Rapid and Comprehensive Screening for Urogenital and Gastrointestinal Schistosomiasis with Handheld Digital Microscopy Combined with Circulating Cathodic Antigen Testing.

Novel methods are required to aid the monitoring of schistosomiasis control and elimination initiatives through mass drug administration. Portable digital and mobile phone microscopy is a promising tool for this purpose. This cross-sectional study evaluated the diagnostic operating characteristics of a converted mobile phone microscope (the SchistoScope) for the detection of Schistosoma haematobium eggs, as determined by community-based field workers and expert microscopists, compared with a field gold standard of light microscopy. Three hundred sixty-five urine samples were evaluated by conventional light microscopy, with 49 (13.4%) positive for S. haematobium. Compared with light microscopy, the sensitivity and specificity of S. haematobium detection by field microscopists trained to use the SchistoScope were 26.5% (95% CI: 14.9-41.1%) and 98.4% (95% CI: 96.3-99.5%), respectively. The sensitivity and specificity of S. haematobium detection by expert microscopists using the SchistoScope was 74% (95% CI: 59.7-85.4%) and 98.1% (95% CI: 95.9-99.3%), respectively, compared with light microscopy. The sensitivity rose to 96.1% and 100% when evaluating for egg counts greater than five and 10 eggs per 10 mL, respectively. A point-of-care circulating cathodic anion (POC CCA) test was used to evaluate Schistosoma mansoni; however, there were too few positive samples to reliably comment on diagnostic characteristics. This study demonstrated that a "urine-only" approach to rapidly screen for schistosomiasis at the point of sample collection can be conducted with mobile phone microscopy (S. haematobium) coupled with POC CCA (S. mansoni). Such an approach may aid in streamlined schistosomiasis control and elimination initiatives.

Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies µl-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.

Accelerated RNA detection using tandem CRISPR nucleases.

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Rapid, point-of-care molecular diagnostics with Cas13.

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/µL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.

Using chaotic advection for facile high-throughput fabrication of ordered multilayer micro- and nanostructures: continuous chaotic printing.

This paper introduces the concept of continuous chaotic printing, i.e. the use of chaotic flows for deterministic and continuous extrusion of fibers with internal multilayered micro- or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (∼102 cm2 cm-3) at high resolution (∼10 µm). This creates structures with extremely high surface area to volume ratio (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. In an exciting new development, we demonstrate a method for scaling down these microstructures by 3 orders of magnitude, to the nanoscale level (∼150 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including-but not limited to-bioprinting of multi-scale layered biological structures such as bacterial communities, living tissues composed of organized multiple mammalian cell types, and fabrication of smart multi-material and multilayered constructs for biomedical applications.

An electrohydrodynamic technique for rapid mixing in stationary droplets on digital microfluidic platforms.

This paper presents an electrohydrodynamic technique for rapid mixing of droplets in open and closed digital microfluidic (DMF) platforms. Mixing is performed by applying a high frequency AC voltage to the coplanar or parallel electrodes, inducing circulation zones inside the droplet which results in rapid mixing of the content. The advantages of the proposed method in comparison to conventional mixing methods that operate based on transporting the droplet back and forth and side to side include 1) a shorter mixing time (as fast as 0.25 s), 2) the use of a fewer number of electrodes, reducing the size of the chip, and 3) the stationary nature of the technique which reduces the chance of cross-contamination and surface biofouling. Mixing using the proposed method is performed to create a uniform mixture after merging a water droplet with another droplet containing either particles or dye. The results show that increasing the frequency, and or the amplitude of the applied voltage, enhances the mixing process. However, actuation with a very high frequency and voltage may result in shedding pico-liter satellite droplets. Therefore, for each frequency there is an effective range of the amplitude which provides rapid mixing and avoids shedding satellite droplets. Also, the increase in the gap height between the two plates (for the closed DMF platforms) significantly enhances the mixing efficiency due to the lower viscous effects. Effects of the addition of salts and DNA to the samples were also studied. The electrothermal effect decreased for these cases, which was solved by increasing the frequency of the applied voltage. To assure the high frequency actuation does not increase the sample temperature excessively, the temperature change was monitored using a thermal imaging camera and it was found that the increase in temperature is negligible.