Methods and Protocols

Escherichia coli O157:H7, a Shiga-toxin-producing E. coli (STEC), is an important pathogen related to foodborne disease that is responsible for a growing number of outbreaks worldwide and has been detected in processed meats, dairy, and fresh vegetables. Although culturing is the gold standard method for detection of this bacterium, molecular methods based on nucleic acid amplification techniques such as PCR are becoming more common because of their rapidity, sensitivity, and specificity. However, to ensure reliable results among the several alternative PCR protocols (e.g., commercial kits and reference methods), different measurement assurance tools, including validated methods, reference materials, and proficiency tests, among others, are required. Herein, we present a digital PCR method validation for E. coli O157:H7 detection and quantification using seven specific gene sequences; this method quantified nucleic acids from different E. coli serotypes, with a detection range of 6.6 to 7900 copies/µL and a repeatability standard deviation over the concentration range of 1% to 13.6%. The relative standard uncertainty was 3.5–14.6%, and the detection limit was 0.27 copies/µL. Subsequently, two batches of a candidate reference material based on E. coli O157:H7 genomic DNA were then produced and characterized for evaluation of copy number concentration with the validated ddPCR method, with assigned values of 164,770 ± 9251 and 172 ± 9 copies/μL. Thus, this study demonstrated the development of a validated method and reference material for dPCR and qPCR detection of E. coli O157:H7, a key STEC responsible for food poisoning. Full article

Using the internet to recruit participants into research trials is effective but can attract high numbers of fraudulent attempts, particularly via social media. We drew upon the previous literature to rigorously identify and remove fraudulent attempts when recruiting rural residents into a community-based health improvement intervention trial. Our objectives herein were to describe our dynamic process for identifying fraudulent attempts, quantify the fraudulent attempts identified by each action, and make recommendations for minimizing fraudulent responses. The analysis was descriptive. Validation methods occurred in four phases: (1) recruitment and screening for eligibility and validation; (2) investigative periods requiring greater scrutiny; (3) baseline data cleaning; and (4) validation during the first annual follow-up survey. A total of 19,665 attempts to enroll were recorded, 74.4% of which were considered fraudulent. Automated checks for IP addresses outside study areas (22.1%) and reCAPTCHA screening (10.1%) efficiently identified many fraudulent attempts. Active investigative procedures identified the most fraudulent cases (33.7%) but required time-consuming interaction between researchers and individuals attempting to enroll. Some automated validation was overly zealous: 32.1% of all consented individuals who provided an invalid birthdate at follow-up were actively contacted by researchers and could verify or correct their birthdate. We anticipate fraudulent responses will grow increasingly nuanced and adaptive given recent advances in generative artificial intelligence. Researchers will need to balance automated and active validation techniques adapted to the topic of interest, population being recruited, and acceptable participant burden. Full article

Over the last few decades, malaria-derived extracellular vesicles (EVs) have gained increasing interest due to their role in disease pathophysiology and parasite biology. Unlike other EV research fields, the isolation of malaria EVs is not standardized, hampering inter-study comparisons. Most malaria EV studies isolate vesicles by the “gold-standard” technique of differential (ultra)centrifugation (DC). Here, we describe in detail an optimized and reproducible protocol for the isolation of malaria-derived EVs by DC. The protocol begins with a description of cultivating high-parasitemia, synchronous P. falciparum cultures that are the source of EV-containing conditioned culture media. The isolation protocol generates two EV subtypes, and we provide details of characterizing these distinct subtypes by analyzing human and parasite proteins by Western blot analysis. We identify some of these proteins as suitable markers for malaria EV subpopulations and subtypes. Full article

The present study proposes the use of Near-Infrared (NIR) spectroscopy as a rapid method for estimating the growth of Spirulina platensis cultures, avoiding any sample manipulation or pretreatment. NIR spectroscopy in diffuse reflectance mode was used on culture volumes as received, with Principal Component Analysis (PCA) and Partial Least Squares (PLS) linear regression, for developing the calibration model in the wavelength range of 1000–2500 nm, in order to choose the appropriate wavelength to estimate the growth of the microalga. The local reflectance maximum at 1062.6 nm, connected with reduced water absorption and scattering effects by the microalga, was identified from PCA as the positive peak in the first loading plot, correlating diffuse reflectance with dilution levels. The calibration curve of diffuse reflectance at 1062.6 nm in response to dilution presented strong linearity, supported by a coefficient of determination (R²) of 0.995. Cross-validation of NIR spectra with a S. platensis culture confirmed the method’s reliability, showing that the growth follows an exponential pattern. The study shows that diffuse reflectance NIR spectroscopy can be used for the rapid monitoring of Spirulina platensis growth. Full article

Background: The aim of this study is to analyze the reproducibility of sample thickness measurements taken by a non-experienced user by comparing a standard digital vernier caliper, with four different protocols, to a specialized thickness gauge. Methods: The current study is a methodological study where we examined the thickness of the porcine arterial wall in the thoracic aorta of six pigs. Two adjacent samples of 10 × 10 mm from each aorta were excised longitudinally from the anterior wall, resulting in twelve specimens. Five protocols were employed to measure the thickness of each sample. In four of these protocols, digital vernier calipers (Multicomp PRO MP012475) were utilized, while the fifth protocol utilized a specialized digital thickness gauge (Mitutoyo 547-500S, Mitutoyo Corp., Kawasaki, Japan). Results: We observed a higher average thickness of the samples during the initial measurement compared to the second measurement (1.11 ± 0.16 vs. 0.94 ± 0.17, p = 0.0319) with the first protocol and smaller values than those determined at the last measurement (0.93 ± 0.15 vs. 1.10 ± 0.15, p = 0.0135) for the third protocol. Further, with the digital vernier calipers, we recorded lower values for all four protocols than for the digital thickness gauge determinations. In addition, we computed the ratio of the thicknesses measured during the first, second, and third measurements to analyze how consistent the values were across the three consecutive measurements, with no difference regarding the third, fourth, and control protocols. Conclusions: The digital thickness gauge offers dependable measurements, regardless of the user’s expertise in assessing tissue thickness, and demonstrates a substantially higher reproducibility when compared to the digital vernier. We also found that taking an average of the thickness measurements from four specific points on each half of the sides or on each diagonal of each corner yielded consistently reliable results over time when using a standard digital vernier caliper instead of a specialized one. Full article

South Africa developed the differentiated service delivery (DSD) model to improve access to healthcare for people living with HIV (PLHIV), especially key populations (KPs) including female sex workers (FSWs) who often face barriers in accessing HIV services. The DSD model, aims to reduce the burden on healthcare users, healthcare workers, and the healthcare system, can significantly benefit this group. However, the success of the DSD model in achieving the desired HIV treatment outcomes for FSWs has been barely evaluated. This paper describes the protocol for evaluation of the DSD model in improving HIV treatment outcomes among FSWs in Gauteng Province of South Africa. Both qualitative and quantitative methods will be utilized to address three study objectives: stakeholder analysis, mapping, and in-depth interviews (objective 1); programme evaluation of the DSD model in selected sites (objective 2); and development of a framework for optimizing the DSD model in improving HIV treatment outcomes (objective 3). Quantitative statistical analysis will be performed using STATA version 17 (College Station, TX, USA). Qualitative analysis will be performed using ATLAS.ti. This study will provide new insights into the utilization of the DSD model among FSWs in South Africa. It will also inform new strategies for the DSD model’s implementation in the country. This study will contribute towards the development of a framework for strengthening the DSD model in improving HIV treatment outcomes among FSWs in Gauteng Province. Full article

Kappaphycus alvarezii is a red seaweed used globally in various biotechnological processes. To ensure the content and stability of its bioactive compounds postharvest, suitable drying protocols must be adopted to provide high-quality raw materials for industrial use. This study aimed to analyze the influence of freeze-drying and oven-drying on the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (FRAP and DPPH assays), total carotenoid content (TC), and lipase (LA) and protease activity (PA) of K. alvarezii samples collected over the seasons in sea farms in southern Brazil. The freeze-drying technique was found to be more effective regarding superior contents of TPC (39.23 to 127.74 mg GAE/100 g) and TC (10.27 to 75.33 μg/g), as well as DPPH (6.12 to 8.91 mg/100 g). In turn, oven-drying proved to be the best method regarding the TFC (4.99 to 12.29 mg QE/100 g) and PA (119.50 to 1485.09 U/g), with better performance in the FRAP (0.28 to 0.70 mmol/100 g). In this way, it appears that the drying process of the algal biomass can be selected depending on the required traits of the biomass for the intended industrial application. In terms of cost-effectiveness, drying the biomass using oven-drying can be considered appropriate. Full article

The valorization of coffee cherry waste through hydrothermal carbonization (HTC) was investigated using various organic and inorganic acid catalysts to produce platform chemicals. This study aimed to evaluate the effectiveness of these catalysts for enhancing reaction rates, improving yields, and promoting selectivity. The results showed that sulfuric acid and adipic acid were the most effective, each resulting in a 20% increase in the total yield, demonstrating the potential of organic acids as efficient catalysts in HTC. Other catalysts, such as benzoic acid and phenylacetic acid, also showed promising results, while butyric acid significantly decreased the total yield. The most abundantly produced platform chemicals were sugars, followed by formic acid, levulinic acid, HMF, and furfural. These findings highlight the potential of coffee cherry waste as a valuable resource for producing key chemicals, and the feasibility of hydrothermal carbonization as a sustainable approach for biomass valorization. This study emphasizes the importance of selecting the appropriate catalysts to optimize the conversion process and maximize the extraction of valuable chemicals. The environmental and economic implications of these findings are significant, as they can contribute to the development of sustainable and efficient biomass utilization technologies that could transform agricultural waste into high-value products while reducing waste and promoting a circular economy. Full article

Continuous advances in neurological research techniques are enabling researchers to further understand the neural mechanisms that regulate energy balance. In this review, we specifically highlight key tools and techniques and explore how they have been applied to study the role of the hypothalamic arcuate nucleus in feeding behaviors. Additionally, we provide a detailed discussion of the advantages and limitations associated with each methodology. Full article

The monitoring of species in a habitat is important to ensure biological diversity. Environmental DNA (eDNA) can infer the presence–absence of species and enable rapid action to avoid threatening factors in ecosystems in the case of non-indigenous species. Loop-mediated isothermal amplification (LAMP) assays for molecular amplification are rapidly gaining popularity in species detection, but LAMP remains an underutilized method for eDNA-based monitoring practices. The most effective combination for successful species monitoring may be the collection of eDNA or biological traces collected by nanofiltration followed by LAMP-based species detection initiatives. Here, we used LAMP analysis to detect the eDNA of Esox lucius (Northern Pike), Anguilla anguilla (European Eel), and Salmo salar (Atlantic Salmon) in Borre Lake and in the Drammen River. The selection of species is based on the categories of regionally invasive species, endangered species, and species of least concern. Two target genes were considered for each species and LAMP primers were designed. Our study showed that LAMP is an effective tool for discovering specific fish eDNA (analysis) to maintain aquatic ecosystems. Full article

Background: Monitoring player mobility in wheelchair sports is crucial for helping coaches understand activity dynamics and optimize training programs. However, the lack of data from monitoring tools, combined with a lack of standardized processing approaches and ineffective data presentation, limits their usability outside of research teams. To address these issues, this study aimed to propose a simple and efficient algorithm for identifying locomotor tasks (static, forward/backward propulsion, pivot/tight/wide rotation) during wheelchair movements, utilizing kinematic data from standard wheelchair mobility tests. Methods: Each participant’s wheelchair was equipped with inertial measurement units—two on the wheel axes and one on the frame. A total of 36 wheelchair tennis and badminton players completed at least one of three proposed tests: the star test, the figure-of-eight test, and the forward/backward test. Locomotor tasks were identified using a five-step procedure involving data reduction, symbolic approximation, and logical pattern searching. Results: This method successfully identified 99% of locomotor tasks for the star test, 95% for the figure-of-eight test, and 100% for the forward/backward test. Conclusion: The proposed method offers a valuable tool for the simple and clear identification and representation of locomotor tasks over extended periods. Future research should focus on applying this method to wheelchair court sports matches and daily life scenarios. Full article

The process of examiner calibration is an essential step in all epidemiological research, as it aims to ensure uniform interpretation, understanding, and application of the instrument to be used. This ensures that the data collected will be valid and reliable. This study aimed to determine the differences in concordance in dental caries calibration across three dental specialties. The population consisted of 45 dentists, divided into three groups: 15 general dentists working in the public sector, 15 dentists specializing in Dental Public Health, and 15 dentists specializing in Restorative and Aesthetic Dentistry. The calibration process was carried out in three stages: theory, calibration using photographs, and calibration on natural teeth, performed by the gold standard. In the first validity process, a statistical difference was only found between the Kappa values of the inter-examiner calibration process using photographs. For the evaluation of teeth, in the second validity process, 33.33% (n = 15) of the participants achieved “almost perfect agreement.” Finally, only 75.56% (n = 34) of the examiners were considered for the reliability report; of this group, 52.94% (n = 18) were in “almost perfect agreement,” and 35.29% (n = 12) were in “substantial agreement.” The validity and reliability of the dental caries experience calibration process did not present significant statistical differences between general dentists in the public sector, dentists specializing in Dental Public Health, and dentists specializing in Restorative and Aesthetic Dentistry. Full article

Polyphenols are responsible for wine colour and astringency, and, as antioxidants, they also have beneficial health properties. In this work, we developed a robust full-scan high-resolution mass spectrometry method for the quantification of 90 phenolic compounds in wine samples (either red, rosé, or white wine), using a UHPLC-Orbitrap^TM system. With this method, we could conduct a detailed analysis of phenolic compounds in red, rosé, and white wines with great selectivity due to sub-ppm mass accuracy. Moreover, accessing the full-scan spectrum enabled us to monitor all the other compounds detected in the sample, facilitating the adaptability of this method to new phenolic compounds if needed. Full article

Maintaining and improving the health and well-being of older people in rural communities through integrated care is essential to address this cohort’s frailty risk. The Indigo 4Ms Tool for health workers is a rural-specific approach to providing care that addresses the common conditions of ageing. With Australian government funding, five small rural health services are implementing the tool. This paper describes the protocol for a hybrid type 2 implementation-effectiveness study to evaluate the tool’s impact on multidisciplinary comprehensive care planning and the implementation strategies that enhance the adoption and sustainability of the tool across diverse rural health settings. Full article

Sucralose stands as the most common non-nutritive sweetener; however, its metabolic effects have sparked significant controversy over the years. We aim to examine the effects of sucralose daily intake on glycemia, subjective appetite, and gut microbiota (GM) changes in subjects with overweight or obesity. In this randomized, crossover, and controlled trial, 23 participants with a body mass index between 25 kg/m² and 39.9 kg/m² will be assigned to one of two interventions to receive either sucralose (2 mg/kg/day equivalent to 40% of the acceptable daily intake) or glucose (control) for 4 weeks, each phase separated by a 4-week washout period. The glycemic response will be determined during a meal tolerance test, subjective appetite will be evaluated using a visual analog scale, and GM changes will be analyzed by next-generation sequencing of the bacterial rRNA 16S gene from fecal samples. All measures will be performed before and after intervention periods. We hypothesize that sucralose supplementation induces changes in glycemic response, subjective appetite, and gut microbiota in overweight and obese participants. This protocol was approved by the Ethics Committee of the UJAT (No. 0721) and was registered in the Australian New Zealand Clinical Trials Registry (ACTRN12621001531808). Full article

Background: Post-stroke epilepsy can reduce patients’ abilities to carry out various activities of daily living. Despite their importance in preventing the onset of post-stroke epilepsy, the prophylactic administration of antiepileptic drugs is controversial due to a lack of high-level clinical research. In this study, we initiated a prospective interventional study of prophylactic antiepileptic drug administration in patients with a subcortical hemorrhage, who are at the highest risk of developing epilepsy after experiencing a stroke. Methods: The study was conducted in a single-center setting and was a single-arm study with no control group; the case entry period started in November 2023 and is due to end in March 2025. Only cases with a subcortical hemorrhage will be included. The treatment regimen used in this study is 2 mg of perampanel per day. Perampanel will be administered for one year, followed by two years of follow-up, for a total study period of three years. The primary endpoint will be the development of epilepsy. Results: Perampanel administration is expected to reduce the incidence of post-stroke epilepsy in comparison to the results of previous reports on the use of alternative treatments. Conclusions: The results of this study will provide new insights into the prevention of post-stroke epilepsy. The relatively small size of this study makes it difficult to provide strong evidence of the efficacy of perampanel, but it may serve as a basis for larger clinical trials. Full article

Background: Telenursing comprises a set of tools and interventions enabling nurses to provide remote care. This study aims to assess the impact of telenursing interventions on the supportive care needs of patients with melanoma and lung cancer who are receiving targeted therapies. Methods: This six-month monocentric, double-arm, randomised, controlled trial study protocol will assess the effect of telenursing on the supportive care needs (primary outcome) in 40 patients (20 in each group) after one month. The secondary outcomes will be monitored at baseline, one, three and six months: supportive care needs (at three and six months), therapeutic adherence, quality of life, usability and satisfaction, performance status, patient-reported outcomes and main adverse events. The SPIRIT guidelines will be used for the reporting. Results: The results from this trial will assess the impact of a telenursing intervention on cancer care. Conclusions: This trial could be a starting point for more extensive studies on telenursing interventions to promote nurses’ skills, as well as the quality and safety of care in patients with cancer, highlighting the impact of more outstanding nursing contributions on cancer care. Trial and Protocol Registration: The study protocol was approved by the relevant Italian Ethics Committee Lazio Area 5 (RS1851/23, 2773; 6 September 2023) and was registered on ClinicalTrials.gov (trial registry number NCT06254196). Full article

Although South Africa was the first country to register and roll out oral pre-exposure prophylaxis (PrEP) biomedical human immunodeficiency virus (HIV) prevention intervention in sub-Saharan Africa (SSA), its uptake remains low, particularly among adolescent girls and young women (AGYW). The uptake of PrEP may have worsened during the Coronavirus disease 2019 (COVID-19) pandemic. Some innovative interventions to improve PrEP uptake among AGYW have been implemented. This study aims to evaluate the effectiveness of PrEP innovative interventions implemented during COVID-19 towards reducing the risk of HIV infection among AGYW in South Africa. An exploratory, descriptive design will be conducted to carry out four study objectives. Firstly, to carry out a systematic review of innovative PrEP interventions implemented during COVID-19 in SSA countries. Secondly, to conduct a stakeholder analysis to identify PrEP stakeholders and interview them on their views on the implemented interventions. Thirdly, to assess the implementation outcomes of the innovative interventions using document reviews and Consolidated Framework for Implementation Research. Fourthly, to develop a framework for an improved PrEP service delivery among AGYW. Qualitative data will be captured in ATLAS.ti software (Technical University, Berlin, Germany) version 23 and analysed via thematic analysis. A statistical software package (STATA) version 18 (College Station, TX, USA) will be used to capture quantitative data and analyse them via descriptive analysis. The generated evidence will be used towards the development of framework, guidelines, and policies to strengthen the uptake of, scale-up, and adherence to PrEP among AGYW. Full article

In recent years, great interest has been focused on the development of highly reproducible 3D in vitro models that are able to mimic the physiological architecture and functionality of native tissues. To date, a wide range of techniques have been proposed to recreate an intestinal barrier in vitro, including synthetic scaffolds and hydrogels, as well as complex on-a-chip systems and organoids. Here, we describe a novel protocol for the generation of an artificial intestine based on the creation of decellularized bio-scaffolds and their repopulation with intestinal stromal and epithelial cells. Organs collected at the local slaughterhouse are subjected to a decellularization protocol that includes a freezing/thawing step, followed by sequential incubation in 1% SDS for 12 h, 1% Triton X-100 for 12 h, and 2% deoxycholate for 12 h. At the end of the procedure, the generated bio-scaffolds are repopulated with intestinal fibroblasts and then with epithelial cells. The protocol described here represents a promising and novel strategy to generate an in vitro bioengineered intestine platform able to mimic some of the complex functions of the intestinal barrier, thus constituting a promising 3D strategy for nutritional, pharmaceutical, and toxicological studies. Full article