The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 μL sample with a pipette. The device, which we call the μSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2–3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus ( n = 62). Moreover, the dynamic range of the sensor extends at least from 10 5.9 virions per mL to 10 10.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (10 5.6 –10 7 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.
more »
« less
The size and culturability of patient-generated SARS-CoV-2 aerosol
Abstract Background Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. Objective We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. Methods Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). Results SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. Significance Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.
more »
« less
- Award ID(s):
- 2032135
- NSF-PAR ID:
- 10296888
- Date Published:
- Journal Name:
- Journal of Exposure Science & Environmental Epidemiology
- ISSN:
- 1559-0631
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare.more » « less
-
more » « less
Abstract Background SARS-CoV-2 is an RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Viruses exist in complex microbial environments, and recent studies have revealed both synergistic and antagonistic effects of specific bacterial taxa on viral prevalence and infectivity. We set out to test whether specific bacterial communities predict SARS-CoV-2 occurrence in a hospital setting.
Methods We collected 972 samples from hospitalized patients with COVID-19, their health care providers, and hospital surfaces before, during, and after admission. We screened for SARS-CoV-2 using RT-qPCR, characterized microbial communities using 16S rRNA gene amplicon sequencing, and used these bacterial profiles to classify SARS-CoV-2 RNA detection with a random forest model.
Results Sixteen percent of surfaces from COVID-19 patient rooms had detectable SARS-CoV-2 RNA, although infectivity was not assessed. The highest prevalence was in floor samples next to patient beds (39%) and directly outside their rooms (29%). Although bed rail samples more closely resembled the patient microbiome compared to floor samples, SARS-CoV-2 RNA was detected less often in bed rail samples (11%). SARS-CoV-2 positive samples had higher bacterial phylogenetic diversity in both human and surface samples and higher biomass in floor samples. 16S microbial community profiles enabled high classifier accuracy for SARS-CoV-2 status in not only nares, but also forehead, stool, and floor samples. Across these distinct microbial profiles, a single amplicon sequence variant from the genus
Rothia strongly predicted SARS-CoV-2 presence across sample types, with greater prevalence in positive surface and human samples, even when compared to samples from patients in other intensive care units prior to the COVID-19 pandemic.Conclusions These results contextualize the vast diversity of microbial niches where SARS-CoV-2 RNA is detected and identify specific bacterial taxa that associate with the viral RNA prevalence both in the host and hospital environment.
-
Abstract The spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.more » « less
-
null (Ed.)Abstract Background Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. Methods Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption–extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity. Results SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods. Conclusions The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41370-021-00376-8
