Sylvia atricapilla). The medial V1 subbranch takes its course along the ventral part of the upper beak to innervate subepidermal layers and the mucosa of the nasal cavity, whereas the lateral V1 subbranch runs along dorsolateral levels until the nostrils to innervate mainly the skin of the upper beak. In the trigeminal brainstem, medial V1 terminals innervate both the dorsal part and the ventral, magnetically activated part of the principal sensory trigeminal brainstem nuclei (PrV). In contrast, the lateral V1 subbranch innervates only a small part of the ventral PrV. The spinal sensory trigeminal brainstem nuclei (SpV) receive topographically ordered projections. The medial V1 subbranch mainly innervates rostral and medial parts of SpV, whereas the lateral V1 subbranch mainly innervates the lateral and caudal parts of SpV. The present findings could provide valuable information for further analysis of the trigeminal magnetic sense of birds.In night-migratory songbirds, neurobiological and behavioral evidence suggest the existence of a magnetic sense associated with the ophthalmic branch of the trigeminal nerve (V1), possibly providing magnetic positional information. Curiously, neither the unequivocal existence, structural nature, nor the exact location of any sensory structure has been revealed to date. Here, we used neuronal tract tracing to map both the innervation fields in the upper beak and the detailed trigeminal brainstem terminations of the medial and lateral V1 subbranches in the night-migratory Eurasian Blackcap ( In birds, the perception of somatosensory information from the facial/beak region is of central importance for a wide range of behaviors. To mediate them, their underlying trigeminal sensory system has undergone a particularly high degree of diversification. In addition to mechanoreception, proprio-, thermo-, chemo-, and nociception, the birds’ trigeminally mediated behavioral repertoire ranges from feeding, tactile exploration of the environment, hatching, grooming, climbing, and nest building  to cognivia the fifth cranial, i.e., trigeminal nerve (N. V.), toward the brain. N. V. splits into ophthalmic (V1), maxillary (V2), and mandibular (V3) branches  to map their respective courses from their distal nerve fiber terminals within the upper beak up to their projections in the trigeminal brainstem complex.Therefore, the aim of the present study was to perform selective neuronal tract-tracing of the medial and lateral V1 subbranches in the long-distance night-migratory songbird Eurasian blackcap (S. atricapilla) were used for this study. All animal procedures were approved by the Animal Care and Use Committees of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit . The birds were wild-caught using mist nets after the breeding season and during autumn migration in the vicinity of University Oldenburg. The birds were housed in pairs in indoor wire cages (102 cm × 50 cm × 40 cm) at the institute’s animal housing facility. They were kept at around 21°C and were exposed to a light regime simulating the natural circannual and circadian light-dark cycle of Oldenburg. Food and water were provided ad libitum.A total of 8 male and 6 female adult Eurasian blackcaps  dissolved in oxygen and administered through a beak mask was used. For medial and lateral V1 tracings, we administered a mix of ketamine hydrochloride  and medetomidine , each at a concentration of 0.1 ml/kg body weight dissolved in 0.9% sodium chloride (NaCl), into the pectoral muscle. For the tracings, the animals were head-fixed. For entire V1 tracings, the nerve was accessed unilaterally through an incision along the dorsal rim of the orbit and careful retraction of the eyeball and oculomotor muscles . This procedure was identical to previous studies, in which V1 was either tracer-labeled or surgically sectioned . Meloxicam  was administered to each bird intramuscularly in the pectoral muscle after 24 and 48 h for post-surgical analgesia. The birds were given 5 days to recover from the surgery and to let the tracer be transported.To visualize the course of V1 and its subbranches, neuronal tract tracer was either injected into the medial V1 subbranch, the lateral V1 subbranch, or the entire V1. Two different types of anesthesia were used to allow accessibility to the respective nerve subbranches: for entire V1 tracings, inhalation of anesthesia with 1–1.5% volume isoflurane CP ectioned . For med2  and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde (PFA) dissolved in phosphate-buffered saline . Brains and beaks were extracted, post-fixed in 4% PFA in PBS for 24 h, and cryoprotected in 30% D(+)-saccharose dissolved in PBS for at least 48 h. Brains and beaks were cut using a freezing microtome . Brains were cut in 40-μm thick slices in the frontal plane in six parallel series and the free-floating slices were collected in 0.1% sodium azide dissolved in PBS. Beaks were cut in 25-μm thick slices in the frontal plane in 10 parallel series, dried on gelatinized glass slides , and stored at −20°C.Immediately after magnetic exposure, birds were deeply anesthetized with pentobarbital (Narcoren The permanent 3′3-diamino-benzidine (DAB) staining method was used to map the detailed course of the nerve (the basis of RRID: AB_258833) overnight at 4°C, with the general neuronal marker monoclonal mouse anti-HuC/HuD antibody  overnight at 4°C, or with a polyclonal rabbit anti-Egr-1 antibody  for 72 h at 4°C.For the permanent DAB staining, parallel series of brain or beak slices were stained together. Brain slices were stained free-floating, beak slices were stained on glass slides. Slices were washed three times for 10 min with Tris-buffered saline . Endogenous peroxidases were saturated with 0.3% hydrogen peroxide for 30 min. After three times of washing for 10 min each in TBS, unspecific binding sites were blocked with 10% normal donkey serum  dissolved in TBS containing 0.3% Triton-X100  for 30 min. Slices were incubated either with a primary polyclonal rabbit anti-CtB antibody . Brain and beak slices were dehydrated in a graded series of alcohol  and cover-slipped with Eukitt . Negative controls were performed on parallel beak slices omitting the primary antibody to distinguish endogenous pigment epithelium from the substrate staining. Brain and beak slices were imaged using light microscopy . Contrast was adjusted with identical settings using the function Enhance contrast of ImageJ  for 120 min at room temperature. After three washing steps for 10 min each in TBS-T, the slices were incubated in an avidin-coupled peroxidase complex  for 60 min at room temperature. After two washing steps of 10 min each in TBS and inactivation of ongoing reactions in 0.1 M sodium acetate for 5 min, peroxidase activity was visualized under continuous visual inspection for 20 min using a 3′3-diamino-benzidine  reaction using glucose oxidase  instead of hydrogen peroxide . The reaRRID: AB_258833) and a monoclonal mouse anti-Tubulin beta 3 (TUBB3) antibody  overnight at 4°C. Slices were washed three times 5 min each in TBS before the TUBB3 signal was enhanced by incubating the slices with a secondary biotinylated horse anti-mouse antibody  for 90 min at room temperature. After three times of 5-min washing each in TBS, primary antibodies were detected with Alexa-conjugated secondary antibodies  on incubation for 90 min at room temperature. Slices were cover-slipped with 4′,6-Diamidin-2-phenylindol  dissolved in DABCO  and Mowiol . Negative controls were performed on parallel beak slices omitting the primary antibodies. Beak slices were imaged using fluorescence microscopy . With ImageJ  for 1 h. Slices were incubated with a polyclonal rabbit anti-CtB antibody . The course of V1 and its subbranches was translated into 2D reconstructions, one depicting the course of the subbranches as a side view  and the Adobe Illustrator 25.4.1 software  and lateral subbranches  and can be tracked almost up to the tip of the beak  to each of the V1 subbranches? Our data indicate that the medial V1 is most likely responsible for mediating somatosensory information from the upper beak since it is the only V1 subbranch that exclusively innervates the dorsal part of PrV . The dorBased on our expectation that any magnetic sensor must be located within the proximity of any of the V1 subbranches, do our data allow us to narrow in on the exact location of any magnetic sensor? At present, the answer is no since our data show that both subbranches innervate different magnetically activated parts of the trigeminal brainstem complex to varying degrees . Thus, bOnly a highly elaborate correlation to function approach, e.g., the analysis of magnetic field-induced neuronal activation and/or a behavioral displacement study after selective ablation of either the medial or lateral V1 subbranch could give further evidence about any specific involvement of one of the V1 subbranches in magnetoreception. However, given the complicated innervation patterns seen here, the estimated number of experimental animals needed to reach a clear conclusion, if any exists that could be obtained from such studies, would exceed our ethical limits, and we have therefore chosen not to perform such studies. In conclusion, the elusive trigeminal-based magnetic sensors could be located anywhere along the entire dendritic field of both V1 subbranches.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Animal Care and Use Committees of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit .DH and HM designed the research. KH, IM, LW-M, BL, AZ, and DH performed experiments. KH, IM, LW-M, and DH analyzed the data. HM provided facilities. KH and DH wrote the first draft of the manuscript, which all co-authors commented on. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early detection and management of hearing loss are important to develop ordinary speaking language and academic skills during childhood. Lack of knowledge by either parents or health care providers could hinder the process of hearing loss diagnosis, such that the intervention will be less effective. There is little evidence about the knowledge and practice of family physicians regarding hearing screening in Saudi Arabia and worldwide.This study aimed to assess family physicians’ knowledge, attitudes, and practices related to hearing loss in children. This in turn will help policy makers and educational institutions to establish and promote a program concerned with screening, diagnosis and intervention of paediatric hearing loss.A cross-sectional descriptive study enrolled 133 family physicians working at primary health centres in Saudi Arabia from March 2020 to September 2020. A self-reported questionnaire was used to assess the knowledge, attitudes, and practices of family physicians concerning hearing loss in children.The majority of the participants were working under the umbrella of the Ministry of Health and around half of them did not screen any child for hearing loss. Despite that, 91.7% indicated the importance of neonatal hearing screening, 70.7% indicate infant candidacy for cochlear implant and only 33.1% know about the existence of the early hearing detection and intervention (EHDI) governmental program in kingdom of Saudi Arabia (KSA). Participants were able to identify factors associated with hearing loss such as a family history of hearing loss (85.6%), meningitis (75%) and craniofacial anomalies (51.5%). The most frequent specialists for patient referrals were ear nose and throat ENT (75.2%) and audiologists (67.7%).This study shows that family physicians have good general background about the benefits of EHDI programs and the management of hearing loss in the paediatric population. However, it also indicated insufficient knowledge in other domains of hearing loss, including assessments and the presence of the EHDI governmental program in KSA. Further actions on the involvement of family physicians in the process of neonatal hearing screening, diagnosis and intervention for hearing impairment are needed. Hearing is essential for the healthy mental and communication development of a child. According to the World Health Organization (WHO), there are 466 million people with hearing disabilities, and 34 million of them are children . Early dThere is a high prevalence of permanent hearing loss in the Saudi paediatric population, documented to be 7.7% ,4. A comAlthough newborn hearing screening (NHS) has provided opportunities for children to be identified directly after birth, the application of such a program is less implemented in developing countries, including Saudi Arabia . GovernmThe knowledge, attitudes and practice (KAP) questionnaire identify what a specific group of individuals knows about certain topics and how they interact towards them. It is composed of three main domains: knowledge, attitudes and practice . Under tThe KAP questionnaire is extensively applied universally by scientists in different health fields for the sake of studying the knowledge, attitudes and practices of healthcare professionals –13, patiRegarding the parents of children with hearing impairment, one study shows that mothers are highly supported by the existence of national neonatal hearing screening. However, there is a necessity for preparing medical workers to enhance the awareness of families on the implication of newborn hearing screening . A studyIn Saudi Arabia, all newborns should be screened for hearing impairment in the hospital before discharge. However, the Ministry of Health statistics showed that screening tests do not cover 30% of newborns . As a reThe current research aimed to determine family physicians’ knowledge and practices related to hearing loss. This is critical since family physicians are considered a direct source of knowledge for parents and the public. They are also responsible for identifying hearing loss, referring patients, and discussing the management plan with parents; therefore, they must have the latest information available related to different facts about hearing loss and have a high level of awareness and knowledge regarding hearing loss risk factors and management options. Knowing the level of physicians’ knowledge will assist policymakers and educational groups in establishing and promoting hearing loss awareness.A cross-sectional descriptive design was deployed in this study. All participants were family physicians working in primary care centres in Saudi Arabia. The enrolment in the study was restricted to include only family medicine physicians without permitting other medical specialists to participate. All family medicine physicians regardless of their experience were invited to participate in the study. A total of 300 participants were recruited as they were collected through networking and snowballing sampling. A paragraph describing the aim and significance of the study was provided, and then participants were asked to give consent and to answer the given questionnaires that explore their knowledge, attitudes, and practices (KAP) about auditory screening in children. The consent was compatible with the Helsinki Declaration of the World Medical Association and was part of a study approved by the Institutional Review Board at King Fahad Medical City, Riyadh, KSA.This study was approved by the Institutional Review Board at King Fahad Medical City Riyadh, KSA (IRB000010471). Before enrolling potential participants, researchers provided information about the purpose and significance of the study. Furthermore, researchers emphasised voluntary participation. Moreover, all participants will be informed about anonymity, confidentiality issues and the option of voluntary termination at any time. At the beginning of the questionnaire, the participants were asked to give their consent before moving forward in filling the questionnaire.The data were collected using a self-reported questionnaire. The questionnaire was adapted from one developed by Moeller, White, and Shisler in 2006 , includiSPSS version 16 was used to analyse data. All nominal and ordinal data are reported as frequencies and percentages, and numerical data were reported in terms of means and standard deviations. The data collected through the questionnaire were analysed using the independent t-test and chi-squared tests with experience in years, sex and specialty level as independent variables. Before that, the Kolmogorov-Smirnov test was applied to check the normality of the data. A comparison-based analysis was then carried out to investigate the interrelationships between knowledge and attitude capabilities among family medicine doctors towards neonatal hearing screening. A p-value of ≤ 0.05 was considered statistically significant in all tests.This study enrolled 133 family physicians; 47.4% of them were specialists and 52.6% were residents. The majority of participants were female , with an age less than 30 years old , and had less than five years of experience . Most participants were practicing their role as family physicians under the umbrella of the Ministry of Health . Around 50% (65/113) of participants did not screen any child for hearing loss, and around 40% (53/113) did not confirm any case of childhood hearing loss during their last five years of experience. Conversely, around 51% (68/113) of participants have screened children for hearing loss during the child’s visit to the well-baby clinic to receive vaccinations, and to assess his or her development, the family doctor asks the parents whether their child underwent a hearing screening test in the hospital. If not, the child is referred to the hospital for the necessary evaluation according to the protocol followed in these institutions. Around 60% of participants (80/113) were able to diagnose one or more children with hearing loss during their last five years of experience .Concerning hearing auditory screening and management, for all participants, 91.7% (122/133) indicated the importance of neonatal hearing screening, 70.7% (94/133) indicated infant candidacy for a cochlear implant and only 33.1% (44/133) knew about the existence of the EIHD governmental program in KSA  was similar to that of specialists  based on the Mann-Whitney U test for their responses on the KAP questionnaire.In our study, participants determined that the most frequent factors associated with permanent hearing loss in children were a family history of hearing loss, meningitis, craniofacial anomalies and congenital syphilis. To a certain extent, such findings were close to those of Campos et al. (2014) that targeted paediatricians and neonatologists and the study by Yerraguntla et al. (2016) that targeted general physicians and medical interns ,29. CampChiefly, all healthcare professionals including family physicians must be aware of the time limits concerning the screening, diagnosis and intervention of hearing loss in paediatric patients. The triad of screening, diagnosis and intervention is essential to be conducted within a specific age to maintain and restore the functioning of their hearing and speech ,31. Our The vast majority (91.7%) of our participants indicated the importance of neonatal hearing screening. Similar findings with a high percentage of positive attitude toward the importance of neonatal screening tests were reported in other studies ,29,32. OIn 2016, screening for hearing loss was included in national screening programs aiming to reveal disorders that are quite common in the Saudi population. Since the hearing screening was recently approved by the Saudi high authorization, family medicine residents and specialists have not yet had sufficient training to improve their knowledge and clinical experience regarding hearing loss and the essence of early detection and management. Additionally, the process of implementing hearing screening programs has covered only 30 hospitals following the Ministry of Health in KSA, leading to the ineffective spread of knowledge about such programs as well as the importance of them among physicians and families. For these reasons, this current study showed that family physicians were not fully familiar with hearing screening and audiological management in KSA. Though approximately more than the half of the respondents, including family medicine residents and specialists, believed that hearing screening is required once the infant is born, they mistakenly think that hearing loss is not a common developmental disorder among infants. On the other hand, the harmful impacts of hearing loss summarized by language disorders, poor academic achievement and psychological issues were defined by most participants.The overall review of the results indicates that family doctors are aware of textbook knowledge about neonatal screening, but they showed a lack of comprehensive understanding of the practical details of paediatric hearing impairment, such as the reasons for conducting hearing screening, specialist referral, gold standard hearing tests, hearing loss interventions and the risk factors of postnatal hearing loss. These findings were consistent with other studies that discussed knowledge regarding the early identification and management of hearing loss in children among health care providers in general , and speMany participants were unconcerned regarding the role of hearing aids along with medication in curing unresolved conductive hearing loss in children. Despite this, clinicians frequently committed to using medications to treat conductive hearing loss, neglecting the necessity to combine antibiotics with hearing aids to treat children suffering from chronic otitis media . ChildreSimilarly, most of the respondents were not aware that children who wore hearing aids or cochlear implants are not capable to demonstrate speech and language competence adequately without speech therapy or hearing rehabilitation . It has Most family physicians were uncertain about the appropriate management when dealing with mild hearing loss in children. Additionally, they doubt the validity of behavioural measurements, such as visual reinforcement audiometry (VRA), to detect hearing impairment in the paediatric population over ABR. However, almost half of the participants agreed to consider hearing amplification as the treatment option for treating children with unilateral hearing loss. The guidelines for hearing aid fitting in children are more extended than those for adults to address unilateral and mild hearing loss patients as candidates for hearing amplification in order to ensure normal language and speech acquisition ,43, follIn clinical practice, the results of this study are useful to determine the weaknesses and strengths in the understanding of family practitioners about universal hearing screening and hearing loss management. This will be extremely effective in highlighting the areas of audiology that need to be considered as educational goals for increasing the knowledge and practice levels of family physicians. For instance, the responses to the study questionnaire revealed family physicians have good knowledge of the assessment, but often fail to properly reach a diagnosis. Although family practitioners are the most in demand medical subspeciality, based on the 2019 report from a physician recruitment institution called Merritt Hawkins , a familIn conclusion, this study reported that family physicians have a good general background about the benefits of EHDI programs and the management of hearing loss in the paediatric population. On the other hand, it further indicated insufficient knowledge in other domains of hearing loss including assessment, the presence of the EHDI governmental program in KSA and best therapeutic approach for persistent otitis media. The failure to acquire this information could be credited to improper implementation of EHDI programs and their limited coverage of certain urban regions in KSA. Thus, this study encourages involving family physicians in the process of neonatal hearing screening, diagnostic evaluation and intervention for hearing impairment in infants and children. As Ministry of Health organizations is responsible for delivering EHDI services, it is important that one of their actions is to reduce the harmful impact of paediatric hearing loss by preparing family practitioners to educate the families regarding the steps to ensure that their children receive adequate management if hearing loss is confirmed.S1 File(XLSX)Click here for additional data file. This paper was transferred from another journal. As a result, its full editorial history  may not be present.15 Feb 2021PONE-D-21-01254Knowledge, Attitude, and Practice of Hearing Screening and Management among Family Physicians in Kingdom of Saudi ArabiaPLOS ONEDear Dr. Alqudah,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Apr 01 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Jorge Spratley, MD, PhDAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. 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Nevertheless there are some comments and suggestions I recommend to be considered during the revision of the paper:I would suggest to include in the title the word children the Knowledge, Attitude, and Practice of Hearing Screening (in children) and Management among Family Physicians in Kingdom of Saudi Arabia, as the investigation relates to Hearing screening in childrenWhen speaking about language, it should be stressed we spoken language is considered (mostly in oposition to sign language or as a complement to sign language?)35: Methods: the total number of family physicians in the KSA that could have been enroled -that eligeble- should be mentioned75: ....it is done before the baby is discharged home127:They are also responsible for the identification of hearing loss and referring patients and discussing with parents the management plan; How exactly are family doctors responsible for the identification of the haering loss? What is their role? do they perform diagnostic tests? are all "refer" newborns referred do the family doctors for further diagnostic tests?139: (related to 35) How many family medicine physicians were actually invited to participate in the study, and what is the universe of family physicians in the KSA ?185:.... Conversely, around 51% (68/113) of participants have screened children for hearing loss and around 60% (80/113) were able to diagnose one or more children with hearing loss during their last five years of experience. How did they screened: did they participate in matternity based screening? and how did they diagnose hearing loss? do they performe diagnostic ABR or other audiological tests?209 : Concernig #CConcerningFigure 2 demonstrates the221: referral options followed by the family physician for children diagnosed with a confirmed hearing loss. Who confirmed the hearing loss? The flowchart of the various stages from screening to intervention proposed in the KSA program should be explained. It seems that after the established diagnose the child goes back to the family doctor and is then referred for audiological intervention?321: the role of surgical treatment in chronic otitis media in children should also be mentioned322-324: this statment is a double negative. Does it mean that children with choclear implant or hearing aids do well without speech theray or hearing rehabilition ?335:unilateral hearing loss or mild hearing loss impairs communication/hearing in noise as well as localizationReviewer #2: The article is quite interesting and practical. It has the potential to help create awareness in the population of family physicians, on the importance of screening and diagnosis of hearing losss. It would be interesting to dive deeper into this question: if physicians have good knowledge of the assessment, but often fail to complete the diagnosis, why does this occur? Is it lack of time or attention? Which programmes or interventions could the authors suggest to improve this intention-action gap?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  19 May 2021please check the file (revision) attached with other documents.Reviewer 1: I have incorporated all of your suggestions into my revision. they were helpful, thank you.Reviewer 2: I have incorporated all of your suggestions into my revision. they were helpful, thank you.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 24 May 2021PONE-D-21-01254R1Knowledge, Attitude, and Practice of Hearing Screening in Children and Management among Family Physicians in Kingdom of Saudi ArabiaPLOS ONEDear Dr. Alqudah,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Jul 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Jorge Spratley, MD, PhDAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):The revised manuscript addesses the reviewers' comments and is now tehcnically sound. However the English used on the new inserts in the paper is quite poor and contrasts with the rest of the paper. As such, the manuscript will only be considered for publication following a careful grammatical revision.[Note: HTML markup is below. Please do not edit.]https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  15 Jun 2021The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. The responses to all comments have been prepared and attached to the applicationAttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 23 Jun 2021PONE-D-21-01254R2Knowledge, Attitude, and Practice of Hearing Screening in Children and Management among Family Physicians in the Kingdom of Saudi ArabiaPLOS ONEDear Dr. Alqudah,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 07 2021 11:59PM. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):The title should be corrected to: Knowledge, Attitude and Management of Hearing Screening in children among Family Physicians in the Kingdom of Saudi ArabiaThe sentence in Abstract Line 39 and Material and Methods line 157 : "Based on the information provided by the Saudi commission for health specialties, the total number of family physicians was 2,700; however, we could not get their contact information from representative authorities, so we followed networking and snowballing sampling techniques and were able to target 300 and recruit 133 family physicians" is redundant to the previous sentence in the same sections and should be removed. Instead, it should be rewritten and sent to the end of the manuscript as a limitation of the study (133 out of 2700 family physicians).Correct grammar in Abstract line 27: "to develop normal spoking 28 language and academic skills during childhood".The new sequencing of the references in the revised main text is now difficult to follow, with numbers in red and black side-to-side[Note: HTML markup is below. Please do not edit.]https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  31 Jul 2021I have incorporated all of your suggestions into my revision. they were helpful, thank you.Attachmentresponse to reviewers.docxSubmitted filename: Click here for additional data file. 12 Aug 2021Knowledge, Attitude and Management of Hearing Screening in children among Family Physicians in the Kingdom of Saudi ArabiaPONE-D-21-01254R3Dear Dr. Alqudah,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Jorge Spratley, MD, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Thanks for the time and effort to correct the manuscript. Congratulations for achieving the level of publication at Plos OneReviewers' comments: 17 Aug 2021PONE-D-21-01254R3 Knowledge, Attitude and Management of Hearing Screening in children among Family Physicians in the Kingdom of Saudi Arabia Dear Dr. Alqudah:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. 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Hearing impairment (HI) is a highly heterogeneous genetic disorder and is classified into nonsyndromic  and syndromic  forms. Variations in GJB2 gene are the leading cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in several populations worldwide. This study was carried out to investigate the prevalence of GJB2 variations in severe-to-profound hearing impaired families of Southern Punjab of Pakistan. Ten families segregating ARNSHL were recruited from different areas of the region. Sanger sequencing of GJB2 coding region was carried out. In two out of ten families, NM_004004:c.*71G>A (p.(Trp24*)) and NM_004004:c.358_360del (p.(Glu120del)) homozygous variants were identified as the cause of hearing loss. Our study showed that GJB2-related hearing loss accounts for at least 20% of all cases with severe-to-profound hearing loss in the Southern Punjab population of Pakistan. Deafness is a prevalent sensory disorder affecting 1 in 650 infants worldwide, with a prevalence of 3.5/1000 in teenagers and 2.7/1000 in children making it the most common hereditary sensory impairment [https://hereditaryhearingloss.org/recessive-loci). Consanguineous marriages are very common in Pakistan, making this population a valuable source for studying genetic disorders [GJB2 variants in prelingual deafness [GJB2 [GJB2 are also the most primary cause of nonsyndromic hearing loss in South Asia [GJB2 frequency has been reported in north of Iran [GJB2 gene have been detected and reported; some are recurrent variations while others are not so prevalent. The variant spectrum diverges significantly among populations and demonstrates ethnic biases, e.g., c.35delG is common among caucasoids with 1 in 51 carrier rate [GJB2 variants in different populations worldwide is significantly high, thereby highlighting the clinical significance of this gene for genetic testing.It is a multifactorial disorder associated with environmental and genetic causes. About 50–60% of deafness is due to genetic factors. Clinically, deafness can be classified into nonsyndromic and syndromic deafness. Nonsyndromic deafness is isolated deafness with no other clinical manifestations, while syndromic deafness is associated with other metabolic or physiological conditions. Nonsyndromic deafness has a prevalence of 70% and is highly heterogeneous . In caseisorders . Moreoveisorders . It is a of Iran . Severalier rate  while caier rate . Carrierier rate  and carrier rate . The oveGJB2 in hearing impaired population of Southern Punjab of Pakistan has not been studied. Therefore, the present study was conducted to determine the prevalence of GJB2 gene in Southern Punjab of Pakistan. Sequencing of coding exon of GJB2 revealed two disease-associated variants; NM_004004:c.*71G>A (p.(Trp24*)) in NKDF01 and NM_004004:c.358_360del (p.(Glu120del)) in NKDF08. This research will help in genetic counselling of these families to avoid carrier to carrier or carrier to affected marriages, which will result in a decrease in the deaf population in Southern Punjab. Moreover, GJB2 variants profiling for the hearing impaired population of Southern Punjab of Pakistan will be a valuable resource for the development of molecular genetic screening tests in the future.Southern Punjab is a loosely defined territory in Punjab Province of Pakistan that encompasses the civil divisions of Bahawalpur, Multan, and Dera Ghazi Khan. It accounts for around 52% of the province’s total land and 32% of its population. It has a population of more than 34,743,590 inhabitants. To the best of our knowledge, the prevalence of GJB2 sequencing.This research work was approved by the Institutional Research Ethics Committee (IREC) of Govt. College University Faisalabad, Pakistan. For this study, 10 punjabi severe to profound deaf families with consanguineous marriages were recruited from Dera Ghazi Khan and Rajanpur districts of South Punjab of Pakistan. Written consent forms were signed by all participants after receiving information about the study. A total of 37 deaf individuals and 72 normal siblings/parents were enrolled for Multiple family members, including elders, were interviewed to obtain medical history and rule out the environmental and syndromic deafness. A physical examination was also carried out to confirm signs and symptoms of night blindness, goiter and skin pigmentation for some of the affected participants. Two affected and one normal individual from each family were subjected to otoscopic examination and pure tone audiometry was performed at various threshold levels ranging from 250 Hz to 4 kHz . Tandem ® polypropylene tubes containing 400 ul of 0.5M EDTA. The genomic DNA was isolated from all blood samples using the non-organic method [10 ml of blood was collected from each person that was properly labelled and stored in 50 ml of Sterilinc method . IsolateTGTGCATTCGTCTTTTCCAG-3’ and 5-‘GGGAAATGCTAGCGACTGAG-3’) were designed, synthesized, and were subsequently used for amplification of the coding exon of GJB2. ExoSAp treatment was applied to the amplified products and sequencing was done using the Big Dye (Big Dye Terminator v3.1 Cycle Sequencing Biosystems® Kit). Genetic Analyzer 3730 (Applied Biosystems Inc) was used to run the sequencing products and data was analyzed with Chromas software version 2.6.6 (Technelysium Pty Ltd).DNA of one affected from each pedigree was subjected to Sanger sequencing. Primers (5‘-GJB2 results in the identification of two reported disease associated variants NM_004004:c.*71G>A (p.(Trp24*)) and NM_004004:c.358_360del (p.(Glu120del)) in NKDF01 and NKDF08 respectively.Sequencing of This family was collected from Dera Ghazi Khan and had nine deaf individuals in four sibships. Eight affected individuals , three unaffected individuals  and their parents  were enrolled for this study .Physical examination did not exhibit the signs and symptoms of skin pigmentation, goiter and night blindness in any individual of this family. Furthermore, deafness in this family was not segregating with any other abnormality. No vestibular dysfunction was observed by tandem gait and rhomberg tests. Pure tone audiometry at 250, 500, 1000, 2000 and 4000 Hz for individuals IV:9 and IV:10 revealed severe to profound hearing loss .GJB2 gene identified a recurrent disease associated variant NM_004004:c.*71G>A (p.(Trp24*)) in deaf individuals of this family , six normal individuals  and their parents  were ascertained .Hearing impairment was segregating without any other anomaly in the affected persons of this family. The signs and symptoms of goiter, skin pigmentation and night blindness in any individual of this family were not recognized. Moreover, tandem gait and romberg tests did not reveal the vestibular dysfunction. Pure tone audiometry demonstrated a severe to profound hearing loss in individuals IV:10 and IV:11 at 250, 500, 1000, 2000 and 4000 Hz . Sanger GJB2 are the leading cause of hearing loss in Pakistani families [GJB2 gene in Pakistani population [GJB2 sequence variations are the primary contributor of deafness in Pakistani individuals. We did not perform the CNV analysis on the deaf families, however sanger sequencing revealed a c.*71G>A (p.(Trp24*)) change in NKDF01 family out of ten severe to profound hearing loss families. Hence, suggests a prevalence of 10% in this population. p.Trp24* frequency is also high in the Indian population reaching up to 95% [Variations in families . Differepulation –16. Our p to 95% –19. Prevp to 95% ,20. HearGJB2 variant c.358_360del (p.(Glu120del)) was observed in NKDF08 family, which exhibited a prevalence of 10% in Southern Punjab. Previously, this change was reported in only one Pakistani family [GJB2 variants, the remaining families will be investigated further and subjected to exome sequencing to identify the causative genes.Another i family . Howeveri family ,22. Morei family . The comGJB2 related hearing loss in severe to profound deaf families is high in Southern Punjab, i.e., 20%. Sequencing of coding exon of GJB2 revealed two disease-associated variants; NM_004004:c.*71G>A (p.(Trp24*)) in NKDF01 and NM_004004:c.358_360del (p.(Glu120del)) in NKDF08 out of ten families. As published earlier, p.Trp24* is the common change in the Pakistani families, while p.Glu120del has been identified in only one Pakistani family. Determination of reported variants along with other frequent variants will help in genetic counselling and family planning for these families, which will result in a decrease in the deaf population in Southern Punjab. Early detection of common GJB2 variants in infants will enable us to adopt the multiple interventional strategies in time. Moreover, GJB2 variants profiling for the hearing impaired population of Southern Punjab of Pakistan will be a valuable resource for the development of molecular genetic screening tests in the future.Our results suggest that the prevalence of S1 Table(DOCX)Click here for additional data file.


This cohort study assesses the association between hearing impairment and decline in physical function in terms of balance and walking endurance in a group of community-dwelling older adults in the US. Is hearing impairment associated with poorer physical function, reduced walking endurance, and faster decline in physical function?In this population-based cohort study of 2956 older adults in the US, participants with hearing impairment had significantly poorer physical function , worse walking endurance , and faster declines in physical function over time compared with those with normal hearing.This study’s findings suggest that because hearing impairment is a prevalent but treatable condition, it may be a target for interventions to slow the decline of physical function associated with aging. Hearing impairment, a common treatable condition, may contribute to poorer physical function with aging.To assess whether hearing impairment is associated with poorer physical function, reduced walking endurance, and faster decline in physical function.In this cohort study, cross-sectional and longitudinal analyses were performed using data from the 2011 to 2019 period of the Atherosclerosis Risk in Communities study, a population-based study of community-dwelling adults at 4 sites in the US.Hearing thresholds (per 10 dB) assessed with pure tone audiometry and categorized as normal hearing or mild, moderate, or severe hearing impairment.Physical function was assessed using the short physical performance battery (SPPB), with composite scores ranging from 0 to 12. A composite score of 6 or less and a score for each component  of 2 or less indicated poor performance. Walking endurance was assessed using a 2-minute fast-paced walk test. Tobit regression models adjusted for sociodemographic factors and medical history were used to calculate the mean differences in SPPB composite scores; logistic regression models, to estimate the odds ratios (ORs) of low SPPB composite and component scores; and linear mixed-effects models, to estimate the mean rate of change in SPPB composite scores over time.Of the 2956 participants  who attended study visit 6 between 2016 and 2017, 1722 (58.3%) were women, and 2356 (79.7%) were White. As determined by pure tone audiometry, 973 (33%) participants had normal hearing, 1170 (40%) had mild hearing impairment, 692 (23%) had moderate hearing impairment, and 121 (4%) had severe hearing impairment. In the Tobit regression model, severe hearing impairment was associated with a lower mean SPPB score  compared with normal hearing. In fully adjusted logistic regression models, hearing impairment was associated with higher odds of low physical performance scores . Over time , participants with hearing impairment had faster declines in SPPB compared with those with normal hearing . In adjusted models for walking endurance, participants with moderate or severe hearing impairment walked a mean distance of –2.81 m  and –5.31 m  than those with normal hearing, respectively, during the 2-minute walk test.In this cohort study, hearing impairment was associated with poorer performance, faster decline in physical function, and reduced walking endurance. The results of the longitudinal analysis suggest that hearing impairment may be associated with poorer physical function with aging. Whether management of hearing impairment could delay decline in physical function requires further investigation. Moreover, studies of the association between hearing impairment and walking endurance—the ability to walk longer distances, an early factor associated with disability15—are scarce and have inconsistent findings.16Associations between self-reported hearing impairment and poorer physical function have been reported previously.Therefore, we investigated the association of hearing impairment with physical function and walking endurance in a cohort of community-dwelling older adults in the US. We hypothesized that participants with hearing impairment would have poorer concurrent physical function and walking endurance and a faster decline in physical function over approximately 8 years compared with participants with normal hearing.17 an ongoing study of community-dwelling adults that enrolled 15 792 participants between 1987 and 1989 at 4 sites in the US . Hearing was assessed during an in-person examination at visit 6 (2016-2017). We used physical function data collected during clinic visits 5 (2011-2013) through 7 (2018-2019). The institutional review board at each site approved the ARIC study, and all participants provided written informed consent. Consent for the ARIC study covered use of the data for all analyses approved by the study publications committee, and data were deidentified. The present study was approved by the institutional review board of the Johns Hopkins Bloomberg School of Public Health. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines for cohort studies.18For this cohort study, we used a population from the Atherosclerosis Risk in Communities (ARIC) study,Among 4003 participants who attended visit 6, 3395 had complete evaluation of physical function. After exclusion of 103 participants with missing information for the hearing assessment and 336 participants with missing information for the covariates of interest , the population for cross-sectional physical function analyses included 2956 participants. Longitudinal data  were available for 2869 participants . Walking endurance of 2961 participants was evaluated at visit 6; 2532 had complete data  and were included in the analytic population for walking endurance analysis.19Pure tone audiometry, assessed at ARIC visit 6 (2016-2017), was completed in a soundproof booth using insert earphones  and an Interacoustics AD629 or Equinox audiometer (Interacoustics A/S). Measurement of air conduction was completed at standard octaves from 500 to 8000 Hz. For each frequency, the hearing threshold was recorded in decibels hearing level (dB HL). We calculated a 4-frequency  pure tone average for each ear and modeled a continuous measure of hearing (better-hearing ear pure tone average [BPTA]) scaled to 10 dB HL, with higher BPTA indicating worse hearing function. Hearing was also categorized according to World Health Organization standards as normal hearing (BPTA≤25 dB HL), mild hearing impairment (BPTA of 26-40 dB HL), moderate hearing impairment (BPTA of 41-60 dB HL), or severe hearing impairment (BPTA>60 dB HL).20 The composite SPPB score  was the sum of the 3 scores. We examined the composite SPPB score continuously and used a binary threshold for poor physical performance (SPPB composite score ≤6).20 For each SPPB component, we also used a binary threshold (SPPB component score ≤2) to indicate poor performance, consistent with prior work  was used to assess physical function, and a fast-paced 2-minute walk (TMW) test was used to measure walking endurance. The SPPB was measured at visits 5, 6, and 7 and included 3 components: balance, gait speed, and chair stands. Each component was scored on a scale of 0 to 4 points, with higher scores indicating better physical function . We calculated gait speed (in meters per second) using the trial with the faster result. For the chair stand test, participants stood up from a chair and sat back down with arms crossed 5 times as quickly as possible. Gait speed and time to complete the chair stands were also examined continuously.The balance test consisted of holding 3 standing positions for 10 seconds each: side-by-side (easiest), semi-tandem, and full-tandem (hardest)The TMW test was administered on a 15.24-m (50-ft) course at visit 6 only. Participants were instructed to walk as fast as they could for 2 minutes. The distance covered was recorded in meters. Participants unable to complete the SPPB 4-m walk test unaided were excluded from the TMW test, leaving 2535 participants for this analysis.17 ; body mass index; occupational noise exposure (very loud sounds for >10 hours/wk in the workplace); and smoking status  were assessed at visit 6. Using data from the annual follow-up interviews, we created a multimorbidity index to control for the presence of chronic conditions. This index was modeled after the Charlson-Deyo Comorbidity Index and included myocardial infarction, stroke, intermittent claudication, heart failure, chronic obstructive pulmonary disease (emphysema or bronchitis), chronic kidney disease, diabetes, and cancer. Stroke and cancer were assigned double weight (counted as 2). No data were available regarding peptic ulcer disease, AIDS or HIV infection, liver disease, or rheumatologic disease. Dementia was not included in the index because we considered it to be a mediator and not a confounder in the association between hearing and physical function. In sensitivity analyses, we tested whether including dementia in the index had any effect on our results. All covariates were assessed at visit 6 and treated as fixed variables.Age; sex; educational level ; race–study site, owing to the correlation between race and site in the ARIC study2 and analysis of variance tests as appropriate. We assessed the cross-sectional associations (at study visit 6) between continuous hearing, by 10-dB HL increments, and physical function and walking endurance using 2 models. Model 1 was adjusted for age, sex, and race–study center, and model 2 was adjusted for those variables as well as educational level, body mass index, occupational noise, smoking status, and the multimorbidity index.We evaluated the differences in demographic characteristics and medical conditions across hearing categories using χBecause of the ceiling effect of the SPPB (approximately 50% of participants had a score ≥10), we used Tobit regression models to calculate the mean differences in SPPB composite scores. We used logistic regression analysis to estimate the odds ratios (ORs) of low SPPB composite scores (≤6) and low scores for each SPPB component (≤2). We used linear regression analysis to assess differences in gait speed and time to complete chair stands as continuous measures.P < .05 was considered statistically significant.We also used linear regression analysis to assess the cross-sectional (visit 6) association between hearing and walking endurance . We performed all analyses using Stata, version 15 (StataCorp LLC). Two-sided For longitudinal analyses  for all hearing groups. Also, among 2520 participants, we examined the association between hearing (per 10-dB HL increments and across hearing categories) and grip strength  using linear regressions adjusted for covariates in model 2. In addition, we built an alternative multimorbidity index including dementia and used it for adjustment in model 2 in lieu of the index without dementia.A total of 2956 participants attended study visit 6 . The meaP < .001), low balance scores , and low gait speed scores . The proportions of participants with a low chair stand score  and the mean (SD) distance covered during the TMW test  were not significantly different across hearing groups.In a descriptive analysis , compareThe differences in continuous measures of SPPB tests  at visit 6, per 10-dB HL increase, and across hearing categories  are presented in When hearing was modeled categorically , in modeIn model 2 , during P = .002) and BPTA × time 2 . Per 10-dB HL higher BPTA, the annual change in SPPB was −0.002 point/y  faster during the first 5 years and −0.008 point/y  faster from year 5 onward. Moreover, during the first 5 years, the rate of decline for those with normal hearing was −0.16 point/y ; in comparison, those with moderate and severe hearing impairment had significantly faster rates of decline  ; interacIn ordinal logistic regression models, the odds of being in the next lower SPPB score were higher with worse hearing for all outcomes, but the OR was not statistically significant for the chair stand test  were clinically meaningful according to previous literature.24 found an association between hearing impairment and poorer physical function (SPPB scores) in a subsample of 250 ARIC participants. In that analysis, there were no associations of hearing impairment with poorer balance and slower gait speed, which might be explained by the smaller sample size. Chen et al12 found associations between hearing impairment and a faster decline in physical function over time in the Health Aging and Body Composition Study. Our findings are consistent with and expand on findings from this prior work. We studied each SPPB component separately, identifying stronger associations between hearing and balance and gait speed than between hearing and chair stands  similar to that (16 m) reported by Valjean et al.16 However, their study was limited to Finnish women, and the findings were not statistically significant.We found an association between hearing and reduced walking endurance. Tests of walking endurance, which correlate with cardiorespiratory fitness,29could contribute to our findings. However, we adjusted for cardiovascular and sociodemographic risk factors, and our estimates were only slightly attenuated. Second, the inner ear hosts both the vestibular and auditory systems. Thus, damage to the inner ear could cause dysfunction in both systems, leading to an association between poorer hearing and balance. Although previous findings suggest that each sensory system has independent risk factors,30 we cannot rule out this mechanism because we were unable to adjust for vestibular function. Third, the association between hearing and physical function may be mediated through reduced cognitive resources,32 depression, social isolation, and reduced life space,36 resulting in less physical activity.38 These mechanisms would be particularly relevant for our findings related to walking endurance. In sensitivity analyses, we observed that when dementia was included in our adjustment variables (part of the multimorbidity index), it attenuated the association between severe hearing impairment and low gait speed scores. This finding is supportive of our theory that cognition is a mediator of the association between hearing and physical function. Fourth, hearing impairment may be associated with reduced physical function via reduced perception of auditory cues from the environment, which contribute to balance.25Multiple mechanisms could explain the associations found in the present study. First, a common cause  of hearing and physical impairment12 Randomized clinical trials to elucidate the effect of hearing aid use on physical function are warranted.39We found no differences in physical function or walking endurance in association with hearing aid use , performance-based tests of physical function and walking endurance, and repeated measures of physical function over 8 years. Our findings were robust to the adjustment for confounders and consistent across different statistical approaches .41; thus, hearing at visit 5 would be correlated with hearing at visit 6. Also, the physical function at visit 5 would be unlikely to affect hearing acuity at visit 6. Moreover, we observed a significant decline in physical function between visit 6 and visit 7 in our models for participants with mild and moderate hearing impairment (This study also has limitations. First, the hearing assessment was conducted at visit 6; thus, in longitudinal analyses, we included measures of physical function that preceded the assessment of hearing. However, age-related hearing impairment progresses slowly at a rate of 1 to 2 dB HL per yearpairment . Second,pairment . Under tOnly 121 participants had severe hearing impairment. The relatively small sample size of this group could have reduced our statistical power to detect differences in physical functioning between participants with normal hearing and severe hearing impairment. However, we detected a significant association across multiple outcomes except in sensitivity analyses, in which the odds of having a low gait speed score were higher for participants with severe impairment. Although the study population included some Black participants, the sample was predominantly White. The lack of other racial/ethnic groups limits the generalizability of our findings. Further research in cohorts that include larger numbers of individuals of other racial/ethnic groups is warranted.In this cohort study, hearing impairment was associated with poorer physical function, reduced walking endurance, and faster decline in physical function over time. Because hearing impairment is amenable to prevention and management, it potentially serves as a target for interventions to slow physical decline with aging.


The intestinal flora is correlated with the occurrence of colorectal cancer. We evaluate a new predictive model for the non-invasive diagnosis of colorectal cancer based on intestinal flora to verify the clinical application prospects of the intestinal flora as a new biomarker in non-invasive screening of colorectal cancer.Subjects from two independent Asian cohorts  were included. A probe-based duplex quantitative PCR (qPCR) determination was established for the quantitative determination of candidate bacterial markers.Prevotella copri (Pc), Gemella morbillorum (Gm), Parvimonas micra (Pm), Cetobacterium somerae (Cs), and Pasteurella stomatis (Ps). A predictive model with good sensitivity and specificity was established as a new diagnostic tool for colorectal cancer. Under the best cutoff value that maximizes the sum of sensitivity and specificity, Gm and Pm had better specificity and sensitivity than other target bacteria. The combined detection model of five kinds of bacteria showed better diagnostic ability than Gm or Pm alone . These findings were further confirmed in the independent cohort II. Particularly, the combination of bacterial markers and fecal immunochemical test (FIT) improved the diagnostic ability of the five bacteria  for patients with colorectal cancer.We screened through the gutMEGA database to identify potential non-invasive biomarkers for colorectal cancer, including Fecal-based colorectal cancer-related bacteria can be used as new non-invasive diagnostic biomarkers of colorectal cancer. Simultaneously, the molecular biomarkers in fecal samples are similar to FIT, have the applicability in combination with other detection methods, which is expected to improve the sensitivity of diagnosis for colorectal cancer, and have a promising prospect of clinical application. In recent years, colonoscopy has been widely used in the diagnosis and screening of colorectal cancer. However, non-invasive screening becomes increasingly important due to the psychological and economic factors of the patients. Although blood/plasma biomarker detection is partially used in some ways, the detection effect is limited, indicating significant differences between studies . SimilarWith the gradual development of intestinal flora research and the gradual popularization of fecal microbiota transplantation (FMT) and other technologies, the detection technology of intestinal flora has been relatively mature . In the Prevotella copri (Pc), Gemella morbillorum (Gm), Parvimonas micra (Pm), Cetobacterium somerae (Cs), and Pasteurella stomatis (Ps)  . Based oThe data, analytical methods, and study materials for the purposes of reproducing the results or replicating the procedures can be made available on request to the corresponding authors.n = 439) were collected from two separate cohorts: cohort I from 2018 to 2020, The First Affiliated Hospital of Soochow University, 318 subjects, consisting of 206 colorectal cancer patients  and 112 normal controls ; and cohort II from 2018 to 2020, The First Affiliated Hospital of Soochow University, 121 subjects, consisting of 67 patients with colorectal cancer  and 54 normal control groups   . When thfemales) . ParticiFecal samples were thawed on ice and DNA extraction was performed using the QIAamp DNA Stool Mini Kit according to the instructions of the manufacturer (Qiagen). Extracts were then treated with DNase-free RNase to eliminate RNA contamination. DNA quality and quantity were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). Primers and probes used in this study are shown in target − Cqcontrol and relative abundance = Power . The nucleotide sequences of primers and probes are shown in qPCR amplification is performed in a 20-μl reaction system of TaqMan Universal Master Mix II  containing 0.3 μmol/L of 96-hole reaction sheets and 0.2 μmol/L of reaction sheet seals. The thermal cycler parameters of an ABI PRISM 7900HT sequence detection system were 95°C 10 min × 45 cycles. Each experiment included positive/reference and negative controls (water as a template). Three measurements were repeated for each sample. The qPCR data were analyzed using sequence detection software  . Data anEach participant received a Medline iFOB  collection tube. Medline iFOB is a qualitative FIT product exempted by the Clinical Laboratory Improvement Act (CLIA). The lower limit of hemoglobin detection set by the manufacturer was 50 g/g. The feces were exposed to ambient temperature for no more than 48 h between emptying and treatment. Each sample was processed in accordance with the instructions of the manufacturer, including verification of activated internal controls. The results of each test were interpreted by two members of the research team .t-test or non-parametric Mann–Whitney U test. All tests were bilateral, and P <0.05 indicated significant statistical differences. In this study, the software R 3.4.3  was used to construct the logistic regression forecasting model, and charts were analyzed using the R software, where the receiver operating feature (ROC) curve was drawn using the Hiplot visual drawing website (https://hiplot.com.cn/). According to the actual flora abundance of the case and predicted situation of the model, the ROC curve was drawn. The area under the curve (AUC) and the differentiation size of the model were also evaluated. The larger the AUC value of the model, the better the prediction effect. Generally, AUC ≥0.8 indicates good differentiation and clinical significance, and AUC <0.6 indicates poor clinical application value. The best truncation value  was determined by the ROC curve and calculated for sensitivity, specificity, diagnostic accuracy, diagnostic ratio, positive prediction rate, positive result likelihood ratio, negative result likelihood ratio at the best cutoff value, and 95% confidence interval. The net gain of the prediction model is evaluated by the predictive model’s decision curve . DCA, as a simple way to evaluate clinical predictive models, diagnostic trials, and molecular markers, is able to better estimate the model for clinical value than AUC by integrating patient or decision-maker preferences into the analysis  for data statistical analysis and logical regression analysis. Classification data were presented by frequency (percentage), chi-square analysis, or Fisher exact probability method. Continuous data were presented as mean ± standard deviation, and group differences were analyzed using analysis . After dhttp://gutmega.omicsbio.info/). Log2 > 3 and Log2 > 3 and P <0.05 were set. The associated abundance differences  were obtained through screening studies. The Venn map showed that there were 13 species of intestinal flora in colorectal cancer and normal human that have significant differences in abundance and 9 species in colorectal cancer and adenoma, where the Pc, Gm, Pm, Cs, and Ps bacteria achieved statistical differences simultaneously in both groups of controls  of 92.00%, and positive predictive value (PPV) of 82.30%. The sensitivity of Pm to this cohort was 93.20%, specificity was 58.04%, NPV was 82.28%, and PPV was 80.33%. This was further validated in the independent cohort II of 67 colorectal cancer patients and 54 healthy controls. The relative abundance of Gm and Pm in colorectal cancer patients remained significantly higher than in the healthy control groups  than other combinations (two to four markers) or Gm (0.799) or Pm (0.791) alone. The relative abundance of the five bacteria in patients with colorectal cancer was significantly higher than that in healthy control groups  can distinguish patients with colorectal cancer from healthy controls, with a sensitivity of 67.96%, specificity of 89.29%, NPV of 60.24%, and PPV of 92.11%, which show better diagnostic performance than Gm or Pm alone .In line with the data after sequencing, the combined use of the bacterial markers tested showed better diagnostic performance than Pm alone Table 1.P < 0.001, Gm or Pm alone. Therefore, the combination of Gm, Pm, Pc, Cs, and Ps improves the diagnostic ability of Gm and Pm to identify colorectal cancer and healthy control groups.The diagnostic performance of the five bacteria was further demonstrated in the independent cohort II. The combination of the five bacteria also showed an increase in AUC . Pairwise comparison of the ROC curve showed that the combinations of five bacteria were significantly superior to Gm or Pm alone for the diagnosis of colorectal cancer (both P < 0.05). FIT was slightly related with TNM staging (P = 0.076), while the relative abundances of the five bacteria or Gm and Pm were independent of TNM staging. Assessment of colorectal cancer detection in the TNM staging subgroup showed that the quantification of bacterial markers was much more sensitive than FIT in stage I. Similarly, in stage II and III, we observed more prominent detection rates by bacteria than FIT . To facilitate the clinical application of the colorectal cancer risk prediction model for the combination of five bacteria, we further constructed a nomogram based on this model . The prediction accuracy of the model was not significantly reduced with the change of the population, which proved the reliability of the colorectal cancer risk model based on the five target bacteria.To further demonstrate the clinical application prospects of the five target bacteria, cohort I was used as the training group to construct the colorectal cancer risk model, and cohort II was analyzed as the testing group to test the feasibility of the cohort I-dependent testing model . The cliIn recent years, colonoscopy has been widely used for colorectal cancer diagnosis and screening. However, non-invasive screening becomes increasingly important due to the psychological and economic factors of the patients. With the gradual development in the study of intestinal microbiota and the gradual promotion of FMT and other technologies, the detection technology of intestinal microbiota has been relatively mature.The occurrence and development of many clinical diseases are closely related to the change of microbiota abundance . The appPc, Gm, Pm, Cs, Ps) with significant changes in abundance during the tumorigenesis of colorectal cancer were searched through the gutMEGA platform  is generally regarded as an oral pathogen, and there is increasing evidence that Fusobacterium infection is common in colorectal carcinoma. It may be a promising biomarker that is predictive of the clinical outcome in patients with CRC  and FIT improved the diagnostic ability for CRC patients, which has broad application prospects and is worthy of further study.In previous research, with CRC . In ordeValidation of potential microbial markers in a large group of people is an important means of non-invasive screening of colorectal cancer in the general population, with high requirements for accuracy, stability, and practicability . AlthougThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. The patients/participants provided their written informed consent to participate in this study.XZ and SS conceived and supervised the project. YY, HN, and XW contributed to the experimental design, prepared the figures, and wrote the manuscript. All authors performed the experiments and interpreted the results. All authors discussed the results and approved the manuscript.This work was supported by the National Nature Science Foundation of China (81974375) and Project of National Science Foundation of Jiangsu Province (BK20161225).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with hearing loss have increased difficulties while navigating communication, especially complex conversations that take place in healthcare settings. Patients with hearing disabilities may often find themselves in noisy reception areas or busy emergency departments where their confidentiality is compromised for the sake of communication, resulting in suboptimal care. The reverse stethoscope technique is a tool that healthcare professionals can use to enhance communications to optimize patient care and maintain confidentiality. We describe a case of an 83-year-old male with bilateral hearing loss whose hearing disability became a major obstacle in his access to healthcare in the inpatient setting. With the help of the reverse stethoscope technique, the patient was able to understand his hospital course, diagnosis, and treatment options and was able to actively participate in making an informed decision. Communication is a foundational aspect of patient-centered care. Effective patient-provider communication allows for the transfer of information regarding medical symptoms, diagnosis, and treatment options and facilitates shared decision-making guided by patients’ values . With stPatients with hearing disabilities are unable to navigate the healthcare system as easily as their hearing-able counterparts. The 2019 Global Burden of Disease Study found that hearing loss is the third leading cause of disability globally; about 1.57 billion people are estimated to suffer from it. In the United States, it is estimated that about one-third of adults from ages 61 to 70 and more than 80 percent of those older than 85 live with some form of hearing loss . The assHearing loss directly impacts patients’ abilities to engage and maintain oral communication and is associated with overall negative health outcomes, increased risk of hospitalization, and mortality . CommuniDespite data on the increasing prevalence of hearing loss amongst an aging population, literature that addresses the serious implications of hearing disabilities on the quality of healthcare is scarce , 2, 4.An 83-year-old male with medical history pertinent for bilateral hearing loss presented to the urgent care clinic for a post-discharge follow-up visit. The patient was recently admitted for post obstructive pneumonia with image findings concerning for malignancy. During the admission, computed tomography angiography of the chest showed obstruction of the left lower segmental bronchus with associated atelectasis - secondary to either mucous plugging vs. underlying malignancy. Discharge documentation indicated that during a conversation to establish goals of care, the patient had indicated that he was not interested in any further workup for malignancy.During the follow-up visit, the recent hospital admission and diagnoses were reviewed with the patient. Although the patient’s hearing aids were intact, it became clear that he was having difficulty with the conversation. The patient revealed that the batteries to his hearing aids had not been working for some time. Communication enhancement techniques such as effective positioning, clear and slow pronunciation, and body language were used to improve the conversation. Even with the use of enhancement techniques, the patient was unable to understand what was being communicated. Finally, the reverse stethoscope technique was employed: after patient consent, the earpieces of the stethoscope were cleaned and placed into his ears. Comfortable and effective placement was ensured. The provider then spoke quietly into the diaphragm of the stethoscope with slow and clear speech, using the tool as a microphone.The reverse stethoscope technique aided with effective communication and ensured confidentiality. After reviewing his recent hospital admission and imaging results, the patient decided to pursue further workup for malignancy and stated that he would be willing to discuss the risk and benefits of associated therapy options.Hearing loss is a medical disability and health disparity: individuals with a sensory deficit will systematically experience greater obstacles to healthcare and are therefore in a position where they may inevitably receive suboptimal care , 5. PrioHealthcare professionals can help to further health equity or to help attain a higher level of health for those with hearing loss by being observant of their patients’ needs and adapting their communication skills to those of their patients , 5. ProvThe COVID-19 pandemic and widespread use of masks have made it increasingly difficult for patients with a hearing disability to communicate as they can no longer rely on the tool of lip-reading and facial expression , 7. In sHearing loss severely limits the ways in which a patient can access healthcare and affects the quality of healthcare received. The purpose of this case report was to highlight one such case: because of his hearing disability, our patient was initially unable to engage in effective communication with his providers which led to a failed understanding of his medical diagnosis and rejection of a therapeutic option. With the help of the reverse stethoscope technique, the patient was able to comprehend his diagnosis and treatment options and was able to actively participate in making an informed decision regarding his care.Hearing loss is a health disparity as patients with hearing disabilities experience increased obstacles to access healthcare and are at an increased risk of receiving suboptimal care. Healthcare professionals can help to attain a higher level of care for those with hearing disabilities by being observant of their patients’ needs. The reverse stethoscope technique is an efficient and effective method to improve the quality of care for hearing-impaired patients while also maintaining patient-physician confidentiality in a busy healthcare setting.


A hallmark of end-of-life (EOL) is reduced functional ability. However, the impact of dementia and other comorbidities on EOL decline for older adults is less well understood. We estimated the effect of having dementia and comorbidities on activity of daily living (ADL) scores using the 2000-2012 Health and Retirement Study. We identified 5,853 people over age 65 who died and predicted monthly ADL impairments in the last 4 years of life controlling for dementia and other characteristics. Stroke and obesity were associated with significantly higher ADL scores, regardless of dementia status. However, if both dementia and either stroke or obesity were present, dementia was associated with significantly higher ADL scores approximately 1-4 years before death. Functional decline occurred closer to death if they had these conditions and no dementia. Differences in function when patients have dementia and comorbidities may affect understanding of survival time and access to appropriate care.


Discrimination is linked to poor health outcomes, but most studies examine young or midlife populations. We assessed associations between discrimination and disability in African Americans. The Detroit Areas Study Everyday Discrimination Scale quantified experiences of interpersonal mistreatment. Separate Cox-proportional hazards models tested the associations between baseline discrimination and incident mobility, activities of daily living (ADLs), and instrumental activities of daily living (IADLs) disability, adjusting for age, sex, education, BMI, smoking, depressive symptoms, and vascular diseases. At baseline, 441, 674, and 469, participants were initially free of mobility, ADL, and IADL disability, respectively, and 257, 185, and 269 new cases of mobility, ADL, and IADL disability were observed over approximately 8.5 years. Discrimination was associated with higher risk of ADL disability  but no other disability type. Everyday discrimination is associated with risk of ADL disability.