Non-invasive Sample Collection Research Articles

Urinary microbiota; Which non-ınvasive urine collection method should we use?

The aim of this study is to establish the optimal non-invasive urine sample collection method for the microbiota studies. Twelve men with bladder carcinoma underwent first voided and midstream urine collection. Urine samples were analysed using V3-V4 regions of bacterial 16s ribosomal RNAs. Bacterial groups with relative abundance above 1% were analysed in first voided urine and midstream urine samples at phylum, class, order and family level. At the genus level, all of the identified bacterial groups' relative abundances were analysed. The statistical significance (P<.05) of differences between first voided and midstream urine sample microbiota was evaluated using the Wilcoxon test. According to the analysis, 8 phyla, 14 class, 23 orders, 39 families and 29 different genera were identified in the first voided and the midstream urine samples. Statistical differences were not identified between first voided and midstream urine samples of all bacteria groups except the Clostridiales at order level (p:0.04) and Clostridia at class level (P: .04). Either first voided or midstream urine samples can be used in urinary microbiota studies as we determined that there is no statistically significant difference between them regarding the results of 16s ribosomal RNA analysis.

The COVID-19 vaccine in women: Decisions, data and gender gap.

The COVID-19 vaccine in women: Decisions, data and gender gap.

Biosensors Used in Dentistry: A Review

A bio-sensor is composed of six elements which include bio-receptor, transfusion element, chemically activeinterface, signal amplifier, signal processor and display. The analyze binds to the immobilized biologicalmaterial and forms a product. The product linked change is then converted by the transducer into electricsignals which can be amplified and measured. Oral fluid bio-sensors have the advantages of easy accessibilityand noninvasive sample collection, making them a novel method in disease diagnostics. However, itslimitations such as less sensitivity and specificity are overcome by the advent of new technologies such asmicrofluidics and nanofluidics. This review article discusses the concepts and applications of biosensors inthe field of dentistry.

Novel methods of collecting and analyzing lung aerosol samples for functional diagnostics of surfactant-dependent states

The development of effective methods for non-invasive collection of lung aerosol samples and studying the surface activity of the obtained native material is relevant for the diagnosis of inflammatory processes of the lungs. In this work, a method based on the capture of pulmonary fluid droplets contained in the form of an aerosol in the air exhaled by a subject is developed and implemented in hardware – electrostatic aerosol trapping system (ESAT). The exhaled air flow is passed through the corona discharge region, where aerosol droplets receive an electric charge and are then transferred to the water surface by electrostatic forces. The lung surfactant contained in the trapped droplets forms an adsorbed layer, the surface properties of which are then investigated by tensiometric methods. Testing of the method and optimization of the installation design were carried out on a model aerosol of a weak electrolyte solution, which allowed measuring the capture efficiency through conducting conductometric measurements. A modified capillary wave method was used to study the surface properties of the lung surfactant. The method of collecting exhaled particles and studying the surface properties of the lung surfactant was tested on a group of healthy volunteers. A comparative study of the surface properties of native material collected by the electrostatic method from exhaled air and obtained during bronchoalveolar flushing in groups of healthy volunteers and patients with pulmonary tuberculosis was conducted.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs.

The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Pre-capture monitoring for a planned translocation of a northern muriqui (&lt;i&gt;Brachyteles hypoxanthus&lt;/i&gt;) female from a forest fragment surrounding Caparaó National Park, Brazil

&#x0D; &#x0D; &#x0D; Conservation translocation may play an important and necessary role in primate conservation as several species are facing an impending extinction risk and population decline. However, translocations are a high-cost endeavor. Therefore, pre-capturing monitoring can be an essential component in the success of primate translocation. In this study, we report the first confirmed sighting of a female Northern Muriqui (Brachyteles hypoxanthus) stranded alone in a small fragment of Brazilian Atlantic Forest, and her pre-capture monitoring for a future planned translocation. Monitoring was realized from June 2018 to July 2019, and included data collection through direct observational and arboreal camera trap methods, and non-invasive collection of faecal samples for additional genetic studies. Our results showed that the female used the total area of the 1-ha forest fragment with an average daily displacement of 59.6 m ± 76.2. The female spent most of the time ‘Resting’, followed by ‘Travelling’ and ‘Feeding’, which did not vary significantly between seasons. Intraspecific activity pattern variations may be related to the amount and quality of the feeding resources available in the forest fragments. No stereotyped behavior or advertising signals were reported during the monitoring. Our study provides insights on solitary primates living in marginal habitats and highlight the importance of pre-capture monitoring to provide important information on the general behavior of the target individuals, on the regular movements and overall home ranges, and to help to guide future translocations.&#x0D; &#x0D; &#x0D;

15335 Real-world utility of a noninvasive gene expression test to rule out primary cutaneous melanoma: A large US registry study

The Pigmented Lesion Assay (PLA) analyzes gene expression to objectively rule out melanoma. The test uses a noninvasive adhesive patch-based sample collection platform that enables guidance on biopsy decisions and elevates pigmented lesion management beyond what can be visually ascertained. The test’s negative predictive value of 99%, and rapid, painless application make it an attractive pre-biopsy solution. It reduces biopsies by 90% while improving care and reducing cost. This registry study (53 US dermatology offices, 90 providers, median patient age 48 years, 60.80% female patients) assesses real-world utility to determine if the PLA changes clinical practice. The PLA assessed 3418 concerning pigmented skin lesions. Three hundred twenty-four lesions (9.48%) were PLA(+) and 3094 (90.52%) were negative. A PLA test result is positive if LINC, PRAME, or both target genes are detected. These molecular pathology findings are known to correspond with histopathology findings of in situ or invasive primary melanoma in 7%, 50%, and 93%, respectively. The 9.48% PLA(+) cases consisted of 5.15% LINC-only, 1.93% PRAME-only, and 2.40% LINC and PRAME double positive cases. Notably, PLA(+) lesions were almost universally surgically biopsied (97.53%), while PLA(−) cases were nearly always monitored and not biopsied (99.94%). These studies demonstrate that the PLA has true clinical value in community-based practices where providers make important decisions based on the test’s results. Pigmented lesions with PLA(+) test results are subjected to surgical biopsies, whereas PLA(−) lesions are followed clinically and not biopsied.

Kinetic profile of N,N-dimethyltryptamine and β-carbolines in saliva and serum after oral administration of ayahuasca in a religious context.

Ayahuasca is a beverage obtained from Banisteriopsis caapi plus Psychotria viridis. B. caapi contains the β-carbolines harmine, harmaline, and tetrahydroharmine that are monoamine oxidase inhibitors and P. viridis contains N,N-dimethyltryptamine (DMT) that is responsible for the visionary effects of the beverage. Ayahuasca use is becoming a global phenomenon, and the recreational use of DMT and similar alkaloids has also increased in recent years; such uncontrolled use can lead to severe intoxications. In this investigation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study the kinetics of alkaloids over a 24 h period in saliva and serum of 14 volunteers who consumed ayahuasca twice a month in a religious context. We compared the area under the curve (AUC), maximum concentration (Cmax ), time to reach Cmax (Tmax ), mean residence time (MRT), and half-life (t1/2 ), as well as the serum/saliva ratios of these parameters. DMT and β-carboline concentrations (Cmax ) and AUC were higher in saliva than in serum and the MRT was 1.5-3.0 times higher in serum. A generalized estimation equations (GEEs) model suggested that serum concentrations could be predicted by saliva concentrations, despite large individual variability in the saliva and serum alkaloid concentrations. The possibility of using saliva as a biological matrix to detect DMT, β-carbolines, and their derivatives is very interesting because it allows fast noninvasive sample collection and could be useful for detecting similar alkaloids used recreationally that have considerable potential for intoxication.

CapsNet-SSP: multilane capsule network for predicting human saliva-secretory proteins

BackgroundCompared with disease biomarkers in blood and urine, biomarkers in saliva have distinct advantages in clinical tests, as they can be conveniently examined through noninvasive sample collection. Therefore, identifying human saliva-secretory proteins and further detecting protein biomarkers in saliva have significant value in clinical medicine. There are only a few methods for predicting saliva-secretory proteins based on conventional machine learning algorithms, and all are highly dependent on annotated protein features. Unlike conventional machine learning algorithms, deep learning algorithms can automatically learn feature representations from input data and thus hold promise for predicting saliva-secretory proteins.ResultsWe present a novel end-to-end deep learning model based on multilane capsule network (CapsNet) with differently sized convolution kernels to identify saliva-secretory proteins only from sequence information. The proposed model CapsNet-SSP outperforms existing methods based on conventional machine learning algorithms. Furthermore, the model performs better than other state-of-the-art deep learning architectures mostly used to analyze biological sequences. In addition, we further validate the effectiveness of CapsNet-SSP by comparison with human saliva-secretory proteins from existing studies and known salivary protein biomarkers of cancer.ConclusionsThe main contributions of this study are as follows: (1) an end-to-end model based on CapsNet is proposed to identify saliva-secretory proteins from the sequence information; (2) the proposed model achieves better performance and outperforms existing models; and (3) the saliva-secretory proteins predicted by our model are statistically significant compared with existing cancer biomarkers in saliva. In addition, a web server of CapsNet-SSP is developed for saliva-secretory protein identification, and it can be accessed at the following URL: http://www.csbg-jlu.info/CapsNet-SSP/. We believe that our model and web server will be useful for biomedical researchers who are interested in finding salivary protein biomarkers, especially when they have identified candidate proteins for analyzing diseased tissues near or distal to salivary glands using transcriptome or proteomics.

ELISA Saliva for Trypanosoma cruzi Antibody Detection: An Alternative for Serological Surveys in Endemic Regions.

Chagas is a neglected disease endemic in Latin America. Vector transmission control had been aggressively performed. Recent entomological surveillance in Brazil has revealed natural infection rates ranging from 0.40% to 0.52%. Although serological surveys are complex to develop, they are important for disease control. In this study, we validated the use of saliva in ELISA commercial kits with a cohort of 100 patients with Chagas disease followed at Hospital das Clinicas in São Paulo, Brazil, and 50 healthy controls. Five ELISA kits for detecting antibodies against Trypanosoma cruzi were tested. The best discrimination between Chagas patients and controls was observed with the Wiener kit, which yielded a sensitivity of 97% and a specificity of 100%. Our findings reveal that the use of saliva may be an alternative to large-scale screening surveys in detecting T. cruzi antibodies; it is a noninvasive sample collection method potentially key to large-scale screening in children.

Developing and Validating a Fast and Accurate Method to Quantify 18 Antidepressants in Oral Fluid Samples Using SPE and LC-MS-MS.

Antidepressant drugs are one of the most widely used medicines for treating major depressive disorders for long time periods. Oral fluid (OF) testing offers an easy and non-invasive sample collection. Detection of antidepressants in OF is important in clinical and forensic settings, such as therapeutic drug monitoring and roadside testing for driving under influence. We developed and validated a comprehensive liquid chromatography-tandem mass spectrometry method for 18 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, cyclobenzaprine, desipramine, desvenlafaxine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, trimipramine, venlafaxine) in oral fluid collected by Quantisal® oral collection devices. One-half milliliter of Quantisal® OF (125μL of neat OF) was submitted to solid-phase extraction. The chromatographic separation was performed employing a biphenyl column in gradient mode with a total run time of 5min. The MS detection was achieved by multiple-reaction monitoring with two transitions per compound. The range for linearity of all analytes was from 10 to 1,000ng/mL, with a limit of detection of 10ng/mL. Intra and inter-day accuracy and precision (n=15) were all within acceptable limits, ±20% error and ±15% relative standard deviation. Analyte recovery at 400ng/mL concentration (n=15) ranged from 91 to 129%. Matrix effect ranged from 73.7 to 157%. The internal proficiency test detected all antidepressants with accuracy ranging from 83.1 to 112.1%. The authentic patient sample showed a percentage difference compared to the previously calculated concentration of 86.3-111%. This method provides for the rapid detection of 18 antidepressants and metabolites in OF, which is readily applicable to a routine laboratory.

Non-Invasive Urine Sampling in Infants: A Review Paper

Good quality urine samples play a crucial role in establishing an early and accurate diagnosis of urinary tract infection in infants. Invasive methods are more reliable but cause pain and discomfort in infants as well as anxiety in clinicians and parents. The current available non-invasive methods are less efficient and produce low quality urine samples being less apprehensive in infants and parents. To date, no studies have been conducted in South East Asia, including Malaysia, to identify the most feasible non-invasive urine sampling method. This literature review aimed to compare various non-invasive urine sample collection methods (bladder stimulation techniques, standard clean-catch urine, urine collection pads and urine bags) which could be more feasible for a district hospital setting in Malaysia. In total, 17 studies were included and reviewed. This review concluded that bladder stimulation techniques are potentially good and feasible alternatives to the current common practices in Malaysia (standard clean-catch urine and catherization) due to its high success rate, fast collection and low contamination rate. There is a higher likelihood to implement these techniques in a tertiary setting if further research on bladder stimulation techniques is found feasible in a less resourceful district hospital setting.

Baseline plasma levels of soluble PD-1, PD-L1, and BTN3A1 predict response to nivolumab treatment in patients with metastatic renal cell carcinoma: a step toward a biomarker for therapeutic decisions

ABSTRACT Despite a proportion of renal cancer patients can experiment marked and durable responses to immune-checkpoint inhibitors, the treatment efficacy is widely variable and identifying the patient who will benefit from immunotherapy remains an issue. We performed a prospective study to investigate if soluble forms of the immune-checkpoints PD-1 (sPD-1), PD-L1 (sPD-L1), pan-BTN3As, BTN3A1, and BTN2A1, could be candidate to predict the response to immune-checkpoint blockade therapy. We evaluated the plasma levels in a learning cohort of metastatic clear cell renal carcinoma (mccRCC) patients treated with the anti-PD-1 agent nivolumab by ad hoc developed ELISA’s. Using specific cut-offs determined through ROC curves, we showed that high baseline levels of sPD-1 (>2.11 ng/ml), sPD-L1 (>0.66 ng/ml), and sBTN3A1 (>6.84 ng/ml) were associated with a longer progression-free survival (PFS) to nivolumab treatment [median PFS, levels above thresholds: sPD-1, 20.7 months (p < .0001); sPD-L1, 19 months (p < .0001); sBTN3A1, 17.5 months (p = .002)]. High sPD-1 and sBTN3A1 levels were also associated with best overall response by RECIST and objective response of >20%. The results were confirmed in a validation cohort of 20 mccRCC patients. The analysis of plasma dynamic changes after nivolumab showed a statistically significant decrease of sPD-1 after 2 cycles (Day 28) in the long-responder patients. Our study revealed that the plasma levels of sPD-1, sPD-L1, and sBTN3A1 can predict response to nivolumab, discriminating responders from non-responders already at therapy baseline, with the advantages of non-invasive sample collection and real-time monitoring that allow to evaluate the dynamic changes during cancer evolution and treatment.

Review—Point-of-Care Urinalysis with Emerging Sensing and Imaging Technologies

Urine testing is a powerful clinical diagnostic tool. The noninvasive collection of samples and wide range of diagnostic targets found in urine makes urinalysis well suited for point-of-care (PoC) monitoring applications. Complete urinalysis testing faces many limitations due to the large quantity of samples processed, the time required for testing, and the labor involved in sample preparation and processing. Development of PoC urinalysis devices with microfluidic technology can enable the detection of infections and monitoring of chronic disease while reducing the demand on testing facilities. In this article, current approaches in clinical urinalysis are reviewed. Emerging sensing and imaging technologies specifically suitable for point-of-care examination of urine samples are discussed with an outlook on the future of point of care urinalysis devices as well as emerging applications enabled by these technologies such as in situ monitoring of Activities of Daily Living (ADL).

Towards Raman spectroscopy of urine as screening tool.

For the screening purposes urine is an especially attractive biofluid, since it offers easy and noninvasive sample collection and provides a snapshot of the whole metabolic status of the organism, which may change under different pathological conditions. Raman spectroscopy (RS) has the potential to monitor these changes and utilize them for disease diagnostics. The current study utilizes mouse models aiming to compare the feasibility of the urine based RS combined with chemometrics for diagnosing kidney diseases directly influencing urine composition and respiratory tract diseases having no direct connection to urine formation. The diagnostic models for included diseases were built using principal component analysis with linear discriminant analysis and validated with a leave-one-mouse-out cross-validation approach. Considering kidney disorders, the accuracy of 100% was obtained in discrimination between sick and healthy mice, as well as between two different kidney diseases. For asthma and invasive pulmonary aspergillosis achieved accuracies were noticeably lower, being, respectively, 77.27% and 78.57%. In conclusion, our results suggest that RS of urine samples not only provides a solution for a rapid, sensitive and noninvasive diagnosis of kidney disorders, but also holds some promises for the screening of nonurinary tract diseases.

Challenges and lessons learned from clinical pharmacogenetic implementation of multiple gene–drug pairs across ambulatory care settings

PurposeIncorporating a patient’s genotype into the clinical decision-making process is one approach to precision medicine. The University of Florida (UF) Health Precision Medicine Program is a pharmacist-led multidisciplinary effort that has led the clinical implementation of six gene–drug(s) pairs to date. This study focuses on the challenges encountered and lessons learned with implementing pharmacogenetic testing for three of these: CYP2D6-opioids, CYP2D6/CYP2C19-selective serotonin reuptake inhibitors, and CYP2C19-proton pump inhibitors within six pragmatic clinical trials at UF Health and partners. MethodsWe compared common measures collected within each of the pharmacogenetic implementations as well as solicited feedback from stakeholders to identify challenges, successes, and lessons learned. ResultsWe identified several challenges related to trial design and implementation, and learned valuable lessons. Most notably, case discussions are effective for prescriber education, prescribers need clear concise guidance on genotype-based actions, having genotype results available at the time of the patient–prescriber encounter helps optimize the ability to act on them, children prefer noninvasive sample collection, and study participants are willing to answer patient-reported outcomes questionnaires if they are not overly burdensome, among others. ConclusionThe lessons learned from implementing three gene–drug pairs in ambulatory care settings will help shape future pharmacogenetic clinical trials and clinical implementations.

The Small RNA Repertoire of Small Extracellular Vesicles Isolated From Donor Kidney Preservation Fluid Provides a Source for Biomarker Discovery for Organ Quality and Posttransplantation Graft Function.

We analyzed whether donor organ preservation fluid contain small extracellular vesicles (sEV) and whether the RNA content of these vesicles could be used as a source for potential biomarkers for posttransplantation kidney function. We provide proof of principle that sEVs are present in preservation fluid, which contain RNAs associated with donor origin. Furthermore, sEV micro RNA profiles could be associated with graft function during the first 7 days posttransplantation, but no significant correlation with DGF could be established based on the current dataset. Overall, the predictive potential of sEV RNA biomarkers together with relatively easy and noninvasive sample collection and analysis methods could pave the way towards universal screening of donor kidney-associated risk for DGF, optimized patient treatment, and subsequently improved short- and long-term graft function and survival.

Current trends in the application of non-invasive genetic sampling to study Neotropical birds: Uses, goals, and conservation potential

The non-invasive collection of biological samples has proven useful to study a diverse array of research topics worldwide. Here, we present a systematic bibliographical synthesis exploring how the non-invasive collection of genetic samples has been used to study avian populations in the Neotropics. We searched international online databases for scientific publications, spanning from 2007 to 2017, to describe the trends and identify the sample types used, species studied, and research questions addressed. The analysis of 21 articles showed that shed feathers were most frequently used (66.7% of articles), followed by carcasses (14.2%), eggs (9.5%), and non-invasively obtained blood (4.8%); one study used both feces and shed feathers. Most studies addressed population genetic issues (38.1%), followed by species identification (28.6%), phylogenetic questions (14.3%), molecular sexing (9.5%), and parentage analyses (9.5%). Brazil produced almost half (47.6%) of the publications retrieved. Despite an increasing interest in using non-invasive sampling to study Neotropical avifauna, its application is still largely concentrated in the most developed countries in this region and to explore a limited number of questions. A more regular use of non-invasive sampling would help advance the knowledge of ecological, behavioral, genetic, and evolutionary aspects of Neotropical birds. Investigating the extent of human–wildlife conflict, such as impact of road-kills, illegal traffic, and collision with aerial infrastructure or unmanned vehicles, is an underexplored avenue of research in which this method could be of much help. Non-invasive genetic sampling can help tackle conservation problems and pave the way to scientifically informed conservation policies in this avian biodiversity hotspot.

Genomic landscape of FNAs positive for medullary thyroid cancer (MTC) and potential impact on systemic therapy.

6087 Background: Systemic therapies targeting specific genomic alterations in advanced MTC are available or under investigation. The Afirma Genomic Sequencing Classifier (GSC) uses RNA sequencing to assess FNA specimens from cytologically indeterminate thyroid nodules, which are also tested for specific molecular aberrations associated with thyroid cancer via a suite of highly accurate malignancy classifiers. This suite can be applied independently to Bethesda V/VI nodules. The Afirma Xpression Atlas (XA) is an additional test that can be combined with Afirma GSC to report nucleotide variants and fusions across 511 cancer-associated genes. Here we report the prevalence and genomic landscape of MTC classifier positive (MTC+) FNA samples. Methods: All Afirma GSC and malignancy classifier tests run in the Veracyte Clinical Laboratory between July 2017 and January 2019 were deidentified and examined for MTC+ cases. Afirma XA was run on all such cases, and all variants and fusions were tabulated. Results: Examination of 29,895 FNAs revealed 90 MTC cases. Of 22,793 Bethesda III cases, 32 (0.14%) were MTC+. Of 5,491 Bethesda IV cases, 33 (0.60%) were MTC+. Provider-ordered testing was done on an additional 16 and 9 MTC cases from Bethesda V and VI nodules, respectively. 58% of all MTC+ samples harbored a RET variant (+/- others), 9% contained a KRAS variant (+/- others), 6% included an HRAS variant, 1% had a BRAF fusion, 1% demonstrated other fusions, and 26% held no variant/fusion. Conclusions: In our cohort, Afirma XA identified a variant or fusion in 74% of MTC+ FNAs. Currently approved or investigational therapies exist for cancers with RET, BRAF and HRAS alterations, suggesting that 64% of our series might be eligible for treatment based on genomic information from FNA. In advanced MTC, noninvasive FNA sample collection at the time of diagnosis may ultimately impact on targeted therapy selection, with the option to repeat FNA testing should the disease progress. Future studies may investigate how finding a genomic alteration by FNA can inform the management of MTC and, in the case of progressive disease, improve our understanding of the mechanisms of disease progression and drug resistance.

Genetic Structure of the Big Summit Herd and Neighboring Wild Horse Populations Inhabiting Herd Management Areas of Oregon

Feral horses roaming North America are distinct not only because of their acclimatization to the wild but also because of their diversified ancestry. The wild horses inhabiting the Ochoco National Forest near Prineville, Oregon, are one of the remaining wild mustang herds and are protected by the Wild Horse and Burro Acts of 1971 and 1978. With deterioration of their rangeland, drought, vegetation conditions, human encroachment, fencing off of grazing lands, competition with wildlife, and other livestock grazing on public lands, a critical balance between conserving the natural ecology of rangelands and protecting and managing the wild horses needs to be monitored. Supplementing traditional visual censuses with genetic analyses can enhance management and conservation efforts because DNA analyses provide insight into the genetic fitness and inbreeding status of the population. The objective of this study was to provide a genetic analysis using noninvasive sample collection methods to assess the genetic health of this small herd and to determine their genetic fitness. A total of 52 individuals from the Big Summit wild horse herd and adjacent herd management areas (HMAs) were genotyped for 17 short tandem repeat (STR) loci, mtDNA haplogroups, and major histocompatibility complex STR loci. Cluster analysis exhibited an admixed population with discernable contribution from Iberian ancestry, the major influence coming from Andalusian and Lusitano breeds. The deficiency of hetero zygosity, a deviation from Hardy–Weinberg equilibrium, together with a positive inbreeding coefficient for the neutral STRs in the Big Summit population suggested a parallel to an “island population” phenomenon leading to loss of genetic diversity within the herd. These findings improve understanding of the genetic structure of feral herds from different HMAs, which in turn will enable enhanced conservation and management strategies.