Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13.

Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets.

Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays.

Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting.

Massively multiplexed nucleic acid detection with Cas13.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection.

Massively parallel screening of synthetic microbial communities.

Microbial communities have numerous potential applications in biotechnology, agriculture, and medicine. Nevertheless, the limited accuracy with which we can predict interspecies interactions and environmental dependencies hinders efforts to rationally engineer beneficial consortia. Empirical screening is a complementary approach wherein synthetic communities are combinatorially constructed and assayed in high throughput.

Changes in the Anterior Lamina Cribrosa Morphology with Glaucoma Severity.

This study was designed to evaluate if primary open angle glaucoma (POAG) and its severity are associated with the shape of the lamina cribrosa (LC) as measured by a global shape index (LC-GSI), or other indices of LC curvature or depth. Optical coherence tomography (OCT) scans of the optic nerve head (OHN) were obtained from subjects with POAG (n = 99) and non-glaucomatous controls (n = 76). ONH structures were delineated, the anterior LC morphology reconstructed in 3D, and the LC-GSI calculated (more negative values denote greater posterior concavity).

Harnessing CRISPR Effectors for Infectious Disease Diagnostics.

Nucleic acid detection is an important method for pathogen identification but can be expensive, have variable sensitivity and specificity, and require substantial infrastructure. Two new methods capitalize on unexpected in vitro properties of clustered regularly interspaced short palindromic repeats (CRISPR) effectors, turning activated nucleases into intrinsic amplifiers of a specific nucleic-acid binding event. These effectors are coupled with a variety of reporters and used in tandem with existing isothermal amplification methods to produce sensitive, sequence-specific pathogen identification in multiple field-deployable formats.

Automated Detection of Iris Furrows and their Influence on Dynamic Iris Volume Change.

We introduced a new method for detecting iris surface furrows and identify its associations with dynamic changes in iris volume in healthy eyes. Swept-source optical coherence tomography was performed on 65 subjects with open angle under light and dark conditions. Iris boundaries were identified and a reconstruction of the anterior iris surface was obtained.

Effect of acute intraocular pressure elevation on the minimum rim width in normal, ocular hypertensive and glaucoma eyes.

Background: To estimate and compare changes in the Bruch's membrane opening-minimum rim width (BMO-MRW) and area in normal, ocular hypertensive and glaucoma eyes following acute elevations in intraocular pressure (IOP).

Methods: The optic nerve heads (ONHs) of 104 subjects (31 normals, 20 ocular hypertension (OHT) and 53 with primary glaucoma) were imaged using Spectral-domain optical coherence tomography (OCT; Spectralis, Heidelberg Engineering, Germany). IOP was raised twice by applying a force (0.

Association of iris crypts with acute primary angle closure.

Purpose: To determine the relationship between iris surface features and acute primary angle closure (APAC) in eyes with angle closure.

Design: Case-control study involving Asian patients diagnosed with previous APAC, primary angle closure suspect (PACS), primary angle closure (PAC) and primary angle closure glaucoma (PACG) at an eye centre in Singapore between August 2012 and January 2015.

Methods: Participants underwent ophthalmic examination and digital slit-lamp iris photography.

Characterization of Choroidal Morphologic and Vascular Features in Young Men With High Myopia Using Spectral-Domain Optical Coherence Tomography.

Purpose: To describe detailed morphologic and vascular features of the choroid in eyes with high myopia.

Design: Cross-sectional study.

Methods: A total of 515 eyes of young men (mean age 21.

DETAILED CHARACTERIZATION OF CHOROIDAL MORPHOLOGIC AND VASCULAR FEATURES IN AGE-RELATED MACULAR DEGENERATION AND POLYPOIDAL CHOROIDAL VASCULOPATHY.

Purpose: To characterize and compare morphologic and vascular features of the choroid in patients with typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) and to determine if PCV subtypes can be identified based on these choroidal features.

Methods: Choroidal features of patients with AMD and PCV recruited from the prospectively planned Asian AMD Phenotyping Study were analyzed. Patients underwent choroidal imaging using spectral-domain optical coherence tomography with enhanced depth imaging.

Characterisation of choroidal morphological and vascular features in diabetes and diabetic retinopathy.

Aim: We aimed to characterise specific morphological and vascular features of the choroid in Indian adults with diabetes and diabetic retinopathy (DR).

Methods: Consecutive participants from the Singapore Indian Eye Study's 6-year follow-up examination underwent choroidal imaging using spectral domain optical coherence tomography (OCT) with enhanced depth imaging. Raw OCT images were loaded on a custom-written application on MATLAB that enabled delineation for detailed morphological and vascular analyses.

Verification of a virtual fields method to extract the mechanical properties of human optic nerve head tissues in vivo.

We aimed to verify a custom virtual fields method (VFM) to estimate the patient-specific biomechanical properties of human optic nerve head (ONH) tissues, given their full-field deformations induced by intraocular pressure (IOP). To verify the accuracy of VFM, we first generated 'artificial' ONH displacements from predetermined (known) ONH tissue biomechanical properties using finite element analysis. Using such deformations, if we are able to match back the known biomechanical properties, it would indicate that our VFM technique is accurate.

Factors Influencing Lamina Cribrosa Microcapillary Hemodynamics and Oxygen Concentrations.

Purpose: To identify and rank the lamina cribrosa (LC) morphologic factors that influence LC microcapillary hemodynamics and oxygen concentrations using computational fluid dynamics (CFD).

Methods: We generated 12,000 'artificial' LC microcapillary networks and predicted blood flow velocities and oxygen concentrations within the microcapillaries using CFD. Across models, we varied the average pore size of the LC (5500 ± 2400 μm2), the microcapillary arrangement (radial, isotropic, or circumferential), the LC diameter (1.

Shape Changes of the Anterior Lamina Cribrosa in Normal, Ocular Hypertensive, and Glaucomatous Eyes Following Acute Intraocular Pressure Elevation.

Purpose: The purpose of this study was to estimate and compare changes in anterior lamina cribrosa (LC) morphology in normal, ocular hypertensive (OHT), and glaucomatous eyes following acute elevations in intraocular pressure (IOP).

Methods: The optic nerve heads (ONHs) of 97 subjects (17 OHT, 19 primary open-angle glaucoma [POAG], 31 primary angle-closure glaucoma [PACG], and 30 normal subjects) were imaged using optical coherence tomography (OCT). Intraocular pressure was raised twice by applying forces to the anterior sclera, using an ophthalmodynamometer.

Iris Crypts Influence Dynamic Changes of Iris Volume.

Purpose: To determine the association of iris surface features with iris volume change after physiologic pupil dilation in adults.

Design: Cross-sectional observational study.

Participants: Chinese adults aged ≥ 50 years without ocular diseases.

Association of iris surface features with iris parameters assessed by swept-source optical coherence tomography in Asian eyes.

Background/aims: To characterise the association of iris surface features (crypts, furrows and colour) with iris volume and curvature assessed by swept-source optical coherence tomography (SSOCT) in Asian eyes.

Methods: Iris crypts (by number and size) and furrows (by number and circumferential extent) were graded from iris photographs. Iris colour was measured by a customised algorithm written on MATLAB (MathWorks, Natick, Massachusetts, USA).

A Global Shape Index to Characterize Anterior Lamina Cribrosa Morphology and Its Determinants in Healthy Indian Eyes.

Purpose: Lamina cribrosa (LC) morphology could be implicated in the progression of glaucoma. To date, no established, quantifiable parameter to assess LC shape in vivo exists. We aim to introduce a new global shape index for the anterior LC (LC-GSI) and to identify associations with ocular factors in a healthy Indian population.