Objective. This study aimed to examine hearing function in children admitted with bacterial meningitis to determine the risk factors for sensorineural hearing loss. Setting. The study was conducted in the audiology unit and paediatric wards of Kenyatta National Hospital. Subjects and Methods. The study involved 83 children between the ages of six months and twelve years admitted with bacterial meningitis. The median age for the children examined was 14. On discharge they underwent hearing testing to evaluate for presence and degree of hearing loss. Results. Thirty six of the 83 children (44.4%) were found to have at least a unilateral mild sensorineural hearing loss during initial audiologic testing. Of the children with hearing loss, 22 (26.5%) had mild or moderate sensorineural hearing loss and 14 (16.9%) had severe or profound sensorineural hearing loss. Significant determinants identified for hearing loss included coma score below eight, seizures, cranial nerve neuropathy, positive CSF culture, and fever above 38.7 degrees Celsius. Conclusions. Sensorineural hearing loss was found to be highly prevalent in children treated for bacterial meningitis. There is need to educate healthcare providers on aggressive management of coma, fever, and seizures due to their poor prognostic value on hearing. Deafness is one of the commonest serious complications of bacterial meningitis in childhood. In developed countries, approximately 10% of survivors of bacterial meningitis are left with permanent sensorineural hearing loss . Other cKenyatta National Hospital, KNH, is Kenya's national referral hospital. Estimates show that an average forty-five children are admitted into its pediatric wards each month with a confirmed diagnosis of bacterial meningitis.Behavioral tests of hearing may be used when an infant reaches the developmental age  of six months. Infants not at this level of development and some of those with more than one disability will need to be tested by otoacoustic emissions (OAEs) and auditory brainstem responses (ABRs). Unfortunately the equipment for these latter two sets was unavailable forcing the study to be carried out in children above 6 months of age using behavioural distraction testing.So far, in KNH no similar study had been undertaken to determine the prevalence, burden, and risk factors for hearing loss following bacterial meningitis in children admitted to KNH. The study involved 83 children  between the ages of six months and twelve years admitted with bacterial meningitis from the pediatric wards, KNH. All cases admitted within twenty-four hours of diagnosis, who met the inclusion criteria, were recruited every weekday evening during the 3-month study period.All participants fulfilled the following criteria: age of six months or older at the time of admission and confirmed diagnosis of bacterial meningitis. Bacterial meningitis was defined according to the World Health Organization (WHO) workbook recommendations based on laboratory findings, symptoms, or signs . Those eA full medical history was documented and factors relating to the patient and prior treatment documented by the principal investigator in the questionnaire. The history included the parents or guardians' assessment of hearing and any history of ear discharge or infection. However, there were no premeningitis audiograms, which is a potential confounding factor and weakness of the study. Otoscopy was done using a Riester hand-held otoscope and its aural speculums and the findings recorded. The children then underwent a thorough physical examination. Hematological and CSF study results were documented. The Glasgow Coma Scale, GCS, was used for the level of consciousness. All these findings were entered in the patient's data entry form. The relevant hearing assessment was done prior to discharge from hospital and two weeks after. This identified and excluded transient hearing loss. All participants' parents or guardians gave informed consent for the study. Five guardians declined to have their children involved in the study. The study protocol was approved by the institutional ethics and review committee of KNH.All patients completed age-appropriate hearing tests carried out by trained audiologists in the ENT audiology section in a sound- proofed booth. This was well lit with minimal littering to minimize the child's distraction. It was also well ventilated and large enough to accommodate the child and parent/guardian, tester, and distractor. The standard ambient noise was 35 dB.Children between six and twenty-four months of age underwent the distraction test, those between twenty-four and thirty-six months of age had the performance tests done, while pure tone audiometry was carried out on children above thirty-six months of age. They were carried out in the following manner.Behavioral Testing. The behavioral testing equipment included toys that were not too bright used by the distractor to distract the child being examined. Three warblers were used: low, mid, and high frequency types. A Manchester rattle was also used to deliver high frequency sounds during behavioral testing. The G-chime was used for mid-frequency sounds and the C-chime for high frequency sound generation.Distraction Test. A distraction test was performed if the infant was sitting and able to turn and locate the source of a sound. It was carried out with the infant sat upon an adult's knee facing forwards where a distractor controlled the infant's attention using toys. The tester introduced the sound signals at high, mid, and moderate frequencies from 45 degrees and one meter behind, at the level of the ear. These were tested separately in order to detect hearing loss restricted to one part of the frequency range. The sounds were introduced at very quiet levels (35 dBA). Care was taken not to give clues as to the tester's position other than the test signal.Performance Tests. The child was shown how to wait until a sound was heard before carrying out an action. Once this could be done, the test was to be performed at a meter distance and from behind. The test was performed using “Go” for low frequencies, “S” for high frequencies, introduced at the quietest voice levels or 2 Warble tones at 500 Hz, 1, or 2 kHz, and 4 kHz introduced at a very quiet level corresponding to normal hearing. The child was said to have “passed” the screen if there were two responses at the quietest level. (1) Pure Tone Audiometry. This was carried out in all children recruited for the study who were above thirty-six months of age. Pure tones  were introduced using headphones and testing carried out by air conduction (500 H–4000 Hz) and bone conduction (500–4000 Hz). Pure tone audiometry was performed using an Interacoustics clinical audiometer . Sound delivery and masking during pure tone audiometry was ensured using TDH-399 headphones.(2) The main outcome measure was presence or absence of sensorineural hearing loss in the children.If the child had made a complete recovery from meningitis, lived far from the hospital, and had no hearing loss, followup was not done.If the child had sequalae that required further management beyond 2 weeks, followup was continued. For those children exhibiting hearing loss, whether conductive or sensorineural, recommendations for management and followup were based on specific test findings and varied accordingly.Descriptive statistics  were calculated to describe the population. The main outcome was degree of sensorineural hearing loss as measured by the age-appropriate hearing test. This had three levels of outcomes: normal, mild/moderate, and severe/profound. The population proportions and the 95% confidence interval, CI, were estimated for each category of outcome, using statistical methods to give more precise estimates. All categorical variables were cross-tabulated with the outcome and Pearson Chi Square computed. Streptococcus pneumoniae (10/17)  admitted with bacterial meningitis during the study period were analyzed.  (10/17) .  no cultured organism;Streptococcus pneumoniae  (10); other  (7)Neisseria meningitidis—4,Haemophilus influenzae—3.On CSF microbiology, three categories were included:Streptococcus pneumoniae, Haemophilus influenza, and Neisseria meningitidis were isolated in 10, 4, and 3 children, respectively. The latter two were classified as “other” microorganisms in comparison with S. pneumoniae. There were no distinctly different clinical presentations among children with S. pneumoniae, H. influenza, and N. meningitidis meningitis. CSF culture findings are illustrated in Only seventeen (20.5%) of CSF specimens examined cultured any bacteria. A significant number of patients enrolled developed sensorineural hearing loss as a sequalae (36/83). The overall prevalence of SNHL loss was estimated using bootstrap with 1000 repetitions to be 43.37% . Of the children with hearing loss, 22 (26.5%) had mild or moderate sensorineural hearing loss, and 14 (16.8%) had severe or profound sensorineural hearing loss. The prevalence of specific categories of SNHL is presented in The results of the univariable ordinary logistic models are presented in In the univariable multinomial logistic regression agitation, fever, seizure, and a positive CSF culture were associated with moderate SNHL, while only seizures were associated with severe SNHL. Estimates could not be obtained for some variables as there were too few numbers within the various categories of the variable.From the final multivariable multinomial regression, seizures and positive CSF culture predicted both moderate and severe SNHLs, while agitation predicted moderate SNHL. Interestingly, the ordered logistic models allowed for the inclusion of more variables which were otherwise dropped out of other models. After adjusting for age and sex, these models suggest that seizures, positive CSF culture, agitation, presence of cranial nerve palsy, and a coma score below eight strongly predicted both moderate and severe SNHL. These models probably provide the most efficient and consistent results. The assumption of proportional odds was not violated by any of the ordered logistic models fitted. Thus, an assumption that there was some ordering in the severity of SNHL seems sensible and appropriate. That is to say that moderate SNHL is proportionally worse than normal, and severe SNHL is proportionally worse than moderate SNHL. Thus, the models provide, for instance, the ordered odds ratio estimate of comparing those with positive CSF culture with those with no growth given that they had a certain severity of SNHL, when other variables are held constant. In this example, those with a positive culture are at an eightfold increased odds of having moderate SNHL compared to those with a negative culture, after controlling for other factors.Age, nutritional status, gender, number of siblings, caregiver level of education, length of illness prior to admission, and stiff neck were not found to be significant risk factors for hearing loss.S. pneumoniae and H. influenzae developed hearing loss, while two of three children with positive culture for N. meningitidis developed hearing loss.All children with positive culture for Lowered CSF glucose and elevated CSF protein were both found to influence development of hearing loss although this did not reach statistical significance.In the present study, children proven by history and CSF findings to have bacterial meningitis were evaluated and found to have a sensorineural hearing loss prevalence of 43.4% 95% CI). This was greater than findings of previous studies. Kutz et al. reported an incidence of 14%  consiste5% CI. ThConsistent with most prior studies, this work did not reveal any relationship between occurrence and severity of hearing loss to the male gender. Forty-nine (59%) of the children evaluated were males, while 34 (41%) were females. There were no differences in the prevalence of hearing loss between the two groups. Kutz et al. showed that being male was a significant independent risk factor for hearing loss .Early age at illness was identified by Grimwood et al. as a significant risk factor for hearing loss, with children suffering from meningitis before twelve months of age performing more poorly than children suffering from meningitis later in infancy and childhood, as well as age-matched controls, on measures of language and reading skills . HoweverStreptococcus pneumonia, Haemophilus influenza, and Neisseria meningitides, were 59%, 23%, and 17.9%, respectively. A study in KNH revealed that in 82% of the cases, the cerebrospinal fluid cultures were bacteriologically positive [S. pneumoniae (45%), N. meningitidis (14%), and H. influenzae (12%). Other studies describe distinctly different clinical presentations between these different causative organisms. This studies' low pick-up rate may have reduced the power to determine the same. Fortnum found no differential risk of hearing impairment by causative agent in one study from Nottingham, UK [Only seventeen 20.5%) of cerebrospinal fluid specimens were reported as positive for microorganism culture. The isolates, positive . Common 0.5% of cS. pneumoniae meningitis developed hearing loss. However, a very low proportion of CSF specimens (20.5%) cultured bacteria and so this may be an overestimate. Richardson et al. showed that the incidence of sensorineural hearing loss in children with S. pneumoniae meningitis was 36% [All children with  was 36% . PatientSeizures and a coma score of less than eight were found to be the most significant determinants for hearing loss. Thirteen children (15.7%) had coma scores of less than eight on admission and all but one developed hearing loss. Seizures occurred in 64 (78%) of the patients. Woolley et al. found no correlation between hearing loss and seizures or hearing loss and altered level of consciousness . SeizureConcurrent cranial nerve neuropathy was found to be a strong predictor for the subsequent development of hearing loss, with all eight of the children 8/83; 10%) with cranial nerve neuropathy developing hearing loss. The only cranial nerve neuropathy found was Abducens nerve palsy. The presence of a cranial nerve neuropathy is certainly a sign of a severe infection and is highly correlated with the development of hearing loss. Of the patients with hearing loss and cranial nerve neuropathy, seventy-one had at least a severe sensorineural hearing loss. Kutz et al. found that 6% of the patients developed a cranial nerve neuropathy, and all but three were found to have hearing loss [/83; 10% S. pneumococcus were found to have a significantly higher CSF protein level. Another potential sequalae of low CSF glucose is direct damage to the cochlea neuroepithelium.Decreased CSF glucose and elevated CSF protein as risk factors for hearing loss did not reach statistical significance. Decreased CSF glucose was the most consistent predictor of hearing loss, as illustrated by Woolley et al. and in previous studies , 16, 19.P = 0.13). Radetsky and Kilpi et al. found that the length of history of nonspecific signs and symptoms did not correlate with outcome [Presence on fever above 38.7 degrees was a significant risk factor for hearing loss with 22/54 children recording these high fevers developing hearing loss. Duration of illness beyond one week prior to admission and caregiver level of education were not found to be significant determinants for hearing loss following bacterial meningitis. Reports of the effect of delay in treatment on hearing outcome differ , 20–22.  outcome , 24. KutMalnutrition was not found to have any significance in the development of hearing loss. Poor nutrition is associated with a poor outcome from meningitis, and a low weight for age was associated with a poor outcome in a study by Molyneux et al. .Hearing loss is highly prevalent in children treated for bacterial meningitis in Kenyatta National Hospital with a prevalence of 43.4%. Strong risk factors for hearing loss following bacterial meningitis include coma score on admission of less than eight, development of seizures, concurrent cranial nerve neuropathy, positive CSF culture, and fever above 38.7 degrees Celsius. This is similar to the findings of studies done elsewhere.Length of illness prior to admission was found not to be a determinant of hearing loss following bacterial meningitis. This is contrary to other studies done.Age at illness, male gender, malnutrition, and primary caregivers' level of education were found not to be significant determinants of hearing loss following bacterial meningitis. The prior three were found in other studies to be significant predictors for the development of hearing loss.Lowered CSF glucose and elevated CSF protein are minimally correlated with development of hearing loss following bacterial meningitis. This is different from other studies that place lowered CSF glucose high on the list of most significant predictors for hearing loss following bacterial meningitis.There exists a need for objective hearing assessment in infants and young children following bacterial meningitis. This should be mandatory in all patients treated for bacterial meningitis.


Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants.Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and  Exploitation of the specificity of antibodies’ recognition of antigenic targets is the core of immunodiagnostic, immunotherapeutic and vaccine technologies. B-cell epitopes, which are recognized by antibodies or B-cells, can be divided into linear or conformational. For linear epitopes of polypeptides, the binding site is typically 10–15 contiguous residues on the antigen’s molecule A different challenge is cross-reactivity between epitopes, that is, those shared between two or more antigens, which otherwise can be distinguished by their type-specific epitopes. Meeting this challenge means teasing out the distinctions between broadly cross-reactive responses, limited cross-reactions among clusters of variants of the same protein, and the truly type-specific responses. More refined understanding of cross-reactive antibody binding between polymorphic antigens could guide the process of selecting the most informative subsets of variants for diagnostics and multivalent subunit vaccines. But is it possible to parse out the limited cross-reactivity from the broad cross-reactive responses?Borrelia burgdorferi. OspC is a surface-exposed lipoprotein that elicits an immunodominant antibody response early in infection ospC genotypes prevalent in any given geographic area range between 10 and 15 One suitable model system to explore these issues is the binding of antibodies to the highly polymorphic protein OspC of the Lyme disease (LD) agent B. burgdorferi. This inference would be potentially useful for clinical management because B. burgdorferi strains, which are definable by their ospC genotypes Despite this evidence of OspC–type specific immunity and for type-specific epitope antibodies, a single OspC type in immunodiagnostic assay preparations has provided for reasonably good sensitivity B. burgdorferiOur approach to this challenge began with development of a protein microarray displaying purified recombinant proteins of several naturally-occurring variants of OspC in North America. Microarrays have been used to probe immune responses to proteomes of several human pathogens Notations and abbreviations used throughout this article are detailed in B. burgdorferi antigens.The 55 patient sera comprised 12 samples from early LD, 25 samples from patients with disseminated and late disease stages, and 18 samples from LD patients with persistent oligoarticular arthritis. All were seropositive by standard criteria for the diagnosis of LD by whole-cell ELISA and then confirmatory immunoblot. Sera from patients were significantly more reactive than sera from controls against each of several antigens. The mean  for array binding in pixels per spot for patient sera and control sera were 11,748  and 2,818  for the B31 strain whole cell lysate; 1,905  and 114 (91 to 147) for Decorin Binding Protein B (DbpB); 5,011  and 416 (251 to 691) for the flagellin FlaB; and 2,818  and 977  for the VlsE outer membrane protein, respectively. 10, rank and binary transforms.The protein microarray developed for this study displayed 23 different OspC variants including types K, A, B, N, and U, which were the most prevalent in nymphal ticks in the northeastern U.S. in a recent survey r was used as an indicator of in vitro antibody cross-reactive binding between OspC proteins, and the r values calculated for each possible pairing populated the cross-reactive antibody binding correlation matrices (DM) shown in 10, rank and binary); the respective numerical r values are available in the Each LD serum sample showed positive antibody binding to more than one OspC type present on the array. The correlation coefficient Pearson’s r from the three matrices. For sera from patients with LD, the OspC pair A, I3 had the highest cross-reactivity value in all three matrices, followed by the pairs I, M; C3, M; C3, E3; H, I3 and C3, I. The remaining 247 OspC pairs had average r values <0.80, with a frequency count for the following ranges: 38 pairs with r values between 0.70–0.80, 94 between 0.60–0.70, 73 between 0.50–0.60, 33 between 0.40–0.50, and 9 between 0.30–0.40. The complete list of pairwise OspC cross-reactivity values for the sera sets studied is provided in r values with mean of near zero, an indication that the correlations found for the observed values are indicative of the range of antibody binding to OspC proteins resulting from the specificity of immune response and not by chance. Histograms of the correlations from the actual data matrices and the randomized matrices are presented in The 20 most cross-reactive OspC pairs are shown in MSA) of the 23 OspC proteins is presented in MSA were used to calculate the sequence identity for each OspC pair, and the resulting values were used to populate the global amino acid sequence identity matrix . On average, OspC proteins shared 72 (68–76)% in their amino acid sequences for the processed protein, with identity ranging from 90% (OspC types F and I3) to 63% (OspC E and OspC L). The values for the complete S-GlobalM matrices are provided in OspC proteins have both conserved and variable regions of amino acid sequences amongst types. The multiple sequence alignment  and the cross-reactive antibody binding (DM) between OspC pairs was calculated, with resulting correlation values for r of 0.16, 0.07, and 0.07, for the log10, rank and binary transforms, respectively. This result was an indication that the degree of amino acid identity shared between two OspC proteins does not account for most of the observed cross-reactive antibody binding between them.The relationship between the global amino acid sequence identity . The highest correlation value (0.34) was obtained for the C-terminal region beginning with the MSA index position 146 (OspC A index position 170). The high correlation associated with the C-terminal portion of the molecule is not sensitive to the precise cut off point used to define the terminal region, as correlation values greater than 0.30 were observed for all C-terminal regions between MSA indices 118 and 153.This analysis was repeated focusing on the N- and C-terminal regions of the OspC molecule, by calculating the correlations between the cross-reactivity matrices and sequence identity matrix using only the terminal regions. MSA index positions 127 and 128 (OspC A indices 152 and 153), the correlations calculated using the N-terminal section produced r values of 0.02, −0.09, and −0.09, for the log10, rank and binary transforms, respectively; whereas for correlations using the C-terminal section, the corresponding values were 0.29, 0.31, and 0.28. This was evidence that the C-terminal region of OspC accounts for much of the cross-reactive antibody binding observed.For the individual cross-reactivity matrices, when the break point was set between S-LocalM) and the cross-reactivity matrices. The highest r values resulted from a window size of 7 residues centered on position 182 in the fifth helix, and were 0.38, 0.39 and 0.30 for the log, rank and binary transforms, respectively. The heat maps summarizing these results are presented in The relationship between regions of more divergent sequence across OspC variants and cross-reactivity between pair members was calculated using local sequence identity matrices , and is located in the center of the fifth and last alpha helix, as highlighted in β atoms of residues 179 and 188 is 14.8 Å, as determined by Chimera UCSF The sequence window of 7 residues in the restricted r values using all-versus-all OspC types. However, when the relationship between local sequence identity and antibody cross-reactivity were calculated for an individual OspC type versus all others, the correlation between cross-reactivity and positions 179 through 188 of the fifth helix is more evident. For instance, for OspC type A, the r values were 0.75, 0.75, and 0.73 for log, rank and binary transforms, and the corresponding values for OspC type D3 were 0.67, 0.65, and 0.60. The central position of the 7-residue window most correlated with antibody cross-reactivity is shown on the solvent-accessible surface model of the 3D structure constructed from the MSA presented in r value for each OspC protein in white boxes; the corresponding source values are provided in Until now all calculations considered S-Local3DM) was generated and correlated with the averaged cross-reactivity matrices. The highest r value, 0.38, was found using a sphere with predicted diameter of 8 Å, which encompassed the polymorphic positions 56, 63, 180, 181, 182, 184, 186, and 188 of the OspC A index. Positions 56 and 63 are part of the first helix, while the remaining 6 positions are in the fifth helix. The fifth helix positions are the same as 6 of the 7 positions (the exception being position 179) that were identified by the sequence scanning approach as being most highly correlated with cross-reactivity. All correlation results using sphere sizes 4 to 40Å are available in To assess the relationship between cross-reactivity and a subset of residues in close proximity to one another in 3-dimensional space, a sequence identity matrix using only the residue cluster  were between I3 and A, further evidence of the immunodominance of the fifth helix over global sequence identity.For two sets of sera examined, from patients with LD and from le pairs . In FiguB. burgdorferi and asked whether cross-reactive antibody binding was influenced by the degree of global identity at amino acid level or by specific smaller regions of the protein. To this end, we performed an automated systematic analysis of the relationship between the variation among subsets of positions adjacent in sequence or 3D space and the experimentally observed antibody cross-reactivity produced by a set of sera from individuals with documented LD. We found that cross-reactivity between specific pairs of OspC proteins is determined by sequence identity at positions 179 through 188 of the C-terminal fifth alpha helix, rather than how much global identity is shared between the pair.We described here a computational protocol for analysis of the binding of antibodies to a diverse population of variants of an antigen presented in an array format. The set of proteins are homologous but diverse enough to feature both type-specific epitopes and cross-reactive epitopes. Accurately distinguishing cross-reactive epitopes from type-specific epitopes on the basis of amino acid sequence is a challenging problem. Our analytic approach automatically generates testable hypotheses regarding which specific sets of residues of the full-length protein comprise immunodominant linear or conformational epitopes. As a model system for development of the protocol, we used 23 variants of the polymorphic OspC surface-exposed protein of B. burgdorferi could bias cross-reactivity results; however, multiple strain infections seem to be uncommon in humans A limitation of the study was that the infecting strain (or strains) for patients with LD was not known. If the infecting type was known for each sample, then the quantitative measure of cross-reactivity between pairs of OspC variants could be calculated more directly using only samples infected with specific types. Additionally, the possibility that patients could be infected with more than one strain of in situ protein at the surface of a living bacterium. Presumably all antibodies directed against an OspC protein are not equal in their effector functions, such as direct neutralization or opsonization. Moreover, as previous studies of strain-specificity of protective immunity have indicated Another limitation of the study was the dependence on an assay that measures binding of antibody to purified protein on a matrix and not to an Thus despite the near ubiquitous reactivity to the conserved N-terminal first helix of the OspC protein Plasmodium falciparum antigens are promising targets for analysis due to the established links between antigen polymorphism and development of resistance only after exposure to many circulating strains, and the ongoing large-scale effort to investigate immune responses of individuals and populations suffering from malaria Although our study represents the broadest effort to determine the immunodominant regions responsible for cross-reactive antibody binding between variants of the OspC protein, the bioinformatics approach described here can be applied in the study of polymorphic immunodominant antigens in other human pathogens. For instance, http://download.igb.uci.edu#ospc.The two types of data that are necessary for performing the cross-reactivity analysis for a set of variants are: (1) quantitative measurements of antibody binding to each variant for multiple patient samples and (2) a multiple sequence alignment of the corresponding sequence variants. For structure scanning, a 3D structure model is also required. The methods for performing the systematic terminal region scanning, sequence scanning, and structure scanning are implemented in a suite of Perl scripts. These scripts, as well as sample input and output files from the OspC project, are publicly available at P. leucopus experimentally-infected with B. burgdorferi isolates HB19, Sh-2-82, IDS, TBO2, WQR and 27577 and the 7 control sera were described in detail in P. leucopus  were inoculated intraperitoneally with fresh CB-17 SCID mice plasma containing host-adapted B. burgdorferi cells. Animals were terminally exsanguinated 5 weeks post-infection. Samples were kept frozen at −80°C until use.The 55 sera from adult patients with different stages of LD and the 25 sera from naïve adults were described in detail previously Sera from human donors were originally collected for other studies for which informed consent had been obtained; patient identifier information had been removed. Rodent serum samples were obtained as described in ospC alleles cloned. ospC ORFs coding for protein sequence without signal peptide were cloned into pXT7 expression vector containing an amino-terminal 10X-Histidine fusion tag, using the in vivo recombination cloning method pLysS E. coli cells transformed with pXT7-ospC plasmids were cultured in Terrific Broth  supplemented with kanamycin until reaching OD600 0.4–0.6. Recombinant protein expression was inducted with IPTG  and further incubation for additional 4 hours. Cells were harvested and the supernadant containing His-tagged OspC fusion protein was incubated with Ni-coupled magnetic beads  for protein purification. Recombinant protein purity was estimated to be 80–90% by densitometry of Coomassie Blue-stained protein bands on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, and concentration was determined by BCA Protein Assay kit . Purified OspC protein samples were aliquoted and stored at −80°C until use. For details on recombinant protein purification, refer to BL21(DE3)Purified recombinant-OspC proteins were printed on nitrocellulose-coated glass FAST slides  using an Omnigrid 100 apparatus , in duplicate spots and in approximately 10 pg and 30 pg of protein per spot. Protein storage buffer alone was printed on the array to serve as a background signal control.P. leucopus) in Protein Array Blocking (PAB) buffer  supplemented with 10% (vol/vol) DH5α E. coli lysate . Incubation and washing procedures are described in Peromyscus leucopus IgG heavy and light chain  were used to detect sera antibody binding to OspC proteins. Probed array slides were scanned in a Perkin Elmer ScanArray Express HT and output RGB TIFF files were quantitated using ProScanArray Express software  with spot-specific background correction. The array data is deposited in NCBI’s Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE45996).Serum samples were diluted 1∶200 (LD patient sera) or 1∶100  the mean, standard deviation, 95% confidence intervals and z-scores of antibody binding intensity to each OspC type were calculated for the LD patient, P. leucopus and respective control sera groups.Inclusion criterion for cross-reactivity analysis was a minimum reactivity corresponding to a z score of 2 to at least one OspC protein. For analysis of antibody binding to OspC proteins on the microarray the following steps were taken: (i) raw values of antibody binding measured as the mean pixel intensity of spots of printed protein were logx the row of all individual raw reactivity values is denoted by xD . For a given pair of OspC variants the Pearson’s correlation r between the corresponding rows of serum reactivity values was used as the quantitative measure of cross-reactive antibody binding. The correlations were calculated using three forms of transformed data: log10 ; rank  to highest reactivity value (55 for human sera or 23 for P. leucopus sera)); and binary . For each of the transforms, all possible pairwise correlations between two OspC types were calculated and saved in the corresponding antibody cross-reactivity matrices: D-logM, D-rank, MD-binaryM. Please refer to Abbreviations section in For a given OspC type MSA) of the OspC proteins was assembled using PSI-BLAST MSA comprises residues 31 to 206 of OspC A using the indexes of Kumaran et al. MSA were used for all global and local amino acid sequence identity calculations and the aligned gaps between OspC pairs were counted as identities.A draft multiple sequence alignment (MSA was constructed using Modeller 9.1 β atoms (Cα atoms for glycine) in the model were used to define inter-residue distances for determining spatial clusters of residues.A 3-dimensional model of the D-log, MD-rank, and MD-binary) were compared to the global sequence identity matrix  and the similarity was summarized by the Pearson’s correlation of the paired matrices: r, r, and r. Similarly, local sequence identity matrices  were calculated systematically using subsets of positions that were adjacent in sequence or in 3-dimensional structure  and compared to the cross-reactivity matrices.The three cross-reactivity correlation matrices , r, and r. The process was repeated for all odd window sizes from 3 to 115 positions. For individual types (e.g. OspC A vs. the other 22 types) the relationship between cross-reactivity and local sequence identity was calculated restricted to corresponding rows of the matrices  and a fixed window size of 7 polymorphic positions.Sequence windows were defined using only the 116 polymorphic positions in polyMSA. Each position was used as the central residue for defining a cluster of residues in 3-dimensional space where membership in the cluster was defined by proximity of less than 4 Å to the central residue. The correlations between the corresponding sequence identity matrix calculated using the residue cluster  and the cross-reactivity matrices were calculated, using distance thresholds of 4 to 40 Å in 4 Å increments.Structural clusters of residues were defined using only the polymorphic positions in Figure S1Box plot of antibody binding to OspC proteins by sera from patients with LD and healthy controls. The intensity of antibody binding to each OspC protein on the microarray is shown for patient (blue) and control (white) sera. Each box indicates the first and third quartiles, and the line inside the box is the median. The 1.5x interquartile range is indicated by the vertical line (whiskers) bisecting the box, and values outside this range are indicated by dots.(EPS)Click here for additional data file.Figure S2Multiple sequence alignment (MSA) of conserved and variable positions among 23 OspC types. Strictly conserved positions are shown in gray; positions with variability are colored according to the number of residues that do not match the consensus residue at a given position. Color scheme is presented in the image key. The portion of the fifth C-terminal helix most correlated with cross-reactivity is outlined with a red box. OspC A index, position of residue in the crystal structure of OspC A MSA index, residue position relative to MSA; Secondary structure, as described for OspC A, showing alpha helices α1 through α5 (EPS)Click here for additional data file.Figure S3Heat maps of correlation between local sequence identity and antibody cross-reactivity for individual OspC types. All results were calculated using a window size of 7 positions (excluding conserved positions). The green to red gradient bar indicates the range of r values observed . White boxes around individual residues indicate the highest r value for each OspC type. Each panel shows results from the calculation performed using the 3 transforms: log10-transformed, rank and binary, on the left, middle and right panels, respectively.(EPS)Click here for additional data file.Figure S4Purified OspC proteins. Coomassie-blue stained SDS-PAGE gels showing the migration of affinity-purified recombinant OspC proteins expressed and purified for this study. The OspC type, designated alphanumerically, is shown on the top of the figure. The migrations of molecular weight markers, in kilodaltons, are shown on the left-most column.(EPS)Click here for additional data file.Table S1Antibody binding by sera from patients with LD and controls to conserved B. burgdorferi proteins and B31 strain whole cell lysate.(DOC)Click here for additional data file.Table S2Individual serum sample information and pixel intensities for antibody binding to OspC proteins on the microarray.(XLS)Click here for additional data file.Table S3Pearson’s correlation r10, rank, binary data transforms and their average .(XLS)Click here for additional data file.Table S4Ranking of pairwise cross-reactivity r values between OspC proteins, calculated for sera from human patients with LD and P. leucopus rodents infected with B. burgdorferi.(XLS)Click here for additional data file.Table S5Pairwise global amino acid sequence identity matrix between 23 OspC proteins , in percentage.(XLS)Click here for additional data file.Table S6Pearson’s r values of correlation between local sequence identity matrix using polymorphic positions and the antibody cross-reactivity matrices .(XLS)Click here for additional data file.Table S7Pearson’s r values between local sequence identity and antibody cross-reactivity matrices for individual OspC types.(XLS)Click here for additional data file.Table S8Pearson’s r values between antibody cross-reactivity matrices and local sequence identity in 3 dimentional space , using distance thresholds of 4 to 40 Å.(XLS)Click here for additional data file.Table S9Sources of genomic template for ospC allele cloning.(DOC)Click here for additional data file.Table S10Nucleotide sequences of PCR primers used for cloning and sequencing of ospC alleles.(DOC)Click here for additional data file.Text S1Detailed information on Materials and Methods.(DOC)Click here for additional data file.


The phantom auditory perception of subjective tinnitus is associated with aberrant brain activity as evidenced by magneto- and electroencephalographic studies. We tested the hypotheses (1) that psychoacoustically measured tinnitus loudness is related to gamma oscillatory band power, and (2) that tinnitus loudness and tinnitus-related distress are related to distinct brain activity patterns as suggested by the distinction between loudness and distress experienced by tinnitus patients. Furthermore, we explored (3) how hearing impairment, minimum masking level, and (4) psychological comorbidities are related to spontaneous oscillatory brain activity in tinnitus patients.Resting state oscillatory brain activity recorded electroencephalographically from 46 male tinnitus patients showed a positive correlation between gamma band oscillations and psychoacoustic tinnitus loudness determined with the reconstructed tinnitus sound, but not with the other psychoacoustic loudness measures that were used. Tinnitus-related distress did also correlate with delta band activity, but at electrode positions different from those associated with tinnitus loudness. Furthermore, highly distressed tinnitus patients exhibited a higher level of theta band activity. Moreover, mean hearing loss between 0.125 kHz and 16 kHz was associated with a decrease in gamma activity, whereas minimum masking levels correlated positively with delta band power. In contrast, psychological comorbidities did not express significant correlations with oscillatory brain activity.Different clinically relevant tinnitus characteristics show distinctive associations with spontaneous brain oscillatory power. Results support hypothesis (1), but exclusively for the tinnitus loudness derived from matching to the reconstructed tinnitus sound. This suggests to preferably use the reconstructed tinnitus spectrum to determine psychoacoustic tinnitus loudness. Results also support hypothesis (2). Moreover, hearing loss and minimum masking level correlate with oscillatory power in distinctive frequency bands. The lack of an association between psychological comorbidities and oscillatory power may be attributed to the overall low level of mental health problems in the present sample. Tinnitus is an auditory percept that does not originate from a physical sound source but is generated within the auditory system. Therefore, a subjective tinnitus is heard only by the affected individual. Cochlear hearing impairment is seen as a permissive if not a necessary condition for tinnitus As a consciously experienced, often continuous, and prominent signal tinnitus should be represented in the spontaneous activation pattern of the cortex. In line with this assumption, magnetoencephalographic (MEG) studies showed that the presence of tinnitus is associated with increased gamma band activity in the auditory cortex (AC) At the same time alpha oscillatory activity is decreased in subjects with tinnitus compared to non-tinnitus controls In the clinical setting a variety of audiological tinnitus characteristics are measured of which tinnitus loudness and tinnitus maskability are particularly important for the patient and the therapist. Tinnitus loudness is determined by different matching procedures, but the results of these measurements are not always satisfactory because they do not necessarily represent the patient's subjectively perceived tinnitus loudness. Minimum masking level on the other hand describes the minimal noise level that is necessary to eliminate the tinnitus perception, and represents a patient's ability to effectively use environmental sound to control the tinnitus perception. While there have been reports on correlates of tinnitus loudness in oscillatory brain activity, electrophysiological correlates of tinnitus maskability and the underlying mechanism remain unclear. In our study we set out to test the following hypotheses on tinnitus and spontaneous oscillatory brain activity:Hypothesis (1): Loudness of the tinnitus sound correlates with gamma band oscillatory power during absence of external auditory stimulation. Conventionally, tinnitus loudness is measured by a variety of audiological matching procedures : Tinnitus loudness and tinnitus-related distress are associated with distinct spontaneous brain oscillations. From patient reports it is evident that tinnitus loudness and tinnitus-related distress are distinct characteristics of the tinnitus In the exploratory part of the present study we focused on the following aspects:(3) We explored the association between oscillatory band power and hearing loss as well as minimum masking level, which are both highly relevant for patients. The relevance of MML has been outlined above and hearing impairment is seen as a permissive, although not sufficient condition for the establishment of tinnitus (4) Finally, we explored how psychological comorbidities that often accompany tinnitus Since gender differences and oversensitivity to external sounds (hyperacusis) might influence resting state EEG power distribution The present study was approved by the ethics committee of the Medical Faculty Mannheim (Ethikkommission II) of Heidelberg University according to the principles expressed in the Declaration of Helsinki. Subjects were acquired by newspaper advertisements and consecutively enrolled in the study. All subjects of the patient and the control group were informed about aim and scope of the study and gave written consent. All participants were males and right handed.Mean age of the 46 tinnitus patients included in the study was 54.8 years (range 22 to 68 years) and it did not differ from that of the control group  subscales indicating unproblematic psychological conditions. Mean age was 50.4 years (range 25 to 62 years), and hearing loss between 0.125 kHz and 16 kHz averaged to  , Table 1Thresholds were measured in 1 dB steps with pure tones at the standard frequencies of the audiogram (range from 0.125 kHz to 10 kHz) and in addition at 11.2 kHz, 12.5 kHz, 14 kHz, and 16 kHz . Mean hearing loss (MHL) was calculated by averaging across all frequencies Uncomfortable loudness thresholds (UCL) were recorded at the standard frequencies. For this purpose the sound pressure level of each pure tone was presented at hearing threshold and its level was increased continuously until the sound became uncomfortable. Subjects indicated UCL by pressing a button.Minimum masking levels (MML) were determined with white noise at the tinnitus ear. In cases of bilateral tinnitus MML was determined for each ear. White noise was presented at hearing threshold and increased in 1 dB step sizes until it masked a subject's tinnitus, which the subject indicated by pressing a button.Tinnitus reconstruction was based on psychoacoustic tinnitus spectra as described earlier Amplitude modulated tinnitus was approximated byAveraged across all tinnitus participants, similarity of the reconstructed tinnitus sound to a patient's own tinnitus reached an average similarity index of Overall five different tinnitus loudness estimates were determined . The firith  see .Third, in order to account for recruitment phenomena Tinnitus-related distress was evaluated with the Tinnitus Questionnaire  and connected with the recording room via a glass window. Participants were seated comfortably with uncrossed arms and legs in an armchair that had a head-rest. They were instructed to relax and to avoid any movements.Eyes were closed during EEG recording, and analysis was confined to resting EEG recorded for 120 s. A cap  with 22 sintered Ag/AgCl surface electrodes was placed at the standard positions of the extended 10–20 system  and referenced to linked ear lobes. The electrooculogram (EOG) was monitored with 4 sintered Ag/AgCl surface electrodes . Impedances were checked to be below 5 kOhm and the sampling rate was set to 512 Hz. EEG signals were acquired by two cascaded 24 bit biosignal amplification units . EEGs were inspected for indicators of sleep such as spindles, enhanced theta oscillations or a slowed alpha rhythm, and only subjects who stayed awake were included.http://chronux.org/ for details). Length of the moving window and step size were set to EEG data were pre-processed and analyzed offline with MATLAB. Slow fluctuations were removed by local linear regression , and tinnitus loudness in dBSL  , and 5.An ANOVA revealed significant differences in theta band power  did not correlate significantly with band power averaged over all electrodes in any frequency band. Likewise, none of the correlations between oscillatory band power and the depression, anxiety or somatization symptom scale scores reached significance .Results of the present study support hypothesis (1) that increasing tinnitus loudness is associated with increasing gamma oscillatory power. This was found to be the case for the tinnitus loudness estimate derived by adjusting the reconstructed tinnitus sound (Hypothesis (2) that tinnitus loudness and tinnitus-related distress are associated with distinct brain activity patterns was also corroborated by the results of the present study. Tinnitus-related distress does not correlate with gamma band power, and significantly correlates with delta band power at locations which differ from those that correlate with tinnitus loudness. Beyond that high-distress tinnitus was associated with significantly higher theta band power compared to low-distress tinnitus.The exploratory analysis (3) and (4) revealed that gamma band power decreases with increasing MHL, and that increasing MML correlates with increasing delta band power. In contrast, psychological symptoms such as depressivity and anxiety did not significantly correlate with oscillatory band power.Recording of spontaneous brain activity revealed increases in oscillatory power in the gamma and delta band related to psychoacoustically determined tinnitus loudness. This increase was significant only when determining tinnitus loudness with the reconstructed tinnitus sound , and gamma oscillations are a component of ERP occurring about 100 ms and 300 ms after sound onset in cat hippocampus, reticular formation and cortex In addition, the tinnitus loudness In summary, whereas both enhanced gamma and delta activity in the  auditory cortex (AC) are associated with tinnitus loudness, only gamma activity is seen as a correlate of the tinnitus percept, and it may be related to attention directed towards and emotions generated by this percept.Increases of tinnitus-related distress correlate with increases of power in the delta band. This correlation loses significance, however, when controlled for general psychological distress (GPD). GPD does not exhibit a significant correlation with delta band activity on its own, which might be due to the circumstance that mean SCL-90-R scores were rather low even though depressivity and anxiety scores were significantly elevated in the present patient group. Besides that, content overlap between the TQ and SCL-90-R questionnaires may obscure the association between delta band activity and tinnitus-related distress when controlling for GPD. In addition, the association between tinnitus loudness and delta band power might have obscured the association between tinnitus-related distress and delta power in the global analysis, although according to the electrode specific analysis it has its maximum in the right hemisphere whereas correlation strength between tinnitus-related distress and delta band activity peaks in the left hemisphere.MEG studies also found enhanced delta band power in tinnitus patients compared to controls Tinnitus patients with high tinnitus-related distress exhibit higher theta oscillatory power than patients with low tinnitus-related distress. Depressiveness itself is associated with enhanced activity in the theta band Similarly, the reports on a correlation between tinnitus-related distress with alpha and beta band activity Minimum masking level (MML) represents the lowest level at which external sounds completely mask the tinnitus percept. Maskability by environmental sound provides the patient with an important, if not the only tool to influence his tinnitus percept, therefore it is of utmost clinical importance to understand the underlying mechanism In particular because of its clinical importance, mechanisms underlying tinnitus masking and their association with brain oscillatory activity should be addressed in further studies.This is the first report that finds a significant correlation between psychophysically determined tinnitus loudness and brain activity. In line with previous reports the tinnitus percept loudness correlates with gamma and delta oscillatory activity, but only when tinnitus loudness is estimated with a novel type of sound derived by tinnitus reconstruction. We report an easy to apply synthesization paradigm to generate this sound, which comprehensively reflects the spectral complexity of the tinnitus percept and for which patients report extraordinary similarity to their tinnitus. Because this novel tinnitus reconstruction achieves better results than other types of sounds, it is suggested to use it for determining tinnitus loudness in future studies.The results of the present study also support the clinically motivated distinction between tinnitus loudness and tinnitus-related distress which were shown to associate with distinct patterns of gamma and delta oscillatory brain activity. Additionally, tinnitus-related distress correlates with theta oscillatory activity which is known to be associated with depressivity and anxiety. The increase of delta oscillatory power together with increasing minimum masking level should be investigated in more detail in the future because of its high clinical importance as a tool to control the tinnitus percept.


Information on the impact of cecal insertion time on colorectal neoplasm detection is limited. Our objective was to determine the association between cecal insertion time and colorectal neoplasm detection rate in colonoscopy screening.We performed a cross-sectional study of 12,679 consecutive subjects aged 40–79 years undergoing screening colonoscopy in routine health check-ups at the Center for Health Promotion of the Samsung Medical Center from December 2007 to June 2009. Fixed effects logistic regression conditioning on colonoscopist was used to eliminate confounding due to differences in technical ability and other characteristics across colonoscopists.The mean cecal insertion time was 5.9 . We identified 4,249 (33.5%) participants with colorectal neoplasms, of whom 1,956 had small single adenomas (<5 mm), 595 had medium single adenomas (5–9 mm), and 1,699 had multiple adenomas or advanced colorectal neoplasms. The overall rates of colorectal neoplasm detection by quartiles of cecal insertion time were 36.8%, 33.4%, 32.7%, and 31.0%, respectively (p trend <0.001).The odds for small single colorectal adenoma detection was 16% lower  in the fourth compared to the first quartile of insertion time (p trend 0.005). Insertion time was not associated with the detection rate of single adenomas ≥5 mm, multiple adenomas or advanced colorectal neoplasms.Shorter insertion times were associated with increased rates of detection of small colorectal adenomas <5 mm. Cecal insertion time may be clinically relevant as missed small colorectal adenomas may progress to more advanced lesions. Early detection and removal of colorectal adenomas by colonoscopy decreases the risk of colorectal cancer , and colHigher adenoma detection rates are associated with more careful examination of the proximal sides of folds and flexures, with adequate distension, with cleaning of pools of fluid and dirty areas, and with time for scope withdrawal ,10. TakiWe conducted a retrospective study of screening colonoscopies at the Center for Health Promotion of the Samsung Medical Center in Seoul, Korea, from December 2007 to June 2009. We restricted the study to male and female participants aged 40 to 79 years old as this is the recommended age range for screening colonoscopy in Korea. From a total of 14,370 consecutive colonoscopies performed at the Center for Health Promotion, we excluded 862 colonoscopies conducted in participants who were <40 or ≥80 years of age, 526 colonoscopies performed for therapeutic (non-screening) purposes, 65 colonoscopies performed outside of the colonoscopy unit, 29 colonoscopies performed in participants with a history of colorectal cancer, inflammatory bowel disease or who had undergone colonic resection, and 113 colonoscopies with incomplete examination due to poor bowel preparation. In subjects who had colonoscopy multiple times during the study period (n = 98), we chose the first colonoscopy results. Because some individuals met more than one exclusion criterion, the final sample size was 12,679 . The study protocol was reviewed and approved by the institutional review board at Samsung Medical Center. The informed consent requirement was exempted by the Institutional Review Board because researchers only accessed retrospectively a de-identified database for analysis purposes.Twenty three board-certified gastroenterologists performed the colonoscopies. Each colonoscopist had performed more than 500 examinations before the study period or before working at the Center for Health Promotion. Colonoscopies were performed after bowel preparation with 4 L polyethylene glycol solution .Cecal insertion time was defined as the time from insertion into the rectum to the time when the colonoscope tip passed to a point proximal to the ileocecal valve so that the base of cecum was visible. Withdrawal time was defined as the time taken for withdrawing the colonoscope tip from the base of cecum to across the anus. Cecal insertion and withdrawal times were recorded immediately after finishing the examination by the colonoscopist. Bowel preparation was assessed as excellent , good , fair , or poor .Colorectal neoplasms were further classified as small single non-advanced adenomas < 5 mm, medium single non-advanced adenomas 5 to <10 mm,and multiple or advanced colorectal neoplasms . Advanced adenoma was defined as a tubular adenoma with diameter ≥10 mm, an adenoma with villous component, or an adenoma with high grade dysplasia. The size of each lesion was estimated using open biopsy forceps.2). Waist circumference was measured at the midpoint between the inferior margin of the last rib and the superior iliac crest in a horizontal plane. Information on history of colorectal polyps, diabetes mellitus, hyperlipidemia, medication use including aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), alcohol drinking and smoking and family history of colorectal cancer were collected using a self-administered questionnaire before endoscopy.A health questionnaire and a detailed physical exam were routinely completed as part of the screening program. Height and weight were measured using an Inbody 720 machine . Body mass index (BMI) was calculated by dividing measured weight (kg) by height squared (m2), waist circumference , family history of colorectal cancer, history of colorectal polyps, diabetes, hyperlipidemia, aspirin use, NSAIDs use, calcium use, alcohol drinking, and smoking .Colorectal adenoma proportions were compared across the quartiles of cecal insertion time. Odds ratios (ORs) and 95% confidence intervals (CIs) for the presence of any colorectal neoplasm for the 3 highest quartiles of insertion time compared to the first quartile were calculated using fixed effects logistic regression conditioned on colonoscopists. These regression models account for data clustering by colonoscopists and eliminate confounding due to differences across colonoscopists. Separate logistic regression models were used for the different colorectal neoplasms. In multivariable models, we adjusted for age , sex, withdrawal time , bowel preparation , BMI  age of study participants was 53.4(7.3) years and 62.9% of study participants were male Table . The proTheir median age of the 23 colonoscopists was 34 years . A majority of the colonoscopists were female (82.6%). The median number of colonoscopies performed by each colonoscopist during the study period was 592 . Before the study period, the 23 colonoscopists had performed a median of 1,400 colonoscopies .Colorectal neoplasms were detected in 4,249 participants .Click here for fileOdds ratios for colorectal neoplasm detection by cecal colonoscopy insertion times .Click here for file


They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from  Tandem repeats (TRs) are sequence repetitions that occur right next to each other. They are typically classified by the length of the tandemly repeated unit into: microsatellites (<10 bp), minisatellites (10–100 bp), and, albeit less widely used, megasatellites (>100 bp) . NucleicProtein TRs are a major element of protein sequences in all domains of life. They are typically encoded by nucleic TRs in coding regions such that a TR unit duplication/loss generally does not involve a frameshift . HoweverHuntingtin leads to Huntington's disease (l = 1), while in fruit flies, polyQ variation in the per gene tunes its circadian clock .The high gain/loss rates of nucleic TRs were proposed to be suggestive of comparable high rates for protein TRs . In partan clock . Other eoperties ; a similIt has been argued that variation in functional TRs may provide a source of genetic variability allowing for fast adaptation , for exaTo study their evolution, the sequence similarity of TR units within a single TR may be analyzed to infer gain/loss events . For exaFor the related problem of gene paralogs arranged in tandem, gene phylogenies  were shown useful for systematic studies of gene gain/loss events . Here, wA). Thus, this approach allows us to recognize TRs that have been conserved since the speciation event. For some proteins, such as the human TORC subunit LST8 (B), the conservation can be traced back deep to the common ancestor of human and yeast. On the other hand, we are also able to identify TRs that have been subject to recent TR unit gains/losses (C). Note that besides TR units gains/losses, other mechanisms such as intragenic conversion may lead to modifications in the TR configuration . An intragenic conversion event may be approximated by a TR unit loss that is immediately followed by a TR unit gain. When referring to gains/losses, we implicitly include such alternative mechanisms in the remainder of this article.Any observed TR region may have been conserved over a long evolutionary time scale, or alternatively, it may have originated very recently through rapid evolution. Without the study of orthologous TR regions from multiple species, it is impossible to deduce whether a TR is of a recent or ancient origin. To shed light on the age of a TR, one requires a mapping of TR unit gains/losses to speciation events, based on alignments of orthologous proteins with TRs from multiple species. This approach has been used to study the evolution of TR regions according to the TR unit number, similar to the analysis of microsatellite DNA data see A. Thus, nit LST8 B, the cos/losses C. Note thttp://www.atgc-montpellier.fr/TRE .In the following, we describe the multispecies analysis of TR unit phylogenies in detail, and introduce criteria to classify TRs according to their mode of evolution. We apply this approach in a proteome wide study on the evolution of human TRs with TR unit length ≥15 aa in comparison with orthologous TRs in 60 other eukaryotic species. For the vast majority of these human TRs, our analyses reveal a surprisingly sustained conservation of TR unit configurations  throughout the eukaryotic kingdom. Finally, we correlate the results with structural features of TRs, as well as functional annotations of TR-containing proteins, to better understand the origin and consequences of the different evolutionary modes. All material and data are available for download at To analyze the evolution of human TRs across the eukaryotic clade, we developed a phylogenetic pipeline, which can be summarized as follows:For each human TR-containing protein, orthology annotations were obtained from the complete proteomes of 60 other eukaryotic species from Ensembl Compara . For eacB), or alternatively, became “separated” through gains or losses of TR units after speciation (fig. 1C).The selected 60 species considered in our study are separated from the human lineage by a wide range of divergences, from chimp (the closest) and mouse, to zebrafish and baker’s yeast (the furthest). Comparisons of TRs in orthologous proteins in all pairs of species allowed us to describe the TR evolution since the speciation events of the species pairs. Taken together, these bispecies comparisons permitted to backtrack the conservation of human TRs throughout the eukaryotic clade. For this purpose, we reconstructed bispecies TR unit phylogenies for all pairs of TR-containing orthologs . These pfig. 1The classification of TRs according to conserved and separated modes of evolution was correlated with functional annotations and other TR characteristics, notably the type of Pfam family.TRs were annotated exhaustively in the human proteome using specifically devised circular profile hidden Markov models (cpHMMs).To annotate consecutive repeat units in sequence data, we have developed cpHMMs based on a sequence profile of a potential TR unit. In our model, the match states describe the consensus positions of the TR unit . In contA set of potential TR unit profiles was obtained from known protein domains and de novo detection of TRs in the human proteome. As the Pfam database  includesFinally, the cpHMMs were used to annotate human TRs. To control the number of false positive TRs, each annotation was statistically validated using a model-based likelihood ratio test . In summTo study the evolution of human protein TRs, we compared them with homologous TRs in orthologous proteins of eukaryotic genomes. Orthology annotations and protein-wide sequence data for 60 other eukaryotic species were obtained from Ensembl Compara . For eacTo systematically determine the mode of TR evolution, we studied bispecies TR unit phylogenies, assuming that all units have descended from an ancestral unit. In particular, for all human versus nonhuman orthologous protein pairs in our data set, we reconstructed phylogenetic histories of TR units similar to those in A and B, we calculated the following statistics:A and B), The TR unit numbers in the two orthologous proteins , , , and  leading to B.The conservation of TR unit order in bispecies cherries, measured by the Kendall rank statistic A and B  TR unit in the orthologous ancestral protein of species B (A). If no TR unit gains or losses occurred since speciation (B), the order and the number of TR units remains the same and the TR unit phylogenies fulfill perfectly conserved between species A and B. Presumably, the numbers of “perfectly conserved” TRs would be underestimated in our analysis, mostly due to errors in phylogenetic reconstruction, and in orthology annotation . To cushion errors in TR annotation and phylogeny reconstruction, we attributed strong TR unit conservation, if ith TR unit in one species may not pair with the ith TR unit in the second species in one case .In one possible evolutionary scenario, no unit duplications or losses occurred in the TR region in neither lineage of two species after speciation: The ancestral TR unit configuration  is then fully preserved in the current day species. Directly after the speciation, the closest relative of any TR unit in the ancestral protein of species pecies B A. If no eciation B, the orn TR units is n = 4, and as low as n = 5 . As discussed earlier, due to errors in tree reconstruction, TR pairs that are “perfectly separated” in reality might not appear separated into two clades on the inferred unit phylogeny. To account for some of these cases, we introduced the following relaxed condition: a pair of TRs exhibited “strong separation” when supplementary fig. S3, Supplementary Material online).The frequency of TR conservation in human proteome was contrasted with the frequency of TR separation, whereby all TR units from one species were more closely related to each other than to any TR unit in the other species, and vice versa. TR separation was evaluated based on the following rules. Two homologous TRs were assumed to exhibit “perfect TR unit separation” if  cf. C. As disIn a more complex scenario, some TR units of the same TR region may be conserved, whereas others may undergo duplications and losses. To account for this case, we attributed “difference in TR unit number” to pairs of TRs with unit number difference ≥4.n TR units is n = 4 and n = 5  to a pair of random TRs with We detected 3,091 nonoverlapping TRs (with ≥4 TR units of length  repeats . In totaA). Similarly, for each human TR-containing gene, we traced back the most distant ortholog that still contained at least four TR units (fig. 3A). We found that almost all TRs were as ancient as the TR-containing gene, and were lost only in rare cases. For example, of all human TR-containing proteins, 90% had an ortholog in a nonmammal species, of which 94% also contained the homologous TR with at least four units. This implies that the TR often is an essential component of the TR-containing protein.fig. 3S. cerevisiae. The TR conservation between human and S. cerevisiae provided strong evidence for the conservation of the human TR since their speciation, thus beyond the first bilaterians. Further, the conservation of a human TR compared with its ortholog in any of the other 60 species indicates that it has been conserved at least since the speciation of chimp and human, thus well beyond the most recent common ancestor of all humans. This definition is cumulative, that is, the number of TRs that are conserved to the root of a given clade  is less than  the number of TRs conserved to the root of any nested clade .To determine evolutionary ranges with evidence for TR conservation, we identified the species furthest to human for which either perfect conservation, or “strong conservation” was detected in the orthologous TR. For example, to establish conservation of a human TR at least to the root of the bilaterians, the human TR was compared with its orthologous TR—if present—in the nonbilaterian species available in our data set, namely A summarizes the numbers of human TRs that have been conserved since different eukaryotic speciation events, ordered from the most recent (human/chimp) to the oldest (human/yeast). We found that 92% of all human protein TRs was strongly conserved, and 90% was perfectly conserved between human and at least one other species in our data set. Surprisingly, such conservation was observed not only within closely related species: 61% of all protein human TRs were strongly conserved since the root of the mammalian clade, while 17% were strongly conserved since the root of the vertebrates. This shows that for the vast majority of TRs their mode of evolution is not marked by high rates of TR unit gain or loss.Figure 3To better understand the functional constraints that require conservation of TRs, we contrasted the subset of human proteins containing TRs that were strongly conserved in at least one species beyond the mammals  with all human TR-containing proteins . We studied the distribution of different TR types, as well as the enrichment of functional annotations for proteins with TRs using GOrilla .TR types strongly conserved at least to the root of all mammals are diverse, spanning 81% of all annotated human TR types. The distribution of different TR types among the proteins with conserved TRs is highly biased. For example, more than half (58%) of all conserved TRs could be attributed to just 5% of all TR types. In other words, a handful of TR types were observed at very high frequencies , whereas the majority of others appeared at low frequencies.Interestingly, for a range of TR types such as the Armadillo repeat (ARM), the HEAT repeat and the PHD-finger, all human TRs have been conserved since the ancestor of mammals. Likely, the unit configuration of these TR types is essential to maintain protein function, be it for structural reasons, or due to the function of certain amino acids on specific TR units.A). To reveal the correlation of TR type and protein function, we linked the results of the GO-enrichment analysis to the TR types found in the protein with enriched functions, as summarized in figure 4A.The high diversity of TR types found for conserved TRs was reflected in the diversity of functions performed by proteins with conserved TR types. A GO-term analysis of these proteins revealed enrichment in diverse biological processes, including prominently, stimuli response, cell adhesion, protein ubiquitination, locomotion, and regulation of development, particularly nervous system development. In terms of molecular function, proteins with conserved TR are enriched in particular in protein binding and catalytic activities A. To revThe TR type with the largest number of conserved TRs was WD40 . WD40 reA).Next to WD40 repeats, TRs with  binding . The sup binding , which i binding A.A). Among the most common TR types in this group were cadherins, ANKs, LRRs, and WD40 repeats (A). Structurally, these TRs are mostly located in the extracellular matrix, or the cytosol, but not within the membrane . Using this approach, for each TR we assessed when it was subjected to different degrees of unit gain/loss. Consequently we found that 5% (or 8%) of all human TRs were completely (or strongly) separated from at least one orthologous mammalian TR. In contrast, 61% of all human TRs were strongly conserved at least since the root of mammals. Note that it is possible that a TR was conserved in the human lineage beyond the ancestor of all mammals, while at the same time strong gains/losses leading to strong separation occurred on the lineage of another mammal after the separation from the human lineage. However, this is expected to be rare (0.5% of human TRs in our data), showing that within a given clade TRs consistently evolve by one single evolutionary mode, but not in a mixed-mode fashion.For different eukaryotic clades, we calculated the number of TRs that have undergone unit changes in at least one species within this clade , and a de novo TR in the NAC-alpha domain-containing protein 1 . In both examples, the TR consisted of almost identical TR units.One other possibility is that TR unit gains/losses do occur, but do not affect the entire TR region. To account for this case, we also annotated pairs of TRs that exhibited a difference in TR unit number: 235 or 8% of all human TRs showed a difference in TR unit number compared with their orthologous TRs in chimp or gorilla, corresponding to 34% of all human TRs that had not been strongly conserved in the same range. In comparison, only 4 or 0.001% of all human TRs showed strong separation compared with their orthologous TR in chimp or gorilla. Thus, in nonconserved TRs, unit mutations often lead to a change in the number of TR units without affecting the entire TR region.B).To shed light on TR characteristics that correlate with strong unit gains/losses, we contrasted the subset of proteins with perfectly or strongly separated TRs in at least one species within the mammals (236 TRs in 230 proteins) with all human TR-containing proteins . Similarly to strongly conserved TRs, we analyzed separated TRs with respect to the distribution of TR types and the GO-term enrichment B.supplementary fig. S6, Supplementary Material online). The family of zinc finger genes has been subject to a massive expansion in vertebrates, accounting for ∼2% of all human genes (B). Most of the zinc-finger-containing genes are arranged in clusters, and evolve through tandem gene duplications and losses , immunoglobulin I-set (PF07679), the calcium-binding EGF (PF07645), and an EGF-like (PF00008) domain repeats. In total, 52 distinct TR types were subject to strong separation in at least one human protein, with 30 of these being de novo annotations. The abundance of de novo detected TRs might imply that the TR types that undergo strong gains/losses in many cases may be relatively rare types, which have possibly appeared recently.In our proteome-wide analyses, most of the TRs were remarkably informative about their duplication history, despite their short sequence. As a result, we were able to classify the majority of human TRs as conserved (68%) with well-preserved TR unit configurations over long evolutionary distances , while only few TRs were separated (8%) with clear evidence of configuration changes in the same range. Below we discuss these sets of TRs, as well as the correlation of their evolutionary mode with TR characteristics including the TR unit number, length, between-unit divergence, as well as the exon structure underlying the TR region.Very few TRs appear to undergo rapid TR unit gains/losses. However, these few identified examples of separated TRs might exhibit variation within populations. Indeed, they include the zinc finger repeat in PRDM9, which carries strong variation in both chimp and human populations , and strPodospora anserina).Beyond these (few) examples our analysis shows that separation of TR units is extremely rare. Strikingly, no case of perfect separation was found between human and chimp or gorilla. In comparison, other types of sequence changes are much more common in this range: ∼70% of all proteins were subject to substitutions (median of two nonsynonymous substitutions per protein), and ∼5% were subject to in-frame indels in a comparison of the human and chimp proteomes . In summInterestingly, more TRs with difference in TR unit number were annotated de novo compared with all TRs . Having fewer Pfam annotations among variable TRs might indicate that de novo TRs are rare (otherwise they should be represented in Pfam), and therefore more likely recent. Indeed, 75% of those variable de novo TRs had no ortholog TR outside mammals, compared with 15% in the complete TR set. Our findings are consistent with a recent hypothesis that TRs may function as a substrate for the formation of new domains and subsequently new genes .B), armadillo (PF00514), and also repeats with other structural configurations such as the EF hand (PF00036)  and the prenyltransferase (PF00432), solenoid repeats such as WD40 (PF00400) B, armadisupplementary table S1, Supplementary Material online). This shows that the majority of TRs are conserved both in terms of the TR sequence and in terms of their unit configuration . Such sequence conservation on two levels is likely to be accompanied by an equally sustained structural conservation of TR regions, which is presumably required to maintain the function of the TR-containing protein.To investigate whether the conservation of a TR unit configuration is generally accompanied by the conservation of the TR unit sequence, we estimated the relative substitution rates in the TR and flanking regions . We found that the substitution rates in the TR region of strongly conserved repeats were on average 2.3 times lower than the rates in the protein sequence flanking the TR, both of which were by an order of magnitude below the respective rates for strongly separated TRs .Many more human TRs are conserved rather than separated . Conserved TRs clearly encompass more distinct TR types compared to separated TRs (∼3:1), although the ratio is lower due to the relatively large number of de novo TRs among the separated repeats. Interestingly, TRs of the same type may be found in proteins with either conserved or separated TRs  development, protein binding, but also catalytic activity. On the other hand, functions of separated TRs were dominated by DNA binding and gene expression regulation (largely due to zinc finger TRs).A). TR units in strongly separated TRs clearly had a lower divergence compared with TR units in strongly conserved TRs. At the same time, we found that substitution rates in the TR regions of strongly separated repeats were on average ten times higher than those for strongly conserved TRs . Taken together, strongly separated TRs had lower between-unit sequence divergence, despite higher substitution rates in the whole TR regions. The following may explain these apparently contradictory results. In strongly separated TRs, due to the elevated rates of TR unit gain/loss, some TR units repeatedly get lost, while others duplicate in identical copies. As long as the substitution rates do not exceed the unit gain/loss rates, the TR units within the TR region will preserve high similarity (or low divergence) with each other . The tendency of separated TRs to contain more units may be grounded in molecular biases: For nucleic tandem repeats, it has been observed that TRs with more units are subject to increased duplication rates  proteins with widely varying numbers of TR units. For example, in conserved human zinc finger TRs unit numbers ranged from (the minimum of) 4 to 39. This holds for TRs that fold into the “beads on a string” structure such as the zinc finger TRs , but alsC). For nucleic microsatellite and minisatellite TRs, for example, TRs with shorter units also have higher TR unit duplication rate . Possibly, as the TR unit length decreases there would be a transition from observing mostly conserved TRs to gradually observing more and more separated TRs. Indeed, frequent insertions/deletions have been observed for homorepeats and dipeptide repeats among primates, including human  we observed a clear decrease in unit conservation, as well as an increase in the relative proportion of strongly separated TRs, compared with TR regions with longer units (ng human . In partng human . On the ng human . Note thD and E).Frequently, exons in TR-containing proteins span multiples of whole TR units . TRs mayfig. 5In our data, 31% of all separated TRs were contained within a single exon. For these, we can exclude the exon-shuffling-like process to explain TR unit changes. On the other hand, for 26% of all separated TRs, one exon corresponded to at most one TR unit. Lacking proximity of the protein TR units in the nucleic sequence, these TRs most likely evolve through an exon shuffling-like process. Another indicator for the mechanism of TR units gains/losses can be derived from the exon structures of multiple TRs of the same TR type. For example, we found that whilst the number of NBPF TR units varied widely (up to 55) in all 14 NBPF-containing proteins, the number of TR units per exon stayed constant (2 or 3). This indicates that NBPF TRs evolve through an exon-shuffling-like process.E), whereas the conserved TRs did not show this behavior. Possibly, for some conserved TRs the presence of multiple exon boundaries rupturing the TR unit structure on the nucleic level may prevent duplications/losses of TR units.Altogether, both mechanisms of unit gains/losses seem to play an important role during the TR evolution. Interestingly, many of the separated TRs either were found to occupy one exon per TR unit, or contained many TR units per exon, exhibiting a roughly bimodal distribution in terms of the maximum number of TR units per exon E, whereal ≥ 15 aa) exhibit sustained conservation deep into the eukaryotic tree, with many TR regions preserved even since the common ancestor of human and yeast. Surprisingly, TR regions are frequently the most conserved part of the protein sequence. Conserved TRs can be found in proteins performing a wide variety of key functions. All together, our observations suggest a pronounced role of protein TRs in the function of the TR-containing protein, indicating that the functional significance of TRs has been underestimated. On the other hand, we found only few TRs with evidence for recent and strong TR unit gains/losses. To better understand the functional and potentially adaptive relevance of this small set of fast evolving protein TRs in the future, a casewise analysis of their function may be of interest.Our genome-wide study of the evolution of human protein TRs demonstrates that despite the common belief that TRs evolve rapidly, large numbers of protein TRs  tandemly repeated PFAM A domains and 2) de novo detections.supplementary fig. S1, Supplementary Material online). Annotated this way, TRs were retained for further analyses if they had at least four TR units  and statistical significance  was used for further analyses. Profile HMMs of de novo TRs were built using HMMER . Again, we refined the de novo based TR annotation of human proteins using cpHMM, and statistically validated all refined TRs . For each TR MSA that contained at least four TR units in both orthologs, we reconstructed bispecies maximum likelihood TR unit phylogenies using PhyML 3.0  genes from other eukaryotes as represented by the Ensembl Compara gene trees. Next, we built MSAs (Multiple Sequence Alignments) for each ortholog pair (hyML 3.0  with defhyML 3.0 .TR unit length (Effective) number of TR units TR unit divergence nce test :  is meaThe number of exons spanning the TR region at least partly.The maximum number of complete TR units in a single exon. The last two statistics relied on the exon structure of the human TR-containing proteins according to Ensembl v.69.We correlated TR classification with a range of TR characteristics . For thihttp://www.atgc-montpellier.fr/TRE .Ensembl protein identifiers were mapped to HGNC symbols. The mapping of HGNC symbols to GO functional annotations, and the enrichment analysis assuming a hypergeometrical model was conducted with Gorilla . All TR-For all pairwise alignments of human–mouse TR-containing orthologs in Ensembl Compara, the evolutionary distances between the TR regions in both species (n units is as low as n = 4 (5), rendering an overestimation of TR conservation unlikely (see the derivation discussed later). In comparison, inference errors in orthology annotation and phylogeny reconstruction are disproportionally more likely to obscure a perfectly conserved TR. Thus, the observed number of conserved TRs is presumably a lower boundary to the actual number of conserved TRs.The probability of falsely assigning perfect conservation to a pair of random TRs with A, and B. Assume Let A and B so that Kendall’s A is always paired with the B).In a phylogeny with perfectly conserved TR units a) all leaves are paired in cherries and b) each cherry groups TR units one from each of a)The probability distribution of model is cn = n) isb)The probability of Thus, the probability that all leaves are paired in cherries A and B is Assumed is a topology with c)A is Assumed is a topology with The formula follows.n TR units is n = 4 (5) (see the derivation later), which is elevated compared with the probability of falsely assigning perfect conservation. Inference errors in phylogeny reconstruction may still tend to cause an underestimation of the number of separated TRs. On the other hand, errors in sequencing and orthology annotation are expected to lead to an overestimation of the number of TRs that are separated or show a difference in TR unit number.The probability of falsely assigning perfect separation to a pair of random TRs with A or B (see fig. 1C for one such configuration). The parsimony score of such a phylogeny is A and B, so that N = Let Since the number of distinct rooted binary trees with aves is   the numbAny perfectly separated bisample TR unit phylogeny has exactly one bipartition separating the tree into two subtrees each of which with leaves representing TR units from only one species either With the results of For Supplementary figures S1–S6 and table S1 are available at Molecular Biology and Evolution online .


Childhood hearing impairment is a common chronic condition that may have a major impact on acquisition of speech, social and physical development. Numerous literature states that injury to the vestibular organs may result in accompanying balance and motor development disorders. But still postural control and motor assessments are not a routine procedure in hearing impaired children. Hence, we aim to provide an overview on motor skill performance and balance in hearing impaired children. Childhood hearing impairment is a significant public health problem, which is associated with long-term academic and communicative difficulties ,2. The pNewborn hearing screening has led to earlier identification and treatment of infants with hearing loss ,8. HowevAs routine screening does not include assessment of balance and motor deficits, physical therapy services are not included in the educational programme, unless obvious neurological or orthopedic disorders are diagnosed. However teachers and parents of these children often report inco-ordination, clumsiness and balance deficits which may hinder the child's optimal performance . MoreoveThe degree of hearing loss explains the severity of hearing impairment. Table American speech-language hearing association has categorized hearing loss based on the part of the auditory system damaged. Accordingly there are three basic types of hearing loss: conductive hearing loss, sensorineural hearing loss, and mixed hearing loss .Results from disruption in any of the mechanism, which conduct sound waves from external canal to the oval window. eg. Outer ear, ear drum or middle ear. It usually involves inability to hear faint sounds.It is the most common type of permanent hearing impairment and results from damage to the inner ear or the nerve pathway from the inner ear to the brain.It is a combination of both conductive and sensorineural hearing impairment.Furthermore, hearing impairment may exist as unilateral or bilateral, and pre-lingual or post-lingual.People have impairment in only one ear.People have impairment in both the ears.It refers to hearing impairment that is sustained prior to the acquisition of language. eg. Congenital, early infancy.It refers to hearing impairment that is sustained after the acquisition of language, which can occur as a result of disease, trauma or a side-effect of a medicine .Davidson et al has proposed an etiologic classification which clarifies the interaction between time of insult, causation and time of expression of hearing loss. Most of the SNHL are idiopathic, and appears to occur almost twice as often as in developed countries. Where the cause is known it may be genetic; pre-natally acquired; peri-natally acquired; post-natally acquired; cranio-facial anomalies; and other ,14.Almost 50% of permanent childhood hearing impairments have a genetic cause and influenced by consanguinity. It may be autosomal dominant, autosomal recessive and sporadic inheritance ,15. ParkPrenatal Infections such as rubella and toxoplasmosis are considered to be the main cause of prenatally acquired hearing impairment. Maternal exposure to alcohol, streptomycin, quinine and chloroquine phosphate may destroy neural elements of the inner ear and contribute to congenital hearing loss . Sever ePerinatal factors such as prematurity, low birth weight, hypoxia, Apgar scores 0-4 at 1 min; and 0-6 at 5 min, ventilation required for five days or more, hyperbilirubinemia requiring transfusion, and admission to the neonatal intensive care unit for 48 hours or longer may predispose to permanent childhood hearing impairment . Davis aThere are many and varied postnatal causes for childhood deafness, which may result in sensorineural or conductive loss or both. The postnatal cause for childhood deafness includes bacterial meningitis, infection , viral labyrinthitis (eg. measles and mumps), recurrent or persistent OME for at least 3 months, complications of otitis media, immunization, genetic causes, and head trauma with loss of consciousness or skull fracture .Hearing is a reflex during the first couple of the month of life. Responses to sound at a threshold are yet to obtain during this period. The only response the child posses during this period is the startle reflex- a response to a loud sound, which is analogous to the protective reflex of hearing in animals. Following this, there is a development of comprehension hearing. The child begins to understand the sound with the help of experience and learning as the cerebral cortex takes over the control of auditory responses, with the simultaneous inhibition of the reflex hearing. Thus the child at the age of six to seven months does not give any response to a loud sound, but turn and look towards a faint sound that the child feels to be pleasant .It is hard to know whether a child has a hearing problem or not. Hearing problems may be suspected if a child does not respond to sound or does not develop language skills appropriately. The National Institute on Deafness and Other Communication Disorders (NIDCD) has listed age-appropriate hearing milestones .Several hierarchies of auditory development have been proposed by numerous researchers and interventionists. Pollack et al has ranked auditory development in four stages .Hearing a sound, pay attention to auditory signals, attend to sounds at a distance, search for and turn to the source of sound.Discriminate the auditory stimuli, monitor the information and modify the speech production.Select a word from group of words spoken, remember and recall information and language make cognitive judgements.Synthesize meaning and respond to spoken language appropriately.It is necessary to understand the neurophysiology of postural control and motor development to get a clear construct of dysfunction. The ability to maintain a static posture  and dynamic posture  is operationally defined as static and dynamic balance respectively. Both the static and dynamic postural control are very important and necessary to execute movement ,24. The Delayed postural development and motor development is a common sensorimotor impairment in profoundly deaf children. The vestibular end-organ and cochlea are closely related both anatomically and functionally. Therefore, injury or trauma prenatally, perinatally, or postnatally may cause damage to one or both systems -30. MoreEarly identification of childhood hearing impairment and prompt intervention are crucial for improved outcomes. Vestibular deficit related impairments and the efficiency of therapy intervention for such impairments in children are only recently documented -41. In sChildhood hearing impairments are a world-wide problem that causes the most serious limitation that can befall a child, as it prevents his optimal development. It has to be viewed as a multifaceted condition as a variety of factors determines the effect of hearing impairment on child's development. The focus of evaluation and treatment for these children is primarily on the language development. Therefore in order to minimize the adverse effects on normal development of hearing disorders, it is crucial to carryout screening examinations and appropriate interventions of balance and motor deficits, which enable early detection of these dysfunctions, which are often either not noticed or under estimated. It is also important to re-evaluate motor function in these children during the course of their childhood in order to assure early intervention.The authors declare that they have no competing interests.Both the authors contributed to the conception of the study and were involved in writing, revising and approving the final draft of the manuscript.