HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

Prediction of Loss of Muscle Mass in Sarcopenia Using Ultrasonic Diaphragm Excursion

February 13, 2022 Lieven Leave a comment

Background: The diagnosis of sarcopenia is based on the mass and function of appendicular skeletal muscle. It is not clear whether diaphragm excursion is related to muscle mass loss. We try to fill the gap by measuring ultrasonic diaphragm excursion during quiet breathing (Dq) and forced deep breathing (Df) and test whether they could predict the muscle mass loss in sarcopenia.

Methods: The subjects are recruited from the elderly patients diagnosed with pulmonary nodules in community physical examination. According to the definition, the subjects were divided into group A (who did not meet the diagnostic criteria for muscle mass loss in sarcopenia) and group B (who met the criteria). Participants were assessed for ultrasonic diaphragm excursion, pulmonary function, and cardiopulmonary exercise testing. Logistic regression was used to assess the correlation between right diaphragm excursion and skeletal muscle mass, and receiver-operating characteristic curve (ROC) was applied to determine the best threshold.

Results: We recruited 64 elderly participants: 52 in group A (39 males) and 12 in group B (8 males). The Df in group A were higher than in group B (6.02 (5.44-6.60) vs. 4.31 (3.53-5.09) cm, P=0.008). The difference also exists in FVC, FEV1.0, PEF, Pimax, WRmax, and VO₂max, but neither in Dq. Logical regression showed that Df was negatively related to muscle mass (B = -0.525, OR = 0.591 (0.378-0.926), P=0.022), even after adjusted age. Based on ROC, a cutoff value of 5.27 cm (AUC = 0.7783, P=0.0028) was selected, and Df ≤ 5.27 cm indicates the increase in odds https://biodas.org/ of existing muscle mass loss.

Conclusion: Ultrasonic diaphragm excursion in forced deep breath is helpful for predicting muscle mass loss in sarcopenia.

Application and Validation of the Tricuspid Annular Plane Systolic Excursion/Systolic Pulmonary Artery Pressure Ratio in Patients with Ischemic and Non-Ischemic Cardiomyopathy

The main aim of this study was to assess the prognostic utility of TAPSE/PASP as an echocardiographic parameter of maladaptive RV remodeling in cardiomyopathy patients using cardiac magnetic resonance (CMR) imaging. Furthermore, we sought to compare TAPSE/PASP to TAPSE. The association of the echocardiographic parameters TAPSE/PASP and TAPSE with CMR parameters of RV and LV remodeling was evaluated in 111 patients with ischemic and non-ischemic cardiomyopathy and cut-off values for maladaptive RV remodeling were defined.

In a second step, the prognostic value of TAPSE/PASP and its cut-off value were analyzed regarding mortality in a validation cohort consisting of 221 patients with ischemic and non-ischemic cardiomyopathy. A low TAPSE/PASP (<0.38 mm/mmHg) and TAPSE (<16 mm) were associated with a lower RVEF and a long-axis RV global longitudinal strain (GLS) as well as higher RVESVI, RVEDVI and NT-proBNP.

A low TAPSE/PASP, but not TAPSE, was associated with a lower LVEF and long-axis LV GLS, and a higher LVESVI, LVEDVI and T1 relaxation time at the interventricular septum and the RV insertion points. Furthermore, in the validation cohort, low TAPSE/PASP was associated with a higher mortality and TAPSE/PASP was an independent predictor of mortality. TAPSE/PASP is a predictor of maladaptive RV and LV remodeling associated with poor outcomes in cardiomyopathy patients.

Preoperative Computed Tomography Angiography Reveals Leaflet-Specific Calcification and Excursion Patterns in Aortic Stenosis

Background: Computed tomography-based evaluation of aortic stenosis (AS) by calcium scoring does not consider interleaflet differences in leaflet characteristics. Here, we sought to examine the functional implications of these differences.

Methods: We retrospectively reviewed the computed tomography angiograms of 200 male patients with degenerative calcific AS undergoing transcatheter aortic valve replacement and 20 male patients with normal aortic valves. We compared the computed tomography angiography (CTA)-derived aortic valve leaflet calcification load (AVLC_CTA), appearance, and systolic leaflet excursion (LE_sys) of individual leaflets. We performed computer simulations of normal valves to investigate how interleaflet differences in LE_sys affect aortic valve area. We used linear regression to identify predictors of leaflet-specific calcification in patients with AS.

Results: In patients with AS, the noncoronary cusp (NCC) carried the greatest AVLC_CTA (365.9 [237.3-595.4] Agatston unit), compared to the left coronary cusp (LCC, 278.5 [169.2-478.8] Agatston unit) and the right coronary cusp (RCC, 240.6 [137.3-439.0] Agatston unit; both P<0.001). However, LCC conferred the least LE_sys (42.8º [38.8º-49.0º]) compared to NCC (44.8º [41.1º-49.78º], P=0.001) and RCC (47.7º [42.0º-52.3º], P<0.001) and was more often characterized as predominantly thickened (23.5%) compared to NCC (12.5%) and RCC (16.5%). Computer simulations of normal valves revealed greater reductions in aortic valve area following closures of NCC (-32.2 [-38.4 to -25.8]%) and RCC (-35.7 [-40.2 to -32.9]%) than LCC (-24.5 [-28.5 to -18.3]%; both P<0.001). By linear regression, the AVLC_CTA of NCC and RCC, but not LCC, predicted LE_sys (both P<0.001) in patients with AS. Both ostial occlusion and ostial height of the right coronary artery predicted AVLC_{CTA, RCC} (P=0.005 and P=0.001).

Conclusions: In male patients, the AVLC_CTA of NCC and RCC contribute more to AS than that of LCC. LCC’s propensity for noncalcific leaflet thickening and worse LE_sys, however, should not be underestimated when using calcium scores to assess AS severity.

Keywords: aortic valve stenosis; calcium; computed tomography angiography; computer simulation; transcatheter aortic valve replacement.

Polysomnographic Plethysmography Excursions are Reduced in Obese Elderly Men

Sleep apnea is a widespread disorder and is defined by the complete or partial cessation of breathing. Obstructive sleep apnea (OSA) is caused by an obstruction in the upper airway while central sleep apnea (CSA) is characterized by a diminished or absent respiratory effort. It is crucial to differentiate between these respiratory subtypes as they require radically different treatments.

Currently, diagnostic polysomnography (PSG) is used to determine respiratory thoracic and abdominal movement patterns using plethysmography belt signals, to distinguish between OSA and CSA. There is significant manual technician interrater variability between these classifications, especially in the evaluation of CSA. We hypothesize that an increased body mass index (BMI) will cause decreased belt signal excursions that increase false scorings of CSA.

The hypothesis was investigated by calculating the envelope as a continuous signal of belt signals in 2833 subjects from the MrOS Sleep Study and extracting a mean value of each of the envelopes for each subject. Using linear regression, we found that an increased BMI was associated with lower excursions during REM sleep (-0.013 [mV] thoracic and -0.018 [mV] abdominal, per BMI) and non-REM (-0.014 [mV] thoracic and -0.012 [mV] abdominal, per BMI). We conclude that increased BMI leads to lower excursions in the belt signals during event-free sleep, and that OSA and CSA events are harder to distinguish in subjects with high BMI. This has a major implication for the correct identification of CSA/OSA and its treatment.

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Centrifuge-free dispersive liquid-liquid microextraction coupled with thin-film microextraction for the preconcentration of molinate in real samples by ion mobility spectrometry

February 13, 2022 Lieven Leave a comment

A tandem microextraction method, centrifuge free dispersive liquid-liquid microextraction and thin-film microextraction (DLLME-TFME), was used for analyzing molinate in environmental samples by ion mobility spectrometry (IMS). Considering the IMS as a competitive detection system, coupling these two popular sample preparation methods reduces the effect of solvent interference and improves the sensitivity of the technique.

Trichloromethane and methanol were used as the extraction, and dispersive solvents for the DLLME method and electrospun polyacrylonitrile/copper-benzene-1,4-dicarboxylic acid fibers were used as a sorbent in the TFME method. Some effective experimental variables influencing the extraction efficiency of an analyte such as type and volume of dispersive and extraction solvents, solution pH, ionic strength, sonication time, and extraction time were studied.

The linear dynamic range of 0.5-50 μg L^-1 and the limit of detection of 0.1 μg L^-1 were obtained under optimized conditions. The relative standard deviations for intra-and inter-day analysis https://biodas.org/ were calculated less than 10%. The present method was used for the determination of molinate in different real samples such as agricultural wastewater, well water, river water, and apple, and the recovery was obtained between 82% and 113%, for the spiked samples.

Acoustofluidic centrifuge for nanoparticle enrichment and separation

Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation.

By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations.

The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.

Quantitative Evaluation of a Telerobotic System for Vascular Ultrasound Measurement on a Short Arm Human Centrifuge

Artificial Gravity generated by Short Arm Human Centrifuges is a promising multi-system countermeasure for physiological deconditioning during long duration space flights. To allow a continuous assessment of cardiovascular hemodynamics during centrifugation, a telerobotic robotic system holding an ultrasound probe has been installed on a Short Arm Human Centrifuge.
A feasibility study was conducted to define the use capabilities and limitations of such a novel method. The objective of this study is to estimate the reproducibility and precision of remotely controlled vascular ultrasound assessment under centrifugation by assessing peripheral vascular diameter and wall distension. Four repeated centrifugation runs of 5 min, with 2.4 g at feet level, were performed including a 15 min rest between each run for a group of eight healthy male volunteers. Vascular diameter and distention were assessed for the common carotid artery (CCA) and the femoral artery (FA) by ultrasound imaging using a 10 MHz linear array probe (Mylab1, Esaote).
Ultrasound measurements were consecutively performed: a) by an expert user in hand-held mode in standing as well as supine position, b) using the telerobotic arm without centrifugation as baseline and c) using the telerobotic arm during centrifugation. Vascular responses were compared between baseline and under centrifugation. Inter-, intra-registration and group variability have been assessed for hand-held and remotely controlled examination.
The results show that intra-registration variability, σ _h , was always smaller than inter-registration variability, σ _m, that is in turned smaller than the inter-subject variability σ _g (σ _h < σ _m < σ _g). Centrifugation caused no significant changes in CCA diameter but a lower carotid distension compared to manual and robotic ultrasound in supine position (p < 0.05). Femoral diameter was significantly decreased in hypergravity compared to robotic sonography without centrifugation.
A good reproducibility and precision of the remotely controlled vascular ultrasound assessment under centrifugation could be demonstrated. In conclusion, arterial wall dynamics can be precisely assessed for the CCA and femoral artery during centrifugation using a telerobotic ultrasound measurement system. Potential improvements to further enhance reproducibility and safety of the system are discussed.

Assessment of bridge natural frequency as an indicator of scour using centrifuge modelling

One of the most prevalent causes of bridge failure around the world is “scour”-the gradual erosion of soil around a bridge foundation due to fast-flowing water. A reliable technique for monitoring scour would help bridge engineers take timely countermeasures to safeguard against failure. Although vibration-based techniques for monitoring structural damage have had limited success, primarily due to insufficient sensitivity, these have tended to focus on the detection of local damage. High natural frequency sensitivity has recently been reported for scour damage.

Previous experiments to investigate this have been limited as a result of the cost of full-scale testing and the fact that scaled-down soil-structure models tested outside a centrifuge do not adequately simulate full-scale behaviour. This paper describes the development of what is believed to be the first-ever centrifuge-testing programme to establish the sensitivity of bridge natural frequency to scour. A 1/60 scale model of a two-span integral bridge with 15 m spans was tested at varying levels of scour. For the fundamental mode of vibration, these tests found up to a 40% variation in natural frequency for 30% loss of embedment.

Models of three other types of foundation, which represent a shallow pad foundation, a deep pile bent and a deep monopile, were also tested in the centrifuge at different scour levels. The shallow foundation model showed lower frequency sensitivity to scour than the deep foundation models. Another important finding is that the frequency sensitivity to “global scour” is slightly higher than the sensitivity to “local scour”, for all foundation types. The level of frequency sensitivity (3.1-44% per scour depth equivalent to 30% of embedment of scour) detected in this experiment demonstrates the potential for using natural frequency as an indicator of both local and global scour of bridges, particularly those with deep foundations.

Microliter ultrafast centrifuge platform for size-based particle and cell separation and extraction using novel omnidirectional spiral surface acoustic waves

Asymmetric surface acoustic waves have been shown useful in separating particles and cells in many microfluidics designs, mostly notably sessile microdroplets. However, no one has successfully extracted target particles or cells for later use from such samples. We present a novel omnidirectional spiral surface acoustic wave (OSSAW) design that exploits a new cut of lithium niobate, 152 Y-rotated, to rapidly rotate a microliter sessile drop to ∼10 g, producing efficient multi-size particle separation.

We further extract the separated particles for the first time, demonstrating the ability to target specific particles, for example, platelets from mouse blood for further integrated point-of-care diagnostics. Within ∼5 s of surface acoustic wave actuation, particles with diameter of 5 μm and 1 μm can be separated into two portions with a purity of 83% and 97%, respectively.

Red blood cells and platelets within mouse blood are further demonstrated to be separated with a purity of 93% and 84%, respectively. These advancements potentially provide an effective platform for whole blood separation and point-of-care diagnostics without need for micro or nanoscale fluidic enclosures.

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Superhydrophobic paper in the development of disposable labware and lab-on-paper devices.

February 13, 2022 Lieven Leave a comment

Traditionally in superhydrophobic surfaces history, the focus has frequently settled on the use of complex processing methodologies using nonbiodegradable and costly materials. In light of recent events on lab-on-paper emergence, there are now some efforts for the production of superhydrophobic paper but still with little development and confined to the fabrication of flat devices.

This work gives a new look at the range of possible applications of bioinspired superhydrophobic paper-based substrates, obtained using a straightforward surface modification with poly(hydroxybutyrate). As an end-of-proof of the possibility to create lab-on-chip portable devices, the patterning of superhydrophobic paper with different wettable shapes is shown with low-cost approaches.

Furthermore, we suggest the use of superhydrophobic paper as an extremely low-cost material to design essential nonplanar lab apparatus, including reservoirs for liquid storage and manipulation, funnels, tips for pipettes, or accordion-shaped substrates for liquid transport or mixing. Such devices take the advantage of the self-cleaning and extremely water resistance properties of the surfaces as well as the actions that may be done with paper such as cut, glue, write, fold, warp, or burn.

The obtained substrates showed lower propensity to adsorb proteins than the original paper, kept superhydrophobic character upon ethylene oxide sterilization and are disposable, suggesting that the developing devices https://biodas.org/ could be especially adequate for use in contact with biological and hazardous materials.

Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents.

Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low μM concentrations, and inhibiting its effects at higher concentrations. Because of zinc’s high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator.

We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine.

Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc.

Labware additives identified to be selective monoamine oxidase-B inhibitors

Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays.

We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B inhibitors.

3D Printing in the Laboratory: Maximize Time and Funds with Customized and Open-Source Labware

3D printing, also known as additive manufacturing, is the computer-guided process of fabricating physical objects by depositing successive layers of material. It has transformed manufacturing across virtually every industry, bringing about incredible advances in research and medicine. The rapidly growing consumer market now includes convenient and affordable “desktop” 3D printers.
These are being used in the laboratory to create custom 3D-printed equipment, and a growing community of designers are contributing open-source, cost-effective innovations that can be used by both professionals and enthusiasts. User stories from investigators at the National Institutes of Health and the biomedical research community demonstrate the power of 3D printing to save valuable time and funding.
While adoption of 3D printing has been slow in the biosciences to date, the potential is vast. The market predicts that within several years, 3D printers could be commonplace within the home; with so many practical uses for 3D printing, we anticipate that the technology will also play an increasingly important role in the laboratory.

3D-Printed Labware for High-Throughput Immobilization of Enzymes

In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme’s immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures.

Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme’s loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist’s time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.

Additive manufactured customizable labware for biotechnological purposes

Yet already developed in the 1980s, the rise of 3D printing technology did not start until the beginning of this millennium as important patents expired, which opened the technology to a whole new group of potential users. One of the first who used this manufacturing tool in biotechnology was Lücking et al. in 2012, demonstrating potential uses 1, 2. This study shows applications of custom-built 3D-printed parts for biotechnological experiments.
It gives an overview about the objects’ computer-aided design (CAD) followed by its manufacturing process and basic studies on the used printing material in terms of biocompatibility and manageability. Using the stereolithographic (SLA) 3D-printing technology, a customizable shake flask lid was developed, which was successfully used to perform a bacterial fed-batch shake flask cultivation. The lid provides Luer connectors and tube adapters, allowing both sampling and feeding without interrupting the process. In addition, the digital blueprint the lid is based on, is designed for a modular use and can be modified to fit specific needs.
All connectors can be changed and substituted in this CAD software-based file. Hence, the lid can be used for other applications, as well. The used printing material was tested for biocompatibility and showed no toxic effects neither on mammalian, nor on bacteria cells. Furthermore an SDS-PAGE-comb was drawn and printed and its usability evaluated to demonstrate the usefulness of 3D printing for everyday labware. The used manufacturing technique for the comb (multi jet printing, MJP) generates highly smooth surfaces, allowing this application.

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Whatman FTA cards versus plasma specimens for the quantitation of HIV-1 RNA using two real-time PCR assays

February 13, 2022 Lieven Leave a comment

Background: Several studies have compared the use of dried blot spot (DBS) as an alternative to plasma specimens, mainly using Whatman 903 cards as filter paper. The aim of this study was to evaluate the use of Whatman FTA card (FTA card) specimens for HIV-1 viral load testing compared to plasma specimens using two real-time PCR assays manufactured by Roche and Abbott.

Methodology: A cross-sectional study was conducted between April 2017 and September 2017 on HIV-1 patients admitted to Yalgado Ouédraogo Teaching Hospital. Paired FTA cards and plasma specimens were collected and analysed using the Abbott Real-Time HIV-1 assay (Abbott) and COBAS AmpliPrep/COBAS TaqMan v2.0 (Roche).

Results: In total, 107 patients were included. No statistical differences (P>0.05) were observed between the mean viral loads obtained from the FTA cards and those of the plasma specimens using the Roche and Abbott assays. In total, 29 samples with Roche and 15 samples with Abbott assay showed discrepant results. At viral loads of ≤1000 copies ml^-1, the sensitivity and specificity of the FTA cards were 78.6 and 100% with Roche, and 92.3 and 95.9% with Abbott, respectively. Both the Roche and Abbott assays showed good correlation and agreement between the FTA cards and plasma values.

Conclusion: Our study demonstrates the feasibility of using FTA card filter paper for HIV-1 viral load testing. However, further studies will be required https://biodas.org/ for the validation of the use of FTA card filter paper in HIV-1 treatment monitoring.

Stability of Human Immunodeficiency Virus Serological Markers in Samples Collected as HemaSpot and Whatman 903 Dried Blood Spots.

Dried blood spots (DBS) are frequently used in clinical testing for biosurveillance, infectious disease and confirmatory testing, and clinical trials, particularly for populations in remote areas. The HemaSpot-HF blood collection device (HS) provides an alternative format to the Whatman 903 cards (903) to simplify sample collection and processing. In this study, the performance of the HS was compared to that of the 903 using previously characterized clinical specimens and HIV seroconversion panels known to exhibit markers of early human immunodeficiency virus (HIV) infection.
HS and 903 samples were prepared and tested by Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay (EIA), GS HIV-1/-2 Plus O EIA, GS HIV-1 Western blot, and HIV-1 Geenius assays. Both HS and 903 performed well for up to 6 months at room temperature, but a marked loss of Western blot and low titer antibody signals from early infection samples was observed in samples stored for 180 days at elevated (37 to 45°C) temperatures and high humidity (95%).
HemaSpot samples placed in sealed bags with additional desiccant were protected from degradation and showed improved signal recovery relative to that of the 903. HS was easier to use than the 903 and showed higher sensitivity and reproducibility for early infection samples and improved stability.

Whatman Protein Saver Cards for Storage and Detection of Parasitic Enteropathogens.

Current methods to identify the etiology of diarrhea require laboratory facilities for storage of pathogens, which is often challenging in low-resource settings. This study evaluated the efficacy of a low-cost method for preserving stool specimens for the detection of parasitic enteropathogens using Whatman 903 protein saver cards (Sigma-Aldrich, St. Louis, MO). Stool samples known to be positive by multiplex real-time polymerase chain reaction for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica parasites were preserved on 232 Whatman cards.

DNA was then extracted from cards using Chelex and Qiagen extraction protocols, and tested for these parasites using multiplex real-time PCR. We included stool samples known to have a higher parasite load (cycle threshold [ct]-value < 30) and those with a lower parasite load (ct values 30-35). Sensitivities and specificities were determined using DNA extracted directly from whole stool samples using Qiagen kits (QIAGEN, Hilden, Germany). For whole stool samples with ct values < 30, preserved directly on Whatman 903 protein saver cards for Giardia analysis, the sensitivity was 100% for both Qiagen and Chelex DNA extraction.

For E. histolytica, this was 100% for sensitivity for Qiagen and 80% for Chelex DNA extractions, and for Cryptosporidium, this was 80% for Qiagen and 50% for Chelex DNA extraction. The specificity was 100% for all parasites for all extraction procedures. Given the high sensitivity for stool samples with higher parasite loads, we recommend the use of the Whatman 903 protein saver card for preserving fecal specimens for the analysis of Giardia and E. histolytica using Qiagen DNA extractions in low-resource settings.

Comparison of stool collection and storage on Whatman FTA Elute cards versus frozen stool for enteropathogen detection using the TaqMan Array Card PCR assay.

The use of Polymerase Chain Reaction (PCR) assays for pathogen detection in travelers’ diarrhea (TD) field studies is limited by the on-site processing and storage requirements for fecal specimens. The objectives of this investigation were to i) characterize the pathogen distribution in deployed military personnel with TD using the TaqMan® Array Card PCR (TAC) on frozen stool and diarrheal smears on Whatman FTA Elute cards (FTA cards), and to ii) compare TAC detection of enteropathogen targets using smeared FTA cards and frozen stool, using TAC on frozen stool as the ‘reference standard’.

Stool samples, obtained from active duty personnel with acute TD enrolled in a field trial, were smeared onto FTA cards and stored at room temperature. A corresponding aliquot of stool was frozen in a cryovial. FTA cards and frozen stool samples were tested at a central lab, using a customized TAC for detection of TD pathogens. 187 paired frozen stool samples and smeared FTA cards were stored for a median of 712 days (IQR 396-750) before testing. Overall detection rates were 78.6% for frozen stool and 73.2% for FTA cards. Diarrheagenic Escherichia coli were the most common bacteria identified. Using the TAC results on frozen stool as the reference, the overall sensitivity and specificity of TAC on FTA cards was 72.9% and 98.0% respectively.

TAC on FTA cards demonstrated a decrease in sensitivity with increasing frozen stool quantification cycle (Cq) (90.0% in FTA cards with a corresponding frozen stool Cq < 30, and 72.9% in samples with a corresponding frozen stool Cq < 35). Our findings support the use and further development of FTA cards in combination with a quantitative PCR assay for enteropathogen detection in TD field studies.

Detection of anti-hepatitis C virus and hepatitis C virus RNA in dried blood spot specimens using Whatman No. 1 filter paper.

Dried blood spot (DBS) specimen simplifies blood collection, processing, storage and shipment and may reduce the cost of testing for hepatitis C virus (HCV) infection. We wanted to see if DBS using a cheap filter paper is reliable alternative to serum for detection of anti-HCV and HCV RNA.

At a tertiary care hospital in Northeast India, we collected 91 paired DBS and serum specimens from patients at risk of HCV infection from July 2014 to June 2015. DBS was collected on Whatman No. 1 filter paper. After processing, the specimens were subjected to anti-HCV detection by a third-generation Enzyme-Linked Immunosorbent Assay (ELISA). The reactive DBS and serum specimens were further subjected to HCV RNA detection by polymerase chain reaction. The results were analysed in paired screen-positive study design.

Anti-HCV was detected in 9 (9.9%) DBS specimens and 10 (10.9%) serum specimens. There was statistically significant (P < 0.0001) correlation between the optical density values of DBS and serum specimens (Pearson r = 0.9181, 95% confidence interval: 0.8781-0.9453). HCV RNA was detected in 5/9 (55.6%) reactive DBS and 9/10 (90.0%) reactive serum specimens. There was no correlation between HCV RNA levels in the DBS and the serum specimens. The relative sensitivity rate and the relative false-positive rate of DBS anti-HCV ELISA were 0.89 and 1.00, respectively.

DBS using Whatman No. 1 filter paper is quite reliable as serum for detection of anti-HCV. It can be useful in effective surveillance. However, it is not suitable for confirmation of chronic HCV infection.

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HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

May 6, 2020 Lieven Leave a comment

The feasibility of producing mitochondrial DNA (mtDNA) knowledge has expanded significantly with the introduction of next-generation sequencing (NGS), particularly in the technology of whole mtDNA genome (mitogenome) sequences. However, the evaluation of those knowledge has emerged as the best problem to implementation in forensics.

To deal with this want, a customized toolkit to be used in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed by means of a collaborative effort between the Armed Forces Medical Examiner System – Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics.

Versatile ion S5XL sequencer for focused subsequent technology sequencing of strong tumors in a scientific laboratory

The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and consists of the interpretation vary required for mtDNA knowledge reporting. AQME additionally integrates an mtDNA haplogroup estimate into the evaluation workflow, which gives the analyst with phylogenetic nomenclature steerage and a profile high quality verify with out the use of an exterior software.

HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform

Supplemental AQME outputs reminiscent of nucleotide-per-position metrics, configurable export information, and an audit path are produced to help the analyst throughout overview. AQME is utilized to straightforward CLC outputs and thus might be integrated into any mtDNA bioinformatics pipeline inside CLC no matter pattern sort, librarypreparation or NGS platform.

An analysis of AQME was carried out to exhibit its performance and reliability for the evaluation of mitogenome NGS knowledge. The research analyzed Illumina mitogenome knowledge from 21 samples (together with related controls) of various high quality and pattern preparations with the AQME toolkit.

A complete of 211 software edits had been routinely utilized to 130 of the 698 complete variants reported in an effort to stick to forensic nomenclature. Although further guide edits had been required for 3 samples, supplemental instruments reminiscent of mtDNA haplogroup estimation assisted in figuring out and guiding these essential modifications to the AQME-generated profile.

Along with profile technology, AQME reported correct haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup project, though phylogenetically shut, recognized a bug that solely impacts partial mitogenome knowledge. Future changes to AQME’s haplogrouping software will deal with this bug in addition to improve the general scoring technique to raised refine and automate haplogroup assignments.

As NGS permits broader use of the mtDNA locus in forensics, the availability of AQME and different forensic-focused mtDNA evaluation instruments will ease the transition and additional help mitogenome evaluation inside routine casework. Toward this finish, the AFMES-AFDIL has utilized the AQME toolbox along with the CLC Genomics Workbench to efficiently validate and implement two NGS mitogenome strategies.

BACKGROUND

Next technology sequencing primarily based tumor tissue genotyping entails complicated workflow and a comparatively longer turnaround time. Semiconductor primarily based subsequent technology platforms assorted from low throughput Ion PGM to excessive throughput Ion Proton and Ion S5XL sequencer. In this research, we in contrast Ion PGM and Ion Proton, with a brand new Ion S5XL NGSsystem for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing efficiency in a scientific laboratory.

METHODS

Eighteen strong tumor samples constructive for numerous mutations as detected beforehand by Ion PGM and Ion Proton had been chosen for research. Libraries had been ready using DNA (vary10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.zero for complete most cancers (CCP), oncomine complete most cancers (OCP) and most cancers hotspot panel v2 (CHPv2) panel as per producer’s directions.

The CHPv2 had been sequenced using Ion PGM whereas CCP and OCP had been sequenced using Ion Proton respectively. All the three libraries had been additional sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data evaluation was carried out using Torrent Suite 4.6 software program on board S5XL and Ion Reporter.

A restrict of detection and reproducibility research was carried out using serially diluted DLD1 cell line.RESULTSA complete of 241 variant calls (235 single nucleotide variants and 6 indels) anticipated in the studied cohort had been efficiently detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was decreased from 4.5 to 2.5 hours with output vary of 3-5 GB (S530) and 8-9.3Gb (S540). Data evaluation time for the Ion S5XL is quicker 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as in comparison with the Ion PGM (3.5-5 h) and Ion Proton (8h). A restrict detection of 5% allelic frequency was established together with excessive inter-run reproducibility.

CONCLUSION

SIon S5XL system simplified workflow in a scientific laboratory, was possible for operating smaller and bigger panels on the similar instrument, had a shorter turnaround time, and confirmed good concordance for variant calls with comparable sensitivity and reproducibility as the Ion PGM and Proton.