Non-invasive Sampling Techniques Research Articles

Salivary molecules as indicators of hydration status

Introduction: Maintaining an appropriate level of hydration is essential to health, especially when doing physical exercise. Thus, the importance of monitoring the before, during and after exercise status. Therefore, knowing and studying the changes and indicators that may provide reliable information on fluctuations in a simple, quick manner, via non-invasive sampling techniques becomes essential. Material and Methods: The data analyzed and processed (α-amylase, K+, Cl-, cortisol, total protein, and osmolality concentration in saliva) in this study are available on the free access research platform for complex physiological signals (https://physionet.org/content/qde/1.0.0/). Ten male subjects voluntarily took part in this study. The median and standard deviation of height, body weight, and age were 179 ±7.5 cm. 79.3 ±9.0 kg. and 25.5 ±3.7 years, respectively. Results: The α-amylase, K+, cortisol, total protein, and osmolality concentration in saliva showed significant variations for moderate dehydration levels. For the cases of Cl- concentration changes in saliva, statistically significant changes can be observed at a much earlier stage (in mild dehydration conditions). Conclusions: The results suggest that Cl- concentration in saliva might be used as an indicator for the hydration status. Therefore, a scale to monitor the hydration status is proposed.

Mitochondrial genetic diversity of the Greater Cane rat (Thryonomys swinderianus) populations from the Eastern Arc Mountains ecosystem, Tanzania.

Management of most herbivorous small mammal species considered to be pests in Africa is still challenging partly because of the paucity of information on their biological traits that would help to manage their destructive impacts. This gap also precludes the potential for tapping species with potential food-value to improving the economy of rural communities through, for example, sustainable game farming programs in Africa. This study investigates the genetic diversity and population demography of the African Greater Cane rat (AGC), a rodent pest of crops and game species inhabiting two isolated blocks of the Eastern Arc Mountains (EAMs), Tanzania to contribute to the species management and conservation. We used non-invasive sampling techniques and DNA sequencing of the D-loop region of MtDNA (515bp) from 46 cane rats (Thryonomys swinderianus) samples to characterize the genetic diversity and structure of the species and potential population threats faced in natural habitats. We found 25 haplotypes:15 from Uluguru and 9 from Udzungwa mountains populations, containing 49 polymorphic regions (32 parsimoniously informative and 17 singleton sites). Haplotype diversity (range: 0.849-0.995) did not differ substantially across populations but the median haplotype diversity for Udzungwa South was overall lower than for other populations. Nucleotide diversity averaged 0.00641, 0.01528, 0.0111 and 0.01313, respectively for Udzungwa South, Udzungwa North, Uluguru Rural and Uluguru Urban, suggesting high genetic diversity within the four populations. Analysis of molecular variance (AMOVA) indicated significantly high genetic differences between the four populations (FST = 0.16, p = 0.00098) whereas neutrality test (FU's Fs) values were negative, indicating historical population expansion. Similarly, the Bayesian skyline analysis indicated a recent demographic expansion suggesting limited bottlenecks in the recent past in this population. Our results show the AGC population in EAMs consists of four distinct populations which have experienced a recent population expansion, especially among the urban population due perhaps to influence of urbanization process that may have favored assisted species movements across the rural-urban landscapes. Future research should focus on understanding impact of geographical isolation on the genetic structure and diversity of this species.

An Extensive Study of Phenol Red Thread as a Novel Non-Invasive Tear Sampling Technique for Proteomics Studies: Comparison with Two Commonly Used Methods.

Tear samples are considered in recent publications as easily, noninvasively collectible information sources for precision medicine. Their complex composition may aid the identification of biomarkers and the monitoring of the effectiveness of treatments for the eye and systemic diseases. Sample collection and processing are key steps in any analytical method, especially if subtle personal differences need to be detected. In this work, we evaluate the usability of a novel sample collection technique for human tear samples using phenol red threads (cotton thread treated with the pH indicator phenol red), which are efficiently used to measure tear volume in clinical diagnosis. The low invasiveness and low discomfort to the patients have already been demonstrated, but their applicability for proteomic sample collection has not yet been compared to other methods. We have shown, using various statistical approaches, the qualitative and quantitative differences in proteomic samples collected with this novel and two traditional methods using either glass capillaries or Schirmer’s paper strips. In all parameters studied, the phenol red threads proved to be equally or even more suitable than traditional methods. Based on detectability using different sampling methods, we have classified proteins in tear samples.

An extensive study of phenol red thread as a novel non-invasive tear sampling technique for proteomics studies: comparison with two commonly used methods

An extensive study of phenol red thread as a novel non-invasive tear sampling technique for proteomics studies: comparison with two commonly used methods

Profiling the Atopic Dermatitis Epidermal Transcriptome by Tape Stripping and BRB-seq.

Tape stripping is a non-invasive skin sampling technique, which has recently gained use for the study of the transcriptome of atopic dermatitis (AD), a common inflammatory skin disorder characterized by a defective epidermal barrier and perturbated immune response. Here, we performed BRB-seq—a low cost, multiplex-based, transcriptomic profiling technique—on tape-stripped skin from 30 AD patients and 30 healthy controls to evaluate the methods’ ability to assess the epidermal AD transcriptome. An AD signature consisting of 91 differentially expressed genes, specific for skin barrier and inflammatory response, was identified. The gene expression in the outermost layers, stratum corneum and stratum granulosum, of the skin showed highest correlation between tape-stripped skin and matched full-thickness punch biopsies. However, we observed that low and highly variable transcript counts, probably due to low RNA yield and RNA degradation in the tape-stripped skin samples, were a limiting factor for epidermal transcriptome profiling as compared to punch biopsies. We conclude that deep BRB-seq of tape-stripped skin is needed to counteract large between-sample RNA yield variation and highly zero-inflated data in order to apply this protocol for population-wide screening of the epidermal transcriptome in inflammatory skin diseases.

Point of care diagnostics and non-invasive sampling strategy: a review on major advances in veterinary diagnostics

The use of point of care diagnostics (POCD) in animal diseases has steadily increased over the years since its introduction. Its potential application to diagnose infectious diseases in remote and resource limited settings have made it an ideal diagnostic in animal disease diagnosis and surveillance. The rapid increase in incidence of emerging infectious diseases requires urgent attention where POCD could be indispensable tools for immediate detection and early warning of a potential pathogen. The advantages of being rapid, easily affordable and the ability to diagnose an infectious disease on spot has driven an intense effort to refine and build on the existing technologies to generate advanced POCD with incremental improvements in analytical performance to diagnose a broad spectrum of animal diseases. The rural communities in developing countries are invariably affected by the burden of infectious animal diseases due to limited access to diagnostics and animal health personnel. Besides, the alarming trend of emerging and transboundary diseases with pathogen spill-overs at livestock-wildlife interfaces has been identified as a threat to the domestic population and wildlife conservation. Under such circumstances, POCD coupled with non-invasive sampling techniques could be successfully deployed at field level without the use of sophisticated laboratory infrastructures. This review illustrates the current and prospective POCD for existing and emerging animal diseases, the status of non-invasive sampling strategies for animal diseases, and the tremendous potential of POCD to uplift the status of global animal health care.

Determining the Dietary Preferences of Wild Asian Elephants (Elephas maximus) in Taman Negara National Park, Malaysia Based on Sex and Age using trnL DNA Metabarcoding Analysis.

The world's largest terrestrial mammal, Asian elephants, are known to have enormous feeding needs. Several factors such as season, sex, age, and daily activities influence the amount of food required by an individual. Generally, captive elephants have a limited choice of food on a daily basis compared with that of elephants in the wild. Elephants in captivity are fed according to a prepared feeding schedule, whereas wild elephants are free to choose the type of plants that they consume in their natural habitat. In the past, ecological observations have been widely used to determine the diet of wild elephants. However, the molecular approach has never been carried out. In the present study, we aimed to; 1) identify the plant diet of wild Asian elephants in Taman Negara National Park (TNNP) according to their sex and age using high-throughput DNA metabarcoding; and 2) determine the dietary formulation of captive elephants based on the generated plant metabarcoding database. DNA was extracted from 24 individual fecal samples collected using noninvasive sampling techniques from TNNP and the National Elephant Conservation Centre (NECC) Kuala Gandah. Seven pooled samples from male adult, female adult, male subadult, female subadult, male juvenile, female juvenile, and captive elephants were amplified and sequenced targeting the trnL region (50-150 base pairs). The CLC Genomic Workbench and PAST 4.02 software were used for data analysis. In total, 24 orders, 41 families, 233 genera, and 306 species of plants were successfully detected in the diet of the Asian elephants. The most abundant plant genera consumed were Sporobolus (21.88%), Musa (21.48%), and Ficus (10.80%). Plant variation was lower in samples from male elephants than in those from female elephants. The plant species identified were correlated with the nutrient benefits required by elephants. Adults and subadults consumed more plant species than were consumed by juvenile elephants. However, there was no significant difference between ages and sexes. The findings of this study can be used as guidance by the Department of Wildlife and National Parks for the management of captive elephants, especially in NECC Kuala Gandah.

Applications of electrochemical biosensors based on 2D materials and their hybrid composites in hematological malignancies diagnosis.

Hematological malignancies encompass a wide variety of severe diseases that pose a serious threat to human health. Given the fact that hematological malignancies are difficult to treat due to their unpredictable and rapid deterioration and high rates of recurrence, growing attention has been paid to their early screening and diagnosis. However, developing a rapid and effective diagnostic tool featuring a noninvasive sampling technique is still extremely challenging. In recent years, novel nanomaterials-based electrochemical biosensors have attracted great interest because of such advantages as simple operation, low cost, fast response, etc. As a kind of rising nanomaterials, two-dimensional materials have excellent electronic and chemical properties, which have been proven to improve the performance of electrochemical biosensors. This review summarizes the applications of different types of electrochemical biosensors (nucleic acid sensors, immunosensors, aptamer biosensors, and cytosensors) based on two-dimensional materials in the detection of biological molecules related to hematological malignancies. Two-dimensional materials-based electrochemical biosensors designed for the diagnosis of leukemia could rapidly detect the target biomolecules at a trace level and show great merits such as wide linear range, low detection limit, high sensitivity, excellent selectivity, and cost-effectiveness. In addition, these biosensors have also achieved satisfactory results in the diagnosis of lymphoma and multiple myeloma. Thus, two-dimensional materials-based electrochemical biosensors are attractive for the early diagnosis of hematological malignancies in clinical practice. Nevertheless, more efforts are still required to further improve the performance of electrochemical biosensors. In this review, we propose the possible main concerns in the design of future two-dimensional materials-based electrochemical biosensors, involving the development of sensors for synchronous detection of diverse target biomolecules, the exploration of other superior two-dimensional materials, the simplification of the sensors fabrication process, the construction of new hybrid structures and how to avoid possible environmental issues.

Environmental DNA approach complements social media reports to detect an endangered freshwater stingray species in the wild

Environmental DNA (eDNA) barcoding has emerged as an important non-invasive sampling technique for the detection of rare and endangered species that can be difficult to sample. Our objective was to develop a low-cost eDNA barcoding approach for the detection of an Endangered freshwater stingray species Fluvitrygon kittipongi in a tropical river system in Peninsular Malaysia. We designed a species-specific primer for a fragment of F. kittipongi cytochrome oxidase subunit I mtDNA (244 bp). The effectiveness of this primer to detect the stingray was evaluated using water samples taken from the upper and middle reaches of the Pahang River coupled with social media reports on sightings of F. kittipongi. Five of 14 water samples tested showed positive PCR amplification for the targeted species. These results represent the first successful application of eDNA to detect freshwater stingrays in Malaysia. Using a combination of freshly obtained carcass samples, social media reporting and target species eDNA detection, this study provides formal occurrence records of F. kittipongi in Malaysia in 3 major watersheds: the Perak, Pahang and Kelantan Rivers.

Comparing noninvasive sampling techniques with standard cannula sampling method for ruminal microbial analysis

Rumen microbes play an important role in the conversion of indigestible plant material to energy and protein in dairy cows. Sampling for ruminal contents via cannula is considered the gold standard technique for microbial analysis, but the technique requires ruminally cannulated animals and specialized animal facilities. The purpose of this study was to determine whether other sampling methods and locations along the digestive tract may serve as noninvasive proxies to the cannula method for microbial analysis. Six ruminally cannulated lactating Holstein dairy cows were adapted to a standard total mixed ration for 2 wk and sampled during the third week. Sampling locations and methods included salivary content, rumination bolus (regurgitated digesta collected from the cow's mouth), feces, and rumen contents via stomach tube and cannula. Stomach tube and cannula samples differ in proportions of solid and liquid material and were therefore separated into whole (as collected), liquid, and solid fractions. Samples were collected at 0 (before feeding), 2, 4, 6, 8, and 12 h after feeding over 2 d. All samples were extracted for total genomic DNA and selected samples for metabolically active DNA (RNA), PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, and analyzed for bacterial diversity using the QIIME2 pipeline followed by statistical analysis in R (https://www.R-project.org/). In DNA-based analysis, at the community level, saliva, rumination bolus, and fecal samples clustered in separate groups, whereas all fractions of stomach tube and cannula samples clustered together, indicating that microbial communities of stomach tube and cannula samples were homogeneous. Rumination bolus samples at 6, 8, and 12 h after feeding clustered with stomach tube and cannula samples, indicating that rumination bolus samples may be an alternative for cannula samples; however, time of sampling is critical for sampling of bolus digesta. Results of the RNA-based analysis of rumination bolus samples and solid samples from cannula and stomach tube at 0 and 6 h after feeding were similar. We concluded that the solid fraction of samples obtained via the stomach tube method may serve as a proxy for the solid fraction of whole ruminal contents obtained via cannula for DNA-based microbial investigations. Both rumination bolus and stomach tube solid samples may serve as proxies for cannula solid samples for RNA-based microbial analysis.

Estimating preharvest density, adult sex ratio, and fecundity of white-tailed deer using noninvasive sampling techniques.

Adult sex ratio and fecundity (juveniles per female) are key population parameters in sustainable wildlife management, but inferring these requires abundance estimates of at least three age/sex classes of the population (male and female adults and juveniles). Prior to harvest, we used an array of 36 wildlife camera traps during 2 and 3 weeks in the early autumn of 2016 and 2017, respectively. We recorded white‐tailed deer adult males, adult females, and fawns from the pictures. Simultaneously, we collected fecal DNA (fDNA) from 92 20 m × 20 m plots placed in 23 clusters of four plots between the camera traps. We identified individuals from fDNA samples with microsatellite markers and estimated the total sex ratio and population density using spatial capture–recapture (SCR). The fDNA‐SCR analysis concluded equal sex ratio in the first year and female bias in the second year, and no difference in space use between sexes (fawns and adults combined). Camera information was analyzed in a spatial capture (SC) framework assuming an informative prior for animals’ space use, either (a) as estimated by fDNA‐SCR (same for all age/sex classes), (b) as assumed from the literature (space use of adult males larger than adult females and fawns), or (c) by inferring adult male space use from individually identified males from the camera pictures. These various SC approaches produced plausible inferences on fecundity, but also inferred total density to be lower than the estimate provided by fDNA‐SCR in one of the study years. SC approaches where adult male and female were allowed to differ in their space use suggested the population had a female‐biased adult sex ratio. In conclusion, SC approaches allowed estimating the preharvest population parameters of interest and provided conservative density estimates.

A New Method for Standardizing Inland Fish Community Surveys: Characterizing Habitat Associated with Small-Bodied Fish Species, Abundance, and Size Distributions in a Highly Modified Estuary

Monitoring fish assemblages in estuary environments is often difficult to standardize due to habitat complexity and gear-specific selectivity. This is further compounded by conventional sampling methods which require fish handling that might cause stress or mortality. To ameliorate these issues, we developed the Single-Platform Aquatic Species and Habitat Sampling System (Platform), an integrated and mobile concentrator net and live box prototype, coupled with a range of physical monitoring equipment. The long-term goal of the Platform is to employ non-invasive sampling techniques such as video and eDNA; however, our aim in this study was to test the Platform’s ability to sample across different habitat types and detect differences in fish assemblages. We investigated the utility of the Platform over a short time period (2 mo) in a relatively small and complex embayment within the highly modified San Francisco Estuary, California. We were able to identify clear physical distinctions among estuary/delta habitat types and detect habitat segregation by ecological groups of sampled fishes with the Platform. The Platform also detected discernable ontogenetic shifts (i.e., size differences) within estuary habitats of commonly observed fish species. These initial results demonstrate the Platform’s ability to contrast fish density, size, and species diversity, which sets the stage to advance more passive monitoring techniques, including video and eDNA methods. The Platform has the potential to fill a methodological gap in non-invasive surveying of small-bodied fish across a range of estuarine habitats, warranting further investigation of potential applications.

Ultra-Fast Electrochemical Sensor for Point-of-Care COVID-19 Diagnosis Using Non-Invasive Saliva Sampling

Point-of-care diagnostic devices that are rapid and reliable remain as an unmet need highlighted by the coronavirus disease (COVID-19) pandemic crisis. The second/third wave of virus spread in various parts of the world combined with new evidence of re-infections and inadequate healthcare facilities demand increased testing rate to diagnose COVID-19 at its core. Although traditional molecular diagnostic tests have served this purpose, there have been shortage of reagents and other supplies at pandemic frontlines. This calls for novel alternate diagnostic processes with potential for obtaining emergency use authorization and that can be deployed in the field at the earliest opportunity. Here, we show an ultra-fast SARS-CoV-2 detection sensor for detecting coronavirus proteins in saliva within 100 milliseconds. Electrochemical oxidation of nickel hydroxide has been controlled using cyclic voltammetry and chronoamperometry techniques for successful detection of SARS-CoV-2. Test results have proven the capability of sensors to quantitatively detect the concentration of virus in blinded analyses. The detection occurs by a process similar to that of SARS-CoV-2 binding onto host cells. The sensor also shows prospects in distinguishing SARS-CoV-2 from other viruses such as HIV. More importantly, the sensor matches the detection limit of the gold standard test for diagnosing early infection. The use of saliva as a non-invasive sampling technique combined with the portability of the instrument has broadened the potential of this sensor.

Biomarkers of asthma.

Biomarkers may be diagnostic of asthma, they may predict or reflect response to therapy or they may identify patients at risk of asthma exacerbation. A biomarker is most often measured in biologic fluids that are sampled using relatively non-invasive sampling techniques such as blood, sputum, urine or exhaled breath. Biomarkers should be stable, readily quantifiable and their measurement should be reproducible and not confounded by other host factors, or the presence of comorbidities. However, asthma comprises multiple molecular endotypes and single, sensitive, specific, biomarkers reflecting these endotypes may not exist. Combining biomarkers may improve their predictive capability in asthma. The most well-established endotypes are those described as type 2 and non-type 2 asthma. Clinical trials established the fraction of exhaled nitric oxide (FeNO) and blood eosinophil counts as key biomarkers of response to corticosteroid or targeted anti-inflammatory therapy in type 2 asthma. However, these biomarkers may have limited value in the management of asthma in real-life settings or routine clinical practice. Biomarkers for type 2 asthma are not well described or validated and more research is needed. Breathomics has provided evidence to propose a number of exhaled volatile organic compounds (VOCs) as surrogate biomarkers for airway inflammatory phenotypes, disease activity and adherence to therapy. Analysis of urinary eicosanoids has identified eicosanoids related to type 2 and non-type 2 inflammation. Future clinical trials will be important in determining how exhaled VOCs or urinary eicosanoid profiles can be used to direct precision treatments. Their future clinical use will also depend on developing simplified instrumentation for biomarker analysis at the point-of-care.

Noninvasive genetic sampling for flying foxes: a valuable method for monitoring demographic parameters

AbstractEstablishing effective wildlife conservation measures requires accurate demographic information such as population size and survival probability: parameters that can be extremely difficult to obtain. This is especially the case for threatened species, which are often rare and sometimes occupy inaccessible areas. While noninvasive genetic sampling (NIGS) techniques are promising tools for providing demographic data, these methods may be unreliable in certain situations. For instance, fecal samples of frugivorous species in tropical areas degrade rapidly, affecting the usability of the genetic material. In this study, we compared (1) NIGS capture–mark–recapture (NIGS‐CMR) with conventional CMR to determine their potential in estimating demographic parameters of fruit bats, and (2) the precision of these demographic parameters and the associated costs given varying sampling designs through simulations. Using Livingstone’s fruit bats (Pteropus livingstonii) fecal samples, microsatellite markers were tested and genotyping success and error rates were assessed. The average genotyping success rate was 77%, and the total genotyping error rate for all loci was low (allelic dropout rate = 0.089, false alleles rate = 0.018). Our results suggested that five loci were required to identify individuals. Simulations showed that monitoring the species over a 9‐yr period with a recapture rate of 0.20 or over a 6‐yr period with a recapture rate of 0.30 seems appropriate to obtain valuable demographic parameters. Overall, in comparison to conventional CMR, NIGS‐CMR offers a better method for estimating demographic parameters and subsequently for conducting long‐term population monitoring in flying foxes due to the fact that (1) sample collection is easy and the level of genotyping errors in the laboratory is low and (2) it is cheaper, less time‐consuming, and less disturbing to individual animals. We strongly advocate an approach that couples a pilot study with simulations as done in this study in order to choose the most efficient monitoring method for a given species or context.

Enhanced Access to the Health-Related Skin Metabolome by Fast, Reproducible and Non-Invasive WET PREP Sampling.

Our skin influences our physical and mental health, and its chemical composition can reflect environmental and disease conditions. Therefore, through sampling the skin metabolome, we can provide a promising window into the mechanisms of the body. However, the broad application of skin metabolomics has recently been hampered by a lack of easy and widely applicable sampling methods. Here, we present a novel rapid, simple, and, most importantly, painless and non-invasive sampling technique suitable for clinical studies of fragile or weakened skin. The method is called WET PREP and is simply a lavage of the skin which focuses on capturing the metabolome. We systematically evaluate WET PREPs in comparison with the non-invasive method of choice in skin metabolomics, swab collection, using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS2) on two complementary chromatographic columns (C18 reversed phase and hydrophilic interaction chromatography). We also integrate targeted analyses of key metabolites of skin relevance. Overall, WET PREP provides a strikingly more stable shared metabolome across sampled individuals, while also being able to capture unique individual metabolites with a high consistency in intra-individual reproducibility. With the exception of (phospho-)lipidomic studies, we recommend WET PREPs as the preferred skin metabolome sampling technique due to the quick preparation time, low cost, and gentleness for the patient.

New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry.

Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.

Girding the loins? Direct evidence of the use of a medieval English parchment birthing girdle from biomolecular analysis.

In this paper, we describe palaeoproteomic evidence obtained from a stained medieval birth girdle using a previously developed dry non-invasive sampling technique. The parchment birth girdle studied (Wellcome Collection Western MS. 632) was made in England in the late fifteenth century and was thought to be used by pregnant women while giving birth. We were able to extract both human and non-human peptides from the manuscript, including evidence for the use of honey, cereals, ovicaprine milk and legumes. In addition, a large number of human peptides were detected on the birth roll, many of which are found in cervico-vaginal fluid. This suggests that the birth roll was actively used during childbirth. This study is, to our knowledge, the first to extract and analyse non-collagenous peptides from a birth girdle using this sampling method and demonstrates the potential of this type of analysis for stained manuscripts, providing direct biomolecular evidence for active use.

Molecular diagnosis of scabies using a novel probe-based polymerase chain reaction assay targeting high-copy number repetitive sequences in the Sarcoptes scabiei genome.

The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.

Incidence of 49 elements in the blood and scute tissues of nesting hawksbill turtles (Eretmochelys imbricata) in Holbox Island

Due to progressive urban development along the Mexican Caribbean coastline, it is crucial to gauge the impact of anthropogenic contamination of marine ecosystems through biomonitoring procedures. In the current study, we quantified the concentration of 49 inorganic elements in the blood and scute tissues of clinically healthy nesting hawksbill sea turtles (Eretmochelys imbricata). The elements were classified into four groups: Group A: essential mineral elements with toxic potential; Group B: non-essential elements with high toxicity; Group C: toxic non-essential minority elements and Trace Elements (TE); and Group D: rare-earth elements (REE) and other TE. Almost all the samples in both tissues showed perceptible levels of all the quantified elements. The only element identified with a correlation between blood and scute was arsenic (As), which could indicate a fast excretion through this type of keratinized tissue. The bio-accumulation of inorganic elements is a complex process, requiring the simultaneous examination of different tissues to evaluate the exposure. Our study reinforces the usefulness of scute tissue as a non-invasive sampling technique for the evaluation of persistent pollutants in marine turtles.