CDKL5 was reported in individuals with the atypical Rett syndrome, a neurodevelopmental disorder, suggesting that CDKL5 plays an important regulatory role in neuronal function. The disease associated with CDKL5 mutation has recently been recognised as CDKL5 deficiency disorder (CDD) and has been distinguished from the Rett syndrome owing to its symptomatic manifestation. Because CDKL5 mutations identified in patients with CDD cause enzymatic loss of function, CDKL5 catalytic activity is likely strongly associated with the disease. Consequently, the exploration of CDKL5 substrate characteristics and regulatory mechanisms of its catalytic activity are important for identifying therapeutic target molecules and developing new treatment. In this review, we summarise recent findings on the phosphorylation of CDKL5 substrates and the mechanisms of CDKL5 phosphorylation and dephosphorylation. We also discuss the relationship between changes in the phosphorylation signalling pathways and the Cdkl5 knockout mouse phenotype and consider future prospects for the treatment of mental and neurological disease associated with CDKL5 mutations.Cyclin-dependent kinase-like 5  is a serine/threonine protein kinase originally identified in 1998 during a transcriptional mapping project of the human X chromosome. Thereafter, a mutation in  CDKL5, has historically been linked to the atypical Rett syndrome. However, recently the disease associated with CDKL5 deficiency was distinguished from the Rett syndrome and identified as a unique disorder called “CDKL5 deficiency disorder (CDD).” In the current review, we discuss molecular- to individual-level analyses of CDKL5 dating back to its discovery.Protein phosphorylation is a chemical modification that plays a crucial role in many pivotal biological processes, including cell division, differentiation, and higher-order neural function. This reaction is catalysed by protein kinases. Defects in genes encoding protein kinases cause a myriad of different diseases –4. CycliMECP2), cyclin-dependent kinase-like 5 , and forkhead box protein G1 (FOXG1) were thought to be the primary genetic drivers of the condition [MECP2 mutation is the most commonly observed cause of the Rett syndrome, the disease caused by the MECP2 mutation is called the typical Rett syndrome, whereas that involving the other genes is historically called the atypical Rett syndrome  [CDKL5 and FOXG1 have resulted in unique diseases that are distinguishable from the Rett syndrome, since the specific symptoms of the disease vary depending on the causative gene involved [CDKL5 cause early life epilepsy [FOXG1 are known to cause characteristic stereotypic movements and severe microcephaly [CDKL5 and FOXG1 mutations are referred to as CDD  and FOXG1 syndrome, respectively. Because an effective treatment for these diseases is yet to be found, elucidation of the molecular signalling pathways controlled by the driver genes is an important prerequisite for the development of viable therapies.The Rett syndrome is an X-linked neurodevelopmental disorder first reported in the 1960s  and is eondition . Becausevariant) . Howeverinvolved –14. For epilepsy , 16, whiocephaly , 18. TheCDKL5 (STK9) was first reported in 1998 as a gene encoding a novel protein kinase situated on the X chromosome. It was discovered during a transcriptional mapping project [CDKL5 mutations and X-linked neurodevelopmental disorders was reported [CDKL5 as the causative gene of atypical Rett syndrome began to emerge [CDKL5 gene have been reported [CDKL5 result in the formation of proteins lacking the C-terminus. The C-terminal region contains a sequence vital to the regulation of the intracellular localisation of CDKL5, and the mutations result in abnormal CDKL5 localisation patterns, as observed in an in vitro transient expression study [CDKL5 expression if a premature stop codon triggers nonsense-mediated decay (NMD) of CDKL5 mRNA. CDKL5 fragment was not detected in the R59X nonsense mutation knockin mice [reported . From 20o emerge . Many nereported –25. As mon study . Furtherkin mice , indicatin vivo knockout analysis in mouse models. In the current review, we will outline the major findings of CDKL5 research and discuss possible future developments.Based on the above, CDKL5 is an important protein kinase strongly associated with CDD. Recently, much research has been dedicated to this protein, including target exploration and STK9, which codes for a serine/threonine (Ser/Thr) protein kinase, was cloned during a transcriptional mapping project of the Xp22 chromosomal region [STK9 shares high similarity with that of enzymes encoded by the CDK gene family [CDKL5. The length of the human CDKL5 is 228 kb, and the gene contains 27 exons. Reports of differential splicing indicate that various isoforms of the protein exist, including one that is strongly expressed in the brain and another expressed abundantly in the testes [In 1998, e family , the gene testes –31.CDKL5 gene product is a Ser/Thr kinase. Its placement in the molecular phylogenetic tree of protein kinases indicates identity with the CDK and mitogen-activated protein kinase (MAPK) families  , 48, we in vitro . Since Din vitro . CDKL5 pin vitro , 52. Thein vitro [NTNG1 is a causative gene of the atypical Rett syndrome [CDKL5-knockdown [Further research has also shown that Ser-631 of the netrin-G1 ligand protein  is phosphorylated by CDKL5 in vitro . NGL-1 iin vitro . Reportssyndrome –56, sincnockdown . Based oin vitro [In 2013, by using a unique substrate search method involving isoelectric focusing electrophoresis , Sekigucin vitro . The phoin vitro . Becausein vitro –60, and in vitro . Finallyin vitro , 63. Thein vitro CDKL5 substrate [N-methyl-D-aspartate (NMDA) receptor, a typical glutamate receptor, HDAC4 ensures the survival of nerve cells and is involved in various aspects of their functions [HDAC8, which plays an important role in neural differentiation, is a candidate causative gene of the atypical Rett syndrome [In 2016, histone deacetylase 4 (HDAC4) was identified as an ubstrate . By cataunctions . Typicalunctions . Furtherunctions . Loss ofunctions  and enhaunctions . Consequunctions . Howeversyndrome , 69. FurCdkl5-knockout neuron [Cdkl5-knockout neurons. Therefore, MAP1S phosphorylation promotes microtubule dynamic instability and inhibits plus-ends growth. A study has demonstrated that CDKL5 exhibited compromised ciliogenesis [in vitro CDKL5 substrate. Moreover, a microtubule-associated protein RP/EB family member 2 (EB2) [in vivo CDKL5 substrates. EB2 is one of the microtubules plus-end tracking proteins that may be involved in microtubule reorganisation [Very recently, two different research groups independently performed comprehensive screens for CDKL5 substrates and identified several candidate protein substrates , 71. Amoogenesis , which mogenesis , disc laogenesis , and Rhonisation . Therefoin vitro. SMAD3 is a transcription factor that plays a central role in the neuronal function mediated by transforming growth factor-β signalling. The authors showed that protein levels of SMAD3 are reduced in the cortex and hippocampus of Cdkl5-knockout mouse [In 2019, Fuchs et al.  found thut mouse . It is tut mouse , SMAD3 m2+/calmodulin-dependent protein kinase II (CaMKII), which preferentially recognises an Arg residue at the P–3 position (consensus sequence: RXXS/T) [One of the reasons for the scarcity of information on the protein substrates of CDKL5 is the lack of knowledge of the substrate recognition mechanism by CDKL5. The structural similarities between CDKL5 and MAPK and CDK initially prompted the researchers to consider it a proline-directed protein kinase . However RXXS/T) , 80; MAP RXXS/T) ; and dua RXXS/T) . However RXXS/T) . These o RXXS/T) . Ser/Thr RXXS/T) . In addi2+/calmodulin-dependent protein kinase kinase (CaMKK) and mitogen-activated protein kinase kinase (MAPKK), respectively [The activity of many protein kinases is regulated through autophosphorylation and phosphorylation by upstream kinases. For example, CaMKI and CaMKIV and MAPK are activated by phosphorylation catalysed by Caectively –85. Simiectively , whereasectively . Finallyectively –90.TEYVAT (phosphorylated amino acids are underlined). While it is believed that these residues are autophosphorylated, whether this process is truly important for kinase activation has long remained an open question [in vitro. Nevertheless, the importance of the Thr residue phosphorylation of the TEY motif remains unknown. In 2010, Chen et al. [in vitro stimulation by brain-derived neurotrophic factor. However, it is unclear whether this phosphorylation occurs at the Thr residue of the TEY motif or elsewhere; further analysis is needed to clarify this issue.CDKL5 contains an activation loop characteristic for MAPK and CDK, which contains the sequence ANYquestion . Recentlquestion  have anan et al.  reportedin vitro. Because DYRK1a is located in a region of chromosome 21 that is critical for the Down syndrome, it is believed to be closely involved in the abnormal neurodevelopment observed in that condition [in vitro NMDA stimulation causes CDKL5 translocation from the nucleus to the cytoplasm [Since the discovery of CDKL5 in 1998, no reports detailing the phosphorylation of CDKL5 by another protein kinase have been published. However, recently, Oi et al.  found thondition , 4. Dyrkondition , and it ytoplasm . The relin vitro NMDA stimulation and that this process is catalysed by protein phosphatase 1 (PP1). As mentioned above, since NMDA receptor-mediated signalling increases cytoplasmic localisation of CDKL5 in the hippocampal neuron [in vitro [Little information is available regarding kinases that phosphorylate CDKL5, and phosphatases that dephosphorylate it. However, in 2015, La Montanara et al.  showed tl neuron , it is bin vitro , suggestTo conclude, many of the mysteries surrounding the regulatory mechanisms of CDKL5 remain unsolved. In addition to the protein kinases that phosphorylate CDKL5, it is possible that the activity of CDKL5 is also regulated by binding proteins, second messengers, and even modulator proteins. The coming years should bring further insight into the mechanisms that regulate CDKL5 activity.CDKL5 mutations have been identified in CDD patients. In particular, recent advancements in the next-generation sequencing technology have enabled highly efficient identification of mutations, and mutation databases such as the Genome Aggregation Database (gnomAD) (http://gnomad.broadinstitute.org/) and the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) have been populated with many examples of such mutations [CDKL5 mutation, resulting in Y177C substitution, in a Japanese patient, and showed that it results in a complete loss of the CDKL5 enzymatic activity [To date, many utations , 94. We activity . Neverthactivity . It is thttp://mecp2.chw.edu.au/), a database that profiles mutations in MECP2, CDKL5, and FOXG1 [Here, we present mutations located in the catalytic domain of CDKL5 (including the Y177C substitution), extracted from RettBASE , a molecule acting downstream of AKT, leading to insufficient neuronal maturation and an increase in the number of apoptotic cells [β inhibitors SB216763 and tideglusib reportedly corrects the maturation deficiencies caused by the loss of Cdkl5 [β has also been reported in the Mecp2-knockout mouse; the phenotype is partially rescued by the inhibition of GSK3β [β through activation of AKT [β pathway is important as a therapeutic target for the Rett syndrome. CDD and the Rett syndrome have several common symptoms, and a novel common candidate drug for CDD and the Rett syndrome might be related to the AKT-GSK3β pathway.Although many mouse models have been analysed, the mechanisms that underpin these phenotypes are still unknown. In the aforementioned paper, Wang et al.  found thic cells . Furtherof Cdkl5 , 108. Fuof GSK3β . In a prof GSK3β . Therefon of AKT , 112. ThCdkl5-knockout mouse experiences severe NMDA-dependent epileptic seizures. As mentioned above, upon NMDA stimulation, CDKL5 is translocated from the nucleus to the cytoplasm, where this system is believed to phosphorylate proteins involved in synapse formation and neurotransmission, such as NGL-1 and Amph1. Further, CDKL5 binds to PSD-95 at the synapse and contributes to synaptic stability [Cdkl5-knockout mice is slightly challenging and needs to be considered. However, knockout and R59X knockin mice showed that CDKL5 deficiency reduced 5-HT2A receptor expression in the cortex [One of the major symptoms of CDD is epilepsy. Recently, Okuda et al.  reportedtability , 114. The cortex  and redue cortex , 115. Fue cortex . TherefoIn this review, we have described the phosphorylation signalling pathways in which CDKL5 participates and outlined the relationship between the CDKL5 signalling pathways and neurodevelopmental diseases. As CDKL5 plays many important roles in the nervous system, gene mutations leading to the loss of its activity are also believed to lead to the onset of severe neurological diseases. In practice, several pathogenic CDKL5 variants exhibit reduced or absence of phosphorylation activity , 41, 44.MECP2 is particularly well known as the primary causative gene of the Rett syndrome, and its relationship with CDKL5 has been the subject of much attention. As mentioned before, CDKL5 has historically been regarded as the causative gene of the atypical Rett syndrome. In CDD patients, seizures and sleep disturbances are more common than in the Rett patients, whereas features of regression and spinal curvature are less common [As shown in s common , 15, 16.s common , 118. Thin vivo substrate, the association between CDKL5 and microtubules has attracted attention of researchers. A previous study showed that CDKL5 deficiency interferes with the IQGAP1/cytoplasmic linker protein of 170 kDa (CLIP170)/Rac1 ternary complex formation and negatively influences the microtubule binding of CLIP170 [β-methoxy-pregnenolone), has been found to rescue morphological defects in CDKL5-deficient neurons by restoring the microtubule association of CLIP170 [As described above, since MAP1S was identified as an  CLIP170 . Further CLIP170 , 119. Th CLIP170 , 119. ThIn conclusion, CDKL5 is strongly related to the pathology of neurodevelopmental disorders. Detailed knowledge of its activity will promote the development of new therapeutic approaches to CDD and further elucidate the mechanisms underlying neuronal development and function. Therefore, an in-depth investigation into CDKL5 is warranted to establish an effective treatment strategy for CDD.


Height, weight, and RMR were measured. Multivariable fractional polynomials (MFP) were used to determine explanatory variables and their functional forms for the model. A variable shrinkage method was used to find the best fit predictor coefficients of the equation. The developed equation was cross-validated on an independent group. Weight and sex code  were identified as reliable independent variables. The new equation developed was RMR  = 284.5 + (13.2 x weight) + (133.0 x sex code). Independent variables of the prediction equation were able to predict 88.5% of the variance. Root mean square error (RMSE) of the prediction equation in validation and cross-validation was 88.11 kcal/day and 79.03 kcal/day, respectively. The equation developed in this study is suitable for predicting RMR in Sri Lankan adults.Resting metabolic rate (RMR) is the key determinant of the energy requirement of an individual. Measurement of RMR by indirect calorimetry is not feasible in field settings and therefore equation-based calculations are used. Since a valid equation is not available for Sri Lankans, it is important to develop a new population-specific equation for field use. The study objective was to develop a new equation for the prediction of RMR in healthy Sri Lankans using a reference method, indirect calorimetry. RMR data were collected from fifty-seven  adults aged 19 to 60 years. They were randomly assigned to validation ( The prevalence of obesity is increasing in Sri Lanka , which iRMR can be measured using direct or indirect calorimetry. However, the use of these methods at field level is not feasible due to high cost and the need for trained personnel . Since cEquations for the prediction of RMR have been developed in different populations during the last century following the early work of Harris-Benedict in 1918. A recent review reporting on 248 prediction equations showed that most of these equations for the prediction of RMR were developed on Western populations . The valCurrently, there is no population-specific equation available for Sri Lankans to predict RMR. It is important to either validate an existing equation on the target population or if the existing equations are not valid, develop a new population-specific equation for the target population. In the present cohort, ten selected equations including Harris-Benedict, Schofield, Mifflin St. Joer, Owen, and WHO and equations developed on Asians and Indians demonstrated significant overestimation in predicting RMR . UnsuitaA total of 57 healthy volunteers  aged between 19 and 60 years, residing in the Western province, were recruited to this cross-sectional study. Sample size was calculated according to Knofczynski and Mundfrom method for prediction model study . Pregnan2 (kg/m2).A single trained investigator did all anthropometric measurements under standard conditions using the International Society for Advancement of Kineanthropometry (ISAK) protocol . Weight ®, Italy). Subjects were instructed to fast for a minimum of 10 hours and also to abstain from alcohol, caffeine, nicotine, and vigorous exercises for 12 hours before the test. Female participants were advised not to schedule their sessions during menstruation days to avoid any effect of menstruation on RMR. A single investigator performed the calorimetric measurements according to standard protocol  + [0.85 × VO2 × 1.11]) × 1440] [The Cosmed Fitmate GS × 1440] . The gasR version 3.6.3. A data-splitting algorithm was used to randomly separate the data into validation and cross-validation datasets in a ratio of 2 : 1. Parameters were compared between validation (n = 38) and cross-validation (n = 19) groups using independent samples t-test. The significance was set at p < 0.05. The validation dataset was used to construct our prognostic model and to estimate the variable coefficients; the cross-validation data were used to validate the model.Data of all fifty-seven participants were included in the final analysis. All statistical analysis was carried out using the statistical package α1, and whether fractional transformation should be performed using α2 [R version 3.6.3 in the validation group.Normality and multicollinearity of the variables were evaluated. Association between dependent and independent variables were assessed by plotting scatter plots with regression line. Binary dummy variables  were used to code sex . Multivausing α2 . Explanaα [Variable shrinkage was used to estimate reliable predictor coefficients of the developed equation to reduce the risk of overfitting. Different penalties were applied to find the equation with the highest prediction accuracy. Predictor coefficients derived from least square regression were brought closer to zero by multiplying by a constant in ridge regression. This method keeps all predictors in the final model. Coefficients were brought to zero by adding or subtracting a constant in least angle selection and shrinkage operator (LASSO) method. This method ensures sparsity of the results by shrinking some coefficients to zero. Elastic net is a hybrid of ridge regression and LASSO by adjusting the values of hyperparameter α .R2 with minimum root mean square error (RMSE). The best model was cross-validated in an independent sample [The best prediction model among different penalty terms was selected by the measure of goodness-of-fit statistic. The best prediction equation should have a high t sample , 24. Blat sample  was usedThe Ethics Review Committee of the Faculty of Medicine, University of Colombo, approved the study protocol (EC-18-68), and all procedures followed were in accordance with the ethics standards of this committee. Informed written consent was obtained from all participants.The general characteristics of the participants in the validation group and cross-validation group are given in r = 0.840, p < 0.001), height , and BMI  of the validation group. Age  did not show a significant correlation with RMR as shown in All the continuous variables were normally distributed. RMR by indirect calorimetry (reference method) was significantly correlated with weight  with 10-fold repeated cross-validation method was used to estimate reliable predictor coefficients for the final model. Best model fit with least RMSE and highest R2 and RMSE obtained for the final model were 0.88, RMSE = 88.11 kcal/day, respectively. Final model was cross-validated in an independent cross-validation sample (n = 19). RMSE obtained for the cross-validation was 79.03. There was no significant difference between RMR by indirect calorimetry and RMR by the preliminary equation . RMSE obtained from the cross-validation group and validation groups were comparable.Prediction coefficients were obtained for best fit elastic net regression model where alpha was 0.1 and lambda was 1. The final model was RMR  = 284.5 + (13.2 x weight) + (133.0 x sex code). r = 0.35, p=0.1417).RMR predicted by the prediction equation was plotted against RMR assessed by the reference method IC . The BlaThe new equation developed and validated for the prediction of RMR in Sri Lankan adults fills a significant void in accurate but simple prediction methods for RMR. The independent variables included in the prediction equation showed a higher degree of association with the dependent variable than the variables that were not included. RMR measured by indirect calorimetry and RMR predicted by the equation correlated well. When the equation was cross-validated in an independent sample, it had a small bias and narrow limit of agreement, indicating very good performance.Cross-validation of existing equations, Harris-Benedict, Schofield, WHO weight, WHO height and weight, Owen, Mifflin St. Jeor, Henry, Liu, Ganpule and Indian equations developed by Soares et al.  and PierMFP approach was adopted in the present study to adjust the continuous variables , allowing for nonlinear functional forms. Weight and gender were the independent variables selected by MFP as the potential variables to predict RMR in this study. Allowing for polynomial functional forms in the present study, height, BMI, and age were not included in the model suggesting that these variables and their polynomials were not significant predictor variables. We used a variable shrinkage method to avoid the effect of overfitting and multicolinearity in the model. Predictor coefficients were tuned by ridge, LASSO, and elastic net regression in order to improve the predictability of the generalized linear model resulting from MFP. Variable shrinkage methods reduce the sampling variation and improve the generalizability of the model. Elastic net regression resulted in the best fit model. Predictor coefficients were estimates for this best fit model (prediction equation). Bias RMR and average RMR were not significantly correlated with each other suggesting that the equation does not result in higher bias for higher RMR and lower bias for lower RMR.n = 60) RMR for an Indian female, and Rao et al. [n = 21) and 1094 ± 238 kcal/day (n = 22) for young Chinese male and female, respectively [The RMR value of 1169 ± 184 kcal/day measured using indirect calorimetry in our study was comparable with values reported for Asians and South Asians. Piers and Shetty  reportedo et al.  reportedectively . It is wectively , 8, 26. ectively , 28. RMRr = 0.840, p < 0.001). This finding is in agreement with almost all the previously published studies on RMR, where body weight accounts for the highest variation in RMR [r = −0.015, p=0.9256) in our study though previous studies have reported its negative correlation with RMR [In this study, weight was the strongest predictor of RMR and had the highest correlation  in prediction accuracy [Most regression equations are based on easily measurable anthropometric parameters  while some are based on body composition parameters , fat mass (FM), and fat-free mass (FFM) . FFM hasaccuracy . Howeveraccuracy .The equation developed in our study was a combined sex code, weight-based equation, which can easily be used at field level. The equation developed by Almajwa and Abulmeat  includedPhysical activity and sedentary behaviour could have an impact on RMR . In our ® desktop indirect calorimeter as the reference method. Fitmate GS® system does not contain a CO2 sensor and it calculates CO2 production from O2 production by assuming 0.85 as the respiratory quotient (RQ). However, scientific evidence indicates that in a steady state, RQ is in the range of 0.84 ± 0.04 [The major limitation of this study is the use of Fitmate GS4 ± 0.04 . TherefoA new population-specific equation for the prediction of RMR was developed for Sri Lankan adults. The new equation performed well in Sri Lankan adults and showed good agreement with measured RMR. Therefore, this equation is suitable for the prediction of RMR in Sri Lankan adults. The newly developed equation is simple; its variables, combined sex code and weight, make it easy to use at field level.


Epidemiological studies identify midlife hearing loss as an independent risk factor for dementia, estimated to account for 9% of cases. We evaluate candidate brain bases for this relationship. These bases include a common pathology affecting the ascending auditory pathway and multimodal cortex, depletion of cognitive reserve due to an impoverished listening environment, and the occupation of cognitive resources when listening in difficult conditions. We also put forward an alternate mechanism, drawing on new insights into the role of the medial temporal lobe in auditory cognition. In particular, we consider how aberrant activity in the service of auditory pattern analysis, working memory, and object processing may interact with dementia pathology in people with hearing loss. We highlight how the effect of hearing interventions on dementia depends on the specific mechanism and suggest avenues for work at the molecular, neuronal, and systems levels to pin this down. Griffiths et al. evaluate candidate explanations for the relationship between hearing loss and dementia. As well as considering common pathology, impoverished input, and resource competition, they develop a novel account based on specific interactions between auditory cognition and dementia pathology in the medial temporal lobe. Hearing loss in midlife has been estimated to account for 9% of cases of dementia, a huge  disease burden given that dementia affects 47 million people worldwide . AcquireThe aim of this article is to examine brain bases for the relationship between hearing loss and dementia. We appraise mechanisms based on common pathology in the cochlea and brain, deterioration of brain resources due to an impoverished acoustic environment, and the diminished availability of cognitive resources that are occupied in support of listening in difficult conditions. A novel mechanism that we propose here, however, is based on a critical interaction between auditory cognitive processing in the medial temporal lobe (MTL) and dementia pathology. This suggested basis is informed by recent work on the role of the MTL in auditory cognition and our current understanding of the relationship between brain activity and the molecular underpinnings of dementia.Mounting evidence supports a link between hearing loss and dementia. The original study used in the longitudinal meta-analysis is instructive . It was Models of the brain mechanism by which hearing loss is linked to dementia must account for the epidemiological findings: an increase in the risk of developing dementia as a function of hearing loss in midlife, even after accounting for vascular risk factors, in which the cases generally meet the criteria for AD or vascular dementia.There are a number of possible mechanisms for the relationship between hearing loss and dementia, summarized in A first possible mechanism is common pathology affecting the cochlea and ascending auditory pathway (causing hearing loss) and the cortex (causing dementia). AD-related pathology has been observed in the retina , but it Vascular pathology can also occur in the cochlea, and this is one of the factors implicated in typical acquired hearing loss . It can A second possible mechanism is that hearing loss leads to the decreased stimulation of cognitive processing. The idea is that auditory deprivation creates an impoverished environment, particularly with the diminishment of speech and language input, that negatively affects brain structure and function. This change in brain structure and function is a risk factor for the subsequent development of dementia.A large number of animal studies, mainly on rodents, have demonstrated changes in brain and behavior deriving from the experience of enriched (as opposed to impoverished) environments. Structural changes can be seen macroscopically  and at the level of synapses, dendrites, somata, axons, glia, and vasculature . The question thus arises as to how the increased use of these resources in the short term contributes to the development of dementia, defined as a cognitive phenotype with specific and enduring pathology, generally based on testing with single tasks. In other words, how does the interfering effect of speech in noise on cognition persist and affect cognitive tests for dementia that are generally carried out without any distracting task? In our view, this mechanism is a more compelling basis for hearing loss as a potential cause for the wide range of cognitive deficits in elderly people  receptor  is a weak candidate, especially in the majority of incident dementia cases that are due to AD. The idea of a predisposition to dementia due to impoverished environment (mechanism 2) is supported by animal data that suggest precise neuronal and anatomical bases and human data that demonstrate structural changes due to hearing loss in cortex that is vulnerable to early AD. Mechanisms 3 and 4 can both be based on the increased use of general cognitive resources, with a substrate including the MTL, under difficult listening conditions. Mechanism 3 is based on increased activity alone, and is also a weak candidate basis for long-term dementia pathology. We favor mechanism 4, in which increased activity interacts with pathology in the MTL. The argument for this is based on auditory cognitive mechanisms that are relevant to difficult listening in the MTL and studies of the distribution of AD pathology. However, there is no direct evidence for the specific interaction between the two in the MTL that we propose. Finally, we should point out that the possible mechanisms are not exclusive. For example, impoverished auditory experience could cause structural changes in the MTL and a decreased cognitive reserve to protect against dementia (mechanism 2), after which there is a specific interaction between activity changes in the MTL and AD pathology (mechanism 4).The mechanisms differ with respect to the predicted effects of intervention that can be tested in human studies . In the We argue above for an effect of hearing loss that may be determined by an interaction between altered use of pattern analysis mechanisms in the MTL and dementia pathology. Those analysis mechanisms are used by different modalities, and one question that arises here is whether hearing loss per se is critical or whether the stimulation of those mechanisms by other modalities may overcome the increased risk. Of possible relevance is the effect of early hearing loss on dementia in prelingually Deaf signers who live in a signing environment— they have the auditory deprivation, but none of the concomitant social deprivation. Diagnosis is problematic in this group, but testing instruments exist , and morThere is an immediate question related to the best auditory processing measures to predict the development of dementia. Further work is required to assess whether measures of hearing loss or more complex measures of auditory cognition are the critical indicators. More complex measures could include speech-in-noise and possibly synthetic stimuli that assess fundamental mechanisms for grouping, segregation, and auditory working memory that may be relevant to speech-in-noise . HoweverA further question relates to types of dementia that are determined by hearing loss. The initial studies used standard criteria for AD and vascular dementia. Here, we developed a model based on the interaction between auditory cognitive mechanisms and dementia pathology that is only relevant to AD, making the distinction important. However, the studies of incident dementia to date have not addressed AD subtypes, and even the large studies included in the meta-analysis  may be uIncreasingly, clinical work has used biomarkers of AD pathology in cerebrospinal fluid  and braiFuture human work has the potential to define the exact nature of the relationship between hearing loss and dementia at the level of the key predictors related to hearing loss and the resultant syndrome. Definition of the underlying neuronal and molecular mechanisms, however, requires animal models. Rodent models of AD  are estaRodent models of deafness and AD, however, are some distance from the patients with hearing loss who may develop dementia and are seen in the clinic. Although hippocampal and MTL organization could be relatively similar between rodents and non-human primates (NHPs) , NHPs prWe started this discussion with a claim about potentially reversible dementia due to hearing loss. This immediately raises the issue of the effect of hearing interventions on the development of dementia. Some hearing aid and cochlear implant studies have measured cognitive outcomes , but theThese studies of treatment are important if our fourth mechanism above is correct. Under this model, changes in brain activity related to difficult listening cause irreversible degenerative damage based on interaction with molecular pathology. In that case, the term “preventable” may be more accurate than the term “reversible,” used by the Lancet Commission . A deepe


Among the 10–13-year-olds, 4PTA was 4.8 dB  and 41.4 dB . For the 14–20-year-olds, the 4PTA was 7.0 dB  and 48.2 dB . On average, hearing thresholds of the hearing impaired group increased, especially at frequencies between 1–3 kHz. In conclusion, 18% of children developed hearing loss, with a maximum hearing loss in the audiograms at 1–3 kHz. The percentage of children with hearing impairment increased from 10% at baseline to 18% at end of trial as did the severity of hearing loss.Most adults with Alport syndrome (AS) suffer from progressive sensorineural hearing loss. However, little is known about the early characteristics of hearing loss in children with AS. As a part of the EARLY PRO-TECT Alport trial, this study was the first clinical trial ever to investigate hearing loss in children with AS over a timespan of up to six years Nine of 51 children (18%) had hearing impairment. Audiograms were divided into three age groups: in the 5–9-year-olds, the 4-pure tone average (4PTA) was 8.9 decibel (dB) ( Alport Syndrome (AS) is a rare genetic disorder of type IV collagen formation leading to progressive renal failure, ocular problems and the development of high-frequency sensorineural deafness ,4,5,6,7.Pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes encoding for type IV collagen cause AS ,3,8. AS Type IV collagen is an important component in the structure and function of respective basement membranes in kidney, cochlear, and eyes. Type IV collagen, composed of six different α chains, assembles into three different heterotrimers , which are tissue-specific . The α1αIn the glomeruli of the kidney, absence or deficiency of α3α4α5 type IV collagen results in decreased mechanical stability and splitting of the GBM, finally leading to hematuria, proteinuria and to progressive renal failure . SimilarHearing loss due to AS has never been described as congenital, and patients would usually pass newborn hearing screening. It is usually first detected by audiometry in late childhood or early adolescence, presenting with bilateral reduction of sensitivity to mid and high frequencies ,19,20,21To clarify whether an even earlier start to therapy is safe and effective, the EARLY PRO-TECT Alport trial (NCT01485978) was initiated in 2012 . The triThe following results are based on 51 patients out of the 66 patients (77%) from the EARLY PRO-TECT Alport trial, from which additional data regarding hearing function were obtained . The 2 fHearing loss was diagnosed in nine of 51 patients (18%), while 39 of 51 children had a normal hearing ability (77%). The audiological report was not conclusive in three patients (6%). A previously normal-hearing eight-year old did not cooperate during audiometric testing; in a three-year old child, pure tone audiometry was not performed but the report suggests reduced amplitudes of otoacoustic emissions. In a third patient, only an ambiguous Transient Evoked Otoacoustic Emissions (TEOAE) without interpretation was transmitted.The youngest child with hearing impairment was a seven-year-old girl with XLAS. Mode of inheritance was X-linked in six and autosomal recessive in three of nine children with hearing loss. Hearing was assessed in eight of nine patients using audiograms. In one patient hearing loss was documented in the medical history without severity. Severity of hearing loss was determined by 4-pure tone average (4PTA) of the better ear . At baseline, one child had a mild hearing loss and four children had a moderate hearing loss. Three children developed hearing loss during the trial. All three patients developed a mild hearing loss, and one of three patients progressed to a moderate hearing loss within two years.p.(?); patient 93_05_06, classified as intermediate, c.2395G>A, p.(Gly799Ser); patient 93_06_08, classified as intermediate, c.2255G>A, p.(Gly752Glu); patient 93_07_06, classified as severe, c.4672C>T, p.(Gln1558 *); patient 93_10_01, classified as severe, deletion exon 22–28, p.(?); patient 93_13_04, classified as intermediate, c.1948G>C, p.(Gly650Arg)). Of the three hearing impaired children with ARAS, one child had a homozygous variant categorized as intermediate, one child had two heterozygous variants categorized as severe and one child had two heterozygous variants categorized as intermediate and less-severe ; patient 93_15_01, compound heterozygous COL4A4, classified as intermediate and less-severe, c.2662G>A and c.4922G>A, p.(Gly888Arg) and p. (Cys1641Tyr); patient 93_16_03, compound heterozygous COL4A3, classified as severe and severe, c.1398del and c.4348C>T, p.(Asp466Glufs * 32) and p. (Arg1450 *)).The genotype–phenotype correlations described for AS are based on the progression of the kidney disease. Half of the six hearing impaired children with XLAS had a variant categorized as severe and the other half had a variant categorized as intermediate  showed a median albuminuria of 45.8 mg albumin/gCrea, while median albuminuria of children with hearing impairment (n = 8) was higher  (p ≤ 0.05) between children with hearing loss and children with normal hearing.As all children with AS in the EARLY PRO-TECT Alport trial were at the early stages of renal disease with normal glomerular filtration rate, renal function was assessed by the amount of albuminuria: in 36 children an audiogram and a test for albuminuria were performed simultaneously. In two patients with normal hearing an albumin excretion was not available at the time of the audiogram. Patients with normal hearing (n/gCrea) . The logAt baseline five of the nine children with hearing loss were in stage I of AS ) and the remaining four hearing impaired children were in stage II of AS . Hearing loss was not observed in children in stage 0 . n = 15) for patients with normal hearing and 43.8 dB (n = 2) with impaired hearing. For the 10–13 years old children, the mean 4PTA was 4.8 dB for children with normal hearing (n = 12) and 41.4 dB for children with impaired hearing (n = 6). For the 14–20 year old children, the mean 4PTA was 7.0 dB  and 48.2 dB . Respectively among the 5–9-year olds, the proportion of hearing loss was lower (12%) compared to the 10–13- and 14–20-year olds (33% and 25%). The mean 4PTA of 14–20 year old children was higher in comparison to the 10–3 year old children. The follow-up of five hearing impaired children showed an annual progression of hearing loss between 0.3 dB up to 9.7 dB.In 38 children , one or several audiograms were available. The audiograms were divided into three age groups, 5 to 9, 10 to 13 and 14 to 20 years . 30 patiThe audiograms showed clear differences between children with and without significant hearing loss . ThreshoThe average tone audiograms of children with hearing loss showed a typical broad mid-cochlear dip with a maximum at 1–3 kHz (trough-type). The curve of the 5–9 year old patients has a maximum dip at 1–2 kHz. In the 10–13 year old patients, the dip spread to 1–4 kHz. In the oldest group of the 14–20 year old patients, there was additional high-frequency hearing loss at 6 and 8 kHz (plateau-type). The maximum hearing loss did not exceed 60 dB.Out of eleven audiograms with a 4PTA > 25 dB, six were classified as symmetrical and five as asymmetrical. From a total of 22 audiograms (eleven per side) from eight children with impaired hearing, the maximum hearing loss was in the mid-frequency range in 64% (14/22), and in 18% (4/22) it was in the high frequency range . In 18% In two patients we were able to analyze the course of hearing impairment over a time-span of 8 years and 4 years: Hearing loss of patient A was found before AS was diagnosed when he was eight years old and was treated with hearing aids. The first audiogram showed a symmetrical broad trough-type with a maximum at 1–2 kHz A. The mePatient B developed bilateral hearing loss during the trial. At the age of 8 years, the audiogram showed mild threshold elevations, but hearing loss was not significant according to WHO classification and TEOAE were present in the right ear for all frequencies and in the left ear at 2–4 kHz. The perception of monosyllabic words (Göttinger speech test 2) was slightly impaired with 80/90/90% correct discrimination 55/65/80 dB. At the age of 10 years, hearing loss had progressed especially in the right ear and hearing aids were prescribed. In addition to renal symptoms and hearing loss, the patient also suffered from dyslalia and hyperopia. Within four years, hearing loss (4PTA) increased by 35 dB (right ear) and 37.5 dB (left ear). The last audiogram at the age of twelve showed a symmetrical hearing loss with a broad trough-type pattern with a maximum at 2 kHz B.The EARLY PRO-TECT Alport trial evaluated the safety and efficacy of early therapy with Ramipril in children with AS. This study was the first clinical trial ever that investigated hearing loss in children with AS (as secondary end-point). The proportion of children with hearing loss in our trial was lower (18%) than described in the literature (about 40% of 11-year-old children with hearing loss) . In adulHearing loss in AS is never congenital and described as first appearing in late childhood or early adolescence ,19,20,21Based on these data, we would suggest audiometric testing in normal-hearing AS patients every three years. Children with minimal threshold elevations (4PTA between 16 and 25 dB) should be followed closely, as they have a higher risk of progression to manifest hearing loss. Hearing impaired patients should be fitted bilaterally with hearing aids for which routine technical and audiometric check-ups are typically performed at least once per year. Rehabilitation with hearing aids is usually successful and. despite the fact that the 2 kHz region is particularly important for speech perception, deficits in language acquisition are exceptional in AS. Our study has several limitations: first, due to limited financial resources in this Government sponsored trial, we only recommended a hearing test including 4PTA at the start and end of the trial and after three years but were not able to include this as part of the official study protocol. This translated to many hearing tests in most of the children, but not to a complete data set. Second, the EARLY PRO-TECT Alport trial included toddlers with a limited ability to perform hearing tests. Finally, the trial included children with AS at very early stages of disease. Therefore, our trial included a number of children, who are likely to develop hearing impairment later during their course of disease and their hearing impairment will develop over time. In conclusion, we expected the number of children with AS and hearing loss to be less in numbers than in a scenario with adult AS-patients.Hearing loss is greatest in the mid-frequency range with maximal hearing loss centered around 2 kHz. Hearing loss in AS used to be described as symmetrical ; howeverRegarding the mechanism of hearing loss, early human (and mouse) temporal bone studies demonstrate primarily atrophy of the stria vascularis, but also loss of inner and outer hair cells, and sometimes neural degeneration, whereas a later study described damage near the basilar membrane ,28,29,30Children with impaired hearing had higher amounts of albuminuria, which corresponds with the association between hearing loss and faster loss of kidney function in AS described in the literature . In our ClinicalTrials.gov, NCT01485978.The primary endpoints on renal function of the EARLY PRO-TECT Alport trial have been published recently . BrieflyChildren with definite diagnosis of AS aged between 24 months and 18 years and normal glomerular filtration rate were included in the trial.Stage 0: Microhematuria without microalbuminuriaStage I: Microalbuminuria: 30–300 mg albumin/g creatinine (gCrea)Stage II: Proteinuria: >300 mg albumin/gCreaStages of AS were defined as:Written informed consent was obtained from all legal representatives and from all patients, who were six years old or older. Children were either randomized, treated with Ramipril or placebo, or openly treated with Ramipril. Children needed to be untreated with an ACEi and to be in stages 0 or I of disease to qualify for randomization. ACEi-pretreated children, children on stage II of disease or those for whom legal representatives denied randomization could be openly treated with Ramipril. During the trial, clinical information about the patient and the medical history of the family were collected using a standardized questionnaire. Family history was considered positive when a family member mentioned any symptoms of hearing loss. All collected data were pseudonymized. In 51 of 66 patients, additional data regarding hearing function was obtained from medical reports from specialists in pediatric audiology or otolaryngology, including medical history and audiograms. The data regarding hearing function was collected in the context of regular patient care (hearing test every three years recommended). A pre-existing diagnosis reported in the medical history or an audiogram with a 4PTA > 25 dB of the better ear was classified as hearing loss. In 38 of 51 children, one or several audiograms were available. Genetic testing for the underlying Alport variant was performed in all children included in this present study.The 4PTA of the better ear was used to classify hearing loss according to the WHO criteria (1997) into mild (26–40 dB hearing loss (HL)), moderate (41–60 dB HL), severe (61–80 dB HL) and profound (>81 dB HL). The audiogram curves were divided into flat-type, trough-type and plateau-type configurations ,21. The Our long-term follow up data originating from a clinical trial confirm that inner ear deafness in children is a very important early sign of AS, which can also be considered to be prognostic factor for progressive kidney disease. Patients with Alport syndrome should have audiometric check-ups to ensure adequate early treatment with hearing aids, and check-ups should start just before elementary school age. In a child with hearing loss and hematuria, genetic testing should exclude or diagnose AS as underlying disease. Future studies should place a special focus on the sociocultural burden and pathogenesis of hearing loss in children with AS, which limits the quality of life before the actual kidney problems show up.


Using a probe-tube microphone to measure and adjust the real-ear performance of the hearing aid to match the prescription target is recommended and widely used in clinical practice. Hearing aid fitting software can approximately match the amplification characteristics of the hearing aid to the prescription without real-ear measurements (REMs), but using REM improves the match to the prescribed target. What is unclear is if the improved match results in a better patient-reported outcome. The primary objective of this review is to determine whether the use of REM improves patient-reported outcomes in adult hearing aid users.The review’s methods are in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines. MEDLINE, EMBASE, PsycINFO, CINAHL, Web of Science and CENTRAL via Cochrane Library will be searched to identify relevant studies. The review’s population of interest will include adults with any degree of sensorineural or mixed hearing loss who have been prescribed with acoustic hearing aids. The included studies should compare REM fitting to the initial fit provided by the manufacturer’s fitting software. Hearing-specific health-related quality of life is the primary outcome but secondary outcomes include self-reported listening ability, speech recognition scores, generic health-related quality of life, hours of use, number of required follow-up sessions and adverse events. Randomised and non-randomised controlled trials will be included. The risk of bias in the included studies will be evaluated using Down and Black’s checklist. The quality of the overall evidence will be assessed using the Grading of Recommendations, Assessment, Development and Evaluations tool.Ethical approval will not be sought because this systematic review will only retrieve and analyse data from published studies. Review results will be published in a peer-reviewed journal and presented at relevant scientific conferences.CRD42020166074. This review will be first to synthesise data that compare two commonly used approaches to fit hearing aids to prescription targets .A variety of outcome measures will be used to compare these two approaches.The implications of this review are important for the emerging category of over-the-counter and direct-to-consumer hearing aids that do not involve real-ear measures.We will include only studies that compare these fitting approaches among adults with sensorineural or mixed hearing loss, because children and those with other types of hearing loss may require different amplification characteristics.Grey literature will not be included .Hearing loss is the most prevalent sensory deficit, affecting more than 5% of the world’s population.5The most prevalent type of hearing loss in adults is sensorineural loss, accounting for more than 90% of all adults with hearing loss in the UK.9In the UK, hearing aids are fitted and prescribed by state-registered hearing health professionals. The selection of the most appropriate hearing aid is based on a combination of audiometric and non-audiometric factors . The optimal amplification characteristics for each hearing aid user are specified according to validated prescription formulae ,Numerous studies have shown that the amplification characteristics approximated by the hearing aid software are inaccurate and significantly deviate from prescription targets.Using REMs to match hearing aid output to prescription targets requires valuable clinical time, which could otherwise be used to deliver alternative health and hearing services .There is no published systematic review examining the evidence on whether the use of REM improves patient-reported outcomes in adult hearing aid users. Determining the effectiveness of hearing aids fitted using REM would help decision-makers and stakeholders either to continue recommending or to abandon routine use of REM during adult hearing aid fittings. This is an urgent knowledge gap for both stakeholders and clinicians, given that the cost of the REM system is relatively high and it consumes a large amount of clinicians’ valuable time. The findings have implications for the emerging category of over-the-counter or direct-to-consumer hearing aids. If fitting hearing aids using REM does not benefit patients, then one of the potential obstacles to people fitting their own hearing aids is overcome. However, if it is found to be beneficial, decision-makers should continue recommending the use of REMs and poorer outcomes will be expected for direct-to-consumer hearing aids.The objective of this review is to systematically evaluate the current evidence on whether the use of REMs to match the hearing aid’s output to one of the validated prescription targets improves outcomes in adult hearing aid users.The protocol of this systematic review has been preregistered with the International Prospective Register of Systematic Reviews. The systematic review’s methods are reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines.16The inclusion and exclusion criteria for studies are reported in accordance with participants, interventions, comparators, outcomes and study designs elements.Participants will include adults (≥18 years old) with any degree of sensorineural or mixed hearing loss. Studies that report only a qualitative description of age and threshold of hearing will also be included. If both children and adults are included in a clinical trial, the study will be excluded unless the data were interpreted and reported independently. Participants with other types of hearing loss  will be excluded.The intervention that will be included comprises conventional acoustic hearing aids that are programmed to a prescription target using a REM system. Assistive listening devices, hearables, personal sound amplification products and direct-to-consumer hearing devices will be excluded. Implantable devices , bone conduction hearing aids or contralateral routing of sound hearing aids will also be excluded.The comparisons of interest are hearing aids that are programmed to the manufacturers’ approximation of the wearers’ hearing loss without REMs .The primary outcome of interest is hearing-specific health-related quality of life .Randomised controlled trials and non-randomised controlled trials will be included. Case reports, conference abstracts, book chapters, dissertations, theses, reviews and clinical guidelines will be excluded.Studies that meet the aforementioned eligibility criteria will be identified using a systematic search strategy of the following databases: MEDLINE, EMBASE, PsycINFO, CINAHL, Web of Science and CENTRAL via Cochrane Library. No search restrictions will be applied in terms of the publication’s language, status and year.The reference lists of the included publications will be manually scanned to identify further studies. Using Google Scholar ‘cited by’ feature, publications that have cited any of the included studies will be screened to identify additional relevant articles. Prior to analysis, the searches will be repeated to identify any other relevant studies if there is a significant delay between searches and a manuscript’s submission for publication (>6 months).The search protocol and methods will be developed by a medical information specialist from Systematic Review Solutions. The search terms will be based on experts’ opinion, free text and controlled terms from Medical Subject Headings, Excerpta Medica Tree and CINAHL headings. The search terms will include many free text terms because truncations may not work well with phrases . The search strategies for all databases are reported in 10.1136/bmjopen-2020-038113.supp1Supplementary dataSearch results, including titles, authors’ details, publication years, publication journals and abstracts, will be extracted to EndNote V.X9 Reference Management software . The same software will also be used to remove any duplicates prior to the initial screening. Next, IA will export the title and abstract of all identified articles into an Excel spreadsheet, so that they can be easily screened against the eligibility criteria. The reason for any article’s exclusion can also be documented and assessed by KM and HD. Each article will be assigned a unique number that is linked to the full details of the article.The title and abstract of all identified studies will be screened independently by IA and KM to determine eligibility for inclusion. A further inspection will be used when there is a discrepancy between the two investigators; this will include assessing the full article. Any disagreement will be resolved by discussion and/or by consulting with the third author (HD). The full text will be retained and inspected by IA and KM for all articles that match the inclusion criteria. Any disagreements will be resolved via discussion or by consulting the third author. Following PRISMA recommendations,https://automeris.io/WebPlotDigitizer) when necessary.IA and KM will independently extract the data from the included studies. Should any discrepancies arise, these will be resolved through discussion or consultation with the third author (HD). The data will be extracted in a predesigned data extraction form adapted from the Cochrane Handbook.The assessment of the risk of bias will be conducted independently by IA, KM and HD. Any disagreements will be resolved using a majority decision. Given the limited number of randomised controlled trials in the field of audiology, it is anticipated that most of the extracted studies will be non-randomised controlled trials; therefore, the Downs and BlackA meta-analysis will be conducted if enough data are available . If the studies used the same continuous outcome measures, the mean differences will be calculated with their 95% CI. When different outcome measures are used, the standardised mean difference will be measured along with 95% CI. If the studies used similar dichotomous outcome measures, the risk ratio will be calculated. If the statistical heterogeneity is low, fixed-effect meta-analyses will be computed; otherwise, only the random-effect meta-analyses will be calculated. For each meta-analysis, the effect size estimate will be calculated using the generic inverse of variance. The effect estimate will be reported along with its 95% CI. Forest plots will be used to present these results. Asymmetrical distribution of continuous outcomes  will be assessed by subtracting the lowest possible value from the mean and then dividing it by the SD. A ratio below 2 or 1 either suggests or indicates a skewed distribution, respectively.If meta-analysis is not appropriate, the risk of bias of each individual study will be assessed and the findings will be systematically reportedThe existence of publication bias will be visually inspected using a funnel plot of the study size or precision as a function of intervention effect estimates.The percentage of variability between studies’ outcomes that is due to heterogeneity rather than random error will be computed using an I² statistics. Given that the absolute threshold of I² is meaningless, the results will be interpreted as either low (0%–40%), medium (41%–60%) or high (61%–100%) heterogeneity.The corresponding authors will be contacted, where necessary, if any of the data are missing. Data that are only presented in graphical forms will be extracted using the detailed method explained in the section Data collection process and data items. In case the SDs are missing and cannot be obtained from the corresponding authors, they will be estimated from the available data . The reasons for the missing data will be assessed to identify whether they are missing at random or not.When possible, subgroup analysis will be performed to determine whether age , severity of hearing loss and experience with hearing aids  are possible sources of heterogeneity.et al.The quality of each outcome measure will be rated as high, moderate, low or very low using a well-developed assessment tool, the Grading of Recommendations, Assessment, Development and Evaluations tool (GRADE).Sensitivity analysis will be administered to assess the robustness of the pooled estimates. That is, studies with a very high risk of bias  will be removed from the quantitative synthesis.Patients and the public were not formally involved in the development of the research question and outcomes. The findings will be displayed on the website of The Manchester Center for Audiology and Deafness. In addition, they will be shared with service user groups within the National Health Service and Action on Hearing Loss, the largest UK charity for deafness.Ethical approval will not be sought because this systematic review will only retrieve and analyse data from published studies. Review results will be published in a peer-reviewed journal, presented at relevant scientific conferences and disseminated on social media.


This article presents an overview of legal, methodological, organisational, financial, structural and technical aspects of the initial audiological measurement of newborns (screening), follow-up (diagnosis) and tracking the results (tracking) within the German newborn hearing screening programme. Universal Newborn Hearing Screening (NHS), as defined in the Children’s Directive , has beeThe aim of this article is to present some legal, methodological, organizational, financial, structural and technical aspects of audiological measurement in newborns (screening), tracking and diagnosis in Germany. The information provided here is based on the standards and protocols specified in the Children’s Directive  and the There were 792,131 live births in Germany in 2016 . We mighIn a narrow sense, newborn hearing screening is typically understood to consist of using a screening test within the first few days of a newborn’s life. This is a test which attempts to detect responses of the auditory system to auditory stimuli using objective methods with a previously-defined statistical probability. It is usually carried out at the maternity/birth centre.the detection of hearing disorders (separately for each ear) (defined as having hearing thresholds of at least 35 dB HL)detection within the first three months of lifethe initiation of therapy before the end of the sixth month of lifeThe Children’s Directive  defines This definition is in broad agreement with international guidelines . If a chTwo automated methods for testing hearing in newborn hearing screening are permitted in Germany: automated auditory brainstem response audiometry (AABR) and automated measurement of transient-evoked otoacoustic emissions (ATEOAE) ,6.With ATEOAE the acoustic response emitted by the outer hair cells in the inner ear in response to broadband auditory stimuli are measured by a microphone located in the auditory canal. AABR uses various auditory stimuli to induce electrophysiological activity in the auditory nerve and the brain stem and is detected using electrodes placed on the scalp.Both test methods are non-invasive and are harmless to the child and his/her hearing. They can be performed on a sleeping or very quiet child. The duration of the measurements themselves, not including preparation time, are on average significantly less than one minute. AABR measurement takes a little longer on average with almost all screening devices, due to the somewhat more complex preparation (mainly attaching the electrodes) and is also slightly more expensive than ATEOAE because of greater consumption of materials .Both procedures have a sensitivity of almost 100% (sensitivity is the percentage of newborns with a hearing disorder for whom the hearing disorder is indeed detected). AABR is regarded as the gold standard for the NHS since it has a higher specificity (the probability that newborns with no hearing disorder are indeed recognised as such) than ATEOAE and can also detect retrocochlear causes of hearing impairment .When used for screening, both test techniques are designed to determine whether the probability of an auditory response to a 35 dB HL stimulus being present exceeds a pre-specified statistical likelihood. If successful, a “pass” result is recorded. If proof of an auditory response is not obtained, it is mandatory to undertake a further test using AABR at the same hospital prior to discharge. A lack of auditory response on this further test requires a full diagnostic audiological investigation (“follow-up”) carried out at a specialist paediatric audiology clinic.A screening result requiring such specialist follow-up in the absence of other risk-factors does not necessarily suggest a high probability of a hearing disorder being present. In practice, a greater number of children fail both of the tests that constitute the NHS than are eventually diagnosed with a hearing loss at the follow-up stage.Newborn hearing screening should be performed during the first three days of life and each ear must be tested separately. This prescribed time frame refers to healthy babies born full term. In addition to the two-stage screening test using ATEOAE at first and AABR second, which is common in some countries, the German guidelines  also allParents/carers should be informed about the medical background of the newborn hearing screening programme and the screening test procedure, and have the right to refuse to have their child screened. The fact that they have been informed of this and the result of the screen should be entered in the child’s personal health record booklet alongside other documented early detection examinations .Measurement can be difficult or even impossible for various reasons, including the presence of vernix caseosa or amniotic fluid in the auditory canal, which can obstruct the conduction of sound to the inner ear. Screening on the second or third day of life gives this fluid chance to clear and is therefore considered an appropriate time frame.Independent of the evaluation of the measurement quality of the methods used, these legal requirements make it necessary for hospitals to have suitable screening devices (see the overview in N = 1008/standard deviation = 90.8/range 1 to 1547) were examined per screener in 2016 [Hearing screening is mainly carried out by nurses or midwives at the maternity/birth centre .At least 95% of children who failed the initial screen should receive an AABR rescreening at the same hospital before being discharged.The proportion of children with findings requiring follow-up after being discharged from hospital  should not exceed 4%.The Children’s Directive  lists thData from the hearing screening project in Westphalia-Lippe , which hAll devices allowing ATEOAE and/or AABR measurements are suitable as screening devices. The most common and suitable devices used in Germany are listed in Newborns have a legal right to newborn hearing screening paid for by the health insurance company. This is included in the remuneration paid to the care provider for an inpatient birth according to the German reimbursement system for hospitals (German Diagnosis Related Groups). Calculated total costs  amount to approximately 13–17 euros per examination . This amIf hearing screening is not carried out at the birth centre, it can be carried out as part of the child’s standard medical check-ups as defined in the Children’s Directive , by the The Institute of the Appraisal Committee , the bodMinimum requirements for medical follow-up centres have been defined by the Ear, Nose and Throat (ENT) Association and the Association of Phoniatricians and Paediatric Audiologists  and their respective scientific societies . These rear microscopy/otoscopytympanometry 226 Hz & 1000 HzTEOAEdiagnostic measurement of auditory brainstem responses (ABR) if necessaryLevel 1 centres comprise established specialists from the above-mentioned specialties or corresponding clinics with adequate facilities, who are able to carry out the following examinations:ear microscopy/otoscopytympanometry 226 Hz and 1000 HzTEOAE, ideally with additional frequency specific measurement of distortion product otoacoustic emissions (DPOAE)frequency-specific ABR with hearing threshold estimation possible for at least 2 frequencies, ideally with click stimuli for latency assessmentbehavioural observation audiometry for ages 0−6 months as a check on the plausibility of objective audiometry resultsLevel 1 + 2 centres are characterised by their specialist pediatric audiological profile and a higher audiological qualification. These are established specialists from the above-mentioned specialties or corresponding clinics with adequate facilities, who can carry out the following examinations:initiation and monitoring of hearing aid usecounselling parents/carerscarrying out or initiating an investigation into the etiology/cause of the hearing impairmentinitiation of early intervention support programmesfurther interdisciplinary diagnostics, if requiredIn addition, where hearing impairment is diagnosed, level 1 + 2 centres are able to carry out or initiate and monitor the following early intervention steps:As a rule, these diagnostic services are funded by health insurance companies.Where test results require follow-up, it is expected that the child and their parents/carers will attend a specialist clinic for medical follow-up as soon as possible, as described in For this purpose, HSCs (or “tracking centres”) coordinate the exchange of information between maternity facilities, parents/carers and follow-up specialists, as shown schematically in In order to ensure that the information from these sources  is correctly merged, many HSPs work with “screening IDs”, a unique number that is assigned to the child by the maternity ward.www.vdhz.org).There are currently 16 hearing screening centers (HSCs) across 13 states in Germany  and the follow-up clinics and the HSC (findings/diagnoses). HSCs are often quite complex and large telematic projects which require considerable additional work by staff members to check screening data, obtain addresses via inquiries at birth centres or registration offices, and sending reminder letters to parents/carers .In contrast to screening itself and specialist medical diagnostics, the Children’s Guideline makes no concrete statements about the structure and financing of the hearing screening centres. As a result, the financing and organisational structure is very different at each one, ranging from direct or indirect state support, financing through service contracts for quality assurance with the participating clinics, to combinations of the use of university resources together with donations or grants.These structural and financial differences have practical implications for the coverage, completeness of data and endpoint of tracking. Coverage depends on whether collaboration with HSCs is mandatory or voluntary for all hospitals, a factor which differs from state to state in Germany. The completeness of the NHS is influenced amongst other things, by the ability to access and compare data held by the HSCs against a birth register or a connection to other early examinations . The endpoint of screening  is in turn influenced by financial and legal framework conditions.Cost analysis of the hearing screening center in Westphalia-Lippe and data from Hessen  show thaThe German NHS programme was evaluated in accordance with the Children’s Guideline on behalf of the G-BA for the cohorts of children born in 2011 and 2012 . The finWhen considering process quality, the main objectives of the NHS were not achieved. The proportion of screened children (82.4% in 2011 and 79.5% in 2012) did not reach the target of 95%. The referral rates in both years (both 5.3%) were above the required 4%. If one looks at the quality of the results, the targeted maximum age of diagnosis (three months) was unfortunately not reached (medians of four and five months respectively). The calculated prevalence of permanent, connatal, bilateral hearing impairment (greater than 20 dB HL) was comparable to other countries, at 1.3 per 1000 live births ,22,23. ICriticism was directed to the low percentage of cases in which the second stage of screening was undertaken following referral for a second test on the first test before discharge from hospital in comparison with the requirements of the Children’s Guideline. This leads to a high rate of children with findings requiring follow-up  and therefore to a greater burden on the follow-up examination centers. One outcome from this evaluation was that the duration before diagnosis and initiation of therapy was significantly reduced with the introduction of nationwide newborn hearing screening. The considerable discrepancies in screening between regions in terms of the process, adherence to legal requirements, documentation and the definition of the required data set suggests that there is scope for improvement of all aspects of the NHS in Germany.In federally-organised countries, such as Germany, the interrelation and overlap of healthcare and data protection responsibilities between the federal government and the individual states, as well as between healthcare providers and funders, can lead to difficulties around the availability of the necessary clinical competencies required for the NHS and problems at the intersections of cooperation. Despite data protection law existing at the federal level, individual states have formed different interpretations of it. This has had concrete effects on the extent and duration of data storage permitted within NHS. Data merging has also been made difficult due to different states having differently defined tracking endpoints and the large number of software packages being used for tracking . The different structures and financing models of the various hearing screening centers have led to each having slightly different responsibilities and objectives. However, this has led to creative solutions being found to problems. The Association of German Hearing Screening Centers is trying to unify such issues, as already described above. This could be achieved by building consensus on uniform definitions  or by providing IT solutions to interface problems.The following section outlines one specific example of a problem which does not occur in centrally-organized projects or countries: tracking across project/state boundaries.If a child has been screened and the result is “refer”, parents/carers are expected to contact a follow-up center. This is usually a facility near the maternity/birth clinic which cooperates with the screening center .If the parents/carers attend a follow-up centre in another area of responsibility , the initial tracking center will not receive any feedback from the follow-up centre and will therefore identify the child as “lost to follow-up”.To solve this problem, a Tracking-ID server was developed  which alWe hope that this solution will be integrated into the tracking software of the various hearing screening centers throughout the country.


On 15 November 2019, the Government of Samoa declared a state of emergency for the country due to a measles epidemic. The following six weeks were marked by closure of schools, prohibition of children at public gatherings, intensive immunization campaigns, curfews, and re-distribution of health resources and services to manage the overwhelming influx of measles patients. The state of emergency was lifted on the 29th of December 2019. According to the Joint WHO/UNICEF Pacific Islands Measles Outbreak Situation Report (8 January 2020), there was a total of 5697 measles cases  [Samoa is now in a state of recovery. Although the measles emergency is over, the health system should now turn its attention to managing the complications that may arise following acute measles infection. This includes the permanent disabilities of hearing, visual, and cognitive impairment -4. It isResearch audiologist (author AK) is on assignment in Samoa for two years, and has previously published a viewpoint in this journal advocating for a public health approach to childhood hearing impairment in the Pacific Islands . The curMeasles outbreaks were also reported in Fiji, Tonga, and American Samoa during October-December 2019. The aim of this viewpoint is to highlight the Samoan experience, and to urge other Pacific Islands nations to review their advocacy, approach, and management for preventable disease and disability. The Sustainable Development Goal (SDG) Project offers an ideal platform to implement strategies aimed at improving immunization programs, as well as improving childhood developmental health through public health service delivery.According to the WHO Global Health Observatory, the first-dose measles immunization coverage among 1-year-old Samoan children in 2018 was only 31%, well below the recommended level of 95%  is a common ear disease that is often present secondary to measles infection . Given tAll measles survivors who present to the ENT Clinic will also receive a hearing assessment by the newly established Audiology Clinic. Children with OM usually present with a temporary hearing loss that will often resolve upon successful management of the ear disease. In addition to OM-related hearing loss, the measles infection itself may also cause permanent damage to the cochlear , and the resulting hearing loss is irreversible and often profound for both ears . Early iThe ENT Clinic used World Hearing Day 2020 (3rd March) to highlight measles as a preventable cause of permanent hearing loss. As well as providing key ear health messages aimed at reducing ear disease and associated hearing loss, this was an opportunity to promote the role of childhood immunizations for protection against childhood mortality, as well as irreversible conditions such as permanent deafness.The ENT Clinic would encourage specialist colleagues to similarly engage and develop a targeted surveillance/screening program for other preventable neurological disabilities. The Ophthalmology Clinic is advocating eye and vision assessments to identify any cases of keratoconjunctivitis, a major complication of measles that can lead to blindness. The current goodwill shown to Samoa to provide assistance in the post-measles recovery period may be maximised to implement long-term public health surveillance/screening programs, especially for children. These aims would contribute to achieving SDGs in Samoa regarding child health, education, and disability.The state of recovery in Samoa is an ideal opportunity to review immunization programs and other health promotion messages. The risk of permanent sensory disability among measles survivors should be addressed during recovery efforts through surveillance/screening programs for any ear/hearing and eye/vision disorders. The tragedy of the measles emergency in Samoa may serve as a catalyst to improve immunization coverage rates and reduce preventable childhood disabilities across the Pacific Islands.


Treatment of emerging contaminants, such as antimicrobials, has become a priority topic for environmental protection. As a persistent, toxic, and bioaccumulative antimicrobial, the accumulation of triclosan (TCS) in wastewater sludge is creating a potential risk to human and ecosystem health via the agricultural use of biosolids. The impact of microwave (MW) pretreatment on TCS levels in municipal sludge is unknown. This study, for the first time, evaluated how MW pretreatment (80 and 160 °C) itself and together with anaerobic digestion (AD) under various sludge retention times  and temperatures (35 and 55 °C) can affect the levels of TCS in municipal sludge. TCS and its potential transformation products were analyzed with ultra-high-performance liquid chromatography and tandem mass spectrometry. Significantly higher TCS concentrations were detected in sludge sampled from the plant in colder compared to those in warmer temperatures. MW temperature did not have a discernible impact on TCS reduction from undigested sludge. However, AD studies indicated that compared to controls (no pretreatment), MW irradiation could make TCS more amenable to biodegradation (up to 46%), especially at the elevated pretreatment and digester temperatures. At different SRTs studied, TCS levels in the thermophilic digesters were considerably lower than that of in the mesophilic digesters. The land application of biosolids is reported as the most sustainable, least expensive, and environmentally accepted disposal option for wastewater sludge management . WastewaTriclosan (TCS) is a broad-spectrum antibacterial and antifungal agent that is effective against both Gram-positive and Gram-negative bacteria as well as yeasts and molds . TCS is Municipal wastewater treatment plants (WWTPs) are generally designed to control easily/moderately biodegradable carbonaceous substances, nutrients and pathogens ; thus, eUntil now, much attention has been paid to the identification and quantification of TCS and its transformation products in aqueous matrices  ,19,37; hAs AD is the most widely used sludge digestion technology, especially for medium- and large-scale WWTPs, it is logical to focus on solutions that can enhance the removal of TCS during anaerobic sludge digestion. In this context, different sludge pretreatment technologies applied prior to AD  have been used for boosting sludge disintegration and improving digestion with several advantages over the conventional AD . ThermalThe main objective of this study was to investigate the occurrence and behavior of the persistent antimicrobial TCS and its potential transformation products in undigested and anaerobically digested municipal sludges, with and without MW pretreatment. In order to achieve these goals, the following tasks were applied: (i) An ultra-high performance liquid chromatography connected to triple quadrupole mass spectrometry (UHPLC-MS/MS) based analytical method was developed for the simultaneous detection and quantification of TCS and its five potential transformation products in sludge samples, and (ii) six bench-scale, semi-continuous flow anaerobic digesters were operated with municipal mixed sludge in order to determine how different MW pretreatment temperatures (80 or 160 °C), digester sludge retention times , and digester operating temperatures (35 or 55 °C) influence the levels of selected target compounds in anaerobically digested sludge. This study also assessed how MW pretreatment itself can affect these compounds in municipal wastewater sludge by batch sludge heating experiments.d-glucuronide, triclosan-O-Sulfate, tetra-III and penta, and 2,3,4-trichlorophenol) considered in this study could be detected in municipal sludge samples . As seen in n = 9 with TCS detected in 100% samples) taken from Canadian WWTPs. The TCS concentrations in the sludge were considerably higher in the samples collected in colder temperatures (4.2 ± 1.2 °C) in comparison to those collected in warmer temperatures (23.4 ± 2.4 °C). The levels of TCS were found between 630–3700 ng/g-dry in the cold season whereas between 250–910 ng/g-dry in the warm season. Tohidi and Cai ), and characterization of digesters performance via conventional parameters. For quantification of the target compounds in control digester feed (mixed sludge), a total of 15 independent undigested mixed sludge samples were analyzed (between the months of December to May). For sample preparation, after sampling, thickened WAS and fermented primary sludge samples were first mixed in the laboratory at a volume % ratio of 67:33, respectively (based on the plant’s operation), then the mixed sample was processed for TCS quantification (p-dioxin), the compounds selected for this study were based on the availability of native standards and their suitability for UHPLC analysis.Once steady-state conditions were reached during each operational phase (after a period corresponding to minimum three SRTs), anaerobically digested sludge samples (fication . Additiofication . Althougd-glucuronide (CAS# 63156-12-7), and triclosan-O-Sulfate (CAS# 68508-18-9) were purchased from Toronto Research Chemicals  while tetra-III (CAS# 63709-57-9) and penta (CAS# 53555-01-4) were obtained from Wellington Laboratories . 2,3,4-trichlorophenol  was acquired through Sigma Aldrich . Isotopically labelled triclocarban-d4  was purchased from C/D/N Isotopes Inc. . Additional information about target analytes are given in TCS  was acquired by Sigma Aldrich . TCS transformation products including triclosan O-β-U.S. EPA Method 1694 with some modifications [Sludge samples were prepared and extracted according to the acid fraction procedure of the ications . A flow ications  and Kor-ications .m/z) of TCS fragment ions as well as the fragment ions of several TCS metabolites made ultra-low detection and quantification limits difficult in such a complex sludge matrix.A Waters™ ACQUITY UHPLC coupled with a Waters™ Xevo TQD triple quadrupole mass spectrometer (MS) equipped with an electrospray ionization (ESI) probe was utilized for the simultaneous detection and quantification of TCS and its transformation products in sludge samples. Various methods for the extraction and detection of TCS and several TCS metabolites have been reported in literature. U.S. EPA 1694 served as the basis for sample extraction and provided a starting point for UHPLC and MS/MS methods. Numerous trials of various aqueous phase additives, UHPLC gradients, mobile phase modifiers, injection volumes, MS conditions etc. were performed to optimize the chromatography (peak shape and compound separation) and to maximize the sensitivity of the MS, while minimizing noise. However, environmental levels of TCS (non-spiked) and the limited biodegradability of TCS during AD resulted in even lower TCS metabolite levels that were challenging to quantify. Furthermore, the low mass to charge ratio  solution and an organic phase of UHPLC grade methanol. Analytes were separated using a Waters™ Ethylene Bridged Hybrid C18  column and matching guard column. MS acquisition was performed in multiple reaction monitoring (MRM) using the negative ion mode (ESI-) for each analyte. Retention times of analytes were within ±15 s of a native compound as per U.S. EPA Method 1694 . Further4 as an internal standard. Good linearity was yielded with a correlation coefficients (R2) ≥ 0.98 for all analytes. The instrument limit of detection (LOD) and limit of quantification (LOQ) were calculated using the signal-to-noise ratio of 3 and 10 or greater in native standards or 9 in samples, respectively. Recoveries for most analytes were within the EPA recommended range of 70%–130% with some exceptions [A multi-point calibration curve was prepared from native compounds to quantify each analyte . Quantification was performed by an isotope dilution technique using isotopically labelled TCC-dceptions . CompounThe daily rate of TCS mass exiting from the conventional and advanced AD system  was calculated with Equation (1):effM = Daily TCS mass loading in the effluent of the digester (ng/d),effQ = Flow rate of anaerobically digested sludge (digestate) (mL/d),effTS = Total solids concentration of anaerobically digested sludge (% by weight), andeffC = TCS concentration in anaerobically digested sludge (ng/g-dry).The reduction percentage of TCS mass loading exiting from MW-pretreated digesters compared to the respective control digesters was then calculated from Equation (2):control digesterM = Daily TCS mass loading in the effluent of the control digester (ng/d), andmicrowave pretreated digesterM = Daily TCS mass loading in the effluent of MW-pretreated digester (ng/d).+B, 2320 B, and 4500-NH3D, respectively. Digester biogas was collected in Tedlar® bags and was measured daily by a U-Tube type manometer, while its composition was analyzed by an Agilent 7820A Gas Chromatograph equipped with a thermal conductivity detector [Total solids, VS, COD, pH, alkalinity, and ammonia were analyzed according to Standard Methods  procedurdetector . Total Vdetector .The experimental data were analyzed with an analysis of variance (ANOVA) considering a 95% confidence interval (α = 0.05), using Minitab™ 17 statistical software.This study assessed the behavior of the antimicrobial TCS in MW-pretreated and anaerobically digested municipal sludges under various process conditions by batch MW pretreatment and semi-continuous flow AD experiments, respectively. The combined effect of AD coupled with MW pretreatment on the occurrence of TCS in wastewater sludge was studied for the first time. All digesters maintained steady-state operation during each of the three SRTs studied. Slight inhibition was observed in the thermophilic digesters operated at the 6-day SRT; nevertheless, these digesters continued producing daily biogas with a typical methane content of 65%. Initial batch pretreatment studies indicated that TCS levels in mixed sludge after MW pretreatment applied at 80 or 160 °C did not significantly change in comparison to un-pretreated sludge. Compared to conventional AD, the combination of anaerobic sludge digestion with MW pretreatment moderately favored the reduction of TCS levels in digested sludge. The advanced thermophilic digesters achieved up to 46% TCS reduction from digested sludge over the respective controls. Compared to the thermophilic temperature, the mesophilic digester temperature was less effective on decreasing TCS levels in digested sludge (up to 37%). Higher TCS concentrations were observed in undigested and anaerobically digested municipal sludges during colder sludge sampling periods. This study confirmed the impacts of seasonal temperature variations on the levels of TCS in municipal wastewater sludge. Although TCS concentrations varied seasonally, MW pretreatment was effective in decreasing TCS levels in anaerobically digested sludge when combined with AD.