regulatory obstacles to become the first authorized over-the-counter COVID-19 test in the United States.

The Ellume COVID-19 home test from Ellume Health (Brisbane, Aus) became the first COVID-19 diagnostic tool authorized for home use by the United States FDA in December 2020. This early pandemic success was built on over ten years of work on the Ellume influenza home test. Ellume overcame critical technology challenges during the development of their influenza test. In addition, it faced a recall of its COVID-19 home test in 2021 due to false positive results. While Ellume initially persevered through the recall and restarted sales in the United States, the combination of the recall and the wide availability of competitors' low-cost over the counter tests in the United States led to Ellume entering voluntary administration in September 2022. This paper traces the history of Ellume and how 10 years of experience with a home influenza test allowed the company to quickly develop the Ellume COVID-19 home test. We will also provide to diagnostic developers the key strategies employed by Ellume to succeed while highlighting the pitfalls that have challenged the company's business success.

SARS-CoV-2 molecular testing and whole genome sequencing following RNA recovery from used BinaxNOW COVID-19 antigen self tests.

Widespread use of over-the-counter rapid diagnostic tests for SARS-CoV-2 has led to a decrease in availability of clinical samples for viral genomic surveillance. As an alternative sample source, we evaluated RNA isolated from BinaxNOW swabs stored at ambient temperature for SARS-CoV-2 rRT-PCR and full viral genome sequencing. 81 of 103 samples (78.6%) yielded detectable RNA, and 46 of 57 samples (80.7 %) yielded complete genome sequences. Our results illustrate that SARS-CoV-2 RNA extracted from used Binax test swabs provides an important opportunity for improving SARS-CoV-2 genomic surveillance, evaluating transmission clusters, and monitoring within-patient evolution.

The critical role of engineering in the rapid development of COVID-19 diagnostics: Lessons from the RADx Tech Test Verification Core.

Engineering plays a critical role in the development of medical devices, and this has been magnified since 2020 as severe acute respiratory syndrome coronavirus 2 swept over the globe. In response to the coronavirus disease 2019, the National Institutes of Health launched the Rapid Acceleration of Diagnostics (RADx) initiative to help meet the testing needs of the United States and effectively manage the pandemic. As the Engineering and Human Factors team for the RADx Tech Test Verification Core, we directly assessed more than 30 technologies that ultimately contributed to an increase of the country's total testing capacity by 1.7 billion tests to date. In this review, we present central lessons learned from this "apples-to-apples" comparison of novel, rapidly developed diagnostic devices. Overall, the evaluation framework and lessons learned presented in this review may serve as a blueprint for engineers developing point-of-care diagnostics, leaving us better prepared to respond to the next global public health crisis rapidly and effectively.

SARS-CoV-2 molecular testing and whole genome sequencing following RNA recovery from used BinaxNOW COVID-19 Antigen Self Tests

Widespread use of over-the-counter rapid diagnostic tests for SARS-CoV-2 has led to a decrease in availability of clinical samples for viral genomic surveillance. As an alternative sample source, we evaluated RNA isolated from BinaxNOW swabs stored at ambient temperature for SARS-CoV-2 rRT-PCR and full viral genome sequencing. 81 of 103 samples (78.6%) yielded detectable RNA, and 46 of 57 samples (80.7 %) yielded complete genome sequences. Our results illustrate that SARS-CoV-2 RNA extracted from used Binax test swabs provides an important opportunity for improving SARS-CoV-2 genomic surveillance, evaluating transmission clusters, and monitoring within-patient evolution.

Multiplatform analyses reveal distinct drivers of systemic pathogenesis in adult versus pediatric severe acute COVID-19.

The pathogenesis of multi-organ dysfunction associated with severe acute SARS-CoV-2 infection remains poorly understood. Endothelial damage and microvascular thrombosis have been identified as drivers of COVID-19 severity, yet the mechanisms underlying these processes remain elusive. Here we show alterations in fluid shear stress-responsive pathways in critically ill COVID-19 adults as compared to non-COVID critically ill adults using a multiomics approach. Mechanistic in-vitro studies, using microvasculature-on-chip devices, reveal that plasma from critically ill COVID-19 adults induces fibrinogen-dependent red blood cell aggregation that mechanically damages the microvascular glycocalyx. This mechanism appears unique to COVID-19, as plasma from non-COVID sepsis patients demonstrates greater red blood cell membrane stiffness but induces less significant alterations in overall blood rheology. Multiomics analyses in pediatric patients with acute COVID-19 or the post-infectious multi-inflammatory syndrome in children (MIS-C) demonstrate little overlap in plasma cytokine and metabolite changes compared to adult COVID-19 patients. Instead, pediatric acute COVID-19 and MIS-C patients show alterations strongly associated with cytokine upregulation. These findings link high fibrinogen and red blood cell aggregation with endotheliopathy in adult COVID-19 patients and highlight differences in the key mediators of pathogenesis between adult and pediatric populations.

Hyperviscosity syndromes; hemorheology for physicians and the use of microfluidic devices.

PURPOSE OF REVIEW: Hyperviscosity syndromes can lead to significant morbidity and mortality. Existing methods to measure microcirculatory rheology are not readily available and limited in relevance and accuracy at this level. In this review, we review selected hyperviscosity syndromes and the advancement of their knowledge using microfluidic platforms. RECENT FINDINGS: Viscosity changes drastically at the microvascular level as the physical properties of the cells themselves become the major determinants of resistance to blood flow. Current, outdated viscosity measurements only quantify whole blood or serum. Changes in blood composition, cell number, or the physical properties themselves lead to increased blood viscosity. Given the significant morbidity and mortality from hyperviscosity syndromes, new biophysical tools are needed and being developed to study microvascular biophysical and hemodynamic conditions at this microvascular level to help predict those at risk and guide therapeutic treatment. SUMMARY: The use of 'lab-on-a-chip' technology continues to rise to relevance with point of care, personalized testing and medicine as customizable microfluidic platforms enable independent control of many in vivo factors and are a powerful tool to study microcirculatory hemorheology.

Subgenomic RNA Abundance Relative to Total Viral RNA Among Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate actively replicating virus, but sgRNA abundance has not been systematically compared between SARS-CoV-2 variants. sgRNA was quantified in 169 clinical samples by real-time reverse-transcription polymerase chain reaction, demonstrating similar relative abundance among known variants. Thus, sgRNA detection can identify individuals with active viral replication regardless of variant.

SARS-CoV-2 Antigenemia is Associated With Pneumonia in Children But Lacks Sensitivity to Diagnose Acute Infection.

BACKGROUND: Nucleocapsid antigenemia in adults has demonstrated high sensitivity and specificity for acute infection, and antigen burden is associated with disease severity. Data regarding SARS-CoV-2 antigenemia in children are limited. METHODS: We retrospectively analyzed blood plasma specimens from hospitalized children with COVID-19 or MIS-C. Nucleocapsid and spike were measured using ultrasensitive immunoassays. RESULTS: We detected nucleocapsid antigenemia in 62% (50/81) and spike antigenemia in 27% (21/79) of children with acute COVID-19 but 0% (0/26) and 15% (4/26) with MIS-C from March 2020-March 2021. Higher nucleocapsid levels were associated with radiographic infiltrates and respiratory symptoms in children with COVID-19. CONCLUSIONS: Antigenemia lacks the sensitivity to diagnose acute infection in children but is associated with signs and symptoms of lower respiratory tract involvement. Further study into the mechanism of antigenemia, its association with specific organ involvement, and the role of antigenemia in the pathogenesis of COVID-19 is warranted.

Clinical evaluation of the Diagnostic Analyzer for Selective Hybridization (DASH): A point-of-care PCR test for rapid detection of SARS-CoV-2 infection.

BACKGROUND: An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing. We evaluated clinical performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC rapid PCR test. METHODS: We conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites where we collected two bilateral anterior nasal swabs and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the "gold standard". RESULTS: We enrolled 315 and analyzed 313 participants with median age 42 years; 65% were female, 62% symptomatic, 75% had received ≥2 doses of mRNA COVID-19 vaccine, and 16% currently SARS-CoV-2 positive. There were concordant results for 307 tests indicating an overall agreement for DASH of 0.98 [95% CI 0.96, 0.99] compared to Xpert. DASH performed at 0.96 [95% CI 0.86, 1.00] sensitivity and 0.98 [95% CI 0.96, 1.00] specificity, with a positive predictive value of 0.85 [95% CI 0.73, 0.96] and negative predictive value of 0.996 [95% CI 0.99, 1.00]. The six discordant tests between DASH and Xpert all had high Ct values (>30) on the respective positive assay. DASH and Xpert Ct values were highly correlated (R = 0.89 [95% CI 0.81, 0.94]). CONCLUSIONS: DASH POC SARS-CoV-2 PCR was accurate, easy to use, and provided fast results (approximately 15 minutes) in real-life clinical settings with an overall performance similar to an EUA-approved laboratory-based PCR.

Nucleocapsid Antigenemia in Patients Receiving Anti-CD20 Therapy With Protracted COVID-19.

Immunocompromised patients with prolonged coronavirus disease 2019 symptoms present diagnostic and therapeutic challenges. We measured viral nucleocapsid antigenemia in 3 patients treated with anti-CD20 immunotherapy who acquired severe acute respiratory syndrome coronavirus 2 infection and experienced protracted symptoms. Our results support nucleocapsid antigenemia as a marker of persistent infection and therapeutic response.

Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

SARS-CoV-2 reliably detected in frozen saliva samples stored up to one year.

Viability of saliva samples stored for longer than 28 days has not been reported in the literature. The COVID-19 pandemic has spawned new research evaluating various sample types, thus large biobanks have been started. Residual saliva samples from university student surveillance testing were retested on SalivaDirect and compared with original RT-PCR (cycle threshold values) and quantitative antigen values for each month in storage. We conclude that saliva samples stored at -80°C are still viable in detecting SARS-CoV-2 after 12 months of storage, establishing the validity of these samples for future testing.

Nucleocapsid Antigenemia Is a Marker of Acute SARS-CoV-2 Infection.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19.

Don't forget about human factors: Lessons learned from COVID-19 point-of-care testing.

During the COVID-19 pandemic, the development of point-of-care (POC) diagnostic testing accelerated in an unparalleled fashion. As a result, there has been an increased need for accurate, robust, and easy-to-use POC testing in a variety of non-traditional settings (i.e., pharmacies, drive-thru sites, schools). While stakeholders often express the desire for POC technologies that are "as simple as digital pregnancy tests," there is little discussion of what this means in regards to device design, development, and assessment. The design of POC technologies and systems should take into account the capabilities and limitations of the users and their environments. Such "human factors" are important tenets that can help technology developers create POC technologies that are effective for end-users in a multitude of settings. Here, we review the core principles of human factors and discuss lessons learned during the evaluation process of SARS-CoV-2 POC testing.

Designing for simplicity: lessons from Mesa Biotech for microfluidic entrepreneurs and early-stage companies.

The COVID-19 pandemic has proven the need for point-of-care diagnosis of respiratory diseases and microfluidic technology has risen to the occasion. Mesa Biotech (San Diego, CA) originally developed the Accula platform for the diagnosis of influenza A and B and then extended the platform to SARS-CoV-2. Mesa Biotech has experienced tremendous success, culminating in acquisition by Thermo Fisher for up to $550m USD. The Accula microfluidics platform accomplished the leap from the lab to commercial product through clever design and engineering choices. Through information obtained from interviews with key Mesa Biotech leaders and publicly-available documents, we describe the keys to Mesa's success and how they might inform other lab-on-a-chip companies.

Assessment of the Abbott BinaxNOW SARS-CoV-2 rapid antigen test against viral variants of concern.

As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here, we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (B.1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.