Non-invasive Sample Collection Research Articles

Fermentation Analytical Technology (FAT): Monitoring industrial E. coli fermentations using absolute quantitative 1H NMR spectroscopy

BackgroundTo perform fast, reproducible, and (absolute) quantitative measurements in an automated manner has become of paramount importance when monitoring industrial processes, including fermentations. Due to its numerous advantages – including its inherent quantitative nature – Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy provides an ideal tool for the time-resolved monitoring of fermentations. However, analytical conditions, including non-automated sample preparation and long relaxation times (T1) of some metabolites, can significantly lengthen the experimental time and make implementation in an industrial set up unfeasible. ResultsWe present a high throughput method based on Standard Operating Procedures (SOPs) and 1H NMR, which lays the foundation for what we call Fermentation Analytical Technology (FAT). Our method was developed for the accurate absolute quantification of metabolites produced during Escherichia coli industrial fermentations. The method includes: (1) a stopped flow system for non-invasive sample collection followed by sample quenching, (2) automatic robot-assisted sample preparation, (3) fast 1H NMR measurements, (4) metabolites quantification using multivariate curve resolution (MCR), and (5) metabolites absolute quantitation using a novel correction factor (k) to compensate for the short recycle delay (D1) employed in the 1H NMR measurements. The quantification performance was tested using two sample types: buffer solutions of chemical standards and real fermentation samples. Five metabolites – glucose, acetate, alanine, phenylalanine and betaine – were quantified. Absolute quantitation ranged between 0.64-3.40 mM in pure buffer, and 0.71-7.76 mM in real samples. SignificanceThe proposed method is generic and can be straight forward implemented to other types of fermentations, such as lactic acid, ethanol and acetic acid fermentations. It provides a high throughput automated solution for monitoring fermentation processes and for quality control through absolute quantification of key metabolites in fermentation broth. It can be easily implemented in an at-line industrial setting, facilitating the optimization of the manufacturing process towards higher yields and more efficiently/sustainable use of resources.

New perspectives on ‘Breathomics’: metabolomic profiling of non-volatile organic compounds in exhaled breath using DI-FT-ICR-MS

Breath analysis offers tremendous potential for diagnostic approaches, since it allows for easy and non-invasive sample collection. “Breathomics” as one major research field comprehensively analyses the metabolomic profile of exhaled breath providing insights into various (patho)physiological processes. Recent research, however, primarily focuses on volatile compounds. This is the first study that evaluates the non-volatile organic compounds (nVOCs) in breath following an untargeted metabolomic approach. Herein, we developed an innovative method utilizing a filter-based device for metabolite extraction. Breath samples of 101 healthy volunteers (female n = 50) were analysed using DI-FT-ICR-MS and biostatistically evaluated. The characterisation of the non-volatile core breathome identified more than 1100 metabolites including various amino acids, organic and fatty acids and conjugates thereof, carbohydrates as well as diverse hydrophilic and lipophilic nVOCs. The data shows gender-specific differences in metabolic patterns with 570 significant metabolites. Male and female metabolomic profiles of breath were distinguished by a random forest approach with an out-of-bag error of 0.0099. Additionally, the study examines how oral contraceptives and various lifestyle factors, like alcohol consumption, affect the non-volatile breathome. In conclusion, the successful application of a filter-based device combined with metabolomics-analyses delineate a non-volatile breathprint laying the foundation for discovering clinical biomarkers in exhaled breath.

Detection of 3-4 Methylenedioxyamphetamine from Drug Abuser’s Fingers and Toenails using Liquid Chromatography with Mass Spectroscopy

information regarding drug abuse and pharmaceutical use. In recent years, drug analysis in human nail clippings has proven its significant value in forensic toxicological applications, identification of in utero drug exposure, monitoring of drug treatment programmes, and therapeutic drug monitoring. Nails have various advantages over conventional matrices (blood and urine), which include a longer detection window (months to years), non-invasive sample collection, and easy storage and transportation. These aspects make nails a very significant matrix for forensic toxicology and therapeutic drug monitoring. Because of the low concentrations of drugs of abuse and pharmaceuticals present in nails and the complexity of the keratinized matrix, analytical techniques need to be more sensitive, and sample preparation is crucial. The aim of the present study is to develop a simple, high-performance liquid chromatography-mass spectroscopy (LC-MS) method for the identification and quantitation of 3,4-methylenedioxyamphetamine (MDA) in fingernail and toenail clippings. Finger and toenail clippings were collected from six users undergoing treatment at a rehab center in Ujjain, M.P., India. Nail clippings were initially decontaminated, then hydrolyzed in 1 M NaOH at 370°C, extracted with ethyl acetate, diluted with methanol, and then subjected to LC-MS analysis. The calibration curve was constructed over the 0.5 to 30 ng/mL concentration range using the MDA reference standard. The limit of detection was calculated at 1.10 ng/mL and the limit of quantification was recorded at 3.67 ng/mL in standard solutions, whereas the respective values in spiked nail clippings were 1.21 and 4.6 ng/mg. The developed method has obtained significant results in original nail clippings with mean concentration ranges of 0.12 ng/mg in fingernails and 0.08 ng/mg in toenails in six abuser samples. The new method developed has been found to be capable of detecting the 3,4-methylendioxyamphetamine MDA drug in nail clippings even after 90 days of drug intake.

Urine-derived stem cell therapy for diabetes mellitus and its complications: progress and challenges.

Diabetes mellitus (DM) is a chronic and relentlessly progressive metabolic disease characterized by a relative or absolute deficiency of insulin in the body, leading to increased production of advanced glycosylation end products that further enhance oxidative and nitrosative stresses, often leading to multiple macrovascular (cardiovascular disease) and microvascular (e.g., diabetic nephropathy, diabetic retinopathy, and neuropathy) complications, representing the ninth leading cause of death worldwide. Existing medical treatments do not provide a complete cure for DM; thus, stem cell transplantation therapy has become the focus of research on DM and its complications. Urine-derived stem cells (USCs), which are isolated from fresh urine and have biological properties similar to those of mesenchymal stem cells (MSCs), were demonstrated to exert antiapoptotic, antifibrotic, anti-inflammatory, and proangiogenic effects through direct differentiation or paracrine mechanisms and potentially treat patients with DM. USCs also have the advantages of simple noninvasive sample collection procedures, minimal ethical issues, low cost, and easy cell isolation methods and thus have received more attention in regenerative therapies in recent years. This review outlines the biological properties of USCs and the research progress and current limitations of their role in DM and related complications. In summary, USCs have shown good versatility in treating hyperglycemia-impaired target organs in preclinical models, and many challenges remain in translating USC therapies to the clinic.

Computer-aided automated detection of kidney disease using supervised learning technique

<p>In this paper, we propose an efficient home-based system for monitoring chronic kidney disease (CKD). As non-invasive disease identification approaches are gaining popularity nowadays, the proposed system is designed to detect kidney disease from saliva samples. Salivary diagnosis has advanced its popularity over the last few years due to the non-invasive sample collection technique. The use of salivary components to monitor and detect kidney disease is investigated through an experimental investigation. We measured the amount of urea in the saliva sample to detect CKD. Further, this article explains the use of predictive analysis using machine learning techniques and data analytics in remote healthcare management. The proposed health monitoring system classified the samples with an accuracy of 97.1%. With internet facilities available everywhere, this methodology can offer better healthcare services, with real-time decision support in remote monitoring platform.</p>

B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device

Abstract Background Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample. Methods According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively. Results The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² > 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² > 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges. Conclusion The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.

Mass Spectrometric Analysis of Urinary N-Glycosylation Changes in Patients with Parkinson's Disease.

Urine is thought to provide earlier and more sensitive molecular changes for biomarker discovery than blood. Numerous glycoproteins, peptides, and free glycans are present in urine through glomerular filtration of plasma, cell shedding, apoptosis, proteolytic cleavage, and exosome secretion. Urine biomarkers have enormous diagnostic potential, and the use of these biomarkers is a long-standing practice. The discovery of non-urological disease biomarkers from urine is also gaining attention due to its non-invasive sample collection and ease of analysis. Abnormal protein glycosylation in plasma or cerebrospinal fluid has been associated with Parkinson's disease, however, whether urine with Parkinson's disease has characteristic glycosylation remains to be explored. Here, we use mass spectrometry-based glycomics and glycoproteomics approaches to analyze urine samples for glycans, glycosites, and intact glycopeptides of urine samples. Reduced abundance of N-glycans was detected at the level of total glycans as well as specific glycosites of glycopeptides. The most abundant N-glycan in urine is S(6)1H5N4F1; S(6)2H5N4 and N4H4F1 are highly present in serum and urine, and 10 biantennary galactosylated N-glycans in the urine of PD patients were significantly decreased. The downregulation of sialylation may be due to the reduction of ST3GAL2. Site-specific N-glycosylation analysis revealed that AMBP, UMOD, and RNase1 have PD-specific N-glycosylation sites. GO and KEGG analysis revealed that N-glycosylation changes may provide clues to identify disease-specific glycosylation biomarkers in Parkinson's disease.

A noninvasive method for whole-genome skin methylome profiling.

Ageing, disease and malignant transformation of the skin are associated with changes in DNA methylation. So far, mostly invasive methodologies such as biopsies have been applied in collecting DNA methylation signatures. Tape stripping offers a noninvasive option for skin diagnostics. It enables the easy but robust capture of biologic material in large numbers of participants without the need for specialized medical personnel. To design and validate a methodology for noninvasive skin sample collection using tape stripping for subsequent DNA -methylation analysis. A total of 175 participants were recruited and provided tape-stripping samples from a sun-exposed area; 92 provided matched tape-stripping samples from a sun-protected area, and an additional 5 provided matched skin-shave biopsies from the same area. Using -enzymatic conversion and whole-genome Illumina sequencing, we generated genome-wide DNA methylation profiles that were used to evaluate the feasibility of noninvasive data acquisition, to compare with established sampling approaches and to investigate biomarker identification for age and ultraviolet (UV) exposure. We found that tape-stripping samples showed strong concordance in their global DNA methylation landscapes to those of conventional invasive biopsies. Moreover, we showed sample reproducibility and consistent global methylation profiles in skin tape-stripping samples collected from different areas of the body. Using matched samples from sun-protected and sun-exposed areas of the body we were able to validate the capacity of our method to capture the effects of environmental changes and ageing in a cohort covering various ages, ethnicities and skin types. We found DNA methylation changes on the skin resulting from UV exposure and identified significant age-related hypermethylation of CpG islands, with a pronounced peak effect at 50-55 years of age, including methylation changes in well-described markers of ageing. These data demonstrate the feasibility of using tape stripping combined with whole-genome sequencing as a noninvasive approach to measuring DNA methylation changes in the skin. In addition, they outline a viable experimental framework for the use of skin tape stripping, particularly when it is performed in large cohorts of patients to identify biomarkers of skin ageing, UV damage and, possibly, to track treatment response to therapeutic interventions.

Quantitative TMT-based proteomics revealing host, dietary and microbial proteins in bovine faeces including barley serpin Z4, a prominent component in the head of beer

There has been little information about the proteome of bovine faeces or about the contribution to the faecal proteome of proteins from the host, the feed or the intestinal microbiome. Here, the bovine faecal proteome and the origin of its component proteins was assessed, while also determining the effect of treating barley, the major carbohydrate in the feed, with either ammonia (ATB) or sodium propionate (PTB) preservative. Healthy continental crossbreed steers were allocated to two groups and fed on either of the barley-based diets. Five faecal samples from each group were collected on Day 81 of the trial and analysed by quantitative proteomics using nLC-ESI-MS/MS after tandem mass tag labelling. In total, 281 bovine proteins, 199 barley proteins, 176 bacterial proteins and 190 archaeal proteins were identified in the faeces. Mucosal pentraxin, albumin and digestive enzymes were among bovine proteins identified. Serpin Z4 a protease inhibitor was the most abundant barley protein identified which is also found in barley-based beer, while numerous microbial proteins were identified, many originating bacteria from Clostridium, while Methanobrevibacter was the dominant archaeal genus. Thirty-nine proteins were differentially abundant between groups, the majority being more abundant in the PTB group compared to the ATB group. SignificanceProteomic examination of faeces is becoming a valuable means to assess the health of the gastro-intestinal tract in several species, but knowledge on the proteins present in bovine faeces is limited. This investigation aimed to characterise the proteome of bovine faecal extracts in order to evaluate the potential for investigations of the proteome as a means to assess the health, disease and welfare of cattle in the future. The investigation was able to identify proteins in bovine faeces that had been (i) produced by the individual cattle, (ii) present in the barley-based feed eaten by the cattle or (iii) produced by bacteria and other microbes in the rumen or intestines. Bovine proteins identified included mucosal pentraxin, serum albumin and a variety of digestive enzymes. Barley proteins found in the faeces included serpin Z4, a protease inhibitor that is also found in beer having survived the brewing process. Bacterial and archaeal proteins in the faecal extracts were related to several pathways related to the metabolism of carbohydrates. The recognition of the range of proteins that can be identified in bovine faeces raises the possibility that non-invasive sample collection of this material could provide a novel diagnostic approach to cattle health and welfare.

In-vial dried urine spot collection and processing for quantitative analyses.

Analysis of dried urine spots (DUSs) is becoming an emerging technique in clinical, toxicological, and forensic chemistry due to the fully non-invasive collection, facile transportation, and simple storage of DUS samples. Correct DUS collection and elution is of the utmost importance because inadequate DUS sampling/processing may have direct consequences on quantitative DUS analyses and these aspects were, for the first time, comprehensively investigated in this contribution. Various groups of endogenous and exogenous species were selected as model analytes and their concentrations were monitored in DUSs collected on standard cellulose-based sampling cards. Strong chromatographic effects were observed for most analytes having a crucial impact on their distribution within the DUSs during sampling. Concentrations of target analytes were up to 3.75-fold higher in the central DUS sub-punch in comparison to the liquid urine. Consequently, substantially reduced concentrations of these analytes were determined in peripheral DUS sub-punches demonstrating that sub-punching, often applied to dried material spots, is not acceptable for quantitative DUS analyses. Hence, a simple, rapid, and user-friendly procedure was suggested, which employed an in-vial collection of a known urine volume on a pre-punched sampling disc (using a low-cost micropipette designed for patient-centric clinical sampling) and in-vial processing of the whole DUS. Excellent accuracy (0.20%) and precision (0.89%) of liquid transfers were achieved by the micropipette, which was also applied to remote DUS collection by laic and expert users. The resulting DUS eluates were analysed by capillary electrophoresis (CE) for the determination of endogenous urine species. The CE results demonstrated no significant differences between the two user groups, elution efficiencies of 88-100% (in comparison to the liquid urine), and precision better than 5.5%.

Determination of diurnal rhythm of salivary corticosterone concentration in the domestic rabbit (Oryctolagus cuniculus f. domestica) using a non-invasive method

There are many advantages associated with the determination of the level of corticosterone in rabbits from saliva, since this is a non-invasive sample collection method that does not affect their welfare and provides a reliable reflection of the state of the animal at a given moment without the results being distorted as they may be, for example, when blood samples are taken. The aim of this study was to determine the diurnal rhythm in the concentration of corticosterone in the saliva of the domestic rabbit. Saliva samples were taken from six domestic rabbits five times during the daytime (at 6:00, 9:00, 12:00, 15:00 and 18:00) over the course of three consecutive days. The levels of corticosterone in the saliva of the individual rabbits displayed a diurnal rhythm during the course of the day, with a significant increase between 12:00 and 15:00 (p < 0.05). No statistically significant difference in the concentrations of corticosterone in the saliva of the individual rabbits was demonstrated. Although the basal value of corticosterone is not known in rabbits and is difficult to determine, the results of our study show the pattern of fluctuations in the concentration of corticosterone in the saliva of rabbits during the daytime.

Ultraviolet B-Rays Induced Gene Alterations and DNA Repair Enzymes in Skin Tissue.

Ultraviolet (UV) radiation leads to deoxyribonucleic acid (DNA) damage and changes in gene expression. Topical DNA repair enzymes in liposomes are capable of undoing this damage. To evaluate gene expression changes induced by ultraviolent B-rays (UVB) light and assess the effect of topical DNA repair enzymes extracted from Micrococcus luteus (M. luteus) and photolyase in modifying these changes. Non-invasive, adhesive patch collection kits were used to sample skin on the right and left post-auricular areas before and 24 hours after UVB exposure (n=48). Subjects applied topical DNA repair enzymes to the right post-auricular area daily for 2 weeks. Subjects returned 2 weeks later for repeat non-invasive skin sample collection. Eight of 18 tested genes demonstrated significant changes 24 hours following UVB exposure. DNA repair enzymes from M. luteus or photolyase had no significant effect on genetic expression compared with the control at 2 weeks post UV exposure. UVB exposure causes acute changes in gene expression, which may play roles in photo-aging damage and skin cancer growth and regulation. While non-invasive gene expression testing can detect UV damage, additional genomic studies investigating recovery from UV damage at different time periods are needed to establish the potential of DNA repair enzymes to minimize or reverse this damage. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.7070.

Tear biomarkers in dry eye disease: Progress in the last decade.

Dry eye disease (DED) is a commonly occurring, multifactorial disease characterized by reduced tear film stability and hyperosmolarity at the ocular surface, leading to discomfort and visual compromise. DED is driven by chronic inflammation and its pathogenesis involves multiple ocular surface structures such as the cornea, conjunctiva, lacrimal glands, and meibomian glands. The tear film secretion and its composition are regulated by the ocular surface in orchestration with the environment and bodily cues. Thus, any dysregulation in ocular surface homeostasis causes an increase in tear break-up time (TBUT), osmolarity changes, and reduction in tear film volume, all of which are indicators of DED. Tear film abnormalities are perpetuated by underlying inflammatory signaling and secretion of inflammatory factors, leading to the recruitment of immune cells and clinical pathology. Tear-soluble factors such as cytokines and chemokines are the best surrogate markers of disease severity and can also drive the altered profile of ocular surface cells contributing to the disease. Soluble factors can thus help in disease classification and planning treatment strategies. Our analysis suggests increased levels of cytokines namely interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-9, IL-12, IL-17A, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α); chemokines (CCL2, CCL3, CCL4, CXCL8); MMP-9, FGF, VEGF-A; soluble receptors (sICAM-1, sTNFR1), neurotrophic factors (NGF, substance P, serotonin) and IL1RA and reduced levels of IL-7, IL-17F, CXCL1, CXCL10, EGF and lactoferrin in DED. Due to the non-invasive sample collection and ease of quantitively measuring soluble factors, tears are one of the best-studied biological samples to molecularly stratify DED patients and monitor their response to therapy. In this review, we evaluate and summarize the soluble factors profiles in DED patients from the studies conducted over the past decade and across various patient groups and etiologies. The use of biomarker testing in clinical settings will aid in the advancement of personalized medicine and represents the next step in managing DED.

Usefulness of Oral Fluid for Measurement of Methylone and Its Metabolites: Correlation with Plasma Drug Concentrations and the Effect of Oral Fluid pH.

The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and correlated with the oral fluid pH using data from our previous study in plasma. Methylone was detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest dose. Oral fluid concentrations of methylone ranged between 88.3-503.8, 85.5-5002.3, 182.8-13,201.8 and 214.6-22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive sample collection.

The Microbial Genetic Diversity and Succession Associated with Processing Waters at Different Broiler Processing Stages in an Abattoir in Australia

The high organic content of abattoir-associated process water provides an alternative for low-cost and non-invasive sample collection. This study investigated the association of microbial diversity from an abattoir processing environment with that of chicken meat. Water samples from scalders, defeathering, evisceration, carcass-washer, chillers, and post-chill carcass rinsate were collected from a large-scale abattoir in Australia. DNA was extracted using the Wizard® Genomic DNA Purification Kit, and the 16S rRNA v3-v4 gene region was sequenced using Illumina MiSeq. The results revealed that the Firmicutes decreased from scalding to evisceration (72.55%) and increased with chilling (23.47%), with the Proteobacteria and Bacteroidota changing inversely. A diverse bacterial community with 24 phyla and 392 genera was recovered from the post-chill chicken, with Anoxybacillus (71.84%), Megamonas (4.18%), Gallibacterium (2.14%), Unclassified Lachnospiraceae (1.87%), and Lactobacillus (1.80%) being the abundant genera. The alpha diversity increased from scalding to chilling, while the beta diversity revealed a significant separation of clusters at different processing points (p = 0.01). The alpha- and beta-diversity revealed significant contamination during the defeathering, with a redistribution of the bacteria during the chilling. This study concluded that the genetic diversity during the defeathering is strongly associated with the extent of the post-chill contamination, and may be used to indicate the microbial quality of the chicken meat.

A Novel Approach on the Use of Samples from Faecal Occult Blood Screening Kits for Metabolomics Analysis: Application in Colorectal Cancer Population.

The incidence of colorectal cancer (CRC) is increasing, and currently it is the third most common cancer. Early CRC diagnosis is still difficult and relies on an invasive colonoscopy and tissue biopsy. The globally observed tendency demands non-invasive, specific, and accurate diagnostic tools for early diagnosis and prognosis. In this work, the main aim was to evaluate for the first time the feasibility of using extracts from the non-invasive sample collection from faecal occult blood (FOB) kits for its use in metabolomics studies taking advantage in this way of the high sensitivity of this technology. Then, a cohort of 131 samples from control individuals (CTL), adenoma (AD) and CRC patients were analysed using a semitargeted approach by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry (UHPLC-ToF-MS). Multivariate and univariate statistical analysis revealed that cholesteryl esters (ChoE) with polyunsaturated fatty acids (PUFAs) together with FOB were relevant metabolites that could clearly separate CRC patients from AD and CTL individuals, whereas the metabolic profiles of CTL and AD were very similar. These results are in agreement with previous findings and reveal the advantage of using the same FOBT samples for several analyses, which would facilitate sample collection and improve direct connection between FOB measurements and metabolomics analysis. Although the sample size and the number of metabolites should be enhanced to cover a wider range of metabolites, alterations in lipid metabolism clearly point out for future perspectives.

Novel Method for the Extraction of DNA from Lamniformes Tooth and Denticle Enamel Suitable for PCR

Extraction of high-quality genomic DNA from Lamniformes tooth fragment and dermal denticle enamel is discussed as a method of identifying individual sharks. We describe a procedure that permits isolation of genomic DNA of satisfactory size and quality for PCR analysis, as well as for most routine cloning applications. This method should allow for the non-invasive collection of genomic samples from Lamniformes.

Assessment of Urinary Biomarkers for Infectious Diseases Using Lateral Flow Assays: A Comprehensive Overview.

Screening of biomarkers is a powerful approach for providing a holistic view of the disease spectrum and facilitating the diagnosis and prognosis of the state of infectious diseases. Unaffected by the homeostasis mechanism in the human body, urine accommodates systemic changes and reflects the pathophysiological condition of an individual. Easy availability in large volumes and non-invasive sample collection have rendered urine an ideal source of biomarkers for various diseases. Infectious diseases may be communicable, and therefore early diagnosis and treatment are of immense importance. Current diagnostic approaches preclude the timely identification of clinical conditions and also lack portability. Point-of-care (POC) testing solutions have gained attention as alternative diagnostic measures due to their ability to provide rapid and on-site results. Lateral flow assays (LFAs) are the mainstay in POC device development and have attracted interest owing to their potential to provide instantaneous results in resource-limited settings. The discovery and optimization of a definitive biomarker can render POC testing an excellent platform, thus impacting unwarranted antibiotic administration and preventing the spread of infectious diseases. This Review summarizes the importance of urine as an emerging biological fluid in infectious disease research and diagnosis in clinical settings. We review the academic research related to LFAs. Further, we also describe commercial POC devices based on the identification of urinary biomarkers as diagnostic targets for infectious diseases. We also discuss the future use of LFAs in developing more effective POC tests for urinary biomarkers of various infections.

Direct Nasal Swab for Rapid Test and Saliva as an Alternative Biological Sample for RT-PCR in COVID-19 Diagnosis.

Accurate and early diagnoses are prerequisites for prompt treatment. For coronavirus disease 2019 (COVID-19), it is even more crucial. Currently, choice of methods include rapid diagnostic tests and reverse transcription polymerase chain reaction (RT-PCR) using samples mostly of respiratory origin and sometimes saliva. We evaluated two rapid diagnostic tests with three specimen types using viral transport medium (VTM) containing naso-oropharyngeal (NOP) swabs, direct nasal and direct nasopharyngeal (NP) samples from 428 prospective patients. We also performed RT-PCR for 428 NOP VTM and 316 saliva samples to compare results. The sensitivity of the SD Biosensor Standard Q COVID-19 antigen (Ag) test kit drastically raised from an average of 65.55% (NOP VTM) to 85.25% (direct nasal samples), while RT-PCR was the gold standard. For the CareStart kit, the sensitivity was almost similar for direct NP swabs; the average was 84.57%. The specificities were ≥95% for both SD Biosensor Standard Q and CareStart COVID-19 Ag tests in all platforms. The kits were also able to detect patients with different variants as well. Alternatively, RT-PCR results from saliva and NOP VTM samples showed high sensitivities of 96.45% and 95.48% with respect to each other as standard. The overall results demonstrated high performance of the rapid tests, indicating the suitability for regular surveillance at clinical facilities when using direct nasal or direct NP samples rather than NOP VTM. Additionally, the analysis also signifies not showed that RT-PCR of saliva can be used as an choice of method to RT-PCR of NOP VTM, providing an easier, non-invasive sample collection method. IMPORTANCE There are several methods for the diagnosis of coronavirus disease 2019 (COVID-19), and the choice of methods depends mostly on the resources and level of sensitivity required by the user and health care providers. Still, reverse transcription polymerase chain reaction (RT-PCR) has been chosen as the best method using direct naso-oropharyngeal swabs. There are also other methods of fast detection, such as rapid diagnostic tests (RDTs), which offer result within 15 to 20 min and have become quite popular for self-testing and in the clinical setting. The major drawback of the currently used RT-PCR method is compliance, as it may cause irritation, and patients often refuse to test in such a way. RDTs, although inexpensive, suffer from low sensitivity due to technical issues. In this article, we propose saliva as a noninvasive source for RT-PCR samples and evaluate various specimen types at different times after infection for the best possible output from COVID-19 rapid tests.

Clinical and Radiologic Factors Associated With Detection of Mycobacterium tuberculosis in Children Under 5 Years old Using Invasive and Noninvasive Sample Collection Techniques-Kenya.

Pediatric tuberculosis (TB) remains a critical public health concern, yet bacteriologic confirmation of TB in children is challenging. Clinical, demographic, and radiological factors associated with a positive Mycobacterium tuberculosis specimen in young children (≤5 years) are poorly understood. We conducted a prospective cohort study of young children with presumptive TB and examined clinical, demographic, and radiologic factors associated with invasive and noninvasive specimen collection techniques (gastric aspirate, induced sputum, nasopharyngeal aspirate, stool, and string test); up to 2 samples were taken per child, per technique. We estimated associations between these factors and a positive specimen for each technique using generalized estimating equations (GEEs) and logistic regression. A median (range) of 544 (507-566) samples were obtained for each specimen collection technique from 300 enrolled children; bacteriologic yield was low across all collection techniques (range, 1%-7% from Xpert MTB/RIF or culture), except for lymph node fine needle aspiration (29%) taken for children with cervical lymphadenopathy. Factors associated with positive M. tuberculosis samples across all techniques included prolonged lethargy (median [range] adjusted odds ratio [aOR], 8.1 [3.9-10.1]), history of exposure with a TB case (median [range] aOR, 6.1 [2.9-9.0]), immunologic evidence of M. tuberculosis infection (median [range] aOR, 4.6 [3.7-9.2]), large airway compression (median [range] aOR, 6.7 [4.7-9.5]), and hilar/mediastinal density (median [range] aOR, 2.9 [1.7-3.2]). Identifying factors that lead to a positive M. tuberculosis specimen in very young children can inform clinical management and increase the efficiency of diagnostic testing in children being assessed for TB.