Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time. IMPORTANCE The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Passively sensing SARS-CoV-2 RNA in public transit buses.

Affordably tracking the transmission of respiratory infectious diseases in urban transport infrastructures can inform individuals about potential exposure to diseases and guide public policymakers to prepare timely responses based on geographical transmission in different areas in the city. Towards that end, we designed and tested a method to detect SARS-CoV-2 RNA in the air filters of public buses, revealing that air filters could be used as passive fabric sensors for the detection of viral presence. We placed and retrieved filters in the existing HVAC systems of public buses to test for the presence of trapped SARS-CoV-2 RNA using phenol-chloroform extraction and RT-qPCR. SARS-CoV-2 RNA was detected in 14% (5/37) of public bus filters tested in Seattle, Washington, from August 2020 to March 2021. These results indicate that this sensing system is feasible and that, if scaled, this method could provide a unique lens into the geographically relevant transmission of SARS-CoV-2 through public transit rider vectors, pooling samples of riders over time in a passive manner without installing any additional systems on transit vehicles.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection.

RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed "Harmony COVID-19," a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.38 SARS-CoV-2 RNA copies/µl and can report in 17 min for high­viral load samples (5000 copies/µl). Harmony detected 97 or 83% of contrived samples with ≥0.5 viral particles/µl in nasal matrix or saliva, respectively. Evaluation in clinical nasal specimens (n = 101) showed 100% detection of RNA extracted from specimens with ≥0.5 SARS-CoV-2 RNA copies/µl, with 100% specificity in specimens positive for other respiratory pathogens. Extraction-free analysis (n = 29) had 95% success in specimens with ≥1 RNA copies/µl. Usability testing performed first time by health care workers showed 95% accuracy.

Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays.

BACKGROUND: Detection of SARS-CoV-2 infections is important for treatment, isolation of infected and exposed individuals, and contact tracing. RT-qPCR is the "gold-standard" method to sensitively detect SARS-CoV-2 RNA, but most laboratory-developed RT-qPCR assays involve complex steps. Here, we aimed to simplify RT-qPCR assays by streamlining reaction setup, eliminating RNA extraction, and proposing reduced-cost detection workflows that avoid the need for expensive qPCR instruments. METHOD: A low-cost RT-PCR based "kit" was developed for faster turnaround than the CDC developed protocol. We demonstrated three detection workflows: two that can be deployed in laboratories conducting assays of variable complexity, and one that could be simple enough for point-of-care. Analytical sensitivity was assessed using SARS-CoV-2 RNA spiked in simulated nasal matrix. Clinical performance was evaluated using contrived human nasal matrix (n = 41) and clinical nasal specimens collected from individuals with respiratory symptoms (n = 110). FINDING: The analytical sensitivity of the lyophilised RT-PCR was 10 copies/reaction using purified SARS-CoV-2 RNA, and 20 copies/reaction when using direct lysate in simulated nasal matrix. Evaluation of assay performance on contrived human matrix showed 96.7-100% specificity and 100% sensitivity at ≥20 RNA copies. A head-to-head comparison with the standard CDC protocol on clinical specimens showed 83.8-94.6% sensitivity and 96.8-100% specificity. We found 3.6% indeterminate samples (undetected human control), lower than 8.1% with the standard protocol. INTERPRETATION: This preliminary work should support laboratories or commercial entities to develop and expand access to Covid-19 testing. Software guidance development for this assay is ongoing to enable implementation in other settings. FUND: USA NIH R01AI140845 and Seattle Children's Research Institute.