Non-invasive Sampling Research Articles

Microbial functional pathways based on metatranscriptomic profiling enable effective saliva-based health assessments for precision wellness

It is increasingly recognized that an important step towards improving overall health is to accurately measure biomarkers of health from the molecular activities prevalent in the oral cavity. We present a general methodology for computationally quantifying the activity of microbial functional pathways using metatranscriptomic data. We describe their implementation as a collection of eight oral pathway scores using a large salivary sample dataset (n = 9350), and we evaluate score associations with oropharyngeal disease phenotypes within an unseen independent cohort (n = 14,129). Through this validation, we show that the relevant oral pathway scores are significantly worse in individuals with periodontal disease, acid reflux, and nicotine addiction, compared with controls. Given these associations, we make the case to use these oral pathway scores to provide molecular health insights from simple, non-invasive saliva samples, and as molecular endpoints for actionable interventions to address the associated conditions.

A reduced SNP panel optimised for non-invasive genetic assessment of a genetically impoverished conservation icon, the European bison.

The European bison was saved from the brink of extinction due to considerable conservation efforts since the early twentieth century. The current global population of > 9500 individuals is the result of successful ex situ breeding based on a stock of only 12 founders, resulting in an extremely low level of genetic variability. Due to the low allelic diversity, traditional molecular tools, such as microsatellites, fail to provide sufficient resolution for accurate genetic assessments in European bison, let alone from non-invasive samples. Here, we present a SNP panel for accurate high-resolution genotyping of European bison, which is suitable for a wide variety of sample types. The panel accommodates 96markers allowing for individual and parental assignment, sex determination, breeding line discrimination, and cross-species detection. Two applications were shown to be utilisable in further Bos species with potential conservation significance. The new SNP panel will allow to tackle crucial tasks in European bison conservation, including the genetic monitoring of reintroduced populations, and a molecular assessment of pedigree data documented in the world's first studbook of a threatened species.

Hair cortisol concentration in finishing pigs on commercial farms: variability between pigs, batches, and farms.

Hair cortisol is a stress indicator and could be used to assess the pigs' exposure to stressors in the weeks/months prior to non-invasive hair sampling. The main aim of this study was to describe the hair cortisol concentration (HCC) variability between individuals within a batch, between farms and between batches within a farm. The secondary aim was to determine how the number of sampled pigs influences the characterization of HCC within a batch. Twenty farrow-to-finish pig farms were recruited considering the diversity of their management practices and health status (data collected). Hair was sampled in two separate batches, 8 months apart. The necks of 24 finishing pigs were clipped per batch the week prior to slaughter. To describe the variability in HCC, an analysis of the variance model was run with three explanatory variables (batch, farm and their interaction). To identify farm clusters, a principal component analysis followed by a hierarchical clustering was carried out with four active variables (means and standard deviations of the two batches per farm) and 17 supplementary variables (management practices, herd health data). We determined how the number of sampled pigs influenced the characterization of HCC within a batch by selecting subsamples of the results. HCC ranged from 0.4 to 121.6 pg/mg, with a mean of 25.9 ± 16.2 pg/mg. The variability in HCC was mainly explained by differences between pigs (57%), then between farms (24%), between batches within the same farm (16%) and between batches (3%). Three clusters of farms were identified: low homogeneous concentrations (n = 3 farms), heterogeneous concentrations with either higher (n = 7) or lower (n = 10) HCC in batch 2 than in batch 1. The diversity of management practices and health statuses allowed to discuss hypotheses explaining the HCC variations observed. We highlighted the need to sample more than 24 pigs to characterize HCC in a pig batch. HCC differences between batches on six farms suggest sampling pigs in more than one batch to describe the HCC at the farm level. HCC variations described here confirm the need to study its links with exposure of pigs to stressors.

Salivary metabolomics in patients with oral lichen planus: a preliminary study based on NMR spectroscopy

ObjectivesThe present preliminary study aimed to investigate the salivary metabolic profile in patients with asymptomatic oral lichen planus (OLP) using nuclear magnetic resonance (NMR) spectroscopy.Material and methodsStimulated whole mouth saliva (SWMS) samples were collected from 15 reticular OLP female patients and 15 from age- and sex-matched controls (HCs). A total of 23 metabolites were identified and quantified. Mann–Whitney’s U test was used to compare the determined concentration salivary metabolite concentrations between OLP patients and the healthy controls.ResultsThe concentration of acetate, methylamine, and pyruvate was elevated, whereas the concentration of tyrosine was decreased in the saliva of OLP patients compared with HCs. To identify a combination of metabolites, multivariate discrimination function analysis (DFA) was conducted. DFA analysis have shown that the most powerful discrimination between the groups was achieved when methylamine and tyrosine were considered as combined biomarkers.ConclusionsSalivary tyrosine was of particular interest and a promising finding for the screening of OLP and its progression. Further longitudinal studies are required to establish it as a reliable salivary biomarker in OLP.Clinical relevanceThe salivary metabolic profiling can describe the pathologic characteristics of OLP on non-invasive saliva samples and NMR analysis. Salivary metabolites provide details to considered early detectors and to impact oral health of OLP patients.

Significance of SDC2 and NDRG4 methylation in stool for colorectal cancer diagnosis

BackgroundRecent studies have identified methylated SDC2 and NDRG4 in colorectal cancer (CRC), however, the diagnostic value of the combined two genes remains undefined. This study aims to investigate the methylation of SDC2 and NDRG4 in stool samples and their application in diagnosis of CRC. MethodsFive groups were enrolled in our study which consisted of CRC (n = 138), advanced adenomas (n = 27), polyp (n = 35), intestinal disease control (n = 150), and healthy individuals (n = 28). Methylation status of SDC2 and NDRG4 in fecal samples were tested with appropriate commercial kits. Primary data were collected and statistical analyses were performed. ResultsThe positive rates of both SDC2 and NDRG4 methylation in stool samples of CRC group were significantly higher (P < 0.001) than those of either group of advanced adenomas, or polyp, or intestinal disease or the healthy control. It was suggested that both methylated SDC2,NDRG4, SDC2/NDRG4 and age were independent risk factors for CRC. The sensitivity of SDC2 and NDRG4 for CRC diagnosis were 73.9 % and 63.0 %, respectively, while SDC2 combined with NDRG4 had a higher sensitivity of 85.5 %. The specificity of SDC2, NDRG4 and SDC2 combined with NDRG4 achieved 91.6 %, 88.3 % and 84.6 %, respectively. The AUC for methylated SDC2 and NDRG4 were 0.828 (95 % CI: 0.780–0.876) and 0.757 (95 % CI: 0.703–0.811), respectively. In contrast, SDC2 combined with NDRG4 improved the AUC to 0.850 (95 % CI: 0.807–0.893). ConclusionsThis research confirmed the significance of detection of SDC2 and NDRG4 methylation by using noninvasive samples of stool. More importantly, attributing to their high level and frequency of methylation in stool, SDC2 and NDRG4 could be promising biomarkers for stool-based method for screening and early diagnosis of CRC, especially when combined.

A Review of the Occurrence of Metals and Xenobiotics in European Hedgehogs (Erinaceus europaeus).

Monitoring data from several European countries indicate that European hedgehog (Erinaceus europaeus) populations are declining, and research exploring the causes of the decline, including exposure to potentially harmful xenobiotics and metals, may inform conservation initiatives to protect this species in the wild. Hedgehogs are ground-dwelling mammals, feeding on a range of insects, slugs, snails, and earthworms, as well as eggs, live vertebrates, and carrion, including carcasses of apex predator species representing higher levels of the food chain. Consequently, hedgehogs come into close contact with contaminants present in their habitats and prey. This review investigated the studies available on the subject of the occurrence of metals and organic xenobiotics in hedgehogs. This study found that a vast range of different pesticides; persistent organic pollutants (POPs), including organochlorine compounds and brominated flame retardants (BFRs); as well as toxic heavy metals could be detected. Some compounds occurred in lethal concentrations, and some were associated with a potential adverse effect on hedgehog health and survival. Due to their ecology, combined with the opportunity to apply non-invasive sampling techniques using spines as sampling material, we suggest that the European hedgehog is a relevant bioindicator species for monitoring the exposure of terrestrial wildlife to potential toxicants in urban and rural environments.

Bio-based ionic liquid filter with enhanced electrostatic attraction for outside filtration and inside collection of viral aerosols

Hazardous biological pathogens in the air pose a significant public environmental health concern as infected individuals emit virus-laden aerosols (VLAs) during routine respiratory activities. Mask-wearing is a key preventive measure, but conventional filtration methods face challenges, particularly in high humidity conditions, where electrostatic charge decline increases the risk of infection. This study introduces a bio-based air filter comprising glycine ionic liquids (GILs) and malleable polymer composite (GILP) with high polarity and functional group density, which are wrapped around a melamine-formaldehyde (MF) resin skeleton, forming a conductive, porous GIL functionized ionic network air filter (GILP@MF). When subjected to low voltage, the GILP@MF composite efficiently captures VLAs including nanoscale virus particles through the enhanced electrostatic attraction, especially in facing high humidity bioaerosols exhaled by human body. The filtration/collection efficiency and quality factor can reach 98.3% and 0.264 Pa−1 at 0.1 m s−1, respectively. This innovative filter provides effective VLA protection and offers potential for non-invasive respiratory virus sampling, advancing medical diagnosis efforts.

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples

BackgroundCurrently, many micromammals are important targets for study. The endangered Galemys pyrenaicus is an outstanding example. Globally, their populations have suffered a substantial decline in last 20 years. In the surveyed area, the capture of desman is legally forbidden due to the high conservation concerns. Reason by non-invasive sampling through faeces is proposed for its monitoring. Furthermore, the confusion between faeces from desman and Mediterranean water shrews must be considered. Thus, the aim of this study was focused on developing RT-PCR assays to determine the presence of Galemys pyrenaicus and N. a. anomalus from non-invasive samples.Methods and resultsThe study was conducted in the mountains of the System Central of Extremadura (Spain). A total of 186 samples were collected from 2018 to 2021 by experts where historically reported and/or our previous studies confirmed their presence. RT-PCR assays using hydrolysis probes were designed to detect genetic material from both desman and Mediterranean water shrews and its specificity was confirmed. The reliability of the method was further assessed by PCR sequencing of mitochondrial Cyb and d-loop, resulting fully compatible with the RT-PCR approach. Intraspecific phylogenetic relationship was reported to improve knowledge about mtDNA variability in the desman from the Central System.ConclusionsWe demonstrated that RT-PCR gives a gold opportunity to further map the species using faeces which minimizes disturbance and reports both population status and individual presence. Cost-effective RT-PCR combined with field-collected faeces allows us to better investigate the full range of occurrence of the species.

Microfluidic-Based Non-Invasive Wearable Biosensors for Real-Time Monitoring of Sweat Biomarkers.

Wearable biosensors are attracting great interest thanks to their high potential for providing clinical-diagnostic information in real time, exploiting non-invasive sampling of biofluids. In this context, sweat has been demonstrated to contain physiologically relevant biomarkers, even if it has not been exhaustively exploited till now. This biofluid has started to gain attention thanks to the applications offered by wearable biosensors, as it is easily collectable and can be used for continuous monitoring of some parameters. Several studies have reported electrochemical and optical biosensing strategies integrated with flexible, biocompatible, and innovative materials as platforms for biospecific recognition reactions. Furthermore, sampling systems as well as the transport of fluids by microfluidics have been implemented into portable and compact biosensors to improve the wearability of the overall analytical device. In this review, we report and discuss recent pioneering works about the development of sweat sensing technologies, focusing on opportunities and open issues that can be decisive for their applications in routine-personalized healthcare practices.

Enabling Lipidomic Biomarker Studies for Protected Populations by Combining Noninvasive Fingerprint Sampling with MS Analysis and Machine Learning.

Triacylglycerols and wax esters are two lipid classes that have been linked to diseases, including autism, Alzheimer's disease, dementia, cardiovascular disease, dry eye disease, and diabetes, and thus are molecules worthy of biomarker exploration studies. Since triacylglycerols and wax esters make up the majority of skin-surface lipid secretions, a viable sampling method for these potential biomarkers would be that of groomed latent fingerprints. Currently, however, blood-based sampling protocols predominate in the field. The invasiveness of a blood draw limits its utility to protected populations, including children and the elderly. Herein we describe a noninvasive means for sample collection (from fingerprints) paired with fast MS data-acquisition (MassIVE data set MSV000092742) and efficient data analysis via machine learning. Using both supervised and unsupervised classification, we demonstrate the usefulness of this method in determining whether a variable of interest imparts measurable change within the lipidomic data set. As a proof-of-concept, we show that the method is capable of distinguishing between the fingerprints of different individuals as well as between anatomical sebum collection regions. This noninvasive, high-throughput approach enables future lipidomic biomarker researchers to more easily include underrepresented, protected populations, such as children and the elderly, thus moving the field closer to definitive disease diagnoses that apply to all.

Comparison between ammonium formate and ammonium fluoride in the analysis of stratum corneum lipids by reversed phase chromatography coupled with high resolution mass spectrometry

Lipids are key constituents of the barrier function in the human stratum corneum (SC), which is the outermost layer of the epidermis and amenable to non-invasive sampling by tape stripping. The three major lipid classes in the SC, i.e., ceramides, fatty acids, and cholesterol, present equimolar concentration. Liquid chromatography coupled with mass spectrometry (LCMS) is elective in profiling lipids in the SC in both positive and negative ion modes. Nevertheless, the latter one allows for the simultaneous detection of the three major epidermal components of the SC. Determination of ceramides in the SC poses analytical challenges due to their wide range of structures and concentrations especially in the case of limited sample amounts. Ammonium formate is a commonly used modifier added to the mobile phase to assist ionization. However, it introduces uncertainty in the identification of ceramides when operating in negative ion mode, even with high resolution MS. We tested the advantages of using fluoride in the lipid profiling of SC and unambiguous identification of ceramides subclasses. The use of fluoride enhanced the ionization of ceramides, regardless the specific substructure, solved misidentification issues, and was successfully applied to the simultaneous detection of all three lipid classes in the human SC.

Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques.

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Traitement guidé des infections à Helicobacter pylori : évolutions des pratiques pour un diagnostic moderne et remboursé ?

AbstractDespite unanimous recommendations from international and national scientific societies and the HAS in 2017 to favor treatment of Helicobacter pylori infections guided by a sensitivity test carried out from gastric biopsies, almost all of the treated patients still receive empirical treatment poorly tolerated and source of the continuous development over forty years of the resistance of this bacteria to antibiotics. It is time for General Practitioners, Gastroenterologists and Biologists to coordinate and mobilize to promote the guided treatment of the most common chronic bacterial infection and thus enter into a process of proper use of antibiotics. The recent reimbursement of PCR detecting H. pylori and the mutations responsible for resistance to clarithromycin should remove one of the obstacles to this guided treatment strategy. We propose a new management algorithm for these infections taking into account the reimbursements recently obtained. Recent technological advances already make it possible to imagine a future integrating sensitivity tests from a non-invasive sample: stools, saving numerous unnecessary fiberoscopies at a time when recourse to a gastroenterologist is difficult. For this near future we propose a new algorithm of what will be the coordinated work of general practitioners, gastroenterologists and biologists.

The frequency of NRAS mutation in stool samples of Iranian colorectal cancers compared to Finnish patients

Abstract Background: Stools from colorectal cancer patients are noninvasive samples that could be used to compare the frequency of hotspot mutations between two different ethnic cohorts. Materials and Methods: We collected stool samples from the Iranian cohort (52 patients and 49 controls) and the Finnish cohort (40 patients and 14 controls). Following stool DNA extraction, we used the AmpliSeq Colon and Lung Cancer panel to prepare DNA libraries before sequencing. Results: The Iranian cohort exhibited 35 hotspot mutations in the BRAF, ERBB4, FBXW7, FGFR1, FGFR3, KRAS, MAP2K, MET, NRAS, PIK3C, SMAD4, and TP53 genes. In the Finnish cohort, 13 hotspot mutations were found in the AKT1, APC, KIT, KRAS, SMO, STK11, and TP53 genes. Mutations in NRAS and FGFR3 were observed only in the Iranian cohort, while APC mutations were exclusive for the Finnish cohort. Conclusion: Genes involved in MAPK and PI3K-MAPK pathways showed a higher frequency of mutations in Iranian patients which may have therapeutic implications.

Individualized lipid profile in urine-derived extracellular vesicles from clinical patients with Mycobacterium tuberculosis infections.

Lipids are a key nutrient source for the growth and reproduction of Mycobacterium tuberculosis (Mtb). Urine-derived extracellular vesicles (EVs), because of its non-invasive sampling, lipid enrichment, and specific sorting character, have been recognized as a promising research target for biomarker discovery and pathogenesis elucidation in tuberculosis (TB). We aim to profile lipidome of Mtb-infected individuals, offer novel lipid signatures for the development of urine-based TB testing, and provide new insights into the lipid metabolism after Mtb infection. Urine-derived extracellular vesicles from 41 participants (including healthy, pulmonary tuberculosis, latent tuberculosis patients, and other lung disease groups) were isolated and individually detected using targeted lipidomics and proteomics technology platforms. Biomarkers were screened by multivariate and univariate statistical analysis and evaluated by SPSS software. Correlation analyses were performed on lipids and proteins using the R Hmisc package. Overall, we identified 226 lipids belonging to 14 classes. Of these, 7 potential lipid biomarkers for TB and 6 for latent TB infection (LTBI) were identified, all of which were classified into diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acid (FFA), and cholesteryl ester (CE). Among them, FFA (20:1) was the most promising biomarker target in diagnosing TB/LTBI from other compared groups and also have great diagnostic performance in distinguishing TB from LTBI with AUC of 0.952. In addition, enhanced lipolysis happened as early as individuals got latent Mtb infection, and ratio of raft lipids was gradually elevated along TB progression. This study demonstrated individualized lipid profile of urinary EVs in patients with Mtb infection, revealed novel potential lipid biomarkers for TB/LTBI diagnosis, and explored mechanisms by which EV lipid raft-dependent bio-processes might affect pathogenesis. It lays a solid foundation for the subsequent diagnosis and therapeutic intervention of TB.

Application of noninvasive sampling technique in mitochondrial genome intraspecific phylogeny of the endangered butterfly, Teinopalpus aureus (Lepidoptera: Papilionidae).

The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.

Addressing Misidentification in Noninvasive DNA Sampling Using Bayesian Approach and Simulations

Addressing Misidentification in Noninvasive DNA Sampling Using Bayesian Approach and Simulations

Development of enzyme-inorganic hybrid nanoflower-modified electrodes and a smartphone-controlled electrochemical analyzer for point-of-care testing of salivary amylase in saliva.

Quantitation of salivary alpha-amylase (sAA) plays a significant role in not only theoretical studies but also clinical practice. This study reports a quantitative point-of-care testing (POCT) system for sAA quantitation anywhere, anytime and by anyone, which consists of customized electrodes and a smartphone-controlled electrochemical analyzer. Organic-inorganic hybrid nanoflowers (NFs) encapsulating α-glucosidase (AG) and glucose dehydrogenase (GDH) have been synthesized and modified onto screen-printed electrodes (SPCEs) to fabricate the customized electrodes. The SPCEs integrated with the smartphone-controlled electrochemical analyzer exhibit good analytical performance for sAA with a low detection limit of 5.02 U mL-1 and a wide dynamic range of 100-2000 U mL-1 using chronoamperometry. The reported POCT system has been successfully demonstrated for quantitation of sAA in clinical saliva samples, and the quantitation results correlated well with those of the Bernfeld method which is extensively used in clinics. More importantly, this study reveals the great potential of sAA as an early warning indicator of abnormal glucose metabolism in obese individuals. Considering the non-invasive saliva sampling process as well as the easy-to-use and cost-effectiveness features of this quantitative POCT system, quantitation of salivary sAA at home by laypersons might become an appealing choice for obese individuals to monitor their glucose metabolism status anytime.

Discerning Endoscopic Severity of Inflammatory Bowel Disease by Scoping the Peripheral Blood Transcriptome

Background and AimsUlcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory bowel diseases (IBD) with an incompletely understood etiology and pathogenesis. Identification of suitable drug targets and assessment of disease severity are crucial for optimal management. MethodsUsing RNA-sequencing, we investigated differential gene expression in peripheral blood samples from IBD-patients and non-inflamed controls, analysed pathway enrichment, and identified genes whose expression correlated with endoscopic disease severity. ResultsNeutrophil degranulation emerged as the most significant pathway across all IBD sample types. Signalling by interleukins was prominent in patients with active intestinal inflammation, but also enriched in CD and UC patients without intestinal inflammation. Nevertheless, genes correlated to endoscopic disease severity implicated the primary cilium in CD patients, and translation and focal adhesion in UC patients. Moreover, several of these genes were located in genome-wide associated loci linked to IBD, cholesterol levels, blood cell counts, and levels of markers assessing liver and kidney function. These genes also suggested connections to intestinal epithelial barrier dysfunction, contemporary IBD drug treatment, and new actionable drug targets. A large number of genes associated with endoscopic disease severity corresponded to non-coding RNAs. ConclusionsThis study revealed biological pathways associated with IBD disease state and endoscopic disease severity, thus providing insights into the underlying mechanisms of IBD pathogenesis as well as identifying potential biomarkers and therapies. Peripheral blood might constitute a suitable non-invasive diagnostic sample type, in which gene expression profiles might serve as indicators of ongoing mucosal inflammation, and thus guide personalized treatment decisions.

Metal in biological samples from electronic cigarette users and those exposed to their second-hand aerosol: a narrative review.

Electronic nicotine delivery systems (ENDS) are gradually becoming more popular, particularly, among today's youth. Despite being marketed as safe by the tobacco industry, the notable absence of regulation in their composition is evident. Both the generated fluids and aerosol exhibit a wide variety of substances that are not yet fully identified. In addition to additives, the aerosol contains metals, the presence of which can be attributed to the excessive heating of metallic filaments used in vaporizing the liquid. This review aimed to identify and describe studies that have assessed metal levels in biological samples obtained from electronic cigarette users and those exposed to their second-hand aerosol. This involved detailing the types and concentrations of metals identified and the biological samples in which the metals were detected. Two independent researchers conducted searches in the MEDLINE and EMBASE databases to identify studies that measured the metal levels in human non-invasive biological samples from electronic cigarette users and second-hand exposure. Data were presented as a narrative review. In total, 18 articles were included in this review. Overall active and passive exposure to ENDS was related to higher levels of many metals, including lead and cadmium, in biological samples. ENDS users, in general, have lower metal concentrations in biological samples compared to the users of combustible cigarettes. The exposure to primary and second-hand e-cigarette aerosol is related to higher metal concentrations in the biological samples. The adverse effects of this exposure on long-term users are yet to be determined.