Hearing loss in the elderly is one of the most incapacitating communication disorders, preventing them to fully perform their tasks in society.This study aimed to determine what is the best tool that together with the audiometric tests better represents the hearing loss reported by the elderly and which frequencies in the audiometric test must be considered to determine the hearing loss degree.Clinical prospective.71 elderly with ages between 60 and 82 years old were assessed. The subjects were submitted to a conventional audiometric assessment and a Hearing Handicap Inventory for the Elderly (HHIE) questionnaire. Three audiometric averages were analyzed and compared with the results obtained in the complete form (HHIE) and in its reduced form (HHIE-S).specificity showed values between 43.5% and 58.5% for HHIE with the different audiometric averages and values between 50% and 63.4% for the HHIE-S.audiometric average pure tone thresholds in the frequencies of 4kHz and 6kHz found in the audiometric assessment did not contribute to the self-reported hearing handicap perception. The correlation between HHIE-S and PTA1 had the best specificity (63.4%) and best positive predictive value (62.5%). Humans are living longer thanks to mankind social, economic and scientific developments. Although a desire of most people, it may result in living with disabilities and dependence. The epidemiological transition resulting from the demographic change has altered the morbi-mortality profile of the populations which went through the population aging process (increase in the ratio of elderly citizens). Possible results are the increase in the prevalence of chronic-degenerative disorders, with its sequelae and complications, causing disability, dependence, the need of care for longer periods even in specialized institutions, and many others such as social support, social networks, loss of social roles, loneliness, solitude, depression, loss of autonomy and of personal meaning, and the lack of meaning for life itself. One major challenge that increased longevity brings us is that of furthering survival with an increasingly better quality of life.Hearing loss causes one of the most disabling communication disorders in the elderly, preventing them from fully performing in society. Of all the sensorial deficits faced by the elderly, communication impairment caused by hearing loss can be one of the most frustrating. It is common to see hearing decline followed by a frustrating speech understanding in the elderly. Senior citizens with hearing impairment have a reduction in auditory sensitivity and a reduction in speech intelligibility at supra-threshold levels, which seriously compromises their verbal communication processes. Since hearing loss starts gradually and does not manifest as a disease, especially in its initial stages, it is not perceived by the patient. It is common for individuals to state they can hear, but can not understand well what is being said. Elderly individuals have a greater difficulty with speech under noise and reverberation; and they also have greater difficulty of complementing their hearing with visual information obtained by means of lip reading. This persistent failure in understanding speech can result in frustration and disheartening, which can lead to resignation.The gold standard used to assess hearing is tonal audiometry, a method which does not analyses qualitatively the hearing loss, and it only classifies the level of hearing loss in the different frequencies tested.In 1982, Ventry and WeinsteinStudies have attempt to analyze and validate instruments that could evaluate to the hearing loss in the elderly and to measure the hearing complaint.Corrêa, Russo Both procedures have been broadly utilized and the HHIE-S is the one most employed because it is more practical; however, it is not well established if the HHIE, even being longer, would not be a more reliable tool to assess the hearing complaint.The proposal of this study was to find out the best instrument which together with the audiometry better represents the elderly patient's complaint, and which tonal audiometry frequencies must be considered in order to assign the degree of hearing loss.This paper was referred to the Ethics in Research Committee for appreciation and was approved under protocol # 0823/06 on June 23, 2006.We evaluated 71 elderly patients, aged between 60 and 82 years, 48 women with mean age of 71.16 years and 23 men, with mean age of 73.91. This study started after approval by the head of the department of this institution where these elderly were assessed.These senior citizens were invited to participate by means of an invitation letter and by agreeing they signed an Informed Consent Form; they came referred to the audiology service by their physician requesting audiologic evaluation and were then invited to undergo the other tests proposed in the study - which were all done in the same day.We used inclusion and exclusion criteria to select the individuals participating in this study. Inclusion criteria were: elderly with ages equal to or above 60 years; elderly individuals with normal hearing and some with mild to moderate sensorineural hearing loss. The exclusion criteria were: severe to profound hearing loss and air conduction hearing loss.All the individuals were submitted to basic audiologic evaluation and the HHIE questionnaire.In the basic audiologic evaluation we performed the following procedures: threshold tonal audiometry; SRT - Speech Reception Threshold - attained by means of trisyllable wordsThe hearing thresholds considered normal were those found up to 25 dB HL (ISO 7566 Standard) in all the sound frequencies assessed, 250, 500, 1000, 2000, 3000, 4000, 6000 and 8000 Hz, based on Silman, Silverman.In order to classify the degree of hearing loss, we adopted the recommendation by DavisThus, for this study, auditory sensitivity was classified and identified for statistical analysis purposes, as “normal hearing” for hearing thresholds of up to 25 dB HL and as “altered hearing” for the mean hearing thresholds above 25 dB HL.The individuals selected for this study were also submitted to the Hearing Handicap Inventory for the Elderly - HHIE questionnaire, used in the format of an interview, in order to list the social and non-auditory aspects resulting from the handicap and hearing loss, which limits or prevents the individual from properly performing his/her daily activities and which compromise his/her family, work and social lives, as observed by Russo.This auditory handicap questionnaire proposed by Ventry, WeinsteinIn order to establish the handicap level we followed the evaluation and scoring criteria proposed by Ventry, Weinstein2: YES = 4 points (%); NO = 0 points (%); SOMETIMES = 2 points (%).Through the answers obtained in this questionnaire, the individuals were distributed in three groups. The criteria followed for this classification was the score obtained in the questionnaire, which follows the classification as shown on After employing the complete questionnaire we extracted the 10 items corresponding to the HHIE-S for analysis, and we obtained the results for each individual regarding the HHIE and HHIE-S.Besides this quantitative classification, the questionnaire was broken down into emotional (E) and social (S) aspects, which were considered for the qualitative analysis in both the procedures, the HHIE and the HHIE-S.In order to realize the elected procedures, we used the following material: 1) Amplaid A321 audiometer with a TDH39 phone and Mx41/AR pad, calibrated according to the EM ISO 389 (1995), ANSI S3.6 91996 and ISO 389-3 (1994) standards. 2) Impedance meter from Interacoustics, model AT 235/425 calibrated according to the ANSI S3.6 – 1996/ISSO 8253-1. 3 norm) and the Hearing Handicap Inventory for the Elderly questionnaire.For sample analysis we used the Anderson-Daling normality test, showed on histogram. We used the chi-squared non-parametric test for independence and in analysis complementation we calculated the sensitivity and specificity statistical values and the predictive value of the elderly hearing complaint in relation to the different audiometric differences .Sensitivity was defined as the percentage of patients complaining of hearing loss among those in whom the audiometric exam showed hearing loss. The specificity was the percentage of patients without hearing complaint among those with normal hearing.The positive and negative predictive values were defined, respectively, as the likelihood of the patient having hearing loss among those with hearing complaints and of presenting normal audiometric values among those without hearing complaints.The results are shown on 5 Tables, according to the text that follows.On The present study correlated the auditory thresholds, SRT, SRPI and different audiometric mean values obtained by means of an audiometric evaluation and age with the elderly hearing complaint by means of the Hearing Handicap Inventory for the Elderly - HHIE, both the complete and the abridged versions.In the present investigation both the HHIE-S score and the HHIE score presented a correlation with the auditory thresholds. There was a statistically significant correlation regarding all the auditory thresholds for the SRT and the SRPI . Both thPinzan-Faria, IórioGates et al.Gates et al.8 reported that the 10 HHIE-S items presented 35% sensitivity and 94% specificity; differently from what we observed in this present study, in which sensitivity varied between 90% and 100%, depending on the audiometric mean value adopted. The global subjective value presented a higher sensitivity (71%); however, lower specificity (71%) than the HHIE-S, the authors concluded that the global value of the hearing loss was more effective than the detailed questionnaire to identify elderly individuals with unknown auditory handicap.For the two auditory complaint assessments (HHIE and HHIE-S) with the different audiometric mean values we found the positive and negative predictive values , Table 5The audiometric mean values involving higher frequencies such as 4kHz and 6kHz, PTA 2 and PTA 3 respectively, have a 100% negative predictive value for the two ways used to assess the auditory complaint, that is, none of the elderly without hearing loss for these audiometric mean values (PTA > 25dB) will have an auditory complaint. This also suggests that the 4kHz and/or 6Khz thresholds can raise the audiometric mean without relevant impact on the 500Hz, 1kHz and 2kHz frequencies, because when the mean value of these thresholds are higher than 25dB there is a 62.5% likelihood of the patient complaining of hearing loss, compared to 32.5% for the PTA 2 and 22.5% for the PTA 3.Sindhusake et al.In the present study we observed a high sensitivity, medium specificity and low positive predictive value (32.5%) when the HHIE-S questionnaire was compared to the mean values of frequencies 500; 1,000; 2,000 and 4,000 kHz (PTA 2 in the present study); this does not depend on the score. Regarding the other audiometric mean values and the use of the entire HHIE, we observed high sensitivity, despite the low positive predictive value when correlated with PTA 1 and PTA2. Nevertheless, Sindhusake et al.Pinzan-Faria, IórioLichtennstein et al.Similarly to the other studies mentioned, the present one studied the correlation between audiometric thresholds with the HHIE-S. Moreover, the present study investigated the correlation between the audiometric thresholds with the HHIE compared to the HHIE-S and its sensitivities and specificities for the three different audiometric mean values from: 500Hz, 1kHz and 2kHz; mean values from 500Hz, 1kHz, 2kHz and 4kHz and mean values from 500Hz, 1kHz, 2kHz, 4kHz and 6kHz.We could then conclude that the inclusion of the 4Khz and 6kHz in the audiometric mean values for hearing assessment purposes did not contribute to the perception of auditory complaints and that both the HHIE and the HHIE-S are good tools to assess the hearing complaint, and the correlation between HHIE-S and PTA1 have the better specificity (63.4%) and the best positive predictive value (62.5%).


Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. To minimize the economic losses caused by this disease, M. hyopneumoniae vaccination is commonly practiced. However, the persistence of M. hyopneumoniae vaccine-induced immunity, especially the cell-mediated immunity, till the moment of slaughter has not been investigated yet. Therefore, on two commercial farms, 25 pigs (n = 50) received a commercial bacterin intramuscularly at 16 days of age. Each month, the presence of M. hyopneumoniae-specific serum antibodies was analyzed and the proliferation of and TNF-α, IFN-γ and IL-17A production by different T cell subsets in blood was assessed using recall assays. Natural infection with M. hyopneumoniae was assumed in both farms. However, the studied pigs remained M. hyopneumoniae negative for almost the entire trial. Seroconversion was not observed after vaccination and all pigs became seronegative at two months of age. The kinetics of the T cell subset frequencies was similar on both farms. Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ T cells were found in blood of pigs from both farms at one month of age but decreased significantly with increasing age. On the other hand, T cell proliferation after in vitro M. hyopneumoniae stimulation was observed until the end of the fattening period. Furthermore, differences in humoral and cell-mediated immune responses after M. hyopneumoniae vaccination were not seen between pigs with and without maternally derived antibodies. This study documents the long-term M. hyopneumoniae vaccine-induced immune responses in fattening pigs under field conditions. Further research is warranted to investigate the influence of a natural infection on these responses.The online version contains supplementary material available at 10.1186/s13567-023-01145-1. Mycoplasma hyopneumoniae (M. hyopneumoniae). It is the primary agent of enzootic pneumonia, endemically present on most farms and responsible for significant economic losses in the pig production worldwide [M. hyopneumoniae, since it can minimize the clinical signs and performance losses, and reduce treatment costs [M. hyopneumoniae vaccination is to raise pathogen-specific immune responses which respond fast and effectively upon a natural M. hyopneumoniae infection. Most commercially available M. hyopneumoniae vaccines for pigs are registered for administration between one and three weeks of age [M. hyopneumoniae-specific immunity varies from one to four weeks and aims to protect against infections during the complete fattening period, until slaughter [One of the most important respiratory pathogens in pigs is orldwide , 2. Vaccnt costs . The goas of age . The onslaughter .+CD8+ T cells, ensuring a fast antibody and cell-mediated immune response after a second contact with the pathogen [M. hyopneumoniae may result in seroconversion and an increase in M. hyopneumoniae-specific antibodies locally in the respiratory tract [M. hyopneumoniae stimulation compared to non-vaccinated animals [+CD8− and CD4−CD8+ T cells, producing INF-γ and TNF-α, were found after M. hyopneumoniae vaccination [M. hyopneumoniae-specific vaccine-induced immunity persists. In most M. hyopneumoniae vaccine efficacy studies, pigs are already challenged a few weeks after vaccination to investigate coughing, immune parameters, pathogen load, and lung lesions [M. hyopneumoniae-specific vaccine-induced cell-mediated immunity from the moment of vaccination until slaughter under field conditions has not yet been investigated.Overall, vaccination or infection of pigs triggers the differentiation of memory B cells and CD4pathogen . Vaccinary tract –9. In va animals . Furthercination . However lesions –16. When lesions –20. To oM. hyopneumoniae vaccination of breeding gilts is a commonly used acclimation practice [Mycoplasma hyopneumoniae-specific cytokine-producing CD4+CD8+ and polyfunctional CD4−CD8− T cells are present after vaccination of breeding animals [M. hyopneumoniae-specific maternally derived antibodies (MDA) and cell-mediated immunity in their blood which can persist till weaning or even longer [M. hyopneumoniae in the presence of (high levels of) MDA, seroconversion is often lacking [M. hyopneumoniae vaccinated piglets compared to non-vaccinated piglets, regardless of the levels of MDA [M. hyopneumoniae vaccination in the presence of high levels of MDA, further differentiation of the T cell subsets and their cytokine production is needed to gain better insights in these cell-mediated immune responses and eventually the influence of a natural infection on these responses.Besides piglet vaccination, practice . Mycopla animals . Pigletsn longer , 22, 23. lacking –26. HoweM. hyopneumoniae vaccination in pigs on two commercial farms. To this end, serum antibodies and proliferation of and cytokine production by different T cell subsets were monitored from the moment of vaccination at 16 days of age until slaughter age. The potential influence of M. hyopneumoniae-specific maternally derived immunity on humoral and cell-mediated vaccine responses in piglets was also investigated.The objective of the present study was to investigate the humoral and cell-mediated immune responses upon M. hyopneumoniae was assumed based on the presence of the pathogen in tracheobronchial swabs (TBS) taken from fattening pigs in the past. Danbred breeding gilts were reared on farm A and purchased on farm B. On farm A, the breeding gilts were vaccinated once against M. hyopneumoniae four weeks prior to first insemination with Ingelvac MycoFLEX® . On farm B, breeding gilts were vaccinated with Stellamune®Mycoplasma  at six months of age upon arrival at the farm and a second time four weeks later. Furthermore, on farm B gilts were also booster vaccinated twice shortly before farrowing. On both farms, sows were not vaccinated against M. hyopneumoniae.Two commercial farrow-to-finish farms were included in the study based on the willingness of the farmer to participate. On both farms, circulation of M. hyopneumoniae with an inactivated whole cell J strain-based bacterin , porcine circovirus type 2 (PCV2)  and porcine reproductive and respiratory syndrome virus (PRRSV) . Pigs were moved to the fattening unit at 9 weeks of age.Farm A practiced a 5-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. The piglets were vaccinated at 16 days of age against M. hyopneumoniae with the same vaccine as in farm A  and PRRSV . The piglets were moved to the fattening unit at 10 weeks of age. On both farms, five breeding animals (two gilts and three sows of mixed parity) were included in the study. The farrowing process was monitored by the main investigator and from each litter, five healthy piglets (birth weight >1 kg) were selected, ear notched and followed up monthly from birth till slaughter (n = 25 piglets / farm). Cross-fostering of the ear notched piglets was not allowed and pigs did not receive antibiotics active against M. hyopneumoniae on both farms during the entire trial.Farm B worked in a 4-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. They were vaccinated at 16 days of age against The study was approved by the Ethical Committee of the Faculty of Veterinary Medicine and the Faculty of Bioscience Engineering, Ghent University . From the sows, colostrum was collected during the farrowing process. Within 6 h after farrowing, a TBS  and blood in a sterile serum tube (clotted blood) were collected from the sows. At two days of age, blood was taken from the piglets in serum tubes. From one to six months of age, blood in sterile serum and EDTA (non-clotted blood) tubes and a TBS were taken from the piglets on a monthly basis. On farm A, pigs were slaughtered at 177 days of age, and therefore, the last sample was taken at 5.5 months of age. On farm B, pigs were sent to the slaughterhouse at 191 days of age so the last sample was taken at 6 months of age. Colostrum and serum samples were stored at -20 °C and TBS samples at -80 °C until further analysis. Non-clotted blood was processed immediately for cell-mediated immunity analysis.M. hyopneumoniae, DNA was extracted from the TBS samples using a commercial kit  and a digital PCR (dPCR) protocol targeting the P102 gene was performed following a previously described protocol [To test for the presence of protocol .Mycoplasma hyopneumoniae-specific antibodies in serum and colostrum samples were analyzed using a commercial indirect ELISA  following the manufacturer’s instructions. Samples were considered positive if the sample to positive (S / P) ratio was higher than 0.40 and negative if the S / P ratio was equal to or lower than 0.40.M. hyopneumoniae J strain bacterin and for each animal a negative medium and positive Concanavalin A (ConA) control were included. The M. hyopneumoniae J strain bacterin was made based on the protocol for production of M. hyopneumoniae F7.2 C bacterin [From the fresh, non-clotted blood, peripheral blood mononuclear cells (PBMCs) were isolated using a Lymphoprep™ density gradient . Isolation, stimulation and staining of the PBMCs were performed as previously described with some minor modifications . Cells wbacterin . To invebacterin . Data weM. hyopneumoniae J strain bacterin to assess proliferation of T cells. The protocol used was described in detail in a previous study [Isolated PBMCs were stimulated in vitro with in-house made us study . For the® Statistics Version 27  to compare results within farms. A descriptive analysis was performed for M. hyopneumoniae-specific antibody levels and the frequency of different T cell subsets. Kolmogorov-Smirnov and Shapiro-Wilk tests were used as tests for normality of the residuals. For normally distributed data, repeated measures analyses of variance were used and post-hoc pairwise comparisons were made with a Bonferroni correction to assess possible differences between successive sampling moments. For not normally distributed data, a non-parametric Friedman test was used together with a Wilcoxon signed rank test with Bonferroni correction to compare the different sampling moments.Statistical analyses were performed using IBM SPSSDescriptive results were given to discuss whether the presence (S / P > 0.40) or absence (S / P ≤ 0.40) of MDA had an influence on the humoral and cell-mediated immunity after piglet vaccination.On each farm, five pigs were excluded during the trial due to death or loss of ear tag. The data of these pigs were included in the analysis till the last correctly documented sampling moment.M. hyopneumoniae DNA. On farm B, all TBS samples from the sows, piglets and fattening pigs tested negative for the presence of M. hyopneumoniae DNA till five months of age. At six months of age, four fattening pigs tested positive for M. hyopneumoniae with 300, 7, 22 and 14 M. hyopneumoniae organisms/µL DNA.On farm A, all TBS samples  tested negative for the presence of M. hyopneumoniae-specific antibodies in serum and colostrum at farrowing , while piglets from seronegative sows were also seronegative (MDA absent)  and 42.3 ± 5.9% (farm B). This frequency significantly decreased at two months of age. On farm A, the frequency stabilized from two months onwards, while on farm B the decrease occurred more gradually  against infection [Serum antibody levels in two-day-old pigs corresponded with the levels of olostrum , 23. Ser studies , 32, 33. studies , 32. Hownfection , 25, 34.M. hyopneumoniae, CD4+CD8− T helper cells, mainly Th1 and Th17 cells, play an important role. Th1 cells produce IFN-γ, which activates macrophages to kill the pathogen, while Th17 cells produce IL-17A to fortify the mucosal barrier [M. hyopneumoniae cells can also be found intracellularly, and given the crucial role of CD4−CD8+ cytotoxic T cells in protecting the host against intracellular pathogens, these cytotoxic T cells might also be important to control M. hyopneumoniae infections [M. hyopneumoniae stimulation, PBMCs were isolated from piglets on both farms. The pattern of T cell subset frequency was similar on these two farms. According to other studies, the percentage of CD4+CD8− T cells is higher than the percentage of CD4−CD8+ T cells at a younger age. With increasing age, the percentage of CD4+CD8− T cells decreases, while the percentage of CD4−CD8+ T cells increases resulting in a decreasing CD4/CD8 ratio over time [+CD8+ T cells. These cells increase with age when pigs have contact with microorganisms, as some of these cells have a pathogen-specific memory function [+CD8+ T cells remained at the same level from two months of age onwards. On farm B, there was a gradual increase till four months of age, like expected, although the percentage of CD4+CD8+ T cells decreased again afterwards. Comparing T cells subsets with other studies remains difficult as the PBMCs in this study were not measured directly after blood sampling, but first incubated for 20 h before measuring the percentage of T cell subpopulations. Mycoplasma hyopneumoniae-specific proliferation of CD3+ T cells persisted till the end of the fattening period, confirming the presence of M. hyopneumoniae-specific T cells in blood of pigs vaccinated at 16 days of age. The presence of M. hyopneumoniae-specific proliferating T cells might be correlated with protection against enzootic pneumonia because previous research demonstrated that the ability of PBMCs to proliferate, after in vitro stimulation with the mitogen ConA, is correlated with the resistance against diseases in pigs [−CD8− T cells were the main T cell subset proliferating on both farms followed by CD4+CD8− T cells with the percentages fluctuating over time. In pigs, most CD4−CD8− T cells bear invariant γδ T cell receptors in contrast to antigen-specific αβ T cells [Actinobacillus pleuropneumoniae, an increase in CD8− γδ T cells was observed [M. hyopneumoniae infections.To protect the host against facultative intracellular and extracellular bacterial pathogens like  barrier –38. Recefections , 35, 39.ver time –42. Piglfunction , 42–44.  in pigs . The CD4 T cells , 42. Upo T cells . Those T T cells . Furtherobserved . Further+CD8+ T cells were significantly decreased compared to the percentages at one month of age. Afterwards, the percentages were variable, they either decreased further or remained stable over time. The same pattern was seen for IL-17A-producing CD4+CD8+ T cells, although not statistically significant. In gilts vaccinated against PRRSV, IFN-γ production by PBMCs was also observed till 42 days post primo vaccination, but decreased afterwards till 147 days post-vaccination [M. hyopneumoniae vaccination has been demonstrated previously [In recall assays, different T cell subsets were assessed for their production of TNF-α, IFN-γ and IL-17A. At two months of age, the percentage of TNF-α-, IFN-γ- and TNF-α/IFN-γ-producing CD4cination . Increaseviously , 10.Chlamydia species, polyfunctional CD4+CD8− T cells are correlated with protection [M. hyopneumoniae also increased the percentages of polyfunctional CD4+CD8− and CD4−CD8+ T cells [+/IFN-γ+ CD4+CD8+ T cells at 24 days post-vaccination, decreasing again towards the end of the study at 56 days post-vaccination [+CD8+ T cells decreased over time. This study focused on circulating blood lymphocytes, but vaccination also induces local M. hyopneumoniae-specific cell-mediated immune responses in lungs and bronchial lymph nodes [M. hyopneumoniae-specific vaccine-induced immunity should also focus on local cell-mediated immune responses. Furthermore, to immunize the piglets only one commercial M. hyopneumoniae vaccine was used in this study. The immune responses can differ between vaccines as they are influenced by the formulation of a vaccine (adjuvant and antigen) and the administration route [M. hyopneumoniae vaccine used, is known to direct the immune response towards a Th1 response [Recently, evidence is accumulating that polyfunctional T cells, which produce more than one cytokine, have a pivotal role for protection and clearance of pathogens. For instance, in pigs infected with otection . Vaccina T cells . In anotcination . In our on route , 52, 53.response .M. hyopneumoniae seronegative and in serum of 20 piglets M. hyopneumoniae-specific MDA were present. Regardless of their MDA status, all pigs were M. hyopneumoniae seronegative at two months of age and seroconversion was not observed at later time points. Our findings correspond with a previous study in which seven-day-old piglets with and without MDA were vaccinated against M. hyopneumoniae [M. hyopneumoniae vaccination in the presence of MDA resulted in higher antibody levels compared to non-vaccinated piglets, but the higher the titer of MDA, the lower the response [M. hyopneumoniae vaccination, M. hyopneumoniae-specific cell-mediated immune responses are induced [M. hyopneumoniae vaccinated pigs regardless of the levels of MDA [M. hyopneumoniae vaccinated pigs with and without MDA at one month of age. Only on farm B, piglets with MDA tended to have higher percentages of TNF-α+ CD4+CD8+ T cells at one month of age compared to piglets without MDA. The relevance of these findings should be further investigated in a study including a larger and equal amount of piglets with and without M. hyopneumoniae-specific MDA. Then also statistical analysis should be performed as this was not done in this study due to the low and unequal number of animals in the group without and with MDA.On each farm, five piglets were eumoniae . In bothresponse , 26. AltM. hyopneumoniae vaccination of piglets was lacking, M. hyopneumoniae-specific cell-mediated immune responses were detected. Polyfunctional cytokine-producing CD4+CD8+ T cells were present till three months of age and proliferating T cells were observed till the end of the fattening period indicating the presence of a pathogen-specific cell-mediated immunity in M. hyopneumoniae vaccinated pigs. The results form the basis for further research assessing the influence of a natural M. hyopneumoniae infection on the vaccine-induced immunity under field conditions.In conclusion, the present study showed that, although seroconversion after Additional file 1. Gating strategy to assess cytokine production by T cells with CytExpert software.Additional file 2. Gating strategy applied on the T cell proliferation assay with CytExpert software.


Mycoplasma hyopneumoniae is the primary pathogen of swine enzootic pneumonia and causes great economic losses to the swine industry worldwide. In China, M. hyopneumoniae seriously hinders the healthy development of the native black pigs. To prevent and treat porcine respiratory disease caused by M. hyopneumoniae, the characteristics of M. hyopneumoniae strain ES-2 isolated from Chinese native black pig lungs with gross lesions at post-mortem were studied for the first time in this study. Strain ES-2 cell was round or oval cells and most sensitive to kanamycin. The diameters of most strain ES-2 cells ranged from 0.4 to 1.0 μm with maximum viability of 1010 CCU/ml. Experimental challenge of animals with strain ES-2 showed respiratory disease could be reproduced, with pneumonic lung lesions evident. Comparative genomics analysis identified that 2 genes are specific to pathogenic M. hyopneumoniae strains, which may be predicted to be a molecular marker. These findings suggest that the study on the characteristics of M. hyopneumoniae strain ES-2 will guide the rapid and accurate drug use in the clinic, and develop a theoretical foundation for accurately diagnosing and treating the infection caused by pathogenic M. hyopneumoniae. Mycoplasma hyopneumoniae (M. hyopneumoniae) is a host-specific microorganism that only infects swine  which is a chronic respiratory disease causing huge economic losses to the pig industry world , M. hyopneumoniae 7422 (GenBank assembly accession: GCA_000427215.1), M. hyopneumoniae 7448 (GenBank assembly accession: GCA_000008225.1), M. hyopneumoniae 168 (GenBank assembly accession: GCA_000183185.1), M. hyopneumoniae 232 (GenBank assembly accession: GCA_000008405.1), M. hyopneumoniae KM014 (GenBank assembly accession: GCA_002257505.1), M. hyopneumoniae TB1(GenBank assembly accession: GCA_002213485.1), and M. hyopneumoniae 11 (GenBank assembly accession: GCA_002193015.1) have been reported , porcine reproductive and respiratory syndrome virus (PRRSV), and so on  ES-2 strain is a China field isolate from the lung with gross lesions of Enshi Black pig. And the lung was from the slaughterhouse. M. hyopneumoniae strain ES-2 was isolated by using Friis liquid medium modified Friis liquid medium and modified Friis solid medium as previously described , and then cut the lung tissue in the centrifuge tube into small pieces with scissors and grind it with a tissue grinder. Subsequently, the supernatants were collected by centrifugation at 5,000 rpm for 10 min, and bacteria in the supernatants were removed by 0.45 μm filtration. The supernatants were transferred into a fresh Friis medium. The mixtures were incubated in 5% CO2 at 37°C for 6 days. On day 6 of culture, M. hyopneumoniae in Friis medium was identified by PCR amplifying the highly conserved 16S rRNA region from M. hyopneumoniae  of kanamycin for planktonic cultures of escribed .M. hyopneumoniae ES-2 strain, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed as previously described with some modifications  which was indicated by the phenol red as previously described .To observe the growth characteristic of ES-2 strain on a solid culture medium, the modified Friis solid medium was prepared as previously described . ES-2 atM. hyopneumoniae inoculation, blood samples were collected from each piglet. Anti-M. hyopneumoniae IgG antibody in sera, PCV-2 and PRRSV in blood were separately detected by the test kit (IDEXXCo.) and PCR was used to confirm that no piglets were exposed to M. hyopneumoniae, PCV-2, or PRRSV. On the 28th day, 4-week-old pigs were weighed and randomly allocated to two different groups. Different groups with 5 pigs in each group were housed in different rooms to avoid the spread of M. hyopneumoniae between groups. All pigs were inoculated and then euthanized as described previously . The complete genome of the M. hyopneumoniae ES-2 strain was sequenced using the PacBio method. The raw reads  were obtained by the third-generation sequencing. Polymerase read quality was 0.876. The reads were assembled de novo using the HGAP2 protocol on the SMRT Portal v2.3.0  was generated based on CDS existence information in 162 mycoplasma strains using amino acid multiple sequence alignment in Roary v3.11.2 (http://sanger-pathogens.github.io/Roary/) with default parameters    was perfM. hyopneumoniae was still identified by PCR . Ultimately, a field M. hyopneumoniae strain was successfully isolated and purified from the lung of Enshi black pigs suffering from typical swine mycoplasma pneumonia. And we named this newly isolated strain M. hyopneumoniae strain ES-2. Meanwhile, this study found that the minimum inhibitory concentration of M. hyopneumoniae ES-2 strain to kanamycin was 2 μg/ml.To get pure M. hyopneumoniae ES-2 strain on solid medium were observed through microscopic in this study. The colonies were found to be very small and granular with irregular margins with their central parts burrowed into the medium, which exhibited a typical “fried egg” appearance  approximately on day 2 postincubation with the corresponding pH of 7.24. After 2 days of culture, the viability in cultures continuously declined. On day 8, only 10 CCU/ml of ES-2 strains viability was detected with the corresponding color shift pH being 6.37. On day 7, color shift pH was minimum (6.37) and stopped declining (Color-changing units (CCU) of liquid medium were commonly used to determine the viability of on day 0 . From daeclining .M. hyopneumoniae could infect pigs of various ages resulting in typical lesions of EP  database. Of these 9 genomes of M. hyopneumoniae strains, the assembly level of 6 genomes  was complete. The other 2 genomes (11 and TB1) were incomplete. The genome size of M. hyopneumoniae strains ranged from 0.8 to 1.0 Mb (M. hyopneumoniae ES-2 strain (GenBank accession number GCA_004768725.1), which was sequenced using the PacBio method, contained a 956,514 bp (GC content 28.36%) single circular chromosome. The total number of genes predicted was 733, and the average gene length was 1,164 bp (mycoplasma reported (http://www.mgc.ac.cn/cgi-bin/VFs/compvfs.cgi?Genus=Mycoplasma) were collected from the virulence factor database to investigate the distribution of virulence-related factors among 9 M. hyopneumoniae strains. By aligning the proteins (identity ≥ 80% and coverage ≥ 80%) from each strain against the reported 25 virulence factors, a total of 11 virulence factors were identified in these genomes. In total, 10 out of 11 virulence factors were found to be present in each of 9 M. hyopneumoniae strains, and some virulence factors had multiple copies in these genomes. The cytoadherence protein (P200)  was unknown.In addition, the above experiments show that mycoplasma species, all 162 complete genomes of different species of mycoplasma strains were collected from the NCBI database. However, no core gene was found in the 162 mycoplasma strains. Therefore, the phylogenetic tree was first constructed based on CDS existence information in 162 mycoplasma strains. The phylogenetic tree showed that 15 porcine mycoplasmas , 2 M. dispar, 1 M. bovoculi, 3 other mycoplasmas form a group; 53 human mycoplasmas form another group; 10 sheep mycoplasmas and 14 bovine mycoplasmas form the third group; 12 chicken mycoplasmas, 2 human mycoplasmas, 2 porcine mycoplasmas, 1 bovine mycoplasma, and 7 other mycoplasmas form the fourth group; and 17 bovine mycoplasmas, 6 chicken mycoplasmas, 53 human mycoplasmas, and 13 other mycoplasmas form the fifth group  at the amino acid level. Venn diagram of the results showed that the number of common genes shared by 4 pathogenic M. hyopneumoniae strains and 1 nonpathogenic M. hyopneumoniae strain J were, respectively, 686 (identity = 0.75), 687 (identity = 0.80), 683 (identity = 0.85), 678 (identity = 0.90), 666 (identity = 0.95), and 521 (identity = 0.99). Excluding the common genes shared by pathogenic and nonpathogenic strains, 12 (identity = 0.75), 10 (identity = 0.80), 12 (identity = 0.85), 13 (identity = 0.90), 12 (identity = 0.95), and 25 (identity = 0.99) genes were found to be shared by all 4 pathogenic M. hyopneumoniae strains at the amino acid level, respectively , which will provide a theoretical basis for the rapid and effective treatment of M. hyopneumoniae ES-2 strain infections.By using the multiple pulmonary-point sampling and continuous passage culturing method, a lack pig . To furtM. hyopneumoniae ES-2 strain, we found that the modified Friis medium was most suitable for the growth of strain ES-2. The maximum viability of ES-2 strain in a modified Friis medium reached 1010 CCU/ml at approximately 2 days of incubation , plays a crucial role in the swine respiratory disease  can be found in the article/The animal study was reviewed and approved by Animal Care and Use Committee of Wuhan Polytechnic University (No: WPU202201002).YZha, BZ, HC, and CT contributed to the conception and design of the study. YZhu organized the database. YZha, BZ, and YZhu performed the statistical analysis. YZha, BZ, and CT wrote the first draft of the manuscript. ML and XW wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (32102674), the National Key R&D Program of China (2017YFD0500202), the Hubei Province Natural Science Foundation for Innovative Research Groups (2016CFA015), the earmarked fund for China Agriculture Research System (CARS-35), and the Hubei Agricultural Science Innovation Centre (2016-620- 000-001-039).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To provide an overview of the main evidence-based recommendations for the diagnosis of hearing loss in children and adolescents aged 0–18 years.Task force members were educated on knowledge synthesis methods, including electronic database search, review and selection of relevant citations, and critical appraisal of selected studies. Articles written in English or Portuguese on childhood hearing loss were eligible for inclusion. The American College of Physicians’ guideline grading system and the American Thyroid Association’s guideline criteria were used for critical appraisal of evidence and recommendations for therapeutic interventions.The topics were divided into 2 parts: (1) treatment of sensorineural hearing loss: individual hearing aids, bilateral cochlear implants, cochlear implants in young children, unilateral hearing loss, and auditory neuropathy spectrum disorder; and (2) treatment of conductive/mixed hearing loss: external/middle ear malformations, ventilation tube insertion, and tympanoplasty in children.In children with hearing loss, in addition to speech therapy, Hearing AIDS (HAs) or implantable systems may be indicated. Even in children with profound hearing loss, both the use of HAs and behavioral assessments while using the device are important. Newborn hearing screening programs have diagnosed profound sensorineural hearing loss in the first few months of life. Initiating auditory rehabilitation, including the use of Hearing AIDS (HAs) before 6 months of age has significantly improved vocabulary, speech intelligibility, general language ability, and socioemotional development.Children who undergo cochlear implantation early in life may achieve similar verbal language skills to those with normal hearing.Although age at the time of cochlear implantation may have only minor long-term effects on general language understanding, it has a lasting effect on more specific and complex skills within the domains of phonetics, expressive vocabulary, grammar, and semantics. These skills depend on the functional specialization of certain networks in the brain that are triggered by sensory input during early sensitive periods of neuronal development.The use of HAs and CIs early in life has rehabilitated many children, who were able to acquire near-normal language. The effectiveness of these interventions is influenced by factors such as maternal education level, duration of daily device use, and nonverbal intelligence.Maximizing hearing in each ear optimizes oral language development. Device options for children with bilateral sensory hearing loss include HAs, CIs, and a combination of both .Unilateral sensorineural hearing loss affects approximately 9% of the population and is less related to aging than bilateral hearing loss. Its prevalence ranges from 0.3 to 1 per 1000 newborns. Approximately 3%–6% of school-age children have some degree of unilateral hearing loss.Children with Single-Sided Deafness (SSD) often struggle with speech perception in noise and sound localization due to lack of binaural auditory input. This outcome has been associated with the absence of binaural hearing effects, such as the squelch effect, the head shadow effect, and binaural summation. SSD has implications for children’s speech language development, cognition, and quality of life and causes psychosocial and behavioral difficulties.Spatial perception is multisensory. The auditory system plays an important role by continuously detecting sounds in the environment, helping with spatial localization. Auditory space constructs are complex and depend on the interaction with signals that dynamically change in terms of frequency spectrum, intensity, and time. The more complex the environment is, the more these signals interact and overlap.Hearing does not involve only sound detection and awareness, but also the management of these complex interactions to provide meaning to these signals. The auditory system is quite sophisticated in fully managing the acoustic information presented to the ears in dynamically changing environments. Listeners can rapidly process this information to identify acoustic stimuli, selectively attending to signals of importance while suppressing competing signals.Spatial hearing depends on the processing of monaural and binaural hearing cues. Although monaural stimulus provides important information and contributes to the ability to determine the distance of a sound source, binaural hearing plays a much larger role. The integration of acoustic information from both ears is essential for spatial hearing and provides critical information for speech processing, localization, differentiation of auditory streams, and perception of fused sounds. Binaural hearing gives rise to a wide array of auditory phenomena due to the integration and processing of differences in arrival time and intensity between the signals at the two ears. The interaural timing difference is the difference in arrival time for a stimulus to reach both ears, being greatest for low frequency signals below 1000 Hz.When a sound is presented to a listener, the ear closest to the signal of interest will detect that sound before the ear farthest from the signal. Likewise, the interaural level difference dictates that the ear closer to a stimulus will receive a more intense signal compared with the contralateral ear.Animal studies have evaluated the effects of unilateral hearing loss during development on different levels of the auditory system — morphology, connectivity, and the response properties of neurons. There was weakening of the representation of the deprived ear and a strengthening of the representation of the intact ear in the cortex.Microtia and congenital aural atresia are malformations that occur in approximately 1 in 10,000–20,000 live births. They are usually unilateral, with a predilection for the right ear, and are more common in boys than in girls (2.5:1). The inner ear and its function are often preserved but can be affected in 10%–20% of cases.The mean incidence of acute otitis media is 10.8 new episodes per 100 person-years,Approximately 98.7 million people have otitis media-related hearing loss, according to the World Report on Hearing,Chronic suppurative otitis media leads to some degree of hearing loss in 50% of cases due to tympanic membrane perforation and changes in the ossicular chain (conductive hearing loss), hair cell damage caused by recurrent infections , or mixed.This systematic review have the purpose to provide an overview of the evidence-based recommendations for the treatment of hearing loss in children and adolescents aged 0–18 years.Between April 28 and 29, 2022, a task force consisting of otolaryngologists, otology specialists, Brazilian Society of Otology  directors, and some SBO members met (in person and remotely) to discuss the topic of this guideline. Each participant in this meeting was tasked with giving a 15-min evidence-based lecture on one of the suggested topics. After the lecture, the participants discussed the topic until reaching a consensus. Each author was asked to write a text with the current literature on the topic, based on evidence and containing the elements discussed during the meeting. A rapporteur prepared the final text, which was reviewed by 4 additional coauthors and the Brazilian Journal of Otorhinolaryngology (BJORL) editor.This guideline is not intended to be a substitute for individual professional judgment. Physicians should always act and decide in a way that they believe is best for their patients, regardless of guideline recommendations. They should also operate within their scope of practice and in accordance with their training. The guidelines represent the best judgment of a team of experienced physicians addressing the scientific evidence for a given topic.The grading system of the American College of Physicians (ACP) was used in this guideline, relating to critical appraisal and recommendations on therapeutic interventions25The American Thyroid Association (ATA) created a diagnostic test appraisal system that included consideration of the following methodological elements: consecutive recruitment of patients representative of clinical practice, use of an appropriate reference gold standard, directness of evidence , precision of diagnostic accuracy measures , and consistency of results across studies using the same test that was also used in this guidelineThe use of HAs and CIs early in life has rehabilitated many children, who were able to acquire near-normal language. The effectiveness of these interventions is influenced by factors such as maternal education level, duration of daily device use, and nonverbal intelligence.Maximizing hearing in each ear optimizes oral language development. Device options for children with bilateral sensory hearing loss include HAs, CIs, and a combination of both .In children with mild-to-moderate bilateral sensorineural hearing loss, the use of HAs should be considered. In the case of severe-to-profound hearing loss, HAs may be insufficient for rehabilitation, and CIs should be considered. Regardless of the degree of hearing loss, HAs should be the first therapy to be considered, even in children with profound hearing loss, before a surgical procedure is indicated. It should be noted that speech assessment and speech therapy are extremely important for children using these devices.Despite treatment, children with mild-to-severe hearing loss using HAs have poorer receptive and expressive language performance, as well as less vocabulary compared with children with normal hearing.The CI is an implantable prosthesis that provides sound information to patients with severe-to-profound sensorineural hearing loss and no functional gain with HAs. It is the most successful sensory prosthesis in the world.The sound is captured by the microphone on the external component, selected, and encoded by the processor. The information is then sent to the transmitter, which sends the encoded sounds to the internal component. The electrodes generate electrical impulses that depolarize the auditory nerve fibers.The cochlear implantation procedure is safe if performed by experienced surgeons. There may be minor  or major  postoperative complications.Before CIs were made available, only approximately 50% of children with severe-to-profound bilateral hearing loss using HAs were able to acquire spoken language compared with children with normal hearing.31Age-appropriate language acquisition in children with CIs has been associated with higher levels of nonverbal intelligence and maternal education, higher levels of residual hearing after surgery, early intervention, a focus on auditory and oral instruction, and the use of novel speech processor technologies.Bilateral implants are the most viable option for verbal language development.Several factors influence the frequency with which children with CIs use their device — degree of sensorineural hearing loss, duration of CI experience, age at the time of surgery, and hearing gain with CI.Studies comparing the benefits of bimodal devices and bilateral CIs on oral language skills (receptive or expressive language) suggest that children with profound bilateral hearing loss should undergo bilateral cochlear implantation. Children with thresholds close to 70 dB in one ear and profound hearing loss in the contralateral ear may benefit from bimodal stimulation. However, they should be closely monitored due to the risk of worsening hearing in the ear using the HA.39Early access to hearing is critical for brainstem auditory system development. The lack of auditory stimulation may affect neural development, causing serious long-lasting effects on auditory development, language acquisition, and cognitive skills.Better linguistic outcomes may be expected for children who undergo early bilateral implantation.Additional intraoperative care is required for patients younger than 12 months. Pediatric patients have significantly lower blood volume compared with the adult population. In children younger than 12 months, the total systemic blood volume is approximately 80 mL/kg. Minor blood losses in the pediatric population can have catastrophic effects. Blood losses <10% in this population can lead to hypovolemia.Although the cochlea and facial recess are adult size at birth, changes occur in facial nerve position and mastoid pneumatization during childhood. In children younger than 12 months, there is lateral and superficial displacement of the facial nerve and semicircular canals, as well as underdevelopment of the mastoid tip.The skin of children younger than 12 months is significantly thinner than the skin of adults. Musculocutaneous flaps should be manipulated with caution.46Regardless of the degree of hearing loss, HAs must be the first therapy to be considered, even in children with profound hearing loss .Auditory rehabilitation should be bilateral and conducted as early as possible. Cochlear implantation should preferably be performed before 1 year of age in children with congenital sensorineural hearing loss .Children should undergo simultaneous cochlear implantation .Cochlear implantation in children younger than 1 year is safe and effective. The rates and nature of major and minor complications are comparable to studies of older children and adults .Patients with profound unilateral hearing loss or SSD have sufficient hearing for communication but struggle with sound localization and speech intelligibility in noise. This negatively impacts development and may cause delayed speech acquisition, poor school performance in up to 59% of children, and behavioral changes.Although the negative implications of SSD are clear, children affected by this condition often have a delay between diagnosis and intervention compared with those with bilateral sensorineural hearing loss.After diagnosis, the school must provide special assistance by placing the child in the front rows, with their better ear facing the teacher. Speech therapy should be conducted if language development is delayed.In recent years, treatment options for unilateral hearing loss have increased. In addition to HAs for partial losses and Contralateral Routing of Signal (CROS) systems for SSD, other rehabilitation options include Bone-Anchored Hearing Systems (BAHSs) and CIs. The benefit of using a device (implantable or not) should be considered when treating these children.Importantly, these treatment options do not provide any binaural hearing benefits. This means that speech discrimination in noisy environments and sound localization remain largely impaired.Traditional options for auditory rehabilitation redirect sound from the affected ear to the normally functioning side. Amplifiers with a CROS system have long been used to redirect sound from the diseased ear to the hearing ear. A microphone is placed on the deaf side to capture sound and transmit it (currently wirelessly) to the receiver on the hearing side. Despite being an option, the use of this device is limited by its high cost. However, using a receiver on the hearing ear may not be an option for some patients, as it disrupts natural hearing acoustics, reducing access to some sounds or modifying monaural level cues on which single-sided listeners become dependent.BAHSs are an alternative in which a microphone placed on the external processor captures sound and transmits it to the temporal bone and inner ear via bone conduction. This allows the hearing ear to remain open and unoccluded while maintaining monaural spectral cues. In addition to requiring surgery, a limitation of bone conduction stimulation for SSD treatment is the reduced signal level that may occur as it travels through the skull, particularly in high frequencies. BAHSs are more effective than HAs with CROS for sound discrimination in noise and quiet.BAHS users often experience reduced hearing benefit and gain over time. The discontinuation rate of BAHS use over a period of 50 months reached 14%, approximately 3% per year.Counseling  and extensive preoperative testing with a soft band BAHS may help patients understand their expectations. The choice of the most suitable device must be determined by the physician, who will discuss the clinical advantages and disadvantages of the devices with the patient and their family after collecting all qualitative and quantitative data derived from a complete audiologic evaluation.The CI is a treatment option for patients with SSD. It restores hearing in the affected year and may provide some hearing benefits that have not been identified with CROS and BAHS use. Patients with SSD using a CI reported improvements in speech perception in the affected ear, hearing in noisy environments, and sound localization compared with those who did not receive any treatmentHearing outcomes of CI users with SSD are not the same as those of children with normal hearing. Study outcomes vary greatly, especially due to the duration of auditory deprivation.A systematic review published in 2020 evaluated 119 children and showed that most of them (79.6%) had improved speech perception in noise after cochlear implantation, which was clinically significant.Prolonged auditory deprivation negatively affects CI results. Based on the maturation of the auditory pathway, the estimated sensitive period for treatment is from 3 to 4 years of age.70Children with SSD were reported to use their CIs between 6 and 12 h per day, similarly to bilateral CI users.Children with asymmetric loss should receive early treatment, given that they develop an aural preference for the hearing ear from an early age, which is difficult to reverse and interrupts their potential to develop binaural hearing .Achieving binaural hearing is always the goal, as it is the most physiological path for CNS development .BAHSs are more effective than HAs with CROS for sound discrimination in noise and quiet in patients with SSD .Children older than 3-years gain little benefit from cochlear implantation in congenital unilateral hearing loss .The contralateral ear should always be reassessed for the risk of hearing loss .The multidisciplinary team should observe some conditions when treating patients with ANSD. Treatment depends on the severity of the impairment and is defined according to each case. The goal is to restore auditory skills with hearing devices. Among available devices, HAs, frequency modulation or similar systems, and CIs are the most relevant ones. The literature shows an advantage of CIs over HAs.Some studies have produced different results with cochlear implantation in 25% of cases, which are dependent on patient conditions such as presence of residual hearing, age at the time of cochlear implantation, duration of sound deprivation before cochlear implantation, cognitive aspects, and factors related to socioeconomic status and anatomic morphology, especially of the cochlear and cochlear nerves.CI users have significantly improved speech perception and auditory performance. Even those with the worst results show a 50% improvement in the recognition of disyllabic words in audiologic tests after cochlear implantation, as well as greater ease of phone use and improved speech understanding in background noise, especially for adults.80The CI may provide positive results even when the lesion site seems to involve the auditory nerve. Molecular markers can be of great assistance when making the final treatment decision. The table below lists the genetic markers associated with better or worse CI outcomes in ANSD.Table 8 shows the genes identified to cause ANSDs and expected CI outcomes In addition to the use of hearing devices, auditory training should be conducted and focus on maximizing the signal-to-noise ratio – improving speech perception in noise with or without the use of a frequency modulation or similar system – always providing adequate counseling and defining the limitations that are more closely related to either postsynaptic or CNS lesions. CI candidates present with variations in auditory nerve function during response to electrical stimulation from the CI. Assessing the integrity of the spiral ganglion and cochlear nerve has become an important focus in predicting CI outcomes. Many studies have attempted to determine whether CI might benefit patients with auditory neuropathy. Auditory neuropathy was initially considered a contraindication to CI. However, after the initial reports of successful cochlear implantation in these patients, larger studies have been conducted and observed that patients with ANSD may benefit from cochlear implantation depending on the lesion site (whether they have neuropathy or synaptopathy).81Precisely determining the site of the lesion causing neuropathy, by using molecular diagnosis combined with electrophysiologic data from electrocochleography as well as intraoperative Electrical ABR (EABR), may help predict postoperative CI outcomes and may assist in the final treatment decision.Another concerning aspect is that OAE response diminishes over time and eventually disappears in approximately 20%–30% of patients.Even in patients with mutations affecting the auditory nerve, cochlear implantation may still be recommended due to better hearing outcomes for most patients .The lesion site is not the only determinant of CI outcome. In addition to clinical factors such as type and duration of deafness, molecular and physiologic factors that may suggest the status of the cochlea, synapse, spiral ganglion, and auditory nerve should also be considered to provide the best possible outcomes for patients .When conducting the initial evaluation of a patient with suspected auditory neuropathy, the possibility of a molecular diagnosis should be considered in addition to electrophysiologic assessment and therapeutic tests in speech therapy, as well as a multidisciplinary discussion involving speech therapists and geneticists .Surgical correction of these malformations is one of the most challenging procedures in otolaryngology. The goals of the procedure are to restore hearing and create an external auditory canal that remains patent and infection-free.88The Jahrsdoerfer grading system is often used to classify patients before surgery.The hearing gain varies depending on the severity of the middle ear malformation and the presence of concomitant malformations in the inner ear. Usually, surgical correction is indicated only in patients who have small malformations associated with a favorable anatomy of the temporal bone.BAHSs represent a revolution in the treatment of patients with middle ear malformations, regardless of the integrity or mobility of the ossicular chain, with a low complication rate. Multiple physiologic mechanisms contribute to hearing via bone conduction. Sound energy is transmitted by skull bone vibrations directly to the cochlea, bypassing the middle ear. Traveling waves propagate along the basilar membrane and stimulate the cochlear nerve. BAHSs can be divided into 4 types:Nonsurgical — bone vibrators that can be mounted on a behind-the-head band , elastic band , adhesive adapter , teeth (Sonitus SoundBite System), or auditory canal (TransEar).Percutaneous — Stimulus occurs via a skin-penetrating abutment coupled to a sound processor .Transcutaneous — a titanium plate is implanted in the temporal bone and a processor is coupled to a magnet that transmits sound through intact skin .Active transcutaneous — an active implant is placed under the skin and muscles of the temporal bone and communicates with the external sound processor wirelessly via radiofrequency .The indication criteria are as follows: (a) unilateral mixed hearing loss: bone conduction threshold — 65 dB (percutaneous), 55 dB (Osia2), and 45 dB (Bonebridge). (b) Speech intelligibility index greater than 60% for open-set recognition of monosyllabic words without HA. (c) Profound unilateral hearing loss: contralateral ear with audiometric thresholds within the normal reference range. (d) Symmetric bilateral conductive or mixed hearing loss: interaural difference between mean bone conduction thresholds at 500, 1000, 2000, and 3000 Hz must not exceed 10 dB and be less than 15 dB at all frequencies. Bilateral implantation is possible. (e) Asymmetric conductive or mixed hearing loss: ideally, BAHS should be placed only unilaterally (on the side with better bone conduction).Children under 5 years of age or patients with contraindications to surgery should use nonsurgical implant systems, to be decided based on physician and family preferences. From this age onward, BAHSs or active middle ear implants can be used.The indication of the type of implantable BAHS depends on the experience of the medical team. Advantages of percutaneous systems include rapid surgery , increased bone conduction amplification capacity, few intraoperative complications, and smaller artifacts in MRI which can be performed in up to 3.0 Tesla scanners. However, patients using these systems have aesthetic complaints due to abutment exposure, in addition to the risk of skin complications and osseointegration failure, especially in young children.93Active transcutaneous systems have the benefit of keeping the skin intact, being more aesthetically acceptable for patients, and providing good hearing gain, despite being smaller than percutaneous systems. To indicate BAHSs, it is important to note that bone conduction thresholds should be evaluated and that they are normally preserved in patients with microtia. Disadvantages include reduced bone conduction amplification capacity, prolonged operative time, and larger artifacts in MRI which can be performed only in 1.5 Tesla scanners.Since 2009, the Vibrant Soundbridge® (VSB) hearing device  has been used in patients with external or middle ear malformations.Surgery must be performed by experienced otologists and in patients with a favorable anatomy due to the high level of difficulty and risk of serious complications. The usually anomalous split facial nerve configuration makes access difficult (even using intraoperative monitoring), and obliteration of the round window may lead to surgical failure.99Mastoid CT and MRI are not required early, especially in unilateral malformations .Children under 5-years of age should use nonsurgical implant systems. From this age onward, BAHSs or active middle ear implants can be used .The indication of an implantable system should be discussed with the family, which should be informed by the surgeon of the benefits of and contraindications to each system .Tympanostomy Tube Placement (TTP) is one of the most common surgical procedures performed in children. A ventilation tube is placed in the tympanic membrane to ventilate the middle ear. Otitis media is one of the most prevalent conditions in childhood, responsible for a large number of primary care visits.The mean incidence is 10.8 new episodes per 100 person-years,Treatment includes analgesics, antipyretics, and antibiotics (if necessary). TTP is indicated for management of serious complications, such as mastoiditis, peripheral facial paralysis, meningitis, petrositis, sigmoid sinus thrombosis, and brain abscess.Recurrent AOM is defined as the occurrence of 3 or more well-documented and distinct episodes of AOM in a 6-month period or 4 or more episodes of AOM in a 12-month period that includes at least 1 episode in the preceding 6-months. TTP should not be performed in children with recurrent AOM without middle ear effusion. However, the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) guidelines include the following exceptions:TTP may also be an option for children with antibiotic allergy or intolerance.Otitis Media with Effusion (OME) is defined as the presence of fluid in the middle ear without signs or symptoms of acute infection. Chronic OME occurs when effusion persists for 3-months or longer from the date of onset (if known) or from the date of diagnosis (if onset is unknown).OME is a common condition, affecting 90% of children before the age of 5-years.107OME persisting for 3-months or longer has been associated with hearing loss, balance disorders, reduced quality of life, and poor school performance.104There is a strong recommendation for the use of pneumatic otoscopy in the diagnosis of OME. In addition, patients with hearing loss, otalgia, or both should be assessed with pneumatic otoscopy to rule out OME.If the diagnosis remains uncertain after pneumatic otoscopy, tympanometry should be performed (strong recommendation).Current guidelines do not recommend the use of systemic or topical intranasal steroids, antibiotics, antihistamines, or decongestants in the treatment of OME due to a lack of evidence showing their effectiveness compared with placebo.The latest AAO-HNS recommendations for TTP in children aged 6-months to 12-years were published in 2022.An observational surveillance interval of 3–6-months should be established in children with OME in order to identify one of the following disease courses: (a) OME resolves spontaneously. (b) Hearing loss is detected. (c) Abnormalities of the tympanic membrane or middle ear are suspected or observed.This surveillance interval allows clinicians to reevaluate children for surgical intervention, avoids complications, and allows for ongoing family counseling. Pediatricians should be aware of these recommendations to ensure adequate treatment for children.Age-appropriate hearing evaluation should be performed when a child becomes a candidate for TTP. Establishing the auditory threshold and identifying the presence of a concomitant sensorineural component helps clinical decision-making about the need for surgery. Children with ventilation tubes should be reevaluated every 3-months.According to the AAO-HNS guidelines, indications for TTP includeWhen TTP is recommended for children under 4-years of age, adenoidectomy should not be performed simultaneously unless there is a clear indication for this surgical procedure, such as chronic adenoiditis or nasal obstruction caused by adenoid hypertrophy. However, in children aged 4-years or older, adenoidectomy with TTP is recommended for the treatment of OME.TTP has been shown to be effective at reducing the prevalence of chronic OME, improving hearing and positively affecting child quality of life.Several ventilation tubes are available on the market, and the choice of tube type should be made in the clinical context of the treatment strategy for the individual child. Ventilation tubes such as Paparella can extrude within 6–12 months, compared with Armstrong tubes which generally extrude within 9–14 months. Long-term tubes are those lasting up to 2 years.Pseudomonas aeruginosa and penicillin-resistant Staphylococcus aureus, with systemic antibiotics being limited to immunocompromised children or those with acute complications, such as cellulitis of adjacent skin and concurrent bacterial infection of the paranasal sinuses or pharynx. However, ear drops should not be used indiscriminately.Perioperative education and family counseling are essential in the care of these patients and allow caregivers to assess the expected duration of tube function, recommended follow-up schedule, and detection of complications.Other reported complications of TTP are obstruction (7%), premature extrusion (4%), granulation tissue (4%), and displacement to the middle ear (0.5%). Myringosclerosis and the associated minimal effects on hearing may account for the long-term sequelae of TTP. Tympanic membrane perforations, which may require repair, are observed in a mean of3% of children after TTP.Water precautions in children with ventilation tubes should not be routinely encouraged unless there is otalgia from water exposure , deep sea diving, or presence of otorrhea.Children with OME should be evaluated periodically. TTP is not indicated for all cases with a less than 3-month diagnosis .Adenoidectomy is not indicated for children under 4-years of age unless there are respiratory symptoms .Patients with recurrent AOM have no indication for routine TTP , with exceptions.There are no swimming restrictions for children with ventilation tubes .The use of ear drops in children with ventilation tubes should include careful consideration, and they cannot be routinely used .Approximately 98.7 million people have otitis media-related hearing loss, according to the World Report on Hearing,Chronic suppurative otitis media leads to some degree of hearing loss in 50% of cases due to tympanic membrane perforation and changes in the ossicular chain (conductive hearing loss), hair cell damage caused by recurrent infections , or mixed.In cases of chronic otitis media with tympanic membrane perforation, tympanoplasty with or without ossicular reconstruction is the treatment of choice and aims to eradicate the infection, close the tympanic perforation, and restore hearing if possible. A systematic review showed that there is improvement in hearing acuity for 7 in every 10 patients undergoing tympanoplasty.Immune system immaturity, Eustachian tube angle and length, recurrent upper respiratory tract infections, and postoperative care are factors suggesting that age may be associated with the surgical success of tympanoplasty, but no statistically significant difference has been observed when comparing ages 4–16 years.120Although anterior perforations have been considered a poor prognostic factor, due to low vascularization and more difficult access, recent studies have produced different results and no longer report the site of perforation as a determinant of poorer outcomes.118Because Eustachian tube function is difficult to measure, assessment of the contralateral ear has been suggested for its evaluation. A normal contralateral ear is a good prognostic factor for the surgical outcome of tympanoplasty, but when altered with tympanic membrane perforation or retraction, it is not necessarily indicative of poor prognosis.Preoperative dry ear time has long been debated as a prognostic factor in tympanoplasty. Some authors have reported better results with dry ears within at least 3-months of surgery, whereas others have observed no difference with wet ears for a period closer to the procedure date.121Because cartilage grafts are often thick, their use may be disregarded due to the possibility of not improving or worsening hearing acuity, although this fact has not been observed. The use of temporalis fascia, perichondrium or cartilage grafts has produced no significant difference in the results of perforation closure or audiometry.When comparing the surgical results for closure of a tympanic membrane perforation and the air-bone gap, there is no significant difference in the choice of lateral (overlay), medial (underlay), or perforation (inlay) graft placement.The use of endoscopic approaches in ear surgery has become increasingly common, leading to less invasive procedures with transcanal access and reducing recovery time and, sometimes, procedure-related costs. Another benefit is superior visualization of the tympanic cavity, enabled by the use of wide-angle lenses. However, the unique anatomic features of the pediatric ear should be considered. Although the tympanic membrane in children is adult size at birth, its angle and the length and size of the internal acoustic meatus differ significantly.124The impact of hearing loss and the potential complications resulting from recurrent middle ear infections are well known, especially in childhood. For these reasons and in view of the available literature, it can be concluded that the treatment of chronic otitis media with tympanic membrane perforation (tympanoplasty with or without ossicular reconstruction) should be started as soon as possible, taking into account the age of the child, status of the ear and Eustachian tube, and the technique with which the surgeon feels most comfortable.There is no level of evidence to contraindicate tympanoplasty considering only the age of the child (Insufficient).Tympanoplasty performed in subtotal perforations or in children with an altered contralateral ear are more likely to fail .There is no consensus on the time required for dry ear before indicating surgery (Insufficient).The type of graft used  does not change the outcome of tympanoplasty .Treatment may include only speech therapy in children with a normal audiogram who present only with a delay in speech development. In children with hearing loss, in addition to speech therapy, HAs or implantable systems may be indicated. Even in children with profound hearing loss, both the use of HAs and behavioral assessments while using the device are important.Children with unilateral hearing loss deserve special attention and should be treated accordingly. This document has provided an overview of the problems that unilateral hearing loss can cause and the importance of binaural hearing. It is up to the medical team to decide whether to refer a patient with profound congenital deafness for cochlear implantation, BAHS, or HA with CROS, but it is important to emphasize that cochlear implantation has better outcomes if performed early. Therefore, even in children with mild-to-moderate unilateral sensorineural hearing loss, HAs should be offered through the Brazilian public health system to prevent CNS alterations that might be permanent.Considering that children with hearing loss, regardless of severity, may also have visual impairment, early ophthalmologic evaluation is imperative.The authors have no financial relationships relevant to this article to disclose.The authors declare no conflicts of interest.