Efficient Laboratory Method Research Articles

Clinicomycological profile of dermatophytosis in a tertiary care teaching hospital, Vadodara

Dermatophytosis is the most common type of cutaneous fungal infection seen in man infecting the superficial keratinized layer of the skin, hair and nails. The aim of the study is to isolate and identify the dermatophytes from clinically suspected cases of dermatophytosis, to determine the frequency of occurrence of various species of dermatophytes, to know the prevalence of age and sex of dermatophytosis in the locality.: The present study was carried out in the Department of Microbiology, Medical College and SSG Hospital, Vadodara. A total of 150 clinically diagnosed cases of dermatophytosis were taken for this study. Skin, hair and nail samples were aseptically collected and were subjected to KOH mount examination and culture. Differentiation of the dermatophyte species was performed based on the macroscopic and microscopic examination of culture and physiological test including urease test.: Tinea corporis (41.3%) was the commonest clinical type with a male to female ratio of 1.05:1. Out of these samples, dermatophytes were isolated in 108 cases with the overall isolation rate of 72%. Seven species of dermatophytes were isolated. 53 (49.1%) was the commonest species followed by 22(20.4%).: Identifying dermatophyte species and knowledge of their host preference and ecology play an important role in treating the patient. Correct and efficient laboratory methods for diagnosis of dermatophytosis is the need of the hour to provide correct and timely treatment and prevent the further spread and chronicity of the disease.

Identification of highly variable sequence fragments in unmapped reads for rapid bacterial genotyping

BackgroundBacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform.ResultsIn routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST’s discriminatory power.ConclusionsA pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.

An Efficient Laboratory Method to Measure Stress-Dependent Tight Rock Permeability With the Steady-State Flow Method

The matrix permeability of tight formations is important for many geological and engineering applications, such as CO2 geological sequestration, disposal of nuclear waste, and production of unconventional hydrocarbon and coalbed methane. In the case of hydrocarbon production, the reservoir permeability decreases with decreasing pore pressure or increasing effective stress. Laboratory characterization of the relationship between matrix permeability and effective stress (and pore pressure) is generally time consuming since the current methods are based on the so-called point-by-point approach that measures one permeability data point only with one test run, and the relationship is generally represented with multiple data points. In this paper, we present a method to determine matrix permeability, its relationship with effective stress, and the Biot coefficient of a tight rock sample with three steady-state flow test runs using large pressure gradients. Unlike the traditional steady-state flow method based on linear flow theories, analytical results based on a nonlinear flow theory are used for determining the related relationship and parameters. The gas properties and permeability along the core sample vary with gas pressure and effective stress, while the traditional method treats them as constants for a given test run. To get the same set of parameters, our method only requires three test runs, and each test run of our method takes a much shorter time than the traditional method because our method is based on nonlinear flow theory and thus allows for the use of large pressure gradients. Comparisons of the permeability measurements with those obtained using traditional methods and numerical simulations demonstrate that our new method can get satisfactory results.

An efficient laboratory method to measure the combined effects of Knudsen diffusion and mechanical deformation on shale permeability

In a shale gas reservoir, the rock matrix has a relatively large porosity and gas in place, but extremely low permeability. Thus, the rock matrix is a bottleneck for shale gas flow from the reservoir to hydraulic fractures and then to the production well. We speculate that the next big thing after hydraulic fracturing for unconventional resources development is to enhance the matrix permeability in an economically feasible way. Consequently, the efficient and accurate characterization of rock matrix permeability in the laboratory is a critical task. The current laboratory techniques for source rock permeability measurement follow a “point-by-point” approach. They need multiple test runs to obtain a permeability-pressure curve, because they can only measure one permeability data point for one test run, and are thus time consuming. The root cause of this “point-by-point” approach is that these laboratory techniques are based on linearized gas flow theory requiring only small pore pressure disturbances to the experiment system. Liu et al. (2019) and this work introduce a new methodology that is based on the nonlinear gas flow theory and allows for direct measurement of the permeability-pressure curve with a single test run. This makes the approach highly time efficient. The feasibility and validity of the methodology are demonstrated in this work based on laboratory measurement results and their consistency with theoretical expectations and other independent measurements. The observed permeability exhibits a complex relationship with pore pressure as a result of the combined effects of Knudsen diffusion and mechanical deformation. For a given confining pressure, the observed permeability initially decreases with pore pressure because of Knudsen diffusion and then increases with pore pressure owing to the mechanical deformation. A rock sample with a lower permeability corresponds to a stronger Knudsen diffusion effect and weaker mechanical diffusion effect. This complex behavior highlights the need to accurately and efficiently measure the combined effects that may have important impacts on shale gas production.

Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing

BackgroundCyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms.ResultsOocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%.ConclusionsDensity gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

Operational utility of the reverse-transcription recombinase polymerase amplification for detection of dengue virus

BackgroundA method for rapid detection of dengue virus using the reverse-transcription recombinase polymerase amplification (RT-RPA) was recently developed, evaluated and made ready for deployment. However, reliance solely on the evaluation performed by experienced researchers in a well-structured and well-equipped reference laboratory may overlook the potential intrinsic problems that may arise during deployment of the assay into new application sites, especially for users unfamiliar with the test. Appropriate assessment of this newly developed assay by users who are unfamiliar with the assay is, therefore, vital.MethodsAn operational utility test to elucidate the efficiency and effectiveness of the dengue RT-RPA assay was conducted among a group of researchers new to the assay. Nineteen volunteer researchers with different research experience were recruited. The participants performed the RT-RPA assay and interpreted the test results according to the protocol provided. Deviation from the protocol was identified and tabulated by trained facilitators. Post-test questionnaires were conducted to determine the user satisfaction and acceptability of the dengue RT-RPA assay.ResultsAll the participants completed the test and successfully interpreted the results according to the provided instructions, regardless of their research experience. Of the 19 participants, three (15.8%) performed the assay with no deviations and 16 (84.2%) performed the assay with only 1 to 5 deviations. The number of deviations from protocol, however, was not correlated with the user laboratory experience. The accuracy of the results was also not affected by user laboratory experience. The concordance of the assay results against that of the expected was at 89.3%. The user satisfaction towards the RT-RPA protocol and interpretation of results was 90% and 100%, respectively.ConclusionsThe dengue RT-RPA assay can be successfully performed by simply following the provided written instructions. Deviations from the written protocols did not adversely affect the outcome of the assay. These suggest that the RT-RPA assay is indeed a simple, robust and efficient laboratory method for detection of dengue virus. Furthermore, high new user acceptance of the RT-RPA assay suggests that this assay could be successfully deployed into new laboratories where RT-RPA was not previously performed.

An efficient laboratory workflow for environmental risk assessment of organic chemicals

In this study, we demonstrate a fast and efficient workflow to investigate the transformation mechanism of organic chemicals and evaluate the toxicity of their transformation products (TPs) in laboratory scale. The transformation process of organic chemicals was first simulated by electrochemistry coupled online to mass spectrometry (EC–MS). The simulated reactions were scaled up in a batch EC reactor to receive larger amounts of a reaction mixture. The mixture sample was purified and concentrated by solid phase extraction (SPE) for the further ecotoxicological testing. The combined toxicity of the reaction mixture was evaluated in fish egg test (FET) (Danio rerio) compared to the parent compound. The workflow was verified with carbamazepine (CBZ). By using EC–MS seven primary TPs of CBZ were identified; the degradation mechanism was elucidated and confirmed by comparison to literature. The reaction mixture and one primary product (acridine) showed higher ecotoxicity in fish egg assay with 96h EC50 values of 1.6 and 1.0mgL−1 than CBZ with the value of 60.8mgL−1. The results highlight the importance of transformation mechanism study and toxicological effect evaluation for organic chemicals brought into the environment since transformation of them may increase the toxicity. The developed process contributes a fast and efficient laboratory method for the risk assessment of organic chemicals and their TPs.

Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens.

A new application of Streptavidin ImmunoCAP® for measuring IgG antibodies against non-available commercial antigens

Enzyme immunoassay has been successfully used for the diagnosis of hypersensitivity pneumonitis (HP), but there are difficulties in standardising this method because of the difficulty in obtaining stable solid phases from a huge variety of heterogeneous antigenic sources. With this in mind, the aim of this work was to demonstrate the usefulness of the standard available fluoro-enzyme-immuno-assay (FEIA) to measure specific IgG to uncommon, commercially unavailable HP-causing antigens. Serum samples from patients suffering from HP caused by canary and pigeon protein exposure with positive immunoprecipitation values, as well as cystic fibrosis patients, patients with respiratory allergies, asymptomatic subjects exposed to canary proteins (canary breeders) and healthy unexposed individuals were evaluated by FEIA using biotinylated serum proteins coupled to streptavidin-activated solid phases. Statistical analyses were performed to compare the different groups and to evaluate the performance of this technique. The group of patients with hypersensitivity pneumonitis caused by exposure to canary proteins showed the highest value of specific IgG (35.70 mg IgG/mL). ROC analysis demonstrated an optimal cut-off value of 8.44 mg IgG/mL with a sensitivity of 100% and a specificity of 91.76%. Comparisons with other groups revealed statistically significant differences. Comparison between ELISA and FEIA results revealed a strong positive relationship between values obtained in both assays. The results demonstrate that Streptavidin ImmunoCAP is a sensitive, specific and efficient laboratory method for routine diagnostic testing of HP caused by protein antigens that are not commercially available.

Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa

In recent publications, it was shown that disease-associated prion protein (PrP(d)) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrP(d) can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrP(d) could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.

Laboratory Methods to Remove Surface Layers from Cereal Grains Using a Seed Scarifier and Comparison with a Barley Pearler

ABSTRACTThe distribution of nutrients and undesirable components within a cereal seed is heterogeneous, and many of the valuable nutrients are located in the outer layers. Efficient laboratory methods to remove these layers will allow for the experimental production of value‐added products. A seed scarifier (SS) was used to abrade barleys, and factors affecting abrading performance and the quality of end fractions including sample charge size, mode of operation, motor speed, and cereal species were investigated. Results showed that the level of surface removal decreased as the sample charge size increased. Compared with a continuous abrading mode (only one abrading cycle per sample), a successive abrading mode (multiple abrading cycles per sample) not only provided a means to collect different fractions of outer layers and remaining kernels, but also minimized kernel breakage and gave more uniform removal of outer layers among seeds. Reduction in motor speeds tended to decrease removal efficiency but significantly prevented kernels from breakage. Subsequent trials with other grain species (wheat, rice, sorghum, and oat) confirmed these findings. Compared with a barley pearler, the SS was easier to operate, produced more repeatable data, and gave more uniform removal from a seed. The SS is an inexpensive and reliable alternative for abrading various species of cereal grains.

Development of a new and efficient laboratory method for processing testicular sperm.

Testicular biopsy specimens contain large amounts of debris that makes sperm pick-up for ICSI more difficult than with epididymal aspirates. We sought to develop improved processing techniques for testicular sperm extraction (TESE). Retrievals were with azoospermic male partner scheduled to undergo percutaneous epididymal sperm aspiration (PESA) and TESE. The study group consisted of 9 retrievals with a new TESE technique (TESE-N). The control group was 21 retrievals with PESA and 3 retrievals with a previous TESE technique (TESE-P). TESE-N eliminated almost all debris, which made ICSI sperm pick-up more rapid. TESE-N, PESA, and TESE-P fertilization (77, 75, and 72%) and ongoing/delivered pregnancy rates per retrieval (67, 76, and 67%) were similar. Our new technique provides for easy removal of debris from TESE specimens and fertilization and pregnancy rates equal to epididymal sperm's. Eliminating debris from TESE specimens allows for rapid sperm pick-up for ICSI, making the procedure more efficient for embryology staff.

Development of a new and efficient laboratory method for processing testicular sperm.

Objective: In recent years, testicular sperm biopsy (TESE), testicular sperm aspiration (TESA) and epididymal sperm have all been used successfully in conjunction with IVF for patients with azoospermia. TESE/TESA specimens require less surgical expertise to obtain than epididymal specimens and can be provided by all staff urologists, thus allowing for greater flexibility in scheduling IVF procedures. Overall, fertilization and ongoing pregnancy rates have tended to be higher with epididymal sperm as compared with testicular sperm. The reasons for this difference are unknown, but may be due to greater difficulty in isolating a clean preparation of motile testicular sperm due to the presence of many different cell types in testicular tissue. These cell contaminants increase the interval for sperm pick-up compared with epididymal aspirates. The purpose of this study was to develop a simple method of processing testicular tissue that would provide a clean preparation of motile sperm and allow sperm pick-up for ICSI with the same relative ease as with epididymal aspirates. Fertilization and pregnancy rates obtained with this new method (TESE-N) were compared with those from Percutaneous Epididymal Sperm Aspiration (PESA) and a previous TESE technique (TESE-P).

A novel route to chlorodimethylsilane

A new efficient laboratory method of preparation of chlorodimethylsilane (Cl(CH3)2SiH) has been elaborated, which is a modification of the Eaborn et al. method (34) and is based on a transsilylation reaction of substituted (amino)dimethylhydrosilanes, R2NSiMe2H (R2 = Me2, Et2, (CH2)n, etc.) with dimethyldichlorosilane (Me2SiCl2). The reaction proceeds at reflux, at 70°C, preferably with an excess of Me2SiCl2. The most important feature of this novel method is a recovery of intermediate (amino)chlorodimethylsilanes (R2NSiMe2Cl), which can be again reduced to R2NSiMe2H. The transsilylation mechanism has been proven by reaction of (diethylamino)methylphenylsilane with Me2SiCl2. The products of this latter reaction are HMePhSiCl and R2NSiMe2Cl, thus a disproportionation mechanism has been excluded. New substituted bis(amino)dimethylsilanes ((R2N)2SiMe2), (amino)dimethylchlorosilanes (R2NSiMe2Cl), and (amino)dimethylhydrosilanes (R2NSiMe2H) have been synthesized and characterized by NMR and IR.Key words: chlorodimethylsilane, (CH3)2SiHCl, synthesis, new laboratory method, transsilylation mechanism.

A novel route to chlorodimethylsilane

A new efficient laboratory method of preparation of chlorodimethylsilane (Cl(CH3)2SiH) has been elaborated, which is a modification of the Eaborn et al. method (34) and is based on a transsilylation reaction of substituted (amino)dimethylhydrosilanes, R2NSiMe2H (R2 = Me2, Et2, (CH2)n, etc.) with dimethyldichlorosilane (Me2SiCl2). The reaction proceeds at reflux, at 70°C, preferably with an excess of Me2SiCl2. The most important feature of this novel method is a recovery of intermediate (amino)chlorodimethylsilanes (R2NSiMe2Cl), which can be again reduced to R2NSiMe2H. The transsilylation mechanism has been proven by reaction of (diethylamino)methylphenylsilane with Me2SiCl2. The products of this latter reaction are HMePhSiCl and R2NSiMe2Cl, thus a disproportionation mechanism has been excluded. New substituted bis(amino)dimethylsilanes ((R2N)2SiMe2), (amino)dimethylchlorosilanes (R2NSiMe2Cl), and (amino)dimethylhydrosilanes (R2NSiMe2H) have been synthesized and characterized by NMR and IR.Key words: chlorodimethylsilane, (CH3)2SiHCl, synthesis, new laboratory method, transsilylation mechanism.

Water Oxidation By Ozone Or Ozone/Hydrogen Peroxide Using The “Ozotest” Or “Peroxotest” Methods

Abstract This article presents a simple, rapid and efficient laboratory method permittingone to simulate the effects of ozonation (OZOTEST method) or of an oxidation by combined ozone/hydrogen peroxide (PEROXOTEST method) on an industrialscale. A critical analysis of the method is made (ozone transfer, validity of the results) and several practical applications are submitted for consideration (ozone demand, oxidation of atrazine and the DOC).

Novel approaches to the preparation and use of monoclonal antibodies.

ONOCLONAL antibodies (MoAbs) are now routinely used in most medical labo­ ratories. This is especially true in transfusion med­ icine where the use of MoAbs has permitted the gradual replacement of many of the human and animal antisera used in the preparation of blood bank reagents· as well as increasing the efficiency of the preparation and testing of blood-derived products, eg, purification of factor VIII,2 virus testing. 3 The impact of MoAbs could be even greater in transfusion medicine because these mol­ ecules are expected to be essential components of new processes (eg, blood cell separation) and ther­ apeutic preparations (eg, human MoAbs) that are presently considered for use in the diagnosis and treatment of blood-related disorders. 4 For many of these applications, the transition from conceptualization to active development work has been made possible by significant recent advances in MoAb technology. It is the objective of this review to summarize some of the progress that has been made recently in the development of more efficient laboratory methods for preparing MoAbs and to illustrate the potential impact and the limitations of the use of these techniques in transfusion medicine and in other fields.

A laboratory method for evaluation of topical anti-inflammatory agents.

Introduction Critical evaluation of the potency of topically applied steroids has been a difficult task. The method of evaluation most often used has been clinical trial in inflammatory dermatoses, using symmetrical sites for paired comparisons. 1-5 This technic is at best a tedious one, and is limited to the comparison of two treatments in each subject. It is difficult to find treatment sites which are exactly comparable, and large numbers of subjects must be used to obtain significant figures. The evaluation of the results of these clinical trials is not precise, and readings must be made by trained personnel. For example, with hydrocortisone, although clinical usage has long since established its usefulness, it may be difficult to distinguish this agent from a placebo in carefully controlled clinical trials. 6 It is clear that an efficient laboratory method is needed to allow for rapid screening of topical anti-inflammatory activity of the

Density, Porosity, and Compaction of Sedimentary Rocks

An efficient laboratory method of obtaining the bulk volume of a chunk sample of rock is explained. The relation between depth of burial and the density, porosity, and compaction of different types of sediment is discussed and data are presented. These relations can be expressed by exponential equations. Compaction as a cause of structure is substantiated by computation and data. A table is given showing the relation in north-central Oklahoma between depth of burial, height of buried hills, and closure resulting from compaction. An approximate idea of the depth of material eroded from a given area may be obtained by density or porosity studies.