Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Relationship between distension-contraction waveforms during esophageal peristalsis: effect of bolus volume, viscosity, and posture.

High-resolution esophageal manometry (HRM) in its current form assesses only the contraction phase of peristalsis. Degree of esophageal distension ahead of contraction is a surrogate of relaxation and can be measured from intraluminal esophageal impedance measurements. The characteristics of esophageal contractions, i.e., their amplitude, duration, velocity, and modulating factors, have been well studied. We studied the effect of bolus volume and viscosity and posture on swallow-induced distension and contraction and the temporal relationship between the two. HRM impedance recordings of 50 healthy subjects with no esophageal symptoms were analyzed. Eight to ten swallows of 5 and 10 mL of 0.5 N saline and a viscous bolus were recorded in the supine and Trendelenburg positions. Custom-built computer software generated the distension-contraction plots and numerical data of the amplitudes of distension (cross-sectional area) and contraction, and the temporal relationship between distension and peak contraction. The hallmarks of distension waveforms are that 1) distension peak, similarly to contraction, travels the esophagus in a peristaltic fashion, and the amplitude of distension increases from the proximal-to-distal direction; 2) the amplitude of distension is greater with 10 mL than with 5 mL and greater in Trendelenburg than in supine posture; and 3) bolus viscosity increases the amplitude of distension and alters the temporal relationship between distension and contraction waveforms. We describe the characteristics of esophageal distension during peristalsis and the relationship between distension and contraction in a relatively large cohort of normal subjects. These data can be used to compare differences between normal subjects and patients with various esophageal motility disorders in future studies.NEW & NOTEWORTHY We studied esophageal distension (surrogate of inhibition) ahead of contraction during peristalsis from intraluminal esophageal impedance measurements. Esophageal distension, similarly to contraction, travels the esophagus in a sequential manner, and the amplitude of esophageal distension increases from proximal to distal direction in the esophagus. Bolus volume, viscosity and posture have significant effects on the amplitude of distension and its temporal relationship with contraction.

High-frequency ultrasound imaging of the anal sphincter muscles in normal subjects and patients with fecal incontinence.

INTRODUCTION: The current "gold standard" to diagnose anal sphincter morphology and disruptions utilizes low-frequency (3-9 MHz) ultrasound (US) imaging techniques that provide a general outline of the sphincter muscles, but not their microstructural details. High-frequency US transducers (7-15 MHz) have been used to study the muscle architecture (direction of muscle fascicles) in the limb muscle. AIMS: The goal of our study was to visualize the microstructural anatomy of the anal sphincters, specifically the external anal sphincter (EAS), using high-frequency US imaging. METHODS: Studies were conducted in asymptomatic female and male subjects and patients with fecal incontinence. US images were acquired using a low-frequency US (3-9 MHz) and high-frequency (7-15 MHz) US transducer. The latter was placed intra-anally to image the anal canal at 12, 9, 3, and 6 o'clock positions. RESULTS: The low-frequency US images revealed the general outline of the anal sphincter muscles. On the other hand, high-frequency imaging visualized muscle fascicles and connective tissue inside the external anal sphincter (EAS). In FI patients, there was loss of muscle fascicles and alteration in the echo-intensity pattern in the region of damaged EAS suggestive of muscle fibrosis. CONCLUSION: High-frequency ultrasound imaging is a powerful tool to visualize the microstructural details of the EAS. Our studies show that damage to the EAS muscle results in the alteration of its myoarchitecture, that is, loss of muscle fascicles and increase in the muscle connective tissue.

Topographical plots of esophageal distension and contraction: effects of posture on esophageal peristalsis and bolus transport.

Each swallow induces a wave of inhibition followed by contraction in the esophagus. Unlike contraction, which can easily be measured in humans using high-resolution manometry (HRM), inhibition is difficult to measure. Luminal distension is a surrogate of the esophageal inhibition. The aim of this study was to determine the effect of posture on the temporal and quantitative relationship between distension and contraction along the entire length of the esophagus in normal healthy subjects by using concurrent HRM, HRM impedance (HRMZ), and intraluminal ultrasound (US). Studies were conducted in 15 normal healthy subjects in the supine and Trendelenburg positions. Both manual and automated methods were used to extract quantitative pressure and impedance-derived features from the HRMZ recordings. Topographical plots of distension and contraction were visualized along the entire length of the esophagus. Distension was also measured from the US images during 10-ml swallows at 5 cm above the lower esophageal sphincter. Each swallow was associated with luminal distension followed by contraction, both of which traversed the esophagus in a sequential/peristaltic fashion. Luminal distension (US) and esophageal contraction amplitude were greater in the Trendelenburg compared with the supine position. Length of esophageal breaks (in the transition zone) were reduced in the Trendelenburg position. Change in posture altered the temporal relationship between distension and contraction, and bolus traveled closer to the esophageal contraction in the Trendelenburg position. Topographical contraction-distension plots derived from HRMZ recordings is a novel way to visualize esophageal peristalsis. Future studies should investigate if abnormalities of esophageal distension are the cause of functional dysphagia. NEW & NOTEWORTHY Ascending contraction and descending inhibition are two important components of peristalsis. High-resolution manometry only measures the contraction phase of peristalsis. We measured esophageal distension from intraluminal impedance recordings and developed novel contraction-distension topographical plots to prove that similar to contraction, distension also travels in a peristaltic fashion. Change in posture from the supine to the Trendelenburg position also increased the amplitude of esophageal distension and contraction and altered the temporal relationship between distension and contraction.

Connectivity of the Superficial Muscles of the Human Perineum: A Diffusion Tensor Imaging-Based Global Tractography Study.

Despite the importance of pelvic floor muscles, significant controversy still exists about the true structural details of these muscles. We provide an objective analysis of the architecture and orientation of the superficial muscles of the perineum using a novel approach. Magnetic Resonance Diffusion Tensor Images (MR-DTI) were acquired in 10 healthy asymptomatic nulliparous women, and 4 healthy males. Global tractography was then used to generate the architecture of the muscles. Micro-CT imaging of a male cadaver was performed for validation of the fiber tracking results. Results show that muscles fibers of the external anal sphincter, from the right and left side, cross midline in the region of the perineal body to continue as transverse perinea and bulbospongiosus muscles of the opposite side. The morphology of the external anal sphincter resembles that of the number '8' or a "purse string". The crossing of muscle fascicles in the perineal body was supported by micro-CT imaging in the male subject. The superficial muscles of the perineum, and external anal sphincter are frequently damaged during child birth related injuries to the pelvic floor; we propose the use of MR-DTI based global tractography as a non-invasive imaging technique to assess damage to these muscles.

Functional morphology of the lower esophageal sphincter and crural diaphragm determined by three-dimensional high-resolution esophago-gastric junction pressure profile and CT imaging.

The smooth muscles of the lower esophageal sphincter (LES) and skeletal muscles of the crural diaphragm (CD) provide a closure/antireflux barrier mechanism at the esophago-gastric junction (EGJ). A number of questions in regard to the pressure profile of the LES and CD remain unclear, e.g., 1) Why is the LES pressure profile circumferentially asymmetric, 2) Is the crural diaphragm (CD) contraction also circumferentially asymmetric, and 3) Where is the LES and CD pressure profile located in the anatomy of the esophagus and stomach? The three-dimensional (3-D) high-resolution esophageal manometry (HRM) catheter can record a detailed profile of the EGJ pressure; however, it does not allow the determination of the circumferential orientation of individual pressure transducers in vivo. We used computed tomography (CT) scan imaging in combination with 3-D EGJ pressure recordings to determine the functional morphology of the LES and CD and its relationship to the EGJ anatomy. A 3-D-HRM catheter with 96 transducers (12 rings, 7.5 mm apart, located over 9-cm length of the catheter, with eight transducers in each ring, 45° apart (Medtronics), was used to record the EGJ pressure in 10 healthy subjects. A 0.5-mm diameter metal ball (BB) was taped to the catheter, adjacent to transducer 1 of the catheter. The EGJ was recorded under the following conditions: 1) end-expiration (LES pressure) before swallow, after swallow, and after edrophonium hydrochloride; and 2) peak inspiration (crural diaphragm contraction) for tidal inspiration and forced maximal inspiration. A CT scan was performed to localize the circumferential orientation of the BB. The CT scan imaging allowed the determination of the circumferential orientation of the LES and CD pressure profiles. The LES pressure under the three end-expiration conditions were different; however, the shape of the pressure profile was unique with the LES length longer toward the lesser curvature of the stomach as compared with the greater curvature. The pressure profile revealed circular and axial pressure asymmetry, with greatest pressure and shortest cranio-caudal length on the left (close to the angle of His). The CD contraction with tidal and forced inspiration increases pressure in the cranial half of the LES pressure profile, and it was placed horizontally across the recording. The CD, esophagus, and stomach were outlined in the CT scan images to construct a 3-D anatomy of the region; it revealed that the hiatus (CD) is placed obliquely across the esophagus; however, because of the bend of the esophagus to the left at the upper edge of the hiatus, the two were placed at right angle to each other, which resulted in a horizontal pressure profile of the CD on the LES. Our observations suggest a unique shape of the LES, CD, and the anatomical relationship between the two, which provides a possible explanation as to why the LES pressure shows circumferential and axial asymmetry. Our findings have implication for the length and circumferential orientation of myotomy incision required for the ablation of LES pressure in achalasia esophagus.NEW & NOTEWORTHY We used computed tomography scan imaging with three-dimensional esophago-gastric junction (EGJ) pressure recordings to determine functional morphology of the lower esophageal sphincter (LES) and crural diaphragm and its relationship to EGJ anatomy. The LES pressure profile was unique with the LES length longer and pressures lower toward the lesser curvature of the stomach, as compared with the greater curvature. Our findings have implications for the length and circumferential orientation of myotomy incision required for the ablation of LES pressure in the achalasia esophagus.

A Predictive Model to Identify Patients With Fecal Incontinence Based on High-Definition Anorectal Manometry.

BACKGROUND & AIMS: Three-dimensional high-definition anorectal manometry (3D-HDAM) is used to assess anal sphincter function; it determines profiles of regional pressure distribution along the length and circumference of the anal canal. There is no consensus, however, on the best way to analyze data from 3D-HDAM to distinguish healthy individuals from persons with sphincter dysfunction. We developed a computer analysis system to analyze 3D-HDAM data and to aid in the diagnosis and assessment of patients with fecal incontinence (FI). METHODS: In a prospective study, we performed 3D-HDAM analysis of 24 asymptomatic healthy subjects (control subjects; all women; mean age, 39 ± 10 years) and 24 patients with symptoms of FI (all women; mean age, 58 ± 13 years). Patients completed a standardized questionnaire (FI severity index) to score the severity of FI symptoms. We developed and evaluated a robust prediction model to distinguish patients with FI from control subjects using linear discriminant, quadratic discriminant, and logistic regression analyses. In addition to collecting pressure information from the HDAM data, we assessed regional features based on shape characteristics and the anal sphincter pressure symmetry index. RESULTS: The combination of pressure values, anal sphincter area, and reflective symmetry values was identified in patients with FI versus control subjects with an area under the curve value of 1.0. In logistic regression analyses using different predictors, the model identified patients with FI with an area under the curve value of 0.96 (interquartile range, 0.22). In discriminant analysis, results were classified with a minimum error of 0.02, calculated using 10-fold cross-validation; different combinations of predictors produced median classification errors of 0.16 in linear discriminant analysis (interquartile range, 0.25) and 0.08 in quadratic discriminant analysis (interquartile range, 0.25). CONCLUSIONS: We developed and validated a novel prediction model to analyze 3D-HDAM data. This system can accurately distinguish patients with FI from control subjects.

Esophageal contractions in type 3 achalasia esophagus: simultaneous or peristaltic?

Absence of peristalsis and impaired relaxation of lower esophageal sphincter are the hallmarks of achalasia esophagus. Based on the pressurization patterns, achalasia has been subdivided into three subtypes. The goal of our study was to evaluate the esophageal contraction pattern and bolus clearance in type 3 achalasia esophagus. High-resolution manometry (HRM) recordings of all patients diagnosed with achalasia esophagus in our center between the years 2011 and 2013 were reviewed. Recordings of 36 patients with type 3 achalasia were analyzed for the characteristics of swallow-induced "simultaneous esophageal contraction." The HRM impedance recordings of 14 additional patients with type 3 achalasia were analyzed for bolus clearance from the impedance recording. Finally, the HRM impedance along with intraluminal ultrasound imaging was conducted in six patients to further characterize the simultaneous esophageal contractions. Among 187 achalasia patients, 30 were type 1, 121 type 2, and 36 type 3. A total of 434 swallows evaluated in type 3 achalasia patients revealed that 95% of the swallow-induced contractions met criteria for simultaneous esophageal contraction, based on the onset of contraction. Interestingly, the peak and termination of the majority of simultaneous esophageal contractions were sequential. The HRM impedance revealed that 94% of the "simultaneous contractions" were associated with complete bolus clearance. Ultrasound image analysis revealed that baseline muscle thickness of patients in type 3 achalasia is larger than normal but the pattern of axial shortening is similar to that in normal subjects. The majority of esophageal contractions in type 3 achalasia are not true simultaneous contractions because the peak and termination of contraction are sequential and they are associated with complete bolus clearance.