Laboratory Practice Research Articles

A United Kingdom-wide audit of the laboratory investigation of primary aldosteronism.

Primary Aldosteronism (PA) is a common but under-recognised cause of secondary hypertension. Early diagnosis with targeted medical and/or surgical intervention is important to prevent irreversible end-organ damage. An Endocrine Society Clinical Practice Guideline was used to define audit standards against which to assess current United Kingdom (UK) laboratory practice. A survey comprising 22 questions, which captured information on screening, confirmatory testing and adrenal vein sampling (AVS), was distributed to all UK Clinical Biochemistry laboratories by the Association for Laboratory Medicine. Consultation with clinical colleagues was encouraged. 50 of 147 laboratories (34.0%) responded, 17 of which provided an analytical service for plasma aldosterone concentration (PAC) and renin, measured as plasma renin activity (PRA) or direct renin concentration (DRC). PRA/DRC, PAC and aldosterone:renin ratios were used to screen for PA. Saline infusion testing was the most common confirmatory test. AVS was used to aid lateralisation. Chemiluminescence immunoassay and liquid chromatography tandem-mass spectrometry were the preferred analytical methods for PAC and PRA/DRC. However, there was considerable variation across centres in respect of reference intervals and cutoffs, which were not fully accounted for by differences in analytical platforms. Although diagnostic algorithms, with pre- and post-analytical support, were in evidence in some centres, these were not universal or always embedded in a multidisciplinary team setting. We observed significant heterogeneity in the laboratory investigation of PA across the UK. Therefore, this work serves as a stimulus for greater collaboration to permit national harmonisation/standardisation of analytical and clinical aspects of UK PA practice.

Hands-On Mastery in Physiological Signal Processing: A Challenge-Based Learning Approach

Abstract Purpose Teaching and learning statistical signal processing in Biomedical Engineering Degrees poses challenges for both students and teachers. Students often perceive signal processing subjects as demanding and somewhat unattractive, with failure rates several times higher than that of other courses. The aim of this work is to address the following question: Are competitions that present real physiological signal processing problems an effective way to teach the practical skills needed in statistical signal processing? Methods We propose to use a problem-driven activity in which students form groups of two or three students and enter a Kaggle-type competition during the whole semester. The competition consists of a real physiological signal problem along with well-defined metrics to establish the performance of the solutions. We measure the effectiveness of the activity with questionnaires together with sentiment and inductive coding analysis that allows evaluation of the acquisition of competencies. Results According to the results obtained in four consecutive editions, students find the challenge to be a better means than traditional laboratory practices for acquiring practical skills, as well as allowing them to better understand theoretical concepts. Students found the activity challenging and demanding but one of the best activities in the whole degree, since it brings the student closer to real practice as biomedical engineers. Conclusion The results support the hypothesis that this type of competition allows students to gain a deeper understanding of the practical aspects of physiological signal processing courses.

A comparison of results of determining the hydrocarbon group-type composition of petroleum samples obtained using various test methods of open-column liquid chromatography

The hydrocarbon group-type composition (so-called SARA-composition) is one of the main characteristics of oil, providing an information about its chemical nature and determining the quality of the obtained petroleum products. In world laboratory practice, data on the hydrocarbon group-type composition of petroleum feedstock are widely used to assess its colloidal stability and compatibility with crude oil, reactivity in various conversion processes, predicting physical properties, etc. Traditionally, the hydrocarbon group-type composition is determined in accordance with standard and research chromatographic methods that differ significantly from each other, which often leads to obtaining incomparable analytical results. In this paper, a comparative analysis of the results of determining the hydrocarbon group-type composition obtained in accordance with two test methods within the open-column liquid chromatography method is carried out for various of petroleum samples of Russian origin (commercial oils of different classes and two petroleum products). Using the standard test method ASTM D4124 (method B) and the research test method developed by JSC VNII NP, the content of identical hydrocarbon groups in the maltene part of petroleum samples was determined: saturated hydrocarbons (SH) and paraffinic-naphthenic hydrocarbons (PNH), naphthenic aromatic (NAH) and aromatic hydrocarbons (AH), polar aromatic hydrocarbons (PAH) and resins (R), respectively. It is shown that with an increase the density and viscosity of petroleum samples, the content of less polar groups (SH and PNH) in their composition decreases and the content of more polar groups (PAH and R) obviously increases, and there is no pronounced distribution pattern of the NAH and AH groups. Statistical processing revealed insignificant differences in the results for «heavy and viscous» oils, despite the large error of those obtained according to the research test method. Obtaining comparable results justifies the use of both test methods not only for the analysis of high-boiling petroleum products, but also for the analysis «heavy and viscous» oil samples.

Challenges for complement functional assays in the clinical laboratory: From test validation to clinical interpretation.

Complement functional assays are essential first-tier tests for a gamut of disorders spanning from inborn errors of the immune system which lead to recurrent severe infections, to angioedema attacks, presentation of autoimmune disease, thrombotic microangiopathies and rare kidney disorders. These assays evaluate the activity of the three complement pathways and specific complement components, which helps in differential diagnosis and monitoring disease progression. The rising use of complement inhibitors for treating complement-mediated thrombotic microangiopathies has heightened the demand for personalized treatment plans and laboratory assessment of complement blockage. However, conducting these assays is challenging due to the labile nature of complement proteins, which necessitates strict handling protocols-prompt processing, cold centrifugation, and preferable storage at -80 °C. Currently, the only FDA-approved complement functional test is the classical pathway activity assay while other tests are categorized as laboratory developed tests (LDTs). Validation of LDTs requires thorough evaluation of precision, accuracy, reference intervals, clinical reportable ranges, analytical sensitivity, and specificity. Achieving harmonization across laboratories is critical but heavily relies on the methodologies and calibrators used. This article discusses the various challenges and limitations associated with complement functional assays, highlighting the need for standardization and improved practices in clinical laboratories.

Pharmacology Students' Perception of the Use of Animals Prior to Laboratory Practice

Aims: Technological development in computing and biochemistry has allowed the development of teaching tools that are useful in the teaching-learning process. In this descriptive-inferential work, the perception of Pharmaceutical Biological Chemistry (QFB) degree regarding the use of animals in their laboratory practices was evaluated, taking as a reference the practice of testing drugs with hypoglycemic effect. Study Design: The design of the study was descriptive inferential. Place and Duration of Study: The study was realized in FES Zaragoza, UNAM, with Biology Pharmaceutical Chemist student careers between february to may in 2024. Methodology: In this work, an evaluation instrument was designed and developed and that was validated by expert criteria and that was applied to 132 students prior to carrying out the practice in the laboratory. The applied evaluation instrument contained 3 questions aimed at knowing this perception of the students. Results: The results present a binomial distribution probability greater than 0.99 in favor of reducing or replacing research animals with computer simulators. Conclusion: The students consider that is possible to replacement or to reduce the animal research in teaching laboratory.

Improvements in skill-based learning in chemical engineering laboratory practicals through the use of flipped classroom methodology assited by video tutorial

Laboratory practicals in the Industrial Chemical Engineering Bachelor’s Degree are a scenario where students can test their hypotheses, and which facilitate the understanding of theoretical concepts, calculation methodologies and the influence of operating conditions. They foster a learning environment based on the essential competencies of 'learning to know', 'learning to do', 'learning to live together' and 'learning to be'. However, these subjects require individualized attention so that our students can fully understand what, how and why they should do. When the groups of students are large, the teacher cannot provide quick advice to everyone and the student may face technical difficulties due to their lack of previous experience in laboratory environments. This fact usually leads to student’s insecurity and poor learning of lab-work skills. This study shows evidence of improvements in learning when implementing the flipped classroom teaching methodology based on the use of video tutorials in experimental subjects. The video tutorials, carefully designed to cover both fundamental concepts and experimental procedures in laboratory practicals, generate a more autonomous learning environment and a more active role of the student in his/her own learning process compared to traditional methods. In fact, 100% of the students rated the overall use of the video tutorials in these laboratory practicals with a score of 4-5 out of 5. It is noticeable that the use of the video tutorials made the students feel more confident in facing their assignments. Furthermore, 92% considered that this teaching innovation should be extended to all the experimental subjects that make up their study programme.

Tissue optical clearing and expanding solution for improved nuclear segmentation

AbstractOver the recent years, fluorescence microscopy has seen substantial progress, transitioning from primarily qualitative analysis to a profoundly quantitative methodology, yet limitations in imaging depth and data quality persist due to tissue opacity. Tissue optical clearing (TOC) methodologies were implemented to overcome this hurdle, enabling the imaging and quantitative analysis of large tissue samples such as entire murine organs. Further improvements, including expansion microscopy merge TOC benefits with improved imaging resolution, converting cells and tissues into tissue‒gel hybrids. Yet, its intricate protocols lead to a remarkable, ∼10‒20× tissue volume expansion, making the acquisition of even thin sections exceedingly challenging in terms of time, labor, and data handling. Here, we present a novel solution, Improved Segmentation by gEntle Expansion (ISEE), for both TOC and gentle (1.6‒1.7×) tissue expansion for significantly improved precision of fluorescent signal segmentation, while maintaining tolerable increase during data acquisition in everyday laboratory practice. When applied to thick ∼40‒200 µm tissue slices, ISEE renders them transparent and ultimately expanded within minutes, in a simple, one‐step process compatible with immunohistochemistry as well as with all major murine organs.

Factors affecting the efficacy of human leukocyte antigen-selected platelet provision: A large retrospective study in the United Kingdom.

A large, retrospective study was designed to interrogate current NHS Blood and Transplant (NHSBT) HLA matching strategies for the provision of HLA selected platelets (HLA SP) and to determine whether additional factors such as ABO blood group matching, patient diagnosis, patient and/or donor age, sex, ethnicity, age of platelet unit at transfusion and possibly seasonal variation also play a role in transfusion efficacy. Data for 56 640 HLA SP transfusions over a 3-year period were collected. Transfusions with missing data for any factor under consideration were excluded, resulting in a cohort of 13 044 transfusions for analysis. Univariable and multivariable regression models were used to determine if any factors influenced an increase in platelet count of ≥10 × 109/L. A stepwise logistic regression was applied, such that each influential factor was adjusted for effects on other factors included in the study. HLA match grade was confirmed as a significant factor in transfusion efficacy, with ABO mismatched units 20% less likely to give an adequate platelet increment (≥10 × 109/L). Platelet donor age, gender and ethnicity were not significant. Conversely, patient diagnosis, ethnicity, gender and age showed significant associations with platelet increments. Some seasonal variation in efficacy of platelet transfusion was also demonstrated. This study has demonstrated the efficacy of HLA SP transfusions in refractory patients with a wide range of diagnoses, the importance of HLA match grade, plus the marginal effect of ABO matching. A wide range of donor-related factors was excluded, while a number of patient-related factors were identified, requiring more extensive investigation in ongoing and independent studies, with implications for donor registry, clinical and laboratory practices.

WHO public health laboratories webinar series – an online platform to disseminate testing recommendations and best practices during health emergencies

Laboratories play a central role in managing public health emergencies. The COVID-19 pandemic imposed unique challenges on global laboratory systems, including testing protocol uncertainties, supply shortages, rapid need for information dissemination, and disruptions to traditional training methods. In response, the WHO established the Public Health Laboratories (PHL) knowledge sharing webinar series whose goals were to respond to the increased demand in up-to-date and reliable information, which WHO is in a unique position to provide. It also aimed to enhance peer-to-peer exchanges across laboratories. This article outlines the PHL webinar series delivery format and presents how the webinar series was received and perceived by participants and how it evolved to support the response to other health emergencies. Contents of the knowledge sharing sessions, as well as attendance, participants’ satisfaction and application of learning were monitored over time using registration forms, satisfaction polls, an annual survey and focus group discussions. From May 2020 to December 2023, 48 sessions attracted 58,688 registrations from 204 countries and territories. Thirty-five sessions featured presentations of WHO guidance, tools or documents and 39 sessions featured country experience sharing. Initially focused on COVID-19, the series became a tool to rapidly disseminate guidance and best practices during new health emergencies and to address cross-cutting topics relevant to the laboratory workforce. Feedback data shows participants found the webinars very useful (86% respondents), reporting knowledge gains in biosafety, quality management, and laboratory practices. The series facilitated knowledge application, with foreseen changes in workplace procedures and training activities (43% respondents). Barriers such as resource limitations, additional training needs, and connectivity issues were frequently identified. Evidence that this knowledge was subsequently applied by participants, such as through changes in workflow, onwards training events and procedural changes further reinforces the efficacy with which the series was able support the laboratory workforce globally in addressing challenges of the COVID-19 pandemic and other health emergencies. The series utilized sessions on cross-cutting topics to run routinely and to keep a high level of engagement with laboratory professionals globally. This enabled it to act as an adaptable tool that was leveraged effectively and quickly during health emergencies for just-in-time learning.

LittleFaceNet: A Small-Sized Face Recognition Method Based on RetinaFace and AdaFace.

For surveillance video management in university laboratories, issues such as occlusion and low-resolution face capture often arise. Traditional face recognition algorithms are typically static and rely heavily on clear images, resulting in inaccurate recognition for low-resolution, small-sized faces. To address the challenges of occlusion and low-resolution person identification, this paper proposes a new face recognition framework by reconstructing Retinaface-Resnet and combining it with Quality-Adaptive Margin (adaface). Currently, although there are many target detection algorithms, they all require a large amount of data for training. However, datasets for low-resolution face detection are scarce, leading to poor detection performance of the models. This paper aims to solve Retinaface's weak face recognition capability in low-resolution scenarios and its potential inaccuracies in face bounding box localization when faces are at extreme angles or partially occluded. To this end, Spatial Depth-wise Separable Convolutions are introduced. Retinaface-Resnet is designed for face detection and localization, while adaface is employed to address low-resolution face recognition by using feature norm approximation to estimate image quality and applying an adaptive margin function. Additionally, a multi-object tracking algorithm is used to solve the problem of moving occlusion. Experimental results demonstrate significant improvements, achieving an accuracy of 96.12% on the WiderFace dataset and a recognition accuracy of 84.36% in practical laboratory applications.

Utilization of Wastewater from Air Conditioner Circulation Systems for Clean Water Energy Efficiency and Industrial Equipment Maintenance

There is significant wasteful use of clean water for operating equipment during field and laboratory practice. Additionally, damage to laboratory equipment has been reported due to the use of untreated water. These issues, along with previous research on managing air conditioner (AC) wastewater at the Lampung State Polytechnic, form the basis for our initiative to utilize AC wastewater as a sustainable resource. Proper management of this abundant water source can support the operation and maintenance of laboratory equipment. The research findings demonstrate that circulating AC wastewater to operate a Soxhlet extraction system for 4.5 hours can reduce clean water consumption by 0.65 cubic meters, equivalent to a cost savings of IDR 43,200. Furthermore, the results indicate improved efficiency, effectiveness, and overall equipment performance when using AC wastewater compared to conventional methods. Contribution to Sustainable Development Goals (SDGs) SDG 6 – Clean Water and SanitationSDG 9 – Industry, Innovation, and Infrastructure:SDG 12 - Responsible Consumption and ProductionSDG 13 - Climate Action

Evaluation of pre-analytical factors impacting urine test strip and chemistry results.

Careful consideration of the pre-analytical process for urine examination is essential to avoid errors and support accurate results and decision-making. Our objective was to assess the impact of various pre-analytical factors on urine test strip and quantitative chemistry results, including stability, tube type, fill volume, and centrifugation. Residual random urine specimens were identified. Stability of 10 urine test strips and 13 quantitative chemistry parameters were assessed at eight time points (2, 4, 6, 8, 24, 48, 72, and 96 h) at room temperature (RT) and 2-8 °C (n=10-20 samples). The effect of additional pre-analytical variables was assessed, including using preservative tubes for urine chemistry as well as preservative tube underfilling and centrifugation on urine test strip results (n=10 samples). Seven of the ten urine tests strips evaluated met the minimal agreement criteria for stability (Cohen's kappa>0.70) across all conditions. A Cohen's kappa value of<0.70 was observed for pH (48 h), glucose (72 h), and protein (96 h) at RT. All 13 urine chemistry analytes remained stable at defined time points and conditions. Underfilling preservative tubes for urine test strips and centrifugation demonstrated no significant effect. The impact of using preservative tubes for urine chemistry was negligible with the exception of sodium and osmolality. These findings highlight the pre-analytical factors that impact urine specimen evaluation and may be useful in informing clinical laboratory practices. Acceptable stability window for urine test strips should be considered in the context of the proportion of pathological samples evaluated.

A Novel Microbe, Immunization Deaths, and Vaccination on Trial: BCG and the Lübeck Disaster of 1930.

Today's controversies of gain-of-function virological research and mRNA COVID vaccination policies had an antecedent nearly a century ago in an event often referred to as "the Lübeck disaster." From April through September 1930, 77 newborn infants in Lübeck, Germany, died after receiving oral BCG immunizations tainted with active human Mycobacterium tuberculosis. The tragedy threatened to end BCG immunizations. BCG and its originators, the French scientists Albert Calmette and Camille Guérin, were exonerated from liability. An inquest uncovered careless laboratory practices that contaminated doses of the vaccine with a human pathogen. The calamity underscored the necessity for exacting standards when handling dangerous infective microbes. The physician and bacteriologist responsible for the immunization program in Lübeck were tried and convicted for negligence, a concrete example of the civic responsibility expected of scientists when an experimental venture inflicts harm on an unwitting public. The example of Lübeck stands as an object lesson on the necessity of an informed, measured approach to any novel treatment. Controversies continue whether BCG vaccination is the preferred public health strategy against tuberculosis. Calmette and Guérin's lasting scientific achievement is the creation of a microbe that over a century has kept its essential features of inciting a vigorous immunological reaction that was tolerated by its host and never regaining its pathogenicity. The features form the basis of modern cancer immunotherapy, where intravesical BCG is first-line therapy against non-muscle invasive bladder cancer.

Evolution of autoimmune diagnostics over the past 10 years: lessons learned from the UK NEQAS external quality assessment EQA programs.

External quality assessment (EQA) programs play a pivotal role in harmonizing laboratory practices, offering users a benchmark system to evaluate their own performance and identify areas requiring improvement. The objective of this study was to go through and analyze the UK NEQAS "Immunology, Immunochemistry and Allergy" EQA reports between 2012 and 2021 to assess the overall level of harmonization in autoimmune diagnostics and identify areas requiring improvement for future actions. The EQA programs reviewed included anti-nuclear (ANA), anti-dsDNA, anti-centromere, anti-extractable nuclear antigen (ENA), anti-phospholipids, anti-neutrophil cytoplasm (ANCA), anti-proteinase 3 (PR3), anti-myeloperoxidase (MPO), anti-glomerular basement membrane (GBM), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA), mitochondrial (AMA), liver-kidney-microsomal (LKM), smooth muscle (ASMA), APCA, and celiac disease antibodies. In the analyzed period, the number in participating laboratories showed an increase for almost all programs. Among solid phase methods, the use of ELISA techniques showed a progressive reduction, while new technologies, such as the fluoroenzymatic immunoassay, chemiluminescence immunoassay, Luminex and immunoblot showed an increased number of users. The number of results complying with the expected negative or positive target slightly increased for almost all antibodies in the last decade. A description of the most frequent causes of mistakes or misinterpretation for each specific test and method is also provided in this study. Although numerous challenges need to be addressed in the area of autoantibody detection to enhance testing quality and attain higher harmonization, the period analyzed revealed that the ever-expanding range of autoantibodies, coupled with the introduction of new tests and methodologies and the advent of automated platforms, has brought about significant changes in autoimmune diagnostics.

An Oversimplified Corrosion Inhibition Experiment Suitable for the Development of the Laboratory Practice for Undergraduate Students

An Oversimplified Corrosion Inhibition Experiment Suitable for the Development of the Laboratory Practice for Undergraduate Students

The small forest: a laboratory for sustainability and regenerative production

El Bosque Pequeño is an innovative project located in Entre Ríos, Argentina, that integrates sustainability, regenerative and circular production, and nature-based solutions. This space, conceived as a sustainability laboratory, combines agricultural, educational and community practices to promote environmental conservation and harmonious coexistence between people and their environment. The proposal focuses on bioclimatic construction, efficient water management, the incorporation of native biodiversity, sustainable beekeeping and agricultural production, and the use of regenerative technologies such as composting and beneficial microorganisms. These practices not only mitigate environmental impact, but also generate applicable knowledge for rural and peri-urban development. From an educational approach, experiential, disruptive and continuous learning is promoted, which fosters cognitive and socioemotional skills, such as critical thinking and teamwork. This model combines cultural and sports activities, such as karate, to reinforce the values of coexistence and co-prosperity, while the preservation of a familiar historical legacy connects participants with their cultural context. El Bosque Pequeño represents an example of how participatory action research can transform a space into a sustainable model, combining education, production and conservation to address current social and environmental challenges.

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of oximes in KIKO mouse plasma.

Chemical warfare nerve agents (CWNAs) are potent and irreversible inhibitors of acetylcholinesterase (AChE). Oxime reactivators are an important part of the standard treatment for CWNA exposure as they can reactivate inhibited AChE. Evaluating the oxime candidates of interest in biological samples requires analytical detection methods and oxime reactivators as a class of compounds have historically been notoriously difficult to isolate, detect and analyze in an analytical laboratory, and there are currently no sensitive or robust analytical detection methods in the literature. The goal of this study was to develop reliable and robust novel extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to detect and quantitate 2-PAM, HI-6, HLö-7, and MMB-4 in a human AChE knock-in, mouse carboxylesterase knock-out (KIKO) mouse in vivo model. This study identified an LC column that achieved retention for all four oxime compounds which is a major advancement over past oxime methods. A unique extraction and chromatographic method was developed for each oxime. The developed methods were sensitive down to 0.5ng/mL for 2-PAM, 50ng/mL for HI-6, and 15ng/mL for both HLö-7 and MMB-4. These methods were validated to meet the Food and Drug Administration (FDA) bioanalytical method validation requirements under Good Laboratory Practice (GLP) conditions. The 4 methods were validated for performance by assessing linearity, sensitivity, precision, accuracy, selectivity, specificity, carryover, extraction recovery, dilution analysis, and stability.

Challenges in the diagnosis and follow-up of Cushing's syndrome: Evaluating LC-MS/MS as a confirmation method for urinary free cortisol analyses.

24-h urinary free cortisol (UFC) measurements are fundamental in the diagnosis and follow-up of Cushinǵs syndrome (CS) and immunoassays (IA) are the most widely used tests for its quantification in clinical laboratory practice. However, their suitability has been questioned mainly due to their limitations concerning analytical specificity. The aim of this research project was to evaluate a novel algorithm for CS diagnosis and follow-up in the clinical laboratory, based on the combination of IA tests with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for UFC quantification. A quantitative LC-MS/MS approach based on liquid-liquid extraction for sample preparation was developed and fully characterized. A population-based reference range was established and the level of agreement for UFC values when compared to IA approach was assessed in patients under CS follow-up or clinical suspicion for hypercortisolism. Significantly higher UFC values were observed for IA when compared to LC-MS/MS approach, therefore population-based reference range was established for the latter (i.e. 4 - 41 µg/day). The clinical application of the herein presented LC-MS/MS approach to be used as a confirmation procedure for CS management was assessed and a high level of agreement with IA UFC values, except in the case of IA undetectable results, was observed. However, IA potential false negative (FN) and false positive (FP) findings were also noted. Higher clinical sensitivity for CS diagnosis / follow-up was observed for LC-MS/MS when compared to IA, supporting the advantage and necessity of implementing LC-MS/MS as a confirmation procedure in the clinical laboratory.

Lamellarity of ultrasound assisted formations of dipalmitoyl-lecithin vesicles.

Formation of unilamellae of fully hydrated dipalmitoylphosphatidylcholine (DPPC) was induced by a horn sonicator from multilamellar vesicles and followed by time-resolved synchrotron small angle X-ray scattering and direct visual morphological investigations by the means of transmission electron-microscopy combined with freeze-fracture. Without incubation the ultrasonication causes continuous increasing in temperature and transformation from the gel to rippled gel structures, then reaching the main transition, the formfactor of unilamellar structure appeared. The ultrasonication resulted in different layer formations at the characteristic temperatures of the gel (20°C), rippled gel (38°C), and liquid crystalline (45°C) phases of the system. At 20°C irregular stacks of multi and oligolamellar lamellae were shaped even after three hours of ultrasonication. At 38 and 45°C the ultrasound induced dominantly unilamellar vesicles (ULVs) in a short time (10 and 3 - 5min, respectively, under typical ultrasound treatments in the general laboratory practice). After the end of the ultrasonication, irregular layer formations with defects structures increased when the temperature of sonication was above the chain melting temperature of the hydrated DPPC system, underlining the importance of optimized sonication processes.

Development and Clinical Detection of Rapid Molecular Diagnostic System for Pathogenic Dermatophytes of Tinea Capitis of Multiple Centres in China.

Tinea capitis remains a common fungal infection in children worldwide. Species identification is critical for determining the source of infection and reducing transmission. In conventional methods, macro- and microscopic analysis is time-consuming and results in slow fungal growth or low specificity. We propose a rapid real-time diagnostic PCR method that allows species-specific identification of dermatophytes, including the Microsporum canis complex, Trichophyton mentagrophytes complex, Trichophyton rubrum complex and Trichophyton tonsurans, in patients with tinea capitis. Hair and scrapings samples were collected from 231 patients with tinea capitis who were positive for fungal elements via direct microscopy with potassium hydroxide. Each sample was subjected to a two-step real-time PCR (RT-PCR) assay, which was designed on the basis of differences in the DNA fragments of the internal transcribed spacer (ITS) and β-tubulin covering the Microsporum canis complex, T. mentagrophyte complex, T. rubrum complex, T. tonsurans, T. verrucosum, T. schoenleinii and N. gypseum. In total, 186/231 samples (80.52%) were positive for fungal culture. The two-step RT-PCR was positive in 215/231 samples (93.07%), among which 179 were culture positive. The combined efficacy was 96.81%, which was significantly different when the RT-PCR assays were performed in parallel with fungal culture. A total of 126 samples (54.55%) were identified as Microsporum canis by fungal culture, among which the positive rate of M. canis complex RT-PCR was 97.62% (123/126). A total of 45 samples were negative for fungal culture, of which 80.0% (36/45) were positive by RT-PCR, and the percentage of M. canis complex-positive samples was 53.33% (24/45). The RT-PCR assays were negative for 16/231 samples, among which 7 were culture positive, including M. canis (n = 3), T. violaceum (n = 3) and N. gypseum (n = 1). We developed a new diagnostic assay system using a rapid real-time TaqMan PCR assay with specific primers that can be applied in routine laboratory practice for hair and skin samples of tinea capitis to detect dermatophytes and increase diagnostic efficiency.