Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Motivation: The motivation for this work was the need to establish a predefined cutoff based on genome copies per ml (GE/ml) rather than Ct, which can vary depending on the laboratory and assay used. A GE/ml-based threshold was necessary to define what constituted ‘low positives” for samples that were included in data sets submitted to the FDA for emergency use approval for SARS-CoV-2 antigen tests. Summary: SARS-CoV-2, the causal agent of the global COVID-19 pandemic, made its appearance at the end of 2019 and is still circulating in the population. The pandemic led to an urgent need for fast, reliable, and widely available testing. After December 2020, the emergence of new variants of SARS-CoV-2 led to additional challenges since new and existing tests had to detect variants to the same extent as the original Wuhan strain. When an antigen-based test is submitted to the Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) consideration it is benchmarked against PCR comparator assays, which yield cycle threshold (CT) data as an indirect indicator of viral load – the lower the CT, the higher the viral load of the sample and the higher the CT, the lower the viral load. The FDA mandates that 10–20% of clinical samples used to evaluate the antigen test have to be low positive. Low positive, as defined by the FDA, are clinical samples in which the CT of the SARS-CoV-2 target gene is within 3 CT of the mean CT value of the approved comparator test’s Limit of Detection (LOD). While all comparator tests are PCR-based, the results from different PCR assays used are not uniform. Results vary depending on assay platform, target gene, LOD and laboratory methodology. The emergence and dominance of the Omicron variant further challenged this approach as the fraction of low positive clinical samples dramatically increased as compared to earlier SARS-CoV-2 variants. This led to 20–40% of clinical samples having high CT values and therefore assays vying for an EUA were failing to achieve the 80% Percent Positive Agreement (PPA) threshold required. Here we describe the methods and statistical analyses used to establish a predefined cutoff, based on genome copies per ml (GE/ml) to classify samples as low positive (less than the cutoff GE/ml) or high positive (greater than the cutoff GE/mL). CT 30 for the E gene target using Cobas® SARS-CoV-2-FluA/B platform performed at TriCore Reference Laboratories, and this low positive cutoff value was used for two EUA authorizations. Using droplet digital PCR and methods described here, a value 49,447.72 was determined as the GE/ml equivalent for the low positive cutoff. The CT cutoff corresponding to 49447.72 GE/ml was determined across other platforms and laboratories. The methodology and statistical analysis described here can now be used for standardization of all comparators used for FDA submissions with a goal towards establishing uniform criteria for EUA authorization.

Introduction: Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described. Methods: We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens. Results: We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit. Conclusion: Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.

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.

Primary bile acid malabsorption is associated with congenital diarrhea, steatorrhea, and a block in the intestinal return of bile acids in the enterohepatic circulation. Mutations in the ileal apical sodium-dependent bile acid transporter (ASBT; SLC10A2) can cause primary bile acid malabsorption but do not appear to account for most familial cases. Another major transporter involved in the intestinal reclamation of bile acids is the heteromeric organic solute transporter alpha-beta (OSTα-OSTβ; SLC51A-SLC51B), which exports bile acid across the basolateral membrane. Here we report the first patients with OSTβ deficiency, clinically characterized by chronic diarrhea, severe fat soluble vitamin deficiency, and features of cholestatic liver disease including elevated serum gamma-glutamyltransferase activity. Whole exome sequencing revealed a homozygous single nucleotide deletion in codon 27 of SLC51B, resulting in a frameshift and premature termination at codon 50. Functional studies in transfected cells showed that the SLC51B mutation resulted in markedly reduced taurocholic acid uptake activity and reduced expression of the OSTα partner protein. Conclusion: The findings identify OSTβ deficiency as a cause of congenital chronic diarrhea with features of cholestatic liver disease. These studies underscore OSTα-OSTβ's key role in the enterohepatic circulation of bile acids in humans.

Background & Aims: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-β (OSTα-OSTβ). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/-mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. Methods: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/-mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/-mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. Results: As early as day 5, Ostα-/-mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid–activated farnesoid X receptor target genes in neonatal Ostα-/-mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2–anti-oxidant responsive genes were increased significantly in neonatal Ostα-/-mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid–induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/-mice. Conclusions: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostβ functions to protect the ileal epithelium against bile acid–induced injury. NCBI Gene Expression Omnibus: GSE99579.

The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasis and after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver. The transcriptional landscape of the epithelium in healthy and regenerating murine livers was investigated, revealing a dynamically fluctuating and heterogeneous YAP transcriptional program. Further analysis uncovered YAP signaling dualism: it is essential in biliary epithelial cells for homeostatic maintenance and in hepatocytes for the regenerative response to injury.

Abstract: Non-alcoholic fatty liver disease (NAFLD) is a major growing worldwide health problem. We previously reported that interruption of the enterohepatic circulation of bile acids using a non-absorbable apical sodium-dependent bile acid transporter inhibitor (ASBTi; SC-435) reduced the development of NAFLD in high fat diet fed mice. However, the ability of ASBTi treatment to impact the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and fibrosis in a diet-induced mouse model remains untested. In the current study, we assessed whether ASBTi treatment is hepatoprotective in the choline-deficient, L-amino acid-defined (CDAA) diet model of NASH-induced fibrosis. Methods: Male C57Bl/6 mice were fed with: (A) choline-sufficient L-amino acid-defined diet (CSAA) (31 kcal% fat), (B) CSAA diet plus ASBTi (SC-435; 60 ppm), (C) CDAA diet, or (D) CDAA diet plus ASBTi. Body weight and food intake were monitored. After 22 weeks on diet, liver histology, cholesterol and triglyceride levels, and gene expression were measured. Fecal bile acid and fat excretion were measured, and intestinal fat absorption was determined using the sucrose polybehenate method. Results: ASBTi treatment reduced bodyweight gain in mice fed either the CSAA or CDAA diet, and prevented the increase in liver to body weight ratio observed in CDAA-fed mice. ASBTi significantly reduced hepatic total cholesterol levels in both CSAA and CDAA-fed mice. ASBTi-associated significant reductions in hepatic triglyceride levels and histological scoring for NAFLD activity were observed in CSAA but not CDAA-fed mice. These changes correlated with measurements of intestinal fat absorption, which was significantly reduced in ASBTi-treated mice fed the CSAA (85 vs. 94%, P < 0.001) but not CDAA diet (93 vs. 93%). As scored by Ishak staging of Sirius red stained liver sections, no hepatic fibrosis was evident in the CSAA diet mice. The CDAA diet-fed mice developed hepatic fibrosis, which was increased by the ASBTi. Conclusions: ASBT inhibition reduced intestinal fat absorption, bodyweight gain and hepatic steatosis in CSAA diet-fed mice. The effects of the ASBTi on steatosis and fat absorption were attenuated in the context of dietary choline-deficiency. Inhibition of intestinal absorption of fatty acids may be involved in the therapeutic effects of ASBTi treatment.

The pronounced choleretic properties of 24-norUrsodeoxycholic acid (norUDCA) to induce bicarbonate-rich bile secretion have been attributed to its ability to undergo cholehepatic shunting. The goal of this study was to identify the mechanisms underlying the choleretic actions of norUDCA and the role of the bile acid transporters. Here, we show that the apical sodium-dependent bile acid transporter (ASBT), organic solute transporter-α (OSTα), and organic anion transporting polypeptide 1a/1b (OATP1a/1b) transporters are dispensable for the norUDCA stimulation of bile flow and biliary bicarbonate secretion. Chloride channels in biliary epithelial cells provide the driving force for biliary secretion. In mouse large cholangiocytes, norUDCA potently stimulated chloride currents that were blocked by siRNA silencing and pharmacological inhibition of calciumactivated chloride channel transmembrane member 16A (TMEM16A) but unaffected by ASBT inhibition. In agreement, blocking intestinal bile acid reabsorption by coadministration of an ASBT inhibitor or bile acid sequestrant did not impact norUDCA stimulation of bile flow in WT mice. The results indicate that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (C T ) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.