Molecular Platform Research Articles

Construction of high content CoMoS active sites through the combination of Co5Mo10 molecular precursors and inert carbon matrix support for deep hydrodesulfurization

The rational design of high sulfurization and abundant CoMoS active phases catalysts is the key to drive the industrialization of hydrodesulfurization (HDS). Herein, we prepared a nano-catalyst with well-dispersed and numerous CoMoS active sites through confining Co3(II)[Co2(III)Mo10O38H4] (Co5Mo10) polyoxometalates (POMs) molecules with high Co/Mo ratio in hollow mesoporous carbon spheres (HMCS). On the one hand, POMs acted as pre-assembled molecular platform can build and regulate the structure of active phases by precisely adjusting the Co/Mo atomic ratio at molecular level. Meanwhile, the intimate interaction of Co-Mo on the POMs precursor also ensures that the Co species mainly exists as Co-promoted MoS2 active phase after the sulfidation process, thus the highly efficient synergism of the bimetals can be fully utilized in the reaction. On the other hand, the mesoporous carbon sphere with homogeneous pore size and high specific surface area can effectively confine the growth of the catalyst to disperse the active metals. The weakening metal–metal interaction property of such inert support enables the S-Co5Mo10/HMCS-10 catalyst to realize higher sulfurization, shorter slab length and higher stacking number. In addition, the abundant mesopores and high pore capacity of support greatly facilitate the mass transfer process and provide sufficient space for the HDS reaction as the nanoreactor. The unique combination of POMs and HMCS enabled the S-Co5Mo10/H10 catalyst to achieve up to 99.2% conversion of dibenzothiophene (DBT) after 4 h of HDS reaction, while obtaining a TOF value of 1.3 × 10−3 s−1 (calculated when keeping the low conversion at 42% after 0.5 h of reaction). The structural orientation of the POMs precursors to the active phases of HDS catalysts was investigated using various characterization methods, and the structure–activity relationship between the catalytic performance and the sulfide active phases was also revealed in this work.

In Vivo Detection of Low Molecular Weight Platform Chemicals and Environmental Contaminants by Genetically Encoded Biosensors.

Genetically encoded biosensor systems operating in living cells are versatile, cheap, and transferable tools for the detection and quantification of a broad range of small molecules. This review presents state-of-the-art biosensor designs and assemblies, featuring transcription factor-, riboswitch-, and enzyme-coupled devices, highly engineered fluorescent probes, and emerging two-component systems. Importantly, (bioinformatic-assisted) strategies to resolve contextual issues, which cause biosensors to miss performance criteria in vivo, are highlighted. The optimized biosensing circuits can be used to monitor chemicals of low molecular mass (<200 g mol-1) and physicochemical properties that challenge conventional chromatographical methods with high sensitivity. Examples herein include but are not limited to formaldehyde, formate, and pyruvate as immediate products from (synthetic) pathways for the fixation of carbon dioxide (CO2), industrially important derivatives like small- and medium-chain fatty acids and biofuels, as well as environmental toxins such as heavy metals or reactive oxygen and nitrogen species. Lastly, this review showcases biosensors capable of assessing the biosynthesis of platform chemicals from renewable resources, the enzymatic degradation of plastic waste, or the bioadsorption of highly toxic chemicals from the environment. These applications offer new biosensor-based manufacturing, recycling, and remediation strategies to tackle current and future environmental and socioeconomic challenges including the wastage of fossil fuels, the emission of greenhouse gases like CO2, and the pollution imposed on ecosystems and human health.

Update on Molecular Diagnostics in Thyroid Pathology: A Review.

Thyroid nodules are quite common, and the determination of a nodule of concern is complex, involving serum testing, radiology and, in some cases, pathological evaluation. For those nodules that raise clinical concern of neoplasia, fine needle aspiration biopsy is the gold standard for evaluation; however, in up to 30% of cases, results are indeterminate for malignancy, and further testing is needed. Advances in molecular testing have shown it to be of benefit for both diagnostic and prognostic purposes, and its use has become an integral part of thyroid cancer management in the United States and in several global nations. After The Cancer Genome Atlas (TCGA) consortium published its molecular landscape of papillary thyroid carcinoma (PTC) and reduced the "black matter" in PTC from 25% to 3.5%, further work ensued to clarify the remaining fraction not neatly attributed to the BRAFV600E-like or RAS-like phenotypes of the TCGA. Over the past decade, commercial molecular platforms have been refined as data accrues, and they increasingly cover most genetic variants of thyroid carcinomas. Molecular reporting focuses on the nodule tested, including related clinical information for that nodule (size of nodule, Bethesda category, etc.). This results in a comprehensive report to physicians that may also include patient-directed, clear language that facilitates conversations about nodule management. In cases of advanced or recurrent disease, molecular testing may become essential for devising an individual therapeutic plan. In this review, we focus on the evolution of integrated molecular testing in thyroid nodules, and how our understanding of tumor genetics, combined with histopathology, is driving the next generation of rational patient management, particularly in the context of emerging small, targetable therapeutics.

Construction and application of a new CRISPR/Cas12a system in Stenotrophomonas AGS-1 from aerobic granular sludge.

Aerobic granular sludge (AGS) is a microbial aggregate with a biofilm structure. Thus, investigating AGS in the aspect of biofilm and microbial attachment at the genetic level would help to reveal the mechanism of granule biofilm formation. In this work, a two-plasmid CRISPR/Cas12a genome editing system was constructed to identify attachment genes for the first time in Stenotrophomonas AGS-1 from AGS. One plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms. Acidaminococcus sp. Cas12a (AsCas12a) was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1. CRISPR/Cas12a-mediated rmlA knockout decreased attachment ability by 38.26%. Overexpression of rmlA in AGS-1 resulted in an increase of 30.33% in attachment ability. These results showed that the modulation of rmlA was an important factor for the biofilm formation of AGS-1. Moreover, two other genes (xanB and rpfF) were knocked out by CRISPR/Cas12a and identified as attachment-related genes in AGS-1. Also, this system could achieve point mutations. These data indicated that the CRISPR/Cas12a system could be an effective molecular platform for attachment gene function identification, which would be useful for the development of AGS in wastewater treatment. This article is protected by copyright. All rights reserved.

IMG-03. HARNESSING COMPUTATIONAL IMAGING, SCALABLE CLOUD-BASED WORKFLOWS, AND MULTI-MODAL DATA ANALYTICS WITH INTEROPERABLE SOFTWARE PLATFORMS IN PEDIATRIC NEURO-ONCOLOGY; THE ROAD AHEAD FOR CHILDREN’S BRAIN TUMOR NETWORK (CBTN)

Abstract Neuroimaging is acquired as part of the clinical standard of care for treatment of brain tumor patients. Capitalizing on this data and integration of imaging with other data types to perform predictive analytics is an increasing need. Here, we review the efforts at CBTN in the past couple of years to establish user-friendly workflows to support the entire imaging data lifecycle and to bridge access to multi-modal datasets with scalable analytics scale, to empower researchers to make breakthrough discoveries that will advance patient care. An automated deep learning-based brain extraction and tumor subregion segmentation model based on a multi-institutional dataset was developed that can reliably segment the MRI scans of children across a variety of brain tumor histologies. This model was subsequently integrated into an end-to-end imaging pipeline for collecting, managing, and analyzing clinically acquired radiology exams of the CBTN consortium with processing of 12,500 exams to date. A public website was also created to allow users to search and explore the dataset based on sequence labels, and clinical and imaging attributes. In addition, we established a scalable workflow and processes for de-identification, ingestion, quality control, and management of digital pathology slides on CBTN (over 8,000 slides to date). Finally, we established interoperability between imaging (Flywheel) and molecular (CAVATICA) platforms deployed in cloud ecosystems which allows streamlined, user-friendly workflows to ingest and harmonize files, perform cohort selection, prepare data using standard processing packages and cloud resources, and conduct analysis on extracted multi-modal feature sets. The described workflow lays the foundation for broad use of imaging studies and access to multi-modal datasets and analytics, with the goal of empowering researchers to make breakthroughs in patient care. The next step is to apply these tools in the context of clinical trials.

Novel organoboron complexes with robust core: Synthesis, functionalization, and subcellular targeting

The construction of novel organoboron complexes with facile synthesis and unique advantages for biological imaging remains a challenge and thus has garnered considerable attention. Herein, we developed a new molecular platform, boron indolin-3-one-pyrrol (BOIN3OPY) via a two-step sequential reaction. The molecular core is robust enough to allow for post-functionalization to produce versatile dyes. When compared to the standard BODIPY, these dyes feature an N,O-bidentate seven-membered ring center, significantly redshifted absorption, and a larger Stokes shift. This study establishes a new molecular platform that provides more flexibility for the functional regulation of dyes.

6-Substituted Triazolyl Benzoxaboroles as Selective Carbonic Anhydrase Inhibitors: In Silico Design, Synthesis, and X-ray Crystallography.

Benzoxaborole is currently a scaffold of great relevance in medicinal chemistry. In 2016, it was reported to be a new and valuable chemotype for designing carbonic anhydrase (CA) inhibitors. Herein, using an in silico design, we report the synthesis and characterization of substituted 6-(1H-1,2,3-triazol-1-yl)benzoxaboroles. 6-Azidobenzoxaborole was described for the first time as a molecular platform to prepare libraries of inhibitors by a copper(I)-catalyzed azide-alkyne cycloaddition via a click chemistry strategy. With inhibition constants below 30 nM, some derivatives, such as compound 20, showed efficacy as selective hCA VII and IX inhibitors. The design hypothesis was validated by crystallographic investigation on the hCA II/20 adduct, which provided explanations over the different inhibition behavior observed against the five evaluated hCA isoforms. Overall, this study identified 20 as a new promising lead compound to develop novel anticancer agents targeting the tumor-associated hCA IX but also potent neuropathic pain relievers targeting hCA VII.

Nature's Needle: Harnessing Bacterial Molecular Syringes

Nature's Needle: Harnessing Bacterial Molecular Syringes

Nanoplasmonic amplification in microfluidics enables accelerated colorimetric quantification of nucleic acid biomarkers from pathogens.

Deployment of nucleic acid amplification assays for diagnosing pathogens in point-of-care settings is a challenge due to lengthy preparatory steps. We present a molecular diagnostic platform that integrates a fabless plasmonic nano-surface into an autonomous microfluidic cartridge. The plasmonic 'hot' electron injection in confined space yields a ninefold kinetic acceleration of RNA/DNA amplification at single nucleotide resolution by one-step isothermal loop-mediated and rolling circle amplification reactions. Sequential flow actuation with nanoplasmonic accelerated microfluidic colorimetry and in conjugation with machine learning-assisted analysis (using our 'QolorEX' device) offers an automated diagnostic platform for multiplexed amplification. The versatility of QolorEX is demonstrated by detecting respiratory viruses: SARS-CoV-2 and its variants at the single nucleotide polymorphism level, H1N1 influenza A, and bacteria. For COVID-19 saliva samples, with an accuracy of 95% on par with quantitative polymerase chain reaction and a sample-to-answer time of 13 minutes, QolorEX is expected to advance the monitoring and rapid diagnosis of pathogens.

Structural annotation, semi-quantification and toxicity prediction of pyrrolizidine alkaloids from functional food: In silico and molecular networking strategy.

Many traditional Chinese herbs contain pyrrolizidine alkaloids (PAs), which have been reported to be toxic to livestock and humans. However, the lack of PAs standards makes it difficult to effectively conduct a risk assessment in the varied components of traditional Chinese medicine. It is necessary to propose a suitable strategy to obtain the representative occurrence data of PAs in complex systems. A comprehensive approach for annotating the structures, concentration, and mutagenicity of PAs in three Chinese herbs has been proposed in this article. First, feature-based molecular networking (FBMN) combined with network annotation propagation (NAP) on the Global Natural Products Social Molecular Networking web platform speeds up the process of annotating PAs found in Chinese herbs. Second, a semi-quantitative prediction model based on the quantitative structure and ionization intensity relationship (QSIIR) is used to forecast the amounts of PAs in complex substrates. Finally, the T.E.S.T. was used to provide predictions regarding the mutagenicity of annotated PAs. The goal of this study was to develop a strategy for combining the results of several computer models for PA screening to conduct a comprehensive analysis of PAs, which is a crucial step in risk assessment of unknown PAs in traditional Chinese herbal preparations.

Myosin and upalpha-actinin regulation of stress fiber contractility under tensile stress

Stress fibers are actomyosin bundles that regulate cellular mechanosensation and force transduction. Interacting with the extracellular matrix through focal adhesion complexes, stress fibers are highly dynamic structures regulated by myosin motors and crosslinking proteins. Under external mechanical stimuli such as tensile forces, the stress fiber remodels its architecture to adapt to external cues, displaying properties of viscoelastic materials. How the structural remodeling of stress fibers is related to the generation of contractile force is not well understood. In this work, we simulate mechanochemical dynamics and force generation of stress fibers using the molecular simulation platform MEDYAN. We model stress fiber as two connecting bipolar bundles attached at the ends to focal adhesion complexes. The simulated stress fibers generate contractile force that is regulated by myosin motors and alpha-actinin crosslinkers. We find that stress fibers enhance contractility by reducing the distance between actin filaments to increase crosslinker binding, and this structural remodeling ability depends on the crosslinker turnover rate. Under tensile pulling force, the stress fiber shows an instantaneous increase of the contractile forces followed by a slow relaxation into a new steady state. While the new steady state contractility after pulling depends only on the overlap between actin bundles, the short-term contractility enhancement is sensitive to the tensile pulling distance. We further show that this mechanical response is also sensitive to the crosslinker turnover rate. Our results provide new insights into the stress fiber mechanics that have significant implications for understanding cellular adaptation to mechanical signaling.

Metaphore Biotechnologies launches with molecular mimicry platform

After 2 years of development at Flagship Pioneering, the start-up Metaphore Biotechnologies emerged on May 16 with $50 million of series A funding. It plans to tackle autoimmune and metabolic diseases, as well as cancer. Metaphore’s approach to drug discovery is to mimic and improve on pharmacophores—abstractions that describe the steric and electronic features needed for bioactive molecules to interact with a biological target and unlock a response in the body. “It all started actually with us asking the question: What if we could mimic any molecular interaction in a programmable way?” says Lovisa Afzelius, Metaphore’s CEO and cofounder. The company condenses the key interaction points of these pharmacophores and then builds a “mold.” This computational framework narrows down the potential molecular solutions from the vast chemical space available, making the optimization problem easier to solve. When building new compounds inside this mold, researchers use machine learning to optimize the

Ultra‐Fast Photonic Digital Polymerase Chain Reaction based on N‐Heterocyclic Carbene Self‐Assembled Monolayer

AbstractA molecular diagnosis of the respiratory syncytial virus (RSV) without bulky and expensive instrumentation is of great importance for the early detection and prevention in a fast‐spreading pandemic. However, the current representative diagnostic methods have the limitation of being time‐consuming, cost, the processing time for polymerase chain reaction (PCR), and inaccurate for lateral flow assay (LFA), representatively. Herein, an integrated photonic digital PCR (dPCR) is developed with high‐velocity photonic scanner for in situ fluorescence detection by introducing the N‐heterocyclic carbene self‐assembled monolayer‐based Au film to prevent the quenching effect. The on‐site rapid molecular diagnostic platform shows the driving of 40 cycles in under 8 min and fluorescence scanning in under 7 min, resulting in a total analysis time within 15 min. In particular, the technology clearly demonstrates the classification of SARS‐CoV‐2 patients and healthy controls (99% in sensitivity, 98.6% in specificity, and 96.4% in accuracy with RdRp gene), comparing with standard RT‐qPCR. This platform can be utilized for prompt point‐of‐care molecular diagnostics in early diagnosis and large‐scale prevention of next pandemic spreading for upcoming infectious diseases and for the distinction diagnosis with other RSV.

Metaphore Biotechnologies launches with molecular mimicry platform

Metaphore Biotechnologies launches with molecular mimicry platform

The feasibility and acceptability of integrating hepatitis C and HIV diagnostic testing on centralized molecular laboratory platforms in Myanmar.

In Myanmar, 1.3 million people have been exposed to hepatitis C (HCV). However, public sector access to viral load (VL) testing for HCV diagnosis remains limited; ten near-point-of-care (POC) devices are available nationally. Myanmar's National Health Laboratory (NHL) has surplus capacity on centralized molecular testing platforms used for HIV diagnostics, presenting an opportunity for integrating HCV testing to expand overall testing capacity. This pilot assessed the operational feasibility and acceptability of HCV/HIV integrated testing implemented with a comprehensive package of supportive interventions. HCV VL samples were collected prospectively from consenting participants at five treatment clinics and tested at Myanmar's NHL (October 2019-February 2020) on the Abbott m2000. To optimize integration, laboratory human resources were bolstered, staff trainings were offered, and existing laboratory equipment was serviced/repaired as needed. Diagnostics data during the intervention period were compared against HIV diagnostics data in the seven months prior. We conducted three time and motion analyses at the laboratory and semi-structured interviews with laboratory staff to assess time needs and program acceptability. 715 HCV samples were processed during the intervention period with an average test processing time of 18 days (IQR: 8-28). Despite adding HCV testing, average monthly test volumes were 2,331 for HIV VL and 232 for early infant diagnosis (EID), comparable to the pre-intervention period. Processing times were 7 days for HIV VL and 17 days for EID, also comparable to the pre-intervention period. HCV test error rate was 4.3%. Platforms utilization increased from 18.4% to 24.6%. All staff interviewed were supportive of HCV and HIV diagnostics integration; suggestions were made for broader implementation and expansion. With a package of supportive interventions, integration of HCV and HIV diagnostics on a centralized platform was operationally feasible, did not adversely impact HIV testing, and was acceptable to laboratory staff. In Myanmar, integrated HCV VL diagnostic testing on centralized platforms may be an important addition to existing near-POC testing in expanding national testing capacity for HCV elimination.

Synthesis and Characterization of Tetraphenylethene AIEgen-Based Push-Pull Chromophores for Photothermal Applications: Could the Cycloaddition-Retroelectrocyclization Click Reaction Make Any Molecule Photothermally Active?

Three new tetraphenylethene (TPE) push-pull chromophores exhibiting strong intramolecular charge transfer (ICT) are described. They were obtained via [2 + 2] cycloaddition-retroelectrocyclization (CA-RE) click reactions on an electron-rich alkyne-tetrafunctionalized TPE (TPE-alkyne) using both 1,1,2,2-tetracyanoethene (TCNE), 7,7,8,8-tetracyanoquinodimethane (TCNQ) and 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ) as electron-deficient alkenes. Only the starting TPE-alkyne displayed significant AIE behavior, whereas for TPE-TCNE, a faint effect was observed, and for TPE-TCNQ and TPE-F4-TCNQ, no fluorescence was observed in any conditions. The main ICT bands that dominate the UV-Visible absorption spectra underwent a pronounced red-shift beyond the near-infrared (NIR) region for TPE-F4-TCNQ. Based on TD-DFT calculations, it was shown that the ICT character shown by the compounds exclusively originated from the clicked moieties independently of the nature of the central molecular platform. Photothermal (PT) studies conducted on both TPE-TCNQ and TPE-F4-TCNQ in the solid state revealed excellent properties, especially for TPE-F4-TCNQ. These results indicated that CA-RE reaction of TCNQ or F4-TCNQ with donor-substituted are promising candidates for PT applications.

Ability of the MeltPro MTB/PZA Assay to Detect Susceptibility to Pyrazinamide in Rifampin-Resistant Tuberculosis Patients.

Prediction of susceptibility to pyrazinamide (PZA) directly from sputum has been challenging. The MeltPro MTB/PZA assay, based on melting curve analysis, can simultaneously detect Mycobacterium tuberculosis and the resistance to PZA from sputum. We aimed to evaluate the MeltPro MTB/PZA assay to predict PZA resistance among rifampin-resistant tuberculosis (RR-TB) patients. We prospectively enrolled RR-TB patients in the registered trials, and their baseline sputum samples were obtained to perform the assay and culture. DNA sequencing of culture isolates was analyzed and used as the reference standard. Sanger sequencing was performed for samples with discrepant results between next-generation sequencing (NGS) and the investigational assay. The main analysis was conducted in the population of patients with interpretable results by both NGS and the assay. A total of 239 patients with RR-TB were screened, and 220 underwent the MeltPro MTB/PZA assay. The assay provided no information for 25 of 220 patients (11.4%). Among the remaining 195 patients, 13 had negative culture or insufficient raw NGS sequencing data, and 15 had indeterminate assay results. A total of 167 patients were included in the main analysis. Against DNA sequencing, the sensitivity, specificity, and negative predictive value of the assay for detecting resistance to PZA were 91.4% (95% confidence interval [CI], 87.1% to 95.6%), 89.9% (95% CI, 85.3% to 94.5%), and 95.2% (95% CI, 91.9% to 98.4%), respectively. In conclusion, the MeltPro MTB/PZA assay is a fast semiautomatic molecular platform to rapidly predict resistance to PZA from sputum and holds promise as a screening tool with satisfactory sensitivity. IMPORTANCE This study evaluated the accuracy of the MeltPro MTB/PZA assay at detecting the presence of PZA resistance through registered clinical trials. Compared to DNA sequencing, the assay had high sensitivity and negative predictive value, suggesting its potential utility as a screening tool in clinical practice. The assay could serve as an ideal primary screening tool in low PZA-resistant M. tuberculosis prevalence settings and could be used as an additional test to identify PZA resistance rapidly and initially in the RR-TB population.

Seasonality and Climatic Factors Associated With Human Rhinovirus/Enterovirus Nasopharyngeal Sample Positivity on Oahu, Hawai'i, 2016-2019.

Globally, human rhinoviruses/enteroviruses (HRVs/ENTs), indistinguishable on many widely available molecular platforms, are among the leading causes of the common cold. Geographic and climatic factors impact the peak activity of these viruses. In temperate climates, the peak activity of HRV occurs during autumn and spring whereas that of ENT occurs during autumn and summer. Both viruses are thought to peak during the rainy season in tropical climates like Hawai'i; however, data remain limited. We describe HRV/ENT seasonality and evaluate the climatic factors associated with peak activity among respiratory viral samples processed on Oahu, Hawai'i. We conducted a retrospective analysis of all respiratory specimens submitted to Tripler Army Medical Center for multiplex polymerase chain reaction testing between May 2016 and May 2019. Among HRV/ENT-positive samples, we recorded the month and year of positivity. Summative monthly positive detection was calculated with peak months above the mean. Associations between temperature, precipitation levels, relative humidity, and wind speed by week and the number of positive samples for HRV/ENT were evaluated using Poisson regression. This analysis was conducted via IRB exempt protocol number 19R18. During our study period, there were 7,143 nasopharyngeal respiratory samples sent for multiplex polymerase chain reaction testing, with 1,572 positive for HRV/ENT (22%). Nineteen percent of respiratory samples positive for HRV/ENT were additionally positive for one or more respiratory pathogens. The majority of HRV/ENT-positive samples arose from children < 5 years of age (n = 959, 61%). Peak months were February, March, May, August, November, and December. After controlling for lagged count and year, average wind speed was the only climatic factor significantly associated with HRV/ENT sample positivity. The peak monthly activity of HRV/ENT was similar to temperate climates with the exception of peak activity in February. Unlike other tropical climates, lower wind speed was associated with increased weekly HRV/ENT positivity and should be further explored as a transmission factor. Our study contributes to understanding the annual variability of HRV/ENT activity in tropical environments, which can inform clinician expectations regarding respiratory viral symptomatology in this region.

Performance of Ultra-Rapid Idylla™ EGFR Mutation Test in Non-Small-Cell Lung Cancer and Its Potential at Clinical Molecular Screening.

The Idylla™ EGFR Mutation Test is an ultra-rapid single-gene test that detects epidermal growth factor receptor (EGFR) mutations using formalin-fixed paraffin-embedded specimens. Here, we compared the performance of the Idylla EGFR Mutation Test with the Cobas® EGFR Mutation Test v2. Surgically resected NSCLC specimens obtained at two Japanese institutions (N = 170) were examined. The Idylla EGFR Mutation Test and the Cobas EGFR Mutation Test v2 were performed independently and the results were compared. For discordant cases, the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 was performed. After the exclusion of five inadequate/invalid samples, 165 cases were evaluated. EGFR mutation analysis revealed 52 were positive and 107 were negative for EGFR mutation in both assays (overall concordance rate: 96.4%). Analyses of the six discordant cases revealed that the Idylla EGFR Mutation Test was correct in four and the Cobas EGFR Mutation Test v2 was correct in two. In a trial calculation, the combination of the Idylla EGFR Mutation Test followed by a multi-gene panel test will reduce molecular screening expenses if applied to a cohort with EGFR mutation frequency >17.9%. We demonstrated the accuracy and potential clinical utility of the Idylla EGFR Mutation Test as a molecular screening platform in terms of turnaround time and molecular testing cost if applied to a cohort with a high EGFR mutation incidence (>17.9%).

Multi-Centered Field Evaluation of a Salmonella spp. Point-of-Care PCR Assay Using Equine Feces and Environmental Samples

The introduction of microfluidic card technology has opened the field for rapid point-of-care (POC) molecular assays, including fecal and environmental Salmonella spp. testing. The purpose of this study was to evaluate a novel POC PCR assay for the detection of Salmonella spp. in feces and environmental samples. A total of 143 fecal samples and 132 environmental samples were collected for POC PCR Salmonella spp. testing as well as qPCR testing. Each sample was inoculated into selenite broth and incubated for 18 to 24 hours. For the POC PCR assay, 14 μl of selenite broth were mixed with 126 μl of PCR reaction mix and pipetted into a microfluidic test card targeting the invA and ttrC gene of Salmonella enterica. For qPCR analysis, 200 µl of the selenite broth were processed for DNA purification and Salmonella spp. testing targeting the invA gene. The overall agreement between the POC PCR Salmonella spp. assay and qPCR assay was 88.1% for feces and 97.0% for environmental samples. Strong agreement and short turn-around-time make the POC device the first molecular diagnostic platform allowing detection of Salmonella spp. in a hospital setting without having to ship out samples to a veterinary diagnostic laboratory. The availability of an accurate POC PCR assay for the detection of Salmonella spp. will enhance the diagnostic capability of equine veterinarians to timely support a diagnosis of salmonellosis and also monitor the environment in order to reduce the risk of nosocomial infections.