IntroductionHearing loss affects people of all ages, and it may become a burden for patients as well as for those around them. It leads to social isolation and impacts the quality of life (QOL). Many studies aim to investigate the outcome of hearing aids as an intervention to treat hearing loss. Our study’s objective is to assess the QOL in participants and investigate the possible factors that have an impact on the outcomes of hearing aid use.MethodsThe study adopted a cross-sectional design and was carried out in the Armed Forces Hospital South Region, a tertiary care center in Aseer Province, Saudi Arabia. Participants were patients who presented to the ENT clinic with a complaint of hearing loss from 2017 to 2019 and who were prescribed hearing aids as their treatment. The study uses the World Health Organization Quality of Life BREF (WHOQOL-BREF) questionnaire and the International Outcome Inventory for Hearing Aids (IOI-HA) to measure the QOL and its determinants in patients using hearing aids.ResultsA total of 210 patients were included in the study. Sensorineural hearing loss (SNHL) was found in 72.2%, and 20% of patients were found to have bilateral hearing loss. Moderate or severe hearing loss was found in 80% of the patients in the worst hearing ear. The overall QOL among the participants was satisfactory, with the highest domain score being the social relationship domain (85.9%). The QOL was significantly higher in participants who were in an intimate relationship (P = 0.02). A positive correlation was found between the IOI-HA scores and the WHOQOL-BREF scores in general health , psychological health , and the overall QOL score .ConclusionHearing aids are a cost-effective intervention that improves QOL and prevents associated comorbidities. Compliance and adherence to hearing aids improve the QOL for patients, as well as for their spouses. Patients suffering from hearing loss while also in an intimate relationship had better QOL scores. A correlation was found in outcomes between the IOI-HA scores and the WHOQOL-BREF scores in general health, psychological health, and overall health. According to the WHO, hearing loss affects approximately 5% of the world’s population and is projected to affect more than 700 million people by 2050 . WorldwiHearing loss can negatively impact a person’s quality of life (QOL), even more so as their better ear worsens ,5. HeariThe objective of our study was to assess the QOL of patients who visited our hospital with hearing loss from 2017 to 2019 and who were prescribed hearing aids to manage their symptoms. We utilized the World Health Organization Quality of Life-BREF (WHOQOL-BREF) questionnaire as a tool to measure patients’ QOL. Our study also aimed to evaluate the outcome of hearing aid use by using the International Outcome Inventory-Hearing Aids (IOI-HA) tool. The IOI-HA is a questionnaire that was designed to determine, from the patient’s perspective, how beneficial hearing aids are as an intervention for their hearing loss . It compStudy designThe study adopted a cross-sectional analysis of QOL and hearing aid use outcomes using the WHOQOL-BREF and IOI-HA questionnaires. Data were collected via a telephone or in-person interview according to the patient’s preference. Patients with significant difficulty in communication because of their hearing loss were offered an interview through the WhatsApp online chatting platform. A detailed explanation of the questionnaire items was given to patients, and the interview took approximately 30 minutes to perform. Informed consent was obtained from all the patients verbally or through the online chatting platform. Data collected from the patient’s electronic file included their basic demographic data, hearing thresholds from their unaided average pure-tune audiometry results , type of hearing loss, and type of hearing aid prescribed to them after the hearing aid fitting process. The questionnaires included questions from the WHOOL-BREF and IOI-HA, as well as questions about general health, comorbidities, and otologic history.Setting and timeframe of researchOur research was carried out in the Province of Aseer, Saudi Arabia, at the Armed Forces Hospital-Southern Region (AFHSR). The AFHSR is one of the largest tertiary public centers in the province that provides free healthcare to military personnel and their families along with civilians. The hospital serves a region containing a population of four million . PatientStudy populationThe study population included patients who visited AFHSR with a complaint of hearing loss with or without any other otologic complaints and who were prescribed conventional hearing aids from 2017 to 2019. Only patients between the ages of 18 and 65 were included in the study. A total of 361 patients were contacted and asked to participate, and 210 patients agreed to participate in the study  . All statistical analyses were performed using two-tailed tests. A P-value of less than 0.05 was considered statistically significant. OOL was assessed using the WHOQOL scale manual, in which scores were converted to a scale from 0 to 10. QOL scores for each domain and overall scores were displayed by the mean, along with the standard deviation. Cross tabulation through one-way analysis of variants (ANOVA) and an independent t-test was used to assess the relationship between QOL, biodemographic data, severity of hearing loss, and type of hearing loss.The participants’ overall IOI-HA score (out of 35) was displayed as the mean with its standard deviation. Correlation analysis was used to assess the nature and significance of the relationship between WHOQOL-BREF domains and IOI-HA scores. Descriptive analysis based on the frequency and percentage of distribution was done for participants’ biodemographic data, hearing loss data, hearing aid usage, and clinical symptoms.Sample descriptionA total of 210 participants were included in the study, and their ages ranged from 18 to 65 years, with a mean age of 48.8 +/- 13.5 years. The sample contained 115 males (54.8%) and 95 females. Regarding the educational level, 126 (60%) had less than a high school education, while 21 (10%) had a university-level education. When the participants were assessed for comorbidities, 65 patients (31%) had diabetes mellitus, 52 (24.8%) had hypertension, 12 (5.7%) had hypothyroidism, and 112 patients (53.3%) had no comorbidities . Sensorineural hearing loss (SNHL) was found in 72.2% of those with right hearing loss and 73.6% of those with left hearing loss. Moderate hearing loss was found in 34.3% of the participants with right hearing loss and in 36.2% of participants with left hearing loss. Severe hearing loss was found in 27.1% of those with right hearing loss and in 24.8% of those with left hearing loss.Behind-the-ear hearing aids were prescribed to 75.7% of participants, while 20% were given in-the-ear hearing aids. Hearing aids were used for more than one year by 135 of the participants  were exposed to acoustic trauma, while 17 (8.1%) were exposed to head trauma. The most common complaints of patients in the two weeks prior to their interview were recent worsening of hearing and tinnitus , followed by the environmental domain (83.3%) and psychological health domain  compared to those who were not in an intimate relationship . Other measured factors did not show a significant association with QOL Table .Quality of hearing without hearing aid use was a significant determinant in QOL scores. Participants who faced only slight difficulty in hearing without a hearing aid showed an average score of 86% compared to 76.5% of those who reported that they could not hear at all without using their hearing aid .Association of QOL and IOI-HAThere was a significant positive correlation between the IOI-HA scores and WHOQOL-BREF scores in the domains of general health  and psychological health , as well as in overall QOL score  was used, it showed results suggesting that using hearing aids can reverse subjective social, emotional, and communication dysfunctions . The meaLotfi et al. reported an improvement in the HHIE score from approximately 66 to 21 in three months following the hearing aid fitting . HoweverWhen Stark et al. used the Short Form Health Survey 36 (SF-36), it showed a significant deterioration in the general health subscale without any remarkable changes in the other subscales . MoreoveThe American Academy of Audiology Task Force on the HRQOL concluded that hearing aid use improved HRQOL by reducing the psychological, social, and emotional effects of SNHL .Intimate relationship and QOL We found that participants who were in an intimate relationship had a better QOL score (82.7%) compared to those who were not . The effects of hearing loss on patients’ intimate partners have been investigated in multiple studies. Patients who were in an intimate relationship disclosed that their hearing loss had an impact on the QOL of their significant other . The mosBrooks et al. also reported that the difficulties experienced by the significant other of a person with hearing loss were correlated with the severity of the hearing loss itself . These dIOI-HA and WHOQOL-BREFWhile the WHOQOL-BREF aims to measure QOL by assessing multiple domains, the IOI-HA reflects the patient’s perspective on the effects of using hearing aids. A positive correlation was found between the IOI-HA scores and the WHOQOL-BREF scores in general health, psychological health, and overall health.Despite the increasing number of studies showing the benefits of hearing aids, Cox et al. reported that only 23% of the elderly with hearing loss were using their hearing aids . FurtherOur study is limited by its cross-sectional nature. Other studies showed equivocal results when alternative tools were used to assess the QoL and the outcomes of hearing aid use. However, it was evident that hearing aid use, when accompanied by proper counseling and appropriate fitting, can have a positive impact on the patient and their intimate partner. Further studies are required to evaluate factors that impact hearing aid use and compliance, especially in young and middle-aged adults. Moreover, policymakers should facilitate hearing aid accessibility and coverage by medical insurance considering their cost-effective and positive impact on patients’ QOL and decreased morbidity.QOL is significantly affected in patients with hearing loss, and multiple studies suggest that hearing aids are a cost-effective intervention that improves QOL. We conclude that hearing loss creates burdens and difficulties for both the patient and their partner. Therefore, involving the patient’s spouse in the management of hearing loss can be beneficial and might influence compliance with hearing aids.Furthermore, a significant correlation was found in outcomes between the IOI-HA scores and WHOQOL-BREF scores in general health, psychological health, and overall health. Moreover, policymakers should prioritize the necessary regulations to make hearing aids accessible and affordable to those in need. Doing so may decrease morbidities associated with hearing loss. Further studies are recommended to investigate factors that would help patients with hearing loss become compliant with their prescribed hearing aids, particularly in young-to-middle-aged patients. Health officials should also increase public awareness of preventable causes of hearing loss.


The aims of this study were to report the audiological characteristics of children with congenital unilateral hearing loss (UHL), examine the age at which the first reliable behavioural audiograms can be obtained, and investigate hearing changes from diagnosis at birth to the first reliable behavioural audiogram.This study included a sample of 91 children who were diagnosed with UHL via newborn hearing screening and had reliable behavioural audiograms before 7 years of age. Information about diagnosis, audiological characteristics and etiology were extracted from clinical reports. Regression analysis was used to explore the potential reasons influencing the age at which first reliable behavioural audiograms were obtained. Correlation and ANOVA analyses were conducted to examine changes in hearing at octave frequencies between 0.5 and 4 kHz. The proportions of hearing loss change, as well as the clinical characteristics of children with and without progressive hearing loss, were described according to two adopted definitions: Definition 1: criterion (1): a decrease in 10 dB or greater at two or more adjacent frequencies between 0.5 and 4 kHz, or criterion (2): a decrease in 15 dB or greater at one octave frequency in the same frequency range. Definition 2: a change of ≥20 dB in the average of pure-tone thresholds at 0.5, 1, and 2 kHz.n = 71) of children had hearing deterioration between the diagnosis and the first behavioural audiogram at one or more frequencies between 0.5 and 4 kHz, with a high proportion of them  developing severe to profound hearing loss. When using the averaged three frequency thresholds , only 26.4% of children (n = 24) in the sample were identified as having hearing deterioration. Applying definition 2 therefore underestimates the proportion of children that experienced hearing changes. The study also reported diverse characteristics of children with or without hearing deterioration.The study revealed that 48 children (52.7% of the sample of 91 children) had their first reliable behavioural audiogram by 3 years of age. The mean age at the first reliable behavioural audiogram was 3.0 years . We found a significant association between children's behaviour and the presence or absence of ongoing middle ear issues in relation to the delay in obtaining a reliable behavioural audiogram. When comparing the hearing thresholds at diagnosis with the first reliable behavioural audiogram across different frequencies, it was observed that the majority of children experienced deterioration rather than improvement in the initial impaired ear at each frequency. Notably, there were more instances of hearing changes (either deterioration or improvement), in the 500 Hz and 1,000 Hz frequency ranges compared to the 2,000 Hz and 4,000 Hz ranges. Seventy-eight percent (The finding that 78% of children diagnosed with UHL at birth had a decrease in hearing loss between the hearing levels at first diagnosis and their first behavioural audiogram highlights the importance of monitoring hearing threshold levels after diagnosis, so that appropriate intervention can be implemented in a timely manner. For clinical management, deterioration of 15 dB at one or more frequencies that does not recover warrants action. Since its implementation, there has been a significant increase in the prevalence of UHL in newborns, rising from approximately 0.3–0.6 per 1,000  to 1–2.4The accurate diagnosis and timely intervention as early as possible are widely recognized as crucial for optimal audiological management of hearing loss configuration, progression, and long-term outcomes in children with hearing loss , 18. Curβ 2 gene (GJB2)   are riske (GJB2) , 25, or e (GJB2) .Due to the challenges associated with obtaining a reliable behavioural audiogram during early childhood, and the variations in reported proportions and potential risks of progressive hearing loss documented in different studies on UHL, there is a need for more evidence to determine the age at which a reliable audiogram can be obtained, as well as to gather more information on the extent of hearing changes in children with UHL. This knowledge will contribute to the development of optimal management and intervention strategies. Considering these challenges and the importance of early intervention, this study aims (1) to describe the audiological characteristics and etiology of a group of children diagnosed with congenital unilateral hearing loss (CUHL); (2) to examine the age at which reliable behavioural audiograms can be obtained and explore potential factors that could delay obtaining these audiograms; and (3) to investigate changes in hearing levels from birth to the first reliable behavioural audiogram, as well as the potential risks of progressive hearing loss.2.2.1.n = 28), Victoria (n = 24), and Queensland (n = 39). All participants were identified with UHL via newborn hearing screening with a subsequent diagnosis of permanent UHL confirmed via electrophysiological hearing tests at diagnostic centres and hospitals. These include tympanometry, distortion product otoacoustic emission (DPOAEs), and TBABRs or ASSRs testing on both ears at a diagnostic centre or hospital for birth dates between March 19, 2014, and February 8, 2018. Following diagnosis of the hearing loss, children were referred to Hearing Australia  to receive further audiological services, which includes ongoing hearing assessments, hearing device fitting and verification. Inclusion criteria for this report included children enrolled in the study on unilateral hearing loss (reported separately) who had (1) a diagnosis of UHL; (2) frequency-specific audiometric thresholds estimated from electrophysiological testing at diagnosis; (3) reliable behavioural audiograms in early childhood at least at one low frequency (0.5 or 1 kHz) and one high frequency (2 or 4 kHz) at Hearing Australia  were as follows: for TBABRs, 500 Hz was adjusted by 10 dB, 1,000 Hz by 10 dB, 2,000 Hz by 5 dB, and 4,000 Hz by 0 dB; for ASSRs, 500 Hz was adjusted by 15 dB, 1,000 Hz by 10 dB, 2,000 Hz by 10 dB, and 4,000 Hz by 10 dB , 40, 41.2.2.2.•clinical note shows reliable behavioural results on the tested frequencies.•must have hearing thresholds at least at one low frequency (0.5 or 1 kHz) and one high frequency (2 or 4 kHz).•must have masked hearing thresholds when required, for air and bone conduction thresholds.Specific decision rules were developed for this study, such as if an audiogram associated with temporary abnormal middle ear function, it was not recorded as a reliable result, and subsequent audiological results were examined accordingly. The date and the detailed audiological results  of the first reliable behavioural audiogram were then recorded for further analysis.The behavioural audiological assessments after diagnosis for all children were performed by clinical paediatric audiologists at Hearing Australia according to the national audiological protocols. The behavioural hearing thresholds were obtained using visual reinforcement audiometry (VROA), conditioned play audiometry (PA), or a combination behavioural method of VROA and PA if needed, depending on the child's age and ability. Serial behavioural audiological results after the time of diagnosis for each child were retrospectively reviewed by experienced research audiologists from clinical records held at Hearing Australia, to identify reliable behavioural audiograms. The definition of a reliable behavioural audiogram for the impaired ear includes:2.2.3.To identify potential factors affecting the age at which reliable behavioural audiograms were obtained, children's demographic and basic audiological information, as well as the etiological details were extracted from available clinical reports of Hearing Australia and provided reports by other diagnostic organizations to Hearing Australia. To gain further insights of clinicians' experiences and challenges on the potential reasons that may affect how early a behavioural audiogram is obtained, we invited clinical audiologists at Hearing Australia to complete an informal online surveyConsidering the suggested reasons from the survey, we reviewed the clinical case notes of earlier testing appointments before obtaining the first reliable behavioural audiogram. We then grouped the comments reported during the actual appointments into three categorised reasons: (1) children's behavioural issues , (2) challenges unrelated to children's behavioural issues , and (3) family availability (such as lost contact or failure to attend scheduled appointments). Cases exhibiting any of these issues were labelled as “Yes”, while those without such issues were labelled as “No”. These three potential reasons and the other four clinical variables  were included in subsequent statistical analysis and discussion on the reasons of the delay in obtaining behavioural audiogram in this report.2.2.4.•If there is no measured threshold at 0.5 kHz, extrapolate the results by decreasing 10 dB from 1 kHz;•The missing threshold at 2 kHz is calculated as the mid value between the 1 and 4 kHz;The three frequencies averaged hearing level (3FAHL) in the impaired ear was then calculated using the thresholds at 0.5, 1, and 2 kHz.The degree of hearing loss in the impaired ear at diagnosis and behavioural audiological assessments was further determined by three-frequency averaged thresholds at 0.5, 1, and 2 kHz , 26. All2.2.5.For the purposes of this study, the changes in hearing were determined by comparing the hearing thresholds measured from the first reliable behavioural audiogram to the baseline hearing thresholds at diagnosis. A positive difference between two thresholds indicated a deterioration in hearing, while a negative difference indicated an improvement. To be consistent with recent literature, the following two definitions were adopted for the analysis of significant deterioration:•Definition 1: a decrease of ≥10 dB at two or more adjacent frequencies between 500 and 4,000 Hz, or a decrease of >15 dB at one octave frequency in the same frequency range , 25, 26.•Definition 2: a change of ≥20 dB in the three frequencies  pure-tone average , 22, 28.Additionally, this study employed a similar version of definition 2 to determine a significant improvement in hearing thresholds. Specifically, an increase of greater than or equal to 20 dB  in the three-frequency average hearing level (3FA HL) was defined as an improvement. Any change that did not meet the criteria for deterioration or improvement  was categorized as stable hearing loss between the estimated baseline hearing threshold and the first reliable behavioural audiogram.2.3.p < 0.05. The statistical analysis was performed using IBM SPSS Statistics for Windows v.29 .To explore the potential factors contributing to the delay in obtaining reliable behavioural audiograms, a multiple regression analysis was conducted. The dependent variable was the age at which the first reliable behavioural audiogram was obtained. The independent variables consisted of 4 clinical variables , as well as 3 additional factors   = 2.63, p = 0.017] with the full set of predictors accounted for 11.3% of total variance in the age of getting the first reliable audiogram. Among these predictors, children's behavioural issues  and the presence or absence of reported ongoing middle ear issues  demonstrated significant associations with the dependent variable. Specifically, children with reported behavioural issues were likely to be older when getting their first reliable audiogram. Conversely, those with reported ongoing middle ear issues tended to be younger when obtaining their first reliable audiogram. However, for the remaining predictors, there was insufficient evidence to demonstrate significant associations with the dependent variable.The results in 3.3.3.3.1.r > 0.75, p < 0.001). The strongest positive correlation between the two measures was observed at 1 kHz , while the lowest correlation was found at 500 Hz  with greatest variability in hearing changes . In F = 2.83, p = 0.039]. Post-hoc analysis indicated that the changes in hearing levels were significantly higher at 500 Hz compared to the other frequencies, followed by 1 kHz, 2 kHz, and the lowest changes were observed at 4 kHz. The main effect of age of getting the behavioural audiogram on the changes in hearing levels , and the interaction between frequency range and age  were not statistically significant.A two-way ANOVA test was conducted to examine the effects of four tested frequencies  and five age ranges on the dependent variable of hearing level changes. The age ranges considered were under 2 years, 2–3 years, 3–4 years, 4–5 years, and over 5 years, which correspond to the age at which the first reliable behavioural audiogram was obtained. The results revealed a statistically significant difference in the overall changes in hearing thresholds among the four frequencies .By analysing the averaged hearing level changes in children with either deteriorated or improved hearing thresholds, it was observed that although the mean values of deterioration or improvement of thresholds were similar across all tested frequencies, the degree of thresholds difference was in general higher for deterioration than for improvement in these children. For example, at 500 Hz, the mean deterioration in hearing thresholds was 28.9 dB, compared to the mean improvement of 18.9 dB. The statistical analysis with independent 3.3.2.n = 71) experienced deterioration at the time of the first reliable behavioural audiogram. Among the 71 children with hearing deterioration, 34 of them demonstrated deterioration by 3 years of age. As shown in n = 24), 23 demonstrated deterioration in the initially impaired ear, and one child was identified as having hearing deteriorated in both ears.As shown in We also observed that a high proportion of children in our study developed severe to profound hearing loss from a milder degree of loss. Specifically, when applying definition 1, out of the total of 71 children who showed progressive hearing loss, 52 of them (73.2%) deteriorated to severe to profound hearing loss, compared to their initial hearing thresholds at the time of diagnosis. When using definition 2, 22 out of the 24 children, an overwhelming majority of them (92%) deteriorated to severe to profound hearing loss during behavioural assessments.3.3.3.n = 12) and severe (n = 14) two groups, more than 70% of the children in any other degrees of hearing loss at diagnosis are likely to remain in the stable group. For example, among the 15 children diagnosed with mild hearing loss, 12 of them (80%) showed no hearing deterioration based on definition 2. This is because definition 2 may not capture further deterioration in certain frequencies. By excluding all children diagnosed with profound hearing loss, whose hearing loss may have already reached the limits of measured hearing thresholds, 60% (51 out of 85) would demonstrate deterioration according to definition 1, while 27.7% (18 out of 85) would show deterioration based on definition 2.Furthermore, the deterioration rates were similar between the group of children fitted with hearing devices and the group without hearing devices, based on both definitions. According to Definition 1, 79% (49 out of 62) of the fitted group showed hearing deterioration, while 21% (13 out of 62) remained stable. In the not fitted group, 76% (22 out of 29) demonstrated deterioration, and 24% (7 out 29) remained stable. Similarly, based on Definition 2, the proportions were similar, with 79% of the fitted group experiencing deterioration, and 21% remaining stable. Among the not fitted group, 76% showed deterioration, and 24% remained stable.Regarding the documented constellation of etiology, using Definition 1, 8 out of 10 children with absent or abnormal auditory nerves, 6 out of the 8 children with CMV, and all the children with LVAS demonstrated further hearing deterioration in at least one frequency. Among the 4 children with syndromic hearing loss, 2 experienced hearing deterioration, while the other 2 did not. The results by using definition 2 showed that the number of children whose documented constellation of etiology  were the same between the two groups of children with and without hearing deterioration. None of the 4 children with syndromic hearing loss showed hearing deterioration. Among the total of 9 children who were born with atresia and/or microtia, both definitions suggested a higher likelihood of stable hearing.4.The primary objective of this study was to report the clinical characteristics in a group of children with congenital unilateral hearing loss. We aimed to gather demographic information, determine the age at which the first reliable behavioural audiogram was obtained, and identify possible factors for any delays in acquiring the audiogram. Another important aspect of the present report was to directly compare the estimated hearing thresholds at the time of diagnosis with the first reliable behavioural audiogram, to investigate the hearing changes between these two measured points. This knowledge will contribute to the development of optimal audiological management, which will enable clinicians to promptly identify any hearing changes and make necessary adjustments to intervention strategies.4.1.n = 10) and ENT malformations (atresia or microtia) (n = 9) accounted for approximately half of the cases with known etiology. Among the 8 children with CMV, 2 were diagnosed with mild or moderate hearing loss, whilst the remaining 6 had severe to profound hearing loss. This finding aligns with previous reports indicating that CMV infection is associated with such hearing loss severity (n = 4) or other syndromes (n = 4). Among these children, the degree of hearing loss at diagnosis ranged from mild to profound, with a significantly higher percentage of moderate hearing loss observed in children with LVAS (3 out of 4 children with LVAS). Notably, 62% of the children in our sample had no reported etiology. Although this finding aligns with previous research reporting a high proportion of UHL cases with unknown etiology or no reported risk factors . Absent/abnormal auditory nerve  identified with UHL through UNHS had their first reliable behavioural audiograms in the impaired ears by 3 years of age, despite a national pediatric clinical protocol recommending that ear-specific behavioural thresholds at all frequencies from 0.5 to 4 kHz should be obtained by 18 months of age for infants with hearing loss ]ing loss . This fiThe regression analysis from this report indicated the effects of degree of hearing loss, etiology, or hearing device fitting were not significant factors influencing the age at which behavioural audiograms were obtained. This differs from the aspects being identified as potential influencing factors by clinicians in an online survey, as detailed in Section Children with middle ear issues tended to have more appointments with clinicians, providing them with more opportunities to have hearing tests at early age. However, ongoing middle ear pathology might also delay the behavioural testing due to the potential for inconsistent test results caused by fluctuations in hearing levels or the need for a recovery period after medical treatment for middle ear conditions. It should be noted that only audiograms taken outside periods of temporary middle ear dysfunction, and meeting other criteria as outlined in Section 4.3.Children's hearing thresholds may experience changes over time, which could be partially due to changes in ear canal acoustics , 54. HowThis study indicates that changes in hearing levels were observed in each testing frequency. These changes could be classified as deterioration to improvement and categorised into a significant change (≥20 dB) or minor change (≥10 dB but <20 dB) at each frequency. The mean hearing deterioration for frequencies at 0.5, 1, 2, and 4 kHz ranged from 23.9 to 28.9 dB, while the mean hearing improvement ranged from 18.8 to 22 dB across the same frequencies see . The anan = 95), 51 (54%) showed progressive HL. Another possible explanation may be that both Fitzpatrick studies compared the diagnostic audiogram with the most recent audiometric assessment. The mean length of time between assessments for children with progressive hearing loss was 64.3 and 50.3 months in the 2023 and 2017 studies, respectively. In contrast, our study only included children with congenital UHL, and we compared the results by examining the initial estimated hearing thresholds with the first behavioural audiogram. This approach allowed us to provide timely support after UNHS, for early identification and intervention in children with hearing deterioration. Using averaged three frequency thresholds  to define deterioration, we only identified 26.4% of children in the sample as having hearing deterioration. This suggests that adopting averaged thresholds to define deterioration may underestimate the proportion of children experiencing hearing changes, which could have direct impact on management decisions.The current study found that 71 (78%) children diagnosed with UHL at birth experienced hearing deterioration between the diagnosis and first behavioural audiogram at one or more frequencies between 0.5 and 4 kHz  experienced progression of hearing loss in at least one frequency. This finding also emphasises the importance of monitoring hearing thresholds at all audiometric frequencies after diagnosis through newborn hearing screening. Early identification of deterioration has direct implications for considerations such as hearing device fitting and adjustments, evaluations of the impact of hearing loss on a child's development and determining appropriate intervention.4.4.One strength of this study is that it only includes children diagnosed with congenital UHL via UNHS, and it conducts a direct comparison between the initial estimated hearing thresholds from electrophysiological measures with the first reliable behavioural audiogram. Both of these methods have been recognised as the gold standard tests for threshold estimation for young infants . In thisThe current study has some limitations that should be considered. Firstly, the current data were drawn from participants in a research study, which may restrict the generalizability of the results to the general population. Secondly, the behavioural audiogram is obtained retrospectively via the Hearing Australia database, which means that certain factors that could influence the test results, such as children's behavioural issues, family's engagement, resources or other challenges during the appointment, might not be recorded in the case notes. This could affect the analysis of the potential factors contributing to the delay in obtaining reliable behavioural audiograms. Additionally, the low variance explained by our regression model indicates that there may be other factors affecting the age at which children obtain their first reliable behavioural audiogram that were not considered in our analysis. Future research could explore potential factors, such as parental and clinician attitudes towards UHL, as well as the frequency of attended appointments. These investigations may inform clinical practice and guide early intervention strategies. Thirdly, identifying potential etiology causes of progressive hearing loss in children with UHL is crucial for clinicians to develop targeted interventions. However, a significant challenge arises from the unknown etiology of many cases in our sample, making the clinical characteristics and risks associated with hearing progression in children with UHL still inconclusive due to the lack of specific information. Close collaboration with ENT specialists may be beneficial in addressing this challenge, as it could facilitate a more efficient acquisition of etiological information. Further research is necessary to investigate risk factors associated with hearing progression in this population. Lastly, the age of the first reliable behavioural audiogram depends on the child and family's availability to attend audiological appointments. As a result, the actual age of hearing deterioration may be even younger than what was reported, but the identification of hearing changes would rely on when the behavioural test was conducted. Additionally, this report does not include follow-up behavioural audiograms after the first reliable audiogram. Future investigations will examine the comparison between the first and subsequent reliable behavioural thresholds to capture the hearing changes in children with UHL during early life.5.The findings from this study contribute to the understanding of the demographic, audiological, and etiological characteristics of children with UHL, highlighting the importance of early monitoring of hearing changes and factors that influence the age at which reliable behavioural audiograms are obtained. By gaining a better understanding of this information and its implications for children's developmental outcomes, clinicians and researchers can strive to optimize early clinical management strategies for children with UHL. Overall, the results indicate that closely monitoring hearing loss after initial diagnosis is essential to ensure optimal interventions are implemented at the earliest age for this target group of children.


Bats exhibited age-related hearing loss. Bats are exposed to immense conspecific noise, but the frequency-dependent hearing loss was not correlated to the noise. Hearing loss is a hallmark of aging, typically initially affecting the higher frequencies. In echolocating bats, the ability to discern high frequencies is essential. However, nothing is known about age-related hearing loss in bats, and they are often assumed to be immune to it. We tested the hearing of 47 wild Egyptian fruit bats by recording their auditory brainstem response and cochlear microphonics, and we also assessed the cochlear histology in four of these bats. We used the bats’ DNA methylation profile to evaluate their age and found that bats exhibit age-related hearing loss, with more prominent deterioration at the higher frequencies. The rate of the deterioration was ∼1 dB per year, comparable to the hearing loss observed in humans. Assessing the noise in the fruit bat roost revealed that these bats are exposed to continuous immense noise—mostly of social vocalizations—supporting the assumption that bats might be partially resistant to loud noise. Thus, in contrast to previous assumptions, our results suggest that bats constitute a model animal for the study of age-related hearing loss. Population aging in humans is an international concern . Aging iMyotis brandtii bat was caught in nature 41 yr after having been first ringed  measured after exposure to noise did not vary significantly from pre-exposure thresholds or from thresholds in control conditions  caught in the wild. These bats rely on ultrasonic echolocation  technique . We examSensorineural hearing loss suggests a pathological condition in either the cochlea or the auditory nerve. The “sensory” component refers to the cochlea, whereas the “neural” component refers to problems primarily in the auditory nerve  or seconABR recordings revealed a clear age-related hearing loss that is more prominent at the higher frequencies. Both the reduced CM amplitudes with age and a reduced stria vascularis (SV) area with age suggest age-related deterioration in the cochlea. The deterioration in neuronal speed of processing, assessed through a suprathreshold temporal analysis of ABRs, might represent neuronal presbycusis.Furthermore, to assess the noise that our bats are typically exposed to in their colony we placed several continuous calibrated microphones in their roost. This procedure revealed that bats are routinely exposed to very high broadband noise levels, which are known to have a negative effect on the auditory system of mammals. Taken together, our results provide the first evidence that bats are susceptible to age-related hearing loss, but show that hearing loss in bats is similar to that observed in humans despite routine exposure to very loud noise.To assess bat hearing thresholds, we played 1-ms tones of 35, 30, 24, 18, 12, and 6 kHz and a 0.1-ms click signal (with most energy up to 10 kHz). Playback intensities spanned from at least 10 dB below to 10 dB above the suspected threshold for each frequency, but not lower than 10 dB sound pressure level  for pure tones (and 20 dB SPL for clicks), and not higher than 90 dB SPL. The hearing threshold at each frequency was defined offline as the lowest intensity at which the peak-to-peak amplitude of the response exceeded a 6.5*SD criterion  and the oldest bats  are up to ∼10 dB . The meaousettus , with thP = 0.018, 0.099, 0.026, 0.037, 0.003, and 0.009 for the frequencies 6, 12, 18, 24, 30, and 35 kHz, respectively, for each frequency, a generalized linear model (GLM) with the hearing threshold set as the explained parameter and age and sex as fixed factors; see the Materials and Methods section).Next, we analyzed the effect of age and sex on hearing thresholds. Sex differences were insignificant, whereas age differences were significant for five of the six frequencies and revealed elevated hearing thresholds in older animals . In addition, the slope at 24 kHz (0.95 ± 0.44 dB threshold elevation per year) was higher than at 18 kHz (0.68 ± 0.3 dB threshold elevation per year) (P = 0.001)  significantly higher at 35 kHz (1.44 ± 0.53 dB threshold elevation per year) and 30 kHz (1.31 ± 0.42 dB threshold elevation per year) than at 24 kHz (0.95 ± 0.44 dB threshold elevation per year), 18 kHz (0.68 ± 0.3 dB threshold elevation per year), 12 kHz (0.77 ± 0.45 dB threshold elevation per year), and 6 kHz (0.87 ± 0.35 threshold elevation per year) (= 0.001) .For further verification of the mechanism underlying hearing deterioration, minimally invasive CM were recorded in response to a 90 dB SPL 0.1-ms click signal that was played at opposite polarities .Analysis of the effect of age and sex on the CM response  revealedTo further evaluate cochlear health, cochlear histology was performed for four female bats . The batP = 0.026, GLM with the hearing thresholds (dB SPL) set as the explained parameter and the SV area as a fixed factor).The hearing thresholds of the bats correlated with their SV area, showing higher (worse) thresholds with smaller SV areas for most of the frequencies, and significantly so for 18 kHz    .We measured the latencies of waves 1, 4, and 5 of each bat’s 30 dB suprathreshold response, which were easier to detect across individuals, and the inter-peak interval (IPI) between wave 1 and wave 4 .P = 0.031 and P = 0.005, respectively, GLM with the latency or IPI set as the explained parameter and age and sex as fixed factors), which represents activity from cranial nerve VIII until the nucleus of the lateral lemniscus (NLL)  . The larus (NLL)  at the ius (NLL) .P = 0.004, GLM with the hearing deterioration (dB SPL per year) set as the explained parameter and the noise level as a fixed factor).Sound recordings in a fruit bat cave revealed almost non-stop exposure to loud conspecific vocalizations. We estimated the noise intensities a bat is exposed to in the roost at two key frequencies (6 and 32 kHz) at windows of 30 s  too experience age-related hearing loss or presbycusis, with a sharper deterioration at the higher frequencies. This represents a typical pattern of age-related hearing loss in mammals  levels of age-related hearing loss  suggest that they might have some special adaptations to cope with very noisy environments. One such adaptation in addition to their middle ear reflex function might be their high defense against oxidative stress , which iWhat is the physiological hearing loss mechanism in fruit bats? We recorded CM as a method to assess OHC functionality . We alsoWe also find evidence of hearing loss at the neuronal level. Our suprathreshold temporal analysis revealed a significant prolongation in the latency of wave 4, and a significant prolonged IPI between wave 1 and wave 4, which represents activity from nerve VIII to the nucleus of the lateral lemniscus (NLL) (through the cochlear nucleus [CN] and the superior olivary complex [SOC]) . Wave laThis deterioration in processing speed is typical of neuronal presbycusis and can result in poorer speech understanding in humans . It mighIn summary, the mechanism behind age-related hearing deterioration in bats appears to involve a mixture of pathologies, at the cochlear level and at the neuronal level. How bats deal with hearing loss despite their crucial reliance on hearing for sensing is yet to be revealed.Rousettus aegyptiacus; 24 female and 23 male) participated in the ABR and CM recordings. The bats were all adults (older than 1 yr), based on their weight and forearm length. Genomic DNA was extracted from wing samples, and methylation data (DNAm) were generated. In addition, cochlear histology was assessed in four females of these bats.47 adult Egyptian fruit bats .The bats’ weight and forearm measurements were taken, and they were anesthetized by subdermal injection of an anesthetization cocktail comprising 0.48 ml water, 0.1 ml Domitor (1 mg/ml), and 0.05 ml midazolam (5 mg/ml). The bats were injected with a 0.25 ml dose of the cocktail and an additional half-dose if needed. Eye moisture drops were given to prevent eye dehydration.Anesthesia is known to affect both the central and peripheral nervous systems  and has To date, with the exception of one study , ABRs inABR recordings followed Rousettus aegyptiacus (Bats were presented with a 0.1-ms click stimulus (with most energy up to 10 kHz), and subsequently with 1-ms tone bursts of 35, 30, 24, 18, 12, and 6 kHz. This set of frequencies encompasses the main hearing range of adult yptiacus . For eacyptiacus . Here, tThe determination of hearing thresholds involves interpretation of the ABR waveform . ABR thrFor each bat, an in-house MATLAB script screened the ABR recordings for each of the frequencies  from the highest to the lowest tested intensity. The ABR system records 10-ms response windows that start with the onset of the auditory signal. The average SD of the first 1 ms from the 10-ms response window was therefore calculated for each intensity. This time window does not include components of the ABR response and should therefore represent the averaged noise. The ABR response to tones was searched in a 3-ms time window between 2.5 and 5.5 ms of the full 10-ms averaged response window. A minimum peak was first detected, and a maximum peak was then detected within a time window of ∼1.5 ms following the minimum peak.The absolute difference between the two peaks was calculated and determined as the peak-to-peak amplitude of the response . If thisEptesicus fuscus), likely because of a combination of stressors  and lacked response to both the higher frequencies and the click signal (tested here up to 90 dB SPL). Its test was repeated a week later with similar results, and it was removed from further analysis. This individual was not the oldest subject—its estimated age was 7.15 yr; whereas the other bats’ ages ranged from 2.44 to 13.9 yr, with a mean of 6.67 ± 3.33 yr. Idiopathic hearing loss in bats was reported recently for the big brown bat . Hearing thresholds are known to represent hair cells’ function, and the purpose of ABR recordings at a fixed suprathreshold of 30 SL is to eliminate the cochlear state and examine neuronal changes only . The meaTo evaluate the function of the OHC, minimally invasive CM were recorded using the same subdermal electrode positioning used for the ABR recordings. Our method is similar to the one used in humans in which the CM response is recorded via surface electrodes . SimilarCM were obtained similar to ABRs, with some modifications:The bats were presented with a 0.1-ms click that was transferred via a 30-cm-long air tube connected on one end to the speaker and serving as an earphone. The other end of the tube was coated with a foam tip and placed at the entrance to the right auditory canal. The intensity at the foam tip end was calibrated to 90 dB SPL, and the signals were played 2,000 times for each of the two polarities separately (condensation and rarefaction). These recordings were then repeated with a similar but blocked tube, in order to rule out possible artifacts of the auditory signal  that areThis control is twofold: first, the CM arrive later in time because of the transmission time in the tube (∼1 ms for a 30-cm tube), compared with artifacts that occur immediately upon signal presentation; and second, artifact responses would also be visible in the blocked tube mode when the auditory signal is played but does not reach the subject’s ear, whereas real CM response from the cochlea will be present only when the tube mode is open.P < 0.0001, GLM with the CM amplitude set as the explained parameter and the insert phone tube mode [open or blocked] as a fixed factor, n = 46).Indeed, averaged CM response was ∼sixfold larger than the possible artifact  and signPeriodic sound waves traveling through air consist of alternating regions of compression  and decompression  of air molecules . “CondenAfter the collection of responses to both polarities (condensation and rarefaction), adding the response to rarefaction to the response to condensation ([condensation + rarefaction]/2) will accentuate the lower frequency components of the response—the ABR; whereas subtracting the response to rarefaction from the response to condensation ([condensation—rarefaction]/2) will bias the higher frequency components by maximizing the spectral response—CM . SubtracCochlear histology was assessed in four bats. The bats were euthanized and perfused with PBSx1. The inner ears were dissected, fixed in 4% PFA at 4°C overnight, and decalcified at 4°C for 8 d in a standard decalcifier. The ears were then processed for paraffin sections using the tissue processor . Paraffin blocks were then made using Histo-Embedder (Leica) and sectioned using a microtome . Cochlea mid-turn cuts were taken in all the samples for consistency. Paraffin serial sections (15 μm) were stained with hematoxylin and eosin using Multistainer (Leica). Slides were imaged using Aperio Slide Scanner (Leica). For quantification of the stria vascularis cross-sectional area, three slides were measured for each ear and averaged using FIJI (ImageJ2). Spiral ganglion neurons and the hair cells of an aged bat (11.3 yr) are shown in To determine the bats’ age, tissue samples were carefully taken from their wings, avoiding puncturing blood vessels. This procedure has been used many times and does not require anesthetization e.g., . HoweverGenomic DNA was extracted from the wing biopsies using DNeasy Blood & Tissue Kit (QIAGEN Ltd.) with one modification to the suggested protocol: at the elution stage, 35 μl of elution buffer was used, in two repetitions, to increase yield. After extraction, all of the samples were quantified using Equalbit dsDNA HS Assay Kit EQ111  using Qubit 2.0 Fluorometer . 30 μl from samples at a concentration of over 10 ng/μl was sent to UCLA Neuroscience Genomics Core.The protocol used to assess the bats’ age was similar to that of DNAm levels were determined by calculating the ratio of intensities between methylated and unmethylated sites, and multispecies epigenetic clock that accurately predicts chronological age was used .The acoustics in a bats’ roost was recorded for 66 h using a calibrated AudioMoth (Open Acoustic Devices). Two AudioMoth devices were placed in crevices where the bats typically perch to record the noise that a bat is exposed to. Maximum levels are a common way to report bats’ call intensities . The maxThe entire frequency spectrum of the conspecific noise exposure was done using recordings in a laboratory colony with 13 Egyptian fruit bats. A calibrated GRAS 40DP 1/8″ microphone (GRAS Sound & Vibration) was placed 105 cm from the cluster of the bats, and 49 recordings with an average of ∼7 s long were taken automatically (over a period of ∼2 h), triggered by the bats’ vocalizations. The raw recordings were filtered at a range of ±2 kHz for each of the frequencies that were tested in the audiogram . Maximum peak-to-peak amplitudes were measured then for each of the 49 recordings, and the average was taken as the noise level. The sound levels at 10 cm were estimated based on the physics of sound propagation.t test with a Bonferroni correction for multiple comparisons.Most of the measurements were statistically analyzed using GLMs fitted in MATLAB. Hearing deterioration rate comparisons between the different frequencies were analyzed by a two-tailed


Most studies on pitch shift provoked by hearing loss have been conducted using pure tones. However, many sounds encountered in everyday life are harmonic complex tones. In the present study, psychoacoustic experiments using complex tones were performed on healthy participants, and the possible mechanisms that cause pitch shift due to hearing loss are discussed.Two experiments were performed in this study. In experiment 1, two tones were presented, and the participants were asked to select the tone that was higher in pitch. Partials with frequencies less than 250, 500, 750, or 1,000 Hz were eliminated from the harmonic complex tones and used as test tones to simulate low-tone hearing loss. Each tone pair was constructed such that the tone with a lower fundamental frequency (F0) was higher in terms of the frequency of the lowest partial. Furthermore, partials whose frequencies were greater than 1,300 or 1,600 Hz were also eliminated from these test tones to simulate high-tone hearing loss or modified sounds that patients may hear in everyday life. When a tone with a lower F0 was perceived as higher in pitch, it was considered a pitch shift from the expected tone. In experiment 2, tonal sequences were constructed to create a passage of the song “Lightly Row.” Similar to experiment 1, partials of harmonic complex tones were eliminated from the tones. After listening to these tonal sequences, the participants were asked if the sequences sounded correct based on the melody or off-key.The results showed that pitch shifts and the melody sound off-key when lower partials are eliminated from complex tones, especially when a greater number of high-frequency components are eliminated.Considering that these experiments were performed on healthy participants, the results suggest that the pitch shifts from the expected tone when patients with hearing loss hear certain complex tones, regardless of the underlying etiology of the hearing loss. Patients with hearing loss may perceive pitch shift when they hear tones; however, this pitch shift does not appear to be common knowledge among otologists and audiologists. Similar cases have been occasionally reported through general or website articles written by doctors or patients themselves. However, only a few scientific papers have detailed the cases of patients with pitch shift related to hearing loss. The lack of studies on pitch shift may be attributed to the difficulty in appropriately evaluating this phenomenon. Some studies have indicated pitch shifts in cases of unilateral hearing loss by using the opposite ear as a reference . HoweverThis study aimed to show that the evaluation of pitch shift by hearing loss can be even more complicated when the types of stimulation tones are considered. Although most studies on pitch shift have been conducted using pure tones , many soWe hypothesized that such complicated characteristics in pitch perception may be related to the pitch shifts noticed by patients with hearing loss. Therefore, we simulated hearing loss in participants with normal hearing in the present study and investigated the condition in which pitches shift from the expected tones on hearing complex tones. In the first part of the study (experiment 1), we examined pitch shifts by having the participants compare several pairs of tones, whereas in the second part of the study (experiment 2), participants were exposed to a melody that might have sounded off-key due to altered pitches to illustrate how such pitch shifts might impact everyday life.A total of 43 participants, comprising 27 men and 16 women aged 19–60 years (mean ± standard deviation [SD] = 40.1 ± 12.9), were included in this study. Among them, five participated in experiment 1 only, four participated in experiment 2 only, and the remaining participated in both experiments. To examine the effect of age on the study outcomes, the participants were divided into two groups according to their age: those who were ≤ 39 years (mean ± SD = 28.4 ± 5.7) and those who were ≥ 40 years (mean ± SD = 51.2 ± 5.9). For experiment 1, 19 participants were included in the younger group, and 20 participants were included in the older group. For experiment 2, 18 participants were included in the younger group, and 20 participants were included in the older group. All participants had normal hearing with no confirmed neurological conditions and confirmed that they were able to hear all the tones used in the experiments. The study protocols were reviewed and approved by the Clinical Research Ethics Committee of Ichimiya Clinic . This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to their inclusion in the study.The stimulation tones were created using the publicly available software Wave Editor TWE . All tones were saved as WAV files (16 bits/44.1 kHz).i.e., tone A) was higher in terms of the frequency of the lowest partial. The frequencies and harmonic ranks of the partials are shown in vs. 1,104 Hz). Only one pair, Y4, consists of pure tones, whereas the other pairs consist of complex tones.Eight tone pairs, which are shown in For experiment 2, tonal sequences were constructed using tones of 500- or 1,000-ms duration, with a 10-ms rise and fall time. Thirteen tones were connected to create a passage of “Lightly Row”. This passage was selected for the experiment because of the participants’ familiarity with the song. The song, which is also known as “Butterfly”, is a popular children’s song in Japan. Similar to experiment 1, the higher and lower components of the harmonic complex tones were eliminated from the complex tones. A total of eight versions were presented in experiment 2. The versions were classified as X1, X2, X3, X4, Y1, Y2, Y3, and Y4 in the same manner as in experiment 1 .An Aspire S3 computer  with a USB audio processor  was used to deliver auditory stimuli binaurally through dynamic headphones  at a comfortable level for the participants, which was approximately 75 dB SPL in all cases.The experiments were performed as described in our previous studies .In experiment 1, the computer monitor showed two buttons that played one of the eight pairs of prepared tones. The participants were asked to compare these tone pairs by clicking on the buttons and select the tone that was higher in pitch. The number of times the participant heard the tone was not predetermined, and the participants were allowed to click on the buttons multiple times before making a decision. Each task was performed twice for each of the eight pairs, resulting in a total of 16 tasks. The order of the tone pairs and the order of the two buttons were randomized.In experiment 2, the computer monitor displayed a button that played one of the eight versions of “Lightly Row”. The participants were asked if the passage they heard sounded correct based on the melody or off-key. The number of times of hearing the tone was not predetermined, and the participants were allowed to click on the buttons multiple times before making a decision. Each task was presented twice; thus, a total of 16 tasks were performed. The order of the tasks was randomized.via a computer monitor during both experiments and were asked to respond to the questionnaire in writing.The participants received written instructions All statistical analyses were performed using EZR version 1.52  , a graphIn experiment 1, the perception was defined as a pitch shift when the participants selected tone A as the tone that is higher in pitch, as the tone they selected was lower in frequency in terms of F0. The participants’ responses were considered to be spectral as they appeared to have responded based on the frequency of partials that make up the tones. Conversely, the perception was defined as an F0 response when they selected tone B as the tone that is higher in pitch. i.e., values for X1, X2, X3, X4, Y1, Y2, Y3, and Y4) for each participant. These values could be −1, 0, or +1 since each task was performed twice.where sp is the number of spectral responses, and f0 is the number of F0 responses. Eight SI values were calculated (p = 0.037), X3 (p = 0.001), and X4 (p = 0.002). The results were similar in the Y series, in which partials with frequencies greater than 1,300 Hz were eliminated. Compared with those for Y1, the SI values were significantly higher for Y3 (p = 0.008) and Y4 (p < 0.001). A comparison between the X and Y series was performed to analyze the effect of eliminating the partials with high frequencies. The SI values were significantly higher when Y1 was compared with X1 (p = 0.005), Y2 was compared with X2 (p = 0.023), Y3 was compared with X3 (p = 0.023), and Y4 was compared with X4 (p < 0.001).The average SI values for all participants are shown in i.e., ambiguous or unambiguous) from the participants. The ambiguous response was significantly higher for X3 (p = 0.027) and X4 (p = 0.027).An ambiguous response was defined as a difference in the participant’s responses to the same task, which had been performed twice. The percentages of these ambiguous responses, with the percentages of unambiguous F0 and spectral responses for each tone pair, are shown in p = 0.003). The SI values were significantly higher for Y2 (p = 0.025), Y3 (p < 0.001), and Y4 (p < 0.001) than those for Y1. Moreover, the SI values were significantly higher when Y3 was compared with X3 (p < 0.001) and Y4 was compared with X4 (p < 0.001). The participants’ ambiguous responses were analyzed similarly as in experiment 1, as shown in p = 0.002) and Y2 (p = 0.020).In experiment 2, the perception was considered as a pitch shift caused by spectral response when the participants judged that the melody sounded off-key. They were analyzed similarly as in experiment 1. The calculated SI values are shown in To examine the effect of age on the study outcomes, the data from experiments 1 and 2 were re-analyzed with the participants divided into two age groups: ≤ 39 years and ≥ 40 years.p-values obtained using Wilcoxon’s signed rank test are shown in the order of ≤ 39 years group and ≥ 40 years group. In experiment 1, the SI values were significantly higher for Y4 than those for Y1 . The SI values were significantly higher when Y4 was compared with X4 . In experiment 2, the SI values were significantly higher for X4 than those for X1 . The SI values were significantly higher for Y3  and Y4  than those for Y1. The SI values were significantly higher when Y3 was compared with X3  and when Y4 was compared with X4 . No significant differences were observed when the ambiguous responses were analyzed.No evident differences were observed between the two age groups for any of the experiments. The SI values that showed large differences when all participants were grouped together regardless of age also showed significant differences within both age groups. The via the removal of partials. Taking into consideration that these experiments enrolled healthy participants, these results suggest that pitch shifts can be perceived when patients with hearing loss hear certain complex tones, regardless of the underlying etiology of the hearing loss. To examine the effect of age on the study outcomes, the data were re-analyzed with the participants divided into two age groups. Although this broad grouping may not capture the detailed age effect, we observed no evident differences between the two age groups. Thus, it is unlikely that age would have a substantial impact on the results of the present study. Low-tone hearing loss of various severities was simulated, which prevented the participants from hearing the lower components of complex tones. The elimination of components with frequencies of less than 250 Hz simulated mild low-tone hearing loss, whereas the elimination of components with frequencies of less than 500, 750, or 1,000 Hz simulated more severe low-tone hearing loss as the values increased. The elimination of high-frequency components can be considered as the simulation of high-tone hearing loss. In addition, it can also be considered to simulate modified sounds that patients may hear in everyday life in certain environments. Sounds are subject to reflections and refractions caused by walls or objects in their paths. Thus, the sound “image” that reaches the ear will differ somewhat from that initially generated. Diffraction occurs at lower frequencies because lower-frequency sounds have longer wavelengths .The results of experiment 1 suggest that when the simulated low-tone hearing loss is mild, many participants do not perceive pitch shift as they perceive the missing fundamental tone. However, when the simulated low-tone hearing loss is more severe, many participants perceive pitch shifts as they cannot perceive the missing fundamental tone. These results were more apparent in the case of the Y series  are reasonable because missing fundamental pitches are generally less distinct when fewer numbers of harmonics are present , whereas some individuals base their judgment on the frequency of the partials that make up the tones  . However present . It may Ambiguous responses were analyzed using the data from our experiment. In the present study, we provisionally defined ambiguous responses as different responses to the same task. Responses were high for both X3 and X4. Although this was a preliminary analysis, based on our results, it can be inferred that the SI values for X3 and X4 were close to 0 as each participant’s response was ambiguous in judging these tasks, rather than dichotomizing the participants’ responses between F0 and spectral responses.Our previous study  presenteExperiment 2 aimed to demonstrate the effect of the pitch shift observed in experiment 1 on everyday life. The results were similar to those of experiment 1 in terms of statistical significance. However, experiments 1 and 2 appear to exhibit pronounced differences. In experiment 2, the SI values indicate more marked F0 responses except for Y3 and Y4. The participants were asked whether the connected tones sounded correct based on the melody or off-key in experiment 2. Such criteria for overall judging might have increased the tendency toward F0 responses. The more unambiguous answers of experiment 2 may also be related to such judging criteria. The abrupt shift towards positive SI values in Y3 and Y4 may be related to the feature of the tonal sequences. Since pure tones were included in Y3 and Y4, it is possible that they may sound off-key. Thus, it is difficult to interpret the results of experiment 2 alone, but combined with the results of experiment 1, a possible scenario is illustrated in Interestingly, the results of the present study can also lead to a completely different interpretation. In the case of some patients with cochlear disorders of low-tone hearing impairment with pitch shift, pure tones may be perceived as off-key, whereas complex tones may not. This phenomenon may be attributed to the fact that these patients are unable to hear low-tone components that should be shifted in pitch, but are able to hear upper harmonics of the complex tones that are not impaired .Since the conditions that construct the tones under which the pitches shift were extreme in this study, they may not be frequently encountered by real patients. We simulated low-tone hearing loss in participants, preventing them from hearing the lower components of complex tones. However, real patients may hear lower components to some extent, unless their hearing loss is severe. Moreover, patients may not remain in an environment where high-frequency components have been eliminated for long periods of time. Thus, the pitch shift we have demonstrated here should only occur occasionally. Clinically, we sometimes see patients who complain of occasional pitch shift perception. This may represent the pitch fluctuation in patients with Meniere’s disease described by We investigated the pitch shift provoked by complex tone stimulation in healthy participants by simulating low-tone hearing loss. We found that the pitch of complex tones shifts when a low tone is not heard, especially in cases when a high tone is also not heard. It is worth noting that the conditions of the tones under which the pitches shifted in this study were extreme and may not be encountered frequently by patients in a real-world setting. However, it may partly explain the pitch shift observed in patients with hearing loss. Since our experiments were performed on healthy participants, we can infer that such pitch shifts can be perceived regardless of the underlying etiology of the hearing loss. Thus, pitch shifts associated with hearing loss should be interpreted with caution.10.7717/peerj.16053/supp-1Table S1Click here for additional data file.10.7717/peerj.16053/supp-2Table S2Click here for additional data file.10.7717/peerj.16053/supp-3Supplemental Information 3Click here for additional data file.


Lawsonia intracellularis is the aetiologic agent responsible for Porcine proliferative enteropathy. However, it is complex to determine whether this intracellular bacterium is the cause of clinical disease due to its high prevalence in the field. In this study, we investigate the agreement between PCR and IHC results for L. intracellularis evaluation, and the infection and co-infection by porcine circovirus type 2 and L. intracellularis in the ilea of pigs presented with suspected proliferative enteropathy in Italy. In swine, the diagnosis of enteric diseases is challenging due to simultaneous presence of one or more microbic agents sharing similar clinical signs and pathological lesions. Therefore, the study of microscopic lesions is crucial in establishing the role of pathogens as causative agents in cases of co-infections; this can be confirmed by demonstrating agent–lesion co-localization. Although PCR is more sensitive than IHC, the latter provides a positive result only when the amount of antigen is significant, and therefore is likely the cause of the enteric pathology. Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease. The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing  Enteric diseases pose a significant challenge in intensive pig farming, not only causing production and economic losses but also impacting animal welfare and necessitating antimicrobial use. These on-farm enteric diseases often involve multiple infections, leading to complex clinical patterns that make it difficult to identify the role of each agent and implement effective control measures . Escherichia coli (ETEC) or Shiga toxin-producing E. coli (STEC). Additionally, viral and parasitic pathogens such as rotavirus, cryptosporidia, and coccidia play a role in the first weeks of life. In the growing and finishing phases, the most significant diseases include salmonellosis, porcine dysentery by Brachyspira hyodysenteriae, spirochetosis by Brachyspira spp., and proliferative enteropathy (PE) by Lawsonia intracellularis and ascaridiosis [Post-weaning, enteric diseases in pigs may be caused by bacteria such as enterotoxigenic ridiosis . L. intracellularis is a Gram-negative bacterium that has been identified as the primary causative agent of enteritides localized in the aboral small intestine of swine, specifically in the ileum [L. intracellularis requires histologic confirmation of the typical lesion, which is represented by the hyperplasia of intestinal crypts. In addition, its co-localization with the presence of the antigen through the immunohistochemistry or immunofluorescence or in situ hybridization [he ileum . This cohe ileum ,4,5. In he ileum ,4,6. It dization  is also L. intracellularis and PCV2 infections are equally associated with growth retardation, emaciation, increased mortality, and diarrhoea in weaned pigs. In addition, these diseases are characterized by lesions that are sometimes macroscopically indistinguishable, such as necrotizing ileitis and colitis [Porcine circovirus-associated diseases (PCVDs) can manifest as both the systemic form of porcine circovirus type 2 (PCV2) disease and the clinical intestinal form, which is referred to as PCV2-enteric disease, PCV2-ED. Both forms of enteritis can be clinically apparent with diarrhoea ,8. L. inL. intracellularis in the development of enteric diseases in swine has been established through field investigations conducted in Denmark [L. intracellularis have been used experimentally to induce PCV-2 associated enteritis in swine [The association between PCV2 and  Denmark . Furtherin swine . Both pain swine .L. intracellularis enteritis with PCR results on ileum of pigs submitted to the laboratories of the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), and the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna (IZSLER), for suspected L. intracellularis proliferative enteropathy, in Italy. Furthermore, this study aims to demonstrate the co-infection L. intracellularis-PCV2 in situ through immunohistochemistry. The purpose of this study is to compare histology and immunohistochemistry (IHC) investigations for the diagnosis of L. intracellularis  and PCR in both acute and chronic cases of L. intracellularis enteritides. A total of 24 farms were enrolled based on anamnesis, which revealed the presence of L. intracellularis, as confirmed by a positive PCR test conducted on stool samples. The selection process for these farms was based on specific criteria, which included the gross appearance of either acute/severe (haemorrhagic/necrotic) or chronic (proliferative) forms of enteritides. The tissue specimens examined are part of a case series collected to define the relationship between the diagnostic gold standard for All samples in the study were taken from animals found dead on the farm. No animals were sacrificed for the purpose.L. intracellularis detection. The selected tract of ileum was formalin-fixed to preserve the samples stored at ambient temperature to maintain their integrity.In order to collect samples, the terminal portion of the ileum located near the ileocecal valve was chosen as it was deemed the most appropriate site for L. intracellularis, Brachyspira hyodysenteriae, and B. pilosicoli on stool pools and, only for L. intracellularis, also on gut wall were performed at the laboratories of the Istituto Zooprofilattico Sperimentale delle Venezie  and the Istituto Zooprofilattico Sperimentale delle Lombardia e dell’Emilia-Romagna .Histopathology and immunohistochemistry have been investigated at the Pathology Service of the Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Italy, while the biomolecular tests (PCR or RT-PCR) for B. hyodysenteriae or B. pilosicoli, according to Gasparrini et al. [Faecal samples were incubated on blood agar culture plates supplemented with rifampin, vancomycin and colistin for 5 days at 37 °C in an anaerobic atmosphere. Evaluation of their hemolytic characteristic (strong and weak hemolysis) allowed the samples to be classified as suspected i et al. . BacteriB. hyodysenteriae or B. pilosicoli. The primers used are reported in A species-specific real-time PCR confirmed the identification of Preliminarily, stool or intestinal scrapings samples were diluted 1:10 in phosphate-buffered saline, PBS (1 g of stool in 9 mL PBS). DNA was extracted from 200 µL of a diluted sample using a MagMAX CORE Nucleic Acid Purification Kit . The total volume for each assay was 25 µL and consisted of: 12.5 µL 2x QuantiNova Probe PCR masterMix , 1.25 µL of each primer , 0.5µL of probe , 2.2 µL of water, 2 µL of EXO IPC mixture, 0.3 µL of Exo IPC DNA  and 5 µL of extracted DNA. Real-time PCR was performed on a CFX-96 System . According to Willems and Reiner , the proL. intracellularis and PCV2. Details of the immunohistochemical protocol used are provided in Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. From each ileum specimen, 3-micron-thick sections were obtained, one stained with hematoxylin-eosin, while the other two underwent immunohistochemical staining for L. intracellularis, and a section of lymph node from a PCV2-SD case positive to PCV2 by both PCR and IHC were used as positive controls.In each immunohistochemical run, a section of ileum previously identified as positive for the presence of The specificity of the immunohistochemical stain has been validated by adding, as the primary reagent, an antibody of the same isotype as the primary antibodies (IgG1) but of irrelevant specificity.A proposed grading system was used to assess the histologic severity of enteritis and crypts hyperplasia . First, L. intracellularis was considered positive in case of brown-coloured granular material in the apical cytoplasm of intestinal crypt cells, and in the cytoplasm of macrophages in the interstitium of intestinal mucosa  for each out of the 28 animals are reported in The results of bacteriological investigations (L. intracellularis was assessed using the Cohen’s Kappa (K) coefficient [Agreement between PCR and IHC results for fficient .The present caseload encompasses 28 ileum samples collected from 24 farms. One farm provided four samples, another provided two samples, and the remaining 22 each provided one sample. Two cases  ranging from 0.72–0.98.  was objectively assessed in two out of four acute cases and two out of 24 chronic cases.Co-infection of Lawsonia intracellularis was detected by immunohistochemistry (IHC) in only 16 out of 28 (57%) examined ilea. Notably, all the samples from the four clinical cases were positive for both IHC and PCR, while only 12 out of the 24 samples from herds with cases of diarrhea and/or suspected subclinical cases were positive to L. intracellularis by IHC. Furthermore, three cases exhibited discordant results with the latter group of samples (PCR+/IHC-). Despite the substantial agreement (K = 0.77) between PCR and IHC results, PCR demonstrated greater sensitivity in diagnosing L. intracellularis infection. This discrepancy could be attributed to the variable amounts of intracellular bacteria present in swine infections, including cases below the limit of detection (LOD) of the immunohistochemical method. Additionally, the sampling strategy that involved analysing one swine in most farms may have limited the probability of detecting cases with detectable antigen levels by IHC. Experimental infection studies have shown that at least five days of intracellular replication are necessary to reach detectable levels of L. intracellularis by IHC or in situ hybridization (ISH). The antigen could be detected up to 14 days post-infection [L. intracellularis PCR and IHC, it is plausible that they represent actual negative cases. Alternatively, due to the segmental distribution of Lawsonia lesions [In the present study, all suspected proliferative enteropathy (PE) cases exhibited hyperplasia of the intestinal crypts. However, the presence of ut of 28 7% examin lesions , the resL. intracellularis-positive samples by IHC in this particular caseload (57%) is substantially higher than the rates reported in similar field investigations in Denmark (18%) [L. intracellularis, which may have influenced the outcomes.Upon comparing the available literature, it has been observed that the percentage of rk (18%) , or in Erk (18%) . This diBrachyspira hyodysenterie, Brachyspira pilosicoli, Salmonella enterica, Escherichia coli, and PCV2 [L. intracellularis infection is not specific, as similar changes can occur in response to mucosa injury or ulceration [L. intracellularis ante-mortem more rapidly and accurately during the first stage of infection [L. intracellularis genome both in stool and in the intestinal mucosa, even when the amounts of microorganisms are so low that it is unlikely to be the actual cause. In contrast, IHC provides a positive result only when the amount of L. intracellularis is significant, and therefore is likely the cause of the enteric pathology. More concordant results can be reached between qPCR and IHC, as a cycle threshold (Ct) less than 20 predicts the presence of lesions and L. intracellularis detected by IHC [Several types of investigation for diagnosing PE in swine are available, each with its strengths and limitations . The cliand PCV2 . The groand PCV2 . Howeverand PCV2 . This exceration . Therefoceration . PCR hasnfection , providinfection . Furtherd by IHC .L. intracellularis; PCR can be used as a screening technique to identify the presence of the microorganism and, in cases of positivity, samples should then undergo further examination by histology and immunohistochemistry for definitive confirmation of L. intracellularis’ role as the actual causative agent.Upon analysis of the findings of our investigation, it appears that using the IHC examination yields more reliable results in terms of identifying the causal diagnosis of the observed enteric pathology. Therefore, it may be beneficial to perform PCR and IHC tests in series to increase the specificity of the causal diagnosis attributed to L. intracellularis was identified in two out of four cases of acute disease and two cases of chronic enteritis, resulting in an overall percentage of four (double-positive) out of 16 IHC Lawsonia-positive cases (25%). The emergence of PCV2 and its associated diseases in the first decade of the 21st century has dramatically changed disease patterns in pig farming, highlighting the pathogenic potential of endemic agents as a direct consequence of the immunosuppressive abilities of PCV2 [L. intracellularis, the literature clearly indicates their respective roles. Recent Danish data show altered infection dynamic and antibody development to PCV2 and L. intracellularis in pigs previously exposed to PCV2 [Salmonella, Brachyspira spp., and/or L. intracellularis [L. intracellularis and PCV2 enteritis in pigs. L. intracellularis enteritis is characterized by crypt hyperplasia, while PCV2 enteritis is marked by histiocytes positive to PCV2 infiltrating the mucosa, sub-mucosa and Peyer’s patches. However, both diseases share features such as depletion and necrosis of Peyer’s patches and monocyte/macrophage infiltration of the lamina propria. Jensen et al. [L. intracellularis-PCV2 co-infection through the use of immunolabelling in 34 out of 64 intestinal samples with suspected PE. At the same time, only PCV2 was detected in 27 out of the remaining 30 cases of enteritis. The authors used a double immunohistochemical staining to show the co-presence of L. intracellularis and PCV2 in macrophages [L. intracellularis in macrophages can explain why the probability of PCV2 replication and associated disease increases in the intestines of L. intracellularis-infected swine. Co-infection with other pathogens or immunomodulation is known to increase the severity of PCV2 infection [L. intracellularis and PCV2 is not surprising. This can support a secondary role of PCV2 in double infection PCV2-L. intracellularis. In the caseload of this study, a higher frequency of acute cases sharing both infections was observed, supporting the hypothesis that when infection by ubiquitous pathogens such as L. intracellularis occurs, it contributes to an increased likelihood of PCV2 replication, creating an opportunity to switch from subclinical to clinical PCV2 disease. However, doubts have been raised about the actual existence of PCV2 intestinal and lung diseases [In this investigation, co-infection of PCV2 and  of PCV2 . PCV2 in of PCV2 , leading to PCV2 . These cllularis ,10,23. Adiseases , as almoL. intracellularis-induced proliferative enteritis underscore the importance of histopathology as the gold standard diagnostic technique for identifying this condition. The more severe and acute cases of PE may be linked to the activity of pathogens that can influence each other. In the case of L. intracellularis and PCV2, both hypotheses of PCV2 as the primary agent causing severe immune system dysfunction, which worsens the pathogenicity of the other agents and the ability of L. intracellularis to prime PCV2 replication, should be considered.Swine enteric diseases, similar to respiratory pathologies, are complex conditions often characterized by the simultaneous presence of one or more microbial agents. Therefore, while reducing antimicrobial use on farms through prophylactic measures is essential, it is equally important to establish tailored diagnostic protocols that can provide a definitive diagnosis and enable targeted control and prophylaxis. This study’s results on the diagnostic pathway for