Lowest Measurable Concentration Research Articles

309. Detection of hemagglutinin H5 influenza A virus sequence in municipal wastewater solids during an outbreak of avian influenza

Abstract Background Influenza is a significant cause of illness and death worldwide. Influenza A virus is responsible for all influenza epidemics, and some strains, such as highly pathogenic avian influenza (HPAI), have zoonotic reservoirs and potential. In 2024, an outbreak of HPAI H5N1 clade 2.3.4.4b, spread to dairy cattle in the United States, raising concerns about potential contributions to wastewater from animal sources. Wastewater surveillance has become a routine tool for monitoring influenza outbreaks due to providing population-level data in near real-time, making it uniquely suited to detection of emerging pathogens. H5 gene concentrations in wastewater solids during cattle outbreaks of avian influenza, TexasFigure 1.Concentrations of IAV M gene target (labeled as IAV, in green) as well as the H5 marker (labeled H5) and associated standard error in units of copies per gram dry weight; if error bars cannot be seen, then they are smaller than the symbols. Open markers are below the lowest measurable concentration (1000 copies per gram dry weight). Methods We developed a hydrolysis probe digital droplet RT-PCR assay for the detection of the H5 hemagglutinin gene of IAV and demonstrated it to be 100% specific and 90% sensitive based on in-silico and in-vitro testing against panels of human viruses including other IAV subtypes. We identified wastewater treatment plants (WWTPs) with aseasonal increases in influenza A concentrations. Clinical data was aggregated from influenza-related emergency health visits and statewide positivity rates and used for comparison to measured influenza concentrations. A timeline of events related an avian influenza outbreak in dairy cattleFigure 2.Top three panels. Concentrations of the IAV M gene in wastewater solids at the three WWTPs in Texas, as measured in the prospective monitoring. Line represents the 5-adjacent sample trimmed average, the symbols show the measured concentrations and the standard errors. Fourth panel from the top shows emergency department visits per week for influenza; PHR 1 represents Texas Public Health Region 1 (containing Potter/Randall Counties) and PHR2/3 represents Public Health Region 2/3 containing Dallas County. Bottom panel shows the presence or absence of the H5 marker in the samples from the three plants. Vertical lines indicate key dates as follows. February 8, 2024 USDA declares ongoing HPAI poultry outbreak, March 7, 2024: Unknown dairy cattle illness first reported, March 20, 2024: Samples collected from dairy cattle in Texas, March 25, 2024: Texas confirms H5N1 in dairy cattle, March 28, 2024: Positive H5N1 specimen collected from human. Results During the monitoring period, 59 WWTPs showed increases in the IAV M gene after the seasonal influenza period that coincided with the emergence of HPAI H5N1 in dairy cattle. Retrospective testing of samples from three WWTPs in Texas revealed the presence of the H5 marker at concentrations approaching those of the IAV M gene. The detection of the H5 marker in wastewater occurred before the official confirmation of H5N1 in dairy cattle and detection of a human case in Texas. Two of the WWTPs in Texas were confirmed to have permitted discharges from animal product processing facilities, including dairies, which is the proposed source of the H5 signal in wastewater. Conclusion This study demonstrates the ability of wastewater monitoring to detect contributions from zoonotic influenza, highlighting the need to consider industrial and agricultural inputs into wastewater systems. The detection of H5 in wastewater could have provided an early warning of the HPAI H5N1 outbreak before official confirmation, illustrating the value of wastewater surveillance when dealing with rapidly emerging pathogens. Disclosures All Authors: No reported disclosures

Toxicology and Pharmacokinetics Study of Intradiscal Injection of Simvastatin in Rabbits.

To test the pharmacokinetics and toxicology of whole organs and tissues after intradiscal injection of simvastatin in rabbits. To provide the information needed to support human clinical trials. Twelve male and twelve female rabbits were randomly divided into four groups: control group (0 mg/ml), low dose group (0.1 mg/ml), medium dose group (1 mg/ml) and high dose group (10 mg/ml). Simvastatin at different concentrations of 10 μl was injected into L3/4, L4/5 and L5/6 intervertebral discs in each group. Poly (ethylene glycol) -poly (lactic-co-glycolic acid) -poly (ethylene glycol) (PEG-PLGA-PEG) polymer as the drug carrier. The pharmacokinetics of blood samples were measured by LC-MS/MS. Cerebrospinal fluid was obtained and the drug concentration was measured. Blood routine, blood biochemistry and urine of all animals were analyzed and evaluated. The heart, kidney, liver and spleen of each animal were observed and weighed. The intervertebral disc tissues were stained with hematoxylin and hematoxylin (H&E), and then qualitatively analyzed by optical microscopy. 28 days after intradiscal injection of simvastatin, 28 days after simvastatin intradiscal injection, there was no significant difference between the weight, food residue, blood routine, blood biochemistry, urine routine results and the weight of each organ in the four groups (p > 0.05). The serum concentration of simvastatin is lower than the lowest measurable concentration. The histological score of the intervertebral disc in the high-dose group was significantly higher than that in the other three groups at 28 days (p < 0.05). Three doses of simvastatin were injected into male and female animals respectively, showing no toxic effects. Microscopic histological evaluation of the intervertebral disc showed that the high dose group (10 mg/ml) had damage to the intervertebral disc tissue.

Association of lead-exposure risk and family income with childhood brain outcomes.

Socioeconomic factors influence brain development and structure, but most studies have overlooked neurotoxic insults that impair development, such as lead exposure. Childhood lead exposure affects cognitive development at the lowest measurable concentrations, but little is known about its impact on brain development during childhood. We examined cross-sectional associations between brain structure, cognition, geocoded measures of the risk of lead exposure, and sociodemographic characteristics in 9,712 9- and 10-year-old children. Here, we show stronger negative associations of living in high lead-risk census tracts in children from lower- versus higher-income families. With increasing risk of exposure, children from lower-income families exhibited lower cognitive test scores, smaller cortical volume, and smaller cortical surface area. Reducing environmental insults associated with lead-exposure risk might confer greater benefit to children experiencing more environmental adversity, and further understanding of the factors associated with high lead-exposure risk will be critical for improving such outcomes in children.

NIMG-70. A LABEL-FREE APPROACH TO ASSESS CHEMOTHERAPY DRUG CONCENTRATION USING CHEMICAL EXCHANGE SATURATION TRANSFER MRI – A FEASIBILITY STUDY

Abstract BACKGROUND Drug delivery is one of the most pressing problems in neuro-oncology as the blood-brain barrier (BBB) uniquely limits penetration of substances into the brain parenchyma. Plasma and CSF drug concentrations are relatively easy to measure, but do not necessarily reflect concentrations in the brain. Furthermore, invasive measurements may be inaccurate in regions of heterogeneous BBB integrity. Advanced non-invasive imaging approaches may help bridge this information gap without the added risk. Chemical exchange saturation transfer (CEST) is an MRI technique that allows in vivo detection of molecules with a suitable hydrogen exchange rate, such as methotrexate (MTX). High-dose MTX is commonly used in oncology and its concentrations in urine (&gt;2000µM), plasma (&gt;1000µM) and enhancing brain tumors (&gt;350µM) are well above the theoretical threshold of CEST. AIM: We aimed to confirm the CEST detectability of MTX in vitro, and to estimate the currently lowest measurable concentrations in solutions mimicking bodily fluids. METHODS We used a 9.4T MRI to assess the spectra of MTX at 37°C at various concentrations (0.1–10.0 mM) in a phosphate-buffered saline (PBS) solution at various pH (6.0–8.0), as well as in synthetic urine at pH 6.2. RESULTS CEST signals attributable to MTX at 1.5 and 2.7ppm were successfully detected in PBS at concentrations as low as 500µM. The optimal field strength was 3.6µT. While increasing pH increased the detection threshold of the 2.7ppm signal, the 1.5ppm signal was minimally affected by pH within physiologic ranges. In synthetic urine, the MTX CEST signal was well-detectable even at concentrations as low as 200µM. CONCLUSIONS These preliminary results suggest that MTX-CEST may be feasible at field strengths achievable in clinical scanners and at MTX concentrations previously measured in enhancing brain tumors treated with high-dose MTX. Further optimization of the technique for in human use is under way.

Monitoring microbial population dynamics at low densities

We propose a new and simple method for the measurement of microbial concentrations in highly diluted cultures. This method is based on an analysis of the intensity fluctuations of light scattered by microbial cells under laser illumination. Two possible measurement strategies are identified and compared using simulations and measurements of the concentration of gold nanoparticles. Based on this comparison, we show that the concentration of Escherichia coli and Saccharomyces cerevisiae cultures can be easily measured in situ across a concentration range that spans five orders of magnitude. The lowest measurable concentration is three orders of magnitude (1000×) smaller than in current optical density measurements. We show further that this method can also be used to measure the concentration of fluorescent microbial cells. In practice, this new method is well suited to monitor the dynamics of population growth at early colonization of a liquid culture medium. The dynamic data thus obtained are particularly relevant for microbial ecology studies.

An electro-optic modulated heterodyne interferometer for measuring low glucose concentration

An electro-optic modulated heterodyne interferometer using the phase-lock technique for measuring the optical rotation angle corresponding to glucose concentration is presented. By inserting a half-wave plate behind the test sample and using the heterodyne interferometric phase-lock technique, it demonstrates a high sensitivity and is capable of measuring low glucose concentration. The validity of the proposed design is demonstrated by the measurements of rotation angles in a half-wave plate sample and the glucose sample, respectively. A correlation coefficient value of 0.999993 indicates a high linear response between the reference rotation angle and the measured rotation angle in a half-wave plate. Moreover, a standard deviation in rotation angle level of 5.9○ × 10−4 has been obtained for glucose solutions with concentrations ranging from 0 to 1.2 g dl−1, with a correlation coefficient value of 0.99989 between the measured rotation angle and the glucose concentration. A standard deviation of rotation angle around 1.5○ × 10−4 is achieved according to the repeatability experiments, and the relative error of rotation angle corresponding to the lowest measurable concentration of 0.01 g dl−1 has been determined to be 5.88%.

Occurrence of ochratoxin A in sweet wines produced in Spain and other countries

A survey for the presence of ochratoxin A (OTA) was undertaken from 2001 to 2005 in 188 samples of sweet wines produced in Spain and in 102 samples originating from other countries: France (n = 49), Austria (9), Chile (9), Portugal (9), Greece (6), Italy (5), Germany (3), Hungary (2), Slovenia (2), Switzerland (2), Canada (1), Japan (1), New Zealand (1), Ukraine (1), South Africa (1) and the USA (1). The analytical method was based on immunoaffinity chromatography clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. The limit of detection (defined as a signal–noise ratio = 3) was estimated to be 0.01 µg l−1. The limit of quantification (0.02 µg l−1) was checked as being the lowest measurable concentration. OTA was detected in 281 out of 290 samples analysed (96.9% positive) at concentrations ranging from 0.01 to 4.63 µg l−1. The overall mean and median levels were estimated to be 0.50 and 0.14 µg l−1, respectively. In Spanish sweet wines OTA was found in 99% of the samples, with mean and median values of 0.65 and 0.19 µg l−1, respectively. The mean value obtained in this study for OTA in the Spanish sweet wines would result in an intake of about 37.5 and 3.2 ng day−1 of OTA for regular consumers and for the overall population, respectively. These figures represent a minor contribution to the provisional tolerable weekly intake (PTWI) or TWI established by the Joint Expert Committee on Food Additives (JECFA) and the European Food Safety Authority: 3.8 and 3.1% for regular consumers; and 0.4 and 0.3% for the whole adult population, respectively.

A simple bubbling system for measuring radon ( 222Rn) gas concentrations in water samples based on the high solubility of radon in olive oil

Based on the different levels of solubility of radon gas in organic solvents and water, a bubbling system has been developed to transfer radon gas, dissolving naturally in water samples, to an organic solvent, i.e. olive oil, which is known to be a good solvent of radon gas. The system features the application of a fixed volume of bubbling air by introducing a fixed volume of water into a flask mounted above the system, to displace an identical volume of air from an air cylinder. Thus a gravitational flow of water is provided without the need for pumping. Then, the flushing air (radon-enriched air) is directed through a vial containing olive oil, to achieve deposition of the radon gas by another bubbling process. Following this, the vial (containing olive oil) is measured by direct use of gamma ray spectrometry, without the need of any chemical or physical processing of the samples. Using a standard solution of 226Ra/ 222Rn, a lowest measurable concentration (LMC) of radon in water samples of 9.4 Bq L −1 has been achieved (below the maximum contaminant level of 11 Bq L −1).

Development of a Sensitive Luminometric Immunoassay for Determining Baseline Seasonal Changes in Serum Vitellogenin Levels in Male Flounder (Pleuronectes Yokohamae)

Vitellogenin is a sensitive biomarker used to study the effects of artificial estrogens in aquatic environments. We developed and optimized a luminometric immunoassay that was able to detect trace amounts of vitellogenin in the serum of male flounder collected from an uncontaminated reference site. The lowest measurable concentration of vitellogenin in the serum was approximately 0.08 ng/ml with purified protein diluted in buffer. The reproducibility of the vitellogenin measurements was determined by the analysis of triplicate samples and found to be about 5.3%, based on serum samples containing vitellogenin at 2.0 ng/ml. This method was successfully applied to samples collected from an uncontaminated reference site for the monitoring of baseline levels of serum vitellogenin in male flounder.

Determination of aniline traces in nitrobenzene by the facilitated proton transfer across the water/nitrobenzene interface

The proposed analytical method is based on the voltammetrical investigation of the interface between two immiscible electrolyte solutions. Transfer of different ions across the interface between water and nitrobenzene can be observed by using cyclic voltammetry. Facilitated transfer of protons from aqueous into nitrobenzene phase can be observed if there is present a suitable organic base like, e.g., aniline in the nonaqueous media. From the peak current the amount of aniline in nitrobenzene can be established. The lowest measurable concentration was 5 × 10 −5 M 4.56 mg of aniline per liter of nitrobenzene.

Reversed-phase liquid-chromatographic simultaneous analysis for thiopental and pentobarbital in serum.

We describe a reversed-phase liquid-chromatographic assay suitable for therapeutic monitoring of thiopental and pentobarbital simultaneously in human serum. The drugs are extracted from serum at pH 6.6 into n-butyl chloride containing thiamylal and barbital as the respective internal standards. The compounds are back-extracted into dilute sodium hydroxide, an aliquot of which is submitted to chromatography. The lowest measurable concentrations are 1.0 mg/L for thiopental and 2.0 mg/L for pentobarbital. The standard curve is linear from 0 to 100 mg/L for both. Between-run CVs are: at 25 mg/L, 4.1% (thiopental) and 3.2% (pentobarbital); at 50 mg/L, 2.8% (thiopental) and 3.4% (pentobarbital). Data on patients receiving thiopental and pentobarbital illustrate use of the method.

Assay of 3-carboxy-antipyrine in urine by capillary gas chromatography with nitrogen selective detection. Some preliminary results in man.

An assay procedure is described for 3-carboxy-antipyrine in urine using gas chromatography with a support coated open tubular capillary column (Carbowax 20M), a solid injection system and nitrogen selective detection. 3-Carboxy-antipyrine was analyzed after derivatization with diazomethane and using 4-bromo-antipyrine as internal standard. Following extraction of the urine samples with a mixture of organic solvents linear calibration curves were obtained in the concentration range of 1--50 microgram/ml. The precision was established as +/- 5.0% (n = 5), and the lowest measurable concentration was 1 microgram/ml at a signal-to-noise ratio of 10:1. Preliminary results in 5 human volunteers showed that after orl administration of 500 mg antipyrine, 3.3 +/- 0.8% of the dose was excreted as 3-carboxy-antipyrine in 52 hours' urine. The compound was excreted completely in the unconjugated form.

Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand's disease.

A solid phase non-competitive immunoradiometric assay (IRMA) has been developed which allows measurement of factor VIII related antigen (VIIIR:AG) levels in normal plasma as low as 2.5 x 10(-4) U/ml. The assay is based on the extraction of VIIIR:AG from test plasma by means of polystrene tubes coated with a specific unlabelled anti-VIIIR:AG rabbit antiserum and subsequent labelling of the extracted antigen with 125I-labelled anti-VIIIR:AG rabbit IgG.

Self-association of the major protein component of bovine serum high density lipoprotein

The major protein component of bovine high density lipoprotein was investigated in solution by fluorescence polarization and ultracentrifugal techniques. A fluorescent derivative of this protein with 1-dimethylaminonaphthalene-5-sulfonyl chloride was employed in the fluorescence experiments. Over the concentration range from 5·10 −7 M to 5·10 −4 M of the protein monomer at pH values from 2 to 11 and ionic strengths from 0.03 to 2.0, at 23 °C, the major protein of bovine high density lipoprotein apoprotein (Apo-HOL-I) was found to exist in a stable aggregated form. The aggregate was not affected by dioxane additions of up to 20% nor by Triton X-100 to 0.2%, but dissociated readily in the presence of 0.07% sodium dodecylsulfate or 6 M urea. At concentrations below 5·10 −7 M, dissociation of the protein aggregate started spontaneously and continued down to 10 −8 M, the lowest measurable concentration. Several physicochemical properties of the major protein of bovine high density lipoprotein were determined in the stable aggregate form. Molecular weight was 104 000 from ultracentrifugal analysis and 80 000 from gel-filtration. Rotational relaxation time was 115 ns at 25 °C, and s 0 20,w was 4.78 s. The results suggest very strong protein-protein interactions ( K d < 10 −7 M) that are not electrostatic in nature. Hydrophobic interactions of a magnitude that could be affected by 20% dioxane or 0.2% Triton X-100 detergent are also excluded. There is saturation of the interaction sites by the aggregation of a few protein monomer units possibly to form a tetramer which is moderately asymmetric (1:4 axial ratio, assuming an ellipsoid of revolution) and relatively rigid. The strong protein-protein interactions in this pure apolipoprotein suggest the possibility of competition of inter-protein associations with protein-lipid interactions in in vitro lipid binding or lipoprotein reconstitution experiments.