Inner ear manifestations in CHARGE: Abnormalities, treatments, animal models, and progress toward treatments in auditory and vestibular structures.

The inner ear contains the sensory organs for hearing and balance. Both hearing and balance are commonly affected in individuals with CHARGE syndrome (CS), an autosomal dominant condition caused by heterozygous pathogenic variants in the CHD7 gene. Semicircular canal dysplasia or aplasia is the single most prevalent feature in individuals with CHARGE leading to deficient gross motor skills and ambulation. Identification of CHD7 as the major gene affected in CHARGE has enabled acceleration of research in this field. Great progress has been made in understanding the role of CHD7 in the development and function of the inner ear, as well as in related organs such as the middle ear and auditory and vestibular neural pathways. The goals of current research on CHD7 and CS are to (a) improve our understanding of the pathology caused by CHD7 pathogenic variants and (b) to provide better tools for prognosis and treatment. Current studies utilize cells and whole animals, from flies to mammals. The mouse is an excellent model for exploring mechanisms of Chd7 function in the ear, given the evolutionary conservation of ear structure, function, Chd7 expression, and similarity of mutant phenotypes between mice and humans. Newly recognized developmental functions for mouse Chd7 are shedding light on how abnormalities in CHD7 might lead to CS symptoms in humans. Here we review known human inner ear phenotypes associated with CHD7 pathogenic variants and CS, summarize progress toward diagnosis and treatment of inner ear-related pathologies, and explore new avenues for treatment based on basic science discoveries.

An alteration in ELMOD3, an Arl2 GTPase-activating protein, is associated with hearing impairment in humans.

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.

Management of Pediatric Tracheocutaneous Fistulae.

Pediatric tracheocutaneous fistulae are best managed initially with secondary closure techniques. This procedure can be performed twice before significant further evaluation should be undertaken. Further studies are still needed with larger patient volumes to power management minutiae for pediatric tracheocutaneous fistulae.

Complications of Cholesteatoma.

Complications of cholesteatoma result from characteristic inflammatory and resorptive processes that erode the structures of the middle and inner ear with potential to spread locally. Common intratemporal complications include hearing loss, facial nerve palsy, labyrinthine fistula, and dysgeusia. Extratemporal complications, though less common, may be life-threatening, and include cerebrospinal fluid leak and encephalocele, meningitis, epidural and intraparenchymal abscesses, subdural empyema, and otic hydrocephalus.

Functional impairment of "helpless" CD8 + memory T cells is transient and driven by prolonged but finite cognate antigen presentation.

Generation of functional CD8 + T cell memory typically requires engagement of CD4 + T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (T E ) and subsequent memory (T M ) CD8 + T cell formation appear impervious to a lack of CD4 + T cell help during priming. Nonetheless, such "helpless" CD8 + T M respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8 + T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8 + T E differentiation is largely normal but a multiplicity of helpless CD8 T M defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8 + T M potential and recall capacity. Our findings provide a unified explanation for helpless CD8 + T cell memory and emphasize an unexpected CD8 + T M plasticity with implications for vaccination strategies and beyond.

HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

Homeostatic turnover of virus-specific memory CD8 T cells occurs stochastically and is independent of CD4 T cell help.

Memory CD8 T cells persist by Ag-independent homeostatic proliferation. To examine the dynamics of this cell turnover, we transferred lymphocytic choriomeningitis virus specific memory CD8 T cells into naive mice and analyzed their in vivo division kinetics longitudinally in individual recipients.Using mathematical modeling, we determined that proliferation of this stably maintained memory CD8 T cell population was homogeneous and stochastic with a small fraction of cells completing division at any given time with an intermitotic interval of 50 d. This homeostatic turnover was comparable between memory CD8 T cells of different viral epitope specificities and also the total memory phenotype (CD44(high)) CD8 T cells. It is well established that CD4 T cell help is critical for maintenance of CD8 T cells during chronic infections, but recent studies have suggested that CD4 T cell help is also required for maintenance of memory CD8 T cells following acute infections. Hence, we assessed the role of CD4 T cells in Ag-independent maintenance of memory CD8 T cells. Consistent with previous reports, we found that memory CD8 T cells declined when transferred into MHC class II-deficient mice. However, their numbers were maintained stably when transferred into CD4 T cell-deficient mice. Interestingly, their homeostatic proliferation, ability to make recall responses, and phenotype were independent of CD4 T cell help because none of these qualities were affected when memory CD8 T cells were transferred and maintained in either MHC class II- or CD4-deficient recipients.

Molecular and clinical studies of X-linked deafness among Pakistani families.

There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132 and PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild-to-profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling and molecular epidemiology of hearing loss among Pakistanis.

Ostial left main stenosis due to takayasu arteritis: multimodality imaging and surgical ostioplasty.

Takayasu's arteritis is an inflammatory process, involving larger blood vessels-namely the aorta and its branches. The majority of these patients present with symptoms of vascular obstruction. We report a case of a 38-year-old Chinese female who presented with one month history of angina pectoris as the initial manifestation. Coronary angiography showed 99% ostial left main coronary stenosis. The diagnosis was first suspected in the operating room by TEE and subsequently supported by laboratory studies and aortic biopsy. The technique of myocardial revascularization was altered and she underwent patch ostioplasty of the left main coronary artery and aortic valve repair to correct aortic regurgitation.

Congenital cytomegalovirus (CMV) for the pediatric otolaryngologist.

Cytomegalovirus (CMV) is a double-stranded DNA virus and a member of the herpesvirus family. It is the most common congenital viral infection. For symptomatic infections, symptoms can vary widely but tends to have a predilection for the central nervous system and for the reticuloendothelial system. Sensorineural hearing loss (SNHL) is by far the most common sequelae of congenital CMV infection. For this reason, it is imperative to understand the screening, diagnosis, and possible treatment options for congenital CMV induced SNHL. This literature review explores the association of CMV with hearing loss, screening for congenital CMV infections, possible treatments options, and the development of a possible vaccine.

CHARGE syndrome in the era of molecular diagnosis: Similar outcomes in those without coloboma or choanal atresia.

CHARGE syndrome (OMIM 214800) is a condition characterized by multisystem involvement with CHD7 pathogenic mutations leading to disease in the majority of patients. Discovery of the molecular cause of CHARGE unmasked a larger phenotypic spectrum than was previously appreciated. Within our interdisciplinary CHARGE syndrome program, we sought to characterize our CHD7-positive CHARGE cohort without coloboma or choanal atresia, highlighting complications and outcomes. We describe 18 individuals with CHD7-confirmed diagnosis from 15 families. The most sensitive finding in the cohort was temporal bone malformations, present in 13/15 individuals. Individuals had an average of 1.6 major features and 3.3 minor features defined by the Blake et al. guidelines. Despite lack of major features or major malformations, the majority of individuals continued to have difficulties with pneumonia, aspiration, secretion management and motility issues that greatly impacted their lives. Our findings illustrate the need for molecular testing and timely recognition given that the major co-morbidities are frequently experienced by patients with the mildest clinical spectrum of CHARGE syndrome.

International Pediatric Otolaryngology Group (IPOG) Consensus Recommendations: Congenital Cholesteatoma.

OBJECTIVE: To provide recommendations to otolaryngologists and allied physicians for the comprehensive management of children who present with signs and symptoms of congenital cholesteatoma. METHODS: A two-iterative Delphi method questionnaire was used to establish expert recommendations by the members of the International Pediatric Otolaryngology Group, on the preoperative work-up, the perioperative considerations, and follow-up. RESULTS: Twenty-two members completed the survey, in 14 tertiary-care center departments representing 5 countries. The main consensual recommendations were: a precise otoscopic description of the quadrants involved, extensive audiological workup (bilateral tonal, vocal audiometry, and BERA), and a CT scan are required. Facial nerve monitoring and a combination of microscope and telescope are recommended for surgical removal. Clinical and audiological follow-up should be pursued yearly for at least 5 years. First MRI follow-up should be done at 18 months postoperatively if the removal violated the matrix. MRI follow-up duration depends on the initial extent of the cholesteatoma. CONCLUSION: The goal of preoperative and follow-up consensus from International Pediatric Otolaryngology Group participants is to help manage infants and children with congenital cholesteatoma. The operative techniques may vary, and experienced surgeons must perform these procedures.

Advanced practice providers and children's hospital-based pediatric otolarynology practices.

INTRODUCTION: Advanced practice providers (APPs), including nurse practitioners and physician assistants, have been deployed in children's hospital-based academic pediatric otolaryngology practices for many years. However, this relationship in terms of prevalence, roles, financial consequences and satisfaction has not been examined. The objective of this study is to explore how APPs impact healthcare delivery in this setting. METHODS: Pediatric otolaryngology chiefs of all academic children's hospitals in the US were electronically surveyed about the ways APPs intersected clinically and financially in their respective practice. RESULTS: A total of 29 of 36 children's hospital-based pediatric otolaryngology practices completed the survey, of which 26 practices (90%) utilized APP. There were large variances within the APP practice cohort in faculty size (mean/median/range = 9.4/8.5/3-29); annual patient visits (mean/median = 18,373/17,600); number of practice site (mean/median/range = 4.3/4/2-9) and number of outpatient APP (mean/median/range = 6.3/5/1-30). No factors (faculty size, annual visits and number of practice sites) differentiated between the APP and non-APP practices. Among APP practices, significant correlation (p<.00001) was observed between size of APP cohort to faculty size and annual visits. 69% of the practices did not differentiate job functions of nurse practitioners and physician assistants. 85% of the practices utilized APPs in all practice sites and 19% utilized APPs in the operating room. 77% of APPs billed independently and 46% had on-site supervision. The most prevalent APP salary bracket based on 0-5, 6-10 and > 11 years of tenure were $76-100K (65%), $100-150K (77%) and $100-150K (86%), respectively. In 46% of the practices, APPs were able to generate enough revenue to cover more than 75% of their salary and 23% of practices generated a profit. 81% of the chiefs ranked the effectiveness of APPs as high (4 and 5) on a 5-point Likert scale. DISCUSSION: The majority of academic pediatric otolaryngology practices employed APPs. Despite the diversity seen in practice complexity, APP functionality and financial impact, most found the APP model to be beneficial in improving patient care, patient access and faculty productivity.

Surgical management of otologic disease in pediatric patients with Turner syndrome.

OBJECTIVES: Previous reports suggest that patients with Turner syndrome have a predisposition for acute and chronic otitis media. However, the role of early or aggressive surgical management of otologic disease has not been explored in the pediatric population. With respect to otitis media in pediatric Turner syndrome patients, we examined (1) the impact of timing and (2) the type of surgical intervention in the treatment of disease. METHODS: Retrospective 10-year review of patients with Turner syndrome and chronic otitis and its surgical management at a single pediatric tertiary institution. RESULTS: One hundred and seventy-eight patients with Turner syndrome were evaluated at our institution from 1997 to 2007. Thirty-two (18.0%) were diagnosed with middle ear disease. Eighteen (10.1%) were referred to otolaryngology for evaluation. Average age at presentation was 4.7 years (range: 1 month to 12 years). The 18 patients referred to otolaryngology required a mean of 16 clinic visits each for otologic symptoms. A mean of 6.7 pressure equalization tubes (PET) were required per patient (range: 0-25). Middle ear effusions (n=14, 78%) along with tympanic membrane retractions and/or perforations (n=10, 55.6%) were the most common otoscopic findings. Patients with tympanic membrane retractions (8/18) required a higher average number of PET (9.1) and cumulatively underwent a total of five tympanoplasty-type procedures. Six ears had evidence of cholesteatoma. Two patients underwent myringoplasty, 6 patients underwent tympanoplasty (33.3%, mean age 11.6 years), and 3 patients (16.7%, mean age 9.4 years) underwent tympanomastoidectomy. Revision procedures were common. Older age at first PET placement was significantly correlated with the need for later tympanoplasty and/or tympanomastoidectomy operations (p<0.036). Tympanoplasty or tympanomastoidectomy patients had their first PET placed on average at 5.2 years as compared to 2.6 years in those not requiring tympanoplasty or tympanomastoidectomy operations. CONCLUSIONS: Recurrent and chronic otitis media is common in patients with Turner syndrome. Once established, disease is recalcitrant and leads to multiple surgical procedures. Early PET insertion is advocated to offset the future necessity of more extensive tympanic procedures.

Relative Preservation of Superior Semicircular Canal Architecture in CHARGE Syndrome.

OBJECTIVES: (1) Describe common patterns of semicircular canal (SCC) anomalies in CHARGE syndrome (CS) and (2) recognize that in CS, the architecture of the superior SCC may be relatively preserved. STUDY DESIGN: This is a retrospective review of temporal bone imaging studies. SETTING: Quaternary care center. SUBJECTS AND METHODS: A sample of 37 patients with CS. All subjects met clinical diagnostic criteria for CS. The presence/absence of anomalies of the middle ear, mastoid, temporal bone venous anatomy, inner ear, and internal auditory canal was recorded. Anomalies of each SCC were considered separately and by severity (normal, dysplasia, aplasia). RESULTS: Thirty-seven subjects (74 temporal bones) were reviewed. Thirty-four (92.0%) patients demonstrated bilateral SCC anomalies. Three (8.0%) had normal SCCs. In patients with SCC anomalies, all canals demonstrated bilateral abnormalities. Thirty-two (86.5%) patients had bilateral horizontal SCC aplasia. These 32 patients also demonstrated posterior SCC aplasia in at least 1 ear. Of 74 temporal bones, 37 (50.0%) had superior SCC dysplasia. All dysplastic superior SCCs showed preservation of the anterior limb. Complete superior SCC aplasia was found in 28 (37.8%) temporal bones. CONCLUSION: SCC anomalies occur with high frequency in CS. Complete absence of the horizontal and posterior canals is typical and usually bilateral. By contrast, the superior SCC often demonstrates relative preservation of the anterior limb.

Dystrophic epidermolysis bullosa with one dominant and one recessive mutation of the COL7A1 gene in a child with deafness.

Dystrophic epidermolysis bullosa can be inherited in autosomal dominant and recessive forms, the former usually expressed as a milder phenotype, although mild forms of recessive dystrophic epidermolysis bullosa can occur. We present a patient who was found to be a compound heterozygote, inheriting a dominant mutation from his father and a recessive mutation from his mother, resulting in a clinically severe case of dystrophic epidermolysis bullosa. Mutations in the gene for collagen VII (COL7A1) have been documented in both types of dystrophic epidermolysis bullosa. Our patient has also been diagnosed with bilateral auditory neuropathy, a disorder coincidentally also mapped to a nearby gene on chromosome 3p21 (the transmembrane inner ear expressed gene, TMIE).

Auditory skills development among children with developmental delays and cochlear implants.

OBJECTIVES: We sought to understand auditory skills outcomes in young children with cochlear implants and developmental delay. METHODS: Children who received cochlear implants at less than 36 months of age were identified via chart review. Their postimplant auditory skills outcomes were measured with the Auditory Skills Checklist. RESULTS: Of 35 children who received cochlear implants before the age of 36 months, 14 children (40%) had additional disabilities or some form of developmental delay. The 12-month postimplant data indicated progress in all groups of children. Children with additional disabilities had the same rate of auditory skills progress as children with no additional disabilities (beta = 9.3 versus 9.3; p = 0.5). However, the children with additional disabilities tended to start at a lower baseline skills set (approximately 6 points lower) on the Auditory Skills Checklist. For children with average developmental quotients (at least 80), the rate of progress was twice that of children with a developmental quotient of less than 80, irrespective of a developmental disability (beta = 9.9 versus 4.8; p = 0.03). Children with a developmental quotient of less than 80 were less likely to gain skills in discrimination and identification after the first postimplant year. CONCLUSIONS: Children with additional disabilities make progress in auditory skills, but may not develop higher auditory skills of identification and comprehension within the first 6 months after implantation. Categorizing children according to a cognitive developmental quotient may provide more predictive ability than does categorizing them by disability type.

Pediatric hearing impairment caregiver experience: impact of duration of hearing loss on parental stress.

OBJECTIVES: Caregivers of children who are deaf/hard of hearing have been reported to have greater stress than caregivers of children with normal hearing. The time of diagnosis is a particularly stressful time and stress levels may change over time based on varying needs at different life events. Thus, we hypothesized that stress experienced by caregivers evolves over time and is impacted by the duration since the diagnosis of hearing loss. METHODS: The 68-item pediatric hearing impairment caregiver experience (PHICE) is a validated questionnaire used to measure stress. The PHICE was administered to 152 caregivers of children with permanent hearing loss. Domain scores were converted into z-scores for analysis of trends of stress over time. RESULTS: Parents of children whose hearing loss was identified more than 60 months ago reported higher stress levels regarding educational aspects of their child's needs as compared to parents of children with less than 24 months or 24-60 months duration since diagnosis. Parents of children diagnosed with hearing loss within the preceding 24 months reported higher stress levels in the area of healthcare than parents of children diagnosed greater than 24 months ago. CONCLUSIONS: Parental stressors change over time with respect to the time of diagnosis of hearing impairment. This phenomenon was observed irrespective of the age of diagnosis of hearing loss. As professionals serving families of children with hearing loss, we should be aware of changing stressors over time and identify the appropriate support services for families to meet those changing needs. By addressing those evolving stressors, the families' ability to support and improve the outcomes for their children who are deaf or hard of hearing may be enhanced.

Language Underperformance in Young Children Who Are Deaf or Hard-of-Hearing: Are the Expectations Too Low?

OBJECTIVE: (1) To examine language performance in the context of cognitive abilities in young children who are deaf or hard-of-hearing and (2) to identify factors associated with having a language underperformance, defined as a gap between the language standard score and the nonverbal IQ (NVIQ) standard score. METHODS: Children 6 to 82 months of age with bilateral hearing loss were enrolled. Language performance was defined as a ratio of language skills relative to cognitive abilities with language underperformance defined as a ratio of language scores to NVIQ <0.85. RESULTS: Among 149 children, approximately half had hearing loss that was clinically classified as mild or moderate, and over one-third received a cochlear implant. Participants had a mean NVIQ in the average range (95.4 [20.3]). Receptive language scores were significantly lower than their NVIQ by 10.6 points (p < .0001). Among children with NVIQs 80 to 100, 62.5% had receptive scores <85 and 50% had a language underperformance (ratio <0.85). Among children with NIVQs >100, 21.1% had receptive scores <85 with 42% having a language underperformance. Children with language underperformance (n = 61, 41.5%) were more likely to have more severe levels of hearing loss, lower socioeconomic status, and be nonwhite. CONCLUSION: Many children early identified with hearing loss continue to demonstrate language underperformance, defined using their cognitive potential. Language deficits have a cascading effect on social functioning in children who are deaf or hard-of-hearing. This study highlights the need to understand a child's cognitive potential to adequately address language needs in existing intervention models.

Pediatric acute mastoiditis in the era of pneumococcal vaccination.

OBJECTIVES/HYPOTHESIS: The objective was to describe trends in the annual prevalence of hospitalization for pediatric acute mastoiditis since introduction of the 7-valent pneumococcal vaccine in 2000 and the 13-valent vaccine in 2010. STUDY DESIGN: Cross-sectional retrospective data analysis. METHODS: The Kids' Inpatient Database from years 2000 to 2012 was analyzed. To determine the annual prevalence of hospitalization for acute mastoiditis, nationally weighted frequencies of hospitalization for children <21 years with acute mastoiditis diagnoses were collected. Trend analysis of hospitalization rates from 2000 to -2012 was performed. RESULTS: From 2000 to 2012, there was no significant trend in hospitalization rates for acute mastoiditis overall (1.38 and 1.43 per 100,000 persons in 2000 and 2012, respectively; P = .86) or by age group. When comparing hospitalization rates at time points 2000 and 2012, children <1 year (4.65 and 3.27 per 100,000 persons, P = .0023) and 1 to 2 years of age (3.95 and 3.18 per 100,000 persons, respectively; P = .0107) demonstrated declines in hospitalization over time. Between 2009 and 2012, hospitalization rates also significantly declined for children aged <1 year (4.50 to 3.27 per 100,000 persons, P = .0056) and 1 to 2 years (4.30 to 3.18 per 100,000 persons, P = .0002) but increased for children 5 to 9 years (1.10 to 1.81 per 100,000 persons, P < .0001) and 10 to 20 years of age (0.41 to 0.72 per 100,000 persons, P < .0001). CONCLUSIONS: Despite introduction of two pneumococcal vaccines, rates of hospitalization for pediatric acute mastoiditis did not decline between 2000 and 2012. Between 2009 and 2012, however, children 0 to 2 years of age showed declining hospitalization rates, possibly reflecting the protective benefit of the 13-valent pneumococcal vaccine. LEVEL OF EVIDENCE: 4. Laryngoscope, 128:1480-1485, 2018.