Samantha A. Byrnes

Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods

Background The global need for disease detection and control has increased effort to engineer point-of-care (POC) tests that are simple, robust, affordable, and non-instrumented. In many POC tests, sample collection involves swabbing the site (e.g., nose, skin), agitating the swab in a fluid to release the sample, and transferring the fluid to a device for analysis. Poor performance in sample transfer can reduce sensitivity and reproducibility. Methods In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices. Transfer efficiency was measured using quantitative PCR (qPCR) for Staphylococcus aureus under conditions representing a range of sampling scenarios: 1) spiking low-volume samples onto the swab, 2) submerging the swab in excess-volume samples, and 3) swabbing dried sample from a surface. Results Excess-volume samples gave the expected recovery for most swabs (based on tip fluid capacity); a polyurethane swab showed enhanced recovery, suggesting an ability to accumulate organisms during sampling. Dry samples led to recovery of ∼20–30% for all swabs tested, suggesting that swab structure and volume is less important when organisms are applied to the outer swab surface. Low-volume samples led to the widest range of transfer efficiencies between swab types. Rayon swabs (63 µL capacity) performed well for excess-volume samples, but showed poor recovery for low-volume samples. Nylon (100 µL) and polyester swabs (27 µL) showed intermediate recovery for low-volume and excess-volume samples. Polyurethane swabs (16 µL) showed excellent recovery for all sample types. This work demonstrates that swab transfer efficiency can be affected by swab material, structure, and fluid capacity and details of the sample. Results and quantitative analysis methods from this study will assist POC assay developers in selecting appropriate swab types and transfer methods.

Programming paper networks for point of care diagnostics

Lateral flow tests (LFTs) are well-suited for rapid point-of-care testing in low resource settings. The wicking action of the paper strip moves the sample and reagents through the device without a need for pumps, but LFTs are typically limited to tests that can be carried out in a single fluidic step. The materials from LFTs can be reconfigured to create paper networks that automatically carry out multi-step fluidic operations, while retaining the same easy-to-use format as a conventional LFT. Here, we describe basic principles of wicking and system-level behavior of paper networks by analogy to electrical circuits. We describe key design principles for a previously-developed 2D paper network (2DPN) and introduce an alternative linear paper network (Pseudo-1DPN) that takes advantage of system-level behavior to perform clean sequential fluid delivery while reducing device running time.

A disposable chemical heater and dry enzyme preparation for lysis and extraction of DNA and RNA from microorganisms

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a Gram-positive bacterium (Staphylococcus aureus) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus. After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Selecting analytical biomarkers for diagnostic applications: a first principles approach

ABSTRACT Introduction: Biomarkers are objective indications of a medical state that can be measured accurately and reproducibly. Traditional biomarkers enable diagnosis of disease through detection of disease-specific molecules, disease-mediated molecular changes, or distinct physiological or anatomical signatures. Areas covered: This work provides a framework for selecting biomarkers that are most likely to provide useful information about a patient’s disease state. Though the authors emphasize markers related to disease, this work is also applicable to biomarkers for monitoring physiological changes such as ovulation or pregnancy. Additionally, the scope was restricted to biomarkers that are amenable to analytical detection across a range of health care levels, including low resource settings. The authors describe trade-offs between biomarkers’ sensitivity/specificity for a disease-causing agent, the complexity of detection, and how this knowledge can be applied to the development of diagnostic tests. This report also details additional assessment criteria for successful tests. Expert commentary: Biomarker selection should primarily be driven by an attempt to answer an explicit clinical question (preferably causative relationship of the biomarker to disease-state), and only then by test development expediency (ease of detection). This framework is useful for stakeholders from test developers to clinicians to identify the trade-offs for diagnostic biomarkers for any use case.

Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRads ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.

A framework for selecting analytical biomarkers: A first principles approach

L are fructose polymers synthesized by a broad range of microorganisms and a limited number of plant species as nonstructural storage carbohydrates and they have potential applications in the pharmaceutical, food, and cosmetic industries. The present study shows a comparative analyses of polysaccharide type Levan biosynthesis in static and shaking fermentation by using Z. mobilis ATCC 10988 strain. All fermentation processes were carried out in Erlenmeyer flasks presenting a capacity of 500 ml and working volume of 100 ml culture medium, in both fermentation types: static and on the rotary shaker, with stirring of 220 rpm, with the temperature maintenance at 32°C for 72 hours. In figure 1, we can find the results obtained by static fermentation for different initial concentrations of sucrose. The best development of the microbial culture in terms of microbial biomass was seen for the initial concentration of 15% sucrose, due probably to the favorable ratio between the source of C and N. The largest amount of polysaccharide was obtained from the experiment with 40% initial sucrose (8.9 g%), but this value is also similar to the experiment with 25% initial sucrose (8.47 g%). The Figure No. 2 shows the results obtained by the stirred fermentation with the strain Z. mobilis ATCC 10988 for different initial concentrations of sucrose. Concerning the content of polysaccharide, if the initial concentration of sucrose in the biosynthesis medium was greater than 20%, the amount of polysaccharide was about 5 g%, without any increase of production in the case of higher concentrations. By comparing the results shown in figures 1 and 2, it can be noticed that the strain Z. mobilis ATCC 10988 performs better concerning the polysaccharide biosynthesis in the frame of a static fermentation, which is not a surprise considering that these bacteria are an optional aerobic one.S rufopilosa, a tsema rowan, is a species of the small ornamental trees in the genus Sorbus and the family Rosaceae found in East Asia. The antioxidant and anticancer effects of S. rufopilosa remain unclear. The objective of this study is to evaluate the antioxidant and anticancer effects of ethanol extract of S. rufopilosa (EESR) and the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells. EESR showed significant antioxidant activity and inhibitory effect on HT29 cell growth in a dose-dependent manner. EESR induced cell cycle arrest at G2/M phase in a dose-dependent manner by modulating the expression of cyclin B, cyclin-dependent kinase 1 (CDK1), and CDK inhibitor p21. EESR-induced apoptosis was associated with the upregulation of p53, a death receptor Fas, a pro-apoptotic protein Bax and the activation of caspase 3, 8, and 9, resulting in the degradation of poly ADP ribose polymerase (PARP). These results suggest that EESR efficiently inhibits proliferation of HT29 by inducing both cell cycle arrest and apoptosis, and may be a possible candidate for the anticancer drug development. Recent Publications: 1. Andreas G (2003) Introduction to apoptosis. ApoReview 2-26. 2. Evan GI, Vousden KH (2001) Proliferation, cell cycle and apoptosis in cancer. Nature 411:342-348. 3. Fulda S (2015) Targeting apoptosis for anticancer therapy. Semin. Cancer Biol. 31:84-88. 4. Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798-4811. 5. Udensi UK, Tchounwou PB (2016) Oxidative stress in prostate hyperplasia and carcinogenesis. J. Exp. Clin. Cancer Res. 35:139.I clinical drug development, it is important to understand the absorption, distribution, metabolism and excretion (ADME) properties of a drug in humans. The micro-tracer study based on the accelerator mass spectrometry (AMS) is an ultrasensitive technique to obtain human ADME profiles with a negligible radiation dose. KD101 is a novel compound under development to treat obesity. The aim of this study was to investigate the absorption, metabolism and excretion properties of KD101 in obese subjects. A randomized, open-label, single-dose, one-treatment, one-period, one-sequence study was conducted in six males with a BMI ≥27, who received KD101 at 400 mg with 3.52 μg of [14C]-KD101 (180 nCi) in the fed state. Plasma, urine and feces samples were collected up to 288 hours post-dose for mass balance and metabolite profiling. Plasma concentrations of KD101 were determined using a validated GC method. Total radioactivity in the samples was determined using AMS. Safety and tolerability was evaluated based on vital signs, adverse events, clinical laboratory tests, and electrocardiography. All of the subjects completed the study with no clinically significant safety issue. Mean total recovery rate (range) was 85.21% (75.3699.01%), consisting of 77.96% (68.31-92.33 %) for urine and 7.26% (5.91-8.51%) for feces, which differed greatly from the preclinical data. Oral absorption of [14C]-KD101 was rapid with the peak plasma concentration reaching at 5.83h post dose, which was consistent with the previous report. In the urine radiochromatogram, five large peaks were identified including a peak represented by the parent compound. KD101 is excreted predominantly through the urine in humans. Many of the excreted materials in the urine were considered metabolites. This study demonstrated effectiveness of the micro-tracer study enabled by AMS in humans to investigate the ADME property of KD101, which hugely differed from that seen in the preclinical animals.Objectives: YH4808 is a highly potent, selective and reversible potassium-competitive acid blocker (P-CAB) on H/K-ATPase under development for the treatment of gastric acid related diseases including gastroesophageal reflux disease and peptide ulcer disease. The objectives of this study were 1) to develop a human PBPK model optimized by human pharmacokinetic (PK) data and 2) to predict the PK profiles of YH4808 using the PBPK model in various clinical settings.Statement of the Problem: High performance liquid chromatography (HPLC, including UHPLC) is one of the most powerful separation techniques capable of providing analysis difficult or impossible with other separation approaches. Thus, chromatographers give particular importance to the design of new efficient stationary phases for HPLC. New trends in chromatographic separations have been directed towards the use of multi-modal stationary phases (MM-SPs) which are high resolution, high selectivity, high loading capacity, high speed, minimal solvent consumption compared with single-modal stationary phases (SM-SPs).Aim: To compare the serum levels of adiponectin, vitamin D, copper and zinc in rheumatoid arthritis (RA) patients and to investigate the relationship between these factors and disease severity. Method: Ninety patients with RA and 30 healthy individuals were participated in this study according to the ACR/ EULAR criteria for RA. Serum concentrations of adiponectin were determined by ELISA, copper and zinc by colorimetric spectrophotometry and vitamin D by HPLC methods. Results: Serum adiponectin and vitamin D were increased and decreased in RA patients, respectively. Adiponectin and disease severity are positively correlated, whereas vitamin D and disease severity are negatively correlated. Adiponectin negatively correlate with vitamin D and positively correlated with disease activity score (DAS). Copper and zinc showed no significant difference between two groups. Conclusion: The definitive roles of adiponectin, vitamin D, copper and zinc is not completely determined. More investigations are needed to deeply explore the impact of them on RA pathophysiology. Finally, we suggest these serum factors as promising diagnostic and therapeutic biomarkers.C stem cells (CSCs) possess characteristics associated with normal stem cells and may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. They involved in drug resistance, metastasis and relapse of cancers can significantly affect tumor therapy. Therefore, it is important to develop specific therapies targeted probe at CSCs for improvement of survival and quality of life of cancer patients. Studies have indicated that the CD166 protein has been considered as a specific marker for colorectal CSCs detection. In addition to monoclonal antibodies, small molecules such as anti-peptides could provide more advantages for CSCs detection in vivo. Here, we attended to design the CD166 antipeptide (CD166ap) to detecting the CSCs in vitro and in vivo. To obtain CSCs, the human colorectal cancer cells (HCT-15) were seeded into selection media (DMEM/F12, 0.4% bovine serum albumin, 2% B27, 5 μg/mL bovine insulin, 4 μg/mL heparin, 20 ng/mL fibroblast growth factor 2, and 20 ng/mL epidermal growth factor) at a density of 1000 cells per mL. Under these conditions, only CSCs and early progenitor cells survive and proliferate, whereas differentiated cells die. Next, we designed a CD166ap (amino acid: KDSEGYESYNGNLGSQC {It is known that human CD6 proteins have the ability to bind the human CD166 proteins specifically (Chappell PE et al., Structure. 2015, 23:1426-1436). Depending on the two proteins binding sites, we designed the amino acid sequence of CD166 anti-peptide. However, the Nand C-terminal amino acid (lysine and cysteine) were added for conjugating with fluorescence, nuclear medicine chelator and Polyethylene glycol (PEG).} and conjugated with fluorescence for CSCs binding assay by flow cytometry and immunofluorescence stain. For in vivo imaging detection, the media-induced CSCs (2×106) were subcutaneous inoculated into the right flank of nude mice (n=5 per each group) and grew for one week. Then, the fluorescence conjugated CD166ap was IV injected into animal model for in vivo imaging system and biodistribution assay. The primary spheres that derived from HCT-15 cells under serum-free conditions and which are highly enriched for CSCs at 48 hours. These induced CSCs overexpressed the reprogramming factors such as OCT4, c-myc, Nanog and anti-apoptosis factor (Survivin). Moreover, they also showed the characteristic of drug resistance compared with cancer cells. In CSCs targeted probe binding assay, the CD166ap and antibody revealed the quite binding capability in CSCs. The in vivo imaging assay, we found that CD166ap specifically targeted to CSCs-induced xenograft model and accumulated in tumor area. In conclusion, we designed a specific probe for CSCs detection in vivo successfully. In addition, the CD166ap may label radioisotope for nuclear medicine imaging and conjugate drug for CSCs therapy in clinical.L (LBL) coating has gained popularity for drug delivery of therapeutic drugs. Herein, we described an approach for enhancing the therapeutic efficiency of the locally administered dexamethasone (Dx) for the treatment of inflammatory bowel disease (IBD). We utilized a LBL-coating technique for alternative coating of Dx microcrystals (DxMCs) with multiple layers of polyelectrolytes composed of poly (allylamine hydrochloride), poly (sodium 4-styrene sulfonate) and Eudragit® S100. The successful deposition of the layers onto DxMCs surfaces were confirmed through zeta potential measurement and confocal laser scanning microscopy, while the surface morphology was investigated through scanning electron microscopy. The drug encapsulation efficiency for LBL-DxMCs was 95% with a mean particle size of 2 μm and negative surface charge of -45 mV. Moreover, in vitro drug release studies showed a minimum release of the drug ( 15%) at an acidic condition during initial first 5 h followed by sustained-release at alkaline condition. For in vivo study, LBL-DxMCs were administered orally to male ICR mice suffering from dextran sulfate sodium-induced colitis. LBL-DxMCs was found to substantially enhance antiinflammatory efficacy of the drug compared to uncoated DxMCs. Macroscopic, histological and biochemical (tumor necrosis factor-α, interleukin-6 and myeloperoxidase) examinations revealed marked improvements of colitis signs in the mice treated with LBL-DxMCs compared with those treated with uncoated DxMCs. Overall, the obtained results demonstrate that LBLDxMCs are an effective and safe colon-targeted delivery system for the treatment of inflammatory bowel disease.Results: Seven RCTs were found in the systematic review (639 patients) and were included in the meta-analysis. There is not clinical difference in the parasitological cure between MBZ and metronidazole (MTZ). The relative risk (RR) is 0.88 (95% CI 0.70-1.10), with a moderate heterogeneity (I2=66%). The prediction interval expected to cover the results of a new trial is wide enough (0.35-2.21) to support both a parasitological relevant difference favoring MBZ and a parasitological relevant difference favoring MTZ.L are fructose polymers synthesized by a broad range of microorganisms and a limited number of plant species as nonstructural storage carbohydrates and they have potential applications in the pharmaceutical, food, and cosmetic industries (1-5). This study presents a comparative analysis of the growth and consumption curves of Z. mobilis NCIB 1163 and Z. mobilis ATCC 10988, levan producing microorganisms. The growth and consumption curves were performed in a liquid medium with a concentration of 5% sucrose and 5% glucose. Thus, the bacterial cells in the exponential growth phase were centrifuged at 12.000 g and the pellet was washed twice with a sterile 0.9% NaCl solution. Finally, the cells were resuspended in 1 ml of physiological saline and they were used as inoculum in 5% liquid medium, which was monitored concerning the fermentable carbohydrate consumption and the growth. The consumption curves were performed under anaerobic conditions (the culture medium was coated with a layer of sterile paraffin oil) by periodical weighing of the medium seeded with different bacterial strains of Z. mobilis and by the graphical representation of weight loss (due to release of carbon dioxide). Exponentially growing cells were used as the inoculum, made at approximately 107 cells/ml. Cell growth was assayed turbidimetrically at a wavelength of 600 nm. The consumption curves of Z. mobilis NCIB 11163 and Z. mobilis ATCC 10988 bacterial strains on complete sucrose substrate medium, 5% under anaerobic conditions (Figure 1) revealed that strains NCIB 11163 and ATCC 10988 exhibit a similarly kinetics of consumptions substrate. The consumption curves of Z. mobilis NCIB 11163 and Z. mobilis ATCC 10988 bacterial strains on complete glucose substrate medium, 5% under anaerobic conditions (Figure 2) show that glycolysis is more intense than hydrolysis of sucrose. From the curves profile it is observed that the strain ATCC 10988 shows a curve having a more pronounced linear decrease.

Enabling lateral transport of genomic DNA through porous membranes for point-of-care applications

Paper-based nucleic acid diagnostics have the potential to translate laboratory assays to simple-to-use, point-of-care devices, but many prototypes of these systems still lack the ability to process realistic samples due to the inability of genomic-sized DNA to move through membranes with small pores. For applications involving pathogen or human gene identification, the ability to fragment and transport DNA would provide more options for device design and broaden the range of applications. To address this challenge, we have developed and characterized a method that combines cell lysis with DNA fragmentation to allow for lateral transport of genomic DNA through commonly-used porous membranes. Additionally, we demonstrate that varying heating time and temperatures allows for control of both lysis and fragmentation based on genome size. These data align with previously published models that describe both DNA denaturation and thermal scission. This level of control allows semi-selective transport of pathogenic DNA, which can reduce the amount of interference from non-target human DNA in downstream applications. This method can be easily automated and is rapid, occurring in less than 10 minutes with one user step.

Electromechanical cell lysis using a portable audio device

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.