A veterinary cerumenomic assay for bovine laminitis identification.

Bovine laminitis disorder results in animal welfare and economic concerns in dairy and beef farms worldwide. However, the affected metabolic pathways, pathophysiologic characteristics, and inflammatory mechanisms remain unclear, hampering the development of new diagnostics. Using cerumen (earwax) as a source of volatile metabolites (cerumenomic) that carry valuable biological information has interesting implications for veterinary medicine. Nonetheless, up to now, no applications of veterinary cerumenomic assays have been made to identify bovine laminitis. This work aims to develop a veterinary cerumenomic assay for bovine laminitis identification that is non-invasive, robust, accurate, and sensitive to detecting the metabolic disturbances in bovine volatile metabolome. Twenty earwax samples (10 from healthy/control calves and 10 from laminitis calves) were collected from Nellore cattle, followed by Headspace/Gas Chromatography-Mass Spectrometry (HS/GC-MS) analysis and biomarker selection in two multivariate approaches: semiquantitative (intensity data) and semiqualitative (binary data). Following the analysis, cerumen volatile metabolites were indicated as candidate biomarkers for identifying bovine laminitis by monitoring their intensity or occurrence. In the semiquantitative strategy, the p-cresol presented the highest diagnostic figures of merit (area under the curve: 0.845, sensitivity: 0.700, and specificity: 0.900). Regarding the binary approach, a panel combining eight variables/volatiles, with formamide being the most prominent one, showed an area under the curve, sensitivity, and specificity of 0.97, 0.81, and 0.90, respectively. In summary, this work describes the first veterinary cerumenomic assay for bovine laminitis that indicates new metabolites altered during the inflammatory condition, paving the way for developing laminitis early diagnosis by monitoring the cerumen metabolites.

Cancer evaluation in dogs using cerumen as a source for volatile biomarker prospection.

Cancer is one of the deadliest diseases in humans and dogs. Nevertheless, most tumor types spread faster in canines, and early cancer detection methods are necessary to enhance animal survival. Here, cerumen (earwax) was tested as a source of potential biomarkers for cancer evaluation in dogs. Earwax samples from dogs were collected from tumor-bearing and clinically healthy dogs, followed by Headspace/Gas Chromatography-Mass Spectrometry (HS/GC-MS) analyses and multivariate statistical workflow. An evolutionary-based multivariate algorithm selected 18 out of 128 volatile metabolites as a potential cancer biomarker panel in dogs. The candidate biomarkers showed a full discrimination pattern between tumor-bearing dogs and cancer-free canines with high accuracy in the test dataset: an accuracy of 95.0% (75.1-99.9), and sensitivity and specificity of 100.0% and 92.9%, respectively. In summary, this work raises a new perspective on cancer diagnosis in dogs, being carried out painlessly and non-invasive, facilitating sample collection and periodic application in a veterinary routine.

Identification of Altered miRNAs in Cerumen of Dogs Affected by Otitis Externa.

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes.

Cerumenogram: a new frontier in cancer diagnosis in humans.

Cancer is the deadliest human disease and the development of new diagnosis methods is important to increase the chances of a cure. In this work it was developed a new method, named here for the first time as cerumenogram, using cerumen (earwax) as a new biomatrix for diagnosis. Earwax samples collected from cancer patients (cancer group) and cancer-free patients (control group) were analyzed by Headspace/Gas Chromatography-Mass Spectrometry (HS/GC-MS), following with multivariate analysis steps to process the raw data generated. In total, 158 volatile organic metabolites (VOMs) were identified in the cerumen samples. The 27 selected as potential VOMs biomarkers for cancer provided 100% discrimination between the cancer and control groups. This new test can thus be routinely employed for cancer diagnoses that is non-invasive, fast, cheap, and highly accurate.

Removal of external ear canal exostoses by piezo surgery: a novel technique.

BACKGROUND: External auditory canal exostoses are known to occur in patients who engage in cold-water sports. Although the majority of patients with exostosis remain asymptomatic, larger lesions can cause wax impaction, conductive hearing loss and predispose to recurrent otitis externa. OBJECTIVE: A novel technique is described of using a piezo saw to excise exostoses that are symptomatic. The piezo saw is used to perform various procedures, but its use in removing exostoses has not been described in the literature. CONCLUSION: Excision of exostoses of the ear canal using a piezo saw is a safe technique and patients have a speedy recovery. This paper describes a new technique for removing exostoses.

Insights into cerumen and application in diagnostics: past, present and future prospective.

Cerumen or earwax is an emerging bio-fluid in clinical diagnosis that has been very little exploited during the past decades in spite of its high diagnostic potential. It is highly abundant in diagnostic biomarkers such as genetic material, lipids, proteins, chemical elements, internal and external metabolites (e.g. hormones, volatile organic compounds, amino acids, xenobiotics etc.) reaching earwax from the blood circulation. Thus, it is able to reflect not only physiology, pathophysiology of the human body but can also detect recent and long term exposure to environmental pollutants, without the need of invasive blood tests and in the same time overcoming many disadvantages faced by using other diagnostic biological fluids. This review discusses the biology, functions, chemistry of earwax, past and current approaches for the study of its chemical composition, emphasizing how a detected variation in its composition can offer information of high clinical value, which can be useful in diagnosis of many diseases such as metabolic disorders and tumours as well as in forensic applications. It also presents details about techniques of sample collection, storage, and analysis. Moreover, it highlights concerns about the use of earwax for diagnostic purposes, which should be addressed to make earwax diagnostics a reality in the future.

Ultrastructure and immunohistochemical characterization of proteins concerned with the secretory machinery in goat ceruminous glands.

The expression of soluble N-ethyl-maleimide sensitive fusion attachment protein receptor (SNARE) proteins in apocrine glands has not been fully elucidated. In addition to performing ultrastructural observation of the ceruminous glands in goats, our study focuses on the demonstration of ß-defensins, SNARE proteins and Rab3D in these glands with the use of immunohistochemical methods. The secretory cells were equipped with two types of vesicles, Golgi apparatus and abundant rough endoplasmic reticulum (ER). Additionally, in some of them, the characteristic concentric structures composed of rough ER were observed in their circum- and infranuclear parts. The expression of phosphorylated inositol requiring enzyme 1a was also detected. These findings may indicate their ability to produce numerous secretory proteins and the maintenance of homeostasis in the glandular cells. Furthermore, ß-defensins were demonstrated as products of the ceruminous glands. The present investigation also revealed the presence of SNARE proteins and Rab3D. It is suggested that these proteins are concerned with the secretory machinery of this gland type.

Earwax metabolomics: An innovative pilot metabolic profiling study for assessing metabolic changes in ewes during periparturition period.

Important metabolic changes occur during transition period of late pregnancy and early lactation to meet increasing energy demands of the growing fetus and for milk production. The aim of this investigation is to present an innovative and non-invasive tool using ewe earwax sample analysis to assess the metabolic profile in ewes during late pregnancy and early lactation. In this work, earwax samples were collected from 28 healthy Brazilian Santa Inês ewes divided into 3 sub-groups: 9 non-pregnant ewes, 6 pregnant ewes in the last 30 days of gestation, and 13 lactating ewes ≤ 30 days postpartum. Then, a range of metabolites including volatile organic compounds (VOC), amino acids (AA), and minerals were profiled and quantified in the samples by applying headspace gas chromatography/mass spectrometry, high performance liquid chromatography/tandem mass spectrometry, and inductively coupled plasma-optical emission spectrometry, respectively. As evident in our results, significant changes were observed in the metabolite profile of earwax between the studied groups where a remarkable elevation was detected in the levels of non-esterified fatty acids, alcohols, ketones, and hydroxy urea in the VOC profile of samples obtained from pregnant and lactating ewes. Meanwhile, a significant decrease was detected in the levels of 9 minerals and 14 AA including essential AA (leucine, phenyl alanine, lysine, isoleucine, threonine, valine), conditionally essential AA (arginine, glycine, tyrosine, proline, serine), and a non-essential AA (alanine). Multivariate analysis using robust principal component analysis and hierarchical cluster analysis was successfully applied to discriminate the three study groups using the variations of metabolites in the two stress states (pregnancy and lactation) from the healthy non-stress condition. The innovative developed method was successful in evaluating pre- and post-parturient metabolic changes using earwax and can in the future be applied to recognize markers for diagnosis, prevention, and intervention of pregnancy complications in ewes.

Earwax: A neglected body secretion or a step ahead in clinical diagnosis? A pilot study.

This work combines the advantages of volatile metabolites profiling as a young growing research field with a non-invasive sampling technique using earwax "a neglected body secretion" for detection and monitoring of biomarkers for diabetes mellitus (types 1 and 2). Earwax samples were collected from 26 diabetic patients of both types, analyzed by headspace gas chromatography mass spectrometry and confronted to the volatile earwax composition of 33 healthy individuals. Data mining analysis was conducted using different models to discriminate the healthy individuals from the diabetic patients and to discriminate between both types of diabetes as well. The model with the best discriminating ability was found to be partial least squares discriminant analysis (PLS-DA) after variable selection. The 6 most important biomarkers were ethanol, acetone, methoxyacetone, hydroxyurea, isobutyraldehyde, and acetic acid. The multivariate model constructed was validated using a test data set and was able to correctly predict all the samples. The receiver operating characteristic (ROC) curves were built for the 6 variables for diabetes types 1 and 2 diagnoses. Among the 6 variables selected, methoxyacetone was the only biomarker able solely to perfectly discriminate between diabetes types 1 and 2. The method is simple, non-invasive, accurate, and highly accepted by patients. SIGNIFICANCE: Our method involves a volatolomic approach by headspace gas chromatography coupled with mass spectrometry as a single analytical technique combined with multivariate data analysis to detect biomarkers of diabetes in earwax samples. Our method was able to discriminate with high accuracy between 33 healthy controls and 26 diabetic patients as well as its types (1 and 2). Our method employing earwax, a "neglected biological matrix" not only has the advantage of non-invasive sampling but also overcomes the limitations of the applied procedures in other biological samples, involving no or minimum sample pretreatment, no external contamination and utilizing a simple sample collection technique.

A Novel Association between the 27-bp Deletion and 538G>A Mutation in the ABCC11 Gene.

A single nucleotide polymorphism in the ABCC11 gene, 538G>A (rs17822931), is known to determine human ear wax type. The G/G and G/A genotypes correspond to the wet type, while the A/A genotype corresponds to the dry type. Another earwax determinant, a 27-bp deletion (Δ27) downstream from the rs17822931 site, is a rare variant that leads to the dry phenotype. In a previous report, we found an individual with the G allele who unexpectedly showed the dry type of earwax, leading to the identification of Δ27. We also demonstrated that the Δ27 allele was present in individuals of Japanese, Thai, native North American, Andean, and Bolivian ancestry but absent in those of European and African ancestry. Here, we assessed the Δ27 allele frequency among Japanese and Ukrainian individuals and identified a novel association between the Δ27 and 538G>A mutations. The Δ27 allele frequency was 0.002 (3/1,520; one individual is heterozygous, and another is homozygous) among Japanese individuals and 0 (0/794) among Ukrainians. We also found a previously unreported homozygous genotype for both the Δ27 and A alleles. Our findings suggest that the Δ27 deletion may have occurred in an ABCC11 gene with the 538G>A mutation.

The Effect of Ethnicity on Human Axillary Odorant Production.

Previous findings from our laboratory highlighted marked ethnic differences in volatile organic compounds (VOCs) from cerumen among individuals of Caucasian, East Asian, and African-American descent, based, in part, on genetic differences in a gene that codes for a transport protein, which is a member of the ATP-binding cassette transporter, sub-family C, member 11 (ABCC11). In the current work, we hypothesized that axillary odorants produced by East Asians would differ markedly from those obtained from individuals of European or African descent based on the pattern of ethnic diversity that exists in ABCC11. Using gas chromatography/mass spectrometry (GC/MS) we examined differences in axillary odorant VOCs among 30 individuals of African-American, Caucasian, and East Asian descent with respect to their ABCC11 genotype. While no qualitative differences in the type of axillary odorants were observed across ethnic groups, we found that characteristic axillary odorants varied quantitatively with respect to ethnic origin. We propose that ABCC11 is not solely responsible for predicting the relative amounts of volatiles found in axillary secretions and that other biochemical pathways must be involved.

Otologic Manifestations of Autosomal Recessive Congenital Ichthyosis in Children.

BACKGROUND: Few studies have investigated ear involvement in nonsyndromic autosomal recessive congenital ichthyosis (ARCI). OBJECTIVES: To assess the type and frequency of otologic manifestations of ARCI in patients under follow-up at the pediatric dermatology department of our hospital. MATERIALS AND METHODS: We prospectively studied the presence of ear pain, ear itching, tinnitus, otitis, cerumen impaction, accumulation of epithelial debris, and hearing loss. Daily hygiene measures, topical treatments, medical-surgical interventions, and frequency of visits to an ear, nose, and throat (ENT) specialist were noted in the patients' medical records. Ear examination and hearing tests were performed in all cases. RESULTS: Ten patients were studied: 2 had a self-healing collodion baby phenotype and 8 had ichthyosis. There was mention of otologic manifestations in the records of all 8 patients with ichthyosis (100%); 6 of these patients (75%) had abnormalities in the external auditory canal examination and 2 (25%) had conductive hearing loss. Our findings are limited by the small number of patients studied, all of whom were younger than 19 years. CONCLUSIONS: The involvement of both dermatologists and ENT specialists in the management of patients with ichthyosis is crucial to ensure the application of the best therapeutic and preventive measures. More studies are needed to assess the prevalence and impact on quality of life of ear involvement in patients with ichthyosis and to determine the optimal interval between ENT visits for these patients.

Ceruminal diffusion activities and ceruminolytic characteristics of otic preparations - an in-vitro study.

BACKGROUND: An in-vitro setup was established in order to determine a) the diffusion activities of eight otic preparations (Aurizon®, Eas Otic®, Epi Otic®, Otifree®, Otomax®, Panolog®, Posatex®, Surolan®) through synthetic cerumen, and b) the ceruminolytic capacity and impregnation effects of these products. The main lipid classes of canine cerumen produced with moderate, non-purulent otitis externa were determined by thin layer chromatography and were subsequently used to produce a standardised synthetic cerumen (SCC). SCC was filled into capillary tubes, all of which were loaded with six commercially available multipurpose otic medications and two ear cleaners, each mixed with two markers in two experimental setups. These two marker compounds (Oil red O and marbofloxacin) were chosen, since they exhibit different physicochemical drug characteristics by which it is possible to determine and verify the diffusion activity of different types of liquids (i.e. the otic preparations). A synthetic cerumen described in the literature (JSL) was also used for comparison as its lipid composition was different to SCC. The diffusion activities of the otic preparations through both types of synthetic cerumen were studied over 24 hours. A second in-vitro experiment determined both the ceruminolytic activity and impregnation effect of the otic preparations by comparing the weight loss or weight gain after repeated incubation of JSL. RESULTS: Canine cerumen is mainly composed of triglycerides, sterol esters, fatty acid esters and squalene. The diffusion experiments showed a high diffusion efficacy along with a high impregnation effect for one test product. All the other products exhibited a lower diffusion activity with a mild to moderate impregnation effect. A mild ceruminolytic activity was observed for the two ear cleaners but not for any of the otic medications. CONCLUSIONS: The present study demonstrates that there are significant differences in the diffusion characteristics and ceruminolytic properties of the eight tested otic preparations.

In-depth proteomic analysis of the human cerumen-a potential novel diagnostically relevant biofluid.

Human cerumen, also called earwax, is a substance secreted by various glands in the outer ear canal. Although the variation of texture and color during otorhinolaryngological diseases is a generally known phenomenon, cerumen as biofluid remains relatively unexplored. However, there is an emerging interest for protein biomarkers which are easily accessible and predictive for diagnostics and therapy outcome. Here we provide a thorough investigation of human cerumen applying two different prefractionation techniques: i) 1D-PAGE prefractionation with subsequent LC-MS/MS, and ii) online SCX-fractionation coupled to LC-MS/MS. Additionally, individual variation was addressed by shotgun LC-MS/MS of specimens from 5 subjects. In total, we identified 11,562 distinct peptides representing 2013 proteins in human cerumen. The in-depth characterization revealed a high complexity of cerumen comparable with other human biofluids such as urine, plasma, or saliva. A probiotic or antibiotic character of cerumen has previously been discussed. In this study we provide further evidence for the important role of cerumen as an antimicrobial barrier and in local immune response, e.g. by assessing high amounts of zinc-alpha-2-glycoprotein. PRACTICAL IMPLICATIONS: Cerumen analysis might have promising potential as diagnostic body fluid for biomarker characterization and disease specific objectives. Disease-associated or infection-specific changes may support diagnostics in otorhinolaryngology and may lead to a better understanding of human cerumen's function in immune response. An easy-to-handle and standardized sample collection and preparation of cerumen can further improve individualized medicine strategies.

Expression of Glut-1, HIF-1α, PI3K and p-Akt in a case of ceruminous adenoma.

OBJECTIVES: Ceruminous adenoma of the external auditory canal (EAC) is a rare type of tumour that is diagnosed histologically. However, the clinical behaviour of these tumours remains controversial. Here, we report a case of ceruminous adenoma of the EAC and expression of a hypoxia marker. CASE REPORT: A 78-year-old man presented with a 6-month history of recurrent otorrhoea in the right ear. Surgery was performed by the transmeatal approach with total removal of the mass. Histopathology revealed a ceruminous adenoma. Tumour cells were positive for CK, S-100 protein, Glut-1, HIF-1α, PI3K and p-Akt. There was no evidence of recurrence at last follow-up 27 months after the operation. CONCLUSIONS: Ceruminous adenoma of the EAC is a rare tumour. The treatment of choice is wide local excision with clear margins. To our knowledge, this is the first report of Glut-1 expression and the PI3K/Akt pathway in ceruminous adenoma of the EAC.

Middle ear and hearing disorders of schoolchildren aged 7-10 years in South Sinai, Egypt.

Hearing loss among schoolchildren in developing countries is reported to be a significant health problem. Data on child hearing loss in South Sinai, a remote governorate of Egypt, are lacking. Middle ear diseases and hearing impairment were assessed among 453 primary-school children aged 7-10 years in South Sinai (906 ears). Otoscopic examination, tympanometry and pure tone audiometry (PTA) were done. Ear disease was found in 27.5% of the ears examined. The commonest cause was secretory otitis media (10.8%), followed by occluded earwax (9.5%). Mild and moderate hearing loss affected 8.5% of the sample, while sensorineural hearing loss affected 2.4%; only 0.4% had moderate and severe hearing loss. Hearing impairment affects 19.3% of this age group in South Sinai. None of the children with hearing impairment had been previously diagnosed or was receiving treatment and support. Hearing and middle ear screening at schools is recommended for early detection and management of middle ear and hearing problems.