Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR.

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots.

Accurate surveillance of coronavirus disease 2019 (COVID-19) incidence requires large-scale testing of the population. Current testing methods require in-person collection of biospecimens by a healthcare worker, limiting access of individuals who do not have access to testing facilities while placing both patients and healthcare workers at risk of exposure to infection. We report the development and validation of a at-home finger-prick dried blood spot collection kit and an analysis method. We demonstrated 100% sensitivity and specificity using at-home collected specimens across the US. Such methods may facilitate the conduct of unbiased serosurveys within hard to reach populations and help reduce the sample collection burden of serological testing on both health care systems and individuals alike.

Sensitive and Specific Detection of SARS-CoV-2 Antibodies Using a High-Throughput, Fully Automated Liquid-Handling Robotic System.

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.

Detection of SARS-CoV-2 neutralizing antibodies with a cell-free PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2-3 hr. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, and to evaluate the impact of various CP processing protocols on CP therapeutic efficacy.

A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots.

Accurate surveillance of coronavirus disease 2019 (COVID-19) incidence requires large-scale testing of the population. Current testing methods require in-person collection of biospecimens by a healthcare worker, limiting access of individuals who do not have access to testing facilities while placing both the patient and healthcare worker at risk of exposure to infection. We report the development and validation of a at-home finger-prick dried blood spot collection kit and an analysis method. We demonstrated 100% sensitivity and specificity using at-home collected specimens across the US. Such methods may facilitate the conduct of unbiased serosurveys within hard to reach populations and help reduce the sample collection burden of serological testing on both health care systems and individuals alike.

Pharmacologic injection treatment of comitant strabismus.

PURPOSE: To report the magnitude and stability of corrections in comitant horizontal strabismus achieved by injecting bupivacaine (BPX, optionally with epinephrine) and botulinum A toxin (BTXA) into extraocular muscles of alert adult subjects with electromyographic (EMG) guidance. METHODS: A total of 55 adults with comitant horizontal strabismus participated in a prospective observational clinical series. Of these, 29 previously had undergone 1 or more unsuccessful strabismus surgeries; 4 had undergone other orbital surgeries. Thirty-one patients with esodeviations received BPX injections in a lateral rectus muscle, some with BTXA in the medial rectus; 24 patients with exodeviations received BPX in a medial rectus muscle, some with BTXA in the lateral rectus muscle. A second treatment (BPX, BTXA, or both) was administered to 27 patients who had residual strabismus after the first treatment. Five patients required additional injections. Clinical alignment was measured at 6 months and yearly thereafter through 5 years' follow-up, with mean follow-up of 28 months. A successful outcome was defined as residual deviation ≤10(Δ). RESULTS: On average, presenting misalignment of 23.8(Δ) (13.4°) was reduced at 28 months by 16.0(Δ) (9.1°), with successful outcomes in 56% of patients. Of patients with initial misalignments ≤25(Δ), 66% had successful outcomes, with corrections averaging 13.2(Δ) (7.5°); of patients with larger misalignments, 40% had successful outcomes, with corrections averaging 20.9(Δ) (11.8°). Corrected alignments were stable over follow-ups as long as 5 years. CONCLUSIONS: Injection treatments resulted in stable, clinically significant corrections in comitant horizontal strabismus. Injection provides a low-cost alternative to incisional strabismus surgery, particularly where it is desirable to minimize surgical anesthesia and avoid extraocular scarring.

Bupivacaine injection remodels extraocular muscles and corrects comitant strabismus.

PURPOSE: To evaluate the clinical effectiveness and anatomic changes resulting from bupivacaine injection into extraocular muscles to treat comitant horizontal strabismus. DESIGN: Prospective, observational clinical series. PARTICIPANTS: Thirty-one comitant horizontal strabismus patients. METHODS: Nineteen patients with esotropia received bupivacaine injections in the lateral rectus muscle, and 12 patients with exotropia received bupivacaine injections in the medial rectus. Sixteen of these, with large strabismus angles, also received botulinum type A toxin injections in the antagonist muscle at the same treatment session. A second treatment was given to 13 patients who had residual strabismus after the first treatment. MAIN OUTCOME MEASURES: Clinical alignment measures and muscle volume, maximum cross-sectional area, and shape derived from magnetic resonance imaging, with follow-up examinations for up to 3 years. RESULTS: At an average of 15.3 months after the final treatment, original misalignment was reduced by 10.5 prism diopters (Δ; 6.0°) with residual deviations of 10Δ or less in 53% of patients. A single treatment with bupivacaine alone reduced misalignment at 11.3 months by 4.7Δ (2.7°) with residual deviations of 10Δ or less in 50% of patients. Alignment corrections were remarkably stable over follow-ups for as long as 3 years. Six months after bupivacaine injection, muscle volume had increased by 6.6%, and maximum cross-sectional area had increased by 8.5%, gradually relaxing toward pretreatment values thereafter. Computer modeling with Orbit 1.8 (Eidactics, San Francisco, CA) suggested that changes in agonist and antagonist muscle lengths were responsible for the enduring changes in eye alignment. CONCLUSIONS: Bupivacaine injection alone or together with botulinum toxin injection in the antagonist muscle improves eye alignment in comitant horizontal strabismus by inducing changes in rectus muscle structure and length.