Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae

The rise in carbapenem-resistant Enterobacteriaceae (CRE) infections has created a global health emergency, underlining the critical need to develop faster diagnostics to treat swiftly and correctly. Although rapid pathogen-identification (ID) tests are being developed, gold-standard antibiotic susceptibility testing (AST) remains unacceptably slow (1–2 d), and innovative approaches for rapid phenotypic ASTs for CREs are urgently needed. Motivated by this need, in this manuscript we tested the hypothesis that upon treatment with β-lactam antibiotics, susceptible Enterobacteriaceae isolates would become sufficiently permeabilized, making some of their DNA accessible to added polymerase and primers. Further, we hypothesized that this accessible DNA would be detectable directly by isothermal amplification methods that do not fully lyse bacterial cells. We build on these results to develop the polymerase-accessibility AST (pol-aAST), a new phenotypic approach for β-lactams, the major antibiotic class for gram-negative infections. We test isolates of the 3 causative pathogens of CRE infections using ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) and demonstrate agreement with gold-standard AST. Importantly, pol-aAST correctly categorized resistant isolates that are undetectable by current genotypic methods (negative for β-lactamase genes or lacking predictive genotypes). We also test contrived and clinical urine samples. We show that the pol-aAST can be performed in 30 min sample-to-answer using contrived urine samples and has the potential to be performed directly on clinical urine specimens

A Multi-Level Fit-Based Quality Improvement Initiative to Improve Colorectal Cancer Screening in a Managed Care Population.

IntroductionColorectal cancer (CRC) is a common but largely preventable disease with suboptimal screening rates despite national guidelines to screen individuals age 50-75. Single-component interventions aimed to improve screening uptake only modestly improve rates; data suggest that multi-modal approaches may be more effective.MethodsWe designed, implemented, and evaluated the impact of a multi-modal intervention on CRC screening uptake among unscreened patients in a large managed care population. Patient-level components included a mailed letter with education about screening options and pre-colonoscopy telephone counseling. For providers, we facilitated communication of screening test results and work-flow for abnormal results. System-level modifications included establishment of a patient navigator, expedited work-up for abnormal results, and stream-lined colonoscopy scheduling. We measured the rate of screening uptake overall, screening uptake by modality, change in the proportion of the population screened, and positive fecal immunochemical test (FIT) follow-up rates in the 1-year study period.ResultsThere were 5093 patients in the intervention cohort. Of these, 33.2% participated in FIT or colonoscopy screening within 1 year of the mailing. A total of 1078 (21.2%) participants completed a FIT and 611 (12.0%) completed a screening colonoscopy. The screening rate in the managed care population increased from 65.1 to 76.6%. Fifty-nine patients (5.5%) had a positive FIT, of which 30 (50.8%) completed a diagnostic colonoscopy.ConclusionMulti-modal interventions can result in substantial improvement in CRC screening uptake in large and diverse managed care populations.Translational impactHealth systems should shift their focus from single-level to multi-level interventions when addressing barriers to CRC screening

Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae

The rise in carbapenem-resistant Enterobacteriaceae (CRE) infections has created a global health emergency, underlining the critical need to develop faster diagnostics to treat swiftly and correctly. Although rapid pathogen-identification (ID) tests are being developed, gold-standard antibiotic susceptibility testing (AST) remains unacceptably slow (1–2 d), and innovative approaches for rapid phenotypic ASTs for CREs are urgently needed. Motivated by this need, in this manuscript we tested the hypothesis that upon treatment with β-lactam antibiotics, susceptible Enterobacteriaceae isolates would become sufficiently permeabilized, making some of their DNA accessible to added polymerase and primers. Further, we hypothesized that this accessible DNA would be detectable directly by isothermal amplification methods that do not fully lyse bacterial cells. We build on these results to develop the polymerase-accessibility AST (pol-aAST), a new phenotypic approach for β-lactams, the major antibiotic class for gram-negative infections. We test isolates of the 3 causative pathogens of CRE infections using ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) and demonstrate agreement with gold-standard AST. Importantly, pol-aAST correctly categorized resistant isolates that are undetectable by current genotypic methods (negative for β-lactamase genes or lacking predictive genotypes). We also test contrived and clinical urine samples. We show that the pol-aAST can be performed in 30 min sample-to-answer using contrived urine samples and has the potential to be performed directly on clinical urine specimens

Loss of the Heparan Sulfate Sulfotransferase, Ndst1, in Mammary Epithelial Cells Selectively Blocks Lobuloalveolar Development in Mice

Considerable evidence indicates that heparan sulfate is essential for the development of tissues consisting of branching ducts and tubules. However, there are few examples where specific sulfate residues regulate a specific stage in the formation of such tissues.We examined the role of heparan sulfation in mammary gland branching morphogenesis, lactation and lobuloalveolar development by inactivation of heparan sulfate GlcNAc N-deacetylase/N-sulfotransferase genes (Ndst) in mammary epithelial cells using the Cre-loxP system. Ndst1 deficiency resulted in an overall reduction in glucosamine N-sulfation and decreased binding of FGF to mammary epithelial cells in vitro and in vivo. Mammary epithelia lacking Ndst1 underwent branching morphogenesis, filling the gland with ductal tissue by sexual maturity to the same extent as wildtype epithelia. However, lobuloalveolar expansion did not occur in Ndst1-deficient animals, resulting in insufficient milk production to nurture newly born pups. Lactational differentiation of isolated mammary epithelial cells occurred appropriately via stat5 activation, further supporting the notion that the lack of milk production was due to lack of expansion of the lobuloalveoli.These findings demonstrate a selective, highly penetrant, cell autonomous effect of Ndst1-mediated sulfation on lobuloalveolar development

Multicenter clinical evaluation of Etest meropenem-vaborbactam (bioMérieux) for susceptibility testing of Enterobacterales (Enterobacteriaceae) and Pseudomonas aeruginosa

Meropenem-vaborbactam (MEV) is a novel carbapenem-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections, including pyelonephritis, in adults. In this study, we evaluated the performance of Etest MEV (bioMérieux, Marcy l\u27Etoile, France) compared to that of broth microdilution for 62

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications

Deep learning-enabled multiplexed point-of-care sensor using a paper-based fluorescence vertical flow assay

We demonstrate multiplexed computational sensing with a point-of-care serodiagnosis assay to simultaneously quantify three biomarkers of acute cardiac injury. This point-of-care sensor includes a paper-based fluorescence vertical flow assay (fxVFA) processed by a low-cost mobile reader, which quantifies the target biomarkers through trained neural networks, all within <15 min of test time using 50 microliters of serum sample per patient. This fxVFA platform is validated using human serum samples to quantify three cardiac biomarkers, i.e., myoglobin, creatine kinase-MB (CK-MB) and heart-type fatty acid binding protein (FABP), achieving less than 0.52 ng/mL limit-of-detection for all three biomarkers with minimal cross-reactivity. Biomarker concentration quantification using the fxVFA that is coupled to neural network-based inference is blindly tested using 46 individually activated cartridges, which showed a high correlation with the ground truth concentrations for all three biomarkers achieving > 0.9 linearity and < 15 % coefficient of variation. The competitive performance of this multiplexed computational fxVFA along with its inexpensive paper-based design and handheld footprint make it a promising point-of-care sensor platform that could expand access to diagnostics in resource-limited settings.Comment: 17 Pages, 6 Figure

Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed optomechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity

Assessment of reproducibility of matrix-assisted laser desorption ionization - Time of flight mass spectrometry for bacterial and yeast identification

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification