Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Plasma and cerebrospinal fluid pharmacokinetics of ondansetron in humans.

AIMS: Changes in serotonergic sensory modulation associated with overexpression of 5-HT3 receptors in the central nervous system (CNS) have been implicated in the pathophysiology of neuropathic pain after peripheral nerve damage. 5-HT3 receptor antagonists such as ondansetron can potentially alleviate neuropathic pain, but have limited effectiveness, due potentially to limited CNS access. However, there is currently limited information on CNS disposition of systemically-administered 5-HT3 receptor antagonists. This study evaluated the cerebrospinal fluid (CSF) disposition of ondansetron, as a surrogate of CNS penetration. METHODS: Fifteen patients were given a single 16 mg intravenous 15 minute infusion of ondansetron, followed by serial blood and a single CSF sampling. Population pharmacokinetic (PK) modelling was implemented to describe the average and individual plasma and CSF profiles of ondansetron. A two-compartmental model was used to capture ondansetron plasma PK with a single CSF compartment to describe distribution to the CNS. RESULTS: The individual model-estimated CSF to plasma partition coefficients of ondansetron were between 0.09 and 0.20. These values were mirrored in the calculated CSF penetration ratios, ranging from 0.08 to 0.26. CONCLUSIONS: After intravenous administration, CSF concentrations of ondansetron were approximately 7-fold lower than those observed in the plasma. A model could be developed to describe individual CSF concentration-time profiles of ondansetron based on a single CSF data point. The low CSF penetration of ondansetron may explain its limited analgesic effectiveness, and affords an opportunity to explore enhancing its CNS penetration for targeting conditions such as neuropathic pain.

Somatic mutations affect key pathways in lung adenocarcinoma.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pretreatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. Moreover, our saliva pretreatment protocol enabled sensitive viral detection by conventional quantitative reverse transcription polymerase chain reaction (qRT-PCR) without RNA extraction. We validated the high performance of these assays on clinical samples and demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.

Differential capture of serum proteins for expression profiling and biomarker discovery in pre- and posttreatment head and neck cancer samples.

INTRODUCTION: A long-term goal of our group is to develop proteomic-based approaches to the detection and use of protein biomarkers for improvement in diagnosis, prognosis, and tailoring of treatment for head and neck squamous cell cancer (HNSCC). We have previously demonstrated that protein expression profiling of serum can identify multiple protein biomarker events that can serve as molecular fingerprints for the assessment of HNSCC disease state and prognosis. METHODS: An automated Bruker Daltonics (Billerica, MA) ClinProt matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer was used. Magnetic chemical affinity beads were used to differentially capture serum proteins prior to MALDI-TOF analysis. The resulting spectra were analyzed using postprocessing software and a pattern recognition genetic algorithm (ClinProt 2.0). An HNSCC cohort of 48 sera samples from 24 patients consisting of matched pretreatment and 6 to 12 month posttreatment samples was used for further analysis. Low-mass differentially expressed peptides were identified using MALDI-TOF/TOF. RESULTS: In the working mass range of 1,000 to 10,000 m/z, approximately 200 peaks were resolved for ionic bead capture approaches. For spectra generated from weak cation bead capture, a k-nearest neighbor genetic algorithm was able to correctly classify 94% normal from pretreatment HNSCC samples, 80% of pretreatment from posttreatment samples, and 87% of normal from posttreatment samples. These peptides were then analyzed by MALDI-TOF/TOF mass spectometry for sequence identification directly from serum processed with the same magnetic bead chemistry or alternatively after gel electrophoresis separation of the captured proteins. We were able to compare this with similar studies using surface-enhanced laser desorption ionization (SELDI)-TOF to show this method as a valid tool for this process with some improvement in the identification of our groups. CONCLUSIONS: This initial study using new high-resolution MALDI-TOF mass spectrometry coupled with bead fractionation is suitable for automated protein profiling and has the capability to simultaneously identify potential biomarker proteins for HNSCC. In addition, we were able to show improvement with the MALDI-TOF in identifying groups with HNSCC when compared with our prior data using SELDI-TOF. Using this MALDI-TOF technology as a discovery platform, we anticipate generating biomarker panels for use in more accurate prediction of prognosis and treatment efficacies for HNSCC.

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%-91% versus 44%-61% for single region QIIME analyses (p < .01, p < .001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively. MVRSION represents a generally applicable tool for taxonomic classification that is superior to single-region 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Eikenella corrodens Sepsis with Cerebrospinal Fluid Pleocytosis in a Very Low Birth Weight Neonate.

We report a case of Eikenella corrodens sepsis associated with CSF pleocytosis in a very low birth weight neonate. A 1000-gram male neonate was born at 27-week gestation due to preterm labor. The patient presented with signs and symptoms of sepsis and was treated for suspected meningitis with intravenous ampicillin and gentamicin for 7 days, with cefotaxime added for three weeks. He had a normal brain MRI at discharge and normal development at 6 months of life. To our knowledge, this is the first case of E. corrodens sepsis and associated meningitis in a very low birth weight neonate.

Validation of a next-generation sequencing assay for clinical molecular oncology.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Injection order effects on efficacy of calcium chloride and sodium tripolyphosphate in controlling the pink color defect in uncured, intact turkey breast.

An experiment was conducted to test sequential injection of sodium tripolyphosphate (STP; 0.5% meat weight basis, mwb) followed by injection with or without addition of calcium chloride (CaCl(2), 500 ppm mwb), and to test the effect of post-injection delay prior to cooking. A second experiment evaluated the impact of injection order and delay time between independent addition of CaCl(2) (500 ppm mwb) and STP (0.5% mwb). Turkey was formulated without an added pink generating ligand (NONE), with nicotinamide (NIC; 0.1% mwb), or with sodium nitrite (NIT; 10 ppm mwb). A white colloid was observed in the extracellular space of treatments containing both STP and CaCl(2.) Addition of CaCl(2) decreased nitrosylhemochrome but did not reduce levels of nicotinamide hemochrome or CIE a(*) values. Injection order or delay between injections did not contribute to controlling the pink defect in cooked, intact turkey breast.