OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic recessive sensorineural hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD). In the present study, we performed OTOF mutation analysis using massively parallel DNA sequencing (MPS). The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population. A total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. The mutation analysis of 68 genes, including the OTOF gene, reported to cause non-syndromic hearing loss was performed using MPS. Thirty-nine out of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF gene. It is assumed that the frequency of hearing loss associated with OTOF mutations is about 1.72% of autosomal recessive or sporadic SNHL cases. Hearing level information was available for 32 of 39 patients with biallelic OTOF mutations; 24 of them (75.0%) showed profound hearing loss, 7 (21.9%) showed severe hearing loss and 1 (3.1%) showed mild hearing loss. The hearing level of patients with biallelic OTOF mutations in this study was mostly severe to profound, which is consistent with the results of past reports. Eleven of the 39 patients with biallelic OTOF mutations had been diagnosed with ANSD. The genetic diagnosis of OTOF mutations has significant benefits in terms of clinical decision-making. Patients with OTOF mutations would be good candidates for cochlear implantation; therefore, the detection of OTOF mutations is quite beneficial for patients, especially for those with ANSD.The  OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the frequent causes of non-syndromic recessive sensorineural hearing loss. To date, more than 160 mutations in OTOF have been reported, and most of the patients with OTOF mutations have stable, prelingual and severe to profound hearing loss. OTOF is also known to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD)[OTOF is mainly expressed in cochlear inner hair cells, and is necessary for synaptic exocytosis at the ribbon synapse[OTOF gene mutation can also present as ANSD.Hearing loss is one of the most frequent congenital sensory disorders, with one out of every 500 newborns having bilateral hearing loss. It is rer (ANSD)–5. ANSD OTOF mutations on the basis of Sanger sequencing[OTOF gene has a large number of exons. In this study, we conducted a genetic analysis of the OTOF gene in 2,265 Japanese hearing loss patients by MPS. The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population.Recently, targeted exon sequencing of selected genes using massively parallel DNA sequencing (MPS) technology has been developed, enabling us to analyze massive amounts of data both relatively quickly and inexpensively improve the molecular diagnostic rate of hearing loss patients –9. Althoequencing, it is bA total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. Hearing loss was evaluated using pure-tone audiometry (PTA) classified by a pure-tone average over 500, 1000, 2000 and 4000 Hz in the better hearing ear. For children who could not undergo PTA, we used an average over 500, 1000, 2000 Hz for either auditory steady stem response (ASSR) or conditioned oriented reflex audiometry (COR), or the response threshold (dBnHL) from ABR. The severity of hearing loss was classified as follows: normal hearing, <25dB; mild hearing loss, 25-39dB; moderate hearing loss, 40-69dB; severe hearing loss, 70-89dB; and profound hearing loss, greater than 90dB. Written informed consent was obtained from all subjects  prior to enrollment in the project. All procedures were approved by the Shinshu University Ethical Committee and the ethical committees of the other participating institutions listed as follows: Hokkaido University, Sapporo Medical University, Akita University, Iwate Medical University, Tohoku University, Tohoku Rosai Hospital, Yamagata University, Fukushima Medical University, Jichi Medical University, Gunma University, Jyuntendo University, Yokohama City University, Tokai University, Mejiro University, National Rehabilitation Center, Nihon University School, Saitama Medical University, Tokyo Medical University, Jikei University, Abe ENT clinic, Toranomon Hospital, Kitasato University, Tokyo Medical Center Institute of Sensory Organs, International University Health and Welfare Mita Hospital, Jichi University Saitama Medical Center, Aichi Children’s Health Medical Center, Chubu Rosai Hospital, Mie Hospital, Kyoto University, Kyoto Prefectural University, Mie University, Shiga Medical Center for Children, Shiga Medical University, Osaka University, Kansai Medical University, Kobe University, Osaka Medical Center and Research Institute for Maternal and Children Health, Hyogo College of Medicine, Okayama University, Kobe City Medical Center General Hospital, Wakayama Medical University, Kouchi University, Hiroshima University, Hiroshima City Hiroshima Citizen Hospital, Yamaguchi University, Ehime University, Kyushu University, Fukuoka University, Kurume University, Nagasaki University, Kanda ENT Clinic, Miyazaki Medical College, Kagoshima University, Ryukyus University.OTOF gene  reported to cause non-syndromic hearing loss , according to the manufacturer’s instructions, for 68 genes including all the exons of the ing loss . The detEmulsion PCR and sequencing were performed according to the manufacturer’s instructions. The detailed protocol was described elsewhere. MPS washttp://evs.gs.washington.edu/EVS/), 3) the Human Genetic Variation Database [The sequence data were mapped against the human genome sequence (build GRCh37/hg19) with a Torrent Mapping Alignment Program. After sequence mapping, the DNA variant regions were piled up with Torrent Variant Caller plug-in software. After variant detection, their effects were analyzed using ANNOVAR software, 12. Thevariants), 4) the variants). All theTo predict the pathogenicity of the missense variants, we used 12 functional prediction software programs including ANNOVAR .trans : PM3 (p.R1856Q or p.R1172Q), 2) they were not found in the control: PM2, 3) the prediction programs scores support their pathogenicity: PP3 and 4) co-segregation with family members with disease: PP1. The mutations judged to be likely benign and variants of uncertain significance are presented in CDH23 mutations; p.V1012A is found with compound heterozygote GJB2 mutations; c.4023+1G>A is found with compound heterozygote SLC26A4 mutations or mitochondrial 3243A>G mutations): BP5 and 4) a reputable source  standards and guidelines. The mutOTOF mutations are shown in All of the patients with biallelic OTOF gene. Hearing level information was available for 32 of the 39 patients with biallelic OTOF mutations; 24 of them (75.0%) had profound hearing loss, 7 (21.9%) had severe hearing loss and 1 (3.1%) had mild hearing loss.Thirty-nine of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF mutations had been diagnosed with ANSD.Only 11 of the 39 patients with biallelic OTOF mutations, with 31 of them (96.9%) not experiencing any episodes of vertigo.Clinical information regarding vertigo was available for 32 of the 39 patients with biallelic OTOF gene. Two patients with heterozygous mutations (p.Y474X and p.R1172Q) showed an ANSD phenotype, and it is strongly suspected that they had OTOF related deafness. Possible explanations for these heterozygous cases are 1) the co-existence of copy number variations, 2) the existence of a second mutation in the exonic region that could not be covered in this study or in regulatory region of OTOF, which was not explored, 3) the contribution to hearing loss of an additional modulatory gene, and 4) the existence of a mutation in another gene  which causes non-syndromic ANSD not examined in this study [OTOF mutations.In this study, 39 (1.72%) of 2,265 SNHL patients compatible with autosomal recessive (including sporadic cases) inheritance carried homozygous or compound heterozygous mutations in the is study , so thatDFNB59 gene , reported to be a cause of non-syndromic ANSD [OTOF and DFNB59) but did not identified any copy number variations in these two patients. Therefore, it is assumed that the frequency of hearing loss patients with OTOF mutations is at least 1.72% among autosomal recessive or sporadic SNHL cases. It was previously reported that OTOF mutations accounted for 1.4–8.3% of non-syndromic hearing loss patients: 2.3% (13/557) in Pakistani[OTOF mutations accounted for 3.2–7.3% of recessive severe to profound SNHL[OTOF mutations, a rate which is comparable with that of our previous report.mic ANSD , was alsPakistani, 3.2%  had profound hearing loss, and 21.9% (7/32) had severe hearing loss. The commonly observed phenotype in patients with OTOF mutations is non-progressive, congenital and severe to profound hearing loss. This is consistent with the results of this study. Genotype-phenotype correlations of OTOF have been discussed in past reports [OTOF gene. Rare cases of temperature-sensitive ANSD, a particular form of ANSD, have been reported in some populations[The hearing levels in patients with biallelic 75.0% 24/2 had pro reports , 23. Pat reports , 25. In OTOF mutations had homozygous p.R1172Q mutations. Eighteen of 39 patients (46.2%) had p.R1172Q with another mutation in compound heterozygosity. In summary, 34 of 39 patients (87.2%) had at least one p.R1172Q mutation. p.R1172Q has been proven to be a founder mutation[OTOF-related hearing loss is a major etiology of hearing loss in the respective country, not only in Japan.As shown in  mutation, c.2905_ mutation, p.V1778 mutation, p.E57X  mutation and p.E1 mutation populatiOTOF mutations lies in its benefits for clinical decision-making. OTOF mutations represent one of the etiologies of ANSD. ANSD is heterogeneous disorder, and the outcomes of cochlear implantation for patients with ANSD vary[OTOF mutations as the OTOF gene mutations result in disruption of the synaptic exocytosis of inner hair cells, and the auditory nerves and spiral ganglions are preserved in patients with these mutations[OTOF mutations are predicted to show good outcomes; therefore, the detection of OTOF mutations is quite beneficial for the patients, especially for those with ANSD.The significance of the genetic diagnosis of ANSD vary, 30. Cocmutations–33. It imutations. PatientOTOF mutations experience episodes of vertigo, and found that 31 of 32 patients (96.9%) with biallelic OTOF mutations had no such episodes. Although the OTOF gene is also expressed in vestibular hair cells, otoferlin-deficient mice show no evidence of apparent vestibular dysfunction based on gross evaluation by vestibular testing [OTOF mutations and episodes of vertigo, and our results also showed that the clinical symptoms of vertigo are rarely observed in OTOF-related hearing loss patients.In this study, we also investigated whether the patients with biallelic atients 9.9% with OTOF mutations were also included among those 154 mutations and were identified in some cases. The pathogenic variants identified in this study will be added to this social health insurance-based genetic testing and further improvement in the diagnostic rate is expected.In Japan, genetic testing for patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, the genetic testing was expanded in 2015 to allow screening for 154 mutations in 19 deafness genes using targeted genomic enrichment with MPS combined with the Invader assay . We prevS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigThe chromatograms of each variant .(TIF)Click here for additional data file.


Congenital cytomegalovirus (cCMV) infection is the most frequent non-genetic cause of sensorineural hearing-loss (SNHL) . It is widely accepted that SNHL at birth, when associated to cCMV symptomatic infection involving the central nervous system, benefits from antiviral therapy started in the neonatal period. Conversely, there is no consensus for antiviral treatment in congenitally infected infants diagnosed with isolated SNHL  at birth.Our aim was to assess the frequency and the auditory outcome of isolated SNHL at birth due to auditory neuropathy (AN)  in infants with cCMV infection.We retrospectively reviewed the clinical history of 60 infants, born at term, with cCMV asymptomatic infection, without additional risk factors for SNHL, and exhibiting bilateral “pass” otoacustic emissions (OAE). None of them underwent antiviral therapy.Hearing thresholds were assessed by means of Auditory Brainstem Responses (ABR). AN affected children were followed up until possible normalization of the hearing thresholds or definitive diagnosis of AN. Each infant diagnosed with monolateral or bilateral AN was classified according to the worst ear threshold.p = 0.007). All AN cases spontaneously recovered a normal auditory threshold over time. The mean length of the audiological follow-up was 32.44 ± 17.58 (SD) months (range 5–60 months).In our population, the first ABR was performed at a mean age of 5.00 ± 2.79 (SD) months and AN was diagnosed in 16/60 (26.67%) infants; in 4 infants the AN was defined as mild , moderate in 11 , and severe in 1 . The mean age at first ABR was 3.69 ± 2.80 (SD) months in the 16 babies with AN and 5.48 ± 2.66 (SD) months in the 44 infants with normal hearing (A delayed maturation of the auditory pathways should be considered when a mild/moderate isolated AN at birth is detected in cCMV infected infants. Prospective studies conducted on larger populations, and with a longer audiological follow-up, are needed to confirm our findings. Congenital cytomegalovirus (cCMV) infection is a leading non-genetic cause of sensorineural hearing-loss (SNHL)  and both sympotmatic and asymptomatic cCMV infections contribute to the burden of SNHL . The benHearing evaluation in infants is usually accomplished by means of Otoacustic Emissions (OAE), to investigate cochlear function, and Auditory Brainstem Responses (ABR). ABR testing is not only able to detect hearing loss associated with cochlear dysfunction but also forms of hearing loss due to problems in conveying the sound information to the brain [auditory neuropathy (AN)]. With AN, a newborn will pass OAE testing but fail ABR testing .In this study, we retrospectively investigate the frequency and the outcome of isolated SNHL at birth due to AN in a selected population of infants with cCMV infection who did not receive antiviral therapy.We retrospectively reviewed the prospectively collected data of a population of infants with cCMV infection, evaluated between January 2011 and June 2018 at the Outpatient Clinic of Congenital-Perinatal Infectious Diseases - Department of Maternal and Child Sciences and Urology - “Sapienza” University of Rome.We included infants born at term  with a cCMV asymptomatic infection and an uneventful clinical course at birth. Namely, neonates suffering from diseases, or undergoing therapies, possibly affecting hearing outcome  were excluded from the study. Other inclusion criteria were: 1) a bilateral “pass” OAE test in the first month of life; 2) a first ABR evaluation within the first 12 months of life , and 3) no antiviral therapy administered.Maternal CMV primary infection was identified and dated according to serological CMV screening performed during pregnancy (CMV IgG seroconversion from negative to positive or low avidity CMV IgG together with CMV IgM antibodies). Congenital infection was established by means of viral culture  or polymerase chain reaction performed on urine within the first 3 weeks of life. Asymptomatic infection was established according to widely agreed procedures . IUGR inAll the infants underwent a first hearing screening within the first month of life , by means of OAE. According to our protocol, infants with a “fail” result are referred within the first month of life to the Department of Sense Organs of our hospital to repeat OAE  and perform ABR if a “fail” OAE result is confirmed. Infants with both a “fail” OAE and a lowered hearing threshold at ABR are candidates for antiviral treatment at our istitution. Infants with cCMV infection exhibiting a “pass” OAE are scheduled for ABR within 3 months of life and then every 6 months until the age of 2 years. After the age of two, the children are tested with play and pure tone audiometry. An otoscopic evaluation of tympanic membrane and of middle ear function trough tympanometry is performed during each follow-up visit.ABR recordings were obtained while sleeping spontaneously and were performed with a GSI Audera device  with a 21 stimuli per second click rate, in an analysis window of 12 msec. The highest intensity reached by this equipment is 100 dB-hearing level (dB-HL). The test implicates the repetition of the stimulus by 10 dB-HL steps, and the lowest intensity at which the V wave is observed, represents the hearing threshold of the subject. In accordance with other studies, our evaluation criterion only included the presence of the V wave , 10. We When the first ABR test was ≤30 dB-HL , subsequent ABRs were not taken into account for the purpose of this study and the infant was categorized as NO-AN; whereas AN was diagnosed (hearing threshold > 30 dB-HL) at the first examination, subsequent ABR were considered until normalization of the hearing threshold (“maturative” AN) or definitive diagnosis of AN (“true” AN). Further ABRs after restoration of a normal auditory threshold were not considered to avoid a definition bias between fluctuations of the auditory threshold and a possible late onset SNHL. However, the auditory threshold at last audiological evaluation was recorded for each AN affected infant.Cognitive, language and motor areas of development were investigated with the Bayley Scales for Infant Development III Ed. (BSID-III) up to age 24 months by a single child neuropsychiatrist (BC). When more than one BSID-III test was available for the same child, the more recent evaluation was considered for the statistical analysis.P value ≤0.05 were considered to be statistically significant. All analyses were performed with IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.Categorical data are expressed as numerical counts and percentages. The Fisher’s exact test was used for comparison between groups for categorical data. For continuous data, the Wilcoxon rank-sum (Mann-Whitney) test was used to compare mean ± standard deviation between groups. Results with a One hundred-three children with cCMV infection were referred to our Outpatient Clinic of Congenital-Perinatal Infectious Diseases in the time interval considered. Thirty of them were excluded due to the presence of at least one exclusion criteria . Thirteen additional children were excluded due to missing audiological follow-up. The group eligible for the study consisted of 60 term infants with asymptomatic cCMV infection, all of them with a bilateral “pass” OAE. All babies were born to mothers who had suffered a primary CMV infection during pregnancy.p = 0.007) lower from the age at first ABR in the 44/60 infants  with normal hearing [5.48 ± 2.66 (SD) months]. Demographical data of the study population were similar between AN and NO-AN groups  months. AN (any degree) was diagnosed in 16/60 (26.67%) infants [22/120 ears (18.33%)] at a mean age of 3.69 ± 2.80 (SD) months. This was significantly , moderate in 11 , and severe in 1  at baseline. In the sixteen AN patients, 22/32 (68.7%) ears were found to be affected by some degree of AN: mild in 8/32 (25.0%), moderate in 13/32 (40.6%), and severe in 1/32 cases (3.1%). No case of profound hearing loss was reported.Further audiological evaluations were considered only in the 16 children diagnosed with AN at first ABR. The timing (mean ± SD) and the auditory performance of these infants, as detected by ABRs and audiometry over time, are illustrated in Table The temporal variations of the auditory thresholds, from AN detection to normalization, for each of the 16 children, are detailed in Fig. All children diagnosed with AN at baseline achieved a normal auditory threshold (≤30 dB-HL) over time; in 14/16 cases  this was accomplished within 18 months of life. In only 1 case  infants aged ≥ 2 years and was normal in all cases (Pure Tone Average ≤ 25 dB-HL); in the remaining 3/16 (18.75%) infants aged < 2 years the last audiological evaluation coincided with the last normal ABR. The mean age at last audiological evaluation was 32.44 ± 17.58 (SD) months (range 5–60 months) in AN infants.When the cognitive, language, and motor areas were investigated by means of BSID-III, no significant differences were detected between groups Table .Table 3cCMV infection is the most common non-genetic cause of SNHL, whereas the term SNHL classically defines a hearing loss possibly due to both cochlear and/or auditory nerve damage.Two recent consensus statements on cCMV infection have recently highlighted the uncertainty surrounding the therapeutic management of isolated SNHL at birth , 5. In fThe aim of our study was to investigate the auditory evolution of children with cCMV asymptomatic infection, not receiving antiviral treatment, who had a diagnosis of isolated SNHL at birth due to AN . In order to emphasize any change in the auditory threshold over time, we choose to rely on the worst ear for severity grading of SNHL, rather than on a functional evaluation based on best-ear assessment .Our study demonstrates that a mild/moderate isolated AN at birth (particularly in the range ≤ 50 dB-HL), is a not negligible event in infants with asymptomatic cCMV infection. Of interest, a normal hearing threshold was always recovered over time, in absence of antiviral treatment, in these cases.p = 0.007) than not affected ones. In this light, also the distribution of AN cases along all trimesters of maternal infection  relevant to the nature of the study itself. Further, though our population was scheduled for ABR within 3 months of life, the resulting mean age at first ABR was substantially higher (5.00 ± 2.79 months). Frequent postponements of audiological appointments by parents and busy waiting lists were the main reasons for this to occur. Finally, the lack of a systematic ABR assessment in the first month of life prevented us an accurate estimate of the burden of AN at birth and, most important, some applicability of these data in the restricted time window available to decide if therapy has to be performed (within 28 days of life) [Despite the above limitations, we believe that this study could raise some important cues worthy of being further verified in prospective, possibly controlled, trials.Infants with mild/moderate SNHL on first ABR and a bilateral pass OAE should be suspected for a maturation delay of the auditory thresholds and, in our opinion, these infants should be possibly retested before any possible therapeutic decision. Severe/profound AN cases were almost absent in our cohort (only 1 ear with severe AN) and this prevented us any meaningful consideration about the possible management of AN of higher degree.OAE, together with ABR, should be part of the initial hearing evaluation of SNHL in cCMV infected infants in order to assess if a cochlear damage, or an auditory neuropathy, is responsible for a hearing loss. In fact, if the clinician relies exclusively on ABR testing to assess the burden of SNHL at birth, this might include, according to our experience, a variable amount of mild/moderate “maturative” AN cases possibly leading to antiviral overtreatment in this population.Besides, testing cCMV infected infants with both OAE and ABR could allow a separate evaluation of both audithory outcome (SNHL due to cochlear damage vs. SNHL due to AN), thus enabling to draw data about a possible, different management between the two types of hearing loss.In cCMV infected infants, a delayed maturation of the auditory pathways should be suspected when a mild/moderate isolated AN at birth is detected. It might be advisible to retest these infants before any possible therapeutic decision.Prospective, possibly controlled trials, conducted on larger populations and with a longer audiological follow-up, are needed to confirm our findings.


H9N2 avian influenza virus is endemic in Egyptian poultry flocks. The role of the live viral vaccines such as LaSota in exaggeration of the clinical picture of H9N2 infection under field conditions is significantly important leading to severe economic losses due to higher mortality and lower growth performance. This experiment was designed to identify the possible interaction between experimental infection with H9N2 virus and NDV live vaccine (LaSota strain) in broiler chickens. Six groups each of 20 broiler chicks were used. Three groups (G1–3) were infected with H9N2 and vaccinated with LaSota, 3 days before, at the same day or 3 days post vaccination (dpv), while the remaining groups (G4–6) were non-vaccinated infected, vaccinated non-infected and non-vaccinated non-infected.P < 0.05) in chicken’s body weight of all H9N2 infected groups from G1 to G4 compared to non-infected control groups, G5 and G6 respectively.The highest mortality rate (37.5%) was noticed in chickens of G1 (H9N2 infected 3 days prior LaSota vaccination). Also, this bird group had the most severe clinical signs, histopathological lesions and the longest viral shedding for 9 days post infection (dpi). In the 2nd and 3rd groups, the mortality rate was the similar (31.2%) with less pronounced clinical signs, histopathological lesions and H9N2 shedding was for only 6 dpi with the least shedding quantity in chickens of G3. The control non-vaccinated infected chickens (G4) had 18.7% mortality with the least degree of clinical signs, lesions and the highest viral shedding quantity but only for 6 dpi. At 35 days of age, there was a statistical significant decrease (It was clear that laSota vaccination significantly affect H9N2 infection in broiler chickens regarding clinical signs, mortality rate, lesions, performance and viral shedding. Staphylococcus aureus, Ornithobacterium rhinotracheale and E. coli or immune suppression, which exacerbates H9N2 influenza A virus infection in chickens [Low pathogenic avian influenza (LPAI) subtype H9N2 virus is widespread worldwide, being endemic in poultry populations in Asia and the Middle East. The first isolation of H9N2 virus in Egypt was obtained from the apparently healthy commercial bobwhite quail flock (A/quail/Egypt/113413v/2011/H9N2) in May 2011 . Later ochickens , 23. Lenchickens . Viral ichickens .In this experiment, we studied the interaction between LPAI subtype H9N2 infection and NDV vaccine (LaSota strain) in broiler chickens regarding clinical signs, body weights, post-mortem lesions, mortality, viral shedding, histopathological lesions and serological response for both viruses.The observed signs in all infected groups with H9N2 virus (G1–4) were depression, decreased feed consumption and respiratory signs. The most severe clinical signs in terms of morbidity were observed in chickens of G1, while the least were in chickens of G4 as the respiratory signs started by sneezing and coughing then exaggerated as rales, nasal discharge, and head swelling till the end of observation period (21 days). Also, white greenish diarrhea was observed from 3rd dpi and subsiding at 11th -12th dpi. In addition, only in G1 exhibited thick oral mucous, which started at 7th dpi and continued until end of observation period.Chickens of G1 suffered from early highest mortality 6/16 birds (37.5%) from 7th to 17th dpi compared to 31.2% (5/16 birds) in both G2 and G3 which began from 8th to 15th and 2nd to 9th dpi, respectively in both groups. While, in G4, the mortality rate was 18.7% (3/16 birds) with delayed start at day 12th dpi and continued only for 3 days.The most prominent PM lesions appeared were tracheitis, pneumonia, air sacculitis, splenomegaly, pancreatitis, enteritis, and nephritis. Chickens of G1 were more affected than other groups in terms of frequency and severity of lesions. No thymus, bursal lesions or caseous plug at tracheal bifurcation were observed.No clinical signs, mortalities or PM lesions were recorded in G5 and G6.P < 0.05) in compare to the birds of G2, G5 and G6 at 21th day of age. At 28th day, all vaccinated and infected chickens  were significantly decreased (P < 0.05) in body weights than G4, G5 and G6. Finally, at 35th day of age the body weights were significantly decreased (P < 0.05) in all H9N2 infected groups from G1 to G4 as 1540, 1509, 1474 and 1605 g, respectively than non-infected control G5 and G6 as 1769 and 1782 g, respectively  lower than G2, G4 or G1 for (with no significant difference between samples of chicken G1 and G4). While, the only significant lower titers (P < 0.05) at 14th dpi were in G1 (4.7) rather than G2, G3 and G4 .For H9N2: At 7th dpi, the HI titers of G3 were significantly (P < 0.05) HI titers were G2, G3 and G5 as 4.2, 4 and 3.9 and 3.7, 3.9 and 4.2, respectively compared to the lowest significant (P < 0.05) titers in G4 at the same dpi as 2.8 and 0.9, respectively  under experimental condition.No clinical signs were observed in G5 and G6 (non-infected groups), while infected and non-vaccinated G4 exhibited mild respiratory signs (without head swelling) and diarrhea. All groups infected and vaccinated with LaSota showed more severe clinical signs started from 3 dpi and continued until the end of the observation period except diarrhea that stopped after 7 days from its appearance. Interference between H9N2 and vNDV was studied by Bonfante, et al.  who repoThe highest severity of signs was recorded specially in chickens of G1 at 3–6 dpi concurrently with high H9N2 viral shedding in this group compared to G4 as detected by rRT-PCR, the results are in agreement with the findings detected by Kwon, et al. , which oRegarding the mortality, the chickens of G3 were suffered from early mortality at 2nd dpi and persisted till 11 dpi while the highest mortality (37.5%) was observed in G1. The least mortality 18.7%) was in chicken G4 began from 12th dpi and continued only for 3 days. Some studies reported no mortality after experimental infection of SPF-chickens with H9N2 , 8, 26..7% was iThis unusual high mortality rate in the present study may be due to the use of commercial broiler and not SPF chicks, which might be naturally carried some bacterial infection such as Mycoplasma and/or E coli, which are known to exaggerate the mortality of H9N2 infection. Li, et al.  found ouAlso, the increase in morbidity and mortality frequency after H9N2 AIV infection could be as a result of viral dissemination and spreading from the nasal cavity, which is the primary site of infection, to the lower respiratory tract, kidneys, intestines and some visceral organs , 28.P < 0.05) at the end of the experiment was reported among all infected groups in compare to G5 and G6, where the vaccinated and infected groups  was more affected than that only infected with H9N2 (G4). Also, the bird weights in G4 significantly (P < 0.05) differ by 9.2 and 9.9% than G5 and G6 respectively. In contrast, Costa-Hurtado, et al. [A significant decrease in body weights  and kidney especially in cases of infected and LaSota vaccinated birds. However, the respiratory organs showed more prominent gross lesions in comparison to other organs. Similar findings were observed by Taubenberger and Morens ; Capua aAlexander  reportedHistopathologically, chickens of G1 had severe of tracheal epithelial degeneration and hyperplasia of the mucosal lining compared to mild to moderate degree in G2, G3 and G4 which may be related to the higher late mortality obtained in this group along with obvious renal glomerulonephritis causing renal failure. This agreed with Aslam, et al. [Also, Doustar, et al.  proved iH9N2 infected birds (G4) also showed marked lymphoid depletion in the form of degeneration of thymocytes, follicular atrophy and cystic follicles of bursa of Fabricius and splenic lymphoid depletion. Similar results were reported by Kwon, et al. . In addiP < 0.05) by the presence of H9N2 suggesting that NDV had insufficient replication to trigger the humoral response even though they were administered the typical vaccination dose of LaSota strain. Another explanation is the immunosuppressive effect of H9N2 that proved histopathologically as degeneration of thymocytes and lymphoid depletion of bursa and spleen. Also, Allawe [Interestingly, the antibody titers against NDV (LaSota- vaccine) were significantly decreased  appeared in G3 versus the highest concentration  in the non-vaccinated and infected group (G4) at 3dpi and 614 virus copy/swab compared to 28,500 virus copy/swab at 6 dpi in both groups respectively, may turn out a possibility of some mechanism of interference between NDV live vaccines and H9N2 viruses in the respiratory tract of broiler chickens.This may be related to the fact that LaSota vaccine and LPAI viruses replicate in cells where there are trypsin-like proteases such as in the upper respiratory and intestinal epithelium, so they might compete for the same target cells or replicate in adjacent cells , 36. LaSAlso, LaSota NDV strain is known to be a weak interferon inducer as part of their low virulent phenotype profile, local interferon production might still be able to interfere with LPAIV replication .The results obtained underline the importance of co-infections, which can either exacerbate clinical disease or affect virus replication by lowering viral titers to under the levels of detection and affecting serological results and in some cases prolonged the time of virus shed which could favor for further transmission and these data was supported by Costa-Hurtado, et al.  and Ge, There was an adverse effect of combined infection with H9N2 and live LaSota vaccination in terms of clinical signs, body weights, mortality, histopathological lesions, viral shedding, and humoral immune response. Here we also recommend to diagnose the presence of H9N2 infection before ND vaccination to avoid to the exaggeration and immunosuppressive effect of H9N2 infection.A total of 130 one-day old commercial broiler chicks Cobb 500 purchased from a local poultry company (NASCO-Egypt) located in Alexandria governorate were used for the experimental infection in this study. All chicken groups were weighted at 14th, 21th, 28th and 35th day of age. Study design and allocating animals to experimental groups are explained in Table All the experimental work, tests and procedures were complied with the general guidelines of Damanhour University and were approved by the Local Ethics Commission of the Animal Health and Welfare of Damanhour University with respect to care of animals under study and all efforts were made to minimize suffering. The Ethical Approval Code was DMU/VetMed-2017/0023.Chicks were housed in galvanized wire cages (40 cm high × 50 cm width × 100 cm length) in (ten chicks each). Fresh feed and water were offered ad-libitum through the experimental period. All chicks were kept under the same managerial, hygienic and environmental conditions. Chicks were fed to cover their requirements according to NRC . The sacVaccines. Birds of G1–4 were vaccinated against NDV by eye drop between days 15th to 21st  at 18th day of age [Egyptian H9N2 strain isolated from broiler chickens in Alexandria Governorate (A/Chicken/Egypt/93/2015 and the GenBank accession No: KY872759) was used as a challenge virus in a dose 0.2 ml/bird of 10y of age .Ten chicks were sacrificed at the first day of age and serum samples were used to determine the level of maternally derived antibodies against H9N2 and NDV using Haemagglutination inhibition test. The remaining 120 chicks were distributed randomly into 6 equal groups of each 20 chicks. Birds were weighed and sacrificed by exsanguination after prior anesthesia.Clinical signs, Mortality rates and Post-mortem (PM) lesions. Birds in all groups were examined daily up to 35 days of age and all disease signs, mortalities and PM lesions of dead or sacrificed birds were recorded.Histopathological examination. Two birds from each group were sacrificed under anesthesia with intravenous injection of sodium pentobarbital (50 mg/kg) at 4th and 9th dpi in groups (1–4) and at 22 and 27 days of age in groups 5 and 6. Samples from trachea, thymus, bursa, spleen and kidney were collected, fixed in 10% formalin solution, stained with Hematoxylin and Eosin (HE) and examined for lesions [barbital 0 mg/kg aHaemagglutination inhibition (HI) test. Serum samples collected at 7th and 14th dpi were tested by HI test for H9N2 and ND antibodies using standard haemagglutination antigens (28 titers) for both viruses. HI test was applied using 4 Haemagglutination Units (HAU) of the relevant antigens and 25 μl of 1% chicken RBCs according to Alexander [Quantification of H9N2 virus shedding by Real-Time RT-PCR (rRT-PCR). Pooled tracheal swabs were collected from 10 chickens in each group just before H9N2 infection then at day 3, 6 and 9 post H9N2 infections. The RNA was extracted using QIAamp viral RNA Mini-kits (QIAGEN 52904- Germany) and the test was performed using rRT-PCR (Strategen 3005P USA) with Quantitect RT-PCR Kit reaction buffer (QIAGEN 204443- Germany) and positive control RNA from A/quail/Egypt/113413v/2011(H9N2). The Sequence of H9 primers and probe as published by Ben Shabat, et al. [, et al.  were useThermal profile was RT at 45 °C for 10 min and 95 °C for 10 min, followed by 40 cycles of PCR at 95 °C for 15 s for denaturation and 60 °C for 45 s for annealing and extension.All the birds were examined daily for the following parameters:p < 0.05.Data of birds’ body weight were subjected to analysis of variance procedures appropriate for a completely randomized design using the GLM procedures of SPSS (2008). The differences among means were determined using the student Newman keuls test. Statements of statistical significance are based on


N-acetylneuraminate, glutaric acid, cystine, 2-methylpropanoate, butanoate and xanthine. As expected, the age of patients was also one of the main discriminant parameters. Metabolic signatures were observed for duration of hearing loss. These findings are promising steps towards illuminating the pathophysiological pathways associated with etiologies of sensorineural hearing loss, and hold open the possibilities of further explorations into the mechanisms of sensorineural hearing loss using metabolomic analysis.Perilymph metabolomic analysis is an emerging innovative strategy to improve our knowledge of physiopathology in sensorineural hearing loss. This study aims to develop a metabolomic profile of human perilymph with which to evaluate the relationship between metabolome and the duration of hearing loss. Inclusion criteria were eligibility for cochlear implantation and easy access to the round window during surgery; patients with residual acoustic hearing in the ear to be implanted were excluded. Human perilymph was sampled from 19 subjects during cochlear implantation surgery. The perilymph analysis was performed by Liquid Chromatography−High-Resolution Mass and data were analyzed by supervised multivariate analysis based on Partial Least-Squares Discriminant Analysis and univariate analysis. Samples were grouped according to their median duration of hearing loss. We included the age of patients as a covariate in our models. Statistical analysis and pathways evaluation were performed using Metaboanalyst. Nineteen samples of human perilymph were analyzed, and a total of 106 different metabolites were identified. Metabolomic profiles were significantly different for subjects with ≤12 or >12 years of hearing loss, highlighting the following discriminant compounds:  Sensorineural hearing loss (SNHL) can negatively affect the development of spoken language, education and social interactions, can reduce quality of life, and limit human communication and socio-professional relations . Around Two of the strongest predictors of CI outcomes are the etiology and duration of deafness ,10. Alth2+ in the HCs is also a source of mitochondrial disturbance, apoptosis and HC death. (3) MAPKs are signaling proteins contributing to the stress-activated protein kinase, a key mediator in oxidative stress and inflammation. (4) Programmed HC death can be mediated by the evoked intracellular pathway or by the extracellular pathway caspase. (5) Infection and inflammation may also induce apoptosis through a response based on innate immunity, promoting the death of pathogens. While many pathways leading to HC and SGN loss have been described and appear to be interlinked, no diagnostic tool is currently available to characterize the lesion site within the cochlea and intracellular mechanisms of SNHL.Cellular mechanisms involved in SNHL are intricately linked. Five main pathways are described by Wong et al. : (1) AccAuditory and language performance with CIs also depend upon the duration of deafness . In caseMetabolomics is an emerging strategy that can be used to find biomarkers that are useful in increasing the knowledge of disease pathogenesis, and may help in diagnosis or prognosis. The development of high throughput analytical methods such as Liquid Chromatography, coupled with High Resolution Mass Spectrometry (LC-HRMS) has enabled numerous health applications for an analysis of biological fluids, cells and tissue . In the The objective of this study was to describe the metabolomic profile of perilymph extracted from patients with SNHL during CI surgery.In total, 106 different metabolites were identified . No outln = 10) and 36 ± 15 years for Group 2 (n = 9). The mean age at cochlear implantation was 38 ± 30 years for Group 1 and 65 ± 19 years for Group 2; a Mann-Whitney test showed no significant difference between the groups for age at implantation (p = 0.062). As different etiologies were observed in the cohort, PCA was used to evaluate the distribution of etiologies within each group; and within each group, the etiologies were similarly distributed.Patients were divided into two groups according to the median duration of hearing loss (12 years). Group characteristics are presented in N-acetylneuraminate, glutaric acid, l-cystine, 2-methylpropanoate, butanoate, xanthine, l-histidine, S-lactate, 4-hydroxy-l-proline, serotonin, 2-deoxy-D-glucose, N-acetyl-l-alanine, l-proline and taurine. As expected, age of patients was the second most important discriminant parameter in the model.Supervised multivariate analysis revealed a good model to discriminate Group 1 and 2 according to the following metabolites  with p < 0.1 and fold change  > 1.2. All metabolites included in multivariate model were also relevant in univariate model but did not reach significance.The volcano plot highlighted only one metabolite  plays a key role in the discrimination between groups according to hearing loss duration.N-acetylneuraminate and the duration of hearing loss. This metabolite is the predominant sialic acid found in mammalian cells, is generally located at the terminal of glycoprotein and glycolipid on the surface of the cell membrane, and is a product of glucose degradation. Elevated rates of this metabolite may be explained by the cell membrane breaking . We would expect elevated levels of this metabolite during ongoing cell death, as in the beginning of progressive hearing loss, and not after a long duration of hearing loss. This hypothesis is difficult to validate, as the direct account of survival hair cells was not performed in this study. Histological study will be necessary to confirm this hypothesis.We found a correlation between By revealing molecular mechanisms of hearing loss and understanding the role of each metabolite in the intricate auditory network, deafness endophenotypes may be better characterized according to metabolic signature, allowing for better understanding of the different pathways involved in hearing loss. The approach is promising, but needs further studies with more patients, especially to investigate the relationship between perilymph metabolomics profiles and SNHL etiologies. A multi-center study would be preferable to recruit adequate numbers of patients with a distribution of etiologies, and will ultimately provide a better understanding of the physiopathology of SNHL. Animal studies, where the age at hearing loss, severity of hearing loss, age at implantation and chronological age can be explicitly controlled, may also provide important insights regarding how these factors may be reflected in the metabolomic profile.n = 2). Thus, 19 patients were ultimately included in the study.Subjects were recruited from the otolaryngology department at University Hospital of Tours. All participants provided written informed consent. The Ethics Committee of the University Hospital of Tours approved the protocol (2015-045). All the patients met criteria for a cochlear implantation. Any patient with a residual acoustic hearing in either ear was excluded from the study. Twenty-one patients who underwent CI surgery from June 2015 to July 2016 were included in the study. Two patients were subsequently excluded during CI surgery due to a poor exposure of the round window (n = 8), presbycusis (n = 5), sudden hearing loss (n = 2), temporal bone fracture (n = 2), Meniere’s disease (n = 1), trauma (n = 1). Audiometric data was collected for at least at one year before implantation, shortly before implantation, and one year after implantation. Before implantation, aided word recognition was <50% correct at 60 dB SPL for all the included patients.Demographic information such as sex, age, onset of hearing loss, duration of hearing loss, duration of hearing aid (HA) use and etiology of hearing loss, was collected as part of the standard CI candidacy evaluation . Among tPatients were grouped according to the median duration of hearing loss  . This pag for 5 min and 0.8 µL were aliquoted.Perilymph samples were collected from patients during CI surgery, before parenteral injection of solumedrol 1mg/kg. After the posterior tympanotomy, the round window was exposed. We collected around 1 µL of perilymph using a 22 g needle through the round window membrane. The perilymph samples were stored in polypropylene tubes at −80 °C immediately after collection and until analysis. Before testing, samples were thawed, centrifuged at 3000× The metabolites found in the perilymph and the metabolic pathways were analyzed for all patients.LC-HRMS analysis was performed using a UPLC Ultimate 3000 system , coupled to a Q-Exactive mass spectrometer  and operated in the positive (ESI+) and negative (ESI−) electrospray ionization modes (one run for each mode). The perilymph analysis method, including the protocols for metabolite extraction and MS methods, are fully described in Mavel et al. . Liquid g for 15 min at 4 °C. The supernatant (95 µL) was evaporated with a SpeedVac concentrator at 40 °C and the dry residue was re-suspended in 100 µL of methanol/water (50:50). Finally the samples were vortexed for 1 min and centrifugated at 20,000× g at 4 °C for 10 min. Quality control (QC) samples were prepared from a mix of all extracted samples.Metabolism of perilymph was quenched and metabolites were extracted with 100 µL of methanol added to 0.8 µL of perilymph. Samples were vortexed for 1 min and stirred for 10 min at 4 °C. The samples were left at −20 °C for 30 min then centrifuged at 15,000× ®, St. Louis, MO, USA) and according the retention time (± 20 s of the standard reference), the molecular mass (within a range of 10 ppm around the reference) and the isotopic ratios. The signal value was calculated using Xcalibur® software version 4.1  by integrating the chromatographic peak area corresponding to the selected metabolite.Metabolites were identified with a library of standard compounds .p-value of the t-tests. The x-axis represents the fold change between the subject groups . The y-axis represents the p-value for t-tests of differences between samples . Correction for multiple test was systematically applied, but results without correction were presented to have an overview of the most promising metabolites in order to give more opportunity to explain pathophysiology with no aim of prediction.The univariate analysis was based on the non-parametric Wilcoxon test using Metaboanalyst (V 2.1). Univariate analysis is represented by a volcano plot combining two relevant information: Fold-change and The classification method was based on unsupervised Principal Component Analysis (PCA) to evaluate distribution of samples and identify outsiders, and supervised analysis based on Partial Least-Squares Discriminant Analysis (PLS-DA) with associated accuracy prediction. Values of Variable Influence on Projection (VIP) represent the importance of the compound (metabolite/lipid) for the PLS-DA models, the score plots show the classified samples, and the loadings characterize the relation between the Y and X variables . We included age of patients as a covariate in our models.This work provides proof of concept for a promising new strategy with which to better understand hearing loss. We used novel approaches toward collecting and analyzing perilymph in patients with SNHL, and found a correlation between the metabolomic profiles of perilymph and the duration of hearing loss.


An 8-year-old boy was referred to the ENT department for further evaluation of right-sided conductive hearing loss. A small cyst anterior to the oval window and fixation of the stapes footplate were observed during an exploratory tympanotomy. The concentration of a perilymph-specific protein, cochlin-tomoprotein (CTP), in the middle ear lavage fluid was measured with an ELISA-based CTP detection kit. The level of CTP in the middle ear lavage fluid before fenestration of the cyst was 0.26 ng/ml (negative), and its level after fenestration was 2.98 ng/ml (positive), confirming the presence of perilymph in the cyst. A small bone dehiscence, considered to be the fissula ante fenestram, was observed anterior to the stapes footplate after removal of the cyst. The CTP detection test results allowed us to confirm that the small bone dehiscence was connected to the inner ear. After superior semicircular canal dehiscence syndrome (SSCD) was first described by Minor et al. , dehisceThe classic symptoms and signs of these syndromes are vertigo, nystagmus induced by loud noises (Tullio phenomenon) or increases in external auditory canal pressure (Hennebert sign), and a characteristic low-frequency air-bone gap with audiometry due to decreased air conduction and increased bone conduction , 5. PatiThese symptoms and signs are not always present in the patients. Thus, surgery is important for making the definitive diagnosis when patients' symptoms and radiological findings suggest OCDS or TWS. However, it is usually hard to confirm that the dehiscence in the otic capsule is connected to the inner ear, even with surgery. In addition, the confirmation of existing perilymph in the middle ear is difficult. Even if fluid leakage is found in the middle ear, it is often difficult to distinguish perilymphatic flow from mucous membrane seepage, cerebrospinal fluid (CSF), or other fluids.COCH gene, has been proven to be a perilymph-specific protein and is used as a diagnostic biochemical marker for perilymph leakage (We treated an 8-year-old boy with right-sided conductive hearing loss caused by an open fissula ante fenestram (FAF) and fixed stapes footplate. During tympanotomy, we identified a cystic lesion anterior to the stapes. The fluid content of the lesion was identified as perilymph using a cochlin-tomoprotein (CTP) detection test. CTP, the shortest isoform of cochlin encoded by the  leakage –10. We pAn 8-year-old boy was referred to our hospital for further evaluation of right-sided conductive hearing loss identified at a health check-up at school . He did 2SO4). The optical density of each sample at 450 nm was then measured. Samples were measured in duplicate, and serially diluted rhCTP (E. coli) was used to generate a standard curve. The concentration of CTP was calculated from the standard curve by using the linear regression method. It takes approximately 2 weeks to obtain the test results  is a slit-like opening anterior to the oval window, an anatomical variation of the bony labyrinth . A FAF mConductive hearing loss implies a mechanical problem in the outer or middle ear. In this case, the patient's outer ear and tympanic membrane were normal. His conductive hearing loss was considered to be due to ossicular malformation or other middle ear malformations. To confirm the diagnosis, we performed an exploratory tympanotomy. The improvement of conductive hearing loss by 15–20 dB at a low frequency  was consTo date, the phenotype of TWS has been reported in association with superior semicircular canal dehiscence (SSCD), posterior canal dehiscence, posterior canal-jugular bulb dehiscence, cochlea-carotid dehiscence, cochlea-facial nerve dehiscence, and wide vestibular aqueduct . NeverthCTP, a 16-kDa short isoform of cochlin, has been identified only in the perilymph . CochlinThe CTP detection test is helpful in evaluating the pathophysiology of hearing loss or vertigo by verifying the existence of perilymph. There are some reports that CT-negative third window syndromes with exactly the same clinical phenotype also exist . Those cWe report the first case of an otic capsule dehiscence confirmed using a CTP ELISA detection test. The CTP detection test is extremely useful to verify the existence of perilymph in the middle ear.Written informed consent was obtained from the legal guardians of the participant for the publication of this case report.TF, TK, KS, and KD reviewed the literature and collected data. TF made figures and wrote a first version of the manuscript. TI, TS, and KD edited the manuscript. All authors contributed to the final version of the manuscript.Saitama Medical University has the patents for the hCTP ELISA test. The development of this hCTP ELISA was performed in collaboration with IBL Inc. under license from (and with) the technical assistance of Saitama Medical University with royalties. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BackgroundChildren with congenital heart disease (CHD) are at increased risk of neurodevelopmental deficits, and the presence of sensorineural hearing loss (SNHL) may further lead to poor language skills acquisition and speech delays. Prevalence of SNHL in the general pediatric population is estimated to be 0.2% at birth to 0.35% during adolescence. Very few studies have attempted to estimate SNHL prevalence in children who have undergone congenital heart surgery.MethodsThis retrospective study aimed to estimate SNHL prevalence in children who underwent congenital heart surgery in our institution and were followed up in our high-risk pediatric cardiology clinics for four years from 2009 to 2013. Data were collected on demographics, preoperative variables, surgical variables, and post-operative variables.ResultsSNHL prevalence in asymptomatic, palliated/repaired CHD patients followed in our high-risk clinics and undergoing routine surveillance was 11.6% (20 of 172 patients with hearing impairment). SNHL prevalence was not statistically higher in single-ventricle patients (17.2%) compared to biventricular patients (14.7%). Inotropic score in the first 24 hours of postoperative period (p=0.05), lowest arterial PaO2 (p=0.003), duration of Lasix drip (p=0), and bolus dose in days (p=0.03) were all found to be statistically significant in the hearing-impaired group. However, using logistic regression, we identified no statistically significant predictors for hearing loss.ConclusionThe results suggest the need for routine audiology screening of all patients with complex CHD, especially those who have undergone neonatal cardiac repair/palliation at less than one year of age, irrespective of risk factors. Survival and life expectancy of children with congenital heart defects have dramatically increased in recent years due to advances in medical and surgical management strategies. Children with congenital heart disease (CHD) are at increased risk of neurodevelopmental deficits , and theThe first purpose of this study was to estimate the prevalence of SNHL in children who underwent congenital heart surgery in our institution and were followed up in our high-risk pediatric cardiology clinics for four years, from 2009 to 2013. The prevalence of SNHL in single-ventricle (SV) patients was also compared to that in biventricular (BiV) patients. Another objective of this study was to identify all possible risk factors associated with SNHL in palliated CHD patients to assess the need for routine hearing screening in patients with complex CHD.We performed a retrospective chart review of patients followed in our high-risk pediatric cardiology clinics from 2009 to 2013 who had routine audiology evaluations. The study design was approved by the institutional review board. Data were collected on patient demographics and preoperative variables such as prematurity and birth weight, presence of genetic syndromes, as well as surgical variables such as total cardiopulmonary bypass time, circulatory arrest time, and cross-clamp time , and clinical seizures. Data collection was performed by a research assistant who was blinded to the hearing test results. Table Post-operative variables were also collected. These included lowest arterial PExclusion criteria were any known genetic syndrome association with SNHL or family history of hearing loss, and any previously documented failed newborn screening. Drug dosages were calculated as cumulative doses in mg/kg based on the weight at the time of each operation. Audiology assessment was performed by an experienced pediatric audiologist using age-appropriate and standard procedures. Young children and developmentally delayed children underwent visually reinforced audiometry, while older children underwent behavioral audiological assessment using pure tone audiometry. All children with test results indicating impaired hearing underwent auditory brain stem response for confirmation. Hearing loss was classified as mild when the hearing loss range in decibels (dB) was 26-40 db. Hearing loss range in dB > 40 dB was grouped as moderate to severe to profound loss . CompariA total of 172 patients were identified, 20 of whom were found to have hearing impairment ranging from mild loss to moderate to severe to profound loss in one or both ears. No patient met the exclusion criteria. The prevalence of SNHL in asymptomatic; palliated/repaired CHD patients followed in our high-risk clinics undergoing routine surveillance was 11.6% (20 with hearing impairment out of 172 patients). The prevalence of SNHL was higher in SV patients (17.2%) compared to BiV patients 14.7%), but there was no statistically significant difference   survivors (2%-4%) . LimitatInner ear ischemia and hypo perfusion are the main proven physiological mechanisms for hearing loss after open heart surgery -12. HypoOur study supports the previously proven etiologies of hypoxia and longer duration of furosemide as strongly associated with SNHL. However, using logistic regression, we identified no single independent predictor as statistically significant for causing hearing loss in complex CHD. But because of the high prevalence of SNHL in this group, the results suggest the need for routine audiology screening of all patients with complex CHD, especially in patients who have undergone neonatal cardiac repair as well as palliation below one year of age, irrespective of risk factors. This screening is necessary to identify children at risk and intervene before SNHL negatively affects their neurodevelopment.


As people grow older, they may experience loss in hearing sensitivity. Age-related hearing loss may negatively affect the patient’s quality of life as it may lead to social isolation. In older patients with cancer, hearing loss can seriously interfere with the patient’s ability to deal properly with all aspects of their disease, and may have a cumulative effect on their already decreased quality of life. Therefore, the proper screening of those conditions is essential in order to optimise the patient’s comfort during and after treatment. This review article aims at providing a concise image of the nature of age-related hearing loss, and provides an overview of the screening methods that could be used in older patients with cancer. Older cancer patients are a vulnerable group of individuals, as they may have developed multiple comorbidities as a consequence of ageing. One of those comorbidities is age-related hearing loss (ARHL), also referred to as presbyacusis or presbycusis. Although its onset and rate of progression may vary widely among individuals, the prevalence of ARHL increases with age. A review by Roth et al. 2005) indicated that at the age of 70, 20% of European women and 30% of European men present with a loss of at least 30 decibel Hearing Loss (dB HL). At the age of 80, numbers increase to 55% of men and 45% of women being affected . In the 005 indicPresbyacusis is known to have a serious impact on patients’ daily life. It not only affects their physical, cognitive, and emotional activities, it also influences patients’ social functioning. As patients lose their hearing, they feel left out as they have difficulties communicating with others. Even without hearing loss, it has been reported that listening effort increases with age as a result of age-related declines in cognitive functioning, hearing sensitivity, and peripheral auditory functions . As a reDespite these difficulties, in cancer patients, who already have a lower quality of life, the optimisation of such a condition is crucial. Therefore, the National Comprehensive Cancer Network (NCCN) guidelines in Older Adult Oncology recommend an assessment of sensory functions such as vision and hearing  as part Over the years, many screening methods for age-related hearing loss have been described. Screening tests were identified by searching the electronic peer-reviewed databases MEDLINE and Web of Science. In a first search, the search strategy was kept broad and consisted only of terms for ‘hearing’  and the term ‘screening’. In a second search, once several screening tools were selected, a more detailed searching strategy was used, as it then consisted out of the term ‘screening’ and the term of the specific screening tool . The reference list of selected manuscripts was then further checked for eligible screening tools. The NCCN Guidelines in Older Adult Oncology recommend using a quick and simple screening tool. The test is performed by whispering the sentence “What is your name?” while standing behind the patient and occluding one ear. The patient fails if he does not hear the sentence. The test is repeated for the contralateral ear ,17. FreeAs the WVT has many problems when accounting for the IRR, researchers have examined other, more objective measures to screen for hearing loss. A popular objective tool is the AudioScope  . The AudA widely used screening questionnaire concerns the Hearing Handicap Inventory for the Elderly (HHIE). Since its development in 1982 by Ventry and Weinstein, the HHIE has gained a lot of interest in terms of screening for hearing loss in an older population . The HHIPure tone audiometry is known as the gold standard to assess hearing loss since its introduction as a clinical tool more than 70 years ago. A necessary requirement for optimal testing is a controlled test environment with ambient noise levels as close to 0 dB sound pressure level (SPL) (ISO 8253) . In thatIn our developing world, eHealth applications are gaining more and more interest. Using information and communication in healthcare can result in improved healthcare access and improved quality of service delivery . SwanepoEarTrumpet, AudCal, and uHear™ all run on iOS devices such as an iPod, iPad, or iPhone. EarTrumpet  was released in the iTunes Store in 2010, and is calibrated with standard Apple earbuds. The test is performed according to the Hughson–Westlake technique and allows basic , comprehensive 0.25–8.0 kHz), or custom testing. Test tones are pulsating with an 0.8-s duration while the silence interval varies between one and two seconds. Masking is done automatically when a difference of at least 35 dB is found between both ears. In a quiet room, 94% : 87–100%) of the thresholds detected with EarTrumpet were within 10 dB of the pure tone threshold obtained with conventional audiometry . Derin e–8.0 kHz,Another measure is otoacoustic emissions (OAEs). OAEs are an objective and fast measure to establish the function of the outer hair cells within the cochlea. They are widely used as a neonatal screening method to establish an audiological diagnosis. When conducting the test, brief acoustic stimuli are produced into the external ear canal. As a result, OAEs can be measured, which are low-volume sounds that originate from the cochlea . OAEs caAnother method to define the patient’s hearing is by use of a speech-in-noise (SPIN) test, as this type of test gives more accurate results in terms of speech intelligibility. SPIN tests give an idea of the patient’s ability to understand meaningful sentences in noisy situations, and provide valuable information, as listeners find themselves surrounded by background noise multiple times a day. The ability to understand speech-in-noise is usually presented as the speech reception threshold, which can be defined as the signal-to-noise ratio that an individual requires in order to understand 50% of the presented speech material . As stanAt present, an optimal screening tool to assess hearing loss in older patients with cancer is lacking. As a result, most geriatric oncology teams do not implement a hearing screening when assessing the CGA. However, the hearing assessment as part of the CGA should not be included to define the patient’s total vulnerability. It should be used as an important informative assessment, as knowing if the patient has a reduced hearing is crucial for cancer patients who need to hear and understand a lot of information on their diagnosis, its treatment, and possible treatment-related side effects. Older patients are very proud of their independence, and in many cases, sensory dysfunctions including vision and hearing loss are one of the first functional declines that they have to deal with. Although wearing glasses is well accepted socially by both young and older people, hearing aids are not that well accepted by many persons. As a result, older patients tend to hide a hearing loss, which, in an onco-geriatric population, may lead to missing essential information about the cancer diagnosis, its treatment, and/or its treatment-related adverse events . If healUntil a validated tool for use within the CGA is available, it is advised to incorporate a subjective assessment of the patient’s hearing as a screening method. If the person conducting the CGA feels that the patient has a hearing loss, this should be mentioned in the CGA report. Ideally, a pop-up regarding this matter should appear when any healthcare provider opens the patient’s medical record. It is not advised to ask the patient about his or her beliefs of their hearing, as our results indicated a large discrepancy between patient-reported hearing loss and objective audiometry results. Objective screening should at this point include pure tone audiometry. Future trials should focus on the development of an eHealth tool providing a SPIN test. Preferably, this SPIN test should be kept short, as listening effort is often increased in older subjects, and assessment of such a longer test could enhance fatigue, which is already present in many cancer patients. Nonetheless, this type of test will provide healthcare professionals with an adequate overview of how the patient experiences listening in daily situations.


Chronic inflammatory demyelinating polyneuropathy (CIDP) is a kind of autoimmune-mediated inflammation and demyelinating disease. The etiology is mainly related to autoimmune dysfunction. The conventional treatments of CIDP have relied on immunomodulation and inhibition therapies such as adrenal cortex hormone, intravenous immunoglobulin (IVIg) and plasma exchange. Hematopoietic stem cell transplantation (HSCT) is known as a novel therapy for autoimmune disorders, which provides the chance to cure CIDP. More than 70 patients with refractory CIDP have received HSCT. The clinical symptoms and electrophysiological examination results of most patients have been improved. However, the treatment still has risks. This review describes the pathogenesis of CIDP and the current conventional treatments, and highlights the application of HSCT in CIDP, including its efficacy and safety. CIDP is mainly a group of clinical syndromes. The course of the disease is chronic progression or recurrence lasting more than 8 weeks. It often manifested as symmetrical weakness of the proximal or distal limbs, loss of sensation and tendon reflexes. The main diagnostic methods include neurophysiological test, cerebrospinal fluid and pathological examination. Most patients often have cerebrospinal fluid protein-cell separation. Neuroelectrophysiological test suggests demyelinating lesions. Nerve biopsy shows segmental demyelination and regeneration, even the formation of “onion bulbs-like” changes. A retrospective study by Karine et al. The pathogenesis is still not very clear. Previous clinical case reports show that the onset of CIDP may be related to the induction of specific infections such as hepatitis B virus, EB virus and hepatitis C virus etc. The main targets of antibodies in CIDP are antigens on myelin and Schwann cells Infiltration of T lymphocytes Macrophages are also the main inflammatory cells and form colonies around blood vessels in nerves Current treatments that have been validated by clinical randomized controlled trials include corticosteroid, plasma exchange and IVIg. Regarding the study of corticosteroid therapy, in a prospective cohort study in 2012 The clinical effect of plasma exchange was confirmed in early clinical trials in 1986 In 2001, Mendell et al. Apart from the above-mentioned first-line treatments, other immunosuppressive and regulatory drugs have also been reported for clinical use and research, such as azathioprine. A clinical randomized trial in 1985 The clinical use of interferon (IFN) has been reported Although there are so many immunosuppressive therapies, they cannot induce long-term, drug-free and symptom-free remission, some patients with CIDP still relapse, even leading to disability and death. Therefore, patients may choose hematopoietic stem cell transplantation as a better treatment option.Hematopoietic stem cell transplantation has been widely used to treat refractory autoimmune diseases, aiming at eradicating autoreactive immune cells and rebuilding a new immune system that is self-tolerant. Relief of clinical symptoms of autoimmune diseases after hematopoietic stem cell transplantation is the result of immune system reconstruction From 2002 to 2018, there are about 70 cases of refractory CIDP treated by HSCT worldwide. In general, the relapse frequency of the patients has decreased with different degrees. The electrophysiological examination results have been significantly improved. The symptoms have diminished, the progress of the disease has been effectively controlled, and some patients have stopped immune regulation or inhibition therapy after HSCT.The earliest reported treatment of CIDP with hematopoietic stem cell transplantation was in 2002. Vermeulen et al. In 2007, Oyama In 2016, R. Kotas From 2002 till now, another 66 patients with refractory CIDP received HSCT treatment. These patients needed to fulfil the definite, probable, or possible EFNS/PNS criteria for CIDP. They were incomplete response or relapse after first-line treatment, and non-response to at least one second-line immunosuppressive drug . After HSCT, most of them have achieved good clinical efficacy. See Table Electrophysiological examination is an important method to evaluate the efficacy of HSCT in patients with CIDP. Specific observation indexes include compound muscle action potential (CMAP), sensory nerve action potential (SNAP) and nerve conduction velocity of limbs. The increase in amplitude of action potential and conduction velocity often indicate remission of the disease.Vermeulen et al. Remenyi et al. In 2009, Mahdi-Rogers et al. In 2013, Press et al. Safety-related indicators of HSCT therapy include adverse reactions, toxic effects and transplant-related mortality (TRM). Adverse reactions and toxic effects often include early adverse reactions and toxic effects (within 100 days after transplantation) and late adverse reactions and toxic effects (after 100 days of transplantation). The former may be related to allergic reaction and side effect induced by immunosuppressive drugs and opportunistic infection after immunosuppression. Common symptoms include fever, infection, abnormal liver function, etc. Bacterial or fungal infection occurs early after HSCT in neutropenia, while long-term hypolymphemia can reactivate latent viruses and other opportunistic infections, thus endangering patients until their immune systems are rebuilt Compared with autologous hematopoietic stem cell transplantation, allogeneic hematopoietic stem cell transplantation may bring more complications and higher mortality Transplant-related mortality is the most important problem in HSCT. In 2010 Gualandi et al. CIDP is an autoimmune disease, which is currently considered to be related to cellular and humoral immune abnormalities. Patients are often characterized by loss of sensation and weakness of limbs. Severe cases may even lead to disability or even death. Conventional treatments include immunosuppression and immune regulation, but some patients still have no response to such treatments, so HSCT is used. So far, more than 70 people have been treated with HSCT. HSCT is the only therapy that can induce long-term, asymptomatic remission in refractory autoimmune diseases and a possible new way to solve the problem of autoimmune regulation abnormalities, so that it provides a new approach and method for refractory CIDP. Judging from the effect of current preclinical tests on CIDP, although large-scale clinical RCT tests are lacking, and cases of relapse and complications related to HSCT have been reported, HSCT is safe and feasible in general. The symptoms and neurological score of most patients improved and a longer-term remission period was obtained. The complications after HSCT include toxic and side effects of conditioning drugs, virus and bacterial infection caused by hypo immunity after transplantation, but most of them can be improved by corresponding treatments. In conclusion, HSCT is a viable treatment option for refractory CIDP.


The vast majority of hearing loss, the most common sensory impairment, and vertigo, which commonly causes falls, both reflect underlying dysfunction of inner ear cells. Perilymph sampling can thus provide molecular cues to hearing and balance disorders. While such “liquid biopsy” of the inner ear is not yet in routine clinical practice, previous studies have uncovered alterations in perilymph in patients with certain types of hearing loss. However, the proteome of perilymph from patients with intact hearing has been unknown. Furthermore, no complete characterization of perilymph from patients with vestibular dysfunction has been reported. Here, using liquid-chromatography with tandem mass spectrometry, we analyzed samples of normal perilymph collected from three patients with skull base meningiomas and intact hearing. We identified 228 proteins that were common across the samples, establishing a greatly expanded proteome of the previously inferred normal human perilymph. Further comparison to perilymph obtained from three patients with vestibular dysfunction with drop attacks due to Meniere’s disease showed 38 proteins with significantly differential abundance. The abundance of four protein candidates with previously unknown roles in inner ear biology was validated in murine cochleae by immunohistochemistry and in situ hybridization: AACT, HGFAC, EFEMP1, and TGFBI. Together, these results motivate future work in characterizing the normal human perilymph and identifying biomarkers of inner ear disease. Fig 1). In addition, a third of the general population in the U.S. report vestibular symptoms such as vertigo, a persistent spinning sensation ..SERPINA3TMPRSS3, a serine protease in which mutations are associated with non-syndromic autosomal recessive deafness (DFNB8/10), inactivation of the protease shortly after birth led to severe and rapid degeneration of the organ of Corti and cochlear and saccular hair cells [Tmprss3 expression in cochlear and vestibular hair cells, supporting cells of the organ of Corti, epithelial cells of the spiral sulcus, and interdental cells [While it is plausible that protease inhibitors play an important role in regulating protease activity in the inner ear, the precise physiological role of AACT in the hearing and balance function has not been reported previously. In a genetically-engineered mouse model carrying mutations in ir cells . These ral cells . InteresHGF are associated with non-syndromic hearing loss, autosomal recessive deafness-39 (DFNB39) [MET is associated with human DFNB97 hearing loss [via viral-mediated intrathecal delivery of human HGF gene led to prevention of kanamycin-induced degeneration of hair cells and SGNs [via coupling with signaling through the c-MET pathway and by reducing levels of reactive oxygen species [Hgfac expression in cochlear cell types that benefit from exogenous HGF application in animal models provides an insight into the mechanism of this therapeutic effect.Hepatocyte growth factor (HGF) is important for human hearing because mutations in the genes encoding this protein and its receptor, MET, are associated with hearing loss. Specifically, non-coding mutations of (DFNB39) , while aing loss . Moreoveing loss . We haveing loss . HGFAC iing loss . HGF is ing loss ,35. Overand SGNs . Elsewhe species . FurtherEFEMP1 gene cause two inherited forms of macular degeneration [Efemp1 RNA expression in the spiral ganglion, spiral ligament, and sensory and supporting cells of the organ of Corti overlaps with the cochlear expression pattern of EGF receptor [EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), also known as Fibulin-3 (FBLN-3), belongs to a family of six proteins associated with the extracellular matrix elastic fibers and basement membranes . We focuneration . EFEMP1 neration . Previouneration . Our finreceptor ,43. WhilTGFBI underlie several types of corneal dystrophies where abnormal deposits of misfolded proteins and amyloid in the corneal stroma lead to visual impairment [Transforming growth factor beta induced (TGFBI), a structural homolog of periostin, is an extracellular matrix protein induced by TGF-β and normally expressed in fibroblasts, keratinocytes, and muscle cells ,45. The pairment . TGFBI ipairment , includipairment , wound hpairment , inflammpairment , tumorigpairment ,52, and pairment .Tgfb1 expression was reported in the embryonic cochlea in the cells of the inner (fibrocyte) compartment of the periotic mesenchyme, whose differentiation and expansion is subsequently regulated by the canonical Wnt signaling pathway [Tgfbi in specific cell types of the adult cochlea validates TGFBI detection in adult human perilymph, and motivates future studies to decipher the role of this protein in cochlear function.Although no prior studies have linked TGFBI expression with the pathophysiology that underlies disorders of hearing or balance,  pathway . Our locA comprehensive network and pathway analysis of the proteome of normal human perilymph highlighted several pathways of interest. The core proteome consisted of largely immunoglobulins, proteases, and protease inhibitors, suggesting that the perilymph is a tightly-controlled, well-balanced system in regulating inner ear function. The acute phase response is a rapid, protective, inflammatory mechanism triggered by a variety of factors. Regulation of inner ear inflammation is likely a critical function of perilymph proteins, as increased inflammatory cytokine production is associated with SNHL in meningitis, otitis media and bone diseases characterized by pathologic remodeling of the otic capsule, including otosclerosis and Paget’s disease ,56. The et al. theorized that inflammation associated with viral infection could affect the stria vascularis, dark cells, and endolymphatic sac in Meniere’s patients [Analysis of the differentially expressed genes in perilymph from patients with normal hearing compared to those with VD yielded two significant networks of interest: one involved in tissue injury and inflammation, and the other associated with energy production. These networks, along with the relative overabundance of enzymes in VD perilymph compared to normal perilymph, may reflect primary etiology or secondary damage to inner ear cells resulting in release of intracellular contents into perilymph. Several bodies of evidence in the literature support the link between inflammation, cellular degeneration and the pathogenesis of MD. Arenberg patients . Furtherpatients . Additiopatients . Finallypatients , and minpatients . The darpatients . When tepatients . Furtherpatients –69. Withpatients , and speOur patients with skull base meningiomas had normal or near-normal hearing. Unlike vestibular schwannomas where hearing is often impaired due to a combination of mechanical nerve compression and tumor-secreted factors, and hearing does not typically improve after tumor removal, hearing improvements have been observed after removal of intracananicular meningiomas. A recent histopathological study on human temporal bones with meningiomas of the IAC showed that cochlear damage is uncommon and hair cell loss is rarely detected . Therefoet al. reported the microRNA (miRNA) profile of human perilymph isolated from patients undergoing cochlear implantation, stapedectomy, or labyrinthectomy [Recently, Shew thectomy . A totalEfemp1, Hgfac, Tgfbi). Findings from this work will motivate the future development of diagnostic and prognostic assays for hearing loss and vestibular dysfunction based on proteomic markers from inner ear fluids.In this report, we have assembled the first proteome of normal human perilymph, collected from people with intact hearing, and performed comparative proteomic analysis of perilymph from patients undergoing labyrinthectomies for disabling vestibular dysfunction due to MD with drop attacks. Using tandem LC-MS/MS, we have significantly expanded the landscape of the known perilymph proteome to over 200 proteins, more than doubling candidate proteins found in previous efforts. We further validated 4 novel protein candidates that had not been previously described in the adult inner ear. We analyzed and defined their precise spatial distribution in the adult murine cochlea by immunohistochemistry (AACT) or fluorescent in-situ hybridization  are shown. A. Acute phase response signaling canonical pathway, B. Liver X receptor (LXR) / retinoid X receptors (RXR) Activation pathway, C. Farnesoid X receptor (FXR) / retinoid X receptors (RXR) Activation pathway. There is an overlap of approximately 21.6% - 26.4% of proteins in the pathway with that in the perilymph samples.The top 3 networks ((TIFF)Click here for additional data file.S2 FigA-C). A. Organismal Injury and Abnormalities, Respiratory Disease, Inflammatory Disease. B. Energy Production, Small Molecule Biochemistry, Developmental Disorder. C. Cell-To-Cell Signaling and Interaction, Cellular Assembly and Organization, Nervous System Development and Function. ERK 1/2, HSP, and APP were nodal molecules in each of the three networks, respectively.The top 3 networks are shown ((TIFF)Click here for additional data file.S3 FigPTA, pure tone average in decibels (dB). WR, Word recognition (%).(TIFF)Click here for additional data file.S4 FigDots represent individual samples.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.