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.

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.

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.

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.

Earwax, osmidrosis, and breast cancer: why does one SNP (538G>A) in the human ABC transporter ABCC11 gene determine earwax type?

One single-nucleotide polymorphism (SNP), 538G>A (Gly180Arg), in the ABCC11 gene determines the type of earwax. The G/G and G/A genotypes correspond to the wet type of earwax, whereas A/A corresponds to the dry type. Wide ethnic differences exist in the frequencies of those alleles, reflecting global migratory waves of the ancestors of humankind. We herein provide the evidence that this genetic polymorphism has an effect on the N-linked glycosylation of ABCC11, intracellular sorting, and proteasomal degradation of the variant protein. Immunohistochemical studies with cerumen gland-containing tissue specimens revealed that the ABCC11 WT protein was localized in intracellular granules and large vacuoles, as well as at the luminal membrane of secretory cells in the cerumen gland, whereas granular or vacuolar localization was not detected for the SNP (Arg180) variant. This SNP variant lacking N-linked glycosylation is recognized as a misfolded protein in the endoplasmic reticulum and readily undergoes ubiquitination and proteasomal degradation, which determines the dry type of earwax as a mendelian trait with a recessive phenotype. For rapid genetic diagnosis of axillary osmidrosis and potential risk of breast cancer, we developed specific primers for the SmartAmp method that enabled us to clinically genotype the ABCC11 gene within 30 min.

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.

Allele frequencies of the ABCC11 gene for earwax phenotypes among ancient populations of Hokkaido, Japan.

Human earwax is classified into wet and dry types, which are determined by a single-nucleotide polymorphism in the adenosine triphosphate-binding cassette, sub-family C11 (ABCC11) gene locus. To investigate the allele frequencies of the ABCC11 locus within ancient populations on the Northern Japanese island of Hokkaido, amplified product-length polymorphisms were analyzed for 50 specimens of the Okhotsk people and 35 specimens of the Jomon and Epi-Jomon people excavated from various archaeological sites of Hokkaido. Of these specimens, 31 Okhotsk and 19 Jomon/Epi-Jomon samples were genotyped successfully. Frequencies of the wet-type allele in the Jomon/Epi-Jomon people, considered a major ancestor of the Ainu, were higher than those of other Northeastern Asian populations, including the modern Ainu. By contrast, in the Okhotsk people, believed to originate from East Siberia, frequencies of the dry-type allele were relatively higher than those in the Ainu and Jomon/Epi-Jomon people. These results suggest that gene flow from the Northeastern Asian Continent to descendants of the Jomon/Epi-Jomon people of Hokkaido through the Okhotsk people occurred, resulting in the establishment of the Ainu.

Japanese map of the earwax gene frequency: a nationwide collaborative study by Super Science High School Consortium.

Wet/dry types of earwax are determined by the c.538G>A single-nucleotide polymorphism (SNP) in the ABCC11 gene; GA and GG genotypes give the wet type and AA the dry type. The Japanese population may have a dual structure comprising descendants of mixtures between the ancient 'Jomon' and 'Yayoi' populations. We hypothesized that the dry type was introduced by the Yayoi people to the Jomon population where the wet type was predominant, and as the mixture of the two populations has not yet been complete, the allele-A frequency (f(A)) would even now be higher along a putative Yayoi man's peopling route within Japanese islands. To know the frequency, a nationwide Super Science High School (SSH) Consortium collected 1963 fingernail samples of pupils/students from at least one high school/university in every prefecture. All further procedures, DNA extraction, SNP genotyping and gene frequency estimation, were carried out by trained SSH pupils. Although the allele-A frequency varied among the 47 prefectures, the Gifu/Kyoto and Okinawa prefectures showed the highest and lowest values, respectively. Areas with high frequencies included Northeastern Kyushu, Northern Shikoku and Kinki districts, showing a belt-like zone, whereas those with low frequencies other than Okinawa were the Southwestern Kyushu, Hiroshima prefecture and Tohoku districts. The f(A) value in Kinki district was statistically higher than those in prefectures westward and east-northward from it. The result may provide another line of evidence supporting a possible route of the Yayoi-man's peopling in Japan.

A strong association of axillary osmidrosis with the wet earwax type determined by genotyping of the ABCC11 gene.

BACKGROUND: Two types of cerumen occur in humans: the wet type with brownish, sticky earwax, and the dry type with a lack of or reduced ceruminous secretion. The wet type is common in populations of European and African origin, while the dry type is frequently seen in Eastern Asian populations. An association between axillary odor and the wet-type earwax was first identified approximately 70 years ago. The data were based on a phenotypical analysis of the two phenotypes among the Japanese by a researcher or by self-declaration of the subjects examined, and were not obtained using definite diagnostic methods. Recently, we identified a single-nucleotide polymorphism (SNP; rs17822931) of the ABCC11 gene as the determinant of the earwax types. In the present study, to determine whether the SNP can serve as a diagnostic marker for axillary osmidrosis (AO), we examined genotypes at rs17822931 in 79 Japanese AO individuals. AO was defined here as a clinical condition of individuals with a deep anxiety regarding axillary odor and had undergone the removal of bilateral axillary apocrine glands. RESULTS: A comparison of the frequencies of genotypes at rs17822931 in the 79 AO individuals and in 161 Japanese from the general population showed that AO was strongly associated with the wet earwax genotype. A total of 78 (98.7%) of 79 AO patients had either the GG or GA genotype, while these genotypes were observed in 35.4% (57/161) of the subjects from the general population (p < 1.1 x 10(-24), by Fisher's exact test). CONCLUSION: The strong association between the wet-earwax associated ABCC11-genotypes (GG and GA) and AO identified in this study indicates that the genotypes are good markers for the diagnosis of AO. In addition, these results suggest that having the allele G is a prerequisite for the axillary odor expression. In other words, the ABCC11 protein may play a role in the excretory function of the axillary apocrine gland. Together, these results suggest that when an AO individual visiting a hospital is diagnosed with dry-type earwax by ABCC11-genotyping, surgical removal of their axillary glands may not be indicated.

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.

Clinical practice guideline: cerumen impaction.

OBJECTIVE: This guideline provides evidence-based recommendations on managing cerumen impaction, defined as an accumulation of cerumen that causes symptoms, prevents assessment of the ear, or both. We recognize that the term "impaction" suggests that the ear canal is completely obstructed with cerumen and that our definition of cerumen impaction does not require a complete obstruction. However, cerumen impaction is the preferred term since it is consistently used in clinical practice and in the published literature to describe symptomatic cerumen or cerumen that prevents assessment of the ear. This guideline is intended for all clinicians who are likely to diagnose and manage patients with cerumen impaction. PURPOSE: The primary purpose of this guideline is to improve diagnostic accuracy for cerumen impaction, promote appropriate intervention in patients with cerumen impaction, highlight the need for evaluation and intervention in special populations, promote appropriate therapeutic options with outcomes assessment, and improve counseling and education for prevention of cerumen impaction. In creating this guideline the American Academy of Otolaryngology-Head and Neck Surgery Foundation selected a panel representing the fields of audiology, family medicine, geriatrics, internal medicine, nursing, otolaryngology-head and neck surgery, and pediatrics. RESULTS: The panel made a strong recommendation that 1) clinicians should treat cerumen impaction that causes symptoms expressed by the patient or prevents clinical examination when warranted. The panel made recommendations that 1) clinicians should diagnose cerumen impaction when an accumulation of cerumen is associated with symptoms, or prevents needed assessment of the ear (the external auditory canal or tympanic membrane), or both; 2) clinicians should assess the patient with cerumen impaction by history and/or physical examination for factors that modify management, such as one or more of the following: nonintact tympanic membrane, ear canal stenosis, exostoses, diabetes mellitus, immunocompromised state, or anticoagulant therapy; 3) the clinician should examine patients with hearing aids for the presence of cerumen impaction during a healthcare encounter (examination more frequently than every three months, however, is not deemed necessary); 4) clinicians should treat the patient with cerumen impaction with an appropriate intervention, which may include one or more of the following: cerumenolytic agents, irrigation, or manual removal other than irrigation; and 5) clinicians should assess patients at the conclusion of in-office treatment of cerumen impaction and document the resolution of impaction. If the impaction is not resolved, the clinician should prescribe additional treatment. If full or partial symptoms persist despite resolution of impaction, alternative diagnoses should be considered. The panel offered as an option that 1) clinicians may observe patients with nonimpacted cerumen that is asymptomatic and does not prevent the clinician from adequately assessing the patient when an evaluation is needed; 2) clinicians may distinguish and promptly evaluate the need for intervention in the patient who may not be able to express symptoms but presents with cerumen obstructing the ear canal; 3) the clinician may treat the patient with cerumen impaction with cerumenolytic agents, irrigation, or manual removal other than irrigation; and 4) clinicians may educate/counsel patients with cerumen impaction/excessive cerumen regarding control measures. DISCLAIMER: This clinical practice guideline is not intended as a sole source of guidance in managing cerumen impaction. Rather, it is designed to assist clinicians by providing an evidence-based framework for decision-making strategies. It is not intended to replace clinical judgment or establish a protocol for all individuals with this condition, and may not provide the only appropriate approach to diagnosing and managing this problem.

Presence of hBD-1 and hBD-2 in human cerumen and external auditory canal skin.

CONCLUSION: Human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) antimicrobial peptides present in the cerumen, which is composed of exfoliated epithelial keratin and gland secretion, might provide the first line of defense against microbes in external auditory canal (EAC) skin. OBJECTIVES: Cerumen (earwax) plays a primary role in protecting the EAC skin and tympanic membrane. Even though the protection by antimicrobial peptides present in the skin secretion has been well established, little is known about the intrinsic role of the peptides in the EAC skin and cerumen. The aim of this study was to examine the presence of important antimicrobial peptides, hBD-1 and hBD-2, in the cerumen and EAC skin. MATERIALS AND METHODS: Cerumen was collected from 20 healthy adults, and the EAC skins were obtained from 12 patients who underwent middle ear surgery with canaloplasty. The presence of hBD-1 and hBD-2 was analyzed using immunohistochemistry and Western blotting. RESULTS: In the immunohistochemical study of the EAC skin, expression of hBD-1 and hBD-2 was observed in both the epithelium and the glands. The presence of hBD-1 and hBD-2 peptides in the cerumen was confirmed by Western blotting.

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.

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.

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.

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.