Unique immune and inflammatory cytokine profiles may define long COVID syndrome.

PURPOSE: Long COVID is estimated to occur in 5-10% of individuals after acute SARS-CoV-2 infection. However, the pathophysiology driving the disease process is poorly understood. METHODS: We evaluated urine and plasma inflammatory and immune cytokine profiles in 33 individuals with long COVID compared to 33 who were asymptomatic and recovered, and 34 without prior infection. RESULTS: Mean urinary leukotriene E4 was significantly elevated among individuals with long COVID compared to asymptomatic and recovered individuals (mean difference 774.2 pg/mL; SD 335.7) and individuals without prior SARS-CoV-2 infection (mean difference 503.1 pg/ml; SD 467.7). Plasma chemokine ligand 6 levels were elevated among individuals with long COVID compared to individuals with no prior SARS-CoV-2 infection (mean difference 0.59 units; SD 0.42). We found no significant difference in angiotensin-converting enzyme 2 antibody levels. Plasma tumor necrosis factor receptor-associated factor 2 (TRAF2) levels were reduced among individuals with long COVID compared to individuals who were asymptomatic and recovered (mean difference = 0.6 units, SD 0.46). Similarly, the mean level of Sarcoma Homology 2-B adapter protein 3 was 3.3 units (SD 1.24) among individuals with long COVID, lower than 4.2 units (SD 1.1) among individuals with recovered, asymptomatic COVID. CONCLUSION: Our findings suggest that further studies should be conducted to evaluate the role of leukotriene E4 as a potential biomarker for a diagnostic test. Furthermore, based on reductions in TRAF2, long COVID may be driven in part by impaired TRAF2-dependent immune-mediated inflammation and potentially immune exhaustion.

Local monitoring of SARS-CoV-2 variants in two large California counties in 2021.

Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to persist due to mutations resulting in newer, more infectious variants of concern. We aimed to leverage an ongoing private SARS-CoV-2 testing laboratory's infrastructure to monitor SARS-CoV-2 variants in two large California counties. Study enrollment was offered to adults aged 18 years or older in Los Angeles County and Riverside County who recently tested positive for SARS-CoV-2 with a polymerase chain reaction (PCR) assay. A cycle threshold value less than or equal to 30 cycles was considered a positive test for sequencing purposes. Within 5 days of study enrollment, clinician-monitored, self-collected oral fluid and anterior nares swab specimens were obtained from participants. Specimens were transported and stored at 8 °C or cooler. Samples underwent ribonucleic acid extraction, library preparation, and sequencing. SARS-CoV-2 lineages were identified using sequencing data. Participant and genomic data were analyzed using statistical tools and visualized with toolkits. The study was approved by Advarra Institutional Review Board (Pro00053729). From May 27, 2021 to September 9, 2021, 503 individuals were enrolled and underwent specimen collection. Of the 503 participants, 238 (47.3%) participants were women, 329 (63.6%) participants were vaccinated, and 221 (43.9%) participants were of Hispanic or Spanish origin. Of the cohort, 496 (98.6%) participants had symptoms at the time of collection. Among the 503 samples, 443 (88.1%) nasal specimens and 353 (70.2%) oral specimens yielded positive sequencing results. Over our study period, the prevalence of the Alpha variant of SARS-CoV-2 decreased (initially 23.1% [95% confidence interval (95% CI): 0-0.49%] to 0% [95% CI 0.0-0.0%]) as the prevalence of the Delta variant of SARS-CoV-2 increased (initially 33.3% [95% CI 0.0-100.0%] to 100.0% [95% CI 100.0-100.0%]). A strain that carried mutations of both Delta and Mu was identified. We found that outpatient SARS-CoV-2 variant surveillance could be conducted in a timely and accurate manner. The prevalence of different variants changed over time. A higher proportion of nasal specimens yielded results versus oral specimens. Timely and regional outpatient SARS-CoV-2 variant surveillance could be used for public health efforts to identify changes in SARS-CoV-2 strain epidemiology.

Detection of persistent SARS-CoV-2 IgG antibodies in oral mucosal fluid and upper respiratory tract specimens following COVID-19 mRNA vaccination.

COVID-19 mRNA vaccines are highly effective at preventing COVID-19. Prior studies have found detectable SARS-CoV-2 IgG antibodies in oral mucosal specimens of participants with history of COVID-19. To assess the development of oral SARS-CoV-2 IgG antibodies among people who received either the Moderna or Pfizer/BioNTech COVID-19 vaccination series, we developed a novel SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) to quantify the concentrations of oral and nasal mucosal SARS-CoV-2 IgG levels. We enrolled 52 participants who received the Moderna vaccine and 80 participants who received the Pfizer/BioNTech vaccine. Oral mucosal specimens were self-collected by participants prior to or on the day of vaccination, and on days 5, 10, 15, and 20 following each vaccination dose and 30, 60, and 90 days following the second vaccination dose. A subset of the cohort provided additional nasal mucosal specimens at every time point. All participants developed detectable oral mucosal SARS-CoV-2 IgG antibodies by 15 days after the first vaccination dose. There were no significant differences in oral mucosal antibody concentrations once participants were fully vaccinated in the Moderna and Pfizer/BioNTech vaccines. Oral or nasal mucosal antibody testing could be an inexpensive and less invasive alternative to serum antibody testing. Further research is needed to understand the duration of detectable oral or nasal mucosal antibodies and how antibody concentrations change with time.

SARS-CoV-2 Specific IgG Antibodies Persist Over a 12-Month Period in Oral Mucosal Fluid Collected From Previously Infected Individuals.

Background: Developing an understanding of the antibody response, seroprevalence, and seroconversion from natural infection and vaccination against SARS-CoV-2 will give way to a critical epidemiological tool to predict reinfection rates, identify vulnerable communities, and manage future viral outbreaks. To monitor the antibody response on a larger scale, we need an inexpensive, less invasive, and high throughput method. Methods: Here we investigate the use of oral mucosal fluids from individuals recovered from SARS-CoV-2 infection to monitor antibody response and persistence over a 12-month period. For this cohort study, enzyme-linked immunosorbent assays (ELISAs) were used to quantify anti-Spike(S) protein IgG antibodies in participants who had prior SARS-CoV-2 infection and regularly (every 2-4 weeks) provided both serum and oral fluid mucosal fluid samples for longitudinal antibody titer analysis. Results: In our study cohort (n=42) with 17 males and 25 females with an average age of 45.6 +/- 19.3 years, we observed no significant change in oral mucosal fluid IgG levels across the time course of antibody monitoring. In oral mucosal fluids, all the participants who initially had detectable antibodies continued to have detectable antibodies throughout the study. Conclusions: Based on the results presented here, we have shown that oral mucosal fluid-based assays are an effective, less invasive tool for monitoring seroprevalence and seroconversion, which offers an alternative to serum-based assays for understanding the protective ability conferred by the adaptive immune response from viral infection and vaccination against future reinfections.

Saliva-Based ELISAs for Effective SARS-CoV-2 Antibody Monitoring in Vaccinated Individuals.

In March 2020, the World Health Organization (WHO) declared a global health emergency-the coronavirus disease 2019 (COVID-19) pandemic. Since then, the development and implementation of vaccines against the virus amidst emerging cases of re-infection has prompted researchers to work towards understanding how immunity develops and is sustained. Serological testing has been instrumental in monitoring the development and persistence of antibodies against SARS-CoV-2 infection, however inconsistencies in detection have been reported by different methods. As serological testing becomes more commonplace, it is important to establish widespread and repeatable processes for monitoring vaccine efficacy. Therefore, we present enzyme linked immunosorbent assays (ELISAs) compatible for antibody detection in saliva as highly accurate, efficacious, and scalable tools for studying the immune response in individuals vaccinated against SARS-CoV-2.

Detection of SARS-CoV-2 Antibodies in Oral Fluid Obtained Using a Rapid Collection Device.

Current commercially available methods for reliably detecting antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain expensive and inaccessible due to the need for whole-blood collection by highly trained phlebotomists using personal protective equipment (PPE). We have evaluated an antibody detection approach using the OraSure Technologies oral antibody collection device (OACD) and their proprietary SARS-CoV-2 total antibody detection enzyme-linked immunosorbent assay (ELISA). We found that the OraSure test for total antibody detection in oral fluid had comparable sensitivity and specificity to commercially available serum-based ELISAs for SARS-CoV-2 antibody detection while allowing for a more accessible form of specimen collection with the potential for self-collection.

ELISA detection of SARS-CoV-2 antibodies in saliva.

To facilitate containment of the COVID-19 pandemic currently active in the United States and across the world, options for easy, non-invasive antibody testing are required. Here we have adapted a commercially available, serum-based enzyme-linked immunosorbent assay (ELISA) for use with saliva samples, achieving 84.2% sensitivity and 100% specificity in a set of 149 clinical samples. This strategy will enable widespread, affordable testing for patients who experienced this disease, whilst minimizing exposure risk for healthcare workers.

A Novel Stool PCR Test for Helicobacter pylori May Predict Clarithromycin Resistance and Eradication of Infection at a High Rate.

Clarithromycin-based regimens are commonly used as a first-line therapy for Helicobacter pylori-positive patients; however, resistance to clarithromycin has led to treatment failures. The aim of this study was to evaluate the feasibility of using stool samples to detect the presence of H. pylori DNA while concurrently detecting mutations associated with resistance to clarithromycin. For this purpose, total DNA was extracted from 294 raw stool specimens from H. pylori-positive and -negative patients. TaqMan real-time PCR amplification was used to detect the presence of H. pylori as well as to predict the phenotype of the organism and the related outcome for patients treated with clarithromycin. Clarithromycin resistance was determined upon analysis of the PCR result. Patients were also tested by a urea breath test and were subjected to esophagogastroduodenoscopy, followed by histology, culture, and a rapid urease test, in order to obtain a consensus patient infection status. Of 294 total stool samples, 227 were deemed true positive. The sensitivity of H. pylori detection by PCR was 93.8%. Of 213 true-positive samples that were sequenced, 36.2% showed point mutations associated with clarithromycin resistance (A2142C, A2142G, A2143G). The final correlation of the mutant genotypes as determined by sequencing with the eradication of infection was 86%. We found that Helicobacter pylori DNA can be detected in human stool specimens with high sensitivity and can therefore be used to determine the presence of the bacterium without obtaining a biopsy sample. Moreover, genotypic resistance to clarithromycin can be predicted without obtaining a biopsy sample, facilitating the choice of the right therapeutic approach.

Automated high multiplex qPCR platform for simultaneous detection and quantification of multiple nucleic acid targets.

Quantitative PCR (qPCR) using real-time detection of amplification is limited to a small number of targets within a single reaction. The ICEPlex system, using our scalable target analysis routine (STAR) technology, was developed to provide an automated, high multiplexing PCR solution. ICEPlex combines PCR thermal cycling with dynamic, sequential amplicon separation by capillary electrophoresis and two-color quantitative detection in a single integrated system. In contrast to probe-based qPCR, ICEPlex directly measures amplicon accumulation through incorporation of labeled primers. Three orders of magnitude of optical detection range and at least 7 logs of detectable target concentration range are demonstrated. The system can separate more than 50 amplicons per color channel, ranging from 100 to 500 bases, providing broad multiplexing capabilities for a wide spectrum of nucleic acid amplification applications. ICEPlex can be used for analysis of viral DNA or RNA targets, detection of genetic variants, and for reverse-transcriptase PCR gene expression panels.

Novel carbamate cholinesterase inhibitors that release biologically active amines following enzyme inhibition.

Conjugation of the phenol derived from rivastigmine with amphetamines gave access to novel carbamate cholinesterase inhibitors. All compounds possessed increased affinity and selectivity for AChE compared to rivastigmine and were orally bioavailable. Compound 4a, incorporating d-amphetamine, caused significant inhibition of cholinesterase in vivo at doses that were well tolerated. Release of amphetamine from 4a was demonstrated following in vitro and in vivo inhibition of cholinesterase. Compound 4a was also effective in alleviating scopolamine induced amnesia in a rat passive avoidance model.

Scalable transcriptional analysis routine--multiplexed quantitative real-time polymerase chain reaction platform for gene expression analysis and molecular diagnostics.

We report the development of a new technology for simultaneous quantitative detection of multiple targets in a single sample. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. STAR demonstrated similar or better sensitivity and precision compared to two commonly used methods, SYBR Green-based and TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction. STAR can be used as a flexible platform for building a variety of applications to monitor gene expression, from single gene assays to assays analyzing the expression level of multiple genes. Using severe acute respiratory syndrome (SARS) corona virus as a model system, STAR technology detected single copies of the viral genome in a two-gene multiplex. Blinded studies using RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly identified all samples containing SARS virus and yielded negative results for non-SARS control samples. Using alternate priming strategies, STAR technology can be adapted to transcriptional profiling studies without requiring a priori sequence information. Thus, STAR technology offers a flexible platform for development of highly multiplexed assays in gene expression analysis and molecular diagnostics.

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse.

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis.

Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.