Totally implantable active middle ear implants (AMEI) provide full-time hearing amplification to those with moderate to severe sensorineural hearing loss. While technology in conventional hearing aids (CHA) has advanced greatly, limitations remain for people with active lifestyles, limited vision or dexterity, and hearing aid fit issues. Furthermore, direct-drive properties of AMEI are thought to provide those with inefficient middle ear transfer functions a distinct advantage in delivering prescribed sound to the cochlea, ultimately improving speech understanding with less distortion. AMEI safety, stability, and efficacy outcomes are well documented and fitting strategies continue to improve. Recent studies show how simple aided speech testing can help predict whether a patient struggling with CHA may instead benefit from an AMEI. Totally implantable AMEI continue to be a viable option for patients who cannot or will not utilize traditional hearing aids. Howevering aids  and rateing aids . Of thosing aids . SeveralFit: discomfort, otitis externa, occlusion effect, poor retention.Use: limited vision, dexterity, self-efficacy, lost devices, frequent battery changes.Lifestyle: Removal for sleep, water wear, athletic endeavors with heavy perspiration.Concurrently, risks of untreated hearing loss are becoming clearer. Accelerated cognitive decline and dementia , increasActive middle ear implants (AMEI) were developed to provide solutions to the above CHA limitations. While four AMEI are commercially available worldwide, only two are totally implantable. This mini review will report the long term device outcomes and updated candidacy considerations for one of these totally implantable AMEI systems.® device, which underwent clinical trials starting in 2000. European CU mark cleared in 2006 and the device was FDA approved in 2010. Uniquely, this AMEI utilizes the tympanic membrane as the natural hearing aid microphone. A piezoelectric sensor on the incus sends ossicular vibration information to a sound processor/battery combination unit, implanted subcutaneously in the postauricular region. The filtered and amplified signal is delivered back to a driver connected to the stapes capitulum, which directly drives the oval window for transduction into the cochlea. This direct drive system contrasts traditional CHA mechanics, which do not account for individual variance in middle ear mechanics affecting the efficiency with which sound reaches the cochlea.The Envoy Medical Corporation, originally called St. Croix Medical, was founded in 1995 in St. Paul, MN, quickly laying groundwork for a totally implantable hearing aid. What developed was the Envoy EsteemOngoing device maintenance includes an outpatient surgery on average every 4.9 years  to replaAdults with moderate to severe sensorineural hearing loss in one or both ears, ≥40% maximum word understanding, and some experience with CHA are the best candidates for this device. AMEI is not a replacement for those with severe to profound hearing loss needing cochlear implant(s). Additional considerations for the Esteem are anatomical and surgical; Imaging is needed to determine if there is ample middle ear space to insert the device and medical consultation is required to ensure patients are healthy enough to undergo an outpatient surgery lasting on average 4.5 h . ContraiAfter establishing audiologic and radiologic candidacy, patients are counseled extensively as to expectations of hearing with a middle ear implant. Existing limitations of current hearing technologies to fully rehabilitate hearing loss must be impressed on the patient who needs to understand that their hearing will not be “normal” and that they will continue to experience difficulties in certain listening situations. The goal of facilitating hearing rehabilitation without an external device and the conveniences, and the conveyed lifestyle benefits  are commThe Esteem surgical procedure is planned as an outpatient procedure unless medically indicated to observe the patient in hospital postoperatively. It requires unique pieces of equipment including a second microscope for laser doppler vibrometry measurements of ossicular movement, a cutting laser and an intracanal microphone assembly to facilitate intraoperative testing. The surgery requires many steps, each of which builds upon earlier ones such that sufficient achievement of each step is required before proceeding. A field clinical engineer performs intraoperative measurements and provides feedback to the surgeon at various steps of the procedure to ensure system integrity. Failure to achieve adequate exposure or transducer alignment can add considerable surgical time. Indeed, a surgeon's first implants often take 6–8 h  requirinThe surgery is done through a postauricular incision large enough to accommodate the amply sized battery/sound processor (SP). A bony trough is drilled through the outer occipitoparietal skull cortex for the SP. A sufficient mastoidectomy is performed to allow the body of the transducers to reside within the mastoid cavity. Adequate exposure requires that bone lateral to the body of the incus is removed and a posterior tympanotomy through the facial recess is performed to facilitate exposure of the lenticular process of the incus and as much of the stapes as is possible to visualize given the anatomic restrictions of the facial nerve and fibrous tympanic annulus.After ensuring adequate native ossicular movement with laser doppler vibrometry, the incudostapedial joint is disarticulated and the lenticular process of the incus is removed with a laser. The transducers are affixed to the skull using flexible stabilizing bars that facilitate adequate positioning of the sensor transducer with the tip over the incus body and the driver transducer tip to the stapes capitulum. The transducer bodies are affixed to the mastoid using a hydroxyapatite cement and the tips of the transducers are attached to the ossicles using a glass ionomer cement . MeasureOngoing data collection pre and post FDA approval has shown device longevity and programmability over time for proper candidates –16. BanaData from 231 ears among these four studies along with extensive phase I and II clinical trial efficacy data , 18 demoWith a wide candidacy range and variable outcomes, it is reasonable to ask if metrics are available to help predict which patients have the most to gain by changing from CHA to AMEI. One metric that has been proposed as predictive of AMEI advantage is aptly named the speech perception gap (SPgap) by Dyer et al.  or earphIn the Shohet et al.  retrospeThe SPgap metric seems to be sensitive to distinct AMEIs audibility advantages, so it is gaining popularity amongst hearing implant manufacturers who argue they can close this performance gap using direct drive properties. It may also be useful to clinicians who want a simple yet sensitive test to identify good audiometric AMEI candidates.Initially, gain prescription for the Esteem required some trial and error, estimating input gain to achieve a certain amount of output aided gain measured in soundfield. Sound processing options have improved since the first generation Esteem setup, and Envoy describes its current sound processing as hybrid: analog signal processing controlled digitally. Also, in 2014 the prescriptive algorithm called SoundFit became available to help clinicians better prescribe Esteem gain based on baseline audiograms. This statistical NAL-RP based formula was developed using data from 540 users' profile settings and correlating gain outcomes. From this prescriptive starting point, clinicians can now fine-tune different program options into the Esteem, just like a CHA. Software developments have also allowed for more transparent device diagnostic testing and parameter programming over time.Widespread adoption of middle ear implants in the United States has met many hurdles some of which have severely limited their adoption. First, and most impactful, has been the absence of Currently Procedural Terminology (CPT) coding that appropriately describes the services being provided which has prevented payment considerations by Centers for Medicare and Medicaid Services (CMS) as well as commercial payers. Second, with limited third-party reimbursement, many surgeons have given up on implanting or even offering these devices to their patients due to device unfamiliarity and discomfort in performing the complex surgical procedure. Lastly, as CHA sound processing technology advances, the perceived sound advantage of direct drive technology over conventional acoustic stimulation through the ear canal may become less perceivable.Standardize audiometric test metrics pre and post intervention to allow meta-analyses of AMEI compared to CHA. Individual clinics may also use these metrics to help identify good AMEI candidates and change treatment course if CHA are not effective.Develop a validated questionnaire for assessing AMEI hearing aid lifestyle benefit. Amongst the many benefits of fully implantable AMEI, freedom from daily device maintenance and confidence gained from 24/7 hearing is described empirically but not measured.Determine if direct drive AMEIs are a suitable, less invasive alternative to hybrid cochlear implants.Re-examine if digital processing mechanisms could be supported by today's implantable battery technology.Compare modern extended bandwidth hearing devices to AMEI output. While AMEI provided more gain than open-fit CHA in the past , 26, thiConsider integrating SPgap testing into standard hearing aid care to help identify patients that may be better served with an AMEI.Envoy Esteem totally implantable AMEI device stability and clinical outcomes have been verified through clinical trials and independent analyses for over 20 years to date. Results consistently show aided hearing thresholds similar to and often better than CHA with notable advantages in mid-frequency gain and superior speech understanding at conversational levels. AMEI should be discussed as an alternative treatment to CHA for proper candidates during hearing device consultations. A simple speech testing metric called the SPgap can be incorporated into busy clinics to help identify good audiometric candidates in addition to those who simply cannot or will not wear CHA. While questionnaires show overall satisfaction with Esteem vs. CHA, opportunities remain to further illuminate why patients prefer the lifestyle advantage of a fully implantable AMEI and “absence of a daily reminder of the disability” (Barbara) .JS and JB contributed to conception and design of the study. JB wrote the clinical aspects and JS the surgical details of the manuscript. Both authors contributed to manuscript revision, read, and approved the submitted version.JS is a member of the Envoy Medical Advisory Board. None of the authors has a financial relationship with Envoy Medical. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rousettus aegyptiacus), raising the question of whether this bat species is a suitable host for IAV. Here, we studied the susceptibility, pathogenesis and transmission of avian and bat-related H9N2 viruses in this new host. In a first experiment, we oronasally inoculated six Egyptian fruit bats with an avian-related H9N2 virus (A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2)). In a second experiment, six Egyptian fruit bats were inoculated with the newly discovered bat-related H9N2 virus (A/bat/Egypt/381OP/2017 (H9N2)). While R. aegyptiacus turned out to be refractory to an infection with H9N2 avian-type, inoculation with the bat H9N2 subtype established a productive infection in all inoculated animals with a detectable seroconversion at day 21 post-infection. In conclusion, Egyptian fruit bats are most likely not susceptible to the avian H9N2 subtype, but can be infected with fruit bat-derived H9N2. H9-specific sero-reactivities in fruit bats in the field are therefore more likely the result of contact with a bat-adapted H9N2 strain.Influenza A viruses (IAV) of subtype H9N2, endemic in world-wide poultry holdings, are reported to cause spill-over infections to pigs and humans and have also contributed substantially to recent reassortment-derived pre-pandemic zoonotic viruses of concern, such as the Asian H7N9 viruses. Recently, a H9N2 bat influenza A virus was found in Egyptian fruit bats ( Low-pathogenic avian influenza viruses (LPAIV) of subtype H9N2 are globally widespread in poultry and endemic in China and Eurasian countries ,2. WhileSturnira lilium) and a South American flat-faced fruit-eating bat (Artibeus planirostris), respectively [Eidolon helvum) in Ghana [Rousettus aegyptiacus) in Egypt. Interestingly, the hemagglutinin (HA) and neuraminidase (NA) encoding segments of this particular virus were closely related to avian H9N2-viruses [The first evidence of influenza A and influenza A-like viruses originating from different bat species was found in 2012 and 2013: two novel influenza A-like viruses, provisionally designated as H17N10 and H18N11, were identified via next-generation sequencing in the feces of a little yellow-shouldered fruit bat  with either a poultry-origin H9N2 strain of clade G1 (A/layer chicken/Bangladesh/VP02-plaque/2016) or the fruit bat-derived strain A/bat/Egypt/381OP/2017 (H9N2). The latter group was cohoused with naïve contact bats (24 h post-infection) to further elucidate H9N2 bat flu virus transmission.In order to specifically investigate susceptibility, pathogenesis and virus shedding, we oronasally infected two groups of Egyptian fruit bats , was obtained from the Friedrich-Loeffler-Institut (FLI) virus repository [Rousettus aegyptiacus) from both experiments were reared at FLI and tested negative for influenza A reactive antibodies via immunofluorescence analysis (IFA) using the inoculation viruses. Six bats in each experiment were anesthetized by inhalation of 5% isoflurane in O2 and oronasally inoculated with 100 µL H9N2 (A/layer chicken/Bangladesh/VP02-plaque/2016) distributed equally into each nostril and mouth at a concentration of 104.7 TCID50 per animal   , respect7 (H9N2) . The six7 (H9N2)  were co-Organ pieces (approximately 2 × 2 × 2 mm) were transferred into 2 mL collection tubes prepared with a stainless-steel bead (diameter 5 mm) and 1 mL of DMEM supplemented with antibiotics . Homogenization was performed using a TissueLyser II instrument  for 2 min at 300 Hz. Supernatants for RNA extractions were acquired following centrifugation at 13,000 rpm for 2 min.RNA extraction of all oral and rectal swabs, as well as organ samples after euthanasia from both experiments was performed via the NucleoMag Vet kit  according to manufacturer’s instructions on a Biosprint 96 platform .RT-qPCRs of organs, oral and rectal swabs in the first experiment (avian origin H9N2) were performed as described before .In order to detect A/bat/Egypt/381OP/2017 (H9N2) viral RNA in the second experiment, a specific primer and probe system was designed . For proBased on RT-qPCR data, selected organs from bats sacrificed at 7 dpi were trimmed for paraffin-embedding: nasal atrium (non-respiratory region), nasal conchae (respiratory and olfactory region), duodenum, jejunum, colon and rectum of inoculated #5 and #6, as well as transmission #11 and #12. Tissue sections were stained with hematoxylin and eosin (HE). Immunohistochemistry for viral antigen detection using a primary antibody against the M protein of IAV (ATCC clone HB-64) was performed on consecutive slides as described earlier . The sevAnimal samples were initially diluted in medium , whereupon 200 µL was transferred into the allantoic cavity of embryonated chicken eggs (three eggs per sample) followed by incubation for five days at 37 °C. Afterwards, the eggs were placed at −80 °C for 30 min to ensure the death of the chicken embryo, whereupon the egg was opened to allow harvesting of the allantoic fluid. The fluid was centrifuged at 3500 rpm for 10 min, followed by a TRIzol LS reagent -based RNA extraction (according to manufacturer’s instructions). Existence of A/bat/Egypt/381OP/2017 (H9N2) viral RNA and thereby evidence for successful propagation of A/bat/Egypt/381OP/2017 (H9N2) was finally verified via RT-qPCR as described before.3.3 TCID50/mL was mixed with the same volume of diluted sera. Each serum was prepared in triplicate in a 96-well plate. After incubation for 2 h at 37 °C, the samples were transferred into a second 96-well plate prepared 24 h earlier containing 100 µL medium on 24 h grown MDCK II cells. Viral replication was assessed after an incubation period of 5 days  via visualization of the cytopathic effect. Validation was achieved by titration of the virus dilutions. In the second experiment, seroconversion was investigated via IFA as well. For that reason, MDCK cells were seeded in a 96-well plate and were grown for 24 h. Afterwards, cells were infected with A/bat/Egypt/381OP/2017 (H9N2) for 24 h followed by cell fixation with 4% paraformaldehyde (PFA), as well as permeabilization with 0.5% Triton-X-100 in PBS. Cells were then stained for 1 h with a dilution series from 1:10–1:256 of the appropriate animal serum, followed by three PBS-washing steps and the incubation with a secondary goat-α-bat IgG (H + L) antibody  for 1 h. Subsequently, cells were washed three times with PBS again, followed by the addition of an Alexa Fluor 488-coupled chicken-α-goat IgG (H + L) third antibody for 1 h to allow visualization under the fluorescence microscope after a final three-times PBS-wash procedure.All serum samples were heat-inactivated at 56 °C for 30 min and animal sera from both experiments were analyzed by a commercial enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against the IAV nucleoprotein (NP) according to manufacturer’s instructions . In addition, a virus neutralization assay was performed in the first experiment. In brief, 50 µL of medium containing H9N2 (A/layer chicken/Bangladesh/VP02-plaque /2016) at a concentration of 10Rousettus aegyptiacus) with 104.7 TCID50 per animal of A/layer chicken/Bangladesh/VP02-plaque /2016 (H9N2), neither clinical signs nor any significant changes in body weight or body temperature could be observed during the experiment. In addition, no viral RNA was detected in organs, oral or rectal swabs. Samples below 10 viral genome copy numbers per mL  were considered negative. At necropsy, no macroscopic lesions were evident. Analysis of sera until 21 dpi by IAV-antibody-ELISA and serum neutralization assay revealed no evidence of seroconversion. Taken together, these observations strongly suggest that this H9N2 subtype of avian origin fails to replicate in Egyptian fruit bats.Following oronasal inoculation of Egyptian fruit bats  per animal of A/bat/Egypt/381OP/2017 (H9N2) established a productive infection in these animals. At 1 dpi, one out of six oral swabs, as well as two rectal swabs from the inoculated animals, were positive for the H9N2 bat flu viral genome using RT-qPCR  viral RNA.Fecal samples of animal cage one were positive for A/bat/Egypt/381OP/2017 (H9N2) viral RNA at 4 and 7 dpi and samples from cage two were positive at 3, 4, 6 and 8 dpi, with maximum viral loads of 4 × 10Histopathology identified an oligo-focal, minimal to mild rhinitis in all animals evaluated at 7 dpi  and 6 days post-contact . Additionally, inoculated animal #6 showed a moderate transmigration of granulocytes and lymphocytes as well as intraluminal cellular debris within the nasolacrimal duct A. ContacSince it has been shown previously that A/bat/Egypt/381OP/2017 (H9N2) efficiently replicates in embryonated chicken eggs , we atteAs shown in As shown by an indirect immunofluorescence assay, all directly inoculated animals sacrificed at day 21 had seroconverted against A/bat/Egypt/381OP/2017 (H9N2), whereas all contact animals remained seronegative . InteresEidolon helvum) [Rousettus aegyptiacus). In addition, the infection dose of 104.7 TCID50 per animal used in this experimental inoculation could be another factor, potentially in combination with the application route. However, oral swabs taken from naturally infected Egyptian fruit bats were more likely to be tested positive for viral RNA than rectal swabs, highlighting that oral epithelial cells of Egyptian fruit bats are able to replicate IAV [Our study shows for the first time that oronasal inoculation using LPAIV of subtype H9N2 (A/layer chicken/Bangladesh/VP02-plaque/2016) does not result in any virus replication or specific seroconversion in an experimental setup with Egyptian fruit bats. This suggests that these fruit bats are not susceptible to an avian-origin H9N2 influenza A virus infection. This was of special interest since there was, contrastingly, a clear indication of H9-specific antibodies in African fruit bats . Therefo helvum) , this obcate IAV .Despite the fact that bat borne H9N2 showed strong genetic similarities to avian H9N2 viruses, the phylogeny revealed that in contrast to the HA and NA, all inner segments were very divergent from the recent AIV ,14. TherOverall, although inoculation of Egyptian fruit bats with A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2) did not result in a productive infection of these animals, inoculation and thereby infection of the same species with A/bat/Egypt/381OP/2017 (H9N2) was successful. First indications for a successful infection were detected by a permanent temperature monitoring of four infected and four contact animals, which only showed subtle differences between both groups, that would have been unnoticed by monitoring of the rectal temperature alone. Therefore, monitoring of the body core temperature independent of any manipulation of the animal is important for challenge experiments where only mild or even no clinical signs are to be expected in the infected animals. Although we were not able to detect viral RNA related to A/bat/Egypt/381OP/2017 (H9N2) in oral/rectal swabs and organ samples from the direct contact animals, we demonstrated mucosal epithelium degeneration via histopathologic analysis in contact animal #6 , indicatIn addition to virus isolation and propagation, we observed that all animals directly inoculated with A/bat/Egypt/381OP/2017 (H9N2) and euthanized at 21 dpi displayed seroconversion. Although we could not demonstrate threshold-exceeding seropositivity of all animals in the ELISA that were seropositive in the IFT, we nevertheless saw obvious differences in the data values of these animals compared to the naïve animals . Taken tRecently published observations concerning the replication of another bat influenza A virus, H18N11, in various animal models proved that H18N11 is being transmitted among and is able to replicate in bats, whereas replication in mice and ferrets was only weakly detectable . Thus, bThis hypothesis could thereby explain why the same species of flying foxes are susceptible to infection with A/bat/Egypt/381OP/2017 (H9N2), but not to A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2). However, due to the rapid adaptation processes of influenza viruses in general, the zoonotic potential of these viruses should not be underestimated and cannot be excluded without further experiments. Therefore, future studies will focus in more detail on the different influenza virus infections of fruit bats and the necessary adaptation processes. Our study provided first insights into the host range of different H9N2 viruses and the adaptation of bat-origin H9N2 to Egyptian fruit bats. Future studies will focus on the minimum infectious dose or the reactivity of the host following bat-origin H9N2 infection.


GJB2 gene. However, patients carrying only one heterozygous pathogenic  GJB2 variant represent a long-lasting diagnostic problem. Interestingly, previous results showed that individuals with a heterozygous pathogenic GJB2 variant are two times more prevalent among those with hearing loss compared to normal-hearing individuals. This excess among patients led us to hypothesize that there could be another pathogenic variant in the GJB2 region/DFNB1 locus. A hitherto undiscovered variant could, in part, explain the cause of hearing loss in patients and would mean reclassifying them as patients with GJB2 biallelic pathogenic variants. In order to detect an unknown causal variant, we examined 28 patients using NGS with probes that continuously cover the 0.4 Mb in the DFNB1 region. An additional 49 patients were examined by WES to uncover only carriers. We did not reveal a second pathogenic variant in the DFNB1 region. However, in 19% of the WES-examined patients, the cause of hearing loss was found to be in genes other than the GJB2. We present evidence to show that a substantial number of patients are carriers of the GJB2 pathogenic variant, albeit only by chance.Hearing loss is a genetically heterogeneous sensory defect, and the frequent causes are biallelic pathogenic variants in the  GJB2 gene (OMIM 121011), causing the DFNB1 type of deafness [GJB2 variant. Additionally, in 2014, Chan et al. published a meta-analysis of 48 case-control studies [GJB2 variant  among patients with hearing loss is twice as high when compared to the normal-hearing population.In neonates, hearing loss is the second most common sensory deficit after color blindness, with an incidence of 1:500–1000 . In addideafness . HoweverGJB2 variants (3). Furthermore, in our previous study, we showed that the frequency of c.35delG heterozygotes in Czech patients with hearing loss is 4%, and the figure is twice as high when compared to a frequency of 1.8% in normal-hearing persons with the monoallelic (heterozygous) c.35delG variant, as presented in the public database GnomAD for the non-Finnish European (NFE) population [The most common cause of hearing loss detected worldwide is the c.35delG pathogenic variant. This variant has frequently been identified in European, North African, Middle Eastern, Asian, and American populations [pulation ,7.GJB2 variant, presumably a small copy number variant (CNV), to compensate for the frequency difference. A similar idea was suggested by del Castillo et al., who discovered a large deletion outside the GJB2 region. The deletion, del(GJB6-D13S1830), is frequent among patients in Spain, France, the United Kingdom, Israel, and Brazil, and could explain the hearing loss in a large number of patients with a heterozygous pathogenic GJB2 variant [Therefore, we hypothesized that some of the patients may be explained by a combination of known monoallelic and some novel pathogenic  variant . Neverth variant .GJB2 gene, an intersection of five of these deletions displays a common interval; this is where the cis regulatory element is probably located, which is important for the regulation of GJB2 gene expression  region have been reported worldwide. In the region outside of the pression  [9,11,12-q12 regie per se ,16,17,18interval . For theGJB2 variant by chance, and that the cause of their hearing loss is on a gene other than the GJB2. Therefore, we performed whole exome sequencing (WES) of 49 patients, 17 of whom we examined in both parts of the project.Our study comprises two parts: The first part examined the second expected pathogenic variant in the DFNB1 (13q11-q12) region, and for that we initially examined 28 patients by a continuous NGS of this region, which covered 0.4 Mb. The comprehensive NGS data analysis included the CNV analysis that used different software to detect the possible deletion as well as the presence of small-scale variants. In the second part, we supposed that a number of patients are only carriers of one heterozygous pathogenic GJB2 testing . Patients’ parents had normal hearing, and an autosomal recessive mode of inheritance was considered the most probable cause. The GJB2 gene (NM_004004.5 coding exon 2 and non-coding exon 1) was examined before the study, and patients with only one heterozygous  pathogenic variant were selected. Prior to the study, the large deletions, del(GJB6D13S1854) and del(GJB6D13S1830), were excluded with PCR or MLPA. The ethics committees of the University Hospital Motol, Prague, Czech Republic and the University Hospital, Bratislava, Slovakia approved the study.The patients selected for this study came from two laboratories dedicated to the genetic testing of hearing loss: the University Hospital Motol in the Czech Republic and the Biomedical Research Center in Slovakia. Patients in whom the non-genetic causes of hearing loss were excluded  were referred by geneticists in order to be investigated for a genetic cause of hearing loss. All patients were unrelated, and the majority of them presented with early/prelingual hearing loss and fulfilled the criteria for GJB2 variant and three, that were homozygotes for the c.35delG  variant. These homozygotes, that carry the biallelic pathogenic GJB2 variant, were used as “negative” controls since it was expected that they would not carry the unknown mutation.For the examination of the DFNB1 (13q11-q12) region, a custom-design SureSelect Target Enrichment kit  was used to continuously cover this region (hg19 reference genome–chr13: 20 697 953-21 105 890). We examined the genomic DNA of 31 Czech patients from which 28 were heterozygotes for pathogenic GJB2 region (chr13: 20 698 553–20 871 892). The WES of 49 patients (29 Czech and 20 Slovak patients) was examined with a Human All Exon V6 kit . Seventeen of the 49 patients were analyzed previously for the DFNB1 (13q11-q12) region.For the haplotype analysis, 29 SNPs from the NGS data of the DFNB1 (13q11-q12) region were selected for the assembly of the haplotype for the Both libraries were sequenced on Hi-Seq platforms . The small sequence changes (single-nucleotide variants and indels) in the NGS data were analyzed with the SureCall  and Genome Analysis Toolkit (GATK) software. Only variants with a frequency of <1.0% in public databases  were evaluated. The pathogenicity of the variants was evaluated according to the following criteria: ACMG criteria modified for genetic hearing loss , previouThe larger sequence variants (CNVs) in the NGS data were analyzed differently for the DFNB1 region, and the WES was based on our own software testing for suitability to each platform. The copy number variant (CNV) analysis of the DFNB1 region was performed with NextGene  and Meta-SV analyses. The Meta-SV analysis tool  was usedhttps://github.com/broadinstitute/gatk/tree/master/scripts/cnv_wdl/germline, accessed on 9 September 2020) in two steps. In the first step, a reference model was prepared from 40 healthy control WES samples (BAMs). In the next step, the reference model was used to perform CNV calling in a cohort of patients (BAMs).The copy number variant (CNV) analysis of the WES was performed with GATK Best Practices for Germline Copy Number Variation . The primers used for the verification of the deletion were placed at a sufficient distance (around 200 bp) from the deletions’ start/end in order to prevent possible allele drop out when the primer, or its part, was placed in the deleted region. Where possible, a segregation analysis of the detected variants was conducted within the family. A deletion larger than 3000 bp was verified by quantitative–comparative fluorescent PCR (QCF PCR) and used to compare a fragment from the deleted region with a reference fragment from a non-deleted region on another chromosome.GJB2. The data were obtained from the results of a routine pre-conception genetic screening for autosomal recessive disorders. Written informed consent for the use of personal particulars as well as genetic information for research purposes was obtained from the individuals.As population-relevant controls, 3278 gamete donors from the Centre for Medical Genetics and the Reproductive Medicine Gennet were analyzed for the carrier frequency of the c.35delG variant in the Using two different approaches of NGS, we examined the DFNB1 (13q11-q12) region in 28 patients and the WES in 49 patients—17 of whom were examined by both approaches.GJB2 variants, with a predominance of the c.35delG variant  pathogenic  variant . The ana variant . Each soAnother five deletions were detected by Meta-analyses. The deletions DS1–DS4 were detected in three patients, and the deletion DS5 in four patients. The length of the deletions (15–130 base pairs) was suitable for verification by Sanger sequencing; however, none of these five deletions (DS1–DS5) were confirmed by Sanger sequencing.GJB2 variant in patient 218517, which was also later confirmed by WES; however, no other pathogenic variant was found. We focused on the GJB2 variants listed in the HGMD professional  with a special interest in the regulatory and splicing regions, yet no disease-causing variant was detected in our samples.Furthermore, in an NGS data analysis of single nucleotide variants (SNVs), we only detected the c.-22-6 A haplotype analysis of the 28 patients and the three controls did not reveal any shared core haplotype among the patients . TherefoOnly the common general haplotype among the controls and patients was detected (chr13 g:20 761 888-20 800 039).GJB2 gene of two patients, which offered a possible explanation for the patients’ hearing loss; these variants are in the UTR5´region of GJB2—the c.-22-2 and c.-22-6. The hearing loss in four other patients was caused by pathogenic biallelic variants in the well-known hearing loss-causing genes: STRC (OMIM 606440); MYO15A (OMIM 602666); SLC26A4 (OMIM 605646); and LOXHD1 (OMIM 613072). Additional variants were detected in five rarely diagnosed genes. In the GPSM2 gene , the homozygous c.858_859delinsGT, p.(Tyr286*) variant was detected in a patient with profound hearing loss. The patient also had hydrocephalus and a dilated ventricular system, although these were not initially connected to hearing loss. In the MPZL2 gene , the homozygous variant c.72del, p.(Ile24Metfs*22) was detected in one patient. In the POU3F4 gene , one male patient with profound hearing loss and normal-hearing parents was found to be hemizygote for the c.607_610del, p.(Gln203Glufs*37) variant. In the SLITRK6 gene , the variant c.1240C>T, p.(Gln414*) in the homozygous state was detected in a patient who suffered from hearing loss, was myopic from two and a half years of age, and needs to wear glasses. In several patients, different variants of uncertain significance (VUS) were detected . The variants c.871T>G, p. and c.881T>C, p.(Ile294Thr), which are both classified as VUS, were detected in a patient with autosomal recessive hearing loss; both are in the trans position and are predicted to affect the splicing. Even without the two patients found to have the second pathogenic variant in the non-coding part of the GJB2, the diagnostic yield of the WES in patients is high. In other words, a hearing loss of 19% in monoallelic patients (9/47) was due to variants in genes other than the GJB2  of the examined patients , with different heterozygous  pathogenic GJB2 variants, detected possible pathogenic variants in 11 patients (22%). The specific variants and genes are summarized in detected . From ththe GJB2 , while tpatients . MoreoveGJB2 variant among patients with hearing loss than there is among the normal-hearing population [GJB2 variant. In a comprehensive study of the DFNB1 (13q11-q12) region among Czech patients with hearing loss, we did not find the pathogenic variant CNV, nor the SNV in the DFNB1 locus. However, WES revealed the cause of hearing loss in 22% of patients.The problems and limitations of diagnosing hereditary hearing loss have been known for some time due to the fact that there is a higher prevalence of individuals with a heterozygous pathogenic pulation . In our GJB2 pathogenic variant, explain the cause of hearing loss in a substantial number of patients with only one heterozygous pathogenic GJB2 variant.The DFNB1 analysis, performed with different software types, revealed six CNV deletions, but we were only able to verify the NG1 deletion with routine genetic methods. The overlapping of the NG1 deletion with the frequent deletion (gssvL29055) suggested convincingly that this variant, which does not interfere with the common interval, is not pathogenic. For the analysis of the DFNB1 region, we chose NGS technology because the resolution for detecting CNV is below the range of chromosomal microarray technology. Despite the fact that NGS is widely used in genetic diagnostics, the CNV analysis from the NGS data is often limited, while chromosomal microarray technology and multiplex ligation-dependent probe amplification (MLPA) are the golden standards for CNV detection . To reduGJB2 variants and tested by NGS. Last year, we published a study where 21% of 197 Czech patients with hearing loss, and where GJB2 pathogenic variants had already been excluded, were clarified by a NGS gene panel. [GJB2 variants were already excluded [In the group of 49 patients investigated by WES, the diagnostic yield was 22%, which is higher than the results of our previous study of patients without pathogenic e panel. . The diaexcluded  to arounexcluded .GJB2. It is possible that these variants affect the splicing. The variants were not detected previously by Sanger sequencing of the coding exon 2. The first 22 nucleotides of the GJB2 exon 2 comprise the untranslated region (UTR). Consequently, this section is not usually covered by Sanger and not included in NGS for variant calling. Data on both variants as pathogenic or probably pathogenic have previously been published. Therefore, we recommend a target examination of this area with Sanger sequencing. It should be used specifically for GJB2 patients with a heterozygous pathogenic GJB2 variant and before the NGS of other hearing-loss genes is performed [Of the variants detected by WES as pathogenic, it was surprising to find the variants c.-22-2 and c.-22-6 in the 5′UTR of erformed ,35.GPSM2, the detected variant c.858_859delinsGT was not reported, although other variants that predicted a premature stop codon were reported as pathogenic in this gene . The GPSM2 variants were reported to cause profound hearing loss DFNB82 or the Chudley–McCullough syndrome, with brain anomalies on MRI, including hypoplasia of corpus callosum. Hydrocephalus was detected in our patient, which is a condition that has previously been reported in some patients with GPSM2 pathogenic variants [MPZL2 gene was reported in several families with the documented disease segregation in the affected members, and with the onset occurring between 3 and 9 years of age. Our patient reported the onset of hearing loss at 10 years of age. Interestingly, this variant is common among the Ashkenazi Jewish (GnomAD 0.38%) population but has also been found in Turkish families [SLITRK6 gene cause deafness and myopia. The variant c.1240C>T, p.Gln414* detected in our patient has previously been suggested to be pathogenic in Amish patients, with the variant being detected as a frequent founder variant among them. Nevertheless, the frequency of this variant in the GnomAD is reported only in the European population (non-Finnish), with a very low frequency of 0.001% [POU3F4 gene is associated with DFNX2 deafness with the X-linked recessive inheritance [GPSM2, MPZL2, and SLITRK6, in which the homozygous rare variants were detected, indicate the high probability of the parents of the affected patients being related/distantly related people.The spectrum of causal genes detected in the present study is broad, and with the exception of well-known genes associated with hearing loss, we detected several specific types. For the variant interpretation, we used the modified ACMG criteria for hearing loss, but most of the variants have previously been reported and proved to be pathogenic . In GPSMvariants . The varfamilies ,38. The f 0.001% ,40. The KARS1 gene cause neurological disorders other than hearing loss. We detected two missense KARS1 variants in a compound heterozygous state in one patient with hearing loss. Only one of these variants was reported in a patient with a complicated neurological phenotype and hearing loss [KARS1 variants could explain the hearing loss in the patient. Both were detected in the trans position and are predicted to affect the splicing; therefore, an additional functional study should be performed in order to clarify their potential pathogenicity. The absence of the second pathogenic variant in the DFNB1 region and the diagnostic yield (22%) of the WES in the patients suggests that the cause of hearing loss may be found in genes other than those related to the GJB2 gene. A possible reason for an excess of the GJB2 heterozygotes among hearing loss patients is the small/limited number of patients included in the previous studies, at least among Czech patients. Moreover, an excess of patients with a heterozygous pathogenic GJB2 variant could be attributed to the limited examination of the pathogenic GJB2 variants. These included not only the first known SNV but also the subsequently discovered deletions, del(GJB6-D13S1830), del(GJB6-D13S1854), and c.-23+1G>A in the non-coding exon 1—an examination of which was not included in the first reports. This supports the 2.2% frequency of c.35delG heterozygotes detected among the 3278 normal-hearing gamete donors. These gamete donors were tested at the Centre for Medical Genetics and Reproductive Medicine Gennet in the Czech Republic (currently unpublished data). The frequency of GJB2 heterozygotes in healthy individuals (2.2%) is more relevant to the Czech population than the GnomAD NFE (1.9%) and is not too far from a previously found frequency of 4% among those with hearing loss. Future studies should focus on other populations and compare the number of individuals with the c.35delG monoallelic variant in those with hearing loss to normal hearing individuals.Variants in the ing loss . Since tGJB2 variant yet, in the general population, the frequency was lower at 2.45%. And in 29.5% of Chinese patients, the pathogenic variants in genes other than GJB2 were detected with WES. If we subtract these clarified patients (29.5%) from the detected frequency (3.89%), we find that the frequency of individuals with a heterozygous pathogenic GJB2 variant with hearing loss versus the general population is almost the same (3.89–1.15%). This indicates that even in the Chinese population, an excess of patients with a monoallelic GJB2 variant is elucidated by the cause of hearing loss in genes other than GJB2, thus making the occurrence of a frequent unknown variant in the DFNB1 region unlikely.Interestingly, results that support our findings come from a Chinese study where it was found that the c.235delC pathogenic variant is very frequent among the Chinese population . In a laGJB2 gene and the common interval into the design of our study, but we could have missed some unknown specific region. Nevertheless, the region examined in this study is consistent with the results of Moisan et al., who characterized the position of GJB2 cis regulatory elements and reported that all four GJB2 enhancers were localized in the 95.4kb common interval [A possible limitation of our study could be the size of the selected DFNB1 region. We included the interval .GJB2 variant. Nevertheless, our study showed that in a substantial number of these patients, the cause of hearing loss can be clarified by pathogenic variants in genes other than the GJB2. We recommend commencing the Sanger sequencing of the GJB2 gene before WES since it is possible to cover the 5´UTR of exon 2 and non-coding exon 1 with Sanger; these regions are covered poorly by WES. The MLPA method should supplement the GJB2 examination of known CNVs. Future studies should focus on additional populations in the diagnostic yield of the WES in patients with a GJB2 monoallelic variant. In the families of patients with a heterozygous pathogenic GJB2 variant, the extended Sanger sequencing and WES could clarify the cause of hearing loss in 22% of the patients while enabling precise genetic counselling and an estimation of the risk of hearing loss recurrence in their relatives.We could not confirm the existence of the presumed, second causal variant in the DFNB1 region that would explain the cause of hearing loss in at least a number of patients with a heterozygous pathogenic


Hearing preservation after cochlear implantation has considerable benefits because the preserved low-frequency hearing in the implanted ear contributes to binaural integration, presumably through the preserved temporal fine structure.The purpose of the study is to gauge the benefits of binaural integration effects (redundancy and squelch) due to preserved low-frequency residual hearing in the implanted ear of cochlear implant users with single-sided deafness. There were 11 cochlear implant users (age 18–61 years old) who had preserved low-frequency hearing in the implanted ear; they had a normal hearing or mild hearing loss in the contralateral ear. Patients were tested with monosyllabic words, under different spatial locations of speech and noise and with the cochlear implant activated and deactivated, in two listening configurations—one in which low frequencies in the implanted ear were masked and another in which they were unmasked. We also investigated how cochlear implant benefit due to binaural integration depended on unaided sound localization ability. Patients benefited from the binaural integration effects of redundancy and squelch only in the unmasked condition. Pearson correlations between binaural integration effects and unaided sound localization error showed significance only for squelch ( In cochlear implant (CI) users the advantage of binaural hearing over the monaural hearing has been extensively studied in terms of improvement in localization and speech discrimination over the monaural condition ,2,3,4,5.However, recent advances in the preservation of low-frequency (LF) hearing in the implanted ear give rise to another possible bilateral arrangement in patients with partial deafness ,8. UnilaFor these patients, the binaural benefit arises from listening to whichever ear has the better signal-to-noise ratio (the “better ear” effect) and from integrating information from both ears. The “better ear” effect depends on the head shadow effect, which provides different intensities to each ear. Listeners make use of the head shadow effect by listening with both ears and deciding which ear has the better SNR and ignoring the other. The “better ear” effect predominates at high frequencies ,11. Binaural redundancy and squelch rely on integrating information received from both ears. Binaural redundancy involves simultaneously processing two signals, one from each ear; since the same signal arrives at each ear with almost the same auditory characteristics, the brain can use overlapping information to better discriminate speech . Redundafine) conveyed through the temporal fine structure of the signal , were examined.To answer the research question of whether unaided sound localization ability is an indicator of the availability of ITDfine, the relationship between binaural benefits and unaided sound localization ability was investigated. In addition, the relationship between binaural benefits and the range of preserved hearing in the implanted ear , a Student’s t-test was used to make pair-wise comparisons of speech outcomes in “CI off” and “CI on” conditions for each test setup . The hypothesis of a normal distribution of the data was evaluated using a Shapiro–Wilk test. The relationships between binaural benefits and unaided sound localization error, and between those and PTAt(10) = 2.8; p = 0.02), redundancy in noise (t(10) = 3.1; p = 0.01), squelch (t(10) = 6.2; p = 0.0001), and “better ear” effect (t(10) = 6.1; p = 0.0001). For the LFmasked–NH configuration, pair-wise comparisons of the “CI off” and “CI on” conditions showed significant difference only for the “better ear” effect (t(10) = 3.0; p = 0.01). For the LF–NH configuration, pair-wise comparisons of the “CI off” and “CI on” conditions showed significant differences for redundancy in quiet  was found only for squelch . squelch c. There An interesting aspect of our study is that the patients differed significantly from those tested in previous studies. Our patients were unique in the way they had successful hearing preservation surgery in one ear and normal or close to normal hearing (NH) in the other ear. Thus, we were able to explore the value of preserved LF hearing in the implanted ear for binaural integration effects of redundancy and squelch, assuming that all binaural cues are available from the NH ear. Generally, when the “CI on” condition was compared to the “CI off” condition, the average level of speech perception improved for redundancy in quiet, redundancy in noise, “better ear” effect, and squelch in the unmasked condition . When preserved LF hearing in the implanted ear was masked, the only significant improvement was for the “better ear” effect. Hence, we have confirmed our hypothesis that bilateral acoustic LF hearing, which is available thanks to hearing preservation in the implanted ear, provides extra benefits via squelch and redundancy effects (over and above the “better ear” effect).env) since stimulating electrical pulses are presented at a fixed, high rate completely unrelated to the sound’s temporal fine structure. Uniquely, however, to enhance temporal information within the electrical stimulation signal, CIs manufactured by the MED-EL company lower the stimulation rate of the low-frequency channels and alter the timing of the pulses to fire at times of zero-crossing of the corresponding band’s signal. Secondly, there is commonly a mismatch between the place of excitation in one ear and in the other. The electrode arrays produced by most CI companies are not fully inserted into the cochlea (an exception being those manufactured by MED-EL). For a low frequency narrowband acoustic input signal, in particular, the CI will stimulate a certain electrode in the cochlea according to its frequency-to-electrode location, which for shallower insertion will not correspond to the cochlear frequency-to-place map of normal hearing. Thus, for SSD CI patients, different places in the two cochleas will usually be stimulated, which will degrade ITDfine cues ). This finding provides additional evidence for the idea that implant recipients with preserved acoustic hearing have continued access to fine structure cues, which should provide greater binaural integration benefits. Additionally, we found there was no correlation between localization ability in the unaided condition and the level of LF hearing (PTA[125–500]). Since both the binaural integration effects of both squelch and localization rely on sensitivity to ITDfine cues, the lack of a significant correlation between squelch and PTA[125–500], and between localization ability and PTA[125–500], suggests there is no relationship between access to ITDfine and the level of LF hearing. This agrees with Strelcyk and Dau [Another interesting observation in the current study was that the binaural integration effect of squelch depends on factors other than audibility. That is to say, squelch and its binaural integration effect is negatively correlated with localization error, although not with the hearing threshold of preserved LF hearing , are also more likely to receive benefits from binaural squelch. According to our testing paradigm, the unaided localization test score in those patients who have CI surgery with hearing preservation can be seen as a marker of LF fine time structure coding. Nevertheless, the level of fine structure accessible to a patient cannot be simply deduced from the hearing thresholds measured postoperatively, which is a conclusion supported by (1) the lack of correlation between CI benefits via a squelch effect in the LF–NH listening configuration and the level of PTA in the implanted ear and (2) the lack of correlation between unaided sound localization error and the level of PTA in the implanted ear.Since SSD CI patients have NH in the non-implanted ear, their better access to fine structure in the implanted ear should translate into greater access to ITDWhen discussing clinical relevance, there are two aspects to consider. Firstly, because the indication criteria for cochlear implants have progressively been extended, the population of CI candidates has increased in range and type of hearing loss . PatientSecondly, due to the large variance in the binaural benefits of SSD CI patients, there is a need for appropriate clinical tests that can be used shortly after implantation to predict possible CI-related benefits that might be achievable in the course of auditory rehabilitation. Results of the current study suggest that in SSD patients with LF hearing preservation, the localization error obtained in the unaided condition can serve as a predictor of the binaural integration effect of squelch and, more generally, of the possible hearing benefits, which stem from accessibility to fine time structure from LF hearing in the implanted ear. The open question is whether this finding can be generalized to other populations of patients with hearing preservation who are EAS users . Future investigation is needed to verify that acoustically based localization ability in EAS users can be used as a marker of sensitivity to fine time structure in the implanted ear and therefore indicates the potential for increased benefits from the integration effects of redundancy and squelch.fine cues. In any particular case, the extent of the improvement does not depend greatly on the threshold of the preserved LF hearing. It seems that in these CI patients, the benefit obtained from squelch is related more to localization error, as measured in the unaided condition. We, therefore, conclude that localization error might serve as a good predictor of the temporal fine structure cues that preserved hearing can provide.The findings of the current study are potentially good news for SSD patients who, after fulfilling the criteria for a CI, end up with preserved LF hearing in the operated ear and NH in the contralateral ear. Our results indicate that it is possible for them to have access to the binaural integration effects of redundancy and squelch and receive additional speech understanding in noise. To our knowledge, the results here provide the first clinical evidence that preserved low-frequency hearing contributes to binaural integration effects of redundancy and squelch, presumably by allowing access to ITDIn brief, hearing preservation after cochlear implantation is beneficial since it preserves low-frequency hearing in the implanted ear, and this can contribute to binaural integration.


IntroductionMucoracea family have an increasing incidence and prevalence. It has always been difficult to diagnose this condition due to various reasons.Invasive fungal infections have always been a major cause of mortality and morbidity and are especially prevalent in the immunosuppressed groups of patients. Members of the Materials and MethodsThis was an observational study carried out jointly by the Department of Microbiology and the Department of Pathology for a duration of one year. All patients who presented in various clinical departments with a high index of clinical suspicion for mucormycosis were included in this study. A total of 186 samples were received from suspected cases of mucormycosis and were all subjected to direct microscopy by potassium hydroxide (KOH), fungal culture, and histopathological examination.ResultsMucormycosis was documented in 33 out of 186 cases on direct microscopy, whereas 21 were positive on fungal culture. Histopathological positivity was reduced with only 11 cases showing aseptate hyphae suggestive of mucormycosis.ConclusionAs these organisms generally do not grow well on routine culture media and with the histopathological results also being not suggestive clearly of mucormycosis, direct microscopy thus becomes more important and essential in the rapid diagnosis of this deadly condition. Mucormycosis is a reemerging fungal infection and has been seen increasingly in immunocompromised patients. It is caused by fungi belonging to the order Mucorales . AlthougIt has always been a challenge to diagnose this condition, and the main prerequisites for an accurate diagnosis are a high index of suspicion and recognition of host factors along with a prompt assessment of clinical manifestations ,4. MicroThis study was carried out for a duration of one year (January 2019-December 2019) in a tertiary care hospital situated in Uttarakhand to determine the prevalence of mucormycosis in the region. A retrospective data analysis was done from the medical records of the patients. All patients who presented in various clinical departments with a high index of clinical suspicion for mucormycosis were included in this study. The enrolled patients comprised of both immunocompromised and immunocompetent individuals. The specimens from the patients were collected in two sterile containers, one with and the other without 10% formalin, and processed in the Department of Microbiology and the Department of Pathology, respectively. The testing of the samples in the Department of Microbiology included direct potassium hydroxide (KOH) examination and fungal culture. The samples were inoculated without crushing in two tubes containing Sabouraud's dextrose agar (SDA) without antibiotics, with one tube incubated at 37°C and the other at 25°C. The cultures were examined for growth daily for the first week and twice a week for the subsequent period. The fungal isolates were finally identified by conventional techniques such as lactophenol cotton blue (LCB) mount. For histopathological examination, tissue sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) stain, and Grocott methenamine silver (GMS) stain.The diagnosis of mucormycosis was confirmed when broad aseptate/sparsely septate, ribbon-like hyphae with right (wide) angle branching were demonstrated in the specimen with or without isolation of mucormycetes. The diagnosis was also confirmed by suggestive histopathological findings.AnalysisData were entered in Microsoft Excel 2021, and analysis was done using the STATA-12. Agreement between the tests was calculated using Kappa. The agreement was classified using standard classification methods. Sensitivity and specificity were calculated using the standard formula with 95% confidence intervals.In the one-year (2019) study period carried out jointly by the Department of Microbiology and the Department of Pathology, a total of 186 clinical samples with a high index of clinical suspicion for mucormycosis were collected. Of these, 60% (111/186) were males and 40% (75/186) were females. The mean age of patients was 40 years. The majority of the samples received were nasal (122), followed by ear (43), mucosa from the sphenoid cavity (5), pus from neck node (3), sputum (2), hard palate biopsy (2), tissue from functional endoscopic sinus surgery (FESS) and cortical mastoidectomy (2), glossectomy (2), submandibular gland (1), post-aural granulation tissue (1), pericardial tissue (1), pus swab from left aryepiglottic growth (1), and biopsy from the left side of the face (1). On direct microscopy by KOH mount, 33 out of 186 (17.2%) turned out to be positive, while 21 out of 186 (11.3%) showed growth on fungal culture. On the other hand, histopathology was positive only in 11 out of the total 186 (6%) cases.Table As per Table During the last 10 years, there has been an enormous surge in the occurrence of mucormycosis cases worldwide, largely as a result of the expansion of the population at risk of having predisposing conditions. In our study, 60% were males and 40% were females, with a mean age of 40 years. Young age males are more prone to acute necrotizing infection. This was in concordance with a study conducted by Bala et al., where the incidence was commoner in males 72%) than in females (28%), and the mean age of the patients was 40.43 years [2% than iRhinocerebral mucormycosis has been found to be the commonest clinical presentation in most of the published literature, and the same was noted in the present study. Similar findings have been observed in a prospective multicenter study and also in another study conducted in the western part of India ,8. A revOn careful analysis of the results, several reasons could be postulated for the low rate of positivity in histopathology as compared with microbiological diagnosis by direct microscopy and culture. One of the reasons could be the sample sent to the Department of Microbiology laboratory and to the histopathology not being from the exact same site and therefore may not be equally representative. It is very challenging to diagnose fungal infections on scanty tissue sections; it requires good expertise . AnotherKOH along with histopathology is recommended by the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium (ECMM/MSG ERC) . KOH micKOH mount microscopy is a reasonably priced and irreplaceable method to make a rapid presumptive diagnosis. It has been for ages the best means to diagnose this fatal condition and provide a presumptive guide to the clinicians in their management. It helps them in initiating the appropriate treatment and therefore saving precious time. Being a point of care test that can be performed immediately, it is the fastest means of getting a presumptive diagnosis. Nevertheless, new molecular and antigenic tools are the need of the hour for the early detection of mucormycosis and therapeutic monitoring.


It was well-documented that extended high-frequency  hearing test could be more sensitive comparing with the conventional measurement on frequency below 8 kHz, regarding the early prediction of auditory damage in certain population. However, hardly any age-specific thresholds of EHF in population with normal hearing ability were observed. This study aims to monitor the age-dependent hearing thresholds at EHF (from 9 to 20 kHz) in healthy hearing population.A total of 162 healthy participants (from 21 to 70 years) with normal conventional pure tone audiograms were recruited and separated into five groups by age. Conventional pure tone average was performed with frequencies from 0.25 to 8 kHz under air conduction and from 0.25 to 4 kHz under bone conduction. EHF audiometry from 9 to 20 kHz was determined under air conduction.The effects of aging on hearing were evident at frequencies above 4 kHz. The hearing thresholds of EHF were less than 26 dB HL before 30 years-olds. Hearing abilities in EHF were deteriorated starting from the 31 ~ 40 group and were most obvious in the 51 ~ 60 group and the 61 ~ 70 group with the maximum thresholds of 75 dB HL. Sensitivity of EHF was inversely proportional to the frequency within each age group, and to age among groups. Subjects under 30 years old were totally responsive up to 16 kHz, and 52.2% could respond to 20 kHz. Meanwhile, no responsiveness was recorded to 20 kHz in the 51 ~ 60 group and even to 18 kHz in the 61 ~ 70 group. No gender differences in hearing threshold was observed within each age group, except an obvious decline at frequencies of 4, 6, 8, and 9 kHz in male participants of the 41 ~ 50 group.Hearing thresholds at EHF from 9 to 20 kHz were more sensitive than at frequencies below 8 kHz for hearing measurement, and aging inversely affected hearing ability at EHF in healthy population. Hearing thresholds at EHF deteriorated with age and raising frequency, while the upper frequency limit decreased with aging. The conventional method of measuring pure tone average (PTA) of hearing thresholds is performed under the frequencies lowing than 8 kHz. However, this conventional audiometry has been suggested not reliable on evaluating auditory damage due to the absence of detecting responses at extended high frequencies (EHF) above 8 kHz. Monitoring EHF responses, on the other hand, could be quite useful in the early prediction of hearing loss in certain populations –4. ReseaStudies with multivariate analysis proved that age was the primary factor of changes to hearing thresholds at EHF , 12. AgiWith the limitations of interpretations that can be made by conventional PTA, EHF audiometry had been performed more often as hearing parameters for clinical and laboratory diagnoses change with research. However, hardly any age-dependent normal audiometric thresholds at EHF from 9 to 20 kHz had been reported up to now. Hence, the present study aimed to provide the data of hearing thresholds at frequencies up to 20 kHz of different age groups in the health population to serve the need.Adults with normal PTA hearing thresholds , aged between 21 to 70 years old, were recruited in the study. Subjects were in a normal state of health, free from all signs or symptoms of ear diseases. They performed ear canal cleaning before testing. No history of noise exposure , smoking, intake of alcohol and ototoxic drugs  , hyperteAudiometric tests were conducted by experienced technicians. Hearing thresholds of each ear at 15 frequencies from 0.25 to 20 kHz were obtained using a pulsed-tone stimulus and modified Hughson-Westlake procedure. Conventional PTA  was performed at octave or semi-octave frequencies under air conduction and at octave frequencies from 0.25 to 4 kHz under bone conduction using a manual audiometer  with TDH-50P supra-aural earphones . EHF audiometry  was determined under air conduction using a calibrated audiometer  with HDA-200 high frequency earphones . The threshold was defined as the lowest decibel level at which the subject responded for at least 50% of the stimulus duration in ascending or descending presentations  . All audp < 0.05 was considered to be significant.Descriptive and quantitative analysis of difference associated with the independent variables were performed using SPSS 15 for Windows. Normality of the distribution was assessed using the Levine test. Comparisons of two independent samples  were conducted either using two-tailed independent samples t-tests if the data were normally distributed revealed by the Levine test, or otherwise using the nonparametric rank-sum test (such as the hearing threshold). When the responsive rate to frequency didn’t exceed 20%, the hearing threshold value was not included. In all tests, A total of 162 healthy participants were enrolled in this study, and grouped into five groups based on their ages: 21 ~ 30 years, 23 participants (46 ears) including 14 males and 9 females; 31 ~ 40 years, 25 participants (50 ears) with 16 males and 9 females; 41 ~ 50 years, 44 participants (88 ears) with 23 males and 21 females; 51 ~ 60 years, 39 participants (78 ears) with 19 males and 20 females; 61 ~ 70 years, 31 participants (62 ears) with 21 males and 10 females. There was no gender difference within each age group, nor individual difference between the right and the left ear on the pure-tone hearing thresholds at the frequencies from 0.25 to 20 kHz.p < 0.05) on the mean thresholds was noticed between the 21 ~ 50 and 51 ~ 70 years old. The mean thresholds at 4 kHz was significantly different from each other when compared in pair , except for the comparison between the 21 ~ 30 and the 31 ~ 40 group. In addition, consistent threshold shifts at the frequencies of 6 and 8 kHz were seen among groups. The hearing thresholds at the frequencies of 6 and 8 kHz were significantly elevated in groups above 31 years old, and showed an age-dependency starting from 51 years old (p < 0.05). These findings suggested that hearing ability of healthy participants declined at the frequencies of 0.25 to 2 kHz from the age of 51, 4 kHz form the age fo 41, and 6 to 8 kHz from the age of 31. Table No reliable gender difference was seen in each group on hearing thresholds at the frequencies from 0.25 to 2 kHz. However, by merging different age groups, a significant difference (p < 0.001) (Table p < 0.05), the same as the situation at the 6 and 8 kHz. At the frequencies of 10 kHz, 11.5 kHz, and 12.5 kHz, the average hearing thresholds between groups were significantly different from each other with the exception of the 31 ~ 40 group comparing with the 41 ~ 50 group. The hearing deterioration at the frequency of 14 kHz was quite similar to the changes at frequencies of 6, 8 and 9 kHz. At the frequency of 16 kHz, the average hearing threshold of 25 dB HL in the 21 ~ 30 group was significantly better than those in 31–60 age groups, and the threshold of 50 dB HL in the 31 ~ 40 group was better than those of 55 dB HL in the 41 ~ 60 age group (p < 0.05). These results showed that hearing at EHF degenerated in subjects starting from their 30s, and dominating in their 50s and older  could be found in the mean thresholds at frequencies of 4, 6, 8, and 9 kHz in the 41 ~ 50 group, suggesting a possibly greater hearing loss in males at their 40s.Moreover, no difference in the means of hearing thresholds between male and female subjects were seen nearly at all frequencies and in all age groups, except that gender differences (Age related hearing loss is often gradual and progressed with subtle changes in an individual’s ability to discriminate high pitches. Meanwhile, some patients also complain about difficulty in understanding speech under noisy environments. Nevertheless, no apparent impact on verbal communication had been observed due to the hearing loss in EHF . TherefoWe examined the hearing thresholds at frequencies from 0.25 to 20 kHz in the population claimed to have normal hearing abilities ranging from 20 to 70 years old in order to observe the potential age-related changes in their auditory system. As the hearing threshold at 4 kHz was obviously increased in adult older than 50 with clinically normal hearing sensitivity, subjects with hearing thresholds lower than 20 dB HL at up to 2 kHz were included in this study. Our data demonstrated similarities in average thresholds at frequencies from 0.25 to 2 kHz in subjects younger than 50 years old. However, the hearing thresholds increased predominantly starting from the age of 41 at 4 kHz, and from the age of 31 at 6 and 8 kHz. Therefore, we might conclude that the onset frequency of effects by aging were 4 kHz.In this study, Fig. Although studies suggested a potential gender difference in the hearing loss, with males earlier than females , 24, WilThe current findings on thresholds at EHF, as well as previous studies describing their use, demonstrate the fact that EHF audiometry should be taken into consideration as a necessary and routine test to diagnose and monitor hearing damage. Early prediction and intervention in the potential population, such as medication dose change or proper use of hearing protection for the ototoxic effect and noise exposure, might ease or even cease the development of hearing loss , 26. FurThis study had limitations. The number of subjects from a single medical center was limited, and it could not fully reflect the epidemiological characteristics of age-related hearing changes. On the other hand, we detected the hearing thresholds at frequencies from 0.25 to 20 kHz in different age groups at the same period. In addition, no long-term follow-up was conducted for the same age group. Future research should involve multicentre cohort study over long term to determine the normal range of EHF deterioration. To establish the age-specific norm, more research is needed as well.This study revealed that hearing thresholds at EHF from 9 to 20 kHz were more sensitive than at frequencies below 8 kHz towards the prediction of hearing loss, and showed clear age-dependency. Both hearing thresholds at EHF and the upper-frequency limit deteriorated with aging and elevation of the frequency. The results of this study also demonstrated reproducibility in measuring hearing thresholds at EHF, indicating that the EHF audiometry might be a reliable tool in monitoring the early etiopathogenesis of auditory diseases.