Need For Multiple Samples Research Articles

Novel SARS‐CoV‐2 Inactivation Formulation Supports Extended COVID‐19 Testing/Surveillance

BackgroundWith the spread of highly infectious strains such as the Delta variant of SARS‐CoV‐2, rapid antigen testing and variant tracking are gaining more attention from infectious disease specialists and the general public as essentials for disease control and prevention amidst the COVID‐19 pandemic. The lateral flow based antigen tests provide fast turnaround of results within minutes, but it’s open ‐faced assay format means that the test operators are more prone to accidental exposure to pathogen‐containing samples. The viral inactivation transport media (ITMs) provide much needed protection against exposure to highly transmissive live viruses during sample collection, transport, and storage. However, the current ITMs were designed for PCR tests and share a harsh chemical formulation that denatures protein analytes, making them incompatible with antigen tests.We developed an innovative viral transport media that is designed for a variety of tests, such as antigen testing and PCR. The new formulation can be used to safely inactivate SARS‐CoV‐2 virus while maintaining compatibility with many different formats beyond rt‐PCR.DesignTo evaluate the viral inactivation performance of the novel inactivation transport media formulation, a cytopathic effect assay was conducted using VERO E6 cells in presence of SARS‐CoV‐2 samples incubated in our novel formulation and log reduction were reported.To assess its compatibility with different SARS‐CoV‐2 related assays, the novel formulation was used as inactivation transport media in antigen, PCR, and NGS based Swab‐Seq tests with known controls.ResultsThe results for the inactivation study confirmed > 3.0 log reduction in viral titer after 30 min exposure in novel transport media formulation and demonstrated 99.5% effectiveness in SARS‐CoV‐2 inactivation. The results for the PCR and antigen test compatibility studies showed comparable and/or superior performance in detection limits for the novel formulation when compared to other sample media. The Swab‐Seq result offers preliminary support on the novel formulation’s compatibility with NGS based assays.ConclusionUsing a novel viral transport medium, we were able to completely inactivate the SARS‐CoV‐2 virus. The new formulation was compatible with both nucleic acid and protein assays while protecting cells from infection. This viral transport media could allow more test types to be done from a single specimen, resulting in safer and reliable outcomes. Additionally, it would reduce the need for multiple samples collected from patients while supporting SARS‐CoV‐2 variant tracking via NGS.

Has the Time Come for Systematic Therapeutic Drug Monitoring of First-Line and WHO Group A Antituberculosis Drugs?

Tuberculosis (TB) is a major global health issue, with approximately 10 million people being infected each year, and is the leading cause of mortality from infectious disease, with 1.5 million deaths a year. Optimal TB treatment requires a combination of drugs for an adequate treatment duration owing to persistent organisms, hardly accessible infection sites, and a high risk of resistance selection. Long-term therapy increases the risk of patients' loss of adherence, adverse drug reactions, and drug-drug interactions, potentially leading to treatment failure. The high interpatient variability of TB drug exposure is another point eliciting interest in therapeutic drug monitoring (TDM) to optimize treatment. Studies reporting clinically relevant exposure thresholds, which might be proposed as targets toward treatment personalization, are discussed. Practical TDM strategies have also been reported to circumvent issues related to delayed drug absorption and the need for multiple samples when evaluating the area under the curve of drug concentrations. The need for treatment individualization is further emphasized because of the development of multidrug-resistant TB or extensively drug-resistant TB. Finally, the willingness to shorten the treatment duration while maintaining success is also a driver for ensuring adequate exposure to TB drugs with TDM. The aim of the present review was to underline the role of TDM in drug-susceptible TB and World Health Organization group A TB drugs.

Favorable Pharmacokinetic Characteristics of Extended-Half-Life Recombinant Factor VIII BAY 94-9027 Enable Robust Individual Profiling Using a Population Pharmacokinetic Approach.

BackgroundProphylaxis with factor VIII (FVIII) should be individualized based on patient characteristics, including FVIII pharmacokinetics. Population pharmacokinetic (popPK) modeling simplifies pharmacokinetic studies by obviating the need for multiple samples.ObjectiveThe objective of this study was to characterize the pharmacokinetics and inter-individual variability (IIV) of BAY 94-9027 in relation to patient characteristics in support of a popPK-tailored approach, including identifying the optimal number and timing of pharmacokinetic samples.MethodsPharmacokinetic samples from 198 males (aged 2‒62 years) with severe hemophilia A, enrolled in BAY 94-9027 clinical trials, were analyzed. Baseline age, height, weight, body mass index, lean body weight (LBW), von Willebrand factor (VWF) level, and race were evaluated. A popPK model was developed and used to simulate pharmacokinetic endpoints difficult to observe from measured FVIII levels, including time to maintain FVIII levels above 1, 3, and 5 IU/dL after different BAY 94-9027 doses.ResultsA one-compartment model adequately described BAY 94-9027 pharmacokinetics. Clearance and central volume of distribution were significantly associated with LBW; clearance was inversely correlated with VWF. Due to the monophasic pharmacokinetics and well-understood IIV sources, identification of patient pharmacokinetics was achievable with sparse blood sampling. Median predicted time to maintain FVIII levels > 1 IU/dL in patients aged ≥ 12 years ranged from 120.1 to 127.2 h after single BAY 94-9027 doses of 45‒60 IU/kg.ConclusionsThis analysis evaluated the pharmacokinetics of BAY 94-9027 and its sources of IIV. Using the model, determination of individual patient pharmacokinetics was possible with few FVIII samples, and a sparse sampling design to support pharmacokinetic-guided dosing was identified.

Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility

BackgroundThe strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy.ResultsThe sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a ‘pooling approach’ can yield a low frequency of ‘missed’ infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools.ConclusionsOur results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in ‘pools-of-five’. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.

Digital Staining of High-Definition Fourier Transform Infrared (FT-IR) Images Using Deep Learning.

Histological stains, such as hematoxylin and eosin (H&E), are routinely used in clinical diagnosis and research. While these labels offer a high degree of specificity, throughput is limited by the need for multiple samples. Traditional histology stains, such as immunohistochemical labels, also rely only on protein expression and cannot quantify small molecules and metabolites that may aid in diagnosis. Finally, chemical stains and dyes permanently alter the tissue, making downstream analysis impossible. Fourier transform infrared (FT-IR) spectroscopic imaging has shown promise for label-free characterization of important tissue phenotypes and can bypass the need for many chemical labels. Fourier transform infrared classification commonly leverages supervised learning, requiring human annotation that is tedious and prone to errors. One alternative is digital staining, which leverages machine learning to map IR spectra to a corresponding chemical stain. This replaces human annotation with computer-aided alignment. Previous work relies on alignment of adjacent serial tissue sections. Since the tissue samples are not identical at the cellular level, this technique cannot be applied to high-definition FT-IR images. In this paper, we demonstrate that cellular-level mapping can be accomplished using identical samples for both FT-IR and chemical labels. In addition, higher-resolution results can be achieved using a deep convolutional neural network that integrates spatial and spectral features.

Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review).

In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. Medical actions include, but are not limited to, early diagnosis, staging, prognosis, anticipation (lead time) and the prediction of therapy responses, as well as follow-up. Historically, the applications of liquid biopsy in cancer have focused on circulating tumor cells (CTCs). More recently, this analysis has been extended to circulating free DNA (cfDNA) and microRNAs (miRNAs or miRs) associated with cancer, with potential applications for development into multi-marker diagnostic, prognostic and therapeutic signatures. Liquid biopsies avoid some key limitations of conventional tumor tissue biopsies, including invasive tumor sampling, under-representation of tumor heterogeneity and poor description of clonal evolution during metastatic dissemination, strongly reducing the need for multiple sampling. On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of cfDNA, the instability of RNA, the low concentrations of certain analytes in body fluids and the confounding presence of normal, as well as aberrant DNAs and RNAs. For these reasons, the analysis of cfDNA has been mostly focused on mutations arising in, and pathognomonicity of, tumor DNA, while the analysis of cfRNA has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression. This review lists some major applicative areas, briefly addresses how technology is bypassing liquid biopsy limitations, and places a particular emphasis on novel, PCR-free platforms. The ongoing collaborative efforts of major international consortia are reviewed. In addition to basic and applied research, we will consider technological transfer, including patents, patent applications and available information on clinical trials aimed at verifying the potential of liquid biopsy in cancer.

Infective endocarditis due to Streptococci and Enterococci: A 3-year retrospective study

Infective endocarditis (IE) is an infection of the heart valves with an aggregation of bacteria in a fibrin plaque called vegetation. This is a retrospective study of all infective endocarditis cases due to alpha haemolytic streptococci and enterococci. All cases of infective endocarditis cases due to alpha haemolytic streptococci and enterococci in a period of three years from 1st January 2010 to 31st December 2012 were included. Isolation of the same organism from more than one set of blood cultures was taken as a confirmed case of infective endocarditis. Clinical and serological parameters were recorded using a proforma. Native valve endocarditis was more common with only five prosthetic valves being involved. Out of 89 clinically suspected cases of IE in the three years from Jan 2010 to Dec 2012, for which blood was sent for culture, 63(70.78%) samples were positive by culture. Of these, 42/63(66.66%) were due to alpha-lytic Streptococci, enterococci and rare gram positive cocci. The rare ones included Enterococcus gallinarum, abiotropha defective, Vagococcus fluvialis and Nutritionally Variant Streptococci(NVS). High level Aminoglycoside resistance(HLAR) was also encountered. The varied and important features of these isolates are discussed. Complications and treatment are described. From a clinical microbiology point of view, the major challenge faced by the microbiologist in diagnosis of IE is proper aseptic collection of sample before starting antibiotics with a need for multiple samples to detect and also to prove the causative organism. Sensitivity reporting can be a difficult task in the context of NVS, HLAR and gram positives that are slow growing. Congestive failure and embolisation occurs even when the antibiotic treatment is successful.When patients go in for complications, it is very rarely due to wrong antibiotics.

Measurements of elastic constants in anisotropic media

Simulation of elastic-wave propagation in rock requires knowledge of the elastic constants of the medium. The number of elastic constants required to describe a rock depends on the symmetry class. For example, isotropic symmetry requires only two elastic constants, whereas transversely isotropic symmetry requires five unique elastic constants. The off-diagonal elastic constant depends on a wave velocity measured along a nonsymmetry axis. The most difficult barrier when measuring these elastic constants is the ambiguity between the phase and group velocity in experimental measurements. Several methods to eliminate this difficulty have been previously proposed, but they typically require several samples, difficult machining, or complicated computational analysis. Another approach is to use the surface (Rayleigh) wave velocity to obtain the off-diagonal elastic constant. Rayleigh waves propagated along symmetry axes have phase and group velocities that are equal for materials with no frequency dispersion, thereby eliminating the ambiguity. Using a theoretical secular equation that relates the Rayleigh velocity to the elastic constants enable determination of the off-diagonal elastic constant. Laboratory measurements of the elastic constants in isotropic and anisotropic materials were made using ultrasonic transducers (central frequency of 1 MHz) for the Rayleigh-wave method and a wavefront-imaging method. The two methods indicated agreement within 1% and 3% for isotropic and transversely isotropic samples, respectively, demonstrating the ability of the Rayleigh-wave method to measure the off-diagonal elastic constant. The surface-wave approach eliminates the need for multiple samples, expensive computational calculations, and most importantly, it removes the ambiguity between the phase and group velocity in the measured data for materials with no frequency dispersion because all measurements are made along symmetry axes.

Assessment of Lead Contamination in Peatlands Using Field Portable XRF

Ombrotrophic peatlands are highly sensitive to atmospheric heavy metal deposition. Previous attempts to quantify peatland lead pollution have been undertaken using the inventory approach. However, there can be significant within-site spatial heterogeneity in lead concentrations, highlighting the need for multiple samples to properly quantify lead storage. Field portable x-ray fluorescence (FPXRF) continues to gain acceptance in the study of contaminated soil, but has not thus far been used to assess peatland lead contamination. This study compares lead concentrations in surface peat samples from the South Pennines (UK) derived using (a) FPXRF in the field, (b) FPXRF in the lab on dried samples and (c) ICP-OES analysis. FPXRF field and lab data are directly comparable when field measurements are corrected for water content, both can be easily used to estimate acid extractable lead using regression equations. This study is a successful demonstration of FPXRF as a tool for a time- and cost-effective means of determining the lead content of contaminated peatlands, which will allow rapid landscape scale reconnaissance, core logging, surface surveys and sediment tracing.

Pressure-induced 0-πtransitions and supercurrent crossover in antiferromagnetic weak links

We compute self-consistently the Josephson current in a superconductor-antiferromagnet-superconductor junction using a lattice model, focusing on $0\ensuremath{-}\ensuremath{\pi}$ transitions occurring when the width of the antiferromagnetic region changes from an even to an odd number of lattice sites. Previous studies predicted $0\ensuremath{-}\ensuremath{\pi}$ transitions when alternating between an even and an odd number of sites for sufficiently strong antiferromagnetic order. We study numerically the magnitude of the threshold value for this to occur, and also explain the physics behind its existence in terms of the phase shifts picked up by the quasiparticles constituting the supercurrent in the antiferromagnet. Moreover, we show that this threshold value allows for pressure-induced $0\ensuremath{-}\ensuremath{\pi}$ transitions by destroying the antiferromagnetic nesting properties of the Fermi surface, a phenomenon which has no counterpart in ferromagnetic Josephson junctions, offering a way to tune the quantum ground state of a Josephson junction without the need for multiple samples.

5-Aminolevulinic acid-induced protoporphyrin IX fluorescence as immediate intraoperative indicator to improve the safety of malignant or high-grade brain tumor diagnosis in frameless stereotactic biopsies

BackgroundFrameless stereotactic biopsies are replacing frame-based stereotaxy as a diagnostic approach to brain lesions. In order to avoid a sampling bias or negative histology, multiple specimens are often taken. This in turn increases the risk of hemorrhagic complications.ObjectiveWe present the use of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence in frameless stereotaxy to improve the procedure duration and yield, and thereby reduce the risk of complications.MethodsPatients with suspected high-grade brain tumors are given 5-ALA 4 h prior to stereotactic biopsy. The biopsy needle is guided to the target using frameless stereotaxy based either on preoperative images or combined with intraoperative MRI sequences. The specimen is illuminated with blue light to look for fluorescence. In case of a positive fluorescence within the tissue sample, no frozen sections are obtained, and no further specimens are taken.ResultsThe samples of 13 patients revealed a positive fluorescence and were histologically confirmed as malignant or high-grade brain neoplasms. four cases were fluorescence-negative, requiring frozen section confirmation and/or multiple samples. In theses cases histology was either nonspecific gliotic changes or low-grade tumors. There were no complications related to the additional use of 5-ALA.Conclusion5-ALA fluorescence in stereotactic biopsies can increase the safety and accuracy of these procedures by reducing sampling errors and eliminating the need for multiple samples and/or frozen section verification, creating a more accurate, faster and safer procedure for cases of suspected malignant or high-grade brain tumors situated in deep or eloquent areas.

Evaluation of Widely Used Diagnostic Tests To Detect West Nile Virus Infections in Horses Previously Infected with St. Louis Encephalitis Virus or Dengue Virus Type 2

Primary West Nile virus (WNV) infections can be diagnosed using a number of tests that detect infectious particles, nucleic acid, and specific IgM and/or IgG antibodies. However, serological identification of the infecting agent in secondary or subsequent flavivirus infections is problematic due to the extensive cross-reactivity of flavivirus antibodies. This is particularly difficult in the tropical Americas where multiple flaviviruses cocirculate. A study of sequential flavivirus infection in horses was undertaken using three medically important flaviviruses and five widely utilized diagnostic assays to determine if WNV infection in horses that had a previous St. Louis encephalitis virus (SLEV) or dengue virus type 2 (DENV-2) infection could be diagnosed. Following the primary inoculation, 25% (3/12) and 75% (3/4) of the horses mounted antibody responses against SLEV and DENV-2, respectively. Eighty-eight percent of horses subsequently inoculated with WNV had a WNV-specific antibody response that could be detected with one of these assays. The plaque reduction neutralization test (PRNT) was sensitive in detection but lacked specificity, especially following repeated flavivirus exposure. The WNV-specific IgM enzyme-linked immunosorbent assay (IgM ELISA) was able to detect an IgM antibody response and was not cross-reactive in a primary SLEV or DENV response. The WNV-specific blocking ELISA was specific, showing positives only following a WNV injection. Of great importance, we demonstrated that timing of sample collection and the need for multiple samples are important, as the infecting etiology could be misdiagnosed if only a single sample is tested.

Rapid bioturbation in equatorial Pacific sediments: evidence from excess 234Th measurements

In order to evaluate bioturbation in equatorial Pacific sediments, profiles of excess 234Th were measured in cores collected during November and December 1992, at six abyssal sites along the JGOFS Equatorial Pacific 140°W transect, from 5°S to 9°N. Excess 234Th (half-life=24.1 days) was detected at all sites. Mean inventories ranged from a low of 0.25 dpm cm −2 at 9°N, to a high of 2.6 dpm cm −2 at 2°N. Eddy-diffusive models of the excess 234Th profiles suggest that, on 100-day timescales, particle mixing is slow at 5°S and 2°S (eddy diffusion coefficient, D b , < 0.4 cm 2 year −1), faster at 0° and 2°N ( D b =0.34–4.8 cm 2 year −1), and fastest at 5°N ( D b =4.7−11 cm 2 year −1). At 9°N, 234Th is found almost entirely in the upper 5 mm of the seabed, and D b values are <0.1 cm 2 year −1. D b values generally increase as POC flux increases, although the linear relationship ( r 2=0.334, p=0.365) displays considerable scatter. However, if the only sites included in the regression are those where 234Th profiles were measured in at least two core sets (i.e. 0°, 2°N, 5°N and 9°N), the linear regression improves substantially ( r 2=0.754, p=0.0012). These results suggest that measurements on a single core may not fully characterize the sediment mixing regime in a particular region, and highlight the need for multiple samples from an individual site. Thorium-234- D b values at 5°S, 2°S and 9°N are in rough agreement with published D b values calculated from the profiles of intermediate half-life radioisotopes, such as 210Pb and 137Cs, in open-ocean abyssal sediments. However, at 0°, 2°N and 5°N, 234Th- D b values are as much as an order of magnitude greater than published 210Pb- D b values. This phenomenon has been observed in other marine environments and may result from age-dependent mixing, non-steady-state mixing or a combination of the two processes. Particle mixing in cores collected at 5°S and 5°N appears to be best described as a largely eddy diffusive process. However, in cores collected at 2°S, 0°, 2°N and 9°N, non-local mixing processes appear to affect 15–30% of the excess 234Th inventory. At these sites, non-local mixing appears to transport recently-deposited material at least 3–4 cm below the sediment-water interface within 100 days of its arrival at the seafloor. The combination of rapid biodiffusive mixing and non-local transport allows recently-deposited, reactive biogenic debris to be rapidly mixed into the seabed. This may affect a wide range of diagenetic processes, including oxygen consumption, dissolution of biogenic debris and organic carbon remineralization. Models of early diagenesis in deep-sea sediments may need to incorporate both non-diffusive transport terms and rapid biodiffusive mixing of recently-deposited particles.

Soil-Landscape Relationships in Virginia: I. Soil Variability and Parent Material Uniformity

Relationships between soil development and parent material or landscape position can often be hidden or confounded by soil variability. Variability in particle size and elemental composition was examined in soils within four representative toposequences in Virginia. The objectives were to partition total variability, describe lateral variability within horizons of the same pedon, and evaluate parent material uniformity. Total variability was partitioned among study sites, landscape positions, horizons, and random effects. Variability attributed to landscape position was minimal (<8%), suggesting that parent material differences or horizon differentiation may be more important in explaining spatial variability in soils than landscape position. Lateral variability was substantially higher in C horizons, than in associated Bt and near-surface horizons. These differences are related to the weathering and pedogenesis occurring in these horizons resulting in an increase in the degree of order of the soil constituents. Mean coefficients of variation ranged from 8 to 34% for particle size data, and 10 to 30% for elemental data, indicating considerable lateral variability and supporting the need for multiple sampling within horizons. Lithologic discontinuities were difficult to recognize without obvious field evidence. Differences in distributions of clay-free sand and Zr, or ratios of Ti/Zr and sand/silt for residual soils were related to differential weathering rates or slight variations in the parent rock.

Experimental Considerations and Limitations in the Application of Ultra High Temperature (2100°C) X-Ray Diffraction

In-situ, high temperature X-ray diffraction (XRD) is an extremely useful tool for studying, monitoring or investigating crystal structure modifications as well as phase transformations in crystalline material during thermal treatments in controlled atmospheres. This technique has been used to investigate the thermal behavior of materials such as carbonate minerals, ceramic fibers, coating pigments, etc. The advantages of such a technique over the conventional practice, where samples are heat treated in a separate oven and then analyzed by XRD include: consistency of sample placement; preservation of high temperature structures to facilitate observation of metastable phases that are unstable upon exposure to outside atmosphere or during cooling; real time monitoring of reactions that occur, and products that are formed at a desired temperature or environment; and need for multiple samples or analysis.

The potassium/sodium ratio in the demonstration of sudden ischemic cardiac death: A critical appraisal

The practical value of measuring the ratio of potassium ion (K+) to sodium ion (Na+) in myocardium as an indicator of early inapparent infarction in sudden cardiac death was assessed using a series of 29 human hearts from selected coroner's autopsies together with experimental material from dogs, including infarcts of 5 min to 4 h duration. Samples for electrolyte analysis were derived from a transverse slice of each heart, taken through both ventricles midway between base and apex, all slices being completely subdivided into a numbered sequence of blocks. Ratios were mapped and compared with macroscopic enzyme staining and histological stains for injured muscle. Detailed examination of coronary arteries was performed on all human cases. Measurement of the K+/Na+ ratio did not detect all human cases of proven acute coronary occlusion and did not unequivocally demonstrate experimental infarcts less than 2 h old. Moreover, all ratios fell with increasing duration of autolysis, emphasizing the need for multiple sampling so that each heart may serve as its own control. As a routine test, therefore, the method is both impracticable and unreliable and as previously used has been subject to misinterpretation.