Fabry disease (FD) is a lysosomal storage disorder resulting in systemic accumulation of globotriaosylceramide resulting in multi-organ dysfunction e.g. cerebral, cardiac, renal and audiologic complications. The audiologic involvement in FD has often been neglected; while not a lethal aspect of the disease, hearing loss can have a significantly negative impact on quality of life.To investigate baseline hearing status of the Danish Fabry cohort prior to treatment, compared to sex- and age-expected hearing levels and correlating hearing to renal and cerebral findings.Retrospective study of baseline hearing status of the Danish Fabry cohort . Air conduction and speech discrimination scores were assessed at 6 frequencies between 0.25–8 kHz bilaterally. Data were collected between 2001–2014 and compiled in STATA using multilinear mixed modelling for statistical evaluation.Hearing thresholds at all frequencies deviated from the expected thresholds of an otologically normal cohort (p<0.001) and ranged 0.5 to 1.5 standard deviations below expected values. In total 29 males and 54 females were included. Hearing loss was more pronounced in the higher frequencies. There was a trend of association between hearing loss and measured glomerular filtration rate (mGFR) (p = 0.084). No association was present between hearing loss and albuminuria (p = 0.90), Fabry related cerebral abnormalities (p = 0.84) and cardiac left ventricular mass index, (LVMi) (p = 0.67) independent of sex. Hearing thresholds were poorer for men compared to women (p = 0.001). Sex differences were present at 0.25, 4 and 8 kHz.Our findings demonstrated significant hearing loss in Danish FD patients before treatment initiation, being more profound than in otologically healthy individuals at all frequencies. Additionally, we observed no association between hearing loss and LVMi, albuminuria or FD cerebral abnormalities, with a trend of association to mGFR.Patients with Fabrys disease have hearing loss of all frequencies and most prominently at high frequencies (4–8 kHz), with no association between the hearing loss and cerebral abnormalities, and cardiac mass but with a trend of association to measured glomerular filtration rate. Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder which affects glycosphingolipid catabolism due to alfa galactosidase deficiency. This results in systemic accumulation of predominantly globotriaosylceramide Gb3) in plasma and lysosomes, resulting in various clinical complications . includi in plasmThe otologic involvement in FD has often been neglected and while not a lethal aspect of the disease, hearing loss can have a significant impact on quality of life. Few studies have shown sensorineural hearing loss in Fabry patients. Hearing loss was found in 16.7% of the women and 46.7% of the men in 37 Japanese Fabry patients . LikewisThe present study aimed to determine the hearing status of the Danish Fabry cohort in 83 patients prior to treatment. The assessments were corrected in accordance to the expected age and sex based hearing decline. Finally, the association between hearing loss and renal, cardiac and cerebral abnormalities as markers of severity of disease was studied.The study was a retrospective cross sectional study of prospectively collected data from the Danish Fabry cohort. The cohort consists of all Danish Fabry probands and their family members found by meticulous family screening since 2001. All patients independent of sex and treatment have been monitored at the University hospital of Copenhagen, Department of Endocrinology in collaboration with other specialist departments in the Fabry Team. Patients diagnosed with FD who had an audiogram prior to treatment were included (n = 83). The population has previously been partly described in other studies –12.Diagnostic criteria included a Fabry positive genotype with or without a reduced alfa galactosidase activity below the lower limit of the reference interval. Based on these criteria the full cohorte comprised 88 patients. All blood related family members were offered genetic testing after a positive diagnosis was confirmed. Audiograms were collected from almost all Fabry diagnosed patients prior to treatment from 2001 to 2014 (n = 83). The most recent audiogram prior to initiation of enzyme replacement therapy was included. If the patients did not receive treatment the most recent audiogram was used.The study was approved by the Danish Health and Medicine Authority (3-3013-667/1/), the Regional Health Research Ethics Committee (H-3-2014-FSP8) and the Danish Data Protection Agency (2014-641-0055).3 is defined as the mean hearing thresholds in dB of 0.5, 1 and 2 kHz. PTA4 is defined as the mean value of hearing thresholds in dB of 0.5, 1, 2 and 4 kHz. PTA6 is defined as the mean hearing thresholds of all 6 frequencies . High frequency PTA4,8 is defined as the mean hearing thresholds in dB of 4 and 8 kHz. Z-PTA is the equivalent but with Z-scores instead of dB.Pure tone average (PTA) is defined as the mean value of hearing thresholds in decibels (dB) hearing level (HL) of multiple frequencies. PTAAn otologically normal person is defined as a person in a normal state of health who at the time of testing is free from excess wax in the ear canals, is without known ear pathology and who has no history of undue exposure to noise.All tests were carried out by pure tone audiometry in both ears of all patients at a frequency span of 250 Hz to 8 kHz. When air conduction values were poorer than 20 dB bone conduction was also determined. Pure-tone audiometry at frequencies 0.25, 0.5, 1, 2, 4 and 8 kHz was carried out in accordance with ISO 8253–1. The modified Hughson-Westlake technique (-10/+5 dB) was employed using a Madsen Astera Audiometer, Madsen Orbiter OB 922 Clinical Audiometer and Interacoustics AC40 Clinical Audiometer, with Sennheiser HDA 200 circumaural earphones. The equipment was calibrated in accordance with IEC 60318–2, ISO 389–5, and ISO 389–8 using a Brüel and Kjaer 2610 measuring amplifier with a 4144 microphone in a 4152 coupler –16. The 4 of the better ear [4. In addition, speech discrimination score was determined by presenting 25 words from a nationally validated prerecorded list contained on a CD. Each ear was assessed individually through the same headset used for pure tone measurements. The score was calculated as the percentage of words the patient could successfully report back to the analyst after each presented word. Speech discrimination scores above 90% were considered normal. All patients could speak Danish.Reports on hearing impairment were given based on WHO grading on hearing impairment . Accorditter ear . AsymmetMeasured glomerular filtration rate (mGFR) values collected within one month of the auditory assessments were included (n = 75). The method for mGFR measurements has been previously described .24-h urine samples were collected by patients at home, the last 24 h before hospital visit. Urine samples were analysed for total albumin content based on the total urine volume and albumin concentration.Transthoracic echocardiographies were performed using a Philips IE 33. Left ventricular (LV) wall thickness and chamber dimensions were measured by two dimensional parasternal images. LV mass (LVM) was calculated by the American Society of Echocardiography equation and indexed to body surface area (LVMi). All cerebral magnetic resonance imaging (MRI) scans performed within six months of the auditory assesment were included (n = 59). The method for MRI has previously been described . All MRIHearing thresholds were standardised as Z-scores calculated based on hearing thresholds of otologically normal controls presented in ISO-7029 . mGFR waThe statistical analyses were performed by multilinear mixed model with Z-scores as dependent variable including independent factors such as left or right ear, sex, frequencies, mGFR, albuminuria,LVMi, A-gal, Gb3 and relevant interactions. Different models were tested by likelihood-ratio tests to find the best model. Where appropiate a t-test was used to detect a difference between means. Results were considered statistically significant at the 95% confidence interval and at p-values less than 5% (P < 0.05). Data has been processed and analysed using STATA 13.0.A total of 83 patients, 29 males and 54 females with audiograms were included. The median age was 35 years , with predominantly females in the upper age groups. One male patient used hearing aids at baseline. All but one male and 41 out of 54 females were of the classic phenotype. Demographic values and general clinical findings are presented in 4 33.8 dB HL) and one female had moderate hearing impairment (PTA4 44 dB HL) .  and two females (Mean PTA4 27 dB HL) exhibited mild hearing loss . No patient had unilateral hearing impairment. The mean high frequency PTA4,8 kHz was 23.2 dB HL for men and 20.0 dB for women.When using WHO’s definition only one male exhibited slight hearing impairment (PTAl 2.4%). . However4 of the left ear at 40 dB HL and a high frequency hearing threshold at 8 kHz of 95 dB HL.Mean speech discrimination scores were 98.9% for men and 99.7% for women. All patients, but one, had normal discrimination scores of both ears. The one male patient had a score of 68% of the left ear. This patient also exhibited a distinct hearing loss with a PTANone of the subjects with hearing loss exhibited air-bone gap when comparing the thresholds for air conduction and bone conduction. Thus, any hearing loses found may be categorised as sensori-neural i.e. cochlear or retrocochlear.6 between classic and non-classic Fabry patients (p = 0.69).Hearing thresholds at all frequencies (250Hz-8kHz) deviated significantly from the expected thresholds of a otologically healthy cohort described in ISO 7029 (p<0.001). Thus, the Fabry cohort exhibited more pronounced hearing loss in comparison to healthy age-matched individuals. Mean Z-scores for men and women ranged from 0.5 to 1.5 standard deviation scores below the population mean . There wNo significant difference was present between the hearing thresholds of the left and right ear in men (p = 0.37 coeff: -0.08 CI95: -0.25–0.09), and women (p = 0.33 coeff: 0.06 CI95: -0.06–0.19), respectively. Due to the absence of asymmetric hearing, results are given based on the mean hearing thresholds for the left and right ears.Baseline mGFR values included 75 observations and albuminuria observations totalled 53. There was a trend of association between Z-scores for hearing thresholds and renal function (p = 0.084), this association was independent of sex (p = 0.71) and being classic or non-classic (p = 0.83). There was no association between albuminuria and Z-scores for hearing thresholds (p = 0.90).Baseline left ventricular mass index totalled 57 measurements. There was no association between LVMi and hearing levels (p = 0.67), independent of sex (p = 0.16).A total of 59 patients had baseline MRI scans, and 15 (25%) had Fabry related abnormalities. There was no association between Fabry related cerebral abnomalities and hearing loss (p = 0.30), independent of sex (p = 0.16).A total of 64 plasma Gb3 and 55 A-gal measurements were included. There was no association between plasma Gb3 and hearing levels (p = 0.15), this was indepedant from the patients sex (p = 0.5). There was no relationship between A-gal activity and hearing levels (p = 0.12), this was independent of sex (p = 0.48).Hearing thresholds at different frequencies of Fabry patients were significantly different between men and women (p = 0.001). The variations in each individual frequency among men and women have been listed in Our findings have demonstrated that FD patients had poorer hearing compared to age-matched otologically normal individuals. Hearing loss was most prominent at high frequencies  but also noticeable at the lowest frequency in males 250Hz). Our findings of all frequency hearing loss was partially in concert with previous studies 0Hz. Our ,22,23.4) three men (27%) and one woman (8%) had mild to profound hearing loss of the more affected ear. Likewise, Germain and colleagues concluded, in accordance to WHO 1980 guidelines, that hearing loss by PTA3 was present in 11 of 22 males [4 of 6.7 dB deviation from the expected and at high frequencies with a mean deviation of 10.0 dB. This indicates that hearing impairment may begin as an early symptom in FD.When Sakurai and colleagues  compared22 males  demonstrInterestingly, a recent study by Rodrigues and colleagues  demonstr3 while the 2013 guidelines based hearing loss on PTA4. Using PTA3 for depicting hearing loss may ultimately result in underreporting hearing loss in FD patients as the majority of available studies as of this date point towards high frequency hearing loss as the largest contributor to hearing loss in Fabry. Using PTA4 as recommended by newer guidelines may yield higher reportage of hearing loss, but still lacks the lowest and highest commonly measured frequency (0.25 and 8kHz). It may therefore be favorable to use a full PTA6 as a mean to report hearing loss in FD, for a more cohesive and clinically inline reportage. No internationally recognised guidelines are currently available to categorise hearing loss from high frequency thresholds.In the present study hearing loss was classified by the recent guidelines from WHO 2013 , which d2, also had mean bilateral PTA3 of >25dB. However, it is worth noting that hearing thresholds were not age and sex corrected in either of these GFR comparisons. In our analysis we found no statistically significant relationship between Z-scores for mGFR and Z-scores for hearing thresholds at all frequencies in PTA3, PTA4 and PTA6, in keeping with two studies [In relation to renal function a study from Köping and colleagues  found anMoreover, Köping et. al.  describeOnly one study has previously shown an association between cerebral white matter lesions (WML) and hearing loss, this is however not a baseline study . The autThe histopathological cause of hearing loss is not quite clear. However, based on the literature no study has found any noticeable air-bone gap in hearing thresholds ,23. ThisNo cure exists for hearing loss in FD patients, however hearing aids may be a useful help for patients with normal speech discrimination scores. In addition, cochlear implants were successfully applied in two male Fabry patients with sensorineural hearing loss . A signiOur data showed less hearing impairment in the FD cohort than seen in other studies. There is no direct explanation for this, but notably the Danish cohort is one of the largest single center cohorts with a near 100% complete family screening. Moreover, all genetically verified FD family members were offered full organ assessment independent of sex or symptoms, why the cohort comprised a higher proportion of females than in other studies, many of them.with few or even no organ manifestations. In addition, 14% of the patients had genetic variations of unknown significance or were of the non-classic variant, further adding to the variability. The Lack of lyso-Gb3 is a limitation, however given this is a baseline investigation most data is from prior the era of lyso-Gb3 measurements.One of the strengths of this study is the value of using Z-scores which standardise data according to age and sex. This is particularly important when studying small groups of different sex and highly varried ages in order to subtract the naturally expected functional decay. This can, however, also be a limitation as it relies highly on accurate standards to produce correct Z-scores. Moreover, this study presents pre-treatment data, which reduced confounding factors when observing the disease itself. Furthermore, with only 83 patients there is a high risk of a type 2 statistical error, which is, however, a given limitation when studying rare disorders. Nevertheless, this is one of the largest studies to date of hearing impairment in FD.Our findings have demonstrated that hearing loss is present in the Danish FD patients and is more profound than in healthy individuals on all measured frequencies. We also demonstrated that hearing loss is most prominent at high frequencies , and that the average Fabry male has more pronounced hearing loss than the females. There was a trend of association between hearing levels and mGFR. There was no association between hearing loss and cardiac left ventricular mass index, ambuminuria, Gb3, A-gal and cerebral abnormalities. More studies of the hearing loss in Fabry patients as well as effect of therapy are needed, preferably by multicenter investigations in order to achieve more statistical power and more reliable results.S1 FileData used in the analysis.(XLSX)Click here for additional data file.


Hearing ability is important for children to develop speech and language skills as they grow. After a mandatory newborn hearing screening, group or mass screening of children at later ages, such as at preschool age, is often practiced. For this practice to be effective and accessible in low-resource countries such as Thailand, innovative enabling tools that make use of pervasive mobile and smartphone technology should be considered.This study aims to develop a cost-effective, tablet-based hearing screening system that can perform a rapid minimal speech recognition level test.An Android-based screening app was developed. The screening protocol involved asking children to choose pictures corresponding to a set of predefined words heard at various sound levels offered in a specifically designed sequence. For the app, the set of words was validated, and their corresponding speech power levels were calibrated. We recruited 122 children, aged 4-5 years, during the development phase. Another 63 children of the same age were screened for their hearing abilities using the app in version 2. The results in terms of the sensitivity and specificity were compared with those measured using the conventional audiometric equipment.For screening purposes, the sensitivity of the developed screening system version 2 was 76.67% (95% CI 59.07-88.21), and the specificity was 95.83% (95% CI 89.77-98.37) for screening children with mild hearing loss . The time taken for the screening of each child was 150.52 (SD 19.07) seconds (95% CI 145.71-155.32 seconds). The average time used for conventional play audiometry was 11.79 (SD 3.66) minutes (95% CI 10.85-12.71 minutes).This study shows the potential use of a tablet-based system for rapid and mobile hearing screening. The system was shown to have good overall sensitivity and specificity. Overall, the idea can be easily adopted for systems based on other languages. Hearing loss in one of the most common disabilities. The World Health Organization estimated that, in 2012, there were 360 million people in the world with disabling hearing loss (5.3% of the world’s population); 328 million (91%) were adults, and 32 million (9%) were children .Hearing testing in children is challenging and usually requires specialist supervision. Depending on the age, each child can have varying capability to cooperate. For newborn up to 3 years of age, objective measurements, such as otoacoustic emission or auditory brainstem response, may be more appropriate . The objMany strategies have been attempted to make the preschool or school-age hearing screening feasible, fast, accurate, and cost-effective; however, to date, these strategies have not managed to meet the target goal -12. The Recent advances in wireless telecommunication have made great changes in public health practice. A mobile app can potentially change a smartphone or a tablet into a medical device. These mobile devices are becoming increasingly powerful while the costs are becoming progressively lower, and the pervasive nature of the existing network answers the accessibility question. Hearing screening and measurement could be considered one of the early apps in the age of mobile or digital health care . Some reThis study was designed to develop an appropriate tool to be used as a hearing screening device for preschool children. The system was implemented on an Android-based tablet, details of which are discussed in the following sections.The study was approved by the Khon Kaen University Ethical Committee for Human Research (HE 571278) and was registered in the Thai Clinical trial (TCTR2014092201). This study was composed of 2 phases. The first phase was the development of the speech audiometry app software and was conducted during 2015-2016. The second phase was the validity of the software and was conducted during 2016-2017. The details of each phase are provided below.To select appropriate words for the screening device, first, an audiologist in our research team chose 36 two-syllable words from an elementary school book that varied in terms of pitch. Corresponding pictures of the words were drawn with the consultation of 3 external audiologists. These words and corresponding pictures were then tried out with 2 groups of 30 preschool children aged between 4 and 5 years. One group was recruited from an urban area, and another group was recruited from a rural area. Parental consents were obtained before the test date. In addition, verbal child assent was also obtained. Each child would first be presented with the set of 6 pictures. There were 6 sets of pictures; these pictures were grouped by category as things, actions, fruits, etc. The researcher pronounced only one word at a time in a random order. A child was asked to point to the picture that represented the word they heard. The number of times the child correctly identified the picture was noted. After completing the 6 sets, the child was again presented with the pictures, this time one by one. They were asked what the picture was and the answer was recorded. The 24 words that 95% of children recognized correctly would be used for the software.All recordings were made in a sound-proof booth at a music studio. A high-sensitivity microphone (Brüel & Kjær type 4188) was positioned approximately 20 cm from the speaker at 0° azimuth and was covered with a 6.5-cm diameter windscreen. The microphone was connected to a sound level meter (B & K model 2239A), and the signal from the linear-weighted AC output of this meter was fed to an analog-to-digital converter  that acquired the mono sounds at a sampling frequency of 44.1 kHz with 16-bit amplitude resolution. A female speaker, who was a professional announcer with 25 years’ experience, enunciated with normal vocal effort, corresponding to approximately 63 decibels sound pressure level  and recorded at a sampling rate of 44.1 kHz.The sound intensity of each word was edited to yield the same average intensity as that of a 1000-Hz calibration tone.The system consisted of a tablet device running 2 Android apps. We used the Samsung Galaxy Tab S 10.1. The tablet was plugged in with an approved headphone to be worn by the child being tested. The app “Calibrate Screening” was used by the support team to provide an Web-based update to the library of calibrated headphones that are selectable within the “Screening” app. The app “Screening” was for the child to perform under the administration of audiologist or trained personnel. The main function of the app is to perform the following test protocol .After completing the test at the end of the screening test, there is a page reporting the scoring. Suggestions and recommendations, should the child fail and have to see a specialist, are also provided.We recruited 2 groups of 60 preschoolers from the northeastern part of Thailand. The first group was tested for the PASS speech audiometer version 1 using the commercial headphone, Creative EP-210. The second group was tested for the PASS speech audiometer version 2 using standard audiometric headphones, TDH39. The same inclusion and exclusion criteria were applied to both groups. The inclusion criteria were as follows: age between 4 and 5 years; use of standard Thai to communicate; and physical health, with the ability to cooperate with a medical examination. Candidates were excluded if they were uncooperative during conditioning play audiometry tests or they had incomplete conditioning play audiometry test results. An otolaryngologist examined the child’s ears without any intervention to clean the external ear. The PASS speech audiometry app tests were performed according to the test protocols in the quiet room.Starting from the right ear, the program randomly picks 1 set (6 pictures) out of an available 4 sets of pictures. The program first randomly outputs a 2-syllable word corresponding to 1 picture of the set at 40 dBHL. It then shows the 6 pictures to a user, who is required to pick the correct picture corresponding to the word from the picture set. The program continues with another word from the set. Only 4 words will be used for each speech level. Users move on to the next speech level if they answer correctly 2 of 3 or 4 times (passing the level) or 4 words out of the 6 have been used. In the latter case, the user is considered to have failed at that speech level. The test continues with the same procedure with lower power speech at 30 dBHL and, finally, at 20 dBHL. Other conditions that are observed are as follows: if the user fails the test at 40 dBHL or 30 dBHL, testing is stopped for the right ear and proceeds to the left ear and if the user passes all 3 levels, testing proceeds to the left ear. For this phase of the trial, we tested the PASS speech audiometry app with standard audiometric headphones (TDH39). The headphones were embedded inside an earmuff with up to 20-dB ambient noise reduction, as it was intended for use in practical situations, such as school or normal rooms, rather than in an audiometric booth or sound-proof room, which are usually required for audiometric measurements. An additional Phono-to-Tip-Ring-Sleeve adaptor was required so that the TDH39 headphones could be plugged to the tablet. Starting from the right ear, the program randomly picks 1 set (6 pictures) out of the available 4 sets of pictures. The program first randomly outputs a 2-syllable word corresponding to 1 picture of the set at 40 dBHL. It then shows the 6 pictures to a child, who is required to select the correct picture corresponding to the word from the picture set. The program continues with another word from the set. The 40-dB sound is presented 4 times for each ear. The test continues with the same procedure with a softer speech at 30 dBHL and, finally, at 20 dBHL. Sounds were presented for each ear 12 times in total. Users will be considered as having passed the test at that speech level if they answer correctly, at least, 2 times out of 4. If a user cannot answer all 4 words correctly at each intensity, the user is considered to have failed at that level. The whole procedure is then repeated for the left ear. The audiologist who performed the test was not involved in the inventor team and was trained to use the PASS speech audiometry. Children were asked to hear the sound in each ear and point to the picture that they heard. The time used for the PASS speech audiometry was recorded.The pediatric audiologist, who was blinded to the results of PASS speech audiometry, performed the conventional play audiometry. Children’s hearing threshold was evaluated with air-conduction pure-tone audiometry at 0.5, 1, 2, 3, and 4 kHz and with spondee words for the speech reception threshold (SRT) measurement in the standard sound-proof room. The procedure’s timing was recorded. Tympanometry was also carried out.The sensitivity and specificity of speech audiometry app to detect a mild hearing loss in either ear  were tested. The mean difference between speech audiometry and conventional play audiometer for each protocol testing was then compared.For this part of the study, 63 children (51% (32/63) boys and 49% (31/63) girls) volunteered. Of these, 57% (36/63) were between 4 years, 7 months and 5 years of age, and 24% (15/63) were between 5 and 5.5 years. Each child would then go through the word selection trial as discussed in the previous section. Having all the results, audiologists finally grouped them into 4 sets of 6. There were 32 boys and 28 girls. The mean age was 4 years and 11 months. Although 2 children had impacted cerumen, 5 had otitis media; 10 children had a unilateral hearing loss, all of which was a mild hearing loss. No bilateral hearing loss was found. Commercial headphones, Creative EP-210 was used. The average time used for the speech audiometry program was 82.9 (SD 24.13) seconds (95% CI 76.67-89.13 seconds). The fastest time was 58 seconds, and the slowest was 195 seconds. The average time used for conventional play audiometry was 11.87 (SD 4.06) minutes (95% CI 10.79-12.87 minutes). The fastest was 6.3 minutes, and the slowest was 32.46 minutes. A total of 63 children participated in the second version trial; there were 22 boys and 38 girls, and the mean age was 4 years and 9 months. Four children had impacted cerumen, 15 had otitis media, and 14 had hearing loss. The unilateral hearing loss was found in 10 children, and bilateral hearing loss was found in 4 children, including 2 with the moderate hearing loss. The average time used for the speech audiometry program was 150.52 (SD 19.07) seconds (95% CI 145.71-155.32 seconds). The average time used for conventional play audiometry was 11.79 (SD 3.66) minutes (95% CI 10.85-12.71 minutes). From 0.5,1,2,4 kHz>25 dBHL, or PTA1,2,4 kHz>25 dBHL as criteria for mild hearing loss, the sensitivity of the PASS speech audiometry app version 2 was greater and the specificity was similar. Increasing the sound stimulus intensity did not improve the sensitivity of the screening. Using the discrimination criterion of 20 dB, the sensitivity of the PASS speech audiometry version 2 was slightly variable among the frequencies of average pure-tone thresholds. If the PTA threshold at 500 Hz was omitted, the sensitivity was increased, and the specificity remained high. The sensitivity to screen mild hearing loss of PTA1,2,4 kHz>20 dBHL was 76.67% (95% CI 59.07-88.21), and the specificity was 95.83% (95% CI 89.77-98.37). Furthermore, the relation of the PTA threshold and SRT for individual ears was compared  and 82.11% (95% CI 73.20-88.52), respectively. When using the SRT >25 dBHL, or PTAcompared .Hearing loss can be preventable and is much more manageable if detected early. To do so, we must equip our medical personnel, especially in low-resource countries, with tools that are appropriate, available, and accessible . The tabThe mobile hearing test apps, such as uHear  and hearFrom the primary result of the PASS version 1 trial using SRT or PTA >20 dB as a criterion of mild hearing loss, the sensitivity of PASS speech audiometry was too low to be used as the hearing screening tool to detect mild hearing loss, though the specificity was very high. The test time was evidently very short, 8.6 times faster than that of conventional play audiometry, an encouraging result. We analyzed the results and considered 2 further changes so that we may learn more about its true potential. First, the test protocol could be modified to find the appropriate number of correct responses to determine pass or fail for each sound pressure level test; this may not be 2 of 4 as in the original protocol. Second, the headphone used in this trial should be the same standard used in conventional pure-tone audiometry for initial reference.1,2,4 >20 dB, the PASS speech audiometry app version 2 showed higher sensitivity and more specificity than version 1. While being able to complete the screening task much faster than when using standard tool, the PASS speech audiometry app version 2 had a sensitivity of 90% and 100%, and a specificity of 93.40% and 79.44% to differentiate children with normal hearing from those with the PTA or SRT >25 dB, respectively. Normally, the SRT is within 5 dB above PTA. Speech discrimination was increased if tested with the suprathreshold of sound stimuli. The PASS app uses the principle that the comprehension of sound will lead children to correctly choose the right picture. The sound stimulus may require higher intensity than that of the stimulus used for finding SRT.Using SRT or PTAFor adults, the pure-tone, air-conduction threshold (PTA) at 500, 1000, and 2000 Hz >25 dBHL is considered to be a mild hearing loss. As mild hearing loss affects the academic performance in young school-age children, the criterion for mild hearing loss is set lower than that of adults . The AmeThe proposed system was based on the screening in Thai words for Thai children; however, we believe that the PASS app is universal and can be easily adopted for screening in other languages as well.In the first trial, we used the same concept of the test protocol that we used in normal practice. If children could respond correctly to half of the test (at least 2 times), we could skip to the next loudness. In the second trial, we tested 4 times for each loudness regardless of the number of correct responses. We found that the cutoff point to determine pass or fail remained, at least, 2 correct responses. The mean test time used for each child in the second trial was twice as much as the time used for each in the first trial. For future improvement in terms of testing time and arrangement as that in the first test protocol may be considered.Another point of consideration would be the selection of headphones. We were able to obtain good results using TDH39 headphones, which have a very flat response and loudness linearity that are important for quality speech measurement. This situation is not always the case for consumer headphones, and we should, therefore, be very specific if possible about which headphones should be used with the app. A pair of THD39 headphones can cost up to US $200, whereas reasonably priced headphones may be 5-10 times less expensive, making them more accessible.As the screening system must be accurate only for specific words at predefined loudness levels, we can simplify the calibration process by only finding the calibration coefficient at those points. This process could be done by adjusting the internal gain at each word-power level so that the output measurement by the sound level meter yields the same dBA as that measured when driving the TDH39 headphones. Masalski et al studied the reference sound level by means of biological calibration on 8620 devices representing models . The refA few limitations of this study deserve mention. First, because of the small sample size and the population that evidently had a low prevalence of hearing loss, some of the 95% CIs were rather wide. Future tests can be designed with increased sample sizes to cover more children with hearing loss. In addition, PASS version 2 was developed as an improvement to PASS version 1. For its trial evaluation, different populations with identical inclusion and exclusion criteria as those of PASS version 1 were used; this fact makes a direct comparison between the 2 trial results less straightforward. Finally, to practically reduce ambient noise to a minimum, an earmuff was used to cover the headphones. In cases where this is not possible, the true effect of using unprotected headphones on the screening performance must be studied.The screening program should add the function of headphone calibration before beginning the test to make it more feasible to accommodate other marketed devices. The test should be modified to make it more attractive to preschool toddlers. It would be ideal if the screening program could be complemented by other audiometry programs to offer more flexibility to the medical professional. Additional options could be standard pure-tone audiometry to study in detail the hearing loss characteristics of each. Conversely, it may be desirable to include a teleconsultant function that can bring a remote audiologist closer to the actual service field to validate hearing loss diagnostics, and any follow-up rehabilitation program can be done as close as possible to the primary practice. Being able to do these steps would address the low accessibility to qualified audiologists, which is a real concern in many countries. All this could be packaged together to form a teleaudiometry service that is an excellent example of mobile health. For the screening program itself, a new test algorithm that reduces the test time even further could be worth exploring. All these aspects are subjects of this work, which we will report in due course.This study demonstrates the potential use of a tablet-based system for rapid and mobile hearing screening. The system was shown to have good overall sensitivity and specificity. The idea can be easily adopted for systems based on other languages.


The current study was aiming to investigate the relation between vitamin D, retinopathy, and hearing loss among type 2 diabetes mellitus (T2DM) patients. Cross-sectional study carried on 638 subjects aged between 20 and 60 years who visited the Endocrinology, Ophthalmology, and ENT Outpatient Clinics of the Medipol Hospital during the period from March 2016 to May 2017. Two audiometers Grason Stadler GSI 61 and Interacoustics AC40 Clinical audiometer were used to evaluate the hearing loss. Risk factors for diabetic retinopathy were evaluated, including age, sex, diabetes duration, glycated hemoglobin (HbA1c), hypertension, and lipid profiles. The mean age  for retinopathy with hearing loss versus normal subjects was 47.7 ±10.2 versus 48.5±9.1. The associated risk factors were significantly higher in T2DM with hearing loss, hypertension (32.6% versus 15.7%), tinnitus (40.0% versus 18.0%), vertigo (59.7% versus 26.8%), and headache (54.9% versus 45.3%), than in normal hearing diabetes. There were statistically significant differences between hearing impairment versus normal hearing for vitamin D , calcium, magnesium, phosphorous, cholesterol, HDL-C, LDL-C, albumin, systolic blood pressure [131.70±9.25 Hg versus 127.73±11.98 Hg], diastolic blood pressure [82.20±8.60 mm Hg versus 79.80±8.20 mm Hg], and microalbuminuria. Multivariate logistic regression analysis revealed that variables such as vertigo, duration of DM, mobile/I pad phone, vitamin D deficiency, sleeping disturbance, headache, frequently TV watching, tinnitus, cigarette smokers, and hypertension were considered at higher risk as a predictors of retinopathy with hearing loss among diabetic patients. Vitamin D deficiency is considered as a risk factor for diabetic retinopathy and hearing loss among diabetic patients. Meanwhile, hyperglycemia could be considered as a modifiable risk factor for diabetic retinopathy; tight glycemic control may be the most effective and important therapy for improving quality of life and substantially reducing the incidence of retinopathy and in T2DM patients. Type 2 diabetes mellitus (T2DM) is a major health problem associated with significant morbidity, mortality, and healthcare expenditure. The etiology of T2DM is complex and includes abnormalities in multiple organs, including liver, skeletal muscle, adipocytes, gut, and beta cell , 2. T2DMRecent studies have reported that vitamin D deficiency is closely related to obesity and increased risk of T2DM –12. TherThis is a cross-sectional study conducted on patients aged from 20 and 60 years who visited the Endocrinology, Ophthalmology, and ENT Outpatient Clinics of the Medipol Hospital during the period from March 2016 to May 2017. IRB ethical approval for this study was obtained from the Medipol International School of Medicine, Istanbul Medipol University, and informed written consent was obtained from patients before the start of the study.The present sample was based on the representative registered 900 patients with diagnosed diabetes. Of the 900 approached registered patient with T2DM, 638 (70.9%) agreed to participate in this study at the Medipol International School of Medicine, Istanbul Medipol University.The patients were considered to have DM if they have a history of DM and are currently taking oral medications for diabetes; we have followed the methods described by Bener et al. 2017  and InteThe study included sociodemographic characteristics, lifestyle habits, BMI, comorbid symptoms, diabetic complications, systolic and diastolic blood pressures, clinical biochemistry serum triglyceride, total cholesterol, high-density lipoprotein (HDL-C) cholesterol, low density lipoprotein (LDL-C) cholesterol, hemoglobin A1c (HbA1c), and fasting glucose levels (FPG) which were collected. Hypertension criteria are defined according to the World Health Organization (WHO)  where syAll the patients with diabetes underwent a complete ophthalmic examination comprised of best-corrected visual acuity, indirect ophthalmoscopy, slit-lamp biomicroscopy, and fundus fluorescein angiography. ETDRS (Early Treatment Diabetic Retinopathy Study)  adaptatiPure tone audiometry is a behavioral test used to measure hearing sensitivity. This measure involves the peripheral and central auditory systems , 15, 20.β-adjusted β), and if the change was more than 10%, the variable was considered as a confounder and retained in the final model. The cut-off value for significance was chosen as 0.05.The statistical analysis was performed by using the Statistical Package for Social Sciences [SPSS]. Student's t-test was used to ascertain the significance of differences between mean values of two continuous variables and confirmed by the nonparametric Mann-Whitney tests to determine if the results differed, indicating lack of normal distribution of the variable. Chi-square test Fisher's exact test (two-tailed) were used to test significance differences between two or more categorical groups. A multivariate logistic regression model was performed to assess the relation between selected lifestyle factors, retinopathy, and presence of hearing loss. Confounders were assessed statistically through change in beta coefficient  DBp , HbA1c , BMI , and physical activity .The results of the present study have revealed high prevalence of hearing impairment in association with retinopathy in T2DM patients. The association between diabetes and eye disease is well recognized with diabetic retinopathy as the most common eye disease in subjects with T2DM . HoweverMore recently, Bener et al. , 6 studiThe current study results are consistent with previous reports about the close relationship between T2DM and high-frequency sensorineural hearing loss , 20, 21 More recent study by Praidou et al.  reportedThe current study reveals strong association between high prevalence of hearing impairment, vertigo, and tinnitus in T2DM patients , 5, 6. TThe present study is not without its limitations. Firstly, this is a cross-sectional design with limits the ability to assess causality. Secondly, only one time point was recorded for the subjects in this study. Thirdly, also, there is possible selection bias as this is not a study of consecutive patients seen at our institution. Although, dietary intake and outdoor exposure data were collected. Additionally, effects from sunlight exposure were minimized in this study as all subjects were enrolled over a twelve-month period. Finally, beside several limitations, the analysis revealed that the overall mean standard deviation of 25(OH)D in this study was statistically strong significant differences between hearing impairment versus normal hearing for vitamin D .The current study results suggests a strong positive association between vitamin D, retinopathy, and hearing impairment among T2DM. Vitamin D deficiency is an independent risk factor for diabetic retinopathy and hearing loss among diabetic patients. Meanwhile, hyperglycemia could be considered as a modifiable risk factor for diabetic retinopathy; tight glycemic control may be the most effective and important therapy for improving quality of life and substantially reducing the incidence of retinopathy and in T2DM patients.


Hearing impairment is one of the most common sensory deficits in humans. Hearing aids are helpful to patients but can have poor sound quality or transmission due to insufficient output or acoustic feedback, such as for high frequencies. Implantable devices partially overcome these issues but require surgery with limited locations for device attachment. Here, we investigate a new optoacoustic approach to vibrate the hearing organ with laser stimulation to improve frequency bandwidth, not requiring attachment to specific vibratory structures, and potentially reduce acoustic feedback. We developed a laser pulse modulation strategy and simulated its response at the umbo (1–10 kHz) based on a convolution-based model. We achieved frequency-specific activation in which non-contact laser stimulation of the umbo, as well as within the middle ear at the round window and otic capsule, induced precise shifts in the maximal vibratory response of the umbo and neural activation within the inferior colliculus of guinea pigs, corresponding to the targeted, modelled and then stimulated frequency. There was also no acoustic feedback detected from laser stimulation with our experimental setup. These findings open up the potential for using a convolution-based optoacoustic approach as a new type of laser hearing aid or middle ear implant. Many of these hearing impaired individuals do not seek help for their hearing loss and can become isolated from society or even lose their jobs. Of the hearing impaired patients who obtain clinical support and receive a hearing aid, a notable proportion of them do not continue to wear and use their hearing devices (up to at least 24%)3. This lack of use, despite remarkable improvements in the technology of the hearing devices, are in part due to their poor performance or discomfort for a portion of these patients3. Several factors contribute to the unsatisfactory outcomes of conventional hearing aids, including insufficient frequency specificity or bandwidth, acoustic feedback issues, and discomfort due to the occlusion effect  with recurrent auditory canal inflammation3.There are about 360 million individuals worldwide who struggle with hearing impairment and have difficulties in communicating on a daily basis4. Several types of these devices can be considered as implantable or partial implantable auditory prostheses, including middle ear implants and various bone-anchored hearing devices. The advantages of these implantable hearing devices are that they can transmit greater energy and broader bandwidth of information to the hearing system compared to conventional hearing aids, especially for patients with severe hearing loss who cannot comfortably wear an earpiece that occludes the ear5. The disadvantage is that they require surgery for placement with possible complications. For example, passive percutaneous bone conduction hearing devices (skin penetrating and bone anchored) such as the Baha®  can have good amplification and sound transmission; however, wound infection around the skin penetrating area6 decreases their acceptance in the hearing impaired population. There are also transcutaneous passive bone conduction hearing devices such as the Baha® Attract System 7 and the SophonoTM8 or active transcutaneous devices such as the BONEBRIDGE 9. However, they have lower sound amplification and modulation capacity due to the transcutaneous interface and can induce pressure issues of the stressed skin between the magnets used to secure the device in its proper location. In cases where bone conduction hearing devices are insufficient, active middle ear implants such as the Vibrant Soundbridge10 or Carina ® 11 provide another option that can lead to satisfying results, but the coupling of the actuator to the middle ear bone structures is challenging in middle ears with pathologies12.In addition to the conventional hearing aids described above that present acoustic amplification to the ear, there are also hearing devices that directly contact and transmit vibrations to the head or hearing structures13. Acoustic hearing aids with an earpiece that occludes the ear could potentially transmit sufficient sound information for these types of patients but occluding the inflamed ear can lead to significant discomfort5. Current middle ear implants may not require ear occlusion but cannot be sufficiently attached or adapted to these damaged structures while conventional bone conduction devices are not well tolerated by patients with the tendency for chronic inflammation around the anchor of the bone conduction system14. Considering these various limitations experienced by many hearing impaired patients, laser stimulation is emerging as a new form of energy that can be sharply focused to activate specific structures or biological tissue and can be applied without the need for directly contacting the targeted vibratory structures17. Laser stimulation could be potentially used to vibrate the ear drum with reduced acoustic feedback since the energy transmitted is light instead of sound and without requiring occlusion of the ear canal. If activation of middle ear structures is required due to peripheral ear damage or inflammation, a laser fiber or bundle of fibers could be inserted into the middle ear cavity and directed towards different mechanical structures without requiring attachment to specific locations that may be damaged or inaccessible in some patients, as is encountered for middle ear implants. Therefore, the use of optical energy as a non-contact stimulation method for the activation of the peripheral hearing organ is a promising solution for hearing impaired patients with these types of limitations, and needs to be further explored.Alternative stimulation strategies are therefore needed for the therapy of hearing deficits and pathologies that are still not sufficiently addressed with the current hearing technology. For example, there are patients with chronic inflammation that induces structural changes of the ear drum and middle ear  that can cause chronic leaking and pain within and around the ear as well as damage of structures involved with sound transmission to the inner ear 19. Different locations along the cochlea and across neurons within each auditory nuclei are sensitive to specific sound frequencies. The hearing organ and the brain are naturally designed to sense and extract different frequency components of sound over time to encode and elicit intelligible hearing perception. This frequency-specific transmission and extraction of sound first begins through the outer ear canal to the vibratory structures of the middle ear through mechanical vibrations. From the middle ear, the sound pressure waves are transmitted to the inner ear through fluid vibrations within the cochlea that activate inner hair cells along the tonotopically organized cochlea, ultimately reaching the auditory brain through the auditory nerve fibers.When developing or implementing a device to improve hearing, one needs to keep in mind the fundamental organizing principle of the hearing organ, which exhibits a spatially ordered frequency coding representation across the cochlea (inner ear) that is maintained up through multiple ascending auditory nuclei within the brain23; (2) activate neuronal structures, such as inner hair cells or auditory nerve fibers, directly with light, which is possible with Infrared Neural Stimulation (INS)24; or (3) transfect the neuronal structures with light sensitive ion channels to become sensitive to laser stimulation, a technique known as optogenetics26. Out of these, INS and optogenetics are appropriate for direct stimulation of the inner ear and the auditory neurons in severe to profound hearing impaired patients. However, the proportion of individuals having conductive, sensorineural or combined hearing loss with residual hearing that can still be activated mechanically is a much larger population, and despite all the improvements in the technology of auditory prostheses, not all patients obtain sufficient or useful hearing. Currently, there are laser-driven hearing devices in which laser is transmitted to a transducer placed at the tympanic membrane (TM),  known as the Earlens device27, or the round window28 to convert the optical signal to mechanical vibrations in those structures. Both devices demonstrate sufficient vibration amplitudes needed by patients with severe hearing impairment for frequencies up to at least 10 kHz. However, these laser devices require placement of a transducer onto a hearing structure that may not be preferred by some patients or clinicians. The only commercially available laser-based hearing device, the Earlens hearing aid, also requires an intact middle ear.Through the perspective of these physical characteristics of transmission of sound energy from the outer ear to the brain, there are several options for introducing light into the system for hearing purposes: (1) transform the light into mechanical energy (optoacoustic) through very quick laser pulses that lead to mechanical rather than thermal perturbations, in order to directly vibrate outer, middle or inner ear structures29. In 2009, Wenzel et al. demonstrated that controlled direct optoacoustic stimulation21 of the inner ear is possible, which was followed up with further experiments demonstrating the ability to directly vibrate structures from the ear drum up to the inner ear without the need for direct contact with the hearing structures21.Therefore, for those individuals not sufficiently benefiting from commercially available hearing devices, a non-contact optoacoustic-based activation approach of the hearing system may offer another hearing option. The first results related to optoacoustic activation of the hearing system was described as artifacts within the inner ear induced through monochrome laser pulses by Friedberger and Ren in 2006There still remain questions on what type of laser pulse patterns to use for optoacoustic stimulation and which peripheral structures to target for sufficient transmission of sound information. For coding speech and other complex signals such as music, one critical question that needs to be addressed based on the fundamental organization of the hearing system described above is if optoacoustic stimulation can induce frequency-specific activation of peripheral hearing structures. This study seeks to answer that question through simulations and experiments in a guinea pig model to demonstrate that laser pulse stimulation of the ear drum and middle ear structures can achieve precise and predictable frequency-specific activation of the auditory system. We were inspired by the amplitude modulation (AM) concept that is well known in radio-frequency engineering, in which a constant carrier frequency could potentially induce vibration waves in the targeted structure that follow the various modulating frequencies Fig. . The actWe amplitude modulated a predetermined laser pulse rate  with different laser modulation rates (LMRs) as shown in Fig. 30, in which the frequency difference between the LPR peak and the sidebands is equal to the LMR. This could be demonstrated across all the LMR/LPR combinations tested  at the entclass1pt{minimaequation )  Fig. . ConsistBesides the fundamental frequency f0, the recorded vibration spectra revealed two other main frequency peaks: at the 2nd harmonic  of the fundamental frequency peak (SNR-f0) with the peak at the second harmonic (SNR-h2) and the peak at the LPR (SNR-LPR) Fig. . SNR-f0 When comparing the variability of the different peaks , the fundamental frequency f0-SNR also demonstrated the least variation having the highest SNR of 27.4 dB SNR +−1.4 dB SE at 10 kHz LMR/32 kHz LPR and lowest SNR of 21.6 dB SNR +−2.3 dB SE at 8 kHz LMR/32 kHz LPR. The h2-SNR demonstrated higher variability than the fundamental frequency f0 peak across the tested LPR/LMR combinations having the highest SNR of 17.4 dB +−2.9 dB SE at 4 kHz LMR/32 kHz LPR and the lowest SNR of 7.4 dB +−2.1 dB SE at 2 kHz LMR/50 kHz LPR. Additionally, the distortions at the LPR also demonstrated a higher variation than the fundamental frequency f0-SNR having the highest SNR of 26.3 dB +−2.1 dB SE at 8 kHz LMR/32 kHz LPR and the lowest SNR of 15.0 dB +−3.9 dB SE at 1 kHz LMR/50 kHz LPR.Overall, these results demonstrate the greater detectability and reliability of the fundamental frequency f0 peak compared to the other peaks in the spectrum and the ability to systematically shift the fundamental frequency f0 peak with our modulated laser pulse approach, based on vibration measurements at the umbo.To further assess the capabilities of achieving frequency-specific activation of the peripheral hearing system with our developed laser pulse sequences applied at the umbo, the activation effects occurring directly in the auditory brain in a live animal was needed. For this validation, we positioned multi-site electrode arrays within the ICC of anesthetized guinea pigs and recorded neural spiking activity in response to laser pulse stimulation at the umbo using different stimulation parameters. Examples representing post stimulus time histograms, PSTHs, are shown in Fig. Consistent with the findings from vibration measurements in explanted specimens, optoacoustic stimulation at the umbo with low LMRs elicited activation of the ICC regions most sensitive to low frequencies, whereas optical stimulation with high LMRs elicited activation of the ICC regions most sensitive to high frequencies. We were able to observe a shift of the activated frequency region of the ICC within each animal in accordance to the predicted, calculated and in explanted specimens tested LMR Fig. . The chaTo demonstrate the reliability of our data regarding the frequency-specific activation with optoacoustic stimulation, we analyzed the correlation between the measured BF and the target modulated frequency (LMR) at the optical threshold. Across 12 animals Fig. , an almoThe optical thresholds were also determined from the PSTH data. Depending on the auditory sensitivity of each animal we observed some variability in the power threshold levels across the tested LMRs, particularly for the 32 kHz LPR Fig. , suggestIn order to provide an estimate of the acoustic level equivalent for optical stimulation, we analyzed the equivalent acoustic sound pressure level (air conduction) that is comparable to optical stimulation with each LPR-LMR combination at 80 mW Fig. . The maxA further advantage of our laser pulse amplitude modulation paradigm is its wide applicability for vibrating different structures in the peripheral auditory system, including the ear drum and structures within the middle ear without needing to be anchored onto or in direct contact with those targeted vibratory structures. Consistent with the results for stimulation at the umbo (ear drum), the applied LMR correlated with the activated BF channels of our ICC electrode for the stimulation at the round window 24 as well as the optogenetic stimulation approach26 have been reported and target patients with severe to profound sensorineural hearing loss . Additionally to INS, the “optophonic” stimulation of the inner ear with near infrared laser light has been observed33 in which sound could be generated by the laser stimulus that vibrated and activated intact auditory sensory cells (hair cells). Another laser approach described by Wenzel et al.17 demonstrated the activation of the inner ear with green pulsed laser light (532 nm) using the optoacoustic effect by delivering very short pulses that induced direct vibrations of the cochlear basilar membrane. In the current study, we have extended this previous work by demonstrating that optoacoustic stimulation can also vibrate structures within the outer and middle ear without requiring direct contact with those targeted structures. By presenting amplitude modulated pulse patterns using a convolution-based method, optoacoustic stimulation can achieve controllable frequency specific activation at the ear drum and more centrally in the ICC, which is a critical requirement for a hearing device.The first non-ablative therapeutic use of laser into the inner ear, the cochlea, was reported in 200434, since the transmitted energy is light instead of sound, acoustic feedback can be greatly reduced for an optoacoustic-based hearing aid, potentially providing greater frequency bandwidth and sound quality to the patients without requiring full occlusion of the ear canal. In cases where there is damage to certain regions of the middle ear pathway , in which the Earlens or conventional middle ear implants may not be suitable, an optoacoustic hearing device could be implanted into the middle ear cavity with a fiber or fiber bundle targeting bone regions beyond the damaged area, especially in cases where there is no reliable location to attach the actuator of a middle ear implant.One advantage over other laser-based hearing devices designed for the outer or middle ear, such as the Earlens, is that the optoacoustic approach would not require a sensor-actuator component be attached to the hearing system . Similar to the Earlens34 and needs to be characterized in a future study. Within the middle ear cavity, the fiber(s) could be embedded in a miniaturized and biocompatible guide tube or casing that is fixed to healthy bony wall within the mastoid and the fiber(s) can be oriented towards different portions of the ossicular chain or bony area in close proximity to the inner ear, round window or surrounding bony structures (e.g. promontorium).There are still several challenges that need to be addressed before an optoacoustic hearing device can be translated into patients. First, an optical fiber or fiber bundle needs to be positioned safely and stably into the ear canal or into the middle ear cavity. One option for the outer ear canal would be to embed the fiber(s) into a vented earpiece that can be inserted into the ear canal or in the inner portions of the concha. The location of the microphone should be positioned as close to the entrance of the ear canal to take advantage of the high-frequency pinna-diffraction, possibly embedding it in the same earpiece, assuming acoustic feedback within the ear canal caused by the vibrating ear drum is minimalSecond, a stimulation strategy is needed that can transmit complex signal patterns, such as speech and music, to the outer and middle ear structures. The fact that we were able to reliably predict the measured vibratory response at the ear drum level using a LTI modelling approach  provides optimism that the results observed in our study with simple pure tones and sinusoid modulated laser pulse sequences may reasonably translate to more complex inputs, in which those complex signals can be modelled as a sum of pure tones in a LTI framework. The brain is undoubtedly a non-linear system, and thus further experiments will still need to be pursued to assess how the responses in the ICC and auditory cortex, as well as hearing perception, actually compares between the complex acoustic inputs and the laser pulse sequences modulated by those complex inputs . Third, we need to determine the safe range of levels of optoacoustic stimulation within the outer and middle ear and if the maximum level is sufficient to provide a wide dynamic range for useful hearing. Finally, we need to calculate the total energy required to drive an optoacoustic hearing device and to minimize overall consumption to ensure the device can last at least for a full day with daily charging of the device overnight.In summary, we have developed and evaluated a laser-based pulse amplitude modulation approach that can provide a versatile and non-contact method to precisely vibrate structures within the peripheral hearing system. This new type of technology could provide an alternative solution for implantable and non-implantable hearing aids by replacing the speaker or the sound transducer  with a non-contact and focused energy transfer modality via laser pulses to be used in hearing impaired patients who are not sufficiently satisfied or aided with current hearing aid technologies.We chose albino guinea pigs  according to the protocols and the procedures approved by the Animal Welfare Office of the University of Saarland and by the Central Veterinary Office of Saarland . We present data from 27 animals, in which 9 animals were used for the umbo vibration measurements in explanted specimens and 18 animals were used for the neural recordings in the inferior colliculus. The guinea pigs weighing 350–900 g were anesthetized with 40 mg/kg ketamine  and 10 mg/kg xylazine  applied intra muscular. For the LDV experiments in extracted specimens the animals were sacrificed in deep anesthesia. For the neural recording experiments the animals were kept anesthetized throughout the experiment.We generated the acoustic- and the trigger-signals on a PC  in the control room Fig. . We tranWe used a 532 nm pulsed Neodymium-doped Yttrium Orthovanadate (Nd:YVO4) laser system . The stimulation laser was operated with a pre-determined laser pulse rate (LPR) of either 32 kHz or 50 kHz. We generated the sinusoid signal with a specific laser modulation rate (LMR) Fig.  as descrTo keep the number of pulses per stimulation unit constant over the different modulated frequencies tested, the number of pulses under the envelope had to vary from one modulated frequency to the other as presented in Table We changed the amplitude of the laser pulses corresponding to the LMR at 1 kHz, 2 kHz, 4 kHz, 8 kHz and 10 kHz from an averaged maximal peak power between 20 and 500 mW Table .Table 3Rvibration measurements in extracted specimens in Fig. The data is presented with line plots or bar plots displaying the mean values and standard error of the mean calculated with MATLAB®. The Statistical Analysis was performed with SPSS . For the We explanted the temporal bones, removed the cartilaginous outer ear canal and exposed the tympanic membrane (TM). To improve the signal to noise ratio (SNR), we placed micron sized glass beads (Ø 50 µm/bead) on the TM. With a custom-made holder we fixed the head, inserted the laser fiber into the outer ear canal and directed it towards the TM. For the vibration recordings in response to the laser stimulation, we placed a scanning laser Doppler vibrometer PSV 500  (LDV) at a distance of 20 cm from the TM. Using the built-in camera of the LDV, the TM could be displayed on the monitor, the measured points were visualized and the recordings could be monitored and controlled Fig. . In ordeFor the acoustic control measurements at the beginning of the LDV recordings in each experiment we applied pure tones  with 0 dB SPL, 60 dB SPL, 70 dB SPL and 80 dB SPL.  and with the laser scanning head that was connected to the laptop running the Polytec GmbH control software Fig. . We perfTo suppress artifacts, we used a bandpass filter between 300 and 12000 Hz and averaged a minimum of 17 runs. We calculated the displacement from the measured velocity data through numerical integration, and determined the single sided amplitude spectra using Fourier transformation. The fundamental frequency as well as the distortions were analyzed.We calculated the SNR at the peaks by dividing the peak frequency  by the same frequency of the noise signal . We then converted the SNR to a dB scale using equation . We disc35, the output of a linear time invariant (LTI) system can be calculated by convoluting the impulse response of the system with an input signal. Based on this idea, we implemented a convolution based model in MATLAB®: We recorded the LDV vibration velocity at the umbo in response to optical stimulation with a 50 µJ laser pulse, which is as an approximation to the Dirac impulse .We considered the LDV velocity recording in response to stimulation with a 50 µJ laser pulse that can be represented as the impulse response of the system using this equation: entclass1pt{minimaequation .37. We placed platinum subdermal needles as a reference signal (vertex) and ground (neck). We then performed the acoustic stimulation to determine the final position of the electrode (for details see Stimuli and recordings) and covered the brain with agar to reduce its swelling, pulsations and drying. We inserted the laser fiber into the outer ear canal and directed it towards the TM using a second micromanipulator  . For other stimulation locations, we directed the fiber towards the round window membrane or the otic capsule at the basal turn level . The LPR was 50 kHz in those set of experiments.For the neural recordings in animals, we extended the initial anesthesia described above with additional 1/4–1/2 of the initial dosage every 30 to 60 minutes to maintain an areflexive state. We injected 50 ml/kg bodyweight saline subcutaneous per day divided in dosages every 1–2 hours. The body temperature was stabilized at 38 °C during the experiment using a DC heating pad. We removed the left auricle and exposed the meatus acusticus externus to attain access to the TM. Additionally, through a retoauricular incision we opened the middle ear (the bulla) and exposed the cochlea. We fixed the head with a custom-made holder, opened the skull and exposed the inferior colliculus. We inserted an A1x16 single shank electrode  in an angle of 45° to the horizontal line using a micromanipulator  with a custom built angle adjusting system in order to position the shank along the tonotopic gradient of the ICC2 and was connected via a custom-made head stage to the biosignal amplifier   from the first one, to prove the reliability of our recordings.The A1x16 electrode had an inter channel spacing of 100 µm and an area of 703 µmia) Fig. . The refWe performed the data analysis in response to the acoustic and the optical stimulation using a spike detection algorithm. Spikes were defined as signal peaks having values with a standard deviation of 3.5 above the background noise. The time at which the spike was detected (timestamp) corresponded to the largest negative peak of that spike  or between 1 and 22 kHz (low frequency electrode position) depending on the probe position of each experiment . We generated the acoustic frequency response map (FRM) online with at least 7 runs per frequency and level pair  from the TSR for each channel. We then normalized the DSR by the maximum spike rate per channel across the presented stimuli and plotted the normalized spike rates as a FRM  to analyze the data and determine the neural response on each channel after acoustic or optical stimulation. The corresponding acoustic BFs were mapped to each histogram channel for further assessment (Supplementary Fig. For the estimation of the acoustic sound pressure level equivalent for pure tone stimulation in comparison to optical stimulation, the ICC spike activity in response to optical stimulation with 80 mW for each LPR-LMR combination was recorded. The DSR was compared to the acoustic level at the same electrode channel after stimulation with the best frequency. We discarded values, if the BF channel at maximal acoustic stimulation differed more than 1 channel from the BF channel at threshold.Custom code for recording and analysis is available from the corresponding authors on reasonable request.Supplementary Figures


Mycoplasma hyopneumoniae (M. hyopneumoniae) infection in weaned piglets. Fifty M. hyopneumoniae-free piglets were assigned at 30 days of age [study day (D)0] to four different groups: 1) negative control group , 2) DON-contaminated group , 3) DON-contaminated and M. hyopneumoniae-inoculated group , 4) M. hyopneumoniae-inoculated group . The piglets were fed the experimental diets ad libitum for five weeks and were monitored during this period and euthanized at day 35 [27 days post infection (DPI)] or 36 (28 DPI). The main parameters under investigation were macroscopic lung lesions (MLL) at euthanasia, respiratory disease score (RDS) from day 8 until day 35, histopathologic lesions and log copies of M. hyopneumoniae DNA detected by qPCR, determined at the day of euthanasia.The mycotoxin deoxynivalenol (DON) is highly prevalent in cereals in moderate climates and therefore pigs are often exposed to a DON-contaminated diet. Pigs are highly susceptible to DON and intake of DON-contaminated feed may lead to an altered immune response and may influence the pathogenesis of specific bacterial diseases. Therefore, the maximum guidance level in feed is lowest in this species and has been set at 900 μg/kg feed by the European Commission. This study aimed to determine the effect of in-feed administration of a moderately high DON concentration (1514 μg/kg) on the severity of an experimental M. hyopneumoniae DNA in the DONMHYO and MHYO group and consequently, no enhancement of the severity of the M. hyopneumoniae infection could be detected in the DONMHYO compared to the MHYO group.No significant difference was obtained for MLL at euthanasia, RDS (8–35), histopathologic lung lesions and log copies of M. hyopneumoniae infection. Further research is needed to investigate the impact of DON on M. hyopneumoniae infections in a multi-mycotoxin and multi-pathogen environment.Under present conditions, the findings imply that feed contaminated with DON (1514 μg/kg) provided to weaned pigs for five weeks did not increase the severity of an experimental  Fusarium graminearum and Fusarium culmorum [Fusarium-produced mycotoxins, of which by toxicological viewpoint the trichothecenes DON and T-2 toxin, zearalenone and fumonisins are the most important [Fusarium species have traditionally been associated with temperate cereals, as these fungi require lower temperatures for growth and mycotoxin production [The mycotoxin deoxynivalenol (DON) is a fungal metabolite produced mainly by culmorum , 2. Fusamportant , have beoduction . Indeed,oduction –7. Extenoduction , 4, 8. Ioduction , 10. In oduction . On top oduction , 12.Salmonella Typhimurium invasion in and passage of the bacterium across the intestinal epithelium [Salmonella Typhimurium in porcine macrophages, indicating the capacity of DON to modulate the innate immune system, and thus to increase the susceptibility of the pig to Salmonella Typhimurium infections [Bordetella bronchiseptica (B. bronchiseptica) and Pasteurella multocida, (P. multocida) were at greater risk to develop pneumonia and had an increase of the extent and severity of the pathological changes compared to dually infected pigs that did not receive fumonisin B1 [P. multocida-infected pigs with a crude extract of fumonisin B1 in a concentration of 500 μg/kg body weight (BW) per day for a period of seven days resulted in the pigs coughing more, in increased bronchoalveolar lavage fluid total cells, macrophages and lymphocytes, and resulted in an increased occurrence of lung lesions compared to the pigs only infected with P. multocida [M. hyopneumoniae infection [It is known that DON can have an impact on the pathogenesis of several bacterial diseases , 13, 14.ithelium . Furtherfections . Deoxynifections , 18. Pignisin B1 . Oral gaultocida . Dietaryultocida  and indunfection .Mycoplasma hyopneumoniae is causing tremendous economic losses in all intensive pig producing countries worldwide [M. hyopneumoniae infections and a high contamination rate of feed with mycotoxins, more specific DON, in Europe [M. hyopneumoniae is high. The present study aimed to investigate the effect of in-feed administration of DON at a moderately high level of 1540 μg/kg feed, on the clinical course of an experimental M. hyopneumoniae infection with two genetically different M. hyopneumoniae strains in weaned piglets.orldwide , despiten Europe . TherefoM. hyopneumoniae-free Rattlerow-Seghers piglets  were included in the study. The herd of origin has been free of M. hyopneumoniae and PRRSv since 2012 based on repeated serological testing, absence of clinical signs and pneumonia lesions, and nested polymerase chain reaction (nPCR) testing on tracheobronchial swabs as previously described [Erysipelothrix rhusiopathiae and Parvovirus before insemination. No vaccinations were administered to the piglets. The piglets were weaned on average at 26 days of age and moved four days later to the experimental facilities of the Faculty of Veterinary Medicine, Ghent University, Belgium. The piglets were individually identified by means of an ear tag. The study design, the different parameters and timing are summarized in Table n = 5): sham-inoculated D8, D9 + control diet, 2) DON-contaminated group : sham-inoculated D8, D9 and DON-diet (1514 μg/kg), 3) DON-contaminated and M. hyopneumoniae-inoculated group : experimentally inoculated with M. hyopneumoniae D8, D9 and DON-diet (1514 μg/kg), 4) M. hyopneumoniae-inoculated group : experimentally inoculated with M. hyopneumoniae D8, D9 + control diet. The number of 15 animals in the treatment groups enabled to find a difference of 5.26 ± 4.7 in the main parameter, namely macroscopic lung lesions (two-sided test) with 95% certainty and a statistical power of 80%. This difference is biologically relevant and was based on previous research in our research group [M. hyopneumoniae negative throughout the study. The different groups were housed in four different facilities equipped with absolute filtered chambers (HEPA U15) in order to avoid cross-infection of M. hyopneumoniae between the different groups. The pigs had free access to drinking water and were fed ad libitum.The study was compliant with all relevant institutional and European standards for animal care and experimentation. The experiment was approved by the Ethics Committee for Animal Experiments of the Faculty of Veterinary Medicine and Faculty of Bioscience Engineering, Ghent University . Fifty escribed . The gilch group . The NCGM. hyopneumoniae: a highly virulent strain F7.2C and low virulent strain F1.12. Both strains had been differentiated and characterized at proteomic level with Sodium-Dodecyl-Sulphate Polyacrylamide gelelectrophoresis (SDS-page) [M. hyopneumoniae strains [M. hyopneumoniae strains, as performed previously by this research group [7 CCU/ml of strain F7.2C on D8 and 7 ml of inoculum containing 107 CCU/ml of strain F1.12A on D9. On both inoculation days, the pigs of the NCG and DON group were endotracheally sham-inoculated with 7 ml of sterile Friis medium. At D35 or D36 of the experiment, the pigs were euthanized using deep anaesthesia by intramuscularly administering 0.3 ml/kg BW of a mixture of tiletamine, zolazepam  and xylazine , followed by exsanguination. For practical reasons and to avoid M. hyopneumoniae contamination of the samples, all pigs of the NCG and DON group were euthanized at D35, followed by five animals of the MHYO group. All the other animals of the MHYO and all animals of the DONMMHYO group were necropsied on D36.The pigs were inoculated with two different strains of DS-page)  and at gDS-page) –29 and uDS-page) , 25, 30. strains , 32, andch group , to mimiFusarium graminearum (F. graminearum) MUCL 42841 (Mycothèque de l’Université catholique de Louvain) was used to produce the DON-culture. The strain was grown in liquid mineral (MIN) medium supplemented with L-arginin as a selective nitrogen source, as previously described by Gardiner et al. [F. graminearum trichothecenes such as nivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin, and results were below the detection limit. Also no zearalenone was detected. Eight l of inoculum were obtained for preparation of 1300 kg of DON-contaminated feed in a concentration aimed at twice the recommended maximum pig feed level of 900 μg/kg or 1800 μg/kg DON. First, 2.67 l of the inoculum was mixed with 10 kg of feed to obtain a thoroughly mixed premix of the DON-contaminated feed. Subsequently, the premix with inoculum was added to 433.3 kg of feed in a feed mill and thoroughly mixed for at least 40 min. The same procedure was repeated twice, to obtain the total amount of 1300 kg of DON-contaminated feed in a concentration of 1800 μg/kg and the feed was collected again in the original 25 kg bags of the feeding company. After preparation of the contaminated feed, a mixed sample originating from three DON-contaminated feed bags was taken and was submitted for LC-MS/MS to obtain the true DON-concentration of the contaminated feed. The results of the LC-MS/MS of the contaminated feed were 407 ± 120 μg/kg DON, 280 ± 100 μg/kg 3-ADON and 827 ± 300 μg/kg 15-ADON, resulting in a total DON and acetylated DON (3-ADON + 15-ADON)-concentration of 1514 μg/kg in the contaminated feed sample. This feed was used in the DON and DONMHYO-groups from D0 until D35/36 of the study.A commercial antibiotic-free diet for weaned piglets was purchased . Before purchasing the feed, a sample of the batch was tested with liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to Monbaliu et al. , 34 for r et al. . After 1From D0 until D35/36 onwards, the piglets were observed daily at 8 a.m. for at least half an hour by the same researcher to assess appetite, faecal consistency and presence of dyspnea and tachypnea. A faecal consistency score was used to evaluate the faeces found on the pen floor before cleaning . Score 0M. hyopneumoniae infection as previously described [The lungs were removed and macroscopic lung lesions (MLL) (D35/36) were determined according to Hannan et al. (1982)  from eacescribed , 39, 40.escribed . This paescribed .M. hyopneumoniae organisms [M. hyopneumoniae DNA of strain F7.2C was used to convert the threshold values to the number of M. hyopneumoniae organisms. Values below the dilution of 1.50 × 101 (1.18 log copies) were considered as negative [Two weeks post inoculation (PI) (D21) of the high virulent strain F7.2C (D8), bronchoalveolar lavage (BAL) fluid from all pigs, while conscious was collected. After snaring the pigs and opening their mouth with a gag, a catheter  was inserted allowing to flush the lungs with 10 ml of sterile phosphate buffered saline (PBS). The PBS fluid was subsequently aspirated. At the day of necropsy (D35/36) the head bronchus of the left part of the lung was flushed with 10 ml of sterile PBS before collection of the histopathological samples. After collection, the BAL fluids were stored at − 70 °C until they were analysed. The DNA was extracted with the DNeasy Blood & Tissue kit  with the DNA Purification protocol for bloods or bloody fluids (spin protocol) on 200 μl of BAL fluid according to the manual instructions and quantitative PCR (qPCR) was performed as previously described to detect the number of rganisms . Brieflynegative , 42.Mycoplasma hyopneumoniae EIA kit, Oxoid Limited, Hampshire, UK) was performed according to the instructions in the protocol manual and as previously described [M. hyopneumoniae in the blood collected at D28, D21 and at necropsy (D35/36). Sera with optical density < 50% of the average value of the OD-buffer control were considered to be positive. All values above or equal to 50% of the average value of the OD-buffer control were classified as negative .A blocking ELISA  with a Bruker Daltonic Microflex LT Biotyper Biotyper MALDI-TOF mass spectrometer by using the direct transfer method and α-cyano-4-hydroxycinnamic acid (HCCA) as matrix, according to the manufacturer’s guidelines. The spectra were obtained and analysed with the MBT Compass software version 3.1. , which included a database of 6903 mean spectra projections.Ten μl of BAL fluid collected at necropsy (D35/36) of each pig was inoculated on Columbia agar supplemented with 5% sheep blood  with a mination . Plates M. hyopneumoniae qPCR-positive samples, M. hyopneumoniae specific AB expressed in OD values and percentage of ELISA M. hyopneumoniae positive samples. These variables were all run in separate models with ‘pig’ as statistical unit and no additional factors included into the model. The normality of the data was investigated by means of descriptive statistics, except for the binary data (M. hyopneumoniae qPCR positive samples and ELISA M. hyopneumoniae positive samples). The parameters BW, ADG and percentage air analysis were normally distributed and a one-way analysis of variance (ANOVA) test was used. In case of the RDS, a repeated measures ANOVA was performed. Scheffé’s post-hoc test was used to make pair-wise comparisons. The qPCR-results and M. hyopneumoniae specific antibodies were not normally distributed and therefore, a non-parametric Kruskal-Wallis test was used, with the Dunn-Bonferroni approach to make pair-wised comparisons, as well for MLL and histopathology results. In case of the normally distributed data, the mean and standard deviation (SD) were reported, in case of the non-parametric data, the median and the interquartile range were reported. All analyses for these parameters were performed with SPSS 23 for Windows . Percentage of seropositive pigs and percentage of pigs testing positive with qPCR in each group were analyzed using binomial logistic regression (R version 3.3.1) [P < 0.05.The independent variable in the statistical analyses was ‘group’: NCG, DON, DONMHYO and MHYO-group. The dependent variables were RDS, weight, ADG, MLL, histopathology and percentage of air, qPCR-results, percentage of n 3.3.1) . The res1–7, RDS 8–35 and RDS 1–35). The RDS from the first inoculation day onwards until euthanasia (RDS 8–35) were 0 ± 0, 0.0071 ± 0.028, 1.04 ± 0.82, 1.14 ± 0.92 for NCG, DON, DONMHYO and MHYO, respectively (P < 0.001). No statistically significant differences were obtained between all groups for D1–7. For D8–35 and D1–35 a statistically significant difference was obtained between the experimentally infected (DONMHYO and MHYO) and non-infected pigs (NCG and DON) (P < 0.001). However, no statistically significant differences were obtained for DONMHYO and MHYO groups in each time period of the study  from D0 onwards for 5 days, according to the product leaflet. The post weaning diarrhoea lasted until D8, D3, D3 and D7 in NCG, DON, DONMHYO and MHYO, respectively. In three groups some pigs with faecal consistency score 3 were noticed throughout the study: in NCG two pigs at D20 and D23 respectively, in DON one pig at D13 and in DONMHYO two pigs at D20 and D23, respectively. In MHYO, normal faecal consistency was observed throughout the study. Coughing was not observed in the NCG. All results of the different time periods for this parameter in the study are shown in Table There were no significant differences between the groups for the parameter BW and the ADG during the different time periods Table .P < 0.001). The histopathological lung lesions were 1.70 ± 0.20, 2.00 ± 0.30, 2.40 ± 0.80 and 2.40 ± 0.90 for the NCG, DON, DONMHYO and MHYO group, respectively (P < 0.001). The percentage of air was 45.94 ± 6.54, 46.85 ± 6.86, 49.75 ± 9.90 and 46.96 ± 8.51 for NCG, DON, DONMHYO and MHYO, respectively (P = 0.25). There were no statistically significant differences between the DONMHYO and MHYO groups for these above-mentioned parameters, however there were statistically significant differences between the experimentally infected (DONMHYO and MHYO) and non-infected pigs (NCG and DON) (P < 0.001), except for the parameter percentage of air analysis (P = 0.25)  and at D35/36 0.80 ± 0.70, 0.29 ± 1.09, 4.05 ± 1.38 and 4.20 ± 1.00 (P < 0.001) for NCG, DON, DONMHYO and MHYO, respectively. There were no significant differences between the DONMHYO and MHYO group, however there were statistically significant differences between the experimentally infected (DONMHYO and MHYO) and non-infected pigs (NCG and DON) (P < 0.001) for D21 and D35/36  and MHYO (1/15) groups. In addition, few colonies of B. bronchiseptica were isolated from one pig of DON (1/15) and four pigs of MHYO (4/15).Few colonies of M. hyopneumoniae DNA in bronchoalveolar lavage fluid) was not statistically different in M. hyopneumoniae infected pigs that received or did not receive DON-contaminated feed. This indicates that ingestion of DON-contaminated feed at a concentration of 1514 μg/kg, exceeding the maximum guidance level for pigs according to EU regulation, did not aggravate the severity of an experimental M. hyopneumoniae-infection in weaned piglets.In the present study, the outcome of the main parameters  coughed, showed lung lesions , seroconverted and M. hyopneumoniae-DNA was detected in BAL fluid at necropsy. The results obtained in the M. hyopneumoniae challenged pigs (DONMHYO and MHYO group) differed significantly from the non-challenged pigs . The latter pigs remained serologically negative throughout the entire study period and no M. hyopneumoniae- DNA was detected in BAL fluid two weeks post-inoculation, nor at necropsy. The obtained data in the challenged groups were comparable with a previous study performed by our research group with the same challenge model [M. hyopneumoniae-strains were not administered on the same day, as the infection model practiced at our research group uses seven ml inoculum containing 107 CCU of the strain/ml [M. hyopneumoniae-strains are grown in the same culture flask. Consequently, it was decided to challenge the piglets on two consecutive days.The challenge infection was successful as all animals in the ge model , demonsttrain/ml . It is nIn the present study, DON was aimed to be administered at two times the maximum guidance level of DON advised by the European commission for pigs (1800 μg/kg feed) . This doIt remains to be elucidated why no impact of DON in the pigs was observed in the present study. The degree of susceptibility of certain breeds/lines to the effects of DON might be one explanation . For insNo feed-refusals were observed in the pigs administered the DON-contaminated feed. The minimum emetic dose for orally distributed DON in pigs is 100 μg/kg , yet vomM. hyopneumoniae-infected pigs administered the DON-supplemented feed did not have a higher RDS compared to the pigs only infected with M. hyopneumoniae. No other studies are available investigating the impact of DON on respiratory tract disease signs in M. hyopneumoniae-infected pigs. However the effect of fumonisin B1 in combination with M. hyopneumoniae or the effect of DON on other pathogens has been studied. Pósa et al., [M. hyopneumoniae-infected pigs, however no firm conclusions could be drawn regarding the difference in coughing between the M. hyopneumoniae-infected pigs with or without fumonisin B1 supplementation in the feed. Halloy et al. [P. multocida-infected pigs and concluded that these pigs coughed more compared to the pigs only infected with P. multocida.The  et al.,  studied y et al.  investigM. hyopneumoniae infections can negatively influence production parameters, such as ADG [M. hyopneumoniae inoculated animals. This result is in agreement with Accensi et al. [M. hyopneumoniae-infected pigs. Rotter et al. [Fusarium mycotoxins from one week onwards. In that study, pigs were fed a diet mixed with naturally contaminated corn  to obtain DON-concentrations of 750, 1500 or 3000 μg DON /kg feed during 28 days. During the first week, the exposed pigs had lower weight gains than the pigs fed a non-contaminated control feed. At the end of the study, however, the overall weight gains did not differ anymore between these groups. This adaptation might be one of the reasons why we did no see an effect of DON on daily growth. The relative low number of pigs, followed up during a limited period of time, which is inherent in experimental infection studies like ours, also makes it difficult to obtain a statistical significant difference in daily growth [M. hyopneumoniae infected group. This finding is in agreement with Pósa et al. [B. bronchiseptica and P. multocida dually-infected groups with or without supplementation of fumonisin B1 in the feed  of three-day-old piglets. Savard et al. (2015) on the other hand, did observe an effect of DON-supplementation in the feed  on macroscopic lung lesions in PRRSv-infected pigs [M. hyopneumoniae infected animals. Similarly, Savard et al. [M. hyopneumoniae infection under the circumstances in this study. Deoxynivalenol has a good distribution in the lung of the pig [M. hyopneumoniae is attached to the cilia of the upper respiratory tract and does not invade the parenchyma of the lung [It is known that h as ADG . Deoxynih as ADG , 56, 57.i et al.  (840 μg i et al.  (1500 μgi et al. . The lati et al. , did notr et al.  observedy growth , 59, 60.a et al. , who neited pigs . In thisd et al.  did not d et al. , 63. It  the pig . Howeverthe lung , 65, 66,the lung , 61, thuM. hyopneumoniae infection in weaned piglets. In the field, however, the impact of DON-contaminated feed on a M. hyopneumoniae-infection might be more expressed, because mostly multi-mycotoxin contamination of the feed occurs [M. hyopneumoniae infections under these multi-pathogen and multi-mycotoxins circumstances and investigating the impact of DON in vitro on M. hyopneumoniae in tracheal explant cells. More research could also focus on factors influencing the effect on DON such as health status, gender, age, and possible genetic resistance of the pigs.No effect was observed of DON contamination in a moderately high dose in the feed on the severity of an experimental d occurs , 52–54, d occurs , 56. Fur