Rapid SARS-CoV-2 Virus Enrichment and RNA Extraction for Efficient Diagnostic Screening of Pooled Nasopharyngeal or Saliva Samples for Dilutions Up to 1:100 Diagnostics 2022 ABSTRACT: As COVID-19 transmission control measures are gradually being lifted, a sensitive and rapid diagnostic method for large-scale screening could prove essential for monitoring population infection rates. However, many rapid workflows for SARS-CoV-2 detection and diagnosis are not amenable to the analysis of large-volume samples. Previously, our group demonstrated a technique for SARS-CoV-2 nanoparticle-facilitated enrichment and enzymatic lysis from clinical samples in under 10 min. Here, this sample preparation strategy was applied to pooled samples originating from nasopharyngeal (NP) swabs eluted in viral transport medium (VTM) and saliva samples diluted up to 1:100. This preparation method was coupled with conventional RT-PCR on gold-standard instrumentation for proof-of-concept. Additionally, real-time PCR analysis was conducted using an in-house, ultra-rapid real-time microfluidic instrument paired with an experimentally optimized rapid protocol. Following pooling and extraction from clinical samples, average cycle threshold (CT) values from resultant eluates generally increased as the pooling dilution factor increased; further, results from a double-blind study demonstrated 100% concordance with clinical values. In addition, preliminary data obtained from amplification of eluates prepared by this technique and analyzed using our portable, ultra-rapid real-time microfluidic PCR amplification instrument showed progress toward a streamlined method for rapid SARS-CoV-2 analysis from pooled samples.

POSTER presented at ASM CVS 2022 Infectious uveitis is a serious, sight-threatening intraocular infection that is commonly caused by a variety of herpesviruses. Molecular testing is performed on intraocular fluids for etiology confirmation and proper clinical management. In many cases, vitreous fluid is collected and diluted in large volumes of buffered saline, which impacts sensitivity of pathogen detection. Nanotrap® particles (Ceres Nanosciences) are hydrogel particles functionalized with affinity baits that capture and concentrate analytes, including viruses such as Zika virus, chikungunya, dengue, HIV-1, influenza, respiratory syncytial virus and coronavirus. Here we explore the use of Nanotrap® particles to capture and concentrate low abundant herpesviruses from diluted porcine vitreous specimens. Nanotrap® particles demonstrated affinity to HSV-1, HSV-2, VZV and CMV in a vitreous matrix (Figure 1). Serially diluted specimens containing virus from 104 to 101 copies/mL were PCR positive after enrichment with Nanotrap® Custom Red Particles. Sensitivity of pathogen detection in the diluted vitreous increased 10-fold when compared with non-enriched specimens. Nanotrap® particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix. Our data demonstrate that these particles can be used for pre-processing of diluted vitreous specimens for improved detection of uveitis-causing herpesviruses.

POSTER presented at ASM CVS 2022 The emergence of novel SARS-CoV-2 variants has highlighted the need for accurate, rapid, and deployable sequencing methods. Sample enrichment technologies are particularly relevant, given the requirement for high viral loads to generate deep read depths and significant genome coverage for accurate mutation detection. Using Nanotrap® Magnetic Virus Particles to capture and concentrate virus prior to RNA extraction improved the sequencing results of SARS-CoV-2 in transport medium samples when compared to RNA extraction methods without enrichment. For contrived samples, using Nanotrap® Magnetic Virus Particles improved viral mapped reads of both extraction methods by 4-fold at 1x106 TCID50/mL and 2-fold at 1x105 TCID50/mL. >> Significant improvements were observed across 10 SARS-CoV-2 positive diagnostic remnant samples using Ceres Method 1. Using Nanotrap® Magnetic Virus Particles with the QIAmp® Viral RNA Mini Kit, viral mapped reads increased by 7-fold and viral genome coverage increased by 52%, on average. >> Significant improvements were observed across 10 SARS-CoV-2 positive diagnostic remnant samples using Ceres Method 2 on a KingFisher System. Using Nanotrap® Magnetic Virus Particles with the MagMAXTM Viral/Pathogen Kit increased viral mapped reads by 42-fold and viral genome coverage by 51%, on average.

Centrifugal Microfluidic Method for Enrichment and Enzymatic Extraction of Severe Acute Respiratory Syndrome Coronavirus 2 RNA Anal. Chem. February 2022 ABSTRACT: The diversification of analytical tools for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is imperative for effective virus surveillance and transmission control worldwide. Development of robust methods for rapid, simple isolation of viral RNA permits more expedient pathogen detection by downstream real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) to minimize stalled containment and enhance treatment efforts. Here, we describe an automatable rotationally driven microfluidic platform for enrichment and enzymatic extraction of SARS-CoV-2 RNA from multiple sample types. The multiplexed, enclosed microfluidic centrifugal device (μCD) is capable of preparing amplification-ready RNA from up to six samples in under 15 min, minimizing user intervention and limiting analyst exposure to pathogens. Sample enrichment leverages Nanotrap Magnetic Virus Particles to isolate intact SARS-CoV-2 virions from nasopharyngeal and/or saliva samples, enabling the removal of complex matrices that inhibit downstream RNA amplification and detection. Subsequently, viral capsids are lysed using an enzymatic lysis cocktail for release of pathogenic nucleic acids into a PCR-compatible buffer, obviating the need for downstream purification. Early in-tube assay characterization demonstrated comparable performance between our technique and a “gold-standard” commercial RNA extraction and purification kit. RNA obtained using the fully integrated μCDs permitted reliable SARS-CoV-2 detection by real-time RT-PCR. Notably, we successfully analyzed full-process controls, positive clinical nasopharyngeal swabs suspended in viral transport media, and spiked saliva samples, showcasing the method’s broad applicability with multiple sample matrices commonly encountered in clinical diagnostics.

bioRxiv, February 2022 ABSTRACT: Monitoring wastewater samples at building-level resolution screens large populations for SARS-CoV-2, prioritizing testing and isolation efforts. Here we perform untargeted metatranscriptomics on virally-enriched wastewater samples from 10 locations on the UC San Diego campus, demonstrating that resulting bacterial taxonomic and functional profiles discriminate SARS-CoV-2 status even without direct detection of viral transcripts. Our proof-of-principle reveals emergent threats through changes in the human microbiome, suggesting new approaches for untargeted wastewater-based epidemiology.

An ultrafast SARS-CoV-2 virus enrichment and extraction method compatible with multiple modalities for RNA detection Analytica Chimica Acta, October 2021 ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 min. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (μRT-PCR), recombinasepolymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale.

APPLICATION NOTE SKU 10XXX SKU 44XXX Download full application note here Key Advantages > Nanotrap® Magnetic Virus Particles improve wastewater sequencing workflows and improve viral pathogen sequencing results. > Enables sequencing of multiple SARS-CoV-2 variants. > Enables wastewater surveillance of multiple virus types from a single sample, including SARS-CoV-2 and Influenza A. Introduction The continuing emergence of novel SARS-CoV-2 variants and other problematic viral species has highlighted the need for accurate, rapid, and deployable sequencing methods. Detection of these viruses through the collection and analysis of municipal wastewater sources is a rapidly evolving detection strategy that has growing utility in the health response to the ongoing SARS-CoV-2 pandemic. Effective sequencing from wastewater typically requires a concentration step or enrichment strategy to alleviate the issues caused by low viral titers and enzyme inhibition inherent to these wastewater samples. Commonly these methods, such as HA filtration, are time consuming and arduous, needlessly limiting the analytical utility of monitoring wastewater. Nanotrap® Magnetic Virus Particles enable a wastewater enrichment strategy that is sensitive, rapid, easy to use, and can be performed manually or in an automated setup. Because it requires no filtration or centrifugation steps, this approach is compatible with medium- and high-throughput settings. Demonstrated previously, Nanotrap® Magnetic Virus Particles are able to capture and concentrate multiple strains of coronavirus (along with other viruses such as Influenza), thus enhancing detection of those viruses, even in co-infection scenarios. Nanotrap® Magnetic Virus Particles can enable automated viral surveillance of wastewater using a standard magnetic bead-based RNA extraction method. Coupled with the Oxford Nanopore Technologies™ MinION™ sequencing platform, Nanotrap® Magnetic Virus Particles increase total viral mapped reads, resulting in greater sequencing depth and total viral coverage. Here, we demonstrate that the Nanotrap® Magnetic Virus Particles workflow can: 1) significantly improve sequencing of low viral titer SARS-CoV-2 positive wastewater samples relative to a standard HA Filter Concentration Method, 2) enable sequencing of multiple SARS-CoV-2 variants, and 3) enrich multiple viral types, including Influenza A, from a single sample.

"Cannabinoids Reduce Extracellular Vesicle Release from HIV-1 Infected Myeloid Cells and Inhibit Viral Transcription" Cells, February 2022 ABSTRACT: Of the 37.9 million individuals infected with human immunodeficiency virus type 1 (HIV-1), approximately 50% exhibit HIV-associated neurocognitive disorders (HAND). We and others previously showed that HIV-1 viral RNAs, such as trans-activating response (TAR) RNA, are incorporated into extracellular vesicles (EVs) and elicit an inflammatory response in recipient naïve cells. Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the primary cannabinoids present in cannabis, are effective in reducing inflammation. Studies show that cannabis use in people living with HIV-1 is associated with lower viral load, lower circulating CD16+ monocytes and high CD4+ T-cell counts, suggesting a potentially therapeutic application. Here, HIV-1 infected U1 monocytes and primary macrophages were used to assess the effects of CBD. Post-CBD treatment, EV concentrations were analyzed using nanoparticle tracking analysis. Changes in intracellular and EV-associated viral RNA were quantified using RT-qPCR, and changes in viral proteins, EV markers, and autophagy proteins were assessed by Western blot. Our data suggest that CBD significantly reduces the number of EVs released from infected cells and that this may be mediated by reducing viral transcription and autophagy activation. Therefore, CBD may exert a protective effect by alleviating the pathogenic effects of EVs in HIV-1 and CNS-related infections.

Scientific Reports, February 2022 ABSTRACT: HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection

Analytical Chemistry, February 2022 ABSTRACT: The diversification of analytical tools for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is imperative for effective virus surveillance and transmission control worldwide. Development of robust methods for rapid, simple isolation of viral RNA permits more expedient pathogen detection by downstream real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) to minimize stalled containment and enhance treatment efforts. Here, we describe an automatable rotationally driven microfluidic platform for enrichment and enzymatic extraction of SARS-CoV-2 RNA from multiple sample types. The multiplexed, enclosed microfluidic centrifugal device (μCD) is capable of preparing amplification-ready RNA from up to six samples in under 15 min, minimizing user intervention and limiting analyst exposure to pathogens. Sample enrichment leverages Nanotrap Magnetic Virus Particles to isolate intact SARS-CoV-2 virions from nasopharyngeal and/or saliva samples, enabling the removal of complex matrices that inhibit downstream RNA amplification and detection. Subsequently, viral capsids are lysed using an enzymatic lysis cocktail for release of pathogenic nucleic acids into a PCR-compatible buffer, obviating the need for downstream purification. Early in-tube assay characterization demonstrated comparable performance between our technique and a “gold-standard” commercial RNA extraction and purification kit. RNA obtained using the fully integrated μCDs permitted reliable SARS-CoV-2 detection by real-time RT-PCR. Notably, we successfully analyzed full-process controls, positive clinical nasopharyngeal swabs suspended in viral transport media, and spiked saliva samples, showcasing the method’s broad applicability with multiple sample matrices commonly encountered in clinical diagnostics.

"Wastewater Surveillance of U.S. Coast Guard Installations and Seagoing Military Vessels to Mitigate the Risk of COVID-19 Outbreaks" medRXiv Feb 2022 ABSTRACT: Military training centers may be high risk environments for the spread of disease such as COVID-19. Individuals arrive after traveling from many parts of the country, live in communal settings, and undergo high-interaction training. A pilot study of wastewater testing was initiated in February, 2021 to determine its feasibility as a sentinel surveillance tool in the U.S. Coast Guard for SARS-CoV-2. Wastewater was analyzed for the presence of two viral genes, N and E, and quantified relative to levels of a fecal indicator virus, Pepper Mild Mottle Virus (PMMoV). A stability control, Bovine Syncytial Respiratory Virus vaccine, was added to samples to assess sample stability and degradation. Wastewater data was validated by comparison with concomitant screening and surveillance programs that identified asymptomatic individuals infected with SARS-CoV-2 by diagnostic testing at on site medical clinics using PCR. Elevated levels of SARS-CoV-2 in wastewater were frequently associated with diagnosed cases, and in several instances, led to screenings of asymptomatic individuals that identified infected personnel, mitigating the risk of spread of disease. Wastewater screening also successfully indicated the presence of breakthrough cases in vaccinated individuals. A method for assessing blackwater from Coast Guard vessels was also developed, allowing detection of SARS-CoV-2 virus in shipboard populations. In one instance, virus was detected in the blackwater four weeks following the diagnosis of a single person on a Coast Guard cutter. These data show that wastewater testing is an effective tool for measuring the presence and prevalence of SARS-CoV-2 in military populations so that mitigation can occur and suggest other diseases may be assessed similarly. As a result, the Coast Guard has established three laboratories with wastewater testing capability at strategic locations and is actively continuing its wastewater testing program.

Journal of Nanobiotechnology, June 21, 2021 ABSTRACT: Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis.