Sample Processing Time Research Articles

Comment on "DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification".

Chen et al (Reports, 17 February 2017, p. 752) highlight an important problem of sequencing artifacts caused by DNA damage at the time of sample processing. However, their manuscript contains several errors that led the authors to incorrect conclusions. Moreover, the same sequencing artifacts were previously described and mitigated in The Cancer Genome Atlas and other published sequencing projects.

DEVELOPMENT OF A MINIATURIZED VERSION OF DICENTRIC CHROMOSOME ASSAY TOOL FOR RADIOLOGICAL TRIAGE.

Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of dicentric chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.

Monitoring of Plant Protein Post-translational Modifications Using Targeted Proteomics.

Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science. One of the most studied PTMs is protein phosphorylation. This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation. Since phosphorylated peptides have a low abundance, usually complex enrichment protocols are required for their research. Initial identification is usually performed with discovery phosphoproteomics, using high sensitivity mass spectrometers, where as many phosphopeptides are measured as possible. Once a PTM site is identified, biological characterization can be addressed with targeted proteomics. In targeted proteomics, Selected/Multiple Reaction Monitoring (S/MRM) is traditionally coupled to simple, standard protein digestion protocols, often omitting the enrichment step, and relying on triple-quadruple mass spectrometer. The use of synthetic peptides as internal standards allows accurate identification, avoiding cross-reactivity typical for some antibody based approaches. Importantly, internal standards allow absolute peptide quantitation, reported down to 0.1 femtomoles, also useful for determination of phospho-site occupancy. S/MRM is advantageous in situations where monitoring and diagnostics of peptide PTM status is needed for many samples, as it has faster sample processing times, higher throughput than other approaches, and excellent quantitation and reproducibility. Furthermore, the number of publicly available data-bases with plant PTM discovery data is growing, facilitating selection of modified peptides and design of targeted proteomics workflows. Recent instrument developments result in faster scanning times, inclusion of ion-trap instruments leading to parallel reaction monitoring- which further facilitates S/MRM experimental design. Finally, recent combination of data independent and data dependent spectra acquisition means that in addition to anticipated targeted data, spectra can now be queried for unanticipated information. The potential for future applications in plant biology is outlined.

Validating outsourced high throughput automated qPCR for increased research outputs from forest pathology trials

Needle diseases of Pinus radiata caused by Phytophthora pluvialis and Phythophthora kernoviae have been increasingly recognised since the discovery of red needle cast in 2008. There is a need for rapid diagnostic screening of numerous samples, but sample processing time, equipment and staff availability limit the throughput and utilisation of diagnostic qPCR analysis in the research environment. Automated and high-throughput capable DNA extraction and real-time PCR provides the opportunity to expand the capacity of research trial analysis and a potential alternative to laborious isolation and plating but must be thoroughly validated before results can be used with confidence. The use of a high-throughput format for qPCR assays targeting Phytophthora pluvialis and Phythophthora kernoviae was validated on a robotic platform, proving to be consistently more sensitive than isolation, achieving qPCR detection down to 1% diluted inoculated material for Phytophthora kernoviae and 10% for Phytophthora pluvialis. Plating results yielded a 60% detection rate of Phythophthora pluvialis in inoculated needle fragments, whereas qPCR yielded a 100% detection on the same material. High throughout automated qPCR can therefore be utilised with confidence in forest pathology research trial analyses in future.

Assessing effects of different processing procedures on the yield of Treponema pallidum DNA from blood

In this work, we employed real-time PCR analysis targeting tp0574 to investigate the effects of different processing procedures on the yield of T. pallidum DNA from blood to improve assay sensitivity. The T. pallidum DNA yields following red blood cell lysis pretreatment were 40.4 times greater from whole blood and 32.4 times greater from residual hematocytes than yields without pretreatment. For the simulated whole-blood experiments, the T. pallidum DNA yields from the lower layer were 2.8, 4.6, 7.3, 12.6, 15.24, 16.7, 65.1 and 73.1 times those from the upper layer following centrifugation at 500×, 1000×, 2000×, 4000×, 5000×, 7000×, 10,000× and 20,000 × g, respectively. However, the T. pallidum DNA yields from blood clots were only 1.0% at different centrifugal forces. The experiment with infected rabbit blood showed results similar to those mentioned above. In addition, sample processing time (within 48 h) and storage temperature (4 °C and 25 °C) did not affect T. pallidum DNA extraction efficiency. The T. pallidum DNA yield can be significantly improved by red blood cell lysis pretreatment and appropriate centrifugation. Furthermore, the T. pallidum DNA extraction yield is greater from whole blood or residual hematocytes from anti-coagulated blood than from plasma, serum or blood clots.

Comparisons of metabolite profile from paired serum and ethylenediaminetetraacetic acid-plasma samples using dry chemistry technology: An emergency department perspective.

BACKGROUND:No data is available evaluating the difference in serum versus plasma sample assay of commonly tested parameters in the emergency department, where the sample processing time can be significantly reduced if plasma is used for analysis instead of conventionally used serum. Hence, this study aimed to evaluate the differences in serum versus plasma sample estimation of commonly evaluated biochemical parameters using dry chemistry technology.MATERIALS AND METHODS:Paired blood samples were collected from a single venipuncture of 405 patients admitted to the emergency department. Dry chemistry autoanalyzer (Vitros-350, Ortho Clinical Diagnostics) was used to process all the samples.RESULTS:Data from 401 patients were analyzed. Percentage differences between serum versus plasma samples for all analytes ranged from 0.0% to 57.44% and were <±4% for a majority of parameters, except uric acid (−6.25%), albumin (+11.90%), chloride (–5.05%), phosphorus (−6.06%), creatine phosphokinase (CPK) total (−57.44%), amylase (−37.53%), lipase (−42.74%), lactate dehydrogenase (LDH) (−8.53%), and C-reactive protein (−7.44%). For albumin, CPK total, amylase, and lipase, the difference between serum and plasma samples was more than the accepted upper range recommended by College of American Pathologists.CONCLUSION:Glucose, urea, creatinine, bilirubin, total protein, serum glutamate-pyruvate transaminase, total cholesterol, high-density lipoprotein cholesterol, triglycerides, sodium, and CPK-mb can be reliably assayed from either serum or plasma samples in emergency/routine practice. CPK total, amylase, and lipase should always be assayed from serum and not plasma due to significant variations. Uric acid, chloride, phosphorous, and LDH only in emergency situations should be assayed from plasma. For routine assays, serum should be preferred.

The role of B7-1 in proteinuria of glomerular origin

The damage and loss of podocytes is a primary hallmark of nephrotic syndrome. In the pursuit of targetable molecules that are involved in podocyte pathophysiology, some studies have identified B7-1 (also known as CD80) as a potential biomarker. Furthermore, B7-1 blockade has been proposed as a podocyte-specific treatment for patients with nephrotic syndrome who have limited therapeutic options, such as those with focal segmental glomerulosclerosis, minimal change disease, diabetic nephropathy and lupus nephritis. In this Perspectives article, we describe and compare supporting and contradicting data on the role of podocyte B7-1 in the pathogenesis of various podocytopathies. Moreover, we highlight crucial issues that should be addressed urgently - such as standardization of sample processing time, material conservation and antibody usage in immunohistochemical protocols - as a clinical trial that is investigating the efficacy of B7-1 blockade in treatment-resistant nephrotic syndrome is ongoing.

Application of BioFire FilmArray Blood Culture Identification panel for rapid identification of the causative agents of ventilator-associated pneumonia

ObjectiveTo evaluate the ability of the BioFire FilmArray Blood Culture Identification (BCID) panel to rapidly detect pathogens producing late-onset ventilator-associated pneumonia (VAP), a severe infection often produced by Gram-negative bacteria. These microorganisms are frequently multidrug resistant and typically require broad-spectrum empiric treatment. MethodsIn the context of an international multicentre clinical trial (MagicBullet), respiratory samples were collected at the time of suspicion of VAP from 165 patients in 32 participating hospitals in Spain, Greece and Italy. Microorganisms were identified using the BCID panel and compared with results obtained by conventional microbiologic techniques. ResultsPseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly identified species, representing 54.7% (70/128) of microorganisms. The BCID panel showed high global specificity (98.1%; 95% confidence interval, 96–100) and negative predictive values (96.6%) and a global sensitivity and positive predictive value of 78.6% (95% confidence interval, 70–88) and 87.3%, respectively, for these microorganisms. Importantly, the BCID panel provided results in only 1 hour directly from respiratory samples with minimal sample processing times. ConclusionsThe BCID panel may have clinical utility in rapidly ruling out microorganisms causing VAP, specifically multidrug-resistant Gram-negative species. This could facilitate the optimization of empiric treatment.

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre‐fractionated by molecular weight cut‐off filtration, digested enzymatically and were analyzed by matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre‐fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose‐dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency‐based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.

Improvements in Tritrichomonas foetus molecular testing.

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

Autoverification process improvement by Six Sigma approach: Clinical chemistry & immunoassay

ObjectiveThis study examines effectiveness of a project to enhance an autoverification (AV) system through application of Six Sigma (DMAIC) process improvement strategies. Design and methodsSimilar AV systems set up at three sites underwent examination and modification to produce improved systems while monitoring proportions of samples autoverified, the time required for manual review and verification, sample processing time, and examining characteristics of tests not autoverified. This information was used to identify areas for improvement and monitor the impact of changes. ResultsUse of reference range based criteria had the greatest impact on the proportion of tests autoverified. To improve AV process, reference range based criteria was replaced with extreme value limits based on a 99.5% test result interval, delta check criteria were broadened, and new specimen consistency rules were implemented. Decision guidance tools were also developed to assist staff using the AV system. The mean proportion of tests and samples autoverified improved from <62% for samples and <80% for tests, to >90% for samples and >95% for tests across all three sites. The new AV system significantly decreased turn-around time and total sample review time (to about a third), however, time spent for manual review of held samples almost tripled. There was no evidence of compromise to the quality of testing process and <1% of samples held for exceeding delta check or extreme limits required corrective action. ConclusionsThe Six Sigma (DMAIC) process improvement methodology was successfully applied to AV systems resulting in an increase in overall test and sample AV by >90%, improved turn-around time, reduced time for manual verification, and with no obvious compromise to quality or error detection.

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods

ABSTRACTSample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

A novel molecular toolkit for rapid detection of the pathogen and primary vector of thousand cankers disease.

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.

Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples.

The process of plate streaking has been automated to improve routine workflow of clinical microbiology laboratories. Although there were many evaluation reports about the inoculation of various body fluid samples, few evaluations have been reported for blood. In this study, we evaluated the performance of automated inoculating system, Previ Isola for various routine clinical samples including blood. Blood culture, body fluid, and urine samples were collected. All samples were inoculated on both sheep blood agar plate (BAP) and MacConkey agar plate (MCK) using Previ Isola and manual method. We compared two methods in aspect of quality and quantity of cultures, and sample processing time. To ensure objective colony counting, an enumeration reading reference was made through a preliminary experiment. A total of 377 nonduplicate samples (102 blood culture, 203 urine, 72 body fluid) were collected and inoculated. The concordance rate of quality was 100%, 97.0%, and 98.6% in blood, urine, and other body fluids, respectively. In quantitative aspect, it was 98.0%, 97.0%, and 95.8%, respectively. The Previ Isola took a little longer to inoculate the specimen than manual method, but the hands-on time decreased dramatically. The shortened hands-on time using Previ Isola was about 6minutes per 10 samples. We demonstrated that the Previ Isola showed high concordance with the manual method in the inoculation of various body fluids, especially in blood culture sample. The use of Previ Isola in clinical microbiology laboratories is expected to save considerable time and human resources.

A novel fast method for aqueous derivatization of THC, OH-THC and THC-COOH in human whole blood and urine samples for routine forensic analyses.

A novel aqueous in situ derivatization procedure with propyl chloroformate (PCF) for the simultaneous, quantitative analysis of Δ9 -tetrahydrocannabinol (THC), 11-hydroxy-Δ9 -tetrahydrocannabinol (OH-THC) and 11-nor-Δ9 -tetrahydrocannabinol-carboxylic acid (THC-COOH) in human blood and urine is proposed. Unlike current methods based on the silylating agent [N,O-bis(trimethylsilyl)trifluoroacetamide] added in an anhydrous environment, this new proposed method allows the addition of the derivatizing agent (propyl chloroformate, PCF) directly to the deproteinized blood and recovery of the derivatives by liquid-liquid extraction. This novel method can be also used for hydrolyzed urine samples. It is faster than the traditional method involving a derivatization with trimethyloxonium tetrafluoroborate. The analytes are separated, detected and quantified by gas chromatography-mass spectrometry in selected ion monitoring mode (SIM). The method was validated in terms of selectivity, capacity of identification, limits of detection (LOD) and quantification (LOQ), carryover, linearity, intra-assay precision, inter-assay precision and accuracy. The LOD and LOQ in hydrolyzed urine were 0.5 and 1.3 ng/mL for THC and 1.2 and 2.6 ng/mL for THC-COOH, respectively. In blood, the LOD and LOQ were 0.2 and 0.5 ng/mL for THC, 0.2 and 0.6 ng/mL for OH-THC, and 0.9 and 2.4 ng/mL for THC-COOH, respectively. This method was applied to 35 urine samples and 50 blood samples resulting to be equivalent to the previously used ones with the advantage of a simpler method and faster sample processing time. We believe that this method will be a more convenient option for the routine analysis of cannabinoids in toxicological and forensic laboratories.

Rapid microwave synthesis of hydroxyapatite phosphate microspheres with hierarchical porous structure

Hierarchical Hydroxyapatite (Ca10(PO4)6(OH)2, HA) microspheres can be applied as microcarriers in cell carriers and drug delivery. However, it is difficult to prepare monodispersed and hierarchical microspheres in short time with conventional method. Here, we report the preparation of hierarchical HA microspheres through a novel microwave-assisted method in several minutes, where similar structured calcium carbonate (CaCO3) microspheres acts as sacrificial hard-templates. The SEM images indicated that surface microstructure and microspheres size were effectively controlled by changing starting parameters such as microwave react time and stirring speed. XRD patterns and BET results further proved that the pure HA microspheres with great specific surface area can be prepared in a short time. The Ca ions release of samples showed that the degradation of HA microspheres decreased with microwave processing time of samples. Drug release of samples showed that on one hand vancomycin release concentration decreased with microwave processing time of samples, on the other hand increased obviously with the lower pH value of buffer solution. Human osteoblasts-like cells were cultured with different concentration of microspheres extracts to investigate in vitro biocompatibility. The results of the CCK-8 assay and Live/Dead staining proved that the cells grew well in all sample extracts, suggesting that cells responded well to the microspheres. Our work provides a novel and rapid way to produce hierarchical HA microspheres for bone repair or drug delivery.

Novel Governance Model for Planning in Pharmaceutical Quality Control Laboratories

A simulation model of pharmaceutical quality control laboratories was employed as a benchmarking platform to estimate the performance of a new facility under alternative governance models. Key performance metrics, such as sample processing time and utilization rates of analytical staff and equipment were computed and analyzed under alternative governance scenarios, in order to evaluate solutions to be implemented in practice. Two frameworks were compared, leading to the conclusion that higher efficiency can be achieved under free-for-all governance, without the need to procure additional resources.

OMNIgene.SPUTUM suppresses contaminants while maintaining Mycobacterium tuberculosis viability and obviates cold-chain transport.

Tuberculosis (TB) diagnostics are centralised, requiring long-distance transportation of specimens in most resource-limited settings. We evaluated the ability of OMNIgene.SPUTUM (OM-S) to obviate cold-chain transport of TB specimens.A two-arm (same-day and after 5 days sample processing) study was conducted to assess contamination rates and Mycobacterium tuberculosis viability in OM-S-treated samples against the standard decontamination procedure (SDP) in Mozambique, using Lowenstein Jensen (LJ) and mycobacterial growth indicator tube (MGIT) culture and molecular bacterial load assay.270 specimens were processed using OM-S and SDP in same-day and 5-day arms. Contamination was lower in OM-S-treated than SDP-treated cultures: 12% versus 15% and 2% versus 27% in the same-day and 5-day arms, respectively. M. tuberculosis recovery in OM-S-treated LJ cultures was 10% and 56% higher in the same-day and 5-day arms, respectively, than SDP-treated cultures, but lower in MGIT (52% and 28% lower in the same-day and 5-day arms, respectively). M. tuberculosis viable count was 1log estimated CFU·mL−1 lower in 5-day OM-S-treated sputa. OM-S was more effective at liquefying sputum with a shorter sample processing time: 22 min for culture.OM-S is simple to use and has demonstrated a high potency to suppress contaminants, maintenance of viability at ambient temperatures and higher M. tuberculosis recovery, particularly in the solid LJ cultures. Optimisation of OM-S to achieve higher MGIT culture positivity and shorter time to result will increase its application and utility in the clinical management of TB.

An Integrated Protocol for the Research and Monitoring of Cutaneous Leishmaniasis

Cutaneous Leishmaniasis is a skin infection which is commonly present in underdeveloped countries. The incidence is particularly high in amazonic countries of Latin-America like Brazil, Colombia and Peru and is usually reported as endemic. In Peru, more than one million people are at risk of infection and approximately 6,000 new cases are detected each year. The present work proposes to integrate a set of control, monitoring and disease quantification procedures in: (1) an automated tool to expedite the analysis in laboratories studying parasiticidal agents and (2) a non-invasive treatment monitoring protocol. The first, consists in the adaptation of an optical microscope KRUSS MBL3100 to perform a fast image capture and automated promastigotes identification. This may be of value to evaluate the effectiveness of various parasiticidal agents. The counting process is performed by an automatic segmentation in the RGB green color space discriminating elements by their area. The second, proposes a protocol for monitoring the evolution of the disease treatment divided into three stages: supra-skin modeling and reconstruction, subcutaneous exploration by textural characteristics and volumetric segmentation. This protocol is performed using a Next Engine HD 3D scanner and a Vevo Visualsonix 2100 ultrasonic scanner. The results show improvements in sample processing time, accuracy and inter- and intra-operational variability. The sensitivity and accuracy of the microscopic identification system was of 97% and 92% respectively. The exactitude and precision error was up to 2% and the sensitivity and specificity went as high as 71% in the 3D reconstruction and texture analysis respectively.

An ELISA based method for quantifying arabinoxylan in wheat flour

Arabinoxylans (AX) are a type of dietary fiber present in cereal grains. Recent studies have shown consuming water-extractable AX (WE-AX) can reduce blood glucose levels and prevent the growth of pathogenic bacteria in the human gut. WE-AX can affect dough quality by increasing baking absorption and reducing gluten formation. Historically, WE-AX has been quantified using the phloroglucinol assay, however, this method is labor intensive and not amendable to large sample sizes. Enzyme-linked immunosorbent assays (ELISAs) quantify molecules through specific antigen-antibody binding. The monoclonal antibody LM11 specifically binds to wheat WE-AX and can be used in an ELISA based quantification. In this study, an ELISA was developed to quantify WE-AX in whole grain flour. Flour WE-AX content was evaluated using ELISA and the phloroglucinol assay in five varieties and two milling methods using a Retsch osilating mill and a Thomas Wiley mill. Moderate correlations were found between assays and milling methods. The ELISA assay was found to reduce sample processing time by 16.5 min. Twenty soft winter wheat varieties were evaluated for WE-AX content using ELISA. The ELISA developed in this study was found to be a highly accurate method of quantifying WE-AX in large sample sizes.