In South Africa, primary health care is the first point of contact with the health system for at least 85% of the population, yet early hearing detection and intervention continues to be elusive in these settings. Nurses at community level may, therefore, be missing an opportunity to identify prelingual infants with hearing losses and alter their developmental trajectory.To determine primary health care nurses’ experiences, practices and beliefs regarding hearing loss in infants.The study was conducted in the eThekwini District of KwaZulu-Natal, South Africa.A descriptive survey was used with quantitative methods of analysis. Fourteen primary health care clinics from the eThekwini district were selected, from which 75 nurses participated by completing a self-administered questionnaire.At least one-third of primary health care nurses had never screened a child for hearing loss, and most clinics did not have access to basic hearing screening equipment or materials. Only 49% of nurses had access to an otoscope, while 31% used the Road to Health Development screener to check for hearing loss. None of the clinics had access to an otoacoustic emission screener nor the Swart questionnaire. Although nurses reported that they would refer to audiology services for some of the risk factors, as indicated on the Joint Committee on Infant Hearing (JCIH) 2007 list, they were less likely to refer if the child was in a neonatal intensive care unit (ICU) longer than five days, had neurodegenerative disorders, meningitis, hyperbilirubinaemia requiring blood transfusion or were undergoing chemotherapy. Less than a third of nurses always referred if the child displayed additional non-JCIH risk factors or those pertinent to the South African context. Approximately 38% reported that communities believed that hearing loss could be because of some form of spiritual or supernatural causes.This study demonstrates that hearing screening and referral practices at primary health care clinics need to be strengthened. Nurses need to be capacitated to conduct basic screening, make necessary referrals, provide information to caregivers and understand community beliefs about hearing loss in order to counsel caregivers appropriately and facilitate the process of early hearing detection and intervention. If problems are identified and infants have a refer result, a diagnostic evaluation needs to be completed by no later than 3 months of age, with interventions by no later than 6 months that include various forms of habilitation, rehabilitation or educational programmes.4 The position statement of the Health Professions Council of South Africa (HPCSA) on EHDI5 supports the above, with slight contextual adjustments in relation to the timeframes for screening, diagnosis and intervention.6 In addition to the initial screening, the infants demonstrating risk indicators for delayed onset and progressive hearing loss must receive ongoing surveillance by caregivers who are informed about the risks and the communication development milestones that need to be observed.7Hearing loss is a commonly occurring birth defect in developing countries.1 The World Health Organization (WHO) states that PHC is accepted as the best model for delivering basic health care, being an appropriate and a cost-effective means of improving the population’s health and a driving force in promoting health care.8 However, limited attention has been afforded to hearing health, especially in primary care settings in developing countries.9 The inadequate provision of good primary ear and hearing care throughout most of sub-Saharan Africa, not only exposes patients to potentially life-threatening complications from ear disease, but results in the common problems of chronic ear disease and disabling deafness.9In South Africa (SA), primary health care (PHC), which is mainly nurse-driven, is the first point of contact with the health system for at least 85% of the South African population.11 The effectiveness and success of programmes that provide for the early detection and intervention of children with hearing loss is contingent upon an interdisciplinary team approach.5 Team members involved should include audiologists; speech-language therapists; nurses; paediatricians; ear, nose and throat specialists (ENTs), community workers and families.5 This multidisciplinary or interprofessional method of shared knowledge and skill for a common goal, such as EHDI, will advance hearing health outcomes, while pooling the necessary resources.12 Findings of research conducted in SA indicate that hearing screening is not happening at the PHC clinics, where most caregivers take their infants for health care services.13There are too few audiologists available in SA to provide an accessible and equitable service at community level. A total of 1800 speech and hearing therapists, audiologists and/or speech therapists, mainly urban-based within the private sector, service a population of over 53 million people in the country, making access to services difficult for most, particularly in rural areas.14 In Cambodia, as an example, it was found that nurses are mainly trained to care for patients presenting with acute illness and to monitor the growth of children, with many lacking training in child development.14 In SA, medical attention is usually sought because of acute illness rather than developmental disabilities and delays, which, together with practitioners’ frequent lack of awareness, results in the at risky behaviours being overlooked.15 Similarly, in a study conducted in Brazil, it was found that most nurses are not trained in the importance of preventing hearing disorders, the identification of risk factors and referring for diagnostic testing.16 In another study conducted in Brazil, over 86% of nurses had not received training and information about referrals for hearing testing in infants.17 However, in a study conducted in Nigeria, hearing screening by PHC nurses was found to be successful, as most children presented for their immunisation visits irrespective of where they are born.18 This is advantageous, as it allows the identification of infants who were not screened at birth because of the limited opportunities for mothers to access universal newborn hearing screening (UNHS), especially in developing contexts. This also allows for the identification of progressive or late onset hearing losses and provides opportunities for caregivers to be sensitised about hearing loss. It can be a measure that is repeated each time the child is immunised, thus also ensuring that any child who has a hearing loss may be identified and loss to follow-up minimised.19 Identifying hearing loss by aligning mothers and/or primary caregiver reports (caregiver-driven) and nurse identification (systems-driven) during immunisation visits may be a viable option in SA, which has a reasonably good immunisation coverage rate, although its timelines need to be improved.20There is limited knowledge about the extent to which the assessment of developmental milestones is part of the medical protocol in developing countries.21 In the absence of hearing screening programmes at the PHC level of care, infants with hearing loss may go undetected for many years, leading to adverse consequences. This age group is particularly vulnerable, as they are unable to communicate using speech and language and cannot answer questions related to their hearing status. The only ‘formalised’ screening for hearing loss by the South African Department of Health (DoH)22 that is generally available, is the voice test and the Swart questionnaire.23 It is important that PHC nurses be very familiar with both these tests to ensure early identification and intervention of hearing loss. According to the DoH,22 it is protocol for nurses to complete a subjective screening test, such as the Swart questionnaire24 or the voice test, with children suspected of having a hearing loss. However, in the absence of knowing their hearing status, such infants may be overlooked.A study conducted in the United States of America showed that nurses trained in ear care can substantially reduce treatment costs, antibiotic usage and referrals to ENT specialists and general practitioners.25 they are in an ideal position to screen for hearing loss. In addition, nurses understand the cultural and linguistic backgrounds of the communities they work in and are knowledgeable about some of the high-risk factors associated with hearing loss.26 Nurses can also serve as intermediaries between caregivers, the doctor and the referral source.26 Furthermore, parents’ or families’ myths, cultural beliefs and societal practices related to the causes and treatment of hearing loss also influence their choice of practitioners and health-seeking behaviours, irrespective of educational level or social standing, and can further delay identifying and intervening for this disability.27Despite the shortage of nurses in the health system and their extensive workloads,25 It is also important to note that societies and cultures have different understandings about what is considered to be normal and disordered,28 making it important for information that is given to the parents and caregivers to be culturally sensitive and appropriate. Additionally, in a diverse and multicultural context, such as SA, the manner in which parents are informed about hearing screening, the available treatment options and who gives them the information is very important.26 Managing and monitoring hearing is within the scope of practice for PHC nurses, as the South African Nursing Council29 notes that they are responsible for maintaining the sensory functions in patients, which includes hearing. Maintaining hearing can be effectively achieved by implementing hearing screening and detection in PHC clinics.Parent and caregiver education is essential in a multicultural setting such as SA, because of the cultural and social stigmatisation that a disability carries.29 This indicates that nurses play an important role in educating mothers and caregivers about pathologies and risk factors related to hearing and hearing loss, as well as counselling patients towards appropriate referrals and keeping appointments that have been made. As nurses execute their role in the overall monitoring of the general health, development and well-being of infants, families become more aware of typical development, including auditory development.4 In the absence of formal screening, nurses need to provide mothers and/or caregivers with information and counselling about hearing loss and make timeous referrals on the presentation of high-risk factors. The DoH contends that the nurse’s role should encompass reviewing the child’s medical records, interviewing the caregiver and completing a physical examination that includes otoscopy and checks for otitis media, conducting a throat examination, checking for neck stiffness and examining the mastoid for pain. They should also complete a hearing screening evaluation, record the results and refer appropriately.22Another area that has been included in the scope of practice for nurses is promoting health and counselling with individuals and groups of people.30 on the quality of care offered to children attending PHC clinics in Johannesburg, it was found with concern that PHC nurses often failed to appropriately assess a child suspected with meningitis, which is a risk factor for hearing loss. Petrocchi-Bartal and Khoza-Shangase31 conducted a study at immunisation clinics in the Northwest and Gauteng provinces with 30 PHC nurses and found that there were no formalised newborn and/or infant hearing screening programmes at any of the clinics. They cited key concerns related to limited training received and budgetary, human resource and equipment constraints. While otoscopes were available, they were only used by 76% of nurses to sometimes conduct otoscopic examinations in children under five years of age, mainly if they presented with upper respiratory tract infections. There was also an inconsistent application of the hearing screening assessment protocol stipulated in the Road to Health Card (RtHC), with the guidelines further stipulating that results obtained for hearing screening be documented on the card to ensure continuity of care for children.In a study conducted by Thandrayen and Saloojee5 One of the roles of the PHC nurse is to record the hearing screening results so that important patient information can be accessed by both the parents and other staff members. Joubert and Casojee32 found that this was not deemed to be necessary information by PHC nurses, who did not adhere to hearing screening or record-keeping practices. The lack of urgency and low priority given to hearing impairment at PHC level may be because of issues related to poverty and the burden of life-threatening diseases, such as the human immunodeficiency virus (HIV) and/or acquired immune deficiency syndrome (AIDS) and tuberculosis.34The HPCSA position statement recommends that each district health system in SA use an integrated information system to manage data.There is a paucity of literature in SA related to how PHC nurses identify and engage with children with hearing loss who attend clinics. Their referrals, based on high-risk factors, as well as information and education provided to caregivers, given the beliefs about the causes of the condition at community level, need clarification. Information obtained will also guide appropriate education, awareness and training programmes for nurses and develop appropriate guidelines for detecting hearing loss in prelingual infants at PHC clinics.The aim of this study was firstly to determine hearing screening experiences, methods and approaches used by PHC nurses to screen for hearing loss in prelingual infants and secondly to determine their referral practices based on high-risk factors and their views on community beliefs about the causes of hearing loss. The findings will have implications for managing prelingual infants with hearing loss at community level.35A descriptive survey design was used with quantitative methods of analysis to enable the researchers to make inferences and learn about a large population by surveying a smaller sample.36 with the provincial health department paying the municipality to render these services.The study participants were PHC nurses working within the eThekwini district in KwaZulu-Natal province, which has a total of 112 PHC provincial and municipal clinics located within it. In SA, the provincial government is responsible for the provision of health care services; however, in some large municipalities, there are municipal clinics that also provide PHC services. This is made possible through a bilateral agreement between the provincial department and the municipality,A multistage (two-stage) sampling strategy was used, firstly to select the clinics and secondly the participants. Based on the estimated numbers of nurses in the facilities, 12 provincial and 18 municipal clinics were selected through simple random sampling. The second stage involved randomly recruiting a minimum of four nurses per clinic to achieve the proposed sample size of 120 from 30 clinics. Nurses of all races, ages (above 18 years), genders and levels of qualification were included, while student nurses were excluded because of their limited work experience.Of the 30 clinics identified, only 14 agreed to participate in the descriptive survey, including the one pilot site. Of the 74 questionnaires that were completed, nine were discarded because of incomplete responses. Another 10 from the pilot were added to the main study, as there were no changes to the questionnaire. The total number of questionnaires accepted for analysis was 75 of the anticipated 120 PHC nurses. These came from eight provincial clinics with 51 nurses, and six municipal clinics with 24 nurses. Some of the reasons cited for non-participation of clinics included staff shortages, that they did not see children with hearing loss, that they declined to participate, and two clinics lost the forms.n = 64), with 48% (n = 36) being between 36 and 50 years and 28% being over 50 years of age. A diploma in general nursing level 6 was held by almost half the nurses , with most  respondents being professional nurses, having received their education at various nursing colleges within the province. Approximately 50% (n = 38) of the nurses had less than 10 years of experience working in PHC, with 36% (n = 27) having over 15 years’ experience.The majority of participants were female 85% (26 and Joubert and Casoojee.32 The risk factors were obtained from the JCIH list of high-risk factors (JCIH 2007),5 HPCSA EHDI position statement (HPCSA 2007) and Olusanya.2 The questionnaire consisted of three sections: Section A obtained demographic information (eight questions), and Section B (Objective 1) explored how the nurses screen children with hearing loss and their experiences (nine questions). It is important to understand current experiences and methods used by nurses to identify hearing loss at PHC and the challenges faced in order to improve best practice.38 This includes availability of screening methods, recording and referral practices and information provided to caregivers. Section C included referrals based on high-risk factors (one question with 25 subquestions) and one open-ended question about the views of nurses about community beliefs regarding the causes of hearing loss. In the absence of UNHS, it is essential for nurses to be aware of the high-risk factors for hearing loss to facilitate referrals for further testing14 and conduct routine checks, especially for late onset or progressive hearing loss.18 Furthermore, nurses need to understand the community beliefs about the causes of hearing loss to enable them to appropriately inform and educate caregivers about the risks and interventions. Studies by a number of authors40 indicate that mothers or caregivers may hold superstitious beliefs about the causes of hearing loss that can delay intervention, with education having been found to be effective in modifying health-seeking behaviour and reducing non-compliance in developing contexts, despite low education and literacy levels.The data collection instrument was developed by the researcher based on the literature and was adapted from the studies by Moodley and Storbeckn = 56 items) was obtained, indicating good internal consistency. The data was processed and analysed using SPSS version 23, while NVivo 11 was used for the open-ended question about nurses’ views of community beliefs regarding the causes of hearing loss, allowing for qualitative analysis.All ethical considerations relevant to the study were adhered to, including signed informed consent, voluntary participation and anonymity. Participants were given two weeks to complete the questionnaires and an additional week in order to improve the response rate. A pilot study was conducted with 10 nurses from one clinic in the eThekwini district to ensure reliability and validity. Cronbach’s alpha was used to determine the internal consistency and reliability of the items contained in the survey questionnaire. A score of 0.887  had never screened a child nor identified a hearing loss. Of the 67% who had screened children, only 28% (n = 21) had screened over 20 children with a possible hearing loss, while the remainder had screened less than 20 in their years of working in a PHC setting. The chi-squared test showed a significant association (p < 0.001) between the years of experience as a PHC nurse and the number of children screened. Recently qualified nurses with fewer than 10 years of work experience tended to screen more children for hearing loss. Half the participants  felt that the best time to screen was every time a child attends the clinic, followed by when they come in for their immunisation checks . No significant association (p = 0.38) was noted between those who had or those who had never screened, and the best time to screen. Most nurses  used the RtHC to record patient information, while the others used the general patient files. In general, when a hearing loss or speech and/or language problem was suspected, referrals were made to various health professionals, including audiologists, ENT specialists, the General Practitioner at the district hospital or clinic and paediatricians.Of the 75 participants, 33% (n = 48) indicated that they provide information about the importance of EHDI to parents or caregivers, while 51% provided information about other professionals and counselling on the effects of hearing loss (n = 30) of the nurses provided feedback to the caregivers about the child’s hearing loss and a third included them in making decisions about their management options.Most nurses 64% (ing loss . Forty pn = 37) indicated that otoscopes were available in 14 clinics, with two to three being available in each clinic, one clinic reporting only having one otoscope and two clinics having none. Three participants had tympanometers and pure tone audiometers, and 20% (n = 15) had the voice test available, with none having the Swart questionnaire  indicated that they did use them, while only 25 participants indicated exactly what they used to screen: 92% (n = 23) used the RtHC that has a developmental screener, one participant spoke to the child and one used a general screener.Regarding their use of any developmental screening charts to assess for hearing, of the 63 who answered this question, 63%  risks emerging from regions in sub-Saharan Africa (Olusanya2), (3) known non-JCIH risk factors (Olusanya2) and (4) SA context risk factors4. The responses are presented in Risk factors were categorised into four groups: (1) those using the JCIH 2007)0075 listOver half of the nurses reported that they would always refer when they encountered at least five of the JCIH 2007 list of high-risk factors for hearing loss . These iThere were fewer referrals made for neonatal ICU admission of more than five days, hyperbilirubinaemia requiring blood transfusion, neurodegenerative disorders, meningitis and chemotherapy. Less than a third of nurses always referred if the child displayed risk factors relevant to developing countries or known non-JCIH risk factors . Most nun = 11); loud noise or high noise exposure or high-pitched noise (9%) (n = 4), untreated infections (9%) (n = 4), trauma or parents hitting child on the ear or cleaning one’s ear with sharp objects and/or eardrum bursting (6%) (n = 3), earphone use (3%)  reported some form of spiritual or supernatural causes of hearing loss, which included rituals not being followed, ancestral spirits, angry ancestors, prayers not performed, parents or family members being punished by ancestors, family did not do well with regard to ancestor matters, beliefs that ancestors want something a child may have (stated by one participant without elaboration), ancestral spirit wants the child to be a sangoma in the future, curse by ancestors and punishment by ancestors because of rituals not being carried out. They also mentioned bewitchment, curses, witchcraft, jealous neighbours, the child being bewitched and blood being impure .Nurses were given an open-ended question to obtain their views about the beliefs of the community pertaining to the causes of hearing loss. Of the 75 nurses, 44 answered this question, with the results being thematically analysed and depicted as a word cloud, with word frequencies  showing 41 38.7% of nurses only indicated one previous encounter with an infant with hearing loss. A study in the Gauteng and Northwest provinces of SA also found that 100% (n = 30) of nurses based at immunisation clinics reported that no formal hearing screening was conducted and that the HPCSA screening guidelines were not followed at the PHC level.13 The authors contend that it is cause of concern, given that this level of care is most accessible to the majority of people, including mothers or caregivers of infants with possible hearing loss.13 The results of the current study indicated that recently qualified nurses screened the most, which could be because of the increased awareness of EHDI programmes in SA.The PHC nurses had very few encounters with children with hearing loss, and the methods used to screen hearing for the condition at community level were inadequate. Similarly, in a study conducted in Nigeria,42 which is very alarming. More than 50% of nurses reported that they provided the parents and/or caregivers with information about other professionals who could assist the children with hearing loss, counselling about the effects of hearing loss and information about EHDI services. Fewer nurses explained the child’s hearing loss and provided management options. Caregiver education was found to be successful in modifying health-seeking behaviour and reducing non-compliance in developing contexts, despite low education and literacy levels.40Over half (54%) of the nurses noted that the best time to screen or identify a possible hearing loss was every time a child attends the clinic or presents for immunisation (22%). However, this is seldom done in practice. Research shows that there are very few opportunities for infant screening in PHC clinics in SA,38 It was deduced that poor attendance was because of parents and/or caregivers not having sufficient knowledge on the importance of hearing in a child’s development. If a refer result is found, nurses should be able to provide the parents or caregivers with immediate counselling on the importance of follow-up appointments and information on possible hearing pathologies.26 Parental support, education and participation in EHDI is crucial to ensure follow-up and after-care services for children with hearing loss, as non-compliance with treatment is common, especially in developing contexts.40Another South African-based study found that children who had been screened and referred did not always attend the follow-up appointments.28 Recommendations that are associated with social stigma or offend cultural norms could result in loss to follow-up.26 As an example, there is considerable stigma attached to taking relatively healthy young children for hearing testing.18 It is essential to enable parents to make informed decisions about their child’s management, making the nurse well positioned to explain the importance of screening, follow-up and intervention.26 In whatever format this information is provided, parents should receive information in a responsive and sensitive manner for them to be amenable to accessing follow-up services.26 They conclude that nurses are often best able to communicate in a culturally appropriate language to make parents feel relaxed and respected for their choices. However, a study conducted by Arnold et al.43 stated that parents felt nurses needed more training regarding what to say to them if their child did not pass the hearing screening.However, the authors do acknowledge that other factors also play a role, such as accessibility and affordability of services, parental cultural beliefs towards childhood deafness and other priorities that mothers and/or caregivers have to deal with. SA is characterised as a multicultural, multilingual country, making it important for professionals to provide services that are culturally and linguistically appropriate.13 in clinics in Gauteng and North-West Provinces, where all participants reported not having access to equipment, citing budgetary and human resource constraints. While it was anticipated that clinics would not have access to tympanometers, pure tone audiometers and otoacoustic emission testing, it was a serious cause of concern that 52.3% of the participants indicated that they did not have an otoscope available at some of the clinics, which implies that this examination is not always conducted on children at these facilities. Petrocchi-Bartal and Khoza-Shangase31 reported that although all their participants reported having an otoscope, approximately 76% indicated that they only used it on some babies. The PHC package outlines the availability of otoscopes as a basic equipment requirement for the inspection of the external auditory meatus and tympanic membrane, and to prevent and detect hearing loss because of otitis media.31 The Swart questionnaire24 and Voice test are two subjective tests recommended by the DoH.22 It is, therefore, cause of concern that 100% of the nurses indicated that they did not have the Swart questionnaire and 76.9% did not have the Voice test. A study on the quality of services provided by clinics in the Johannesburg area also showed that the Swart questionnaire was only conducted 14% of the time and the Voice test only 7%.44 This has serious implications for the early detection of hearing loss and its further management.Based on the non-availability of equipment, it is clear that none of the clinics provided any formal screening. Similar findings were reported by Petrocchi-Bartal and Khoza-Shangase34 There remains a lack of emphasis on early screening and identifying disability throughout Africa, which results in few referrals and limited interventions.26 One of the main challenges is the lack of funding for screening programmes, as emphasis is placed on reducing the high mortality rate from the burden of communicable diseases.26 While reducing the high mortality rates from these diseases such as HIV and AIDS and tuberculosis (TB) in SA is important, Moodley and Storbeck26 further stated that ‘it is likely that a reduction in the mortality rate could result in an increase in the number of children who survive with disabilities’ (p. 29). This shows the need for funding to be given to screening programmes in order to improve the overall health of the paediatric population in SA.The lack of EHDI services has been reported by many researchers, and it is often regarded as a low priority and of little importance.22 Late referrals or no referrals for infants presenting with hearing loss is problematic, as the lack of timeous intervention represents a threat to their future quality of life.14 The DoH22 guidelines state that PHC nurses are to record the results of the hearing screening completed during an immunisation session on the RtHC.31 However, only 54% of nurses in the current study stated that they recorded the results as required, despite this card needing to be shown to the nurse at every PHC clinic visit.24 Recording the child’s progress is important, as it creates a partnership between the parent and/or caregiver and the health professional and thus promotes patient care and follow-up appointments.24 It is also important to note that constant recording in the RtHC will aid ‘a national information system that meets the requirements for hearing screening record-keeping, as recommended in the HPCSA 2007 position statement’ (p. 27).32Every nurse is in a position to make appropriate referrals when hearing problems are detected, as well as to ensure proper record-keeping practices, and should keep a record of the management decisions and referrals made.26 A study by Kanji and Opperman6 found that a large number of participants were not effectively followed up on for hearing screening, and while this may be because of poor return rates it could also be attributed to poor record-keeping practices. Diagnostic follow-up and intervention serve as critical ethical markers in the hearing screening process and provide a means of recording outcomes, such as the overall efficiency and effectiveness of screening programmes.45The lack of importance given to early screening and identification of disability in Africa may suggest that training and experience in the area of hearing loss is limited.26 Many health care professionals are unaware of the significance of appropriate interdisciplinary referrals.42 This was also found to be the case in the study by Casojee,46 who noted a general lack of compliance with referral protocols. The author thus concludes that this not only presents a stumbling block for health systems development but is also contradictory to the principles of EHDI.46 According to the DoH,22 nursing staff should keep a record of all patients seen and all referrals made. PHC nurses do not adhere to record-keeping or the referral practices31 required by the DoH,22 which hinders quality patient care.Nurses and audiologists, as well as families and community workers, are part of the collaborative interdisciplinary team required to service the population with hearing loss and those at risk to ensure that affected children receive an appropriate quality of service.47 found the most frequent risk factor to be neonatal ICU admission (28.1%) and prematurity (15.1%), with the most common postnatal risk factor being neonatal jaundice. Nurses in this current study were also less likely to always refer if the child had meningitis, yet this was the second most common risk factor for postnatal hearing loss.47Nurses must be aware of the risk factors for hearing loss in order to facilitate EHDI at community level. The HPCSA (2007) states that even if a child passes the screening, if they exhibit risk factors for a progressive, late-onset hearing loss or other auditory pathologies that can lead to a speech and language delay, the child should be monitored, and the parents informed of the risks and provided with information on the developmental milestones. In this current study, nurses indicated that they were less likely to refer if a child was in neonatal ICU for more than 5 days and had low birthweight that was usually related to prematurity or neonatal jaundice. However, a retrospective record review of paediatric cochlea implant recipients conducted by Le Roux et al.48 focussed on the nature of hearing loss and associated risk profile and found that hyperbilirubinaemia was one of the most prevalent risk factors. Other risk factors included birthweight less than 1500 grams, syndromes present, congenital infection, craniofacial defect and bacterial meningitis.48 In some countries in sub-Saharan Africa and Southeast Asia, non-JCIH risk factors such as consanguinity and sickle cell anaemia are more common, although they are related to specific racial or ethnic groups.2Swanepoel, Johl and Pienaar2 However, the author contends that the precise means of how these affect hearing, especially in infancy, is yet to be determined. Less than one-third of nurses in this current study always referred if the child displayed risk factors emerging from developing countries and known non-JCIH risk factors, necessitating greater education and awareness of these risk factors. Children who have disabilities, or present with risk factors for disabilities, also require services from a number of health professionals,49 and this needs to be facilitated as early as possible. For the South African context, diseases such as maternal HIV and/or AIDS and TB need to be considered, as these infections can lead to hearing loss because of their association with low birthweight, prematurity, bacterial meningitis, viral encephalitis and cytomegalovirus.50 HIV and/or AIDS, as well as TB are, therefore, important risk factors for hearing loss, along with hyperbilirubinaemia, congenital rubella, congenital syphilis and a family history of permanent hearing loss.Emerging risk factors from developing countries, such as Nigeria, include maternal hypertensive disorders in pregnancy, non-elective caesarean delivery, unskilled attendant at delivery and under-nutrition.26 It was encouraging to note that nurses would always refer or refer most times for most of the JCIH risk factors and otitis media. It is evident that they need to be provided with information and education about other high-risk factors still pertinent to the SA context, such as low birthweight, prematurity, asphyxia and exposure to HIV and/or AIDS. Thus, nurses will be able to obtain case history information from the parent or caregiver and, as a result of being aware of the risk factors, will monitor the child’s development during visits for immunisation. They will also use the information to plan and organise appropriate hearing prevention and promotion awareness activities and referrals based on high-risk factors for hearing loss. A study by Aires et al.17 revealed that nurses are only aware of some risk factors for hearing loss but that to effectively conduct the preceding activities they will require more information and training.In developed countries, however, certain risks have been minimised or eliminated because of improved health services but are still very relevant in developing contexts, such as those related to low birthweight and birth asphyxia, making it essential for early identification procedures to be coordinated for efficient outcomes.5 Some of these factors include jaundice, respiratory distress syndrome, neonatal ICU admissions and infections, such as cytomegalovirus.5 It can therefore be deduced that the nurses who are aware of children who present with these risk factors in the community clinics, have the opportunity to screen them for a hearing loss. Expanding PHC nurses’ knowledge will enable them to provide more effective services for the children in their clinics. A policy document that examined the skills needed in the PHC system stated that in community health settings, nurses should have knowledge of otitis media.51 This is essential, as a large proportion of children present with otitis media and conditions such as HIV and/or AIDS, are a contributing factor towards middle-ear pathologies. The current study found that 62% of nurses did refer if otitis media was present. This is important as poor socio-economic conditions mean that recurrent otitis media is a reality for many children in Africa.52 In a study conducted by Biagio et al.53 regarding paediatric otitis media at a PHC clinic, the prevalence of chronic suppurative otitis media was 6.6%. Evidence suggests that poorly managed or unmanaged otitis media can lead to permanent hearing loss.54 Nurses do receive training in the structure of the ear, otitis media, hearing problems and the effects of hearing losses on speech and language development, although its extent is unclear.55 However, in a study conducted in SA, nurses expressed an interest in learning more about managing hearing loss at PHC level.55Furthermore, it is reported that 50% of children with a hearing loss have identified risk factors that present at birth or within the early years of life.56 This indicates why it is essential for nurses to provide education and information to mothers and/or caregivers about the causes and risk factors for hearing loss, as well as the importance of EHDI and follow-up. In SA, where peoples’ beliefs are buried deep within their cultures, it is important that health care professionals take cognisance of these beliefs so that they may be incorporated into information dissemination.39 De Anrade and Ross57 conducted a study on beliefs and practices of Black South African traditional healers regarding hearing impairment and noted some of the causes of hearing loss to be related to ancestors, noise, congenital factors, bewitchment and blood impurities.Differences in health beliefs, perceptions of illness and healing methods between the patient and the provider can be a barrier to accepting services.57 it is not surprising that communities tend to share similar beliefs about the traditional and non-traditional causes of hearing loss, as was noted in the current study. There is widespread stigma associated with having a child with a hearing loss, who may be considered as a bad omen who will bring misfortune to the family.40 Some communities regard unnatural causes of hearing loss to include sorcery, spirits, ancestors and failure to perform certain rites and rituals.57 The current study also found that there were references to ancestors, rituals, bewitchment and curses. According to Olusanya18 it is not uncommon for permanent childhood hearing loss (PCHL) to be attributed to supernatural causes or superstitious beliefs. In a study conducted by Swanepoel and Almec39 at least 57% of mothers held at least one superstitious belief regarding the cause of infant hearing loss. Parents may thus also make use of spiritual healing or traditional medicine, which can be potentially harmful.18 These factors possibly account for caregivers not returning for follow-up appointments.38 However, in the current study there was also mention of causes of hearing loss such as genetics, exposure to loud sounds, untreated ear infections, trauma because of sharp objects used to clean the ears and use of earphones. Olusanya, Luxon and Wirz58 attribute the poor knowledge displayed by mothers and caregivers to deficiencies in health education during the antenatal period, which needs to be addressed.As 8 out of 10 Black South Africans, seek services from traditional healers,Research evidence supports that as the UNHS is unlikely to be attained in the near future in the SA context, other feasible options need to be developed, revisited or even redesigned to ensure that early identification happens at the PHC level. The study limitations included the small sample size, which may affect the generalisability of the findings. In addition, the beliefs of nurses regarding the causes of hearing loss may not be the same as the communities they serve, which may have introduced information bias in the study. The responses were based on nurses’ self-reports of their practices and not their observed practices.This study demonstrates that hearing screening and referral practices at primary health care clinics need to be strengthened. Despite the challenges relating to screening for hearing loss, PHC nurses should conduct some form of early hearing detection at community level, given that this is most accessible to most people and where mothers and/or caregivers take their infants for health care. Child health in SA is one of the key imperatives of the PHC package, and while preventative care is currently limited to immunisation against diseases and conducting growth monitoring, it should include developmental screening. In this PHC context, nurses can be given basic skills during their training by members of the multidisciplinary team to manage hearing loss at community level and thereby reduce the frequency and chronicity of ear disease and disabling deafness.


Nerve biopsy was suggestive in 3/9 (33%), with magnetic resonance imaging supportive in 9/20 (45%). Twenty-one children received immuno-modulatory treatment at first presentation, of which majority  received IVIG (immunoglobulin) monotherapy with 13 (68%) showing a good response. 8 children received second line treatment with either IVIG or steroids or plasmapharesis (PE) and 4 needed other immune-modulatory agents. During a median follow-up of 3.6 years, 9 (43%) had a monophasic course and 12 (57%) had a relapsing–remitting course. At last paediatric follow up 7 (33%) were off all treatment, 9 (43%) left with no or minimal residual disability and 6 (28%) children were walking with assistance (n = 3) or were non-ambulant (n = 3). Our review highlights challenges in the diagnosis and management of paediatric CIDP. It also confirms that certain metabolic disorders may mimic CIDP.Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a treatable chronic disorder of the peripheral nervous system. We retrospectively studied 30 children with a suspected diagnosis of CIDP. The diagnosis of CIDP was compared against the childhood CIDP revised diagnostic criteria 2000. Of the 30 children, five did not meet the criteria and four others met the criteria but subsequently had alternative diagnosis, leaving a total of 21 children  with CIDP as the final diagnosis. Thirteen children presented with chronic symptom-onset (>8 weeks). The majority presented with gait difficulties or pain in legs ( Goals of diagnosis and management are to control the inflammation and therefore prevent axonal loss and the resulting disability. Although in some the course is monophasic with complete recovery, in others it can be slowly-progressive, or relapsing-remitting resulting in prolonged morbidity and sometimes permanent disability CIDP is less common in children than adults and hence knowledge of the clinical characteristics, response to treatment, and prognosis are often based on several small paediatric series. In this series we attempt to describe the presentation, diagnosis, response to treatment and long-term outcome of childhood CIDP.2Children seen in the neuromuscular service at a single tertiary unit from 1992 to 2015 with a suspected diagnosis of CIDP were identified from our neuromuscular and neurophysiology databases and their medical records were reviewed.Revised diagnostic criteria for childhood CIDP proposed by Nevo et.al. Published reference values for normal paediatric sensory and motor nerve conduction values were used Information was obtained on age of onset, time to maximum disability, symptoms at presentation; disease course, immuno-modulatory treatment used and disease activity at last follow up. An attempt was made to identify atypical features in the suspected CIDP cases that later had a diagnostic revision. Clinical deficit was quantified using the Modified Rankin scale MRS;  [2] at p3n = 25) met the criteria; ten met the confirmed CIDP criteria and 15 met the possible CIDP criteria. Five cases that did not meet the criteria had a later diagnostic revision to GBS in four cases and a likely genetic cause in the other. Amongst twenty-five that met the criteria four subsequently had alternative diagnosis and were excluded. A total of 21 cases were established to have CIDP and these are detailed as belowThirty children with CIDP suspicion were identified  and thei3.1n = 13). Two children included in the chronic group had leg pains and foot deformity for more than a year and then became non-ambulant acutely. The interval between symptom onset and maximum disability ranged from <1–44 months. Preceding infectious illness was present in less than half (n = 11).The mean age of onset was 7 years and 3 months; range 2–16 years. Symptom onset was acute (<4 weeks) in five, three had a sub-acute (4–8 weeks) and more than 50% had a chronic (>8 weeks) onset (n = 11); all subsequently developed 4 limb involvement. All children were areflexic with one hyporeflexic at presentation. 3/21 (14%) had bulbar weakness with weak cry or swallowing/speech difficulty with two others having cranial nerve involvement with anisocoria/optic neuritis and bilateral lower facial weakness, respectively.The majority of children presented with gait difficulties (3.2n = 8), and both demyelinating and axonal changes in four cases.Electrophysiology studies done in all the twenty-one children who met the CIDP diagnostic criteria were reviewed; of whom 12 met the electrodiagnostic criteria for CIDP. Parameters that were looked into included presence or absence of conduction block, sensory and motor nerve conduction velocities, distal CMAP latency and F wave latency. A minimum of four to five nerve were studied. In these 12 children that met the electrodiagnostic criteria for CIDP the study was demyelinating in majority (Rest of the 9 cases that met the CIDP criteria (confirmed or possible) did not meet all the required major or supportive criteria to meet the electrodiagnostic criteria for CIDP; of these two had only axonal changes.n = 18/19; 94%) met the CSF criteria Lumbar puncture was performed in all but 2 cases and majority (n = 10) with pre-existing, early or progressive foot deformity and/or poor response to treatment. These included comprehensive 56 gene inherited neuropathy panel (INP) in three and were negative in all cases.Genetic testing for inherited neuropathies was done in half of the cases (Neuroimaging of spine was done in 20 and 9 (45%) had thickening and/or enhancement of cauda equina nerve root . Two oth3.3n = 19; 90%) received IVIG monotherapy with 68% (n = 13/19) showing a good response after first dose. IVIG was then gradually weaned and eventually 6/13 (46%) children with good response to IVIG were off treatment irrespective of disease course (monophasic or relapsing) over a mean FU of 5.5 years (range 1.1–13.7 years). 5 children had side effects including headache, nausea, vomiting and transient high blood pressure but continued on therapy. Two children were started on steroids as first line with partial response in one and no response in other.All twenty-one children received immuno-modulatory treatment as first line therapy, of which majority  had a monophasic course and 57% (n = 12) had a relapsing–remitting course. Of the 12/21 children with relapsing-remitting course, three evolved to chronic progressive. The majority had 2–5 relapses (n = 6) and three children each had 1 or more than 5 relapses (n = 3) with 3 weeks to 24 months interval between relapses.43% (n = 10) and prednisolone was used in the other two. 4 children in this group continued with relapse or progression and were trialed on other immuno-modulatory therapy including plasma exchange (PE), azathioprine, cyclophosphamide and mycophenolate, none of them had a good response except PE in one. Cyclophosphamide was trialed in one child for ten courses with minimal response and no motor worsening. Three children remain on mycophenolate and IVIG infusions with minimal response.IVIG was trialed as first line therapy for relapse in the majority of cases  were off all treatment, of these, two were cured as per CDAS classification (stable and off treatment for more than 5 years) and 5 others were in remission (stable and off treatment for less than 5 years). A total of six (28%) still had unstable active disease and were on treatment.At last paediatric follow up 33% (n = 9) were left with no or minimal residual disability.On reviewing the MRS scores  13 child3.5Supplementary Table S2 highlights the findings in individual cases that led to diagnostic revisions. These included findings like early onset, pre-existing wasting/contractures, associated co-morbidities like development delay, epilepsy, chromosomal deletions, cognitive decline and abnormal investigations like high CK or abnormal metabolic profile, poor or no response to IVIG therapy.n = 2) and GBS (n = 4). The diagnosis of CIDP is thus very challenging and needs frequent re-evaluation.Of the total 30 cases with initial suspicion of CIDP, 19  had a reTwo children in our cohort were later diagnosed with MNGIE and MLD respectively. Both these children met the clinical, CSF and electrophysiological criteria for demyelination and managed as CIDP initially. A routine screen for urine organic acids was abnormal in one and led to a genetic diagnosis of MNGIE two years after presentation.ARSA gene. MRI brain at 2.6 years showed typically tigroid pattern of leukodystrophy with bilateral dark thalami with thickening and enhancement of 3rd, 5th, 7th and 8th cranial nerve and cauda equina nerve roots.The other child with MLD presented with unsteady gait and loss of ambulation at 13 months and showed a partial response to IVIG. Nerve biopsy showed presence of granular storage material in cytoplasm of myelinating schwann cell and was crucial in leading to the diagnosis of MLD. Meanwhile, her symptoms progressed with cognitive decline and upper motor neuron signs. Arylsulfatase A activity was very low in white blood cells, and the diagnosis was subsequently genetically confirmed with compound heterozygous mutations in the Two siblings born to non-consanguineous parents were thought to have a congenital onset of CIDP. History of polyhydramnios and reduced fetal movements suggested antenatally onset disorder. The index case was born following foetal distress, needing CPAP support for two days. She was profoundly hypotonic, weak with poor antigravity movements and areflexia. At four weeks of age her NCS demonstrated absent sensory nerve action potentials (NAP), motor conductivity (cV) 1–2 m/s and dispersed compound motor action potentials (CMAP). She was treated with 4–5 courses of IVIG following which she had gradual normalization of clinical examination and NCS at 9.5 months of age. Her younger sibling presented similarly with hypotonia and weakness and responded well to IVIG therapy, continuing to make improvement. Her mother when tested did not have a neuropathy.Two children initially thought to have CIDP were later considered to have a genetic etiology; one among them (Pt 8) presented at 4 years with only lower limb, distal involvement with tip toe gait and thereafter rapidly progressive bilateral equinovarus and calf wasting. Nerve biopsy showed a chronic picture of re and demyelination with no active inflammation, more suggestive of a genetic cause. The other child (Pt 15) had an early presentation and developed a progressive disease course with marked foot deformity. A nerve biopsy was declined. Both the children did not respond to therapy with negative genetic testing including inherited neuropathy panel.4We are presenting data of CIDP cohort from a single tertiary center in UK. McMillan et al have previously reported on 30 American children with CIDP and combined this with data from 11 previous case series (1980–2009) to provide a comprehensive review of 143 childhood CIDP cases Based on the revised diagnostic criteria for childhood CIDP proposed by Nevo et al. n = 9) in our group had diagnostic revision due to relapse, progression or pre-existing wasting or contractures. Due to the chronic nature of symptoms genetic neuropathies can mimic CIDP and can be equally challenging to differentiate the two. Shabo et al. The overlap between GBS and acute onset CIDP and between inherited neuropathy and CIDP were the two main common themes in differential diagnosis in our group. Difficulties in diagnosis of GBS and the acute onset CIDP is known  of our cohort diagnosed as CIDP on re-evaluation suggesting the challenges in diagnosis of this treatable condition.A clinical suspicion of CIDP along with appropriate tests, treatment, regular follow up and serial electrodiagnostics helped to confirm the diagnosis in our cohort. In cases where there were atypical features either in history, investigations or progression, the diagnosis was revised. This is shown by 52% , which were initially diagnosed as CIDP. This has been reported in literature. Bedlack et al. In our study the two siblings thought to have congenital CIDP had a later diagnostic revision to GBS. Congenital presentations of CIDP are rare but have been reported before Cranial nerve involvement has been reported in children with CIDP and can cause considerable diagnostic confusion. We as clinicians need to be aware of this presenting feature in CIDP. Costello et al. In our study the treatment response to first line IVIG therapy was deemed as good in 68% of cases; this is comparable to the existing literature with a combined value of 79% from 135 total reported cases by McMillan et al. McMillan et al. 5Our review highlights the challenges in the diagnosis and management of paediatric CIDP. In general this is a rare diagnosis but clinical suspicion, early recognition and investigation with prompt management can result in better outcomes of this potentially treatable neuropathy. Our review also highlights that various differential diagnoses must be considered in cases where the disease course changes or new signs evolve.


Although sympathetic nerve activity and thermogenic capacity of BA is downregulated under fasting conditions in mice, fasting-dependent structural changes and fluid kinetics of BAT remain unknown. Here we show that the fasting induces fine and reversible structural changes in the non-parenchymal region in murine BAT with widened intercellular spaces and deformed collagen bands as revealed by electron microscopy. Interestingly, a newly introduced near infrared fluorescent probe of single-walled carbon nanotubes (CNTs) coated with phospholipid polyethylene glycol (PLPEG) easily demonstrated enhanced vascular permeability in BAT by the fasting. PLPEG-CNTs extravasated and remained in intercellular spaces or further redistributed in parenchymal cells in fasted mice, which is a previously unknown phenomenon. Thus, PLPEG-CNTs provide a powerful tool to trace fluid kinetics in sub-tissue levels.Brown adipose tissue (BAT), which is composed of thermogenic brown adipocytes (BA) and non-parenchymal components including vasculatures and extracellular matrix, contribute to the maintenance of body temperature. BAT distribution is detected by positron emission tomography-computed tomography (PET/CT) using  In 18F-FDG-PET/CT, BAT signals are enhanced by cold stimulations. Although cold stimuli up-regulate the activity of sympathetic nerves, which is the major regulator of BAT activity in mice, sympathomimetics fail to enhance 18F-FDG signals in human BATs1. In addition, human BAT signal in 18F-FDG-PET/CT is increased by fasting2 while the activity of BAT-governing sympathetic nerves is lowered under fasting conditions in mice3. Despite these discrepant findings, human and murine BATs share substantial similarities in gene expression profiles4 and distributions in the body5. Brown adipocytes (BA) utilize lipids as the primary energy source, whereas signals in 18F-FDG-PET/CT reflect their glucose-uptake activities, but not the rate of their energy expenditure per se. Therefore, signal intensities in 18F-FDG-PET/CT in BAT would not always reflect their thermogenic capacities and certain fine morphological changes might possibly take place to enhance glucose accumulations in BAT independently of the level of sympathetic nerve activity.Brown adipose tissue (BAT) exerts anti-obesity and anti-diabetes effects, and therefore, is attracting attention as a new target of the drug discovery for the treatment of metabolic syndrome. Currently, In adipose tissues in general, fasting induces shrinkage of lipid droplets reflecting enhanced lipolysis to supply materials for gluconeogenesis. When we examined the histology of BAT specimens in starved mice, we determined not only the reduction of lipid droplet sizes and numbers but also alteration in the structure of intercellular spaces. Although altered interstitial structures may reflect the change in the fluid kinetics in the sub-tissue level, it takes considerable time and labor to find out such fine morphological changes by histological examinations. Moreover, real-time tracing of fine structural changes cannot be carried out by histological observations using fixed specimens.7 is known to emit fluorescence in the near infrared (NIR) region around 1000–1400 nm8, and works as an excellent probe when coated with phospholipid polyethylene glycol (PLPEG-CNT) for vascular imaging by following reasons. Light in this region can penetrate deeply into the body due to its low absorption and is weakly scattered by biomolecules. In addition, biomolecule-derived auto-fluorescence level is low in this wavelength region. Accordingly, CNT NIR-fluorescence-based bio-imaging techniques have an advantage that they show high signal-to-noise ratios12. Due to the high bio-affinity property of PLPEG and the small size of CNT, PLPEG-CNT shows very low binding affinities to biomolecules13, which is suitable to image the vascular systems17 and, further expected to find the fluid kinetics in sub-tissue levels.A new probe of single-walled carbon nanotubes (CNTs)Therefore, PLPEG-CNT was administrated into starved mice to trace the blood flow, and possibly, interstitial fluid in BAT. Surprisingly, intravenously administrated PLPEG-CNT fluorescent probe revealed that the vasculature permeability in BAT was specifically enhanced by the fasting. This unexpected finding presents a potential of PLPEG-CNT as a new tool to examine fluid kinetics in sub-tissue levels.18. To further evaluate the impact of the character of the coated materials, we re-performed whole-body imaging using CNT coated with phospholipid-based compound of PLPEG  12 minutes  and axillary BAT (aBAT) Fig.  than nonThe fasting-depending up-regulation of the signal intensity of BAT was further confirmed by measuring the CNT-NIR-fluorescence intensity of the tissues dissected at PIT 5 h and PIT 1d. We determined that BATs of the fasted mice were significantly brighter than those of non-fasted mice Fig. .Figure 3From these findings, we hypothesized that iBAT-selective imaging for the fasted mice was attributed to certain histological alterations, which induced the prolonged retention of PLPEG-CNT in iBAT. Morphological examinations revealed that the structure of reticular fiber (type III collagen) was disarranged Fig.  and the Collectively, fasting induced unique and distinctive fine morphological changes in iBAT especially in its interstitial regions.The fasting-induced structural changes in BAT were significantly alleviated at PIT 1d as inferred from the histological observation (Supporting Data 3), suggesting that the tissue structure changes caused by the fasting would be a reversible process.Mmp1 mRNA was undetected and Mmp14 expression level was not affected by the fasting  were not reduced by the fasting, suggesting that the disorders of the gap junction and collagen bands were not resulted from downregulated synthesis of these proteins. Autophagic degradation of Cx4321 does not seems to be mainly involved in the induction of iBAT structure disorders either, because the expressions of autophagy-related genes (Atg5) was not increased by fasting  3, which is known to digest collagen type III, was significantly increased by the 20 hours-fasting, while ing Fig. . These dgenes Atg was not To examine whether there were any changes in the fluid flow conditions in iBAT, the histology of iBAT of PLPEG-CNT-administrated mice were examined by NIR fluorescence microscopy.Coinciding with the iBATs imaging results shown in Fig. The arteriole and its proximate areas in iBAT were further examined at higher magnification Fig. . In addiExtravasation of PLPEG-CNTs from the arteriole and the capillary in iBAT suggested by the optical microscopic observation results Fig.  were furIn vitro observations, PLPEG-CNTs showed the appearance of fibers, which corresponds to the individual CNTs with diameters of about 1 nm, or-bundle-like appearances (Supporting Data 6). For in vivo observations, the tissue slice (5 μm thick) used for optical microscopic observations  of aBAT (aBAT-LNs) Fig. . There wFasting enhanced extravasation of fluid through degenerated basal membranes of capillary/arteriolar walls as demonstrated by disarranged or broken collagen bands in the extracellular matrix in BAT of fasted mice Figs –7, which22 using dataset of GeneAtlas MOE430, gcrma23, Mmp3 expressions in BAT (259.57 ± 120.44) are significantly higher than 3 x median value (3 × 6.3 = 18.9). Indeed, BAT is the 8th-highest Mmp3 mRNA-expressing tissue. Interestingly, lymph node (794.36 ± 447.47) is the 4th-highest tissue. The reason why Mmp3 expression was upregulated in iBAT by fasting is unknown, which remains to be elucidated by future studies.The widening of inter-cellular spaces and disorders of collagen bands may be attributed, at least in part, to the fasting-induced proteolytic events, as confirmed by the increase of Mmp3 mRNA levels. According to the gene expression data that are stored in BioGPS database24, which is not the case in our study because the mRNA expression levels of Vegfa and Vegfb did not increase by the fasting . The BAT-ECs are highly unique in several points. Firstly, they serve as a progenitor of brown adipocyte (BA) in adult mice29, and leptin increases adipose tissues capillary permeability30, which needs further confirmation.The accumulation of PLPEG-CNTs in iBAT of the fasted mice re-increased when the refeeding started , which is reported to be scarcely absorbed by PLPEG-CNTs13. Thus, it is reasonable that the PLPEG-CNTs were not detected in the capillary endothelial cells in adipose tissues including BAT.In contrast to the case of CNTs coated with an amphiphilic and biocompatible polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB-CNT), which specifically accumulates in the capillary endothelial cells in adipose tissues18. PLPEG-CNTs, however, hardly absorb apolipoproteins (Supporting Data 12). It may be possible that hepatic stellate cells ingested PLPEG-CNTs and PMB-CNTs simply by a physical reason since they are nanometer-sized small particles with fibrous structures. The precise mechanism of PLPEG-CNTs accumulation in hepatic stellate cells will be the subject of future research.Besides BATs and lymph nodes, PLPEG-CNTs accumulated in the hepatic stellate cells (Supporting Data 13–15). As we previously reported, PMB-CNT was ingested by hepatic stellate cells probably by apolipoproteins transferred to PMB-CNT in plasma18F-FDG-PET/CT. Glucose is up-taken via transporter proteins existing on the plasma membrane of BA. By preconditioning with cold stimulations 31 glucose uptake by BA is significantly enhanced due to upregulated activity of the sympathetic nerve, which activates AMP-dependent protein kinase (AMPK), thus enhancing membrane translocation of Glut4 protein to augment glucose uptake by BA. Since BAT are intensively innervated by the sympathetic nerve system, 18F-FDG-PET/CT provides the “BAT over WAT imaging”. Not only by acute cold stimulations, cold acclimation enhances the activity of BAT to up-regulate its thermogenic activity for an adaptation to cold environments35. Moreover, the thermogenic activity of BAT is even enhanced under postprandial conditions to expend a calorie surplus toward the optimal energy balance in a living body37. On the other hand, BAT activity is repressed under fasted conditions by the decline of sympathetic nerve activity in mice, contributing to saving energy expenditures by suppressing thermogenesis-mediated energy loss38. The discrepancy between enhanced signal intensities in 18F-FDG-PET/CT in humans and lowered thermogenesis activities of BAT in mice under fasted conditions remains unsolved. Our finding that the vascular permeability in BAT is enhanced in fasted mice suggests that the fasting-depending 18F-FDG-PET/CT signal enhancement in humans might possibly be attributed at least in part, to the enhanced 18F-FDG extravasation in BAT.Currently, the BAT imaging is generally performed by With an advantage of high diffusiveness throughout the body, PLPEG-CNTs will provide an effective tool to trace fine fluid kinetics changes in various tissues of a living body.18F-FDG-PET/CT signal enhancement for human BAT reflects increments in permeability in BAT vessels. Owing to their high diffusivity and the advantage that they emit NIR fluorescence, PLPEG-CNTs have a possibility to be an effective tool to trace fluid flow in sub-tissue levels, which is a challenge as a new application of PLPEG-CNTs in addition to the conventional whole-body vascular imaging.Fasting induced disarrangement of collagen bands in the extracellular matrix and widening of the intercellular space in murine BAT. In addition to these, the vascular permeability was enhanced, which was determined by using PLPEG-CNT as a NIR fluorescent probe to trace blood and fluid flow in sub-tissues level. Namely, intravenously administered PLPEG-CNTs accumulated more intensively in BAT in fasted mice than ad lib fed mice, where PLPEG-CNTs extravasated and re-distributed in parenchymal areas as well as non-parenchymal cells. These findings strongly suggest that fasting-induced About 100 mg of CNTs  in 100 mL of an aqueous solution of sodium cholate  were homogenized by using an ultrasonic homogenizer  at 30% amplitude for for 3 h and centrifuged at 50 000 rpm (210 000 g) for 2 hours with an angle rotor . 80% of the supernatant was collected and centrifuged again at the same condition, and 70% of the resulting supernatant was collected. To concentrate CNTs and exchange the dispersant in the solution, centrifugal filter unit was used . To the 10 ml of the supernatant, 2 ml of 1% phospholipid polyethylene glycol aqueous solution )-5000], SUNBRIGHT® DSPE-050PA, NOF American Corporation, New York, USA) was added and concentrated using the filter unit at 2330 g to ~6 ml. 6 ml of water was added to the concentrate and filtered again. This process was repeated 5–7 times. Finally, the solution was concentrated to 2 ml which contained 0.5 mg/ml CNTs and 1% PLPEG . The optical absorption spectra and PL spectra were measured with a UV-vis-NIR spectrometer  and a spectrofluorometric equipment  equipped with a liquid nitrogen-cooled InGaAs array detector, respectively.18. A lamp  was placed obliquely above the mouse, and the NIR camera , above the mouse. An objective lens  and 1000 nm long-pass filter were used to exclude lights other than CNT fluorescence. The NIR camera exposure time was 100 ms.A home-built imaging system was used for the mouse whole body imaging and paraffin-embedded tissues as described previously18. It was equipped with a Xe lamp , a single-band bandpass filter , and a 785 nm laser single-edge laser-flat dichroic beam splitter . The specimen fluorescence passed through the 785 nm laser single-edge laser-flat dichroic beam splitter, an 808 nm best-value long-pass edge filter , and a 1100 nm long-pass filter , before reaching the NIR camera. This equipment also had the halogen lamp and CCD camera , by which visible-light transmission images were taken.The near infrared fluorescence microscopy was carried out using an epi-illumination-type optical microscope  was introduced in detail elsewhereFor NIR fluorescence microscopy, the formalin-fixed tissues were stained with nuclear fast red (NFR). NIR fluorescent intensity of the NFR reagent was low enough to observe the CNT fluorescence image by 708 nm excitation. To observe collagen bands, the formalin-fixed tissues were stained by silver impregnation.18, nude mice  were used. We used the nude mice because hairs of hairy mice diffuse light and make the imaging of mice body difficult. If hairy mice were used, hairs must be removed for imaging, when the skin were damaged and PLPEG-CNTs could accumulate at the damaged-skin site, influencing the biodistribution of PLPEG-CNTs. Before the PLPEG-CNTs injection, the mice were acclimatized to the institute laboratory for 2–3 weeks, and PLPEG-CNT was injected in mice from tail veins. Dose was 0.2 mL/mouse (about 5 mg/kg CNT/body weight). The mice were anaesthetized with isoflurane inhalation solution  for the whole-body imaging with the NIR camera. After the imaging, the mice were euthanized by sampling blood from the vena cava. The adipose tissues and organs were collected, which fixed using formalin or glutaraldehyde.As similar to the previous reportMice number used in the whole-body imaging was n = 3–5, and the histology study, n = 5.a.All the animal experiments were performed in accordance with the regulations approved by the Animal Care and Use Committee of National Institute of Advanced Industrial Science and Technology and Hokkaido University.ActB as an internal standard. Briefly, 2 µg of total RNA was reverse transcribed using an oligo (dT) 15-adaptor primer and M-MLV reverse transcriptase . Real-time PCR was performed using a fluorescence thermal cycler , with SYBR Green  employed as a double-strand DNA-specific dye, in accordance with the manufacture’s protocol. Primers used are listed in Supporting Data 16.Total RNA was extracted using RNAiso  according to the manufacture’s protocol. The mRNA levels were measured quantitatively by real-time RT-PCR, and normalized using mRNA expression of PLPEG-CNT  was mixed with 50% diluted murine sera (125 mL) in a 1.5 mL tube, which was kept rotated at 4 °C overnight. The reaction supernatants were subjected to Western blotting studies using a rabbit polyclonal anti-apolipoprotein B antibody , a rabbit polyclonal anti-apolipoprotein E antibody , or a rabbit monoclonal anti-bovine serum albumin antibody , along with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody . In some experiments, Can Get Signal® Immunoreaction Enhancer Solution  was used. Chemiluminescence reactions were performed using ECL Western blotting detection reagents  or SuperSignal™ West Dura Extended Duration Substrate . Visualisation and intensity measurements of protein bands were performed by development on X-ray films or using a C-DiGit® chemiluminescent Western blot scanner .18. Briefly, the tissue blocks with sizes of 1–2 mm were fixed with glutaraldehyde, stained with an aqueous solution containing 1% OsO4, embedded in in epoxy resin (Epon812), and sliced (thickness 80 nm) for the TEM observations. These observations were performed with an acceleration voltage of 80 kV .The detailed methods were described elsewhereSupplementary Information


Age-related hearing loss is associated with cognitive decline and has been proposed as a risk factor for dementia. However, the mechanisms that relate hearing loss to cognitive decline remain elusive. Here, we propose that the impairment of the cochlear amplifier mechanism is associated with structural brain changes and cognitive impairment. Ninety-six subjects aged over 65 years old  were evaluated using brain magnetic resonance imaging, neuropsychological and audiological assessments, including distortion product otoacoustic emissions as a measure of the cochlear amplifier function. All the analyses were adjusted by age, gender and education. The group with cochlear amplifier dysfunction showed greater brain atrophy in the cingulate cortex and in the parahippocampus. In addition, the atrophy of the cingulate cortex was associated with cognitive impairment in episodic and working memories and in language and visuoconstructive abilities. We conclude that the neural abnormalities observed in presbycusis subjects with cochlear amplifier dysfunction extend beyond core auditory network and are associated with cognitive decline in multiple domains. These results suggest that a cochlear amplifier dysfunction in presbycusis is an important mechanism relating hearing impairments to brain atrophy in the extended network of effortful hearing. Age-related hearing loss or presbycusis is characterized by bilateral progressive hearing loss and impaired speech understanding, especially in noisy environments . AccordiAlthough epidemiological and clinical studies confirm the relationship between hearing loss and cognitive decline, the mechanisms that relate them remain elusive . The patThere are two types of receptor cells in the mammalian cochlea: inner hair cells (IHC) and outer hair cells (OHC). While, IHCs are related to sensory transduction of acoustic vibrations, OHCs are responsible for the cochlear amplifier mechanism, which allows high sensitivity and sharp frequency selectivity to low-intensity sounds . In natuHere we hypothesized that a cochlear amplifier dysfunction in presbycusis – an OHC loss – is associated with cognitive decline and structural brain changes in the elderly population. We studied the possible associations between cochlear OHC function (measured by distortion product otoacoustic emissions [DPOAE]), cognitive performance, and brain structure  in a Chilean cohort of elders without dementia (ANDES) and with different levels of hearing loss.All procedures were approved by the Ethics Committee of the Clinical Hospital of the University of Chile, permission number: OAIC752/15. All subjects gave written informed consent in accordance with the Declaration of Helsinki.For this cross-sectional prospective study, a total of 142 patients aged ≥ 65 years (screened between 2016 and 2018) from Recoleta’s primary health public center located in the city of Santiago (Chile) were evaluated for recruitment. According to the inclusion and exclusion criteria of the Auditory and Dementia study (ANDES), 96 subjects were included for neuropsychological, audiological and brain imaging assessments. Inclusion criteria were the following: patients older than 65 years without dementia, which was evaluated by a mini-mental state examination (MMSE) score ≥ 24 and preserved functionality measured by the Pfeffer activities questionnaire . Exclusi®). In addition, in order to rule out conductive hearing loss, bone conduction thresholds were measured at 0.25, 0.5, 1, 2, 3 and 4 kHz. All audiometric data presented in the present work refer to air conduction thresholds. The PTA at 0.5, 1, 2 and 4 kHz were calculated for each subject in both ears. Subjects were classified according to their hearing level: normal hearing (≤20 dB), mild presbycusis (>20 and ≤35 dB), and moderate presbycusis (>35 dB) based on the PTA of the better hearing ear. Moderate presbycusis subjects were referred to the Chilean program for hearing aids for elders >65 years.All the audiological evaluations were carried out by an experienced audiologist in a double wall soundproof room located in the Otolaryngology Department of the Clinical Hospital of the University of Chile. Air conduction pure tone audiometric (PTA) hearing thresholds were evaluated at 0.125, 0.25, 0.5, 1, 2, 3, 4, 6 and 8 kHz for each subject in both ears using a clinical audiometer , presenting eight pairs of primary tones  in each ear at eight different 2f1–f2 frequencies: 707, 891, 1122, 1414, 1781, 2244, 2828 and 3563 Hz. n = 96), without eliminating subjects with no detectable DPOAEs (and no measurable amplitude in dB SPL), which is common in elderly subjects.Otoacoustic emissions are sounds emitted by the OHCs of the inner ear that can be measured through sensitive microphones . DPOAEs All subjects and their relatives were evaluated by a neurologist during a structured interview and graded according to their cognitive complaints using the clinical dementia rating scale . Cogniti®, Erlangen, Germany) equipped with a head volume coil. T1-weighted magnetization-prepared rapid gradient echo (T1-MPRAGE) axial images were collected, and parameters were as follows: time repetition (TR) = 2300 ms, time echo (TE) = 232 ms, matrix = 256 × 256, flip angle = 8°, 26 slices, and voxel size = 0.94 × 0.94 × 0.9 mm3. T2-weighted turbo spin echo (TSE)  and fluid attenuated inversion recovery (FLAIR)  were also collected to inspect structural abnormalities. A total of 440 images were obtained during an acquisition time of 30 min per subject.Neuroimaging data were acquired by a MAGNETOM Skyra 3-Tesla whole-body MRI Scanner . The function “recon-all” creates gross brain volume extents for larger-scale regions : total gray and white matter, subcortical gray matter, brain mask volume, and estimated total intracranial volume.To determine the structural brain changes of controls and presbycusis individuals, we measured the volume and thickness of bilateral cortical regions. FreeSurfer (version 6.0)Additionally, we measured the cortical thickness in native space using FreeSurfer tools. We calculated the cortical thickness of each mesh of vertices by measuring the distance between the point on one surface and the closest conforming point on the opposite surface. Then we measured the average of the two values calculated from each side to the other . Based oSPSS software version 20.0 was used to perform statistical analysis of demographic data, audiometric measurements, neuropsychological test results and structural MRI data. The ANCOVA method was used to compare cognitive performances and volume and thickness differences between the three tested groups including age, sex, and years of education as covariables. Pearson correlations were performed according to the distribution of data.p = 0.01  to contrast the difference of correlations between the studied groups using PTA as predictor for both volume and thickness. Age, education and sex were adjusted as covariates. The results were corrected for the multiple comparison using a significant alpha value of p = 0.01 . Regionsch space . In addin = 96, 63 female) was 73.6 ± 5.3 years [mean ± standard deviation (SD)] with an average educational level of 9.5 ± 4.2 years of schooling (mean ± SD). The audiometric hearing thresholds were normal in 32 subjects, while 44 and 20 individuals were considered as mild and moderate presbycusis, respectively. None of the 96 subjects used hearing aids at the moment of recruitment. A summary of demographic data, including age, sex, education, hearing level, smoking, and cardiovascular risk factors is presented in The mean age of the recruited subjects (n = 31); (ii) mild and moderate presbycusis  with preserved cochlear amplifier (PCF) (n = 33); and (iii) mild and moderate presbycusis  with cochlear amplifier dysfunction (<5 detectable DPOAE) (CD) (n = 31). One subject could not be classified within these criteria as he presented normal hearing with cochlear amplifier dysfunction and was excluded for further analyses.Next, in addition to the classification based on the cochlear amplifier status, we used the degree of hearing loss (PTA) to divide subjects into three groups: (i) Controls: normal hearing  with preserved cochlear amplifier function (≥5 detectable DPOAE)  = 3.92, p = 0.02) and postcentral gyri  = 3.92, p = 0.02), parahippocampus  = 4.55, p = 0.01) and amygdala  = 3.31, p = 0.04). Tukey post hoc tests showed significant differences between the CD and controls in the volume of precentral and postcentral gyri and parahippocampus. The only significant difference in brain volume between PCF and CD groups was obtained from parahippocampus  using ANCOVA models adjusted by age, sex and education. Structural volumes were normalized by the total estimated intracranial volume. The ANCOVA models for brain volumes yielded significant differences between the three groups in the precentral  = 3.04, p = 0.04), PCC F = 4.17, p = 0.01), precentral gyrus  = 4.00, p = 0.02), postcentral gyrus  = 6.43, p = 0.002) and parahippocampus  = 3.3, p = 0.04). Tukey post hoc tests showed that the thickness of precentral and postcentral gyri in the CD group were significantly reduced compared to that of the control group, while the thickness of ACC and PCC in the CD group were thinner than those of the PCF group  shown in red and blue colors in Next, in order to isolate the effect of hearing levels and cochlear amplifier dysfunction on brain structures, we ran GLM analyses. The GLM contrasted the difference of associations between PCF and CD groups using PTA as predictor for both volume and thickness. This method can effectively select voxels that correlate with PTA when contrasting PCF and CD groups. The whole brain analyses of volume and thickness related to cochlear amplifier dysfunction revealed significant correlations between PTA and different brain regions . Consequently, the majority of the study’s subjects have only mild to moderate presbycusis. Another limitation of the present work is that DPOAE loss, aging and brain changes could be associated with other peripheral and central auditory impairments that might affect cognition. These factors include IHC damage, auditory-nerve fiber loss and central auditory processing abnormalities, which could also be associated with DPOAE loss. Finally, the three groups are not perfectly matched by age and PTA. We control this limitation using these and other variables as covariates when needed. This control has its limitations, however, given our conditions, main conclusions should not be affected (see Supplementary Materials In non-demented patients with mild to moderate presbycusis, cochlear amplifier dysfunction was associated with brain atrophy and cognitive decline. We found structural changes in non-auditory brain areas related to the extended language processing network: the anterior and posterior cingulate cortices, sensorimotor areas and the parahippocampus. The dysfunction of this network is related to impairments in cognitive domains beyond auditory processing. Future longitudinal studies should consider measuring DPOAE, as a potentially early biomarker of cognitive decline and structural brain alterations.All procedures were approved by the Ethics Committee of the Clinical Hospital of the University of Chile, permission number: OAIC752/15. All subjects gave written informed consent in accordance with the Declaration of Helsinki.CB analyzed the data and wrote the manuscript. RV and SSM analyzed the data. AL, BM, and MM performed the data acquisition. CD and PD performed experimental design and data analysis, and edited the manuscript.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.