A Review of Regulatory Frameworks for Biobanking in Southeast Asia.

Southeast Asian countries are at the forefront of public health pressures due to a confluence of factors such as population growth, urbanization, environmental pollution, and infectious diseases (re)emergence. Therefore, the ability to be able to conduct research addressing local and regional needs is of paramount importance. As such, biobanking activities, the standardized collection of biological samples, and associated data, developed over the past few decades supporting ongoing biomedical and clinical research, as well as surveillance are of critical importance. However, the regulatory landscape of biobanking is not widely understood and reported, which this narrative review aims to address for the ASEAN member states. It is evident that there are specific regulatory arrangements within each ASEAN member state, which though may be sufficient for the current level of operations, are unlikely to support a regional sharing of biological samples, data, and eventually benefits from the conducted research. Additionally, legacy and often-overlapping regulatory frameworks exist, which raise the need of an eventual consolidation under a single framework. Thus, this field requires further study as well as the creation of viable, practical proposals that would allow for biobanking harmonization and thus the exchange of biological samples and data to be achieved regionally, if not further afield.

Parasitological Examination of the Digestive System of Wild Boar from a Practical Point of View-Endoparasitological Sampling under Field Conditions.

From 2015 to 2023, we conducted a comprehensive study in the 11,893-hectare hunting area managed by the Marcal-Bitvaközi Hunting Company, characterised by its substantial wild boar population. The research was carried out across various settings, including a free-range wild boar garden during large-scale hunts and free-living areas during individual hunts. We examined 216 wild boars in total, with 173 individuals from free-living areas and 43 from free-range areas. Throughout the sample collection process, we encountered numerous technical challenges that are infrequently detailed in the professional literature, often mentioned only tangentially. This oversight in existing publications neglects the significance of addressing field sampling difficulties, which are crucial for ensuring the precision and accuracy of research. This paper details the equipment requirements, sampling methodologies, and practical solutions to streamline fieldwork. While our primary focus was on endoparasitic infections of the stomach and small intestine, the described methodologies and findings are broadly applicable to research involving all internal organs.

Sample Collection and Processing in Volatile Organic Compound Analysis for Gastrointestinal Cancers.

Volatile organic compounds have drawn significant attention in recent years as a novel tool for non-invasive detection of a wide range of diseases, including gastrointestinal cancers, for which the need for effective, affordable, and non-invasive screening methods is substantial. Sample preparation is a fundamental step that greatly influences the quality of results and the feasibility of wide-range applications. This review summarizes sampling methods used in studies aiming at testing the diagnostic value of volatile organic compounds in gastrointestinal cancers, discussing in detail some of the recent advancements in automated sampling techniques. Finally, we propose some directions in which sample collection and processing can improve for VOC analysis to be popularized in clinical settings.

Hair collection protocol in 12-month-old infants.

Purpose: Most studies assessing hair cortisol were conducted with adults. As specific guidelines for infant hair collection are lacking, we developed a hair collection protocol for 12-month-old infants and assessed its acceptability and feasibility. Results: Out of the total (N = 45), 95.6 % (n = 43) of caregivers consented to the procedure, while one caregiver did not consent (2.2 %), and another requested the procedure to be halted before required amount of hair had been reached (2.2 %). Furthermore, two (4.4 %) infants did not have enough hair for collection. There was no attrition due to infant fussiness/crying. Discussion: We learned five lessons which can help to enhance reproducibility, mother's consent, and mother-infant comfort and acceptance of the procedure. The first lesson is to have the infant sit on the caregiver's lap to ensure the infant feels safe and remains relatively still. The second is to reassure caregivers by showing hair samples representing the amount to be cut as well as by clarifying no unaesthetic gaps would be visible. The third is to caress the infant's head to habituate them to the hair manipulation and to make soap bubbles as distractors. The fourth is to take extra care when securing the lock of hair for cutting because the infant scalp is thin and malleable. The fifth is to place a precision scale in the collection room to ensure the necessary weight is reached. Conclusion: Our hair collection protocol developed for 12-month-old infants was deemed feasible and acceptable, filled an important literature gap concerning the absence of published protocols for infants, and will contribute to increase the replicability and collection efficiency for other research teams.

A comparative study of sampling methods in the detection of esophageal cancer-related microbiota.

Esophageal cancer (EC) is a multifaceted disease. Our understanding of the involvement of esophageal microbiota in its pathogenesis and progression is limited, which is due to the lack of proper endoscopic sampling methods. Hereby, we conducted a comparative analysis of paired samples obtained through endoscopic brushing and cytosponge, aiming at assessing the feasibility of using cytosponge as a minimally invasive sampling way for studying esophageal microbiota. Our findings suggest that cytosponge sampling yielded significantly superior community richness and diversity compared to endoscopic brushing in both controls (non-cancerous) and EC individuals. The analysis of beta-diversity revealed distinct microbial community pattern in the genus diversity between the two sampling methods, underscoring the importance of selecting appropriate sampling methods to effectively characterize the esophageal microbiota. Specifically, Lactococcus and Serratia showed higher abundance in the samples collected by endoscopic brushing, while Alloprevotella and Leptotrichia were more enriched in the samples collected by cytosponge. These differences in dominant microbes were associated with metabolic pathways that particularly were related to host inflammation, such as pyruvate and glucose metabolisms. Notably, the phylogenetic levels of the microbiota indicated varied explanatory power for different detection purposes. This study underscores the substantial impact of sampling method selection on the acquisition of esophageal microbiota associated with the EC development, encompassing considerations of both abundance and diversity. This highlights the significance of selecting an appropriate sampling method for investigating the esophageal microbial status and studying the micro-environment in EC-related individuals. IMPORTANCE: This study addresses a critical issue in esophageal cancer study by comparing two different sampling methods, endoscopic brushing and cytosponge, for investigating the esophageal microbiota. Our work highlights the suitability of the cytosponge technique as a minimally invasive sampling method for studying the esophageal microbiota and emphasizes the importance of selecting an appropriate sampling method to characterize the microbial community. Our findings have significant implications for advancing the understanding of the role of the esophageal microbiota in cancer development and will inform future research and clinical approaches in this field.

Development of Aptamers for RNase Inactivation in Xtract-Free™ Sample Collection and Transport Medium.

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses.

Evaluation of fecal sample collection methods for feline gut microbiome profiling: fecal loop vs. litter box.

Introduction: Microbial population structures within fecal samples are vital for disease screening, diagnosis, and gut microbiome research. The two primary methods for collecting feline fecal samples are: (1) using a fecal loop, which retrieves a rectal sample using a small, looped instrument, and (2) using the litter box, which collects stool directly from the litter. Each method has its own advantages and disadvantages and is suitable for different research objectives. Methods and results: Whole-genome shotgun metagenomic sequencing were performed on the gut microbiomes of fecal samples collected using these two methods from 10 adult cats housed in the same research facility. We evaluated the influence of collection methods on feline microbiome analysis, particularly their impact on DNA extraction, metagenomic sequencing yield, microbial composition, and diversity in subsequent gut microbiome analyses. Interestingly, fecal sample collection using a fecal loop resulted in a lower yield of microbial DNA compared to the litterbox method (p = 0.004). However, there were no significant differences between the two groups in the proportion of host contamination (p = 0.106), virus contamination (p = 0.232), relative taxonomy abundance of top five phyla (Padj > 0.638), or the number of microbial genes covered (p = 0.770). Furthermore, no significant differences were observed in alpha-diversity, beta-diversity, the number of taxa identified at each taxonomic level, and the relative abundance of taxonomic units. Discussion: These two sample collection methods do not affect microbial population structures within fecal samples and collecting fecal samples directly from the litterbox within 6 hours after defecation can be considered a reliable approach for microbiome research.

Analyzing factors affecting positivity in drive-through COVID-19 testing: a cross-sectional study.

BACKGROUND: Demand for COVID-19 testing prompted the implementation of drive-through testing systems. However, limited research has examined factors influencing testing positivity in this setting. METHODS: From October 2020 to March 2023, a total of 1,341 patients, along with their clinical information, were referred from local clinics to the Sasebo City COVID-19 drive-through PCR center for testing. Association between clinical information or factors related to the drive-through center and testing results was analyzed by Fisher's exact test and logistic regression models. RESULTS: Individuals testing positive exhibited higher frequencies of upper respiratory symptoms; cough (OR 1.5 (95% CI 1.2-1.8), p < 0.001, q = 0.005), sore throat (OR 2.4 (95% CI 1.9-3.0), p < 0.001, q < 0.001), runny nose (OR 1.4 (95% CI 1.1-1.8), p = 0.002, q = 0.009), and systemic symptoms; fever (OR 1.5 (95% CI 1.1-2.0), p = 0.006, q = 0.02), headache (OR 1.9 (95% CI 1.4-2.5), p < 0.001, q < 0.001), and joint pain (OR 2.7 (95% CI 1.8-4.1), p < 0.001, q < 0.001). Conversely, gastrointestinal symptoms; diarrhea (OR 0.2 (95% CI 0.1-0.4), p < 0.001, q < 0.001) and nausea (OR 0.3 (95% CI 0.1-0.6), p < 0.001, q < 0.001) were less prevalent among positives. During omicron strain predominant period, higher testing positivity rate (OR 20 (95% CI 13-31), p < 0.001) and shorter period from symptom onset to testing (3.2 vs. 6.0 days, p < 0.001) were observed compared to pre-omicron period. Besides symptoms, contact history with infected persons at home (OR 4.5 (95% CI 3.1-6.5), p < 0.001, q < 0.001) and in office or school (OR 2.9 (95% CI 2.1-4.1), p < 0.001, q < 0.001), as well as the number of sample collection experiences by collectors (B 7.2 (95% CI 2.8-12), p = 0.002) were also associated with testing results. CONCLUSIONS: These findings underscore the importance of factors related to drive-through centers, especially contact history interviews and sample collection skills, for achieving higher rates of COVID-19 testing positivity. They also contribute to enhanced preparedness for next infectious disease pandemics.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Current Trends and Challenges of Microbiome Research in Prostate Cancer.

PURPOSE OF REVIEW: The role of the gut microbiome in prostate cancer is an emerging area of research interest. However, no single causative organism has yet been identified. The goal of this paper is to examine the role of the microbiome in prostate cancer and summarize the challenges relating to methodology in specimen collection, sequencing technology, and interpretation of results. RECENT FINDINGS: Significant heterogeneity still exists in methodology for stool sampling/storage, preservative options, DNA extraction, and sequencing database selection/in silico processing. Debate persists over primer choice in amplicon sequencing as well as optimal methods for data normalization. Statistical methods for longitudinal microbiome analysis continue to undergo refinement. While standardization of methodology may help yield more consistent results for organism identification in prostate cancer, this is a difficult task due to considerable procedural variation at each step in the process. Further reproducibility and methodology research is required.

LC-MS/MS determination of atropine toxicity: Pre-analytical effect of blood collection tube and analytical matrix.

Atropine (ATR) intoxication is a recurrent case in emergency departments. The diagnosis is dependent on clinical evaluation and is supported by analytical assessment. The assay is limited by the rapid degradation/metabolism of ATR into TRP as well as the preanalytical factors impairing correct detection and diagnosis. In this study, an HPLC-MS/MS method was optimized for the simultaneous determination of ATR and TRP. The effect of analytical matrix and the impact of blood-collection tube type on the ATR analytical signal were investigated. Separation was achieved using water: 0.01% formic acid acidified methanol (40: 60, v/v) as a mobile phase and Inertsil® C18 column (5 µm; 4.6*150 mm) as a stationary phase. The retention-times were 2.6 and 6.5 min for ATR and TRP, respectively. A chromatographic shift (0.4 min) in ATR peak, but not TRP, was observed in biological samples from neat ones. The best analytical signal was observed when heparinized blood collection tubes were employed. The method was linear, accurate and precise in the ATR toxicity range enabling the detection of ATR intoxication down to a concentration of 0.1 ng/mL by applying a simple sample clean-up procedure. In conclusion, an HPLC-MS/MS method for the simultaneous determination of ATR and TRP is presented. The method highlights the chromatographic shift of ATR peak in biological samples that may induce false-negative detection and poses TRP as an alternative toxicological marker for ATR toxicity. Meanwhile the study recommends heparin tubes for blood-sample collection.

Modified Procedure for Simple and Rapid Collection of Zebrafish Fluid Samples for Biochemical Analyses.

This study addresses the challenge of collecting blood samples from zebrafish for biochemical analysis. Traditional methods are cumbersome due to low blood flow and rapid coagulation. Based on a previously published technique, we simplified the process by applying an anticoagulant solution directly to the incision site. The modified protocol involves immersing the fish in an ice bath, making a cross-sectional incision, and immediately applying anticoagulant solution. Centrifugation of the specimens provides a streamlined and efficient approach to zebrafish fluid sample collection, compatible with classic biochemical marker analyses.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 µL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

A novel method for sampling subgingival microbiome: a comparative metatranscriptomic study.

The subgingival microbiome has been implicated in oral and systemic diseases such as periodontitis and Alzheimer's disease. However, subgingival sampling is challenging. We developed a novel method of sampling the subgingival microbiome by rotationally swabbing the supragingival area, named subgingival-P (for proxy) samples. We sampled and metatranscriptomically analyzed subgingival and subgingival-P samples of three different teeth in 20 individuals. The subgingival-P samples were comparable to the subgingival samples in the relative abundances of microorganisms and microbial gene expression levels. Our data demonstrate that the novel method of collecting and analyzing the subgingival-P samples can act as a proxy for the subgingiva, paving the way for large and diverse studies investigating the role of the subgingival microbiome in health and disease.

Effects of variation in sample storage conditions and swab order on 16S vaginal microbiome analyses.

IMPORTANCE: The composition of the human vaginal microbiome has been linked to a variety of medical conditions including yeast infection, bacterial vaginosis, and sexually transmitted infection. The vaginal microbiome is becoming increasingly acknowledged as a key factor in personal health, and it is essential to establish methods to collect and process accurate samples with self-collection techniques to allow large, population-based studies. In this study, we investigate if using AssayAssure Genelock, a nucleic acid preservative, introduces microbial biases in self-collected vaginal samples. To our knowledge, we also contribute some of the first evidence regarding the impacts of multiple swabs taken at one time point. Vaginal samples have relatively low biomass, so the ability to collect multiple swabs from a unique participant at a single time would greatly improve the replicability and data available for future studies. This will hopefully lay the groundwork to gain a more complete and accurate understanding of the vaginal microbiome.

Effect of multiple loading FIT sample collection devices on faecal haemoglobin concentration.

BACKGROUND: Faecal immunochemical tests (FIT) are routinely used in colorectal cancer (CRC) screening programmes around the world. More recently, quantitative FIT has been recommended to help triage patients presenting to primary care with symptoms suggestive of CRC. Participants collect faecal samples using sampling probes which are inserted into sample collection devices (SCDs) containing preservative buffer. The SCDs have an internal collar designed to remove excess sample. The aim of this study was to investigate the impact of multiple loading on faecal haemoglobin concentration (f-Hb) using SCDs of four FIT systems. METHODS: Pools of f-Hb negative samples were spiked with blood, homogenised and loaded into SCDs 1, 3 and 5 times, with insertion of the sampling probes into the SCDs with and without mixing between loads. The f-Hb was measured using the relevant FIT system. The percentage change in f-Hb for multiple loads was compared with a single load for each system for the mixed and unmixed groups. RESULTS: The p values show a significant difference (p < 0.05) in the mass and f-Hb for the mixed and unmixed group, for 1-3 and 1-5 loads for all systems. The median percentage change in f-Hb for the mixed is higher than the unmixed group. CONCLUSION: This study showed that multiple loading does significantly increase the f-Hb in the SCDs.

Quantitative Analysis of a Pilot Transwell Barrier Model with Automated Sampling and Mathematical Modeling.

In the preclinical phase of drug development, it is necessary to determine how the active compound can pass through the biological barriers surrounding the target tissue. In vitro barrier models provide a reliable, low-cost, high-throughput solution for screening substances early in the drug candidate development process, thus reducing more complex and costly animal studies. In this pilot study, the transport properties of TB501, an antimycobacterial drug candidate, were characterized using an in vitro barrier model of VERO E6 kidney cells. The compound was delivered into the apical chamber of the transwell insert, and its concentration passing through the barrier layer was measured through the automated sampling of the basolateral compartment, where media were replaced every 30 min for 6 h, and the collected samples were stored for further spectroscopic analysis. The kinetics of TB501 concentration obtained from VERO E6 transwell cultures and transwell membranes saturated with serum proteins reveal the extent to which the cell layer functions as a diffusion barrier. The large number of samples collected allows us to fit a detailed mathematical model of the passive diffusive currents to the measured concentration profiles. This approach enables the determination of the diffusive permeability, the diffusivity of the compound in the cell layer, the affinity of the compound binding to the cell membrane as well as the rate by which the cells metabolize the compound. The proposed approach goes beyond the determination of the permeability coefficient and offers a more detailed pharmacokinetic characterization of the transwell barrier model. We expect the presented method to be fruitful in evaluating other compounds with different chemical features on simple in vitro barrier models. The proposed mathematical model can also be extended to include various forms of active transport.

Driving under the influence of cannabis: A 5-year retrospective Italian study.

INTRODUCTION: Cannabis consumption is associated with driving impairment and increased crash risk, endangering road safety. Toxicological analyses play a fundamental role in detecting a recent consumption of psychoactive substances. The aim of this study was to examine the concentration of cannabinoids in blood samples of driving-under-the-influence (DUI) offenders in order to investigate whether delayed sample collection affects the toxicological assessment of the offenders. MATERIALS AND METHODS: An observational retrospective study was performed using anonymized toxicological data referring to cannabis-related DUI offenders involved in road traffic accidents (RTA) or apprehended by the police from 1 January 2017-31 December 2021 archived at Legal Medicine and Toxicology Department of the University Hospital of Padova, Italy. RESULTS: In a total sample of 318 drivers, 143 blood samples tested positive for tetrahydrocannabinol (THC) and metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), and 173 blood samples were positive for THC-COOH with THC negative. In the first group, the mean concentrations of THC and THC-COOH were 4.05 ng/mL and 28.29 ng/mL, respectively. In THC-negative cases, the mean THC-COOH concentration was 7.3 ng/mL. The time elapsed between the event and sample collection varied from 15 min to 7 h (mean 2 h 29 min). The average estimated time elapsed after consumption of cannabinoids was 3 h 7 min (Model I) and 2 h 36 min (Model II). CONCLUSIONS: The present research discussed the main difficulties in the toxicological evaluation of drivers under the influence of Cannabis. Issues related to the time between RTA and sample collection, the laws and legal limits in force in various Countries were presented.

Unravelling the brain metabolome: A review of liquid chromatography - mass spectrometry strategies for extracellular brain metabolomics.

The analysis of the brain extracellular metabolome is of interest for numerous subdomains within neuroscience. Not only does it provide information about normal physiological functions, it is even more of interest for biomarker discovery and target discovery in disease. The extracellular analysis of the brain is particularly interesting as it provides information about the release of mediators in the brain extracellular fluid to look at cellular signaling and metabolic pathways through the release, diffusion and re-uptake of neurochemicals. In vivo samples are obtained through microdialysis, cerebral open-flow microperfusion or solid-phase microextraction. The analytes of potential interest are typically low in concentration and can have a wide range of physicochemical properties. Liquid chromatography coupled to mass spectrometry has proven its usefulness in brain metabolomics. It allows sensitive and specific analysis of low sample volumes, obtained through different approaches. Several strategies for the analysis of the extracellular fluid have been proposed. The most widely used approaches apply sample derivatization, specific stationary phases and/or hydrophilic interaction liquid chromatography. Miniaturization of these methods allows an even higher sensitivity. The development of chiral metabolomics is indispensable, as it allows to compare the enantiomeric ratio of compounds and provides even more challenges. Some limitations continue to exist for the previously developed methods and the development of new, more sensitive methods remains needed. This review provides an overview of the methods developed for sampling and liquid chromatography-mass spectrometry analysis of the extracellular metabolome.

Application of Single Particle ICP-MS for the Determination of Inorganic Nanoparticles in Food Additives and Food: A Short Review.

Due to enhanced properties at the nanoscale, nanomaterials (NMs) have been incorporated into foods, food additives, and food packaging materials. Knowledge gaps related to (but not limited to) fate, transport, bioaccumulation, and toxicity of nanomaterials have led to an expedient need to expand research efforts in the food research field. While classical techniques can provide information on dilute suspensions, these techniques sample a low throughput of nanoparticles (NPs) in the suspension and are limited in the range of the measurement metrics so orthogonal techniques must be used in tandem to fill in measurement gaps. New and innovative characterization techniques have been developed and optimized for employment in food nano-characterization. Single particle inductively coupled plasma mass spectrometry, a high-throughput nanoparticle characterization technique capable of providing vital measurands of NP-containing samples such as size distribution, number concentration, and NP evolution has been employed as a characterization technique in food research since its inception. Here, we offer a short, critical review highlighting existing studies that employ spICP-MS in food research with a particular focus on method validation and trends in sample preparation and spICP-MS methodology. Importantly, we identify and address areas in research as well as offer insights into yet to be addressed knowledge gaps in methodology.