Methods Protoc., Volume 5, Issue 6 (December 2022) – 17 articles

The rate of caesarean section (C/S) has been increasing globally. The proposed study aims to evaluate the effects of ultrasonography (USG) and health education in reducing unnecessary C/S among pregnant women. The secondary objectives are to increase antenatal care (ANC) and post-natal care (PNC) visit rates, increase USG use, increase institutional (hospitals and clinics) delivery, reduce delivery related complications and reduce the number of still births. This is a prospective, open-label, two-arm cluster randomized controlled trial (RCT). A total of 288 pregnant women are enrolled from two urban and two rural hospitals in Bangladesh. Women were screened during their first ANC visits, then written informed consent was taken from the participants. Women in intervention hospitals receive eight ANC visits, two additional USG, and health education eight times during their ANC visits. In contrast, participants at control hospitals receive usual care consisting of four ANC visits and two USG during their ANC visits. The primary outcome is to compare the rate of unnecessary C/S during PNC visits. This study obtained approval from the Institutional Review Board of North South University (2021/OR-NSU/IRB/0804), Bangladesh in November 2021 and was registered in clinicalTrails.gov (#NCT05135026). Full article

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption–desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required. Full article

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials. Full article

Acute respiratory infections (ARIs) need to be better understood and treated, as they are critical to public health, especially during crises such as the SARS-CoV2 pandemic. These are the most abundant infections in the general population and are seen primarily in primary care by general practitioners (GPs). Many different viruses are involved, according to epidemic variations. Viral co-detections account for a significant proportion of ARIs in hospital cohorts. The objective of the ECOVIR cohort was to study viral co-detections by setting up a biobank of respiratory tract samples from patients consulting their general practitioner for ARI symptoms. We report here on the course of the study: the design, the conduct, and the difficulties encountered. ECOVIR (Etude des CO-detections VIrales dans les prélèvements Respiratoires) was a prospective, multicenter cohort conducted in France during two epidemic seasons (2018–2019 and 2019–2020). We recruited GPs. Each GP investigator (GPI) saw patients weekly for examination, clinical data collection, and nasopharyngeal swabbing. Each sample was sent to the virology unit for biobanking and molecular analysis. Clinical and sociodemographic data were collected 7 days after inclusion. ECOVIR involved 36 GPIs. Patients with symptoms of an ARI were included (n = 685). The median number of inclusions was 16 patients per GPI over both seasons (IC_25–75% [4.75; 27]). Patients aged 18 to 64 years were the most numerous (57%), followed by children (30%), and the elderly (13% over 65 years). This age distribution emphasizes the young adult and middle-aged population. Residents participated in the project and called patients on day 7 to obtain clinical and sociodemographic data. Our study triggered the creation of an original network, which plans to establish a functional link between research and primary health care. Primary care is unfortunately poorly represented in research protocols, particularly in respiratory infections, even though it is a cornerstone of our French health care system, as demonstrated every day in this period of crisis. Full article

This study assessed the relationship between posterior permanent dentition and radiographic changes of the mandibular condyle. This descriptive, cross-sectional study was conducted on 300 panoramic radiographs of patients over 40 years of age (188 females and 112 males). Panoramic radiographs were evaluated for condylar changes such as flattening, subcortical sclerosis, subcortical cyst, erosion, osteophytes, and generalized sclerosis. Presence of muscle pain and temporomandibular joint (TMJ) pain and sounds, and history of TMJ trauma were also assessed. The occlusal scheme of posterior teeth was analyzed according to the Eichner’s index. The frequency of condylar changes was calculated in the right and left sides, and their association with posterior permanent dentition was analyzed by the Chi-square test (alpha = 0.05). The frequency of flattening, muscle pain, TMJ sounds, and erosion was 11.7%, 9.7%, 5.7%, and 3.7% in the right side, respectively. The frequency of flattening, muscle pain, erosion, and subcortical cyst was 12%, 9.3%, 5%, and 5% in the left side, respectively. The frequency of bilateral muscle pain, flattening, TMJ sounds, and TMJ pain was 18%, 16.7%, 11.7%, and 9.3%, respectively. Cases with TMJ trauma, generalized sclerosis, and osteophytes were few. According to the Eichner’s index, most patients with condylar changes had classes A and B, and a smaller percentage had class C. No significant difference was noted between healthy individuals and those with condylar changes regarding dentition patterns. No relationship existed between condylar changes and posterior permanent dentition pattern. Full article

Transcriptome-wide analysis of RNA-binding partners is commonly achieved using UV crosslinking and immunoprecipitation (CLIP). Individual-nucleotide-resolution CLIP (iCLIP)enables identification of the specific position of the protein–RNA interaction. In addition to RNA-binding proteins (RBPs), microRNA (miRNA)–mRNA interactions also play a crucial role in the regulation of gene expression. Argonaute-2 (Ago2) mediates miRNA binding to a multitude of mRNA target sites, enabling the identification of miRNA–mRNA interactions by employing modified Ago2-CLIP protocols. Here, we describe an Ago2-specific CLIP protocol optimized for the use of small quantities of cell material, targeting endogenous Ago2 while avoiding possible methodological biases such as metabolic labeling or Ago2 overexpression and applying the latest advances in CLIP library preparation, the iCLIP2 protocol. In particular, we focus on the optimization of lysis conditions and improved radioactive labeling of the 5′ end of the miRNA. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although COVID-19 is a viral illness, many patients admitted to hospital are prescribed antibiotics, based on concerns that COVID-19 patients may experience secondary bacterial infections, and the assumption that they may respond well to antibiotic therapy. This has led to an increase in antibiotic use for some hospitalised patients at a time when accumulating antibiotic resistance is a major global threat to health. Procalcitonin (PCT) is an inflammatory marker measured in blood samples and widely recommended to help diagnose bacterial infections and guide antibiotic treatment. The PEACH study will compare patient outcomes from English and Welsh hospitals that used PCT testing during the first wave of the COVID-19 pandemic with those from hospitals not using PCT. It will help to determine whether, and how, PCT testing should be used in the NHS in future waves of COVID-19 to protect patients from antibiotic overuse. PEACH is a retrospective observational cohort study using patient-level clinical data from acute hospital Trusts and Health Boards in England and Wales. The primary objective is to measure the difference in antibiotic use between COVID-19 patients who did or did not have PCT testing at the time of diagnosis. Secondary objectives include measuring differences in length of stay, mortality, intensive care unit admission, and resistant bacterial infections between these groups. Full article

Engaging in physical movement has a number of mental and physical health benefits, and yet 45% of Australia’s population do not meet the recommended guidelines for physical activity. The current study aims to develop an online habit-based intervention designed to reduce sedentary behavior within the workplace, using environmental cues to instigate simple behavioral changes. Participants in this study will include full time office workers who self-report as having a highly sedentary job and work from either a commercial office, home office, or a mixture of both. Participants will complete a habit-based intervention over a four-week period designed to reduce sedentary behavior by increasing habitual responses to simple physical movement behaviors cued by their environment. Analysis will involve mixed methods ANOVAs to test the efficacy of the intervention. A successful intervention will show a reduction in sedentary behavior as a response to habitual simple physical movement behaviors. Full article

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (K_d = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z’-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases. Full article

UV filters (UVFs) are widely used in personal care and in industrial products for protection against photodegradation. In recent years, their potential toxicological and environmental effects have received growing attention. Due to their excessive use, their residue levels in the environment are gradually increasing and they tend to accumulate on biological wastewater treatment sludge. The utilization of sludge as fertilizer could be one of the main routes of UVF contamination in the environment. Therefore, the development of a reliable and sensitive method of analyzing their trace level residues in waste sludge samples is of great importance. The success of the method largely depends on the sample preparation technique in such complex matrices. This study presents a rapid, sensitive and green analysis method for eight UVFs in sludge samples, selected for their rather low no-observed-effect concentrations (NOEC). For this purpose, the QuEChERS methodology was coupled with in-port derivatization for subsequent detection of the targeted UVFs via GC–MS/MS. The analysis time was substantially shortened using this method, and reagent utilization was also reduced. The method was validated in the sludge samples, and high recovery (66–123%) and low RSD values (<25.6%) were obtained. In addition, major contributing uncertainty sources and expanded uncertainties were determined. Full article

Maxillary canine impaction is an increasing dental anomaly and is often related to other dento-skeletal anomalies. The aim of this work is to support the clinician in evaluating the relationship between a displaced maxillary canine and clinical (the features of lateral incisors)/skeletal (ponticulus posticus and sella turcica bridging) anomalies through orthopanoramic radiographs, lateral cephalograms, and plaster casts to identify the parameters that best predict maxillary canine impaction. A retrospective observational study was carried out on the analysis of the medical records, radiographic findings (panoramic radiographs and lateral cephalograms), and plaster casts of 203 orthodontic patients divided into a case group, with at least one impacted maxillary canine, and a control group, without an impaction. A chi-square test and logistic regression analysis were used to analyze the data. A statistically significant association was found between the impaction of the maxillary canine and the female sex, the bridging of the sella turcica, the ponticulus posticus calcification, and the anomaly of the lateral incisor; a logistic regression revealed that these significant variables were found to be positive predictors of impacted maxillary canines, particularly in reference to the impaction in the palatal area. Finding one of these clinical and radiographic elements can represent a predictive sign of the possible impaction of the maxillary canine. Full article

The drug combination of rosuvastatin (ROS) and ezetimibe (EZE) is used to treat hypercholesterolemia. In this work, a simultaneous electrochemical examination of ROS and EZE was conducted for the first time. The electrochemical determination of ROS and EZE was carried out using adsorptive stripping differential pulse voltammetry (AdSDPV) on a glassy carbon electrode (GCE) in 0.1 M H₂SO₄. The effects of the pH, scan rate, deposition potential, and time on the detection of ROS and EZE were analyzed. Under optimum conditions, the developed sensor exhibited a linear response between 1.0 × 10⁻⁶ M and 2.5 × 10⁻⁵ M for EZE and 5.0 × 10⁻⁶ M, and 1.25 × 10⁻⁵ M for ROS. The detection limits for ROS and EZE were 3.0 × 10⁻⁷ M and 2.0 × 10⁻⁶ M, respectively. The developed sensor was validated in terms of linear range, accuracy, precision, the limit of determination (LOD), and the limit of quantification (LOQ), and it was evaluated according to ICH Guidelines and USP criteria. The proposed method was also used to determine ROS and EZE in human urine and serum samples, which are reported in terms of recovery studies. Full article

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. Nanodrop^TM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process. Full article

Introduction: Exposure to natural hazards such as fire, drought, floods, and earthquakes can have negative impacts on physical and mental health and wellbeing. The social and structural factors contributing to individual and community vulnerability also influence responses to disaster and the resulting consequences on health and wellbeing. Experiencing disasters like bushfires amplifies the impacts of inequality, magnifying existing disparities and contributing to additional psychological burdens of grief, trauma and adaptive challenge. There is a need to understand how vulnerability can influence responses to disaster, and to identify factors that develop and foster resilience in the context of increasing disasters and vulnerability. Materials and Methods: This protocol will describe the methodology of two scoping reviews: the first will describe the mental health outcomes of vulnerable populations after droughts and bushfires; the second will identify and describe strategies that promote community resilience in vulnerable populations in the context of a disaster. A thorough search will be conducted in relevant databases. Studies will be limited to English language. The reviews will be reported using the 22-item checklist for the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Methodological quality of the included papers will be assessed using the Joanna Briggs Institute’s critical appraisal tools. Results & Conclusions: The two scoping reviews described in this protocol will have broad relevance in the context of increasing and intensifying disasters, and will especially consider the compounded impact of disaster on vulnerable communities. Findings will contribute directly to the design and implementation of solutions to improve post-disaster health and wellbeing and community resilience. Full article

Introduction: Dental caries in the adult population that require preventive and therapeutic treatment are generally neglected in rural communities. The determination of the effectiveness of the application of 38% silver diamine fluoride (SDF) in arresting caries lesions when combined with atraumatic restorative treatment (ART) is very important, as it serves as a preventive and restorative procedure to regain the function of the permanent dentition. The assessment of optimal SDF application with ART, in comparison with ART alone, in managing cavitated carious lesions in a pragmatic setting, is the need of the hour to recommend optimal dental care, especially in rural settings which have minimal access to comprehensive dental care. Methods and Analysis: The clinical trial will enrol 220 adults (18–65 years) with cavitated carious lesions attending the Amrita School of Dentistry in the Ernakulam district, India. This study is a randomized, controlled trial with a 1:1 allocation ratio in two parallel groups. Study arm 1 will receive 38% SDF application and ART, and study arm 2 will receive ART only. A digital radiograph will be taken immediately after restoration (baseline) as well as at the end of the 6th month for evaluation of caries arrest. The assessment of the survival of the restoration will be done on the 7th day, 30th day, and at the end of the 6th month. The final analysis would include both the tooth and person levels. Ethics and Dissemination: This trial adheres to the principles of the Declaration of Helsinki and the guidelines of the Indian Council of Medical Research (ICMR). This study protocol has been approved by the Institutional Review Board. This trial has been registered prospectively with the Clinical Trial Registry of India (Registration No: CTRI/2021/12/038816). Full article

Sabbaticals should be a main feature of every academic career. This article provides some personal insights from experience along with an analysis of the benefits to the home and host institutions. Furthermore, the paper includes extensive and easy-to-follow timelines and guides for one’s own sabbatical. It is hoped that this article will expand the use of sabbaticals for everyone’s benefit in academia. Full article